Effects of quercetin and genistein on egg quality, lipid profiles, and immunity in laying hens.

BACKGROUND: After the peak laying stage, laying hens become susceptible to lipid accumulation and inflammatory reactions. The objective of this experiment was to examine the impact of quercetin and genistein on egg quality and lipid profiles in laying hens. A total of 240 Hy-Line Brown hens were randomly assigned to three dietary treatments. Each treatment had eight replicates, with ten hens in each replicate, and the hens were aged between 46 and 56 weeks. The test diets consisted of a corn-soybean meal-based basal diet, a basal diet supplemented with 300 mg kg-1 quercetin, and a basal diet supplemented with 300 mg kg-1 quercetin and 40 mg kg-1 genistein. RESULTS: Results showed that, separately, supplemental quercetin significantly improved egg quality (eggshell strength, albumen height, and Haugh unit, P < 0.05) and reduced the deposition of abdominal fat (P < 0.05). Our findings also showed that, separately or as a combination, supplemental quercetin and genistein significantly increased eggshell thickness (P < 0.05), decreased the levels of lipids in serum (low-density lipoprotein cholesterol, total cholesterol, total triglycerides, and non-esterified fatty acids, P < 0.05) and significantly increased serum immunoglobulins A and G (P < 0.05), and promoted the expression of splenic immune-related genes (IgA and IL-4, P < 0.05). CONCLUSION: This study confirmed that supplemental quercetin and genistein, either separately or in combination, can enhance eggshell thickness, lipid profiles, and immune function in aging hens. Moreover, both quercetin alone and quercetin + genistein exhibited similar abilities to lower lipid levels and improve immune function. © 2023 Society of Chemical Industry.

Dietary genistein increases microbiota-derived short chain fatty acid levels, modulates homeostasis of the aging gut, and extends healthspan and lifespan.

Age-related gastrointestinal decline contributes to whole-organism frailty and mortality. Genistein is known to have beneficial effects on age-related diseases, but its precise role in homeostasis of the aging gut remains to be elucidated. Here, wild-type aging mice and Zmpste24-/- progeroid mice were used to investigate the role of genistein in lifespan and homeostasis of the aging gut in mammals. A series of longitudinal, clinically relevant measurements were performed to evaluate the effect of genistein on healthspan. It was found that dietary genistein promoted a healthier and longer life and was associated with a decrease in the levels of systemic inflammatory cytokines in aging mice. Furthermore, dietary genistein ameliorated gut dysfunctions, such as intestinal inflammation, leaky gut, and impaired epithelial regeneration. A distinct genistein-mediated alteration in gut microbiota was observed by increasing Lachnospira abundance and short-chain fatty acid (SCFA) production. Further fecal microbiota transplantation and dirty cage sharing experiments indicated that the gut microbiota from genistein-fed mice rejuvenated the aging gut and extended the lifespan of progeroid mice. It was demonstrated that genistein-associated SCFAs alleviated tumor necrosis factor alpha-induced intestinal organoid damage. Moreover, genistein-associated propionate promoted regulatory T cell-derived interleukin 10 production, which alleviated macrophage-derived inflammation. This study provided the first data, to the authors' knowledge, indicating that dietary genistein modulates homeostasis in the aging gut and extends the healthspan and lifespan of aging mammals. Moreover, the existence of a link between genistein and the gut microbiota provides a rationale for dietary interventions against age-associated frailty.

Regulation of the Paneth cell niche by exogenous L-arginine couples the intestinal stem cell function.

Although previous studies show that exogenous nutrients regulate the stem cell function, little is known about the effects of L-arginine on intestinal stem cells (ISCs). In this study, we utilize mice, small intestinal (SI) organoids, and ISC-Paneth cell co-cultured models to clarify the role of L-arginine in ISC function. We find that exogenous L-arginine is essential for ISCs proliferation and intestinal epithelial renewal. Our data show that Paneth cells, a critical component of the ISCs niche, augment the ISCs function in response to L-arginine. Moreover, enhanced the expression of Wnt3a in Paneth cells, which is a ligand of the Wnt/ß-catenin signaling pathway, mediates the effects of L-arginine on ISCs function. Pre-treatment with L-arginine enhances the ISCs pool and protects the gut in response to injury provoked by murine tumor necrosis factor α (TNF-α) and 5-Fluorouracil (5-FU). Our findings establish that the regulation of Wnt3a in the Paneth cell niche by exogenous L-arginine couples ISCs function and favours a model in which the ISCs niche couples the nutrient levels to ISCs function.

