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In-Vivo Effects of Canary (Serinus Canary) Semen Diluted with Different Semen Extenders on Eggs in Artificial Insemination

Yıl 2024, Cilt: 9 Sayı: 1, 60 - 66, 31.03.2024
https://doi.org/10.35229/jaes.1377519

Öz

In this study, the effect of Dulbecco's Phosphate Buffered Saline (DPBS) and Dulbecco's Modified Eagle's Medium (DMEM) extenders on artificial insemination was determined.. For this purpose, the number of fertile eggs, the total number of eggs, the number of hatched eggs, and the number of hatchlings from fertile eggs were determined in the study. From these values, the fertility rate (%), egg hatch rate (%) and fertile egg hatch rate (%) were calculated. Twelve male and female canaries (Gloster breed; 1-2 years old) were used in each group. In the study, three groups, one control (C) and the other two treatment groups (T-1, T-2) were formed. Female canaries in group C were directly inseminated without using any extenders. Females in the T-1 group were diluted 1:1 with DPBS; females in the T-2 group were inseminated with semen diluted 1:1 with DMEM. Although there was a significant difference between the groups in terms of fertility rate (P<0.05), egg-laying rate and fertile egg-laying rate were not different in terms of parameters (P>0.05). The highest fertility rate (61.81%) was in group C; the lowest was in the T-1 (29.17%) group. (P<0.05). Using a DPBS extender, it was revealed that the fertility rate was negatively affected in insemination. Due to the limited number of studies on this subject, it is thought that it will be essential to investigate DPBS and DMEM diluents at different rates and methods in future studies

Etik Beyan

Dear Managing Editor, I would like to submit the manuscript entitled “In-Vivo Effects of Canary (Serinus Canary) Semen Diluted with Different Semen Extenders on Eggs in Artificial Insemination” in JAES This manuscript neither the entire nor any part of its ‎content has been published or being considered for publication elsewhere. All ‎authors were fully involved in the study and preparation of the manuscript and ‎they have read and approved this manuscript and take responsibility for its ‎contents. We would like to thank the reviewers, especially the editor, who will make all the evaluations. Sincerely. Dr. Arda Onur ÖZKÖK

