130 In Vitro Production of Hybrid Desert Bighorn × Domestic Sheep Embryos Using Frozen–Thawed Epididymal Semen from a Hunter-Harvested Ram

Abstract

Although considered a species of least concern by the International Union for Conservation of Nature red list, the Desert Bighorn sheep (Ovis canadensis nelsoni) is listed in Appendix II of CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) in Mexico, due to population size and the lack of protected areas. Postmortem epididymal sperm collected from a hunter-harvested Desert Bighorn sheep ram in Mexico, with an unofficial Safari Club International score of 197 2/8 and an estimated 5.5 years old, were used to evaluate the in vitro production (IVP) of embryos using postmortem-collected ram sperm. Testicles with epididymides were placed in the refrigerator ~45 min after harvest. Sperm were extracted from each epididymis and assessed separately for total motility (TM), progressive motility (PM), and membrane integrity using a phase contrast microscope. The sperm suspension was obtained from the distal end of both epididymides and cryopreserved 12 h postmortem using triladyl with egg yolk. Membrane integrity and morphology were evaluated using Eosin-Nigrosin stain. Sperm DNA fragmentation was analysed using the Halomax kit (Halosperm SL, Madrid, Spain) with fluorescence microscopy. Centrifugation with density gradient PureSperm (Nidacon International, Mölndal, Sweden) was used to remove dead sperm and debris before IVF. Ovaries were collected from Domestic sheep (Ovis aries) at a local slaughterhouse. The maturation medium was TCM-199 with Earle’s salts and a modified Tris-buffered medium was used for fertilization. Frozen straws of sperm from the Desert Bighorn ram were thawed for 45 s at 37°C. Sperm were diluted with modified Tween medium B with milk powder (mTBM) to a final concentration of 5 × 10⁶ cells mL⁻¹. The gametes were co-incubated for 18 h under previously described conditions. The cumulus cells were mechanically removed from zygotes and grown using a co-culture with granulosa cells in sequential media SOF1-SOF2. With regard to sperm collection, epididymis 1 produced 29 straws of sperm (0.25 mL, 136 × 10⁶ sperm mL⁻¹) and epididymis 2 produced 32 straws of sperm (0.25 mL, 68 × 10⁶ sperm mL⁻¹). The sperm sample used for IVF had TM of 60% and PM of 30%. Live dead staining of fresh sperm showed 68% live (i.e. intact cell membranes) and 28% post-thaw. Regarding DNA integrity, only 2% of sperm had DNA fragmentation at 0 h. Of 15 Grade 1 oocytes used for IVF, 4 cleaved (27%), with 1 developing to blastocyst stage (25%). The results show that frozen–thawed epididymal sperm collected from a recently deceased Desert Bighorn ram can provide a valuable source of sperm for IVP of embryos. These results also provide new information on Desert Bighorn sheep reproductive parameters for use in health assessment, or reproduction and conservation management through gene banking and assisted reproductive techniques.