Background: High-fat diets (HFD) are known to enhance feed conversion ratio in broiler chickens, yet they can also result in hepatic fat accumulation. Bile acids (BAs) and gut microbiota also play key roles in the formation of fatty liver. In this study, our objective was to elucidate the mechanisms through which BA supplementation reduces hepatic fat deposition in broiler chickens, with a focus on the involvement of gut microbiota and liver BA composition. Results: Newly hatched broiler chickens were allocated to either a low-fat diet (LFD) or HFD, supplemented with or without BAs, and subsequently assessed their impacts on gut microbiota, hepatic lipid metabolism, and hepatic BA composition. Our findings showed that BA supplementation significantly reduced plasma and liver tissue triglyceride (TG) levels in 42-day-old broiler chickens (P < 0.05), concurrently with a significant decrease in the expression levels of fatty acid synthase (FAS) in liver tissue (P < 0.05). These results suggest that BA supplementation effectively diminishes hepatic fat deposition. Under the LFD, BAs supplementation increased the BA content and ratio of Non 12-OH BAs/12-OH BAs in the liver and increased the Akkermansia abundance in cecum. Under the HFD, BA supplementation decreased the BAs and increased the relative abundances of chenodeoxycholic acid (CDCA) and cholic acid (CA) in hepatic tissue, while the relative abundances of Bacteroides were dramatically reduced and the Bifidobacterium, Escherichia, and Lactobacillus were increased in cecum. Correlation analyses showed a significant positive correlation between the Akkermansia abundance and Non 12-OH BA content under the LFD, and presented a significant negative correlation between the Bacteroides abundance and CA or CDCA content under the HFD. Conclusions: The results indicate that supplementation of BAs in both LFD and HFD may ameliorate hepatic fat deposition in broiler chickens with the involvement of differentiated microbiota–bile acid profile pathways. Graphical

Background: L-citrulline (L-Cit), a non-protein amino acid, has been implicated in several physiological functions including anti-inflammatory, anti-oxidative, and hypothermic roles, however, there is a paucity of information with regards to its potential in poultry production.Methods: This study was designed to investigate the effects of dietary L-Cit supplementation on the production performance, nitric oxide production, and antioxidant status of laying hens during summer period. Hy-Line Brown laying hens (n = 288, 34 weeks old) were allotted to four treatment, 6 replicates of 12 chickens each. Dietary treatments of control (basal diets), 0.25%, 0.50% and 1.00% L-Cit supplementation were fed to chickens for eight (8) weeks. Production performance, free amino acid profiles, nitric oxide production, and antioxidant properties were measured. Blood samples were collected at the 4^thand 8^thweeks of the experiment.Results: Air temperature monitoring indicated an average daily minimum and maximum temperatures of 25.02 °C and 31.01 °C respectively. Dietary supplementation with L-Cit did not influence (P > 0.05) the production performance, and rectal temperature of laying hens. Egg shape index was increased (P < 0.05) with increasing levels of L-Cit. Serum-free content of arginine, citrulline, ornithine, tryptophan, histidine, GABA, and cystathionine were elevated, but taurine declined with L-Cit diets. Plasma nitric oxide (NO_x) concentration was highest at 1% L-Cit. Likewise, nitric oxide synthase (NOS) activity for total NOS (tNOS) and inducible NOS (iNOS) were upregulated with increasing L-Cit levels, although, tNOS was not affected at the 4^thweek. Anti-oxidant enzymes including catalase and superoxide dismutase (SOD) were increased with L-Cit supplementation, however, SOD activity was unchanged at 4^thweek, while total anti-oxidant capacity increased at the 8^thweek. L-Cit supplementation attenuated the extent of lipid peroxidation, and also inhibited glutathione peroxidase activity.Conclusion: Dietary L-Cit supplementation modulated systemic arginine metabolism, nitric oxide synthesis, antioxidant defense system, and increased the egg shape index of laying hens during the summer season. 1% L-Cit supplementation proved most effective in potentiating these effects and may be adopted for feed formulation strategies.

In this study, chlorogenic acid (CGA) was injected into the amniotic cavity of chicken embryos to study the effects of in ovo feeding of CGA on the antioxidant capacity of postnatal broilers. On the 17th day of embryonic age, a total of 300 healthy broiler fertile eggs with similar weights were randomly subjected to five groups as follows; in ovo injection with 0.5 ml CGA at 4 mg/egg (4CGA) or 7 mg/egg (7CGA) or 10 mg/egg (10CGA), or sham-injection with saline (positive control, PC) or no injection (negative control, NC). Each group had six replicates of ten embryos. Six healthy chicks with similar body weights hatched from each replicate were selected and reared until heat stress treatment (35°C ± 1°C, 8 h/d) at 28–42 days of age. The results showed that there was no significant difference in the hatching rate between the groups (p > 0.05). After heat stress treatment, 4CGA group showed an improved intestinal morphology which was demonstrated by a higher villus height in the duodenum and a higher villus height/crypt depth ratio in the jejunum, compared with the NC group (p < 0.05). The antioxidant capacity of chickens was improved by in ovo feeding of CGA since 4CGA decreased the plasma content of malondialdehyde (MDA) (p < 0.05), whereas, it increased the superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) activities compared with NC group (p < 0.05). Also, the MDA content of the different injection groups had a quadratic effect, with the 4CGA group having the lowest MDA content (P_quadratic < 0.05). In the duodenum, 4CGA injection significantly increased the mRNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (H O -1), glutathione synthetase (GSS), and SOD1 compared to the NC and PC groups (p < 0.05). The mRNA expressions of glutathione reductase (GSR) and GPX7 were significantly increased in all CGA-treated groups compared with the PC group (p < 0.05), while the mRNA expression of CAT was significantly increased by 4CGA group than the NC group (p < 0.05). The mRNA expressions of epigenetic-related genes, ten eleven translocation 1 and 2 (Tet1 and Tet2), and DNA-methyltransferase 3 alpha (DNMT3A) in the duodenum of 4CGA injected group was significantly increased compared with the NC and PC groups (p < 0.05). The mRNA expressions of Nrf2, SOD1, and Tet2 showed a significant quadratic effects with the 4CGA group having the highest expression (P_quadratic < 0.05). In conclusion, in ovo feeding of CGA alleviated heat stress-induced intestinal oxidative damage. Injection with CGA of 4 mg/egg is considered most effective due to its actions in improving intestinal antioxidant capacity, especially in the duodenum. The antioxidant effects of in ovo CGA on postnatal heat-stressed broilers may be related to its regulation of epigenetic mechanisms. Thus, this study provides technical knowledge to support the in ovo feeding of CGA to alleviate oxidative stress in postnatal heat-stressed broilers.

