Arginine alleviates Clostridium perfringens α toxin-induced intestinal injury in vivo and in vitro via the SLC38A9/mTORC1 pathway.

Introduction: Clostridium perfringens α toxin is a main virulence factor responsible for gut damage in animals. Arginine is a functional amino acid exhibiting significant immunoregulatory activities. However, the effects and immunoregulatory mechanisms of arginine supplementation on α toxin-induced intestinal injury remain unclear. Methods: In vivo, 256 male Arbor Acres chickens were randomly assigned to a 2×2 factorial arrangement, involving diet treatments (with or without 0.3% arginine supplementation) and immunological stress (with or without α toxin challenge). In vitro, IEC-6 cells were treated with or without arginine in the presence or absence of α toxin. Moreover, IEC-6 cells were transfected with siRNA targeting mTOR and SLC38A9 to explore the underlying mechanisms. Results and discussion: The results showed that in vivo, arginine supplementation significantly alleviated the α toxin-induced growth performance impairment, decreases in serum immunoglobulin (Ig)A and IgG levels, and intestinal morphology damage. Arginine supplementation also significantly reduced the α toxin-induced increase in jejunal proinflammatory cytokines interleukin (IL)-1ß, IL-6 and IL-17 mRNA expression. Clostridium perfringens α toxin significantly decreased jejunal mechanistic target of rapamycin (mTOR) and solute carrier family 38 member 9 (SLC38A9) mRNA expression, while arginine supplementation significantly increased mTOR and SLC38A9 mRNA expression. In vitro, arginine pretreatment mitigated the α toxin-induced decrease in cell viability and the increase in cytotoxicity and apoptosis. Arginine pretreatment also alleviated the α toxin-induced upregulation of mRNA expression of inflammation-related cytokines IL-6, C-X-C motif chemokine ligand (CXCL)10, CXCL11 and transforming growth factor-ß (TGF-ß), as well as apoptosis-related genes B-cell lymphoma-2 associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-extra large (Bcl-XL) and cysteinyl aspartate specific proteinase 3 (Caspase-3) and the ratio of Bax to Bcl-2. Arginine pretreatment significantly increased the α toxin-induced decrease in mTOR, SLC38A9, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) and ribosomal protein S6 kinase (S6K) mRNA expression. Knockdown SLC38A9 and mTOR largely abrogated the positive effects of arginine pretreatment on α toxin-induced intracellular changes. Furthermore, SLC38A9 silencing abolished the increased mTOR mRNA expression caused by arginine pretreatment. In conclusion, arginine administration attenuated α toxin-induced intestinal injury in vivo and in vitro, which could be associated with the downregulation of inflammation via regulating SLC38A9/mTORC1 pathway.

Parental betaine supplementation promotes gosling growth with epigenetic modulation of IGF gene family in the liver.

Betaine is widely used as a feed additive in the chicken industry to promote laying performance and growth performance, yet it is unknown whether betaine can be used in geese to improve the laying performance of goose breeders and the growth traits of offspring goslings. In this study, laying goose breeders at 39 wk of age were fed basal (Control, CON) or betaine-supplemented diets at low (2.5 g/kg, LBT) or high (5 g/kg, HBT) levels for 7 wk, and the breeder eggs laid in the last week were collected for incubation. Offspring goslings were examined at 35 and 63 d of age. The laying rate tended to be increased (P = 0.065), and the feed efficiency of the breeders was improved by betaine supplementation, while the average daily gain of the offspring goslings was significantly increased (P < 0.05). Concentrations of insulin-like growth factor 2 (IGF-2) in serum and liver were significantly increased in the HBT group (P < 0.05), with age-dependent alterations of serum T3 levels. Concurrently, hepatic mRNA expression of the IGF gene family was significantly increased in goslings derived from betaine-treated breeders (P < 0.05). A higher ratio of proliferating cell nuclear antigen (PCNA)-immunopositive nuclei was found in the liver sections of the HBT group, which was confirmed by significantly upregulated hepatic expression of PCNA mRNA and protein (P < 0.05). Moreover, hepatic expression of thyroxine deiodinase type 1 (Dio1) and thyroid hormone receptor ß (TRß) was also significantly upregulated in goslings of the HBT group (P < 0.05). These changes were associated with significantly higher levels of global DNA 5-mC methylation, together with increased expression of methyl transfer genes (P < 0.05), including betaine-homocysteine methyltransferase (BHMT), glycine N-methyltransferase (GNMT), and DNA (cytosine-5-)-methyltransferase 1 (DNMT1). The promoter regions of IGF-2 genes, as well as the predicted TRß binding site on the IGF-2 gene, were significantly hypomethylated (P < 0.05). These results indicate that gosling growth can be improved by dietary betaine supplementation in goose breeders via epigenetic modulation of the IGF gene family, especially IGF-2, in the liver.

