Neonatal Chicks Research Articles

Central GABAA receptor mediates taurine-induced hypothermia and possibly reduces food intake in thermo-neutral chicks and regulates plasma metabolites in heat-exposed chicks.

The aim of this study was to examine the central action of taurine on body temperature and food intake in neonatal chicks under control thermoneutral temperature (CT) and high ambient temperature (HT). Intracerebroventricular injection of taurine caused dose-dependent hypothermia and reduced food intake under CT. The mRNA expression of the GABAA receptors, GABAAR-α1 and GABAAR-γ, but not that of GABABR, significantly decreased in the diencephalon after central injection of taurine. Subsequently, we found that picrotoxin, a GABAAR antagonist, attenuated taurine-induced hypothermia. Central taurine significantly decreased the brain concentrations of 3-methoxy-4-hydroxyphenylglycol, a major metabolite of norepinephrine; however, the concentrations of serotonin, dopamine, and the epinephrine metabolites, 3,4-hydroxyindoleacetic acid and homovanillic acid, were unchanged. Although hypothermia was not observed under HT after central injection of taurine, plasma glucose and uric acid levels were higher, and plasma sodium and calcium levels were lower, than those in chicks under CT. In conclusion, brain taurine may play a role in regulating body temperature and food intake in chicks through GABAAR. The changes in plasma metabolites under heat stress suggest that brain taurine may play an important role in maintaining homeostasis in chicks.

Possible role of corticosterone in proteolysis, glycolytic, and amino acid metabolism in primary cultured avian myotubes incubated at high-temperature conditions

Excess glucocorticoid secretion induces oxidative damage and muscle proteolysis and modulates glucose and lipid metabolism. It is known that the high-temperature (HT) treatment enhances corticosterone (CORT) secretion, muscle proteolysis, and mitochondrial reactive oxygen species (mtROS) generation in chickens. The present study investigated the co-effects of CORT on proteolysis and mtROS production, together with glucose, fatty acid, and amino acid metabolism in HT-treated cells. Myoblast cells were isolated from the major pectoralis muscle of five 0- or 1-day-old neonatal chicks and were precultured at 37°C/CO2 conditions for 48 h to reach subconfluent (80%–90%) conditions. Cells were then reseeded onto a 6- or 24-well microplate for the subsequent measurement, followed by the culture under a control temperature (37°C, control) or HT (41°C) conditions for 1 or 6 h. The HT-treated cells were cocultured with physiologically relevant concentrations of CORT (20 ng/mL in dimethyl sulfoxide). The HT treatment decreased cellular protein content (P < 0.05) and increased atrogin-1 mRNA levels and mtROS generation levels compared to the control group (P < 0.05), whereas HT/CORT co-treatment did not induce changes in either parameter. The mRNA level of glucose transporter-1 was decreased in HT-treated cells compared to that in normal cells (P < 0.05), and the decrease was increased in the CORT co-treatment (P < 0.05). While HT treatment did not alter pyruvate dehydrogenase kinase-4 mRNA level, the level was increased in the CORT co-treatment compared to the control and HT-treated cells (P < 0.05). Neither HT nor HT/CORT treatments altered the mRNA levels of fatty acid oxidation-related factors, carnitine palmitoyl transferase-1, and cluster of differentiation 36. The study conducted a metabolic analysis using gas chromatography-mass spectrometry. The results showed that HT/CORT-treated cells had decreased intracellular citrate and α-ketoglutarate levels (P < 0.05) and increased extracellular alanine and amino acid that have gluconeogenic properties, as well as increased aspartate, isoleucine, serine, methionine, and threonine levels (P < 0.05) compared to HT-treated cells. These results suggest that CORT may not affect proteolysis and mtROS production but can suppress pyruvate oxidation and promote alanine production in HT-treated chickens.

Research Note: Application of an Escherichia coli spray challenge model for neonatal broiler chickens

