Large Animal Review 4 - 2021 by E.V. Soc. Cons. a r.l.

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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

04/21

Bimonthly, Year 27, Number 4, August 2021

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 Fatty Acids as Biomarkers of Metabolic Diseases in Dairy Cows identified through Thin Layer Chromatography and Gas Chromatographic Techniques (TLC-GC) • Central corneal thickness increases with age in cattle

OVINE • The relationship between placental characteristics and lamb birth weight in Akkaraman Turkish native sheep breed

SWINE • La minaccia globale della peste suina africana in una prospettiva One Health

EQUINE • “Iron and fire”: history and advances of mule shoeing

POULTRY • Effects of short-term and combined use of thyme powder and aqueous extract in broilers

CASE REPORTS BOVINE • Hereditary zinc deficiency syndrome in a calf

OVINE • Fetal retention due to unilateral partial uterine horn torsion in a ewe

SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

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INDEX

BOVINE

Anno 27, numero 4, Agosto 2021

ROSSELLA TESSARI, ELISA MAZZOTTA, FRANCESCA BLASI, MASSIMO MORGANTE, TAMARA BADON, SILVIA BEDIN, GIORGIA FABBRI, ANASTASIA LISUZZO, BARBARA CONTIERO, ENRICO FIORE, 187 MICHELE BERLANDA

Rivista indicizzata su: CAB ABSTRACTS e GLOBAL HEALTH IMPACT FACTOR (2021): 0.417 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 Managing Editor: Matteo Gianesella

Milk Fatty Acids as Biomarkers of Metabolic Diseases in Dairy Cows identified through Thin Layer Chromatography and Gas Chromatographic Techniques (TLC-GC)

Central corneal thickness increases with age in cattle BUSRA KIBAR KURT, ÖMER KURT, KAĞAN KOCASARI, TOLGA MERIÇ DÜMBEK, MEHMET ERDEM, ROJDA YİĞİT, EMRE GÜRDAL

195

OVINE The relationship between placental characteristics and lamb birth weight in Akkaraman Turkish native sheep breed UĞUR ŞEN

201

Technical Editor: Enrico Fiore 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.

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

SWINE La minaccia globale della peste suina africana in una prospettiva One Health FRANCESCA CIMINO, CARMEN MARESCA, MARIA LUISA MARENZONI, FRANCESCO FELIZIANI

Direttore Responsabile Antonio Manfredi Stampa Press Point - Via Cagnola, 35 20081 Abbiategrasso (MI) - Tel. 02/9462323

EQUINE “Iron and fire”: history and advances of mule shoeing GIANLUCA CELANI, DOMENICO ROBBE, PRISCO MARTUCCI, LUCIO PETRIZZI, PAOLA STRATICÒ, AUGUSTO CARLUCCIO

Prezzo di copertina: € 10,00. La rivista è inviata a tutti i veterinari interessati ai settori degli animali da reddito con il versamento di € 52,00 per l’Italia; € 62,00 per l’Estero. Servizio abbonamenti: Tel. 0372/403507. Ai Soci SIVAR in regola con il pagamento della quota associativa, la rivista è inviata gratuitamente in quanto la quota è comprensiva dell’abbonamento alla rivista stessa.

215

POULTRY Effects of short-term and combined use of thyme powder and aqueous extract on growth performance, carcass and organ characteristics, blood constituents, enzymes, immunity, intestinal morphology and fatty acid profile of breast meat in broilers MAJID BELALI, ALIREZA SEIDAVI AND MEHRDAD BOUYEH

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. Palazzo Trecchi - 26100 Cremona Ufficio Pubblicità: Paola Orioli Tel. 0372/403539 - E-mail: info@sivarnet.it

209

223

CASE REPORTS BOVINE

Hereditary zinc deficiency syndrome in a calf ZAFER USTA, SILKE DUDERSTADT, TERESA PUNSMANN, MARTIN HÖLTERSHINKEN, MARION HEWICKER-TRAUTWEIN, OTTMAR DISTL

233

OVINE Fetal retention due to unilateral partial uterine horn torsion in a ewe EMSAL SİNEM ÖZDEMİR SALCI, ABID HUSSAIN SHAHZAD

237

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R. Tessari et al. Large Animal Review 2021; 27: 187-193

Milk Fatty Acids as Biomarkers of Metabolic Diseases in Dairy Cows identified through Thin Layer Chromatography and Gas Chromatographic Techniques (TLC-GC)

187

ROSSELLA TESSARI1, ELISA MAZZOTTA1, FRANCESCA BLASI2, MASSIMO MORGANTE1, TAMARA BADON1, SILVIA BEDIN1, GIORGIA FABBRI1, ANASTASIA LISUZZO1, BARBARA CONTIERO1, ENRICO FIORE1, MICHELE BERLANDA1,* 1

Department of Animal Medicine, Production and Health, University of Padua, Viale dell’Università 16, 35020 Legnaro (PD), Italy Department of Pharmaceutical Sciences, Via San Costanzo, 06126, University of Perugia, Perugia, 06123, Italy

SUMMARY In the transition period an excessive mobilization of adipose tissue in high milk production dairy cows predisposes to metabolic diseases as subclinical ketosis. The aim of this research was to identify the association between the concentration of milk fatty acids and the elevated plasmatic value of Non Esterified Fatty Acid (NEFA) for the diagnosis of excessive lipomobilization in dairy cows using Thin Layer Chromatography and Gas Chromatographic Techniques (TLC-GC). Fifty-four multiparous Holstein-Friesian dairy cows in the first phase of lactation were enrolled in the study. Blood samples from the coccygeal vein were collected and Non-Esterified Fatty Acids (NEFA) was evaluated in laboratory of University of Padua. Milk samples (40 mL) were taken at the evening milking from each bovine enrolled in the trial. Animals were divided into two groups on the basis of blood NEFA: healthy animals (NEFA-0) with a value of NEFA ≤ 0.57 mEq/L and sick animals (NEFA-1) with a value of NEFA > 0.57 mEq/L. Milk fatty acids concentrations have been evaluated in 4 lipid classes: Free Fatty Acids (FFA), Cholesterol Esters (CE), Phospholipids (PL), and Triglycerides (TG). Data were analysed using SAS system software (version 9.4; SAS Institute Inc., Cary, NC, USA). The General Linear Model (GLM) analysis was performed for repeated measurements in order to evaluate the differences in the composition of milk fatty acids related to the four lipid fractions in function of two different NEFA blood concentrations (NEFA0 vs NEFA-1). The results showed the following statistical significance (p ≤ 0.05) in the milk lipid classes: two fatty acids were significant in CE, one fatty acid was significant in FFA, nine fatty acids were significant in TG and one fatty acid was significant in PL. These milk fatty acids, with predictive value for the development of metabolic disorder, could be considered valuable new biomarkers.

KEY WORDS Transition period, non-esterified fatty acid, milk fatty acids, lipid classes, thin layer-gas chromatographic techniques.

INTRODUCTION In the transition period the energy demand for fetal growth and the lactogenesis, results in an energy deficiency1. This energy disequilibrium is expressed through a negative energy balance (NEB) aggravated by the reduced of Dry Matter Intake (DMI). The NEB leads to a release of Non-Esterified Fatty Acids into the blood as a result of an enhancement of lipolysis2,3. NEFA are biomarkers of excessive lipid mobilization in high milk production dairy cows. The increased plasma concentration of NEFA is associated with a grater incidence of metabolic disorder such as ketosis and fatty liver4. Under normal energy balance conditions, the plasma concentration of NEFA in dairy cows is less than 0.2 mEq/L5. In prepartum, the blood concentration of NEFA begins to increase up to a threshold, which

Corresponding Author: Michele Berlanda (michele.berlanda@unipd.it).

should not exceed 0.29 mEq/L. Value of NEFA higher than the cut-off indicates an increased risk of developing metabolic pathologies. After calving the threshold is greater than 0.57 mEq/L6,7. Within the first 5-6 weeks of lactation, the plasma concentration of NEFA should stabilize around a value of 0.300 mEq/L in healthy dairy cows8. The excessive mobilization of adipose tissue precedes subclinical - clinical ketosis development and mobilized fatty acids (FA) are incorporated in milk fat. Changes in milk FA composition might be an earlier indicator of hyperketonemia9,10. The milk lipid concentration higher than 4.8% is indicative of a severe NEB, and consequently of a high blood NEFA concentration11. Lipids in milk derive either from the ex novo synthesis at the mammary gland level or from the uptake into bloodstream. The substrates used for the ex-novo synthesis are acetic acid and butyric acid. Short and medium chain fatty acids (C4:0 - C14:0) and 50% of fatty acids with a hydrocarbon chain consisting of 16 carbon atoms, are synthesized in the epithelial cells of mammary gland12,13. The uptake of fatty acids by the mammary gland

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Milk Fatty Acids as Biomarkers of Metabolic Diseases in Dairy Cows identified through Thin Layer Chromatography

from the bloodstream provides part of the fatty acids with 16 carbon atoms and all the Long Chain Fatty Acids (LCFA)14. The different plasma fatty acids, mobilized from the lipolysis of the adipose tissue, contribute to composition of the milk lipid profile15. NEFA, mostly released into circulation from body reserves, are C16:0, C18:0, and C18:1 ω 916,17 and their uptake by the mammary gland is directly proportional to their blood concentration12. A percentage variable from 96 to 98% of total milk lipids derives from the class of Triglycerides (TG) which constitute cell membranes. The residual percentage is compound of Phospholipids (PL), Cholesterol (CO) and Cholesterol Esters (CE)18. The content of Free Fatty Acids (FFA) is very limited19. The milk fatty acid present in high concentration are Saturated (SFA- 70%) and Monounsaturated (MUFA-25%)20,21. Oleic acid (C18:1 ω 9) is the main MUFA present in milk and is also used as a biomarker of excessive concentration of NEFA in the blood17. Myristic acid (C14:0), Palmitic acid (C16:0) and Stearic acid (C18:0) are the SFA present in greater quantities in milk21. Polyunsaturated Fatty Acids (PUFA) are more concentrated in the lipid fractions of PL and CE, associated with High Density Lipoprotein (HDL) for transport in the bloodstream. The uptake by the mammary gland of lipids from HDL is very low, and for this reason PUFA have lower concentrations in milk, about 5% of the total12,20. Dairy cows after calving, present SFA in lower concentrations but these fatty acids increase up to the twelfth week when the energy balance improves. On the other hand, MUFA, mostly represented by C18:1 ω 9, increase the amount in the first phase of lactation and the decrement occurs with the improvement of the NEB22,23. High concentrations of Unsaturated Fatty Acids in milk (MUFA and PUFA) and low concentrations of SFA indicate energy imbalance23. Moreover, the lactation stage has a significant impact on the lipid profile of milk24. The high uptake of LCFA by the mammary gland in the first lactation phase negatively affects the ex novo synthesis of fatty acids23. LCFA, reduce the ex-novo synthesis through the inhibition of the enzyme acetyl-CoA carboxylase22. Indeed, the increase in the lipid profile of LCFA and the decrease in Short Chain Fatty Acids (SCFA) in postpartum are indicative of a negative energy balance23. The lipid profile of the fatty acids belonging to the 4 lipid classes changes as the energy balance enhance throughout the lactation25. The goals of our research were to identify a new method not requiring blood sampling that could be incorporated into the daily milking routine and the identification of new biomarkers for the early diagnosis of excessive lipid mobilization and subclinical-clinical ketosis. In order to achieve these aims we examined the correlation between concentrations of fatty acids in milk with elevated NEFA concentrations during the first phase of lactation. The evaluation of the fatty acids concentrations in milk could allow the implementation of nutritional and management strategies in order to avoid the development of metabolic diseases.

tion period. The aim of Bovine OMICS Project was to identify new biomarkers of metabolic disorder. In the first study of Fiore et al., plasma fatty acids were evaluated on the basis of blood BHB, subsequently Fiore et al., investigated the fatty acids in milk always based on the blood value of BHB26,27,28. In this article we analyzed the milk fatty acids considering the value of blood NEFA.

Animals Fifty-four Holstein Frisian dairy cows enrolled in this study, were the same as those sampled in the research of Fiore et al27. The farm, located in Northern Italy (45° 36’ N. 11° 40’ E. 23 m above sea-level), consisted of a single building, with a free housing system and cubicle surface were equipped with a rubber mattress. The group of animals enrolled were in early postpartum, with an average of 26.5 ± 1.5 days in milk (DIM) as the development of ketosis is more frequent between the second and seventh week of lactation29. All animals had a dry period of 55 ± 5 days and steaming-up was not carried out. Total Mixed Ration (TMR) and the chemical composition of the lactation diet were reported in the study by Fiore et al.26 and water was available ad libitum. All animals were evaluated by the Veterinarian of University of Padua (Italy) and were clinically healthy. Animals had not developed mastitis during ongoing lactation prior to sampling. Bovine did not present clinical signs of mastitis and SCC (Somatic cell count) was also used to define udder health status30. All animals had SCC values lower than 200.000 cells/mL during ongoing lactation.

Experimental design

MATERIALS AND METHODS

Blood sampling was collected using coccygeal vein puncture. In order to select cows with metabolic disorders, before the blood sample, the BHB was measured using the Nuova Biomedical Express (Nuova Biomedical, Runcorn, United Kingdom) digital reader with specific BHB test strips (Stat Strip Ket, Nova Biomedical). Three samples were collected with the vacutainer system for each enrolled cow. The first aliquot was taken using a tube containing EDTA (5 ml; Terumo Venoject, Leuven, Belgium) and the other two aliquots were stored in Venosafe tubes containing Clot Activator (9 ml; Terumo Venosafe, Leuvel, Belgium). Milk samples were taken at the evening milking from each bovine enrolled in the trial. For sterile sampling, the operator was equipped with disposable gloves to minimize any potential risk of contamination and induction of mastitis. After cleaning, drying and disinfection of the teat, the first 2 foremilk were discarded by manual milking. Then, 40 mL was aseptically collected in sterile plastic tubes (50 mL, Vetrotecnica S.r.l., Padova, Italy). The biological material collected was transported to the Clinical Diagnostic Laboratory of the Department of Animal Medicine, Production and Health (MAPS) of the University of Padua (Italy) stored at 4°C. The blood samples containing Clot Activator were centrifuged in the laboratory (Heraeus Labofouge 400, Thermo Scientific, Milan, Italy). Finally, serum and milk samples were stored at -20° for subsequent biochemical analysis.

The data analyzed for this article were collected in the context of the Bovine OMICS Project (supported by the University of Padua), based on the analysis through TLC-GC of the plasma lipid profile and milk lipid profile of dairy cows in the transi-

Serum biochemistry and plasma gas chromatography (GC) were performed in the Clinical Diagnostic Laboratory of MAPS Department.

Blood analysis

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R. Tessari et al. Large Animal Review 2021; 27: 187-193

The Serum was assessed employing automatic clinical chemistry analyser (BT3500 Biotecnica Instrument SPA, Rome, Italy). Serum NEFA (Non-Esterified Fatty Acids) concentration was measured with the NEFA RX Monza test colorimetric method (Randox, Crumlin, UK). On the basis of NEFA concentrations obtained in the laboratory, animals were divided into two different groups: Group NEFA-0 had 33 animals with NEFA concentration lower than 0.57 mEq/L and Group NEFA-1 had 21 animals with a NEFA quantity higher than 0.57 mEq/L. This cut-off was chosen based on the work of Ospina (2010); this value indicates excessive lipomobilization and can be used as a threshold for monitoring dairy cows at risk of postpartum diseases7.

189

Table 1 - Mean value (±SEM) of NEFA, days in milk (DIM), body condition score (BCS), parity, and daily milk yield. Parameters

NEFA-0 (n = 33)

NEFA 1 (mEq/L)

NEFA-1 (n = 21)

Correlation (p-value)

0.23 ± 0.10

0.83 ± 0.43

<0.001

DIM 2

28.34 ± 12.44

25.76 ± 14.96

<0.001

BCS 3

2.75 ± 0.21

2.89 ± 0.15

<0.001

Parity

2.58 ± 1.86

2.57 ± 1.40

NS 4

Milk (kg/day)

28.40 ± 7.38

30.71 ± 8.43

<0.001

: non-esterified fatty acid; 2: day in milk; 3: body condition score; 4: not significant.

Milk analysis In order to perform the GC in the milk, the samples underwent three stages of treatment including 1) extraction of lipids from the milk 2) separation of the lipid classes by Thin Layer Chromatography (TLC) 3) methylation of the carbon chain. The method used for the quantification of milk fatty acids was the same used in the studies of Fiore et al.26,27,28 in accordance with the study of Carnielli et al.31. Before starting these procedures, the samples were mixed with the internal standards (C9-C15 or C17) of every lipid class. Finally, for each milk sample 30 fatty acids were obtained per lipid classes: FFA (free fatty acids), PL (phospholipids), TG (triglycerides) and CE (cholesterol esters). The fatty acids methyl esters were separated and quantified in splitless mode by GC using a TRACE GC/MS (Thermo Quest, Milan, Italy) equipped with a flame ionization detector (FID) and a polar fused-silica capillary column (Capillary Column Omegawax, 30 m× 0.25 mm × 0.2 µm film). Helium was used as the carrier gas at a flow rate of 1 ml/min. Data for plasma fatty acid were calculated in mg/dL.

Statistical analysis Blood biochemical parameters and milk fatty acid methyl ester data were analyzed using the SAS system software (version 9.4; SAS Institute Inc., Cary, NC, USA). The General Linear Model (GLM) analysis was performed for repeated measurements in order to evaluate the differences in the composition of milk fatty acids in the four lipid fractions within the two classes (NEFA-0 - vs NEFA-1). Statistical significance was set at p ≤ 0.05.

RESULTS The mean values (±SEM) regarding NEFA, Day in Milk (DIM), Body Condition Score (BCS), parity and yield milk produced (kg/day) for all enrolled animals based on the two classes of NEFA (NEFA-0 vs NEFA-1) are presented in Table 1. The mean value of NEFA concentration was 0.23 ± 0.10 mEq/L and 0.83 ± 0.43 mEq/L for NEFA-0 and NEFA-1 group, respectively. The different composition of fatty acids in the four lipid classes of FFA, CE, PL and TG was assessed through analyzes performed in TLC-GC technique. The mean value of the 30 milk fatty acid relating to these four classes of lipids (FFA, CE, PL and TG) was compared between the two groups with different blood concentrations of non-esterified fatty acid (NEFA0 vs NEFA-1). In the Table 2 are shown the mean values and the standard er-

Table 2 - Milk Fatty Acids mean values with SE related to the lipid class of Cholesterol Ester (TLC-GC). Free Fatty Acids (CE) mg/dL

NEFA-0 (n = 33)

SE NEFA-0

NEFA-1 SE NEFA-1 Correlation (n = 21) (p-value)

1.72

0.37

1.46

0.42

0.99

0.16

0.80

0.18

C10

1.50

0.17

0.89

0.19

0.023

C12

4.46

0.56

3.38

0.64

C14

1.51

0.24

0.99

0.27

C14:1 ω 5

1.24

0.57

0.68

0.65

C16

3.56

1.15

5.70

1.31

C16:1 ω 7

0.78

0.26

0.68

0.30

C18

1.39

0.19

1.24

0.21

C18:1 ω 9

0.88

0.21

0.85

0.25

C18:2 ω 6

0.31

0.04

0.29

0.05

C18:3 ω 6

0.06

0.03

0.09

0.03

C18:3 ω 3

0.18

0.12

0.14

0.13

C20:2 ω 6

0.11

0.03

0.13

0.03

C20:3 ω 6

0.04

0.01

0.03

0.01

C20:4 ω 6

0.09

0.03

0.05

0.04

C20:3 ω 3

0.18

0.06

0.12

0.07

C20:5 ω 3

0.16

0.04

0.05

C22

0.03

0.01

0.03

0.01

C22:1 ω 9

0.09

0.02

0.04

0.03

C22:2 ω 6

0.04

0.02

0.03

0.02

C22:4 ω 6

0.36

0.15

0.53

0.17

C22:5 ω 3

0.06

0.02

0.05

0.02

C22:6 ω 3

0.11

0.04

0.19

0.05

C23

0.19

0.05

0.28

0.06

C24

0.13

0.03

0.11

0.03

C24:1 ω 9

0.04

0.01

0.05

0.01

C16 DMA

8.46

0.40

8.57

0.46

mg/dl

78.40

6.46

70.81

7.37

NS: not significant.

ror (SE) of the different milk fatty acids related to the lipid class of CE. A statistically significant difference between NEFA-0 and NEFA-1 was found for C10:0 (p = 0.023) and C20:5 ω 3 (p = 0.05).

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Milk Fatty Acids as Biomarkers of Metabolic Diseases in Dairy Cows identified through Thin Layer Chromatography

Table 3 - Milk Fatty Acids mean values with SE related to the lipid class of Phospholipids (TLC-GC). Free Fatty NEFA-0 Acids (n = 33) (PL; mg/dL)

SE NEFA-0

NEFA-1 SE NEFA-1 Correlation (n = 21) (p-value)

Table 4 - Milk Fatty Acids mean values with SE related to the lipid class of Free Fatty Acids (TLC-GC). Free Fatty NEFA-0 Acids (n= 33) (FFA; mg/dL)

SE NEFA-0

NEFA-1 SE NEFA-1 Correlation (n= 21) (p-value)

0.40

0.16

0.80

0.20

2.65

0.72

3.09

0.88

0.27

0.12

0.25

0.15

6.92

1.07

5.75

1.32

C10

1.04

0.33

0.52

0.42

C10

8.64

1.77

5.45

2.16

C12

4.42

1.69

2.87

2.19

C12

9.25

1.24

7.77

1.52

C14

2.68

0.78

1.83

1.01

C14

7.35

1.42

6.29

1.74

C14:1 ω 5

0.12

0.04

0.07

0.06

C14:1 ω 5

0.25

0.07

0.15

0.09

C16

7.55

1.79

6.44

2.32

C16

1.92

0.46

1.12

0.56

C16:1 ω 7

0.07

0.08

0.27

0.10

C16:1 ω 7

23.99

4.27

22.82

5.23

C18

3.53

0.41

4.57

0.53

C18

6.15

0.80

5.85

0.98

C18:1 ω 9

3.70

1.18

3.31

1.52

C18:1 ω 9

3.16

1.25

2.29

1.53

C18:2 ω 6

0.65

0.18

1.01

0.23

C18:2 ω 6

0.38

0.16

0.23

0.20

C18:3 ω 6

0.04

0.01

0.03

0.01

C18:3 ω 6

0.11

0.03

0.06

0.04

C18:3 ω 3

0.07

0.02

0.04

0.03

C18:3 ω 3

0.07

0.05

0.01

0.06

C20

0.11

0.02

0.17

0.03

C20

0.09

0.01

0.07

0.02

C20:1 ω 9

0.03

0.01

0.06

0.01

C20:1 ω 9

0.02

0.01

0.04

0.02

C20:2 ω 6

0.03

0.04

0.12

0.05

C20:2 ω 6

0.13

0.03

0.07

0.03

C20:3 ω 6

0.10

0.02

0.20

0.02

C20:3 ω 6

0.14

0.03

0.11

0.04

C20:4 ω 6

0.10

0.04

0.16

0.05

C20:4 ω 6

0.05

0.02

0.07

0.02

C20:3 ω 3

0.02

0.003

0.02

0.004

C20:3 ω 3

0.02

0.01

0.02

0.01

C20:5 ω 3

0.05

0.02

0.06

0.03

C20:5 ω 3

0.04

0.01

0.02

0.01

C22

0.66

0.12

0.90

0.16

C22

0.07

0.03

0.05

0.03

C22:1 ω 9

0.03

0.01

0.02

C22:1 ω 9

0.02

0.01

C22:2 ω 6

0.01

C22:2 ω 6

0.38

0.12

0.08

0.15

C22:4 ω 6

0.12

0.05

0.06

C22:4 ω 6

0.16

0.05

0.03

0.06

0.05

C22:5 ω 3

0.03

0.02

0.07

0.03

C22:5 ω 3

0.09

0.04

0.01

0.05

C22:6 ω 3

0.06

0.02

0.13

0.03

0.05

C22:6 ω 3

0.21

0.07

0.21

0.09

C23

0.28

0.07

0.42

0.09

C23

0.29

0.06

0.28

0.08

C24

0.64

0.08

0.82

0.10

C24

0.08

0.01

0.06

0.01

C24:1 ω 9

0.24

0.16

0.07

0.21

C24:1 ω 9

0.05

0.01

0.04

0.01

C16 DMA

1.14

0.16

1.30

0.21

C16 DMA

2.38

0.29

1.97

0.35

mg/dl

41.87

9.70

39.21

12.53

mg/dl

75.43

11.63

64.23

14.25

NS: not significant.

