| Intestinal microorganisms and intestinal barriers play an important role in the health,growth and development of piglets.The objective of this study was to investigate the stress resistance of native swine(Mashen pigs)from the perspective of intestinal microorganisms and intestinal barrier,and to provide theoretical reference for studying the intestinal microecological mechanism of piglets.In present study,Mashen and Jinfen White pigs were used as experimental animals.The intestinal contents(jejunum,ileum,colon),intestinal tissues(duodenum,jejunum,ileum,cecum,colon)and serum were taken from piglets at the age of 1 day,28 and 70 days.The intestine microbial diversity was analyzed using 16 S rRNA gene high-throughput sequencing technology,HE staining method was used to observe the structure and morphology of the intestinal tract,PAS staining was used to observe the number of goblet cells in the intestinal tract,immunohistochemistry was used to detect the relative expression level of intestinal tight junction protein,ELISA was used to detect the content of immune indicators in serum,q PCR was used to detect the expression of intestinal barrier related genes,and the contents of SCFA were detected by gas chromatography.The results showed that:The length of villi of the two breeds piglets in 1 day was extremely significantly higher than that at the age of 28 days and 70 days(P<0.01),and the area and crypt depth of small intestinal villi in 70 days piglets were significantly higher than that at the age of 1 day and 28 days piglets(P<0.05).The villi length/crypt depth of the two breeds decrease extremely significantly with increasing age(P<0.01).The intestinal villi area and crypt depth of Mashen pigs were significantly higher than those of Jinfen White pigs(P<0.05).The villi length/crypt depth of 28 days Mashen pigs was significantly higher than Jinfen White pigs(P<0.05).The number of goblet cells in the small intestine of two breeds at the age of 28 and 70 days was extremely significantly higher than that at the age of 1 day(P<0.01).The number of goblet cells and intestine of Mashen pigs were higher than that of Jinfen White pigs in each period.The number of goblet cells in the small intestine was extremely significantly different at the age of 28 days(P<0.01).The expression level of intestinal tight junction protein(ZO-1,Occludin)in 1 day piglets was significantly higher than that in 70 days piglets(P<0.05),and the m RNA level of tight junction protein(ZO-1,E-cadherin)was similar result.The expression levels of TNF-α,IL-6 and IL-1β m RNA in Mashen pigs at different stages and intestines were significantly higher than those in Jinfen White pigs(P<0.05).The concentration of LPS in the serum of 1 day and 28 days piglets was significantly higher than that of 70 days piglets(P<0.05).The concentration of TNF-α increased with the growth of piglets.The concentration of TNF-α at the age of 70 days Mashen pigs was extremely significantly higher than that in 1 day and 28 days pigs(P<0.01).The concentrations of DAO in the serum of two breeds of piglets increased at first and then decreased extremely significantly(P<0.01),but there was no difference between 1 day and 70 days piglets(P>0.05).The concentration of D-LA and IL-6 in the serum of Jinfen White pigs increased gradually,reaching the maximum at the age of 28 days,and the highest at the age of 70 days.The content of SCFAs in colon of Mashen piglets was extremely significantly higher than that of Jinfen White pigs(P<0.01).The content of SCFAs in the colon of Jinfen White pigs increased first and then decreased with time,and the contents of acetic acid,propionic acid,butyric acid and valeric acid were significantly different among different ages(P<0.05).The contents of acetic acid,propionic acid and butyric acid in the colon of Mashen pigs increased gradually with the passage of time,and there were extremely significantly differences of each stage(P<0.01).In summary,Mashen piglets had stronger intestinal barrier function,digestion and absorption ability than Jinfen White piglets.The predominant small intestinal microflora of the two breeds of piglets were Firmicutes,Proteobacteria and Bacteroidetes,and the main genera were Clostridium sensu stricto 1,Pseudomonas,Lactobacillus and Streptococcus.With the growth and development of piglets,the diversity and structure of small intestinal microflora changed significantly,and age was an important factor.The specific flora in the small intestine of Mashen piglets was significantly less than that of Jinfen White pigs(P<0.05),and a fiber metabolizing bacterium sinibacillius was found in the small intestine of Mashen pigs.Association analysis showed that LPS,TNF-α,IL-6,DAO,D-LA,crypt depth were significantly correlated with intestinal flora(P<0.05),while the villi had no significant correlation with intestinal flora(P>0.05).Clostridium was positively correlated with LPS,D-LA and DAO(P<0.05),and negatively correlated with IL-6 and TNF-α(P<0.05).There was a significant positive correlation between Lactobacillus and crypt depth(P<0.05).The Firmicutes and the Bacteroidetes are the dominant bacteria,and the main genera were Lactobacillus,Prevotella,Bacteroides and Clostridium sensu stricto 1.There was no significant difference in intestinal microbial diversity of piglets at birth.The microbial diversity increased significantly with the growth and development of piglets(P<0.05),and basically stabilized at the stage of piglet conservation.The specific flora showed significant difference with the increase of age at different stages in two breeds.The proportion of Lactobacillus and Prevotella increased significantly after weaning.Association analysis showed that LPS,TNF-α,IL-6 were significantly correlated with intestinal flora(P<0.05),while the DAO and D-LA had no significant correlation with intestinal flora(P>0.05).LPS was significantly positively correlated Pseudomonas,Eubacterium fissicatena,etc.,and negatively correlated with three types of Prevotella,Pseudobutyrivibrio and Faecalibacterium(P<0.05).The associated flora of TNF-α was opposite to LPS(P<0.05).IL-6was significantly positively correlated Lactobacillus,Streptococcus,Prevotella UCG 003,Ruminococcusc UCG 005(P<0.05),and negatively correlated with Alistipes and Prevotella(1and 9)(P<0.05). |
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