Do sheep embryos develop better in the laboratory when cultured together rather than alone?

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This in vitro experimental culture study aimed to develop a serum-free system supporting high levels of cleavage and blastocyst formation from sheep zygotes developed in vitro. The researchers tested the effects of amino acids, ammonium, vitamins, and group culture on sheep zygote development in Synthetic Oviduct Fluid (SOF) medium supplemented with BSA (32 mg/ml).

When embryos were cultured singly in the same drop of medium for 6 days, adding amino acids did not affect the percentage arrested at the 8–16-cell stage: 43% in SOF and 41% in SOF plus amino acids.

However, with all Eagle’s amino acids present, replacing the medium every 48 h to alleviate ammonium toxicity significantly reduced developmental arrest to 6% (p < 0.05). Blastocyst cell number increased from 52 cells in SOF to 105 cells in SOF plus amino acids (p < 0.01), and the percentage reaching the blastocyst stage increased from 29% to 67% (p < 0.05). With medium renewal every 48 h, nonessential amino acids and glutamine also significantly reduced developmental arrest (p < 0.05).

In SOF containing all Eagle’s amino acids and renewed every 48 h, embryos cultured singly, in groups of two, and in groups of four had blastocyst hatching rates of 47%, 31%, and 79%, respectively, and mean blastocyst cell numbers of 105, 136, and 173. The abstract reports significant increases in hatching and mean cell number across these culture conditions, although the hatching rate for pairs was lower than for single embryos. After culture in groups of four, blastocyst cell numbers were equivalent to those of in vivo-developed controls (160 cells) and significantly greater than those of blastocysts developed in serum (103 cells; p < 0.01).

Analysis of blastocyst metabolism per cell showed that amino acids did not affect glucose uptake or lactate production, whereas adding amino acids and vitamins significantly increased both measures (p < 0.01). A similar response was observed in blastocysts developed in serum.

After culture with amino acids, sheep blastocysts produced significantly more ammonium than mouse blastocysts. The authors regarded this as indirect evidence that ruminant embryos use amino acids to a greater extent than rodent embryos.

The authors concluded that ammonium impairs sheep embryo development in culture and that maximal development to the blastocyst stage occurs with all Eagle’s amino acids present. Eagle’s amino acids significantly increase blastocyst formation, hatching, and cell number without affecting glucose uptake or lactate production per cell. Vitamins do not affect embryo morphology but significantly increase glucose uptake and lactate production per cell. The authors also concluded that sheep embryos produce one or more factors that stimulate their development in culture.

Culturing sheep zygotes in groups of four with Eagle’s amino acids for 6 days, with medium renewal every 48 h, resulted in 95% blastocyst development, a 79% hatching rate, and 173 cells per blastocyst. The study concluded that a serum-free culture system without somatic cells can achieve in vitro cleavage rates equivalent to those in vivo.