Department of Animal Reproduction & Artificial Insemination, Veterinary Research Institute, National Research Centre, Cairo, Egypt.
Accredited (ISO 17025) Embryo and Genetic Resources Conservation Bank in National Research Centre (NRC), Cairo, Egypt.
Reprod Biol Endocrinol. 2024 Apr 5;22(1):39. doi: 10.1186/s12958-024-01209-7.
In livestock breeding, oocyte cryopreservation is crucial for preserving and transferring superior genetic traits. This study was conducted to examine the additional effect of melatonin to maturation and vitrification media on the in vitro developmental capacity, mitochondrial distribution, and intensity of buffalo oocytes. The study involved obtaining ovaries from a slaughterhouse and conducting two phases. In the first phase, high-quality oocytes were incubated in a maturation medium with or without 10M melatonin for 22 h (at 38.5°C in 5% CO). Matured oocytes were fertilized in vitro and cultured in SOF media for seven days. In the second phase, vitrified in vitro matured oocytes were stored in vitrified media (basic media (BM) containing a combination of cryoprotectants (20% Ethyl Glycol and 20% Dimethyl sulfoxide), with or without melatonin, and then stored in liquid nitrogen. Normal vitrified/thawed oocytes were fertilized in vitro and cultured as described. Finally, the matured oocytes from the fresh and vitrified/thawed groups, both with and without melatonin, were stained using DAPI and Mitotracker red to detect their viability (nuclear maturation), mitochondrial intensity, and distribution using a confocal microscope. The study found that adding 10M melatonin to the maturation media significantly increased maturation (85.47%), fertilization rate (84.21%)cleavage (89.58%), and transferable embryo (48.83%) rates compared to the group without melatonin (69.85%,79.88%, 75.55%, and 37.25% respectively). Besides that, the addition of melatonin to the vitrification media improved the recovery rate of normal oocytes (83.75%), as well as the cleavage (61.80%) and transferable embryo (27.00%) rates when compared to the vitrified TCM group (67.46%, 51.40%, and 17.00%, respectively). The diffuse mitochondrial distribution was higher in fresh with melatonin (TCM + Mel) (80%) and vitrified with melatonin (VS2 + Mel groups) (76.70%), Furthermore, within the same group, while the mitochondrial intensity was higher in the TCM + Mel group (1698.60) than other group. In conclusion, Melatonin supplementation improves the developmental competence and mitochondrial distribution in buffalo oocytes in both cases(in vitro maturation and vitrification).
在畜牧业中，卵母细胞的冷冻保存对于保存和转移优良的遗传特性至关重要。本研究旨在探讨褪黑素在成熟和玻璃化培养基中的添加对水牛卵母细胞体外发育能力、线粒体分布和强度的影响。该研究涉及从屠宰场获得卵巢，并进行两个阶段的实验。在第一阶段，将高质量的卵母细胞在含有或不含有 10M 褪黑素的成熟培养基中培养 22 小时（在 38.5°C 和 5% CO 下）。成熟的卵母细胞在体外受精，并在 SOF 培养基中培养七天。在第二阶段，将体外成熟的卵母细胞玻璃化保存于玻璃化培养基中（基础培养基（BM）中含有两种冷冻保护剂（20%乙二醇和 20%二甲基亚砜），添加或不添加褪黑素，然后储存在液氮中。正常的玻璃化/解冻卵母细胞在体外受精，并按上述方法培养。最后，使用 DAPI 和 Mitotracker red 对新鲜组和玻璃化/解冻组的成熟卵母细胞进行染色，使用共聚焦显微镜检测其活力（核成熟）、线粒体强度和分布。研究发现，与不添加褪黑素的组相比，在成熟培养基中添加 10M 褪黑素可显著提高成熟率（85.47%）、受精率（84.21%）、卵裂率（89.58%）和可移植胚胎率（48.83%）（分别为 69.85%、79.88%、75.55%和 37.25%）。此外，在玻璃化培养基中添加褪黑素可提高正常卵母细胞的回收率（83.75%），以及卵裂率（61.80%）和可移植胚胎率（27.00%），与玻璃化 TCM 组相比（分别为 67.46%、51.40%和 17.00%）。新鲜的添加褪黑素的组（TCM+Mel）（80%）和玻璃化的添加褪黑素的组（VS2+Mel）（76.70%）中，线粒体的弥散分布更高。此外，在同一组中，TCM+Mel 组的线粒体强度高于其他组（1698.60）。综上所述，褪黑素的添加可以提高水牛卵母细胞在体外成熟和玻璃化过程中的发育能力和线粒体分布。
