Embryonic Cadaverine Signaling: Implications for Plasma Free Amino Acid and Skeletal Muscle Energy Metabolism in Newly Hatched Chicks.

Cadaverine is a bioactive substance derived from lysine degradation by lysine decarboxylase and has gained attention for its physiological effects. Studies in rodents have revealed its role as a cell growth regulator, particularly intestinal bacterial-produced cadaverine. However, the nutritional and physiological roles of cadaverine during the embryonic period remain unclear, especially considering the immature state of the gut microbiota and digestive functions during this stage. This study explored the potential functions of cadaverine as a nutritional and metabolic signal during chicken embryonic development. Experiments were conducted using an in ovo administration method to evaluate the effects of nutritional bioactive substances on developing chicken embryos. Although there were no observable changes in body or organ weights of newly hatched chicks following in ovo cadaverine administration to day 18 chick embryos, plasma tryptophan, Nτ-methylhistidine, and Nπ-methylhistidine concentrations decreased and the gene expression of insulin/insulin-like growth factor 1 signaling in skeletal muscle was upregulated. These findings imply that cadaverine influences tryptophan metabolism and skeletal muscle catabolism during the embryonic period, suggesting its role as a bioactive factor contributing to energy metabolism signaling in skeletal muscle.

Quantification of Nτ -Methylhistidine and Nπ-Methylhistidine in Chicken Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

The concentration of Nτ-methylhistidine in plasma provides an index of skeletal muscle protein breakdown. This study aimed to establish a quantitative method for measuring the concentrations of Nτ-methylhistidine and its isomer Nπ-methylhistidine in chicken plasma, using liquid chromatography-tandem mass spectrometry with stable isotope dilution analysis. The acceptable linear ranges of detection were 1.56-50.00 µmol/L for Nτ-methylhistidine and 0.78-25.00 µmol/L for Nπ-methylhistidine. The proposed method detected changes in the plasma levels of Nτ-methylhistidine and Nπ-methylhistidine in response to fasting and re-feeding. These results suggest that the method developed in this study can be used for the simultaneous measurement of Nτ-methylhistidine and Nπ-methylhistidine in chicken plasma.

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.

Cryopreservation Competence of Chicken Oocytes as a Model of Endangered Wild Birds: Effects of Storage Time and Temperature on the Ovarian Follicle Survival.

For the conservation of endangered avian species, developing gamete preservation technologies is essential. However, studies in oocytes have not been widely conducted. In this study, assuming that the ovaries are transported to a research facility after death, we investigated the effect of ovary storage on oocytes for the purpose of cryopreserving avian female gametes by using a chicken as a model of endangered avian species. After excision, the ovaries were stored at either a low temperature (4 °C) or room temperature for 1-3 days. Ovarian follicles stored under different conditions for each period were examined by neutral red staining, histology, and gene and protein expression analysis. In addition, the pH of the storage medium after preserving the ovaries was measured. Then, ovarian tissues were vitrified to determine the cryopreservation competence. Storing the ovarian tissues at 4 °C kept the follicles viable and morphologically normal for 3 days with slow decline. In contrast, although different storage temperature did not influence follicle viability and morphology after only 1 day of storage, ovarian tissues stored at room temperature rapidly declined in structurally normal follicles, and viable follicles were rarely seen after 3 days of storage. Gene and protein expression analysis showed that apoptosis had already started on the first day, as shown by the higher expression of CASP9 under room temperature conditions. Furthermore, high expression of SOD1 and a rapid decline of pH in the storage medium under room temperature storage suggested the influence of oxidative stress associated with low pH in this condition on the follicle survivability in hen ovarian tissues. Our cryopreservation study also showed that ovarian tissues stored at 4 °C could recover after cryopreservation even after 3 days of storage. The described storage conditions and cryopreservation methods, which preserve chicken follicle survival, will lay the foundation of ovarian tissue preservation to preserve the fertility of wild female birds.

Metabolism of Imidazole Dipeptides, Taurine, Branched-Chain Amino Acids, and Polyamines of the Breast Muscle Are Affected by Post-Hatch Development in Chickens.

