THE EFFECTS OF POLYCHLORINATED BIPHEYNLS (PCBs) ON AVIAN CARDIAC DEVELOPMENT

Polychlorinated biphenyls (PCBs) are a class of synthetic organochlorines that are thermally stable, resistant to degradation, and persistent in the environment as a result of bioaccumulation and intermittent redistribution through trophic levels. These compounds were sold commercially as mixtures in the twentieth century and later banned due to their biological toxicity. There are 209 known PCB congeners, each with different toxicities and physical properties that cause a variety of adverse health effects. Moreover, the effects of PCB mixtures vary with exposure concentrations, PCB congener toxicity, and species sensitivity. However, limited information is available about the impact of PCBs on the development of the embryonic cardiovascular system. There is a major site of contamination along the upper Hudson River in New York; wildlife in that region have shown evidence of exposure to PCBs. The purpose of this research was to determine the impact of embryonic exposure to a PCB mixture and a single congener, both found in the upper Hudson River on the developing avian cardiovascular system. In study 1, tree swallow eggs (Tachycineta bicolor) were dosed with PCB 77 and incubated to hatch. Similarly, domestic chicken eggs (Gallus domesticus) were dosed with the PCB mixture at embryonic day zero and incubated to hatch in study 2. Eggs were monitored through incubation; other measures were taken at hatch along with tissue collection. Results showed that embryonic exposure to PCBs resulted in an absence of the ventricular wall compact layer and hypertrabeculation in tree swallow hatchlings in spite of no effect on survival. Embryonic exposure to a PCB mixture in domestic chickens resulted in compact layer absence as well as additional cardiomyopathies, including absence of the ventricular wall trabeculated layer, ventricular chamber dilation, abnormal heart wall and septal formations, and arrhythmias during embryonic development. In study 3, embryonic exposure to a PCB mixture was studied at Hamburger Hamilton stages 10, 16, and 20. Embryonic exposure to a PCB mixture resulted in abnormal proliferation of cardiomyocytes early in heart development. Dose-dependent mortality occurred in chicken embryos exposed to the PCB mixture. These results support other findings demonstrating PCB effects on the cardiovascular system. Further, these data showed dramatic adverse effects of the PCB mixture as well as a single congener found in the region of the upper Hudson River on the developing avian cardiac system

Out of sight but not out of mind:The role of the embryo in hormone mediated maternal effects

In the last few decades, the study of maternal effects, as being strongly suggested by its name, has been substantially investigated from the perspective of mothers in terms of mechanisms and its evolutionary consequences. In addition, when it comes to the maternal effects on the offspring, the studies have been and still are more often than not focusing on the post-natal stage. However, recently, several studies have suggested that embryos are also playing an active and important role that may potentially induce a mother-offspring conflict by heavily converting maternal androgens to other hormones. Yet, this also has led to many unknowns. Therefore, in this thesis, I focused on the role of the embryo in determining its own development under the influence of the mother. In particular, I explored 3 important unknowns about the embryonic metabolism of maternal androgens which are: 1) to what extent do embryos can affect the hormonal environment they are exposed to during early development, 2) how does such a hormonal environment affect their prenatal development, and 3) are embryos able to control their early hormonal environment depending on the context in which they develop

Immediate and protracted transcriptional response of muscle tissue to transient variation of incubation temperature in broilers

Eggshell temperature is a critical factor, which can possibly influence immediate and/or long-term gene transcription and expression. Manipulation of temperature (low 36.8°C and high 38.8°C relative to control 37.8 °C) at two specific periods of embryonic development day (ED7-10 or ED10-13) was carried out and microarray-based gene expression profiles were explored in hind-limb and breast muscles. The overall results show tissue-specific patterns of transcriptional changes depending on temperature, day of the treatment and indicate immediate and long-term responses to the aforementioned treatment factors. Interestingly, the high temperature modification at early ED7-10 showed a profound immediate response based on the number of differentially expressed genes (DEGs), while the effect the low temperature was observed more at late ED10-13 compared to early ED7-10. Moreover, the low temperature modification at ED10-13 relative to ED7-10 and compared to other treatment conditions resulted in more DEGs at the day 35 post-hatch sampling stage regarded as long-term effects and implying an involvement of epigenetic mechanisms. microRNA (miRNA) expression analysis was performed to understand post-transcriptional regulation. In silico functional analysis of DEGs and differential miRNAs suggests that acute response to high temperature at ED7-10 for both muscle tissue types elevated mRNA transcripts related to cell maintenance, organismal development, and survival ability such as FABP1 in hind-limb and SMAD3 in breast muscle. Down-regulation of miR-199a-5p, miR-1915, miR-638 in hind-limb muscle and up-regulation of miR-133 in breast muscle were treatment specific and positively influence size of body and myogenesis, respectively. Low temperature condition at ED 10-13 affected reduction on programmed cell death and possibly gained mass of skeletal muscle in hind-limb samples (e.g. NR1H3), while pathways of accelerated cardiovascular system, skeletal-muscular, and connective tissue development were over-represented via the RUNX2 gene in breast. Interestingly, down regulation of let-7, miR-93 and miR-130c in breast was associated with diminished size of bone in type II muscle. Thermal interventions during incubation initiate immediate and delayed transcriptional responses that are specific for timing and direction of treatment. For miRNAs the study shows substantial immediate alterations, whereas late miRNA response was small. The mechanisms mediating considerable phenotypic plasticity contribute to the biodiversity and broaden the basis for managing poultry populations

