Background: Mitochondria are considered the powerhouse of cells, and skeletal muscle cells are no exception. However, information regarding muscle mitochondria from different species is limited. Methods: Different muscles from cattle, pigs and chickens were analyzed for mitochondrial DNA (mtDNA), protein and oxygen consumption. Results: Bovine oxidative muscle mitochondria contain greater mtDNA (p < 0.05), protein (succinate dehydrogenase, SDHA, p < 0.01; citrate synthase, CS, p < 0.01; complex I, CI, p < 0.05), and oxygen consumption (p < 0.01) than their glycolytic counterpart. Likewise, porcine oxidative muscle contains greater mtDNA (p < 0.01), mitochondrial proteins (SDHA, p < 0.05; CS, p < 0.001; CI, p < 0.01) and oxidative phosphorylation capacity (OXPHOS, p < 0.05) in comparison to glycolytic muscle. However, avian oxidative skeletal muscle showed no differences in absolute mtDNA, SDHA, CI, complex II, lactate dehydrogenase, or glyceraldehyde 3 phosphate dehydrogenase compared to their glycolytic counterpart. Even so, avian mitochondria isolated from oxidative muscles had greater OXPHOS capacity (p < 0.05) than glycolytic muscle. Conclusions: These data show avian mitochondria function is independent of absolute mtDNA content and protein abundance, and argue that multiple levels of inquiry are warranted to determine the wholistic role of mitochondria in skeletal muscle.

The scientific community needs objective measures to appropriately assess animal welfare. The study objective was to assess the impact of housing system on novel physiological and behavioral measurements of animal welfare for laying hens, including secretory and plasma Immunoglobulin (IgA; immune function), feather corticosterone (chronic stress), and attention bias testing (ABT; anxiety), in addition to the well-validated tonic immobility test (TI; fearfulness). To test this, 184 Bovan brown hens were housed in 28 conventional cages (3 birds/cage) and 4 enriched pens (25 birds/pen). Feces, blood, and feathers were collected 4 times between week 22 and 43 to quantify secretory and plasma IgA and feather corticosterone concentrations. TI tests and ABT were performed once. Hens that were from cages tended to show longer TI, had increased feather corticosterone, and decreased secretory IgA at 22 weeks of age. The caged hens fed quicker, and more hens fed during the ABT compared to the penned hens. Hens that were in conventional cages showed somewhat poorer welfare outcomes than the hens in enriched pens, as indicated by increased chronic stress, decreased immune function at 22 weeks of age but no other ages, somewhat increased fear, but reduced anxiety. Overall, these novel markers show some appropriate contrast between housing treatments and may be useful in an animal welfare assessment context for laying hens. More research is needed to confirm these findings.

Exposure to high ambient temperature has detrimental effects on poultry welfare and production. Although changes in gene expression due to heat exposure have been well described for broiler chickens, knowledge of the effects of heat on laying hens is still relatively limited. In this study, we profiled the transcriptome for pectoralis major muscle (n = 24) and liver (n = 24), during a 4-week cyclic heating experiment performed on layers in the early phase of egg production. Both heat-control and time-based contrasts were analyzed to determine differentially expressed genes (DEGs). Heat exposure induced different changes in gene expression for the two tissues, and we also observed changes in gene expression over time in the control animals suggesting that metabolic changes occurred during the transition from onset of lay to peak egg production. A total of 73 DEGs in liver were shared between the 3 h heat-control contrast, and the 4-week versus 3 h time contrast in the control group, suggesting a core set of genes that is responsible for maintenance of metabolic homeostasis regardless of the physiologic stressor (heat or commencing egg production). The identified DEGs improve our understanding of the layer’s response to stressors and may serve as targets for genetic selection in the future to improve resilience.

