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Divergent selection of broilers for water conversion ratio has established and high-(HWE) and low- water efficient (LWE) broiler lines. Two 2 × 2 factorial experiments were conducted to assess the gene expression profile of systems involved in renal water homeostasis. In Exp. 1, male and female HWE and LWE broilers were individually phenotyped between 4 and 6 wks of age to determine growth performance and water conversion ratio (g water intake/g body weight gain). Kidney samples were obtained from 5 males and 5 females from each line. In Exp. 2, HWE and modern random bred (MRB) broilers were placed in 12 controlled-environmental chambers (2 floor pens/chamber, 6 chambers/line, 11 birds per pen, 132 birds/line) on day of hatch. The broilers were brooded at thermoneutral temperatures from 0 to 4 wks. From 4 to 7 wks, broilers were maintained at thermoneutral (TN, 25 °C) or exposed to cyclic heat stress (HS, 35 °C, 8h/day) conditions. Body weight, feed intake, and water intake were recorded. Kidney samples were collected, flash frozen in liquid nitrogen, and kept at -80 °C for gene expression analysis. Data were analyzed by Two-way ANOVA and means compared by Tukey's HSD multiple comparison test. Molecular analyses from Exp. 1 showed that the renal expression of arginine vasopressin (AVP), angiotensinogen (AGT), angiotensin II receptor type 1 and 2 (AT1/2), sodium-potassium ATPase subunit B1 (ATP1B1), and aquaporin 3 (AQP3) were upregulated in HWE compared to the LWE line. In contrast, mRNA expression of mesotocin receptor (MTR), AT1/2, AQP1/2, and occludin were significantly higher in females than in males. In Exp. 2, target genes were regulated in environment and/or line-dependent manner. The renal expression of heat shock proteins 70 and 90, AVP receptor 2 (AVPR2), AGT, renin, AT1/2, and AQP1was significantly upregulated in HS compared to TN birds, however AVPR2 expression was significantly higher in HWE compared to MRB birds. Together, the up-regulation of AVP, the renin-angiotensin system (RAS), and AQP in HWE, female, or under HS conditions suggests a better renal water reabsorption to support water use efficiency.

In the broiler industry, intensive genetic selection has been placed on muscle growth which has undesirably led to increased fat accretion. Models of chicken preadipocyte differentiation in vitro have conventionally used incubators without the ability to control oxygen (O2) tension; thus, the cells are exposed to atmospheric (∼20-21%) O2, which is supraphysiological compared to the O2 tension within adipose tissue. The objective of this study was to investigate embryonic broiler preadipocyte differentiation at different O2 tensions, including atmospheric (20%), physiological (5%), and hypoxic (1%). Culture at 1% O2 resulted in increased abundance of HIF1α, a canonical protein stabilized during hypoxia, thus confirming effectiveness of the treatment. Increased accumulation of lipid was observed in preadipocytes cultured in adipogenic differentiation medium compared to the control medium. When considering oxygen tension, lipid accumulation was increased in preadipocytes that were cultured in differentiation medium at 20% O2 compared to 5% or 1% O2. Furthermore, abundance of transcripts related to fatty acid transport and adipogenesis, fatty acid binding protein 4 (FABP4) and peroxisome proliferator-activated receptor gamma (PPARγ), were increased in differentiated preadipocytes cultured at 20% O2 compared to 5% or 1% O2. Abundance of transcripts related to lipid synthesis and oxidation, acyl-CoA synthetase long chain family member 1 (ACSL1) and carnitine palmitoyltransferase 1A (CPT1A), were increased in the differentiation cultures compared to the control cultures. Abundance of glutathione peroxidase 4 (GPX4) was increased in all the differentiation cultures compared to the controls, regardless of oxygen tension; however, differences in the abundance of other antioxidant enzymes were not observed. Overall, exposure to atmospheric oxygen tension promotes lipid accumulation within chicken preadipocytes, which may need to be considered when developing in vitro models of this process.

