Maternal riboflavin deficiency (RD) induces embryonic death in poultry. The underlying mechanisms, however, remain to be established and an overview of molecular alterations at the protein level is still lacking. We investigated embryonic hepatic proteome changes induced by maternal RD to explain embryonic death. A total of 80 45-week-old breeding female ducks were divided into two groups of 40 birds each, and all birds were raised individually for 8 weeks. All the female ducks received either a RD or a riboflavin adequate (control, CON) diet, which supplemented the basal diet with 0 or 10 mg riboflavin /kg of diet respectively. The riboflavin concentrations of maternal plasma and egg yolk, as well as egg hatchability declined markedly in the RD group compared to those in the CON group after 2 weeks, and declined further over time. The hepatic proteome of E13 viable embryos from 8-week fertile eggs showed that 223 proteins were upregulated and 366 proteins were downregulated (> 1.5-fold change) in the RD group compared to those in the CON group. Pathway analysis showed that differentially expressed proteins were mainly enriched in the fatty acid beta-oxidation, electron transport chain (ETC), and tricarboxylic acid (TCA) cycle. Specifically, all the proteins involved in the fatty acid beta-oxidation and ETC, as well as six out of seven proteins involved in the TCA cycle, were diminished in the RD group, indicating that these processes could be impaired by RD. Maternal RD leads to embryonic death of offspring and is associated with impaired energy generation processes, indicated by a number of downregulated proteins involved in the fatty acid beta-oxidation, ETC, and TCA cycle in the hepatic of duck embryos. These findings contribute to our understanding of the mechanisms of liver metabolic disorders due to maternal RD.

Riboflavin is an essential micronutrient and a precursor of flavin mononucleotide and flavin adenine dinucleotide for maintaining cell homeostasis. Riboflavin deficiency (RD) induces cell apoptosis. Endoplasmic reticulum (ER) stress is considered to induce apoptosis, and C/EBP homologous protein (CHOP) is a key pathway involved in this process. However, whether RD-induced apoptosis is mediated by ER stress and the CHOP pathway remains unclear and needs further investigation. Therefore, the current study presents the effect of RD on ER stress and apoptosis in the human hepatoma cell line (HepG2). Firstly, cells were cultured in a RD medium (4.55 nM riboflavin) and a control (CON) medium (1005 nM riboflavin). We conducted an observation of cell microstructure characterization and determining apoptosis. Subsequently, 4-phenyl butyric acid (4-PBA), an ER stress inhibitor, was used in HepG2 cells to investigate the role of ER stress in RD-induced apoptosis. Finally, CHOP siRNA was transfected into HepG2 cells to validate whether RD triggered ER stress-mediated apoptosis by the CHOP pathway. The results show that RD inhibited cell proliferation and caused ER stress, as well as increased the expression of ER stress markers (CHOP, 78 kDa glucose-regulated protein, activating transcription factor 6) (p < 0.05). Furthermore, RD increased the cell apoptosis rate, enhanced the expression of proapoptotic markers (B-cell lymphoma 2-associated X, Caspase 3), and decreased the expression of the antiapoptotic marker (B-cell lymphoma 2) (p < 0.05). The 4-PBA treatment and CHOP knockdown markedly alleviated RD-induced cell apoptosis. These results demonstrate that RD induces cell apoptosis by triggering ER stress and the CHOP pathway.

• Dietary riboflavin supplementation improves breast meat quality of ducks. • Dietary riboflavin supplementation increases muscle n3-PUFA and PG contents. • Dietary riboflavin supplementation increases two odorants contents of muscle. • Dietary riboflavin supplementation enhances muscle antioxidant capacity. • Dietary riboflavin supplementation activates mitochondrial aerobic respiration.

