Large Animal Review - Year 26, Number 5, October 2020 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

05/20

Bimonthly, Year 26, Number 5, October 2020

LAR Large Animal Review

ISSN: 1124-4593

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

ORIGINAL ARTICLES BOVINE • Effect of the administration of different levels of solid feed on production performance, welfare, health status and antibiotic use in veal calves • Effects of pain and nonsteroid antiinflammatory drugs (NSAIDS) after abomasal displacement operations • Oxidative status along different stages of pregnancy in dairy cows CAPRINE • Investigating erythrocyte membrane lipid and protein oxidation with Na+/K+ATPase activity in caprine Anaplasmosis

22° CONGRESSO 2020

IN STREAMING DALLE 15 ALLE 18: MERCOLEDÌ 7 OTTOBRE GIOVEDÌ 8 OTTOBRE VENERDÌ 9 OTTOBRE GIOVEDÌ 15 OTTOBRE VENERDÌ 16 OTTOBRE

SWINE • Space allowance and piglets survival rate in the farrowing crate POULTRY • Broiler’s performance and carcass characteristics improvement by prebiotic supplementation CASE REPORTS BOVINE • Su di un caso di malformazione doppia (cefalotoracopagia) nel vitello

SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

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INDEX

ORIGINAL ARTICLES

Anno 26, numero 5, Ottobre 2020 Rivista indicizzata su: CAB ABSTRACTS e GLOBAL HEALTH IF (2019): 0.299

Editor in chief: Massimo Morgante

BORGO, RICCARDO COMPIANI, GIANLUCA BALDI, LUCIANA ROSSI, LUIGI BERTOCCHI

203

Effects of pain and nonsteroid anti-inflammatory drugs (NSAIDS) after abomasal displacement operations of cattle

Managing Editor: Matteo Gianesella

EROL HANIFI, EROL MUHARREM, IZCI CELAL

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.

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

Effect of the administration of different levels of solid feed on production performance, welfare, health status and antibiotic use in veal calves for white meat production SILVIA GROSSI, CARLO ANGELO SGOIFO ROSSI, GIUSEPPE

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

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)

BOVINE

213

Oxidative status along different stages of pregnancy in dairy cows RAFFAELE LUIGI SCIORSCI, MADDALENA MUTINATI, MARIAGRAZIA PICCINNO, EDOARDO LILLO, ANNALISA RIZZO

223

CAPRINE

Investigating erythrocyte membrane lipid and protein oxidation with Na+/K+ATPase activity in caprine Anaplasmosis SALIM ILKAYA, YETER DEGER, BEKIR OGUZ, UGUR OZDE

231

SWINE

Space allowance and piglets survival rate in the farrowing crate ELEONORA BUOIO, ANNAMARIA COSTA

239

Direttore Responsabile Antonio Manfredi Stampa Press Point - Via Cagnola, 35 20081 Abbiategrasso (MI) - Tel. 02/9462323 Spedizione Poste Italiane SPA - Spedizione in A.P. D.L. 353/2003 (Conv. in L. 27/02/2004 N. 46) Art. 1, Comma 1, DCB Piacenza

AMENI ASKRI, AZIZA RAACH-MOUJAHED, NACEUR M’HAMDI, ZIED MAALAOUI, HAJER DEBBABI

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 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.

POULTRY

Broiler’s performance and carcass characteristics improvement by prebiotic supplementation 249

CASE REPORTS BOVINE

Su di un caso di malformazione doppia (cefalotoracopagia) nel vitello MARILENA BOLCATO, PONTES JACINTO JOANA GONÇALVES, DAVIDE BOLOGNINI, ARCANGELO GENTILE

259

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S. Grossi et al. Large Animal Review 2020; 26: 203-210

Effect of the administration of different levels of solid feed on production performance, welfare, health status and antibiotic use in veal calves for white meat production

203

S. GROSSI*1, G. BORGO2, R. COMPIANI1, G. BALDI1, L. ROSSI1, L. BERTOCCHI3, C.A. SGOIFO ROSSI1 1

University of Milan, Faculty of Veterinary Medicine, Department of Veterinary Science for Health, Animal Production and Food Safety, Milan 20133, Italy 2 Vercelli Group, Formigliana 13030, Italy 3 IZSLER “Bruno Ubertini”, Brescia 25124, Italy

SUMMARY The study aimed at evaluating the effect of five different levels of solid feed administration on production performance, animal welfare, health status and antibiotic use in veal calves for white meat production. A total of 1750 male Friesian calves (49.61±2.06 kg initial live weight) were randomly divided into five different dietary treatments (193±2.59 days). The five experimental groups differed in terms of amount of solid feed (SF) and milk replacer (MR) administered during the entire fattening period: i) “Solid feed 180 kg” (SF180); ii) “Solid feed 225 kg” (SF225); iii) “Solid feed 270 kg” (SF270); iv) “Solid feed 330 kg” (SF330); v) “Solid feed 380 kg” (SF380). Growth performances were evaluated as final live weight (FW) and average daily gain (ADG). At slaughter, dressing percentage (DP), hot carcass weight (HCW), carcass colour classification, and percentage of calves with a carcass weight lower than 110 kg, were recorded. Welfare and health status were assessed in terms of morbidity, mortality, number of calves moved into hospital pen, haemoglobin value and the incidence of calves with haemoglobin level lower than 7.25 g/dL (4.5 mmol/L) at 80 days of breeding. Antibiotic use was included (grams of active principle/head). The results showed a positive effect of increasing solid feed in terms of production performance and health status. A solid feed administration higher than 225 and 330 kg, significantly improved the ADG (SF180: 1.12±0.08 kg/d; SF225: 1.13±0.08 kg/d; SF270: 1.17±0.07 kg/d; SF330: 1.19±0.06 kg/d and SF380: 1.23±0.07 kg/d) and the FW (SF180: 265.61±14.78 kg; SF225: 268.97±15.35 kg; SF270: 279.34±13.40 kg; SF330: 276.31±11.07 kg and SF380: 286.13±14.62 kg). Similar trends were found in HCW, with calves from the SF180 and SF225 groups showing the significantly lowest weights (SF180: 142.47±6.39 kg; SF225 143.66±7.07 kg), followed by SF270 and SF330 groups (SF270: 148.78±6.57 kg; SF330: 146.28±9.45 kg), and calves from SF380 the highest weight (SF380: 150.65±9.43 kg). The increase in solid feed administration reduced the incidence of carcasses in the «white» colour category, while it increased in the pinkish category. Administration of solid feed above 270 kg, significantly reduced morbidity and mortality, with lower values in groups SF380 (morbidity 20.00% and mortality 3.71%) and SF330 (morbidity 22.85% and mortality 4.29%) compared to groups SF270 (morbidity 25.71% and mortality 5.14%), SF225 (morbidity 28.57% and mortality 5.71%) and SF180 (morbidity 28.57% and mortality 5.43%). Consequently, a consumption of solid feed above 270 kg significantly reduced the use of antibiotics (SF180: 120.83±45.10 g/head; SF225: 124.11±45.46 g/head; SF270: 118.97±50.25 g/head; SF330: 105.57±41.67 g/head; SF380: 103.70±40.30 g/head). Higher solid feed administration significantly increased the haemoglobin level, with less calves with haemoglobin lower than 7.25 g/dL in group SF270 compared to SF225 and SF180 and in group SF330 and SF380 groups compared to SF270. The results of the study show that the administration of solid feed oriented towards an ad libitum availability, significantly improves both welfare and growth performance, in agreement with the law’s indications aimed at maximizing welfare and reducing the consumption of antibiotics in livestock production.

KEY WORDS Veal calves; solid feed; antibiotics; welfare; performances.

INTRODUCTION Worldwide, surplus male dairy calves are mainly used for veal production in specialized fattening units under intensive rearing conditions, to produce white meat. Europe raises about 6 million calves per year for veal meat, resulting in more than 1000 thousand tonnes of carcass weight, and Netherlands, Spain, France and Italy are the main producing countries, contributing

Corresponding Author: Silvia Grossi (silvia.grossi1994@libero.it)

respectively for 25.7%, 24.8%, 18.9% and 9.1% to the total veal meat yearly production1. Outside the EU, veal is also produced in the United States (Indiana, Michigan, New York, Ohio, Pennsylvania and Wisconsin) (6% of the global production), Canada (4%), Australia (4%) and New Zealand (3%)2. In the United States special-fed veal represents a 1-billion-dollar industry3. Traditionally, veal calves for white meat production, were feed for the hole fattening period with a low iron milk replacer and low amount of solid feed, to obtain the typical pale meat. This kind of management has been strongly criticized for poor animal welfare4, because a too low amount of solid feed, limits

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Effect of the administration of different levels of solid feed on production performance, welfare...

the physiological development of the forestomach, and increase oral stereotypies, such as chewing, licking, tongue rolling5,6, and digestive disorders, due to abomasal damages and hairballs formation in the rumen7,8,9. EU directive specified that veal calves from 8 to 20 weeks of age should be provided daily with an increasing quantity of at least 50 to 250 g of solid feed4, but several evidences reported that this minimum amount of solid feed is still not enough to meet the real needs of the calves during the entire breeding period. Recent studies showed that the increase in the quantity of solid feed, even at six times higher than recommended, significantly reduced unnatural oral behaviours10. Also, calves free to choose the feed to eat, reconstituted milk, concentrate or long fodder, increased, between 3 and 6 months of age, from 25.0 ± 4.7% to 47.1 ± 2.1% the total amount of dry matter ingested from concentrate at the expense of milk, reaching a voluntary daily consumption of solid feed equal to 3205.5 ± 175 g of dry matter11. Berends et al. (2012a)12 showed that an increase in the amount of solid feed inclusion in the diet, led to an improvement in growth performance. Limitation in increasing solid feed administration is due to the increase in iron uptake that can alter the pale colour of the meat, impairing its marketability. However, Brscic et al. (2014)13, found that feeding 140 kg of solid feed on dry matter basis in 201 days rearing period, did not affect carcass colour. Increasing solid feed administration without negative effect on carcass colour might reduce the production cost enhancing welfare14. Moreover, digestion disorders and not optimal physiological status also increases antibiotic consumption and antimicrobial use, and veal sector is one of the highest in drugs use of all food producing animal sectors15,16. For those reasons the correct balance between milk replacer and solid feed is fundamental to improve welfare but also to reduce the antimicrobial use, avoiding any negative potential effect on meat colour. Thus, the aim of this study was to investigate the effect of different levels of solid feed and milk replacer administration on production parameters, health status and antibiotic use in white veal calves in an intensive rearing system.

MATERIALS AND METHODS The study did not require any approval by the ethical board since no additional calf manipulations were needed. Calf haemoglobin was assessed on the blood samples routinely collected by the farm veterinary to comply with the minimum threshold value set by the European Directives on calf protection4.

Farm and housing facilities The study was carried out in commercial farm, located in Piemonte region in the north of Italy. According with the national and international laws, calves were housed individually for no more than the first eight weeks of life, and then in pens of five animals each4. The space allowance per animal was in line with legislation, both for the single cages (1.30 m length and 0.80 m width) and for the group pens (> 1.8 m2/head). Calves were housed on slatted (< 2.5 cm) wooden floor, with a correct dimensioning of the manger (< 0.5 m/head). Calves were allowed to have social contact also in the individual cages thanks to the lateral partitions of cages that are provided with slotted fences. Fresh water is always available. Feeding plan consists in two milk replacer meals (07:00 am 07:00

pm), and two solid feed administration after milk meal, starting from the second week of fattening The characteristics of milk replacer are reported in Table 1. All the calves receive the milk replacers in a bucket and the daily amount was delivered in two equal meals. The solid feed (Table 2), was administered in a dedicated bucket, starting from the second week of fattening and in quantities greater than the minimum amount required by law4. The solid feed was a mash between flakes cereals, meals cereals, meals protein raw materials, mineral and vitamin mix and pre-chopped and cleaned wheat straw. The quantities for milk replacer and solid feed were adjusted daily.

Animals and experimental protocol A total of 1750 two weeks old Holstein Friesian male calves, were enrolled for the trial. At the arrival, all the calves were weighted (average initial weight of 49.61±2.06 kg) and randomly divided into five treatment groups (Table 3). All the calves were monitored for the entire fattening period, which lasted 193±2.59 days. All the calves received the same type of milk replacers (MR) and also the same fibrous solid feed (SF). The five treatments groups involved 5 different increasing levels of solid feed administration, and a simultaneous parallel reduction of the milk replacer (Table 3).

Sanitary protocol Animals were treated individually only after clinical signs of disease. Upon reaching specific morbidity thresholds, the entire group underwent metaphylactic mass treatment. Antibiotics administered were chosen by the veterinary staff of the farm accordingly with the Government and European guide lines to avoid antimicrobial resistant development.

Table 1 - Milk replacer characteristics. Parameter, on as fed Humidity, %

Milk replacer 6.00

Crude protein, %

20.00

Fat, %

18.30

Crude cellulose, %

0.40

Lactose, %

45.20

Starch, %

2.28

Ash, %

7.39

Iron, ppm

13.00

Table 2 - Solid feed characteristics. Parameter, on as fed

Solid feed

Humidity, %

9.80

UFC, unit

1.10

Crude protein, %

14.50

Fat, %

4.45

NDF, %

17.25

Starch, %

45.65

Sugar, %

2.54

Ash,%

3.95

Iron, ppm

31.75

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S. Grossi et al. Large Animal Review 2020; 26: 203-210 Table 3 - Groups and treatments.

Table 4 - Total solid feed and milk replacer consumption.

Group

N° of calves

Initial weight, kg

Feeding treatment

SF180

350

48.97±2.12

180 kg solid feed, 360 kg milk replacer

SF225

350

49.37±2.34

205

225 kg solid feed, 330 kg milk replacer

SF270

350

49.66±1.62

270 kg solid feed, 300 kg milk replacer

SF330

350

50.23±2.02

330 kg solid feed, 265 kg milk replacer

SF380

350

49.81±2.19

380 kg solid feed, 235 kg milk replacer

Detected parameters Growth performance and health On day0 (arrival), and three days before slaughter, all animals were weighed. The average daily gain (ADG) was then calculated. The total milk replacer and solid feed intakes were detected daily on the basis of the quantities effectively consumed every day. Then, the feed conversion rate (FCR) was calculated as follow:

Group

Milk, tot kg/head

Solid Feed, tot kg/head

SF180

345

170

SF225

320

225

SF270

300

270

SF330

270

335

SF380

240

400

Carcass characteristics and meat colour Carcass weight was evaluated on hot carcass (HCW) and then dressing percentages (DP) were calculated from the following formula: Dressing % = (HCW/live weight) x 100. Carcasses colour were assessed on cooled carcass at 24 hours post mortem, by a certified expert in carcass evaluation, using a 5-point scale, were 1 was the optimal colour (white) and 5 the worst and darkest colour (dark rose).

Statistical analysis Data were analysed with the GLM (General Linear Model), procedure of SAS (version 9.3 - SAS Inst. Inc., Cary, NC, USA). The level of significance to indicate differences stated in the ANOVA model were P<0.05.

FCR = Total MR intake (kg)+Total SF intake (kg) Weight gain (kg)

RESULTS AND DISCUSSION

Animals were inspected twice a day by the farm veterinary and technicians trained staff. Every case of morbidity was recorded as well as mortality. Every medical treatment, either individual or mass treatments, was reported. The grams of antibiotic used per head was then calculated on the basis of the concentration of the pharmaceutical product, the doses administered, and the number of treatments done. The calves that needed to be moved to the hospital pen were also registered. At the slaughterhouse the incidence of calves with a carcass weight lower than 110 kg, was recorded. Haemoglobin level Calves’ haemoglobin levels and percentage calves with a level of haemoglobin lower than 7.25 g/dL were also used as health indicators. Haemoglobin concentration was assessed by the farm Veterinary to comply with the threshold value set by the European Directives (haemoglobin level must to be over than 7.25 g/dL or 4,5 mmol l-1)4. Haemoglobin was measured, for each calf, on blood samples collected at day80 of the fattening cycle, from the jugular vein after the morning feeding, and using vacutainer tubes containing K3 EDTA. On the basis of the haemoglobin level, calves with risk of becoming anaemic were treated with iron.

Solid feed and milk replacer intake Solid feed and milk replacer intake for the different groups, are reported in Table 4. Data showed an excellent correspondence with the theoretical amount planned by the protocol and the amount consumed along the study. Coherently with the objectives of the study, animals in the groups with the highest amount of solid feed were able to intake a higher amount of solid feed then predicted. Gottardo et al. (2000)17 highlighted that veal calves are motivated to eat solid feed in an amount that clearly exceeded the amount recommended by animal welfare regulation directives. Webb et al. (2014)11 showed that calves from 3 to 6 months of age, free to choose between long fodder, concentrate or reconstituted milk, increased the voluntary intake of concentrate at the expense of milk, reaching a daily consumption of solid feed higher than 3.2 kg of dry matter.

Growth performance Growth performance and feed conversion rate are reported in Table 5. Calves from the SF180 group showed the lowest ADG (1.12±0.08) while the ADG significantly increased (P<0.05), as the solid feed intake increases, reaching the highest growth in the SF380 group (1.23±0.07). Significant differences were found between SF180 and SF225 in comparison to SF270 and

Table 5 - Growth performance. Group

Fattening, day

Initial weight, kg

Final weight, kg

ADG, kg/head/day

FCR

SF180

193

48.97±2.12

265.61±14.78a

1.12±0.08a

2.38

SF225

194

49.37±2.34

268.97±15.35

1.13±0.08

2.48

SF270

197

49.66±1.62

279.34±13.40b

1.17±0.07b

2.48

SF330

190

50.23±2.02

276.31±11.07

1.19±0.06

2.68

SF380

192

49.81±2.19

286.13±14.62c

1.23±0.07c

2.71

*a,b,c= P<0.05

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Table 6 - Health status: mortality, morbidity, antibiotic use, calves with carcass weight lower than 110 kg, calves moved to the hospital pen. Group

Morbidity, %

Mortality, %

Antibiotic, g/head

HCW <110 kg, %

Moved to hospital pen, %

SF180

28.57a

5.43a

120.83±45.10a

6.00

1.71

SF225

28.57

5.71

124.11±45.46

7.14

2.00

SF270

25.71a

5.14a

118.97±50.25a

6.00

1.71

SF330

22.85

4.29

105.57±41.67

7.14

1.43

SF380

20.00b

3.71c

103.70±40.30b

5.14

1.43

*a.b.c = P<0.05

SF330 groups, and between those and SF380. The difference in average daily gain between SF180 and SF380 calves was 110 g/head/day. As a result, also the final weight was statistically improved by the increasing of solid feed intake, following a pattern similar to the average daily gain. Those results are in line with previous researches that reported an improvement in ADG in calves fed with a great amount of solid feed, starting from the early phases of fattening period12. Early administration of solid feed in correct amount, improved rumen development, triggering rumen fermentation and activity that led to volatile fatty acids production, especially butyrate, fundamental for the rumen papillae development12,18. Also, the physical structure of solid feed contributed to the muscular development and expansion of ruminal volume19, and then to rumen efficiency10,18,19. Berends et al. (2012b)20, in a study carried out on calves housed in metabolic chambers, also found a better nitrogen and energy retention when solid feed was fed in comparison to calves fed only with milk replacer, but also when the amount of solid feed administered increased. As the solid feed intake increases, the feed conversion rate (FCR) increased numerically, reporting a slight, numerical, negative effect of increased level of solid feed administration on feed efficiency. In fact, the SF180 group showed the lowest FCR (2.38) while increased gradually in the other groups, with only a numerical difference, with the SF380 showing the highest (2.71). Besides improving rumen activity and functionality, the increase in solid feed intake lowered the feed efficiency. In spite of it, improving rumen development and functionality enhances animal welfare and health, resulting in better growing performances and allows to use, in an efficient way anyway, different feed sources, more typical for a ruminant, that are not in competition with human nutrition. This can help in increasing the sustainability of the hole system.

Health status Health status along the entire rearing period is reported in Table 6. The administration of higher amount of solid feed had a statistically positive effect on the main health indices as morbidity, mortality, and antibiotic consumption. Indeed, the increase of solid feed showed a significantly lower incidence of morbidity in group SF380 (20%), and SF330 (22.85%), followed by groups SF270, SF225 and SF180 (25.71%, 28.57% and 28.57%, respectively). Same trend was observed in mortality rate, with a reduction to the increase of solid feed administration, and the lowest significant value in group SF380 (3.71%) compared to all the others groups. Also, calves in group SF 330 (4.29%) showed a significantly lower mortality then calves in groups SF270, SF225 and SF180 (5.14%, 5.71% and 5.43%, respectively). As a reflection of the lower morbidity and mortality, the an-

tibiotic use significantly decreased as the consumption of solid feed increases. The groups SF180, SF225 and SF270 showed the highest antibiotic consumption per head (120.83±45.10, 124.11±45.46 and 118.97±50.25 g/head, respectively), while the groups SF330 and SF380 the lowest (105.57±41.67 and 103.70±40.30 g/head respectively) (P<0.05). These results are consistent with those of Cozzi et al. (2002)21, that reported a lower number of medical treatments for respiratory and gastrointestinal diseases in veal calves fed with higher amount of solid feed. The improvement in health condition, increasing the solid feed administration, can be explained by the improvement in rumen development and activities, stress conditions reduction, and higher level of haemoglobin, with less risk of sub clinical and clinical anaemia22,23. Indeed, a greater availability of solid feed allows the rumen to an early and correct development, implementing natural behaviours such as rumination, with a significant reduction in chronic stress. Chronic stress strongly impairs immune system, increasing the probability for calves to contract pathologies, and then, increases antibiotics treatments24. Regarding the risk of anaemia, in Table 7 are reported the results of haemoglobin levels and incidence of calves with haemoglobin level lower than 7.25 g/dL. Average haemoglobin significantly increased as the solid feed intake increases. Statistical differences were found between groups SF180 and SF225 (8.38±0.74 and 8.42±0.63g/dL, respectively), and groups SF330 and SF380 (8.83±0.61 and 8.99±0.43 g/dL, respectively). In addition, no calves showed haemoglobin level lower than 7.25 g/dL in the groups SF330 and SF380, while groups SF180 and SF225, showed an incidence of calves with haemoglobin level lower than 7.25 g/dL, close to 10%. Increasing the solid feed administration along the fattening period from 225 kg to 270 kg, significantly reduced by 4 times the incidence of calves with haemoglobin lower than 7.25 g/dL. These results are in line with the findings of previous researches that established a positive effect of solid feed intake on the

Table 7 - Health status: haemoglobin. Group

Haemoglobin at d80, average, g/dL

Haemoglobin <7.25 g/dL, %

SF180

8.38±0.74a

10.57a

SF225

8.42±0.63

9.14a

SF270

8.62±0.59ab

2.00b

SF330

8.83±0.61

0.00c

SF380

8.99±0.43b

0.00c

*a.b.c = P<0.05

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S. Grossi et al. Large Animal Review 2020; 26: 203-210

209

Table 8 - Slaughtering performance. Group

DP, %

HCW, kg

SF180

53.64±0.58a

SF225

53.41±0.33

SF270

53.26±0.60ab

SF330

52.94±0.71

SF380

52.65±0.88b

Colour 1, %

Colour 2, %

Colour 3, %

Colour 4, %

Colour 5, %

142.47±6.39a

35.15a

36.28

20.85

7.72a

0.00

143.66±7.07

32.01

34.57

148.78±6.57bc

26.57bc

32.00

146.28±9.45

25.71

150.65±9.43c

27.42bc

23.42

10.00

0.00

26.29

15.14bc

0.00

29.73

25.71

18.57

0.28

30.28

25.16

17.14c

0.00

*a.b.c = P<0.05

haemoglobin level and in reducing the risk of anaemia in white veal calves, thanks to the iron content present in solid feed21,22. Studies also showed that pyloric lesions, the most common type of abomasal lesions in veal calves that contribute to increase morbidity and mortality, are reduced by the increase of solid feed intake9. No statistically significant differences were found in terms of calves moved to the hospital pen and in calves with a final carcass weight lower than 110 kg, signs of animals with poor health conditions and growth performance (Table 6).

