Mohammad A. Bahry

Acute heat stress up-regulates neuropeptide Y precursor mRNA expression and alters brain and plasma concentrations of free amino acids in chicks

Heat stress causes an increase in body temperature and reduced food intake in chickens. Several neuropeptides and amino acids play a vital role in the regulation of food intake. However, the responses of neuropeptides and amino acids to heat-stress-induced food-intake regulation are poorly understood. In the current study, the hypothalamic mRNA expression of some neuropeptides related to food intake and the content of free amino acids in the brain and plasma was examined in 14-day-old chicks exposed to a high ambient temperature (HT; 40±1 °C for 2 or 5 h) or to a control thermoneutral temperature (CT; 30±1 °C). HT significantly increased rectal temperature and plasma corticosterone level and suppressed food intake. HT also increased the expression of neuropeptide Y (NPY) and agouti-signaling protein (ASIP) precursor mRNA, while no change was observed in pro-opiomelanocortin, cholecystokinin, ghrelin, or corticotropin-releasing hormone precursor mRNA. It was further found that the diencephalic content of free amino acids - namely, tryptophan, leucine, isoleucine, valine and serine - was significantly higher in HT chicks with some alterations in their plasma amino acids in comparison with CT chicks. The induction of NPY and ASIP expression and the alteration of some free amino acids during HT suggest that these changes can be the results or causes the suppression of food intake.

Central administration of neuropeptide Y differentially regulates monoamines and corticosterone in heat-exposed fed and fasted chicks.

Recently, we demonstrated that brain neuropeptide Y (NPY) mRNA expression was increased in heat exposed chicks. However, the functions of brain NPY during heat stress are unknown. This study was conducted to investigate whether centrally administered NPY affects food intake, rectal temperature, monoamines, stress hormones and plasma metabolites in chicks under high ambient temperatures (HT). Five or six-day-old chicks were centrally injected with 0, 188 or 375pmol of NPY and exposed to either HT (35±1°C) or a control thermoneutral temperature (CT; 30±1°C) for 3h whilst fed or fasted. NPY increased food intake under both CT and HT. NPY reduced rectal temperature 1 and 2h after central administration under CT, but not under HT. Interestingly, NPY decreased brain serotonin and norepinephrine concentrations in fed chicks, but increased concentrations of brain dopamine and its metabolites in fasted and fed chicks, respectively. Plasma epinephrine was decreased by NPY in fed chicks, but plasma concentrations of norepinephrine and epinephrine were increased significantly by NPY in fasted-heat exposed chicks. Furthermore, NPY significantly reduced plasma corticosterone concentrations in fasted chicks. Plasma glucose and triacylglycerol were increased by NPY in fed chicks, but triacylglycerol declined in fasted NPY-injected chicks. In conclusion, brain NPY may attenuate the reduction of food intake during heat stress and the increased brain NPY might be a potential regulator of the monoamines and corticosterone to modulate stress response in heat-exposed chicks.

Chronic oral administration of pine bark extract (flavangenol) attenuates brain and liver mRNA expressions of HSPs in heat-exposed chicks

Exposure to a high ambient temperature (HT) can cause heat stress, which has a huge negative impact on physiological functions. Cellular heat-shock response is activated upon exposure to HT for cellular maintenance and adaptation. In addition, antioxidants are used to support physiological functions under HT in a variety of organisms. Flavangenol, an extract of pine bark, is one of the most potent antioxidants with its complex mixture of polyphenols. In the current study, chronic (a single daily oral administration for 14 days) or acute (a single oral administration) oral administration of flavangenol was performed on chicks. Then the chicks were exposed to an acute HT (40±1°C for 3h) to examine the effect of flavangenol on the mRNA expression of heat-shock protein (HSP) in the brain and liver. Rectal temperature, plasma aspartate aminotransferase (AAT), a marker of liver damage, and plasma corticosterone as well as metabolites were also determined. HSP-70 and -90 mRNA expression, rectal temperature, plasma AAT and corticosterone were increased by HT. Interestingly, the chronic, but not the acute, administration of flavangenol caused a declining in the diencephalic mRNA expression of HSP-70 and -90 and plasma AAT in HT-exposed chicks. Moreover, the hepatic mRNA expression of HSP-90 was also significantly decreased by chronic oral administration of flavangenol in HT chicks. These results indicate that chronic, but not acute, oral administration of flavangenol attenuates HSP mRNA expression in the central and peripheral tissues due to its possible role in improving cellular protective functions during heat stress. The flavangenol-dependent decline in plasma AAT further suggests that liver damage induced by heat stress was minimized by flavangenol.

