Rat one-cell embryos recovered from naturally mated females were cultured in modified hamster embryo culture medium 1 without amino acids. In the presence of 0.4 mmol phosphate l⁻¹ (NaH₂PO₄), no embryos developed beyond the two-cell stage, regardless of the presence of 5.0 mmol glucose l⁻¹. This inhibition was dose dependent at very low concentrations of phosphate in the medium supplemented with 7.5 mmol glucose l⁻¹ and osmolarity adjusted to 244 mosmol; development to the blastocyst stage was not inhibited at 0.001–0.01 μmol phosphate l⁻¹, but development to the morula and four-cell stages was markedly inhibited at 0.1 and 1.0 μmol phosphate l⁻¹. In the medium without phosphate, glucose did not inhibit or promote development to the morula stage, but adequate concentrations of glucose were necessary for the development of morulae to the blastocyst stage; the percentage of one-cell embryos that developed to the blastocyst stage at 7.5 mmol glucose l⁻¹ (67%) and 10.0 mmol glucose l⁻¹ (60%) were not statistically different from the percentage at 5.0 mmol glucose l⁻¹ (46%), but was significantly greater than the percentage at 2.5 mmol l⁻¹ (33%). When osmolarity of the medium with 5.0 mmol glucose l⁻¹ was varied by adjusting the amount of NaCl added, more (82–98%) of the one-cell embryos developed to the four-cell stage at 212–276 mosmol, but development was greatly inhibited at 304 mosmol. Development to the blastocyst stage was largely dependent on osmolarities; at 244 mosmol, 61% of embryos developed to the blastocyst stage, although this percentage was not significantly different from the percentage (43%) at 264 mosmol.

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 Gin or NEAA. The mean number of cells in blastocysts developed in the medium with EAA + Gin + 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.

The aim of this study was to examine the effect of thyroxine on ovulation in immature rdw rats and the fertilization and development of the eggs. Serum thyroxine concentrations at 30 days of age were significantly lower in rdw rats than in normal rats (P < 0.001), and greatly increased after thyroxine replacement therapy (P < 0.001). Although few eggs (1–5 ± 1–2) were obtained from immature rdw rats treated with gonadotrophins alone, females treated with gonadotrophins and thyroxine ovulated significantly more eggs (85 ± 5). As a control, normal littermates ovulated 21–45 eggs when treated with gonadotrophins alone, and 68 eggs when administered with gonadotrophins and thyroxine. Of the eggs collected from rdw rats treated with gonadotrophins and thyroxine, and inseminated with spermatozoa from mature F₁ males, 98% were penetrated and in almost all (99%) of these eggs, male and female pronuclei formed. Forty-seven per cent of the pronuclear eggs developed to the blastocyst stage in vitro. After transfer to recipients, 21% (14/66) of one-cell and 22% (8/37) of two-cell embryos developed to offspring, and 62% (8/13) of pups were of rdw/rdw genotype. The average body weight (6.9 versus 7.8 g) of offspring derived from one-cell embryos was lower than that for two-cell embryos. The morulae and blastocysts did not develop to term, although 41% implanted in the uterine horns of recipients. In conclusion, in immature rdw rats, superovulation was induced by gonadotrophins combined with thyroxine therapy and the superovulated oocytes were fertilized and developed in vitro and developed to term after embryo transfer.