The ionome and proteome landscape of aging in laying hens and relation to egg white quality.

The ionome is essential for maintaining body function and health status by participating in diverse key biological processes. Nevertheless, the distribution and utilization of ionome among different organs and how aging impacts the ionome leading to a decline in egg white quality remain unknown. Thus, we used inductively coupled plasma mass spectrometry (ICP-MS) to analyze 35 elements and their isotopic contents in eight organs of laying hens at 35, 72, and 100 weeks. Moreover, the magnum proteome, amino acids in egg white, and egg white quality were analyzed in laying hens at three different ages using 4D proteomics techniques, an amino acid analyzer, and an egg quality analyzer. Across the organs, we identified varying distribution patterns among macroelements (Mg24, Ca43/44, K39, and P31), transition metals (Zn64/66, Cu63/65, Fe56/57, and Mn55), and toxic elements (Pb208, Ba137, and Sr86). We observed an organ-specific aging pattern characterized by the accumulation of toxic elements (Pb208, Ba137, and Sr86) and calcification in the small intestine. Additionally, a decrease in the utilization of essential trace elements selenium (Se78/82) and manganese (Mn55) was noted in the oviduct. By analyzing ionome in tandem with egg quality, egg white amino acids, and proteome, we unveiled that the reduction of selenium and manganese concentrations in the magnum during the aging process affected amino acid metabolism, particularly tryptophan metabolism, thereby inhibiting the amino acid synthesis in the magnum. Furthermore, it accelerated the senescence of magnum cells through necroptosis activation, leading to a decline in the albumen secretion function of the magnum and subsequently reducing egg white quality. Overall, this study provides insights into the evolution of 35 elements and their isotopes across 8 organs of laying hens with age. It also reveals the elemental composition, interactions, and utilization patterns of these organs, as well as their correlation with egg white quality. The present study highlights the significance of ionome and offers a comprehensive perspective on the selection of ionome for regulating the aging of laying hens.

Construction of low intestinal bacteria model and its effect on laying performance and immune function of laying hens.

The objective of this study was to establish a low-bacteria intestinal model in chickens, and then to investigate the characteristics involving in immune function and intestinal environment of this model. A total of 180 twenty-one-week-old Hy-line gray layers were randomly allocated into 2 treatment groups. Hens were fed with a basic diet (Control), or an antibiotic combination diet (ABS) for 5 weeks. Results showed that the total bacteria in the ileal chyme were significantly dropped after ABS treatment. Compared with the Control group, the genus-level bacteria such as Romboutsia, Enterococcus, and Aeriscardovia were reduced in the ileal chyme of the ABS group (P < 0.05). In addition, the relative abundance of Lactobacillus_delbrueckii, Lactobacillus_aviarius, Lactobacillus_gasseri, and Lactobacillus_agilis in the ileal chyme were also descended (P < 0.05). However, Lactobacillus_coleohominis, Lactobacillus_salivarius, and Lolium_perenne were elevated in the ABS group (P < 0.05). Beyond that, ABS treatment decreased the levels of interleukin-10 (IL-10) and ß-defensin 1 in the serum, as well as the number of goblet cells in the ileal villi (P < 0.05). Additionally, the genes mRNA levels of the ileum such as Mucin2, Toll-like receptors 4 (TLR4), Myeloid differentiation factor 88 (MYD88), NF-κB, IL-1ß, Interferon-gama (IFN-γ), IL-4 and the ratio of IFN-γ to IL-4 were also down-regulated in the ABS group (P < 0.05). In addition, there were no significant changes about egg production rate and egg quality in the ABS group. In conclusion, dietary supplemental antibiotic combination for 5 weeks could establish a low intestinal bacteria model of hens. The establishment of a low intestinal bacteria model did not affect the egg-laying performance, while caused immune suppression in laying hens.

Soya saponin fails to improve the antioxidation and immune function of laying hens with antibiotics treated.

Soya saponin (SS) helps to improve antioxidant and immune function of body, and intestinal bacteria might play an important role here. In the present study, the co-occurring network of the ileal flora was analyzed with 50 mg/kg SS supplemented to the diet, and Romboutsia was found to have evolved into a dominant flora. In addition, the co-occurring network of the flora was changed with the combined antibiotic treated, and the unidentified-cyanobacteria developed into the dominant flora, whereas the relative abundance of Romboutsia was dropped. Dietary SS failed to elevate the relative abundance of Romboutsia with antibiotics treated, at the same time, it was not helpful for the antioxidant and immune function of laying hens. While dietary SS had a little help on the egg-laying performance. Intestinal bacteria did play a key role in the biological functions of SS on laying hens. In conclusion, SS failed to improve the antioxidation and immune function of laying hens with antibiotics treated.