l-Leucine acts as a potential agent in reducing body temperature at hatching and affords thermotolerance in broiler chicks

Thermal manipulation (TM) of incubation temperature causes metabolic alterations and contributes to improving thermotolerance in chicks post hatching. However, there has been no report on amino acid metabolism during TM and the part it plays in thermotolerance. In this study, we therefore first analyzed free amino acid concentrations in the embryonic brain and liver during TM (38.6°C, 6h/d during embryonic day (ED) 10 to ED 18). It was found that leucine (Leu), phenylalanine and lysine were significantly decreased in the embryonic brain and liver. We then chose l-Leu and other branched-chain amino acids (l-isoleucine (L-Ile) and l-valine (l-Val)) for in ovo injection on ED 7 to reveal their roles in thermoregulation, growth, food intake and thermotolerance in chicks. It was found that in ovo injection of l-Leu, but not of l-Ileu or l-Val, caused a significant decline in body temperature at hatching and increased food intake and body weight gain in broiler chicks. Interestingly, in ovo injection of l-Leu resulted in the acquisition of thermotolerance under high ambient temperature (35±1°C for 180min) in comparison with the control thermoneutral temperature (28±1°C for 180min). These results indicate that the free amino acid concentrations during embryogenesis were altered by TM. l-Leu administration in eggs caused a reduction in body temperature at hatching, and afforded thermotolerance in heat-exposed young chicks, further suggesting that l-Leu may be one of the key metabolic factors involved in controlling body temperature in embryos, as well as in producing thermotolerance after hatching.

L-Citrulline acts as potential hypothermic agent to afford thermotolerance in chicks

Recently we demonstrated that L-citrulline (L-Cit) causes hypothermia in chicks. However, the question of how L-Cit mediates hypothermia remained elusive. Thus, the objective of this study was to examine some possible factors in the process of L-Cit-mediated hypothermia and to confirm whether L-Cit can also afford thermotolerance in young chicks. Chicks were subjected to oral administration of L-Cit along with intraperitoneal injection of a nitric oxide synthase (NOS) inhibitor, NG-nitro-l-arginine methyl ester HCl (L-NAME), to examine the involvement of NO in the process of hypothermia. Food intake and plasma metabolites were also analyzed after oral administration of L-Cit in chicks. To examine thermotolerance, chicks were orally administered with a single dose of L-Cit (15mmol/10ml/kg body weight) or the same dose twice within a short interval of 1h (dual oral administration) before the exposure to high ambient temperature (35 ± 1°C) for 180min. Although the rectal temperature was reduced following administration of L-Cit, L-NAME caused a greater reduction. L-NAME reduced total NO2 and NO3 (NOx) in plasma, which confirmed its inhibitory effect on NO. A single oral administration of L-Cit mediated a persistent state of hypothermia for the 300min of the study without affecting food intake. It was further found that plasma glucose was significantly lower in L-Cit-treated chicks. Dual oral administration of L-Cit, but not a single oral administration, afforded thermotolerance without a significant change in plasma NOx in chicks. In conclusion, our results suggest that L-Cit-mediated hypothermia and thermotolerance may not be involved in NO production. L-Cit-mediated thermotolerance further suggests that L-Cit may serve as an important nutritional supplement that could help in coping with summer heat.

