2025 年 62 巻 論文ID: 2025008
From the perspective of animal welfare, freedom from hunger and thirst is an undeniable requirement for the poultry industry. Regulatory mechanisms underlying drinking behavior have not yet been identified in chickens; however, the regulation of osmolality and water intake appears to be closely related. This study clarified whether wet feeding affects appetite, osmolality, and stress-related gene expression in the hypothalami of chicks. In Experiment 1, the effects of different wet feed percentages on the growth of broiler chicks were examined. Wet feeds were prepared by mixing either 0.25 g (20% wet feed), 0.667 g (40% wet feed), or 1.5 g (60% wet feed) of distilled water per g of commercial feed. Then, the wet feeds were offered to 4-day-old broiler chicks until 42 d of age. Forty percent wet feed significantly increased body, breast, and leg weights. In Experiment 2, 7-day-old broiler chicks were given either commercial starter feed or 40% wet feed until 21 d of age. Again, weights of the body, breasts, and legs were significantly increased by wet feeding. The total amount of water loss in the individual waterers was significantly decreased by wet feeding. No significant changes were observed in mRNA levels of the genes encoding appetite-regulatory peptides (neuropeptide Y and α melanocyte-stimulating hormone), osmoregulatory peptides (vasotocin and mesotocin), or stress-related peptides (corticotrophin-releasing factor) in the chicken hypothalamus. Overall, 40% wet feed improved growth without inducing thirst or hunger in broiler chicks. These findings suggest that wet feeding contributes to both meat yield and animal welfare during broiler production.
Freedom from hunger and thirst is an important requirement of the poultry industry[1]. Modern broiler chicks show hyperphagia; thus, they eat more feed and grow faster than layer chicks[2]. However, broiler chicks do not show higher expression of orexigenic neuropeptide genes or lower expression of anorexigenic neuropeptide genes in the hypothalamus, the central regulatory site of food intake, compared to that of layer chicks[3,4,5,6]. Therefore, it is likely that modern broiler chicks do not feel hungry when fed ad libitum.
Recent studies have demonstrated that wet feeding improves broiler growth. The addition of water to wheat-based feed (1.3 g/g feed) significantly improves feed intake and body weight gain in broiler chicks[7,8]. The addition of water to corn-based feed (1.2–1.3 g/g feed) significantly improves feed intake and body weight gain in broiler chicks in normal[9,10] and hot climate conditions[11]. In these studies, the feed conversion ratio was not influenced by wet feeding. These findings suggest that broiler chicks prefer to consume wet feed, which increases their body weight. Additionally, because wet feeding contains both water and feed, it may satiate hunger and quench thirst in broiler chicks. However, the effects of wet feeding on appetite-, osmolality-, and stress-related gene expression in the central nervous system of chicks have not yet been examined.
In chickens, neuropeptide Y (NPY) and α-melanocyte-stimulating hormone (α-MSH) play important roles in appetite regulation. For example, central administration of NPY potently stimulates food intake in chicks[5], and 12 h of fasting significantly increases mRNA levels of hypothalamic NPY in broiler chicks[12]. There is also evidence that mRNA levels of hypothalamic NPY and proopiomelanocortin (POMC) are significantly altered in hungry chickens[13,14]. Moreover, central administration of α-MSH potently suppresses food intake in chicks[6], and transcriptional and pathway analyses of the hypothalamus of newly hatched chicks suggest that POMC, a precursor of α-MSH, plays an important role in the central regulation of feeding[15]. Corticotrophin-releasing factor (CRF) is a stress marker frequently used in chicks[16,17,18]. Therefore, NPY and POMC mRNA levels in the hypothalamus may be markers of hunger in chicks, whereas CRF mRNA levels in the hypothalamus may reflect stress.
In humans, increased blood osmolality is probably the most important homeostatic signal for drinking[19]. Vasotocin, the equivalent of mammalian vasopressin, and mesotocin have osmoregulatory functions in birds[20]. For example, 8 h of dehydration significantly increases hypothalamic vasotocin mRNA levels in chicks[21]. Intravascular administration of a hypertonic saline solution significantly elevates vasotocin levels and reduces mesotocin levels in hen blood[22]. Intraperitoneal injection of[23] or drinking[24] hypertonic saline significantly elevates blood vasotocin levels in chicks[23]. Intravascular injection of vasotocin and mesotocin significantly decreases urine volume in hens, but a higher dose of mesotocin significantly increases urine volume[25]. Peripheral administration of vasotocin in hens[26] and central administration of mesotocin in chicks[27] suppresses water intake. Therefore, vasotocin and mesotocin mRNA levels in the hypothalamus may reflect body fluid balance in chicks.