Effects of Dietary Astragalus Polysaccharide Supplementation on the Th17/Treg Balance and the Gut Microbiota of Broiler Chickens Challenged With Necrotic Enteritis.

This study aimed to investigate the effects of dietary astragalus polysaccharide (APS) supplementation on the immune function, gut microbiota and metabolism of broiler chickens challenged with necrotic enteritis (NE). Two hundred forty Arbor Acres broiler chicks (one day old) were randomly assigned using a 2 × 2 factorial arrangement into two groups fed different levels of dietary APS (0 or 200 ppm of diet) and two disease challenge groups (control or NE challenged). The results showed that NE infection significantly increased FCR, mortality rate, Th17/Treg (Th17 cells% in blood and ileum, Th17/Treg, IL-17 and IL-17/IL-10 in blood), NO, lysozyme activity and IL-1ß in blood, intestinal immune cell proportion and activity (Tc%, Treg% and monocyte phagocytic activity in ileum), intestinal inflammatory cytokines (TLR2, NF-κB, TNF-α and IL- 6) gene expression levels, and the number of Clostridium perfringens in cecum. NE infection significantly reduced body weight gain, thymus index, lymphocyte proliferation activity in blood and ileum, villus height and V/C in jejunum, Th cells% and Mucin2 gene expression in ileum. Dietary APS supplementation significantly increased body weight, feed intake, proportion of immune cells (T cells in blood and Tc, Treg in ileum), lymphocyte proliferation activity, V/C in jejunum, and ZO-1 gene expression in ileum. Dietary APS supplementation significantly reduced FCR and mortality rate, Th17/Treg, Th17%, intestinal pathology scores, intestinal inflammatory cytokine gene expression levels, and the number of Clostridium perfringens in cecum. In addition, broilers challenged with NE significantly increased Staphylococcus and Turicibacter and reduced α diversity of microbiota in ileum. Dietary APS supplementation significantly increased α diversity, Romboutsia, Halomonas, propionic acid, butyric acid, formononetin, taurine, cholic acid and equol and downregulated uric acid, L-arginine and serotonin in ileum. Spearman's correlation analysis revealed that Romboutsia, Turicibacter, Staphylocpccus, Halomonas, Streptococcus, Escherichia-Shigella, Prevotella, uric acid, L-arginine, jerivne, sodium cholate and cholic acid were related to inflammation and Th17/Treg balance. In conclusion, APS alleviated intestinal inflammation in broilers challenged with NE probably by regulating intestinal immune, Th17/Treg balance, as well as intestinal microbiota and metabolites.

Dietary soya saponin improves the lipid metabolism and intestinal health of laying hens.

Soya saponin (SS) is a natural active substance of leguminous plant, which could improve lipid metabolism and regulate immune function. Intestinal flora might play a key role in the biological functions of SS. The objective of this study was to measure the effects of dietary SS on immune function, lipid metabolism, and intestinal flora of laying hens with or without antibiotic treated. The experiment was designed as a factorial arrangement of 3 dietary SS treatments × 2 antibiotic treatments. Birds were fed a basal diet (CON) or a low-SS diet (50 SS) containing 50 mg/kg SS, or a high-SS diet (500 SS) containing 500 mg/kg SS. Birds were cofed with or without antibiotics. The growth experiment lasted for 10 wk. Results showed that birds fed the 50 mg/kg SS diet tended to have lower abdominal fat rate. The gene expression of liver X receptor-α (LxR-α) in liver and serum total cholesterol (TC) were dropped, and the gene expression of acyl-CoA thioesterase 8 (ACOT8) in liver were upregulated. Compared with CON group, the levels of lysozyme, IL-10, and transforming growth factor (TGF-ß) in the serum were elevated as along with gene expression of IL-10, TGF-ß, and LYZ in ileum of both 50 and 500 SS group. However, the level of secretory immunoglobulin A (sIgA) and Mucin-2 in the ileum were downregulated in the 500 SS group. Additionally, Lactobacillus and Lactobacillus gasseri were the dominant bacteria in the 50 SS group, whereas the relative abundance of Lactobacillus was dropped in the 500 SS group. With combined antibiotics treatment, the α-diversity of bacteria was reduced, and the biological effects of SS were eliminated. In conclusion, the lipid metabolism, immune function, and intestinal flora of the laying hens were improved with the dietary supplementation of 50 mg/kg SS. But dietary 500 mg/kg SS had negative effects on laying hens.

