| An Partial Deletion Method was used in this study to investigate the effects of partial deleting lysine(Lys),methionine(Met)and threonine(Thr)on growth perfermance,rumen environment,nitrogen metabolism,liver metabolism and gene expression,and to obtain the limiting sequences and optimal pattern of Lys,Met and Thr in diet of 3 to 6 months old dairy female calves.The experiment was conducted for 90 days with 72 dairy female calves aged about 84 day-old fed in restricted amounts.Following a 7-day adaptive period,calves were allocated to 4 groups according to date of birth and body weight: theoretical amino acid balanced treatment(PC),30% Lys reducing treatment(PCLys),30% Met reducing treatment(PCMet)and 30% Thr reducing treatment(PCThr).Amino acids additives were added as rumen protected amino acids.Part 1: Effects of reducing partial Lys,Met and Thr levels on growth development,dietary nutrients digestion and metabolism in dairy female calvesThe objective of this part was to evaluate the effects of reducing partial dietary Lys,Met and Thr level on growth performance,and nutrients digestion and metabolism in 3-6 month-old dairy female calves.Body weight,body size and length of nipples were measured on 1,30,60 and 90 day.Six healthy calves were selected from each group at the age of 132-138 and 173-179 days for twice metabolism tests.Feces,urine and feeds samples were collected to determine the apparent digestibility of nutrients and nitrogen metabolism.Dry matter intake was recorded daily throughout the whole trial period.The results showed that reducing dietary Lys level significantly decreased Gain/Feed in 3 to 6 month-old calves(P<0.05).Average daily gain(ADG)of PCLys group was the lowest among four groups(P>0.05).ADG and Gain/Feed were not influenced by reducing Met and Thr level in diets for 90 to 180 day-old calves(P>0.05).Reducing dietary Lys and Thr level decreased fecal score(P<0.05)of calves.Reducing Lys,Met and Thr levels in diets did not affect body size and apparent nutrients digestibility of calves(P>0.05).Retained N of intake N and biological value of N were significantly decreased via reducing dietary Lys level(P<0.05).According the nitrogen retention and growth development,Lys was the first limiting amino acid,followed by Met and Thr.On the basis of the theoretical amino acid balanced diet,reducing dietary Lys level had adverse effect on growth performance and nitrogen metabolism,and reducing dietary Thr level could deteriorate diarrhea,while reducing dietary Met level had no effects on calves.We calculated the optimal pattern of Lys,Met and Thr based on nitrogen retention.The optimal pattern of Lys,Met and Thr were 100:29:53 of 135-138 day-old and 100:31:57 of 176-179 day-old.The limiting sequence of amino acids was Lys>Met>Thr.Part 2: Effects of reducing partial Lys,Met and Thr levels on liver metabolism and gene expression in dairy female calvesThis part aimed to investigate the effects of reducing partial Lys,Met and Thr levels of diets on liver metabolism and gene expression in dairy female calves using RNA-sequencing and untargeted LC-MS metabolomics.Three healthy calves were selected from each group on 90 day and their liver samples were obtained for biopsy and used to measure non-targeted metabolomics and transcriptomics.Network and pathway analyses revealed that noradrenaline,adenosine 5’-monophosphate,acetyl-CoA,and coenzyme A were significantly decreased,including those involved in the regulating of lipolysis in adipocytes and fatty acid degradation,were downregulated(P < 0.05).We also identified eight significantly differentially expressed genes(SDEGs),among which ADRB2,WFDC2,and CLDN4 were associated with inhibition of lipolysis,and CCRN4 L,FOSL2,and ARG2 were associated with inhibiting lipid synthesis(P < 0.05).Correlation tests showed that coenzyme A was strongly correlated with SDEGs(0.82≤|r|≤0.96).AcetylCoA and adenosine 5’-monophosphate were strongly correlated with CCRN4L(0.90≤|r|≤0.92),indicating a strong correlation between the changes in SDEGs and these metabolites.In conclusion,reducing Met and Thr not affected liver function.Reducing Lys affected lipid metabolism in the liver by inhibiting lipolysis and lipid synthesis in calves.Part 3: Effects of reducing partial Lys,Met and Thr levels on rumen fermentation and bacterial community composition of dairy female calvesThe present part was aimed to estimate the effects of partial reducing dietary Lys,Met and Thr levels on the rumen fermentation and bacterial community composition of dairy female calves.Five healthy calves in feeding experiment were selected from each group on 90 day to sample rumen fluid and used to rumen fermentation parameters,and microbial communities.The results showed that reducing Lys,Met and Thr levels did not affect rumen fermentation parameters and alpha diversity of the microbiota(P > 0.05).However,the relative abundance of Sharpea,[Ruminococcus] gauvreauii group and [Eubacterium] coprostanoligenes group belonging to Firmicutes were significantly decreased by reducing Lys level of diet(P < 0.05).The relative abundance of Succinivibrionaceae was significantly decreased by reducing Thr level of diet(P < 0.05).In conclusion,reducing partial Lys,Met and Thr levels did not affect the rumen fermentation pattern.In summary,reducing thirty percent Lys level of diet led to the imbalance of amino acid.It is characterized by inhibiting the expression of arginase,which induced the increase of discharge of urea nitrogen and the decrease of biological value of nitrogen and feed efficiency.Furthermore,reducing Lys level disturbed lipid metabolism in the liver by inhibiting lipolysis and lipid synthesis.However,reducing dietary Met or Thr level had no affect on gowth development of claves.According to the growth performance and nitrogen retention,the sequence of three limiting amino acids of corn0alfalfa-soybean with 1.21% Lys for 3 to 6 months old calves was Lys>Met>Thr.The optimal pattern of dietary Lys,Met and Thr level was 100:29:53 of 135-138 days of age and 100:31:57 of 176-179 days of age. |
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