Avian pathogenic Escherichia coli (E. coli) is an opportunistic pathogen often introduced to neonatal chicks during the hatching process. This commensal bacterium, particularly as a pioneer colonizer of the gastrointestinal tract, can have substantial implications in the rearing of poultry because of reduced flock performance. In order to mimic the effects of the natural bacterial bloom present during the hatch, a seeder challenge model was developed to expose neonatal chicks to virulent E. coli. On day 20 of embryogenesis, selected early hatched chicks (n = 18/hatcher) were briefly removed and sprayed challenged with saline (vehicle) or E. coli at 1 × 107 colony-forming unit (CFU)/chick (exp 1) and 2.5 × 107 CFU/chick (exp 2). These challenged chicks were returned to the hatcher to serve as seeders to transmit the pathogen to the indirect challenged or contact chicks (n = 195/hatcher). For two 7-d experiments, the efficacy of transmission was evaluated via enteric bacterial recovery, body weight gain (BWG), and mortality. For exp 1 and exp 2, significantly (P < 0.0001) more gram-negative bacteria were recovered from the seeder and contact gastrointestinal samples than the negative control samples on day of hatch. In addition, there was a reduction (P < 0.05) in 7-d BWG and significantly (P < 0.0001) higher mortality in the contact-challenged chicks than the negative control chicks in both exp 1 and exp 2. These data suggest that this challenge model could be used to evaluate different methods of controlling the bacterial bloom that occurs in the hatching environment.

Interaction of Central Glutamatergic and Histaminergic Systems on Food Intake Regulation in Layer Chickens.

This study purposed to discover the connection between the central glutamatergic and histaminergic systems on feeding behavior in layer chickens. In the first experiment, chicks obtained intracerebroventricular (ICV) injections of saline (control solution), α-FMH (250 nmol), glutamate (300 nmol), and α-FMH + glutamate. Experiments 2-6 were comparable to the first experiment, apart from the birds being injected with chlorpheniramine (histamine H1 receptor antagonist, 300 nmol), famotidine (histamine H2 receptor antagonist, 82 nmol), and thioperamide (histamine H3 receptor antagonist, 300 nmol) instead of α-FMH. In Experiment five, experimental groups were divided into (A) control solution, (B) MK-801 (N-methyl-D-aspartate receptor antagonist, 15 nmol), (C) histamine (300 nmol) and (D) MK-801 + histamine. Experiments 6-10 and Experiment five were similar apart from the ICV injections of CNQX (AMPA receptor antagonist, 360 nm), UBP-302 (Kainate receptor antagonist, 390 nm), AIDA (mGluR1 antagonist, 2 nmol), LY341495 (mGluR2 antagonist, 150 nmol), and UBP1112 (mGluR3 antagonist, 2 nmol) given instead of MK-801. Afterward, cumulative food intake was recorded at30, 60, and 120 minutes after the injection process. According to the results, ICV injection of glutamate considerably reduced food intake (p<0.05). Co-injection of α-FMH + glutamate and/or chlorpheniramine + glutamate reduced the hypophagic influence of glutamate (p<0.05), whereas thioperamide + glutamate augmented glutamate-induced hypophagia in neonatal chicks (p<0.05). Co-injection of MK-801 + histamine or UBP-302 + histamine reduced the hypophagic influence of the histamine (p<0.05), whereas LY341495 + histamine augmented the hypophagic influence of the histamine (p<0.05). Given the results, it is suggested that the effect of the connection between these systems on the process of food intake regulation is mediated by H1 and H3 histamines as well as NMDA, Kainate, and mGluR2 glutamate receptors in neonatal layer chickens.

Aerosol delivery of synthetic DNA containing CpG motifs in broiler chicks at hatch under field conditions using a commercial-scale prototype nebulizer provided protection against lethal Escherichia coli septicemia

Synthetic DNA containing CpG motifs (CpG-ODN) are potent innate immune stimulators in neonatal and adult broiler chickens against bacterial septicemia. We have recently demonstrated that intrapulmonary (IPL) delivery of CpG-ODN as microdroplets under laboratory conditions can protect neonatal chickens against lethal Escherichia coli septicemia. The objectives of this study were to develop a commercial-scale poultry nebulizer (CSPN) that can deliver CpG-ODN as microdroplets in neonatal broiler chicks in the hatcheries and study the efficacy of CSPN in inducing immune-protective effects under different environmental conditions in 2 geographical locations in Canada. Three field experiments were conducted in commercial poultry hatcheries during different seasons of the year in Saskatchewan and British Columbia, Canada. Neonatal broiler chicks (n = 8,000/experiment) received CpG-ODN by the IPL route in the CSPN chamber for 30 min, and control chicks received distilled water (DW) for 30 min. Broiler chicks (CpG-ODN—240 chicks/experiment and DW—40 chicks/experiment) were randomly sampled from all locations of the CSPN after nebulization and challenged with a lethal dose of E. coli to examine the CpG-ODN nebulization induced protection. We found a significant level (P < 0.05) of protection in broiler chicks against E. coli challenge, suggesting that the newly built CSPN successfully delivered CpG-ODN via the IPL route. We found that when the CSPN was maintained at humidex 28°C or below and relative humidity (RH) between 40 and 60%, neonatal birds were significantly (P < 0.05) protected against E. coli septicemia after IPL delivery of CpG-ODN. By contrast, protection in chicks was adversely affected when the CSPN was maintained at the humidex of 29°C or higher and RH of 70%. Overall, the present study successfully built a CSPN for CpG-ODN delivery in chicks at the hatchery and revealed that the temperature, humidity, and humidex were critical parameters in CSPN for efficient delivery of CpG-ODN.

