Effects of egg yolk level, penetrating cryoprotectant, and pre-freeze cooling rate, on the post-thaw quality of stallion sperm

The current study determined the effect of the egg-yolk (phospholipid source) level (egg yolk [20% EY] vs. skim-milk + egg yolk [SM + 2% EY]), cryoprotectant (glycerol [Gly] vs. glycerol + methylformamide [Gly + MF]), and pre-freeze cooling rate (-0.1 vs. -1 vs. -5 °C/min) on post-thaw stallion sperm quality. In Experiment 1, ejaculates (n = 27) from 9 stallions (3 ejaculates each) with varied sperm quality (High, Average, or Low) were frozen in EY-Gly, SMEY-Gly, EY-Gly + MF, or SMEY-Gly + MF extenders. Sperm in each group were cooled from 22° to 5°C using either -0.1 °C/min or -1 °C/min linear cooling rates prior to freezing. In Experiment 2, ejaculates (n = 24) from 12 stallions (2 ejaculates each) with High or Average sperm quality were frozen in EY-Gly, EY-Gly + MF, or in BotuCrio (BC) extenders. Sperm in each group were cooled from 22° to 5°C using either -1 or -5 °C/min linear cooling rates prior to freezing. In Experiment 1, for stallions with High or Average sperm quality, either cooling rate generally resulted in lower sperm quality for the SMEY-based extenders than for the EY-based extenders (P 0.05). Stallions with Low sperm quality were unaffected by any experimental treatment (P 0.05). In Experiment 2, a -5 °C/min cooling rate yielded lower sperm quality in BC than in EY-Gly or EY-Gly + MF groups (P 0.05); however, a -1 °C/min cooling rate yielded similar sperm quality among these treatments (P 0.05). In summary, the phospholipid level in the freezing extender and the pre-freeze cooling rate, but not the penetrating cryoprotectant, affected the post-thaw quality of stallion sperm.