Aim: To investigate the effect of cryopreservation on the plasma membrane integrity in the head and tail regions of individual sperm, and the relationship between intact cryopreserved sperm and its motility and zona-free hamster oocyte penetration rate. Methods: The eosin Y exclusion and the hypoosmotic swelling tests were combined to form a single test (HOS-EY test) to identify the spermatozoa with four types of membrane integrity. Results: After cryopreservation, there was a marked decline in the percentage of spermatozoa with Type IV membrane integrity (head membrane intact/tail membrane intact), and a significant increase in those with Type I (head membrane damaged/tail membrane damaged) and Type III (head membrane damaged/tail membrane intact) membrane integrity (n=50, P<0.01). The value of Type III integrity had a wide range of variability, whereas Type II (head membrane intact/tail membrane damaged) was uncommon after thawing. A high correlation was observed between the percentage of Type IV integrity and sperm motility (n=50, r=0.74, P<0.01). However, the values of Type IV integrity were usually lower than those of post-thaw motility in most cryopreserved samples. The value of Type IV integrity did not correlate with the sperm penetration rate (n=25, r=0.22, P>0.05). Conclusion: (1) The HOS-EY test has the advantage of showing four patterns of membrane integrity in individual spermatozoon; (2) Cryopreservation causes a significant membrane rupture in the head and tail regions of spermatozoa; Type III is the main transitional state of membrane cryodamage; (3) Cryodamage to head and tail membrane may occur independently; the presence of an intact tail membrane does not necessarily indicate the intactness of head membrane. (4) Intact membranes are closely related to post-thaw motility, but do not reflect the fertilizing potential.

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