Choline And Methionine Regulate Nutrient Balance And Health Of Transition Dairy Cows: Responses And Potential Mechnisms

The negative multi-nutrient balance is one of the most typical characteristics in transition dairy cows.To meet the nutrient requirements,cows usually metabolize the body deposition of fat,protein and other nutrients.The mobilization can lead to the occurrence of metabolic diseases,such as ketosis and fatty liver,and induce oxidative stress and immunosuppression,thereby decreasing the liver function and whole body health.Due to the beneficial effects in regulating the hepatic one-carbon circle,lipid oxidation and transport,and body health,the choline and methionine(Met)has attracted more and more attention.However,more experiments are still needed for some unclear points in existed studies: 1)temporal dynamics of nutrient balances,physiological biomarkers and associated comprehensive indexes;2)the rumen-protected choline(RPC)and rumen-protected methionine(RPM)are supplemented into the diets without considering the dietary CP level.Moreover,a systems research with multi-parameter,multi-level and enough-depth is highly needed;3)the potential mechanisms and key signaling pathways are not entirely known.Hence,we systematically assessed the temporal dynamics of nutrient balance and physiological indicators in transition dairy cows(Exp.1).Accordingly,the regulation of nutritional metabolism by dietary RPC and RPM were studied in various perspectives,exploring maternal energy metabolism and health(Exp.2),maternal protein balance and postpartum lactation performance(Exp.3)as well as body size indexes and nutritional physiology of neonatal calves(Exp.4).Afterwards,we established an in vitro NEFA-induced LO2 hepatocyte model(Exp 5),then the signaling pathways and regulatory factors were studied(Exp.6).The above trials aimed to provide theoretical basis and technological reference for the applications of RPC and RPM in transition dairy cows.Exp.1 The assessment of nutrient balances and temporal dynamics of blood metabolic indicators in transition dairy cowsThis trial was conducted to better understand the dynamic adaptations of nutrient balances and physiological indicators of the transition dairy cows,as well as to provide basic data for follow-up trials and nutritional practice.Fourteen multiparous healthy Chinese Holstein dairy cows were chosen after drying milk,and were given the same diet during thewhole dry period(far-off and close-up),containing 1.51 Mcal/kg net energy for lactation(NEL)and 11.92% CP(dry matter basis,the same as below).The dietary physically effective neutral detergent fiber(peNDF)and rumen degradable starch(RDS)were peNDF1.18 =28.71%,peNDF8.0 = 18.58%,and RDS = 20.17%,respectively.Therefore,the dietary carbohydrate balance index(CBI)could be calculated as CBI1.18 = peNDF1.18/RDS = 1.42 and CBI8.0 = peNDF8.0/RDS = 0.92,respectively.After calving,cows were transferred to a lactation diet containing 1.69 Mcal/kg NEL and 15.02% CP,and peNDF1.18 = 24.24%,peNDF8.0 = 16.60%,RDS = 18.06%,CBI1.18 = 1.34,and CBI8.0 = 0.92,respectively.The far-off period was an adaptive phase for the diet,and the trial period lasted from prepartum period(close-up,-21 d to 0 d)to the postpartum period(0 to 21 d).Samples were collected daily or weekly for further analysis,calculation and model prediction.We found that: 1)the balance values of NEL,metabolizable protein(MP)and metabolizable glucose(MG)during the prepartum period stayed above zero.The negative balance values appeared after calving,and the nadir appeared at 7 d,during which the balance values for NEL,MP and MG were5.76 Mcal/d,254.42 g/d,and 387.54 g/d,respectively;2)With the approaching of parturition,dietary intakes of DM,NEL,MP and MG kept decreasing,and compared with-21 d,the intakes at 0 d decreased by 39.27%,39.30%,39.45% and 32.82%,respectively.Ditetary intakes came to recover after parturition;3)From-21 to 21 d,the blood metabolic parameters and comprehensive indexes were in accordance with quadratic curves,and extreme points appeared