In the present study, a screening experiment was conducted to examine the effect of different percentages of water in feed on the growth of broiler chicks. Next, the effects of 40% wet feeding on the growth and gene expression of hypothalamic peptides were examined in chicks.
This study was approved by the Institutional Animal Care and Use Committee and performed according to the Kobe University Animal Experimentation Regulations (2019-03-01). One-day-old male chicks (Ross 308) were purchased from a local hatchery (Yamamoto Co., Ltd., Kyoto, Japan). They were provided free access to water and a commercial chick starter diet (22.0% crude protein (CP) and 3,050 kcal/kg metabolizable energy; Nichiwa Sangyo Co. Ltd., Kobe, Japan).
In Experiment 1, 4-day-old chicks were allocated based on body weight into four experimental groups with four replicates (eight chicks per group and two chicks per cage) and fed the experimental diets. The chicks in the control group received commercial starter feed until 21 d of age and then commercial grower feed until 42 d of age (19.0% CP and 3,200 kcal/kg metabolizable energy; Nichiwa Sangyo Co. Ltd., Kobe, Japan). The chicks in the other three groups received wet feeds prepared by mixing either 0.25 g (20% wet feed), 0.667 g (40% wet feed), or 1.5 g (60% wet feed) of distilled water per g of commercial feed (Fig. 1). Wet feed was immediately provided to the chicks after mixing every morning. The remaining feed was discarded the following morning. At 42 d of age, chicks were euthanized by decapitation under carbon dioxide anesthesia. The skin was removed and the breasts and legs, consisting of the thigh and drumstick, were excised and weighed.
Feed forms of experimental feeds. Wet feeds were prepared by mixing either 0.25 g (20% wet feed), 0.667 g (40% wet feed), or 1.5 g (60% wet feed) of distilled water per g of commercial starter (A) or grower (B) feed.
In Experiment 2, male broiler chicks were reared under the same conditions as those in Experiment 1. At 7 d of age, chicks were allocated, based on body weight, into two experimental groups of four replicates (eight chicks per group, two chicks per cage) and given commercial starter feed or 40% wet feed until 21 d of age. The weight of the waterer in each cage was measured daily and the decrease of water in each waterer was calculated. At 21 d of age, chicks were euthanized by decapitation under carbon dioxide anesthesia. The skin was removed and the breasts and legs were excised and weighed. The hypothalamus was dissected from the brain by referring to a stereotaxic atlas[28] and stored using RNA later tissue storage reagent (Sigma-Aldrich, St. Louis, MO, USA) for real-time polymerase chain reaction (PCR) analysis. The septopalliomesencephalic tract, the third cranial nerves, the distance of 1.5 mm from midline, and the dorsal section from the anterior commissure to 1.0 mm ventral to the posterior commissure were used as landmarks of the hypothalamus.
Real-time PCR analysisTotal RNA was extracted from tissues using Sepazol-RNA I Super G (Nacalai Tesque, Inc., Kyoto, Japan). First-strand cDNA was synthesized from total RNA using Rever Tra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo Co. Ltd, Osaka, Japan). mRNA levels were quantified for each primer using TB Green Premix Ex Taq II (Tli RNase H Plus; Takara Bio Inc., Otsu, Japan), according to the supplier’s recommendations, and quantified based on the relative standard curve method using the ABI 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Primer sequences for ribosomal protein S17 (RPS17) have been described in a previous study[29]. The following primer sequences were used for NPY (NM_205473), POMC (NM_001031098), CRF (AJ621492), vasotocin (X55130), and mesotocin (AB194408): NPY sense, 5ʹ-CTT GTC GCT GCT GAT CTG -3ʹ, NPY antisense, 5ʹ-GCC TCA GAG CCG AGT AGT-3ʹ; POMC sense, 5ʹ-AGA TGG AGA AGG GTT GGA A-3ʹ, POMC antisense, 5ʹ-CGT TGG GGT ACA CCT TGA-3ʹ; CRF sense, 5ʹ-CTC CCT GGA CCT GAC TTT-3ʹ, CRF antisense, 5ʹ-CCT CAC TTC CCG ATG ATT-3ʹ; vasotocin sense, 5ʹ-TCC GGG CAC ACT CAG CAT-3ʹ, vasotocin antisense, 5ʹ-ATG TAG CAG GCG GAG GAC AA-3ʹ; mesotocin sense, 5ʹ-TGG CTC TCT CAG CTT GTT AT-3ʹ, and mesotocin antisense, 5ʹ-GGC ACG GCA CGC TTA CC-3ʹ. The mRNA levels of target genes were normalized to those of RPS17.
Data analysisAll statistical analyses were performed using Excel 2013 (Microsoft, USA) with the add-in software Statcel 3 (OMS, Tokyo, Japan). Data from Experiment 1 were analyzed using Dunnett’s test. Data from Experiment 2 were analyzed using the Student’s t-test.