Calcium and phosphorus homeostasis is crucial for the performance and bone health of laying hens. The calcium and phosphorus transporters play an important role in calcium and phosphorus absorption, reabsorption, and excretion. In the present study, Hy-Line Brown layers were sampled at brooding period (1, 4, 6 wk), growing and developing period (12, 18 wk) and laying period (20, 28, 80 wk) respectively, and the calcium transporters CaBP-D28k and PMCA1b and phosphorus transporters NPt2a and NPt2b were respectively measured in duodenum, jejunum, ileum and kidney. The result showed that serum calcium increased (P < 0.0001) and phosphorus level fluctuated (P = 0.0019), while alkaline phosphatase activity decreased with age (P < 0.0001). The mRNA and protein expressions of CaBP-D28k in small intestine elevated after maturity (P ≤ 0.0001). In contrast, the PMCA1b mRNA showed a trend to increase with age in jejunum (P = 0.0059) and ileum (P = 0.0825) whereas there was a decrease for PMCA1b protein in 12-18 wk (P ≤ 0.0009). The peak of NPt2b mRNA were observed at 28 wk in duodenum (P = 0.0001) and jejunum (P = 0.0622) and 1 wk in ileum (P < 0.0001). The NPt2b protein expression reached the top point at 4 or 6 wk and 20 wk and decreased to the lowest point around 12 wk (P ≤ 0.0850). In kidney, CaBP-D28k mRNA was not influenced by age (P = 0.4999), while PMCA1b highly expressed in 6-12 wk (P = 0.0003). The protein expressions of CaBP-D28k (P = 0.0148) and PMCA1b (P = 0.0003) decreased with age and lowly expressed in 12-18 wk and increased thereafter. In contrast, NPt2a expression increased steadily with age and decreased at 80 wk (P < 0.0001). In conclusion, the expressions of intestinal calcium and phosphorus transporters were changed by age, intestinal CaBP-D28k and renal NPt2a showed a dramatic increase after maturity, which coincide with the increased calcium and phosphorus requirement for egg production.

Intestinal inflammation induced by heat stress is an important factor restricting the healthy growth of broilers. The aim of this study was to evaluate the effect of chicken embryo thermal manipulation (39.5 ℃ and 65 % RH for 3 h daily during 16-18 th embryonic age) on intestinal inflammation in broilers under postnatal heat stress and to investigate whether transient receptor potential V4 (TRPV4) plays a role in this process. Our results suggest that broilers with embryo thermal manipulation experience could delay the rising of rectal temperature during postnatal heat stress (P < 0.05), and had better production performance (P < 0.05), intestinal morphological parameters (P < 0.05) and higher expression of tight junction related genes (P < 0.05). The increased serum lipopolysaccharide (LPS) content, activation of nuclear factor-kappa B (NF-κB) signaling pathway and the increased expression of pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor alpha (TNF-α) in jejunum during postnatal heat stress were alleviated by embryo thermal manipulation (P < 0.05). Postnatal heat stress induced an increase in mRNA and protein expression of TRPV4 in jejunum (P < 0.05), but had no effect on broilers which experienced embryo thermal manipulation (P > 0.05). Inhibition of TRPV4 reduced LPS-induced Ca²⁺ influx and restrained the activation of NF-κB signaling pathway and the expression of downstream pro-inflammatory cytokines (P < 0.05). The expression of DNA methyltransferase (DNMT) in the jejunum of broilers exposed to postnatal heat stress was increased by embryo thermal manipulation (P < 0.05). The DNA methylation level of TRPV4 promoter region was detected, and the results showed that embryo thermal manipulation increased the DNA methylation level of TRPV4 promoter region (P < 0.05). In conclusion, Chicken embryo thermal manipulation can alleviate jejunal inflammation in broilers under postnatal heat stress. This may be due to the decreased circulating LPS or the increased DNA methylation level in the promoter region of TRPV4, which inhibits TRPV4 expression, thereby reducing Ca²⁺ influx, and finally alleviating inflammation by affecting NF-κB signaling pathway. The work is an attempt to understand the mechanism involved in alleviation of adverse effects of heat stress during postnatal life through prenatal thermal manipulation and to reveal the important role of epigenetics.