Nondestructive Chicken Egg Fertility Detection Using CNN-Transfer Learning Algorithms

This study explores the application of CNN-Transfer Learning for nondestructive chicken egg fertility detection. Four models, VGG16, ResNet50, InceptionNet, and MobileNet, were trained and evaluated on a dataset using augmented images. The training results demonstrated that all models achieved high accuracy, indicating their ability to accurately learn and classify chicken eggs’ fertility state. However, when evaluated on the testing set, variations in accuracy and performance were observed. VGG16 achieved a high accuracy of 0.9803 on the testing set but had challenges in accurately detecting fertile eggs, as indicated by a NaN sensitivity value. ResNet50 also achieved an accuracy of 0.98 but struggled to identify fertile and non-fertile eggs, as suggested by NaN values for sensitivity and specificity. However, InceptionNet demonstrated excellent performance, with an accuracy of 0.9804, a sensitivity of 1 for detecting fertile eggs, and a specificity of 0.9615 for identifying non-fertile eggs. MobileNet achieved an accuracy of 0.9804 on the testing set; however, it faced challenges in accurately classifying the fertility status of chicken eggs, as indicated by NaN values for both sensitivity and specificity. While the models showed promise during training, variations in accuracy and performance were observed during testing. InceptionNet exhibited the best overall performance, accurately classifying fertile and non-fertile eggs. Further optimization and fine-tuning of the models are necessary to address the limitations in accurately detecting fertile and non-fertile eggs. This study highlights the potential of CNN-Transfer Learning for nondestructive fertility detection and emphasizes the need for further research to enhance the models’ capabilities and ensure accurate classification

K-MEANS CLUSTERING FOR EGG EMBRYO'S DETECTION BASED-ON STATISTICAL FEATURE EXTRACTION APPROACH OF CANDLING EGGS IMAGE

This research discusses the detection of embryonic eggs using the k-means clustering method based on statistical feature extraction. The processes that occur in detection are image acquisition, image enhancement, feature extraction, and identification/detection. The data used consisted of 200 egg image data, consisting of 100 test data and 100 new test data. The acquisition process uses a smartphone camera by capturing candled egg objects. The results of image acquisition become a reference in the process of image enhancement and feature extraction using Statistical Feature Extraction. The statistical feature extraction applied is the Gray Level Co-occurrence Matrix (GLCM) method, which consists of 6 features, namely Energy, Contrast, Entropy, Variance, Correlation, and Homogeneity. The results of feature extraction (6 features) are grouped by the K-means Clustering method. The clustering process uses Euclidean distance calculations to determine the proximity of features. The results of grouping and testing give the best average results with an accuracy of ≈ 74% from several test samples

Otsu Method for Chicken Egg Embryo Detection based-on Increase Image Quality

Detection of chicken egg embryos using image processing has limitations and needs some processes for improvement. By human vision, the previous process used binoculars and candling using light/beams directed at the chicken eggs in the incubator. In this study, we propose the application of image segmentation using the Otsu method in detecting chicken egg embryos. This method uses image segmentation with increased image quality (preprocessing) by several methods such as resizing, grayscaling, image adjustment, and image enhancement. These processes produce a better image and can be used for input in the segmentation process. In addition, this study compares several segmentation methods in detecting chicken egg embryos, such as thresholding, Otsu basic, and k-means clustering. The results show that our proposed method produced segmentation images to detect chicken egg embryos of 200 datasets images. This method has a faster process and can create a uniform segmentation than other methods. However, other methods can also detect chicken egg embryos. The method’s accuracy proposed in this study increased by 1.5% compared to other methods. In addition, the resulting SSIM value has a percentage close to and more than 90%, which means that the segmentation of the results obtained can be used to detect chicken egg embryos