Enterococcus faecium PNC01 isolated from the intestinal mucosa of chicken as an alternative for antibiotics to reduce feed conversion rate in broiler chickens.

BACKGROUND: The development and utilization of probiotics had many environmental benefits for replacing antibiotics in animal production. Bacteria in the intestinal mucosa have better adhesion to the host intestinal epithelial cells compared to bacteria in the intestinal contents. In this study, lactic acid bacteria were isolated from the intestinal mucosa of broiler chickens and investigated as the substitution to antibiotic in broiler production. RESULTS: In addition to acid resistance, high temperature resistance, antimicrobial sensitivity tests, and intestinal epithelial cell adhesion, Enterococcus faecium PNC01 (E. faecium PNC01) was showed to be non-cytotoxic to epithelial cells. Draft genome sequence of E. faecium PNC01 predicted that it synthesized bacteriocin to perform probiotic functions and bacteriocin activity assay showed it inhibited Salmonella typhimurium from invading intestinal epithelial cells. Diet supplemented with E. faecium PNC01 increased the ileal villus height and crypt depth in broiler chickens, reduced the relative length of the cecum at day 21, and reduced the relative length of jejunum and ileum at day 42. Diet supplemented with E. faecium PNC01 increased the relative abundance of Firmicutes and Lactobacillus, decreased the relative abundance of Bacteroides in the cecal microbiota. CONCLUSION: E. faecium PNC01 replaced antibiotics to reduce the feed conversion rate. Furthermore, E. faecium PNC01 improved intestinal morphology and altered the composition of microbiota in the cecum to reduce feed conversion rate. Thus, it can be used as an alternative for antibiotics in broiler production to avoid the adverse impact of antibiotics by altering the gut microbiota.

Targeted Isolation of Antioxidant Constituents from Plantago asiatica L. and In Vitro Activity Assay.

Plantago asiatica L. is widely distributed in Eastern Asia and a commonly used drug in China, Korea, and Japan for diuretic and antiphlogistic purposes. In this experiment, the present study was performed to isolate antioxidant molecules based on the DPPH scavenging activity assay and discover the bioactive compounds which contributed to performing the function of Plantago asiatica L. Each faction was chosen for further isolation guided by DPPH scavenging activity assay. Afterwards, two potential bioactive molecules, aesculetin and apigenin, were isolated for in vitro antioxidant activity in cells. Hydrogen-peroxide-induced oxidative stress led to decreased cell viability, impaired intercellular junction, and damage to the cell membrane and DNA. Furthermore, aesculetin ameliorated decreased cell viability induced by hydrogen peroxide via upregulation of antioxidant related genes, and apigenin also protected against H2O2 mainly by improving the glutathione (GSH) antioxidant system, such as increasing the activity of glutathione peroxidase (GPX), glutathione reductase (GR), and the ration of GSH/glutathione disulfide (GSSG). Above all, these findings suggest that aesculetin and apigenin may be bioactive compounds for antioxidant function in Plantago asiatica L.

Zinc Supplementation, via GPR39, Upregulates PKCζ to Protect Intestinal Barrier Integrity in Caco-2 Cells Challenged by Salmonella enterica Serovar Typhimurium.

Background: Zinc has been shown to improve intestinal barrier function against Salmonella enterica serovar Typhimurium (S. typhimurium) infection, but the mechanisms involved in this process remain undefined.Objective: We aimed to explore the roles of G protein-coupled receptor (GPR)39 and protein kinase Cζ (PKCζ) in the regulation by zinc of intestinal barrier function.Methods: A Transwell Caco-2 monolayer was pretreated with 0, 50, or 100 µM Zn and then incubated with S. typhimurium for 0-6 h. Afterward, cells silenced by the small interfering RNA for GPR39 or PKCζ were pretreated with 100 µM Zn and incubated with S. typhimurium for 3 h. Finally, transepithelial electrical resistance (TEER), permeability, tight junction (TJ) proteins, and signaling molecules GPR39 and PKCζ were measured.Results: Compared with controls, S. typhimurium decreased TEER by 62.3-96.2% at 4-6 h (P < 0.001), increased (P < 0.001) permeability at 6 h, and downregulated (P < 0.05) TJ protein zonula occludens (ZO)-1 and occludin by 104-123%, as well as Toll-like receptor 2 and PKCζ by 35.1% and 75.2%, respectively. Compared with S. typhimurium-challenged cells, 50 and 100 µM Zn improved TEER by 26.3-60.9% at 4-6 h (P < 0.001) and decreased (P < 0.001) permeability and bacterial invasion at 6 h. A total of 100 µM Zn increased ZO-1, occludin, GPR39, and PKCζ 0.72- to 1.34-fold (P < 0.05); however, 50 µM Zn did not affect ZO-1 or occludin (P > 0.1). Silencing GPR39 decreased (P < 0.05) zinc-activated PKCζ and blocked (P < 0.05) the promotion of zinc on epithelial integrity. Furthermore, silencing PKCζ counteracted the protective effect of zinc on epithelial integrity but did not inhibit GPR39 (P = 0.138).Conclusion: We demonstrated that zinc upregulates PKCζ by activating GPR39 to enhance the abundance of ZO-1, thereby improving epithelial integrity in S. typhimurium-infected Caco-2 cells.

