| With the advance of modern genetics techniques and the improvement of sow reproductive performance,artificial rearing system has become increasingly used in commercial pig production.Studies have found that artificial rearing caused high incidence of diarrhea,increased mortality,and seriously harmed welfare in piglets.Fecal microbiota transplantation(FMT)is a method for curing diseases by transplanting fecal microbiota from a healthy donor into the recipient’s intestine to reshape the intestinal microbiota structure.However,whether FMT can relieve diarrhea in artificially-reared piglets remains to be further investigated.Therefore,this study selected artificially-reared piglets as the research object,evaluated the effects of artificial rearing on welfare based on physiological indicators and behavioral expression,and applied 16 s r RNA and RNA-seq technologies to clarify the mechanism underlying artificial rearing-induced diarrhea.Fecal microbiota from sow-reared piglets was transplanted,and the mechanism by which FMT alleviated diarrhea in artificially reared piglets was explored.This study helps elucidate the effects of artificial rearing on the welfare and health of piglets and provides a theoretical basis for FMT as a feasible approach to mitigate diarrhea in piglets.Part Ⅰ: Effects of artificial rearing on growth performance and welfare status of pigletsIn this experiment,a total of 30 Duroc×Min sows and 240 Yorkshire×Duroc×Min newborn piglets were selected and randomly assigned to three groups: the control group(CON group),artificially-reared piglets from 21 days of age(AR21 group),and artificially-reared piglets from 7days of age(AR7 group).The trial period lasted for 36 days.We weighted piglets,observed normal behaviors(drinking milk,eliminating,exploring,playing,and fighting)and abnormal behaviors(belly nosing and oral manipulation of conspecifics),assessed the area of tear staining,and measured cortisol concentration and immune indicators(Ig G,Ig A,CRP,IL-1β,IL-4,IL-6,IL-10,and TNF-α).The results were as follows:(1)The average daily gain and body weight of artificially-reared piglets were significantly lower than those of the sow-reared piglets(P<0.05).(2)During the entire behavioral observation period,the AR7 piglets displayed significantly fewer drinking milk,exploring,and playing(P<0.05)but significantly more belly nosing and oral manipulation of conspecifics(P<0.05).The AR21 piglets exhibited significantly fewer drinking milk and playing(P<0.05)but significantly more belly nosing(P<0.05).(3)At 21 days,the AR7 piglets were more likely to defecate in the rest area.(4)The tear staining scores of artificially-reared piglets were significantly higher than those of the sow-reared piglets(P<0.05).(5)Compared to the sow-reared piglets,the AR7 piglets had significantly higher concentrations of cortisol,CRP,IL-1β,IL-4,IL-6,and TNF-α(P<0.05),but significanltly lower concentrations of Ig G,Ig A,and IL-10(P<0.05).The AR21 piglets had significantly higher levels of CRP,IL-4,and IL-6(P<0.05)but significanltly lower IL-10(P<0.05).These results indicate that artificial rearing from 7 and 21 days of age can cause stress in piglets,which has a negative impact on growth performance,welfare status,and immune function.The presence of sows can help piglets to establish a fixed elimination area earlier.Part II: Study on the mechanism of artificial rearing-caused diarrhea of pigletsIn this experiment,10 piglets were randomly selected from each group.The contents and mucosa of the ascending colon were collected.Colonic microstructure was observed using H&E staining and PAS staining.The colonic microbiota were detected using 16 s r RNA sequencing.Differentially expressed genes were detected by RNA-seq and then verified by RT-q PCR and Western blot.The results were as follows:(1)The diarrhea rate of artificially-reared piglets was significantly higher than that of sowreared piglets(P<0.05).(2)Compared with the sow-reared piglets,the richness and diversity of colonic microbiota were significantly decreased in artificially-reared piglets(P<0.05).The microbiota composition was changed,and the carbohydrate metabolism pathway was significantly up-regulated.(3)Compared with sow-reared piglets,artificially-reared piglets showed obvious microstructural changes in the colon,especially leukocyte infiltration.The number of goblet cells was significantly reduced in the colon of AR7 piglets(P<0.05).(4)Differentially expressed genes,including tight junction proteins,NF-κB pathway-related genes,cytokines,and SLC family members were screened by RNA-seq.These genes were significantly enriched in signaling pathways related to inflammation,immunity,and nutrient digestion and absorption.