Successful pregnancy using stallion semen stored at 17ºC for 6 days
Keywords:
Dilution, extender, stallion, semen, pregnancy
Abstract
Aim of this study was to assess the effectiveness of Beyond® in maintaining sperm motility at 17ºC with ‘poor cooler’ stallion semen and achieving pregnancy after artificial insemination. An ejaculate was diluted in EquiPlus (Minitube) and divided into 2 aliquots. One was kept at 5ºC (control sample), whereas the other was centrifuged, further diluted in Beyond® and kept at 17°C in a semen cooler (Minitube) for 10 days. Total motility (TM) and membrane integrity were assessed daily. For the control sample, TM was 65 and 35% after 24 hours and 48 hours, respectively, whereas the sample diluted with Beyond® still had a TM of 40% after 10 days. Six days after storage at 17°C, a mare was inseminated; a healthy colt was born 344 days after ovulation. We are reporting the first successful pregnancy after storing semen from a poor cooler stallion for 6 days at 17°C.
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References
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27. Graham JK: Principles of cooled semen. In: McKinnon AO, Squires EL, Vaala et al: editors. Equine Reproduction. 2nd edition, Ames; Wiley: 2011. p. 1308–1315.
28. Cuervo-Arango J, Nivola K, Vaihkonen L, et al: The effect of storage temperature of stallion semen on pregnancy rates. J Equine Vet Sci 2015;35:611–616. doi: 10.1016/j.jevs.2015.06.003
2. Kowalczyk A, Czerniawska-Piątkowska E, Kuczaj M: Factors influencing the popularity of artificial insemination of mares in Europe. Animals 2019;9:460. doi: 10.3390/ani9070460
3. Price S, Aurich J, Davies-Morel M, et al: Effects of oxygen exposure and gentamicin on stallion semen stored at 5 and 15 degrees. Reprod Domest Anim 2008;43:261–266. doi: 10.1111/j.1439-0531.2007.00888.x
4. Trentin JM, Rodrigues MF, Pessoa GA, et al: Viability of pony stallion semen in different temperature and dilution. Acta Sci Vet 2017;45:1482.
5. Varner DD, Blanchard TL, Love CL, et al: Effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters. Theriogenology 1987;28:709–723.doi: 10.1016/0093-691X(87)90288-3
6. Clulow J, Gibb Z: Liquid storage of stallion spermatozoa – Past, present and future. Anim Reprod Sci 2022;24:107088. doi: 10.1016/j.anireprosci.2022.107088
7. Gibb Z, Clulow JR, Aitken RJ, et al: First publication to describe a protocol for the liquid storage of stallion spermatozoa for 7 days. J Equine Vet Sci 2018;66:37–40. doi: 10.1016/j.jevs.2018.05.016
8. Menezes Nunes M, Munhoz ML, Gallelli MF, et al: Case report: adjusting semen freezing and insemination protocols to achieve pregnancies from an Arabian horse stallion with poor frozen sperm quality. Anim Reprod 2024;21:39.
9. Morris L, Harteveld R, Gibb Z: A simplified fixed-time insemination protocol using frozen-thawed stallion spermatozoa stored at 17°C for up to 24 h before insemination. Equine Vet J 2024;56:688–696. doi: 10.1111/evj.14096
10. Varner D, Love C, Blanchard T, et al: Breeding-management strategies and semen-handling techniques for stallions-case scenarios. Proc Am Assoc Equine Pract 2010; p. 215–226.
