Effect of heat stress on the hypothalamic expression of water channel- and noncoding RNA biogenesis-related genes in modern broilers and their ancestor red jungle fowl.

Genetic selection for high growth rate has resulted in spectacular progress in feed efficiency in chickens. As feed intake and water consumption (WC) are associated and both are affected by environmental conditions, we evaluated WC and its hypothalamic regulation in three broiler-based research lines and their ancestor jungle fowl (JF) under heat stress (HS) conditions. Slow growing ACRB, moderate growing 95RB, fast growing MRB, and JF were exposed to daily chronic cyclic HS (36 °C, 9 h/d) or thermoneutral temperature (24 °C). HS increased WC in the MRB only. Arginine vasopressin (AVP) mRNA levels were decreased by HS in the MRB. Within the renin-angiotensin-aldosterone system (RAAS) system, renin expression was increased by HS in the JF, ACRB, and 95RB, while angiotensin I-converting enzyme (ACE), angiotensin II receptors (type 1, AT1, and type 2, AT2) were affected by line. The expression of aquaporin (AQP2, 7, 9, 10, 11, and 12) genes was upregulated by HS, whereas AQP4 and AQP5 expressions were influenced by line. miRNA processing components (Dicer1, Ago2, Drosha) were significantly different among the lines, but were unaffected by HS. In summary, this is the first report showing the effect of HS on hypothalamic water channel- and noncoding RNA biogenesis-related genes in modern chicken populations and their ancestor JF. These results provide a novel framework for future research to identify new molecular mechanisms and signatures involved in water homeostasis and adaptation to HS.

Effect of heat stress on the hypothalamic expression profile of water homeostasis-associated genes in low- and high-water efficient chicken lines.

With climate change, selection for water efficiency and heat resilience are vitally important. We undertook this study to determine the effect of chronic cyclic heat stress (HS) on the hypothalamic expression profile of water homeostasis-associated markers in high (HWE)- and low (LWE)-water efficient chicken lines. HS significantly elevated core body temperatures of both lines. However, the amplitude was higher by 0.5-1°C in HWE compared to their LWE counterparts. HWE line drank significantly less water than LWE during both thermoneutral (TN) and HS conditions, and HS increased water intake in both lines with pronounced magnitude in LWE birds. HWE had better feed conversion ratio (FCR), water conversion ratio (WCR), and water to feed intake ratio. At the molecular level, the overall hypothalamic expression of aquaporins (AQP8 and AQP12), arginine vasopressin (AVP) and its related receptor AVP2R, angiotensinogen (AGT), angiotensin II receptor type 1 (AT1), and calbindin 2 (CALB2) were significantly lower; however, CALB1 mRNA and AQP2 protein levels were higher in HWE compared to LWE line. Compared to TN conditions, HS exposure significantly increased mRNA abundances of AQPs (8, 12), AVPR1a, natriuretic peptide A (NPPA), angiotensin I-converting enzyme (ACE), CALB1 and 2, and transient receptor potential cation channel subfamily V member 1 and 4 (TRPV1 and TRPV4) as well as the protein levels of AQP2, however it decreased that of AQP4 gene expression. A significant line by environment interaction was observed in several hypothalamic genes. Heat stress significantly upregulated AQP2 and SCT at mRNA levels and AQP1 and AQP3 at both mRNA and protein levels, but it downregulated that of AQP4 protein only in LWE birds. In HWE broilers, however, HS upregulated the hypothalamic expression of renin (REN) and AVPR1b genes and AQP5 proteins, but it downregulated that of AQP3 protein. The hypothalamic expression of AQP (5, 7, 10, and 11) genes was increased by HS in both chicken lines. In summary, this is the first report showing improvement of growth performances in HWE birds. The hypothalamic expression of several genes was affected in a line- and/or environment-dependent manner, revealing potential molecular signatures for water efficiency and/or heat tolerance in chickens.

Effect of Cyclic Heat Stress on Hypothalamic Oxygen Homeostasis and Inflammatory State in the Jungle Fowl and Three Broiler-Based Research Lines.

