Alleviating Effect Of Metformin On Follicular Retardation In The Low-yield Laying Hen

The egg production capacity of laying hens depends on the number of follicles in the ovary and whether the follicles are well developed.About less than 5%of the follicles in the ovaries of poultry can develop into the hierarchical follicle stage and be ovulated,and many follicles are lost during development.Those follicles that do not continue to develop and ovulate are called atretic follicles.Commercial hens reach their peak laying period when they are about 280 days old.At this time,the laying rate of hens can reach95%.After a high yield period of one year,the laying rate has dropped drastically.The main reason for the decrease in egg production rate of low-yield hens is the decrease in the number of prehierarchical follicles（PHFs）in the ovary and the increase in the number of atresia follicles.The main reason causing the atresia of PHFs is apoptosis of follicular granulosa cells（GCs）.This experiment took Hyline white hens as the research object.On the one hand,it studied the specific mechanism of poultry follicular atresia,and on the other hand,it explored the regulatory effect of metformin on GCs apoptosis,alleviating the atresia of the follicles of D580 hens,and making the PHFs develop to the next grade and finally ovulates.This study provides a new idea for improving the production of poultry eggs.1.Endoplasmic reticulum stress and decreased yolk deposition capacity were involved in the follicular atresia of D580 hensThe level of egg production depends on the number of follicles that can develop to hierarchical follicles in the ovary and the number of atresia follicles,while the number of PHFs in the ovaries of the laying hens directly determines the laying potential of the laying hens.First,we compared the morphology of follicles in high-yield hens（280days old,D280）and low-yield hens（580 days old,D580）by HE staining,immunofluorescence chemical staining（IF）and transmission electron microscope and other morphological methods,and found that the number of Caspase-positive cells in D580-PHFs and atretic follicles wan significantly increased when compared with D280-PHFs.In addition,endoplasmic reticulum（ER）stress occurred in D580-PHFs and GCs of atretic follicles,the expression of GRP78 and CHOP protein increased,and the contact sites between the ER and mitochondria was decreased.It suggests that ER stress may be one of the factors of follicular cell apoptosis.In addition,580-PHFs and the follicle-stimulating hormone receptor and estrogen alpha receptor expression in granulosa cells of atretic follicles were lower than D280-PHFs.More importantly,occludin protein was significant in these two types of follicles.At the same time,the high-density lipoprotein,low-density lipoprotein,free fatty acids and estrogen in the plasma of D280 and D580 hens found that these four indicators have varying degrees of decline.These evidences suggest that the yolk production and deposition ability of D580 hens are lower than that of D280 hens.2.Metformin inhibits follicular atresia in D580 hens by coordinating the cooperation of calcium transportation and insulin signaling pathway and improving the ability of yolk depositionDue to the poor morphology of the atretic follicles in the physiological state after being cultured in vitro,using them as the object of in vitro experiments to explore the mechanism of follicular atresia and specific measures to alleviate the follicular atresia has brought a certain degree of difficulty.This article established follicular atresia in vitro model by using H₂O₂,setting four groups of different concentrations of H₂O₂,and compared with the atretic follicles under physiological conditions.It was verified from three levels whether the model was successfully established（the ability of yolk deposition,hormone receptor expression and cell apoptosis）,and finally determined that 1 m M H₂O₂ was the most appropriate additive dose.The physiological indexes of follicles treated with this concentration of H₂O₂ were closest to those of atretic follicles.In addition,in exploring measures to alleviate follicular atresia,we found that continuous feeding of metformin at a dose of 62.5 mg/Kg for 7 days could significantly increase the serum estradiol content of low-laying hens.The addition of 2 m M to the atretic follicles in vitro could inhibit the atresia of follicles to some extent.This was specifically manifested in three aspects:（1）The metformin could regulate the calcium ion transport between the endoplasmic reticulum and mitochondria to avoid apoptosis caused by the imbalance of calcium ion homeostasis in the endoplasmic reticulum and mitochondrion;（2）metformin inhibited the expression of downstream apoptotic factor BAD through coordination the collaboration of calcium and insulin signal pathways,leading to decreased cell apoptosis;（3）metformin could promote the expression of VLDLR in follicular GCs and reduce the expression of occludin to improve the yolk deposition ability of follicles.In summary,at the first,this article compared the differences of follicles between D280 and D580 hens in morphology and molecular biology,and measured the levels of hormones and yolk precursor substances in serum and plasma.It was found that PHFs in D580 hens were significantly reduced,and atretic follicles were increased.At the same time,the ability of formation yolk was reduced.Subsequently,this study explored the molecular reason of follicular atresia and found that the apoptosis of GCs caused by ER stress and the decline of the follicular yolk deposition capacity were the main reasons.At the same time,it established an in vitro follicular atresia model by H₂O₂ and found that metformin could inhibited the follicular atresia of D580 hens by coordinating the cooperation of calcium transportation and insulin signaling pathway and improving the ability of yolk deposition.This article provides a theoretical basis for whether to use Galega orientalis Lam（metformin is a guanidine derivative of the extract of the falega orientalis Lam）as a feed additive to increase the egg production of low-yield hens,and provides a new idea for delaying the atresia of low-laying hens.