Central NPY‐Y5 sub‐receptor partially functions as a mediator of NPY‐induced hypothermia and affords thermotolerance in heat‐exposed fasted chicks

Exposure of chicks to a high ambient temperature (HT) has previously been shown to increase neuropeptide Y (NPY) mRNA expression in the brain. Furthermore, it was found that NPY has anti‐stress functions in heat‐exposed fasted chicks. The aim of the study was to reveal the role of central administration of NPY on thermotolerance ability and the induction of heat‐shock protein (HSP) and NPY sub‐receptors (NPYSRs) in fasted chicks with the contribution of plasma metabolite changes. Six‐ or seven‐day‐old chicks were centrally injected with 0 or 375 pmol of NPY and exposed to either HT (35 ± 1°C) or control thermoneutral temperature (CT: 30 ± 1°C) for 60 min while fasted. NPY reduced body temperature under both CT and HT. NPY enhanced the brain mRNA expression of HSP‐70 and ‐90, as well as of NPYSRs‐Y5, ‐Y6, and ‐Y7, but not ‐Y1, ‐Y2, and ‐Y4, under CT and HT. A coinjection of an NPYSR‐Y5 antagonist (CGP71683) and NPY (375 pmol) attenuated the NPY‐induced hypothermia. Furthermore, central NPY decreased plasma glucose and triacylglycerol under CT and HT and kept plasma corticosterone and epinephrine lower under HT. NPY increased plasma taurine and anserine concentrations. In conclusion, brain NPYSR‐Y5 partially afforded protective thermotolerance in heat‐exposed fasted chicks. The NPY‐mediated reduction in plasma glucose and stress hormone levels and the increase in free amino acids in plasma further suggest that NPY might potentially play a role in minimizing heat stress in fasted chicks.

Oral administration of a medium containing both D-aspartate-producing live bacteria and D-aspartate reduces rectal temperature in chicks

ABSTRACT 1. The aim of this study was to investigate the effects on the rectal temperature of young chicks of the oral administration of a medium that contained both live bacteria that produce D-aspartate (D-Asp) and D-Asp. 2. In Experiment 1, chicks were subjected to chronic oral administration of either the medium (containing live bacteria and 2.46 μmol D-Asp) or water from 7 to 14 d of age. Plasma-free amino acids as well as mitochondrial biogenic gene expression in the breast muscle were analysed. In Experiment 2, 7-d-old chicks were subjected to acute oral administration of the above medium or of an equimolar amount of D-Asp to examine their effect on changes in rectal temperature. In Experiment 3, after 1 week of chronic oral administration of the medium, 14-d-old chicks were exposed to either high ambient temperature (HT; 40 ± 1°C, 3 h) or control thermoneutral temperature (CT; 30 ± 1°C, 3 h) to monitor the changes in rectal temperature. 3. Chronic, but not acute, oral administration of the medium significantly reduced rectal temperature in chicks, and a chronic effect also appeared under HT conditions. 4. Chronic oral administration of the medium significantly reduced the mRNA abundance of the avian uncoupling protein (avUCP) in the breast muscle, but led to a significant increase in avian adenine nucleotide translocator (avANT) mRNA in the same muscle. 5. (a) These results indicate that the medium can reduce body temperature through the decline in avUCP mRNA expression in the breast muscle that may be involved in reduced mitochondrial proton leaks and heat production. (b) The increase in avANT further suggests a possible enhancement of adenosine triphosphate (ATP) synthesis.