Different fat-to-fiber ratios by changing wheat inclusion level impact energy metabolism and microbial structure of broilers.

Introduction: Dietary nutrient content is crucial for energy metabolism and development of gut microbiota. Herein, this study aimed to explore the effects of fat-to-fiber ratios on nutrient transporter, energy metabolism and gut microbiota when ingredients composition was altered. Methods: A total of 240 as-hatched broiler chickens were randomly assigned into three groups including low fat-high dietary fiber (LF-HD), medium fat-medium dietary fiber (MF-MD) and high fat-low dietary fiber (HF-LD), with diets being iso-protein, and broilers were offered the same commercial diets from 21 to 42 d. The data were analyzed using one-way ANOVA of SPSS. Results and Discussion: Results showed that HF-LD diet significantly increased glucose content and decreased triglyceride in serum of broilers (p < 0.05). The mRNA abundance of jejunal gene involved in glucose transporter and tricarboxylic acid (TCA) cycle was significantly increased in broilers fed with HF-LD diets. Compared with LF-HD, HF-LD had a lower abundance of Anaerofilum and CHKCI001, and an increased proportion of beneficial bacteria such as Alistipes, Catenibacillus, Intestinimonas, Lactobacillus, and Peptococcus (p < 0.05). Functional prediction of these microbial changes indicated that HF-LD diet drove caecal microbiota to participate in carbohydrate metabolism and TCA cycle (p < 0.05). Dietary HF-LD-induced microbiota changes were positively correlated with growth performance of broilers (p < 0.05). Therefore, HF-LD diet increased glucose transporters and energy metabolism in intestine and shaped microbial structure and metabolic pathways, which may benefit the growth performance of broilers.

Broiler Age Differently Affects Apparent Metabolizable Energy and Net Energy of Expanded Soybean Meal.

Accurately determining the energy values of ingredients is crucial for meeting energy requirements and achieving maximum production performance of animals. This study was conducted to measure the available energy values of three expanded soybean meals (ESBMs) for Arbor Acres male broilers from 14 to 16 day and 28 to 30 day using the difference method. A corn-soybean basal diet was formulated, and test diets were developed with 25% ESBMs as substitutes for energy-yielding ingredients. A completely randomized design was used for determining heat production and energy balance of broilers in 12 open-circuit respiration chambers, with six replicates per group. Prior to measurement, four (14 to 16 day) or two (28 to 30 day) birds per chamber were given a 4-day adaption to diets and chambers. The period lasted for 3 days to determine the apparent metabolizable energy (AME), nitrogen balance, gas exchanges, and heat production. Broilers fed test diets with 25% ESBM exhibited higher nitrogen intake (p < 0.05), nitrogen excreta (p < 0.05), and increased energy deposition as protein irrespective of age (p < 0.05). Furthermore, results showed that AME, nitrogen corrected AME (AMEn), and net energy (NE) values of 3 ESBMs averaged 10.48, 8.93, and 6.88 MJ/kg for broilers from 14 to 16 day, while averaged 11.91, 10.42, and 6.43 MJ/kg for broilers from 28 to 30 day. Broilers from 28 to 30 day showed significantly higher AMEn values but lower NE/AME values of ESBMs compared with those from 14 to 16 day (p < 0.05). Therefore, age-dependent energy values of a single ingredient should be considered in feed formulations to optimize economic returns.

Dual-sgRNA CRISPRa System for Enhanced MK-7 Production and Salmonella Infection Mitigation in Bacillus subtilis natto Applied to Caco-2 Cells.