In Experiment 1, the effects of different percentages of water in the feed on the body weight and meat yield of broiler chicks were examined to evaluate the optimum water content in the feed and to improve the growth of chicks. As shown in Table 1, 40% and 60% wet feeding significantly increased body and leg weights, and 40% wet feeding significantly increased breast weight, but decreased the leg weight ratio. These results suggest that 40% wet feeding significantly increases meat yield in broiler chickens.
| Wet feed | |||||||||||||
| Control | 20% | 40% | 60% | ||||||||||
| Body weight (g) | |||||||||||||
| 4-day-old | 80.6 | ± | 1.3 | 80.9 | ± | 1.2 | 80.9 | ± | 1.5 | 81.1 | ± | 1.2 | |
| 11-day-old | 223.6 | ± | 33.4 | 253.6 | ± | 20.4 | 265.2 | ± | 28.2 | 236.7 | ± | 42.4 | |
| 18-day-old | 598.3 | ± | 20.4 | 664.9 | ± | 58.4 | 722.4 | ± | 57.4 | 673.8 | ± | 89.2 | |
| 21-day-old | 797.9 | ± | 28.2 | 880.9 | ± | 83.2 | 982.2 | ± | 72.1 | 906.3 | ± | 110.6 | |
| 28-day-old | 1359.3 | ± | 42.4 | 1432.0 | ± | 139.7 | 1703.3 | ± | 83.3 | 1542.5 | ± | 155.4 | |
| 35-day-old | 1984.0 | ± | 218.5 | 2199.5 | ± | 171.4 | 2489.3 | ± | 97.2* | 2381.5 | ± | 191.2 | |
| 42-day-old | 2413.8 | ± | 242.9 | 2730.1 | ± | 164.2 | 3024.9 | ± | 164.2* | 2974.9 | ± | 171.1* | |
| Breasts weight (g) | 430.0 | ± | 81.6 | 550.6 | ± | 78.8 | 606.3 | ± | 52.6* | 570.0 | ± | 65.0 | |
| Breasts weight (g/g body weight) | 0.177 | ± | 0.002 | 0.199 | ± | 0.002 | 0.200 | ± | 0.009 | 0.191 | ± | 0.013 | |
| Legs weight (g) | 486.3 | ± | 44.7 | 528.8 | ± | 36.1 | 571.3 | ± | 37.7* | 604.9 | ± | 24.6* | |
| Legs weight (g/g body weight) | 0.202 | ± | 0.004 | 0.194 | ± | 0.004 | 0.189 | ± | 0.005* | 0.204 | ± | 0.009 | |
Data are expressed as means ± SEM of four replicates in each group. * Significance with respect to the control group (P < 0.05).
In Experiment 2, the effects of wet feeding on growth, water intake, and hypothalamic gene expression were examined in 21-day-old broiler chicks. The 40% wet feed significantly increased body weight, as well as the weight of the breasts and legs at 21 d of age, compared to that of the control group (Table 2). The total decrease in the amount of water in waterers was significantly reduced by wet feeding (Table 2). However, there were no significant differences in mRNA levels of genes related to appetite regulation (NPY and POMC), osmoregulation (vasotocin and mesotocin), or the stress response (CRF) (Table 2). These results suggest that broiler chicks did not feel hungry, thirsty, or stressed when fed wet feed. It is also likely that broiler chicks were satisfied with small amounts of water when they obtained water from wet feed.
| Control | Wet feed | ||||||
| Body weight (g) | |||||||
| 7-day-old | 142.4 | ± | 1.8 | 142.5 | ± | 1.5 | |
| 14-day-old | 438 | ± | 8.5 | 470.8 | ± | 2.7* | |
| 21-day-old | 926.3 | ± | 27.7 | 1030.1 | ± | 15.9* | |
| Breast weight (g) | 126.3 | ± | 3.6 | 151.5 | ± | 3.1* | |
| Breast weight (g/g body weight) | 0.136 | ± | 0.003 | 0.147 | ± | 0.004 | |
| Leg weight (g) | 153.7 | ± | 3.1 | 170.8 | ± | 3.0* | |
| Leg weight (g/g body weight) | 0.166 | ± | 0.004 | 0.166 | ± | 0.002 | |
| Decreased weight of waterer (g) | 3719 | ± | 119 | 2920 | ± | 52* | |
| Hypothalamic gene expression | |||||||
| Neuropeptide Y | 1.00 | ± | 0.16 | 0.97 | ± | 0.15 | |
| Proopiomelanocortin | 1.00 | ± | 0.12 | 1.54 | ± | 0.43 | |
| Vasotocin | 1.00 | ± | 0.09 | 0.89 | ± | 0.20 | |
| Mesotocin | 1.00 | ± | 0.26 | 0.79 | ± | 0.21 | |
| Corticotrophin releasing factor | 1.00 | ± | 0.07 | 0.83 | ± | 0.03 | |
Data are expressed as means ± SEM of four replicates in each group. * Significance with respect to the control group (P < 0.05).
