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Effects of Intraoral, Intracrop, and Intragastric Administration of Copper Sulfate on Vomiting, Retching Behavior, and Nausea-Like Responses in Chicks
2026 年 63 巻 論文ID: 2026019
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2026 年 63 巻 論文ID: 2026019
Nausea and vomiting are induced by environmental distress and pain; understanding their etiology can improve animal welfare in modern farm operations. Oral administration of CuSO4 induces nausea and vomiting in some mammalian species and in pigeons. Although oral administration of CuSO4 induces retching in chicks, whether it causes true vomiting or nausea-like responses remains unknown. This study examined whether intraoral, intracrop, and intragastric administrations of CuSO4 induced vomiting, retching, and nausea-like responses in chicks (Gallus gallus domesticus). Food intake, crop emptying rate, and voluntary activity were used as indices of nausea-like responses. CuSO4 limited food intake, irrespective of the administration route, and markedly reduced the crop-emptying rate. Voluntary activity remained unaffected by intraoral administration, but decreased following intracrop and intragastric administrations. Four of the six chicks exhibited retching after intraoral administration of CuSO4; whereas intracrop and intragastric administrations failed to elicit this behavior. Similarly, CuSO4 failed to cause any vomiting. These findings indicate that CuSO4 does not act as an emetic agent in chicks; rather it induces nausea-like responses. Route-dependent effects further suggest that exposure within the oral cavity is required for the excretion of CuSO4, possibly serving as a protective behavioral mechanism to reject irritant compounds before they enter the lower gastrointestinal tract.
Modern animal rearing systems must consider animal welfare, thereby eliminating any sources of environmental stress or pain and providing optimal feed and water. However, quantifying the internal state of distress in animals remains challenging. True vomiting is a coordinated reflex that results in the expulsion of gastric contents; whereas nausea is an unpleasant sensation that precedes vomiting[1]. Physiological stressors such as infection, food poisoning, and motion sickness cause vomiting and nausea[1]. Identifying emetic and nausea-like indices can be useful for detecting environmental distress and pain. Therefore, elucidating the mechanisms underlying vomiting and nausea may contribute to improving animal welfare.
Various pharmacological agents have been used to induce vomiting and nausea[1]. Copper sulfate (CuSO4) is an emetic agent that stimulates the mucosal membranes of the gastrointestinal tract. Oral administration of CuSO4 causes true vomiting in dogs, cats, ferrets, and house shrews[2,3,4,5]. In contrast, in rats and mice, it causes nausea characterized by anorexia, delayed food passage, and behavioral depression, because rodents possess anatomical constraints in the diaphragm and esophagus that render them incapable of vomiting[6,7,8,9]. Additionally, oral CuSO4 induces pica, an adaptive behavioral response marked by the targeted ingestion of non-nutritive materials such as kaolin[6]. This evidence indicates that, depending on the species, CuSO4 administration can induce both vomiting- and nausea-like responses.
Among birds, CuSO4 is known to induce vomiting in pigeons[10,11], even though the diaphragm and the structure of the digestive tract are different from those of mammals[12,13]. However, only a few studies have been conducted on chickens, despite their importance in global poultry production. Preliminary evidence indicates that oral CuSO4 induces retching behavior in both neonatal and adult chickens; however, it fails to trigger complete expulsion associated with true vomiting[14,15]. These findings suggest that the CuSO4-induced responses in chickens may differ from those observed in pigeons. Nausea-like responses to CuSO4, as well as the region of the upper gastrointestinal tract responsible for CuSO4-induced retching, remain poorly characterized.
The primary objective of this study was to examine the localized effect of intraoral CuSO4 administration on vomiting and nausea in chicks (Gallus gallus domesticus), as well as the effect of intracrop and intragastric CuSO4 administrations on the sensitivity of the gastrointestinal tract to this compound.
Animal experiments were approved by the Committee for Animal Care and Use of Ehime University, Japan (No. 08-o5-10), and conducted in accordance with Ehime University Animal Experiment Regulations.