K-means Segmentation Based-on Lab Color Space for Embryo Egg Detection

The hatching process also influences the success of hatching eggs beside the initial egg factor. So that the results have a large percentage of hatching, it is necessary to check the development of the embryo at the beginning of the hatching. This process aims to sort eggs that have embryos to remain hatched until the end. Maximum checking is done the first week in the hatching period. This study aims to detect the presence of embryos in eggs. Detection of the existence of embryos is processed using segmentation. Egg images are segmented using the K-means algorithm based on Lab color images. The results of the images acquisition are converted into Lab color space images. The results of Lab color space images are processed using K-means for each color. The K-means process uses cluster k=3, where this cluster divided the image into three parts, namely background, eggs, and yolk eggs. Yolk eggs are part of eggs that have embryonic characteristics. This study applies the concept of color in the initial segmentation and grayscale in the final stages. The results of the initial phase show that the image segmentation results using k-means clustering based on Lab color space provide a grouping of three parts. At the grayscale image processing stage, the results of color image segmentation are processed with grayscaling, image enhancement, and morphology. Thus, it seems clear that the yolk segmented shows the presence of egg embryos. Based on this process and results, K-means segmentation based on Lab color space can be used for the initial stages of the embryo detection process. The evaluation uses MSE and MSSIM, with values of 0.0486 and 0.9979; this can be used as a reference that the results obtained can indicate the detection of embryos in egg yolk.Comment: 11 pages, 6 figures, ICoSiET Conference 202

K-Means Segmentation Based-on Lab Color Space for Embryo Detection in Incubated Egg

The quality of the hatching process influences the success of the hatch rate besides the inherent egg factors. Eliminating infertile or dead eggs and monitoring embryonic growth are very important factors in efficient hatchery practices. This process aims to sort eggs that only have embryos to remain in the incubator until the end of the hatching process. This process aims to sort eggs with embryos to remain hatched until the end. Maximum checking is done the first week in the hatching period. This study aims to detect the presence of embryos in eggs. Detection of the existence of embryos is processed using segmentation. Egg images are segmented using the K-means algorithm based on Lab color images. The results of the image acquisition are converted into Lab color space images. The results of Lab color space images are processed using K-means for each color. The K-means process uses cluster k=3, where this cluster divides the image into three parts: background, eggs, and yolk. Egg yolks are part of eggs that have embryonic characteristics. This study applies the concept of color in the initial segmentation and grayscale in the final stages. The initial phase results show that the image segmentation results using k-means clustering based on Lab color space provide a grouping of three parts. At the grayscale image processing stage, the results of color image segmentation are processed with grayscaling, image enhancement, and morphology. Thus, it seems clear that the yolk segmented shows the presence of egg embryos. Based on this process and results, the initial stages of the embryo detection process used K-means segmentation based on Lab color space. The evaluation uses MSE and MSSIM, with values of 0.0486 and 0.9979; this can be used as a reference that the results obtained can detect embryos in egg yolk. This protocol could be used in a non-destructive quantitative study on embryos and their morphology in a precision poultry production system in the future

Deciphering the genetic background of quantitative traits using machine learning and bioinformatics frameworks

