Improved in vitro bovine embryo development and increased efficiency in producing viable calves using defined media.
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 8 mg/mL BSA, or 0.1mg/mL PVA and 8 mg/mL BSA+2.77 mM myo-inositol or 0.1mg/mL PVA+2.77 mM 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 2.77 mM 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 100 ng/mL EGF concentrations, the maximal effect was observed with 10 ng/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.
Lim
Kwang Taek
KT
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea.
Jang
Goo
G
Ko
Kyung Hee
KH
Lee
Won Wou
WW
Park
Hee Jung
HJ
Kim
Jung Joo
JJ
Lee
So Hyun
SH
Hwang
Woo Suk
WS
Lee
Byeong Chun
BC
Kang
Sung Keun
SK
D000818:Animals; D000831:Animals, Newborn; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D002417:Cattle / Q000196:embryology* / Q000502:physiology; D003470:Culture Media / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004624:Embryo Transfer / Q000662:veterinary*; D047108:Embryonic Development / Q000187:drug effects / Q000502:physiology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D018507:Gene Expression Regulation, Developmental; D051272:Glucose Transporter Type 1 / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D007370:Interferon Type I / Q000235:genetics; D011247:Pregnancy; D011256:Pregnancy Outcome; D011257:Pregnancy Proteins / Q000235:genetics; D018873:Pregnancy Rate; D012333:RNA, Messenger / Q000378:metabolism; D017527:Receptor, IGF Type 2 / Q000235:genetics
D003470:Culture Media; D051272:Glucose Transporter Type 1; D007370:Interferon Type I; D011257:Pregnancy Proteins; D012333:RNA, Messenger; D017527:Receptor, IGF Type 2; C020947:interferon tau; D004815:Epidermal Growth Factor; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases