Anti-Heat Stress Effect Of Macleaya Cordata Extract On Mice And Yellow Feather Broilers

With global warming,animal production’s heat stress is becoming more serious.Heat stress severely reduces poultry performance and affects animal welfare,resulting in substantial economic losses.Therefore,mitigating the negative effects of heat stress on animals is critical to animal production and welfare.Some natural plant extracts with antibiotic effects can not only replace the antibacter ial and anti-inflammatory effects of antibiotics,reduce bacterial resistance,reduce the impact of animal antibiotic residues on human body,but also play an anti-stress role.Therefore,the development of natural plant extracts to alleviate heat stress will be of great significance to the poultry industry.The purpose of this experiment was to study the relieving effects and mechanisms of Macleaya cordata extract on heat stress-induced mice and yellowfeathered broilers.Experiment 1 selected 18 Kunming mice and randomly divided them into three groups:control group(CON,25°C,n=6),heat stress group(HS,42°C,n=6),and heat stress plus extract group(HSMCE,42°C,n=6).The CON and HS groups were intragastrically administered with normal saline 0.2 m L/g body weight/day for 14 days.The HS-MCE group was intragastrically administered 5 mg MCE/kg/day for14 days.Heat stress(12:00~14:00,2 h/d)was performed on days 8-14.In the second experiment,360 male yellow feather broilers(28 days old)with uniform body weight were randomly divided into five groups,namely the control group(CON,24 ± 2 °C),the heat stress group(HS,35 ± 2 °C),and the low dose Macleaya cordata extract group(HS250,35 ± 2°C),medium dose Macleaya cordata extract group(HS500,35 ± 2°C),high dose Macleaya cordata extract group(HS1000,35 ± 2°C),6 replicates per group,12 chicks per replicate.CON and HS groups were fed the basal diet for 14 consecutive days,HS250,HS500,and HS1000 group s were fed basal diet plus 250,500 and 1000 mg/kg Macleaya cordata extract for 14 consecutive days,respectively.Heat stress lasted for 14 days(09:30~17:30,8 h/d,28~42 d).In the experiment,biochemical kits were used to detect common blood biochemical indicators,ELISA kits were used to detect stress hormone indicators and inflammatory factor levels,and the mechanism of Macleaya extract to alleviate heat stress in broilers was explored through the combined analysis of 16 S r DNA sequencing and metabonomics.The main results are as follows:1.Heat stress increased the serum AST,ALT,and liver CORT,HSP70,MDA,IL-1β,and IL-6 levels(p < 0.05),decreased the liver SOD concentration(p < 0.05),and the liver tissue morphology showed obvious vacuolization.Furthermore,heat stress increased hepatic HSP70 mRNA abundance but decreased hepatic Nrf2,CAT,and SOD mRNA abundance(p < 0.05).Macleaya cordata extract significantly reduced serum AST,ALT levels,and liver CORT,HSP70,MDA,IL-1β,and IL-6 levels in heat-stressed mice(p < 0.05)and increased liver SOD concentration(p < 0.05),alleviated the morphological damage of liver tissue.In addition,Macleaya cordata extract decreased HSP70 mRNA abundance and increased Nrf2,CAT,and SOD mRNA abundance in the liver of heat-stressed mice(p < 0.05).2.Heat stress damaged the morphological of mouse jejunum,significantly decreased jejunal villus height,VH/CD ratio,Claudin-1 and Occludin mRNA abundance(P < 0.05),and significantly increased mouse jejunal IL-1β,IL-6,and TNF-α mRNA abundance(P < 0.05).In addition,heat stress also led to disturbance of the intestinal microbiota in mice,increasing the microbiota’ alpha diversity(Shannon index,P < 0.05),resulting in abnormal community structure,decreasing the Firmicutes and Lactobacillus abundance(P < 0.05),while increasing Bacteroidetes,Muribaculaceae,Prevotellaceae,Oscillospiraceae,and Alloprevotella abundance(P < 0.05).Predicted gut microbiota metabolic pathway changes showing microbial metabolism in different environments,carbon metabolism,ABC transporters,ribosomes,purine metabolism,quorum sensing,amino sugar and nucleotide sugar metabolism,glycolysis/gluconeogenesis,starch and sucrose metabolism,pyrimidine metabolism,aminoacyl-t RNA biosynthesis,and pyruvate metabolism were significantly reduced under heat stress conditions.Macleaya cordata extract improved heat stress-induced mouse jejunum tissue morphology,increased jejunal villus height,VH/CD ratio,Occluding and ZO-1 mRNA abundance(P < 0.05),decreased jejunum Crypt depth,mRNA abundance of IL-1β and IL-6(P < 0.05).In addition,Macleaya cordata extracts regulated intestinal microbiota homeostasis and reduced the alpha diversity of the microbiota(Ace,Chao,Shannon index,P < 0.05),improved the microbiota structure,alleviated the changing trend of intestinal flora abundance in heat-stressed mice regardless of at the phylum or genus level.Macleaya cordata extract ameliorated abnormal metabolic levels in heat-stressed mice.Spearman correlation analysis found that changes in gut microbiota were correlated with serum oxidative indexes.3.Heat stress increased the rectal temperature,serum ACTH,CORT,TC,LDL-C,IL-1b,IL-6,TNF-aconcentrations,and the activities of AST,ALT,and LDH(P < 0.05);decreased the Final BW,ADFI,ADG,half evisceration rate,full evisceration rate,and pectoral muscle rate,serum ALP,GLU,T3 concentration,and T3/T4 ratio in yellow-feathered broilers(P < 0.05).The rectal temperature in the HS1000 group decreased compared with the HS group at 8 days(P < 0.05).At 14 days,Macleaya cordata extracts decreased the serum ACTH concentration(P < 0.05)and increased the serum ALP activity in the HS250 group(P <0.05);decreased the rectal temperature,serum ACTH,CORT,AST,and ALT concentration(P < 0.05),increased Final BW,ADFI,ADG,half evisceration rate,serum ALP and GLU,T3 concentration and T3/T4 ratio in the HS500 group(P < 0.05);decreased rectal temperature,serum ACTH,CORT,Cre concentrations and AST,ALT,LDH activities(P < 0.05),increased Final BW,ADFI,ADG,half evisceration rate,complete evisceration rate,chest muscle rate,and serum ALP,GLU,T3 concentration and T3/T4 ratio in the HS1000group(P < 0.05).There had no significant effect on serum IL-6,and TNF-a concentrations in the HS250 and HS500 groups(P(29)0.05);but significantly decreased the serum IL-1b concentration(P < 0.05),and had no significant effect on IL-6 and TNF-a concentrations in the HS1000 group(P(29)0.05).4.Heat stress decreased the spleen and liver index,blood Mon,Lym and serum CD4,IgG,IgM levels,spleen GSH concentration,CAT activity in yellow feather broilers(P < 0.05),increased the blood Het levels,H/L ratio,spleen MDA concentration,IL-1b and TNF-a mRNA abundance(P < 0.05).Macleaya cordata extract increased the blood Lym level and the spleen IL-1b mRNA expression level(P < 0.05),and decreased the H/L ratio in the HS250 group(P < 0.05);and increased the spleen index,blood Lym and serum IgG level(P < 0.05),and decreased the blood Het level,H/L ratio,spleen MDA concentration,IL-1b,IL-6 and TNF-α mRNA abundance in the HS500 group(P < 0.05);and increased spleen and liver index,blood Mon,Lym,serum CD4,IgG,IgM levels,spleen GSH content and CAT activity(P < 0.05),decreased blood Het levels,H/L ratio,and spleen MDA concentration,IL-1b,IL-6,TNF-a mRNA abundance in the HS1000 group compared with the HS group(P < 0.05).5.Heat stress damaged the structure of the duodenum,jejunum,and ileum and decreased their villus height,and VH/CD ratio(P < 0.05),and increased jejunal crypt depth,serum DAO,and D-Lac levels in the yellow-feathered broiler(P < 0.05).In addition,heat stress decreased the intestinal barrier-related genes Claudin,Occludin,ZO-1,Mucin,and antioxidant-related genes Nrf2,HO-1,SOD1,SOD2,NQO1,and GSTA3 mRNA abundance(P < 0.05),and increased the jejunal HSP70,HSP90 and inflammation-related genes IL-1b,IL-6,TNF-α,NF-kB mRNA abundance in yellow feather broilers(P < 0.05).1000 mg/kg Macleaya cordata extract improved the morphology of the duodenum,jejunum,and ileum,and increased their villus height and VH/CD ratio(P < 0.05),and decreased jejunal crypt depth,serum DAO and D-Lac concentrations in heat-stressed yellow-feathered broilers(P < 0.05).In addition,Macleaya cordata extract increased Claudin,Occludin,Nrf2,HO-1,SOD1,NQO1,and GSTA3 mRNA abundance(P < 0.05),and decreased jejunal HSP70,HSP90,IL-1b,IL-6,TNF-α and NF-kB mRNA abundance(P < 0.05).6.Based on the combined analysis of gut microbiota 16 S sequencing and metabolomics,we found that heat stress increased Bacteroidetes,Bacteroidaceae,and Bacteroides relative abundance(P < 0.05)and decreased Firmicutes,Lachnospiraceae,and Alistipes relative abundance(P < 0.05)in the yellow-feathered broilers.The HS group was dominated by Bacteroidetes,Bacteroidales,Bacteroidaceae,Bacterodia,and Bacteroides.Heat stress reduced the alpha diversity(Chao and Ace indices,P < 0.01),resulting in disordered microbiota structure and changing the metabolic composition of cecal contents in yellow-feathered broilers.Sixteen metabolic pathways,including D-glutamine and D-glutamate metabolism,steroid biosynthesis,niacin and nicotinamide metabolism,fructose and mannose metabolism,and pyruvate metabolism were involved in the negative effects of heat stress on yellow-feathered broilers.1000 mg/kg Macleaya cordata extract decreased the relative abundances of Bacteroidetes,Bacteroidaceae,and Bacteroides in yellow feather broilers(P < 0.05),and increased the relative abundance of Firmicutes,Rikenellaceae,and Alistipes(P <0.05).The HS1000 group was marked by Alistipes,Rikenellaceae,Mogibacterium,Butyrivibrio,and Lachnospira.Macleaya cordata extract increased the alpha diversity(Chao,P < 0.05;Ace,P < 0.05;Shannon and Simpson,P < 0.01),and alleviated heat stress-induced flora structural disturbances and abnormal metabolism in the heat-stressed yellow-feathered broilers.Pyruvate metabolism,glycolysis/gluconeogenesis,glyoxylate,dicarboxylic acid metabolism,tyrosine metabolism,and purine metabolism are involved in the process of Macleaya cordata extract in the regulation of heat stress.Spearman correlation analysis found that differential metabolites were associated with changes in gut microbiota.In conclusion,Macleaya cordata extract improved intestinal flora homeostasis and changed intestinal flora metabolism,which further alleviated the negative effects of heat stress on yellow-feathered broilers.This experiment confirmed that Macleaya cordata extract alleviated heat stress-induced liver,jejunum damage and flora disturbance in mice,improved immune function,antioxidant capacity,intestinal barrier function,and ameliorated flora homeostasis and metabolism in heat-stressed yellow-feather broilers,ultimately improving production performance.