Morphological and Hematological Responses to Hypoxia During Development in the Japanese quail, Coturnix coturnix

Hypoxic responses in quail development differ depending upon stage, duration and level of oxygen partial pressure of embryo. Incubation was switched to/from 110mmHg partial pressure (hypoxia), to/from 150mmHg (normoxia) during different stages in development, and control was incubated in normoxia throughout. Hatchability and embryo survival resulted in no hatchlings in continuous hypoxia. Responses to various hypoxic exposures throughout development resulted in recovery/repair of hypoxic damage by hatch. Heart and body mass, beak and toe length, hemoglobin, and hematocrit were measured to determine embryo responses to hypoxia during development at days 10, 15, and hatch. Hypoxia seemed to have the most deleterious effects on eggs in continuous hypoxia. Collectively, data indicate critical developmental windows for hypoxia susceptibility, especially during mid-embryonic development

Eggs and chicken embryos as potential sentinels for flock and hatchery health

Tese de Doutoramento em Ciências Veterinárias, especialidade Sanidade AnimalSuccessful hatching of strong, healthy chicks with a potential for good productive and reproductive performance depends on the assurance of as normal and ideal as possible embryonic development. The latter, however, can be negatively affected by a series of factors intrinsic to either the egg, its handling, or the incubator. This project aimed at collecting and interpreting essential and extensive information regarding embryonal response to physical challenges (temperature, relative humidity and carbon dioxide concentration) in the incubation environment, to infectious challenge with vertically transmitted Salmonella serovars Enteritidis and Typhimurium and, ultimately, to apply the knowledge obtained to a field survey of a commercial hatchery. For this purpose, eggs were incubated under different, controlled environments or after infection with a vertically transmitted pathogen, and an extensive list of parameters used to monitor subsequent changes in embryonic behavior, metabolism, growth and viability. The information gathered was then applied to a sample of non-viable eggs incubated at a major hatchery. Overall, the effects of manipulating temperature were more prominent than those of manipulating humidity and carbon dioxide. The most significantly affected parameters pertained to embryo metabolism, embryo weight, mortality and malformation rates. Changes in the development of the lymphoid follicles in the bursa of Fabricius and of the glandular epithelium of the gizzard were also found. Vertical infection with Salmonella was associated with higher embryonic mortality rates. No changes in performance or growth were observed in infected but viable embryos. The results obtained during the field survey did not directly correlate to the variables studied. However, the issues detected allowed for the designation of critical control points and opportunities for performance improvement that could have otherwise gone undetected at the hatchery and at the breeder farm, highlighting the importance of embryodiagnosis in the monitoring of hatchery and flock health.A operação eficiente e lucrativa de uma exploração industrial produtora de espécies aviárias (Gallus gallus) depende, entre outras coisas, do acesso a pintos saudáveis e com bom potencial produtivo, obtidos por incubação artificial de ovos de um bando reprodutor. É com a incubação destes ovos, levada a cabo em grande escala por máquinas altamente especializadas, que se inicia o desenvolvimento das aves, tendo esta influência directa sobre a viabilidade e futura performance do pinto eclodido. Vários factores podem intervir negativamente no sucesso da incubação, estando estes relacionados com o ovo em si, com o seu manuseamento ou com a própria incubadora. Por essa razão, este trabalho teve por objectivo adquirir, coligir e interpretar informação essencial sobre a resposta embrionária a alterações físicas do ambiente de incubação (temperatura, humidade relativa (RH) e concentração de dióxido de