ABSTRACT: Heat stress (HS) and water scarcity are significant challenges to sustainable poultry production worldwide. It is, therefore, critical to identify effective strategies to prevent, withstand, or adapt to these challenges. After four generations of divergent selection for water efficiency, the present study was undertaken to determine the effect of HS on meat quality and muscle myopathy incidences in high (HWE)- and low (LWE)-water efficient broilers. Day-old male chicks (240 chicks/line) were allotted randomly by line and body weight-matched groups to 12 controlled-environmental chambers (2 pens/chamber). At d29, birds were exposed to 2 environmental conditions (thermoneutral (TN), 25°C; or cyclic HS, 36°C, 9h/d) in a 2 × 2 factorial design. On d49, birds were processed, carcass parts were weighed, meat quality and muscle myopathy incidence were assessed. Processing data were analyzed by Two-way ANOVA and Tukey's HSD multiple comparison test, and frequency of muscle myopathy score between groups was determined using Chi-square and Fisher's exact test. Significance was set at P < 0.05. As no significant environment by line interaction was discerned, the 2 main factors were analyzed separately. High water efficient birds had significantly higher tender- and leg quarter (LQ)-weight as well as carcass without giblet (WOG), chilled carcass WOG (CWOG), wing, LQ, and rack yields compared to their LWE counterparts. Both abdominal fat content and yields were significantly greater in LWE than HWE chickens. Chronic HS exposure significantly decreased dock, WOG, fat, CWOG, breast, tender, wing, and LQ weights as well as breast yield. HWE chickens had a significantly lower b* value compared to the LWE birds and HS significantly reduced the drip loss and the b* value compared to TN condition. Compared to LWE, HWE birds had higher and lower incidence of severe woody breast (WB) and white striping (WS) under TN and HS, respectively. HS reduced the incidence of both myopathies in both lines. In conclusion, the genetic selection for water efficiency seems to improve carcass yield, reduce fat content, and decrease the breast b* value. HWE birds had higher incidences of WB and WS under TN, which is reversed under HS conditions.

ABSTRACT: Mitigating the negative effects of heat stress (HS) is a critical challenge for the global poultry industry. This study evaluated the thermoregulatory potential of 3 in ovo delivered bioactive substances using selected gut health parameters. Eggs were incubated and allotted to 5 groups, and respective bioactive substances delivered. These groups included—the noninjected, in ovo saline, in ovo folic acid (FA), in ovo probiotics (P), and in ovo essential oil (EO). At hatch, chicks were assigned to 5 new posthatch treatment combinations, including A) Negative control (NC)—noninjected eggs offered a basal corn-wheat-soybean diet, B) Antibiotics—NC + 0.05% bacitracin methylene disalicylate, C) In ovo FA—eggs injected with FA + NC diet, D) In ovo probiotics—eggs injected with probiotics + NC diet, E) In ovo + in-water EO—eggs injected with EO and supplied EO via drinking water + NC diet. Birds were raised for 28 d in 8 replicate cages/treatment (6 birds/cage) and exposed to either a thermoneutral (24°C ± 0.2) or HS challenge (31°C) condition from d 21 to d 28. The in ovo delivered FA and EO treatments reduced (P < 0.001) hatchability by at least 26% compared to NC. Induced HS reduced (P < 0.001) total plasma protein, total antioxidant capacity, and villus width in the duodenum and jejunum. Independent of HS and compared to NC, the in ovo + in-water EO treatment resulted in (P < 0.05) at least a 15% increase in villus height: crypt depth across the 3 gut sections. The in ovo + in-water EO treatment also increased the relative mRNA expression of intestinal barrier-related genes (Claudin1,3,4, Occludin, Zona occludens-2, and Mucin 2). Under HS, the in ovo + in-water EO treatment recorded a 3.5-fold upregulation of amino acid transporter gene (SLC1A1), compared to NC. Subject to further hatchability optimization, the in ovo + in-water delivery of EO show potential to afford broiler chicken thermotolerance.

