Research Note: Morphine influences circulating and tissue concentrations of met-enkephalin and proenkephalin (PENK) expression and plasma concentrations of corticosterone in chickens.

The effects of the administration of the opioid agonist, morphine, on plasma and tissue concentrations of Met-enkephalin were determined in 14 wk old female chickens. In addition, effects of morphine on proenkephalin (PENK) expression were examined. Plasma concentrations of Met-enkephalin were reduced 10 minutes after morphine administration. Plasma concentrations of peptides that contain Met-enkephalin motifs were decreased 30 minutes after morphine administration. Tissue concentrations of Met-enkephalin tended to be depressed following morphine administration. Adrenal concentrations of PENK peptides containing Met-enkephalin motifs were decreased in chickens challenged with morphine. Expression of PENK in the anterior pituitary gland and adrenal glands were decreased in morphine treated compared to control pullets. In contrast, plasma concentrations of corticosterone were elevated 10 min after morphine treatment. Morphine also induced changes in mu (µ) opioid receptors and delta (δ) opioid receptors in both anterior pituitary tissue and adrenal tissues.

Effects of Restraint Stress on Circulating Corticosterone and Met Enkephalin in Chickens: Induction of Shifts in Insulin Secretion and Carbohydrate Metabolism.

This study examined the effects of acute restraint stress in the presence or absence of naltrexone on the circulating concentrations of insulin, glucose, Met-enkephalin and corticosterone in 14-week-old chickens [design: 2 sex × 2 stress/non-stress × 2 +/- naltrexone]. In chickens (five male and five females per treatment) subjected to restraint for 30 min, there were increases in the plasma concentrations of corticosterone and Met-enkephalin. The plasma concentrations of insulin and glucose were also increased in the chickens during restraint. Moreover, there were increases in the plasma concentrations of insulin and glucose in the chickens. The patterns of expression of the proenkephalin gene (PENK) in both the anterior pituitary gland and the adrenal gland were very similar to that of plasma Met-enkephalin. There were relationships between the plasma concentrations of corticosterone, Met-enkephalin, insulin and glucose after 30 min of restraint. The effects of naltrexone treatment on both untreated and stressed chickens were also examined, with naltrexone attenuating the stress-induced increases in the plasma concentrations of corticosterone, Met-enkephalin and glucose but not in those of insulin. The present study demonstrates that stress increases insulin secretion in chickens but also induces insulin resistance.

Avian opioid peptides: evolutionary considerations, functional roles and a challenge to address critical questions.

The present review considers the putative hormonal opioid peptides in birds. In birds and all other vertebrates, there are four opioid related genes encoding a series of peptides. These genes are, respectively, proenkephalin (PENK), prodynorphin (PDYN), pronociceptin (PNOC) and proopiomelanocortin (POMC). Proenkephalin (PENK) encodes Met- and Leu-enkephalin together with peptides containing met enkephalin motifs in birds, mammals and reptiles. Proopiomelanocortin (POMC) encodes ß endorphin together with adrenocorticotropic hormone (ACTH), and melanocyte stimulating hormone (MSH). Prodynorphin (PDYN) encoding dynorphins A and B with α- and ß-neoendorphins together intermediate polypeptides across the vertebrates. Pronociceptin (PNOC) encodes nociceptin together with possibly putative avian nocistatin and a non-opioid peptide derived from the C terminal of pronociceptin. There is a high degree of identity in the sequences of enkephalin peptides, dynorphin-A and B and nociceptin in birds and, to a less extent, across vertebrates. The opioid peptides exert effects related to pain together with other biological actions such as growth/development acting via a series of opioid receptors. What is unclear, particularly in birds, is the biological roles and interactions (additivity, antagonistic and synergistic) for the individual opioid peptides, the processing of the prohormones in different tissues and the physiological relevance of the different peptides and, particularly, of the circulating forms.