Kaynakça

  • Almubarak, A.M.,Kim, W., Abdelbagi, N.H., Balla, S.E., Yu, I.J. & Jeon, Y. (2021). Washing solution and centrifugation affect kinematics of cryopreserved boar semen. Journal of Animal Reproduction and Biotechnology, 36, 65-75. DOI: 10.12750/JARB.36.2.69
  • Bakst, M.R. & Cecil, H.C. (1997). Techniques for semen evaluation, semen storage, and fertility determination. In: Poultry Science Association, 2nd edn, Murray R. Bakst & Julie A. Long, Buffalo, Minnesota, p. 11-15.
  • Blanco, J.M., Wildt, D.E., Höfle, U., Voelker, W. & Donoghue, A.M. (2009). Implementing artificial insemination as an effective tool for ex situ conservation of endangered avian species. Theriogenology, 71, 200-213. DOI: 10.1016/j.theriogenology.2008.09.019
  • Coutteel, P. (2003). Veterinary aspects of breeding management in captive passerines. In Seminars in Avian and Exotic Pet Medicine, 12, 3-10. DOI: 10.1053/saep.2003.127879
  • Cramer, E.R., Ålund, M., McFarlane, S.E., Johnsen, A. & Qvarnström, A. (2016). Females discriminate against heterospecific sperm in a natural hybrid zone. Evolution, 70, 1844-1855. DOI: 10.1111/evo.12986
  • Cramer, E.R., Rowe, M., Eroukhmanoff, F., Lifjeld, J.T., Sætre, G.P. & Johnsen, A. (2019). Measuring sperm swimming performance in birds: effects of dilution, suspension medium, mechanical agitation, and sperm number. Journal of Ornithology, 160, 1053-1063. DOI: 10.1007/s10336-019-01672-9
  • Dumpala, P.R., Parker, H.M. & McDaniel, C.D. (2006). The effect of semen storage temperature and diluent type on the sperm quality index of broiler breeder semen. International Journal of Poultry Science, 5, 838-845. DOI: 10.3923/ijps.2006.838.845
  • Donoghue, A.M. & Wishart, G.J. (2000). Storage of poultry semen. Animal Reproduction Science, 62, 213-232. DOI: 10.1016/S0378-4320(00)00160-3
  • Faul, F., Erdfelder, E., Lang, A.G. & Buchner, A. (2007). G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behavior Research Methods, 39, 175-191.
  • Fischer, D., Schneider, H., Meinecke-Tillmann, S., Wehrend, A. & Lierz, M. (2020). Semen analysis and successful artificial insemination in the St. Vincent amazon (Amazona guildingii). Theriogenology, 148, 132-139. DOI: 10.1016/j.theriogenology.2020.03.004
  • Gee, G.F. (1995). Artificial insemination and cryopreservation of semen from nondomestic birds. In: Proceeding of the 1st Symposium on the Artificial Insemination in Poultry, Savoy, P262- 280.
  • Gee, G.F., Bertschinger, H., Donoghue, A.M., Blanco, J. & Soley, J. (2004). Reproduction in nondomestic birds: physiology, semen collection, artificial insemination and cryopreservation. Avian and Poultry Biology Reviews, 15, 47-101. DOI: 10.3184/147020604783637435
  • Hall, C.A., Potvin D.A. & Conroy, G.C. (2023). A new candling procedure for thick and opaque eggs and its application to avian conservation management. Zoo Biology, 42, 296-307. DOI: 10.1002/zoo.21730
  • Hassan, K.H. (2009). Fertility of broiler males and females at various ages by using artificial insemination. Diyala Agricultural Sciences Journal, 1, 25-30.
  • Hudelson, K.S. (1996). A review of the mechanisms of avian reproduction and their clinical applications. In Seminars in Avian and Exotic Pet Medicine, 5, 189-198. DOI: 10.1016/S1055-937X(96)80027-2
  • Humann-Guilleminot, S., de Montaigu, C.T., Sire, J., Grünig, S., Gning, O., Glauser, G., Vallat A. & Helfenstein, F. (2019). A sublethal dose of the neonicotinoid insecticide acetamiprid reduces sperm density in a songbird, Environmental Research, 177, 108589. DOI: 10.1016/j.envres.2019.108589
  • Hobbs, R.J., Upton, R., Keogh, L., James, K., BaxterGilbert, J. & Whiting, M.J. (2021). Sperm cryopreservation in an Australian skink (Eulamprus quoyii). Reproduction, Fertility and Development, 34, 428-437. DOI: 10.1071/RD21031
  • Kleven, O., Fossøy, F., Laskemoen, T., Robertson, R.J., Rudolfsen, G. & Lifjeld, J.T. (2009). Comparative evidence for the evolution of sperm swimming speed by sperm competition and female sperm storage duration in passerine birds. Evolution, 63, 2466-2473. DOI: 10.1111/j.1558- 5646.2009.00725.x
