Effects Of Prepartum Dietary Anionic Salt And Calcium Supplementation On Calcium Homeostasis And Lipid Metabolism Of Transition Dairy Cows

Transition period is a critical stage in production cycle of dairy cow.Owing to the influence of huge physiological changes during this period,for instance,rapid fetal development,parturition and lactation initiation,nutrients intake can be suppressed and not enough to meet requirement of nutrients.Thus cows are prone to negative energy balance(NEB),bringing about mobilization of body tissue,especially adipose tissue.However,hepatic damage and accumulation of ketone bodies may result from excessive lipid mobilization which make ketosis and fatty liver become high incidence diseases among transition cows.Ca plays an important role in muscle contraction,and cell signal transduction.Hypocalcemia that frequently occurred around parturition can further affect postpartum feed intake recovery,postpartum BW loss and incidence of other diseases such as ketosis,suggesting a potential relationship between blood Ca and body tissue mobilization.Anionic salt supplementation in prepartum diet has been reported to improve postpartum blood calcium of dairy cows,but how anionic salt and its combination with high Ca addition act on blood calcium homeostasis to regulate calcium metabolism,and affect lipid metabolism of transition dairy cows need further research.Therefore,taking transition Holstein cows as research object,this study was carried out to compare production performance,blood metabolites and Ca-regulating hormone of cows with different postpartum blood Ca levels.Further investigation about the effects of prepartum anionic salt and Ca supplementation on the functioning pathway that anionic salt and Ca supplementation regulate calcium metabolism through Ca homeostasis mechanism,and their further effects on lipid metabolism of transition dairy cows was conducted in order to improve metabolic adaptation to transition period.Exp.1 A comparative study on production performance,blood metabolites and calcium-regulating hormones of transition cows with different serum calcium levels following parturition.Among 30 Holstein cows with parity=3,body weight of 811.7±72.1 kg being selected as research animals,8 detected with serum Ca levels in range of 1.38?2.0 mmol/L at 24 h postpartum were divided into low blood Ca group(LC,n=8).According to similar initial body weight(BW)and body condition scoring(BCS),8 cows with normal serum Ca levels were selected as normal serum calcium group(2.0?2.5 mmol/L,NC,n=8).The trial period for each cow was from-28 d relative to expected calving date to 28 d postpartum.Same close-up and lactation TMR diet were fed to both groups.From our results:(1)The SCH(Subclinical Hypocalcemia)incidence among experimental cows was26.67%.BW loss of LC cows in post-perinatal period was 24.76 kg higher than that of NC,and milk yield was significantly lower(P<0.05).(2)Among calcium-regulating hormones,serum 1,25(OH)₂D₃ level of LC cows at 24h postpartum was significantly lower than NC(P<0.05),and strong ions difference(SID)was significantly higher(P<0.05).(3)After calving,serum cholesterol(CHOL),total protein(TP)and globulin(GLB)levels of LC dairy cows were significantly lower than those of NC(P<0.05),and non-esterified fatty acid(NEFA)and hydroxybutyric acid(BHBA)were significantly higher(P<0.05).Correlation analysis showed that serum Ca level in transition period was negatively correlated with NEFA and BHBA levels(r=-0.49,P<0.01;r=-0.26,P<0.05).These results suggest that subclinical hypocalcemic cows had lower production performance,higher perinatal diseases incidence,lower levels of 1,25(OH)₂D₃ and higher SID.Postpartum blood Ca level was significantly negatively correlated with lipid metabolites NEFA and BHBA,and cows with low serum calcium showed higher lipid mobilization and ketone production.Exp.2 Effects of prepartum dietary anionic salt and calcium