Table 3 shows the mean values and the standard error (SE) of the different milk fatty acids related to the lipid class of PL. A statistically significant difference between NEFA-0 and NEFA1 was found for C22:6 ω 3 (p = 0.05). Table 4 shows the mean values and the standard error (SE) of the different milk fatty acids related to the lipid class of FFA. Comparing the two groups NEFA-0 and NEFA-1 only C22:4 ω 6 was statistically different (p = 0.05). Data of the mean values and the standard error (SE) of the different milk fatty acids related to the lipid class of TG are presented in Table 5. A statistically significant difference between NEFA-0 and NEFA-1 was found for C14:1 ω 5 (p = 0.043), C16:0 (p = 0.05), C16:1 ω 7 (p = 0.013), C18:1 ω 9 (p = 0.05), C18:3 ω 3 (p = 0.05), C20:1 ω 9 (p = 0.05), C22:0 (p = 0.05) and C22:4 ω 6 (p = 0.011).

DISCUSSION In order to identify, among milk fatty acids, new biomarkers with predictive function in the development of metabolic pathologies, we divided the recruited animals according to plasmatic NEFA. Plasma NEFA concentrations are considered to be important risk factors for the development of metabolic disease32. Mann et al. (2016), assessed the variation in milk fatty acid concentrations in dairy cows with different blood NEFA values, using as threshold value 1.0 mmol/L. The main biomarkers selected in the study mentioned above were Pentadecanoic acid (C15:0), Palmitoleic acid (C16:1 ω 9), C18:1 ω 9 and also the ratio C16:1 ω 9/ C15:0 and C18:1 ω 9/ C15:010. Differently from Mann et al., our study, for the first time, evaluated the difference between the fatty acids distinguished by lipid

Tessari_imp_ok 27/07/21 14:39 Pagina 191

R. Tessari et al. Large Animal Review 2021; 27: 187-193 Table 5 - Milk Fatty Acids mean values with SE related to the lipid class of Triglycerides (TLC-GC). Free Fatty NEFA-0 Acids (n= 33) (TG; mg/dL)

SE NEFA-0

NEFA-1 SE NEFA-1 Correlation (n= 21) (p-value)

29.21

4.30

31.13

5.37

28.26

3.75

27.53

4.67

C10

51.70

6.70

48.13

8.36

C12

39.09

5.98

46.92

7.46

C14

92.19

7.50

87.16

9.36

C14:1 ω 5

4.37

0.69

6.65

0.86

0.043

C16

275.82

24.59

352.24

30.67

0.05

C16:1 ω 7

11.73

1.93

19.70

2.41

0.013

C18

96.57

13.13

110.07

16.37

C18:1 ω 9

205.30

32.62

311.04

40.69

0.05

C18:1 ω 7

13.26

1.64

14.91

2.05

C18:2 ω 6

25.51

3.14

29.73

3.92

C18:3 ω 6

0.96

0.11

1.03

0.14

C18:3 ω 3

3.64

0.43

4.88

0.54

0.05

C20

0.96

0.19

1.22

0.23

C20:1 ω 9

0.66

0.25

1.35

0.31

0.05

C20:2 ω 6

0.45

0.05

0.48

0.06

C20:3 ω 6

0.71

0.09

0.66

0.11

C20:4 ω 6

1.61

0.27

2.21

0.33

C20:3 ω 3

0.12

0.02

0.12

0.03

C20:5 ω 3

2.17

0.59

1.00

0.74

C22

0.38

0.07

0.15

0.09

0.05

C22:1 ω 9

0.07

0.02

0.07

0.02

C22:2 ω 6

0.02

0.01

0.03

0.01

C22:4 ω 6

0.20

0.04

0.02

0.05

0.011

C22:5 ω 3

0.39

0.06

0.45

0.07

C22:6 ω 3

0.19

0.04

0.11

0.05

C23

0.58

0.18

1.02

0.23

C24

0.13

0.02

0.11

0.03

C24:1 ω 9

0.16

0.02

0.15

0.03

C16 DMA mg/dl

1.25

0.20

1.16

0.25

943.37

95.13

1170.45

118.65

NS: not significant.

class of belonging: CE, PL, FFA and TG. The variation of fatty acid concentrations within the lipid fractions of milk, could be used as a marker to predict a state of hyperketonemia9. In table 1, statistically significant difference (p < 0.001) was highlighted between healthy (NEFA-0) and sick animals (NEFA-1) with regards to concentration of plasmatic NEFA (0.23 ± 0.10 mEq/L vs 0.83 ± 0.43 mEq/L); while no statistically significant difference was detected regarding the number of lactations between the two groups (2.58 ±1.86 vs 2.57 ±1.40). Animals with an excessive BCS have a more reduction in the voluntary ingestion of dry matter (DMI) during the transition period33. The reduction of DMI leads to a greater activation of the biochemical process of lipolysis and an increase of plasma fatty acids concentrations33. As expected, BCS score was significantly (p < 0.001) higher in dairy cows with high blood con-

191

centrations of NEFA (NEFA-1, 2.89 ± 0.15) than in healthy cows (NEFA-0, 2.75 ± 0.21). In the study of Gillund et al. (2010), dairy cows that developed a metabolic disorder in post-partum, such as ketosis, had a greater BCS score at calving than a healthy bovine34. In our study, repeated measures of BCS to calculate the loss of nutritional status have not been evaluated, but we expected a higher progressive reduction of the BCS score in NEFA-1 group than bovine with normal value of NEFA. Indeed, BCS drops abruptly the first 42 DIM and it is relatively constant thereafter in animals with normal concentration of BHB. In ketotic bovine the BCS continues a negative decrease until around 90 DIM34. Milk production generated a significant difference (p < 0.001) between the healthy group (NEFA-0, 28.40 ± 7.38 kg/day) and sick animals (NEFA-1, 30.71 ± 8.43 kg/day). When the energy demand for milk production exceeds the available energy, the lipolysis is activated with the release of NEFA into the bloodstream35. Subsequently, we analyzed the acid composition of each lipid class comparing the two groups of animals (NEFA-0 vs NEFA1). In CE lipid class, a reduction of milk fatty acids has been observed in cows with blood NEFA concentrations higher than 0.57 mEq/L compared to healthy animals. Table 2 showed that total fatty acids decrease their concentrations as blood NEFA increase (NEFA-0 = 78.40 mg/dL; NEFA-1 = 70.81 mg/dL). The ruminants present a decrease of the enzyme Lecithin Cholesterol Acyl-Transferase (LCAT) after calving36,37. LCAT is a serum enzyme, synthesized in the liver, that catalyzes the transfer of fatty acids from lecithin to cholesterol producing esterified cholesterol (CE)37. In the study of Nakagawa and Katoh (1998), animals in ketosis presented a greater decrease in blood concentrations of LCAT and CE compared to healthy cows38. The decrease in blood concentrations could explain their reduced uptake by the mammary gland12. Also, in our data, C10:0 (p = 0.023) and C20:5 ω 3 (p = 0.05), significant milk fatty acids, show decreasing concentrations as the blood NEFA increase. The milk lipid fraction of PL is predominantly constitute of saturated LCFA and saturated medium chain fatty acids (MCFA) such as Lauric acid (C12:0), C14:0, C16:0 and C18:0 and unsaturated LCFA like C18:1 ω 9 and Linoleic acid (C18:2 ω 6)39. Also, in our study a higher concentration of these fatty acids was noted in the lipid fraction of PL. Furthermore, dairy cows with a normal NEFA value (NEFA-0) have higher concentrations of these fatty acids than cows with excessive mobilization of adipose tissue (NEFA-1) (Table 3). HDL and Low-Density Lipoproteins (LDL) transport PL into the bloodstream12. Dairy cows with fatty liver in post-partum, resulting from excessive mobilization of fat reserves, have lower concentrations of PL associated with HDL compared to animals with normal fat infiltration in the liver40. This further reduces uptake by the mammary gland12. Therefore, the decrease in milk fatty acids in PL lipid class was expected in cows with greater alteration of lipid metabolism (NEFA-1). Contrary, in our data, healthy animals had total fatty acid values equal to 41.87 mg/dL compared to bovine with high concentrations of NEFA where the value was 39.21 mg/dL. In BEN, the plasma fatty acids increase their concentrations as blood BHB and NEFA concentrations increase27. Contrary to plasma concentrations, our study showed that milk fatty acids, belonging to FFA lipid class, tended to decrease with in-

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192

Milk Fatty Acids as Biomarkers of Metabolic Diseases in Dairy Cows identified through Thin Layer Chromatography

creasing blood NEFA. In table 4, the total fatty acids in healthy animals (NEFA-0, 75.43 mg/dL) were greater than the total fatty acids present in the NEFA-1 group (65.23 mg/dL). It is essential to note how concentration of Arachidonic acid (C22: 4 ω 6), necessary precursor for the endogenous synthesis of inflammatory mediators, decreased significantly (p = 0.05) between healthy cows (0.16 mg/dL) to animals in a negative state of excessive lipid mobilization (0.03 mg/dL). Further studies are needed to evaluate the variation of fatty acids in the FFA lipid fraction especially what concerns eicosanoids, precursors of inflammation mediators41. A variable concentration from 96 to 98% of the milk lipid composition is constituted by TG18. Total concentrations of TG in the NEFA-0 group were 943.37 mg/dL whereas, NEFA-1 group had a value of 1170.45 mg/dL. Analyzing the lipid fraction of TG, we observed that most of the significant fatty acids were higher in the group of animals affected by excessive lipid mobilization (NEFA-1) with respect to the NEFA-0 group (healthy animals). The increase of these lipid fractions is indicative of an energy imbalance11. In the present study 5 out of the six MUFA and PUFA (TG class) increased their concentrations with increasing of plasma NEFA (Table 5) in accordance to the study of Vrankovic et al. (2017), in which milk concentrations of MUFA and PUFA are higher in dairy cows with higher NEB23. Myristoleic acid (C14:1 ω 5, p = 0.043), Palmitic acid (C16:1 ω 7, p = 0.013), α-Linolenic acid (C18:3 ω 3, p = 0.05) e Gondoic acid (C20:1 ω 9, p = 0.05) had higher concentrations in the NEFA-1 group than in the NEFA-0 group. Specifically, Oleic acid (C18:1 ω 9) had a statistically different (p ≤ 0.05) concentration of 205.30 mg/dL in the NEFA-0 group while in sick animals the quantity is equal to 311.04 mg/dL. Oleic acid in milk fat is a good indicator of NEB10,17,42 both in the early lactation phase and in any other period in which the animal is anorexic or suffering from ketosis9. Indeed, Oleic acid is the main fatty acid contained in the adipose tissue and released during the periods of NEB33. Dairy cows, affected by subclinical ketosis, often present high quantities of C18:1 ω 9 in milk fat, which could potentially correlate with a deficiency of the enzyme acyl-CoA dehydrogenase9. Moreover, animals in subclinical ketosis and in NEB have lower concentrations of Medium Chain Saturated Fatty Acids (MCSFA) and SFA in milk than healthy cows9,23. In our study C22:0 decreased significantly (p = 0.05) with increasing NEFA value (NEFA-0, 0.38 mg/dL; NEFA-1, 0.15 mg/dL), as in available literature, whereas C16:0 had higher concentrations (p = 0.05) in the NEFA-1 group (352.24 mg/dL) compared to the NEFA-0 group (275.82 mg/dL)23. The higher level of concentration of C16:0 could be explained by the increase of mobilization of fat reserves that occurred in dairy cows with a negative energy balance17. Moreover, in the present study was examined the different chemical composition of significant fatty acids, evaluating the presence of double bounds. Predictive values include 3 SFA and 9 UFA, that were divided in four MUFA and five PUFA. According to Vrankovi et al. (2017), although not distinguishing fatty acids for individual lipid fractions, showed that the concentration of all MUFA increased in dairy cows affected by a more intense NEB23. In our study C14:1 ω 5, C16:1 ω 7, C18:1 ω 9 and C20:1 ω 9, fatty acids with predictive function for the development of metabolic disorder, had higher concentrations in the NEFA-1 group and belonged to the TG lipid class.

As in available literature, PUFA, C20:6 ω 3 and C18: 3 ω 3, showed an increasing trend when the concentrations of blood NEFA increase23. Conversely, C22:4 ω 6 (FFA class and TG class) and C22:5 ω 3 (CE class) exhibited an anomalous trend, decreasing their concentrations with increasing lipid mobilization. This abnormality in the PUFA class could be caused due to different composition of the diet of recruited animals. Feeding largely affects the content of LCFA in milk21,43. Diets enriched with rapeseed oil or linseed oil lead to an increase in PUFA in milk44. In the study of Rutkowska et al. (2015), soybeans increase the content of PUFA ω 3 and PUFA ω 6 whereas rapeseed seeds cause an increase in the proportion of C18:2 ω 9 and C18:1 ω 945. Therefore, a limitation of the present study was the diet of enrolled animals.

CONCLUSIONS In our study, the trend of milk fatty acids in four lipid classes (CE, PL, FFA and TG) with different blood concentrations of Non-Esterified Fatty Acid was comprehensively described and 12 predictive parameters were identified. The variation in the composition of plasma fatty acids in the first lactation phase confirmed the applicability of TLC-GC as a new method, not requiring blood sampling, that could be incorporated into the daily milking routine. Finally, we concluded that the main fatty acids with high predictive power for the diagnosis of excessive lipomobilization were MUFA belong to TG lipid fraction such as C14:1 ω 5, C16:1 ω 7, C18:1 ω 9 and C20:1 ω 9.

Funding This work was financially supported by 2 grants of University of Padova, Italy (SID Berlanda 2016 - Prot. IDs = BIRD169974/16; and SID Fiore 2019 Prot. IDs = BIRD195883/19).

Ethical approval Ethics statement approved by Animal Care and Use Committee of University of Padua (number 91/2019 - “BovineOmics” Projects).

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B. Kibar Kurt et al. Large Animal Review 2021; 27: 195-198

Central corneal thickness increases with age in cattle

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BUSRA KIBAR KURT1, ÖMER KURT2, KAĞAN KOCASARI1, TOLGA MERIÇ DÜMBEK1, MEHMET ERDEM1, ROJDA YİĞİT1, EMRE GÜRDAL3 1 2 3

University of Aydin Adnan Menderes, Faculty of Veterinary Medicine, Surgery Department Uzman Veterinary Clinic, Aydın, Turkey Arif Gürdal Dairy Farm, Aydın, Turkey

SUMMARY The aim of this study was to identify the physiological reference values for central corneal thickness (CCT) and the age-related changes in CCT in healthy dairy cattle. The age of 50 dairy cows was recorded in days and the CCT values of both eyes were measured using an ultrasonographic pachymeter device. The mean age of the cows was 781.38 days and the mean CCT value was 799.44 µm. There was a positive correlation between age and CCT (p<0.001, y = 751.34 + 0.0616x, R2=0.2104). Knowing the corneal thickness and age-related changes is important physiologically and pathologically. Medical treatment costs in eye diseases, injury due to extra procedure, decreased weight gain, decreased milk production and the transfer of animals that do not respond to treatment cause economic loss. In literature searches, no studies were found on this subject, except for a few studies (one of them postmortem) conducted about 25 years ago. The present study is the first study conducted on dairy cattle in the same environmental conditions in an organic farm. According to the presented study corneal thickness increases with age. In conclusion, corneal thickness in cattle changes over their lifetime and is similar to that reported in other mammals. Knowing the agerelated physiological change of corneal thickness has diagnostic, epidemiological and pathological importance.

KEY WORDS Bovine, cattle, central corneal thickness, cornea, ultrasound pachymeter, pachymetry.

INTRODUCTION The integrity of the optic surface against threats coming from outside the cornea is important to be able to perform the act of seeing. The structure of the corneal epithelium, and the cellular and chemical components of the conjunctiva and tear film layer protect the corneal surface against pathogen agents and micro-organisms1. Light passes into the eye because of the clear and avascular structure of the cornea. Refracting light rays passing through the lens are transformed to electrical and chemical energy by reaching the retina. The signals obtained are analysed through transmission to the brain by the optic nerve and are perceived as an image. As the external surface of the cornea is convex, it protects intra-ocular organs, maintains intra-ocular pressure, and provides light refraction2 The cornea consists of 5 layers, which from inner to outer are the epithelial layer, the lamina limitans anterior, stroma layer, Descemet membrane, and endothelial layer. The corneal surface has a specific structure. Although cells in the corneal epithelium are non-keratinised, they are named keratocytes, and are accepted as the continuation of the bulbar conjunctiva. Keratocytes are multi-layered. The corneal epithelium is formed of 3 different types of cells. These are 2-3 rows of surface cells, 2-3 rows of wing cells and 1 row of a columnar basal cell layer. Only basal cells show proliferation activity 3,4. The lamina limitans anterior is a thin and uniform structure of the epithelium expressed by basal cells. The Descemet

Corresponding Author: Busra Kibar Kurt (busrakibar@yandex.com) (busra.kibar@adu.edu.tr)

membrane is the basal membrane of the epithelium. The endothelial layer is formed of a single layer of cells, and the anterior chamber is bordered 5. Corneal thickness was first measured by Blix 6 on human eye but until the mid 20th century, no studies were conducted related to corneal thickness. In 1951, David Maurice 7 designed the pachymeter and for human cornea thereafter, many studies of corneal thickness were conducted. In two studies on bovine corneal thickness, it was reported that measurements were made by ultrasound after slaughter 8,9. The biological variations of corneal thickness depend on changes in the amount of collagen fibrils and interfibrillar substance which form the corneal stromal tissue in both human and veterinary ophthalmology. In healthy individuals, corneal thickness is therefore, a measure of tissue mass and corresponding biomechanical parameters such as bending rigidity10. There are many methods of measuring corneal thickness. The ultrasonographic pachymeter device makes contact with the cornea and measures corneal thickness with the principle of ultrasonography. In literature, there are many comparative studies in veterinary and human subjects 11-14. The ultrasonographic pachymeter device is extremely sensitive with up to 0.004 mm difference in repeated measurements 15 . Corneal thickness shows a difference according to species and even according to breed. Age-related changes are seen in both animal and human corneal thickness 1,4. It has been reported that there is an increase in corneal thickness due to stromal hydration. This means that based on the data, corneal thickness can be used as an index of endothelial damage16. Decreased corneal thickness is seen in many corneal dystrophies and secondary to ulcerative inflammatory diseases of various origins.

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Central corneal thickness increases with age in cattle

It is thought that in these situations, the decrease in corneal thickness is caused by loss or re-distribution of corneal tissue. The thickness of the cornea is not only related to physiology and pharmacology, but variations in normal thickness are also of diagnostic, epidemiological and pathological importance. Therefore, great interest has been shown in sensitive and accurate measurement methods. Diseased eyes can cause a great deal of pain to patients, as those who have personally experienced corneal erosions or other painful ocular conditions will attest. In caring for cattle, treatments should be directed at preventing ocular disease whenever possible. The purpose of this study was to identify the physiological reference values for central corneal thickness (CCT) and the agerelated changes in CCT in healthy dairy cattle.

Figure 2 - Distribution of the central corneal thickness values for the left and right eyes.

MATERIAL AND METHODS The study was conducted on the Arif Gürdal Dairy Farm with the approval of ADÜ - HADYEK (64583101/2020/095). Evaluation was made of 100 eyes of 50 healthy cattle aged 389-2970 days. The measurements for each eye were obtained using ultrasonographic pachymetry to determine CCT. All the measurements were taken by the same surgeon on the same day between 11:00 am and 3:00 pm (Figure 1). Ophthalmological examination was performed before the cattle were included in the study. The ophthalmological examination and CCT measurements were made in head-lock barriers. All cattle eyes were free of inflammation or other ocular disease. Ultrasonographic pachymeter (Pocket II One Touch Ultrasound Pachymeter) utilizes ultrasound energy to measure CCT. The probe was gently touched to the central cornea without pressure or indentation. The device automatically calculates the average value of 5 consecutive measurements.

Statistical Analysis Data obtained in the study were analyzed statistically using SPSS vn.22 software (Statistical Package for Social Sciences), Jamovi (1.1.9) and Microsoft Excel programs. Conformity of the data to normal distribution was examined with the Shapiro Wilk test and the Levene test. Measurements of the right and left eyes were evaluated with the Dependent Samples t-test and correlations between age and CCT with the Pearson Correlation test. A value of p<0.05 was accepted as statistically significant.

Figure 3 - Central corneal thickness and age distribution.

RESULTS All cattle eyes were free of inflammation or other ocular disease. Measurements were taken of 100 eyes of 50 cattle. The study population consisted of healthy dairy cattle. The ages of the cattle varied between 389-2970 days and CCT between 425960 µm. The results are shown in detail in Tables 1 and 2. The mean age of the cattle was 781.38 days. The mean CCT value was 799.44 µm. The mean CCT value was 806.58 µm (range 425-960 µm) for the right eyes and 792.30 µm (range 491-938 µm) for the left eyes (Figure 2). According to the Dependent Samples t-test, no statistically significant difference was determined between the right and left eyes in respect of CCT (p=0.363) (Tables 1, 2). The age distribution of the CCT of these Table 1 - Central corneal thickness (CCT, µm) values in the left and right eyes. Right CCT (µm)

Left CCT (µm)

Mean

806,58

792,3

Std. error mean

11,99

12,81

Minimum

425

491

Maximum

960

938

Table 2 - The average central corneal thickness (CCT, µm) and age of the animals. Age (day)

Figure 1 - Central corneal thickness measurements.

CCT (µm)

100

Mean

781,38

799,44

Std. error mean

92,74

8,76

Minimum

389

425

Maximum

2970

960

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B. Kibar Kurt et al. Large Animal Review 2021; 27: 195-198

Figure 4 - Scatterplot demonstrating the age and corneal thickness values of the cattle.

subjects is summarized in Figure 3. A significant positive correlation was observed between age and CCT in these measurements of 50 dairy cows (p<0.001, y = 751.34 + 0.0616x, R2=0.2104) (Figure 4). According to these results, it is seen that the cornea gets thicker as the age increases. Based on the analyses, right and left CCT are similar.