AnimalsOne-day-old male layer chicks (G. gallus domesticus, White Leghorn) were purchased from a local hatchery (Minami Iyo Yokei, Ehime, Japan) and raised in a room maintained at 30 °C under continuous light. A commercial diet (crude protein: 23%, metabolizable energy: 3050 kcal/kg; JA Zen-Noh Kumiai Feed Co. Ltd., Tokyo, Japan) and water were provided ad libitum. The chicks were transferred to an experimental cage for at least two days before each acclimation experiment and were individually caged one day prior to testing. Before the experiments, body weight was measured and the chicks were allocated to experimental groups to ensure a uniform body weight across treatment groups.
Drugs and administrationsAll administrations were administered between 05:00 and 12:00. CuSO4·5H2O (Nacalai Tesque Inc., Kyoto, Japan) was dissolved in ultrapure water. For intraoral administrations, 200 mg of CuSO4 per kg body weight (200 mg/kg) was administered in 100 μL into the oral cavity using a micropipette. For intracrop administrations, a silicone tube (70 mm length, 5 mm diameter) was inserted into the crop, and 200 mg/kg CuSO4 (500 μL) was injected. For intragastric administrations, a stainless-steel feeding needle (80 mm length, 1.2 mm diameter, with a silicone-covered tip) was inserted into the proventriculus, and 200 mg/kg CuSO4 was injected (200 μL). Equivalent volumes of ultrapure water were injected in each control treatment.
Experiment 1: Effect of CuSO4 on vomiting and food intakeFor the intraoral administration study, 9-day-old chicks were administered vehicle only (control) or 200 mg/kg CuSO4ad libitum. The administered dose of CuSO4 was selected based on a previous study[14]. The chicks were returned to their home cages and a sheet of white paper was placed beneath each cage to monitor the incidence of vomiting. A pre-weighed feeder was then provided and food intake was measured at 1, 2, 3, 4, 5, and 6 h after administration using a digital balance with a precision of 1 mg. The incidence of vomiting was verified by checking the white paper during each food intake measurement.
For intracrop and intragastric administration studies, 7-day-old chicks were injected with vehicle only or 200 mg/kg CuSO4 under ad libitum feeding conditions. Food intake was measured as previously described.
Experiment 2: Effect of CuSO4 on vomiting and crop emptyingThe crop-emptying rate was measured as previously reported[15]. Nine-day-old chicks, food-deprived for 15 h to clear residual ingesta from the crop, were injected intraorally with vehicle only or 200 mg/kg CuSO4. Immediately following administration, the chicks were directly gavaged with approximately 2 g of feed slurry into the crop. The feed slurry was prepared by mixing 40% powdered diet with 60% distilled water on a weight basis. Following gavage, the chicks were returned to their individual cages, and feed and water were withheld. A sheet of white paper was placed beneath each cage to monitor vomiting. Two hours after gavage, the chicks were euthanized via CO2 inhalation, after which the crops were exposed, the upper and lower esophagi were clamped, and the crops were excised. The total content of each crop was recovered, dried at 55 °C for 48 h, and then air-dried for 24 h. The air-dried slurry was weighed using a digital balance with a precision of 1 mg and the corresponding wet slurry weight was calculated based on the dry weight. The weight of the slurry emptied from the crop through the lower esophagus was calculated by subtracting the weight of the slurry remaining within the crop from the total weight of the administered slurry. The crop-emptying rate was expressed as the percentage of slurry emptied from the crop. Vomiting was visually assessed by the presence of expelled gastric contents on the underlying white paper and confirmed by the absence or presence of regurgitated material during periodic observations.
For intracrop and intragastric administration studies, 8- and 9-day-old chicks fasted for 15 h were injected with vehicle only or 200 mg/kg CuSO4, and the crop-emptying rate was investigated as described above.
Experiment 3: Effect of CuSO4 on vomiting and voluntary activityNine-day-old ad libitum-fed chicks were intraorally injected with vehicle only or 200 mg/kg CuSO4. The chicks were then returned to their home cages and a sheet of white paper was placed beneath the cages to assess the occurrence of vomiting. Voluntary activity was measured every 30 min for 6 h using a passive pyroelectric infrared detector (NS-AS02; Neuroscience Inc., Osaka, Japan) connected to a DAS-64 system (Neuroscience Inc.). After measurement, the occurrence of vomiting was verified by checking the underlying white paper. Chick behavior was recorded for 30 min using a digital video camera to quantify the number of retching episodes.
