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Improved in vitro bovine embryo development and increased efficiency in producing viable calves using defined media.

Lim KT, Jang G, Ko KH, Lee WW, Park HJ, Kim JJ, Lee SH, Hwang WS, Lee BC, Kang SK

Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea; Embyro Research Center, Seoul Dairy Corporation, Gyeonggi 476-851, Republic of Korea.

In this study, we developed a defined culture medium that supported improved in vitro bovine embryo development and calving rate after embryo transfer (ET). In vitro-matured bovine oocytes from abbatoir-derived ovaries from Korean native, HanWoo cattle were fertilized with frozen-thawed spermatozoa and embryos were cultured in two-step culture media. In Experiment 1, embryos were cultured in media supplemented with 8mg/mL BSA, or 0.1mg/mL PVA and 8mg/mL BSA+2.77mM myo-inositol or 0.1mg/mL PVA+2.77mM myo-inositol. Although defined culture media containing PVA supported lower developmental competence compared to undefined media (containing BSA; 8% versus 34%, respectively), defined culture media containing 2.77mM myo-inositol increased rates of blastocyst formation up to 28%. In Experiment 2, the effect of energy substrate (1.5mM glucose or 1.2mM phosphate) in PVA-myo-inositol defined culture medium on in vitro embryo development was investigated. Defined culture media containing PVA, myo-inositol and phosphate supported better embryo development to blastocysts compared to medium supplemented with both glucose and phosphate (43% versus 31%). In Experiment 3, the effect of epidermal growth factor (EGF) in PVA+myo-inositol-phosphate two-step culture medium on in vitro embryo development was investigated. Among 0, 1, 10 and 100ng/mL EGF concentrations, the maximal effect was observed with 10ng/mL EGF (52% blastocyst formation). In Experiment 4, total cell number and calving rate were compared between defined PVA-myo-inositol-phosphate-EGF medium and undefined medium containing BSA, glucose and phosphate. No differences in total cell number of blastocysts obtained from the two groups were observed, however, the rate of viable offspring production was increased using the defined culture medium, compared to the undefined culture medium. In Experiment 5, the relative abundance of mRNA transcripts [interferon-tau (If-tau), glucose transporter-1 (glut-1) and insulin like growth factor 2 receptor (Igf2r)] were analyzed in blastocysts derived from undefined or defined culture media. Gene expression of If-tau, glut-1 was significantly increased in defined culture medium compared to undefined medium. In conclusion, chemically defined culture media without BSA or FBS improved developmental competence of in vitro cultured bovine embryos and delivery of viable calves after ET.

Published 15 December 2006 in Theriogenology, 67(2): 293-302.
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