Intracerebroventricular injection of L-arginine and D-arginine induces different effects under an acute stressful condition

Central administration of L-arginine was reported to attenuate stress responses in neonatal chicks. The present study aimed to elucidate the differential effects of centrally administered L-arginine and its enantiomer, D-arginine, on the stress response in chicks and the associated mechanisms. Intracerebroventricular injection of L-arginine attenuated acute isolation stress by inducing sleep-like behavior, while central administration of D-arginine potentiated the stress response, reducing the time spent standing motionless with eyes open and increasing distress vocalizations compared to the control. The brain concentrations of amino acids and monoamines following L- and D-arginine administration during stress were also determined. L-Arginine significantly increased the mesencephalic L-glutamine concentration. D-Arginine administration did not affect the levels of L-arginine or other amino acids in the examined brain regions. 3,4-Dihydroxyphenylacetic acid (DOPAC) level and dopamine (DA) metabolic rate (DOPAC/DA) were significantly higher in the diencephalon in the D-arginine group compared to the L-arginine group, while the mesencephalic DA level was significantly lower in the D-arginine group compared to the control. In vitro experiment using the brain slice culture demonstrated that extracellular perfusion of D-arginine significantly elevated the mRNA expression level of monoamine oxidase B, the major enzyme involved in DA metabolism, in the locus coeruleus region of the brainstem. In conclusion, in neonatal chicks, central administration of D-arginine exerted a stimulant effect on the stress response, in contrast to the stress-attenuating effects of L-arginine, partly through an effect on brain dopaminergic metabolism and not through competition with the L-stereoisomer.

New Bioink Derived from Neonatal Chicken Bone Marrow Cells and Its 3D-Bioprinted Niche for Osteogenic Stimulators.

This study examined whether neonatal chicken bone marrow cells (cBMCs) could support the osteogenesis of human stromal cells in a three-dimensional (3D) extracellular bioprinting niche. The majority (>95%) of 4-day-old cBMCs subcultured 5 times were positive for osteochondrogenesis-related genes (Col I, Col II, Col X, aggrecan, Sox9, osterix, Bmp2, osteocalcin, Runx2, and osteopontin) and their related proteins (Sox9, collagen type I, and collagen type II). LC-MS/MS analysis demonstrated that cBMC-conditioned medium (c-medium) contained proteins related to bone regeneration, such as periostin and members of the TGF-β family. Next, a significant increase in osteogenesis was detected in three human adipose tissue-derived stromal cell (hASC) lines, after exposure to c-medium concentrates in 2D culture (p < 0.05). To evaluate biological function in a 3D environment, we employed the cBMC-derived bioactive components as a cell-supporting biomaterial in collagen bioink, which was printed to construct a 3D hASC-laden scaffold for observing osteogenesis. Complete osteogenesis was detected in vitro. Moreover, after transplantation of the hASC-laden structure into rats, prominent bone formation was observed compared with that in control rats receiving scaffold-free hASC transplantation. These results demonstrated that substance(s) secreted by chick bone marrow cells clearly activated the osteogenesis of hASCs in 2D- or 3D-niches.

Synergistic effects of the early administration of Lactobacillus kefiranofaciens DN1 and Kluyveromyces marxianus KU140723-05 on the inhibition of Salmonella Enteritidis colonization in young chickens