  • Lara, C.E., Taylor, H.R., Holtmann, B., Johnson, S.L., Santos, E.S., Gemmell, N.J. & Nakagawa, S. (2020). Dunnock social status correlates with sperm speed, but fast sperm does not always equal high fitness. Journal of Evolutionary Biology, 33, 1139-1148. DOI: 10.1111/jeb.13655
  • Lüpold, S., Linz, G.M. & Birkhead, T.R. (2009). Sperm design and variation in the New World Blackbirds (Icteridae). Behav Ecol Sociobiol, 63, 899-909. DOI: 10.1007/s00265-009-0733-6
  • Madeddu, M., Marelli, S., Abdel Sayed, A., Mosca, F., Cerolini, S. & Zaniboni, L. (2022). Assessment of sperm viability and computer-assisted motility analysis in budgerigars (Melopsittacus undulatus): effect of several in vitro processing conditions. Veterinary Medicine International, 2022, 5997320. DOI:10.1155/2022/5997320
  • McDiarmid, C.S., Hurle, L.L., Le Mesurier, M., Blunsden, A.C. & Griffith, S. C. (2022). The impact of diet quality on the velocity, morphology and normality of sperm in the zebra finch Taeniopygia guttata. J Exp Biol., 225(9), jeb243715, DOI: 10.1242/jeb.243715
  • Mohan, J., Sharma, S.K., Kolluri, G., Tyagi, J.S. & Kataria, J.M. (2015). Selection of diluent for short term preservation of guinea fowl semen. Asian Journal of Animal and Veterinary Advances, 10, 360-364. DOI: 10.3923/ajava.2015.360.364
  • Mohan, J., Sharma, S.K., Kolluri, G. & Dhama, K. (2018). History of artificial insemination in poultry, its components and significance. World's Poultry Science Journal, 74, 475-488. DOI: 10.1017/S0043933918000430
  • Pearlin, V., Mohan, J., Tyagi, J.S., Gopi, M., Kolluri, G., Prabakar, G. & Shanmathy, M. (2021). Efficiency of different diluents and dilution rates on the fertilization potential of chicken spermatozoa. Indian Journal of Animal Research, 55, 139-144. DOI: 10.18805/IJAR.B-3940
  • Piaček, V., Zukal, J., Seidlova, V., Heger, T., Němcová, M., Přibyl, M., Vitula, F. & Pikula, J. (2020). Fresh semen characteristics in captive accipitrid and falconid birds of prey. Acta Veterinaria Brno, 89, 291-300. DOI: 10.2754/avb202089030291
  • Purohit, G.N., Duggal, G. P., Dadarwal, D., Kumar, D., Yadav, R.C. & Vyas, S. (2003). Reproductive biotechnologies for improvement of buffalo: The current status. Asian-Australasian Journal of Animal Sciences, 16, 1071-1086. DOI: 10.5713/ajas.2003.1071
  • Rakha, B.A., Ansari, M. S., Akhter, S., Hussain, I. & Blesbois, E. (2016). Cryopreservation of Indian red jungle fowl (Gallus gallus murghi) semen. Animal Reproduction Science, 174, 45-55. DOI: 10.1016/j.anireprosci.2016.09.004
  • Roiter, Y.S. & Konopleva, A.P. (2019). Universal biotechnological medium for sperm dilution during poultry artificial insemination. In: IOP Conference Series: Earth and Environmental Science, P 315. DOI: 10.1088/1755- 1315/315/4/042020
  • Schmoll, T. & Kleven, O. (2016). Functional infertility in a wild passerine bird. Ibis, 158, 670-673. DOI: 10.1111/ibi.12376
  • Shinde, A.S., Mohan, J., Sastry, K.V.H., Singh, R.P., Chouhan, L. & Tyagi, J.S. (2013). CARI diluent for short-term preservation of Kadaknath chicken semen. Indian Journal of Poultry Science, 48, 240-243.
  • Tabachnick, B.G., Fidell, L.S. & Ullman, J.B. (2019). Using Multivariate Statistics (Seventh Ed.). Pearson.
  • Trivedi, A.K., Rani, S. & Kumar, V. (2006). Control of annual reproductive cycle in the subtropical house sparrow (Passer domesticus): evidence for conservation of photoperiodic control mechanisms in birds. Frontiers in Zoology, 3, 1- 12. DOI: 10.1186/1742-9994-3-12
  • Whiteaker, M. (1979). Gloster Canary, AFA Watchbird, 6, 15-15.
  • Yang, Y., Zhang, Y., Ding, J., Ai, S., Guo, R., Bai, X. & Yang, W. (2019). Optimal analysis conditions for sperm motility parameters with a CASA system in a passerine bird, Passer montanus. Avian Research, 10, 1-10. DOI: 10.1186/s40657-019- 0174-5
  • Yenilmez, F. (2020). Canary Production. Turkish Journal of Agriculture-Food Science and Technology, 8, 941-944. DOI: 10.24925/turjaf.v8i4.941- 944.3197
  • Zambelli, D. & Cunto, M. (2022). Artificial insemination in queens in the clinical practice setting: protocols and challenges. Journal of Feline Medicine and Surgery, 24, 871-880. DOI: 10.1177/1098612X221118756.

