Study Of The Regulation Mechanism Of Guanidinoacetic Acid On Rumen Fermentation And Related Metabolism Of Tissue In Beef Cattle

Guanidinoacetic acid（GAA,or Glycocyamine）is a precursor of creatine.Creatine（Cr）plays the vital role of energy buffer in the muscle of livestock.Our previous research showed that the supplementation of GAA in the diet could significantly improve the growth performance of Jinjiang cattle,reduce the feed consumption and improve the feed conversion rate.Rumen is a unique digestive organ of ruminants.The health and functional changes of rumen have an important impact on the growth performance of ruminants.At present,there are few reports on the effect of guanidinoacetic acid on rumen fermentation function,and the regulation mechanism is still unclear.In this study,Jinjiang cattle were selected as experimental animals.Combined with serial experiments in vitro and in vivo,this study was systematically performed:（1）Degradation of GAA in rumen;（2）Effects of GAA on rumen fermentation parameters,microbial diversity and colony structure;（3）Effects of dietary GAA on functional and metabolomic profiles and ATP（adenosine triphosphate）synthesis mechanism of rumen microorganisms;（4）Effects of GAA on enzyme activities and metabolites in target organs such as muscle,liver and kidney of Jinjiang cattle.This study applied the advanced techniques as ion chromatography determination in（1）,16S r DNA sequencing in（2）,metagenomic library-based technique and metabolomic profiles of rumen microorganisms in（3）and（4）.The aim of this study was to clarify the effect of GAA on ruminal fermentation function and metabolism in beef cattle,with the hope to provide references for the application of GAA in beef cattle feedlots.ExperimentⅠ:Degradation rate of GAA incubated in vitro for 24 h.The purpose of this experiment was to investigate whether GAA could be degraded in rumen.In this experiment,the content of GAA in the control group（0 mg GAA+500 mg diet substrate）and the 4.0 g/kg GAA group（2 mg GAA+500 mg diet substrate）incubated for 0 h and 24 h were determined by ion chromatography UV detection,and the degradation rate of GAA was calculated.The results showed that the degradation rate of GAA incubated in vitro for 24 hours was 6.39～31.29%（mean 12.69%）.The results suggest that GAA can be partially degraded in the rumen,and the GAA may be utilized by rumen microorganisms,which may affect the rumen microbial microorganisms and the function of fermentation.ExperimentⅡ:Effects of GAA on rumen fermentation parameters and rumen microbial diversity（in vitro）The results of experimentⅠshowed that GAA could be degraded by rumen microorganisms,but the effect of GAA on fermentation function in cattle’s rumen was still unclear.The aim of this experiment was to study the effects of different doses of GAA on ruminal gas production,fermentation parameters and rumen microorganisms.Furthermore,the optimal dosage of GAA supplemented in diet was screened out by all tested indexes.There were 5 groups:control group,2.0 g/kg GAA,4.0 g/kg GAA,8.0 g/kg GAA and 16.0g/kg GAA,with 5 replicates in each group in serial experiments.The rumen fermentation parameters:in vitro dry matter degradation rate（IVDMD）,gas production（GP）,p H value,ammonia nitrogen（NH₃-N）concentration,microbial crude protein（MCP）concentration and volatile fatty acids（VFA）were measured after each in vitro fermentation was suspended at 8 h,12 h,24 h,36 h,48 h and 72 h.The results of experimentⅡsuggested that（1）Supplementing 2.0～4.0 g/kg GAA in diet received the optimal effects in all rumen fermentation parameters.（2）According to the multiple-factor AE index（MFAEI）of the additive value of the single-factor AE index（SFAEI）including the parameters of IVDMD,GP,p H,NH₃-N,VFA and MCP;the effect of supplementing 4.0 g/kg GAA was rectified as the best in vitro.（3）Compared with the control,4.0 g/kg GAA significantly reduced theαdiversity andβdiversity（P<0.01）at 36 h incubation.The 16S r DNA results indicated that the relative abundance of Firmicutes,Fusobacterium,Succinivibrio,Anaerovibrio,Clostridium,Prevotella,Selenomonas and Streptococcus were significantly enhanced when the diet was supplemented with 4.0 g/kg GAA（P<0.05）at 36 h incubation.With correlated gene annotation on COG data base,the results revealed that supplementing GAA in diet significantly accelerated the reproductive division of rumen bacteria,promoted the production of MCP,enhanced the bioavailability,transport and metabolism of amino acids,nucleic acids,lipids,coenzymes and other secondary