The goal of this experiment was to measure the physiological response of individual laying hens exposed to heat stress (HS). Performance, egg quality, body temperature (BT), and blood chemistry of laying hens were individually recorded before and after various intervals of daily cyclic HS. In total, 407 18-week-old W-36 parent-line laying hens (Hy-Line International, Dallas Center, IA) were housed individually in battery cages. After an acclimation period, baseline data were collected from 22 to 24-wk before the hens were subjected to a daily cyclic HS consisting of 7 h at 35°C returning to 30°C for the remaining 17 h/D from 24 to 28-wk of age. Eggs were collected and individually weighed daily. Feed intake (FI), egg production (EP), egg weights, egg mass, BW, and feed efficiency (FE) (g egg/kg FI) were calculated over 2-wk time periods. Eggs were collected for quality assessment the day before HS began, the 2nd day of HS, and on a weekly basis throughout the 4-wk HS. Blood was collected and BT measured the day before heat HS was initiated, on the first day of HS, and again at 2 and 4-wk of HS. Blood PCO2 and iCa decreased, and blood pH increased within 4 to 6 h of HS (P ≤ 0.01). Shell weights decreased with acute HS, possibly due to the reduction in blood iCa (P ≤ 0.01). After 4-wk of HS the blood pH returned to pre-HS levels but iCa remained decreased (P ≤ 0.01). Shell weights remained low and Haugh units decreased after 2 and 4-wk of HS (P ≤ 0.01). Feed efficiency was increased and FI, EP, and BW decreased by 2-wk of HS and remained low through 4-wk (P ≤ 0.01). The cyclic HS had a significant effect on the performance, egg quality, and blood chemistry over the 4-wk HS.

BackgroundHeat stress negatively affects the welfare and production of chickens. High ambient temperature is considered one of the most ubiquitous abiotic environmental challenges to laying hens around the world. In this study, we recorded several production traits, feed intake, body weight, digestibility, and egg quality of 400 commercial white egg-laying hens before and during a 4-week heat treatment. For the phenotypes that had estimated heritabilities (using 600k SNP chip data) higher than 0, SNP associations were tested using the same 600k genotype data.ResultsSeventeen phenotypes had heritability estimates higher than 0, including measurements at various time points for feed intake, feed efficiency, body weight, albumen weight, egg quality expressed in Haugh units, egg mass, and also for change in egg mass from prior to heat exposure to various time points during the 4-week heat treatment. Quantitative trait loci (QTL) were identified for 10 of these 17 phenotypes. Some of the phenotypes shared QTL including Haugh units before heat exposure and after 4weeks of heat treatment.ConclusionsEstimated heritabilities differed from 0 for 17 traits, which indicates that they are under genetic control and that there is potential for improving these traits through selective breeding. The association of different QTL with the same phenotypes before heat exposure and during heat treatment indicates that genomic control of traits under heat stress is distinct from that under thermoneutral conditions. This study contributes to the knowledge on the genomic control of response to heat stress in laying hens.

Heat stress has a large negative impact on poultry around the world in both intensive and small-scale production systems. Better understanding of genetic factors contributing to response to high ambient temperatures would provide a basis to develop strategies for alleviating negative impacts of heat on poultry production. The objective of this work was to characterize the genetic control (heritability estimate and quantitative trait loci (QTL)) of blood chemistry components before and after exposure to acute and chronic high ambient temperature in a commercial egg laying line Hy-Line W-36 female parent line mature hens were exposed to 4 wk of daily cyclic heat exposure. Blood was collected pre-heat, on the first day of heat, and 2 and 4 wk post heat initiation and analyzed immediately using an i-STAT® hand-held blood analyzer. Thirteen blood components were quantified at the 4 time points: pH, pCO2, pO2, HCO3, TCO2, sO2, iCa, Na, K, base excess, glucose, "hematocrit" (estimated from blood electrical conductivity, BEC), and "hemoglobin" (calculated from BEC). Heritabilities were estimated using genomic relationship information obtained from 600k SNP chip data. All 13 parameters exhibited a significant change after 5 h of heat exposure and most did not return to pre-heat levels throughout the duration of the study. Eight parameters (base excess, glucose, hemoglobin, HCO3, hematocrit, K, pCO2, TCO2) had heritability estimates differing from zero at one or more time points (0.21 to 0.45). The traits with significant heritability would be good candidates for use as biomarkers in a selection program if they are correlated with traits of economic importance that are more difficult to measure. QTL were identified for nine of the traits at one or more time point. These nine traits, however, did not have significant heritability estimates suggesting that while some QTL have been identified their effects are generally small.