supplementation on production performance,calcium homeostasis and lipid metabolism of transition dairy cows.84 Holstein late pregnant dairy cows with parity=2?5 and BW 771.70±94.08 kg were selected and divided into 28 block as randomized block design,according to milk yield of previous lactation,initial BCS,parity and date relative to expected calving.3 cows in each block were randomly assigned into three different prepartum diet groups:control group(CON,basal diet):DCAD+10.11 m Eq/100 g,Ca 0.4%,anionic salt group(ND,adding anionic salt):DCAD-24.40 m Eq/100g,Ca 0.4%,anionic salt high Ca group(NDCA,with anionic salt and Ca supplementation):DCAD-24.44 m Eq/100g,Ca 1.95%,containing 28cows in each group.Same lactation TMR was fed to all cows after parturition.Trial period for each cow was from 28 d before expected calving date to 28 d postpartum.From our result,(1)Prepartum DMI of NDCA cows was significantly higher than that of ND(P<0.05).At postpartum stage,compared with CON,ND diet increased DMI by 5.64%.NDCA increased DMI by 7.65%(P<0.05),which also significantly enhanced nutrient intake of NE_L,CP,NDF,ADF,Ca,P(P<0.05)and apparent digestibility of gross energy(GE),DM,CP(P<0.05).Cows consumed NDCA and ND diets had lower post-perinatal BW loss which were reduced by 33.60%and 24.19%when compared with CON.And BCS loss in NDCA was significantly lower than other groups(P<0.05).Colostral fat and lactose percentages of NDCA cows were significantly increased(P<0.05),and SCC was decreased(P<0.05).Milk lactose content of cows in ND and NDCA was significantly increased and SCC was decreased(P<0.05).Post-perinatal milk yield of NDCA cows was significantly higher than other groups(P<0.05).(2)The blood Ca level of cows fed with ND and NDCA diet was significantly increased during transition period(P<0.05).Incidence of subclinical hypocalcemia(Ca<2.0 mmol/L)was decreased(CON 26.39%,ND 17.48%,NDCA 18.75%)and milk fever incidence was lowest in NDCA.The prepartum urinary Ca excretion was significantly increased by ND and NDCA(P<0.05).ND,NDCA cows had urinary p H in range from 5.5 to 6.0 prepartum and at calving,and significantly lessened blood HCO₃^-,SID and p H(P<0.05),resulting in change of acid base balance.Among Ca-regulating hormones,PTH and PTH related peptide(PTHr P)was not affected by ND or NDCA,but 1,25(OH)₂D₃ and vitamin D binding protein(VDBP)were significantly higher than CON(P<0.05).Bone mobilization activity biomarkers:collagen type?c-terminal peptide(CTX I),hydroxyproline(HYP)at 24 h postpartum were significantly greater(P<0.05),and BGP:CTX I reduced in ND and NDCA(P<0.05).Meanwhile,Ca apparent digestibility was significantly increased in ND and NDCA(P<0.05).(3)Postpartum NEB of cows in ND and NDCA was significantly increased(P<0.05),and EB of NDCA cows at 1 wk postpartum was 17.66%higher than CON and 6.37%higher than ND.Among lipid metabolism related hormones,Adiponectin(APN,P<0.05)was decreased and Insulin-like Growth Factor I(IGF-I)hormone secretion were increased significantly by ND and NDCA.In addition,NDCA significantly increased Insulin(INS,P<0.05)and decreased Glucagon(GC)secretion(P<0.05).NEFA levels were significantly lower in ND and NDCA(P<0.05).(4)Lipogenesis related enzymes:Fatty Acid Synthase(FAS)and Acetyl-coenzyme Carboxylase(ACC)activities were significantly increased in ND and NDCA(P<0.05),and ACC of NDCA cows was the highest among treatments(P<0.05).Among lipid oxidation and lipolysis related enzymes,activities of Hormone Sensitive Lipase(HSL)and Acyl Coenzyme A oxidase(ACOX)of ND and NDCA cows were significantly reduced(P<0.05),and activities of HSL and ACOX in NDCA were lower than other groups(P<0.05).Moreover,in fatty acids oxidation,fatty acid activation molecular and enzyme:coenzyme A and Acyl Co A synthetase(ACS),transporters:carnitine and acylcarnitine,and oxidation products:acetyl Co A,BHBA and ACAC(acetyl acetate)concentration were significantly reduced in NDCA(P<0.05).For liver lipid transport lipoprotein,NDCA cows had greater very low density lipoprotein(VLDL)and apolipoprotein(Apo B100)levels(P<0.05),suggesting enhancement of hepatic lipid transportation.NDCA further reduced liver impairment biomarkers,alkaline phosphatase(ALP),aspartate aminotransferase(AST),total bilirubin(TB)levels(P<0.05).