| Starch is the main energy source for ruminants,which plays an important role in promoting the higher performance of animals.The energy efficiency of starch digestion sites differs in the rumen and small intestine,and starch digestion in the small intestine is much efficiency than in the rumen.More starch digestion in the rumen could result in metabolic disorders,and therefore more starch digestion in the small intestine can improve the feed energy value and be helpful for host.However,small intestinal starch digestion in ruminant is limited,averaging 60%,and the lack of pancreatic α-amylase was the first limited factor.Previous studies have shown that leucine could stimulate the synthesis and secretion of adult ruminants pancreatic α-amylase,and accelerate the development of child ruminants.But whether leucine could further improve the small intestinal starch digestion is not clear,and the detailed mechanism of leucine’s effect should also be clarified.In addition,the potential influences of the surgery on pancreatic functions could not be ruled out,and how to evaluate the small intestinal starch digestion and exclude the surgery influence also should be settled.Duodenal starch flux could affect the pancreatic α-amylase secretion,and what is the effect of leucine when duodenal starch flux is different? Thus,the present study was firstly evaluated the effect of rumen-protected leucine(RP-Leu)on the small intestinal starch digestion in Holstein heifers via the parameter of blood glucose concentration,the apparent total tract starch digestion,ruminal fermentation profiles,fecal fermentation and microbiota composition.Secondly,we evaluated the different effect of RP-Leu and rumen-protected lysine(RP-Lys)on small intestinal starch digestion,and explored the molecular mechanism of leucine and lysine on stimulating the synthesis of pancreatic α-amylase in pancreas acinar cells.Eventually,the relationship between the effect of leucine on improving small intestinal starch digestion and small intestinal starch flux amount was studied.The whole studies were explored in order to provide a theoretical basis and practical application for improving production performance and feed efficiency of ruminants via amino acids nutrition regulating methods.Exp.1 The effect of rumen-protected leucine on the small intestinal starch digestion in Holstein heiferFourteen male Holstein heifers were randomly assigned to two groups with seven calves per group.The calves in the control group received the basal diet only,while the calves in the other group were fed the basal diet supplemented with 36 g/d RP-Leu.The experiment lasted 4 w,and the last week was sampling week.Rumen fluid,fecal and blood sample was collected during the sampling period,mainly measuring the ruminal and fecal fermentation profiles,blood biochemical index,and fecal microbiota composition with 16 S r RNA gene sequencing technology.RP-Leu supplementation didn’t influence the dry matter intake,apparent total tract starch digestion and ruminal fermentation profiles(P > 0.05),but significantly improved the blood glucose concentration and fecal p H(P < 0.05),and decreased the molar proportion of fecal propionate(P < 0.05).Meanwhile,RP-Leu supplementation changed the fecal microbiota composition,and it improved the fibrolytic genera(Pseudobutyrivibrio,Butyrivibrio,Ruminiclostridium＿5 and Lachnospiraceae＿NK3A20＿group)abundance and decreased the amylolytic genus of Faecalibacterium(P < 0.05).Therefore,we concluded that the small intestinal starch amount was equal,and the hindgut starch amount digestion was decreased in the RP-Leu group,and exogenous glucose absorption was increased,which indicating that RP-Leu had improved starch digestion in the small intestine.Exp.2 the different effect of rumen-protected leucine and rumen-protected lysine on the small intestinal starch digestion in Holstein heiferThe Exp.1 showed that RP-Leu could improve the small intestinal starch digestion.In order to distinguish the leucine effecting as molecular signaling or precursor of protein synthesis,the present study chose lysine recording as limited amino acids as control to compare the different effect of leucine and lysine.Fifteen male Holstein heifers were randomly assigned to three groups with five calves per group.The calves in the control group(CON)received the basal diet only,while calves in the RP-Leu group or RP-Lys group were supplemented with 36 g/d RP-Leu and 56 g/d RP-Lys(equal molar to RP-Leu),respectively.The experiment lasted 4 w,and the last week was sampling week.Rumen fluid,fecal and blood sample was collected during the sampling period,mainly measuring the ruminal and fecal fermentation profiles,blood biochemical index,and fecal microbiota composition with 16 S r RNA gene sequencing technology.Both RP-Leu and RP-Lys had no effect on the dry matter intake,apparent starch total tract digestion and ruminal fermentation profiles(P > 0.05).Compared