Feeding behavior is a complex trait that is regulated by various hypothalamic neuropeptides and neuronal populations (nuclei). Understanding the physiological regulation of food intake is important for improving nutrient utilization efficiency in agricultural species and for understanding and treating eating disorders. Knowledge about appetite in birds has agricultural and biomedical relevance and provides evolutionary perspective. I thus investigated hypothalamic molecular mechanisms associated with appetite in broilers, layers, chicken lines selected for low (LWS) or high (HWS) body weight, and Japanese quail, which provide a unique perspective to understanding appetite. Broiler-type chicks have been genetically selected for rapid growth and consume much more feed than do layer-type chicks which have been selected for egg production. Long-term selection has caused the LWS chicks to have different severities of anorexia while the HWS chicks become obese, thus making these lines a valuable model for metabolic disorders. Lastly, the Japanese quail have not undergone as extensive artificial selection as the chicken, thus this model may provide insights on how human intervention has changed the mechanisms that regulate feeding behavior in birds. This research involved applying a variety of different treatments including fasting and refeeding, diets differing in macronutrient composition, and/or central administration of neuropeptide Y, xenopsin, neuropeptide K, oxytocin, mesotocin, gonadotropin-inhibitory hormone, and prolactin-releasing peptide, after which I measured feeding behavior and various aspects of hypothalamic physiology. I measured nuclei activation in hypothalamic appetite-associated regions including the lateral hypothalamus, paraventricular nucleus, ventromedial hypothalamus, dorsomedial nucleus, and arcuate nucleus and I measured gene expression of various appetite-associated factors in the whole hypothalamus and individual nuclei. These data provided information about the regions of the brain involved in mediating effects on appetite and the molecular pathways involved in the effect on appetite. There were differences in dose threshold sensitivity to various injected factors in the different stocks, differential responses to fasting and refeeding, and differences in nuclei and genes that were activated in response to the various treatments. These data provide valuable insights on the molecular mechanisms that are associated with the short-term regulation of feeding behavior and pathways that may be genetically stock-dependent. / PHD

Mammalian and avian species differ in some appetite-related aspects including how and which neurotransmitters and hormones regulate appetite. The objective of this research was to determine how two satiety-inducing neuropeptides regulate feeding behavior in avian models. Neuropeptide FF (NPFF) was intracerebroventricularly (ICV) injected into Japanese quail and decreased food intake at a dose of 32 nmol. NPFF-injected quail had increased expression levels of hypothalamic melanocortin subtype 3 receptor and decreased expression levels of neuropeptide Y receptor subtype 1 mRNAs compared to vehicle-injected controls. In a second study, gastrin was ICV injected into broiler chicks and decreased food intake at a dose of 500 ng (0.12 nmol). There was increased c-Fos immunoreactivity in the lateral hypothalamus (LH), paraventricular nucleus (PVN), arcuate nucleus, nucleus of the solitary tract, and area postrema at 1 h post-injection. Although a variety of genes were measured in those activated nuclei, there were only differences in melanin-concentrating hormone mRNA in the LH and corticotropin-releasing factor (CRF) mRNA in the PVN, suggesting that CRF signaling was involved in the hypothalamic response to gastrin. However, co-injection of gastrin and astressin, a CRF receptor antagonist did not affect gastrin-induced suppression of food intake, implying that the CRF receptors may not be directly associated with gastrin-induced satiety. Identifying the molecular pathways that mediate the effects of anorexigenic neuropeptides in birds will lead to the development of novel treatment options for appetite-related diseases and increased understanding of factors that affect production efficiency in commercial poultry and survival/resource allocation in wild birds. / Master of Science

