IN-VITRO MATURATION AND FERTILIZATION OF EQUINE OOCYTES RECOVERED DURING THE BREEDING-SEASON

The aim of this study was to develope an efficient and reproducible pr ocedure for in vitro maturation (IVM) and fertilization (IVF) in the h orse. Cumulus-oocyte complexes (COCs) recovered from the ovaries of ma res slaughtered during the breeding season were morphologically evalua ted, and those showing a compact cumulus and homogeneously appearing c ytoplasm were selected for culture. Effects on the maturation of estro us mare serum (EMS) versus estrous cow serum (ECS) as medium supplemen t were also evaluated (Experiment 1). In Experiment 2, the fertilizati on of in vitro matured oocytes with frozen-thawed semen separated by s wim-up and treated with heparin was carried out to determine the effec ts on fertilization of 1) increasing sperm concentrations (1x10(6), 5s 10(6) and 1x10(7) sperm cells/ml), 2) IVM medium supplementation with EMS or ECS and 3) partial cumulus mass removal before insemination. Fo rty-nine percent of the collected oocytes (335/683) showed a compact c umulus and homogeneous ooplasm and thus were selected for culture. In Experiment 1, high nuclear maturation rates were observed in both EMS (82%,32/39) and ECS (87.5%,56/64) groups, with no statistically signif icant difference. In Experiment 3, the percentage of normal fertilizat ion (2 polar bodies, 2 pronuclei and sperm tail) was similar for all 3 tested sperm concentrations (12.9%,4/31; 15.2%,9.59 and 15.5%,9/58). No advantage in using the homologous serum in IVM medium was noted in terms of fertilization (12.2%, 5/41 with EMS vs 12.9%, 4/31 for ECS). However, significantly higher fertilization rates were obtained after partial cumulus removal compared with that of oocytes fertilized with a whole cumulus (32.6%, 14/43 vs 12.2%, 5/41; P<0.05). The incidence o f polyspermic fertilization was low under all culture conditions (0 to 2.4%). In a replicate in which the oocytes fertilized after the cumul us removal were further cultured for 72 h two embryos, one at the 2-ce ll stage and the other at the 4-cell stage, could be obtained. These r esults indicate that, in the horse, the cumulus can be partially remov ed to increase the fertilization and of compact-cumulus oocytes recove red during the breeding season using frozen-thawed, heparin-treated se men.