Copy number variation (CNV) is an important polymorphism that is associated with a wide range of traits in human, wild and livestock species. In chicken, an important source of animal protein and a developmental model organism, CNV is associated with several phenotypes and evolutionary footprints. However, identification and characterization of CNV inheritance on chicken genome lacks further investigation. We screened CNVs in chicken using two distinct populations with known pedigree. In 826 broilers we identified 25,819 CNVs (4,299 deletions and 21,520 duplications) of which 21,077 were inherited, 201 showed no inheritance and 4,541 were classified as de novo CNVs. In 514 F2 animals (layer and broiler cross) we identified 21,796 CNVs (2,254 deletions and 19,543 duplications) of which 18,230 were inherited, 587 not inherited and 2,979 were classified as de novo CNVs. After a strict filtering step to remove potential false positives and negative CNVs, only 220 (4.84%) and 430 (14.43%) de novo CNVs remained in the broiler and F2 populations, respectively. A total of 33.11% (50 out of 151) of the inherited CNVs identified in ten animals were validated by sequencing data. From the validated CNVs, 64% had more than 80% of their size (bp) validated. A total of 59% and 48.8% were classified as novel CNVs regions (CNVRs) in the broiler and F2, respectively. Considering the Bonferroni-corrected p-values for multiple testing and statistically significant p-values ≤ 0.01, we found two CNV segments significantly associated with breast weight, one with breast weight yield, six with breast meat weight, 18 CNV segments with breast meat yield, four with breast filet weight and two with breast yield. These CNV segments that were significantly associated overlapped with 181 protein-coding genes. The CNVseg 300, that was associated with all traits and encompass six CNVRs, overlapped a total of 26 protein-coding genes. Among these genes, the gene MYL1 (Myosin Light Chain 1) is expressed in the fast skeletal muscle fibers, and the genes MLPH (Melanophilin), PRLH (Prolactin Releasing Hormone) and RAB17 (Member RAS Oncogene Family), that were associated with the lavender phenotype (feather blue-grey color) and regulation of homeothermy and the metabolism. The present study improves our knowledge about CNV in the chicken genome and provides insight in the distribution and of different classes of CNVs, i.e. inherited and de novo CNVs, in two experimental chicken populations. In addition, the genome-wide association analyses were the first performed on broiler population with breast muscle traits, that are important characteristics for poultry production. The GWAS results allow us to understand the probably relationship between some genes and CNVRs that are significantly associated with breast muscle traits. / A variação de número de cópias (CNV) é um polimorfismo importante que está associado a uma ampla gama de características em seres humanos, espécies selvagens e domésticas. Em frango, que é uma importante fonte de proteína e considerado um modelo biológico, CNVs foram associados a vários fenótipos e passos evolutivos. No entanto, nenhum estudo foi realizado para a identificação e caracterização da herança da CNV no genoma da galinha. Identificamos as CNVs no genoma da galinha usando duas populações experimentais e com pedigree conhecido: uma população de frangos de corte e uma F2. Em 826 frangos de corte, identificamos 25.819 CNVs (4.299 deleções e 21.520 duplicações), dos quais 21.077 foram herdados, 201 não foram herdados e 4.541 foram CNVs denominados de novo. Em 514 animais F2, identificamos 21.796 CNVs (2.254 deleções e 19.543 duplicações) das quais 18.230 foram herdadas, 587 não foram herdadas e 2.979 foram de novo CNVs. Após a etapa de filtragem nos de novo CNVs, apenas 220 (4,84%) e 430 (14,43%) permaneceram nas populações de frango de corte e F2, respectivamente. Um total de 33,11% (50 de 151) das CNV identificadas por dados de genotipagem em dez animais foram validados por dados de sequenciamento. Dos validados, 64% tinham mais de 80% do tamanho (pb) validados. Um total de 59% e 48,8% foram classificados como novas regiões de CNVs (CNVRs) nas populações de frango de corte e F2, respectivamente. Considerando os p-values corrigidos por Bonferroni para testes múltiplos e estatisticamente significativos (≤ 0,01), encontramos dois segmentos de CNV significativamente associados ao peso do peito, um ao rendimento de peso de peito, seis ao peso de carne de peito, 18 ao rendimento de carne de peito, quatro ao peso de filé de peito e dois ao rendimento do filé de peito. Esses segmentos de CNV significativamente associados estão sobrepostos com 181 genes codificadores de proteínas. O CNVseg 300, que foi associado a todas as características e abrange seis CNVRs, foram sobrepostos a um total de 26 genes codificadores de proteínas. Entre estes genes, o gene MYL1 (Myosin Light Chain 1) é expresso nas fibras rápidas do músculo esquelético, e os genes MLPH (Melanophilin), PRLH (Prolactin Releasing Hormone) e RAB17 (Member RAS Oncogene Family), que foram anteiromente associados ao fenótipo de cor azul acinzentado de penas e à regulação da homeotermia e do metabolismo. O presente estudo melhora o conhecimento sobre CNVs no genoma de frango, especialmente sobre a distribuição de CNV herdadas, não herdadas e de novo, em duas populações experimentais de frango. Além disso, a associação genômica foi a primeira realizada na população de frangos de corte com características do músculo do peito, que são muito importantes para a avicultura. Os resultados do GWAS nos permitem compreender a provável relação entre alguns genes e CNVRs que foram significativamente associados às características do músculo do peito.

