A study was made of three methods of washing bull, boar and ram spermatozoa, using as criteria contamination with free-lying cytoplasmic droplets, sperm motility and leakage of glucose-phosphate isomerase and hexokinase into the medium. The methods used were: washing in a Ringer-fructose-phosphate medium, washing in a hypoosmotic phosphate medium and washing with balanced media in the cold after slow cooling and storage overnight at 4° C in a milk diluent. Only by using the last method was it possible to remove adequately the freelying cytoplasmic droplets ; this method also caused least damage to the sperm suspensions.
The problems of washing spermatozoa with respect to cell damage and leakage of enzymes are discussed in the light of the present findings. The evidence suggests that glucose-phosphate isomerase and hexokinase leak from the cytoplasmic droplets as well as from the spermatozoa during washing. Hypo-osmotic media appear to cause an immediate and marked release of these enzymes from the droplets.
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