This study optimized the menaquinone-7 (MK-7) synthetic pathways in Bacillus subtilis (B. subtilis) natto NB205, a strain that originated from natto, to enhance its MK-7 production. Utilizing mutation breeding, we developed NBMK308, a mutant strain that demonstrated a significant 117.23% increase in MK-7 production. A comprehensive transcriptome analysis identified two key genes, ispA and ispE, as being critical in MK-7 synthesis. The dual-sgRNA CRISPRa system was utilized to achieve precise regulation of ispA and ispE in the newly engineered strain, A3E3. This strategic modulation resulted in a significant enhancement of MK-7 production, achieving increases of 20.02% and 201.41% compared to traditional overexpression systems and the original strain NB205, respectively. Furthermore, the fermentation supernatant from A3E3 notably inhibited Salmonella invasion in Caco-2 cells, showcasing its potential for combating such infections. The safety of the dual-sgRNA CRISPRa system was confirmed through cell assays. The utilization of the dual-sgRNA CRISPRa system in this study was crucial for the precise regulation of key genes in MK-7 synthesis, leading to a remarkable increase in production and demonstrating additional therapeutic potential in inhibiting pathogenic infections. This approach effectively combined the advantages of microbial fermentation and biotechnology, addressing health and nutritional challenges.

Long-term supplementation of genistein improves immune homeostasis in the aged gut and extends the laying cycle of aged laying hens.

Aging is associated with alterations in gut function, including intestinal inflammation, leaky gut, and impaired epithelial regeneration. Rejuvenating the aged gut is imperative to extend the laying cycle of aged laying hens. Genistein is known to have beneficial effects on age-related diseases, but its precise role in homeostasis of the aged gut of laying hens remains to be elucidated. In this study, 160 45-wk-old Hyline Brown laying hens were continuously fed a basal diet or a diet supplemented with 40 mg/kg genistein until they reached 100 wk of age. The results revealed that long-term genistein supplementation led to an improvement in the egg production rate and feed conversion ratio, as well as an increase in egg quality. Moreover, the expression levels of senescence markers, such as ß-galactosidase, P16, and P21, were decreased in the gut of genistein-treated aged laying hens. Furthermore, genistein ameliorated gut dysfunctions, such as intestinal inflammation, leaky gut, and impaired epithelial regeneration. Treg cell-derived IL-10 plays a crucial role in the genistein-induced regulation of age-related intestinal inflammation. This study demonstrates that long-term consumption of genistein improves homeostasis in the aged gut and extends the laying cycle of aged laying hens. Moreover, the link between genistein and Treg cells provides a rationale for dietary intervention against age-associated gut dysfunction.

Effects of Aspirin Eugenol Ester on Liver Oxidative Damage and Energy Metabolism in Immune-Stressed Broilers.

The aim of this study was to investigate the effects of aspirin eugenol ester (AEE) on liver oxidative damage and energy metabolism in immune-stressed broilers. In total, 312 broilers were divided into 4 groups (saline, LPS, SAEE, and LAEE). Broilers in the saline and LPS groups were fed a basal diet; the SAEE and LAEE groups had an added 0.01% AEE in their diet. Broilers in the LPS and LAEE groups were injected with lipopolysaccharides, while the saline and SAEE groups were injected with saline. Results showed that AEE increased the body weight, average daily gain, and average daily feed intake, as well as decreasing the feed conversion ratio of immune-stressed broilers. AEE protects against oxidative damage in immune-stressed broiler livers by elevating the total antioxidant capacity, superoxide dismutase activity, and glutathione S-transferase alpha 3 (GSTA3) and glutaredoxin 2 (GLRX2) expression, while decreasing malondialdehyde content. AEE lessened inflammation by reducing prostaglandin-F2α production and prostaglandin-endoperoxide synthase 2 (PTGS2) and interleukin-1beta (IL-1ß) expression. AEE decreased oxidative phosphorylation rates by increasing succinic acid levels and lowering both adenosine diphosphate (ADP) levels and ceroid lipofuscinosis neuronal 5 (CLN5) expression. AEE modulated the metabolism of phenylalanine, tyrosine, lipids, and cholesterol by reducing the phenyllactate and L-arogenate levels, lowering dopachrome tautomerase (DCT) and apolipoprotein A4 (APOA4) expression, and increasing phenylpyruvic acid and dopa decarboxylase (DDC) expression. In summary, AEE can effectively alleviate liver oxidative damage and energy metabolism disorders in immune-stressed broilers.

Aspirin eugenol ester affects ileal barrier function, inflammatory response and microbiota in broilers under lipopolysaccharide-induced immune stress conditions.

