Expression and localization of the neuropeptide Y-Y4 receptor in the chick spleen: mRNA upregulation by high ambient temperature.

High ambient temperatures (HT) can increase diencephalic neuropeptide Y (NPY) expression, and central injection of NPY attenuates heat stress responses while inducing an antioxidative state in the chick spleen. However, there is a lack of knowledge about NPY receptor expression, and its regulation by HT, in the chick spleen. In the current study, male chicks were used to measure the expression of NPY receptors in the spleen and other immune organs under acute (30 vs. 40 ± 1°C for 3 h) or chronic (30 vs. 40 ± 1°C for 3 h/day for 3 days) exposure to HT and in response to central injection of NPY (47 pmol, 188 pmol, or 1 nmol). We found that NPY-Y4 receptor mRNA was expressed in the spleen, but not in other immune organs studied. Immunofluorescence staining revealed that NPY-Y4 receptors were localized in the splenic pulp. Furthermore, NPY-Y4 receptor mRNA increased in the chick spleen under both acute and chronic exposure to HT. Central NPY at two dose levels (47 and 188 pmol) and a higher dose (1 nmol) did not increase splenic NPY-Y4 receptor mRNA expression or splenic epinephrine under HT (35 ± 1°C), and significantly increased 3-methoxy-4-hydroxyphenylglycol (MHPG) concentrations under HT (40 ± 1°C). In conclusion, increased expression of NPY-Y4 receptor mRNA in the spleen under HT suggest that Y4 receptor may play physiological roles in response to HT in male chicks.

Comparison of oral administration of fructose and glucose on food intake and physiological parameters in broiler chicks.

Like glucose, fructose is a monosaccharide, but the mechanisms of its absorption and metabolism in the body are very different between the 2 molecules. In this study, we investigated the effects of oral administration of glucose and fructose on food intake, diencephalic gene expression, and plasma metabolite concentrations in broiler chicks. The animals used in this study were 4-day-old male broiler chicks (Ross 308). They were given glucose, fructose (200 mg/ 0.5 mL/ bird), or a similar volume of distilled water orally after 6 h fasting. After treatment, measurements of food intake (at 0, 30, and 60 min), and blood glucose as well as insulin concentrations were measured over time; however, diencephalic (hypothalamus) gene expression and plasma metabolites were measured at 30 min. The results showed that glucose administration suppressed food intake, but fructose administration did not suppress food intake and it was at the same level as distilled water administration. In addition, fructose administration did not increase plasma glucose and insulin levels as did glucose administration. In the diencephalon, expression levels of genes related to the melanocortin system were unaffected by the treatment, while gene expression levels related to intracellular energy regulation, such as AMP-activated protein kinase were affected by the glucose treatment in the fasted chicks. These results suggest that fructose administration does not suppress feeding behavior as a result of possible reduction in the energy levels in the diencephalon and associated energy metabolism.

Intracerebroventricular injection taurine changes free amino acid concentrations in the brain and plasma in chicks.

Brain amino acid metabolism has been reported to regulate body temperature, feeding behavior and stress response. Central injection of taurine induced hypothermic and anorexigenic effects in chicks. However, it is still unknown how the amino acid metabolism is influenced by the central injection of taurine. Therefore, the objective of this study was to investigate the changes in brain and plasma free amino acids following central injection of taurine. Five-day-old male Julia layer chicks (n = 10) were subjected to intracerebroventricular (ICV) injection with saline or taurine (5 µmol/10 µL). Central taurine increased tryptophan concentrations in the diencephalon, and decreased tyrosine in the diencephalon, brainstem, cerebellum, telencephalon and plasma at 30 min post-injection. Taurine was increased in all the brain parts after ICV taurine. Although histidine and cystathionine concentrations were increased in the diencephalon and brainstem, several amino acids such as isoleucine, arginine, methionine, phenylalanine, glutamic acid, asparagine, proline, and alanine were reduced following central injection of taurine. All amino acid concentrations were decreased in the plasma after ICV taurine. In conclusion, central taurine quickly changes free amino acid concentrations in the brain and plasma, which may have a role in thermoregulation, food intake and stress response in chicks.

Effect of neonatal isolation on responses to subsequent exposure to isolation stress in young chickens.