DISCUSSION The aim of this study was to establish the normal CCT values of dairy cattle and to investigate the relationship between CCT and age. The ultrasound pachymeter device is widely preferred for the measurement of CCT as it is inexpensive, easy-to-apply and repeatable. Of the techniques available, the ultrasound pachymeter shows the least variance and may therefore be the most accurate method 17. Contact with the cornea is the greatest disadvantage of this method, and placement of the probe at the correct angle without pressing the cornea is important for accurate measurement 18. In comparisons of pachymetry and other CCT measurement methods, there has been reported to be no difference in the results, but unlike other methods, pachymetry does not require sedation 17,19,20. For these reasons, ultrasound pachymetry was selected for use in this study. In previous studies in literature, CCT in cattle has been reported as 805 µm 21, 1015 µm 22, 725-936 µm 23. In the current study of 50 dairy cows aged 389-2970 days, the mean CCT was determined as 799.44 µm, ranging from 425 µm to 960 µm. This study was planned on the hypothesis that there could be a correlation between age and corneal thickness in cattle. Just as in all living creatures, tissues in cattle show age-related changes 24. The repair capacity of corneal endothelial cells is known to be extremely limited. Knowledge of the normal values and age-related changes is necessary not only to determine the physiological limits, but also to reveal abnormal conditions. Previous studies have shown that corneal thickness changes depending on age 25-27. It has been reported in previous human studies that a decrease is seen in corneal thickness as age increases. Although corneal thickness varies according to species and race, the

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mean corneal thickness is in the range of 0.5-0.8 mm. The periphery is generally thicker than the centre. The cornea in elderly animals can thicken up to 0.9 mm because of endothelial cell dysfunction. In studies performed on cattle eyes collected after slaughter, values of 1.29 ± 0.03 mm8 and 1.4±0.011 mm9 were reported. In cats, the cornea continues to develop until the age of 1-2 years. CCT increases together with age, and has been reported to be 0.55 mm at 16 weeks and 0.57 mm at 67 weeks. In dogs, there is thinning in the cornea up to 6 weeks of age, after which thickening develops together with age and the mean thickness is 0.56 mm1,4 25,2833 . While corneal thickness has been reported to be thinner in young llamas than in older llamas, no significant age-related difference has been observed in alpacas 34. Studies of horses 35,36 , sheep 37, dogs 38,39, and cats 40 have shown an age-related increase in corneal thickness measurements. Another study reported a negative correlation between age and corneal thickness in horses 41. In this study, the mean CCT value was 799.44 µm and the average age was 781.38 days. The results of the current study showed a positive correlation between age and corneal thickness in dairy cattle, which was similar to the findings of other animal studies. The accumulation of new material in Descemet’s membrane over their lifetime may partially explain this finding 16. The ophthalmologic disease in the dairy farms can result in significant economic losses to producers. Along with economic impacts, the ophthalmologic disease can lead to individual pain and suffering and therefore negatively affects animal welfare 42. In corneal diseases, the increase in corneal thickness is due to hydration. A decrease in corneal thickness is seen in corneal dystrophy and ulcerative inflammatory diseases. The thickness of the cornea is not only related to physiology and pharmacology, but a change in normal thickness is also of diagnostic, epidemiological and pathological importance43. Therefore, it is important that the corneal thickness is measured definitively and accurately. There were some limitations in conducting out study due to the farm conditions. Only 50 cattle over 1 year old were included and under 1 year old could not be evaluated. However, the data obtained from the study clearly showed that cornea thickness is related to age. In this context, this study revealed the need of both to evaluate the corneal thickness measured starting from the weekly age and whether gender has an effect on the corneal thickness has to be evaluated. Knowing CCT and its increase with age is important for diagnosing ocular diseases such as corneal damage and determining the prognosis. In pathological conditions such as hypoxia, keratitis, laceration, corneal thickness goes beyond physiological levels16. According to results from the study at different ages it is evident that different corneal thickness. It will be useful to know physiological data in terms of provide animal welfare and preventing economic losses. The presented study will contribute to the literature as it has been conducted on live cattle, unlike other studies on cattle eyes 8,9. There are studies in literature that have shown a decrease in the density of endothelial cells when an age-related increase is forming in corneal thickness 16,44. Knowing that corneal thickness increases with age is of diagnostic importance. However, there is a need for further studies to fully clarify the mechanism of age-related increase in thickness.

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24. Chen K.H., Azar D., Joyce N.C. (2001). Transplantation of adult human corneal endothelium ex vivo: A morphologic study. Cornea. 25. Rao S.K., Sen P.R., Fogla R., Gangadharan S., Padmanabhan P., Badrinath S.S. (2000). Corneal endothelial cell density and morphology in normal Indian eyes. Cornea. 26. Nemesure B., Wu S.Y., Hennis A., Leske M.C. (2003). Corneal thickness and intraocular pressure in the Barbados Eye Studies. Archives of Ophthalmology. 27. Niederer R.L., Perumal D., Sherwin T., McGhee C.N.J. (2007). Age-related differences in the normal human cornea: A laser scanning in vivo confocal microscopy study. British Journal of Ophthalmology. 28. Lekskul M., Aimpun P., Nawanopparatskul B., Bumrungsawat S., Trakulmungkijkarn T., Charoenvanichvisit J., et al. (2005). The correlations between Central Corneal Thickness and age, gender, intraocular pressure and refractive error of aged 12-60 years old in rural Thai community. Journal of the Medical Association of Thailand = Chotmaihet thangphaet. 88, 175-179. 29. Abib F.C., Barreto J. (2001). Behavior of corneal endothelial density over a lifetime. Journal of Cataract and Refractive Surgery. 30. Sanchis-Gimeno J.A., Lleó-Pérez A., Alonso L., Rahhal M.S., MartínezSoriano F. (2005). Corneal endothelial cell density decreases with age in emmetropic eyes. Histology and Histopathology. 31. Galgauskas S., Norvydaite D., Krasauskaite D., Stech S., Ašoklis R.S. (2013). Age-related changes in corneal thickness and endothelial characteristics. Clinical Interventions in Aging. 8, 1445-1450. 32. Foster P. (1998). Central corneal thickness and intraocular pressure in a Mongolian population,. Ophthalmology. 105(6), 969-973. 33. Rüfer F., Schröder A., Bader C., Erb C. (2007). Age-Related Changes in Central and Peripheral Corneal Thickness. Cornea. 26(1), 1-5. 34. Andrew S.E., Willis A.M., Anderson D.E. (2002). Density of corneal endothelial cells, corneal thickness, and corneal diameters in normal eyes of Ilamas and alpacas. American Journal of Veterinary Research. 35. Ramsey D.T., Hauptman J.G., Petersen-Jones S.M. (1999). Corneal thickness, intraocular pressure, and optical corneal diameter in Rocky Mountain Horses with cornea globosa or clinically normal corneas. American Journal of Veterinary Research. 36. Pirie C.G., Alario A.F., Barysauskas C.M., Gradil C., Uricchio C.K. (2014). Manual corneal thickness measurements of healthy equine eyes using a portable spectral-domain optical coherence tomography device. Equine Veterinary Journal. 46(5), 631-634. 37. Coyo N., Peña M.T., Costa D., Ríos J., Lacerda R., Leiva M. (2016). Effects of age and breed on corneal thickness, density, and morphology of corneal endothelial cells in enucleated sheep eyes. Veterinary Ophthalmology. 19(5), 367-372. 38. Gwin R.M., Lerner I., Warren J.K., Gum G. (1982). Decrease in canine corneal endothelial cell density and increase in corneal thickness as functions of age. Investigative Ophthalmology and Visual Science. 39. Hoehn A.L., Thomasy S.M., Kass P.H., Horikawa T., Samuel M., Shull O.R., et al. (2018). Comparison of ultrasonic pachymetry and Fourier-domain optical coherence tomography for measurement of corneal thickness in dogs with and without corneal disease. Veterinary Journal. 40. Franzen A.A., Pigatto J.A.T., Abib F.C., Albuquerque L., Laus J.L. (2010). Use of specular microscopy to determine corneal endothelial cell morphology and morphometry in enucleated cat eyes. Veterinary Ophthalmology. 13(4), 222-226. 41. Van Der Woerdt A., Gilger B.C., Wilkie D.A., Strauch S.M. (1995). Effect of auriculopalpebral nerve block and intravenous administration of xylazine on intraocular pressure and corneal thickness in horses. American Journal of Veterinary Research. 42. Irby N.L., Angelos J.A. (2018). Ocular Diseases. Rebhun’s Diseases of Dairy Cattle: Third Edition. 43. Ehlers N., Hjortdal J. (2004). Corneal thickness: measurement and implications. Experimental Eye Research. 78(3), 543-548. 44. Coyo N., Peña M.T., Costa D., Ríos J., Lacerda R., Leiva M. (2016). Effects of age and breed on corneal thickness, density, and morphology of corneal endothelial cells in enucleated sheep eyes. Veterinary Ophthalmology. 19(5), 367-372.

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Uğur Şen.; Large Animal Review 2021; 27: 201-207

The relationship between placental characteristics and lamb birth weight in Akkaraman Turkish native sheep breed

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UĞUR ŞEN1 1

Ondokuz Mayis University, Faculty of Agriculture, Department of Agricultural Biotechnology, 55139, Samsun, Turkey

SUMMARY Introduction - The size and nutrient transfer capacity of the placenta plays a central role in determining the prenatal growth trajectory fetus, resulting in alteration birth weight. Aim - The aim of this study was to determine the relationship between placental characteristics and lamb birth weight in Akkaraman sheep breed. Materials and methods - The 63 single born Akkaraman male lambs with low (n= 18), moderate (n= 26) and high (n= 19) birth weight (BW) and their placentas were used as experimental materials. Placental weight (PW), the numbers (TCN) and weights (TCW) of cotyledon were determined. Length (CL), depth (CDe), diameter (CDia) and volume (CV) of cotyledons were also measured with an electronic digital compass. The total cotyledon surface area (TCSA), placental efficiency (PE), cotyledon efficiency (CE), volumetric cotyledon efficiency (VCE) and cotyledon density (CD) were calculated for each ewe. Results - Placental weight (PW), cotyledon weight, total and small cotyledon number (SCN), total weight, and the number of large cotyledon in lambs with low BW were lower than moderate and high BW (p < 0.05). High BW lambs had higher PE, CE, and VCE values than moderate and low BW (p < 0.05). Additionally, positive correlations were observed between BW, CE, and VCE (p < 0.05). A significant relation was calculated between BW and LCN (p < 0.05). The mortality rate of high lambs BW was lower (p < 0.05) than moderate and low BW until weaning. Discussion - Lamb BW changed with placental characteristics, and lambs with higher BW were found to have better placental components. Conclusions - The results of the present study revealed that placental components affect BW in Akkaraman lambs and poor placental characteristics may have a significant impact on survival until weaning.

KEY WORDS Placenta, fetal development, birth weight, mortality, Akkaraman sheep.

INTRODUCTION It is known that the deficiencies in sheep production and management systems increase mortality rates in newborns and also cause serious economic losses 1. In this respect, it is important to define physiological characteristics of offspring at birth and the postpartum period, to examine the adaptation the adaptability of the offspring and to better address current breeding or management problems as well as to develop the new sheep production strategies 2,3,4,5. The placenta is an organ that provides the passage of nutrients, respiratory gases such as oxygen and carbon dioxide, and waste materials between maternal and fetal systems through blood circulation 6. Sheep placenta has multiple cotyledonary structures and the placenta, where the fetus is located, is connected to the uterus wall by the embryo originated cotyledons, which combine with the protrusions on the uterus endometrium called the caruncle 2. Placentomes is consisted by the combination of

Corresponding Author: Uğur Şen (ugur.sen@omu.edu.tr)

cotyledon and caruncle, on the placenta ensure the blood circulation between dam and offspring, transferring nutrients to the fetus and removing the metabolic wastes produced by the fetus through the bloodstream 7. Therefore, the functional ability of the placenta is directly related to the number and size of the placentomes, which can be affected by both maternal and fetal factors 7,8. Previous studies in farm animals showed that placental growth occurs before fetal growth, and there is a positive relationship between placental mass and offspring weight at birth 8,9,10,11. The majority of the placental growth and development occur in the first trimester part of gestation and there is no change in dry matter content of the placenta following the first period in sheep 7,10 . The placenta reaches its maximum size during the first trimester part of gestation, but the fetus gained only 10% of its birth weight 8,10. Therefore, abnormalities or insufficient in placental development can directly affect fetal growth and development, or even the survival of the fetus. Moreover, the size and nutrient delivery capacity of the placenta play a critical role on the birth weight, growth, postnatal viability, adult productivity and health of newborn 8. Fetal growth in the last quarter of pregnancy in sheep is influenced by maternal nutrition level 12, nutrient transfer capacity

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The relationship between placental characteristics and lamb birth weight in Akkaraman Turkish native sheep breed

of the placenta to the fetus 7 and blood flow from the uterus to the placenta 13. Placental development is one of the most important prenatal factors affecting the birth weight of lambs 2,6,11. Placental weight is becoming more important for explaining the fetal weight variation that occurs when late and middle pregnancy periods are compared 14. Low birth weight increases postpartum mortality, while high birth weight causes dystocia resulting in decrease maternal care and increasing maternal mortality 15. Previous studies have reported that postnatal survival rate of lambs with low birth weight were lower than lambs with normal birth weight and slower to stand up and try to suck on their dam after birth 16,17. Moreover, placental features can be used as an important indicator in the assessment of postpartum infant mortality and survival in small ruminants 6,18. Additionally, it is known that insufficient placental development negatively affects fetal brain development and nervous system development. Placental insufficiency causes decrease development and number of nerve cells of the fetus and negatively affects the signaling mechanism of nerve cells with other cells 19,20 . These effects can lead to impaired neuromotor development of the offspring after birth and may increase the offspring’s time to stand up, but also reduce the willingness to try to suck on their dam. These situations show that the placenta not only plays an important role in adequate fetal development but also significantly affect fetal brain development, which will have possible effects on postpartum behavior, such as stand up and suckling of offspring 2. Akkaraman sheep breed is commonly raised indigenous sheep breed in Turkey and they constitute approximately 45% of the total sheep population 21. Akkaraman has high survival capacity and resistant to malnutrition due to a fat-tailed sheep breed 22. This breed has a high adaptability to the harsh climate, disease resistance, poor pasture and severe conditions 23. Male lambs of this breed are used as fattening material for meat production, and they makes up an important source of red meat production under harsh climate conditions 24. As a result, placental features may affect birth weight, survival rate until weaning, daily weight gain, live weight at the end of fattening, which affects the profitability of meat sector. Therefore, the aim of this study was to determine the relationship between placental characteristics, lamb birth weight and postnatal survival until weaning in Akkaraman Turkish native sheep breed.

MATERIAL AND METHODS The experimental procedures were approved by the Local Animal Care and Ethics Committee of Kirsehir Ahi Evran University, Kirsehir, Turkey, ensuring compliance with EC Directive 86/609/EEC for animal experiments. The study was done during the breeding season (September to March) in Turkey. A total of 63 male singleton lambs with low (n= 18), moderate (n= 26) and high (n= 19) birth weight (BW) from Akkaraman ewes, which at least in second parturition and ranging from 2 - 3 years of age, and their placenta were used as experimental materials. Low and high BW lambs were determined as lambs with one standard deviation difference of the average considering the average BW of all lambs born in the same flock and period. BW of singleton born male Akkaraman lambs (low; 3.88 ± 0.20, moderate; 4.88 ± 0.19 and high; 5.72 ± 0.16) are presented in Figure 1.

a b c

Figure 1 - Birth weights of singleton born male Akkaraman lambs. Different letters in the same color bars indicate significant difference (p<0.05).

a-b

All ewes were raised at a private farm in Kirsehir, Turkey (38° 55’ 56.8” N, 34° 10’ 45.6” E and 985 m above sea level) and they were allowed to graze daily 5 h during gestation. Additionally, the ewes were offered average concentrate at 100 g and wheat straw of 1 kg per day during the last third of gestation. The BW of singleton male lambs was determined within 5 hours following birth and the placentas, which were drop as naturally, were collected as a whole after delivery. Placental weight (PW) was determined with discharged placental fluid before weighing. The numbers (TCN) and weights (TCW) of cotyledons, which were dissected from the chorioallantois were also determined. Length (CL), depth (CDe), diameter (CDia) and volume (CV) of cotyledons were measured with an electronic digital compass. Cotyledons were then classified as small (<10 mm diameter), medium (10-30 mm diameter) and large (>30 mm diameter). The total cotyledon surface area (TCSA) was calculated after the measurements of all the cotyledons in individual placenta as cm2 with following formulae; radius squared of cotyledon [((cotyledon width + cotyledon length) / 4)2] × 3.14 (π) × TCN. Additionally, placental efficiency (PE; lamb BW / PW), cotyledon efficiency (CE; lamb BW / TCSA), volumetric cotyledon efficiency (VCE; CV / PW) and cotyledon density (CD; TCN / per gram PW) were calculated for each ewe. Following lambing, lambs were kept in the pen with their dam for 15 days in order to suck enough colostrum. After this period, lambs were treated with protection against internal and external parasites, and they were allowed to go to the pasture with their dam until 90 days of weaning age. In addition to pasture, the ewes were fed average concentrate at 50 g and wheat straw of 1 kg per day during the lactation period. Mortality rates of lambs in the BW groups up to weaning was calculated by ratio the number of lambs died until weaning to the number of lambs born. Placental characteristics and postnatal survival rate of lambs with different BW were analyzed using a completely randomized design by the General Linear Model procedure of the SPSS package program. Significant differences between means were tested using Duncan’s test and results were computed as mean ± SE. Statistical significance was considered at p < 0.05 and p < 0.01. Kruskall-Wallis H test was performed to analyze the effect of lamb birth weight on vital status until weaning survival. Relationships between variable traits for discrete data were

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determined with Pearson correlation analysis at the 95% confidence interval.

RESULTS Placental components of Akkaraman male lambs with low, moderate and high BW are presented in Table 1. The significant differences in terms of placental weight, total and average cotyledon weights were found and lambs with low BW had lower placental weight, total and average cotyledon weight than those of moderate and high BW (p < 0.05). There were no significant differences between lamb BW groups in terms of medium and small cotyledon weights, but large cotyledon weight of lambs with low BW were lower than high BW lambs (p < 0.05). Cotyledon characteristics of Akkaraman male lambs with low, moderate and high BW are presented in Table 2. Low BW lambs

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had higher (p < 0.05) total cotyledon and small cotyledon numbers compared to lambs with moderate and high BW, but they had lower (p < 0.05) large cotyledon numbers. There were no significant differences between lamb BW groups in terms of size measurements of placental cotyledons except for small cotyledon length. Low BW lambs had lower (p < 0.05) small cotyledon length than those of moderate BW. Various efficiency features of placenta and cotyledon of Akkaraman male lambs with low, moderate and high BW are presented in Table 3. Placental efficiency of high BW lambs was higher (p < 0.05) than lambs with low and moderate BW. Similarly, high BW lambs had higher (p < 0.05) cotyledon, volumetric cotyledon and average volumetric cotyledon efficiency values than those of low BW. There were no significant differences between lamb BW groups in terms of cotyledon volume and cotyledon surface area features. Pearson correlation coefficients of placental characteristics and birth related factors in Akkaraman male lambs are presented

Table 1 - Placental components of Akkaraman male lambs with low, moderate and high birth weight. Birth weights Traits (g)

Low

Moderate

High

326.3 ± 30.1b

370.8 ± 38.5a

392.4 ± 16.9a

TCW

77.50 ± 4.12b

93.20 ± 6.19a

93.56 ± 10.70a

ACW

1.10 ± 0.07b

1.632 ± 0.12a

1.514 ± 0.15a

SCW

14.50 ± 1.18

13.20 ± 1.75

16.68 ± 3.64

MCW

37.80 ± 2.88

35.38 ± 3.21

42.06 ± 2.88

LCW

25.20 ± 3.41b

38.31 ± 5.08a

41.2 ± 10.7a

a,b Different superscript letters in the same line indicate a significant difference (p < 0.05) PW = placental weight, TCW = total cotyledon weight, ACW = average cotyledon weight, SCW = small cotyledon weight, MCW = medium cotyledon weight, LCW = large cotyledon weight

Table 2 - Cotyledon characteristics of Akkaraman male lambs with low, moderate and high birth weight. Birth weights Traits (mm)

Low

Moderate a

High b

TCN

71.00 ± 1.68

58.59 ± 2.89

61.33 ± 4.20b

SCN

32.00 ± 1.50a

18.71 ± 2.25b

20.00 ± 5.06b

MCN

29.18 ±2.66

26.24 ±2.11

26.83 ± 1.70

LCN

9.82 ±0.980

13.65 ±1.60

14.50 ±3.01a

0.26 ± 0.02

0.19 ± 0.02

0.28 ± 0.1

ACDia

19.95 ± 0.45

22.72 ± 0.67

21.09 ± 0.53

ACL

24.92 ± 0.58

28.59 ± 0.72

26.69 ± 0.69

ACDe SC CDia

CDe

3.62 ± 0.11

4.11 ± 0.10

3.87 ± 0.122

9.11 ± 0.30

11.75 ± 1.03

10.13 ± 0.41

20.09 ± 0.36

23.06± 0.10

20.99 ± 0.51

30.76 ± 0.35

33.45 ± 0.10

32.26 ± 0.838

16.65b ± 0.35

20.55 ± 0.98a

17.66 ± 0.39ab

25.24 ± 0.47

28.12 ± 0.99

26.58 ± 0.61

32.88 ± 0.57

37.08 ± 1.05

35.82 ± 1.04

2.97 ± 0.17

3.28 ± 0.15

2.76 ± 0.12

3.60 ± 0.16

4.03 ± 0.15

4.19 ± 0.16

4.28 ± 0.19

5.01 ± 0.16

4.65 ± 0.25

a,b Different superscript letters in the same line indicate a significant difference (p < 0.05) TCN = total cotyledon number, SCN = small cotyledon number, MCN = medium cotyledon number, LCN = large cotyledon number, CD = cotyledon density, ACDia = average cotyledon diameter, ACL = average cotyledon length, ACDe = average cotyledon depth, SC = small cotyledon, MC = medium cotyledon, LC = large cotyledon

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The relationship between placental characteristics and lamb birth weight in Akkaraman Turkish native sheep breed

Table 3 - Various efficiency features of placenta and cotyledon of Akkaraman male lambs with low, moderate and high birth weight. Birth weights Traits (g)

Low

Moderate b

High b

14.25 ± 0.98

16.27 ± 2.15

24.14 ± 5.63a

14.39 ± 0.64b

20.32 ± 1.40ab

23.58 ± 3.39a

VCE

44.32 ± 2.88

50.89 ± 4.34

64.6 ± 10.33a

AVCE

0.62 ± 0.04b

0.86 ± 0.07ab

1.04 ± 0.17a

TCV

90.05 ± 6.6

108.53 ± 10.69

112.29 ± 16.16

ACV

1.29 ± 0.10

1.88 ± 0.17

1.76 ± 0.22

TCSA

270.28 ± 10.32

255.49 ± 15.86

285.58 ± 28.53

ACSA

3.86 ± 0.16

4.38 ± 0.20

4.51 ± 0.34

a,b

Different superscript letters in the same line indicate a significant difference (p < 0.05). PE = placental efficiency, CE = cotyledon efficiency, VCE = volumetric cotyledon efficiency, AVCE = average volumetric cotyledon efficiency, TCV = total cotyledon volume, ACV = average cotyledon volume, TCSA = total cotyledon surface area, ACSA = average cotyledon surface area.

in Table 4. The correlation coefficients of BW groups for placental characteristics and birth-related factors were pooled and presented in the table. There were positive correlation between BW and CE (r = .420; p < 0.05) and BW and VCE (r = .422; p < 0.05). Positive correlations were calculated between PW and LCN (r = .488; p < 0.05) and PW and ACDia (r = .356; p < 0.05), while negative correlations were found between PW and SCN (r = -.405; p < 0.05), PW and CD (r = -.641; p < 0.01) and PW and PE (r = -.694; p < 0.01). Negative correlations were obtained between the LCN and SCN (r = -.495; p < 0.05) and MCN and CE (r = -.342; p < 0.05). Positive correlations were calculated between SCN and TCSA (r = .385; p < 0.05), SCN and CD (r = .701; p < 0.01) and SCN and PE (r = .471; p < 0.05), while negative correlations were found between SCN and CE (r = -.475; p < 0.05), and SCN and VCE (r = -.451; p < 0.05). Positive correlations were obtained between ACDia and ACL (r = .636; p < 0.01), ACDia and ACSA (r = .879; p < 0.01), ACDia and TCSA (r = .737; p < 0.01) and ACDia and TCV (r = .587; p < 0.05), while negative correlations were calculated between ACDia and CE (r = -.559; p < 0.05), and ACDia and VCE (r = -.534; p < 0.05). Positive correlations were calculated between ACL and ACSA (r = .923; p < 0.01), ACL and TCSA (r = .536; p < 0.05) and ACL and TCV (r = .542; p < 0.05), while negative correlations were found between ACL and CE (r = .349; p < 0.05) and ACL and VCE (r = -.404; p < 0.01). A positive correlations was obtained between ACDe and TCV (r = .751; p < 0.01), but a negative correlations was calculated between ACDe and VCE (r = -.421; p < 0.05). A positive correlation was calculated between ACSA and TCV (r = .618; p < 0.01), while negative correlations were found between ACSA and CE (r = -.455; p < 0.05) and ACSA and VCE (r = -.477; p < 0.01). There was positive correlation between CE and VCE (r = .889; p < 0.01), but a negative correlations was calculated between CE and TCV (r = -.506; p < 0.05). There were positive correlation between CD and PE (r = .918; p < 0.01) and TCV and CE (r = .889; p < 0.01). The vital status of male Akkaraman lambs (death or alive) with low, moderate and high birth weight until weaning age are presented in Figure 2. The significant differences in terms of survival rates until weaning were found between the lamb BW groups (p < 0.05). As expected, lambs with high BW had lower death rate (5.26%) and higher alive rate (94.74%) compare to lambs with moderate (15.4% and 84.6%) and low BW (16.7% and 83.3%) until 90 days of weaning age.

b b

a b

Figure 2 - The vital status of male Akkaraman lambs (death or alive) with low, moderate and high birth weight until weaning age. ab Different letters in the same color bars indicate significant difference (p<0.05).

