Lim Kwang Taek, Jang Goo, Ko Kyung Hee, Lee Won Wou, Park Hee Jung, Kim Jung Joo, Lee So Hyun, Hwang Woo Suk, Lee Byeong Chun, Kang Sung Keun
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea.
Theriogenology. 2007 Jan 15;67(2):293-302. doi: 10.1016/j.theriogenology.2006.07.011. Epub 2006 Sep 15.
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.
在本研究中，我们开发了一种特定培养基，该培养基能支持体外培养的牛胚胎发育得到改善，并提高胚胎移植（ET）后的产犊率。从韩国本土韩牛屠宰场采集的卵巢中获取体外成熟的牛卵母细胞，用冻融精子进行受精，胚胎在两步培养基中培养。在实验1中，胚胎在补充有8mg/mL牛血清白蛋白（BSA）、或0.1mg/mL聚乙烯醇（PVA）以及8mg/mL BSA + 2.77mM肌醇、或0.1mg/mL PVA + 2.77mM肌醇的培养基中培养。尽管与未定义培养基（含BSA）相比，含PVA的特定培养基支持的发育能力较低（分别为8%和34%），但含2.77mM肌醇的特定培养基使囊胚形成率提高至28%。在实验2中，研究了PVA - 肌醇特定培养基中能量底物（1.5mM葡萄糖或1.2mM磷酸盐）对体外胚胎发育的影响。与同时添加葡萄糖和磷酸盐的培养基相比，含PVA、肌醇和磷酸盐的特定培养基支持胚胎更好地发育至囊胚（43%对31%）。在实验3中，研究了PVA + 肌醇 - 磷酸盐两步培养基中表皮生长因子（EGF）对体外胚胎发育的影响。在0、1、10和100ng/mL EGF浓度中，10ng/mL EGF观察到最大效果（囊胚形成率52%）。在实验4中，比较了特定的PVA - 肌醇 - 磷酸盐 - EGF培养基和含BSA、葡萄糖和磷酸盐的未定义培养基之间的囊胚总细胞数和产犊率。两组获得的囊胚总细胞数未观察到差异，然而，与未定义培养基相比，使用特定培养基可提高活仔畜的生产比率。在实验5中，分析了从未定义或特定培养基获得的囊胚中mRNA转录本[干扰素 - τ（If - τ）、葡萄糖转运蛋白 - 1（glut - 1）和胰岛素样生长因子2受体（Igf2r）]的相对丰度。与未定义培养基相比，特定培养基中If - τ、glut - 1的基因表达显著增加。总之，不含BSA或胎牛血清（FBS）的化学成分明确的培养基提高了体外培养牛胚胎的发育能力以及ET后活犊牛的产出率。
