Intestinal Microbiota In Response To Dietary Copper Levels Affecting The Growth And Health Of Rats And Suhuai Suckling Piglets

Since the 1950s,antibiotic growth promoters(AGPs)are widely used in swine production as feed additives,which not only improved the performance and health of animals,but also exacerbated the antibiotic resistance of bacteria.First,in 2006,many countries in the European Union(EU)banned the use of antibiotic agents as growth promoters in animal husbandry,the high-level copper was widely used as the alternative measure,the 170-175 mg·kg-1 copper can be used in piglets from early feeding to 12-week-old according to the EU regulations.However,in recent years,a growing body of studies find that high-level copper is able to share genetic elements with antibiotic resistant genes,which may promote the spread of antibiotic resistance.Hence,the rationality of high-level copper in diets need to be received more concern.In china swine production,the antibiotic and high-level copper are coexisting and common used as growth promoter in feed,the effect of high-level copper on pig’s health and porcine bacterial resistance is more complicated.In this study,we conduct the experiments as following:(i)rats and suckling piglets were used as the model;(ⅱ)the ionomic,metabolomics,and gut microbial composition were analyzed to investigate the intestinal microbiota in response to dietary copper levels affecting animal growth and health.Our study here provides scientific basis for the reasonable additive dosage of copper in feed when the antibiotics are withdrawn from swine production in China.1 Effect of dietary copper level on the growth and intestinal health of SD ratsNumerous countries in the EU banned the use of antibiotic agents as growth promoters of animal husbandry ten years ago,but no significant decrease in the number of antibiotic resistant bacteria is observed,rationalization of high-level copper as growth promoters in animal feed becomes more attention.Sixty male SD rats were randomly divided into three groups:6,120 and 240 mg·kg-1 Cu group,which were allowed free access to deionized water and purified diets.The feed intake and body weight were recorded,serum biochemical parameters and the morphology,oxidative biomarkers,antioxidant enzymes activity of intestinal tissues were also analyzed.These parameters were measured to investigate the effect of dietary copper level on the growth and intestinal health of SD rats model.The results showed that the average daily gain(ADG)tended to increase in high-level copper(120 and 240 mg·kg-1)during the first four weeks(P=0.07),while it tended to decrease in the last four weeks(P=0.08).The average daily feed intake(ADFI)and feed conversion ratio(FCR)showed similar variation,but the differences were not significant.The serum insulin,ghrelin,TNF-α,and IL-6 were increased in 240 mg·kg-1 Cu group(P<0.05),no significant differences were observed in both serum copper content and copper transport related proteins.The copper content was significantly increased in ileac and colonic digesta when rats feed with high level(120 and 240 mg·kg-1)copper diet(P<0.01).The ileac villus height(VH)and crypt depth(CD)were significantly decreased in 240 mg·kg-1 Cu group(P<0.05),the ratio of VH/CD was increased in 6 and 240 mg·kg-1 Cu group(P<0.05).The jejunal and ileac nitric oxide(NO)(P<0.05),the ileac superoxide dismutase(SOD)(P<0.05),Catalase(CAT)(P=0.08),and glutathione(GSH)(P=0.07)were decreased in high level(120 and 240 mg·kg-1)copper group,the jejunal oxidized glutathione(GSSG)was increased(P<0.05).In summary,the dietary copper level was capable to promote growth in the short-term,while might inhibit the growth with further addition.Moreover,long-term intake of high level copper(120 and 240 mg·kg-1)diets resulted in significant accumulation of copper in the ileac and colonic digest,which changed the status of oxidative stress and reduced the antioxidant capacity of the intestinal tissue,also breaking the balance between the generation and removal of reactive oxygen species(ROS)and causing the inflammatory response.These results indicated that high copper level might take an adverse effect on the health of the rats.2 Effect of dietary copper level on the hepatic copper metabolism and oxidative status of SD ratsThe liver is the main organ that metabolizes and stores