Physiological responses to heat stress in two genetically distinct chicken inbred lines

Physiological responses to heat stress in two genetically distinct chicken inbred lines Abstract High ambient temperature is one of the most important environmental factors negatively impacting poultry production and health. Genetics is an important contributor in mitigating the stress response to heat. Two genetically distinct highly inbred lines of similar body size (Leghorn and Fayoumi) were characterized for phenotypic differences in response to heat. At 14 days of age, birds were exposed to 38°C with 50% humidity for 4 hours, then 35°C until the conclusion of the experiment. Non-treated individuals were kept at 29.4°C for the first week and then 25°C throughout the experiment. Birds in the heat-stress group were inoculated at day (d) 21 with Newcastle disease virus (NDV) La Sota strain to investigate the effects of heat stress and NDV infection. Thirteen blood parameters were measured using the iSTAT blood analyzer at three stages: 4 h, 6 d, and 9 d post heat-stress treatment, representing acute heat (AH) exposure, chronic heat (CH1) exposure, and chronic heat exposure after virus infection (CH2), respectively. Most blood parameters were significantly changed with heat stress in Leghorns at AH and in Fayoumis at CH1 and CH2. The Leghorn line had significant acute responses with disrupted acid-base balance and metabolic disorders. The heat-resilient Fayoumis maintained a relatively well-balanced acid-base balance. The current study provides the comprehensive profile of biomarker signatures in blood associated with heat tolerance and suggests that PO2, TCO2, HCO3, and base excess can be served as potential biomarkers that can be used to genetically improve heat tolerance in poultry. INTRODUCTION Similar to other homoeothermic animals, chickens maintain a constant body temperature over a wide range of ambient temperatures (Deeb and Cahaner, 1999). However, with the global climate change, poultry production is increasingly impacted by high ambient temperatures, especially in developing countries where poultry are mainly reared in environments without temperature control. Once ambient temperatures exceed the tolerable range, birds have difficulties with maintaining their homeostatic body temperature, which is classified as “heat stress” (Lara and Rostagno, 2013). Heat stress results in physiological changes, and subsequent negative effects on production performance in poultry. Heat stress is one of the most important environmentally influenced factors that can negatively affect poultry production and health (Ayo et al., 2011). With heat stress, laying hens typically suffer from significant reductions in production traits such as egg weight, shell thickness, and rate of production (Wolfenson et al., 2001). Fast-growing broilers maintain higher internal body temperatures than layers, and high mortality rates, low growth rate, and reduced meat quality and body weight have been observed under heat stress (Muiruri and Harrison, 1991). Heat stress also affects the immune response in poultry. Immunosuppression has been observed in both broilers and layers during exposure to a heat stress environment (Padgett and Glaser, 2003). Reduced lymphoid organ weight and lower levels of circulating antibodies, including specific IgM and IgY, were detected in heat-stressed birds compared to non-treated birds (Felver-Gant, 2012). Further, antibody response and phagocytosis of macrophages were repressed in chickens under heat stress (Niu et al., 2009). Heat stress also affects physiological parameters including chemistry and blood gas parameters in animals and the responses vary depending on the time and degree of thermal challenge (Boddicker et al., 2014). Obtaining accurate and reliable measurements of these parameters used to present a great challenge in animal farming until the recent development of a point of care, portable blood analyzer, i.e., the i-STAT (Abbott Laboratories, San Diego, CA) handheld blood analyzer (Martin et al., 2010). Physiological parameters associated with host response to heat stress can be immediately measured, minimizing alterations in the blood-gas and electrolyte values as a result of sample handling and transportation (Kaneko et al., 2008). The i-STAT has been used to assess blood clinical chemistries in chickens and reference ranges have been established for broiler breeders (Martin et al., 2010). Depending on the purpose, different i-STAT cartridges can be selected. In our study, the CG8+ cartridge was used to evaluate heat-stress-related parameters in blood. Genetics plays a significant role in affecting host response to heat stress in poultry (Felver-Gant, 2012). The divergent selection in Leghorns has resulted in chicken lines with different levels of heat tolerance (Wilson et al., 1975). In addition, fast-growing broilers were more sensitive to heat stress than slow-growing broiler strains (Yunis and Cahaner, 1999). Genetic resistance to high ambient temperatures in chickens can be inherited from their ancestors (Lu et al., 2007). Indigenous chicken breeds in tropical areas have been documented to have higher heat stress tolerance compared to other breeds (Soleimani and Zulkifli, 2010). Two genetically distinct highly inbred chicken lines (Leghorn GB2 and Fayoumi M43) maintained at Iowa State University, whose inbreeding coefficients were more than 99.99% (Zhou and Lamont, 1999), were used in the current study. Thus, genetic variations within the line are negligible. The Fayoumis were originally imported from Egypt and they had undergone natural selection for heat tolerance (Lamont et al., 2014). Based on previous reports, Fayoumi birds are more resistant to both disease and heat stress (Bjorkquist et al., 2015; Wang et al., 2014). A broiler (heat-susceptible) × Fayoumi (heat-resistant) advanced intercross line (AIL) was used to fine-map quantitative trait loci (QTL) using the 600 K Affymetrix chicken single nucleotide polymorphism (SNP) array to investigate genetic influence of host response to heat stress in chickens (Van Goor et al., 2015). Several QTL regions had been identified including one major QTL for breast muscle yield under heat stress which accounted for more than 24% of the genetic variation in this study. Thirty-five QTLs were identified to be associated with the response to heat stress. In addition, association of physiological changes on blood components and genomic regions were identified on the same chicken AIL line, which may serve as markers for genetic selection to enhance heat tolerance in poultry (Van Goor et al., 2016). Most recently, the Fayoumi and broiler breeds were also used to study host transcriptome response to heat and Lipopolysaccharide (LPS) stimulation using RNA-seq (Van Goor et al., 2017) and genes and signal pathways associated with heat stress and LPS identified can be served as additional biomarkers for heat tolerance. Based on these studies, some heat tolerant favorable alleles might be fixed in Fayoumi breed, which can be utilized for genetic selection. We hypothesize that Leghorns are relatively heat susceptible while Fayoumis are more heat tolerant. The current study is a part of a US Agency for International Development sponsored program (Feed the Future Innovation Lab for Genomics to Improve Poultry) to improve food security in Africa by enhancing resistance to Newcastle disease and heat stress in chickens (http//:gip.ucdavis.edu). Our major focus in the current study was to characterize physiological responses to heat stress in these two diverse highly inbred lines and develop candidate diagnostic biomarkers for potential genetic selection to improve heat tolerance in poultry. MATERIALS AND METHODS Experimental Populations One hundred and eleven birds, 55 Leghorns and 56 Fayoumis, were housed in two temperature and humidity-controlled isolators, and birds were provided with ad libitum access to food and water. On day (d) 1 of age, 30 Leghorn and 31 Fayoumi birds were randomly chosen as the treatment groups and housed in one isolator and the rest of birds were used as the non-treated groups in another isolator. The two lines were mixed in each isolator. From 14 days of age to the end of the experiment (41 days of age), the heat-treated groups were exposed to continuous heat stress of 38°C for the first 4 hours, and then decreased to 35°C, while the non-treated groups were maintained at 29.4°C for the first week and 25°C throughout the whole experiment. On d 21, birds in the heat-treated groups were inoculated with 107 EID50 Newcastle Disease virus (NDV) La Sota strain through both ocular and nasal passages (50 μL per nostril and eye). The animal experiment was performed according to the guidelines approved by the Institutional Animal Care and Use Committee, University of California, Davis (IACUC #17853). Blood Parameters Measurements Blood gases and chemistry parameters were measured in all birds at d14 (4 hour post-heat-stress treatment, acute phase (AH)), d20 (6 days post-treatment, chronic phase 1 (CH1)) and d23 (9 days post-treatment, chronic phase 2 (CH2)), respectively. Meanwhile, six birds per line per group were euthanized for tissue collection (RNA-seq analysis on separate study) at d14 and d23. Approximately 1 mL of blood was collected into a heparinized syringe (2,000 unit/mL heparin in PBS, Sigma-Aldrich, St. Louis, MO) for each bird, and then blood samples were analyzed immediately using an i-STAT Portable Blood Analyzer. The i-STAT cartridge (CG8+) measured 13 different parameters including four chemistry/electrolyte parameters (concentrations of sodium (Na+), potassium (K+), ionized calcium (Ca2+), and glucose (Glu)); seven blood gas parameters (blood pH, carbon dioxide partial pressure (PCO2), oxygen partial pressure (PO2), total carbon dioxide (TCO2), bicarbonate (HCO3), BE, and oxygen situation (sO2)), and two hematologic parameters: hematocrit (Hct or packed cell volume (PCV)), and hemoglobin (Hb). TCO2 measures the total carbon dioxide and is calculated from pH and PCO2. Measurement of TCO2 is useful to assess HCO3 concentration (Ungerer et al., 1990). Base excess (BE) can be estimated from HCO3 and pH. Statistical Analysis The i-STAT parameters were analyzed using the following linear model for each time point separately in JMP 13 software (SAS Institute, 2016), since population size continued to decrease due to tissue collections at each time point.   \begin{equation*}Y = \mu + T + L + T \times L + e\end{equation*} Where Y is the dependent variable of various parameters, μ is the population mean of the measurements, T is the fixed effect associated with the heat stress treatment, L is the fixed effect associated with the genetic line, T × L is the interaction effect of treatment, and line and e is the random error effect. All measurements in this study were represented as least square mean ±SE. P < 0.05 was considered statistically significant. All figures were generated by Prism 6 GraphPad software (La Jolla, CA). RESULTS Blood Parameter Measurements Lease square means and P values for each factor were listed in Table 1 for all blood parameters of both genetic lines at each time point. Physiological responses to heat stress between the two genetic lines and within each genetic line were compared based on the blood parameter measurements. At the AH stage, six (K+, iCa2+, pH, PCO2, PO2, and sO2) out of thirteen parameters had significant T × L interactions (P < 0.05). For the rest seven parameters, all main genetic line effects were significant except Glu. While only two parameters (Na+ and BE) were significant on main treatment effects. At the CH1 stage, seven (K+, Glu, PCO2, TCO2, HCO3, PO2, and BE) out of thirteen parameters had significant T × L interactions (P < 0.05). Significant main treatment effects were identified on four parameters (pH, sO2, Hct, and Hb) and no main genetic line effects were observed. At the CH2 stage with 2 days post inoculation (DPI) NDV inoculation, five parameters (Glu, TCO2, HCO3, PO2, and sO2) had significant T × L interactions (P < 0.05). The main treatment effect was identified on pH and BE; and there were three parameters (Na+, pH, and BE) had main genetic line effects. For blood parameters having significant T × L effects, line effects in treated and non-treated groups and treatment effects within genetic lines were further analyzed. Table 1. Lease square means and P values of blood parameters with Heat stress treatment.   LS mean ± SE  P values    Blood parameter  LC  LT  FC  FT  Trt  Line  Line*Trt  Acute Heat  Na+ (mmol/L)  142.21 ± 0.31A  140.8 ± 0.27B  142.78 ± 0.31A  142.48 ± 0.28A  0.0045  0.0002  0.0619  K+ (mmol/L)  4.90 ± 0.13A  4.31 ± 0.11C  4.52 ± 0.13BC  4.66 ± 0.12AB  0.0702  0.90  0.0036  iCa2+ (mmol/L)  0.89 ± 0.05B  1.09 ± 0.04A  1.03 ± 0.05A  1.03 ± 0.04A  0.0347  0.3557  0.0270  Glu (mg/dL)  218.96 ± 6.08B  238.50 ± 5.44A  222.61 ± 6.22AB  225.38 ± 5.54AB  0.0585  0.4185  0.1534  pH  7.47 ± 0.015B  7.49 ± 0.017B  7.48 ± 0.017B  7.59 ± 0.015A  0.0002  0.0035  0.0047  PCO2 (mmHg)  25.55 ± 1.02BC  26.70 ± 0.93B  30.35 ± 1.04A  23.46 ± 0.93C  0.0043  0.4272  0.0001  TCO2(mmol/L)  19.50 ± 0.65C  21.21 ± 0.59BC  23.26 ± 0.66A  22.62 ± 0.59AB  0.3941  0.0001  0.0626  HCO (mmol/L)  18.68 ± 0.64C  20.46 ± 0.58B  22.30 ± 0.65A  21.95 ± 0.58AB  0.2450  0.0001  0.0842  PO2 (mmHg)  61.38 ± 1.38A  55.37 ± 1.24B  55.83 ± 1.14B  56.45 ± 1.26B  0.0447  0.0948  0.0139  sO2 (%)  92.54 ± 0.73AB  90.21 ± 0.66C  90.57 ± 0.75BC  93.21 ± 0.66A  0.8273  0.4675  0.0006  BE (mmol/L)  −4.88 ± 0.77C  −2.90 ± 0.70BC  −1.17 ± 0.78AB  0.21 ± 0.70A  0.0250  0.0001  0.6866  Hct (%PCV)  16.77 ± 0.69AB  18.45 ± 0.61A  16.39 ± 0.68B  15.90 ± 0.61B  0.3640  0.0257  0.0969  Hb (g/dL)  5.70 ± 0.24AB  6.28 ± 0.21A  5.56 ± 0.23B  5.40 ± 0.21B  0.3378  0.0218  0.0968  Chronic Heat 1  Na+ (mmol/L)  142.78 ± 0.38B  143.18 ± 0.39AB  143.53 ± 0.42AB  143.79 ± 0.33A  0.3925  0.0770  0.8547  K+ (mmol/L)  4.90 ± 0.11A  4.58 ± 0.11BC  4.43 ± 0.12C  4.83 ± 0.09AB  0.7019  0.3176  0.0013  iCa2+ (mmol/L)  0.88 ± 0.06A  0.86 ± 0.06A  1.00 ± 0.06A  0.91 ± 0.05A  0.3240  0.1301  0.5635  Glu (mg/dL)  201.44 ± 6.92A  215.18 ± 7.12A  216.20 ± 7.58A  201.00 ± 5.99A  0.9159  0.9667  0.0403  pH  7.41 ± 0.02B  7.47 ± 0.02AB  7.44 ± 0.02B  7.50 ± 0.02A  0.0076  0.1313  0.9783  PCO2 (mmHg)  26.93 ± 1.26B  24.24 ± 1.29BC  31.34 ± 1.43A  23.35 ± 1.09C  0.0001  0.1725  0.0410  TCO2(mmol/L)  17.78 ± 0.71B  18.24 ± 0.73B  22.14 ± 0.81A  19.17 ± 0.62B  0.0858  0.0005  0.0203  HCO (mmol/L)  16.98 ± 0.70B  17.52 ± 0.73B  21.26 ± 0.80A  18.30 ± 0.61B  0.0943  0.0007  0.0167  PO2 (mmHg)  59.17 ± 1.90A  59.29 ± 1.95A  51.07 ± 2.08B  60.17 ± 1.64A  0.0176  0.0610  0.0208  sO2 (%)  89.61 ± 0.92B  92.35 ± 0.95A  87.14 ± 1.04B  92.63 ± 0.80A  0.0001  0.2127  0.1460  BE (mmol/L)  −7.67 ± 0.86C  −6.12 ± 0.88BC  −2.93 ± 0.97A  −4.83 ± 0.74AB  0.8377  0.0009  0.0499  Hct (%PCV)  21.00 ± 0.71A  16.63 ± 0.74B  20.53 ± 0.76A  17.13 ± 0.60B  0.0000  0.9812  0.4958  Hb (g/dL)  7.14 ± 0.24A  5.66 ± 0.25B  6.99 ± 0.26A  5.85 ± 0.21B  0.0000  0.9176  0.4756  Chronic Heat 2 (+ NDV 2 DPI)  Na+ (mmol/L)  143.05 ± 0.31B  142.87 ± 0.29B  144.06 ± 0.32A  144.21 ± 0.28A  0.9601  0.0021  0.5791  K+ (mmol/L)  4.72 ± 0.11A  4.93 ± 0.10A  4.93 ± 0.11A  4.94 ± 0.10A  0.2829  0.3139  0.3266  iCa2+ (mmol/L)  0.84 ± 0.05B  0.98 ± 0.04A  0.95 ± 0.05AB  0.95 ± 0.04AB  0.1403  0.4094  0.1395  Glu (mg/dL)  184.58 ± 