Effect Of N-carbamylglutamate Research Articles

Effects of Dietary N-carbamylglutamate on Growth Performance, Apparent Digestibility, Nitrogen Metabolism and Plasma Metabolites of Fattening Holstein Bulls.

Simple SummaryIt is critical to find an effective and safe feed additive to improve the utilization of nitrogen by ruminants. N-carbamylglutamate (NCG), a structural analogue of N-acetylglutamate, may have the potential to improve the utilization of nitrogen by bulls. Therefore, the effects of adding different levels of NCG on the growth performance, nutrient digestibility, nitrogen metabolism and plasma metabolites of fattening Holstein bulls were investigated in this study. The addition of NCG increased concentrations of plasma Arg, Ile, Val, total essential amino acid and total nonessential amino acid, which in turn resulted in higher N utilization and CP digestibility for growth and, therefore, higher average daily gain in NCG-fed bulls.N-carbamylglutamate (NCG), a structural analog of N-acetylglutamate, improves nitrogen utilization in dairy cows. However, the effects of NCG on bulls are unknown. The purpose of the current research was to investigate the effects of adding different amounts of NCG on growth performance, nutrient digestibility, nitrogen metabolism and plasma metabolites of fattening Holstein bulls. Twenty-four Holstein bulls with similar body weights (BW, 408 ± 21.9 kg) and ages (450 ± 6.1 d; all mean ± SD) were selected for the feeding trial. After 2 weeks of adaptation, bulls were blocked by BW and age and subsequently randomly assigned to 1 of 4 groups: (1) CON group (control diet), (2) L group (supplementation with 20 mg/kg BW NCG), (3) M group (supplementation with 40 mg/kg BW NCG), or (4) H group (supplementation with 80 mg/kg BW NCG). The addition of NCG linearly and quadratically increased the average daily gain (CON vs. L vs. M vs. H = 1.03 vs. 1.19 vs. 1.40 vs. 1.26 kg/d) (p < 0.05), feed conversion ratio (CON vs. L vs. M vs. H = 11.92 vs. 9.22 vs. 7.76 vs. 8.62) (p < 0.05), crude protein digestibility (CON vs. L vs. M vs. H = 64.3 vs. 63.8 vs. 67.7 vs. 65.8%) (0.05 < p < 0.10), N retention (p < 0.05) and N utilization (p < 0.05) of bulls, whereas the contents of fecal N (0.05 < p < 0.10) and urinary N (0.05 < p < 0.10) in NCG-fed bulls linearly decreased compared with those in CON bulls. Bulls fed NCG showed a quadratic increased plasma nitric oxide (p < 0.05) concentration. Furthermore, Arg (p < 0.05), Ile (p < 0.05), Val (p < 0.05), Ala (p < 0.05), Glu (p < 0.05), Ser (p < 0.05), total essential amino acid (p < 0.05) and total nonessential amino acid (p < 0.05) concentrations linearly and quadratically increased with increasing doses of NCG. In contrast, plasma urea (p < 0.05) and ammonia (p < 0.05) concentration linearly and quadratically decreased with increasing doses of NCG. Overall, the addition of NCG increased plasma Arg, Ile, Val, TEAA and TNEAA concentration, which in turn resulted in a higher N utilization and, therefore, higher average daily gain in NCG-fed bulls, providing baseline data for the widespread application of NCG in beef cattle production.

Effects of N-carbamylglutamate on steroidogenesis and relative abundances of mRNA transcripts in pig placental trophoblasts

