Development Potential of Mouse Embryos Conceived in Vitro and Cultured in Ethylenediaminetetraacetic Acid with or without Amino Acids or Serum1

Abstract

Mouse ova were inseminated in vitro in modified Earle''s balanced salts solution (EBSS) supplemented with 10 or 100 .mu.M EDTA and 4 mg/ml BSA. After 4 h exposure to sperm, the ova were transferred to five different culture conditions based on albumin-free EBSS supplemented with 10 .mu.M EDTA minus or plus amino acids, or with 100 .mu.M EDTA minus or plus amino acids, or with human cord serum. After 44 h of culture, four-cell embryos from each culture group were transferred in cohorts of five into the left oviduct of pseudopregnant recipients (13-16 per culture condition). Two-cell embryos developed in vivo were similarly transferred to a separate group of recipients to serve as controls. The pregnancy rates following transfer of embryos cultured in 10 .mu.M EDTA minus or plus amino acids or in 100 .mu.M EDTA plus amino acids (38%, 43%, and 50%, respectively) were not significantly different from those of the in vivo control group (43%). The pregnancy rates following transfer of embryos cultured in 10 .mu.M EDTA minus amino acids (21%) or plus cord serum (8%) were significantly lower (p < 0.01) than those of the other groups. The overall yield of fetuses from total embryos transferred was significantly higher (p < 0.01) for the groups developed in 100 .mu.M EDTA plus amino acids (29%) and in vivo (26%) compared with embryos developed in 10 or 100 .mu.M EDTA with no amino acids, 10 .mu.M EDTA plus amino acids, or 100 .mu.M EDTA plus cord serum (15%, 15%, 9%, and 3%, respectively). Average fetal weights of all the in vitro groups 15 days after transfer were significantly (p < 0.03) lower than those of the in vivo control groups (264 .+-. 13 mg) except for the group cultured in 100-.mu.M EDTA plus amino acids (223 .+-. 16 mg). Thus, the developmental parameters of embryos cleaved in 100 .mu.M EDTA plus amino acids were the most similar to those of the in vivo controls. In summary, these results show that (1) neither albumin nor amino acids are absolutely required during early cleavage stages for embryo viability; (2) free amino acids during early cleavages in vitro are beneficial to the postimplantation viability of embryos in the presence of adenquate concentration of EDTA: (3) human cord serum is harmful to embryos cultured to the four-cell stage even in the presence of EDTA; and (4) postimplantation development of mouse embryos conceived in vitro is a specific and sensitive test for the "normality" of early cleavage culture conditions.