Aims: The aim of this study was to investigate the effects of aspirin eugenol ester (AEE) on ileal immune function in broilers under lipopolysaccharide (LPS)-induced immune stress. Methods: Two hundred and forty one-day-old male Arbor Acres chicks were randomly divided into four groups (saline, LPS, saline + AEE and LPS + AEE) with six replicates of ten broilers each. The saline group and LPS group were fed the normal diet, while the other two groups received normal diet plus 0.1 g/kg AEE. Broilers in the LPS and LPS + AEE groups were injected intraperitoneally with 0.5 mg/kg B.W LPS in saline for seven consecutive days beginning at 14 days of age, while broilers in the saline and saline + AEE groups were injected with saline only. Results: The results showed that AEE improved the ileal morphology and increased the ratio of villus height to crypt depth of immune-stressed broilers. LPS-induced immune stress significantly reduced the expression of the genes for the tight junction proteins occludin, zonula occludens-1 (ZO-1), claudin-1 and claudin-2, in the ileum, while AEE significantly up-regulated the expression of these genes. Compared with the saline group, the LPS-treated chickens showed significantly increased mRNA expression of the inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-10 (IL-10), cyclooxygenase-2 (COX-2), and microsomal Prostaglandin E Synthesase-1 (mPGES-1) in the ileum, while they were significantly decreased by AEE supplementation. In addition, analysis of the ileal bacterial composition showed that compared with saline and LPS + AEE groups, the proportion of Firmicutes and Lactobacillus in the LPS group was lower, while the proportion of Proteobacteria and Escherichia-Shigella was higher. Similarly, Line Discriminant Analysis Effect Size (LEfSe) analysis showed that compared with the LPS group, Brevibacillus was dominant in the saline group, while the LPS + AEE group was rich in Rhizobium, Lachnoclostridium, Ruminococcaceae, Faecalibacterium, Negativibacillus, Oscillospiraceae, and Flavonifractor. Conclusion: These results indicate that dietary supplementation with 0.1 g/kg AEE could protect the intestinal health by improving the intestinal villus morphology, enhancing the expression of tight junction genes and alleviating inflammation to resist the immune stress caused by LPS stimulation in broilers, and the mechanism may involve COX-2-related signal transduction and improved intestinal microbiota composition.

Effect of dietary aspirin eugenol ester on the growth performance, antioxidant capacity, intestinal inflammation, and cecal microbiota of broilers under high stocking density.

This study was designed to examine the impact of aspirin eugenol ester (AEE) on the growth performance, serum antioxidant capacity, jejunal barrier function, and cecal microbiota of broilers raised under stressful high density (HD) stocking conditions compared with normal density broilers (ND). A total of 432 one-day-old AA+ male broilers were randomly divided into 4 groups: normal density (ND, 14 broilers /m2), high density (HD, 22 broilers /m2), ND + AEE, and HD + AEE. The results of the study revealed a significant decrease in the growth performance of broiler chickens as a result of HD stress (P < 0.05). The total antioxidant capacity (T-AOC) in serum demonstrated a significant decrease (P < 0.05) at both 28 and 35 d. Conversely, the serum level of malondialdehyde (MDA) exhibited a significant increase (P < 0.05). Dietary supplementation of AEE resulted in a significant elevation (P < 0.05) of serum GSH-PX, SOD and T-AOC activity at both 28 and 35 d. Moreover, exposure to HD stress resulted in a considerable reduction in the height of intestinal villi and mRNA expression of tight junction proteins in the jejunum, along with, a significant elevation in the mRNA expression of inflammatory cytokines (P < 0.05). However, the administration of AEE reversed the adverse effects of HD-induced stress on villus height and suppressed the mRNA expression of the pro-inflammatory genes, COX-2 and mPGES-1. Additionally, the exposure to HD stress resulted in a substantial reduction in the α-diversity of cecal microbiota and disruption in the equilibrium of intestinal microbial composition, with a notable decrease in the relative abundance of Bacteroides and Faecalibacterium (P < 0.05). In contrast, the addition of AEE to the feed resulted in a notable increase in the relative abundance of Phascolarctobacterium and enhanced microbial diversity (P < 0.05). The inclusion of AEE in the diet has been demonstrated to enhance intestinal integrity and growth performance of broilers by effectively mitigating disruptions in gut microbiota induced by HD stress.

Effects of dietary chlorogenic acid on cecal microbiota and metabolites in broilers during lipopolysaccharide-induced immune stress.

