Dinkins M B, Stallknecht D E, Brackett B G
Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7389, USA.
Anim Reprod Sci. 2001 Mar 30;65(3-4):205-13. doi: 10.1016/s0378-4320(01)00075-6.
Infectious viruses bind more tenaciously to the zonae pellucidae of in vitro produced bovine embryos than to zonae of in vivo derived embryos. Currently, the International Embryo Transfer Society recommends that all in vivo derived embryos be subjected to a rigorous washing procedure in combination with exposure to trypsin to remove viruses adherent to the zonae. In contrast to in vivo derived embryos, this method is not effective for disinfecting in vitro produced embryos. Our hypothesis was that a more potent, non-specific protease from Streptomyces griseus (S. griseus) would provide a more effective treatment for virus removal from in vitro produced bovine embryos. Bovine oocytes were matured, fertilized, and cultured in completely defined in vitro conditions. Zygotes were washed according to the procedure outlined by the International Embryo Transfer Society, replacing trypsin with the experimental protease. Experimental incubations were with 0.1% (4 units/ml) protease for 0, 30, 45, 60 and 75s intervals. Embryos were able to withstand exposure to this enzymatic treatment for only 45s before their developmental potential was significantly reduced; 60s exposure was detrimental (P<0.05). Oocytes were exposed to epizootic hemorrhagic disease virus serotype 2 (EHDV-2, 10(6) TCID(50)/ml) during in vitro maturation. Resulting zygotes were washed according to the International Embryo Transfer Society procedure and either exposed to trypsin or protease. Exposure to EHDV-2 prevented cumulus expansion and markedly reduced embryonic development (P<0.05). There were no differences in development among virus exposed groups receiving no treatment or treatment with trypsin or protease. However, proportions of infected embryos were reduced after protease treatment versus positive controls and trypsin treated embryos.
与体内来源胚胎的透明带相比，感染性病毒与体外生产的牛胚胎的透明带结合更紧密。目前，国际胚胎移植协会建议对所有体内来源的胚胎进行严格的洗涤程序，并结合胰蛋白酶处理，以去除附着在透明带上的病毒。与体内来源的胚胎不同，这种方法对体外生产的胚胎消毒无效。我们的假设是，来自灰色链霉菌（S. griseus）的一种更强效的非特异性蛋白酶将为从体外生产的牛胚胎中去除病毒提供更有效的处理方法。牛卵母细胞在完全确定的体外条件下成熟、受精并培养。受精卵按照国际胚胎移植协会概述的程序进行洗涤，用实验蛋白酶代替胰蛋白酶。实验孵育分别在0、30、45、60和75秒的间隔内使用0.1%（4单位/毫升）的蛋白酶。胚胎在其发育潜能显著降低之前，仅能承受45秒的这种酶处理；60秒的暴露是有害的（P<0.05）。卵母细胞在体外成熟期间暴露于2型流行性出血病病毒（EHDV-2，10(6) TCID(50)/毫升）。产生的受精卵按照国际胚胎移植协会的程序进行洗涤，然后要么暴露于胰蛋白酶，要么暴露于蛋白酶。暴露于EHDV-2会阻止卵丘扩展并显著降低胚胎发育（P<0.05）。在未接受处理或接受胰蛋白酶或蛋白酶处理的病毒暴露组之间，发育情况没有差异。然而，与阳性对照组和胰蛋白酶处理的胚胎相比，蛋白酶处理后感染胚胎的比例有所降低。