at 0 or 7 d.In these days,body reserves were seriously metabolized,and the neuroendocrine were significantly changed.The liver function and body health were dramatically decreased around calving.The above results indicated that the negative balances of NEL,MP and MG only appeared after calving;however,the metabolic indicators,organ functions and body health started changing before calving.Hence,regulatory strategies are absolutely essential for prepartum dairy cows.Exp.2 Effects of rumen-protected choline and rumen-protected methionine on energy balance and health of transition dairy cowsThe objective of this trial was to study the effects of dietary RPC(15 g/d,calculated as choline chloride)and/or RPM(15 g/d,calculated as DL-Met)supplementation on energy balance(EB)and health of transition dairy cows.Forty-eight multiparous healthy Chinese Holstein dairy cows were chosen after drying milk,and were given the same basal diet before calving.After calving,another basal diet for lactation were fed to cows.The ingredients and nutrient composition for both prepartum and postpartum diets were the same as Exp.1.With the approaching of-21 d,cows were pair-assigned into one of the four groups with 12 cowsper group.Groups were Control(basal diet),RPC + basal diet,RPM + basal diet,and RPC +RPM + basal diet.The trial lasted for 42 days,from-21 d to 21 d.Supplementation of RPC increased dry matter intake(DMI)of transition dairy cows at 14 d,while RPM did not affect the DMI.Both RPC and RPM promoted the postpartum EB,but the prepatum EB stayed unchanged.Both of the additives decreased the plasma concentrations of non-esterified fatty acids(NEFA)and β-hydroxybutyric acid(BHBA),and increased the concentrations of glucose and very low density lipoprotein(VLDL).Supplementation of RPC improved the liver activity index(LAI)and liver functionality index(LFI)of transition dairy cows,and tended to improve the revised quantitative insulin sensitivity check index(RQUICKI).The RPM showed a tendency to improve LFI and RQUICKI.Both RPC and RPM increased the total antioxidant capacity(T-AOC),the glutathione peroxidase(GSH-Px)activity and vitamin E concentration in the plasma,as well as the CD4+ to CD8+ ratio(CD4+/CD8+)of T lymphocyte in the peripheral blood.Besides,RPC and RPM decreased the plasma concentrations of malondialdehyde(MDA),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α).Results demonstrated that dietary supplementation of RPC and RPM could attenuate the negative energy balance,and enhance the liver function and health of transition dairy cows.Comprehensively analyzing the above data and some other results in chapter Ⅲ,the RPC is better than RPM in regulating the energy metabolism of transition dairy cows.Exp.3 Effects of rumen-protected choline and rumen-protected methionine on protein balance,amino acid metabolism and postpartum lactation performance of transition dairy cowsThis experiment was conducted to explore the effects of RPC and/or RPM supplementation on protein balance,amino acid metabolism and postpartum lactation performance of transition dairy cows.The experimental design and animal management were the same as Exp.2.Considering the whole transition period,the RPM tended to promote the MP balance of dairy cows,while RPC showed no effect on MP balance.In addition,both RPC and RPM tended to promote MP balance at 14 d.Supplementation of RPM decreased plasma concentrations of 3-methylhistidine(3-MH)and urea nitrogen.The RPC decreased plasma 3-MH level,but did not affect urea nitrogen.Both additives increased or tended to increase plasma total amino acids(TAA),total essential amino acids(TEAA),Met,leucine(Leu)and taurine(Tau).Both RPC and RPM increased 4% fat-corrected milk(FCM)yield(22.70,23.42,23.31 and 23.94 kg/d for Control,RPC,RPM and RPC+RPM,respectively)and milk fat percentage.Supplementation of RPM could increase milk protein percentage,while RPC did not change milk protein percentage.Besides,the RPM increased theconcentration of histidine(His),and showed a tendency to increase milk Met;the RPC