Response of the hen ovary to eCG treatment: Insight into morphology and expression of genes related to steroidogenesis and vitellogenesis.

The present study aimed to examine the effect of equine chorionic gonadotropin (eCG) treatment on the chicken ovarian folliculogenesis and steroidogenesis. The expression of vitellogenesis-related genes in the liver was also investigated. Laying hens were injected with 75 I.U./kg of body weight/0.2 mL of eCG, once a day for 7 successive days. On day 7 of the experiment hens, including control hens which were receiving vehicle, were euthanized. The liver and ovarian follicles were harvested. Blood was collected daily through the whole experiment. The eCG treatment resulted in the cessation of egg laying after 3 or 4 days. The eCG-treated hens had heavier ovaries with a higher number of yellowish and yellow follicles arranged in a non-hierarchical way in contrast to ovaries of control hens. Moreover, these birds had elevated plasma estradiol (E2) and testosterone (T) concentrations. The molar ratios of E2:progesterone (P4) and T:P4 were increased in chickens injected with eCG. Real-time polymerase chain reaction revealed changes in mRNA abundances of steroidogenesis-associated genes (StAR, CYP11A1, HSD3ß, and CYP19A1) in ovarian follicles: white, yellowish, small yellow, and the largest yellow preovulatory (F3-F1) as well as VTG2, apoVLDL II, and gonadotropin receptors in the liver. In general, the abundances of gene transcripts were higher in eCG-treated hens than in control hens. Western blot analyses showed an elevated abundance of aromatase protein in the prehierarchical and small yellow follicles of eCG-treated hens. Unexpectedly, there was presence of both FSHR and LHCGR mRNA in the liver and the level of expression was shifted in eCG-treated hens. In summary, eCG treatment leads to disruption of the ovarian hierarchy with accompanying changes in circulating steroids and ovarian steroidogenesis.

Discontinuities in understanding follicular development, the ovulatory cycle and the oviposition cycles in the hen: Advances, opportunities, slow downs and complete stops.

There has been considerable progress in understanding follicular development, the ovulatory cycle and the oviposition cycles in the hen. In particular, there have been tremendous advances in understanding follicular development and recruitment of follicles to the hierarchy of large yellow follicles. There is a need to continue to follow the earlier literature while employing present techniques. Early work allowed an understanding of the ovulation and oviposition cycles. Models for ovulation were developed. However, while these have no passed the test of time, there is no present model that fully accounts to the cycles. Earlier work employed ahemoral light cycles to examine ovulation and oviposition cycles. Recent work has demonstrated that clock genes are expressed in the ovary. The control of incubation by prolactin has been largely elucidated in turkeys. There is evidence that other endocrine glands influence female reproduction in birds including the adrenal cortex, thyroid and pineal. However, there is much that remains to be fully understood.

A generic avian physiologically-based kinetic (PBK) model and its application in three bird species.

Physiologically-based kinetic (PBK) models are effective tools for designing toxicological studies and conducting extrapolations to inform hazard characterization in risk assessment by filling data gaps and defining safe levels of chemicals. In the present work, a generic avian PBK model for male and female birds was developed using PK-Sim and MoBi from the Open Systems Pharmacology Suite (OSPS). The PBK model includes an ovulation model (egg development) to predict concentrations of chemicals in eggs from dietary exposure. The model was parametrized for chicken (Gallus gallus), bobwhite quail (Colinus virginianus) and mallard duck (Anas platyrhynchos) and was tested with nine chemicals for which in vivo studies were available. Time-concentration profiles of chemicals reaching tissues and egg compartment were simulated and compared to in vivo data. The overall accuracy of the PBK model predictions across the analyzed chemicals was good. Model simulations were found to be in the range of 22-79% within a 3-fold and 41-89% were within 10- fold deviation of the in vivo observed data. However, for some compounds scarcity of in-vivo data and inconsistencies between published studies allowed only a limited goodness of fit evaluation. The generic avian PBK model was developed following a "best practice" workflow describing how to build a PBK model for novel species. The credibility and reproducibility of the avian PBK models were scored by evaluation according to the available guidance documents from WHO (2010), and OECD (2021), to increase applicability, confidence and acceptance of these in silico models in chemical risk assessment.

