Large Animal Review 3-2021 by E.V. Soc. Cons. a r.l.

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

03/21

Bimonthly, Year 27, Number 3, June 2021

LAR

Large Animal Review

ISSN: 1124-4593

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

ORIGINAL ARTICLES BOVINE • Evaluation of the effect of artificial collapse on the viability of vitrified bovine blastocysts • Digestibility and sorting of hay-based total mixed rations employed in the Parmigiano-Reggiano area as affected by dietary particle size distribution • In vitro maturation of bovine oocytes may using royal jelly as protein source in the culture media

OVINE • Serum cardiac troponin I concentrations in ewes diagnosed with parturient paresis: correlation with blood ionized calcium and conventional cardiac enzymes

SWINE • Enteropatia proliferativa da Lawsonia intracellularis nel suino

EQUINE • Study of postnatal growth of mule and donkey foals sired by the same jackass

CASE REPORTS BOVINE • Management of post-urethral urinary obstruction due to struvite uroliths in a female buffalo calf (Bubalus bubalis)

POULTRY • First isolation of Salmonella Duisburg from quail flock

SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

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INDEX

Anno 27, numero 3, Giugno 2021

BOVINE Evaluation of the effect of artificial collapse on the viability of vitrified bovine blastocysts KUBRA KARAKAS ALKAN, METEHAN OZKAN, MEHMET RIFAT VURAL, MUSTAFA KAYMAZ

Rivista indicizzata su: CAB ABSTRACTS e GLOBAL HEALTH IF (2019): 0.299

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Digestibility and sorting of hay-based total mixed rations employed in the Parmigiano-Reggiano area as affected by dietary particle size distribution

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

MARICA SIMONI, ARMANDO CANEPA, GRAZIANO ROSARIO PITINO, AFRO QUARANTELLI, FEDERICO RIGHI

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In vitro maturation of bovine oocytes may using royal jelly as protein source in the culture media EMRE ŞİRİN, MEHMET KURAN

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

Consiglio direttivo SIVAR 2020-2023 Mario Facchi (Presidente) Daniele Gallo (Presidente Senior) Alberto Ferrero (Vice-Presidente) Michela Conterbia (Segretario) Vito Loconte (Tesoriere) Alessandro Federici (Consigliere) Osvaldo Parolin (Consigliere) Chiara Musella (Consigliere) Mattia Bottacini (Consigliere) Giuseppe Argiolas (Consigliere) Edizioni SCIVAC Palazzo Trecchi - 26100 Cremona Tel. 0372/460440 Iscrizione registro stampa del Tribunale di Cremona n. 299 del 25/9/1995

OVINE Serum cardiac troponin I concentrations in ewes diagnosed with parturient paresis: correlation with blood ionized calcium and conventional cardiac enzymes KENAN ÇAĞRı TÜMER, MEHMET ÇALIŞKAN, TARıK ŞAFAK

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SWINE Enteropatia proliferativa da Lawsonia intracellularis nel suino GIULIA D’ANNUNZIO, ROBERTO BARDINI, FABIO OSTANELLO, GIUSEPPE SARLI

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EQUINE Study of postnatal growth of mule and donkey foals sired by the same jackass AUGUSTO CARLUCCIO, ALBERTO CONTRI, ALESSIA GLORIA, DOMENICO ROBBE, GIORGIO VIGNOLA

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

CASE REPORTS

Spedizione Poste Italiane SPA - Spedizione in A.P. D.L. 353/2003 (Conv. in L. 27/02/2004 N. 46) Art. 1, Comma 1, DCB Piacenza Concessionaria esclusiva per la pubblicità E.V. Soc. Cons. a r.l. Palazzo Trecchi - 26100 Cremona Ufficio Pubblicità: Paola Orioli Tel. 0372/403539 - E-mail: info@sivarnet.it 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.

BOVINE Management of post-urethral urinary obstruction due to struvite uroliths in a female buffalo calf (Bubalus bubalis) KALLEMUCHIKAL MANIKANDAN MANJUSHA, KHAN SHARUN, ELANGOVAN KALAISELVAN, ROHIT KUMAR, ABHISHEK CHANDRA SAXENA, PRAKASH KINJAVDEKAR, ABHIJIT MOTIRAM PAWDE, AMARPAL 175

POULTRY First isolation of Salmonella Duisburg from quail flock OZGE ARDICLI, SERPIL KAHYA DEMIRBILEK, HAVVA KURNAZ, KAMIL TAYFUN CARL

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K.K. Alkan et al. Large Animal Review 2021; 27: 115-121

Evaluation of the effect of artificial collapse on the viability of vitrified bovine blastocysts

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KUBRA KARAKAS ALKAN1*, METEHAN OZKAN2, MEHMET RIFAT VURAL2, MUSTAFA KAYMAZ2 1

Selcuk University, Faculty of Veterinary Medicine, Department of Obstetrics and Gynaecology, Konya, Turkey Ankara University, Faculty of Veterinary Medicine, Department of Obstetrics and Gynaecology, Ankara, Turkey

SUMMARY The present study aimed to evaluate the viability of the embryos following freezing and warming by vitrification after artificial collapse in bovine embryos produced in vitro. In vitro maturation, fertilization, and culture procedures were performed using oocytes obtained from ovaries collected from slaughterhouses. Embryonic development was evaluated and recorded. In total, 289 blastocysts were obtained after in vitro production, and 61.94% (179/289) of the obtained blastocysts were graded as Code I (excellent or good) quality. Only Code I embryos were used in the study and 60 of these blastocysts were artificially collapsed (Group 1) and 60 of them used as control (Group 2). Blastocoelic fluid of the blastocysts from group 1 was aspirated by entering through trophoblast cells using microinjection pipettes with a micromanipulator system. Thereafter, blastocysts from both groups were vitrified and warmed with ethylene glycol and glycerol-based protocols and embryonic development was monitored for 24 hours. The post-warm rate of re-expanded blastocyst was 96.66% (58/60) and 91.66% (55/60) in Group 1 and 2, respectively (P > 0.05). The viability rates at 24 hours were 91.66% (55/60) and 78.33% (47/60) (P > 0.05), and hatching rates were 65% (39/60) and 11.66% (7/60) (P < 0.05) in Group 1 and 2, respectively. Consequently, it was found that in vitro produced blastocysts can be vitrified after artificial collapse and embryo development and viability rates following warming are quite high.

KEY WORDS Artificial collapse, blastocyst, bovine; embryo viability, vitrification.

INTRODUCTION Embryo transfer is an assisted reproductive technology that involves the transfer of embryos obtained from high-yield donors to recipients1,2. Embryo production can be performed in both in vivo and in vitro conditions3. According to the International Embryo Technology Society (IETS), it has been reported that the total annual number of embryos produced in vivo and in vitro is 1.5 million, with greater in vitro embryo production compared with in vivo production4. Cryopreservation of embryos is an important step after both in vivo and in vitro production of an embryo2,5. This technique can be readily used if there is no favorable condition for embryo transfer, the time of transfer is not proper for recipients, and a large number of embryos have been obtained and transport or storage of the embryos is required. Moreover, cryopreservation allows the conservation of genetic resources of endangered animals and the establishment of a gene bank6,7. The

• A preliminary report of these data was presented at the 3rd International Congress on Advances in Veterinary Science and Technics (ICAVST), in September 2018. Corresponding Author: Kubra Karakas Alkan (kubrakarakas@gmail.com).

obtained embryos can be frozen using controlled freezing (slow freezing) methods or vitrification. Vitrification is the most preferred cryopreservation method worldwide because it is cheap and fast and easily applicable8,9. Compared with slow freezing, vitrification is used more often because it results in greater enhancement of post-warm re-expansion and hatching rates of embryos produced in vitro6,10. The embryo reached the blastocyst stage is the optimal time for cryopreservation because of its high cell count and differentiation11. Similar to slow freezing, the initial condition in vitrification is cellular dehydration. Water is excreted through osmosis from the water channels called the aquaporin of the embryo exposed to hypertonic cryoprotectant substances. The embryo rapidly shrinks owing to the outflow of water in the embryo and the entry of cryoprotectants into the cell. The second osmosis occurs when the embryo begins to be exposed to subzero degrees after achieving an intercellular fluid balance. However, because of the fluid-filled cavity called blastocoel, the blastocyst is sensitive to freezing. It has been emphasized that the survival ability of the embryo decreases after vitrification owing to the increased volume of the blastocoel. As the blastocoel volume increases, the influx of cryoprotectants into the blastocyst becomes insufficient, leading to an increased risk of ice crystal formation. The liquid remaining in the blastocoel reduces the survival ability of the embryo during thawing6,11. Owing to these reasons, when the embryos obtained from humans

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Evaluation of the effect of artificial collapse on the viability of vitrified bovine blastocysts

need to be frozen, artificial collapse (AC) is routinely performed as first mentioned by Vanderzwalmen et al. (2002)12 and it has been reported that subsequent vitrification procedures reduced DNA damage in the embryo and that post-thaw re-expansion and ability of survival were more successful12-17. In this point of view the applicability of this method in in vitro produced bovine embryos was tried to be determined. Also, the present study evaluated the effect of artificial collapse prior to vitrification on the viability, development, and hatching process of the in vitro produced embryos following warming.

MATERIAL AND METHODS The study was approved by the Local Ethics Committee (Ankara University Local Ethics Committee for Animal Experiments, Approval Number: 2016-5-73).

Collection of bovine ovaries and oocyte retrieval Ovaries of Holstein breed cows were collected from a slaughterhouse in Ankara, Turkey and transferred to the laboratory in 0.9% isotonic NaCl solution (Deva, Turkey) containing 50 µg/ml gentamicin (Sigma-Aldrich, USA) at 20°C for a maximum of 4 hours. The ovaries were washed at least twice with 0.9% NaCl solution to remove the surrounding tissues, blood, and transport medium. Washed ovaries were left in isotonic solution until oocyte aspiration. The follicle fluid containing the cumulus-oocyte complexes (COCs) was aspirated from the 28-mm diameter peripheral follicles found in the ovaries using an 18G needle with a pressure adjustable (180-200 mmHg) aspiration pump. The collected follicle fluids were taken into conical centrifuge tubes. These tubes were kept for 5-10 minutes at the end of the aspiration procedure to allow the oocytes to settle to the bottom. COCs accumulated at the bottom of the tubes were washed two times in medium containing 1% calf serum (CS, Sigma-Aldrich, USA) + Ringer’s solution (Polifarma, Turkey) to separate them from other cells in the tubes. After washing was completed, the medium containing COCs was transferred into 90-mm diameter Petri dishes and examined under a stereomicroscope. The obtained COCs were evaluated as previously described18.

In vitro maturation COCs detected under the microscope were collected and placed into 60-mm Petri dishes containing medium (1% CS + Ringer’s solution). Collected COCs were transferred into 35mm Petri dishes containing 25 mM HEPES-buffered M-199 (Sigma-Aldrich, USA) + 5% CS + 0.02 mg FSH (FolltropinV, Vetoquinol, Canada) + antibiotic (maturation medium) af-

ter washing at least five times at different points of the 60 mm Petri dishes. Then, COCs were taken into maturation drops (5 µl for each oocyte) and each drop contained 20 oocytes. Oocytes were matured in an incubator at 38.5°C with 5% CO2 for 2224 hours at maximum humidity.

In vitro fertilization Frozen semen of an only one Holstein bull was used for sperm treatment in the study. Before in vitro fertilization, Percoll (Sigma-Aldrich, USA) gradient technique was used to select motile spermatozoa19. The final sperm concentration was determined as 3 × 106/ml. Afterward, the sperm suspension prepared for in vitro fertilization was taken into 35-mm Petri dishes in drops and covered with paraffin liquid. The COCs extracted from the maturation medium were washed twice in Brackett and Oliphant’s solution containing 10 mg/ml BSA (SigmaAldrich, USA), then taken into drops contained fertilization medium (Tyrode lactate solution supplemented with BSA, Sodium pyruvate, penicillin, streptomycin) and cultured in an incubator at 38.5°C with 5% CO2 at maximum humidity for 1820 hours (Day 0).

In vitro culture After fertilization, presumptive zygotes were washed in embryo culture medium. Denudation was performed for the removal of the cumulus cells and sperm. Presumptive zygotes that were completely peeled from the cumulus cells were then transferred into the culture medium. CR1aa + 5% CS + 0.25 mg/ml linoleic acid albumin (Sigma-Aldrich, USA) culture medium were used for embryo culture20. Presumptive zygotes taken into embryo culture medium drops were cultured in an incubator at 38.5°C with 5% CO2 at maximum humidity for 7 days.

Evaluation of obtained embryos Day-7 embryos after in vitro culture were evaluated according to the IETS criteria21. Accordingly, the codes for embryo quality were graded as follows: Code I (excellent or good) represents symmetrical and spherical embryo mass, uniform blastomeres in size, color, and density, and ≥85% intact cellular material; Code II (fair) represents ≥50% intact cellular material and vital embryonic mass, moderate irregularity of the blastomeres in size, color, and density, and ≤25% fragmentation; Code III (poor) represents marked irregularity of the blastomeres in size, color, and density and ≥25% intact cellular material and vital embryonic mass; Code IV (death or degenerated) represents degenerated embryos and unfertilized oocytes. Code I quality blastocysts were randomly selected; 60 of these embryos were used for AC (Group 1), and 60 were used only for vitrification without AC (Group 2, control).

Artificial Collapse Table 1 - Re-expansion, viability and hatched rates in blastocysts following vitrified/warmed. Number of Blastocysts (n)

Re-expanded Viable Hatched blastocysts blastocysts blastocysts (%) (%) (%)

Group 1 (AC)

58 (96.6)

55 (91.6)

39 (65)

Group 2 (Control)

55 (91.7)

47 (78.3)

7 (11.6)

>0.05

<0.05

On day 7/8 of embryo development in the Group 1, blastocoelic fluid was aspirated with a micromanipulator (Olympus, IX73, Japan) and microinjection pipette (Eppendorf TransferMan 4R, Germany) in embryo culture medium22. During this procedure, all of the fluid was aspirated by a microinjection pipette inserted between the trophoblast cells without interference with the inner cell mass until the blastocoel was completely collapsed. After that, the blastocysts immediately transferred into vitrification solution. No manipulation was applied to the embryos of the control group.

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K.K. Alkan et al. Large Animal Review 2021; 27: 115-121

Vitrification of the embryos Embryos in both groups were cryopreserved by vitrification. First, embryos were transferred into the vitrification solution (VS)-1 (10% Glycerol + 0.1 M Sucrose + 1% BSA) and kept for 5 minutes. Afterward, embryos were taken into the VS-2 (10% Glycerol + 10% Ethylene Glycol + 0.2 M Sucrose + 2% BSA) and kept in this solution for 5 minutes; finally, following the transfer to the VS-3 (10% Glycerol + 10% Ethylene Glycol + 0.3 M Sucrose + 3% BSA), embryos were pulled to the 0.25 ml straws (one straw for one embryo) within 1 minute and the open part of the straw was closed by a plug. Later, the straw was placed into liquid nitrogen and vitrification was completed.

Warming of the embryos For devitrification, the straws protected in the liquid nitrogen were warmed in two phases, for 10 seconds in the air and 20 seconds in a 30°C water bath. Subsequently, to purify the toxic level of sucrose in the embryo, they were initially transferred into a Petri dish containing mD-PBS (Sigma-Aldrich, USA) 0.5 M sucrose CS and washed for 5 minutes. Then, washing was done with lesser concentrations of mD-PBS + 0.25 M sucrose + 20% CS solution for 5 minutes. In the final stage, equilibration was performed in mD-PBS + 20% CS solution for 5 minutes. After devitrification, embryos were placed into 25 µl drops of M-199 + 20% FCS + 0.1 mM β-mercaptoethanol (SigmaAldrich, USA) for culture and incubated at 38.5˚C with 5% CO2 for 24 hours. During the 24-hour incubation period, the development of embryos was observed under a stereomicroscope. During incubation, the re-expansion of embryos and achievement of the advanced embryonic developmental stage (expanded, hatched embryo) were used to determine the viability of these embryos. In addition, embryos were followed up in terms of hatching during the first 24 hours.

Statistical analysis For discrete and continuous variables, descriptive statistics were given. In addition, the homogeneity of the variances, which is one of the prerequisites of parametric tests, was checked through Levene’s test. The assumption of normality was tested via the Shapiro-Wilk test. To compare the differences between two groups, Student’s t test was used when the parametric test prerequisites were fulfilled, and the Mann Whitney-U test was used when such prerequisites were not fulfilled. The data were evaluated via SPPS 25 (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.). P<0.05 and P<0.01 were taken as significance levels.

RESULTS In total, 289 blastocysts were obtained in the study. 179 of these embryos were evaluated as Code 1 (61.94%), 51 of them as Code 2 (17.65%) and 39 of them as Code 3 (13.49%). Only Code I quality embryos were used in the study and 60 of these embryos were included in group 1 and 60 in group 2. After the vitrified embryos were warmed according to the procedure, they were transferred back to the culture medium and the developmental processes were evaluated for 24 hours. The rates of re-expansion, viability, and hatched blastocysts at the end of 24 hours are given in Table 1. The re-expansion of embryos and achievement of the advanced embryonic developmental stage during incubation indicated the viability of

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these embryos. No statistical difference was found between reexpanded and viable blastocyst rates in the AC and control groups. However, the rate of hatched blastocysts was higher in the AC group compared to the control group (P <0.05).

DISCUSSION Intracellular ice formation or high concentration compounds used during cryopreservation affects the transcription of developmentally important genes and the viability of the cells, thus reducing the survival ability of the embryo and the rate of conception9,10. One of the methods that can be used to eliminate these problems is to reduce the blastocoelic fluid before vitrification12. Artificial collapse of the blastocyst before vitrification is a new approach to improve the viability of the blastocyst after warming. This technique helps to reduce the damage caused by ice crystal formation by reducing the amount of fluid in the blastocoel15. Several studies have reported higher rates of re-expansion, viability, and hatching of the embryos frozen or thawed after the blastocoelic fluid was reduced12,14,15,23. This study evaluated the effect of this method, which was previously used in humans, on development of bovine embryos produced in vitro. According to the obtained findings, the re-expansion rates after vitrified/warmed were 96.7% and 91.6% in Group 1 and 2, respectively (P > 0.05). The reason for the lack of difference between the re-expansion rates is thought to be due to the recovery of blastocoelic fluid following devitrification in both groups. However, it is thought that there were problems in the steps after re-expansion in the control group. Returning to the pre-freezing phase is very critical for the embryos to continue their development after freezing/thawing24. There is a close relationship between the success of vitrification and the expansion of the blastocoel because the large blastocoel before freezing causes insufficient cryoprotectant permeation and dehydration12. Kovacic et al. (2018)25 have reported that the blastocyst volume inside the zona pellucida was significantly lower in the group with artificial collapse following warming than in the control group. However, the large volume of blastocysts in the control group is thought to be owing to partially persistent fluid accumulation before vitrification. However, this persistent fluid may have a toxic effect on the embryo after warming25. Desai et al. (2008)15 have reported greater cell damage in blastocysts without artificial collapse than in collapsed blastocysts. This may be because of insufficient dehydration of the blastocoel and a slower rate of recovery/re-expansion of non-collapsed blastocyst15. In the present study, survival rates after vitrified/warmed were 91.6% and 78.3% in groups 1 and 2, respectively (P > 0.05). Although there was no statistical difference between the survival rates of the embryos in both groups, the survival rate in the AC group was higher than in the control group. The reason for this low blastocyst viability rate in the control group is thought to be due to the ice crystals formed during vitrification. Because blastocoelic fluid influence the permeation of cryoprotectant during vitrification, and this cause intracellular ice crystal formation. Also, Ha et al. (2010)26 and Min et al. (2013)27 have found that artificial collapse treatment increases the survival rate in cattle. In previous studies by Desai et al. (2008)15 and Levi-Setti et al. (2016)28 in humans and Kazemi et al. (2016)29 and Pooyanfar et al. (2018)30 in mice, it was re-

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ported that the survival rate did not improve by artificial collapse of blastocysts. However, in these studies, artificial collapse was performed with the microneedle technique. In contrast, studies have reported that artificial collapse with laser pulse leads to an increase in the survival rate31,32. Moreover, Van Landuyt et al. (2015)23, Darwish and Magdi (2016)33, and Kovacic et al. (2018)25 have noted increased survival rates after artificial collapse using a laser pulse. This may be owing to the lower probability of damage to the trophectoderm cells of the embryo with a laser pulse33. However, Wang et al. (2017)32 have reported that artificial collapse of blastocysts using a microneedle and laser pulse did not affect the survival rates of the embryos. In the present study, the rates of hatched embryos were quite high in Group 1 compared with Group 2 (65% vs. 11.6%, respectively; P < 0.05). The higher rate of hatched blastocyst in the AC group is thought to be due to the higher survival rate in this group and the lower rate of damaged cells. Because embryos with a lower rate of damaged cells continue to divide and reach the hatched stage. Thus, vital cells and contractions are essential for the hatching of the embryo. However, damaged cells observed in the embryos subjected to standard vitrification may preclude the hatching process. The lesser number of damaged and apoptotic cells in embryos subjected to artificial collapse can facilitate the hatching process25,27,30. Min et al. (2014)34 found that the implementation of forced collapse prior to vitrification increased the hatching rate after warming in their study on cattle. Ha et al. (2010)26 reported that the hatching rates of the embryos at the 12th hour after warming in the group with artificial collapse prior to vitrification and in the control group were 51.7% and 0%, respectively. In previous studies by Cao et al. (2014)31 and Van Landuyt et al. (2015)23 in humans and Kazemi et al. (2016)29 in mice, it was reported that higher hatching rates in embryos were observed with artificial collapse. Therefore, it has been established that the implementation of artificial collapse before vitrification is effective in increasing the hatching rates after warming.

3. 4. 5. 6.

8. 9.

10.

11. 12.

13.

14.

15.

16.

17.

18.

CONCLUSIONS

19.

The reduction of blastocoelic fluid by a micromanipulator in in vitro produced bovine embryos has no statistically significant effect on the re-expansion and survival of post-warm embryos; however, improved hatching rate was observed in the group with artificial collapse. Therefore, the implementation of artificial collapse before freezing of embryos produced in vitro by vitrification may positively affect post-warm embryo development and hatching. Nevertheless, future studies involving cattle are needed to evaluate the effect of this procedure on the conception rate after the transfer.

20.

21. 22.

23.

Acknowledgements This study was supported by The Scientific Research Projects Chieftaincy of Ankara University (Project No: 15A0239003).

24.

References 1.

Hasler J.F. (2004). Factors influencing the success of embryo transfer in cattle. Page 66 in Proc. Proceedings of the World Buiatrics Congress, Québec, Canada. Mapletoft R.J., Bo G. (2016). Bovine embryo transfer. In: International Veterinary Information Service, Ed. Mapletoft R.J., Bo G., 1st ed., IVIS, Ithaca, USA.

25.

26.

Gordon I. (2004). Reproductive technologies in farm animals. 1st ed., Cambridge: CABI, Oxford, UK. Viana J. (2019). 2018 statistics of embryo collection and transfer in domestic farm animals. Embryo Transfer Newletter, 32: 14-26. Arav A. (2014). Cryopreservation of oocytes and embryos. Theriogenology, 81: 96-102. Do V.H., Walton S., Taylor-Robinson A.W. (2014). Benefits and constraints of vitrification technologies for cryopreservation of bovine in vitro fertilized embryos. JVSAH, 2(4): 1-5. Ferré L.B., Kjelland M.E., Strøbech L.B., Hyttel P., Mermillod P., Ross P.J. (2019) Review: Recent advances in bovine in vitro embryo production: reproductive biotechnology history and methods. Animal, 25: 1-14. Vajta G. (2000). Vitrification of the oocytes and embryos of domestic animals. Anim Reprod Sci, 60-61: 357-364. Sanches B.V., Zangirolamo A.F., da Silva N.C., Morotti F., Seneda M.M. (2017). Cryopreservation of in vitro-produced embryos: Challenges for commercial implementation. Anim Reprod, 14: 521-527. Reyes J.N., Jaramillo L. (2016). Cryopreservation method and composition of the vitrification solution affect viability of in vitro bovine embryos. Rev Colomb Cienc Pec, 29: 130-137. Liebermann J., Conaghan J. (2013). Artificial collapse prior blastocyst vitrification: Improvement of clinical outcomes. JC Embrology, 16: 107-119. Vanderzwalmen P., Bertin G., Debauche C., Standaert V., Van Roosendaal E., Vandervorst M., Bollen N., Zech H., Mukaida T., Takahashi K., Schoysman R. (2002). Births after vitrification at morula and blastocyst stages: Effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod, 17: 744-751. Hiraoka K., Hiraoka K., Kinutani M., Kinutani K. (2004). Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Hum Reprod, 19: 2884-2888. Mukaida T., Oka C., Goto T., Takahashi K. (2006). Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod, 21: 3246-3252. Desai N., Szeptycki J., Scott M., AbdelHafez F.F., Goldfarb J. (2008). Artificial collapse of blastocysts before vitrification: Mechanical vs. laser technique and effect on survival, cell number, and cell death in early and expanded blastocysts. Cell Preserv Technol, 6: 181-189. Raju G.A., Jaya Prakash G., Murali Krishna K., Madan K. (2009). Vitrification of human early cavitating and deflated expanded blastocysts: Clinical outcome of 474 cycles. J Assist Reprod Genet, 26: 523-529. Kaymaz M., Onur G., Ozdemir M., Karakas K., Yagci IP. Reprodüktif sürü saglıgında yardımcı üreme teknolojilerinin kullanımı. Turkiye Klinikleri J Vet Sci Obstet Gynecol-Special Topics, 1(1): 86-99. Kobayashi S. (2007). Manual for ovum pick-up and in vitro fertilization. 1st ed., Japan International Cooperation Agency, Japan. Takahashi Y., Kanagawa H. (1998). Effects of glutamine, glycine and taurine on the development of in vitro fertilized bovine zygotes in a chemically defined medium. J Vet Med Sci, 60: 433-437. Somfai T., Imai K., Kaneda M., Akagi S., Watanabe S., Haraguchi S., Mizutani E., Dangnguyen T.Q., Inaba Y. (2011). The effect of ovary storage and in vitro maturation on mRNA levels in bovine oocytes; a possible impact of maternal ATP1A1 on blastocyst development in slaughterhouse-derived oocytes. J Reprod Develop, 57: 723-729. Bó G.A., Mapletoft R.J. (2013). Evaluation and classification of bovine embryos. Anim Reprod, 10: 344-348. D’Alessandro A., Federica G., Palini S., Bulletti C., Zolla L. (2012). A mass spectrometry-based targeted metabolomics strategy of human blastocoele fluid: A promising tool in fertility research. Mol Biosyst, 8: 953-958. Van Landuyt L., Polyzos N.P., De Munck N., Blockeel C., Van De Velde H., Verheyen G. (2015). A prospective randomized controlled trial investigating the effect of artificial shrinkage (collapse) on the implantation potential of vitrified blastocysts. Hum Reprod, 30: 2509-2518. Lin R., Feng G., Shu J., Zhang B., Zhou H., Gan X., Wang C., Chen H. (2017). Blastocoele re-expansion time in vitrified-warmed cycles is a strong predictor of clinical pregnancy outcome. J Obstet Gynaecol Re, 43: 689695. Kovacic B., Taborin M., Vlaisavljevi V. (2018). Artificial blastocoel collapse of human blastocysts before vitrification and its effect on re-expansion after warming - a prospective observational study using time-lapse microscopy. J Obstet Gynaecol, 36: 121-129. Ha A.N., Cho S.J., Deb G.T., Bang J., Kwon T.H., Choi B.H., Kong I.K. (2010). Effect of the artificial shrinkage on the development of vitrified

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the expression of klf4 gene in mouse embryos. Vet Res Forum, 9: 87-92. 31. Cao S., Zhao C., Zhang J., Wu X., Guo X., Ling X. (2014). Retrospective clinical analysis of two artificial shrinkage methods applied prior to blastocyst vitrification on the outcome of frozen embryo transfer. J Assist Reprod Genet, 31: 577-581. 32. Wang C., Feng G., Zhang B., Zhou H., Shu J., Lin R., Chen H., Wu Z. (2017). Effect of different artificial shrinkage methods, when applied before blastocyst vitrification, on perinatal outcomes. Reprod Biol Endocrin, 15: 1-6. 33. Darwish E., Magdi Y. (2016). Artificial shrinkage of blastocoel using a laser pulse prior to vitrification improves clinical outcome. J Assist Reprod Genet, 33: 467-471. 34. Min S.H., Kim J.W., Lee Y.H., Park S.Y., Jeong P.S., Yeon J.Y., Park H., Chang K.T., Koo D.B. (2014). Forced collapse of the blastocoel cavity improves developmental potential in cryopreserved bovine blastocysts by slow-rate freezing and vitrification. Reprod Domest Anim, 49: 684-692.