Dietary folic acid supplementation improves semen quality and spermatogenesis through altering autophagy and histone methylation in the testis of aged broiler breeder roosters.

The aging phenomenon often exerts a significant reduction in the reproduction performance of aged animals. The objective of this project was to investigate the effects of dietary Folic acid (FA) supplementation on the reproductive performance of aged broiler breeder roosters. A total of 16 aged ROSS 308 broiler breeder roosters (50-week-old) were randomly divided into two groups. The treatments were basal diet (CON), a basal diet supplemented with 10 mg/kg Folic acid (FAS) for four weeks. At the end of the experiment, semen quality, histopathological studies, serum concentrations of testosterone and relative mRNA and protein expressions of testes were evaluated. The results showed that dietary FA supplementation dramatically improved semen quality of aged roosters, manifested by increasing semen volume, sperm concentration, sperm motility, and sperm membrane functional integrity. Furthermore, seminiferous tubule epithelial height (SEH) and testis scores were increased by dietary supplementation with FA. Dietary FA also remarkably augmented the transcription level of spermatogenesis-related gene (CREM, PCK2, DDX4, and GDNF). No significant differences were observed in serum concentrations of testosterone between FAS and CON groups. We noted significant upregulation Beclin-1 and ATG5 protein expressions, and the ratio of LC3-Ⅱ/Ⅰ, as well as significant downregulation of p-mTOR protein expressions in testicular tissue of aged roosters with FA supplementation. In addition, dietary FA supplementation significantly increased the protein expression of H3K9me2 and reduced the protein expression of H3K27me2. In summary, dietary FA supplementation improved the testicular autophagy through the mTOR-signaling pathway, and altered histone methylation in the testis. Dietary supplementation with FA can ameliorate semen quality and spermatogenesis of aged roosters.

Dietary genistein supplementation alters mRNA expression profile and alternative splicing signature in the thymus of chicks with lipopolysaccharide challenge.

Genistein is abundant in the soybean products, which exerts prominent effects on immune function. Little information is available about the effect of dietary genistein on thymic transcriptome, especially when suffering from lipopolysaccharide challenge. In this study, 180 one-day-old male broilers were randomly allocated to 3 groups: nonchallenged chicks given a basal diet (CON), and lipopolysaccharide-challenged chicks fed a basal diet (LPS), or lipopolysaccharide-challenged chicks fed a basal diet supplemented with 40 mg/kg genistein (GEN). Lipopolysaccharide injection induced thymocyte apoptosis and inflammatory reactions in the chicks. The results showed dietary genistein significantly reduced the percentage of CD3+ T lymphocytes by 10.04% and CD4+/CD8+ T lymphocyte ratio by 21.88% in the peripheral blood induced by lipopolysaccharide injection (P < 0.05). In addition, genistein significantly reduced the thymus index by 50% and apoptotic index by 12.34% induced by LPS challenge (P < 0.05). Transcriptomic analysis identified 1,926 DEGs (1,014 upregulated and 912 downregulated, P < 0.05) between GEN and LPS groups, which altered the mRNA expression profile and signaling pathways (Toll-like receptor, and NOD-like receptor signaling pathway) in the thymus. Furthermore, 5 splicing (AS) isoforms of the Drosophila Disabled-2 (DAB2) gene were detected, which were significantly upregulated in the GEN group compared with that in the LPS group. In summary, dietary genistein supplementation altered the RNA expression profile and AS signatures in the thymus, and alleviated immune response against lipopolysaccharide challenge.

Dietary mulberry-leaf flavonoids supplementation improves liver lipid metabolism and ovarian function of aged breeder hens.