The effect of dietary fats on reproductive performance, egg quality, fatty acid composition of tissues and egg yolk, and prostaglandin levels in embryonic tissues in Japanese quail (Coturnix, coturnix japonica) were evaluated. The addition of 5.0% chicken fat (CHX), hydrogenated soybean oil (HSBO), menhaden fish oil (MENH) or soybean oil (SBO) to the maternal and paternal diet altered tissue and yolk composition of the hens and tissue composition of the males to reflect the dietary source. Comparisons were made to chicken fat which served as a control. Feeding MENH increased the omega three fatty acid concentrations in liver, heart, testicle, and yolk. Feeding HSBO and SBO increased the omega six fatty acid concentrations in yolk. Feeding MENH and SBO increased the total monosaturated fatty acids in yolk. The concentrations of polyunsaturated fatty acids were increased by feeding MENH and SBO. Feeding MENH decreased egg production. Feeding MENH and HSBO decreased hatchability. Feeding MENH decreased specific gravity of eggs at day 30, 60, and 90. Feeding CHX increased specific gravity at day 30, 60 and 120. In addition, feeding CHX increased chick weight. Feeding SBO decreased early embryonic death. There were no consistent differences noted in tissue prostaglandin levels of embryos from hens on the differing diets. / Master of Science

Appetite is a complex behavior which can be influenced by factors within the animal's body as well as the environment around it. Internal factors include hormonal and nutrient concentrations found in the blood stream and subsequent neuropeptide and neurotransmitter signaling in the hypothalamus. External factors, such as high ambient temperature (HAT), can indirectly affect appetite regulation through other neuroendocrine systems such as the hypothalamo-pituitary-adrenal (HPA) axis. Understanding the physiological responses to endogenous factors and HAT exposure in birds will have implications in both the agricultural and biomedical fields. Thus, the purpose of this dissertation research was to explore the hypothalamic molecular mechanisms associated with food intake in broiler type chickens and Japanese quail, and the effect of HAT exposure on food intake in broiler type chickens. Broiler type chickens have undergone intense artificial selection for traits that promote rapid growth, consequently driving them to consume feed incessantly. It is hypothesized that broiler type chickens lack a mechanism that signals satiety, causing them to eat significantly more than layer type chickens. Selection for rapid growth of meat (muscle tissue) has made the broiler more susceptible to the deficits associated with HAT exposure, as animals composed primarily of muscle dissipate less heat while also producing more heat than those composed of fat. The Japanese quail have undergone relatively minor artificial selection compared to the chicken, suggesting that use of this model may provide insight into the mechanisms of appetite regulation in wild-type bird species. This research involved administrating appetite associated factors into the avian brain via an intracerebroventricular (ICV) injection including gastrin releasing peptide, vasoactive intestinal polypeptide, neuropeptide AF, and prostaglandin D2. Additionally, I explored the effects of HAT on food intake, and on the efficacy of several ICV administered appetite associated factors including neuropeptide Y, corticotropin releasing factor and α-melanocyte stimulating hormone. After treatment administration, I measured changes in food intake and behavior, activation of hypothalamic nuclei including the arcuate nucleus, dorsomedial nucleus, lateral hypothalamus, paraventricular nucleus, and the ventromedial nucleus, and the nucleus of the hippocampal commissure. I then measured changes in gene expression in both whole hypothalamic samples and specific hypothalamic nuclei. The data from non-HAT associated studies provided information on the hypothalamic nuclei which respond to the various appetite associated factors and the molecular mechanisms mediating changes in appetite. The data from the HAT study provided information on the hypothalamic nuclei involved in the avian response to HAT exposure, and the molecular mechanisms involved in the effect on food intake. Overall, these data provide insight on the mechanisms associated with short-term regulation of appetite, and pathways associated with stress and food intake. / Doctor of Philosophy / Appetite regulation can be affected by factors both in the body and out in the environment. Understanding how both internal and external factors affect appetite regulation can have positive implications in both the agriculture industry as well as the biomedical field. In agriculture, animals exposed to high ambient temperatures often exhibit several deficits including immunosuppression, decreased body weight, and ultimately an increased risk of mortality. It is hypothesized that the factor linking negative wellbeing to heat exposure is a reduction in food intake. Animals aside, the prevalence of eating disorders has doubled worldwide every 6 years since the year 2000. These numbers have increased even more during the recent COVID-19 pandemic. In order to improve the wellbeing of both humans and animals exposed to stressing stimuli, it is imperative we understand how individual appetite associated factors affect food intake, and how external stressors can impact the normal physiology of the hypothalamus. Thus, the purpose of this dissertation was to elucidate the hypothalamic mechanisms mediating appetite regulation using broiler type chickens and Japanese quail as models. Related pathways and molecular mechanisms were explored for several appetite associated factors including gastrin releasing peptide, vasoactive intestinal polypeptide, neuropeptide AF, and prostaglandin D2. Additionally, the effect of high ambient temperature on food intake, on the efficacy of several appetite associated factors including neuropeptide Y, corticotropin releasing factor and α-melanocyte stimulating hormone, and the hypothalamic pathways and molecular mechanisms mediating heat-induced anorexia were assessed.