For intracrop and intragastric administration studies, 10-day-old ad libitum-fed chicks were injected with vehicle only or 200 mg/kg CuSO4, and voluntary activity assessment and video recordings were performed as described above.
Statistical analysisData on food intake and voluntary activity were statistically analyzed using a two-way mixed model of analysis of variance with respect to administration and time. A t-test was used as a post-hoc test at each time point. Data on the crop-emptying rate were analyzed using t-tests. Data on the number of retching events were analyzed using the Mann–Whitney U-test. Data are expressed as means ± standard error of the mean (SEM), and statistical significance was set at P < 0.05. The number of chicks in each group is reported for each figure.
None of the chicks administered CuSO4 via administration route exhibited vomiting.
Intraoral administration of CuSO4 markedly suppressed food intake [F(1,12) = 49.1, P < 0.05], and a significant interaction was observed between CuSO4 treatment and time [F(5,60) = 44.7, P < 0.05]. Chicks injected with CuSO4 showed reduced food intake at all time points (Fig. 1A).
Effect of (A) intraoral, (B) intracrop, and (C) intragastric administrations of CuSO4 on food intake by chicks under ad libitum feeding conditions. Data are expressed as means ± standard error of the mean (SEM) for 9 chicks per group in the intraoral study; 8 (control) or 9 (200 mg/kg) chicks per group in the intracrop study; and 7 chicks per group in the intragastric study. *P < 0.05 compared to the vehicle-only control.
Similarly, food intake decreased following intracrop administration of CuSO4 [F(1,12) = 58.8, P < 0.05] at all time points (Fig. 1B), revealing a significant interaction between CuSO4 and time [F(5,60) = 59.3, P < 0.05].
A significant inhibitory effect on food intake [F(1,12) = 104.0, P < 0.05] was observed also upon intragastric CuSO4 administration at all time points, with a significant interaction between CuSO4 and time [F(5,60) = 61.0, P < 0.05]; it almost completely abolished feeding behavior (Fig. 1C).
After 6 h, intraoral, intracrop, and intragastric administrations of CuSO4 reduced food intake to 14.0%, 18.5%, and 1.4% of the control, respectively.
Experiment 2: Effect of CuSO4 on vomiting and crop emptyingConsistent with the observations in Experiment 1, CuSO4 administration failed to induce vomiting during the observation period, irrespective of administration route.
In the intraoral administration study, baseline feed slurry gavage volumes remained highly uniform between groups (control, 2.26 ± 0.01 g; CuSO4, 2.24 ± 0.00 g). Administration of CuSO4 notably reduced the crop-emptying rate by 49.0% compared with the control value (Fig. 2A).
Effect of (A) intraoral, (B) intracrop, and (C) intragastric administrations of CuSO4 on the crop-emptying rate in chicks. Data are expressed as means ± standard error of the mean (SEM) for 7 chicks per group in the intraoral study, 9 chicks per group in the intracrop study, and 6 chicks per group in the intragastric study. Groups with different letters indicate statistically significant differences (P < 0.05).
In the intracrop administration study, equivalent amounts of feed slurry were gavaged across treatment groups (control, 2.24 ± 0.01 g; CuSO4, 2.25 ± 0.00 g). The crop-emptying rate was significantly decreased by CuSO4, dropping to 44.8% of the control value (Fig. 2B).
Similarly, in the intragastric administration study, pre-administered slurry weights exhibited no variation between groups (control, 2.25 ± 0.01 g; CuSO4, 2.24 ± 0.00 g). However, intragastric administration of CuSO4 markedly reduced the crop-emptying rate to 6.6% of the control value (Fig. 2C).
Experiment 3: Effect of CuSO4 on vomiting and voluntary activityNo chicks exhibited vomiting after administration of CuSO4 via any administration route.
Voluntary activity was not affected by the intraoral administration of CuSO4 [F(1,10) = 2.6, P = 0.14], and no significant interaction between CuSO4 treatment and time was observed [F(11,110) = 0.6, P = 0.86] (Fig. 3A). Administration of CuSO4 did not affect voluntary activity at any time point. Notably, four of the six chicks injected intraorally with CuSO4 showed retching behavior, reflecting an increase in the number of retching episodes (Table 1). The supplementary movie shows the typical retching behavior of the chicks.
