2026 年 63 巻 論文ID: 2026003
From an animal welfare perspective, thirst avoidance is an important criterion for the poultry industry. Neuropeptide Y (NPY) and α−melanocyte-stimulating hormone (α-MSH) play critical roles in regulating food intake, which is closely related to water intake in mammals and chickens. This study aimed to clarify the role of appetite-regulating neuropeptides in controlling thirst in chicks. In Experiment 1, 7-day-old male chicks were allocated to two groups and deprived of water for 24 h. Chicks were intracerebroventricularly injected with saline or the peptide. Water intake was measured at 60 and 120 min after injection under feed-deprived conditions. α-MSH significantly suppressed thirst-induced water intake, whereas NPY exhibited no effect. In Experiment 2, 8-day-old male chicks were allocated to two groups and intracerebroventricularly injected with either saline or peptide under ad libitum drinking conditions. NPY significantly increased water intake, whereas α-MSH had no effect. In Experiment 3, 8-day-old male chicks were allocated to two groups and euthanized after 0 or 3 h of water deprivation. Diencephalon mRNA levels of NPY and proopiomelanocortin (the gene encoding α-MSH) were not affected by water deprivation. Our findings suggest that chick NPY and α-MSH function as thirst-inducing and thirst-quenching peptides, respectively. Both control water intake through post-translational regulation.
Avoiding thirst is an important criterion of animal welfare for the poultry industry[1]. Angiotensin II, a hyperosmotic signal, stimulates drinking in mammals and birds[2]; its effect is attenuated by the opioid receptor antagonist naloxone[3]. However, a recent study by our group showed that intracerebroventricular administration of opioid peptides, including Met-enkephalin, β-endorphin, nociception, dynorphin B, and endomorphins did not induce water intake in chicks[4].
In vertebrates, neuropeptide Y (NPY) and α-melanocyte-stimulating hormone (α-MSH) play critical roles in the regulation of food intake, as orexigenic and anorexigenic neuropeptides, respectively[5,6,7]. Furthermore, there is a significant positive correlation between feed and water intake in chickens[8]. In mammals, eating triggers thirst in anticipation of food absorption[9]. In chicks, the central administration of NPY increases food intake, but not water intake[10,11], suggesting that NPY can quench thirst. In contrast, central administration of α-MSH suppresses food intake without affecting water intake[12], suggesting that α-MSH induces thirst.
In the present study, we examined the effects of NPY and α-MSH on water intake of chicks under feed-deprived conditions, a situation not examined before. Our findings suggest that NPY and α-MSH may be involved in thirst regulation in chicks.
Because male layer chickens cannot be used for commercial egg production, they were euthanized immediately after hatching. To avoid wasting valuable life, male layer chickens are commonly used for research in avian physiology. In contrast, meat-producing broiler chicks grow very fast and show hyperphagia, making them unsuitable for similar research purposes. In this study, day-old male chicks (White Leghorn) were purchased from a local hatchery (Japan Layer K.K., Gifu, Japan). They were provided free access to water and a commercial chick starter diet (Nichiwa Sangyo Co., Ltd., Kobe, Japan). The chicks were reared in electrically heated cages under a 23 h/1 h light/dark cycle. The temperature was kept at 31 ± 2 °C during the test period. This study was approved by the Institutional Animal Care and Use Committee and was conducted according to the Kobe University Animal Experimentation Regulations (2022–06-02, Experiment 1 and 2; 2023-0603, Experiment 3).
Chicken NPY and α-MSH were purchased from Peptide Institute. Inc. (Osaka, Japan). Based on our previous studies[13,14], 100 pmol NPY and 50 pmol α-MSH were injected into each chick, as described below.
Experiment 1. Effect of central administration of peptides on water intake in chicks under thirst-inducing conditionTwenty-eight 7-day-old chicks were weighed and allocated to two groups based on body weight (14 birds per group). The chicks were given free access to feed, but not water, for 24 h prior to peptide injection. The peptides were dissolved in a 0.85% (w/v) saline solution containing 0.1% (w/v) Evans Blue. Either vehicle or one of the peptides was administered intracerebroventricularly as 10-μL injections as detailed previously[15]. Water intake was measured at 60 and 120 min under fasting conditions, as previously described[4]. At the end of the experiment, chicks were euthanized by decapitation. Injection was confirmed by detecting the presence of Evans Blue dye in the lateral ventricle[16]. Data from chicks not showing Evans Blue staining in the lateral ventricle or those that spilled water were omitted.
