27 Cryopreservation of bovine semen using cell-permeating antioxidant and protein-free extender

Effective semen extenders are those with defined composition that promote sperm longevity. Generation of reactive oxygen species during semen cryopreservation results in sperm membrane lipid peroxidation and reduced longevity. Recent studies demonstrated that small cell permeating dimethyl tyrosine conjugated peptides, such as SS-31, translocate to the mitochondria and scavenge excess reactive oxygen species. The present study was conducted to test the hypothesis that the addition of SS-31 improves the post-thaw quality and fertility potential of bovine semen. The effect of SS-31 was tested in combination with two extenders: (1) conventional tris-egg yolk-glycerol (TEYG, control) and (2) cholesterol-cyclodextrin+tris-glycerol (CC+TG, a defined protein-free extender). Fifteen ejaculates were collected from 5 Black Angus bulls. Ejaculates were diluted to 400×106 spermmL−1 with tris-citric acid buffer, and treated with 0, 50, or 100 µmolmL−1 SS-31 for 15min at 32°C. Semen aliquots were diluted further with TEYG, 0.5mgmL−1 CC+TG, or 1mgmL−1 CC+TG extender to a final concentration of 50×106 spermmL−1. Semen was then cooled to 4°C and frozen in a programmable freezer. Post-thaw sperm motion parameters were evaluated at 0, 2, 4, 6 and 24h with CASA. Semen fertility was determined by fixed-time AI using Hereford-cross cows (n=100). Synchronized cows were inseminated once with semen extended in TEYG, 1mgmL−1 CC+TG, or 1mgmL−1 CC+TG + 100 µmolmL−1 SS-31. Pregnancy was diagnosed by ultrasonography at 27 days post-insemination. Post-thaw sperm motion parameters were compared by ANOVA for repeated-measures, and pregnancy rates were compared using binomial linear mixed-model ANOVA. No differences in sperm motion parameters were detected among SS-31 treatments within extenders. Semen extended in TEYG or 1mgmL−1 CC+TG had greater total and progressive motilities at 0 and 2h post-thaw than semen extended in 0.5mgmL−1 CC+TG (P<0.05). Pregnancy rates after fixed-time insemination did not differ among semen extender groups [TEYG: 15/25 (60%), 1mgmL−1 CC+TG: 19/34 (56%), 100 µmolmL−1 SS-31+1mgmL−1 CC+TG: 21/37 (57%)]. Addition of the cell permeating antioxidant SS-31 did not improve post-thaw semen quality or fertility. The CC+TG extender was as robust as conventional egg yolk extender in protecting bovine sperm during cryopreservation. This research was supported by NSERC Canada, the Government of Saskatchewan, and Agriculture and Agri-Food Canada.