Factors affecting motion characteristics of frozen-thawed stallion spermatozoa

Abstract

Five experiments were conducted to evaluate damage incurred in each processing step for cryopreservation of stallion spermatozoa. In Experiment 1, semen was centrifuged for 9 centrifugation times and the percentage of spermatozoa recovered after each treatment was calculated and spermatozoal motion characteristics analysed. Recovery of spermatozoa was > or = 80% when spermatozoa were centrifuged for > or = 10 min. Experiment 2 evaluated spermatozoa cryopreserved at 5 different concentrations in each of 2 extenders (skim milk-egg yolk-glycerol, SM-EYG; and lactose-EDTA, LAC). In SM-EYG, TMOT and PMOT were higher at spermatozoal concentrations of 20, 200 and 400 x 10(6)/ml (51%/41%, 52%/44%, 50%/43%, respectively) than for samples frozen at > or = 800 x 10(6) spermatozoa/ml (41%/35%, 32%/27%; P < 0.05). Spermatozoa frozen in LAC at a concentration of 20 x 10(6)/ml resulted in the highest TMOT and PMOT (43% and 30%, respectively, P < 0.05). The effect of freezing rate on motion characteristics of spermatozoa was evaluated in Experiment 3. The VCL of spermatozoa frozen in SM-EYG was the only parameter affected by freezing rate (P < 0.05). Experiment 4 evaluated motion characteristics after cryopreservation of spermatozoa in different sized straws (0.5 or 2.5 ml) in each of 2 extenders (SM-EYG and LAC). In SM-EYG, PMOT (38%) and VCL (109 microns/s) were highest when spermatozoa were frozen in 0.5 ml straws (P < 0.05). In Experiment 5, spermatozoa thawed immediately after cryopreservation or thawed after storage in liquid nitrogen for 24-48 h were evaluated. There was no effect of length of storage in liquid nitrogen on spermatozoal motion characteristics (P < 0.05). Experiment 6 evaluated the effects of cooling time to 5 degrees C (0, 2.5 and 5 h) on motion characteristics of spermatozoa cryopreserved in 2 extenders (SM-EYG and LAC). TMOT and PMOT were effected by cooling time, and there was a cooling-time-by-extender interaction (P < 0.05). In SM-EYG, TMOT and PMOT were higher if spermatozoa were cooled to 5 degrees C prior to initiation of freezing than if freezing was initiated at 20 degrees C (P < 0.05). A suggested protocol for cryopreservation of stallion spermatozoa would include: 1) centrifugation at 400 g for 14 to 16 min; 2) extension at 23 degrees C with SM-EYG to 400 x 10(6) spermatozoa/ml; 3) cool to 5 degrees C for 2.5 h; 4) package in 0.5 ml straws at 5 degrees C; 5) freeze in liquid nitrogen vapour at -160 degrees C; and 6) thaw for 30 s in 37 degrees C water.