| Flammulina velutipes stem-base（FVS）is a byproduct consisting of the base of the stalk and a small amount of bran produced during the production of golden mushroom.The research group found that FVS as a dietary alternative can regulate ovarian development and lipid metabolism in peak laying hens,and has no adverse effects on growth and production performance.In view of the important role of the"liver-blood-ovary"reproductive signaling axis in egg production and the unclear mechanism of the effect of FVS on egg performance during the late peak laying period.Therefore,in this study,Hyland Brown hens were randomly divided into four treatment groups:blank control group（basal diet）,2%FVS group（basal diet+20 g/kg FVS）,4%FVS group（basal diet+40 g/kg FVS）and 6%FVS group（basal diet+60 g/kg FVS）.The rations are formulated according to the principle of equal energy and equal nitrogen,the FVS diet was used to replace a certain proportion of maize and soybean meal to alleviate the decline in antioxidant capacity and reproductive system of laying hens from 47 weeks of age（47w）to 67 weeks of age（67w）.The antioxidant status of the reproductive system,the degree of ovarian tissue apoptosis,the levels of reproductive hormones and their receptors,parameters related to yolk precursor synthesis and lipid metabolism,morphological observations of the liver and ovaries,production performance and egg quality were measured to investigate the possible mechanisms of FVS as a functional feed substitute for maintaining reproductive homeostasis in aging laying hens during the late peak laying period.The results are showed as follows:1.At the late peak of egg production（47-67w）,the egg production rate of hens in the 2%FVS group was significantly higher than that of the CON group（P<0.05）;the mean egg weight and feed-to-egg ratio remained consistent among the treatment groups.Regarding organ indices,there were no differences in liver indices between the CON and all FVS groups（P>0.05）;in addition,we observed a significant increase in ovarian indices and preovulatory grade follicle indices and a significant decrease in abdominal fat percentage in both the 4%and 6%FVS groups（P<0.05）.In terms of egg quality,all FVS groups significantly improved eggshell thickness（P<0.05）without any adverse effects on eggshell strength,Haugh units,egg shape index and protein height.These results indicate that FVS can replace a certain percentage of the basal diet to achieve the effect of maintaining reproductive organ levels,production performance and egg quality in hens.2.At both 47 and 67 weeks of age,the FVS group was able to increase the levels of total superoxide dismutase,glutathione peroxidase and total antioxidant capacity in the liver,serum and ovaries of laying hens to varying degrees and significantly reduced the malondialdehyde（MDA）content（P<0.05）.In addition,further analysis of antioxidant-related gene and protein expression levels by real-time fluorescence quantitative PCR and immunoblotting assay verified that FVS could improve the antioxidant status of liver and ovary of laying hens by activating the Nrf2-Keap1/ARE signaling pathway.3.H&E staining results showed that lipid and inflammatory cell infiltration was more pronounced in the liver of the 47 and 67-week-old CON groups,whereas the number of lipid droplets was reduced in the FVS group and inflammation was slightly reduced.Overall,hepatocyte nuclei and cytoplasm were more evenly distributed in all groups,with intact hepatocyte membranes and normal tissue structure.Further analysis of liver function impairment was followed by measuring glutamic aminotransferase（AST）and glutamic alanine aminotransferase（ALT）indexes in the liver,and the FVS group was able to reduce AST and ALT levels in the liver extremely significantly（P<0.01）.The results indicated that FVS intake had a significant alleviating effect on liver function damage in laying hens.4.The results of H&E staining showed that the number of follicles in the ovaries of 47-week-old laying hens was high,and good development of primordial follicles（PF）and growing follicles could be clearly observed,with good follicular filling and clear boundaries of the granulosa cell membrane layer（GC）and basement membrane,but a small number of atretic follicles（AF）were present in all of them.As the hens continued to age,the number of follicles in the ovaries became significantly smaller at 67 weeks of age.Compared with the CON group,the ovarian medulla structure and GC appeared to be more dense and the number of AF was relatively small in the FVS group,and we hypothesized that FVS might have a mitigating effect on ovarian decline.Therefore,we recorded the number of PF and the preovulatory grade follicle index,and the results showed significantly higher values in the 4%and 6%FVS groups（P<0.05）.In addition,by measuring ovarian apoptosis-related genes and proteins in laying hens,it was found that the FVS group was able to significantly reduce the expression levels of pro-apoptotic Bax and Caspase3（P<0.05）and significantly increase the expression level of anti-apoptotic Bcl-2（P<0.05）,verifying that FVS was able to alleviate Bax/Bcl-2 and Caspase3 signaling pathways by regulating apoptosis levels in the ovary.5.At 47 weeks of age,serum estradiol（E2）,follicle stimulating hormone（FSH）and luteinizing hormone（LH）levels were significantly higher in the FVS group than in the CON group（P<0.05）,while P4 levels were not significantly different（P>0.05）,as determined by ELISA assay.At 67 weeks of age,the FVS group was able to significantly increase serum E2,P4,FSH,and LH concentrations（P<0.05）.In addition,In addition,the gene expression levels of various hormone receptors were measured,and ER-α/βin the liver and FSHR and LHR m RNA expression levels in the ovaries were significantly increased in the FVS group（P<0.05）.6.The levels of yolk precursors and their receptors in laying hens were measured by ELISA assay,and the levels of very low density lipoprotein（VLDLy）and yolk proteinogen（VTG）produced in the liver were significantly increased in the FVS group compared with the CON group（P<0.05）,and the levels of very low density lipoprotein receptor（VLDLR）in the ovaries were significantly increased（P<0.05）.Analysis of the m RNA abundance related to yolk precursors and their receptor synthesis showed that Apo B,Apo VLDLII,VTGII and VLDLR m RNA expression was significantly higher in the FVS group than in the CON group（P<0.05）.Subsequent measurements of triglyceride（TG）and total cholesterol（T-CHO）levels in liver and serum revealed that TG and T-CHO levels were significantly lower in the FVS group than in the CON group of laying hens（P<0.05）.The ability of FVS to significantly increase liver fatty acid synthase,peroxisome proliferator-activated receptor-α/γand microsomal triglyceride transport protein m RNA expression levels were significantly increased by FVS（P<0.05）.Taken together,FVS was able to enhance the antioxidant capacity of liver,serum and ovary,reduce ovarian apoptosis,and maintain the homeostasis of hepatic lipid metabolism to promote reproductive hormone secretion and follicular development through regulating the Nrf2-Keap1/ARE signaling pathway.In addition,the expression level of m RNA related to yolk precursor synthesis was significantly increased,which ultimately delayed the decline of egg production performance in aging hens,verifying the potential hepatoprotective effects of FVS as a functional feed alternative,promoting the transport and exchange of multiple substances through the liver-blood-ovary axis and maintaining reproductive homeostasis in aging hens to some extent. |
