Hongrong Wang

Effects of Arginine Concentration on the In Vitro Expression of Casein and mTOR Pathway Related Genes in Mammary Epithelial Cells from Dairy Cattle

Arginine (Arg) is a conditionally-essential amino acid that is taken up by bovine mammary gland in excess of its output in milk. In this study we evaluated the effects of Arg concentration on the expression of casein and signaling pathway-related genes in mammary epithelial cells. The treatments (applied for 24 h) were designed to be devoid of Arg 0X (control; 0.00 mg/L), resemble the profile of Arg in casein (Arg 1X; 278.00 mg/L), be deficient [Arg 0.25X (69.50 mg/L) and Arg 0.5X (139.00 mg/L)], or be in excess of the amount in casein [Arg 2X (556.00 mg/L), Arg 4X (1,112 mg/L), and Arg 8X (2,224 mg/L)]. The expression of CSN1S, CSN3 and mTOR in the experimental groups was higher than those of the control group (P<0.05). Except for Arg 0.25X and Arg 8X (P>0.05), the expression of CSN1S2, CSN2 and JAK2 in other experimental groups was higher (P<0.05) than those in the control group. Except for Arg 8X (P>0.05), the expression of STAT5 in the other experimental groups was higher than those of the control (P<0.05). It also was observed that except for Arg 0.5X, the S6K expression was higher in other experimental groups than the control (P<0.05). In contrast, except for Arg 0.25X the other experimental groups resulted in lower 4EBP1 expression than the control (P<0.05). Among groups, the expression of CSN1S1, CSN1S2, CSN2, CSN3, JAK2, STAT5, mTOR and S6K gene was highest with Arg 2X (P<0.05); the reverse was true for 4EBP1 gene, with the lowest expression in this group (P<0.05). Taken together, Arg appears to play an important role in the transcriptional regulation of casein genes and mTOR-related genes in bovine mammary epithelial cells.

Arginine Relieves the Inflammatory Response and Enhances the Casein Expression in Bovine Mammary Epithelial Cells Induced by Lipopolysaccharide

As one of functional active amino acids, L-arginine holds a key position in immunity. However, the mechanism that arginine modulates cow mammary inflammatory response in ruminant is unclear. Therefore, this study was conducted to investigate the effects of L-arginine on inflammatory response and casein expression after challenging the bovine mammary epithelial cells (BMECs) with lipopolysaccharide (LPS). The cells were divided into four groups, stimulated with or without LPS (10 μg/mL) and treated with or without arginine (100 μg/mL) for 12 h. The concentration of proinflammatory cytokines, inducible nitric oxide synthase (iNOS), mammalian target of rapamycin (mTOR), and Toll-like receptor 4 (TLR4) signaling pathways as well as the casein was determined. The results showed that arginine reduced the LPS-induced production like IL-1β, IL-6, TNF-α, and iNOS. Though the expression of NF-κB was attenuated and the mTOR signaling pathway was upregulated, arginine had no effect on TLR4 expression. In addition, our results show that the content of β-casein and the total casein were enhanced after arginine was supplemented in LPS-induced BMECs. In conclusion, arginine could relieve the inflammatory reaction induced by LPS and enhance the concentration of β-casein and the total casein in bovine mammary epithelial cells.

Effects of different dietary concentrate to forage ratio and thiamine supplementation on the rumen fermentation and ruminal bacterial community in dairy cows

