The aim of this study was to investigate the effects of Clostridium butyricum (C. butyricum) on the performance, serum lipid metabolism, muscle morphology, meat quality, and fatty acid profiles of Peking ducks. A total of 1,500 Peking ducks were randomly divided into five groups with five replicates and were fed a non-antibiotic basal diet (Control) or a basal diet supplemented with either 200, 400, or 600 mg/kg of C. butyricum (2.0 × 109 CFU/g) or 150 mg of aureomycin/kg for 42 d. Compared with the control group, supplementation with C. butyricum increased the average daily weight gain but reduced the feed/gain ratio from 1 to 42 d of age. Similarly, dietary C. butyricum increased the activities of antioxidant enzymes but decreased the malondialdehyde (MDA) and lipid metabolites concentration. C. butyricum supplementation increased the muscle pH value at 45 min postmortem, the redness of the meat, and the contents of inosine acid (IMP) and intramuscular fat (IMF) in Peking ducks. By contrast, C. butyricum supplementation lowered the lightness, drip loss, and the shear force of breast meat. Supplementation with C. butyricum increased the concentrations of essential amino acids and flavor amino acids, as well as arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and total polyunsaturated fatty acids (PUFA) in breast muscle. Dietary C. butyricum could positively improve performance, lipid metabolism, meat quality, and the amino acid and fatty acid composition in a dose-dependent manner. Therefore, C. butyricum is proposed as a feasible alternative feed additive for the production of healthier Peking duck meat with favorable properties.
Subclinical ketosis (SCK) in dairy cows, a common metabolic disorder during the peripartal period, is accompanied by systemic inflammation. Excessive release of azurophil granule (AG) contents during degranulation of polymorphonuclear neutrophils (PMN) could contribute to systemic inflammation in SCK cows. Although the increase in blood free fatty acids (FFA) in SCK cows may promote AG degranulation from PMN, the underlying mechanisms are unclear. Thirty multiparous cows (within 3 wk postpartum) with similar lactation numbers (median = 3, range = 2-4) and days in milk (median = 6, range = 3-15) were classified based on serum β-hydroxybutyrate (BHB) level as control (n = 15, BHB < 0.6 mM) or SCK (n = 15, 1.2 mM < BHB < 3.0 mM). Cows with SCK had greater levels of serum haptoglobin, serum amyloid A, IL-1β, IL-6, IL-8 and tumor necrosis factor-α. These proinflammatory factors had strong positive correlations with myeloperoxidase (MPO), a marker protein of PMN AG, whose content was greater in the serum of SCK cows. Both the number of AG and the protein abundance of MPO were lower in PMN isolated from SCK cows. Additionally, we found a greater ratio of blood CH138A + /CD63 high cells and greater mean fluorescence intensity of CD63 on the PMN membrane, further confirming the greater degree of AG degranulation in cows with SCK. In vitro FFA dose response (0, 0.3, 0.6, 1.2, and 2.4 mM for 4 h) and time course (0, 0.5, 1, 2, and 4 h with 0.6 mM) experiments were performed on PMN isolated from control cows. The increase in MPO content in extracellular supernatant resulting from those experiments led to the selection of 0.6 mM FFA for 1 h duration as conditions for subsequent studies. After FFA treatment, release of intracellular MPO was increased along with increased levels of CD63 mean fluorescence intensity on the PMN membrane, confirming that FFA promoted degranulation of AG. In addition, FFA treatment increased reactive oxygen species (ROS) production by PMN, an effect that was attenuated by incubation with diphenyleneiodonium chloride (DPI), a NADPH oxidase-derived ROS inhibitor. The mitochondrial-derived ROS inhibitor carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) did not affect ROS in response to FFA treatment. Treatment with FFA increased p47 phosphorylation and mRNA abundance of NCF1, NCF2, and CYBB in PMN. Furthermore, DPI, but not FCCP, dampened the degranulation of PMN AG induced by FFA in vitro. These data suggested that the degranulation of AG in PMN induced by FFA was mediated by NADPH oxidase-derived ROS. As verified ex vivo, PMN from SCK cows had greater levels of ROS, phosphorylation of p47, and mRNA abundance of NCF1, NCF2, and CYBB. Overall, the present study revealed that high blood concentrations of FFA in SCK cows induce the production of NADPH oxidase-derived ROS, thereby promoting degranulation of AG in PMN. The stimulatory effect of FFA on the release of AG content during degranulation, especially MPO, provides a new insight into the systemic inflammation experienced by peripartal co...
Ketosis in dairy cows often occurs in the peripartal period and is accompanied by immune dysfunction. High concentrations of β-hydroxybutyrate (BHB) in peripheral blood during ketosis inhibits the release of neutrophil extracellular traps (NET) and contributes to immune dysfunction. However, the mechanisms whereby BHB affects NET release remains unclear. In this study, 5 healthy peripartal dairy cows (within 3 wk postpartum) with serum BHB concentrations <0.6 mM and glucose concentrations >3.5 mM were used as blood donors. Blood samples were collected before feeding, and the isolated polymorphonuclear neutrophils were incubated with 3 mM BHB for different times. Inhibition of Cit-H3 (citrullinated histone 3) protein abundance, a marker of NET activation, in response to BHB was used to determine an optimal incubation time for in vitro experiments. Four hours was selected as the optimal duration of BHB treatment. Phorbol-12-myristate-13-acetate (PMA) was used to induce the release of NET in vitro. The BHB treatment with or without PMA treatment decreased protein abundance of Cit-H3 and PAD4 (arginine deiminase 4) and increased neutrophil elastase. Immunofluorescence and scanning electron microscope analyses revealed that BHB treatment inhibited PMA-induced NET release. The BHB treatment also decreased double strain DNA content in the supernatant, further confirming the inhibitory effect of BHB on NET release. Furthermore, BHB treatment decreased the level of intracellular reactive oxygen species (ROS), phosphorylation level of p47, and protein abundance of Rac2, suggesting that BHB-induced NET inhibition may have been caused by decreased NADPH oxidase-derived ROS. The phosphorylation level of phosphoinositide 3-kinase (PI3K), an important upstream regulator of NADPH oxidase, was attenuated by BHB treatment. To confirm the involvement of PI3K signaling pathway in BHB-induced NET inhibition, 740Y-P, a potent activator of PI3K signaling pathway, was used. Data indicated that 740Y-P relieved the inhibitory effects of BHB on ROS production and NADPH oxidase activation. Importantly, as revealed by immunofluorescence and scanning electron microscopy analyses, 740Y-P also dampened the inhibitory effect of BHB on NET release and the protein abundance of Cit-H3 and PAD4. Overall, the present study revealed that high concentration of BHB impairs NET release through inhibiting PI3K-mediated NADPH oxidase ROS production. These findings help partly explain the immune dysfunction in cows experiencing negative energy balance or ketosis in early lactation.
Peripartum dairy cows experience negative energy balance, characterized by high concentrations of blood free fatty acids (FFA) and immune dysfunction. Palmitic acid (PA), the most abundant saturated fatty acid in cow blood, is not only an energy precursor, but causes cellular dysfunction when in excess. Neutrophil extrareleasing neutrophils. Overall, these data demonstrate PA inhibits NET release by suppressing autophagic flux, which provides information for understanding the immune dysfunction in postpartum cows.
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