Evaluation of chilled and frozen-thawed canine spermatozoa using a zona pellucida binding assay

Zona pellucida binding assays provide information about the fertilizing abi lity of spermatozoa. A zona-binding assay for canine spermatozoa using inta ct, denuded homologous oocytes has not been evaluated previously, in the pr esent study, an assay using canine oocytes derived from frozen-thawed ovari es was evaluated using three types of semen: fresh untreated; killed; and a 50:50 mixture of untreated and killed spermatozoa. The assays were perform ed on 3 x 20 oocytes for each sperm treatment, using semen from pooled ejac ulates (0.5 x 10(6) spermatozoa in each 50 Cll droplet containing five oocy tes). There was a significant difference (P < 0.001) between all treatments . Thereafter, the same procedure was used to evaluate methods of chilling a nd freeze-thawing of canine semen. There was a trend (P = 0.067) for more s perm binding after 1 day of chilling compared with after 4 days of chilling . Semen samples frozen using an extender (with or without the addition of E quex STM paste) were evaluated. Equex had a significant (P = 0.034) positiv e effect on the capacity of the spermatozoa to bind to the zona pellucida. In conclusion, the addition of a zona pellucida binding assay to establishe d in vitro tests should give a better estimate of the damage caused by the various procedures when developing new techniques for chilling and freeze-t hawing. Furthermore, the present study showed that chilling for 4 days tend ed to reduce the zona-binding capacity of the spermatozoon, and that Equex STM paste had a beneficial effect on the capacity of the frozen-thawed sper matozoon to bind to the zona pellucida.