11. Gibb Z, Aitken RJ: Recent developments in stallion semen preservation. Equine Vet Sci 2016;43:29–36. doi: 10.1016/j.jevs.2016.06.006
12. Bedford-Guaus S: Transported stallion semen and breeding mares with cooled or frozen-thawed semen. Clin Tech Eq Pract 2007;6:239–248. doi: 10.1053/j.ctep.2007.09.003
13. Newcombe JR, Cuervo-Arango J: The effect of time of insemination with fresh cooled transported semen and natural mating relative to ovulation on pregnancy and embryo loss rates in the mare. Reprod Domest Anim 2011;46:678–681. doi: 10.1111/j.1439-0531.2010.01728.x
14. Brinsko SP, Crockett EC, Squires EL: Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage. Theriogenology 2000;54:129–136. doi: 10.1016/S0093-691X(00)00331-9
15. Segabinazzi L, Carmo MT, Freitas-Dell’Aqua CP, et al: Post-cooling semen processing and sperm re-suspension as an alternative method to circumvent poor semen cooling in stallions. Equine Vet J 2024;56:697–710. doi: 10.1111/evj.14085
16. Cecere JT: Eosin-nigrosin staining in the evaluation of sperm. In: Dascanio J, McCue P: editors. Equine Reproductive Procedures. 2nd edition, Ames; Wiley: 2021. p. 373–376. doi: 10.1002/9781119556015.ch132
17. Samper JC, Plough T: Techniques for the insemination of low doses of stallion sperm. Reprod Domest Anim 2010;45:35–39. doi: 10.1016/j.theriogenology.2005.05.012
18. Troedsson MHT: Uterine clearance and resistance to persistent endometritis in the mare. Theriogenology 1999;52:461–471. doi: 10.1016/S0093-691X(99)00143-0
19. McCue P: Ultrasound examination of the pregnant mare. In: Dascanio J, McCue P: editors. Equine Reproductive Procedures. 2nd edition, Ames; Wiley: 2021. p. 257–262.
20. Curran S: Fetal sex determination in cattle and horses by ultrasonography. Theriogenology 1992;37:17–21. doi: 10.1016/0093-691X(92)90244-L
21. LeFrapper L, Walston BS, Whisnant CS: Comparison of various extenders for storage of cooled stallion spermatozoa for 72 hours. J Equine Vet Sci 2010;35:200–204. doi: 10.1111/j.1439-0531.2006.00828.x
22. Novello G, Podico G, Segabinazzi LGTM, et al: Stallion semen cooling using native phosphocaseinate-based extender and sodium caseinate cholesterol-loaded cyclodextrin-based extender. J Equine Vet Sci 2020;92:103104. doi: 10.1016/j.jevs.2020.103104
23. Papin J, Stuhtmann G, Martinsson G, et al: Stored stallion sperm quality depends on sperm preparation method in INRA82 or INRA96. J. Equine Vet Sci 2021;98:103367. doi: 10.1016/j.jevs.2020.103367
24. Rečková Z, Filipčík R, Soušková K, et al: The efficiency of different types of extenders for semen cooling in stallions. Anim Biosci 2022;35:670–676. doi: 10.5713/ab.21.0300
25. Prell MJ, McCue PM, Moffett PD, et al: Motility and fertility evaluation of thawed frozen stallion semen after 24 hours of cooled storage. J Equine Vet Sci 2020;90:102983. doi: 10.1016/j.jevs.2020.102983
26. Gibb Z, Aitken RJ: The impact of sperm metabolism during in vitro storage: the stallion as a model. BioMed Res Int 2016;2016:9380609. doi: 10.1155/2016/9380609
27. Graham JK: Principles of cooled semen. In: McKinnon AO, Squires EL, Vaala et al: editors. Equine Reproduction. 2nd edition, Ames; Wiley: 2011. p. 1308–1315.
28. Cuervo-Arango J, Nivola K, Vaihkonen L, et al: The effect of storage temperature of stallion semen on pregnancy rates. J Equine Vet Sci 2015;35:611–616. doi: 10.1016/j.jevs.2015.06.003
Published
2025-11-28
How to Cite
Munhoz , M., Nunes , M., Gallelli , M., Carneiro , G., & Miragaya , M. (2025). Successful pregnancy using stallion semen stored at 17ºC for 6 days. Clinical Theriogenology, 17. https://doi.org/10.58292/CT.v17.13213
Section
Technique Reports
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