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Abstract Background Circular RNAs (circRNAs) are stable, covalently closed non-coding RNAs formed by reverse splicing of precursor mRNA. They play critical roles in various biological processes, including insulin secretion and metabolism. However, their function in avian skeletal muscle's response to insulin remains poorly understood. This study aimed to comprehensively identify insulin-responsive circRNAs and explore their temporal and breed-specific regulation in poultry. Results Using strand-specific RNA sequencing (ssRNA-Seq) on the pectoralis muscles of both Arbor Acres (AA) broilers and Silky fowls following insulin administration (5 IU/kg.BW, PBS as control). We identified 2,027 muscle circRNAs. Insulin-responsive circRNAs were detected in Silky fowls (29) and broilers (45) at 120 min, and in broilers (20) at 15 min post-injection. These circRNAs are enriched in processes such as exocrine pancreas development, response to exogenous stimuli, and regulation of intracellular signal transduction, likely mediated through a circRNA-miRNA network. Fewer insulin-responsive circRNAs were shared between time points in broilers (1) or between breeds (3) at 120 min. We further characterized a conserved insulin-responsive circRNA (circINSR), formed by exon 2 of the Insulin Receptor (INSR). The circINSR showed a similar spatiotemporal expression pattern to INSR, but exhibited distinct changes post-insulin administration. In broilers, INSR expression was dynamically modulated, while circINSR was downregulated only at 15 min (P

Abstract Background Heat stress (HS) incidence is associated with the accumulation of reactive substances, which might be associated with bone loss. N-Acetylcysteine (NAC) exhibits strong antioxidants due to its sulfhydryl group and being as the precursor for endogenous glutathione synthesis. Therefore, interplay between oxidative stress and bone turnover of broilers and the effects of dietary NAC inclusion on antioxidant capability and “gut-bone” axis were evaluated during chronic HS. Results Implementing cyclic chronic HS (34 °C for 7 h/d) evoked reactive oxygen species excessive production and oxidant stress, which was accompanied by compromised tibia mass. The RNA-seq of proximal tibia also revealed the enrichment of oxidation–reduction process and inflammatory outbursts during HS. Although no notable alterations in the growth performance and cecal microbiota were found, the diet contained 2 g/kg NAC enhanced the antioxidant capability of heat-stressed broiler chickens by upregulating the expression of Nrf2 in the ileum, tibia, and bone marrow. Simultaneously, NAC tended to hinder NF-κB pathway activation and decreased the mRNA levels of the proinflammatory cytokines in both the ileum and bone marrow. As a result, NAC suppressed osteoclastogenesis and osteoclast activity, thereby increasing osteocyte-related gene expression. Furthermore, the inclusion of NAC tended to increase the ash content and density of the whole tibia, as well as improve cortical thickness and bone volume of the diaphysis. Conclusions These findings HS-mediated outburst of oxidant stress accelerates bone resorption and negatively regulates the bone quality of tibia, which is inhibited by NAC in broilers. Graphical abstract

Supplementing the diet of broiler with N-acetyl-L-cysteine (NAC) has been shown to support small intestinal integrity, and as a precursor of L-cysteine, it may also support antioxidant functions. As heat-stressed chickens suffer from intestinal disturbances caused by oxidative stress and at the same time increase water consumption, it was hypothesised that NAC supplemented via the drinking water could reinforce finisher broiler productivity under heat stress. Hence, three hundred 15-day-old male Ross 308 broilers were assigned to three treatments until 39 days of age: thermoneutral (TN), heat-stressed (HS), or HS + NAC. NAC was administered to the drinking water at 1000 mg/L starting on day 20. At 22 days, a chronic cyclic heat stress model was initiated in the HS and HS + NAC groups. On day 39, birds were euthanized and sampled for physiological measurements. Heat stress significantly decreased weight gain and feed intake from 22 to 39 days but did not affect feed efficiency. Rectal temperature and respiration rate significantly increased in heat-stressed broilers on days 23, 30, and 38. Also, ileal mucosal malondialdehyde (MDA) and plasma MDA levels were significantly higher in the HS group and the HS + NAC group, respectively, as compared to the TN group, whereas MDA levels in lung tissue were lower in both HS groups. Small intestinal villus height was significantly lower and serum FITC-d levels were significantly higher in the HS group compared to the TN group, without the effect of NAC supplementation. Furthermore, hepatic gene transcript of glutamate-cysteine ligase catalytic subunit was upregulated under heat stress conditions which may reflect higher glutathione synthesis, whereas in HS + NAC birds cystathionine-beta synthase-like (CBSL) mRNA level was dramatically reduced, indicating decreased transsulfuration activity. In conclusion, the administration of 1000 mg/L NAC in drinking water did not alleviate the reduced performances of heat-stressed finisher broilers. Nevertheless, it resulted in the downregulation of CBSL expression, suggesting a potential methionine-sparing effect.