Supplementation of N-carbamylglutamate (NCG) improves gestation outcomes, with increased piglet within-litter uniformity of birth weight and reduced peripheral steroid concentrations in pregnant sows and ewes. It was hypothesized that the effect of NCG on placental function results from direct effects on the placental trophoblasts. There, therefore, was investigation of the effects of NCG on pig placental trophoblast (pTr) steroidogenesis, mRNA transcript abundance, and cell proliferation in vitro. The pTr were treated with NCG in serum-free medium for 24–48 h. Treatment with NCG inhibited pTr progesterone, androstenedione, testosterone (all P < 0.01), and estradiol (P < 0.05) production, whereas it promoted (P < 0.05) pTr proliferation. Treatment with NCG suppressed (P < 0.05) the relative abundances of CYP11A1, CYP19A1, and CASP3 and increased abundances of CCDN1 (P < 0.01) and CDK4 (P < 0.05) mRNA transcripts in pTr, whereas NCG treatment had no effect (P > 0.10) on relative abundances of StAR, HSD17B4, or HSD3B mRNA transcripts. Treatments with NCG can increase pTr cell numbers of sows through upregulating CCND1 and CDK4 and suppressing CASP3 mRNA transcript abundances, while modulating steroidogenesis through effects on CYP11A1 and CYP19A1 mRNA transcript abundances. It is concluded that NCG may have a direct action on pTr and may regulate placental function by suppressing pTr differentiation as a consequence of lesser steroid synthesis while promoting pTr proliferation and inhibiting apoptosis in sows.

Improvement of eggshell quality by dietary N-carbamylglutamate supplementation in laying chickens

Egg quality defects seriously reduce the quality grade and increase egg breakage in egg marketing activities. In this study, the effect of N-carbamylglutamate (NCG) on eggshell quality was investigated by evaluating calcium absorption and calcification in laying hens. A total of 30 newly hatched female Hy-Line chicks were randomly assigned to the control group (basal diet) and treatment group (basal diet supplemented with 1% NCG). At 25 wk, eggs from each group were obtained to assess egg quality parameters. Blood samples were collected for analysis of mineral, hormone, and amino acids levels at 16 h after laying egg. Uterine tissues were removed and fixed in 4% neutral paraformaldehyde solution or kept in liquid nitrogen for mineral determination, quantitative PCR, and Western blot. Results showed that the egg quality (eggshell thickness, strength and percentage, egg specific gravity, and eggshell effective thickness) was significantly increased while effective thickness of mastoid layer, width of mastoid gap, and mammillary knobs were significantly decreased by dietary NCG supplementation (P < 0.05). The levels of minerals (Ca, P, Fe, Mg, Na, K) in eggshell, plasma, and uterus were remarkably elevated (P < 0.05). Meanwhile, the concentrations of calcium metabolism–related hormones (17β-estradiol, parathyroid hormone, and calcitonin) were increased in the NCG group (P < 0.05). Moreover, expression of calbindin 1, carbonic anhydrase 2, ovalbumin, ovotransferrin, ovocleidin-17, ovocleidin-116, and clusterin mRNAs, as well as calbindin 1 and ATP2A1 proteins in uterus, duodenum, and kidney, was all upregulated in hens fed with NCG (P < 0.05). In addition, the number of blood vessels in the uterus, height of uterine mucosal folds, villus length in endometrium, and areas of uterine mucosal folds were significantly increased in the NCG group (P < 0.05). In conclusion, dietary 1% NCG supplementation during 0 to 25 wk can improve eggshell quality through changes in endometrial morphology, expression of calcium metabolism–related genes, and secretion of related hormones to promote eggshell formation in the laying hens.

Effect of dietary N-carbamylglutamate on development of ovarian follicles via enhanced angiogenesis in the chicken

N-carbamylglutamate (NCG), an analogue of N-acetyl-L-glutamate (NAG), can increase arginine synthesis in mammals and improve the reproductive performance. However, the effect of NCG on poultry laying performance is still unclear. This study investigated the effect of dietary NCG on development of chicken ovarian follicles. The dosage and timing for NCG administration were evaluated for its effect on follicular development. Results showed that supplementation with 1% NCG in the diet for 14 D led to accelerated development of growing follicles (over 60 μm in oocyte diameter) and significantly increased feed intake and feed efficiency. Plasma amino acids (AA) analysis showed that feeding with 1% NCG significantly increased of plasma AA levels. RNA-seq analysis revealed that NCG supplementation upregulated expression of genes related to angiogenesis and cell proliferation, but downregulated expression of apoptosis-related genes. Meanwhile, RT-qPCR and Western blot analysis validated the RNA-seq results. Moreover, NCG enhanced plasma NO level; upregulated expression of PKG-I, Raf1, and p-p38; and increased angiogenesis of the ovaries. In conclusion, dietary NCG (1% for 14 D) can promote development of ovarian follicles by increasing angiogenesis in ovaries of the chicken.

