| During the growth process of livestock and poultry,excessive deposition of body fat will not only have an important impact on the health of the host,inflammation and its complications will occur frequently,fertility will decrease,and feed rewards will increase,but also affect the meat quality of livestock and poultry.In this study,metabolomics technology combined with high-throughput sequencing technology was used to analyze the relationship between intestinal microbes and their metabolism and fat deposition in livestock and poultry breeding,and to reveal the correlation and mechanism of pig intestinal microbes and their metabolism and fat deposition.It is of great significance to the healthy production of pigs and the improvement of meat quality,in order to achieve the purpose of regulating meat quality.This research is divided into three parts:Experiment 1: Establishment of Jinhua Pig Animal Models with Different Body Fat Percentage and Comparison of PhenotypesA total of 35 healthy Jinhua pigs with the same batch,30 days of weaning age,and body weight of 4.87±0.93 kg were selected for feeding in this study.At the day of age 270,and their body weight,backfat thicknessand ribeye areas were measured,and the colon contents and serum were collected.According to the ratio of backfat thickness/ ribeye areas,the top 7 ratios were the high body fat percentage group(H group,High Group),and the lower 7 ratios were the low body fat percentage(L group,Low group),and then the serum and colon contents of 7 Jinhua pigs in the high body fat rate group and the low body fat rate group were measured for short-chain fatty acid content.The results showed that Jinhua pigs with different body fat percentage:(1)There were significant differences in the backfat thickness and ribeye areas.The backfat thickness of the H group was 17.12% higher than that of the L group(P<0.01),and the ribeye areas was 9.97% lower than that of the L group(P<0.05);(2)There were differences in acetate acid,propionate acid,butyrate acid and isobutyrate acid in the coloncontents.The acetate acid in the H group was 25.63% lower than that in the L group(P<0.05),and the propionate acid was 33.98% lower than that in the L group(P<0.05),butyrate acid was 53.01% lower than that of L group(P<0.01),and isobutyrate acid was 37.83% lower than that of L group(P<0.01);(3)Serum acetate acid,butyrate acid and isobutyrate acid were significantly different,H The acetate acid in the group was 39.74% lower than that in the L group(P<0.05),the butyrate acid was 52.41% lower than that in the L group(P<0.05),and the isobutyrate acid was 59.07% lower than that in the L group(P<0.01).Experiment 2: Comparison analysis of Gut Microbiota of Jinhua Pigs with Different Body Fat PercentageThe contents in the colon of Jinhua pigs in group H and group L were taken for PCR amplification,and the amplified products were analyzed by 16 S r RNA and ITS high-throughput sequencing.The results showed that:(1)There were differences in the total number and diversity of bacteria,fungi and archaea species in the colon contents of Jinhua pigs with different body fat percentage.(2)PCA analysis showed that different clusters,indicating that there were differences in the bacterial community structure between the two group.(3)Based on phylum and genus levels,bacteria have 16 phyla and 247 genera,fungi have 5 phyla and 86 genera,and archaea have 3 phyla and 50 genera.Firmicutes,Basidiomycota and Euryarchaeota are the dominant phyla of colon bacteria,fungi and archaea,respectively.The genus-level bacteria were mainly Lactobacillus,Clostridium sensustricto 1 and Bacteroidales S24-7 group＿norankand other 30 kinds of bacteria.The fungi are mainly 20 species of fungi including Naganishia,Kazachstania and Piromyces.The archaea are mainly 20 kinds of archaea,including Methanobrevibacter,Euryarchaeota＿norank and Methanomicrobiales＿norank.