Large Animal Review - Year 26, Number 6, December 2020

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Poste Italiane spa - Spedizione in A.P. - D.L. 353/2003 (conv. in L. 27/02/2004 N. 46) art. 1, comma 1, DCB Piacenza - Concessionaria esclusiva per la pubblicità: E.V. Soc. Cons. a r.l. - Cremona

06/20

Bimonthly, Year 26, Number 6, December 2020

LAR Large Animal Review

ISSN: 1124-4593

LARGE ANIMAL REVIEW is ranked in Citation Index (SciSearch®) Journal Citation Reports/Science Edition and CAB ABSTRACTS

ORIGINAL ARTICLES BOVINE • Milk yield and quality characteristics of an endangered Italian cattle breed: the Pontremolese • Monitoring and benchmarking antibiotic usage of Italian beef farms: a pilot study • Effect of melatonin implantation on haematological parameters in anestrus lactating buffalo during summer season under tropical conditions • Comparative study on the potency of trivalent vaccine of foot and mouth disease in different cattle breeds and triggered immune response signaling pathway OVINE • Effects of omega-3 and omega-6 fatty acids on some reproductive parameters in ewes CAPRINE • Valutazione della sostenibilità ambientale della produzione di latte e formaggio caprino mediante approccio LCA RUMINANTS • Antibacterial and repellent activities of Hypericum perfoliatum (St. John’s Wort) on different bacterial strains and anatomical tissues of Ovine and Bovine species SWINE • SNPs discovery in RRLs from DNA pools of Nero Siciliano pigs with extreme and divergent phenotypes for the Back Fat Thickness (BFT) tract POULTRY • Effect of dietary supplementation of Panax ginseng leaf extract on production performance and egg quality of hens at the beginning of their laying period EQUINE • Comparison of repeatable and random regression models for genetic parameter estimation on Thoroughbreds REVIEWS BOVINE • Malattie congenite del sistema nervoso del bovino EQUINE • Equine laminitis. New insights into the pathogenesis CASE REPORTS CAMELID • Pregnancy toxemia and lipid mobilization syndrome in two alpaca (Vicugna pacos) at 6 and 10 months of gestation

SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

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INDEX

ORIGINAL ARTICLES

Anno 26, numero 6, Dicembre 2020 Rivista indicizzata su: CAB ABSTRACTS e GLOBAL HEALTH IF (2019): 0.299

N

MINA MARTINI , IOLANDA ALTOMONTE, FRANCESCA GABRIELLI, ACHILLE GUASTALLI, FEDERICA SALARI

Editor in chief: Massimo Morgante Editorial Board 2019-2021: Anna Rita Attili - Roberto Bardini Francesca Bonelli - Marta Brscic Marco Colombo - Vincenzo Cuteri Antonella Dalle Zotte - Enrico Fiore Giovanni Franzo - Matteo Gianesella Elisabetta Giudice - Paolo Moroni Davide Ranucci - Antonia Ricci Giuseppe Stradaioli - Erminio Trevisi

LINDSEY LORENZO LAZZARINO, ALBERTO FERRERO, GIORGIA VEZZARO, VALENTINA MARIA MERLINO

275

Effect of melatonin implantation on haematological parameters in anestrus lactating buffalo during summer season under tropical conditions

Technical Editor: Enrico Fiore

Consiglio direttivo SIVAR 2020-2023 Mario Facchi (Presidente) Daniele Gallo (Presidente Senior) Alberto Ferrero (Vice-Presidente) Michela Conterbia (Segretario) Vito Loconte (Tesoriere) Alessandro Federici (Consigliere) Osvaldo Parolin (Consigliere) Chiara Musella (Consigliere) Mattia Bottacini (Consigliere) Giuseppe Argiolas (Consigliere)

267

Monitoring and benchmarking antibiotic usage of Italian beef farms: a pilot study

Managing Editor: Matteo Gianesella

LARGE ANIMAL REVIEW è una rivista bimestrale pubblicata per favorire l’aggiornamento dei veterinari che si dedicano alla prevenzione e alla cura delle malattie degli animali da reddito e alla qualità e salubrità dei prodotti derivati.

BOVINE Milk yield and quality characteristics of an endangered Italian cattle breed: the Pontremolese

TAMER RAMADAN, RAKESH SHARMA, SUSHIL PHULIA, INDERJEET SINGH

283

CAPRINE

j

Valutazione della sostenibilità ambientale della produzione di latte e formaggio caprino mediante approccio LCA STEFANIA CELOZZI, SILVANA MATTIELLO, MONICA BATTINI, GIOVANNI BAILO, LUCIANA BAVA, ALBERTO TAMBURINI, IRENE VALSECCHI, MADDALENA ZUCALI 293

SWINE

O

Edizioni SCIVAC Palazzo Trecchi - 26100 Cremona Tel. 0372/460440 Iscrizione registro stampa del Tribunale di Cremona n. 299 del 25/9/1995

SNPs discovery in RRLs from DNA pools of Nero Siciliano pigs with extreme and divergent phenotypes for the Back Fat Thickness (BFT) tract ANNA MARIA SUTERA, ALESSANDRO ZUMBO, IRENE SAPIENZA, GIUSEPPE TARDIOLO, ENRICO D’ALESSANDRO

301

Direttore Responsabile Antonio Manfredi Stampa Press Point - Via Cagnola, 35 20081 Abbiategrasso (MI) - Tel. 02/9462323 Spedizione Poste Italiane SPA - Spedizione in A.P. D.L. 353/2003 (Conv. in L. 27/02/2004 N. 46) Art. 1, Comma 1, DCB Piacenza Concessionaria esclusiva per la pubblicità E.V. Soc. Cons. a r.l.

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REVIEWS

N

BOVINE Malattie congenite del sistema nervoso del bovino ARCANGELO GENTILE, JOANA JACINTO GONÇALVES PONTES, CINZIA BENAZZI, MARILENA BOLCATO 305

CASE REPORTS CAMELID Pregnancy toxemia and lipid mobilization syndrome in two alpaca (Vicugna pacos) at 6 and 10 months of gestation LAKAMY SYLLA, MARTINA CROCIATI

317

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SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

SAVE the DATE CORSO PER VETERINARIO AZIENDALE AI SENSI DEL DM 7 DICEMBRE 2017 Settore ovino e caprino 23-26 Febbraio 2021 FAD - Dalle 14 alle 18 Accreditamento ECM

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M. Martini et al. Large Animal Review 2020; 26: 267-272

Milk yield and quality characteristics of an endangered Italian cattle breed: the Pontremolese

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N

MINA MARTINI1,2, IOLANDA ALTOMONTE3, FRANCESCA GABRIELLI4, ACHILLE GUASTALLI5, FEDERICA SALARI1 1

Dipartimento di Scienze Veterinarie, University of Pisa, Pisa, Italy Centro Interdipartimentale di Ricerca Nutraceutica e Alimentazione per la Salute (NutraFood), University of Pisa, Pisa, Italy 3 Centro di Ricerche Agro-Ambientali «Enrico Avanzi», University of Pisa, Pisa, Italy 4 Ambito Territoriale di Caccia Massa, Massa Carrara, Italy 5 Associazione Regionale Allevatori Toscana, Sesto Fiorentino (Firenze), Italy 2

SUMMARY The Pontremolese cow is a relic breed at risk of extinction. It reached a minimum number of heads bred in 2008 (14 heads) and more recently a slight increase was recorded in the number of individuals bred to about 70 heads. To date according to the FAO, the situation of the breed remains critical.

Given its great rusticity, the Pontremolese is used for the cow-calf breeding system in marginal areas and for the production of a typical local single-breed cheese.

This study characterizes for the first time the quality and nutritional characteristics of the Pontremolese cow’s milk in order to enhance its production.

Milk samples were taken from Pontremolese cows and analyzed at 30, 60 and 90 days of lactation.

Milk was characterized by a protein content of 3.42% ± 0.344 and a fat content of. 3.5% ± 1.779. The average diameter of the fat globules was 4.63 µm ± 1.279, while the analysis of the fatty acid profile showed an average content of 69% saturated fatty acids, 31% unsaturated fatty acids, and an omega-6 / omega-3 ratio of 4.14. In addition, the milk had a higher fat desaturation index (29.25) than that reported in the literature for Bruna and Italian Friesian cattle. During the first 90 days of lactation, no statistically significant changes were observed in the quality of the milk, or in the fatty acid profile. Increases were found in the lipid content, the fat globule diameter and in the content of C15:0, C18:1 t9, C18:1 t11, of C18:3 n3 and total omega 3. Similarities in the fat and protein content were observed Reggiana cows, which are traditionally used for the production of Parmigiano Reggiano cheese. These similarities could be related to the hypothesis that traces the origin of the breed back to a variety of the Reggiana cow.

KEY WORDS Pontremolese cow, milk quality, fatty acids, desaturase index.

INTRODUCTION Highly selected breeds have grown in numbers at the expense of local cattle breeds, which have become endangered or extinct 1.

The excessive specialization of the livestock sector is a major cause of biodiversity losses, for instance in Europe the Friesian breed represents about 60% of livestock 2, the loss of biodiversity also leads to an impoverishment of local products.

The survival of local breeds is mainly due to their ability to adapt to environments characterized by soil and climatic conditions which are unsuitable for cosmopolitan breeds. The ability of local breeds to adapt to environmental or climate conditions is important when considering climate change. In addition, these breeds can play a socio-cultural, landscape, and natural role in promoting the region they originate from.

Corresponding Author: Iolanda Altomonte (altomonte@vet.unipi.it).

In Italy there are currently 16 autochthonous cattle breeds at risk of extinction (www.associazionerare.it). The increasing demand by consumers for typical local products provides a strong incentive for the enhancement of local breeds.

The area of origin of the Pontremolese cow is Tuscany (central Italy), but in the past, the breed was also widespread in the provinces of La Spezia and Parma, and in other provinces of northern Italy. The origin of this breed has been traced back to the Parmigiana cow, a variety of the Reggiana bred in the hills. Some scholars have shown its affinity with local cattle (Bardigiana, Valtarese, Cornigliese cows) from Emilia Romagna that are now extinct and are all linked to an Iberian population that once settled on the hilly and mountainous areas of the Apennines.

The Pontremolese is a triple-aptitude breed, which showed qualities of robustness and rusticity, and in the past was used to transport marble in the Carrara quarries, or for work on the plough. This breed was also used for meat and milk production, but it decreased in number due to the competition of the more productive Alpine Brown cows (www.aia.it).

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Milk yield and quality characteristics of an endangered Italian cattle breed: the Pontremolese

The Pontremolese is a relic breed at risk of extinction. It reached a minimum number of heads bred in 2008 (14 heads) and more recently a slight increase was recorded in the number of individuals bred to about 70 heads (www.fao.org). It is reared in small farms mainly in the Tuscan-Emilian Apennines. To date according to the FAO, the situation of the breed remains critical.

Given its great rusticity, the Pontremolese is used for the cowcalf breeding system in marginal areas. It was also included among the breeds whose meat has been allocated the Designation of Origin, «Beef of Garfagnana and Valle del Serchio» 3.

Currently Pontremolese milk is used for the production of a typical local single-breed cheese. Since the quality of milk plays an important role in all dairy products, particularly for niche ones, the aim of this study was to characterise the quality and nutritional characteristics of Pontremolese milk for the first time in order to revitalize the interest in this breed and the local cheese and to strengthen the importance of the breed in the area.

MATERIALS AND METHODS Animals and sampling Individual milk samples were taken from the morning milking at 30, 60 and 90 days of lactation from seven Pontremolese cows (about 10% of the breed population) for a total of 16 milk samples (approx. 500 ml each), analysed in duplicate.

The cattle were reared in one farm and the management system was based on indoor farming, the animals were fed a traditional diet based on dry forage and concentrate (Table1); a mechanical milking system was adopted.

Chemical and physical quality of milk and somatic cell counts Total nitrogen, caseins, dry matter and ash were determined according to the methods of the Association of Official Analytical Chemists 4.

Fat and lactose contents were analysed by infrared analysis using a fully automatic milk analyser (MilkoScan™ 7 RM; Italian Foss Electric, Padua, Italy).

The pH was assessed by the potentiometric method using a Thermo Fisher Scientific Inc. pH meter (Waltham, USA).

The somatic cell count (SCC) was evaluated by the fluor-optoelectronic method (Fossomatic Italian Foss Electric, Padova, Italy).

Milk fat extraction was performed following Rose-Gottlieb’s method and methyl esters of fatty acids were prepared according to Christie 4. All solvents and reagents were of analytical grade and were purchased from Sigma Aldrich (Milan, Italy).

A PerkinElmer Clarus 480 (PerkinElmer, Norwalk, CT, USA) equipped with a flame ionisation detector and a capillary column (ThermoScientific TR-FAME 60 m × 0.25 mm ID; film thickness 0.25 m, Fisher Scientific, Loughborough, Leicestershire, UK) was used. C5:0 was used as internal standard. The peak areas of individual FAs were identified using a FA standard injection (Food Industry FAME Mix – Restek Corporation, Bellefonte, PA, USA) and quantified as the percentage of total FAs. The helium carrier gas flow rate was 1 mL/min. The oven temperature program level 1 was 50°C held for 5 min; level 2 was 50 to 140°C at 3°C/min, then held for 2 min; and level 3 was 140 to 240°C at 1°C/min, then held for 10 min. The injector

Table 1 - Chemical composition of grass hay and concentrate offered to the cows.

Components

DM

Grass hay

Commercial concentrate

g/100g

88

91.6

g/100g of DM

9.8

20.74

Fat

2.5

3.93

Crude fiber

33

9.06

Ash

9

6.55

Na

nd

0.22

Crude protein

Abbreviations: DM: dry matter; n.d.: not determined.

Trace element compounds and vitamins of the commercial feed (per kg of DM): 196 mg of iron carbonate [II], 54 mg of copper sulphate pentahydrate, 141 mg of zinc oxide, 147 mg of manganese oxide, 1.2 mg of sodium selenite, 6.5 mg of anhydrous calcium iodate, 3275 IU of vitamin A, 2183 IU of vitamin D3, 40.93 mg of vitamin E, 0.14 mg of vitamin B1, 1.09 mg Vitamin B6, 0.049 mg of vitamin B12, 109 mg of niacinamide, 76 mg of Choline Chloride, 27 mg of Betaine anhydrous.

and detector temperatures were set at 270 and 300°C, respectively.

Milk fatty acids were grouped as saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA).

To evaluate the nutritional properties, the atherogenic (AI) and thrombogenic (TI) indices were calculated as suggested by Ulbricht and Southgate 5, while the total desaturation index was calculated according to Schennink et al.6.

Total desaturation index = (C10:1 + C12:1 + C14:1 c9 + C16:1 c9 + C18:1 c9 + CLA c9, t11)/(C10:1 + C12:1 + C14:1 c9 + C16:1 c9 + C18:1 c9 + CLA c9, t11 + C10:0 + C12:0 + C14:0 + C16:0 + C18:0 + C18:1 t11) × 100.

Morphometric analysis of milk fat globules was evaluated according to the direct method by Martini et al. 7. The globules were grouped into three sizes: small globules (SG) with a diameter of <2 μm, medium-sized globules (MG) with a diameter from 2 to 5 μm, and large globules (LG) with a diameter of >5 μm.

Statistical analysis Mean and standard deviation of the chemical and physical qualTable 2 - Chemical, physical characteristics and somatic cell count in Pontremolese cow’s milk (mean and standard deviations).

-

Mean

SD

pH

6.69

0.236

Dry matter (%)

12.71

1.505

Protein (%)

3.42

0.344

Casein (%)

2.67

0.232

Fat (%)

3.5

0.779

Lactose (%)

4.61

0.573

Ash (%) Somatic cell count *1000

0.78

0.091

154.13

129.041

Diameter of MFG (µm)

4.63

1.279

SG (%)

21.27

15.200

MG (%)

39.67

19.707

LG (%)

39.06

22.303

MFG: milk fat globules; SG: small globules; MG: medium globules; LG: large globules; SD: standard deviation.

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M. Martini et al. Large Animal Review 2020; 26: 267-272

ity of the milk and the somatic cell count were calculated.

In addition, the results were analysed using ANOVA for repeated measurements, considering the lactation period (30, 60 and 90 days) as the fixed effect and the subject as a random effect. Least significance means were compared by the t-test. Significant differences were considered at P ≤ 0.05. Statistical analysis was carried out using JMP software 8.

RESULTS The average milk yield produced by the morning milking of the cows was 8.00 L ± 1.60.

The average physical chemical composition of milk and the SCC are shown in Table 2. Milk had an average total protein content of 3.42% ± 0.344, while of the total proteins, on average 78% consisted of caseins (Table 2).

In terms of the lipid fraction, fat was on average 3.5% ± 1.779, and the mean diameter of the fat globules was 4.63 µm ± 1.279, in addition approximately 80% of the globules were larger than 2 microns.

Analysis of the milk fatty acid profile (Table 3) showed an average content of 69% saturated fatty acids of, 31% unsaturated fatty acids, and an omega-6 / omega-3 ratio of 4.14.

In the first 90 days of lactation, the milk samples did not show significant changes in the physico-chemical parameters (Table 4), although an increasing trend in the lipid content was found, as well as an increasing trend in the diameter of the fat globules, and in the pH and somatic cell count.

In terms of the fatty acids (Table 5), no significant differences were found in the period of the study, although increasing trends were found in the content of C15:0, C18:1 t9, C18:1 t11 of C18:3 n3 and total omega 3; while decreasing trends were found in C18:2 c9, c12 and total omega 6 and omega-6 / omega-3 ratio.

269

Table 3 - Fatty acid profile, nutritional and desaturation indexes in Pontremolese cow’s milk (mean and standard deviations).

Fatty acids (g/100g of total fatty acids)

Mean

SD

C10:0

Capric acid

2.61

0.809

C11:0

Undecylic acid

0.05

0.035

C12:0

Lauric acid

3.05

0.935

C13:0

Tridecylic acid

0.12

0.039

C14:0

Myristic acid

11.64

2.101

C14:1

Myristoleic acid

1.18

0.328

C15:0

Pentadecylic acid

1.55

0.364

0.45

0.138

Palmitic acid

32.67

4.366

Palmitoleic acid

1.84

0.482

Margaric acid

0.83

0.246 0.179

C15:1 C16:0 C16:1 n7 C17:0 C17:0

-

0.46

C18:0

Stearic acid

11.13

3.347

C18:1 t9

Elaidic acid

0.15

0.077

C18:1 t11

Vaccenic acid

0.95

0.407

C18:1 c9

Oleic acid

21.91

5.253

-

0.4

0.145

C18:2 t9,12 (n6) C18:2 c9,12

Linoleic acid

1.98

0.333

C18:3 c6,9,12

γ-Linolenic acid

0.04

0.011

C18:3 c9,12,15

0.211

α-Linolenic acid

0.39

C20:0

Arachidic acid

0.22

0.095

C18.2 c9, t11

Rumenic acid

0.68

0.186

C20:1

Gondoic acid

0.25

0.063

C21:0

Heneicosylic acid

0.05

0.028

C20:2

Eicosadienoic acid

0.04

0.014

Dihomo-γ-linolenic acid

0.11

0.029

DISCUSSION

C20:4 c5,8,11,14

Arachidonic acid

0.001

0.001

C20:3 c11,14,17

Eicosatrienoic acid

0.14

0.041

The average milk yield per milking was lower than cosmopolitan breeds, which are selected according to the milk yield, but comparable to those reported for the Reggiana 9.

The protein content, which is known to influence the milk clotting ability, together with lactose and pH, showed similar values to those in the literature for cow’s milk 10,11. The average total milk protein content detected during the period analysed was between the values reported for Italian Friesian (www.anafi.it) and Italian Brown cows (www.anarb.it) (3.35% and 3.6% respectively).

Although breed is only one of the factors to influence the quantity and quality of fat content, our results showed more similar values to those reported for the Italian Friesian (www.anafi.it) and lower than the Italian Brown (www.anarb.it) (3.7% and 4.0% respectively).

In Pontremolese milk, similarities in fat and protein contents have been reported in several autochthonous breeds from northern Italy and Reggiana cows (fat: 3.51%; protein: 3.38%) 9. The milk compositional similarities between the Pontremolese and Reggiana breeds could be linked to origin of the breed being traced back to a variety of Reggiana traditionally used for the production of Parmigiano Reggiano cheese.

The average diameter of the fat globules was 3.5–5.5 μm, which is within the range of findings reported for cow’s milk 12. A ge-

C22:0

Behenic acid

0.1

0.048

C22:1

Erucic acid

0.04

0.02

Decosapentaenoic acid

0.01

0.006

C23:0

Tricosylic acid

0.04

0.009

C22:2

-

0.06

0.03

C24:0

Lignoceric acid

0.06

0.033

C24:1

Nervonic acid

0.01

0.006

C22:5 n3

Decosapentaenoic acid

0.11

0.036

C22:6 c4,7,10,13,16,19

Docosahexaenoic acid

0.01

0.006

C20:3 c8,11,14

C20:5 c5,8,11,14,17

Omega 3

0.65

0.24

Omega 6

2.54

0.395

SFA (%)

68.78

6.238

MUFA (%)

27.25

5.743

PUFA (%)

3.97

0.714

UFA/SFA ratio

0.47

0.139

Omega-6/omega-3 ratio

4.14

0.997

Atherogenic Index

2.87

0.934

Thrombogenic Index

3.40

0.880

Desaturation index

29.25

6.474

SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; UFA = unsaturated fatty acids; SD: standard deviation.

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Milk yield and quality characteristics of an endangered Italian cattle breed: the Pontremolese

Table 4 - Chemical, physical characteristics and somatic cell count in Pontremolese cow’s milk during the first 90 days of lactation.

30

Days of lactation 60

RMSE

P

90

pH

6.63

6.7

6.72

0.253

0.925

Dry matter (%)

12.83

12.46

12.91

1.601

0.546

Protein (%)

3.45

3.32

3.52

0.357

0.503

Casein (%)

2.71

2.52

2.79

0.214

0.491

Fat (%)

3.20

3.34

3.8

0.891

0.375

Lactose (%)

4.77

4.64

4.74

0.297

0.772

Ash (%)

0.75

0.70

0.78

0.091

0.850

Somatic cell count *1000

143

153.67

165.8

138.25

0.338

Diameter of MFG (µm)

4.67

4.07

5.66

1.228

0.223

SG (%)

20.26

30.03

6.42

0.305

0.078

MG (%)

43.63

37.58

33.26

20.596

0.728

LG (%)

36.10

32.39

60.32

21.156

0.187

MFG: milk fat globules; SG: small globules; MG: medium globules; LG: large globules; RMSE=root mean square error

netic component appears to exist for the average MFG size in bovine milk 13, and breed is one of the multiple factors affecting the average MFG size 12.

The milk fat globule size is an interesting trait because it affects the technological and sensory properties and nutritional quality of the milk and milk products 14,15. In addition, milk fat globules affect the stability of the creaming rate, and their size could alter the moisture content and texture of cheese 16 .

The average somatic cell count (SCC) detected in our study was lower than the limit set by the EU regulation 17 and compatible with the good health of the udder.

An optimal somatic cell count is important both in terms of hygiene and production. In fact, cows with an SCC greater than 250000 are highly likely to be infected on at least one udder quarter, which indicates mastitis in the herd, also at a sub-clinical level 18.

From a production point of view, a high SCC is negatively correlated with milk production, lactose, fat and casein, with deleterious effects on the cheesemaking 19.

The atherogenic index (AI) and thrombogenic index (TI) take into account the effects that single FAs might have on human health and, in practice, on the probability of increasing the incidence of pathogenic phenomena such as atheroma and/or thrombus formation. Low AI and TI milk indices are considered more beneficial for health.

Results on the nutritional indices of Pontremolese cows (Table 3) showed that the atherogenic index (2.87) was between the values calculated in the literature for the Italian Friesian compared to the Italian Brown cow (2.80 and 3.03 respectively) 20,21.

On the other hand the thrombogenic index (3.40) was closer to Brown than Friesian cows (3.29 and 2.84 respectively).

The desaturation index is related to the contribution of unsaturated fatty acids in milk 21, which is linked to the dietary supply of polyunsaturated fatty acids (PUFAs) and subsequent rates of biohydrogenation in the rumen, but also to the action of the steraoyl-CoA desaturase (Δ9-desaturase) of the mammary gland. Steraoyl-CoA desaturase activity consists in desaturating saturated fatty acids by converting them into the corresponding monounsaturated fatty acids 22.

Interest in the desaturation indices derives from the fact that

it generally shows higher heritability than individual fatty acids as found both in Brown 21 and in Canadian Holstein cows 23.

Desaturation indices are of interest both to evaluate the health characteristics of milk, since reducing the total intake of SFAs is recommended in the diet 24, as well as to modulate the unsaturation of fatty acids through selective strategies.

The desaturation index in Pontremolese cows (29.25) was higher than reported in the literature for Italian Brown cows (26) 21 , and more similar to Canadian and Italian Friesian breeds (about 29 and 30 respectively) 23,20.

The omega-6 / omega-3 ratio was 4.14, which is within the range of cow’s milk, again closer to the values in Friesian milk (3.52) than in Brown cow’s milk (6.81) 21,20.

In Pontremolese milk (Table 3), the average SFA and MUFA percentages were in agreement with the literature on cow’s milk 25.

Despite the presence of saturated fatty acids, milk and dairy products also provide potentially bioactive fatty acids such as vaccenic acid (VA), rumenic acid (C18:2 c9, t11) and essential fatty acids such us alpha linolenic acid (C18:3 c9, 12, 15) and linoleic acid (C18:2 c9, 12) 26.

It has been reported that the presence of C18 unsaturated fatty acids in milk may help prevent cardiovascular disease, atherosclerosis, and other chronic diseases in consumers 27.

In Pontremolese milk, the percentages of oleic, rumenic and linoleic acids were in agreement with the literature on Friesian cow’s milk 20, and the average content of rumenic acid was similar to the value reported in another Italian indigenous breed, the Burlina (0.66%) 28.

As for the lactation phase, an increasing although not significant lipid content trend was found as lactation progressed. This was also followed by a similar trend in the average diameter, in agreement with the positive relation between the percentage of fat and the diameter of the milk fat globules observed in other studies 13,15.

Regarding milk fatty acid profile, its changes during the early lactation can be predictive of the energy balance of the cows and of cows suffering hyperketonemia 29.

In this study, no significant changes were found in the fatty acids profile during the 90 days of lactation even if an increasing trend

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271

Table 5 - Fatty acid profile, nutritional and desaturation indexes in Pontremolese cow’s milk during the first 90 days of lactation.

Fatty acids (g/100g of total fatty acids)

30

Days of lactation 60

RMSE

P

90

C10:0

Capric acid

2.6

2.72

2.49

0.862

0.905

C11:0

Undecylic acid

0.04

0.06

0.06

0.036

0.611

C12:0

Lauric acid

2.95

3.2

2.95

0.996

0.891

C13:0

Tridecylic acid

0.13

0.13

0.13

0.039

0.492

C14:0

Myristic acid

11.44

12.06

11.34

2.227

0.842

C14:1

Myristoleic acid

1.12

1.21

1.21

0.35

0.899

C15:0

Pentadecylic acid

1.4

1.57

1.68

0.371

0.513

0.51

0.4

0.45

0.139

0.451

Palmitic acid

31.12

34.36

32.19

4.434

0.482

C15:1 C16:0 C16:1 n7

Palmitoleic acid

1.67

1.93

1.92

0.5

0.651

C17:0

Margaric acid

0.87

0.78

0.83

0.26

0.833

C17:1

-

0.48

0.46

0.46

0.192

0.978

C18:0

Stearic acid

12.61

9.95

11.05

3.382

0.452

C18:1 t9

Elaidic acid

0.11

0.14

0.2

0.073

0.189

C18:1 t11

Vaccenic acid

0.88

0.98

0.99

0.434

0.910

C18:1 c9

Oleic acid

22.46

20.6

22.93

5.525

0.701

-

0.39

0.44

0.35

0.15

0.624

Linoleic acid

2.09

1.94

1.93

0.349

0.714

C18:3 c6,9,12

γ-Linolenic acid

0.04

0.04

0.05

0.012

0.576

C18:3 c9,12,15

α-Linolenic acid

0.34

0.4

0.42

0.223

0.817

C20:0

Arachidic acid

0.25

0.19

0.24

0.098

0.608

C18:2 c9,t11

Rumenic acid

0.61

0.74

0.68

0.19

0.520

C20:1

Gondoic acid

0.25

0.23

0.27

0.066

0.636

C21:0

Heneicosylic acid

0.04

0.04

0.05

0.03

0.893

C18:2 t9,12 (n6) C18:2 c9,12

C20:2

Eicosadienoic acid

0.04

0.04

0.04

0.014

0.847

Dihomo-γ-linolenic acid

0.12

0.1

0.12

0.029

0.464

C20:4 c5,8,11,14

Arachidonic acid

0.001

0.001

0.001

0.001

0.950

C20:3 c11,14,17

Eicosatrienoic acid

0.15

0.14

0.13

0.042

0.611

C22:0

Behenic acid

0.11

0.09

0.11

0.05

0.601

C22:1

Erucic acid

0.04

0.03

0.05

0.019

0.319

Decosapentaenoic acid

0.01

0.01

0.01

0.006

0.739

C20:3 c8,11,14

C20:5 c5,8,11,14,17 C23:0

Tricosylic acid

0.04

0.04

0.04

0.01

0.559

C22:2

-

0.06

0.05

0.06

0.032

0.865

C24:0

Lignoceric acid

0.07

0.05

0.07

0.035

0.773

C24:1

Nervonic acid

0.01

0.01

0.01

0.006

0.359

C22:5 n3

Decosapentaenoic acid

0.1

0.1

0.12

0.037

0.679

C22:6 c4,7,10,13,16,19

Docosahexaenoic acid

0.01

0.01

0.01

0.006

0.926

Omega 3

0.61

0.66

0.69

0.256

0.900

Omega 6

2.64

2.53

2.45

0.416

0.756

SFA (%)

68.5

69.99

67.6

6.608

0.833

MUFA (%)

27.54

25.99

28.48

6.059

0.791

PUFA (%)

3.96

4.02

3.92

0.766

0.975

UFA/SFA ratio

0.47

0.44

0.49

0.148

0.849

Omega-6/omega-3 ratio

4.36

4.23

3.83

1.044

0.715

Atherogenic Index

2.75

3.13

2.70

0.951

0.732

Thrombogenic Index

3.36

3.60

3.20

0.937

0.775

Desturation index

29.57

27.94

30.54

6.848

0.818

SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; UFA = unsaturated fatty acids; RMSE = root mean square error

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Milk yield and quality characteristics of an endangered Italian cattle breed: the Pontremolese

was observed for C18:1 t-9.

An increasing omega 3 trend was also found and it was described

by Bilal et al 23 in Canadian Holstein, which was accompanied by a decreasing trend in the omega-6: omega-3 ratio.

According to a review by Simopoulos 30 , this ratio should be reduced in the diet since it is one of the most important dietary factors in the prevention of obesity, along with physical activity.

CONCLUSIONS To the best of our knowledge this is the first study characterizing the milk of the Pontremolese relic breed. Similarities with the Reggiana breed were highlighted in the fat and protein content of Pontremolese milk, and the milk also had a higher desaturation index than Brown and Friesian cows.

The similarities observed with Reggiana milk could be linked to the hypothesis that traces the origin of the Pontremolese back to a variety of Reggiana cow, whose milk was traditionally used for the production of Parmigiano Reggiano cheese. Although further investigations are needed in this regard, the similarities observed with Reggiana milk seem to indicate that the use of this milk in dairy processing could be one way of revitalising this indigenous breed.

References 1. Altomonte I., Salari F., Neglia A., Martini M. (2016). Milk yield and quality characteristics of Cinisara and Modicana cows reared on a farm in the province of Palermo (Sicily-Italy). Large Anim Rev, 22:251-254.

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Linee guida per la conservazione e la caratterizzazione della biodiversità vegetale, animale e microbica di interesse per l’agricoltura. Piano Nazionale sulla Biodiversità di Interesse Agricolo, Inea, Roma.

3. Dal Prà A., Zanon A. (2013). Antiche razze da salvare, al via un progetto regionale. Agricoltura. Dicembre 2013:70-71.

4. AOAC. 2000. Official methods of analysis, 17th ed., Association of Official Analytical Chemists, Arlington, VA. 5. Ulbricht T.I.V., Southgate D.A.T. (1991). Coronary heart disease: seven dietary factors. Lancet, 338:985–992.

6. Schennink A., Heck J.M.L., Bovenhuis H., Visker M.H.P.W., Van Valenberg H.J.F., Van Arendonk J.A.M. (2008). Milk fatty acid unsaturation: genetic parameters and effects of stearoyl-CoA desaturase (SCD1) and acyl CoA: diacylglycerol acyltransferase 1 (DGAT1). J Dairy Sci, 91:21352143.

7. Martini M., Cecchi F., Scolozzi C. (2006). Relationship between fat globule size and chemical and fatty acid composition of cow’s milk in mid lactation. Ital J Anim Sci, 5:349-358.

8. SAS (2002). Institute JMP user's guide. Version 5.0 SAS Institute Inc., Cary, NC. 9. Gandini G., Maltecca C., Pizzi F., Bagnato A., Rizzi R. (2007). Comparing local and commercial breeds on functional traits and profitability: the case of Reggiana dairy cattle. J Dairy Sci, 90: 2004-2011.

10. De Marchi M., Fagan C.C., O’Donnell C.P., Cecchinato A., Dal Zotto R., Cassandro M., Penasa M., Bittante G. (2009). Prediction of coagulation properties, titratable acidity, and pH of bovine milk using mid-infrared spectroscopy. J Dairy Sci, 92:423-432.

11. Gottardo P., Penasa M., Righi F., Lopez-Villalobos N., Cassandro M., De Marchi M. (2017). Fatty acid composition of milk from Holstein Friesian, Brown Swiss, Simmental and Alpine Grey cows predicted by midinfrared spectroscopy. Ital J Anim Sci, 16:380-389.

12. Martini M., Salari F., Altomonte I. (2016). The macrostructure of milk lipids: the fat globules. Crit Rev Food Sci Nutr, 56:1209-1221.

13. Fleming A., Schenkel F.S., Chen J., Malchiodi F., Ali R.A., Mallard B., Sargolzaei M., Corredig M., Miglior F. (2017). Variation in fat globule size in bovine milk and its prediction using mid-infrared spectroscopy. J Dairy Sci,100:1640-1649.

14. Martini M., Altomonte I., Salari F. (2013). Evaluation of the fatty acid profile from the core and membrane of fat globules in ewe's milk during lactation. LWT-Food Sci Technol, 50:253-258.

15. Martini M., Altomonte I., Bortoluzzi Moro A., Caneppele C., Salari F. (2017). Influence of fat content on quality of cow's milk. Ital J Food Sci, 29:138-144.

16. Michalski M.C., Gassi J.Y., Famelart M.H., Leconte N., Camier B., Michel F., Briard V. (2003). The size of native milk fat globules affects physic-chemical and sensory properties of Camembert cheese. Lait, 83:131–143.

17. Regulation (EC) 853/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific hygiene rules for food of animal origin. Official Journal of the European Union L 139 of 30 April 2004.

18. Burgess K. (2010). Key requirements for milk quality and safety: a processor’s perspective. In: Woodhead Publishing Series in Food Science, Technology and Nutrition, Improving the Safety and Quality of Milk, Ed. Mansel W. Griffiths, 64-84, Woodhead Publishing.

19. Bobbo T., Ruegg P.L., Stocco G., Fiore E., Gianesella M., Morgante M., Pasotto D., Bittante G., Cecchinato A. (2017). Associations between pathogen-specific cases of subclinical mastitis and milk yield, quality, protein composition, and cheese-making traits in dairy cows. J Dairy Sci, 100: 48684883.

20. Conte G., Serra A., Cremonesi P., Chessa S., Castiglioni B., Cappucci A., Bulleri E., Mele M. (2016). Investigating mutual relationship among milk fatty acids by multivariate factor analysis in dairy cows. Livest Sci, 188:124132.

21. Pegolo S., Cecchinato A., Mele M., Conte G., Schiavon S., Bittante G. (2016).

Effects of candidate gene polymorphisms on the detailed fatty acids profile determined by gas chromatography in bovine milk. J Dairy Sci, 99:45584573.

22. Dewhurst R.J., Shingfield K.J., Lee M.R.F., Scollan N.D. (2006). Increasing the concentrations of beneficial polyunsaturated fatty acids in milk produced by dairy cows in high-forage systems. Animal Feed Science and Technology, 131:168-206.

23. Bilal G., Cue R.I., Mustafa A.F., Hayes J.F. (2014). Effects of parity, age at calving and stage of lactation on fatty acid composition of milk in Canadian Holsteins. Can J Anim Sci, 94: 401-410.

24. WHO (World Health Organization) (2018). Draft guidelines on saturated fatty acid and trans-fatty acid intake for adults and children. Public Consultation May to June 2018. https://extranet.who. int/dataform/ upload/surveys/666752/files/Draft%20WHO%20SFA-TFA%20guidelines_ 04052018%20Public%20Consultation(1).pdf 25. Soyeurt H., Dardenne P., Dehareng F., Bastin C., Gengler N. (2008). Genetic parameters of saturated and monounsaturated fatty acid content and the ratio of saturated to unsaturated fatty acids in bovine milk. J Dairy Sci, 91:3611-3626.

26. Bainbridge M.L., Cersosimo L.M., Wright A.D., Kraft J. (2016). Content and composition of branched-chain fatty acids in bovine milk are affected by lactation stage and breed of dairy cow. PLoS One, 11:0150386.

27. Bai C., Cao Q., Khas-Erdene A.C., Gao P., Zhang Y., Zhang T. (2018). Combined effects of oleic, linoleic and linolenic acids on lactation performance and the milk fatty acid profile in lactating dairy cows. Animal, 12:983989.

28. Niero G., Visentin G., Ton S., De Marchi M., Penasa M., Cassandro M. (2016). Phenotypic characterisation of milk technological traits, protein fractions, and major mineral and fatty acid composition of Burlina cattle breed. Ital J Anim Sci, 15:576-583.

29. Fiore E., Blasi F., Morgante M., Cossignani L., Badon T., Gianesella M., Contiero B., Berlanda M. (2020). Changes of milk fatty acid composition in four lipid classes as biomarkers for the diagnosis of bovine ketosis using bioanalytical Thin Layer Chromatography and Gas Chromatographic techniques (TLC-GC). J Pharmaceut Biomed, 188, 113372.

30. Simopoulos A.P. (2016). An increase in the omega-6/omega-3 fatty acid ratio increases the risk for obesity. Nutrients, 8:128.

Webography http://www.associazionerare.it http://www.aia.it/CMSContent/Registro%20Anagrafico%20Bovini.pdf http://www.fao.org/dad-is/browse-by-country-and-species/en http://www.anafi.it/ http://www.anarb.it

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SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

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Monitoring and benchmarking antibiotic usage of italian beef farms: a pilot study

275

N

LINDSEY LORENZO LAZZARINO1, ALBERTO FERRERO2, GIORGIA VEZZARO3, VALENTINA MARIA MERLINO4 1

Research Fellow, Dep. of Agricultural, Forest and Food Sciences (DISAFA), University of Torino, 10095 Grugliasco, Italy 2 Veterinary Practitioner, Council Member, SocietĂ Italiana Veterinari per Animali da Reddito (SIVAR), 26100 Cremona, Italy 3 Research Fellow, Dep. of Veterinary Science, University of Torino, 10095 Grugliasco, Italy 4 Phd Student, Dep. of Agricultural, Forest and Food Sciences (DISAFA), University of Torino, 10095 Grugliasco, Italy

SUMMARY Beef production in Italy represents an important economic sector. Antimicrobials are administered to cattle to treat various pathological disorders such as respiratory diseases. The One Health approach promotes the rational use of antimicrobial drugs to contrast the antimicrobial resistance (AMR). The present study constitutes the first attempt to analyse antibiotic consumption of the beef cattle sector in the region of Piedmont (North-West of Italy).

The goal of our study was to assess the antibiotic use, in both quantitative and qualitative terms, of a sample of beef farms with a software that would then enable us to set benchmark levels for the considered sample.

To this aim, the antibiotic usage of ten intensive beef farms in the two-year period was recorded and analysed. For each farm that is part of the panel analysed, data about its annual antibiotic usage in 2017 and 2018, subdivided between the various antibiotic commercial products utilised, was recorded directly by the veterinarians that manage the selected farm. These data was then used as an input for the specific software, developed by the Italian Society of Veterinarians operating in the Livestock Sector (Sivar), to measure the antibiotic consumption of each selected beef farm and calculate the DDD/y (Defined Daily Dose per year), in mg/kg/day, of a singular farm.

The results of data elaboration were discussed considering a DDD/y benchmark system, specifically designed for the considered farmâ&#x20AC;&#x2122;s panel, based as close as possible to the one already use by the Netherlands Veterinary Medicines Institute (SDa). The DDD/y benchmark levels, at the basis of the considered one, were developed to fit the specific farming conditions of the intensive Piedmontese beef cattle sector, in accordance with the veterinarians that manage the selected farms. The main results showed an average DDD/y value of the considered panel of farms equal to 2.876 considering the two-years period. The DDD/y ranged from 0.150 to 7.409 for the singular farm in one year. Differences about the relative use of different classes of antibiotics were detected between farms. Furthermore, three farms out of ten fell out of the highest set benchmark level in at least one year of the biennium analysed.

Further studies will be needed to assess whether the benchmark levels set in the current pilot study can be extended to all the Piedmontese beef farming sector.

KEY WORDS Beef, antibiotic, antimicrobial, Defined Daily Dose (DDD), Piedmont.

INTRODUCTION The rational use of antibiotics in the livestock sector is a major concern in nowadays health management. Furthermore, this approach has a direct positive impact on the antibiotic resist-

Corresponding Author: Lindsey Lorenzo Lazzarino (lindsey.lazzarino@edu.unito.it).

ant bacteria selection risk. It has been proven that bacteria traits resistant to antibiotics can be selected in livestock farms thus setting a starting point to work on to reduce the risk in question1.

The beef cattle sector is usually less interested in measuring antibiotic consumption than the dairy one. This is probably due to the antibiotic usage in the meat supply chain does not have a direct negative effect on the income of beef farmers, as instead happens on milk production. In fact, the antibiotic con-

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Monitoring and benchmarking antibiotic usage of italian beef farms: a pilot study

sumption on dairy cows ordinarily results directly in higher quantities of milk not sellable. In this context, reference levels of antibiotic consumption in beef cattle farms should be set to reduce their use. To achieve this, the first step is to measure the actual use of antibiotics at farm level. This is important, primarily, for two reasons: to promote a more rational use of antibiotics in general and, more specifically, to reduce to the minimum the use of those classes of antibiotics which are of primary importance in human care (cephalosporins of 3rd and 4th generation and fluoroquinolones)2. The antibiotic classes in question are used in the farming sector because they work in livestock health management as well as in human medicine2,3. The rational use of antibiotics at farm level, by enabling farms to achieve a more accurate usage that can result in limiting their overall consumption, could reduce the selective pressure on pathogens in the farming environment. This must be done to try to avoid the development of antibiotic-resistant traits which, in the worst case scenario, may transmit their new resistance to human pathogens. To this end it is necessary to achieve a rational level of antibiotic consumption as close to zero as possible. Since the selective pressure on bacteria resulting from the use of antibiotics cannot be completely avoided, the effectiveness of life-saving antibiotics must be preserved to ensure human safety. The problem arising from the selection of antibiotic-resistant bacteria is perceived as such not only by the medical sector, but also by the agricultural sector. Indeed, many stakeholders recognise the need to reduce the overall use of antibiotics at farm level as a priority2.

In the current study a software designed to measure antibiotic consumption as DDD/y (Defined Daily Dose per year), which was developed by the Italian Society of Veterinarians operating in the Livestock Sector (Sivar), was used to analyse antibiotic consumption at farm level. The software in question is available to all Veterinarians that are Sivar members. Studies of antibiotic consumption in the Italian livestock sector that use DDD indexes are quite recent in Italy4,5,6. The DDD/y index, which considers the posology of each commercial antibiotic product, does not only give us primarily a measure of antibiotic consumption, but a risk index of selecting antibiotic resistant bacteria at farm level. For the dairy sector, however, many veterinarians have already used this tool to assess the level of antibiotic consumption at farm level.

The key to approach the problem of antibiotic resistance remains the One Health concept7,8. At this purpose, this work has been conducted to investigate the consumption of antibiotics at farm level, as this is one of the levels at which antibiotic-resistant microorganisms can develop and subsequently spread to other health care sectors8.

This research represents the first pilot study to analyse the consumption of antibiotics on cattle farms in Piedmont. Currently, no standard has been set to assess the consumption of antibiotics in the cattle breeding sector by Italian regulators.

MATERIALS AND METHODS Ten intensive beef farms specialised in the only fattening of beef bulls and located in the Po plain area between the cities of Turin and Cuneo (Piedmont, Italy) were involved in the research for data collection. The low number of farms included in the study is strictly related to farmersâ&#x20AC;&#x2122; availability to take part in the research. The panel consistency is however adequate to achieve

the objectives of the current pilot study. The selected farms have been chosen to be representative of the Piedmontese intensive beef farming production system in terms of number of animals fattened (on average 294 beef bulls, ranging from 43 to 572 animals per farm), of production cycle and of animals characteristics (breed and final weight at slaughter). The Piedmontese beef farming sector is based on a high degree of intensity, a long fattening cycle and a relevant incidence of animal imports. In the Italian Po plain, due to the high pressure of the livestock sector on a limited agricultural area, animals are not ordinarily raised extensively on pasture and beef cattle are sold to the slaughterhouse usually aging between sixteen and eighteen months. Furthermore, a relevant percentage of beef animals farmed in Piedmont are bought, just after weaning, directly from France. In 2017, according to statistical data provided by the national Italian livestock Register (Anagrafe Nazionale Zootecnica), 212,898 bovines were imported in Piedmont from other countries and the nearly 96% from France.

For each analysed farm, antibiotic usage data was collected relatively to the two-year period 2017-2018 in order to compare the difference in consumption between different years. The list of all antibiotic administrations of the biennium under analysis, together with the number of medicine packages used and the corresponding actual dosages of each administration, were recorded, directly by veterinarians, for each considered farm.

This data was employed by the Sivar software to calculate the DDD/y values of each farm. The first step in the software calculation process consisted in obtaining the partial DDD/y value of every commercial antibiotic (named (DDD/y)p) by applying the formula (a): (a) where: - Active Ingredient refers to the total amount of active ingredient in milligrams; - DDDA is the reference dosage calculated for every commercial antibiotic, measured as mg/kg/day, DDDA values are based on the corresponding EMA ones9; - Number of Animals is the actual herd consistency in the year under analysis - Standard Weight is the standard weight of the animal category under analysis.