Mulberry-leaf flavonoids (MF), extracted from mulberry leaves, exert antioxidant and hypolipidemic effects. The purpose of this experimental study was to investigate the effects of dietary MF on the ovarian function and liver lipid metabolism of aged breeder hens. We used 270 (60-weeks-old) Qiling breeder hens randomly assigned in 3 treatments with supplemental dietary MF doses (0, 30, 60 mg/kg). The results showed that dietary MF significantly improved the egg-laying rate, followed by the reduced feed conversion rate (FCR) (p < 0.05). However, there is no obvious difference in hatchability and fertilised eggs hatchability among the three groups (p > 0.05). The level of T-CHO, LDL-C and AKP in serum was reduced, and the HDL-C concentrations were increased by dietary MF (p < 0.05). MF treatment also improved the antioxidant capacity and reduced the apoptotic index of the ovary (p < 0.05). Additionally, dietary MF significantly increased the serum estradiol (E2) levels (p < 0.05) and the transcription level of CYP19A1 and LHR in the ovary (p < 0.05). Dietary MF enhanced fatty acid ß-oxidation in the liver via up-regulating the mRNA expressions of PPARα and CPT-I (p < 0.05). Moreover, the HMF group significantly decreased mRNA expressions of SREBP-1c (p < 0.05) and increased mRNA expressions of ERα, VTG-Ⅱ and ApoB in the liver (p < 0.05). In conclusion, dietary MF could improve the reproduction performance of aged breeder hens through improving ovary function and hepatic lipid metabolism.

Dietary mulberry-leaf flavonoids improve the eggshell quality of aged breeder hens.

Eggshell quality is subject to a significant decline in the late laying period, which results in huge economic losses. The purpose of this study was to investigate the effects of dietary mulberry-leaf flavonoids (MF) on the eggshell quality of aged breeder hens. A total of 270 (60-week-old) Qiling breeder hens were randomly assigned to 3 treatments with supplemental dietary MF doses (0, 30, and 60 mg/kg). The results showed that dietary MF improved the eggshell thickness and strength, following the reduced broken egg ratio (P < 0.05). Histological analysis showed that dietary MF increased glandular density and luminal epithelium height in the shell gland (P < 0.05). MF treatment reduced the apoptotic index of the shell gland, following by improved antioxidant capacity (P < 0.05). The protein expression of Caspase 3 was down-regulated, and Nrf2 was up-regulated by dietary MF (P < 0.05). Furthermore, calcium (Ca) content in the serum and shell gland, as well as the activity of Ca2+-ATPase in the shell gland were increased by dietary MF (P < 0.05). Ca transport-related genes (ESRα, ESRß, KCNA1, OPN, CABP-28K and CDH6) in the shell gland were upregulated by dietary MF treatment (P < 0.05). In conclusion, dietary MF could ameliorate the eggshell quality of aged hens by improving antioxidative capability and Ca deposition in the shell gland of uterus.

Dietary hawthorn-leaves flavonoids improves ovarian function and liver lipid metabolism in aged breeder hens.

Hawthorn-leaves flavonoids (HF), extracted from hawthorn leaves, were reported to exert antioxidant, anti-inflammatory and hypolipidemic properties. The aim of our study was to investigate the effects of dietary HF on the reproduction performance and liver lipid metabolism of aged breeder hens. A total of 270 aged Qiling breeder hens (60-wk-old) were randomly divided into 3 treatments: 1) basic corn-soybean diet (CON); 2) basic corn-soybean diet supplemented with 30 mg/kg HF (LHF); 3) basic corn-soybean diet supplemented with 60 mg/kg HF (HHF). The results showed that supplemented HF significantly improved the egg-laying rate and hatching rate of aged breeder hens (P < 0.05). HF treatment reduced the serum TG, T-CHO and L-LDL levels (P < 0.05), and upregulated the mRNA expressions of ESR1, ESR2, VTGⅡ, ApoB, and ApoVI in the liver (P < 0.05). Serum estrogen levels in HF treated groups were elevated compared with the CON group (P < 0.05). In the HHF group, the number of the primordial follicles was higher in comparison with the CON group (P < 0.05). Furthermore, dietary supplementation with HF improved the activity of antioxidant enzymes (T-AOC, GSH-Pχ) (P < 0.05), following with the reversed ovarian apoptosis and morphological damage. In addition, 60 mg/kg dietary HF upregulated the protein expression of PCNA and Nrf2 in the ovary (P < 0.05). In summary, dietary supplementation with HF could improve the reproduction performance through regulating liver lipid metabolism and improving ovarian function in aged breeder hens.

Dietary alpha-lipoic acid supplementation improves spermatogenesis and semen quality via antioxidant and anti-apoptotic effects in aged breeder roosters.