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M. Simoni et al. Large Animal Review 2021; 27: 123-132

Digestibility and sorting of hay-based total mixed rations employed in the ParmigianoReggiano area as affected by dietary particle size distribution

123

MARICA SIMONI*, ARMANDO CANEPA, GRAZIANO ROSARIO PITINO, AFRO QUARANTELLI, FEDERICO RIGHI Dipartimento di Scienze Medico-Veterinarie. Università degli Studi di Parma, via del Taglio 10, 43126 Parma, Italy

SUMMARY Introduction- Together with chemical composition, also the physical form of the Total Mixed Ration (TMR) has been demonstrated to affect dairy cattle digestive physiology and productive performance. However, few studies have been performed on the hay-based diets typically administered to dairy cattle in specific regions like the Parmigiano-Reggiano cheese production area. Moreover, almost none of these studies focused on the relationship between particle size distribution and diet digestion parameters. This relationship needs to be investigated together with the effect of particle size on sorting, in order to provide recommendation on the particle size distribution to be achieved to improve the efficiency of use of these hay-based diets. Aim- The aim of the present trial was to investigate the relationship between TMR particle size distribution, diet digestibility and sorting of hay-based diets in lactating dairy cows. Materials and methods- Five farms located in the Parmigiano-Reggiano production area were involved in the study. Three sampling procedures were performed in each farm with 15 day intervals at 0, 12 and 24 hours after TMR delivery. Five fecal samples were collected 12 hours after feed distribution from fresh healthy lactating cows (60 to 90 days in milk). Physical, chemical and digestibility analyses were performed on the TMR samples. Particle size distribution was determined using the Penn State Particle Separator (PSPS) and total tract apparent dry matter digestibility (ttaDMDe) and total tract apparent neutral detergent fibre digestibility (ttaNDFDe) were estimated using undigested NDF (uNDF-residual NDF after 240 h of fermentation) as a marker in both diet and feces. Dietary uNDF was calculated as weighted average of the uNDF determined on TMR samples collected at the 3 intervals assuming a 60% TMR intake in the first 12 hours after distribution. The relationship between the dietary residues retained on each sieve of the PSPS and ttaDMDe and ttaNDFDe were studied through a curve fitting procedure. The effect of particle size distribution at feed delivery on sorting was also investigated. Results and discussion- The distribution of the TMR particles, expressed as percentage of the total mass, on the 3 screens and bottom pan was on average 12.1%, 25.2%, 35.1% and 27.4% (Upper-U-, Medium-M-; Lower-L-; and Bottom -B- respectively). The estimated digestibilities were the highest when the U sieve residues ranged between 10 -15% (DMD: 68.35% DM; NDFD: 52.76% NDF); the M sieve residue was around 25% (DMD: 68.73% DM; NDFD: 52.27% NDF), the L sieve ranged from 35 to 40% (DMD: 66.56% DM; NDFD: 50.60% NDF) and the proportion of particles retained in the bottom pan was around 40% (DMD: 68.36% DM; NDFD: 51.57% NDF). Aside from the general sorting against longer particles, the farm with the lowest geometric mean value in the delivered TMR (farm 4: 3.92 mm) shows an increase, after 12 h, in the proportion of particles held in the B. The increased proportion of the biggest particles in the diet (>19 mm) retained on the U sieves was directly related to the variation in the particle size distribution after 12 h. Conclusions- Particle size seems to affect both digestibility and sorting parameters. A careful preparation of the hay-based TMR diet, considering the suggested values of particle size distribution, may improve the efficiency of its degradation and digestion.

KEY WORDS Dairy cows, diet; Penn State Particles Separator, physical characteristics.

INTRODUCTION The Parmigiano-Reggiano consortium disciplinary obliges farmers to use specific feeds providing in the same time indications about the amount of their use in the attempt to regu-

Corresponding Author: Marica Simoni (marica.simoni@unipr.it).

late diet composition and quality (https://www.parmigianoreggiano.com/it/consorzio-disciplinare-normative/) affecting the final products organoleptic and nutritional properties1. Thus, the diets fed in the Parmigiano-Reggiano cheese making area consisting of all dry hay, usually with a high proportion of alfalfa. No fermented forages or feeds can be fed and the majority of the forage should be harvested within the region. The TMR preparation using unchopped dry forages lead to higher variability in dietary roughage particle size and this

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Digestibility and sorting of hay-based total mixed rations employed in the Parmigiano-Reggiano...

in turn deeply affect rumination and passage rate, the latter being connected to the achievement of a critical particle size. Aside from the chemical parameters generally employed to evaluate diet adequacy also the physical form of the Total Mixed Ration (TMR) has been, in fact, demonstrated to deeply affect feeding behaviour (e.g. sorting), digestion process as well as milk quality and yield 2, 3. Several parameters and tools have been developed to describe these characteristics of the diet. Among them, the most important are the physical effective NDF (peNDF) and dietary particle size distribution measurement. The former is partially dependent on the second and has been demonstrated to relate with chewing activity, digestion and passage rate 4-6. Similarly, the particle size distribution affects chewing activity, saliva production, ruminal pH, fermentation processes, feeding behaviour and passage rate exerting a direct effect on feeds and fiber degradation5. In this scenario, the use of the Penn State Forage Particle Separator (PSPS) represents a good field tool in order to measure TMR particle size distribution, to study its relationship with the productive parameters and to monitor the sorting activity2 in the attempt to improve the efficiency of the digestion process. Several studies have been performed evaluating the effects of peNDF and particle size distribution on intake, digestibility and performance, mainly in silage based diets, but also in high forage based diets4, 7. In many cases, the particle size reduction demonstrated a positive correlation with the NDF digestibility4, 8, and along with dietary NDF content and quality (expressed also by its undigestible fraction - uNDF9) could explain the 41% variability of the total tract NDF digestibility8. However, few studies have been performed in hay-based diets, which are typically administered in specific regions like the Parmigiano-Reggiano area. In this area the number of farmers feeding cows with the TMR technique has been increasing in the last 20 years, but there are few indications regarding the dietary physical characteristics. In this case, in fact, the particle size distribution was studied mainly in relationship to its effect on the peNDF2, on the potential sorting after distribution10 and on the productive performance1,2. The relationship between the particle size distribution and the digestion parameters of hay-based diets needs therefore to be investigated in order to provide indications on the optimization of diet exploitation by the animals. The effect of this factor on sorting needs however to be taken into account when recommendations have to be provided. The aim of the present work was to investigate the relationship between Total Mixed Ration (TMR) particle size distribution, diet digestibility and sorting in lactating high producing dairy cows.

MATERIALS AND METHODS The present study complied with Italian legislation on animal experimentation and ethics (DL 04/03/2014 n. 26). The study was conducted on 5 dairy farms located in the Parmigiano-Reggiano cheese production area, specifically in the provinces of Modena (44°46’33’’ N, 10°50’59’’ E and 44°44’2.4’’ N, 10°50’45.6’’ E) and Reggio Emilia (44°53’45.6’’ N, 10°34’44.4’’ E; 44°43’26.4’’ N, 10°43’1.2’’ E and 44°37’1.2’’ N, 10°40’33.6’’ E). The trial was performed during spring, over a period of two months. The herds ranged from 60 to 200 lactating Holstein cows receiving TMR ration (Table 1) once a day. The farms were

visited on three days with 15 days interval. During each visit the following information and samples were collected: productive parameters data (herd average milk yield and composition); number of lactating cows; amount of TMR distributed and refused. On each day, 3 sampling sessions were conducted, namely at feed delivery and after 12 and 24 hours. During each session, TMR and faecal samples were collected for physical and chemical evaluations (TMR) and for the estimation of the digestibility. In particular, faecal samples were collected only 12 hours after feed distribution since this interval could be representative of the average faeces excreted daily 11, 12 . Regarding to TMR, at delivery and after 12 hours three samples of 1000 g (as fed) were collected on the feeding line: at the beginning, in the middle and at the end of the feed bunk for a total of 3 replicates per 2 sampling time per day per farm. Every TMR sample was divided in two homogeneous subsamples: one for the physical evaluation; one for chemical and nutritional laboratory analysis. The physical evaluation of the diet was performed using the Penn State Particle Separator (PSPS) according to the procedure described by Kononoff et al.13 and Lammers et al.14 on a total of 90 samples (3 replicates x 2 sampling time x 3 day visits x 5 farms). The chemical analyses were performed by Near Infrared Reflectance Spectroscopy (NIRS) including dry matter (DM), ash, crude protein (CP), soluble protein (SolP), ether extract (EE), neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), starch and total sugars. During the second visit of the day (12 hours after feed distribution), 5 fecal samples, from fresh, healthy lactating cows (60 to 90 DIM) were collected for the determination of the undigested neutral detergent fiber at 240 hours (uNDF240) as a marker to estimate the total tract apparent dry matter digestibility (ttaDMDe) and total tract apparent NDF digestibility (ttaNDFDe). The uNDF240 was determined both in diet and feces by 240 hours in vitro fermentation based on Goering and Van Soest15 method, modified as reported by Sgoifo Rossi et al.16. The same methodology was employed for the determination of diet and NDFD at 24 hours of fermentation. The total digestible nutrient at 3 times maintenance intake (TDN3x) was calculated for each dietary sample according to NRC (2001). The ttaDMDe and ttaNDFDe were calculated for each cow by using average dietary uNDF from TMR samples collected at the three daily intervals as described by Righi et al.17. In particular, the digestibilities were estimated by assuming a 60% of ration consumption in the first 12 hours after distribution and the remaining 40% between 12 and 24 hours following feed delivery, according to the circadian feeding behaviour described by Harvatine18. The dietary uNDF used for digestibility calculation was thus estimated as weighted average of the mean dietary uNDF between delivery and 12 hours after feed distribution and the mean dietary uNDF between the latter interval and the 24 hours after feed delivery. After 24 hours from TMR distribution, the herd total intake was estimated by the difference between the amount of feed delivered and orts (Total intake = weight of distributed TMR - weight of the 24 hours residue), whereas the individual dry matter intake was calculated by dividing the total intake by the number of the lactating animals, accounting for moisture. The same calculation was repeated at each daily visit (total of 3) during the whole trial.

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Table 1 - Ingredients included in the TMR diets administered to the herds involved in the study (%DM).

Farm 3

12.0

20.0

7.2

2 cut alfalfa hay

9.7

14.3

20.0

50.5

38.5

3rd cut alfalfa hay

9.7

26.0

8.5

13.6

4.2

4.3

Corn grain meal

23.2

20.6

28.6

18.9

26.9

Corn grain flacked

8.4

4.1

3.8

Barley grain meal

7.6

4.1

Barley grain flacked

3.8

Mixed hay nd

4 cut alfalfa hay Wheat straw

Wheat grain meal

5.8

6.2

7.7

Soybean flacked

2.3

1.9

Soybean expeller

6.2

Soybean meal

Wheat bran

2.0

Soybean hulls

2.1

Beet pulp

4.6

2.1

Concentrate mix1

10.1

29.1

10.5

7.5

9.6

MinVit

1.5

2.2

Forage (%)

39.83

40.36

44.17

64.12

50.00

Concentrate (%)

60.17

59.64

55.83

35.88

50.00

Forage:Concentrate

0.66

0.68

0.79

1.79

1.00

Concentrate mix of farm 1 (% DM): barley ground, 43.0; wheat bran, 21.9; corn flacked, 21.9; molasses, 13.2. Concentrate mix of farm 2 (% DM): wheat bran, 27.8; beet pulp, 25.3; corn meal, 14.9; soybean meal, 13.0; sunflower meal, 12.8; molasses, 6.1. Concentrate mix of farm 3 (% DM): soybean meal, 57.2; sunflower meal, 19.0; vitamin-mineral premix, 14.2, molasses, 9.2. Concentrate mix of farm 4 (% DM): wheat bran, 43.0; linseeds extruded, 34.8; vitamin-mineral premix, 22.2. Concentrate mix of farm 5 (% DM): beet pulp, 50.6; soybean hulls, 16.3; linseed extruded, 12.0; vitamin-mineral premix, 9.5; molasses, 7.3; wheat bran, 4.2. Three samples per farm were collected.

From milk yield and milk composition, the energy corrected milk was calculated using the equation reported in Comino et al.3 as follows: ECM = 0.327 x milk lbs + 12.97 x fat lbs + 7.21 x protein lbs and values were then converted to kg. Sorting was measured as variation of the retained material proportion on each sieve and the bottom pan expressed as absolute value of the difference from the initial value - the latter being measured in each farm in the 3 positions of the feed bunk in the 3 days considered - at 12 hours after feed delivery. These variations (%) were then regressed over the initial value (%) of each sieve and of the bottom pan to obtain the relative curves. Statistical analysis was performed using the SPSS for Windows software package (version 26.0; SPSS Inc., Chicago, IL). The differences between chemical composition parameters in the diets, milk yield and milk composition, as well as ECM, feed efficiency (FE), peNDF, geometric mean length (Xgm) and geometric standard deviation (Sgm) were evaluated through the ANOVA one-way procedure using the farm as fixed factor, and the different parameters measured as dependent variables. The amount of the residue retained on each sieve was expressed as a percentage of the total sample amount (as fed) and compared between farms through the univariate procedure of the General Linear Model using the farm as fixed factor, and farm visit and position in the feed bunk as random effects. The amount of the residue on each sieve were also regressed over the ttaDMDe and ttaNDFDe values in order to establish the relation-

ship between feed particle size distribution and diet digestibility. The correlations between ttaDMDe and ttaNDFDe and compositional parameters of the diets were previously tested in order to exclude any significant interference derived from diet composition on the mentioned regressions.

RESULTS The chemical composition of the TMRs supplied in the farms involved in the present study is reported in table 2. Differences were found in CP (ranging from 15.74 to 18.15% DM; P≤0.001), EE (ranging from 2.65 to 4.76% DM; P≤0.001), SolP (ranging from 5.23 to 6.19% DM; P=0.010), starch (ranging from 19.84 to 23.31% DM; P=0.001), NDF (ranging from 30.51 to 35.25% DM; P≤0.001), ADF (ranging from 19.58 to 24.29% DM; P≤0.001) and uNDF (ranging from 29.82 to 40.16% NDF; P≤0.001) levels. No differences were found in NSC levels (ranging from 35.06 to 38.03% DM) and in TDN and NDFD values, that ranged between 60.62 and 64.03% of DM and from 37.17 to 45.53% of NDF respectively. Table 3 reports data on feed intake, milk productivity and FE. Farm 1 showed the highest DMI (24.02 kg), milk yield (43 kg) and ECM (41.32 kg), and one of the highest FE, all these parameters were significantly different among farms (P≤0.001). The highest FE (1.77) was found in farm n°4 where the lowest DMI (19.20 kg) and intermediate milk yield and ECM (33.94 kg and 34.00 kg, respectively) were obtained. The farm 5 showed

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Digestibility and sorting of hay-based total mixed rations employed in the Parmigiano-Reggiano...

Table 2 - Chemical composition of the TMR diets administered to the herds involved in the study (data are expressed as least square means).

Farm1 3

DM (% as fed)

67.73

70.78

Ash (% DM)

10.4

9.17

CP (% DM)

18.15c

EE (% DM)

3.2b

SEM

P-value

75.9

74.87

77.66

1.393

0.146

9.8

10.23

10.2

0.228

0.455

15.74a

17.32b

17.29b

17.17b

0.161

<0.001

2.65a

3.38b

4.76d

4.09c

0.121

<0.001

Chemical characteristics2

SolP (% DM)

5.72

Starch (% DM)

20.14a

5.47

5.6

19.84a

20.9a

6.19

5.23

0.091

0.010

20.1a

23.31b

0.161

0.001

Sugars (% DM)

4.48

4.47

3.83

4.11

3.57

0.312

0.308

NSC (% DM)

35.06

37.2

36.86

36.67

38.03

0.351

0.098

NDF (% DM)

33.2c

35.25d

32.65bc

31.06ab

30.51a

0.383

<0.001

ADF (% DM)

23.06bc

24.29c

22.37b

20.16a

19.58a

0.366

<0.001

ADL (% DM)

5.04

5.15

4.95

5.22

4.89

0.061

0.411

TDN3x (% DM)

62.03

60.62

62.13

64.03

63.26

0.452

0.152

NDFD (% NDF)

38.08

40.52

45.53

37.17

42.98

1.139

0.123

NE l (Mcal/Kg)

1.45ab

1.38a

1.45ab

1.51b

1.48b

0.015

0.048

uNDF240 (%NDF)

40.16c

34.06ab

30.85a

37.55bc

29.82a

0.887

<0.001

a-d: Means within a row without a common superscript letter differ for p<0.05. Total number of samples analysed = 45 DM, dry matter; CP, crude protein; EE, ether extract; NSC, non-starch carbohydrate; NDF, neutral detergent fibre; ADF, acid detergent fibre; ADL, acid detergent lignin; TDN3x, Total Digestible Nutrients at 3 times maintenance intake, calculated according to NRC (2001); NDFD, in vitro NDF digestibility at 24 hours; uNDF240, undigestible NDF expressed as a percentage on NDF. 2

Table 3 - Dry matter intake (DMI), milk production and energy corrected milk (ECM), milk composition, and feed efficiency (FE) of the herds involved in the study (data are expressed as least square means).

1 DMI, kg/d Milk Yield (kg/d)

Farm1 3

SEM

P-value

24.02e

21.63c

19.72b

19.20a

23.47d

0.520

≤0.001

29.50b

1.426

≤0.001

43.00

38.04

29.00

33.94

Fat (%)

3.60

3.74

3.69

3.83

4.53

0.093

0.081

Protein (%)

3.43a

3.31a

3.33a

3.59b

3.20a

0.025

0.010

ECM (kg/d)

41.32b

36.85c

27.96a

34.00ac

31.19b

1.425

≤0.001

1.33a

0.047

≤0.001

1.72

1.70

1.42

1.77

a-e: Means within a row without a common superscript letter differ for p<0.05. Total number of data recorded per farm = 15

the lowest FE (1.33), medium-high level of intake and lower milk production compared to the other farms. However, the latter farm showed the numerically highest milk fat proportion (4.53%; P=0.081) with the lowest percentage of protein (3.2%; P=0.010), leading to an intermediate level of ECM (31.19 kg/d). Faecal uNDF was different in the farms considered (P≤0.001) with farm 5 showing the lowest value compared to farms 1, 3 and 4. The distribution of the TMR particles in the considered diets are reported in table 4, as a percentage of the total mass. The values obtained on the 3 sieves and on the bottom pan were on average 12.1%, 25.2%, 35.1% and 27.4% (Upper-U-, Middle-M, Lower-L- and Bottom-B- respectively). The highest proportion of particles retained in the U sieve (size >19 mm) was found in the farms 3 and 5 (P≤0.001), which also showed the lowest amount of particles in the M sieve (particle size between 8 and 19 mm; P≤0.001). Farm 2 revealed the lowest value for the U and the numerically highest proportion of particles in

the M sieve. Farms 2 and 4 showed the highest proportion of particles retained in the L sieve (P≤0.001). Farms 1 and 3 exhibited the highest proportion of particles smaller than 1.18 mm (B pan) compared to the farms 2 and 5 (P=0.003). The peNDF1.18 of the farm 2 was the highest (P≤0.001), while farm 4 showed the lowest value for the peNDF8 (P=0.014). Farm 5 showed longer particles compared to farms 4, 1 and 2 (P=0.011). The highest ttaDMDe and ttaNDFDe were found in farm 4 (P≤0.001). The lowest ttaDMDe was observed in farms 2 and 3, whereas, the lowest ttaNDFDe was observed for farm 2 (P≤0.001) as reported in the table 4. Based on the regressions between the proportions of particles retained on each sieve and the estimated digestibility parameters, it appeared that, both ttaDMDe and ttaNDFDe reached the highest levels when the U sieve residue was at 12% (Graph 1), the proportion of particles retained on the M sieve was 25% for the ttaDMDe and 24% for the ttaNDFDe (Graph 2) and

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Graphic 1 - Variation of the estimated total tract apparent dry matter digestibility (ttaDMDe - continuous line) and of the estimated total tract apparent neutral detergent fibre digestibility (ttaNDFDe dotted line) as a function of the proportion of particles retained on the upper screen of the Penn State Particle Separator (% as fed).

Graphic 2 - Variation of the estimated total tract apparent dry matter digestibility (ttaDMDe - continuous line) and of the estimated total tract apparent neutral detergent fibre digestibility (ttaNDFDe dotted line) as a function of the proportion of particles retained on the medium screen of the Penn State Particle Separator (% as fed).

Graphic 3 - Variation of the estimated total tract apparent dry matter digestibility (ttaDMDe - continuous line) and of the total tract apparent neutral detergent fibre digestibility estimated (ttaNDFDe dotted line) as a function of the proportion of particles retained on the lower screen of the Penn State Particle Separator (% as fed).

Graphic 4 - Variation of the estimated total tract apparent dry matter digestibility (ttaDMDe - continuous line) and of the total tract apparent neutral detergent fibre digestibility estimated (ttaNDFDe dotted line) as a function of the proportion of particles collected in the bottom pan of the Penn State Particle Separator (% as fed).

the proportion of particles on the L sieve was 37%. Concerning the residues on the B, ttaDMDe and ttaNDFDe were depressed when the proportion of particles were respectively equal to 28% and 25% (Graph 3 and 4 respectively). The variation of the proportions describing the dietary particle distribution after 12 hours from the feed delivery is reported in table 5. In three out of four farms the particles retained on the U sieve increased (P<0.05) and in farm 2 the value after 12 hours was numerically higher even if not significantly. No differences were observed in the M sieve with exception for the farm 1 which showed an increase of the particles retained on this sieve (P≤0.001). A decrease in the proportion of particles retained on the L sieve was observed in the majority of the farms after 12 hours from TMR distribution (P<0.05) and no effect was observed for this sieve in farm 1. Contrasting results, dependently to the considered farm, were observed concerning the proportion of particles retained in the bottom pan. In fact, farms 1 and 3 showed a decreased proportion of particles held in the bottom pan (P=0.091 and P≤0.001 respectively), while no effects were observed in farms 2 and 5. Moreover, an increased proportion of particles was observed in farm 4 diet (P<0.05). In graph 5, the variation of the particle size distribution dur-

ing the first 12 hours after feed administration is showed in relation with the amount of particles retained on each sieve and in the B pan at the delivery in the feed bunk. The increased pro-

Graphic 5 - Relations between the cumulative variation (Δ) of particles size distribution in the first 12 hours after feed delivery and the proportion of particles retained on each sieve and in the bottom pan of the Penn state Particle Separator at the feed delivery.

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Digestibility and sorting of hay-based total mixed rations employed in the Parmigiano-Reggiano...

Table 4 - Estimated total tract apparent dry matter and neutral detergent fiber digestibilities (ttaDMDe; ttaNDFDe) and particle size distribution measured through the Penn State Particle Separator, of the total mixed diet fed to the cows of the studied herds (data are expressed as least square means). 1

Farm1 3

18.78c

7.44ab

SEM

p-value

19.11c

1.161

≤0.001

Physical characteristics2 10.22b

19 mm

4.89a

19.0- 8.0 mm

27.44

29.44

21.22

25.33

22.78

0.566

≤0.001

8.0- 1.18 mm

31.56a

41.56b

29.89a

40.22b

32.33a

0.881

≤0.001

<1.18 mm

30.67

23.89

29.67

27.11

25.89

0.659

0.003

peNDF1,18 (% NDF)3

23.01a

26.71b

22.84a

22.72a

22.63a

0.372

≤0.001

peNDF8 (% NDF)4

12.53b

12.1b

13.1b

10.17a

12.76b

0.303

0.014

Xgm mm5

4.06a

4.09a

4.57ab

3.92a

5.01b

0.116

0.011

Sgm mm

3.41

2.94

3.74

3.10

3.58

0.054

≤0.001

Estimated total tract apparent digestibility7 ttaDMDe (%DM)

66.11b

60.80a

61.81a

70.89c

65.23b

0.620

≤0.001

ttaNDFDe (%NDF)

47.41b

40.69a

47.13b

55.46c

45.02ab

0.578

≤0.001

1 A-D: Means within a row without a common superscript letter differ for p<0.05. 2Total number of samples : 45; 3peNDF 1.18: physically effective NDF, ration NDF multiplied by amount of DM > 1.18 mm; 4peNDF 8: physically effective NDF, ration NDF multiplied by amount of DM > 8.0 mm; 5Xgm: geometric mean length; 6Sgm: geometric standard deviation; 7 Total number of samples: 75.

portion of the bigger particles (retained on the U sieve) is directly related to the variation in the particle size distribution after 12 hours, even if this relationship does not appear to be strong (R2=0.341). Any relationship was found with the amount of particles retained on the other sieves and in the B pan.