Exposure to high temperatures is known to impair immune functions and disease resistance of poultry. Characterizing changes in the transcriptome can help identify mechanisms by which immune tissues, such as the thymus, respond to heat stress. In this study, 22-day-old chickens from two genetic lines (a relatively resistant Fayoumi line and a more susceptible broiler line) were exposed to acute heat stress (35 °C) and/or immune simulation with lipopolysaccharide (LPS; 100 µg/kg). Transcriptome responses in the thymus were identified by RNA-sequencing (RNA-seq). Expression of most genes was unaffected by heat and/or LPS in the Fayoumi line, whereas these treatments had more impact in the broiler line. Comparisons between the broiler and Fayoumi transcriptomes identified a large number of significant genes both at homeostasis and in response to treatment. Functional analyses predicted that gene expression changes impact immune responses, apoptosis, cell activation, migration, and adhesion. In broilers, acute heat stress changed thymic expression responses to LPS and could impact thymocyte survival and trafficking, and thereby contribute to the negative effects of high temperatures on immune responses. Identification of these genes and pathways provides a foundation for testing targets to improve disease resistance in heat-stressed chickens.

Exposure to heat stress suppresses poultry immune responses, which can increase susceptibility to infectious diseases and, thereby, intensify the negative effects of heat on poultry welfare and performance. Identifying genes and pathways that are affected by high temperatures, especially heat-induced changes in immune responses, could provide targets to improve disease resistance in chickens. This study utilized RNA-sequencing (RNA-seq) to investigate transcriptome responses in the bursa of Fabricius, a primary immune tissue, after exposure to acute heat stress and/or subcutaneous immune stimulation with lipopolysaccharide (LPS) in a 2 × 2 factorial design: Thermoneutral + Saline, Heat + Saline, Thermoneutral + LPS and Heat + LPS. All treatments were investigated in two chicken lines: a relatively heat- and disease-resistant Fayoumi line and a more susceptible broiler line.

In this experiment, producing layers were exposed to 4 weeks of cyclic heat to better understand the effects of heat stress on the gene expression in the cecal tonsil (CT). CT is the immune tissue located at the junction between the ileum and the ceca within the digestive tract. CT has been suggested to have a role in the regulation of the cecal content, which may have an impact on nutrient absorption. Knowledge of specific changes in gene expression at the CT highlights potential mechanisms that the tissue utilizes to alleviate the effect from heat stress.

