Interaction of extender composition and freezing method for effective semen cryopreservation in the North American river otter (Lontra canadensis)

Abstract Semen cryopreservation and storage in genome resource banks (GRBs), in combination with artificial insemination (AI), could be invaluable for genetic management and conservation of endangered otter species. For any applied conservation benefit, effective methods for otter sperm processing and cryopreservation first must be established. In this study, our objective was to develop an effective semen cryopreservation method for the North American river otter, evaluating the effect of extender composition (i.e., glycerol concentration, Equex STM paste supplementation) and freezing protocol (timing of glycerol addition, pre-freeze cooling rate, freezing/packaging method) on post-thaw sperm motility, longevity and acrosome status. Semen was collected from 14 otters housed at 9 zoos, and following cryopreservation in an egg-yolk based extender, thawed to assess sperm motility and acrosome status immediately post-thaw and during 6 h of in vitro culture. Results indicated that extender containing 4% glycerol was preferable (p   0.05) post-thaw sperm parameters. Treatments with extender containing Equex and frozen by pelleting on dry ice showed greater (p