DISCUSSION The diets were typical of the Parmigiano-Reggiano cheese making area, and were similar to the hay-based diets described by Comino et al.3. Indeed, as reported in table 1, the diets considered in the present study were characterized by a forage to concentrate ratio (F:C) ranging from 0.66 to 0.79 with the only exception of farm 4 which included higher levels of forage (F:C= 1.79). In general, the forage portion comprised a big amount of alfalfa hay from different cuts, providing both fiber and proteins. In addition, diets 1, 3 and 5 were comprised of mixed hay, and rations of farms 3 and 5 included also wheat straw as roughage. The diets considered included on average 22.78% of corn meal, as a main source of starch, whose level ranged from 18.9 to 28.6% of the diet DM. Additionally, a customized concentrate mix was supplied in all cases to fulfil the lactating cattle requirements. Generally, the composition of the diets was similar among the farms considered. The highest dietary CP content observed in the farm 1 might be related to the highest milk production yielded by the relative herd, in agreement with results from Comino et al.3. The EE content was the highest in farm 4 and the lowest in farm 2. The high level of EE in farm 4 was probably one of the reasons for the high net energy of lactation (NEl) observed, despite the higher F:C in this diet. In fact, farm 4 ration contained the highest amount of forages that could have led to the rumen filling which justify the lowest DMI. Moreover, the high forage proportion in the same diet could have improved the acetic acid and butyric acid production in the ru-

men, along with a higher microbial protein synthesis, both reflected in the milk composition as high milk fat and protein levels. However, dietary EE and CP content are not considered as major variables affecting the total tract digestibility, which is investigated in the present trial. The starch content was the highest in farm 5 but from a practical point of view, the level observed can be considered comparable to the values of the other farms. Farm 2 showed the highest level of NDF content in the diet, while the other farms were similar. Despite the slight differences in terms of chemical composition between the studied rations, TDN3x and NDF digestibility of the complete diets were unaffected, indicating that the differences found in the total tract digestibility are mainly due to the particle size distribution. The proportion of the TMR particle size distribution was comprised in the same ranges identified by other authors in the Parmigiano-Reggiano area1,2,8. In particular, they were similar to the ranges reported by Fustini et al.10 which were 0.0 to 19.6% for U, from 14.0 to 50.0% for M, from 22.9-46.0 for L and from 13.6 to 53.2 for the B. In the present study the diets were comparable for their peNDF, and close to the value of peNDF1.18 of 20-22% recommended by Mertens6 in almost all cases. An exception was observed in farm 2 which showed the highest value having 75.89% of the particles longer than 1.18 mm and the highest NDF content in the diet, resulting in a peNDF1.18 of about 27% of NDF. Moreover, peNDF8 was the lowest in farm 4 diet which, in fact, showed the highest content of particles retained in the lower screen. The same diet showed the numerically lowest geometric mean length of the particles. Despite the possible effect of this parameter on the DMI7, that was not investigated in the present study, some authors demonstrated that a decrease in TMR particle length, increased nutrients digestibility4, 19 and milk production8 though reducing the milk protein content4. Our results partially agree with those of Stojanovic et al.4 and Haselmann et al.19 since the farm with the lowest dietary value of Xgm of particles size showed the highest ttaDMDe, ttaNDFDe and milk protein lev-

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131

Table 5 - Variation of the particle size distribution at 12 hours after feed delivery (values are expressed as %). Diet 1

Sieve1

T0 (%)2

T12 (%)3

SEM

p-value

10.22

6.67

1.144

0.130

27.44

33.33

1.147

≤0.001

31.56

32.78

0.573

0.414

30.67

27.33

1.163

0.091

4.89

8.00

1.467

0.183

29.44

28.44

0.664

0.507

41.56

38.44

1.293

0.042

23.89

25.33

0.642

0.458

18.78

32.89

3.067

≤0.001

21.22

18.78

1.120

0.110

29.89

25.00

1.064

0.002

29.67

23.00

1.146

≤0.001

7.44

12.44

0.962

0.036

25.33

23.33

0.642

0.188

40.22

33.11

1.645

≤0.001

27.11

31.11

1.204

0.044

19.11

28.33

1.918

≤0.001

22.78

24.89

0.538

0.165

32.33

24.89

1.253

≤0.001

25.89

23.00

1.189

0.142

U: upper (19.0 mm), M: middle (8.0 mm), L: lower (1.18 mm) and B: bottom pan; 2 Proportion of particles at feed delivery; 3 Proportion of particles 12 hours after feed delivery. Total number of samples: 45 at each time point.

els. This could be related to a higher content of particles retained on the lower sieve: these are represented, usually, by the finest forage particles -leaves and small stems scraps- and concentrate fragments -particles of pellets, flakes and shreds- generated by a more intense mixing and chopping procedure and characterized by high digestibility. The presence of these residues, given by particles over the critical size, associated to the low intake observed is expected to increase the ruminal retention time with an improvement in the extent of digestion. The latter could be also enhanced by the relatively high ratio between the surface area for the microbial attack and the mass of the particles, thus leading to a feed efficiency raise. The study of the relationship between the proportion of residual particles retained on each sieve and the ttaDMDe and ttaNDFDe showed a relationship between the residues collected on the M screen and the digestion parameters. The latter showed very weak relationships with particles retained on the U screen. Whereas, the amount of residues reaching L screen and the bottom pan did not show specific relationship with the digestibility parameters; particles smaller than 1.18 mm are, in fact, poorly retained in the rumen but exploited in the fermentation process20. Some indications can be, however, obtained by the examination of the graphic representation of the mentioned regressions. Apart from the specific optimal value for each sieve residue stated in the results section, it appears that the ttaDMDe and ttaNDFDe were generally higher when the U sieve residues ranged between 10 and 15%, the amount of particles on the M sieve were around 25%, the proportion of particles on the L sieve ranged from 35 to 40% and the percentages of residues on the B were around 40%. The number of observations was however limited and these values should be considered as pre-

liminary indications. Based on these results, it could be speculated that these proportions of particles in the diets can improve the ruminal mat formation optimizing, accordingly to Zebeli et al.5, ruminal environment stimulating chewing and ruminal motility and promoting particle retention, modulating in the same time the passage rate. Concerning sorting, an increase in the proportion of the particles bigger than 19 mm (retained on the U screen) was observed in the majority of the farms after 12 hours from the TMR distribution. Four farms showed a significant reduction in the percentage of particles retained on the L sieve within a general decline in the proportion of the smallest particles retained (<8 mm). The highest variation in the smallest particles was observed in the farms 3 and 5 whose percentage of particles longer than 8 mm were higher in the initial TMR. These results apparently indicated a sorting effect against longer particles in favour of small particles according to several studies21, 22. However, the farm with the lowest Xgm in the TMR at the delivery was the only one showing an increase in the proportion of particles held in the bottom pan after 12 hours, highlighting the importance of the dietary particles size also in the feeding behaviour. In general, the proportion of particles bigger than 19 mm in the TMR was positively related with the relative variation of particle size distribution during the first 12 hours after feed delivery. This is particularly evident in farms 3, 4 and 5, indicating that the higher initial presence of bigger particles was responsible for the feed sorting of the diet. Consistently to the results from Leonardi and Armentano22, that found a strong correlation between the proportion of particles retained on the 26 mm screen and the feed sorting, in the present study cows generally sorted in favour of smaller particles than the longer ones.

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Digestibility and sorting of hay-based total mixed rations employed in the Parmigiano-Reggiano...

CONCLUSIONS 8.

Total mixed ration particle size seems to affect both digestibility and sorting parameters. The estimated total tract digestibilities of both DM and NDF, in alfalfa hay-based TMR diets typical of the Parmigiano-Reggiano cheese production area, appear higher when the proportion of particles retained in the U sieve ranges between 10 and 15%, is around 25% in the M residues, ranges between 35 and 40% in the L and is around 40% in the bottom pan. The U sieve residues particle are the main responsible for the feed sorting after meal distribution. A careful preparation of the hay-based TMR diet, considering the suggested values of particle size distribution, may improve the efficiency of its degradation and digestion.

10.

11.

12.

ACKNOWLEDGMENTS

13.

The authors gratefully acknowledge the farmers and the technicians involved in this study for their support.

References 1.

Summer A, Formaggioni P, Franceschi P, Di Frangia F, Righi F, & Malacarne M (2017) Cheese as Functional Food Food Technol Biotechnol 55(3) 277-289, https://doi.org/10.17113/ft. Righi F, Quarantelli A, Tonelli L, Renzi M, & Gandolfi B (2007) Use of Penn State Particle Separator for the evaluation of total mixed rations typical of Parmigiano Reggiano cheese production area Ital J Anim Sci 6(SUPPL. 1) 347-349, https://doi.org/10.1073/pnas.030539197. Comino L, Righi F, Coppa M, Quarantelli A, Tabacco E, & Borreani G (2015) Relationships among early lactation milk fat depression, cattle productivity and fatty acid composition on intensive dairy farms in Northern Italy Ital J Anim Sci 14(3) 350-361, https://doi.org/10.4081/ ijas.2015.3656. Stojanovic B, Grubic G, Djordjevic N, Glamocic D, Bozickovic A, & Ivetic A (2012) Efectos producidos por diferentes fibras físicamente efectivas en la alimentación de vacas en su primera fase de lactación Spanish J Agric Res 10(1) 99-107, https://doi.org/10.5424/sjar/2012101-159-11. Zebeli Q, Aschenbach JR, Tafaj M, Boguhn J, Ametaj BN, & Drochner W (2012) Invited review: Role of physically effective fiber and estimation of dietary fiber adequacy in high-producing dairy cattle J Dairy Sci 95(3) 1041-1056, https://doi.org/10.3168/jds.2011-4421. Mertens DR (1997) Creating a System for Meeting the Fiber Requirements of Dairy Cows J Dairy Sci 80(7) 1463-1481, https://doi.org/10.3168/jds. S0022-0302(97)76075-2. Kononoff PJ, & Heinrichs AJ (2003) The effect of reducing alfalfa haylage particle size on cows in early lactation J Dairy Sci 86(4) 1445-1457,

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https://doi.org/10.3168/jds.S0022-0302(03)73728-X. Tafaj M, Zebeli Q, Baes C, Steingass H, & Drochner W (2007) A metaanalysis examining effects of particle size of total mixed rations on intake, rumen digestion and milk production in high-yielding dairy cows in early lactation Anim Feed Sci Technol 138(2) 137-161, https://doi.org/10.1016/j.anifeedsci.2007.06.020. Righi F, Simoni M, Visentin G, Manuelian CL, Currò S, Quarantelli A, & De Marchi M (2017) The use of near infrared spectroscopy to predict faecal indigestible and digestible fibre fractions in lactating dairy cattle Livest Sci 206 105-108, https://doi.org/10.1016/j.livsci.2017.10.006. Fustini M, Heinrichs AJ, Palmonari A, & Formigoni A (2016) CASE STUDY: Farm characteristics and total mixed ration particle size issues on Parmigiano Reggiano farms in northern Italy Prof Anim Sci 32(6) 869873, https://doi.org/10.15232/pas.2016-01550. Mambrini M, & Peyraud JL (1997) Retention time of feed particles and liquids in the stomachs and intestines of dairy cows. Direct measurement and calculations based on faecal collection Reprod Nutr Dev 37(4) 427442, https://doi.org/10.1051/rnd:19970404. Dufreneix F, Faverdin P, & Peyraud JL (2019) Influence of particle size and density on mean retention time in the rumen of dairy cows J Dairy Sci 102(4) 3010-3022, https://doi.org/10.3168/jds.2018-15926. Kononoff PJ, Heinrichs AJ, & Buckmaster DR (2003) Modification of the Penn State Forage and total mixed ration particle separator and the effects of moisture content on its measurements J Dairy Sci 86(5) 1858-1863, https://doi.org/10.3168/jds.S0022-0302(03)73773-4. Lammers BP, Buckmaster DR, & Heinrichs AJ (1996) A Simple Method for the Analysis of Particle Sizes of Forage and Total Mixed Rations J Dairy Sci 79(5) 922-928, https://doi.org/10.3168/jds.S0022-0302(96)76442-1. Goering HK, & Van Soest PJ (1970) Forage fiber analyses (Apparatus, reagent, procedures and some applications): Agriculture Handbook No. 379 USDA-ARS. Sgoifo Rossi CA, Compiani R, Baldi G, Taylor SJ, Righi F, Simoni M, & Quarantelli A (2019) Replacing sodium bicarbonate with half amount of calcareous marine algae in the diet of beef cattle Rev Bras Zootec 48 1-12, https://doi.org/10.1590/rbz4820180129. Righi F, Simoni M, Malacarne M, Summer A, Costantini E, & Quarantelli A (2016) Feeding a free choice energetic mineral-vitamin supplement to dry and transition cows: Effects on health and early lactation performance Large Anim Rev 22(4) 161-170. Harvatine KJ (2012) Circadian Patterns of Feed Intake and Milk Composition Variability In: Proc. 21st Tri-stage Dairy Nutr. Conf. April 43-55. Haselmann A, Zehetgruber K, Fuerst-Waltl B, Zollitsch W, Knaus W, & Zebeli Q (2019) Feeding forages with reduced particle size in a total mixed ration improves feed intake, total-tract digestibility, and performance of organic dairy cows J Dairy Sci 102(10) 8839-8849, https://doi.org/ 10.3168/jds.2018-16191. Martz FA, & Belyea RL (1986) Role of Particle Size and Forage Quality in Digestion and Passage by Cattle and Sheep J Dairy Sci 69(7) 1996-2008, https://doi.org/10.3168/jds.S0022-0302(86)80626-9. Miller-Cushon EK, & DeVries TJ (2017) Feed sorting in dairy cattle: Causes, consequences, and management J Dairy Sci 100(5) 4172-4183, https://doi.org/10.3168/jds.2016-11983. Leonardi C, & Armentano LE (2003) Effect of quantity, quality, and length of alfalfa hay on selective consumption by dairy cows J Dairy Sci 86(2) 557-564, https://doi.org/10.3168/jds.S0022-0302(03)73634-0.

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E. Şirin et al. Large Animal Review 2021; 27: 135-141

In vitro maturation of bovine oocytes may using royal jelly as protein source in the culture media

135

EMRE ŞİRİN1*, MEHMET KURAN2 1

Kırşehir Ahi Evran University, Faculty of Agriculture, Department of Agricultural Biotechnology, 40100, Kırşehir, Turkey Ondokuz Mayıs University, Faculty of Agriculture, Deparment of Agricultural Biotechnology, 55100, Samsun, Turkey

SUMMARY The present study investigated the effect of using royal jelly (RJ) as protein source for the culture media that would be used in the nuclear maturation stage of bovine oocytes. Bovine ovaries were collected from local slaughterhouse and then the cumulus oocyte complexes (COCs) were recovered from visible antral follicles (2 to 8 mm) by aspiration method. The obtained COCs were examined under an inverted microscope. COCs with uniform cytoplasm and homogeneous distribution of cumulus cells were selected for in vitro maturation. COCs were randomly incubated in tissue culture media–199 (TCM-199) with 10% royal jelly (10RJ, n=179) and 10% fotal calf serum (0RJ, n=172 oocytes) for 22h at 39 ºC under 5% CO2 in humidified air at 95%. The nuclear maturation stages were determined by examining the oocytes under the inverted microscope. The proportion of oocytes reaching metaphase-I (MI) stage in the 0RJ and 10RJ groups was 19% and 20%, respectively. The rate of oocytes reaching the anaphase-I (AI) stage in both groups was determined as 2%. On the other hand, 1% of the oocytes developed up to the telephase-I (TI) stage in both groups. The maturation rate in 10RJ media (78%) was similar when compared with 0RJ media (77%). Methaphase-II (MII) stage oocytes the 10RJ media did not affect the expansion rates of cumulus cells when compared to 0RJ media. Similarly, the ratios in first polar bodies and the maturated oocytes cleaved to 2- cell 48h post activation and were not affected by the use of 10RJ in the culture media. Therefore, these results suggest that royal jelly (%10) can be used as a protein source in the in vitro maturation (IVM) of bovine oocytes. This study has shown that it will contribute to the studies to be carried out by identifying different protein sources in the in vitro maturation stage. The present study investigated the effect of using RJ as protein source for the culture media that would be used in the nuclear maturation stage of bovine oocytes.

KEY WORDS Bovine, oocytes, IVM, royal jelly, parthenogenetic activation.

INTRODUCTION In recent years, while new biotechnologies have been developed that can help animal breeding, some technologies have started to spread to application areas. These biotechnologies include estrus synchronization, artificial insemination, in vitro embryo production, cloning and Multiple Ovulation Embryo Transfer (MOET). The aim of these reproductive biotechnologies is to increase the number of offspring obtained from selected males and females and to accelerate genetic progression by shortening the interval between generations1. The genetic progression rate of males was increased by artificial insemination. Especially in female cattle, the period between generations is longer due to the generation of one generation per year. For this reason, there is a greater need for reproductive biotechnology in increasing the rate of genetic progression by shortening the time between genetics in cattle. One of the technologies developed to obtain more than one offspring per year in females is in vitro embryo production technology2.

Corresponding Author: Emre Şirin (emre.sirin@ahievran.edu.tr).

In vitro embryo production consists of in vitro maturation, in vitro fertilization and in vitro culture stages and this technology is known as a biotechnology to increase the genetic progression rate in females. While this reproductive biotechnology developed to increase the rate of genetic progress in animal breeding is being put into practice, it is also necessary to increase the efficiency of the technologies developed. Genetic progression rate has been increased with in vitro embryo production biotechnology. It has been shown that the birth weight of calves obtained by in vitro embryo production is higher than those produced in vivo3, 4. With the implementation of this biotechnology, which is aimed to increase the rate of genetic progress, various negativities may occur. Due to the increase in birth weight, the incidence of caesarean section, dystocia5 and large offspring syndrome6 also increases. In addition, protein sources used in the in vitro embryo production process lead to the transfer of various pathogens and some agents whose effects are not fully known7. Therefore, in order to eliminate such negativities affecting the success of in vitro embryo production, it is necessary to determine the protein sources that will not cause such adversities in the culture environment. For these reasons, this study aimed to investigate whether 10RJ can be used as a protein source in the culture medium for in vitro maturation of bovine oocytes.

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In vitro maturation of bovine oocytes may using royal jelly as protein source in the culture media

MATERIALS AND METHODS Ovaries and oocytes collection The research material consists of cattle ovaries obtained from different breeds. Ovaries were placed in a salt solution with phosphate tampon (PBS) at 39 °C, which was prepared prior to the experiment, and then they were taken to the laboratory inside this solution. Intra-follicular fluid was collected from follicles 2-8 mm in diameter on the ovaries using a syringe with an 18 g needle diameter.

the inverted microscope. The oocytes detected as 1st polar body were transferred to each compartment of the four-well culture dishes by placing approximately 1 ml of 3: 1 acetic acid: ethanol mixture and the oocytes were fixed at +4 °C for at least 24 hours. Oocytes were placed on a slide, stained using 10-15 µl aceto orcein and covered with coverslip. Following these procedures, prepared slides were examined under a inverted microscope. Thus, the nuclear maturation stages of oocytes were determined.

Oocytes maturation

Parthenogenetic activation

COCs were examined under a microscope. A mixture of 100 ml Hepes buffered tissue culture media (HTCM-199, Sigma, M7528), 1 ml penicillin streptomycin and 10 mg L-glutamine (Sigma, G3126) was stored at +4 °C. 19 ml of this mixture was taken and 1 ml of Fetal Bovine Serum (FBS) (5% v/v, Sigma, F4135) was added on it and the temperature was brought to 35 °C. The COCs examined under the microscope were then transferred to this mixture. The COCs, which were then transferred to the oocyte search medium, were morphologically evaluated with the aid of a microscope, and only oocytes with smooth cytoplasm and nonatretic cumulus cells lining the zona pellucida and with enough cumulus cells around were selected for maturation. These selected oocytes were washed twice in oocyte search medium. Later, two different culture media were prepared. The first of these is bicarbonate buffered culture medium with serum addition. This culture medium is mixed with 1 ml of FBS, 100 µl penicillin (50 IU / ml) -streptomycin (50 µg / ml) and 20 µl sodium puree (22 µg/ml, Sigma, S8636) into 9 ml bicarbonate buffered tissue culture medium (TCM-199, Sigma, M4530). This mixture (0RJ) has been gassed for approximately 2 hours in a humid atmosphere containing 5% CO2 at 39 °C before use. The second culture medium is a culture medium with RJ added as a protein source. Before this culture medium was prepared, the crude protein analysis of RJ was made according to the Weende analysis method and the crude protein content of RJ was determined as 17.3%. Culture medium supplemented with RJ, 2 g royal jelly is dissolved in 20 ml Hepes buffered medium, then 200 µl penicillin (50 µl / ml) -streptomycin (50 µg / ml) and 40 µl sodium pyruvate (22 µg / ml) are added to stock solution was prepared. After taking 1 ml of this stock solution, 9 ml of bicarbonate buffered culture medium (50 IU penicilline, 50 µg streptomycin, 22 µg sodium pyruvate per ml) was added. Thus, a culture medium containing 10RJ was prepared. The using both maturation mediums (0RJ and 10RJ) were drops (45 µl) prepared. These drops are covered with 2-3 ml of mineral oil (Sigma, M8410). These drops have been gassed for 2 hours at 39 °C, 5% CO2 and 95% humidity. Morphologically normal oocytes were transferred into drops with 10 oocytes per 5 µl. These oocytes were matured at 39 °C, 5% CO2 and 95% humidity for an average of 22 hours. 172 oocytes in the 0RJ group and 179 oocytes in the 10RJ group were subjected to in vitro maturation. In order to remove the cumulus cells, oocytes were kept for 5-7 minutes in 1 ml Hepes buffered culture medium containing 100 Units /ml hyaluromidase (Sigma, H3506) enzyme at room temperature. The oocytes from which the cumulus cells were removed, detected 1st polar bodies under

Oocytes were subjected to a two-step parthenogenetic activation. In the first stage (ethanol activation), Hepes buffered culture medium (50 µl FBS and 10 µl penicillin-streptomycin per ml) was prepared. This mixture was passed through a 0.20 µm diameter filter. Later, to this mixture, 7% ethanol (152 µl, 92% absolute ethanol) was added. Oocytes were treated with the prepared solution at room temperature for 5 minutes. Following this process, oocytes were then washed two times in the culture medium with Hepes tampon. In the second step (activation with Cycloheximide and Cytochalasin-B), 10 µl of Cycloheximide and 10 µl of cytochalasin-B were dissolved in 1.5 ml of Hepes buffered culture medium (no serum and antibiotics were added, filtered). Drops of 45 µl were prepared from this mixture and they were gassed in the incubator for a certain time after they were covered with mineral oil. Oocytes were transferred to drops following activation with ethanol and incubated for 6 hours at 39 °C, in an atmosphere containing 5% CO2 and 95% humidity. After 6 hours of incubation, oocytes were washed three times in Hepes buffered culture medium and transferred into previously prepared culture medium containing granulosa cells. Then, the oocytes were continued to be incubated at 39 °C, in an atmosphere containing 5% CO2 and 95% humidity. The division rates of oocytes were determined at 72 hours following the activation process.

Statistical analysis Statistical analysis of data obtained from the experiment was conducted by using ki-square method (Minitab 13.0).

RESULTS In vitro maturation rates The maturation rates obtained in this study conducted on the effect of adding RJ as a protein source to the culture medium on the in vitro maturation parameters of bovine oocytes are also given in Table 1. The effect of adding 10RJ as a protein source to the culture medium on the rate of oocytes reaching MII (χ2 = 0.836) was found to be insignificant (P> 0.05). In vitro maturation rates of bovine oocytes were found to be 77% for 0RJ group and 78% for 10RJ group.

Cumulus expansion rates The effect of adding 10RJ to the culture medium as a protein source on the cumulus expansion of bovine oocytes is given in Table 2. The effect of adding 10RJ as a protein source to the culture medium on the rate of in vitro cumulus expansion (χ2 = 0.215) was found to be insignificant (P> 0.05).

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Table 1 - Effect of adding RJ as a protein source in culture medium on in vitro maturation of bovine oocytes. Nuclear maturation degree (%) Culture

No of oocytes

MII

Unidentified

0RJ (n/n)

172

20 (28/139)

2 (3/139)

1 (1/139)

77 (107/149)

10RJ (n/n)

179

19 (27/141)

2 (3/141)

1 (1/141)

78 (110/141)

Unidentified: Number of oocytes for which it is not determined at which stage of nuclear maturation. MI: Metaphase-I, AI: Mainphase-I, TI: Telaphase-I, MII: Metaphase-II.

Table 2 - Expansion rates of cumulus cells in culture media using royal jelly and serum as protein sources. Culture

No of Oocytes

No of Cumulus Expansion

Expansion Rate (%)

0RJ

172

167

10RJ

179

169

Table 3 - Polar body numbers that can be observed in oocytes matured in culture media using 10RJ and 0RJ as a protein source. Culture

No of Oocytes

No of Cumulus Expansion

Expansion Rate (%)

0RJ

172

10RJ

179

Table 4 - Cleavage rates following parthenogenetic activation of bovine oocytes matured in vitro. No of oocytes analysed

PB* number

No of oocytes divided

0RJ

117

10RJ

122

Culture

PB*: Number of oocytes for which polar bodies (1st polar body) are determined.

The impact that RJ as protein source to the culture environment, on the expansion of in vitro cumulus cells (χ2 = 0,215) created was found to be insignificant (P>0.05). Following the in vitro maturation of bovine oocytes, rates of in vitro cumulus expansion were found to be 97% for 0RJ group and 94% for 10RJ group.

Ratio of first polar body 1st polar body numbers detected in bovine oocytes matured in vitro by adding serum 0RJ and 10RJ as protein sources to the culture medium are given in Table 3. There was no difference in 1st body numbers observed between 10RJ and 0RJ groups (P> 0.05). Observable ratio of 1st polar bodies was 39% (69/179) in the 10 RJ group and 40% (69/172) in the 0RJ group.

Parthenogenetic activation of bovine oocytes The findings obtained in this study to determine the effect of royal jelly and serum (fetal calf serum) used as protein sources in culture medium in in vitro maturation on the division rates

following parthenogenetic activation of bovine oocytes are given in Table 4. The effect of adding royal jelly as a protein source to the culture medium on the cleavage rates determined at the 48th hour in granulosa monalayer co-culture following in vitro maturation (χ2 = 0.437) was found to be insignificant (P> 0.05). Following in vitro maturation, the division rate of bovine oocytes subjected to parthenogenetic activation was 36% in the 10RJ group and 42% in the 0RJ group.