Quantitative Morphometric, Physiological, and Metabolic Characteristics of Chickens and Mallards for Physiologically Based Kinetic Model Development.

Physiologically based kinetic (PBK) models are a promising tool for xenobiotic environmental risk assessment that could reduce animal testing by predicting in vivo exposure. PBK models for birds could further our understanding of species-specific sensitivities to xenobiotics, but would require species-specific parameterization. To this end, we summarize multiple major morphometric and physiological characteristics in chickens, particularly laying hens (Gallus gallus) and mallards (Anas platyrhynchos) in a meta-analysis of published data. Where such data did not exist, data are substituted from domesticated ducks (Anas platyrhynchos) and, in their absence, from chickens. The distribution of water between intracellular, extracellular, and plasma is similar in laying hens and mallards. Similarly, the lengths of the components of the small intestine (duodenum, jejunum, and ileum) are similar in chickens and mallards. Moreover, not only are the gastrointestinal absorptive areas similar in mallard and chickens but also they are similar to those in mammals when expressed on a log basis and compared to log body weight. In contrast, the following are much lower in laying hens than mallards: cardiac output (CO), hematocrit (Hct), and blood hemoglobin. There are shifts in ovary weight (increased), oviduct weight (increased), and plasma/serum concentrations of vitellogenin and triglyceride between laying hens and sexually immature females. In contrast, reproductive state does not affect the relative weights of the liver, kidneys, spleen, and gizzard.

Quantitative Comparison of Avian and Mammalian Physiologies for Parameterization of Physiologically Based Kinetic Models.

Physiologically based kinetic (PBK) models facilitate chemical risk assessment by predicting in vivo exposure while reducing the need for animal testing. PBK models for mammals have seen significant progress, which has yet to be achieved for avian systems. Here, we quantitatively compare physiological, metabolic and anatomical characteristics between birds and mammals, with the aim of facilitating bird PBK model development. For some characteristics, there is considerable complementarity between avian and mammalian species with identical values for the following: blood hemoglobin and hemoglobin concentrations per unit erythrocyte volume together with relative weights of the liver, heart, and lungs. There are also systematic differences for some major characteristics between avian and mammalian species including erythrocyte volume, plasma concentrations of albumin, total protein and triglyceride together with liver cell size and relative weights of the kidney, spleen, and ovary. There are also major differences between characteristics between sexually mature and sexually immature female birds. For example, the relative weights of the ovary and oviduct are greater in sexually mature females compared to immature birds as are the plasma concentrations of triglyceride and vitellogenin. Both these sets of differences reflect the genetic "blue print" inherited from ancestral archosaurs such as the production of large eggs with yolk filled oocytes surrounded by egg white proteins, membranes and a calciferous shell together with adaptions for flight in birds or ancestrally in flightless birds.

Antioxidant and antimicrobial activities of phytonutrients as antibiotic substitutes in poultry feed.

Globally, there is increasing demand for safe poultry food products free from antibiotic residues. There is thus a need to develop alternatives to antibiotics with safe nutritional feed derivatives that maximize performance, promote the intestinal immune status, enrich beneficial microbiota, promote health, and reduce the adverse effects of pathogenic infectious microorganisms. With the move away from including antibiotics in poultry diets, botanicals are among the most important alternatives to antibiotics. Some botanicals such as fennel, garlic, oregano, mint, and rosemary have been reported to increase the poultry's growth rate and/or feed to gain ratio. Botanicals' role is assumed to be mediated by improved immune responses and/or shifts in the microbial population in the intestine, with the elimination of pathogenic species. In addition, modulation of the gut microbiota resulted in various physiological and immunological responses and promoted beneficial bacterial strains that led to a healthy gut. There is thus a need to understand the relationship between poultry diets supplemented with botanicals and good health of the entire gastrointestinal tract if we intend to use these natural products to promote general health status and production. This current review provides an overview of current knowledge about certain botanicals that improve poultry productivity by modulating intestinal health and reducing the negative impacts of numerous pathogenic bacteria. This review also describes the efficacy, negative effects, and modes of action of some common herbal plants applied in poultry as alternatives to reduce the use of antibiotics.

