Effect of cryopreservation on zona - Binding capacity of canine spermatozoa in vitro

The hemizona assay (HZA) was used as a functional test for zona pellucida b inding capacity of fresh and frozen-thawed canine spermatozoa. We investiga ted 30 ejaculates from 3 dogs with sperm motility >70% and sperm concentrat ion >5.10(8) cells per ejaculate with up to 20% abnormal and dead spermatoz oa. Fifteen ejaculates were each divided into 2 portions: one portion was u sed for analysis of fresh semen, the other for cryopreserved semen. On the day of the experiments, in vitro-matured canine oocytes were bisected into 2 equal hemizonae. One half of the hemizonae were coincubated with fresh ca pacitated (control) spermatozoa, the other half of:the hemizonae were coinc ubated with frozen-thawed (tested) spermatozoa at final concentration of 1 to 2x10(6) cells/mL in 200 mu L droplets of BSA-supplemented Toyoda, Yokoja ma and Hoshi (TYH) medium at 37 degrees C, 5% CO2 for 1 h. Sperm suspension s were examined kinesigraphically for post capacitation type of movement. T he Student's t-test was used to compare differences between semen parameter s. The data on; HZA binding activity of fresh and frozen-thawed canine seme n were analyzed by ANOVA and then by the Newman Keuls multiple range method . The results showed no differences in the initial:semen quality parameters among the 3 dogs. After thawing, the semen from Dog land Dog 2 demonstrate d relatively uniform sperm parameters, while in Dog 3 sperm motility, and v iability and the percentage of morphologically normal spermatozoa were sign ificantly decreased. The binding activity of frozen-thawed spermatozoa from the 3 dogs was significantly reduced (29.40+/-9.02, 18.60+/-3.30, 8.20+/-4 .49) compared with that (107.20+/-19.22, 109.80+/-0.75, 78.0+/-12.47; P<0.0 1) of fresh spermatozoa. The results showed that semen samples with similar sperm parameters prior to cryopreservation displayed different sperm zona- binding capacity after freezing. The HZI (value of sperm binding capacity o f frozen-thawed vs fresh semen samples) was higher in Dog 1 (27.43) than in Dog 2 (16.90) or Dog 3 (10.40), and thus confirmed the variation of zona b inding activity after thawing between dogs. The freezability of individual dog semen is discussed. In conclusion HZA may be a valuable tool for evalua ting the post-thaw fertilizing ability of canine spermatozoa. (C) 1999 by E lsevier Science Inc.