Necrotic enteritis (NE) has reemerged as a significant problem as a result of growing restrictions of antibiotics in agricultural animal production and increasing concerns over antibiotic resistance in human pathogens. To enhance our understanding of host-pathogen immunobiology in NE, transcriptional analysis was conducted to compare changes in NE-induced intestinal transcripts and to identify immune-related genes whose expression are associated with NE disease resistance using two genetically disparate Fayoumi chicken lines, M5.1 and M15.2. NE was induced by co-infection of Eimeria maxima and Clostridium perfringens using an established disease model and two major NE-induced clinical signs, body weight loss and intestinal lesions, were measured in two inbred Fayoumi chicken lines, M5.1 and M15.2. In the clinical criteria, line M5.1 chickens were more resistant to NE compared to line 15.2 birds. Although they have the same genetic background, these two chicken lines are genetically disparate at their major histocompatibility complex (MHC) and this difference was reflected in the differential expression patterns of several inflammatory genes such as suppressor of cytokine signaling 3 (SOCS3), interleukin 8 (IL8), nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, zeta (NFKBIZ), serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1 (SERPINF1), and gap junction protein, alpha 1, 43kDa (GJA1) between NE-afflicted and uninfected chickens. These results will lead to increased insights on the NE disease resistance mechanisms and the role of host genes controlling host immune response to C. perfringens.

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Valine and Tryptophan play a role of being a precursor for protein synthesis. Glycation is a non-enzymatic reaction causing dehydrating condensation between the carbonyl group of glucose and the α-amino group of amino acids. Glycation of tryptophan forms two types of glycated compounds, Amadori product and (1R,3S)-1-(D-gluco-1,2,3,4,5-pentahydroxypentyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (PHP-THβC). In case of valine, only Amadori product is formed by glycation. These compounds are lack of the α-amino group, which might not make glycated amino acids be a precursor for protein synthesis. It is known that glycation proceeds more readily in chickens than in mammals because hyperglycemia and high body temperature are features in avian species. In this study, therefore, we attempted to evaluate nutritional value of glycated valine and tryptophan as a precursor for protein synthesis. Myoblasts from chicken embryos were incubated in valine or tryptophan-free medium supplemented with or without insulin-like growth factor-I (IGF-I). In this cell culture system, the effect of glucose-valine Amadori product supplemented in the valine deficient medium was compared to that of supplemented valine. The effect of PHP-THβC supplementation was also examined. Protein synthesis and degradation of myoblasts were measured using a radioactive tracer, [³H]-phenylalanine. Protein synthesis of chicken embryo myoblasts was not affected by the addition of glucose-valine Amadori product into valine-deficient medium. When IGF-I was added in the medium, and the addition of PHP-THβC into tryptophan-deficient medium increased protein synthesis of chicken embryo myoblasts, but the extent of elevation of protein synthesis was lower than that in case of tryptophan supplementation into tryptophan-deficient medium. In the absence of IGF-I, PHP-THβC did not increase protein synthesis. These results suggest that valine-glucose Amadori product and PHP-THβC does not have nutritional value for being a precursor of protein synthesis.

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Heat stress (HS) stimulates mitochondrial reactive oxygen species (ROS) production and protein degradation in skeletal muscle. The present study investigated the stimulatory effects of HS-induced mitochondrial ROS production on the ubiquitin-proteasome protein degradation system in primary cultured avian muscle cells. Cells were isolated from the breast muscle of neonatal chicks, and then grown for 48 h. Thereafter, the cells were subjected to 37°C or 41°C (HS). Exposure to 6 h of HS treatment significantly decreased the cellular protein content compared to that of normal cells, an effect was completely suppressed by the addition of a proteasome-specific inhibitor. Whereas the mRNA levels of the 20S proteasome C2 subunit, which is one of the subunits of the 26S proteasome, did not change at any time during HS treatment (1, 3, 6 h), the mRNA levels of atrogin-1 and muscle ring-finger protein 1, both of which are muscle-specific ubiquitin ligases, increased after 1 h of HS but then decreased to near-normal values with time. Intracellular ROS production (the sum of H₂O₂, hydroxyl radicals, peroxyl radicals, peroxynitrite) did not change in the 1 h HS-exposed cells, but was significantly increased after 3 h and 6 h of HS. Mitochondrial superoxide production was significantly increased after 1 h of HS, which might increase the mRNA expression of ubiquitin ligase in muscle cells. In cells pretreated with 4-hydroxy TEMPO, which is able to decrease mitochondrial superoxide production, the increases in mitochondrial superoxide production and ubiquitin ligase mRNA levels observed after 1 h of HS were suppressed. The protein content of these cells was not decreased, which was observed after the longest period of HS (6 h). These findings suggest that mitochondrial superoxide production may play an important role in activating the ubiquitin-proteasome system, probably via the induction of ubiquitin ligases, in HS-exposed muscle cells.