Suni Tohumlamada Farklı Semen Sulandırıcılarla Sulandırılan Kanarya (Serinus Canary) Spermasının Yumurtalar Üzerine In-Vivo Etkileri

Yıl 2024, Cilt: 9 Sayı: 1, 60 - 66, 31.03.2024
https://doi.org/10.35229/jaes.1377519

Öz

Bu çalışmada, Dulbecco'nun Fosfat Tamponlu Salini (DPBS) ve Dulbecco'nun Modifiye Eagle's Medium (DMEM) sulandırıcılarının suni tohumlama üzerindeki etkisini belirlendi. Bu amaçla çalışmada döllü yumurta sayısı, toplam yumurta sayısı, kuluçkadan çıkan yumurta sayısı ve döllü yumurtalardan çıkan yavru sayısı belirlenmiştir. Bu değerlerden doğurganlık oranı (%), yumurtadan çıkış oranı (%) ve verimli yumurtadan çıkış oranı (%) hesaplandı. Her grupta 12 adet erkek ve dişi kanarya (Gloster cinsi; 1-2 yaşında) kullanıldı. Çalışmada biri kontrol (C) ve diğer iki tedavi grubu (T-1, T-2) olmak üzere üç grup oluşturuldu. C grubundaki dişi kanaryalar herhangi bir sulandırıcı kullanılmadan doğrudan tohumlandı. T-1 grubundaki dişiler DPBS ile 1:1 oranında seyreltildi; T-2 grubundaki dişiler DMEM ile 1:1 seyreltilmiş meni ile tohumlandı. Gruplar arasında doğurganlık oranı açısından önemli bir fark olmasına rağmen (P<0.05), yumurtlama oranı ve verimli yumurtlama oranı parametreler açısından farklı değildi (P>0.05). En yüksek doğurganlık oranı (%61,81) C grubundaydı; en düşük oran T-1 (%29,17) grubundaydı. (P<0.05). DPBS sulandırıcı kullanılarak tohumlamada doğurganlık oranının olumsuz etkilendiği ortaya çıktı. Bu konuda yapılan çalışmaların sınırlı sayıda olması nedeniyle gelecek çalışmalarda DPBS ve DMEM seyrelticilerinin farklı oran ve yöntemlerle araştırılmasının önemli olacağı düşünülmektedir.