Impact of experimentally induced bacterial chondronecrosis with osteomyelitis (BCO) lameness on health, stress, and leg health parameters in broilers.

Stress and lameness negatively affect the health, production, and welfare of broilers. Bacterial chondronecrosis with osteomyelitis (BCO) is a leading cause of stress and lameness in commercial broilers. External changes in skin temperature related to changes in blood flow can be detected with infrared thermography (IRT), offering a noninvasive tool to assess the health of animals. This study compared physiological and noninvasive measures of stress and lameness in clinically healthy and lame male broiler chickens between 25 and 56 d. Birds were raised in pens within separate environmental chambers containing either litter flooring (sound) or wire flooring, with the latter established to induce BCO lameness (lame). Physiological and noninvasive measures of stress and lameness were collected: body weight, (BW), relative bursa weight, core body temperature, corticosterone (CORT) concentrations in serum and feathers, surface temperatures of the head (eye and beak) and leg (hock, shank, and foot) regions by infrared thermography (IRT), leg blood oxygen saturation (leg O2), and BCO lesion severity scores of tibial head necrosis (THN) and femoral head necrosis (FHN). Lame birds exhibited greater FHN and THN lesion severities, core body temperatures, and serum CORT (P < 0.05), but had lower BW, relative bursa weight, leg O2, and IRT surface temperatures of the beak, hock, shank, and foot compared with sound birds (P < 0.05). The difference in THN lesion severity between sound and lame birds decreased with age. Linear relationships between leg O2 with IRT leg surface temperatures were positive and negative between leg O2 with BCO lesion severity (P < 0.05). There were negative correlations between serum CORT with hock, shank and foot temperatures (P < 0.001), indicating that BCO is stressful. These results indicate that birds lame from BCO are stressed, have reduced oxygen saturation of blood in their legs, and that IRT surface temperatures can be used as noninvasive indicators of stress and lameness in broilers.

Avian Physiology: Are Birds Simply Feathered Mammals?

There are marked differences between the physiology of birds and mammals. These reflect the evolutionary distance between the two classes with the last common ancestor estimated as existing 318 million years ago. There are analogous organ systems in birds and mammals. However, marked differences exist. For instance, in the avian gastro-intestinal tract, there is a crop at the lower end of the esophagus. This functions both to store feed and for microbial action. The avian immune system lacks lymph nodes and has a distinct organ producing B-lymphocytes, namely the bursa Fabricius. The important of spleen has been largely dismissed until recently. However, its importance in both innate and specific immunity is increasingly recognized. There is a major difference between birds and mammals is the female reproductive system as birds produce large yolk filled eggs. The precursors of the yolk are synthesized by the liver. Another difference is that there is a single ovary and oviduct in birds.

Broiler stress responses to light intensity, flooring type, and leg weakness as assessed by heterophil-to-lymphocyte ratios, serum corticosterone, infrared thermography, and latency to lie.