5.91C  220.09 ± 5.37A  197.06 ± 6.07BC  200.71 ± 5.26B  0.0009  0.5440  0.0062  pH  7.42 ± 0.02B  7.37 ± 0.01C  7.49 ± 0.02A  7.45 ± 0.01B  0.0025  0.0000  0.9765  PCO2 (mmHg)  27.04 ± 1.09AB  28.47 ± 0.99A  26.85 ± 1.12AB  25.22 ± 0.97B  0.9265  0.1033  0.1471  TCO2(mmol/L)  18.16 ± 0.61B  17.39 ± 0.56B  21.39 ± 0.63A  18.08 ± 0.55B  0.0009  0.0013  0.0338  HCO (mmol/L)  17.36 ± 0.59B  16.56 ± 0.53B  20.47 ± 0.60A  17.16 ± 0.52B  0.0005  0.0015  0.0295  PO2 (mmHg)  57.89 ± 1.37A  54.43 ± 1.24AB  51.11 ± 1.41B  54.88 ± 1.22A  0.9081  0.0180  0.0073  sO2 (%)  89.63 ± 0.90A  87.00 ± 0.82B  88.83 ± 0.92AB  89.75 ± 0.80A  0.3224  0.2605  0.0426  BE (mmol/L)  −7.16 ± 0.70BC  −8.74 ± 0.64C  −2.94 ± 0.72A  −6.79 ± 0.62B  0.0001  0.0000  0.0954  Hct (%PCV)  17.06 ± 0.64A  16.17 ± 0.56A  16.44 ± 0.64A  17.17 ± 0.55A  0.8943  0.7505  0.1838  Hb (g/dL)  5.89 ± 0.21A  5.49 ± 0.19A  5.59 ± 0.21A  5.81 ± 0.18A  0.6512  0.9686  0.1171    LS mean ± SE  P values    Blood parameter  LC  LT  FC  FT  Trt  Line  Line*Trt  Acute Heat  Na+ (mmol/L)  142.21 ± 0.31A  140.8 ± 0.27B  142.78 ± 0.31A  142.48 ± 0.28A  0.0045  0.0002  0.0619  K+ (mmol/L)  4.90 ± 0.13A  4.31 ± 0.11C  4.52 ± 0.13BC  4.66 ± 0.12AB  0.0702  0.90  0.0036  iCa2+ (mmol/L)  0.89 ± 0.05B  1.09 ± 0.04A  1.03 ± 0.05A  1.03 ± 0.04A  0.0347  0.3557  0.0270  Glu (mg/dL)  218.96 ± 6.08B  238.50 ± 5.44A  222.61 ± 6.22AB  225.38 ± 5.54AB  0.0585  0.4185  0.1534  pH  7.47 ± 0.015B  7.49 ± 0.017B  7.48 ± 0.017B  7.59 ± 0.015A  0.0002  0.0035  0.0047  PCO2 (mmHg)  25.55 ± 1.02BC  26.70 ± 0.93B  30.35 ± 1.04A  23.46 ± 0.93C  0.0043  0.4272  0.0001  TCO2(mmol/L)  19.50 ± 0.65C  21.21 ± 0.59BC  23.26 ± 0.66A  22.62 ± 0.59AB  0.3941  0.0001  0.0626  HCO (mmol/L)  18.68 ± 0.64C  20.46 ± 0.58B  22.30 ± 0.65A  21.95 ± 0.58AB  0.2450  0.0001  0.0842  PO2 (mmHg)  61.38 ± 1.38A  55.37 ± 1.24B  55.83 ± 1.14B  56.45 ± 1.26B  0.0447  0.0948  0.0139  sO2 (%)  92.54 ± 0.73AB  90.21 ± 0.66C  90.57 ± 0.75BC  93.21 ± 0.66A  0.8273  0.4675  0.0006  BE (mmol/L)  −4.88 ± 0.77C  −2.90 ± 0.70BC  −1.17 ± 0.78AB  0.21 ± 0.70A  0.0250  0.0001  0.6866  Hct (%PCV)  16.77 ± 0.69AB  18.45 ± 0.61A  16.39 ± 0.68B  15.90 ± 0.61B  0.3640  0.0257  0.0969  Hb (g/dL)  5.70 ± 0.24AB  6.28 ± 0.21A  5.56 ± 0.23B  5.40 ± 0.21B  0.3378  0.0218  0.0968  Chronic Heat 1  Na+ (mmol/L)  142.78 ± 0.38B  143.18 ± 0.39AB  143.53 ± 0.42AB  143.79 ± 0.33A  0.3925  0.0770  0.8547  K+ (mmol/L)  4.90 ± 0.11A  4.58 ± 0.11BC  4.43 ± 0.12C  4.83 ± 0.09AB  0.7019  0.3176  0.0013  iCa2+ (mmol/L)  0.88 ± 0.06A  0.86 ± 0.06A  1.00 ± 0.06A  0.91 ± 0.05A  0.3240  0.1301  0.5635  Glu (mg/dL)  201.44 ± 6.92A  215.18 ± 7.12A  216.20 ± 7.58A  201.00 ± 5.99A  0.9159  0.9667  0.0403  pH  7.41 ± 0.02B  7.47 ± 0.02AB  7.44 ± 0.02B  7.50 ± 0.02A  0.0076  0.1313  0.9783  PCO2 (mmHg)  26.93 ± 1.26B  24.24 ± 1.29BC  31.34 ± 1.43A  23.35 ± 1.09C  0.0001  0.1725  0.0410  TCO2(mmol/L)  17.78 ± 0.71B  18.24 ± 0.73B  22.14 ± 0.81A  19.17 ± 0.62B  0.0858  0.0005  0.0203  HCO (mmol/L)  16.98 ± 0.70B  17.52 ± 0.73B  21.26 ± 0.80A  18.30 ± 0.61B  0.0943  0.0007  0.0167  PO2 (mmHg)  59.17 ± 1.90A  59.29 ± 1.95A  51.07 ± 2.08B  60.17 ± 1.64A  0.0176  0.0610  0.0208  sO2 (%)  89.61 ± 0.92B  92.35 ± 0.95A  87.14 ± 1.04B  92.63 ± 0.80A  0.0001  0.2127  0.1460  BE (mmol/L)  −7.67 ± 0.86C  −6.12 ± 0.88BC  −2.93 ± 0.97A  −4.83 ± 0.74AB  0.8377  0.0009  0.0499  Hct (%PCV)  21.00 ± 0.71A  16.63 ± 0.74B  20.53 ± 0.76A  17.13 ± 0.60B  0.0000  0.9812  0.4958  Hb (g/dL)  7.14 ± 0.24A  5.66 ± 0.25B  6.99 ± 0.26A  5.85 ± 0.21B  0.0000  0.9176  0.4756  Chronic Heat 2 (+ NDV 2 DPI)  Na+ (mmol/L)  143.05 ± 0.31B  142.87 ± 0.29B  144.06 ± 0.32A  144.21 ± 0.28A  0.9601  0.0021  0.5791  K+ (mmol/L)  4.72 ± 0.11A  4.93 ± 0.10A  4.93 ± 0.11A  4.94 ± 0.10A  0.2829  0.3139  0.3266  iCa2+ (mmol/L)  0.84 ± 0.05B  0.98 ± 0.04A  0.95 ± 0.05AB  0.95 ± 0.04AB  0.1403  0.4094  0.1395  Glu (mg/dL)  184.58 ± 5.91C  220.09 ± 5.37A  197.06 ± 6.07BC  200.71 ± 5.26B  0.0009  0.5440  0.0062  pH  7.42 ± 0.02B  7.37 ± 0.01C  7.49 ± 0.02A  7.45 ± 0.01B  0.0025  0.0000  0.9765  PCO2 (mmHg)  27.04 ± 1.09AB  28.47 ± 0.99A  26.85 ± 1.12AB  25.22 ± 0.97B  0.9265  0.1033  0.1471  TCO2(mmol/L)  18.16 ± 0.61B  17.39 ± 0.56B  21.39 ± 0.63A  18.08 ± 0.55B  0.0009  0.0013  0.0338  HCO (mmol/L)  17.36 ± 0.59B  16.56 ± 0.53B  20.47 ± 0.60A  17.16 ± 0.52B  0.0005  0.0015  0.0295  PO2 (mmHg)  57.89 ± 1.37A  54.43 ± 1.24AB  51.11 ± 1.41B  54.88 ± 1.22A  0.9081  0.0180  0.0073  sO2 (%)  89.63 ± 0.90A  87.00 ± 0.82B  88.83 ± 0.92AB  89.75 ± 0.80A  0.3224  0.2605  0.0426  BE (mmol/L)  −7.16 ± 0.70BC  −8.74 ± 0.64C  −2.94 ± 0.72A  −6.79 ± 0.62B  0.0001  0.0000  0.0954  Hct (%PCV)  17.06 ± 0.64A  16.17 ± 0.56A  16.44 ± 0.64A  17.17 ± 0.55A  0.8943  0.7505  0.1838  Hb (g/dL)  5.89 ± 0.21A  5.49 ± 0.19A  5.59 ± 0.21A  5.81 ± 0.18A  0.6512  0.9686  0.1171  Note: LC: Leghorn non-treated group; LT: Leghorn treated group; FC: Fayoumi non-treated group; FT: Fayoumi treated group; Trt: treatment effects; Line: line effects; Line*Trt: line and treatment interaction effects; Different superscript letters within row represent significant differences (P < 0.05). View Large Effects of Heat Stress between Genetic Lines Without heat stress, the two genetic lines generally had 7 out of 13 parameters significantly difference due to their genetic background which were iCa2+, K+, PCO2, TCO2, HCO3, BE, and PO2 on d14 (Figure 1); 6 out of 13 parameters (K+, PCO2, TCO2, HCO3, BE, and PO2) on d 20 (Figure 2); and 6 out of 13 parameters (Na+, pH, TCO2, HCO3, PO2, and BE) on d 23 (Figure 3). Across all time points, Leghorn birds had significantly higher PO2 and lower TCO2, HCO3, and BE than Fayoumi birds. They also had higher K+ and lower levels of PCO2 than Fayoumis on both d14 and d20 and higher pH and lower Na+ levels than Fayoumis on d23. Figure 1. View largeDownload slide Blood parameter differences between non-treated genetic lines at 14 days of age. Blood parameter differences between non-treated genetic lines at day 14. *indicated significant pairwise difference between lines (P < 0.05) **; indicated P < 0.01, and ***indicated P < 0.001. Figure 1. View largeDownload slide Blood parameter differences between non-treated genetic lines at 14 days of age. Blood parameter differences between non-treated genetic lines at day 14. *indicated significant pairwise difference between lines (P < 0.05) **; indicated P < 0.01, and ***indicated P < 0.001. Figure 2. View largeDownload slide Blood parameter differences between non-treated genetic lines at 20 days of age. Blood parameter differences between non-treated genetic lines at day 20. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01. Figure 2. View largeDownload slide Blood parameter differences between non-treated genetic lines at 20 days of age. Blood parameter differences between non-treated genetic lines at day 20. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01. Figure 3. View largeDownload slide Blood parameter differences between non-treated genetic lines at 23 days of age. Blood parameter differences between non-treated genetic lines at day 23. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 3. View largeDownload slide Blood parameter differences between non-treated genetic lines at 23 days of age. Blood parameter differences between non-treated genetic lines at day 23. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. With heat treatment, more parameters (8 out of 13) were significantly different between the two genetic lines at the AH stage, which were Na+, K+, pH, PCO2, sO2, BE, Hct, and Hb (Figure 4). One week later at the CH1 stage, there were no differences in parameters between the Fayoumi and Leghorn lines. At the CH2 stage (after NDV infection), Leghorn birds had higher Glu and PCO2 and lower Na, sO2, pH, and sO2 levels than Fayoumis (Figure 5). Figure 4. View largeDownload slide Blood parameter differences between genetic lines at the acute heat stress stage. Blood parameter differences between heat-stress treated genetic lines at the acute heat stage. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 4. View largeDownload slide Blood parameter differences between genetic lines at the acute heat stress stage. Blood parameter differences between heat-stress treated genetic lines at the acute heat stage. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 5. View largeDownload slide Blood parameter differences between genetic lines at the chronic heat stress and NDV infection stage. Blood parameter differences between heat stress and NDV treated genetic lines at the chronic heat 2 stage. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 5. View largeDownload slide Blood parameter differences between genetic lines at the chronic heat stress and NDV infection stage. Blood parameter differences between heat stress and NDV treated genetic lines at the chronic heat 2 stage. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Effects of Heat Stress within Genetic Lines At the AH stage Leghorn birds had more vigorous physiologic responses than Fayoumis with acute heat stress. Comparing with the non-treated groups, there were 7 parameters changed in the Leghorns upon heat stress, which were Na+, K+, iCa2+, Glu, HCO3, PO2, and sO2 (Figure 6); while only 3 parameters (pH, PCO2 and sO2) were significantly changed in Fayoumi birds (Figure 7). Heat stressed Leghorns had significantly lower Na+, K+ and higher iCa2+, Glu than the non-treated birds (Figure 6). There were no differences in blood chemistry/electrolytes in Fayoumi birds at the AH stage (Figure 7). For the five CO2 associated parameters, the HCO3 level in the heat stress Leghorns was the only one increased comparing with non-treated leghorns and there was no change in the rest four parameters (Figure 6). Higher pH and lower PCO2 levels were observed in heat stressed Fayoumis than non-treated ones (Figure 7). For the two oxygen associated parameters PO2 and sO2, Leghorns had significantly lower PO2 and sO2 with heat stress treatment (Figure 6), while heat stressed Fayoumis had higher sO2 levels at AH stage (Figure 7). For the two hematology parameters, Hct and Hb, there were no differences in Hct and Hb levels between heat stressed and non-treated groups of both genetic lines (Figure 6 and 7). Figure 6. View largeDownload slide Phenotypic responses to heat stress at the acute heat stage in the Leghorn line. Blood parameter differences between acute heat stressed and non-treated birds in the Leghorn line. *indicated significant pairwise difference (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 6. View largeDownload slide Phenotypic responses to heat stress at the acute heat stage in the Leghorn line. Blood parameter differences between acute heat stressed and non-treated birds in the Leghorn line. *indicated significant pairwise difference (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 7. View largeDownload slide Phenotypic responses to heat stress at the acute heat stage in the Fayoumi line. Blood parameter differences between acute heat stressed and non-treated birds in the Fayoumi line. **indicated significant pairwise difference (P < 0.01); ***indicated P < 0.001. Figure 7. View largeDownload slide Phenotypic responses to heat stress at the acute heat stage in the Fayoumi line. Blood parameter differences between acute heat stressed and non-treated birds in the Fayoumi line. **indicated significant pairwise difference (P < 0.01); ***indicated P < 0.001. At the CH Stages At the CH1 stage, between heat stressed and non-treated birds, only 4 parameters (K+, sO2, Hct, and Hb) were significantly changed in Leghorn birds (Figure 8), compared to 9 parameters (K+, pH, PCO2, TCO2, HCO3, PO2, sO2, Hct, and Hb) changed responding to heat stress in Fayoumis (Figure 9). At the CH2 stage, which was 2 DPI with NDV, Leghorn birds had significant changes in iCa2+, Glu, pH, and sO2 (Figure 10) and Fayoumi birds had more blood gas parameters changed including pH, TCO2, HCO3 PO2, and BE comparing with non-treated birds (Figure 11). Figure 8. View largeDownload slide Phenotypic responses to heat stress at chronic heat 1 stage in the Leghorn line. Blood parameter differences between chronic heat stressed and non-treated birds in the Leghorn line at the CH1 stage. *indicated significant pairwise difference (P < 0.05); ***indicated P < 0.001. Figure 8. View largeDownload slide Phenotypic responses to heat stress at chronic heat 1 stage in the Leghorn line. Blood parameter differences between chronic heat stressed and non-treated birds in the Leghorn line at the CH1 stage. *indicated significant pairwise difference (P < 0.05); ***indicated P < 0.001. Figure 9. View largeDownload slide Phenotypic responses to heat stress at chronic heat 1 stage in the Fayoumi line. Blood parameter differences between chronic heat stressed and non-treated birds in the Fayoumi line at the CH1 stage. *indicated significant pairwise difference (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 9. View largeDownload slide Phenotypic responses to heat stress at chronic heat 1 stage in the Fayoumi line. Blood parameter differences between chronic heat stressed and non-treated birds in the Fayoumi line at the CH1 stage. *indicated significant pairwise difference (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 10. View largeDownload slide Phenotypic responses to heat stress at chronic heat 2 stage in the Leghorn line. Blood parameter differences between chronic heat stressed and non-treated birds in the Leghorn line at the CH2 stage with NDV infection. *indicated significant pairwise difference (P < 0.05); ***indicated P < 0.001. Figure 10. View largeDownload slide Phenotypic responses to heat stress at chronic heat 2 stage in the Leghorn line. Blood parameter differences between chronic heat stressed and non-treated birds in the Leghorn line at the CH2 stage with NDV infection. *indicated significant pairwise difference (P < 0.05); ***indicated P < 0.001. Figure 11. View largeDownload slide Phenotypic responses to heat stress at chronic heat 2 stage in the Fayoumi line. Blood parameter differences between chronic heat stressed and non-treated birds in the Leghorn line at the CH1 stage with NDV infection. *indicated significant pairwise difference (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 11. View largeDownload slide Phenotypic responses to heat stress at chronic heat 2 stage in the Fayoumi line. Blood parameter differences between chronic heat stressed and non-treated birds in the Leghorn line at the CH1 stage with NDV infection. *indicated significant pairwise difference (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. At the CH1 stage, K+ was lower in the heat stressed Leghorns, while higher in heat stressed Fayoumis compared to non-treated birds (Figures 8 and 9). At the CH2 stage, heat stressed Leghorn birds kept higher levels of iCa2+ and Glu compared to non-treated Leghorns (Figure 10), however, there were no significant changes in parameters between heat stressed and non-treated Fayoumis during this stage (Figure 11). With the exception of a lower pH level at the CH2 stage, CO2 associated blood gas parameters were similar between heat stressed and non-treated Leghorns at both CH1 and CH2 stages (Figures 8 and 10). However, heat stressed Fayoumis kept a higher pH level than non-treated ones at CH1 and the pH level went down to lower than non-treated Fayoumis at CH2 (Figures 9 and 11). Consequently, in Fayoumis, heat stressed