In this study, we aimed to assess the feasibility of the lactic acid bacterium Lactobacillus kefiranofaciens DN1 (LKF_DN1) and the yeast Kluyveromyces marxianus KU140723-05 (KMA5), recently isolated from kefir, as probiotics. Specifically, we evaluated the effect of early administration of these 2 microbes on the inhibition of Salmonella Enteritidis (SE) colonization in neonatal chicks. We also examined the effects of exposure of chicks to probiotics before SE exposure on the reduction in the number of gut SE. A total of 108 1-day-old specific-pathogen-free male layer chicks were used for 3 independent experiments. The experimental chicks were randomly divided into 6 groups (negative control: basal diet [BD] without probiotics and SE; positive control: BD; probiotic group [PG] 1: BD + LKF_DN1; PG2: BD + KMA5; PG3: BD + LKF_DN1 + KMA5; and PG4: BD+ a commercial product IDF-7), all of which, except negative control, were coadministered with SE strain resistant to rifampicin (SERR). We found that the administration of LKF_DN1 and/or KMA5 reduced the number of viable cells of the SERR strain in chicks by up to 1.90 log10, relative to positive control chicks. Compared with late administration (day [D] 10 and D11), early administration (D1 and D2) of the probiotics was more effective in reducing SERR cell numbers in the gut. Furthermore, we detected no significant difference in the reduction of gut SERR cell numbers in chicks from the same groups exposed to the probiotics at D10 and D11 before and after administration with SERR. Collectively, our findings indicate that, as dietary additives, LKF_DN1 and KMA5 showed potential probiotic activity in chicks. Moreover, the combination of the lactic acid bacteria and/or yeast strain was found to rapidly reduce SE numbers in the chick gut and showed a prolonged inhibitory effect against SE colonization. We, thus, propose that the administration of these 2 probiotics, as early as possible after hatching, would be considerably effective in controlling SE colonization in the guts of chicks.

Clove essential oil in the sanitation of fertile eggs

The aim of this study was to evaluate the efficacy of sanitizing fertile eggs with clove essential oil as an alternative to paraformaldehyde; effects on the reduction in eggshell microbial count, incubation yield, and neonatal chick quality were measured. A total of 1,460 brown fertile eggs with a mean weight of 58.64 ± 0.49 g (from 37-wk-old CPK [Pesadão Vermelho] breeder hens) were collected under aseptic conditions and randomly distributed into 4 treatments (nonsanitized and sanitized with grain alcohol, clove essential oil, and paraformaldehyde) before incubation. The count of total aerobic mesophilic bacteria was significantly lower after spraying with clove essential oil (2.30 ± 0.24 log10 CFU/mL) than on nonsanitized eggs (3.49 ± 0.34 log10 CFU/mL) or on eggs sprayed with grain alcohol (3.09 ± 0.14 log10 CFU/mL) but did not differ significantly from the count in the paraformaldehyde group (2.23 ± 0.29 log10 CFU/mL). The hatchability of fertile eggs differed significantly between the studied treatments. The mean values for the eggs treated with clove essential oil (84.69 ± 1.65%) and paraformaldehyde (81.87 ± 3.92%) were statistically similar but were higher than the negative control (74.03 ± 3.58%) and grain alcohol (73.59 ± 2.87%) values. In the Pasgar© score assessment, it was determined that the clove essential oil (9.21 ± 0.89) had a superior effect on the physical quality of the chicks compared with the effects of the other treatments. Clove essential oil is effective and safe for eggs intended for incubation. Its use as an alternative to paraformaldehyde in the sanitation of fertile eggs is strongly recommended.

The Effect of RFamide-Related Peptide-3 (RFRP-3 or NPVF) on Food Intake in Neonatal Chickens: The Role of MC3/MC4 and CRF1/CRF2 Receptors

RFamide-related Peptide-3(RFRP-3) plays a key role in appetite regulation. The current study aimed to determine the effect of RFRP-3(Neuropeptide VF) on feeding behavior and its interaction with the melanocortin and corticotropin systems on food intake in the neonatal broiler-type chickens. In experiments 1 and 2, chickens received intracerebroventricular (ICV) injection of control solution besides RFRP-3 (4, 8, and 16 nmol) and RF9 (NPFF receptors antagonist; 4, 8 and 16 nmol) respectively to investigate the effective doses of RFRP-3 and RF9. Experiments 3–8 have been designed to investigate the mediatory role of CRF1/CRF2 and MC3R/MC4R on food intake induced by RFRP-3 in chickens. Finally, in experiments 8–11, the synergistic effect between the MC3R and MC4R with RFRP-3 have been investigated via ICV co-injection of sub-effective doses of RFRP-3 + γ1-MSH and PG-931. Then, cumulative food intake was recorded at 30, 60, and 120 min after injection. According to the results, co-injection of astressin-B and RFRP-3, significantly attenuated hypophagic effect of RFRP-3; while astressin2-B reversed this effect of RFRP-3 (p < 0.01). Besides, co-administration of SHU9119 and MCL0020 significantly attenuated the hypophagia induced by RFRP-3 in chickens (p < 0.01). However, co-administration of RFRP-3 and PG-931 synergistically decreased food intake in neonatal chicken (p < 0.01). These results suggested hypophagic effect of RFRP-3 is possibly mediated via corticotropin CRF2 and melanocortin MC4R in neonatal broiler chickens.

