Lactation Performance,health And Reproduction Of Dairy Cows Influenced By Supplementation Of N-carbamoylglutamate And Mechanism Involved

Arginine(Arg)is an essential amino acids(AA)for dairy cows and plays an important role in regulating biological functions.However,the natural Arg would be degraded in the rumen,and it is expensive relatively and easy to produce antagonism with other AAs.Alternatively,N-carbamoylglutamate(NCG)is a structural analog of N-acetylglutamate,which is essential in endogenous Arg synthesis.Thus,NCG is regarded as an enhancer of Arg,and extensively studied in monogastric animals.However,limited systematic studies were conducted on NCG in dairy cows.Therefore,the objective of this study was to determine the effects of dietary NCG on milk production at different lactation stages of dairy cows.On this basis,the effects of NCG were evaluated on dairy cows and related mechanism were approached from the aspects of physiological metabolism,immune health and reproductive performance at transition period when the improvement effect was the best on lactation performance.The results obtained would provide basic and practical data for NCG application as a new nutritional strategy for the healthy feeding of dairy cows.PART1Lactation performance of dairy cows influenced by supplementation with NCG at different lactation stagesThirty transition,30 early-lactation and 30 mid-lactation dairy cows were selected for three feeding experiment,respectively.Each experiment was divided into two groups without(control)or with 20g NCG per day based on parity,body weight,body condition scores,and lactation performance of cows and was carried out by randomized blocks design.The transition experiment started at 3 week before calving and lasted until 3 weeks after calving.The experiment for early lactating cows started at 4 week and ended at 100 days in milk(14 week).The experiment period of mid-lactation was from 25 week to 34 week after calving.Dry matter intake(DMI)was recorded for two consecutive days every other week,and milk yield was recorded,and milk samples were collected for analysis of milk composition weekly.Blood samples were taken from the coccygeal vein of each cow and centrifuged for collection of plasma,and then analyzed for blood paramaters and AA composition.The results obtained are as followings.In transition cows,DMI tended to be greater(P=0.06)in cows fed NCG than in control.The yield of milk was greater in NCG-fed cows(P<0.01)than in control cows.Milk urea nitrogen concentration was lower(P<0.01)in the NCG cows than in the CON cows,but no differences were found in contents of milk protein and fat between two treatments.The concentrations of blood urea nitrogen(P<0.01),triglyceride(P=0.09),and ?-hydrobutyrate(P=0.05)in plasma were lower in the cows fed NCG than those of CON-cows.In contrast,the plasma concentrations of glucose(P=0.05),cholesterol(P=0.10),nitric oxide(NO,P<0.01)and NO synthase(T×W;P=0.06)were greater in the NCG-fed cows than those of CON cows.Compared with the CON cows,NCG-fed cows had greater mammary plasma flow(P=0.04)and AA supply(P<0.05),respectively.In the early lactation,no significant difference was observed in DMI and milk composition between two groups.However,the milk yield was higher after feeding NCG for 6 weeks in the NCG cows(P=0.06,T×W),with lower somatic cell counts in these cows(P<0.01).The plasma concentrations of total protein concentration(P=0.02)and globulin(P=0.04)were higher and alanine aminotransferase(P=0.06,T× W)was lower in the NCG-fed cow than the control,suggesting the improved liver function in the NCG-fed cows.In addition,the plasma concentration of NO(P=0.08)and NOS(P=0.05),and concentrations of Arg(P=0.06),Pro(P=0.04),Gly(P=0.08)and Ser(P=0.05)were higher or tended to be higher in the NCG group compared than control cows.No differences were observed in other AA and total AA between two groups.In mid-lactation cows,both DMI and milk yield were not affected by addition of NCG(P>0.05).The cows administered with NCG had an increased milk fat content(P=0.04).The milk protein content tended to be higher in the NCG group,compared with that of the control(P=0.08).The addition of NCG increased the concentration of NO(P<0.01)and total NO synthase(P<0.01);and the concentration of total bilirubin(P=0.07)tended to increase.The addition of NCG increased the AA content of dairy cows,with higher concentrations of Arg(P<0.01),Glu(P<0.01)and Pro(P=0.05).In short