Kaushik Kalpana, Krishna Kavya, Johnson P, Gupta P S P, Nandi S, Mondal S, Suganthi R U, Nikhil Kumar Tej J
ICAR-National Institute of Animal Nutrition and Physiology, Bengaluru, India.
Department of Biotechnology, Jain University, Bengaluru, India.
Reprod Domest Anim. 2023 Jul;58(7):903-911. doi: 10.1111/rda.14364. Epub 2023 Apr 27.
The present study aimed to evaluate the effect of α-tocopherol on viability, lipid peroxidation and the expression of apoptosis, stress and development-related genes in the vitrified sheep secondary follicles. Ovarian secondary follicles (200-300 μm) were isolated and distributed separately to the vitrification treatment and supplemented with 5 mM, 10 mM, 20 mM and 30 mM of α-tocopherol (while the control fresh group was without vitrification and supplementation of α-tocopherol). After a week, the follicles were thawed and evaluated for follicular viability by trypan blue dye exclusion method, lipid peroxidation and gene expression studies. The results showed that the vitrification with 10 and 20 mM of α-tocopherol positively affected (p < .05) the viability of vitrified follicles in comparison with vitrified ones without α-tocopherol but the higher concentration of α-tocopherol, i.e., 30 mM negatively affected the viability (p < .05) in comparison with the 10 and 20 mM of α-tocopherol groups. The malondialdehyde (MDA) levels were significantly (p < .05) higher in the vitrified without α-tocopherol group in comparison to the vitrified with 20 mM of α-tocopherol group. The expression of apoptotic-related gene, BCL2L1 was significantly higher in 10 mM α-tocopherol group compared to the control fresh and CASPASE 3, 9 expressions were significantly higher in the vitrified group when compared to the vitrified with 10 mM α-tocopherol group. Expressions of BAX, BAD, BAK, BMP-15 and GDF-9 showed no significant difference among the groups. The mRNA expression of SOD1 was significantly higher in the vitrified without α-tocopherol group when compared to other groups. We conclude that the supplementation of 10 and 20 mM α-tocopherol in vitrification solution was the efficient vitrification procedure for the vitrification of ovine secondary follicles.
本研究旨在评估α-生育酚对玻璃化处理的绵羊次级卵泡的活力、脂质过氧化以及凋亡、应激和发育相关基因表达的影响。分离出卵巢次级卵泡（200 - 300μm），分别进行玻璃化处理，并添加5mM、10mM、20mM和30mM的α-生育酚（而对照新鲜组不进行玻璃化处理且不添加α-生育酚）。一周后，将卵泡解冻，通过台盼蓝染料排斥法评估卵泡活力，并进行脂质过氧化和基因表达研究。结果表明，与未添加α-生育酚的玻璃化卵泡相比，添加10mM和20mMα-生育酚的玻璃化处理对玻璃化卵泡的活力有积极影响（p < 0.05），但与10mM和20mMα-生育酚组相比，较高浓度的α-生育酚（即30mM）对活力有负面影响（p < 0.05）。与添加20mMα-生育酚的玻璃化组相比，未添加α-生育酚的玻璃化组丙二醛（MDA）水平显著更高（p < 0.05）。与对照新鲜组相比，10mMα-生育酚组中凋亡相关基因BCL2L1的表达显著更高，与添加10mMα-生育酚的玻璃化组相比，玻璃化组中CASPASE 3和9的表达显著更高。BAX、BAD、BAK、BMP - 15和GDF - 9的表达在各组之间无显著差异。与其他组相比，未添加α-生育酚的玻璃化组中SOD1的mRNA表达显著更高。我们得出结论，在玻璃化溶液中添加10mM和20mMα-生育酚是绵羊次级卵泡玻璃化的有效方法。