carbono), assim como à infecção com agentes patogénicos com transmissão vertical conhecida (Salmonella enterica vars. Enteritidis e Typhimurium). Estes ensaios foram conduzidos em laboratório, em ambiente cuidadosamente controlado. Numa segunda fase, o conhecimento obtido através de experimentação laboratorial foi aplicado na análise de uma amostra de ovos incubados provenientes de um centro de incubação industrial, com o objectivo de verificar a compatibilidade dos dados experimentais com os dados obtidos em situação real. Com vista a estudar a influência de alterações no ambiente de incubação sobre o desenvolvimento embrionário, procedeu-se à incubação de 2300 ovos specificpathogen free, dividos em dez grupos diferentes. O primeiro grupo foi incubado sob condições controlo (temperatura - 37.8ºC; RH - 50-55% durante os primeiros 18 dias de incubação e 60-65% durante os últimos 3 dias) e os restantes cinco grupos incubados sob: A) temperatura elevada constante (38.9ºC) e RH padrão; B) temperatura inferior constante (36.7ºC) e RH padrão; C) temperatura e RH padrão com um período (3 h) de elevação da temperatura (38.9ºC) aos dias 3 e 18 de incubação; D) temperatura e RH padrão com um período (3 h) de redução da temperatura (36.7ºC) aos dias 3 e 18 de incubação; E) humidade relativa elevada constante (63%) e temperatura padrão; F) humidade relativa inferior constante (43%) e temperatura padrão; G) humidade relativa e temperatura padrão com um período (3 h) de elevação da RH (63%) aos dias 3 e 18 de incubação; H) temperatura padrão com um período (3 h) de redução da RH (43%) aos dias 3 e 18 de incubação; I) ausência de renovação de ar/concentração de CO2 elevada constante durante incubação sob temperatura e RH padrão. Para investigar a influência da infecção com microrganismos transmitidos verticalmente, foram utilizados 576 ovos specific-pathogen free, divididos por oito grupos, quatro grupos controlo e quatro grupos inoculados. O primeiro grupo tratado foi inoculado com 105 CFU/ml de uma cultura de referência de S. enterica var. Enteritidis (CECT-4300) em NaCl (0,9%) estéril. O segundo grupo foi inoculado com 102 CFU/ml (dose de inoculação semelhante à concentração destas bactérias previamente reportada em ovos naturalmente infetados) da mesma cultura de referência. O terceiro grupo foi inoculado com 105 CFU/ml de uma cultura de referência de S. enterica var. Typhimurium (CECT-443) em NaCl (0,9%) estéril. Finalmente, o quarto grupo foi inoculado com 102 CFU/ml da mesma cultura de referência. Cada grupo tratado foi incubado juntamente com um grupo controlo inoculado com NaCl (0.9%) estéril. A presença ou ausência de Salmonella em cada grupo foi confirmada através de PCR multiplex convencional. Em todos os ensaios, foi retirada uma amostra de ovos da incubadora a cada 48 horas de incubação para análise dos efeitos das diferentes manipulações sobre o desenvolvimento embrionário. Uma extensa lista de parâmetros relacionados com o comportamento, metabolismo, crescimento e viabilidade dos embriões foi depois analisada. Em relação ao estudo de campo, foi efetuada análise morfológica, histológica e microbiológica de uma amostra de 51 ovos não viáveis, obtidos após 21 dias de incubação, num centro de incubação industrial. Relativamente aos ensaios de manipulação do ambiente físico de incubação, os resultados obtidos permitiram concluir que as alterações de temperatura, em qualquer direção, exerceram efeitos mais marcados sobre o desenvolvimento embrionário do que as alterações na humidade relativa ou na concentração de CO2 no interior da incubadora, enquanto que estas duas últimas variáveis produzem efeitos na sua maior parte sobreponíveis. Dos parâmetros monitorizados, aqueles que mais consistentemente apresentaram alterações encontravam-se relacionados com o comportamento e metabolismo embrionários, nomeadamente com a diminuição do metabolismo embrionário como consequência do stress induzido pela manipulação. O crescimento embrionário, representado pelo peso do embrião e por parâmetros alométricos, foi igualmente afetado, tendo-se verificado que as alterações de temperatura resultaram em embriões mais