All the values of the partial (DDD/y)p (expressed in mg/kg/day) calculated for each commercial antibiotic for each farm were summed by the software to obtain the overall DDD/y value of the singular farm. Consequently, at the end of the calculation process, the DDD/y value is expressed in milligrams of antibiotic administered on average in a single day of the year to one average kilogram of body weight of all the animals farmed in one year (mg/kg/day).

The Sivar software groups all beef cattle into just one category of animals, all weighting 600 kg. Rearing farms are not included into the Sivar software.

In order to obtain the farm DDD/y value relative to a singular antibiotic class, the (DDD/y)p values of all commercial antibiotics that contains the same active ingredient, that have been used in one year, are summed together. If commercial antibiotic formulations with more than one active ingredient are present, the software calculates each antibiotic class separately. Different antibiotic classes are grouped as follows: beta-lactams,

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macrolides, tetracyclines, quinolones, phenicols, aminoglycosides, lincosamides, sulfonamides, cephalosporins of 1st & 2nd generation, cephalosporins of 3rd & 4th generation, pleuromutilis, rifaximins and glycopeptides.

The classification of critically important antibiotics that was considered in the present study is the one utilised by the Netherlands Veterinary Medicines Institute (SDa) in its annual reports, the most recent of which was released in 201810. This was done to enable us to set our benchmarking system as close as possible to the SDa one. Our choice is also linked to the fact that when the Sivar software was developed in 2014, it was set as close as possible to the antibiotic management system in place in the Netherlands.

The Dutch equivalent of the DDD/y index (incorporated into the Sivar software) is the DDDANAT (Defined Daily Dose Animal) which is calculated with the same formula (a) and on a year base as well.

Despite the Sivar system has been set as close as possible to the Dutch one (by adopting the same index calculated as shown in (a)), the reference weights of the two systems are different: the Sivar software attributes an average weight of 600 kg to one average fattening bull, while the Dutch system only of 500 kg.

This choice is probably linked to the average final weight of bulls of specialized beef breeds typically fattened in Italy, which is considerably higher than the average one of beef breeds reared in Northern Europe. Consequently, 600 kg probably represents a good estimate of the average weight of a single animal during the fattening process in Italy, especially since this data is in accordance with the statistics provided by the Italian Institute of Statistics (ISTAT-Istituto Nazionale di Statistica).

Since our benchmark system was set as close as possible to the Dutch SDa one, it was necessary to determine the following thresholds: the upper limit of the Target zone, under which the DDD/y values of farms should be; the bottom limit of the Action zone above which a DDD/y value by a beef farm is automatically followed by sanctions, if there are no overt medical justifications for it. The DDD/y values scored by farms, that fall between the upper limit of the Target zone and the bottom limit of the Action zone are included into the Signalling zone.

By falling into the Signalling zone a farm is not necessarily exposed to sanctions.

Benchmark levels for dairy farms used by the Dutch SDa were proved to be applicable to the Sivar software calculation to benchmark Piedmontese dairy farms11.

Since the Sivar software and the Dutch SDa adopt the same formula to calculate the DDD/y value of one beef farm, the benchmark levels for Piedmontese beef farms were set lower than the Dutch ones for dairy farms (Dutch benchmark levels for dairy farms are set at a DDD/y value equal to 4 and 6). This is due to there is no medical reason to justify a higher level of antibiotic use, on average per one kilogram of live weight, for fattening beef bulls higher than the level of antibiotic used, always on average per one kilogram of live weight, for dairy cows and lactating calves. This is so because, due to milking management of dairy cows and sanitary problems connected with lactating calves, these animal categories are more prone to infections and diseases than fattening beef bulls reared in the modern and up to date farms of the Nord of Italy. In the calculation process of the DDD/y value of one farm, which is the same for the Dutch SDa and the Sivar software, for dairy farms are considered the antibiotic usage of dairy cows, heifers and lactating calves wile

277

for beef farms is considered only the antibiotic usage of fattening beef bulls.

The actual benchmark levels set at a DDD/y value equal to 3 and 5 were determined empirically with the help of the veterinary practitioners that manage the beef farms that make up the panel. The upper limit of the target zone was set at a DDD/y value of 3 as veterinarians have stated that it is impossible for them to bring this value below 3 for all cattle farms they manage, especially with the tools at their disposal. For example, the use of antimicrobial resistance testing is not a viable option as the available laboratories take too many days to provide the results of these tests. This means that waiting for the results before prescribing appropriate antibiotics can lead to the death of sick animals. The lower limit of the zone of action is set at a DDD/y of 5 because the causes that lead farms to exceed this limit have always been declared solvable with the diagnostic tools available to them by farm veterinarians. If the DDD/y value of one farm in between 3 and 5 it falls into the Signalling zone, which requires further analyses of the singular farm antibiotic management strategy.

It is also important to underline whether differences in antibiotic usage are present between beef farms with higher antibiotic usage and those with a lower one. To achieve this, the average DDD/y values of the antibiotic classes considered by the Sivar software, of the three farms with higher antibiotic usage and of the three with a lower one, were analysed.

RESULTS In Figure 1 the overall DDD/y values of every beef farm under analysis are reported. The comparison between the two considered years and set benchmark levels are included.

Five farms out of ten fall out of the estimated Target zone in 2017. In the same year only one beef farm has a DDD/y value higher than five (DDD/y = 5.034).

In 2018 three of the five farms that exceeded the DDD/y = 3 benchmark in 2017, are well under the same level. Curiously, the two farms with the highest DDD/y value of the panel in 2017, increased their antibiotic usage consistently. The farm F08 underwent an even higher increase in antibiotic usage in 2018, than the other two beef farms in question (F09 and F10).

In 2017 only half of the farms of the panel managed to keep their DDD/y value under three. In 2018 however, it can be detected a reduction in antibiotic consumption that brings a total of seven farms under the benchmark level in question. However, the three farms which scored higher DDD/y values (as average DDD/y values of the biennium under analysis), used more antibiotics in 2018 than in 2017.

In Figure 2 the average DDD/y values of the different classes of antibiotics used by farms are reported.

Beta-lactams antibiotic was the antibiotic class most used in 2017, followed by macrolides, tetracyclines and quinolones. The four classes of antibiotic mentioned, as an all, amount to 80% of total antibiotic usage in the same year. In 2018, beta-lactams alone, represent on average 48% of the total DDD/y of the average beef farm of the panel. Beta lactams usage increases consistently in 2018 while the use of macrolides, tetracyclines and quinolones decreased.

Figure 3 represents the antibiotic usage, in 2017, of the three beef farms with lower DDD/y values and of the three farms with higher ones, respectively.

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Figure 1 - DDD/y values of the panel in 2017 and 2018, with benchmark levels.

Figure 4 represents the average antibiotic usage in 2018 of the three beef farms with the lowest DDD/y value and of the three farms with the highest, respectively.

In both groups, beta-lactams and macrolides were the classes of antibiotics which are more used in 2017 as well as in 2018.

In farms with lower DDD/y values, however, macrolides are more used than beta-lactams. Beef farms with higher DDD/y values utilise more beta-lactams than macrolides but it is important to underline that their average DDD/y value for macrolides is actually higher than the one of farms, which scored a lower total DDD/y value.

No relevant differences between the usages of different classes of antibiotics are detected between the two years. When overall antibiotic consumption increases, the use of every antibiotic class typically increases, even if not always of the same relative amount.

DISCUSSION In this research, the semaphore structure, utilised in the Netherlands since 2012, was adopted to reduce in an efficient way the pressure on selecting antibiotic resistant bacteria at farm level. The upper limit of the Target zone (green) was set at DDD/y=3, while the border that separates the Signalling zone (yellow) and the Action zone (red) were set at DDD/y=5.

To maintain the effectiveness of a benchmark system on the long run it should be periodically adapted12. Under the current conditions, the benchmark levels set in this work seem to guarantee the highest reduction in overall antibiotic usage, as a result of a more rational use, in the Piedmontese beef farming sector.

No beef farm that makes up the panel analysed used any cephalosporins of 1st & 2nd generation, cephalosporins of 3rd & 4th generations, pleuromutilins, rifaximins or glycopeptides, in

Figure 2 - Average DDD/y values of the panel for every antibiotic class: 2017 and 2018.

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Figure 3 - Antibiotic usage of farms with lower and higher DDD/y value: 2017.

the biennium 2017-2018. They also utilised a very low amount of quinolones (fluoroquinolones are included), the use of which underwent a reduction amounting at -44.3% in 2018 with respect to 2017.

Such a low usage of the antibiotics of the classes in question is the result of the voluntary work, of the veterinarians that manage the beef farms analysed. They dedicate their efforts to rationalise the use of antibiotic classes of primary importance to human health: this enabled the farms of the panel to reach consumption levels, of the classes of antibiotics in question, equal to zero in both the analysed years.

Veterinarians are often aware of the pivotal role they play in reducing the risk of selecting bacteria resistant at farm level14,15. Even if dedicated protocols, specifically designed by veterinarians, result in an increase in the workload for farmers, the benefits they provide have been proved to outnumber the inevitable increase in complexity of the resulting farm management15.

If the number of veterinarians working in the livestock sector who decide to voluntarily undertake the discussed approach were to increase, then the risk of selecting antibiotic-resistant bacteria could be significantly reduced by a more rational use of antibiotics. This would be achievable with the approach analysed even in the absence of specific regulations. Indeed, in the United States, for example, despite the lack of national policies on antimicrobials, a consistent spread of a voluntary approach has helped the legislator to start tackling the problem16.

Another potential driver that can help spreading the use of the DDD/y Sivar system are animal welfare certifications, which were proved to be high valued by consumers17. If a DDD/y system like the Sivar one, were to be incorporated into the assessment process of animal welfare certifications, it can also become a stimulus to lower antibiotic usage levels despite the lack of specific regulations on a national level18,19. However, as all historic data from European countries, that have dealt with the

Figure 4 - Antibiotic usage of farms with lower and higher DDD/y value: 2018.

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same problem in the past (such as Sweden, Denmark, Germany and the Netherlands), have shown, no great result on the rational usage of antibiotics can be expected without a dedicated regulation system put into place by central authorities20,21,22.

CONCLUSIONS

6.

7.

8.

The developed system for antibiotic usage management and control, which was based on the Sivar software designed to calculate the DDD/y value, seems to be able to guarantee the maximum reduction in the overall use of antibiotics on the selected panel of cattle farms, which is possible under current conditions. Being set as close as possible to the one officially in force in the Netherlands, it can also help to build a common antibiotic resistance prevention system. In Italy no mandatory system to decrease antibiotic consumption at farm level is currently adopted. However, veterinarians that are Sivar members can decrease the risk of developing antibiotic resistant bacteria, in the beef farms in which they operate, by utilizing the DDD/y based Sivar software on a voluntary basis.

Since the current work constitutes the first attempt to study antibiotic usage of Piedmontese intensive beef farms, further studies will be needed to analyse antibiotic consumption considering an higher number of beef farms. This would make it possible to assess the changes to the developed benchmark system that may be needed to ensure its effectiveness in different contexts, regional and national. Reducing the risk of selecting antibiotic resistant bacteria is an ongoing process that plays a key role in the application of the One Health approach. By promoting a more rational use of antibiotics, the agricultural sector can do its part to ensure global health.

9.

10.

11.

12.

13.

14.

15.

16.

17.

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2.

3.

4.

5.

Teuber, M. (2001). Veterinary use and antibiotic resistance. Current opinion in microbiology, 4(5), 493-499.

Redding, L. E., Bender, J., & Baker, L. (2019). Quantification of antibiotic use on dairy farms in Pennsylvania. Journal of dairy science, 102(2), 14941507.

World Health Organization. (2017). Critically important antimicrobials for human medicine: 5th revision 2016. WHO Advisory Group on Integrated Surveillance of Antimicrobial Resistance (AGISAR).

Scoppetta F., Mandolini N. A., Petrocchi S., Pelagalli G., Mattozzi C., Gallitri M., Baiguini A., Pierucci P., Perugini G., Capuccella M. (2018). Analisi farmaco epidemiologica “Defined Daily Doses-based” sull’uso di antibiotici nel settore suinicolo in un’area vasta della regione Marche: un nuovo strumento per la farmacosorveglianza e la farmacovigilanza. Large Animal Review 2018; 24: 157-165.

Caucci C., Di Martino G., Schiavon E., Garbo A., Soranzo E., Tripepi L., Stefani A. L., Gagliazzo L., Bonfanti L. (2018). Impact of bovine respiratory disease on lung lesions, slaughter performance and antimicrobial usage in French beef cattle finished in North-Eastern Italy. Italian Journal of Animal Science, 17(4), 1065-1069.

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Tarakdjian, J., Capello K., Pasqualin D., Santini A., Cunial G., Scollo A., Mannelli A., Tomaso A., Vonesch N., Di Martino G., (2020). Antimicrobial use on Italian Pig Farms and its Relationship with Husbandry Practices. Animals, 10(3), 417.

European Commission (EC), (2017). A European One Health Action Plan against Antimicrobial Resistance (Amr). https://ec.europa.eu/health/ amr/sites/amr/files/amr_action_plan_2017_en.pdf., Accessed 10 February 2019.

Magouras, I., Carmo, L. P., Stärk, K. D., & Schüpbach-Regula, G. (2017).

Antimicrobial Usage and-Resistance in Livestock: Where Should We Focus?. Frontiers in veterinary science, 4, 148.

European Medicines Agency, (2015). Principles on Assignment of Defined Daily Dose for Animals (DDDA) and Defined Course Dose for Animals. http://www.ema.europa.eu/does/en_GB/document_library/Scientific_guidline/2015/03/WC500184369.pdf. Accessed 10 February 2019.

SDa Autoriteit Diergeneesmiddelen (2018). Usage of antibiotics in agricultural livestock in the Netherlands in 2017. https://cdn.i-pulse.nl/autoriteitdiergeneesmiddelen/userfiles/Publications/engels-def-rapportage2017.pdf Accessed 10 February 2019.

Lazzarino L., Massaglia S., Ferrero A., (2019). Dairy farms sustainable antibiotic usage monitored to extimate the risk of selecting antibiotic resistant bacteria at farm level, Italy: first results. Quality - Access to Success, 20(S2), pp. 343-350.

Hommerich, K., Ruddat, I., Hartmann, M., Werner, N., Käsbohrer, A., & Kreienbrock, L. (2019). Monitoring Antibiotic Usage in German Dairy and Beef Cattle Farms A Longitudinal Analysis. Frontiers in veterinary science, 6, 244.

Speksnijder, D. C., Jaarsma, A. D. C., Van Der Gugten, A. C., Verheij, T. J., & Wagenaar, J. A. (2015). Determinants associated with veterinary antimicrobial prescribing in farm animals in the Netherlands: a qualitative study. Zoonoses and public health, 62, 39-51.

Speksnijder, D. C. (2017). Antibiotic use in farm animals: supporting behavioural change of veterinarians and farmers. Utrecht University.

Massaglia, S., Merlino, V. M., Borra, D., Verduna, T., Renna, M., & Rambozzi, L. (2018a). Impact of Swine Ascariasis on Feeding Costs and Revenues in Farms associated with the Italian PDOs Dry-Cured Hams Industry. Calitatea, 19(167), 146-154.

Maron, D. F., Smith, T. J., & Nachman, K. E. (2013). Restrictions on antimicrobial use in food animal production: an international regulatory and economic survey. Globalization and health, 9(1), 48.

Merlino, V. M., Borra, D., Girgenti, V., Dal Vecchio, A., & Massaglia, S. (2018). Beef meat preferences of consumers from Northwest Italy: Analysis of choice attributes. Meat science, 143, 119-128.

Merlino V. M., Borra D., Verduna T., Massaglia S., (2017). Household Behavior with Respect to Meat Consumption: Differences between Households with and without Children. Veterinary sciences, 4(4), 53.

Massaglia, S., Merlino, V., & Borra, D. (2018b). Marketing strategies for animal welfare meat identification: Comparison of preferences between millennial and conventional consumers. Calitatea-Acces La Succes, 19(S1), 305-311.

Bager, F., Aarestrup, F. M., & Wegener, H. C. (2000). Dealing with antimicrobial resistance-the Danish experience. Canadian Journal of Animal Science, 80(2), 223-228.

Hayes, D. J., Jensen, H. H., & Fabiosa, J. (2002). Technology choice and the economic effects of a ban on the use of antimicrobial feed additives in swine rations. Food Control, 13(2), 97-101.

Ungemach, F. R., Müller-Bahrdt, D., & Abraham, G. (2006). Guidelines for prudent use of antimicrobials and their implications on antibiotic usage in veterinary medicine. International Journal of Medical Microbiology, 296, 33-38. https://apps.who.int/iris/bitstream/handle/10665/255027/9789241512220eng.pdf?sequence=1&isAllowed=y», Accessed 10 February 2019.

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Effect of melatonin implantation on haematological parameters in anestrus lactating buffalo during summer season under tropical conditions

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N

TAMER RAMADAN1*, RAKESH SHARMA2, SUSHIL PHULIA2, INDERJEET SINGH2 1

2

Animal Production Research Institute, Agricultural Research Center, 4 Nadi El-Said, 12311 Dokki, Giza, Egypt Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, 125001, Hisar, Haryana, India

SUMMARY The purpose of this study was to investigate the effects of melatonin implantation in Murrah buffaloes on some haematological parameters during summer season under tropical conditions. Twelve lactating Murrah buffaloes were divided into control and treated groups of six animals each. Treated buffaloes were implanted with melatonin (18 mg melatonin / 50 kg body weight) at the base of left ear. Blood samples for measuring haematological parameters collected biweekly between 0 and 84 days were analysed by haematology analyzer. Melatonin treatment had no effect on all haematology parameters during summer season under tropical conditions. With the advancement of melatonin treatment increases (P < 0.05) in mean corpuscular hemoglobin at day 70 after treatment and in count of mean corpuscular hemoglobin concentration and red blood cell distribution with-coefficient of variation at day 84 were found. In addation, melatonin revealed an increase (P < 0.05) in mean platelet volume at days 28 and 56 after treatment. On the other hand, the advancement of melatonin treatment exhibited gradual decrease (P < 0.05) in neutrophil, platelets and plateletcrit count 84 days after treatment. Other heamatological parameters were not affected by advancement of day after treatment. In general, melatonin implantation did not affect the haematological parameters to counteract the summer stress conditions in lactating buffaloes under tropical conditions.

KEY WORDS Melatonin implantation, Lactating buffalo, Haematology, Summer season.

INTRODUCTION Buffaloes are short day breeders with reproductive efficiency adversely influenced by unfavorable biometeorological factors1.

Stress, the obvious reaction of the animal, disturbs body homeostasis causing detrimental effects. Livestocks undergo various types of stresses such as physical, nutritional, chemical, psychological and thermal. Among all, heat stress is the most concerning constraint on animal production nowadays under the ever changing climatic scenario. It is the perceived discomfort and physiological strain associated with exposure to the uttermost hot temperature. It stimulates sort of complex responses which are fundamentals in the preservation of cell survival.

Among many adverse effects, heat stress causses the aberration of reproductive functions, oxidative stress, enzymatic dysfunction and electrolyte imbalances. These physiological adjustments are essential to maintain normal body funuctions and to prevent hyperthermia2.

Melatonin has been reported to play a fundamental role in the biology of body cells3 and to influence a wide range of physi-

Corresponding Author: Tamer Ramadan (tamereweda1174@Yahoo.com).

ological processes for all organs and cells of the body4. However, the effect of melatonin on blood parameters is controversial and what is available in literature is contradictory. Karimungi and Joshi5 reported that melatonin treatment once or twice daily for two weeks induced a decrease in the red blood cells (RBCs) count and in the erythrocyte indices (MCV, MCH and MCHC) of rats. Durotoye and Rodway6 found that subcutaneous implants of melatonin led to reduction in RBCs count and packed cell volume (PCV) in ewes, while increased MCV. Out-of-season breeding in deep anestrous buffaloes, requests the use of melatonin7,8. Melatonin is a hormone produced and stored in the pineal gland during the day and secreted during the dark, starting after sunset and ending at sunrise. It controls the reproductive rhythm in diverse livestock species, like goats and sheep (short-day species), and horses (long-day species), especially at higher latitudes9. Melatonin-mediated pathways regulate GnRH pulsatibility and, therefore, the activity of the reproductive neuroendocrine axis. It also modulates prolactin secretion by acting on the hypophysis.

Melatonin has been shown to be a highly effective antioxidant and free radical scavenger10. By virtue of its antioxidant properties melatonin, quenches the oxidants including nitric oxide, arrests lipid peroxidation, and acts synergistically with other classic antioxidants such as glutathione peroxidase (GPx), su-

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peroxide dismutase (SOD), vitamin E, and selenium11. The main function of melatonin is to serve as an antioxidant to protect organisms from ubiquitous oxidative stresses. It is considered to be more effective than glutathione (GSH) and mannitol in scavenging free radicals. It was found that melatonin has the ability to neutralize damaging reactive oxygen species (ROS) and reduce lipid peroxide concentrations and DNA damage, thereby, improving the viability of germ cells12. There is a limited data available on the effect of melatonin implantation on the haematology prameters of lactating buffaloes.

The aim of the present study was to analyse the effects of melatonin implantation on some blood haematology parameters in anestrus lactating buffaloes to serve as indicators of their sustainability to the expenditure of melatonin treatment for preventing summer-induced decline in body functions.

MATERIALS AND METHODS The present study was conducted at the animal farm of the Central Institute for Research on Buffaloes, Hisar, India (29° 10 ’ N, 75° 41’ E), using anestrus lactating buffaloes during the outof-breeding season (from June to September). All procedures and experimental protocols were conducted in accordance with the «Guide for the Care and Use of Agricultural Animals in Research and Teaching»13.

2 × 4 mm absorbable melatonin implants (18 mg melatonin ⁄ implant, Regulin, CEVA Animal Health Limited, Chesham, Buckinghamshire, UK) at the base of left ear using an implanter.

Total implants inserted to each animal were calculated on the basis of body weight (one implant ⁄ 50 kg)16. These implants were designed to release melatonin for at least 60 days, although their functionality can extend to more than 100 days without disturbing the endogenous secretion of melatonin17.

Blood haematology parameters Blood samples were collected via jugular venipuncture into a heparinized vial (at 06:00 a.m.). On both treated and control groups biweekly blood sampling were taken throughout the experiment between days 0 to 84. Collected blood samples were analysed for estimation of haematological parameters which included count of red blood cells (RBC), white blood cells (WBC), lymphocytes (LY), monocytes (MO), Neutrophil, Eosinophil, Basophil, percentage of lymphocytes (LY %), monocytes (MO %), Neutrophil (%), Eosinophil (%), Basophil (%), hemoglobin (Hb), hematocrit (HCT/PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width-coefficient of variation (RDWCV), platelets (PLT), plateletcrit (PCT), mean platelet volume (MPV), and platelet distribution width (PDWC) using hematology analyzer (HA-22-CLINDIAG).

Animals and management

Statistical analysis

Twelve lactating Murrah buffalo (parity: 2-4, body condition score: 4-5, milk yield 7-9 kg/day and body weight: 400-500 kg) at day 65-70 of lactation were used in the present study. The study was conducted during the hot-humid months from June to first of September when ambient temperatures and relative humidity ranged from 35 to 45°C and 35 to 80%, respectively. Daily maximum and minimum temperatures and Relative humidity (RH) were recorded at Department of Agriculture Meteorology, Chaudhary Charan Singh Haryana Agricultural University, Hisar, Haryana. The daily records of humidity and temperature were obtained from the closest meteorological station, approximately 10 km far from the farm. The temperature-humidity index (THI) was calculated from the equation14.

THI= (1.8 * AT + 32) - [(0.55-0.0055 * RH) × (1.8 * AT-26)], where AT = average temperature (°C), RH = Relative humidity (%). The mean values of temperature and THI in different weeks are described in Figure 1. The experimental period was divided into: low (< 70); moderate (70-80) or high (> 80) THI. Lactating buffaloes were confined for the entire period of study to a barn with access to an open sheltered space. They were subjected to teasing and observed for visual signs of estrus twice per day for one hour but they failed to exhibit estrus characteristices. They were fed on roughage and concentrate supplement according to their body weight requirements15.

Chaffed green fodder and wheat straw were also offered. Water was accessibly available at all times. Animals were free from diseases and were clinically normal with a healthy appearance.

Lactating buffaloes were milked twice a day, morning (04:00 a.m.) and evening (03:00 p.m.).

All data records were tested for normality with the Shapiro-Wilk (W) test from the UNIVARIATE procedure18, and results indicated that all data were distributed normally (W > 0.90). Data for the effect of melatonin was analyzed using PROC MIXED of SAS (SAS Inst., Inc., Cary, NC) for repeated measures. Data for the effect of melatonin implantation on blood heamatology were analyzed by adapting the following model: Yijk = µ + Ti + Dj + ( T ×D) ij + eijk , where Yijk is the observed value of the dependent variable determined from a sample taken from each animal, µ is the overall mean, Ti is the fixed effect of the ith treatment (i = 1,2), Dj is the fixed effect of the jth day (j = day 0:day 84), (T×D) ij is the first-order interaction between treatments and days and eijk is the residual error. Significant differences among means within each classification were tested using least square differences 0.05.

Experimental design Lactating buffaloes were randomly allocated to melatonin nonimplanted (control) and implanted (treated) groups (n = 6 each). In melatonin-treated group, animals were administered

RESULTS Means of ambient temperature, relative humidity and THI during the experimental period were 32.55 ± 2.56 ºC, 60.81 ± 18.08 % and 83.14 ± 1.78 % respectively (Figure 1). Haematological parameters of anestrus lactating buffalo as affected by melatonin implantation are displayed in Tables 1 and 2. Melatonin implantation had no effect on all haematological parameters during summer season under tropical condition. After melatonin implantation gradual decrease (P < 0.05) take place in neutrophil, PLT and PCT (Figure 2). On the other hand, with the advancement of treatment an increase (P < 0.05) in MCH was found at day 70 after treatment. Also, increase (P < 0.05) in counts of MCHC and RDW-CV was observed at day 84 and in MPV at days 28 and 56 after melatonin implantation (Figure 3). Other heamatological parameters were not affected by

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THI

Relative Humidity (%)

Ambient temperature (%)

T. Ramadan et al. Large Animal Review 2020; 26: 283-290

Weeks Figure 1 - The weekly average ambient temperature (A), relative humidity (B) and temperature-humidity index (THI) over the experimental period.

advancement of day after melatonin implantation of lactating buffalo during summer season under tropical condictions.

DISCUSSION The significance of reference values on haematological indices was recognised as being useful in determining the general health status of animals19, an aid for differential diagnosis of clinical conditions and for monitoring response to therapy20. Although

melatonin implantation had many beneficial effects on improving reproductive performance in hiefers and lacating buffaloes7,8 and on semen quality of buffalo bulls21 it had no obvious effects on heamatology of lactating buffaloes.

The blood system is sensitive to temperature changes and is an important indicator of physiological responses to stressors. Several factors such as species, breed, sex, age, nutrition, diseases, physiological stage and seasonal variations can affect the pattern of haematological values22,23. Quantitative and morphological changes in blood cells are associated with heat stress. Other stud-

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Figure 2 - Effect of day after melatonin treatment on some blood haematological parameters neutrophils (109/L), platelets (109/L) and PCT (%) of control (â??) and treated (â&#x2014;?) groups of lactating buffalo during summer season (Least square means Âą SEM).

ies show the variations in hematocrit values, mean erythrocytes count and hemoglobin24,25. Poor nutrition, which occurs in animals under long-term heat stress, reduces the number of erythrocytes and hemoglobin level, resulting in a decrease of red blood cells in the bloodstream26. With the rise in environmental temperature, the animal loses liquids through the respiratory tract, reducing the blood plasma volume and increasing the concentration of hematocrit. If physical exertion is prolonged, dehydration occurs, and thus, loss of fluids by the evaporative

process, results in more hematocrit increase. Thermal stress may cause hyperthermia and potentially have several physiological side effects. It is known to alter the homeostatic mechanisms of animals resulting in impaired erythropoiesis.

In the present study, mean values for all haematological parameters were situated in the normal physiological limits without statistical significant difference between implanted and unimplanted animals. However, the mean RBCs and Hb were found to be numerically lower after melatonin implantation.

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Figure 3 - Effect of day after melatonin treatment on some blood haematological parameters MCH (Pg), MPV (fL), MCHC (g/dl) and RDWCV (%) of control (❍) and treated (●) groups of lactating buffalo during summer season (Least square means ± SEM).

The variation in RBCs may be attributed to the fact that the high environmental temperature increases the animals oxygen consumption through increased respiration rate. The high oxygen intake increases the partial pressure of oxygen in blood, decreases erythropoiesis, which in turn reduces the number of circulating RBCs and Hb values27. Kumar et al.28 also recorded low mean Hb in summer stressed Beetal goats as compared to pre-summer values.

No literature concerning the effect of melatonin implantation

on haematological parameters in lactating buffaloes during summer season are available. However, Anwar et al.29 found that melatonin treatment in rats increased RBCs, Hb and PCV numerically. On the other hand, Durotoye and Rodway6 reported that implants of melatonin in ewes reduced RBCs count and PCV. The average values for Hb varied insignificantly in conrtol (10.18±0.23 g/dL) and treated groups (9.83±0.23 g/dL).

However, the level of hemoglobin was influenced by the season with the highest values obtained in summer season23. Also,

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Table 1 - Effect of melatonin treatment on blood haematological parameters of lactating buffalo during summer season (Least square means ± SEM).

Parameter

Treatment (T) Control Melatonin

SEM

RBCs (10 12 /L)

6.84

Hb (g/dL)

10.18

9.83

0.23

WBC (10 9 /L)

8.98

8.92

0.30

6.77

0.16

Lymphocytes (10 9 /L)

4.90

4.41

0.26

Monocytes (10 9 /L)

0.27

0.26

0.10

Neutrophil (10 9 /L)

4.28

4.13

0.16

Eosinophil (10 9 /L)

0.09

0.08

0.01

9

Basophil (10 /L)

0.011

0.012

0.01

HCT (%)

36.43

35.80

0.79

MCV (fL)

53.26

52.97

0.48

MCH (Pg)

14.75

14.68

0.12

MCHC (g/dl)

27.93

27.97

0.14

RDW-CV (%)

20.45

20.71

0.19

204.25

210.07

7.09

0.19

0.20

0.01

9

PLT (10 /L) PCT (%) MPV (fL)

9.82

9.85

0.14

PDWC (%)

36.37

36.28

0.39

Fagiolo et al.30 reported higher Hb values in lactating buffaloes, during the summer season (13.62 g/dL) compared to winter (11.37 g/dL).

A plausible explanation for the decrease in Hb levels during thermal stress could be the increased attack of reactive oxygen molecules on the erythrocyte membrane which is rich in lipid content, and ultimate lysis of RBC or inadequate nutrient avail-

ability for Hb biosynthesis due to decreased voluntary intake as the animal consumes less feed under heat stress31. During summer stress a significant depression in Hb levels may also be due to haemodilution effect where more water is infused into the circulatory system for evaporative cooling32. At high temperature, peripheral vasodilation and redistribution of cardiac output are associated with expansion of blood volume and result in haemodilution33.

Moreover, the increase in Hb concentration obtained in the control group of the present study may be attributed either to its direct stimulatory effect on bone marrow29 or indirectly through stimulation of some cytokines, which have a powerful stimulatory effect on bone marrow cells proliferation. Another possible explanation for the increase in Hb levels after goat exposure to sunshine and melatonin treatment during thermal stress could be the protection of bone marrow from damage by free radicals due to melatonin antioxidant effect34. The average percentages of hematocrit (36.43±0.79% in control and 35.80±0.79% in treated buffaloes) were between those in summer (40.75%) and those in winter period (32.63%) as reported

by Fagiolo et al.30 and Enculescu et al.23.

Total WBC count in the current study were similar to those reported by Enculescu et al.23 and Garkal et al.22. No available literature could be traced regarding the effect of melatonin on this parameter in lactating buffaloes treated with melatonin.

Hasin et al.35 showed that melatonin treatment was found to induce a significant increase in total leucocyte count in goats and these results are consistent with those previously reported showing that administration of melatonin increased the TLC in broiler chicks36, rats29 and squirrels37. The precise mechanism responsible for this increase is not clear. However, several mechanisms could be involved in this respect. One possible mechanism may be the direct action of melatonin either on bone marrow38 or on lymphatic tissue39 to accelerate leukocytogenesis40. The second possible mechanism may be an indirect ac-

Table 2 - Effect of day after melatonin treatment on blood haematological parameters of lactating buffalo during summer season (Least square means ± SEM).

Parameter 0 RBC (10

12

/L)

14

28

Day ( D) 42

56

70

84

7.03

7.00

6.94

6.75

6.64

6.68

6.60

Hb (g/dL)

9.70

10.09

10.05

9.97

9.85

10.27

10.12

WBC (10 9 /L)

9.24

9.26

8.70

8.20

8.54

9.72

8.99

Lymphocytes (10 9 /L)

4.31

5.50

4.85

4.11

4.21

4.69

4.92

9

Monocytes (10 /L)

0.30

0.19

0.21

0.23

0.28

0.31

0.31

Neutrophil (10 9 /L)

5.24a

4.07b

3.74b

3.95b

4.25b

4.25b

3.94b

Eosinophil (10 9 /L)

0.06

0.11

0.09

0.08

0.09

0.07

0.06

Basophil (10 9 /L)

0.011

0.010

0.010

0.013

0.010

0.016

0.011

HCT (%)

36.37

36.45

36.98

36.39

35.84

35.93

34.87

MCV (fL)

54.08

52.91

52.50

53.00

52.91

53.91

52.50

b

b

b

b

ab

a

15.43

14.70b

MCH (Pg)

14.62

14.43

14.44

14.53

14.88

MCHC (g/dl)

27.15 d

27.34 d

27.63 cd

27.58cd

28.29bc

28.59ab

29.06a

b

b

b

b

b

RDW-CV (%)

19.67

19.80

20.19

20.18

20.56

20.65

23.01 a

PLT (10 9 /L)

307.10a

241.75b

181.50c

173.42c

166.17c

187.71c

192.47c

PCT (%)

0.31

MPV (fL) PDWC (%) a,b

a

b

b

c

c

cd

0.17

cd

0.16

0.14d

0.24

0.19

0.19

9.99b

10.17ab

10.75a

10.52ab

10.83a

8.15c

8.41c

35.88

36.35

37.23

36.01

35.79

36.41

36.60

Within a row, means with different superscript differ (P < 0.05).

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289

tion through reduction of corticosterone hormone, which is associated with the elevation of the leukocytic count29. A third possible mechanism may be the protective effect of melatonin on bone marrow from damage by the free radicals due to its antioxidant effect34.

The advancement of day of treatment revealed significant increase in total lymphocytes, Eosinophils and Basophils percentages in days 42, 84 and 70 in lactating buffalo. Similarly, administration of melatonin increased the total leukocytic count and lymphocyte percentage in broiler chicks36, rats29, immature chicks41 and squirrels37. Rai and Haldar42 reported that daily subcutaneous injection of melatonin increased significantly the lymphocyte count in adult male squirrels, while pinealectomy decreased it along with percent lymphocyte count in peripheral blood and bone marrow. The WBC counts of birds injected subcutaneously with 40 mg melatonin/kg BW/day for 7 days were significantly higher than the WBC counts of saline-injected birds41. The increase in neutrophil counts after day 0 of the experiment commencement could be attributed to lactational stress leading to the release of endogenous corticosteroids43. The monocyte counts recorded in this study were in accordance with those of Ellah et al.44 in heifers and of Ali and Shukla45 in normal cyclic post-partum buffaloes.

Advancement of day of treatment showed an increase in MCH and MCHC of lactating buffalo towards the end of experiment.

Lee et al.46 described a significant decrease in haematocrit value and RBC counts in dairy cows exposed to high temperatures.

This decrease was probably caused by a rise in erythrocyte destruction; haemodilution effect could also participate here, because more water was transported to the circulatory system for evaporative cooling. Nadia47 indicated that heat stress decreases MCH and MCHC, and increases MCV value in heat stressed Japanese quail. Nonetheless, the values of MCV, MCH, and MCHC did not vary significantly between groups of Murrah buffaloes in the current study. The increased RDW-CV at day 84 of the experiment was probably due to enhanced erythropoiesis. On the other hand, gradual decrese in total neutrophils, PLT and PCT with advancement of day of treatment were obtained. Current findings for PLT counts were in tune with those recorded by Das et al.48 on lactating Mehsani buffaloes.

Little is known about the antioxidant defense mechanisms in anestrus buffalo. Oxidative stress, caused by different metabolic processes, is controlled by various antioxidant defense mechanisms including antioxidant enzymes49. Ramadan et al.8 reported that melatonin implantation increased superoxide dismutase (SOD) activity in anestrus lactating buffalo during non-breeding season. Therefore, it stimulates the activities of enzymes involved in metabolizing reactive oxygen species (ROS) and preserves cell membrane fluidity. Indeed, melatonin was shown to be twice as potent as vitamin E in removing peroxyl radicals50, and it is more effective in scavenging hydroxyl radicals than glutathione and mannitol51. Melatonin also protects and stimulates the activities of antioxidant enzymes such as SOD52 which acts as antioxidant that catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide.

terpretation of haematological parameters in lactating buffaloes.

The responsiveness of lacataing baffalo to melatonin implantation in summer season showed non significant differencses in all blood prameters. No significant deviations in different haematological indices of lactating buffalo during summer seasons after melatonin treatment were declared.

CONCLUSIONS

14.

The values obtained for haematological parameters in melatonin treated and untreated buffalo groups were within normal physiological limits. These values could be useful for in-

Acknowledgements This research was funded by a research grant awarded by CV Raman International Fellowship for African Researchers under Post-Doctoral Fellowship, New Delhi, India.

Compliance with ethical standards All applicable international, national, and/or institutional guidelines for the care and use of animals were followed and the unnecessary discomfort to the animals was avoided.

Conflict of interest statement The authors declare no conflict of interest.

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L’AGGIORNAMENTO VETERINARIO

ON DEMAND

Quando vuoi e ovunque tu sia, l’aggiornamento sempre a tua disposizione.

Ramadan_imp_ok 11/12/20 10:06 Pagina 292

Celozzi imp_ok 11/12/20 10:06 Pagina 293

S. Celozzi et al.; Large Animal Review 2020; 26: 293-298

Valutazione della sostenibilità ambientale della produzione di latte e formaggio caprino mediante approccio LCA

293

j

STEFANIA CELOZZI, SILVANA MATTIELLO, MONICA BATTINI, GIOVANNI BAILO, LUCIANA BAVA, ALBERTO TAMBURINI, IRENE VALSECCHI, MADDALENA ZUCALI* Dipartimento di Scienze Agrarie e Ambientali - Produzione, Territorio, Agroenergia, Università degli Studi di Milano, Via Giovanni Celoria 2, 20133 Milano, Italy

SUMMARY Although the contribution to greenhouse gas emissions of dairy goat breeding has a limited weight in respect to the whole livestock sector, it is yet paramount to reduce the carbon footprint in order to limit the impact on climate change. The objective of this study was to quantify the environmental impact of milk production in 8 dairy goat farms in Lombardy region (Northern Italy) by using a Life Cycle Assessment approach; the focus was set on critical points of the production process. Environmental impact results were very variable, ranging from 1.12 to 3.05 kg CO2 eq/kg fat and protein corrected milk. Farms with a different milk production level (946 and 1260 kg per goat/year) showed similar levels of kg CO2 eq emitted (1.46 vs 1.48). This suggests that the intensification of milk production alone is not enough to make the goat milk production system more efficient: in fact, appropriate feeding strategies, in particular the increase of feed self-sufficiency and a rational management of livestock effluents, can also help to reduce the emission load of goat milk production. Our study confirms that enteric methane emissions and purchased feed are the main drivers of environmental impact of goat milk production, although with a different weight of these hotspots, according to the production context. During goat cheese production, only 10% of greenhouse gas emissions depend on cheese making process per se, whereas the major impact (90%) is due to goat milk production. Farms that are characterized by extensive systems had worse environmental results expressed by kg fat and protein corrected milk; when 1 ha of usable agricultural area is considered as functional unit, these farms obtain better environmental results, because their impact is diluted on a higher soil availability. This study has highlighted that, although there are processes such as enteric methane emission in which the intervention of farmer is limited, there are management strategies that can contribute to reduce the environmental impact of goat milk production.

KEY WORDS Environmental impact, Dairy goat production, Life Cycle Assessment.

INTRODUZIONE La sostenibilità di un processo è un concetto complesso, al quale si fa fatica a dare una definizione. Ancor più complessa è la valutazione della sostenibilità dei processi biologici, quali sono quelli che caratterizzano la produzione zootecnica. Quest’ultima è una filiera lunga e con molti attori, che include molteplici prodotti, sottoprodotti e scarti. Ad aumentare la complessità dell’analisi di tale processo produttivo vi è poi l’aspetto del management aziendale, che varia da azienda ad azienda, così come l’attenzione alla registrazione dei dati che caratterizzano il processo produttivo. In generale, possiamo affermare che una produzione zootecnica sostenibile fa riferimento ad un contesto di produzione efficiente, sicuro e che mira a proteggere, migliorare e soprattutto preservare a lungo termine l’ambiente naturale, le condizioni sociali ed economiche degli allevatori, i loro dipendenti e le comunità locali, salvaguardando la salute e il benessere di tutti gli animali allevati. Le emissioni di gas ser-

Corresponding Author: Maddalena Zucali (maddalena.zucali@unimi.it)

ra rappresentano, soprattutto in ambito scientifico, uno fra i metodi più utilizzati per esprimere il carico ambientale di un processo, sebbene si tratti solo di una delle possibili misure adottabili. La sostenibilità di un processo può essere considerata anche attraverso altri punti di vista, per esempio in relazione alla biodiversità, ai servizi di mantenimento del territorio, ai servizi sociali e fra quest’ultimi la conservazione delle tradizioni locali.

A livello globale la zootecnia ha un peso del 14,5% sul totale delle emissioni antropiche; il settore ovino e caprino impatta per il 6,5% sulle emissioni di gas climalteranti imputate al settore zootecnico1. A livello italiano questo dato è sicuramente più basso, se si considera che l’impatto ambientale dell’intero settore agricolo rappresenta il 7% delle attività antropiche, in termini di emissione di gas ad effetto serra². Rispetto al passato tuttavia, si sta assistendo ad un aumento delle aziende intensive, con possibile peggioramento dell’impatto ambientale di questo comparto zootecnico. Pertanto, se si vuole mitigare il cambiamento climatico, anche il settore caprino deve ridurre la sua impronta carbonica. Anche l’allevatore può trarne dei vantaggi, come quello di indentificare i punti critici della propria azienda, anche da un punto di vista economico. Vi

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può essere inoltre l’opportunità di valorizzare il prodotto (diversi sono gli esempi di marchi di dichiarazione ambientale di prodotto), e difenderlo da attacchi mediatici.

Il metodo più comunemente utilizzato per valutare il carico ambientale della produzione di latte è quello dell’LCA (Life Cycle Assessment), che consente di identificare e quantificare i consumi di materia ed energia e le emissioni nell’ambiente³. Tale metodo permette da una parte di valutare il processo produttivo nella sua totalità, identificandone i punti critici, e dall’altra di comparare processi diversi (ad esempio biologico vs convenzionale) o che portano alla produzione di alimenti differenti. Si tratta di uno strumento che può essere utile non solo all’allevatore per prendere decisioni a livello aziendale, ma anche ai politici per prendere decisioni a livello locale e nazionale.

Nonostante ciò, tale metodo non è in grado di considerare la totalità degli impatti ambientali che si generano dalla produzione di un alimento, non quantificando ad esempio l’impatto sulla biodiversità, sui servizi di mantenimento del territorio, sui servizi sociali, ecc. I risultati sono inoltre fortemente dipendenti dalle scelte di calcolo utilizzate, come ad esempio quella dell’unità funzionale (ossia l’unità di prodotto o di superficie sulla quale viene ripartito l’impatto) e non sono di facile comprensione. L’impatto ambientale può essere valutato considerando differenti fattori, come le emissioni di gas serra, l’acidificazione, l’eutrofizzazione, l’uso del suolo e quello dell’energia da fonti non rinnovabili: fra queste, la prima è senz’altro quella più utilizzata.

La produzione di alimenti utilizza risorse ambientali e determina l’immissione nell’ambiente di gas e prodotti con potere inquinante. Nell’allevamento dei ruminanti, e dunque anche nella produzione del latte di capra, le fermentazioni enteriche e la produzione degli alimenti rappresentano i principali hotspot, ossia i processi che contribuiscono all’impatto ambientale.

Lo scopo dello studio è stato quello di quantificare l’impatto ambientale della produzione di latte di capra in un campione di aziende lombarde mediante metodo LCA, al fine di individuare i punti critici del processo produttivo.

senti in allevamento: capre in lattazione, caprette, capretti e becchi.

I risultati di impatto ambientale sono stati espressi come chilogrammi di anidride carbonica equivalente emessa in atmosfera e sono stati ripartiti su a) un chilogrammo di latte corretto per grasso e proteina (kg CO2 eq/kg latte corretto); b) un chilogrammo di formaggio; c) un ettaro di superficie aziendale.

Al fine di quantificare il latte corretto per grasso e proteina è stata utilizzata la seguente equazione dell’INRA: FPCM = MilkDEL (0.26 + 0.1352 fat% + 0.079 prot%) Dove: MilkDEL è il latte totale consegnato per anno e per azienda (kg/anno) fat% è la percentuale in grasso del latte consegnato (%) prot% è la percentuale in proteina grezza del latte consegnato (%) Considerando come unità funzionale il chilogrammo di formaggio è stato preso in considerazione un “equivalente formaggio medio”, un ipotetico formaggio semi stagionato con resa al 12%, che ha consentito di paragonare le diverse produzioni a livello aziendale e fra le aziende. Il sottoprodotto principale del processo di caseificazione è rappresentato dal siero, che viene somministrato ai suini in 6 aziende su 7; una sola azienda non dichiara la modalità di smaltimento di questo sottoprodotto.

Per il latte è stato utilizzato un metodo di allocazione fisico, al fine di ripartire l’impatto ambientale tra latte e carne prodotti in azienda, secondo quanto suggerito da IDF 20158. Per il formaggio è stata presa in considerazione un’allocazione per sostanza secca, che permette di ripartire l’impatto in funzione del contenuto di sostanza secca del formaggio e del siero. La percentuale di sostanza secca del siero dolce (6%) è stata ottenuta dal database del United States Department of Agriculture (USDA, 20199).

RISULTATI MATERIALI E METODI Lo studio ha visto il coinvolgimento di 8 aziende commerciali di capre da latte, situate in 7 differenti province della regione Lombardia.

