144 EFFECTS OF CO-CULTURE WITH FIBROBLASTS AND OVIDUCT CELLS ON IN VITRO PRODUCTION OF PORCINE EMBRYOS

Cell co-culture during in vitro maturation or embryo culture has been reported as a method to improve the efficiency of maturation or embryo development (Kidson et al. 2003 Theriogenology 59, 1889; Romar et al. 2005 Anim. Reprod. Sci. 85, 287). Here, we present the impact of different methods of co-culture with mouse embryonic fibroblasts or oviduct epithelial cells on in vitro embryo production in pigs. Cumulus–oocyte complexes (COC) were collected from follicles with diameter larger than 3 mm and used for in vitro embryo production based on the method of Kikuchi et al. 2002 (Biol. Reprod. 66, 1033) with minor modifications. There were 8 groups; group 1: maturation and embryo culture without cell co-culture (control group); group 2: maturation in the presence of fibroblasts; group 3: embryo culture in the presence of fibroblasts; group 4: both maturation and embryo culture in the presence of fibroblasts; group 5: maturation in the presence of oviduct cells; group 6: embryo culture in the presence of oviduct cells; group 7: both maturation and embryo culture in the presence of oviduct cells; group 8: both maturation and embryo culture in the presence of both fibroblast and oviduct cells. In vitro maturation (IVM) was carried out at 39oC under 5% CO2 in air for 44 h using NCSU-37 as basic medium. Matured oocytes were inseminated using epididymal frozen semen in IVF medium modified Pig-FM supplemented with 2 mM caffeine and 5 mg mL–1 bovine serum albumin (Kikuchi et al. 2002). The percentage of cleaved embryos and percentage of cleaved embryos which developed to the compact morula and early blastocyst stage were recorded. Results were analysed by one-way ANOVA followed by Dunnett’s test. To investigate the effects of co-culture with mouse embryonic fibroblasts and oviduct epithelial cells on oocyte maturation, some COCs cultured in groups 1, 2, 5, and 8 were fixed to assess their nuclear maturation to the metaphase II stage. The rate of matured oocytes in the groups 2, 5, and 8 was 76.85 ± 3.39% (n = 102), 79.11 ± 3.75% (n = 64), and 81.84 ± 3.93% (n = 66), respectively; these rates were increased significantly compared to the group 1 (55.87 ± 1.88%, n = 94; P < 0.05). The effect of co-culture on the fertilization and embryo development is shown in Table 1. Our results indicate that co-culture increases the rates of embryonic cleavage in all groups by comparison with the control group. However, a significant increase in the rate of morula-blastocyst was only observed when embryos were co-cultured with fibroblasts or when both maturation and culture were performed in co-culture with either fibroblasts or oviduct cells (groups 3, 4, 7, and 8). The most important increase in morula-blastocyst rate was recorded for the group of embryos co-cultured with fibroblasts (group 3). In conclusion, the co-culture with fibroblast or oviduct cells during maturation can improve oocyte maturation and cleavage rate, while co-culturing the embryos with fibroblasts seems sufficient to improve both the cleavage and the morula-blastocyst rates. Table 1. Effect of co-culture on the fertilization and embryo development in pig Supported by a grant from the NAFOSTED VN.