A subacute ruminal acidosis (SARA) model was induced gradually by increasing the proportion of dietary concentrate to evaluate the effect of thiamine supplementation on the structure of bacterial community in dairy cows. Three Holstein dairy cows with rumen cannula were randomly assigned to a replicated 3 × 3 Latin square design trial and received three diets during three successive 21-day periods in each square. The three dietary treatments were as follows: a low-concentrate diet (control), a high-concentrate SARA-induced diet (SARA) and a high-concentrate SARA-induced diet with 180 mg thiamine/kg DM (SARA+thiamine). Real-time–polymerase chain reaction assay was used to quantify the population variation of SARA-related ruminal bacteria in these cows. The results showed that SARA was induced gradually when cows were fed with the high-concentrate diets. The mean ruminal pH value was higher in the control cows than in those of SARA and SARA+thiamine groups, the mean was decreased in cows fed on SARA diet, and the depression was alleviated by supplemented thiamine and the difference was significant (P 0.05). It was concluded that thiamine supplementation to high-concentrate diets at concentrations of 180 mg/kg DM could help alleviate SARA by increasing rumen pH and balancing the population of lactic acid-producing and -consuming bacteria.

Effects of n-6:n-3 polyunsaturated fatty acid ratio on heterophil:lymphocyte ratio and T lymphocyte subsets in the peripheral blood of the Yangzhou gosling

The objective of this study was to determine the effects of dietary n-6:n-3 polyunsaturated fatty acid (PUFA) ratios on heterophil:lymphocyte (H:L) ratios and T cell subsets in the peripheral blood of the growing Yangzhou goose. A total of 160 healthy Yangzhou geese (0.407 ± 0.023 kg), aged 21 d, were randomly divided into 4 groups consisting of diets with different n-6:n-3 PUFA ratios: 12:1, 9:1, 6:1, and 3:1. White blood cell counts and H:L ratios were measured at 42, 56, and 70 d of age; T lymphocyte subsets were also analyzed using monoclonal antibodies and flow cytometry simultaneously. The results showed that differences were found in white blood cells between groups (P < 0.05) but not within groups (P > 0.05), and white blood cells of 70-d-old geese were significantly higher than those of other ages within the same group. The H:L ratios of all groups decreased as age increased, and differences were found in 6:1 and 3:1 groups between ages (P < 0.05). As for the comparison of H:L ratio between treatments, 6:1 and 3:1 were lower than the other 2 groups at 42 or 70 d of age (P < 0.05), and 3:1 was lower than the other 3 groups at 56 d of age (P < 0.05). Moreover, H:L ratios had a tendency to decrease with the decrease of n-6:n-3 PUFA ratios. Furthermore, cluster of differentiation (CD) 3(+) and CD8(+) increased gradually, whereas CD4(+) and CD4(+):CD8(+) ratios first increased and then decreased as age increased. Differences were detected in T cell subsets among ages (P < 0.05). In addition, CD4(+) and CD4(+):CD8(+) ratios were affected remarkably by n-6:n-3 PUFA ratios but CD3(+) and CD8(+) were not, and 6:1 had much higher CD4(+) percentage and CD4(+):CD8(+) ratio compared with the other groups (P < 0.05). Taken together, diets containing low n-6:n-3 PUFA ratios could decrease H:L ratios in the peripheral blood. Additionally, CD4(+) percentage and CD4(+):CD8(+) ratio were much higher in the 6:1 group, which could be relevant for improving positive immune responses for Yangzhou goslings from 42 to 70 d of age.

Effects of Glucose and Starch on Lactate Production by Newly Isolated Streptococcus bovis S1 from Saanen Goats.

