DEEP-FREEZING RAM SPERMATOZOA: THE EFFECTS OF MILK, YOLK-CITRATE AND SYNTHETIC DILUENTS CONTAINING SUGAR

in Reproduction

Summary.

In a factorial experiment skim milk, egg-yolk-citrate and synthetic diluents composed of fructose or lactose, sodium chloride and phosphate buffer containing 3·0% w/v of a lyophilized preparation of non-dialysable solids from (cow) milk were used as diluents for deepfreezing ram spermatozoa and for incubating spermatozoa at 37° C after thawing. All samples of semen were diluted forty-fold before freezing. Egg-yolk-citrate was inferior to skim milk as a diluent for freezing spermatozoa and for incubating spermatozoa after thawing. Of the two synthetic diluents, lactose synthetic was better for freezing spermatozoa whilst fructose synthetic was better for incubating spermatozoa after thawing. Only spermatozoa frozen in the lactose synthetic diluent and resuspended after thawing in the fructose synthetic survived incubation at 37° C for 2 hr as well as spermatozoa frozen and incubated in skim milk diluents.

Two factorial experiments compared egg-yolk-citrate and skim milk as diluents for freezing semen diluted from ten- to forty-fold. At ten-fold dilution, revival was much the same after freezing in milk or egg-yolk-citrate. A twenty- or forty-fold dilution was better than a tenfold dilution in milk, but revival was depressed at these higher dilution rates after freezing in egg-yolk-citrate. When semen was frozen at a tenfold dilution it was advantageous to resuspend spermatozoa in a diluent free of glycerol after thawing. A diluent based on Krebs—Henseleit— Ringer solution containing 0·5% w/v of non-dialysable milk solids was better for incubating spermatozoa after thawing than egg-yolk-citrate or milk.

A period of 5 hr equilibration at 5° C before freezing was better than 30 min equilibration.

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