The Appropriate Ratio Of Dietary Lysine And Threonine For 6 To 9 Months Old Holstein Heifers

An Amino Acid Partial Deletion Method was used in this experiment to study the effects of deleting Lys,Met and Thr on growth performance,rumen environment,nitrogen metabolism and liver metabolome,and to obtain the limiting sequences and appropriate pattern of lysine?Lys?,methionine?Met?and threonine?Thr?in diet of 6 to 9 months old Holstein heifers.The experiment was conducted for three months with 72 Holstein heifers?average 5.5±0.5 months old;200±9.0 kg of body weight?.Following an initial 15d adaptation period,heifers were allocated to one of four groups:theoretical amino acid,positive control?PC?;30%Lys deleted group?PD–Lys?;30%Met deleted group?PD–Met?,and 30%Thr deleted group?PD–Thr?.Experimental animals were fed corn–soybean–alfalfa based total mixed rations?TMR?supplemented with Lys,Met,and Thr?ruminal bypass?.Part 1:Effects of partial deleting Lys,Met and Thr on growth performance and serum hormone concentrations of 6 to 9 months old Holstein heifersThe experiment was conducted to evaluate the effects of deleting Lys,Met and Thr on the growth performance and serum hormone concentrations of 6 to 9 month-old Holstein heifers.Individual TMR intake was recorded daily.Body weight and body size were measured at 30 days interval,and blood samples were collected at same time by jugular venipuncture to determine concentrations of hormone.The results indicated that body weight and dry matter intake were not influenced?P>0.05?by group.Average daily gain and feed conversion efficiency of 8 to 9 months old heifers were lower?P<0.05?in both PD–Lys and PD–Met group than those in PC group.The serum insulin level in PD–Met group was lower?P<0.05?than that in PC group,and the serum estradiol level in PD–Lys group was lower?P<0.05?than that in PC group.Deleting 30%of Lys and Met caused the imbalance of amino acid,which led to some decrease of average daily gain and feed conversion efficiency,as well as the imbalance of estrogen and insulin secretion of 6 to 9 months old heifers.According to growth performance,the limiting sequence of three amino acids was Lys>Met>Thr.Part 2:Effects of partial deleting Lys,Met and Thr on rumen environment of 6 to 9 months old Holstein heifersThe experiment was conducted to determine whether deleting Lys,Met and Thr would affect the absorption of amino acids in small intestine by altering the production of microbial protein?MCP?.Rumen fluid was collected at 30 days interval to determine ruminal fermentation parameters and microflora.The results showed as follows:no significant differences of pH value,NH₃-N concentration,MCP yields were found?P>0.05?among groups,but the MCP yields in PD-Met group tended to be lower?P<0.1?than that in PC group.In addition,the molar ratio of propionic acid was higher?P<0.05?in PD-Met group than that in PC group,resulting in a lower ratio of acetate/propionate?P<0.05?in PD-Met group compared to PC group.The values of Chao and Ace of microbial diversity were reduced?P<0.05?as Met level decreased,but the abundance of Prevotella₇ was significantly increased?P<0.05?in PD-Met group.No significant effects on rumen microflora and composition was observed?P>0.05?after the deletion of Lys and Thr.The results indicated that deleting 30%of rumen bypass Lys and Thr did not affect rumen fermentation parameters and rumen microbial growth of growing heifers,so the AA levels of small intestine were affected by rumen bypass amino acid.However,deleting Met led to decrease of Microbial abundance in the rumen,as well as the increases of Prevotella₇ abundance and propionic acid ratio.Part 3:Effects of partial deleting Lys,Met and Thr on liver metabolome of 6 to 9 months old Holstein heifersThe experiment was conducted to evaluate the effects of amino acids levels on metabolic mechanism of heifers,and to provide theoretical support for the research on amino acid metabolism of growing cows.At the end of the trial,five heifers in each group were selected to take 2 g liver samples by biopsy for determination of liver metabolites.The results showed that there were thirteen different metabolites contents in PD–Lys group compared with the PC group,among which five phosphatidylcholines and three ceramides were significantly down-regulated?VIP>1,P<0.05?;arachidonic acid and eicosenoic acid were significantly up-regulated?VIP>1,P<0.05?;L-aspartic acid,L-histidine and L-leucine were significantly up-regulated?VIP>1,P<0.05?.Three important pathways were obtained,namely,Sphingolipid metabolism,Histidine metabolism Alanine,aspartic acid and glutamate metabolism and Glycerophospholipid metabolism pathways.Deleting Met and Thr levels had no impact on glycolipid metabolism,but they resulted in increase of aspartic acid synthesis?VIP>1,P<0.05?.Phospholipids metabolism is enhanced after deleting 30%of Lys,resulting in the increase of pro-inflammatory factors and the decrease of anti-inflammatory factors.In addition,lack of Lys can enhance protein catabolism;inhibit histidine metabolism;increase the production of aspartic acid.Part 4:Limiting sequence and appropriate pattern of Lys,Met and Thr for 6 to 9 months old Holstein heifersIn order to determine the limiting sequence and appropriate pattern of Lys,Met and Thr,the experiment was conducted to evaluate the effect of nitrogen metabolism after deleting three amino acids.Blood were collected at 30 days interval to determine serum urea nitrogen content.Digestion and metabolism trial was conducted at the last week of the experiment with 4 days of adaptation period and3 days of feces and urine samples collection period.The results indicated that the PD–Lys group and PD–Met group had higher levels of urea nitrogen,total excreted N,and serum urea N?P<0.05?than those of PC group,which showed that nitrogen retention and utilization rate significantly decreased?P<0.05?as dietary Lys and Met levels decreased.But no differences were found?P>0.05?between PD-Thr group and PC group.It shows that deleting 30%of Lys and Met will lead to the imbalance of amino acid,resulting in the increase of serum urea N content and urine N emissions,as well as the decrease of nitrogen retention.According to nitrogen retention,Lys was the first limiting AA,followed by Met and Thr,respectively.The optimal pattern of Lys,Met and Thr in the diet was100:32:57.In summary,deleting Lys and Met levels led to the imbalance of amino acid.It is characterized by increasing aspartic acid synthesis and accelerating the urea synthesis in liver,which resulted in increase of serum urea nitrogen and urine nitrogen,and decrease in nitrogen retention.Finally,deleting Lys and Met levels caused the decrease of growth performance.Moreover,deleting 30%Lys resulted in the increase of pro-inflammatory factors and the decrease of anti-inflammatory factors.According to the growth performance and nitrogen retention,the sequence of three limiting amino acids for 6 to 9 months old heifers fed corn–soybean–alfalfa based diets was Lys?29?Met?29?Thr.The optimal pattern of Lys,Met and Thr based on nitrogen retention was100:32:57.