The aim of the present study was to investigate effects of isolation at an early age on behavioral and physiological responses of chickens to an isolation challenge at two weeks of age. Birds were assigned to a control group or to one of three treatments where chicks were isolated for 5 min per day. The groups were 1) no isolation (control); 2) early isolation (EI; 2 to 4 days of age); 3) late isolation (LI; 5 to 7 days of age); or 4) full isolation (FI; 2 to 7 days of age). All groups of chicks were challenged with isolation for 5 min at two weeks of age, with distress vocalizations (DV), stepping and jumping behavior measured. Hypothalamic and blood samples were collected at the end of isolation challenges. There were no significant differences between groups in body weight gain at 2 weeks of age. Latency of jump was lower in the LI group compared with the control group, but DV and number of steps were not affected by isolation treatment during the neonatal period. There were no significant differences among groups in plasma glucose or FFA concentrations. Gene expression for hypothalamic corticotropin-releasing hormone, was lower in the EI than the control group, with no differences in expression between control and LI or control and FI groups. There were no significant differences among groups in the expression of arginine vasotocin, thyrotropin-releasing hormone, neuropeptide Y, proopiomelanocortin, and orexin genes. These results suggest that isolation in the first week of life may affect responses to isolation of chicks when they are older, and that there may be a critical period of several days for this effect to occur.

mRNA Expressions of Methylation Related Enzymes and Duration of Thermal Conditioning in Chicks.

DNA methylation regulates gene expression by modifying the nucleosome structure of DNA, without altering the gene sequence. It has been reported that DNA methylation reactions are catalyzed by several enzymes. In chickens, thermal conditioning treatment affects the central DNA methylation levels. The purpose of this study was to clarify the changes in DNA methylation and demethylation factors during thermal conditioning in the hypothalamus of 3-day-old chicks. Male chicks (3-days old) were exposed to 40±0.5°C as a thermal conditioning treatment for 1, 2, 6, 9, or 12 h. The control chicks were kept in a thermoneutral zone (30±0.2°C). After thermal conditioning, the mRNA levels of DNA methyltransferase (DNMT)-1, -3a, -3b, and ten-eleven translocation (TET)-1, -2, and -3 in the hypothalamus were measured by q-PCR. The mRNA levels of DNMT-3a and TET-1 were increased by thermal conditioning. Moreover, the expression level of TET-1 increased with the loading time of the thermal conditioning. The gene expressions of DNMT-1, DNMT-3b, TET-2, and TET-3 were not affected by thermal conditioning. Since DNMT-3a is a catalyst for de-novo DNA methylation and TET-1 catalyzes the oxidation of methylated cytosine, it is suggested that the thermal conditioning increased the activation of DNA methylation and demethylation factors, which occur in the hypothalamus of neonatal chicks.

Effects of Thermal Conditioning and Folic Acid on Methylation of the BDNF Promoter Region in Chicks.

This study aimed to investigate the effects of thermal conditioning and folic acid on the methylation levels of the avian brain-derived neurotrophic factor (BDNF) promoter region at the M3 and M9 positions in the early life of broiler chicks. In Experiment 1, male broiler chicks (day 3 of life) were orally injected with methyl cellulose solution with or without folic acid (25 mg). The chicks in the heat-treatment groups were immediately exposed to a high ambient temperature (40±0.5°C) for 12 h, while chicks in the non-heat treatment groups were left in the thermoneutral zone (30±0.5°C). The groups were as follows: 1) no thermal conditioning group without folic acid (control), 2) thermal conditioning group without folic acid, 3) no thermal conditioning group with folic acid, and 4) thermal conditioning group with folic acid. In Experiment 2, treatments were similar to those in Experiment 1, except for the usage of female chicks. After the treatments, the methylation levels of the BDNF promoter in chicks were determined using semiquantitative PCR. There were no significant differences between groups in the levels of methylation at the M3 position in both males and females as a result of thermal conditioning and folic acid treatment. Interestingly, significant effects of thermal conditioning and folic acid treatment on methylation at the M9 position were found. BDNF methylation levels at M9 significantly decreased following thermal conditioning, while folic acid suppressed demethylation in both male and female chicks. These data suggest that folic acid and thermal conditioning affects DNA methylation patterns in the central nervous system of chicks, regardless of sex.

Single nucleotide polymorphism in avian uncoupling protein gene is associated with thermoregulation in chicks.