Il metodo LCA è stato implementato per valutare l’impatto ambientale delle produzioni aziendali; sono state prese in considerazione le emissioni di gas serra (o “Carbon Footprint” = impronta carbonica) come categoria d’impatto. La fase di inventario ha visto la raccolta dei dati sulle produzioni e sugli acquisti effettuati in un anno dalle aziende zootecniche; queste informazioni sono state raccolte sottoponendo gli allevatori a questionari. Le emissioni di gas ad effetto serra sono state quantificate utilizzando il software SIMAPRO e il metodo di caratterizzazione ILCD (International Life Cycle Data system) 2011 Midpoint V1.03 - ISO 14040/445. Le emissioni di metano enterico (CH4) sono state stimate con il metodo TIER 2, applicando un’equazione INRA (2018)6 che tiene conto della digeribilità della razione, mentre per le emissioni durante la stabulazione e dai reflui seguendo le linee guida elaborate nel 2006 dall’Intergovernmental Panel on Climate Change (IPCC)7. Le emissioni sono state calcolate per ogni categoria di animali pre-

In tabella 1 vengono mostrate le caratteristiche principali delle aziende, che sono state coinvolte nella fase dimostrativa del progetto DEMOCAPRA - Divulgazione partecipativa di modelli gestionali sostenibili per l’allevamento della capra da latte in Lombardia mediante strumenti innovativi, finanziato dalla regione Lombardia (FEASR - Programma di Sviluppo Rurale 2014 - 2020 Misura 1 Operazione 1.2.01). I valori soglia presentati in questo paragrafo seguono le indicazioni dei tecnici Aral del team DEMOCAPRA. Il numero medio di capre per azienda nel 2018 (137) è stato più alto della media delle aziende italiane nel 2016 (78,7 capre/azienda10). In sei aziende sono allevate razze cosmopolite (Camosciata delle Alpi o Saanen), mentre in due aziende sono allevate anche razze autoctone come la Nera di Verzasca (azienda F) e Nera di Verzasca e meticce (azienda H). L’orientamento produttivo riscontrato nelle aziende era diverso: tre aziende trasformavano il 70% del latte prodotto e vendevano il restante 30%; l’intera produzione aziendale veniva invece trasformata in quattro aziende e venduta interamente in una soltanto. La prolificità ha fatto registrare un numero medio di capretti nati per parto inferiore al valore soglia che, fa-

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Tabella 1 - Caratteristiche medie delle aziende analizzate.

Variabili

Carico di bestiame

Unità

Media

Deviazione standard

Min

Max

UBA*/ha

1,08

0,84

0,05

2,85

Capi munti fuori stagione

%

8

22

0

62

Capi in lattazione lunga

%

4,0

8,0

0

21,0

Fertilità ai parti**

%

83

10

69

96

n capretti/parto

1,68

0,20

1,45

1,95

%

22

11,4

2,0

36

Prolificità Tasso di rimonta Mortalità capretti

%

7

7

1

19

Mortalità capre

%

4

3

0

10

Capi improduttivi

%

3

3

0

7

Superficie totale

ha

56,7

103,8

9,75

310

Superficie a pascolo

% della SAU*** totale

17

26

0

65

Superficie a erba medica

% della SAU*** totale

19

33

0

82

Autosufficienza foraggera

%

74

19

50

100

*UBA= Unità di Bestiame Adulto **Fertilità ai parti calcolata come parti/capi alla monta in 7 aziende (l’azienda G non disponeva del dato del numero di parti) ***SAU= Superficie Agricola Utile

cendo riferimento alle indicazioni dei tecnici del team DEMOCAPRA11, dovrebbe essere ≥ 1,8. La mortalità delle caprette è stata appena sotto al valore limite dell’8%11 ad indicare un aspetto di criticità nella gestione dei primi momenti di vita degli animali. La mortalità delle capre adulte è stata invece entro limiti ritenuti accettabili (valore soglia <10%)11. Il valore medio dei capi improduttivi è stato al di sotto del 10%, valore oltre il quale devono essere ricercate le cause della scarsa efficienza produttiva11. Più di un terzo della SAU è stata utilizzata per il pascolo e la produzione di erba medica; l’autonomia foraggera media si è rivelata infatti essere piuttosto elevata.

Come si può osservare nelle Figure 1 e 4, l’emissione di gas climalteranti delle 8 aziende è stata assai variabile, passando da 1,12 a 3,05 kg di CO2 equivalenti/kg latte corretto. Questi risultati di impatto ambientale non sempre sono stati influenzati dal livello di produzione di latte individuale (espresso in

kg di latte per capra/anno), anche se si può notare un trend interessante. Le aziende C e D hanno mostrato un impatto ambientale simile (1,46 e 1,48 kg di CO2 equivalenti/kg latte corretto rispettivamente), nonostante abbiano presentato livelli produttivi differenti (946 e 1.260 kg di latte per capra/anno).

Valutando le aziende in termini di latte consegnato annualmente (Figura 2), si è potuto osservare che l’azienda G, che ha mostrato la produzione di latte annua più elevata (259.956 litri di latte prodotto/anno), ha fatto anche registrare il secondo peggior risultato dal punto di vista dell’impatto ambientale (2,24 kg di CO2 equivalenti/kg latte corretto). L’azienda H, caratterizzata da un regime di allevamento semi estensivo e dalla presenza nel gregge anche di razze locali (Nera di Verzasca e meticce), ha conseguito l’impatto ambientale più elevato (3,05 kg di CO equivalenti/kg latte corretto), sebbene la sua produzione di latte sia stata di poco superiore a quella dell’azienda C, che

Figura 1 - Produzione di latte individuale e impatto ambientale.

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Figura 2 - Impatto ambientale della produzione di latte aziendale in confronto al livello produttivo annuale.

invece ha avuto un carico emissivo decisamente più modesto (1,46 kg di CO2 equivalenti/kg latte corretto). Ciò è da ricondurre prevalentemente ad un acquisto rilevante di concentrati da parte dell’azienda H e dunque a uno scarso sfruttamento del pascolo.

Le emissioni di metano enterico alla stalla e l’acquisto di alimenti sono risultati essere i principali driver dell’impatto ambientale della produzione di latte di capra. Nonostante ciò, aziende con impatto simile sull’ambiente (1,12 e 1,57 kg di CO2 equivalenti/kg di latte corretto per l’azienda A ed E, rispettivamente) hanno mostrato un diverso “peso” di queste voci di impatto, come mostrato nella Figura 3. Il processo che più degli altri si rende responsabile dell’impatto ambientale dell’azienda E è rappresentato dalle emissioni di metano enterico, seguite dall’acquisto di alimenti. Un’altra voce importante di impatto è rappresentata dalle emissioni di campo, a evidenziare una gestione non ottimale della fertilizzazione organica e minerale.

Anche nell’azienda A le emissioni di gas climalteranti sono sta-

te dovute ai medesimi hotspot, ma con il peso maggiore attribuito all’acquisto di alimenti, determinato da un elevato livello di concentrati della razione e una minore autosufficienza alimentare di questa azienda. In entrambe le aziende, lo stoccaggio delle deiezioni zootecniche ha rappresentato un ulteriore punto critico in termini ambientali, essendo la quarta voce che ha influenzato le emissioni in aria di gas serra.

I dati di impatto ambientale ripartito per chilogrammo di formaggio vengono riportati in Figura 4 per le sette aziende che hanno trasformato interamente o in massima parte (70%) il latte prodotto (ad eccezione dunque dell’azienda G, che ha venduto tutto il latte). Sebbene l’impatto ambientale della produzione di formaggio di capra sia risultato essere superiore a quello del latte in termini di chilogrammi di CO2 equivalenti emessi in atmosfera, le differenze che sono state riscontrate tra le varie aziende relativamente all’emissione di gas climalteranti espressa per chilogrammo di formaggio sono state analoghe a quelle espresse per chilogrammo di latte corretto (Figure 1

Figura 3 - Processi responsabili dell’emissione di gas climalteranti nelle aziende A ed E.

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Figura 4 - Impatto ambientale delle aziende analizzate, ripartito per le unità funzionali considerate.

e 4). Ciò è dovuto al fatto che circa il 90% dell’impatto ambientale della produzione del formaggio è dovuto alla produzione del latte; solo un rimanente 10% è dovuto alla corrente elettrica utilizzata in caseificio e ad altri input come il sale e i detergenti per la pulizia in caseificio. Questi risultati di impatto ambientale non si allontanano molto da quelli della produzione di Grana Padano DOP, dove il 95,3% dell’impatto ambientale è imputabile alla produzione della materia prima latte, mentre il 3,93% è dovuto al processo di caseificazione. Il restante 0,78% è dovuto alla stagionatura¹2.

Considerando come unità funzionale l’ettaro di superficie aziendale, i risultati di impatto ambientale sono cambiati notevolmente (Figura 4). La Superficie Agricola Utile (SAU) media delle 8 aziende analizzate è risultata essere pari a 56,7 ettari, ma ha presentato un’ampia variabilità (circa 10 ettari nell’azienda D vs 310 ettari nell’azienda H). L’azienda H, che ha mostrato l’impatto ambientale più elevato se espresso per kg di CO2 eq/kg di latte corretto, ha mostrato il minor valore di emissioni di gas climalteranti per ettaro di superficie, poiché l’ampia disponibilità di suolo ha consentito di “diluire” tale impatto.

DISCUSSIONE I risultati di impatto ambientale sono stati dapprima ripartiti sul chilogrammo di latte corretto; questa unità funzionale consente di avere una qualità del latte standardizzata e quindi risultati comparabili tra le varie aziende. Al pari di quanto evidenziato in molti studi scientifici13, l’impatto ambientale è risultato inversamente proporzionale alla produzione di latte individuale, ma sono state evidenziate situazioni aziendali differenti che suggeriscono come le scelte gestionali e alimentari possano avere un effetto molto importante sull’impatto ambientale della produzione lattea. Per lo stesso motivo, non sempre le aziende che annualmente consegnano più latte (che sono generalmente quelle con dimensioni maggiori del gregge) hanno conseguito i migliori risultati da un punto di vista ambientale. Anche la razza allevata può influire sull’impatto ambientale, in quanto razze più produttive, come la Saanen o la

Camosciata, presentando produzioni più elevate, permettono di ripartire le emissioni su un maggior numero di kg di latte; viceversa, razze meno produttive, come ad esempio la Nera di Verzasca, allevata nell’azienda H, forniscono risultati peggiori dal punto di vista dell’impatto in termini di kg di CO2 equivalenti/kg latte corretto, ma rappresentano una risorsa positiva per la sostenibilità delle produzioni animali dal punto di vista della biodiversità zootecnica14. I risultati del presente studio hanno confermato le emissioni di metano enterico alla stalla e l’acquisto di alimenti quali principali drivers dell’impatto ambientale della produzione di latte di capra15 16, sebbene con un diverso peso di queste voci di impatto a seconda del contesto aziendale considerato. In particolare, una minor autosufficienza alimentare aziendale e l’acquisto di sostanze proteiche dal mercato, come ad esempio la farina di estrazione di soia, hanno comportato un aumento importante delle emissioni di gas climalteranti, in quanto tale materia prima proviene in gran parte dal Brasile, dove la soia viene coltivata su terreni dove prima era presente foresta. Occorre tuttavia considerare che vi sono processi nei confronti dei quali è più facile intervenire rispetto ad altri: tra questi troviamo le emissioni dagli stoccaggi, così come le emissioni di campo e gli alimenti acquistati. La riduzione dell’emissione di metano enterico è sicuramente più difficile da contenere, essendovi una quota fissa di metano imputabile al metabolismo delle capre. L’incremento della produzione di latte individuale e l’adozione di corrette strategie alimentari e gestionali possono però consentire di ridurre anche il peso ambientale di questo processo fisiologico, con un conseguente miglioramento dell’impatto dell’intero processo produttivo. La ripartizione dell’impatto ambientale in funzione della produzione di formaggio di capra è interessante, in quanto prende in considerazione l’impatto del prodotto finale, che potrebbe essere influenzato dal tipo di formaggio e dalle tecniche di caseificazione. Tale impatto è risultato essere superiore a quello della produzione di latte, con un andamento ad esso sovrapponibile. Ciò si spiega con il fatto che la maggior parte dei kg di CO2 equivalenti emessi in atmosfera nel processo di produzione di formaggio di capra è da imputare alla produzione del latte. L’impiego dell’unità di superficie

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come unità funzionale è considerato da diversi autori interessante quando la disponibilità di superficie aziendale è un fattore limitante. La ripartizione per ettaro ha permesso di evidenziare l’effetto “diluizione” sulla superficie aziendale, che ovviamente può risentire fortemente della tipologia di allevamento (intensivo vs estensivo).

Analogamente a quanto riscontrato da altri autori (17 e 18) infatti, le aziende caratterizzate da un sistema gestionale estensivo o semi-estensivo (come quello dell’azienda H) sono risultate essere caratterizzate da un impatto ambientale per ettaro di superficie inferiore rispetto a quello di aziende intensive o semi-intensive (sistema gestionale che caratterizza invece le altre 7 aziende analizzate). Nelle aziende con diverso grado di estensivizzazione, inoltre, la presenza di pascolo, se ben gestito, rappresenta anche un modo per ridurre l’acquisto di alimenti.

Bibliografia 1.

2.

3.

4.

5.

6.

7.

CONCLUSIONI Sebbene nei confronti di processi come l’emissione di metano enterico l’allevatore abbia un margine di intervento limitato, alcune scelte aziendali possono sicuramente contribuire a ridurre il carico emissivo della produzione del latte di capra.

L’incremento della produzione di latte dev’essere infatti accompagnato da scelte alimentari corrette, che devono puntare a ridurre l’acquisto di concentrati in un’ottica di aumento dell’efficienza alimentare. Particolare attenzione dev’essere rivolta all’autoproduzione della quota proteica della razione. Parimenti la gestione delle emissioni dagli stoccaggi e la distribuzione delle deiezioni in campo necessitano di particolare cura.

L’utilizzo dell’ettaro di superficie come unità funzionale consente di valorizzare le aziende caratterizzate da un sistema estensivo che, producendo meno latte, risultano essere penalizzate quando ad essere considerato è il chilogrammo di latte prodotto.

RINGRAZIAMENTI Si ringraziano gli allevatori che sono stati coinvolti nel progetto PSR DEMOCAPRA; un grazie particolare è rivolto alla società agricola Cascina Bagaggera, che il 13 novembre 2019 ha ospitato la giornata dimostrativa sulla sostenibilità ambientale dell’allevamento caprino. Il presente studio è stato realizzato nell’ambito del progetto DEMOCAPRA - Divulgazione partecipativa di modelli gestionali sostenibili per l’allevamento della capra da latte in Lombardia mediante strumenti innovativi, finanziato dalla Regione Lombardia (FEASR - Programma di Sviluppo Rurale 2014 - 2020 Misura 1 Operazione 1.2.01).

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ISPRA, Annuario dei dati ambientali 2013. www.isprambiente.gov.it visitato novembre 2019.

IDF, 2015. A common carbon footprint approach for dairy. The IDF guide to standard lifecycle assessment methodology for the dairy sector. In the Bulletin of the IDF No 479/2010. International Dairy Federation, Brussels, Belgium.

McClelland S.C., Arndt C., Gordon D.R., Thoma G. (2018). Type and number of environmental impact categories used in livestock life cycle assessment: A systematic review. Livest. Sci. 209, 39-45.

International Reference Life Cycle Data System (ILCD), 2011. HandbookRecommendations for Life Cycle Impact Assessment in the European context, 2011. EUR 24571 EN. Luxemburg. Publications Office of the EU. INRA, 2018. INRA feeding system for ruminants. Wageningen Academic Publisher, Wageningen, the Netherland 640 pp.

IPCC, 2006. Emissions from livestock and manure management. In: IPCC, Eggleston, H., Buendia, L., Miwa, K., Ngara, T., Tanabe, K. (Eds.), IPCC Guidelines for National Greenhouse gas Inventories. Volume 4: Agricolture, Forestry and Other Land Use. Chapter 10. Institute for Global Environmental Strategies, Hayama, Japan.

IDF, 2015. A common carbon footprint approach for dairy. The IDF guide to standard lifecycle assessment methodology for the dairy sector. In the Bulletin of the IDF No 479/2010. International Dairy Federation, Brussels, Belgium.

United States Department of Agriculture (USDA), www.fas.usda.com visitato novembre 2019.

Associazione Italiana Allevatori (AIA), www.aia.it visitato novembre 2019.

DEMOCAPRA (2020) Schede tecniche DEMOCAPRA. Università degli Studi di Milano & Associazione Regionale Allevatori della Lombardia, Milano Bava L., Bacenetti J., Gislon G., Pellegrino L., D’Incecco P., Sandrucci A., Tamburini A., Fiala M., Zucali M. (2018). Impact assessment of traditional food manifacturing: The case of Grana Padano cheese. Sci. Total. Environ.

626, 1200-1209.

Beukes, P.C., Gregorini, P., Romera, A.J., Levy, G., Waghorn, G.C. (2010).

Improving production efficiency as a strategy to mitigate greenhouse gas emissions on pastoral dairy farms in New Zealand. Agric. Ecosyst. Environ. 136, 358-365.

Loszach S., Bianco E., Bovolenta S. (2008) Biodiversità e zootecnia montana in Friuli Venezia Giulia. Quaderno SOZOOALP, 5: 279-285.

Marino R., Atzori A.S., D’andrea M., Iovane G., Trabalza-Marinucci, M., Rinaldi, L., 2016. Climate change: Production performance, health issues, greenhouse gas emissions and mitigation strategies in sheep and goat farming. Small Rumin. Res. 135, 50-59 Zucali M., Lovarelli D., Celozzi S., Bacenetti J., Sandrucci A., Bava L. (2020).

Management options to reduce the environmental impact of dairy goat milk production. Livest. Sci. 231.

Robertson, K., Symes, W., Garnham, M., 2015. Carbon footprint of dairy goat milk production in New Zealand. J. Dairy Sci. 98, 4279–4293.

Gutiérrez-Peña, R., Mena, Y., Batalla, I., Mancilla-Leytón, J.M., 2019. Carbon footprint of dairy goat production systems: A comparison of three contrasting grazing levels in the Sierra de Grazalema Natural Park (Southern Spain). J. Environ. Manag. 232, 993-998.

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A.M. Sutera et al. Large Animal Review 2020; 26: 301-304

SNPs discovery in RRLs from DNA pools of Nero Siciliano pigs with extreme and divergent phenotypes for the Back Fat Thickness (BFT) tract

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ANNA MARIA SUTERA1, ALESSANDRO ZUMBO1, IRENE SAPIENZA1, GIUSEPPE TARDIOLO1, ENRICO D’ALESSANDRO1 1

Department of Veterinary Sciences, Division of Animal Production, University of Messina, Messina, Italy.

Via Palatucci, snc, 98168 - Messina (Italy)

SUMMARY Fat deposition is a key biological process that has implications in pig economic management. This process affects carcass quality and aptitude for the typical product productions such as salami, sausages and of cured ham. This study aimed to detect single nucleotide polymorphisms (SNPs) that could be associated with the backfat thickness (BFT) tract in Nero Siciliano pigs. The Food and Agriculture Organization (FAO) has expressed concern about the lack of interest in local breeds compared to highoutput animals and conservation programs have been implemented by various countries worldwide. Genomic DNA from two groups of Nero Siciliano pigs with divergent phenotypes for BFT was pooled and digested with BsuRI (HaeIII) restriction enzyme for preparation of reduced representation libraries (RRLs). The two RRLs produced 4124595 (BFT+) and 4052107 (BFT-) sequenced reads, were mapped on Sus scrofa reference genome (Sscrofa 11.1 assembly). SNP calling was performed using SNAPE, a software that implements a Bayesian approach for SNP calling in pooled samples. 47,791 putative SNPs were called by SNAPE, of these 32,235 (67.4%) were polymorphic while 15,556 (32.5%) were monomorphic. Of all SNPs detected in this study, 22 showed enriched alleles in one or the other RRLs. These SNPs, some of these localised in genes involved in fat metabolism, might be potential markers associated with BFT in Nero Siciliano pig.

In this study, we identified SNPs potentially associated with BFT that might be utilized for applications in breeding programs.

Attitude to high-fat deposition (in particular in neck, withers and back) for the Nero Siciliano pig is known and our results could contribute to explain the biology of fat metabolism in this breed.

KEY WORDS Nero Siciliano pig; Back Fat Thickness; SNPs; RRLs; DNA pooling; DNA sequencing.

INTRODUCTION In the swine production, the deposition of fat is a biological process of fundamental importance for the know implications on the efficiency of animal’s growth and on the technological and nutritional characteristics of meat products with significant implications also on business management1. Although in the last year’s admirable efforts have been made to recover the extremely threatened biodiversity of pigs, today only a few Italian local breeds are able to withstand the competition with foreign commercial breeds (i.e. the Large White, Landrace and Duroc) and/or with other commercial crossbreeds today widespread in the market which combine high reproductive efficiency, good growth performances, good quality of carcasses and excellent quality of meat, are the genetic types on which the production of heavy pigs is based in Italy2. Nero Siciliano pig, also known as ‘Nero dei Nebrodi’, is a local pig breed reared on the island of Sicily mainly under extensive management3.

Nero Siciliano dates back to ancient times. However, the genetic pool of the breed seems to have been formed mainly during the last few centuries4. The breed is known for its aptitude in

Corresponding Author: Enrico D’Alessandro (edalessandro@unime.it)

the accumulation of fat in the region of the neck, back and withers5. The remarkable adipogenic capacity of the unimproved breeds is reported in the literature6,7. The production aspects and the characteristics of the carcass are complex are partly genetically determined. Although the heritability of these traits is low, increased knowledge of the genome represents an important tool for introducing significant innovations in the management of farms, to better understand their genetic structure8.

SNPs represent the primary functional basis of genetic variability, which is reflected in the phenotypic differences between and within the breeds. In case of the identified polymorphisms represent exclusive selective imprints of the breed taken in consideration, these markers can be applied efficiently to identify the most favourable genetic variants for the improvement of quantitative traits, or in the identification and traceability of products.9 Taking advantage from the sequenced genome of the pig and its reference assembly (Sscrofa 11.1) it is now possible to use next generation sequencing (NGS) platforms to further investigate the level and extent of genetic variability in different breeds and populations10.The Ion Torrent technology is a promising NGS platform that is based on a semiconductor detection of pH variation during the sequencing process that can be applied in different experimental approaches in which a medium-high throughput is needed11.

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The aim of this study was to detect SNPs that could be associated with the BFT in the Nero Siciliano pig, considering the great importance of fat deposition for the production of hams, and in the known technological effects in the seasoning phase12.

To perform our investigation the reduced-representation libraries (RRLs) method was used. For the construction of the libraries, the DNA of various pigs was grouped into two Reduced Representation Libraries were obtained by enzymatically digest DNA pools constructed from pigs with an extreme and divergent average of the BFT.

using BWA-short13 , BWA-long14 and SSAHA15. SNP calling was obtained using SNAPE16. SNAPE input files (PILEUP format) were obtained using Samtools v.0.1.417,18. To map gene positions and to predict the effect of each substitution was used Variant effect predictor (VEP) tool (http://www.ensembl.org/Sus scrofa/Tools/VEP)19 and to evaluate if missense mutations could have deleterious effects on the translated proteins was used SIFT20. Fisher’s exact test was computed for each alternative genomic position covered by a minimum depth of 3x to evaluate differences in allele frequency derived by the number of alternative reads between the two RRLs. All the positions with PFisher < 0.05 were also visually inspected with IGV (IntegrativeGenomicsViewer) software.

MATERIAL AND METHODS All the procedures used in this research were in compliances with the European guidelines for the care and use of animals in research (Directive 2010/63/EU 2010).

The study was carried out on 192 Nero Siciliano pigs coming from different swine farms, divided into two homogeneous groups for number (96 pigs), sex (half male and half female), age (10 months) and live weight (95 kg± 5 kg). All pigs were registered in National Herd book of the National Pig Breeder Association (ANAS). The animals were slaughtered on reaching the average live weight of 92 ± 5 kg. From the measurements were extrapolated the averages of the BFT and used for the generation of the extreme and divergent groups, each of 50 pigs (50 with the most negative BFT and 50 with the most positive BFT). Average and standard deviation of BFT of the pigs in the negative and positive tails were 25,70 ± 2,97 mm and 34,40 ± 4,65 mm respectively.

Genomic DNA (gDNA) was extracted from blood sample of two groups of Nero Siciliano pigs with divergent phenotype for average of BFT using the Wizard® Genomic DNA Purification Kit (Promega Corporation, Italy) and quantified by Qubit 2.0 fluorometer with Qubit dsDNA HS Assay Kit (Thermo Fisher, Italy). Genomic DNA digested with BsuRI (HaeIII) restriction enzyme for preparation of reduced representation libraries (RRLs). For each library, 200 ng of DNA gel-purified was enzymatically fragmented by Ion Shear™ Plus 10X Reaction Buffer and Ion Shear™ Plus Enzyme Mix II (Thermo Fisher Scientific). Then, the following steps occurred: the adapters ligation, the nick-repair to make the covalent ligation on both strands (Ion Xpress™ Plus Fragment Library Kit, Thermo Fisher Scientific), and a further purification step of the ligated DNA. Obtained DNA material was size-selected using E Gel® SizeSelect™ Agarose Gel (Thermo Fisher Scientific) and bands corresponding to 200bp of inserts were collected and quantified by qPCR using a StepOnePlus Real-Time PCR System (Life Technologies). Libraries were clonally amplified in emPCR and enriched on Ion OneTouch™ 2 System (Thermo Fisher Scientific) using Ion PGM™ Hi- Q™ OT2 kit (Thermo Fisher Scientific). Each library was loaded on an Ion 318™ Chip V2 BC, previous evaluation of the templating efficiency with the Ion Sphere™ Quality Control kit and the Qubit® 2.0 Fluorometer, and then sequenced on the Ion PGM™ System with Ion PGM™ Hi‐Q™ Sequencing Kit. The reads from the two RRLs were checked with FAstaQC program, processed using the Ion Torrent Suite v3.6 software (Life Technologies) and cleaned with Trimmomatic v. 0.36, then mapped on the reference genome (Sscrofa 11.1; GCA_000003025.6) with TMAP 3.6 ALIGNER (https://github.com/iontorrent/TS/tree/master/Analysis/TMAP)

RESULTS AND DISCUSSION The two RRLs produced 4124595 (BFT+) and 4052107 (BFT-) sequenced reads, which after cleaning (filtering and trimming) were mapped on Sus scrofa reference genome (Sscrofa 11.1 assembly). Only reads with mapping quality score> 20 were retained for subsequent analysis. SNP calling was performed using SNAPE, a software that implements a Bayesian approach for SNP calling in pooled samples. 47,791 putative SNPs were called by SNAPE, of these 32,235 (67.4%) were polymorphic while 15,556 (32.5%) were monomorphic. The analysis performed with VEP showed that most of the identified variants (about 74%) were already present in dbSNPs (release 151), whereas about 26% were novels. The SNPs was identified in the transcribed regions. Among these located in the coding regions of the genome, 96 were synonymous mutations, 277 missense, 10 stop-gained (e.g. in ADH6, PGM2, TMA16, MRPL1 genes), 9 stop lost (e.g. in WDR25, HECTD1) and 1 stop retained (in ENSSSCG00000036380). Among the aforementioned missense mutations, 38 were classified as deleterious by SIFT. These mutations have been noted in genes that play a role in processes correlated to lipid metabolism (such as MIGA1 and CUBN) and in various cellular mechanisms such as in the activation of the innate immune response (TLR10), in the neuronal migration (SLIT2), and in the apoptotic signal (DTHD1) (data not showed). SNAPE was also used to estimate, from the count of alternative reads, the allelic frequency of each polymorphic genomic position, among the 452288 in common between the two RRLs. Fischer’s exact test was performed to evaluate differences in allele frequency estimates as determined by alternative read count between the two libraries. Of all SNPs detected in this study, 22 showed enriched alleles in one or the other RRLs for PFisher < 0.05 (Table. 1). The SNPs identified were located in autosomal chromosomes in which candidate genes (SSC1, SSC6, SSC12, SSC14, SSC18) and Quantitative Trait Loci (QTLs) (SCC12, SCC14) for BFT have been mapped (Table. 2).

Among the 22 identified variants about 31% were intergenic variants, about 31% were located in the intronic regions, 4% were downstream variants and 3% upstream. The downstream and upstream variants were located in annotated six genes with known functions such as Melanocortin-4 receptor (MC4R), Fatty acid synthase (FASN), Phosphodiesterase 6C (FFAR4), Stearoyl-CoA desaturase (SCD), whereas the intronic variants in genes Leptin (LEP) and Leptin Receptor (LEPR). In particular, MC4R gene is a member of the superfamily of G-protein-coupled receptors (GPCRs), which affects body weight, energy homeostasis and food intake in humans and mice and the

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Table 1 - SNPs showed PFisher < 0.05 between two RRLs (Positive Library: BFT+, Negative Library: BFT-) BFT+ Chr

Genomic postion

BFT-

Reference

Alt

N_Ref

N_Alt

N_Ref

N_Alt

PFischer

1

4,553

A

GA

0

6

8

2

2,86E-02

1

1,082

G

TG

0

7

7

1

4,82E-02

1

284,340

T

TA

29

4

17

7

4,05E-02

1

124,584,196

C

CG

17

2

6

5

1,28E-02

1

160,773,173

A

AG

8

1

0

3

3,37E-02

2

123,750,235

G

GA

13

0

1

4

1,70E-02

2

123,751,016

T

TC

1

8

13

3

4,65E-02

6

956,438

G

GA

9

0

4

6

3,35E-02

6

145,607,675

C

CA

11

1

3

7

3,55E-02

6

145,631,685

G

AG

13

1

4

9

1,27E-02

6

145,654,695

C

CT

15

2

2

4

3,63E-02

6

146,893,537

A

GA

8

0

4

7

1,87E-02

6

67,619,980

G

TG

27

13

17

32

1,03E-02

7

75,888,372

T

TA

11

0

3

7

1,25E-02

12

993,860

T

TG

32

3

8

14

2,63E-02

13

31,684,656

G

GA

29

0

11

2

3,69E-02

14

105,011,727

C

CT

8

13

10

1

4,37E-02

14

111,463,097

C

TC

1

15

7

2

1,29E-02

15

155,198,719

C

CG

0

6

7

1

4,96E-02

15

155,253,410

C

CA

0

7

8

0

2,95E-02

16

36,782,695

T

GT

8

43

12

21

3,27E-02

18

20,123,273

C

CT

5

49

57

13

1,29E-02

Chr = Chromosome number; Genomic position on the Scrofa 11.1; Reference = Reference nucleotide on Sccrofa 11.1 reference genome; N_Ref = number of reads supported reference nucleotide; Alt = alternative variant; N_Alt = Number of alternative reads of reference nucleotide

Asp298Asn polymorphism of the MC4R gene is associated with backfat thickness in different commercial cross-bred pigs21.

FASN gene codes for a protein that has an essential role in the synthesis of long-chain fatty acids, starting from acetyl CoA and using malonyl-CoA as a carbon 2 donor22. This gene has been mapped to chromosome 1223, in a region that includes numerous QTLs for traits related to the adiposity of the carcass (http://aaa.animalgenome.org/cgi- bin/gbrowse/pig/?name = Chr.12). LEP (Leptin) gene codes for a 146 amino acid protein, mainly expressed in adipose tissue24. This hormone acts on the hypothalamic receptor by modulating the sense of satiety and therefore regulating body weight and energy balance. Several mutations at this locus have been associated with traits related to average daily weight gain, intramuscular fat and back fat thickness25,26 in the Casertana, Duroc, Large White and Meishan breeds, SCD gene is a candidate gene for fatty acid com-

position. It is located on pig SSC14 in a region where quantitative trait loci (QTL) for fatty acid composition were previously detected in a Duroc purebred population27. LEPR gene plays an important role in the regulation of fat deposition and other commercially important traits in pigs and this regulation is known to be breed-specific28. Balatsky et al., (2018) suggest that LEPR SNP c.2856C > T can be considered as a genetic marker for subcutaneous fat deposition and average daily weight gain in Ukrainian Large White pigs.

CONCLUSIONS Fatness-related traits, in particular the backfat thickness, are very important in pig production since they influence meat quality and technological processes adopted for long matured prod-

Table 2 - Significant SNP for back fat thickness allocated in candidate gene.

CHR

PosSNP

GENE

Position Gene

Acc. Num.

1

160,773,173

MC4R

160,772,013-160,774,124

397359

6

146,893,537

LEPR

146,802,297-146,896,152

396836

12

993,860

FASN

992,405-997,560

397561

14

105,011,727

FFAR4

105,010,974-105,122,628

100135678

14

111,463,097

SCD

111,461,570-111,478,033

396670

18

20,123,273

LEP

20,106,867-20,124,071

396832

Chr = chromosome number; PosSNP = nucleotide position on the Sscrofa 11.1 reference genome of the SNP having PFisher < 0.05 ; *Pos SNP located within QTL regions

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ucts such as cured ham. For valorisation of Nero Siciliano’s productions, a request to label the fresh meat with the Protected Denomination of Origin (PDO) was issued in 2005 while for Nero Siciliano’s cured ham the request for the PDO has been started in 2011. In this study, we identified SNPs potentially associated with BFT that might be utilized for applications in breeding programs. Attitude to high-fat deposition (in particular in neck, withers and back) for the Nero Siciliano pig is known and our results could contribute to explain the biology of fat metabolism in this breed.

References 1. D’Alessandro E., Sapienza I., Giosa D., Giuffrè L., Zumbo A. (2019). In silico analysis of meat quality candidate genes among Nero Siciliano, and Italian heavy pigs genomes. Large Animal Review, 25(4):137-140.

2. (ANAS) Associazione Nazionale Allevatori Suini (2016.) Notizie: La selezione per il prosciutto DOP. Una sfida tecnica per un orizzonte dilungo periodo. Retrieved on 15 January 2018 from http://www.anas.it/circolari/20170001A.PDF Google Scholar.

3. Guastella A.M., Criscione A., Marletta D., Zuccaro A., Chies L., Bordonaro S. (2010). Molecular characterization and genetic structure of the Nero Siciliano pig breed. Genet Mol Biol, 33:650-656 4. D’Alessandro E., Sottile G., Sardina M.T., Criscione A., Bordonaro S., Sutera A.M., Zumbo A., Portolano B., Mastrangelo S. (2020). Genome wide analyses reveal the regions involved in the phenotypic diversity in Sicilian pigs. Anim Genet, 51(1):101-105. doi: 10.1111/age.12887 5. Chiofalo L. (2007). Nero Siciliano pig. Proceedings of 6th International Symposium on the Mediterranean Pig. Capo d’Orlando (ME), Italy.

6. Maiorano G. (2009). Swine production in Italy and research perspectives for the local breeds. Slovak J Anim Sci, 42 (4): 159-166.

7. Chiofalo B., Lo Presti V., Savoini G., D’Alessandro E., Chiofalo V., Liotta L. (2011). Nucleotides in Brolier chicken diet: Effect on breast muscles quality. Czech J. Food Sci, 29(4):308-317.

8. D’Alessandro E., Giosa D., Sapienza I., Giuffrè L., Aiese C. R., Romeo O., Zumbo A. (2019). Whole genome SNPs discovery in Nero Siciliano pig. Genet Mol Biol, 42(3):594-602. doi.org/10.1590/1678-4685-GMB2018-0169 9. Fontanesi L., D’Alessandro E., Scotti E., Liotta L., Crovetti A., Chiofalo V., Russo V. (2010). Genetic heterogeneity and selection signature at the KIT gene in pigs showing different coat colours and patterns. Anim Genet, 41:478-492.

10. Bosse M., Megens H.J., Frantz L.A., Madsen O., Larson G., Paudel Y., Duijvesteijn N., Harlizius B., Hagemeijer Y., Crooijmans R.P., Groenen M.A. (2014). Genomic analysis reveals selection for Asian genes in European pigs following human-mediated introgression. Nature Commun, 5:43922014.

11. Rothberg J.M., Hinz W., Rearick T.M., et al. (2011). An integrated semiconductor device enabling non-optical genome sequencing. Nature, 475:348-352.

12. Bosi P., Russo V. (2004). The production of the heavy pig for high quality processed products Ital J Anim Sci, 3:309-321.

13. Li H and Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754-1760.

14. Li H., Durbin R. (2010) Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics, 26: 589-595.

15. Ning Z., Cox A.J., Mullikin J.C. (2001). SSAHA: a fast search method for large DNA databases. Genome Research 11, 1725-9.

16. Raineri E., Ferretti L., Esteve-Codina A., Nevado B., Heath S., Pérez-Enciso M. (2012). SNP calling by sequencing pooled samples. BMC Bioinformatics, 13:239 17. Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G.,Durbin.(2009).The sequence alignment/Map format and SAM tools. Bioinformatics, 27:863-864.

18. Li H. (2011). A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics, 27(21):2987-93.

19. McLaren W., Pritchard B., Rios D., Chen Y., Flicek P., Cunningham F. (2010). Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor. Bioinformatics, 26:2069-2070.

20. Ng P.C., Henikoff. S. (2011). Predicting deleterious amino acid substitutions. Genome Res, 1:863-874.

21. Lyadskiy, I. K., Getya, A. A., Pochernyaev, K. F. (2011). Association of the Asp298Asn polymorphism in the mc4r gene with back fat thickness in pigs of the large white breed. Cytology and Genetics, 45(2):106-109.

22. Menendez J.A., Vazquez-Martin A., Ortega F.J., Fernandez-Real J.M. (2009).

Fatty acid synthase: Association with insulin resistance, type 2 diabetes, and cancer. Clin Chem, 55:425-438.

23. Muñoz G., Alcazar E., Fernandez A., Barragan C., Carrasco A., de Pedro E., Silio L., Sanchez J.L., Rodriguez M.C. (2011). Effects of porcine MC4R and LEPR polymorphisms, gender and Duroc sire line on economic traits in Duroc X Iberian cross are pigs, Meat Science, 88:169-173.

24. D’Andrea M., Pilla F., Giuffra E., Waddington D., & Archibald A. L. (2008).

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25. Chao Z., Wang F., Deng C.Y., Wei L.M., Sun R.P., Liu H.L., Liu Q.W., Zheng X.L. (2012). Distribution and linkage disequilibrium analysis of polymorphisms of MC4R, LEP, H-FABP genes in the different populations of pigs, associated with economic traits in DIV2 line, Mol Biol Rep, 19:111120.

26. Hirose K., Ito T., Fukawa K., Arakawa A., Mikawa S., Hayashi Y., Tanaka K., (2014): Evaluation of effects of multiple candidate genes (LEP, LEPR, MC4R, PIK3C3, and VRTN) on production traits in Duroc pigs. Anim Sci J, 85:198-206.

27. Uemoto, Y., Nakano, H., Kikuchi, T., Sato, S., Ishida, M., Shibata, T., Suzuki, K. (2012). Fine mapping of porcine SSC14 QTL and SCD gene effects on fatty acid composition and melting point of fat in a Duroc purebred population. Anim Genet, 43(2):225-228.

28. Balatsky, V., Oliinychenko, Y., Sarantseva, N., Getya, A., Saienko, A., Vovk, V., & Doran, O. (2018). Association of single nucleotide polymorphisms in leptin (LEP) and leptin receptor (LEPR) genes with backfat thickness and daily weight gain in Ukrainian Large White pigs. Livestock science, 217:157-161.

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Malattie congenite del sistema nervoso del bovino

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ARCANGELO GENTILE, JOANA GONÇALVES PONTES JACINTO*, CINZIA BENAZZI, MARILENA BOLCATO Dipartimento di Scienze Mediche Veterinarie, Alma Mater Studiorum - Università di Bologna

RIASSUNTO Le malattie congenite del sistema nervoso centrale dei vitelli rappresentano un’evenienza solo apparentemente rara. Fra le altre cose, l’assenza di strutturati piani di sorveglianza ed il basso valore che spesso caratterizza i vitelli maschi impediscono una puntuale segnalazione ed un tempestivo invio di soggetti ammalati a centri diagnostici o di ricerca. Se si aggiunge che per molte di queste patologie la conferma diagnostica è possibile solo con una attenta necroscopia o addirittura con un esame istologico, ecco che si giustifica la sottostima che accompagna questo capitolo nosologico. Quanto detto sopra vale, in effetti, in generale per tutte le malattie congenite; in un lavoro datato 1973, le malattie congenite del sistema nervoso vennero comunque stimate nell’ordine del 20% di tutte le malattie presenti alla nascita.

I disturbi neurologici congeniti possono essere dovuti ad agenti teratogeni esterni o a difetti genetici ereditari. I primi - fra i quali si annoverano agenti biologici, chimici, fisici, metabolico-nutrizionali - possono agire direttamente sui processi di sviluppo embrionale oppure provocare alterazioni nell’espressione di un gene.

Da parte loro, i difetti genetici ereditari - prevalentemente rappresentati da mutazioni autosomiche recessive - si basano su errori innati del metabolismo per mancanza e/o disfunzioni di ormoni, recettori, enzimi, proteine strutturali, neurotrasmettitori, canali ionici. L’aumentato grado di consanguineità della popolazione bovina, dovuto all’uso intensivo di alcune linee genetiche, ha aumentato la frequenza di alcune varianti alleliche portatrici di tare genetiche, fra le quali alcune riguardano proprio malattie del sistema nervoso centrale.

Le cause genetiche comprendono anche le c.d. mutazioni “de novo”, ovvero quelle varianti patogene non “ereditarie”, che si verificano o a livello delle cellule germinali di uno dei due genitori, o compaiono nelle primissime fasi dell’embriogenesi.

I difetti caratterizzati da deformità delle strutture nervose sono definiti “malformazioni congenite”, e sono il risultato di disturbi dello sviluppo tissutale quali: agenesia (completa assenza di un organo), aplasia (mancato sviluppo di un organo), ipoplasia (ridotto sviluppo di un organo), disrafismo (fallimento nella fusione sagittale), mancata involuzione con persistenza di strutture embrionali, mancata divisione di tessuti embrionali, atresia (mancata formazione di una cavitazione/lume), displasia (anormale organizzazione strutturale cellulare di un tessuto o organo), ectopia (errore morfogenetico di posizionamento di un tessuto o organo), distopia (errore morfogenetico in cui un tessuto o organo rimangono in una posizione avuta durante una delle fasi dello sviluppo). Per quanto riguarda i difetti caratterizzati da alterazioni innate delle funzioni fisiologiche o dei processi metabolici, essi, a seconda delle disfunzioni che provocano, possono anche non essere evidenti alla nascita, ma lasciarsi riconoscere solo in fasi più tardive della vita. Ciò nonostante devono essere considerati come problemi congeniti e non interpretati come patologie acquisite.

Obiettivo dell’articolo è non solo illustrare i più comuni difetti congeniti del sistema nervoso centrale del vitello, ma anche stimolare i veterinari operanti sul campo (e di riflesso gli allevatori) ad una maggiore attenzione nei confronti di questo capitolo nosologico, eventualmente considerando l’invio degli animali a centri diagnostici e/o di ricerca al fine di favorire lo studio e l’approfondimento delle cause e delle eventuali possibilità di trattamento e/o prevenzione.

PAROLE CHIAVE Malattie congenite, sistema nervoso, vitello.

INTRODUZIONE L’aumentato conferimento agli scriventi - associato a ripetute segnalazioni - di vitelli affetti da patologie nervose a carattere congenito ha spinto ad innalzare il livello di attenzione e di studio nei riguardi di tale capitolo della nosologia veterinaria.

Se vale la difficoltà di avvalersi di affidabili valutazioni epidemiologiche di frequenza e prevalenza, rimane il fatto che non

Corresponding Author: Joana Gonçalves Pontes Jacinto (joana.goncalves2@studio.unibo.it).

può sfuggire la sensazione che le patologie nervose del giovane bovino - vuoi su base genetica, infettiva o metabolica - rappresentino comunque un motivo di preoccupazione per il mondo allevatoriale. Vari fattori penalizzano in negativo la stima della loro presenza: citiamo, fra gli altri, l’eliminazione “a priori” di animali nati morti o deceduti nelle primissime fasi di vita, la “pigrizia” a segnalare soggetti già evidentemente infausti nella evoluzione, la “percezione” che l’investimento temporale ed economico della segnalazione ed invio di animali così affetti a centri di ricerca possa non avere un ritorno per l’economia aziendale, la non sempre facile disponibilità (e capillarità sul territorio) di strutture di ricerca disponibili per indagini così

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specifiche come quelle relative al sistema nervoso, la lunghezza dei necessari tempi di risposta. Si aggiunga a ciò che molti difetti congeniti sono rilevabili solo con un accurato esame necroscopico o con specifiche indagini istopatologiche.

Per malattia congenita si intende un processo patologico presente dalla nascita, dal latino congenĭtus, composto di con- e genĭtus, participio passato di gignĕre «generare»: seppure datato nel tempo, si ritiene meritevole la citazione del contributo di Greene e coll. (1973), in cui si stimò che i difetti congeniti del sistema nervoso rappresentassero il 20% di tutti i difetti congeniti del bovino.

Per quanto riguarda la natura, le patologie congenite comprendono processi malformativi, difetti di neurotrasmissione (canalopatie), disfunzioni metaboliche, malattie da accumulo, forme tumorali e patologie post-traumatiche. Per alcune di loro i sintomi possono non essere presenti esattamente alla nascita, ma comparire in un periodo successivo (normalmente comunque nelle primissime settimane di vita): trattandosi in ogni caso di difetti innati, non crediamo peccato grave considerarle congenite.

Le malformazioni possono avvenire in qualunque fase dello sviluppo del sistema nervoso, delle quali ricordiamo: a) la formazione del tubo neurale e della cresta neurale a partire dal neuroectoderma; b) la differenziazione del tubo neurale nelle tre componenti primarie del sistema nervoso intracranico (il prosencefalo, da cui si svilupperanno la corteccia cerebrale, i nuclei della base, il sistema limbico ed il talamo; il mesencefalo, da cui si svilupperà la parte craniale del tronco encefalico; il rombencefalo, da cui si svilupperanno la parte caudale del tronco encefalico con ponte e midollo allungato, ed il cervelletto) e nelle diverse parti del midollo spinale (neuroni sensitivi, interneuroni, cellule gliali, e motoneuroni); c) la proliferazione e migrazione delle cellule della cresta neurale per la formazione del sistema nervoso periferico; d) la proliferazione, migrazione e definitiva differenziazione delle cellule all’interno del tubo neurale verso la loro definitiva localizzazione specifica finale; e) la crescita assonale con l’organizzazione delle connessioni e la formazione delle sinapsi; f) la selezione e la programmazione delle cellule da eliminare (apoptosi).

I processi sopra descritti, e quindi gli sviluppi anatomo-funzionali ad essi conseguenti, possono essere turbati sia da agenti teratogeni esterni (di tipo biologico, tossico, chimico, fisico, metabolico) sia da errori nella decodifica dei tratti genomici (malattie genetiche, eventualmente ereditarie).