birds had significantly lower PCO2, TCO2, and HCO3 levels at the CH1 stage (Figure 9) and much lower levels of TCO2, HCO3, and BE at the CH2 stage (Figure 11). The difference of the oxygen related parameters was only identified on sO2 in Leghorns at both CH1 and CH2 stage, in which the sO2 levels of heat stressed Leghorns were higher than non-treated birds at the CH1 stage (Figure 8) and lower at the CH2 stage (Figure 10). However, heat stressed Fayoumis had higher PO2 and sO2 at the CH1 stage (Figure 9) and higher PO2 at the CH2 stage (Figure 11). The two genetic lines responded similarly at CH stages on hematology parameters. At the CH1 stage, Hct and Hb were significantly lower in heat stressed birds compared to non-treated birds observed in both lines (Figures 8 and 9). There were no differences in hematologic parameters between heat stress and non-treated birds at the CH2 stage for both genetic lines (Figures 10 and 11). DISCUSSION Genetic background plays important roles in heat stress resistance in poultry (Lara and Rostagno, 2013). Different chicken breeds or strains have varied tolerance to heat stress (Deeb and Cahaner, 1999; Yalcin et al., 2001; Cahaner et al., 2008; Islam and Nishibori, 2009). Generally, native or indigenous chicken breeds in the tropical areas have better potential to adapt to changes in ambient temperatures (Soleimani and Zulkifli, 2010). Commercial breeds such as fast growing broilers and high production egg layers are more sensitive to heat stress (Yunis and Cahaner, 1999; Soleimani and Zulkifli, 2010). The Fayoumi birds used in our present study were originally imported from Egypt to the United States in 1954 due to their disease resistance (Deeb and Lamont, 2002) and had evolved with a genetic adaptation to high ambient temperatures (Van Goor et al., 2017). Heat treatment had less impact on Fayoumi birds in a genotype-environment interaction study that revealed significant negative impact of heat on commercial broilers (Essam et al., 2007). Breed, age, management, and nutritional factors have significant influences on blood chemistry measurements in chickens. Previously established reference ranges for blood chemistry parameters by i-STAT in broiler breeder hens (Martin et al., 2010) and in pullet and laying hens (Schaal et al., 2016) suggested that i-STAT can serve as a reliable tool to evaluate metabolic disturbance during the heat stress in chickens. There were physiological differences observed between Fayoumi and Leghorn lines in blood chemistries under the normal ambient temperature at the three data collection time points (Figures 1 to 3), which could be due do genetic differences between the two lines. Heat stress treatment expanded the differences between these two genetic lines at the AH stage (Figure 4), and adapted to heat environment after one week at the CH1 stage without significant differences (data not shown), there were then a number of different parameters with both heat stress and NDV inoculation three days later (Figure 5). We assume that Fayoumi is more resilient to heat stress than the Leghorn line as the Fayoumi originated from Egypt, a hot climate region. Additional study in our group from another experiment in a Hy-Line Brown population with the same experiment design revealed a significantly positive correlation between these blood parameters before the heat treatment and growth rate with heat stress (data not published). Heat treatment resulted in more differences between two genetic lines at the AH stage than the chronic stages, which suggested that these two lines had distinct responses in the course of heat stress. Readouts of blood parameters from Fayoumis could be associated with heat tolerance in chickens. Within each genetic line between the heat treated and non-treated groups, Leghorn birds had much more rapid responses than Fayoumis at the AH Stage (7 incidences vs. 3 incidences). At the CH1 stage, the opposite situation was observed in that Leghorns had less responses than Fayoumi birds (4 vs. 9). At the CH2 stage with NDV infection, a similar number of parameters were significantly changed in the two lines (4 vs. 5). Most of blood parameters were significantly changed in the opposite directions with the heat stress between the two genetic lines or significantly changed in only one line (Table 2). Table 2. Differences in blood parameters within genetic lines between heat stress and non-treated groups.   Heat stress (HS) vs. non-treated (NT)    Fayoumi  Leghorn    AH  CH1  CH2  AH  CH1  CH2  Na+  NS  NS  NS  -  NS  NS  K+  NS  +  NS  -  -  NS  iCa2+  NS  NS  NS  +  NS  +  Glu  NS  NS  NS  +  NS  +  pH  +  +  -  NS  NS  -  PCO2  -  -  NS  NS  NS  NS  TCO2  NS  -  -  NS  NS  NS  HCO3  NS  -  -  +  NS  NS  BE  NS  NS  -  NS  NS  NS  PO2  NS  +  +  -  NS  NS  sO2  +  +  NS  -  +  -  Hct  NS  -  NS  NS  -  NS  Hb  NS  -  NS  NS  -  NS    Heat stress (HS) vs. non-treated (NT)    Fayoumi  Leghorn    AH  CH1  CH2  AH  CH1  CH2  Na+  NS  NS  NS  -  NS  NS  K+  NS  +  NS  -  -  NS  iCa2+  NS  NS  NS  +  NS  +  Glu  NS  NS  NS  +  NS  +  pH  +  +  -  NS  NS  -  PCO2  -  -  NS  NS  NS  NS  TCO2  NS  -  -  NS  NS  NS  HCO3  NS  -  -  +  NS  NS  BE  NS  NS  -  NS  NS  NS  PO2  NS  +  +  -  NS  NS  sO2  +  +  NS  -  +  -  Hct  NS  -  NS  NS  -  NS  Hb  NS  -  NS  NS  -  NS  Note: NS, not significant between heat stress and non-treated groups; -, heat stressed birds lower than the non-treated birds (P < 0.05); +, heat stressed birds higher than the non-treated birds (P < 0.05). View Large As a heat resilient chicken line, we hypothesize that Fayoumi birds are more elastic in regulating their physiological response to adapt to heat stress, as indicated from changes in their measured blood parameters, which allowed them to suffer less from the heat stress and maintain their homeostasis. Details of these parameters related to physiological response to heat stress are discussed as follows. Electrolytes/Chemistries With the heat stress, the balance of blood electrolytes can be disrupted due to large amounts of water intake and disordered homeostasis (Ait-Boulahsen et al., 1995). Many researchers have reported that electrolyte supplements can help birds to alleviate the effects of heat stress (Sahin et al., 2009; Majekodunmi et al., 2013). Three electrolytes, Na+, K+, and iCa2+ were measured in this study. With the heat treatment, significant loss of Na+ and K+ at the AH stage and even less K+ at the CH1 stage were observed in Leghorn birds (Supplementary Figure S1 to S2) in other reports (Olanrewaju et al., 2006; Olanrewaju et al., 2007). On the other hand, within the Fayoumi line, electrolyte levels in the blood were maintained very well at the AH and CH2 stage, except a rise of K+ observed at the CH1 stage in heat stressed Fayoumi (Supplementary Figure S3). During heat stress, losing electrolyte balance is a major problems in chickens (Borges et al., 2004). The capability to maintain electrolyte balance is a key factor in maintaining a higher performance (Gamba et al., 2015). Supplementation with electrolytes such as Na+ and K+ is one of approaches used to alleviate the impact of heat stress on the performance of chickens (Ait-Boulahsen et al., 1995; Ahmad and Sarwar, 2006). Comparing with Leghorn birds, Fayoumis were able to keep a better electrolyte balance during the heat stress treatment. Fayoumi birds were able to raise the K+ level up at the CH1 stage, which could be one of their mechanisms to preserve higher performance and have higher tolerance to heat stress. Three dynamic forms of calcium exist in the blood, calcium bounded to protein, calcium complexed with organic acid, or ionized calcium (iCa2+) (Odom et al., 1986). Free iCa2+ in blood was mostly used for bone mineralization (Sgavioli et al., 2016) or used by the shell gland (Odom et al., 1986). Many studies have reported significant reduction in blood iCa2+ during heat stress (Odom et al., 1986), however a few studies also reported that heat stress did not reduce blood iCa2+ (Samara et al., 1996). In the current study, iCa2+ levels of heat stressed Leghorn birds were significantly elevated in both AH and CH2 stages (Supplementary Figure S4). As Leghorn line was originally composed of US commercial layer, maintaining a higher iCa2+ level could be critical for them to have better performance in their later lives. Meanwhile, CH2 stage was a combination of both heat stress and NDV infection. Calcium levels were able to be increased by hepatitis C virus infection in humans (Paracha et al., 2013). Therefore, the elevation of iCa2+ could also be due to NDV inoculation at CH2 in our study. There were no significant differences in glucose levels between the two genetic lines before exposure to heat. Significant glucose differences between the two genetic lines were found at the CH2 stage with heat exposure and NDV infection, in which Fayoumis had lower glucose levels in blood than Leghorns. With heat stress, Leghorn birds significantly increased their glucose levels compared to non-treated birds (Supplementary Figure S5). Blood glucose concentration may be altered due to various stressors including heat stress (Soleimani et al., 2011). It has been reported that heat stress can result in increase of corticosteroids in the blood plasma, which then subsequently causes an increased blood glucose concentration (Ayo et al., 2011). Heat-stressed animals can use glucose as a fuel by increasing hepatic glucose output through glycogenolysis and gluconeogenesis (Rhoads et al., 2013). Fayoumi birds had lower glucose levels than Leghorns with chronic heat stress in the current study. Therefore, we speculate that Fayoumi birds might depress glycogenolysis and gluconeogenesis metabolisms to “wait out” heat stress, which could be a physiologic mechanism to be resilient to heat stress. Blood Gases There were seven blood gas parameters in which pH, PCO2, TCO2, HCO3, and BE are closely related with each other and are also associated with blood CO2; sO2 is calculated based on PO2 and both of them are associated with blood oxygen (O2). pH, PCO2, TCO2, HCO3, and BE CO2 related parameters are indicators of the acid-base balance in blood. Maintaining a stable acid-base balance is a beneficial responses for chickens to be tolerant to high ambient temperature (Gamba et al., 2015). PCO2 is a major component used to evaluate ventilation and respiratory response of the acid-base status (Ardiaca et al., 2013). During heat-stress treatment, birds with more CO2 loss via the lungs reduced the PCO2 in the blood (Sandercock et al., 2001; Borges et al., 2003; Ahmad and Sarwar, 2006; Yalcin et al., 2008; Angell and Seymour, 2015), subsequently, the decreased PCO2 resulted in increased plasma pH with an alkalosis. TCO2 measures the total carbon dioxide and can be calculated from pH and PCO2. Both TCO2 and HCO3 can be used in the evaluation of the acid-base imbalance (Hamm et al., 2013). As a major metabolic component of the acid-base balance (Ahmad et al., 2008), increased HCO3 also caused metabolic alkalosis (Ahmad and Sarwar, 2006; Angell and Seymour, 2015). Blood BE is calculated by the combination of pH and HCO3, and is one of indicators of non-respiratory metabolic disturbance of the acid-base status (Ardiaca et al., 2013). Based on readouts of PCO2 and pH, Fayoumis had significant respiratory alkalosis at both AH and CH1 stages, but not the CH2 stage (Supplementary Figures S6 to S7), while the Leghorn had no significant changes except a decrease of pH at the CH2 stage. Based on readouts of HCO3 and BE, Fayoumis had significant chronic metabolic acidosis (Supplementary Figures S8 to S9), while Leghorns had significant acute metabolic alkalosis (Supplementary Figures S10). These results were partially consistent (consistent in HCO3 in the chronic phase, and in the opposite direction in PCO2) with a previous heat stress study in which naked neck (heat tolerant) and non-naked neck Rhode Island Red layers were used for evaluating blood gas changes with acute (1 hour) and chronic heat stress (8 wk) (Pech-Waffenschmidt et al., 1995). The inconsistency could be due to different experimental designs and different genetic lines used. In summary, heat tolerant birds (Fayoumi) had phenotypes of early respiratory alkalosis and late metabolic acidosis, while heat susceptible birds (Leghorn) had phenotypes of early metabolic alkalosis. At the CH1 stage, Fayoumi birds were able to lower TCO2 and HCO3 to compensate the respiratory alkalosis. However, this was accomplished at the CH2 stage by having a lower pH and normal PCO2. On the other hand, Leghorn birds had metabolic alkalosis at the AH stage, then stopped responding at the CH1 stage without any changes and with a decrease in the pH at the CH2 stage. NDV inoculation could be a contributing factor to late metabolic acidosis in Fayoumies and pH decrement in Leghorns. To our knowledge, this is the first report in which the dynamic signature of blood gas parameters were used to characterize heat tolerant/susceptible birds. PO2, sO2, and Hematology (Hct and Hb) The other two blood gas parameters PO2 and sO2 are mostly associated with the blood O2 levels. PO2 is a sensor of pulmonary ventilation (Bollen et al., 2005). The sO2 measures the amount of O2 binding to Hb. Fayoumi birds demonstrated much stronger physiological function and elasticity with increased sO2 and PO2 in responding to heat stress than Leghorns. Hct is normally used to determine and monitor the fractional volume of red blood cells (Leypoldt et al., 1995), which is one of the key indicators of the body hydration state as well as the blood's capability to transport oxygen (Fair et al., 2007). One of the heat stress symptoms in poultry is the reduction of blood Hct and Hb concentration, which is a consequence of overhydration due to too much water intake (Hilman P.E., 2000; Puvadolpirod and Thaxton, 2000; Zulkifli et al., 2009). During heat stress, when the temperature difference between birds and environment is decreased, passive temperature reduction (radiation, conduction, and convention) cannot be used for birds to lower core body temperature (Freeman et al., 1999). Therefore, heat loss by hyperventilation is facilitated, which results in an increase of water evaporation through respiration, and drinking additional amounts of water is needed to restore the osmotic balance (Ryder et al., 2004). This subsequently caused reduced Hct and Hb due to hematic dilution (Cristina et al., 2004). Results from our current study were mostly consistent with others by having lower levels of Hct and Hb at the CH1 stage in both genetic lines. Hct and Hb levels were similar between non-treated genetic lines across all time points. However, in the AH stage, heat stressed Leghorn birds had significantly higher Hct and Hb levels than heat stressed Fayoumis (Supplementary Figures S11 to S12). It could because that Leghorn birds slightly increased their Hct (P = 0.072) and Hb (P = 0.062) levels with heat stress due to suffering from dehydration at the AH stage. In summary, the present study provided the comprehensive profile of biomarker signatures in blood associated with heat tolerance in the two genetically distinct chicken highly inbred lines. The Leghorn line had a significant acute response to heat, while the Fayoumi had limited early response, but more significant chronic responses during the acclimation to the stress. Specifically, during the heat stress, Leghorn birds had