| Inositol is a kind of bioactive substance,which can improve the production performance and health status of monogastric animals and aquatic animal as feed additive.This experiment explores the effects of inositol on rumen fermentation function,rumen microorganisms,blood indicators,rumen and blood metabolite groups through in vitro rumen simulation fermentation experiments and animal feeding experiments,in order to further elucidate the laws and mechanisms of inositol regulating nutrient metabolism in sheep,and provide a theoretical basis for the development of inositol as a new functional feed additive for ruminants.Experiment 1: To study the effects of different doses of inositol on rumen fermentation parameters,rumen microorganisms,and metabolites in sheepDesign six levels of inositol supplementation doses,namely low dose groups(50,100,200 mg/kg DM)and high dose groups(400,800,1600 mg/kg DM),collect sheep rumen fluid,use in vitro rumen simulation fermentation technology system,collect samples with fermentation time of 3,6,12,24,and 48 hours,and measure rumen fermentation parameters(p H,ammonia nitrogen concentration,volatile fatty acids,total gas production),Collect samples with fermentation time of 6,24,and 48 hours to determine the dry matter digestibility and crude protein digestibility of in vitro fermentation substrates.Collect rumen contents with fermentation time of 12 and 48 hours to determine microbial diversity and rumen metabolites.The results showed that the in vitro dry matter digestibility(IVDMD),in vitro crude protein digestibility(IVCPD),and ammonia nitrogen(NH3-N)concentrations in the low dose group(50,100,200 mg/kg DM)were significantly higher than those in the control group and the high dose group(400,800,1600 mg/kg DM)(P<0.05);Among them,the p H value of the 800,1600 dose inositol supplementation groups was significantly higher than that of the 200,400 mg/kg DM inositol supplementation groups(P<0.05),and was significantly higher than that of the 50,100 mg/kg DM inositol supplementation groups(P<0.01).At 24,48 h of fermentation,the p H value of the high dose groups(400,800,1600 mg/kg DM)was significantly higher than that of the low dose groups(50,100,200 mg/kg DM)(P<0.05);After 3-12 h of fermentation in vitro,the concentration of total volatile fatty acids(TVFA)in the 100 mg/kg DM inositol supplemented group was significantly higher than that in the control group and the 400 mg/kg DM inositol supplemented group(P<0.05),and was significantly higher than that in the 800,1600 mg/kg DM inositol supplemented groups(P<0.01);The concentrations of acetic acid and propionic acid in the 50,100 mg/kg DM inositol supplemented groups were significantly higher than those in the control group and the800,1600 mg/kg DM inositol supplemented groups after in vitro fermentation for 3,6,12 h(P<0.05);The concentration of isobutyric acid in the 50,100,200,400 mg/kg DM inositol supplemented groups was significantly higher than that in the 800,1600 mg/kg inositol supplemented groups at 3,6,12 h of in vitro fermentation(P<0.01);The butyric acid concentration in the 50,100 mg/kg DM inositol supplemented groups was significantly higher than that in the 800,1600 mg/kg DM inositol supplemented groups(P<0.01),and significantly higher than that in the 800,1600 mg/kg DM inositol supplemented groups(P<0.05)at 6,12 h of fermentation in vitro;The concentration of isovaleric acid in the 50,100,200,400 mg/kg DM inositol supplemented groups was significantly higher than that in the 1600 mg/kg DM group and extremely significantly higher than that in the 800 mg/kg DM inositol supplemented group after 3 h fermentation in vitro,and the concentration of isovaleric acid in the 100,200,400mg/kg DM inositol supplemented groups was significantly higher than that in the 800,1600 mg/kg DM inositol supplemented groups after 6 h fermentation in vitro(P<0.05);The concentration of valeric acid in the 100 mg/kg DM inositol added group was significantly higher than that in the control group and the 400,800,1600 mg/kg DM inositol added group after 3,6,12 h of fermentation in vitro(P<0.01),while the concentration of valeric acid in the 200 mg/kg DM inositol added group was significantly higher than that in other inositol added groups after 24 h of fermentation in vitro(P<0.01).Inositol can effectively regulate the rumen internal environment and enhance the abundance and diversity of microbial communities.The 100,200 mg/kg DM inositol supplementation groups can significantly increase the abundance of feed protein decomposing related bacteria such as Prevotella,Succinolytic Bacteria,and