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Effects of Central Administration of Opioid Peptides, Vasotocin, Mesotocin, and Corticotrophin-Releasing Factor on Water Intake in Chicks
2025 Volume 62 Article ID: 2025011
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2025 Volume 62 Article ID: 2025011
Freedom from thirst is an undeniable requirement of the poultry industry. However, the regulatory mechanisms underlying water intake in chicks are not yet fully understood. In humans, increased blood osmolality is probably the strongest signal for drinking. Angiotensin II, a hyperosmotic signal, induces water intake in chickens; this effect is attenuated by an opioid receptor antagonist. Vasotocin and mesotocin appear to have osmoregulatory functions in chicken. Dehydration activates brain corticotrophin-releasing factor (CRF) neurons in rats, and the central administration of CRF induces water intake in rabbits. This study aimed to clarify the effects of neuropeptides such as opioid peptides, vasotocin, mesotocin, and CRF on water intake to identify thirst-inducing neuropeptides in chicks. Eight-day-old male chicks were intracerebroventricularly injected with saline or the peptides. Water intake was measured 120 min after the injection under feed-deprived conditions. Intracerebroventricular administration of Met-enkephalin (a δ-opioid receptor agonist), β-endorphin (a δ-opioid receptor agonist), and nociception (a κ-opioid receptor and opioid receptor-like protein agonist) significantly suppressed water intake in chicks, whereas dynorphin B (a κ-opioid receptor agonist) and endomorphin-1 and 2 (μ-opioid receptor agonists) did not affect water intake. Intracerebroventricular administration of vasotocin, mesotocin, and CRF significantly suppressed water intake in chicks. Our findings suggest that none of the neuropeptides used in this study function as thirst-inducing peptides in the central nervous system of chicks.
From an animal welfare perspective, freedom from thirst is an essential requirement in the poultry industry[1]. In mammals, increased blood osmolality is a major effect of drinking[2]. The subfornical organ and organum vasculosum of the lamina terminalis (OVLT) neurons sense osmolality and project to the paraventricular nucleus of the hypothalamus (PVN) and the supraoptic nucleus (SON)[2]. Opioid peptides and neuropeptides expressed in the PVN neurons, such as vasotocin, mesotocin, and corticotrophin-releasing factors (CRF), may be involved in the generation of thirst in chickens. However, the effects of these peptides on water intake in chicks have not been fully clarified.
Subcutaneous administration of angiotensin II, a hyperosmotic signal, increases water intake in chickens[3]; this effect is attenuated by intramuscular administration of the opioid receptor antagonist naloxone[4]. Bu et al. identified the preferential receptors of chicken opioid peptides, including enkephalins, β-endorphin, dynorphins, and nociception by using an in vitro reporter assay[5]. Central administration of 1 nmol endomorphin-2 reverses memory loss induced by a μ-opioid receptor antagonist in chicks[6,7]. Endomorphin-1 and -2, mammalian μ-opioid receptor-specific agonists[8], induce angiogenesis in chicken embryos; these effects are suppressed by an opioid receptor antagonist[9]. Therefore, these opioid peptides are likely to be involved in regulating chicken water intake.
Vasotocin and mesotocin, equivalents of mammalian vasopressin and oxytocin, respectively, have osmoregulatory functions in birds. For example, urine volume was significantly decreased by vasotocin, but increased by mesotocin, when administered intravascularly in hens[10]. Osmotic stress significantly increases hypothalamic vasotocin mRNA expression[11], elevates blood vasotocin[12,13,14], and reduces blood mesotocin[12] levels in chickens; however, in feed-deprived rats, central administration of vasopressin increases water intake in a dose-dependent manner[15], whereas oxytocin receptor antagonists increase water intake[16]. The effects of peripheral or central administration of vasotocin or mesotocin on water intake have been examined in hens[17], European starlings[18], and chicks[19]. However, all these avian experiments were carried out under ad libitum feeding conditions. This may be confounding because food intake is one of the causes of thirst; moreover, the central administration of vasotocin and mesotocin suppresses food intake in chicks[20]. Therefore, the effects of central administration of vasotocin and mesotocin on water intake should be examined in chicks under feed-deprived conditions.
