Effects And Mechanism Of Dietary Branched-Chain Amino Acids On Milk Fat Branched-Chian Fatty Acids Synthesis In Dairy Cows

Branched-chain fatty acids(BCFAs)are an important class of fatty acids in milk fat,which have important physiological functions such as anti-inflammatory and anti-tumor.Milk is the main source of human dietary BCFA.It is of great significance to explore effective nutritional regulation methods to increase the content of BCFA in milk fat and then increase the intake of human dietary BCFA to maintain human health.Branched-chain fatty acids(BCAAs)are important precursors for BCFA synthesis,based on this,this study focused on the regulation of BCAA on BCFA content in milk,and carried out the following four parts:Experiment 1: The effects of parities and days in milk(DIM)on milk fat BCFAOne hundred and fifty-seven Holstein cows with different parities(1,2,3 and >3)and DIM(≤ 21 DIM,21<DIM≤100,100<DIM≤200 and >200)were selected to investigate the effect of parity and DIM on milk fat effect of BCFA content.The experiment lasted for 3 weeks,and the milk samples of each cow were collected on the last 3 days of the last week and the content of BCFA was determined.The results showed that both parity and lactation stage had certain effects on BCFA in milk fat of dairy cows.The proportion(g/100 g fatty acids)of BCFA in milk fat of the 1st and 2nd parity cows was higher than that of the other parity cows,and the BCFA production(g/d)of the 2nd parity cows was also relatively high.The proportion of BCFA was highest during the >200 DIM period,however its production was relatively high during the 21<DIM≤100 and 100<DIM≤200 periods.Experiment 2: Correlation between BCFA content in rumen,blood and milk fat and its synthetic precursor BCAA contentTwenty-seven healthy Holstein dairy cows with similar or same body weight(630±27)kg/d,parity(3.6±0.3),DIM(170±18)d and milk yield(25.7±3.1)kg/d were selected and raised in the same pen and fed the same diet for 4 weeks.The milk,rumen fluid and blood samples were collected for the analysis of BCFA,and the BCAA content in rumen was also determined.The results showed that: 1)BCAA in the rumen could be found in the rumen with a significant(P<0.05)or extremely significant(P<0.01)correlation,and most of the above correlations were moderately strong;BCFA with significant(P<0.05)or extremely significant(P<0.01)correlations were found in plasma,and the above correlations were mostly weak or moderately strong;3)There is a weak correlation between rumen BCAA and BCFA in milk 4)There was a moderately strong correlation between rumen and BCFA in milk.In summary,altering the content of synthetic BCFA precursors in the rumen may be a potential nutritional regulation strategy to increase OBCFA content in milk.Experiment 3: The regulatory effect and mechanism of BCAA on the synthesis of BCFA in the rumen in vitroIn this study,the rumen in vitro fermentation technology was used to explore the regulation and mechanism of BCAA,the precursor of BCFA synthesis by rumen microorganisms,on the content of BCFA in fermentation broth.A completely randomized experimental design was used,with no added control group(CON)and experimental group,and the experimental group added 1,2,and 4 m M leucine(Leu),isoleucine(Ile)and valine(Val),respectively,with 5 replicates for each treatment,and a total of 3experiments were performed as replicates.The fermentation was terminated after 24 h,and the fermentation broth was collected to determine the BCFA content,activities of BCFA synthesis-related enzymes,microbial composition,and changes in metabolites.The results showed that BCAA,the precursor of BCFA synthesized by rumen microorganisms,had a certain regulatory effect on the content of BCFA in the rumen fermentation broth in vitro.The addition of Leu at different levels linearly increased the contents of iso-13:0,iso-15:0,iso-16:0 and ΣBCFA in the fermentation liquid;the addition of Ile increased the contents of iso-14:0,iso-16:0,iso-17:0 and ΣBCFA;the addition of Val increased the content of iso-14:0 and iso-15:0.Leu,Ile and Val all increased the activities of BCAT and BCFAS;16S r RNA analysis showed that Firmicutes may be the dominant phylum for BCFA synthesis using Leu,while the Bacteroidetes may be the dominant phylum that utilizes Ile and Val to synthesize BCFA.The results of metabolomic KEGG pathway topology analysis showed that the addition of Leu regulated metabolic pathways such as glycerophospholipid metabolism,purine metabolism and AA metabolism;the addition of Ile regulated metabolic pathways such as AA metabolism,purine metabolism and aminoacyl-t RNA biosynthesis;The addition of Val regulated metabolic pathways such as AA metabolism,nitrogen metabolism and aminoacyl-t RNA biosynthesis.Experiment 4: Effect of dietary Leu supplementation on BCFA content in milk fat of lactating cowsTwenty healthy Holstein cows with similar or same parity(1.7±1.0),DIM(172±24)d and milk yield(28.1±5.9)kg/d were selected and divided into 2 groups(n=10),namely the control group(CON)and the Leu supplemented group(LEU-80).The CON group was fed the basal diet,and the LEU-80 group was supplemented with 80 g of Leu daily.The trial was divided into 6 weeks,a 2-week pre-trial period and a4-week trial period.Milk samples from each cow were collected during the last week of the experimental period for the determination of BCFA concentrations.Compared with the CON group,the LEU-80 treatment group increased the total BCFA concentration in milk fat by 27.8%,with iso-13:0(24.3 μg/m L vs 34.9 μg/m L),iso-15:0(133.0 μg/m L vs 173.5 μg/m L),iso-16:0(40.5 μg/m L vs 60.4 μg/m L),iso-17:0(117.5 μg/m L vs 142.2 μg/m L)and anteiso-17(107.9 μg/m L vs 133.0 μg/m L)concentration were increased by 43.6%,30.5%,49.1%,21.0% and 23.3%,respectively.The results of this study showed that the addition of Leu to the diet could effectively increase the concentration of BCFA in milk fat of dairy cows.