Gutiérrez-Añez Juan Carlos, Lucas-Hahn Andrea, Hadeler Klaus-Gerd, Aldag Patrick, Niemann Heiner
Medical-Surgical Department, College of Veterinary Medicine, University of Zulia, Maracaibo, Venezuela; Institute of Farm Animal Genetics, (FLI), Mariensee, Germany.
Institute of Farm Animal Genetics, (FLI), Mariensee, Germany.
Theriogenology. 2021 Feb;161:285-293. doi: 10.1016/j.theriogenology.2020.12.011. Epub 2020 Dec 11.
Bovine oocytes from prepubertal donors have been used for in vitro embryo production to decrease the generation interval. However, reduced cumulus-oocyte competence, mainly attributed to increased apoptosis, has been observed in oocytes/embryos collected from prepubertal donors. Here, we investigated the effects of the potent antioxidative molecule melatonin on cumulus-oocyte competence and embryo development in prepubertal and adult dairy cattle in vitro. A total of fifteen Holstein Friesian calves, six to ten months old (7.6 ± 1.34 months of age). And fifteen adult cows with one to four calvings (2.3 ± 0.96 calvings) were enrolled as ovum pick up (OPU) donors in this study. Cumulus-oocyte complexes (COCs) were cultured either in the presence or absence of melatonin (0.01 nM). The proportion of cleavage stages, blastocysts, and advanced blastocysts was determined. Embryo quality was assessed via differential staining to determine the total embryonic cells and allocation to the inner cell mass (ICM) and trophectoderm (TE) cells. Melatonin treatment yielded a greater percentage of blastocysts compared to the control group, i.e. oocytes from both adult cows (P = 0.0485; 24.8 ± 3.5% vs. 16.0 ± 3.4%, respectively), and from prepubertal donors (P = 0.0007; Melatonin 23.1 ± 5.1% vs. Control: 11.1 ± 3.5%). Adult cows had significantly (P = 0.0370) greater advanced blastocyst rates than those found in the prepubertal group (13.9%± vs. 7.0±%, respectively). Additionally, the number of ICM, total cells, and the ratios ICM: Total, ICM: TE, respectively, were greater (P < 0.05) after melatonin treatment compared with the control group (39.1 ± 2.8, 98.6 ± 5.7, 0.4 ± 0.01, and 0.7 ± 0.04 vs. 27.3 ± 2.9, 81.2 ± 5.8, 0.34 ± 0.01, and 0.52 ± 0.04, respectively). Blastocysts derived from adult cows had a greater number of TE (P = 0.01) and total embryonic cells (P = 0.0095) compared to the prepubertal donor group (63.5 ± 3.2 and 101.05 ± 4.8 vs. 48.9 ± 4.3 and 78.8 ± 6.5, respectively). Nevertheless, embryonic cell counting in embryos derived from prepubertal COCs equated to that observed from adult donors after melatonin exposure. In conclusion, these results indicate that the presence of melatonin during in vitro maturation improves cumulus-oocyte competence, embryo development, and quality by increasing the allocation of embryonic cells to the ICM compartment and the total number of embryonic cells in both adult and prepubertal bovine donors.
来自青春期前供体的牛卵母细胞已被用于体外胚胎生产，以缩短世代间隔。然而，在从青春期前供体收集的卵母细胞/胚胎中，已观察到卵丘-卵母细胞能力下降，这主要归因于细胞凋亡增加。在此，我们研究了强效抗氧化分子褪黑素对青春期前和成年奶牛体外卵丘-卵母细胞能力及胚胎发育的影响。本研究共纳入15头6至10个月大（7.6±1.34月龄）的荷斯坦弗里生犊牛和15头有1至4次产犊经历（2.3±0.96次产犊）的成年母牛作为采卵（OPU）供体。卵丘-卵母细胞复合体（COCs）在有或无褪黑素（0.01 nM）的情况下进行培养。测定了分裂期、囊胚和扩张囊胚的比例。通过差异染色评估胚胎质量，以确定总胚胎细胞数以及内细胞团（ICM）和滋养外胚层（TE）细胞的分配情况。与对照组相比，褪黑素处理产生的囊胚百分比更高，即来自成年母牛的卵母细胞（P = 0.0485；分别为24.8±3.5%和16.0±3.4%）以及来自青春期前供体的卵母细胞（P = 0.0007；褪黑素组为23.1±5.1%，对照组为11.1±3.5%）。成年母牛的扩张囊胚率显著高于青春期前组（分别为13.9%±和7.0%±，P = 0.0370）。此外，与对照组相比，褪黑素处理后ICM数量、总细胞数以及ICM:总细胞、ICM:TE的比例均更高（P < 0.05）（分别为39.1±2.8、98.6±5.7、0.4±?0.01和0.7±0.04，对照组分别为27.3±2.9、81.2±5.8、0.34±0.01和0.52±0.04）。与青春期前供体组相比，成年母牛来源的囊胚具有更多的TE细胞（P = 0.01）和总胚胎细胞（P = 0.0095）（分别为63.5±3.2和101.05±4.8，青春期前供体组分别为48.9±4.3和78.8±6.5）。然而，青春期前COCs来源的胚胎在褪黑素处理后的胚胎细胞计数与成年供体来源的胚胎相当。总之，这些结果表明，体外成熟过程中褪黑素的存在通过增加胚胎细胞向内细胞团部分的分配以及成年和青春期前牛供体中胚胎细胞的总数，提高了卵丘-卵母细胞能力、胚胎发育和质量。