In dieser Doktorarbeit habe ich zwei Ansätze verfolgt, mit denen genetische Mechanismen, welche quantitativen Merkmalen zugrunde liegen, aufgezeigt und bestimmt werden können. In diesem Zusammenhang lag mein Fokus auf der Entwicklung effizienter Methoden um Genotyp-Phänotyp Assoziationen zu identifizieren. Durch diese lassen sich im Weiteren regulatorische Mechanismen beschreiben, welche phänotypische Unterschiede zwischen Individuen verursachen. Im ersten Ansatz habe ich Schlüsselmechanismen der Genregulation untersucht, welche die Entwicklung der Bruchfestigkeit von Eierschalen steuern. Das Ziel war es zeitliche Unterschiede der Signalkaskaden, welche die Eierschalen Bruchfestigkeit im Verlauf eines Vogellebens regulieren, zu detektieren. Hierfür habe ich die Bruchfestigkeit zu zwei verschiedenen Zeitpunkten innerhalb eines Produktionszyklus betrachtet und die Genotyp-Phänotyp Assoziationen mithilfe eines Random Forest-Algorithmus bestimmt. Für die Analyse der entsprechenden Gene wurde ein etablierter systembiologischer Ansatz verfolgt, mit dem genregulatorische Pathways und Master-Regulatoren identifiziert werden konnten. Meine Ergebnisse zeigen, dass einige Pathways und Master-Regulatoren (z.B. Slc22a1 und Sox11) gleichzeitig in verschiedenen Legephasen identifiziert wurden, andere (z.B. Scn11a, St8sia2 oder der TGF-beta Pathway) speziell in lediglich einer Phase gefunden wurden. Sie stellen somit altersspezifische Mechanismen dar.Insgesamt liefern meine Ergebnisse (i) signifikante Einblicke in altersspezifische und allgemeine molekulare Mechanismen, welche die Eierschalen-Bruchfestigkeit regulieren und bestimmen; und (ii) neue Zuchtziele, um die Bruchstärke von Eierschalen vor allem in späteren Legephasen zu erhöhen und somit die Eierschalen Qualität zu verbessern. In meinem zweitem Ansatz, habe ich die Methode der Random Forests mit einer Strategie zur Signaldetektierung kombiniert, um robuste Genotyp-Phänotyp-Beziehungen zu identifizieren. Ziel dieses Ansatzes war die Verbesserung der Effizienz der Einzel-SNP basierten Assoziationsanalyse. Genomweite Assoziationsstudien (GWAS) sind ein weit verbreiteter Ansatz zur Identifikation genomischer Varianten und Genen, die verantwortlich sind für Merkmale, welche von Interesse sowohl für den akademischen als auch den wirtschaftlichen Sektor sind. Trotz des langjährigen Einsatzes verschiedener GWAS-Methoden stellt die zuverlässige Identifikation von Genotyp-Phänotyp-Beziehungen noch immer eine Herausforderung für viele quantitative Merkmale dar. Dies wird hauptsächlich durch die große Anzahl genomischer Loci begründet, welche lediglich einen schwachen Effekt auf das zu untersuchende Merkmal haben. Daher lässt sich Hypothese aufstellen, dass genomische Varianten, welche zwar einen geringen, aber dennoch realen Einfluss ausüben, in vielen GWAS-Ansätzen unentdeckt bleiben. Zur Behandlung dieser Unzulänglichkeiten wird in der Arbeit ein zweistufiges Verfahren verwendet. Zunächst werden kubische Splines für Teststatistiken und genomische Regionen angepasst. Die Spline-Maxima, welche höher als die zu erwartenden zufallsbasierten Maximalwerte ausfallen, werden als quantitative Merkmals-Loci (QTL) eingestuft. Anschließend werden die SNPs in diesen QTLs, basierend auf ihrer Assoziationsstärke mit den Phänotypen, durch einen Random Forests-Ansatz priorisiert. Im Rahmen einer Fallstudie haben wir unseren Ansatz auf reale Datensätze angewendet und eine plausible Anzahl, teilweise neuartiger, genomischer Varianten und Genen identifiziert, welche verschiedenen Qualitätsmerkmalen zugrunde liegen.In this thesis, I developed two frameworks that can help highlight the genetic mechanisms underlying quantitative traits. In this regard, my focus was to design efficient methodologies to discover genotype-phenotype associations and then use these identified associations to describe the regulatory mechanism that affects the manifestation of phenotypic differences among the individuals. In the first framework, I investigated key regulatory mechanisms governing the development of eggshell strength. The aim was to highlight the temporal changes in the signaling cascades governing the dynamic eggshell strength during the life of birds. I considered chicken eggshell strength at two different time points during the egg production cycle and studied the genotype-phenotype associations by employing the Random Forest algorithm on genotypic data. For the analysis of corresponding genes, a well established systems biology approach was adopted to delineate gene regulatory pathways and master regulators underlying this important trait. My results indicate that, while some of the master regulators (Slc22a1 and Sox11) and pathways are common at different laying stages of chicken, others (e.g., Scn11a, St8sia2, or the TGF-beta pathway) represent age-specific functions. Overall, my results provide: (i) significant insights into age-specific and common molecular mechanisms underlying the regulation of eggshell strength; and (ii) new breeding targets to improve the eggshell quality during the later stages of the chicken production cycle. In my second framework, I combined the Random Forests and a signal detection strategy to identify robust genotype-phenotype associations. The objective of this framework was to improve on the efficiency of single-SNP based association analysis. Genome wide association studies (GWAS) are a well established methodology to identify genomic variants and genes that are responsible for traits of interest in all branches of the life sciences. Despite the long time this methodology has had to mature the reliable detection of genotype-phenotype associations is still a challenge for many quantitative traits mainly because of the large number of genomic loci with weak individual effects on the trait under investigation. Thus, it can be hypothesized that many genomic variants that have a small, however real, effect~remain unnoticed in many GWAS approaches. Here, we propose a two-step procedure to address this problem. In a first step, cubic splines are fitted to the test statistic values and genomic regions with spline-peaks that are higher than expected by chance are considered as quantitative trait loci (QTL). Then the SNPs in these QTLs are prioritized with respect to the strength of their association with the phenotype using a Random Forests approach. As a case study, we apply our procedure to real data sets and find trustworthy numbers of, partially novel, genomic variants and genes involved in various egg quality traits.2021-10-1