Aims: The aim of this study was to investigate the effects of chlorogenic acid (CGA) on the intestinal microorganisms and metabolites in broilers during lipopolysaccharide (LPS)-induced immune stress. Methods: A total of 312 one-day-old Arbor Acres (AA) broilers were randomly allocated to four groups with six replicates per group and 13 broilers per replicate: (1) MS group (injected with saline and fed the basal diet); (2) ML group (injected with 0.5 mg LPS/kg and fed the basal diet); (3) MA group (injected with 0.5 mg LPS/kg and fed the basal diet supplemented with 1,000 mg/kg CGA); and (4) MB group (injected with saline and fed the basal diet supplemented with 1,000 mg/kg CGA). Results: The results showed that the abundance of beneficial bacteria such as Bacteroidetes in the MB group was significantly higher than that in MS group, while the abundance of pathogenic bacteria such as Streptococcaceae was significantly decreased in the MB group. The addition of CGA significantly inhibited the increase of the abundance of harmful bacteria such as Streptococcaceae, Proteobacteria and Pseudomonas caused by LPS stress. The population of butyric acid-producing bacteria such as Lachnospiraceae and Coprococcus and beneficial bacteria such as Coriobacteriaceae in the MA group increased significantly. Non-targeted metabonomic analysis showed that LPS stress significantly upregulated the 12-keto-tetrahydroleukotriene B4, riboflavin and mannitol. Indole-3-acetate, xanthurenic acid, L-formylkynurenine, pyrrole-2-carboxylic acid and L-glutamic acid were significantly down-regulated, indicating that LPS activated inflammation and oxidation in broilers, resulting in intestinal barrier damage. The addition of CGA to the diet of LPS-stimulated broilers significantly decreased 12-keto-tetrahydro-leukotriene B4 and leukotriene F4 in arachidonic acid metabolism and riboflavin and mannitol in ABC transporters, and significantly increased N-acetyl-L-glutamate 5-semialdehyde in the biosynthesis of amino acids and arginine, The presence of pyrrole-2-carboxylic acid in D-amino acid metabolism and the cecal metabolites, indolelactic acid, xanthurenic acid and L-kynurenine, indicated that CGA could reduce the inflammatory response induced by immune stress, enhance intestinal barrier function, and boost antioxidant capacity. Conclusion: We conclude that CGA can have a beneficial effect on broilers by positively altering the balance of intestinal microorganisms and their metabolites to inhibit intestinal inflammation and barrier damage caused by immune stress.

Genome-Wide Analysis of lncRNA and mRNA Expression in the Uterus of Laying Hens during Aging.

Eggshell plays an essential role in preventing physical damage and microbial invasions. Therefore, the analysis of genetic regulatory mechanisms of eggshell quality deterioration during aging in laying hens is important for the biosecurity and economic performance of poultry egg production worldwide. This study aimed to compare the differences in the expression profiles of long non-coding RNAs (lncRNAs) and mRNAs between old and young laying hens by the method of high-throughput RNA sequencing to identify candidate genes associated with aging in the uterus of laying hens. Overall, we detected 176 and 383 differentially expressed (DE) lncRNAs and mRNAs, respectively. Moreover, functional annotation analysis based on the Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) databases revealed that DE-lncRNAs and DE-mRNAs were significantly enriched in "phosphate-containing compound metabolic process", "mitochondrial proton-transporting ATP synthase complex", "inorganic anion transport", and other terms related to eggshell calcification and cuticularization. Through integrated analysis, we found that some important genes such as FGF14, COL25A1, GPX8, and GRXCR1 and their corresponding lncRNAs were expressed differentially between two groups, and the results of quantitative real-time polymerase chain reaction (qPCR) among these genes were also in excellent agreement with the sequencing data. In addition, our study found that TCONS_00181492, TCONS_03234147, and TCONS_03123639 in the uterus of laying hens caused deterioration of eggshell quality in the late laying period by up-regulating their corresponding target genes FGF14, COL25A1, and GRXCR1 as well as down-regulating the target gene GPX8 by TCONS_01464392. Our findings will provide a valuable reference for the development of breeding programs aimed at breeding excellent poultry with high eggshell quality or regulating dietary nutrient levels to improve eggshell quality.

Effects of Bacillus subtilis Natto NB205 and Its Mutant NBMK308 on Egg Quality in Aging Laying Hens.

