Amirov A R, Krivokharchenko A S, Ivanova L B, Vil'ianovich L I
All-Russian Research Institute of Physiology, Biochemistry, and Nutrition of Agricultural Animals, Russian Academy of Agricultural Sciences, Borovsk, Russia.
Ontogenez. 1998 Jul-Aug;29(4):254-7.
We have examined possible use of the follicle-stimulating hormone (FSH) for the induction of superovulation in pigs and studied the effect of biopsy of preimplantation pig embryos on their survival in vitro. Superovulation was induced by injecting FSH twice daily over a period of four days for a total dose of 25 units. per animal. Pigs receiving pregnant mare serum gonadotropin (PMSG) according to the standard scheme served as the control. The experiments demonstrated that FSH produces significantly better estrus figures as compared with PMSG (100% and 60%, respectively; p < 0.05). The mean number of ovulations per donor was also better in the FSH group, i.e., 36.5 as compared with 17.3 in the control group (p < 0.05). The mean number of embryos obtained from one donor was higher in FSH treated animals as compared with the animals that received PMSG (29.2 vs. 19.3), however, this difference was not statistically significant. Biopsy of preimplantation pig embryos was conducted by a "manual" technique without a micromanipulator; middle blastocysts collected at days 5-6 after insemination were the most convenient for this operation. Embryos at these developmental stages contained a well developed inner cell mass, which allows separation of trophoectodermal cells only, thus minimizing damage to the embryo. The number of cells in dissected fragments did not depend on the stage of development, and even smallest fragments (4 blastomeres) were sufficient for genome analysis with the aid of PCR. The survival of embryos in vitro after biopsy practically did not differ from that of control intact embryos. Thus, we demonstrated the effective use of FSH for the induction of superovulation in pigs; we also determined the developmental stages which are most convenient for conducting biopsies and developed a technique for preimplantation biopsy, which allows genome analysis of embryos without any decrease of their survival in vitro.
我们研究了促卵泡激素(FSH)在诱导猪超数排卵中的潜在用途,并研究了植入前猪胚胎活检对其体外存活的影响。通过在四天内每天注射两次FSH来诱导超数排卵,每只动物的总剂量为25单位。按照标准方案接受孕马血清促性腺激素(PMSG)的猪作为对照。实验表明,与PMSG相比,FSH产生的发情率明显更高(分别为100%和60%;p<0.05)。FSH组每个供体的平均排卵数也更好,即36.5个,而对照组为17.3个(p<0.05)。与接受PMSG的动物相比,接受FSH处理的动物从一个供体获得的胚胎平均数量更高(29.2个对19.3个),然而,这种差异没有统计学意义。植入前猪胚胎的活检采用“手工”技术,无需显微操作器;授精后第5-6天收集的中期囊胚最适合此操作。处于这些发育阶段的胚胎含有发育良好的内细胞团,这使得仅分离滋养外胚层细胞成为可能,从而将对胚胎的损伤降至最低。解剖碎片中的细胞数量不取决于发育阶段,即使是最小的碎片(4个卵裂球)也足以借助PCR进行基因组分析。活检后胚胎在体外的存活率与对照完整胚胎的存活率实际上没有差异。因此,我们证明了FSH在诱导猪超数排卵中的有效应用;我们还确定了最适合进行活检的发育阶段,并开发了一种植入前活检技术,该技术允许对胚胎进行基因组分析,而不会降低其体外存活率。