Experiment 2. Effect of central administration of peptides on water intake in chicks under ad libitum drinking conditionTwenty-eight 8-day-old chicks were weighed and allocated to two groups based on body weight (14 birds per group). The chicks were given free access to food and water before peptide injection. Either the vehicle or one of the peptides was administered intracerebroventricularly, feed was removed, and water intake was measured as described in Experiment 1.
Experiment 3. Effect of water deprivation on mRNA levels of neuropeptide Y and proopiomelanocortin in the chicken diencephalonTwelve 7-day-old chicks were weighed and allocated to two groups based on body weight (six birds per group). After 0 or 3 h of water deprivation, the chicks were euthanized by decapitation and their diencephalons were collected. The mRNA levels of NPY and proopiomelanocortin (POMC, the gene encoding α-MSH) were analyzed as described previously[17].
Data analysisAll data were analyzed by Student’s t-test using Microsoft Excel 2016 (Microsoft Corporation, Redmond, WA, USA). Statistical significance was defined at P < 0.05.
Under thirst-inducing conditions, intracerebroventricular administration of α-MSH significantly decreased water intake within 60 min (Fig. 1a); whereas NPY had no significant effect (Fig. 1b). Conversely, under ad libitum drinking conditions, α-MSH did not show a significant effect (Fig. 1c); whereas NPY significantly increased water intake at 60 and 120 min after administration (Fig. 1d). These results suggest that α-MSH and NPY may function as thirst-quenching and thirst-inducing peptides in chicks, respectively.
Effect of intracerebroventricular administration of α-melanocyte-stimulating hormone (α-MSH) and neuropeptide Y (NPY) in chicks on water intake.
The experiments were conducted under thirst-inducing (a and b) or ad libitum drinking (c and d) conditions. Data are expressed as the mean ± SEM of replicates in each group. The number of chicks is shown in parentheses. Asterisks indicate significance with respect to the control group (*P < 0.05; **P < 0.01).
We previously showed that more than 3 h of water deprivation significantly suppressed food intake in 8-day-old layer chicks[17]. Given that NPY and α-MSH are appetite-regulating peptides, we hypothesized that suppression of food intake induced by long-term water deprivation might affect NPY and POMC expression. No effect on NPY or POMC mRNA levels in the diencephalon was detected after short-term (3 h) water deprivation (Fig. 2). This indicates that water deprivation may alter protein levels and localization of NPY and α-MSH in the brain and, hence, post-translational regulation of thirst.
Effect of water deprivation for 0 or 3 h on the mRNA levels of neuropeptide Y (NPY) and proopiomelanocortin (POMC) in the chicken diencephalon. Data are expressed as the mean ± SEM of replicates in each group (n = 6).
The effect of centrally administered NPY on food and water intake has been examined in mammals and chicks; however, results remain inconsistent. On the one hand, intracerebroventricular administration of NPY significantly increases food and water intake in rabbits[18] and rats[19]. On the other hand, it significantly increases food intake but suppresses water intake in mice[20]. In chicks, intracerebroventricular administration of NPY significantly increases food intake but does not affect water intake[10,11]. However, NPY-induced water intake was observed in feed-deprived rabbits[18] and chicks (Fig. 1d). All experiments were conducted under ad libitum drinking conditions. Notably, intracerebroventricular administration of NPY significantly suppresses water intake in feed-deprived mice after 18 h of water deprivation[20]. Thirst can be influenced by many factors, such as ambient temperature and humidity, salt intake, and physical activity[21,22]. Therefore, it is likely that the response to NPY administration among homeothermic animals is both species-specific and dependent on experimental conditions.
Central administration of α-MSH suppressed food intake, but did not affect water intake in genetically selected low- or high-body-weight broiler chicks under ad libitum drinking conditions[12]. In the present study, α-MSH suppressed water intake after 24 h of water deprivation. Therefore, thirst-induced water intake may be blocked by α-MSH in chicks. In rats, central administration of α-MSH did not affect water intake after 24 h of water deprivation[23]. Therefore, unlike rats, α-MSH may function as a thirst-quenching neuropeptide in chicks.