Slaughtering performance and carcass colour Data about slaughtering performance and carcass colour are reported in Table 8. In agreement with growth performance, the increase in solid feed administration significantly increased carcass weights. Groups SF180 and SF225 showed the lightest carcasses (142.47±6.39 kg and 143.66±7.07) and calves in group SF380 the heaviest (150.65±9.43 kg). A slight reduction in the dressing percentage is visible as the feed intake increases, with the group SF380 showing the significantly lowest dressing percentage (52.65±0.88%). The increase in solid feed administration also affected carcass colour, and then the carcass distribution in the different colour classes. Higher level of solid feed corresponded to a statistical lower percentage of carcasses in the first colour category and, correspondingly, to a significant higher percentage of carcasses in the fourth category (P<0.05). Those result are mainly due to the higher iron content of the solid feeds that lead to a higher level of myoglobin in muscles, as demonstrated from several studies21,22, and confirmed by the haemoglobin levels found in the calves from different groups. Studies showed that the increase in solid feed administration in veal calves for white meat production, doesn’t affect the meat palatability traits, highlighting the absence of negative effects on consumer palatability satisfaction25,26.

CONCLUSIONS Results of the present study highlight that increasing the solid feed administration in veal calves for white meat production, effectively improve growth performance and animal welfare and health. Administration of solid feed in larger and increasing quantities, significantly reduce morbidity, mortality and the antimicrobial use. Considering the small negative effects found on carcass colour, the increase in solid feed administration in veal calves for white meat production, can much better satisfy the consumers demand with a product with higher value in terms of welfare and antibiotics use.

References 1. EUROSTAT (2019). https://ec.europa.eu/eurostat/statistics-explained/index.php?title=Agricultural_production_-_livestock_and_meat&oldid=427096#Veal_and_beef 2. Brown R., Claxton R. (2011). Global veal market overview presentation. In: Proceedings of the 5th International Veal Conference. 19-20 May. Noordwijk aan Zee. the Netherlands. 3. Soehnlen M.K., Aydin A., Lengerich E.J., Houser B.A., Fenton G.D., Lysczek H.R., Burns C.M., Byler L.I., Hattel A.L., Wolfgang D.R. (2011). Blinded, controlled field trial of two commercially available Mycoplasma bovis bacterin vaccines in veal calves. Vaccine, 29(33):5347-5354. 4. European Commission, (2008). Council Directive of 18 December 2008 concerning minimum standards for the protection of calves (codified version), 2008/119/EC. In: Official Journal, L 10, 15/01/2009, pp 7-13. 5. Mattiello S., Canali E., Ferrante V., Caniatti M., Gottardo F., Gozzi G., Andrighetto I., Verga M. (2002). The provision of solid feeds to veal calves: II. Behaviour, physiology, and abomasal damage. J Anim Sci, 80:367-375. 6. Webb L.E., Bokkers E.A.M., Heutinck L.F.M., Engel B., Buist W.G., Rodenburg T.B., Stockhofe-Zurwieden N., van Reenen C.G. (2013). The effect of roughage type, amount and particle size on veal calf behaviour and gastrointestinal health. J Dairy Sci, 96:7765-7776. 7. Leruste H., Brscic M., Cozzi G., Kemp B., Wolthuis-Fillerup M., Lensink B.J., Bokkers E.A.M., van Reenen C.G. (2014). Prevalence and potential influencing factors of non-nutritive oral behaviours of veal calves on commercial farms. J Dairy Sci, 97:7021-7030. 8. Morisse J.P., Huonnic D., Cotte J.P., Martrenchar A. (2000). The effect of four fibrous feed supplementations on different welfare traits in veal calves. Anim Feed Sci Technol, 84:129-136. 9. Bus J.D., Stockhofe N., Webb L.E. (2019). Invited review: abomasal damage in veal calves. J Dairy Sci, 102(2):943-960. 10. Webb L.E., Bokkers E.A.M., Engel B., Gerrits W.J.J., Berends H., Van Reenen C.G. (2012). Behaviour and welfare of veal calves fed different amounts of solid feed supplemented to a milk replacer ration adjusted for similar growth. Appl Anim Behav Sci, 136:108-116. 11. Webb L.E., Jensen M.B., Engel B., Van Reenen C.G., Gerrits W.J.J., De Boer I.J.M., Bokkers E.A.M. (2014). Chopped or long roughage: What do calves prefer? Using cross point analysis of double demand functions. PLoS One, 9:1. 12. Berends H., van Reenen C.G., Stockhofe-Zurwieden N., Gerrits W.J.J. (2012a). Effects of early rumen development and solid feed composition on growth performance and abomasal health in veal calves. J Dairy Sci, 95:3190-3199. 13. Brscic M., Prevedello P., Stefani A. L., Cozzi G., Gottardo F. (2014). Effects of the provision of solid feeds enriched with protein or nonprotein nitrogen on veal calf growth, welfare, and slaughter performance. J Dairy Sci, 97:4649-4657. 14. Mollenhorst H., Berentsen P.B.M., Berends H., Gerrits W.J.J., de Boer I.J.M. (2015). Economic and environmental effects of providing increased amounts of solid feed to veal calves. J Dairy Sci, 99:2180-2189. 15. Pardon B., Catry B., Dewulf J., Persoons D., Hostens M., De Bleecker K., Deprez P. (2012). Prospective study on quantitative and qualitative antimicrobial and anti-inflammatory drug use in white veal calves. J Antimicrob Chemother, 67:1027-1038. 16. Jarrige N., Cazeau G., Morignat E., Chanteperdrix M., Gay E. (2017). Quantitative and qualitative analysis of antimicrobial usage in white veal calves in France. Prev Vet Med, 144. 158-166. 17. Gottardo F., Cozzi G., Segato S., Fregolent G., Berzaghi P., Andrighetto

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Effect of the administration of different levels of solid feed on production performance, welfare... I. (2000). Effect of the type of solid feed on the production performance in vivo and on the characteristics of the carcass of white meat calves. Atti della SocietĂ Italiana delle Scienze Veterinarie (Italy), 54:483-484. Berends H., van den Borne J.J.G.C., Stockhofe-Zurwieden N., Gilbert M.S., Zandstra T., Pellikaan W.F., van Reenen C.G., Bokkers E.A.M., Gerrits W.J.J. (2015). Effects of solid feed level and roughage-to-concentrate ratio on ruminal drinking and passage kinetics of milk replacer, concentrates and roughage in veal calves. J Dairy Sci, 98:5621-9. Baldwin R.L., McLeod K.R., Klotz J.L., Heitmann R. N. (2004). Rumen development, intestinal growth and hepatic metabolism in the pre- and postweaning ruminant. J Dairy Sci, 87:E55-E65. Berends H., van den Borne J.J.G.C., Alferink S.J.J., van Reenen C.G., Bokkers E.A.M., Gerrits W.J.J. (2012b). Low-protein solid feed improves the utilization of milk replacer for protein gain in veal calves. J Dairy Sci, 95:6654-6664. Cozzi G., Gottardo F., Mattiello S., Canali E., Scanziani E., Verga M., Andrighetto I. (2002). The provision of solid feeds to veal calves: I.

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Growth performance, forestomach development, and carcass and meat quality. J Anim Sci, 80:357-366. Morisse J.P., Cotte J.P., Huonnic D., Martrenchar A. (1999). Influence of dry feed supplements on different parameters of welfare in veal calves. Anim Welf, 8:43-52. Prevedello P., Moro L., Brscic M., Gottardo F., Stefani A. (2009). Trend overtime of total haemoglobin. iron metabolism and trace minerals in veal calves fed high amounts of two different solid feeds. Ital J Anim Sci 8:184-186. Stull C.L., McDonough S.P. (1994). Multidisciplinary approach to evaluating welfare of veal calves in commercial facilities. J Anim Sci, 72(9):2518-2524. Xiccato G., A. Trocinoa, P.I. Queaquea, A. Sartoria, A. Carazzolo (2002). Rearing veal calves with respect to animal welfare: effects of group housing and solid feed supplementation on growth performance and meat quality. Livestock Production Science 75:269-280. Ngapo M.T. GariĂŠpy C. (2006). Factors affecting the meat quality of veal. J Sci Food Agric 86:1412-1431.

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Effects of pain and nonsteroid anti-inflammatory drugs (NSAIDS) after abomasal displacement operations of cattle

213

HANIFI EROL1*, MUHARREM EROL2, CELAL IZCI3 1 2 3

Department of Surgery, Faculty of Veterinary Medicine, Erciyes University, Kayseri-Turkey Department of Surgery, Faculty of Veterinary Medicine, Balıkesir University, Balıkesir-Turkey Department of Surgery, Faculty of Veterinary Medicine, Selçuk University, Konya-Turkey

SUMMARY Displacement of abomasum (DA) is one of the most common non-infectious disease in dairy cattle and multifactorial condition characterized by gas and fluid building up in the abomasum. A number of medical and mechanical technique are used for treatment of DA, but the most effective method is surgery. The aim of this study was to evaluate post-operative pain caused by DA operations and to compare the effects of ketoprofen, flunixin meglumine and acetaminophen. In total, 24 Holstein dairy cattle (3-8 years old; 5 ± 1.6 years) were used as subjects for this study. All animals were subjected to similar environments and feeding conditions. The cows were examined for postpartum diseases such as mastitis, retention secundinarium, hoof problems and ketosis and the animals diagnosed of these diseases were excluded. Right flank laparotomy was carried out on all subjects. In the pre and post-operavite periods, adrenaline, noradrenaline, cortisol, body temperature (BT), heart rate (HR), respiratory rate (RR) and rumen contraction (RC) were recorded. The statistical differences were evaluated for laboratory and clinical parameters. The significant differences were found in plasma adrenaline, noradrenaline and serum cortisol levels between groups’ evaluations. Generally, the measured BT, HR, RR and RC values were higher in control group than study groups. In abdominal surgeries very little research has focused on the differences in type, magnitude and time course between somatic and visceral pain. In cattle the surgical treatment methods of DA include left flank, right flank and right paramedian approaches. Pain from these procedures can be similar to laparotomies, however the procedures require the manipulation and suturing of the abomasum or periabomasal tissue, which may cause additional pain. In this study, flunixin meglumine was found to be more effective than ketoprofen in postoperative pain control. As a result of this study, it was concluded that it is necessary to provide effective postoperative analgesia after DA operations in cattle for the animals’ welfare and treatment of anormal physiological function.

KEY WORDS Abomasum, cattle, displacement, pain.

INTRODUCTION Displacement of the abomasum (DA) is one of the most common non-infectious disease in dairy cattle. DA is a multifactorial disease characterized by gas and fluid building up in the abomasum, and according to which side the abomasum moves, can occur as left displacement of the abomasum (LDA) or right displacement of the abomasum (RDA). DA has also been associated with other diseases such as retained placenta, metritis and ketosis1-3. A number of medical and mechanical technique are used for treatment of DA, but the most effective method is surgery. Pain is sensory process that results from tissue damage and has two components (sensory and emotional). A stimulus which causes deterioration of tissue integrity is defined as a noxious stimulus. Therefore, any surgical procedure in the living organism is a noxious stimulus4,5. In general, abdominal surgery on cattle is performed with local anesthesia. The short lasting effects of local anesthetic agents require the animals to be given drugs for postoperative pain control. Therefore, in order to decrease postoperative pain, drugs should be selected very carefully in the postoperative period.

Corresponding Author: Hanifi Erol (drhaneroll@yahoo.com)

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in veterinary practice for postoperative pain control 6,7. The analgesic effects of NSAIDs are dependent on the inhibition of the cyclooxygenase (COX) enzyme, which catalyzes the formation of prostaglandins and prostanoids (thromboxane and prostacyclin) from arachidonic acid. The COX enzyme has two isoforms: (COX-1 and COX-2). COX-1 regulates the renal blood flow and gastric mucus production and COX-2 allows the prostaglandins and prostanoids in damaged and inflamed tissues to occur 8-10. Ketoprofen is a COX-1 inhibitor and has analgesic, antipyretic and anti-inflammatory effects. Flunixin meglumine is widely used in viseral and colic pains, inhibits both COX-1 and COX2, but is more selective on COX-19,11,12. Acetaminophen inhibits the COX-1 and COX-2 enzymes but the anti-inflammatory effect of it is poor. Given the low incidence of gastrointestinal side effects with acetaminophen, it is recommended to use this drug together with other NSAIDs to increase their analgesic effects 13,14 . Although many aspects of DA have been investigated, the pain and effects of NSAIDs have not been sufficiently investigated in the postoperative period. Studies carried out on pain and NSAIDs in cattle generally focus on lameness, liver biopsy and dehorning12,15-17 . In the present study, various NSAIDs i.e. ketoprofen, flunixin meglumine and acetaminophen were ad-

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Effects of pain and nonsteroid anti-inflammatory drugs (NSAIDS) after abomasal displacement operations of cattle

ids were administered intravenously (I.V.) for 2 days when necessary. Feeding was initiated 24 h after surgery. NSAIDs were not administered to animals in the CG. In the GI, after the extraction of gas and fluids, 50 mg/kg of diluted paracetamol was injected into the abomasum during the operation. In GII, after the extraction of gas and fluids, 50 mg/kg diluted paracetamol was injected into the abomasum during the operation, and 2.2 mg/kg of flunixin meglumine (Filumed, Alke, Turkey) was administered IM for three days after the operation. In GIII, after the extraction of gas and fluids 50 mg/kg diluted paracetamol was injected into the abomasum during the operation, and 3 mg/kg of ketoprofen (Ketobay, Bayer, Turkey) was administered IM for three days after the operation. Before local anesthesia, a 20-gauge intravenous catheter was placed in the right jugular vein for blood sample collection. Blood samples of total volume of 11 mL were collected into heparinized (3 ml) and clot activator tubes (8 ml) at preoperative (PO), end of operation (EO), post-operative (POS) 1, 2, 4, 6, 24, 48 and 72 h periods. Body temperature (BT), heart rate (HR), respiratory rate (RR) and rumen contraction (RC) were recorded at the same times. The plasma and serum samples were centrifuged (Nüve, NF 200, Turkey) at 2349 g for 10 min at room temperature and stored at -18 oC until biochemical analysis. Adrenaline and noradrenaline were measured from plasma samples using a high performance liquid chromatography method (Shimadzu, Prominence Modular LC20A HPLC, Japan), and cortisol was measured from serum samples using chemoluminescence method (ILAB 300 Plus, Italy).

ministred after DA operation, to control the postoperative pain and stress caused by DA operations and their effects were compared.

MATERIALS AND METHODS Animals The study was approved by the Local Ethics Committee of Selçuk University (Decision no: 2009/024). In total, 24 Holstein dairy cattle (3-8 years old; 5 ± 1.6 years) were included, consisting of 7 undergoing surgery for RDA and 17 for LDA. All animals were subjected to similar environments and feeding conditions. The cows were examined for postpartum diseases such as mastitis, retention secundinarium, hoof problems and ketosis and the animals diagnosed of these diseases were excluded. Routine physical examination and laboratory and specific DA tests (peritoneal and abomasal fluids analysis) were carried out before surgery carried out.

Experimental desing Animals were randomly divided into four groups, with each group containing six cattle. Group I (GI) consisted of one animal with RDA and five animals with LDA, group II (GII) consisted of six animals with LDA, group III (G III) consisted of two with RDA and four with LDA and the control group (CG) consisted of four with RDA and two with LDA. After the preparation of the surgical field, 20 ml local anesthetic, 2% lidocaine (Adokain, Sanovel, Turkey) was infiltrated into the right paralumbar fossa of the animals and a right flank laparotomy was performed on all subjects. During the operations conducted on the animals with LDA, gas and fluid contents were extracted from the abomasum, and omentopexy was performed on the right incision line with horizontal mattress sutures on the abdominal wall. After omentopexy, the abdominal cavity was closed routinely. During the operations conducted on the animals with RDA, the abomasal contents were aspirated and toggle pins were inserted into the abomasum and paramedian abomasopexy was performed 5 cm to the right of the ventral line. After the paramedian abomasopexy, the abdominal cavity was closed routinely. Intramuscularly (I.M.) penicillin-streptomycin (5 ml/100 kg, RedipenS, Ceva, Turkey) was administered for one week after surgery. All animals were housed in warm stalls with dry bedding. For rehydration and the correction of electrolyte imbalances, flu-

Statistical analysis All analysis were performed with the Minitab Release 12.1 package program. A Wilcoxon’s rank test was used to assess the significances of difference in intra-group evaluations, and the Kruskal-Wallis followed by Mann-Whitney U tests were performed for camparison between groups. Values of P < 0.05 were considered statistically significant. Results were presented as the mean ± standard deviation (SD).

RESULTS The plasma adrenaline values are shown in Table 1. In general, the adrenaline levels were increased at EO and POS 1 h. The highest values were seen in the CG at POS 1h. In intra-group comparison of adrenaline in CG, there were significant differ-

Table 1 - Plasma adrenaline concentration (Mean ± SD) (pg/ml) (n=6). Time

GI ¥

7.90 ± 1.87

9.90 ± 10.31* *

G II *

G III

11.10 ± 3.39

5.50 ± 2.23

10.80 ± 2.60*

12.25 ± 3.24*

6.50 ± 1.02¥

14.75 ± 3.69* a

14.80 ± 6.74¥ a

POS 1 h

18.65 ± 20.89 a

14.95 ± 5.78 a

7.80 ± 4.62 a

POS 2 h

12.35 ± 4.95¥ b

13.10 ± 3.79* a

5.20 ± 1.82*

10.65 ± 3.46

POS 4 h

6.75 ± 1.68

12.85 ± 4.34

6.45 ± 0.87¥

10.90 ± 2.61*

POS 6 h

6.50 ± 8.06¥

13.90 ± 4.02*

4.60 ± 1.74**

14.00 ± 4.40* b

POS 24 h

6.75 ± 10.93

12.80 ± 7.45

6.40 ± 2.41

14.25 ± 2.67 a

POS 48 h

8.40 ± 15.08

11.75 ± 3.48

7.75 ± 3.12

12.35 ± 4.80

POS 72 h

8.95 ± 3.91

12.15 ± 4.41

6.30 ± 2.21

PO; preoperative, EO; end of operation, POS; post-operative, a, b; in same column is the significant difference in groups (P < 0.05), cant difference between groups (P < 0.05).

12.80 ± 5.02 *,¥,**

; in same line is the signifi-

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Table 2 - Plasma noradrenaline level (Mean ± SD) (pg/ml) (n=6). Time

54.6 ± 27.2

50.1 ± 67.3 *

G II

G III

35.45 ± 18.36

42.2 ± 32.9

45.75 ± 20.6

30.70 ± 23.96

30.6 ± 26.2 a

47.70 ± 19.3

POS 1 h

80.3 ± 10.43 a

52.00 ± 28.2 b

30.2 ± 28.8 a

54.90 ± 44.2¥ a

POS 2 h

73.2 ± 32.0* b

40.40 ± 19.17¥

39.9 ± 15.73¥

42.60 ± 29.9¥

POS 4 h

60.0 ± 18.22 c

47.95 ± 10.50 bc

35.6 ± 14.72

25.70 ± 15.3

POS 6 h

61.6 ± 95.2 c

48.70 ± 13.96 bc

35 ± 16.20

34.40 ± 18.1

POS 24 h

58.3 ± 88.8

61.70 ± 238.0 a

32.8 ± 16.41

45.15 ± 20.9

POS 48 h

64.4 ± 96.6 c

46.85 ± 15.66 bc

33.5 ± 12.46

24.50 ± 45.4

POS 72 h

68.4 ± 71.6 c

43.30 ± 13.94 c

26.5 ± 4.92 a

59.00 ± 25.5 a

PO; preoperative, EO; end of operation, POS; post-operative, a, b, c; in same column is the significant difference in groups (P < 0.05), *,¥,**, ; in same line is the significant difference between groups (P < 0.05).