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Several peptides show different effects on food intake between broiler and layer chicks. For example, the central administration of β-melanocyte-stimulating hormone decreased food intake in layer chicks but not in broiler chicks. The central administration of agouti-related protein increased food intake in layer chicks but not in broiler chicks. We previously reported that glucagon and neuromedin U (NMU) suppress food intake in layer chicks when administered centrally. However, the effects of these peptides on food intake in broiler chicks have not been examined. In the present study, we thus examined whether these peptides also show anorexigenic effects in broiler chicks. The intracerebroventricular administration of glucagon significantly suppressed food intake and increased plasma glucose concentration in broiler chicks. The hyperglycemic effect of glucagon had been shown in layer chicks in our previous studies. Intracerebroventricular administration of NMU significantly suppressed food intake and induced wing-flapping behavior in broiler chicks as well as in layer chicks. All our findings demonstrate that glucagon and NMU function as anorxigenic peptides in both broiler and layer chicks.

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The objective of the present study was to investigate the effectiveness of a non-antibiotic growth promoter (Non-AGP) which is a combination of phytomolecules, direct-fed microbials (DFMs) and organic acids on performance, serum enzyme activities, immune response and visceral organ weights of commercial broiler chickens. A total of 375 day-old Ross 308 broiler chickens (mixed sex) were obtained from a local hatchery, weighed, and randomly allotted to 5 treatment groups with 5 replicates of 15 birds each. They received different treatments as basal diet (control); control plus 400 mg/kg bacitracin methylene disalicylate (BMD); control plus 0.05% Non-AGP (Non-AGP1); control plus 0.1% Non-AGP (Non-AGP2) and control plus 0.1% commercial probiotic (PRO). Chicks fed BMD resulted in maximum feed efficiency and a significant (P≤0.05) increase in the body weight, relative weights of liver, kidney and pancreas and Newcastle disease (ND), Infectious Bursal Disease (IBD) and Avian Influenza (AI) titers at d 42. Dietary inclusion of Non-AGP at the level of 0.1% into the basal diet proved its efficiency equal to BMD fed chicks, whereas addition of a probiotic at the level of 0.1% showed its applicability similar to the control group. It is concluded that the tested Non-AGP product at 0.1% but not 0.05% in the current study may be a suitable replacement for BMD in poultry diets.

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Heat stress has negative effects on biological defense mechanisms such as the immune response in chickens, and organs of the immune system, such as the spleen, are atrophied by heat stress in broiler chickens. In order to assess heat stress-induced functional changes of immune mechanisms in the spleen of broiler chickens, expression of genes encoding splenic cytokines (Th1 type, Th2 type, and pro-inflammatory cytokines) was analyzed. Heat exposure at 34°C for 15 days significantly induced the spleen involution, increased interleukin (IL)-4 and IL-12 expression and decreased interferon (IFN)-γ. However, expression of IL-6, 10, 13, and 18 was not affected. Heat stress reduced feed intake, which may affect the spleen weight and cytokine expression. Therefore, a pair-fed group at 24°C (24PF) was included in which chickens received the same amount of feed as those in the group at 34°C. Spleen weight was not affected by the reduction in feed intake. IL-4 expression in the 24PF group was higher than that in the control group. Further, IFN-γ expression increased and IL-12 expression was not affected by the reduction of feed intake, suggesting that the feed intake reduction induced by heat stress does not modulate splenic cytokine expression in broiler chickens. These data suggest that heat stress induces spleen involution and affects the expression of splenic cytokines such as IL-12 and IFN-γ in broiler chickens, but not through the feed intake reduction.

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Growth, known as the relation between liveweight and age, is explained mathematically by functions that have parameters with biological meaning. These parameters are used to describe growth pattern over time and to estimate the expected weight of animals at specific ages. Animal growth generally follows a sigmoidal pattern and several nonlinear functions have been used to describe it. This study was carried out to estimate the parameters of logistic, Gompertz and Richards growth curve models in a nondescript chicken breed population from southern Italy to determine the goodness of fit. Male and female birds were weighed weekly starting from two to twenty-four weeks of age. Based on our dataset, chickens showed a slow-growth pattern. All the growth functions evaluated were easily fitted to the observed data by nonlinear regression; our findings showed that Gompertz model fitted liveweight data very well both for male and female birds being the best model for studying the growth of our animals. Nevertheless, the four-parameter Richards function provided also a good fit of the data. Success in studying the growth characteristics of our nondescript chicken breed will contribute to define appropriate feeding regimens and to develop selection programme.

