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Survival Rates of Rapidly Frozen Bovine Spermatozoa

1960; Elsevier BV; Volume: 43; Issue: 8 Linguagem: Inglês

10.3168/jds.s0022-0302(60)90294-0

1529-9066

James J. Kennelly, R.S. Hoyt, R.H. Foote, R.W. Bratton,

Reproductive biology and impacts on aquatic species

Subsamples of semen from each of two ejaculates from each of 12 bulls were extended with a yolk-citrate-glycerol extender, equilibrated 5 to 6 hr. at 5 ° C., and placed directly in a freezing bath at temperatures of +5, 0, --5, --10, --15, --20, --25, --30, --35, and --40 ° C. The freezing bath was cooled at the rate of 0.8 ° C. per minute from +5 to --15 ° C., at a rate of 5 ° C. per minute from --15 to --40 ° C., and at the rate of l0 ° C. per minute from --40 to --75 ° C. The percentage of motile spermatozoa in ampules subsequently stored in a mechanical freezer at --85 ° C. for 60 days were, in order of decreasing temperature of the freezing bath at the time of transfer, 29, 21, 24, 27, 33, 36, 35, 35, 35, and 35.From these data it is concluded that time can be saved and a higher percentage of motile spermatozoa recovered by transferring the semen abruptly from +5 ° C. to a freezing bath ranging from --20 to --40 ° C.Early work on the freezing of bull spermatozoa by Polge and coworkers (10,11,12) indicated that for maximum survival of sperm the rate of cooling between +5 and --15 ° C. should not exceed 2 ° C. per minute, but that the rate could be increased to 10 ° C. per minute from --15 to --79 ° C. Abrupt cooling from +5 to --79 ° C. resulted in poor survival.Other workers (3, 7, 8, 13) have obtained highest sperm survival by cooling at rates ranging from 1 to 4 ° C. per minute down to --79 ° C.This procedure is generally tedious, time-consuming, and not easily regulated manually.Luyet and Keane (6) froze spermatozoa to --195 ° C. in a two-step procedure.When the terminus of the first step was --20 ° C. nearly all the spermatozoa died, as contrasted with 75% survival when the terminus of the first step was --27 ° C. Bialy and Smith (1) reported that a higher percentage of spermatozoa survived when they were frozen to --79 ° C. in a two-step procedure, with the first terminus at --20 ° C. than when they were frozen by the slow cooling procedure used as the control.A two-step procedure, with the first terminus at --15 ° C., was no better than the control.Recent studies by Larson and Graham (5) and Polge ( 9) indicate that the terminus of the first step should be lower when storage is to be at --195 ° C. than when it is to be at --79 ° C. The results sug: gested, that once the critical ranges were better established, a faster, simpler, and less harmful freezing procedure could be developed.The studies reported here were undertaken to establish the optimum temperature of the first terminus in a two-step freezing procedure with subsequent storage at --85 ° C.EXPERIMENTAL PROCEDURE Design of experiment.Two ejaculates of semen were obtained from each of 12 bulls owned by the New York Artificial Breeders' Coop., Inc.Each of the 24 ejaculates was processed and frozen at ten different rates, and subsequently