The fact that sensory modalities do not become functional at the same time raises the question of how sensory systems and their particular experiential histories might influence one another. Few studies have addressed how modified stimulation to earlier-emerging modalities might influence the functioning of relatively later-developing modalities. Previous findings have shown that enhanced prenatal tactile and vestibular (proximal) stimulation extended and delayed normal patterns of auditory and visual responsiveness to species-typical maternal cues in bobwhite quail respectively. Although these results were attributed to the increased amount of sensory stimulation, these results may be a function of when prenatal augmented proximal exposure took place. To address this issue the present study exposed groups of bobwhite quail embryos to equivalent amounts of augmented tactile and vestibular stimulation either at a time when a later-emerging modality (auditory or visual) was beginning to functionally emerge or when it had already functionally emerged. Results indicate that differences in the timing of augmented tactile and vestibular stimulation led to differences in subsequent auditory and visual responsiveness. Embryos were unable to learn a maternal call prior to hatching when enhanced proximal stimulation coincided with auditory functional emergence implicating a deficit in auditory functioning, but did learn a maternal call when enhanced proximal stimulation occurred after auditory functional emergence. Augmented proximal stimulation that coincided with visual functional emergence did not appear to influence normal visual responsiveness, but when proximal stimulation occurred after visual emergence, chicks displayed an accelerated approach response to species-typical visual cues. These findings support the view that the timing of enhanced stimulation to earlier-emerging modalities is important, and have meaningful implications for intersensory theory and research. / Ph. D.

Early perceptual learning capacity has been shown to correspond with the relative status of emergent sensory systems throughout prenatal and postnatal development. It has also been shown that young infants can learn perceptual information during perinatal development. However, the exact nature of the relationship between prenatal and postnatal perceptual development and the role of early experience on learning ability have yet to be examined. The present study examined how auditory learning capacity in bobwhite quail chicks is affected by the interrelationship between the developing auditory and visual systems in late prenatal/early postnatal development. Chicks were provided with auditory information during the period immediately prior to or the period following hatching. In addition, visual experience was either provided or attenuated during both the prenatal and postnatal periods. Findings revealed that chicks postnatally exposed to 10 min/hr of maternal auditory stimulation in lighted conditions required 72 hr exposure to the call in order to learn that bobwhite maternal call (Experiments 1A and 1B). Control chicks who experienced the prenatal egg-opening procedure demonstrated no naive preference for two individual variants of the bobwhite maternal assembly call (Experiment 2). However, embryos who received 10 min/hr of prenatal visual stimulation, or who were reared in prenatal darkness successfully learned a maternal call with only 24 hr of postnatal exposure (Experiments 3A and 3C). Embryos who received prenatal visual and postnatal darkened rearing conditions (a mismatch between prenatal and postnatal experience) showed deficits in postnatal auditory learning (Experiment 3B). Embryos who were exposed to 10 min/hr of prenatal maternal auditory stimulation and 10 min/hr of nonconcurrent visual stimulation remembered the maternal call into later ages of postnatal development than in previous studies when reared in lighted or darkened postnatal conditions (Experiments 4A and 4B). However, when all prenatal and postnatal visual experience were both removed from embryos' and chicks' environments, deficits in prenatal auditory learning and postnatal memory were observed (Experiment 4C). These results indicate that prenatal and postnatal learning in bobwhite quail occur differently, that mismatches in prenatal and postnatal experience interfere with postnatal auditory learning, and that prenatal learning and postnatal memory are affected by the amount of visual stimulation present within chicks' environmental milieu. In the broader scheme, these results provide further evidence that the auditory and visual systems are linked during early development and support an ecological perspective of learning and memory. / Ph. D.