ABSTRACT When ruminants are fed high-concentrate diets, Streptococcus bovis proliferates rapidly and produces lactate, potentially causing rumen acidosis. Understanding the regulatory mechanisms of the metabolism of this species might help in developing dietary strategies to alleviate rumen acidosis. S. bovis strain S1 was newly isolated from the ruminal fluid of Saanen dairy goats and then used to examine the effects of glucose and starch on bacterial metabolism and gene regulation of the organic acid-producing pathway in cultures at a pH of 6.5. Glucose or starch was added to the culture medium at 1 g/liter, 3 g/liter (close to a normal range in the rumen fluid), or 9 g/liter (excessive level). Lactate was the dominant acid produced during the fermentation, and levels increased with the amount of glucose or starch in a dose-dependent manner (P < 0.001). The production of formate and acetate in the fermentation media fluctuated slightly with the dose but accounted for small fractions of the total acids. The activities of lactate dehydrogenase (LDH) and α-amylase (α-AMY) increased with the starch dose (P < 0.05), but the α-AMY activity did not change with the glucose dose. The relative expression levels of the genes ldh, pfl (encoding pyruvate formate lyase), ccpA (encoding catabolite control protein A), and α-amy were higher at a dose of 9 g/liter than at 1 g/liter (P < 0.05). Expression levels of pfl and α-amy genes were higher at 3 g/liter than at 1 g/liter (P < 0.05). The fructose 1,6-diphosphate (FDP) concentration tended to increase with the glucose and starch concentrations. In addition, the S. bovis S1 isolate fermented glucose much faster than starch. We conclude that the quantities of glucose and soluble starch had a major effect on lactate production due to the transcriptional regulation of metabolic genes. IMPORTANCE This work used a newly isolated S. bovis strain S1 from the rumen fluid of Saanen goats and examined the effects of glucose and soluble starch on organic acid patterns, enzyme activity, and expression of genes for in vitro fermentation. It was found that lactate was the dominant product from S. bovis strain S1, and the quantities of both glucose and starch in the medium were highly correlated with lactate production and with the corresponding changes in associated enzymes and genes. Therefore, manipulating the metabolic pathway of S. bovis to alter the dietary level of readily fermentable sugar and carbohydrates may be a strategy to alleviate rumen acidosis.

Effects of supplementation of rumen-protected choline on growth performance, meat quality and gene expression in longissimus dorsi muscle of lambs

This study determined the effects of rumen-protected choline (RPC) on growth performance, blood lipids, meat quality and expression of genes involved in fatty-acid metabolism in young lambs. A total of 24 Dorper × Hu lambs (about 20 kg body weight) were kept in individual pens and fed diets with 0%, 0.25%, 0.50% and 0.75% RPC for 60 d. Supplementation of 0.25% RPC increased average daily gain of lambs, whereas treatments had no significant effect on feed intake. The pH values of meat were increased at 0.25% RPC and both, dripping loss and shear force of meat, were significantly decreased in RPC-supplemented lambs. No significant changes were observed for dressing percentage and intramuscular fat. RPC supplementations had no significant effect on the concentrations of triglycerides and cholesterols in serum, but the concentration of high-density lipoprotein was decreased at 0.50% RPC and that of low-density lipoprotein was increased at 0.75% RPC. In m. longissimus dorsi, the expressions of cluster of differentiation 36 (CD36), acetyl-CoA carboxylase (ACC) and fatty-acid synthase (FASN) genes were increased at 0.25% RPC. Supplementation of 0.75% RPC increased the expressions of lipoprotein lipase (LPL) and FASN genes, decreased the expression of ACC gene and had no effect on CD36 gene. The results of this study showed that supplementation of 0.25% RPC could promote growth performance of lambs and improve meat quality. This may be mediated by effects on blood lipid profiles and the metabolism of fatty acids in skeleton muscles. However, the beneficial effects of 0.25% RPC supplementation need to be validated with a larger number of animals. Higher doses, particularly 0.75% RPC, showed adverse effects on live weight gain and ACC expression.

Effects of Dietary l-Arginine and N-Carbamylglutamate Supplementation on Intestinal Integrity, Immune Function, and Oxidative Status in Intrauterine-Growth-Retarded Suckling Lambs