Heat stress (HS) is devastating to poultry production sustainability due its detrimental effects on performance, welfare, meat quality, and profitability. One of the most known negative effects of HS is feed intake depression, which is more pronounced in modern high-performing broilers compared to their ancestor unselected birds, yet the underlying molecular mechanisms are not fully defined. The present study aimed, therefore, to determine the hypothalamic expression of a newly involved pathway, hypoxia/oxygen homeostasis, in heat-stressed broiler-based research lines and jungle fowl. Three populations of broilers (slow growing ACRB developed in 1956, moderate growing 95RB from broilers available in 1995, and modern fast growing MRB from 2015) and unselected Jungle fowl birds were exposed to cyclic heat stress (36°C, 9 h/day for 4 weeks) in a 2 × 4 factorial experimental design. Total RNAs and proteins were extracted from the hypothalamic tissues and the expression of target genes and proteins was determined by real-time quantitative PCR and Western blot, respectively. It has been previously shown that HS increased core body temperature and decreased feed intake in 95RB and MRB, but not in ACRB or JF. HS exposure did not affect the hypothalamic expression of HIF complex, however there was a line effect for HIF-1α (P = 0.02) with higher expression in JF under heat stress. HS significantly up regulated the hypothalamic expression of hemoglobin subunits (HBA1, HBBR, HBE, HBZ), and HJV in ACRB, HBA1 and HJV in 95RB and MRB, and HJV in JF, but it down regulated FPN1 in JF. Additionally, HS altered the hypothalamic expression of oxygen homeostasis- up and down-stream signaling cascades. Phospho-AMPKThr172 was activated by HS in JF hypothalamus, but it decreased in that of the broiler-based research lines. Under thermoneutral conditions, p-AMPKThr172 was higher in broiler-based research lines compared to JF. Ribosomal protein S6K1, however, was significantly upregulated in 95RB and MRB under both environmental conditions. HS significantly upregulated the hypothalamic expression of NF-κB2 in MRB, RelB, and TNFα in ACRB, abut it down regulated RelA in 95RB. The regulation of HSPs by HS seems to be family- and line-dependent. HS upregulated the hypothalamic expression of HSP60 in ACRB and 95RB, down regulated HSP90 in JF only, and decreased HSP70 in all studied lines. Taken together, this is the first report showing that HS modulated the hypothalamic expression of hypoxia- and oxygen homeostasis-associated genes as well as their up- and down-stream mediators in chickens, and suggests that hypoxia, thermotolerance, and feed intake are interconnected, which merit further in-depth investigations.

Ileal microbial composition in genetically distinct chicken lines reared under normal or high ambient temperatures.

BACKGROUND: Heat stress (HS) has negative effects on poultry productivity, health and welfare resulting in economic losses. Broiler chickens are particularly susceptible to HS due to their high metabolic rate and rapid growth. The commensal intestinal bacterial populations have an important physiological role in the host and could ameliorate the negative effect of HS on the host. Thus, the aim of this study was to compare changes in the ileal (IL) microbiota in four different broiler lines during HS. RESULTS: Day-old broiler chicks from Giant Jungle Fowl (JF), Athens Canadian Random Bred (ACRB), 1995 Random Bred (L1995), and Modern Random Bred (L2015) lines were raised under thermoneutral (TN) conditions until day (d) 28. On d 29 birds were subjected to TN (24 °C) or chronic cyclic HS (8 h/d, 36 °C) condition till d 56. On d 56 two birds per pen were euthanized, and IL luminal content (IL-L) and mucosal scrapings (IL-M) were collected for bacterial DNA isolation. Libraries were constructed using V3-V4 16S rRNA primers and sequenced using MiSeq. DNA sequences were analyzed using QIIME2 platform and SILVA 132 database for alpha and beta diversity, and taxonomic composition, respectively. Functional property of microbiota was predicted using the PICRUSt 2 pipeline and illustrated with STAMP software. Shannon index was significantly elevated in IL-M under HS. ß-diversity PCoA plots revealed separation of microbial community of L2015-TN from JF-TN, JF-HS, ACRB-TN, and ACRB-HS in the IL-M. PERMANOVA analysis showed a significant difference between microbial community of L1995-HS compared to ACRB-HS and JF-TN, L1995-TN compared to ACRB-HS and JF-TN, L2015-HS compared to ACRB-HS and ACRB-TN, L2015-HS compared to JF-TN, L2015-TN compared to ACRB-HS and JF-TN, and ACRB-HS compared to JF-TN in the IL-L. The impact of HS on microbial composition of IL-M was more prominent compared to IL-L with 12 and 2 taxa showing significantly different relative abundance, respectively. Furthermore, differences in microbiota due to the genetic line were more prominent in IL-M than IL-L with 18 and 8 taxa showing significantly different relative abundance, respectively. Unlike taxonomy, predicted function of microbiota was not affected by HS. Comparison of L2015 with JF or ACRB showed significant changes in predicted function of microbiota in both, IL-M and IL-L. Differences were most prominent between L2015 and JF; while there was no difference between L2015 and L1995. CONCLUSIONS: These data indicate the genetic line × temperature effect on the diversity and composition of IL microbiota. Moreover, the data showcase the effect of host genetics on the composition of IL microbiota and their predicted function. These data are of critical importance for devising nutritional strategies to maintain GIT microbial balance and alleviate the negative effects of HS on broiler chickens' performance and health.