I virus ricoprono un ruolo importante come agenti teratogeni. Fra questi primo richiamo va fatto per il virus della diarrea virale bovina (BVDV), con azione patogena sul feto sia immunocompetente che non immunocompetente. Seppure non tutti presenti in Europa, il gruppo degli Orthobunyavirus sta acquisendo sempre maggiore importanza: ecco allora l’Akabane virus (AKAV), il virus Aino (AV), il virus Shamonda (SHAV), il virus Peaton (PRAV) ed il virus Schmallenberg (BSV), quest’ultimo ben circolante anche sul territorio italiano. Fortemente patogeni per il feto sono anche il virus Blue Tongue (BTV) ed i virus erpetici (BHV); per questi ultimi le lesioni encefaliche rimangono spesso trascurate trattandosi di animali abortiti o nati morti per la gravità delle lesioni in altri organi.

Neospora caninum può causare difetti congeniti del SN; tuttavia il suo ruolo come agente causale di sindromi nervose non

è ancora ben documentato.

Fra le piante tossiche sicura azione teratogena hanno Conium maculatum, Nicotina glauca, lupino e sorgo, soprattutto durante il primo trimestre di gestazione.

Per quanto riguarda i difetti di neurotrasmissione, le disfunzioni metaboliche e le malattie da accumulo, si tratta per lo più di alterazioni su base genetica: esse possono essere dovute sia a mutazioni insorte “de novo” nella spermiogenesi/oogenesi dei genitori (mosaicismo) o nell’embrione stesso (eventualmente per effetto di un agente teratogeno), sia a varianti genetiche ereditate da uno (malattia genetica a trasmissione autosomica dominante) o da entrambi i genitori (malattia genetica a trasmissione autosomica recessiva). A tale proposito va ricordato come l’intensa selezione genetica che hanno subito molte razze abbia fortemente ridotto la c.d. “grandezza effettiva della popolazione”, ovvero il numero di individui in grado di riprodursi (nello specifico zootecnico, i tori utilizzati per la riproduzione) provocando l’incremento del livello di consanguineità ed incrementando il rischio dell’affermazione di varianti deleterie nella popolazione, dapprima nascoste in individui eterozigoti e poi manifestatesi fenotipicamente in quelli omozigoti.

Nel novero nosologico delle malattie congenite sono infine da citare i tumori che, seppure rari, possono potenzialmente svilupparsi da qualunque elemento neurale e le conseguenze di parti distocici.

Di seguito verranno presentate le principali malattie congenite del sistema nervoso del bovino, portando, laddove possibile, supporto iconografico derivante dall’esperienza diretta.

Malformazioni e malattie delle strutture intracraniche La mancata chiusura della porzione craniale del tubo neurale (disrafia)2 è alla base delle diverse manifestazioni di anencefalia (assenza di encefalo), una alterazione incompatibile con la vita con vitelli nati morti o che lo divengono subito dopo3.

Poiché nell’esperienza personale il tronco encefalico è sempre presente e strutture prosencefaliche e cerebellari sono comunque in qualche modo abbozzate, per questi casi la definizione più appropriata sarebbe di ipoplasia prosencefalica. Il cranio può essere appiattito o normalmente sviluppato. In quest’ultimo caso lo spazio lasciato libero dagli emisferi a/ipoplasici viene riempito da liquido cefalorachidiano. Si può parlare, così, di idranencefalia, ma anche di idrocefalo esterno, idrocefalo compensatorio, idrocefalo ex vacuo, idrocefalo normotensivo. Poiché il neurocranio deriva embriologicamente dal tubo neurale, l’anencefalia può essere associata a cranioschisi (cranium bifidum) e protrusione di meningi a livello di linea mediana. Se il difetto di formazione coinvolge anche la ghiandola pituitaria, ci si può trovare di fronte a gestazione prolungata (Figura 1), anomalo sviluppo della corteccia surrenalica ed ipotricosi.

L’associazione idranencefalia-artrogriposi è la classica manifestazione dell’infezione da AKAV, uno dei più potenti agenti teratogeni dei ruminanti domestici, largamente distribuito in alcune regioni dell’Australia4. L’idranencefalia è stata associata anche ad infezione intrauterina da BVDV e BSV5,6.

Il difetto di chiusura mediana della scatola cranica (cranioschisi, cranium bifidum) è responsabile di situazioni di meningocele, erniazione di un “sacco” meningeo contenente liquido cefalorachidiano, coperto o meno da cute (Figura 2). Nel caso il sac-

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Figura 1 - Ipoplasia prosencefalica in vitello partorito morto all’undicesimo mese di gravidanza. Sezione sagittale di preparato congelato. Si noti la praticamente completa assenza del prosencefalo mentre ben riconoscibili sono il tronco encefalico ed il cervelletto. Si noti anche l’appiattimento della regione frontale del cranio.

co meningeo erniato dovesse contenere anche tessuto cerebrale, si parla, allora, di meningoencefalocele2.

Porencefalia è il termine utilizzato per indicare la presenza di una cisti, a contenuto liquido, nel tessuto cerebrale, correlata soprattutto alla azione diretta di virus sul tessuto neuronale immaturo e sulle cellule neurogliali, che determina la distruzione del parenchima e quindi la formazione di una cavitazione intraparenchimatosa.

L’idrocefalo interno è l’accumulo del liquido cefalorachidiano a livello dei ventricoli cerebrali, dovuto normalmente ad un’alterata circolazione con ostacolo al deflusso ed aumento della pressione intraventricolare (idrocefalo ostruttivo o idrocefalo congenito iperteso). La dilatazione del terzo ventricolo e dei ventricoli laterali (Figura 3) determinano l’atrofia delle strutture prosencefaliche adiacenti. La stenosi del dotto mesencefalico o acquedotto di Silvio (via di comunicazione fra terzo e quarto ventricolo) è la causa più frequente di questo difetto (idrocefalo iperteso non comunicante). L’aumento della pressione intracranica nel cranio fetale prima della saldatura delle ossa è responsabile dell’aumento della circonferenza del cranio, con il caratteristico aspetto a cupola (Figura 4). L’idrocefalo interno è stato associato ad infezioni da BVDV, SBV, BTV e Neospora caninum6 così come a tare genetiche autosomiche recessive (razze Hereford, White Shorthorn, Jersey, Dexter, Holstein-Fresian)5. Un leggero idrocefalo è presente anche nella c.d. “artrogriposi multipla”, un difetto scheletrico ereditario della razza Angus, causato da una mutazione recessiva7. Il termine idrocefalo neuropatico indica, invece, un difetto ereditario recessivo dei vitelli Angus, già conosciuto come “water-head”, caratterizzato da un enorme allargamento della scatola cranica, fino alle dimensioni di un pallone da pallavolo o da basket. Le ossa della testa sono deformate, con scarsa organizzazione delle placche ossee che tendono a disgregarsi quando la cavità cranica viene aperta. Questa risulta repleta di fluido e senza tessuto cerebrale riconoscibile.

Anche il midollo spinale è dilatato e parimenti senza tessuto nervoso identificabile. Al contrario della testa, il resto del corpo è notevolmente ridotto7.

Dal punto di vista clinico i difetti prosencefalici sono caratterizzati da modificazioni comportamentali, quali riduzione dello stato cosciente, tendenza a procedere in circolo, pressioni con la testa ed eventualmente crisi epilettiformi. Anche la postu-

Figura 2 - Meningocele in vitello di tre giorni di età. a) si noti la protrusione fluttuante sulla regione frontale; b) si noti il sacco meningeo erniato scoperto dalla cute dal quale, con una siringa, si sta allontanando liquido cefalorachidiano completamente limpido: c) si noti l’encefalo visibile in profondità dopo l’apertura delle meningi. Da tutte le tre immagini si comprende la mancata chiusura sagittale delle ossa frontali.

ra, i riflessi posturali, il movimento e la propriocezione conscia possono essere compromessi. Il coinvolgimento dei motoneuroni superiori e delle vie generali della propriocezione sono da considerarsi come conseguenza della malformazione della porzione caudale del diencefalo. Da parte loro, i danni del tronco encefalico sono caratterizzati da depressione grave fino al coma (per interessamento della formazione reticolare, mesencefalo o ponte), alterazioni posturali e deficit di movimento; particolare significato diagnostico è, poi, da attribuire ai deficit dei nervi cranici.

L’ipoplasia cerebellare è un difetto del SNC piuttosto comune negli allevamenti con circolazione di BVDV. L’insulto patogeno è da ricondurre all’esposizione virale del feto nel pe-

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Figura 3 - Idrocefalo interno in soggetto vissuto fino a due mesi e mezzo di età. Il vitello non è mai stato in grado di assumere o mantenere la stazione quadrupedale. Si noti la dilatazione del ventricolo laterale dell’emisfero cerebrale di sinistra.

riodo tra i 90 e i 170 giorni di gestazione5. La malformazione è il risultato sia di una ipoplasia che di una atrofia. Infatti, la distruzione dello strato germinale esterno determina l’assenza dello strato granulare neuronale mentre l’azione necrotizzante e la risultante infiammazione provocano degenerazione ed infine atrofia delle altre strutture del parenchima cerebellare8. Il grado di ipoplasia è molto variabile e dipende dall’età gestazionale e dalla virulenza (Figura 5). Anche altri virus6 possono provocare ipoplasia cerebellare (SBV, BTV, AKAV, AV); nella razza Shorton è descritta anche una responsabilità genetica9. Considerata l’importanza del cervelletto nel coordinamento del movimento, le malformazioni cerebellari sono caratterizzate da decubito, opistotono, oscillazioni e movimenti incontrollabili della testa, tremori intenzionali, iperriflessia. Se in grado di mantenere la stazione, il vitello aumenta la base d’appoggio, trema e durante la locomozione presenta atassia ed ipermetria. Lo stato del sensorio è normalmente conservato. I vitelli possono inoltre essere ciechi (sindrome oculocerebellare) per difetti dell’occhio (microftalmia e/o cataratta), della retina (displasia retinica), del nervo ottico (neurite o atrofia) o per lesioni intrinseche del cervelletto stesso (cecità apparente). La diagnosi definitiva post mortem si basa sulla osservazione del volume cerebellare, sia in termini assoluti che relativi (esprimendo il peso del cervelletto come percentuale sul peso totale dell’encefalo, fisiologicamente tra 9,7% e 11,8%)5.

Un’altra sindrome cerebellare, probabilmente ereditaria e dovuta ad un gene dominante a penetranza incompleta, è stata descritta nella razza Angus, e nei suoi incroci, con il nome di atassia e convulsione familiare. I sintomi possono variare da semplice rigidità fino a crisi convulsive con collasso, opistotono ed eventualmente morte. Nel giro di due o tre mesi la malattia assume la forma di una sindrome atassica con spasticità ed ipermetria10. Le lesioni caratteristiche includono vacuolizzazione, necrosi e perdita delle cellule di Purkinje con formazione di rigonfiamenti assonali nello strato granulare cerebellare11.

Ipermetria ereditaria è il termine utilizzato per indicare una sindrome neurologica congenita caratterizzata da ipermetria bilaterale simmetrica. Nei bovini Shorton è stata descritta come difetto ereditario autosomico recessivo12. I soggetti più gravemente affetti cadono e mostrano tremori di testa e collo. Sebbene i segni clinici richiamino una sindrome cerebellare, l’assenza di evidenze anatomo-istopatologiche, così come ultra-

Figura 4 - Idrocefalo interno in vitello di tre giorni di età. a) si noti il profilo del cranio; b) si noti l’atrofia delle strutture prosencefaliche dovute all’accumulo del liquido nel terzo ventricolo e nei ventricoli laterali (sezione sagittale di preparato congelato). Clinicamente il vitello presentava forte depressione sensoriale e cecità dovuta a concomitante microftalmia.

Figura 5 - Ipoplasia cerebellare in vitello di tre giorni di età forzato al decubito laterale con opistotono. Si noti la praticamente completa assenza del cervelletto. L’afflosciamento della parte caudale del telencefalo denuncia la concomitante presenza di idrocefalo interno.

strutturali, a livello di tutte le strutture nervose, non ha consentito di risalire al meccanismo patogenetico.

Fra le lesioni congenite del cervelletto è da includere anche il medulloblastoma cerebellare, tumore primitivo neuroectodermico del sistema nervoso centrale, probabilmente derivante da residui cellulari dello strato granulare esterno del cervelletto2.

I sintomi sono quelli tipici delle malformazioni cerebellari. Considerato che trattasi di tumore potenzialmente occupante spa-

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zio, altri segni clinici possono essere dovuti a fenomeni compressivi delle strutture adiacenti, per es. il tronco encefalico (Figura 6)13.

Le malformazioni di Arnold-Chiari raggruppano anomalie di sviluppo della fossa cranica caudale. Le caratteristiche principali sono date da erniazione e dislocazione del cervelletto e dell’encefalo caudale attraverso il foramen magnum, che risulta più largo del normale, fin nel canale vertebrale craniale (Figura 7). I lobi occipitali possono essere estesi caudalmente ed i relativi giri orientati parallelamente con le fessure longitudinali. Clinicamente i vitelli sono vigili ma in decubito permanente ed incapaci di coordinare il tronco con gli arti nell’assunzione della stazione. L’esame ultrasonografico attraverso la finestra atlanto-occipitale evidenzia lo slittamento del cervelletto attraverso il foramen magnum14. La causa di tale

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Figura 7 - Malformazione di Arnold-Chiari in vitello nato morto. Si noti l’erniazione del cervelletto attraverso un foramen magnum più grande del normale (# cervelletto; * midollo spinale). Il vitello mostrava altre malformazioni, tra le quali la spina bifida e l’assenza della coda.

malformazione è ancora sconosciuta: a differenza della medicina umana e di quella canina, non ci sono al momento evidenze di ereditarietà del difetto. La malformazione di ArnoldChiari può essere anche causa di idrocefalo interno e può essere associata a situazioni mielodisplasiche (soprattutto spina bifida e/o siringo-idromielia).

Malformazioni e malattie delle strutture extracraniche

Figura 6 - Medulloblastoma in vitello di 11 giorni di età. a) si noti l’opistotono nel soggetto posizionato in decubito sternale; b) si noti lo strabismo divergente dovuto al fenomeno compressivo del tumore sul tronco encefalico a livello dei nuclei del nervo trocleare; c) si noti la massa tumorale coinvolgente la parte destra del cervelletto.

Mielodisplasia15 è il termine omnicomprensivo utilizzato per indicare una malformazione del midollo spinale: nel vitello gli aspetti più frequenti sono primariamente la spina bifida ed il complesso siringo-idromielia e, secondariamente, la duplicazione del midollo spinale (diplomielia e diastematomielia) e l’ipoplasia segmentale.

La spina bifida, o disrafismo spinale, è la mancata chiusura dell’arco (o degli archi) vertebrali al di sopra del midollo spinale. È un tipico esempio di difetto multiforme integrato del tubo neurale, in cui coesistono malformazioni di differenti strutture anatomiche: midollo spinale, vertebre, muscolatura paravertebrale e la coprente cute. Sulla base delle strutture coinvolte si riconoscono due forme: la spina bifida occulta e la spina bifida aperta.

La spina bifida occulta è la mancata chiusura del solo arco vertebrale, senza alterazioni della componente nervosa. La porzione dorsale della vertebra è sostituita da tessuto fibroso ed è ricoperta dai muscoli epiassiali e dalla cute integra. In maniera caratteristica, proprio all’altezza del difetto vertebrale, la cute può presentare anomalie del tipo pelo a ciuffi o alopecia o depressione. I soggetti colpiti di solito non mostrano segni clinici e la diagnosi è accidentale (Figura 8).

Nella spina bifida aperta la fessurazione della struttura ossea è accompagnata da una anomalia dei muscoli epiassiali, delle meningi e/o dello stesso midollo spinale. Mancando una copertura rigida osseo-muscolare le meningi possono protrudere, assumendo un aspetto cistico: se la spina bifida cistica contiene solo liquido cefalorachidiano si parla di meningocele, se contiene anche il midollo spinale di meningomielocele. Un sottile strato cutaneo sarà l’unica struttura di copertura anche se, in casi estremi, si può arrivare ad una rachischisi completa, con

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Figura 9 - Malformazione del tipo perosomus elumbis/asacratus in vitello meticcio nato morto. Si noti lo scarso sviluppo del cinto pelvico (a) e l’assenza completa delle vertebre del tratto lombare, sacrale e coccigeo (b). A motivo della mancanza della impalcatura scheletrica lombo-sacrale, il cinto pelvico era connesso alla parte anteriore del corpo solo da strutture molli (muscoli del tronco, cute).

Figura 8 - Spina bifida in vitello di due giorni di età. a) si noti la sproporzione di sviluppo fra cinto toracico (normale) e cinto pelvico (anchilosato e con grave atrofia muscolare). L’animale era costretto al decubito laterale ma aveva un sensorio conservato; b) si noti la lesione in corrispondenza della regione lombo-sacrale rappresentata da una membrana meningea priva di copertura cutanea; c) si noti la apertura dorsale del canale vertebrale a livello delle ultime vertebre lombari e del sacro, e l’assenza di una struttura riconducibile al midollo spinale, sostituito solo da fasci nervosi. Il vitello presentava anche una lesione del tipo malformazione di Arnold-Chiari.

esposizione del tessuto neurale, eventualmente senza meningi (mieloschisi).

Le conseguenze cliniche della spina bifida dipendono dall’entità e dalla localizzazione del difetto, comunque di volta in volta caratterizzate da difficoltà propriocettive, atassia, spasticità da coinvolgimento dei motoneuroni superiori, paresi da lesione dei motoneuroni inferiori, disfunzioni urogenitali. Nei casi di completa rachischisi la perdita di liquido cefalorachidiano dallo spazio subaracnoideo può comportare perdita di elettroli-

ti (iponatremia e ipocloremia) così come rischi di meningomielite settica.

La spina bifida è stata descritta in associazione ad altre malformazioni quali dicefalia, idrocefalia, sindrome di ArnoldChiari, palatoschisi, fusione renale, cifo-scoliosi, lordosi, artrogriposi posteriore, neuromiodisplasia, anurìa ed il complesso dei c.d. perosomi16. Fra tutte queste merita un richiamo il c.d. perosomus elumbis/asacratus. Si tratta di un difetto di sviluppo caratterizzato da parziale o completa agenesia delle vertebre della regione lombare, sacrale e coccigea associata a pari agenesia del corrispettivo tratto di midollo spinale. Questa malformazione causa un accorciamento del tronco dell’animale ed una atrofia delle strutture muscolo-scheletriche caudali, specialmente degli arti posteriori. Tipicamente la metà craniale dell’animale, dalla testa al torace, ha dimensioni fisiologiche mentre la metà caudale, per l’assenza di tratti vertebrali, è di dimensioni ridotte.

La mancanza della struttura ossea lombare o lombosacrale fa sì che gli arti posteriori siano attaccati direttamente alle vertebre toraciche oppure mantenuti connessi alla parte anteriore del corpo solo da strutture molli (muscoli del tronco, cute) (Figura 9). Gli arti anteriori sono normali mentre quelli posteriori mostrano deformità flessorie ed atrofia muscolare neurogena17. La causa del perosomus elumbis/asacratus rimane ancora sconosciuta18.

Con i termini idromielia e siringomielia ci si riferisce ad un insieme di anomalie del midollo spinale caratterizzate dalla pre-

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Figura 10 - Siringo-idromielia in vitello di 9 giorni. a) si noti l’incapacità ad assumere la stazione quadrupedale per la paralisi (con spasticità) del treno posteriore; b) si noti la cavitazione cistica del midollo spinale localizzata a livello del tratto T13-L1.

senza di un accumulo cistico di liquido (Figura 10). L’idromielia è una dilatazione del canale centrale contenente liquido cerebro-spinale rachidiano e rivestita dall’ependima; la siringomielia è una cavitazione tubuliforme neoformata del neuroparenchima

Figura 11 - Ipoplasia segmentale nello stesso vitello della figura 10. a) si noti il restringimento a clessidra del tratto L3-L4 e la mancanza dei relativi nervi spinali di sinistra; b) si noti l’associazione ipoplasia e idro-siringomielia.

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parimenti riempita di liquido ma rivestita da cellule gliali. Poiché spesso le cavità idromieliche si aprono nel parenchima o viceversa le cavitazioni siringomieliche confluiscono nel canale centrale, è accettato il termine omnicomprensivo di idro-siringomielia, laddove le cavitazioni possono essere rivestite in parte da cellule gliali ed in parte da ependima19.

L’ipoplasia o disgenesia segmentale del midollo spinale è una rara forma di mielodisplasia in cui l’anomala morfogenesi del midollo spinale, probabilmente secondaria ad insulti focali sull’organo in via di formazione, ha carattere limitato ad uno o più segmenti ed è generalmente associata a fenomeni idro-siringomielici (Figura 11) così come a difetti delle corrispondenti vertebre.

Lo sdoppiamento del midollo spinale, evento non così raro, è indicato con il termine di diplomielia se le due unità midollari sono contenute - normalmente con comunanza di parte della sostanza bianca - in un unico avvolgimento meningeo (Figura 12), con il termine di diastematomielia se ogni midollo ha una sua propria guaina meningea ed un suo proprio canale vertebrale, separato da strutture ossee compatte20.

Per quanto riguarda l’aspetto clinico delle mielodisplasie sopracitate, esso è fortemente condizionato dalla localizzazione e dalla gravità della malformazione spinale: in linea di massima, comunque, i sintomi più comuni comprendono decubito, debolezza, tetra/paraparesi, atassia, dismetria, anomalie posturali e alterazione dei riflessi spinali. Nel caso di localizzazione lombosacrale, la diagnosi clinica può trovare perfezionamento con l’indagine ultrasonografica21,22.

Ad accompagnare gli aspetti neurologici sopradescritti, il deficit di innervazione motoria dei muscoli scheletrici può provocare la c.d. artrogriposi neuropatica7.

Il problema del difetto dell’innervazione motoria dei muscoli scheletrici è anche la causa della atrofia muscolo spinale (SMA), una patologia ereditaria, autosomica recessiva, progressiva e letale riportata nella razza Bruna23. Anche se i segni clinici compaiono dopo alcune settimane di vita, per la sua patogenesi la SMA può considerarsi una malattia congenita. Infatti, l’ipotesi più accreditata collega l’atrofia dei muscoli ad un aberrante fenomeno di apoptosi dei motoneuroni inferiori che, presente già durante la vita fetale, si estrinseca clinicamente solo dopo la nascita, quando raggiunge un grado tale da impedire dapprima il mantenimento della stazione e poi la funzionalità respiratoria. Debolezza simmetrica degli arti posteriori, difficoltà locomotoria e leggera dispnea compaiono a partire dalla 2a-4a settimana di vita per peggiorare inesorabilmente fino al decubito sternale ed alla morte per l’atrofia dei muscoli respiratori. Istologicamente si rileva degenerazione assonale del midollo spinale, neuronofagia, degenerazione e perdita dei motoneuroni delle corna ventrali del midollo spinale (soprattutto della regione brachiale e lombo-sacrale) associata a degenerazione vacuolare del mesencefalo e della corteccia motoria centrale. Forme di SMA sono state in seguito riportate anche in altre razze, fra le quali la Frisona e, recentemente, in vitelli Blonde D’Aquitaine tetraparetici sin dalla nascita24. Gli studi eseguiti sui casi di SMA nei vitelli di razza Frisona hanno evidenziato una diminuita espressione della sinaptofisina nei motoneuroni proponendo come ipotesi patogenetica alternativa del danno ai motoneuroni la perdita delle terminazioni presinaptiche piuttosto che il fenomeno apoptotico sopra citato25.

La dismielinizzazione spinale è un disturbo neurologico congenito, ereditario, autosomico recessivo26 che colpisce i vitelli di razza Bruna ed i suoi incroci con la American Brown Swiss.

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Stati di tremore congenito trovano nelle infezioni intrauterine da virus BVD una delle cause più frequenti27. Queste forme patologiche sono caratterizzate da tremori ed oscillazioni di tronco e arti, movimenti incontrollati (tremori intenzionali) della testa, incapacità ad assumere e/o mantenere la stazione quadrupedale, dovuti a fenomeni di ipomielinizzazione neurassiale. Sindromi di tremore congenito non ricondotte ad infezione da BVD sono state associate anche a mielopatia congenita spongiforme28 e ad assonopatie del midollo spinale29,30.

Un’altra assonopatia degenerativa congenita (sindrome di Demetz) è stata descritta nella razza Grigio Tirolese e riconosciuta ereditabile con meccanismo autosomico recessivo. Gli animali colpiti hanno difficoltà a mantenere la stazione quadrupedale, assumendo eventualmente una postura a cavalletto con ampia base d’appoggio, perdita dell’equilibrio, paraparesi deambulatoria con atassia soprattutto degli arti posteriori. I segni clinici compaiono entro il mese e mezzo di vita e vanno via via peggiorando, costringendo il ricorso ad una macellazione di opportunità verso gli 8-10 mesi di vita. La malattia è istologicamente caratterizzata da degenerazione walleriana assonale bilaterale simmetrica ed astrogliosi soprattutto del tratto spinocerebellare dorsale e del fascicolo gracile del midollo spinale31.

Da notare come l’assonopatia degenerativa congenita della razza Grigio Tirolese sia clinicamente molto simile alla mieloencefalopatia progressiva degenerativa della razza Bruna (sindrome weaver), un disturbo ereditario che però compare in età più avanzata. La sindrome weaver è caratterizzata da degenerazione assonale, vacuolizzazione della sostanza bianca e da degenerazione e/o riduzione numerica delle cellule del Purkinje nel cervelletto32.

A conclusione di questo capitolo, ricordiamo - quanto meno per l’importanza che possono avere nei ragionamenti diagnostico differenziali - gli esiti di parti distocici. Fra questi spiccano le lesioni della colonna vertebrale e gli stiramenti dei nervi soprattutto del cinto pelvico.

Difetti di neurotrasmissione (canalopatie) Figura 12 - Diplomielia in un vitello di 40 giorni di età. a) si noti la duplicazione della sostanza grigia, osservabile nel tratto di midollo da L1 a S1; b) si noti la duplicazione della sostanza grigia osservabile con l’esame istologico, che meglio evidenzia la presenza di due canali centrali; c) si noti la presenza di due puntini iperecogeni, corrispondenti ai due canali centrali, all’esame ecografico eseguito a livello della finestra acustica lombo-sacrale (L6-S1).

Essa è caratterizzata dall’incapacità, sin dalla nascita, ad assumere la stazione quadrupedale, a fronte di uno stato mentale e di grandi funzioni organiche pienamente conservate. Si tratta, anche se con diversi gradi di espressione, di una forma di tetraparesi spastica: la tendenza al decubito laterale con opistotono, l’iperestensione degli arti posteriori e l’esagerata risposta al riflesso flessorio validano questa affermazione diagnostica di sindrome. La malattia è dovuta ad un processo di ipomielinizzazione e dismielinizzazione simmetrica della sostanza bianca del midollo spinale, soprattutto a livello di tratto ascendente del funicolo gracile, tratto ascendente dorsale spino-cerebellare e tratto discendente solco-marginale, soprattutto a livello di intumescenze cervicale e lombare.

La mioclonia congenita ereditaria dei vitelli Polled Hereford è un disordine ereditario a carattere autosomico recessivo dovuto a deficit del recettore per la glicina (GlyR), proteina della membrana postsinaptica contenente un canale anionico selettivo per il cloro. Quando la glicina si lega ai recettori glicinergici il canale si apre e permette al cloro di passare per diffusione passiva dall’esterno all’interno della membrana. La funzione della proteina è di controllare la scarica dei motoneuroni a livello di midollo spinale e di encefalo, con funzione quindi inibitoria. Un suo difetto si manifesta clinicamente con iperestesia e scariche miocloniche della muscolatura scheletrica, che possono comparire spontaneamente o in seguito a stimolazione tattile, visiva e uditiva. Quando vengono stimolati ad assumere la stazione quadrupedale i vitelli mostrano iperestensione della muscolatura degli arti e tremori. In passato la malattia era conosciuta come edema neuroassiale7,33. Un caso di mioclonia congenita è stato successivamente riportato anche in un vitello di razza Frisona tedesca34. Una disfunzione del recettore glicinergico (GlyR) è parimenti responsabile della distonia muscolare congenita di tipo II descritta nei vitelli di razza Blu Belga, malattia ereditaria a carattere autosomico recessivo. I soggetti colpiti presentano gravi attacchi mioclonici in

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seguito a stimoli acustici o tattili e di solito giungono a morte entro poche ore dalla nascita35.

La sindrome miastenica congenita della razza Brahman, malattia ereditaria autosomica recessiva, è dovuta alla assenza della subunità epsilon del recettore colinergico nicotinico (nAChR), proteina postsinaptica della giunzione neuromuscolare contenente un canale cationico che permette il passaggio all’interno della cellula di ioni Na+ causando la depolarizzazione responsabile della eccitazione cellulare della muscolatura volontaria. I sintomi, che possono essere presenti alla nascita o comparire dopo qualche settimana di vita, sono caratterizzati da debolezza muscolare e progressiva difficoltà a mantenere o ad assumere la stazione quadrupedale36.

Malattie metaboliche La citrullinemia bovina, riportata in vitelli di razza Holstein, è un disturbo congenito del metabolismo, ereditario e letale, dovuto ad un deficit dell’enzima argininosuccinato sintetasi (ASS), l’enzima che nel ciclo dell’urea converte la citrullina in urea. Le conseguenti ipercitrullinemia ed iperammoniemia sono responsabili di sintomi neurologici quali depressione del sensorio, deambulazione afinalistica, pressioni con la testa, cecità apparente, che nel giro di pochi giorni possono associarsi a deficit propriocettivi, atassia, decubito, convulsioni fino al coma ed alla morte. Poiché durante la gravidanza l’ammonica in eccesso viene rimossa dalla madre in virtù della circolazione materno-fetale, il vitello alla nascita è completamente normale per cominciare, comunque, a manifestare i segni dell’“intossicazione” già in prima o seconda giornata di vita37,38.

Un’altra sindrome neurologica da disturbo del metabolismo, ereditata con carattere autosomico recessivo, è la malattia delle urine a sciroppo d’acero, riportata nella razza Polled Shorton e Polled Hereford. I segni neurologici comprendono depressione, postura a cavalletto con ampia base d’appoggio, atassia, tremori ripetitivi della testa, eventualmente decubito, opistotono, spasmi tetanici post stimolazione e convulsioni. La malattia è inesorabilmente letale entro pochi giorni dalla nascita. Il caratteristico odore di sciroppo d’acero delle urine è responsabile del nome assegnato alla malattia. Istologicamente è presente edema cerebrale e degenerazione spongiforme della sostanza bianca7,39. La dismetabolia è dovuta alla carenza dell’enzima mitocondriale alfa-chetoacido deidrogenasi (BCKDH) che provoca una elevata concentrazione, sia ematica che tissutale (ed urinaria), di aminoacidi a catena ramificata, ovvero leucina, isoleucina e valina, e degli alfa-chetoacidi prodotti dal loro metabolismo.

Come nota diagnostico differenziale dei disturbi metabolici, oltre a quanto sopra menzionato, sindromi neurologiche peri/neonatali possono essere dovute a situazioni di ipotermia, ipoglicemia e ipossia. In questi casi una scrupolosa raccolta anamnestica sulla genealogia e sullo svolgimento del parto può essere di forte aiuto. Nel ragionamento diagnostico, poi, non devono mai mancare gli stati setticemici.

Malattie da accumulo Fra le malattie congenite da accumulo nel vitello annoveriamo l’α-mannosidosi, la β-mannosidosi, la glicogenosi, la gangliosidosi e la lipodistrofia neuronale, tutte su base gene-

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tica ereditaria autosomica40. Per molte di queste patologie i sintomi compaiono dopo la nascita, ma, come già anticipato, trattandosi comunque di difetti innati possono essere incluse fra le malattie congenite.

L’ α-mannosidosi, descritta nelle razze Angus, Murray Grey e Galloway, è un difetto ereditario autosomico recessivo del catabolismo glicoproteico dovuto a diminuita attività dell’enzima lisosomiale α-mannosidasi, enzima deputato alla scissione del mannosio (monosaccaride) da oligosaccaridi ricchi di esso. Ne risulta un accumulo lisosomiale di oligosaccaridi indigeribili ricchi di mannosio e N-acetilglucosamina. I vitelli presentano debolezza fin dalla nascita, ma segni clinici più specifici compaiono solo dopo alcuni mesi. Si tratta di una sindrome cerebellare con tremori intenzionali, postura a cavalletto con ampia base d’appoggio, atassia, ipertermia. I sintomi tendono gradualmente a peggiorare fino a costringere il vitello ad un decubito permanente. Un altro segno caratteristico è l’aggressività che compare quando gli animali vengono contenuti. All’esame autoptico si rileva lieve idrocefalo interno e, istologicamente, vacuolizzazione dei neuroni, dei macrofagi e di altre cellule somatiche41,42.

La β-mannosidosi, descritta nella razza Salers, è un altro difetto ereditario autosomico recessivo del catabolismo glicoproteico dovuto a diminuita attività dell’enzima lisosomiale βmannosidasi, enzima coinvolto nell’ultima fase della degradazione degli oligosaccaridi, in particolare nella scissione dei disaccaridi contenenti mannosio. La conseguenza finale è un accumulo di disaccaridi e trisaccaridi indigeribili che hanno un mannosio terminale legato con un legame β-alla N-acetilglucosamina. I segni clinici, presenti sin dalla nascita, comprendono arti anteriori divaricati, andatura in circolo, testa abbassata, eventualmente decubito permanente, tremori. Nel quadro clinico sono, poi, riportati deformità del cranio, prognatismo e fimosi palpebrale. All’esame autoptico si rileva lieve idrocefalo interno e, istologicamente, vacuolizzazione dei neuroni e di varie cellule somatiche. La grave vacuolizzazione delle cellule epiteliali della tiroide differenzia questa patologia lisosomiale dalla α-mannosidosi, in cui le cellule della ghiandola non sono così gravemente colpite42,43.

Il termine glicogenosi raggruppa una serie di difetti enzimatici che determinano un accumulo di glicogeno nelle cellule.

Nelle razza Brahman e Shorton, e nella loro derivata Droughtmaster, si tratta di un difetto ereditario autosomico recessivo dell’enzima α-glicosidasi lisosomiale, l’enzima che catalizza il glicogeno in glucosio, e prende il nome di glicogenosi generalizzata o di tipo II o malattia di Pompe44,45. Sebbene il danno dell’accumulo sia maggiormente manifesto a livello di muscoli, con miopatia vacuolare della muscolatura scheletrica e cardiaca, la contemporanea presenza di lesioni del tipo di degenerazione walleriana a livello di midollo spinale e nervi periferici consente di inserire questa glicogenosi nelle malattie del sistema nervoso. Clinicamente, oltre alla debolezza muscolare sono presenti segni neurologici quali incoordinazione, incespicamento, barcollamenti, difficoltà ad assumere la stazione e decubito. Nonostante si tratti di difetto innato, solitamente la sintomatologia compare a qualche mese di età con aspettativa di vita sotto l’anno. A titolo informativo, esclusivamente muscolare, invece, è la glicogenosi di tipo V descritta nella razza Charolais, basata sul difetto dell’enzima miofosforilasi (malattia di McArdle) e caratterizzata da intolleranza all’esercizio, mialgia e mioglobinuria.

L’accumulo lisosomiale di gangliosidi nei neuroni è alla base

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della gangliosidosi GM1, descritta in vitelli di razza Frisona e dovuta al difetto dell’enzima β-galattosidasi. I GM1 gangliosidi sono componenti glicolipidici delle membrane cellulari e rivestono un ruolo fondamentale nella plasticità neuronale e nei processi riparativi, così come nel rilascio di neurotropine nell’encefalo. Il loro accumulo porta a vacuolizzazione e degenerazione neuronale40,46. I segni clinici, che compaiono dopo alcuni mesi di vita, includono scarso accrescimento, incoordinazione motoria posteriore, riluttanza al movimento, postura a cavalletto con ampia base d’appoggio, cecità. L’aspettativa di vita è di 6-9 mesi.

L’accumulo di lipopigmenti fluorescenti nei neuroni sta alla base della lipofuscinosi ceroide neuronale, descritta nella razza Devon con carattere autosomico recessivo. Nonostante il difetto sia innato, i sintomi compaiono a circa 12 mesi di età, con progressiva atassia, andatura in circolo, pressioni con la testa, riduzione e poi perdita della vista, tremori, “ritardo mentale”. Patognomonico è il diffuso accumulo intralisosomiale di pigmenti granulari di lipofuscine ceroidi sia nelle cellule gangliari retiniche che nelle cellule del sistema nervoso centrale ed ancora di altri tessuti. Si associano fenomeni degenerativi cerebrali e cerebellari del tipo di degradazione della sostanza grigia cerebro-corticale, atrofia cerebro-corticale, atrofia dell’ippocampo, atrofia del cervelletto, dilatazione dei ventricoli laterali47,48.

sis (complete absence of an organ), aplasia (absence of an organ with retention of the organ rudiment), hypoplasia (incomplete development of an organ), dysraphic anomaly (failure of opposed structures to undergo adhesion and fusion), involution failure (persistence of an embryonic structure that normally disappears during development), division failure (incomplete cleavage of embryonic tissues owing to lack of programmed cell death), atresia (failure of an organ rudiment to form a lumen), dysplasia (abnormal organization of cells in a tissue), ectopia (error of morphogenesis in which an organ is located outside its correct anatomic site), dystopia (error of morphogenesis in which an organ is retained at a site where it resided during a stage of development). The majority of these disorders involve also the nervous system.

The defect characterized by inborn alteration of physiological functions or metabolic processes may not be expressed or recognized until later in life, so being erroneously interpreted as acquired diseases.

The article presents the most common congenital defects of the nervous system in calves. Due to the fact that for the majority of the congenital diseases the exact cause remains unknown, it is intention of this article to prompt farmers and veterinarians to consider - whenever possible - the submission of such cases to diagnostic centers for further studies and investigation.

SUMMARY

KEY WORDS

Bovine congenital diseases of the nervous system

Congenital diseases, nervous system; calf.

Bibliografia In calves, congenital (condition existing at or before birth) disorders of the nervous system are not rare: in the past they were estimated to account for about 20% of all congenital defects.

Unfortunately, the scarcity of surveillance programs and the low economical value of young animals hinder submission of clinical cases to diagnostic centers; hence their real prevalence goes underestimated. Moreover, many defects can be identified only by necropsy or histopathology.

Congenital neurological conditions may be due to external teratogens or to genetic defects.

Teratogens are biologic, toxic, chemical, physical or metabolic and can act directly on the developmental processes or can alter patterns of gene expression, inhibit cell interactions, or block morphogenetic cell movements. Genetic defects are mostly represented by autosomal inherited recessives and are usually based on inborn errors of metabolism and on lack or dysfunction of hormones, receptors, enzymes, structural proteins, neurotransmitters and ion channels (metabolic defects, storage defects, disturbed neurotransmission, channelopathies). The increased use of intensive breeding programs, based on the widespread use of selected elite sires, and the consequent increase of the “inbreeding grade”, has allowed the homozygous presence of recessive variants - responsible for malformation - in the inbred progeny of many breeds.

Genetic causes includes also «de novo» pathogenic variants that may occur in one of the germ cells (sperm or egg) of one of the parents, or arise in the fertilized egg itself during early embryogenesis.

The defects characterized by structural deformities of the nervous structures are termed “congenital malformation” and are the result of disorders of tissue development such as: agene-

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Pregnancy toxemia and lipid mobilization syndrome in two alpaca (Vicugna pacos) at 6 and 10 months of gestation LAKAMY SYLLA*, MARTINA CROCIATI Department of Veterinary Medicine, University of Perugia, Via San Costanzo 4, 06126 Perugia - Italy

SUMMARY Primary ketosis, or pregnancy toxemia, is an uncommon feature of pregnancy in camelids compared to small ruminant species.

In severely debilitated females, induction of abortion or parturition should be considered to alleviate the syndrome. This case report describes the clinical findings and medical treatment approach of ketosis in two pregnant alpacas (Vicugna pacos) referred to the University Veterinary Teaching Hospital (OVUD) of the Department of Veterinary Medicine of the University of Perugia, Italy.

The first patient was a 7-years old female alpaca 6-months pregnant, 80 kg body weight and BCS 3.25, with a 5 days history of anorexia and regurgitation. At initial examination the body temperature was 38.6° C , with a glycaemia of 341 mg/dL. Ultrasound examination of the abdomen revealed no fetal heart activity. On the third day from admission the animal started to feed and the temperature decreased to 37.9° C. On the same day 100 µg of prostaglandin agonist (Estrumate®) were administered IM. On the fourth day of admission, appetite increased and abortion of a dead fetus was noticed. On fifth day of admission, temperature was 37.5° C, the alpaca showed good appetite and rumination, feces were normal and fetal membranes were expelled.

Due to the improved general condition the alpaca was dismissed.

The second patient was a 3-year old female alpaca, 10-months pregnant, 65 kg body weight and BCS 3.0, which was referred with lethargy, anorexia and signs of colic. At presentation, glycaemia was 141 mg/dL and rumen activity was absent. Ultrasound examination revealed live and vital fetal parameters (heart rate; 110 bpm). A blood sample was taken and, once centrifuged, the serum appeared milky and triglycerides reached 1208 mg/dL. The general condition of the animal improved with fluid and supportive therapy. At day 4 of admission a morphologically normal, female cria was born; however, no suction reflex was present within the first 1.5 hours. The cria was bottle-feed with bovine frozen colostrum, but the general condition constantly declined; failure of passive transfer was noticed through biochemistry profile. Since no response to supportive therapy was achieved, the cria was euthanized at 36 hours of life. Dam’s condition improved and, after expulsion of fetal membranes, the alpaca was dismissed.

Prognosis for camelids whit fat mobilization syndrome and pregnancy toxemia depends on the timing of diagnosis and intervention. In both reported cases, our approach showed good results in preserving the survival of the dams, while one fetus was diagnosed as already dead upon initial clinical exam, and the other one died within 48h from birth.

KEY WORDS Pregnancy toxemia, hyperlipemia, induction of abortion, Vicugna pacos.

INTRODUCTION Pregnancy toxemia is an uncommon disease of pregnancy in camelids, and is often secondary to other diseases. A case of primary ketosis was reported in a 3-year-old alpaca at the 11th month of pregnancy that presented a 2-week period of anorexia, lethargy, and weight loss together with ketonuria, hyperglycemia, hypokalemia, metabolic acidosis, and increased serum liver enzymes1. Conversely, in severely debilitated females, induction of abortion or parturition should be considered to alleviate the syndrome2.

Insufficient energy intake during or at the end of pregnancy is usually responsible for increased body fat mobilization, liver lipidosis and ketonemia. Indirect evidence of a lipid disor-

Corresponding Author: Sylla Lakamy (lakamy.sylla@unipg.it).

der may be obtained by measuring liver function. Camelids with any of the following abnormalities such as γ-glutamyl transferase (GGT) greater than 60 IU/L, aspartate aminotransferase (AST) greater than 500 IU/L, sorbitol dehydrogenase (SDH) greater than 50 IU/L, NEFA greater than 1 mEq/L, beta-hydroxybutyrate (BHB) greater than 5 mg/dL, bile acids greater than 30 mg/dL should be considered at high risk. Direct evidence of hepatic lipidosis may be obtained by biopsy, but severely affected camelids occasionally die during or shortly after this procedure. At necropsy, gross lipidosis may be visible as well3.

General assessment of renal function, protein and electrolyte concentrations are recommended, especially in camelids on IV fluids. Refractometers may not provide accurate estimates of blood protein in camelids with severe hyperlipemia.

This case report describes the clinical findings and medical approach to ketosis in a 6-months and a 10-months pregnant alpacas (Vicugna pacos) reared in two different farms.

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Pregnancy toxemia and lipid mobilization syndrome in two alpaca (Vicugna pacos) at 6 and 10 months of gestation

First case The first patient was a 7-years old female alpaca 80 kg body weight, BCS of 3.00, 6-months pregnant. She was referred at 9.30 PM to the University Veterinary Teaching Hospital (OVUD) of the Department of Veterinary Medicine of the University of Perugia, Italy, with a 5 days history of anorexia and regurgitation. In the days previous to the admission, the alpaca underwent medical treatment for gastric ulcer with Sucralfate®, antibiotic therapy (Baytril® 5 mg/kg) and intravenous fluid-therapy (Ringer Lactate), as established by the veterinary practitioner in charge of the farm.

On presentation at the OVUD, the patient was lethargic and in sternal recumbence; body temperature was 38.6° C, heart rate was 80 bpm and respiratory rate was 48 breaths per minute.

Mucous membranes were normal, slight de-hydration was present, together with complete C1 compartment stasis. Venous blood sample from oral mucosa revealed glucose concentration of 341 mg/dL through field glucometer. The patient received an IM injection of 40 IU of human insulin (Humulin®), but the response was unsatisfactory: the animal was still hyperglycemic at the following control, which was carried out one hour later (241 mg/dL). The animal received 3 L of Ringer Lactate through intravenous infusion overnight for stabilization.

For the following days, the treatment protocol consisted in the administration of Sucralfate® at the dose of 0.5 gr per OS four times a day and IV fluid therapy with Ringer Lactate (2 L per day).

The day after admission, body temperature was 38.9° C. An ultrasound examination of the abdomen was performed through a machine equipped with a 3-8 MHz convex transducer (MyLab 30 Gold, Esaote, Genova, Italy). Fetal annexes, amniotic and allantois fluids were normal but fetal heart activity was absent.

Thus, fetal death was confirmed. Other abdominal viscera appeared normal. Venous blood sample was obtained from the jugular vein for a complete biochemistry profile (Table 1). A diagnosis of ketosis and lipid metabolism imbalance was proposed.

Table 1 - Biochemistry profile of the first adult female pregnant alpaca referred to the OVUD. Biochemistry profile

Value

Reference limits4,5

Cholesterol (mg/dL)

63.0

15.5 - 88.9

Triglycerides (mg/dL)

126.0

10.62 - 45.14

BHB (mmol/L)

0.93

< 0.48

NEFA (mmol/L)

0.82

< 0.8

BUN (mg/dL)

86.0

21.62 - 60.66

Creatinine (mg/dL)

2.53

1.0 - 2.4

Glucose (mg/dL)

161.0

90.0 - 149.0

Total proteins (g/dL)

6.3

5.7 - 7.2

Albumin (g/dL)

3.15

2.9 - 4.3

Calcium (mg/dL)

8.4

4.2 - 9.0

Phosphorous (mg/dL)

2.3

4.5 - 7.3

AST (IU/L)

575.0

137.0 - 391.0

ALP (IU/L)

297.0

32.0 - 167.0

GGT (IU/L)

20.0

13.0 - 50.0

CPK (IU/L)

928.0

56.0 - 662.0

LDH (IU/L)

1612.0

10.0 - 695.0

Moderate response to the insulin was seen, since glycaemia was 161 mg/dL, and insulin administration was interrupted, but the animal was still anorectic. The treatment plan was refined by adding a non-steroidal antinflammatory drug (Rimadyl® 4 mg/kg SC) and 2 gr of yeasts per OS diluted in warm water.