Effect of (A) intraoral, (B) intracrop, and (C) intragastric administrations of CuSO4 on voluntary activity in chicks. Data are expressed as means ± standard error of the mean (SEM) for 6 chicks per group and study. *P < 0.05 and †P < 0.10 compared to the vehicle-only control.
| Groups | Number of retching chicks/ Total chicks | Time after injection (min) | |||
| 0–10 | 10–20 | 20–30 | |||
| Intraoral | |||||
| 0 mg/kg | 0/6 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | |
| 200 mg/kg | 4/6 | 64.8 ± 40.3† | 7.7 ± 6.5 | 8.0 ± 7.4 | |
| Intracrop | |||||
| 0 mg/kg | 0/6 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | |
| 200 mg/kg | 0/6 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | |
| Intragastric | |||||
| 0 mg/kg | 0/6 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | |
| 200 mg/kg | 0/6 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 | |
Data are expressed as means ± standard error of the mean (SEM). †P < 0.10 compared to the vehicle only control (0 mg/kg).
In contrast, intracrop administration of CuSO4 substantially decreased voluntary activity [F(1,10) = 18.7, P < 0.05]; however, no significant interaction between CuSO4 treatment and time was observed [F(11,110) = 0.8, P = 0.64] (Fig. 3B). Intracrop administration of CuSO4 substantially reduced voluntary activity at 1.5, 2, 4, and 5 h after administration and did not elicit retching behavior in any of the chicks (Table 1).
Voluntary activity was markedly reduced following intragastric administration of CuSO4 [F(1,10) = 41.7, P < 0.05], accompanied by a significant interaction between CuSO4 treatment and time [F(11,99) = 1.9, P = 0.03] (Fig. 3C). CuSO4 tended to decrease the voluntary activity at 1 h and remained markedly decreased thereafter until the end of the observation period. None of the chicks exhibited retching behavior (Table 1).
After evaluating total (6 h) voluntary activity, chicks subjected to intraoral, intracrop, and intragastric administrations of CuSO4 maintained 77.0%, 65.7%, and 41.1% of baseline voluntary activity, respectively, compared with the corresponding control values (Fig. 3).
Oral administration of CuSO4 induces nausea and vomiting in several mammalian species and in pigeons[2,3,4,5,10,11]. However, in the present study, CuSO4 did not induce vomiting in chicks, echoing a recent study from our group on cisplatin, another emetic agent[16]. Therefore, chicks may not vomit in response to emetic agents.
Because vomiting is a primary physiological response used to exclude toxic feed from the gastrointestinal tract, the inability to vomit may represent a survival disadvantage. Rodents do not vomit, which can be explained in two ways[17]. First, rodents nibble and consume smaller amounts during frequent feeding bouts, allowing for more time to detect toxic foods, whereas many other animals consume larger amounts during discrete meals. Second, rodents may have evolved robust detoxification mechanisms capable of handling small toxic loads.
The diaphragm and esophageal reverse peristalsis play important roles in mammalian vomiting[18]. Rodents possess a less muscular diaphragm, a longer descending esophagus, and a differently shaped stomach compared to animals capable of vomiting, such as dogs, cats, ferrets, and house musk shrews[17].
Birds lack a diaphragm[12], and their gastrointestinal tract differs substantially from that of mammals. For example, most birds have a crop in the esophagus and two distinct specialized stomach regions (the proventriculus and gizzard). In contrast, pigeons, which are known to vomit, produce milk within the crop to feed their young[13] and possess a specific mechanism for regurgitation and vomiting. Owls also eject pellet-like indigestible prey components from their stomachs[19]. Although certain avian species do vomit, the underlying mechanisms differ from those in mammals. Chickens do not exhibit true vomiting but rely on retching[11], thus providing a useful model for distinguishing CuSO4-induced nausea-like responses from retching. In our preliminary observations, chicks injected with CuSO4 exhibited slurry leakage from the mouth after euthanasia with CO2. This finding supports the hypothesis that chicks actively control crop contents to prevent reflux into the mouth.