DISCUSSION In vitro maturation rates In this study, it was determined that adding royal jelly to the culture medium had no negative effect on in vitro maturation of oocytes. It has also been demonstrated that royal jelly can be used as a protein source in culture systems in the in vitro maturation of bovine oocytes. In most in vitro maturation studies, serum and serum albumin are used as protein sources in culture medium. Protein sources of animal origin such as serum

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In vitro maturation of bovine oocytes may using royal jelly as protein source in the culture media

and serum albumin may contain various pathogens in their bodies7. Therefore, the use of these protein sources of animal origin in the culture medium is limited. In addition, when serum is used as a protein source in culture media, due to various abnormalities in the metabolism and development of embryos can occur large offspring syndrome8. For these reasons, commercial (CPRS-3 and Ultroser-G) products obtained from serum by various methods have been developed. Even when these commercial products were used as a source of protein in culture media, the results were not exactly as expected9. In addition, culture media without protein were developed, but the desired results were not obtained from these culture media. In this study, the use of royal jelly, which is a natural protein source, in in vitro maturation in culture medium was tried for the first time. It was observed that the in vitro maturation rates obtained in culture media using royal jelly as a protein source were similar to the in vitro maturation rates obtained in culture media using protein sources such as serum and serum albumin. In addition, royal jelly does not contain various risks such as serum and serum albumin. The in vitro maturation rates obtained in our study were similar to the in vitro maturation rates in culture media using other protein sources. For these reasons, it can be said that royal jelly can be used as a protein source instead of serum and serum albumin in in vitro maturation. In addition, the in vitro maturation rates obtained in different studies using serum as a protein source in culture media are similar to the in vitro maturation rates obtained in our study10, 11, 12. In summary, these results show that royal jelly can be used as a protein source in culture media.

Cumulus expansion ratios It has been determined that there is no difference in the use of 0RJ or 10RJ as a protein source in the culture medium in terms of cumulus expansion. Bovine oocytes are surroundered by few rows of cumulus cell masses13. These cells nourish the oocyte and provide key products for it to develop. The small number or absence of the cumulus cell mass surrounding the oocyte has a negative effect on the embryo after fertilization14. The expansion of the cumulus cells, which is an indicator of the maturation of the oocyte, is seen at the 18th hour of the culture, and this expansion has a significant effect on the oocyte reaching MII15. In our study, since it was determined that bovine oocytes matured in culture media using serum and royal jelly as protein sources have similar cumulus expansion rates, it has been an indicator that royal jelly can be used as a protein source in culture medium instead of serum in vitro maturation. In addition, it was observed that the cumulus expansion rates obtained in studies using serum as a protein source in culture media were lower than the cumulus expansion rates obtained in our study16. These results showed that it is possible to use royal jelly as a protein source in culture medium in terms of cumulus expansion, which is effective in the development of oocytes to the maturation stage.

Number of first polar body observable It was determined that the use of 0RJ or 10RJ in the culture medium did not cause a difference in terms of polar body number. The oocyte undergoes a number of structural changes as it progresses towards the MII stage17. One of these structural

changes is that the 1st polar body becomes apparent. The 1st polar body usually forms between 18-21st hours of in vitro maturation18. Some changes seen in the oocyte maturation process are related to the time required for the release of the 1st polar body. Chromosomes localize to the peripheral space following the release of the first polar body15. Following this process, chromosomes once again flow towards the dense areas and develop towards the MII stage. The ratios of 1st polar body obtained in a study using serum as a protein source in the culture medium are similar to the rates obtained in our study19. Considering the number of 1st polar bodies detected in our study, it has shown that royal jelly can be used instead of serum as a protein source in the culture medium.

Parthenogenetic activation of bovine oocytes It was determined that bovine oocytes matured in culture media using 0RJ and 10RJ as protein sources had similar division rates following parthenogenetic activation. The resumption of development of oocytes waiting at the MII stage is provided by fertilization or parthenogenetic activation20, 21. Oocyte activation is regulated by specific calcium signal22. It was determined that oocytes matured in culture media using royal jelly as a protein source and oocytes matured in culture media using serum as protein source have similar division rates. The division rate obtained in a study23 conducted on the activation of bovine oocytes was found to be similar to the division rate obtained in our study. As a result, it has been shown that the addition of royal jelly as a protein source to the culture medium in the in vitro maturation of bovine oocytes can be divided up to 2-cell and later stages as in the serum addition. According to the results obtained, it has been shown that in vitro matured oocytes can be divided as in vitro mature oocytes in culture media where serum and serum albumin are used as protein sources in culture media where royal jelly is added as protein source.

CONCLUSION In conclusion, the successful maturation of bovine oocytes in culture media using royal jelly instead of serum and the detection of their cleavage following parthenogenetic activation is a first in this field. It is necessary to investigate the growth potential until the blastocyst stage and pregnancy capacity of oocytes that are matured using royal jelly as a protein source in the culture medium.

ACKNOWLEDGEMENT This article was produced from Emre SIRIN’s master thesis.

References 1. Machatkova M., Jokesova E., Horky F., Krepelova A. (2000). Utilization of the growth phase of the first follicular wave for bovine oocyte collection improves blastocyst production. Theriogenology, 54: 543-550. 2. Lohuis M.M. (1995). Potential benefits of bovine embryo-manipulation technologies to genetic improvement programs. Theriogenology, 43: 51-60.

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E. Şirin et al. Large Animal Review 2021; 27: 135-141 3. Kruip T., den Daas J. (1997). In vitro produced and cloned embryos: effects of pregnancy, parturation, and offspring. Theriogenology, 47: 4352. 4. Van Wagtendonk-de Leeuw A.M., Mullaart B.J.G., den Daas J. (1998). Abnormal offspring Following in vitro production of bovine preimplantation embryos. Theriogenology, 49: 883-894. 5. Behboodi E., Anderson G.B, BonDurant R.H., Cargill S.L., Kreuscher B.R., Medrano J. F., Murray J.D. (1995). Birth of large calves that developed from in vitro-derived bovine embryos. Theriogenology, 44: 227-232. 6. Young L.E., Sinclair K.D. (1998). Wimut I. Large offsprin syndrome in cattle and sheep. Rev Reprod, 3: 155-163. 7. McEvoy T., Sinclair G., Young K. D., Wilmut I., Robinson J.J. (2000). Large offsprin sendrome and other consequences of ruminant embryo cultre in vitro: revelance to blastocyst culture in human. Human Fertility, 3: 238-246. 8. Sinclair K.D., Maxfield E.K., Robinson J.J., Maltin C.A., McEvoy T.G., Dune L.D., Young L.E., Broadbent P.J. (1997). Culture of sheep zygotes can alter fetal growth and devolopment. Theriogenology, 47: 380. 9. Duque P., Gomez E., Diaz E., Facal N., Hidalgo C., Diez C. (2003). Use of two replacements of serum during bovine embryo culture in vitro. Theriogenology, 59: 889-899. 10. Ali A., Sirard M.A. (2002). Effect of the absence or presence of various protein supplements on further de.velopment of bovine oocytes during in vitro maturation. Biol Reprod, 66: 901-905. 11. Geshi M., Takenouchi N., Yamauchi E. (2000). Effects of sodium pyruvate in nonserum maturation medium on maturation, fertilization, and subsequent development of bovine oocytes with or without cumulus cells. Biol Reprod, 63: 1730-1734. 12. Ocana-Quero J. M., Pinedo-Merlin M., Moreno-Millan M. (1999). Influence of follicle size, medium, temperature and time on the incidence of diploid bovine oocytes matured in vitro. Theriogenology, 51 (3), 667-672.

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13. Crozet N. (1984). Ultrastructure aspects of in vivo fertilization in the cow. Gamete Res, 10: 241- 251 14. Blondin P., Sirard M.A. (1995). Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes. Mol Reprod Dev, 41: 54-62 15. Hyttel P., Xu K.P., Smith S., Greve T. (1986). Ultrastructure of in-vitro oocyte maturation in cattle. J Reprod Fertil, 78: 615-625 16. Lorenzo P.L., Illera M.J., Illera J.C., Illera M. (1994). Enhancement of cumulus expansion and nuclear maturation during bovine oocyte maturation in vitro by the addition of epidermal growth factor and insulilike growth factor I. J Reprod Fertil, 101: 697-701. 17. Duranthon V., Renard J.P. (2001). The developmental competence of mammalian oocytes: A convenient but biologically fuzzy concept.Theriogenology, 55: 1277-1289. 18. King W.A., Bousquet D., Greve T., Goff A.K. (1986). Meiosis in bovine oocytes matured in vivo and in vitro. Acta Vet Scand, 27: 267-279. 19. Stojkovic M., Buttner M., Zakhartchenko V., Brem G., Wolf F. (1998). A reliable procedure for differential staining of in vitro produced bovine blastocyst: comparison of tissue culture medium 199 and Menezo’s B2 medium. Anim Reprod Sci, 50:1-9. 20. Presice G. A., Yang X. (1994). Nuclear dynemics of parthenogenesis of bovine oocytes matured in vitro for 20 and 40 hours and activated with combine d ethanol and cycloheximide treatment. Mol Reprod Dev, 37: 61-68. 21. Ware C.B., Barnes F.L., Maiki-Laurila M., First N.L. (1998). Age dependence of bovine oocyte activation. Gamet Res, 22: 265-275. 22. Taylor C., Lawrence T., Kingsland Y.M., Biljan C.R., Cuthberston K.S.D. (1993). Oscillation in intracelluler free calcium induced by spermatozoa in human oocytes at fertilization. Hum Reprod, 8: 2147-2179. 23. Otoi T.K.Y., Koyoma N., Tachikawa S. (1996). Developmen of in vitro matured bovine oocytes activated with low temparature. Theriogenology, 45: 153.

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K.Ç. Tümer et al. Large Animal Review 2021; 27: 143-147

Serum cardiac troponin I concentrations in ewes diagnosed with parturient paresis: correlation with blood ionized calcium and conventional cardiac enzymes

143

KENAN ÇAĞRI TÜMER1*, MEHMET ÇALIŞKAN1, TARIK ŞAFAK2 1 2

Department of Internal Medicine, Faculty of Veterinary Medicine, Fırat University, Elazığ, Turkey Department of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Fırat University, Elazığ, Turkey

SUMMARY Calcium is an essential mineral for cardiac muscle excitation-contraction coupling and relaxation, and hypocalcemia results in a decrease in myocardial contractility force and an increase in cardiac cell permeability. Therefore, myocardial damage can occur in ewes with parturient paresis, a metabolic disease caused by decreased blood ionized calcium (iCa) concentrations. This study aimed to investigate the occurrence of myocardial damage in ewes diagnosed with parturient paresis by measuring serum cardiac troponin I (cTnI) concentrations as well as to determine whether a correlation exists among cTnI, iCa, and conventional enzymes, namely, myocardial band of creatine kinase (CK-MB), creatine kinase (CK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). Twelve ewes diagnosed with parturient paresis (iCa 1.0 mmol/L) and ten healthy control ewes (iCa ≥ 1 mmol/L) were used in this study. To analyze the blood iCa, beta-hydroxybutyrate (β-OHB), and serum cTnI, CK-MB, CK, AST, and LDH concentrations, we collected venous blood samples from the vena jugularis of the ewes. Serum cTnI concentrations were significantly increased in the patient group (1.11 ± 0.62 ng/mL) compared with those in the control group (0.02 ± 0.01 ng/mL). The serum concentrations of CK-MB, CK, AST, and LDH were also higher in the patient group than in the control group. Moreover, cTnI was positively correlated with AST (r = 0.639; P = 0.001), LDH (r = 0.553; P = 0.008), and CK (r = 0.598; P = 0.003). However, a significant negative correlation was detected between cTnI and iCa (r = −0.867; P = 0.001). The results showed that myocardial damage occurred in ewes with parturient paresis.

KEY WORDS Cardiac troponin I, ewe, ionized hypocalcemia, parturient paresis.

INTRODUCTION Parturient paresis is an acute-onset metabolic disturbance in pregnant and lactating ewes and is characterized by tetany, ataxia, incoordination, recumbency, and coma.1 This condition is mainly caused by a decrease in blood ionized calcium (iCa) concentrations due to increased calcium (Ca) demands for fetal skeleton mineralization during late gestation.2 Ca is necessary in many physiological functions in the body. For instance, Ca plays a role in muscle contractions.3 Apart from its function in skeletal muscle, Ca plays a critical role in cardiac muscle excitation-contraction coupling and relaxation. In cardiac muscle, action potential induces the opening of L-type channels, allowing the flow of extracellular Ca into the myocytes . This phenomenon results in the Ca-induced release of Ca from the sarcoplasmic reticulum and in the increased intracellular Ca concentration. Subsequently, Ca binds to the tro-

Corresponding Author: Kenan Çağrı Tümer (kctumer@firat.edu.tr).

ponin-tropomyosin complex and activates the formation of actin-myosin cross-bridges.4,5 Regardless of its cause, hypocalcemia reduces the myocardial contractility force.6 The occurrence of hypocalcemia-related myocardial dysfunction is well recognized in human patients, and many of the cases are associated with chronic hypocalcemia.7 However, a closer look on the literature on hypocalcemia-related myocardial damage in veterinary science revealed the limited number of studies conducted on ruminants. In these studies, the presence of myocardial damage in cattle with hypocalcemia was evaluated by using cardiac markers or by examining macroscopic and microscopic changes in the myocardium.8-10 To our knowledge, no previous research has investigated the presence of myocardial damage in relation to parturient paresis in ewes. Thus, this study aimed to investigate the presence of myocardial damage in ewes diagnosed with parturient paresis by measuring serum cardiac troponin I (cTnI) concentrations and to determine whether a correlation exists among cTnI, iCa, and conventional enzymes, namely, myocardial band of creatine kinase (CK-MB), creatine kinase (CK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH).

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Serum cardiac troponin I concentrations in ewes diagnosed with parturient paresis

MATERIALS AND METHODS Animals This study was approved by the Fırat University Local Ethics Committee on Animal Experimentation (14.05.2020, 2020/7). The ewes whose owners signed a consent form were enrolled in this study. Twelve pregnant and lactating ewes that were admitted to the Fırat University Veterinary Teaching Hospital and were diagnosed with parturient paresis comprised the patient group. Of the 12 ewes, three were in their first week of lactation and the rest were in their last month of pregnancy. The ewe breeds in this group were Akkaraman (n = 10) and Awassi (n = 2). The ewes in this group met the following criteria: a blood iCa concentration of less than 1.0 mmol/L11, a blood beta-hydroxybutyrate (β-OHB) concentration of less than 0.8 mmol/L12, and being in their late pregnancy or early lactation stage. The control group consisted of 10 ewes that were clinically healthy and had a blood iCa concentration above 1.0 mmol/L and a blood β-OHB concentration below 0.8 mmol/L. Of the 10 ewes, 5 ewes were in their last month of pregnancy and 5 were in their early lactation stage. The ewe breeds in the control group were Akkaraman (n = 5) and Awassi (n = 5). Ewes with anemia, systemic inflammation, and any pathological conditions associated with pregnancy based on the clinical and hematological findings were excluded from this study.

Hematological, biochemical, and venous blood gas-electrolyte analysis Blood samples for the hematological, biochemical, blood βOHB, and venous blood gas-electrolyte analysis were extracted from the vena jugularis of all the ewes and placed into tubes containing EDTA (BD Vacutainer, Plymouth, UK), into serum tubes with clot activator (BD Vacutainer), and into an injector containing electrolyte-balanced dry heparin (Pico 50, Radiometer, Copenhagen, Denmark), respectively. Packed cell volume (PCV) was measured using the spun method, and the total white blood cell (WBC) counts were determined by a hemocytometer. Venous pH and sodium, potassium, chloride (Cl), and iCa concentrations were analyzed using a bench-top blood gas analyzer (ABL80 Flex Basic, Radiometer). A pointof-care analyzer (Freestyle Optium NeoH, Abbott, Alameda, CA, USA) was used to measure the whole blood β-OHB concentration. Serum CK-MB, CK, AST, LDH, magnesium (Mg),

and phosphorus (P) concentrations were determined with an automatic chemiluminescence immunoassay system (Advia Centaur XP, Siemens Healthcare Diagnostics, Malvern, PA, USA). A human-based cTnI analyzer (Advia Centaur TnI-Ultra, Siemens Healthcare Diagnostics) was used to measure serum cTnI concentrations.

Statistical analysis Statistical differences between groups were assessed using SPSS21 (IBM Corp., Armonk, NY, USA). The Shapiro-Wilk test was performed to determine whether the variables were normally distributed. Data are presented as mean ± standard deviation. An independent sample t-test was used to determine the statistical difference between groups. Moreover, correlation was tested using Pearson’s correlation coefficient test. A Pvalue of less than 0.05 was considered to indicate statistical significance.

RESULTS Clinical and hematological findings The results are shown in Table 1. The mean ages for the patient group and control group were 4.75 ± 0.89 and 3.50 ± 1.19, respectively. The ewes in the patient group showed a different clinical presentation, as follows: they displayed flaccid paralysis, lateral recumbency, and absence of pupillary light reflex (n = 2); they were able to stand when supported, and they displayed sternal recumbency and absence of pupillary light reflex (n = 6); they were able to stand when supported but showed severe muscle tremors (n = 2); and they were able to stand when supported but showed stiff gait (n = 2). Rectal temperature did not differ between the groups (P = 0.549). However, the heart rate increased significantly (P = 0.013) in the patient group compared with that in the control group. No statistical differences in PCV and WBC counts were observed between the groups.

Venous blood gas and electrolyte results The results are shown in Table 2. No significant differences were observed between the groups in terms of venous blood pH and partial CO2 pressure (pCO2). In the patient group, blood Cl concentrations were significantly decreased (P = 0.001) compared with those in the control group. The mean blood iCa con-

Table 1 - Mean, standard deviation (S.D), median, minimum and maximum values of the rectal temperature, heart rate, PCV, and WBC counts in the patient group (n=12) and control group (n=10). Variables

Unit

Rectal Temperature ˚C Heart Rate beats/min. PCV % WBC × 103/µL

Descriptive Statistics

Groups

Mean

S.D

Median

Minimum

Maximum

Control

38.81

0.33

38.85

38.20

39.30

Patient

38.62

0.90

39.95

36.70

39.60

Control

98.00

7.65

97.00

110

Patient

12.08

26.85

112.50

180

Control

33.40

3.16

Patient

31.58

3.67

30.50

Control

7.08

1.50

7.05

5.2

9.7

Patient

7.64

1.49

7.50

5.9

10.5

PCV: packed cell volume; WBC: white blood cell.

0.549

0.013

0.234

0.393

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Table 2 - Mean, standard deviation (S.D), median, minimum and maximum values of the venous blood gas and electrolytes in the patient group (n=12) and control group (n=10). Variables

Unit

Venous pH pCO2

mmHg

Sodium mmol/L Potassium mmol/L Ionized Calcium mmol/L Chloride mmol/L Phosphorus mg/dL Magnesium mg/dL

Descriptive Statistics

Groups

Mean

S.D

Median

Minimum

Maximum

Control

7.43

0.33

7.43

7.39

7.49

Patient

7.47

0.68

7.47

7.37

7.58

Control

37.56

2.72

37.95

32.00

41.30

Patient

35.40

6.42

33.75

30.00

53.90

Control

148.60

3.83

147.50

145

156

Patient

146.58

5.82

147.00

133

155

Control

4.23

0.41

4.29

3.25

4.86

Patient

3.65

0.78

3.59

2.64

5.30

Control

1.22

0.07

1.24

1.10

1.32

Patient

0.69

0.18

0.67

0.43

0.98

Control

108.60

0.96

108.50

107.00

110.00

Patient

102.75

5.32

102.00

95.00

115.00

Control

4.32

1.24

4.20

2.41

7.00

Patient

7.10

2.82

7.05

3.10

14.10

Control

2.36

0.41

2.26

1.80

3.27

Patient

2.35

0.36

2.34

1.71

2.95

0.188

0.336

0.360

0.049

0.001

0.003

0.009

0.948

Table 3 - Mean, standard deviation (S.D), median, minimum and maximum values of the serum cTnI, CKMB, CK, AST, LDH, and blood βOHB concentrations in the patient group (n=12) and control group (n=10). Variables

Unit

cTnI ng/mL CKMB U/L CK U/L AST U/L LDH U/L β-OHB

mmol/L

Descriptive Statistics

Groups

Mean

S.D

Median

Minimum

Maximum

Control

0.02

0.01

0.02

0.01

0.04

Patient

1.11

0.62

1.07

0.14

2.27

Control

151.69

45.00

153.44

73.07

215.42

Patient

239.85

120.21

202.03

104.31

509.85

Control

172.20

144.43

144.50

648

Patient

400.17

103.81

398.50

223

648

Control

87.30

15.49

86.50

115

Patient

169.17

42.66

170.00

253

Control

451.40

52.52

449.50

371

571

Patient

809.23

266.94

800

443

1292

Control

0.23

0.13

0.20

0.10

0.50

Patient

0.34

0.20

0.30

0.10

0.70

centration in the patient group was 0.69 ± 0.18 mmol/L whereas that in the control group was 1.22 ± 0.07 mmol/L. Also, serum P concentration was significantly higher (P = 0.009) in the patient group than in the control group.

Biochemical results The results are shown in Table 3. The mean serum cTnI concentration was significantly increased (P = 0.001) in the patient group relative to that in the control group. Also, the mean concentrations of the other cardiac markers were increased in the patient group. iCa concentrations were significantly negatively correlated with CK-MB (r = −0.458; P = 0.032), CK (r = −0.694; P = 0.001), AST (r = −0.750; P = 0.001), and LDH (r = −0.677; P = 0.001). The most evident negative correlation was detected between iCa and serum cTnI concentrations (r = −0.867;

0.001

0.041

0.001

0.157

P = 0.001). By contrast, serum cTnI concentrations were positively correlated with serum AST (r = 0.639; P = 0.001), LDH (r = 0.553; P = 0.008), and CK (r = 0.598; P = 0.003) concentrations, but no correlation was observed between cTnI and CKMB. In terms of mean serum β-OHB concentration, no significant difference was found between the groups.

DISCUSSION In this study, we evaluated the presence of myocardial damage in ewes with parturient paresis by measuring the serum concentrations of cTnI and of the conventional enzymes CK-MB, CK, AST, and LDH. The results indicated the occurrence of myocardial damage in ewes with parturient paresis. Studies have

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Serum cardiac troponin I concentrations in ewes diagnosed with parturient paresis

shown the presence of myocardial lesions and the increased cardiac markers in cattle with clinical hypocalcemia.8-10 However, to our knowledge, no prior studies have investigated myocardial damage in ewes with parturient paresis. The clinical manifestation of parturient paresis may vary and is strongly associated with both the circulating Ca concentration and the stage of the disease.1,13,14 Additionally, ewes that have multiple fetuses are more prone to develop parturient paresis during late gestation.14 Moreover, studies have reported various clinical manifestations of parturient paresis, including depression, incoordination, muscular weakness, muscular tremor, recumbency, flaccid paralysis, twisted or extended head, increased respiration, increased heart rate, ruminal tympany, and decreased reflexes.15,16 In the present study, the ewes in the patient group showed clinical signs similar to those previously reported. Ca, Mg, and P metabolisms are closely related. When blood iCa concentration decreases, the parathyroid glands are stimulated to secrete parathyroid hormone (PTH). PTH secretion results in increased reabsorption of Ca from kidneys, intestines, and bones. While PTH secretion increases the reabsorption of Ca, it also increases P excretion via the kidney and saliva and raises the renal threshold of Mg. Consequently, hypocalcemic animals tend to be hypophosphatemic and hypermagnesemic.3,6 There have been numerous studies investigating the serum Mg and P concentrations in sheep with parturient paresis. Woldemeskel et al. reported no difference in serum concentrations of Mg and P between hypocalcemic and normocalcemic pregnant ewes.16 In another study, the researchers reported a normal serum P concentration and a decreased serum magnesium concentration in hypocalcemic sheep.15 Contrary to the previous reports, we found hyperphosphatemia and normal serum Mg concentration in our patient group. The possible explanations for the increased serum P concentration are that the PTH and calcitriol might have increased the serum P concentration as they stimulated P absorption from bones and intestines and that the high dietary P concentration might have contributed to the increased serum P concentration.6 Troponins are regulatory and structural proteins found in both cardiac and skeletal muscles. A troponin complex consists of three subunits, namely, troponin T, troponin C, and troponin I (TnI). TnI has three isoforms; the cardiac isoform of TnI (cTnI) differs from the two other TnI isoforms found in skeletal muscle based on the additional 32 amino acids at its N-terminal region. This feature of cTnI renders it as a gold standard in the determination of myocardial damage.17,18 In veterinary medicine, circulating cTnI concentrations are evaluated in different diseases that lead to primary or secondary myocardial damage.19-24 Increased cTnI concentrations were also reported in sheep with pregnancy toxemia, acute ruminal lactic acidosis, babesiosis, experimental salinomycin toxication, white muscle disease, and foot and mouth disease.12,25-29 In our study, we found a significantly higher serum cTnI concentration in the patient group than in the control group, consistent with the previous findings. In addition to measuring serum cTnI concentration, we also measured other cardiac biomarkers, namely, CK, CK-MB, AST, and LDH. Although these markers are commonly used in research studies, their use in clinical practice is limited in both human and veterinary medicine because of their lack of sensitivity and specificity for cardiac tissue.30 The results for CK-MB, CK, AST, and LDH concentrations indicated an obvious muscle damage in the patient group. How-

ever, it is improper to attribute the increase of these enzymes to cardiac muscle damage because these enzymes demonstrate a high activity in skeletal muscle.31 Also, parturient paresis causes neuromuscular dysfunction, and it is highly possible that the increase in these enzymes is due to recumbency.32 Although the role of Ca during muscle excitation-contraction coupling and relaxation is well recognized, the pathophysiological mechanism of myocardial damage during hypocalcemia has not yet been fully understood. Some researchers have reported that myocardial damage is reversible during hypocalcemia and that circulating cTnI concentration decreased and cardiac contractility improved after an appropriate treatment.33,34 Also, it has been shown that hypocalcemia increases cellular permeability by keeping the cellular membrane pore gates open.35 One of the possible mechanisms of troponin release from cardiac myocytes into the circulation is the increased cellular permeability.23 In the present study, the possible cause of increased serum cTnI concentration in ewes with parturient paresis is the increased permeability of the cell membrane of cardiac myocytes.

CONCLUSION Although parturient paresis is not as common in ewes as in cattle, it may occur in ewes during late pregnancy and during early lactation. The presence of myocardial damage in the hypocalcemic ewes may affect the prognosis of the disease. Future research should consider the potential prognostic value of serum cTnI changes in hypocalcemic ewes.

References 1.

