Effects Of Long-chain Fatty Acid On Pig Growth Performance And Study Of Intestinal Absorption Mechanism

Long-chain fatty acids(LCFAs)are the most abundant fatty acids in feeds.On the one hand,LCFA can be used as efficient substances for energy supply and energy storage in the body.On the other hand,LCFA participates in biomembrane constitution,cell apoptosis,and regulation of various genes,which are important for neurological development,intestinal health,and immunity.In animal farming,it is generally believed that supplementing LCFA into feed can improve the growth performance of commercial pigs.Also,LCFA plays a crucial role in health condition and development of weaning piglets.However,the influential factors of growth promoting effects and the underlying mechanism is not understood yet.As the fundamental step for LCFA exerting functions the intestinal uptake mode of LCFA still remains unknown.Hence,this study is focused on the effects of LCFA on pig growth performance and the LCFA uptake mechanism.Firstly,we introduced meta-analysis method to comprehensively and systematically collect and screen all the LCFA-related pig trials published in recent 20 years.We quantitatively analyzed the effects of LCFA supplementation on pigs,the influential factors,and the underlying mechanism.Secondly we built piglet and cell models showing reduced LCFA absorption caused by Enterotoxigenic Escherichia coli(ETEC)infection.We screened out genes that affect fatty acid absorption by changes in expression levels before and after infection.With respect to confirm the functions of targeted genes and the upregulating pathways,we manipulated the level of gene expression and phosphorylation by means including plasmid transfection and chemical treatment combined with fatty acid absorption detection.The major results and conclusions are as follow:(1)Effects of LCFA supplementation on the growth performance of pigs:a meta-analysisWe searched the PubMed and Web of Science databases(articles published from Jan 1st,2000,to Sep 30th,2018;restricted to English)and compared LCFA-supplemented diets with control diets.We retrieved 2,346 studies,18 of which(1,314 pigs)were eligible for our analysis.We used a random-effects model to calculate the weighted mean differences(WMDs)and 95%confidence intervals(CIs).The analyses included comprehensive analysis,regression analysis,subgroup analysis,sensitivity analysis,and publication bias analysis.Our results support that LCFA supplementation in feed improves the average daily gain(ADG)and gain:feed(G:F)ratio of both growers and finishers,whereas LCFA supplementation leads to a reduction in the average daily feed intake(ADFI)of finishers.Moreover,for finishers,only a high LCFA concentration(approximately 5%)is capable of enhancing the ADG and G:F ratio and decreasing the ADFI,whereas whether distillers’ dried grains with solubles are supplemented in basal diet and the saturation level of fatty acids have little effects on the ADG,ADFI and G:F ratio of finisher pigs.These findings indicate that the positive effects of LCFA supplementation on pig growth performance are resulted from increasing energy density,lowering the gastric emptying speed and enhancing the amino acid digestibility.In the next studies we should apply a higher LCFA concentration to explore the absorbing mechanism under growth promoting conditions.(2)LCFA absorption by pig intestinal epithelia upon ETEC infectionA total of 12 head 28-day-old weaning piglets were randomly divided into 2 groups with 6 duplicates in each.After 4-day preliminary feeding,on day 5 and day 6 the treatment group was gavaged with PBS containing ETEC and the control group was gavaged with the same volume of PBS.During day 7 to day 9,fecal consistency score was recorded consistently.After fasting overnight on day 9,all piglets were gavaged with 10 mL olive oil on day 10 and serum and jejunum samples were collected.Afterwards we found:(1)the ETEC-treated piglets developed symptoms of diarrhea;(2)the content of D-lactic acid in the serum increased;(3)the contents of serum LCFAs including C16:0,C18:1,C20:3,C20:4 and the total compromised,whereas the content of C14:0 whose chain length is similar to medium chain fatty acid(MCFA),was not affected;(4)the western-blot assays of jejunum samples exhibited the expression of tight junctions(claudin-1,occludin,ZO1)was hindered,the expression of fatty acid transport protein 4(FATP4)was compromised while fatty acid binding protein 1-3(FABP1-3)and cluster of differentiation 36(CD36)was not influenced,and we observed upregulated phosphorylation of extracellular signal-regulated kinase 1/2(ERK1/2)and peroxisome proliferator-activated receptor γ(PPARγ)together with the lowered expression of non-phosphorylated PPARy.Our results indicate that ETEC infection would cause abnormal absorption of LCFAs in piglets.The transportation and upstream regulation mechanism of fatty acid intestinal absorption is related to the expression of FATP4 and the phosphorylation levels of ERK1/2 and PPARγ.(3)LCFA absorption by pig intestinal epithelial cells upon ETEC infectionWe treated polarized IPEC-J2 cells with ETEC to establish an in vitro model.After 28-days polarization culture,IPEC-J2 cells formed a single confluent monolayer with dense microvilli on the cell surface.At the same time,the absorbing ability of LCFA C16:0 was greatly improved.But as for MCFA C12:0,the absorbing ability was not affected.The effects of ETEC infection on polarized IPEC-J2 cells included:(1)impaired intestinal barrier and occurrence of inflammation;(2)the absorption of LCFA C16:0 was disturbed,whilst absorption of MCFA C12:0 was not impeded;(3)the expression of FATP4 was inhibited,together with phosphorylated ERK1/2 and PPARγ.These results based on polarized IPEC-J2 cells are in consistence with the results from piglets.(4)Regulation role of ERK1/2-PPARγ-FATP4 pathway in LCFA absorption by pig intestinal epithelial cellsBased on the results from in vivo and in vitro models,we used polarized IPEC-J2 cells to explore the regulation role of ERK1/2-PPARγ-FATP4 pathway in LCFA absorption.We firstly knocked down FATP4 and found that the absorption of LCFA C16:0 decreased whereas MCFA C12:0 remained in the same level.Furthermore.we treated cells with ERK1/2 inhibitor U0126 and PPARy antagonist T0070907,respectively.It was found that ERK1/2 could activate cascaded phosphorylation of downstream PPARy and reduce the content of non-phosphorylated PPARy,thereby inhibiting the expression of FATP4 combined with impaired absorption of LCFA C16:0 by intestinal epithelial cells.These results indicate that ERK1/2 can inhibit the expression of FATP4 gene by inducing the phosphorylation of PPARγ and simultaneously reducing the content of non-phosphorylated PPARy,thereby negatively affecting FATP4-dependent intestinal LCFA absorption.In summary,this study focuses on the effects of LCFAs on pig growth performance and the underlying LCFA intestinal absorption mechanism.Firstly,through our meta-analysis,we systematically prove that LCFAs can promote the growth performance by increasing dietary energy levels,lowering the gastric emptying speed and enhancing the amino acid digestibility.Secondly,we used in vivo and in vitro models and confirmed that the absorption of LCFA by the pig intestine depends on FATP4,in which the cascaded phosphorylation of ERK1/2-PPARy can negatively regulate the absorption of LCFA by interfering FATP4 synthesis.This study will provide scientific assistance and new direction for the application of LCFAs in pig production and the research in intestinal absorption.