Effect of Cyclic Heat Stress on Feeding-Related Hypothalamic Neuropeptides of Three Broiler Populations and Their Ancestor Jungle Fowl.

Heat stress (HS) has been increasingly jeopardizing the sustainability of the poultry production. Moreover, modern high-performing chickens are far less able to withstand HS than their predecessors due to higher growth rate and metabolic rates. Performance losses caused by HS are mainly ascribed to decreases in feed consumption. Since feed intake is tightly controlled by the hypothalamic centers of hunger and satiety, we sought to determine the effect of chronic cyclic HS on the expression of feeding-related hypothalamic neuropeptides (FRHN) in unselected chickens (i.e., the ancestor junglefowl-JF) and three broiler lines from diverse stages of genetic selection (i.e., the slow growing ACRB, the moderate growing 95RN, and the fast growing MRB). From 29 to 56 days, birds (n = 150 birds for each population) were subjected to either thermoneutral (TN, 25°C) or cyclic heat stress (HS, 36°C, 0900-1,800 h) conditions. Molecular data were analyzed by two-way ANOVA with interaction between the main factors, namely environmental temperature and line. The expression of major FHRN, like neuropeptide Y, agouti-related peptide, proopiomelanocortin, and cocaine and amphetamine regulated transcript remained unchanged. However, melanocortin receptor 1 exhibited a line-dependent decreasing trend from JF to MRB under both TN and HS (p = 0.09), adiponectin expression showed a distinct trend toward significance with 95RB exhibiting the highest mRNA level irrespective of the environmental temperature (p = 0.08), and JF had a greater mRNA abundance of visfatin than ACRB under TN (p < 0.05). The hypothalamic integration of circadian information, acclimation to long-lasting HS exposure, stable hypothalamic pathways unaffected by evolution and genetic selection, focus on mRNA abundances, and use of the entire hypothalamus masking gene expression in specific hypothalamic nuclei are all possible explanations for the lack of variations observed in this study. In conclusion, this is the first assessment of the impacts of heat stress on feeding-related hypothalamic neuropeptides of chicken, with a valuable and informative comparison between the ancestor junglefowl and three differently performing broiler lines.

Effects of Cyclic Chronic Heat Stress on the Expression of Nutrient Transporters in the Jejunum of Modern Broilers and Their Ancestor Wild Jungle Fowl.