significant electrolyte disruption, while Fayoumis had well-balanced electrolytes, even with enhanced potassium. In addition, Fayoumis had significant respiratory alkalosis and chronic metabolic acidosis, while Leghorns had significant acute metabolic alkalosis. Collectively, the above results suggested potential distinct acute responses between Fayoumi and Leghorn is responsible for determining resilient birds. Finally, significant basal level differences between Fayoumi and Leghorn in PO2, TCO2, HCO3, and BE provided potential biomarkers that can be used to genetically improve heat tolerance in poultry. The insights generated from this study have laid a solid foundation in understanding the genetic control of heat tolerance and susceptibility in poultry. Further investigation on the underlying molecular and cellular mechanisms is warranted. SUPPLEMENTARY DATA Supplementary data are available at Poultry Science online. ACKNOWLEDGEMENTS The study is supported by US Agency for International Development (USAID) AID-OAA-A-13-00080. This study is made possible by the generous support of the American people through USAID. The contents are the responsibility of the Feed the Future Innovation Lab for Genomics to Improve Poultry and do not necessarily reflect the views of USAID or the United States Government. This project was partially supported by Multistate Research Project NE-1334 (CA-D-ASC-2156-RR), NC1170 (CA-D-ASC-2155-RR), from the USDA Cooperative State Research, Education, and Extension Service. REFERENCES Ahmad T., Khalid T., Mushtaq T., Mirza M. A., Nadeem A., Babar M. E., Ahmad G.. 2008. Effect of potassium chloride supplementation in drinking water on broiler performance under heat stress conditions. Poult. Sci.  87: 1276– 1280. Google Scholar PubMed  Ahmad T., Sarwar M.. 2006. Dietary electrolyte balance: implications in heat stressed broilers. World Poult. Sci. J.  62: 638– 653. Ait-Boulahsen A., Garlich J. D., Edens F. W.. 1995. Potassium chloride improves the thermotolerance of chickens exposed to acute heat stress. Poult. Sci.  74: 75– 87. Google Scholar CrossRef Search ADS PubMed  Angell L., Seymour C.. 2015. A simple approach to blood gas analysis: the essentials. Veterinary Nursing J.  30: 5. Google Scholar CrossRef Search ADS   Ardiaca M., Bonvehi C., Montesinos A.. 2013. Point-of-care blood gas and electrolyte analysis in rabbits. Vet. Clin. North Am. Exot. Anim. Pract.  16: 175– 195. Google Scholar CrossRef Search ADS PubMed  Ayo J. O., Obidi J. A., Rekwot P. I.. 2011. Effects of heat stress on the well-being, fertility, and hatchability of chickens in the northern Guinea savannah zone of Nigeria: a review. ISRN Vet. Sci.  2011: 838606. Google Scholar CrossRef Search ADS PubMed  Bjorkquist A. G. R., Rothschild M. F., Persia M. E., Ashwell C., Schmidt C., Lamont S. J.. 2015. Genetic Markers Found for Response to Heat Stress in Chickens in Animal Industry ReportIowa state university. Boddicker R. L., Seibert J. T., Johnson J. S., Pearce S. C., Selsby J. T., Gabler N. K., Lucy M. C., Safranski T. J., Rhoads R. P., Baumgard L. H., Ross J. W.. 2014. Gestational heat stress alters postnatal offspring body composition indices and metabolic parameters in pigs. Plos One.  9. doi ARTN e11085910.1371/journal.pone.0110859. Bollen C. W., van Well G. T., Sherry T., Beale R. J., Shah S., Findlay G., Monchi M., Chiche J. D., Weiler N., Uiterwaal C. S., van Vught A. J.. 2005. High frequency oscillatory ventilation compared with conventional mechanical ventilation in adult respiratory distress syndrome: a randomized controlled trial [ISRCTN24242669]. Crit. Care.  9: R430– 439. Google Scholar CrossRef Search ADS PubMed  Borges S. A., Fischer da Silva A. V., Ariki J., Hooge D. M., Cummings K. R.. 2003. Dietary electrolyte balance for broiler chickens exposed to thermoneutral or heat-stress environments. Poult. Sci.  82: 428– 435. Google Scholar CrossRef Search ADS PubMed  Borges S. A., Fischer da Silva A. V., Majorka A., Hooge D. M., Cummings K. R.. 2004. Physiological responses of broiler chickens to heat stress and dietary electrolyte balance (sodium plus potassium minus chloride, milliequivalents per kilogram). Poult. Sci.  83: 1551– 1558. Google Scholar CrossRef Search ADS PubMed  Cahaner A., Ajuh J. A., Siegmund-Schultze M., Azoulay Y., Druyan S., Zarate A. V.. 2008. Effects of the genetically reduced feather coverage in naked neck and featherless broilers on their performance under hot conditions. Poult. Sci.  87: 2517– 2527. Google Scholar CrossRef Search ADS PubMed  Cristina D., P. M., Cureu I., Braslasu M. C.. 2004. Interaction between the diet, temperature and some blood constituents in Roso hens. Archiva Zootechnica . 7: 5. Deeb N., Cahaner A.. 1999. The effects of naked neck genotypes, ambient temperature, and feeding status and their interactions on body temperature and performance of broilers. Poult. Sci.  78: 1341– 1346. Google Scholar CrossRef Search ADS PubMed  Deeb N., Lamont S. J.. 2002. Genetic architecture of growth and body composition in unique chicken populations. J. Hered . 93: 107– 118. Google Scholar CrossRef Search ADS PubMed  El-Gendy Essam A., Nassar M. K., Mostageer A.. 2007. Genotype-environment interaction in relation to heat tolerance in chickens 2. variation in juvenile growth of warm region's oriented breeds. Int. J. Poult. Sci.  6: 322– 328. Google Scholar CrossRef Search ADS   Fair J., Whitaker S., Pearson B.. 2007. Sources of variation in haematocrit in birds. Ibis . 149: 535– 552. Google Scholar CrossRef Search ADS   Felver-Gant J. N. 2012. Genetic variations alter physiological responses following heat stress in 2 strains of laying hens. Poult. Sci.  91: 1542– 1551. Google Scholar CrossRef Search ADS PubMed  Freeman M. L., Borrelli M. J., Meredith M. J., Lepock J. R.. 1999. On the path to the heat shock response: Destabilization and formation of partially folded protein intermediates, a consequence of protein thiol modification. Free Radical Bio. Med . 26: 737– 745. Google Scholar CrossRef Search ADS   Gamba J. P., Rodrigues M. M., Neto M. G., Perri S. H. V., Faria M. J. D., Pinto M. F.. 2015. The strategic application of electrolyte balance to minimize heat stress in broilers. Braz. J. Poult. Sci.  17: 237– 245. Hamm L. L., Hering-Smith K. S., Nakhoul N. L.. 2013. Acid-base and potassium homeostasis. Semin. Nephrol.  33: 257– 264. Google Scholar CrossRef Search ADS PubMed  Hilman P.E., N. R. S., N. R. A. V. T. 2000. physiological responses and adaptations to hot and cold environments. Pages 71 in Physiology in Liverstock . Y. M. K. ed. Islam M. A., Nishibori M.. 2009. Indigenous naked neck chicken: a valuable genetic resource for Bangladesh. World Poult. Sci. J.  65: 125– 138. Google Scholar CrossRef Search ADS   Kaneko J. J., Harvey J. W., Bruss M. L.. 2008. Clinical Biochemistry of Domestic Animals Sixth Edition Preface . Clinical Biochemistry of Domestic Animals, 6th Edition: Ix– Ix. doi 10.1016/B978-0-12-370491-7.00031-3. Google Scholar CrossRef Search ADS   Lamont S. J., Coble D. J., Bjorkquist A., Rothschild M. F., Persia M., Ashwell C., Schmidt C.. 2014. Genomics of heat stress in chickens. Proc. 10th World Congress of Genetics Applied to Liverstock Production , Canada. Lara L. J., Rostagno M. H.. 2013. Impact of Heat Stress on Poultry Production. Animals (Basel) . 3: 356– 369. Google Scholar CrossRef Search ADS PubMed  Leypoldt J. K., Cheung A. K., Steuer R. R., Harris D. H., Conis J. M.. 1995. Determination of circulating blood volume by continuously monitoring hematocrit during hemodialysis. J. Am. Soc. Nephrol . 6: 214– 219. Google Scholar PubMed  Lu Q., Wen J., Zhang H.. 2007. Effect of chronic heat exposure on fat deposition and meat quality in two genetic types of chicken. Poult. Sci.  86: 1059– 1064. Google Scholar CrossRef Search ADS PubMed  Majekodunmi B. C., Sokunbi O. A., Ogunwole O. A., Adebiyi O. A.. 2013. Influence of electrolytes and ascorbic acid supplementation on serum and erythrocytic indices of broiler chickens reared in a hot environment. Afr. J. Agric. Res.  8: 7. Martin M. P., Wineland M., Barnes H. J.. 2010. Selected blood chemistry and gas reference ranges for broiler breeders using the i-STAT handheld clinical analyzer. Avian Dis . 54: 1016– 1020. Google Scholar CrossRef Search ADS PubMed  Muiruri H. K., Harrison P. C.. 1991. Effect of roost temperature on performance of chickens in hot ambient environments. Poult. Sci.  70: 2253– 2258. Google Scholar CrossRef Search ADS PubMed  Niu Z. Y., Liu F. Z., Yan Q. L., Li W. C.. 2009. Effects of different levels of vitamin E on growth performance and immune responses of broilers under heat stress. Poult. Sci.  88: 2101– 2107. Google Scholar CrossRef Search ADS PubMed  Odom T. W., Harrison P. C., Bottje W. G.. 1986. Effects of thermal-induced respiratory alkalosis on blood ionized calcium levels in the domestic hen. Poult. Sci.  65: 570– 573. Google Scholar CrossRef Search ADS PubMed  Olanrewaju H. A., Thaxton J. P., Dozier W. A.3rd, Branton S. L.. 2007. Electrolyte diets, stress, and acid-base balance in broiler chickens. Poult. Sci.  86: 1363– 1371. Google Scholar CrossRef Search ADS PubMed  Olanrewaju H. A., Wongpichet S., Thaxton J. P., Dozier W. A., Branton S. L.. 2006. Stress and acid-base balance in chickens. Poult. Sci.  85: 1266– 1274. Google Scholar CrossRef Search ADS PubMed  Padgett D. A., Glaser R.. 2003. How stress influences the immune response. Trends Immunol.  24: 444– 448. Google Scholar CrossRef Search ADS PubMed  Pech-Waffenschmidt V., Bogin E., Avidar Y., Horst A.. 1995. Metabolic and biochemical changes during heat stress in relation to the feathering degree of the domestic hen. Avian Pathol.  24: 33– 44. Google Scholar CrossRef Search ADS PubMed  Pracha U.Z., Fatima K., Alqahtani M., Chaudhary A., Abuzenadah A., Damanhouri G., Qadri I.. 2013. Oxidative stress and hepatitis C virus. Virol J.  10: 251. Google Scholar CrossRef Search ADS PubMed  Puvadolpirod S., Thaxton J. P.. 2000. Model of physiological stress in chickens 1. Response parameters. Poult. Sci.  79: 363– 369. Google Scholar CrossRef Search ADS PubMed  Rhoads R. P., Baumgard L. H., Suagee J. K., Sanders S. R.. 2013. Nutritional interventions to alleviate the negative consequences of heat stress. Adv. Nutr.  4: 267– 276. Google Scholar CrossRef Search ADS PubMed  Ryder A. A., Feddes J. J. R., Zuidhof M. J.. 2004. Field study to relate heat stress index to broiler performance. J. Appl. Poult. Res.  13: 493– 499. Google Scholar CrossRef Search ADS   Sahin K. S., Sahin N., Kucuk O., Hayirli A., Prasad A. S.. 2009. Role of dietary zinc in heat-stressed poultry: A review. Poult. Sci.  88: 2176– 2183. Google Scholar CrossRef Search ADS PubMed  Samara M. H., Robbins K. R., Smith M. O.. 1996. Environmental heat stress does not reduce blood ionized calcium concentration in hens acclimated to elevated temperatures. Poult. Sci.  75: 197– 200. Google Scholar CrossRef Search ADS PubMed  Sandercock D. A., Hunter R. R., Nute G. R., Mitchell M. A., Hocking P. M.. 2001. Acute heat stress-induced alterations in blood acid-base status and skeletal muscle membrane integrity in broiler chickens at two ages: Implications for meat quality. Poult. Sci.  80: 418– 425. Google Scholar CrossRef Search ADS PubMed  Schaal T. P., Arango J., Wolc A., Brady J. V., Fulton J. E., Rubinoff I., Ehr I. J., Persia M. E., O'Sullivan N. P.. 2016. Commercial Hy-Line W-36 pullet and laying hen venous blood gas and chemistry profiles utilizing the portable i-STAT(R)1 analyzer. Poult. Sci.  95: 466– 471. Google Scholar CrossRef Search ADS PubMed  Sgavioli S., Santos E. T., Domingues C. H. F., Quadros T. C. O., Castiblanco D. M. C., Andrade-Garcia G. M., Amoroso L., Naas I. A., Garcia R. G., Baraldi-Artoni S. M.. 2016. Effect of high incubation temperature on the blood parameters of layer chicks. Braz. J. Poult. Sci.  18: 41– 47. Soleimani A. F., Zulkifli I.. 2010. Effects of high ambient temperature on blood parameters in red jungle fowl, village fowl and broiler chickens. J. Anim. Vet. Adv.  9: 1201– 1207. Google Scholar CrossRef Search ADS   Soleimani A. F., Zulkifli I., Omar A. R., Raha A. R.. 2011. Physiological responses of 3 chicken breeds to acute heat stress. Poult. Sci.  90: 1435– 1440. Google Scholar CrossRef Search ADS PubMed  Ungerer J. P. J., Ungerer M. J., Vermaak W. J. H.. 1990. Discordance between measured and calculated total carbon-dioxide. Clin. Chem.  36: 2093– 2096. Google Scholar PubMed  Van Goor A., Ashwell C. M., Persia M. E., Rothschild M. F., Schmidt C. J., Lamont S. J.. 2016. Quantitative trait loci identified for blood chemistry components of an advanced intercross line of chickens under heat stress. BMC Genomics.  17: 287. Google Scholar CrossRef Search ADS PubMed  Van Goor A., Ashwell C. M., Persia M. E., Rothschild M. F., Schmidt C. J., Lamont S. J.. 2017. Unique genetic responses revealed in RNA-seq of the spleen of chickens stimulated with lipopolysaccharide and short-term heat. PLoS One.  12: e0171414. Google Scholar CrossRef Search ADS PubMed  Van Goor A., Bolek K. J., Ashwell C. M., Persia M. E., Rothschild M. F., Schmidt C. J., Lamont S. J.. 2015. Identification of quantitative trait loci for body temperature, body weight, breast yield, and digestibility in an advanced intercross line of chickens under heat stress. Genet. Sel. Evol.  47. doi ARTN 9610.1186/s12711-015-0176-7. Wang Y., Lupiani B., Reddy S. M., Lamont S. J., Zhou H.. 2014. RNA-seq analysis revealed novel genes and signaling pathway associated with disease resistance to avian influenza virus infection in chickens. Poult. Sci.  93: 485– 493. Google Scholar CrossRef Search ADS PubMed  Wilson H. R., Wilcox C. J., Voitle R. A., Baird C. D., Dorminey R. W.. 1975. Characteristics of White Leghorn chickens selected for heat tolerance. Poult. Sci.  54: 126– 130. Google Scholar CrossRef Search ADS PubMed  Wolfenson D., Bachrach D., Maman M., Graber Y., Rozenboim I.. 2001. Evaporative cooling of ventral regions of the skin in heat-stressed laying hens. Poult. Sci.  80: 958– 964. Google Scholar CrossRef Search ADS PubMed  Yalcin S., Cabuk M., Bruggeman V., Babacanoglu E., Buyse J., Decuypere E., Siegel P. B.. 2008. Acclimation to heat during incubation. 1. Embryonic morphological traits, blood biochemistry, and hatching performance. Poult. Sci. 87: 1219– 1228. Yalcin S., Ozkan S., Turkmut L., Siegel P. B.. 2001. Responses to heat stress in commercial and local broiler stocks. 1. Performance traits. Br. Poult. Sci.  42: 149– 152. Google Scholar CrossRef Search ADS PubMed  Yunis R., Cahaner A.. 1999. The effects of the naked neck (Na) and frizzle (F) genes on growth and meat yield of broilers and their interactions with ambient temperatures and potential growth rate. Poult. Sci.  78: 1347– 1352. Google Scholar CrossRef Search ADS PubMed  Zhou H., Lamont S. J.. 1999. Genetic characterization of biodiversity in highly inbred chicken lines by microsatellite markers. Anim. Genet.  30: 256– 264. Google Scholar CrossRef Search ADS PubMed  Zulkifli I., Al-Aqil A., Omar A. R., Sazili A. Q., Rajion M. A.. 2009. Crating and heat stress influence blood parameters and heat shock protein 70 expression in broiler chickens showing short or long tonic immobility reactions. Poult. Sci.  88: 471– 476. Google Scholar CrossRef Search ADS PubMed  © 2017 Poultry Science Association Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Physiological responses to heat stress in two genetically distinct chicken inbred lines