The goose industry plays important roles in economics, cultures, and ecosystems, yet the low laying and growth rates of many indigenous breeds hinders the development of the goose farming. Betaine, an important methyl donor, is commonly used as a feed additive in livestock and poultry to enhance animal growth. Dietary supplementation of betaine in laying hens or gestational sows has been reported to promote the growth of their offspring. Here, we sought to investigate whether and how dietary betaine supplementation affects the growth and development of offspring goslings. In this study, goose breeders, both male and female, were fed a basal diet supplemented respectively with 0, 2.5, or 5 g/kg betaine for 7 wk. Goslings hatched from the breeder eggs of different groups were raised under the same standard condition for assessing the growth performance. Parental betaine increases the growth rate of offspring goslings with decreased DNA methylation on the IGF-2 gene promoter and increased expression of the IGF-2 gene in the liver. These results provide scientific evidence for the inter-generational effect of betaine on gosling growth.

Changes in gene expression in the intestinal mucus of broilers with woody breast myopathy.

Previous work has shown that dietary treatments affect woody breast (WB) incidence differently, which indicates that gut conditions such as gut barrier function, inflammation, and oxidative stress are likely related to WB. In this study, dietary supplementation with antibiotics (bacitracin) or probiotics (Bacillus subtilis) was investigated for their effects on the expression of transcripts related to gut barrier function, inflammation, and oxidative stress in the mucus lining of the jejunum from broilers with or without WB. A split-plot experimental design was used in this study. The dietary treatments served as the main plot factor and the breast muscle condition was the subplot factor. On d 41, jejunum mucus was collected from 1 bird from each of 3 replicate pens in each 3 dietary treatment groups that exhibited WB and an additional bird that contained a normal breast (3 biological replicates/treatment/phenotype; 3 × 3 × 2, total N = 18). Total RNA was extracted using a commercial RNA extraction kit. The expression levels of CLDN1, MUC6, TLR2A, TLR2B, TLR4, IFN-γ, IL-1ß, IL-8L1, IL-10, NOS2, and SOD were determined using 2-step RT-qPCR analysis. The gene expression difference in ΔCt values was determined after normalizing with the chicken 18S rRNA gene. When the significant differences occurred between treatments, the relative fold change was calculated using the ΔΔCt method and the significance level was calculated. The PROC GLM procedure of SAS 9.4 was used, and the level of significance was set at P ≤ 0.05. There were no significant interactive effects between diet and the breast muscle condition on the expression of any of the genes tested. However, birds with WB exhibited higher MUC6 (P < 0.0001) gene expression levels than birds with normal breast muscles. In addition, the expression of SOD decreased in birds that were fed the antibiotic diet when compared to birds that were fed the probiotic diet (P = 0.014). In conclusion, WB identified in broilers tested in the current study is attributed to increased expression of mucin, indicating a correlation between WB incidence and gel-forming mucin secretion and pathogen signaling.

Betaine supplementation alleviates corticosterone-induced hepatic cholesterol accumulation through epigenetic modulation of HMGCR and CYP7A1 genes in laying hens.

Excessive corticosterone (CORT) exposure could cause hepatic cholesterol accumulation in chickens and maternal betaine supplementation could decrease hepatic cholesterol deposition through epigenetic modifications in offspring chickens. Nevertheless, it remains uncertain whether providing betaine to laying hens could protect CORT-induced hepatic cholesterol accumulation via epigenetic mechanisms. This study aimed to examine the effects of dietary betaine on plasma and hepatic cholesterol contents, expression of cholesterol metabolic genes, as well as DNA methylation on their promoters in the liver of laying hens exposed to CORT. A total of 72 laying hens at 130 d of age were randomly divided into 3 groups: control (CON), CORT, and CORT+betaine (CORT+BET) groups. The experiment lasted for 35 d. Chickens in CON and CORT groups were fed a basal diet, whereas the CORT+BET group chickens were fed the basal diet supplemented with 0.1% betaine for 35 d. On d 28 of the experiment, chickens in CORT and CORT+BET groups received daily subcutaneous injections of CORT (4.0 mg/kg body weight), whereas the CON group chickens were injected with an equal volume of solvent for 7 d. The results showed that CORT administration led to a significant increase (P < 0.05) in the contents of cholesterol in plasma and liver, associated with activation (P < 0.05) of sterol regulatory element binding transcription factor 2 (SREBP2), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), lecithin-cholesterol acyltransferase (LCAT) and low-density lipoprotein receptor (LDLR) genes expression, and inhibition of cholesterol-7-alpha hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1) genes expression in the liver compared to the CON. In contrast, CORT-induced up-regulation of HMGCR mRNA and protein abundances and downregulation of CYP7A1 mRNA and protein abundances were completely normalized (P < 0.05) by betaine supplementation. Besides, CORT injection led to significant hypomethylation (P < 0.05) on HMGCR promoter and hypermethylation (P < 0.05) on CYP7A1 promoter. Moreover, dietary betaine rescued (P < 0.05) CORT-induced changes in methylation status of HMGCR and CYP7A1 genes promoters. These results indicate that dietary betaine addition protects laying hens from CORT-induced hepatic cholesterol accumulation via epigenetic modulation of HMGCR and CYP7A1 genes.

Molecular cloning, subcellular localization, and rapid recruitment to DNA damage sites of chicken Ku70.