Table 3 - Serum cortisol level (Mean ± SD) (nmol/ml) (n=6). Time

G II

G III

1.21 ± 2.51

1.42 ± 1.91

0.36 ± 0.40

1.50 ± 2.22

5.90 ± 4.68* b

5.52 ± 2.46* a

1.07 ± 8.59**

3.48 ± 6.24¥ a

POS 1 h

9.58 ± 3.34* a

5.43 ± 4.34** a

1.80 ± 7.40 a

3.60 ± 5.28¥ a

POS 2 h

4.60 ± 3.25 bc

3.33 ± 4.76 b

0.29 ± 4.29

1.38 ± 2.09¥

POS 4 h

3.36 ± 2.74* c

3.38 ± 3.08* b

0.48 ± 2.36

0.15 ± 4.13

POS 6 h

2.73 ± 2.62 d

1.58 ± 1.18

0.48 ± 1.21

0.66 ± 1.32

POS 24 h

0.79 ± 2.89

1.83 ± 1.63

0.34 ± 1.24

0.46 ± 0.38

POS 48 h

1.01 ± 2.52

0.94 ± 0.99

0.30 ± 2.47

0.91 ± 0.98

POS 72 h

0.80 ± 0.60

1.07 ± 0.41

0.27 ± 0.76

0.15 ± 1.54

PO; preoperative, EO; end of operation, POS; post-operative, a, b, c, d; in same column is the significant difference in groups (P < 0.05), *,¥,**, ; in same line is the significant difference between groups (P < 0.05).

ences detected between POS 1 and POS 2 h, and between POS 1h, 2 h and the other time points. In GI at POS 1h and 2 h, in GII at POS 1h and 2 h, in GIII at EO and POS 1, 6, 24 h values of adrenaline were different compared to other times. When comparing the groups to each other, signficantly higher values of adrenaline were found in GI and GIII than in the other groups at PO, EO, POS 1, 2, and 6 h. Plasma noradrenaline values are presented in Table 2. In the CG, POS values were higher than those of PO and EO. Significant differences were recorded between POS 1h, 2 h and POS 4, 6, 48 and 72 h. In GI, significant differences were seen at POS 1, 24 and 72 h and between POS 4, 6 and 24 h. In GII, the plasma noradrenaline values of EO, POS 1 and 72 h were significantly lower than other times. In GIII, POS 1 and 72 h values were significantly higher than at the other time points. Between groups comparison of noradrenaline statistical differences were recorded at POS 1, 2 and 4 h in all groups. Serum cortisol concentrations were significantly increased at EO and POS 1 h in all groups (Table 3). The highest value was seen at POS 1 h in the CG. Compared to the other time points, significant differences were recorded at EO, POS 1, 4 and 6 h in the CG. Significant differences of the cortisol concentration were found at EO, POS 1 h and POS 2, 4 h in GI, at POS 2 h in GII and at POS 1, 2 h in GIII. After POS 6 h the serum cortisol values decreased in all groups. In the comparison of serum cortisol levels between the groups, significant differences were observed at EO, POS 1, 2 and 4 h. At these times the serum cortisol levels of the CG and GI were higher than GII and GIII. The HR of all groups are shown in Table 4. The EO, POS 1, 2

and 4 h values were significantly higher than the other times in the CG. In GI and GII, significant increases of the HR were observed at EO. In GIII, the measured values of HR at EO and POS 1 h were higher and significantly different than other time points. Between groups comparasion of HR, significant differences were detected at EO, POS 1, 2 h time points in CG, GI and GII, GIII, with CG and GI values higher than GII and GIII. The RR values of the groups are shown in Table 5. The RR values of EO, POS 1, 2 and 4 h were significantly higher in the CG. There were no significant differences recorded in GI. In GII and GIII the values of EO were higher and different than other time points. There were no sitatistical differences recorded for BT and RC values in intra group and between group evaluations (Table 6, 7).

DISCUSSION The stress hormones such as adrenocorticotropic hormone (ACTH), cortisol, adrenaline and noradrenaline are secreted from the pituitary gland, adrenal cortex, adrenal medulla and sympathetic nerve endings18,19. These hormones increase the adaptation power of living organisms to various stress factors. Pain is a stress in the post-operative period. An increase of stress hormone levels and metabolic changes are seen in this period18-20. The present study is the first to investigate the pain and the effects of NSAIDs’ on the postoperative period after DA operations. In this study the changes in the stress hormone levels and the effects of

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Effects of pain and nonsteroid anti-inflammatory drugs (NSAIDS) after abomasal displacement operations of cattle

Table 4 - Heart rate (HR/min) (Mean ± SD) (n=6). Time

G II

G III

66.0 ± 6.05

77.0 ± 23.69

65.5 ± 12.04

64.5 ± 11.03

77.5 ± 8.82 a*

82.0 ± 22.72 a*

69.0 ± 5.15 a

67.0 ± 6.09 a

POS 1 h

75.5 ± 10.84 a*

75.0 ± 14.63*

65.5 ± 4.94

67.5 ± 5.15 a

POS 2 h

73.5 ± 9.54 a*

77.0 ± 16.75*

66.5 ± 2.73

65.0 ± 3.71

POS 4 h

69.0 ± 13.64

71.0 ± 17.55

66.5 ± 13.86

60.0 ± 12.50

POS 6 h

68.0 ± 10.79

73.0 ± 11.29

69.0 ± 5.60

58.0 ± 4.90

POS 24 h

66.0 ± 9.11

77.0 ± 11.66

54.0 ± 4.28

54.5 ± 4.62

POS 48 h

58.5 ± 7.63

68.0 ± 10.27

57.0 ± 9.09

53.0 ± 8.85

POS 72 h

62.5 ± 6.43

61.5 ± 9.30

60.0 ± 10.45

55.0 ± 9.25

PO; preoperative, EO; end of operation, POS; post-operative, a; in same column is the significant difference in groups (P < 0.05), *; in same line is the significant difference between groups (P < 0.05).

Table 5 - Respiratory rate (RR/min) measurement (Mean ± SD) (n=6). Time PO

G II

G III

27.5 ± 6.26

33.0 ± 16.08

31.5 ± 7.00

25.0 ± 6.34

41.0 ± 4.92 a *

36.0 ± 17.59

36.0 ± 5.51 a

34.0 ± 7.66 a

POS 1 h

40.0 ± 6.83 a *

35.5 ± 14.92

34.0 ± 4.73

31.0 ± 7.53

POS 2 h

37.5 ± 10.46 a

35.5 ± 16.99

32.0 ± 4.27

28.5 ± 5.06

POS 4 h

37.0 ± 7.69 a

32.0 ± 15.92

33.0 ± 4.86

27.0 ± 3.83

POS 6 h

33.0 ± 5.43

33.0 ± 10.25

31.0 ± 9.83

30.5 ± 5.62

POS 24 h

28.0 ± 4.88

29.0 ± 7.48

31.0 ± 6.11

29.0 ± 6.25

POS 48 h

28.0 ± 6.31

26.5 ± 6.40

31.0 ± 7.57

25.5 ± 7.29

POS 72 h

26.0 ± 3.95

25.0 ± 6.94

32.0 ± 5.24

24.0 ± 6.38

Table 6 - Body temperature (BT, oC) measurement (Mean ± SD) (n=6). Time PO

G II

G III

38.45 ± 0.40

38.35 ± 0.70

38.60 ± 0.81

38.15 ± 0.73

38.50 ± 0.39

38.40 ± 0.87

39.15 ± 0.83

38.60 ± 0.66

POS 1 h

38.57 ± 0.27

38.65 ± 0.95

39.00 ± 0.87

38.55 ± 0.70

POS 2 h

38.45 ± 0.12

38.50 ± 0.46

38.80 ± 0.67

38.30 ± 0.66

POS 4 h

38.42 ± 0.16

38.00 ± 0.85

38.65 ± 0.55

38.20 ± 0.74

POS 6 h

38.17 ± 0.39

38.35 ± 0.82

38.75 ± 1.67

38.10 ± 1.15

POS 24 h

38.20 ± 0.21

38.00 ± 0.56

38.65 ± 0.93

38.35 ± 0.84

POS 48 h

39.22 ± 0.35

39.00 ± 0.31

38.50 ± 0.59

38.70 ± 0.62

POS 72 h

38.20 ± 0.25

38.10 ± 0.89

39.00 ± 0.46

38.30 ± 0.77

PO; preoperative, EO; end of operation, POS; post-operative.

Table 7 - Rumen contraction (RC/5 min) measurement (Mean ± SD) (n=6). Time PO

G II

G III

3.0 ± 0.84

3.5 ± 1.26

3.5 ± 0.81

3.0 ± 0.83

3.0 ± 0.89

3.0 ± 1.17

3.0 ± 1.63

3.0 ± 0.75

POS 1 h

3.5 ± 1.37

3.0 ± 0.98

3.0 ± 1.09

3.0 ± 0.41

POS 2 h

3.5 ± 0.81

3.0 ± 1.18

3.0 ± 1.09

3.0 ± 0.52

POS 4 h

3.5 ± 0.81

3.0 ± 1.23

3.5 ± 0.98

3.0 ± 0.75

POS 6 h

4.0 ± 0.75

3.5 ± 1.21

4.0 ± 0.75

3.0 ± 0.84

POS 24 h

3.5 ± 0.55

3.5 ± 0.81

4.0 ± 1.60

3.0 ± 0.52

POS 48 h

3.0 ± 0.63

3.5 ± 1.37

4.0 ± 1.03

3.0 ± 1.37

POS 72 h

4.0 ± 0.89

4.0 ± 0.75

3.5 ± 0.98

PO; preoperative, EO; end of operation, POS; post-operative.

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H. Erol et al. Large Animal Review 2020; 26: 213-220

NSAIDsâ&#x20AC;&#x2122; on postoperative pain were detected in dairy cattle. NSAIDs create analgesic and anti-inflammatory effects by reducing prostaglandin synthesis through the inhibition of COX-1 and COX-2 enzymes in the peripheral tissue and central nervous system. The measurement of COX proteins after stimulus is the evidence of systemic delivered COX inhibitors. COX-2 inhibitors have rapid activity after surgical intervention. They show their antihyperalgesic actions by not requiring any induction after surgery. This observation argues for a site within the central nervous system wherein this isozyme is constitutively expressed21. The inhibition of COX enzymes due to NSAIDs may have more immediate impact on pain by inhibiting prostaglandin production in the periphery compared to the selective compounds of them22. Dopamine, adrenaline, noradrenaline and cortisol have been shown to be good indicators of stress and pain in animals23,24. Adrenaline and noradrenaline are amino acid-derived hormones that are increased in situations of fear and anxiety19. In the present study, adrenaline and noradrenaline levels increased at EO and POS 1 h in all groups. These increases were more evident in the CG than other groups. Using NSAIDs in GI, GII and GIII reduced the increases of the adrenaline and noradrenaline hormones. These data support the use of NSAIDs in the treatment of postoperative pain. However, after POS 2 h, the adrenaline and noradrenaline levels decreased with the most significant reductions occurring in GII and GIII. The stress hormone (adrenaline, noradrenaline and cortisol) values of GII were lower than GIII. This suggests that flunixin meglumine is more selective and effective on the COX enyzmes than ketoprofen. Lees et al. (1996)25 stated that the half-life of flunixin meglumine in cattle is 23 h. On the other hand, the half-life of ketoprofen was reported as 2-3 h by Grisneaux et al. (2003)26 and Whay et al. (2005)9. At POS 1, 2, 4, 6, 24, 48 and 72 h, the adrenaline and noradrenaline values were higher in GIII than GII. These findings showed that the half-life of flunixin meglumine and ketoprofen may be different but the findings supported the powerful effectiveness of the flunixin meglumine on postoperative pain. In the current study acetaminophen was used in GI without any other NSAID. The adrenaline values of GI were lower than the CG at POS 1 h, but higher at POS 4, 6, 24, 48 and 72 h. However, the measured noradrenaline values did not exhibit the same changes at the same time points. The noradrenaline values were higher in GI than the CG. These findings showed that the effect of acetaminophen on adrenaline and noradrenaline was not the same. The use of acetaminophen with other NSAIDs did not change the effects of them. Cortisol is the main glucocorticoid hormone that is released in response to stress including pain. Cortisol has been measured in animals to estimate the effects of different procedures causing pain such as abdominal surgery27. Mudron et al. (2007)15 operated on cattle with LDA using the omentopexy technique and measured serum cortisol levels before and 15, 30 and 60 min after the operation. They found statistical differences between the cortisol measurements taken before and after the operation. Milligan et al. (2004)11 compared the effects of ketoprofen on the dehorning of calves and measured their cortisol levels. They detected lower cortisol levels in the ketoprofen groups. In the present study, the cortisol levels were found very high at POS 1 h compared to PO, and continued to increase at POS 6 h in the CG. In GI, the cortisol levels between EO and POS 24 h were

219

higher than those at PO, and were statistically different at EO, POS 1, 2, 4 and 6 h. At the same times, the cortisol values were lower in GII and GIII compared to CG and GI. The decreases of cortisol levels were more evident in GII and the statistical difference was recorded at POS 1 h. The comparison of cortisol levels between GII and GIII confirmed that flunixin meglumine was more efficacious in reducing pain and stress parameters. The clinical responses to pain include changes in the cardiovascular and respiratory please zoom the 27 to system27.The assessment of physiological parameters is generally used in conjunction with behavioural assessment methods to assess postsurgical pain, although various studies have reported a poor correlation between physiological data28. Recent studies suggest to measure the HR for the assessment of sympathetic and parasympathetic activation29. GrĂ¸ndahl-Nielsen et al. (1999)30 found that the HR increased during 3.5 h, after hot-iron disbudding was conducted on calves without analgesic. Tachycardia, increased RR and ruminal hypomotility are the clinical symtoms of DA in cattle31,32. In the present study, HR and RR increased at EO and there was no statistical difference detected in RC. In the individual group evaluation significant differences were recorded at EO, POS 1 h, 2 h in the CG, and at EO in GI, GII and GIII. In the comparisons between groups statistical differences were found at the same times (EO, POS 1 and 2 h). HR and RR were high in the CG and GI compared to other groups. It was also found that the measured stress hormones were increased parallel to the increase of HR and RR. The reducing effects of NSAID on pain affects the clinical pain parameters such as HR, RR, RC. This situation was supported by the measurement values of GII and GIII. Abdominal surgeries require the skin and abdominal muscles to be cut in order to access the peritoneal cavity, and manipulation or destruction of internal tissue. However, very little research has focused on the differences in type, magnitude and time course between somatic and visceral pain. In cattle the surgical treatment methods of DA include left flank, right flank and right paramedian approaches. Pain from these procedures can be similar to laparotomies, however the procedures require the manipulation and suturing of the abomasum or periabomasal tissue, which may cause additional pain5. Wittek et al. (2008)33 observed an improvement in RC rate on the day after abomasal correction surgery in animals that received flunixin meglumine compared to the control group. However, there were no statistical differences found in BT and RC in the present study. This was thought to be caused by different surgical techniques and types of DA, as in the present study, cattle with LDA and RDA were operated on. Newby et al. (2013)34 operated on cattle with LDA using the abomasopexy technique and applied ketoprofen during the postoperative period. They emphasized that the administration of ketoprofen following the operation did not appear to have any benefits for pain management. Flunixin meglumine was more effective compared to ketoprofen in postoperative pain control in the present study. In this aspect, this study supported the study of Newby et al. (2013)34 with the difference being that the subjects of the present study were comprised of cattle with RDA as well as LDA in the ketoprofen group (GIII), and with LDA in the flunixin meglumine group (GII). Different operation techniques, animal species, types of DA, perioperative assessment, using preanesthetic agents and intra abomasal acetaminophen can change the values of pain parameters. The results of the present study were consistent with the literature5,35,36.

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Effects of pain and nonsteroid anti-inflammatory drugs (NSAIDS) after abomasal displacement operations of cattle

CONCLUSION In conclussion, flunixin meglumine was found to be more effective in postoperative pain control. A high increase in the pain parameters was seen between EO and POS 1 h period in all groups. Therefore, the timing of administration and the choice of NSAID should be well determined. As a result of this study, it was concluded that it is necessary to provide effective postoperative analgesia after DA operations in cattle for animals’ welfare and treatment of anormal physiological functions.

ACKNOWLEDGEMENT This study was supported by Scientific Research Projects Coordination Unit of Selçuk University. Project number: 09401083.

References 1. Sickinger M., Leiser R., Failing K., Doll K. (2008). Evaluation of differences between breeds for substance P, vasoactive intestinal polypeptide, and neurofilament 200 in the abomasal wall of cattle. Am J Vet Res, 69: 1247-1253. 2. Doll K., Sickinger M., Seeger T. (2009). New aspects in the pathogenesis of abomasal displacement. Vet J, 181: 90-96. doi: 10.1016/j.tvjl. 2008.01.013 3. Durgut R., Sagkan Ozturk A., Ozturk O.H., Guzel M. (2016). Evaluation of oxidative stress, antioxidant status and lipid profile in cattle with displacement of the abomasum. Ankara Univ Vet Fak Derg, 63:137-141. 4. Sorkin L., Wallace M. (1999). Acute pain mechanisms. Surg Clin North Am, 79: 213-229. 5. Walker K.A., Duffield T., Weary D.M. (2011). Identifying and preventing pain during and after surgery in farm animals. Appl Anim Behav Sci, 135: 259-265. doi: 10.1016/j.applanim.2011.10.021 6. Vinuela-Fernandez I., Jones E., Welsh E.M., Fleetwood-Walker S.M. (2007). Pain mechanisms and their implication for the management of pain in farm and companion animals. Vet J, 174:227-239. doi: 10.1016/j.tvjl.2007.02.002 7. Bomzon A. (2011). Pain and stress in cattle: a personal perspective. Isr J Vet Med, 66: 2-20. 8. Deneuche A.J., Dufayet C., Goby L., Fayolle P., Desbois C. (2004). Analgesic comparison of meloxicam or ketoprofen for orthopedic surgery in dogs. Vet Surg, 33: 650-660. doi: 10.1111/j.1532-950X.2004.04088.x 9. Whay H.R., Webster A.J.F., Waterman-Pearson A.E. (2005). Role of ketoprofen in the modulation of hyperalgesia associated with lameness in dairy cattle. Vet Rec ,157: 729-733. doi: 10.1136/vr.157.23.729 10. Martin T.J., Buechler N.L., Eisenach J.C. (2006). Intrathecal administration of a cylcooxygenase-1, but not a cyclooxygenase-2 inhibitor, reverses the effects of laparotomy on exploratory activity in rats. Anesth Analg, 103: 690-695. doi: 10.1213/01.ane.0000226093.46973.39 11. Milligan B.N., Duffield T., Lissemore K. (2004). The utility of ketoprofen for alleviating pain following dehorning in young dairy calves. Can Vet J, 45: 140-143. 12. Stilwell G., Lima M.S., Broom D.M. (2008). Effects of nonsteroidal anti-inflammatory drugs on long-term pain in calves castrated by use of an external clamping techniques following epidural anesthesia. Am J Vet Res, 69: 744-750. doi: 10.2460/ajvr.69.6.744. 13. Edwards J.E., McQuay H.J., Moore R.A. (2002). Combination analgesic efficacy: Individual patient data meta-analysis of single-dose oral tramadol plus acetaminophen in acute postoperative pain. J Pain Symptom Manage, 23:121-130. doi: 10.1016/s0885-3924(01)00404-3 14. Schecter W., Bongard F., Gainor B., Weltz D., Horn J. (2002). Pain control in outpatient surgery. J Am Coll Surg, 195: 95-104. 15. Mudron P., Herzog K., Höltershinken M., Rehage J. (2007). Effects of abdominal surgery on thiobarbituric acid reactive substances and plasma anti-oxidative capacity in dairy cows. Journal of veterinary medicine. J Vet Med A Physiol Pathol Clin Med, 54: 441-444. doi: 10.1111/j.1439-0442.2007.00992.x 16. Thüer S., Mellema S., Doherr M.G., Wechsler B., Nuss K., Steiner A. (2007). Effect of local anaesthesia on short- and long-term pain induced by two bloodless castration methods in calves. Vet J, 173: 333-342. doi: 10.1016/j.tvjl.2005.08.031

17. Laven R.A., Lawrence K.E., Weston J.F., Dowson K.R., Stafford K.J. (2008). Assessment of the duration of the pain response associated with lameness in dairy cows, and the influence of treatment. N Z Vet J, 56: 210-217. doi: 10.1080/00480169.2008.36835 18. Jasmin L., Tien D., Janni G., Ohara P.T. (2003). Is noradrenaline a significant factor in the analgesic effect of antidepressants? Pain, 106:3-8. doi: 10.1016/j.pain.2003.08.010 19. Queyras A., Carosi M. (2004). Non-invasive techniques for analysing hormonal indicators of stress. Ann Ist Super Sanita, 40: 211-221. 20. Sahinduran S., Albay M.K. (2006). Haematological and biochemical profiles in right displacement of abomasum in cattle. Rev Med Vet, 157: 352-356. 21. Svensson C.I., Yaksh T.L. (2002). The spinal phospholipase-cyclooxygenase-prostanoid cascade in nociceptive processing. Annu Rev Pharmacol Toxicol, 42: 553-583. doi: 10.1146/annurev.pharmtox.42.092401. 143905 22. Ochroch E.A., Mardini I.A., Gottschalk A. (2003). What is the role of NSAIDs in pre-emptive analgesia? Drugs, 63: 2709-2723. doi: 10.2165/00003495-200363240-00002 23. Almeida T.F., Fantoni D.T., Mastrocinque S., Tatarunas A.C., Imagawa V.H. (2007). Epidural anesthesia with bupivacaine, bupivacaine and fentanyl, or bupivacaine and sufentanil during intravenous administration of propofol for ovariohysterectomy in dogs. J Am Vet Med Assoc, 230: 45-50. doi: 10.2460/javma.230.1.45 24. Ndlovu T., Chimonyo M., Okoh A.I., Muchenje V. (2008). A comparison of stress hormone concentrations at slaughter in Nguni, Bonsmara and Angus steers. African J Agric Res, 3: 96-100. 25. Lees P., Delatour P., Foster A., Foot R., Baggot D. (1996). Evaluation of carprofen in calves using a tissue cage model of inflammation. Brit Vet J, 152:199-211. 26. Grisneaux E., Dupuis J., Pibarot P., Bonneau N.H., Charette B., Blais D. (2003). Effects of postoperative administration of ketoprofen or carprofen on short- and long-term results of femoral head and neck excision in dogs. J Am Vet Med Assoc, 223:1006-1012. doi: 10.2460/javma.2003.223.1006 27. Landa L. (2012). Pain in domestic animals and how to assess it: a review. Vet Med, 57: 185-192. 28. Price J., Catriona S., Welsh E.M., Waran N.K. (2003). Preliminary evaluation of a behaviour-based system for assessment of postoperative pain in horses following arthroscopic surgery. Veterinary Anaesthesia and Analgesia, 30: 124-137. doi: 10.1046/j.1467-2995.2003.00139.x 29. Von Borell E., Langbein J., Despres G., Hansen S., Leterrier C.L., Marchant Forde J.N., Marchant Forde N., Minero M., Mohr E., Prunier A., Valance D., Veisser I. (2007). Heart rate variability as a measure of autonomic regulation of cardiac activity for assessing stress and welfare in farm animals-A review. A review PhysiolBehav, 92: 293-316. doi: 10.1016/j.physbeh.2007.01.007 30. Grøndahl-Nielsen C., Simonsen H.B., Lund J.D., Hesselholt M. (1999). Behavioural, endocrine and cardiac responses in young calves undergoing dehorning without and with use of sedation and analgesia. Vet J, 158: 14-20. doi: 10.1053/tvjl.1998.0284 31. Goetze L., Müller M. (1990). The therapy of hypovolemic shock in cows with right-sided abomasal displacement. Zentralbl Vet A, 37: 300-309. 32. El-Attar H.M., Yassein M., Abd El-Raof Ghanem M.M. (2007). Alterations in the clinical, hematological and biochemical pictures in abomasal displacement in cows in Egypt. Vet Med J, 102-109. 33. Wittek T., Tischer K., Fürll M., Constable P.D. (2008). Effect of preoperative administration of erythromycin or flunixin meglumine on postoperative abomasal emptying rate in dairy cows undergoing surgical correction of left displacement of the abomasum. J Am Vet Med Assoc 232: 418-423. doi: 10.2460/javma.232.3.418. 34. Newby N.C., Pearl D.L., LeBlanc S.J., Leslie K.E., von Keyserlingk M.A.G., Duffield T.F. (2013). The effect of administering ketoprofen on the physiology and behavior of dairy cows following surgery to correct a left displaced abomasum. J Dairy Sci, 96: 1511-1520. doi: 10.3168/ jds.2012-5566 35. Coetzee J.F., Gehring R., Bettenhausen A.C., Lubber B.V., Toerber S.E., Thomson D.U., Kulkanich B., Apley M.D. (2007). Attenuation of acute plasma cortisol response in calves following intravenous sodium salicylate administration prior to castration. J Vet Pharmacol Ther, 30: 305313. doi: 10.1111/j.1365-2885.2007.00869.x 36. Coetzee J.F., Lubbers B.V., Toerber S.E., Gehring R., Thomson D.U., White B.J., Apley M.D. (2008). Plasma concentration of substance P and cortisol in beef calves after castration or simulated castration. Am J Vet Res, 69: 751-762. doi: 10.2460/ajvr.69.6.751

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Oxidative status along different stages of pregnancy in dairy cows

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R.L. SCIORSCI, M.MUTINATI, M. PICCINNO, E.LILLO, A. RIZZO Department of Veterinary Medicine, University of Bari “Aldo Moro”, Strada p.le per Casamassima, km 3, 70010 Valenzano (Bari), Italy

SUMMARY Introduction: The oxidant-antioxidant balance play a crucial role in the development of a stable pregnancy in dairy cows. The oxidant system is represented by the reactive oxygen species (ROS), which are indispensable molecules in different processes, like embryo implantation, uterine and cervical modification during the last phase of gestation. On the other hand, it’s fundamental to counterbalance oxidative stress, mostly in periods in which their levels can reach particularly high concentrations such as during early and late pregnancy. This task is prerogative of the antioxidant system, a complex of vitamins, enzymes and oligoelements, capable of transforming oxygen radicals into non-radical compounds. Between them, β-carotene stands out mainly for its antioxidant skills, but also for the important role played during pregnancy. Aim: This study aims to evaluate serum β-carotene and Reactive Oxygen Species concentrations along different stages of pregnancy (from 30-60 days to 240-270 days), in Friesian dairy cows. Materials and methods: 80 healthy, pregnant Friesian cows were enrolled in this study and, based on gestational age, the cows were divided into 8 groups of 10 animals each. Blood was sampled and the obtained serum was used to dose β-carotene and ROS, by means of photometric analytical systems. Results and discussion: Results show that blood β-carotene concentrations undergo a progressive, slow reduction from 30-60 days of pregnancy, until 180-210 days, when they significantly decreased, whereas ROS levels increase, especially at 180-210 days. Both the reduction of β-carotene and the rise of ROS can be dangerous during pregnancy. Indeed, the potential risk of oxidative stress in the periparturient cow and in its embryo has been described. Conclusion: Therefore, the results of this study support the growing evidence that an adequate β-carotene integration in the diet of the parturient cow should be recommended in order to curb oxidative stress both in the mother and the fetus.

