Evaluation Of N-carbamoylglutamate As Arginine Enhancer And Its Effect On Rumen Fermentation,Lactation Performance And Nitrogen Utilization In High Yielding Lactating Dairy Cows

High yielding animals fed diet rich in protein impairs the ureagenesis and causes ammonia toxicity, consequently decreasing feed intake and milk production in dairy cows. The arginine (Arg) plays an important role in ureagenesis. As a precursor of polyamines, creatine and nitric oxide (NO), it exerts many beneficial effects on animal body and enhance angiogenesis, lactogenesis, finally leading to the improvement in nitrogen (N) metabolism, reproduction, lactation, immunity and growth of animals. Due to its high rumen degradation and its high price feeding rumen protected Arg seems to be uneconomical. Alternatively, NCG is structural analogue of N-acetylglutamate (NAG), which activates key enzymes carbamoyl phosphate synthase1(CPS1) of both urea cycle and Arg synthetic pathway) has been potentially substituted for Arg in non-ruminant animal. However, limited information is available on efficacy of NCG in dairy cows.1. Development of methods for Arginine and N-carbamoylglutamate determination in rumen fluid (Trail.1)No method has been available for the determination of NCG in rumen fluid. The Arg could be analyzed by amino acid (AA) analyzer, but it is expensive and not convenient. The purpose of this study was to build up simple and accurate method for the determination of Arg concentration in rumen fluid by spectrophomter and NCG by Ion chromatography. For determination of Arg in rumen fluid a Sakaguchi reaction method was used. Temperature, time and absorbance were optimized in the procedure based on Sakaguchi reaction. Color consistency remained4-6min. The optimum temperature (0-5℃) was observed for maximum optical density (0.663) at wave length of (500nm). Minimum Arg that could be determined in rumen fluid by spectrophotometer was4-5μg/mL. No significant (P>0.05) difference was observed between two results derived from spectrophotometer and AA analyzer methods. For the determination of NCG in rumen fluid an Ion chromatography method was used. One mmol/L with or without NCG was incubated in rumen fluid for0,2,4,6,12and24h. The effects of the different chromatographic parameters on the separation were investigated. Detection limits of NCG in rumen fluid during different incubation periods were175.3±1.63,165.9±1.35,164.9±4.28,163.2±1.92and156.4±4.27μg/ml during2,4,6,12and24hours. The relative standard deviation of the retention time and peak area were less than0.88%and4.71%, respectively. The developed method is suitable for determination of NCG in rumen fluid. Thus, the spectrophotometer method of Arg determination in rumen fluid based on Sakaguchi reaction is easy, accurate, and economical and could be useful in learning Arg metabolism in the rumen. To our knowledge, this is first study for determination of Arg by spectrophomter and NCG by Ion chromatography in rumen fluid.2. Evaluation of Arginine and N-carbamoylglutamate degradation in rumen and their effect on rumen fermentation in vitro (trai.2.)The purpose of this study was to determine the rumen degradation of NCG in comparison with Arg, and to investigate their effect on rumen fermentation in in vitro. Rumen fluids were collected from3rumen-fistulated cows and then incubated with Arg or NCG at1mmol/L in a glass syringe system at39℃for24h. The control treatment was given neither Arg nor NCG. Gas production was recorded, and pH at2,4,6,12, and24h was determined. At12and24h, the measurements were also made for ammonia N, volatile fatty acids and microbial crude protein (MCP) yield on purine quantification basis. At24h, the proportion of Arg and NCG degradation in rumen fluid was100.0and17.8%, respectively. Gas production and the acetate to propionate ratio increased in groups treated with Arg and NCG, compared with the control (P<0.01). Ammonia N concentration was higher (P<0.01) in the Arg group than in the NCG and control groups. The results of pH value was significantly altered among the treatment (P<0.01) at12and 24h, but it was with in the normal range of (6.41-6.54). The MCP concentration diminished in Arg and NCG groups, in comparison with the control (P<0.01). In conclusion, the effects of Arg and NCG on rumen fermentation were numerically relatively similar. Rapid degradation of Arg in rumen is a nutritionally wasteful process. Thus, Arg should be spared from rumen degradation, while NCG could be fed to ruminant without need for coating.3. The effects of N-carbamoylglutamate on lactation performance and nitrogen utilization in high yielding Chinese Holstein dairy cows (Trail.3)The objectives of this study were to see the effects of feeding NCG on blood metabolite, metabolizable protein, rumen MCP, milk production and N utilization in high yielding Chinese Holstein dairy cows. Sixty multiparous cows were blocked based on days in milk (78±17.3d; mean±SD) and milk yield (41.9±7.9kg; mean±SD) and were randomly allocated to receive one of4dietary treatments supplemented with50%of NCG at0(control),20,40or60g/d/cow, the actual predicted available amount being0,10,20and30g/d, respectively. Milk yield was recorded weekly. Dry matter intake, milk composition, plasma variables and urea N content in plasma, urine and milk were determined every other week. Blood samples were collected from the coccygeal vein and plasma was frozen at (-20℃) for later analysis. The spot urine samples were collected from all cows for determination of urea N and rumen MCP synthesis by purine derivates every other week. The metabolizable protein (MP) was measured by intestinal digestible protein by incubating the feed samples in the rumen for16h and analyzed protein content after Invitro incubation. The initial and final live body weight (BW) and body conditions score (BCS) were measured. The dry matter intake was not changed across the treatments. Cows fed20g/d NCG showed trends of (P<0.07) higher milk yield (40.2vs.38.1kg/d) compared to control, and significantly (P<0.01) increased the content and yield of milk protein. Addition of20g/d NCG showed higher (P<0.08) trends of milk total solid content, and30g/d NCG significantly (P<0.05) has higher milk lactose content than control. Yield of milk fat, lactose and total solid and somatic cell count were not different among all treatments. No significant differences were observed in the plasma variables of (total protein, β-hydroxybutyric acid, non-estertified fatty acid, triglyceride, glucose, albumin and globulin) across the treatments. Dietary NCG linearly increased (P<0.01) plasma nitric oxide and decreased ammonia N. Compared with control, plasma Arg concentration was increased by1.13,10.4and16.0%in cows fed10,20and30g/d NCG, respectively. The ratio of Lys:Met were3.82,3.88,3.12and3.32in cows fed0,10,20and30g/d NCG respectively. The MCP synthesis in rumen were not significantly changed across the treatments, but MP utilization showed quadratic higher trends (P <0.06) in cows fed20g/d than that of control. Urea N concentration in milk, plasma and urine linearly decreased (P<0.03) with NCG addition, with the lowest value for20g of NCG/d. The N utilization showed higher trends (P<0.07) in cows fed20g/d NCG (0.289vs0.261) compared with control, indicating reduce N excretion in environment. Cows fed20g/d NCG linearly increased (P<0.01) the BCS compared to control, respectively. It is inferred that supplementation of20g/d NCG altered the plasma metabolite and milk contents, thereby improved lactation performance and N utilization of high yielding dairy cows.In summary, this study provides convenient methods for Arg analysis in rumen fluid by spectrophotometer and NCG determination by Ion chromatography. Because of its lower rumen degradation, NCG can be used in ruminant diet without need for protection. Supplementation of NCG to high yielding Chinese Holstein dairy cows increased yield of milk and milk composition and improved N utilization, which are attributed to alteration in plasma metabolite and balanced absorbable AA profile through efficient urea cycle regulation and partially higher MP utilization. The optimum level obtained is at20g/d NCG in basal diet. The higher level of30g/d might have some deleterious effect on dairy cows performance...