(5)As for immunity,postpartum serum Ig A and M levels of cows consumed ND and NDCA were significantly increased(P<0.05).Lipopolysaccharide(LPS)and inflammatory cytokines Interleukin 1?(IL-1?),IL-6 and Tumor Necrosis Factor?(TNF-?)were significantly reduced(P<0.05).NDCA further reduced them on 7 d postpartum(P<0.05).Cows fed with NDCA had the lowest incidence of diseases related to hypocalcemia and infection and adverse health score among treatments.The above results shows that prepartum anionic salt supplementation could improve postpartum feed intake recovery and alleviate NEB of transition dairy cows.Addition of anionic salt with high Ca significantly increased postpartum DMI,nutrients intake and digestibility.BW and BCS loss were thereby reduced and lactation performance was improved.Periparturient blood Ca was increased significantly and occurrence of hypocalcemia was reduced by anionic salt addition.It changed acid-base balance of body fluid and promoted hydroxylation of 1,25(OH)₂D₃ in Ca homeostasis mechanism,the latter further alleviated Ca decline of transition cows from bone Ca mobilization and Ca absorption in digestive tract.With increase on lipogenesis hormones and enzymes,anionic salts improved lipogenesis activity.Regarding to lipolysis,anionic salt addition reduced lipolysis hormones and enzymes activities,product NEFA level and fatty acid activation.Moreover,anion salt with high Ca further reduced lipolysis enzymes,as well as transporter,enzymes,ketones production in fatty acid oxidation process.It also increased hepatic lipid transporter and alleviated liver impairment.The anionic salt addition consequently enhanced immune function and reduced inflammation of transition cows.The incidence of postpartum diseases incidence was lowest in cows fed with anionic salt with high Ca addition.Exp.3 The effects of prepartum dietary anionic salt and calcium supplementation on serum metabolome of transition dairy cows.Based on the results of improvement of NEB and lipid metabolism in transition dairy cows in Exp.2,the objective of present study was to further explore the internal metabolic pathway about effects of dietary anionic salt and calcium addition on lipid metabolism of dairy cows through metabolomics analysis.8 cows in CON,ND and NDCA groups were randomly selected for this experiment,and serum metabolomics of experimental cows at 24h postpartum were analyzed by LC-MS method.From our results,(1)ND and NDCA significantly reduced serum levels of long chain fatty acids(LCFA,P<0.05).NDCA diet significantly affected the Glycerophospholipid metabolism pathway(P<0.05)where choline and 1-Acyl-sn-glycero-3-phosphoserine were reduced(P<0.05),and Glycerophosphocholine was significantly higher(P<0.05)which are involved in transportation of hepatic lipid.It reduced fatty acid metabolites Methylbutyryl Carnitine,3-Hydroxyl-3-Methylglutaric acid and BHBA levels as well(P<0.05).Compared with ND,NDCA diet further reduced serum LCFA:Sebacic acid,10z-19 acid concentrations significantly(P<0.05).(2)Postpartum Pantothenate,Co A biosynthesis pathway of ND cows was significantly influenced.Serum Pantothenic acid involved in fatty acids oxidation was significantly decreased(P<0.05),while Niacinamide involved in lipogenesis were significantly increased(P<0.05)by ND and NDCA diets.(3)NDCA significantly affected Tricarboxylic acid metabolism pathway(TCA)and Glyoxylate and Dicarboxylate metabolism pathway(P<0.05)where concentrations of Aconite,Citric acid,Succinic acid and Malic acid were significantly increased(P<0.05).The metabolic pathway of TCA and Glyoxylate and Dicarboxylate metabolism was strengthened contributing to utilization of fatty acids oxidation products.