During postmortem metabolism, muscle pH gradually declines to reach an ultimate pH near 5.6 across most meat species. Yet, broiler pectoralis major (P. major) muscle generates meat with high ultimate pH (pH ∼ 5.9). For better understanding of the underlying mechanism responsible for this phenomenon, we evaluated the involvement of breast muscle chilling on the extent of postmortem metabolism. Broiler breast muscles were either subjected to chilling treatment (control) or left at room temperature (RT) for 120 min. P. major muscle from the RT treatment had lower ultimate pH, greater glycogen degradation and lactate accumulation. While these findings suggest that carcass chilling can contribute to the premature termination of postmortem metabolism, chilling did not fully explain the high ultimate pH of P. major muscle. Our results also revealed that glucose-6-phosphate (G6P) was very low at 24 h, and therefore we hypothesized that G6P was limiting. To test this hypothesis, muscle samples from P. major and porcine longissimus lumborum (LL) muscle were homogenized into a reaction buffer that mimics postmortem glycolysis with or without 0.5 mg/mL isolated mitochondria. While samples containing porcine LL muscle reached the normal level of ultimate pH, P. major muscle samples reached a value similar to that observed in vivo even in the presence of excess G6P, indicating that G6P was not limiting. Mitochondria enhanced the glycolytic flux and pH decline in systems containing muscle from both species. More importantly, however, was that in vitro system containing chicken with mitochondria reached pH value similar to that of samples containing LL muscle without mitochondria. To investigate further, phosphofructokinase (PFK) activity was compared in broiler P. major and porcine LL muscle at different pH values. PFK activity was lower in P. major muscle at pH 7, 6.5, and 6.2 than LL muscle. In conclusion, carcass chilling can partially contribute to the high ultimate pH of broiler P. major muscle, while low PFK activity and mitochondria content limit the flux through glycolysis.

Climate change and disease have large negative impacts on poultry production, but little is known about the interactions of responses to these stressors in chickens. Fayoumi (heat and disease resistant) and broiler (heat and disease susceptible) chicken lines were stimulated at 22 days of age, using a 2x2x2 factorial design including: breed (Fayoumi or broiler), inflammatory stimulus (lipopolysaccharide (LPS) or saline), and temperature (35°C or 25°C). Transcriptional changes in spleens were analyzed using RNA-sequencing on the Illumina HiSeq 2500. Thirty-two individual cDNA libraries were sequenced (four per treatment) and an average of 22 million reads were generated per library. Stimulation with LPS induced more differentially expressed genes (DEG, log2 fold change ≥ 2 and FDR ≤ 0.05) in the broiler (N = 283) than the Fayoumi (N = 85), whereas heat treatment resulted in fewer DEG in broiler (N = 22) compared to Fayoumi (N = 107). The double stimulus of LPS+heat induced the largest numbers of changes in gene expression, for which broiler had 567 DEG and Fayoumi had 1471 DEG of which 399 were shared between breeds. Further analysis of DEG revealed pathways impacted by these stressors such as Remodelling of Epithelial Adherens Junctions due to heat stress, Granulocyte Adhesion and Diapedesis due to LPS, and Hepatic Fibrosis/Hepatic Stellate Cell Activation due to LPS+heat. The genes and pathways identified provide deeper understanding of the response to the applied stressors and may serve as biomarkers for genetic selection for heat and disease tolerant chickens.

Energy is an expensive component of poultry diets with lipids providing a concentrated energy source to meet these needs. Three separate experiments with broilers (15 d of age), turkeys (15 d of age) and laying hens (60 wk of age) were conducted to determine the AMEn content of 10 lipids with varying fatty acid and free fatty acid concentrations and to compare these values to predicted values based on previously published equations. A corn-soybean meal (broilers and turkeys) or corn-soybean meal-distillers dried grains with solubles (layers) control diet was formulated with test diets created by mixing the control diet with 0, 3, 6, or 9% (broilers and turkeys) or 0, 2, 4, and 6% (laying hens) lipid. Experimental diets were fed over a 5-d acclimation period followed by a 48 h total excretion collection period with AMEn of the diets calculated based upon the GE, nitrogen, and titanium dioxide in the feed and excreta samples. The pen-mean AMEn of each diet was regressed on percentage lipid inclusion level using linear regression with the slope of the line representing the AMEn of each lipid source. As expected, the AMEn values varied widely among lipid sources and species, with broilers having a greater AME compared to turkeys and layers. In general, saturated fatty acids (C14: 0, C16: 0, and C18: 0) were negatively correlated to AMEn while unsaturated fatty acids (namely C18: 2) were positively correlated to AMEn. Consequently, the unsaturated: saturated ratio, the polyunsaturated fatty acids: saturated and monounsaturated fatty acids ratio, and iodine value tended to be positively related to AMEn. Compared to broilers and turkeys that were able to efficiently utilize energy from corn oil (purified and crude DDGS corn oil) relative to soybean oil, laying hen AMEn values for corn oils were reduced in comparison to soybean oil. These data indicate that energy values of lipid differ widely due to source and appear to differ relative to a particular species.

