Bacillus subtilis is one of the most popular commercial probiotics used in farm animal production. However, its potential mechanisms are not very clear. The aim of this study was to investigate the effects of dietary Bacillus subtilis on intestinal histomorphology, innate immunity, microbiota composition, transcriptomics, and related metabolomics. Twenty-four 48-week-old Lohman Pink-shell laying hens were randomly divided into two groups: a basic diet and the basic diet supplemented with Bacillus subtilis (0.5 g/kg) for a 9-week experiment. At the end of the experiment, tissues of the duodenum, ileum, and jejunum as well as cecal content of each bird were collected for microstructure, PCR, transcriptome, metabolome, and 16S rRNA analyses. The results showed that dietary Bacillus subtilis supplement had no effect on the intestinal microstructure. However, Bacillus subtilis increased mRNA expression of tight junction protein occludin (P < 0.05), while reduced mRNA expression of lipopolysaccharide-induced TNF factor (P < 0.01) in the duodenum. Moreover, transcriptomic results indicated that most of Bacillus subtilis supplement-induced differential genes were associated with inflammation and immunity, including cytochrome b-245 beta chain, transferrin, and purinergic receptor P2X 7, resulting in a decrease in Malondialdehyde level (P < 0.05) in the duodenum. In addition, at the genus level, Bacillus subtilis supplement enriched the potential beneficial bacteria, Candidatus_Soleaferrea (P = 0.02) but inhibited the harmful bacteria including Lachnospiraceae_FCS020_group, Ruminiclostridium, Lachnospiraceae_UCG-010, and Oxalobacter. Metabolomic results revealed that N-Acetylneuraminic acid and ADP were increased by fed Bacillus subtilis. These results suggest that dietary Bacillus subtilis could inhibit gut inflammation and improve antioxidative status and barrier integrity of the duodenum via regulating gut microbial composition in laying hens.

The aim of this study was to investigate the effects of osteocalcin (OCN) on fatty liver hemorrhagic syndrome (FLHS) in aged laying hens. Thirty 68-week-old White Plymouth laying hens were randomly assigned into conventional single-bird cages, and the cages were randomly allocated into one of 3 treatments (n = 10): normal diet (ND + vehicle, ND + V), high-fat diet (HFD + vehicle, HFD + V), and HFD + OCN (3 μg/bird, 1 time/2 d, i.m.) for 40 d. At day 30, oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT) were performed. At the end of experiment, the hens were euthanized followed by blood collection. The plasma aspartate transaminase (AST), alkaline phosphatase (ALP), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using an automatic biochemistry analyzer. Pathological changes in the liver were examined under both light and transmission electron microscopes. The plasma inflammatory factors including interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-α) were analyzed by ELISA, and the gene expressions of these inflammatory factors in the liver were analyzed by real-time PCR. The level of oxidative stress was evaluated using malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) assay kits, respectively. The results showed that HFD + V hens had more severe liver hemorrhage and fibrosis than ND + V hens (P < 0.05). The ultramicrostructural examination showed that hepatocytes of HFD + V hens exhibited necrotic pyknosis showing great intracellular electron, mitochondrial swelling, shrunk nucleus, and absence of autolysosomes. Osteocalcin mitigated HFD + V–induced pathological changes in aged laying hens. High-fat diet + OCN hens had higher insulin sensitivity; lower liver concentrations of MDA (P = 0.12) but higher GSH-Px (P < 0.05); and lower blood TNF-α concentrations (P < 0.05) and mRNA expressions (P < 0.05) than HFD + V hens. These results suggest OCN functions in preventing the FLHS process in old laying hens through inhibiting excessive energy diet-induced metabolic disorder, oxidative stress, and related pathological damage.

This study was to investigate the effects of Bacillus subtilis on production performance and bone pathophysiological characteristics of layers. Twenty-four 48-week-old Lohmann Pink-shell laying hens were randomly divided into two groups: a basic diet (control) and the basic diet mixed with Bacillus subtilis (0.5 g/kg) for a 60-day trial. Statistically, independent-sample t-test was used to assess the treatment differences. The results showed that Bacillus subtilis supplementation improved the percent of marketable eggs (p < 0.05) with reduced numbers of broken and soft-shelled eggs but had no effects on egg weight, height of albumen, yolk color, and Haugh unit (p > 0.05). Bacillus subtilis supplement also elevated maximum load (p = 0.06), maximum stress (p = 0.01), stiffness (p < 0.01), and Young’s modulus (p < 0.01) but suppressed maximum strain (p = 0.06) in the femur. In addition, compared with control birds, phosphorous concentration (p < 0.01) was reduced in serum at day 61 but increased in the femur (p < 0.05) in Bacillus subtilis fed birds. Bacillus subtilis fed birds also had lower magnesium concentrations in both femur (p = 0.04) and feces (p = 0.09). Furthermore, Bacillus subtilis increased plasma estrogen concentration (p = 0.01) and femur TNF receptor superfamily member 11b (OPG) expression (p < 0.05) but reduced plasma IL-1 (p < 0.01) and TNF-α (p < 0.01) concentrations. These results indicate that Bacillus subtilis could be used as a health promotor to reduce overproduction-induced inflammation and associated bone damage and to increase marketable egg production. The data provide evidence for developing a management strategy to use Bacillus subtilis as a feed additive to improve marketable egg production and health and welfare status of laying hens.

