| Goose fatty liver,rich in lecithin and unsaturated fatty acids,is a high-end healthy food and waterfowl product.It has a large market at home and abroad,and the market is expanding with the improvement of living standard of customers.Landes goose is the main breed for foie gras(or fatty liver)production.In the process of overfeeding,the feed containing high-energy maize is digested and absorbed by intestinal tract,followed by transformation of oversupplied energy into fat,which is deposited in the liver and other tissues.The intestinal tract is the interface between animal body and nutrients and the interface between animal body and intestinal microbes.It can affect the physiological functions of tissues and organs in the body through the intestine-liver axis.In addition to the basic function,nutrient digestion and absorption,the intestinal tract also has the functions of endocrine and multiple barriers.When the structures and functions of intestinal tract and intestinal bacteria are in disorder,diseases may occur in the body.However,in the development of goose fatty liver,the adaptive changes of intestinal structure and transcriptome in response to overfeeding have not been systematically elucidated.The purpose of this study was to reveal the adaptive changes of intestinal structure and transcriptome in response to overfeeding by comparing the histological structures and transcriptomes of different intestinal segments(jejunum,ileum and cecum)between the overfeeding and non-overfeeding groups of geese at early(12 days)and late(24 days)stages of overfeeding,and by combining the results from the determination on the content of plasma lipopolysaccharide(LPS)with some of the data from metabolomics analysis and metagenomics analysis of intestinal microbes.The contribution of the changes to the development of goose fatty liver was discussed.The main results are as follows:(1)The average body weight,liver weight and the ratio of liver to body weights in the overfed geese were increased significantly(P<0.01),compared to the control geese.They were 6.87 kg,830.19 g and 12.09%,respectively,at 24 days of overfeeding.This indicated that the effect of overfeeding was obvious and induced a large amount of changes in the bodies of the overfed geese.Indeed,the histological analysis of intestinal structure showed that intestinal tract had obvious adaptation to overfeeding.First of all,HE staining analysis showed that the intestinal villi were long,contact and orderly,the intestinal crypts were clear,arranged closely and orderly,and there was no special pathological phenomenon and injury,suggesting that the intestinal tract was not damaged by overfeeding.However,compared with the control group,both the villus height and villus-to-crypt ratio were significantly increased in the ileum,the depth of the crypt was significantly increased and the villus-to-crypt ratio was significantly decreased in the jejunum at early stage of overfeeding,the villus height of the jejunum and ileum and the villus-to-crypt ratio of the ileum were significantly increased at late stage of overfeeding,while the villus height of the cecum was significantly decreased.The findings indicated that both the early and late stages of overfeeding promoted the proliferation of epithelial cells,digestion and absorption of the ileum.This promotion was also seen in the jejunum at late stage of overfeeding.In contrast the inhibitive effect of overfeeding was shown in the jejunum at the early stage of overfeeding and in the cecum at the late stage of overfeeding.Therefore,the enhanced adaptive function of ileum in response to overfeeding may be the main contributor to the enhancement of nutrient digestion and absorption,which is crucial to the development of goose fatty liver.The measurement of plasma LPS content in the overfed and control geese showed that there was no significant change between the two groups at both early and late stages of overfeeding(F>0.05),which further indicated that the integrity of intestinal structure was not affected by overfeeding,providing a protective mechanism for goose fatty liver against inflammation.(2)The transcriptomes of jejunum,ileum and cecum from the overfed and control geese at the early and late stages of overfeeding were sequenced.The results showed that 1,572 differentially expressed genes(DEGs)were identified between the two groups of geese(711 genes up-regulated,and 861 genes down-regulated in the overfeeding vs control groups).There were 102,176 and 132 DEGs in the jejunum,ileum and cecum,respectively,at the early stage of overfeeding,and 100,325 and 737 DEGs at the late stage of overfeeding.KEGG pathway analysis showed that DEGs in all intestinal segments were enriched in 97 pathways,including 13,17 and 5 pathways in jejunum.ileum and cecum at the early stage of overfeeding,and 8,22 and 32 pathways at the late stage of overfeeding.In general,DEGs are lainly enriched in the pathways involved in multiple amino acid metabolisms,lipid metabolisl,immune/inflammation,endocrine,signal transduction and other biological processes.GO analysis showed that DEGs in all intestinal segments were included in 22 GO categories,including 5,3 and 3 GO categories in the jejunum,ileum and cecum at the early stage of overfeeding,and 7,0 and 4 GO categories at late stage of overfeeding.Generally,GO categories of DEGs were mainly involved in cytoplasmic region,cortex,multiple nutrient binding,immune/inflammation,enzyme activity,defense response,etc.