The purpose of the present study was to investigate the effects of dietary alpha-lipoic acid (ALA) supplementation on the reproductive performance of aged breeder roosters. Sixteen 50-wk-old ROSS 308 breeder roosters were randomly allocated to two groups: roosters received a basal diet (CON), or a basal diet supplemented with 300 mg/kg of ALA (ALA). The results indicated that dietary ALA supplementation significantly increased sperm concentration, motility, viability, and membrane functional integrity. ALA also dramatically increased seminiferous tubule epithelial height (SEH) and testis scores. The ALA group had a higher serum concentration of testosterone than the CON group. ALA supplementation remarkably increased total antioxidant capacity (T-AOC), the enzyme activities of glutathione peroxidase (GPx), and catalase (CAT) in the testes; following a decrease in malondialdehyde (MDA) levels. In addition, we noted significant upregulation of Nrf2 mRNA and protein expression of and mRNA expression of its Downstream Genes (GPx1, NQO1, and GCLC), as well as significant downregulation of Keap1 mRNA expression in testicular tissue of aged roosters with ALA supplementation. The protein expression of Caspase 3 was downregulated and the protein expression of proliferating cell nuclear antigen (PCNA) was upregulated by ALA supplementation. The mRNA expression of spermatogenesis-related genes (ER1, AKT1, and Cav1) were markedly augmented in the ALA group compared with the CON group. In conclusion, dietary ALA supplementation enhanced the testicular antioxidant capacity through the Nrf2-signaling pathway, exerted anti-apoptotic effects, and improved the reproductive performance of aged roosters.

Dietary stevioside supplementation increases feed intake by altering the hypothalamic transcriptome profile and gut microbiota in broiler chickens.

BACKGROUND: Stevioside (STE) is a widely used sweetener. Despite the fact that chickens are insensitive to sweetness, dietary STE supplementation could increase the feed intake of broiler chickens. Stevioside might regulate the feeding behavior through functional mechanisms other than its high-potency sweetness. The present study was aimed to elucidate the potential sweetness-independent mechanism of an STE-induced orexigenic effect using the broiler chicken and considering the hypothalamic transcriptome profile and gut microbiome. RESULTS: The analysis of RNA-Seq identified 398 differently expressed genes (160 up-regulated and 238 down-regulated) in the hypothalamus of the STE-supplemented group compared with the control group. Cluster analysis revealed several appetite-related genes were differentially expressed, including NPY, NPY5R, TSHB, NMU, TPH2, and DDC. The analysis of 16S rRNA sequencing data also indicated that dietary STE supplementation increased the relative abundance of Lactobacillales, Bacilli, Lactobacillus, and Lactobacillaceae. Meanwhile, the proportion of Ruminococcaceae, Lachnospiraceae, Clostridia, and Clostridiales was decreased after dietary supplementation with STE. CONCLUSION: Dietary STE supplementation promoted feed intake through the regulation of the hypothalamic neuroactive ligand-receptor interaction pathway and the alteration of intestinal microbiota composition. This study provides valuable information about the sweetness-independent mechanism of the STE-induced orexigenic effect using the broiler chicken (which is insensitive to sweetness) as the animal model. © 2020 Society of Chemical Industry.

Alpha-lipoic acid improves the reproduction performance of breeder hens during the late egg-laying period.

Alpha-lipoic acid (ALA), a multifunctional antioxidant, can promote fatty acid mobilization, energy expenditure and scavenge free radicals. The effects of dietary ALA on the reproductive performance of breeder hens were investigated in the current study. In the 5-week experiment, 180 54-week-old Qiling breeder hens were randomly divided into three treatments with five replicates and supplemented with three levels of ALA (0, 300 and 600 mg/kg) in the basic corn-soya bean meal diets. 600 mg/kg ALA treatment group (HLA) significantly improved the eggshell thickness and strength (p < .05). ALA-treated groups improved egg-laying rate compared with the CON group, but with no statistically significant difference (p > .05). The levels of HDL-C, ALB and estradiol (E2) of the serum in the HLA group were elevated compared with the CON group (p < .05). In addition, ALA (600 mg/kg) treatment exhibited a reduced level of serum AST and TG (p < .05). Dietary ALA increased the activity of hepatic lipase in liver (p < .05). Supplemental 600 mg/kg ALA also improved the SOD activity and total antioxidant capacity level, along with a decreased MDA in ovarian tissue (p < .05). Furthermore, the mRNA expressions of ESR1, ESR2, VTG2 and ApoB in the liver and FSHR in follicles were upregulated in the HLA group (p < .05). In conclusion, dietary supplementation with 600 mg/kg ALA during the late egg-laying period could improve lipid metabolism and reproductive performance of breeder hens.