FK506-binding protein 5 (FKBP5) is a negative regulator of the glucocorticoid response and may play an important role in regulating metabolic homeostasis in birds. However, limited information is available regarding its role in avian species. This study aimed to clarify the spatiotemporal characteristics of chicken FKBP5 and investigate the effects of exogenous stimuli on its expression. Real-time quantitative PCR (RT-qPCR) was utilized to analyze the spatiotemporal expression patterns of FKBP5 in chickens. Additionally, the impact of exogenous stimuli, including fasting, energy restriction, and insulin/pyruvate/glucose injections, on FKBP5 expression in the pectoral muscle was investigated. The results showed that FKBP5 is broadly expressed in various bird tissues, with dominant expression in insulin-sensitive tissues. The FKBP5 levels in birds are dramatically modulated during development in insulin-sensitive tissues, with the highest expression observed in striated muscle and liver at embryonic day 19 (E19) (P < 0.01). The basal level of FKBP5 in the pectoral muscle of broilers is higher than that in Silky chickens. Insulin administration leads to a rapid drop in blood glucose levels in both breeds, with a slower recovery in AA broilers (P < 0.01). However, FKBP5 expression in the pectoral muscle initially shows a slight drop, followed by a sharp increase over time after insulin administration (P < 0.01). Additionally, breed heterogeneity in FKBP5 expression was observed in the pectoral muscle upon insulin stimulation, with broilers showing stronger insulin sensitivity compared to Silky chickens. A 24-hour fasting period downregulated blood glucose levels (P < 0.01) and FKBP5 expression in the pectoral muscle of AA broilers (P < 0.01). A 30% energy restriction over three weeks slightly reduced blood glucose levels (P = 0.075) and significantly decreased FKBP5 levels in the pectoralis muscle of broilers. In addition, pyruvate increased blood glucose and pectoralis FKBP5 transcription levels at 60 min (P < 0.01), whereas glucose increased blood glucose levels but had no effect on FKBP5 expression. Overall analysis showed a negative correlation between FKBP5 expression in the pectoral muscle and blood glucose levels (ρ = −0.405, P < 0.001). In conclusion, FKBP5 is a key player in the metabolic regulation of birds, with its expression being highly dynamic and tissue-specific. Insulin regulates FKBP5 expression in pectoral muscles in a time-dependent manner, while pyruvate increases FKBP5 levels and fasting/energy restriction reduces FKBP5 mRNA expression in the pectoral muscles.

ABSTRACT: Glucose Transporter 4 (GLUT4) is a crucial protein facilitating glucose uptake and metabolism across cell membranes in mammals. However, information on GLUT4 in birds has historically been limited. In this study, we investigated the dynamic expression profile of chicken GLUT4 using real-time quantitative PCR (RT-qPCR) and examined its potential effects and mechanisms via GLUT4 overexpression and RNA sequencing (RNA-seq) in chicken primary skeletal muscle satellite cells (CP-SMSCs). Our results demonstrated that chicken GLUT4 is differentially expressed across tissues, with predominant expression in skeletal muscles, and across developmental stages of CP-SMSCs, with notable upregulation during the phases of cell proliferation and early differentiation. Notably, 0.1 μM insulin for 60 min significantly elevated the expression of GLUT4 in CP-SMSCs (P < 0.05). GLUT4 overexpression in CP-SMSCs promoted cell proliferation, as evidenced by Cell Counting Kit-8 (CCK-8) (P < 0.05) and 5-Ethynyl-2′-Deoxyuridine (EDU) assays (P < 0.05), and enhanced glucose consumption following 0.1 μM insulin treatment (P < 0.05). However, it inhibited glucose consumption 12 h after the addition of 5 g/L glucose (P < 0.05). After overexpressing GLUT4, we identified 302 differentially expressed genes (DEGs) in CP-SMSCs, with 134 upregulated and 168 downregulated. These DEGs are primarily enriched in pathways such as oxidative phosphorylation, ribosome, cardiac muscle contraction, ATP metabolic processes, and mitochondrial protein complexes. Specifically, in the enriched oxidative phosphorylation pathway, the upregulated DEGs (12) encode mitochondrial proteins, while the downregulated DEGs (6) are nuclear genome-derived. The ribosomal pathway is predominantly inhibited, accompanying with the downregulation of the translocase of outer mitochondrial membrane 7 (TOMM7)/translocase of inner mitochondrial membrane 8 (TIMM8A) complex responsible for mitochondrial protein transport, and a reduction in 28S (LOC121106978) and 18S (LOC112533601) ribosomal rRNAs. In conclusion, chicken GLUT4 is dynamically modulated during development and acts as an insulin responder that significantly regulates cellular glucose uptake and cell proliferation. This regulation occurs mainly through enhancing the mitochondrial oxidative phosphorylation and inhibiting ribosomal pathway.

ABSTRACT: Animal aggression is one of the most conserved behaviors. Excessive and inappropriate aggression was a serious social concern across species. After long-term selection under strict stress conditions, Henan gamecock serves as a good model for studying aggressive behavior. In this research, we constructed a Henan game chicken backcross population containing 25% Rhode Island Red (RIR), and conducted brain transcriptomics and serum metabolomics analyses on Henan gamecock (HGR) through its comparison with its female encounters (HGH) and the male backcross birds (BGR). The study revealed that seven differential metabolites in serum and 172 differentially expressed genes in the brain were commonly shared in both HGR vs. HGH and HGR vs. BGR comparisons. They exhibited the same patterns of modulation in Henan gamecocks, following either HGH < HGR > BGR or HGH > HGR < BGR style. Therein, some neurological genes involving in serotonergic and dopaminergic signaling were upregulated, while the levels of many genes related with neuro-immune function were decreased in Henan gamecock. In addition, many unknown genes specifically or highly expressed in the brain of the Henan gamecock were identified. These genes are potentially key candidates for enhancing the bird's aggression. Multi-omics joint analysis revealed that tyrosine metabolism and neuroactive ligand-receptor interaction were commonly affected. Overall, our results propose that the aggressiveness of Henan gamecocks can be heightened by the activation of the serotonergic-dopaminergic metabolic process in the brain, which concurrently impairs the neuroimmune system. Further research is needed to identify the function of these unknown genes on the bird's aggressive behavior.