In mammals, dehydration activates CRF neurons to drive the hypothalamus-pituitary-adrenal axis[21,22]. Central administration of CRF significantly increases water intake in rabbits[23]. Intraperitoneal administration of 3M NaCl significantly elevates plasma corticosterone levels in chicks, suggesting that the hypothalamus-pituitary-adrenal axis is activated by osmotic stress[24]. CRF is the central regulator of the HPA axis and stress-induced behavior in chicks[25,26]. Intracerebroventricular CRF administration did not influence water intake in chicks after 24 h of fasting[27], nor did it suppress food or water intake in Japanese quail[28]. The reason for this discrepancy may be differences in feeding conditions. Therefore, we measured the effects of central CRF administration on water intake in chicks under short-term feed-deprived conditions, specifically for opioid peptides, vasotocin, mesotocin, and CRF. Our findings suggest that the neuropeptides chosen for analysis in this study may not function as thirst-inducing neuropeptides in the central nervous system of chicks.
Day-old male chicks (White Leghorn) were purchased from local hatcheries (Japan Layer K.K., Gifu, Japan) and provided with free access to water and a commercial chick starter diet (NICHIWA SANGYO Co., Ltd., Kobe, Japan). Chicks were reared in electrically heated cages with a 23-h/1-h light-dark cycle 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 Kobe University Animal Experimentation Regulation (2022–06-02).
Chicken dynorphin B, nociception, and β-endorphin were purchased from Biologica Co. (Nagoya, Japan) as custom peptides. (Ile8)-oxytocin (mesotocin) was purchased from NACALAI TESQUE, Inc. (Kyoto, Japan). Human CRF, endomorphin-1, and endomorphin-2 were purchased from the PEPTIDE INSTITUTE. Inc. (Osaka, Japan). Vasotocin and Met-enkephalin were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Neuropeptide administrationCentral administration of 1 nmol endomorphin-2[6,7] and 1-5 nmol nociception[29] had significant effects in chicks. Central administration of 0.5-1.5 nmol β-endorphin induced significant hyperphagia in birds[30,31]. Bu et al. reported that Met-enkephalin, dynorphin B, and nociceptin activate δ-opioid receptor-, κ-opioid receptor-, and opioid receptor-like protein-mediated signaling pathways, respectively, with a similar range of EC50 values[5]. Based on these findings, the effect of 1 nmol opioid peptides on water intake was examined.
Masunari et al. showed that central injection of either 1 nmol vasotocin or mesotocin induced behavioral changes in chicks[20]. A central injection of 200 pmol CRF suppressed food intake in chicks[32]. Based on these findings, the effects of 1 nmol vasotocin and mesotocin and 200 pmol CRF on water intake were examined.
Neuropeptide central administration and measurementTwenty-eight seven-day-old chicks were weighed and divided into two groups based on body weight (14 birds each). Chicks were given free access to feed but were deprived of water for 24 h. 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 intracerebroventricularly administered as described by Davis et al.[33], in a volume of 10 μl. Chicks were placed in individual cages (150 mm × 250 mm × 150 mm), and water intake was measured 120 min after treatment. Water was supplied using a plastic waterer (78 mm × 58 mm × 77 mm) without feeding. Waterers filled halfway with water were pre-weighed and placed in cages. Control waterer samples were placed next to the cages. At each time point, the waterers were weighed using an electric digital balance (readability: 10 mg). The water intake was calculated as:
Water intake = (The amount of water decrease in the chick’s waterer) − (The amount of water decrease in the control waterer).
At the end of the experiment, chicks were euthanized by decapitation. Injection was verified by the presence of Evans Blue dye in the lateral ventricle[34]. Data from chicks lacking this staining were omitted. Data from chicks that spilled water were omitted.
