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

TLDR

The study aimed to develop a serum‑free culture system that supports high cleavage and blastocyst formation from in‑vitro‑derived sheep zygotes. The authors tested Synthetic Oviduct Fluid supplemented with BSA and all Eagle’s amino acids, renewing the medium every 48 h to reduce ammonium toxicity, and compared single versus group embryo culture, also evaluating nonessential amino acids and glutamine. Medium renewal every 48 h reduced arrested embryos to 6 % and doubled blastocyst rates from 29 % to 67 %, increased mean cell numbers (105 to 105 cells), and group culture of four embryos produced blastocysts with 160 cells, matching in‑vivo controls and exceeding serum‑based culture.

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).