Regression of placental characteristics on birth weight was performed by linear regression, and although the regression model was found statistically significant, the coefficient of expression (R2) was determined as 0.139. SCN and TCW were excluded from regression model because of null coefficient and the resulting regression model; BW= 4.355 + 0.001 × PW + 0.005 × LCW + 0.003 × MCW + 0.045 × SCW + 0.037 × LCN + 0.019 × MCN - 0.023 × TCN. To determine the effective variables stepwise variable selection procedure was applied. A significant relation was calculated between BW and LCN and this model found as statistically significant with determination coefficient of p < 0.05. After elimination of the variables obtained regression model was; BW= 4.251 + 0.05 × LCN (Figure 3).

DISCUSSION Previous studies show that the nutrient intake during gestation especially the second trimester, in which term placental growth occur in sheep, affects the placental development and resulting in alteration lamb BW 8,11,14. Additionally, past studies indicated that the lambs with low BW had a higher mortality rate until weaning 16,25 and it was taken long to try to stand up and suck on the dam 17. Ocak et al. 4 reported that the par-

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Large cotyledon numbers

Uğur Şen.; Large Animal Review 2021; 27: 201-207

Birth weight (g) Figure 3 - Correlation between birth weight and large cotyledon numbers for 63 lambs born to Akkaraman Turkish native sheep breed.

ity did not affect the BW of lamb, but Dwyer et al. 2 reported that lamb BW increased as the number of parity increased, and younger ewes had lower BW lambs than mature. Similarly, Sen and Onder 6 reported that the yearling goats in the first party had a lower kid BW than those of adults. Although breeding conditions and parity number of the Akkaraman sheep breed, which are used as experimental animal, were nearly similar, there were differences in terms of lamb BW and placental characteristics. Therefore, the current study was based on these basic differences, and it is thought that the differences in placental characteristics may cause differences in lamb BW. Previous studies have reported that placental weight, which is one of the indicators of growth deficiency of the fetus, can be associated with cotyledon weight and number 6. Dwyer et al.2 observed a difference in the placental characteristics of lambs with relatively different BW obtained from sheep with different parity numbers. Same authors reported that lambs with higher BW from sheep with more parity numbers had the heav-

205

ier placenta and average cotyledon weights. Similarly, Konyali et al. 26 reported that BW and placental weight increased or decreased together in goats. On the contrary, Ocak et al. 4 did not observe a relationship between placental weight and BW of lambs obtained from sheep with different number of parity. In the present study, it has been determined that Akkaraman lambs with low BW have lower PW, TCW and ACW than those of lambs with moderate and high BW. Additionally, it was determined that the lambs with high BW had higher weight of large, medium and small and total cotyledons than low BW lambs. These results might suggest that differences in BW of lambs with same parity of ewes maybe due to variations of the placental development. Differences in BW of Akkaraman lambs with same parity cannot be explained by only the variation of placental development. Therefore, future researches on histological components are necessary to define the variations of placentas. The newborn mortality and survival rate until weaning age are important indicators of sustainability in livestock farming. Postnatal lamb mortality, which is an important factor for profitability in sheep production, varies between 7-51% around the world 3. Importance factors for the postnatal mortality rate are multiple births, dystocia, BW, ambient temperature, maternal care, placental insufficiency, breed and flock management 6,27,28,29. Ocak et al. 4,30 determined that there is a relationship between placental characteristics and postnatal mortality, and also they reported that the postnatal survival rate of lambs born to different sheep breeds were between 5-6% until weaning. Additionally, Mellor and Stafford 27 indicated that postnatal mortality of the offspring in livestock is associated with the growth and development of the placenta during pregnancy. In the present study, postnatal mortality rate of lambs was between 5-16% until weaning. However, it was observed that the survival rates of lambs with high BW were found higher than that of low and moderate BW lambs from birth to weaning. These results indicated that the Akkaraman lambs, which have heavier various cotyledons, with higher birth weight than flock

Table 4 - Pearson correlation coefficients of placental characteristics and birth related factors in Akkaraman male lambs. PW

BW PW LCN MCN SCN ACDia ACL ACDe ACSA TCSA CE CD PE TCV

.108

TCN

LCN

MCN

SCN

ACDia

ACL

ACDe ACSA TCSA

CE *

-.202

.232

-.215

-.262

.200

.240

.185

.265

.091

.420

.054

.488*

.196

-.405*

.356*

.184

-.050

.285

.290

-.187

-.268

TCV

VCE

.050

.301

.188

.422*

-.641** -.694**

.119

-.155

-.495

.032

.052

-.247

.042

.080

.022

-.248

-.245

-.129

.149

-.003

.212

-.040

-.036

.082

.310

-.342*

-.187

-.309

.182

-.310

-.017

-.172

.043

-.136

.385*

-.475*

.701**

.471*

.281

-.451*

.636**

.156

.879**

.737**

-.559*

-.100

-.107

.587*

-.534*

.304

.923

.536

-.349

-.089

.032

.542

-.404*

.266

.091

.000

.130

.231

.751**

-.421*

-.477* -.712**

.675

-.455

-.115

-.033

.618

-.796**

.197

.034

.707**

.109

-.506

.889**

.918**

.235

-.207

-.171

.197

.005 -.729**

BW= birth weight, PW = placental weight, TCN = total cotyledon number, SCN = small cotyledon number, MCN = medium cotyledon number, LCN = large cotyledon number, ACDia = average cotyledon diameter, ACL = average cotyledon length, ACDe = average cotyledon depth, ACSA = average cotyledon surface area, TCSA = total cotyledon surface area, CE = cotyledon efficiency, CD = cotyledon density, PE = placental efficiency, TCV = total cotyledon volume, VCE = volumetric cotyledon efficiency. * p < 0.05, ** p < 0.01.

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The relationship between placental characteristics and lamb birth weight in Akkaraman Turkish native sheep breed

average have a higher survival rate until weaning. The attachment of cotyledons to the uterus takes place between 25 - 30 days of gestation in sheep, and growth restrictions (insufficient main feeding, abnormal environmental conditions and abnormal endocrinal activity etc.) during this period may affect the number of placental cotyledons 7,11. Moreover, restrictive factors in the last period of gestation can affect the morphology and size of cotyledons rather than the number 7,31. Dwyer et al. 2 reported that lambs with higher BW had fewer cotyledons. However, Konyali et al. 26 observed that there was a positive relationship between kid BW and cotyledon number in the goat placenta. Similarly, Sen and Onder 6 show that as the BW increases, the number of cotyledons on the placenta increase as well. We observed that the number of total and small cotyledons was higher in lambs with low BW than lambs with moderate and high BW. On the other hand the number of large cotyledons was higher in lambs with high BW than those with low and moderate BW. In the study, cotyledon number with same parity seems to be the most important indicator for different BW. Previous studies show that the morphological characteristics of cotyledons on the placenta and the exchange between the maternal and fetal circulation are highly related 7,11. Ocak et al.4 indicated that singleton and twin lambs with different BW had similar diameter, length and depth in placental cotyledons. However, Ocak et al. 30 reported that twin kids had higher cotyledon diameter, length, and depth than single born in Damascus goats. Moreover, Ocak and Önder 29 showed that parity did not affect the cotyledon diameter and depth of kids, but it affects cotyledon length. Similarly, it was observed that small cotyledons were longer in lambs with moderate BW than lambs with high BW in the current study. This result may indicate that cotyledonal measurements were related to lamb BW and there were a difference in the length of small cotyledons. Uterus capacity is defined as the total placental tissue mass and also placenta efficiency has been calculated as an index of uterine capacity 20. Placenta efficiency is used as an important parameter for the assessment of offspring development during gestation in multiple birth farm animals such as pigs and sheep 2, 32 . However, it has been reported that the determination of the TCSA and the calculation of the CE give more precise and reliable results in determining the differentiation in placental development, its functional ability and its effect on the development of offspring 30. Although it is difficult to measure the placental surface area in sheep, determining of the cotyledonal surface area and cotyledon density in the placenta is a strong indicator of the link between dam and offspring and it is a useful tool for predicting adequate placental development during gestation 6,7,30. In the present study placental efficiency of lambs with high BW was higher than lambs with low and moderate BW. Similarly, cotyledon efficiency and volumetric cotyledon efficiency of lambs with high BW were observed to be higher than lambs with low BW. Previous studies reported that parity did not affect PE and CD 4, but twin-born lambs had higher rate of PE and CD than those of singleton 4,30. Ocak et al. 3 reported that female lambs with low BW had a lower rate of placental efficiency and cotyledon density than male lambs with higher BW. Also, Sen and Onder 6 reported that there was a difference in cotyledon efficiency of kids with different BW. The results of the current study show that PW, PE, CE and CE are directly related to BW of Akkaraman male lambs and these results support the conclusion of previous studies 2,3,4,6,11,30.

Previous studies reported that there was significant relationship between BW and PW in different species 2,6,33. However, there was no significant correlation between pooled lamb BW and PW in the present study. The results of the present study are consistent with the results of Ocak et al. 4,30. Pooled data of Akkaraman male lambs with different birth weight showed that there were positive correlations among BW, CE and VCE placental characteristics. These observations are in agreement with the argument of past studies 4,6. In the current study relationships were determined among PW and some cotyledon type and dimensions as positively, but a negative correlation was observed between PE and CD measuremets. These observations are consistent with the findings reported by Ocak and Onder29, Ocak et al. 30 and Sen and Onder 6. In the current study, we observed that lamb BW changed with placental characteristics and lambs with higher BW were found to have better placental components. Although, Akkaraman sheep breed was raised in the same management and feeding procedure, the differences in the BW of lambs might be due to different placental development. Additionally, the results of the current study indicated that the placental characteristics significantly affected the postnatal survival of the lambs in relation to BW in Akkaraman sheep breed, which is an importance native breed of Turkey. All these results suggest that placental development during gestation can affect the future productivity of the lambs in relation considering BW in Akkaraman sheep breed. Future studies may enable the determination of the possible effects of placental characteristics on the subsequent productivity characteristics such as meat, milk and wool yield, and the development of new breeding strategies by looking at the placental characteristics of lambs.

Acknowledgments The author is grateful to Prof. Hasan ÖNDER and Dr. Emre SIRIN for their technical support in the placental measurements and statistical analysis. The author acknowledges the financial support by the Kirsehir Ahi Evran University Scientific Research Projects Coordination Unit (Project no.: PYO-ZRT.4001.15.004) to carry out this study.

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21. TurkStat. (2020). Turkish Statistical Institute. Livestock Statistics, available at, http,//www.turkstat.gov.tr last access, August 24, 2020. 22. Aksoy Y, Uğurlu M, Önenç A, Şirin E, Şen U, Çiçek Ü, Ulutaş Z, Kuran M. (2018). Meat production characteristics of Turkish native breeds, I. Fattening, slaughter and carcass traits of lambs. South African Journal of Animal Science, 484: 665-672. 23. Aksoy Y, Uğurlu M, Önenç A, Şirin E, Şen U, Çiçek Ü, Ulutaş Z, Kuran M. (2019). Meat production characteristics of Turkish native breeds, II. meat quality, fatty acid, and cholesterol profile of lambs. Archives Animal Breeding, 62: 41-48. 24. Sirin, E, Aksoy, Y, Ugurlu, M, Cicek, U, Onenc, A, Ulutas, Z, Sen, U, Kuran, M. (2017). The relationship between muscle fiber characteristics and some meat quality parameters in Turkish native sheep breeds. Small Ruminant Research, 150: 46-51. 25. Gama LT, Dickerson GE, Young LD, Leymaster KA. (1991). Effects of breed, heterosis, age of dam, litter size, and birth weight on lamb mortality. Journal of Animal Science, 69: 2727-2743. 26. Konyali A, Tölü C, Daş G, Savaş T. (2007). Factors affecting placental traits and relationships of placental traits with neonatal behaviour in goat. Animal Reproduction Science, 97: 394-401. 27. Mellor DJ, Stafford KJ. (2004). Animal welfare implications of neonatal mortality and morbidity in farm animals. The Veterinary Journal, 168: 118-133. 28. Kerslake JI, Everett-Hinks JM, Campbell AW. (2005). Lamb survival: A new examination of an old problem. New Zealand Society of Animal Production, 65: 13-18. 29. Ocak S, Onder H. (2011). Placental traits and maternal intrinsic factors affected by parity and breed in goats. Animal Reproduction Science, 128: 45-51. 30. Ocak S, Ogun S, Gunduz Z, Onder H. (2014). Relationship between placental traits and birth related factors in Damascus goats. Livestock Science, 161: 218-223. 31. Wu G, Bazer FW, Wallace JM, Spencer TE. (2006). Board-invited review, intrauterine growth retardation, implications for the animal sciences. Journal of Animal Science, 84: 2316-2337. 32. Mesa H, Safranski Tj, Johnson RK, Lamberson WR. (2003). Correlated response in placental efficiency in swine selected for an index of components of litter size. Journal of Animal Science, 81(1): 74-79. 33. Echternkamp SE. (1993). Relationship between placental development and calf birth weight in beef cattle. Journal of Animal Science, 32: 1-13.

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FRANCESCA CIMINO¹*, CARMEN MARESCA², MARIA LUISA MARENZONI¹, FRANCESCO FELIZIANI² 1 2

Dipartimento di Medicina Veterinaria, Università degli Studi di Perugia Istituto Zooprofilattico Sperimentale Umbria e Marche ‘‘Togo Rosati‘”

RIASSUNTO La Peste Suina Africana (PSA), African Swine Fever (ASF) rappresenta una delle più importanti malattie dei maiali sia per l’ampia diffusione geografica che per l’enorme impatto che determina nell’industria suinicola. È inserita nella lista delle malattie notificabili dell’Organizzazione Mondiale della Sanità (OIE), insieme a tutte le altre malattie che costituiscono un pericolo per la Sanità animale e pubblica. L’incessante diffusione dell’African Swine Fever virus (ASFV), insieme con la sua capacità di adattarsi ai nuovi ambienti, la sua elevata resistenza nell’ambiente e nei prodotti biologici e la mancanza di un vaccino, rendono difficile la sua eradicazione. La malattia è rimasta confinata in Africa fino alla fine degli anni ’50, fin quando il virus (genotipo I) ha fatto la sua comparsa in Portogallo nel 1957. Nel 2007 l’ASFV (genotipo II) ha raggiunto la regione del Caucaso, da dove ha fatto il suo ingresso nell’Unione Europea (UE) nel 2014, espandendosi progressivamente in diversi Stati Membri tra cui: Bulgaria, Slovacchia, Estonia, Ungheria, Lettonia, Lituania, Polonia, Romania e più recentemente Germania. L’infezione si è presentata anche nella Repubblica Ceca e successivamente in Belgio, dove è stata eradicata con successo. L’esperienza legata alle conseguenze di questa infezione offre molti spunti multidisciplinari; la malattia ha infatti ripercussioni negli ambiti più disparati, da quello socio-economico fino a quello medico, passando per l’ambito psicologico e culturale; tenendo conto di quanto appena esposto lo scopo del presente lavoro è quello di proporre un approccio One Health per il controllo della PSA in quanto, sebbene non sia una infezione trasmissibile all’uomo, implica uno stretto legame tra malattie degli animali e dell’uomo.

PAROLE CHIAVE Peste Suina Africana; incessante diffusione; adattamento; essenza multidisciplinare; One Health; perdita economica.

INTRODUZIONE La Peste Suina Africana (PSA) è una malattia infettiva virale dei suidi, causata dall’African Swine Fever virus (ASFV), un virus a DNA a doppio filamento, appartenente alla famiglia Asfarviridae (African Swine Fever and related virus), genere Asfivirus. Essa è altamente contagiosa, non è trasmissibile all’uomo, può colpire sia il maiale domestico sia il cinghiale (biologicamente la stessa specie, Sus scrofa) ed è solitamente letale¹. Questa malattia può determinare danni considerevoli alle produzioni zootecniche suine, sia direttamente a causa della elevata mortalità, sia indirettamente per le restrizioni del commercio nazionale ed internazionale di suini e dei prodotti derivati. La PSA causa gravi conseguenze socio-economiche nei Paesi in cui è diffusa; in virtù della sua capacità a raggiungere proporzioni epidemiche tali da mettere a repentaglio la sicurezza degli scambi tra le nazioni, è ritenuta una delle malattie transfrontaliere più importanti¹. Per tale motivo, è inserita nell’elenco delle malattie infettive di primaria importanza e soggette a notifica obbligatoria, sulla base dell’articolo 5, comma 1, del Regolamento dell’Unione Europea (UE) 429/2016². Descritta per la prima volta in Kenya nel 1921, in seguito all’introduzione dell’allevamento del maiale domestico³, la malattia è ritenuta endemica nell’Africa sub-sahariana. La prima notifica di PSA fuori dal continente africano si verificò in Portogallo nel 1957 a causa di rifiuti alimentari infetti trasporta-

Corresponding Author: Francesca Cimino (francescaa.c@libero.it).

ti da aerei di linea, con cui vennero alimentati suini allevati nei pressi dell’aeroporto di Lisbona. Di seguito, l’infezione fu segnalata in diversi Paesi europei, tra cui Spagna, Francia, Italia, Belgio e Paesi Bassi. A cavallo degli anni ’70 comparve in Brasile, Cuba, Repubblica Dominicana e Haiti². Nel 2007, focolai di infezione sono emersi in Georgia, Armenia, Azerbaijan, così come in Russia Europea, Ucraina e Bielorussia; dal Caucaso la malattia si è diffusa nell’Unione Europea, fino a presentarsi, alla fine del 2019, in dieci stati membri: Lituania, Polonia, Lettonia, Estonia, Romania, Ungheria, Bulgaria, Belgio, Slovacchia e Serbia4. In Sardegna la PSA è rimasta endemica dalla sua comparsa nell’isola nel 1978 fino ai più recenti periodi, nonostante l’applicazione di misure di controllo sempre più serrate5. L’avanzamento ulteriore dell’infezione in nuovi territori come l’Asia e le difficoltà nel contrastare l’infezione, nonostante le numerose misure di controllo intraprese dai vari Stati colpiti, rendono questa malattia una minaccia attuale all’economia e al benessere degli animali e dell’uomo a livello globale. Sebbene sia meno contagiosa rispetto ad altre malattie transfrontaliere come l’afta epizootica, la PSA ha un notevole impatto negativo nei più disparati ambiti, a volte inaspettati: da quello socio-economico a quello medico e psicologico. Lo scopo di questo lavoro è quello di proporre un approccio “One Health” nei confronti della malattia, proprio per la sua “natura” multidisciplinare. L’approccio One Health riconosce il legame tra la salute degli esseri umani, degli animali e l’ambiente in una visione olistica, che abbraccia più discipline, ma solitamente riservata alle zoonosi. Per dimostrare come la lotta alla PSA, secondo questa prospettiva, possa rappresentare una strategia vincente, in grado di dare risultati decisivi, migliori di quel-

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li già ottenuti negli anni, il presente studio intende fornire un’analisi delle gravi conseguenze che la diffusione della PSA ha causato e può causare tuttora, sulla base di dati scientifici, ma anche di notizie provenienti da fonti scientifiche meno accreditate, ma più veloci e a diffusione maggiore.

tica prima che nell’uomo8. Il vantaggio economico di tale metodo è già stato dimostrato per le zoonosi, come ad esempio per la West Nile in Emilia-Romagna, dove l’esperienza di sorveglianza umana e animale integrata ha determinato una più efficiente risoluzione del problema, oltre a un risparmio economico9.

Approccio One Health collegato alla problematica PSA La visione One Health, introdotta all’inizio del 2000, si basa sul riconoscimento che la salute umana, la salute degli animali e la salute dell’ecosistema siano strettamente dipendenti l’una dall’altra e rappresenta l’approccio ideale per raggiungere la salute globale6. L’American Veterinary Medical Association ha definito l’approccio One Health come “l’insieme degli sforzi collaborativi di più discipline che operano localmente, a livello nazionale e globale per raggiungere una salute ottimale per persone, animali ed ambiente”. Solitamente questo approccio è applicato alle malattie zoonotiche che rappresentano una minaccia per la salute globale, determinando ingenti perdite economiche. È opinione condivisa che l’interazione tra uomini, animali e ambiente dovrebbe essere considerata come un sistema dinamico unico in cui quattro sono gli elementi chiave: • le attività umane; • il sistema agro-alimentare; • la globalizzazione del commercio di animali e prodotti di origine animale (componente geografica); • la fauna selvatica e i fattori ambientali (ecologia) (Figura 1). Riconoscere le interconnessioni tra i diversi Paesi del mondo, notevolmente aumentate in seguito alla globalizzazione dei commerci e degli spostamenti delle persone, potrebbe determinare una svolta per quanto riguarda il controllo delle malattie transfrontaliere e quindi anche della PSA7. L’approccio multidisciplinare integrato One Health non è certamente qualcosa di estraneo agli aspetti che caratterizzano la PSA; l’importanza di tale approccio è stata sottolineata anche dalla World Bank, che ha stimato una riduzione dei costi in caso di rilevazione anticipata di una malattia emergente (e dei problemi che ne conseguono) in vettori, bestiame o fauna selva-

MATERIALI E METODI Strategia di ricerca delle fonti dei dati La ricerca delle fonti dei dati è stata eseguita esaminando i database Pubmed, CAB Abstracts e Scopus, oltre ad alcune specifiche riviste scientifiche, che rappresentano fonti di informazioni accreditate. Ulteriori documenti sono stati invece trovati mediante ricerca sul web e successivo screening delle numerose informazioni circolanti relative all’argomento di interesse. La ricerca sul web è stata fatta utilizzando il motore di ricerca “Google”, usando come parole chiave “ASF economic impact” OR “ASF economic damage” OR “pig production” OR “ASF famine”, estendendo la ricerca a tutti i campi e alla sezione “Notizie”, per trovare informazioni più recenti. Su questa base si sono voluti raccogliere dati ed informazioni sull’impatto che la PSA ha avuto e che sta avendo tuttora nel settore socio-economico, nell’ambito psicologico e culturale, e in campo medico. Dopo una prima selezione basata sui titoli ed abstract, sono stati raccolti numerosi articoli da quotidiani, settimanali, riviste, ecc. È stata effettuata una seconda revisione sugli articoli selezionati, in base agli scopi e agli obiettivi della tesi, al fine di includerli o escluderli nel presente elaborato.

RISULTATI Ambiti di maggior impatto della PSA In base alla ricerca sopramenzionata, emerge che la PSA può esercitare una pressione negativa su diversi ambiti che si riflettono sulla salute, sulle attività o semplicemente sulle condizioni di benessere dell’uomo. I settori più gravemente colpiti sono ovviamente l’industria suinicola ed il settore socioeconomico in generale, seguiti dall’area medica, da quella psicologica e culturale e dall’alimentazione. Di seguito sono riportati alcuni degli effetti documentati che la comparsa di tale patologia ha determinato nelle aree colpite da PSA.

Ambito socio-economico

Figura 1 - Componenti della interazione tra uomini, animali e ambiente. Fonte: Calistri P., Iannetti S., Danzetta M. L., Narcisi V., Cito F., Di Sabatino D., Bruno R., Sauro F., Atzeni M., Carvelli A., Giovannini A. (2013). The components of ‘One World - One Health’ Approach. Transbound Emerg Dis, 60: 4-13.

