Effects Of Melatonin On Ovary Granulosa Cells Of Laying Hens In Late Laying Period

The laying performance of laying hens is closely related to the development of follicles.The whole process of follicular development is accompanied by the growth of granular cells.Ovarian granulosa cells play an important role in regulating the development,maturation and atresia of layer follicles.Therefore,in order to explore the relationship among melatonin,ovarian granulosa cells and egg production performance,the effects of melatonin on the antioxidant and anti-apoptotic properties of chicken ovarian granulosa cells were studied in vivo and in vitro.The main results are as follows:1.Cultivation and identification of chicken ovarian granulosa cells.The results showed that the chicken ovarian granulosa cells in the culture system of M199 basal medium+1%ITS+1%double-antibody were fusiform or polygonal and grew in good condition.PCR identification of specific FSH gene showed that the expected band appeared at 191 bp The cells were confirmed to be granulosa cells by sequencing;The immunofluorescence results show that the specific FSHR protein is distributed in the cytoplasm.Compared with DAPI staining,it could be seen that the positive rate of FSHR was above 98%,so the purity of cultured granule cells was above 98%.The results of the growth curve of chicken ovarian granulosa cells showed that the logarithmic growth period of the cells was 18～72 h.2.Effects of melatonin on granulosa cells of chicken ovarian:(1)The effect of melatonin on the relative expression levels of MT1,MT2,FSHR and LHR mRNA in ovarian granulosa cells was determined by fluorescence quantitative PCR,and the second messenger cAMP and E2 were detected by ELISA kits.The results showed that the relative expression of MT1、MT2 and LHR mRNA and the amount of E2 in the 1 ng/mL melatonin treatment group were significantly increased(p<0.05);in the 5 ng/mL melatonin treatment group,cAMP production was significantly reduced by 13.16%(p<0.05),and FSHR mRNA was inhibited about 3 times(p<0.05).(2)The effects of melatonin on ROS,T-AOC and SOD in chicken ovarian granulosa cells were determined by ELISA.The results showed that The results showed that:in 1 ng/mL melatonin treatment group,the ROS accumulation was decreased by 18.67%(p<0.05),and T-AOC was increased by 71%(p<0.05);in 0.1 ng/mL melatonin treatment group,SOD activity was increased by 40.46%(p<0.05).(3)The CCK-8 method was used to determine the growth of chicken ovarian granulosa cells,and the fluorescence quantitative PCR was used to determine the mRNA expression levels of anti-apoptotic protein Bcl-2 and pro-apoptotic proteins Bax and Caspase-3.The results showed that when the granulosa cells were treated with 1 ng/mL melatonin,the OD value at 450 nm was the highest and the cells grew well.The Bax mRNA expression was significantly reduced by 5 times(p<0.05),and the Bcl-2 mRNA expression was significantly increased by about 3 times(p<0.05).When the granulosa cells were treated with 0.1 ng/mL melatonin for 24 h,the Caspase-3 mRNA expression was also significantly increased by about 2 times(p<0.05).3.Animal experiment:Laying hens at the end of laying(about 550 days old)were divided into two groups(control group and test group).The experimental group was fed with 1 mg melatonin per 1 kg for 4 weeks.The results showed that the laying rate of the control group was decreased by 1.62%compared with that before feeding(p<0.05).The egg production rate of the experimental group was increased by 0.54%compared with that before feeding,and the difference was not significant(p>0.05).The egg breaking rate of the control group was increased by 1.59%compared with that before feeding(p<0.05).The egg breaking rate of the experimental group was decreased by 0.26%,and the difference was not significant(p>0.05).It showed that melatonin affected the secretion of hormones FSH and LH and the expression of receptors by acting on the receptors MT1 and MT2,and promoted the secretion of estrogen,thereby improving the quality of granulosa cell development and promoting the development of follicles.Melatonin could also reduce the level of ROS,increase the activity of antioxidant enzymes through its antioxidant capacity,reduce the stimulation of oxidative stress on the development of follicles,improve the environment for follicular development,and promoted the development of follicles to the next level.Melatonin promotes the proliferation of granular cells by inhibiting the expression of pro-apoptotic factors Bax,Caspase-3 and stimulating the expression of anti-apoptotic factor Bcl-2 mRNA,thereby inhibiting follicular atresia.It showed that exogenous feeding of melatonin can maintain or even increase the laying rate of laying hens at the end of laying in vivo.It provided a basis for the use of melatonin to delay ovarian senescence and improve the performance of laying hens.