Effects of N-carbamylglutamate and arginine on steroidogenesis and proliferation of pig granulosa cells in vitro

Results of in vivo studies indicate dietary N-carbamylglutamate (NCG) and arginine (ARG) can enhance reproductive performance in gilts. It was hypothesized that both NCG and ARG will alter hormone-induced estradiol (E2) production by granulosa cells (GC), explaining why these compounds could improve reproductive performance in pigs. The objective of these studies, therefore, was to evaluate the direct effects of NCG and ARG on porcine GC proliferation and steroidogenesis, using an in vitro cell culture system. The GC from small (SM; 1–5 mm) and large (LG; >5 mm) pig follicles were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing 500 ng/mL of testosterone (as an E2 precursor), and NCG or ARG at various doses in the presence of either follicle-stimulating hormone (FSH; 30 ng/mL), insulin-like growth factor-1 (IGF1; 30 ng/mL), or both. Numbers of GC were determined at the end of the experiment and concentrations of progesterone (P4) and E2 in culture medium were determined. Results indicated that LG-follicle GC were more responsive to NCG and ARG than SM-follicle GC. Specifically, in LG-follicle GC, NCG inhibited (P < 0.05) basal and FSH-induced P4 and E2 production but stimulated cell numbers; whereas ARG inhibited FSH-induced E2 production and cell numbers. In SM-follicle GC, treatment with NCG and ARG decreased IGF1 plus FSH induced P4 production, but E2 production and cell proliferation were not affected. These studies indicate that NCG and ARG may directly affect follicular function in pigs.

Effects of dietary N-carbamylglutamate supplementation on growth performance, tissue development and blood parameters of yellow-feather broilers

The effects of N-carbamylglutamate (NCG) on the growth performance, tissue development, and blood parameters of broilers are unknown. In this study, 2 linked experiments were conducted to investigate the effects of 4 graded dietary levels and 3 dietary stages of NCG in a Chinese indigenous yellow-feather broiler breed during 2 growth phases: 1 to 18 d and 19 to 36 d. The dietary levels of NCG were 0.05%, 0.10%, 0.15%, and 0.20%, and dietary stages were designed to add NCG during the starter stage or grower stage or throughout the experimental period. At the age of 18 d, graded doses of NCG from 0.05 to 0.20% in the diet produced quadratic (P < 0.05) positive responses in body weight, width of intermuscular fat cingulum, liver weight, serum blood urea nitrogen, and serum low-density lipoprotein as well as linear (P < 0.05) positive responses in albumin serum concentration. The average feed per gain and mortality were unaffected by dietary NCG levels. Among 3 dietary treatments, only NCG dietary treatments throughout the experimental period improved the body weight and daily weight gain linearly (P < 0.05). The daily weight gain under the 3 dietary treatments used indicated that the most fitting dose is 0.1% NCG among the 4 dietary levels of NCG (P < 0.05). At this dose, muscle weight increased, whereas subcutaneous adipose as well as the serum contents of uric acid, triglyceride, and albumin decreased. Considering the growth performance and tissue development under the conditions used in this study, the best-fit model for NCG requirements of Chinese yellow-feather broilers was estimated from regression analysis to be 0.09 to 0.12% dietary NCG treatments during the grower stage. The modified blood parameters indicated that NCG dietary effects on broiler growth may be accompanied by modified homeostasis of arginine metabolism, lipid deposition, protein synthesis, and immune response.

N-carbamylglutamate restores nitric oxide synthesis and attenuates high altitude-induced pulmonary hypertension in Holstein heifers ascended to high altitude

