Effects Of Dietary Supplementation With Guanidinoacetic Acid On Small Intestinal Nutrient Flux,absorption And Organismal Creatine Metabolism In Sheep

Guanidinoacetic acid(GAA)is the only precursor for the synthesis of creatine in animals.Guanidinoacetic acid has positive significance for animal energy metabolism.The effects of guanidineacetic acid on livestock production and its possible mechanisms of action have been extensively studied,and the main effects of GAA on ruminant livestock such as cattle and sheep are to improve weight gain and feed conversion efficiency.However,the results of the current studies on its mechanism of action still do not explain well the weight gain and feed conversion efficiency produced by GAA.Hence,it is necessary to continue to conduct in-depth studies on the mechanism of GAA’s effect on animal metabolism.In this paper,the effects of exogenous GAA on rumen metabolism,small intestine nutrient flow,small intestine absorption,liver creatine synthesis and muscle creatine metabolism and its possible mechanisms of action were investigated in sheep during the growing season to enrich the theory of GAA metabolism and provide scientific theoretical basis and technical support for the application of GAA in sheep production.Experiment I: Effect of dietary guanidinoacetic acid supplementation on the concerntration of guanidinoacetic acid-related metabolites in plasma and urine of sheepThe experiment investigated the effect of supplementation with guanidinoacetic acid(GAA)on blood and urine guanidinoacetic acid-related metabolites in sheep before and after feeding.Twelve healthy Kazakh rams with an average body weight of 27.35±0.58 kg at 3 months of age were divided into control and GAA groups(n=6/group)according to body weight.The GAA group was fed a trial with 1000 mg/kg guanidinoacetic acid per kg of basal diet,taking into account the results of the reported studies.The results showed tha,1)the 0～12 h after feeding plasma GAA mean levels were significantly higher in the GAA group compared to the control group(P<0.05).Plasma creatine levels were significantly higher in the GAA group at 8 h after feeding(P<0.05).2)Creatinine urinary excretion was highly significant in the GAA group compared to the control group(P<0.01).Therefore,under the conditions of this study,the supplementation of GAA in the diet increased the plasma guanidinoacetic acid and creatine levels in sheep and increased the excretion of creatinine in the urine.This initially reflects the ability of sheep to utilize exogenous guanidinoacetic acid.Experiment II: Effect of dietary supplementation with guanidinoacetic acid on rumen metabolism and duodenal nutrient flux in sheepThis experiment explores the extent to which GAA is degraded in the rumen and explores the effect of GAA on rumen metabolism and nutrient flow into the duodenum of sheep.Twenty-four 5-month-old Kazakh rams with rumen fistula and proximal duodenal fistula of similar weight(34.29±1.95)kg were selected(n=6/group).The test lambs were fed a basal diet without creatine and guanidinoacetic acid,with 0,550,1100 and 1650 mg/kg of guanidinoacetic acid per kg of basal diet in each group.The effect of supplemental guanidinoacetic acid on ruminal guanidinoacetic acid flow and rumen metabolism and nutrient digestion in the small intestine of sheep was investigated using a non-isotopic marker(Li,Cr-EDTA)method for the determination of surimi flow.The results showed that GAA rumen passage was 54%～85% with the addition of 550～1650 mg/kg DM GAA to the diet.Ruminal ammonia nitrogen levels were increased(P<0.05),ruminal fluid p H was increased linearly(Linear,P<0.05)and rumen fluid acetic acid concentration decreased linearly(P<0.05)at 2 h post-feeding as the GAA content of the diet increased in sheep compared to the 0 mg/kg group.Ruminal fluid propionic acid concentrations were significantly lower in the 550 mg/kg and 1650 mg/kg groups at 0 h,2 h and 0～8 h of feeding(P<0.05).Ruminal fluid butyric acid content was significantly higher(P<0.05)in the 1650mg/kg group at 0 h,4 h,8 h and 0～8 h mean values of feeding.Total VFA levels increased significantly(P<0.05)in the 1650 mg/kg group at 4 h post-feeding and decreased significantly(P<0.05)in the 550mg/kg group at 6 h post-feeding.Linear increase in total reducing sugars content into the small intestine(Linear,P<0.05).Experiment III: Effect of dietary guanidinoacetic acid supplementation on sheep growth performance and organism creatine metabolismThis experiment investigated the effects of guanidinoacetic acid supplementation on growth performance,guanidinoacetic acid absorption and creatine-related metabolism in sheep.Twenty-four healthy Kazakh rams of similar weight(27.35±0.58)kg at 3 months of age were randomly divided into four groups of six animals each(n=6/group).The test rams were fed a basal diet without creatine and guanidinoacetic acid,and each group was supplemented with 0,550,1100 and 1650 mg/kg guanidinoacetic acid per kg of basal diet.The results showed that 1)The mean daily weight gain of sheep showed a linear increase(P<0.05)with the level of GAA in the diet from 1 to 45 d during the test period.Mean daily weight gain from 1 to 45 d was significantly higher in the 1650 mg/kg group compared to the 0 mg/kg group(P<0.05),and jugular venous plasma INS and IGF-I were significantly higher in the 1650 