ABSTRACT: Lameness is a leading cause of animal welfare and production concerns for the poultry industry as fast-growing, high-yielding broilers seem more susceptible to bone disease and infections. A major limitation to the study of these disorders is the lack of a chicken immortalized chondrocyte cell. Primary cell isolation is a valid and complex method for establishing a relevant in vitro model for diseases. In this study, isolation and high-density culturing of primary chondrocytes form 1-d old chicks was followed by confirmation of cell type, identification of optimal phenotypic expression, and evaluation of cells functionality. mRNA expression, as well as protein production and secretion, of COLI, COLII, Sox9, ACAN, and COLXA1 on day 3 (d3), d7, d11, d14, d18, and d21 in culture showed that avian growth plate chondrocytes under these conditions exhibit optimal phenotypes from d3 to d7. This is evident by a shift from COLII dominant expression in early-culture to COLI dominant expression by late-culture in conjunction with a loss of other chondrocyte markers Sox9, ACAN, and COLXA1. Additionally, morphological changes seen through live cell imaging coincide with the shift of phenotype in mid- to late-culture periods indicating a dedifferentiated phenotype. The functionality of the cultured cells was confirmed using Brefeldin-A treatment which significantly reduced secretion of COLII by d7 chondrocytes. These results provide a foundation for future research utilizing avian primary chondrocytes with optimal phenotypes for disease modeling or passaging.

ABSTRACT: Commercial broilers have been selected for high growth rate and productivity; however, this has negatively impacted their susceptibility to heat stress (HS). Insight into the molecular mechanisms underlying this vulnerability can help design targeted strategies for improvement of HS tolerance. Red blood cells (RBC) and white blood cells (WBC) were isolated from red jungle fowl and 4 lines of commercial modern broilers. Lines A and B are considered standard-yielding lines, whereas Lines C and D are high-yielding. Cells were cultured at either 37°C or 45°C for 2 h to induce heat stress (HS). Gene expression of cytokines, chemokines, and inflammasome components were measured. Heat shock proteins 27 and 70 (HSPs) in RBC were significantly affected by line (P < 0.05), whereas HSP27 and 60 were affected by temperature (P < 0.05). In WBC, there was a significant line effect on HSP gene expression (P < 0.05), and a significant increase (P < 0.05) in HSP90 in Line D in HS compared to TN conditions. In RBC, there was a main effect of HS on TNFα, CCL4, and CCLL4 (P < 0.05). HS significantly increased IL-8L1 (>30-fold, P < 0.0001) in Line C. Inflammasome genes (NLRP3, NLRC5 and NLRC3) were significantly affected by the line studied (P < 0.05). In WBC, the effect of line was significant for all cytokines, chemokines, and inflammasome components studied (P < 0.05). To examine the mechanical properties of isolated RBC from the 4 commercial lines and jungle fowl, RBC were placed into nematic liquid crystals, where Lines B and D were the most strained, and Line A and the jungle fowl were the least strained. Together, these findings indicate not only the dynamic nature of circulating cells, but the differences in the stress and inflammatory response among commercially available lines and their common ancestor. These profiles have the potential to serve as a future marker for stress responses in broilers, though further study is warranted.

ABSTRACT: Autophagy is a cell survival and homeostasis mechanism involving lysosomal degradation of cellular components and foreign bodies. It plays a role in bone homeostasis, skeletal diseases, and bacterial infections as both a cell-survival or cell-death pathway. This study sought to determine if autophagy played a role in bacterial chondronecrosis with osteomyelitis (BCO). BCO is a prominent cause of lameness in modern broilers and results from bacterial infection of mechanically stressed leg bone growth plates. The protein and gene expression of key autophagy machinery was analyzed in both normal and BCO-affected broilers using real-time qPCR and immunoblot, respectively. Gene expression showed a significant downregulation of key target signatures involved in every stage of autophagy in BCO-affected bone, such as ATG13, SQSTM1 (p62), ATG9B, ATG16L, ATG12, LC3C, and RAB7A. Additionally, protein expression for LC3 was also significantly lower in BCO. An in vitro study using human fetal osteoblast cells challenged with BCO isolate, Staphylococcus agnetis 908, showed a similar dysregulation of autophagy machinery along with a significant decrease in cell viability. When autophagy was inhibited via 3-methyladenine or chloroquine, comparable decreases in cell viability were seen along with dysregulation of autophagy machinery. Together, these results are the first to implicate autophagy machinery dysregulation in the pathology of BCO.