The second day of admission the general condition slightly worsened: temperature increased to 39.1° C, hyperglycemia recurred and therapy was changed to 60 IU of insulin (Caninsulin®) IM, due to the major sensitivity of camelids to canine insulin6®. Slow IV infusion of 500 mL of glucose 5% solution was started, together with 5 mL IV of vitamin-B complex (Dobetin-B1® 10 mg/kg). Four hours later, glycaemia was within normal range.

On third day of admission the animal started to feed, temperature decreased to 37.9° C, even if no abortion occurred.

Thus, 100 µg of prostaglandin F2 (Estrumate®) was administered IM. Vital parameters were monitored at 30-min interval.

After prostaglandin injection heart rate immediately increased to 60 bpm while respiratory rate raised to 39 breaths per minute, then normalization occurred one hour later.

On fourth day of admission, appetite increased and abortion was noticed. The animal was kept in the hospital in order to verify expulsion of fetal membranes, thus two IM injections of 20 IU of oxytocin (Pitocina®) at an interval of 12 hours were given.

On fifth day of admission, temperature was 37.5° C, the alpaca showed good appetite and rumination, feces were normal and fetal membranes were expelled. Due to the improved condition of the animal and to the normalization of glycaemia, the female alpaca was dismissed.

Second case The second patient was a 3-year old female alpaca, 65 kg body weight with a BCS of 3.0, at the 10th month of pregnancy, which was referred to the OVUD at evening time, with a history of lethargy, anorexia and colic symptoms. The veterinary practitioner in charge of the farm already started a therapy based on rumen-stimulatory drug (Zoocolagogo®) and Sucralfate® three times a day. At presentation at the OVUD the animal was slightly lethargic, body temperature was 37.9° C, heart rate was 56 bpm and breath rate was 48 per minute. A peripheral venous blood drop was obtained and used for glucose stick (glycaemia: 141 mg/dL). Rumen activity was almost absent.

Ultrasound examination of the abdomen revealed a live and vital fetus, with heart rate of 110 bpm and spontaneous movements. No abnormalities were identified in the other abdominal organs. An IV fluid therapy was started with 1 L Ringer Lactate and 0.5 L NaCl 0.9% overnight.

The day following admission, fluid therapy was maintained and associated to vitamin-B complex (Dobetin-B1®, 6 mL IV and Metabolase®, 8 mL IM). Blood cell count and biochemistry profile were obtained (Tables 2 and 3). Once centrifuged, serum appeared milky, as shown in Figure 1. General condition of the animal improved and the alpaca started to feed and drink, to ruminate and to eliminate urine and feces.

At day 4 after admission a presumably premature female cria of 5.5 kg weight was born. The cria was morphologically normal, assumed standing position within 2 hours, but no suction reflex was present within the first 1.5 hours. A venous catheter was placed into the right jugular vein for hydration. When the owners provided bovine frozen colostrum, 2 hours later, the cria started spontaneously to suckle milk from the bottle. Every

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L. Sylla, M. Crociati; Large Animal Review 2020; 26: 317-320 Table 2 - Blood cell count of the second adult female pregnant alpaca referred to the OVUD. Blood cell count

Value

Reference limits4,5

WBC (103/µL)

11.59

5.7 - 32.9

RBC(103/µL)

11.83

9.1 - 13.8

Hgb (g/dL)

13.9

10.4 - 17.0

Htc (%)

23.9

24 - 36

MCV (fL)

20.2

21.8 - 28.9

MCH (pg)

11.7

10.6 - 12.7

MCHC (g/dL)

58.2

39.3 - 46.8

Plt (103/µL)

439

200 - 600

Table 3 - Biochemistry profile of the second adult female pregnant alpaca referred to the OVUD. Biochemistry profile

Value

Reference limits4,5

Cholesterol (mg/dL)

169.0

15.5 - 88.9

Triglycerides (mg/dL)

1208.0

10.62 - 45.14

BHB (mmol/L)

0.55

< 0.48

NEFA (mmol/L)

1.07

< 0.8

BUN (mg/dL)

30.0

21.62 - 60.66

Creatinine (mg/dL)

1.67

1.0 - 2.4

Glucose (mg/dL)

113.0

90.0 - 149.0

Total proteins (g/dL)

8.0

5.7 - 7.2

Calcium (mg/dL)

8.9

4.2 - 9.0

Phosphorous (mg/dL)

7.9

4.5 - 7.3

154.0

144.0 - 155.0

3.6

4.0 - 5.7

118.0

97.0 - 111.0

Sodium (mEq/L) Potassium (mEq/L) Chloride (mEq/L)

two hours, the cria was bottle-fed with 60 mL of colostrum. The day after, due to the lack of elimination of the meconium, 2.25 gr of pediatric glycerol diluted in 60 mL of warmed soap water was used as an enema, with success.

The general condition of the cria started to decline. A biochemistry, hemogasanalysis and hematocrit profile of the cria were obtained (Tables 4 and 5), which confirmed hypo-proteinemia and failure of passive transfer of immunity. A venous blood collection (100 mL) was performed from the jugular vein of the dam for haemo-transfusion. Whole blood was transferred

Table 4 - Blood cell count of the neonate alpaca cria.

Blood cell count

Value

Reference limits4,5

WBC (103/µL)

2.30

5.7 - 32.9

RBC(103/µL)

10.31

9.1 - 13.8

Hgb (g/dL)

10.9

10.4 - 17.0

Htc (%)

21.5

24 - 36

MCV (fL)

20.9

21.8 - 28.9

MCH (pg)

10.6

10.6 - 12.7

MCHC (g/dL)

50.7

39.3 - 46.8

Plt (103/µL)

430

200 - 600

Neutrophils (%)

87.9

49 - 65

Lymphocytes (%)

10.4

21 - 25

Monocytes (%)

1.7

0-5

Eosinophils (%)

0.0

6 - 22

Basophils (%)

0.0

0 - 0.5

Table 5 - Biochemistry profile and haemogas analysis of the neonate alpaca cria.

Biochemistry profile and hemogasanalysis BUN (mg/dL)

Value

Reference limits4,5

43

21.62 - 60.66

Creatinine (mg/dL)

2.26

1.0 - 2.4

Glucose (mg/dL)

121

90.0 - 149.0

Total proteins (g/dL)

4.4

5.7 - 7.2

Albumin (g/dL)

3.14

2.9 - 4.3

Calcium (mg/dL)

10.4

4.2 - 9.0

AST (IU/L)

101.0

137.0 - 391.0

ALP (IU/L)

549.0

32.0 - 167.0

GGT (IU/L)

54.0

13.0 - 50.0

CPK (IU/L)

35.0

56.0 - 662.0

LDH (IU/L)

1083.0

10.0 - 695.0

pH

7.37

pCO2 (mmHG)

42.9

pO2 (mmHG)

354

Be (mmol/L)

-1

3

HCO (mEq/L)

24.4

TCO2 (mEq/L)

26

SatO2 (%) Sodium (mEq/L) Potassium (mEq/L)

Figure 1 - Milky and dense aspect of the blood serum of the second female pregnant alpaca referred to the OVUD.

319

100 154.0

144.0 - 155.0

4.1

4.0 - 5.7

to the cria at 45 mL/kg/h speed. An ultrasound examination of the cria abdomen revealed a C1 fluid accumulation, thus a gastric tubing was performed, C1 was emptied and an IV supportive fluid therapy associated to sodium bicarbonate was carried. Glycaemia was monitored every 2 hours through stick glucometer and corrected when necessary by infusion of bolus of 33% glucose diluted in a saline solution. Due to the non-responsiveness to therapy, the cria was euthanized at 36 hours of life.

Dam’s condition improved in terms of appetite and rumination and fecal output rapidly reached normality. After assessing complete expulsion of fetal membranes, the female alpaca was dismissed, at day 7 after admission.

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Pregnancy toxemia and lipid mobilization syndrome in two alpaca (Vicugna pacos) at 6 and 10 months of gestation

DISCUSSION Pregnancy toxemia arises in ruminants from negative energy balance, thus causing lipid mobilization, liver lipidosis and ketosis7. However, camelids are slightly different by the digestive and metabolic point of view: glycaemia is higher and ketones are lower compared to true ruminants. Finally, it is not completely clear how much camelids rely on ketogenesis to support energy metabolism8. In addition to simple starvation or competition for food, various stressors may promote lipolysis. These include transport, extreme temperatures, hypoproteinemia, and systemic diseases. Concerning hypoproteinemia, a possible explanation is that gluconeogenesis also relies on amino acids as substrates, especially during periods of anorexia9.

The two patients herein reported belonged to two different farms, so it was difficult to relate the onset of pregnancy toxemia to a herd-related condition. However, it is possible that competition could lead to lesser feed intake in less dominant animals.

Some Authors report that abnormal lipid metabolism is associated to pregnancy toxemia due to fat mobilization, however other studies report that only one-third of the affected camelids show increased serum triglycerides, thus representing a low-sensitive determination8. However, in case of intensive lipid mobilization, blood serum could appear milky and dense, as reported in the second patient.

Induction of parturition or cesarean section are sometimes suggested for dams affected by pregnancy toxemia. In the first alpaca referred to our hospital, fetal death was identified. As no signs of abortion were noticed, induction of fetal expulsion was obtained in 24 hours after a single injection of prostaglandin agonist. Since camelids show intense enzymatic pathways for gluconeogenesis but relatively poor enzymatic pathways for ketogenesis, we hypothesized that pregnancy-associated negative energy balance induced lipid mobilization.

The second alpaca was referred to the OVUD close to the end of the pregnancy, with less severe clinical signs and with a live fetus, although fat mobilization was more intensive as demonstrated by dense and milky serum and blood biochemistry findings. Supportive medical therapy and spontaneous parturition contributed to a rapid recovery of the dam. Although the cria was born alive, it developed failure of passive transfer of immunity and the therapeutic approach herein presented failed to improve its clinical condition.

Prognosis for camelids with pregnancy toxemia depends on the timing of diagnosis and intervention. In both the cases reported, our approach showed good result in preserving the survival of the dams. The fetus from the first alpaca was diagnosed as already dead soon after admission, while the other one died within 48h from birth.

Improved feeding management for pregnant alpacas could be useful in preventing insufficient energy intake, fat mobilization and pregnancy toxemia. Ensuring animals a good quality-feed, considering hierarchy and providing easy access to feed for less dominant animals could represent an easy-to-implement strategy at the farm level, together with disease and parasite control.

Funding No third-party funding or support was received in connection with this study or the writing or publication of the manuscript.

Conflict of Interest The authors declare that there were no conflicts of interest.

References 1.

2.

3.

4.

5.

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7.

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Seeger T, and Walter J. Ketosis and hyperlipemia in a female alpaca. A case report. Tierarztl. Prax. Ausgabe G Grosstiere - Nutztiere, 2008, 36:333337.

Bravo PW, Bazan PJ, Troedsson MH, Villalta PR, and Garnica JP. Induction of parturition in alpacas and subsequent survival of neonates. J. Am. Vet. Med. Assoc. 1996, 209:1760-2.

Cebra C, Anderson DA, Tibary A. Van Saun RJ, Johnson LW. 2014. Llama and Alpaca care: Medicine, reproduction, nutrition and herd health, 1st Ed.pp 537-551.

Fowler ME, Zinkl JG. (1989) Reference ranges for hematologic and serum biochemical values in llamas (Lama glama). Am. J. Vet. Res.50:204953.

Foster A, Bidewell C, Barnett J, Sayers R. Haematology and biochemistry in alpacas and llamas. In practice 2009;31:276-281.

Waitt LH, Cebra CK. Characterization of hypertriglyceridemia and response to treatment with insulin in llamas and alpacas: 31 cases (19952005). J Am Vet Med Assoc, 2008, 232(9):1362-1367.

Tornquist SJ, Van Saun RJ, Smith BB, Cebra CK, Snyder SP. Hepatic lipidosis in llamas and alpacas: 31 cases (1991-1997). J Am Vet Med Assoc, 1999, 214(9):1368-1372.

Anderson DE, Constable PD, Yvorchuk KE, Anderson NV, St-Jean G. and Rock L. Hyperlipemia and ketonuria in an Alpaca and a Llama. J Vet Intern Med 1994, 8(3):207-211.

Cebra CK, Tornquist SJ. Effects of epinephrine and insulin on blood biochemical constituents in llamas and alpacas, Am J Vet Res 2004, 65:16921696.

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M.Z. Khan et al. Large Animal Review 2020; 26: 321-328

Comparative study on the potency of trivalent vaccine of foot and mouth disease in different cattle breeds and triggered immune response signaling pathway

321

N

MOHAMMAD ZEB KHAN1#, XUWEN GAO1, XIAOLONG FAN1, LIYING SONG1, MUJEEB-UR-REHMAN MEMON2, DILDAR HUSSAIN KALHORO2, IQRA BANO2, SAQIB ALI FAZILANI2, YIJING LI1, YIGANG XU1, LIJIE TANG1* 1

Heilongiang Key laboratory for animal disease control and pharmaceutical development, department of preventive Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Harbin-150030, China 2 Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tandojam-70060, Pakistan

SUMMARY The Foot and Mouth Disease (FMD) is a very common disease which causes infection in almost all cloven-footed animals including cattle, camel, goat, and sheep. The objectives of current study were to determine the prevalence of FMD in different breeds of Khyber Pakhtunkhwa, Pakistan and to evaluate and compare the potency of locally available trivalent vaccine against FMD in Achai (local breed of Khyber Pakhtunkhwa) and exotic pure breeds i.e. Holstein Friesian and Jersey. In addition, to observed the immune signaling pathway and the involvement of IL-17 cytokine response. A total of 135 sera samples were collected from Achai, Jersey and Holstein Friesian breeds to know the strains of Foot and mouth disease (FMD) circulating in different cattle breeds. The strain-based prevalence against O, Asia 1 and A antibodies against all three tested strains was found. In Holstein Frisian O strain (33.33%) was the highest circulating FMD viral strain followed by Asia1 and A. In Jersey breed, O strain (26.66%) was significantly higher followed by A and Asia 1. Whereas, in Achai breed, O strain (13.33%) was also found to be the highly prevalent FMD viral strain followed by Asia 1. Furthermore, potency of FMD vaccine, prepared at the University of Veterinary and Animal Sciences Lahore, Pakistan besides Deccivac water-based, Germany were used in Cattle, and the potency of the vaccine was evaluated through Complement Fixation Test. The data revealed non-significant difference (p>0.05) among the three different groups when evaluated at day 30 and significant difference (p>0.05) at day 60 and 90 post vaccination. The difference in antibody titers was found non-significant difference (p>0.05) between FMD trivalent preparation of UVAS besides water-based Deccivac on 90th day of immunization screening. A higher titer was recorded in Achai cattle breed followed by Holstein Friesian and Jersey of UVAS made vaccine and water base Desivac vaccine trialed. In addition, initial involvement of IL-17 immune response was observed among challenged groups and the result was supported by IgY antibodies, that showed significantly higher ratio at day 30, 60 and 90.

KEY WORDS Foot and Mouth Disease, Cattle, IL-17 immune response, Strains, Prevalence, Vaccine.

INTRODUCTION The Foot and Mouth Disease (FMD) is life threatening viral disease which causes infection in almost all cloven-footed animals including camel, cattle, goat, and sheep. This viral infection is also reported in some wild animals like deer, Asian elephants and wild boar1. It causes a considerable loss in terms of decreased productivity2. Aphthovirus is causative agent of FMD which belong to family Picornaviridae RNA, non-enveloped and icosahedral virus having a nucleocapsid with diameter 27 to 30 nm, which is surrounded by four capsid coats of proteins enu-

Corresponding Author: Lijie Tang (tanglijie@163.com).

merated as VP1, VP2, VP3, and VP4. The FMD has seven major immunological serotypes Southern African Territories (SAT) 1, SAT 2, SAT 3, A, O, C and Asia3.

Foot and Mouth Disease is endemic in Pakistan and its neighboring countries, imposing a substantial negative impact on livestock industries4. Cattle and buffalo are a major part and occupy a premier place in the livestock industry, contributing significantly to the economy of the world. Their populations are threatened by a number of health hazards, among the most notable of which are Foot and Mouth Disease. These diseases inflict substantial losses in terms of reduced productivity5. The most prevalent serotype of FMD virus in Pakistan is O type followed by Asia1 and A, respectively whiles serotype C has also been reported sporadically in 1954, 1963 and 19956,7. Efforts to control FMD in Pakistan have been limited by the lack of resources available in the country, and many features of the

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Comparative study on the potency of trivalent vaccine of foot and mouth disease in different cattle breeds

transmission and control of FMD in Pakistan are yet to be elucidated. Traditional practices, such as unregulated animal movement between nomadic and transhumant herds, limits the effectiveness of veterinary services. Foot and Mouth Disease vaccines are not used systematically in Pakistan, and thus attributes such as the efficacy, potency, and strain composition of the vaccines are uncertain6.

Buffalo and cattle are the main contributor in the livestock sector and play a vital role directly and indirectly in the livestock sector contributing drastically to world economy. In Pakistan, the breeds of cattle are Red Sindhi, Thari, Rohjan, Dhanni, Lohani, Bhag Nari, Sahiwal, Achai2. Globally FMD is the main concern in improvement of the socio-economic situation8. Vaccination is the most proper method for the control of disease in China, India and certain African countries where FMD is endemic. The World Organization for Animal Health (OIE) officially documented Argentina and Uruguay as free countries from FMD with the proper schedule vaccination and management9. The efficacy of vaccination program will largely depend on the quality (purity, safety, and potency) and suitability of the chosen vaccine as well as selection of vaccine on basis of suitable adjuvants like oil or gel-based. The choice of adjuvant in vaccine development determines its effectiveness or ineffectiveness.

The adjuvant can be classified on the basis of chemical nature, origin and physical properties as well as on the basis of activation of innate or adaptive immune response10. The OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals describes two methods for assessing FMD vaccine potency in cattle, namely the Ph. Eur. 50% protective dose (PD50) test and the South-American “Protection against Podal Generalization” (PPG) tests OIE11. In addition, relations between pathogen PAMPs and TLRs trigger a torrent of indicating actions that result in the stimulation of dendritic cells and cellular reactions, such as the difference of naive T helper (Th) cells into mature effector Th1, Th2, Th17, and Treg cells12,13. These signals actions terminate in the secretion of different cytokines, including proinflammatory interleukin (IL)-1 Th cells, interferon-γ (IFNγ) by Th1 cells, IL13 by Th2 cells, IL-17 by Th17 cells, and transmuting growth factor (TGF)-β and IL-10 by Treg cells14,15. Cytokines are the effector fragments that linkage information among cells of the immune system.

Currently in Pakistan, various types of inoculations are used for FMD but still outbreaks of FMD have been reported in various regions even in vaccinated animals which points towards the facts to be investigated. Keeping in view the above-discussed problem, the current study was intended to determine the prevalence of FMD in District Swat Pakistan and evaluate the potency of locally/imported prepared trivalent vaccine in Achai, Jersey and Holstein Friesian breed. Furthermore, it illuminated the host role in characterization of antibodies and immune response of different cytokines in the vaccinated animals.

MATERIAL AND METHODS Study Area and Epidemiological Study The study was conducted in Khyber Pakhtunkhwa, Pakistan.

Data for the study was collecting on the basis of Tag number, sex, age, breeds, previous vaccination, manure disposal and interaction of animals with each other.

Experimental design A total number of 36 animals of 1-3 years of age from Achai (A), Jersey (B) and Holstein Friesian (C) breeds were used to determine vaccine potency. Each group was comprised of 12 animals which were divided into 3 subgroups, A1, A2 A3, B1, B2, B3, C1, C2, and C3. Each subgroup A1, B1, and C1 were comprised of 4 animals, and was injected with manufacturer recommended dose of oil-based vaccine, A2, B2, and C2 subgroups were injected according to the manufacturer recommended dose of water-based vaccine while; subgroups A3, B3 and C3 were kept as control.

Prevalence percentage The total number of 135 sera samples, 45 samples from Achai, 45 samples from Jersey and 45 from Holstein Friesian breeds were collected from suspected animals of those farmers visited in the study area respectively. Data were collected regarding the suspected risk factors of these animals through a questionnaire. The sample size was calculated by considering the expected Seroprevalence to be 8-10% based on previous studies conducted in Pakistan16 with confidence limits at 95%.

Vaccine procedure and Sample collection Sampling was started by day 0 i.e. preparing dosage in all three assemblies, each group contains 4 animals and the booster dose was given after 30 days. The Immunization of animals was performed with a master line sterile BD syringe in the early morning. The Inoculation was directed via both intramuscular as well as subcutaneous routes. The 1st blood sample was collected on day 0 before priming. The 2nd, 3rd, and 4th sampling was collected after 30, 60 and 90 days respectively. All vacutainers were placed into dry ice bags and were moved instantly to Veterinary research and disease investigation center Balogram Swat, Pakistan and kept at 4 for 24 hours. The serum was separated via centrifugation. The serum samples were properly labeled and stored in Ultra Cold Freezer at -20 for additional processing.

The Complement Fixation Test (CFT) The CFT was performed to measure the anti-FMD O antibody titer from serum samples from vaccinated cattle as per recommendations of11 with little modification.

Enzyme-Linked Immunosorbent Assay (ELISA) Post challenged groups of Achai, Jersey and Holstein Friesian breeds, IgY antibodies were detected at day 30, 60 and 90 via using ELISA kit, (Meilian Biological Technology Co., Ltd.) by follow the manufacturer’s instruction. Briefly, at day 30, 60 and 90 post challenged of vaccine to Achai, Jersey and Holstein Friesian breeds, their blood were collected and preceded for getting plasma and serum samples. Next, samples were examined in triplicate; every ELISA plate contained their own standards.

Dilution for IgY 1:20,000 as a detection of antibody. Afterward, the samples were incubated with tetramethylbenzidine for 30 min (Sigma), reaction was stopped via 2 M H2SO4 (55 µL/well). Primary wavelength for read was 450 nm by using an ELX 800 universal micro plate reader (BIO-RAD Model 680) and absorbance was measured at 490 nm. To compute antibody concentration, the mean of the duplicate values for each sample was calculated. The mean value of the blanks was subtracted from the measured antibody concentration to account for non-

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Table 1 - PCR and RT-qPCR primers list with references.

Primers

Target gene

Bp

Reference or source

F-5,-GGTGGTGCTAAGCGTGTTAT-3,

GAPDH

264

K01458

IFN-γ

259

Y07922

IL-2

256

AF000631

IL-4

140

DQ852343

IL-10

145

AF0680058

IL-12

588

19; 169(1-2): 82-92.

IL-17

292

AJ493595

R-5,-ACCTCTGTCATCTCTCCACA-3, F-5,- AGCTGACGGTGGACCTATTATT-3, R-5,- GGCTTTGCGCTGGATTC-3, F-5, -TCTGGGACCACTGTATGCTCT-3, R-5, -ACACCAGTGGGAAACAGTATCA-3, F-5,-AGAGCTCGGTGACCTCAGAC-3 R-5,-CTTGCATGGCGGTCTTTAG-3 F-5,-GAACTCCCTGGGGGAAAAC-3 R-5,-GGCTTTGTAGACGCCTTCCT-3 F-5, -CTCAGCAGTTGGTCATCTCC-3, R-5, -CACTGCCTTCCTGACACTCC-3, F-5,-CTCCGATCCCTTATTCTCCTC-3,

specific binding, after read blank adjusted data were transferred to an Excel file. The concentration of the standard and their absorbance value for each plate was created; each sample concentration of the antibody was expressed as micrograms /milliliter.

Cytokines expression by Quantitative real-time polymerase chain reaction (RT-qPCR) RNA was isolated from all samples of challenged groups with control by using the TRIzol Plus RNA Purification kit (Invitrogen) following the manufacturer’s instructions, and then eluted 100 ul RNase-free water to determine concentration, transparency and concentration of RNA were measured spectrophotometrically at an optical density of 260 nm, via NanoDrop spectrophotometer ND-2000, (Thermofisher, MA) and verified the purity of RNA via the ratio of absorbance at 260 nm / 280 nm17. To reverse transcribed into cDNA using MMLV reverse transcriptase (Promega). The cDNA was analyzed

by Quantitative real time PCR (RT-qPCR). The oligonucleotide primers used for analyses, GAPDH and specific target genes primers are shown in supplement (Table 1). Briefly, RTqPCR Light cycler 96 (Roche, Switzerland) was used. Total 10 µl mix comprised 4.5 µl of SYBR Green Supermix (Bio-Rad, Hercules) 0.5 µl of each primer, forward and reverse, 1 µl of cDNA and 3.5 distilled water. Standard curve was created by using log10 diluted standard RNA, and individual’s levels of transcript were standardized to those of GAPDH analyzed by the Q-gene program. For normalize RNA levels, the mean threshold cycle (Ct) values for products amplification were calculated by pooling values from all samples in that experiment.

Every samples relative level was calculated by pfaffls method18.

Statistical analysis The test result for each animal was recorded in a Microsoft Excel spreadsheet. Descriptive statistical analysis and multiple logistic regressions were being carried out using State software version 9 (State Corp., College Station, Texas, USA). The true prevalence was calculated using a Win Episcope 2.0 (University of Edinburgh, UK). Data for vaccine potency was analyzed by statistical package for social science (SPSS) version 20.0. Oneway ANOVA was done to calculate the antibody response of each inoculation. Additionally, Chi-square assessment was done to observe the bond among qualitative variables. P-value < 0.05 was set as a level of significance.

RESULTS The point prevalence of FMD was significantly higher in exotic breeds as depicted in table 2. Amongst the exotic breed’s prevalence of FMD was statistically higher in Holstein Friesian than Jersey (33.33%). The lowest FMD prevalence was recorded in Achai cattle breed in the present study area.

The O strain prevalence was found significantly higher than Asia-1 and A in all three breeds of cattle (Table 3).

A significant difference of potency in the sense of antibody titer level was recorded amongst different cattle breeds (Table 4).

A higher titer was recorded in Achai cattle breed followed by Holstein Friesian and Jersey. Also, a statistically significant variation of cumulative geo means titer was recorded against Desivac vaccine trialed in three different cattle breeds each group having 4 animals. A stronger titer was recorded in Jersey followed by Holstein Friesian and Achai. No variation of CGMT was recorded among control group.

Table 2 - Prevalence of FMD in different cattle breeds in Khyber Pakhtunkhwa, Pakistan.

Breeds

Sample Size

Positive

Prevalence (%)

Holstein Friesian

45

19

42.22%

Jersey

45

15

33.33%

Achai

45

07

15.5%

P-Value 0.027

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Table 3 - Prevalence of different (FMD) Disease of virus strain circulates in different cattle breeds in, Khyber Pakhtunkhwa, Pakistan.

Breeds

Asia 1 Prevalence

O Prevalence

A Prevalence

Holstein Friesian

9 (20.00%)b

15 (33.33%)a

5 (11.11%)c

c

a

b

Jersey

5 (11.11%)

12 (26.66%)

8 (17.77%)

Achai

05 (11.11%)b

6 (13.33%)a

5 (11.11%)b

P-Value 0.041

Table 4 - Comparison of different vaccine potency in different cattle breeds.

Vaccine type

Overall mean Titre of different Vaccines Achai Breed

UVAS Made

a

9.0

P-Value

Jersey

7.74b

a

b

7.41 c

Holstein Friesian

c

0.043

Desivac

5.39

9.13

8.70

0.037

Control

1.12

1.24

1.37

0.729

Comparison of anti-FMDV antibody titer of various groups

Comparison of gel oil and waterbased

The difference among the three different groups of Achai, Jersey and Holstein Friesian administered with FMDV vaccines was significant that is p≤0.05. Group A (UVAS made primed with Gel Oil-based enhanced with Gel Oil Grounded Vaccine via subcutaneous-intramuscular paths respectively) group B (water-based Vaccine via intramuscular route respectively) group C (left as a control). Group A, B, and C resulted in cumulative geometric mean titer CGMT±SD values of 15.31±1.20, 12.20±1.691 and 1.22±0.71 respectively (Figure 1).

There was a significant difference seen between the adjuvants of vaccine i-e P≤0.05. The animals immunized through priming and boosting dosage of gel oil grounded preparation displayed pointedly increased, the anti-FMD antibody titer as compared to the animals vaccinated with water. Moreover, the CGMT±SD of gel with oil and with water were 15.31±1.20, 8.07±4.04 correspondingly (Figure 3).

Comparison of two vaccines

There was significant difference i-e P≤0.05 between the groups vaccinated with various routes of administration.

The animals immunized with priming dosage with the subcutaneous route and by means of boosting dose intramuscular displayed meaningfully increased anti-FMD antibody titer at the 90th day of immunization associated with those vaccinated intramuscularly for both priming’s as well as boosting displayed CGMT±SD 15.31±1.20 and 10.51±2.19 respectively (Figure 4).

The antibody titers obtained were subject to SPSS Version 20.0.0 for the calculation of ANOVA. The results revealed that there was no imperative variation P≥0.05 among the antibody mixtures of FMD trivalent inoculation of UVAS and waterbased (Desivac) vaccine at 90th day of immunization screening CGMT±SD ratios of 12.05±.68 and 7.71±5.05 correspondingly (Figure 2- 3).

Figure 1 - Comparison of mean anti-FMD virus antibodies. The difference among the three different groups of Achai, Jersey and Holstein Friesian administered with FMDV vaccines was significant that is p≤0.05.

Comparison of the route of administration

Figure 2 - Comparison of days’ post-vaccination. There was nonsignificant difference P≥0.05 between the antibody titers of FMD trivalent vaccine of UVAS and water based (Desivac) vaccine at 90th day of inoculation showing CGMT ±SD values of 12.05±.68 and 7.71±5.05 respectively.

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M.Z. Khan et al. Large Animal Review 2020; 26: 321-328

Figure 3 - Comparison of Gel Oil base and water base. Animals inoculated with priming and boosting dose of gel oil based vaccine showed significantly high anti FMD antibody titer than animals inoculated with water for both priming and boosting. CGMT±SD of gel with oil and water with water were 15.31±1.20, 8.07±4.04 respectively.

Figure 4 - Comparison of different vaccine administration. There was significant difference i-e P≤0.05 between the groups vaccinated with various routes of administration. The animals inoculated with priming dose through s/c followed by boosting dose I/m showed significantly high anti FMD antibody titer at 90th day of inoculation compared with those inoculated intramuscularly for both priming and boosting showing CGMT±SD 15.31±1.20 and 10.51±2.19 respectively.

Moreover, the animals immunized by means of oil-based vaccines for both priming as well as boosting via S/c showed marked significant decline in the anti-FMD antibody titer.

Comparison of different serotypes There was a non-significant difference i-e P≥0.05 between the anti-FMD antibody titers of different three serotypes of FMD. The serotypes are serotype “O”, “serotype Asia 1” and Serotype “A” showing CGMT±SD values 10.08±5.46, 10.49±5.49 and 9.08±4.73 respectively (Figure 5).

Cytokine Gene Expression and IgY titer Immune response amongst challanged groups was determined via expression levels of cytokines, cytokine IL-17 and IFNγ, expression was significanltly (P < 0.05) upregulated at day 30, 60 and 90 that showed theirs initial immune response

325

Figure 5 - Comparison among different serotypes of FMD. There was non-significant difference i-e P≥0.05 among the anti FMD antibody titers produced in response of different three serotypes of FMD. The serotypes are serotype “O”, “serotype Asia 1” and Serotype “A” showing CGMT±SD values 10.08±5.46, 10.49±5.49 and 9.08±4.73 respectively.

as compared with unchallenged group. Furthermore, IL-2, IL4, 1L-10 and IL-12 were unregulated in challenged groups at day 30 period but showed significantly upregulated at day 60 and 90 as compared with control (Figure 6 and 7). In adding, humoral adaptive immune response specified that greater levels of antibodies against FMD were identified in the plasma of challenged breeds as compared with control (Figure 7) that showed their suggestive evidence and indication that IL-17 are mainly involved in initial mediating immune response. Among different routes the IgY titer significantly higher in challenged groups at day 30, 60 and 90 that supported the results of cytokines. In addition, gel oil grounded preparation presented increased the IgY titer as compared to the animals vaccinated with water. Moreover, ELISA test supported the results of cytokines.

DISCUSSION In this study, 3 different serotypes ‘O’ ASIA 1’ and ‘A’ of (FMD) Disease virus were carried under the study to find out the response of vaccine against (FMD) disease. The results of the research proved no significant variation (p≥0.05) between the (FMD) antibody titers displayed in reply of 3 diverse serotypes of FMD. The serotypes are serotype “O”, “serotype Asia 1” and Serotype “A” showing CGMT±SD ranges 10.08±5.471, 10.49±5.49 and 9.08±4.73 respectively. The findings of the current study are in agreement with the results of19,20 that used different serotypes of the FMD viruses for safety evaluation and antibody titers of the vaccines. The different serotypes were combined in vaccines were also reported in their study. In present study the titers of antibody of every (FMD) Disease serotypes were observed similarly in all studies cases. It can be concluded that there was no significant difference among the effect of antibody titer of vaccine. The study of21 tested FMDV quadrivalent PEG concentrated vaccine with double oil emulsion that was tested in goats. The serum neutralization titers were 2.76, 2.94, 3.0 and 3.22 for serotypes O, A, C and Asia 1 after 90-day post-vaccination, while, AGS vaccine, gave titers 1.02, 1.68, 1.02 and 1.27 for O, A, C and Asia 1, respectively. During current

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Figure 6 - Expression levels of cytokines, cytokine IL-17 and IFNγ, expression was significanltly (P < 0.05) upregulated at day 30, 60 and 90 that showed their initial response as compared with unchallenged group. Furthermore, IL-2, IL-4, IL-10 and IL-12 were unregulated in challenged groups at day 30 period but showed significantly upregulated at day 60 and 90 as compared with control.

experiment various responses of immune were observed significant (P≥0.05) statistically. The mention difference in the immune response was observed because of the addition of oil adjuvant in vaccination that increased time duration of the potency of immunity as compared to the water-based vaccination. Moreover, the vaccine which was gel based showed rapid antibody titers that are more important during early response of vaccination is required in case of outbreak. The strain-based prevalence against O, Asia 1 and A antibodies against all the three tested strains were found. O strain prevalence was found significantly higher than Asia-1 and A. In Holstein Friesian O was found the highest-circulating FMD viral strain followed by Asia1

Figure 7 - IgY level at day 30, 60 and 90. Each value is represented in triplicate±SD. significantly difference (P≤ Value 0.05).

and A. In Jersey breed O was found significantly higher followed

by A and Asia 1. While in Achai FMD viral strain O was also found to be the highly prevalence FMD viral strain followed

by Asia 1 and A respectively. The present study is in line with the previous studies of22,23 who stated that in Pakistan, the most prevalent serotypes are O, Asia-I and A respectively. Same statement has been reported by the6, who had reported that the distribution of various (FMD) Disease serotypes virus in different areas of Pakistan showed that O serotype was detected high prevalence serotype followed by Asia 1 and A (F, D) disease serotype C. Furthermore,24 found highest proportion of positive samples of serotype O, followed by serotype A and serotype Asia-1 respectively.

The data revealed non-momentous alteration (p>0.05) between the three dissimilar groups in which one was be kept as control directed with FMDV inoculations when assessed at day 30 and noteworthy alteration (p<0.05) at day 60 and 90 post-immunization. Group 1, 2 and 3 displayed (CGMT±SD) ratios of 15.31±1.20, 12.20±1.69 and 1.22±0.70 respectively. Moreover, the analysis of modification displayed momentous alteration (p<0.05) within the antibodies among assemblies on days 60 and 90. In addition on day 60 in assembly 1, inoculated by means of UVAS gel adjuvant shot for priming and oil adjuvant by way of booster dosage, uppermost average range was seen tracked through group 2, inoculated through water grounded Deccivac vaccine. Moreover, the average ratio of antibodies was decreased in Group 3 and was found once the antibody titer was calculated at day 90 alike outline was detected for antibodies.

The outcomes are in line as described by25 who used inactivated

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M.Z. Khan et al. Large Animal Review 2020; 26: 321-328

FMD virus suspension was mixed with aluminum hydroxide gel induced detectable level of anti-FMD virus antibodies in rabbits. They found that antibodies in serum appeared earlier in the rabbits when vaccinated with Aluminum Hydroxide gel (AHG-FMDV) vaccine as compared to those vaccinated with Lanolin (LAN-FMDV) and Montanide Oil Base (OBFMDV) vaccines. The adjuvants such as aluminum hydroxide gel and oils are still used in vaccine production as a base of vaccine formulations. Gel type adjuvant is commonly used in vaccines of veterinary importance. Liquid paraffin was commonly used as mineral oil (oil adjuvant) in animal vaccines. The emulsifying agents like Mannide monooleate (Arlacel-A) and Sorbitan monooleate (Span- 80) were used in 10% concentration in vaccines in the research26.

The reliability of different inoculations mainly rests on the emulsifier concentration27,28. The gel was least toxic for animal tissues, hence suitable for use in bovine, caprine, equine and even in human vaccines, while oil adjuvant vaccine induces a vaccine depot at the site of inoculation hence causes inflammation due to irritation, and recruiting lymphocytes and antigenpresenting cells at the injection site26 used oil- or AI (OH) 3/ saponin-adjuvanted vaccines in an experiment that demonstrated potency and competency of vaccine inducing antibodies in first week of administration in cattle. Dissimilar results in diverse species of animals were described28. Found that water FMD inoculation gave minute duration of resistance and antibody titer quickly fall although oil adjuvant FMD vaccines had lengthier period of immunity.

The difference in antibody titers in the current study was found non-significant (P>0.05) between the antibody titers of FMD trivalent inoculation of UVAS and Deccivac water-based at 90th day of vaccination viewing (CGMT±SD) ratios of 12.05±.68 and 7.71±5.05 respectively. There was a momentous difference (p<0.05) among the adjuvants of inoculation. The animals immunized with priming dosage of gel grounded vaccine tracked via oil-based boosting displayed significantly increased antiFMD antibody titer as compared to the animals immunized with oil for mutually priming as well as boosting. The (CGMT±SD) of gel by means of oil and water with water was 15.31±1.20, 8.07±4.04 correspondingly. The water grounded inoculations were proceeding less efficacious than the oil-adjuvant inoculations of cattle and sheep. The advancement in vaccine construction is focused on the assortment of the appropriate adjuvant that can elaborate increased as well as prolonged immunity.

Further, we hypothesize that the various functions and characteristics of the different cytokines and their immune regulatory molecules that secreted by the various subsets of T-helper cells that previous described by different authors28-15. Generally, when stimulated via macrophages and/or dendritic cells, T-helper cells typically produce IL-1 and other pro-inflammatory cytokines that stimulate their differentiation into Th1, Th2, and Th17. In present study, at day 30, IL-17 expression was upregulated. Beside, activated Th1 cells primarily produced IFNγ, and sometimes IL-2, that are very important for protection against intracellular pathogens via activation and promote of phagocytes and production of complement-fixation antibodies14. In this study, IFN-γ showed significant effect from day 30, while IL-10 and IL-12 showed no significant difference at day 30, whereas at day 60 and 90 found significantly difference.

Moreover, IL-4 and IL-2 showed unregulated difference among breeds at day 30, while showed upregulated at day 60

327

and 90. This may indicate the IL-17 cells play initial role and involvement in the rising of immune responses. Further, the presence of high levels antibodies in challenged breeds and upregulated IL-17 at three-time period also supports the results of cytokines expression. These results showed that IL-17 cells are primarily involved in signaling mechanism underlying the inflammatory responses. This study makes positive linked with previous reported by15-30,31.

CONCLUSIONS It is concluded that point prevalence of FMD was significantly higher and exotic breeds i.e., Holstein Friesian and Jersey, O strain prevalence was found significantly higher than Asia1 and A in Holstein Friesian, Jersey and Achai. Holstein Friesian is the most susceptible breed as compared to Jersey and Achai cattle breed. The University of Veterinary and Animal Sciences (UVAS) made an oil base vaccine via subcutaneous-intramuscular route showed best produced higher anti-FMDV antibody titers results against FMD as compared to Decivac water-based vaccine. The present study on seroprevalence of Foot and Mouth Disease in cattle is essential for further epidemiological studies to develop effective disease control strategies, particularly in the areas where animal movement is not restricted. Moreover, these results suggest that any forthcoming plans to moderate vaccine against FMD should be particularly based on their impending to induce Th17 (IL-17) cytokine excretion.

CONFLICT OF INTEREST There is no conflict of interest to declare.

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Effects of omega-3 and omega-6 fatty acids on some reproductive parameters in ewes

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l

ERCAN SOYDAN1*, MEHMET KURAN1, NUH OCAK2, SEDAT YILDIZ3, ZAFER ULUTAĹ&#x17E;4 1

Ondokuz Mayis University, Agriculture Faculty, Department of Agricultural Biotechnology, Samsun, Turkey Ondokuz Mayis University, Agriculture Faculty, Department of Animal Science, Samsun, Turkey 3 Inonu University, Medicinal Faculty, Department of Physiology, Malatya, Turkey 4 Omer Halisdemir University, Faculty of Agricultural Sciences and Technologies, Department of Animal Production and Technologies, NiÄ&#x;de, Turkey 2

SUMMARY n-6 and n-3 fatty acid families act as nutraceuticals to complement the sequential processes of follicle and embryo development.

However, there is a lack of information on effect of dietary supplementation of n-6 and n-3 fatty acids on different reproductive events in the sheep. Accordingly, in this study, the effect of supplementation of n-6 PUFA rich SoyPreme (SP) or n-3 PUFA rich Flaxtech (FT) on plasma hormone concentrations and some ovarian activity in the sheep were studied. Following the first detected estrus, a total of 44 ewes were allocated into either bazal diet (C, n = 22) or SP (n = 22) treatments until next estrus (pre-mating). At the second estrus, the ewes were mated and again randomly allocated to either the C or FT allowance until day 15 (post-mating; mating = day 0). Hence, there were four nutrition treatments; CC (n = 11), SPC (n = 11), SPFT (n = 11) and CFT (n = 11). Blood samples were collected to monitor plasma hormon levels. Ewes were slaughtered on 16th day after mating, and the numbers and weights of corpora lutea (CL) and follicles were recorded. Plasma progesterone (P<0.05) and PGFM (P<0.01) concentrations including basal and peak PGFM in the SP ewes during pre-mating period were higher than those of the C ewes.

The number of CL were higher in the SPFT ewes compared to the CC and SPC ewes (P<0.05). While the number of small follicules in the SPC, CFT and SPFT ewes were lower than those of CC, large follicles in the SPC and CFT ewes were lower than those of CC (P<0.05). It was concluded that short-term (15-17 days) changes in dietary n-6 and n-3 supplementation can have a beneficial effect on plazma hormon consentration and ovarian activity during pre-mating and post-maing, respectively, in ewes.

KEY WORDS Sheep, nutrition, PUFA, ovarian activity, PGFM.

INTRODUCTION Increasing the litter size by maximizing the ovulation rate and minimizing post-mating wastage are one of the most important approaches to increase the reproductive performance of the small ruminats1. Reproductive performance in domestic ruminants is influenced by dietary fat used to enhance reproductive status, as previously reviewed2-3. It is well accepted that dietary fat directly affects dairy ewes and fertility 3-5. Therefore, nutrition influences ruminant fertility directly and indirectly.

The direct effect relate to the supply of specific nutrients required for the processes of oocyte and spermatozoa development, ovulation, fertilization, embryo survival and the establishment of pregnancy, whereas the indirect influence is circulating concentrations of the hormones and other nutrientsensitive metabolites that are required for the success of these processes6. Indeed, the positive effects of some nutritients such as fat and energy supplementation on improvements in re-

Corresponding Author: Ercan Soydan (esoydan@omu.edu.tr)

production of domestic ruminants are well documented3. A number of these documents have primarily focussed on the effects of total dietary fat and energy balance3,5,6, rather than specific effects of n-3 or n-6, especially in the sheep.

Fatty acids of the n-6 and n-3 families act as nutraceuticals, altering innate immune responses and subsequent gene expression within the uterus to complement the sequential processes of follicle and embryo development and survival of the embryo and fetus. The n-37-8 and n-6 fatty acids9-10 were reported to have effects on progesterone and prostaglandin release in cattle6,8,11,12 , sheep13,14 and goat5. The n-6 fatty acids increase the synthesis of prostaglandine series 27,14 while n-3 fatty acids increase the progesterone synthesis15. In recently, it has been reported that ALA affects prepubertal sheep embryo quality associated with alteration of releasing reproductive hormones16 and supplementation of n-3 fatty acid increases the number of preovulatory follicles and ovulation rate, decreases the metabolites of serum prostaglandin F2 and E2 during the window of pregnancy recognition5.

Ruminants obtain their unsaturated fatty acid needs from the green forages in pasture land17. Incorporation of fresh grasses into diets is vital to sustain the dietary ideal n-6/n-3 ratio18.

However, in autumn season, i.e, mating season in most parts

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Effects of omega-3 and omega-6 fatty acids on some reproductive parameters in ewes

of the World, pasture lands are poor in vegetation with respect to the specific nutrient such as fatty acids19. This situation can negatively affect reproduction in sheep. To avoid this, enriching the diets with special nutrients affect fertility positively. However, it is still not clear whether changing the fatty acid composition in diet before or during mating may affect reproduction parameters in sheep or not. In previous studies, the effect of n-6 and especially n-3 fatty acids, polyunsaturated fatty acids (PUFA) were found to be important for various reproductive processes, especially as steroid hormone and prostaglandin precursors5,16. However, there has been a lack of information related to the dietary n-3 and n-6 supplementations in sheep during follicular and luteal phases on ovarium activities, corpus luteum activities, embryo viability, pregnancy rate, concentration of reproduction hormones. Thus, it was hypothesized that shortterm changes of n-6 and n-3 diet during pre- and post-mating period respectively could enhance not only the uterine environment and oocyte quality, but also ovarium activity, independent of a secondary effect on production or metabolism modifications. Therefore, the aim of this study was to determine the effect of supplementation of n-6 PUFA rich SoyPreme (SP) or n-3 PUFA rich Flaxtech (FT) on plasma hormones concentration and ovarium activity in the sheep during pre- and post-mating period.

MATERIALS AND METHODS Animals and diets The study was carried out during the breeding season at the experimental farm of the Gaziosmanpasa University, Tokat, Turkey (40°31’ N, 36°53’ E and 650 m above sea level). The ethical approval was received by Ondokuz Mayis University Ethical Commity. A total of 44, 4 year old multiparous Karayaka ewes with an average weight of 43.26 ± 3.97 kg were used. All ewes were fed on control diet composed of 65% forage and 35% concentrate feed at maintenance level20 over an estrus cycle pe-

Table 1 - Composition and nutrient content of basal diet (g/kg fed basis).

Fatty acid (%)

SoyPreme

Flaxtech

C 16:0

11

5.5

C 18:0

4

3.6

C 18:1

22

16.4

C 18:2 (n-6)

53

16.2

C 18:3 (n-3)

8

55.3

Others**

2

3.0

Provided from product cataloge ** Values below 1% were classified as “others”.

riod during pre-mating and post-mating.

