Laboratory of Embryology and Reproductive Biotechniques-FAVET-UFRGS, Porto Alegre (RS), Brazil.
J Virol Methods. 2013 Feb;187(2):443-8. doi: 10.1016/j.jviromet.2012.11.029. Epub 2012 Dec 5.
The aim of this study was to evaluate the capacity of three semen processing techniques, Percoll gradient centrifugation, Swim-up and a combination of Swim-up and Percoll gradient centrifugation, to reduce the viral load of bovine viral diarrhea virus (BVDV) in experimentally infected semen samples. The evaluation was performed using two approaches: first, searching for the presence of virus in the processed samples (via virus titration and RT-PCR) and second, ascertaining the possible interference on in vitro embryo production. The sperm count and DNA integrity (Comet assay) of the processed samples were analyzed (Experiment 1). The amount of virus in the processed samples was determined by titration in cell culture (Experiment 2). The samples processed by Swim up/Percoll gradient centrifugation were utilized for in vitro embryo production, and the embryos produced were tested for BVDV by RT-PCR (Experiment 3). Sperm concentration, Comet assay and embryo production were analyzed by chi-squared tests (P<0.05). There was a significant difference between sperm separation techniques when the sperm count and Comet assay were analyzed. The sperm count obtained from the Swim up/Percoll gradient centrifugation group was lower than that obtained in either of the two other groups (Swim up and Percoll gradient centrifugation), and the Comet assay showed that the combination of the two semen processing techniques (Swim up/Percoll gradient) produced a 1.1% prevalence of Comet level 2, which was not observed in the other groups. The BVDV titer (10(6.68)TCID(50)/mL) added to experimentally infected semen samples decreased after Percoll gradient centrifugation to 10(2.3)-10(1)TCID(50)/mL; for the Swim up group, the titer range was 10(3.3)-10(1.87)TCID(50)/mL, and in the Swim up/Percoll gradient centrifugation group, BVDV was undetectable. The decreases in titer varied from 99.9% in the Swim up-processed group to 100% in the Swim up/Percoll gradient centrifugation group. In vitro embryo production displayed similar blastocyst development rates among all groups, and RT-PCR was negative for the produced embryos. The data showed that the combination of Swim up/Percoll gradient centrifugation promoted the elimination of BVDV from the semen samples without damaging spermatozoa cells and also allowed successful in vitro embryo production free of BVDV. Hence, the risk of BVDV contamination is negligible for the embryo recipient.
本研究旨在评估三种精液处理技术，即 Percoll 梯度离心、精子泳动和精子泳动与 Percoll 梯度离心联合应用，对牛病毒性腹泻病毒（BVDV）在实验感染精液样本中的病毒载量的降低效果。采用两种方法进行评估：首先，通过病毒滴定和 RT-PCR 检测处理后样本中病毒的存在情况；其次，确定其对体外胚胎生产的可能干扰。分析了处理后样本的精子计数和 DNA 完整性（彗星试验）（实验 1）。通过细胞培养中的滴定法确定处理后样本中的病毒量（实验 2）。利用精子泳动/Percoll 梯度离心处理的样本进行体外胚胎生产，并通过 RT-PCR 检测生产的胚胎是否存在 BVDV（实验 3）。采用卡方检验（P<0.05）分析精子分离技术的精子计数和彗星试验。当分析精子计数和彗星试验时，精子分离技术之间存在显著差异。精子泳动/Percoll 梯度离心组的精子计数低于其他两组（精子泳动组和 Percoll 梯度离心组），彗星试验显示两种精液处理技术的组合（精子泳动/Percoll 梯度离心）产生了 1.1%的彗星水平 2 的流行率，而其他两组则没有观察到这种情况。将 BVDV 滴度（10(6.68)TCID(50)/mL）加入到实验感染的精液样本中，经 Percoll 梯度离心后降低至 10(2.3)-10(1)TCID(50)/mL；对于精子泳动组，滴度范围为 10(3.3)-10(1.87)TCID(50)/mL，而在精子泳动/Percoll 梯度离心组中，BVDV 无法检测到。滴度降低率从精子泳动处理组的 99.9%到精子泳动/Percoll 梯度离心组的 100%不等。体外胚胎生产显示所有组的囊胚发育率相似，且生产的胚胎 RT-PCR 结果均为阴性。数据表明，精子泳动/Percoll 梯度离心联合应用促进了精液样本中 BVDV 的清除，而不损害精子细胞，同时也允许无 BVDV 的体外胚胎生产。因此，对于胚胎接受者来说，BVDV 污染的风险可以忽略不计。