In aging laying hens, reproductive changes reduce egg quality. Bacillus subtilis natto (B. subtilis) is a versatile bacterium with high vitamin K2 content, providing health benefits for animals and humans. This study investigated the effect of B. subtilis natto NB205 and its mutant NBMK308 on egg quality in aging laying hens. Results showed that NB205 and NBMK308 supplementation significantly improved albumen height (p < 0.001), Haugh units (p < 0.05), and eggshell thickness (p < 0.001) compared to the control group. Supplementation also increased ovalbumin expression, regulated tight junction (TJ) proteins, reduced pro-inflammatory cytokine levels, and improved the health and productivity of aging laying hens by regulating key apoptosis-related genes in the magnum part of the oviduct. There were differences in the expression of vitamin K-dependent proteins (VKDPs) in the magnum between NB205 and NBMK308, but no significant differences in the improvement of egg quality. Supplementation with NB205 and NBMK308 can improve egg quality in aging laying hens.

Dietary Supplementation with Chlorogenic Acid Enhances Antioxidant Capacity, Which Promotes Growth, Jejunum Barrier Function, and Cecum Microbiota in Broilers under High Stocking Density Stress.

Chlorogenic acids (CGA) are widely used as feed additives for their ability to improve growth performance and intestinal health in poultry. However, whether dietary CGAs could reverse the impaired intestinal condition caused by high stocking density (HD) in broiler chickens is unknown. We determined the effect of dietary CGA on growth, serum antioxidant levels, jejunum barrier function, and the microbial community in the cecum of broilers raised under normal (ND) or HD conditions. HD stress significantly decreased growth and body weight, which was restored by CGA. The HD group showed increased serum malondialdehyde, an oxidative byproduct, and decreased SOD and GSH-Px activity. CGA reduced malondialdehyde and restored antioxidant enzyme activity. HD stress also significantly decreased jejunal villus length and increased crypt depth. Compared with ND, the expression of tight-junction genes was significantly decreased in the HD group, but this decrease was reversed by CGA. HD also significantly upregulated TNF-α. Compared with ND, the cecal microbiota in the HD group showed lower alpha diversity with increases in the harmful bacteria Turicibacter and Shigella. This change was altered in the HD + CGA group, with enrichment of Blautia, Akkermansia, and other beneficial bacteria. These results demonstrated that HD stress decreased serum antioxidant capacity, inhibited the development of jejunal villi, and downregulated expression of tight-junction genes, which increased intestinal permeability during the rapid growth period (21 to 35 days). Dietary CGA enhanced antioxidant capacity, improved intestinal integrity, and enhanced beneficial gut bacteria in chickens raised under HD conditions.

Restricted feeding regimens improve white striping associated muscular defects in broiler chickens.

The current study investigated the effects of intermittent feeding (IF) and fasting strategies at different times post-hatch on muscle growth and white striping (WS) breast development. In the first trial, 32 one-day-old Abor Acre broilers were fed ad libitum (AL) for 3 d post-hatch and then randomly allotted into 4 feeding strategies including AL, 1h-IF group (1 h IF, 4 times feeding/d, 1 h each time), 1.5h-IF (1.5 h IF, 4 times feeding/d, 1.5 h each time), and fasting (1d acute fasting, 6 d free access to feed) groups and fed for 7 d. Although angiogenic genes including VEGFA, VEGFR1, and VEGFR2, and myogenic genes including MYOG and MYOD were upregulated (P < 0.05), the breast muscle satellite cell (SC) number and PAX7, MYF5 expression were decreased by the IF strategies (P < 0.05). One-day fasting at 6 d of age also upregulated angiogenic genes and MYOD expression (P < 0.05), downregulated MYF5 expression (P < 0.05), but did not change SC number (P > 0.05). In the second trial, 384 one-day-old birds were fed AL for 1 wk and then randomly allotted to the above 4 feeding strategies starting at 8 d of age until 42 d of age. Similarly, IF and fasting strategies upregulated the expression of angiogenic and myogenic genes (P < 0.05). Both 1h-IF and 1.5h-IF increased breast muscle SC number (P < 0.05). At slaughter, breast muscle fiber diameter of 1.5h-IF was smaller but the SC number was larger than that of the birds fed AL (P < 0.05). The IF and fasting strategies prevented WS development, and reduced breast WS scores and triglyceride content (P < 0.05) without changing the body weight (P > 0.05). Fasting and 1h-IF reduced the expression of adipogenic genes ZNF423 and PDGFRα (P < 0.05). Moreover, IF and fasting strategies reduced fibrosis in breast muscle and reduced skeletal muscle-specific E3 ubiquitin ligases (TRIM63 and MAFBX) (P < 0.05). Fasting significantly reduced CASPASE-3 in breast muscle (P < 0.05). In conclusion, IF starting in the first week decreases SC number. Compared to AL, IF or fasting promotes muscular angiogenesis, increases SC number, prevents muscle degeneration, and prevents the development of WS without impairing the growth performance of broiler chickens.

