Division of Applied Life Science (BK21 Program), Graduate School of Gyeongsang National University, Jinju, Gyeongsangnam-do, Republic of Korea.
Dairy Cattle Improvement Center, National Agricultural Cooperation Federation, Goyang-si, Gyeonggi-do, Republic of Korea.
Theriogenology. 2014 Mar 15;81(5):675-82.e1. doi: 10.1016/j.theriogenology.2013.11.010. Epub 2013 Nov 21.
The production of embryos with a preselected sex sperm is important in the livestock industry. In this study, we examined the efficiency of producing female embryos by intracytoplasmic sperm injection (ICSI) with flow cytometry sorted (ssICSI) and unsorted (usICSI) bovine sperm, and their developmental competence in vitro. For comparison, bovine embryos were also produced by in vitro fertilization (IVF) with sorted (ssIVF) and unsorted (usIVF) bovine sperm. The semen used in this study was from a bull selected for its high fertility and blastocyst developmental competence among four bulls. We first examined and compared pronuclear (PN) formation and cleavage rates of the produced embryos among the treatment groups. Our results demonstrated that PN formation rates (judged by two pronucleus [2PN]) and cleavage rates in ssIVF group (23.1% and 43.6%) were lower than those in the usIVF (71.1% and 71.6%), usICSI (73.1% and 92.8%) and ssICSI (75% and 79.1%) groups, respectively (P < 0.05). Moreover, the blastocyst formation rate in the ssIVF group was less than those in the usIVF, usICSI, and ssICSI groups (2.7% vs. 30.2%, 28.7% and 24.7%, respectively; P < 0.05). Importantly, we reported that the blastocyst formation rate in the ssICSI group was similar to that in the usICSI group, which indicated that ICSI can rescue the damage introduced to sperm by flow cytometry-mediated sex-sorting. Of note, we achieved a blastocyst formation rate in the ssICSI group to be comparable with the usIVF group. We then examined embryo quality by counting the number of normal and apoptotic cells in blastocysts. It was found that, despite the fact that blastocyst formation rate in the ssIVF group was significantly lower than those in the usIVF, usICSI and ssICSI groups, there was no difference in total and apoptotic cell numbers among these groups (P > 0.05). Finally, karyotyping analysis demonstrated that the proportion of female embryos in the ssICSI and ssIVF groups was 100%, whereas it was 58.8% and 57.8% in the usIVF and usICSI groups, respectively. In conclusion, ICSI with flow cytometry sorted bovine sperm provides an alternative approach to produce embryos with predetermined sex.
通过胞浆内精子注射（ICSI）结合流式细胞分选（ssICSI）和未分选（usICSI）牛精子生产具有预选性别的胚胎在畜牧业中很重要。在这项研究中，我们研究了通过体外受精（IVF）结合流式细胞分选（ssIVF）和未分选（usIVF）牛精子生产牛胚胎的效率，以及它们的体外发育能力。为了比较，我们还通过体外受精（IVF）结合流式细胞分选（ssIVF）和未分选（usIVF）牛精子生产牛胚胎。本研究使用的精液来自于在 4 头公牛中选择的一头具有高繁殖力和囊胚发育能力的公牛。我们首先检查并比较了各处理组中产生胚胎的原核（PN）形成和卵裂率。我们的结果表明，ssIVF 组的原核形成率（通过两个原核[2PN]判断）和卵裂率（23.1%和 43.6%）均低于 usIVF 组（71.1%和 71.6%）、usICSI 组（73.1%和 92.8%）和 ssICSI 组（75%和 79.1%）（P<0.05）。此外，ssIVF 组的囊胚形成率低于 usIVF、usICSI 和 ssICSI 组（2.7%比 30.2%、28.7%和 24.7%；P<0.05）。重要的是，我们报告说，ssICSI 组的囊胚形成率与 usICSI 组相似，这表明 ICSI 可以挽救流式细胞仪介导的性别分选对精子造成的损伤。值得注意的是，我们使 ssICSI 组的囊胚形成率与 usIVF 组相当。然后，我们通过计数囊胚中正常和凋亡细胞的数量来检查胚胎质量。结果发现，尽管 ssIVF 组的囊胚形成率明显低于 usIVF、usICSI 和 ssICSI 组，但这些组之间的总细胞和凋亡细胞数量没有差异（P>0.05）。最后，核型分析表明，ssICSI 和 ssIVF 组中雌性胚胎的比例为 100%，而 usIVF 和 usICSI 组分别为 58.8%和 57.8%。总之，流式细胞仪分选牛精子的 ICSI 为生产预定性别的胚胎提供了一种替代方法。