Data analysisAll data were analyzed using Student’s t-test and Microsoft Excel 2016 (Microsoft Corporation, Redmond, WA, USA). Statistical significance was defined as P < 0.05.
Central administration of Met-enkephalin, β-endorphin, and nociception significantly suppressed water intake in chicks, whereas the dynorphin B and endomorphins had no effect (Fig. 1). Central administration of the osmoregulatory peptides vasotocin and mesotocin (Fig. 2), as well as CRF (Fig. 3), also significantly suppressed water intake. These findings suggest that none of these neuropeptides function as thirst-inducing peptides in chicks.
Effects of intracerebroventricular administration of opioid peptides on water intake. Data are presented as means ± SEM of replicates in each group. The number of chicks are shown in parentheses. Asterisks indicate significance with respect to the control group (* P < 0.05; ** P < 0.01).
Effects of intracerebroventricular administration of vasotocin and mesotocin on water intake in chicks. Data are presented as means ± SEM of replicates in each group. The number of chicks are shown in parentheses. Asterisks indicate significance with respect to the control group (* P < 0.05; ** P < 0.01).
Effects of intracerebroventricular administration of corticotrophin-releasing factors (CRF) on water intake in chicks.
Data are presented as means ± SEM of replicates in each group. The number of chicks is shown in parentheses. Asterisks indicate significance with respect to the control group (** P < 0.01).
In the present study, none of the opioid peptides stimulated water intake in chicks. Chicken opioid. Opioid receptors are widely distributed in the chicken brain[5]. In mammals, many brain regions, including the anterior cingulate cortex, insular cortex, bed nucleus of the stria terminalis, and central amygdala have been implicated in the central representation of thirst[2]. In the present study, the peptides were injected into the lateral and third ventricles, suggesting that they had diffused around the ventricles. Therefore, more local and precise administration of opioid peptides to specific brain areas may be required to clarify their potential role(s), if any, in regulating water intake in chicks. Further studies are required to examine the effects of multiple doses of opioid peptides at multiple time points.
Naloxone, a δ-, κ-, and μ-opioid receptor antagonist[35,36], attenuated angiotensin II-induced water intake in chicks[4]. In the present study, δ-opioid receptor agonists (Met-enkephalin and β-endorphin) and an opioid receptor-like protein agonist (nociception) suppressed water intake in chicks, whereas a κ-opioid receptor agonist (dynorphin B) and mammalian μ-opioid receptor agonists (Endomorphin-1 and 2) did not influence water intake. In chicks, central administration of β-endorphin and nociception has been shown to significantly increase food intake[29,31]. Central administration of a δ-opioid receptor agonist increased food intake[37]. In addition, central administration of a μ-opioid receptor agonist decreased food intake, whereas a κ-opioid receptor agonist increased it[37]. These findings suggest that the effects of opioid peptides observed in the present study are unrelated to appetite regulation. Interestingly, central administration of U-50488H, a δ-opioid receptor agonist, stimulated food intake[37], but central administration of dynorphin suppressed wet mash intake[38]. In the present study, dynorphin B suppressed water intake. These findings suggest that, at least in chicks, δ-opioid receptor stimulation preferentially increases feed intake but not water intake. Therefore, it is possible that δ the-opioid receptor is involved in the stimulation of feeding and the suppression of drinking in different manners.
Central administration of vasotocin and mesotocin significantly suppressed water intake in chicks. In rats, there is evidence that oxytocin functions as a water intake-inhibiting peptide[16,39,40]. Therefore, the central role of mesotocin in regulating water intake may be conserved between mammals and birds. On the other hand, unlike mammals, vasotocin does not induce water intake in chicks, suggesting that it functions differently from mammalian vasopressin. However, rout of vasotocin injection may show different effects. For example, intracerebroventricular administration of the satiety hormone peptide YY (PYY)3-36 induced food intake[41], whereas intra-arcuate injection of PYY3-36 suppressed food intake[42]. The mRNA of chicken vasotocin receptor were expressed in the OVLT[24], which is involved in vasopressin-induced thirst generation in mice[43]. Therefore, further study is needed to examine the effect of vasotocin administration into OVLT on water intake in chicks.