Aging is associated with detrimental bone loss, often leading to fragility fractures, which may be driven by oxidative stress. In this study, the outcomes of comparing the differences among young, adult and aged C57BL/6J mice found that the trabecular bone volume was significantly lower in the aged mice compared to young mice, and the bone characteristics were significantly correlated with the oxidative status. To counteract the adverse effects of aging, methyl sulfonyl methane (MSM), a stable metabolite of dimethyl sulfoxide, was used to supplement the drinking water (400 mg/kg/day) of the aged mice (73 weeks old) for 8 weeks. The MSM supplementation improved the maximum load, bone microarchitecture, and mRNA levels of osteocyte-specific genes in the tibia. Furthermore, MSM reduced the serum level of the C-terminal telopeptide of type I collagen, a marker of bone resorption, and downregulated the mRNA levels of genes related to osteoclast proliferation and activity. MSM also decreased the levels of pro-inflammatory cytokines in both the serum and bone marrow. Importantly, the MSM-treated mice exhibited an enhanced antioxidant status, characterized by increased glutathione peroxidase (GPx) activity and glutathione concentration in plasma, erythrocytes and bone marrow. These improvements were linked to the activation of the nuclear factor E2 related factor 2 (Nrf2) pathway and its downstream antioxidant gene expression, including that of superoxide dismutase and GPx. These findings suggested that age-related bone loss is closely tied to oxidative stress, and MSM supplementation effectively reverses bone loss by mitigating oxidative stress-mediated bone resorption.

IntroductionThe study aimed to investigate the potential effects of varying wheat levels in broiler diets on growth performance, intestinal barrier, and cecal microbiota.MethodsDay-old male broilers were fed the same diet until 10 d of age. Then they were randomly assigned to 1) the low-level wheat group, where inclusion of 15.0% and 25.0% wheat in the grower and finisher diet, respectively, 2) the medium-level wheat group with 30.0% and 40.0% of wheat in the grower and finisher periods; and 3) the high-level wheat dietary group, in which the grower and finisher diets contained 55.77% and 62.38% of wheat, respectively.ResultsDietary treatments unaffected the body weight at 39 d, whereas incorporating high wheat in diets significantly increased the feed intake and reduced the feed conversion ratio from 10 to 39 d (p

The compromised egg quality and leg abnormality during the end of the laying cycle (after 40 weeks) have been leading to poor animal welfare and substantial economic losses. Therefore, the effects of fermented calcium (Ca) butyrate, produced by fermentation by Clostridium butyricum, on production, eggshell quality, and tibial property of hens were explored. A total of 192 Hy-line brown laying hens at 50-week-old were assigned to a basal diet or the basal diet with 300 mg/kg of the fermented Ca butyrate from 50 to 58 weeks of age. Each treatment had 6 replicates with 16 hens each. The diet supplemented with 300 mg/kg fermented Ca butyrate notably increased egg weight, ovarian follicle number, and eggshell strength (P = 0.072) as compared to the basal diet, which were associated with cytokine secretion, toll-like receptor signaling pathways, and intestinal immunity based on the RNA-seq data from the granulosa. Dietary Ca butyrate inclusion decreased the expression of ileal tumor necrosis factor-alpha and serum pro-inflammatory cytokine concentration, as well as increased the content of serum immunoglobulin A when compared to the basal diet (both P 0.05). In addition, the diet with fermented Ca butyrate reduced the number of osteoclasts in the proximal tibia and the level of C-terminal cross-linked telopeptide of type I collagen, a bone resorption marker (P

Multiply adverse effects including declines in production performance and excessive fat deposition were noticed with the extension of the laying cycle in hens, which are pertinent to animal welfare and human food safety. This study aimed to investigate the effect of dietary supplementation of bile acids (BAs) on production performance and lipid metabolism in late-phase laying hens. A total of 144 70-week-old hens were distributed into three treatments with eight replicates per treatment, including the basal diet with 0 (Ctrl), 95.01 (Low-BA), and 189.99 mg/kg (High-BA) of porcine BAs, respectively. The test period was from 70 to 75 weeks. The supplementation of BAs did not significantly alter laying performance during the trial, whereas it increased (p < 0.05) the total follicles compared to the Ctrl diet. The eggs from the hens fed the BA diet exhibited increased (p > 0.05) relative weight of eggshell and yolk color than those that consumed the Ctrl diet. There were no significant changes following BA treatment regarding the serum lipid profile. Dietary BA treatment reduced the total triglyceride in livers to different extents, resulting in the decreased diameter and area of vacuoles in liver tissues. The low-dose BA treatment decreased the mRNA levels of fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD), while promoting the expression of lipoprotein lipase (LPL) compared to the Ctrl group (both p < 0.05). Of note, the expressions of farnesoid X receptor (FXR), apical sodium-dependent bile acid transporter (ASBT), and ileum bile acid-binding protein (IBABP) were notably downregulated (p < 0.05) by the low-dose BA treatment. Dietary BA treatment had no apparent effects on laying performance, whereas it increased the follicle frequency, eggshell weight, and yolk color. Moreover, a diet containing 95.01 mg/kg of BAs depressed ileal BA resorption and hepatic fatty deposition by reducing lipogenesis and promoting lipolysis, which may have a beneficial effect on the liver in late-phase layers.