KEY WORDS β-carotene; oxidative stress; pregnancy; dairy cow.

INTRODUCTION Pregnancy is characterized by high energy requirements and an increase in oxygen consumption by both placenta and fetus1. It is precisely in such period that an increase in Reactive Oxygen Species (ROS) generation occurs due to both maternal and fetal metabolism2. Physiological ROS levels play a crucial role during pregnancy, modulating uterine function, luteal development, embryogenesis, embryonic attachment to the endometrium and both fetal and placental development. On the other hand, high ROS levels may represent a potential risk factor for both maternal and fetal health2,3 . In order to reduce ROS-related damage, living organisms have developed a complex mechanism of antioxidant defense consisting of enzymes (superoxide-dysmutase, catalase, glutathione-peroxidase), vitamins (A and E) and some oligoelements (Copper, Zinc, Selenium, Manganese)4.

Corresponding Author: Raffaele Luigi Sciorsci (raffaeleluigi.sciorsci@uniba.it).

Retinol (Vit A) has in particular been defined as “scavenger”, i.e. a molecule endowed with the capability of reacting with free radicals so as to give rise to chemically stable byproducts5. Domestic herbivores do not take Vit A as such at pasture or with feeding, since this vitamin is present in plants only in its precursor form, carotenoids. β-carotene is, among carotenoids, the one which most transforms into Vit A6, via activation of some enzymes located in the gut mucosa7. Vit A is essential for many biological functions to occur, such as cell growth and differentiation, epithelial protection, immune competence, eye function, besides playing a protective role against free radicals, oxidizing them. It has in vitro been demonstrated that Vit A enhances cell-to-cell communication, stimulating the synthesis of connexin, and therefore of gap-junctions8, which are important, in the uterus, for labor induction. Deficits in Vit A and/or β-carotene levels around parturition have been associated with a reduction in reproductive performances and with an increase in uterine inertia, calving paresis, mastitis, placental retention, pathological mammary edema and late restoration of full cyclicity after calving9,10. Bovine dietary ration should include a daily β-carotene intake of 0.18 mg/kg B.W. (76 UI/kg B.W. of Vit A)11. β-carotene lev-

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Oxidative status along different stages of pregnancy in dairy cows

els ranging between 200 and 300 µg/100 ml should be considered normal; sub-normal levels should range between 100 and 200 µg/100 ml, whereas levels ranging between 9 and 100 µg/100ml indicate a deficit in β-carotene. As to Vit A, physiological values range between 25 and 80 µg/100 ml, sub-normal values range between 7 and 12 µg/100 ml and values lower than 7 µg/100 ml indicate a deficit in Vit A content12. Oxidative stress occurs when ROS generation is not adequately counterbalanced by antioxidant activity13,14 and it is among the main factors inducing pregnancy alterations15-17. Given these premises, this study aims at evaluating blood βcarotene and ROS concentrations during pregnancy in dairy cows.

MATERIAL AND METHODS Declarations Ethical Guidelines Committee This study was performed in accordance with the ethical guidelines of the animal welfare committee and with the statute of the ‘Aldo Moro’ University of Bari, concerning the use of clientowned animals under clinical investigation. Procedures with animals were performed following good veterinary practice for animal welfare according to national laws in force (EU Directive 2010/63/EU; Italian Legislative Decree 116/92). Informed owner consent was obtained.

Soon after blood sampling, centrifugation was performed at 3000xg for 10 minutes at room temperature (Centrifuge, XC2000) and serum was stored in eppendorf (1.5 mL) until analytical determination. β-carotene was dosed with ICheckTM Carotene Photometer (BioAnalyt, GmbH, Germany). Specifically, 0.4 mL of serum were taken with a graduate syringe and were put into a vial containing a patented solvent mixture (iExTM Carotene vial extraction). Soon after, the vial with the sample was vigorously shaken for 10 seconds and then placed on a horizontal plane to allow the solution to sediment for 5 minutes until phase separation. Afterwards, the vial was placed in a photometer. The results are expressed in mg/L and enable the β-carotene content to be defined as: insufficient (<1.5 mg/L, equivalent to 150 µg/dL), marginal (1.5-3.5 mg/L, equivalent to 150-350 µg/dL) or optimal (>3.5 mg/L, equivalent to 350 µg/dL), with a linear range of 0.15-25.0 mg/L. ROS serum concentrations were obtained by means of a photometric analytical system (FREE®, Diacron, Parma, Italy). FREE® measures reactive oxygen metabolites (ROMs), a variety of free radicals characterized by an odd number of electrons around the external orbital of oxygen. ROMs react with a chromogen which, if correctly buffered, forms a colored compound that can be measured photometrically (maximum absorbance peak at 505 nm). Once the absorbance value is determined, the instrument automatically converts the data into the appropriate arbitrary Carr Unit (1 U.Carr. = 0.08 mg H2O2/100 mL).

Procedure

Statistical analysis

80 healthy, pregnant Friesian cows were enrolled in this study. The cows were between their first and third lactation and had a BCS ranging between 3 and 3.5. The cows were kept in a dairy farm in the South of Italy, with a consistency of 530 animals and a mean milk production of 30 kg/day. The farm has installed a cooling system combining low pressure misting and forced ventilation, along the feeding line. The cows were under a semiintensive breeding system and were fed pasture and unifeed (13 kg of concentrate, 6 kg of graminaceous forage and 10 liters of water/cow/day). Dried cows were fed pasture, 1 kg of supplementary feed for dried cows, dry forage and water ad libitum. All cows received basal diet, with vitamins and minerals supplements, formulated to meet their requirements in different phase of lactation. A complete clinical exam (rectal palpation and ultrasonography, SonoSite MicroMaxx Bothell WA, USA with linear probe set at 7.5 MHz), was used to check the gestational age, in accordance with Hughes and Davies18. Based on gestational age (expressed in days), all the cows enrolled in this study were divided into 8 groups of 10 animals each: • 30-60 • 60-90 • 90-120 • 120-150 • 150-180 • 180-210 • 210-240 • 240-270 A venous blood sample taken from the coccygeal vein into vacutainer serum refrigerated tubes was used to dose β-carotene and ROS. Blood samples were taken at the same time in the morning from cattle before feeding.

All data obtained underwent statistical analysis using the program IBM SPSS Statistics 19, through one way ANOVA and post hoc least significant difference test. Statistical significance was set at p< 0.05.

RESULTS Serum concentrations (Media± s.d.) of β-carotene and ROS found in the pregnant cows at different gestational ages are shown on Table 1. Table 1 - β-carotene (µg/dL) and Reactive Oxygen Species (ROS) (U.Carr) concentrations (Media±s.d.) in the different gestational ages in the dairy cow. GESTATIONAL AGE (days)

β-carotene (µg/dL)

ROS (U.Carr)

30-60

196.25±31.12 A

94.56±5.87 D

60-90

195.00±24.59 A

100.89±8.55

90-120

174.50±25.40 A

119.25±14.58

120-150

174.63±19.65 A

120.85±8.25

150-180

173.75±13.76 A

126.96±11.56

180-210

171.38±44.21 A

121.87±8.36

210-240

109.13±23.56 B

128.59±10.23 E

240-270

107.88±12.28 B

147.25±15.26 E

The trend of β-carotene blood concentrations underwent a progressive, slow reduction throughout pregnancy duration, whereas ROS blood levels increased. In particular, levels of blood β-carotene content remained sta-

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ble from 30-60 days of pregnancy, until 180-210 days, when they significantly decreased. Conversely, blood ROS levels slightly increased from 30-60 days of pregnancy to 180-210 days, when they underwent a sharp increase until term. As to β-carotene, values were marginal, i.e. they ranged between 150 and 350 µg/dL, until 180-210 days of pregnancy; afterwards, their concentration declined to insufficient levels (<150 µg/dL).

DISCUSSION AND CONCLUSION β-carotene plays a pivotal role in the delicate and complex phase of the beginning of gestation, in cows; this vitamin is necessary for protecting luteal parenchyma from oxidative damage and promoting luteal functional16 as well as embryonic implantation and development19. Moreover β-carotene is crucial for maintaining adequate blood progesterone (P4) levels; this vitamin in fact enhances pituitary luteinizing hormone (LH) secretion, leading to an increase in gonadic P4 synthesis. Furthermore β-carotene protects the activity of the enzyme P450 side chain cleavage (scc), which, in turn, leads to the transformation of cholesterol into P4, in steroidogenic cells20. These are the reasons why β-carotene blood levels are required to be constantly high throughout pregnancy. β-carotene blood content should also be kept constantly sustained in the last pregnancy period, i.e. close to term, since it has been demonstrated in vitro that carotenoids promote cell to cell communication, stimulating connexin synthesis and an increase in gap-junctions, necessary for uterine contraction to occur. On the contrary, in this study, a sharp decrease in β-carotene blood levels occurred between 210 and 270 days of pregnancy. This decrease could be due both to the shift to the dry period and to a likely high β-carotene consumption in an attempt to counteract the oxidative stress known to occur at the fetomaternal interface as pregnancy goes on and the placenta gets older1,21. Pregnancy is known, in fact, to be characterized by sustained ROS generation, from its beginning, until term16,17,21. During pregnancy embryonic and fetal growth are associated with an increase in both placental and maternal metabolism which, in turn, leads to an increase in ROS generation, mainly deriving from an increase in lipid peroxidation, and, potentially, to oxidative stress22. At the beginning of gestation, when implantation occurs, the increase in reactive oxygen species is functional to embryonic development and growth, since ROS mediate embryonic physiological signaling pathways; furthermore ROS play a pivotal role in promoting neaoangiogenesis and apoptosis, thus enhancing the invasion of the maternal tissues, necessary for a physiological implantation to take place23,24. Notwithstanding these important functions, the first period of gestation is very critical, since the embryo may easily become prone to oxidative damage. It has in fact been shown that oxidative stress, if not properly counterbalanced, may impair gamete and embryo viability, leading to the arrest of embryo development to two cell stage16 . As pregnancy went on, maternal β-carotene and ROS blood concentrations showed an opposite trend, the former decreasing, the latter increasing, strengthening the hypothesis that the increase in ROS generation due to embryonic and fetal growth as well as to the accelerated placental metabolism might have been adequately kept under control by the reducing effect of

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the investigated antioxidant vitamin. Starting from the period between 90 and 120 days of gestation, when β-carotene and ROS mean blood levels were 174.50±25.40 µg/dL and 119.25±14.58 U. Carr, respectively, a plateau phase was observed, lasting until 180-210 days of gestation, i.e. through mid-gestation, in which, only a progressive fetal growth occurs, without the occurrence of crucial events such as implantation or calving. In the last phase of gestation, the highest ROS blood levels were found, compared to all the other phases investigated. This is an expectable event, in accordance with numerous research works, both in human and veterinary medicine, showing that in the last third of pregnancy an increase in reactive oxygen species occurs. This increase is mediated by a progressive increase in estrogen secretion by the maternal organism, which, in turn, promotes a massive arrival of leukocytes (mainly PMN and macrophages) in the utero-cervical tissue, as well as the release of an amount of pro-inflammatory cytokines, functional to trigger uterine contraction and cervical ripening and remodeling25-29 . In this study, the statistically significant increase in ROS found at term, might have depleted maternal blood β-carotene sources which, were found to be almost half compared to what had been observed at the beginning of pregnancy. Many Authors described the potential risk of oxidative stress in the periparturient cow as being an excess of reactive oxygen species and, if not properly counterbalanced, a potential harmful factor contributing to the development of mastitis or cystic ovarian disease (COD)16,26,29,30. The results of this study support the growing evidence that an adequate β-carotene integration in the diet of the parturient cow should be recommended in order to curb the dangerous effect of uncontrolled ROS generation and oxidative stress both in the mother and the unborn.

Conflict of interest statement The authors declare that there are no conflicts of interest.

References 1. Al-Gubory K.H., Fowler P.A., Garrel C. (2010). The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. Int J Biochem Cell B, 42: 1634-1650. 2. Aurouseau B., Durand D., Gruffat D. (2006). Gestation linked radical oxygen species fluxes and vitamins and trace mineral deficiencies in the ruminant. Reprod, Nutr, Dev, 46: 601-620. 3. Burton G.J., Hempstock J., Jauniaux E. (2003). Oxygen, early embryonic metabolism and radical mediated embryopathies. Reprod Biomed Online, 6: 84-96. 4. Anderson D., Phillips B.J. (1999). Comparative in vitro and in vivo effects of antioxidants. Food Chem Toxicol, 37: 1015-1025. 5. Stratton S.P., Liebler D.C. (1997). Determination of singlet oxygen –specific versus radical mediated lipid peroxydation in photosensitized oxidation of lipidic bilayer: effect of beta-carotene and alpha-tocopherol. Biochemistry 36: 12911-12920. 6. Bendich A. (1990). Antioxidant micronutrients and immune responses. Ann N Y Acad Sci, 587: 163-172. 7. Chew D.P., Wong T.S., Michel J.J. (1993) Uptake of orally administered β-carotene by blood plasma, leukocytes, and lipoproteins in calves. J Anim Sci, 71: 730-736. 8. Bertram J.S., Bortkiewicz H. (1995). Dietary carotenoids inhibit neoplastic transformation and modulate gene expression in mouse and human cells. Am J Clin Nutr, 62: 1327-1336. 9. Johnston L.A., Chew B.P. (1984). Peripartum changes in plasma and milk vitamin A and β-carotene among dairy cows with and without mastitis. J Dairy Sci, 67: 1832-1839.

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Oxidative status along different stages of pregnancy in dairy cows

10. Michal J.J., Heirman L.R., Wong T.S., Chew B.P., Frigg M., Volker L. (1994). Modulatory effects of dietary β-carotene on blood and mammary leukocyte function in periparturient dairy cows. J Dairy Sci 77: 1408-1421. 11. Weiss W.P. (1998). Requirements of fat-soluble vitamins for dairy cows: A Review. J Dairy Sci, 81: 2493-2501. 12. Akar Y., Gazioglu A. (2006). Relationship between Vitamin A and Beta carotene levels during the postpartum period and fertility parameters in cows with and without retained placent. Bull Vet Inst Pulawy, 50: 93-96. 13. Kawashima C., Kida K., Schweigert F.J., Miyamoto A. (2009). Relationship between plasma β-carotene concentrations during the peripartum period and ovulation in the first follicular wave postpartum in dairy cows. Anim Reprod Sci, 111: 105-111. 14. Grace N.D., Knowles S.O. (2012). Lack of production response in grazing dairy cows supplemented with long-acting injectable vitamin B12. N Z Vet J, 60: 95-99. 15. Celi P., Di Trana A., Claps S. (2010). Effect of plane of nutrition on oxidative stress in goats during the peripartum period. Vet J, 184: 95-99. 16. Rizzo A., Roscino M.T., Binetti F., Sciorsci R.L. (2012). Roles of reactive oxygen species in female reproduction. Reprod Domest Anim, 47: 344352. 17. Mutinati M., Piccinno M., Roncetti M., Campanile D., Rizzo A., Sciorsci R.L. (2013). Oxidative stress during pregnancy in the sheep. Reprod Domest Anim, 48: 353-357. 18. Hughes E., Davies, D. (1989). Practical uses of ultrasound in early pregnancy in cattle. Vet Rec, 124: 456. 19. Ledgard A.M., Lee R.S., Peterson A.J. (2009). Bovine endometrial legumain and TIMP-2 regulation in response to presence of a conceptus. Mol Reprod Dev, 76: 65-74. 20. Young F.M., Luderer W.B., Rodgers, R.J. (1995). The antioxidant betacarotene prevents covalent cross-linking between cholesterol side-chain cleavage cytochrome P450 and its electron donor, adrenodoxin, in

bovine luteal cells. Mol Cell Endocrinol, 109: 113-118. 21. Sciorsci R.L., Rizzo A. (2012). Riproduzione e stato ossidativo nella pecora. Qualità della carne dell’agnello: effetti della somministrazione parenterale di antiossidanti nella pecora gravida. Grafiche Vito Radio Editore, Putignano (BA), ISBN: 9788896766057. 22. Myatt L. (2010). Review: reactive oxygen and nitrogen species and functional adaptation of the placenta. Placenta, 31: S66-S69. 23. Abrahams V.M., Kim Y.M., Straszewski S.L., Romero R., Mor G. (2004). Macrophages and apoptotic cell clearance during pregnancy. Am J Reprod Immunol, 51: 275-282. 24. Covarrubias L., Hernandez-Garcia D., Schnabel D., Salas-Vidal E., Castro-Obregon S. (2008). Function of reactive oxygen species during animal development: passive or active? Dev Biol, 320: 1-11. 25. Frangogiannis N.G., Smith C.W., Entman M.L. (2002). The inflammatory response in myocardial infarction. Cardiovasc Res, 53: 31-47. 26. Rizzo A., Mutinati M., Spedicato M., Minoia G., Trisolini C., Jirillo F., Sciorsci R.L. (2008). First demonstration of an increased serum level of reactive oxygen species during the peripartal period in the ewes. Immunopharm Immunot, 30: 741-746. 27. Appiah I., Milovanovic S., Radojicic R., Nikolic-Kokic A., Orescanin-Dusic Z., Slavic M., Trbojevic S., Skrbic R., Spasic M.B., Blagojevic D. (2009) Hydrogen peroxide affects contractile activity and antioxidant enzymes in rat uterus. Br J Pharmacol, 158: 1932-1941. 28. Sordillo L.M., Contreras G.A., Aitken S.L. (2009). Metabolic factors affecting the inflammatory response of periparturient dairy cows. Anim Health Res Rev, 10: 53-63. 29. Bernabucci U., Ronchi B., Lacetera N., Nardone A. (2005). Influence of body condition score on relationship between metabolic status and oxidative stress in periparturient dairy cows. J Dairy Sci, 88: 2017-2026. 30. Rizzo A., Minoia G., Trisolini C., Spedicato M., Jirillo F., Sciorsci R.L. (2009). Reactive oxygen species: involvement in follicular cyst etiopathogenesis. Immunopharm Immunot, 31: 631-635.

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Salim Ilkaya et al. Large Animal Review 2020; 26: 231-237

Investigating Erythrocyte Membrane Lipid and Protein Oxidation with Na+/K+ATPase Activity in Caprine Anaplasmosis

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SALIM ILKAYA1, YETER DEGER1, BEKIR OGUZ2*, UGUR OZDEK3 1 2 3

Van Yuzuncu Yil University, Faculty of Veterinary Medicine, Department of Biochemical, Van, Turkey Van Yuzuncu Yil University, Faculty of Veterinary Medicine, Department of Parasitology, Van, Turkey Van Yuzuncu Yil University, Vocational School of Health Services, Van, Turkey

SUMMARY Anaplasmosis is an infectious disease that is caused by the genus Anaplasma belonging to the family Anaplasmataceae in the order Rickettsiales and is seen in the mammals inhabiting tropical and subtropical climate zones. Anaplasma ovis and A. phagocytophilum are the most remarkable species causing anaplasmosis in goats. Anaplasmosis induces both cellular and humoral immunity. As animals develop a long-term immunity against the disease, it becomes difficult to treat the disease. Immunity develops mainly depending on premunition. Increased osmotic fragility of erythrocytes in animals infected with anaplasmosis has been found, and it has been reported that this may be developed by various immune-mediated mechanisms including oxidative damage. Moreover, it may also be associated with high cell membrane ATPase activity and erythrocyte morphological changes. The aim of this study was to evaluate the effect of anaplasmosis on erythrocyte membrane malondialdehyde (MDA), advanced oxidation protein products (AOPP), Sodium-potassium adenosine 5â&#x20AC;&#x2122;-triphosphatase (Na+/K+ATPase), and hematological and biochemical parameters in goats. For this purpose, 45 male hair goats (35 infected and 10 healthy) that were brought to the slaughterhouse of Van Metropolitan Municipality were enrolled in the study. In infected goats, the hematological and biochemical concentrations of RBC, Hb, Hct, MCV, MCH, MCHC as well as serum total protein, albumin, total cholesterol and TIBC were found significantly lower, however concentrations of of WBC, globulin, total bilirubin, direct bilirubin, triglyceride, and iron as well as enzyme activities of AST, ALT, GGT were found higher when compared to the healthy goats (p<0.05). In addition, the MDA and AOPP levels were markedly increased in erythrocyte membrane from infected animals while the Na+/K+ATPase enzyme activity was significantly decreased compared to the the healthy goats (p<0.05).Therefore, it can be concluded that oxidative stress in erythrocyte membrane may play an important role in the pathogenesis of anemia in caprine anaplasmosis.