mg/kg group(P<0.05).Jugular vein plasma GAA and creatine levels increased linearly with increasing GAA levels in the diet,reaching significant levels in both the 1100 mg/kg and1650 mg/kg groups(P<0.05).mean feed intake was significantly lower(P<0.05)in the 46～100 d and1～100 d GAA-added groups,and mean daily gain and mean feed-to-weight ratio were not significantly different(P> 0.05).Jugular vein plasma GAA levels showed a secondary trend with increasing GAA levels in the diet(P=0.10),with the highest in the 550 mg/kg group.2)Portal vein plasma GAA levels showed a secondary trend with increasing GAA levels in the diet.Compared with the 0 mg/kg group,portal vein guanidinoacetic acid was significantly higher in the 550 mg/kg and 1100 mg/kg groups(P<0.05).The relative expression levels of SLC6A6 and SLC6A8 m RNA in jejunal mucosa were significantly lower in the 1650 mg/kg group(P<0.05),and the relative expression levels of SLC6A8 m RNA in ileal mucosal tissue were significantly lower(P< 0.05).Hepatic venous plasma creatine levels showed secondary changes,with the highest in the 550 mg/kg group(P<0.05).Portal venous plasma glucose levels increased significantly in the 1100 mg/kg and 1650 mg/kg groups(P<0.05).Portal plasma essential amino acid levels were significantly higher in the 550 mg/kg and 1100 mg/kg groups(P<0.05);total amino acid levels were significantly higher in the 550 mg/kg,1100 mg/kg and 1650 mg/kg groups(P<0.05).Experiment IV: Effect of dietary supplementation with guanidinoacetic acid on amino acid and glucose levels in sheep chyme and portal plasmaThis experiment was conducted to determine the effect of dietary guanidinoacetic acid on the nutrient content of small intestinal chyme and portal vein nutrients in sheep,and to investigate the effect of dietary guanidinoacetic acid on amino acid and glucose absorption in sheep.The experimental design was the same as in Experiment III.The results showed that 1)the content of basic amino acids(including arginine,histidine and lysine)in the small intestinal chyme increased linearly(Linear,P<0.05),the content of total acidic amino acids and aspartic acid in the small intestinal chyme changed quadratically(Quadratic,P<0.05)and the content of soluble non-essential amino acids and total amino acids in the chyme increased linearly(Linear,P<0.05)as the level of guanidinoacetic acid added to the diet increased.The total acidic amino acid and aspartic acid content of the small intestinal chyme showed a quadratic change(Quadratic,P<0.05)and the soluble non-essential amino acid and total amino acid content of the chyme increased linearly(Linear,P<0.05).The total alkaline amino acid and lysine levels in portal plasma varied quadratically(Quadratic,P<0.05)with increasing levels of guanidinoacetic acid in the diet,with the highest levels in the 550 mg/kg group and the lowest in the 0mg/kg group.Portal plasma essential amino acid levels showed a quadratic change(Quadratic,P<0.05),with the lowest levels in the 0 mg/kg group and the highest levels in the 1100 mg/kg group.The total amino acid content of portal plasma showed a quadratic trend(Quadratic,P=0.05),with the highest level in the 550 mg/kg group and the lowest level in the 0 mg/kg group.2)Compared to the 0 mg/kg group,the ileal chyme soluble glucose content was significantly lower in the 1100 mg/kg and 1650 mg/kg groups(P<0.05).The portal plasma glucose levels were significantly higher in the 1100 mg/kg and 1650mg/kg groups(P<0.05).Experiment V: Transcriptome-based study of the effect of guanidinoacetic acid on gene expression in the small intestinal mucosa of sheepIn this experiment,we used transcriptome analysis to analyse the effect of guanidinoacetic acid on gene expression in the small intestinal mucosa,to investigate the mechanism of guanidinoacetic acid’s effect on the small intestinal mucosa,and to further enrich the theoretical basis of guanidinoacetic acid’s regulation of absorption,transport and metabolism in the sheep’s small intestine.Based on the results of Experiment III,the 0 mg/kg group and the 550 mg/kg group were selected for differential expression analysis of the mucosa of the small intestine(duodenum,jejunum and ileum)of the two groups using transcriptomics analysis.The results showed that the significant entry for GO analysis in the 550 mg/kg group was ATPase activity(MF)in duodenum and significant entry in ileum: DNA-dependent ATPase activity(MF)(P<0.05,Padj<0.05).Duodenal differential genes involved pathway significant changes in:anti-folate resistance,ABC transport,and bile secretion(P<0.05,Padj<0.05).The jejunal DEGs involved significant pathway changes in retinol metabolism,pentose and glucuronide interconversion,and folate biosynthesis(P<0.05,Padj<0.05).In summary,the passage rate of guanidinoacetic acid in the rumen of sheep was 54%～84% in diets supplemented with 550～1650 mg/kg.The dietary supplementation of guanidinoacetic acid increases the flow of reducing sugars into the duodenum and the amino acid content in small intestinal chyme,and improves the functional activity of the small intestine ATPase and energy metabolism,and promoting the absorption of essential amino acids in the small intestine,and increases the level of creatine in the circulating blood,as well asstimulates the secretion of insulin and IGF-I to promote the growth of lambs during the growing period.