Effects Of Macleaya Cordata Extracts On Laying Performance,Intestinal Microbiota And Physical Health In Laying Hens

Macleaya cordata is one of important herbal medicine in China and its main active ingredient is an alkaloid.In recent years,macleaya cordata extracts are widely used in livestock because of its biological characteristics such as antibacterial,anti-inflammatory,antioxidant and insecticides.However,the effect of macleaya cordata extracts on laying performance and egg quality of laying hens and its effect mechanism had been rarely studied.The current study aims to investigate the mechanism and impact of macleaya cordata extracts on laying performance,egg quality,intestinal health and ovarian hormone levels in Jing-white laying hens,and to provide a basis for the application of macleaya cordata extracts in laying hens breeding.270 of 202-day-old Jing-white hens were randomly divided into 3 groups,with 6replicates（15 hens per replicate）after a 7-days adaptation period.The hens in control（CTR）group were fed with basal diet without macleaya cordata extracts（McE）,while the hens in the remaining groups were received 0.6 mg/kg feed（McE-Ⅰ）or 1.2 mg/kg feed（McE-Ⅱ）McE for 35 days,and then the laying hens were slaughtered and the samples were collected.The results were as follows:1.Laying performance and egg qualityCompared with the CTR group,the average egg weight and laying rate in McE-Ⅰgroup were significantly increased（P<0.05）,while feed to egg ratio was significantly decreased（P<0.05）,and the eggshell strength,protein height,eggshell thickness,yolk weight and yolk ratio in McE-Ⅰgroup were significantly increased（P<0.05）.There was no difference between the control group and McE-Ⅱgroup.The above results indicated that only 0.6mg/kg feed macleaya cordata extracts could improve laying performance and egg quality of Jing-white laying hens.Therefore,the subsequent experiment was focused on the 0.6mg/kg macleaya cordata extracts for experimental analysis and marked as McE.2.Ovarian hormone levelsCompared with the CTR group,McE significantly increased the serum follicle-stimulating hormone（FSH）content（P<0.05）,but had no significant effect on the luteinizing hormone（LH）and progesterone（Prog）contents（P>0.05）.Moreover,compared with the CTR group,the mRNA expression levels of ESR-α,ESR-β,FSHR and GnRH in ovarian in McE group were significantly increased（P<0.05）,while the expression levels of INH were significantly decreased（P<0.05）.These results indicated that the supplementation of macleaya cordata extracts can improve laying performance of laying hens by regulating the secretion of laying hormones.3.Physical barrier function of intestinal mucosaThe results of jejunum microvillus electron microscopy showed that McE increased the microvillus height and intercellular junctions（tight junctions,adhesion junctions and desmosomes）in jejunum.In addition,McE increased the mRNA expression of tight junction proteins related genes such as ZO-1,claudin-1,occludin and adhesive protein MUC-2（P<0.05）,thus improving the physical barrier function of jejunum mucosa.4.Antioxidant capacityMcE significantly increased the activities of glutathione peroxidase（GSH-Px）,superoxide dismutase（SOD）and total antioxidant capacity（T-AOC）（P<0.05）,while significantly decreased malondialdehyde（MDA）level（P<0.05）.Compared with the CTR group,the mRNA expression levels of CAT,SOD,GPx and HO-1 in liver and Nrf-2,GPx and HO-1 in jejunum mucosa in McE group were significantly up-regulated（P<0.05）.The above results indicated that McE could increase the antioxidant capacity of laying hens.5.Apoptosis and autophagyCompared with the CTR group,the mRNA expression levels of apoptosis factors caspase 3,caspase 8,p53 and BID in jejunum mucosa of layers in McE group were significantly down-regulated（P<0.05）,and the level of anti-apoptotic factor Bcl-2 was up-regulated（P<0.05）.Compared with the CTR group,the autophagy genes pink-1,Parkin,LC-3,ATG-3,ATG-5,ATG-9 and BECN-1 in liver and the levels of LC-3 and ATG-3 in jejunum mucosa in the McE group were increased（P<0.05）,while the expression level of p62in intestinal mucosa was significantly decreased（P<0.05）.The above results indicated McE could induce the autophagy in liver and intestine of laying hens.6.Immune functionCompared with the CTR group,the mRNA expression levels of inflammatory factors IL-1β,IL-2,IL-8 and IFN-γin liver and IL-1β,IL-2,IL-8,IFN-γ,IL-17 and IL-18 in jejunum mucosa in McE group were significantly decreased（P<0.05）,while IL-4 and IL-10 in liver and IL-4,IL-10 and IL-13 in jejunum mucosa were significantly increased（P<0.05）.In addition,the mRNA expression levels of TRAF-6 and NF-κB in liver and TRAF-6,NF-κB,MAPK,TLR-2 and TLR-4 in jejunum mucosa were down-regulated（P<0.05）.The above results indicated McE could improve immune function of laying hens.7.Other serum related indicesThe contents of triglyceride（TG）,glucose（Glu）and the activity of aspartate aminotransferase（AST）in serum were significantly decreased by McE（P<0.05）.The contents of total protein（TP）,calcium ion(Ca²⁺)and alkaline phosphatase（AKP）were significantly increased（P<0.05）.The results suggested that the supplementation of macleaya cordata extracts can significantly improve biochemical metabolism and reduce liver injury of laying hens.8.Cecal microbiota and short-chain fatty acidsThe results of 16S high-throughput sequencing of cecal contents showed that indices of ACE,Simpson and PD＿whole＿tree in intestinal flora in McE group were significantly higher（P<0.05）than those in the control group,and theα-diversity of intestinal flora increased in McE group.Microbial community principal coordinate analysis（PCoA）based on Bray-Curtis distance showed significant differences between the two groups（P<0.05）,which was consistent with the results of non-metric multidimensional scaling analysis（NMDS）.There were significant differences in the structure and abundance of intestinal microbiota between the two groups.At the phylum level,the abundance of Cyanobacteria in the intestinal tract of laying hens tended to be increased（P=0.089）in McE group.At the genus level,the relative abundance of Ruminococcaeae＿UCG-005 was significantly increased（P<0.05）and the abundance of Lachnospiraceae were significantly decreased（P<0.05）.LEFSE analysis found that the Helicobacter、Helicobateraceae、Ruminococcaceae＿UCG＿005、Bacteroridales、and Clostridiales＿vadin BB60 were enriched in McE group,while Lachnospiraceae、Bacteroides and Ruminococus were enriched in CTR group.The short chain fatty acids（SCFAs）assay found that the concentrations of acetic acid,propanoic acid,butyric acid and isovaleric acid in the caecum in the McE group were significantly increased compared with the control group（P<0.05）.Functional microbiota predictions found that the abundance of bacteria related to amino acid anabolism and secondary metabolite biosynthesis in McE group was significantly increased（P<0.05）.Correlation analysis showed that Ruminococcaceae＿UCG-005was positively correlated with SOD activity,FSHR levels,isovaleric acid and propanoic acid content,while negatively correlated with INH levels.The above results implyed that macleaya cordata extracts could improve the intestinal microbiota composition and structure,increase the content of SCFA,finally promoted intestinal health.In conclusion,dietary supplemented with 0.6mg/kg feed macleaya cordata extracts can improve laying performance and egg quality of Jing-white laying hens by regulating intestinal microbiota and enhancing autophagy,antioxidant,anti-apoptosis,intestinal barrier and immune functions and promoting the secretion of laying-related hormonse.