KEY WORDS AOPP, Caprine Anaplasmosis, Goat, MDA, Na+/K+ATPase.

INTRODUCTION Anaplasmosis is an infectious disease that is caused by Anaplasma species and leads to significant losses in meat, fleece and milk production in tropical and subtropical climate zones with inadequate feeding conditions. Anaplasma species are obligate intracellular rickettsial pathogens that have a size of 0.3-1 micron and no cytoplasm and are localized in the erythrocytes of hosts and close to their membrane1. Ovine and caprine anaplasmosis are generally associated with Anaplasma ovis and A. phagocytophilum and this disease is transmitted biologically by various tick species (Boophilus spp., Rhipicephalus spp., Hyalomma spp., Ixodes spp. and Dermacentor spp.) and mechanically by some diptera species (Tabanid spp., Stomoxys spp. and Melophagus ovinus)2. The clinical symptoms of the disease vary based on the gen-

*Corresponding Author: Bekir OGUZ (bekiroguz@yyu.edu.tr)

eral condition, breed, and age of the infected animal. In sheep and goats, acute anaplasmosis starts with general depression, numbness, and fever of 40-41Â°C and develops with weight loss, progressing anemia, dehydration, and hepatitis along with a rapid decrease in milk yield3. Anaplasmosis induces both cellular and humoral immunity. As animals develop a long-term immunity against the disease, it becomes difficult to treat the disease. Immunity develops mainly depending on premunition4. It has been reported that the osmotic fragility of erythrocytes increases in the animals infected with anaplasmosis and this may be developed by several immune-mediated mechanisms including oxidative damage5 and also may be associated with high cell membrane ATPase activity and erythrocyte morphological changes6. A great number of studies have focused on the effect of reactive oxygen species in the pathogenesis of parasitic infections and their cellular defense mechanisms. Some studies have revealed the effects of anaplasmosis on oxidative markers and antioxidants 5,7; however, there has been no report on erythrocyte membrane lipid peroxidation product malondialdehyde (MDA), advanced oxidation protein products (AOPP) and sodi-

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Investigating Erythrocyte Membrane Lipid and Protein Oxidation with Na+/K+ATPase Activity in Caprine Anaplasmosis

Statistical analysis

um-potassium adenosine 5’-triphosphatase (Na+/K+ATPase) in the goats infected by Anaplasma species. This study was conducted in order to reveal the effect of anaplasmosis on erythrocyte membrane MDA, AOPP, and Na+/K+ATPase as well as hematological and biochemical parameters in infected goats.

The data was statistically analyzed using SPSS 22 program. Statistical differences between the groups were evaluated using Mann-Whitney “U” test. The obtained results were given as X±SE. The value of p<0.05 was accepted as statistically significant.

MATERIALS AND METHODS

RESULTS

A total of 35 male hair goats diagnosed with anaplasmosis upon clinical and laboratory findings, and 10 healthy male hair goats, all of which had an average age of between 1.5 and 2.0 years and were brought to the slaughterhouse of Van Metropolitan Municipality, were used as the study material. Thin blood smear was prepared using the blood sample obtained from ear tip of each goat, thereafter these smears were stained using Giemsa method and examined using a light microscope at x100 magnification in terms of anaplasmic forms. Also, blood samples were taken from jugular vein of the animals into EDTA tubes and into tubes without anticoagulant agent. The blood samples taken into the tubes without anticoagulant agent were centrifuged at 840 g for 10 minutes in order to obtain serum samples. Commercial c-ELISA kit (Anaplasma antibody test kit, c-ELISA, no: 282- 2VMRD-USA) was used in these serum samples in order to determine the anaplasma antibodies. The determinations of serum total protein, albumin, globulin, total bilirubin, direct bilirubin, triglyceride, cholesterol, total iron binding capacity (TIBC) and iron levels as well as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma glutamyl transferase (GGT) enzyme activities were performed using a commercial kit in a modular auto analyzer (Roche, Germany). In the blood samples taken into EDTA tubes, red blood cell (RBC), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and white blood cell (WBC) parameters were determined in the blood count device (Vet. Wasson MC- 1200). Then, these blood samples were centrifuged at 4000xg+4°C for 5 minutes, plasma were separated, and the remaining erythrocyte pellet was used for extraction of erythrocyte membrane8. In the erythrocyte membrane obtained, MDA, AOPP, Na+ /K+ATPase and protein analyses were performed. MDA determination in erythrocyte membrane was performed based on Ohkawa et al.,9 method. This method is based on formation of a pink-colored chromogen by MDA, a lipid peroxidation product, which reacts when heated with thiobarbituric acid in acidic environment. The absorbance of this colored complex at 532 nm is directly proportional to MDA concentration. For AOPP measurement, the method used by Witko-Sarsat et al.10 was applied by making some revisions. This method is based on the spectrophotometric measurement of AOPP in the acidic environment in the presence of potassium iodide that allows to show maximum absorption at 340 nm. In membrane suspension, Na+/K+ATPase activity measurement was performed using the commercial kit that works based on biotin double antibody sandwich technique (Goat Na+/K+ATPase, Catalog No: YLA0092GO). The protein content of erythrocyte membrane was determined by Lowry method, in which bovine serum albumin is used as standard11.

When the hematological and biochemical parameters were compared, it was determined that the RBC, Hb, Hct, MCV, MCH, MCHC, total protein, albumin, and total cholesterol levels of the infected group decreased and their WBC, globulin, total bilirubin, direct bilirubin, triglyceride, iron, TIBC levels and AST, ALT, and GGT enzyme activities increased compared to the values of the healthy group (p<0.05). When the parameters in erythrocyte membrane were examined, it was determined that the erythrocyte membrane MDA and AOPP levels of the infected group increased and their Na+/K+ATPase enzyme activity decreased compared to the values of the healthy group (p<0.05).

DISCUSSION The most important species leading to ovine and caprine anaplasmosis in the eastern Turkey are A. ovis and A. phagocytophilum. The disease is diagnosed by using microscopic examination and serological or molecular methods12. In anaplasmosis infections, changes are observed in the blood parameters of the host in relation with the duration and severity of disease. Hematologic disorders such as anemia, thrombocytopenia, and leukopenia are frequently observed in animals infected with anaplasmosis 13,14. Although the certain mechanism of anemia is not clear, the infected erythrocytes become hemolyzed. This is associated with the direct damage of lipid peroxidation, increasing during the course of the disease, to membrane structure as well as the weak antioxidant defense.6,15Present study revealed a significant decrease in RBC, Hct, and Hb values in the goats with anaplasmosis15,16,17,18,19. The decrease of these parameters may be resulting from intravascularly hemolyzed erythrocytes, damaged due to pyrogens released by the Anaplasma species, and failure to remove these erythrocytes from circulation by reticuloendothelial system (RES)16,18. Additionally, some studies reported no significant difference in these parameters in the A. ovis-infected goats5,20. It has been stated that in the anaplasma infection, the decrease in hemoglobin and erythrocyte indices generally causes macrocytic hypochromic15,16, normochromic normocytic5 and macrocytic normochromic17 anemia in small ruminants. In the present study, MCH, MCHC, and MCV values, accompanying the change of hematocrit values in the infected goats decreased significantly, which may indicate that microcytic hypochromic anemia developed in these animals. It was determined in this study that there was a significant increase in WBC count of the infected animals5,18,20. This increase may be associated with the activation of lymphoid organs and chronic antigenic stimulation depending on the presence of anaplasma21. On the other hand, Ahmadi-hamedani et al.,16 determined that WBC count decreased. The blood parameters such as urea, creatinine, cholesterol, triglyceride, albumin, globulin, total pro-

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Salim Ilkaya et al. Large Animal Review 2020; 26: 231-237

tein, total lipid, and bilirubin and also the important enzymes such as amylase, AST, ALT, ALP, and CK are crucial parameters for determining biochemical profile. Based on these parameters, important information is obtained on the pathogenesis and prognosis of the disease22,23. Parasites cause significant changes in the biochemical profile of their hosts 16. In the previous studies, it was determined that anaplasmosis caused an increase in AST, GGT24, ALT, and ALP25 enzyme activities in infected animals. Differently from the foregoing results, it was determined that AST activity decreased15 and ALT activity did not change26. In this study, ALT, AST and GGT enzyme activities, which are specific for liver, significantly increased in the infected goats. Hypoxia developing in anaplasmosis due to anemia may increase activity of these enzymes by causing liver damage 27. The measurement of bilirubin caused by the decomposition of erythrocytes is used in the diagnosis and treatment of the liver, hemolytic, hematologic, and metabolic disorders22. In the present study, it was determined that bilirubin concentrations significantly increased in infected goats15,24. This significant increase in bilirubin levels may be associated with the hemolysis of the parasitic erythrocytes and hepatic function disorder15. Differently from these results, it has been reported in the studies that total bilirubin level decreases26 or remains within the physiological limits25. Many diseases affect the concentration, distribution or synthesis rates, degradation or excretion of serum proteins albumin, and globulin synthesized by liver. The changes in total protein value, fractional distribution and A/G ratio are generally used to assess the disorders of the protein metabolism22,23. Previous studies reported that serum total protein and globulin levels significantly increased in the animals infected with anaplasmosis15,26. However, other studies indicated that the increase in the

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total protein24,25 and albumin15,24 levels was not significant in infected animals. On the other hand, Kumar et al.,26 found that the serum albumin levels of the infected animals decreased. In this study, it was determined that while the serum total protein and albumin levels of the infected goats decreased, their globulin levels increased. The decreased albumin level may be due to hepatic impairment or due to the fact that it is an acute phase protein. On the other hand, the increase in serum globulin level may be associated with increased γ-globulin as a response to antigenic stimulation. The blood lipid profile of ruminants changes based on ration, age, gender, pregnancy, genetics, season, lactation, and liver and gallbladder diseases28. The serum cholesterol and triglyceride levels in anaplasma infection reveal different results. While Ahmadi-hamedani et al.,24 determined that there was no significant difference between healthy and infected goats in terms of cholesterol and triglyceride levels, Khaki et al.,15found a significant decrease in their cholesterol level. The results of this study showed that the triglyceride level significantly increased but the cholesterol level significantly decreased in the infected goats. High triglyceride level may be associated with the adipose tissue lipolysis that stimulates its production in the liver. And the low total cholesterol level may be due to the fact that its normal synthesis interrupts based on liver damage. Iron is an essential element for many living creatures and has a vital role. 60-70% of iron in the organism is included in the hemoglobin in erythrocytes. TIBC is a measure of the transferrin concentration in blood29. It has been determined that the serum iron level increases significantly in the sheep infected with anaplasmosis due to anemia and the decrease in TIBC level is not significant15. On the other hand, Jalali et al.,5 found that the increase in the iron level was not significant in the goats infected with A. ovis. It was

Table 1 - Hematological and biochemical parameters in healthy and anaplasmosis goats. Parameters

Healthy group (n = 10) X ± SE)

Infected group (n = 35) (X ± SE)

WBC (m/mm-3)

8.69±0.41

11.93±0.80*

-3

Hematological

Biyochemical

RBC (M/mm )

15.31±0.63

6.41±0.21*

Hb (g/dl-1)

9.79±0.43

5.56±0.30*

Hct (%)

29.39±1.24

14.24±0.69*

MCV (fl)

20.14±0.79

11.77±0.33*

MCH (pg)

5.96±0.2

4.23±0.18*

MCHC (g/dl-1)

35.10±1.08

27.82±0.81*

AST (U/L-1)

100.85±9.57

189.42±18.68*

ALT (U/L-1)

20.00±1.07

37.00±4.78*

-1

GGT (U/L )

40.71±0.81

96.14±16.02*

Albumin (g/L-1)

29.57±1.34

24.71±0.75*

-1

Globulin(g/dL )

43.14±2.26

63.42±4.91*

T protein (g/L-1)

92.14±5.78

69.14±2.52*

T bilirubin (mg/dL )

0.66±0.03

0.92±0.03*

D bilirubin (mg/dL-1)

0.36±0.01

0.53±0.02*

-1

T cholesterol (mgvdL )

91.86±7.69

52.37±1.95*

Triglyceride (mg/dL-1)

16.43±1.89

31.71±6.74*

Iron (µg/dL-1)

108.28±7.08

141.00±8.95*

TIBC (µg/dL-1)

149.14±16.21

254.71±25.51*

Compared to healty group: *p<0.05. WBC: Leukocyte, RBC: Erythrocyte, Hb: Haemoglobin, Hct: Haematocrit, MCV: Mean corpuscular volume, MCH: Mean corpuscular haemoglobin, MCHC: Mean corpuscular haemoglobin concentration, ALT: Alanine aminotransferase, AST: Aspartate aminotransferase, GGT: Gamma glutamyltransferase, TIBC: Total iron-binding capacity, T: Total, D: Direct

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Investigating Erythrocyte Membrane Lipid and Protein Oxidation with Na+/K+ATPase Activity in Caprine Anaplasmosis

Table 2 - Erythrocyte membrane MDA and AOPP levels with Na+/K+ATPase enzyme activity in healthy and anaplasmosis goats. Parameters

Healthy group (n = 10) (X ± SE)

Infected group (n = 35) (X ± SE)

MDA (nmol/g-1 protein)

4.01±0.19

5.91±0.23*

AOPP (nmol/g-1 protein)

0.41±0.01

0.54±0.02*

Na+/K+ ATPase (nmol/mg-1 protein)

3.33±0.18

2.97±0.16*

Compared to healty group: *p<0.05. MDA: Malondialdehit, AOPP: Advanced oxidation protein products, Na+/K+ ATPase: Sodium-potassium adenosine 5’triphosphatase

determined in the present study that the serum iron level significantly increased and TIBC level significantly decreased in the infected goats. The increase in the iron level is probably due to the iron releasing from intravascular hemolysis of erythrocytes. The decreased TIBC level may be associated with the decrease of transferrin, which is a negative acute phase reactant, due to the inflammation developing as a result of disease. In the host cells infected by different parasite species, the amount of reactive oxygen species (ROS) increases and therefore, cell and tissue damages occur. ROS induces the oxidation of polyunsaturated fatty acids in biological systems and leads to the formation of lipid peroxidation products. MDA is a lipid peroxidation product. The analysis of MDA levels is used to measure the lipid peroxidation level and free radical levels. As erythrocyte membrane is exposed to continuous high oxygen concentration which is rich in polyunsaturated fatty acids, it is highly sensitive to lipid peroxidation30. No data has been found on the MDA level of erythrocyte membrane in the goats infected with anaplasmosis. However, it was determined that MDA level increased in the plasma of the cattle infected with anaplasmosis30,31 and it did not change in the serum of sheep6. It was found in this study that anaplasma infection elevated the MDA level of erythrocyte membrane in goats. This increase may be associated with the lipid peroxidation induced based on the increase of ROS generating during the infection. Also, increasing number of iron ions might have aggravated the oxidative damage, as a strong oxidative catalyst forming free radicals. The oxidation of proteins as a result of the covalent modification directly with ROS or indirectly with the secondary products of oxidative stress has a role in the etiology or progress of several disorders and diseases. AOPP, included in the protein oxidation products, is used in determining the oxidation level32. Upon the literature review, no data was found on the AOPP levels of erythrocyte membrane in goats injected with anaplasmosis. However, it was determined that serum AOPP level increased in malaria33,34 and babesiosis35. It was found in this study that anaplasma infection elevated the AOPP level of erythrocyte membrane. This increase may be due to the fact that it caused protein oxidation characterized by the changes in the structure and functions of ROS in erythrocyte membrane. Na+/K+ATPase is a membrane-associated transport enzyme responsible for maintaining the membrane integrity and the ion balance in cell. Na+/K+ATPase, directly or indirectly, controls numerous basic cellular functions and the regulation of this enzyme is effective in the etiology of various pathological processes36. Also, it takes place on the top among the enzymes

that are affected by the formation of ROS that damages cells37. Although there has been no study evaluating the Na+/K+ATPase activity of erythrocyte membrane in the infections caused by Anaplasma species, it has been determined that Na+/K+ATPase activity decreases in the infections caused by malaria species37,38,39 and Babesia ovis39. Although the mechanisms underlying the inhibition of Na+/K+ pump are unclear, Staines et al.,38 reported that many changes occurred in the physical and chemical characteristics of erythrocyte after malaria infection, and any of them may change the activity of the endogenous transport systems. In addition, it was asserted that new permeability pathways (NPPs) induced by parasites may affect the activity of the endogenous carriers39. In this study, it was determined that the Na+/K+ATPase activity of erythrocyte membrane decreased in goats infected with anaplasmosis. The Na+/K+ATPase activity loss considered to develop due to the above reasons may accelerate the hemolysis of erythrocytes by leading to the disruption of intracellular ionic balance.

CONCLUSIONS The results have revealed that there are significant changes in hematological and biochemical parameters and anemia and oxidative stress are common complications of anaplasmosis in ruminants. In addition, this study showed an increase in MDA and AOPP levels and a decrease in Na+/K+ATPase activity in the erythrocyte membrane in goats infected with anaplasmosis. Therefore, it can be concluded that oxidative stress in erythrocyte membrane may play an important role in the pathogenesis of anemia in goats infected with anaplasmosis.

ACKNOWLEDGEMENTS This study was sponsored by Van Yuzuncu Yil University Scientific Research Project Fund and registered under Project No: TYL-2018-7273.

CONFLICT OF INTEREST The authors have declared no conflict of interest.

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25. Hornok S., Elek V., de la Fuente J., Naranjo V., Farkas R., Majoros G., Földvári G. (2007). First serological and molecular evidence on theendemicity of Anaplasma ovis and A. marginale in Hungary. Vet Microbiol, 122(34): 316-322. 26. Kumar A., Vihan V.S., Sharma H.N. (2010). Haematological and biochemical effects of tick infestation in common Indian Goat. Adv Biores,1(1): 164 -169. 27. Liang S., Ma H.Y., Zhong Z., Dhar D., Liu X., Xu J., Koyama Y., Nishio T., Karin D., Karin G., Mccubbin R., Zhang C., Hu R., Yang G., Chen L., Ganguly S., Lan T., Karin M., Kisseleva T., Brenner D.A. (2019). NADPH oxidase in liver macrophages promotes ınflammation and tumor development in mice. Gastroenterology ,156(4):1156-1172. 28. Sengul Y., Mert H., Mert N. (2017). Determination of serum lipid profile and lipoprotein levels of sheep with naturally acute babesiosis. Van Vet J, 28 (1): 1-4. 29. Hoffbrand A.V., Moss P.A.H., Pettit J.E. (2006). Eritropoiesis and general aspects of anemia. In: Hoffbrand A.V., Moss P.A.H., Pettit J.E. (Eds.). Essential Hematology. Oxford: Blackwell publishing, 12-28. 30. Esmaeilnejad B., Tavassoli M., Asri-Rezaei S., Dalir-Naghadeh B., Malekinejad H., Jalilzadeh-Amin G., Arjmand J., Golabi M., Hajipour N. (2012). Evaluation of antioxidant status and oxidative stress in sheep naturally infected with Babesia ovis. Vet Parasitol, 185: 124-130. 31. El-Ashker M., Hotzel H., Gwida M., El-Beskawy M., Silaghi C., Tomaso H. (2015). Molecular biological identification of Babesia, Theileria, and Anaplasma species in cattle in Egypt using PCR assays, gene sequence analysis and a novel DNA microarray.Vet Parasitol, 30(3-4):329-34. 32. Kayalı R., Telci A., Cakatay U., Karaca C., Akcay T., Sivas A., Altug T. (2003). Oxidative protein damage parameters in plasma in chronic experimental diabetes in rats. Eur J Med Res, 8: 307-312. 33. Nhabomba A.J., Guinovart C., Jimenez A., Manaca M.N., Quintó L., Cisteró P., Aguilar R., Barbosa A., Rodríguez M.H., Bassat Q., Aponte J.J., Mayor A., Chitnis C.E., Alonso P.L., Dobaño C. (2014). Impact of age of first exposure to Plasmodium falciparum on antibody responses to malaria in children: a randomized, controlled trial in Mozambique. Malar J, 13:121. 34. Zhang G., Skorokhod O.A., Khoo S.K., Aguilar R., Wiertsema S., Nhabomba A.J., Marrocco T., McNamara-Smith M., Manaca M.N., Barbosa A., Quintó L., Hayden C.M., Goldblatt J., Guinovart C., Alonso P.L., Dobaño C., Schwarzer E., LeSouëf P.N. (2014) Plasma advanced oxidative protein products are associated with anti-oxidative stress pathway genes and malaria in a longitudinal cohort. Malaria J, 3: 133-134. 35. Baldissera M.D., Sousa K.C.M., André M.R., Guarda N.S., Moresco R.N., Herrera H.M., Machado R.Z., Jaques J.A.S., Tinucci-Costa M., Silva A.S. (2015).Nitric oxide, protein oxidation and total antioxidant levels in serum of dogs naturally infected by Ehrlichia canis, Leishmania infantum and Babesia vogeli. Acta Sci Vet, 43: 1320. 36. Akbulut S. (2008). The investigation of the effects of erythropoietin application on Na+-K+ ATPase enzyme (E.C.3.1.6.37) activities in patients with diabetic polyneuropathy. Phd thesis. Konya University Science Institution. Konya, Turkey. 37. Nikolic M., Stanic D., Antonijevic N., Niketi V. (2004). Cholesterol boundto hemoglobin in normal human erythrocytes: a new form of cholesterol in circulation. Clin Biochem, 37: 22-26. 38. Staines H.M., Ellory J.C., Kirk K. (2001). Perturbation of the pump-leak balance for Na+ and K+ in malaria-infected erythrocytes. Am J Physiol Cell Physiol, 280, 1576-1587. 39. Yur F., Yazar M., Deger Y., Dede S. (2010). Na+/K+ ATPase activity in sheep with natural Babesiosis. Acta Vet Brno, 79: 233-236.

