This study aimed to examine the role of thiamine in the local inflammation of ruminal epithelium caused by high-concentrate diets. Eighteen mid-lactating (148 ± 3 d in milk; milk yield = 0.71 ± 0.0300 kg/d) Saanen goats (body weight = 36.5 ± 1.99 kg; body condition score = 2.73 ± 0.16, where 0 = emaciated and 5 = obese) in parity 1 or 2 were selected. The goats were randomly divided into 3 groups (n = 6/group): (1) control diet (concentrate: forage 30:70), (2) high-concentrate diet (HC; concentrate: forage 70:30), and (3) highconcentrate diet with 200 mg of thiamine/kg of dry matter intake (THC; concentrate: forage 70:30). Goats remained on experimental diets for 8 wk. On the last day of 8 wk, ruminal and blood samples were collected to determine ruminal parameters, endotoxin lipopolysaccharide, and blood inflammatory cytokines. Goats were slaughtered to collect ruminal tissue to determine gene and protein expression of toll-like receptor 4 (TLR4) signaling pathways. Thiamine supplementation increased ruminal pH (6.03 vs. 5.42) compared with the HC group. Propionate (21.08 vs. 31.61 mM), butyrate (12.08 vs. 19.39 mM), lactate (0.52 vs. 0.71 mM), and free lipopolysaccharide (42.16 vs. 55.87 × 10 3 endotoxin units/mL) concentrations in ruminal fluid were lower in THC goats compared with HC goats. Similar to plasma interleukin 1β (IL-1β) concentration (209.31 vs. 257.23 pg/mL), blood CD8 + percentage (27.57 vs. 34.07%) also decreased in response to thiamine. Compared with HC goats, THC goats had lower ruminal epithelium activity of the enzymes myeloperoxidase and matrix metalloproteinase (MMP) 2 and 9. In contrast to HC, THC had downregulated mRNA expression of nuclear factor-κB (NFKB), TLR4, IL1B, MMP2, and MMP9 in ruminal epithelium. Thiamine supplementation led to lower relative protein expression of IL-1β, NF-κB unit p65, and phosphorylated NF-κB unit p65 in ruminal epithelium. Taken together, these results suggest that thiamine supplementation mitigates HC-induced local inflammation and ruminal epithelial disruption.

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 peNDF (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 peNDF content. The body frame size (including withers height, body length, and heart girth) of the heifers was not increased significantly by enhanced peNDF content. Ruminal pH and ammonia concentration were both increased with increasing dietary peNDF content. The ruminal total volatile fatty acid concentration and percentage of acetate and butyrate profiles were not significantly affected by dietary peNDF content. However, the enhanced peNDF 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 peNDF 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 peNDF content. The apparent digestibility of acid detergent fiber and crude protein was improved with an increasing content of dietary peNDF. The results suggest that an optimal or advisable dietary particle size and peNDF 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 peNDF content is the most suitable for 8- to 10-mo-old Holstein heifers.

No abstract

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.

(), a core clock gene, encodes a circadian rhythm protein which has been shown to control mammary metabolism in rodents. Whether regulates milk component synthesis such as α-casein protein in bovine mammary cells is unknown. Thus, we used gene silencing technology to determine if silencing could affect α-casein synthesis and cell growth in cultured primary bovine mammary epithelial cells (BMEC). The BMEC were established by enzymatic digestion of mammary tissue from mid-lactation cows. A transient-transfection technique was used to insert a small interfering RNA (siRNA) oligonucleotide specific for to inhibit transcription. Control and siRNA-transfected cells were cultured for 48 h. qRT-PCR and ELISA analysis showed that silencing enhanced the synthesis of 2 kinds of α-casein ( < 0.05) through upregulating the mRNA level of and ( < 0.01). Furthermore, the 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) results demonstrated that cell proliferation was not affected ( > 0.05). These data led us to hypothesize that PER2 protein may potentially play an important role in the control of milk protein synthesis and, hence, represents a target that can be used to regulate protein synthesis rate during lactation.