Prenatal sensory stimulation can have facilitative or interfering effects upon subsequent perceptual learning and development in bobwhite quail. Exposure to moderate amounts of unimodal prenatal sensory stimulation has been shown to accelerate early intersensory responsiveness, while exposure to concurrent prenatal bimodal sensory stimulation has been shown to interfere with perceptual learning and development. An immediate mechanism that may underlie this developmental intersensory interference is the arousal level of the organism associated with exposure to prenatal bimodal stimulation. Concurrent bimodal stimulation is known to elicit significantly higher levels of behavioral arousal and heart rate in bobwhite quail embryos. This study investigated the possibility that increased arousal associated with prenatal bimodal stimulation could have enduring effects upon subsequent postnatal behavioral organization and perceptual abilities in bobwhite quail. Subjects were exposed to one of three prenatal stimulation regimes: (a) concurrent bimodal (auditory/visual) stimulation, (b) unimodal auditory stimulation, or (c) no supplemental stimulation. Chicks exposed to concurrent prenatal bimodal stimulation demonstrated significantly greater levels of behavioral activity as well as decreased social behavior in the open-field when compared to unimodal auditory subjects and controls. Additionally, prenatal bimodal exposure may have led to a failure to utilize multimodal maternal cues in determining species-specific perceptual preferences in the days following hatching. All exposure groups demonstrated postnatal auditory learning of a maternal call, thus no interference effect was found for concurrent prenatal bimodal stimulation on postnatal auditory learning. These results suggest that concurrent prenatal bimodal stimulation has enduring effects upon postnatal behavioral arousal that may impact perceptual responsiveness of bobwhite quail in the days following hatching. / Ph. D.

Previous research has demonstrated that augmented prenatal sensory stimulation can influence the emergence of normal or species-typical patterns of intersensory perception. For example, unusually early visual experience can produce a facilitative effect on subsequent postnatal perceptual responsiveness, while substantially augmented prenatal visual stimulation can interfere with early postnatal responsiveness. In constructing a link between early experience and neuronal plasticity, it has been established that unusual visual experience can produce measurable changes in post-synaptic structures, particularly dendritic morphology, in brain areas responsible for vision. In avian species, the brain area responsible for vision is the visual Wulst, thought to be analogous to the mammalian visual cortex. This study examined the effects of differing amounts of augmented prenatal visual stimulation on the plasticity of neurons in the visual Wulst and on subsequent postnatal visual responsiveness to maternal cues in bobwhite quail chicks. Results revealed that the pattern of neuronal organization and postnatal behavior was influenced by the amount of prenatal visual experience subjects were provided. Specifically, chicks exposed to 240 min of prenatal visual stimulation during the last 24 hr prior to hatching had neurons with significantly fewer spines/10 mm dendrite and displayed accelerated patterns of species-typical visual responsiveness. In contrast, chicks provided 900 min of prenatal visual stimulation had more complex neurons (including more spines, longer dendrites, and more branches) and failed to display normal species-specific visual responsiveness in the days following hatching. These results suggest that neuronal organization in the bobwhite Wulst proceeds in a selective fashion, molded by experience, and appears to influence early perceptual development and organization during the perinatal period / Ph. D.