Stress and leg weakness are detrimental to broiler production, health, and welfare. Traditional methods to evaluate stress may be stressful to the bird because they are invasive and require handling and restraint. Two studies examined the effects of light intensity and flooring on the following in broilers: 1) traditional methods for assessing stress using heterophil-to-lymphocyte ratios and serum corticosterone (CORT) concentrations, 2) noninvasive measures of stress from infrared thermography (IRT) eye and beak surface temperatures, and 3) latency-to-lie (LTL) test times of birds tested individually and in groups of 5. Day-of-hatch male broiler chicks were placed into 6 pens (N = 120 chicks/pen). At 1 wk, pens were allocated to 3 light intensity treatments (2, 5, or 10 lux). At 4 wk, half of the birds from each pen were moved to a pen with wire flooring and the same light intensity. At 1, 4, 5, and 8 wk, blood samples were collected and IRT images of the heads of 5 clinically healthy broilers from each pen were captured. In study 2, IRT images of the heads of birds that became lame in the wire flooring pens were taken. There were no treatment effects on the LTL times of birds tested in groups or individually (P > 0.05). On day 56 in study 1, birds on wire flooring had elevated heterophil-to-lymphocyte ratios and CORT concentrations (P ≤ 0.002) and depressed IRT eye and beak temperatures (P < 0.0001). In both studies, there were negative correlations between CORT concentrations and IRT beak surface temperatures (P < 0.05). Lame birds had lower IRT eye and beak surface temperatures than sound birds (P ≤ 0.004), and the IRT beak surface temperatures of lame birds were lower than their eye surface temperatures (P = 0.004) in study 2. These studies indicate that the IRT surface temperatures of the eye, and more distinctly of the beak, can be used as sensitive noninvasive indicators of stress.

Effect of transportation and shackling on plasma concentrations of corticosterone and heterophil to lymphocyte ratios in market weight male turkeys in a commercial operation.

There is limited information on the effects of stress and/or physiological manipulation on either plasma concentrations of corticosterone (CORT) and/or heterophil: lymphocyte (H : L) ratios in turkeys. The present studies examine the effects of catching/transportation/lairage in a holding shed and shackling on plasma concentrations of CORT and H : L ratios in male market weight turkeys. Plasma concentrations of CORT were increased after transportation and lairage but not further elevated by shackling, irrespective of its duration up to 240 s. In one study, there were increased H : L ratios following catching/placing birds into transportation cages/transportation/lairage. In one study, H : L ratios declined following shackling. It is concluded that while moving turkeys from the farm to immediately before the shackling line is stressful, shackling for up to 4 min was not perceived as more stressful in turkeys. There were also differences between farms/houses for both plasma concentrations of CORT and H : L ratios.

Effects of putative stressors and adrenocorticotropic hormone on plasma concentrations of corticosterone in market-weight male turkeys.

There is limited information on the effects of stress and/or physiological manipulation on plasma concentrations of corticosterone (CORT) in turkeys. Under basal conditions, there was evidence for episodic release of CORT in turkeys. The present studies determine the effects of handling, herding, herding, the administration of Escherichia coli endotoxin, and challenge with turkey adrenocorticotropic hormone (ACTH) on plasma concentrations of CORT in market-weight male turkeys. Plasma concentrations of CORT were increased after challenge with turkey ACTH, handling together with saline injection or herding (moving birds from one pen to another). There were no effects on plasma concentrations of CORT of the following putative stressors: handling per se, endotoxin challenge, or of placing in an inverted position on simulated shackles.

Opioid-like peptides and ghrelin mitigation of bariatric results depends on obesity level.