Effects of dietary sweeteners supplementation on growth performance, serum biochemicals, and jejunal physiological functions of broiler chickens.

The objective of this study was to investigate the effects of dietary 3 kinds of sweeteners supplementation on growth performance, serum biochemicals, and jejunal physiological functions of broiler chickens for 21 D. A total of one hundred ninety-two 1-day-old male Ross 308 broiler chicks were randomly divided into 4 treatments with 6 replicates for each treatment. The treatments were basal diet (CON), a basal diet supplemented with 250 mg/kg stevioside (STE), a basal diet supplemented with 100 mg/kg sucralose (SUC), and a basal diet supplemented with 600 mg/kg saccharin sodium (SAC). All birds were housed in 3-level battery cages. The results showed that dietary STE supplementation increased (P < 0.05) growth performance, serum total protein, serum albumin, and jejunal antioxidant capacity of broiler chickens. Both SUC and SAC supplementation decreased (P < 0.05) serum total protein and albumin. Dietary SAC supplementation impaired the intestinal integrity, permeability, and mucus layer of the jejunum in broiler chickens. In addition, SAC supplementation elevated (P < 0.05) the transcription expression level of jejunal bitter taste receptors and induced excessive jejunal apoptosis. Our data suggest that STE could be potentially applied as a growth-promoting and antioxidant feed additive in broiler chickens. Whereas, dietary supplementation with high level SAC has side-effects on the jejunal physiological functions of broiler chickens.

Dietary genistein supplementation protects against lipopolysaccharide-induced intestinal injury through altering transcriptomic profile.

Genistein is abundant in the corn-soybean meal feed. Little information is available about the effect of dietary genistein on the intestinal transcriptome of chicks, especially when suffering from intestinal injury. In this study, 180 one-day-old male ROSS 308 broiler chickens were randomly allocated to 3 groups, with 4 replicates (cages) of 15 birds each. The treatments were as follows: chicks received a basal diet (CON), a basal diet and underwent lipopolysaccharide-challenge (LPS), or a basal diet supplemented with 40 mg/kg genistein and underwent LPS-challenge (GEN). LPS injection induced intestinal injury and inflammatory reactions in the chicks. Transcriptomic analysis identified 7,131 differently expressed genes (3,281 upregulated and 3,851 downregulated) in the GEN group compared with the LPS group (P adjusted value < 0.05, |fold change| > 1.5), which revealed that dietary genistein exposure altered the gene expression profile and signaling pathways in the ileum of LPS-treated chicks. Furthermore, dietary genistein improved intestinal morphology, mucosal immune function, tight junction, antioxidant activity, apoptotic process, and growth performance, which were adversely damaged by LPS injection. Therefore, adding genistein into the diet of chicks can alter RNA expression profile and ameliorate intestinal injury in LPS-challenged chicks, thereby improving the growth performance of chicks with intestinal injury.

Dietary l-arginine supplementation ameliorates inflammatory response and alters gut microbiota composition in broiler chickens infected with Salmonella enterica serovar Typhimurium.

This study was conducted to investigate the effects of dietary arginine (Arg) supplementation on the inflammatory response and gut microbiota of broiler chickens subjected to Salmonella enterica serovar Typhimurium. One hundred and forty 1-day-old Arbor Acres male birds were randomly assigned to a 2 × 2 factorial arrangement including diet treatment (with or without 0.3% Arg supplementation) and immunological stress (with or without S. typhimurium challenge). Samples were obtained at 7 D after infection (day 23). Results showed that S. typhimurium challenge caused histopathological and morphological damages, but Arg addition greatly reduced these intestinal injuries. S. typhimurium challenge elevated the levels of serum inflammatory parameters, including diamine oxidase, C-reactive protein, procalcitonin, IL-1ß, IL-8, and lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITNF) homolog. However, Arg supplementation decreased the serum procalcitonin, IL-1ß, IL-8, and LITNF concentration. S. typhimurium challenge significantly increased jejunal IL-1ß, IL-8, IL-10, and IL-17 mRNA expression and tended to upregulate IL-22 mRNA expression, but Arg supplementation remarkably reduced IL-8 mRNA expression, tended to downregulate IL-22 mRNA expression, and dramatically elevated IFN-γ and IL-10 mRNA expression. In addition, sequencing data of 16S rDNA indicated that the population of Proteobacteria phylum; Enterobacteriaceae family; Escherichia-Shigella, and Nitrosomonas genera; and Escherichia coli and Ochrobactrum intermedium species were more abundant, but the population of Rhodocyclaceae and Clostridiaceae_1 families and Candidatus Arthromitus genus were less abundant in the ileal digesta of birds with only S. typhimurium infection when compared with the controls. Treatment with Arg in birds subjected to S. typhimurium challenge increased the abundances of Firmicutes phylum, Clostridiaceae_1 family, Methylobacterium and Candidatus Arthromitus genera but decreased the abundance of Nitrosomonas genus and Rhizobium cellulosilyticum and Rubrobacter xylanophilus species as compared with the only S. typhimurium-challenged birds. In conclusion, Arg supplementation can alleviate intestinal mucosal impairment by ameliorating inflammatory response and modulating gut microbiota in broiler chickens challenged with S. typhimurium.

