EFFECT OF CRYOPRESERVATION PROTOCOLS ON THE SPERM KINEMATICS AND VITAL SEMEN ATTRIBUTES IN FROZEN-THAWED MARWARI STALLION SPERM
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Authors
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Thirumala Talluri Rao
ICAR-NRC Equine, Hisar
Keywords:
Marwari stallion, Cryopreservation, automated controlled rate freezing, vapour freezing; Semen qualityAbstract
Studies involving the effects of various procedures and techniques of cryopreservation and their impact on conception rates in horse breeding programs have been previously reported in the literature. The current study aims to compare the efficiency of the automated system (controlled-rate freezer) and the conventional vapour freezing system (manual system) for freezing the stallion semen after cooling at 4 °C and to evaluate the efficiency by studying their post-thaw sperm kinematics and sperm characteristics. Sixty-four ejaculates from eight adult fertile stallions were used in this study. Ejaculates from each stallion were collected twice a week for four weeks. Each ejaculate was extended with Lactose-Glucose-DMF extender and the samples were then packed into 0.5 ml straws, which were divided into two parts: one for cryopreservation in a controlled-rate automated freezer and the other for a conventional vapour freezing system. Semen samples were evaluated for sperm motility, liveability, acrosome integrity, plasma-membrane integrity and mitochondrial membrane potential at fresh, equilibrated and frozen-thawed stages of cryopreservation. Though there was non-significant difference in post-thaw total sperm motility and plasma membrane integrity, however, viability, acrosome integrity and mitochondrial membrane integrity of the stallion spermatozoa were significantly (P<0.05) higher in the automated controlled freezing method in comparison to the conventional vapour freezing system. The kinematic parameters were also recorded be significantly (P<0.05) higher in the automated in comparison to the manual system of freezing. In conclusion, the conventional vapour and automated freezing methods allow significant differences in results for sperm kinematic and vital sperm parameters. The conventional vapour freezing system allows cost reduction with the cryopreservation procedure and is easy to handle and transport, making it suitable for use in breeding projects.
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References
1. Al-Essawe E M, Johannisson A, Wulf M, Aurich C and Morrell J M. 2018. Improved cryosurvival of stallion spermatozoa after colloid centrifugation is independent of the addition of seminal plasma. Cryobiology 81: 145-52.
2. Allai L, Benmoula A, da Silva M M, Nasser B and El Amiri B. 2018. Supplementation of ram semen extender to improve seminal quality and fertility rate. Animal Reproduction Science 192: 6-17.
3. Arangasamy A, Legha R, Yash Pal Y P, Bansal R, Singh J and Sharma R. 2008. Conception rate of Marwari stallions semen cryopreserved by custom freezing.
4. Ashrafi I, Kohram H, Naijian H, Bahreini M and Mirzakhani H. 2011. Effect of controlled and uncontrolled cooling rate on motility parameters of cryopreserved ram spermatozoa. BMC Research Notes 4: 1-6.
5. Binyameen M, Saleem M and Riaz A. 2019. Recent trends in bovine reproductive biotechnologies. CABI Reviews(2019): 1-10.
6. Bustamante-Filho I, Pederzolli C, Sgaravatti A, Mattos R, Dutra-Filho C and Jobim M. 2006. Activity of glutathione peroxidase and catalase in stallion semen during cryopreservation. In Animal Reproduction Science, pp 70-73: ELSEVIER RADARWEG 29, 1043 NX AMSTERDAM, NETHERLANDS.
7. Clulow J, Mansfield L, Morris L, Evans G and Maxwell W. 2008. A comparison between freezing methods for the cryopreservation of stallion spermatozoa. Animal Reproduction Science 108(3-4): 298-308.
8. Forero‐Gonzalez R A, Celeghini E C C, Raphael C, Andrade A F C, Bressan F F and Arruda R P d. 2012. Effects of bovine sperm cryopreservation using different freezing techniques and cryoprotective agents on plasma, acrosomal and mitochondrial membranes. Andrologia 44: 154-59.