This study investigated the effects of dietary l-arginine (Arg) and N-carbamylglutamate (NCG) supplementation on intestinal integrity, immune function, and oxidative status in intrauterine-growth-retarded (IUGR) suckling lambs. A total of 48 newborn Hu lambs of normal birth weight (CON) and IUGR were allocated randomly into four groups of 12 animals each: CON, IUGR, IUGR + 1% Arg, or IUGR + 0.1% NCG. All lambs were raised for a period of 21 days from 7 to 28 days after birth. The Arg or NCG group exhibited improved ( p < 0.05) final body weights compared to that of the IUGR group. In comparison to the IUGR lambs, the apoptotic percentage was lower ( p < 0.05) in the ileum of IUGR lambs supplemented with Arg and NCG. In addition, in comparison to IUGR, the concentrations of protein carbonyl and malondialdehyde were lower ( p < 0.05) and the reduced glutathione (GSH) concentration and ratio of GSH/oxidized glutathione were greater ( p < 0.05) in the jejunum, duodenum, and ileum of IUGR + 1% Arg or 0.1% NCG lambs. In comparison to the IUGR group, the mRNA abundance of myeloid differentiation factor 88, toll-like receptor 9, toll-like receptor 4, interleukin 6, and fuclear factor-κB was lower ( p < 0.05) and the mRNA abundance of superoxide dismutase 1, B-cell lymphoma/leukaemia 2, zonula occludens-1 (ZO-1), and occludin was greater in the ileum of the IUGR lambs supplemented with Arg or NCG. Furthermore, the protein abundance of ZO-1 and claudin-1 in the ileum was greater ( p < 0.05) in the IUGR + 1% Arg or 0.1% NCG lambs. The results show that Arg or NCG supplementation improves the growth, intestinal integrity, immune function, and oxidative status in IUGR Hu suckling lambs.

Effects of dietary physically effective neutral detergent fiber content on the feeding behavior, digestibility, and growth of 8- to 10-month-old Holstein replacement heifers

The objective of this study was to evaluate the effects of dietary physically effective neutral detergent fiber (peNDF) content on the feeding behavior, digestion, ruminal fermentation parameters, and growth of 8- to 10-mo-old dairy heifers and to predict the adequacy of dietary fiber in growing dairy heifers. Twenty-four Holstein dairy heifers (245 ± 10.8 d of age, 305.6 ± 8.5 kg initial live weight) were randomly divided into 4 treatments with 6 replicates as a completely randomized design. During the 60-d period with a 10-d adaptation, heifers were offered 1 of 4 diets, which were chemically identical but included different peNDF8.0 (particle size is >8 mm and <19 mm) content (% DM): 10.8, 13.5, 18.0, or 19.8%, which was achieved by chopping forage into different lengths (fine = 1 cm, short = 3 cm, medium = 5 cm, and long = 7 cm). The concentrate and silage were mixed and fed restrictedly and exclusive of forage (Chinese ryegrass hay) were offered ad libitum. The body weight and frame size of the heifers were measured every 15 d during the experimental period. Samples of the rumen content (2 h after the morning feeding) were taken for pH, ammonia, and volatile fatty acid determination. The dry matter intake and average daily gain of the heifers were not significantly affected by peNDF8.0 content. The body frame size (including withers height, body length, and heart girth) of the heifers was not increased significantly by enhanced peNDF8.0 content. Ruminal pH and ammonia concentration were both increased with increasing dietary peNDF8.0 content. The ruminal total volatile fatty acid concentration and percentage of acetate and butyrate profiles were not significantly affected by dietary peNDF8.0 content. However, the enhanced peNDF8.0 content led to a decrease in the propionate percentage. The ratio of acetate to propionate in the 13.5% treatment was highest among the treatments. Increasing the particle size and dietary peNDF8.0 content resulted in increased eating and chewing time but had no effect on rumination time. Heifer total eating and chewing time and eating and chewing time per kilogram of dry matter intake were increased with increasing dietary peNDF8.0 content. The apparent digestibility of acid detergent fiber and crude protein was improved with an increasing content of dietary peNDF8.0. The results suggest that an optimal or advisable dietary particle size and peNDF8.0 content improves chewing activity, rumen fluid pH, and ruminal fermentation. The data based on feeding behavioral and growth responses of heifers as well as rumen fermentation and digestion by improving total eating and chewing time indicate that 18.0% dietary peNDF8.0 content is the most suitable for 8- to 10-mo-old Holstein heifers.

