Effect And Mechanism Of Dietary Rice Bran Oil Supplementation To Improve Growth Performance,intestinal Barrier And Gut Microbiota Of Early-weaned Piglets

In large-scale and intensive pig production,early weaning strategies are generally adopted to maximize sow utilization efficiency,which leads to anorexia,intestinal damage and oxidative stress,intestinal inflammation and intestinal microbiota imbalance as the main symptoms Early weaning stress in piglets.As China expands the scope of banning antibiotics for disease resistance and growth promotion and limits the addition of copper and zinc,it is imminent to find alternatives to antibiotics to relieve intestinal damage and intestinal microbiota disturbance caused by early weaning stress in piglets.Rice bran oil(RBO)is a polyphenol-rich by-product of rice processing,mainly composed of tocotrienols and tocopherols,which have antioxidant and anti-inflammatory properties in vivo and in vitro.The main components of rice bran oil used in this experiment were tocopherols and tocotrienols,which were 4.932 μmol/L α-tocopherol,9.740 μmol/Lα-tocotrienol,16.465 μmol/L β-tocotrienol,5.390 μmol/L γ-tocotrienol,and 8.865 μmol/Lδ-tocotrienol.In order to explore the effect and mechanism of adding rice bran oil to the diet to improve the growth performance,intestinal barrier and intestinal microbiota of early-weaned piglets,this study first explored the improvement of the growth performance,intestinal barrier and intestinal microbiota of early-weaned piglets by different additive doses of RBO The effect of microbiota and its mechanism;then the effect and mechanism of optimal dietary RBO supplementation level on lipopolysaccharide(LPS)-induced intestinal dysfunction in early weaned piglets were studied.The main experiments and their findings are as follows.Part 1 Effects of dietary rice bran oil supplementation to improve growth performance,gut barrier and gut microbiota in early weaned piglets168 piglets(21 ± 1 day old)were randomly divided into 4 groups(7 replicates in each group,6 piglets in each replicate,male and female half and half),received basal diet(Ctrl),basal diet + 100 mg/kg RBO(RBO1),basal diet + 200 mg/kg RBO(RBO2)or basal diet +300 mg/kg RBO(RBO3)treatment,the test period is 21 days.Subsequently,7 piglets in each group were randomly sacrificed to collect serum,jejunum,ileum,colon tissue and feces.The research results are as follows:(1)Adding different proportions of RBO to the diet can significantly reduce the 4th day of early weaned piglets(P < 0.05),the 8th day(P < 0.01)and day 12(P < 0.05)diarrhea score.200 mg/kg RBO and 300 mg/kg RBO could significantly increase the ADFI of early weaned piglets(P < 0.05)and ADG(P < 0.01),but only 200 mg/kg The RBO group can reduce the feed-to-meat ratio(P < 0.05).(2)In the jejunum tissue,compared with the control group,200 mg/kg RBO significantly increased the villi length of jejunal tissue(P < 0.05),significantly decreased the crypt depth of jejunal tissue(P < 0.01),and increased the ratio of villi length/crypt depth(P < 0.05).In the ileum tissue,compared with the control group,200 mg/kg RBO could significantly increase the length of villi in ileum tissue(P < 0.05),but had no significant effect on the crypt depth and ratio of villi length/crypt depth in ileum tissue.(3)In the jejunum tissue,compared with the control group,200 mg/kg RBO can reduce pro-inflammatory factor TNF-a,IL-1β,IL-6 m RNA expression trend,but no significant difference,only 200 mg/kg RBO significantly reduced pro-inflammatory factors IL-1β(P < 0.05,Fig.2-3B)m RNA expression,and significantly increased antiinflammatory factors Expression levels of IL-10(P< 0.05)m RNA.In ileum and colon tissue,200 mg/kg RBO can significantly increase anti-inflammatory factors IL-10(P< 0.05)m RNA levels,on pro-inflammatory factors TNF-a,IL-1β,IL-6 There was no significant effect on m RNA expression.(4)RBO treatment groups(P < 0.01)could significantly increase the concentration of T-AOC in serum,and significantly increase the concentration of SOD in serum.add 1 00mg/kg RBO and 2 00 mg/kg RBO significantly reduced the concentration of MDA in serum of early weaned piglets.add 2 00 mg/kg RBO significantly increased the concentration of specific immune indicators Ig G,Ig A,Ig M and non-specific immune indicators PDB-1,LZM(P < 0.01).