Avian uncoupling protein (av-UCP) is a key protein for thermoregulation in poultry. A single nucleotide polymorphism (SNP) in the av-UCP gene has been reported in chickens. The purpose of the current study was to clarify the association between this av-UCP gene mutation and thermoregulation in chickens. Wild and mutant type chicks for the av-UCP gene SNP (g. 1270 of the av-UCP gene exon 3 with C to T substitution and amino acid substitution) were exposed to high ambient temperature. Rectal temperature, radiation temperature on the body surface, and the expression of heat dissipation behavior (wing drooping and panting) during heat exposure were measured. In addition, oxygen consumption rate in the thermoneutral zone in wild and mutant type chicks was measured. Changes in wing temperature during heat exposure in wild-type chicks were lower than those in mutants. The latency of continuous wing drooping during heat exposure in wild-type chicks was shorter than in mutant chicks. It was also found that the SNP in the av-UCP gene caused reduced oxygen consumption. These results suggest that the av-UCP gene mutation affects thermoregulation, especially heat production, in chickens.

Application of a simple measuring method to evaluate the fecal microflora of dairy cows in the summer season.

OBJECTIVE: The effect of seasonality needs to be considered in designing future studies because global warming has caused a rise in ambient temperatures. The objective of the present study is to investigate the effect of high ambient temperatures on fecal score and fecal microflora in dairy cows during summer. MATERIALS AND METHODS: During the 7 days before the sampling of feces, the daily mean temperatures were 19.9°C in early summer and more than 27.5°C in late summer. Fecal samples were collected from the rectum of cows and the fecal score was evaluated on a 4-point scale. The equalized samples were used to extract the genomic deoxyribonucleic acid (DNA) of the bacteria (Escherichia coli, Salmonella, Lactobacillus, and Bifidobacterium). RESULTS: There was no significant difference in fecal scores between the sampling times in early and late summer. In the populations of the bacteria, there was no significant difference between sampling days in the DNA level of Salmonella, and E. coli in late summer increased to more than three times the level in early summer. However, both levels of Lactobacillus and Bifidobacterium in early summer significantly decreased after 2 months. CONCLUSION: These data suggest that the increase in temperature in late summer may adversely affect the populations of bacteria in the intestinal environment of dairy cows. In addition, the method used in the present study was sufficient to evaluate the changes in internal and external environmental conditions of dairy cattle.

Liver metabolomic analysis in broiler chicks: Profiling the metabolites after oral administration of l-citrulline.

Hypothermia is directly linked to metabolism; however, it is still unknown how the overall metabolism is altered by oral administration of hypothermic agent, l-citrulline (l-Cit). The present study aimed to determine the characteristics of liver metabolites of chicks orally administered l-Cit to provide a greater understanding of its metabolism. Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) and liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS) were conducted on liver samples after oral administration of l-Cit. A total of 361 liver metabolites were identified. Although a small number of samples were used for each group, a principal component analysis and heatmap patterns confirmed that the composition of metabolites could be segregated from each other. Of the 361 compounds detected in the liver, 41 compounds, including amino acids related to the Cit-arginine (Arg) cycle, argininosuccinic acid, Arg, ornithine, and Cit, as well as gamma aminobutyric acid, glycine, histidine, and nicotinamide adenine dinucleotide were abundant in l-Cit-treated livers. In contrast, 24 compounds containing fatty acids, amino acids, and cyclic adenosine monophosphate were lower in the l-Cit group. These data imply that the active Cit-Arg cycle, TCA cycle metabolism, and a low activity in fatty acid metabolism occur in l-Cit-treated broiler chicks.

Oral administration of L-citrulline changes the concentrations of plasma hormones and biochemical profile in heat-exposed broilers.

We examined the effects of oral administration of L-citrulline (L-Cit) on plasma metabolic hormones and biochemical profile in broilers. Food intake, water intake, and body temperature were also analyzed. After dual oral administration (20 mmol/head/administration) of L-Cit, broilers were exposed to a high ambient temperature (HT; 30 ± 1°C) chamber for 120 min. Oral administration of L-Cit reduced (p < .001) rectal temperature in broilers. Food intake was increased (p < .05) by heat stress, but it was reduced (p < .05) by L-Cit. Plasma levels of 3,5,3'-triiodothyronine, which initially increased (p < .0001) due to heat stress, were reduced (p < .01) by oral administration of L-Cit. Plasma insulin levels were increased by heat exposure (p < .01) and oral L-Cit (p < .05). Heat stress caused a decline (p < .05) in plasma thyroxine. Plasma lactic acid (p < .05) and non-esterified fatty acids (p < .01) were increased in L-Cit-treated heat-exposed broilers. In conclusion, our results suggest that oral L-Cit can modulate plasma concentrations of major metabolic hormones and reduces food intake in broilers.

Av-UCP single nucleotide polymorphism affects heat production during cold exposure in chicks.

