LUNAM University, Oniris, Nantes-Atlantic National College of Veterinary Medicine, Food Science and Engineering, Nantes, France.
Theriogenology. 2012 Feb;77(3):644-51. doi: 10.1016/j.theriogenology.2011.09.004. Epub 2011 Oct 19.
For each of the five fertilization trials of the experiment, frozen semen was prepared for in vitro capacitation at a concentration of 1 × 10(7) spz/ml and divided into three groups. One group was used as a control, while the two others were inoculated with 100 μl/ml of either culture medium from non-infected cells (placebo group) or cell culture medium containing virus at a concentration of 10(5) TCID(50)/ml (infected group). A total of 789 oocytes were used for IVF. For each of the five trials a group of oocytes were used as a non-infected control and were found to be caprine arthritis-encephalitis virus (CAEV) free. The other oocytes were divided in two equal batches. Oocytes in the first batch were in vitro fertilized with CAEV infected sperm (infected group) and the second batch were fertilized with CAEV non-infected sperm (placebo and control groups). After IVF, the zygotes of each group were washed 12 times. The CAEV genome was not detected (using RT-PCR) in the washing media of either the control or placebo groups from each trial. In contrast, the first three washing media from the infected group were consistently found to be positive for the CAEV genome (5/5), whereas subsequent washing media were CAEV-free (P < 0.05). Zygotes obtained using all semen groups tested negative for both the provirus and genome of CAEV. These results clearly show that the first four washes were sufficient to remove viral particles from CAEV infected fertilization media and that CAEV-free embryos can be produced by IVF using spermatozoa infected in vitro by CAEV.
在该实验的五次受精试验中，每份冷冻精液均在浓度为 1×10(7)spz/ml 的条件下进行体外获能，并分为三组。一组作为对照组，另外两组分别接种浓度为 10(5)TCID(50)/ml 的病毒培养液（感染组）或不含感染细胞的培养液（安慰剂组）100 μl/ml。共使用了 789 个卵母细胞进行 IVF。五次试验中的每一次，一组卵母细胞均作为非感染对照，且被证实无山羊关节炎脑炎病毒（CAEV）。其他卵母细胞被分为两等份。第一份卵母细胞与 CAEV 感染的精子进行体外受精（感染组），第二份卵母细胞与 CAEV 非感染的精子进行受精（安慰剂和对照组）。IVF 后，每组的受精卵均进行 12 次洗涤。在各试验中，从对照组和安慰剂组的洗涤液中均未检测到 CAEV 基因组（使用 RT-PCR）。相比之下，感染组的前三个洗涤液始终为 CAEV 基因组阳性（5/5），而后续的洗涤液均不含 CAEV（P<0.05）。使用所有精液组获得的受精卵均对 CAEV 的前病毒和基因组检测均为阴性。这些结果清楚地表明，前四次洗涤足以从 CAEV 感染的受精培养液中去除病毒颗粒，并且可以通过使用 CAEV 体外感染的精子进行 IVF 来产生无 CAEV 的胚胎。
