Submission Number: BAR-4-5-3

Abstract Number: 98

EFFECT OF EXTRACTION METHODS ON CRYOPRESERVATION OF CANINE EPIDIDYMAL SPERM.

Barbara Durrant*, Dianne Amodeo, Autumn Anderson and Mary Ann Olson

Center for Reproduction of Endangered Species, Zoological Society of San Diego, San Diego, CA, USA ¹

Abstract:
Sperm of endangered species can be rescued by post-mortem epididymal extraction. The domestic dog is an excellent model for developing epididymal sperm cryopreservation techniques for exotic carnivores. These experiments compared cryosurvival of domestic dog sperm collected by two epididymal sperm extraction methods: 1) MC, epididymides were minced, then filtered to separate sperm from tissue; 2) FL, the vas was flushed with DME and sperm was collected from the incised cauda epididymis. In Experiment 1, one epididymis from each of six pairs of testes was minced while the other was flushed. Initial motility (MOT), speed of progression (SOP), and membrane integrity (initial live, IL) was recorded. Initial motility score (IMS) was calculated as MOT x SOP2. Sperm was extended with Test-Yolk buffer (TY) and cooled in cryovials for 30 min at 4C before the addition of glycerol for a final concentration of 4%. Vials were frozen over liquid nitrogen vapor at 12.8C/min, and stored for a minimum of 10 days before thawing in a 37C waterbath for 1 minute. Sperm was evaluated for %IL and % IMS immediately after thawing (T0), washed by centrifugation and resuspended in DME. Sperm was again evaluated for %IL and %IMS after 30 min (T30) and 60 min (T60) at 37C. ANOVA revealed that FL sperm exhibited a higher (p<.05) %IL at T0, T30, and T60 than MC sperm. %IMS was not significantly affected by collection method. In Experiment 2 six pairs of testes were treated as described in Experiment 1. FL sperm was pooled and frozen separately from a pool of MC sperm. In addition, Experiment 2 compared final glycerol concentrations of 2.2%, 2.5%, 3.0%, 3.2%, and 4.0%. There was no interaction between glycerol concentration and sperm extraction method, and 3.2% glycerol increased %IL (p<.01) over all other concentrations. %IL was greater at T0 (p<.05) in FL sperm compared to MC sperm and %IMS was higher in FL than MC at T0 and T30 (both p<.01). Although the detrimental effect of tissue mincing on cryosurvival of canid sperm is not known, these data suggest that vas flushing is the preferred method of epididymal sperm extraction for all carnivores. .

Keywords: cryopreservation, canid, epididymal, sperm

This abstract is being presented at: 8:00 AM in session:
Cryopreservation