The feed additive Availa-ZMC was investigated for the ability to reduce lameness in broilers using 2 alternative models for inducing lameness. The mixture of organic trace minerals was effective in reducing lameness by 20% in the wire flooring model and 25% in the litter flooring model with the bacterial challenge. Lameness in both models is overwhelmingly attributable to bacterial chondronecrosis with osteomyelitis. The reduction in lameness was associated, at least in part, with enhanced intestinal barrier integrity mediated by elevated expression of tight junction proteins and stimulation of bactericidal killing of adherent peripheral blood monocytes obtained from the birds treated with Availa-ZMC. Lameness is a major animal welfare concern in broiler production. The wire flooring model and litter flooring model with the bacterial challenge are effective models for evaluation of management strategies for mitigating infectious causes of lameness.

Woody breast (WB) myopathy is a major concern and economic burden to the poultry industry, and for which, there is no effective solution because of its unknown etiology. In a previous study, we have shown that phytase (Quantum Blue, QB) reduces the WB severity by 5% via modulation of oxygen homeostasis-related pathways. As WB has been suggested to be associated with lipid dysmetabolism, we aimed to determine the effect of QB on WB and breast muscle fatty acid profile. Male broilers were subjected to 6 treatments (96 birds/treatment): a nutrient adequate control group (PC), the PC supplemented with 0.3% myo-inositol (PC + MI), a negative control (NC) deficient in available P and Ca by 0.15 and 0.16%, respectively, the NC fed with QB at 500 (NC+500 FTU), and 1,000 (NC+ 1,000 FTU) or 2,000 FTU/kg of feed (NC+2,000 FTU). Woody breast and white striping scores were recorded, and fatty acid profiles were determined using gas liquid chromatography. Woody breast-affected muscles exhibited a significant higher incidence of white striping as liquid chromatography analysis reveals an imbalance of fatty acid profile in the breast of WB-affected birds with a significant higher percent of saturated fatty acids (SFA, myristic [14:0], pentadecanoic [15:0], and margaric [17:0]) and monounsaturated fatty acids (myristoleic [14:1], palmitoleic [16:1c], 10-trans-heptadecenoic [17:1t], oleic [18:1c9], and vaccenic [18:1c11]), and lower content of polyunsaturated fatty acids (PUFA) and omega-3 (P

The purpose of this study was to evaluate heat stress as a model to induce gastrointestinal leakage in broiler chickens. On the day of hatch, 320 chicks were allocated into 8 environmental chambers, 4 thermoneutral (TN) and 4 continuous heat stress (HS). Each chamber was divided into 2 pens containing separate feeders and water jugs (8 replicates per treatment, 20 birds/pen). The environment was established to simulate production setting as best possible for the first 21 D. A gradual reduction of temperature from 32°C to 24°C with relative humidity at 55 ± 5% was adopted for the first 21 D. At the time of HS, the HS groups were exposed to 35°C from Day 21 to 42, while thermoneutral ones were maintained at 24°C from Day 21 to 42. Chickens were equipped with a Thermochron temperature logger for continuous monitoring of core body temperature. The environmental temperature and relative humidity were continuously recorded. Fluorescein isothiocyanate–dextran (FITC-d) was orally gavaged to 2 chickens/replicate (n = 16) randomly selected on days 21, 28, 35, and 42. After 1 h of oral gavage, blood samples were collected to determine the passage of FITC-d. Tibias were removed from all chickens to evaluate break strength only on 21 D and 42 D (before HS and at the end of the trial). Performance parameters were evaluated weekly from 21 D to the end of the trial. Body temperature was significantly (P

