FACTORS AFFECTING MOTION CHARACTERISTICS OF FROZEN-THAWED STALLION SPERMATOZOA

Five experiments were conducted to evaluate damage incurred in each pr ocessing step for cryopreservation of stallion spermatozoa, In Experim ent 1, semen was centrifuged for 9 centrifugation times and the percen tage of spermatozoa recovered after each treatment was calculated and spermatozoal motion characteristics analysed, Recovery of spermatozoa was greater than or equal to 80% when spermatozoa were centrifuged for greater than or equal to 10 min, Experiment 2 evaluated spermatozoa c ryopreserved at 5 different concentrations in each of 2 extenders (ski m milk-egg yolk-glycerol, SM-EYG; and lactose-EDTA, LAG), In SM-EYG, T MOT 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 sam ples frozen at greater than or equal to 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%, r espectively, P<0.05), The effect of freezing rate on motion characteri stics of spermatozoa was evaluated in Experiment 3. The VCL of spermat ozoa frozen in SM-EYG was the only parameter affected by freezing rate (P<0.05). Experiment 4 evaluated motion characteristics after cryopre servation of spermatozoa in different sized straws (0.5 or 2.5 ml) in each of 2 extenders (SM-EYG and LAG), In SM-EYG, PMOT (38%) and VCL (1 09 mu m/s) were highest when spermatozoa were frozen in 0.5 ml straws (P<0.05). In Experiment 5, spermatozoa thawed immediately after cryopr eservation or thawed after storage in liquid nitrogen for 24-48 h were evaluated, There was no effect of length of storage in liquid nitroge n on spermatozoal motion characteristics (P<0.05). Experiment d evalua ted the effects of cooling time to 5 degrees C (0, 2.5 and 5 h) on mot ion characteristics of spermatozoa cryopreserved in 2 extenders (SM-EY G and LAG), 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 in itiation of freezing than if freezing was initiated at 20 degrees C (P <0.05). A suggested protocol for cryopreservation of stallion spermato zoa would include: 1) centrifugation at 400 g for 14 to 16 min; 2) ext ension at 23 degrees C with SM-EYG to 400 x 10(6) spermatozoa/ml; 3) c ool 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.