Sykes A.R. (2007). Deficiency of mineral macro-elements. In: Diseases of Sheep, Ed. Aitken I.D.,4th ed., 363-377, Blackwell, Oxford, UK. 2. Oetzel G.R., Goff J.P. (2009). Milk fever (Parturient Paresis) in cows, ewes and doe goats. In: Current Veterinary Theraphy in Food Animal Practice, Ed. Anderson D., Rings M., 5th ed., 132-134, Saunders, St. Louis, MO, USA. 3. Schenck P.A., Chew D.J., Nagode L.A., Rosol T.J. (2012). Disorders of calcium: Hypercalcemia and hypocalcemia. In: Fluid, Electrolyte, and Acid-Base Disorders in Small Animal Practice, Ed. DiBartola S.P., 4th ed., 120-194, Saunders, St. Louis, MO, USA. 4. Bers D.M. (2002). Cardiac excitation-contraction coupling. Nature, 415: 198-205. 5. Eisner D.A., Caldwell J.L., Kistamás K., Trafford A.W. (2017). Calcium and excitation-contraction coupling in the heart. Circ Res, 121 (2): 181-195. 6. Goff J.P. (2015). Minerals. In: Dukes’ Physiology of Domestic Animals, Ed. Reece W.O., 568-586, Blackwell, Iowa. 7. Newman D.B., Fidahussein S.S., Kashiwagi D.T.,Kennel K.A. Kashani K.B, Wang Z., Altayar O., Murad M.H. (2014). Reversible cardiac dysfunction associated with hypocalcemia: a systematic review and metaanalysis of individual patient data. Heart Fail Rev, 19 (2): 199-205. 8. Basbuğan Y., Yüksek N., Altuğ N. (2015). Significance of homocysteine and cardiac markers in cattle with hypocalcemia. Turk J Vet Anim Sci, 39 (6): 699-704. 9. Yamagishi N., Naito Y. (1997). Calcium metabolism in hypocalcemic cows with myocardial lesion. J Vet Med Sci, 59 (1): 71-73. 10. Yamagishi N., Ogawa K., Naito Y. (1999). Pathological changes in the myocardium of hypocalcaemic parturient cows. Vet Rec, 144 (3): 6772. 11. Scott P. (2013). Hypocalcaemia in ewes. Livestock, 18 (1): 37-40. 12. Souza L.M., Mendonça C.L., Assis R.N., Oliveira Filho E.F., Gonçalves D.N.A., Souto R.J.C., Soares P.C., Afonso J.A.B. (2019). Cardiac biomarkers troponin I and CK-MB in ewes affected by pregnancy toxemia. Small Rumin Res, 177: 97-102.

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K.Ç. Tümer et al. Large Animal Review 2021; 27: 143-147 13. Cockcroft P.D., Whiteley P. (1999). Hypocalcaemia in 23 ataxic/recumbent ewes: clinical signs and likelihood ratios. Vet Rec, 144 (19): 529-532. 14. Elias E., Shainkin-Kestenbaum R. (1990). Hypocalcaemia and serum levels of inorganic phosphorus, magnesium parathyroid and calcitonin hormones in the last month of pregnancy in Awassi fat-tail ewes. Reprod Nutr Dev, 30 (6): 693-699. 15. El-Khodery S., El-Boshy M., Gaafar K., Elmashad A. (2008). Hypocalcaemia in Ossimi sheep associated with feeding on beet tops (Beta vulgaris). Turk J Vet Anim Sci, 32 (3): 199-205. 16. Woldemeskel M., Eneyew M., Kassa T. (2000). Study on ovine hypocalcemia in ewes in central Ethiopia. Rev Med Vet, 151 (4): 345-350. 17. Wells S., Sleeper M. (2008). Cardiac troponins. J Vet Emerg Crit Car (San Antonio), 18 (3): 235-245. 18. Sarko J., Pollack C.V. (2002). Cardiac troponins. J Emerg Med, 23 (1): 57-65. 19. Fartashvand M., Nadalian M.G., Sakha M., Safi S. (2013). Elevated serum cardiac troponin I in cattle with theileriosis. J Vet Intern Med, 27 (1): 194-199. 20. Karapınar T., Tümer K.Ç., Çalışkan M., Yılmaz Ö. (2019). Case report: serum cardiac troponin I concentration in three calves with doxycycline overdose. Rev Med Vet, 170 (1-3): 43-45. 21. Varga A., Angelos J.A., Graham T.W., Chigerwe M. (2013). Preliminary investigation of cardiac troponin I concentration in cows with common production diseases. Journal of Veterinary Internal Medicine 2013; 27 (6): 1613-1621. 22. Varga A, Schober K, Holloman C, Stromberg P, Lakritz J et al. Correlation of serum cardiac troponin I and myocardial damage in cattle with monensin toxicosis. J Vet Intern Med, 23 (5): 1108-1116. 23. Langhorn R., Willesen J.L. (2016). Cardiac troponins in dogs and cats. J Vet Intern Med, 30 (1): 36-50. 24. Karapinar T., Dabak D.O., Kuloglu T., Bulut H. (2010). High cardiac troponin I plasma concentration in a calf with myocarditis. Can Vet J, 51 (4): 397-399.

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25. Hajimohammadi A., Rajaian H., Khaliji E., Nazifi S., Ansari-Lari M. (2014). Serum cardiac troponin I as a biomarker in cardiac degeneration following experimental salinomycin toxicosis in sheep. Vet Arh, 81 (4): 41-51. 26. Kırbaş A., Baydar E., Kandemir F.M., Dorman E., Kızıl O., Yıldırım B.A. (2014). Evaluation of serum cardiac troponin I concentration in sheep with acute ruminal lactic acidosis. Vet Arh, 84 (4): 355-364. 27. Karapınar T., Eröksüz Y., Beytut E., Sözdutmaz I., Eröksüz H., Dabak M. (2012). Increased plasma cardiac troponin I concentration in lambs with myocarditis. Ve Clin Pathol, 41 (3): 375-381. 28. Kılınç Orunç Ö., Göz Y., Yüksek N., Başbuğan Y., Yılmaz A.B., Ata A.D. (2015). Determination of serum cardiac biomarkers and plasma D-dimer levels in anemic sheep with babesiosis. Turk J Vet Anim Sci, 39: 606-610. 29. Tunca R., Erdoğan H.M., Sözmen M., Çitil M., Devrim A.K., Erginsoy S., Uzlu E. (2009). Evaluation of cardiac troponin I and inducible nitric oxide synthase expressions in lambs with white muscle disease. Turk J Vet Anim Sci, 33 (1): 53-59. 30. O’Brien P.J. (2008). Cardiac troponin is the most effective translational safety biomarker for myocardial injury in cardiotoxicity. Toxicology, 245 (3): 206-218. 31. Braun J.P., Trumel C., Bézille P. (2010). Clinical biochemistry in sheep: A selected review. Small Rumin Res, 92 (1-3): 10-18. 32. Oetzel G.R. (1988). Parturient paresis and hypocalcemia in ruminant livestock. Vet Clin North Am Food Anim Pract, 4 (2): 351-364. 33. Aguiara P., Cruz D., Rodrigues R.F., Peixoto L., Araújo F., Soares J.L.D. (2013). Hypocalcemic cardiomyopathy. Rev Port Cardiol, 32 (4): 331335. 34. Gupta P., Tomar M., Radhakrishnan S., Shrivastava S. (2011). Hypocalcemic cardiomyopathy presenting as cardiogenic shock. Ann Pediatr Cardiol, 4 (2): 152-155. 35. Tripp A. (1976). Hyper and Hypocalcemia. Am J Nurs, 76 (7): 11421145.

SCARICA LA APP L’informazione veterinaria on line

È GRATUITA

Iscrizione Pubblico Registro della Stampa del Tribunale di Cremona n. 375 - 11/10/2001

di Anmvi Oggi

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Enteropatia proliferativa da Lawsonia intracellularis nel suino

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GIULIA D’ANNUNZIO1, ROBERTO BARDINI2, FABIO OSTANELLO1, GIUSEPPE SARLI1* 1 2

Dipartimento di Scienze Mediche Veterinarie, Alma Mater Studiorum - Università di Bologna Trouw Nutrition Italia S.p.A.

RIASSUNTO L’enteropatia proliferativa del suino (proliferative enteropathy - PE), denominata anche ileite, è causata da Lawsonia intracellularis, un batterio intracellulare obbligato. La PE è una patologia a trasmissione oro-fecale che si manifesta soprattutto nella fase di magronaggio ed è responsabile di consistenti perdite economiche negli allevamenti intensivi. I danni economici sono causati dalla riduzione dell’incremento ponderale e dell’indice di conversione dell’alimento e dall’aumento della mortalità e dei soggetti di scarto. La PE è endemica in numerosi Paesi con prevalenze di aziende ed animali infetti che, in Europa, superano il 90% e il 40%, rispettivamente. Nel suino, la patologia è caratterizzata da un ispessimento della mucosa intestinale dovuto alla proliferazione incontrollata delle cellule delle cripte intestinali accompagnata dall’inibizione, ad opera di L. intracellularis, della maturazione e della differenziazione delle cellule caliciformi secretorie e delle cellule assorbenti. La conseguenza inevitabile è la riduzione dell’assorbimento dei nutrienti e la perdita di aminoacidi e proteine nel lume intestinale, con conseguente diarrea. La PE si manifesta con due forme cliniche principali: 1) la forma acuta (enteropatia proliferativa emorragica - PHE) che si osserva principalmente in animali dai 4 ai 12 mesi d’età, caratterizzata da una diarrea sanguinolenta e da elevata mortalità (fino al 50%) e, 2) la forma cronica (adenomatosi intestinale - PIA) che colpisce suini dalle 6 alle 20 settimane d’età, e il cui sintomo principale è rappresentato da una diarrea con feci pastose. Sono state descritte anche altre due forme cliniche a bassa incidenza: a) l’enterite necrotica (NE), espressione di una forma di enteropatia proliferativa cronica complicata da infezioni secondarie che esita in un’estesa necrosi coagulativa dell’epitelio intestinale e, b) l’ileite regionale (RI), risultante dalla guarigione delle lesioni dovute a NE e caratterizzata da deposizione di tessuto di granulazione e ispessimento della tonaca muscolare. La diagnosi indiretta, che viene generalmente eseguita utilizzando un test ELISA, consente di valutare l’eventuale esposizione dei suini a L. intracellularis, mentre la diagnosi diretta (realizzata impiegando test biomolecolari qualitativi: PCR o quantitativi: qPCR, immunoistochimica - IHC) permette di valutare se l’infezione è in atto. Analogamente a quanto accade per altre forme patologiche del suino, la semplice messa in evidenza di L. intracellularis nelle feci non rappresenta un criterio diagnostico valido nei confronti di PE. La corretta procedura diagnostica prevede la quantificazione del numero di microrganismi/grammo di feci e la messa in evidenza di L. intracellularis all’interno delle lesioni intestinali. La profilassi e il controllo si basano sull’applicazione di rigide misure di lavaggio e disinfezione che consentono di ridurre la contaminazione ambientale tra un ciclo produttivo e l’altro e l’applicazione di misure di biosicurezza interna. Particolare attenzione va riservata all’alimentazione, che dovrebbe garantire l’equilibrio della microflora intestinale tramite un corretto rapporto tra proteina altamente digeribile e frazione di fibra, con il supporto di integratori probiotici e prebiotici. In una logica di uso consapevole del farmaco, la somministrazione di massa di antibiotici durante la fase critica del magronaggio dovrebbe essere limitata ai soli gruppi con sintomatologia clinica, implementando invece la profilassi vaccinale.

PAROLE CHIAVE Lawsonia intracellularis; enteropatia proliferativa; suino.

INTRODUZIONE Le patologie enteriche dei suini, in particolare quelle che si manifestano durante lo svezzamento, sono molto comuni e spesso di natura infettiva. In genere, i virus svolgono un ruolo patogeno principale negli animali giovani, mentre in quelli in accrescimento alcuni batteri e protozoi sono spesso responsabili di diarrea, con conseguente riduzione dell’incremento ponderale medio giornaliero (IPMG)1.

Corresponding Author: Giuseppe Sarli (giuseppe.sarli@unibo.it)

L’enteropatia proliferativa del suino (proliferative enteropathy - PE), è una patologia tipica della fase di magronaggio, responsabile di consistenti perdite economiche negli allevamenti intensivi dovute alla riduzione dell’IPMG, dell’indice di conversione dell’alimento e all’aumento della mortalità e dei soggetti di scarto2. Stime realizzate in diversi Paesi indicano che la PE può causare una riduzione dell’1,2% degli utili dell’allevamento e perdite variabili da 1,2 a 2,5 €per ogni suino svezzato e da 2,2 a 7,8 €per ogni suino da ingrasso con sintomatologia2. Il microrganismo responsabile, Lawsonia intracellularis, è un batterio intracellulare obbligato, Gram-negativo, non sporigeno, microaerofilo3, localizzato, in vivo, nel citoplasma apicale del-

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le cellule epiteliali delle cripte intestinali. Nel suino, la patologia è caratterizzata da un ispessimento della mucosa intestinale causato dalla proliferazione incontrollata delle cellule delle cripte intestinali accompagnata all’inibizione, ad opera di L. intracellularis, della maturazione e della differenziazione delle cellule caliciformi secretorie e delle cellule assorbenti. La conseguenza inevitabile è la riduzione dell’assorbimento dei nutrienti e la perdita di aminoacidi e proteine nel lume intestinale, condizioni prodromiche allo sviluppo di diarrea4. Le conoscenze sulla patogenesi della PE sono limitate sia dalla difficoltà a coltivare in vitro, su substrati cellulari, colture pure di L. intracellularis5, sia dalla mancanza di modelli in vitro in grado di riprodurre le lesioni proliferative6. Sebbene i focolai di PE vengano segnalati a livello globale, è stato stimato che meno di 25 isolati di L. intracellularis siano stati coltivati con successo e mantenuti in vitro in tutto il mondo. Di questi isolati, solo 15 sono stati saggiati per la loro sensibilità agli antimicrobici7. In questa review vengono presentati gli aspetti principali dell’enteropatia proliferativa da Lawsonia intracellularis nel suino, compresi quelli recentemente riportati nel corso della 1st International conference on Lawsonia intracellularis - The Gordon Lawson Memorial Symposium (The Royal (Dick) School of Veterinary Studies, Edinburgh, UK, 26 e 27 settembre 2019) (Figura 1).

EPIDEMIOLOGIA L’infezione da L. intracellularis è diffusa nei suini domestici di tutto il mondo, con evidenza di forme cliniche soprattutto negli allevamenti intensivi8. In alcuni Paesi Europei (Germania, Danimarca, Spagna, Olanda e Regno Unito), la prevalenza di allevamenti infetti varia dal 6,7 al 93,7%, mentre la prevalenza intra-aziendale varia dallo 0,7 al 43,2%2. Tuttavia, per la corretta interpretazione di questi dati occorre prendere in considerazione alcuni fattori quali l’età dei soggetti esaminati e la diversa sensibilità e specificità dei test diagnostici utilizzati nei diversi studi. Inoltre, la semplice dimostrazione della presenza di L. intracellularis nelle feci dei suini non indica necessariamente una condizione di malattia9. È stata dimostrata la positività sierologica anche nei suini selvatici10, 11, che potrebbero rappresentare un fattore di rischio per gli allevamenti suinicoli all’aperto12. L’infezione naturale o sperimentale da L. intracellularis e le lesioni intestinali proliferative sono sporadicamente riportate anche in numerose specie diverse dal suino (criceto, cavallo, ratto, coniglio, furetto, volpe, cane, pecora, cervo, ratiti e primati non umani), sebbene non sia chiaro il ruolo di questi animali nell’epidemiologia della patologia del suino. In particolare, forme di malattia simili a quelle osservate nel suino sono state ampiamente descritte nel criceto da laboratorio e nel puledro13. Nonostante l’alto grado di omologia genetica tra gli isolati di L. intracellularis provenienti da specie animali diverse14, Vannucci et al.15 segnalano una potenziale differenza negli isolati di L. intracellularis ottenuti da suino e cavallo. È stato ipotizzato che i diversi ceppi di L. intracellularis possano possedere una sorta di specie-specificità. Sampieri et al.16 hanno dimostrato che i conigli sono più recettivi agli isolati di origine equina rispetto alle varianti suine mentre i criceti, al contrario, sono risultati più sensibili alle varianti di origine suina ri-

spetto a quelle di origine equina. I diversi ceppi sono quindi fenotipicamente identici ma è possibile differenziarli geneticamente (sia quelli provenienti da specie diverse, sia all’interno della stessa specie), individuando i polimorfismi del DNA mediante la quantificazione del numero di ripetizioni in tandem (Variable Number of Tandem Repeats - VNTR)17 (Figura 1a). La trasmissione di L. intracellularis è di tipo oro-fecale ed avviene per via diretta e indiretta attraverso il contatto con feci di animali infetti, o attraverso contatto intraspecifico18. Sperimentalmente, la dose infettante è relativamente bassa (108 microrganismi)9 mentre la quantità di L. intracellularis eliminata può superare il valore di 106 microrganismi/grammo di feci19. Sperimentalmente è stato dimostrato che i suini si possono infettare anche attraverso il contatto con feci di topi infetti facendo ipotizzare che questi animali possano rivestire un ruolo importante nell’epidemiologia di L. intracellularis negli allevamenti suinicoli20 (Figura 1b). L’eliminazione fecale inizia circa una settimana post-infezione (p.i.), raggiunge il picco a 3 settimane e permane, in molti soggetti, per 4 settimane. In alcuni animali è stato osservato che l’eliminazione fecale si protrae in maniera intermittente fino a 12 settimane dimostrando quindi che L. intracellularis è in grado di colonizzazione a lungo termine l’ospite21 (Figura 1c). Questo aspetto è di particolare importanza considerando che nelle infezioni subcliniche gli animali clinicamente sani possono eliminare per lungo tempo il microrganismo nell’ambiente, condizionandone la persistenza in allevamento, anche a fronte di una resistenza ambientale di L. intracellularis di circa 2 settimane a 5-15°C. Gli animali con infezione subclinica presentano comunque una scarsa massa corporea in conseguenza della riduzione dell’IPMG22. Negli allevamenti, la malattia si può manifestare in forme diverse in funzione del management aziendale, della dose infettante e del livello immunitario degli animali. Negli allevamenti a flusso continuo, l’infezione di solito si verifica poche settimane dopo lo svezzamento, presumibilmente in coincidenza con il declino dell’immunità passiva. La presenza di immunità materna giustificherebbe anche la mancata trasmissione di L. intracellularis in sala parto23. Questa dinamica temporale di infezione può essere ritardata dalla somministrazione di antimicrobici nelle prime settimane dopo lo svezzamento. In questo caso, la malattia compare nelle fasi di accrescimento e ingrasso. Negli allevamenti multisito, dove si applica la stretta separazione dei gruppi di suini (all-in/all-out, AIAO), l’infezione da L. intracellularis può comparire nei suini in accrescimento fino all’età di 14-20 settimane24.

PATOGENESI I fattori di virulenza responsabili dell’ingresso di L. intracellularis negli enterociti non sono stati ancora chiaramente individuati. Tuttavia, prove sperimentali in vitro hanno dimostrato che il processo di internalizzazione del batterio prevede una sua stretta adesione alla superficie cellulare, seguita dall’ingresso attraverso vacuoli prodotti dalla invaginazione della membrana cellulare e dal rilascio del patogeno nel citoplasma delle cellule25. Lawsonia intracellularis è in grado di infettare sia gli entero-

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citi maturi sia le cellule epiteliali immature delle cripte intestinali: a 12 ore dall’infezione sperimentale per via orale, il patogeno è già presente nel citoplasma degli enterociti maturi, all’apice dei villi del piccolo intestino. Da 5 a 28 giorni p.i., L. intracellularis è presente nel citoplasma di cellule poco differenziate delle cripte intestinali, causando un’infezione persistente. A questo stadio dell’infezione, le cellule epiteliali delle cripte continuano a proliferare ma non raggiungono la maturazione, dando così luogo alla proliferazione adenomatosa caratteristica della PE24. Nel citoplasma delle cellule, L. intracellularis è associata a ribosomi liberi e mitocondri e, tramite il meccanismo conosciuto come “parassitismo energetico”, sfrutta il pool energetico dell’ospite attraverso lo scambio di ADP batterico con ATP dell’ospite6. Nonostante il continuo trasporto d’ossigeno attraverso le membrane mitocondriali, L. intracellularis riesce a sopravvivere all’interno della cellula grazie ad un meccanismo di protezione dallo stress ossidativo che coinvolge l’enzima superossido dismutasi Cu-Zn (sodC) e la diossigenasi, resistendo ai metaboliti dello scoppio respiratorio15, 26. La replicazione intracellulare di L. intracellularis è direttamente associata alla proliferazione degli enterociti27, ma non sono ancora del tutto chiari i meccanismi mediante i quali L. intracellularis è in grado di determinare tale proliferazione. Grazie alla caratterizzazione del profilo di espressione genica degli enterociti infetti è stato osservato che le cellule proliferanti hanno una significativa attivazione della trascrizione del DNA, della biosintesi proteica e dei geni Rho che agiscono sulla fase G1 del ciclo cellulare15. L’attivazione di questo meccanismo è già conosciuta nell’oncogenesi e nella promozione della proliferazione cellulare (Figura 1d). Considerando che alcune ciclomoduline batteriche possono attivare patologicamente le proteine Rho28, è stato ipotizzato che possa esserci un meccanismo simile anche alla base dell’induzione della proliferazione da parte di L. intracellularis6. Durante l’infezione è stata osservata la sotto-espressione di numerosi geni i cui prodotti sono espressi nelle membrane apicali degli enterociti e sono coinvolti nell’assorbimento di nutrienti in quanto trasportatori di membrana responsabili dell’assorbimento di carboidrati, aminoacidi, lipidi e vitamina B12. L’infezione intracellulare influenza inoltre la secrezione di elettroliti, diminuendo l’espressione del gene del canale del cloruro (CLCA1). Questa riduzione dell’assorbimento di nutrienti, accompagnata dalla secrezione di elettroliti, è alla base del malassorbimento e della patogenesi della diarrea. L. intracellularis è quindi direttamente responsabile della mancata differenziazione degli enterociti immaturi oltre che della loro proliferazione che è probabilmente indotta dalla mancata regolazione della fase G1 del ciclo cellulare dell’ospite15. Con il progredire dell’infezione, L. intracellularis può essere osservata all’interno dei macrofagi situati nella lamina propria, anche dopo la sua clearance dalle cellule epiteliali dell’intestino (Figura 2). È quindi probabile che i macrofagi abbiano un ruolo nella diffusione dell’infezione1. La risposta immunitaria umorale e cellulo-mediata è evidenziabile a partire da 2 settimane p.i. e, in alcuni animali, persiste fino a circa 3 mesi p.i. Il picco sierico di IgG si osserva alla terza settimana p.i. a cui fa seguito una graduale riduzione. IgA specifiche sono evidenziabili a livello intestinale a partire dalla terza settimana p.i.24. È stato dimostrato che suini che hanno superato l’infezione sperimentale risultano protetti nei confronti della colonizzazione intestinale e della sintomatologia

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clinica se esposti nuovamente al patogeno. La risposta immunitaria specifica nei confronti di L. intracellularis è quindi proteggente (per almeno 10 settimane) nei confronti di un’eventuale re-infezione29. La progenie di scrofe gravemente interessate dalla patologia non è completamente protetta nei confronti della PE23. Alcune osservazioni sperimentali indicano inoltre che L. intracellularis è in grado di modulare la risposta immunitaria, consentendo l’infezione persistente delle cellule epiteliali delle cripte. Studi in vivo hanno costantemente dimostrato una sottoregolazione dei geni correlati alla risposta immunitaria. È stata inoltre osservata, nei suini infetti, una riduzione del numero di linfociti T e B24.

FORME CLINICHE L’enteropatia proliferativa del suino si può presentare sia in forma clinica sia in forma subclinica (Figura 3). Le forme cliniche principali sono rappresentate da una forma acuta (enteropatia proliferativa emorragica, proliferative hemorrhagic enteropathy - PHE) che si manifesta soprattutto in animali di 4-12 mesi d’età ed è caratterizzata da una diarrea sanguinolenta e da elevata mortalità (fino al 50%) e da una forma cronica (adenomatosi intestinale, porcine intestinal adenomatosis - PIA) che colpisce suini dalle 6 alle 20 settimane d’età, e il cui sintomo principale è rappresentato da una diarrea con feci pastose. Sono state descritte anche altre due forme cliniche a bassa incidenza: l’enterite necrotica (necrotic enteritis - NE), espressione di una forma di enteropatia proliferativa cronica complicata da infezioni secondarie che esita in un’estesa necrosi coagulativa dell’epitelio intestinale e l’ileite regionale (regional ileitis - RI), risultante dalla guarigione delle lesioni dovute a NE e caratterizzata da deposizione di tessuto di granulazione e ispessimento della tonaca muscolare24, 30. Clinicamente, la PHE è caratterizzata dalla morte improvvisa dei soggetti interessati, preceduta da diarrea emorragica e anemia. La PIA può essere invece di più difficile identificazione soprattutto quando la diarrea è assente o si manifesta solo nei soggetti in cui le lesioni intestinali sono più gravi. Nelle forme croniche clinicamente evidenti, i suini presentano lieve o moderata diarrea da densa ad acquosa e di colore verdognolo, associata ad anoressia di grado variabile e ridotto accrescimento, nonostante il consumo di alimento sia nella norma24. La forma subclinica di PE è quella più comune nei suini in accrescimento, anche se di difficile riconoscimento in quanto gli unici sintomi sono rappresentati dalla riduzione dell’IPMG e dalla difformità di accrescimento all’interno del gruppo24, 31 (Figura 1e).

LESIONI ANATOMOPATOLOGICHE La lesione macroscopica tipica della PE è l’ispessimento della mucosa intestinale che si verifica a causa della proliferazione delle cellule delle cripte intestinali. Nei suini in accrescimento affetti dalla forma cronica (PIA), le lesioni si osservano principalmente a carico della porzione terminale dell’ileo, lungo i 10 cm a monte della valvola ileo-ciecale, e a livello di cieco (Figura 1f). Nei casi gravi le lesioni possono coinvolgere anche il

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Figura 1 - Aspetti principali dell’enteropatia proliferativa da Lawsonia intracellularis nel suino enfatizzati nel corso della 1st International conference on Lawsonia intracellularis - The Gordon Lawson Memorial Symposium (The Royal (Dick) School of Veterinary Studies, Edinburgh, UK, 26 e 27 settembre 2019).

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Le lesioni istologiche comuni a tutte le forme di PE sono la proliferazione adenomatosa dell’epitelio delle cripte del piccolo intestino e delle ghiandole nella mucosa del grosso intestino. Le cripte intestinali si presentano iperplastiche, con epitelio costituito da enterociti immaturi e cellule disposte su più piani a formare un epitelio pseudostratificato al posto di quello tipico monostratificato colonnare (Figura 4). Le cellule caliciformi possono essere rare o del tutto assenti32. Tramite opportune colorazioni istologiche è possibile rilevare la presenza di L. intracellularis a livello intracellulare.

DIAGNOSI

Figura 2 - Positività immunoistochimica per L. intracellularis nel citoplasma dei macrofagi a livello di lamina propria della mucosa intestinale.

digiuno e il colon spirale. Indipendentemente dalla localizzazione, la mucosa si presenta ispessita e arricciata in pieghe longitudinali e trasversali, assumendo il tipico aspetto “cerebroide”. Nei casi complicati da infezioni secondarie che esitano in forme di enterite necrotica (NE), all’ispessimento della mucosa si associa la presenza di membrane fibrino-necrotiche sulla superficie della mucosa intestinale che possono staccarsi ed essere rinvenute libere nell’intestino o nelle feci diarroiche. Nella forma acuta emorragica (PHE), si osservano dilatazione intestinale e ispessimento della parete causato dalla presenza di edema e dalla proliferazione della mucosa. Il lume dell’ileo può contenere uno o più coaguli frammisti a detriti fibrinonecrotici; nel retto si possono osservare feci dall’aspetto catramoso contenenti sangue misto ad alimento indigerito24.

