Variation in sperm cryosurvival is not modified by replacing the cryoprotectant glycerol with ethylene glycol in bulls Short running title: Cryoprotectants fail to modify sperm cryosurvival differences

Abstract

Breed and sire differences in sperm cryosurvival have been noted, with negative implications for sperm cryobanking and assisted reproduction programs. This study hypothesised that these differences could be modified by using lower molecular weight cryoprotectants. Therefore, the effect of replacing glycerol (GLY) with ethylene glycol (EG) on differential cryosurvival of semen from two Sanga cattle breeds (Mashona vs. Tuli) was determined. Three to five ejaculates were collected from each of ten bulls (3-8 years) by electro-ejaculation, diluted in three Tris-egg yolk extenders (Triladyl®, 7% GLY-based and 7% EG-based) and evaluated for sperm motility, viability and morphology at three time periods (fresh – 0 h, pre-freeze – 4 h and post-thaw). Tuli bulls produced larger (11.8±0.31 ml vs. 8.5±0.38 ml) and more concentrated ejaculates of lower fresh semen quality. Breeds differed across time for motility and morphology, but not viability. Mashona bull semen had significantly higher motility and normal morphology values at each sampling time. Bulls classified as poor freezers had lower concentration (0.70±0.09 × 109 sperm/ml vs. 1.37±0.10 × 109 sperm/ml), sperm motility index (SMI, 35.0±3.4 % vs. 67.8±2.1 %) and viability (69.7±1.1 % vs. 75.7±1.0 %) compared to good freezers. Maintenance of semen quality by GLY and EG did not differ between breeds, poor and good freezers, or age groups. The interaction breed by extender across time did not reach statistical significance for all variables. The study revealed that bull and breed variation in sperm quality and cryosurvival is not modified by replacing GLY with EG, suggesting that cryostress tolerance of sperm may be under control of mechanisms other than differential response to GLY cytotoxicity.