FACTORS AFFECTING THE INVITRO DEVELOPMENT TO BLASTOCYSTS OF BOVINE OOCYTES MATURED AND FERTILIZED INVITRO

The effects of media (TCM199 vs. synthetic oviduct fluid, SOF), sera (foetal calf serum, FCS vs. human serum, HS), gas atmosphere (5% CO2 in air vs. 5% CO2, 5% O2 and 90% N2) and coculture with bovine oviduct epithelial cells (cells vs. no cells) on the in-vitro development of in-vitro matured and fertilized bovine oocytes were examined. Immature oocytes surrounded with compacted cumulus cells were cultured for 24 h in TCM199 supplemented with 10% FCS, 10-mu-g follicle-stimulating hormone (FSH)/ml and 10-mu-g luteinizing hormone (LH)/ml, 1-mu-g oestradiol/ml, and 1 x 10(6) granulosa cells at 39-degrees-C under 5% CO2 in air. In-vitro fertilization was performed with frozen-thawed, heparin-treated (100-mu-g/ml, 15 min) spermatozoa from 2 bulls. Oocytes were incubated with 2.5 x 10(6) spermatozoa/ml for 24 h and then cultured in one of 16 treatments for 7 days. Cleavage (2-8-cell) and development to blastocysts were recorded on Days 2 and 7, respectively, after the start of culture. SOF was superior to TCM199 for cleavage (P < 0.01), development to blastocysts (P < 0.001) and for proportion of cultured ova resulting in blastocysts with at least 60 or at least 100 nuclei (P < 0.001). FCS was superior to HS for the development to blastocysts (P < 0.001) and 5% oxygen was superior to air for the proportion of ova reaching at least 60 cells (P < 0.01). For cleavage and development to blastocysts, there was an interaction between serum and cells (P < 0.01). In the presence of cells, ova preferred FCS; in their absence, serum had little effect. A third-order interaction between medium, cells and gases for percentage of ova developing to blastocysts (P < 0.001) and percentage developing to at least 60 nuclei (P < 0.01) showed that, without cells, 5% oxygen was advantageous in both media. With cells, gas had little effect on development in SOF, but ova in TCM199 developed most readily in air. The highest incidence of development to blastocysts in TCM199 (22% of ova cultured) occurred when ova were cultured with cells, FCS and air. The best development in SOF occurred with cells, FCS and either air (32%) or 5% oxygen (27%); or no cells, 5% oxygen and either FCS (31%) or HS (26%). The results show that the choice of culture conditions has a marked effect on the development of in-vitro matured and fertilized cattle oocytes. We choose to culture in SOF + 5% oxygen + FCS or HS routinely as this system is easy to set up, since it does not involve coculture and the effect of culture additives is not confounded by effects on cells and embryos.