BackgroundHigh-altitude pulmonary hypertension (HAPH) is a life-threating condition for animals in high altitude, and disturbance of endothelial nitric oxide (NO) synthesis contributes to its pathogenesis. N-carbamylglutamate (NCG), which enhances arginine synthesis, promotes endogenous synthesis of NO. In this study, we determined the effects of NCG on alleviating HAPH in Holstein heifers that ascended to Tibet (Lhasa, 3,658 m).MethodsExp. 1, 2,000 Holstein heifers were transported from low elevation (1,027 m) to Lhasa. After being exposed to hypoxia for 1 yr, Holstein heifers were assigned to a healthy group (Control, n = 6) with mean pulmonary hypertension (mPAP) < 41 mmHg, and an HAPH affected group (HAPH, n = 6) with mPAP > 49 mmHg. Lung tissues were collected to evaluate histopathological changes and the expression of endothelial nitric oxide synthase (eNOS). Exp. 2, ten healthy heifers and 10 HAPH affected heifers were supplemented with NCG (20 g/d per heifer) for 4 wk. Physiological parameters were determined and blood samples were collected on d − 1 and d 28 of the feeding trial.ResultsExpression of eNOS in small pulmonary arteriole intima was higher in the healthy than HAPH group (P = 0.006), whereas HAPH group had significantly thicker media and adventitia than healthy group (all P < 0.05). The mRNA of eNOS and protein level of eNOS were higher in the lungs of heifers in the healthy group than in the HAPH group (both P < 0.001), whereas endothelin-1 protein levels were higher in HAPH group than in the healthy group (P = 0.025). NCG supplementation decreased mPAP and ammonia (both P = 0.001), whereas it increased the expression of eNOS, arginine, and plasma NO (all P < 0.05).ConclusionsThe expression of eNOS was decreased in Holstein heifers with HAPH. NCG supplementation decreased mPAP through the restoration of eNOS and endogenous NO synthesis.

Effects of N-carbamylglutamate and L-arginine on steroidogenesis and gene expression in bovine granulosa cells

Feeding N-carbamylglutamate (NCG) and arginine (ARG) improves reproductive measures in pigs and reduces systemic steroid levels in pregnant ewes. We hypothesized that the effects of NCG and ARG on reproduction were due to direct effects on the ovary. Thus, the objectives of this study were to investigate the effects of NCG and ARG on granulosa cell (GC) steroidogenesis, gene expression, and cell proliferation in vitro. GC were collected from small (1–5mm) bovine follicles and treated in vitro with NCG or ARG in serum-free medium for 24h to 48h. Both NCG and ARG inhibited (P<0.05) IGF1- and FSH-induced GC estradiol production but only NCG inhibited (P<0.05) progesterone production. In contrast, NCG and ARG increased (P<0.05) GC numbers induced by IGF1 and FSH. NCG inhibited (P<0.05) StAR, CYP11A1 and CYP19A1 mRNA abundance in small-follicle GC, whereas ARG had no effect (P>0.10) on StAR, CYP11A1 or CYP19A1 mRNA abundance. We conclude that NCG and ARG may act directly on GC and therefore may regulate ovarian function by slowing follicular differentiation via inhibiting IGF1 action, and steroid synthesis while stimulating GC proliferation in cattle.

Effects of N-carbamylglutamate and L-arginine on gonadotrophin-releasing hormone (GnRH) gene expression and secretion in GT1-7 cells.

Recent studies have shown that N-carbamylglutamate (NCG) and arginine (ARG) supplementation improves reproductive performance in livestock. The objectives of the present study were to evaluate the effects of NCG and ARG on GT1-7 cell gonadotrophin-releasing hormone (GnRH) secretion, gene expression and cell proliferation. GT1-7 cells were treated in vitro with different concentrations of NCG (0-1.0mM) or ARG (0-4.0mM) in serum-free medium for 12 or 24h. For GnRH secretion and cell proliferation, GT1-7 cells were more sensitive to NCG than ARG. NCG treatment after 12h increased cell numbers and inhibited GnRH secretion in a dose-dependent manner (P<0.05), although there was no significant effect of NCG on these parameters after 24h culture. ARG treatment decreased GnRH secretion after 24h (P<0.05), whereas it had no effect after 12h. GT1-7 cells express GnRH, Kiss-1 metastasis-suppressor (Kiss1), G-protein coupled receptor 54 (GPR54), neuronal nitric oxide synthase (nNOS) and estrogen receptor α (ERα) genes. High concentrations of NCG (1.0mM) and ARG (4.0mM) inhibited (P<0.05) GnRH and nNOS mRNA abundance in GT1-7 cells. ARG treatment decreased Kiss1 and increased ERα mRNA abundance. Thus, high concentrations of NCG (1.0mM) and ARG (4.0mM) may act both directly and indirectly to regulate GnRH neuron function by downregulating genes related to GnRH synthesis and secretion to slow GnRH production while stimulating GT1-7 cell proliferation.