Negli ultimi anni il consumo di prodotti animali, tra cui quelli suinicoli, sta crescendo rapidamente nei Paesi più ricchi, con il risultato di un aumento della produzione di bestiame in quei Paesi, unitamente ad un aumento delle importazioni ed esportazioni. Sebbene l’economia globale tragga vantaggio in questa situazione, la possibilità di diffusione di malattie animali come la PSA si intensifica10. La Cina rappresenta il principale Paese produttore di carne suina, essendo responsabile di circa metà della sua produzione globale, per cui l’insorgenza e la costante presenza di una malattia altamente letale quale la PSA determina devastanti conseguenze nel settore socio-economico. L’attuale epidemia cinese di PSA si sta verificando in un momento critico per quanto riguarda lo scenario commerciale dovuto alle tensioni politiche e commerciali tra Cina e Stati Uni-

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ta causa dell’aumento dei prezzi della carne di maiale (Figura 3) ¹³. Per quanto riguarda la situazione italiana, la PSA sta mettendo a rischio la filiera suinicola, basata principalmente sull’industria della trasformazione. Proprio lo scorso anno ASSICA, l’associazione delle industrie italiane delle carni trasformate e dei salumi, ha lanciato l’allarme, ribadendo la necessità di un adeguamento dei prezzi sui prodotti finali in seguito al rialzo del costo della carne suina, per evitare il blocco dell’intero sistema di produzione14. Da quanto evidenziato fino ad ora si può affermare che la PSA è l’epidemia più grave che abbia mai coinvolto il settore zootecnico mondiale e che rappresenta una questione di elevata priorità su scala globale poiché sta mettendo a dura prova l’intera economia mondiale15. Figura 2 - Cambiamenti nei prezzi della carne suina cinese dal 31 dicembre 2017 al 6 febbraio 2020. Fonte: Mason-D’Croz D., Bogard J. R., Herrero M., Robinson S., Sulser T. B., Wiebe K., Willenbockel D., Godfray H. C. J. (2020). Modelling the global economic consequences of a major African swine fever outbreak in China. Nat food, 1(4): 221-228.

ti legate anche all’aumento delle tariffe sulle importazioni di carne di maiale dagli USA alla Cina dal 12 al 62% e alla pandemia da SARS-Cov2. Negli anni precedenti uno studio di Mason-D’Croz et al. (2020) ha utilizzato un modello economico globale del sistema alimentare per esaminare ed esplorare le potenziali conseguenze di un grave shock produttivo nell’industria cinese dei suini, causato da una grave epidemia di PSA, sul mercato globale delle carni suine. Il modello prevede l’interruzione della fornitura di maiali in Cina e una domanda non soddisfatta che determina un aumento dei prezzi di altre carni. Considerando la produzione di carne suina in Cina nel 2018, che ammontava a circa 55 milioni di tonnellate all’anno (MT), ed ipotizzando una riduzione dei suini che porta a un suo calo tra 10 e 40 MT, si è stimato un aumento dei prezzi mondiali della carne suina del 17-85%. Nel modello i prezzi più alti della carne suina riducono la domanda della stessa in tutti i Paesi, compresa la Cina, mentre determinano un aumento della sua produzione nei Paesi al di fuori della Cina, compensando in questo modo una parte delle perdite della Cina e, di conseguenza, la riduzione della produzione suina mondiale. I consumatori, inoltre, sono portati a sostituire la carne di maiale con alimenti alternativi e l’aumento della loro domanda porta ad un aumento della loro produzione su scala mondiale, la quale determina un aumento, seppur minore rispetto al precedente, dei loro prezzi. Un ulteriore effetto delle variazioni dei prezzi dei prodotti alimentari e dei modelli di domanda-offerta è rappresentato dalla ridistribuzione dei beni nel settore agricolo. In Cina, ad esempio, si potrebbe verificare un calo della domanda di colture per mangimi (ad esempio, mais e soia), utilizzate nell’industria suinicola in favore di quella ad esempio del grano, mentre nei Paesi in cui la produzione di suini è aumentata si registrerebbe un aumento della loro domanda¹¹. Nell’UE il settore suinicolo rappresenta uno dei comparti produttivi principali dal punto di vista economico, costituendo l’8,5% della produzione totale dell’industria agro-zootecnica e il 50% della produzione totale di carne¹². La richiesta di carne suina da parte della Cina nel 2019 è sta-

Ambito psicologico-culturale Lo studio di Mason-D’Croz et al. (2020) ha fornito una stima dei cambiamenti che la PSA potrebbe causare sui redditi e sul benessere delle famiglie dei Paesi colpiti, principalmente in Cina. I risultati mostrano che in caso di una riduzione tra il 20 e l’80% della produzione suina il benessere delle famiglie in Cina diminuirebbe tra lo 0,12 e lo 0,78%, influenzando prevalentemente i piccoli allevatori, con un calo del reddito delle famiglie dello 0,3-1,8%. Il calo del benessere registrato in Cina potrebbe comunque caratterizzare altri Paesi colpiti dalla PSA magari con diversi livelli di intensità a seconda delle generali condizioni economiche e sociali. Fanno eccezione i Paesi ad alto reddito dell’Oceania (Australia e Nuova Zelanda), i quali trarrebbero un leggero beneficio dall’aumento dei prezzi della carne suina e delle materie prime causato dalla diffusione della PSA, essendo due dei maggiori esportatori di carne suina¹¹. Per quanto riguarda i fattori culturali legati alla PSA e che da essa possono essere condizionati, un esempio può essere rappresentato dalla Sardegna centro-orientale, dove era frequente la presenza di suini non identificati, il loro commercio illegale, unitamente al pascolo brado. Tutti questi fattori, derivanti da retaggio culturale, hanno favorito la persistenza della PSA16. Recentemente l’azione di forte contrasto a queste pratiche ha fornito un contributo notevole alla lotta alla diffusione dell’infezione, nonostante l’iniziale forte opposizione di una parte della popolazione locale.

Figura 3 - Un venditore di carne suina al mercato di Chengdu, a sud ovest della Cina. Fonte: Bottarelli, M. (2019) In Cina è emergenza febbre suina: già morti 50 milioni di maiali. Si rischia una crisi alimentare globale. Available at: https://it.businessinsider.com/febbre-suina-in-cina-milioni-di-maiali-morti-crisi-alimentare-globale/.

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Ambito medico-farmaceutico Pensando alla produzione suinicola, non sempre si considerano i prodotti medicali, che derivano dal suino. Ad esempio, la maggior parte delle eparine disponibili sono prodotte da aziende cinesi a partire dalla mucosa suina; appare chiaro come la PSA possa avere conseguenze gravi anche in campo sanitario. L’epidemia di PSA in Cina causa un graduale esaurimento delle disponibilità di eparina per il resto del mondo17. Dato il suo vasto e importante impiego in campo medico, un mancato accesso ad essa costituisce un rischio generalizzato. La ricerca di valide alternative terapeutiche, la riduzione degli sprechi, la regolazione del suo utilizzo in base alla priorità di trattamento medico, oltre all’applicazione di un sistema di distribuzione in situazioni di crisi, sono provvedimenti che contribuirebbero a mitigare la carenza di eparina e che necessitano del coinvolgimento di tutte le parti interessate, inclusi infermieri, farmacisti, fornitori commerciali e dirigenti tecnici ed amministrativi18. Quanto appena detto per l’eparina potrebbe verificarsi anche per altri prodotti medicali di origine suina, quali l’insulina.

Ambito alimentare Lo studio di Mason-D’Croz et al. (2020) ha stimato anche gli effetti della PSA sulla diversità alimentare ed assunzione di alimenti e quindi sul consumo calorico. I risultati hanno mostrato una flessione negativa della disponibilità calorica nella maggior parte dei Paesi a causa dell’aumento dei prezzi; nei Paesi con un reddito elevato (Asia orientale) ciò non causerebbe gravi conseguenze sulla salute umana, nei Paesi a basso reddito, come il Vietnam e le Filippine, gli effetti negativi sarebbero rilevanti. Le ripercussioni maggiori si avrebbero probabilmente in India, dove l’aumento del prezzo del grano, uno dei più importanti alimenti di base in questo Paese, deriverebbe da un aumento dei prezzi della carne suina. Uno scenario completamente differente è rappresentato dall’Africa orientale e meridionale e dall’America centrale, in cui il mais è un alimento base. In questi Paesi la ridotta domanda di mais come mangime per gli animali ne ridurrebbe i prezzi e ciò porterebbe ad un aumento della disponibilità calorica.

Ambito comunicazione: informazione, disinformazione e fake news Nel caso specifico della PSA, considerando le implicazioni sociali ed economiche, la comunicazione è essenziale non soltanto quando si è già verificato un focolaio epidemico, ma anche e soprattutto in funzione della prevenzione della malattia, della informazione e accettazione delle misure di controllo da intraprendere. La Commissione Europea suggerisce i canali informativi da utilizzare al fine di una comunicazione rapida ed efficace, tra cui articoli di giornale, riviste specializzate, media regionali e locali, eventi e seminari di formazione ed informazione, web e social media, brevi video e animazioni, poster e volantini. Alcuni esempi sono stati già forniti dall’EFSA (https://www.efsa.europa.eu/it/topics/topic/african-swine-fever) e dal Ministero della Salute. La comunicazione in emergenza, di cui la Pubblica Amministrazione (PA) rappresenta l’organo di riferimento, è uno strumento di Salute Pubblica. La PA, in modo tempestivo e trasparente, ha il dovere di inviare informazioni puntuali e accreditate, derivanti dalla collaborazione tra esperti scientifici ed esperti della comunicazione, al fine di evitare fenomeni di disinformazione12,19.

Un uso corretto ed appropriato dei social networks, a cui maggiormente si rivolgono i comuni cittadini, da parte della PA, diventa quasi una necessità e potrebbe venire in aiuto per garantire una comunicazione tempestiva.

DISCUSSIONE E CONCLUSIONI La PSA si presenta seguendo scenari molto diversi, essendo l’ASFV capace di sopravvivere nelle più svariate condizioni ambientali. In merito agli effetti della PSA, data la sua lenta ma costante diffusione globale, sono ormai disponibili diverse pubblicazioni relative all’impatto che la malattia ha avuto, sta avendo e avrà in futuro sul settore socio-economico. Pochi sono ancora invece gli studi sugli effetti che la PSA determina in altri ambiti come quello psicologico e culturale, nell’area medica o sull’alimentazione. Questi settori di ricerca sono ancora poco esplorati rispetto ad esempio, l’eziologia, la diagnosi, o alla valutazione del rischio di introduzione e/o diffusione dell’infezione. Se l’approccio One Health venisse applicato alla PSA, gli aspetti non convenzionalmente trattati potrebbero essere approfonditi e si potrebbe disporre così di analisi del rischio più accurate. Certamente, la questione principale legata ad un’eventuale incursione della PSA rimane l’impatto nell’ambito sociale ed economico. La carne suina rappresenta, insieme all’allevamento avicolo, la fonte proteica a più veloce produzione e, conseguentemente, l’aumento della popolazione mondiale negli ultimi anni ha causato una crescita esponenziale del consumo di carne suina e prodotti suinicoli. In questo scenario la PSA può avere un effetto devastante: le perdite economiche sono prioritariamente legate all’elevata letalità dell’infezione da ASFV, ma anche alle misure di controllo dell’infezione, ed in particolare allo stamping out, che rimane lo strumento principale da adottare in caso di focolaio; non sono però da sottovalutare i danni indiretti legati al divieto di movimentazione di suini e prodotti derivati in caso di epidemia. L’impatto economico maggiore, in questo momento, sta coinvolgendo il continente asiatico ed in particolare la Repubblica Popolare Cinese. L’epidemia di PSA in questo Paese sta mettendo in crisi l’intero sistema di produzione e sta determinando un aumento della domanda di importazioni di carne di maiale, con conseguenze e ripercussioni a livello globale. L’aumento del prezzo della carne suina ha interessato, di riflesso, anche altri continenti, in particolare Europa e America, che sono i principali esportatori di questi prodotti. Risulta evidente che la persistenza in forma endemica della PSA influenzi negativamente il reddito di diverse categorie di lavoratori e non solo nelle zone colpite; questo danno diretto porta inevitabilmente anche ad altre importanti conseguenze, compromettendo lo stato di benessere generale di intere famiglie a causa delle ridotte prospettive di mantenimento del tenore di vita o semplicemente della perdita di sicurezze acquisite attraverso investimenti in settori economici messi in crisi dal diffondersi dell’epidemia o per la perdita di posti di lavoro. In campo medico, la fornitura di eparina, prodotta in quasi totalità da aziende cinesi a partire dalla mucosa dei suini, è ormai a rischio proprio a causa dell’epidemia di PSA in Cina, rappresentando un grave problema per la salute di tutte le persone che necessitano di questo presidio terapeutico. La PSA è considerata una “malattia degli animali” e, certamente

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anche nel nostro paese è necessario aumentare la consapevolezza degli stakeholders riguardo l’impatto economico che ne deriva. Il presente lavoro dimostra comunque come la problematica non investa solo il settore zootecnico, su cui ormai gli effetti devastanti sono già stati descritti. Quanto sopra menzionato suggerisce che un approccio One Health, che includa interventi sul sistema zootecnico e agroalimentare, sulla globalizzazione del commercio di animali e prodotti derivati, sulle attività umane collegate, direttamente o indirettamente, ai fattori di rischio della diffusione dell’infezione, e che coinvolga l’aspetto “ecologico”, potrebbe rappresentare una strategia vincente nella lotta alla malattia o nel contenimento dei suoi effetti. Recentemente è stata rimarcata la necessità di un tale approccio nei confronti della PSA in una comunicazione pubblicata dalla rivista Transboundary and Emerging Diseases; gli autori, considerando le ripercussioni della malattia sul benessere generale allo stesso modo di quelle determinate dalla pandemia da SARS-Cov2, propongono l’Agenda 2030 per lo Sviluppo Sostenibile, composta da 17 obiettivi (“Sustainable Development Goals”) e approvata dall’Assemblea Generale delle Nazioni Unite, come supporto per ricercatori e politici nella guida di soluzioni One Health a lungo termine, efficaci e sostenibili, nei confronti della PSA20. Nonostante al momento l’approccio nei confronti della PSA appaia ancora molto limitato rispetto alla prospettiva proposta e alle sue potenzialità, l’impiego di quest’ultima risulta non solo applicabile alla PSA, ma anche impellente.

THE GLOBAL THREAT OF AFRICAN SWINE FEVER IN A ONE HEALTH PERSPECTIVE SUMMARY African swine fever (ASF) is one of the most important swine diseases, both for its wide geographical spread and for the relevant impact that it has on pig industry; it is included in the list of notifiable diseases of the World Organization for Animal Health (OIE), together with all the other diseases that represent a danger to animal and public health. The constant spread of African Swine Fever virus (ASFV), together with its ability to adapt to different environments, its high resistance in the environment and biological products and the lack of a vaccine, make it difficult to eradicate. The disease was confined to Africa until the end of the 1950s, until the virus belonging to genotype I appeared in Portugal in 1957. In 2007, the ASFV (genotype II) reached the Caucasus region and entered the European Union (EU) in 2014, reaching progressively several Member States such as: Bulgaria, Slovakia, Estonia, Hungary, Latvia, Lithuania, Poland, Romania, and Germany. The infection also appeared in the Czech Republic and Belgium, but it was successfully eradicated. The knowledge about the consequences of this infection evidences many multidisciplinary aspects: the disease has consequences in many areas, from the socio-economic one to the medical one, passing through the psychological and cultural sphere. The aim of this paper is to propose a One Health approach for ASF because of its strict linkage between animal and human health as well, although it is not transmissible to humans.

KEY WORDS African swine fever; constant spread; ability to adapt; multidisciplinary essence; One Health; economic loss.

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logia veterinaria, 2. ed, 449-450, UTET Scienze Mediche, Torino. EFSA (2020). Scientific topic: Il benessere animale al macello | Autorità europea. EFSA J. Gallardo M. C., Reoyo A. d. l. T., Fernández-Pinero J., Iglesias I., Muñoz M. J., Arias M. L. (2015). African swine fever: a global view of the current challenge. Porcine Health Managt, 1(1): 21. Istituto Superiore di Sanità (ISS) (2020). One Health. Available at: https://www.iss.it/one-health (Accessed: 25 June 2020). Calistri P., Iannetti S., Danzetta M. L., Narcisi V., Cito F., Di Sabatino D., Bruno R., Sauro F., Atzeni M., Carvelli A., Giovannini A. (2013). The components of ‘One World- One Health’ Approach. Transbound Emerg Dis, 60: 4-13. Zinsstag J., Crump L., Schelling E., Hattendorf J., Maidane Y. O., Ali K. O., Muhummed A., Umer A. A., Aliyi F., Nooh F., Abdikadir M. I., Ali S. M., Mausezhal D., de White M. B. G., Cordon Rosales C., Castillo D. A., McCracken J., Abakar F., Cercamondi C., Emmenegger S., Maier E., Karanja S., Bolon I., de Castaneda R. R., Bonfoh B., Tschopp R., Probst-Hensch N., Cissé G. (2018). Climate change and one health. FEMS Microbiol Lett, 365 (11). Paternoster G., Martins S. B., Mattivi A., Cagarelli R., Angelini P., Bellini R., Santi A., Galletti G., Pupella S., Marano G., Copello F., Rushton J., Stark K. D. C., Tamba M. (2017). Economics of One Health: Costs and benefits of integrated West Nile virus surveillance in Emilia Romagna. PloS ONE, 12 (11): e0188156. Alumbaugh J. (2019). The Global Threat of African Swine Fever. Farm Journal’s Pork. Mason-D’Croz D., Bogard J. R., Herrero M., Robinson S., Sulser T. B., Wiebe K., Willenbockel D., Godfray H. C. J. (2020). Modelling the global economic consequences of a major African swine fever outbreak in China. Nat food, 1(4): 221-228. European Commission (2020). Strategic approach to the management of African Swine Fever for the EU. Brussels. Burset G. (2019). La PSA in Cina trascina il prezzo dei suini a livello mondiale. Available at: https://www.3tre3.it/articoli/la-psa-in-cinatrascina-il-prezzo-dei-suini-a-livello-mondiale_8920/. G.d.O. (2019). L’allarme di Assica: col boom dei prezzi delle carni filiera suinicola a rischio. Agrisole. Anmvi Oggi (2020). La PSA minaccia la produzione mondiale di carne suina. Mur L., Atzeni M., Martínez-López B., Feliziani F., Rolesu S., SanchezVizcaino J. M. (2016). Thirty-Five-Year Presence of African Swine Fever in Sardinia: History, Evolution and Risk Factors for Disease Maintenance. Transbound Emerg Dis. 63(2): e165-e177. Vilanova E., Tovar A., Mourão P. (2019). African Swine Fever in China: risk of a global shortage of heparin. Rosovsky R. P., Barra M. E., Roberts R. J., Parmar A., Andonian J., Suh L., Algeri S., Biddinger P. D. (2020). When Pigs Fly: A Multidisciplinary Approach to Navigating a Critical Heparin Shortage. Oncologist, 25(4): 334-347. Iorio E. (2020). Comunicazione in emergenza: ecco qual è il ruolo della PA. Innovazione nella Pubblica Amministrazione e Forum PA (FPA). Available at: https://www.forumpa.it/open-government/comunicazione-pubblica/comunicazione-in-emergenza-il-ruolo-della-pa-per-uninformazione-certificata/. Villanueva-Cabezas J. P., Rajkhowa A., Campbell A. J. D. (2020). One Health needs a vision beyond zoonoses. Transbound Emerg Dis.

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GIANLUCA CELANI1,*, DOMENICO ROBBE1, PRISCO MARTUCCI2, LUCIO PETRIZZI1, PAOLA STRATICÒ1, AUGUSTO CARLUCCIO1 1

Veterinary Teaching Hospital, Faculty of Veterinary Medicine, University of Teramo, Località Piano d’Accio, 64100 Teramo, Italy Military Veterinary Centre, Grosseto, Italy

SUMMARY During the last decades in Europe and the United States there has been a renewed interest in the use of the mule. This review summarizes the scarce information available in the veterinary literature on the anatomical particularities and shoeing techniques of the mule’s hoof that have an important impact on the health and welfare of the animal. Compared with the horse the mule’s hoof has a distinctive upright dorsal wall angle and a broken forward hoof-pastern axis; the cartilages of the distal phalanx are much more developed towards the palmar/plantar parts of the hoof; the inclination degree of the hoof wall to the ground, at the toe, the quarters and heels is almost vertical; the sole has elevated moisture content in its deep layers and the horn tubules are more evident; the coronary dermis together with the large coronary groove are higher; the laminar dermis of the wall segment is less extended; the mule’s hoof is smaller, longer and narrower. The mule’s shoes display a web of greater and uniform thickness over their entire extension compared to the horse’s. The toe of the front shoe is rounded, fitted slightly wider and turned up; and its outline fitting is marginally wider on the toe and on the external branch. The front shoe coverage, therefore, is slightly wider at the toe, and gently decreases to the toe quarters, heel quarters and heels; moreover, the internal branch is narrower than the outside. The hind shoe shows the same characteristics of the front one, plus some distinct features such as a larger blunt toe, equal full outline fitting proportions, and identical branch coverage.

KEY WORDS Muline, hoof, anatomy, farriery, shoe.

INTRODUCTION In the past, mules have assisted man in daily activities from agriculture, to trade, to military service. In recent decades, the mechanization of farming and the demobilization of some local mountain armies have contributed to a significant decline in the mule population. Recently, there has been a renewed interest in the use of this animal to carry lumber within parks and for trekking tourism activities1. In zootechnical terminology, the mule is a domestic equine hybrid resulting from a cross between a mare (female horse) and a jack (male donkey) while the hinny is the offspring of a jennet (female donkey) and a stallion (male horse)1,2. From the donkey, the mule inherits an ideal hoof structure and conformation to support agricultural work or face paths on steep and inaccessible terrains. Hinnies play an important role in drier areas and are used mainly as work animal. Over time, the anatomical peculiarities of the hoof and the types of work required of the animal have lead the art of farriery to a high degree of specialization. The purpose of this paper is to provide an overview of the scarce information available in the veterinary literature about functional anatomy and shoeing techniques of the mule’s hoof compared with the horse.

Corresponding Author: Gianluca Celani (gcelani@unite.it).

ANATOMY OF THE MULE’S HOOF: BASIC DIFFERENCES COMPARED WITH THE HORSE In the art of farriery (Mascalcia in Italian) the distal end of the digit is called the “hoof“ (colloquial terms: fore-foot and hind-foot). In anatomical language, the middle-distal extremity of the equine limb is the manus in the thoracic limb (carpus, metacarpus and digit) and the pes in the pelvic limb (tarsus, metatarsus and digit)3,4,5,6. They are the anatomical regions of greatest interest for shoeing, although a general examination of the animal should not be neglected by focusing exclusively on the distal extremity of the limb. The evaluation of overall conformation, the correct alignment of the skeletal components of the limb, and movement at different gaits are essential. In equestrian terminology, the term “hoof ” or “ungula” refers to the horny hoof capsule as well as all the structures within: the sensitive dermis (corium), digital cushion (frog and bulb portions), distal phalanx (coffin bone), most of the collateral cartilages of the distal phalanx (lateral and medial foot cartilages), distal interphalangeal (coffin) joint, distal parts of the middle phalanx (short pastern bone), distal sesamoid (navicular) bone, podotrochlear bursa (navicular bursa), tendons of insertion of the common digital extensor and deep digital flexor muscles, and numerous ligaments, blood vessels, and nerves7,8,9,10,11,12,13. The mules and donkeys usually have a distinctive upright dorsal hoof wall angle and often a broken forward hoof-

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Figure 1 - Lateral view of the mule hoof, left hindlimb (punta = toe, mammella = toe quarter, quarto = heel quarter, tallone = plantar heel).

pastern axis14,15. The cartilages of the distal phalanx are much more developed towards the palmar/plantar parts of the hoof compared with the horse4. The almost vertical inclination degree of the hoof wall to the ground, at the toe, the quarters and heels is always less accentuated than in the horse4. When the hoof is on the ground, it is possible to identify different areas on the external surface of the hoof capsule: the lateral view shows the coronary border, the perioplic band, the heel bulbs and the wall. The hoof wall can be divided into the dorsal toe, the toe quarters (medial and lateral), the heel quarters (medial and lateral), and the palmar or plantar heels (Figure 1). In the mule, the perioplic band (stratum externum of the hoof wall) presents a particular development, extending beyond the heels, up to their angle of inflection (bar of the heel)3,4. The solar surface of the hoof includes the bearing margin of the wall, the sole, the frog, the bars, and a well-defined zone called the white line (zona alba ungulae). The white line represents the transition between the horn of the hoof wall and the horn of the sole and is an important anatomical landmark for the farrier (Figure 2). The sole of the mule has an elevated moisture content in its deep layers, and the horn tubules are more evident than in the horse4. The coronary dermis (corium coronae) supplies the epidermis that forms the thickest layer of the hoof wall (stratum medium), and together with the large coronary groove are higher than in the horse4. The inner layer of the hoof wall (stratum internum or lamellatum) consists of several primary epidermal lamellae (laminar horn) which extending down perpendicularly from the distal border of the coronary groove, dovetail with the dermal lamellae (laminar

dermis) in a very strong union (interdigitations of epidermal and dermal laminae)12,16. The sole dermis (corium solae) is thinner and covered with long papillae. The distal end of each dermal lamella raises many papillae known as the terminal papillae. The horn tubules produced from these papillae together with the laminar horn originate the white zone, which is the dividing line of epidermal sole-wall junction16 and is used as a guide for positioning nails with shoeing. In the mule, relative to the parietal surface of the distal phalanx the laminar dermis of the wall segment (corium parietis) is less extended than in the horse, with a lower average ratio 1:2 than 1:3, 1:4 in the horse; these laminae even if less numerous are thicker and more vascularized4. In the horse there are approximately 550 primary dermal laminae, 450 in the mule and 350 in the donkey4. The growth of the hoof wall progresses at the rate of about 8 mm per month, with a range between 3.98 and 13.6 mm all around the coronet. On average, complete hoof wall renewal takes approximately 8-16 months at the toe, 6-10 months at the toe quarters and 4-6 months at the heel quarters7. In the horse, the lateral and medial side of the wall are quite oblique with a convergence in a proximal direction, and the angles to the ground of the dorsal hoof wall and of the wall at the heel are parallel with about 47 degrees and in continuity with the inclination of dorsal aspect of the pastern; the shape of the hoof in its solar aspect is almost rounded in the horse. The mule has inherited the conformation and the structure of the hoof from the donkey. The mule’s hoof is smaller, longer and narrower compared to the horse: the lon-

Figure 2 - Solar surface of the mule hoof, left forelimb (1 toe, 2 medial and lateral toe quarters, 3 medial and lateral heel quarters, 4 palmar heels, 5 bulbs of the heels, 6 frog, 7 medial and lateral groove.

