Enhanced Rates of Cleavage and Development for Sheep Zygotes Cultured to the Blastocyst Stage in Vitro in the Absence of Serum and Somatic Cells: Amino Acids, Vitamins, and Culturing Embryos in Groups Stimulate Development1

Abstract

The aim of this study was to develop a serum-free culture system that could support high levels of cleavage and blastocyst formation from sheep zygotes developed in vitro. To this end, we investigated the effects on sheep zygote development of amino acids, ammonium, vitamins, and culture of embryos in groups in Synthetic Oviduct Fluid (SOF) medium supplemented with BSA (32 mg/ml). The inclusion of amino acids in the culture medium had no effect on the percentage of embryos arrested at the 8–16-cell stage when embryos were cultured singly in the same drop of medium for 6 days (43% in SOF; 41% in SOF+amino acids). However, in medium containing all Eagle’s amino acids, replacing the culture medium every 48 h to alleviate ammonium toxicity significantly decreased the number of arrested embryos (6%; p < 0.05) and significantly increased blastocyst cell number (52 cells in SOF; 105 cells in SOF+amino acids; p < 0.01) and the number of embryos developing to the blastocyst stage (29% in SOF; 67% in SOF+amino acids; p < 0.05). When the medium was renewed every 48 h, nonessential amino acids and glutamine also significantly decreased the number of arrested embryos (p < 0.05). Culturing embryos singly or in groups in SOF medium with all Eagle’s amino acids that was renewed every 48 h resulted in significant increases in blastocyst hatching and mean cell number (47%, 31%, and 79%; 105, 136, and 173 cells for embryos cultured singly, in groups of 2, and in groups of 4, respectively). After culture in groups of 4, blastocyst cell numbers were equivalent to in vivo-developed controls (160 cells) and significantly greater than those developed in serum (103 cells; p < 0.01). Analysis of blastocyst metabolism, expressed on a per-cell basis, revealed that amino acids did not affect either glucose uptake or lactate production, whereas the addition of amino acids and vitamins resulted in a significant increase in both parameters (p < 0.01). A similar response was observed in serum-derived blastocysts. Ammonium production by sheep blastocysts after culture in the presence of amino acids was significantly greater than that produced by mouse blastocysts, indirect evidence that ruminant embryos utilize amino acids to a greater extent than do rodent embryos. In summary, the data demonstrate that 1) sheep embryo development in culture is impaired by ammonium; 2) maximal development to the blastocyst occurs in the presence of all Eagle’s amino acids; 3) Eagle’s amino acids significantly increase blastocyst formation, hatching, and cell number, but do not affect glucose uptake or lactate production per cell; 4) vitamins do not affect embryo morphology but do significantly increase glucose uptake and lactate production per cell; 5) sheep embryos produce a factor(s) that stimulates their development in culture. The culture of sheep zygotes in groups of 4 in the presence of Eagle’s amino acids for 6 days, with the medium renewed every 48 h, resulted in 95% blastocyst development, 79% hatching rate, and a blastocyst cell number of 173. It is therefore possible to obtain in vitro cleavage rates equivalent to those in vivo, in the absence of somatic cells in a serum-free culture system.