Furthermore, the administration of CuSO4 markedly reduced food intake (Fig. 1) and the crop-emptying rate (Fig. 2). Although intraoral administration of CuSO4 did not affect voluntary movement, intracrop and intragastric administrations markedly decreased voluntary movement (Fig. 3). In rats, intragastric administration lowered food intake[6]; whereas intraoral administration of CuSO4 reduced gastric emptying[7,8]. Moreover, intraoral administration of CuSO4 increased immobility time during tail suspension and forced swim tests in mice[9]. Because these parameters serve as established indices of nausea in animals incapable of vomiting, the administration of CuSO4 appears to induce nausea-like responses in chicks.
CuSO4 remarkably suppressed food intake, voluntary activity, and crop-emptying rate when injected into the proventriculus. In dogs, the stomach pylorus is more sensitive to CuSO4 than the antrum, and the duodenum is even more sensitive to CuSO4 in triggering vomiting[20,21]. Therefore, the proventriculus and lower gastrointestinal tract of chicks are highly susceptible to harmful stimuli. The crop and proventriculus may act as gatekeepers to prevent the entry of feed into the proventriculus and lower digestive tract during gastrointestinal distress. To this end, food intake was nearly abolished following intragastric administration of CuSO4, thereby preventing additional ingesta from entering the irritated proventriculus.
A key novel finding of this study is that retching behavior was induced exclusively by intraoral exposure to CuSO4 (Table 1); whereas postoral administration failed to elicit this response, despite stronger suppression of feeding, gastrointestinal motility, and voluntary activity. This dissociation indicates that retching in chicks is not simply a consequence of visceral discomfort, but is critically dependent on sensory detection within the oral cavity. These findings suggest that in contrast to mammals, where emesis is primarily driven by gastrointestinal and central mechanisms, chickens rely on a pre-ingestive rejection system to eliminate potentially harmful substances before they enter the lower gastrointestinal tract. However, as some chicks receiving intraoral administration of CuSO4 did not exhibit retching (Table 1), the actual mechanism underlying this response will require further elucidation.
The route-dependent effects observed in this study suggest the involvement of distinct peripheral sensory pathways. In particular, the induction of retching exclusively after intraoral administration of CuSO4 indicates that sensory inputs from the oral cavity, potentially mediated by trigeminal afferents[22], play a critical role in the response. In contrast, the pronounced suppression of food intake, crop emptying, and voluntary activity following intracrop and intragastric administration suggests the activation of post-ingestive signaling pathways, possibly involving vagal afferents that convey visceral information[23]. Additionally, bioactive peptides, such as cholecystokinin, glucagon-like peptide-1, and glucagon-like peptide-2, might be related to the effects of CuSO4 because their receptors are expressed in the crop or proventriculus in chickens[24,25,26]. As these peptides reduce food intake in chicks[27,28,29], they may mediate the nausea-like responses caused by CuSO4. Such differential activation of the oral versus gastrointestinal sensory systems may underlie the dissociation between retching behavior and nausea-like responses observed in chicks. It is possible that the injected CuSO4 was absorbed in the intestine as copper and sulfate ions, thereby systemically inducing nausea-like responses. Further studies involving systematic administration of CuSO4 will clarify the actual mechanism of CuSO4-induced nausea-like responses.
In summary, the present study demonstrated that the administration of CuSO4 does not induce true vomiting, but reliably elicits nausea-like responses in chicks. Additionally, physiological sensitivity to CuSO4 varies across distinct segments of the gastrointestinal tract. Further studies are warranted to clarify the exact mechanisms underlying nausea-like responses in chicks.
All animal experiments were approved by the Committee for Animal Care and Use of Ehime University, Japan (No. 08-o5-10), and conducted in accordance with Ehime University Animal Experiment Regulations.
The authors received no specific grant for the research described in this article.
We greatly appreciate the advice provided by Ms. M. Aoki.
Tetsuya Tachibana conceived the present study and experimental design, conducted the experiments, analyzed the data, and wrote the paper; Kaoru Nakamura conducted the experiments and analyzed the data; Sakirul Khan designed the experiments, discussed the results, and edited the paper; Mark A. Cline discussed the results and edited the paper.
The authors declare no conflicts of interest.
The authors declare that no artificial intelligence (AI) or AI-assisted technologies were used in this study.
The online version contains supplementary material available at https://doi.org/10.2141/jpsa.2026019
Typical retching behavior in chicks receiving intraoral administration of CuSO4.