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gitudinal diameter always exceeds the transverse one of 1/3, 1/4 or even 1/2 giving the appearance to the muline hoof of a quadrilateral4. It is a narrow hoof with a less rounded toe, a very large, concave and moist sole with more distinct horn tubules, a shorter frog, wider at the base and with narrower grooves and a more developed perioplic band4. The main function of the hoof is to support the weight of the animal (weight-bearing mechanism) and minimize the effects of concussion (anti-concussion mechanism) in the standing position and during locomotion17. The mule’s hoof has an ideal structure to bear the weight and the pressures with respect to the size of the animal, the hardened terrain and the natural gaits. The hoof of this hybrid represents a perfect balance between strength/hardness of the hoof wall and extreme elasticity that together guarantee the remarkable safety during the stance phase of the mule’s limb. This great elasticity in a rigid hoof wall is facilitated by the angulation of bony structures, the presence of the support ligaments, the bulbar portion of the digital cushion and the collateral cartilages of the distal phalanx, which extending more palmarly/plantarly, allow lateral movements for shock absorption4. Other structures improve the hoof shock absorption mechanism (heel expansions): particularly the frog portion of the digital cushion which is rich in fat, the tactile corpuscles, the sweat glands whose duct open into the central groove of the frog making it more elastic with their secretion, and the close interdigitation of epidermal and dermal laminae that presents an increasingly adhesion proceeding from the region of the toe to the heel quarters4,8. During the midstance phase under maximal loading of fetlock joint, it is

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possible to observe changes in the shape of the hoof that occur in different areas with diverse modalities and timing. The first variation is the outwards expansion of the heel quarters, followed by a narrowing of the coronary edge in front, then a reduction of the height of the hoof simultaneously with the sinking of the heels and finally a flattening and a sinking of the sole7,17.

THE BASICS OF FARRIERY Farriery, synonymous with shoeing, is the science and art of applying a metal rim/bar or plate on the palmar/plantar surface (bearing or ground or solar side) of the hoof to provide protection and secure the sole. The single term “farriery” or “shoeing”, however, comprises four very specific phases that require different skills and basic knowledge for excellent results: removing the shoe, trimming the hoof, forging the shoe and placement and application of the new shoe (real shoeing)6,17,18,19. Adequate hoof protection of horses, donkeys and mules used in agriculture, transport and army service is of fundamental importance for the greater wear imposed on this structure. In fact, the Ancient Greeks and the Romans used hipposandals, heavy iron horseshoes secured to the feet by cords or leather straps. It is possible to trace the birth of farriery to the Celts and Gauls who were known to nail-on iron shoes to horse’s feet. Beside its practical and therapeutic implications, it immediately distinguished itself as a real art. The forge is the realm of the farrier, with a «siderotechnic» facility where the forging procedure takes place (Figure 3) and an open working area where to remove the shoe, trim the hoof and re-shoe the animal.

Forging procedures: shoemaking

Figure 3 - The master farrier Giovanni Carluccio’s forge.

The forging process is the manufacturing of the shoe by hand after an initial assessment of the hoof conformation. The farrier chooses the most suitable bars, which in ancient times were made from waste material, such as chains of ships or cutouts of armor. The bars can be made of pure metal such as iron, steel, copper and aluminum or of their alloys. The best material is homogeneous, ductile, weldable and mild, like iron. However when iron is overheated becomes hard and not easily weldable. The type of cooling influences iron hardness: if spontaneous (air) the iron is softer, or if quenched in cold water it becomes harder. The bar is heated to the correct temperature with several heatings or firings that take place on a coal burning forge with the use of bellows; the shoe is forged with a hammer and held with tongs. With the first and second firings, the outside branch and then the slightly shorter inside one are bent. The same heatings will be used to start the lower nail hole openings, applying the stamp to the ground surface of the hot shoe and then to calibrate the upper nail openings with the hammer and back pritchel from the hoof or ground surface of the shoe. However, before the back pritchelling (back punching), after the stamp has been used it is desirable to remove the bottom piece of metal of each hole and fully penetrate the shoe with the pritchel on the ground surface of the shoe. In the mule since the hoof wall at quarters and heel is almost vertical the nail holes often are punched upright17. At the end of cooling, the shoe will be completed by rounding the edges with a few file passes. The farrier’s skill can be evident from how many

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Figure 4 - Forelimb and hindlimb mule shoes.

times the hammer is used before and then the file to obtain an optimal quality shoe (Figure 4).

Removing the shoe To be reshod, the old shoe must be removed first before the novel shoeing. The initial step is to straightened or cut off the clenches with a buffer. Then the nail can be withdrawn with the use of a nail puller or placing the jaws of the pincer (shoe puller) between the shoe and the hoof starting at the heel, which is levered forward, toward the toe exposing the head of nail that can be grasped individually with the pincer.

Trimming the hoof Both for first shoeing and re-shoeing, the hoof must be trimmed before applying the shoe. To restore the proportions and physiological inclinations of the hoof, the wall, the sole and the frog will be trimmed with the intent to make the sole as parallel as possible to the solar surface of the distal phalanx (plane of pedal bone); therefore more hoof wall will be removed at the toe than quarters or heels, where the deformation occurs. During the hoof trimming the balance and hoof-pastern axis should be ideally maintained straight but this is not always possible in mules and donkey20. For a first rough trimming, the hoof cutter (hoof nipper) and toe knife are used. The irregularities of the palmar/plantar surface will be eliminated using a drawing knife and a hoof rasp. With the hot shoeing (hot fitting or hot setting) technique, the shoe is adjusted and fitted more accurately (Figure 5).

Placement and application of the shoe: fitting and nailing on the shoe The shoeing procedure is the first farriery practice for an animal that has never been shod before, while the re-shoeing procedure is the practice of removing the old shoe and applying a new one in a shod animal. Choosing when to shoe young animals for the first time, the reduction of the normal lateral movements of the hoof wall at the heel and the blood circulation within the foot due to shoeing must be considered. Therefore it is advisable not to proceed with shoeing before 24 months of age. Sometimes in order to avoid excessive wear of the hoof capsule during the activity it is necessary to shoe a horse at a very early age for

Figure 5 - Hot fitting.

working purposes. In these cases, to allow the lateral expansion of the heel, the farrier foresight will be to not put nails in the shoe behind the quarter of the hoof wall. The re-shoeing interval time depends on both the shoes’ extent of wear and individual hoof wall growth rate; the redundancy of one of these components commits to re-shoe the animal. Considering that the anticipation of re-shoeing damages the horny structures of the hoof due to excessive nailing, and the delay can modify the hoof conformation hindering heel expansion due to rigidity of the old horn, the re-shoeing procedure is usually performed every five to six weeks. Depending on the method of fitting a hot or cold shoe on a levelled bearing surface of the hoof wall, shoeing can be executed with a hot or cold technique4,6,17,18. To fit correctly, a hot shoe is applied at red dull heat for sufficient time on a well-prepared bearing surface to ensure an absolute coaptation between shoe and hoof. For cold shoeing, a level shoe will fit adequately on a trimmed hoof with the help of measuring and balancing hoof tools, for example, a hoof gauge and the T-square. However the ability of the farrier lies in the capability to measure the hoof at a «glance». In any case a measurement of the hoof must also be performed in hot shoeing before forging the shoe. Before shoeing an animal, the farrier must study the conformation of its feet and limbs, both standing squarely and in motion, observing the animal from the front, the side and behind to assess the correct alignment of the skeletal com-

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ponents of the limb, the movement and the placement of the hoof as it strikes the ground both at rest and at different gaits. After hoof trimming and forging the shoe, the latter must be placed on the hoof bearing surface to evaluate the exact adherence between the two surfaces (surface fitting), and the adaptation of the external edge of the shoe to the perimeter of the hoof wall (outline fitting: close or wide)4,17,19. The shoe must be steady by pressing alternately from one side or the other, the heel of the shoe should extend correctly behind the bearing surface and the branches of the shoe must be equidistant from the central groove of the frog. The shoe must be placed on the solar surface of the hoof with congruency between the shoe’s and the hoof ’s toe, and the frog lying centrally between the two heels of the shoe. Subsequently with the shoe held in this fitted position, the nails are driven, the first nail at the outside toe quarter and then at the inside. When the hoof and shoe outline edges correspond, the remaining nails are driven alternately on each side of the heel quarters and finally on the toe, otherwise the farrier moves the shoe in the correct position with light hammer blows on the left or right branch. Once the nail is driven, the most important act is to immediately bend the tip exiting the hoof wall approximately 2 cm above the junction of the shoe and the hoof; then the turned over nails ends must be cut off and bent into the small indentations of the hoof wall horn created with the gouge (clench trenching process) into which the clenches can be sunk (clenching process). The farrier’s ability will also have a cosmetic result: the clench line should be straight and parallel to the ground or to the coronet (Figure 6) otherwise the clench line will «make music». Finally, the irregularities of the solar edge of the hoof in contact with the shoe are smoothed with the file side of the rasp.

MULE-SHOEING In ancient times the small dimension of the mule’s hoof was considered insufficient to support the work, so the shoeing provided shoes larger than the hoof, to give more stable shoeing and ground contact. It was also believed that this practice promoted the impulse of movement. Over time it has been noted that the dimensions and the structural characteristics of the muline hoof were optimal

Figure 6 - Clench line at finishing off.

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for the work that required walking steep and rocky paths between hills and mountains. However, the use of a good coverage (wide-webbed shoes) has been preserved to increase the duration of the shoeing and to facilitate the passage on soft soils where the animal could easily sink with hooves with a reduced bearing surface and a notably concave sole. It is important to consider whether the intended use of the mule is for ordinary service or heavy draft. For the latter, shoes fitted wider than the hoof on the toe and heel, were used to increase the ground contact and promoting efficient impulse while preventing slipping3,4,21. The mule’s shoes display a web of greater and uniform thickness over their entire extension compared to those of horses. The toe of the front shoe is rounded, fitted slightly wider and turned up (“set toe”); its outline fitting is marginally wide on the toe and on the external branch3,4,21. The front shoe coverage, therefore, is slightly wider at the toe, and gently decreases to the toe quarters, heel quarters and heels; moreover the internal branch is narrower than the outside4. The front shoe must cover the heels and never extend behind them. The choice of making a shoe with calkins is at the discretion of the farrier. The nail holes, from six to eight, are placed and angled away from the outer edge (coarse holing) and are designed to use nails with short wide shanks. The hind shoe shows the same characteristics of front shoe and some distinct properties; such as a larger blunt toe (“square toe”), equal full outline fitting proportions, and identical branches coverage3,4,21; the nail holes are stamped more towards the heels to not impair the toe, and the calkins are always fixed. In mules, to achieve the best surface fitting between the shoe and hoof wall, the hot shoeing technique is preferred. Three main types of shoe have been identified and designed to adapt shoeing to the different uses of the mule: the “square shoe” with a blunt shape of the toe (“square-toe-shoe”) suitable for draft mules, the “round shoe” with a wide round toe bent up (“rocker-toe-shoe”) for mules used in agriculture, and finally the “Florentine shoe” for pack mules used in the Maritime Alps and the Genoese Apennines. The latter shoe is heavy and difficult to hand forge3,4,21. Sometimes in winter, the use of a shoe with an additional nail hole may be preferred for the eventual insertion of a non-slip nail. To help the leakage of water between the shoe and the sole, some farriers make a notch at the toe on the palmar/plantar edge of the hoof; other farriers prefer to forge shoes with a web narrow on the heels compared to the rest of the branches and bend-up behind the heels to protect them and avoid excessive grip causing injuries to the hoof and increased risk of a lost shoe3,4. In the military field, for the mules used in mountain artillery, in the Alpine’s Infantry and in Bersaglieri Regiments and other military districts, the same indications of shoeing the mules for civil service have also been adopted, applying the calkins both on the hind and the front shoes, and using the same shoeing method regardless the type of use for which each mule was enrolled in the army. In the practice of modern mule shoeing, the methods for protecting the hoof capsule with nails or adhesives are similar to that of the horse. The shoe may be made of metal (steel, aluminum and titanium), synthetic polymers, or various composites of the two materials22. Typically, each mule shoe must be hand-forged or machine-made horseshoes

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must be customized. However, in some countries, shoes specific for mules are manufactured in different sizes (“Mule” and “Mule Heel”, St. Croix Forge, USA; “Ferro da mulo ramponato”, Viali, Blacksmith, IT).

8. 9.

CONCLUSIONS This review based on scarce scientific evidence obtainable in the veterinary literature provides an overview on functional anatomy of muline hoof and mule-shoeing techniques. Furthermore the principles of farriery are discussed in its basic traits. This mule-specific knowledge is critical for equine practitioner and professional farrier as it has an important impact on the health and welfare of the animal. The present study has been carried out in the framework of the Project “Demetra” (Dipartimenti di Eccellenza 2018 - 2022, CUP_C46C18000530001), funded by the Italian Ministry for Education, University and Research.

10. 11. 12.

13.

14. 15. 16. 17.

References 18. 1. 2.

3. 4. 5. 6.

Campbell Smith D. (2009). The book of mules: selecting, breeding, and caring for equine hybrids, The Lyons Press, Guilford, USA. Burnham S.L. (2002). Anatomical differences of the donkey and mule. Pages 102-109 in Proc of the Annual Convention of the AAEP, Orlando, FL, USA. Bossi V. (1926). Trattato di mascalcia, Casa Editrice Francesco Vallardi, Milano, IT. Mensa A. (1950). Podologia. UTET, Torino, IT. Muttini A., Petrizzi L. (1995). Podologia appunti di lezione, Società Editrice Esculapio, Bologna, IT. Blasio V. (2012). La scuola italiana nell’arte del ferrare: mascalcia e tec-

19.

20. 21. 22.

niche di ferratura equine, Equitare, Siena, IT. Leisering A.G., Hartman H.M. (1895). Il piede del cavallo sotto il rapporto della anatomia, della fisiologia e della ferratura, Ed. Lungwitz, A., 8th ed., Eds. Baldoni A., Caradonna G.B., 1st italian ed., Editore Agnelli, Milano, IT. Fogliata G. (1910). Ippopodologia volume primo, Casa Editrice Francesco Vallardi, Milano, IT. Davies H.M.S., Philip C. (2007). Gross anatomy of the equine digit. In: Equine podiatry, Eds. Floyd A.E., Mansmann R.A., 1-24, Saunders Elsevier, St Louis, USA. Ashdown, R.R., Done, S.H. (2011). Color atlas of veterinary anatomy volume 2 the horse, 2nd ed., Mosby Elsevier Ltd, St Louis, USA. Budras, K.D., Sack, W.O., Röck, S. (2011). Anatomy of the horse, 6th ed., Schlutersche Verlagsgesellschaft, Hannover, DE. Fails A.D. (2020). Functional anatomy of the equine musculoskeletal system. In: Adams & Stashak’s lameness in horses, Ed Baxter G.M., 7th ed., 1-65, Wiley-Blackwell, West Sussex, UK. König E.H., Liebich H.G. (2020). Veterinary anatomy of domestic animals textbook and colour atlas, 7th ed., Georg Thieme Verlag, Stuttgart, DE. Thiemann A., Rickards K. (2013). Donkey hoof disorders and their treatment. In Practice, 35:134-140. Thiemann A., Poore L.A. (2019). Hoof disorders and farriery in the donkey. Vet. Clin. North Am. Equine Pract., 35:643-658. Pollit C.C. (2016). The illustrated horse’s foot: a comprehensive guide, Elsevier, St Louis, USA. Hickman J., Humphrey M. (1988). Hickman’s farriery: a complete illustrated guide, 2nd ed., JA Allen & Co Ltd, Ramsbury, UK. Colles C., Ware R. (2010). The principles of farriery, 1st ed., JA Allen & Co Ltd, Ramsbury, UK. O’Grady S.E. (2020). Principles of trimming and shoeing. In: Adams & Stashak’s lameness in horses, Ed. Baxter G.M., 7th ed., 1095-1111, Wiley-Blackwell, West Sussex, UK. Bourassi M., Kay G. (2005). Techniques de maréchalarie courante chez l’ànes et chez le mulet. Pratique Vétérinaire Equine, 148:15-19. Chiari E. (1927). Elementi di podologia, 4th ed, UTET, Torino, IT. Lutz-Ferdinand L., Burkhard R. (2012). Hufpflege und –beschlag bei eisen und maultieren. In: Der huf: lehrbuch des hufbeschlages, Eds. Lutz-Ferdinand L., Burkhard R., 1st ed., 318-324, Georg Thieme Verlag: Stuttgart, DE.

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Effects of short-term and combined use of thyme powder and aqueous extract on growth performance, carcass and organ characteristics, blood constituents, enzymes, immunity, intestinal morphology and fatty acid profile of breast meat in broilers

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MAJID BELALI1, ALIREZA SEIDAVI1* AND MEHRDAD BOUYEH1 1

Department of Animal Science, Rasht Branch, Islamic Azad University, Rasht, Iran

SUMMARY This experiment was performed to evaluate the effects of Thyme Extract (TE) and Thyme Powder (TP) on growth performance, carcass and organ characteristics, blood parameters, enzymes, immune system, intestinal morphology and fatty acid profile of breast meat in broilers. The experiment was based on a completely randomized design with 5 treatments, 4 replications and 10 Ross 308 male broilers in each replication for 42 days. Experimental treatments included aqueous extract of thyme (50 and 100 ppm) and thyme powder (100 and 150 mg/kg/feed) which were used in combination with the basal diet (control). The effect of treatments was analyzed by analysis of variance (SAS) and the means were compared at 5% probability level with Duncan’s multiple range test. The results showed that, in the final period of experiment, different levels of thyme aqueous extract and thyme powder had a significant difference on daily weight gain, feed intake and conversion ratio improvement (P<0.05) so that the highest means were related to treatment TE (50 ppm) - TP-(150 mg/kg). The effect of different levels of thyme aqueous extract and thyme powder had a significant difference on European index, economic value and total weight (P<0.05), which had the highest mean of TE (50 ppm) -TP (150 mg/kg). Different levels of thyme aqueous extract and thyme powder had significant differences on the relative weight of thymus, bursa of Fabricius, live weight, ventricular fat and pancreas (P<0.05). The effect of different levels of thyme aqueous extract and thyme powder on the performance of the immune system of broilers was not significant (P> 0.05). The effect of different levels of thyme aqueous extract and thyme powder on the performance of the immune system of broilers was not significant (P>0.05). The highest percentage of unsaturated fatty acids was related to high levels of thyme powder and extract, meaning that the highest mean was related to TE (100 ppm) -TP (250 mg/kg) and the lowest mean was related to TE (0 ppm). TP (0 mg/kg). Based on the results of the present study, the use of TE 50 ppm, TP 150 mg/kg level is recommended to supplement the diet of Ross 308 broilers.

KEY WORDS Medicinal plants, thyme, chick, growth, breast meat, antibody.

INTRODUCTION The use of drugs and chemicals as growth stimulants in livestock and poultry diets has been officially banned by the European Union since 2006. In recent years, the use of natural and organic substances as alternatives has increased sharply following the imposition of legal restrictions on the use of drugs and chemicals. Studies have shown that herbs and their products, known as phytobiotics, are good alternatives to growth-promoting antibiotics in the diet or drinking water of broilers (Griggs et al., 2005; Grashorn, 2010; Hashemi et al. 2010; Windisch et al. 2008). The use of antibiotics has been banned due to drug resistance in humans, drug retention in the carcass, and disturbance of the normal intestinal microflora unless necessary for treatment. In many parts of the world there are recommendations to discourage the use of growth-promoting antibiotics, so to

Corresponding Author: Alireza Seidavi(alirezaseidavi@iaurasht.ac.ir)

compensate for this reduction in growth, it is necessary to find suitable alternatives. In recent years, the use of aromatic plants and their extracts as potential growth stimulants has attracted much attention (Ghazanfari et al. 2015). Medicinal plants due to factors such as high economic value and low cost of production, little effect on the environment, few side effects compared to chemical drugs and antibiotics and reduced relative resistance to pathogens. Some of the beneficial properties of medicinal plants are related to the presence of secondary metabolites such as phenolic compounds, essential oils and saponins. (Tipu et al. 2006). Thyme (Thymus vulgaris L) is a herbaceous, aromatic plant belonging to the mint family. Thymol and carvacrol are important active ingredients, but other substances such as paracetamol, linalool and cineole, flavonoids, terpenes, spicy compounds and a number of other active ingredients are found (Sharififar et al. 2007). Addition of thyme essential oil to the diet or drinking water of broilers has led to weight gain and improved feed conversion ratio (Alcicek et al. 2004). Cross et al. (2007) reported that thyme oil had a positive effect on the performance

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of broilers. Al-Kassie, (2009) investigated the effect of thyme and cinnamon extracts at 100 and 200 mg in broilers and reported that growth performance traits (daily weight gain, feed intake and feed conversion ratio) were significantly higher for birds that received these two extracts than the control group, and the higher levels of extracts showed higher results than lower levels. Herandez et al. (2004) showed that feeding plant extracts such as thyme cause faster growth, improve intestinal digestion, starch digestibility, dry matter utilization of diets and carcass traits in broilers. Thyme extract has high antioxidant properties that, in addition to reducing blood lipids, can play a role in inhibiting LDL oxidation. Carvacrol reduces plasma triglyceride concentrations. The use of carvacrol has been reported to stimulate the growth and proliferation of lactobacilli, and lactobacilli play an important role in improving blood parameters and lowering serum lipids (Esteve et al. 2000). The most important phenolic compounds in thyme include thymol and carvacrol. This plant has antibacterial, antifungal and anti-coccidiotic properties that are attributed to thymol and carvacrol Mikaili et al. (2010). The presence of beneficial microflora has been shown to increase villi length, crypt and intestinal cell proliferation, but pathogenic bacteria produce toxic compounds such as am-

monia, destroying the epithelial layer and increasing cell transformation to regenerate atrophic cells. Villi height decreases and intestinal crypt depth increases (Bakkali et al. 2008). Thyme causes the secretion of digestive enzymes such as amylase and chymotrypsin and increases the amount of food intake by increasing absorption through intestinal villi (Denli et al. 2004). There are limited results regarding the simultaneous evaluation of the effects of thyme powder and its extract in the diet of broilers in the short term and to investigate the possibility of reducing the cost of diet, treatments were used in the short term. The present experiment was performed to evaluate the effects of thyme powder and its extract on growth performance, carcass and gastrointestinal characteristics, blood parameters, immune system, intestinal morphology and profile of breast fatty acids in broiler chickens.