La diagnosi diretta di laboratorio, che consente di valutare la presenza di L. intracellularis nelle feci o in campioni di tessuti intestinale, può essere eseguita utilizzando tecniche di (Polymerase Chain Reaction) PCR qualitativa, PCR quantitativa (qPCR), PCR multiplex o l’immunoistochimica (IHC). La ricerca di anticorpi specifici nel siero o nei fluidi orali, che fornisce informazioni relative all’esposizione dei suini al patogeno, viene eseguita utilizzando tecniche immuno-enzimatiche (enzyme-linked immunosorbent assay; ELISA), l’immunofluorescenza (immunofluorescence assay; IFA), o l’immunoperossidasi (immunoperoxidase monolayer assay; IPMA)31. La conferma del sospetto clinico di PE può essere ottenuta mediante l’osservazione, durante l’esame post-mortem, delle lesioni macroscopiche tipiche della patologia, associata alla rilevazione di quelle microscopiche. L’IHC, mediante la quale gli antigeni di L. intracellularis vengono messi in evidenza all’interno delle lesioni grazie all’impiego di anticorpi monoclonali specifici18 (Figura 5), è la tecnica che garantisce la maggiore sensibilità e specificità. Per questo motivo, l’IHC rappresenta il gold standard per la diagnosi di PE (Figura 1g).

Figura 3 - Classificazione delle forme cliniche e subcliniche di enterite proliferativa del suino (PE)

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Figura 4 - Cripte iperplastiche del piccolo intestino: cellule epiteliali delle cripte disposte su più piani a formare un epitelio pseudostratificato (ematossilina - eosina, 40×).

Nelle aziende in cui l’infezione è endemica, i soggetti con forma subclinica di PE possono essere individuati rilevando la presenza intermittente di L. intracellularis nelle feci di animali con ridotto accrescimento. L’individuazione del DNA batterico nelle feci indica una condizione di infezione attiva ma è necessaria un’indagine diagnostica più completa per stabilire il reale stato di salute della mandria o l’eventuale associazione di L. intracellularis con altre malattie enteriche che possono mimare la presentazione clinica di PE quali colite da Brachispira spp., salmonellosi, colibacillosi, gastroenterite trasmissibile (TGE) e la diarrea causata dal Circovirus suino tipo 2 (PCV2)24. La sensibilità analitica delle tecniche biomolecolari è aumentata considerevolmente negli ultimi anni soprattutto grazie alla messa a punto di saggi Real-Time PCR, che sono in grado di fornire un esito positivo anche con basse quantità di L. intracellularis (100 microrganismi/grammo di feci). A causa di questa elevata sensibilità analitica, la semplice dimostrazione della presenza di L. intracellularis nelle feci può non essere indicativa della presenza in allevamento di problematiche economicamente significative causate da PE. Allo stesso modo, considerando la distribuzione segmentale delle lesioni da PE, occorre interpretare con cautela un risultato negativo all’IHC. È preferibile, quando possibile, esaminare con l’IHC un numero maggiore di segmenti intestinali per dimostrare in maniera più efficiente l’infezione da L. intracellularis nel contesto di lesioni tipiche localizzate24 (Figura 1g). La gravità dei sintomi è proporzionale al livello di escrezione di L. intracellularis nelle feci33 e l’aumento della concentrazione del patogeno è significativamente associato alla riduzione dell’IPMG mentre l’escrezione di basse quantità di L. intracellularis non sembra avere alcun effetto sull’accrescimento. L’aumento di un logaritmo della quantità di L. intracellularis eliminata con le feci raddoppia la probabilità che un suino presenti un basso tasso di accrescimento 19. In particolare, quando vengono rilevati oltre 106 patogeni/g di feci, ciò costituisce un fattore di rischio significativo nei confronti di un incremento ponderale non soddisfacente19. I risultati dei test qPCR permettono quindi di mettere in relazione la quantità di L. intracellularis presente nelle feci con le conseguenze economiche della malattia (riduzione dell’IPMG)19, o con la presenza di più gravi lesioni macroscopiche e istologiche34. Ad esempio, Burrough et al.35 utilizzando una PCR semi-quantitativa hanno definito che valori di threshold cycle (Ct) <20 (corrispondenti a

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Figura 5 - Positività immunoistochimica per L. intracellularis nella porzione apicale delle cellule delle cripte intestinali (63×; inserto: 40×).

una quantità di L. intracellularis superiore a 104/g di feci) sono correlati ad alti valori di score all’esame IHC di campioni di tessuto intestinale (Figura 1h). Quando i valori di Ct sono compresi tra 20 e 30, il risultato deve essere interpretato prendendo in considerazione l’anamnesi clinica per escludere che altri agenti eziologici siano responsabili dei segni clinici presenti nell’animale esaminato. Per valori di Ct >30, nel 95% degli animali esaminati il risultato dell’IHC era negativo, suggerendo la scarsa probabilità che L. intracellularis fosse la causa della diarrea osservata nell’animale esaminato. La positività alla PCR di campioni fecali di animali clinicamente sani sottolinea l’importanza delle forme subcliniche di PE. Questa osservazione deve essere presa in considerazione nella definizione di corretti protocolli diagnostici che consentano di implementare gli interventi terapeutici mirati per il contenimento efficace della PE, anche alla luce della necessità di un uso consapevole e razionale degli antimicrobici in allevamento. Nei macelli di alcuni Paesi del Nord Europa è già operativo un sistema di sorveglianza delle lesioni intestinali causate da L. intracellularis che prevede l’esame in parallelo, utilizzando qPCR, esame istologico ed immunoistochimico, di tratti di ileo prossimi alla valvola ileo-ciecale. I risultati vengono ritenuti ottimali per stimare la presenza di forme subcliniche in allevamento36.

PROFILASSI E CONTROLLO La profilassi e il controllo della PE si basano su quattro tipologie di interventi (misure di igiene ambientale e biosicurezza interna; alimentazione; impiego di antibiotici; vaccinazione) che possono essere implementati singolarmente o in associazione in funzione delle caratteristiche epidemiologiche della malattia in allevamento e della tipologia dell’azienda.

Misure di igiene ambientale e biosicurezza interna L’applicazione di rigorosi protocolli di pulizia e disinfezione e di procedure AIAO sono fondamentali per diminuire la prevalenza di PE37. Numerosi disinfettanti (sali quaternari d’ammonio, aldeidi, agenti ossidanti) inattivano L. intracellularis in 10-30 minuti e possono essere impiegati per ridurre la contaminazione dell’ambiente. È probabile che l’ambiente della mag-

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gior parte degli allevamenti di suini sia contaminato da quantità elevate di L. intracellularis presente nel materiale fecale che permane all’interno delle strutture anche dopo le procedure di pulizia e disinfezione oppure sulle attrezzature o altri fomiti. Lo spostamento di materiale fecale da aree contaminate ad altre aree della stessa azienda si verifica più comunemente nelle aziende a sito singolo. In queste condizioni, a temperature medio-basse, L. intracellularis può sopravvivere per circa 2 settimane. È quindi possibile che la contaminazione ambientale possa causare l’infezione dei nuovi gruppi di suini introdotti in queste strutture. L’infezione può diffondersi con velocità diverse e con variazioni nell’età di insorgenza della malattia sia in allevamenti diversi, sia nella stessa azienda, sia tra gruppi diversi all’interno dello stesso edificio38. Il trasferimento di feci contaminate si può realizzare anche tramite vettori animati quali insetti e roditori entrati a contatto con le feci di suino. Sperimentalmente è stato inoltre dimostrato che i topi si possono infettare e trasmettere L. intracellularis a suini esposti alle loro feci20. Pertanto, i roditori possono avere un ruolo importante nell’introduzione, nel mantenimento e nella trasmissione di L. intracellularis. Ne consegue che la derattizzazione rappresenta una importante misura per il controllo della PE. L’assenza, anche per un periodo di anni, di forme cliniche di PE in un allevamento convenzionale non è garanzia di indennità da L. intracellularis. Gli animali che provengono da tali allevamenti possono essere responsabili dell’introduzione del patogeno in popolazioni naïve, spesso seguita da un focolaio epidemico di forme emorragiche acute di PE e successivamente endemizzazione di forme croniche24.

Alimentazione La somministrazione di colture pure di L. intracellularis a suini germ-free non è in grado di indurre l’infezione che si realizza, invece, somministrando il contenuto intestinale di animali infetti39. Questa osservazione suggerisce che le caratteristiche dell’ambiente intestinale e la composizione quali-quantitativa del microbiota possano influenzare la sopravvivenza di L. intracellularis o modulare la sua patogenicità. La composizione della dieta e la sua granulometria possono anche influenzare le probabilità di infezione40. Ad esempio, le diete pellettate sono associate a livelli più elevati di L. intracellularis nel microbiota ileale rispetto a quelle non pellettate41. Per valutare l’eventuale effetto delle materie prime, sono stati condotti anche alcuni test in vivo: tre diverse diete a diversa inclusione percentuale di distiller di cereali non hanno modificato la situazione, mentre diete a base di orzo e avena hanno ridotto l’incidenza di forme cliniche rispetto alle diete di controllo. Anche l’orzo intero non macinato e le diete macinate grossolanamente hanno ridotto la frequenza di comparsa di forme cliniche da L. intracellularis. L’influenza che questi aspetti dell’alimentazione hanno sulle probabilità di sviluppo di forme cliniche da L. intracellularis è simile a quella osservata anche per altri patogeni intestinali quali Salmonella42. La loro implementazione permette quindi di controllare contemporaneamente più patologie. Sempre in relazione alle materie prime, basandosi anche sulle ultime ricerche in vitro, si devono prendere in considerazione alcuni fattori generali che valgono per tutte le diete correlate a qualsiasi patologia del grosso intestino. I fattori chiave sono due: sarebbe preferibile utilizzare fonti proteiche ad altissima digeribilità, in modo che la massima quantità possibile di pro-

teina venga assorbita a livello del piccolo intestino, riducendo al minimo il flusso in transito da questa porzione intestinale verso il grosso intestino. L’eventuale quota indigerita, una volta raggiunti ileo e cieco-colon, non farebbe altro che fermentare e squilibrare la microflora intestinale inducendo la presenza di grandi quantità di batteri indesiderati che altererebbero la funzionalità della mucosa intestinale. Allo stesso modo la fibra, che deve essere a sua volta identificata in diverse frazioni, modifica la composizione della popolazione batterica dell’intestino ed agisce sulla motilità intestinale. A differenza delle proteine, la quota strutturale identificata come componente della parete cellulare (lignina, cellulosa, emicellulosa, pentosani, pectine) e definita come “lentamente fermentescibile”, dovrebbe essere in grado di raggiungere in quantità le porzioni più distali dell’intestino in modo da condizionarne la motilità, stimolando soprattutto le contrazioni longitudinali, e selezionare una popolazione batterica più utile alla corretta funzionalità dell’apparato digerente. Tuttavia, l’impiego di diete con elevata quantità di questa frazione non ha fornito risposte definitive. Per quanto riguarda gli additivi, tutti i probiotici e prebiotici che, in modo aspecifico, concorrono a rinforzare l’adesione delle giunzioni “T” tra gli enterociti e/o mantengono integre la lunghezza dei villi e la profondità delle cripte, possono essere di aiuto per migliorare lo stato sanitario di tutto l’apparato intestinale. A questo proposito, in Spagna, alcune filiere hanno effettuato test in vivo sulla presenza di L. intracellularis in feci di suini Iberici allevati all’aperto, rilevando un aumento sistematico e significativo nei valori di Ct relativi alla quantificazione, mediante qPCR, di L. intracellularis nelle feci, testando come additivi pannello di colza idrolizzato, segale fermentata e colture del fungo Agaricus subrufescens, utilizzati con ottimi risultati sia nell’alimentazione suina sia in quella umana per il controllo di Salmonella. Tra gli acidi organici, l’aggiunta di acido formico non ha comportato alcun miglioramento della prevalenza di forme cliniche da L. intracellularis, mentre dosi di acido lattico del 2,4% sembrano avere qualche effetto positivo, fermo restando la sua impossibilità di applicazione per l’eccessivo costo addizionale. In ogni caso, i meccanismi con i quali la dieta o il microbiota intestinale influenzano la colonizzazione intestinale di L. intracellularis rimangono ancora poco chiari.

Impiego di antibiotici Gli antimicrobici sono utilizzati a scopo terapeutico per controllare i focolai di PE, permettendo di ridurre rapidamente la progressione dell’epidemia. In passato, gli antimicrobici sono stati tuttavia utilizzati anche in senso profilattico, mediante somministrazione di massa nell’alimento o nell’acqua di abbeverata. La scelta del farmaco è fondamentale per ottenere il migliore risultato possibile. Tuttavia, le informazioni sulla sensibilità in vitro di L. intracellularis sono scarse. La ragione principale di questa mancanza di informazioni è rappresentata dalla difficoltà di isolare L. intracellularis dai campioni fecali o intestinali degli animali infetti. L. intracellularis è un batterio intracellulare obbligato, replica solo all’interno di cellule coltivate in vitro e non può essere isolato con le comuni tecniche batteriologiche. Ne consegue che i metodi standard di valutazione della sensibilità antimicrobica non possono essere utilizzati ma vanno adattati tenendo in considerazione le specificità di questo microrganismo7. L’isolamento richiede personale esperto e diversi mesi per ottenere una coltura pura di L. intracel-

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lularis. Di conseguenza, la valutazione della sensibilità in vitro del ceppo di L. intracellularis responsabile del focolaio di PE è impossibile da ottenere in tempo utile. Sperimentalmente, alte dosi di tiamulina, tilosina e clortetraciclina sono in grado di prevenire l’infezione da L. intracellularis se somministrate in maniera continuativa nel mangime43. Tuttavia, una volta sospesa la somministrazione di questi antibiotici, i suini rimangono immunologicamente naïve e sono pienamente recettivi42, 43. Inoltre, l’uso prolungato di antibiotici può aumentare la possibilità che altre specie batteriche sviluppino antibiotico-resistenza. In passato sono state anche applicate alcune strategie terapeutiche in grado di prevenire le forme cliniche di malattia ma di consentire l’infezione da L. intracellularis e permettere quindi lo sviluppo di una immunità attiva nei confronti di una eventuale re-infezione. La somministrazione di antibiotici con l’alimento veniva sospesa per 12-18 giorni e poi pulsata nel mangime o in acqua a dosi elevate per 2-4 giorni ogni 2-3 settimane per prevenire la comparsa delle forme cliniche. Durante il periodo di mancata somministrazione di antibiotici i suini si infettavano e sviluppavano una immunità specifica43. La preoccupazione dell’opinione pubblica relativa allo sviluppo di fenomeni di antibiotico-resistenza e la necessità di un uso consapevole dei farmaci ha portato a individuare strategie alternative per controllare la PE e per ridurre l’uso profilattico degli antibiotici. Le attuali strategie relative alla riduzione dei fenomeni di resistenza limitano considerevolmente l’impiego in senso profilattico degli antibiotici, prevedendo il trattamento dei soli casi singoli o dei gruppi di animali sintomatici individuati mediante un corretto percorso diagnostico. In queste situazioni, vengono considerati efficaci tilosina, enrofloxacina, tetracicline, tiamulina e tilmicosina mentre sono considerati non efficaci penicilline, bacitracina, neomicina, virginiamicina e ionofori. L’individuazione, mediante la valutazione dell’incremento dei titoli anticorpali degli animali, dell’arco di tempo in cui si sviluppa l’infezione all’interno dell’allevamento consente di stabilire con relativa precisione il momento in cui iniziare la somministrazione degli antibiotici che dovrà essere realizzata circa 3 settimane prima del picco di sieroconversione (Figura 1i).

Vaccinazione L’infezione naturale da L. intracellularis produce una risposta immunitaria sia umorale (IgG e IgA locali mucosali) che cellulo-mediata che è il presupposto da raggiungere con la vaccinazione per contenere l’infezione da L. intracellularis. È disponibile da tempo un vaccino vivo attenuato, somministrabile oralmente, in genere nell’acqua di abbeverata o nell’alimento in broda. Negli animali vaccinati le lesioni da PE sono meno gravi, la quantità di L. intracellularis nelle feci si riduce e migliora la risposta immunitaria specifica cellulo-mediata, 44 . Nella mucosa ileale dei suini vaccinati è stata inoltre osservata la presenza di IgG e IgA specifiche. Valutazioni di campo indicano che la vaccinazione è economicamente vantaggiosa e che la quantità di antibiotici utilizzata negli allevamenti affetti da PE si riduce45. Trattandosi di un vaccino vivo attenuato, è consigliabile interrompere la somministrazione di qualsiasi antibiotico almeno 3 giorni prima e 3 giorni dopo la vaccinazione. Dal 2016 è commercialmente disponibile un vaccino inattivato, somministrabile in un’unica dose per via intramuscolare a partire da 3 settimane di età. Anche per questa tipologia di vac-

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cino è stata dimostrata una risposta sierologica specifica oltre alla riduzione della gravità delle lesioni da PE e della quantità di L. intracellularis presente nelle feci47.

CONCLUSIONI A quasi 40 anni dalla dimostrazione che il microrganismo oggi denominato Lawsonia intracellularis è il responsabile di forme di adenomatosi intestinale nel suino ed in altre specie, numerose questioni restano ancora aperte. Le particolarità di questo batterio non consentono un approccio diagnostico classico e questo condiziona sia la corretta valutazione della prevalenza della malattia sia la valutazione di eventuali fenomeni di antibiotico-resistenza. Inoltre, la presenza di eliminatori asintomatici costringe, come per altri agenti eziologici di malattie del suino (es. PCV2, Mycoplasma hyopneumoniae), al ricorso a tecniche diagnostiche che siano in grado non tanto di rilevare la semplice presenza dell’agente eziologico ma di valutarne la quantità all’interno di escreti/secreti o nel contesto delle lesioni anatomopatologiche specifiche. È verosimile ritenere che, in alcuni contesti, queste oggettive difficoltà portino a sottostimare la reale importanza sanitaria ed economica dell’enterite proliferativa o ad orientare il controllo mediante la somministrazione profilattica di antibiotici. Per queste ragioni, il controllo della PE dovrebbe basarsi su una corretta valutazione epidemiologica, su un uso maggiormente responsabile dei farmaci, su misure di profilassi diretta anche di tipo alimentare e, dove necessario, sulla pianificazione di interventi vaccinali.

PORCINE PROLIFERATIVE ENTEROPATHY CAUSED BY LAWSONIA INTRACELLULARIS SUMMARY Porcine Proliferative Enteropathy (PE or ileitis) is an infectious enteric disease caused by the intracellular pathogen Lawsonia intracellularis (LI). PE is endemic in many countries and causes severe economic losses in swine production system worldwide due to reduction of daily weight gain, reduction of feed conversion ratio and increase of mortality and swine waste. In Europe, the prevalence of infected farms and infected animals is more than 90% and 40%, respectively. In PE, intestinal mucosa is thickened by uncontrolled proliferation of intestinal crypt cells while secretory cells and absorbent cells are decreased in number because LI prevents their maturation. Diarrhea is the consequence, due to reduced absorption and loss of amino acid and protein in intestinal lumen. Clinical forms are divided into acute (proliferative hemorrhagic enteropathy - PHE) and chronic form (porcine intestinal adenomatosis - PIA). Acute form affects animals from 4 to 12 weeks of age and is characterized by high mortality (>50%) and hemorrhagic diarrhea. Chronic form affects swine of 6-20 weeks of age and is characterized by pasty diarrhea. Based on morphological findings, two other forms are reported: necrotic enteritis (NE) and regional ileitis (RI). The first is a chronic form complicated by secondary infection that result in coagulative necrosis of intestinal epithelium. Healing of necrotic enteritis lesions results in both thickening of muscular layer of intestinal wall and granulation tissue deposition, both of which are typical findings of RI. In-

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direct diagnosis (e.g. ELISA) assess the exposure to L. intracellularis while direct diagnosis (PCR, qPCR, Immunohistochemistry -IHC) assess the current infection. Effective diagnosis is obtained comparing quantitation of microorganism/gram of feces with the detection of L. intracellularis within intestinal lesion. Prophylaxis and control of proliferative enteropathy are based on biosecurity measures combined with strict washing and disinfection measures to reduce environmental contamination. Proper nutrition management helps to ensure the balance of intestinal microflora by the use of highly digestible protein, by correct intake of fiber fraction and with probiotic and prebiotic supplements. To limit subclinical forms of disease, vaccination should replace antibiotic treatments which instead should be reserved only for symptomatic groups of pig. KEY WORDS Lawsonia intracellularis; proliferative enteropathy; swine.

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G. D’Annunzio et al. Large Animal Review 2021; 27: 149-163 finish swine herds. Pol J Vet Sci, 18: 825-831. 38. Hammer J.M. (2004). The temporal relationship of fecal shedding of Lawsonia intracellularis and seroconversion in field cases. J Swine Health Prod, 12: 29-33. 39. McOrist S., Mackie R.A., Neef N., Aitken I., Lawson G.H. (1994). Synergism of ileal symbiont intracellularis and gut bacteria in the reproduction of porcine proliferative enteropathy. Vet Rec, 134: 331-332. 40. Boesen H.T., Jensen T.K., Schmidt A.S., Jensen B.B., Jensen S.M., Moller K. (2004). The influence of diet on Lawsonia intracellularis colonization in pigs upon experimental challenge. Vet Microbiol, 103: 35-45. 41. Molbak L., Johnsen K., Boye M., Jensen T.K., Johansen M., Moller K., Leser T.D. (2008). The microbiota of pigs influenced by diet texture and severity of Lawsonia intracellularis infection. Vet Microbiol, 128: 96-107. 42. Andres V.M., Davies R.H. (2015). Biosecurity measures to control Salmonella and other infectious agents in pig farms: a review. Compr Rev Food Sci Food Saf, 14: 317-335. 43. Collins A.M. (2013). Advances in Ileitis Control, Diagnosis, Epidemiology and the Economic Impacts of Disease in Commercial Pig Herds. Agri-

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culture, 3: 536-555. 44. Nogueira M.G., Collins A.M., Donahoo M., Emery D. (2013). Immunological responses to vaccination following experimental Lawsonia intracellularis virulent challenge in pigs. Vet Microbiol, 164: 131-138. 45. Bak H. Rathkjen P.H. (2009). Reduced use of antimicrobials after vaccination of pigs against porcine proliferative enteropathy in a Danish SPF herd. Acta Vet Scand, 51: 1. 46. Jacobs A.A.C., Harks F., Hazenberg L., Hoeijmakers M.J.H., Nell T., Pel S., Segers R. (2019). Efficacy of a novel inactivated Lawsonia intracellularis vaccine in pigs against experimental infection and under field conditions. Vaccine, 37: 2149-2157. 47. Roerink F., Morgan C.L., Knetter S.M., Passat M.H., Archibald A.L., AitAli T., Strait E.L. (2018). A novel inactivated vaccine against Lawsonia intracellularis induces rapid induction of humoral immunity, reduction of bacterial shedding and provides robust gut barrier function. Vaccine, 36: 1500-1508. 48. Marcato P.S. (2015) Patologia sistematica veterinaria. Edagricole, Bologna.

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Study of postnatal growth of mule and donkey foals sired by the same jackass

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AUGUSTO CARLUCCIO1, ALBERTO CONTRI2, ALESSIA GLORIA1*, DOMENICO ROBBE1, GIORGIO VIGNOLA1 1 2

Faculty of Veterinary Medicine, University of Teramo, Loc. Piano d’Accio, 64100 Teramo, Italy Faculty of Biosciences and Technologies for Agriculture Food and Environment, University of Teramo, via Balzarini 1, 64100 Teramo, Italy

SUMMARY Mules have been enrolled for agricultural work in national parks or islands and hard-to-reach places in which the use of machinery is forbidden or not feasible. Despite the importance of this animal, limited information is reported regarding gestation, parturition, and post-natal growth in mule. To evaluate the effects of the pregnancy length, birthweight, and fetal sex on neonatal health and growth in mule and donkey foals born from Heavy Draft mares (n=15) and Martina Franca jennies (n=18) inseminated with the same jackass until 30 months of age. This study was conducted on 15 healthy Heavy Draft mares, 4 to 6 years old and weighing 640 to 730 kg, and 18 healthy Martina Franca jennies 4 to 6 years old and weighing 390 to 420 kg. All animals presented normal reproductive history such as cyclicity, pregnancy, and foaling. The semen used for artificial insemination was collected from the same 7 years old 485 kg bodyweight Martina Franca jackass of proven fertility. Pregnancy length in the mares bearing mule foal (339 ± 9 days) was similar to that reported in the equine gestation and was longer in female mules (347 ± 4.4 days) than males (331 ± 5.46 days) (P < 0.05). In donkey, pregnancy length was significantly longer in male foals (373.7 ± 10.4 days) than females (366.9 ± 5.6 days) (P < 0.01). The mean birth weight was higher in mules compared to donkey foals (50.6 ± 4.83 vs 33.4 ± 5.7 kg) and was greater in male mules than in females (53.95 ± 3.2 kg and 46.6 ± 2.96 kg, respectively; P < 0.05). Mule foal’s weight increased faster than donkey foals, despite a similar height at withers at different time points. Pregnancy length affected birthweight in both mule and donkey foals, and birthweight was related to postnatal growth in both mule and donkey foals. The findings reported in this manuscript could optimize the maternal-foal management of this equine hybrid.

KEY WORDS Horse, mule; Martina Franca donkey, breeding, postnatal growth.