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ELEONORA BUOIO, ANNAMARIA COSTA* Department of Health, Animal Science and Food Safety (VESPA), Faculty of Veterinary Medicine, Università degli Studi di Milano, via Celoria 10, 20133 Milan; via dell’Università 6, Lodi, Italy

SUMMARY Perinatal mortality represents an important cause of economic loss in pig production and the most critical phase is represented by the occupation time in the farrowing room before weaning. A wide number of studies report that the high mortality rate in farrowing depends on the combination of many parameters: genetics, environment/design of housing system, nutritional status, infectious diseases and the maternal attitude of the mother towards new-born piglets and litter size. Among these factors, crushing represents the 18% of healthy piglets. This study has the aim 1) to compare the crushing rate of piglets in farrowing, in conventional (CFC) and welfare farrowing crates (WFC); 2) to evaluate the effect of a larger space allowance in WFC after the 15th d age of piglets. The trial took place in 2019 a piggery lodging 2500 sows (Landrace x Large White), in Northern Italy, in South west Lombardy. This farm adopts Animal Welfare guidelines to improve sow conditions during gestation and farrowing. In the first part of the experimental study, 329 sows lodged in CFC and 293 sows housed in the WFC were considered. In the second part, 71 sows lodged in WFC were involved to assess the effect of space allowance on piglets’ crushing rate. The first trial showed that the number of total crushed piglets was higher in WFC (1.17 vs 0.95, P<0.05) with significant consistency from d 3 to weaning (0.40 vs 0.32, P<0.05). The second trial of the study showed that the management strategy to provide more space allowance to sows in the WFC unit increased the crushing rate of piglets (0.06 vs 0.23, P<0.05). In conclusion, the WFC crates, representing an improvement compared to the conventional ones, in terms of welfare for sows, showed a higher crushing rate in comparison with the CFC, and that a larger space in the farrowing unit, from d 15 to the weaning, lowered furtherly piglet survival rate. In the present study, the availability of a larger area, allowed a higher crushing rate by the sows, for the rolling behaviour and movements in the welfare farrowing units. These results show that, even if farrowing systems with no or only temporary confinement of sows guarantee welfare conditions for sows, the benefits for piglets remain a controversy for the lower survival before weaning induced by crushing.

KEY WORDS Farrowing crate, piglets crushing rate, space allowance.

INTRODUCTION In modern pig farming, the only possible action to improve farm productivity is to act to improve the ratio weaned piglets/sow per year, avoiding piglets’ losses during farrowing. Perinatal mortality represents an important cause of economic loss in pig production1, and the most critical phase is represented by the occupation time in the farrowing room before weaning. Literature widely reports studies demonstrating that the high mortality rate in farrowing depends on the combination of many parameters: genetics, environment, design of housing system, nutritional status, infectious diseases and the maternal attitude of the mother towards the new-born piglets and litter size2,3.

*Corresponding Author: Annamaria Costa (annamaria.costa@unimi.it).

In the farrowing unit, approximately 50% of the pre-weaning death losses occur in the first three days of life, as a result from failure of the piglets to avoid the sow4. At this regard, preventing the deaths of the new-born from crushing, representing 18% of healthy piglets5, is the key to improve their survival during farrowing. The number of crushed piglets by the mother is linked to the sudden movements of the sows, which, combined with the great difference in size between mother and son, can cause suffocation and serious injuries, with consequent death of the new born piglets. The adequate design of the farrowing crate, limiting the movements of sows and avoiding her occupation of the nest and of the creep area, can reduce the possibility for the piglets to be crushed6. In the last decade, the concept of farrowing crate raised societal concerns for animal welfare and public interest moved for

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alternative rearing techniques in intensive animal husbandry, because of the restricted area available for the sow in the farrowing crate, that is detrimental for sow welfare7,8. Following these societal concerns, innovative solutions were and are conceived, to improve the welfare conditions of reared animals, and to maintain high productivity in intensive farms, as the farrowing crates adoption: in the last times, the free farrowing systems are slowly adopted in piggeries, to improve the physiological and behavioural condition of sows. Nevertheless, uptake of non-crated farrowing systems in piggeries is limited, for the higher crushing rate of piglets, while the farrowing crate represents advantages for piglet survival and farm productivity, for the lower crushing rate9, the European Food Safety Authority in 2007 has expressed caution towards the adoption of farrowing pens for the increased risk of higher mortality for crushing in loose housing systems10. The adoption of modified farrowing crates, larger than the conventional ones, can represent an advantage for farrowing housing, favouring the movements and the exhibition of behavioural patterns of lodged sows, reaching the same results of conventional crates, if well managed11.

For the above listed reasons, the aim of this study was to:

positioned in the front area and it is equipped with a IR lamp to kept warm piglets during resting time. Before the delivery, the floor of the pen around the sow is covered with paper strips and the nest is covered with a paper mat to preserve new-born piglets from high air flows coming from the pit under the floor.

The welfare farrowing crates (WFC) The Welfare farrowing unit is an innovative farrowing area with a total surface of 5.62 m2. The nest area is 1.04 m2 wide, the sow area is equipped with a removable gate, in order to enlarge it from 2.39 m2 to 2.80 m2, when the gate is removed. The movement area is 1.5 m2, the mat positioned in the pens has a total area of 3.84 m2 (see Figure 2). The difference between the WFC and the CFC crates consists in the total area available to the animals, with the further possibility to remove the gate for further enlarging the sow area to improve her welfare conditions. The floor is composed of cracked «plastic» tiles of two types: the figures 2 show the yellow tiles, characterized by resistance to high pressure exerted by considerable weight, define the space for the sow; the blue tiles, define the nest area of the piglets, less resistant to pressure insults. In these boxes there is also the

• Compare the crushing rate of piglets in conventional and innovative “welfare” farrowing crates • Evaluate the further space allowance in the “welfare” farrowing crates, from d 15 to weaning, on piglet survival rate

MATERIALS AND METHODS Location The trial took place in a piggery lodging 2500 sows (Landrace x Large White), in Northern Italy, Lombardy. This farm adopts Animal Welfare guidelines to improve sow conditions during gestation and farrowing. The farrowing section is composed in • Two facilities, with 8 farrowing rooms equipped with 48 Conventional Farrowing Crates (CFC). • Two facilities with 17 farrowing rooms equipped with Welfare Farrowing Crates (WFC), structured and designed for more space allowance for sows in the farrowing pen. Eleven farrowing rooms are composed of 24 WFC and 6 rooms of WFC.

The conventional farrowing crates (CFCs) The CFCs are designed in order to cover a total area of 4.42 m2, the nest area is 90 cm2 wide, the total pen area ranges from 1.14 to 1.71 m2 wide (see Figure 1). These farrowing crates can be enlarged from 60 to 90 cm, according to the size of the sow. This aspect is fundamental for piglet survival rate, since if the crate is too large, the probability for the mother to crush piglets rises, while if the crate is too narrow, the milking could be difficult for the new-born. The CFC is equipped with containment bars to avoid the climbing of the sow, and inclined bars, arranged on the lower beam, to contain the sow’s movements that could be a risk for piglets. The farrowing pen has a cast-ironed slatted floor, one nipple for the sow and one for the piglets (see Figure 1). The nest is

b Figure 1a and 1b - Section and picture of the conventional farrowing crate (CFC).

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• • • • • • •

the day of delivery the parity of sows the number of piglets born alive the number of piglets born dead the number of piglets mummified number of dead piglets, for infections or pathologies number of crushed piglets - piglets crushed at d 1 - piglets crushed from d 1 to d 3 - piglets crushed from d 3 to the weaning (d 28) • number of weaned piglets The second part of the trial was aimed to evaluate the effect of a higher space allowance in the WFC crates on piglets crushing rate, starting from d 15 up to weaning time. For this purpose, other 71 WFCs were considered, in 36 crates the gate dividing sow from piglets was kept, 35 crates were “opened” removing the gate limiting the sow area, from d 15. For this purpose, the following parameters were recorded for each sow of both group: • the day of delivery • the parity of sows • the number of piglets born alive • the number of piglets born dead • the number of piglets mummified • number of dead piglets (for infections or pathologies) • number of crushed piglets - piglets crushed at d 1 - piglets crushed from d 1 to d 3 - piglets crushed from d 3 to d 15 - piglets crushed from d 15 to the weaning (d 28) • number of weaned piglets

b Figure 2a and 2b - Section and picture of the welfare farrowing crate (WFC).

possibility of arranging mats to improve the well-being of the sow, not adopted during the trial for the excessive manure retainment on the crate floor. From personal observation, the presence of the mat, more comfortable than the bare floor, led the sows to a more abrupt descent for laying down, increasing the risk for piglets of being crushed. The nest area is located on the front-right portion of the unit, equipped with an IR lamp to warm the piglets. At the time of delivery, the nest is set up, adding paper strips, with the aim to stimulate the sow to prepare the nest for delivery, allowing her to manifest its natural instinct, and avoiding the development of stereotyped and abnormal behaviour, indicators of stress. The strip paper also allows drying of piglets, at the time of birth, thus avoiding their cooling and the onset of pathologies.

Data Collection The trial was conducted in 2019, considering 329 sows lodged in CFCs and 293 sows housed in the WFCs. The first part of this trial was aimed at comparing the piglets crushing rate in the two farrowing crate types. For this purpose, the following parameters were recorded for each sow of both groups:

Statistical analysis Data were submitted to Proc Freq of the SAS statistical package 9.4, 2019 to analyze the sows distribution in crates. Data related to the first trial were submitted to variance analysis (Proc GLM of the SAS statistical package 9.4, 2019) in order to evaluate the effect of farrowing crate type and parity, considering the interaction of these two parameters on piglets crushing rate. For the second trial, data were proceeds through variance analysis (Proc GLM of the SAS statistical package 9.4, 2019) to evaluate the effect of the higher space allowance for sows, in the WFS and parity, from d 15 to the weaning on piglets survival.

RESULTS Trial 1: Piglets survival, CFCs vs WFCs Table 1 shows the number and percentage of sows lodged in CFCs and WFCs, according to parity. A total of 967 sows were grouped as primiparous sows (Parity 1), multiparous at the second delivery (parity 2), sows from the 3rd to the 8th delivery (parity 3-8), and sows from 9th to 14th delivery (Parity 9-14), for statistical purpose. Figure 3 shows the number of piglets (according to parity of sows) born alive, born dead, mummies and weaned after cross fostering of litters. The number of born alive piglets was affected by parity (P<0.001); the number of born dead piglets was depending on parity (P<0.05) and crate type (P<0.01). The number of mum-

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DISCUSSION

Table 1 - Distribution of sows in the two crate types, according to parity. Percentage

Parity WFC

FREQ

% 2

3-8

9-14

Total

This study was aimed at evaluating the survival rate of piglets in welfare farrowing crates, characterized by greater space allowance for the sow. The first trial showed that the number of total crushed piglets was higher in WFCs (1.17 vs 0.95, P<0.05) with significant consistency from d 3 to weaning (0.40 vs 0.32, P<0.05). The second trial of the study showed that the management choice to provide more space allowance to sows in the WFC units increased the crushing rate of piglets (0.06 vs 0.23, P<0.05). These results put in evidence the importance of crate design and space allowance in the farrowing rooms. The introduction of conventional farrowing crates in piggeries happened in 60s, converting the free farrowing pens to reduce piglet crushing by limiting the sow movement. The adoption of conventional farrowing crate contributed to the decrease of the number of crushing8,12. However, although the farrowing crates adoption, the crushing rate still represents a great part of mortality before weaning1,3,13 in piggeries. Dead born piglets and mummies depend on several factors that can be linked to how the gestation phase took place, the possible presence of pathologies, or birth defects such as miscarriages or physiological problems that led to an impossibility in the correct foetal growth. In general, postpartum mortality in farrowing is caused by starvation (40-50%), crushing (20-30%), low vitality (5-20%), genetics (0-10%), diseases (0-15%) and other causes, as, for example poor maternal attitude of the sow (5-15%), uncomfortable environment/design or microclimate2,14. In particular, an important cause of piglet death during farrowing is the combination of respiratory and gastroenteric pathologies, often due to malabsorption of colostrum and therefore insufficient transmission of maternal immunity. The main cause of the increase in mortality over the years, however, is also the litter size, in fact the piglets are often underweight and with low energy reserves, in addition the competition for colostrum and milk produces limited weight gain, and therefore a possible increase in mortality: as the litter size increases from 6-8 to 16-19 piglets, neonatal mortality increases from 10-15% to about 45%15. Amongst deaths of liveborn piglets, crushing is by far the major cause16. The risk for crushing can be summarized in three groups: environment, piglet conditions and sow. Physical environment, or structural characteristics17, and management are factors18, that can account for mortality rate

Crate type CFC

Total

159

106

265

16.44%

10.96%

27.4%

154

9.2%

6.72%

15.93%

229

207

436

23.68%

21.41%

45.09%

112

6.2%

5.38%

11.58%

537

430

967

55.53%

44.47%

100%

mified piglets was higher in primiparous sows (P<0.05); while the number of weaned ones was higher in CFC (P<0.01). Figure 4 shows the number of crushed piglets in the two types of farrowing crates, according to parity and type of crate. Crushed at d 1 was influenced by parity (P<0.05) and type of crate (P<0.01). The number of total crushed piglets was higher in WFC (P<0.05). The interaction parity for crate type was not significant, then it was deleted from the analysis, the analysis considering parity and crate type, is shown in Table 2. Table 3 reports the statistical significance of data.

Trial 2: Increasing space allowance in WFC from d 15 to weaning This second investigation was conducted on 71 sows lodged in the WFC crates, 36 crates were kept unvaried for surface area available for the sow (WFCC), in 35 crates the gate was removed up to provide 2.80 m2 of area to the sow (WFCO). Results reported in Table 4 show that opening the crates at d 15 to weaning, aimed at providing a larger area for sow welfare affected the crushing rate of piglets (0.06 vs 0.23; P<0.05), the number of weaned was significantly higher in closed WFCs (P<0.05), parity had no effect on piglet survival rate. Moreover, considering the values of piglets crushed during the first 15 days in the 71 WFCs as an overall mean (1.31 piglets), the practice to provide more space allowance to sows resulted detrimental to piglet survival (1.58 vs 1.31; P<0.05).

Table 2 - Parameters collected in WFCs and CFCs on piglet performance and crushing rate. Piglets

WFC

SEM

CFC

SEM

Born alive

11.71

0.21

11.95

0.23

Born dead

1.27

0.08

0.91

0.09

Mummies

0.35

0.05

0.25

0.06

Crushed at d 1

0.20

0.03

0.15

0.03

Crushed from d1 to d 3

0.57

0.06

0.48

0.06

Crushed from d 3 to weaning

0.40

0.04

0.32

0.04

Total crushed

1.17

0.08

0.95

0.08

Weaned

9.99

0.08

10.51

0.08

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Figure 3 - Piglets born alive, born dead, mums and weaned according to parity of sows.

Table 3 - Significance level of processed data considering parity and type of crate (*: P<0.005, **:P<0.01; ***:P<0.001). Piglets

Parity

Born alive

***

Born dead

Type of crate

***

Mummies Crushed at d 1

Crushed from d 1 to d 3 Crushed from d 3 to weaning

Total crushed

Weaned

***

ranging between 11% and 13%, when including a previous 78% of losses due to stillbirths19. In addition, other environmental aspects may be involved, as the presence of a sliding floor that can induce an abrupt falling of the sow on the piglets13. The organization and the design of the farrowing unit can play an important role in piglet survival at weaning time6, for example the area for the resting of the piglet near the sow can lead to crushing20. With regard to piglet condition, low vitality17 can contribute to crushing, as hypothermia, hypoxia, and low body weight conditions21. In the farrowing pen and crate, what contributes to increase the probability of crushing is the difficulty of walking of the new-born, especially in the first days of age, combined with the

Table 4 - Means of the piglet parameters evaluated in WFCs, kept closed and opened at d 15 up to weaning, for increasing sow area and improving sow movements and area. WFCC (36 sows) Variable

Mean

SEM

Parity

3.69

Piglets Born alive

12.47

Piglets Born dead Piglets Mummified

WFCO at d 15 (35 sows) Mean

SEM

Overall mean of the two groups

3.21

5.74

3.18

4.72

3.30

13.06

3.64

12.76

0.56

0.73

0.74

0.95

0.65

0.06

0.33

0.14

0.43

0.10

Crushed at d 1

0.00

Crushed from d 1 to d 3

1.17

1.59

0.54

0.82

0.85

Crushed from d 3 to d 15

0.36

0.76

0.54

0.66

0.45

Crushed up to d 15

1.53

1.95

1.09

1.15

1.31

Crushed from d 15 to weaning

0.06

0.23

0.43

Total crushed

1.58

2.02

1.31

1.16

Weaned

10.19

1.09

9.51

1.34

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246

Space allowance and piglets survival rate in the farrowing crate

Figure 4 - Number of piglets crushed at d 1, from d 1 to d 3, from d 3 to weaning, and total crushed during farrowing in WFCs and CFCs.

considerable disproportion with the mother’s size. At the end, crushing by the sow is a predominant cause of death in crates and pens13, for her movements and her rolling behaviour, that can produce 18-36% crushing rate22, up to 6575% as reported by Weary et al23. Sow’s risk movements could be prevented through the adoption of alternative farrowing systems, Mazzoni et al.24 found that sows housed in the up and down designed farrowing crates reported the lower crushing mortality rate if compared to conventional crates. Another important factor in crushing is the presence of sow’s legs injuries and lameness, in a recent study, front injuries were found to be more important if compared to those detected on rear legs in crushing rate during farrowing, probably for the difficulty of the sow in lifting and turning to the rest position25. In the present study, the availability of a larger area, allowed a higher crushing rate by the sows, for the rolling behaviour and movements in the welfare farrowing units. These results show that, even if farrowing systems with no or only temporary confinement of sows guarantee welfare conditions for sows, the benefits for piglets remain a controversy, with regard to crushing26.

CONCLUSIONS The WFC crates, representing an improvement compared to the conventional ones in terms of welfare for sows, showed a higher crushing rate in comparison with the CFC. A larger space in the WFC farrowing unit, from d 15 to the weaning, furtherly lowered piglet survival rate. These results highlight the necessity to conduct further studies considering welfare farrowing system properly designed, considering also the economic losses related to the use of the described crates.

References 1. Marchant J.N, Rudd A.R., Mendl M.T., Broom D.M., Meredith MJ, Corning S, Simmins PH. (2000). Timing and causes of piglet mortality in alternative and conventional farrowing systems. Vet Rec. 147:209-214. 2. Weary DM, Phillips PA, Pajor EA, Fraser D, Thompson BK. (1998). Crushing of piglets by sows: effects of litter features, pen features and sow behaviour. Appl Anim Behav Sci. 61:103-111. 3. Anderson, I. L., G. M. Tajet, I. A. Haukvik, S. Kongsrud, and K. E. Bøe. (2007). Relationship between postnatal piglet mortality, environmental factors and management around farrowing in herds with loose-housed, lactating sows. Acta Agric. Scand. A Anim. Sci. 57:38-45 4. Danholt L, Moustsen VA, Nielsen MBF, Kristensen AR. (2011). Rolling behaviour of sows in relation to piglet crushing on sloped versus level floor pens. Livest Sci. 141:59-68. 5. English P.R., Smith W.J. (1975) Some causes of death in neonatal piglets. Veterinary Annual 15, 95-104. 6. Hales J., Moustsen V.A., Devreese A.M., Nielsen M.B.F. and Hansen C.F. (2015) Comparable farrowing progress in confined and loose housed hyper-prolific sows. Livest. Sci. 171:64-72. 7. Marchant, J. N., D. M. Broom, and S. Corning. (2001). The influence of sow behaviour on piglet mortality due to crushing in an open farrowing system. Anim. Sci. 72:19-28. 8. Jarvis S., D’Eath R.B., Fujita K. (2005). Consistency of piglet crushing by sows. Anim Welfare. 14:43-51. 9. Weber, R.; Keil, N.M.; Fehr, M.; Horat, R. (2007). Piglet mortality on farms using farrowing systems with or without crates. Anim. Welf. 16, 277-279. 10. European Food Safety Authority. (2007). Animal health and welfare aspects of different housing and husbandry systems for adult breeding boars, pregnant, farrowing sows and unweaned piglets1. Scientific Opinion of the Panel on Animal Health and Welfare. The EFSA J.572, 1-13. 11. Baxter, E. M., A. B. Lawrence, and S. A. Edwards. (2012). Alternative farrowing accommodation: Welfare and economic aspects of existing farrowing and lactation systems for pigs. Animal 6:96-117. 12. Ostovic M, Pavicic Z, Tofant A, Kabalin AE, Mencik S, Potocnjak D, Antunovic B. (2012). Sow parity, body length, postural changes and piglet crushing. Veterinarski Archiv. 82:319-326. 13. Damm B. I., B. Forkman and Pedersen L. J. (2005) Lying down and rolling behaviour in sows in relation to piglet crushing. Appl. Anim. Behav. Sci. 90:3-20 14. Jarvis S., D’Eath R.B., Fujita K. (2005) Consistency of piglet crushing by sows. Anim Welfare. 14:43-51.

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Eleonora Buoio, Annamaria Costa; Large Animal Review 2020; 26: 239-247 15. Blasco A., Bidanel J. P., and Haley C. S. (1995). Genetics and Neonatal Survival. In: M.A. Varley (ed.) The Neonatal Pig. Development and Survival, pp 17-38. CAB International, Wallington, Oxon, UK]. 16. Sala V., Fantinati P., (2012). Implicazioni manageriali e strutturali nello schiacciamento dei suinetti. Summa Animali da reddito. 29 (6): 30-34. 17. Andersen I.L., Haukvik I.A., Bøe K. (2009) Drying and warming immediately after birth may reduce piglet mortality in loose-housed sows. Animal. 3:592-597 18. Fangman, T. J., and S. F. Amass. (2007). Postpartum care of the sow and neonates. 784-788 in Current Therapy in Large Animal Theriogenology, 2nd ed. R. S. Youngquist and W. R. Threlfall, ed. Saunders, St. Louis, MI 19. Kirkden R.D., Broom D.M., Andersen I.L. (2013) Invited review: piglet mortality: management solutions. J Anim Sci. 91:3361-3389 20. Vasdal G, Glaerum M, Melisova M, Boe KE, Broom DM, Andersen IL. 2010. Increasing the pigs’ use of the creep area-a battle against biology? Appl Anim Behav Sci. 125:96-102.

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21. Milligan, B. N., D. Fraser, and D. L. Kramer. (2001). Birth weight variation in the domestic pig: effects on offspring survival, weight gain and suckling behaviour. Appl. Anim. Behav. Sci. 73:179-191 22. Vieuille C., Berger F., Le Pape G., Bellanger D. (2003) Sow behaviour involved in the crushing of piglets in outdoor farrowing huts - a brief report. Applied Animal Behaviour Science 80, 109-115] 23. Weary D.M., Pajor E.A., Fraser D., Honkanen A. (1996). Sow body movements that crush piglets: a comparison between two types of farrowing accomodation. Applied Animal Behaviour Science 49, 149-158. 24. Mazzoni C., Scollo A., Righi F., Bigliardi E., Di Ianni F., Bertocchi M., Parmigiani E., Bresciani C. (2018). Effects of three different designed farrowing crates on neonatal piglets crushing: preliminary study. Italian Journal of Animal Science. 17(2): 505-510. 25. Sala V., Gusmara C., C. Zolin C., Costa A. (2019). Piglets crushing rate related to sow foot lesions in the farrowing room. Large Animal Review. 25: 55-60. 26. Hoy, S. (2013). Freilaufbucht: Noch nicht praxisreif! Top Agrar. 6, 8-11.