Evaluation of in ovo Bacillus spp. based probiotic administration on horizontal transmission of virulent Escherichia coli in neonatal broiler chickens

Evaluation of in ovo Bacillus spp. based probiotic administration on horizontal transmission of virulent Escherichia coli in neonatal broiler chickens

Mucosal delivery of CpG-ODN mimicking bacterial DNA via the intrapulmonary route induces systemic antimicrobial immune responses in neonatal chicks

The transition to antibiotic-free poultry production in the face of pathogenic threats is a very challenging task. We recently demonstrated that mucosal delivery of CpG-ODN alone by the intrapulmonary route (IPL) has potential as an effective alternative to antibiotics in neonatal chicks against Escherichia coli septicemia. How exactly mucosal delivery of CpG-ODN elicits, protective antibacterial immunity remained poorly understood. In this study, CpG-ODN or saline was delivered via the intrapulmonary route to day-old chicks (n = 80/group) using a compressor nebulizer in an acrylic chamber (1 mg/mL CpG-ODN for 15 minutes). In the first part of the study, two days after mucosal CpG-ODN delivery, 40 chicks from each group were challenged subcutaneously with 1 × 105 cfu (n = 20) or 1 × 106 cfu (n = 20) of E. coli and the mortality pattern was monitored for seven days. We found significantly higher survival, better clinical conditions and lower bacterial loads in chicks that received mucosal CpG-ODN. To explore the mechanisms behind this protective immunity, we first looked at the kinetics of the cytokine gene expression (three birds/ group/ time for 10 time-points) in the lungs and spleens. Multiplex gene analysis demonstrated a significant elevation of pro-inflammatory cytokine genes mRNA in the CpG-ODN group. Interleukin (IL)-1β robustly upregulated many folds in the lung after CpG-ODN delivery. Lipopolysaccharide-induced tumor necrosis factor (LITAF) and IL-18 showed expression for an extended period in the lungs. Anti-inflammatory cytokine IL-10 was upregulated in both lungs and spleen, whereas IL-4 showed upregulation in the lungs. To investigate the kinetics of immune enrichment in the lungs and spleens, we performed flow cytometry, histology, and immunohistochemistry at 24, 48 and 72 hrs after CpG-ODN delivery. CpG-ODN treated lungs showed a significant enrichment with monocytes/macrophages and CD4+ and CD8+ T-cell subsets. Macrophages in CpG-ODN treated group demonstrated mature phenotypes (higher CD40 and MHCII expression). Importantly, mucosal delivery of CpG-ODN via the intrapulmonary route significantly enriched immune compartment in the spleen as well, suggesting a systemic effect in neonatal chicks. Altogether, intrapulmonary delivery of aerosolized CpG-ODN orchestrates protective immunity against E. coli septicemia by not only enhancing mucosal immunity but also the systemic immune responses.

Effect of TLR agonist on infections bronchitis virus replication and cytokine expression in embryonated chicken eggs

Avian infectious bronchitis (IB) is an acute, highly infectious and contagious viral disease of chickens caused by avian infectious bronchitis virus (IBV) belonging to the genus Coronavirus and family Coronaviridae. It can affect all age groups of birds. The toll-like receptors (TLRs) are a major class of innate immune pattern recognition receptors that have a key role in immune response and defense against various infections.The TLRs are essential for initiation of innate immune responses and in the development of adaptive immune responses. An in ovo model was employed to study the antiviral activity of TLR ligands (Pam3CSK4, LPS and CpG ODN) on replication of IBV. It was hypothesized that optimum dose and specific timing of TLR ligands may reduce viral load of IBV in specific pathogen free (SPF) embryonated chicken eggs (ECEs). Further, the mechanism involved in the TLR-mediated antiviral response in chorioallantoic membrane (CAM) of ECEs was investigated. The ECEs of 9–11 days old were treated with different doses (high, intermediate and low) of TLR-2 (Pam3CSK4), TLR-4 (LPS) and TLR-21 (CpG ODN) ligands. In addition, to know the timing of TLR ligand treatment, six time intervals were analyzed viz. 36, 24 and 12 h prior to infection, time of infection (co-administration of TLR ligands and avian IBV) and 12 and 24 h post-IBV infection. For studying the relative expression of immuno-stimulatory genes (IFN-α, IFN-β, IFN-γ, IL-1β, iNOS and OAS) in CAM, TLR ligands were administered through intra-allantoicroute and CAM were collected at 4, 8 and 16 h post treatment. The results demonstrated that intermediate dose of all the three TLR ligands significantly reduced virus titers and used in the present study. However, the LPS reduced virus titer pre- and post-IBV infection but Pam3CSK4 and CpG ODN reduced only pre-IBV infection. Further analysis showed that TLR ligands induced IFN-γ, IL-1β and IFN stimulated genes viz. iNOS and OAS genes in CAM. The present study pointed towards the novel opportunities for rational design of LPS as immuno-stimulatory agent in chickens with reference to IBV. It may be speculated that in ovo administration of these TLR ligands may enhance resistance against viral infection in neonatal chicken and may contribute towards the development of more effective and safer vaccines including in ovo vaccines.