MATERIALS AND METHODS This study was conducted in one of the broiler farms located in Masal, Iran. The average weight of 45 ± 2 g with control treatment was used in a total of 5 treatments and four replications and 10 chickens per replication for 42 days. Two levels of thyme powder (150 and 250 mg/kg) and two levels of aqueous thy-

Table 1 - Ingredients, chemical composition, and energy of the diets (from 1 to 42d of age).

1 The amount of vitamins and minerals per kg of the final diet: vitamin A, 9000 IU; vitamin D3, 3000 IU; vitamin E, 18 IU; vitamin K3, 3 mg; vitamin B1 (Thiamine), 1/8 mg; vitamin B2 (Riboflavin), 6 mg; vitamin B6 (Pyridoxine), 3 mg; vitamin B12 (Cyanocobalamin), 0/012 mg; vitamin B3 (Niacin), 30 mg; vitamin B9 (Folic acid),1 mg; vitamin H3 (Biotin), 0/24mg; vitamin B5 (Pantothenic acid),10 mg; 500 mg; Choline,100 mg; Mn,100 mg; Zinc, 80 mg; Iron,10 mg; Cu,1 mg; I, 0.2 mg; Selenio.

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me extract (at levels of 50 and 100 ppm) in the diet in the first 24 days of feeding were applied as follows. The treatments were: Treatment 1: Aqueous extract of thyme (0 ppm) + Thyme powder (0 mg/kg) Treatment 2: Aqueous extract of thyme (50 ppm) + Thyme powder (150 mg/kg) Treatment 3: Thyme aqueous extract (50 ppm) + Thyme powder (250 mg/kg) Treatment 4: Thyme aqueous extract (100 ppm) + Thyme powder (150 mg/kg) Treatment 5: Aqueous extract of thyme (100 ppm) + thyme powder (250 mg/kg) which was used in combination with the basic diet. Aqueous extract of thyme and thyme powder made by Zarghani Pharmaceutical Company (Sabzevar, Iran) were purchased and used based on the desired concentrations. Diets were adjusted according to the poultry nutritional requirements table containing the minimum nutrients recommended in the Ross 308 strain feeding guide Manual, (2012) (Table 1). Chickens were reared in 1 × 1 m cages on a cellulose roll bed for 42 days. Each cage had a cylindrical feeder and a manual feeder. The temperature of the breeding hall decreased to 33 degrees Celsius in the first days and then gradually to 23 degrees Celsius on the 18th day of breeding and then continued until the end of the period. Environmental conditions were similar for all groups (20 pens) and included 23 hours of light exposure and one hour of darkness, the humidity of the hall was 65 to 70%. Access to water and feed was similar and free during the rearing period. In addition, the birds were vaccinated against infectious bronchitis (10th day of age), Newcastle (4th, 21st and 35th days of age) and Infectious Bursal disease (12th day of age) (NDV; Viscerotropicvelogenic strain). All vaccines were obtained from Razi Serum and Vaccine Institute (Karaj, Iran).

Economic growth performance and returns The weight gain of chickens per pen at periods of 1 to 10, 11 to 24 and 25 to 42 days was calculated by a digital scale with an accuracy of 0.01 g. At the end of each period (initial 1 to 10, growth 11 to 24 and final 25 to 42) the amount of feed left was weighed and subtracted from the amount of feed given at the beginning of each period, to calculate the amount of feed consumed. Feed conversion ratio was calculated by dividing feed intake by weight gain for days 1 to 10, 11 to 24, 25 to 42 and the whole period. Ghoreyshi et al. (2019) used the following formula to measure the European production index. European production index: Average live weight (g) × Retention rate/Food conversion ratio × Number of breeding days × 10 The following formula was used to measure the cost of feed per kilogram of live chicken. The daily price of thyme powder and thyme aqueous extract used was calculated separately for each diet and placed in the formula. Cost of feed per kilogram of live chicken = (Weight of a chicken at 42 days in kilograms/feed price during 42 days for each chicken in Rials)

Characteristics of carcasses and digestive organs At the end of the experiment, after two hours of starvation, 2 birds were slaughtered from each replicate, weighing close to the average, by a digital scale with an accuracy of ± 0.1 g and stuffed carcass weight, full carcass weight, empty carcass weight, breast weight, weight thigh, wing weight as well as the

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weight of internal organs (pancreas, heart, gills, spleen, bursa of Fabricius, liver, ventricular fat, weight of duodenum, geogenome and ileum) were measured (Zahirian et al. 2019).

Blood serum parameters and digestive enzymes At the end of the experiment (42 days old), 2 birds weighing close to the average were randomly selected and blood samples were taken from the wing vein of 5 ml. The samples were centrifuged at room temperature at 5000 rpm for 3 minutes (5702, Eppendorf, Germany) and serum was separated and transferred to microtubes and transferred to the laboratory. The serum was stored at minus 20 ° C until blood metabolites were measured. Serum was thawed at room temperature followed by glucose, triglyceride, cholesterol, total protein, albumin, globulin, creatine kinase, lactate dehydrogenase, VLDL (High-density lipoprotein), HDL (high-density lipoprotein), LDL (Lowdensity lipoprotein) alanine transferase and alkaline phosphatase were measured. These parameters were measured with Pars test kits and by autoanalyzer (Hitachi brand model 917/Japan) based on Gholami et al. (2020).

Immune response To evaluate humoral safety, broilers were immunized against sheep erythrocytes (SRBC according to the theory of Lerner et al. (1971)). To prepare an SRBC injection suspension, blood samples were taken from 3 sheep and poured into jars containing EDTA. Rinses were washed three times in PBS saline phosphate buffer, and at the end of a suspension, 2% SRBC was prepared in PBS. All the above steps were performed under sterile conditions. Then it was injected into the vein of the above solution 7 and 14 days after the first and second injections and blood samples were taken on days 35 and 42 (Gore and Qureshi, 1997). SRBC was measured by hemagglutination method. To measure the antibody titer, V-shaped pellets for microhemagglutination were prepared, which have 96 wells in 12 columns (1 to 12) and 8 rows (A to H). Van derzipp method was used to measure total antibody. According to this method for measuring Total anti-SRBC, ul 50 of the serum sample was mixed with ul 50 saline phosphate buffer (PBS) inside the microtiter plate and in the next step, 50% of 2% SRBC suspension solution was added to each well and then placed at room temperature for 4 to 5 hours. Titers were expressed based on log2. The highest rate of complete agglutination was expressed (Pourhossein et al. (2015). In addition, the birds were vaccinated against Newcastle disease and influenza (NDV; Viscerotropicvelogenic strain). All vaccines were obtained from Razi Serum and Vaccine Institute (Karaj, Iran). NDV) and influenza Blood samples were taken from 2 birds per pen on 28 and 42 days and then hemagglutination inhibition (HI) test according to OIE standard was performed on Newcastle and influenza serum titers, first 25 μl PBS. It was poured into all nests, then 25 microliters of bird serum was diluted in the first nest and diluted to the last nest. The mechanical shaker was placed and the microplate was placed at 25 ° C for 30 minutes, then 25 μl of 1% red blood cells was added to all the lumps and the microplate was placed on the mechanical shaker again for 15 seconds. The microplate was then placed at 25 ° C for 30 minutes and the results were recorded. Pastel was used for HI test. The titles were diluted based on log2. The 1% red blood cells used were also obtained from SPF chickens. On day 42, for the total white blood cell count and their differential count, 2 birds

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Table 2 - Growth performance mean (±SEM) of Ross 308 broilers at starter, grower, finisher and whole periods of age fed diets containing the different levels of thyme extract and thyme powder from 1st-24th days of age.

* Means within each column of dietary treatments with no superscript letter or at least one common superscript letter do not differ significantly (P>0.05); SEM: Standard Error of Means

were collected from each pen and their blood was transferred to tubes containing anticoagulant. Blood cells were determined by Cell staining, differentiation and eye counting were performed under a light microscope (Golrokh et al. 2015).

Intestinal morphology At 42 days of age, immediately after slaughter, tissue sections of the intestine (1 cm from the middle part of the intestinal jejunum) were cut and placed immediately in cans containing 10% formalin. After three steps of formalin replacement and stabilization of tissue samples, 5 mm sections were cut with a sterile surgical blade and the tissue sections were prepared by hematoxylin and eosin (HE) method. Xylol (10 minutes each), 96% alcohol (5 minutes), 100% alcohol (5 minutes) and hematoxylin (10 minutes), once inserted into an alcohol container and then, three steps of rinsing with distilled water, eosin (3 min), 96% and 100% alcohol were placed in containers and finally clarification was performed in two xylol containers, and then the slides were placed under a microscope with a graduated lens and the villi height and crypt depth were examined. (Ganjeh et al. 2016). Histomorphometric indices were studied using hematoxylin and eosin staining in the jejunum, which were the total thickness of the intestinal wall from the base of the hairs to the serous layer, the length of the hairs, the thickness of the hairs, the ratio of the length of the hairs to the depth of the crypts, the thickness of the epithelium.

Profile of fatty acids of breast meat Determination of breast fatty acid profile was performed by extracting 10 g of breast fat from 1 chicken in each treatment. At first, the fat samples were mixed well with 100 ml of methanol: chloroform (2: 1) solution for about 3 to 4 hours. The puri-

fied samples were then mixed with 25 ml of sodium chloride saturated solution in a decanter funnel. In the next step, the chloroform phase containing the fat was removed by filter paper impregnated with potassium anhydrous sulfate. The filtered sample was dried under vacuum by a rotating operator to leave only fat. After this step, 10 mg of extracted fat was stirred well with 2 ml of potassium hydroxide, 2 ml of normal methanol and 7 ml of -n hexane, then the resulting samples were centrifuged for 10 minutes. In the next step, the sample remained stationary for 5 minutes to separate the top phase. Then about one microliter of the supernatant was injected into the gas chromatography apparatus to evaluate the profile of fatty acids and the amount of the above fatty acids was expressed as a percentage of total fatty acids (Tavakoli et al. 2020).

Statistical analysis At the end of the experiment, the obtained data were statistically analyzed using analysis of variance by SAS statistical software. The mean of treatments was compared at 5% probability level with Duncan’s multiple range test. The design used in this experiment was completely random.

RESULTS AND DISCUSSION Growth performance The results of using different levels of thyme aqueous extract and thyme powder on the performance of broilers are given in Tables 2 and 3. In the period of 1 to 10 days, different levels of thyme aqueous extract and thyme powder did not differ significantly in feed intake and conversion ratio improvement (P> 0.05), but there was a significant difference in daily weight gain (P<0.05, which was the highest mean of treatment). TE (50

Table 3 - Economic performance mean (±SEM) of Ross 308 broilers at 42 days of age fed diets containing the different levels of thyme extract and thyme powder from 1st-24th days of age.

Means within each column of dietary treatments with no superscript letter or at least one common superscript letter do not differ significantly (P>0.05); SEM: Standard Error of Means

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ppm) - TP (150 mg/kg) and the lowest mean was related to TE (0 ppm) -TP (0 mg/kg). In the period of 11 to 24 days, different levels of thyme aqueous extract and thyme powder did not differ significantly in improving the conversion ratio (P> 0.05) but there was a significant difference in daily weight gain and feed intake (P<0.05) in the period of 25 to 42 days. There was no significant difference between thyme aqueous extract and thyme powder on daily weight gain, feed intake and conversion ratio (P> 0.05). In the period of 1 to 42 days, different levels of thyme aqueous extract and thyme powder had a significant difference on daily weight gain, feed intake and conversion ratio improvement (P<0.05), which was the best mean for TE (50 ppm) -TP (150 mg/kg) treatment and the weakest mean was related to TE (0 ppm) -TP (0 mg/kg) treatment i.e. the control group. The effect of different levels of thyme aqueous extract and thyme powder was significantly different on European index, economic value and total weight of the period (P<0.05). Disadvantages include the presence of harmful microbes in the gastrointestinal tract, increased degradation of proteins and amino acids in the gastrointestinal tract due to the deamination activity of microbes, and increased rate of their decomposition due to the secretion of substances such as microbial urease enzyme. Medicinal plants reduce the microbial population of the gastrointestinal tract, thus slowing down the breakdown of proteins and amino acids in the digestive tract and absorbing larger amounts of them, thereby improving feed conversion ratio (Lee et al. 2003b). Antioxidants in medicinal plants, by protecting the intestinal villi, improve the absorption of nutrients and thereby improve the performance of the bird, which is consistent with the results of the present experiment (Manzanillo et al. 2001). In an experiment, chickens that received alcoholic extract of thyme in drinking water had the highest weight gain (Abdulkarimi et al. 2011), which is consistent with the results of

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the present experiment. This result may be related to antimicrobial and stimulant properties. Digestion of thyme extract with its low pH. Manzanillo et al. (2001) also reported that beneficial antioxidant compounds of medicinal plants protect intestinal villi by improving nutrient uptake, thereby improving bird performance. Kalantar et al. (2011) also used thyme essential oil in drinking water and at the end of the experiment, they reported the best conversion ratio of 0.2% thyme, which is consistent with the results of the present experiment. Rahimi et al. (2011) reported that supplementing the diet of broilers with 0.1% thyme extract improved feed conversion ratio compared to the control group, which is consistent with the results of the present experiment. Hamdieh et al. (2013) and Okac et al. (2008) did not observe a significant difference in feed intake in any of the experimental courses by adding dried thyme powder and thyme essential oil to the diet of broilers.

Characteristics of carcasses and some organs The effect of experimental treatments on carcass characteristics is shown in Tables 4 and 5. The results showed that the use of different levels of aqueous extract of thyme and thyme powder had a significant difference on the relative weight of thymus, bursa of Fabricius, live weight, ventricular fat and pancreas (P<0.05). It has been reported that the consumption of medicinal plants stimulates the growth of immune organs of broilers and causes a significant increase in their weight (Takahashi et al. 2000). The presence of bioactive compounds in thyme probably stimulates cell proliferation in these organs. The bursa of Fabricius, thymus, and spleen are among the organs of the immune system, and improving the weight of each can improve the condition of the bird’s immune system. Perhaps the higher relative weight in the bursa of Fabricius and

Table 4 - Mean (±SEM) of economically relevant carcass characteristics of Ross 308 broilers at 42 days of age fed diets containing the different levels of thyme extract and thyme powder from 1st-24th days of age.

Means within each column of dietary treatments with no superscript letter or at least one common superscript letter do not differ significantly (P>0.05); SEM: Standard Error of Means

Table 5 - Mean (±SEM) of organ characteristics of Ross 308 broilers at 42 days of age fed diets containing the different levels of thyme extract and thyme powder from 1st-24th days of age.

Means within each column of dietary treatments with no superscript letter or at least one common superscript letter do not differ significantly (P>0.05); SEM: Standard Error of Means

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thymus indicates the effect of thyme extract on the bird’s immune status. Hamdieh et al. (2013), Abazari et al. (2011), Rahimi et al. (2011), using a mixture of plant essential oils did not observe a significant effect on the relative weight and carcass components, which is consistent with the results of the present experiment. The effect of thyme alcoholic extract supplement on growth performance and some carcass traits was investigated and it was reported that the relative wing weight of chickens that received the 4% level was significantly higher than the chickens that did

not receive. Thyme extract in drinking water significantly increased the relative weight of breast and wings. Abdulkarimi et al. (2011) concluded that consumption of alcoholic thyme extract in drinking water improved the performance and relative weight of broiler chickens which does not agree with the results obtained in the present experiment.

Intestinal parts The effect of experimental treatments on different parts of the intestine is shown in Table 6. The use of different levels of aque-

Table 6 - Mean (±SEM) of intestinal segments of Ross 308 broilers at 42 days of age fed diets containing the different levels of thyme extract and thyme powder from 1st-24th days of age.

Means within each column of dietary treatments with no superscript letter or at least one common superscript letter do not differ significantly (P>0.05); SEM: Standard Error of Means

Table 7 - Blood constituents mean (±SEM) of Ross 308 broilers at 42 days of age fed diets containing the different levels of thyme extract and thyme powder from 1st-24th days of age.

Means within each column of dietary treatments with no superscript letter or at least one common superscript letter do not differ significantly (P>0.05); SEM: Standard Error of Means

Table 8 - Liver enzymes mean (±SEM) of Ross 308 broilers at 42 days of age fed diets containing the different levels of thyme extract and thyme powder from 1st-24th days of age.

Means within each column of dietary treatments with no superscript letter or at least one common superscript letter do not differ significantly (P>0.05); SEM: Standard Error of Means

Table 9 - Immune response mean (±SEM) of Ross 308 broilers fed diets containing the different levels of thyme extract and thyme powder from 1st-24th days of age.

Means within each column of dietary treatments with no superscript letter or at least one common superscript letter do not differ significantly (P>0.05); SEM: Standard Error of Means

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Table 10 - Immunity related organ mean (±SEM) of Ross 308 broilers at 42 days of age fed diets containing the different levels of thyme extract and thyme powder from 1st-24th days of age.

Means within each column of dietary treatments with no superscript letter or at least one common superscript letter do not differ significantly (P>0.05); SEM: Standard Error of Means

ous extract of thyme and thyme powder had a significant difference on the weight ratio of rectum, jejunum and colon (P<0.05), which had the highest mean level TE (0 ppm) - TP (0 mg/kg). The use of a mixture of thyme and peppermint increased the relative weight of the ileum numerically compared to the control (Denli et al. (2004), which did not match the results of the present experiment.

Blood parameters and liver enzymes Data on the effect of thyme aqueous extract and thyme powder on blood parameters are given in Table 7. The results showed that the use of different levels of thyme aqueous extract and thyme powder on the blood parameters of broilers was not significant (P> 0.05). Tymorizade et al. (2010) reported a significant decrease in blood cholesterol and triglyceride levels in experiments on broilers using thyme extract, which did not match the results of the present experiment. Demir, et al. (2003) studied the effect of powder of several medicinal plants (garlic, thyme, cinnamon and oregano) on the hematological values of broilers and reported that these extracts did not have a significant effect on the concentration of plasma triglycerides of broilers that is consistent with the results of the present experiment. The effect of different levels of thyme aqueous extract and thyme powder on liver enzymes is shown in Table 8. The results showed that the effect of different levels of thyme aqueous extract and

thyme powder had a significant difference on alanine transaminase, lactate dehydrogenase and creatine kinase of broilers in the whole period (P<0.05). The antioxidant properties of thyme seem to reduce the destructive oxidative effect of the toxin on the liver and reduce cholesterol, triglycerides and liver enzymes due to the inhibitory effect of these extracts on key enzymes such as HMG-COA reductase. They have also been implicated in lipid and cholesterol production (Sarica, et al. 2005).

Immune System The effect of different levels of aqueous extract and thyme powder on the function of the humoral immune system in response to SRBC antigen injection and antibody titers against Newcastle virus and influenza are shown in Tables 9 and 10. The results showed that the use of different levels of thyme aqueous extract and thyme powder on the performance of the immune system of broilers was not significant (P> 0.05). Beheshti et al. (2010) reported the use of 2% of a mixture of thyme, mint and savory in the diets of laying hens improved the performance of blood parameters and safety, which does not match the results of the present experiment. Silymarin Phytosomes in the seed plant Silybum marianum or phenolic compounds in the leaves of thyme (such as thymol and carvacrol) have not been able to significantly change the titers of Newcastle disease and influenza, Gambro and bronchitis from the present experiment (Lee et al. 2003a).

Table 11 - Morphological indices of jejunum Ross 308 broilers in 42-day diets containing different levels of thyme extract and thyme powder from 1st-24th days of age.

Table 12 - Profile of breast fatty acids Ross 308 broilers in 42-day diets containing different levels of thyme extract and thyme powder from 1st-24th days of age.

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Intestinal morphology Based on the data obtained in this study, which is shown in Table 11, the highest villi length, crypt depth, layer thickness and villi length were observed relative to the crypt depth using TE (100 ppm) -TP (250 mg/kg). The highest villi width was observed in TE (50 ppm) -TP (150 mg/kg). Garcia et al. (2007) reported that the use of herbs in the diet increased villi height in broilers. The researchers suggested that

by introducing herbs into the diet, the total population of harmful bacteria in the intestinal wall was reduced, thereby reducing the production of toxic compounds and damaging the cells of the intestinal lining, so that the villi became longer and the crypt deepened. This reaction can cause changes in the morphology of the gut. Garcı a et al. (2007) at 42 days of age did not observe any significant differences between plant extract treatment contain-

Thyme extract (50 ppm)-thyme powder (150 mg/kg)

Thyme extract (50 ppm)-thyme powder (250 mg/kg)

Thyme extract (100 ppm)-thyme powder (150 mg/kg)

Figure 1 - Morphological image of jejunum Ross 308 broilers in diets on day 42 with diets containing different levels of thyme extract and thyme powder from 1st-24th days of age.

Thyme extract (100 ppm)-thyme powder (250 mg/kg)

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ing cinnamaldehyde, carvacrol and capsaicin and control treatment with villi length and crypt depth in the jejunum, which matched the results of the present experiment. Khattak et al. (2014) reported that they used different levels of plant essential oils (basil, cumin, bay leaf, oregano, tea and thyme) in the diet of broilers and found that at the level of 300 g/ton the width of cecal villi and area level also increased. The height of villi in cecum of chickens fed a diet supplemented with 100 μm of plant essential oil increased compared to the control group (291 μm vs. 320 μm). (P<0.05). Cecum had a significant increase (P<0.01) in chickens receiving treatments of 200, 300 and 500 mg of essential oil per kg of feed compared to the control, which is consistent with the results of the present experiment.

Profile of fatty acids of breast meat The percentage of fatty acids in breast muscle tissue is shown in Table 12. The results showed that the lowest percentage of three saturated fatty acids, myristic acid palmitic acid and stearic acid and also the highest level of unsaturated fatty acids was related to the highest treatment of thyme extract, except palmitoleic acid, cis-8-11-14-eicosatrienoic acid. Cis-11,14-eicosadienoicacid that the highest percentage was observed in low levels of this extract. The results of using thyme powder showed that the highest saturated fatty acid, myristic acid, and stearic acid belonged to its highest treatment, except palmitic acid, which belonged to the lowest treatment. The results of interaction between powder and extract showed that the lowest saturated fatty acids myristic acid and stearic acid were related to TE (50 ppm) - TP (150 mg/kg) level. Lee et al. (2003) also found that the amount of linoleic acid in adipose tissue increased with thyme supplementation in the diet, which is consistent with the results of the present experiment.

CONCLUSIONS In general, in the present study, it can be concluded that the use of thyme powder and thyme extract in the diet of Ross 308 broiler chickens in the short term improved feed consumption, daily weight gain, total period conversion ratio and production index. Although it did not have much effect on blood parameters, it had a positive effect on liver enzymes and also led to an improvement in the immune system and a reduction in ventricular fat, thus improving the quality of meat. The use of high levels of thyme powder and thyme extract improved the morphological evaluation parameters of intestinal jejunum. The use of high levels of thyme powder and thyme extract increased saturated fatty acids and decreased some saturated fatty acids, which have health benefits. Therefore, according to the results of this experiment, it is recommended that thyme powder and thyme extract be used as two antioxidant, antimicrobial and inexpensive growth stimulants.

ACKNOWLEDGMENTS This manuscript is based on a PhD thesis presented by the first author to Rasht Branch, Islamic Azad University, Rasht, Iran. Financial support by Rasht Branch, Islamic Azad University, grant number 17.16.4.18418 is gratefully acknowledged.

