The objective of this study was to determine the effects of dietary soy saponin (SS) on the antioxidant and immune functions of laying hens. Two hundred seventy 22-week-old Hy-line gray layers were randomly allocated into three treatment groups: a control group (Control) fed a basal diet with low soybean meal and groups supplemented with 50 and 500 mg/kg SS (50 SS and 500 SS). After ten weeks, eight chickens from each treatment group were anesthetized and sacrificed to collect tissue samples. In the 50 and 500 SS groups, results showed that the levels of superoxide dismutase (SOD) in serum and spleen were elevated, and the content of malondialdehyde (MDA) in serum decreased. The mRNA levels of genes such as NF-E2-related factor 2 (Nrf-2) in the ileum and Nrf-2 and SOD in the spleen were also upregulated. In addition, the skin irritation index of phytohemagglutinin (PHA), the number of serum white blood cells, and lymphocytes were elevated in the two groups. At the same time, the number of monocytes in the blood increased in the 50 SS group,and it was significantly higher in the 500 SS group. In addition, the mRNA levels of lysozyme (LYZ) and IFN-γ in the spleen were upregulated, similar to the mRNA levels of zinc finger protein A20 (A20) in the ileum. Furthermore, the mRNA levels of NF-κB and IL-6 in the ileum were downregulated. In conclusion, with supplementation of 50 and 500 mg/kg SS in low soybean meal diets, the antioxidant, and immune functions of laying hens were improved. More importantly, the target for SS to exert biological effects on laying hens may be in the intestine and spleen tissues.

Production of pimpled or sandpaper-shelled eggs (SE) is a major problem in aged hens. Probiotics can improve eggshell quality; however, the relationship between SE production and gut bacteria remains unclear. Here, 1200 450-d-old Hy-line hens were assigned to four groups (300 hens each), with the control group fed basal diet and treatment groups fed basal diet plus 500, 1000, and 1500 mg/kg of Clostridium butyricum and Bacillus subtilis, respectively. After 4 weeks, probiotics significantly decreased the SE rate from 42.51% to 28.02%. To address why probiotics reduced SE rate, the hens that only produced normal eggs (NE) or SE based on a 2-week assessment were assigned to three groups (NE, SE, and SEP groups; 10 hens each), with the NE and SE groups fed a basal diet and SEP group fed a basal diet plus 1000 mg/kg probiotics. After 4 weeks, ileal tissues from eight birds/group were collected for histomorphological and gene expression analyses, and the ileal content was collected from five birds/group for 16S rDNA sequencing analysis. The data showed that probiotics significantly increased the villus length and ratio of villus length to crypt depth. Quantitative PCR analysis indicated that there were no significant differences in the expression of genes related to tight junctions, nutrient transport, and calcium absorption among the groups (except TRPV6, P<0.001). The 16S rDNA sequencing analysis indicated that the alpha-diversity of gut bacteria in the SEP group was the highest among the groups. The Firmicutes phylum was dominant in the NE and SEP groups, whereas the Proteobacteria phylum was dominant in the SE group. Together, these results suggest that probiotics can significantly influence the intestinal structure and composition of the intestinal microbiota, which may lead to a reduction in the SE rate in aged hens.

