Storing stallion sperm in SpermSafe™ at 17°C may improve fertility by reducing mPTP formation

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.