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Ameni Askri et al. Large Animal Review 2020; 26: 249-256

Broiler’s performance and carcass characteristics improvement by prebiotic supplementation

249

AMENI ASKRI*1a, AZIZA RAACH-MOUJAHED1b, NACEUR M’HAMDI1b, ZIED MAALAOUI2, HAJER DEBBABI1a 1a

Department of Agro Food Industries, UR17GR01 PATIO; 1bDepartment of Animals’Sciences; National Agronomic Institute of Tunisia, University of Carthage, 43 Av. Charles Nicolle, Tunis 1082 2 Arm & Hammer Animal Nutrition, North Africa, Tunis, Tunisie, Adresse, Tunis 1002

SUMMARY The objective of the present study was to assess the influence of the supplementation of the increasing levels of Saccharomyces cerevisiae-derived prebiotic on broiler’s diets on their growth performances and carcass characteristics. A total of 192 male chicks Arbor Acres were randomly distributed into four dietary treatments with six replicates each and were housed in cages (8 birds/cage). Dietary mixtures in the experiments were as follows: the control group (T0) received the basal diet, and the experimental groups (T1, T2, and T3) received a basal diet supplemented with 1; 1.5 and 2 g/kg of prebiotic, respectively. Growth performances are measured; Weight Gain (WG), ADG (Average Daily Gain), Daily Feed Intake (DFI), and Feed Conversion Ratio (FCR) throughout the trial period. The carcass quality was also studied. It was observed that prebiotic supplementation enhanced the body growth rate. On the final day of the experiment, the body weight was significantly increased (P<0.01) in the treated groups in comparison with that of the control group. The highest achieved chicken body weight was in treatment T3 (2278.73±188 g) which was followed by treatment T1 (2215.73±179 g) with statistically significant differences (P <0.05). In carcass, the highest yield was recorded in dietary treatment T2 (76.21 %) which was statistically significant (P <0.05) higher compared to the control group (74.25%). Also, the supplementation of prebiotic to broiler’s diet decreases significantly the small intestine weight compared with the control (60.9±9.29 vs 65.7±10.17 g). In conclusion, our study has shown that the supplementation of the increasing levels of Saccharomyces cerevisiae-derived prebiotic in a broiler diet can improve growth performance.

KEY WORDS Broiler, carcass characteristics, levels, feed conversion ratio, prebiotic.

INTRODUCTION The use of antibiotics as growth promoters (AGPs) in poultry nutrition has been associated with the fast-growing nature of broiler chickens (Puva a et al., 2013; Sarica et al., 2005). Although, Donoghue (2003) affirmed that chicken reared with the addition of antibiotics achieved good performance but their potential side effects became a real public health global problem. Antibiotics lead to drug resistance in bacteria and drug residues in poultry products (Issa and Omer, 2012). Therefore, the wish to decrease the usage of antibiotics in animal production, replacements have been developed, such as probiotics, prebiotics, synbiotics, and herbal medicines (Castanon, 2007). Prebiotics were successfully used in the broiler diet as potential alternatives to antibiotics. By definition, prebiotics is non-digestible food ingredients fermented by intestinal microbiota. It beneficially affects the host by stimulating selectively the growth and/or activity of one or a limited number of bacteria in the colon (Gibson and Roberfroid,1995). Optimal characteristics of prebiotic were described by Patterson and Burk-

Corresponding Author: Ameni Askri (askria.ing@gmail.com)

holdar (2003): (1) prebiotics should not be hydrolyzed by animal gastrointestinal enzymes, (2) prebiotics cannot be absorbed directly by cells in the gastrointestinal tract, (3) prebiotics selectively enrich one or limited numbers of beneficial bacteria, (4) prebiotics alter the intestinal microbiota and their activities and (5) prebiotics improve luminal or systemic immunity against pathogen invasion. Several in vivo studies have shown that dietary supplementation of prebiotic had beneficial effects on productive traits and gut health. Prebiotics stimulate the proliferation of beneficial bacteria, inhibit the colonization of pathogenic bacteria, improve nutrient absorption, promote growth rate and feed utilization efficiency (Pourabedin et al., 2015; Mathlouthi et al, 2012). Commercial prebiotics is mainly obtained by enzymatic processes, impacting their cost of production and therefore their price for the farmers (Hajati and Rezaei, 2010). A preliminary study conducted by Askri et al. (2018) indicated that the administration of Saccharomyces cerevisiae-derived prebiotic to broilers could enhance growth performances, but has altered meat sensory quality. This study, therefore, was planned with the basic objective to optimize the inclusion levels of commercial prebiotic AVIATOR® in broiler diet for improving growth performance and carcass characteristics when prebiotic was removed one week before slaughter.

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Broiler’s performance and carcass characteristics improvement by prebiotic supplementation

Table 1 - Ingredient and nutritive values of the basal diet (g/kg). Ingredients (%)

Starter (d1-14)

Grower-Finisher (d15-42)

Corn

Soybean meal

Mineral and vitamin mixture Anticoccidial Total

None

100

Calculated nutrient Content MEC(Kcal/Kg)

2900

2970

Crude Protein %

20.5

19.5

Crude fiber %

Ash %

6.5

Fat %

Calcium %

0.9

Available Phosphorus %

0.67

0.66

Methionine %

0.5

0.44

Threonine %

0.8

0.78

Tryptophan %

0.3

0.25

Mineral mixture supplied (mg·kg-1 of diet): CF1: Mn. 80; Fer. 50; Cu. 25; Zn. 65; Co. 0.2; Se. 0.3; I. 1.2/ CF2: Mn. 70; Fer. 40; Cu. 20; Zn. 52; Co. 0.16; Se. 0.24; I. 0.69. BVitamin mixture supplied per kg of diet: CF1: Vit A. 13000 IU; Vit D3. 3500 IU; Vit E. 40 mg/ CF2: Vit A. 10400 IU; Vit D3. 2800 IU; Vit E. 32 mg. C ME: metabolizable energy.

cerevisiae such as mannan oligosaccharides (MOS), mannose, beta-glucans, and galactosamines. Following results found by Askri et al (2018), the prebiotic was removed one week before slaughter to avoid meat sensory quality alteration. The dietary treatments were: The control group received a basal diet (T0) without prebiotic. The experimental groups (T1, T2, and T3) received a basal diet supplemented with, respectively, 1; 1.5 and 2 g/kg of prebiotic. All experimental diets had the same nutrient level.

Measurements Performances Broiler chickens were weighed individually each week at the same time. Daily Feed intake (DFI) was calculated, during the whole experiment for each treatment, by the following mathematical formula: DFI (g/d/b =

Feed supplied (g)-Feed refused (g)

The average daily weight gain (ADG) was calculated as follow: ADG (g/d/b) =

Final Body Weight (g)-Initial Body Weight (g)

MATERIALS AND METHODS All procedures related to animal care, handling, and sampling were conducted under the approval of the Official Animal Care and Use Committee of National Agronomic Institute of Tunisia (protocol N° 05/15) before the initiation of research and followed the Tunisian guidelines.

Birds and housing This experiment was carried out in the poultry unit of the National Agronomic Institute of Tunisia. One hundred and ninety-two male day-old chicks from the “Arbor Acres” strain (average body weight: 45.53 ± 3.59 g) were used in the current trial over 42 days. All birds were individually identified, weighed, divided into four groups and were housed in individual cages. There were six replicates for each group with 8 chicks per cage. All birds were vaccinated against Newcastle Disease, Infectious Bronchitis, and Gumboro. The room temperature was gradually decreased from 33°C at day 3 to 24°C until the end of the experiment and continuous light was provided. Feed and water were supplied ad libitum throughout the experiment.

Dietary treatments The basal diet composition is presented in Table 1. It was composed of corn and soybean meal and was formulated according to the nutritional requirements for chickens (National Research Council, 1994). All chicks were fed starter and growerfinisher diets from 1 to 14 d and 15 to 42 d age, respectively. All diets were given in the floury form (fine particles) and did not contain antimicrobial growth promoters or coccidiostats. The prebiotic AVIATOR® is based on a yeast culture and products of the enzymatic hydrolysis of the yeast wall: Saccharomyces

Number of days (d)

And the feed conversion ratio (FCR) were calculated subsequently: FCR (g/g) =

Ethical considerations

Number of days (d)

Daily Feed Intake (DFI) Average Daily Gain (ADG)

Carcass characteristics At the end of the experiment, all birds had fasted for a period of 12 h with only water allowed. Birds were weighed individually and slaughtered by Halal Muslim method. Afterward, broiler organs including gizzard, liver, and heart were then extracted carefully. For the gizzards, after removing the surrounding fat, they were then opened and the contents were removed. All organs were weighed jointly. Thus, all eviscerated carcasses were refrigerated at 4°C for 24 h and weighed individually to calculate the eviscerated carcass yield (ECY). After cutting, chicken muscles (breast and thighs) were also weighed. Eviscerated carcass yield (%) =

Eviscerated carcass weight Live weight at slaughter

× 100

Data analysis A cage was the experimental unit for performance traits while the individual bird was the experimental unit for carcass and organ characteristics. Data were analyzed using the GLM general factorial ANOVA procedure using SAS 9.1.3 Statistical Analysis Software for Windows (SAS Institute: Cary, NC, USA, 2008). Prior analysis the residuals of the traits were tested for normal distribution. Dunnet’s test was applied to compare every mean to a control mean. Statistical significance was considered at P < 0.05. Additionally, regression (linear, cubic and quadratic) models were run to study dose-dependent responses.

RESULTS At arrival, birds showed an average body weight of 45.53 ± 3.59

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Table 2 - Effects of prebiotics on productive traits (WG, ADG, FI and FCR) in broilers on 42nd day of the experiment. Parameters T0 (Control)

T1 (1 g/kg)

T2 (1.5 g/kg)

T3 (2 g/kg)

P-value (ANOVA)

P-values of regression model Linear

Quadratic

Cubic

d 7-7 WG (g/b)

76.51±7.82

77.03±10.74

78.92±8.91

78.49±9.27

0.139

0.171

0.088

0.129

ADG (g/d/b)

10.93±1.12

11.00±1.53

11.27±1.27

11.21±1.31

0.317

0.131

0.320

0.321

DFI (g/d/b)

12.27±1.51

12.17±1.34

12.72±1.42

11.74±1.70

0.628

0.264

0.427

0.654

FCR (g/g)

1.21±0.05

1.12±0.05

1.13±0.09

1.04±0.13

0.892

0.798

0.867

0.892

d 7-14 WG (g/b) ADG (g/d/b)

169.33±16b ab

24.18±2.31

161.73±22c

162.29±20c

175.54±14a

0.043

0.047

0.038

0.031

25.07±2.11a

0.037

0.041

0.032

0.028

23.10±3.23

23.18±2.97

DFI (g/d/b)

37.28±4.7

36.47±3.7

34.38±2.5

37.81±1.8

0.044

0.054

0.042

0.045

FCR (g/g)

1.54±0.15a

1.59±0.18a

1.49±0.12ab

1.51±0.16ab

0.048

0.051

0.046

0.042

WG (g/b)

267.66±52c

275.45±43b

296.22±19a

d 14-21

277.86±15b

0.041

0.054

0.037

0.039

ADG (g/d/b)

38.23±7.45

39.35±6.22

42.31±2.81

39.69±2.15

0.039

0.047

0.052

0.038

DFI (g/d/b)

71.41±7.6b

74.52±10.931a

70.53±8.31b

60.73±3.71c

0.044

0.043

0.048

0.042

0.048

0.040

0.039

FCR (g/g)

1.91±0.25

1.94±0.16

1.67±0.19

1.53±0.13 d 21-28

362.26±52b

366.93±61ab

367.45±24ab

391.41±32a

0.048

0.042

0.034

0.031

51.75±7.51

52.41±8.73

52.49±3.55

55.91±4.64a

0.039

0.041

0.055

0.032

DFI (g/d/b)

108.19±14

107.79±20

102.34±11

105.63±11

0.050

0.047

0.043

0.039

FCR (g/g)

2.09±0.08a

2.11±0.64a

1.96±0.28b

1.90±0.36b

0.042

0.051

0.047

0.046

WG (g/b)

454.03±62b

422.15±38c

426.27±29c

0.051

0.045

0.043

0.038

ADG (g/d/b)

64.86±8.90

60.30±5.44

60.89±4.15

67.65±10.21

0.046

0.047

0.049

0.043

DFI (g/d/b)

138.43±18a

124.37±12b

115.49±16b

133.01±11a

0.043

0.044

0.053

0.035

0.049

0.047

0.052

0.043

WG (g/b) ADG (g/d/b)

d 28-35

FCR (g/g)

2.14±0.18

2.07±0.24

473.55±71a a

1.92±0.34

1.98±0.17 d 35-42

WG (g/b) ADG (g/d/b)

487.05±118ab

533.77±56a

460.11±51b

531.73±119a

0.047

0.052

0.046

0.039

75.96±17.09a

0.043

0.047

0.040

0.041

69.57±16.94

76.25±8.03

74.59±9.62

DFI (g/d/b)

105.67±9

128.33±9

108.34±14

118.07±11

0.045

0.051

0.047

0.036

FCR (g/g)

1.61±0.48b

1.69±0.21a

1.71±0.35a

1.62±0.38b

0.042

0.052

0.049

0.045

WG (g/b)

1816.83±310.12b

1837.08±232.48b

1791.29±155.36b

1928.61±262.76a

d 1-42

0 .034

0 .046

0 .028

0 .017

ADG (g/d/b)

43.25±7.38

43.74±5.53

42.46±4.08

45.91±6.25

0 .005

<0.001

DFI (g/d/b)

78.86±9.37a

80.60±9.74a

73.96±9.11b

77.83±6.98a

0 .048

0 .051

0 .043

0 .035

0 .057

0 .0 48

0 .041

0 .038

FCR (g/g)

1.73±0.20

1.74±0.24

1.64±0.23

1.61±0.22

WG= Weight gain (g/b); ADG= Average Daily Gain (g/d/b); DFI= Daily Feed Intake (g/d/b); FCR= Feed conversion ratio (g/g) Means within a row with different superscripts are significantly different (p<0.05). Values represent the Mean ± SEM of six replicates.

a–c

g. The results relative to performance parameters are presented in Table 2. During the starter period, the weight gain (WG) of prebiotic-supplemented birds did not significantly differ when compared with the control group (P=0.139). Moreover, no significant difference was noticed regarding feed intake (FI; P=0.628) and feed conversion ratio (FCR; P= 0.892) between birds fed increasing doses of prebiotic and control ones. Nevertheless, at week 3 the FI of the group receiving 2 g of prebiotic was significantly reduced as compared to the control group (P<0.05; 60.73 vs 71.41). Besides, FCR was significantly lower (P<0.05) in birds supplemented 2 g/kg of prebiotic (1.53) in comparison with the control group (1.91). At week 5 results showed a significant difference in FI between the control group

and the group receiving 1.5 g of prebiotic: the treated group presented a lower FI (138.43 vs 115.49). At the end of the experiment, results showed that the prebiotic supplementation had a significant effect on weight gain (P<0.05). Furthermore, the group receiving 2 g of prebiotic presented a higher weight gain compared to the control group, respectively 1928.61 and 1816.83 g. The average daily gain (ADG) of the treated group (2 g) was significantly (P=0 .005) higher (45.91 g) than the control group (43.25). Concerning the FI, results showed a significant difference between control and different groups fed prebiotic (P<0.05). Remarkably, WG was distinctly greater with the incorporation of 2 g of prebiotic in the broiler diet. Also, our study showed that FCR was significantly improved

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Table 3 - Effects of prebiotic supplementation on carcass and organs characteristics. Parameters Weight at slaughter (g) Hot Eviscerated Carcass (g) Hot Carcass yield (%) Cold Eviscerated Carcass (g) Cold Carcass yield (%)

T0 (Control)

T1 (1 g/kg)

T2 (1.5 g/kg)

T3 (2 g/kg)

P-Value (ANOVA)

p-values of regression model Linear Quadratic Cubic

2154.41±189b

2215.73±179a

2087.35±184b 2278.73±188a

0.024

0.035

0.026

0.024

1598.53±144 74.25±3.36b

1673.26±187

75.47± 5.69ab a

1589.52±144b 1727.36±175 76.21±3.90a b

0.026

0.043

0.045

0.026

76.13±4.86a

0.047

0.041

0.036

0.048

1542.17±143b

1614.52±179

1514.32±140

1642.84±78

0.035

0.029

0.044

0.035

71.63±3.49b

72.82±5.37a

72.62±4.37a

72.38±4.45a

0.043

0.058

0.047

0.038

Thighs (g)

440.57±69

Breast (g)

502.60±48b a

470.80±54

452.70±45

475.13±35

0.036

0.034

0.025

0.021

546.00±75a

509.00±61b

551.00±48a

0.027

0.038

0.029

0.022

Liver (g)

39.06±14

39.45±10

38.54±11

37.62±12

0.049

0.064

0.043

0.039

Heart (g)

9.98±1.69

11.77±1.78

10.78±3.34

10.87±2.23

0.082

0.079

0.074

0.069

Gizzard (g)

50.61±9.13

48.46±10.14

45.46±9.34

46.76±7.87

0.025

0.029

0.038

0.028

Gastrointestinal tract (g)

65.70±10.17a

65.80±8.56a

60.66±10.47b

60.90±9.29b

0.038

0.042

0.036

0.027

Small Intestine (cm)

178.76 ±0.19

183.42±0.17

172.63±0.10

170.16±0.24

0.497

0.643

0.438

0.392

Weight at slaughter (g); Hot Eviscerated Carcass (g); Hot Carcass yield (%); Cold Eviscerated Carcass (g); Cold Carcass yield (%); Thighs (g); Breast (g); Liver (g); Heart (g); Gizzard (g); Gastrointestinal tract (g); Small intestine (cm). Eight birds were evaluated from each group. a–c Means within a row with different superscripts are significantly different (p<0.05). Values represent the Mean ± SEM of six replicates.

(P=0.048; 1.53 g/g) by the administration of the highest dose of prebiotic during week 3. The effects of prebiotic on internal organs weight and carcass are presented in Table 3. Differences have been recorded when comparing the values for the different treatments to the control group on 42nd days of age. The highest achieved a weight of chicken at slaughter was observed in treatment T3 (2278.73±188 g) which was followed by treatment T1 (2215.73±179 g) with statistically significant differences (P= 0.024) compared to control group (T0). Treatments with the addition of prebiotics (T1, T3) achieved eviscerated carcass weight of 1673.26±187 g and 1727.36±175 g and which were statistically significantly (P<0.05) higher than the eviscerated carcass of broilers in control group (T0) (1598.53±144 g) and T2 (1589.52±144 g). The highest cold eviscerated carcass was observed in broilers in treatments T3 ( 1642.84±78 g). The cold carcass yield ranged from 71.63% for the control group (T0) to 72.82% for T1. Similarly, the weights of the thighs (475.13±35 g) and breast (551±48 g) were concluded to be the highest (P < 0.05) in the broiler’s receiving a basal diet complemented with 2 g of prebiotic. Regarding, the liver weight, the highest average value was noted in the control group (T0) compared to experimental broilers (P< 0.05). Nevertheless, no significant difference in heart weight among treatment group broilers (P=0.082) was observed. For the gizzard and gastrointestinal tract weight, a significant decrease (P< 0.05) was noticed in supplemented prebiotic broilers.

DISCUSSION Several researchers have demonstrated the positive effects of prebiotic supplementation on growth performances. Our results are in agreement with those of Bednarczyk et al. (2016) that indicated prebiotics addition could significantly increase body weight gain during the first three weeks. The result showed that chickens fed prebiotic supplementation had better final body weight in comparison with those received only basal diet. These results are in agreement with those of Biggs et al. (2007), Taherpour et al. (2009) and Murshed et al. (2015). Moreover,

the current study confirm results found by Askri et al. (2019) highlighted that this prebiotic should be present in broiler diet during the whole period for optimum growth performance. Nevertheless, many studies demonstrated that prebiotics had no significant effects on body weight, body weight gain, feed conversion ratio and feed intake (Mountzouris et al., 2007; Morales-López et al., 2009 and Houshmand et al., 2012a). The beneficial effects of prebiotic on FCR are in good agreement with previous studies (Oliva Das et al., 2017; Ahmed et al., 2015 and Mokhtari et al., 2015). On the other hand, Sohail et al. (2012) and Sherif et al. (2012) noted that the usage of prebiotic in broiler diet had no significant effect on feed intake and feed conversion ratio. Also, Midilli et al. (2008) observed no significant improvement in productive traits. Our study showed that the prebiotic administration impacted positively the carcass of broilers and the relative weight of some internal organs. Indeed, the cold carcass yield was more than the value observed by Abdel-Raheem and Abd-Allah (2011) who reported 64.45 to 70.68% in broilers at 42 days. Our findings are in agreement with those of Li and Zhang (2007) who stated that the use of prebiotic in broiler’s diet improves the breast muscle. Likewise, a study conducted by Maiorano et al., (2017) showed that birds supplemented with prebiotics had a higher breast muscle weight. Also, the latest researches found that prebiotic administration had a positive effect on breast muscle weight (Dankowiakowska et al., 2019; Tavaniello et al., 2018). In contrast, Wang et al. (2015) reported no significant effects of prebiotic-supplemented to broiler diet on breast muscle. Our results support the findings of Parsa, (2018); Wang and Gu. (2010); Çınar et al., (2009) and Mateova et al., 2008 who confirmed the growth-promoting effect of prebiotics supplementation. Likewise, Wang et al. (2015) found the highest liver weight when prebiotic was added at 0.13%. However, some other researchers held opposite views and stated that adding prebiotic to broilers diet did not affect liver and heart weight (Houshmand et al. 2012b; Li and Zhang, 2007; Bozkurt et al. 2008). These results agree with the findings of Waqas et al. (2018) who reported that all the carcass parameters including breast, liv-

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Ameni Askri et al. Large Animal Review 2020; 26: 249-256

er, heart and gizzard weight presented significant (P≤0.05) variations among supplemented prebiotic groups. Contrarly, Baurhoo et al. (2007) found no significant effect of different prebiotic supplementation on liver, heart and gizzard weights. On the other hand, results revealed no significant effect of prebiotic supplementation on intestinal weight corroborating the findings of Hosseini et al. (2016). Well established evidence by many researchers (Çınar et al., 2009; Lutfullah et al., 2011) showed that dietary containing additives reduced intestine weight and length. According to the above analysis, the results of group T3 broilers were optimal. Consequently, the optimum adding levels of dietary prebiotic were 2 g/kg.

CONCLUSION The presented data showed that the supplementation of Saccharomyces cerevisiae-derived prebiotic in broiler diet has a positive result on productive traits and in the improvement of broilers carcass yield. The use of prebiotics in the feeds for broilers determined the improvement of the slaughter yield by 1.9% for the supplemented group compared to the control group. These results confirm the favorable effects of prebiotic « AVIATOR®» on meat production. However, further investigations are needed to evaluate meat quality traits and consumers acceptance.

DATA AVAILABILITY The data sets are available upon request from the corresponding author.