with RP-Lys and CON,RP-Leu significantly increased the blood glucose concentration and fecal p H(P < 0.05),but RP-Lys didn’t work(P > 0.05).In addition.fecal cellulolytic family(Ruminococcaceae and Bacteroidales＿BS11＿gut＿group)tended to be increased by RP-Leu(0.05 < P < 0.1),but not RP-Lys(P > 0.05).In total,the results proved that RP-Leu,but not RP-Lys could improve the small intestinal starch digestion,which suggested that differences in the effect of improving starch digestion existed between leucine and lysine.Exp.3 The molecular mechanism of leucine and lysine on stimulating the pancreatic α-amylase synthesis and secretion in pancreas acinar cellsBased on the results of Exp.2,this study was conducted to explore the different effect of leucine and lysine on pancreatic amylase synthesis and secretion in molecular mechanism.This experiment was conducted by adding different concentration of leucine(negative control,positive control,0.45,0.90,3.60,7.20 m M)and lysine(negative control,positive control,0.50,1.00,4.00,8.00 m M)on the culture media respectively,and the incubation time was 60 min.The amylase activity and the phosphorylation of key protein factor were measured with kit and Western-blot technology.The results showed that 3.6 m M leucine could significantly increase the pancreatic α-amylase activity and the phosphorylation of the p70S6 K and 4EBP1 which were the key protein factor of m TOR(P < 0.05),but lysine had no effect on the α-amylase activity and the phosphorylation of m TOR pathway within the concentration set in this experiment(P > 0.05).Therefore,we concluded that leucine could promote the formation of a transcriptional initiation complex and then increases the synthesis and secretion of protein in the pancreatic acinar cells.Exp.4 Effects of rumen-protected leucine supplementation and dietary rumen-escape starch level on small intestinal starch digestion in Holstein heiferBase on the above three experiments,we could conclude that leucine can promote the pancreatic α-amylase synthesis and secretion acting as molecular signaling,further improved small intestinal starch digestion,and duodenal starch flux could affect the pancreatic α-amylase secretion.The present study was conducted to explore the interaction effect of RP-Leu and small intestinal starch flux.Twelve male Holstein calves were randomly allocated into four groups with three calves per group in a 2 × 2 factorial crossover design.The two factors were rumen-escape starch(RES,high or low level)and RP-Leu supplementation(0 or 36 g/d).The RES was prepared as coarsely ground corn(highly rumen escape rate)or steam-flaked corn(360 g/L density,low rumen escape rate).The experiment included 2 periods,and each period lasted 4 w,and the last week was sampling week.Rumen fluid,fecal and blood sample was collected during the sampling period,mainly measuring the ruminal and fecal fermentation profiles,blood biochemical index,and fecal microbiota composition with 16 S r RNA gene sequencing technology.L-RES significantly increased the apparent total tract starch digestion and ruminal total volatile fatty acids and the molar proportion of ruminal propionate and fecal acetate(P < 0.05),and had a tendency to decrease fecal total volatile fatty acids and the molar proportion of fecal butylate(0.05 < P < 0.1).L-RES diet increased the relative abundance of fecal fibrolytic microbiota(Bacteroidetes,Spirochaetae and Ruminococcaceae),and decreased the relative abundance of fecal amylolytic microbiota(Succinivibrionaceae,Succinivibrio,Prevotellaceae＿UCG-003 and Clostridiaceae＿1)(P < 0.05),but didn’t affect the blood glucose concentration(P > 0.05).RP-Leu supplementation had no effect on the apparent total tract starch digestion and ruminal fermentation profiles(P > 0.05),but resulted in significant increases in blood glucose concentration in both the H-RES and L-RES groups(P < 0.05),but at 2 different magnitudes: 19.8% versus 12.5% increase.the fecal butyrate molar proportion decreased by 23.85 and 12.33% in the H-RES and L-RES groups,respectively,with RP-Leu supplementation compared with the control group.RP-Leu supplementation significantly increased the relative abundance of fibrolytic microbiota(P < 0.05).Interaction effects existed in fecal butyrate molar proportion and fibrolytic microbiota(Bacteroidaceae and Ruminococcaceae UCG-005)between RP-Leu and RES(P < 0.05).Therefore,RP-Leu could improve small intestinal starch digestion and interaction effects existed between RP-Leu and RES.This study showed that Leucine promoted the pancreatic α-amylase synthesis and excretion,and further increased the small intestinal starch digestion in Holstein heifers through activating m TOR signaling pathway as a molecular signaling.Meanwhile,within the limited duodenal starch fluxing range,the effect of leucine on improving starch digestion was stronger in higher starch fluxing. |
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