ABSTRACT: Woody breast (WB) condition has created a variety of challenges for the global poultry industry. To date, there are no effective treatments or preventative measures due to its unknown (undefined) etiology. Several potential mechanisms including oxidative stress, fiber-type switching, cellular damage, and altered intracellular calcium levels have been proposed to play a key role in the progression of the WB myopathy. In a previous study, we have shown that WB is associated with hypoxia-like status and dysregulated oxygen homeostasis. As satellite cells (SC) play a pivotal role in muscle fiber repair and remodeling under stress conditions, we undertook the present study to determine satellite cell fate in WB-affected birds when reared in either normoxic or hypoxic conditions. Modern random bred broilers from 2015 (n = 200) were wing banded and reared under standard brooding practices for the first 2 wk post-hatch. At 15 d, chicks were divided in 2 body weight-matched groups and reared to 6 wk in either control local altitude or hypobaric chambers with simulated altitude of 6,000 ft. Birds were provided ad libitum access to water and feed, according to the Cobb recommendations. At 6 wk of age, birds were processed and scored for WB, and breast samples were collected from WB-affected and unaffected birds for molecular analyses (n = 10/group). SCs were isolated from normal breast muscle, cultured in vitro, and exposed to normoxia or hypoxia for 2 h. The expression of target genes was determined by qPCR using 2−∆∆Ct method. Protein distribution and expression were determined by immunofluorescence staining and immunoblot, respectively. Data were analyzed by the Student's t test with significance set at P < 0.05. Multiple satellite cell markers, myogenic factor (Myf)-5 and paired box (PAX)-7 were significantly decreased at the mRNA and protein levels in the breast muscle from WB-affected birds compared to their unaffected counterparts. Lipogenic-and adipogenic-associated factors (acetyl-CoA carboxylase, ACCα; fatty acid synthase, FASN, malic enzyme, ME; and ATP citrate lyase, ACLY) were activated in WB-affected birds. These data were supported by an in vitro study where hypoxia decreased the expression of Myf5 and Pax7, and increased that of ACCα, FASN, ME, and ACLY. Together, these data indicate that under hypoxic condition, SC change fate by switching from a myogenic to an adipogenic program, which explains at least partly, the etiology of the WB myopathy.

Heat stress (HS) is a critical concern to the poultry industry as it affects both productivity and well-being. Various managerial and nutritional strategies have been proposed to mitigate the negative effects of HS in chickens, with plant-based additives showing promise. Recently, we reported the positive effect of a phytogenic feed additive (PFA) on growth performance in HS birds. Owing to the antioxidant nature of these compounds, we sought to further explore the effect of PFA on whole blood circulating chemokines, cytokines, and inflammasomes in HS broilers. Broilers (600 males, 1 d) were randomly assigned to 12 environmental chambers, subjected to 2 environmental conditions (12 h cyclic heat stress, HS, 35°C vs. thermoneutral condition [TN], 24°C) and fed 3 diets (control, PFA-C 250 ppm, PFA-C 400 ppm) in a 2 × 3 factorial design. After 21 d of cyclic HS, blood samples were collected for target gene expression analysis. HS upregulated the expression of superoxide dismutase 1 (SOD1) and downregulated glutathione peroxidase-3 (GPX-3), and there was diet × temperature interaction for SOD2, GPX-1, and GPX-3, where gene expression was increased by PFA-C250 during HS but was unchanged for PFA-C400. Plasma total antioxidant capacity (TAC) and malondialdehyde (MDA) content were increased by HS. Gene expression of interleukin-18 (IL-18) was decreased by HS, without further effect of PFA. HS increased tumor necrosis factor α (TNFα), but this effect was mitigated by PFA-C400. C-C motif chemokine ligands 4 and 20 (CCL4 and CCL20) showed a similar pattern to TNFα, with PFA-C400 ameliorating the negative effect of HS. The nucleotide-binding, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome was decreased by HS and further lowered by PFA-C400, but the nucleotide-binding oligomerization domain, leucine-rich repeat, and CARD domain containing 3 (NLRC3) and nucleotide-binding, leucine-rich repeat containing X1 (NLRX1) inflammasomes were increased by PFA under TN conditions, with no effects of HS. Heat shock proteins (HSP) and heat shock factors (HSF) were unaffected by PFA or HS. Together these data indicate that gene expression of circulating inflammatory factors are dysregulated during HS, and supplemental dietary PFA may be protective.