Ku70 is a multifunctional protein with pivotal roles in DNA repair via non-homologous end-joining, V(D)J recombination, telomere maintenance, and neuronal apoptosis control. Nonetheless, its regulatory mechanisms remain elusive. Chicken Ku70 (GdKu70) cDNA has been previously cloned, and DT40 cells expressing it have significantly contributed to critical biological discoveries. GdKu70 features an additional 18 amino acids at its N-terminus compared to mammalian Ku70, the biological significance of which remains uncertain. Here, we show that the 5' flanking sequence of GdKu70 cDNA is not nearly encoded in the chicken genome. Notably, these 18 amino acids result from fusion events involving the NFE2L1 gene on chromosome 27 and the Ku70 gene on chromosome 1. Through experiments using newly cloned chicken Ku70 cDNA and specific antibodies, we demonstrated that Ku70 localizes within the cell nucleus as a heterodimer with Ku80 and promptly accumulates at DNA damage sites following injury. This suggests that the functions and spatiotemporal regulatory mechanisms of Ku70 in chickens closely resemble those in mammals. The insights and resources acquired will contribute to elucidate the various mechanisms by which Ku functions. Meanwhile, caution is advised when interpreting the previous numerous key studies that relied on GdKu70 cDNA and its expressing cells.

N-carbamylglutamate supplementation induces functional egg production in layers by modulating liver transcriptome profiles.

Eggs rich in polyunsaturated fatty acids (PUFA), known as functional eggs, are animal products deemed beneficial to human health and possess high economic value. The production of functional eggs involves supplementing exogenous additives with the ability to regulate lipid metabolism. As N-Carbamylglutamate (NCG) serves as an endogenous arginine synthesizer, and arginine acts as the substrate for the formation of nitric oxide (NO), the biological function of NCG is partially mediated by NO. NO is a key regulatory molecule in lipid metabolism, suggesting that NCG may also have the ability to modulate lipid metabolism. In order to assess the capacity of NCG in regulating liver lipid metabolism and its potential application in producing functional eggs, we conducted a study to investigate the effects of dietary supplementation of NCG on production performance, serum, and liver NO levels, yolk fatty acid composition, and the liver transcriptome of layers. In this study, we utilized 30 layers of the Jinghong No.1 breed, all aged 45 wk. All the birds were randomly divided into 2 groups. Each group had 5 replicates, and each replicate had 3 birds. We provided them with different diets: one group received the basic diet, and the other group's diet was supplemented with 0.08% NCG. The experiment lasted for 14 wk. The results did not reveal any positive impact of NCG on production performance. However, NCG supplementation elevated NO levels in serum and liver, along with an increase in yolk PUFA, ω-3, and ω-6 fatty acids. Liver transcriptome analysis identified 124 upregulated differentially expressed genes (DEGs) and 43 downregulated DEGs due to NCG supplementation. Functional annotation using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database highlighted 3 upregulated DEGs (CPT1A, MOGAT1, and CHKA) and 2 downregulated DEGs (FASN and ETNPPL) associated with lipid metabolism. Pathway enrichment analysis revealed that CPT1A was enriched in the AMPK signaling pathway and the PPAR signaling pathway, while FASN was enriched in the AMPK signaling pathway. Thus, CPT1A and FASN are potential functional genes related to lipid metabolism facilitated by NCG supplementation. In summary, our study suggests that NCG supplementation modulates liver lipid metabolism, leading to the production of functional eggs in layers.

A comparative study on the effect of limestone particle size on performance, ileal digestibility of calcium and phosphorus, and bone characteristics in broilers and pullets.

1. The effects of limestone particle size on growth performance, gastrointestinal tract (GIT) traits, ileal morphology, duodenal gene expression of calbindin, apparent ileal digestibility coefficients (AIDC) of calcium (Ca) and phosphorus (P) and tibia characteristics in broilers and pullets were assessed in broilers and pullets. These birds have different growth rates and likely different responses to parameters, such as particle size.2. A total of 240 chicks aged one day, 120 Ross 308 female broilers, and 120 Hy-Line pullets were allocated randomly into four treatments in a 2 × 2 factorial arrangement with two bird types (broilers vs. pullets) and two limestone particle sizes (<0.5 mm versus 1-2 mm) to give six replicates containing 10 chicks in each from 1 to 21 d of age.3. Feed intake and weight gain were greater (P < 0.001) and feed per gain (FCR) was better (P < 0.001) in broilers compared to pullets from 1 to 21 d of age. Greater villus width (P < 0.01), villus height (P < 0.001) and crypt depth (P < 0.01) were seen for broilers compared to pullets.4. Pullets fed coarse Ca particles had higher calbindin gene expression at 21 d of age (P = 0.05). Both AIDC of Ca and P were higher (P < 0.001) in broilers compared to pullets. The AIDC of Ca from 0.463 to 0.516 was increased (P < 0.05) by feeding coarse limestone particles. A significant interaction was found between bird type and limestone particle size (P < 0.01), where pullets fed coarse Ca particles had higher bone P concentration in tibia than broilers.5. Broilers had better ileum absorptive capacity and growth performance compared to pullets. The AIDC of Ca and P was higher in broilers than in pullets. Increased limestone particle size elevated villus height, AIDC of Ca and concentration of P in the tibia.

