| Precision nutrition is proposed to improve production efficiency and welfare of animals through personalized nutrition intervention.A prerequisite for precision nutrition achievement is to understand the inter-individual heterogeneity in nutrition metabolism and its mechanism.Methionine(Met)is the first limiting amino acid(AA)for lactating dairy cows,thus supplementing rumen-protected Met(RPM)has been perceived as an efficient approach to improve the lactation performance of dairy cows.However,increasing evidences demonstrated that the lactation performance of dairy cows exhibited differences in response to RPM among studies.Various studies have investigated the causality for this difference in response to RPM at population level,but were unable to elucidate the detailed mechanisms for such individual differences.Therefore,this study firstly investigated the effects of supplementary RPM on lactation performance and rumen fermentation in dairy cows by a dose-effect experiment,and determined the optimal supplemented dose of RPM(Part 1).The individual differences in lactation performance in response to optimal dose of RPM was identified in a selfcontrolled trial(Part 2).to illuminate the mechanisms explaining such individual response,we further selected two groups of cows showing significantly different responses for downstream analyses on the rumen fermentation and microbiome(Part 3)and mammary gland metabolism(Part 4)before and after RPM supplementation.1.The effects of supplementing RPM on lactation performance and rumen fermentation in lactating dairy cows(Part 1)Sixty healthy mid-lactating Holstein dairy cows(milk yield = 34.0 ± 5.77 kg/d,days in milk = 168 ± 12.7,parity = 2.87 ± 0.96)were blocked into 15 groups and were randomly assigned to 1 of the 4 treatments: 0(control group,CON),15,30,and 60g/d/cow of RPM(Met content = 77.6%)supplementation.The experiment was conducted over a 12-wk period,recorded milk yield and collected milk sample for two consecutive days per week,and dry matted intake was measured for two consecutive days every two weeks.Plasma and rumen fluid samples were collected at wk 6 and wk12.Compared with the CON,no significant differences were found in dry matter intake,milk yield,and milk composition in cows supplemented with RPM(P > 0.10),but numerically the energy corrected milk(ECM)yield in 30 g/d group was highest and significantly higher than that of 60 g/d group(P < 0.05).in individual level,a total 19 cows had an increased milk yield and 27 cows had a decreased milk yield compared with control.No significant effect of RPM was observed on rumen fermentation in dairy cows(P > 0.10).Compared with the CON,the plasma concentration of Met,Pro,nonessential AA and total AA were significantly higher(P < 0.05)and the blood urea nitrogen concentration was significantly lower(P < 0.05)in 30 g/d group.The malondialdehyde concentration was significantly lower in 60 g/d group(P < 0.05).Overall,supplementation of RPM had no significant effects on lactation performance and rumen fermentation because of the large individual differences among dairy cows.Supplementation of 30 g/d RPM can improve the AA metabolism and the optimal supplemental dose of RPM was estimated to be 22.7 g/d according to the regression equation established by milk yield and RPM supplement dosage.2.Study on inter-individual variability in the response to RPM in lactating dairy cows(Part 2)A total of 120 healthy mid-lactating Holstein dairy cows(milk yield = 33.6 ± 6.50kg/d,days in milk = 111 ± 11.9,parity = 1.6 ± 0.70)were selected and used in a selfcontrolled design trial.The trial lasted for 13 weeks,with the first 5 weeks as the adaptation period.in the determination period of 8 weeks,all the cows were added at20 g/d RPM(Met content = 80%).Milk yield was recorded daily,milk sample was collected weekly,and dry matter intake was estimated for two consecutive days every two weeks.The sample of plasma,feces and urine were collected at wk 0 and wk 8.After supplementing RPM,the response in lactation performance of individual dairy cow varied largely,and the change range(coefficient of variation)of ECM,milk protein content,milk fat content and milk lactose content of individual cows were-6.08 ~ 9.08kg/d(231%),-0.46 ~ 0.62%(3343%),-1.06 ~ 1.45%(218%),and-0.34 ~ 0.49%(1036%),respectively.Based on the statistical power analysis,10 cows with positive response(PR)and 10 cows with limited response(LR)were selected for follow-up analysis.The lactation performance of PR cows was improved(P < 0.05),while that of LR cows kept declining(P < 0.05).The apparent digestibility of crude protein(P < 0.01)and dry matter(P = 0.10)of PR cows significantly increased or had an increasing trend,while all nutrients apparent digestibility of LR cows were no significant change(P >0.10).Milk protein(nitrogen)yield increased(P < 0.01)in PR cows,while in LR cows urinary nitrogen concentration(P = 0.02)and yield increased(P = 0.03),and milk protein(nitrogen)yield(P = 0.01)was significantly decreased.Meanwhile,the plasma glucose(P <0.01)and blood urea nitrogen concentration reduced(P = 0.02),the ?