216 IN VITRO OOCYTE MATURATION, FERTILIZATION, AND CULTURE AFTER LAPAROSCOPIC OVUM PICK-UP IN AN ENDANGERED GAZELLE (GAZELLA DAMA MHORR)

Abstract

The recovery of immature oocytes followed by in vitro maturation, fertilization and culture (IVMFC) allows the rescue of biological material of great genetic value for the establishment of genetic resource banks. Studies have been carried out on endangered Mohor gazelle sperm cryopreservation (Garde et al. 2003 Biol. Reprod. 69, 602-611), but there are no studies on oocytes in this species. The purpose of this work was to develop a protocol for ovarian stimulation for the recovery of oocytes and subsequent IVMFC. The study was conducted using six reproductively mature female Mohor gazelles from the breeding herd at the Estacion Experimental de Zonas Aridas. Animals were synchronized by insertion of controlled progesterone internal drug release (CIDR) devices for 14 days and removal of the devices on the day of ovum pickup (OPU). Follicular growth was stimulated by a total of 5.28 mg of oFSH (Ovagen, ICP, Auckland, New Zealand) given in four equal doses every 12 h. OPUs were performed (Berlinguer et al. 2004 Theriogenology 61, 1477-1486) on Day 15 from the beginning of treatment, and follicles were aspirated with a syringe and a 25G needle using TCM199-HEPES with 50 ¼g/mL streptomycin, 50 IU/mL penicillin, 0.1% polyvinyl alcohol, and 15 IU/mL heparin. Degenerated oocytes and those with expanded cumulus were removed. Oocytes were cultured in TCM-199 plus 10% FCS, 10 ¼g/mL ovine FSH/LH, 1 ¼g/mL estradiol, and 0.1 mg/mL glutamine at 38.5°C under 5% CO₂/air and maximum humidity. Spermatozoa were cryopreserved in Tes-Tris with 5% egg yolk and 6% glycerol, and selected by swim-up in SOF medium. After 24 h sperm-oocyte coincubation (sperm concentration: 1 × 10⁶/mL) in SOF with 2% estrus sheep serum under 5% CO₂ 5% O₂ 90% N₂, presumptive zygotes were transferred to SOF with 0.4% BSA and amino acids under 5% CO₂, 5% O₂ 90% N₂ and cultured for 4 days. Oocytes and embryos were stained with Hoechst 33342 and propidium iodide (1 ¼g/mL each) and visualized under a fluorescence microscope. A total of 35 oocytes were recovered from 56 punctured follicles (62.5%). This recovery rate was similar to those in wildlife in earlier reports, but more studies are needed to improve hormonal stimulation and oocyte harvesting. Out of 29 cumulus-oocyte complexes matured in vitro, 3.5% were found at GV and 6.9% at MI; 20.7% were degenerated and 68.9% had advanced to MII. Fertilization and cleavage rates were 40% and 30%, respectively, of matured oocytes. Out of eight zygotes, six showed cleavage (ranging from 2 to 8 cells). None of the developing embryos progressed to the blastocyst stage, suggesting the existence of a developmental block and the need to improve culture conditions. Although more trials will help to improve IVMFC, this study demonstrates for the first time the feasibility of in vitro fertilization with frozen-thawed semen of in vitro matured oocytes collected by OPU from FSH-stimulated endangered gazelles.

This work was supported by the Spanish Ministry of Education and Science (REN 2003-11587) and Acciones Integradas (HI20030336).