ABSTRACT: Heat stress (HS) in poultry has deleterious effects on intestinal development and barrier function, along with inflammatory outbursts. In the present study, chronic HS reduced body weight of broilers and activated mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) /nuclear factor kappa B (NF-κB) signaling pathways to elicit the inflammatory cytokine response in jejunum. Subsequently, this study investigated the protective effects of the Malt1 inhibitor on the intestine of broilers under HS conditions. The 21-day-old male broilers were allocated to 8 pens housed in HS room (34°C for 7 h/d) until 28 d of age. During this period, 4 birds were selected from each heat-stressed pen and received intraperitoneal injection of 20 mg/kg body weight Mepazine (a Malt1 inhibitor) or the equivalent volume of phosphate buffer saline (PBS) every other day. When compared to PBS broilers, birds received Mepazine injection exhibited increased relative weight and higher villus height in jejunum (both P < 0.05). Mepazine treatment also increased (P < 0.05) the mRNA of zonula occludens-1 (ZO-1), claudin-1, and cadherin 1 of jejunum, which was companied by the reduced caspase-3 transcription under HS condition. Meanwhile, the gene expression levels of toll-like receptor 4 (TLR4), Malt1, NF-κB, interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) in the jejunum were significantly downregulated by Mepazine administration (P < 0.05). Although there were no significant differences in the relative weight of the thymus and bursa, the transcription levels of T helper 1 (Th1)- and Th17-related cytokines were lower in thymus of birds injected with Mepazine. The cytokines of Treg cytokine transforming growth factor beta (TGF-β) and forkhead box protein P3 (Foxp3) in both the thymus and bursa were not influenced. These results suggest that inhibition of Malt1 protease activity can protect intestinal integrity by promoting the production of tight junction proteins and attenuating NF-κB-mediated intestinal inflammation response under HS conditions.

The glucose metabolism of poultry draws wide attention as they have nearly twice the fasting blood glucose than that of mammals. To define the relationship between glucose metabolism and breed of chicken, the outcomes from different growth rate chickens showed that Arbor Acres (AA) broilers, a well-known fast-growing breed, had a lower fasting blood glucose concentration and glucose clearance rate when compared to Silky chickens, a Chinese traditional medicinal chicken with black skin and a slow growth rate. Moreover, AA broilers had a relatively slow rise in blood glucose in response to oral glucose solution than the Silky chickens on 21 and 42 d (P

Replacing corn with different levels of wheat in the iso-energy and -protein diet of broilers and the impacts on growth performance and intestinal homeostasis of broilers under the condition of supplying the multienzyme complex were evaluated in this study. A total of 480 10-day-old male broilers were assigned randomly to the low-level wheat group (15% wheat and 35.18% corn), the medium-level wheat group (30% and 22.27%), and the high-level wheat group (55.77% wheat without corn) until 21 d. The different levels of wheat supplementation did not affect hepatic function, serum glycolipid profile, or bone turnover. The replacement of corn with 55% wheat in the diet of broilers increased the body weight at 21 d and feed intake during 10 to 21 d (both p < 0.05), with a comparable feed conversion ratio. Compared with the low-wheat group, the dietary addition of medium or high wheat levels notably increased the ratio of villus height to crypt depth in the duodenum (p < 0.05) and the ileal villus height (p < 0.05). Meanwhile, the supplementation of medium and high wheat in the diet increased the proportion of Bacteroidetes, and a diet with high wheat proportion elevated the content of Firmicutes when compared to the low-level wheat group (both p < 0.05). In addition, the diet containing 30–55% wheat enhanced the anti-inflammatory capability in both the ileum and the serum. These findings suggest that the replacement of corn with 55% wheat in the diet improved the growth performance of 21-day-old broilers, which might be linked to the alteration in intestinal morphology and cecal microbiota.

Abstract Background Escherichia coli (E. coli) infection in humans and animals usually comes with gut dysbiosis, which is potential culprit to skeletal health, it is still unclear to whether diet interfered gut microbiome changes can be a protective strategy to bone loss development. Here, the effects of resistant starch from raw potato starch (RPS), a type of prebiotic, on E. coli-induced bone loss and gut microbial composition in meat ducks were evaluated. Results The results showed that dietary 12% RPS treatment improved bone quality, depressed bone resorption, and attenuated the pro-inflammatory reaction in both ileum and bone marrow. Meanwhile, the 12% RPS diet also increased the abundance of Firmicutes in E. coli-treated birds, along with higher production of short-chain fatty acids (SCFAs) especially propionate and butyrate. Whereas addition of β-acid, an inhibitor of bacterial SCFAs production, to the drinking water of ducks fed 12% RPS diet significantly decreased SCFAs level in cecum content and eliminated RPS-induced tibial mass improvement. Further, treatment with MI-2 to abrogate mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) activity replicated the protective role of dietary 12% RPS in E. coli-induced bone loss including reduced the inhibition on nuclear factor κB (NF-κB) inflammasome activation, decreased bone resorption, and improved bone quality, which were correlated with comparable and higher regulatory T cells (Treg) frequency in MI-2 and 12% RPS group, respectively. Conclusions These findings suggested that the diet with 12% RPS could alleviate E. coli-induced bone loss in meat ducks by changing the gut microbial composition and promoting concomitant SCFAs production, and consequently inhibiting Malt1/NF-κB inflammasome activation and Treg cells expansion. Graphical Abstract