The effect of the application of copper waterline on the performance and gut health of aged laying hens was evaluated in this study. Forty-eight 70-week-old laying hens were divided into two groups (three replicates of eight hens each): control and copper (Cu) groups provided with normal polyvinyl chloride (PVC) waterline or Cu waterline. The laying performance was measured during the four-week period of the experiment. The intestinal antioxidant status and the microbiota diversity of the cecal content were determined. Moreover, a bacteriostasis test on Escherichia coli and Salmonella enteritidis was conducted after inoculation in waterline and hens, respectively. The water Cu²⁺ content was increased by Cu waterline compared to the control (P<0.05). Cu waterline had no detectable effect on most production performances, however, it increased the egg weight (P<0.05). Cu waterline increased the Cu level in the eggshell. Cu level in excreta increased with time, especially in the final two weeks, however, there was no significant change in fecal Cu excretion. The lipid peroxidation product malondialdehyde content in ileum decreased (P<0.01), while the activities of CuZn-superoxide dismutase (SOD) of ileum and glutathione peroxidase (GSH-PX) activity of jejunum and ileum increased after Cu treatment. The relative abundance and richness of cecal microbiota increased after Cu treatment (P<0.05). Cu waterline changed the microbial composition, including the increased proportion of Methanocorpusculum, Paludibacter, and decreased proportion of Fucobacterium, Anaerobiospirillum, and Campylobacter. The colonization of E. coli and S. enteritidis in Cu waterline was suppressed by Cu treatment, indicating that Cu waterline had potential antibacterial properties. The result suggests that Cu waterline could inhibit the colonization of pathogenic microorganisms such as E. coli and Salmonella and facilitate the enrichment of cecal microbiota diversity.

We aimed to compare the combinatorial effect of 3,4,5-trihydroxybenzoic acid (THB) and oregano extracts (OE) with THB alone on the growth performance and elimination of deleterious effects in coccidiosis-infected broilers. A total of 210 one-day-old broilers were randomly assigned to one of five dietary treatments, with six replicates each, for 35 days. Dietary treatments were: 1) non-challenged, non-treated (NC); 2) challenged, non-treated (PC); 3) PC+Salinomycin (0.05 g/kg; AB); 4) PC+THB (0.1 g/kg; THB); and 5) PC+THB+OE (0.1 g/kg; COM). On day 14, all groups except for NC were challenged with a 10-fold dose of Livacox^® T anticoccidial vaccine to induce mild coccidiosis. All treatments significantly improved (P<0.05) body weight, average daily gain, and average daily feed intake, compared to PC, on days 21, 28, and 35. However, all treatments significantly reduced (P<0.05) the feed conversion ratio of PC by more than 14.60% on day 35, 11.76% during growing period, and 10.36% through the entire period. Broilers receiving anticoccidial treatments had 54.23% and 51.86% lower lesion scores (P<0.05) at 4 and 7 days post-infection, respectively, compared to PC. Additionally, the villus height of COM was significantly longer (P<0.05) than that of THB. Although the molecular action of COM remains unclear, OE addition to THB reduced the shedding of oocysts better than THB alone (P<0.05, 9-11 days post-infection). Most importantly, COM effectively minimized the mortality of challenged birds from as high as 11.90% (PC) to 0%, a level similar to NC and AB, while THB maintained a mortality of 2.38%. In conclusion, the anticoccidial effect of THB can be enhanced by the addition of OE for better animal performance and the elimination of deleterious effects from coccidiosis-infected broilers for 35 days.

Increasing food loss and waste (FLW) is a global problem, and efforts are being made to use waste food as potential livestock feed material. The amount of self-supplied feed is lower in Japan than in other countries, and the government recommends FLW use for animal feed. Sake (Japanese rice wine) is a traditional alcoholic beverage. During the sake manufacturing process, large amounts of squeezed solids or “lees” (sake lees) are generated. Sake lees are nutritious and functional, but are prone to spoilage. In this study, we investigated whether sake lees should be mixed with animal feed immediately or after drying. To assess the usefulness of sake lees as a poultry feed ingredient and determine the effect of sake lees on intestinal immunity, we performed a feeding trial with three treatments: a raw sake lees (RSL) diet, dried sake lees (DSL) diet, and control diet. Three-week-old broilers were fed these diets (n＝8 per group) for two weeks. We then calculated feed efficiency and performed RT-qPCR to assess the effects of diet on intestinal immunity. The growth performance in the RSL diet group was equivalent to that in the control diet group. The DSL diet became difficult for broilers to eat, resulting in decreased growth performance. In the ileum of RSL-diet broilers, the mRNA expression levels of TGF-β1 and avian β-defensin (AvBD)12 were significantly increased compared to those of control diet broilers (p<0.05), and a significant correlation was observed between the two genes (p<0.05). Our results indicated that sake lees should not be dried and should be mixed immediately with feed, and this sake lees when fed to chicken activates the intestinal immunity. However, sake lees have a lower fat content than corn, and it is thus important to combine sake lees with high-energy feed.