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The aim of the investigation was to determine influence of L-histidine dietary supplementation (P1 0.0%, P2 0.1%, P3 0.2% and P4 0.3%), hybrid line (Cobb 500 and Hubbard Classic) and gender on meat quality and carnosine concentration in chicken breast and thigh muscles. The research was carried out on 184 one day old broiler chickens as 4×2×2 factorial design. Chicken live weight, carcass weight, dressing percentage, absolute and relative shares of main body parts in the carcass, pHi, pHu, colour, drip loss, cooking loss and share force of breast muscle, and carnosine concentration in breast and thigh muscles were determined. L-histidine dietary supplementation significantly affected live weight, carcass weight, weight of drumsticks and thighs, backs and wings, share of back and the CIE a* value (P<0.05), as well as the content of carnosine in breast muscle (P＝0.003). Hybrid line influenced relative shares of main body parts in the carcasses, except for wings, and the CIE a* value of breast muscle (P<0.001). Chicken gender affected live weight of chickens, carcass weight, as well as weight of main parts (P<0.001), except for breast. The results of the investigation showed that dietary L-histidine significantly increased carnosine content in broiler breast muscle. Cobb 500 female chickens of the P4 group had higher carnosine concentrations in breast muscle by 156.61 and male chickens by 150.49 μg/g of tissue than the control group. Cobb 500 broiler chickens deposited more carnosine in meat than Hubbard Classic broilers.

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A 5×2×2 factorial experiment was conducted using 300 Ross 305 broiler chicks aged 3 h after removing from hatcher to examine the effects on physiological adaptive parameters of post hatch fasting (PHF; 0, 12, 24, 36, and 48 h), intrayolk sac (IYS) inoculation of olive oil (0 or 0.7 ml) and sex. The weight loss percentage was significantly greater in the chicks received IYS inoculation of 0.7 ml olive oil than non-injected birds at 24, 36 and 48 h age. Yolk residual utilization was 0.0664 and 0.1011 g/h in the injected and non-injected birds, respectively, over the PHF periods of 12 to 48 h. Independent of sex, liver weight was increased (P<0.001) and breast (P<0.05) and leg percentage (P<0.05) were decreased as PHF extended from 12 to 48 h with no difference between the injected and non-injected chicks (P>0.05). Serum GLU level was decreased by 36.03 and 40.31 mg/dl in the injected and non-injected birds (P>0.05), respectively. Serum TP, ALB, CRE, URE and UA levels were significantly increased over the extended periods of fasting up to 48 h. Serum very low density lipoproteins concentration was greater in the olive oil-injected birds compared with non-injected chicks at 48 h (P<0.05). The chicks received olive oil inoculation showed greater serum triglycerides and P concentrations than non-injected birds at 36 and 48 h of age. It was concluded that IYS inoculation of 0.7 ml olive oil did not aid the increased energy demands in the fasted neonate chicks resulting in greater weight loss and diverged metabolic responses presumably due to intensified ketosis.

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To examine the inhibitory effect of whole grain paddy rice diet (WPR) feeding on the colonization of Campylobacter jejuni in the cecum of broiler chicks, we performed the following examination. Sixty female broiler chicks (14 days of age) were allocated into two groups, thirty birds were fed a ground corn diet (GC) as a control group, and 30 chicks were fed WPR as an examination group. After feeding with the different diets for 16 days, six chicks were selected from each group (12 chicks in total), and each chick was inoculated with 2×10³ colony-forming units of C. jejuni GTC03263. Forty-eight hours after the bacterial inoculation (the birds continued to be fed with the corresponding diets after the bacterial inoculation), the chicks (six from each group) were killed and dissected to detect bacterial colonization in the cecum. The other six chicks were dissected to weigh the chick gizzard and to measure the pH of its contents. No bacterial colonization was observed in the cecum of chicks in the WPR group, whereas bacteria were found in the cecum of five of six chicks in the GC group. The average ratios of gizzard weight to body weight of the WPR-fed chicks was significantly higher than that of the GC-fed chicks, whereas the average pH value of the gizzard contents was not different between the two groups. These results suggest that WPR feeding in broiler chicks has a positive effect on development of the gizzard muscle and grinding activity of the gizzard. Increased grinding activity may eliminate the regional differences in pH within the gizzard, resulting in less bacterial survival in the gizzard and then less bacterial colonization in the cecum of WPR-fed chicks than in GC-fed chicks.