Practical book on biology

УЧЕБНО-МЕТОДИЧЕСКИЕ ПОСОБИЯПРАКТИКУМЫБИОЛОГИЯИНОСТРАННЫЕ СТУДЕНТЫДОВУЗОВСКАЯ ПОДГОТОВКАКЛЕТКИРАЗМНОЖЕНИЕНАСЛЕДСТВЕННОСТЬИЗМЕНЧИВОСТЬ ГЕНЕТИЧЕСКАЯВИРУСЫБАКТЕРИИВОДОРОСЛИЛИШАЙНИКИГРИБЫРАСТЕНИЯЖИВОТНЫЕЧЕЛОВЕКА РАЗВИТИЕМОРФОГЕНЕЗВ практикуме рассматриваются сущность жизни, уровни организации живого, биология и физиология клетки, размножение организмов, индивидуальное развитие, наследственность и изменчивость, вирусы, бактерии, водоросли, лишайники, грибы, растения, животные, особенности строения и физиологии человека. Пособие включает 34 темы практических и 5 итоговых занятий. Каждое практическое занятие состоит из вводной части материала, цели занятия, вопросов, основных понятий и терминов для подготовки к занятию, 20 тестов для проверки уровня знаний по теме, литературы для подготовки и материалов для выполнения практической работы. Итоговые занятия включают цель, вопросы для подготовки, литературу и материалы для выполнения практической работы

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

The avian lingual and laryngeal apparatus within the context of the head and jaw apparatus, with comparisons to the mammalian condition: Functional morphology and biomechanics of evaporative cooling, feeding, drinking, and vocalization

© Springer International Publishing AG 2017. All rights reserved. The lingual and laryngeal apparatus are the mobile and active organs within the oral cavity, which serves as a gateway to the respiratory and alimentary systems in terrestrial vertebrates. Both organs play multiple roles in alimentation and vocalization besides respiration, but their structures and functions differ fundamentally in birds and mammals, just as the skull and jaws differ fundamentally in these two vertebrate classes. Furthermore, the movements of the lingual and laryngeal apparatus are interdependent with each other and with themovements of the jaw apparatus in complex and littleunderstood ways. Therefore, rather than updating the existing numerous reviews of the diversity in lingual morphology of birds, this chapter will concentrate on the functionalmorphological interdependences and interactions of the lingual and laryngeal apparatus with each other and with the skull and jaw apparatus. It Will