(5)Pearson correlation analysis found that there was a close interaction between bacteria,fungi and archaea of Jinhua pigs with different body fat percentage.Clostridium sensustricto 1 and Rhizopus were negatively correlated with almost all archaea(Rho=-0.26—-0.60),and Turicibacter was closely related to Clostridium sensustricto1,Christensenellaceae R-7 group,[Eubacterium]coprostanoligenes group and Romboutsia showed a strong positive correlation(Rho=0.75-0.82).Methanimicrococcus and Methanomicrobiales＿norank were negatively correlated with most genera(Rho=-0.06—-0.73),and Bacteroides had the strongest positive correlation with backfat thickness and ribeye areas,respectively.The Turicibacter,the most negatively related genera were Sulfophobococcus,Romboutsia and Fusarium,respectively.Experiment 3: Comparison analysis of Serum Metabolomes of Jinhua Pigs with Different Body Fat PercentageBlood samples from Jinhua pigs in group H and group L were collected respectively,and serum samples were obtained by centrifugation at 3000 r/min for 10 min.Non-target metabolomic analysis of Jinhua pig serum with different body fat percentage.The results showed:(1)The total particle flow chart showed that there were differences in the metabolites in serum,with obvious metabolic physiological changes,and the OPLS-DA score chart further showed their obvious separation and distinction.(2)In the positive ion mode,there were 23 significantly different identified metabolites and 85 significantly different unidentified metabolites between the H and L groups;in the negative ion mode,there were 59 significant differences between the H and L groups.Different identified metabolites and 268 significantly different unidentified metabolites.(3)In the positive ion mode,32 serum metabolites were significantly up-regulated or up-regulated,and 76 serum metabolites were significantly down-regulated or down-regulated;in the negative ion mode,222 serum metabolites were significantly up-regulated or tended to be up-regulated.105 serum metabolites were significantly down-regulated or down-regulated.(4)Set a threshold(LDA>2,P<0.05)to screen out 24 metabolite biomarkers.Among them,6 metabolites are serum marker metabolites in the high lipid rate group,such as TG(15:0/22:2/O-18:0),PE(22:5/16:0),4-acetylamino Butyric acid,etc.;18 metabolites are serum marker metabolites in the low-fat rate group,such as PC(22:1/15:0),pentadecanoic acid and PE(22:6/P-18:1),etc.;(5)Pearson correlation analysis showed that serum metabolites with the strongest positive correlation with backfat thickness and ocular muscle area were 4-Acetamidobutanoic acid and Cer(d18:0/16:0),respectively,and serum metabolism with the strongest negative correlation was The substances were Schidigeragenin C and PE(22:5/16:0),respectively.There was a significant negative correlation between backfat thickness and Schidigeragenin C(Rho=-0.634,P=0.015),and a significant positive correlation between eye muscle area and Cer(d18:0/16:0)(Rho =0.770,P=0.001).(6)In the positive ion mode,the number of metabolic pathway disorders in the H and L groups is 7.The reason for the metabolic pathway disorder is the change of metabolites,such as glycerophospholipid metabolism,linoleic acid metabolism and other 4 metabolic pathways.In addition,the metabolites involved in these metabolic pathways are PE(22:5/16:0),4’-Demalonylsalvianin,respectively.In negative ion mode,there are 4 metabolic pathways including arginine and proline metabolism and primary bile acid biological metabolism.The metabolites involved were 4-Acetamidobutanoic acid,Hydroxyproline,respectively.In summary,these key metabolic pathways are divided into various lipid metabolism and bile acid metabolism.Lipid metabolism includes glycerophospholipid metabolism,linoleic acid metabolism,alpha-linolenic acid metabolism and arachidonic acid metabolism and primary bile acid metabolism,metabolites involved in these metabolic pathways may be used as biomarkers for regulating lipid metabolism.Conclusion: Differences was found in gut microbiota,short-chain fatty acids and serum metabolites of Jinhua pigs with different body fat percentage,and there is a certain correlation between with backfat thickness and ribeye areas,indicating that the changes in gut microbiota and metabolites may be associated with host fat deposition certain correlation.Three gut microbiotas,Bacteroides,Turicibacter and Romboutsia,SCFAs and PE(22:5/16:0)may be key bacteries and metabolites regulating lipid metabolism.Furthermore,glycerophospholipids metabolic pathways may be the key metabolic pathways regulating lipid metabolism. |
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