Pseudomonas(P<0.05).In the metabolite detection results,organic acids and their derivatives were the main metabolites.In the inositol addition group,differential metabolites such as azelaic acid,lysine,glycerol,D-glyceric acid,phenylpropionic succinic acid,2-deoxybutyric acid,and allyl malonic acid were significantly upregulated compared to the control group.The differential metabolites were mainly concentrated in Glyceride metabolism,Valine,leucine and isoleucine biosynthesis,and Alanine,aspartate and glutamate metabolism.significantly increased the metabolic level of hydrocinnamic acid in the rumen in the 50,200 mg/kg DM inositol supplemented groups after 12 h in vitro fermentation,while the 100,200 mg/kg DM inositol supplemented groups significantly reduced the metabolic level of sucrose in the rumen(P<0.05);After 48 h of in vitro fermentation,the 50,100 mg/kg DM groups significantly increased the metabolic levels of 1,2,4-butanotriol and 1,4-cyclohexanedione in the rumen,the 50,200 mg/kg DM groups significantly increased the metabolic level of succinate semialdehyde,and the 50,100,200 mg/kg DM groups significantly reduced the metabolic level of Carbobenzyloxy-L-leucine degr1 in the rumen(P<0.05).Based on the above results,the additive doses of inositol of 0,50,100mg/kg DM were selected as the additive doses for animal experiments.Experiment 2: To study the effects of different doses of inositol on rumen fermentation parameters,rumen microorganisms and metabolites,blood indicators and blood metabolites in sheepAccording to the results of the in vitro test,the addition doses of inositol of 0,50,100 mg/kg DM were selected,and 3×3 In the Latin square feeding experiment design,sheep with permanent rumen fistulas were used as experimental animals.Inositol was infused into the rumen before daily feeding during the formal period of the experiment.Rumen fluid samples and sheep blood samples were collected at 0,2,4,8,12 h,and 4h on the first,fourth,and seventh days of the formal period,respectively.Rumen fermentation parameters,rumen microbial diversity,rumen metabolites,blood biochemical indicators,blood immune indicators,blood oxidative stress indicators and blood metabolites were measured.The results showed that in the blood indexes,the increase of inositol infusion days could increase the blood density of High density lipoprotein,and promote the increase of Ig G,Ig M and IL4 levels in sheep blood.In addition,inosidol could improve the bacterial community structure in rumen,optimize the ratio of Bacteroidetes to Firmicutes,and significantly increase the abundance of Prevotella and Succiniclasticum bacteria(P<0.05).Metabolomics technology was used to detect the effects of inositol on rumen metabolites of sheep.The main differential metabolites were organic acids and their derivatives,such as proline,D-α-glycerophosphoric acid,L-lysine,L-tyrosine and phenylacetic acid.These differential metabolites were mainly involved in the enrichment of ABC transporters,biotin metabolism and phenylalanine metabolism pathway.In addition,100 mg/kg DM inositol supplementation group could improve the metabolism level of phenylacetic acid on the 7th day of inositol infusion,and the differential metabolites were mainly concentrated in phenylalanine metabolism pathway.In the results of blood metabolites,inositol was mainly involved in the lipid metabolism of sheep.The differential metabolites of 5Z-Dodecenoic acid,(10E,12Z)-(9S)-9-Hydroperoxyoctadeca-10,12-dienoic acid,palmitoleic acid,linoleic acid oxidation products,inosine and n-butylphthalein in the inositol addition group were significantly upregulated compared with the control group.Differential metabolites were mainly concentrated in fatty acid biosynthesis,arginine biosynthesis,glutathione metabolism,tyrosine metabolism and other metabolic pathways.Among them,50mg/kg DM dose group significantly increased the metabolic levels of palmitolic acid and inosine in blood.On the 7th day of inositol infusion,inosine addition group decreased the metabolic levels of glycocholic acid in blood.These results indicate that inositol has the potential to regulate sterol.Based on the results of this study,inositol can effectively regulate the diversity of rumen microorganisms,increase the abundance of feed protein decomposition related bacteria,optimize rumen fermentation function,enhance animal immune levels,improve animal health,and increase the metabolic levels of beneficial substances such as phenylacetic acid and palmitoleic acid.It is a suitable choice as a new feed additive for ruminants. |
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