179 A simplified insemination protocol for frozen-thawed stallion semen stored for 24 hours after thawing

Insemination of mares with frozen semen typically involves intensive reproductive management. Sieme et al. (2003 Theriogenology 60, 1153–1164) reported that a single frozen-thawed semen insemination of 800 million frozen-thawed spermatozoa within 12 h of ovulation produced pregnancy rates of 44.7%. Morris et al. (2003 Equine Vet. J. 35, 197–201) achieved high pregnancy rates of (64%–67%) when mares were inseminated either conventionally or hysteroscopically with 14 million frozen-thawed spermatozoa at a fixed time. When the insemination dose was 3 million frozen-thawed spermatozoa, hysteroscopic insemination was better than conventional insemination. New developments in liquid storage of fresh stallion spermatozoa at room temperature (Clulow and Gibb 2022 Anim. Reprod. Sci. 247, 107088) have improved the longevity of frozen-thawed spermatozoa. The objective of our study was to determine the fertility of thawed semen, stored for 6, 16, or 24 h in a modified SpermSafe™ diluent at 17°C when mares are inseminated at a fixed time after ovulation induction. The fertility of frozen-thawed semen from three commercial stallions was evaluated after insemination of 42 mares at 6 (n = 13), 16 (n = 16), and 24 (n = 13) hours after thawing. The insemination dose was prepared by thawing 3 × 0.5 mL straws of semen at 37°C and processed through a microfluidic device (VetMotl Inc., USA) to select a viable subpopulation of spermatozoa suspended in a modified SpermSafe™ longevity diluent and stored at 17°C for 6, 16, or 24 h. At 40 h after ovulation induction with BioRelease^® Deslorelin, the mares were inseminated using deep horn insemination to deliver the insemination dose which contained ~3 million total spermatozoa ipsilateral to the side of ovulation. Embryo recovery was performed 8 days after ovulation. Embryos were sexed by PCR. Statistical analysis was performed using General Linear Model followed by Bonferroni comparison in Minitab v20. There was a significant beneficial effect of processing the thawed semen with the microfluidic device on total sperm motility (P < 0.001). Sperm total and progressive motility were maintained at >55% for 24 h after thawing. There was no difference in sperm total motilty at 6, 16, and 24 h post-thaw. The overall embryo recovery rate was 52% (n = 42). The percentage of embryo recovery from the mares in each treatment group was as follows: 6 h (54%), 16 h (69%), and 24 h (31%). There was an effect of time of insemination on the sex ratio of the embryos (chi-square, d.f. 1, P = 0.042). This study confirms that storage of thawed stallion semen in modified SpermSafe™ at 17°C will produce satisfactory embryo recovery rates after low-dose, deep uterine insemination at 40 h after ovulation induction.

This research was funded by the New Zealand Equine Research Foundation.