To define the relationship between dietary nutrient density, calcium (Ca), and meat quality in meat ducks. A total of 288 male Cherry Valley SM3 medium ducklings were fed a common standard starter diet until d 14. At 15 d of age, ducks were randomly divided into 2 treatment groups and fed either a conventional diet or a low nutrient density (LND) diet. Compared with the conventional diet, the energy was reduced in the LND diet by 8.6% and 16.8% in grower (15 to 35 d) and finisher (36 to 56 d) phases, respectively, while other essential nutrients were kept proportionate to energy. The LND diet decreased the shear force (P

Objective Diet acidification supplementation is known to influence intestinal morphology, gut microbiota, and on phosphorus (P) utilization of broilers. Alterations in intestinal barrier and microbiota have been associated with systemic inflammation and thus regulating bone turnover. Hence the effect of acidifier addition to drinking water on tibia mass and the linkages between intestinal integrity and bone were studied. Methods One-d-old male broilers were randomly assigned to normal water (control) or continuous supply of acidified water (2% the blend of 2-hydroxy-4-methylthiobutyric acid, lactic, and phosphoric acid) group with 5 replicates of 10 chicks per replicate for 42 d. Results Acidification of drinking water improved the ash percentage and calcium content of tibia at 42 d. Broilers receiving acidified water had increased serum P concentration compared to control birds. The acidified group showed improved intestinal barrier, evidenced by increased wall thickness, villus height, the villus height to crypt depth ratio, and upregulated mucin-2 expression in ileum. Broilers receiving drinking water containing mixed organic acids had a higher proportion of Firmicutes and the ratio of Firmicutes and Bacteroidetes, as well as a lower population of Proteobacteria. Meanwhile, the addition of acidifier to drinking water resulted in declined ileal and serum proinflammatory factors level and increased immunoglobulin concentrations in serum. Concerning bone remodeling, acidifier addition was linked to a decrease in serum C-terminal cross-linked telopeptide of type I collagen and tartrate-resistant acid phosphatase reflecting bone resorption, whereas it did not apparently change serum alkaline phosphatase activity that is a bone formation marker. Conclusion Acidified drinking water increased tibia mineral deposition of broilers, which was probably linked with higher P utilization and decreased bone resorption through improved intestinal integrity and gut microbiota and through decreased systemic inflammation.

ABSTRACT: Glucose transporter protein 4 (GLUT4) plays an important role in regulating insulin-mediated glucose homeostasis in mammals. Until now, studies on GLUT4 have focused on mammals mostly, while chicken GLUT4 has been rarely investigated. In this study, chicken GLUT4 mRNA sequences were obtained by combining conventional amplification, 5′- and 3′- rapid amplification of cDNA ends technique (RACE), then bioinformatics analysis on its genomic structure, splicing pattern, subcellular localization prediction and homologous comparisons were carried out. In addition, the distribution of GLUT4 was detected by RT-qPCR in bird′s liver and striated muscles (cardiac muscle, pectoralis and leg muscle) at different ages, including embryonic day 14 (E14), E19, 7-day-old (D7), D21 and D49 (n = 3–4). Results showed that chicken GLUT4 gene produced at least 14 transcripts (GenBank accession No: OP491293-OP491306) through alternative splicing and polyadenylation, which predicted encoding 12 types of amino acid (AA) sequences (with length ranged from 65 AA to 519 AA). These proteins contain typical major facilitator superfamily domain of glucose transporters with length variations, sharing a common sequence of 59 AA, and were predicted to have distinct subcellular localization. The dominant transcript (named as T1) consists of 11 exons with an open reading frame being predicted encoding 519 AA. In addition, analyzing on the spatio-temporal expression of chicken GLUT4 showed it dominantly expressed in pectoralis, leg muscles and cardiac muscle, and the mRNA level of chicken GLUT4 dramatically fluctuated with birds′ development in cardiac muscle, pectoralis and leg muscles, with the level at D21 significantly higher than that at E14, E19, and D49 (P < 0.05). These data indicated that chicken GLUT4 undergoes complex alternative splicing events, and GLUT4 expression level in striated muscle was subjected to dynamic regulation with birds′ development. Results indicate these isoforms may play overlapping and distinct roles in chicken.

