Clinic for Cattle, University of Veterinary Medicine, Hanover, Germany.
Theriogenology. 2012 Jul 1;78(1):132-9. doi: 10.1016/j.theriogenology.2012.01.028. Epub 2012 Mar 22.
Although sex-sorted sperm have been used for AI and IVF for over a decade there is still need to improve the technology as the results are highly variable. The goal of the present study was to assess the effect of seminal plasma and seminal plasma proteins as a supplement to sorted sperm on subsequent embryonic development, as a beneficial effect of these substances has been reported. In vitro matured oocytes were fertilized in vitro with either unsorted sperm (n = 215; Group 1), bulk sorted sperm (n = 226; Group 2), bulk sorted sperm extended in the presence of 1% seminal plasma (n = 185; Group 3) or bulk sorted sperm supplemented with seminal plasma proteins (4 mg mL(-1); n = 254; Group 4). An additional group of oocytes (n = 307; Group 5) was fertilized with the semen of another bull routinely used for IVF and served as a laboratory standard control. Subsequently, the presumptive zygotes were cultured for 8 days under standard conditions (SOFaa, 39 °C, 5% CO(2), 5% N(2)). Cleavage rates were assessed on day 3 p.i. (post insemination; group 1: 30.5 ± 14.7%; group 2: 28.8 ± 9.8%; group 3: 20.8 ± 14.9%; group 4: 25.7 ± 8.2%; group 5: 54.8 ± 11.5%). Development rates were documented on days 7 p.i. (group 1: 7.3 ± 6.6%; group 2: 5.6 ± 3.1%, group 3: 6.2 ± 7.7%, group 4: 6.7 ± 5.9%, group 5: 20.2 ± 6.9%) and 8 p.i. (group 1: 8.9 ± 7.0%; group 2: 6.0 ± 2.9%; group 3: 8.6 ± 11.3%; group 4: 7.8 ± 6.2%; group 5: 23.3 ± 7.8%), respectively. Significant differences among cleavage and development rates could only be seen for Group 5 compared to all other groups. However, this difference between Groups 1-4 vs. Group 5 regarding the development rates on Day 8 could not be detected when assessing the development rates on base of the number of cleaved embryos instead of the number of oocytes fertilized (group 1: 31.4 ± 17.2%; group 2: 26.0 ± 21.0%; group 3: 33.3 ± 19.05%; group 4: 26.6 ± 17.8%; group 5: 42.6 ± 11.3%). The relative abundance of six different developmentally important gene transcripts (G6PD, HSP1A1, SLC2A3, BAX, BCL2L1, DNMT3A) was determined using single Day 8 expanded blastocysts of all five groups. No significant differences were seen among the embryos of the five groups. Our results show that neither the bulk sorting procedure nor the addition of seminal plasma or seminal plasma proteins, respectively, affected cleavage and development rates when sperm from a specific bull was used. Additionally, sorting and subsequent exposure of sperm to either seminal plasma or seminal plasma proteins did not influence mRNA expression in bovine IVP embryos.
尽管用于 AI 和 IVF 的性别分选精子已经使用了十多年，但仍需要改进该技术，因为结果高度可变。本研究的目的是评估精液和精液蛋白作为补充物对分选精子后续胚胎发育的影响，因为已有报道称这些物质具有有益作用。体外成熟的卵母细胞在体外与未分选的精子（n=215；第 1 组）、批量分选的精子（n=226；第 2 组）、在存在 1%精液的情况下扩展的批量分选的精子（n=185；第 3 组）或用精液蛋白补充的批量分选的精子（4mg mL(-1)；n=254；第 4 组）受精。另外一组卵母细胞（n=307；第 5 组）用常规用于 IVF 的另一头公牛的精液受精，作为实验室标准对照。随后，将假定的受精卵在标准条件下（SOFaa，39°C，5%CO(2)，5%N(2))培养 8 天。第 3 天（受精后）评估卵裂率（第 1 组：30.5±14.7%；第 2 组：28.8±9.8%；第 3 组：20.8±14.9%；第 4 组：25.7±8.2%；第 5 组：54.8±11.5%）。第 7 天（第 1 组：7.3±6.6%；第 2 组：5.6±3.1%，第 3 组：6.2±7.7%，第 4 组：6.7±5.9%，第 5 组：20.2±6.9%）和第 8 天（第 1 组：8.9±7.0%；第 2 组：6.0±2.9%；第 3 组：8.6±11.3%；第 4 组：7.8±6.2%；第 5 组：23.3±7.8%）记录发育率。仅第 5 组与所有其他组之间的卵裂和发育率存在显著差异。然而，当根据分裂胚胎的数量而不是受精卵母细胞的数量评估第 8 天的发育率时，第 1-4 组与第 5 组之间的差异（第 1 组：31.4±17.2%；第 2 组：26.0±21.0%；第 3 组：33.3±19.05%；第 4 组：26.6±17.8%；第 5 组：42.6±11.3%）无法检测到。使用所有五组的单个第 8 天扩展囊胚确定了六个不同发育重要基因转录物（G6PD、HSP1A1、SLC2A3、BAX、BCL2L1、DNMT3A）的相对丰度。五组胚胎之间未见显著差异。我们的结果表明，当使用特定公牛的精子时，无论是批量分选过程还是添加精液或精液蛋白，都不会影响卵裂和发育率。此外，精子的分选和随后暴露于精液或精液蛋白均不会影响牛 IVP 胚胎的 mRNA 表达。