Kaynakça

  • Almubarak, A.M.,Kim, W., Abdelbagi, N.H., Balla, S.E., Yu, I.J. & Jeon, Y. (2021). Washing solution and centrifugation affect kinematics of cryopreserved boar semen. Journal of Animal Reproduction and Biotechnology, 36, 65-75. DOI: 10.12750/JARB.36.2.69
  • Bakst, M.R. & Cecil, H.C. (1997). Techniques for semen evaluation, semen storage, and fertility determination. In: Poultry Science Association, 2nd edn, Murray R. Bakst & Julie A. Long, Buffalo, Minnesota, p. 11-15.
  • Blanco, J.M., Wildt, D.E., Höfle, U., Voelker, W. & Donoghue, A.M. (2009). Implementing artificial insemination as an effective tool for ex situ conservation of endangered avian species. Theriogenology, 71, 200-213. DOI: 10.1016/j.theriogenology.2008.09.019
  • Coutteel, P. (2003). Veterinary aspects of breeding management in captive passerines. In Seminars in Avian and Exotic Pet Medicine, 12, 3-10. DOI: 10.1053/saep.2003.127879
  • Cramer, E.R., Ålund, M., McFarlane, S.E., Johnsen, A. & Qvarnström, A. (2016). Females discriminate against heterospecific sperm in a natural hybrid zone. Evolution, 70, 1844-1855. DOI: 10.1111/evo.12986
  • Cramer, E.R., Rowe, M., Eroukhmanoff, F., Lifjeld, J.T., Sætre, G.P. & Johnsen, A. (2019). Measuring sperm swimming performance in birds: effects of dilution, suspension medium, mechanical agitation, and sperm number. Journal of Ornithology, 160, 1053-1063. DOI: 10.1007/s10336-019-01672-9
  • Dumpala, P.R., Parker, H.M. & McDaniel, C.D. (2006). The effect of semen storage temperature and diluent type on the sperm quality index of broiler breeder semen. International Journal of Poultry Science, 5, 838-845. DOI: 10.3923/ijps.2006.838.845
  • Donoghue, A.M. & Wishart, G.J. (2000). Storage of poultry semen. Animal Reproduction Science, 62, 213-232. DOI: 10.1016/S0378-4320(00)00160-3
  • Faul, F., Erdfelder, E., Lang, A.G. & Buchner, A. (2007). G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behavior Research Methods, 39, 175-191.
  • Fischer, D., Schneider, H., Meinecke-Tillmann, S., Wehrend, A. & Lierz, M. (2020). Semen analysis and successful artificial insemination in the St. Vincent amazon (Amazona guildingii). Theriogenology, 148, 132-139. DOI: 10.1016/j.theriogenology.2020.03.004
  • Gee, G.F. (1995). Artificial insemination and cryopreservation of semen from nondomestic birds. In: Proceeding of the 1st Symposium on the Artificial Insemination in Poultry, Savoy, P262- 280.
  • Gee, G.F., Bertschinger, H., Donoghue, A.M., Blanco, J. & Soley, J. (2004). Reproduction in nondomestic birds: physiology, semen collection, artificial insemination and cryopreservation. Avian and Poultry Biology Reviews, 15, 47-101. DOI: 10.3184/147020604783637435
  • Hall, C.A., Potvin D.A. & Conroy, G.C. (2023). A new candling procedure for thick and opaque eggs and its application to avian conservation management. Zoo Biology, 42, 296-307. DOI: 10.1002/zoo.21730
  • Hassan, K.H. (2009). Fertility of broiler males and females at various ages by using artificial insemination. Diyala Agricultural Sciences Journal, 1, 25-30.
  • Hudelson, K.S. (1996). A review of the mechanisms of avian reproduction and their clinical applications. In Seminars in Avian and Exotic Pet Medicine, 5, 189-198. DOI: 10.1016/S1055-937X(96)80027-2
  • Humann-Guilleminot, S., de Montaigu, C.T., Sire, J., Grünig, S., Gning, O., Glauser, G., Vallat A. & Helfenstein, F. (2019). A sublethal dose of the neonicotinoid insecticide acetamiprid reduces sperm density in a songbird, Environmental Research, 177, 108589. DOI: 10.1016/j.envres.2019.108589
  • Hobbs, R.J., Upton, R., Keogh, L., James, K., BaxterGilbert, J. & Whiting, M.J. (2021). Sperm cryopreservation in an Australian skink (Eulamprus quoyii). Reproduction, Fertility and Development, 34, 428-437. DOI: 10.1071/RD21031
  • Kleven, O., Fossøy, F., Laskemoen, T., Robertson, R.J., Rudolfsen, G. & Lifjeld, J.T. (2009). Comparative evidence for the evolution of sperm swimming speed by sperm competition and female sperm storage duration in passerine birds. Evolution, 63, 2466-2473. DOI: 10.1111/j.1558- 5646.2009.00725.x
  • Lara, C.E., Taylor, H.R., Holtmann, B., Johnson, S.L., Santos, E.S., Gemmell, N.J. & Nakagawa, S. (2020). Dunnock social status correlates with sperm speed, but fast sperm does not always equal high fitness. Journal of Evolutionary Biology, 33, 1139-1148. DOI: 10.1111/jeb.13655