CpG-ODN Induces a Dose-Dependent Enrichment of Immunological Niches in the Spleen and Lungs of Neonatal Chicks That Correlates with the Protective Immunity against Escherichia coli.

Immunoprotective function of oligodeoxynucleotides containing CpG motifs (CpG-ODN) has been demonstrated in neonatal chickens against common bacterial pathogens such as E.coli and Salmonella sp. Our recent study reported that CpG-ODN administration enriches immune compartments in neonatal chicks. However, a causal relationship between CpG-ODN-induced immune enrichment and protective mechanisms remains unestablished. In this study, we investigated in ovo administered CpG-ODN-mediated immune cell recruitment in the immunological niches in lymphoid (spleen) and nonlymphoid (lungs) organs using various doses of CpG-ODN and examined whether the immunological profiles have any correlation with immunoprotection against E.coli infection. Eighteen-day-old embryonated eggs were injected with either 5, 10, 25, and 50 μg of CpG-ODN or saline (n = ~40 per group). On the day of hatch (72 hr after CpG-ODN treatment), we collected the spleen and lungs (n = 3‐4 per group) and examined the recruitment of macrophages/monocytes, their expression of MHCII and CD40, and the number of CD4+ and CD8+ T-cell subsets in the immunological niches in the spleen and lungs using flow cytometry. We observed the dose-dependent recruitment of immune cells, wherein 25 μg and 50 μg of CpG-ODN induced significant enrichment of immunological niches in both the spleen and the lungs. Four days after the CpG-ODN treatment (1-day after hatch), chicks were challenged with a virulent strain of E. coli (1 × 104 or 1 × 105 cfu, subcutaneously). Clinical outcome and mortality were monitored for 8 days postchallenge. We found that both 25 μg and 50 μg of CpG-ODN provided significant protection and reduced clinical scores compared to saline controls against E. coli infection. Overall, the present study revealed that CpG-ODNs orchestrate immunological niches in neonatal chickens in a dose-dependent manner that resulted in differential protection against E. coli infection, thus supporting a cause and effect relationship between CpG-ODN-induced immune enrichment and the antibacterial immunity.

Interaction between leptin and glutamatergic system on food intake regulation in neonatal chicken: role of NMDA and AMPA receptors

Objective: The aim of the current study was to determine the possible interaction of the central leptin and Glutamatergic systems on feeding behavior in neonatal 3-hours food deprived (FD3) broilers chickens.Methods: In experiment 1, FD3 chicken received intracerebroventricular (ICV) injection of control solution (group i) and 2.5, 5 and 10 µg of Leptin (groups ii–iv). In experiment 2, FD3 chicken were ICV injected with (group i) control solution and groups ii–iv with 2.5, 5 and 10 nmol of AG-490 (JAK2 antagonist). In experiment 3, injections were (i) control solution, (ii) Leptin (10 µg), (iii) AG-490 (2.5 nmol) and (iv) Leptin + AG-490. In experiment 4, broiler chickens were ICV injected with (i) control solution, (ii) Leptin (10 µg), (iii) MK-801 (NMDA glutamate receptors antagonist; 15 nmol) and (iv) Leptin + MK-801. Experiments 5–9 were similar to experiment 1, except chicken were ICV injected with CNQX (AMPA receptor antagonist, 390 nmol), UBP-302 (Kainate receptor antagonist, 390 nmol), AIDA (mGluR1 antagonist, 2 nmol), LY341495 (mGluR2 antagonist, 150 nmol) and UBP1112 (mGluR3 antagonist, 2 nmol) instead of MK-801. Then, food intake was measured until 120 min after injection.Results: ICV injection of leptin (2.5, 5 and 10 µg) significantly decreased food intake in a dose dependent manner (p < 0.05). Also, ICV injection of the JAK2 antagonist (2.5, 5 and 10 nmol) had hyperphagic effect in chicken (p < 0.05). Co-administration of leptin + AG-490, partially decreased leptin-induced hypophagia in broiler chicken (p < 0.05). In addition, co-injection of leptin + MK-801 significalty inhibited leptin-induced hypophagia in neonatal chicken (p < 0.05). Also, co-administration of leptin + CNQX partially attenuated hypophagic effect of leptin in chicken (p < 0.05).Conclusion: The results of present study suggest that leptin has hypophagic effect in neonatal chicken and this effect is probably mediated via NMDA and AMPA glutamatergic receptors.