9. Gil L, Galindo-Cardiel I, Malo C, González N and Álvarez C. 2013. Effect of Cholesterol and Equex‐STM Addition to an Egg Yolk Extender on Pure Spanish Stallion Cryopreserved Sperm. International Scholarly Research Notices 2013(1): 280143.
10. Heckenbichler S, Deichsel K, Peters P and Aurich C. 2011. Quality and fertility of cooled-shipped stallion semen at the time of insemination. Theriogenology 75(5): 849-56.
11. Henry M, Snoeck P and Cottorello A. 2002. Post-thaw spermatozoa plasma membrane integrity and motility of stallion semen frozen with different cryoprotectants.
12. Jhamb D, Talluri T R, Sharma S, Juneja R, Nirwan S S, Yadav D, Pargi K K, Tanwar A, Kumar P and Kumar R. 2023. Freezability and fertility rates of stallion semen supplemented with Trehalose in lactose extender. Journal of Equine Veterinary Science 126: 104293.
13. Johnson L, Weitze K, Fiser P and Maxwell W. 2000. Storage of boar semen. Animal Reproduction Science 62(1-3): 143-72.
14. Kaeoket K, Tantiparinyakul K, Kladkaew W, Chanapiwat P and Techakumphu M. 2008. Effect of different antioxidants on quality of cryopreserved boar semen in different breeds. Thai J Agri Sci 41(1-2): 1-9.
15. Kumar S, Millar J and Watson P. 2003. The effect of cooling rate on the survival of cryopreserved bull, ram, and boar spermatozoa: a comparison of two controlled-rate cooling machines. Cryobiology 46(3): 246-53.
16. Loomis P. 1993. Factors affecting the success of artificial insemination with cooled, transported semen.
17. Loomis P and Graham J. 2008. Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Animal Reproduction Science 105(1-2): 119-28.
18. Loomis P R. 2006. Advanced methods for handling and preparation of stallion semen. Veterinary Clinics: Equine Practice 22(3): 663-76.
19. Maziero R R D, Guasti P N, Monteiro G A, Avanzi B R, Hartwig F P, Lisboa F P, Martin I and Papa F O. 2013. Evaluation of sperm kinetics and plasma membrane integrity of frozen equine semen in different storage volumes and freezing conditions. Journal of Equine Veterinary Science 33(3): 165-68.
20. Morel M C D. 2015. Equine reproductive physiology, breeding and stud management: CABI Wallingford UK.
21. Morel M C D. 2020. Equine reproductive physiology, breeding and stud management: Cabi.
22. Nascimento J, Raphael C F, Andrade A F C d, Alonso M, Celeghini E C C and Arruda R P d. 2008. Effects of sperm concentration and straw volume on motion characteristics and plasma, acrosomal, and mitochondrial membranes of equine cryopreserved spermatozoa. Journal of Equine Veterinary Science 28(6): 351-58.
23. Oliveira R, Duarte F and Gambarini M. 2018. Comparison between the conventional and automated systems for frozen cooled equine semen. Animal Reproduction (AR) 12(2): 324-27.
24. Pal Y, Arangasamy A, Legha R, Singh J, Bansal R, Khurana S and Tandon S. 2011. Freezability and fertility of Marwari stallion semen. Indian Journal of Animal Sciences 81(5): 445.
25. Pal Y and Legha R. 2009. Seminal characteristics of Marwari stallions. Indian Veterinary Journal (India) 86(9).
26. Pal Y, Legha R and Tandon S. 2009. Comparative assessment of seminal characteristics of horse and donkey stallions. Indian Journal of Animal Sciences 79(10): 1028.
27. Pickett B and Shiner K. 1994. Recent developments in artificial insemination in horses. Livestock Production Science 40(1): 31-36.
28. Pukazhenthi B S, Johnson A, Guthrie H D, Songsasen N, Padilla L R, Wolfe B A, da Silva M C, Alvarenga M A and Wildt D E. 2014. Improved sperm cryosurvival in diluents containing amides versus glycerol in the Przewalski’s horse (Equus ferus przewalskii). Cryobiology 68(2): 205-14.
29. Raval K, Paul N, Nag P, Elango K, Pal Y, Legha R, Talluri T and Kumaresan A. 2022. Asthenozoospermic stallions tend to have high acrosome reacted spermatozoa as evidenced by dual fluorescent staining assay.
