子宫细胞单层培养系统在显微操作牛胚胎中的应用。

Use of a uterine-cell monolayer culture system for micromanipulated bovine embryos.

作者信息

Voelkel S A, Amborski G F, Hill K G, Godke R A

机构信息

Animal Science Department, Louisiana State University, Baton Rouge, LA 70803 USA.

出版信息

Theriogenology. 1985 Sep;24(3):271-81. doi: 10.1016/0093-691x(85)90218-3.

DOI:10.1016/0093-691x(85)90218-3

PMID:16726080

Abstract

The objective of this study was to evaluate the growth of micromanipulated bovine embryos in two in vitro culture systems. Sixty ova (day 7 from estrus) were collected in Dulbecco's phosphate-buffered saline (PBS), with 2% fetal calf serum, and transferred to a PBS holding medium containing 10% fetal calf serum to prepare for micromanipulation. Forty embryos (morula to expanded blastocyst stages) were selected for embryo splitting using a modified microsurgery procedure. Thirty-nine of these embryos were successfully bisected into demi-embryos (DE) and the halves allotted by post-manipulation quality grades into one of two treatment groups (Trt). DE in Trt A were cultured in Ham's F-10 medium with 10% FCS (HF-10) while the remaining DE halves from each embryo were cocultured in HF-10 on a monolayer of endometrial fibroblasts (8 x 10(4) viable fibroblast cells plated three days prior to culture) in Trt B. Embryo development, recorded at 12-hour intervals, was evaluated by a split-plot analysis of variance. Results indicated that embryo viability decreased (P<0.001) over time in culture. Overall viability was greater (P<0.001) for DE in Trt B than in Trt A, with a significant (P<0.05) Trt x Time interaction, indicating that embryo viability decreased more rapidly across time in HF-10 than in the monolayer coculture system. The percentage of DE developing at 12, 24, 36, 48, 60 and 72 hours in culture was: 44%, 41%, 33%, 28%, 21% and 18% for Trt A and 69%, 69%, 69%, 67%, 62% and 62% for Trt B. Fourteen of the DE in Trt B attached to fibroblast monolayer and initiated trophoblastic outgrowth and four additional DE remained viable for up to 17.5 days in vitro as intact blastocysts. These findings are the first reported that demonstrate that the zona-free bovine DE will develop during in vitro culture. Also, the bovine endometrial fibroblast monolayer system proved to be excellent for both short term (</=12 hours) and long term (up to 72 hours) culture of halved bovine embryos.

摘要

本研究的目的是评估微操作牛胚胎在两种体外培养系统中的生长情况。在含有2%胎牛血清的杜氏磷酸盐缓冲盐水（PBS）中收集60个卵子（发情后第7天），并将其转移至含有10%胎牛血清的PBS保存培养基中，为微操作做准备。使用改良的显微手术程序，选择40个胚胎（桑椹胚至扩张囊胚阶段）进行胚胎分割。其中39个胚胎成功分割为半胚（DE），并根据操作后质量等级将两半分配到两个处理组（Trt）之一。Trt A中的DE在含有10%胎牛血清的Ham's F-10培养基（HF-10）中培养，而来自每个胚胎的其余DE半胚在Trt B中与子宫内膜成纤维细胞单层（培养前三天接种8×10⁴个活的成纤维细胞）共同培养于HF-10中。每隔12小时记录胚胎发育情况，并通过裂区方差分析进行评估。结果表明，培养过程中胚胎活力随时间下降（P<0.001）。Trt B中DE的总体活力高于Trt A（P<0.001），且存在显著的（P<0.05）Trt×时间交互作用，表明在HF-10中胚胎活力随时间下降的速度比在单层共培养系统中更快。培养12、24、36、48、60和72小时时Trt A中发育的DE百分比分别为：44%、41%、33%、28%、21%和18%，Trt B中分别为：69%、69%、69%、67%、62%和62%。Trt B中有14个DE附着于成纤维细胞单层并开始滋养层生长，另外4个DE作为完整囊胚在体外存活长达17.5天。这些发现是首次报道表明无透明带牛DE在体外培养期间会发育。此外，牛子宫内膜成纤维细胞单层系统被证明对于分割后的牛胚胎短期（≤12小时）和长期（长达72小时）培养均非常出色。