ABSTRACT: Methyl sulfonyl methane (MSM) is available as a dietary supplement for human and has been associated with multiple health benefits such as reduction of oxidative stress. Heat stress (HS) is an environmental stressor challenging poultry production and known to inflict oxidative stress. We hypothesized that dietary MSM could attenuate HS-induced detrimental effects in broilers mediated by enhancement of antioxidant defenses. Hence, seven hundred ninety-two 1-day-old male Ross 308 broilers were allocated to 3 dietary treatments composed of corn-soybean meal diets with 0 (Ctrl), 1, or 2 g/kg MSM, with 12 replicates (22 birds each) per treatment for 39 d and subjected to a chronic cyclic HS model (temperature of 34°C and 52–58% relative humidity for 6 h daily) from d 24 to 39. MSM at 1 and 2 g/kg linearly increased daily gain and decreased feed-to-gain ratio compared with Ctrl in the grower phase (d 10–21, both P < 0.05). In the finisher phase (d 21–39) none of the performance and carcass indices were affected by treatment (P > 0.05). Nonetheless, data suggest reduced mortality by feeding MSM during HS. Also, during HS the diets with graded levels of MSM resulted in reduced rectal temperatures (P < 0.05) along with linearly decreased panting frequency on d 24 (P < 0.05). MSM supplemented birds showed a trend for linearly decreased thiobarbituric acid reactive substances of breast meat upon simulated retail display (P = 0.078). In addition, MSM administration linearly decreased lipid oxidation in plasma (d 25 and 39, P < 0.05) and breast muscle at d 23 (P < 0.05), concomitantly with linearly increased glutathione levels in erythrocytes (d 23 and 39, P < 0.05; d 25, P < 0.1) and breast muscle (d 23, P < 0.05; d 39, P < 0.1). In conclusion, MSM increased growth performance of broilers during grower phase, and exhibited positive effects on heat tolerance mediated by improved antioxidant capacity in broilers resulting in lower mortality in finisher phase.

Abstract Background Alterations in ambient temperature have been associated with multiple detrimental effects on broilers such as intestinal barrier disruption and dysbiosis resulting in systemic inflammation. Inflammation and 25-hydroxycholecalciferol (25-OH-D3) have shown to play a negative and positive role, respectively, in the regulation of bone mass. Hence the potential of 25-OH-D3 in alleviating heat induced bone alterations and its mechanisms was studied. Results Heat stress (HS) directly induced a decrease in tibia material properties and bone mass, as demonstrated by lower mineral content, and HS caused a notable increase in intestinal permeability. Treatment with dietary 25-OH-D3 reversed the HS-induced bone loss and barrier leak. Broilers suffering from HS exhibited dysbiosis and increased expression of inflammatory cytokines in the ileum and bone marrow, as well as increased osteoclast number and activity. The changes were prevented by dietary 25-OH-D3 administration. Specifically, dietary 25-OH-D3 addition decreased abundance of B- and T-cells in blood, and the expression of inflammatory cytokines, especially TNF-α, in both the ileum and bone marrow, but did not alter the diversity and population or composition of major bacterial phyla. With regard to bone remodeling, dietary 25-OH-D3 supplementation was linked to a decrease in serum C-terminal cross-linked telopeptide of type I collagen reflecting bone resorption and a concomitant decrement in osteoclast-specific marker genes expression (e.g. cathepsin K), whereas it did not apparently change serum bone formation markers during HS. Conclusions These data underscore the damage of HS to intestinal integrity and bone health, as well as that dietary 25-OH-D3 supplementation was identified as a potential therapy for preventing these adverse effects.

Essential oils have been proven to exert multiple effects on growth performance, production quality, and health status in poultry nutrition, which is dependent on the component and/or dose of essential oils. Diets with the optimal combination of essential oils might be able to improve the performance traits and welfare of laying hens. Therefore, this study was conducted to evaluate the effects of dietary essential oils, which are composed of cinnamaldehyde with carvacrol or thymol, on performance, egg quality, and intestinal health in post-peak laying hens. A total of 384, 50-week-old Hy-line brown laying hens were randomly divided into three groups with 8 replicates of 16 birds each: (1) a basal diet (Ctrl), (2) a basal diet with 100 mg/kg of essential oils consisting of 4.5% cinnamaldehyde with 13.5% carvacrol (CAR+CIN), and (3) a basal diet containing 100 mg/kg of essential oils composed of 4.5% cinnamaldehyde with 13.5% thymol (THY+CIN). The CAR+CIN diet increased the feed consumption from 52 to 55 weeks more than the Ctrl and the THY+CIN diet. Compared with the Ctrl group, the addition of essential oils decreased the dirty egg rate (P = 0.07) in the whole trial period. Regarding egg quality, the birds that received the CAR+CIN and THY+CIN diets increased the eggshell strength (P = 0.099) or Haugh unit (HU, p = 0.03) at 54 weeks, respectively. Supplementation of both CAR+CIN and THY+CIN diets significantly increased the ratio of villus height to crypt depth in the duodenum through increasing villus height and decreasing crypt depth as well as upregulated the mRNA abundances of duodenal occluding and cadherin (P < 0.05). However, the treatment with dietary essential oils did not notably change the proportion of cecal microbiota and bacterial diversity. This study suggested that dietary supplementation of cinnamaldehyde with carvacrol or thymol, the active components of essential oils, could promote egg quality in post-peak laying hens, which might be associated with improved intestinal development and barrier.