The n-6 was fed as SoyPreme (Boregaard UK, Warrington, UK), a heat-treated product of xylose and cracked soybean. The n3 was fed as Flaxtech (Flaxtech, Virtus Nutrition, USA) a calcium salt of flaxeed and Ca salt containing 84% fat and 9% Ca. This process reduces the degradability of the protein and protects the PUFAs from biohydrogenation in the rumen8.

Synchronisation of ewes and allocation into experimental groups The estrus cycles of ewes were synchronized using a 14-day treatment of progestagen impregnated vaginal sponges (40 mg Florogestone Acetate; Chronogest®, Intervet) combined with PGF2 and GnRH injections. After synchronization, the ewes detected with referance heat by teaser ram were allocated to treatment groups without mating. The days detected reference estrus (day 16 pre-mating; -16) considered as starting experiment. Animals used in the experiment were waited until they show natural estrus. Then the animals were mated (mating; day 0) and allocated to feeding programme as planed in the experimental design. A 16-d (an estrus cycle period) feeding programme prior to mating and a 15-d feeding programme after mating were applied. During estrus cycle period, sheep were allocated to 2 groups fed basal diet (control) and n-6 diet, respectively. Following the first detected estrus, a total of 44 ewes were allocated into either bazal diet (C, n = 22) or SP (n = 22) treatment until next estrous (pre-mating). At the second estrus, the ewes were mated, and again randomly allocated to either the C or FT allowance until day 15 (post-mating; mating = day 0). Hence, there were four nutrition treatments; CC (n = 11), SPC (n = 11), SPFT (n = 11) and CFT (n = 11).

Blood sampling Blood samples were taken 3-d intervals for plasma progesterone, daily for estradiol-17 analysis from the 15th day of estrus cycle to 3rd day after mating, and two hourly for prostaglandin (PGFM) from 13rd day to 16th day of natural estrus cycle. The blood samples in heparinized tubes were placed on ice and immediately centrifuged 15 min at 4000 rpm at 4 °C, frozen and stored at -20 °C until hormones assays. On day 16 post mating, ewes were slaughtered humanly to take ovary of each ewe to be transported to laboratory at 37 °C in PBS. The weights of corpus luteum and ovarium were recorded after isolation using scissors and forceps.

Hormone assays Plasma progesteron and estradiol were measured by enzyme immunoassay using kit (DRG Instruments GmbH International, Marburg, Germany; progesteron: EIA-1561, estradiol: EIA-

Table 3 - Concentrates given to the control and treatment groups (%).

Concentrates

Table 2 - Forages used in the study (% DM).

Control

Omega-3 (n-3)

Omega-6 (n-6)

Wheat

35.5

31.5

28.3

Canola meal

28.0

22.3

20.0

(%)

Megalac

12.2

-

5.0

Grass hay

23.3

Soypass

24.3

22.3

-

Corn silage

76.2

SoyPreme

-

-

46.7

Beet Pulp

4.0

Flaxtech

-

23.8

-

Forages

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331

Table 4 - Nutrient contents of mixtures used in C and treatment groups (%).

Nutrient

Forage

Control

Omega-3 (n-3)

Omega-6 (n-6)

As-fed

DM

As-fed

DM

As-fed

DM

As-fed

DM

DM

41.00

100

90.60

100

93.65

100

92.12

100

OM

37.98

92.63

83.23

91.87

81.52

87.05

87.16

94.62

CP

2.33

5.70

22.54

24.88

23.80

25.41

24.02

26.07

EE

0.32

7.80

13.32

14.70

15.90

16.98

13.83

15.01

CF

16.25

39.63

5.84

6.45

3.61

3.85

6.27

6.80

NFE

19.08

46.53

41.53

45.84

38.21

40.81

43.04

46.74

Ash

3.02

7.37

7.37

8.13

12.13

12.95

4.96

5.38

ADF

20.70

50.39

13.75

15.18

8.79

9.40

11.81

12.82

NDF

30.20

73.56

22.17

24.47

15.07

16.09

21.12

22.93

ME, kcal/kg

1713

3630

3817

3632

DM: Dry matter, OM: Organic matter, CP: Crude protein, EE: Ether extracts, CF: Crude fibre, NFE: Nitrogen free extracts, A: Ash, ADF: Acid detergent fibre, NDF: Neutral detergent fibre, ME: Metabolisable energy.

2693). Tests were adjusted according to the sheep experiment as the standards used in these test kits were prepared in human sera. For this purpose, sheep blood plasma treated with active coal (Activated-charcoal stripped) was prepared. Standard curves were formed by adding progesterone or estradiol (SIGMA) in different ratios in this plasma from which steroid hormones were removed by using charcoal. The intra- and inter-assay coefficents of variation were 8.8%, 18.1% and 0.1 ng/ml for progesteron, and 9.6%, 14.4% and 10.0 pg/ml for estradiol, respectively. The stable metabolite of PGF2α13,14 dihydryo-15 keto prostaglandin F2α (PGFM) was measured by enzyme immunoassay using a kit (Cayman Chemical Company, USA). The intra- and inter-assay coefficents of variation for this metabolite were 9.6%, 15.4% and 7.8 pg/ml, respectively.

Table 5 - Fatty acid contents of forages and concentrates used in C, n-3 and n-6 groups (%).

C chain

Forage

Control

Omega-3 (n-3)

Omega-6 (n-6)

C14:0

1.49

-

0.57

0.64

C16:0

33.86

15.24

7.26

18.41

Statistical analysis GLM procedures of SPSS were used to evaluate the effects of treatment on parametric data with comparing them Duncan Multiple Range Test while non-parametric data regarding on degenerated and CL counts were tested by Khi Square (χ2) in the same software (Windows version of SPSS, release 10.0). All other variables (P4, E2 and PGFM) were analyzed using a linear mixed model (MIXED procedure) for repeated measurements. Permutation test, a nonparametric method which was not affected by suppositions, was used to evaluate the effects of n-3 and n-6 fatty acids due to the fact that variance analyse results were not dependable as the curves related to the ovulation rate, small and large follicule counts, ovarium and CL weights did not show normal distribution21 Advanced pairwise comparison permutation tests were used to investigate the source of variation among the averages. Data were presented as means ± standard error.

RESULTS Ovarium activity

C16:1 (n-7)

1.08

0.00

0.95

0.37

C18:0

6.02

1.83

1.29

4.55

C18:1 (n-9)

18.18

63.48

24.33

30.27

C18:2 (n-6)

15.04

18.03

19.95

35.21

C18:3 (n-3)

12.01

1.42

40.50

8.32

C18:4 (n-3)

-

-

0.69

0.26

C20:0

1.78

-

-

0.42

C20:1

2.88

-

-

0.50

C20:2

1.47

-

0.29

-

-

-

0.82

-

C20:5

1.34

-

1.16

0.53

C22:1 (n-9)

4.86

-

0.95

0.52

C20:3 (n-6)

C22:6 (n-3) n-6/n-3 ratio

-

-

1,24

0.64

1.25

12.69

0.49

3.81

The effects of short-term variation of diet n-6 and n-3 contents during pre- and post-mating periods on CL numbers, ovulation rates and weights of ovarium and CL in ewes were presented in Table 6. CL numbers formed following ovulation and degenerated during slaughter were found higher in C+C and n-6+C groups compared to those in n-6+n-3 and C+n-3 (P<0.05), indicating that the groups which were not fed n-3 diet had degenerated CLs. Ovarian weights were found higher in sheep fed n-6+n-3 diet compared to those fed C+n-3 diet (P<0.05). Short-term changes in n-6 and n-3 contents of diets during pre- and post-mating periods did not cause any differences in terms of CL weights among the experimental groups.

But, the the average weight of the CLs in n-6+n-3 group tend to be higher compared to the other groups (P>0.05).

Follicle numbers The effects of short-term variation of diet n-6 and n-3 contents during pre- and post-mating periods on follicule counts and sizes in sheep are presented in Table 7. Small follicule counts

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Table 6 - CL numbers, ovulation rates, ovarian weights and CL weights in sheep fed n-6 diets during pre-mating period and n-3 diets during post-mating periods (n=11).

Groups

CL number

Ovulation rate

Ovarian weights (g)

CL weights (g)

C+C

0.40b (4/10)

0.91

1.80±0.25ab

0.56±0.15

n-6+C

0.27b (3/11)

1.00

1.93±0.08ab

0.53±0.07

C+n-3

0.67a (6/9)

0.82

1.47±0.07b

0.56±0.06

n-6+n-3

0.73a (8/11)

1.00

2.02±0.19a

0.81±0.09

Table 7 - Small, large and total follicle numbers in sheep fed C and n-6 diets during pre-mating period and C and n-3 diets during postmating periods.

Treatment Groups

Small follicle numbers (1-3 mm)

Large follicle numbers (>3 mm)

Total follicle numbers

C+C

13.0±1.16a

4.27±0.68a

17.27±1.62a

n-6+C

8.27±1.49b

1.27±0.41b

9.55±1.67b

C+n-3

b

8.73±1.18

b

2.27±0.47

11.00±1.27b

n-6+n-3

9.0±1.38b

2.36±0.54ab

11.36±1.21b

a,b

a,b : Averages with different letters in the same column are statistically different (P<0.05).

: Averages with different letters in the same column are statistically different (P<0.05).

Figure 1 - Plasma progesterone concentrations in sheep fed basal diet (control) and n-6 diet (n-6) during pre-mating period (P<0.05).

were lower in ewes fed n-6+C, C+n-3 and n-6+n-3 diets compared to those in sheep fed C+C diet (P<0.05). Large follicle numbers were lower in ewes fed n-6+C and C+n-3 diets compared to those in sheep fed C+C diet (P<0.05). Total follicle numbers were found lower in sheep fed n-6+C, C+n-3 and n6+n-3 diets compared to those in sheep fed C+C diet (P<0.05).

Hormone levels Dietary n-6 treatment increased (P< 0.05) plasma progesterone

concentration level over the 9th, 12th and 15th days in ewes fed on n-6 diet during pre-mating period compared to control (7.31±0,64 vs 5.22±0.49). Plasma progesterone concentrations were 6.30 and 8.41 ng/ml in C+C and n-6+C groups at 12th day following mating and these values decreased to 5.03 and 7.11 ng/ml at 15th day (Figure 1). Plasma progesterone concentrations increased from 5.82 and 7.16 ng/ml at 12th day to 6.54 and 7.17 ng/ml at 15th day in C+n-3 and n-6+n-3 groups, respectively (Figure 2). Short-term variations in n-6 and n-3 contents

Figure 2 - Plasma progesterone concentrations during post-mating period in treatment groups.

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333

Figure 3 - Plasma estradiol concentrations of control goup C(____) and sheep fed n-6 during pre-mating and n-3 after mating period (------) (P> 0.05).

Figure 4 - Plasma PGFM concentrations in sheep fed C() and n-6 () diets during premating period (P<0.01).

of diets during pre- and post-mating periods did not cause any differences in terms of plasma progesterone concentrations among the experimental groups.

The effects of short-term variation of diet n-6 contents during pre- mating period on plasma estradiol content are presented in Figure 3. Plasma estradiol concentrations during mating period (-1, 0, +1) were found as 41.30±4.87 pg/ml and 43.72 pg/ml in ewes fed diets rich in n-6 and those fed C diet, respectively, during pre-mating period (Figure 3). Plasma estradiol concentrations in -1., 0. and +1 days were found as 43.16±7.59, 45.99±6.85 and 41.89±8.45 pg/ml in sheep fed C diet at pre-mating period and 44.07±9.04, 41.35±9.08 vs 38.76±7.72 pg/ml in sheep fed n-6 diet, respectively (Figure 3).

Short-term variations in n-6 contents of diets during pre-mating period did not cause any differences in terms of plasma estradiole concentrations among experimental groups.

Plasma PGFM concentrations in sheep fed n-6 diets during premating period were given in Figure 4. The plasma PGFM concentration in sheep fed n-6 diet during pre-mating period (656.854±73.44 pg/ml) was found higher compared to that in sheep fed C diet (252.15±35.91 pg/ml) (P<0.01) (Figure 4).

Basal and peak plasma PGFM concentrations were found as 176.71 and 523.47 pg/ml (P<0.01) and 679.58 vs 1487.71

(P<0.01) for sheep fed C diet and for those fed n-6 diet during pre-mating period (Figure 5).

DISCUSSION The results of the present study indicate that short-term (1517 days) changes in dietary n-6 and n-3 supplementation can have a beneficial effect on plazma hormon consentration and ovarian activity during pre-mating and post-maing, respectively, in ewes. These results support the idea that fats in diet can influence reproduction positively by altering both ovarian follicle and corpus luteum function via improved energy status and by increasing precursors for the synthesis of reproductive hormones such as steroids and prostaglandins2, 3,8,16. In the present study, the SoyPreme and Flaxtech as a source of protected n-6 and n-3 FA was chosen in order to maximize the proportion of n-3 PUFA and n-6 PUFA in plasma. It has been reported that the rumen-protected form as in the present study is sufficient to enable a significant increase in plasma between 7-30 days after feeding, even with the moderate amounts in the diet8. It is known that some scientific articles on use of the protected oil as a PUFA source in ruminant animal diets to enhance

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Effects of omega-3 and omega-6 fatty acids on some reproductive parameters in ewes

Figure 5 - Basal (ď Ż) and peak ( ) plasma PGFM concentrations in sheep fed C and n-6 diets during pre-mating period (A, B; a, b:P<0.01).

reproductive status contain also some data such as: blood fatty acid concentrations before and after insemination, non-fertilization or early embryo mortality on 17 d after insemination and body weight change during supplementation periods. Unfortunately, these physiological traits were not investigated in the present study. The aim of the present study was to determine dietary supplementation of n-6 and n-3 fatty acids whether enhance plasma hormone concentrations and ovarian activity rather than modify blood fatty acid concentrations of ewes.

To ensure a specific effect of n-3 FA and n-6 FA, a basal diet without oil, especially PUFA was chosen as the control, because among the PUFA, we have focused on the effect of short-term (15-17 days) changes in n-6 and n-3 FA supplementation.

Our findings did not support the idea that n-3 supplementation during post-mating period inhibit prostaglandin secretion and increase the progesterone secretion by stimulating the luteal and steoroidogenic activity. Kuran et al. (1999) reported that luteal cells produced higher amounts of progesterone in in vitro conditions in ewes fed diets supplemented with palmitic acid protected with Ca soaps22. Thatcher et al. (1995) reported that high linoleic acid diets stimulated progesterone synthesis due to their effects on the luteal cells in corpus luteum23. Cholesterol side chain cleavage, the first step in progesterone synthesis, was unchanged by n-3 supplementation24. Thus, the lacking effect of n-3 fatty acids on progesterone synthesis can be explained by this mechanism.

The differences among the studies might be caused by the experimental design, animal species, fatty acid amount and source (fish oil vs linseed oil), n-3:n-6 ratio or n-3/other fatty acids (n-7, n-9 etc.) ratio, the protection types of fatty acids from biohydrogenation in the rumen (formaldehit treatment vs Ca-soap treatment). Indeed, progesterone synthesis decreased when the luteal cells incubated with n-3 fatty acids in cows25 and plasma progesterone synthesis decreased in cows fed diets rich in n-3 fatty acids6,12,26,27. Moussavi et al. (2007) reported that protected fish oil, a source of n-3, led to an increase in n-3 fatty acid amount in uterus endometrium and consequently a decrease in n-6/n-3 ratio28. Moreover, there are some studies indicating that progesterone concentrations were not affected following the incubation of cow endometrium cells with different n-3 sources29. Therefore, results of our study are in accordance with previous works.

The results of the present study on the plasma PGF2Îą concentration of ewes are conflicting literature related to the effects of n-6 fatty acid supplementation on blood PGFM concentrations11,30. Indeed, Chassagne and Bornouin (1992) reported that linoleic acid/linolenic acid ratio in diet had significant effect on reproduction functions and that decrease in this ratio lowered the prostaglandine synthesis and activity31. Linoleic and arachidonic acids are considered as limiting precursors in the PGF2Îą synthesis14,32. In the same study, an increase in PGF2 synthesis was found in placental tissues and uterus endometrium in ewes fed high linoleic acid ratio. Higher PGF2 concentrations in ewes fed high linoleic acid compared to the control group might be attributed to the fact that n-6 fatty acids increased arachidonic acid concentrations in blood circulation.

Mattos et al. (2000) and Robinson et al. (2002) found similar results in their studies in which SoyPreme was used in cattle6,33.

The results on plasma estradiol concentration are agreed with suggestions of Lammoglia et al. (1997) in cows and of Elmes et al. (2005) in ewes14,34. Conversely, Robinson et al. (2002) reported that n-6 supplementation in cows had no effect on plama estradiol concentration6. Because of the conflicts between studies there is no precise judgment related to the mechanism of the effects of n-6 supplementation on plasma estradiol concentration.

Our results on small and large follicle numbers and follicle sizes show that treatments aimed at increasing the amount and qualities of animal products by changing the n-6 and n-3 fatty acid amounts and ratios might negatively affect follicle growth. Lower large follicle numbers in ewes fed n-3 diet compared to those in ewes fed control diet might be attributed to the fact that n3 fatty acids suppress follicle growth by stimulating the negative feedback mechanism between GnRH and FSH due to their (n-3 fatty acids) increasing effects on luteal activity of corpus luteum. This stuation may be explained the results with regard to plasma progesterone level and active corpus luteum numbers of the present study.

Negative effects of both fatty acids on small and large follicle numbers and sizes might be caused by the amount of fatty acids and ratios between these fatty acids (n-3:n-6). Stanko et al.

(1997) reported that vegetable oil supplementation below 4% of diet dry matter led to maximum follicular growth35. Chassagne and Bornouin (1992) reported that linoleic acid/linolenic

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Ercan Soydan et al.; Large Animal Review 2020; 26: 329-336

acid (n-6:n-3) had significant effect on reproduction functions31.

In this study, total fat amount is at the level of 5.75% of diet DM. The findings of the present study on follicler numbers and sizes and ovulation rate are inconsistent with those obtained in many studies6,36,37. This inconsisteny might be caused by the differences in experimental designs, physiological phase, fatty acid amounts and sources and/or diet’s linoleic acid/linolenic acid contents.

The results on CL numbers may be related to the contributing effect of n-3 supplementation during post-mating period on the viability of the corpus luteum. n-3 fatty acids achieve this contribution by inhibiting the PGF2α synthesis in the uterus endometrium. n-3 supplementation decreased or suppressed PGF2α secretion by decreasing COX-2 proteins38, by changing n-3/n-6 ratio in uretus endometrium28 and/or by decreasing the arachidonic acid synthesis30. Mattos et al. (2000) reported that n-3 fatty acids inhibited PGF2� synthesis in uterus endometrium cells33. n-3 fatty acids might have stimulated luteal cells in the corpus luteum to secrete higher amounts of progesterone. Consequently, the presence of a strong feedback mechanism between progesterone and PGF2� might have affected CL activity and viability positively.

Early embryonic deaths are among the most significant factors which limit the optimum reproduction performance in livestock. The incidence of embrionic losses were 30-40% in the first 3 weeks of pregnancy in ewes and 70-80% of these are between 8th and 16th days. Moreover, it was suggested that most of the early embrionic losses occurred due to the luteal cells’ insufficient functions and fertility can be increased 20% by stimulating the luteal activity39. Cam and Kuran (2004) and Cam et al. (2004) claimed that progesterone levels can be increased by enhancing luteal activity via hormonal applications and consequently embrionic deaths can be diminished39,40. Degenerated CL counts during post-mating period is one of the most important indicators of embrionic deaths. Thus, lower degenerated CL counts in groups fed n-3 diets during post-mating period supports the findings that blood progesterone synthesis increases15,41-43 and consequently incidence of early embrionic deaths decreases39. In present study, while plasma progesterone concentrations began to diminish from the 12th day after mating in ewes fed control diet, it progressively increased in ewes fed n-3 diet. Our results thus indicate that n-3 fatty acid supplementation during post-mating period may affect the viability of CL and so may diminish the incidence of early embrionic losses.

Our results on the plasma progesterone, PGFM and estradiol concentrations showed that short-term variations of fatty acid composition during pre-mating and post-mating period decreased both small and large follicle numbers, increased the CL counts and did not affect the ovulation rate. It can be said that n-3 supplementation during post-mating period might decrease the incidence of embrionic losses due to its positive influence on CL numbers. These results indicate that fatty acid contents of diet at mating are important especially in terms of enhancing the pregnancy rate.

In conclusion, short-term (15-17 days) changes in dietary n6 and n-3 supplementation can have a beneficial effect on plasma hormon concentration and ovarian activity during pre-mating and post-maing, respectively, in ewes.

Conflict of Interest None of the authors have any conflict of interest to declare.

335

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16. Ghaffarilaleh V., Fouladi-Nashta A., Paramio M.T. (2014). Effect of αlinolenic acid on oocyte maturation and embryo development of prepubertal sheep oocytes. Theriogenology, 82: 686-696.

17. Geay Y., Bauchart D., Hocovette J. F., Culioli J. (2001). Effect of nutritional factors on biochemical, structural and metabolic characteristics of muscles in ruminants, consoquences on dietetic value and sensorial qualities of meat. Reprod Nutr Dev, 41: 1-26.

18. Garipoglu A.V., Ocak N. (2003). Ruminant etlerinin yağ asitleri içerikleri üzerine beslemenin etkileri. 2. Ulusal Hayvan Besleme Kongresi, KonyaTürkiye, pp.263-266.

19. Ocak N., Cam M.A., Kuran M. (2006). The influence of pre-and post-mating protein supplementation on reproductive performance in ewes maintained on rangeland. Small Ruminant Res, 64: 16-21.

20. NRC (1985) Nutrient requirements of sheep. 6th Edition. National Re-

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22. Kuran M., Onal A. G., Robinson J. J., Mackie K., Speake B. K., McEvoy T. G. (1999). A dietary supplement of calcium soaps of fatty acids enhances luteal function in sheep. J Anim Sci 69: 385-393.

23. Thatcher W.W., Meyer M.D., Danet-Desnoyers G. (1995). Maternal recognition of pregnancy. J Reprod Fertil Supplement, 49: 15-28.

24. Chin E.C., Naddafy J.M., Cheng Z., Brickell J. S., Wathes D.C., Abayasekara D.R.E. (2006). Endocrine Abstract, 11, 750.

25. Hinckley T., Clark R.M., Bushmich S.L., Milvae R.A. (1996). Long chain polyunsaturated fatty acids and bovine luteal cell function. Biol Reprod, 55: 445-449.

26. Petit H.V., Dewhurst R.J., Proulx J.G., Khalid M., Haresign W. (2001). Twagiramungu H. Milk production, milk composition, and reproductive function of dairy cows fed different fats. Can J Anim Sci, 81: 263-271.

27. Petit, H.V. (2002). Digestion, milk production, milk composition, and blood composition of dairy cows fed whole flaxseed. J Dairy Sci, 85: 1482-1490.

28. Moussavi A.R., Gilbert R.O., Overton T. R., Bauman D.E., Butler W.R. (2007). Effects of feeding fish meal and n-3 fatty acids on ovarian and uterine responses in early lactating dairy cows. J Dairy Sci, 90: 145-154.

29. Palin M.F., Brochu-Gaudreau K., Beaudry D., Small J., Petit H.V. (2005).

Effects of feeding flaxseed on cyclooxygenase 2 (Cox-2) and peroxisome proliferator-activated receptors (PPAR) delta and gamma mRNA levels at the time of maternal recognition of pregnancy in Holstein cows. Biol Reprod, SI: 142-142.

30. Cheng Z., Elmes M., Kirkup S.E., Abayasekara D.R., Wathes D.C. (2004).

Altediet of prostaglandin production and agonist responsiveness by n6 polyunsaturated fatty acids in endometrial cells from late-gestation ewes.

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31. Chassagne M., Bornouin J. (1992). Circulating PGF2α and nutritional parameters at parturition in dairy cows with and without retained placenta: relation to prepartum diet. Theriogenology, 38: 407-418.

32. Elmes M., Tew P., Cheng Z., Kirkup S.E., Abayasekara D.R.E, Calder P. C., Hanson M. A., Wathes D.C., Burdge W.G. (2004). The effect of dietary supplementation with linoleic acid to late gestation ewes on the fatty acid

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Lammoglia M. A., Willard S. T., Hallford D. M., Randel R. D. (1997). Effects of dietary fat on follicular development and circulating concentrations of lipids and insulin on follicular development and circulating concentrations of lipids, insulin, progesterone, estradiol 17b, 13,14-dihydro15-keto-prostaglandin F2a and growth hormone in estrous cyclic Brahman cows. J Anim Sci, 75: 1591-1600.

Stanko R.L., Fajersson P., Corver L.A., Williams G.L. (1997). Follicular growth and metabolic changes in beef heifers fed incremental amounts of polyunsaturated fat. J Anim Sci 75: 223.

Ryan D.P., Spoon R.A., Williams G.L. (1992). Ovarian follicular characteristics, embryo recovery and embryo viability in heifers fed high-fat diets and treated with FSH. J Anim Sci, 70: 3505-3511.

Abayasekara D.R.E., Wathes D.C. (1999). Effects of altering dietary fatty acid composition on prostaglandin synthesis and fertility. Prostaglandins Leukot Essent Fatty Acids, 61: 275-287.

Naddafy J.M., Chin E.C., Brickell J.S., Cheng Z., Wathes D.C., Abayasekara D.R. (2006). Endocrine Abstract, 12: 101.

Cam M.A., Kuran M., Selcuk E. (2004). GnRH uygulamasının koyunlarda plazma progesteron konsantrasyonu ve döl verimine etkisi. Turk J Vet Anim Sci, 28: 1065-1070.

Cam M.A., Kuran M. (2004). Effects of single injection of hCG or GnRH on day 12 post-mating on fetal growth and reproductive performance of sheep. Anim Reprod Sci, 80: 81-90.

Mann G.E., Lamming G. E. (2001). Relationship between maternal endocrine environment, early embryo development and inhibition of the luteolytic mechanism in cows. Reproduction, 121: 175-180.

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E. Giudice et al. Large Animal Review 2020; 26: 337-340

Antibacterial and repellent activities of Hypericum perfoliatum (St. John’s Wort) on different bacterial strains and anatomical tissues of Ovine and Bovine species

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ELISABETTA GIUDICEa*, CLAUDIA GIANNETTOa, CHIARA CRINÒa, GAETANO SALERNOb, FRANCESCA ARFUSOa, MARIA RIZZOa, FILIPPO GIARRATANAa, ANNASTELLA FALCONEa, SIMONA DI PIETROa a

Department of Veterinary Sciences, University of Messina, Polo Universitario SS. Annunziata 98168, Messina, Italy b Freelance Veterinary Professional, Italy

SUMMARY St. John’s Wort (Hypericum perfoliatum, SJW) is an herbaceous medical plant that grows abundantly and spontaneously in Mediterranean area. Since ancient times, SJW has been used to treat different kind of mental and physical diseases, and for its antiseptic, antinflammatory and antibacterial properties. In this study, we investigated the antibacterial and repellent activities of Hypericum perfoliatum oil on different animal samples in vitro and ex vivo. The flowering aerial parts of Hypericum perfoliatum were obtained from a cultivation Mediterranean area in Italy; fresh flowering aerial parts of Hypericum perfoliatum were left for maceration in a transparent jar inside extra virgin olive oil under daylight. The oily part was then removed by filtering and placed in dark brown jars with hermetic plastic stoppers. The inhibitory activity was evaluated on six different bacterial strains: 2 strains of Staphylococcus aureus, 2 strains of Listeria monocytogenes, 1 strain of Salmonella tiphymurium and 1 strain of Escherichia coli O157:H7. Agar gel diffusion technique was used with four different concentrations of the compound (5, 10, 15 and 20 µL). As a control, a sterile disc, not soaked with the oil, was placed on each plate. The effect of the oil against the selected strains was evaluated by measuring the halo of inhibition around the discs soaked with Hypericum perfoliatum oil. To establish the repellent activity, different animal samples were brushed once with the oil and kept outdoors under natural environmental conditions for one week. Only Staphylococcus aureus was susceptible to Hypericum perfoliatum oil, even at the lowest concentration used. For the other bacterial strains no inhibition was observed. A single application of the oil was effective against different species of insects, unlike untreated samples. The results achieved in this study confirm that Hypericum perfoliatum possesses antibacterial properties only against Gram positive bacteria and a strong repellent activity against insects.

KEY WORDS Antibacterial activity, Hypericum perfoliatum, repellent activity, St. John’s wort, ovis aries, bos taurus.

INTRODUCTION St. John’s Wort (SJW) is an herbaceous medical perennial plant, belonging to the family of Hypericaceae, genus Hypericum (APG III classification)1; this common denomination is used both for Hypericum perforatum (H. perforatum) and Hypericum perfoliatum (H. perfoliatum). SJW grows on dry lands, as high as 1200-1600 meters above sea level, in almost all of Europe, in North Africa, West Asia and many other parts of the world. The flowering season is between May and August.

Since ancient times, SJW has been used to treat mild to severe forms of depression, anxiety and in psychiatric illness in general2, as a topical remedy for skin wounds, abrasions and burns, and for its antiseptic3, antiviral, antitumor, antiangiogenic, antinflammatory4 and antibacterial properties. The most common side effects reported after using SJW are photosensitivity5 and chemical interaction with some drugs6. Even if sever-

Corresponding Author: Elisabetta Giudice (egiudice@unime.it)

al constituents of the aerial part have been recognized as therapeutic in these plants7 the greatest medical and therapeutic activity is manly comprised in two substances: hyperforin and hypericin. Hyperforin is the major lipophilic constituent, even if it is unstable and susceptible to oxidative degradation. It seems to play the greatest role in the antidepressant effect of SJW2.

However, it also shows anti-inflammatory, anticarginocenic8 and antimicrobial effects.

Hypericin is a lipophilic and hydrophobic naphthodianthrone, which is known to be a non specific protein kinase inhibitor, nontoxic and nonmutagenic, with antiviral, antineoplastic9, antidepressant10 and antimicrobial effects.

Furthermore, six Hypericum essential oils, in particular the monoterpene and sequiterpene fractions, also showed antimicrobial effects against Gram-positive Bacillus subtilis (B. subtilis) and Staphilococcus aureus (S. aureus)11. No notable activity against Gram-positive Enterococcus fecalis (E. fecalis) and Gram-negative Escherichia coli (E. coli) was detected11. In other studies, the antibacterial, against both Gram-positive and Gram-negative bacteria, and antifungal activities have been described3,12-13. SJW has also shown antibacterial activity against different strains of mycobacteria14 and Methicillin-Resistant

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Antibacterial and repellent activities of Hypericum perfoliatum (St. John’s Wort)

Staphylococcus aureus (MRSA)15. The different effectiveness against Gram positive and Gram negative bacteria could depend on the different structure of cell walls; the lack of an outer lipopolysaccharide membrane in the Gram-positive bacteria may allow increased permeability of Hypericum metabolites into cells16. Moreover, the alcoholic extracts were found to be more active than aqueous solutions3.

Regarding Hypericum perfoliatum, a recent study17 reported a high content in polyphenols (gallic, chologenic and caffeic acid, rutin, hyperoside and quercitin) and antimicrobial activity against S. aureus, Pseudomonas aeruginosa (P. aeruginosa), Bacillus cereus (B. cereus), E. coli and Chronobacter sakazakii (C. sakazakii) of chloroform and chloroform-methanol extracts.

Although the antibacterial activity against several bacterial pathogens of SJW is well known, few studies have been carried out on the antibacterial properties of Hypericum perfoliatum1719 and, to the authors’ knowledge, no scientific work exists on its repellent activity against insects in animal biological samples.

In view of such considerations, the aim of this study was to evaluate the in vitro antibacterial properties of a phytotherapeutic product from Hypericum perfoliatum against the most common agents, and the repellent activity against insects on different anatomical tissues of Ovine and Bovine species.

MATERIALS AND METHODS Experimental material and preparation of SJW oil The flowering aerial parts of Hypericum perfoliatum were obtained from a cultivation Mediterranean area of Sicily, Italy (37°43’N, 13°26’E) and the crude oily extract was prepared according to an ancient Sicilian recipe. Fresh flowering aerial parts of Hypericum perfoliatum (300 g) were left for maceration for 40 days in a transparent jar inside extra virgin olive oil (1 litre) (Biancolilla-Nocellara, Sicily) under daylight. The oily part was then removed by filtering through sterile gauzes to obtain a clear filtrate and placed in dark brown jars with hermetic plastic stoppers, kept in a dark place20.

In vitro antibacterial activity In vitro antibacterial activity was investigated by evaluating the inhibitory activity on different bacterial strains. 2 strains of Staphylococcus aureus (S. aureus), 2 strains of Listeria monocytogenes (L. monocytogenes), 1 strains of Salmonella tiphymurium (S. tiphymurium) and 1 strains of Escherichia coli (E. coli) O157:H7 were chosen. All the strains had been previously isolated from various food products. The strains were enriched in Brain Heart Infusion (BHI) (Oxoid Ltd., Basinkstoke, Hampshire, England) broth and incubated at 37° C for 24 hours.

Each culture was adjusted in order to reach an optical density of 0.5 McFarland.

Agar gel diffusion technique was used to assess the effectiveness of SJW against the selected strains. Four different concentrations of the compound (5, 10, 15 and 20 µL) were tested. Muller Hinton Agar plates (MHA) (Oxoid Ltd., Basinkstoke, Hampshire, England) were inoculated with each strain suspension in BHI broth and sterile discs (Biolife, Milan, Italy), soaked with SJW oil were placed on the surface of MHA. Plates were then incubated at 37° C for 24 h. As a control, a sterile disc, not soaked with the oil, was placed on each plate.

The inhibitory activity was evaluated by measuring the halo of inhibition around the discs.

Repellent activity against insects To establish the repellent activity of the same ointment, anatomical tissue (head, fetlock, muscle, testicle and mammary gland) of various species (ovine, bovine) were brushed once with the oil and, then, kept outdoors (sunrise at 04.33, sunset at 19.19 over the study period) uncovered, at natural environmental conditions. Thermal and hygrometric records were carried out for the whole study by means of a data logger (Gemini, UK), and they followed the normal seasonal pattern for the place (minimum and maximum mean temperature between 20.3° C and 27.3° C, mean relative humidity of 59.3%). Other samples, similar in dimension, without Hypericum perfoliatum oil treatment, were kept in the same condition. Samples were checked for one week, every hour during the first day and then three times a day.

RESULTS In vitro antibacterial activity The two strains of S. aureus were susceptible to Hypericum perfoliatum oil, with halos of inhibition from 3 mm, with the lowest concentration (5 µL), to 6 mm, with the highest concentration (20 µL) used. However, for the other strains evaluated in the study, no halo of inhibition was detected even with the highest concentration of the oil. In each plate, no halo of inhibition was found around the control disc (Figure 1).

Repellent activity against insects A single application of the oil was effective in repelling various species of insects (Table 1). During the observation period, no insects had landed on the surface of the treated animal biological samples. On the other hand, the untreated samples were strongly targeted by various species of insects (flies, horseflies, bees, wasps and ants). Furthermore, it should be mentioned that the treated meat gradually dried out, forming a superficial crust, without signs of putrefaction, which however appeared in the untreated samples, from the second day of observation (Figure 2).

Table 1 - Visual inspection of the Diptera (flies and horseflies) presence on the anatomical tissue of Ovine and Bovine species, 8 hours after treated with Hypericum perfoliatum oil and without treatment.

Species Anatomical tissue

Ovine

Bovine

Number of Diptera (flies and horseflies) Treated with oil No treated with oil

Head

Absence

10

Fetlock

Absence

15

Muscle

Absence

7

Testis

Absence

5

Mammary gland

Absence

6

Head

Absence

12

Fetlock

Absence

9

Muscle

Absence

14

Testis

Absence

6

Mammary gland

Absence

9

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Figure 1 - Inhibitory action of the alcoholic extract of Hypericum perfoliatum on crops of two strains of Staphylococcus aureus (A and B): inhibition halo between 12 and 18 mm. Four different concentrations of the compound (5, 10, 15 and 20 ÂľL) were tested.

DISCUSSION The results obtained in the present study show that Hypericum perfoliatum possesses antibacterial properties against Gram positive bacteria (S. aureus), whereas no oil activity against Gram negative (S. typhimurium and E. coli O157:H7) and L. monocytogenes was observed. The results obtained are in line with previous studies on Hypericum perfoliatum antimicrobial activity17,19 regarding S. aureus. However, Del Monte et al.17 also demonstrated Hypericum perfoliatum antibacterial activity against E. coli. This could be connected to the different type of extract used or to a different sensitivity of E. coli O157:H7 compared to the other strains.

Considering that Staphylococcus spp. is one of the most common cutaneous bacteria and often responsible for skin infections, this could explain the effectiveness of SJW ointment in wound healing21-24. Furthermore, the demonstrated and long lasting repellent activity against insects in general, and especially against Diptera (flies and horseflies), could represent a useful and cheap aid for skin lesion management, especially in

A

animals living outdoors, to prevent the onset of myasis and to reduce the risk of Summer Seasonal Recurrent Dermatitis (SSRD, sweet itch) in horses.

Although the literature reports that the stability of SJW oil should last for an indefinite period, our product showed a reduction in effectiveness after one year, compared to fresh oil.

It is advisable, therefore, to renew it annually to increase the effectiveness of the medication.

Considering the small number of bacterial strains tested in this study, further scientific work should be carried out on a larger and more varied number of bacterial species. Moreover, further evaluations should be made to evaluate the antibacterial activity of Hypericum perfoliatum oil in vivo skin infections caused by Gram positive bacteria, and to demonstrate the absence of interference with anti-doping investigation in sport horses and of those residues in food production animals that could be dangerous for human health. Finally, more studies should be carried out in order to establish the possible toxic effect of these extracts.

B

Figure 2 - Representative example of anatomical tissue (ovine mammary gland) 8 hours after treated with Hypericum perfoliatum oil (A) and without treatment (B). Red arrows indicate Diptera (flies and horseflies).

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Antibacterial and repellent activities of Hypericum perfoliatum (St. John’s Wort)

CONCLUSIONS The use of SJW in veterinary practice should be better studied. Its extraction technique is economical and easy to perform and the extracted product shows a remarkable stability over time. For this reason, it could be a good remedy for use in biological farming, with a positive effect in terms of environmental impact and drug resistance. Our results improve the knowledge on the effects of Hypericum perfoliatum extract, in contrast to the more well-known Hypericum perforatum25-26, present in some commercial products. The findings of this study seem to suggest that SJW could be a good antibacterial and repellent remedy. In in vivo infections, it could be used to make treatment of infection easier, cheaper and less dangerous for human and animal health.

CONFLICT OF INTEREST The authors declare that there is no conflict of interests regarding the publication of this article.

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2. Caccia S., Gobbi M. (2009) St’s John Wort components and brain: uptake, concentrations reached and the mechanisms underlying pharmacological effects. Cur. Drug Metab, 10: 1055-1065.

3. Saddiqe Z., Naeem I., Maimoona A. (2010) A review of the antibacterial activity of Hypericum perforatum L. J. Ethnopharmacol, 131: 511521.

4. Meinke M.C., Schanzer S., Haag S.F., Casetti F., Muller M.L., Wofle U., Kleemann A., Lademann J., Schempp C.M. (2012). In vivo photoprotective and anti-inflammatory effect of hyperforin is associated with high antioxidant activity in vitro and ex vivo. Eur J Pharm Biopharm, 81: 346-350.

5. Onoue S., Seto Y., Ochi M., Inoue R., Ito H., Hatano T., Yamada S. (2011) In vitro photochemical and phototoxicological characterization of major constituents in St. John’s Wort (Hypericum perforatum) extracts. Phytochemistry, 72: 1814-1820.

6. Fukunaga K., Orito K. (2012) Time-course of St John’s Wort on the pharmacokinetics of cyclosporine in dogs: interactions between herbal extracts and drugs. J Vet Pharmacol Ther, 35: 446-451.

7. Khan A.U., Gilani A.H., Rehman N.U. (2011) Pharmacological studies on Hypericum perforatum fractions and constituents. Pharm Biol, 49: 46-56.

8. Koeberle A., Rossi A., Bauer J., Dehm F., Verotta L., Northoff H., Sautebin L., Werz O. (2011) Hyperforin, an anti-inflammatory constituent from St. John’s Wort, inhibits microsomal prostaglandin E2 synthase-1 and suppresses prostaglandin E2 formation in vivo. Front Pharmacol, 2: 1-10.

9. Lu W.D., Atkins W.M. (2004). A novel antioxidant role for ligand behavior of glutathione S-transferases: attention of the photodynamic effects of hypericin. Biochemistry-US. 43, 12761-12769.

10. Wang Y., Shi X., Qi Z. (2010). Hypericin prolongs action potential duration in hippocampal neurons by acting on K+ channels. Br J Pharmacol, 159: 1402-1407.

11. Maggi F., Cecchini C., Cresci A., Coman M.M., Tirillini B., Sagratini G., Papa F., Vittori S., (2010) Chemical Composition and Antimicrobial Activity of the Essential Oils from Several Hypericum Taxa (Guttiferae) Growing in Central Italy (Appennino Umbro-Marchigiano). Chem Biodivers, 7: 447-466.

12. Kizil G., Toker Z., Özen H.Ç., Aytekin Ç. (2004) The Antimicrobial Activity of Essential Oils of Hypericum scabrum, Hypericum scabroides and Hypericum triquetrifolium. Phytother Res, 18: 339-341.

13. Sarkisian S.A., Janssen M.J., Matta H., Henry G.E., LaPlante K.L., Rowley D.C. (2012) Inhibition of Bacterial Growth and Biofilm Production

by Constituents from Hypericum spp. Phytother Res, 26: 1012-1016.

14. Mortensen T., Shen S., Shen F.A., Walsh M.K., Sims R.C., Miller C.D. (2012) Investigating the effectiveness of St John’s Wort Herb as an Antimicrobial Agent against Mycobacteria. Phytother Res, 26: 1327-1333.

15. Zuo G.Y., An J., Han J., Zhang Y.L., Wang G.C., Hao X.Y., Bian Z.Q. (2012) Isojacareubin from the Chinese Herb Hypericum japonicum: Potent Antibacterial and Synergistic Effects on Clinical Methicillin-Resistant Staphylococcus aureus (MRSA). Int J Mol Sci, 13: 8210-8218.

16. Tian F., Li B., Ji B., Yang J., Zhang G., Chen Y., Luo Y. (2009). Antioxidant and antimicrobial activities of consecutive extracts from Galla chinensis: The polarity affects the bioactivities. Food Chem 11: 173-179.

17. Del Monte D., De Martino L., Marandino A., Fratianni F., Nazzaro F., De Feo V. (2015) Phenolic content, antimicrobial and antioxidant activities of Hypericum perfoliatum L. Ind Corps Prod, 74: 342-347.

18. Benkiki N., Kabouche Z., Tillequin F., Vérité P., Chosson E., Seguin E. (2003). A new polyisoprenylated phloroglucinol derivative from Hypericum perfoliatum (Clusiaceae). Z. Naturforsch. C, 58: 655-658.

19. Nogueira T., Medeiros M.A., Marcelo-Curto M.J., Garcia-Perez B.E., Luna-Herrera J., Costa M.C. (2013). Profile of antimicrobial potential of fifteen Hypericum species from Portugal. Ind Crops Prod, 47: 126131.

20. Campanini E. (2012) Dizionario di fitoterapia e piante medicinali, third ed. Tecniche Nuove, Milano.

21. Pris caru A.I., Andri oiu C.V., Andriescu C., H vârneanu E.C., Popa M., Motoc A.G.M., Sava A. (2013). Evaluation of the wound-healing effect of a novel Hypericum perforatum ointment in skin injury. Rom. J Morphol Embryol, 54: 1053-1059.

22. Sayar H., Gergerlioglu N., Seringec N., Ozturk P., Bulbuloglu E., Karaba G. (2014) Comparison of efficacy of topical phenytoin with hypericin in second-degree burn wound healing: An experimental study in rats. Med Sci Monit Basic Res, 20: 36-46.

23. Suntar I.P., Akkol E.K., Yilmazer D., Baykal T., Kirmizibekmez H., Alper M., Yesilada E. (2010) Investigations on the in vivo wound healing potential of Hypericum perforatum L. J. Ethnopharmacol, 127: 468-477.

24. Giudice E., Crinò C., Salerno G., Rizzo M., Levanti M, Di Pietro S. (2017). Evaluation of wound healing activity of St. John’s Wort (Hypericum perfoliatum) in horses. Comp Clin Pathol; 26: 611-615.

25. Galeotti N. (2017) Hypericum perforatum (St John’s wort) beyond depression: A therapeutic perspective for pain conditions. J Ethnopharmacol; 200: 136-146.

26. Yücel A., Kan Y., Yesilada E., Akın O. (2017) Effect of St. John’s wort (Hypericum perforatum) oily extract for the care and treatment of pressure sores; a case report. J Ethnopharmacol, 196: 236-241.

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Merve Isil Sen Mutlu, Arda Yildirim; Large Animal Review 2020; 26: 341-348

Effect of dietary supplementation of Panax ginseng leaf extract on production performance and egg quality of hens at the beginning of their laying period

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MERVE ISIL SEN MUTLU, ARDA YILDIRIM* Department of Animal Science, Faculty of Agriculture, University of Tokat Gaziosmanpasa, Tokat, Turkey

SUMMARY A study was conducted to determine the effect of ginseng (Panax ginseng C.A. Meyer) leaf on egg production and egg quality characteristics of hens at the beginning of their laying period. Eighty commercial Atak-S brown layers at the age of 20 weeks were randomly allocated to one of four treatments with four replicates of five hens per treatment in a completely randomized design. The birds were fed standard layer diets control (0 mg/kg), 50 mg/kg, 100 mg/kg or 150 mg/kg Panax ginseng leaf extract (PGLE) for 12 weeks period. Laying performance was assessed by recording egg production, egg weight daily; feed intake and feed efficiency weekly and egg quality biweekly. Statistical analysis of the research results proved influence of PGLE supplementation on the weight of eggs (P <0.05), but they did not have any influence on body weight, feed intake and feed efficiency (P >0.05). Overall, there were no differences among groups in external egg quality parameters (P >0.05), while significant increases were observed in albumen and yolk index of internal egg quality. The trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant power (FRAP) and total phenolics concentration of PGLE were 606.3±0.948 mmol trolox/kg, 15.99±0.232 mmol TEAC/kg and 1.11±0.02 g gallic acid equivalents (GAE)/kg, respectively. As a result of research, it could be considered that up to 150 mg PGLE/kg in a laying hen’s diet did not change the egg production performance of layers and there is only a positive effect on egg weight and eggshell redness increase which could contribute to profitability.

KEY WORDS Atak-S, egg production, egg quality, laying hen, Panax ginseng.