tended to increase milk His concentration,but the effect on milk Met concentration was not significant.In summary,dietary supplementation of RPC and RPM could promote the protein balance,decrease the mobilization of body protein,modulate amino acid metabolism,and improve postpartum lactation performance of transition dairy cows.Furthermore,the regulatory effects of RPM on amino acid metabolism seemed to be slightly better than RPC.Exp.4 Effects of prepartum supplementation of rumen-protected choline and rumen-protected methionine on body measurement parameters and blood metabolic biomarkers of transition dairy cowsTo explore the potential benefits of maternal RPC and/or RPM supplementation on fetus growth and development,nutritional physiology and health,on the basis of Exp.2 and 3,we analyzed the calf birth weight and body size index,and the blood biomarkers of physiological metabolism,liver function,antioxidant and immune status of neonatal calves.The experimental design and the management of the prepartum maternal dairy cows were the same as Exp.2.After the cows calved,the calf birth weight and body size were measured,and blood sample were taken from the jugular vein before they were available to colostrum.The RPC increased the rump width,while the RPM increased the hip width.Neither RPC nor RPM affected the calf birth weight and other body size parameters.Supplementation of RPC tended to increase plasma glucose concentration,while the RPM showed a tendency to increase plasma urea nitrogen.Plasma concentrations of NEFA,BHBA,VLDL and 3-MH were not changed by RPC or RPM.The RPM improved plasma levels of Met and cysteine(Cys)in neonatal calves,and tended to improve the Tau level.The RPC tended to improve the levels of Met and Cys,but did not influence the plasma Tau level.Both RPC and RPM increased or tended to increase the plasma concentration of insulin-like growth factor 1(IGF-1),but neither of them influence the other endocrine parameters.Maternal supplementation of RPC reduced the plasma TNF-α concentration,and tended to improve the plasma T-AOC as well as the CD4+/CD8+ of peripheral T lymphocytes in neonatal calves.The maternal RPM presented a tendency to reduce TNF-α concentration and increase T-AOC in the plasma of neonatal calves,but had no effect on the CD4+/CD8+.Therefore,maternal supplementation of RPC and RPM in prepartum dairy cows could: 1)modulate the protein metabolism,and change the plasma amino acid profiles of neonatal calves;2)improve plasma IGF-1 and glucose(only RPC)concentration;3)improve the antioxidant status and immune function.Whether the above alterations could benefit the subsequent growth and development of calves needs to be further discovered.Exp.5 Effects of NEFA and BHBA on energy and lipid metabolism,oxidative stress and inflammatory response of in vitro cultured LO2 hepatocytesIn this trial,we studied the effects of BHBA and NEFA on hepatic energy and lipid metabolism,oxidative stress and inflammation using LO2 hepatocytes,where an in vitro NEFA-induced hepatocyte model was established for further research.Firstly,the LO2 hepatocytes were treated with different BHBA or NEFA doses for 24 h(6 replicates for each treatment),then cell proliferation,redox status,and gene expressions of adenosine monophosphate-activated protein kinase α(AMPKα)and TNF-α were detected.The doses of BHBA and NEFA were 0,0.5,1.0,1.5,2.0,and 4.0,respectively.With the increasing concentrations of BHBA and NEFA,the cell proliferation,antioxidant capacity and AMPKαexpression were decreased gradually,while the TNF-α expression was increased.Accordingly,1.5 mmol/L BHBA and 1.5 mmol/L NEFA were selected for the subsequent experiments.Secondly,the other hepatocytes were divided into 4 groups(Control,1.5 mmol/L BHBA,1.5mmol/L NEFA,and 1.5 mmol/L BHBA+1.5 mmol/L NEFA)with 6 replicates per group.Cells were incubated for 24 h for further analysis.Stimulation by 1.5 mmol/L BHBA,1.5 mmol/L NEFA or both,to a certain degree,changed the gene expressions of AMPKα,liver X receptorα(LXRα),peroxisome proliferator-activated