Leg problems often result from the rapid weight gain and poor bone quality in modern ducks, leading to a high risk of fractures and continuous pain. We hypothesized that improving bone quality in combination with delaying weight gain via a low nutrient density (LND) diet probably reverses these skeletal abnormalities. Studies indicated that 25-hydroxycholecalciferol (25-OH-D3), a vitamin D3 metabolite, is effective in treating bone-related disorders. Therefore, Exp. 1 evaluated the effects of 25-OH-D3 on tibial mass of meat ducks. Male meat ducklings were fed a standard nutrient density diet (containing a regular vitamin regimen) without or with 25-OH-D3 at 0.069 mg/kg for 35 d. The results showed that 25-OH-D3 supplementation improved the mineral content, microarchitecture and mechanical properties of tibias, and this companied by a decreased serum bone resorption marker and a concomitant decrement in osteoclast-specific marker genes expression. Subsequently, Exp. 2 was conducted to examine the impacts of 25-OH-D3 incorporating an LND diet on tibial quality of ducks under 2 different vitamin regimens (regular and high). Ducklings were allocated to a 2 × 2 factorial arrangement with 2 kinds of vitamin premixes and without or with 25-OH-D3 at 0.069 mg/kg in LND diets. The high premix had higher levels of all vitamins except biotin than the regular premix. The results demonstrated that high vitamin diets exhibited more significant effects than regular vitamin diets on inhibiting bone turnover and increasing minerals deposition. Tibial mineral content, microarchitecture, and strength of birds under the regular vitamin regimen were increased by 25-OH-D3 supplementation; However, these positive effects were not observed in ducks under the high vitamin regimen. To conclude, 25-OH-D3 supplementation improves tibial mass by suppressing osteoclast-mediated bone resorption in meat ducks, and this positive impact only was observed in regular but not high vitamin regimen when birds fed an LND diet.

ABSTRACT: Fatty liver hemorrhagic syndrome is characterized by hepatic damage and hemorrhage impairing animal welfare in birds, which was well-known to be moderately relieved through dietary choline chloride supplementation in laying hens. Chinese herb has been proven to exert a positive role on hepatic health in human and rodents. Here, we investigated the effect of herbaceous mixture (HM), which consists of Andrographis paniculate, Silybum marianum, Azadirachta Indica, and Ocimum basilicum (2:3.5:1:2), on the hepatic lipid metabolism and health status in laying hens. A total of 240 Hy-line Brown hens (389-day-old) were randomly fed the basal diet with 0 mg/kg choline chloride (negative control, NC), 1,000 mg/kg choline chloride (control, Ctrl), or 300 mg/kg HM for 28 d. Birds fed HM diet exhibited lower serum triglyceride (TG) and low-density lipoprotein cholesterol concentration, and higher high-density lipoprotein cholesterol level than those received NC and Ctrl diets (P < 0.05). When compared to control and NC group, the diets with HM decreased the contents of total cholesterol and TG in liver, as well as upregulated the mRNA abundance of hepatic hormone-sensitive lipase and lipoprotein lipase. Meanwhile, the hepatic area and diameter of steatosis vacuoles were also decreased by dietary HM administration (P < 0.05), which accompanied by decreased serum alanine aminotransferase activity (P < 0.05). Birds fed HM diets enhanced the hepatic antioxidative capacity than those received NC and Ctrl diet. Dietary HM depressed the mRNA level of inflammatory cytokine as compared to NC but not Ctrl group. Collectively, the diet with 300 mg/kg HM has a favorable effect in decreasing the lipid deposition and protecting liver injury by alleviating hepatic oxidant stress and inflammation in post-peak laying hens.

ABSTRACT: The cytidine diphosphate diacylglycerol synthases (CDSs) gene encodes the cytidine diphosphate-diacylglycerol (CDP-DAG) synthase enzyme that catalyzes the formation of CDP-diacylglycerol from phosphatidic acid. At present, there are no reports of CDS2 in birds. Here, we identified chicken CDS2 transcripts by combining conventional RT-PCR amplification, 5′ rapid amplification of cDNA ends (RACE), and 3′ RACE, explored the spatio-temporal expression profiles of total CDS2 and the longest transcript variant CDS2-4, and investigated the effect of exogenous insulin on the mRNA level of total CDS2 via quantitative RT-PCR. Four transcripts of chicken CDS2 (CDS2-1, -2, -3, and -4) were identified, which were alternatively spliced at the 3′-untranslated region (UTR). Both total CDS2 and CDS2-4 were prominently expressed in adipose tissue, and exhibited low expression in liver and pectoralis of 49-day-old chickens. Regarding the spatio-temporal expression patterns of CDS2 in chicken, total CDS2 exhibited a similar temporal expression tendency with a high level in the later period of incubation (embryonic day 19 [E19] or 1-day-old) in the brain, liver, and pectoralis. While CDS2-4 presented a distinct temporal expression pattern in these tissues, CDS2-4 levels peaked at 21 d in the brain and pectoralis, while liver CDS2-4 mRNA levels were highest at the early stage of hatching (E10). Total CDS2 (P < 0.001) and CDS2-4 (P = 0.0090) mRNA levels in the liver were differentially regulated throughout the development of the chicken. Total CDS2 levels in the liver of Silky chickens were higher than that of the broiler in the basal state and after insulin stimulation. Exogenous insulin significantly down-regulated the level of total CDS2 at 240 min in the pectoralis of Silky chickens (P < 0.01). In conclusion, chicken CDS2 isoforms with variation at the 3′-UTR were identified, which was prominently expressed in adipose tissue. Total CDS2 and CDS2-4 presented distinct spatio-temporal expression patterns, that is they were differentially regulated with age in brain, liver, and pectoralis. Insulin could regulate chicken CDS2 levels in a breed- and tissue-specific manner.