Heat stress (HS) has been reported to disrupt nutrient digestion and absorption in broilers. These effects may be more prominent in fast-growing chickens due to their high metabolic activity. However, the underlying molecular mechanisms are not yet fully elucidated. Hence, the current study aimed to evaluate the effect of chronic HS on jejunal nutrient transport in slow- (Athens Canadian Random Bred, ACRB from 1950), moderate- (The 1995 random bred, 95RAN), rapid- (modern broilers, modern random bred, MRB) growing birds and their ancestor wild jungle fowl (JF). One-day male chicks (n = 150/line) were placed by line in environmentally controlled chambers and kept under the same industry-standard environmental conditions until d28. On d29, an 8-h daily cyclic HS (36°C) was applied to half of the chambers, which lasted until d55, while keeping the rest under thermal neutral (TN, 24°C) conditions. Jejunum tissues were collected for morphology assessment and molecular analysis of carbohydrate-, amino acid-, and fatty acid-transporters. MRB exhibited the highest body weight (BW) followed by 95RAN under both conditions. HS decreased feed intake (FI) in MRB and 95RAN, which resulted in lower BW compared to their TN counterparts; however, no effect was observed in ACRB and JF. MRB showed a greater villus height (VH) to crypt depth (CD) ratio under both environmental conditions. Molecular analyses showed that glucose transporter (GLUT) 2, 5, 10, and 11 were upregulated in MRB compared to some of the other populations under TN conditions. HS downregulated GLUT2, 10, 11, and 12 in MRB while it increased the expression of GLUT1, 5, 10, and 11 in JF. GLUT2 protein expression was higher in JF compared to ACRB and MRB under TN conditions. It also showed an increase in ACRB but no effect on 95RAN and MRB under HS conditions. ACRB exhibited greater expression of the EAAT3 gene as compared to the rest of the populations maintained under TN conditions. HS exposure did not alter the gene expression of amino acid transporters in MRB. Gene expression of CD36 and FABP2 was upregulated in HS JF birds. Protein expression of CD36 was downregulated in HS JF while no effect was observed in ACRB, 95RAN, and MRB. Taken together, these data are the first to show the effect of HS on jejunal expression of nutrient transporters in three broiler populations known to represent 70 years of genetic progress in the poultry industry and a Red Jungle Fowl population representative of the primary ancestor of domestic chickens.

Intestinal Barrier Integrity in Heat-Stressed Modern Broilers and Their Ancestor Wild Jungle Fowl.

High environmental temperature has strong adverse effects on poultry production, welfare, and sustainability and, thereby, constitutes one of the most challenging stressors. Although colossal information has been published on the effects of heat stress on poultry productivity and gut health, the fundamemntal mechanisms associated with heat stress responses and intestinal barrier function are still not well defined. The aim of the present study was, therefore, to determine the effects of acute (2 h) heat stress on growth performance, gut integrity, and intestinal expression of heat shock and tight junction proteins in slow- (broilers of the 1950's, ACRB), moderate- (broilers of 1990's, 95RAN), rapid-(modern broilers, MRB) growing birds, and their ancestor wild jungle fowl (JF). Heat stress exposure significantly increased the core body temperature of 95RAN and MRB chickens by ~0.5-1°C, but not that of JF and ACRB compared to their counterparts maintained at thermoneutral conditions. Heat stress also depressed feed intake and increased serum fluorescein isothiocyanate-dextran (FITC-D) levels (P < 0.05) in modern broilers (95RAN and MRB) but not in JF and ACRB, indicating potential leaky gut syndrome. Molecular analyses showed that heat stress exposure significantly up regulated the duodenal expression of occludin (OCLN) and lipocalin (LCN2) in ACRB, zonula occludens (ZO-2), villin1 (VIL1), and calprotectin (CALPR) in 95 RAN, and only CALPR in MRB compared to their TN counterparts. In the jejunum however, heat stress down regulated the expression of PALS1-associated tight junction protein (PATJ) in ACRB, 95RAN, and MRB, and that of cadherin1 (CDH1) in MRB. In the ileum, heat stress significantly down regulated the expression of OCLN in 95 RAN, ZO-1 in MRB, gap junction protein alpha1 (GJA1) in JF, and VIL1 in ACRB compared to their TN counterparts. In summary, this is the first report, to our knowledge, showing that tight junction protein expression is environmental-, genotype-, and intestinal segment-dependent and identifying molecular signatures, such as CDH1, CALPR, and ZO-1, potentially involved in leaky gut syndrome-induced by heat stress in MRB.