Keskintepe L, Morton P C, Smith S E, Tucker M J, Simplicio A A, Brackett B G
Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens 30602, USA.
Zygote. 1997 Aug;5(3):261-5. doi: 10.1017/s0967199400003701.
Experiments were undertaken to develop intracytoplasmic sperm injection (ICSI) to produce caprine embryos out of the normal breeding season. Oocytes were obtained from 2-6 mm ovarian follicles at slaughter. Selected oocytes with two to four layers of cumulus cells were incubated in 1 ml of H-TCM199 supplemented with 10 micrograms each of oFSH and bLH (NHPP, NIDDK, NICHD, USDA) and 20% fetal bovine serum (FBS) in a thermos (38.5 degrees C) for 4.5 h during transportation. Then, oocytes were transferred into 75 microliters of freshly prepared maturation medium under paraffin oil and a mixture of 5% O2, 5% CO2 and 90% N2. Approximately 26 h after recovery oocytes were denuded by incubation with hyaluronidase (100 IU/ml) and pipetting and held at 38.5 degrees C for 90 min. Spermatozoa frozen in egg yolk extender were thawed in a 37 degrees C water bath for 15 s. Motile fractions were selected by swim-up, then incubated for 90 min in TALP with 10 micrograms heparin/ml. Each oocyte was positioned with its first polar body at 6 or 12 o'clock by a holding pipette. Sperm (1 microliter) were added to 10 microliters medium containing 10% polyvinylpyrrolidone. A sperm cell was aspirated into a pipette, and then injected head-first into the cytoplasm of an oocyte maintained in H-TCM199 + 20% FBS at 37 degrees C. Injected oocytes were transferred to HM and, after 90 min, cultured in 50 microliters of BSA-free synthetic oviduct fluid plus polyvinyl alcohol, citrate and non-essential amino acids. Results demonstrate that caprine blastocysts can be produced outside the breeding season by the use of frozen-thawed semen and injection of sperm cells with broken tails into ova followed by culture in defined medium.
开展了实验以开发胞浆内单精子注射（ICSI）技术，用于在正常繁殖季节之外生产山羊胚胎。在屠宰时从2 - 6毫米的卵巢卵泡中获取卵母细胞。将挑选出的带有两到四层卵丘细胞的卵母细胞置于1毫升添加了10微克促卵泡素（oFSH）和促黄体素（bLH）（美国国立卫生研究院、美国国立儿童健康与人类发展研究所、美国农业部国家激素和垂体项目）以及20%胎牛血清（FBS）的H - TCM199中，在保温瓶（38.5摄氏度）中运输4.5小时。然后，将卵母细胞转移到石蜡油下75微升新配制的成熟培养基中，并置于5%氧气、5%二氧化碳和90%氮气的混合气体环境中。回收后约26小时，用透明质酸酶（100 IU/ml）孵育并吹打去除卵丘细胞，将卵母细胞置于38.5摄氏度下90分钟。用蛋黄稀释液冷冻保存的精子在37摄氏度水浴中解冻15秒。通过上游法挑选出活动精子部分，然后在含10微克/毫升肝素的输卵管液（TALP）中孵育90分钟。用固定吸管将每个卵母细胞的第一极体置于6点或12点位置。将1微升精子加入到10微升含10%聚乙烯吡咯烷酮的培养基中。用吸管吸取一个精子细胞，然后将其头部先端注入到37摄氏度下保存在H - TCM199 + 20% FBS中的卵母细胞胞质内。将注射后的卵母细胞转移到改良的人类输卵管液（HM）中，90分钟后，在50微升不含牛血清白蛋白（BSA）的合成输卵管液加聚乙烯醇、柠檬酸盐和非必需氨基酸的培养基中培养。结果表明，在繁殖季节之外，通过使用冻融精液、将断尾精子细胞注射到卵母细胞中并在特定培养基中培养，可以生产山羊囊胚。