summary,the concentration of Arg and NO increased with NCG addition at all three lactation stages.However,responses of lactation performance to NCG addition were varying at different stages,which may be attributed to different AA requirements and physiological metabolism of the cows at different stages.Therefore,the effects of NCG on health and reproduction of perinatal cows was further investigated.PART 2Effects of dietary NCG on the health and immunity of transitional dairy cowsThe thirty transition cows in PART1 were used in the current study.The number of diseases of cows was recorded according to the diagnosis.Blood samples were collected from the coccygeal vein of each cow at week-3,-1,0(the calving date),1,and 3 relatives to calving date for analysis of biomarkers of inflammation and oxidative stress.In addition,the polymorphonuclear was collected in week 1 relative to calving day and analysed for the functional genes.Compared with that of CON-cows,the NCG-cows tended to be lower in the mastitis incidence(P=0.06).Serum amyloid protein A concentration was lower(P=0.04,T× W),concentrations of interleukin 6(P=0.08,T× W)and tumor necrosis factor-?(P=0.08,T×W)tended to be lower at week 1 and 3 in NCG-fed animals.Blood concentration of malondialdehyde(P=0.05,TXW)in week 0,1,and 3,and reactive oxygen species(P=0.04,T×W)in week 1 was lower in NCG-cows.The concentrations of glutathione peroxidase(P=0.05,T×W)and total antioxidant capacity(P<0.06,TxW)were higher in NCG-fed cows than in control.Expression of genes related to Arg and glucose transporters(SLC6A14,SLC7A1,SLC7A6,SLC7A7,SLC7A9,SLC2A1)as well as apoptosis/survival genes(FAS,CASP9,CASP2,BAK1)were not affected by the NCG.However,expression of S100A9(P=0.05)and MMP9(P=0.10)were down-regulated,while expression of ITGB2(P=0.08),XBPP1(P=0.06)and CLEC6A(P=0.03)were up-regulated in NCG-cows than in the control.Overall,these results indicated that feeding NCG during transition stage reduced the mastitis incidence by improving polymorphonuclear functions and alleviating inflammation and oxidative stress of postpartum dairy cows.PART 3Effects of dietary NCG on the reproduction of dairy cowsIn this part,the thirty transition cows used in PART2 were included.The birth weight of newborn calves was recorded,and the blood sample was collected from jugular vein at birth and 24 h after feeding colostrum for analysis of AA compositions,plasma variables,NO,NOS and IgG concentrations.On the calving day,the colostrum was collected to determine the concentration of IgG.Additionally,the placenta was collected,and analysed for the abundance of expression of functional genes relative to transporter of Arg,glucose and fatty acid,angiogenesis factors,and the mTOR signal pathway.In the cows fed NCG,birth weight of newborn calves was significantly higher(37.3 kg vs.32.2 kg;P<0.01),and plasma concentration of Arg tended to be higher(P=0.07),and concentration of IgG on day 1 post-calving was higher(P=0.04),in relative to the control.Addition of NCG improved the mRNA abundance of SLC2A3(P<0.01)and NOS3(P<0.01)of placenta and activated the mTOR signal pathway.To further investigated the mechanism with which NCG affects the reproductive performance of dairy cows,the plasma metabonomic was conducted.The main metabolic pathways that were affected by NCG were linoleic acid metabolism,alanine,aspartate and glutamate metabolism,arginine and proline metabolism,and the tricarboxylic acid cycle.In short summary,feeding NCG may increase birth weight of the newborn calves,which may be attributed to improved health of dairy cows,increased nutrient supply,and improved nutrient transport capacity of placenta,ultimately improved the health status of both transition dairy cows and the calves.In summary,the effects of dietary addition of NCG varied at different lactation stages,with the best improvement observed at the transition period of dairy cows.The improved lactation performance is attributed to the enhanced synthesis of Arg and NO,and consequently increased AA supply and imprived AA metabolism.Addition of NCG may improve the functions of polymorphonuclear and alleviate the inflammation and oxidative stress,leadin to the improved cows' health.Additional function of NCG included the promoted placental angiogenesis,increased glucose transportation and enhanced the mTOR signal pathway,which may improve AA and energy metabolism of the fetus,increase the body weight of calves and improve dairy cows reproduction.