Bovine oocytes in secondary follicles grow in medium containing bovine plasma after vitrification.

There has been no culture system that supports the growth of bovine oocytes for more than 2 weeks. In the present study, bovine secondary follicles were cultured for 4 weeks, and the effects of supplemented protein components and FSH in the culture medium on the growth of the oocytes were examined. The effect of vitrification of secondary follicles on the subsequent oocyte growth was also examined. Secondary follicles (150 to 200 µm in diameter) containing growing oocytes (approximately 60 µm in diameter) were dissected from ovaries and cultured in a medium supplemented with FSH (0, 25 or 50 ng/ml) and one of the following four kinds of protein components: bovine serum albumin (BSA), bovine plasma (BPL), fetal calf serum (FCS) and bovine follicular fluid (BFF). In BSA- and BPL-supplemented media with 0 or 25 ng/ml FSH, more than 50% of follicles showed no degenerative signs during culture, and oocytes significantly increased in size after 4 weeks (P<0.05). Higher percentages of granulosa cell-enclosed oocytes were recovered from the follicles cultured in BPL-supplemented media with 0 and 25 ng/ml FSH, and the oocytes grew to 90 µm or more in diameter. In FCS- and BFF-supplemented media, FSH increased the numbers of degenerating follicles. Next, vitrified-warmed secondary follicles were cultured in BPL-supplemented medium. One third of the follicles showed no degenerative signs, and the oocytes increased in diameter to 88.8 ± 3.1 µm after 4 weeks of culture. These results suggest that a BPL-supplemented medium supports oocyte growth in bovine secondary follicles for 4 weeks, even after vitrification and warming of the follicles.

Development of vitrified bovine secondary and primordial follicles in xenografts.

The objective was to evaluate the effect of various vitrification conditions on the morphology of bovine secondary and primordial follicles, and to use xenografting to confirm their developmental ability. Secondary follicles were placed in vitrification solution containing 15% (v:v) ethylene glycol (EG), 15% (v:v) dimethyl sulfoxide (DMSO), 20% (v:v) fetal calf serum (FCS), and 0, 0.25, or 0.5 M sucrose at room temperature for 1 or 30 min, or at 4 degrees C for 30 min before being plunged into liquid nitrogen (LN(2)). Ovarian tissues with primordial follicles were equilibrated in a solution containing 7.5% EG, 7.5% DMSO, and 20% FCS for 5 or 15 min, and then treated with a vitrification solution (15% EG, 15% DMSO, and 20% FCS) containing 0 or 0.5 M sucrose at room temperature for 1 min, and then plunged into LN(2). One week later, follicles and tissues were warmed, and morphology assessed histologically. Secondary follicles vitrified in sucrose-free solution had more oocytes with shrinkage of the nucleus and abnormal cytoplasm relative to those vitrified in sucrose-containing solution. When primordial follicles were equilibrated for 5 min and vitrified in sucrose-free solution, the percentage of morphologically normal primordial follicles was higher than in the other groups (P < 0.05). After 4 wk and 6 mo of xenografting of vitrified-warmed secondary and primordial follicles, respectively, in SCID mice, follicles developed to the antral stage and oocytes grew. In conclusion, bovine secondary follicles were successfully cryopreserved in sucrose-containing vitrification solutions and maintained their ability to develop to the antral stage and grow oocytes, whereas primordial follicles vitrified in sucrose-free solution maintained their morphology and developed to the antral stage, with oocyte growth.

Bovine oocytes grown in serum-free medium acquire fertilization competence.

We previously found that bovine oocytes 90-99 microm in diameter in early antral follicles grew to nearly their final size in serum-free medium, with some of the oocytes acquiring the nuclear competence to reach the second metaphase. In the present study, we examined the competence of the fertilization and pre-implantational development of the oocytes grown in serum-free medium. Bovine early antral follicles, 0.4-0.7 mm in diameter, were collected mechanically using fine forceps, embedded in collagen gels, and cultured in serum-free medium for 16 days. Grown oocytes which were enclosed by granulosa cells and did not show disintegrated ooplasm were recovered as normal oocytes, were transferred to the maturation medium, and then inseminated with spermatozoa. Ten to 12 h after insemination, 28% (41/145) of the oocytes were penetrated by spermatozoa. Of the penetrated oocytes, 18 (12%) formed a female and a male pronuclei, and 10 (7%) had a female pronucleus and an enlarged sperm head. Among the abnormally penetrated oocytes (13/41), 10 were penetrated by multiple spermatozoa and 3 were penetrated by a spermatozoon at the first metaphase stage. Of the 106 inseminated oocytes grown under serum-free conditions, 8 oocytes had cleaved and developed to the 2-cell stage 48 h after insemination, and 3-4-cell embryos and 5-8-cell embryos were observed after 72-96 h. However, no embryo developed to the blastocyst stage within 8 days. These results indicate that bovine oocytes grown in serum-free medium can be fertilized, but acquire insufficient embryonic development competence under the employed culture conditions.