(5)Compared with sow-reared piglets,the expression of tight junction proteins ZO-1,Claudin-2,and JEAP in the colon of artificially-reared piglets were significantly reduced(P<0.05);TLRs pathway-related genes TNFSF11,TNF-α,IL-1β,TLR2,TLR4,and My D88 was significantly increased(P<0.05),IL-1Ra was significantly decreased(P<0.05);NF-κB pathway-related genes NF-κB,COX-2,PTGEs,and i NOS significantly up-regulated(P<0.05),the expression of IκBαsignificantly down-regulated(P<0.05);the expression of cytokines IL-2,IL-6,IL-17 and IFN-γ was significantly increased(P<0.05);the expression of SLC family members SLC51 A,SLC51B,SLC9A3,SLC26A3 and SLC2A5 were significantly decreased(P<0.05).These results indicate that artificial rearing alters colonic microbiota in piglets,activates the NF-κB pathway,promotes the secretion of pro-inflammatory cytokines,regulates the expression of the SLC family members,affects solute transport,and increases diarrhea rate in piglets.Part III: Effects of FMT on growth performance and welfare status of pigletsIn this experiment,a total of 18 Duroc×Min sows and 144 Yorkshire×Duroc×Min newborn piglets were selected and randomly divided into three groups: the control group(CON group),artificially-reared piglets(AR group),and FMT-treated piglets(FMT group).The trial period lasted for 36 days.Healthy piglets without specific pathogens were selected as experimental donors.Sows reared these donor piglets until weaning.Feces were collected and made into fecal microbiota capsules.From 7 days of age,AR and FMT piglets were artificially fed with milk replacer.At 1-11 days of age,the piglets in the FMT group were fed with fecal microbiota capsules.We measured body weight,observed normal behaviors(drinking milk,eliminating,exploring,playing,and fighting)and abnormal behaviors(belly nosing and oral manipulation of conspecifics),and analyzed cortisol concentration.The results were as follows:(1)After being FMT,the average daily gain and body weight at 35 days of age increased significantly(P<0.05).(2)Compared with the artificially-reared piglets,there were no significant differences in the behaviors of drinking milk,exploring,playing,belly nosing,and oral manipulation of conspecifics(P＞0.05).(3)Compared with the artificially-reared piglets,the cortisol concentration in the FMT group decreased significantly(P<0.05).There results indicate that FMT can improve growth performance and welfare status of artificially-reared piglets to some extent.Part IV: Effects of FMT on colonic microstructure,microbiota,and immune response in pigletsIn this experiment,6 piglets were randomly selected from each group.The contents and mucosa of the ascending colon were collected.Colonic microstructure was observed by H&E staining and PAS staining.The colonic microbiota were detected using 16 s r RNA sequencing.Differentially expressed genes were screened by RNA-seq and then verified by RT-q PCR and Western blot.Differential metabolites in intestinal contents were detected using non-targeted metabolomics.A model of IPEC-J2 cells treated with 5-HIAA and LPS was established.Cell viability was measured using a CCK-8 kit,and the expression of tight junction proteins and cytokines were detected using RT-q PCR,Western blot,and immunofluorescence staining.The results were as follows:(1)After being FMT,the diarrhea rate of artificially-reared piglets significantly decreased(P<0.05).(2)FMT changed the composition of piglet colonic microbiota,and the relative abundance of Bifidobacterium significantly increased(P<0.05)(3)FMT significantly improved artificial rearing-induced colon damage and reduced neutrophils.Goblet cells number and MUC2 expression significantly increased(P<0.05).(4)Differentially expressed were significantly enriched in the IL-17 signaling pathway.(5)Compared with the artificially-reared piglets,tight junction proteins ZO-1,Claudin-1,Claudin-4,Occludin,and E-cadherin in the colon of FMT piglets were significantly increased(P<0.05);TLRs pathway-related genes IL-1β,TNF-α,TLR2,and TLR4 were significantly downregulated(P<0.05);JNK pathway-related genes MEKK1,MEKK4,MKK4,MKK7,JNK1,JNK2,c-Jun,and c-Fos were significantly down-regulated(P<0.05);the cytokines IL-6,IL-17 and IFN-γwere significantly decreased(P<0.05),but the IL-2 and IL-10 were significantly increased(P<0.05);the chemokines CXCL2,CXCL8,CXCL10 and CCL2 was significantly decreased(P<0.05);MMP family members MMP1,MMPP9,and MMP13 decreased significantly(P<0.05);CYP family members CYP1A1 and CYP1B1 was significantly upregulated(P<0.05).(6)Non-targeted metabolomics analysis showed that differential metabolites were significantly enriched in the tryptophan metabolic pathway.(7)The combined analysis of 16 s r RNA and non-targeted metabolomics showed a significant positive correlation between Bifidobacterium and the tryptophan metabolite 5-HIAA.(8)After being FMT,the concentrations of 5-HT and 5-HIAA in the colon of artificially-reared piglets were significantly increased(P<0.05).(9)In IPEC-J2 cells,5-HIAA can alleviate LPS-induced inflammatory response,which manifested by a significant increase in cell viability(P<0.05),a significant increase in the expression of CYP1A1 and CYP1B1(P<0.05),significant increases of tight junction proteins Claudin-1,Claudin-4,Occludin,and E-Cadherin(P<0.05),a decrease in the expression of cytokines TNF-α,IL-1β,IL-6 and IL-17 as well as a increase of IL-10(P<0.05).These results indicate that FMT can change the composition of the colonic microbiota and its metabolites,promote tryptophan metabolism and the production of 5-HIAA,enhance the intestinal mucosal barrier function,inhibit the activation of JNK pathway and the expression of MMPs,reduce the secretion of pro-inflammatory cytokines and chemokines,and alleviate diarrhea in artificiallyreared piglets. |
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