Effects of osmolarity and amino acids in a chemically defined medium on development of rat one-cell embryos

Rat one-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (R1ECM) under different experimental conditions. When the osmolarity of the medium with a reduced concentration (63.8 mmol l-1) of NaCl was varied by adding different amounts of D-sorbitol, more (79-91%) of the one-cell embryos developed to the four-cell stage at 212-278 mosmol than at 306 mosmol (13%). The greatest proportions of morulae (74%) and blastocysts (60%) were obtained at 246 mosmol. When the medium was supplemented with amino acids in various combinations and the osmolarity adjusted to about 246 mosmol, more (80-98%) of the embryos developed to the morula stage. More blastocysts were obtained in medium supplemented with glutamine (Gln: 80%), minimal essential medium (MEM) essential amino acids (EAA) (90%), Gln+EAA (83%), EAA+MEM nonessential amino acids (NEAA) (83%) or EAA+Gln+NEAA (90%) than in medium without amino acids (59%). Few (3-10%) hatching or hatched blastocysts were observed 120 h after the start of culture in the medium with EAA plus Gln or NEAA. The mean number of cells in blastocysts developed in the medium with EAA+Gln+NEAA was 46.7 +/- 7.2. When a total of 82 morulae or early blastocysts that had developed in culture were transferred to eight pseudopregnant rats on day 4, six recipients into which 62 embryos were transferred maintained their pregnancies beyond day 23, although no deliveries had occurred by day 25 or 26. When the rats were killed, 42 (68%) implantation sites and eight (13%) full-term fetuses with no gross abnormality were observed in the uterine horns.