metabolites,and improved the biogenesis of bacterial plasma membrane（P<0.05）.In a word in ExperimentⅡ,guanidinoacetic acid had a significant affect on the rumen fermentation parameters,microbial diversity and colony structure in rumen.The optimal addition range of GAA is 2.0～4.0 g/kg in beef cattle.ExperimentⅢ:Effects of dietary GAA on rumen microbial metagenome and metabolome（in vivo）.The results of experimentⅡshowed that the optimum supplementation range of GAA was 2.0～4.0 g/kg in vitro.The previous results of our study showed that dietary 2.0 g/kg GAA supplementation had the best effects in the 52-d feeding trial（in vivo）.The aim of experimentⅢwas to explore the molecular mechanism of GAA regulating the function of rumen fermentation in beef cattle.The metagenome and metabolome of rumen microorganisms in Jinjiang beef cattle regulated by GAA were excavated by omics technology to clarify the molecular mechanism of GAA on rumen microorganisms.The results showed that:（1）Microorganisms and diversity:the supplementation of GAA in diet promoted the proliferation of fiber carbohydrate（FC）decomposing bacteria Firmicutes and Clostridium,as well as the proliferation of non-fiber carbohydrate（NFC）decomposing bacteria Bacteroides and Alistipes.Forteen species of bacteria with anaerobic respiration characteristics,such as Clostridium kluyveri,were screened out.（2）Annotation of gene function:the supplementation of GAA increased the gene abundance of 5 glycoside hydrolases[EC 3.2.1.-]annotated in the carbohydrate active enzyme database（CAZy）.The supplemention of GAA increased the expression of genes related to nutrient transport and metabolism,and the primary dehydrogenase,oxidoreductase and molecular motor proton pump ATPase within the ETC（electron transport chain）of rumen bacteria annotated in the KEGG database（P≤0.05）.（3）The results of metabolomics showed that the metabolism of amino acids and fatty acids in rumen microorganisms were significantly enhanced with the supplementation of GAA in the diet,which promoted the synthesis of MCP（P≤0.05）.（4）Omics annotation:there was high correlation between Firmicutes and the raw material sources（ammonia nitrogen and VFA）,and the energy sources（ATP）utilized in rumen MCP synthesis.There was high correlation between Firmicutes and acid detergent fiber digestibility（ADFD）,dry matter digestibility（DMD）.Firmicutes（phylum level）was the the rumen bacteria with the highest correlation between metagenome and metabolites,and so as Clostridium（genus level）.In a word in ExperimentⅢ,supplementation of GAA promotes rumen fermentation and SLP,meanwhile improves anaerobic respiration and ETP,which plays an important role in improving MCP yield and promoting beef growth.ExperimentⅣ:Effects of GAA on enzyme activities and metabolites in target organs such as muscle,liver and kidney of Jinjiang cattleThe aim of this experiment was to study the effect of GAA on the biochemical metabolism of beef cattle.After slaughtering 10 beef cattle used in experimentⅢ,the longissimus thoracis（LT）muscles,Semitendinosus（SM）muscles,livers and kidneys of Jinjiang cattle were collected to make homogenate to study the effect of GAA on the metabolism of beef cattle.The results showed that the content of muscle glycogen（MG）,laminin（LN）,type IV collagen（CV-Ⅳ）and the activities of creatine kinase（CK）and lactate dehydrogenase（LDH）were significantly regulated in beef（P<0.05）;The activities of glutamyl transpeptidase（GGT）,adenosine deaminase（ADA）,hydroxybutyrate dehydrogenase（HBDH）and alanine aminotransferase（ALT）and the content of urea（Urea）and creatinine（CRE）in liver and kidney were significantly regulated（P<0.05）.The results suggested that 2.0 g/kg GAA significantly promoted the production of creatine and nitrogen retention,enhanced the energy buffer and efficient utilization function of beef cattle,promoted the activities of amino acid metabolism and nucleic acid metabolism related enzymes of beef cattle（the host of rumen microbes）,and improved the function of energy buffering in the hosts.In conclusion,GAA could be partially degraded by rumen microorganisms.Supplementing GAA in diet could improve the function of rumen fermentation,influence the rumen microbes,improve the energy efficiency and anaerobic respiration function of rumen microbes,increase ATP and MCP production in rumen fluid.In addition,supplementing GAA in diet could also promote amino acid metabolism and nucleic acid metabolism and improve the energy buffering function in beef cattle,increase the growth of the beef cattle.