INTRODUCTION: Bariatric surgery, as the only effective treatment of obesity, has strong effects on the metabolism, and nervous and endocrine systems. Thus, based on the different opinions about the efficaciousness of morbid obesity treatments, the aim of the present study was to estimate the association of serum ghrelin and Met-enkephalin (native, five amino acids and cryptic, precursor of enkephalin) concentrations with body mass index (BMI) value in bariatric patients within 30 postoperative days. MATERIAL AND METHODS: The study was performed on 38 female patients divided into two groups: I - BMI lower than 40 kg/m² (n = 18) and II - BMI higher than 40 kg/m² (n = 20). Blood was taken before (-24 h), and 72 h and 30 days after the sleeve gastrectomy. Routine haematological, anthropometric, and metabolic parameters as well as thyroid-stimulating hormone (TSH), ghrelin, and Met-enkephalin values were measured in all patients. RESULTS: There were statistically significant differences between the two groups before the surgery in terms of TSH, both forms of Metenkephalin, triglycerides concentrations, and activity of alanine transaminase (ALT), gamma glutamyltransferase (GGTP), and C-reactive protein (CRP). After 72 h, the serum levels of cryptic Met-enkephalin and CRP, and activity of enzymes varied between the two groups of patients. Thirty days after the surgery, some metabolic and immune parameters were still different in both female groups in favour of patients with lover BMI. However, significant differences were noticed in the levels of ghrelin (increase), and native (decrease) and cryptic Met-enkephalins (increase). CONCLUSIONS: The activity of endogenous peptides in bariatric patients is connected with the degree of obesity. Ghrelin level increases are negatively correlated with native Met-enkephalin changes shortly after bariatric surgery. The interplay of ghrelin and opioids might be considered as a predictor of postoperative weight loss success.

Isolation stress impacts Met-enkephalin in the hypothalamo-pituitary-adrenocortical axis in growing Polish Mountain sheep: a possible role of the opioids in modulation of HPA axis.

It was hypothesized that there is cross-talk between the classical constituents of the hypothalamo-pituitary-adrenocortical axis (HPA) and Met-enkephalin in the HPA axis. The study examined effects of isolation stress, sex, and age on concentrations of native Met-enkephalin and pro-enkephalin (PENK) gene expression in tissues of the HPA (hypothalamus, pituitary gland and adrenal cortex) in 3-, 6- and 9-month old female and male lambs. In addition, the effects of isolation stress on in vitro release Met-enkephalin from fragments of the hypothalamus or adrenal cortex were examined. Isolation stress was followed by decreases in the concentration of Met-enkephalin in both the pituitary gland and adrenal cortex. There were also increases in the hypothalamic concentration of Met-enkephalin together with increases in PENK gene expression in the HPA in 6- and 9-months old females and males. There were reductions in release of Met-enkephalin from hypothalamic and adrenocortical tissue in vitro after isolation stress. In the presence of naltrexone, there were increases in basal release in vitro of Met-enkephalin from hypothalamic tissue from control and stressed female lambs but a decrease in tissue from stressed male lambs. In a somewhat similar manner, the presence of naltrexone was associated with increases in the basal release of Met-enkephalin from adrenocortical tissue from control female lambs but a decrease with tissue from stressed female and both stressed and control male lambs. Lay summary The present studies examine the impact of isolation stress on Met-enkephalin in growing female and male lambs. The results clearly showed the involvement of Met-enkephalin modulation of the psychological stress response in growing female and male lambs.

Thermal Micro-Environment during Poultry Transportation in South Central United States.

This observational study was conducted to characterize the thermal micro- climate that broilers experienced in commercial poultry transporters under various weather conditions and typical management practices in the South Central USA. We continuously monitored temperature and relative humidity in 45 interior locations of 28 fully-loaded commercial trailers over 2 year spans from 2015⁻2016 in South Central USA. In the cold season, double boarding of the exterior area of the transport modules maintained temperatures at least 8 °C warmer than ambient temperatures as low as -16 °C. Overall, temperature at all locations decreased as transporters traveled from farms to processing plants during winter trips with double boards. In the hot season, assistance by evaporative cooling during on-farm loading resulted in interior temperatures within ± 2 °C of ambient conditions (up to 36 °C) during road transport. In the summer months, trailers uniformly gained 2 °C as vehicles travelled for 45 min from farms to plants. Apparent equivalent temperatures of the monitored summer trips averaged 80.5 °C, indicating possible heat stress conditions based on the thermal comfort zones defined by literature index values. For longer trips, cooling assistance on the farms may be insufficient to prevent temperatures from rising further into extremely hot conditions in the transporters, leading to a dangerous thermal environment.