PARENT SESSION
Preimplantation Embryo/Fetal Development

DEVELOPMENT OF PORCINE ACTIVATED OOCYTES AND CLONED EMBRYOS IN G1.2/G2.2 MEDIUM.

Miyoshi, Kazuchika^1,2, Rzucidlo, S. Jacek¹, Stice, Steven¹, ^{1 2}

ABSTRACT- To optimize culture conditions for porcine cloned embryos, we compared developmental ability of activated oocytes cultured in NCSU-23, G1.2 or G1.2/G2.2 (the oocytes were transferred from G1.2 to G2.2 at 3 days after culture) medium. In vitro matured oocytes were activated by applying two direct current pulses of 75 V/mm for a duration of 60 sec at intervals of 5 sec, incubated in NCSU-23 or G1.2 supplemented with 7.5 g/ml cytochalasin B for 2 h and transferred into 100 l of each medium. The oocytes were examined for cleavage and blastocyst formation at 2 and 7 days after culture, respectively. The percentages of cleaved oocytes in G1.2 (60.3 ± 4.3%) and G1.2/G2.2 (53.8 ± 2.3%) were significantly (P<0.05) higher than that in NCSU-23 (31.8 ± 3.4%). Similarly, the blastocyst formation rates were improved (P<0.05) by culturing the oocytes in G1.2 (14.1 ± 3.6%) and G1.2/G2.2 (15.0 ± 2.2%) compared to that in NCSU-23 (5.6 ± 3.4%). When the oocytes were cultured in G1.2/G2.2, three hatched blastocysts were observed. There were no significant differences in the mean number of cells in blastocysts cultured in different media. Although some oocytes were cultured in NCSU-23 with the osmolarity reduced to the same as that of G1.2, the development was not improved. Cloned embryos were produced by transferring adult skin fibroblasts into enucleated oocytes. After activation and culture in G1.2/G2.2, 38.2 ± 4.2 and 7.6 ± 0.5% of them cleaved and developed to blastocysts, respectively. The mean number of cells in the blastocysts was 45.1 ± 6.3 with a range from 20 to 77. These results indicate that G1.2/G2.2 is more effective for culture of porcine activated oocytes than NCSU-23 and can support development to blastocysts of porcine cloned embryos.

KEY WORDS: pig, activation, nuclear transfer, culture medium