Effects of Selenium Yeast on Egg Quality, Plasma Antioxidants, Selenium Deposition and Eggshell Formation in Aged Laying Hens.

Internal egg and eggshell quality are often deteriorated in aging laying hens, which causes huge economic losses in the poultry industry. Selenium yeast (SY), as an organic food additive, is utilized to enhance laying performance and egg quality. To extend the egg production cycle, effects of selenium yeast supplementation on egg quality, plasma antioxidants and selenium deposition in aged laying hens were evaluated. In this study, five hundred and twenty-five 76-week-old Jing Hong laying hens were fed a selenium-deficient (SD) diet for 6 weeks. After Se depletion, the hens were randomly divided into seven treatments, which included an SD diet, and dietary supplementation of SY and sodium selenite (SS) at 0.15, 0.30, and 0.45 mg/kg to investigate the effect on egg quality, plasma antioxidant capacity, and selenium content in reproductive organs. After 12 weeks of feeding, dietary SY supplementation resulted in higher eggshell strength (SY0.45) (p < 0.05) and lower shell translucence. Moreover, organs Se levels and plasma antioxidant capacity (T-AOC, T-SOD, and GSH-Px activity) were significantly higher with Se supplementation (p < 0.05). Transcriptomic analysis identified some key candidate genes including cell migration inducing hyaluronidase 1 (CEMIP), ovalbumin (OVAL), solute carrier family 6 member 17 (SLC6A17), proopiomelanocortin (POMC), and proenkephalin (PENK), and potential molecular processes (eggshell mineralization, ion transport, and eggshell formation) involved in selenium yeast's effects on eggshell formation. In conclusion, SY has beneficial functions for eggshell and we recommend the supplementation of 0.45 mg/kg SY to alleviate the decrease in eggshell quality in aged laying hens.

Effects of dietary chlorogenic acid on intestinal barrier function and the inflammatory response in broilers during lipopolysaccharide-induced immune stress.

Immune stress exerts detrimental effects on growth performance and intestinal barrier function during intensive animal production with ensuing serious economic consequences. Chlorogenic acid (CGA) is used widely as a feed additive to improve the growth performance and intestinal health of poultry. However, the effects of dietary CGA supplementation on amelioration of the intestinal barrier impairment caused by immune stress in broilers are unknown. This study investigated the effects of CGA on growth performance, intestinal barrier function, and the inflammatory response in lipopolysaccharide (LPS) mediated immune-stressed broilers. Three hundred and twelve 1-day-old male Arbor Acres broilers were divided randomly into 4 groups with 6 replicates of thirteen broilers. The treatments included: i) saline group: broilers injected with saline and fed with basal diet; ii) LPS group: broilers injected with LPS and fed with basal diet; iii) CGA group: broilers injected with saline and feed supplemented with CGA; and iv) LPS+CGA group: broilers injected with LPS and feed supplemented with CGA. Animals in the LPS and LPS+CGA groups were injected intraperitoneally with an LPS solution prepared with saline from 14 d of age for 7 consecutive days, whereas broilers in the other groups were injected only with saline. LPS induced a decrease in feed intake of broilers during the stress period, but CGA effectively alleviated this decrease. Moreover, CGA inhibited the reduction of villus height and improved the ratio of villus height to crypt depth in the duodenum of broilers 24 and 72 h after LPS injection. In addition, dietary CGA supplementation significantly restored the expression of cation-selective and channel-forming Claudin2 protein 2 h after LPS injection in the ileum. LPS enhanced the expression of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in the small intestine, but this enhancement was blocked by CGA supplementation. The expression of interleukin-10 (IL-10) increased with LPS injection and CGA promoted the production of IL-10. CGA addition downregulated the expression of intestinal interleukin-6 (IL-6) of broilers under normal rearing conditions. However, CGA supplementation upregulated the expression of IL-6 of broilers 72 h after LPS injection. The data demonstrate that dietary supplementation with CGA alleviates intestinal barrier damage and intestinal inflammation induced by LPS injection during immune stress thereby improving growth performance of broilers.