To explore metabolic characteristics during the post-hatch developmental period, metabolomic analyses of breast muscle and plasma were performed in chickens. The most significant growth-related changes in metabolite levels were observed between seven and 28 days of age. Some of these metabolites are essential nutrients or reported as growth-promoting metabolites. In the muscle, two imidazole dipeptides-carnosine and its methylated metabolite, anserine-increased with the development. These dipeptide levels may be, in part, regulated transcriptionally because in the muscle mRNA levels of carnosine synthase and carnosine methylation enzyme increased. In contrast, taurine levels in the muscle decreased. This would be substrate availability-dependent because some upstream metabolites decreased in the muscle or plasma. In branched-chain amino acid metabolism, valine, leucine, and isoleucine decreased in the muscle, while some of their downstream metabolites decreased in the plasma. The polyamines, putrescine and spermidine, decreased in the muscle. Furthermore, mRNA levels associated with insulin/insulin-like growth factor 1 signaling, which play important roles in muscle growth, increased in the muscle. These results indicate that some metabolic pathways would be important to clarify metabolic characteristics and/or growth of breast muscle during the post-hatch developmental period in chickens.

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.

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.

Glycyl-l-glutamine attenuates NPY-induced hyperphagia via the melanocortin system.

This study aimed to determine whether glycyl-l-glutamine (Gly-Gln; ß-endorphin (30-31)), a non-opioid peptide derived from ß-endorphin processing, modulates neuropeptide Y (NPY)-induced feeding and hypothalamic mRNA expression of peptide hormones in male broiler chicks. Intracerebroventricular injection of NPY (235 pmol) generated a hyperphagic response in ad libitum chicks within 30 min. Co-administration of Gly-Gln (100 nmol) attenuated this response, inducing a 30 % decrease. This was not attributable to Gly-Gln hydrolysis because co-administration of glycine (Gly) and glutamine (Gln) had no effect on NPY-induced hyperphagia. Gly-Gln injected alone also showed no effect. The hypothalamic pro-opiomelanocortin mRNA expression in the co-injection group was significantly higher than that in the NPY alone group. These data indicate that endogenous Gly-Gln may contribute to regulate feeding behavior via the central melanocortin system in chicks and acts as a counter regulator of the neural activity in energy metabolism.

Effect of Stereotaxic Surgery of the Third Ventricle on Growth Performance in Neonatal Chicks.

Feeding behavior and energy metabolism are precisely regulated by humoral and/or neural factors in the central nervous system. In particular, nuclei, such as the arcuate nucleus, ventromedial hypothalamic nucleus, and lateral hypothalamic area located near the third ventricle of the hypothalamus are the centers of feeding and energy metabolism in various vertebrate species, including chickens. In this study, we evaluated the effects of cannulation of the third ventricle on chick growth and feeding behavior in the neonatal stage, to develop a method for local and chronic central nervous system-mediated energy metabolism. Referring to the chick brain atlas, a guide cannula was inserted into the third ventricle of the chick under anesthesia immediately after hatching using a stereotaxic instrument. The chicks that recovered from anesthesia were bred for 11 days under normal feeding management conditions, and then feed intake amount, body weight gain, and metabolic tissue weight were measured. The effects of direct stimulation of the third ventricle with 2-deoxy-D-glucose on the expression level of the immediate-early gene, cFOS, and feed intake in 5-day-old chicks were also evaluated. There were no differences in feed intake, body weight gain, and metabolic tissue weight between 11-day-old cannulated and control chicks. The expression of cFOS mRNA in the ventromedial hypothalamic nucleus was higher than that in the amygdala after the third ventricular administration of 2-deoxy-D-glucose. Additionally, direct third ventricular injection of 2-deoxy-D-glucose attenuated the feeding behavior of chicks for a while. Overall, we speculate that the technique is effective for local and/or chronic stimulation of the nucleus near the third ventricle of the chick hypothalamus, which is important for feed and energy metabolism regulation.

Effects of In Ovo Vitamin D3 Injection on Subsequent Growth of Broilers.

