Background: Megasphaera elsdenii is an ecologically important rumen bacterium that metabolizes lactate and relieves rumen acidosis (RA) induced by a high-grain-diet. Understanding the regulatory mechanisms of the lactate metabolism of this species in RA conditions might contribute to developing dietary strategies to alleviate RA.Methods: Megasphaera elsdenii was co-cultured with four lactate producers (Streptococcus bovis, Lactobacilli fermentum, Butyrivibrio fibrisolvens, and Selenomonas ruminantium) and a series of substrate starch doses (1, 3, and 9 g/L) were used to induce one normal and two RA models (subacute rumen acidosis, SARA and acute rumen acidosis, ARA) under batch conditions. The associations between bacterial competition and the shift of organic acids’ (OA) accumulation patterns in both statics and dynamics manners were investigated in RA models. Furthermore, we examined the effects of substrate lactate concentration and pH on Megasphaera elsdenii’s lactate degradation pattern and genes related to the lactate utilizing pathways in the continuous culture.Results and Conclusion: The positive growth of M. elsdenii and B. fibrisolvens caused OA accumulation in the SARA model to shift from lactate to butyrate and resulted in pH recovery. Furthermore, both the quantities of substrate lactate and pH had remarkable effects on M. elsdenii lactate utilization due to the transcriptional regulation of metabolic genes, and the lactate utilization in M. elsdenii was more sensitive to pH changes than to the substrate lactate level. In addition, compared with associations based on statics data, associations discovered from dynamics data showed greater significance and gave additional explanations regarding the relationships between bacterial competition and OA accumulation.

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.

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.

A previous in vitro study revealed that Arg elicits positive effects on casein synthesis through alterations of the Arg-ornithine pathway in bovine mammary epithelial cells. The main purpose of this work was to determine the effects of arginase inhibition using N-hydroxy-nor-l-arginine (nor-NOHA) on milk protein synthesis in vivo. Six healthy Chinese Holstein cows with similar body weight (550.0 ± 20 kg; means ± standard deviation), parity (4), body condition score (3.0), milk yield (21.0 ± 1.0 kg), and days in milk (80 ± 2) were selected and randomly assigned to 3 treatments in a replicated 3 × 3 Latin square design with 22 d for each period (7 d for infusion and 15 d for washout). The treatments were (1) control: saline infusion; (2) nor-NOHA: infusion of 125 mg/L of nor-NOHA; (3) nor-NOHA + Arg: infusion of 125 mg/L of nor-NOHA with 9.42 g/L of Arg. The activity of enzymes related to Arg metabolism, milk protein synthesis, and expression of AA transporters was determined. The infusion of nor-NOHA decreased the activity of arginase but had no effect on the activity of ornithine decarboxylase and nitric oxide synthase in serum, and these responses were the same at the gene expression level in mammary gland. In addition, the infusion of nor-NOHA also reduced protein and fat synthesis in milk but had no effect on milk yield. When Arg was infused with nor-NOHA, the activity of total arginase, ornithine decarboxylase, and nitric oxide synthase, and the concentration of casein, protein, and fat in milk did not change compared with the nor-NOHA group, but the milk protein yield, the expression of some Arg transporters (SLC7A5 and SLC7A8), and milk yield increased. Overall, results verified previous in vitro findings indicating that synthesis of casein protein is closely regulated by the Arg-ornithine pathway in bovine mammary gland.

Cereal grains treated with organic acids were proved to increase ruminal resistant starch and can relieve the risk of ruminal acidosis. However, previous study mainly focussed on acid-treated barley, the effects of organic acid-treated corn is still unknown. The objectives of this study were to evaluate whether feeding ground corn steeped in citric acid (CA) would affect ruminal pH and fermentation patterns, milk production and innate immunity responses in dairy goats. Eight ruminally cannulated Saanen dairy goats were used in a crossover designed experiment. Each experimental period was 21 day long including 14 days for adaption to new diet and 7 days for sampling and data collection. The goats were fed high-grain diet contained 30% hay and 70% corn-based concentrate. The corn was steeped either in water (control) or in 0.5% (wt/vol) CA solution for 48 h. Goats fed CA diet showed improved ruminal pH status with greater mean and minimum ruminal pH, and shorter (P<0.05) duration of ruminal pH<5.6 and less area of ruminal pH<5.6, 5.8 and 6.0. Concentration of total volatile fatty acid and molar proportion of propionate were less but the molar proportion of acetate was greater (P<0.05) in goats fed the CA diet than the control diet. Concentration of ruminal lipopolysaccharide (LPS) was lower (P<0.05) and that of lactic acid also tended (P<0.10) to be lower in goats fed CA than the control. Although dry matter intake, actual milk yield, yield and content of milk protein and lactose were not affected, the milk fat content and 4% fat-corrected milk tended (P<0.10) to be greater in goats fed CA diet. For the inflammatory responses, peripheral LPS did not differ, whereas the concentration of LPS binding protein and serum amyloid A tended (P<0.10) to be less in goats fed CA diet. Similarly, goats fed CA diet had less (P<0.05) concentration of haptoglobin and tumour necrosis factor. These results indicated that feeding ground corn treated with CA effectively improved ruminal pH status, thus alleviated the risk of ruminal acidosis, reduced inflammatory response, and tend to improve milk yield and milk fat test.