The results of Experiment 1 suggested that 40% wet feeding was suitable and stable for improving broiler chicken growth (Table 1). The addition of 1.2-fold[10] or 1.3-fold[9] more water to the diet significantly increases the body weight of broilers. However, the addition of 1.3-fold more water to the diet does not affect the body weight of mixed-sex broiler chicks[30]. In addition, 1.4-fold more water significantly decreases body weight in male broiler chicks[31]. Therefore, excessive water inclusion should be avoided to improve broiler chicken growth.
In Experiment 1, the leg weight ratio significantly decreased during 40% wet feeding, although the body, breast, and leg weights increased. One possible explanation for this is that increased body weight might influence leg growth. Sanotra et al. have reported that the risk of leg problems is significantly influenced by body weight[32]. Furthermore, Liu et al. have reported that leg problems in broiler chickens are improved by a nutritionally balanced diet[33]. In the current study, the average body weight of the 40% wet-fed group was 1.25-fold higher than that of the control group (Table 1). Therefore, modifying the nutrient composition of 40% wet feed may further increase the meat yield of broiler chickens.
Food intake during the early post-hatch period affects subsequent growth in broiler chicks[34,35]. In the present study, wet feed was provided from 4 d of age in Experiment 1, but from 7 d of age in Experiment 2, and the growth-promoting effect of wet feeding was observed earlier in Experiment 2 (14-day-old) than in Experiment 1 (35-day-old). These results suggested that the growth-promoting effects of wet feeding did not occur during the early post-hatching period in broiler chicks. The reason for this remains unclear. However, chicks fed a mash diet show a significantly lower water/feed intake ratio from 2 to 4 d of age, whereas chicks fed a crumble diet show a significantly lower water/feed intake ratio from 4 to 6 d of age[36]. Hence, the suitable water/feed intake ratio might change during the neonatal period, which might explain the difference between Experiments 1 and 2. Thus, it might be better to begin 40% wet feeding at 7 d of age than at 4 d of age.
The feed conversion ratio may be improved in broiler chicks by employing the appropriate feed. For example, broiler chicks fed pellet feed have better feed-to-gain ratios than those of chicks fed crumble-type feed, and both groups’ exhibit better feed-to-gain ratios than chicks fed mash-type feed from 1–21 d of age[37]. Lv et al. have reported that crumble-type feed improves growth performance during the starter phase in broiler chicks, compared to the growth performance of chicks fed mash-type feed[38]. As shown in Fig. 1, 40% and 60% wet feed does not perform as well as mash-type feed when compared with 0% and 20% wet feed. In addition, previous studies have demonstrated that more than 50% wet feeding increases feed intake and body weight gain without affecting the feed conversion ratio in broiler chicks[7,8,10,11]. Overall, 40% wet feed may be more suitable to improve the feed conversion ratio in broiler chicks, similar to pellet- or crumble-type feed, than that of mash-type feed.
Osmotic challenge significantly elevates blood vasotocin levels[22,23] or hypothalamic vasotocin expression[24] in chickens. Water deprivation significantly affects hypothalamic mRNA and blood levels of vasotocins in chicks[21]. The antidiuretic functions of vasopressin and vasotocin in the kidneys are conserved in mammals and birds[20]. However, vasotocin suppresses water intake in birds under ad libitum feeding conditions[26,39], whereas vasopressin neurons in the suprachiasmatic nucleus of the hypothalamus are involved in inducing water intake in mammals[40]. The intracerebroventricular administration of vasotocin suppressed water intake in chicks under feed-deprived conditions (unpublished data). Therefore, the physiological role of vasotocin in birds might differ from that in mammals. Further studies are needed to identify thirst-inducing mechanisms in the chicken brain.
In conclusion, wet feeding significantly promoted growth in growing broiler chickens, but did not have significant effects on mRNA levels of hypothalamic peptides related to appetite and osmoregulation. These results suggest that wet feeding is a desirable feeding method from the perspective of animal welfare as it increased poultry productivity without the stress of thirst or hunger.
Yuji Taniguchi, Sei-ichi Hinomoto, and Hiroshi Kamisoyama designed the experiments; Tomoya Matsunami, Yuhui Zhang, and Kazuhisa Honda conducted the experiments and analyzed the data; Yuhui Zhang and Tomoya Matsunami drafted the manuscript; and Takaoki Saneyasu and Kazuhisa Honda supervised the experiments and edited the manuscript.
The authors declare no conflicts of interest.