  • Lüpold, S., Linz, G.M. & Birkhead, T.R. (2009). Sperm design and variation in the New World Blackbirds (Icteridae). Behav Ecol Sociobiol, 63, 899-909. DOI: 10.1007/s00265-009-0733-6
  • Madeddu, M., Marelli, S., Abdel Sayed, A., Mosca, F., Cerolini, S. & Zaniboni, L. (2022). Assessment of sperm viability and computer-assisted motility analysis in budgerigars (Melopsittacus undulatus): effect of several in vitro processing conditions. Veterinary Medicine International, 2022, 5997320. DOI:10.1155/2022/5997320
  • McDiarmid, C.S., Hurle, L.L., Le Mesurier, M., Blunsden, A.C. & Griffith, S. C. (2022). The impact of diet quality on the velocity, morphology and normality of sperm in the zebra finch Taeniopygia guttata. J Exp Biol., 225(9), jeb243715, DOI: 10.1242/jeb.243715
  • Mohan, J., Sharma, S.K., Kolluri, G., Tyagi, J.S. & Kataria, J.M. (2015). Selection of diluent for short term preservation of guinea fowl semen. Asian Journal of Animal and Veterinary Advances, 10, 360-364. DOI: 10.3923/ajava.2015.360.364
  • Mohan, J., Sharma, S.K., Kolluri, G. & Dhama, K. (2018). History of artificial insemination in poultry, its components and significance. World's Poultry Science Journal, 74, 475-488. DOI: 10.1017/S0043933918000430
  • Pearlin, V., Mohan, J., Tyagi, J.S., Gopi, M., Kolluri, G., Prabakar, G. & Shanmathy, M. (2021). Efficiency of different diluents and dilution rates on the fertilization potential of chicken spermatozoa. Indian Journal of Animal Research, 55, 139-144. DOI: 10.18805/IJAR.B-3940
  • Piaček, V., Zukal, J., Seidlova, V., Heger, T., Němcová, M., Přibyl, M., Vitula, F. & Pikula, J. (2020). Fresh semen characteristics in captive accipitrid and falconid birds of prey. Acta Veterinaria Brno, 89, 291-300. DOI: 10.2754/avb202089030291
  • Purohit, G.N., Duggal, G. P., Dadarwal, D., Kumar, D., Yadav, R.C. & Vyas, S. (2003). Reproductive biotechnologies for improvement of buffalo: The current status. Asian-Australasian Journal of Animal Sciences, 16, 1071-1086. DOI: 10.5713/ajas.2003.1071
  • Rakha, B.A., Ansari, M. S., Akhter, S., Hussain, I. & Blesbois, E. (2016). Cryopreservation of Indian red jungle fowl (Gallus gallus murghi) semen. Animal Reproduction Science, 174, 45-55. DOI: 10.1016/j.anireprosci.2016.09.004
  • Roiter, Y.S. & Konopleva, A.P. (2019). Universal biotechnological medium for sperm dilution during poultry artificial insemination. In: IOP Conference Series: Earth and Environmental Science, P 315. DOI: 10.1088/1755- 1315/315/4/042020
  • Schmoll, T. & Kleven, O. (2016). Functional infertility in a wild passerine bird. Ibis, 158, 670-673. DOI: 10.1111/ibi.12376
  • Shinde, A.S., Mohan, J., Sastry, K.V.H., Singh, R.P., Chouhan, L. & Tyagi, J.S. (2013). CARI diluent for short-term preservation of Kadaknath chicken semen. Indian Journal of Poultry Science, 48, 240-243.
  • Tabachnick, B.G., Fidell, L.S. & Ullman, J.B. (2019). Using Multivariate Statistics (Seventh Ed.). Pearson.
  • Trivedi, A.K., Rani, S. & Kumar, V. (2006). Control of annual reproductive cycle in the subtropical house sparrow (Passer domesticus): evidence for conservation of photoperiodic control mechanisms in birds. Frontiers in Zoology, 3, 1- 12. DOI: 10.1186/1742-9994-3-12
  • Whiteaker, M. (1979). Gloster Canary, AFA Watchbird, 6, 15-15.
  • Yang, Y., Zhang, Y., Ding, J., Ai, S., Guo, R., Bai, X. & Yang, W. (2019). Optimal analysis conditions for sperm motility parameters with a CASA system in a passerine bird, Passer montanus. Avian Research, 10, 1-10. DOI: 10.1186/s40657-019- 0174-5
  • Yenilmez, F. (2020). Canary Production. Turkish Journal of Agriculture-Food Science and Technology, 8, 941-944. DOI: 10.24925/turjaf.v8i4.941- 944.3197
  • Zambelli, D. & Cunto, M. (2022). Artificial insemination in queens in the clinical practice setting: protocols and challenges. Journal of Feline Medicine and Surgery, 24, 871-880. DOI: 10.1177/1098612X221118756.
Toplam 37 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Konular Veteriner Bilimleri (Diğer)
Bölüm Makaleler
Yazarlar

Arda Onur Özkök 0000-0001-9932-3608

Erken Görünüm Tarihi 19 Mart 2024
Yayımlanma Tarihi 31 Mart 2024
Gönderilme Tarihi 17 Ekim 2023
Kabul Tarihi 8 Şubat 2024
Yayımlandığı Sayı Yıl 2024 Cilt: 9 Sayı: 1

Kaynak Göster

APA Özkök, A. O. (2024). In-Vivo Effects of Canary (Serinus Canary) Semen Diluted with Different Semen Extenders on Eggs in Artificial Insemination. Journal of Anatolian Environmental and Animal Sciences, 9(1), 60-66. https://doi.org/10.35229/jaes.1377519


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