Developmental competence of ovum pick up derived Sahiwal oocytes in maturation media supplemented with cysteamine and melatonin

Antioxidants, cysteamine, and melatonin have an important role in mitochondrial membrane potential (ΔΨM), in vitro nuclear maturation, and the developmental competence of oocytes. A comprehensive study was planned to investigate the effect of cysteamine 50 µM and melatonin 10-9 mol L-1 as antioxidants on ΔΨM, in vitro nuclear maturation, and developmental competence of ovum pick-up (OPU) derived Sahiwal oocytes. Culturable grade OPU-derived Sahiwal oocytes were divided into three in vitro maturation groups cultured in TCM-199 supplemented with cysteamine 50 µM, melatonin 10-9 mol L-1, and TCM-199 alone, for assessing nuclear maturation by Lamin/ DAPI and developmental competence of oocytes. ΔΨM was assessed by JC-1 staining in the pre-maturation group and post-maturation cysteamine, melatonin, and control groups. Red to green ratio of fluorescence intensity on JC-1 staining was higher (p < 0.05) in melatonin (1.19±0.04) and cysteamine (1.09±0.04) supplementation groups as compared to control (0.81±0.10) and pre-maturation (0.71±0.03) groups. ΔΨM improved post-maturation in all the treatment and control groups compared to the pre-maturation group (0.71±0.03). Melatonin supplementation improved (p < 0.05) M-II stage oocytes (6.5±0.65, 68.13 percent) as compared to cysteamine supplemented (5.25±0.25, 55.63 percent) and control (4.75±0.25, 50.63 percent) groups. The COC expansion rate was higher in the antioxidant-supplemented group. Fertilization rate, cleavage rate, and blastocyst rate were higher (p < 0.05) in the melatonin-supplemented group (92.31, 59.17, and 20.56 percent) as compared to cysteamine supplemented (82.96, 41.48 and 11.39 percent) and control (75.28, 27.59 and 5.19 percent) groups, respectively. In conclusion, cysteamine and melatonin supplementation as antioxidants in the in vitro maturation media improved (p < 0.05) ΔΨM. Significant improvement in MII stage oocytes, cleavage, and blastocyst rate in OPU-derived Sahiwal cattle oocytes by supplementation of melatonin to the IVM medium compared to cysteamine supplemented and control groups. Melatonin improved both cytoplasmic and nuclear maturation thereby improving the developmental competence of OPU-derived Sahiwal oocytes. Cysteamine supplementation improved the cytoplasmic maturation and not the nuclear maturation and developmental competence of OPU derived Sahiwal oocytes.

抗氧化剂、半胱胺与褪黑素对线粒体膜电位（mitochondrial membrane potential, ΔΨM）、卵母细胞体外核成熟及发育潜能具有重要调控作用。
本研究计划开展一项综合性实验，探究浓度为50 μM的半胱胺与10⁻⁹ mol·L⁻¹的褪黑素这两种抗氧化剂，对卵母细胞采集（ovum pick-up, OPU）获得的萨希瓦尔牛卵母细胞的ΔΨM、体外核成熟及发育潜能的影响。
将符合培养级别的OPU来源萨希瓦尔牛卵母细胞分为3组体外成熟体系，分别在添加50 μM半胱胺、10⁻⁹ mol·L⁻¹褪黑素的TCM-199培养基，以及仅添加基础TCM-199的培养基中培养；采用Lamin/DAPI染色评估核成熟情况，并检测卵母细胞的发育潜能。
成熟前及成熟后（半胱胺组、褪黑素组与对照组）的卵母细胞ΔΨM通过JC-1染色进行检测。结果显示，褪黑素组（1.19±0.04）与半胱胺组（1.09±0.04）的JC-1染色荧光强度红/绿比显著高于对照组（0.81±0.10）与成熟前组（0.71±0.03）（p < 0.05）。与成熟前组（0.71±0.03）相比，所有处理组与对照组的卵母细胞在成熟后ΔΨM均得到改善。
褪黑素组的M-II期卵母细胞占比（6.5±0.65，68.13%）显著高于半胱胺组（5.25±0.25，55.63%）与对照组（4.75±0.25，50.63%）（p < 0.05）。抗氧化剂添加组的卵丘-卵母细胞复合体（cumulus-oocyte complexes, COC）扩展率更高。
褪黑素组的受精率、卵裂率与囊胚率分别为92.31%、59.17%与20.56%，均显著高于半胱胺组（82.96%、41.48%与11.39%）与对照组（75.28%、27.59%与5.19%）（p < 0.05）。
综上，在体外成熟（in vitro maturation, IVM）培养基中添加半胱胺与褪黑素这两种抗氧化剂，可显著提升卵母细胞的ΔΨM（p < 0.05）。与半胱胺添加组及对照组相比，在IVM培养基中添加褪黑素，可显著提升OPU来源萨希瓦尔牛卵母细胞的M-II期卵母细胞占比、卵裂率与囊胚率。褪黑素可同时改善胞质成熟与核成熟，进而提升OPU来源萨希瓦尔牛卵母细胞的发育潜能。半胱胺添加仅能改善OPU来源萨希瓦尔牛卵母细胞的胞质成熟，无法提升其核成熟与发育潜能。