Gut microbiota interfered with using prebiotics may improve bone mass and alleviate the onset of bone problems. This study aimed to investigate the beneficial effect of resistant starch from raw potato starch (RPS) on bone health in meat ducks. Response to the dietary graded level of RPS supplementation, both tibia strength and ash were taken out linear and quadratic increase and positively correlated with increased propionate and butyrate levels in cecal content. Moreover, further outcomes of gut microbiota and micro-CT analysis showed the beneficial effect of RPS on bone mass might be associated with higher Firmicutes proportion and the production of short-chain fatty acids (SCFAs) in the cecum. Consistent with improving bone mass, SCFAs promoted phosphorus absorption, decreased the digestive tract pH, and enhanced intestinal integrity, which decreased the expression of pro-inflammatory genes in both gut and bone marrow, and consequently depressed osteoclastic bone resorption mediated by inflammatory cytokines. These findings highlight the importance of the “gut-bone” axis and provide new insight into the effect of prebiotics on bone health.

ABSTRACT: The first purpose of this study was to reveal the distribution of the angel wing (AW) of geese. Our data showed that the total incidence of AW was 6.67% in 150-day-old White Zhedong (ZD) geese, the occurrence of AW in left wing is higher than that in right wing and bilateral wing than unilateral wing (both P < 0.01). In 70-day-old Hybrid-Wanxi (HW) geese, the total incidence of AW was 8.86%, with similar incidence rate between unilateral and bilateral. The sex has not apparently affected the incidence of AW in both ZD and HW geese. To explore the potential relationship between wing type with body weight, organ index, bone characteristic, or blood biochemical parameters in 70-day-old HW geese. We found that the body weight and organ index were similar between normal wing (NW) and AW geese. The length for the humerus, metacarpal and phalanx, and the phalanx weights, as well as the angle between the humerus and the radial ulna (HRU) in NW geese were pronounced greater than that in AW geese (P < 0.05). Furthermore, the angel wing was strongly associated with lower platelet size indicators. Collectively, AW affected the wing bone length, phalanx weight, and HRU, and the occurrence of AW may be related with dysfunctional platelet activation in geese.

Roles of plant-derived cinnamaldehyde, carvacrol, and thymol in the gut and bone health of laying hens was evaluated in the present study. After acclimation for 2 weeks, a total 384 of 52-week-old laying hens were allocated into three groups for 6 weeks: (1) basal diet group (Ctrl), (2) combination of cinnamaldehyde with carvacrol group (CAR+CIN), and (3) blend of cinnamaldehyde with thymol (THY+CIN). The dietary essential oil level was 100 mg/kg. Each treatment group had eight replicate pens (16 birds/pen). The stiffness and ultimate load of the tibiae from both the CAR+CIN and THY+CIN groups were higher than that of the Ctrl group (p < 0.05), along with comparable tibia ash, calcium, and phosphorus content among groups. At the same time, the manipulation of essential oils upregulated the transcription abundances of intestinal barrier proteins to varying degrees, whereas the experimental treatment failed to affect the composition in phyla of cecal microbiota. When compared to the Ctrl group, birds fed the CAR+CIN and THY+CIN diet displayed decreased bone resorption markers, reduced interleukin-1 concentrations, and increased transforming growth factor beta levels in serum. These findings suggest that cinnamaldehyde with carvacrol or thymol in feed of hens could enhance intestinal barrier and improve the mechanical properties of tibiae through structural modelling but not increase the mineral density, which might be involved in suppressing inflammation-mediated bone resorption.

Growing concern for public health and food safety has prompted a special interest in developing nutritional strategies for removing waterborne and foodborne pathogens, including Salmonella. Strong links between manganese (Mn) and intestinal barrier or immune function hint that dietary Mn supplementation is likely to be a promising approach to limit the loads of pathogens in broilers. Here, we provide evidence that Salmonella Typhimurium (S. Typhimurium, 4 × 108 CFUs) challenge-induced intestinal injury along with systemic Mn redistribution in broilers. Further examining of the effect of dietary Mn treatments (a basal diet plus additional 0, 40, or 100 mg Mn/kg for corresponding to Mn-deficient, control, or Mn-surfeit diet, respectively) on intestinal barrier and inflammation status of broilers infected with S. Typhimurium revealed that birds fed the control and Mn-surfeit diets exhibited improved intestinal tight junctions and microbiota composition. Even without Salmonella infection, dietary Mn deficiency alone increased intestinal permeability by impairing intestinal tight junctions. In addition, when fed the control and Mn-surfeit diets, birds showed decreased Salmonella burdens in cecal content and spleen, with a concomitant increase in inflammatory cytokine levels in spleen. Furthermore, the dietary Mn-supplementation-mediated induction of cytokine production was probably associated with the nuclear factor kappa-B (NF-κB)/hydrogen peroxide (H2O2) pathway, as judged by the enhanced manganese superoxide dismutase activity and the increased H2O2 level in mitochondria, together with the increased mRNA level of NF-κB in spleen. Ingenuity-pathway analysis indicated that acute-phase response pathways, T helper type 1 pathway, and dendritic cell maturation were significantly activated by the dietary Mn supplementation. Our data suggest that dietary Mn supplementation could enhance intestinal barrier and splenic inflammatory response to fight against Salmonella infection in broilers.