Transformation of Arg to nitric oxide and ornithine (Arg-Orn) constitutes the main route of Arg metabolism in mammals. The primary objective of this work was to determine the effects of inhibiting the Arg-Orn pathway via N ω-hydroxy-nor-l-arginine (nor-NOHA) on health of lactating cows. Furthermore, we also explored the effect of Arg-Orn inhibition on the efficiency of nitrogen utilization to find support for previous research that showed the inhibition of Arg-Orn inhibited milk protein synthesis. Six healthy Chinese Holstein cows of similar body weight (550.0 ± 20 kg), parity (4.0 ± 0), body condition score (3.0 ± 0), milk yield (21.0 ± 1.0 kg), and days in milk (80 ± 2 d) 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) saline infusion (control); (2) infusion of 125 mg/L of nor-NOHA; and (3) infusion of 125 mg/L of nor-NOHA with 9.42 g/L of Arg. Dry matter intake, apparent digestibility of nutrients, urinary N, N in milk, and blood indices of metabolism and immune function were determined. Compared with the control, the infusion of nor-NOHA had no effect on the concentrations of cholesterol, high-density lipoproteins, IgA, IL-1β, tumor necrosis factor-α, and alanine transaminase. In addition, the dry matter intake, apparent digestibility of N, and the concentration of milk protein N in the Nor-NOHA did not differ from the control; however, the infusion of nor-NOHA and Arg resulted in greater concentrations of high-density lipoprotein, IgA, IL-1β, and tumor necrosis factor-α, and lower concentrations of cholesterol in serum compared with the control. Moreover, the addition of Arg to cows infused with nor-NOHA increased the concentration of nitrate (the indicator of nitric oxide) in serum and was associated with greater milk protein N production due to greater milk yield compared with those infused with nor-NOHA. Overall, the results indicated important roles of Arg in immunity and mammary N utilization, whereas a minor role of the Arg-Orn pathway in these physiologic processes was found.

This experiment was conducted to investigate the effects of certain long-chain fatty acid combinations on ruminal microbial community in vitro. Three cows fitted with permanent ruminal cannulas were used to provide rumen liquor for the in vitro trail. The treatments were fatty acid combinations of stearic acid, oleic acid, linoleic acid and linolenic acid, which respectively were group A (calcium palmitate, control), B (1.5%, 1.0%, 0.5% and 1.5%, acetic type fermentation), C (1.5%, 1.0%, 1.5% and 1.0%, propionic acid type fermentation), D (1.0%, 1.5%, 1.5% and 0.5%, butyric acid type fermentation). Three goats fitted with rumen cannula were used to provide the culture medium was collected for the measurement of the rumen microorganism in vitro culture. Culture medium was collected at 0, 3, 6, 12, 18 and 24 h. The results demonstrated that, except for Ruminococcus albus, all the other bacterial genus had significant differences between groups (P < 0.05). The Fibrobacter succinogenes and Ruminococcusflavefaciens, Clostridium proteoclasticum and Ruminobacter amylophilus were higher in group B; Butyrivibrio fibrisolvens, Megasphaera elsdenii and Ruminococcus bromii were higher in group C; while Pseudobutyrivibrio ruminis was higher in group D. Different higher species in different treatments described above related to their fermentation type. It was also observed that, bacteria were the dominant flora in rumen and Megasphaera elsdeniiwere the dominant species in bacterial fraction no matter what the treatment is. It was therefore concluded that, certain fatty acid combinations have remarkable effects on the amount of ruminal specific bacteria species,mainly related to their fermentation type.

The expression of CSN3, hormone receptor, the expression of genes regulating the mTOR, JAK–STAT signal pathways, and the relative content of к-casein as well as total casein were determined in the present study to explore the mechanism of the effect of lactogenic hormones on milk-protein synthesis in bovine mammary epithelial cells. The results showed that apoptosis of the cells was increased by inhibitor LY294002, while the expressions of genes encoding PKB, Rheb, PRAS40 and S6K1 in the mTOR signal pathway, JAK2, STAT5A in the JAK–STAT signal pathway, and genes encoding INSR, PRLR, NR3C1 and CSN3 were all downregulated, and the relative contents of κ-casein and total casein were decreased in the mammary epithelial cells compared with those in the control group. Comparatively, the inhibitory effects of AG-490 were more profound than those of LY294002, and the double block using both inhibitors had a greater effect than the single block. The CSN3 gene expression was downregulated and the content of milk casein was decreased by the inhibitors. In addition, the expression of the hormone receptor genes was downregulated. Our results suggest that lactogenic hormones, via their receptors in the membrane, regulated the JAK–STAT and m-TOR signal pathways, and affected cell proliferation and apoptosis, leading to changes in milk-protein synthesis.