308 EFFECT OF GONADOTROPIN TREATMENT ON OOCYTE COLLECTION AND IN VITRO FERTILIZATION IN THE BAN MINIPIG

The Ban minipig is a local breed characterized by small ovaries with a scant number of follicles available for in vitro maturation (IVM). The combination of eCG and hCG has been used successfully to control estrus in pig breeding programs. In this paper we present the first results of IVF in this breed in comparison with 2 types of oocyte preparation: (1) from animals not receiving gonatotropin treatment (group 1, n = 9); and (2) from animals receiving an injection of mixed pregnant mare serum gonadotropin and hCG, 300 IU/animal, for 3 days before oocyte collection (group 2, n = 4). All animals were 1 to 3 years old and with body weights that varied from 8 to 12 kg. At the time of collection, the ovaries were observed for follicle development; the cumulus–oocyte complexes (COCs) were aspirated using a 18-gauge needle. COCs of categories A (with more than 4 layers of cumulus cells) and B (with 2 to 4 layers of cumulus cells) were collected and matured in vitro as described previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) at 39�C under 5% CO2 in air. Matured oocytes with expanding cumulus cells were inseminated using male Ban minipig epididymal semen frozen by the methods reported by Kikuchi et al. (1998 Theriogenology 50, 615–623). The frozen–thawed spermatozoa were pre-incubated for 1 h in modified medium-199 adjusted to pH 7.8 in the incubator at 37�C. The capacitated spermatozoa were diluted and added to drops of fertilization medium (Fig-FM; Suzuki et al. 2002 Int. J. Androl. 123, 135–142) containing oocytes; the final concentration of sperm was 106/mL. After 3 h of co-incubation, attached spermatozoa and cumulus cells were removed from oocytes and the oocytes were the cultured in vitro as described previously (Kikuchi et al. 2002). The results obtained from 4 replicates showed that the number of follicles with a diameter larger than 2 mm and the rates of oocytes categorized as A and B were significantly lower (P < 0.05; ANOVA test) in the nontreated animals (0.0 and 67.5%, respectively) than in the treated group (25.5 and 87.1%, respectively). The rates of oocytes with a clearly expanding cumulus obtained after IVM were 78.6 (n = 136) and 88.1% (n = 101) for groups 1 and 2, respectively. The rates of cleaved embryos and embryos developed to the compact morula stage were 47.2 and 9.1% (n = 39), respectively, for group 1; and 89.1 and 18.8% (n = 101), respectively, for group 2. In conclusion, gonadotropin treatment before the collection of oocytes is recommended for application of IVM–IVF to local Ban minipigs. This work was supported by a grant from the VAST-Japan Society for the Promotion of Science Project.