Coccidiosis is a costly parasitic disease to the poultry industry with multiple control methods being explored to reduce its effect . This study evaluated the effects of a direct-fed microbial (DFM) provided as a dietary supplement over a 4-wk period on responses of broilers to a coccidiosis challenge. In total, 1,056 day-old male chicks were randomly assigned to 8 replicate pens (22 birds/pen) of 6 treatments in a 3 × 2 factorial arrangement including 3 DFM levels (zero, low, high) with or without a 10× coccidiosis vaccine as a challenge on d 15. Performance was assessed weekly, coccidian pathology at d 21, and body composition at d 28 using dual energy x-ray absorptiometry (DXA). Data were analyzed using 2-way ANOVA with LS Means to separate means with significance assigned at P ≤ 0.05. Lesion scores were significantly reduced in the duodenum of the DFM-low birds and jejunum of DFM-high birds compared with other groups, no lesion differences were observed in the ileum and ceca. As expected, the coccidiosis challenge main effect resulted in higher FCR and lower BWG and FI over the 28-d trial . Dietary treatment had little effect on overall performance; however, those birds on the DFM-low diet had lower fat and higher lean percentages than those of other groups without the challenge . Also, 28-d DXA analysis revealed that body fat and lean tissue were reduced in the challenged groups, a potential indication of differential resource allocation to the defense response to an enteric infection rather to performance . Further investigation of immunity and microbial profiling may provide additional clues of the mechanistic functions of this DFM .

A direct fed microbial was evaluated using a broiler avian pathogenic Escherichia coli (APEC) challenge . The product is a single strain of Lactobacillus plantarum (GB-LP1) . In total, 432 d-old male broiler chicks were randomly assigned to 8 replicates of 9 birds for 6 treatments. Treatments included a non-challenged positive control (PC), a challenged non-treated negative control (NC), challenged with an antibiotic (BMD), and challenged with 0.05, 0.10, or 0.20% GB-LP1. Chicks were housed in raised-wire cages in an environmentally controlled room for the 28-d period with ad libitum access to feed and water. Challenge protocol included oral gavage with 108 cfu of APEC in 0 .5 mL on d 7 . Mortality were recorded daily and all dead birds were necropsied. Feed intake and body weight were recorded to calculate mortality corrected feed conversion ratio (FCR). On d 14, 3 birds/pen were euthanized to determine spleen weight (SW). Data were analyzed using one-way ANOVA with protected LS means to separate means with significance assigned at P = 0.05. Body weight was not affected by treatment, but APEC challenge increased FCR over the PC (P = 0.05), with inclusion of BMD returning FCR to PC values . GB-LP1 treatment did not improve FCR and the 0.10% GB-LP1 resulted in FCR that was higher than the NC (P = 0.05). There were 7 mortality attributed to APEC in the infected non-treated NC and no mortalities in the non-infected PC birds. Treatment of birds with BMD, 0.05 and 0.20% GB-LP1 resulted in moderate reductions in mortality (5 each), but the 0.10% GB-LP1 resulted in only 2 mortality attributed to APEC challenge . At D14, SW (% of body weight) was increased by APEC (P = 0.05) with the BMD and 0.05 and 0.20% GB-LP1 returning SW to an intermediate weight. Interestingly, the 0.10% GB-LP1 resulted in the highest spleen weight at 14 d. In conclusion, these data suggest that 0.10% GB-LP1 might be upregulating the immune response (increased spleen weight and FCR) of birds resulting in increased survivability when challenged with APEC.