(3)Real-time quantitative PCR was used to validate the transcriptomic data.The data indicated that 8 out of 9 genes were differentially expressed between the control and overfeeding groups,which reflected the reliability of the transcriptome analysis.Among the DEGs obtained by transcriptome analysis,there were some DEGs related to inflammation and tight junction.For example,the expression of FGB,C8A and SOCS3 in intestinal tract was significantly decreased(P<0.05),which suggested that overfeeding inhibited the occurrence of intestinal inflammatory response.The expression levels of ZO-1(TJP1)and OCLN in cecum for tight junction were not significantly different between the overfeeding and control geese,indicating that overfeeding did not damage the integrity of intestinal structure,which provided a good barrier for the body.(4)KEGG analysis of intestinal transcriptome also revealed that the DEGs were enriched in sphingolipid metabolism pathway in the ileum and cecum of the overfed and control geese at the late stage of overfeeding.In the ileum,sphingolipid synthesis was increased(SPTLC3),ceramide synthesis was decreased(CERS1)and ceramide degradation was increased(ASAH2).In the cecum,sphingolipid synthesis was decreased(SPTLC1)and ceramide synthesis was increased(CERS6).Furthermore,metagenomics analysis of intestinal microbes showed that the relative abundance of the sphingolipid metabolism-involving bacteria,Bacteroides,in the jejunum and ileum of the overfed goose was significantly increased at the late stage of overfeeding,while that in the cecum was significantly decreased.In addition,metabolomics analysis of intestinal contents showed that the levels of DL-dihydrosphingosine,phytosphingosine and D-erythro-sphingosine in intestinal contents were affected by overfeeding.The levels of the sphingosines in the jejunum were significantly increased at the early stage of overfeeding,but decreased at the late stage of overfeeding;the levels of the sphingosines in the ileum were decreased at the early and late stages of overfeeding;and the levels of the sphingosines in the cecum were increased at the early stage of overfeeding.Consistently,ethanolamine(or cholamine)and ethanolamine phosphate,the substrates involved in the synthesis of sphingolipids,were also significantly different between the overfeeding and control groups.That is to say,the relative concentration of ethanolamine iin the ileum was decreased significantly at the early and late stages of overfeeding,but increased significantly in the cecum at the late stage of overfeeding,and the relative concentration of O-phosphate ethanolamine was decreased in the jejunum and increased in the cecum at the late stage of overfeeding.These findings suggested that sphingolipid metabolism played an important role in the development of goose fatty liver from three aspects,including intestinal transcriptome,intestinal bacteria and intestinal metabolites.The effect of overfeeding on sphingolipid metabolism may help to explain the response of ileum and cecum to overfeeding,i.e.ileal proliferation,the increase of ileal villus height,the cecal atrophy,the decrease of cecal villus height,and the deepening of cecal crypts,and explain no inflammation occurred in the intestine and liver.In conclusion,the integrity of intestinal structure is good during the development of goose fatty liver,which provides a protection against inflammation induced by intestinal bacteria invading the body.Overfeeding promoted the proliferation of intestinal epithelial cells,the increase of villus height and villus-to-crypt ratio in ileum,and the atrophy of cecum,the decrease of villus height and the deepening of crypt in cecum.Ileum is the main contributor of nutrient digestion and absorption in the overfed geese.The change in the expression of inflammation or tight junction-related genes indicates that there is no inflammation in the intestinal tract of the overfed geese at the molecular level,and the integrity of intestinal structure is not damaged by overfeeding,providing a protective mechanism against inflammation in goose fatty liver.Intestinal transcriptome analysis demonstrates that overfeeding mainly affected the pathways involved in multiple amino acid metabolism,lipid metabolism,immune inflammation,endocrine,signal transduction.Sphingolipid metabolism may play an important role in the effect of overfeeding on intestinal structure and the development of fatty liver.This study is helpful to elucidate the changes of intestinal structure and the mechanism by which the changes influence the development of fatty liver in Landes goose during overfeeding. |
