The present study was carried out to investigate the development of bovine embryos fertil-ized in vitro up to blastocyst using CRlaa medium.
The cumulus free in vitro fertilized ova were randomly assigned to 6 groups and then these were cul-tured for 7 days. The groups were 1) CRLaa; 2) CR1aa+ 5 % calf serum (CS) ; 3) CRLaa+ 5% CS+bovine oviductal epithelial cells (OVC) ; 4) TCM-199; 5) TCM-199+ 5% CS; and 6) TCM-199+ 5% CS+OVC.
For the 6 groups, developmental rates (24.1 and 22.1%) of embryos cultured in CRlaa+ 5 % CS and CRlaa+ 5 % CS+OVC up to blastocyst stage were higher (P<0.05) than the other culture systems. The total cell numbers of embryos cultured in CRlaa+ 5 % CS and CRlaa+ 5 % CS+OVC at blastocyst stage were 100.1 ± 27.7 and 97.2 ± 31.4, respectively, and there is no significant difference.
In conclusion, our experiment clearly demonstrated that CRlaa require serum nutrients for development of cumulus free early bovine embryos to blastocyst stage, and that CR1aa+ 5 % CS is beneficial in the development in vitro under the condition of the absence of co-culture with oviductal epithelial cells.

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The present study was carried out to investigate the parthenogenetic development rate into blastocyst of in vitro matured bovine oocytes.
In Experiment 1, in vitro mature oocytes without cumulus cells for 30 h were activated by electric pulse (EP; 150 V/mm, 90 μsec) or Ca-ionophore (IA; 5 μM, 5 min) treatment, and were cultured in TCM-199 + 5 % calf serum (CS) supplemented with cytochalasin D for 1018 h. These ova were cultured in CR1aa + 5 %CS for 7 d. Developmentrates up to blastocyst of ova activated by EP or IA treatment were 7 % and 9 %, respectively.
In Experiment 2, in vitro matured oocytes without cumulus cells for 24 h were treated with EP (at 24 h from the beginning of in vitro maturation:100 V/mm, 90 μsec) + cycloheximide (CY; 24 to 30 h), EP (24 h) + CY (24 to 30 h) + IA (30 h) or IA (24 h) + EP (25 h) + CY (24 to 30 h). The activated ova were treated with cytochalasin D (24 to 42 h) and then were cultured in CRlaa + 5 %CS for 7 d. The development rates up to blastocyst were 37% (EP+CY), 49% (EP+CY+IA) or 50% (IA+EP+CY), respectively.
It was demonstrated that bovine activated oocytes can develop to the blastocyst stage. Moreover electric pulse+Ca ionophore+cycloheximide treatment is the optimal activation method for in vitro matured bovine oocytes.

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[in Japanese]

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[in Japanese]

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[in Japanese]

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[in Japanese]

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We assessed the effect of the developmental stage of donor embryos on in vitro development of nuclear transplants in cattle. Nuclear donor embryos were obtained from in vitro fertilization and culture. The average cell numbers per donor embryo 68, 92, 116, and 140 h after insemination were 13.6, 13.5, 23.2, and 40.5, respectively. Enucleated oocytes were cultured 33 h for maturation and activated by electrical stimulation; activated oocytes were then cultured for an additional 9 h. Blastomeres from donor embryos were fused with enucleated oocytes by exposure to electric pulses. Nuclear transplants were then cultured with bovine oviductal epithelial cells. The fusion rate of 82-96% did not vary among the donor stages. The proportions of nuclear transplants that cleaved to the 2-and 8-cell stage were 73-87% and 22-53%, respectively. Maximal transplant development was obtained with donor embryos cultured for 116 h after insemination; 35% developed to blastocysts. This rate was significantly (P<0.01) higher than for donor embryos cultured for 68 h (13%), 92 h (3%), or 140 h (12%) after fertilization. These results indicate that developmental stage and cell cycle of donor embryos affect in vitro development of nuclear transplants.

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The present study was carried out to establish in vitro fertilization system with high rates of sperm penetration and male pronucleus formation in pig. The major results were summarized as follows. 1) Preservation of ejaculated spermatozoa for 24 h at 15 C promoted the ability of the spermatozoa to penetrate oocytes matured in vitro. 2) Frozen-thawed epididymal spermatozoa can penetrate oocytes matured in vivo and in vitro. Piglets were obtained from in vitro fertilization of in vivo matured oocytes with frozen-thawed epididymal spermatozoa. 3) Numbers of follicle cells surrounding oocytes and concentrations of progesterone in the maturation medium significantly affect meiotic progression of oocytes to the second metaphase and male pronuclear formation after in vitro fertilization. 4) When oocytes were cocultured with follicle shells in a non-static culture system, both rates of maturation and male pronuclear formation were high irrespective of the number of follicle cells surrounding the oocyte. 5) In vitro matured oocytes with high and low rates of male pronuclear formation showed the same protein synthesis as in vivo matured oocytes both before and after in vitro fertilization. After 6 h of insemination, the decrease in the 46K and 25k bands and the increase in the 22k band were observed. 6) Glutathione levels in oocytes were related to rates of male pronuclear formation after in vitro fertilization. Piglets were born from in vitro fertilization of oocytes matued in the medium containing cysteine with ejaculated spermatozoa.
These findings indicate that the number of cells surrounding oocytes, the concentration of progesterone in the maturation medium, the maturation culture system and glutathione levels in oocytes are important for nomal maturation of oocytes resulting in high rates of male pronuclear formation after in vitro fertilization.

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