BACKGROUND:Heat stress in poultry results in considerable economic losses and is a concern for both animal health and welfare. Physiological changes occur during periods of heat stress, including changes in blood chemistry components. A highly advanced intercross line, created from a broiler (heat susceptible) by Fayoumi (heat resistant) cross, was exposed to daily heat cycles for seven days starting at 22 days of age. Blood components measured pre-heat treatment and on the seventh day of heat treatment included pH, pCO2, pO2, base excess, HCO3, TCO2, K, Na, ionized Ca, hematocrit, hemoglobin, sO2, and glucose. A genome-wide association study (GWAS) for these traits and their calculated changes was conducted to identify quantitative trait loci (QTL) using a 600 K SNP panel.RESULTS:There were significant increases in pH, base excess, HCO3, TCO2, ionized Ca, hematocrit, hemoglobin, and sO2, and significant decreases in pCO2 and glucose after 7 days of heat treatment. Heritabilities ranged from 0.01-0.21 for pre-heat measurements, 0.01-0.23 for measurements taken during heat, and 0.00-0.10 for the calculated change due to heat treatment. All blood components were highly correlated within measurement days, but not correlated between measurement days. The GWAS revealed 61 QTL for all traits, located on GGA (Gallus gallus chromosome) 1, 3, 6, 9, 10, 12-14, 17, 18, 21-28, and Z. A functional analysis of the genes in these QTL regions identified the Angiopoietin pathway as significant. The QTL that co-localized for three or more traits were on GGA10, 22, 26, 28, and Z and revealed candidate genes for birds' response to heat stress.CONCLUSIONS:The results of this study contribute to our knowledge of levels and heritabilities of several blood components of chickens under thermoneutral and heat stress conditions. Most components responded to heat treatment. Mapped QTL may serve as markers for genomic selection to enhance heat tolerance in poultry. The Angiopoietin pathway is likely involved in the response to heat stress in chickens. Several candidate genes were identified, giving additional insight into potential mechanisms of physiologic response to high ambient temperatures.

Agriculture provides excellent model systems for understanding how selective pressure, as applied by humans, can affect the genomes of plants and animals. One such system is modern poultry breeding in which intensive genetic selection has been applied for meat production in the domesticated chicken. As a result, modern meat-type chickens (broilers) exhibit enhanced growth, especially of the skeletal muscle, relative to their legacy counterparts. Comparative studies of modern and legacy broiler chickens provide an opportunity to identify genes and pathways affected by this human-directed evolution. This study used RNA-seq to compare the transcriptomes of a modern and a legacy broiler line to identify differentially enriched genes in the breast muscle at days 6 and 21 post-hatch. Among the 15,945 genes analyzed, 10,841 were expressed at greater than 0.1 RPKM. At day 6 post-hatch 189 genes, including several regulators of myogenic growth and development, were differentially enriched between the two lines. The transcriptional profiles between lines at day 21 post-hatch identify 193 genes differentially enriched and still include genes associated with myogenic growth. This study identified differentially enriched genes that regulate myogenic growth and differentiation between the modern and legacy broiler lines. Specifically, differences in the ratios of several positive (IGF1, IGF1R, WFIKKN2) and negative (MSTN, ACE) myogenic growth regulators may help explain the differences underlying the enhanced growth characteristics of the modern broilers.

Transcriptome analysis by RNA-seq has emerged as a high-throughput, cost-effective means to evaluate the expression pattern of genes in organisms. Unlike other methods, such as microarrays or quantitative PCR, RNA-seq is a target free method that permits analysis of essentially any RNA that can be amplified from a cell or tissue. At its most basic, RNA-seq can determine individual gene expression levels by counting the number of times a particular transcript was found in the sequence data. Transcript levels can be compared across multiple samples to identify differentially expressed genes and infer differences in biological states between the samples. We have used this approach to examine gene expression patterns in chicken and human cells, with particular interest in determining response to heat stress.