Intracerebroventricular Injection of L-Pipecolic Acid Exerts Hypnotic Effects Without Activating NMDA Receptors in Neonatal Chicks under Social Isolation-induced Stress.

L-Pipecolic acid is an intermediate of L-lysine catabolism. Its central injection exerted a hypnotic effect on the brain, which was partially mediated by the activation of γ-aminobutyric acid-A and γ-aminobutyric acid-B receptors. L-Proline has also been shown to exert a similar effect on N-methyl-D-aspartate receptors. Furthermore, L-pipecolic acid is known as L-homoproline, and both L-pipecolic acid and L-proline belong to the imino acid group; therefore, it is plausible that they share certain commonalities, including similar functions. However, the role of N-methyl-D-aspartate receptors with respect to the effects of L-pipecolic acid has not been examined yet. In the present study, the relationship between N-methyl-D-aspartate receptors and the central function of L-pipecolic acid was investigated in neonatal chicks. The behavioral postures for active wakefulness and standing/sitting motionless with eyes opened were significantly affected after intracerebroventricular injection of L-pipecolic acid; whereas, sitting motionless with head drooped (sleeping posture) was significantly enhanced. However, the N-methyl-D-aspartate receptor antagonist, MK-801, did not affect these changes. In conclusion, the central administration of L-pipecolic acid did not exert hypnotic effects through the activation of N-methyl-D-aspartate receptors in neonatal chicks. These results suggest that the imino group is not a determinant for activating N-methyl-D-aspartate receptors.

Effect of Stereotaxic Surgery of the Third Ventricle on Growth Performance in Neonatal Chicks.

Feeding behavior and energy metabolism are precisely regulated by humoral and/or neural factors in the central nervous system. In particular, nuclei, such as the arcuate nucleus, ventromedial hypothalamic nucleus, and lateral hypothalamic area located near the third ventricle of the hypothalamus are the centers of feeding and energy metabolism in various vertebrate species, including chickens. In this study, we evaluated the effects of cannulation of the third ventricle on chick growth and feeding behavior in the neonatal stage, to develop a method for local and chronic central nervous system-mediated energy metabolism. Referring to the chick brain atlas, a guide cannula was inserted into the third ventricle of the chick under anesthesia immediately after hatching using a stereotaxic instrument. The chicks that recovered from anesthesia were bred for 11 days under normal feeding management conditions, and then feed intake amount, body weight gain, and metabolic tissue weight were measured. The effects of direct stimulation of the third ventricle with 2-deoxy-D-glucose on the expression level of the immediate-early gene, cFOS, and feed intake in 5-day-old chicks were also evaluated. There were no differences in feed intake, body weight gain, and metabolic tissue weight between 11-day-old cannulated and control chicks. The expression of cFOS mRNA in the ventromedial hypothalamic nucleus was higher than that in the amygdala after the third ventricular administration of 2-deoxy-D-glucose. Additionally, direct third ventricular injection of 2-deoxy-D-glucose attenuated the feeding behavior of chicks for a while. Overall, we speculate that the technique is effective for local and/or chronic stimulation of the nucleus near the third ventricle of the chick hypothalamus, which is important for feed and energy metabolism regulation.

Suppression of GABAergic transmission in the spinal dorsal horn induces pain-related behaviour in achicken model of spina bifida.