This study was undertaken to evaluate the milk protein response when cows were supplied a balanced AA profile and to determine whether a deficiency of Leucine (Leu) or Arginine (Arg) had a negative effect on milk protein. Eight mid-lactation Holstein cows were randomly assigned to 5-day continuous jugular infusions of saline (CTL), EAA mixture prepared on the profile of casein and supplied (in % of lysine (Lys)) 100% of Lys, 33.3% of methionine (Met), 110.2% of Leu, 43.6% of Arg, 50.8% of threonine (Thr), 81.6% of valine (Val), 69.7% of isoleucine (Ile), 61.4% of phenylalanine (Phe) and 34.2% of histidine (His) (Casein, 160 g/d), EAA mixture excluding Leu (-Leu, 163 g/d) or EAA mixture excluding Arg (-Arg, 158 g/d) in a duplicated 4 × 4 Latin square design with four infusion periods separated by 7-day interval period. The basal diet supplied 1.6 Mcal NE and 94.4 g MP per 1 kg DM to meet requirements for lactation. The Casein treatment provided a balanced supply (in % of MP) of 10.3% Leu and 5.3% Arg, whereas in the two subsequent -Leu and -Arg treatments, the concentration of Leu and Arg was reduced to 8.4 and 4.6% respectively. Dry matter intake (15.4 kg/day) was not affected by treatments. The Casein treatment increased milk yield (14.9%, p < 0.001), milk protein yield (120 g, p < 0.001) and milk protein efficiency (0.03, p = 0.099) than CTL treatment. However, the -Leu treatment decreased the responses of above-measured parameters by 6.25%, 70 g, 0.05 (p < 0.06) (compared with Casein). These effects of Leu were related to decreased Leu concentration and improved concentration of Ile and Val in plasma. The -Arg treatment decreased the plasma Arg concentration than the Casein treatment, whereby resulted in the decrease of milk yield (5.7%, p = 0.073), milk protein yield (60 g, p = 0.011) and milk protein efficiency (0.04, p = 0.037). In conclusion, supply of EAA profile of casein can increase the lactation production in dairy cows, and 8.6% of Leu in MP partly limits the milk protein response when the requirements of Lys, Met and His were met. The level of Arg at 4.6% MP is not deemed to an ideal profile, as evidenced by decreased milk protein efficiency.

The main purpose of this work was to determine the effect of arginase inhibition via N-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 [Dulbecco's 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.

BackgroundEnhancing the post-ruminal supply of arginine (Arg), a semi-essential amino acid (AA), elicits positive effects on milk production. Our objective was to determine the effects of Arg infusion on milk production parameters and aspects of nitrogen (N) absorption and utilization in lactating dairy cows. Six lactating Chinese Holstein cows of similar body weight (508 ± 14 kg), body condition score (3.0 ± 0), parity (4.0 ± 0), milk yield (30.6 ± 1.8 kg) and days in milk (20 ± 2 d) were randomly assigned to 3 treatments in a replicated 3 × 3 Latin square design with 21 d for each period (1 week for infusion and 2 weeks for washout). Treatments were 1) Control: saline; 2) Arg group: saline + 9.42 g/L L-Arg; 3) Alanine (Ala) group: saline + 19.31 g/L L-Ala (iso-nitrogenous to the Arg group). Milk production and composition, dry matter intake, apparent absorption of N, profiles of amino acids (AA) in blood, urea N in urine, milk, and blood, and gene expression of AA transporters were determined.ResultsCompared with the Control or Ala group, the infusion of Arg led to greater expression of AA transporters (SLC7A2 and SLC7A8) and apparent uptake of free AA in the mammary gland, and was accompanied by greater milk yield, milk protein yield and milk efficiency (calculated by dividing milk yield over feed intake), together with lower concentration of urea N [regarded as an indicator of N utilization efficiency (NUE)] in blood and milk. Furthermore, in the cows infused with Arg, the NUE was higher and the concentration of urea N in urine was lower than those in the Ala group, although no differences were detected in NUE and urea N in urine between the Control and Arg group. The infusion of Ala had no effect on those indices compared with the Control.ConclusionsOverall, enhancing the post-ruminal supply of Arg via the jugular vein had a positive effect on the synthesis of milk protein at least in part by increasing gene expression of some AA transporters and uptake of free AA by mammary gland.