Li Jingchun, Zhu Sibing, He Xianjing, Sun Rui, He Qianyu, Gan Yi, Liu Shengjun, Funahashi Hiroaki, Li Yanbing
Department of Animal Science, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, China.
Department of Animal Science, Graduate School of Environmental and Life Science, Okayama University, Tsushima-Naga, Okayama, Japan.
Theriogenology. 2016 Apr 15;85(7):1211-8. doi: 10.1016/j.theriogenology.2015.12.001. Epub 2015 Dec 13.
Viable sperm from sex-sorted semen without centrifugal treatment was separated by a microfluidic sperm sorter (MFSS) for IVF to improve in vitro embryo production of dairy cattle. The MFSS was originally developed to isolate motile human sperm by two laminar flows in the micro-channel (there are four chambers in an MFSS. Chamber A is the inlet for semen, chamber B is the inlet for the medium, chamber C is the exit chamber for motile sperm, and chamber D is the outlet for nonmotile sperm). Sex-sorted sperm were adjusted to 1 × 10(7) spermatozoa/mL (2 million cells/dose, sperm motility was 30% above after thawing). In a first experiment, diluted sex-sorted semen was mixed with modified Medium199(mM199) containing 5-mM caffeine for 5 minutes, resulting in variations in sperm concentration and quality parameters at chambers A, C, and D. In a second experiment, medium containing sperm from three MFSS chambers was collected and mitochondrial activity of the sperm was determined by flow cytometry, the relative activity of sperm mitochondria in chamber C (1.56 ± 0.03) was the highest in three observation areas (P < 0.05). Thus, sperm motility and mitochondrial activity of sperm was high in chamber C. In a third experiment, different concentrations of sperm were added to chamber A and dairy cattle IVM oocytes were placed in chamber C, where motile spermatozoa will accumulate, with mM199 containing 5-mM caffeine for 5 minutes, and then cultured in caffeine-free mM199 for 8 hours. The results showed that sperm penetration rate, the monospermic penetration rate, and blastocyst rate of the 10 × 10(6) group (10 × 10(6) sperm/mL) were higher than in the 1 × 10(6) and 5 × 10(6) groups (P < 0.05). In the last experiment, we compared sperm penetration in the MFSS-IVF system with a modified standard IVF method (cocultured in droplets for 8 hours). The normal fertilization index (the ratio of monospermic oocytes to the number of oocytes examined) 8 hours after insemination was higher in the MFSS-IVF system than the modified standard IVF system (P < 0.05). Developmental competence of fertilized oocytes to the blastocyst stage was also higher in the MFSS-IVF system (40.12% ± 2.61%) than the modified standard IVF technique (24.55% ± 4.54%). These results demonstrate that a short coculture of dairy cattle oocytes with isolated motile sex-sorted spermatozoa gradually accumulated in the MFSS device improves the efficiencies of normally produced fertilized embryos and blastocyst formation.
通过微流控精子分选仪（MFSS）对未经离心处理的性别分选精液中的活精子进行分离，用于体外受精，以提高奶牛的体外胚胎生产效率。MFSS最初是为通过微通道中的两个层流来分离活动的人类精子而开发的（一个MFSS有四个腔室。腔室A是精液入口，腔室B是培养基入口，腔室C是活动精子的出口腔室，腔室D是非活动精子的出口）。将性别分选的精子调整至1×10⁷精子/mL（每剂量200万个细胞，解冻后精子活力高于30%）。在第一个实验中，将稀释的性别分选精液与含有5 mM咖啡因的改良199培养基（mM199）混合5分钟，导致腔室A、C和D处的精子浓度和质量参数出现变化。在第二个实验中，收集来自三个MFSS腔室的含精子培养基，并通过流式细胞术测定精子的线粒体活性，腔室C中精子线粒体的相对活性（1.56±0.03）在三个观察区域中最高（P<0.05）。因此，腔室C中的精子活力和线粒体活性较高。在第三个实验中，向腔室A中添加不同浓度的精子，并将奶牛体外成熟（IVM）卵母细胞置于腔室C中（活动精子将在此处聚集），与含有5 mM咖啡因的mM199混合5分钟，然后在不含咖啡因的mM199中培养8小时。结果显示，10×10⁶组（10×10⁶精子/mL）的精子穿透率、单精子穿透率和囊胚率高于1×10⁶和5×10⁶组（P<0.05）。在最后一个实验中，我们将MFSS-体外受精（IVF）系统中的精子穿透情况与改良的标准IVF方法（在液滴中共培养8小时）进行了比较。授精8小时后的正常受精指数（单精子受精的卵母细胞与检查的卵母细胞数量之比）在MFSS-IVF系统中高于改良的标准IVF系统（P<0.05）。MFSS-IVF系统中受精卵母细胞发育至囊胚阶段的能力也高于改良的标准IVF技术（40.12%±2.61%）（24.55%±4.54%）。这些结果表明，奶牛卵母细胞与在MFSS装置中逐渐聚集的分离活动的性别分选精子进行短时间共培养，可提高正常产生的受精卵和囊胚形成的效率。
