| Gut microbiota which is mainly consisted of Firmicutes and Bacteroidetes divisions has been shown to affect fat storage and energy harvesting of host.Previous studies mainly focused on the flora in the feces or digesta, reflecting flora of the intestine lumen not the intestinal mucosa flora. Mucosa flora composition was significantly different from intestine lumen.Duroc x Meishan and DLY pigs has different capable of fat deposition. The amount of fat deposition and the intestinal flora of the two kinds of rossbred pigs were investigated. Then the correlation between intestinal mucosa flora and the amount of fat deposition was studied. And the correlation between intestinal mucosa flora and the expression level of TLR4was studied. The effect of different microbial modulator on intestinal mucosa flora, the amount of fat deposition, blood biochemical parameters, the inflammatory factor gene and fat metabolism gene expression were investigated. The mechanism that the gut microbiota regulates fat storage in pigs was also investigated. Four experiments were conducted as follows:EXP.1The correlation between intestinal mucosa flora and the amount of fat deposition of pigsTo study the relationship between intestinal mucosa flora and the amount of fat deposition, the amount of fat deposition and the intestinal flora of Duroc x Meishan(DM) and DLY pigs were investigated. Six DM pigs and Six DLY pigs(about20kg) were feed the same corn-soybean meal. Each pig was removed from the test on the day it most nearly approximated110kg. Every pig from each treatment were slaughtered, and the backfat depth, longissimus muscle area, leaf lard weight and intramuscular fat content of pigs were measured. Ileum mucosa were collected for detecting the16S rDNA gene sequence of total bacterial, Firmicutes and Bacteroidetes divisions using the RT-PCR method. Results of the experiment indicated that:(1) DLY pigs trial period was significantly shorter than the DM pigs(P<0.01), average daily gain was significantly higher than DM pigs (P<0.01), while the average daily feed intake was significantly lower than DM pigs (P <0.05), F/G was significantly lower than DM pigs (P<0.01). Leaf lard weight was no significant different between DM and DLY pigs. The yield of carcass, average backfat depth, longissimus muscle area, intramuscular fat content of DM pigs were significantly higher than DLY pigs (P<0.01).(2)The copy numbers of total bacterial,Firmicutes and Bacteroidetes divisions of ileum contents from both kind pigs were significantly higher than ileum mucosa(P<0.01). The copy numbers of Firmicutes divisions of ileum contents from DM pigs were significantly higher than DLY pigs(P<0.05). There were no significant different of total bacterial, Firmicutes and Bacteroidetes divisions of ileum mucosa between two kinds of rossbred pigs. The copy numbers of Firmicutes and Bacteroidetes divisions of ileum contents and ileum mucosa frome DM pigs were significantly higher than DLY pigs(P<0.01).(3)Both kind pigs having the sum of percents of Firmicutes and Bacteroidetes divisions of ileum contents and ileum mucosa which were about85%. Firmicutes of ileum contents from both kind pig has the largest proportion(P<0.01) while Bacteroidetes of ileum mucosa has the largest proportion(P<0.01).(4) The relative abundance of Firmicutes, Firmicutes/Bacteroides ratio of gut lumenal contents and intestinal mucosa were positively correlated to average backfat(.P<0.01,.P<0.01), while the relative abundance of Bacteroides and average backfat thickness negatively correlated (.P<0.01).EXP.2The relationship between ileum mucosa flora and plasma LPS level, TLR4, TLR5gene expression level of pigsThe relationship between ileum mucosa flora and plasma LPS level, TLR4, TLR5gene expression level was studied,animal and the experimental design were the same to EXP.1. After slaughter, ileum mucosa and subcutaneous fat were collected for measuring the copy numbers of Firmicutes and Bacteroidetes divisions and expression level of TLR4and TLR5. Results of the experiment indicated that:TLR4mRNA expression levels of intestinal mucosa and subcutaneous fat from DM pigs were significantly higher than DLY pigs (P<0.05). TLR5mRNA expression levels of intestinal mucosa from DM pigs was no significant different from DLY pigs. TLR4mRNA expression levels of subcutaneous fat from both kind pigs was significantly higher than intestinal mucosa(P<0.01). TLR4mRNA expression levels of intestinal mucosa and subcutaneous fat were positive correlated to intestine Firmicutes/Bacteroidetes, The coefficient of determination R2were0.7437and0.7739respectively.EXP.3The effect of the of microbial regulators on pig performance, carcass quality and mucosal floraThe EXP.1proved that the ratio of Firmicutes/Bacteroidetes was positively correlated to fat deposition amount, and the coefficient of determination was higher than the one of ileum contents. To further validate this relationship, the effect of colistin sulfate, Bacillus and white fungus spores fermentation products on pig production performance, carcass quality and mucosa flora were investigated.24pigs (20±0.63kg) were randomly allotted into4dietary treatments with6replicate pens per treatment and1pig per pen:(1) control diets containing no antibiotic (C group),(2) colistin sulfate added to the control diets (CS group),(3) Bacillus added to the control diets (BS group),(4) TFS added to the control diets (TFS group). Each pig was removed from the test on the day it most nearly approximated110kg.4pigs from each treatment were slaughtered, and the backfat depth, longissimus muscle area, leaf lard weight, intramuscular fat content and the copy number of total bacterial, Firmicutes and Bacteroidetes of ileum mucosa were measured. Results of the experiment indicated that:(1) The colistin sulfate, probiotics and white fungus spores fermentation products improved pig performance.