Kharche S D, Pathak J, Agarwal S, Kushwah B, Sikarwar Aks
ICAR-CIRG, Makhdoom, Farah, Mathura, UP, India.
Reprod Domest Anim. 2016 Aug;51(4):611-7. doi: 10.1111/rda.12701. Epub 2016 May 11.
The aim of the present investigation was to study the effect of calcium ionophore activation on blastocyst production following intracytoplasmic sperm injection (ICSI) in in vitro-matured Caprine oocytes. A total of 470 in vitro-matured oocytes were selected and randomly divided in to three groups. Cumulus oocyte complexes (COCs) recovered by slicing the Caprine ovaries were matured in TCM199 supplemented with 10% foetal bovine serum (FBS) + 10% follicular fluid + FSH (5 μg/ml) + LH (10 μg/ml) + estradiol (1 μg/ml) + EGF (10 ng/ml) + BSA (3 mg/ml) for 27 h in humidified atmosphere at 38.5°C with 5% CO2 in CO2 incubator. After 27 h of culture, selected COCs (n = 470) were separated from cumulus cells by treating with 0.1% hyaluronidase enzyme and passing repeatedly through a fine pipette and randomly divided into three groups. In group 1, (n = 168) matured oocytes were injected with injection micropipette without sperm as control. In group 2, (n = 152) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette. In group 3, (n = 150) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette and then activated with 5 μm Ca ionophore for 5 min. The oocytes of all groups were then culture in RVCL media for embryo development. The cleavage rate was observed after 48-72 h of injection. The cleavage rate and blastocyst production in group 1, 2 and 3 were 0.00 and 0.00, 18.42 and 3.57 and 61.33% and 16.30%, respectively. The result indicated that mechanical activation failed to induce cleavage in in vitro-matured Caprine oocytes, whereas chemical activation of intracytoplasmic sperm-injected in vitro-matured Caprine oocytes showed significantly higher cleavage rate and blastocyst production as compare to non-activated oocytes.
本研究的目的是探讨钙离子载体激活对体外成熟的山羊卵母细胞胞浆内单精子注射（ICSI）后囊胚产生的影响。共选取470个体外成熟的卵母细胞，随机分为三组。通过切割山羊卵巢回收的卵丘卵母细胞复合体（COCs）在补充有10%胎牛血清（FBS）+10%卵泡液+促卵泡素（FSH，5μg/ml）+促黄体生成素（LH，10μg/ml）+雌二醇（1μg/ml）+表皮生长因子（EGF，10ng/ml）+牛血清白蛋白（BSA，3mg/ml）的TCM199中，于38.5°C、5%二氧化碳的湿润培养箱中培养27小时。培养27小时后，用0.1%透明质酸酶处理并反复通过细吸管，将选取的COCs（n = 470）与卵丘细胞分离，随机分为三组。第1组（n = 168），将成熟卵母细胞用注射微吸管注射但不注射精子作为对照。第2组（n = 152），将获能精子通过注射微吸管注入体外成熟卵母细胞的细胞质中。第3组（n = 150），将获能精子通过注射微吸管注入体外成熟卵母细胞的细胞质中，然后用5μm钙离子载体激活5分钟。然后将所有组的卵母细胞在RVCL培养基中培养以进行胚胎发育。注射后48 - 72小时观察卵裂率。第1组、第2组和第3组的卵裂率和囊胚产生率分别为0.00和0.00、18.42和3.57以及61.33%和16.30%。结果表明，机械激活未能诱导体外成熟的山羊卵母细胞发生卵裂，而体外成熟的山羊卵母细胞胞浆内单精子注射后的化学激活与未激活的卵母细胞相比，显示出显著更高的卵裂率和囊胚产生率。
