Effect Of Gossypol On Microorganism And Metabolism In The Rumen Of Sheep By Using Omics Analysis

Gossypol is a toxic secondary metabolite found in cotton plants.The presence of gossypol hinders the use of cotton by-products as feed ingredients.The large amounts of microorganism in the rumen of ruminants play an important role in adaption or degradation of gossypol.In the process of rumen microorganism degradation or metabolism of gossypol,which microorganism and metabolites were effected by gossypol is still unclear.This study was aimed to evaluate the effect of gossypol on rumen microorganisms of sheep,and to study the mechanism of gossypol degradation by gossypol degradation bacteria by using omics method.It will provide support data for further research on the mechanism of rumen microorganism tolerance(biodegradation)to gossypol.The experimental were shown as follows:Experiment 1:Effects of gossypol on rumen microorganism diversity,function and metabolism in sheepEight sheep with permanent rumen fistula were randomly divided into 2 groups,4 in each group,the trial group and control group.Dietary gossypol inclusions in the gossypol group were 600 and 1200 mg per animal per day during the first(days 1?27)and subsequent(days 28?47)stages.The sheep in the control group were fed basal diet.Rumen fluid samples at days 5,10,15,20,32,37,42 and 47 were collected to detected the change of the concentrations of volatile fatty acid and gossypol,the number of protozoa,the diversity,function and metabolic of rumen microorganisms.The results of volatile fatty acid and gossypol concentrations,the number of protozoa in the rumen of sheep showed that supplementation with gossypol had no significant effect on total volatile fatty acid concentration in the rumen fluid of sheep(P>0.05).The concentration of gossypol in the rumen fluid were increased with the dose of the gossypol,and the gossypol concentration did not change significantly through with the same amount of gossypol supplementation.Gossypol significantly reduced the numbers of total protozoa in rumen fluid at day 20(P<0.05),but there was no significant effect at other time points(P>0.05).The results of bacterial diversity in rumen fluid showed that compared with control group,supplementation with gossypol significantly increased the richness of bacteria in the rumen fluid at day 20(P<0.05),and significantly increased the richness and diversity of bacteria at day 37(P<0.05).But there were no significant effect on the diversity and richness of bacteria at day 47(P>0.05).At the phylum level,gossypol significantly affected rumen bacteria communities with a lower relative abundance of the spirochaetes phylum(P<0.05).At the genus level,supplementation with gossypol significantly increased the relative abundance of Ruminococcaceae_NK4A214 and significantly decreased the relative abundance of Sphaerochaeta at day 20(P<0.05).Gossypol had no significantly affect on the relative abundance of bacterial at day 37,and gossypol significantly increased the relative abundance of Ruminobacter and Succinimonas at day 47.The results indicated that supplementation with gossypol significantly affected on the richness of bacteria in the rumen fluid at day 20 and 37,but had no effected on the diversity and richness of bacteria at day 47.It had no significant effect on the relative abundance of dominant bacteria in the rumen fluid of sheep.The results of fungi diversity showed that supplementation with gossypol significantly increased the diversity of fungi at day 37(P<0.05),but there was no significantly effect on the diversity and richness of fungi at day 20 and 47(P>0.05);At the genus levels,gossypol significantly increased the relative abundance of Mucor at day 37 and the relative abundance of Metschnikowia at day 47.The results indicated that there was no significant effect on fungi diversity and richness at day 20 and 47,gosypol significantly increased fungi diversity at day 37,but there is no significant effect on the relative abundance of dominant fungi.The results of protozoa diversity showed that gossypol had no significant effect on the diversity and richness of protozoa in rumen fluid at different time points;Gossypol significantly increased the relative abundance of Ophryoscolex at day 37(P<0.05),but had no significant effect on the structure of other protozoa genus in rumen fluid(P>0.05).The results indicated that gossypol has no effect on diversity,richness and structure of protozoa in rumen fluid of sheep.Metagenomics was used to test the effect of gossypol on rumen microorganisms function.The results showed that most of the genes in the CAZy annotation were annotated to glycoside hydrolase,and the metastat analysis of functional differences between the groups had a significant difference of in the top 35 genes,of which xylanase and endo-?