Spina bifida aperta (SBA), one of the most common congenital malformations, causes various neurological disorders. Pain is acommon complaint of patients with SBA. However, little is known about the neuropathology of SBA-related pain. Because loss of g-aminobutyric acid GABAergic neurons in the spinal cord dorsal horn is associated with pain, we hypothesised the existence of crosstalk between SBA-related pain and alterations in GABAergic transmission in the spinal cord. Therefore, we investigated the kinetics of GABAergic transmission in the spinal cord dorsal horn in achicken model of SBA. Neonatal chicks with SBA exhibited various pain-like behaviours, such as an increased number of vocalisations with elevated intensity (loudness) and frequency (pitch), reduced mobility, difficulty with locomotion, and escape reactions. Furthermore, the chicks with SBA did not respond to standard toe-pinching, indicating disruption of the spinal cord sensorimotor networks. These behavioural observations were concomitant with loss of GABAergic transmission in the spinal cord dorsal horn. We also found apoptosis of GABAergic neurons in the superficial dorsal horn in the early neonatal period, although cellular abnormalisation and propagation of neuro-degenerative signals were evident at middle to advanced gestational stages. In conclusion, ablation of GABAergic neurons induced alterations in spinal cord neuronal networks, providing novel insights into the pathophysiology of SBA-related pain-like complications.

Interaction Between Oxytocin and Opioidergic System on Food Intake Regulation in Neonatal Layer Type Chicken

The aim of the current study was to determine possible interaction of central oxytocin and opioidergic system on food intake regulation in neonatal layer-type chicken. In experiment 1, FD3 chicken ICV injected with control solution, oxytocin (10 µg), β-FNA (µ receptor antagonist, 5 µg) and oxytocin (10 µg) + β-FNA were injected. Experiments 2–6 were similar to experiments 1, except chicken injected with nor-BNI (κ receptor antagonist, 5 µg), NTI (δ receptor antagonist, 5 µg), DAMGO (µ receptor agonist, 62.25 pmol), U-50488H (κ receptor agonist, 10 nmol), DPDPE (δ receptor agonist, 20 pmol) instead of β-FNA. In experiment 7, control solution, DAMGO (125 pmol), d(CH2)5Tyr(Me)-[Orn8]-vasotocin (oxytocin antagonist, 5 µg) and DAMGO + d(CH2)5Tyr(Me)-[Orn8]-vasotocin were ICV injected to FD3 chicken. Experiments 8 and 9 were similar to experiments 7, except chicken injected with U-50488H (30 nmol) and DPDPE (40 pmol) instead of DAMGO. Then, cumulative food intake was recorded at 30, 60 and 120 min after injection. According to the results, ICV injection of the oxytocin (10 µg) significantly decreased food intake compared to control group (P < 0.05). Co-injection of the oxytocin + β-FNA and oxytocin + U-50488H significantly decreased hypophagic effect of the oxytocin (P < 0.05). While, co-injection of the oxytocin + nor-BNI or oxytocin + DAMGO significantly amplified hypophagic effect of the oxytocin in chicken (P < 0.05). In addition, ICV injection of DAMGO (125 pmol) significantly decreased cumulative food intake compared to control group (P < 0.05). However, co-addministration of the DAMGO + (CH2)5Tyr(Me)-[Orn8]-vasotocin significantly decreased hypophagic effect of the DAMGO (P < 0.05) in chicken. These results suggested there are interconnection between oxytocin and opioidergic system on central food intake regulation, which mediates via µ and κ opioidergic receptors in neonatal layer-type chicken.

Hepatic Mitochondrial Oxidative Metabolism and Lipogenesis Synergistically Adapt to Mediate Healthy Embryonic-to-Neonatal Transition in Chicken

During the normal embryonic-to-neonatal development, the chicken liver is subjected to intense lipid burden from high rates of yolk-lipid oxidation and also from the accumulation of the yolk-derived and newly synthesized lipids from carbohydrates. High rates of hepatic lipid oxidation and lipogenesis are also central features of non-alcoholic fatty liver disease (NAFLD) in both rodents and humans, but is associated with impaired insulin signaling, dysfunctional mitochondrial energetics and oxidative stress. However, these adverse effects are not apparent in the liver of embryonic and neonatal chicken, despite lipid burden. Utilizing comprehensive metabolic profiling, we identify that steady induction of hepatic mitochondrial tricarboxylic acid (TCA) cycle and lipogenesis are central features of embryonic-to-neonatal transition. More importantly, the induction of TCA cycle and lipogenesis occurred together with the downregulation of hepatic β-oxidation and ketogenesis in the neonatal chicken. This synergistic remodeling of hepatic metabolic networks blunted inflammatory onset, prevented accumulation of lipotoxic intermediates (ceramides and diacylglycerols) and reduced reactive oxygen species production during embryonic-to-neonatal development. This dynamic remodeling of hepatic mitochondrial oxidative flux and lipogenesis aids in the healthy embryonic-to-neonatal transition in chicken. This natural physiological system could help identify mechanisms regulating mitochondrial function and lipogenesis, with potential implications towards treatment of NAFLD.