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The expression of atrogin-1/MAFbx, a muscle-specific E3 ubiquitin ligase, is high under catabolic conditions that result in muscle atrophy. We previously showed that expression of atrogin-1/MAFbx was expressed in skeletal, smooth and cardiac muscles of chickens and its expression in skeletal and smooth muscles was regulated by nutritional conditions. In this study, the effects of fasting and refeeding on the expression of atrogin-1/MAFbx and other proteolytic-related genes, including 20S proteasome C2 subunit, m-calpain large subunit, and cathepsin B, in cardiac muscle of broiler chickens were investigated. Chickens were fasted for 24 h, and refed for 2 h. Atrogin-1/MAFbx mRNA expression was increased by fasting, but subsequent refeeding reduced the increase. The mRNA expression of the 20S proteasome C2 subunit and m-calpain large subunit mRNA was not affected by fasting or refeeding. Cathepsin B mRNA expression was increased by fasting, but unlike atrogin-1/MAFbx mRNA expression, the increase was not reduced by refeeding. These results demonstrate the muscle-specific ubiquitin ligase atrogin-1/MAFbx gene expression in cardiac muscle as well as skeletal and smooth muscles of chicken is regulated by food deprivation and nutritional supply.

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The effect of hay bales and perches as environmental enrichment material was investigated on the behavior, heterophil/lymphocyte (H/L) ratio, footpad dermatitis (FPD), and productivity of Japanese broilers in a commercial farm. About 21,500 birds were allocated to 4 houses: male versus female×control versus treatment house. We observed maintenance behavior, the utilization of hay bales and perches, and the H/L ratio of birds at 3, 5, and 8 weeks of age. FPD was scored and productivity was calculated at about 60 days after hatching. Compared to birds in the control houses, birds in the treatment houses invested more time in stand-resting and moving behaviors (P<0.01, for each behavior) and less time feeding, drinking, and sit-resting (P<0.01, for each behavior). Females used the hay bales and perches more than the males (P<0.1 and P<0.01, respectively). The activity and utilization of hay bales and perches decreased with age (P<0.01, for each behavior, respectively). The H/L ratio of birds in the treatment houses was lower than that in the control houses (P<0.01). The prevalence of FPD was milder in the treatment houses compared to the control houses for females (P<0.01), but not for males. In conclusion, hay bales and perches represent effective enrichment materials, allowing broilers to express normal behavior, alleviating stress in the younger and lighter birds of both sexes, and reducing FPD in female birds using the hay bales and perches more than males.

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Some amino acids are important regulators of key metabolic pathways and necessary for several physiological functions. However, little is know about thermoregulatory functions of amino acids. In this study, therefore chicks were either centrally or orally administered with L-citrulline (L-Cit), L-arginine (L-Arg) or L-ornithine (L-Orn) to monitor changes in rectal temperature. In Experiment 1, the amino acids (L-Cit, L-Arg and L-Orn) were administered into the left ventricle of the chicks by intracerebroventricular (i.c.v.) injection at a dose of 1 μmol/10 μl to monitor the effects of these amino acids on rectal temperature during 120 min of the experimental period. In Experiment 2, chicks received the same amino acids by oral administration at a dose of 15 mmol/10 ml/kg body weight. In Experiment 3, chicks received three doses of L-Cit (3.75, 7.5 or 15 mmol/10 ml/kg body weight) by oral administration. I.c.v. injection with any of the amino acids studied did not alter body temperature, but oral administration of L-Cit significantly reduced body temperature. Importantly, the highest does effectively reduced body temperature. These results suggest that peripheral L-Cit has a hypothermic function in chicks, which may be a new candidate to minimize high body temperature in poultry during summer heat stress.

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Fig. 1.　Avian species designated as “nationally endangered species of wild fauna and flora” under the conservation and propagation programs. (A) Short-tailed albatross (Diomedea albatrus), (B) Japanese Crested Ibis (Nipponia nippon), (C) Japanese crane (Grus japonensis), (D) Blakiston’s fish owl (Ketupa blakistoni blakistoni), (E) Golden eagle (Aquila chrysaetos japonica), (F) Okinawa woodpecker (Sapheopipo noguchii), (G) White’s thrush (Zoothera dauma major), (H) Amami Woodcock (Scolopax mira), (I) Common Murre, Guillemot (Uria aalge inornata), (J) Tufted Puffin (Lunda cirrhata), (K) Okinawa Rail (Gallirallus okinawae), (L) White-tailed eagle (Haliaeetus albicilla albicilla), (M) Steller’s sea eagle (Haliaeetus pelagicus), (N) Japanese wood pigeon (Columba janthina nitens), (O) Ptarmigan (Lagopus mutus japonicas)

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