ACKNOWLEDGMENTS The authors thank the National Agronomic Institute of Tunisia and the company Arm&Hammer Animal Nutrition for financial support.

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PUBBLICAZIONE ARTICOLI LARGE ANIMAL REVIEW I medici veterinari interessati alla pubblicazione di articoli scientifici sulla rivista “LARGE ANIMAL REVIEW” devono seguire le indicazioni contenute nel file Istruzioni per gli autori consultabili al sito www.largeanimalreview.com INFORMAZIONI: Segreteria di Redazione - largeanimalreview@sivarnet.it

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M. Bolcato et al. Large Animal Review 2020; 26: 259-264

Su di un caso di malformazione doppia (cefalotoracopagia) nel vitello

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MARILENA BOLCATO1, JOANA GONÇALVES PONTES JACINTO1, DAVIDE BOLOGNINI2, ARCANGELO GENTILE1 1 2

Dipartimento di Scienze Mediche Veterinarie, Alma Mater Studiorum - Università di Bologna Libero professionista, Castelfranco Emilia, Modena

RIASSUNTO La cefalotoracopagia è una rara forma di mostruosità doppia in cui i gemelli si presentano congiunti a livello di testa e torace; colonna vertebrale, arti e pelvi sono invece separati e gli organi interni possono presentare gradi variabili di duplicazione. Casi di gemelli congiunti sono stati descritti in tutte le specie domestiche, con incidenza maggiore nel bovino. Il caso presentato riguarda gemelli congiunti di sesso femminile, estratti a termine di gravidanza, già morti, per mezzo di taglio cesareo che mostravano una sola testa normoconformata, un ulteriore padiglione auricolare, un abbozzo di stoma, due toraci fusi tramite due sterni, due colonne vertebrali, otto arti e due code. Gli organi interni erano rappresentati da una seconda laringe ectopica, un cuore, due polmoni trasposti, un unico canale esofago-tracheale, un fegato megalico, due apparati prestomacali, due abomasi, due intestini, due milze, due uteri, quattro reni e due vesciche. La presente documentazione si va ad aggiungere alle sporadiche segnalazioni sull’argomento rappresentando una variante morfologica non precedentemente descritta. Le cause eziopatogenetiche, così come l’ontogenesi di queste mostruosità, non sono state ancora del tutto chiarite. È convinzione degli autori però che solo la segnalazione di tali evenienze, e la conseguente percezione della loro reale incidenza, potranno incidere significativamente sui dati relativi alla prevalenza e, auspicabilmente, sull’individuazione delle cause.

PAROLE CHIAVE Bovino, cefalotoracopago, Octopus, gemelli congiunti, co-gemello parassita acardiaco.

INTRODUZIONE Seppure allo stato delle cose statistiche epidemiologiche su eventi malformativi nella specie bovina non siano disponibili, è convinzione degli scriventi che, in generale, il numero delle malattie congenite - di tipo malformativo o di tipo disfunzionale - sia da considerare fortemente sottostimato. Ne è ragione non tanto (o non solo) la difficoltà diagnostica, ma anche e soprattutto la loro mancata segnalazione a strutture di ricerca: vuoi per motivi di reticenza culturale, vuoi per distrazione da parte di altri problemi apparentemente più impellenti, vuoi per accettazione di una perdita già avvenuta e non meritevole di ulteriore danno da spese diagnostiche, vuoi per difficoltà logistiche nell’inviare materiale patologico. Dalla serie delle mostruosità (“anomalia tanto grave da essere da sola sufficiente a far profondamente differire l’individuo che ne è affetto dall’individuo normale”) di cui abbiamo avuto esperienza in tempi recenti, riportiamo un caso di duplicazione del tipo dei “gemelli siamesi”, derivante, come tutti i difetti morfologici congeniti, cioè presenti dalla nascita, da un anomalo sviluppo embrionale. Il meccanismo di sviluppo dei gemelli congiunti (termine più appropriato rispetto alla definizione di siamesi) non è stato ancora compreso in modo completo; le ipotesi più accreditate sono che essi derivino o dall’incompleto perfezionamento della divisione embrionale (fissione) di due gemelli monozigoti (che

Corresponding Author: Marilena Bolcato (marilena.bolcato2@unibo.it).

avviene nei primi 13 giorni dopo il concepimento), oppure dalla (ri)fusione di due embrioni di gemelli, sempre monozigoti, già separatisi (siamo, quindi, indicativamente fra il 14° ed il 15° giorno dopo il concepimento)1,2,3,4. I gemelli congiunti possono essere simmetrici (completi o siamesi) o asimmetrici (incompleti). Nel primo caso entrambi gli individui hanno approssimativamente le stesse dimensioni e, fatta eccezione per le aree di fusione, sono quasi normali. Il punto di congiunzione è alla base della nomenclatura: craniopaghi (uniti a livello di cranio), cefalotoracopaghi (uniti a livello di testa e torace), toracopaghi (uniti a livello di torace), onfalopaghi (uniti a livello di addome), rachipaghi (uniti a livello di schiena), pigopaghi (uniti a livello di sacro) ed ischiopaghi (uniti a livello di regione pelvica)1,2. I gemelli parapaghi sono caratterizzati da una fusione latero-laterale, che si estende dal diaframma alla pelvi, con diversi gradi di coinvolgimento di cranio e torace e che si distinguono, a loro volta, in diprosopi (due facce), dicefali (due teste e due facce), ditoracici (due teste e due toraci)4. Nel caso dei vitelli asimmetrici, un individuo è quasi normale e l’altro (gemello parassita) è più piccolo, sviluppato in modo incompleto e “funzionalmente” dipendente dal primo5. Mostruosità doppie sono state descritte in tutte le specie domestiche: nella pecora, nella capra, nel maiale, più di rado nel cane, nel gatto e nel cavallo6,7,8,9,10,11. L’incidenza maggiore si rinviene nella specie bovina, dove alcune indagini le riportano in una percentuale tra l’1,9% ed il 17,5% delle malformazioni congenite5. In questa specie la duplicazione più frequente è quella toracopagica (64-75%). Tutte le forme rappresentano un rischio per il parto, anche se le fusioni del tipo craniopago, nel

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Figura 1 - Visione craniale della creatura mostruosa posizionata, con riferimento al “gemello 1” (quello “dotato” della testa), in decubito sternale. La linea punteggiata indica la colonna vertebrale del “gemello 1”; le stelline sono in corrispondenza con lo sterno assegnato al “gemello 2”; gli asterischi delineano la colonna vertebrale del “gemello 2”.

caso di impegno in sequenza del canale del parto, possono godere di una maggiore facilità di esecuzione11. Da un punto di vista eziologico, oltre alla predisposizione genetica, di volta in volta sono stati chiamati in causa fattori meccanici (manipolazione degli embrioni), fisici (esposizione a radiazioni ionizzanti), chimici (etanolo, vincristina, anestetici locali e generali, colchicina, acetaldeide, ormoni), ambientali e alimentari12,13. Nello specifico della specie bovina, sono stati riconosciuti come agenti teratogeni anche l’ipertermia, l’indebita pressione durante la palpazione rettale nella diagnosi precoce di gravidanza e la manipolazione di embrioni14.

DESCRIZIONE DEL CASO Quanto segue riguarda un caso di gemelli congiunti, entrambi di sesso femminile, estratti a termine di gravidanza, già morti, per mezzo di taglio cesareo. Il peso complessivo della creatura era di 43 kg. Per maggiore facilità di comprensione delle aberrazioni, i due soggetti di seguito verranno denominati “gemello 1” e “gemello 2”.

Figura 2 - Visione laterale, dopo asportazione della pelle, della creatura mostruosa posizionata, con riferimento al “gemello 1” (quello “dotato” della testa), in decubito sternale. La linea punteggiata indica la colonna vertebrale del “gemello 1”; le stelline sono in corrispondenza con lo sterno assegnato al “gemello 2”; gli asterischi delineano la colonna vertebrale del “gemello 2”.

Figura 3 - Visione laterale, dopo asportazione della pelle e dei piani muscolari del tronco, della creatura mostruosa posizionata, con riferimento al “gemello 1” (quello “dotato” della testa), in decubito sternale. Mancano anche i due arti dei costati convergenti sullo sterno del “gemello 2”. Si noti la ben evidente colonna vertebrale del “gemello 1”. Le stelline sono in corrispondenza con lo sterno assegnato al “gemello 2”; gli asterischi delineano la colonna vertebrale del “gemello 2”.

All’esame esterno la mostruosità era caratterizzata da una sola testa grossolanamente normoconformata connessa, tramite unica articolazione atlanto-occipitale, a due distinte colonne vertebrali. Non essendo possibile riconoscere ad uno dei gemelli la proprietà dell’unica testa, l’abbiamo assegnata arbitrariamente al “gemello 1” (Figura 1). Un padiglione auricolare ed un abbozzo di stoma erano presenti a circa 10 cm dalla convergenza delle colonne vertebrali, cranialmente ad uno dei due sterni di cui era dotato l’animale. Queste due strutture sono state attribuite al “gemello 2”. Seguivano due tronchi attaccati a livello dei costati; a questi erano poi connessi gli arti toracici, nel numero di quattro. La zona di unione somatica si estendeva fino alla regione ombelicale, che rappresentava il punto di passaggio fra parte congiunta e parte divisa dei due corpi. I cinti pelvici, infatti, erano completamente indipendenti e normo sviluppati. Nel totale si contavano, così, quattro arti pelvici. Lo scuoiamento dell’animale consentiva di meglio definire per quanto possibile in considerazione del gravissimo dismorfismo - la conformazione delle strutture scheletriche e di assegnare, ad ogni gemello, i rispettivi arti. Si notava così che a fronte di due colonne vertebrali distinte, coste e sterni dei due gemelli erano strutturati in modo da circoscrivere un’unica cavità o ambiente toracico: nel dettaglio di una situazione comunque difficilmente rappresentabile, la parete costale di sinistra del “gemello 1” si articolava, tramite uno sterno (apparentemente attribuibile al “gemello 2”), alla parete costale destra del “gemello 2” (Figure 2 e 3), la cui parete costale di sinistra a sua volta si articolava, tramite un altro sterno opposto (apparentemente attribuibile al “gemello 1”), alla parete costale di destra del primo gemello (Figura 4). Per rendere meglio l’idea, il tutto come se le due pareti costali di ogni gemello non si fossero “chiuse” sul proprio sterno ma si fossero “abbracciate” con quelle del fratello per il tramite di due strutture sternali; quale degli sterni fosse dell’uno o dell’altro non era possibile stabilirlo. In corrispondenza delle rispettive pareti costali, poi, si inserivano i cinti scapolari e, quindi, gli arti. L’ambiente toracico era diviso dall’addome per mezzo di due diaframmi fusi medialmente e conteneva un unico organo cardiaco e, per quanto riguarda l’apparato respiratorio, un unico polmone destro ed un unico polmone sinistro, entrambi comunque

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IL PORTALE DEL VETERINARIO

DI FIDUCIA

Il portale del Veterinario di Fiducia è una piattaforma multimediale rivolta a Medici Veterinari che svolgono attività clinica e manageriale negli allevamenti italiani. È gestito dalla SIVAR (Società Italiana Veterinari per Animali da Reddito - Federata ANMVI) che ne è anche proprietaria. Tutti i dati vengono trattati ai sensi della normativa sulla privacy. Il portale contiene: – notizie – materiali didattici – DES (Database Epidemiologico Sanitario) – DDD (Database per il Monitoraggio degli Antibiotici) – forum di discussione

DES

DDD

DEFINED DAlLY DOSE Software sperimentale che consente di calcolare le quantità di antibiotici somministrati sui propri allevamenti

CODICE EGO

➜

➜ DATABASE EPIDEMIOLOGICO SANITARIO Raccoglie dati sanitari ed epidemiologici di cui dispone solo il veterinario d’azienda

AREA LIBERA DEL PORTALE Alcune sezioni e funzioni (es. notizie e materiali didattici sono di libero accesso e non richiedono l’inserimento di credenziali (né password né username EGO). AREA RISERVATA DEL PORTALE Alcune sezioni e funzioni sono accessibili solo utilizzando il proprio Codice Ego (username e password) dopo averne richiesto l’attivazione alla casella: vetdifiducia@anmvi.it oppure info@sivarnet.it. Sono in area riservata le seguenti funzionalità: – DES (Database Epidemiologico Sanitario) – DDD (Database per il Monitoraggio degli Antibiotici) – forum di discussione – alcuni materiali didattci

Se sei un Socio SIVAR in regola con la quota annuale, puoi richiedere di essere abilitato scrivendo a: vetdifiducia@anmvi.it oppure info@sivarnet.it

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M. Bolcato et al. Large Animal Review 2020; 26: 259-264

Figura 4 - Visione laterale della creatura mostruosa in posizione supina se riferita al “gemello 1” (quello “dotato” della testa). La croce indica lo sterno assegnato al “gemello 2”; gli asterischi delineano la colonna vertebrale del “gemello 2”.

atelettasici e privi, prossimalmente, di apparato bronchiale principale. Il cuore, di forma globosa e contenuto in un unico pericardio, presentava una comunicazione interventricolare e pervietà del dotto di Botallo. Le quattro camere erano ben distinte, nonostante i due ventricoli avessero lo stesso spessore. Per quanto riguarda i polmoni, il loro posizionamento non consentiva di assegnarli all’uno od all’altro gemello: infatti il polmone sinistro era alloggiato in prossimità della parete costale destra del “gemello 1”, il polmone destro in prossimità della parete costale sinistra del “gemello 2”. Al centro dell’ambiente toracico, era presente un tubo del diametro di un pugno, che, per la continuità distale con la cavità addominale, era riconducibile all’esofago; poiché, però, prossimalmente si continuava nella regione cervicale dove entrava in comunicazione sia con la faringe che con la laringe, a questa struttura era riconosciuta una fusione con la trachea, confermata anche dalla presenza di abbozzi di anelli cartilaginei. Nella porzione più distale, poi, era annesso un rudimento di una seconda laringe: la posizione di questa era “curiosamente” adiacente al già citato abbozzo di stoma posto esternamente. Passato il diaframma l’esofago si divideva per continuarsi in due strutture prestomacali complete. Al pari di quello toracico anche l’ambiente addominale era unico. Fatto salvo un solo e comunque abnorme fegato - al quale si dirigevano due distinte vene ombelicali che si univano poco prima di immettervisi - tutti gli altri organi mostravano una tendenza alla fisiologica rappresentazione, sia morfologica che numerica: due erano le milze, quattro i reni, due gli uteri, due le vesciche. Due erano anche gli apparati prestomacali, gli abomasi e gli intestini. Per questi ultimi, però, si notava una comunanza del mesodigiuno ed un’ectopia dell’ansa spirale del colon nel “gemello 2”. Le sopra citate vene ombelicali confluivano in un unico anello ombelicale che, come già accennato, rappresentava il punto di demarcazione fra la parte di tronco congiunto e le due restanti parti di tronco disgiunte. A seguire, i due cinti pelvici erano entrambi nella norma, e vi erano associati normali sfinteri anali, vagine e vulve.

DISCUSSIONE E CONCLUSIONI Nella complessità della mostruosa aberrazione che abbiamo voluto documentare, il difetto è definibile come una gemellarità

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congiunta del tipo cefalotoracopago (cioè uniti nella parte superiore del corpo, nel nostro caso regione ombelicale compresa). Per quanto riguarda la parte cefalica, seppure apparentemente deradelfo, cioè con una sola testa, in effetti il “mostro” era dotato di un secondo abbozzo di testa, rappresentato da un padiglione auricolare ed uno stoma ai quali era contrapposta, nell’ambiente toracico, una rudimentale laringe. A differenza dei craniopaghi, dotati comunque di due teste unite o a livello frontale o parietale, i cefalotoracopaghi presentano normalmente una sola testa, eventualmente con due facce più o meno formate. Queste possono essere o contrapposte e complete (“janus“) o contrapposte ma con una delle due poco sviluppata o costituita dalle sole orecchie (“iniope”)2,4,5. A quest’ultimo aspetto, con uno sviluppo facciale ancora più rudimentale ed un solo orecchio, si avvicinava il nostro caso. Per quanto riguarda il tronco, la conformazione ed i rapporti reciproci delle due colonne vertebrali e dei rispettivi toraci, articolati a due sterni tramite emitoraci opposti, testimoniavano una fusione toracopagica cui in parte si erano associati i visceri toracici ed addominali. I gemelli congiunti cefalotoracopaghi, oltre alle varie anomalie scheletriche, presentano malformazioni degli organi interni incompatibili con la vita. Le anomalie più frequenti, riscontrate tanto in medicina veterinaria che in quella umana, sono localizzate lungo la linea mediana a testimonianza della difettosa duplicazione longitudinale dell’embrione2,4. È riportato in letteratura2 che quando i gemelli sono uniti a livello di torace e/o addome, sia sul piano mediano (toracopaghi, onfalopaghi) sia latero-lateralmente (parapaghi), è possibile che si verifichino situazioni di situs inversus, condizione in cui gli organi che normalmente si trovano nel lato destro del corpo sono invece situati nel sinistro e viceversa. Nel nostro caso, i gemelli mostravano segni di situs inversus toracico considerato che il polmone destro era alloggiato in un emitorace sinistro ed il polmone sinistro in un emitorace destro. Per quanto riguarda il cuore, differentemente da quanto maggiormente descritto in letteratura2, il nostro cefalotoracopago ne presentava uno unico. Pur in assenza di elementi probatori, abbiamo assegnato arbitrariamente il cuore al “gemello 1”, quello che, con pari libertà interpretativa, abbiamo riconosciuto come “dotato” della testa. La dipendenza del “gemello 2” dall’organo centrale del circolo dell’altro gemello per il suo sviluppo e la sua vita fetale, gli è valsa la definizione di co-gemello parassita acardiaco2. Questa situazione è spesso correlata alla presenza di un solo fegato, enormemente aumentato di volume2, come effettivamente accaduto nei nostri gemelli. Ancora, tra le varie anomalie, fusioni e duplicazioni che queste mostruosità possono presentare, a livello di apparato digerente e respiratorio è spesso rilevabile un’anomala differenziazione del setto esofago-tracheale, che nel nostro caso aveva portato alla formazione di un unico canale esofago-tracheale, con una faringe ed una laringe complete ed un addizionale rudimento laringeo ectopico. Nell’uomo, è riportata una predominanza di gemelli congiunti di sesso femminile, la cui eziopatogenesi rimane ignota2. In questi casi, soprattutto quando la fusione coinvolge la porzione caudale del corpo, innumerevoli possono essere le dismorfie degli apparati genitale ed urinario2. Nel nostro caso, considerato anche che il punto di fusione non coinvolgeva la regione pelvica, il tratto genitourinario delle due femmine si presentava normoconformato e ciascun gemello era dotato di propri reni, vescica, utero e vagina.

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Su di un caso di malformazione doppia (cefalotoracopagia) nel vitello

La presente documentazione si va ad aggiungere alle segnalazioni sull’argomento rappresentando una variante morfologica non precedentemente descritta. Le mostruosità doppie sono evenienze sporadiche in tutti i mammiferi e molti aspetti di tipo eziopatogenetico ed ontogenetico sono ancora controversi e solo al livello di ipotesi. Nel nostro caso, purtroppo, non è stato possibile risalire, neanche in termini di sospetto, ad una di quelle ipotesi causali di cui si è dato cenno in precedenza. Rimane valida la considerazione, però, che solo l’emersione di tali evenienze (e questo vale per tutte le malformazioni) e la conseguente percezione della loro reale incidenza, possono rappresentare motivazione e spinta ad uno studio più approfondito e quindi giustificazione di investimento di risorse ed impegno da parte del mondo scientifico, oggi, purtroppo, fortemente condizionato non tanto da genuino interesse speculativo ma da motivazioni economiche che portano a trascurare malattie apparentemente di scarso impatto. A chiusura di tale scritto, ci permettiamo di esortare i veterinari operanti nella pratica a dare valore ad ogni evento malformativo, favorendo, anche attraverso la sensibilizzazione degli allevatori, la segnalazione a centri di ricerca. Ne potranno trarre giovamento i dati relativi alla prevalenza e, auspicabilmente, l’individuazione delle cause.

two tails, two ani and two vulve normally developed were noticed. The dissection enabled to better understand the skeletal conformation. In particular, the left thoracic wall of twin 1 was linked to the right thoracic wall of twin 2 through one of the two sterna. The right thoracic wall of twin 1 was linked to the left thoracic wall of twin 2 through the other sternum. Within the thorax, a single heart, two transposed unaerated lungs (situs inversus) and a severe megaesofagus, fused with the trachea, were present. In the abdominal cavity a single but enlarged liver was present. Two umbilical veins, entering the body from a unique umbilical ring, divided before entering the liver. Each twin had its own rumen, reticulum, omasum and abomasum as well as its own jejunum, ileum, caecum, colon and rectum. The two mesojejunum were conjuncted. An ectopia of the spiral loops of the colon was also present in twin 2. Two spleens, two uteri, four kidneys and two bladders were also detected. The lack of reliable information on the prevalence of such kind of malformation motivated the Authors to present this case. The reporting of malformed calves constitutes an indispensable step for improving the knowledge and recognize the etiology of these diseases.

❚ A case of double malformation (cephalothoracopagus) in a calf

Bovine, cephalothoracopagus, Octopus, conjoined twins, acardiac parasitic co-twin.

SUMMARY The mechanism of the development of conjoined twins is not completely understood, and such far two main hypotheses can be considered: fission and fusion. The fission hypothesis proposes that conjoined twins arise from the incomplete division of a single fertilized ovum and thus they are the result of the failure of the separation of monozygotic twins; the fusion hypothesis proposes that conjoined twins arise from the re-fusion of two previously already separated monozygotic twins. Various authors have described the teratogenic influence of genetics and environmental (viruses, plants, drugs) factors suspected of causing duplication defects. The definition of the different aspects of this malformation is based on the localization of the conjunction: “craniopagus” is the conjunction of the heads, “cephalothoracopagus” of the entire upper part of the body, “thoracopagus” of the thorax, “omphalopagus” of the abdominal wall, “rachipagus” of the back, “pigopagus” of the sacral region, “ischiopagus” of the pelvic region. Parapagus is the term used to indicate a latero-lateral fusion. The aim of this study is to present an interesting case of conjoined twins in bovine. Morphological aspects, etiopathogenetic and possible classification of the conjoined twinning are also discussed. A stillborn female Holstein conjoined twins (twin 1 and twin 2) were referred for clinicopathological examination. External examination showed a single head linked by a unique atlantooccipital joint to two separated vertebral columns. An additional auricular pinna and a primitive stoma were also present. Upper part of the twins bodies were joined through the fusion of the thoracic walls resulting in a four-sided thorax with two sterna. Four forelimbs were linked to this thorax. The conjunction of the trunk extended until the umbilical region; caudally, two identical and symmetrical bodies presenting four hindlimbs,

KEY WORDS

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