leves, enquanto as alterações de humidade relativa e CO2 produziram embriões de tamanho semelhante mas peso superior aos dos restantes tratamentos. À análise histológica, os embriões sujeitos a alterações contínuas de temperatura apresentaram ainda atraso na formação do tecido linfoide da bolsa de Fabricius e alterações celulares com distorção da arquitetura do epitélio glandular da moela, estas últimas em embriões sujeitos a aumentos de temperatura). Os embriões sujeitos a modificações da humidade relativa produziram efeitos temporários sobre o desenvolvimento do tecido hepático. As lesões microscópicas encontradas em animais mortos durante a incubação não permitiram o diagnóstico fiável da(s) causa(s) imediata de morte. Nenhum dos tratamentos produziu elevação significativa dos níveis de mortalidade, à exceção da incubação sob temperaturas inferiores ao normal. Dos restantes tratamentos, a elevação da humidade relativa resultou num aumento da mortalidade no final do período de incubação. Ainda relativamente à viabilidade embrionária, verificou-se que apenas as manipulações da temperatura de incubação resultaram num aumento significativo na incidência de malformações espontâneas. Finalmente, é de notar que, para todas as variáveis estudadas, a alteração contínua de uma variável produziu efeitos mais pronunciados e duradouros, enquanto a alteração pontual e temporária de uma variável produziu efeitos mais limitados, tanto em amplitude como em duração. Ao contrário dos ensaios de manipulação do ambiente de incubação, a inoculação de ovos specific-pathogen free não incubados com serovars de Salmonella enterica e sua posterior incubação não produziu alterações significativas no comportamento, metabolismo ou desenvolvimento embrionários. A inoculação de S. Enteridis, tanto em inóculo alto como em inóculo baixo, resultou num aumento significativo da taxa de mortalidade, sendo que esta se encontrou limitada aos primeiros 7 dias de incubação. A inoculação com qualquer dos inóculos de S. Typhimurium produziu, igualmente, níveis de mortalidade superiores comparados com os controlos, limitados aos primeiros dias de incubação, mas inferiores aos produzidos por S. Enteritidis. As lesões microscópicas observadas em animais encontrados mortos não permitiram o diagnóstico fiável da(s) causa(s) imediata de morte, sendo que a única diferença significativa encontrada se restringiu a alteração da afinidade tintoral dos tecidos provenientes de embriões infetados, indicativa de decomposição mais acelerada provavelmente devido a maior proliferação bacteriana. Contudo, nenhum dos embriões viáveis inoculados apresentou qualquer alteração macroscópica ou histológica significativa e em nenhum destes animais se observaram agregados bacterianos à análise microscópica, apesar da presença de infeção confirmada por métodos de biologia molecular. Os resultados obtidos sugeriram, ainda, que ambos os serovars de Salmonella utilizados apresentam tropismo para o embrião, possuindo a capacidade de não só sobreviver dentro do ovo, mas também de atravessar compartimentos orgânicos em direção ao embrião. Estas observações evidenciam algumas das razões para a eficiência demonstrada por estes serovars (e particularmente S. Enteritidis) na transmissão vertical e estabelecimento de portadores assintomáticos. Em relação ao estudo de campo, a análise dos ovos não viáveis não evidenciou diminuição de performance ou viabilidade embrionária devido a qualquer das variáveis estudadas em laboratório. Contudo, o exame externo, macroscópico dos ovos, assim como a sua análise microbiológica evidenciaram problemas na seleção e calibração dos ovos para incubação, assim como na possível higiene e práticas de recolha dos ovos na exploração de reprodutores, sinalizando potenciais pontos de intervenção futura com vista ao melhoramento da performance de incubação que de outra forma não seriam detetados. Finalmente, este trabalho permitiu a aquisição de informação essencial para a compreensão da resposta embrionária aos desafios da incubação, permitindo ainda confirmar a importância do embriodiagnóstico na monitorização da saúde da incubadora e do bando