Loading next page...
 
/lp/ou_press/physiological-responses-to-heat-stress-in-two-genetically-distinct-v6XOh0J0bM
Publisher
Oxford University Press
Copyright
© 2017 Poultry Science Association Inc.
ISSN
0032-5791
eISSN
1525-3171
D.O.I.
10.3382/ps/pex363
Publisher site
See Article on Publisher Site

Abstract

Abstract High ambient temperature is one of the most important environmental factors negatively impacting poultry production and health. Genetics is an important contributor in mitigating the stress response to heat. Two genetically distinct highly inbred lines of similar body size (Leghorn and Fayoumi) were characterized for phenotypic differences in response to heat. At 14 days of age, birds were exposed to 38°C with 50% humidity for 4 hours, then 35°C until the conclusion of the experiment. Non-treated individuals were kept at 29.4°C for the first week and then 25°C throughout the experiment. Birds in the heat-stress group were inoculated at day (d) 21 with Newcastle disease virus (NDV) La Sota strain to investigate the effects of heat stress and NDV infection. Thirteen blood parameters were measured using the iSTAT blood analyzer at three stages: 4 h, 6 d, and 9 d post heat-stress treatment, representing acute heat (AH) exposure, chronic heat (CH1) exposure, and chronic heat exposure after virus infection (CH2), respectively. Most blood parameters were significantly changed with heat stress in Leghorns at AH and in Fayoumis at CH1 and CH2. The Leghorn line had significant acute responses with disrupted acid-base balance and metabolic disorders. The heat-resilient Fayoumis maintained a relatively well-balanced acid-base balance. The current study provides the comprehensive profile of biomarker signatures in blood associated with heat tolerance and suggests that PO2, TCO2, HCO3, and base excess can be served as potential biomarkers that can be used to genetically improve heat tolerance in poultry. INTRODUCTION Similar to other homoeothermic animals, chickens maintain a constant body temperature over a wide range of ambient temperatures (Deeb and Cahaner, 1999). However, with the global climate change, poultry production is increasingly impacted by high ambient temperatures, especially in developing countries where poultry are mainly reared in environments without temperature control. Once ambient temperatures exceed the tolerable range, birds have difficulties with maintaining their homeostatic body temperature, which is classified as “heat stress” (Lara and Rostagno, 2013). Heat stress results in physiological changes, and subsequent negative effects on production performance in poultry. Heat stress is one of the most important environmentally influenced factors that can negatively affect poultry production and health (Ayo et al., 2011). With heat stress, laying hens typically suffer from significant reductions in production traits such as egg weight, shell thickness, and rate of production (Wolfenson et al., 2001). Fast-growing broilers maintain higher internal body temperatures than layers, and high mortality rates, low growth rate, and reduced meat quality and body weight have been observed under heat stress (Muiruri and Harrison, 1991). Heat stress also affects the immune response in poultry. Immunosuppression has been observed in both broilers and layers during exposure to a heat stress environment (Padgett and Glaser, 2003). Reduced lymphoid organ weight and lower levels of circulating antibodies, including specific IgM and IgY, were detected in heat-stressed birds compared to non-treated birds (Felver-Gant, 2012). Further, antibody response and phagocytosis of macrophages were repressed in chickens under heat stress (Niu et al., 2009). Heat stress also affects physiological parameters including chemistry and blood gas parameters in animals and the responses vary depending on the time and degree of thermal challenge (Boddicker et al., 2014). Obtaining accurate and reliable measurements of these parameters used to present a great challenge in animal farming until the recent development of a point of care, portable blood analyzer, i.e., the i-STAT (Abbott Laboratories, San Diego, CA) handheld blood analyzer (Martin et al., 2010). Physiological parameters associated with host response to heat stress can be immediately measured, minimizing alterations in the blood-gas and electrolyte values as a result of sample handling and transportation (Kaneko et al., 2008). The i-STAT has been used to assess blood clinical chemistries in chickens and reference ranges have been established for broiler breeders (Martin et al., 2010). Depending on the purpose, different i-STAT cartridges can be selected. In our study, the CG8+ cartridge was used to evaluate heat-stress-related parameters in blood. Genetics plays a significant role in affecting host response to heat stress in poultry (Felver-Gant, 2012). The divergent selection in Leghorns has resulted in chicken lines with different levels of heat tolerance (Wilson et al., 1975). In addition, fast-growing broilers were more sensitive to heat stress than slow-growing broiler strains (Yunis and Cahaner, 1999). Genetic resistance to high ambient temperatures in chickens can be inherited from their ancestors (Lu et al., 2007). Indigenous chicken breeds in tropical areas have been documented to have higher heat stress tolerance compared to other breeds (Soleimani and Zulkifli, 2010). Two genetically distinct highly inbred chicken lines (Leghorn GB2 and Fayoumi M43) maintained at Iowa State University, whose inbreeding coefficients were more than 99.99% (Zhou and Lamont, 1999), were used in the current study. Thus, genetic variations within the line are negligible. The Fayoumis were originally imported from Egypt and they had undergone natural selection for heat tolerance (Lamont et al., 2014). Based on previous reports, Fayoumi birds are more resistant to both disease and heat stress (Bjorkquist et al., 2015; Wang et al., 2014). A broiler (heat-susceptible) × Fayoumi (heat-resistant) advanced intercross line (AIL) was used to fine-map quantitative trait loci (QTL) using the 600 K Affymetrix chicken single nucleotide polymorphism (SNP) array to investigate genetic influence of host response to heat stress in chickens (Van Goor et al., 2015). Several QTL regions had been identified including one major QTL for breast muscle yield under heat stress which accounted for more than 24% of the genetic variation in this study. Thirty-five QTLs were identified to be associated with the response to heat stress. In addition, association of physiological changes on blood components and genomic regions were identified on the same chicken AIL line, which may serve as markers for genetic selection to enhance heat tolerance in poultry (Van Goor et al., 2016). Most recently, the Fayoumi and broiler breeds were also used to study host transcriptome response to heat and Lipopolysaccharide (LPS) stimulation using RNA-seq (Van Goor et al., 2017) and genes and signal pathways associated with heat stress and LPS identified can be served as additional biomarkers for heat tolerance. Based on these studies, some heat tolerant favorable alleles might be fixed in Fayoumi breed, which can be utilized for genetic selection. We hypothesize that Leghorns are relatively heat susceptible while Fayoumis are more heat tolerant. The current study is a part of a US Agency for International Development sponsored program (Feed the Future Innovation Lab for Genomics to Improve Poultry) to improve food security in Africa by enhancing resistance to Newcastle disease and heat stress in chickens (http//:gip.ucdavis.edu). Our major focus in the current study was to characterize physiological responses to heat stress in these two diverse highly inbred lines and develop candidate diagnostic biomarkers for potential genetic selection to improve heat tolerance in poultry. MATERIALS AND METHODS Experimental Populations One hundred and eleven birds, 55 Leghorns and 56 Fayoumis, were housed in two temperature and humidity-controlled isolators, and birds were provided with ad libitum access to food and water. On day (d) 1 of age, 30 Leghorn and 31 Fayoumi birds were randomly chosen as the treatment groups and housed in one isolator and the rest of birds were used as the non-treated groups in another isolator. The two lines were mixed in each isolator. From 14 days of age to the end of the experiment (41 days of age), the heat-treated groups were exposed to continuous heat stress of 38°C for the first 4 hours, and then decreased to 35°C, while the non-treated groups were maintained at 29.4°C for the first week and 25°C throughout the whole experiment. On d 21, birds in the heat-treated groups were inoculated with 107 EID50 Newcastle Disease virus (NDV) La Sota strain through both ocular and nasal passages (50 μL per nostril and eye). The animal experiment was performed according to the guidelines approved by the Institutional Animal Care and Use Committee, University of California, Davis (IACUC #17853). Blood Parameters Measurements Blood gases and chemistry parameters were measured in all birds at d14 (4 hour post-heat-stress treatment, acute phase (AH)), d20 (6 days post-treatment, chronic phase 1 (CH1)) and d23 (9 days post-treatment, chronic phase 2 (CH2)), respectively. Meanwhile, six birds per line per group were euthanized for tissue collection (RNA-seq analysis on separate study) at d14 and d23. Approximately 1 mL of blood was collected into a heparinized syringe (2,000 unit/mL heparin in PBS, Sigma-Aldrich, St. Louis, MO) for each bird, and then blood samples were analyzed immediately using an i-STAT Portable Blood Analyzer. The i-STAT cartridge (CG8+) measured 13 different parameters including four chemistry/electrolyte parameters (concentrations of sodium (Na+), potassium (K+), ionized calcium (Ca2+), and glucose (Glu)); seven blood gas parameters (blood pH, carbon dioxide partial pressure (PCO2), oxygen partial pressure (PO2), total carbon dioxide (TCO2), bicarbonate (HCO3), BE, and oxygen situation (sO2)), and two hematologic parameters: hematocrit (Hct or packed cell volume (PCV)), and hemoglobin (Hb). TCO2 measures the total carbon dioxide and is calculated from pH and PCO2. Measurement of TCO2 is useful to assess HCO3 concentration (Ungerer et al., 1990). Base excess (BE) can be estimated from HCO3 and pH. Statistical Analysis The i-STAT parameters were analyzed using the following linear model for each time point separately in JMP 13 software (SAS Institute, 2016), since population size continued to decrease due to tissue collections at each time point.   \begin{equation*}Y = \mu + T + L + T \times L + e\end{equation*} Where Y is the dependent variable of various parameters, μ is the population mean of the measurements, T is the fixed effect associated with the heat stress treatment, L is the fixed effect associated with the genetic line, T × L is the interaction effect of treatment, and line and e is the random error effect. All measurements in this study were represented as least square mean ±SE. P < 0.05 was considered statistically significant. All figures were generated by Prism 6 GraphPad software (La Jolla, CA). RESULTS Blood Parameter Measurements Lease square means and P values for each factor were listed in Table 1 for all blood parameters of both genetic lines at each time point. Physiological responses to heat stress between the two genetic lines and within each genetic line were compared based on the blood parameter measurements. At the AH stage, six (K+, iCa2+, pH, PCO2, PO2, and sO2) out of thirteen parameters had significant T × L interactions (P < 0.05). For the rest seven parameters, all main genetic line effects were significant except Glu. While only two parameters (Na+ and BE) were significant on main treatment effects. At the CH1 stage, seven (K+, Glu, PCO2, TCO2, HCO3, PO2, and BE) out of thirteen parameters had significant T × L interactions (P < 0.05). Significant main treatment effects were identified on four parameters (pH, sO2, Hct, and Hb) and no main genetic line effects were observed. At the CH2 stage with 2 days post inoculation (DPI) NDV inoculation, five parameters (Glu, TCO2, HCO3, PO2, and sO2) had significant T × L interactions (P < 0.05). The main treatment effect was identified on pH and BE; and there were three parameters (Na+, pH, and BE) had main genetic line effects. For blood parameters having significant T × L effects, line effects in treated and non-treated groups and treatment effects within genetic lines were further analyzed. Table 1. Lease square means and P values of blood parameters with Heat stress treatment.   LS mean ± SE  P values    Blood parameter  LC  LT  FC  FT  Trt  Line  Line*Trt  Acute Heat  Na+ (mmol/L)  142.21 ± 0.31A  140.8 ± 0.27B  142.78 ± 0.31A  142.48 ± 0.28A  0.0045  0.0002  0.0619  K+ (mmol/L)  4.90 ± 0.13A  4.31 ± 0.11C  4.52 ± 0.13BC  4.66 ± 0.12AB  0.0702  0.90  0.0036  iCa2+ (mmol/L)  0.89 ± 0.05B  1.09 ± 0.04A  1.03 ± 0.05A  1.03 ± 0.04A  0.0347  0.3557  0.0270  Glu (mg/dL)  218.96 ± 6.08B  238.50 ± 5.44A  222.61 ± 6.22AB  225.38 ± 5.54AB  0.0585  0.4185  0.1534  pH  7.47 ± 0.015B  7.49 ± 0.017B  7.48 ± 0.017B  7.59 ± 0.015A  0.0002  0.0035  0.0047  PCO2 (mmHg)  25.55 ± 1.02BC  26.70 ± 0.93B  30.35 ± 1.04A  23.46 ± 0.93C  0.0043  0.4272  0.0001  TCO2(mmol/L)  19.50 ± 0.65C  21.21 ± 0.59BC  23.26 ± 0.66A  22.62 ± 0.59AB  0.3941  0.0001  0.0626  HCO (mmol/L)  18.68 ± 0.64C  20.46 ± 0.58B  22.30 ± 0.65A  21.95 ± 0.58AB  0.2450  0.0001  0.0842  PO2 (mmHg)  61.38 ± 1.38A  55.37 ± 1.24B  55.83 ± 1.14B  56.45 ± 1.26B  0.0447  0.0948  0.0139  sO2 (%)  92.54 ± 0.73AB  90.21 ± 0.66C  90.57 ± 0.75BC  93.21 ± 0.66A  0.8273  0.4675  0.0006  BE (mmol/L)  −4.88 ± 0.77C  −2.90 ± 0.70BC  −1.17 ± 0.78AB  0.21 ± 0.70A  0.0250  0.0001  0.6866  Hct (%PCV)  16.77 ± 0.69AB  18.45 ± 0.61A  16.39 ± 0.68B  15.90 ± 0.61B  0.3640  0.0257  0.0969  Hb (g/dL)  5.70 ± 0.24AB  6.28 ± 0.21A  5.56 ± 0.23B  5.40 ± 0.21B  0.3378  0.0218  0.0968  Chronic Heat 1  Na+ (mmol/L)  142.78 ± 0.38B  143.18 ± 0.39AB  143.53 ± 0.42AB  143.79 ± 0.33A  0.3925  0.0770  0.8547  K+ (mmol/L)  4.90 ± 0.11A  4.58 ± 0.11BC  4.43 ± 0.12C  4.83 ± 0.09AB  0.7019  0.3176  0.0013  iCa2+ (mmol/L)  0.88 ± 0.06A  0.86 ± 0.06A  1.00 ± 0.06A  0.91 ± 0.05A  0.3240  0.1301  0.5635  Glu (mg/dL)  201.44 ± 6.92A  215.18 ± 7.12A  216.20 ± 7.58A  201.00 ± 5.99A  0.9159  0.9667  0.0403  pH  7.41 ± 0.02B  7.47 ± 0.02AB  7.44 ± 0.02B  7.50 ± 0.02A  0.0076  0.1313  0.9783  PCO2 (mmHg)  26.93 ± 1.26B  24.24 ± 1.29BC  31.34 ± 1.43A  23.35 ± 1.09C  0.0001  0.1725  0.0410  TCO2(mmol/L)  17.78 ± 0.71B  18.24 ± 0.73B  22.14 ± 0.81A  19.17 ± 0.62B  0.0858  0.0005  0.0203  HCO (mmol/L)  16.98 ± 0.70B  17.52 ± 0.73B  21.26 ± 0.80A  18.30 ± 0.61B  0.0943  0.0007  0.0167  PO2 (mmHg)  59.17 ± 1.90A  59.29 ± 1.95A  51.07 ± 2.08B  60.17 ± 1.64A  0.0176  0.0610  0.0208  sO2 (%)  89.61 ± 0.92B  92.35 ± 0.95A  87.14 ± 1.04B  92.63 ± 0.80A  0.0001  0.2127  0.1460  BE (mmol/L)  −7.67 ± 0.86C  −6.12 ± 0.88BC  −2.93 ± 0.97A  −4.83 ± 0.74AB  0.8377  0.0009  0.0499  Hct (%PCV)  21.00 ± 0.71A  16.63 ± 0.74B  20.53 ± 0.76A  17.13 ± 0.60B  0.0000  0.9812  0.4958  Hb (g/dL)  7.14 ± 0.24A  5.66 ± 0.25B  6.99 ± 0.26A  5.85 ± 0.21B  0.0000  0.9176  0.4756  Chronic Heat 2 (+ NDV 2 DPI)  Na+ (mmol/L)  143.05 ± 0.31B  142.87 ± 0.29B  144.06 ± 0.32A  144.21 ± 0.28A  0.9601  0.0021  0.5791  K+ (mmol/L)  4.72 ± 0.11A  4.93 ± 0.10A  4.93 ± 0.11A  4.94 ± 0.10A  0.2829  0.3139  0.3266  iCa2+ (mmol/L)  0.84 ± 0.05B  0.98 ± 0.04A  0.95 ± 0.05AB  0.95 ± 0.04AB  0.1403  0.4094  0.1395  Glu (mg/dL)  184.58 ± 5.91C  220.09 ± 5.37A  197.06 ± 6.07BC  200.71 ± 5.26B  0.0009  0.5440  0.0062  pH  7.42 ± 0.02B  7.37 ± 0.01C  7.49 ± 0.02A  7.45 ± 0.01B  0.0025  0.0000  0.9765  PCO2 (mmHg)  27.04 ± 