Low-Se Diet Increased Mitochondrial ROS to Suppress Myoblasts Proliferation and Promote Apoptosis in Broilers via miR-365-3p/SelT Signaling Axis.

microRNA (miRNA) controls the post-transcriptional translation of mRNA to affect the expression of many genes participating in functional interaction pathways. Selenoproteins are characterized by their antioxidant activity, wherein selenoprotein T (SelT) is an essential membrane-bound selenoprotein serving as a guardian of intracellular homeostasis. During muscle development and regeneration, myoblasts enter the cell cycle and rapidly proliferate. However, the role of SelT in muscle development and selenium (Se) deficiency-induced muscle damage remains poorly investigated. This study established Se deficient broiler models, chicken embryos models, and cultured chicken primary myoblasts in vitro. We showed that Se deficiency induced skeletal muscle damage in broilers, promoted miR-365-3p expression, and downregulated the level of SelT, significantly. The absence of SelT led to the accumulation of mitochondrial superoxide and downregulated mitochondrial dynamics gene expression, which, in turn, induced the disruption of mitochondria potential and blocked the oxidative phosphorylation (OXPHOS) process. Limited ATP production rate caused by mitochondrial ROS overproduction went along with cell cycle arrest, cell proliferation slowness, and myocyte apoptosis increase. Using Mito-TEMPO for mitochondrial ROS elimination could effectively mitigate the above adverse reactions and significantly restore the proliferation potential of myoblasts. Moreover, we identified miR-365-3p, a miRNA that targeted SelT mRNA to inhibit myoblast proliferation by disrupting intracellular redox balance. The omics analysis results showed that Se deficiency led to the significant enrichment of "cell cycle", "oxidative stress response", and "oxidative phosphorylation" pathway genes. Finally, we proved that the effect of the miR-365-3p/SelT signaling axis on muscle development did exist in the chicken embryo stage. In summary, our findings revealed that miR-365-3p was involved in broiler skeletal muscle damage in Se deficiency by targeting SelT, and SelT, serving as an intracellular homeostasis guardian, resisted mitochondrial oxidative stress, and protected ATP generation, promoting myoblast proliferation and inhibiting apoptosis. This study provides an attractive target for the cultivated meat industry and regenerative medicine.

Transcriptome analysis of hypothalamus and pituitary tissues reveals genetic mechanisms associated with high egg production rates in Changshun green-shell laying hens.

BACKGROUND: Changshun green-shell laying hens are unique to the Guizhou Province, China, and have high egg quality but relatively low yield. Egg production traits are regulated by the hypothalamus-pituitary-ovary axis. However, the underlying mechanism remains unclear. Thus, we conducted RNA sequencing of hypothalamic and pituitary tissues from low- and high-yielding Changshun green-shell laying hens to identify critical pathways and candidate genes involved in controlling the egg production rate. RESULTS: More than 39 million clean reads per sample were obtained, and more than 82% were mapped to the Gallus gallus genome. Further analysis identified 1,817 and 1,171 differentially expressed genes (DEGs) in the hypothalamus and pituitary, respectively. Nineteen DEGs were upregulated in both the hypothalamus and pituitary of high-yielding chickens. The functions of these DEGs were mainly associated with ion transport or signal transduction. Gene set enrichment analysis revealed that the pathways enriched in the hypothalamus were mainly associated with gonadotropin-releasing hormone (GnRH) secretion, neurotransmitter release, and circadian rhythms. The pathways enriched in the pituitary were mainly associated with GnRH secretion, energy metabolism, and signal transduction. Five and four DEGs in the hypothalamus and pituitary, respectively, were selected randomly for qRT-PCR analysis. The expression trends determined via qRT-PCR were consistent with the RNA-seq results. CONCLUSIONS: The current study identified 19 DEGs upregulated in both the hypothalamus and pituitary gland, which could provide an important reference for further studies on the molecular mechanisms underlying egg production in Changshun green-shell laying hens. In addition, enrichment analysis showed that GnRH secretion and signal transduction, especially neurotransmitter release, play crucial roles in the regulation of egg production.

Research Note: Transcriptome analysis reveals differentially expressed genes regulated muscle development in Pekin ducks during dietary threonine deficiency.

To investigate the underlying molecular mechanism of threonine (Thr) regulation on the development of breast muscle in Pekin ducks, 240 male Pekin ducks at 1 d of age were fed a Thr deficiency diet (Thr-D), Thr sufficiency diet (Thr-S), or Thr excess diet (Thr-E) for 21 d. The results showed that Thr-D reduced body weight (BW), average weight gain (ADG), and average feed intake (ADFI), and increased the feed/gain (F/G) in Pekin ducks (P < 0.05), and Thr-E did not affect BW, ADG, ADFI, or F/G (P > 0.05), compared with Thr-S. The diameter and cross-sectional area of the breast muscle fibers in the Thr-S group were larger than those in the Thr-D group (P < 0.05). RNA sequencing revealed 1,300 differential expressed genes (DEGs) between the Thr-D and Thr-S groups, of which 625 were upregulated and 675 were downregulated by Thr-D. KEGG analysis showed that the upregulated genes were enriched in mTOR, FoxO, Wnt, fat digestion and absorption, and other signaling pathways. The downregulated genes were enriched in the MAPK signaling, glycolysis/gluconeogenesis, adipocytokine signaling, and biosynthesis of unsaturated fatty acids signaling pathways. The genes of Wnt family member 3a (Wnt3a), myogenin, myozenin 2, and insulin like growth factor 2 mRNA binding protein were upregulated, and platelet derived growth factor subunit B, PDGF receptor beta and Wnt4 were downregulated by Thr deficiency, which involving in muscle development. Our findings indicated that Thr increased breast fiber size, perhaps because Thr affected the proliferation and differentiation of satellite cells in breast muscle of ducks after hatch. Our results provide novel insights into new understanding of the molecular mechanisms underlying breast muscle development in ducks subjected to dietary Thr.

