Comparison of different osmolalities and egg-yolk composition in processing media for the cryopreservation of red wolf ( Canis rufus) sperm

Abstract

Successful cryopreservation of sperm and the maintenance of a sperm-based genome resource bank have been identified as priorities for the recovery of the endangered red wolf ( Canis rufus). The objectives were to improve sperm processing and to determine the relative timing of damage to red wolf sperm during freezing and thawing. Fresh ejaculates ( n = 37) from adult red wolves ( n = 15, aged 2–13 y) were collected via electroejaculation and subjected to cooling, freezing and thawing in four TRIS-egg-yolk extender treatments varying in osmolality (∼305 mOsm versus ∼350 mOsm) and egg-yolk composition (0.8 μm-filtered versus unfiltered). Ejaculates were evaluated for sperm percentage motility, forward progressive motion, and morphological characteristics immediately upon collection and following extension, cooling (prior to freezing) and thawing. Although no single treatment consistently produced superior results, sperm suspended in ∼305 mOsm extenders exhibited slight losses in motility post-thawing (13 and 7%). Also, sperm suspended in ∼350 mOsm extenders tended to have slower rates of decline in motility in vitro post-thawing than those stored in ∼305 mOsm extenders ( P = 0.55). Finally, extenders incorporating unfiltered egg yolk exhibited a slightly larger ratio of absent to partial acrosomes than did sperm frozen in extenders prepared with clarified egg yolk. For ∼350 mOsm extenders, most motility loss occurred during the cooling rather than freezing and thawing. In conclusion, these data contribute to knowledge regarding cryopreservation of red wolf sperm.