The aim of this study was to evaluate the effects of B. subtilis-fermented products (BSFP) on growth performance, intestinal inflammatory gene expression, and cecal microbiota community in broilers challenged with dextran sulfate sodium (DSS) in a 14-day experiment. A total of 32, 1-day-old male broiler chickens (Ross 308), were randomly divided into four groups of eight birds per group and reared individually (n＝8). The treatments consisted of a control diet without supplementation and DSS challenge, control diet plus 1.5% DSS, control diet plus 1 g/kg BSFP in combination with 1.5% DSS, and control diet plus 3 g/kg of BSFP in combination with 1.5% DSS. The results showed that BSFP supplementation (1 and 3 g/kg) partially improved body weight and average daily gain in broilers under DSS challenge. Relative to DSS treatment alone, BSFP supplementation dose-dependently increased the body weight of broilers at 7 days of age, with the average daily gain being at 1 to 7 days of age. BSFP supplementation (1 and 3 g/kg) alleviated intestinal inflammatory gene expression in broilers under DSS challenge. The richness and evenness of bacterial species in cecal digesta increased in a dose-dependent manner in the groups treated with BSFP (1 and 3 g/kg) in combination with DSS challenge, compared with the control group. Unweighted principal coordinate analysis indicated distinct clusters separating the group treated with 3 g/kg of BSFP in combination with DSS challenge from the other three groups. The abundance of short-chain fatty acid-producing bacteria (genus Ruminococcaceae_unclassified) increased and that of mucin-degrading bacteria (genus Ruminococcus torques group) decreased in the cecal digesta of broilers fed 3 g/kg of BSFP, compared with the control group. In conclusion, BSFP supplementation dose-dependently improved growth performance, reduced gut inflammation, and regulated the cecal microbiota of broilers exposed to DSS challenge during the starter phase.

The current study aimed to evaluate the efficacy of selenium nanoparticles (SeNPs), combined or loaded with chitosan (COS), in broiler chickens reared under standard management protocols. The parameters under investigation were production performance, organ development, components of the intestinal barrier, and ileal microbial count. Two hundred and forty day-old chicks were raised in five groups, with each group containing eight replicates (n＝6/replicate). The control group received a basal diet whereas the other four groups received basal diets supplemented with SeNPs (0.5 mg/kg), COS (200 mg/kg), SeNPs+COS (0.5 mg/kg SeNPs + 200 mg/kg COS), and SeNPs-loaded COS (SeNPs-L-COS) (200 mg/kg) respectively. On day 35, two birds/replicate were sampled to collect the viscera under investigation. The results revealed that dietary inclusion of SeNPs-L-COS increased (p<0.05) the body weight gain and improved (p<0.05) feed conversion ratio. Similarly, SeNPs-L-COS supplementation increased (p<0.05) the small intestinal villus surface area as well as the count of acidic goblet cells and intraepithelial lymphocytes when compared with the control group. Whereas the total goblet cell count was higher (p<0.05) in the small intestines of both the SeNPs+COS and SeNPs-L-COS groups. Microbial analysis of ileal contents also revealed an increase (p<0.05) in Lactobacilli species count with a concurrent decrease (p<0.05) in Escherichia coli count in the SeNPs-L-COS group when compared with the COS and control groups. Based on the results of the current trial, we can conclude that supplementation with SeNPs-L-COS is a superior combination for promoting the gut health and performance of broilers.

It has been reported that the supplementation of chicken diet with polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) affects the qualities of eggs and meat. Previous studies have shown that a functional fatty acid taste receptor, G protein-coupled receptor 120 (GPR120), is broadly expressed in chicken oral and gastrointestinal tissues, and chickens have a gustatory perception of oleic acid, which is a chicken GPR120 agonist. The aim of this study was to elucidate the role of chicken GPR120 in response to PUFAs in chicken diets. Ca²⁺ imaging analyses revealed that chicken GPR120 was activated by AA, EPA, and DHA in a concentration-dependent manner. These results suggest that chickens can detect PUFAs via GPR120 in the oral and gastrointestinal tissues, implying that chickens have a gustatory perception of PUFAs.