OBJECTIVE: Uncoupling protein one (UCP1) is involved in thermogenesis, especially in non-shivering heat production. In chickens, a single nucleotide polymorphism (SNP) of the av-UCP (avian UCP) gene has been reported to be associated with body weight gain and increased abdominal fat. The purpose of this study was to examine the relationship between the av-UCP gene SNP and heat production in chicks. METHODS: C/C and T/T male chicks (Rhode Island Red) of av-UCP gene SNP (g. 1270, C > T) were exposed to a low temperature environment (16 °C for 15 min) and their physiological responses were compared. RESULTS: After cold exposure, mean rectal temperatures of C/C chicks were higher than those of T/T chicks. In pectoral muscle, genes expression of av-UCP and carnitine palmitoyltransferase-1 were higher in C/C chicks than T/T chicks. Hypothalamic expression levels of thyrotropin-releasing hormone and proopiomelanocortin genes were higher in C/C chicks than T/T chicks. Expression of hypothalamic corticotropin-releasing hormone, arginine vasotocin, brain-derived neurotrophic factor and neuropeptide Y genes did not differ between C/C and T/T chicks. In addition, plasma free fatty acid levels in C/C chicks were lower than those of T/T chicks. CONCLUSION: These results suggest that the av-UCP gene SNP affects non-shivering heat production via the hypothalamo-pituitary-thyroid axis and fatty acid metabolism in the chicken.

Adenosine 5'-monophosphate induces hypothermia and alters gene expressions in the brain and liver of chicks.

The adenosine A1 receptor is important for body temperature regulation in mammals; however, little is known about its function in avian species. In this study, we investigated the effects of the adenosine A1 receptor agonist and antagonist (adenosine 5'-monophosphate [5'-AMP] and 8 p-sulfophenyl theophylline [8-SPT], respectively) on thermoregulation in chickens. Male chicks were used in this study. After administration of 5'-AMP and 8-SPT, the rectal temperature, plasma metabolites, and gene expressions in the hypothalamus and liver were measured. The rectal temperature was reduced by peripheral administration of 5'-AMP, and the hypothermic effect of 5'-AMP was attenuated by central injection of 8-SPT in chicks. In the hypothalamus, the mRNA level of the agouti-related protein (AgRP) was increased by 5'-AMP administration, whereas it was suppressed by 8-SPT. The plasma levels of free fatty acid were elevated in 5'-AMP-treated chicks and that elevation was suppressed by the 8-SPT treatment. The gene expression of proopiomelanocortin in the hypothalamus was affected by 8-SPT. Nevertheless, the gene expressions of the thermoregulation-related genes, such as the thyrotropin-releasing hormone, were not affected by 5'-AMP and 8-SPT. Hepatic gene expressions related to lipid intake and metabolism were suppressed by 5'-AMP. However, the gene expression of the uncoupling protein was upregulated by 5'-AMP. Based on these results, birds, like mammals, will undergo adenosine A1 receptor-induced hypothermia. In conclusion, it is suggested that 5'-AMP-mediated hypothermia via the adenosine A1 receptor may affect the central melanocortin system and suppress hepatic lipid metabolism in chickens.

The use of behavioral tests of fearfulness in chicks to distinguish between the Japanese native chicken breeds, Tosa-Kukin and Yakido.

The aim of this study was to investigate the differences in fearfulness between two Japanese native chicken breeds, Tosa-Kukin (TOK) and Yakido (YKD). In a tonic immobility test, TOK breed chicks showed longer duration and lower induction number compared with YKD. The number of peeps in YKD in an isolation test was lower than that in TOK, whereas there were more bouts of peeping in YKD than in TOK. In a manual restraint test, YKD showed more active responses and initiated peeps and struggling earlier than TOK. The three behavioral tests all indicated that YKD are less fearful than TOK chickens. A latent structures discriminant (OPLS-DA) analysis was used to identify behavioral parameters that contributed to the differences between the breeds. The major parameters were duration and number of inductions in the tonic immobility test and number of struggle in the manual restraint test. These results suggest that three behavioral tests can be used together to evaluate fearfulness of Japanese native breeds of chickens.

Repeated thermal conditioning during the neonatal period affects behavioral and physiological responses to acute heat stress in chicks.