INTRODUCTION As a result of the prohibition of the use of antibiotics, a major problem has arisen in the egg industry in order to achieve effective production from healthy flocks such as improving the growth, feed utilization in poultry and reducing mortality1.

Thus, alternative feed additives can be added to rations to improve poultry health, production performance and egg quality2,3. For this purpose, feed additives, which are used to increase yield or support animal health, should not have negative effects on both animal and consumer health. Indeed, the most suitable additives are natural additives derived from aromatic plants.

Thus, herbal extracts, mixtures and essential oils are considered as alternative natural products and studies have been investigated on animal product performance and quality4,5.

There are many bioactive compounds in the structures of plants.

Essential oils derived from medicinal and aromatic plants give characteristics to plants which have antioxidant, antimicrobial, digestive and appetite enhancing properties. Their use in poultry compound feeds has recently become widespread due to their positive effects on performance in various animal species.

*Corresponding Author: Arda Yildirim (arda.yildirim@gop.edu.tr).

But in practical terms, the use of herbal extracts is very limited in the feeding of modern-day chickens. There are many medicinal and aromatic plants and herbal extracts that may be an alternative to antibiotics as a natural growth promoter and one of them is the ginseng plant and its extract6,7. Ginseng has been used in China, Korea and Japan for about 2000 years and its usage area has spread all over the world in recent years. It is a plant belonging to the family of Araliaceae8,9. Saponin glycosides (ginsenosides) are the most important phytochemicals of ginseng and more than 60 ginsenosides were isolated in various ginseng species. Apart from these, essential oils, sterols, flavonoids, polysaccharides, polyacetylenes, vitamins (B and D group), enzymes and minerals are other active substances isolated10,11. The pharmacological effects of ginseng have been reported in the central nervous, endocrine, immune and cardiovascular system12,13. In addition, Panax ginseng is widely used as a physical, chemical and biological resistance enhancer14, regulating blood lipid and decreasing blood sugar levels15, cancer prevention16, liver, kidney and heart protection and immunostimulatory effects17.

Digestion in poultry is completed in a shorter period due to the high metabolic activity and short passage times of nutrients from the digestive system. Thus, ginseng may have many advantages, including the bioactive compounds contained in it, which could improve digestion effects in poultry. Panax gin-

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seng extract could be used as a natural feed additive in poultry feed due to the phenolic compounds contained in it because of its strong antioxidant, anti-radical, antimicrobial, mineral and high vitamin content7.

There are many researches about botanical, chemical, biological, ecological characteristics and cultivation of ginseng in the world with its usability in animal husbandry, food and pharmaceutical industry. However, there are not enough studies about the use of Panax ginseng leaf extract in the diets of laying hens. The present research was conducted to determine the effects of supplementing graded levels of a commercial PGLE on egg production performance, egg quality and some blood serum biochemical parameters in laying hens at 20 weeks of age.

MATERIALS AND METHODS Ethics statement and Location This study was approved by the Animal Experimentation Ethics Committee (Process no. 309/2011 HADYEK-041) of Tokat Gaziosmanpa a University. Animals in this experiment were cared under the guidelines stated in the Guide for the Care and Use of Atak-S brown hybrid laying hens in the Poultry Research Institute, Ankara, Turkey. The study was carried out at the research and application farm poultry unit of Tokat Gaziosmanpa a University, Turkey, situated at 40°19’51.95” N, 36°28’31.23” E and 856 m above sea level.

Animals and housing conditions The eighty commercial Atak-S brown (Turkish native hybrid) layers at the age of 20 weeks were randomly allocated to one of four treatments with four replicates of five hens in each group.

The current experiment was conducted with completely randomized design. The five birds per replicate were kept in a wire cages (450 cm2 per bird) in a windowed poultry house at a light regimen of 16 h light and 8 h dark. Prior to the experiment, the birds were given a two week adaptation period before the trial began and the trial lasted 12 weeks. The treatments were

a basal diet as control and three levels of supplementation, 50, 100 and 150 mg/kg diet of a commercial PGLE (Batch No: GRE110508, Dried, 100% Natural), supplied by Changsha Herbway Biotech Co. Ltd. (China). The PGLE contained 80.7% ginsenosides, as determined by Changsha Herbway Biotech UV using spectrophotomeric analysis. Laying hens had ad libitum access to water and diets in a mash form throughout the experimental period. The composition of feeds was analysed in the laboratory according to AOAC18 procedures. The experimental basal diets were isonitrogenous and isoenergetic and were formulated to meet or slightly exceed the nutrient requirement of laying hens according to the NRC19. The ingredients and calculated nutrient level of the basal diet are shown in Table 1.

Three portable temperature and relative humidity loggers (HOBO U12-012 T/RH logger, temperature measuring range between -20 ºC and 70 ºC with ±0.35 ºC accuracy; relative humidity range of 5%-95% with ±2.5% accuracy) were used to monitor indoor temperature and relative humidity at animal level. Data were recorded every 60 min and downloaded weekly throughout the experiment. The average temperature, relative humidity and light intensity of the entire experimental period were 15.74±0.129 ºC, 48.15±6.410% and 14.76±3.828% lux, respectively.

Egg production performance and egg quality characteristics Hens were weighed individually at the beginning and the end of the experiment. Feed consumption was recorded weekly and calculated as g per hen per day. Viability was observed visually and recorded daily throughout the entire experimental period. The value of feed efficiency was calculated as g feed per g egg. The first egg was laid within 23 weeks of age in all the groups. Eggs from each replicate were collected twice a day (09:00 - 11:00 and 14:00 - 16:00) and weighed with an electronic balance at the same time every day to calculate hen-day egg production ((Number egg produced / number live hens) x 100; HDP), hen-house production ((Number egg produced / number live hens in initial experiment) x 100; HHP), egg weight

Table 1 - Composition and nutrient content of basal diet (g/kg fed basis).

Ingredients

Analysed nutrient composition

Maize

294

Dry matter

898

Wheat

277

Crude protein (CP)

168

Soybean meal (47% CP)

92

Crude fat

Barley

30

Ash

Sunflower meal (36% CP) Full-fat soybean Soybean oil

80 100 24

Crude fibre

36 136 67

Calculated nutrient composition Calcium

36

Available phosphorus

4.0

14

Lysine

8.0

83

Methionine

4.5

Sodium bicarbonate

1

Dicalcium phosphate Marble powder Salt

2

Methionine+cystine

Vitamin-mineral premix*

3

ME (MJ/kg)**

7.6 11.72

* Each kg of vitamin-mineral premix contained: 4800000 IU vitamin A; 1200000 IU vitamin D3; 12000000 IU vitamin E; 1600 mg vitamin K3; 1200 mg vitamin B1; 2400 mg vitamin B2; 12000 mg vitamin B3; 4000 mg vitamin B5; 2000 mg vitamin B6; 20000 mg vitamin C; 6 mg vitamin B12; biotin; 400 mg folic acid; 120000 mg choline; 2000 mg Cu; 24000 mg Fe; 32000 mg Mn; 60 mg Se; 24000 mg Zn; 200 mg Co; 800 mg I. **Dry matter, crude protein and crude ash were analysed according to established procedures AOAC18 and other nutrient compositions are calculated based on NRC19 data of feedstuffs nutrient tables.

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and egg mass ((weekly egg number in replicate x average egg weight)/100, EM) from 20 weeks to 32 weeks of age20,21. In addition, age at point-of-lay, first egg weight, age (day) at 10%, 20%, 30% and 50% egg production were recorded. Thirty two eggs were randomly collected from each experimental group on the last day of every two-week period to assess egg quality. Immediately after sampling, eggs were assayed for quality.

An average of three measurements (3 times) taken at the equator, blunt edge and pointed edge of the egg were recorded for lightness (L*), redness (a*), and yellowness (b*) of the eggshell, using a Minolta CR400 chromameter (Konica Minolta Sensing Inc., Osaka, Japan). The psychometric colour terms such as, Hue (tan-1 b*/a*; H), C* ((a*2+b*2)1/2) and the total colour difference over time ((L*2+a*2+b*2)½; ΔE*) were used in order to evaluate the colour changes of the eggshell. The egg quality traits included specific gravity, eggshell breaking strength, shell thickness, yolk colour and albumen pH. All eggs were weighed individually. The egg shape index was calculated using the following formula: shape index=(width/length) × 10022.

Egg specific gravities were determined from graded salt solutions ranging from 1.069 to 1.099 with gradations of 0.003, as described by Hamilton23. After that, shell breaking strength was measured using a shell strength device with a spiral pressure system (Fujihara, Saitama, Japan), and the values were expressed in kg/cm2. Subsequently, the egg was broken on a glass plate with a waiting period of 5 min to measure the albumen and yolk heights using a tripod micrometer, the long and short diameters of albumen, and diameter of yolk using the digital calliper with a sensitivity of 0.001 mm. A Haugh unit was calculated according to the following formula: Haugh unit (HU) = 100 log [albumen height (mm) + 7.57- 1.7× egg weight0.37 (g)]24.

Albumen index (%) = [Albumen height / (long diameter of albumen + short diameter of albumen / 2) × 100; AI]; yolk index (%) = (yolk height / yolk diameter) x 100; YI22. Egg surface area (ES) in cm2 was calculated for each egg using the following equation suggested by Nordstrom and Ousterhout25: 3.9782 x egg weight0.70. The shell weight (SW) was calculated with the following formula specified by Harms et al.26: (2.0341 x egg weight) - [(2.1014 x egg weight) / specific gravity]. Egg albumen pH values were measured with digital pH meters (Sartorious PP15, AG Weender Landstrasse 94 PP108, Goettingen, Germany). Shell thickness was measured as an average of three measurements taken at the equator, blunt edge and pointed edge of the egg without membrane using the calliper. The yolk colour was determined with a DSM27 yolk colour fan (DSM Nutritional Products Ltd., Basel, Switzerland), which ranges from a pale yellow at score 1 to a dark orange at score 15, according to the CIE standard colorimetric system.

343

PGLE antioxidant activity and total phenolics assay Total antioxidant activity was measured using Oyaizu’s28 ferric reducing antioxidant power (FRAP) assay method. The antioxidant capacity assay was carried out with a Spectronic Genesys 5 spectrophotometer by the improved ABTS•+ method, as described by Re et al.29. A total phenolic constituent of PGLE was performed, employing the literature methods involving Folin-Ciocalteu reagent and gallic acid as standard30.

Statistical analysis Data in the tables are presented as arithmetic means and standard error of means (SEM). The data were analysed by SPSS31 16.0 software for Windows (Inc. Chicago, IL. USA). The differences between groups were determined by one-way ANOVA test. Duncan’s multiple-range tests were performed according to the following model: Yij = µ + Ti + eij where Y denotes the dependent variable; µ denotes the mean; T is the effect of treatment; and e is the random residual error term. All values were presented as means and standard error mean; the significance levels were set at P <0.05.

RESULTS The total phenolic content, TEAC value and FRAP value of PGLE (dry matter) were 1.11±0.02 g gallic acid equivalents (GAE)/kg, 606.3±0.948 mmol trolox/kg and 15.99±0.232 mmol TEAC/kg, respectively.

The performance of the laying hens was not influenced by PGLE intake, as shown by the absence of differences in body weight, body weight gain, viability, feed intake, and feed efficiency among treatments in the Table 2 (P >0.05).

The egg mass, egg production (hen-house and hen-day), morning and afternoon egg yield were also not influenced by feed intake containing different level PGLE when compared with the control (Table 3). No differences were observed in laying hens fed the experimental diets in external and internal egg quality (Table 4). The results demonstrated that levels of PGLE did not affect overall SI, shell breaking strength, egg shell thickness, shell weight and egg surface area, yolk colour, albumen pH and HU (P >0.05) except for AI (P <0.05) and YI (P <0.01), which were higher when 50 mg PGLE/kg and 100 mg PGLE/kg were included in diets. The results of measurements of the eggshell colour L*, a*, b*, Hue, C* and ΔE* are presented in Table 5.

Some serum blood parameters of laying hens fed dietary PGLE at 32 weeks of age are summarized in Table 6.

Blood serum At the age of 32 weeks, approximately 5 ml of blood were taken from the wing vein (Vena cutanea ulnaris) of each hen (32 in total) and were collected in a glass tube (16 mm x 100 mm), kept on ice and transferred to the laboratory. Blood samples were left to stand at room temperature for clotting, and then the serum was obtained by centrifugation at 3000 x g for 5 min.

The resultant serum (supernatant) was collected in 1.5 mL eppendorf tubes and stored at -20 ºC for biochemical assays. The concentrations of serum glucose, cholesterol and triglyceride were measured, using commercial kits (Cat. No. GLU0102, TC0102 and TG0102) on an auto-analyser (Mindray BS series analysers, Hamburg, Germany).

DISCUSSION The total phenolic content, TEAC value and FRAP value of PGLE (dry matter) are higher than those reported by Hu and Kitts32; Saumya and Mahaboob Basha33 and Yıldırım et al.3. Kim et al.34 determined that the total phenolic concentration of ginseng extract was 7.78 g/kg in the ethyl acetate fraction. Deng et al.35 have found that the total phenolic, FRAP and TEAC values of ginseng fresh leaf in lipolytic fraction were 9.25±0.17 mg/g GAE, 10.40±0.44 µmol/g Fe (II) and 10.57±0.17 µmol/g Trolox, respectively. The total phenolic content and antioxidant activity of ginseng may vary depending on the type of plant

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Table 2 - Body weight, feed intake and feed efficiency of laying hens fed on diets supplemented with PGLE. PGLE1 in diet (mg/kg)

Parameters

Initial body weight (g)

0

50

100

150

SEM2

P

1374

1377

1377

1372

7.84

ns

Final body weight (g)

1753

1785

1810

1843

16.17

ns

Body weight gain (g)

369.5

408.2

449.7

471.9

23.30

ns

85.0

100.0

100.0

100.0

2.72

ns

Viability (%) Feed intake (g/hen/day) 0 - 4 weeks

84.9

96.6

92.7

94.9

1.72

ns

4 - 8 weeks

129.6

129.1

135.9

132.2

2.84

ns

8 - 12 weeks

132.8

138.8

139.3

138.1

2.51

ns

Overall

114.5

121.5

121.4

121.7

2.11

ns

0 - 4 weeks

6.62

7.27

6.37

7.22

0.70

ns

4 - 8 weeks

3.57

2.95

2.80

2.99

0.19

ns

8 - 12 weeks

2.41

2.53

2.46

2.54

0.06

ns

Overall

3.56

3.46

3.32

3.48

0.09

ns

Feed efficiency (g feed/g egg)

1

PGLE, Panax ginseng leaf extract; 2 Mean of standard error; P > 0.05.

and the extraction method used. In addition, the total phenolic content and antioxidant capacities among ginseng varieties affected by cultivation practices, cultivation conditions, climate and geographic origin35,36. The antioxidant capacity of ginseng extract may have the potential to be used as an effective dietary antioxidant to prevent oxidative stress-related diseases suggested

by Oh et al.37. The results in the current study is similar to the results reported in previous research3 for panax root extract and demonstrate the antioxidant potential of PGLE, evidenced by

its free radical scavenging and ferric reducing abilities. This supports the use of the plant in traditional medicine. Moreover, when consumed as plant extract, PGLE may contribute to the total intake of dietary antioxidants like other panax ginseng extracts.

The lack of information on the effects of Panax ginseng plant products, especially in laying hens, the possibility of comparing the findings of the study with other studies was limited. Catalan38 reported that the commercial Panax ginseng added to the

Table 3 - Effects of dietary PGLE levels on egg productivity performance traits of laying hens.

PGLE1 in diet (mg/kg)

Parameters 0

50

100

150

SEM2

P

47.7 54.9a 58.8 56.3a

51.4 56.2ab 59.8 57.7ab

50.6 55.1a 58.5 56.3a

49.9 57.3b 60.7 58.7b

0.55 0.33 0.35 0.38

ns * ns *

18.3 40.1 55.1 39.1

16.5 44.4 54.9 43.9

21.9 48.7 56.9 44.7

20.2 45.1 54.8 44.3

1.82 2.11 0.87 1.40

ns ns ns ns

Oviposition time Morning egg weight (g) Afternoon egg weight (g)

57.3a 53.0

58.5ab 55.8

56.9a 53.8

59.5b 54.7

0.38 0.54

* ns

Hen-house egg production (%) 0 - 4 weeks 4 - 8 weeks 8 - 12 weeks Overall

36.8 60.7 79.1 64.5

32.3 77.9 91.8 76.2

43.2 78.6 86.9 76.3

39.4 78.6 90.2 75.5

3.76 4.03 3.03 3.18

ns ns ns ns

Hen-day egg production (%) 0 - 4 weeks 4 - 8 weeks 8 - 12 weeks Overall

38.5 73.1 93.8 69.5

32.4 79.0 91.8 76.2

43.5 88.3 97.2 79.6

39.4 78.6 90.2 75.5

3.75 3.74 1.53 2.59

ns ns ns ns

Morning hen-day egg yield (%) Afternoon hen-day egg yield (%)

53.8 15.7

54.0 22.2

66.3 13.4

62.0 13.4

2.89 1.37

ns ns

Egg weight (g) 0 - 4 weeks 4 - 8 weeks 8 - 12 weeks Overall Egg mass (g/hen/day) 0 - 4 weeks 4 - 8 weeks 8 - 12 weeks Overall

1

PGLE: Panax ginseng leaf extract; 2 Mean of standard error; Means within a row followed by the different superscripts differ significantly. * P <0.05.

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Table 4 - Effects of dietary PGLE levels on means of external and internal egg quality characteristics.

PGLE1 in diet (mg/kg)

Parameters 0 Shape index Specific gravity (g/cm3) 2

Shell breaking strength (kg/cm ) Egg shell thickness (µm) Shell weight (g)

100

150

SEM2

P

76.3

76.0

76.4

77.2

0.21

ns

1.090b

1.087a

1.089ab

1.090b

0.00

*

2.51

2.13

2.24

2.28

0.07

ns

351.3

343.7

339.7

352.4

2.00

ns

6.38

6.61

6.42

6.61

0.07

ns

Surface area (cm2)

66.98

67.77

66.84

67.81

0.31

ns

Yolk colour

10.59

9.75

9.57

10.32

0.15

ns

8.69

8.71

8.71

8.69

0.01

ns

a

b

ab

ab

Albumen pH Albumen index

12.35

13.90

0.20

*

Yolk index

45.78a

45.96a

50.44b

46.38a

1.35

**

93.85

92.96

92.32

93.85

0.95

ns

Haugh unit (score) 1

50

13.00

12.78

2

PGLE: Panax ginseng leaf extract; Mean of standard error; Means within a row followed by the different superscripts differ significantly. * P <0.05; ** P <0.01

Table 5 - Change in colour of eggshell [lightness (L*), redness (a*), yellowness (b*)]1 PGLE2 in diet (mg/kg)

Parameters 0

50

100

150

SEM3

P

L*

66.94

65.79

65.67

66.38

0.27

ns

a*

12.50a

13.62b

13.78b

13.20ab

0.17

*

b*

25.29

25.11

26.27

24.70

0.31

ns

Hue

63.59

61.35

62.10

61.74

0.32

ns

C*

28.26

28.62

29.72

28.07

0.32

ns

ΔE*

72.75

71.87

72.23

72.24

0.20

ns

1

A higher L* value means lighter colour; a higher a* value means a redder colour; a higher b* value means a more yellow colour. 2PGLE, Panax ginseng leaf extract; 3 Mean of standard error; Means within a row followed by the different superscripts differ significantly. * P <0.05.

Table 6 - Effect of PGLE supplementation on some serum biochemical parameters in laying hens at end of experiment.

PGLE1 in diet (mg/kg)

Parameters 0

1

50

100

150

SEM2

P

Glucose (mg/dL)

306.8

302.2

294.2

298.9

2.83

ns

Cholesterol (mg/dL)

159.7

152.2

149.3

154.8

6.85

ns

Triglyceride (mg/dL)

1414.3

1687.6

1544.9

1709.1

67.63

ns

PGLE: Panax ginseng leaf extract; 2 Mean of standard error; P >0.05

diet of the laying hens had no effect on the body weight, body weight gain and feed intake and was consistent with the present research finding. Albeit, in agreement with our previous study on Atak-S laying hens3, the panax ginseng root extract supplementation no changed the values of body weight, body weight gain, feed intake, and feed efficiency during 12 weeks laying period. The current research displayed that viability was higher in laying hens fed diets supplemented with the PGLE than in those fed the control diet and it was similar with the finding of Yıldırım et al.3. This suggests that up to 150 mg PGLE/kg in a diet can be used as an ingredient without any adverse effect on production performance in laying hens or at 11.7 mg/bird/day or 100 mg/kg of body weight.

The lower egg production and egg mass (EM) between 0 and 4 weeks might be because that all hens unexpectedly laid during weeks 22 and 23. Similar to our previous research3, all pa-

rameters were similar in the graded PGLE supplemented group compared with the control group, except that egg production of the 50 and 150 mg PGLE/kg groups was higher than the control, though did not reach statistical significance. These results are consistent with the findings of Ao et al.39, who reported no positive effects on egg production and feed intake in laying hens fed different levels of fermented red ginseng extract. In disagreement with that, Jang et al.40 observed that fermented wild ginseng culture by-product increased egg production which may be attributed to the improvement in health status of birds fed supplemented diets. This inconsistency might be due to the use of different ginseng sources, and different methods of preparation of the ginseng products and strains used in the experiment3. In addition, the level of PGLE supplementation might not have been enough to cause an improvement in egg productivity performance. Otherwise the PGLE supplementation

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increased egg weight and morning (oviposition time) egg weight (57.6 vs. 56.3 g; P <0.05 and 58.3 vs. 57.3 g; P <0.05) as compared to laying hens fed the control diet in overall. This performance variable improved in linear fashion as dietary PGLE supplementation increased. Supplementation of PGLE increases egg weight which may be associated to its active constituents.

It could be hypothesized that PGLE increased egg weight more than the control treatment. Therefore, the PGLE used in this work contains some components, like phenolic acids and antioxidant capacity, which have been implicated in other works as various pharmacological effects14, physiological function and immunity17. The egg weight values in our experiment are in line with the finding of Jang et al.40 and Osfor41, who found that the significant weight increase observed in their trials. In contradiction, some researchers3,38,39,42 illustrated that the PGLE supplementation to diets had no effect on the egg weight of the laying hens. Egg weight and egg shell quality characteristics vary according to the oviposition time. These results agree with previous results3,43,44 that eggs laying early in the morning are heavier than those laid during the day. In addition, the morning henday egg yield was very high than the afternoon in all groups, but still heavy eggs were laid in the morning.

This finding agrees with Yıldırım et al.3, who indicated that AI and YI parameters were not affected by Panax ginseng root extract groups in 20-32 week old laying hens. Therefore, it is thought that the difference detected in both parameters may be closely related to egg weight. Specific gravity significantly decreased when dietary 50 mg/kg PGLE supplementation to diets laying hens. In the current study, specific gravity data consistent with the findings of Yıldırım et al.3 who determined the medium specific gravity ranged from 1.087 to 1.092 in 32 week old Atak-S laying hens. Hens fed with diet supplemented with PGLE had similar values of SI, eggshell thickness, shell weight, surface area, yolk colour and HU score, but lowest values of shell breaking strength compared to control group (P >0.05). SI is a method used to determine egg shape. The SI in ideal eggs is 74% in terms of commercial and hatching properties. If the SI value is greater than 76%, the eggs are round, 72-76% is normal, and <72% is longer45. In the experiment, the group which received 150 mg/kg PGLE was found to be more than 76% in terms of SI and the other groups were classified as normal SI. Yıldırım et al.3 reported that the SI values (76.2-77.4) of the laying hens fed with Panax ginseng root extract supplement were similar to our findings. Eggs shorter and longer than normal eggs are not desirable by the consumer and they cause economic losses during transportation and marketing. To reduce the chances of breaking eggs at the marketing and transportation stages, the shell thickness of the eggs is expected to be at least 330 μm and more46. In this context, according to our findings related to the egg thickness close to each other in the groups (339.7-352.4 μm), thus it was among the desired minimum values. These results are consistent with those of several studies3,38,39,40,42 who found that various ginseng supplemented to laying chicken diets had no significant effect on the egg shell weight. The findings agree with those reported by Ao et al.39, who found no significant effect of dietary fermented red ginseng extract supplemented on HU, eggshell thickness and egg breaking strength parameters in ISA brown laying hens. By contrast, Yıldırım et al.3 reported that the paler yolk at inclusions of 100 mg Panax ginseng root extract/kg and 150 mg Panax ginseng root extract/kg suggested xanthophylls interacted with ginsenosides of Panax ginseng root extract. Thus, this condition

can be explained by ginsenoside content variability. Albumen quality, which is an important indicator of egg freshness, has a great significant in egg processing industry47. The pH is a useful means for describing changes in albumen quality over time during storage claimed by Silversides and Villeneuve48. However, albumen pH is not affected by PGLE groups and can be used to measure egg freshness. Overall albumen pH ranged from 8.69 to 8.71, which is in agreement with previous data, such as 8.67 for Atak-S laying hens3 and 8.29 to 8.39 for 30 week old Dekalb white laying hens2.

Hen nutrition is an area that needs to be tested in relation to eggshell color49. Overall L*, b*, Hue, C* and ΔE* did not differ among treatments during the entire experiment. However, the present study demonstrates that the dietary PGLE supplementation significantly improved the a* values of egg in laying hens in both 50 and 100 mg/kg groups when compared to control group. Supplementing PGLE to diet, increased (P <0.05) the redness of the eggshell and came close the other among groups, but this effect was not reflected in the same way in the other parameters (Hue, C* and ΔE*). However, a possible mechanism of action could be due to any factor that promotes liver function, subsequent lipid metabolism, synthesis of protoporphyrin IX in the shell gland and deposition of pigment in the eggshell49. This fact agrees with the results of Odabasi et al.50, who indicated that a decrease in pigmentation was associated with a decrease in the amount of redness (a*) in the eggshell.

The lighter the shell in colour, the less redness it contains, and this negative correlation becomes stronger as the hen ages over the first 10 months of the laying cycle claimed by the same researchers. The present study, L* was numerically lower in PGLE groups than in the control. Hence, the lighter coloured eggshells (higher L*) would have less redness (a*). The averages of the overall measurements at three points on the same egg were also very close to those of the one point measurement.

The results indicated high uniformity of eggshell pigmentation.

Yıldırım et al.3 determined the average L*, a* and b* values of brown eggshell from 32 weeks old hens to be between 65.0 and 66.4, 13.4 and 14.1, 24.9 and 26.8 for brown eggs, respectively. In the present study, the same parameter values were between 65.7 and 66.9; 12.5 and 13.8; 24.7 and 26.3, respectively for AtakS brown egg. This is consistent with previous results, with same age effect. Shell colour is usually affected by genetic background, the season of lay, nutrition, housing systems, diseases, all kinds of stimulus, and drug, etc.49,51.

While assessing the health status of poultry fed diets with unconventional feeds, serum biochemical parameters play an important role in providing useful information about the metabolic alterations of organs and tissues52. Melesse et al.53 and Hussein et al.54 reported that several factors cause a considerable influence on serum biochemical parameters of chickens such as feed additives, genotype and environmental temperature. The supplementation of PGLE to laying hens diets had no significant effect on the serum glucose, cholesterol, and triglyceride concentrations. However, serum cholesterol and glucose was numerically lower in PGLE groups than in the control. This consistent with Ao et al.39, who observed that serum cholesterol concentrations varied from 176 mg/dL to 185 mg/dL and there were no significant differences when various levels of fermented red ginseng extract were supplemented. Muwalla and Abuirmeileh55 and Yokozawa et al.56 found that the addition of dietary ginseng decreased the level of blood lipids. On the other hand, total cholesterol concentration was decreased by fermented gin-

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seng culture in laying hens demonstrated by Jang et al.40. However, this effect was not confirmed in the present study, probably because of the low levels of supplementation3. Overall, the data from the current study supported the contention that laying hen diets can contain up to 150 mg/kg PGLE without affecting measures of hen productivity or specific measures of egg and eggshell quality.

CONCLUSIONS The results of this study showed that overall, dietary supplementation of PGLE to laying hens’ diet improved the egg weight and eggshell redness at the beginning of the laying period, but it had no adverse effects on the health status of the hens, external egg quality and some serum biochemical measurements.

Future research needed to explain the mechanism, significance and the use of PGLE as a source of bioactive substance and to determine the welfare of laying hens in different management conditions, including various stress factors such as environmental and physiological stresses, dietary ingredients and nutrient content for the poultry feed industry.

ACKNOWLEDGEMENT This study was supported as a master thesis project by the Research Fund of Tokat Gaziosmanpa a University (Project No: BAP/2012/42). The authors are thankful to the staff in the Department of Animal Science, Faculty of Agriculture, University of Gaziosmanpa a, Tokat, Turkey.

AUTHOR’S CONTRIBUTION AY conceived and designed the study, AY and MISM conducted the analyses and contributed to the data collection, drafted the manuscript. All authors read and approved the final manuscript.

CONFLICT OF INTEREST STATMENT The authors declare that they have no competing interests.

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Comparison of repeatable and random regression models for genetic parameter estimation on Thoroughbreds

349

Ă&#x201C;

UMIT COSKUN1, HASAN Ă&#x2013;NDER1* AND SAMET HASAN ABACI1 1

Ondokuz Mayis University, Faculty of Agriculture, Department of Animal Science, 55139, Samsun, Turkey

SUMMARY Introduction - The combination of multiple factors (track, year, ages, etc.) is effective in achieving the maximum level of race performance in Thoroughbreds.

Aim - The aim of this study is to estimate and compare genetic parameters on the number of race success characteristics in Thoroughbreds with random regression models (RRM) and repeatable animal models (RAM) with a different number of repetitions.

It was also aimed to investigate which number of observation points would be sufficient for genetic parameter estimation for Thoroughbred.

Materials and methods - As data, 111312 test day race completion time (sec) records of 13625 Thoroughbreds raced taken from the Jockey Club of Turkey between 2005 and 2016 were used. Competition performances were compared with different measurements using the same repeatability model. Variance components of Thoroughbreds were obtained by using RRM and RAM using DFREML and WOMBAT package, respectively.

Results and discussion - When AIC and BIC values were examined, it was observed that the values in RRM were lower than RAM method for ten races. According to Akaike Weights results, while the fifth race shows 35.56% better fit than the fourth race in the model. The AW values of other number of races showed less superiority; thus, 5th and 6th races can be preferred over its competitors in terms of Kullback-Leibler discrepancy.

Conclusion - Our results showed that number of race of five were sufficient to estimate genetic parameters for Thoroughbred horse. Also, RRM method can be preferred compared to RAM method.

KEY WORDS Heritability, Random regression, REML, Repeatable animal models, Thoroughbreds.

INTRODUCTION The contributions of horse races are made in every community and culture from the 17th century to the present day and have been improved by selection of horseracing performance for about 300 years. While horses are bought and sold at very high prices, high-performing horses in the races are making significant gains to their owners1, 2.

The Thoroughbred horse breed was established in England in the early 1700s based on crosses between stallions of Arabian origin and indigenous mares3. The Thoroughbred horse breed is used in a variety of sports such as running, jumping and hunting. In equestrian sports, Thoroughbred horses have a very important place for flat racing for speed at middle - distances between 1400 m and 2400 m all around the world4. Thoroughbred racing industry and its professionals are now even more capable of selecting the most appropriate horses for breeding and racing5. The statistical power of these studies has also been restricted by the statistical methods and computational resources, making it difficult to include all necessary environ-

Corresponding Author: Hasan Onder (hasanonder@gmail.com).

mental factors in the analyses and potentially biasing estimates6.

There are many types of methods for predicting genetic parameters, such as the animal model or the sire model restricted maximum likelihood (REML), best linear unbiased prediction (BLUP), random regression (RR), and Bayesian methods1, 2, 7-11. To use in dairy cattle breeding for the analysis of test day production records random regression models (RRM) has been introduced by Henderson (1982) and Laird and Ware (1982) as mentioned by Schaeffer11. During the last 20 years, longitudinal data analyzed by RRM are used extensively on many economical traits at different animal breeding scenarios1, 10, 12-14 . Due to the improved modelling of variances and genetic parameters, RRM is a good choice for modelling the traits which are measured repeatedly for each individual, but change gradually and continually in time8, 15.

This study aims to estimate and compare genetic parameters on the number of race success Thoroughbreds with random regression models (RRM) using L(3,3) legendre polynomials and repeatable animal model with restricted maximum likelihood (REML) method. Gomez et al8 used the L(2,2) model, while Buxadera and da Mota12 used the L(3,2) model. In our study, we used the L(3,3) model with fixed residual variance, which is used extensively in other species10,13. It was also aimed to investigate which number of observation points would be sufficient for genetic parameter estimation for Thoroughbred.

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Comparison of repeatable and random regression models for genetic parameter estimation on Thoroughbreds

MATERIALS AND METHODS Materials As data, 111312 test day race completion time (sec) records of 13625 Thoroughbreds taken from the Jockey Club of Turkey between 2005 and 2016 were used after exclusion. Only animals with two to ten repeated measurements per animal were used. Some animals whose parents were not reached and the animals that were recorded for less than two races were excluded from the study (number of excluded records were 3361). The average percentage of known parental grandparent was 21.4% and maternal grandparent was 43.7%. The average inbreeding was calculated as 0.07. The race tracks type, the race year and the age of the horse were fixed, and the race tracks distance were taken as covariates. Sample size for the number of races were 13625, 12880, 12126, 11383, 10674, 10051, 9510, 8973 and 8464 from two races to ten races, respectively. In the model where variance elements are estimated included the fixed effects; race tracks (3 types), the race year (12 years) and the age (9 different ages) of the horses and the race tracks distance (16 different distances from 800m to 2400m with increasing 100m) were taken as covariates.

Methods Repeatable animal model approach using sire and dam pedigree information was used. The following model was used to apply the RRM1.

eijkl ~ N(0, Ď&#x192;e2).

In RAM, heritability (h2) was calculated from genetic parameters (additive genetic effect / phenotypic effect) obtained by REML method.

To compare the models, Akaike information criterion (AIC), Bayesian information criterion (BIC) and Log likelihood values were used12. The DXMMR option running under the DFREML program for the RRM and the WOMBAT package program for the repeatable animal models were used.

RESULTS The values of the variance components obtained by the RRM and RAM methods are given in Table 1. According to the findings obtained with the RAM method, the additive genetic effect value varied between 3.651 and 4.066, while it varied between 4.014 and 5.165 with the RRM method. When the permanent environmental effect values are examined, it varied between 2.310 and 2.899 according to the RAM methods, while it varied between 2.209 and 7.045 in the RRM method. When the methods were examined according to phenotypic effects, values between 13.938 and 17.267 were observed in RAM methods, while values were found between 14.776 and 20.015 in RRM method.

DISCUSSION here; Yijkl: observation value of the horse l at race track ith at race year of jth and age of kth, RTi: ith race tracks (sand, grass, artificial grass); RYj: jth race year (2005-2016); HAk: kth horse age (218); đ?&#x203A;˝đ?&#x2018;&#x161;: mth fixed regression coefficients for horse j; tij: ith test day of the horse j; x(m) (tij): mth covariates (race tracks distance: from 800 to 2400 (with 100m increase); Îąjm: mth additive genetic random regression coefficients for horse j; Pjm: mth permanent environmental random regression coefficients for horse j; Ď&#x2022;m: mth polynomial evaluated for the race tij; KB, KA and KP are the order of fitted fixed, random additive and random permanent regression coefficients; eijkl: random residual effect for yijkl.

In RRM, the (co) variances of the random regression coefficients and the heritability values (additive genetic effect / phenotypic effect) were estimated by the REML method. The estimates were obtained using the AI-REML algorithm, thus avoiding the problems of possible local maximum estimates â&#x20AC;&#x153;without derivativeâ&#x20AC;?. Third-order Legendre polynomials have been used to define the structure of (co)variance between observations of the same individual.

The individual repeatable animal model used to estimate the components of variance and heritability are presented below:

here; Yijkl: observation value of the horse l at race track ith at race year of jth and age of kth, Âľ: population mean, RTi: the effect of ith race track, RYj: the effect of jth race year, HAk: the effect of kth horse age, b: scalar coefficient for race completion time, Xijkl: race time for animal l in ijtth sub group, XĚ&#x201E;: mean of the race completion time for the population, ađ?&#x2018;&#x2122;: is the random animal effect of Thoroughbreds l.,: is the random permanent environmental effect of Thoroughbreds đ?&#x2018;&#x2122;. and eijkl: random error term

Estimated heritability values were similar with the results of Buxadera and da Mota12, but higher than the estimates of da Gama, Borquis7. Orhan and Kaygisiz16 found the heritability of race completion time as 0.05 which was the lowest estimates of the trait, this may have occurred from their sample size which had 3184 races. Faria, Maiorano14 found the heritability of race completion time from 0.45 to 0.56, which is higher than the results of this study. This difference may have occurred as a result of the race track distances they used. The highest heritability was observed in 5th and 6th races which shows that the 5th and 6th races were enough to comment on the horseâ&#x20AC;&#x2122;s racing performance.

When the AIC and BIC values were examined in Table 4, it was observed that the values of the RRM method were lower than those of the RAM method for 10 race. Therefore, RRM method can be preferred instead of RAM method. Many researchers recommended the use of RRM8, 11, 12, 17-21.

To determine the adequate number of race for genetic parameter estimations, value of the heritability and Akaike Weights (AW), in the AIC sense that minimizes the Kullback-Leibler discrepancy, Wagenmakers and Farrell22 interpreted together. According to AW results, while the fifth race shows 35.56% better fit than the fourth race in the model, the sixth race shows 23.64% better fit than the fifth model. The AW values of other number of races showed less superiority; thus, 5th and 6th races can be preferred over its competitors in terms of KullbackLeibler discrepancy. And also heritability values were found as higher at these number of races.

CONCLUSIONS Within the conditions of this research, it became evident that

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U. Coskun et al. Large Animal Review 2020; 26: 349-352

351

Table 1 - Values of variance components obtained by RRM and RAM methods.

Methods

Number of races

Additive Genetic Effect

Permanent Environmental Effect

Residual Effect

Phenotypic Effect

RAM

2 3 4 5 6 7 8 9 10

3.651 3.991 3.961 3.969 4.066 4.059 4.015 4.007 4.016

± ± ± ± ± ± ± ± ±

0.253 0.240 0.232 0.228 0.227 0.226 0.224 0.224 0.224

2.617 2.310 2.485 2.592 2.605 2.684 2.754 2.837 2.899

± ± ± ± ± ± ± ± ±

0.201 0.178 0.170 0.165 0.163 0.161 0.160 0.160 0.160

7.669 ± 0.093 8.387 ± 0.073 9.045 ± 0.065 9.236 ± 0.058 9.525 ± 0.055 9.813 ± 0.052 10.066 ± 0.050 10.246 ± 0.049 10.351 ± 0.047

13.938 14.688 15.491 15.797 16.197 16.556 16.835 17.091 17.267

± ± ± ± ± ± ± ± ±

0.149 0.140 0.137 0.133 0.133 0.132 0.130 0.130 0.131

RRM

2 3 4 5 6 7 8 9 10

4.014 4.449 4.799 5.042 5.165 5.161 5.031 4.782 4.427

± ± ± ± ± ± ± ± ±

0.234 0.231 0.261 0.300 0.334 0.361 0.383 0.409 0.454

2.209 2.645 3.231 3.715 4.006 4.174 4.449 5.222 7.045

± ± ± ± ± ± ± ± ±

0.164 0.163 0.187 0.213 0.233 0.247 0.263 0.299 0.379

8.543 8.543 8.543 8.543 8.543 8.543 8.543 8.543 8.543

14.766 15.637 16.573 17.300 17.714 17.878 18.023 18.547 20.015

± ± ± ± ± ± ± ± ±

0.134 0.136 0.155 0.177 0.193 0.205 0.218 0.243 0.303

± ± ± ± ± ± ± ± ±

0.044 0.044 0.044 0.044 0.044 0.044 0.044 0.044 0.044

Heritability estimates for RAM and RRM on the number of races were given in Table 2. The values of heritability estimates were moderate and close to each other, RRM estimates were higher when compared to RAM estimates. Additive genetic correlations were nearly two times higher than phenotypic correlations (Table 3) estimated by RRM among the number of race. This finding was an evidence that genetic factors have greater influence than the environmental factors.

Table 2 - Heritability estimates for RAM and RRM on number of races.

Number of races

RAM

RRM

Heritability

Permanent Environmental Effect Ratio

Heritability

Permanent Environmental Effect Ratio

2

0.262 ± 0.017

0.188 ± 0.015

0.284 ± 0.014

0.150 ± 0.011

3

0.272 ± 0.015

0.157 ± 0.013

0.289 ± 0.013

0.169 ± 0.011

4

0.256 ± 0.014

0.160 ± 0.011

0.291 ± 0.014

0.195 ± 0.012

5

0.251 ± 0.013

0.164 ± 0.011

0.291 ± 0.015

0.215 ± 0.013

6

0.251 ± 0.013

0.161 ± 0.010

0.288 ± 0.016

0.226 ± 0.013

7

0.245 ± 0.012

0.162 ± 0.010

0.279 ± 0.017

0.233 ± 0.014

8

0.238 ± 0.012

0.164 ± 0.010

0.257 ± 0.017

0.247 ± 0.015

9

0.234 ± 0.012

0.166 ± 0.010

0.221 ± 0.018

0.282 ± 0.016

10

0.233 ± 0.012

0.168 ± 0.010

0.271 ± 0.019

0.352 ± 0.018

Heritability values estimated by RRM had increasing trend until the number of six races and then it tended to decrease; however, the heritability values estimated by RAM had a decreasing trend after the second race.

Table 3 - Additive genetic (below diagonal) and phenotypic (above diagonal) correlations estimated by RRM among the number of race.

2 2

3

4

5

6

7

8

9

10

0.428

0.424

0.414

0.400

0.379

0.350

0.309

0.254

0.464

0.463

0.454

0.434

0.403

0.354

0.288

0.492

0.488

0.472

0.442

0.392

0.321

0.509

0.499

0.473

0.427

0.358

0.515

0.498

0.460

0.400

0.516

0.493

0.446

0.521

0.495

3

0.998

4

0.992

0.998

5

0.985

0.994

0.999

6

0.975

0.988

0.995

0.999

7

0.964

0.979

0.989

0.995

0.999

8

0.950

0.968

0.981

0.990

0.995

0.999

9

0.933

0.954

0.970

0.981

0.989

0.995

0.999

10

0.911

0.936

0.954

0.968

0.979

0.987

0.994

0.540 0.998

While the estimated additive genetic correlations were found to be high, phenotypic correlations were found to be too low. The maximum phenotypic correlation was obtained as 0.54, show medium relation between the number of races 9 and 10. Results of the comparison criteria for RRM and RAM methods are given in Table 4. Considering the data of 10 races at the same time in both methods, AIC and BIC values obtained from the RRM method were found to be lower than the RAM method. However, considering the smaller number of races, the AIC and BIC values of the RAM methods were found to be lower.

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Comparison of repeatable and random regression models for genetic parameter estimation on Thoroughbreds

Table 4 - Comparison criteria for RRM and RAM methods.

Method

Number of parameters

Number of records

Log Likelihood

AIC

BIC

RRM

13

111312

-192780.0

385586.0

385711.1

RAM-2

3

27250

-47472.9

94951.7

94976.4

RAM-3

3

40131

-70004.6

140015.2

140041.0

RAM-4

3

52257

-91902.8

183811.6

183838.2

RAM-5

3

63640

-111711.3

223428.5

223455.7

RAM-6

3

74314

-130719.1

261444.2

261471.8

RAM-7

3

84365

-148852.5

297710.9

297738.9

RAM-8

3

93875

-166077.4

332160.8

332189.1

RAM-9

3

102848

-182250.6

364507.2

364535.8

RAM-10

3

111312

-197283.7

394573.5

394602.3

the heritability estimates were higher and more reliable estimated by RRM than RAM method. Use of RRM brings an opportunity to successfully select stallions which could be selected as sires for the next generation because the random regression procedure provides further information for the selection process. Thus, the use of random regression as a tool in the evaluation of race horse performance can be highly recommended. Also, our results showed that 5 races were sufficient to estimate genetic parameters for Thoroughbred horse because the sample size was high enough to state that expression.

9.

10.

11.

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CONFLICT OF INTERESTS The authors declare that they have no conflict of interests.

13.

ACKNOWLEDGEMENTS The authors wish to thank the Jockey Club of Turkey for assistance. This study was a short summary of first author’s MSc thesis.

14.

15.

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Abaci S. H, Coskun U., Onder H. (2019). Comparison of Some random regression models for racing performances of the British racing horses in Turkey. BSJ Agri., 2 (1):6-9.

Tozaki T., Miyake T., Kikuchi M., Kakoi H., Hirota K., Kusano K., Ishikawa Y., Nomura M., Kushiro A., Nagata S. (2019). Heritability estimates of fractures in Japanese Thoroughbred racehorses using a non linear model. J. Anim. Breed. Genet. 136 (3): 199-204. http://doi.org/10.1111/jbg.12387 Binns M., Boehler D., Bailey E., Lear T., Cardwell J., Lambert, D. (2012).

Inbreeding in the Thoroughbred horse. Anim. Genet., 43 (3): 340-342. doi:10.1111/j.1365-2052.2011.02259.x Paksoy Y., Ünal N. (2019). Multivariate analysis of morphometry effect on race performance in Thoroughbred horses. R. Bras. Zootec., 48. e20180030. doi:10.1590/rbz4820180030 Velie B., Hamilton N., Wade C. (2015a). Heritability of racing performance in the Australian Thoroughbred racing population. Anim Genet., 46 (1): 23-29. doi:10.1111/age.12234 Velie B., Hamilton N., Wade C. (2015b). Performance selection for Thoroughbreds racing in Hong Kong. Equine Vet. J., 47 (1): 43-47. doi:10.1111/evj.12233 da Gama M. P. M., Borquis R. R. A., de Araújo Neto F. R., de Oliveira H. N., Fernandes G. M., da Mota, M. D. S. (2016). Genetic parameters for racing performance of Thoroughbred horses using Bayesian linear and Thurstonian models. J. Equine Vet. Sci., 42, 39-43. doi:10.1016/j.jevs.2016.03.02 Gómez M., Molina A., Menendez-Buxadera A., Valera M. (2011). Estimation of genetic parameters for the annual earnings at different race dis-

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tances in young and adult Trotter Horses using a Random Regression Model. Livest. Sci., 137 (1-3): 87-94. doi:10.1016/j.livsci.2010.10.004 Gómez M., Valera M., Molina A. (2010). Genetic analysis of racing performance of trotter horses in Spain. Livest. Sci., 127(2-3): 197-204. doi:10.1016/j.livsci.2009.09.012.