Effect of Glucose Supplementation on Apoptosis in the Pectoralis major of Chickens Raised under Thermoneutral or Heat Stress Environment.

Reduced feed intake during heat stress (HS) disrupts glucose homeostasis, thereby resulting in endoplasmic reticulum (ER) stress and triggering apoptosis in chickens. We hypothesize that glucose supplementation could reduce apoptosis in chickens raised under HS. This study comprised 456 28-day-old broiler chickens randomly assigned to four treatment combinations under glucose supplementation and HS. The treatments were TN0, TN6, HS0, and HS6 with two glucose levels (0% and 6%) and two temperature levels (25 °C (thermoneutral-TN) and 35 °C (8.00 AM to 8.00 PM, (HS)). After 7 days post-HS, the blood glucose level for the HS6 group was higher than for TN0, TN6, and HS0. We studied the mRNA expression of genes and caspase-3 activity in the four experimental groups. The expressions of GCN2, ATF4, CHOP, and FOXO3a increased during HS regardless of glucose supplementation, while PERK and MAFbx increased only under HS with glucose supplementation. We show that under TN conditions, glucose supplementation led to a significant increase in cellular apoptosis in the Pectoralis (P.) major. However, under HS with glucose, the level of apoptosis was similar to that of chickens raised under TN conditions with no glucose supplementation. The utility of glucose to curtail apoptosis under HS should be tested under other intense models of HS.

Flaxseed Supplementation in Chicken Feed Accelerates Salmonella enterica subsp. enterica Serovar Enteritidis Clearance, Modulates Cecum Microbiota, and Influences Ovarian Gene Expression in Laying Hens.

Salmonella is a foodborne pathogen that poses a serious threat to both human and animal health and food safety. Flaxseed is rich in unsaturated fatty acids; has anti-metabolic syndrome, anti-inflammatory, and neuroprotective properties; and may be a potential source of feed additives. To investigate the impact of flaxseed on Salmonella-infected laying hens, we administered Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) after adding flaxseed to the feed of laying hens (15% [750 mg/kg]). S. Enteritidis colonization was reduced and its clearance was accelerated from the laying hens. Furthermore, flaxseed supplementation mitigated the damage to the ileum caused by S. Enteritidis. We analyzed alterations in intestinal flora through 16S rRNA amplicon sequencing. S. Enteritidis infection increased the abundance of Akkermansia and triggered the host inflammatory response. Conversely, the addition of flaxseed to the feed increased the abundance of beneficial intestinal bacteria, such as Lactobacilli and Bacteroides. Ovarian health is important for egg production performance in laying hens and our findings indicate that S. Enteritidis can persist in the ovaries for an extended period. Therefore, we further performed transcriptome sequencing analysis of ovarian tissues on day seven after S. Enteritidis infection. S. Enteritidis infection leads to altered ovarian gene expression, including the downregulation of lipid metabolism and growth and development genes and the upregulation of host immune response genes in laying hens. The upregulation of genes associated with growth and development may have stimulated ovarian growth and development.

Effects of melittin on production performance, antioxidant function, immune function, heat shock protein, intestinal morphology, and cecal microbiota in heat-stressed quails.

The purpose of this study was to investigate the effects of melittin on production performance, antioxidant function, immune function, heat shock protein, intestinal morphology, and cecal microbiota of heat-stressed quails. A total of 120 (30-day-old) male quails were randomly divided into 3 groups. Each group consisted of 4 replicates with 10 birds per replicate. The ambient temperature of the control group (group W) was 24°C ± 2°C. The heat stress group (group WH) and the heat stress + melittin group (group WHA2) were subjected to heat stress for 4 h from 12:00 to 16:00 every day, and the temperature was 36°C ± 2°C for 10 d. The results showed that compared with the group W, heat stress significantly decreased growth performance, serum and liver antioxidative function, immune function, intestinal villus height (VH) and villus height-to-crypt depth ratio (VH/CD), and cecal microbiota Chao and ACE index (P < 0.05). The crypt depth (CD) in the small intestine, and HSP70 and HSP90 mRNA levels in the heart, liver, spleen, and kidney were significantly increased (P < 0.05). Dietary melittin significantly increased growth performance, serum and liver antioxidative function, immune function, intestinal VH and VH/CD, and cecal microbiota Shannon index in heat-stressed quails (P < 0.05). Melittin significantly decreased small intestinal CD, and HSP70 and HSP90 mRNA levels in the viscera (P < 0.05). Furthermore, dietary melittin could have balanced the disorder of cecal microbiota caused by heat stress and increased the abundance and diversity of beneficial microbiota (e.g., Firmicutes were significantly increased). PICRUSt2 functional prediction revealed that most of the KEGG pathways with differential abundance caused by high temperature were related to metabolism, and melittin could have restored them close to normal levels. Spearman correlation analysis showed that the beneficial intestinal bacteria Anaerotruncus, Bacteroidales_S24-7_group_norank, Lachnospiraceae_unclassified, Shuttleworthia, and Ruminococcaceae_UCG-014 increased by melittin were positively correlated with average daily feed intake, the average daily gain, serum and liver superoxide dismutase, IgG, IgA, bursa of Fabricius index, and ileum VH and VH/CD. In sum, our results demonstrate for the first time that dietary melittin could improve the adverse effects of heat stress on antioxidant function, immune function, heat shock protein, intestinal morphology, and cecal microbiota in quails, consequently improving their production performance under heat stress.