Central administration of NPY did not affect water intake[11] or drinking behavior[24] in genetically selected low- and high-body-weight broiler chicks at 5 days of age. Kuenzel et al.[10] reported that NPY stimulated food intake without affecting water intake in broiler chicks. In the present study, NPY increased water intake in 8-day-old chicks under feed-deprived conditions. Although significant orexigenic effects of NPY have been observed in both broiler and layer chicks[11,13,24], intensive genetic selection has resulted in many physiological differences[25]. Therefore, the impact of NPY on water intake may differ between broiler and layer chicks, suggesting the need for further studies under feed-deprived conditions.
In mammals, osmotic stress triggers thirst and diuresis[9]. In rats, osmotic stress induces NPY mRNA expression in the hypothalamic paraventricular and supraoptic nuclei, but not in the arcuate nucleus[26]. In contrast, osmotic stress suppresses NPY mRNA expression in the hypothalamic arcuate nucleus in mice[27]. Notably, these changes are thought to cause the release of the antidiuretic hormone vasopressin in both rats and mice[26,27]. Thus, the regulatory mechanism underlying NPY-mediated antidiuresis may differ between rats and mice. In chickens, the relationship between NPY and vasotocin, an avian equivalent of vasopressin, remains unclear. Given that short-term water deprivation did not affect NPY expression in the diencephalon of chicks, the role of NPY in each hypothalamic nucleus and in controlling diuresis warrants further study.
Angiotensin II stimulates drinking in mammals and birds[2], and the subfornical organ and vasculosum lamina terminalis in the brain have been implicated in the control of water intake elicited by angiotensin II in mammals[9] and chicks[28]. In mammals, excitatory lamina terminalis neurons stimulate vasopressin release from the paraventricular hypothalamus into the bloodstream via their axon terminals in the posterior pituitary gland[9]. Avian vasotocin, expressed in the paraventricular regions of the hypothalamus[29], is similarly released into the bloodstream[30]. However, vasopressin and vasotocin trigger also an anti-diuretic response in mammals[9] and birds[30]. These networks may play important roles in the maintenance of body fluids in response to osmotic stress; although evidence suggests that NPY and α-MSH are not involved in this system. For example, hypertonic saline injection did not affect mRNA levels in the hypothalamus of chicks[17]. Central administration of vasotocin suppresses water intake[4]. However, vasotocin induces wing flapping behavior in chicks[31]; whereas NPY and α-MSH do not[22]. Interestingly, acute heat stress significantly increased NPY mRNA levels in the diencephalon of chicks, although food intake was significantly decreased[32]. While water intake was not measured during heat stress experiments, NPY may play a key role in such circumstances in chicks. Hypothalamic POMC is upregulated during incubation in laying hens[33,34], indicating that α-MSH may quench thirst. Further studies are required to determine the physiological role of NPY and α-MSH in body fluid maintenance in chicks.
Preventing thirst is critical not only for maintaining animal welfare but also for efficient meat and egg production[1]. Water scarcity negatively affects animal rearing[35,36]. As climate change puts a strain on freshwater resources, water-efficient poultry production will ensure not only sustained yields but also adequate animal welfare[37]. Recently developed water-efficient broiler chicks[20] may not feel as thirsty as normal broiler chicks. In this study, we identified the functions of NPY and α-MSH in the regulation of thirst in chicks. Identification of the mechanisms underlying thirst regulation in chickens may provide new strategies for genetic selection by poultry producers.
In conclusion, central administration of NPY significantly increased water intake, whereas α-MSH suppressed it. Short-term water deprivation did not affect NPY or POMC expression. Our findings suggest that NPY and α-MSH function as thirst-inducing and thirst-quenching peptides, respectively, and mediate post-translational regulation of water intake in chicks.
This study was supported by JSPS KAKENHI (grant number 24K01907).
Nnamdi Godswill Dialoke, Kaoruko Murata, Yuhui Zhang, and Kazuhisa Honda conducted the experiments and analyzed the data; Yuji Taniguchi, Sei-ichi Hinomoto, and Kazuhisa Honda designed the experiments; Nnamdi Godswill Dialoke wrote the manuscript; Junya Takegaki, Takaoki Saneyasu, and Kazuhisa Honda edited the manuscript.
The authors declare no conflicts of interest.
