| Effects of different sera, hormones and big follicular fluid (BFF) on the maturation of bovine oocytes were investigated. With or without BFF, the maturation rates of oocytes cultured in the maturation media added with oestrus cow serum (OCS) and fetal bovine serum (FBS) were significantly higher than that of oocytes matured in medium with neonatal calf serum (NCS) (85.1%, 83.5% vs. 57.7%, with BFF; 83.1%, 82.9% vs. 53.0%, without BFF; P<0.01). The results suggest that OCS and FBS can improve oocyte maturation effectively and NCS is inferior to OCS and FBS for bovine oocyte maturation in our experimental systems. There is no significant difference between the effect of low quality hormones (FSH 10 ~tg/ml and LH 20 jig/mI) and high quality HMG (0.075 U/mI and 0.075 U/mI) on bovine oocyte maturation.2.The cleavage and development of bovine parthenogenetic oocytes activated by different chemical procedures were investigated. Matured oocytes were grouped into 9 treatments randomly: (1) ionomycin + 6-DMAP (ID); (2) ionomycin + 6-DMAP + cytochalasin 13(CCB) (IDC); (3) ionomycin + cycloheximide(CHX) (IX); (4) ionomycin + CHX + CCB (IXC); (5) 7% ethanol + 6-DMAP (ED); (6) ethanol + 6-DMAP + CCB (EDC); (7) ethanol + CHX (EX); (8) ethanol + CHX + CCB (EXC); (9) Sr2~+CCB (SC). The treatment time for ionomycin or 7% ethanol, 6-DMAP ?CCB, CHX ?CCB and Sr2~+CCB were 5 mm, 4 h, 5 h and 10 h, respectively. The cleavage rate of oocytes in SC (30.1%) was significantly lower than those in other treatments (53.0-64.6%) after oocytes were cultured for 24 h. The cleavage rates were 72.0%, 75.5%, 81.3%, 85.1%, 81.7%, 82.4%, 74.4%, 78.1% and 5 1.0% respectively after oocytes were cultured for 36 h, and the development rates to blastocyst stage were 18.2%, 24.5%, 7.2%, 11.9%, 11.1%, 19.4%, 6.7%, 12.2% and 2.0% respectively after cultured for 8 days. The blastocyst developmental percentages of ID, IDC and EDC parthenogenetic embryos were significantly higher than those of IX, IXC, ED, EX, EXC and SC (P<0.0 1). These results indicate that bovine oocytes can be activated effectively by ID, IDC and EDC. Ionomycin has strong activative effect compared with ethanol, while 6-DMAP is more effective than cycloheximide in activating bovine oocytes. Cytochalasin B is advantage to the development of bovine parthenogenetic embryos. Oocytes cultured in the 0.2mM 6-DMAP medium bears lower-129-blastocyst developmental potential than those in 2mM 6-DMAP and low concentration of 6-DMAP cannot activate bovine oocytes efficiently.3.Activated with 5 jiM lonomycin for 5 mm and 2mM 6-DMAP+5 jig/mi CCB for 4 h, matured oocytes were cultured in TALP+M199 (1:2) (TM), BMOC+M199 (1:2) (BM) and CRlaa, respectively. After cultured for 24 h and 36 h, the cleavage rate of oocytes were similar for all three treatments. Cultured for 8 days, the blastocyst development rate of parthenogenetic oocytes cultured in TM, BM and CRlaa were 18.2%, 21.4% and 29.3%, respectively. CRIaa was superior to TM for the blastocyst development of parthenogenetic embryos significantly (P<0.0I), but there was no difference with BM. These results suggest that CRlaa and BM are suitable for in vitro culture of parthenogenetic oocytes. EGF and IGF had no enhancement effect on the cleavage and development of parthenogenetic oocytes when there were added in the culture medium. Exposuring to Hoechst 33342 and UV radiation had deleterious effects on the cleavage and development of parthenogenetic oocytes and significantly decreased the oocyte cleavage rate.4.Using the conventional tissue culture technique, adult skin fibroblast cells were established for 2 Hostein dairy cows and I Angus cow. Skin fibroblasts were frozen in 8% DMSO in serum using simple method.5.Matured oocytes were enucleated at 22-23 h of maturation, and donor cells were transferred into the enucleated oocytes by electric pulses or intracytoplasmic nuclear injection (ICNI). After activated with ionomycin and 6-DMAP (or 6-DMAP+CCB), reconstituted embryos were cultured in different culture...
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