(5)In the jejunum tissue,add 1 00 mg/kg RBO significantly increased the expression of Claudin-1(P < 0.05)m RNA,adding 2 00 mg/kg RBO significantly increased the expression of Claudin-1(P < 0.001),ZO-1(P < 0.01),adding 3 00 mg/kg RBO Significantly increased Occludin Expression levels of m RNA(P < 0.05).In colon tissue,add 2 00 mg/kg RBO significantly increased the expression levels of Claudin-1(P < 0.01),Occludin(P < 0.01),ZO-1(P < 0.05)m RNA.add 2 00 mg/kg RBO significantly increased the protein expression levels of Claudin-1 in jejunum tissue(P < 0.05)and Occludin in colon tissue(P < 0.05).Summary : Adding different doses of RBO to the diet can improve the growth performance of early weaned piglets,reduce the rate of diarrhea,reduce the production of pro-inflammatory factors,increase the level of anti-inflammatory factors,and increase the tight junction proteins Occludin and Claudin-1 in jejunum and colon tissues The expression of m RNA increases the protein levels of tight junction proteins Occludin and Claudin-1,and improves the antioxidant capacity of intestinal tissue,thereby improving intestinal damage caused by weaning stress in piglets.Part 1I Effects of dietary rice bran oil supplementation to alleviate the disturbance of microbiota in the jejunum and colon of early weaned pigletsThis part of the animal experiment is the same as the first part,using 16 S r DNA sequencing to analyze the microbiota in the jejunum and colon contents of piglets,and measure the concentration of short-chain fatty acids in the jejunum and colon contents.The research results are as follows:16S r DNA sequencing of the jejunum and colon contents of early-weaned piglets covered most of the microbiota,and the results of PCo A analysis of microbiota β-diversity showed that different doses of RBO could lead to changes in the jejunum and colonic microbiota of early-weaned piglets,and form different clusters with the Ctrl group,among which the RBO2 group has the most significant treatment results;both can improve the content microbiota diversity index Feature,Chao 1,ACE,Shannon,Simpson,PD＿whole＿tree and Coverage of the jejunum and colon to varying degrees,but did not reach the significance level.(2)In the contents of the jejunum,add 200 mg/kg RBO significantly increased propionate concentrations but had no significant effect on acetate and butyrate.In colonic fecal contents,adding different doses of RBO significantly increased the concentrations of acetate,propionate and butyrate in colonic fecal samples.(3)The dominant bacteria Collinsella at the species level Aerofaciens were significantly positively correlated with Claudin-1 and villus height,and significantly negatively correlated with MDA.Acetic acid was significantly negatively correlated with MDA;propionic acid was significantly positively correlated with T-AOC and SOD,butyric acid was significantly positively correlated with T-AOC and villi height/crypt depth,and significantly negatively correlated with MDA.Summary: Add different doses to the diet RBO may improve the intestinal barrier function and antioxidant capacity of piglets by improving the structure of intestinal microbiota at the genus and species levels and promoting the metabolites of SCFAs of intestinal microbiota.Part III Effects and mechanism of dietary rice bran oil supplementation to relieve LPS-induced intestinal damage and gut microbial dysbiosis in pigletsThis part of the animal experiment is the same as the first part,7 piglets from each group of Ctrl and RBO2 groups were treated with lipopolysaccharide(LPS)(100 μg /kg BW)(LPS group and RBO+LPS group respectively),meanwhile,the piglets of Ctrl group Seven piglets were treated with vehicle saline(Ctrl group).Four hours later,all treated piglets were sacrificed for collection of serum,jejunal tissue and feces.The research results are as follows:(1)Compared with the control group,the LPS group could significantly increase the pro-inflammatory factors TNF-a(P < 0.01),IL-1β(P < 0.01),IL-6 The expression level of m RNA(P < 0.05),supplemented with 2 00 mg/kg RBO can significantly reduce the gene expression levels of pro-inflammatory factors TNF-a(P < 0.05),IL-1β(P < 0.05),IL-6(P < 0.01),and significantly increase the anti-inflammatory factor IL-10 The expression level of m RNA(P < 0.01).