30. Ricketts S. 1993. Evaluation of stallion semen. Equine Veterinary Education 5(5): 232-37.
31. Roca J, Hernández M, Carvajal G, Vázquez J and Martinez E. 2006. Factors influencing boar sperm cryosurvival. Journal of Animal Science 84(10): 2692-99.
32. Samper J. 2001. Management and fertility of mares bred with frozen semen. Animal Reproduction Science 68(3-4): 219-28.
33. Samper J C. 2008. Equine breeding management and artificial insemination: Elsevier Health Sciences.
34. Sancho S, Casas I, Ekwall H, Saravia F, Rodriguez-Martinez H, Rodriguez-Gil J, Flores E, Pinart E, Briz M and Garcia-Gil N. 2007. Effects of cryopreservation on semen quality and the expression of sperm membrane hexose transporters in the spermatozoa of Iberian pigs. Reproduction 134(1): 111-21.
35. Soni Y, Talluri T, Kumar A, Ravi S, Mehta J and Tripathi B. 2019. Effects of different concentration and combinations of cryoprotectants on sperm quality, functional integrity in three Indian horse breeds. Cryobiology 86: 52-57.
36. Talluri T, Bhardwaj A, Mehta S and Pal Y. 2020. Efficacy of evaluation of mitochondrial membrane potential in equine spermatozoa using JC-1. The Indian Journal of Animal Sciences 90(12): 1599-601.
37. Talluri T, Jhamb D, Paul N, Mehta S, Singh J, Dedar R, Legha R and Pal Y. 2023. Cryopreservation and freezability of epididymal and ejaculated stallion spermatozoa.
38. Talluri T R, Arangasamy A, Ravi S K and Pal Y. 2012. Hypo-osmotic swelling test for quality evaluation of fresh and frozen semen quality in horses. Indian Vet. J 89(11): 68-70.
39. Talluri T R, Kumar S, Dedar R K, Mehta S and Bhattacharya T K. 2025. Raj-Sheetal: India's first horse foal born through vitrified embryo transfer. Current Science (00113891) 128(3).
40. Talluri T R, Kumar S, Pal Y, Legha R A, Dedar R K and Bhattacharya T K. 2024a. Raj-Prathama: India's first Marwari filly born through embryo transfer technology. Current Science (00113891) 126(3).
41. Talluri T R, Kumaresan A, Paul N, Elango K, Raval K, Nag P, Legha R A and Pal Y. 2024b. Heterologous seminal plasma reduces the intracellular calcium and sperm viability of cryopreserved stallion spermatozoa. Biopreservation and Biobanking 22(1): 82-87.
42. Talluri T R, Mal G and Ravi S K. 2017. Biochemical components of seminal plasma and their correlation to the fresh seminal characteristics in Marwari stallions and Poitou jacks. Veterinary World 10(2): 214.
43. Talluri T R, Ravi S, Singh J, Legha R, Pal Y, Gupta A, Singh R and Tripathi B. 2016. Some reproductive parameters of Zanskari mares reared in an organized farm under tropical climate. Ind J Anim Sci 86: 163-67.
44. Tejpal K, Mehta J, Ravi S, Talluri T, Kumar A, Kumar A and Yogesh S. 2016. Cryosurvival of Marwari stallion sperm in different extenders. Indian Journal of Animal Sciences 86(12): 1396-400.
45. Tejpal T, Mehta J, Ravi S, Swati Ruhil S R, Talluri T, Ashok Kumar A K and Diler Singh D S. 2017. Study of certain physical parameters of fresh semen in Marwari horse.
46. Vishwanath R and Shannon P. 2000. Storage of bovine semen in liquid and frozen state. Animal Reproduction Science 62(1-3): 23-53.
47. Voss J, Pickett B and Squires E. 1981. Stallion spermatozoal morphology and motility and their relationship to fertility. Journal of the American Veterinary Medical Association 178(3): 287-89.
48. Watson P F. 2000. The causes of reduced fertility with cryopreserved semen. Animal Reproduction Science 60: 481-92.
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