copper,to investigate the effect of dietary copper level on the copper metabolism and oxidative status of rats,sixty male SD rats were randomly divided into three groups:6,120 and 240 mg·kg-1 Cu group.After eight weeks feeding trial,the liver was surgically sampled.The hepatic morphology,copper content,copper transport-related genes mRNA expression,oxidative biomarkers and antioxidant enzymes activity were analyzed.The results showed that obvious structural damages of hepatocytes were observed in high-level copper(120 and 240 mg·kg-1)diet.The dietary copper level had no significant effect on the hepatic copper content,hyderoxyl raica,protein carbonyl,nitric oxide(NO),malonaldehyde(MDA),and 8-OH-dG(P>0.05).However,the hepatic superoxide dismutase(T-SOD)(P<0.05),Mn superoxide dismutase(Mn-SOD)(P<0.05),glutathione peroxidase(GSH-px)(P<0.05)and oxidized glutathione(GSSG)(P<0.05)were remarkably increased in high-level(120 and 240 mg·kg-1)copper diet.The hepatic CuZn superoxide dismutase(CuZn-SOD)was notably increased in 120 mg·kg-1 Cu group(P<0.05).Additionally,the mRNA expression of Ctrl was significantly down regulated in 240 mg·kg-1 Cu group compared with other groups(p<0.05).The mRNA expression of Sodl(P<0.05),Mt1a(P<O.05),Cp(P<0.05),and Ccs(P=0.09)genes were up regulated in 120 mg·kg-1 Cu group.The results of the correlation analysis showed that hepatic copper content significantly positively correlated with Ccs and Cp genes mRNA expression,hepatic superoxide dismutase(SOD)and CuZn superoxide dismutase(CuZn-SOD)(P<0.05),negatively correlated with serum IL-6 level(P<0.05).In summary,long-term intake of high-level(120 and 240 mg·kg-1)copper induced the morphological damage in hepatocytes,the expression of Ctr1 was down regulated in order to control copper uptake of hepatocytes in 120 mg·kg-1 Cu group,the mRNA expression of Mt1a and Cp were up regulated to induce the increase of hepatic antioxidant ability,copper resistance and maintenance of copper homeostasis.Dietary 240 mg·kg-1 copper was only able to regulate the expression of Ctrl gene in hepatocytes,while without improving the tolerance of hepatocytes to high-level copper.Hence,the damage of liver cells is serious in 240 mg·kg-1 copper.3 Effect of dietary copper level on the gut microbiota and its correlation with inflammatory cytokines and oxidative status in SD ratsLong-term intake of high-level copper leading to significant accumulation of unabsorbed copper in intestinal digesta of rats,this accumulation also affects the gut microbiota,which plays an important role in animal health.In this study,the 16S rRNA pyrosequencing was designed and used to investigate the effect of dietary copper level on the diversity of the fecal microbial community and the correlation between the serum inflammatory cytokines and oxidative status in SD rat models.Sixty male SD rats were randomly divided into three groups:6,120 and 240 mg·kg-1 Cu group.After eight weeks feeding trial,fecal samples were collected.The results showed that the microbial richness and diversity indices were significantly increased in high level(120 and 240 mg·kg-1)copper group(P<0.05).Compared with 6 mg·kg-1 Cu group,the butyrate producing bacteria(Dorea,Blautia,Coprococcus),intestinal health-related bacteria(Christensenellaceae,Oscillibacter,Alistipes),and potential pathogens(Erysipelotrichaceae,Parabacteroides)were remarkably increased in high level(120 and 240 mg·kg-1)copper groups(P<0.05).Correlation analysis showed that the microbial richness and diversity indices were not only positive correlated with the copper content in the rat ileac and colonic digesta,but also positive correlated with serum TNF-α,negative correlated with jejunal nitric oxide(NO)(P<0.05).The microbial richness index was negative correlated with ileac T-SOD and Mn-SOD(P<0.05).Furthermore,the abundance of Anaerotruncus was positive correlated with serum TNF-α,negative correlated with jejunal nitric oxide(NO),ileac T-SOD and Mn-SOD(P<0.05),while Parasutterella had an opposite correlation.The abundance of OTU402(Coprococcus)and OTU52(Bacteroides vulgatus)were positive correlated with serum TNF-α(P<0.05),OTU99(Parabacteroides),and OTU273(Alistipes)were negative correlated with serum TNF-α(P<0.05).The abundance of Blautia(OTU88)and OTU69(Christensenellaceae)were positive correlated with jejunal and ileac nitric oxide(NO),ileac