Effects Of Tea Polyphenols On Methane Production,Forage Degradation And Rumen Epithelial Cells Transport In Rumen Of Dairy Cows

In this study,the effects of tea polyphenols on methane production,forage degradation and rumen epithelial cells transport in dairy cows were systematically analyzed by using multi-omics technology through in vitro and vivo experiments.The aim is to provide theoretical reference for the precise application of tea polyphenols in ruminant production.1 Effect of tea polyphenols on methane production in rumen of dairy cows in vitro and its regulation mechanismIn this experiment,the effects of chlorogenic acid,tannic acid,tea polyphenols and brown algae polyphenols on methane production and forage degradation rate were studied by the in vitro method,and 16S high-throughput sequencing was used to explore the microbial mechanism of polyphenols regulating methane production.The single factor design was used in this experiment and the addition levels of polyphenols were 0%,1%,2%,3%and 4%of the substrate（dry matter basis）.Five replicates were set for each addition level and five blanks were set for each batch to correct gas production and methane production.The results showed that:compared with the control group,the addition of different levels of tea polyphenols could significantly reduce methane（CH₄）production（P<0.05）,and the addition of 4%tea polyphenols could significantly reduce the content of ammonia nitrogen（NH₃-N）（P<0.05）.When the tannic acid level was 1%,the yield of CH₄ significantly decreased（P<0.05）,while the tannic acid level was 4%,the yield of CH₄ significantly increased（P<0.05）.The content of NH₃-N was significantly decreased when the addition level of brown algae polyphenols was 4%（P<0.05）.The addition of 2%chlorogenic acid increased the yield of CH₄（P<0.05）,while the addition of 3%and 4%chlorogenic acid significantly decreased the concentration of CH₄（P<0.05）.Different levels of tea polyphenols could significantly improve the degradation rate of dry matter（DM）and neutral detergent fiber（NDF）（P<0.05）.When tannic acid level was 4%,the degradation of DM was significantly reduced（P<0.05）.Adding brown algae polyphenols and chlorogenic acid had no significant effect on the degradation of DM,NDF and ADF in vitro（p>0.05）.The effects of polyphenols on methane generation and feed degradation rate were analyzed comprehensively,and the optimum level was 1%of tea polyphenols.Therefore,the level of 1%tea polyphenols was selected for follow-up experiments.The effects of tea polyphenols（1%DM）on VFA production and microflora in rumen were studied by ion chromatography and 16S high-throughput sequencing.The results showed that the ratio of acetate to propionate was significantly reduced of tea polyphenols group（P<0.05）.Tea polyphenols can improve the degradation rate of forage by increasing the relative abundance of Fibrobacter in rumen.Tea polyphenols inhibit the production of methane by reducing the relative abundance of unclassified_dArchaea methanogens and Pseudoentodinium and g_Balantioides protozoa.2 Effects of tea polyphenols on the degradation rate of forage and the adhesion of rumen microorganismsThe effects of tea polyphenols on the dynamic degradation of nylon bags in forage were studied by feeding cows with tea polyphenols.The experiment was divided into two groups:control group and tea polyphenols group,with 3 Holstein cows with permanent fistulas in each group.The cows of control group were fed the basic diet,and the cows of tea polyphenols group were fed the basic diet with 1%DM tea polyphenols.Scanning electron microscope（SEM）was used to observe the adhesion of rumen microorganisms to forage at different degradation time.16S high-throughput sequencing and metagenomics technology were used to analyze the changes of community structure and function of adhesion microorganisms on forge during rumen degradation.Nylon bags were placed in the rumen of dairy cows through fistula before morning feeding,and then they were removed in batches after staying in the rumen for 0.5 h,2 h,6 h,12 h,24 h,48 h,72 h.Rumen fluid was collected before morning feeding（0 h）and at each sampling time point to determine the pH of rumen fluid.The results showed that the pH of the rumen fluid in the control group and the tea polyphenols group were similar.The pH of the rumen fluid of two groups of dairy cows