INTRODUCTION The mule, as the hybrid of the female horse and male donkey, is the most popular equine hybrid, and in the past 5,000 years, millions of mules were reared to sustain human activities1. The reason for this success could be related to the physical performances, similar to those of the horse, together with the meekness, typical of the donkey. These characteristics have favored the introduction of the mule in agricultural work in developing countries or in delicate ecosystems, such as national parks or islands, where mechanization is not feasible. Interestingly, Allen reported that at least the horse, the donkey, and the mule (E. mulus mulus; 2n = 63) within the equine family can accept, gestate, carry to term, give birth to, and rear successfully truly xenogenetic extra-specific foals. In that case, the xenogenetic extra-specific pregnancies were obtained

Corresponding Author: Alessia Gloria (agloria@unite.it)

by between-species embryo transfer techniques2. Fetal development is affected by several factors such as genetic, environmental, and maternal factors3. As demonstrated by Allen et al.4 and Wilsher and Allen3, the transfer of the nutrients mediated by the placenta is positively correlated with the area of the placenta3,4. This may explain how an increase in foal weight occurs at birth when an embryo of a small breed is transferred to a larger breed. Tischner and his colleagues similarly highlighted the influence of maternal size upon foal growth when they used embryo transfer to gestate Polish Konig pony foals in the uteri of larger draught-type mares5. Several authors have shown how foals of small breed born heavier than usual if gestated in the uteri of larger mares and vice versa, besides this increased size at birth persist to adulthood5. In the Arabian horse, the birth height was found correlated with the adult height, suggesting the use of this parameter as a predictor for the adult size6. Differently to other domestic animals, in equid species, the postnatal growth rate received limited attention. In a previous study conducted on Thoroughbred foals, the growth curve from birth

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Study of postnatal growth of mule and donkey foals sired by the same jackass

to weaning was described7. At birth, no differences were described between Thoroughbred colts and fillies in birthweight and at weaning, but the birthweight accounted the 16% of the variability of weaning weight, suggesting a relevant role of this parameter on the future foal growth. No information was reported in the literature regarding the growth curve in the mule. The present study aimed at reporting the growth curves for bodyweight, height at withers, chest circumference, and cannon bone circumferences of mule and donkey foals, born after artificial insemination (AI) using only one jackass with proven fertility, between birth and 30 months of age. At the same time, the effect of the foal sex and pregnancy length on the birth weight and the post-natal growth rate were also evaluated.

MATERIALS AND METHODS Animals and data collection This study was conducted on n = 15 Heavy Draft mares and n = 18 Martina Franca jennies. The criteria of inclusion were age between 4 and 6 years, the weight between 630 and 730 kg for the mares, and between 380 to 420 kg for the jennies, the parity (2 previous foalings), the month of ovulation (April to May) and feed management. All animals presented normal reproductive history regarding cyclicity, pregnancy, and foaling. The semen used for the inseminations was collected from the same Martina Franca jackass. The Jackass was of proven fertility, 7 years old, and weighing 485 kg (Figure 1). The animals were housed in the Veterinary Teaching Farm of the University of Teramo (Italy). All the animals were reared in open paddocks with free access to a shed. Daily, mares and jennies received standard hay and water ad libitum and commercial equine fodder (4 and 6 kg for jennies and mares, respectively). Jackass was kept in an individual 5 x 5 m2 box with access to an outdoor paddock and received 10 kg of standard hay supplemented with 3 kg of commercial balanced stallion fodder twice daily. The procedures described in this study have been performed in agreement with the Italian legislation concerning animal care (DL n.116, 27/01/1992). Oestrus detection was performed as previously reported8. At detection of first signs of estrous in mares and jennies, an uter-

ine sterile swab was performed. Those animals with bacterial growth were excluded from the trial. The ovarian follicular growth was monitored by transrectal ultrasonography twice daily from heat onset until the day of ovulation, with a Concept 2000 ultrasound machine (Dynamic Imaging Limited, Livingston, Scotland, UK) equipped with a 7.5-MHz linear probe. From the visualization of a follicle of 30 mm in size, jennies and mares were subjected to artificial insemination every 48 hours until ovulation. Semen was collected by a Missouri artificial vagina, and sperm concentration and objective progressive motility were measured as previously reported9. Insemination doses (15 mL) were prepared by diluting raw semen with INRA 96 extender (IMV Technologies, L’Aigle, France) to achieve 800 x 106 progressive spermatozoa/dose. The ovulation day was defined as the day the dominant follicle disappeared on the ovary cortex, on a daily successive ultrasound monitoring. Pregnancy diagnosis was carried out by transrectal ultrasound on day 14 post ovulation and confirmed on day 45. All the jennies and the mares were monitored monthly throughout gestation to evaluate fetal viability and wellness. At foaling, each foal was clinically examined and birth weight and sex were recorded. In this study, only foals born by spontaneous foaling and without obstetric interventions were included. Within five minutes after birth, the APGAR score was evaluated as previously reported10. After fetal membrane expulsion, the fetal membranes were weighted. In all cases, pregnancy length (PL), calculated from ovulation to foaling, was recorded. On the day of foaling, the quality of the colostrum was evaluated with a Brix refractometer (HR-150N, Optika, Bergamo, Italy). Values below Brix score 15 were considered suggestive of poor colostral quality.

Postnatal growth measurements Each foal was weighed (W) and measured for height at withers (HW), chest circumference (CC) immediately caudal to the withers (Figure 2), and cannon bone circumference (CAC), measured immediately distal to the metacarpal tuberosity at the proximal end of the cannon bone, at the day of birth and then monthly until 6 months of age, and at 12, 18, 24, and 30 months of age.

Statistical analysis All morphometric parameters are reported as mean ± standard deviation (SD). The coefficient of the linear regression was cal-

Figure 1 - Martina Franca stallion.

Figure 2 - Measurement of height in withers and chest circumference in mule foals.

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culated for each measurement, to estimate the growth of the specific parameter, namely weight (cW), height at withers (cHW), chest circumference (cCC), and cannon bone circumference (cCAC). Differences between all the growth measurements and the calculated growth trend for each parameter were compared using the general linear model (GLM) based on the univariate ANOVA. The month and sex were considered as fixed factors. Correlations between the PL, the birth weight, and the cW, cHW, cCC, and cCAC were compared with Pearson’s correlation test. Differences were considered significant when P < 0.05. Statistical analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA).

RESULTS The number of estrus/pregnancy was 1.8 for Heavy Draft mares, and 1.5 for jennies, without significant differences (P > 0.05). The mean duration of the estrus was 5.3 ± 0.8 and 6.5 ± 0.6 days for Heavy Draft mares and Martina Franca jennies, respectively (P > 0.5). At ovulation, the mean follicular diameter was 4.8 ± 0.4 cm for Heavy Draft mares and 4.3 ± 0.3 cm for jennies. The pregnancy diagnosis was performed on day 14 post ovulation, and the embryo vesicle was spherical in both Heavy Draft mares and jennies. The mean diameter at diagnosis was 1.6 ± 0.2 cm and 1.5 ± 0.3 cm for mares and jennies. All mules (7 females and 8 males) and donkey foals (7 females and 11 males) were born at term and by spontaneous eutocic foaling. The mean PL was 338.82 ± 9.32 days for mulebearing mares and 368.4 ± 11.5 days for jennies (P < 0.01). The mule sex affected PL in mares since it was longer in females (347 ± 4.4) than males (331 ± 5.46) (P < 0.05). Conversely, donkey’s PL was significantly longer for males compared to females foals, with mean values of 373.7 ± 10.4 and 366.9 ± 5.6 days, respectively, (P < 0.01). In all cases, the APGAR score evaluated within five minutes after birth was 9.3 (range 8 to 10). No differences were found between the APGAR score in mule and donkey foals, nor between males and females (P >

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0.05). All the newborns followed a normal clinical course throughout the evaluation period. The placental weight for mule foals was 5.6 ± 0.48 kg, without differences between colts and fillies (5.6 ± 0.4 kg and 5.2 ± 0.6 kg, respectively; P >0.05). For donkey foals, the placental weight was 3.1 ± 0.3 kg, without differences between colts, and fillies (3.6 ± 0.4 kg and 3.7 ± 0.2 kg, respectively; P > 0.05). Differences between placental weight in mule and donkey foals reflected the difference in birthweight. The Brix refractometry of the colostrum, collected the day of parturition, was 26.2 ± 2.1 % for Heavy Draft mares and 24.6 ± 3.4 % for jennies. The mean W, HW, CC, and CAC of mules and donkey foals from birth to 30 months of age were reported in Table 1. The mean birth weight, evaluated before first suckling, was 50.6 ± 4.83 kg for mules, with differences between males 53.95 ± 3.2 kg and females 46.6 ± 2.96 kg (P < 0.05), and 33.4 ± 5.7 kg for donkey foals, with no differences between males (32.4 ± 4.1 kg) and females (33.2 ± 2.5 kg) (P > 0.05). The mule foal growth in weight was faster than the donkey foal (Figure 3), as corroborated also by the cW value (14.9 ± 0.6 and 11.8 ± 0.9 for the mules and the donkey foals, respectively). The HW measured for the mules at foaling was 94 ± 1 cm, with similar values for the males (93 ± 1 cm) and the females (96 ± 3 cm, P > 0.05). In the donkeys at foaling the HW was (86.7 ± 4.9 cm) with similar values for the males (84.8 ± 4.3 cm) and female foals (88.3 ± 3.6 cm, P > 0.05). The cHW was similar in both the species, as shown in Figure 4. Although a similar height at withers between the species, the higher weight recorded in the mule foals at each time-point was the result of the higher increase in CC and CAC (Figure 5). The mules’ CC measured at foaling was (88 ± 1 cm), with similar values in both males and females (87.4 ± 1.1 cm, and 88.7 ± 1.3 cm, respectively; P > 0.05; Figure 6). In the donkey at foaling, the CC was 66.9 ± 4.5 cm with no differences between males (66.3 ± 2.7 cm) and females (67.2 ± 3.4 cm; P > 0.05). The coefficient of the linear regression of the chest circumference appeared higher in the mule (3.236) than in the donkey (2.348; P < 0.05). Similarly to the CC, the CAC of mules at foaling was

Table 1 - Morphometric parameters (weight - W; height at wither - HW; chest circumference - CC; cannon bone circumference - CAC) measured at the different time-points in donkey and mule foals from the same jackass. W (kg)

HW (cm)

CC (cm)

CAC (cm)

Donkey foal

Mule foal

Donkey foal

Mule foal

Donkey foal

Mule foal

Donkey foal

Mule foal

Month

Mean±SD

33.4±5.7a

50.6±4.8b

86.7±4.9a

94±4.4a

66.9±4.5a

88.2±4.1b

10.5±1.1a

13.1±0.6b

51.1±7.2a

83.2±7.5b

94.2±5.7a

104.1±3.1b

77.9±4.2a

98.5±3.6b

11.6±1.1a

13.5±0.6b

12.6±1.2

14.3±0.9b

13.8±1.1a

14.7±0.9a

71.5±10.6

88.9±11.8a a

111.4±7.8

100.6±4.8

110.6±2.8

140.7±6.4b

107.7±5.3a

114.3±2.6a

88.7±5.1

95.6±5.2a a

106±3.3

112±3.7b b

15.2±1a

102.3±13.1

166.2±5.8

112.2±4.7

117.5±2.8

101.3±4.7

116.5±4.9

15.1±1

121.7±17.4a

190.7±6.2b

115.4±5.2a

120±2.6a

106.1±4.9a

119.6±5.4b

15.9±1.2a

15.5±1a

138.1±17.9

208.5±5.4

119.1±5.8

122.9±3.4

113.4±5.2

123.6±6.8

16.5±1.1

16±1.1a

193.7±24.6a

293.2±11b

132.7±6.3a

127.2±3.8a

127.3±7.1a

147.2±12b

17.8±1.3a

17.1±0.7a

240.2±28.1

373±27.7

277.3±31.2a

449.7±49b

306.4±23.1

516.2±59.4

134.2±6.1

131.6±5

138.9±6.8a

135.4±6a

141.7±6.1

139.1±6.8

136.4±7.1

152.7±13.8

18.2±1.2

18.1±0.8a

143.4±5.2a

157.4±14.5b

18.7±0.9a

18.9±0.9a

147.1±4.6

For each parameter and at each time, values with different superscript (a/b) differ significantly (P < 0.05)

163.4±15.7

19.3±1.3

19.7±1a

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Study of postnatal growth of mule and donkey foals sired by the same jackass

Figure 3 - Graphical representation of the weight (kg) at the different time-points (month) in mule (dark gray) and donkey foals (light gray), and the respective trend lines.

(13.1 ± 0.2 cm), with similar values for the males (13 ± 0.3 cm) and the females (13.3 ± 0.3 cm; P > 0.05). The donkey CAC at foaling was 10.5 ± 1.1 cm, with similar values for the males (9.7 ± 1.5 cm) and the females (10.9 ± 1.2 cm; P > 0.05). The increase of the parameter, summarized by the coefficient of the linear regression, was higher for mule (0.392) than donkey foals (0.208; P < 0.05). Significant correlations between PL and birthweight in both mule and donkey foals (R = 0.789, P = 0.004, and R = 0.723, P = 0.008, respectively) were found. Moreover, PL was significantly correlated with the coefficient of the linear regression of W (R = 0.813, P = 0.002 and R = 0.784, P = 0.003 for mule and donkey foals respectively), and the coefficient of the linear regression of the HW (R = 0.603, P = 0.026, and R = 0.634, P = 0.0022 for mule and donkey foals respectively). A significant correlation was found between the birthweight and the coefficient of the linear regression of the weight in both mule (R = 0.639; P = 0.001) and donkey foals (R = 0.684; P = 0.001).

DISCUSSION In this study, the pregnancy length calculated from ovulation day to foaling in the mares bearing mule foals was 339 days. The value was similar to those reported in other studies11,12. The PL in the mule was also similar to the values reported for mares13-15. The PL in the donkey reported in the present study was

369 days. The PL was within the normal range reported by our group in the Martina Franca donkey16,17 and was consistent with that reported in other donkey breeds18. The data about jennies confirmed the finding that PL is longer in the donkey8,16 compared to mares. Few studies evaluated the relationship between PL and birthweight of the equine foal. A previous study found a relationship between PL and placental weight until 6.5 kg, while the correlations were lost after this threshold, suggesting a limiting effect only for low-size placenta on fetal growth19. In the donkeys and mules considered in the present study, although on a limited number of animals, the effect of the PL appeared more relevant to determine the foal weight at birth, since in both these species significant correlations were found between these parameters. In donkey an increased stereological complexity of the microcotyledons was found, suggesting a reduced efficiency of the placenta in this species16. This could explain the increased PL recorded in this species, but also the higher dependence of the foal birthweight to the PL. Unfortunately, no information is available in the literature regarding the relationship between mule placenta and mule foal birthweight. In the present study, the foal sex influenced the duration of PL in jennies, with longer PL in male than in female donkey foals, in agreement with our previous finding in the same donkey breed20. Similar findings were reported in mare bearing a male foal13,21,22. Conversely, in mares bearing mules embryos, the PL was significantly longer for females compared to males. In a pre-

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Figure 4 - Graphical representation of the height in withers (cm) at the different time-points (month) in mule (dark gray) and donkey foals (light gray), and the respective trend line.

vious study on mule PL, the gestation was slightly longer in mares bearing female mule, even if the difference was not significant12. The reason for such effect of fetal sex on the equid PL is unclear. It was hypothesized that the endocrinology of parturition could be affected differently by the hormones produced by the male and female fetus21,23, but the conclusive demonstration was not yet reported in equids. Unfortunately, no information regarding the fetal and foal endocrinology of the mule

Figure 5 - Representative image of a Heavy Draft mare with her mule.

was reported, thus specific studies are needed to elucidate this specific aspect. Several studies showed that age and parity affect birthweight significantly24,26. Although these parameters are closely correlated, a recent study using a stratified and multivariate approach clarified that the parity rather than the age affects the foal birthweight, in particular between the first and the second foaling18. Furthermore, Robles et al. reported that primiparous mares showed a reduced foal weight to the placenta ratio compared to multiparous mares26. The reduced chorionic volume associated with the microcotyledon density detected in the placentas

Figure 6 - Representative image of mules.

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Study of postnatal growth of mule and donkey foals sired by the same jackass

collected from primiparous mares seems to be responsible for this phenomenon19,24. As in the herein study, we recruited exclusively third foaling mares, foal’s birthweight was not affected. The donkey birthweight recorded in the present study was consistent with previous data reported in Martina Franca donkey foals10,20,27. The mule weight at birth recorded in the present study was consistent with the values reported by Paolucci et al.11, regardless of the different weight of mares considered (Heavy Draft mares in the present study, Italian Standardbred mares in the previous one). In the horse’s genus Equus, an epitheliochorial non-invasive placenta, diffuse on the entire contact surface is present1,28. As a consequence, the nutrition available for fetal development depends on the volume of the uterus, which is in turn related to the mare’s size29. Allen et al., using the embryo-transfer between ponies and Thoroughbreds, showed that the maternal size affects fetal growth and the mass, gross area, and volume of the allantochorion4. However, the evident effect on the fetal growth reported in that trials could be due to the large difference in the breeds used. More recently, Peugnet et al. reported no differences between saddlebred embryos developed in draft mares compared with their control developed in saddlebred mares29, similarly to our findings. In the present study, the foal weight growth after birth was significantly higher in mule than in donkey foal. This higher weight appeared related to the increase in transverse diameters, measured using the chest circumference and the cannon bone circumference, rather than the height at withers, similar in Martina Franca donkey and mule. This difference agrees with the expected morphology of the mule, which express the physical characteristic of the horse parent1. In the mule, the foal sex affected the bodyweight at birth and the following growth rate. A similar trend was reported previously in a study on 1992 Thoroughbred foals13. On the other hand, Morel et al. found no significant differences in weight at foaling and weaning between Thoroughbred colt and fillies, even if males appeared heavier of about 1.7 kg7. Although a similar increased weight trend in both sexes, this difference agrees with the evidence that the adult male is heavier than the female one. In the present study, the mule foal weight increased faster than donkey foal, and this difference could be related to several factors. An increased milk availability for the mule foals could be a factor since the milk yield increases with the mare size29. The milk intake, however, could only partially explain this difference, since it was found relevant for the equine growth within the first 4 weeks after foaling, while there was no relationship between milk intake and foal growth between 4 and 8 weeks30. Thus, other genetic or metabolic factors could affect this difference. In both the categories, the birthweight positively affects the weight that the subjects reached at 30 months, confirming previous findings in some studies26. In conclusion, the mule foal was born after a PL similar to that of the horse foal, with longer PL in the female mule foals. The birthweight was affected by the PL, and it could be considered an important factor to predict the future growth of the mule foal since the birthweight was significantly correlated with the growth rate and the bodyweight at 30 months.

CONFLICT OF INTEREST The authors declare that they have no conflict of interest.

ACKNOWLEDGMENTS This work was part of the research supported by the Assessorato alle Politiche Agricole, Regione Puglia (Italy). The present study was conducted in the framework of the Project “Demetra” (Dipartimenti di Eccellenza 2018 - 2022, CUP_C46C18000530001), funded by the Italian Ministry for Education, University and Research.

References 1. Allen W.R., Short R.V. (1997). Interspecific and extraspecific pregnancies in equids: anything goes. J Heredity, 88: 384-392. 2. Allen W.R., (1982). Embryo tranfer in the horse. In Mammalian Egg Transfer. (Ed. CE Adams) pp. 135-154. (Florida CRC). 3. Wilsher S., Allen W.R. (2012). Factors influencing placental development and function in the mare. Equine Vet J, 44: 113-119. 4. Allen W.R., Wilsher S., Turnbull C., Stewart F., Ousey J., Rossdale P.D. (2002.) Influence of maternal size on placental, fetal and postnatal growth in the horse. I. development in utero. Reproduction, 123: 445453. 5. Tischner M., Klimczak M. (1989.) The development of Polish ponies born after embryo transfer to large recipients. Equine Vet J, 8: 62-63. 6. Reed R., Dunn N. (1977). Growth and development of the Arabian horse. In: Proceedings of the 5th equine nutrition physiology symposium; Apr 28-30. pp. 76-98. (St. Louis, Missouri USA). 7. Morel P.C., Bokor A., Rogers C.W., Firth E.C. (2007.) Growth curves from birth to weaning for Thoroughbred foals raised on pasture. The New Zealand Vet J, 55: 319-325. 8. Contri A., Robbe D., Gloria A., De Amicis I., Veronesi M.C., Carluccio A. (2014). Effect of the season on some aspects of the estrous cycle in Martina Franca donkey. Theriogenology, 81: 657-661. 9. Gloria A., Contri A., De Amicis I., Robb D., Carluccio A. (2011). Differences between epididymal and ejaculated sperm characteristics in donkey. Anim Reprod Sci, 128, 117-122. 10. Veronesi M.C., Gloria A., Panzani S., Sfirro M.P., Carluccio A., Contri A. (2014). Blood analysis in newborn donkeys: hematology, biochemistry, and blood gases analysis. Theriogenology, 82: 294-303. 11. Paolucci M., Palombi C., Sylla L., Stradaioli G., Monaci M. (2012). Ultrasonographic features of the mule embryo, fetus and fetal-placental unit. Theriogenology, 77: 240-252. 12. Boakari Y.L., Alonso M.A., Riccio A.V., Fernandes C.B. (2019.) Are mule pregnancies really longer than equine pregnancies? Comparison between mule and equine pregnancies. Reprod Dom Anim, 54: 823827. 13. Hintz H.F., Hintz R.L., Lein D.H., Van Vleck L.D. (1979). Length of gestation periods in Thoroughbred mares. J Equine Med Surg, 3: 289292. 14. Vincent S.M., Evans M.J., Alexander S.L., Irvine C.H.G. (2014). Establishing normal foaling characteristics in Standardbred mares in New Zealand. J Equine Vet Sci, 34: 217e9. 15. Mariella J., Iacone E., Lanci A., Merlo B., Palermo C., Morris L., Castagnetti C. (2018). Macroscopic characteristics of the umbilical cord in Standardbred, Thoroughbred, and Warmblood horses. Theriogenology, 113: 166-170. 16. Veronesi M.C., Villani M., Wilsher S., Contri A., Carluccio A. (2010). A comparative stereological study of the term placenta in the donkey, pony and Thoroughbred. Theriogenology, 74: 627-631. 17. Gloria A., Veronesi M.C., Carluccio R., Parrillo S., De Amicis I., Contri A. (2018). Biochemical blood analysis along pregnancy in Martina Franca jennies. Theriogenology, 115: 84-89. 18. Fielding D. (1988). Reproductive characteristics of the jenny donkey Equus asinus: a review. Trop Anim Health Prod, 20: 161-166. 19. Elliott C., Morton J., Chopin J., Elliott C. (2009). Factors affecting foal birth weight in Thoroughbred horses. Theriogenology, 71: 683-689. 20. Carluccio A., Gloria A., Veronesi M.C., De Amicis I., Noto F., Contri A. (2015). Factors affecting pregnancy length and phases of parturition in Martina Franca jennies. Theriogenology, 84: 650-655. 21. Davies Morel M.C., Newcombe J.R., Holland S.J. (2002). Factors affecting gestation length in the Thoroughbred mare. Animl Reprod Sci, 74: 175-185. 22. Dicken M., Gee E.K., Rogers C.W., Mayhew I.G. (2012). Gestation length and occurrence of daytime foaling of Standardbred mares on two stud farms in New Zealand. The New Zealand Vet J, 60: 42-46. 23. Jainudeen R.M., Hafez E.S.E. (2000). Gestation, prenatal physiology and parturition. In: (Hafez ESE, Hafez B, editors). Reproduction in

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A. Carluccio et al. Large Animal Review 2021; 27: 165-173 farm animals., William and Wilkins pp.140-155. (Lippincott, Maryland, USA) 24. Wilsher S., Allen W. (2002). The influences of maternal size, age and parity on placental and fetal development in the horse. Theriogenology, 58: 833-835. 25. Wilsher S., Allen W. (2003). The effects of maternal age and parity on placental and fetal development in the mare. Equine Vet J, 35: 476-483. 26. Robles M., Dubois C., Gautier C., Dahirel M., Guenon I., Bouraima-Lelong H., Viguié C., Wimel L., Couturier-Tarrade A., Chavatte-Palmer P. (2018). Maternal parity affects placental development, growth and metabolism of foals until 1 year and a half. Theriogenology, 108: 321-330. 27. Carluccio A., Contri A., Gloria A., Veronesi M.C., Sfirro M.P., Parrillo S., Robbe D. (2017). Correlation between some arterial and venous

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blood gas parameters in healthy newborn Martina Franca donkey foals from birth to 96 hours of age. Theriogenology, 87: 173-178. 28. Doreau M., Boulot S. (1989.) Recent knowledge on mare milk production: a review. Livest Prod Sci, 22: 213-235. 29. Peugnet P., Wimel L., Duchamp G., Sandersen C., Camous S., Guillaume D., Dahirel M., Dubois C., Reigner F., Berthelot V., Chaffaux S., Tarrade A., Serteyn D., Chavatte-Palmer P. (2014). Enhanced or reduced fetal growth induced by embryo transfer into smaller or larger breeds alters post-natal growth and metabolism in pre-weaning horses. PLoS ONE, 9:e102044. 30. Doreau M., Boulot S., Martin-Rosset W., Robelin J. (1986). Relationship between nutrient intake, growth and body composition of the nursing foal. Reprod Nutr Dev, 26: 683-69.

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Management of post-urethral urinary obstruction due to struvite uroliths in a female buffalo calf (Bubalus bubalis)

175

KALLEMUCHIKAL MANIKANDAN MANJUSHA1#, KHAN SHARUN1*#, ELANGOVAN KALAISELVAN1, ROHIT KUMAR1, ABHISHEK CHANDRA SAXENA1, PRAKASH KINJAVDEKAR1, ABHIJIT MOTIRAM PAWDE1, AMARPAL1 1

Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India.

SUMMARY Obstructive urolithiasis is an economically relevant disease of the ruminants. Male buffalo calves are most frequently affected compared to females because of their anatomical peculiarities. The occurrence of obstructive urolithiasis in females is a rare finding and requires documentation. A three-month-old female buffalo calf was presented with history of oliguria and stranguria in the past two days. General clinical examination identified that the animal was dull and restless with a rough hair coat, and congested mucus membrane. Abdominal palpation identified the presence of an enlarged urinary bladder. Based on history and clinical signs the condition was diagnosed as urinary tract obstruction. The hematological changes identified were neutrophilia and monocytosis and the serological parameters were within the normal range. Microscopic examination of the collected urine following sedimentation revealed the presence of numerous struvite calculi. It was decided to manage the case conservatively rather that going for invasive surgical procedure such as tube cystotomy. High resistance was encountered when a urinary catheter was passed through the urethra in the post-urethral region. The obstruction was relieved when the catheter was carefully maneuvered into the urethra. The animal was treated with parenteral antibiotics and oral ammonium chloride therapy following which the calf made an uneventful recovery.

KEY WORDS Urinary obstruction; post-urethral; struvite calculi; buffalo; bubalus bubalis.

INTRODUCTION Obstructive urolithiasis is a rare finding in female buffalo calves as compared to males. Anatomical peculiarities of the male urinary tract predispose them to calculi formation, especially in animals castrated before puberty. The health status and body condition of the animal solely depend on the duration of illness and the patency of urinary bladder1. Diet is one of the major contributing factors in calculi development along with other physiological and management factors. Struvite is the major calculi detected in ruminants due to excessive feeding of concentrate as compared to roughage2. Diet rich in magnesium and phosphorus that is low in calcium and potassium predisposes to the formation of struvite calculi3. Occurrence of silica, oxalate, and carbonate calculi in urine are considered to be incidental findings in ruminants4. Tentative diagnosis of obstructive urolithiasis can be made from history, clinical signs, per rectal or abdominal palpation, urinalysis, radiography, ultrasonography and haemato-biochemical examination5. In mild cases, the animals can be treated by using tranquilizers and antispasmodics6. Various treatment modalities, both medical and surgical have been developed in almost all the species for the management of urolithiasis7,8. Despite sophisCorresponding Author: Khan Sharun (sharunkhansk@gmail.com / sharunkhan@ivri.res.in). #Both K. M. Manjusha and Khan Sharun equally contributed to the work and therefore considered first authors.

ticated surgical techniques and various supportive treatments prognosis of urolithiasis in bovine still remains unpredictable9. Medical management is optional depending on the condition of animal, clinical signs, severity, site of occurrence, number of calculi, and the economic status of the owner. Rupture of bladder may occur in delayed cases or due to administration of diuretics in ruminants2. Time is an important factor that decides the prognosis of obstructive urolithiasis in ruminants. The present paper describes about post urethral urinary obstruction in a female buffalo calf and its conservative management.

