Developmental competence of frozen-thawed blastocysts from fair-quality bovine embryos cultured with beta-mercaptoethanol.

Two-hundred-and-thirty-one fair-quality embryos at the compacted morula stage collected from 89 superovulated cows were cultured in TCM199 or Brinster's BMOC-3 medium with or without 100 microM beta-mercaptoethanol (beta-ME). After 24 h culture, a total of 142 fair-quality embryos developed to the blastocyst stage, of which 106 were subsequently frozen with 1.8 M ethylene glycol. The mean cell number and development rates of frozen-thawed blastocysts from the fair-quality embryos cultured in TCM199 containing beta-ME were higher than those of the fair-quality embryos directly frozen without culture. The pregnancy rates obtained with frozen blastocysts from fair-quality embryos tended to be lower than those of non-cultured fresh fair-quality embryos and cultured fresh blastocysts. These results indicate that the inclusion of beta-ME in pre-freezing culture media improve the development of frozen-thawed blastocysts from fair-quality embryos, but not the pregnancy rate.

Relationship between dead cells and DNA fragmentation in bovine embryos produced in vitro and stored at 4 degrees C.

DNA fragmentation and its relationship with dead cells were examined in bovine blastocysts produced in vitro and stored at 4 degrees C for 1-5 days. Survival and development to the hatching and hatched blastocyst stage decreased with increasing storage time. Both were significantly lower at 72 hr than at 48 hr. None of the embryos stored for 120 hr developed to the hatching or hatched blastocyst stage. The proportion of dead cells per embryo increased progressively as the time of storage increased, until 69% of embryonic cells were dead after 120 hr of storage. There was no significant difference between the proportions of DNA fragmentation per embryo stored for 0 and 24 hr (12% vs 16%). However, the proportion of DNA fragmentation in embryos stored for longer than 48 hr was significantly greater than that in embryos stored for less than 24 hr. There were no significant differences among those stored for longer than 48 hr (28-33%). These results suggest that the reduced developmental competence of bovine embryos stored at 4 degrees C is characterized by necrotic change rather than apoptotic change.

Development to live young from bovine small oocytes after growth, maturation and fertilization in vitro.

Bovine oocytes (90 to 99 microns in diameter) were isolated from early antral follicles (0.5 to 0.7 mm in diameter). Cumulus-oocyte complexes (COC) with pieces of parietal granulosa were embedded in collagen gels and cultured for 14 d. After in vitro growth culture, oocytes recovered from the collagen gels were further matured, fertilized and cultured in vitro, and then were transferred to recipient cows. After 14 d of growth culture, 37% of the oocytes (203/556) showed normal morphology in the collagen gels. The mean diameter of the oocytes was 110.1 +/- 6.0 microns, significantly larger (P < 0.01) than before growth culture (94.8 +/- 2.7 microns), and 77% were at the germinal vesicle stage while 23% had undergone germinal vesicle breakdown. After 24 h of maturation culture followed by insemination, 27% of in vitro-grown oocytes reached the second metaphase, and 42% of the oocytes were normally fertilized. After insemination, 18.2% of in vitro-grown oocytes cleaved and 3.7% developed to the blastocyst stage. Three blastocysts obtained from in vitro-produced 90- to 99-micron oocytes were transferred to 3 recipients. One recipient subsequently became pregnant and delivered a live calf on Day 277. These results demonstrated for the first time that 90 to 99-micron oocytes from early antral follicles can complete growth and acquire full developmental competence in vitro so that live young can be produced after maturation, fertilization, subsequent culture in vitro, and transfer to recipient cows.