Effect of Frozen-Thawed Granulosa Cells and Serum Additives on Individual Bovine Embryo Development

Individually cultured bovine embryos have shown a reduced ability to proceed through development to the blastocyst stage when compared to cultures containing multiple embryos. The origin of this deficit is not entirely clear, although it is suspected to be due to a dilution of autocrine/paracrine factors secreted by the zygotes. Individual cell culture is sometimes necessary, as in the case of low oocyte numbers, or when individual embryo identification is needed. Thus, the aim of this project was to evaluate the effects on individual bovine embryo development of adding granulosa cells and/or two types of serum to the culture media. Our hypothesis was that addition of granulosa cells, 10% fetal bovine serum, knockout SR serum replacer, or a combination of serum and cells would improve development to the blastocyst stage of individually cultured bovine embryos up to the level of development seen in group culture. Ovaries were obtained from a local abattoir and all visible follicles aspirated. The recovered oocytes were washed, evaluated and selected for integrity of cumulus cell layers, and individually matured in maturation medium for 23±1 h. Oocytes were then moved to individual fertilization droplets and 25,000 frozen-thawed motile sperm were added to each droplet and co-cultured for 18 h. Then, the presumptive zygotes were randomly divided into six treatment groups with a 3x2 design. A culture of frozen-thawed mitomycin-c treated bovine granulosa cells was established in the bottom of droplets in half of the treatment groups. Both groups, with and without granulosa cells, were further divided into three serum treatment groups. The embryo culture media (synthetic oviductal fluid) was altered by the addition of 10% fetal bovine serum, Knockout SR serum replacer, or not modified. Embryos were assessed on day 8 post-fertilization to determine blastocyst development, and on days 10-11 postfertilization to evaluate hatching success. A separate group of oocytes was matured, fertilized, and cultured in a group (n=10) as a standard with which to compare the treatment groups. The results showed that none of the blastocyst rates of the six treatment groups were statistically different from each other (avg. 4.7%), with the group culture being significantly higher than all of them (21.7%) (one-way ANOVA). When aggregated, none of the interventions had a significant effect on blastocyst rate in individual culture. However, a trend (p=0.06) was found in the hatching rate between blastocysts cultured in media including cells (71.4%) and those cultured without cells (18.2%). This suggests that although the addition of granulosa cells did not increase the number of blastocysts formed, it increased the health and viability of the ones which did form.