Intestinal microbiota functions such as an endocrine organ to regulate host physiological homeostasis and behavioral exhibition in stress responses via regulating the gut–brain axis in humans and other mammals. In humans, stress-induced dysbiosis of the gut microbiota leads to intestinal permeability, subsequently affecting the clinical course of neuropsychiatric disorders, increasing the frequency of aggression and related violent behaviors. Probiotics, as direct-fed microorganism, have been used as dietary supplements or functional foods to target gut microbiota (microbiome) for the prevention or therapeutic treatment of mental diseases including social stress-induced psychiatric disorders such as depression, anxiety, impulsivity, and schizophrenia. Similar function of the probiotics may present in laying hens due to the intestinal microbiota having a similar function between avian and mammals. In laying hens, some management practices such as hens reared in conventional cages or at a high stocking density may cause stress, leading to injurious behaviors such as aggressive pecking, severe feather pecking, and cannibalism, which is a critical issue facing the poultry industry due to negative effects on hen health and welfare with devastating economic consequences. We discuss the current development of using probiotic Bacillus subtilis to prevent or reduce injurious behavior in laying hens.

The aim of this study was to examine effects of various daylight exposure during the 24-h light-dark (L-D) cycle on growth performance, skeletal health, and welfare state in broilers. Environmental photoperiod and related circadian clock, the 24-h L-D cycle, are important factors in maintaining productive performance, pathophysiological homeostasis, and psychological reaction in humans and animals. Currently, various lighting programs as management tools for providing a satisfactory environmental condition have been used in commercial broiler production. Four hundred thirty-two 1-day-old Rose 308 broiler chicks were assigned to 24 pens (18 birds/pen). The pens were randomly assigned to 1 of 4 thermal and lighting control rooms, then the birds were exposed to (n = 6): 1) 12L, 2) 16L, 3) 18L, or 4) 20L at 15 d of age. Lighting program effects on bird body weight, behavioral patterns, bone health, and stress levels were evaluated from d 35 to d 45, respectively. The birds of 12L as well as 16L groups, reared under short photoperiods close to the natural 24-h L-D cycle, had improved production performance, leg bone health, and suppressed stress reaction compared to the birds of both 18L and 20L groups. Especially, 12L birds had heavier final body weight and averaged daily weight gain (P < 0.05), higher BMD and BMC with longer and wider femur (P < 0.05), lower H/L ratio (P < 0.05), and more birds reached the observer during the touch test (P < 0.05) but spent shorter latency during the tonic immobility test (P < 0.05). Taken together, the data suggest that supplying 12 h as well as 16L of daily light improves performance and health while decreasing stress levels in broilers, making it a potentially suitable approach for broiler production.

Osteocalcin (OCN) has a function in preventing fatty liver hemorrhagic syndrome (FLHS) in poultry. The aim of this study was to investigate the effects of OCN on fat emulsion stimulated chicken embryonic hepatocytes and related signaling pathways. The primary chicken embryonic hepatocytes were isolated from the incubated 15-day (E15) pathogen free eggs and cultured with dulbecco's modified eagle medium (DMEM). After the hepatocyte density reached 80%, the cells were divided into 5 groups: control group (CONT), fat emulsion group (FE, 10% FE, v/v), FE with ucOCN at 1 ng/mL (FE-LOCN), 3 ng/mL (FE-MOCN), and 9 ng/mL (FE-HOCN). In addition, 2 mM N-Acetyl-L-cysteine (NAC) a reactive oxygen species (ROS) scavenger, and 5 μM SP600125, a Jun N-terminal kinase (JNK) inhibitor, were added separately in to the DMEM with 10% FE to test effects of FE on the function of ROS-JNK signal pathway. The number of hepatocytes, cell ultra-microstructure, viability, and apoptosis were detected after 48 h treatment, and the protein expressions and enzyme concentrations were detected after 72 h treatment. The results showed that, compared to the control group, FE increased the triglyceride (TG) concentration and lipid droplets (LDs) in chicken embryonic hepatocytes (P < 0.05), and induced hepatocytic edema with obviously mitochondrial swelling, membrane damage, and cristae rupture. FE also decreased ATP concentration, increased ROS concentrations and mitochondrial DNA (mtDNA) copy number, promoted inflammatory interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α) concentrations and hepatocytic apoptosis rate, and raised phospho-c-Jun N-terminal kinase (p-JNK) protein expressions. Compared to the FE group, ucOCN significantly increased hepatocyte viability, reduced hepatocytic TG concentrations and LDs numbers, and alleviated hepatocytic edema and mitochondrial swelling. Furthermore, ucOCN significantly decreased ROS concentrations, increased ATP concentrations, reduced IL-1, IL-6, TNF-α concentrations and hepatocytic apoptosis rate, and inhibited p-JNK protein expressions (P < 0.05). NAC had the similar functions of ucOCN reduced the ROS concentration and inhibited the TNF-α protein expression and p-JNK/JNK ration. Similarly, SP600125 reduced p-JNK/JNK protein expression, IL-1, IL-6, TNF-α, and TG concentrations without effects on ROS concentration and hepatocytic apoptosis. These results suggest that ucOCN alleviates FE-induced mitochondrial damage, cellular edema, and apoptosis of hepatocytes. These results reveal that the functions of ucOCN in reducing fat accumulation and inflammatory reaction in chicken embryonic hepatocytes are mostly via inhibiting the ROS-JNK signal pathway.