Heart rate and oxygen consumption during the critical prenatal period in chicken embryos (Gallus gallus): Influence of light cues and the onset of pulmonary ventilation.

To examine if a rhythm can be entrained in either heart rate or oxygen consumption in late stage embryos (days 17-19.5) with light as a zeitgeber, chicken embryos were incubated in complete darkness (D:D) and 12:12 light:dark cycle (L:D). Light had no impact on oxygen consumption (390 µL O2∙min-1∙egg-1) but increased heart rate for non-internally pipped embryos (260 to 270 beats∙min-1 during light cycle). Oxygen consumption increased independent of pipping while heart rate increased (255 to 265 beats∙min-1) in D:D embryos due to pipping. A light-induced rhythm or effect occurred in heart rate but not oxygen consumption, suggesting heart rate and oxygen consumption may be uncoupled

THE FUNCTIONAL PROFILES OF CHICKEN EGGS INCUBATED UNDER MONOCHROMATIC LIGHTING

Poultry production remains susceptible to significant infectious disease threats such as Avian Flu, and Newcastle Disease Virus (NDV), which threaten the supply of poultry production. My dissertation research addresses this challenge by leveraging avian circadian biology to improve responses to vaccines to enhance poultry performance. The central hypothesis is that specific visible light wavelengths would enhance circadian rhythm development in ovo, leading to improved immune responses. I addressed an essential question regarding the effect of providing photoperiods with different wavelengths (Blue, Green, and White) on circadian rhythm development and its interplay with the immune response following the NDV challenge in chick embryos using the RNAseq technology. Our results showed that incubating chicken embryos under blue light 450nm was most efficient in entraining the circadian rhythm in lung tissue, compared to white light or dark treatment. Blue light showed a specific impact on skeletal muscle, regulation of striated muscle contraction, Glycerolipid metabolism, and development of neurons. The white light incubation led to a photo-acceleration stimulant effect on epidermal growth factor receptor signaling pathway, ErbB signaling pathway, MAPK signaling pathway, and Insulin signaling pathway were upregulated in white light non-challenged treatment. The response to NDV challenging showed a distinct transcriptome profile between blue and white light. The blue light showed a potent innate immune response, targeting viral replication, clearly pointing to antiviral response. The white light showed less immune response, but more pronounced cell proliferation and metabolic state, suggesting photo-acceleration as the primary process, particularly T cell development, T cell homeostasis, and T lymphocytes' quantity, suggests a rapid or ongoing transition between innate and adaptive immunity. This observation paves the way to photo-accelerated effect of providing white light during chicken egg incubation on organismal development and immune response. It is noteworthy that unvaccinated white light incubated chicks hatched 6-8 h earlier than other blue or dark incubation at the organism level. In conclusion, this study is the first to generated high-resolution RNASeq evidence demonstrating the effect of lighting color background on the circadian rhythm development and modulation of the innate immune response of chicks’ embryos challenged with NDV

Development of Cardiovascular Regulation in Embryos of the Domestic Fowl (Gallus Gallus), with a Partial Comparison to Embryos of the Desert Tortoise (Gopherus Agassizii)

In adult vertebrates, cardiovascular regulation is accomplished by numerous systems with neural, hormonal and local components responsible for the majority of regulation. These regulatory components work in concert to maintain the essential function of blood perfusion to adult tissues. Given the essential nature of this function it is therefore surprising that the development of cardiovascular regulation during gestation is poorly understood. The majority of what is known is based on a single vertebrate model, the fetal lamb. The fetal lamb has been used in multiple studies due to the clear clinical applications and has been pivotal in understanding the onset of regulation in developing vertebrates. However, study on the fetal lamb is limited to the latter 40% of gestation and has the added complication of an in-utero developmental strategy. Therefore the primary focus of this dissertation was to characterize basic cardiovascular regulation in the chicken embryo to provided the needed information for it's use an alternative to the fetal lamb. Developing chicken embryos rely on both alpha and beta adrenergic tones to maintain normal heart rate and arterial blood pressure during incubation. However, on day 21, just prior to hatch, these animals lose both tones on arterial pressure suggesting the onset of adult regulation. Cholinergic tone, however, was absent throughout chicken development indicating that it must mature during the neonatal life. Adult cardiovascular reflexes become apparent late in chicken development with a clear baroreflex specifically operating initially on day. However, an adult response to changes in ambient gas tension was absent during incubation suggesting embryos possess unique regulatory systems that are absent in adult chickens. This mechanism is comprised entirely of adrenergic systems with no cholinergic action during change in ambient gas tension. Similar developmental patterns were determined in embryos of the desert tortoise suggesting fundamental differences between in-utero and ex-utero developing vertebrates

Convective oxygen transport during development in embryos of the snapping turtle Chelydra serpentina

This article describes a study investigating the maturation of convective oxygen transport in embryos of the snapping turtle (Chelydra serpentina). Measurements included: mass, oxygen consumption, heart rate, blood oxygen content and affinity and blood flow distribution at 50%, 70% and 90% of the incubation period