Effect of Bacillus toyonensis BCT-7112T supplementation on growth performance, intestinal morphology, immune-related gene expression, and gut microbiome in Barbary ducks.

This study aimed to investigate the effect of Bacillus toyonensis BCT-7112T supplementation on growth performance, intestinal morphology, immune-related gene expression, and the cecal microbiota of meat ducks. A total of 150 one-day-old male Barbary ducks were divided into 3 groups with 5 replicates (n = 10 ducks per replicate) by completely randomized design and offered diets supplemented with the commercial product Toyocerin (containing 1 × 109B. toyonensis BCT-7112T viable spores/g product) at the levels of 0, 500, or 1,000 mg/kg (0, 500, or 1,000 ppm), respectively, for 8 wk. The results showed that although ducks in the 500 ppm B. toyonensis BCT-7112T group displayed numerically better values (e.g., weight gain and feed conversion ratio) than those in the control group, the growth performance of ducks fed diets supplemented with B. toyonensis BCT-7112T did not differ significantly from that of the control group (P > 0.05). There were no significant differences in the intestinal mucosal morphology of ducks across the experimental groups (P > 0.05). However, ducks in the 500 ppm B. toyonensis BCT-7112T group showed a trend of greater values, for example, villus height per crypt depth of duodenum (P = 0.16) and ileum (P = 0.12) compared with those in the control group. The relative expression of immune-related genes, for example, interferon (IFN) and interleukin-6 (IL-6) in the meat duck spleen was significantly lower in both B. toyonensis BCT-7112T groups at 14 d and 35 d than in the control group (P < 0.05). Beta diversity analysis of the cecal microbiota of ducks in either the 500 ppm or the 1,000 ppm B. toyonensis BCT-7112T group showed to have higher diversity than that in the control group, where at the phylum level, Bacteroidetes was the most abundant, followed by Firmicutes, and at the genus level, Bacteroides, Fusobacterium, and Ruminococcaceae were the top 3 most abundant genera. In conclusion, our study demonstrates that 500 ppm supplementation with B. toyonensis BCT-7112T in duck diets can reduce proinflammatory cytokine gene expression, improve immunological function, and increase the variety of microbial communities in the ceca of meat-type ducks.

Molecular evidence for the involvement of alternative splicing in jejunum epithelial cells in broiler chickens fed dietary supplementation with olive mill wastewater.

There is no doubt that alternative splicing is conserved in chickens and mammals, but evaluating the effects of nutrition on alternative splicing in chickens is crucial in a wide range of fields. Although the olive diet has been extensively studied in human, mouse, and chicken systems, little is known about its impact on chicken alternative splicing systems. Hence, the current study aimed to assess the effect of feeding polyphenol-enriched olive mill wastewater to female broiler chickens via alternative splicing by analyzing high-throughput sequencing raw reads of RNA utilizing genomics and bioinformatics methodologies. It also aimed to look for differences in isoform expression and discover molecular functions and biological processes linked to differentially transcribed genes. The findings of our study revealed that 51 genes involved in isoform switching and alternative splicing events were not used evenly. This is due to the reduced use of ATSS in olive mill wastewater groups compared to control groups. Furthermore, the gene ontology analysis revealed that 25 GO terms were enriched in biological processes, 16 GO terms were enriched in molecular function, and 25 GO terms were enriched in cellular components. Kinase and adenylyltransferase activities were significantly enriched in terms. The molecular analysis presented herein provides valuable insight into the role of phenolics in alternative gene-splicing mechanisms in chickens, demonstrating how an industrial waste product can be repurposed as a feed supplement with a satisfactory outcome.

Evaluating the impact of organic chromium on hepatic phospholipid fatty acid molecular species, transcription of genes associated with lipid metabolism and oxidative status in broiler chickens fed flaxseed.