This study was conducted to investigate the influence of in ovo vitamin D3 (Vit D3) administration on growth of broiler chickens when Vit D3 was dissolved in soybean oil. Sixty Ross broiler eggs were incubated at 37.8°C and >60% relative humidity. Distilled water, soybean oil, or Vit D3 (60 IU / 0.5 mL) dissolved in soybean oil, was administered in ovo on Day 18 of incubation. Seven days after hatching, chicks were sexed, and 12 birds (six female and six male) close to the average body weight (BW) of each treatment were selected and their BW continuously recorded until 28 days of age, then sacrificed. Liver and pectoral muscle were collected to determine the mRNA expression of IGF-1 and IGF-1 receptor, and the length of tibia was measured. There were no significant differences in BW, liver weight, or pectoral muscle weight between the groups. However, an interaction was observed between treatments and sexes in the tibia length. In comparison among only males, tibia length in the Vit D3 with oil group was longer than that of the control, but not different from that of the oil group. The same tendency was observed in the hepatic IGF-1 mRNA expression in chicks of either sex, with this effect only being observed after the treatments and not in the control. On the other hand, there was an interaction between treatments and sexes in the mRNA expression of IGF-1 receptor, which was highest in the Vit D3 with oil group in females, but not in males. These results indicated that the in ovo administration of Vit D3 affected IGF-1 receptor mRNA expression without growth.

Effects of Dietary Methionine or Arginine Levels on the Urinary Creatinine Excretion in Broiler Chicks.

Two experiments were conducted to evaluate the usefulness of urinary creatinine levels as a criterion for the estimation of protein and amino acid requirements in poultry. Here we studied the effects of dietary precursor levels of creatinine, methionine and arginine, on urinary creatinine excretion in experiments. Both experiments used 15 Chunky broilers chicks that were 8 days old. The chicks were assigned to three dietary groups, with five chicks each, and were fed an experimental diet for 7 days. The experimental diets mainly consisted of corn and soybean meal, and contained deficient, adequate, or excessive methionine and arginine levels in experiments 1 and 2, respectively. Excreta were collected for the last 3 days of the feeding trial, and chicks were terminated by dislocation of the neck at the end of the feeding trial to collect their livers. Creatinine concentration in the excreta and hepatic l-arginine-glycine amidinotransferase (AGAT) activities were determined. Urinary creatinine levels increased with increasing both dietary methionine and arginine levels from deficient to adequate recommended by Japanese feeding standard (P<0.05), and then remained constant in experiments 1 and 2, respectively. The hepatic AGAT activity decreased when both dietary creatinine precursors levels were increased from deficient to adequate levels (p<0.05), and then remained constant. These results suggested that creatinine excretion was changed with both increasing dietary methionine and arginine, dose-dependently.

A genetically female brain is required for a regular reproductive cycle in chicken brain chimeras.

Sexual differentiation leads to structural and behavioural differences between males and females. Here we investigate the intrinsic sex identity of the brain by constructing chicken chimeras in which the brain primordium is switched between male and female identities before gonadal development. We find that the female chimeras with male brains display delayed sexual maturation and irregular oviposition cycles, although their behaviour, plasma concentrations of sex steroids and luteinizing hormone levels are normal. The male chimeras with female brains show phenotypes similar to typical cocks. In the perinatal period, oestrogen concentrations in the genetically male brain are higher than those in the genetically female brain. Our study demonstrates that male brain cells retain male sex identity and do not differentiate into female cells to drive the normal oestrous cycle, even when situated in the female hormonal milieu. This is clear evidence for a sex-specific feature that develops independent of gonadal steroids.

Comparisons of insulin related parameters in commercial-type chicks: Evidence for insulin resistance in broiler chicks.