Effects of High-Dose Genistein on the Hypothalamic RNA Profile and Intestinal Health of Female Chicks.

Genistein is abundant in animal feed. In this study, the side effects of high-dose genistein on intestinal health and hypothalamic RNA profile were evaluated. Chicks exposed to high-dose genistein by intraperitoneal injection (416 ± 21, 34.5 ± 2.5) and feed supplementation (308 ± 19, 27.2 ± 2.1) both showed a reduced body weight gain and feed intake in comparison with the control group (261 ± 16, 22.7 ± 1.6, P < 0.01). In comparison with the control (22.4 ± 0.5, 33.3 ± 2.4), serum levels of albumin and total protein were decreased after high-dose genistein injection (21.6 ± 0.5, 31.8 ± 1.6) and diet supplementation (20.6 ± 0.9, 29.9 ± 2.5, P < 0.001). Interestingly, the genistein diet presented the chick hypothalamus with downregulated expression of bitter receptors (TAS1R3, P < 0.05). Meanwhile, it upregulated the expressions of TAS2R1 (P < 0.05) and downstream genes (PLCB2 and IP3R3) in the ileum (P < 0.05). Accordingly, high-dose dietary genistein reduced villus height and the abundance of Lactobacillus, along with the increased abundance of pathogenic bacteria in the ileum (P < 0.05). Furthermore, transcriptomic analysis identified 348 differently expressed genes (168 upregulated and 224 downregulated) in the high-dose dietary genistein treated group in comparison with the control (P < 0.05, |log2FoldChange| > 0.585). Therefore, high-dose dietary genistein altered the hypothalamic RNA profile and signal processing. Cluster analysis further revealed that high-dose dietary genistein significantly influenced apoptosis, the immune process, and the whole synthesis of steroid hormones in the hypothalamus (P < 0.05). In conclusion, high-dose dietary genistein altered the hypothalamic RNA profile and intestinal health of female chicks.

RNA Expression Profile and Alternative Splicing Signatures of Genistein-Treated Breeder Hens Revealed by Hepatic Transcriptomic Analysis.

Little information has been available about the influence of dietary genistein (GEN) on hepatic transcriptome of laying broiler breeder (LBB) hens. The study is aimed at broadening the understanding of RNA expression profiles and alternative splicing (AS) signatures of GEN-treated breeder hens and thereby improving laying performance and immune function of hens during the late egg-laying period. 720 LBB hens were randomly allocated into three groups with supplemental dietary GEN doses (0, 40 mg/kg, and 400 mg/kg). Each treatment has 8 replicates of 30 birds. Dietary GEN enhanced the antioxidative capability of livers, along with the increased activities of glutathione peroxidase and catalase. Furthermore, it improved lipid metabolic status and apoptotic process in the liver of hens. 40 mg/kg dietary GEN had the better effects on improving immune function and laying performance. However, transcriptome data indicated that 400 mg/kg dietary GEN did negative regulation of hormone biosynthetic process. Also, it upregulated the expressions of EDA2R and CYR61 by the Cis regulation of neighbouring genes (lncRNA_XLOC_018890 and XLOC_024242), which might activate NF-κB and immune-related signaling pathway. Furthermore, dietary GEN induced AS events in the liver, which also enriched into immune and metabolic process. Therefore, the application of 40 mg/kg GEN in the diet of breeder hens during the late egg-laying period can improve lipid metabolism and immune function. We need to pay attention to the side-effects of high-dose GEN on the immune function.