-hydroxybutyric acid concentration increased(P = 0.05)in PR cows.The plasma concentration of globulin(P = 0.05)and creatinine(P < 0.01)increased and total antioxidant status decreased(P < 0.01)in LR cows.These results indicate that there exists relatively large inter-individual variation in the response to RPM in dairy cows.The changes in apparent digestibility,nitrogen metabolism and plasma parameters were not consistent between different responding cows,which may be one of the reasons for the different responses to RPM in the individual dairy cows.3.Study on difference of rumen fermentation and microorganism in dairy cows with different responses to RPM(Part 3)For the 20 dairy cows selected in Part 2,the rumen contents were collected at wk0 and wk 8 to analyze the fermentation parameters,to estimate the microbial protein yield,and to analyze the microbial diversity and composition using 16 S r RNA sequencing.in PR cows,the rumen concentration of total volatile fatty acids(P = 0.03),acetate(P < 0.01)and propionate(P = 0.05)and microbial protein yield(P = 0.01)significantly increased,while the molar ratio of isobutyrate(P = 0.10),valerate(P <0.01)and isovalerate(P = 0.06)significantly decreased or had a decreasing trend.in LR cows,the rumen valerate concentration(P = 0.03)and molar ratio of butyrate(P =0.04),valerate(P < 0.01)and isovalerate(P = 0.01)significantly decreased.in PR cows,the relative abundance of 40 bacterial taxa was increased(LDA > 2),and that of 27 bacterial taxa was significantly reduced(LDA > 2)from the phylum level to the genus level.At the genus level,the relative abundance of several bacterial(Lachnospiraceae NK3A20 group(P = 0.01),Acetitomaculum(P = 0.05),Ruminococcus gauvreauii group(P = 0.01),unclassified(f)Lachnospiraceae(P = 0.01)),for which the fermentation products contained acetate and propionate,was significantly increased.However,the relative abundance of 24 bacterial taxa was significantly increased(LDA > 2)in LR cows,and that of 34 bacterial taxa was significantly decreased(LDA > 2)from the phylum level to the genus level.At the genus level,the relative abundance of several bacterial(Prevotellaceae UCG-003(P = 0.03),unclassified(f)Prevotellaceae(P = 0.02)and Lachnospiraceae XPB1014 group(P = 0.01)),for which the fermentation products include acetate and propionate,were significantly decreased.These results indicated that the changes in the rumen fermentation were inconsistent between PR cows and LR cows,which may be caused by the different abundance change in several acetate-and propionate-producing bacteria,partially accounting for the different response to RPM in the individual cows.4.Study on difference of mammary gland metabolism in dairy cows with different responses to RPM(Part 4)This part focused on the differences in mammary gland metabolism between PR and LR dairy cows by comparison of the variables before and after RPM supplementation.The sample of milk,coccygeal artery and vein blood were collected at wk 0 and 8 to analyze the free AA concentration to reflect the uptake and utilization of AA by the mammary glands.Milk metabolome was analyzed to reflect the metabolism of the mammary glands.in PR cows,the mammary plasma flow was increased(P = 0.05);the arterial supply,mammary intake,clearance rate and the ratio of mammary intake to milk output of most of AA were significantly increased or had an increasing trend(P < 0.10),suggesting that the mammary utilization of AA has been improved in these cows.in LR cows,the mammary plasma flow did not change(P =0.38).The arterial supply,mammary intake,clearance rate and the ratio of mammary intake to milk output of most of AA did not change(P > 0.10),while that of cysteine decreased or tended to decrease(P < 0.10),indicating that Met may be partitioned in cysteine anabolism.in PR cows,the relative concentration of biotin was significantly increased(VIP > 1,FC > 2),and the metabolites differentially enriched were purine and pyrimidine metabolic pathways that were closely related to the proliferation of mammary cells,suggesting that improvement in the mammary AA utilization may be related to the proliferation of mammary cells.However,the relative concentration of LMet was decreased(VIP > 1,FC < 0.5),and the metabolites differentially enriched was the central carbon metabolism in cancer and lysine degradation pathways in LR cows,suggesting that Met may be utilized by non-lactation-related metabolism,and ultimately resulting in the limited response in lactation performance.Overall,the mammary utilization of Met is not consistent between PR and LR cows,which may account for the differences in mammary uptake and utilization of AA,and ultimately result in the inter-individual response.in summary,this study observed the obvious individual differences in response of lactation performance to RPM in dairy cows,and explored the possible causes of the response differences from the perspectives of rumen fermentation and mammary gland metabolism.The differences in the abundance of several rumen bacterial that produce acetate and propionate may affect the change of rumen fermentation and ultimately result in the variation in lactation performance response.in addition,the difference in the metabolic utilization of Met by the mammary gland may affect the mammary AA uptake and utilization,resulting in the difference response in lactation performance.These results from the current study can provide knowledge for in-depth understanding of the effect of Met on lactation performance in dairy cow and contribute to the realization of precision feeding in the future. |
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