T-SOD and Mn-SOD(P<0.05).The abundance of Blautia(OTU368),OTU402(Coprococcus),and OTU145(Oscillibacter)were negative correlated with jejunal,ileac nitric oxide(NO),and ileac T－SOD(P<0.05).In summary,the accumulation of unabsorbed copper affected fecal microbial richness and diversity,by changing the abundance of certain bacteria and the dominant status.These variations might further induce oxidative status unbalances and inflammatory response.Thus,high-level copper(120 and 240 mg·kg-1)in diet might have an adverse effect on the health of the rats.4 Effect of dietary copper level in antibiotic free creep feed on the health and ionomic profiles of suckling pigletsIn this study,antibiotic withdrew from creep feed to investigate the effect of dietary copper level in antibiotic free creep feed on the growth performance,serum biochemical parameters,ion balance of Suhuai suckling piglets.The effect of dietary copper level on the suckling piglets’ health was further discussed by correlation analysis.One hundred and seventy-two piglets from 18 multiparous Suhuai sows(inseminated twice)were blocked by their expected farrowing dates and assigned to three groups:6,20,and 300 mg·kg-1 Cu(CuSO4).All 14 days’ piglets were fed a com-soybean base diet,feed and water were available ad libitum for all piglets during the 40-d animal trial.The results showed that,compared with 6 mg·kg-1 Cu group,the average daily gain(ADG)(P<0.05)and average daily feed intake(ADFI)(P<0.05)were significantly increased in 300 mg·kg-1 Cu group during d 14 to 28,while the average daily gain(ADG)(P<0.05)and feed conversion rate(FCR)(P<0.05)were notably decreased during d 29 to 40.The diarrhea rate was significantly decreased in 300 mg·kg-1 Cu group compared with other groups(P<0.05).Moreover,compared with 6 mg·kg-1 Cu group,the malonaldehyde(MDA)(P<0.05),alanine aminotransferase(ALT)(P<0.05),aspartate transaminase(AST)(P<0.05),total bile acid(TBA)(P<0.05)were dramatically decreased in 20 mg·kg-1 Cu group;while the growth hormone(GH)(P<0.05),TNF-α(P<0.05),total bile acid(TBA)(P<0.05),Albumin(P<0.05),metallothionein(P<0.05)were significantly increased,also the SOD tended to reduce in 300 mg·kg-1 Cu group(P=0.09).Furthermore,compared with 6 mg·kg-1 Cu group,the hair,and serum Mg were significantly increased in 20 mg·kg-1 Cu group(P<0.05),the hair Na and K,serum Ca,P and fecal Cu were significantly increased in 300 mg·kg-1 Cu group(P<0.05);compared with 20 mg·kg-1 Cu group,the hair Na and K were remarkably increased(P<0.05),Mg,P,and Mn were notably decreased in 300 mg·kg-1 Cu group(P<0.05).The significantly positive correlation between each element was observed(P<0.05),the interaction between macro(Ca,Mg,P,Na,K)and micro(Cu,Mn,Zn)elements were disappeared in 300 mg·kg-1 Cu group.The hair Na and K were positively correlated with IGF-1 and total antioxidant capacity(T-AOC)(P<0.05),negatively correlated with TNF-α and metallothionein(P<0.05).The Fe of hair,serum and feces were positive correlated with metallothionein,alanine aminotransferase(ALT),and Albumin(P<0.05),negative correlated with malonaldehyde(MDA)(P<0.05).The Cu of hair,serum and feces were negative correlated with malonaldehyde(MDA),total bile acid(TBA)and blood urea nitrogen(BUN)(P<0.05).In summary,dietary high level(300 mg·kg-1)copper could only promote growth in short term,but it was lack of a significant growth response over the entire experimental period.Also,the hepatorenal function and redox balance were impaired.The FCR was higher in 20 mg·kg-1 Cu group,which could effectively improve the antioxidant ability and protect tissues from oxidative damage compared with 6 and 300 mg·kg-1 Cu groups,the diarrhea rate was in the acceptable range.The unbalance of Na,K and Fe,Zn in 300 mg·kg-1 Cu group,which leading to redox unbalance and hepatorenal dysfunction.5 Effect of dietary copper level in antibiotic free creep feed on gut microbiota and its correlation with inflammatory and oxidative status in suckling pigletsGut microbiota plays a crucial role in animal health,which needs to be more considered when we investigate the effect of dietary copper level on suckling piglets’ health.In this study,the 16S rRNA pyrosequencing was also designed and used to investigate the effect of dietary copper level on gut microbiota of suckling piglets.The experimental design was consistent with