OBJECTIVE: The aim of the present study was to investigate the effects of repeated thermal conditioning (RTC) at an early age on physiological and behavioral responses in chicks. METHODS: Birds were assigned to one of the four treatments in which the RTC was exposure to 40 °C for 15 min daily. The treatments were 1) no thermal conditioning (control); 2) early exposure group (EE; RTC from 2 to 4 days of age); 3) later exposure group (LE; RTC from 5 to 7 days of age); or 4) both early and later exposure (BE; RTC from 2 to 7 days of age). All groups of chicks were challenged with high ambient temperature (40 °C for 15 min) at two weeks of age. RESULTS: During heat challenge, initiation times of dissipation behaviors (panting and wing-drooping) were measured. Rectal temperature and respiration rate were measured after and before heat challenge. Hypothalamic samples and blood were collected at the end of heat challenges. Initiation times of dissipation behaviors and rectal temperature were not affected by the treatments. Increases in respiration rate in response to heat challenge were suppressed by early RTC treatment. There was no clear pattern of glucose levels in relation to thermal conditioning, whereas plasma corticosterone levels were decreased by early treatment (EE and BE groups). Hypothalamic thyrotropin releasing hormone gene expression was suppressed by early and later thermal conditioning and suppressed further by both early and later exposure. Neuropeptide Y gene expression in the BE group was lower than in the other groups, with a similar trend for corticotropin releasing hormone expression. CONCLUSION: Our results suggest that the effect of repeated thermal conditioning on the central thermoregulatory system depends on the number of times that chicks experienced conditioning. In addition, repeated thermal conditioning has greater effects on the acquisition of thermotolerance when conditioning occurs in chicks of two to four days of age in comparison with chicks of five to seven days of age.

The effect of facial hair whorl position and raising environment on the temperament of the Chinese Yellow cattle in Shinjang Uyghur Aptonom Rayoni, China.

OBJECTIVE: The objective of this study is to determine whether there is a relationship between hair whorl position and temperament in Chinese Yellow cattle using a questionnaire. Also, the effect of the raising environment is investigated. MATERIALS AND METHODS: A total of 122 Chinese Yellow cattle were surveyed in 3 village areas and four grassland areas in the northern area of China. For each cattle, an investigator asked the care-person about each item of a temperament questionnaire and determined the facial hair whorl position of each cattle. The location of hair whorl was categorized as low, middle, or high in relation to the eyes. RESULTS: The overall distribution was 20.5% high, 58.2% middle, 13.1% low, 6.6% double, and 1.6% no hair whorl. There was no significant difference between the grassland and village area cattle in the distribution of whorl position. The scores of grassland areas were significantly higher than those of village areas in terms of "Retentive memory", "Sensitivity," and "Timidity" and lower in "Docility" and "Fortitude." The scores for the high position tended to be higher than those for the middle plus low positions in terms of "Adaptability" and "Obedience." On the other hand, there was a tendency for a score for the high position to be lower than the other positions in "Excitability." A significant interaction between area and whorl position was observed in "Friendliness to cattle." CONCLUSION: These findings suggest that hair whorl location may be useful in predicting the temperament in cattle, but temperament can be influenced by environment and/or handlings.

Effects of Thermal Conditioning on Changes in Hepatic and Muscular Tissue Associated With Reduced Heat Production and Body Temperature in Young Chickens.

Effects of increased summer temperatures on poultry production are becoming more pronounced due to global warming, so it is important to consider approaches that might reduce heat stress in chickens. Thermal conditioning in chickens in the neonatal period can improve thermotolerance and reduce body temperature increases when birds are exposed to high ambient temperature later in life. The objective of this study was to investigate physiological and molecular changes associated with heat production and hence body temperature regulation under high ambient temperatures in thermally conditioned chicks. Three-day-old broiler chicks (Chunky) were thermally conditioned by exposure to a high ambient temperature (40°C) for 12 h while control chicks were kept at 30°C. Four days after the treatment, both groups were exposed to 40°C for 15 or 90 min. The increase in rectal temperature during 90 min of exposure to a high ambient temperature was less in thermally conditioned than control chicks. At 15-min of re-exposure treatment, gene expression for uncoupling protein and carnitine palmitoyletransferase 1, key molecules in thermogenesis and fatty acid oxidation, were significantly higher in pectoral muscle of control chicks but not conditioned chicks. Hepatic argininosuccinate synthase (ASS) decreased and hepatic argininosuccinate lyase (ASL) increased after reexposure to a high temperature. The concentrations of hepatic arginosuccinic acid, and ASS and ASL expression, were upregulated in conditioned chicks compared with the control chicks, indicating activity of the urea cycle could be enhanced to trap more energy to reduce heat production in conditioned chicks. These results suggest thermal conditioning can reduce the increase in heat production in muscles of chickens that occurs in high ambient temperatures to promote sensible heat loss. Conditioning may also promote energy trapping process in the liver by altering the heat production system, resulting in an alleviation of the excessive rise of body temperature.