The aim of this study is to elucidate whether insulin acts differentially within the central nervous system (CNS) of two types of commercial chicks to control ingestive behavior. Male layer and broiler chicks (4-day-old) were intracerebroventricularly (ICV) injected with saline or insulin under satiated and starved conditions. Feed intake was measured at 30, 60 and 120 min after treatment. Secondly, blood and hypothalamus were collected from both chick types under ad libitum feeding and fasting for 24 h. Plasma insulin concentration was measured by time-resolved fluoro-immunoassay. Hypothalamic insulin receptor mRNA expression levels were measured by quantitative RT-PCR. The ICV injection of insulin significantly inhibited feed consumption in layer chicks when compared with saline (P<0.05), but not broiler chicks (P>0.1). Plasma insulin concentration of both chick types significantly decreased following 24 h of fasting, while insulin concentrations in the broiler chicks were significantly higher compared to the layers fed under ad libitum conditions. Hypothalamic insulin receptor mRNA expression levels were significantly lower (P<0.05) in broiler chicks than in layer ones under ad libitum feeding. Feed deprivation significantly decreased insulin receptor mRNA levels in layer chicks (P<0.01), but not in broiler chicks (P>0.1). Moreover, plasma insulin concentrations correlated negatively with hypothalamic insulin receptor protein expression in the two types of chicks fed ad libitum (P<0.05). These results suggest that insulin resistance exists in the CNS of broiler chicks, possibly due to persistent hyperinsulinemia, which results in a down-regulation of CNS insulin receptor expression compared to that in layer chicks.

Localization of hypothalamic insulin receptor in neonatal chicks: evidence for insulinergic system control of feeding behavior.

Feeding behavior is managed by various neuropeptides and/or neurotransmitters within the central nervous system in vertebrates. It is proposed that central insulin acts as the negative-feedback regulator of appetite via the central melanocortin system in neonatal chicks. The present study investigated the localization of insulin receptors in the chick hypothalamus using immunohistochemistry. Immunostaining revealed hypothalamic neuron expressing insulin receptors in the paraventricular nucleus, ventromedial hypothalamus, lateral hypothalamus and infundibular nucleus, the avian equivalent of the mammalian arcuate nucleus. Additionally, double-staining immunohistochemistry in the infundibular nucleus revealed the presence of insulin receptors in both α-melanocyto stimulating hormone and neuropeptide Y neurons. Immunohistological analysis indicates that the insulinergic system in the chick hypothalamus contributes to feeding behavior and this system regulates both anorexigenic and orexigenic neuropeptides. Furthermore, the mechanisms of central insulin induced-feeding behavior contributes to the regulation of the melanocortin system in the chick infundibular nucleus.

The number of taste buds is related to bitter taste sensitivity in layer and broiler chickens.

The relationship between taste sensitivity and the number of taste buds using a bitter tastant, quinine hydrochloride, was investigated in White Leghorn, Rhode Island Red, and broiler chickens. The White Leghorn and Rhode Island Red strains were able to perceive 2.0 mmol/L quinine hydrochloride, but the taste sensitivity of Rhode Island Red chickens was higher than that of White Leghorn chickens. Broiler chickens perceived 0.5 mmol/L quinine hydrochloride. The number of taste buds in the White Leghorn strain was the lowest, then the Rhode Island Red strain, with the number of taste buds highest in the broiler chickens. The number of taste buds was well correlated with bitter taste sensitivity. Therefore, we suggest that the number of taste buds is a vital factor in the perception of bitter taste and may be useful in selecting appropriate feeds for chickens.

Mutual information and boson radius in a c=1 critical system in one dimension.

We study the generic scaling properties of the mutual information between two disjoint intervals, in a class of one-dimensional quantum critical systems described by the c=1 bosonic field theory. A numerical analysis of a spin-chain model reveals that the mutual information is scale invariant and depends directly on the boson radius. We interpret the results in terms of correlation functions of branch-point twist fields. The present study provides a new way to determine the boson radius, and furthermore demonstrates the power of the mutual information to extract more refined information of conformal field theory than the central charge.

Effect of nociceptin/orphanin FQ on feeding behavior and hypothalamic neuropeptide expression in layer-type chicks.