Flaxseed is a rich source of α-linolenic acid (ALA, 18:3 n-3) and can be used to enrich chicken tissues with n-3 fatty acids (FA). However, antinutritional factors in flaxseed compromise the live performance of birds coupled with increased oxidative stress. Chromium (Cr) is a trace element with antioxidant properties. It is hypothesized that Cr supplementation will affect the hepatic total lipid profile, phospholipid n-3 and n-6 FA molecular species, lipid oxidation products, and transcription of genes associated with lipid metabolism in broiler chickens fed flaxseed. Ninety (n = 90), day-old Cornish cross chicks were fed a corn-soybean meal-based diet containing 0% flaxseed (CTR), 10% flaxseed (FLAX), and FLAX + 0.05% organic Cr (FLAXCr) for 42 d. The chicks were kept in 18 pens with 5 chicks per pen. For all response variables, the effect of dietary treatments were compared separately using SAS 9.4. P values were considered significant at ≤0.05. Total lipids, saturated FA, long-chain (≥20C) n-6 FA were reduced while total n-3 FA and long-chain n-3 FA were higher in the liver of FLAX and FLAXCr than CTR (P < 0.05). Hepatic phosphatidylcholine (PC) and phosphatidylethnolamine (PE) n-3 species (36:5, 38:6) were higher in FLAX and FLAXCr compared to CTR (P < 0.05). On the contrary, n-6 species in PC (36:4, 38:4) and PE (38:4) were lower in FLAX and FLAXCr compared to CTR (P < 0.05). Addition of Cr to a flaxseed-containing diet led to an increase in PE 36:4 (P < 0.05). A decrease in the transcription of ELOVL6 gene involved in de novo lipid synthesis was observed in FLAXCr (P = 0.01). An increase in the transcription of genes involved in FA oxidation (ACAA2, ACOX1) was observed in FLAX compared to FLAXCr (P = 0. 05; P = 0.02). A trend for a decrease in the transcription of FADS2 and HMGCS1 was observed in FLAXCr than CTR and FLAX (P = 0.06; 0.08). Transcription of other genes involved in de novo lipid synthesis (FASN, PPARA), FA oxidation (CPT1A, CPT2, ACAA1), and oxidative stress response (GPX1, NQO11, GSTA2, SLC40A1, NFE2L2) were not affected by the diets (P > 0.05). Lipid peroxidation products measured as thiobarbituric acid reactive substances (TBARS) in liver was reduced in FLAXCr than CTR (P < 0.05) and was not different from FLAX (P > 0.05). Serum cholesterol and aspartic aminotransferase were reduced in FLAX and FLAXCr compared to CTR (P < 0.05). The serum glucose level was decreased in FLAX compared to CTR (P < 0.05) and a trend in decrease was noticed in FLAXCr vs. CTR (P = 0.10). Serum TBARS were higher in CTR and FLAXCr compared to FLAX (P < 0.05). In conclusion, flaxseed supplementation enhances total and long-chain n-3 FA while reducing total lipids, saturated, and n-6 FA in the liver. Supplementing Cr along with flaxseed increased n-6 FA species in the hepatic PE and decreased the transcription of genes involved in FA oxidation and lipid synthesis.

Transcriptomic analysis of mechanism underlying the effect of induced molting on semen quality and reproductive performance in aged Houdan roosters.

The reproductive performance of breeder roosters has significant economic importance in the poultry industry. Breeder roosters have severely reduced semen quality with age and will be at risk of culling in the following years. In order to extend the use of breeder roosters, we drew on the induced molting model of hens and selected 35 Houdan roosters aged 50 wk for induced molting. By comparing the body weight, testicular weight, semen quality, and reproductive performance before and after induced molting, we found that induced molting could restore the body weight and testicular weight to the levels before molting (P > 0.05). At the same time, it significantly improved sperm motility (P < 0.05) and also improved reproductive performance such as fertilization rate and hatching rate. To further reveal the mechanism underlying the effects of induced molting on semen quality and reproductive performance in aged Houdan roosters, we collected testes from 3 periods: 1 d before fasting (F0), 15 d after fasting (F15), and 32 d after recovery feeding (R32) for transcriptome sequencing analysis. A total of 5,671 genes were detected in F0, F15, and R32, and trend analysis of the 5,671 differential genes showed 2 significant trends (profile 5 and profile 2). KEGG enrichment analysis of the genes in the 2 profiles, revealed significantly enriched pathway regulation of actin cytoskeleton. In the regulation of actin cytoskeleton pathway, we found a protein kinase gene (SRC) and a senescence gene (ROCK2). SRC was highly expressed at F15, leading to the phosphorylation of key substrates, which in turn disrupted the Sertoli cell spermatid connection and the spermiogenesis process, resulting in no mature spermatozoa produced from F15, SRC expression was inhibited at R32, the expression level was reduced, and mature spermatozoa reappeared. The senescence gene ROCK2 was highly expressed at F15 compared to F0 and R32, which may have been responsible for inducing senescence atrophy in the testes.

α-Linolenic acid-regulated testosterone biosynthesis via activation of the JNK-SF-1 signaling pathway in primary rooster Leydig cells.