(2)Compared with the control group,the LPS group can significantly increase the DAO(P < 0.05)and MDA(P < 0.05)contents in the serum of early weaned piglets,and significantly reduce the CAT(P < 0.05),SOD(P < 0.05),the concentration of T-AOC(P < 0.001).Compared with the LPS group,supplemented with 2 00 mg/kg RBO can significantly reduce the content of DAO(P < 0.05)and MDA(P < 0.05)in serum of early weaned piglets,and significantly increase CAT(P < 0.05),SOD(P < 0.05),T-AOC(P< 0.0 01)concentration.(3)Compared with the control group,the LPS group significantly reduced the serum Ig M(P < 0.05)content of early weaned piglets,and significantly reduced the serum LZM(P < 0.05)concentration of piglets;compared with the LPS group,supplemented with 200mg/kg RBO can significantly increase the content of Ig A(P < 0.05)and Ig M(P < 0.05)in serum,and significantly increase the concentration of β-defensin-1(P < 0.05)and LZM(P < 0.05).(4)The diversity of β-PCo A showed that the LPS group and the RBO+LPS group formed obvious clusters with the control group.The analysis of Alpha diversity results showed that compared with the control group,LPS reduced the indicators Observed species,Chao1,ACE,but had no significant effect on Shannon,Simpson and Coverage;compared with the LPS group,supplemented with 200 mg/kg RBO can significantly increase Chao1 index,but has no significant effect on Observed species,ACE,Shannon,Simpson and Coverage index.(5)Compared with the control group,the stimulation of LPS group can increase the function of amino acid metabolism,cell function,digestive system,lipid metabolism,metabolism,transport and decomposition of terpenoids and polyketones in the intestinal microbiota of early weaned piglets Metabolism,biodegradation and metabolism of xenobiotics,but reduces the ability to metabolize carbohydrates.Compared with the LPS group,dietary supplementation with RBO significantly increased carbohydrate metabolic function,excretory system,biodegradation,and metabolism of xenobiotics,but decreased biosynthesis of other secondary metabolites,energy metabolism,environmental adaptation,and glycan synthesis.Biosynthesis and Metabolism.At the same time,compared with the control group,the LPS group significantly reduced the concentration of short-chain fatty acids propionate and butyrate in feces.Dietary supplementation with RBO significantly increased fecal short-chain fatty acid concentrations including acetate,propionate,and butyrate compared with LPS group.Collinsella dominant bacteria at species level Aerofaciens were significantly positively correlated with Claudin-1 and villus height,and significantly negatively correlated with MDA.The dominant bacterium Clostridiaceae at the genus level was significantly positively correlated with the height of jejunal villi,and significantly negatively correlated with MDA.ACE,which represents the alpha diversity index of the microbiota,was positively correlated with Claudin-1.Acetic acid was significantly positively correlated with villi length and the ratio of villi length to crypt depth in jejunal intestinal tissue and was significantly negatively correlated with DAO.Propionic acid was significantly positively correlated with T-AOC,villi height and the ratio of villi height to crypt depth,and significantly negatively correlated with DAO.Butyric acid was significantly positively correlated with Occludin,Claudin-1,villi height and villi height and crypt depth,and was significantly negatively correlated with DAO and MDA.Summary: Adding 200mg/kg RBO to the diet can effectively prevent LPS-induced intestinal damage,oxidative stress and jejunal microbiota imbalance.Conclusion: Adding different doses of RBO to the diet can reduce the production of pro-inflammatory factors in the intestine of piglets,increase the level of anti-inflammatory factors in the intestine,increase the expression of tight junction proteins Occludin and Claudin-1 m RNA in the jejunum and colon tissues,and increase the tight junction.The protein levels of Occludin and Claudin-1 can improve the structure of intestinal microbiota at the genus and species levels,promote the metabolites of SCFAs of intestinal microbiota,and then improve the intestinal barrier function,increase the antioxidant capacity of intestinal tissue,and finally reduce the risk of early weaning Piglet diarrhea and improved growth performance in early weaned piglets.The test results showed that adding 200 mg/kg of RBO is the optimal dose.