dropped to the lowest after 6 h feeding.The degradation rate of DM in the diet were significantly increased at 6 h and 12 h（P<0.05）;the NDF degradation rate of 6 h and 72 h were significantly increased in the tea polyphenols group（P<0.05）;the ADF degradation rate of 2 h and 72 h were significantly increased in the tea polyphenol group（P<0.05）.SEM images confirmed the dynamic degradation of forage throughout its incubation in the rumen.16S high-throughput sequencing results indicated that the addition of tea polyphenols in the diet affected the adhesion of microorganisms on forage and accelerated the adhesion of microorganisms such as Ruminococcaceae_UCG-010 and Rikenellaceae_RC9_gut_group to the forage.The results of Metagenomics analysis showed that the corresponding functions of microorganisms attached to forage were GHs,GTs,CEs,CBMs,AAs and PLs.The relative abundance of CE1 and GH78 was significantly increased by adding tea polyphenols to the diet（P<0.05）.Spearman correlation analysis showed that CE1 and GH78 were positively correlated with DM,NDF and ADF degradation rate of forage（P<0.05）.Therefore,tea polyphenols may improve the fiber degradation ability by affecting the relative abundance of CE1 and GH78.3 Effects of tea polyphenols on the interaction between rumen microorganisms and host in dairy cowsIn this study,16S high-throughput sequencing and iTRAQ proteomics techniques were used to systematically analyze the relationship among tea polyphenols,rumen microflora and host.This experiment was divided into two groups:control group and tea polyphenols group,with 3 Holstein cows with permanent fistulas in each group.The cows of control group were fed on the basic diet,and the cows of tea polyphenols group was fed on the basic diet with 1%DM tea polyphenols.The results showed that tea polyphenols significantly increased T-AOC and GSH-Px in blood（P<0.05）,and significantly decreased NH₃-N in rumen fluid（P<0.05）.Through LefSe analysis,it was found that Rikenellaceae_RC9_gut_group,Ruminococcaceae_NK4A214_group and Butyrivibrio₂ were significantly enriched in the rumen fluid samples of dairy cows in the tea polyphenols group:Fusobacterium was significantly enriched in the rumen fluid of dairy cows in the control group.The spearman correlation analysis showed that Rikenellaceae_RC9_gut_group and Christensenellaceae_R7_group were significantly positively correlated with propionate（P<0.05）,but significantly negatively correlated with acetate/propionate ratio（P<0.05）.In this experiment,199 differential proteins were screened from the control group and the tea polyphenol group,including 112 up-regulated Proteins and 87 down-regulated proteins.The results showed that the differentially expressed proteins in rumen epithelium cells were enriched in the function of transporting active molecules through the GO analysis.Compared with the control group,tea polyphenols group COIII,S100A8,FABP1,SLC2A8 and SLC29A1 proteins were significantly up-regulated（P<0.05）,while proteins such as HBB,RAB4A,RBP4,LOC 107131172,HBA and ZFYVE19 were significantly down-regulated（P<0.05）.Spearman correlation analysis was used to analyze the relationship between transporter activity proteins and rumen fermentation parameters.The results showed that FABP1 was positively correlated with propionate content（P<0.05）,while RAB4A was negatively correlated with propionate content（P<0.05）but positively correlated with NH₃-N content（P<0.05）.Therefore,tea polyphenols could enhance the transport capacity of rumen epithelium cells to nutrients by increasing the expression of S100A8,FABP1,SLC2A8 and SLC29A1.In conclusion,tea polyphenols could inhibit the production of methane in rumen and reduced the loss of feed energy by affecting the diversity and community structure of bacteria,protozoa and methanogens in rumen.By feeding tea polyphenols,the adhesion of fiberdegrading bacteria to forage was accelerated,the rumen degradation rate of forage was increased,and the feed conversion efficiency was improved.Tea polyphenols could affect the rumen microflora,changed the rumen fermentation mode,enhanced the transport capacity of the rumen epithelium cells of the host,and improved the utilization efficiency of feed.In production practice,tea polyphenols may be used to enhance the utilization efficiency of nutrients,and improve the production performance of dairy cows.