-1,4-xylanase may be involved in gossypol degradation.After eggNOG database comparison,the most annotated genes were unknown functions,followed by carbohydrate transport and metabolism.The LEFse analysis results showed that the relative abundance related to cytoskeleton was significantly reduced,and the relative abundance of functional genes related to translation,ribosomal structure and biogenesis,carbohydrate transport and metabolism,inorganic ion transport and metabolism were significantly increased.Metastat analysis of KEGG showed that gossypol significantly reduced the relative abundance of cell motility.These results indicate that gossypol may inhibit the growth of microorganisms in rumen fluid by demage cell structure and reducing cell motility.Metabolomics of rumen fluid was detected by LC-MS method.The results showed that in the positive ion mode,1839 metabolites were detected in samples,32 metabolites were significantly altered between gossypol and control group(17 up-regulated and 15 down-regulated).In negative ion mode,a total of 1120 metabolites were detected.20 metabolites were significantly altered between gossypol and control group(12 up-regulated and 8 down-regulated).Differential metabolites are enriched in phenylalanine,tyrosine and tryptophan biosynthesis,polycyclic aromatic hydrocarbon degradation,benzoate degradation.The results indicate that gossypol degradation by rumen microorganisms may be the same as that of other aromatic hydrocarbons.Experiment 2:study on isolation,identification and degradation mechanism of gossypol-degrading bacteriaThe screen medium were using gossypol as the sole carbon source,and was used to screen gossypol-degrading bacteria.Identification of gossypol-degrading bacteria by traditional andmolecular biological methods.The results showed that the gossypol-degrading bacteria GD2,GD5,GD8 were isolated from rumen fluid of sheep.The colony characteristics of 3 bacteria were round and white in the screening medium.Gram staining revealed gram-negative.The 16S rDNA sequencing and analysis showed that the three gossypol-degrading bacteria were Klebsiella,Enterobacter sp.and kosakonia.After gossypol and three gossypol-degrading bacteria co-cultured for 72 h,the degradation rates of the three gossypol-degrading bacteria to gossypol reached 31.67%,33.74%,and 18.33%,respectively.RNA sequencing was used to test the effect of gossypol on the gene expression of Enterobacter sp.GD5.The results showed that after 72 h of culture,gossypol to have significant influence on 132 metabolites,104 up-regulated and 28 down-regulated.The GO enrichment results showed that the differential genes were mainly enriched in biological process and molecular function,which significantly affected homeostatic process.Further analysis revealed that supplementation with gossypol significant increased the gene expression related to anti-oxidative stress in sp.GD5.KEGG enrichment pathway result showed that gossypol significantly affected pyruvate metabolism.The results indicated that gossypol induced oxidative stress and resulting with increased expression of anti-oxidative stress genes in Enterobacter sp.GD5,which may be the reason why Enterobacter sp.GD5 tolerated or degraded gossypol.LC-MS method was used to detect the changes of metabolites in Enterobacter sp.GD5 after supplementation with gossypol.The results showed that 9 metabolites were significantly difference between gossypol and control group(4 up-regulated and 5 down-regulated).In negative ion mode,there are 6 differential metabolites,of which 4 up-regulated and 2 down-regulated.The significant differentce metabolites enriched in glutathione metabolism under the positive and negative ion mode,and it was significantly enriched in the biosynthesis of amino acids in negative ion mode.The results indicated that Enterobacter sp.GD5 through regulated glutathione metabolism to degradation gossypol.In summary,supplementation with gossypol had no significant effect on the dominant genus of bacteria,fungi,and protozoa in the rumen fluid of sheep,gossypol significantly affects the relative abundance of cell motility,and significantly affects metabolism related to the metabolism of aromatic hydrocarbons.In vitro tests have shown that the oxidative stress of was induced by the gossypol and resulting with increased expression of anti-oxidative stress genes in Enterobacter sp.GD5.Metabolomics results also showed that gossypol affected glutathione metabolism.These results provide surport data for further research on microbial tolerance or biodegradation gossypol,and provide theoretical support for the rational use of cotton by-products.