Case history and clinical findings A 3-month-old female buffalo calf was presented to the surgery division of Referral Veterinary Polyclinic, IVRI, Bareilly, with the history of oliguria and stranguria in the past two days. On general clinical examination, the calf had a rough hair coat and turgid skin with sunken eyes. The calf exhibited frequent efforts to urinate and intermittent dribbling of urine was observed (Figure 1). The condition was diagnosed as obstructive urolithiasis based on the history and clinical signs. History indicated that the calf was exclusively fed on wheat bran diet as reported by the owner. Hematological parameters revealed marked neutrophilia and monocytosis. Biochemical analysis revealed marked increase in the serum creatinine and blood urea nitrogen (BUN) value indicating azotemia (Table 1).

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Management of post-urethral urinary obstruction due to struvite uroliths in a female buffalo calf (Bubalus bubalis)

Table 1 - Hematological and serological parameters of the buffalo calf with obstructive urolithiasis on the day of the presentation. Hematological parameters Day 0

Reference rangea

RBC count (× 10 / L)

5.44

5.0-10.0

PCV (%)

38.55

24-46

Parameter 6

Key findings

Hemoglobin (g/dl)

12.6

8.0-15.0

Total WBC count (× 103/ L)

11.5

4.0-12.0

Neutrophil (%)

15-33

Neutrophilia

Lymphocyte (%)

45-75

Lymphopenia

Monocyte (%)

0-8

Monocytosis

Eosinophil (%)

0-20

Basophil (%)

0-2

Platelets (× 103/ L)

230

100-800

Day 0

Reference rangea

6.54

6.7–7.5

Serological parameters Parameter Total protein (g/dl) Albumin

3.1

2.5-3.8

Globulin

3.44

3.0-3.5

Total bilirubin (mg/dl)

0.2

0-1.6

AST (IU/L)

60-125

Key findings

BUN (mg/dl)

48.2

10-25

Azotemia

Creatinine (mg/dl)

3.04

0.5-2.2

Azotemia

Hematology and serum biochemical reference ranges, The Merk Veterinary Manual - 11th edition (2016)

DISCUSSION Urinary calculi can occur anywhere in the urinary tract but it mostly depends upon the animal species. The occurrence of obstructive urolithiasis is rare in female calves compared to males. Knowledge on diet plays an important role in the predicting the nature of stone. A thorough investigation of the history should always precede the clinical examination. Young ruminants are mostly affected with this debilitating condition2. The condition is caused by multiple etiologies including sex, age, type of feed, hormonal imbalances, season, reduced water intake, genetic makeup, early castration before sexual maturation, hypovitaminosis A, and the affections of bladder and urethra10. Tiruneh (2004) found that high level of oxalates and silica in

Figure 1 - Female buffalo calf presented with post-urethral urinary obstruction. The urine was constantly dribbling from the vulva (yellow arrow).

pasture plants were considered as major factors deciding the formation of uroliths in grazing animals11. Moreover, provision of concentrate ration that is typically rich in phosphorus, or an imbalance in the calcium and phosphorus ratio, were stated as major causes of urolithiasis in feedlot animals. A calcium phosphorus imbalance in diet will precipitate the development of phosphate calculi due to high urinary excretion of

Figure 2 - Numerous calculi which are refractile and colorless along with erythrocytes and renal epithelial cells. The presence of 6 to 8 sided prisms and rectangles with coffin lid appearance is suggestive of struvite calculi (yellow arrow). In general, crystals can be orthorhombic, equant, wedge like, short prismatic, tabular, hemimorphic.

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177

Acknowledgments The authors are thankful to the Director, Indian Veterinary Research Institute, Izatnagar (UP) and Head, Surgery Division, Indian Veterinary Research Institute, Izatnagar (UP) for the facilities provided.

Funding No substantial funding to be stated.

Disclosure statement All authors declare that there exist no commercial or financial relationships that could, in any way, lead to a potential conflict of interest.

References Figure 3 - (a) Multiple uroliths were found towards the base of the tail after relieving the obstruction. (b) Macroscopic struvite uroliths expelled through the urine.

phosphorus3. The shortage of drinking water could further act as a precipitating factor in the development of urolithiasis. Sharma et al. (2005) conducted study on twelve bovine calves suffering from urolithiasis. The haemato-biochemical parameters showed increased values of Hb, PCV, TLC, neutrophils, BUN, creatinine, inorganic phosphorus and potassium. According to the study, these parameters returned towards normal levels after surgical intervention and fluid therapy12. Ruminants are the most susceptible group of animals that are affected by obstructive urolithiasis and is extensively reported in male sheep, goat and cattle4. Rupture of the bladder occur as a sequela to the complete obstruction of urethra due to lodgment of calculi13. Early signs of urethral obstruction include severe pain that leads to stranguria14. Sandy calculi which initially get lodged in the urethra may later lead result in complete urethral obstruction in young animals15. The treatment is based on history, clinical signs and the type of calculi with surgical correction being the supreme and ultimate choice.

4. 5.

10.

CONCLUSION Obstructive urolithiasis should be considered as an emergency condition in ruminants that requires immediate intervention. Stage of clinical presentation and condition of the animal is important in deciding the prognosis. Since the condition is having multifactorial etiology i.e. nutritional, physiological, and management factors, the treatment should be directed to resolve all these factors. Recurrence of calculi can occur either due to failure of treatment or due to lack of proper care. Furthermore, the formation of stones within the urinary tract is not a specific disease but is a potential complication of many different disorders. Therefore, a thorough knowledge about the pathogenesis of the condition is required for efficient and costmanagement.

11.

12.

13.

14.

15.

Kushwaha R.B., Amarpal K.P., Aithal H.P., Kinjavdekar P., Pawde, A.M. (2014). Clinical appraisal of 48 cases of obstructive urolithiasis in buffalo calves treated with tube cystostomy and urethrotomy. Adv Anim Vet Sci, 2(2): 106-110. Amarpal K.P., Aithal H.P., Pawde A.M., Pratap K., Gugjoo, M.B. (2013). A retrospective study on the prevalence of obstructive urolithiasis in domestic animals during a period of 10 years. Adv Anim Vet Sci, 1(3): 8892. Radostitis O.M., Blood D.C., Gray G.C. Hinchcliff K.W. (2005). Veterinary Medicine a text book of the disease of cattle, sheep, pig, goat and horse. Bailliere Tindall, London, 1877. Makhdoomi D.M., Ghazi M.A. (2013). Obstructive urolithiasis in ruminants- A review. Vet World, 6: 233-238. Parrah J.D., Hussain S.S., Moulvi B.A., Makhdoomi D.M., Buchoo, B.A., Malik H.U. (2011). Ultrasonographic diagnosis of obstructive urolithiasis in calves. Indian J Vet Surg, 32(2): 117-20. Elisa M., Ermilio M.C., Smith. (2011). Treatment of Emergency Conditions in Sheep and Goats. Vet Clin North America: Food Anim Pract, 27 (1): 33-45. Dubey A., Pratap K., Amarpal, Aithal H.P., Kinjavdekar P., Singh, T., Sharma M.C (2006). Tube Cystotomy and chemical dissolution of urethral calculi in goats. Indian J Vet Surg, 27(2): 98-103. Janke J.J., Osterstock J.B., Washburn K.E., (2009). Use of Walpole’s solution for treatment of goats with urolithiasis: 25 cases (2001-2006). J Am Vet Med Assoc, 234: 249–252. Sharma A.K., Mohindroo J., Aithal H.P. (2009). Physiological, urological changes and surgical management of urethal obstruction in bovine 25 cases (2001-2006). J Am Vet Med Assoc, 234: 249-252. Udall R.H. Chow F.H. (1969). The etiology and control of urolithiasis. Adv Vet Sci Comp Med, 13: 29-57. Tiruneh R. (2004). Minerals and Oxalate content of feed and water in relation with ruminant urolithiasis in Adea district, central Ethiopia. Revue De Med Vet, 155(5): 272-77. Sharma A.K., Mogha J.V., Singh G.R., Amarpal A., Aithal H.P. (2005). Clinico-physiological and haematobiochemical changes in urolithiasis and its management in bovine. Indian J Anim Sci, 75: 1131-1134. Rajesh K., Sharun K., Naveen K.V., Sasikala R., Mohd A.B., Pawde A.M., Amarpal K. P. (2019). Management of obstructive urolithiasis in a bullock by urethrotomy. J Pharmacognosy and Phytochemistry, 8(3): 20802082. Monoghan M.L., Boy, M.G. (1990). Ruminants Renal System: Diseases of the renal system. In: Large Animal Internal Medicine. C. V. S. Mosby Company, Philadelphia, 888-890. Gugjoo M.B., Zama M.M.S., Amarpal K.P., Mohsina A., Saxena A. C., Sarode I.P. (2013). Obstructive urolithiasis in buffalo calves and goats: incidence and management. J Adv Vet Res, 3: 109-113.

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First isolation of Salmonella Duisburg from quail flock

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OZGE ARDICLI, SERPIL KAHYA DEMIRBILEK, HAVVA KURNAZ, KAMIL TAYFUN CARLI Department of Microbiology, Faculty of Veterinary Medicine, Bursa Uludag University, 16059, Bursa, Turkey

SUMMARY The first isolation of Salmonella enterica subsp. enterica serovar Duisburg (S. Duisburg) (4,12,[27]:d:e,n,z15) from quails was presented in this case report. Internal organs and ileocecal parts of intestines were collected from quails at 20-day old age in the flock (total of 150 quails) located in South Marmara region of Turkey. Isolation was performed according to International Organization for Standardization Method 6579. Regarding the identification of Salmonella-suspected colonies, API 20E test strips and Phoenix 100 ID/AST system were used. Serotyping of the isolate was undertaken using the slide serum agglutination test. Minimum inhibitory concentration results showed that Salmonella isolate was susceptible to all the tested antimicrobials. Although the prominent species is chicken in poultry, quail breeding increases its importance and extensiveness. Therefore this study may be useful not only for current antibiotic practices in quail breeding but also for further studies on avian microbiology.

KEY WORDS Quail, Salmonella, serotyping, S. Duisburg, antibiotic susceptibility.

INTRODUCTION

CASE PRESENTATION

Salmonella serotypes are significant bacterial pathogens for poultry worldwide. Vertical (from breeder stocks to their chicks via eggs) and lateral transmission (from environment contaminated with faeces from infected chickens to healthy chickens) occur in Salmonella infections. Moreover, the infection has a high transmission capability among individuals with respect to feed, drinking water or environmental sources. Therefore prevention strategies including good monitoring and screening programs are essential in the flock to preclude economic losses1. Salmonella genus has more than 2500 serotypes and has a wide host range which comprises several animal species within mammals, birds and reptiles1. A number of studies in Turkey have shown that Salmonella infections are endemic in many parts of the country and Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) and S. Infantis are the most common serotypes2,3. Observations of the remaining serotypes are rare, such as S. Thompson, S. Agona and S. Duisburg. Worldwide, S. Duisburg was isolated from cattle egrets4, chicken carcasses5, turkey6, badger, cattle, sheep, pig7, lizard8, and drinking water9. However, no information about this pathogen is available in quails. Hence, to the best of authors’ knowledge, this is the first report indicating the presence of the S. Duisburg from ileocecal part of the intestines in quail flock.

Samples were collected from egg-laying Japanese quail (Coturnix coturnix japonica) flock (a total of 150 quails) that suffered from diarrhoea. The flock was located in Bursa, Turkey and the birds were housed in cages with dimensions of 100x45x20 cm. Each cage consists of 8-10 quails which were fed with the same commercial diet. The birds were provided with 16 h of light in a ventilated room at approximately 22-24 °C ambient temperature. No vaccinal program was applied. In the flock, the mortality rate was 20%. Four quails at 20-day old age were necropsied and samples from lung, liver, kidney, heart and ileocecal part of intestines were collected for culture method. Ileocecal parts of intestines were pooled into one sample. Concerning necropsy findings, there were no lesions in the internal organs including lung, liver, kidney, and heart. However, there were signs for enteritis such as gross lesions and swelling in intestines. In order to investigate the presence of bacteria that could be the potential cause of clinical signs, initially, internal organs were examined by standard bacteriological method. In this context samples were inoculated on two 5% blood agars (GBL, Turkey), a MacConkey (MC; Merck, Darmstadt, Germany) agar and a Mycoplasma agar (Oxoid, CM0401, England). One blood agar and Mycoplasma agar were incubated at 37 °C in microaerophilic atmosphere while the others were incubated in an aerobic atmosphere. Results revealed that there was no Salmonella agent in internal organs nor was there any other bacteria. Isolation and identification of Salmonella from ileocecal parts of intestines were performed according to International Organization for Standardization Method 657910. Briefly, 1 g of the sample was pre-enriched in 9 ml of buffered peptone wa-

Corresponding Author: Ozge Ardicli (ozgeyilmaz@uludag.edu.tr)

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ter (BPW; Merck, Darmstadt, Germany) and incubated at 37 °C for 18 h. After incubation, 0.1 ml of pre-enrichment broth culture was transferred into modified semi-solid Rappaport Vassiliadis agar (MSRV; Oxoid, CM1112) and incubated at 41.5 °C for 24 h. Afterwards, a loop, full of MSRV culture, was streaked separately onto the surface of Xylose Lysine Desoxycholate agar (XLD; Oxoid, CM0469) and MC agar. They were incubated overnight at 37 °C. Following incubation, colonies were analysed to identify typical morphology of Salmonella. Typical Salmonella colonies are pink-edged and black centred on XLD agar, while, pale translucent on MC agar. Identification of suspected Salmonella colonies was performed by biochemical tests according to their urease activity (Urea Agar Base, Oxoid, CM0053), triple sugar utilization and hydrogen sulphide (H2S) formation (Triple Sugar Iron Agar, Oxoid, CM0277), and lysine decarboxylase activity (Lysine Iron Agar, Oxoid, CM0381). Final identification was performed using both API 20E test strips (bioMerieux, Marcy L’Etoile, France) and Phoenix 100 ID/AST system (Becton Dickinson Co., Sparks, Md.). The NMIC/ID-433 panel was processed in a Phoenix 100 ID/AST system according to the manufacturer’s directions. The existence of Salmonella spp. was substantiated by the abovementioned test protocols. Minimum inhibitory concentration (MIC) results were evaluated by using Phoenix AST panel for amikacin (8-32 µg/mL), gentamicin (2-8 µg/mL), ertapenem (0.25-1 µg/mL), imipenem (0.25-8 µg/mL), meropenem (0.125-8 µg/mL), cefazolin (4-32 µg/mL), cefuroxime (4-16 µg/mL), ceftazidime (1-8 µg/mL), ceftriaxone (1-4 µg/mL), cefepime (1-8 µg/mL), ceftolozane-tazobactam (1/4-4/4 µg/mL), ampicillin (4-16 µg/mL), amoxicillin-clavulanate (2/2-16/2 µg/mL), ampicillin-sulbactam (1/8-8/8 µg/mL), piperacillintazobactam (4/4-16/4 µg/mL), colistin (1-4 µg/mL), trimethoprim-sulphamethoxazole (2/38-8/152 µg/mL), ciprofloxacin (0.0625-1 µg/mL), levofloxacin (0.5-2 µg/mL), and tigecycline (0.5-2 µg/mL). Isolate, identified as Salmonella, was susceptible to all the tested antimicrobials. Salmonella isolate was serotyped according to the Kauffmann-White-Le Minor Scheme11. Identification of the O and H group antigens was performed by slide agglutination tests by means of O4, O12, O27, Hd antisera (Statens Serum Institute, Copenhagen, Denmark) and He, Hz15 antisera (Denka Seiken Co., Ltd. Tokyo, Japan). Thus, the isolate was serotyped as Duisburg with the antigenic formula 4,12,[27]:d:e,n,z15.

DISCUSSION Salmonellosis is one of the major bacterial diseases in poultry industry and it frequently involves in outbreaks and cases of foodborne diseases which cause millions of human infections and even deaths12. The consumption of contaminated poultry products has been reported to be the most important reason for Salmonella infections in humans3. Therefore, studies on salmonellosis should be carefully considered not only for poultry industry but also the humans’ health. It is clear that the main component of poultry is chicken but other species, such as quail, turkey, duck, geese, and etc., are significant sources of both meat and egg production worldwide. These species have also been reported to be the potential carriers of Salmonella spp. which serve as the sources of exposure or infection for humans. However in the literature there is limited information about Salmonella infections in quail. In this study S. Duisburg was first-

ly identified in quails. This serotype, was first isolated by Korell and Seeliger13 from a child with diarrhoea in 1953. Although S. Duisburg is not a common serotype in poultry, there are few studies indicating the presence of S. Duisburg observed in salmonellosis cases6,14. In the present study S. Duisburg was isolated from ileocecal part of the intestines in quails. It is important to note that the knowledge on the existence of Salmonella serovars in quail is rather limited compared to prominent avian species such as chicken and turkey. In this context, there are some studies reporting Salmonella spp. in quails but the determination of serovars was not performed in these studies. For instance, in the study by Omoshaba et al.15, four hundred cloacal swabs of quail birds were collected in Nigeria, and in all, Salmonella was isolated from 14 (3.5%) cloacal swabs. In addition Palanisamy and Bamaiyi16 indicated that the prevelance of Salmonella spp. was 11.11% in the three quail farms located in Malaysia. Jahan et al.17 suggested that the overall prevalence of Salmonella spp. in quails was found to be 13.33%. On the other hand, McCrea et al.18 performed a detailed analysis to investigate the existence of Salmonella spp. and Campylobacter spp. among the various avian species including squab, quail, guinea fowl, duck, poussin (young chicken), and free-range broiler chickens. However, their results showed that there were no Salmonella isolates in the samples obtained from guinea fowl and quail flocks. Similarly, Dipineto et al.19 reported that there was no detection of Salmonella spp. in common quails cloacal swab samples. In the literature, there are several studies on the isolation of Salmonella serovars including S. Virchow, S. Meleagridis, S. Typhimurium in Japanese quails20, S. Typhimurium, S. Typhimurium variant Copenhagen, and S. Hadar in live commercial quails and carcasses, and S. Paratyphi in the flock environment21,22. Moreover, Boroomand et al.23 investigated Enterobacteriaceae responsible for early mortality in Japanese quail chicks and the results showed that 78% of them were infected with Escherichia coli (E. coli), S. Ruzizi, S. Typhimurium. These authors reported that E. coli and Salmonella spp. are the major causes of early mortality in newly-hatched Japanese quail chicks. In this study antimicrobial susceptibility was evaluated by MIC analysis and the results revealed that S. Duisburg was 100% susceptible to all tested antimicrobials including amikacin, gentamicin, ertapenem, imipenem, meropenem, cefazolin, cefuroxime, ceftazidime, ceftriaxone, cefepime, ceftolozanetazobactam, ampicillin, amoxicillin-clavulanate, ampicillin-sulbactam, piperacillin-tazobactam, colistin, trimethoprim-sulphamethoxazole, ciprofloxacin, levofloxacin, and tigecycline. Unlike our results, many studies showed the presence of multidrug resistant Salmonella in quails. In this respect, Omoshaba et al.15 reported that the Salmonella isolates exhibited variable rates of resistance to antimicrobials, as follows: ampicillin (100%), tetracycline (100%), doxycycline (100%), sulphamethoxazole (92.9%), nalidixic acid (85.8%), ceftazidime (78.6%), neomycin (64.3%), streptomycin (50%) and gentamicin (28.6%). However all the isolates were susceptible to ciprofloxacin. In a similar vein, Jahan et al.17 suggested that all Salmonella isolates were found to be multidrug resistant (100% resistant to erythromycin and tetracycline and 90% to colistin sulphate). On the other hand Palanisamy and Bamaiyi16 found that Salmonella isolates were resistant to ampicillin only. Antibiotic resistance, which can affect the severity of salmonellosis, makes the infections much more difficult to treat be-

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cause it reduces the number of effective antibiotics and causes delayed or inadequate therapies5,14. The resistance formation can be prevented by prudent use of antibiotics supported by antibiogram tests before drug administration or avoiding incorrect use of antibiotics in livestock as well as poultry. The 100% susceptibility to tested antibiotics observed in this study may be a result of limited utilization of the mentioned antimicrobials in quails. Consequently, the present study is the first report of S. Duisburg in quail. Further detailed epidemiological and molecular studies are essential on the determination of observation frequency of this serovar in various avian species such as quails.

References 1. Gast R.K., JR Porter R.E. (2020). Salmonella infections. In: Diseases of poultry, Eds. Swayne D.E., Boulianne M., Logue C.M., McDougald L.R., Nair V., Suarez D.L., 14th ed., 717-753, Wiley-Blackwell, USA. 2. Kahya S., Kesin Tu B., Temelli S., Carli K.T., Eyigor A. (2014). Detection of Salmonella from Layer Flocks and Typing of the Isolates. Kafkas Univ. Vet. Fak. Derg, 20: 939-944. 3. Temelli S., Kahya S., Zafer A., Carli K.T., Eyigor A. (2015). Presence of Salmonella in retail grade a eggs determined by the International Organization for Standardization 6579 method and a LightCycler polymerase chain reaction system. Ankara Univ. Vet. Fak. Derg, 62: 125-132. 4. Silva M.A., Fernandes É.F.S.T., Santana S.C., Marvulo M.F.V., Barros M.R., Vilela S.M.O., Reis E.M.F., Mota R.A., Silva J.C.R. (2018) Isolation of Salmonella spp. in cattle egrets (Bubulcus ibis) from Fernando de Noronha Archipelago. Brazil. Braz. J. Microbiol, 49: 559-563. 5. Bada-Alambedji R., Fofana A., Seydi M., Akakpo A.J. (2006). Antimicrobial resistance of Salmonella isolated from poultry carcasses in Dakar (Senegal). Braz. J. Microbiol, 37: 510-515. 6. Pedersen K., Hansen H.C., Jørgensen J.C., Borck B. (2002). Serovars of Salmonella isolated from Danish turkeys between 1995 and 2000 and their antimicrobial resistance. Vet Rec,150: 471-474. 7. Euden P. (1990). Salmonella isolates from wild animals in Cornwall. Br. Vet. J, 146: 228-232. 8. Gorski L., Jay-Russell M.T., Liang A.S., Walker S., Bengson Y., Govoni J., Mandrell R.E. (2013). Diversity of pulsed-field gel electrophoresis pulsotypes, serovars, and antibiotic resistance among Salmonella isolates from wild amphibians and reptiles in the California central coast. Foodborne Pathog Dis, 10: 540-548. 9. Rolland D., Block J. (1980). Simultaneous concentration of Salmonella and enterovirus from surface water by using micro-fiber glass filters. Appl. Environ. Microbiol, 39: 659-661.

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10. International Organization For Standardization (ISO), ISO 6579: 2002 (2002). Microbiology of food and animal feeding stuffs–horizontal method for the detection of Salmonella spp. Geneva, Switzerland. 11. Grimont P.A.D., Weill F.X. (2007). Antigenic formulae of the Salmonella serovars. 9th ed., 9: 1-166, WHO collaborating centre for reference and research on Salmonella, Paris, France. 12. Mead P.S., Slutsker L., Griffin P.M., Tauxe V. (1999). Food-related illness and death in the United States reply to Dr. Hedberg. Emerg. Infect. Dis, 5: 841. 13. Korell O., Seeliger H. (1954). New type of Salmonella B group; Salmonella 4, 12: d: e, n, z15. Zentr. Bakteriol., Parasitenk., Abt. I. Orig, 161: 421422. 14. Maka Ł., Maćkiw E., Ścieżyńska H., Pawłowska K., Popowska M. (2014). Antimicrobial susceptibility of Salmonella strains isolated from retail meat products in Poland between 2008 and 2012. Food Control, 36: 199-204. 15. Omoshaba E., Olufemi O.F., Ojo O., Sonibare A., Agbaje M. (2017). Multidrug-resistant Salmonellae isolated in Japanese quails reared in Abeokuta, Nigeria. Trop. Anim. Health. Prod, 49: 1455-1460. 16. Palanisamy S., Bamaiyi P.H. (2015). Isolation and antibiogram of Salmonella spp. from quails in a farm from Kelantan, Malaysia. J. Vet. Adv, 5: 1191-1198. 17. Jahan S., Zihadi M.A.H., Nazir K.N.H., Islam M.S., Rahman M.B., Rahman M. (2018). Molecular detection and antibiogram of Salmonella spp. from apparently healthy Japanese quails of three different quail farms in Mymensingh. J. Adv. Vet. Anim. Res, 5: 60-66. 18. McCrea B., Tonooka K., Vanworth C., Boggs C., Atwill E.R., Schrader J.S. (2006). Prevalence of Campylobacter and Salmonella species on farm, after transport, and at processing in specialty market poultry. Poult. Sci, 85: 136-143. 19. Dipineto L., Russo T.P., Gargiulo A., Borrelli L., De Luca Bossa L.M., Santaniello A., Buonocore P., Menna L.F., Fioretti A. (2014). Prevalence of enteropathogenic bacteria in common quail (Coturnix coturnix). Avian Pathol, 43: 498-500. 20. Al-Nakhli H.M. (2005). Occurrence of Paratyphoid Infection Among japanese quails (Coturnix coturnix japonica) in Saudi Arabia. J. Biosci, 12: 59-66. 21. Sander J., Hudson C.R., Dufour-Zavala L., Waltman W.D., Lobsinger C., Thayer S.G., Otalora R., Maurer J.J. (2001). Dynamics of Salmonella contamination in a commercial quail operation. Avian Dis, 45: 1044-1049. 22. Rocha-E-Silva R.C., Cardoso W.M., Teixeira R.S.C., Albuquerque Á.H., Horn R.V., Cavalcanti C.M., Lopes E.S., Gomes Filho V.J.R. (2013). Salmonella Gallinarum virulence in experimentally-infected Japanese quails (Coturnix japonica). Braz. J. Poultry Sci, 15: 39-45. 23. Boroomand Z., Jafar R., Gharibi D., Kazemi K. (2018). An Investigation into Enterobacteriaceae Responsible for Early Mortality in Japanese Quail Chicks and Their Antibiotic Susceptibility Patterns. Arch. Razi Inst, 73: 277-285.

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