In vivo and in vitro protective effect of arginine against intestinal inflammatory response induced by Clostridium perfringens in broiler chickens.

BACKGROUND: Necrotic enteritis is a widespread disease in poultry caused by Clostridium perfringens. We previously reported that dietary arginine supplementation protected the intestinal mucosa of broiler chickens with necrotic enteritis, but the related protective mechanisms remain unclear. The in vivo trial was designed as a 2 × 2 factorial arrangement to evaluated the effects of arginine supplementation on inflammatory responses, arginine transporters, arginine catabolism and JAK-STAT signalling pathway in broiler chickens challenged with C. perfringens or without C. perfringens. Furthermore, we validated the in vivo results using intestinal epithelial cells of chicken embryos. RESULTS: C. perfringens infection markedly increased gut gross pathological and histopathological lesion scores, promoted liver C. perfringens invasion, reduced serum arginine levels, and elevated jejunal mucosal lysozyme activities (P < 0.05), but these effects were significantly reversed by arginine supplementation in vivo (P < 0.05). The challenge significantly increased serum procalcitonin levels, jejunal mucosal iNOS activities and jejunal IL-6, TGF-ß3, cationic amino acid transporter (CAT)-1, and CAT-3 mRNA expression (P < 0.05), whereas arginine supplementation significantly reduced jejunal IFN-γ, IL-1ß, IL-6, IL-10, TGF-ß3, and CAT-3 mRNA expression (P < 0.05). Arginine supplementation significantly attenuated the C. perfringens challenge-induced increases in jejunal iNOS, arginase 2, arginine decarboxylase, arginine:glycine amidinotransferase, JAK1, JAK3, STAT1, and STAT6 mRNA expression (P < 0.05). The in vitro experiment showed that C. perfringens challenge markedly increased cellular cytotoxicity and the mRNA expression of IL-1ß, IL-8, IL-10, CAT-1 and CAT-3 (P < 0.05), which were significantly reversed by 50 µmol/L and/or 400 µmol/L arginine pre-treatment (P < 0.05). CONCLUSIONS: Arginine prevented C. perfringens challenge-induced circulated arginine deficiency, normalized intestinal arginine transport and catabolism, down-regulated JAK-STAT signalling pathway and attenuated the inflammatory response, which exerted protective effects on the intestine of broiler chickens.

Supplementing Genistein for Breeder Hens Alters the Fatty Acid Metabolism and Growth Performance of Offsprings by Epigenetic Modification.

The experiment was designed to clarify the effect and molecular mechanism of maternal genistein (GEN) on the lipid metabolism and developmental growth of offspring chicks. Laying broiler breeder (LBB) hens were supplemented with 40 mg/kg genistein (GEN), while the control group was fed with the low-soybean meal diet. The offspring chicks were grouped according to the mother generation with 8 replicates each. Hepatic transcriptome data revealed 3915 differentially expressed genes (DEGs, P adjusted < 0.05, fold change > 1.5 or fold change < 0.67) between chicks in the two groups. Maternal GEN activated the GH-IGF1-PI3K/Akt signaling pathway, which promoted the developmental processes and cellular amino acid metabolic processes, as well as inhibited the apoptotic process. GEN treatment significantly increased the weight gain, breast muscle percentage, and liver index in chicks. PANTHER clustering analysis suggested that maternal GEN enhanced the antioxidant activity of chicks by the upregulation of gene (SOD3, MT1, and MT4) expression. Accordingly, the activities of T-AOC and T-SOD in the liver were increased after GEN treatment. The overrepresentation tests revealed that maternal GEN influenced the glycolysis, unsaturated fatty acid biosynthesis, acyl-coenzyme A metabolism, lipid transport, and cholesterol metabolism in the chick livers. Hepatic cholesterol and long-chain fatty acid were significantly decreased after GEN treatment. However, the level of arachidonic acid was higher in the livers of the GEN-treated group compared with the CON group. Moreover, GEN treatment enhanced fatty acid ß-oxidation and upregulated PPARδ expression in the chick liver. ChIP-qPCR analysis indicated that maternal GEN might induce histone H3-K36 trimethylation in the promoter region of PPARδ gene (PPARD) through Iws1, methyltransferases. It also induced histone H4-K12 acetylation at the PPARD promoter through MYST2, which activated the PPAR signaling pathways in the chick livers. In summary, supplementing LBB hens with GEN can alter lipid metabolism in the offspring chicks through epigenetic modification and improve the antioxidative capability as well as growth performance.