As a functional fatty acid, α-linolenic acid (ALA) is essential in promoting animal testosterone biosynthesis. This study investigated the effects of ALA on testosterone biosynthesis and the possible mechanism underlying the signaling pathway in primary Leydig cells of the rooster. METHODS: Primary rooster Leydig cells were treated with ALA (0, 20, 40, or 80 µmol/L) or pretreated with a p38 inhibitor (50 µmol/L), a c-Jun NH2-terminal kinase (JNK) inhibitor (20 µmol/L), or an extracellular signal-regulated kinase (ERK) inhibitor (20 µmol/L) before ALA treatment. Testosterone content in the conditioned culture medium was detected using an enzyme-linked immunosorbent assay (ELISA). The expression of steroidogenic enzymes and JNK-SF-1 signaling pathway factors was detected using real-time fluorescence quantitative PCR (qRT-PCR). RESULTS: Supplementation with ALA significantly increased testosterone secretion within culture media (P < 0.05), and the optimized dose was 40 µmol/L. Compared with the control group, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA expression significantly increased (P < 0.05) in the 40 µmol/L ALA group; 17-hydroxylase/c17-20 lyase (P450c17) and p38 mRNA expressions were not significantly different in the 40 µmol/L ALA group; ERK and JNK mRNA expressions were significantly upregulated (P < 0.05) in 40 µmol/L ALA group. In the inhibitor group, testosterone levels were significantly downregulated (P < 0.05). Compared with the 40 µmol/L ALA group, StAR, P450scc, and P450c17 mRNA expressions were significantly decreased (P < 0.05), and 3ß-HSD mRNA expression in the p38 inhibitor group did not change; StAR, P450scc, and 3ß-HSD mRNA expressions were significantly decreased (P < 0.05), and P450c17 mRNA expression in ERK inhibitor group did not change; StAR, P450scc, 3ß-HSD, and P450c17 mRNA expressions were significantly decreased (P < 0.05) in JNK inhibitor group. Additionally, the increased steroidogenic factor 1 (SF-1) gene expression levels induced by ALA were reversed when the cells were pre-incubated with JNK and ERK inhibitors. The levels in the JNK inhibitor group were significantly lower than those in the control group (P < 0.05). CONCLUSION: ALA may promote testosterone biosynthesis by activating the JNK-SF-1 signaling pathway to upregulate StAR, P450scc, 3ß-HSD, and P450c17 expression in primary rooster Leydig cells.

Nano-Selenium inhibited antibiotic resistance genes and virulence factors by suppressing bacterial selenocompound metabolism and chemotaxis pathways in animal manure.

Numerous antibiotic resistance genes (ARGs) and virulence factors (VFs) found in animal manure pose significant risks to human health. However, the effects of graphene sodium selenite (GSSe), a novel chemical nano-Selenium, and biological nano-Selenium (BNSSe), a new bioaugmentation nano-Se, on bacterial Se metabolism, chemotaxis, ARGs, and VFs in animal manure remain unknown. In this study, we investigated the effects of GSSe and BNSSe on ARGs and VFs expression in broiler manure using high-throughput sequencing. Results showed that BNSSe reduced Se pressure during anaerobic fermentation by inhibiting bacterial selenocompound metabolism pathways, thereby lowering manure Selenium pollution. Additionally, the expression levels of ARGs and VFs were lower in the BNSSe group compared to the Sodium Selenite and GSSe groups, as BNSSe inhibited bacterial chemotaxis pathways. Co-occurrence network analysis identified ARGs and VFs within the following phyla Bacteroidetes (genera Butyricimonas, Odoribacter, Paraprevotella, and Rikenella), Firmicutes (genera Lactobacillus, Candidatus_Borkfalkia, Merdimonas, Oscillibacter, Intestinimonas, and Megamonas), and Proteobacteria (genera Desulfovibrio). The expression and abundance of ARGs and VFs genes were found to be associated with ARGs-VFs coexistence. Moreover, BNSSe disruption of bacterial selenocompound metabolism and chemotaxis pathways resulted in less frequent transfer of ARGs and VFs. These findings indicate that BNSSe can reduce ARGs and VFs expression in animal manure by suppressing bacterial selenocompound metabolism and chemotaxis pathways.

Effects of ß-carotene supplementation in the diet of laying breeder hens on the growth performance and liver development of offspring chicks.

This experiment was conducted to evaluate the growth performance, growth regulating factors, and liver morphology of chicks hatched from egg-laying breeding hens dietary supplemented with additives (ß-carotene). Hy-line breeding hens were allocated into three groups with three replicates/group. The dietary treatments were as follows: basal diet as a control (Con), basal diet supplemented with 120 (ßc-L) or 240 (ßc-H) mg/kg of ß-carotene diet. After 6 weeks, the eggs were collected and incubated. The hatched chicks were fed the same diet. The results showed that chicks in the ßc-L group increased in body weight at 21 days (p < 0.01). At 42 days, chicks in the ßc-H group showed a significant increase in tibia length (p < 0.05). The liver index increased in the ßc-L and ßc-H groups at 7 days (p < 0.05). Serum HGF (7, 14, 21, and 42 days) and leptin (14 days) were significantly increased in the group supplemented with ßc. Hepatic GHR (14 days), IGF-1R (14 days), and LEPR (21 days) mRNA expression were significantly increased. In addition, there was an increase in PCNA-positive cells in the liver of chicks in the ßc group. In conclusion, the addition of ß-carotene to the diet of laying breeder hens was more advantageous in terms of growth performance and liver development of the offspring.