The inner perivitelline layer, separated from laid chicken eggs, was investigated as readily available material for studying the spermatozoa–egg interaction in vitro. This layer was found to have a similar response to hydrolysis by spermatozoa as the inner perivitelline layer from ovulated and follicular ova, in terms of the numbers of points of hydrolysis made by spermatozoa during a 5 min incubation at 40°C. Initiation of hydrolysis of the inner perivitelline layer was found to occur within 2.5 min, after which the size, but not the number of holes, increased with time. The frequency of the points of hydrolysis per unit area of the inner perivitelline layer was positively correlated with the concentration of spermatozoa in the incubation medium. The perivitelline hydrolysis assay was able to detect more damaged spermatozoa in samples that had been either stored at 5°C or cryopreserved in liquid N2 than did other tests of sperm quality, which are known to overestimate the fertilizing ability of stored avian semen.
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