(4)ND and NDCA diets significantly reduced serum levels of Alanine and amino acid degradation metabolites(P<0.05),and affected Arginine,Proline metabolic pathway significantly(P<0.05).The Urea cycle was weaken where Arginine and Cutrilline,N(2)-Acetyl-L-Ornithine concentrations were decreased in NDCA.NDCA diet further increased Methionine and Glutamine levels(P<0.05),and promoted synthesis of Tyrosine(P<0.05).(5)Bromarker analysis showed that 5 metabolites including Glutamine,Pyrrolidone carboxylic acid,Uracil,Uridine and Pantothenic acid could be used as biomarkers for the effect of anionic salt addition to prepartum diet on postpartum metabolism pf daity cows.Creatine,Palmitoyl glycine,Glycerolipatidylcholine,L-Alanine,L-Methionine,N(2)acetyl-l-Ornithine,Nicotinamide and Pantothenic acid could be the biomarkers for the effect of anionic salt and high Ca supplementation on postpartum metabolism of dairy cows. These results suggested that anionic salt reduced LCFA derived from lipid mobilization,decreased pantothenate biosynthesis in Pantothenate,Co A biosynthesis pathway which is precursor of Co A involved in fatty acid oxidation activation.Anionic salt with high Ca addition further reduced serum LCFA concentrations and their oxidation products,and enhanced Glycerophosphocholine metabolism in glycerophospholipid metabolic pathway which are involved in hepatic lipid transportation.Meanwhile,it promoted TCA cycle metabolic pathway and glyoxalate-dicarboxylate metabolic pathway,contributing to utilization of lipid oxidation products.In conclusion,subclinical hypocalcemia could reduce production performance,increase perinatal diseases incidence,and deteriorate lipid mobilization of transition dairy cows.With anionic salt supplementation to prepartum diet,feed intake recovery in fresh period and milk quality was increased.The anionic salt with high Ca addition further improved postpartum nutrients intake and apparent digestibility,thus lessened postnatal BW and BCS loss and increased lactation performance.Anionic salt changed acid-base balance,and promoted hydroxylation of 1,25(OH)₂D₃ in Ca homeostasis mechanism,resulting in increased bone reabsorption and Ca apparent digestibility.Anionic salt addition replenished periparturient blood Ca drop of transition dairy cows via bone Ca mobilization and Ca absorption in digestive tract.Incidence of hypocalcemia was lowered.Further analysis revealed that anion salt supplementation alleviated postpartum NEB.Accompanied with increases of lipogenesis related hormones secretion,enzyme activities and Niacinamide,the lipogenesis was promoted by anionic salt supplementation.On the other hand,lipolysis regulating hormones,enzymes and product LCFA were reduce by anionic salt.Meanwhile,pantothenate,Co A Biosynthesis pathway producing pantothenic acid and Co A involved in fatty acid oxidation activation was lowered.Consequently,lipid mobilization was suppressed.In addition,cows given anionic salt with high Ca supplementation had further lower lipolysis enzymes activities,LCFA,as well as transporters,oxidation enzyme activity,products and ketone levels in fatty acids oxidation process.The metabolic pathway analysis shown that it improved lipoprotein and Choline Glycerophosphocholine metabolism in glycerophospholipid pathway involved in hepatic lipid transportation,and TCA cycle metabolic pathway and glyoxalate-dicarboxylate metabolic pathway involved in utilization of fatty acid oxidation products.As a result,it alleviated accumulations of hepatic impairment and ketones.With improvements of postpartum Ca homeostasis and lipid metabolism of transition dairy cows,immunity was enhanced and inflammation was reduced.Peripartum disease incidences were lowest in cows fed with anionic salt with high Ca addition which promoted healthy adaptation to transition period of dairy cows.