The pungency induced by spices and herbs plays an important role in food choice and appetite, and it is suggested that adding spices and herbs to feed as natural alternatives to antibiotics has beneficial effects in poultry farming. However, our knowledge of the chemosensory perception of herbal compounds in chickens is limited. Transient receptor potential ankyrin 1 (TRPA1) is involved in the sensory perception of various herbal compounds. Here, we performed calcium imaging and electrophysiological analyses using cells transiently expressing chicken TRPA1 (cTRPA1) and identified two novel cTRPA1 ligands—eugenol and thymol. In a behavioral assay, chickens responded to cTRPA1 ligands, including eugenol, thymol, cinnamaldehyde, carvacrol, and allyl isothiocyanate. These results provide evidence that chickens have a functional TRPA1 channel and chemosensory perception of various herbal compounds.

This study was conducted to investigate the effects of increasing stocking density under suitable environmental conditions on the performance and ileal microbiota of broilers. A total of 108 Arbor Acres male broilers (28 days old) were allocated to a normal stocking density (NSD, normal stocking density; 31 kg/m²) and a maximum allowed stocking density group (MSD, maximum stocking density; 39 kg/m²). All birds were reared at a constant temperature of 21°C. At 42 days of age, bacterial DNA was extracted from ileal content, and the V3-4 hypervariable region of 16S rRNA was amplified. Increasing stocking density had no significant effect on average daily gain, average daily feed intake, and feed conversion ratio (P>0.05). The alpha and beta diversities of the ileal microbiomes did not differ significantly between the NSD and MSD groups; however, increasing stocking density altered the composition of ileal microbiota. The relative abundance of Lactobacillales, including Lactobacillus, Enterococcus, and Streptococcus, significantly decreased in MSD broilers, compared with NSD broilers. The present results suggest that even under suitable environmental conditions, an increase in stocking density to a level of 39 kg/m² may disturb the composition of ileal microbiota in broilers. Further studies are needed to determine the reasons and the potential consequences for animal health and physiology.

The present study aimed to determine whether lactic acid content is associated with the intensity of the sour taste of Choshu-Kurokashiwa broth. Chicken broth was prepared using breast (pectoralis major) and leg (thigh + drumstick) meat of male and female Choshu-Kurokashiwa and broilers. These broths were assessed by a screened and trained panel and analyzed for sour taste substances (lactic, citric, pyruvic, malic, succinic, acetic, phosphoric, aspartic, and glutamic acids) and pH. The sensory sour taste was significantly higher in the Choshu-Kurokashiwa breast broth than in the broiler breast broth (P<0.001), and no significant difference was observed in the leg broths among the breeds (P>0.05). Choshu-Kurokashiwa breast broth had a significantly higher lactic acid content than broiler breast broth (P<0.001). The leg meat broth of male Choshu-Kurokashiwa had a significantly lower lactic acid content than that of female Choshu-Kurokashiwa and broiler leg meat (P<0.01). The sensory sour taste score was significantly and positively correlated with lactic acid content in the breast broth (P<0.001), but not in the leg broth (P>0.1). No other organic acids were detected. Phosphoric acid and glutamic acid contents were higher in broiler broth than in Choshu-Kurokashiwa broth for both breast and leg meat (P<0.001). In the breast broth, the aspartic acid content was not significantly different (P>0.1), and in the leg broth, it was higher in broiler and female Choshu-Kurokashiwa broth than in male Choshu-Kurokashiwa broth (P<0.001). The present study suggests that high lactic acid content contributes to the sour taste of the Choshu-Kurokashiwa breast broth and demonstrated that the lactic acid content is an essential indicator for determining the sour taste of Choshu-Kurokashiwa breast meat.