1.09AB  28.47 ± 0.99A  26.85 ± 1.12AB  25.22 ± 0.97B  0.9265  0.1033  0.1471  TCO2(mmol/L)  18.16 ± 0.61B  17.39 ± 0.56B  21.39 ± 0.63A  18.08 ± 0.55B  0.0009  0.0013  0.0338  HCO (mmol/L)  17.36 ± 0.59B  16.56 ± 0.53B  20.47 ± 0.60A  17.16 ± 0.52B  0.0005  0.0015  0.0295  PO2 (mmHg)  57.89 ± 1.37A  54.43 ± 1.24AB  51.11 ± 1.41B  54.88 ± 1.22A  0.9081  0.0180  0.0073  sO2 (%)  89.63 ± 0.90A  87.00 ± 0.82B  88.83 ± 0.92AB  89.75 ± 0.80A  0.3224  0.2605  0.0426  BE (mmol/L)  −7.16 ± 0.70BC  −8.74 ± 0.64C  −2.94 ± 0.72A  −6.79 ± 0.62B  0.0001  0.0000  0.0954  Hct (%PCV)  17.06 ± 0.64A  16.17 ± 0.56A  16.44 ± 0.64A  17.17 ± 0.55A  0.8943  0.7505  0.1838  Hb (g/dL)  5.89 ± 0.21A  5.49 ± 0.19A  5.59 ± 0.21A  5.81 ± 0.18A  0.6512  0.9686  0.1171    LS mean ± SE  P values    Blood parameter  LC  LT  FC  FT  Trt  Line  Line*Trt  Acute Heat  Na+ (mmol/L)  142.21 ± 0.31A  140.8 ± 0.27B  142.78 ± 0.31A  142.48 ± 0.28A  0.0045  0.0002  0.0619  K+ (mmol/L)  4.90 ± 0.13A  4.31 ± 0.11C  4.52 ± 0.13BC  4.66 ± 0.12AB  0.0702  0.90  0.0036  iCa2+ (mmol/L)  0.89 ± 0.05B  1.09 ± 0.04A  1.03 ± 0.05A  1.03 ± 0.04A  0.0347  0.3557  0.0270  Glu (mg/dL)  218.96 ± 6.08B  238.50 ± 5.44A  222.61 ± 6.22AB  225.38 ± 5.54AB  0.0585  0.4185  0.1534  pH  7.47 ± 0.015B  7.49 ± 0.017B  7.48 ± 0.017B  7.59 ± 0.015A  0.0002  0.0035  0.0047  PCO2 (mmHg)  25.55 ± 1.02BC  26.70 ± 0.93B  30.35 ± 1.04A  23.46 ± 0.93C  0.0043  0.4272  0.0001  TCO2(mmol/L)  19.50 ± 0.65C  21.21 ± 0.59BC  23.26 ± 0.66A  22.62 ± 0.59AB  0.3941  0.0001  0.0626  HCO (mmol/L)  18.68 ± 0.64C  20.46 ± 0.58B  22.30 ± 0.65A  21.95 ± 0.58AB  0.2450  0.0001  0.0842  PO2 (mmHg)  61.38 ± 1.38A  55.37 ± 1.24B  55.83 ± 1.14B  56.45 ± 1.26B  0.0447  0.0948  0.0139  sO2 (%)  92.54 ± 0.73AB  90.21 ± 0.66C  90.57 ± 0.75BC  93.21 ± 0.66A  0.8273  0.4675  0.0006  BE (mmol/L)  −4.88 ± 0.77C  −2.90 ± 0.70BC  −1.17 ± 0.78AB  0.21 ± 0.70A  0.0250  0.0001  0.6866  Hct (%PCV)  16.77 ± 0.69AB  18.45 ± 0.61A  16.39 ± 0.68B  15.90 ± 0.61B  0.3640  0.0257  0.0969  Hb (g/dL)  5.70 ± 0.24AB  6.28 ± 0.21A  5.56 ± 0.23B  5.40 ± 0.21B  0.3378  0.0218  0.0968  Chronic Heat 1  Na+ (mmol/L)  142.78 ± 0.38B  143.18 ± 0.39AB  143.53 ± 0.42AB  143.79 ± 0.33A  0.3925  0.0770  0.8547  K+ (mmol/L)  4.90 ± 0.11A  4.58 ± 0.11BC  4.43 ± 0.12C  4.83 ± 0.09AB  0.7019  0.3176  0.0013  iCa2+ (mmol/L)  0.88 ± 0.06A  0.86 ± 0.06A  1.00 ± 0.06A  0.91 ± 0.05A  0.3240  0.1301  0.5635  Glu (mg/dL)  201.44 ± 6.92A  215.18 ± 7.12A  216.20 ± 7.58A  201.00 ± 5.99A  0.9159  0.9667  0.0403  pH  7.41 ± 0.02B  7.47 ± 0.02AB  7.44 ± 0.02B  7.50 ± 0.02A  0.0076  0.1313  0.9783  PCO2 (mmHg)  26.93 ± 1.26B  24.24 ± 1.29BC  31.34 ± 1.43A  23.35 ± 1.09C  0.0001  0.1725  0.0410  TCO2(mmol/L)  17.78 ± 0.71B  18.24 ± 0.73B  22.14 ± 0.81A  19.17 ± 0.62B  0.0858  0.0005  0.0203  HCO (mmol/L)  16.98 ± 0.70B  17.52 ± 0.73B  21.26 ± 0.80A  18.30 ± 0.61B  0.0943  0.0007  0.0167  PO2 (mmHg)  59.17 ± 1.90A  59.29 ± 1.95A  51.07 ± 2.08B  60.17 ± 1.64A  0.0176  0.0610  0.0208  sO2 (%)  89.61 ± 0.92B  92.35 ± 0.95A  87.14 ± 1.04B  92.63 ± 0.80A  0.0001  0.2127  0.1460  BE (mmol/L)  −7.67 ± 0.86C  −6.12 ± 0.88BC  −2.93 ± 0.97A  −4.83 ± 0.74AB  0.8377  0.0009  0.0499  Hct (%PCV)  21.00 ± 0.71A  16.63 ± 0.74B  20.53 ± 0.76A  17.13 ± 0.60B  0.0000  0.9812  0.4958  Hb (g/dL)  7.14 ± 0.24A  5.66 ± 0.25B  6.99 ± 0.26A  5.85 ± 0.21B  0.0000  0.9176  0.4756  Chronic Heat 2 (+ NDV 2 DPI)  Na+ (mmol/L)  143.05 ± 0.31B  142.87 ± 0.29B  144.06 ± 0.32A  144.21 ± 0.28A  0.9601  0.0021  0.5791  K+ (mmol/L)  4.72 ± 0.11A  4.93 ± 0.10A  4.93 ± 0.11A  4.94 ± 0.10A  0.2829  0.3139  0.3266  iCa2+ (mmol/L)  0.84 ± 0.05B  0.98 ± 0.04A  0.95 ± 0.05AB  0.95 ± 0.04AB  0.1403  0.4094  0.1395  Glu (mg/dL)  184.58 ± 5.91C  220.09 ± 5.37A  197.06 ± 6.07BC  200.71 ± 5.26B  0.0009  0.5440  0.0062  pH  7.42 ± 0.02B  7.37 ± 0.01C  7.49 ± 0.02A  7.45 ± 0.01B  0.0025  0.0000  0.9765  PCO2 (mmHg)  27.04 ± 1.09AB  28.47 ± 0.99A  26.85 ± 1.12AB  25.22 ± 0.97B  0.9265  0.1033  0.1471  TCO2(mmol/L)  18.16 ± 0.61B  17.39 ± 0.56B  21.39 ± 0.63A  18.08 ± 0.55B  0.0009  0.0013  0.0338  HCO (mmol/L)  17.36 ± 0.59B  16.56 ± 0.53B  20.47 ± 0.60A  17.16 ± 0.52B  0.0005  0.0015  0.0295  PO2 (mmHg)  57.89 ± 1.37A  54.43 ± 1.24AB  51.11 ± 1.41B  54.88 ± 1.22A  0.9081  0.0180  0.0073  sO2 (%)  89.63 ± 0.90A  87.00 ± 0.82B  88.83 ± 0.92AB  89.75 ± 0.80A  0.3224  0.2605  0.0426  BE (mmol/L)  −7.16 ± 0.70BC  −8.74 ± 0.64C  −2.94 ± 0.72A  −6.79 ± 0.62B  0.0001  0.0000  0.0954  Hct (%PCV)  17.06 ± 0.64A  16.17 ± 0.56A  16.44 ± 0.64A  17.17 ± 0.55A  0.8943  0.7505  0.1838  Hb (g/dL)  5.89 ± 0.21A  5.49 ± 0.19A  5.59 ± 0.21A  5.81 ± 0.18A  0.6512  0.9686  0.1171  Note: LC: Leghorn non-treated group; LT: Leghorn treated group; FC: Fayoumi non-treated group; FT: Fayoumi treated group; Trt: treatment effects; Line: line effects; Line*Trt: line and treatment interaction effects; Different superscript letters within row represent significant differences (P < 0.05). View Large Effects of Heat Stress between Genetic Lines Without heat stress, the two genetic lines generally had 7 out of 13 parameters significantly difference due to their genetic background which were iCa2+, K+, PCO2, TCO2, HCO3, BE, and PO2 on d14 (Figure 1); 6 out of 13 parameters (K+, PCO2, TCO2, HCO3, BE, and PO2) on d 20 (Figure 2); and 6 out of 13 parameters (Na+, pH, TCO2, HCO3, PO2, and BE) on d 23 (Figure 3). Across all time points, Leghorn birds had significantly higher PO2 and lower TCO2, HCO3, and BE than Fayoumi birds. They also had higher K+ and lower levels of PCO2 than Fayoumis on both d14 and d20 and higher pH and lower Na+ levels than Fayoumis on d23. Figure 1. View largeDownload slide Blood parameter differences between non-treated genetic lines at 14 days of age. Blood parameter differences between non-treated genetic lines at day 14. *indicated significant pairwise difference between lines (P < 0.05) **; indicated P < 0.01, and ***indicated P < 0.001. Figure 1. View largeDownload slide Blood parameter differences between non-treated genetic lines at 14 days of age. Blood parameter differences between non-treated genetic lines at day 14. *indicated significant pairwise difference between lines (P < 0.05) **; indicated P < 0.01, and ***indicated P < 0.001. Figure 2. View largeDownload slide Blood parameter differences between non-treated genetic lines at 20 days of age. Blood parameter differences between non-treated genetic lines at day 20. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01. Figure 2. View largeDownload slide Blood parameter differences between non-treated genetic lines at 20 days of age. Blood parameter differences between non-treated genetic lines at day 20. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01. Figure 3. View largeDownload slide Blood parameter differences between non-treated genetic lines at 23 days of age. Blood parameter differences between non-treated genetic lines at day 23. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 3. View largeDownload slide Blood parameter differences between non-treated genetic lines at 23 days of age. Blood parameter differences between non-treated genetic lines at day 23. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. With heat treatment, more parameters (8 out of 13) were significantly different between the two genetic lines at the AH stage, which were Na+, K+, pH, PCO2, sO2, BE, Hct, and Hb (Figure 4). One week later at the CH1 stage, there were no differences in parameters between the Fayoumi and Leghorn lines. At the CH2 stage (after NDV infection), Leghorn birds had higher Glu and PCO2 and lower Na, sO2, pH, and sO2 levels than Fayoumis (Figure 5). Figure 4. View largeDownload slide Blood parameter differences between genetic lines at the acute heat stress stage. Blood parameter differences between heat-stress treated genetic lines at the acute heat stage. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 4. View largeDownload slide Blood parameter differences between genetic lines at the acute heat stress stage. Blood parameter differences between heat-stress treated genetic lines at the acute heat stage. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 5. View largeDownload slide Blood parameter differences between genetic lines at the chronic heat stress and NDV infection stage. Blood parameter differences between heat stress and NDV treated genetic lines at the chronic heat 2 stage. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 5. View largeDownload slide Blood parameter differences between genetic lines at the chronic heat stress and NDV infection stage. Blood parameter differences between heat stress and NDV treated genetic lines at the chronic heat 2 stage. *indicated significant pairwise difference between lines (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Effects of Heat Stress within Genetic Lines At the AH stage Leghorn birds had more vigorous physiologic responses than Fayoumis with acute heat stress. Comparing with the non-treated groups, there were 7 parameters changed in the Leghorns upon heat stress, which were Na+, K+, iCa2+, Glu, HCO3, PO2, and sO2 (Figure 6); while only 3 parameters (pH, PCO2 and sO2) were significantly changed in Fayoumi birds (Figure 7). Heat stressed Leghorns had significantly lower Na+, K+ and higher iCa2+, Glu than the non-treated birds (Figure 6). There were no differences in blood chemistry/electrolytes in Fayoumi birds at the AH stage (Figure 7). For the five CO2 associated parameters, the HCO3 level in the heat stress Leghorns was the only one increased comparing with non-treated leghorns and there was no change in the rest four parameters (Figure 6). Higher pH and lower PCO2 levels were observed in heat stressed Fayoumis than non-treated ones (Figure 7). For the two oxygen associated parameters PO2 and sO2, Leghorns had significantly lower PO2 and sO2 with heat stress treatment (Figure 6), while heat stressed Fayoumis had higher sO2 levels at AH stage (Figure 7). For the two hematology parameters, Hct and Hb, there were no differences in Hct and Hb levels between heat stressed and non-treated groups of both genetic lines (Figure 6 and 7). Figure 6. View largeDownload slide Phenotypic responses to heat stress at the acute heat stage in the Leghorn line. Blood parameter differences between acute heat stressed and non-treated birds in the Leghorn line. *indicated significant pairwise difference (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 6. View largeDownload slide Phenotypic responses to heat stress at the acute heat stage in the Leghorn line. Blood parameter differences between acute heat stressed and non-treated birds in the Leghorn line. *indicated significant pairwise difference (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 7. View largeDownload slide Phenotypic responses to heat stress at the acute heat stage in the Fayoumi line. Blood parameter differences between acute heat stressed and non-treated birds in the Fayoumi line. **indicated significant pairwise difference (P < 0.01); ***indicated P < 0.001. Figure 7. View largeDownload slide Phenotypic responses to heat stress at the acute heat stage in the Fayoumi line. Blood parameter differences between acute heat stressed and non-treated birds in the Fayoumi line. **indicated significant pairwise difference (P < 0.01); ***indicated P < 0.001. At the CH Stages At the CH1 stage, between heat stressed and non-treated birds, only 4 parameters (K+, sO2, Hct, and Hb) were significantly changed in Leghorn birds (Figure 8), compared to 9 parameters (K+, pH, PCO2, TCO2, HCO3, PO2, sO2, Hct, and Hb) changed responding to heat stress in Fayoumis (Figure 9). At the CH2 stage, which was 2 DPI with NDV, Leghorn birds had significant changes in iCa2+, Glu, pH, and sO2 (Figure 10) and Fayoumi birds had more blood gas parameters changed including pH, TCO2, HCO3 PO2, and BE comparing with non-treated birds (Figure 11). Figure 8. View largeDownload slide Phenotypic responses to heat stress at chronic heat 1 stage in the Leghorn line. Blood parameter differences between chronic heat stressed and non-treated birds in the Leghorn line at the CH1 stage. *indicated significant pairwise difference (P < 0.05); ***indicated P < 0.001. Figure 8. View largeDownload slide Phenotypic responses to heat stress at chronic heat 1 stage in the Leghorn line. Blood parameter differences between chronic heat stressed and non-treated birds in the Leghorn line at the CH1 stage. *indicated significant pairwise difference (P < 0.05); ***indicated P < 0.001. Figure 9. View largeDownload slide Phenotypic responses to heat stress at chronic heat 1 stage in the Fayoumi line. Blood parameter differences between chronic heat stressed and non-treated birds in the Fayoumi line at the CH1 stage. *indicated significant pairwise difference (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 9. View largeDownload slide Phenotypic responses to heat stress at chronic heat 1 stage in the Fayoumi line. Blood parameter differences between chronic heat stressed and non-treated birds in the Fayoumi line at the CH1 stage. *indicated significant pairwise difference (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 10. View largeDownload slide Phenotypic responses to heat stress at chronic heat 2 stage in the Leghorn line. Blood parameter differences between chronic heat stressed and non-treated birds in the Leghorn line at the CH2 stage with NDV infection. *indicated significant pairwise difference (P < 0.05); ***indicated P < 0.001. Figure 10. View largeDownload slide Phenotypic responses to heat stress at chronic heat 2 stage in the Leghorn line. Blood parameter differences between chronic heat stressed and non-treated birds in the Leghorn line at the CH2 stage with NDV infection. *indicated significant pairwise difference (P < 0.05); ***indicated P < 0.001. Figure 11. View largeDownload slide Phenotypic responses to heat stress at chronic heat 2 stage in the Fayoumi line. Blood parameter differences between chronic heat stressed and non-treated birds in the Leghorn line at the CH1 stage with NDV infection. *indicated significant pairwise difference (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. Figure 11. View largeDownload slide Phenotypic responses to heat stress at chronic heat 2 stage in the Fayoumi line. Blood parameter differences between chronic heat stressed and non-treated birds in the Leghorn line at the CH1 stage with NDV infection. *indicated significant pairwise difference (P < 0.05); **indicated P < 0.01, and ***indicated P < 0.001. At the CH1 stage, K+ was lower in the heat stressed Leghorns, while higher in heat stressed Fayoumis compared to non-treated birds (Figures 8 and 9). At the CH2 stage, heat stressed Leghorn birds kept higher levels of iCa2+ and Glu compared to non-treated Leghorns (Figure 10), however, there were no significant changes in parameters between heat stressed and non-treated Fayoumis during this stage (Figure 11). With the exception of a lower pH level at the CH2 stage, CO2 associated blood gas parameters were similar between heat stressed and non-treated Leghorns at both CH1 and CH2 stages (Figures 8 and 10). However, heat stressed Fayoumis kept a higher pH level than non-treated ones at CH1 and the pH level went down to lower than non-treated Fayoumis at CH2 (Figures 9 and 11). Consequently, in Fayoumis, heat stressed birds had significantly lower PCO2, TCO2, and HCO3 levels at the CH1 stage (Figure 9) and much lower levels of TCO2, HCO3, and BE at the CH2 stage (Figure 11). The difference of the oxygen related parameters was only identified on sO2 in Leghorns at both CH1 and CH2 stage, in which the sO2 levels of heat stressed Leghorns were higher than non-treated birds at the CH1 stage (Figure 8) and lower at the CH2 stage (Figure 