Short communication: Arginase inhibition reduces the synthesis of casein in bovine mammary epithelial cells

The main purpose of this work was to determine the effect of arginase inhibition via Nω-hydroxy-nor-l-arginine (nor-NOHA) on casein synthesis in bovine mammary epithelial cells (BMEC). Passage 2 BMEC isolated from dairy cows were seeded to 6-well plates and randomly divided into 4 treatments: (1) control [Dulbeccos modified Eagle medium:Nutrient Mixture F-12 medium (DMEM/F12)]; (2) nor-NOHA (DMEM/F12 + 1 mmol/L nor-NOHA); (3) nor-NOHA + arginine (DMEM/F12 + 1 mmol/L nor-NOHA + 3.2 mmol/L Arg); and (4) nor-NOHA + ornithine (DMEM/F12+ 1 mmol/L nor-NOHA + 1 mmol/L Orn). Then, we determined the activity of enzymes related to Arg metabolism and casein synthesis in BMEC and the proliferation of cells. The addition of nor-NOHA reduced the activity of arginase and ornithine decarboxylase but had no effect on the activity of nitric oxide synthase, and these responses were the same at the gene expression level. In addition, supplementation of nor-NOHA in BMEC reduced cellular proliferation and casein synthesis. Addition of Arg to nor-NOHA resulted in cellular proliferation and casein synthesis similar to that of nor-NOHA alone. In contrast, addition of Orn to the medium with nor-NOHA increased the synthesis of casein and cellular proliferation compared with Nor-NOHA. In conclusion, suppression of the Arg-arginase-Orn pathway reduced casein synthesis and cellular proliferation, which indicated that this pathway is an important regulator of the synthesis of casein in BMEC.

Jugular arginine infusion relieves lipopolysaccharide-triggered inflammatory stress and improves immunity status of lactating dairy cows

The objective of this study was to evaluate the effects of jugular l-Arg infusion on performance and immune function during lipopolysaccharide (LPS)-induced inflammation of lactating dairy cows. Eight Holstein cows (multiparous, 608.8 ± 31.5 kg) at mid-lactation were randomly assigned to 5-d jugular infusions of control (saline), Arg (3 g/h), LPS (0.033 μg/kg per h), and LPS + Arg (0.033 μg/kg per h of LPS and 3 g/h of Arg) in a replicated 4 × 4 Latin square design with 4 infusion periods separated by 10-d noninfusion periods. Jugular solutions of saline, Arg, LPS, and LPS + Arg were continuously infused using peristaltic pumps for approximately 6 h/d during infusion periods. Milk yield was measured on each day of the infusion period. Milk samples were obtained on the last 2 d of each infusion period, and blood samples were obtained on the last day of each infusion period before infusion (0 h) and at 3 and 6 h. We found that the jugular LPS infusion significantly increased serum concentrations of IL-1β, IL-6, tumor necrosis factor, inducible nitric oxide synthase, and lipopolysaccharide binding protein, whereas Arg attenuated the increase in IL-6 and inducible nitric oxide synthase levels and tended to decrease the lipopolysaccharide binding protein level. Arginine alleviated the decrease in dry matter intake and milk fat yield and the increase of somatic cell count induced by LPS. Total casein in milk was decreased during the LPS-induced inflammation period, and jugular Arg infusion significantly increased the content of total casein. In contrast, lactalbumin in milk increased during the LPS-induced inflammation period, whereas jugular Arg infusion significantly decreased the content of lactalbumin. The concentrations of plasma Gly, Thr, Ile, Leu, Arg, Phe, and total free AA were significantly decreased by LPS treatment, but Arg attenuated this tendency. These results indicated that jugular Arg infusion (18 g/d) has protective effects on relieving inflammatory stress and improving immunity status triggered by LPS. In conclusion, Arg could attenuate inflammatory stress and improve milk performance of lactating dairy cows. This protective effect may be due to the ability of Arg to suppress LPS effects and improve immunity status.