title: Evaluation of chilled and frozen-thawed canine spermatozoa using a zona pellucida binding assay.
abstract: Zona pellucida binding assays provide information about the fertilizing ability of spermatozoa. A zona-binding assay for canine spermatozoa using intact, denuded homologous oocytes has not been evaluated previously. In the present study, an assay using canine oocytes derived from frozen-thawed ovaries was evaluated using three types of semen: fresh untreated; killed; and a 50:50 mixture of untreated and killed spermatozoa. The assays were performed on 3 x 20 oocytes for each sperm treatment, using semen from pooled ejaculates (0.5 x 10(6) spermatozoa in each 50 microliter droplet containing five oocytes). There was a significant difference (P < 0. 001) between all treatments. Thereafter, the same procedure was used to evaluate methods of chilling and freeze-thawing of canine semen. There was a trend (P = 0.067) for more sperm 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 Equex STM paste) were evaluated. Equex had a significant (P = 0.034) positive effect on the capacity of the spermatozoa to bind to the zona pellucida. In conclusion, the addition of a zona pellucida binding assay to established in vitro tests should give a better estimate of the damage caused by the various procedures when developing new techniques for chilling and freeze-thawing. Furthermore, the present study showed that chilling for 4 days tended 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 spermatozoon to bind to the zona pellucida.
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Centre for Reproductive Biology in Uppsala, Swedish University of Agricultural Sciences (SLU), Box 7039, SE-750 07 Uppsala, Sweden.
authors.forename: B
authors.initials: B
authors.lastname: Ström Holst
authors.forename: B
authors.initials: B
authors.lastname: Larsson
authors.forename: C
authors.initials: C
authors.lastname: Linde-Forsberg
authors.forename: H
authors.initials: H
authors.lastname: Rodriguez-Martinez
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods; D003080:Cold Temperature; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004285:Dogs; D005069:Evaluation Studies as Topic; D005260:Female; D016018:Least-Squares Analysis; D008297:Male; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000502:physiology*; D013997:Time Factors
chemical_list: D003451:Cryoprotective Agents; D003902:Detergents