10). However, heat stressed Fayoumis had higher PO2 and sO2 at the CH1 stage (Figure 9) and higher PO2 at the CH2 stage (Figure 11). The two genetic lines responded similarly at CH stages on hematology parameters. At the CH1 stage, Hct and Hb were significantly lower in heat stressed birds compared to non-treated birds observed in both lines (Figures 8 and 9). There were no differences in hematologic parameters between heat stress and non-treated birds at the CH2 stage for both genetic lines (Figures 10 and 11). DISCUSSION Genetic background plays important roles in heat stress resistance in poultry (Lara and Rostagno, 2013). Different chicken breeds or strains have varied tolerance to heat stress (Deeb and Cahaner, 1999; Yalcin et al., 2001; Cahaner et al., 2008; Islam and Nishibori, 2009). Generally, native or indigenous chicken breeds in the tropical areas have better potential to adapt to changes in ambient temperatures (Soleimani and Zulkifli, 2010). Commercial breeds such as fast growing broilers and high production egg layers are more sensitive to heat stress (Yunis and Cahaner, 1999; Soleimani and Zulkifli, 2010). The Fayoumi birds used in our present study were originally imported from Egypt to the United States in 1954 due to their disease resistance (Deeb and Lamont, 2002) and had evolved with a genetic adaptation to high ambient temperatures (Van Goor et al., 2017). Heat treatment had less impact on Fayoumi birds in a genotype-environment interaction study that revealed significant negative impact of heat on commercial broilers (Essam et al., 2007). Breed, age, management, and nutritional factors have significant influences on blood chemistry measurements in chickens. Previously established reference ranges for blood chemistry parameters by i-STAT in broiler breeder hens (Martin et al., 2010) and in pullet and laying hens (Schaal et al., 2016) suggested that i-STAT can serve as a reliable tool to evaluate metabolic disturbance during the heat stress in chickens. There were physiological differences observed between Fayoumi and Leghorn lines in blood chemistries under the normal ambient temperature at the three data collection time points (Figures 1 to 3), which could be due do genetic differences between the two lines. Heat stress treatment expanded the differences between these two genetic lines at the AH stage (Figure 4), and adapted to heat environment after one week at the CH1 stage without significant differences (data not shown), there were then a number of different parameters with both heat stress and NDV inoculation three days later (Figure 5). We assume that Fayoumi is more resilient to heat stress than the Leghorn line as the Fayoumi originated from Egypt, a hot climate region. Additional study in our group from another experiment in a Hy-Line Brown population with the same experiment design revealed a significantly positive correlation between these blood parameters before the heat treatment and growth rate with heat stress (data not published). Heat treatment resulted in more differences between two genetic lines at the AH stage than the chronic stages, which suggested that these two lines had distinct responses in the course of heat stress. Readouts of blood parameters from Fayoumis could be associated with heat tolerance in chickens. Within each genetic line between the heat treated and non-treated groups, Leghorn birds had much more rapid responses than Fayoumis at the AH Stage (7 incidences vs. 3 incidences). At the CH1 stage, the opposite situation was observed in that Leghorns had less responses than Fayoumi birds (4 vs. 9). At the CH2 stage with NDV infection, a similar number of parameters were significantly changed in the two lines (4 vs. 5). Most of blood parameters were significantly changed in the opposite directions with the heat stress between the two genetic lines or significantly changed in only one line (Table 2). Table 2. Differences in blood parameters within genetic lines between heat stress and non-treated groups.   Heat stress (HS) vs. non-treated (NT)    Fayoumi  Leghorn    AH  CH1  CH2  AH  CH1  CH2  Na+  NS  NS  NS  -  NS  NS  K+  NS  +  NS  -  -  NS  iCa2+  NS  NS  NS  +  NS  +  Glu  NS  NS  NS  +  NS  +  pH  +  +  -  NS  NS  -  PCO2  -  -  NS  NS  NS  NS  TCO2  NS  -  -  NS  NS  NS  HCO3  NS  -  -  +  NS  NS  BE  NS  NS  -  NS  NS  NS  PO2  NS  +  +  -  NS  NS  sO2  +  +  NS  -  +  -  Hct  NS  -  NS  NS  -  NS  Hb  NS  -  NS  NS  -  NS    Heat stress (HS) vs. non-treated (NT)    Fayoumi  Leghorn    AH  CH1  CH2  AH  CH1  CH2  Na+  NS  NS  NS  -  NS  NS  K+  NS  +  NS  -  -  NS  iCa2+  NS  NS  NS  +  NS  +  Glu  NS  NS  NS  +  NS  +  pH  +  +  -  NS  NS  -  PCO2  -  -  NS  NS  NS  NS  TCO2  NS  -  -  NS  NS  NS  HCO3  NS  -  -  +  NS  NS  BE  NS  NS  -  NS  NS  NS  PO2  NS  +  +  -  NS  NS  sO2  +  +  NS  -  +  -  Hct  NS  -  NS  NS  -  NS  Hb  NS  -  NS  NS  -  NS  Note: NS, not significant between heat stress and non-treated groups; -, heat stressed birds lower than the non-treated birds (P < 0.05); +, heat stressed birds higher than the non-treated birds (P < 0.05). View Large As a heat resilient chicken line, we hypothesize that Fayoumi birds are more elastic in regulating their physiological response to adapt to heat stress, as indicated from changes in their measured blood parameters, which allowed them to suffer less from the heat stress and maintain their homeostasis. Details of these parameters related to physiological response to heat stress are discussed as follows. Electrolytes/Chemistries With the heat stress, the balance of blood electrolytes can be disrupted due to large amounts of water intake and disordered homeostasis (Ait-Boulahsen et al., 1995). Many researchers have reported that electrolyte supplements can help birds to alleviate the effects of heat stress (Sahin et al., 2009; Majekodunmi et al., 2013). Three electrolytes, Na+, K+, and iCa2+ were measured in this study. With the heat treatment, significant loss of Na+ and K+ at the AH stage and even less K+ at the CH1 stage were observed in Leghorn birds (Supplementary Figure S1 to S2) in other reports (Olanrewaju et al., 2006; Olanrewaju et al., 2007). On the other hand, within the Fayoumi line, electrolyte levels in the blood were maintained very well at the AH and CH2 stage, except a rise of K+ observed at the CH1 stage in heat stressed Fayoumi (Supplementary Figure S3). During heat stress, losing electrolyte balance is a major problems in chickens (Borges et al., 2004). The capability to maintain electrolyte balance is a key factor in maintaining a higher performance (Gamba et al., 2015). Supplementation with electrolytes such as Na+ and K+ is one of approaches used to alleviate the impact of heat stress on the performance of chickens (Ait-Boulahsen et al., 1995; Ahmad and Sarwar, 2006). Comparing with Leghorn birds, Fayoumis were able to keep a better electrolyte balance during the heat stress treatment. Fayoumi birds were able to raise the K+ level up at the CH1 stage, which could be one of their mechanisms to preserve higher performance and have higher tolerance to heat stress. Three dynamic forms of calcium exist in the blood, calcium bounded to protein, calcium complexed with organic acid, or ionized calcium (iCa2+) (Odom et al., 1986). Free iCa2+ in blood was mostly used for bone mineralization (Sgavioli et al., 2016) or used by the shell gland (Odom et al., 1986). Many studies have reported significant reduction in blood iCa2+ during heat stress (Odom et al., 1986), however a few studies also reported that heat stress did not reduce blood iCa2+ (Samara et al., 1996). In the current study, iCa2+ levels of heat stressed Leghorn birds were significantly elevated in both AH and CH2 stages (Supplementary Figure S4). As Leghorn line was originally composed of US commercial layer, maintaining a higher iCa2+ level could be critical for them to have better performance in their later lives. Meanwhile, CH2 stage was a combination of both heat stress and NDV infection. Calcium levels were able to be increased by hepatitis C virus infection in humans (Paracha et al., 2013). Therefore, the elevation of iCa2+ could also be due to NDV inoculation at CH2 in our study. There were no significant differences in glucose levels between the two genetic lines before exposure to heat. Significant glucose differences between the two genetic lines were found at the CH2 stage with heat exposure and NDV infection, in which Fayoumis had lower glucose levels in blood than Leghorns. With heat stress, Leghorn birds significantly increased their glucose levels compared to non-treated birds (Supplementary Figure S5). Blood glucose concentration may be altered due to various stressors including heat stress (Soleimani et al., 2011). It has been reported that heat stress can result in increase of corticosteroids in the blood plasma, which then subsequently causes an increased blood glucose concentration (Ayo et al., 2011). Heat-stressed animals can use glucose as a fuel by increasing hepatic glucose output through glycogenolysis and gluconeogenesis (Rhoads et al., 2013). Fayoumi birds had lower glucose levels than Leghorns with chronic heat stress in the current study. Therefore, we speculate that Fayoumi birds might depress glycogenolysis and gluconeogenesis metabolisms to “wait out” heat stress, which could be a physiologic mechanism to be resilient to heat stress. Blood Gases There were seven blood gas parameters in which pH, PCO2, TCO2, HCO3, and BE are closely related with each other and are also associated with blood CO2; sO2 is calculated based on PO2 and both of them are associated with blood oxygen (O2). pH, PCO2, TCO2, HCO3, and BE CO2 related parameters are indicators of the acid-base balance in blood. Maintaining a stable acid-base balance is a beneficial responses for chickens to be tolerant to high ambient temperature (Gamba et al., 2015). PCO2 is a major component used to evaluate ventilation and respiratory response of the acid-base status (Ardiaca et al., 2013). During heat-stress treatment, birds with more CO2 loss via the lungs reduced the PCO2 in the blood (Sandercock et al., 2001; Borges et al., 2003; Ahmad and Sarwar, 2006; Yalcin et al., 2008; Angell and Seymour, 2015), subsequently, the decreased PCO2 resulted in increased plasma pH with an alkalosis. TCO2 measures the total carbon dioxide and can be calculated from pH and PCO2. Both TCO2 and HCO3 can be used in the evaluation of the acid-base imbalance (Hamm et al., 2013). As a major metabolic component of the acid-base balance (Ahmad et al., 2008), increased HCO3 also caused metabolic alkalosis (Ahmad and Sarwar, 2006; Angell and Seymour, 2015). Blood BE is calculated by the combination of pH and HCO3, and is one of indicators of non-respiratory metabolic disturbance of the acid-base status (Ardiaca et al., 2013). Based on readouts of PCO2 and pH, Fayoumis had significant respiratory alkalosis at both AH and CH1 stages, but not the CH2 stage (Supplementary Figures S6 to S7), while the Leghorn had no significant changes except a decrease of pH at the CH2 stage. Based on readouts of HCO3 and BE, Fayoumis had significant chronic metabolic acidosis (Supplementary Figures S8 to S9), while Leghorns had significant acute metabolic alkalosis (Supplementary Figures S10). These results were partially consistent (consistent in HCO3 in the chronic phase, and in the opposite direction in PCO2) with a previous heat stress study in which naked neck (heat tolerant) and non-naked neck Rhode Island Red layers were used for evaluating blood gas changes with acute (1 hour) and chronic heat stress (8 wk) (Pech-Waffenschmidt et al., 1995). The inconsistency could be due to different experimental designs and different genetic lines used. In summary, heat tolerant birds (Fayoumi) had phenotypes of early respiratory alkalosis and late metabolic acidosis, while heat susceptible birds (Leghorn) had phenotypes of early metabolic alkalosis. At the CH1 stage, Fayoumi birds were able to lower TCO2 and HCO3 to compensate the respiratory alkalosis. However, this was accomplished at the CH2 stage by having a lower pH and normal PCO2. On the other hand, Leghorn birds had metabolic alkalosis at the AH stage, then stopped responding at the CH1 stage without any changes and with a decrease in the pH at the CH2 stage. NDV inoculation could be a contributing factor to late metabolic acidosis in Fayoumies and pH decrement in Leghorns. To our knowledge, this is the first report in which the dynamic signature of blood gas parameters were used to characterize heat tolerant/susceptible birds. PO2, sO2, and Hematology (Hct and Hb) The other two blood gas parameters PO2 and sO2 are mostly associated with the blood O2 levels. PO2 is a sensor of pulmonary ventilation (Bollen et al., 2005). The sO2 measures the amount of O2 binding to Hb. Fayoumi birds demonstrated much stronger physiological function and elasticity with increased sO2 and PO2 in responding to heat stress than Leghorns. Hct is normally used to determine and monitor the fractional volume of red blood cells (Leypoldt et al., 1995), which is one of the key indicators of the body hydration state as well as the blood's capability to transport oxygen (Fair et al., 2007). One of the heat stress symptoms in poultry is the reduction of blood Hct and Hb concentration, which is a consequence of overhydration due to too much water intake (Hilman P.E., 2000; Puvadolpirod and Thaxton, 2000; Zulkifli et al., 2009). During heat stress, when the temperature difference between birds and environment is decreased, passive temperature reduction (radiation, conduction, and convention) cannot be used for birds to lower core body temperature (Freeman et al., 1999). Therefore, heat loss by hyperventilation is facilitated, which results in an increase of water evaporation through respiration, and drinking additional amounts of water is needed to restore the osmotic balance (Ryder et al., 2004). This subsequently caused reduced Hct and Hb due to hematic dilution (Cristina et al., 2004). Results from our current study were mostly consistent with others by having lower levels of Hct and Hb at the CH1 stage in both genetic lines. Hct and Hb levels were similar between non-treated genetic lines across all time points. However, in the AH stage, heat stressed Leghorn birds had significantly higher Hct and Hb levels than heat stressed Fayoumis (Supplementary Figures S11 to S12). It could because that Leghorn birds slightly increased their Hct (P = 0.072) and Hb (P = 0.062) levels with heat stress due to suffering from dehydration at the AH stage. In summary, the present study provided the comprehensive profile of biomarker signatures in blood associated with heat tolerance in the two genetically distinct chicken highly inbred lines. The Leghorn line had a significant acute response to heat, while the Fayoumi had limited early response, but more significant chronic responses during the acclimation to the stress. Specifically, during the heat stress, Leghorn birds had significant electrolyte disruption, while Fayoumis had well-balanced electrolytes, even with enhanced potassium. In addition, Fayoumis had significant respiratory alkalosis and chronic metabolic acidosis, while Leghorns had significant acute metabolic alkalosis. Collectively, the above results suggested potential distinct acute responses between Fayoumi and Leghorn is responsible for determining resilient birds. Finally, significant basal level differences between Fayoumi and Leghorn in PO2, TCO2, HCO3, and BE provided potential biomarkers that can be used to genetically improve heat tolerance in poultry. The insights generated from this study have laid a solid foundation in understanding the genetic control of heat tolerance and susceptibility in poultry. Further investigation on the underlying molecular and cellular mechanisms is warranted. SUPPLEMENTARY DATA Supplementary data are available at Poultry Science online. ACKNOWLEDGEMENTS The study is supported by US Agency for International Development (USAID) AID-OAA-A-13-00080. This study is made possible by the generous support of the American people through USAID. The contents are the responsibility of the Feed the Future Innovation Lab for Genomics to Improve Poultry and do not necessarily reflect the views of USAID or the United States Government. This project was partially supported by Multistate Research Project NE-1334 (CA-D-ASC-2156-RR), NC1170 (CA-D-ASC-2155-RR), from the USDA Cooperative State Research, Education, and Extension Service. REFERENCES Ahmad T., Khalid T., Mushtaq T., Mirza M. A., Nadeem A., Babar M. E., Ahmad G.. 2008. Effect of potassium chloride supplementation in drinking water on broiler performance under heat stress conditions. Poult. Sci.  87: 1276– 1280. Google Scholar PubMed  Ahmad T., Sarwar M.. 2006. Dietary electrolyte balance: implications in heat stressed broilers. World Poult. Sci. J.  62: 638– 653. Ait-Boulahsen A., Garlich J. D., Edens F. W.. 1995. Potassium chloride improves the thermotolerance of chickens exposed to acute heat stress. Poult. Sci.  74: 75– 87. Google Scholar CrossRef Search ADS PubMed  Angell L., Seymour C.. 2015. A simple approach to blood gas analysis: the essentials. Veterinary Nursing J.  30: 5. Google Scholar CrossRef Search ADS   Ardiaca M., Bonvehi C., Montesinos A.. 2013. Point-of-care blood gas and electrolyte analysis in rabbits. Vet. Clin. North Am. Exot. Anim. Pract.  