Onder H., Sen U., Takma C., Ocak S., Abaci S. H. (2015). Genetic parameter estimates for growth traits in Saanen kids. Kafkas Univ. Vet. Fak. Derg., 21 (6): 799-804. doi: 10.9775/kvfd.2015.13407 Schaeffer L. (2004). Application of random regression models in animal breeding. Livest. Prod. Sci., 86 (1-3): 35-45. doi:10.1016/S03016226(03)00151-9 Buxadera A. M., da Mota, M. D. S. (2008). Variance component estimations for race performance of thoroughbred horses in Brazil by random regression model. Livest. Sci., 117 (2-3): 298-307. doi:10.1016/j.livsci.2007.12.027 Cruz A., Menéndez-Buxadera A., Gutiérrez G., Morante R., Burgos A., Gutiérrez J. P. (2020). Genetic (co)variance across age of fiber diameter and standard deviation in Huacaya alpacas, estimated by repeatability, multi-trait and random regression models. Livest. Sci., 231: 103863. doi:10.1016/j.livsci.2019.103863 Faria R. A. S., Maiorano A. M., Correia L. E. C. d. S., Santana Jr, M. L., Silva, J. A. I. I. V. (2019). Time class for racing performance of the Quarter Horse: Genetic parameters and trends using Bayesian and multivariate threshold models. Livest. Sci., 225: 116-122. doi:10.1016/j.livsci.2019.05.013 Meyer K. (2004). Scope for a random regression model in genetic evaluation of beef cattle for growth. Livest. Prod. Sci, 86(1-3): 69-83. doi:10.1016/S0301-6226(03)00142-8 Orhan H., Kaygisiz, A. (2010). Genetic and Environmental parameters effecting racing performance of Turk-Arabian Horses raised at Anatolian state farm. Asian J. Anim. Vet. Adv., 5, 112-119. doi:10.3923/ajava.2010.112.119 Bartolomé E., Menéndez Buxadera A., Molina A., Valera, M. (2018). Plasticity effect of rider-horse interaction on genetic evaluations for Show Jumping discipline in sport horses. Anim. Breed. Genet., 135 (2): 138-148. doi:10.1111/jbg.12315 Bussiman, F. d. O., Perez, B., Ventura, R. V., Peixoto, M., Curi, R., Balieiro, J. C. d. C. (2018). Pedigree analysis and inbreeding effects over morphological traits in Campolina horse population. Animal, 12(11): 22462255. doi:10.1017/S175173111800023X Gómez M., Menendez-Buxadera A., Valera M., Molina A. (2015). A reaction norm model approach to estimate the genetic effect of temperature on sportive performance of trotter horses. J. Anim. Breed. Genet., 132(3): 256-267. doi:10.1111/jbg.12118 Kim N. Y., Son J. K., Cho I. C., Shin S. M., Park S. H., Seong P. N., Woo J. H., Park N. G., Park H. B. (2018). Estimation of genetic parameters for temperament in Jeju crossbred horses. Asian-Australas J. Anim. Sci., 31(8): 1098-1102 doi:10.5713/ajas.17.025 Posta J., Komlósi I., Mihók S. (2009). Breeding value estimation in the Hungarian Sport Horse population. Vet. J., 181 (1): 19-23. doi: 10.1016/j.tvjl.2009.03.006 Wagenmakers E. J., Farrell S. (2004). AIC model selection using Akaike weights. Psychon. Bull. Rev., 11 (1): 192-196. doi:10.3758/BF03206482

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Equine laminitis.

New insights into the pathogenesis.

A review

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Ó

PAOLO STEFANO MARCATO1, ANTONELLA PERILLO2 1 2

Departement of Veterinary Medical Sciences. Alma Mater Studiorum - University of Bologna, Italy Departement of Veterinary Medicine. University of Bari Aldo Moro, Italy

SUMMARY Laminitis is typical of Ungulates but it is especially significant in horses. The most prevalent clinical signs (≥70%) are difficulty turning and a short/stilted or lame walk. Very severe, highly acute laminitis cause the third phalanx to detach from the hoof wall and to drop ventrally inside the hoof, favoured both by the destruction of the dermal-epidermal junction, as well as by a hyperplasia of the epidermal laminae that produce a horny growth (keraphylocele) acting as a wedge. Four main risk factors correspond to four types of laminitis: 1) sepsis /SIRS (systemic inflammatory response syndrome) related laminitis; 2) endocrinopatic laminitis; 3) pasture-associated laminitis; 4) supporting limb laminitis, experimentally linked to hoof lamellar hypoxia. Type 1 correlates with sepsis from Gram-negative polymicrobial bacteria (postpartum metritis with retained placenta, colic, proximal enteritis, volvulus and enterocolitis). The systemic inflammatory events that occur in type 1 laminitis coincide with marked increase in digital lamellar expression of a variety of inflammatory mediators and with activation of extracellular matrix metalloproteinases (MMPs). The most common form of laminitis, endocrinopatic laminitis (type 2), may occurr secondary to metabolic diseases (equine metabolic syndrome, Cushing’s disease), usually in obese horses and ponies, and is exacerbated in animals that graze lush pastures (type 3 laminitis). The unifying pathogenic factor in types 2 and 3 laminitis, is hyperinsulinemia with insulin toxicity. The main failure in these cases is the loss of the adherence of the basal epithelial cells in the epidermal lamellae to the underlying dermal lamellae through the separation of the dermo-epidermal attachment at the basement membrane (BM) level. The early and key cytomorphological pathology points to lamellar cell stretching, suggesting cytoskeletal deformation with weakening and elongation of the lamellar epithelial cells, which translate into alteration of their tensegrity. These events may well induce some secondary alteration in the structure and/or elasticity of the BM, with cytoskeletal disengagement and loss of the hemidesmosomes and thus a further relaxation of the lamellae followed by loss of adhesion of the layer of basal epithelial lamellar cells with the underlying BM. The lamellae are sparsely populated with insulin receptors (insR), whereas IGF-1 receptors (IGF-1R) are abundant. However insulin is unlikely to directly bind and to activate equine IGF-1R in vivo, even at high physiological concentrations. An indirect mechanism through which insulin could activate IGF-1R should be envisaged in the displacing IGF-1 from IGF-binding proteins (IGFBPs) such as IGFBP7 or fragments of IGFBP3, thereby increasing free IGF-1 concentrations, or in a direct action on a very small population of lamellar InsR.

KEY WORDS Endotoxaemic laminitis, endocrinopatic laminitis, hyperinsulinemia, lamellar cell stretching.,cytoskeletal mechanics.

INTRODUCTION The equine foot is a miracle of bioengineering produced by the forces of evolution, but becomes a common site of disease and injury when subjected to the demands of human domestication, being mainly the site of a significant and predominant disorder called pododermatitis1. A widespread, sporadic, aseptic pododermatitis, is typical of Ungulates, and can affect cattle, sheep and goats, rarely pigs, but it is especially significant in horses, the species to which the present review refers. This pododermatitis is called laminitis (founder, fourbure, infosura) and is a specific condition of the foot that can produce lameness. The terms

Corresponding Author: Paolo Stefano Marcato (paolostefano.marcato@unibo.it)

«laminitis» and «founder» are used interchangeably. However, in horses, founder usually refers to a chronic (long-term) condition associated with rotation of the third (distal) phalanx (coffin bone), the catastrophic result of laminitis3, whereas acute laminitis refers to symptoms associated with a sudden initial attack, including pain and inflammation of the dermal lamellae (lamellar dermis). Dermal lamellae form, together with the epidermal/insensitive lamellae of the inner hoof wall with which they interlock, the suspensory apparatus of the third phalanx1, in other words where the horny wall, by its vertical keratophyl laminae, is fused with the podophyl laminae of the keratogenous layer.

Laminitis has been a recognized disease, since early Greek times,

by Xenophon of Athens (380 BCE)2 .

The main site of this process is the inner layer of the wall (stratum lamellatum) or the cornifying laminar epithelium (hard ker-

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Equine laminitis. New insights into the pathogenesis. A review Table 1 - Possible causes or precipitating factors in equine laminitis (from Baxter G.M.7).

atin or hard horn) of the hoof, i.e. the laminar or lamellar corium (dermis) or dermal lamellae (Figure 1). It consists of a lesion involving the complex interdigitation system of the keratinized lamellae which ensure that there is a firm bond between the epidermal hoof wall and the bone of the third phalanx.

Damage to the lamellae can cause this interdigitation to fail and the underlying third phalanx to separate from the wall3 4 5. At the base, there is damage to the dermo-epidermal interface of the lamellae that causes failure of the attachment of epidermal lamellae, connected to the hoof wall, to the dermal lamellae, attached to the distal phalanx.

This lamellar separation, with failure of the attachment between the inner hoof wall and the distal phalanx, usually occurs in horses as a sequela to gastrointestinal disorders resulting from carbohydrate overload, colic, enterotoxaemia, systemic and metabolic diseases, especially equine metabolic syndrome*6.

The pathogenetic mechanisms involved in the onset of the laminitis are different on the basis of theories that propose inflammatory, vascular, enzymatic, metabolic or traumatic factors7. With regard to two mechanisms that in the past have enjoyed much favour, i.e. inflammation and digital vascular dysfunction, there is debate over which is the primary or whether they are interdependent and have a simultaneous onset, bearing in mind that digital blood flow always plays a crucial role in triggering laminitis.

In the USA, it has been estimated that 15% of horses develop laminitis during their lifetime and 75% of affected horses that are admitted to university referral hospitals are eventually euthanatized. Moreover, in the USA the annual incidence of equine laminitis is 2% but rises to around 5% in spring-summer8. Almost half of all cases occur in animals at pasture. Foot lameness in the horse is the most prevalent and frequent medical issue, affecting about 11% of the general equine population in the UK in 20119.

Major insights have been gained into the pathophysiology of the condition only in the last 40 years, thanks to the development of several experimental models of laminitis10.

Table 1 shows different possible causes or precipitating factors in equine laminitis7. Although much is known about equine laminitis, more is being discovered from cellular and molecular studies about the equine limbs and the disorders they are prone to.

Acute laminitis. In acute laminitis, there is a significant local increase in hoof wall temperature, a sharp pain in the hooves on hammer testing, and hypertension of the digital and collateral arteries11. Laminitis can involve the front or hind or all four limbs.

In horses, it is found mainly in the forelimbs, while in cattle it affects the hind limbs and inner hooves. At a behavioural level (Figure 2), affected animals show prolonged leg resting and are reluctant to move at all, weight-bearing exclusively on the heel of the affected hoof so as to provide relief for the anterior parts where the pain is more intense. In addition, the animals exhibit hyperpnea

Figure 1 - Drawing of a sagittal section of equine hoof. Schematic diagram illustrating the entire structure of the horse hoof. (Figure from Al-Agele R. et al.9).

Figure 2 - Acute laminitis. Typical stance of a horse with laminitis of both forelimbs. The horse, when standing, may well lean back on to its hind feet in order to relieve the pressure on its front feet.

1. Carbohydrate overload a. excess grain intake b. lush pasture (grass laminitis) c. feed change to high energy legume 2. Endotoxemia, sepsis, shock a. colitis b. proximal enteritis c. small intestinal strangulation/obstruction d. retained placenta, metritis, abortion e. septicemia or toxemia from any cause 3. Excessive unilateral weight bearing (support laminitis) a. severe lameness b. rehabilitation of fracture repair 4. Management a. ingestion of cold water by overheated horse b. unconditioned horse worked on hard surface (concussion or road laminitis) c. overweight horses or ponies d. trimming horses too short e. black walnut wood shavings 5. Miscellaneous a. treatment with corticosteroids b. hypothyroidism c. high estrogen plants d. continuous estrus in mares e. allergic-type reactions to certain medications.

CLINICAL SIGNS AND PATHOLOGY

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Figure 3 - Schematic drawings of laminitic hoofs. (A) The detached pedal bone (third phalanx) can rotate (arrow). (B) Pedal bone is forced downwards to protrude through the sole (arrows).

and tachycardia, and sometimes symptoms of toxic shock. The most prevalent owner reported clinical signs (≥70%) are difficulty turning and a short/stilted or lame walk12.

Affected horses may have recurrent episodes and sometimes have to be euthanized due to permanent damage occurring in the hoof.

Indeed, once the devastating pathological cascade of laminitis is under way, the anatomical dislocations are so overwhelming that there is little hope that the foundered foot can be restored to normal.

Examining a section of a horse’s hoof shows only considerable congestion of the dermal lamellar interface and sometimes hemorrhagic phenomena. The skin of the coronary sulcus may be swollen by edema. In very severe, highly acute cases the disintegration of the dermal-epidermal junction can cause the third phalanx to detach from the hoof wall and to drop ventrally inside the hoof (rotation of the third phalanx in the opposite direction to the dorsal wall of the hoof or distal dislocation of the third phalanx), while a distinct depression and hemorrhagic effusion appear in the coronary sulcus13.

In the horse, the distance between the dorsal aspect of the third phalanx and the outer wall of the hoof must be less than 18 mm.

An increase in this distance is caused by an inflammatory swelling of the laminae with hemorrhage and oedema.

The lamellar tissue which holds the pedal bone in place and properly aligned, starts to lose consistency, then fails as the flexor tendon pulls on the phalanges and the downward rotation of the pedal bone begins, its tip tending to wedge itself under the sole.

Deformation of the hoof occurs mainly under the sole that bulges from concave to convex. If rotation of the third phalanx continues, its tip can eventually penetrate the sole of the foot. Sinking is less common and much more severe. It results when a significant failure of the interdigitation between the sensitive and insensitive laminae around a significant portion of the hoof occurs.

Chronic laminitis. Long-term cases usually are the consequence of one or more attacks of the acute form. In chronic laminitis, the horse rests on the back of the hoof of affected limbs thus deforming the hoof, raising the heel together with a sometimes conspicuous elongation (known as a «Turkish slipper») and deformation of the front part of the hoof wall14. The sole, too, is deformed and can show extensive avulsion at the white line. Moreover, in severe cases, there may be a continuous solution in the anterior portion of the sole into which the sole margin of the third phalanx has moved. This is due to the fact that when the third (or distal) phalanx is lowered towards the sole, it compresses it, thinning it, deforming it and, in severe cases, even perforating it (Figure 3).

Figure 4 - Three parasagittal sections of equine hoofs, normal (A) and laminitic (B, C). B-C: Sequence of the rotation and sinking process. B. The term rotation has commonly been used when the dorsal surface of the pedal bone (third phalanx) stretches or separates its distal (bottom) attachment from the hoof capsule and appears to rotate downward. C. Sinking. This condition occurs when the pedal bone loses most of its attachment to the hoof capsule and moves distally (downward) in the hoof capsule. If rotation of the third phalanx continues, its tip can eventually penetrate the sole of the foot. PB = Pedal bone (coffin bone, third phalanx). Yellow line = Indicates the extension of displacement of pedal bone and of hyperplasia of lamellar dermis forming a wedge in C. (Figure from ELPO, modified).

Ventral deviation, in a flexor direction, of the distal phalanx, is favoured both by the destruction of the dermal-epidermal junction, as well as by a hyperplasia of the epidermal laminae that produce a horny growth (keraphylocele), which, by progressively filling the residual space between the third phalanx and the inextensible hoof wall, acts as a wedge between these two parts (Figures 4 and 13). In the hoof, irregular strips of horny growth are also characteristic (so-called laminitic or founder rings) (Figure 14).

In order to explain chronic laminitis, it is believed that certain hyperreactive subjects suffer exacerbations, including subclinical ones, of acute laminitic episodes following exposure to antigenic stimulation from vaccinations or environmental allergens (hypothesis of immunological hyperreactivity). Where such hyperreactivity is not linked to autoimmune components, in reference to the strong induction of chemokines for neutrophils and to the persistent presence of neutrophils, these cells cause the per-

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Figure 5 - PAS stained histological section of normal hoof lamellae highlighting the basement membrane (arrows). Closely adherent to the SEL (Secondary epidermal lamella) basal cells, basement membrane (arrows) shows as a dark unbroken line. Connective tissue fills the secondary dermal lamellae (SDL). PEL = Primary epidermal lamella, anuclear, keratinised. Bar = 10 Âľm.

petuation of the inflammatory state and tissue alteration60.

MAIN RISK FACTORS There are four main risk factors for laminitis15: 1) diseases involving sepsis or endotoxemia; 2) metabolic diseases from endocrine origin; 3) lush pastures (pasture-associated laminitis) especially in ponies; 4) protracted trauma (load-bearing laminitis, supporting limb laminitis).

Cases of laminitis during sepsis or endo (entero) toxaemia cor-

Figure 7 - PAS stained histological section of grade 2 (moderate) laminitis. The basement membrane (BM) appears as a partially continuous line stained dark grey (compare with the dark unbroken line of the preceding figure 5 of control normal lamellar tissue). At the tips of the pointed secondary epidermal lamellae (SEL) the BM has continued the process seen in figure 6 by lifting from the underlying basal cells to form empty, teat-shaped caps (single arrowheads).

The BM has disappeared from the crypts between the SEL bases (double arrowheads). There is a reduced amount of connective tissue between the SELs that clump together to form amorphous BMfree masses, on either side of the lamellar axis. PEL = Primary epidermal lamella. (Figure from Pollitt C.C.64, modified).

relate with sepsis from Gram-negative polymicrobial bacteria, and include postpartum metritis with retained placenta, colic (proximal enteritis, volvulus) and enterocolitis. The systemic inflammatory events that occur in sepsis /SIRS (systemic inflammatory response syndrome) related laminitis coincide with marked increase in lamellar expression of a variety of inflammatory mediators including the cyclooxygenase (COX) enzyme, which catalyses the biosynthesis of prostanoids (prostaglandins, prostacyclin and thromboxane) from arachidonic acid. These results suggest that COX 2 and its metabolites are involved in the

Figure 6 - Laminitic hoof. Ultrastructure of the onset of acute laminitis in a secondary epidermal lamella. Early separation of the basement membrane (BM) by disconnection of anchoring filaments of hemidesmosomes. Dark arrow points to normal looking hemidesmosomes still having anchoring filaments (arrowhead) attaching the lamellar epidermal basal cell (LEBC) to the lamina densa (LD) of the basement membrane (BM). White arrow points to fading and disappearing hemidesmosomes as laminitis starts. D = Dermis. Transmission electron micrograph. Bar = 200 nm. (Figure from de Laat M.A. and Pollitt C.C.62, modified).

Figure 8 - (A) Photomicrograph of hoof lamellar dermis histology from normal horse. SEL = Secondary epidermal lamellae. PEL = Primary epidermal lamella. EE.

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Figure 9 - (B) Photomicrograph of hoof lamellar dermis histology from laminitic horse. Acute lamellar pathology 48h post-induction of euglycaemic hyperinsulinaemia. Cell stretching: SELs (Secondary epidermal lamellae) are lengthened, attenuated and distorted. PEL = Primary epidermal lamella. The s.c. cell stretching is also due to simultaneus acceleration of cellular necrosis-proliferation cycle. Cellular pathology precedes leucocyte infiltration and basement membrane pathology (fig. 6 and 7), indicating that the latter changes may be secondary or downstream events in hyperinsulinaemic laminitis.

EE. (Figure from de Laat et al23, modified ).

initiation of pathological changes seen in sepsis associated events such as sepsis related laminitis16.

Endocrinopathic and grazing-associated laminites are the most common forms. Hormonal disorders, with their consequent metabolic alterations, are responsible for up to 90% of the laminitis cases occurring in animals at pasture.

Most cases of laminitis appear in horses and especially ponies feeding on grass (hence the term pasture laminitis)19 and usually occur in obese horses and ponies and are exacerbated in animals that graze lush pastures. The risk of developing pasture laminitis lies in the dynamic interaction between predisposing factors (equine metabolic syndrome*, Cushingâ&#x20AC;&#x2122;s disease) and environmental conditions, particularly the abundance of non-structural carbohydrates contained in pasture fodder.

Through analysis of High density single nucleotide polymorphism (SNP) genotype data it was determined that eight measured biochemical traits associated with equine metabolic syndrome (EMS) were moderately to highly heritable in Welsh ponies and Morgan horses17.

Laminitis cases subsequent to protracted or biomechanical traumas are mainly linked to bearing excessive weight on a limb for long periods due to an inability to use the contralateral limb where it has been immobilized due to severe lameness, orthopedic procedures or paralysis.

357

of the secondary lamellae, attributed to a mechanical disorder of the hoof as a sequela to disease, while others have focussed on the lamellar and sublamellar regions with findings of hemorrhage, perivasal infiltrates, oedema, alterations of the vascular endothelium, the presence of platelets and fibrin emboli. Cytoplasmic vacuolations, nuclear pyknosis and the loss of cellular integrity, being obvious expressions of necrosis27, and the separation between dermal and epidermal components at the basement membrane (Figure 6), in the early stages of laminitis, are all commonly regarded as changes to the lamellar epidermis.

During the acute phase of laminitis, there is a tendency for the basement membrane to lose stainability, especially at the base of the secondary epidermal lamellar cells26 (Figure 7). Moreover, at the top of these there are bistratified bullous residues of the detached basement membranes, while as the lesion progresses, detachment occurs with total loss of connection, leaving just a few bullous lesions.

Immunohistochemistry has shown the loss of key lamellar basement membrane components such as collagen IV and laminin.

Key factors in laminitis are the loss of the anchoring filament protein laminin-332 (Ln-332, one of the main laminin isoforms of the epithelial basement membrane) and the numerical reduction in laminin-associated hemidesmosomes connecting the basement membrane to epithelial cells 47 48 (Figure 6).

In some cases of hyperacute laminitis, no separation of basal cells from the basement membrane is observed, but the key histopathological event is epidermal necrosis27.

Lesions of the lamellae during the acute phase of laminitis manifest as a sequela to a pathology unconnected with the hoof. In fact, laminitis is associated, as an epiphenomenon, with a variety of predisposing conditions - excessive intake of cereals or fodder rich in non-structural carbohydrates (simple sugars, starch and fructan, a polymer of fructose molecules), i.e. highly digestible carbohydrates, consumption of black walnut wood shavings, colitis, pneumonia, retained placenta, metritis. Over half of the cases of laminitis are associated with alterations of the distal part of the intestine.

Researchers have used these pathological findings on the one hand to correlate them to the severity of lameness and to disease duration, and on the other to support, reject or advance etiopathogenetic hypotheses.

PATHOGENESIS OF LAMINITIS The main failure in laminitis cases is the disruption of the structural integrity of the dermo-epidermal bond of the digital lamellae, i.e. loss of the adherence of the basal epithelial cells in the epidermal lamellae to the underlying dermal lamellae19 . But this stage in the pathology of the digit can be reached through vascular, enzymatic, inflammatory or mechanical mechanisms, or by a combination thereof.

According to the histopathology of submural tissues20 21 22 23 24 25 26 , some have focused on the structural alterations to the lamellar interface, such as the bevelling and the alterations to the axis

Figure 10 - (A) High magnification histology of hoof normal lamellar dermis. The secondary epidermal lamellae (SEL) are uniform in length and have rounded tips. The lamellar basement membrane is closely apposed to the SEL perimeter (white arrowheads). EE.

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Figure 11 - (B) High magnification histology of lamellar dermis in laminitic horse. Severe lengthening and distortion of lamellar architecture. SELs (Secondary epidermal lamellae) are lengthened, attenuated with pointed tips, s.c. cell stretching (black arrowheads).

Wavy strands of basement membrane, no longer apposed to basal cells, are present at SEL tips (white arrowheads). Many basal cell nuclei are oval-shaped but oriented parallel to the SEL axis instead of perpendicular. EE. (Figure from Walsh D.M. et al.37, modified).

Metabolic-toxic hypothesis The metabolic-toxic pathogenetic hypothesis proposes that the initial insult to the hoof is directed against the cornifying epithelium of the lamellar interface. In this context, one or more factors of haematic origin (circulating toxins or enzymes associated with a primary disease not originating in the hoof) could act by altering a biochemical step that is critical for the vitality of the lamellar epidermis or for normal cornification.

Supporters of this metabolic-toxic hypothesis8 claim the severity of dermo-epidermal alterations is evidence that epidermal cells are the target of haematogenous triggers. They also suggest that the other epidermal and dermal changes observed in horses with laminitis are secondary consequences of inflammatory processes or depend on mechanical defects of the affected digits. The haematogenous triggering factors and their role in the onset of laminitis would converge in the dysfunctional regulation of the enzymes responsible for maintaining the attachment of the basement membrane to the basal epithelial cells. The morphological evidence of splitting of the basement membrane is seen as supporting this hypothesis and the consequent dysadesion between the basement membrane and the basal cells of the epidermal lamellae (Figure 6) are regarded as the effect of activating extracellular matrix metalloproteinases (MMPs)28 29 .

Evidence for the idea that reperfusion injury could also contribute to the development of laminitis comes with a biphasic episode of lamellar ischaemia interrupted by a period of hyperemia in the early stages of experimentally-induced laminitis in horses treated with black walnut extract (Juglans nigra), that mimics a systemic inflammatory reaction syndrome30. Endothelial vascular changes, the presence of platelets, fibrin thrombi and hemorrhage are considered to be signs of injury to digital blood flow.

In essence, the dysadhesion of the lamellar basement membrane is regarded as a later step in the pathogenesis of acute laminitis, while the primary cause of laminitis becomes vascular hypoperfusion with regressive alterations of lamellar epidermal cells.

The discovery of a new neurotransmitter system mediated by the neuromedin U peptide, which effectively and potently induces constriction of the equine common digital vein without affecting arterial tone, contributed to an understanding of the factors regulating digital blood flow. And this confirms existing knowledge that digital veins are more sensitive and reactive than digital arteries to vasoconstrictor agents (noradrenaline, 5-hydroxytryptamine, endothelin-1) and vasodilators (β-adrenergic agonists) and is consistent with the regional haemodynamic regulation of the hoof which is mainly controlled by the venous compartment. The vasculature of the hoof wall displays specific angio-adaptations to high pressure and tensile load31. However, his unique structural and functional features, such as high hydrostatic pressure and numerous arteriovenous anastomoses, make digital blood flow in the horse more susceptible and vulnerable to dysfunction. Haemodynamic disorders responsible for regional hypoxia-ischemia can also induce an upregulation-activation of destructive proteinases such as extracellular matrix metalloproteinases (MMP-2, -9 and other MMPs) in digital laminae.

Damage to the basement membrane and consequent lamellar separation may result. Such damage can also lead in the hoof to vessel hyperpermeability, to diapedesis of neutrophils and to inflammation.

Hormonal hypothesis (Endocrinopathic laminitis) As mentioned above, laminitis is a pathological syndrome in which the effects of hormonal dysfunctions (endocrinopathic

Vascular hypothesis The other hypothesis, of a vascular pathogenesis of laminitis (the vascular theory, based on a model of ischaemia-reperfusion injury), is based on the histopathological data in which epidermal changes during the onset of laminitis are considered to be consequences of a blood flow disorder occurring before the onset of lameness. The resulting phenomenon of ischaemia/hypoperfusion followed by reperfusion could result in damage to epidermal cells and an inflammatory reaction with secondary onset of hyperemia and secondary structural impairment due to alteration (destruction and displacement) of the lamellar basement membrane. The damage to the cells of the basal layer, considered to be necrotic27, is also documented by an increase in apoptotic and regenerative mitotic phenomena in these cells8.

Figure 12 - Horse's foot with chronic laminitis. X-rays (radiograph) showing rotated pedal bone (PB) and chronic remodeling of the pedal bone and hoofâ&#x20AC;&#x2122; sole. (Figure from canberraequinehospital.com.au/).

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Figure 13 - (A) Parasagittal section of normal hoof. (B) Parasagittal section of severe chronic laminitic hoof. Pedal bone rotated and forced downwards and the sole has become convex. The pedal bone shows remodelling and atrophy. K = keraphylocele forming wedge as the distal pedal bone rotates and displaces distally. PB = Pedal bone (third, distal phalanx). >>>> = Direction of rotation of pedal bone. ET = Extensor tendon. FT = Flexor tendon. WL = White line. LD = Lamellar Dermis. (Figure from Marcato P.S.63, modified).

laminitis) are great. Equine endocrinopathic laminitis, associated with conditions such as equine metabolic syndrome* (EMS), pituitary pars intermedia dysfunction (PPID), and exogenous corticosteroid administration, is the most common type of laminitis encountered in equine veterinary practice32. Common to these disorders appears to be disturbed glucose and insulin regulation, now termed insulin dysregulation (ID).

In a research on horses and ponies with acute laminitis, an endocrinopathy was identified in 94% of the cases. Of these endocrinopathies, equine metabolic syndrome (EMS) was the most prevalent underlying disease occurring in 82% of cases, whereas pituitary pars intermedia dysfunction (PPID) was present in ~38% of the animals33.

Pasture-associated endocrinopathic laminitis is common, and occurs most frequently in spring and summer, suggesting that particular pasture conditions may exacerbate the syndrome. In horses predisposed to laminitis, although the triggering factor is represented mostly by pasture34, there is a background endocrinopathy in up to 90% of cases and usually in overweight animals.

The unifying pathogenic factor, in endocrinopathic laminitis (including equine metabolic syndrome* and Cushing’s disease) and in so-called pasture laminitis, is hyperinsulinemia35 36. When horses digest sugar and starch in the gastrointestinal tract, and absorb glucose, they also release insulin from the pancreas to aid glucose uptake into the cells, which makes the cells sensitive to insulin. When horses eat large amounts of non-structural carbohydrates [such as those in the CHO experiment (administering a CarboHydrate Overload) and in certain lush pastures], the cells become insulin resistant. In such cases, the horse is said to have insulin resistance (IR). This decreases glucose uptake from the blood by the cells, normally enhanced by insulin. And this depletes the supply of glucose, or its metabolization in cells, including those of the foot.

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Insulin toxicity appeared to be a key factor in triggering equine laminitis. The onset of laminitis is associated with plasma insulin that exceeds 100 µIU/ml [normal range = 8 to 30 µIU /ml]37.

Insulin has vasoregulatory effects, which is considered one of the plausible links between IR and laminitis. In response to insulin, vasodilation normally occurs due to increased synthesis of nitric oxide (NO) by endothelial cells. However, insulin can also induce vasoconstriction by stimulating endothelin-1 (ET-1) synthesis and activating the sympathetic nervous system. Activation of IGF-1Rc [the insulin-like growth factor (IGF)-1 (tyrosine kinase) receptor] stimulates at least two different signaling pathways in vascular endothelial cells23. NO is secreted when the phosphatidylinositol 3-kinase (PI3K) pathway is activated, whereas activation of the mitogen-activated protein kinase (MAPK) pathway causes the release of ET-1. It is known that IR states involve inhibition of the NO synthesis pathway by PI3K, while the MAPK pathway may be overstimulated due to compensatory hyperinsulinemia, leading to an increase in ET-1 synthesis. Therefore, as NO production decreases, vasoconstriction can be promoted in the insulin-resistant animal and this alters the ability of the vessels to respond to stimulation. Histological examination in horses in the pre-clinical phase of insulin-induced laminitis (Figure 9) have shown that significant changes in the lamellae occur before the onset of clinical symptoms.

Gene expression and immunohistochemistry studies have indicated that the lamellae are sparsely populated with insulin receptors, whereas IGF-1 receptors (IGF-1R) are abundant, suggesting that the action of insulin may be mediated by insulin binding to the IGF-1R. Radioligand-binding studies using 125I-IGF1 and 125I-insulin confirmed an abundance of high-affinity IGF1R in lamellae (KD 0.16 nM, Bmax 243 fmol/mg protein)46. However, the affinity of insulin for binding to the lamellar IGF-1R (Ki 934 nM) was >5,800 fold less than that of IGF-1, suggesting that insulin is unlikely to bind and to activate equine IGF1R in vivo, even at high physiological concentrations. Moreover there was no evidence to support the presence of insulin/IGF1 hybrid receptors in lamellae. These findings suggest that insulin does not act directly through IGF-1 receptors and that an alternative theory is required to explain the mechanism of insulin action in laminitis.

In conclusion, insulin is capable of binding to equine IGF-1 re-

Figure 14 - A mare’s hoof with chronic endocrinopathic laminitis resulting from repeated episodes of acute laminitis, as evidenced by multiple “founder rings” (white arrowheads) around the hoof wall, which generally indicates a laminitis of long duration. (Figure from alleghenyequine.net).

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ceptors at very high concentrations, but these concentrations exceed the levels reached in horses and ponies in vivo, even in the most severely insulin-dysregulated animals.

According to Nanayakkara et al.43 the mechanism by which insulin causes laminitis remains still unknown, but an indirect action via stimulation of the IGF-1 receptor appears to be highly unlikely.

However, Patterson-Kane et al.15 suggest that one indirect mechanism through which insulin could activate IGF-1R should be envisaged in the displacing IGF-1 from IGF-binding proteins (IGFBPs) such as IGFBP7 or fragments of IGFBP3, thereby increasing free IGF-1 concentrations, or in a direct action on a very small population of lamellar InsR. This would explain the apparent causal link between hyperinsulinaemia and laminitis and it would be consistent with the presence of mitotic figures in the epithelial cells examined from the tissues of horses exposed to supra-physiological intravenous infusions of insulin.

Future work will determine how IGF-1 receptor-mediated cell stimulation might lead to weakening and elongation of the secondary epidermal lamellae (lamellar epithelial cell stretching).

Whether there is a direct hormonal influence on cellular cytoskeletal mechanics will likely necessitate in vitro experimentation with superior lamellar cell/explant culture models.

EXPERIMENTAL MODELS OF LAMINITIS The pathogenetic mechanisms of laminitis struggle to find unequivocal explanations, but there is little doubt that experimental models of laminitis have led to significant progress in understanding.

Sepsis /SIRS (systemic inflammatory response syndrome) related laminitis. In well-tested experimental models reproducing laminitis, oral administration of either carbohydrate (starch or oligofructose) or black walnut heartwood extract is used8. In carbohydrate overload, a disturbance of the intestinal bacterial flora can be detected along with an alteration of intestinal permeability, a sign of structural tissue alteration. Furthermore, many feel that laminitis results in the release of biologically active products, derived both from bacteria and from the host. Development of laminitis as a result of starch overload, suggests that changes in intestinal microbiota may also influence diseases outside of the equine gastro-intestinal tissue61.

The experimental model of laminitis that involves administering a carbohydrate overload, known as the CHO model, is the one that most realistically simulates clinical cases of sepsis-related laminitis (e.g. enterocolitis, occlusion of the large intestine, acute metritis) and also those resulting from so-called pasture laminitis50. Indeed, carbohydrate overload causes a reduction in caecum pH, an increase in bacteria which produce lactic acid, destroy Gram-negative bacteria which release vasoactive endotoxins and a substantial alteration of the cecal mucosal barrier resulting in absorption of pathogenic bacterial molecules, including endotoxins.

Bacterial endotoxins are capable of causing haemodynamic alterations in the vascular bed of the hoof by modifying the local quali-quantitative balance of vasoactive substances or the responsiveness to them of the vessels or variously interfering with the functionality of receptor systems that regulate vasomotility. This can occur through endothelium-dependent increase of digital artery relaxation mediated by β-adrenergic agonist51.

It is also believed that the rapid development of bacteria of the genus Streptococcus in the cecum and colon, observed in parallel with laminitis induced with carbohydrate overload, can directly cause laminitis through the production of exotoxins capable of activating matrix metalloproteinases (MMPs) present in the lamellar structure. Activated MMPs can break down hemidesmosomes, which are essential components of the basement membrane, ultimately causing detachment of the basement membrane from the basal epidermal cells33.

Thus, the experiment confirms that nutrition can simulate the laminitis that occurs as a result of systemic inflammation usually as a sequela to inflammatory processes and destruction of microbial populations in the large intestine (so-called inflammatory laminitis).

Administering more starch than the small intestine can digest causes indigestible materials to pass through to the large intestine. Moreover, fructans, although partly subject to acid hydrolysis or fermentation in the anterior intestine, are thought to still pass in considerable quantities and relatively unmodified into the large intestine where, due to intense proliferation of amylolytic and saccharolytic bacteria producing lactic acid, pH is greatly reduced, leading to mucosal damage.

So-called inflammatory laminitis is also characterized by activation of neutrophilic granulocytes and their migration from the blood into the tissues of the lamellae where they make a significant contribution to the alteration of these structures52. The importance of neutrophils in the laminitis development phase and the systemic nature of the inflammatory process are confirmed by the presence of neutrophil elastase (a protease released by azurophilic granules) in the plasma, skin and lamellar tissues.

Furthermore, granulocyte elastase is an enzyme that acts by disintegrating the basement membrane of the lamellae. Pro-inflammatory cytokines (chemokines CXCL1, CXCL6 and CXCL8) play a key role in granulocyte activation, migration and degranulation53. Maximum granulocyte infiltration and epithelial alteration occur at the onset of laminitis. However, in experimental inflammatory laminitis, the increase in concentration of chemokines in the lamellae comes before the accumulation of granulocytes and lesion of the lamellae have reached their peak.

This shows that the intervention of granulocytes is the cause of, and not a reaction to, the degradation of the basement membrane and the structural alteration of the lamellar tissue.

Also in the experimental Black walnut extract (BWE) model of laminitis, the inflammatory process in the lamellae is very evident and documented by the increase in proinflammatory cytokines, chemokines, cyclooxygenase (COX)-254 and the endothelial adhesion molecules needed for the adhesion and diapedesis of leukocytes 30. In these experimental conditions of systemic inflammation, several affected organs, such as the stomach and skin, are made up of epithelial tissues and therefore it is plausible that extensive degradation of the components of their basement membranes is also required. However the pathology is more manifest and harmful in the equine foot as the dysadhesion-dislocation of the lamellar basement membrane, a phenomenon that is only detected in the hoof in the case of laminitis, is favoured by the weight-bearing on the hoof and by the locomotor forces that under normal conditions the hoof would be able to withstand without damage.

Endocrinopatic laminitis. Digital lamellae from insulin treated ponies were attenuated and elongated with many epidermal basal cells (EBC) in mitosis49. Unlike carbohydrate induced laminitis in horses there was no global separation at the lamel-

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lar dermal/epidermal interface among ponies. Sporadic EBC basement membrane (BM) separation was associated with the proximity of infiltrating leucocytes. Lamellar proliferation may be an insulin effect through its mitogenic pathway. Aberrant lamellar mitosis may lengthen and weaken the lamellar, distal phalanx attachment apparatus and contribute to the clinical signs that developed. Examination by TEM showed excessive waviness of the basement membrane zone and pointed tips of some secondary epidermal lamellae, an ultrastructural lesion typical of laminitis. The average number of hemidesmosomes/microm of basement membrane was decreased and their distance from the centre of the lamina densa of the basement membrane was increased49.

Insulin weakens the structural integrity of equine lamellar explants and an ex vivo model for evaluation of hyperinsulinaemia induced lamellar failure was established. The histological evaluation of explants of digital lamellae incubated with insulin validated the structural failure that occurs through the separation of the dermo-epidermal attachment at the basement membrane (BM) level38 39. Ultrastructural study62 reveals that in the acute phase of insulin-induced laminitis the BM zone is extensively disorganized with loss of uniformity of the lamina lucida and lamina densa, reduction, fragmentation and disorientation of hemidesmosomes (HDs), and cytoskeletal disengagement of the HDs contributing to the weakening of the dermoepidermal junction and lamellar failure (Figure 6). HD loss is also accounted by disassembly of HDs during excessive cellular proliferation, secondary to hyperinsulinemia.

In experimentally-induced laminitis with prolonged insulin infusion, leukocyte infiltration of the lamellae is very low or absent, but there is nevertheless a link between hyperinsulinemia and inflammation as the MAPK (mitogen-activated protein kinase)-dependent signaling pathway is implicated in neutrophil migration/diapedesis40. As mentioned above, MAPK is stimulated by insulin and correlated with insulin-stimulated cell proliferation in various tissues, and is also activated by various cytokines and inflammatory mediators.

Other studies32 also provide evidence of inflammatory signaling (pro-inflammatory cytokines and chemokines detected) within the digital lamellae of horses experiencing prolonged supraphysiologic hyperinsulinemia by euglycemic hyperinsulinemic clamp (EHC) technique. However, it seems unlikely that this signaling, possibly elicited through exposure to increased concentrations of glucose, insulin (acting through insulin receptor and/or IGF-1 receptor), leptin, or other metabolic intermediates, represents established, “classic” inflammation (where leukocyte emigration is a hallmark of inflammation) as a primary pathophysiologic mechanism involved in endocrinopathic laminitis.

Since in agreement with previous reports, very little to no evidence of leukocyte emigration into lamellar tissue was observed in response to this model. Rather, the lamellar inflammatory signaling may instead be an evidence of “cross talk between metabolic regulatory signaling pathways and inflammatory pathways” occurring in response to altered intracellular concentrations of energetic metabolites, which has been documented to occur in multiple cell types in vitro and in vivo41. Moreover, the lack of significant lamellar inflammatory cell infiltrates accompanying this model suggests that the source of pro-inflammatory mediators detected within lamellar tissue is likely to be the major cell type in this tissue, the lamellar keratinocyte.

Equine laminitis also involves epidermal cell proliferation, in this case in the epidermal basal cells of the digital lamellae. Hyper-

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insulinemia produces a more hyperplastic lesion where proliferation and distortion (stretching) of lamellar epidermal basal cells results in lamellar thinning and lengthening (Figure 9). The proliferative component of the response suggests a growth factor type role for insulin in the instigation of laminitis.

In insulin-dysregulated ponies, mean, post-prandial EGF concentrations were found almost three times higher than in healthy ponies, but the same authors in an investigation of the equine epidermal growth factor (EGF) system have found no evidence of an increased expression (neither gene nor protein) of the lamellar EGFR (Epithelial grow factor receptor) during experimentally-induced laminitis42. EGFR is unlikely to be a pathogenic factor in insulin-associated laminitis pathophysiology, but it might play a role, at least in part, in epidermal repair.

Although the EGFR does not appear to play a major pathogenic role in hyperinsulinemic laminitis, the significance of increased EGF in insulin-dysregulated ponies deserves further investigation.

Very few insulin receptors (InsR) were identified in the lamellae43, and more importantly they are not located on the epidermal basal cells44. Therefore the mechanism by which insulin causes cell proliferation is not known. However, in other species, insulin at high concentrations is known to directly stimulate receptors for insulin-like growth factor-1 (IGF-1), which is a powerful cell mitogen. Reorganisation of the cytoskeleton can occur in response to receptor activation, largely studied in the context of cancer cell motility, migration and epithelial to mesenchymal transition (EMT)45.

Support limb laminitis. A work has established an experimental model to study preferential weight bearing and initial results have suggested that lamellar hypoxia may act in the pathogenesis of supporting limb laminitis18.

Further experimental mechanistic research is warranted to explore any causal relationship(s) between hyperinsulinaemia and cellular changes15. Investigations should focus on: (1) the earliest changes, possible invisible histologically or ultra-structurally; (2) analyses of specific tissue compartments using NGS (Next Generation Sequencing) and/or proteomics, in particular of basal lamellar epithelial cells; and (3) investigation of possible links between insulin, IGF-1R, cellular tensegrity, inflammatory mediators, and BM composition/structure.

CONCLUSIONS Laminitis, rather than a distinct morbid entity, must be better considered a clinical syndrome associated with (1) a systemic inflammatory response syndrome (SIRS), inflammatory laminitis, or with (2) a systemic pathology with endocrinopathy, endocrinopathic laminitis, or (3) associated, much less frequently, with protracted traumatism or biomechanical causes, load laminitis, support limb laminitis.

Studies in the Europe and USA agree that endocrine-associated laminitis (endocrinopathic laminitis) is the predominant form of 90% in horses presenting primarily with lameness 55.

The main endocrinopathic disorders that result in laminitis are the pituitary pars intermedia dysfunction (PPID) and/or other endocrine diseases (today collectively called equine metabolic syndrome - EMS -), the first characterized by obesity, insulin resistance and laminitis35.

Instead of the pathogenetic definition of laminitis as a primary and serious pathology of the basement membrane 48 62, current

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investigations on the predominant endocrinopathic laminitis propose as a key early pathophysiological event a variable subclinical phase centered on the stretching and elongation of the cells of the epidermal lamellae (lamellar epithelial cell stretching)23 (Figures 9 and 11).

In endocrinopathic laminitis, hyperinsulinemia is expected to alter the metabolism of basal epidermal cells, not with a direct effect on cells, but through the activating binding of insulin with the IGF-1Rc receptor [insulin-like growth factor (IGF)-1(tyrosine kinase) receptor] also present on epidermal cells56. As has been observed in other cell types, the IGF-1Rc could interact dynamically also with integrins, intercellular adhesion molecules, and cytoskeleton. The alteration of the cytoskeleton of the cells of the secondary epidermal lamellae62 would reduce their ability to support normal biochemical stress on the lamellae57. The cytoskeleton is in fact an arbiter of the shape and mechanical properties of the cell with microtubules and actin filaments that act respectively as compression-resistant elements and as traction components.

To complete the IGF-1Rc intervention there is also the regulation of two important signaling pathways: p44 / 42 MAPK (mitogen-activated protein kinase) and PI3K (phosphatidylinositol 3-kinase) / Akt, the latter also capable of regulate cell adhesion, by dissolving hemidesmosomes58 (Figure 6), and altering the normal organization of the cytoskeleton. In turn, the activation by the two pathways of proteins, via phosphorylation of serine, threonine and tyrosine, can converge in the downstream activation of mTORC1 complex and RPS6 29, proteins both involved in producing epithelial cell dysplasia.

Cytomorphological alterations can result from damage to the cytoskeleton, such as loss of perpendicular orientation of the nuclei of the secondary epidermal lamellae with respect to their basement membrane, assumption of the ovoid shape of the nuclei that from apical become more central in the cytoplasm (Figure 11), while the nucleoli become prominent and with more random orientation. And the weakening and elongation (stretching) of the lamellar epithelial cells (which translate into alteration of their tensegrity, an architectural system in the structures that stabilizes them) may well induce some secondary alteration in the structure and / or elasticity of the basement membrane, and thus a further relaxation of the lamellae followed by loss of adhesion of the layer of basal epithelial lamellar cells with the underlying basement membrane59.

The basal lamellar epithelial cells participate in the suspension of the distal phalanx on the stratum internum of the hoof capsule and have firmly maintained hooks with their basement membrane. Therefore the first motive that begins to compromise the suspension system of the distal phalanx in the hoof causing its descent is the alteration of the basal epithelial lamellar cells which determines their detachment from the basement membrane.

—————* The term equine metabolic syndrome was first introduced in 2002 to propose that obesity, insulin resistance and laminitis were all components of a single clinical syndrome identified in horses and foals. Similarly to the human syndrome of the same name, equine metabolic syndrome is a condition of insulin resistance that derives from the hypersecretion of tissue cortisol (under the effect of 11-β-hydroxysteroid-dehydrogenase) present in adipose tissue, without any pituitary involvement (equine Cushing’s syndrome). The associated clinical signs are a tendency to obesity and the notable prevalence of chronic laminitis in middle-aged animals. Diagnosis is based on clinical data and confirmation of persistent hyperinsulinemia and hyperglycaemia after intravenous administration of a glucose bolus.

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