In the present study, central administration of CRF suppressed water intake, suggesting that chicken CRF does not function as a thirst-inducing peptide. Intracerebroventricular administration of CRF in rabbits increased the intake of both water and 0.5 M NaCl[23]. In contrast, CRF injection into the lateral parabrachial nucleus (LPBN) in rats suppressed the intake of both water and 2% NaCl[44]. The reason for this apparent discrepancy remains unclear. However, it may be due to species or injection differences. In chickens, CRF receptors 1 and 2 are widely distributed in the brain[45,46]. Aman et al. reported that the intraperitoneal administration of 3M NaCl significantly decreased CRF receptor 1 mRNA levels in the chicken PVN[24]. The expression of CRF receptors in the LPBN has not yet been reported in chickens[24]. However, further studies are needed to examine the effect of CRF administration to the PVN on water intake in chicks.
Central administration of vasotocin and mesotocin induces wing flapping and voluntary activity in chicks[20,25]. Central administration of CRF significantly induced vocalization and locomotive activity[25,26]. Wing-flapping behavior and vocalization were also observed in the present study. Therefore, it is not clear whether the suppression of water intake is induced by behavioral changes or quenching of thirst. Clarification of chick physiological thirst indicators is required for future studies.
In the present study, 8-day-old layer chicks were used because layer chicks are widely used in behavioral experiments. To the best of our knowledge, the opposite effect of central administration of neuropeptides on behavioral changes between chick types or ages has not been reported, although resistance to several neuropeptides has been observed[47,48,49]. In fact, central administration of CRF suppresses food intake in both layers and broiler chicks during the neonatal[25,50] and later stages[27]. Mesotocin induces preening behavior in neonatal broilers[19] and layers[20]. Nociceptin induces feeding behavior in neonatal broilers[51] and layers[29]. However, further studies of varying ages and types are required to clarify the physiological roles of neuropeptides, especially those of the endomorphins and dynorphin B.
The relationship between CRF, vasotocin, mesotocin, and opioid peptides in the regulation of water intake is unclear. However, Masunari et al. suggested that the vasopressin-4 receptor is involved in behavioral changes induced by central administration of vasotocin and mesotocin in chicks[20]. In contrast, CRF receptors are involved in CRF-induced behavioral change in chicks[32]. Tachibana et al. reported that vasotocin induced wing flapping, whereas CRF induced vocalization[25]. These findings suggest that the mechanism underlying vasotocin- and mesotocin-induced behaviors differ from that of CRF in chicks. Sasaki et al. suggested that the κ-opioid receptor is involved in chick hyperosmotic stimulation and angiotensin II injection-induced vasotocin release[52]. However, central administration of dynorphin B, a κ-opioid receptor agonist, did not induce wing flapping in the present study (data not shown). Met-enkephalin, β-endorphin, and nociceptin suppressed water intake but did not induce vocalization or wing-flapping behavior (data not shown). Therefore, it is likely that Met-enkephalin, β-endorphin, and nociceptin are not involved in CRF-, vasotocin-, or mesotocin-induced behavioral changes in chicks.
In conclusion, none of the candidate opioid peptides induced water intake in chicks. Osmoregulatory peptides (vasotocin and mesotocin) and stress-related peptides (CRF) suppressed water intake in chicks. These findings suggested that none of the neuropeptides used in this study function as thirst-inducing peptides in the chick central nervous system.
This study was supported by JSPS KAKENHI (grant number 24K01907).
Yuhui Zhang and Kaoruko Murata conducted experiments and analyzed data; Kazuhisa Honda designed experiments; Yuhui Zhang wrote the manuscript; and Junya Takegaki, Takaoki Saneyasu, and Kazuhisa Honda edited the manuscript.
The authors declare no conflicts of interest.
