Effects Of Guanidineacetic Acid On Rumen Fermentatio,Microflora Structure And Function In Vitro

This experiment investigated the effects of adding different levels of guanidineacetic acid（GAA）on rumen in vitro gas production,gas composition,rumen fermentation parameters,bacterial community structure and function by an in vitro method.The experiment was conducted in a one-way design with 0%（control,CON）,0.2%（GAA02）,0.4%（GAA04）,0.6%（GAA06）and 0.8%GAA（GAA08）added to the fermentation substrate,and three replicates were set up in each group.Ruminal fluid was collected from four Simmental cows for in vitro fermentation,gas production was recorded at each time point during fermentation,theoretical maximum gas production was predicted and gas production rate was calculated,fermentation gas was collected and gas composition was determined.After 48 h of fermentation,the p H,ammonia nitrogen（NH₃-N）,microbial protein（MCP）and volatile fatty acid（VFA）concentrations of the fermentation broth were measured.The microflora of V3-V4 variable regions was determined using 16S r RNA high-throughput sequencing technology,and the structural changes of the flora were analyzed,and the related functions were analyzed using PICRUSt1 according to the Kyoto Encyclopedia of Genes and Genomes（KEGG）.Rumen fermentation results:There was no significant difference in total gas production,theoretical maximum gas production,gas production rate and gas composition at 48 h of in vitro fermentation with GAA addition（P>0.05）.Rumen propionic acid concentration and MCP content increased significantly（P<0.05）,isovaleric acid concentration decreased significantly（P<0.05）,while p H,NH₃-N,TVFA,isobutyric acid,butyric acid,and valeric acid concentrations did not change significantly（P>0.05）.Results of flora structure analysis:The Shannon index was significantly lower in the GAA02 group（P<0.05）,and the addition of different levels of GAA had no significant effect on the Chao index（P>0.05）.PCA analysis showed a significant separation of the five groups of samples.At the phylum level,the relative abundance of the phylum Bacteroidota was significantly increased in the GAA08 group（P<0.05）.At the genus level,the relative abundance of the genera norank＿f＿F082 and Papillibacter was significantly higher in the GAA06 group,and the relative abundance of the genera Prevotella and Prevotellaceae＿UCG-001 was significantly higher in the GAA08 group（P<0.05）.Results of the functional analysis of the microflora:The metabolic pathways of Flagellar assembly and Bacterial chemotaxis were significantly higher in the GAA02 group（P<0.05）.The metabolic pathways of Mismatch repair and Nucleotide excision repair were significantly lower in the GAA08 group than in the other groups（P<0.05）.The metabolic pathways of Bile secretion and Protein digestion and absorption was significantly higher in the GAA08 group than in the other groups（P<0.05）.In summary,addition of 0.2%GAA reduced the microflora diversity and improved the metabolic pathways for Flagellar assembly and Bacterial chemotaxis.The addition of 0.8%GAA increased MCP and propionic acid concentrations and decreased isovaleric acid concentrations in rumen fermentation parameters,and increased the metabolic pathways of Bacteroidota,Prevotella and Prevotellaceae＿UCG-001.Enhancing metabolic pathways for Bacterial bile secretion and Protein digestion and absorption,and weakened metabolic pathways for Mismatch repair and Nucleotide excision repair.