Many aspects of biological function are affected by hormones, from physiology to behavior, and the synthesis and release of hormones in vertebrates are regulated by the endocrine axes of control. A growing body of research shows that the mechanisms underlying the endocrine axes of control are complex and interconnected, with many hormones having multiple effects, and with many interactions between axes. In this study, I examined the effects of decreased thyroid function on the glucocorticoid stress response in Japanese quail, a potential interaction between the hypothalamic-pituitary thyroid (HPT) and hypothalamic-pituitary adrenal (HPA) axes of control. I used the thyroid inhibitor ammonium perchlorate (AP) for 2 weeks and 5 weeks to induce two states of decreased thyroid function: a thyroid challenged state, in which birds have depleted thyroidal T4 content, but still maintain euthyroid (normal) concentrations of plasma T4, and a hypothyroid state, in which birds have depleted thyroidal T4 content and decreased concentrations of plasma T4. Thyroid function was assessed by measuring plasma T4 concentrations, thyroidal T4 content, and thyroid gland mass. I took blood samples from birds both immediately prior to and immediately following a 30 minute confinement and agitation stressor to evaluate the effects of decreases in thyroid function on basal and stress-induced plasma corticosterone and plasma T4 concentrations. I found two key results: First, although baseline levels of plasma corticosterone were unchanged, the corticosterone stress response was significantly blunted in both the thyroid challenged and hypothyroid birds as compared to controls. This finding suggests that the HPT and HPA axes are functionally connected in birds, and other evidence suggests this connection is likely at the pituitary or hypothalamic level. Second, in hypothyroid birds, plasma T4 concentrations were elevated (into the euthyroid range) in response to the experimental stressor, although no change in plasma T4 was observed in thyroid challenged or control birds. This finding suggests that plasma T4 may have a permissive role in mounting a stress response. / Master of Science in Life Sciences

The relationships among body weight, body composition, adipose tissue cellularity and the onset of sexual maturity were studied in Japanese quail (Coturnix coturnix japonica). In an initial experiment, body composition and sexual maturity were examined in response to photoperiodic and hormonal manipulations. Attempts were made to modify body composition, and thereby age at sexual maturity, by feeding diets containing PTU (4-phenyl-2 thiouracil), by imposing a daily photoperiod (IP) of four hours, by the subcutaneous implantation of estrogen to females and testosterone to males, and by the intramuscular injection of turkey growth hormone and anti-sera to turkey growth hormone. The quail were sacrificed at 63 days of age and body composition was examined. Quail subjected to the IP treatment failed to enter sexual maturity by 63 days of age, while about 50% of the controls receiving a photoperiod of 14 hours were sexually mature at this age. The failure of these quail to mature was associated with a reduction in body fat. The effects of the other treatments on sexual maturity, however, were not consistently associated with differences in body weight and/or body composition. In the second phase of this study, adipocyte hyperplasia was examined in relation to sexual maturity. Total DNA concentrations in the stromal and lipocyte fractions of collagenase-digested abdominal fat depots were determined on a chronological age basis from 28 to 240 days of age. In both males and females, significant increases in abdominal fat weights at ages beyond the age at sexual maturity were associated with increased DNA concentrations in abdominal fat adipose tissue. These results suggested that mature Japanese quail were capable of hyperplastic fat deposition. In the final phase of this study, the incorporation of methyl-³H-thymidine into DNA of lipid and non-lipid fractions of collagenase-digested abdominal fat from both ad libitum and restricted-fed (70% of ad libitum intake) quail was examined. Regardless of the feeding regime and stage of maturity, substantial radioactivity was recovered from both the stromal and lipocyte fractions when the quail were examined 24 hours after the administration of tritiated thymidine. When quail were examined five days after the administration of tritiated thymidine, an apparent migration of radioactivity from stromal to lipocyte DNA occurred. It was concluded that this pattern represented mitotic activity of adipocyte progenitor cells located in non-lipid fractions of adipose tissue, and the subsequent maturation of these cells into lipocytes as they accumulated lipid. The hypothesis that adipocyte progenitors reside in both the stromal and lipocyte fraction, however, was not disproven. Nevertheless, the similarity of results obtained prior to, at, and subsequent to the onset of lay indicated that adipocyte hyperplasia contributes to increases in adipose tissue mass in mature Japanese quail. / Ph. D.