l-Ornithine is a potential acute satiety signal in the brain of neonatal chicks

Recently, we observed that neonatal chicks exhibit feeding behavior characterized by frequent food intake and short resting intervals, with changes detected in the brain amino acid and monoamine concentrations. In this study, we aimed to clarify further the relationship between the appetite of neonatal chicks and brain amino acid metabolism. In Experiment 1, changes were investigated in free amino acids in the brain under conditions of regulated appetite induced by fasting and subsequent short-term re-feeding. Chicks (5 days old) were distributed into four treatment groups--namely, fasting for 3h, and fasting for 3h followed by re-feeding for 10, 20 or 30 min. Brain samples were collected after treatment to analyze free amino acid concentrations. Amino adipic acid and proline in all brain parts as well as arginine and ornithine in all brain parts--except mesencephalic arginine and cerebellar ornithine--were increased in a time-dependent manner following re-feeding. In Experiment 2, we further examined the effect of exogenous administration of some amino acids altered in association with feeding behavior in Experiment 1. We chose L-arginine and its functional metabolite, L-ornithine, to analyze their effects on food intake in chicks. Intracerebroventricular injection (2 μmol) of L-ornithine, but not L-arginine, significantly inhibited food intake in neonatal chicks. In Experiment 3, we found that central injection of L-ornithine (2, 4, and 6 μmol) dose-dependently suppressed food intake in chicks. These results suggested that L-ornithine may have an important role in the control of food intake as an acute satiety signal in the neonatal chick brain.

Reduction in voluntary food intake, but not fasting, stimulates hypothalamic gonadotropin-inhibitory hormone precursor mRNA expression in chicks under heat stress

Heat stress is an issue of rising concern across the globe. Recently, we found that mRNA expression of gonadotropin-inhibitory hormone (GnIH), an orexigenic neuropeptide, was increased in the heat-exposed chick brain when food intake was reduced. The aim of the current study was to examine mRNA expression of GnIH and of the glucocorticoid receptors (GRs) in the hypothalamus as well as the plasma corticosterone (CORT) and metabolites in 14-d-old chicks exposed to a high ambient temperature (HT; 40 ± 1 °C for 1 or 5 h) or a control thermoneutral temperature (CT; 30 ± 1 °C), either with free access to food or fasted. Heat stress caused a voluntary reduction of food intake and reduced plasma triacylglycerol concentration, but increased rectal temperature and plasma CORT and glucose concentrations (P < 0.05). Heat stress also increased (P < 0.05) the expression of diencephalic GnIH mRNA in chicks when they reduced food intake voluntarily, but did not do so under fasting conditions. Although the expression of GR mRNA was not altered as a result of heat stress, its expression was decreased (P < 0.05) in fasted chicks at 5 h in comparison with fed chicks. In addition, the rectal temperature of fasted chicks was lower than that of fed chicks under both CT and HT. In conclusion, voluntary reduction of food intake caused an increase in brain GnIH mRNA expression, plasma CORT, and body temperature in chicks under heat stress. Interestingly, brain GnIH mRNA expression was not induced by heat stress in fasted chicks and was not accompanied by a decrease in rectal temperature. These results suggest that the increased expression of brain GnIH mRNA in chicks under heat stress could be a consequence of a mechanism mediated by the voluntary reduction of food intake, but that it is not a consequence of fasting.

Cortisol responses of goldfish (Carassius auratus) to air exposure, chasing, and increased water temperature

Fish can respond to stimuli from the internal or external environment with activation of the hypothalamo-pituitary-interrenal (HPI) axis and the secretion of cortisol. Stimuli that activate the HPI axis of fish include short term air exposure and increases in water temperature. The present study was conducted to determine how quickly cortisol concentrations increase in goldfish subjected to an increase in water temperature, and to compare the response to an increase in water temperature with responses to other stimuli. Plasma cortisol concentrations varied widely between individual goldfish, with concentrations ranging from 9.1 to 516.0 ng/mL in goldfish on the day of arrival from the supplier. Mean cortisol concentrations in undisturbed goldfish were low (4.5 ± 1.0 ng/mL). Mean cortisol concentrations in fish exposed to air for 3 min and in fish that experienced chasing for 10 min were markedly elevated 15 min after the beginning of the stimuli (132.6 ± 31.0 and 121.1 ± 23.9 ng/mL respectively). Mean cortisol concentrations in fish that experienced an increase in water temperature rose to 22.2 ± 7.6 ng/mL after 15 min, declined to <10 ng/mL at 30 and 60 min then increased and were elevated (79.0 ± 10.8 ng/mL) at 240 min. Cortisol measurements can be used to indicate the responsiveness of fish to changes in water temperature and goldfish will be a convenient study species for the development of studies of plasticity in responses of fish to increases in water temperature that are happening due to climate change.