(2) Variety of microbial modulator showed no significant effect on yield of carcass and leaf lard weight.The colistin sulfate significantly reduced the pig backfat thickness (P<0.01), and probiotics and white fungus spores fermentation products significantly improved pig backfat depth, longissimus muscle area and intramuscular fat content.(3)There were no significantly differences among the copy numbers of total bacterial,Firmicutes and Bacteroidetes. Colistin sulfate had no effect on the rate of Firmicutes/total bacterial and Firmicutes/Bacteroidetes, but had trends of reducing the rate of Firmicutes/total bacterial, Firmicutes/Bacteroidetes and increasing the rate of Bacteroidetes/total bacterial. Probiotic significantly increased the rate of Firmicutes/total bacterial(P<0.05) and had trends of reducing the rate of Bacteroidetes/total bacterial and increasing the rate of Firmicutes/Bacteroidetes. White fungus spores fermentation products significantly reduced the rate of Bacteroidetes/total bacterial. The BS and TSF goup had higher rate of Firmicutes/total bacterial than CS group(P<0.05). The TSF goup had the lowest rate of Bacteroidetes/total bacterial than CS group(P<0.01).EXP.4Effect of microbial modulator on pig serum indicators inflammatory factors and fat metabolism gene expression levelsThe EXP.3verified the conclusion of the EXP.1, to study the pathway of intestinal flora affecting fat deposition, the effect of microbial regulators on pig blood biochemical indicators, the expression levels of fat metabolism-related genes and the inflammatory cytokines were investigated. Animals and experimental design were same to EXP.3. Vena cava blood were sampled for determination of plasma LPS, serum glucose, insulin, leptin, and lipid levels when pigs have weights up to20kg,50kg and110kg. Subcutaneous fat and intestinal mucosa were sampled for determination of inflammatory factors and fat metabolism gene expression levels. Results of the experiment indicated that:(1)The probiotic significantly increased the level of plasma lipopolysaccharide, colistin sulfate and white fungus spores fermentation products trend to increase plasma lipopolysaccharide levels.(2)Microbial modulator has different effect on blood indicators of different weight stage pigs, but showed some trend:the colistin sulfate raise plasma glucose and insulin levels, and lower serum leptin levels; probiotics reduce plasma glucose levels, serum leptin levels; TSF lower plasma glucose and insulin levels, serum leptin levels. Colistin sulfate trend to reduce the pig serum total triglycerides, cholesterol, free fatty acids; probiotics trend to increase serum triglycerides, total cholesterol; TSF trend to reduce110kg stage pig serum triglycerides and total cholesterol.(3) Microbial modulator has different effect on inflammatory factor gene expression levels of different tissue, but generally speaking, colistin sulfate reduce TLR4, TNF-a, IL1β and IL6expression levels, probiotics increase TLR4, TNF-a, IL1β and IL6expression levels, and TSF increase TLR4, TNF-a, IL1β.(4)The colistin sulfate reduced the pig fat synthesis gene expression, increased lipolysis and fat transport related gene expression; probiotics increased fat synthesis genes expression, reduce lipolysis gene expression; TSF increase pig fat decomposition related gene expression levels, reduce fat synthesis gene expression.In summary, the results of this study showed that:(1) The total bacteria, relative abundance of Firmicutes and Bacteroides from ileal mucosa were significantly lower than the lumenal(2) The effect of Firmicutes/Bacteroides ratio on backfat thickness is stronger than Firmicutes/total bacteria ratio; Lleum mucosa Firmicutes/Bacteroides ratios has stronger correlation to fat deposition than ileal digesta in pig(3) Porcine plasma LPS levels and ileum Firmicutes/Bacteroides ratio were positively correlated; TLR4gene expression levels in porcine adipose tissue and the Ileum mucosa were positively correlated to intestinal mucosa Firmicutes/Bacteroides ratio(4) According to the results of Exp.1, CS decrease backfat thickness is consistent with decreasing Firmicutes/Bacteroides ratio; BS increase backfat thickness is consistent with increasing Firmicutes/Bacteroides ratio; TSF increase backfat thickness is inconsistent with decreasing Firmicutes/Bacteroides ratio (5) Under the action of different probiotics backfat thickness and Firmicutes/Bacteroides variation are not entirely consistent with plasma LPS, TLR4gene changes(6) Colistin sulfate, Bacillus may influence the number and abundance of Firmicutes and Bacteroides in pig intestine, thus affecting the trial pig backfat and intestinal inflammatory cytokines gene expression, fat metabolism key enzyme gene expression, and ultimately affect the ability of fat deposition.The Tremella fuciformis ferment substance affect the role of fat deposition has nothing to do with ileal flora.. |
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