Diets enriched with phytogenic feed additives (PFA) such as AV/HGP/16 premix (AVHGP), Superliv concentrate premix (SCP), and bacteriostatic herbal growth promotor (BHGP) with essential oils have been shown to improve feed efficiency (FE) in broilers. This FE improvement was achieved via modulation of hypothalamic neuropeptides, which results despite feed intake reduction, in increased breast yield without changes in body weight compared to the control group. To gain further insights into the mode of action of these PFA, the present study aimed to determine the potential involvement of signaling pathways associated with lipid and protein metabolism. One day-old male Cobb 500 chicks were randomly assigned into 1 of 4 treatments, comprising 8 replicates per treatment in a completely randomized design. The dietary treatments included a basal diet (control) or 0.55 g/kg diet of AVHGP, SCP, or BHGP. The birds had ad libitum access to water and feed. On day 35, after blood sampling, the liver, abdominal adipose tissue (AT), and breast muscle samples were collected. The levels of phosphorylated mechanistic target of rapamycin (mTOR)Ser2481 as well as its levels of mRNA and those of its downstream mediator RPS6B1 were significantly upregulated in the muscle of the PFA-fed groups compared with the control group. In the liver, the phosphorylated levels of acetyl-CoA carboxylase alpha at Ser79, the rate-limiting enzyme in fat synthesis, was significantly induced in the PFA-fed groups compared with the control group, indicating a lower hepatic lipogenesis. The hepatic expression of hepatic triglyceride lipase (LIPC) and adipose triglyceride lipase (ATGL) was significantly upregulated in the AVHGP-fed group compared with the control group. These hepatic changes were accompanied by a significant downregulation of hepatic sterol regulatory element-binding protein cleavage-activating protein in all the PFA groups and an upregulation of peroxisome proliferator–activated receptor alpha and gamma in the SCP-fed compared with the control group. In the AT, the mRNA abundances of ATGL and LIPC were significantly increased in both SCP- and BHGP-fed birds compared with the control group. Together these data indicate that PFA improve FE via modulation of muscle mTOR pathway and hepatic lipolytic/lipogenic programs, thus, favoring muscle protein synthesis and lowering hepatic lipogenesis.

The therapeutic utility of liver X receptor (LXR) agonists in treating atherosclerosis is limited by an undesired accumulation of triglycerides in the blood and liver. This effect is caused by an increase in the transcription of genes involved in fatty acid synthesis. Here, we show that the primary bile acid, chenodeoxycholic acid (CDCA), antagonizes the stimulatory effect of the synthetic LXR agonist, T0-901317, on the expression of acetyl-coenzyme A carboxylase-α (ACCα) and other lipogenic enzymes in chick embryo hepatocyte cultures. CDCA inhibits T0-901317-induced ACCα transcription by suppressing the enhancer activity of a LXR response unit (−101 to −71 bp) that binds LXR and sterol-regulatory element binding protein-1 (SREBP-1). We also demonstrate that CDCA decreases the expression of SREBP-1 in the nucleus and the acetylation of histone H3 and H4 at the ACCα LXR response unit. The CDCA-mediated reduction in ACCα expression is associated with a decrease in the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and small heterodimer partner and an increase in the expression of fibroblast growth factor-19 (FGF-19). Ectopic expression of FGF-19 decreases T0-901317-induced ACCα expression. Inhibition of p38 mitogen-activated protein kinase (MAPK) and/or extracellular signal-regulated kinase (ERK) suppresses the effects of CDCA on the expression of ACCα, SREBP-1, PGC-1α, and FGF-19. These results demonstrate that CDCA inhibits T0-901317-induced ACCα transcription by suppressing the activity of LXR and SREBP-1. We postulate that p38 MAPK, ERK, PGC-1α, and FGF-19 are components of the signaling pathway(s) mediating the regulation of ACCα gene transcription by CDCA.