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Hereditary zinc deficiency syndrome in a calf

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ZAFER USTA1*, SİLKE DUDERSTADT2, TERESA PUNSMANN2, MARTIN HÖLTERSHINKEN3, MARİON HEWİCKER-TRAUTWEIN4, OTTMAR DISTL2 1

Department of Genetics, Faculty of Veterinary Medicine, Burdur Mehmet Akif Ersoy University, Istiklal Yerleşkesi, 15030 Burdur/Turkey Institute of Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bünteweg 17p, 30559 Hannover/Germany Clinic for Cattle, University of Veterinary Medicine Hanover, Bischofsholer Damm 15, 30173 Hannover/Germany Institute of Pathology, University of Veterinary Medicine Hanover, Bünteweg 17, 30559 Hannover/Germany

SUMMARY Bovine Hereditary Zinc Deficiency (BHZD) is an autosomal recessive disorder. A twin calf with signs consistent with BHZD. The coat was dull. Due to the typical skin lesions veterinary treatment with zinc was started. During treatment the skin lesions did not get worse but a recovery was not gained either. The lesions got bigger when the treatment was interrupted. The co-twin calf did not show any signs of BHZD. After amplifying a product of 294 base pair (bp) including the putatively causal SLC39A4 mutation the product was incubated with the restriction enzyme HpHI. A 148- and 147-bp product could be detected which was expected for the wildtype sequence. A 337-bp product including the putative PLD4 causal mutation was amplified. The G to A mutation (rs378824791) leads to the loss of the restriction site of the enzyme HpyCH4III. The putative causal mutation was not proven using this restriction fragment length polymorphism (RFLP). Further sequence analyses are needed to clarify the mutation causing BHZD in the present case. The present case also shows that there might be further mutations in Fleckvieh cattle responsible for BHZD.

KEY WORDS BHZD; Cattle; PLD4; SLC39A4.

INTRODUCTION Bovine Hereditary Zinc Deficiency (BHZD) is an autosomal recessive disorder. Due to this condition zinc is inadequately absorbed by the intestines. Impressive consequences are severe skin lesions in mainly mechanically stressed areas1. The disease is also known as hereditary parakeratosis, Adema disease or lethal trait A461. Affected Holstein calves are born without clinical symptoms. Clinical abnormalities manifest at the age of four to eight weeks. These include loss of epidermis associated with desquamation and incrustation. At the beginning these lesions can be found especially around the muzzle, peri-ocular and auricular1. Furthermore, alopecia, growth retardation and insufficiency of the immune system can be seen2. At an advanced stage of the disease, the affected animals show decreased food intake and are dejected and dehydrated1. Histological examination reveals acantholysis in the stratum spinosum and persistent cell nuclei. Beside these findings, affected animals are emaciated and show hypotrophy of thymus, spleen and lymph nodes1. In Holstein cattle a causal mutation in SLC39A4 has been identified3. A G>A SNP (single nucleotide polymorphism) in intron 10 leads to a deletion of exon 10 of SLC39A4. BHZD in Holstein calves resulting from mutations in SLC39A4 can be ameliorated by highly-dosed oral zinc supplementation. In contrast, affected Fleckvieh calves did not respond to oral zinc supplementation4. In Fleckvieh calves with signs similar to BHZD, Jung et al. (2014) found a putatively causal mutaCorresponding Author: Zafer Usta (zusta@mehmetakif.edu.tr).

tion (rs378824791) in the phospholipase D family member 4 encoding gene (PLD4) using genome-wide re-sequencing of the affected Fleckvieh calves. The mutation is predicted to lead to a premature stop codon with a shortened protein and disturbed protein activity. In addition, zinc deficiency-like (ZDL) syndrome is an inherited defect of Fleckvieh calves, with striking similarity to BHZD in a other study. However, the causative mutation in a phospholipase D4 encoding gene (PLD4) shows no connection to zinc metabolism5.

MATERIALS AND METHODS A twin calf with signs consistent with BHZD was submitted to the Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover. It descended from a Fleckvieh cow and a Blonde d’Aquitaine bull. The full pedigree of the affected calf could not be obtained. The Blonde d’Aquitaine bull was not known to have sired calves with hereditary diseases. The Fleckvieh cow had several calvings with normal calves. At the beginning the twin calves developed equally. After four weeks one of the calves showed skin lesions, growth retardation and reduced feed intake. Its behaviour was very calm. Genomic DNA was isolated using 500 μl EDTA blood by standard ethanol fraction. Precipitation was achieved by 6 M NaCl, 70% ethanol, and 100% ethanol (Carl Roth) in consecutive steps according to standard protocols. To identify the putatively causal mutations in PLD4 and SLC39A4 primer pairs based on the cow reference sequences of Ensembl and NCBI in UMD3.1 were designed.

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Table 2 - Primer sequences for amplification the BHZD-associated mutation within SLC39A4 using the restriction enzyme HpHI.

Table 1 - Primer sequences for amplification the BHZD-associated mutation within PLD4 using the restriction enzyme HpyCH4III.

RESULTS

The amplified product of PLD4 had a size of 373 base pairs (bp). The product was incubated with the restriction enzyme HpyCH4III overnight at 37 °C. In the amplicon with the mutation (rs378824791) the enzyme will not produce any restriction fragment. In a PCR-product without the mutation, restriction fragments of 43 bp and 330 bp can be detected (Table 1). The restriction digestion with HpyCH4III was analyzed on a 3% agarose gel stained with ethidium bromide. The amplified product of SLC39A4 had a size of 294 bp. This product was incubated with the restriction enzyme HpHI overnight at 37°C. In a PCR-product without the mutation, restriction fragments of 148 bp and 147 bp can be detected (Table 2). The mutation leads to a loss of the restriction site. The restriction digestion with HpHI was analyzed on a 2% agarose gel stained with ethidium bromide.

The calf showed a very calm behaviour (Figure 1). The coat was dull. Due to the typical skin lesions veterinary treatment with zinc was started. During treatment the skin lesions did not get worse but a recovery was not gained either. By owner information the lesions got bigger when the treatment was interrupted. The twin calf did not show any signs of BHZD. Blood samples of the affected animal, its twin and the dam were taken. After euthanasia the affected calf was examined in the Institute of Pathology. Respiratory (10 per minute) and heart rate (54 per minute) were slightly below the reference range. Mucous membranes were bright. The slightly sunken eyes indicated a light dehydrated condition. The lymph nodes (lnn.) subiliaci and cervicales superficiales were slightly enlarged. Caudal the left olecranon a 25 x 15 centimetres (cm) alopecic area with loss of epithelium was found. Cranially the shoulder joint a barky and scaly skin lesion with a diameter of 1 cm was found. In the inguinal region the skin was alopecic and scaly in a 30

Figure 1 - A twin calf with signs consistent with BHZD.

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Figure 2 - Haematoxylin and eosin-stained skin section from the shoulder region of fore leg with hyperplastic epidermis and marked parakeratotic hyperkeratosis.

x 20 cm large area. Alopecic and scaly skin surrounded anus and vulva. The muzzle was surrounded by scaly and alopecic skin with a width of 0.5 cm (Figure 2). The skin over the entire body appeared scaly and exfoliated. It was fed with milk substitute. Hey, water and electrolytes were available ad libitum. The feed intake was very poor. Laboratory analysis of blood showed a slight haemoconcentration. Although high doses of zinc were supplemented, the zinc level was at the lower limit of reference range. Because of the bad condition it was euthanized at the age of 61 days. Histopathological examination of the present calf revealed infiltration of lnn. mesenterici, thymus, abomasum and small intestine with eosinophilic granulocytes. In addition, a purulent inflammation of the abomasum was found. The thymus had a size of 18 x 5 x 2 cm. All skin lesions were characterized by a moderate parakeratotic hyperkeratosis with spongiosis (Figure 2). Furthermore, a perivascular to diffuse dermatitis was present. Lymphocytes, macrophages, eosinophil granulocytes and plasma cells, in some cases with Russell bodies, were involved in this process. The epidermis was slightly hyperplastic (Figure 2). In the skin of the forelimb the excretory ducts were slightly dilated.

DISCUSSION Affected calves in previous reports can be recomposed by permanent practice of complementary zinc given at high doses2,6. In our calf, due to the typical skin lesions veterinary treatment with zinc was started. During treatment the skin lesions did not get worse but a recovery was not gained either. Lesions are mostly described by parakeratosis and dermatitis, and occur in areas of permanent skin flexion or in areas especially subjected to erosion. Hence, lesions are most extensive around the mouth, eyes, base of the ear, joints, and lower parts of the thorax, abdomen and limbs7. Similar to these symptoms, the table appears. Parakeratosis and ulceration of the intestinal tract occurs and is usually meant clinically as stomatitis. Therefore, a decrease in eating capacity and growth retardation seen1. Similarly, growth retardation, reduced feed intake, very calm behaviour are the most obvious findings in this study. Lymph nodes, thymus and gut-associated lymphoid tissue are hypoplastic, and affected animals are immunosuppressed due to impaired function of the immune system7,8,9. Animals often develop bronchopneumonia. Lesions are progressive and,

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if left untreated, calves die within 4 to 8 weeks after primary symptoms are observed10. In this study, due to typical skin lesions veterinary treatment with zinc was started. During treatment the skin lesions did not get worse but recovery was not gained either. The lesions became larger when the treatment was interrupted. Mutations in SLC39A4 are known to cause defects resembling the phenotypic appearance of the present case in various species including cattle3. After amplifying a product of 294 base pair (bp) including the putatively causal mutation the product was incubated with the restriction enzyme HpHI. A 148- and 147bp product could be detected which was expected for the wildtype sequence. The causal mutation for BHZD in Holstein cattle was not detected. In Fleckvieh, a causative nonsense mutation in a PLD4 was identified4. A 337-bp product including the putative causal mutation was amplified. The G to A mutation (rs378824791) leads to the loss of the restriction site of the enzyme HpyCH4III. The putative causal mutation was not proven using this restriction fragment length polymorphism (RFLP). In addition, zinc deficiency-like (ZDL) syndrome is an inherited defect of Fleckvieh calves, with striking similarity to BHZD in a other study5.

CONCLUSIONS Further sequence analyses are needed to clarify the mutation causing BHZD in the present case. The present case also shows that there might be further mutations in Fleckvieh cattle responsible for BHZD.

References 1.

Machen M.T., Montgomery T., Holland R., Braselton E., Dunstan R., Brewer G. Yuzbasiyan-Gurkan V. (1996). Bovine hereditary zinc deficiency: lethal trait A 46. J Vet Diagn Invest, 8(2): 219-227. 2. Brummerstedt E., Basse A., Flagstad T., Andresen E. (1977). Animal model of human disease. Acrodermatitis enteropathica, zinc malabsorption. Am J Pathol, 87(3): 725. 3. Yuzbasiyan-Gurkan V. Bartlett E. (2006). Identification of a unique splice site variant in SLC39A4 in bovine hereditary zinc deficiency, lethal trait A 46: An animal model of acrodermatitis eneteropathica. Genomics, 88(4): 521-526. 4. Jung S., Pausch H., Langenmayer M.C., Schwarzenbacher H., MajzoubAltweck M., Gollnick N.S., Fries R. (2014). A nonsense mutation in PLD4 is associated with a zinc deficiency-like syndrome in Fleckvieh cattle. BMC genomics, 15(1): 623. 5. Langenmayer M.C., Jung S., Majzoub-Altweck M., Trefz FM., Seifert C., Knubben-Schweizer G., Fries R., Hermanns W., Gollnick NS. (2018). Zinc deficiency-like syndrome in Fleckvieh calves: clinical and pathological findings and differentiation from bovine hereditary zinc deficiency. J Vet Intern Med, 32(2): 853-859. 6. Kroneman J., Mey G.J.W.v.d., Helder A. (1975). Hereditary zinc deficiency in Dutch Friesian Cattle. Zbl Vet Med A, 22: 201-208. 7. Agerholm J.S. (2007). Inherited Disorders in Danish Cattle. APMIS Suppl, 122(115): 32-33. 8. Brummerstedt E., Andresen E., Basse A., Flagstad T. (1974). Lethal trait A 46 in cattle. Immunological investigations. Nord Vet-Med, 26: 279-293. 9. Perryman L.E., Leach D.R., Davis W.C., Mickelsen W.D., Heller S.R., Ochs H.D., Ellis J.A., Brummerstedt E. (1989). Lymphocyte alterations in zincdeficient calves with lethal trait A46. Vet Immunol Immunopathol, 21:239248. 10. Brummerstedt E. (1981). Lethal trait A46 in cattle - a review. In Brummerstedt E (ed.): Papers dedicated to Professor Johannes Moustgaard. The Royal Danish Agricultural Society, Copenhagen 240-245.

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E.S. Özdemi̇r Salci et al. Large Animal Review 2021; 27: 237-239

Fetal retention due to unilateral partial uterine horn torsion in a ewe

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EMSAL SİNEM ÖZDEMİR SALCI1, ABID HUSSAIN SHAHZAD2 1

Department of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Bursa Uludag University, 16059 - Bursa/Turkey Department of Clinical Science, University of Veterinary and Animal Sciences, Lahore Jhang Campus, 35200, Pakistan

SUMMARY Uterine torsion has low incidence in small ruminants, which causes the dystocia. This presented report describes, for the first time, partial foetal retention due to the dystocia resulting from unilateral partial uterine horn torsion in a ewe. A pregnant Merinos breed ewe was presented with the history of dystocia. Anamnesis pointed out that a half foetus was removed and cranial parts of the foetus were in uterus. Clinically, abdominal distension and pain were observed. Vaginal examination revealed an opened cervix, and a left sided located fetal part was felt by palpation; however, it was not possible to reach the foetus due to uterine horn torsion. Radiography showed the remained cranial parts of the foetus. Considering the clinical and radiological findings, caesarean section was performed. Abdominal exploration revealed 270 degrees clockwise rotated left uterine horn, which was at cranial ½ part of the uterine horn. After the removing of the foetal parts, all incisions were sutured routinely. Analgesic and antibiotic medications were recommended to the owner. It was informed that ewe was healthy at postoperative ten days.

KEY WORDS Dystocia, ewe, foetal retention, partial uterine horn torsion.

INTRODUCTION Uterine torsion is defined as the twisting of the uterus on its long axis that is a remarkable finding for ewe1,2,3. In ruminants, this condition is encountered where it involves rotation of this semicircle on its transverse axis, similar to intestinal volvulus3. Incidence of dystocia on account of uterine torsion is about 4.4% in ewes4. The cause of the low incidence rate of uterine torsion is little practiced involvement in small ruminants, and difficult to distinguish the case for diagnosis and treatment1. The main etiological factors responsible for uterine torsion are: suspension with a broad ligament supporting the bending of the uterus and making the gravid uterus more unstable and prone to torsion in ruminants3. Uterine torsion causing dystocia can be either partial or total, and usually seen on most caudal portion of uterus as partial. Its assessment is made on the basis of degrees of torsion. If the torsion is <180º vaginal palpation of foetus is possible. At the terminal stages of pregnancy, colic like signs are obvious and labour contractions are observed following sings of lambing1,5. Unlike large ruminants, where diagnosis and evaluation of extent of uterine torsion is easy, it is difficult to diagnose in ewe by vaginal examination due to the closure of the cervix3,6,7. Thus, the treatment plan consists of rotating per vaginal foetus, surgical detorsion and caesarean section depending on the degree of torsion of the uterus3. Although some reports are available about uterine horn

Corresponding Author: Emsal Sinem Özdemir Salci (ssalci@uludag.edu.tr).

torsion2,8, this case is the first report of the partial foetal retention due to the dystocia resulting from unilateral partial uterine horn torsion in a ewe.

CASE PRESENTATION A 3-year-old, multiparous pregnant Merinos breed ewe was presented with the history of dystocia to Faculty Clinics of Veterinary Medicine, Bursa Uludag University. According to narrated history, ewe started the lambing signs 3 days ago and gave one lamb without any manual aid. At 2nd days after first delivery, straining started again, but no lamb was delivered. The owner suspected foetal retention due to ongoing strain and checked to birth canal for possible lamb. And, he palpated a second foetus, and he was able to bring only the caudal part of the foetus (both the posterior limbs and the caudal abdomen). He could not remove the cranial part of the foetus, and second half of foetus could not be delivered spontaneously, as well. Considering to this anamnesis, a half foetus was in uterus. On clinical examination, the ewe was lying on lateral side, and vulva was swollen and enlarged with reddish brown discharge. The ewe had 39.2°C rectal temperature, normal mucosal colour, 2 seconds capillary refilling time, rapid and shallow respiration and inability to stand up. Abdominal palpation revealed distension and pain. On radiographical examination, remaining parts of the foetus were observed at the caudal abdominal cavity as shown in Figure 1. Vaginal examination revealed that cervix was fully dilated and there was no abnormality at this level, which might create any stenosis of the vagina leadings to dystocia. Per-vaginal examination indicated that the cra-

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Fetal retention due to unilateral partial uterine horn torsion in a ewe

It was seen that the cranial ½ of the left uterine horn rotated approximately 270 degrees clockwise, and the uterus colour was changed from the torsion site. After the torsion was corrected, the incision was made in the longitudinal direction to the uterus, and the remaining cranial parts of the lamb were taken out (Figure 2). Two goblets containing 500 mg chlortetracycline HCl (Devamisin, Vetas, Turkey) were placed in the uterine horn and the incision line on uterus was sutured continually (Figure 3). After the last intraabdominal exploration, all anatomical structures of the left flank were closed routinely. Postoperatively, flunixin meglumine (1.1 mg/kg QD) (Fluvil, Vilsan, Turkey) was administered intravenously as analgesic, and amoxicillin clavulanic acid (10 mg/kg BID) (Synulox, Pfizer, Turkey) was given intramuscularly as antibiotic. These drugs were recommended for the next 4 days to the owner, and then the ewe was discharged. After ten days, it was informed by the owner that ewe was healthy and skin sutures were removed.

Figure 1 - This radiological view points out the remaining parts of the fetus (in dashed ellips) in the abdominal cavity.

nial parts of the foetus were at left sided location, but removing is impossible due to uterine horn torsion. Peripheral blood samples were taken for haematological analysis, and all parameters were within the reference ranges. The clinical and radiological results were informed to the owner and caesarean section was decided. The ewe was sedated with xylazine HCl (Alfazine, Alfasan/Egevet, Turkey) (1 mg/kg, im.) and then the peripheral vein was catheterized and 0.9% isotonic NaCl solution was given pre-, per- and postoperatively. After the shaving of the left flank region, ewe was brought to the lateral position on the operation table, and then surgical preparation of the operation area and local anaesthetic infiltration with lidocaine HCl (Adokain, Sanovel, Turkey) were performed. Incisions and dissections from left flank region were made to approach the abdominal cavity. In the exploration, a congestive enlarged left uterine horn was observed. The appearance of the other uterine horn was normal.

Figure 2 - The appearance of the remaining cranial parts of the fetus in the uterine horn torsion after removal.

DISCUSSION There is little information about to prevalence of dystocia in ewe, because many reasons are responsible for this such as irregular record keeping and treatment expenses9,10. Dystocia due to uterine torsion is rare in ewes, and it is commonly defined as the revolving or twisting of the uterus3. Mortality rate in ewe is 3.8% on account of dystocia in sheep10. Here, partial uterine horn torsion and its clinical and intraoperative results were reported in a ewe. Although the aetiology of uterine torsion is unclear, factors such as uterine contractions, foetal movements,

Figure 3 - Intraoperative view of the detorsioned uterine horn after suturing. Arrows show the torsioned part. R: Right uterine horn, L: Left uterine horn.

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E.S. Özdemi̇r Salci et al. Large Animal Review 2021; 27: 237-239

rough handling during gestation, decreased tone of the gravid uterus, flaccid uterine walls and long flaccid mesometrium have been considered in gravid animals11. In this presented case, based on the anamnesis, it was predicted that after the first offspring was removed, torsion was formed in the left uterine horn as a result of continuation of uterine contractions, and during the intervention of labour, the offspring could not be removed due to torsion, and foetal retention occurred. In ewe with uterine torsion, initial examination may be too late to save the lambs or ewe3. Diagnosis is particularly difficult in pregnant ewes, as in such cases; it is impossible to detect alterations of any kind in the vagina due to the closure of the cervix5. In general, the torsion occurs at the level of the cervix. Diagnosis of the uterine horn torsion in the sheep is difficult; as vaginal and rectal access is limited2. In our case, despite the cervical opening, dystocia was interfered by the owner and the caudal part of the foetus was removed. In the per-vaginal examination, it was felt that the cranial part of the foetus was directed to the left and it was impossible to remove it manually due to uterine horn torsion. In uterine problems, a complete blood count can be helpful in determining the severity of the problem and its prognosis12. In cases with uterine torsion, mortality is typically high, probably due to prolonged parturition associated with the healthy condition and subsequent foetal hypoxia13. In parallel with the clinical findings and images during the surgical exploration, the haematological findings of the presented case were within the reference ranges. As with large ruminants, the rotation method is not suitable for the correction of uterine torsion, as the torsion direction in sheep is not fully understood3. The torsion is successfully corrected by surgical intervention through left flank caesarean section3,5. Thus, the left flank surgical approach was planned to correct the uterine horn torsion and to perform the caesarean section in the ewe. During intraabdominal exploration, it is still valuable method for the assessment of uterine torsion by spiral folds formation6. It is reported that surgical intervention is the method of choice for the treatment of uterine torsion on the basis of high success rate in corrected cases as 94.5%14. In intraabdominal exploration, colour change and torsion in the uterus were seen, and in our case, a 270-degree clockwise torsion was determined in the cranial 1/2 part of the left uterine horn. Following to correction of the torsion, the cranial parts of the foetus were removed from the uterine horn. The general condition of the ewe was good after surgery.

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CONCLUSIONS In conclusion, uterine torsion is the cause of dystocia, and especially in cases with partial uterine torsion, emergency caesarean section should be planned after clinical diagnosis, since the foetus cannot survive.

References 1.

3. 4. 5.

6. 7.

9. 10. 11. 12.

13.

14.

Arthur G.H., Noakes D.E., Pearson H., Parkinson T.J. (2001). Veterinary reproduction and obstetrics. 8th ed., 229-244, WB Saunders Company Ltd., London. Castillo J.M., Dockweiler J.C., Cheong S.H., Diel de Amorim M. (2018). Pyometra and unilateral uterine horn torsion in a sheep. Reprod Dom Anim, 53(1): 274-277. Ijaz A., Talafha A.Q. (1999). Torsion of the uterus in an Awassi ewe. Aust Vet J, 77(10): 652-653. Ali A.M.H. (2011). Causes and management of dystocia in small ruminants in Saudi Arabia. J Agric Vet Sci, 4(2): 95-108. Kacprzak K.J., Jurka P., Max A., Czerniawska-Piatkowska E., Bartyzel B.J. (2014). Etiology, symptoms and treatment of uterine torsion in domestic animals. Folia Pomer Univ Technol Stetin Agric Aliment Pisc Zootech, 315(32): 21-30. Frazer G. S., Perkins N. R., Constable P. D. (1996). Bovine uterine torsion: 164 hospital referral cases. Theriogenology, 46(5): 739-758. Wehrend A., Bostedt H., Burkhardt E. (2002). The use of trans-abdominal B mode ultrasonography to diagnose intra-partum uterine torsion in the ewe. Vet J, 164(1): 69-70. Larsonberg E.L., Pearson L.K., Campbell A.T. (2013). Uterine horn torsion associated with a mummified fetus in a ewe. Clinical Theriogenology, 5(3): 409. McSporran K.D., Buchanan R., Fielden E.D. (1977). Observations on dystocia in a Romney flock. N Z Vet J, 25(9): 247-251. Scott P.R. (2005). The management and welfare of some common ovine obstetrical problems in the United Kingdom. Vet J, 170(1): 33-40. Erlwanger K.H., Costello M.A., Meyer L.C. (2011). Uterine torsion in a Spraque Dawley rat (Rattus norvegicus). J S Afr Vet Assoc, 82(3): 183-184. Ali A., Derar R., Hussein H.A., Abd Ellah M.R., Abdel-Razek A.Kh. (2011). Clinical, hematological and biochemical findings of uterine torsion in buffaloes (Bubalus bubalis). Anim Reprod Sci, 126(3-4): 168-172. Lyons N., Gordon P., Borsberry S., Macfarlane J., Lindsay C., Mouncey J. (2013). Clinical forum: Bovine uterine torsion: a review. Livestock, 18(1): 18-24. Minkov T., Prvanov P., Kosev K. (1998). Diagnosis, nonsurgical and surgical correction of uterine torsion in sheep. Veterinarna Meditsina, 4(34): 213-216.

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