chapter 4.The results showed that dietary copper level did not affect the richness and diversity indices of fecal microbiota(P>0.05),and the relative abundance of Enterococcus,Escherichia coli,Lactobacillus,Staphylococcus(P>0.05).Compared with 6 and 20 mg·kg-1 Cu group,the abundance of fecal Fibrobacteres and Streptococcus were remarkably increased,while the Coprococcus,Acidaminococcus,and Roseburi were significantly decreased in 300 mg·kg-1 Cu group(P<0.05).Correlation analysis showed that the abundance of Lachnospiraceae(NK4A136 and FCS020 group)were positive correlated with malonaldehyde(MDA)and albumin(p<0.05),negative correlated with total antioxidant capacity(T-AOC)(P<0.05).The abundance of Ruoinococcus was positive correlated with TNF-α(P<0.05).The abundance of Halomonas and Methanobrevibacter were positive correlated with TNF-α,albumin,and blood urea nitrogen(BUN)(P<0.05).The abundance of Corynebacterium was positive correlated with TNF-α and albumin(P<0.05).In summary,the abundance of butyrate producing bacteria were reduced in 300 mg·kg-1 Cu group,leading to a rise of potential pathogens(Streptococcus),and changes in abundance of certain bacteria that related to inflammatory response,oxidative state,and the hepatorenal function.These results suggested that high level copper might play an adverse role in animal health.6 Effect of dietary copper level in antibiotic free creep feed on the fecal metabolites and its correlation with ion balance and gut microbiota in suckling pigletsIn this study,non-targeted metabolome was designed and used to investigate the effect of dietary copper level on the fecal metabolites of suckling piglets.The experimental design was consistent with chapter 4.Metabolome analysis by GC-MS showed that a total of 91 metabolites were detected and 44 metabolites were identified as the potential significant different metabolites among three groups,including amino acids,carbohydrates,fatty acids,polyols,and organic acids.Compared with 20 and 300 mg·kg-1 Cu group,the essential amino acids(leucine,phenylalanine,methionine,and arginine),conditionally essential amino acid(proline and tyrosine),monosaccharide(arabinose and mannose),and sugar phosphates(glucose-6-phosphate,mannose-6-phosphate and fructose-6-phosphate)were dramatically up-regulated in 6 mg·kg-1 Cu group(P<0.05).Additionally,dietary copper level significantly affected the carbohydrate metabolism(galactose metabolism and gluconeogenesis),metabolism of small molecules containing N(protein biosynthesis,urea cycle,and metabolism of arginine,proline,alanine,phenylalanine,tyrosine,methionine)(P<0.05).Regarding the correlation between ion and metabolites,the hair Na,K and Cu were negatively correlated with purine metabolism(Inosine),phenylalanine and tyrosine metabolism as well as mitochondrial electron transport chains(fumaric acid)(P<0.05),the serum Ca,Mg,and P were correlated with urea cycle,arginine and proline metabolism(ornithine and arginine)(P<0.05).Moreover,fecal Cu was significantly correlated with betaine metabolism(methionine),biosynthesis of pantothenic acid and coenzyme A(pantothenic acid)(P<0.05).Concerning the correlation between microbiota and metabolites,the relative abundance of Prevotellaceae UCG-004 was correlated with galactose metabolism(glucose,mannose,inositol)and glucose-alanine cycle(glucose)(P<0.05),the abundance of Coprococcus,Acidaminococcus,and Ruminococcus(OTU18)were correlated with carbohydrate metabolic pathways(citric acid cycle,Malate-aspartate shuttle,gluconeogenesis,pentose phosphate pathway,and glycolysis)(P<0.05),the abundance of Streptococcus were negative correlated with small molecules containing N pathways(protein biosynthesis,and metabolism of phenylalanine,proline,methionine,glycine,serine,threonine)(P<0.05).In summary,the dietary copper level significantly altered the composition of metabolites in the hindgut of suckling piglets.The absorption and digestion rate of protein and carbohydrate reduced in 6 mg·kg-1 Cu group.High-level(300 mg·kg-1)copper in the diet changed the elements in hair,serum and feces,which made the ion status unbalance and further affected the protein,carbohydrate,fat,and energy metabolism.Changes in the abundance of certain bacteria impacted the microbial metabolism pathways and metabolic homeostasis in piglets,which also had an adverse effect on the health.