Nociceptin/orphanin FQ (N/OFQ) was identified in 1995 as the endogenous ligand for the orphan G(i)/G(o)-coupled opioid receptor-like 1 receptor (NOP(1)). Exogenous N/OFQ increases food intake in mammals, but its effect and mode of action in chicks are not fully known. We report herein that N/OFQ (5.0 nmol) has a stimulatory effect on food intake in layer-type chicks over a 2-h period after intracerebroventricular (icv) injection. Thirty minutes after central injection of N/OFQ (5.0 nmol) the concentration of agouti-related protein (AGRP) mRNA in the diencephalon increased, while cocaine- and amphetamine-regulated transcript (CART) mRNA decreased. However, concentrations of neuropeptide Y, proopiomelanocortin and glutamate decarboxylase mRNAs, and of catecholamines and excitatory amino acids were not affected. Simultaneous administration of alpha-melanocyte stimulating hormone (alpha-MSH: 1.0 pmol), a competitor of AGRP, completely blocked the orexigenic effect of N/OFQ (5.0 nmol). These data suggest that N/OFQ functions in layer chicks as an orexigenic peptide in the central nervous system, and that the AGRP and the CART neurons may mediate this function, as in mammals.

Effects of N-terminal fragments of beta-endorphin on feeding in chicks.

It is known that N-terminal fragments of beta-endorphin have biological activities, such as an antagonism effect of beta-endorphin (1-31) on the secretion of hormones or thermoregulation in mammals. We studied the effects of the N-terminal fragments on feeding behavior in male broiler chicks. Intracerebroventricular administration of beta-endorphin (1-27) (0.4nmol) stimulated feeding behavior compared with saline control during the 60-min experimental period. beta-Endorphin (1-17) (2.0nmol) also increased food intake at 30min postinjection. Co-injection of either beta-endorphin (1-27) or (1-17) was effective in reducing full-length beta-endorphin-induced feeding in chicks. These data suggest that the N-terminal fragments of beta-endorphin act as a partial agonist, and may regulate the activity of the central opioidergic system in chicks.

Micro-opioid receptor agonist diminishes POMC gene expression and anorexia by central insulin in neonatal chicks.

Pro-opiomelanocortin (POMC) neurons in the hypothalamus are direct targets of peripheral satiety signals, such as leptin and insulin in mammals. The stimulation of these signals activates hypothalamic POMC neurons and elevates POMC-derived melanocortin peptides that inhibit food intake in mammals. On the other hand, it has been recognized that beta-endorphin, a post-translational processing of POMC, acts in an autoreceptor manner to the micro-opioid receptor (MOR) on POMC neurons, diminishing POMC neuronal activity in mammals. Recently, we found that central insulin functions as an anorexic peptide in chicks. Thus, the present study was done to elucidate whether beta-endorphin affects the activation of POMC neurons by insulin in neonatal chicks. Consequently, quantitative real-time PCR analysis shows that intracerebroventricular (ICV) injection of insulin with beta-endorphin significantly decreases brain POMC mRNA expression when compared with insulin alone. In addition, co-injection of MOR agonist (beta-endorphin or [d-Ala2, N-MePhe4, Gly5-ol]-enkephalin (DAMGO)) significantly attenuates insulin-induced hypophagia in chicks. These data suggest that beta-endorphin regulates the activity of the central melanocortin system, and its activation may provide an inhibitory feedback mechanism in the brain of neonatal chicks.

Central insulin suppresses feeding behavior via melanocortins in chicks.

Growing evidence suggests that insulin interacts with both orexigenic and anorexigenic peptides in the brain for the control of feeding behavior in mammals. However, the action of central insulin in chicks has not yet been identified. In the present study, we investigated the effects of central injection of insulin on feeding behavior in chicks. Intracerebroventricular (ICV) administration of insulin, at doses that do not influence peripheral glucose levels, significantly inhibited food intake in chicks. Central injection of insulin in chicks significantly increased expression of pro-opiomelanocortin (POMC) mRNA, and decreased that of neuropeptide Y (NPY) mRNA. Finally, co-injection of the melanocortin antagonist (SHU9119 or HS014) prevented the reduction in food intake caused by ICV administration of insulin. These data suggest that insulin functions in chicks as an appetite-suppressive peptide in the central nervous system, and that the central melanocortin system mediates this anorexic effect of insulin, as in mammals.