Abstract
Stallions with sperm that do not tolerate cooling have limited commercial potential. Cooling increases sperm intracellular calcium (Ca2+i; White, Reproduction, Fertility and Development. 1993;5:639-58), which in human spermatozoa triggers formation of the mitochondrial permeability transition pore (mPTP), leading to apoptosis (Treulen et al., Human Reproduction. 2015;30:767-76). However, the mechanism of sperm mPTP formation is yet to be described in the horse. As such, this study investigated whether mPTP formation occurs in stallion spermatozoa following Ca2+i increase and whether storing spermatozoa at higher temperatures can minimize Ca2+i increase and mPTP formation, thereby improving fertility. This would be investigated by alternatively storing sperm at 17°C in SpermSafe™ (BREED Diagnostics), a medium designed for liquid-storage at higher temperatures. Experiment 1 utilised three ejaculates from each of three pony stallions (n=9) to investigate the relationship between Ca2+i and mPTP formation. Semen was collected using an artificial vagina (AV) and extended 2:1 (extender:semen) using EquiPlus (Minitube). Spermatozoa were isolated from seminal plasma and extender using single-layer colloidal centrifugation before being resuspended to 20 × 106/ml in Biggers, Whitten, and Whittingham medium. Spermatozoa were pre-loaded with calcein-AM (fluorescent mPTP probe) and exposed to various doses of ionomycin to induce the Ca2+i increase. Data were checked for normaldistribution (Shapiro-Wilk test), with oneway ANOVA (treatment effect) and Dunnets test (comparing treatments and the control) used to identify significant differences, and ‘stallion’ used as a blocking term. The mPTP formed (loss of calcein fluorescence) following ionomycin-induced Ca2+i increase at 5 nM (100±16.4 AFU vs. 44.3±8.3 AFU for control and 5 nM ionomycin, respectively; P≤0.001), indicating that stallion spermatozoa do undergo mPTP formation in response to elevated Ca2+i. Experiment 2 utilised a fertile (83% per-cycle conception rate [PCCR] with fresh semen; n=6), 5-year-old Warmblood stallion with a history of poor fertility using cooled semen. Semen was collected and extended as described above and was washed via cushioned centrifugation. Sperm pellets were resuspended to 50 × 106/ml in either EquiPlus (cooled in a standard commercial shipper) or in SpermSafe (stored at 17°C in an EquOcyte shipper) for 24 h. Due to the logistical constraints of commercial practice, split ejaculates could not be utilized, so each sample was a separate ejaculate. Fixed-time AI was performed 24 h following semen collection and ovulation induction. Pregnancy rates of 0% (0/6) and 67% (8/12) were achieved following AI with cooled vs. SpermSafe-stored spermatozoa, respectively (Chi Square; P≤0.01). In conclusion, these data suggest that storing stallion sperm at 17°C in SpermSafe before AI may improve fertility in stallions with poor cooling tolerance, potentially due to reduced mPTP formation.
Original language | English |
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Number of pages | 1 |
DOIs | |
Publication status | Published - 2023 |
Event | The International Symposium on Equine Reproduction 2023: ISER XIII - Paraná, Brazil Duration: 09 Jul 2023 → 12 Jul 2023 https://www.iserxiii-brazil.com/ https://www.iserxiii-brazil.com/wp-content/uploads/2023/06/ISERXIII-Oral-and-Poster-Programmes-Handbook.pdf (Program and handbook) |
Conference
Conference | The International Symposium on Equine Reproduction 2023 |
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Country/Territory | Brazil |
City | Paraná |
Period | 09/07/23 → 12/07/23 |
Other | It has been over 29 years since the International Symposium on Equine Reproduction (ISER) was held in South America, in Caxambu, Brazil. In 2023, ISER returns to a continent and country which not only has the fourth largest equine herd in the world, with 5.5. million horses, but has a long standing history involving the breeding of equids. Horses, donkeys or mules are used for sport, leisure and work activities. Brazil and Argentina are also at the forefront of equine reproduction research, particularly assisted reproductive technologies such as embryo transfer, cloning, intracytoplasmic sperm injection and semen sexing. People in this vast continent are passionate about their horses and also know how to party, with music, dancing and delicious food all part of their unique culture. |
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