16: 175– 195. Google Scholar CrossRef Search ADS PubMed  Ayo J. O., Obidi J. A., Rekwot P. I.. 2011. Effects of heat stress on the well-being, fertility, and hatchability of chickens in the northern Guinea savannah zone of Nigeria: a review. ISRN Vet. Sci.  2011: 838606. Google Scholar CrossRef Search ADS PubMed  Bjorkquist A. G. R., Rothschild M. F., Persia M. E., Ashwell C., Schmidt C., Lamont S. J.. 2015. Genetic Markers Found for Response to Heat Stress in Chickens in Animal Industry ReportIowa state university. Boddicker R. L., Seibert J. T., Johnson J. S., Pearce S. C., Selsby J. T., Gabler N. K., Lucy M. C., Safranski T. J., Rhoads R. P., Baumgard L. H., Ross J. W.. 2014. Gestational heat stress alters postnatal offspring body composition indices and metabolic parameters in pigs. Plos One.  9. doi ARTN e11085910.1371/journal.pone.0110859. Bollen C. W., van Well G. T., Sherry T., Beale R. J., Shah S., Findlay G., Monchi M., Chiche J. D., Weiler N., Uiterwaal C. S., van Vught A. J.. 2005. High frequency oscillatory ventilation compared with conventional mechanical ventilation in adult respiratory distress syndrome: a randomized controlled trial [ISRCTN24242669]. Crit. Care.  9: R430– 439. Google Scholar CrossRef Search ADS PubMed  Borges S. A., Fischer da Silva A. V., Ariki J., Hooge D. M., Cummings K. R.. 2003. Dietary electrolyte balance for broiler chickens exposed to thermoneutral or heat-stress environments. Poult. Sci.  82: 428– 435. Google Scholar CrossRef Search ADS PubMed  Borges S. A., Fischer da Silva A. V., Majorka A., Hooge D. M., Cummings K. R.. 2004. Physiological responses of broiler chickens to heat stress and dietary electrolyte balance (sodium plus potassium minus chloride, milliequivalents per kilogram). Poult. Sci.  83: 1551– 1558. Google Scholar CrossRef Search ADS PubMed  Cahaner A., Ajuh J. A., Siegmund-Schultze M., Azoulay Y., Druyan S., Zarate A. V.. 2008. Effects of the genetically reduced feather coverage in naked neck and featherless broilers on their performance under hot conditions. Poult. Sci.  87: 2517– 2527. Google Scholar CrossRef Search ADS PubMed  Cristina D., P. M., Cureu I., Braslasu M. C.. 2004. Interaction between the diet, temperature and some blood constituents in Roso hens. Archiva Zootechnica . 7: 5. Deeb N., Cahaner A.. 1999. The effects of naked neck genotypes, ambient temperature, and feeding status and their interactions on body temperature and performance of broilers. Poult. Sci.  78: 1341– 1346. Google Scholar CrossRef Search ADS PubMed  Deeb N., Lamont S. J.. 2002. Genetic architecture of growth and body composition in unique chicken populations. J. Hered . 93: 107– 118. Google Scholar CrossRef Search ADS PubMed  El-Gendy Essam A., Nassar M. K., Mostageer A.. 2007. Genotype-environment interaction in relation to heat tolerance in chickens 2. variation in juvenile growth of warm region's oriented breeds. Int. J. Poult. Sci.  6: 322– 328. Google Scholar CrossRef Search ADS   Fair J., Whitaker S., Pearson B.. 2007. Sources of variation in haematocrit in birds. Ibis . 149: 535– 552. Google Scholar CrossRef Search ADS   Felver-Gant J. N. 2012. Genetic variations alter physiological responses following heat stress in 2 strains of laying hens. Poult. Sci.  91: 1542– 1551. Google Scholar CrossRef Search ADS PubMed  Freeman M. L., Borrelli M. J., Meredith M. J., Lepock J. R.. 1999. On the path to the heat shock response: Destabilization and formation of partially folded protein intermediates, a consequence of protein thiol modification. Free Radical Bio. Med . 26: 737– 745. Google Scholar CrossRef Search ADS   Gamba J. P., Rodrigues M. M., Neto M. G., Perri S. H. V., Faria M. J. D., Pinto M. F.. 2015. The strategic application of electrolyte balance to minimize heat stress in broilers. Braz. J. Poult. Sci.  17: 237– 245. Hamm L. L., Hering-Smith K. S., Nakhoul N. L.. 2013. Acid-base and potassium homeostasis. Semin. Nephrol.  33: 257– 264. Google Scholar CrossRef Search ADS PubMed  Hilman P.E., N. R. S., N. R. A. V. T. 2000. physiological responses and adaptations to hot and cold environments. Pages 71 in Physiology in Liverstock . Y. M. K. ed. Islam M. A., Nishibori M.. 2009. Indigenous naked neck chicken: a valuable genetic resource for Bangladesh. World Poult. Sci. J.  65: 125– 138. Google Scholar CrossRef Search ADS   Kaneko J. J., Harvey J. W., Bruss M. L.. 2008. Clinical Biochemistry of Domestic Animals Sixth Edition Preface . Clinical Biochemistry of Domestic Animals, 6th Edition: Ix– Ix. doi 10.1016/B978-0-12-370491-7.00031-3. Google Scholar CrossRef Search ADS   Lamont S. J., Coble D. J., Bjorkquist A., Rothschild M. F., Persia M., Ashwell C., Schmidt C.. 2014. Genomics of heat stress in chickens. Proc. 10th World Congress of Genetics Applied to Liverstock Production , Canada. Lara L. J., Rostagno M. H.. 2013. Impact of Heat Stress on Poultry Production. Animals (Basel) . 3: 356– 369. Google Scholar CrossRef Search ADS PubMed  Leypoldt J. K., Cheung A. K., Steuer R. R., Harris D. H., Conis J. M.. 1995. Determination of circulating blood volume by continuously monitoring hematocrit during hemodialysis. J. Am. Soc. Nephrol . 6: 214– 219. Google Scholar PubMed  Lu Q., Wen J., Zhang H.. 2007. Effect of chronic heat exposure on fat deposition and meat quality in two genetic types of chicken. Poult. Sci.  86: 1059– 1064. Google Scholar CrossRef Search ADS PubMed  Majekodunmi B. C., Sokunbi O. A., Ogunwole O. A., Adebiyi O. A.. 2013. Influence of electrolytes and ascorbic acid supplementation on serum and erythrocytic indices of broiler chickens reared in a hot environment. Afr. J. Agric. Res.  8: 7. Martin M. P., Wineland M., Barnes H. J.. 2010. Selected blood chemistry and gas reference ranges for broiler breeders using the i-STAT handheld clinical analyzer. Avian Dis . 54: 1016– 1020. Google Scholar CrossRef Search ADS PubMed  Muiruri H. K., Harrison P. C.. 1991. Effect of roost temperature on performance of chickens in hot ambient environments. Poult. Sci.  70: 2253– 2258. Google Scholar CrossRef Search ADS PubMed  Niu Z. Y., Liu F. Z., Yan Q. L., Li W. C.. 2009. Effects of different levels of vitamin E on growth performance and immune responses of broilers under heat stress. Poult. Sci.  88: 2101– 2107. Google Scholar CrossRef Search ADS PubMed  Odom T. W., Harrison P. C., Bottje W. G.. 1986. Effects of thermal-induced respiratory alkalosis on blood ionized calcium levels in the domestic hen. Poult. Sci.  65: 570– 573. Google Scholar CrossRef Search ADS PubMed  Olanrewaju H. A., Thaxton J. P., Dozier W. A.3rd, Branton S. L.. 2007. Electrolyte diets, stress, and acid-base balance in broiler chickens. Poult. Sci.  86: 1363– 1371. Google Scholar CrossRef Search ADS PubMed  Olanrewaju H. A., Wongpichet S., Thaxton J. P., Dozier W. A., Branton S. L.. 2006. Stress and acid-base balance in chickens. Poult. Sci.  85: 1266– 1274. Google Scholar CrossRef Search ADS PubMed  Padgett D. A., Glaser R.. 2003. How stress influences the immune response. Trends Immunol.  24: 444– 448. Google Scholar CrossRef Search ADS PubMed  Pech-Waffenschmidt V., Bogin E., Avidar Y., Horst A.. 1995. Metabolic and biochemical changes during heat stress in relation to the feathering degree of the domestic hen. Avian Pathol.  24: 33– 44. Google Scholar CrossRef Search ADS PubMed  Pracha U.Z., Fatima K., Alqahtani M., Chaudhary A., Abuzenadah A., Damanhouri G., Qadri I.. 2013. Oxidative stress and hepatitis C virus. Virol J.  10: 251. Google Scholar CrossRef Search ADS PubMed  Puvadolpirod S., Thaxton J. P.. 2000. Model of physiological stress in chickens 1. Response parameters. Poult. Sci.  79: 363– 369. Google Scholar CrossRef Search ADS PubMed  Rhoads R. P., Baumgard L. H., Suagee J. K., Sanders S. R.. 2013. Nutritional interventions to alleviate the negative consequences of heat stress. Adv. Nutr.  4: 267– 276. Google Scholar CrossRef Search ADS PubMed  Ryder A. A., Feddes J. J. R., Zuidhof M. J.. 2004. Field study to relate heat stress index to broiler performance. J. Appl. Poult. Res.  13: 493– 499. Google Scholar CrossRef Search ADS   Sahin K. S., Sahin N., Kucuk O., Hayirli A., Prasad A. S.. 2009. Role of dietary zinc in heat-stressed poultry: A review. Poult. Sci.  88: 2176– 2183. Google Scholar CrossRef Search ADS PubMed  Samara M. H., Robbins K. R., Smith M. O.. 1996. Environmental heat stress does not reduce blood ionized calcium concentration in hens acclimated to elevated temperatures. Poult. Sci.  75: 197– 200. Google Scholar CrossRef Search ADS PubMed  Sandercock D. A., Hunter R. R., Nute G. R., Mitchell M. A., Hocking P. M.. 2001. Acute heat stress-induced alterations in blood acid-base status and skeletal muscle membrane integrity in broiler chickens at two ages: Implications for meat quality. Poult. Sci.  80: 418– 425. Google Scholar CrossRef Search ADS PubMed  Schaal T. P., Arango J., Wolc A., Brady J. V., Fulton J. E., Rubinoff I., Ehr I. J., Persia M. E., O'Sullivan N. P.. 2016. Commercial Hy-Line W-36 pullet and laying hen venous blood gas and chemistry profiles utilizing the portable i-STAT(R)1 analyzer. Poult. Sci.  95: 466– 471. Google Scholar CrossRef Search ADS PubMed  Sgavioli S., Santos E. T., Domingues C. H. F., Quadros T. C. O., Castiblanco D. M. C., Andrade-Garcia G. M., Amoroso L., Naas I. A., Garcia R. G., Baraldi-Artoni S. M.. 2016. Effect of high incubation temperature on the blood parameters of layer chicks. Braz. J. Poult. Sci.  18: 41– 47. Soleimani A. F., Zulkifli I.. 2010. Effects of high ambient temperature on blood parameters in red jungle fowl, village fowl and broiler chickens. J. Anim. Vet. Adv.  9: 1201– 1207. Google Scholar CrossRef Search ADS   Soleimani A. F., Zulkifli I., Omar A. R., Raha A. R.. 2011. Physiological responses of 3 chicken breeds to acute heat stress. Poult. Sci.  90: 1435– 1440. Google Scholar CrossRef Search ADS PubMed  Ungerer J. P. J., Ungerer M. J., Vermaak W. J. H.. 1990. Discordance between measured and calculated total carbon-dioxide. Clin. Chem.  36: 2093– 2096. Google Scholar PubMed  Van Goor A., Ashwell C. M., Persia M. E., Rothschild M. F., Schmidt C. J., Lamont S. J.. 2016. Quantitative trait loci identified for blood chemistry components of an advanced intercross line of chickens under heat stress. BMC Genomics.  17: 287. Google Scholar CrossRef Search ADS PubMed  Van Goor A., Ashwell C. M., Persia M. E., Rothschild M. F., Schmidt C. J., Lamont S. J.. 2017. Unique genetic responses revealed in RNA-seq of the spleen of chickens stimulated with lipopolysaccharide and short-term heat. PLoS One.  12: e0171414. Google Scholar CrossRef Search ADS PubMed  Van Goor A., Bolek K. J., Ashwell C. M., Persia M. E., Rothschild M. F., Schmidt C. J., Lamont S. J.. 2015. Identification of quantitative trait loci for body temperature, body weight, breast yield, and digestibility in an advanced intercross line of chickens under heat stress. Genet. Sel. Evol.  47. doi ARTN 9610.1186/s12711-015-0176-7. Wang Y., Lupiani B., Reddy S. M., Lamont S. J., Zhou H.. 2014. RNA-seq analysis revealed novel genes and signaling pathway associated with disease resistance to avian influenza virus infection in chickens. Poult. Sci.  93: 485– 493. Google Scholar CrossRef Search ADS PubMed  Wilson H. R., Wilcox C. J., Voitle R. A., Baird C. D., Dorminey R. W.. 1975. Characteristics of White Leghorn chickens selected for heat tolerance. Poult. Sci.  54: 126– 130. Google Scholar CrossRef Search ADS PubMed  Wolfenson D., Bachrach D., Maman M., Graber Y., Rozenboim I.. 2001. Evaporative cooling of ventral regions of the skin in heat-stressed laying hens. Poult. Sci.  80: 958– 964. Google Scholar CrossRef Search ADS PubMed  Yalcin S., Cabuk M., Bruggeman V., Babacanoglu E., Buyse J., Decuypere E., Siegel P. B.. 2008. Acclimation to heat during incubation. 1. Embryonic morphological traits, blood biochemistry, and hatching performance. Poult. Sci. 87: 1219– 1228. Yalcin S., Ozkan S., Turkmut L., Siegel P. B.. 2001. Responses to heat stress in commercial and local broiler stocks. 1. Performance traits. Br. Poult. Sci.  42: 149– 152. Google Scholar CrossRef Search ADS PubMed  Yunis R., Cahaner A.. 1999. The effects of the naked neck (Na) and frizzle (F) genes on growth and meat yield of broilers and their interactions with ambient temperatures and potential growth rate. Poult. Sci.  78: 1347– 1352. Google Scholar CrossRef Search ADS PubMed  Zhou H., Lamont S. J.. 1999. Genetic characterization of biodiversity in highly inbred chicken lines by microsatellite markers. Anim. Genet.  30: 256– 264. Google Scholar CrossRef Search ADS PubMed  Zulkifli I., Al-Aqil A., Omar A. R., Sazili A. Q., Rajion M. A.. 2009. Crating and heat stress influence blood parameters and heat shock protein 70 expression in broiler chickens showing short or long tonic immobility reactions. Poult. Sci.  88: 471– 476. Google Scholar CrossRef Search ADS PubMed  © 2017 Poultry Science Association Inc.

Journal

Poultry ScienceOxford University Press

Published: Mar 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 12 million articles from more than
10,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Unlimited reading

Read as many articles as you need. Full articles with original layout, charts and figures. Read online, from anywhere.

Stay up to date

Keep up with your field with Personalized Recommendations and Follow Journals to get automatic updates.

Organize your research

It’s easy to organize your research with our built-in tools.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

Monthly Plan

  • Read unlimited articles
  • Personalized recommendations
  • No expiration
  • Print 20 pages per month
  • 20% off on PDF purchases
  • Organize your research
  • Get updates on your journals and topic searches

$49/month

Start Free Trial

14-day Free Trial

Best Deal — 39% off

Annual Plan

  • All the features of the Professional Plan, but for 39% off!
  • Billed annually
  • No expiration
  • For the normal price of 10 articles elsewhere, you get one full year of unlimited access to articles.

$588

$360/year

billed annually
Start Free Trial

14-day Free Trial