Plasticity of the neuromuscular system in response to changes in dactyly

Changes in limb morphology enabled the terrestrial lifestyle of tetrapods, as they adapted to different types of locomotion. The distal part of the limb – called the autopod, and encompassing the carpals/tarsals, metacarpals/metatarsals, and digits (fingers and toes) – in particular, went through drastic morphological changes during tetrapod evolution. Both digit patterns, as well as digit numbers, were modified during this process. While fossil data of stem-group tetrapods show that some ancestors possessed up to eight digits on one extremity, this number was reduced to five at the basis of the crown tetrapod group. Today, there is no known natural tetrapod population that surpasses the so-called ‘pentadactyl state’, and having more than five fingers is considered a pathology referred to as ‘polydactyly’. In the limb, not only the skeletal structure has to be modified during digit gains or losses, but also the soft tissues – like, e.g., the neuromuscular system - has to follow the skeletal changes, to form a fully functional unit. While polydactyly and the molecular alterations leading to it have been extensively studied at the skeletal level, little is known about the accompanying changes in the neuromuscular system. The present work aims to bridge this gap, by studying the plasticity of the neuromuscular system in response to changes in digit numbers. First, we evaluated muscle and nerve patterns in the periphery of polydactyl limbs. Then, on a molecular and cellular level, we attempted to understand how muscle-specific motor neuron pools are modified in polydactyl individuals. We used chicken embryos, where in-ovo manipulation of the limb can result in mirror-image duplication of the digits, thus providing an ideal and well-established model for our study. Using wholemount immunostaining of nerves and muscles followed by light sheet microscopy, we first reconstructed a 3D developmental time series of control wings and legs. There, our main finding uncovered a rotated pattern of the main nerve branches between wings and legs. Moreover, challenging the system with additional digits demonstrated a differential response of muscles and nerves in polydactyl limbs. Namely, while muscles seemed able to perfectly follow the pattern of skeletal mirror-image duplications, only two of the three main nerve branches responded and split to innervate the duplicated muscles. These intriguing results motivated us to turn our attention toward the central nervous system. The cell bodies of limb-innervating motor neurons reside in the spinal cord and are organized into small subsets, so-called motor neuron pools. Each pool innervates one specific muscle, and the survival and maturation of these pools largely depend on target-muscle derived factors. The observed changes in innervation patterns pointed toward a potential modification of motor neuron survival and pool identity, a rationale we set out to explore in the second half of this thesis. First, we showed that motor neuron numbers in the rostral LMC are decreased in the spinal cord of polydactyl individuals, due to increased cell death. This phenomenon was correlated to changes in the muscle patterns of the forearm, and the modification of fast versus slow muscle fibre composition in the limb, which might impair efficient innervation. In order to gain insights into the motor neuron pool compositions and transcriptomes, we performed, for the first time in chicken embryos, single-cell RNA-sequencing of spinal cord tissue. The resulting data identified putative digit-specific motoneuron pool markers but indicated only minor differences in cell-type distribution between polydactyl and control neural tubes. However, the traditional emulsion-based method might have failed to capture high enough numbers of motor neurons. Accordingly, we focused on a different, more direct approach, for an in-depth study of motor neuron pools innervating native and duplicated muscles in polydactyl embryos. To this aim, we developed a method combining ex-ovo retrograde axonal labelling, manual purification of neurons, and the highly sensitive Smart-seq2 single-cell RNA-sequencing technique. Our results showed that our method is efficient in extracting embryonic motor neurons, that it captures a high number of genes per cellular transcriptome, and therefore represents a valuable tool for studying motor circuit formation. Preliminary comparative transcriptomic analysis of neurons coming from two motor neuron pools – innervating the EMR and FDQ wing muscles - revealed unique pool-specific signatures and further validated our technique. Collectively, in the present thesis work, we describe the plasticity of the neuromuscular system in response to polydactyly, and how muscles and nerves integrate changes in the skeletal patterning. Furthermore, we show that changes in muscle identity and shifts in muscle fibre composition can affect motor neuron survival. Also, we developed a technique to study in great detail the transcriptomes of muscle-specific motoneurons, and we intend to use this methodology to better understand neuronal wiring and molecular muscle-neuron cross-talk during circuit formation. Our data provide the basis for further developmental studies and offer a framework for medical research, in order to better understand the etiologies of human polydactyl phenotypes