32 An approach to invitro oocyte maturation and fertilization in threatened Saharan Dorcas gazelle (Gazella dorcas osiris) using frozen-thawed epididymal sperm cells
Authors: Ruiz-Conca M. , Gardela J. , Álvarez-Rodríguez M. , Fernández-Bellon H. , López-Béjar M. 
Published: January 2021

The Dorcas gazelle (Gazella dorcas), in the family Bovidae, is one of the smallest species of the genus Gazella. Its population is decreasing and it is classified as vulnerable by the International Union of Conservation of Nature Red List of Threatened Species. The subspecies Saharan Dorcas gazelle (G. dorcas osiris), originating in the central-western Saharan and Sahel regions, is almost extinct in the wild because of over-hunting. Thereon, assisted reproductive techniques (ARTs) could help in its population management. Here, we performed invitro maturation (IVM) of oocytes recovered from an adult female of Saharan Dorcas gazelle (10 years old) to attempt IVF with frozen-thawed epididymal sperm cells from an adult male (9 years old) of the same subspecies, following protocols for ARTs in cattle. Epididymal sperm was obtained after postmortem examination by cannulation of vas deferens with commercial semen extender Gent A (Minitub). Motility features were assessed using a computer-assisted sperm analysis system (Proiser SL), and viability was evaluated using eosin/nigrosin staining. After collection, viability (88.3%), total (17.8%) and progressive (8.7%) motility were assessed. A total of 820.5×106 sperm cells was obtained and diluted (1:1) in a semen extender with permeable cryoprotectants (Gent B, Minitub). Then, the sample was refrigerated in straws (200×106 mL−1; 0.5mL) at 4°C for 1h and exposed to liquid nitrogen vapors (LN2) for 10min before being directly plunged into and kept on LN2. A total of 35 oocytes were recovered by slicing, 27 of which were selected for IVM (24h, 38.5°C, 5% CO2 in humidified air). Motile sperm were obtained by centrifugation of thawed semen (10min, 1000×g) by performing a density gradient (BoviPure, Nidacon). Post-thawing viability (72.9%) and total (14%) and progressive (4%) motility were evaluated just before IVF. After IVM, 17 oocytes were incubated for IVF for 20h with 1×106 thawed sperm cells mL−1 (although the first polar body extrusion was observed only in 5 of the 17 oocytes before IVF; 29.4%). Also, 7 oocytes selected for IVM were vitrified and kept for the genetic bank, and 3 were fixed. Thereafter, all oocytes that underwent IVF were incubated for invitro embryo culture (38.5°C, 5% CO2, 5% O2 in humidified air) to avoid discarding any potential zygote. Cleavage was assessed at 48h post-IVF. From those, only 2 oocytes successfully achieved IVF out of the 5 that showed first polar body extrusion (40%), although cleavage was asymmetric and embryos did not progress. The rest of oocytes (12) did not achieved IVM, IVF, or cleavage. Results suggest that optimal protocols and appropriate individualized conditions for vulnerable nondomestic ungulates are required for successful ARTs. Future efforts are needed to improve preservation strategies for the gametes/embryo conservation of endangered species.
JG and MRC contributed equally to this work. JG is supported by AGAUR (2018 FI_B 00236) and MRC by FPU (Training for Academic Staff) (FPU15/06029).