receptor α(PPARα)and sterol-regulatory element binding protein-1c(SREBP-1c),thereby the downstream target genes,involved in the process of fatty acid oxidation,synthesis and TG transport,were decreased.Besides,both BHBA and NEFA inhibited the Nrf2(nuclear factor erythroid 2-related factor 2)antioxidant signaling pathway,and activated the NF-κB(nuclear factor kappa B)inflammatory signaling pathway.Results indicated that high concentrations(1.5 mmol/L)of BHBA and/or NEFA could inhibit the energy and lipid metabolism,decreased cellular antioxidant capacity,and induced inflammatory response in LO2 hepatocytes.As a consequence,the 1.5 mmol/L NEFA were chosen to establish the LO2 hepatocyte model,simulating the hepatic environment during body fat mobilization.Exp.6 The regulation of energy and lipid metabolism by choline and methionine in the NEFA-induced LO2 hepatocyte modelOn the basis of previous animal experiments and the LO2 hepatocyte model,this trial was conducted to investigate the effects of choline(as choline chloride,the same as below)and Met on expressions(mRNA and protein)of key genes and regulators in the process of carbohydrates and lipid metabolism of hepatocytes.The other objective was to confirm the role of AMPKα signaling pathway in the above processes.We used a culture medium without containing choline and Met to precisely control the concentrations of choline and Met.Beforetreating,cells were cultured in the customized medium(without fetal bovine serum)for 6 h starvation.After a concentration screening test(see the 7th chapter),50 μmol/L choline and300 μmol/L Met were selected for the follow-up trials.We set up 4 treatments with 6replicates per treatment,Control(without choline and Met),choline(50 μmol/L,without Met),Met(300 μmol/L,without choline),and choline 50 μmol/L+Met 300 μmol/L.To explore the role of AMPKα,the BML-275(10 μmol/L,an AMPKα inhibitor)was added into the culture system in another trial(6 replicates as well).All the cells were treated for 24 h,after which biochemical parameters and gene expressions(mRNA and protein)were detected.We found that: 1)both choline and Met supplementation promoted the protein phosphorylation of AMPKα(p-AMPKα/AMPKα),increased the PPARα expression,and reduced the expressions of LXRα and SREBP-1c,thereby changed the expressions of downstream target genes.The expressions of key genes associated with fatty acid oxidation and apolipoprotein synthesis were increased,and genes involved in the synthesis of fatty acids and ketone bodies were declined;2)choline and Met enhanced the cellular antioxidant capacity of LO2 hepatocytes,activated the Nrf2 pathway,and reduced the mRNA and protein expressions of TNF-α;3)when 10 μmol/L BML-275 was added,the p-AMPKα/AMPKα was significantly reduced,and regulatory responses of choline and Met on the energy and lipid metabolism(regulatory factors and target genes)was remarkably decreased.To sum up,choline and/or Met could modulate the energy and lipid metabolism through AMPKα signaling pathway and some regulatory factors(LXRα,PPARα and SREBP-1c),thereby promoting fatty acid oxidation and apolipoprotein synthesis and inhibiting the synthesis of fatty acids and ketone bodies in NEFA-induced LO2 hepatocyte model.Besides,both choline and Met benefitted the hepatic redox status of hepatocytes,and might attenuate the oxidative and inflammatory injury.In conclusion,choline and Met could activate AMPKα signaling pathway and change the expressions of some regulatory factors(LXRα,PPARα and SREBP-1c)to modulate the energy and lipid metabolism in NEFA-induced LO2 hepatocyte model,decreasing the lipid accumulation of hepatocytes.Dietary supplementation of RPC and RPM could,to a certain extent,modulate the metabolic processes of energy,lipid and amino acids.These two rumen bypass additives promoted the liver and whole body health,alleviated the negative balances of NEL and MP,and improved postpartum lactation performance in transition dairy cows.Moreover,the RPC and RPM benefitted the nutritional physiology of neonatal calves.