Background: Although methionine (Met), the first-limiting dietary amino acid, has crucial roles in growth and regulation of lipid metabolism in ducks, mechanisms underlying are not well understood. Therefore, the objective was to use dietary Met deficiency to investigate the involvement of Met in lipid metabolism and fat accumulation of Pekin ducks. Methods: A total of 150 male Pekin ducks (15-d-old, 558.5 ± 4.4 g) were allocated into 5 groups (6 replicates with 5 birds each) and fed corn and soybean meal-based diets containing 0.28%, 0.35%, 0.43%, 0.50%, and 0.58% Met, respectively, for 4 weeks. Met-deficient (Met-D, 0.28% Met) and Met-adequate (Met-A, 0.43% Met) groups were selected for subsequent molecular studies. Serum, liver, and abdominal fat samples were collected to assess the genes and proteins involved in lipid metabolism of Pekin ducks and hepatocytes were cultured in vivo for verification. Results: Dietary Met deficiency caused growth depression and excess fat deposition that were ameliorated by feeding diets with adequate Met. Serum triglyceride and non-esterified fatty acid concentrations increased (P < 0.05), whereas serum concentrations of total cholesterol, low density lipoprotein cholesterol, total protein, and albumin decreased (P < 0.05) in Met-D ducks compared to those in Met-A ducks. Based on hepatic proteomics analyses, dietary Met deficiency suppressed expression of key proteins related to fatty acid transport, fatty acid oxidation, tricarboxylic acid cycle, glycolysis/gluconeogenesis, ketogenesis, and electron transport chain; selected key proteins had similar expression patterns verified by qRT-PCR and Western blotting, which indicated these processes were likely impaired. In vitro verification with hepatocyte models confirmed albumin expression was diminished by Met deficiency. Additionally, in abdominal fat, dietary Met deficiency increased adipocyte diameter and area (P < 0.05), and down-regulated (P < 0.05) of lipolytic genes and proteins, suggesting Met deficiency may suppress lipolysis in adipocyte. Conclusion: Taken together, these data demonstrated that dietary Met deficiency in Pekin ducks resulted in stunted growth and excess fat deposition, which may be related to suppression of fatty acids transportation and hepatic catabolism.

An experiment was conducted to investigate the effects of dietary riboflavin levels on reproductive performance, riboflavin status, and antioxidant status of laying duck breeders, to estimate the requirement of this vitamin for duck breeders. Different levels crystalline riboflavin (0, 2.5, 5, 10, and 15 mg/kg) were supplemented to a corn-soybean-corn gluten meal basal diet to produce 5 dietary treatments with different analyzed total riboflavin levels (1.48, 3.20, 6.30, 11.71, and 16.83 mg/kg). A total of 80 White Pekin duck breeders aged 40 wk were allotted to 5 dietary treatments of 16 birds each (8 replicates per treatment and 2 breeders per replicate), and all birds were raised individually for 9 wk. At the end of the experiment, reproductive performance, tissue riboflavin concentrations, and antioxidant status of White Pekin duck breeders were measured. The results showed that body weight, egg weight, egg production, and egg fertility were not affected by dietary riboflavin levels. However, among all of the laying duck breeders, the birds fed the basal diet without riboflavin supplementation had the lowest egg hatchability, plasma riboflavin, egg yolk riboflavin, and egg albumen riboflavin (P < 0.001). In addition, the duck breeders fed the basal diet without riboflavin supplementation showed the lowest antioxidant capacity indicated by greatest plasma malondialdehyde (MDA) content and lowest reduced glutathione content, total superoxide dismutase (T-SOD) activities, and total antioxidant capacity in both plasma (P < 0.001) and egg yolk (P < 0.001). These results revealed that dietary riboflavin supplementation improved the reproductive performance and antioxidant status of the duck breeders. According to the broken-line model, the riboflavin requirements (based on dietary total riboflavin) of laying duck breeders in terms of the egg hatchability, plasma riboflavin, egg yolk riboflavin, egg albumen riboflavin, plasma T-SOD activity, and plasma MDA content were 3.19, 7.42, 3.88, 7.44, 6.45, and 8.84 mg/kg, respectively.