Copy number variation (CNV) is an important polymorphism that is associated with a wide range of traits in human, wild and livestock species. In chicken, an important source of animal protein and a developmental model organism, CNV is associated with several phenotypes and evolutionary footprints. However, identification and characterization of CNV inheritance on chicken genome lacks further investigation. We screened CNVs in chicken using two distinct populations with known pedigree. In 826 broilers we identified 25,819 CNVs (4,299 deletions and 21,520 duplications) of which 21,077 were inherited, 201 showed no inheritance and 4,541 were classified as de novo CNVs. In 514 F2 animals (layer and broiler cross) we identified 21,796 CNVs (2,254 deletions and 19,543 duplications) of which 18,230 were inherited, 587 not inherited and 2,979 were classified as de novo CNVs. After a strict filtering step to remove potential false positives and negative CNVs, only 220 (4.84%) and 430 (14.43%) de novo CNVs remained in the broiler and F2 populations, respectively. A total of 33.11% (50 out of 151) of the inherited CNVs identified in ten animals were validated by sequencing data. From the validated CNVs, 64% had more than 80% of their size (bp) validated. A total of 59% and 48.8% were classified as novel CNVs regions (CNVRs) in the broiler and F2, respectively. Considering the Bonferroni-corrected p-values for multiple testing and statistically significant p-values ≤ 0.01, we found two CNV segments significantly associated with breast weight, one with breast weight yield, six with breast meat weight, 18 CNV segments with breast meat yield, four with breast filet weight and two with breast yield. These CNV segments that were significantly associated overlapped with 181 protein-coding genes. The CNVseg 300, that was associated with all traits and encompass six CNVRs, overlapped a total of 26 protein-coding genes. Among these genes, the gene MYL1 (Myosin Light Chain 1) is expressed in the fast skeletal muscle fibers, and the genes MLPH (Melanophilin), PRLH (Prolactin Releasing Hormone) and RAB17 (Member RAS Oncogene Family), that were associated with the lavender phenotype (feather blue-grey color) and regulation of homeothermy and the metabolism. The present study improves our knowledge about CNV in the chicken genome and provides insight in the distribution and of different classes of CNVs, i.e. inherited and de novo CNVs, in two experimental chicken populations. In addition, the genome-wide association analyses were the first performed on broiler population with breast muscle traits, that are important characteristics for poultry production. The GWAS results allow us to understand the probably relationship between some genes and CNVRs that are significantly associated with breast muscle traits. / A variação de número de cópias (CNV) é um polimorfismo importante que está associado a uma ampla gama de características em seres humanos, espécies selvagens e domésticas. Em frango, que é uma importante fonte de proteína e considerado um modelo biológico, CNVs foram associados a vários fenótipos e passos evolutivos. No entanto, nenhum estudo foi realizado para a identificação e caracterização da herança da CNV no genoma da galinha. Identificamos as CNVs no genoma da galinha usando duas populações experimentais e com pedigree conhecido: uma população de frangos de corte e uma F2. Em 826 frangos de corte, identificamos 25.819 CNVs (4.299 deleções e 21.520 duplicações), dos quais 21.077 foram herdados, 201 não foram herdados e 4.541 foram CNVs denominados de novo. Em 514 animais F2, identificamos 21.796 CNVs (2.254 deleções e 19.543 duplicações) das quais 18.230 foram herdadas, 587 não foram herdadas e 2.979 foram de novo CNVs. Após a etapa de filtragem nos de novo CNVs, apenas 220 (4,84%) e 430 (14,43%) permaneceram nas populações de frango de corte e F2, respectivamente. Um total de 33,11% (50 de 151) das CNV identificadas por dados de genotipagem em dez animais foram validados por dados de sequenciamento. Dos validados, 64% tinham mais de 80% do tamanho (pb) validados. Um total de 59% e 48,8% foram classificados como novas regiões de CNVs (CNVRs) nas populações de frango de corte e F2, respectivamente. Considerando os p-values corrigidos por Bonferroni para testes múltiplos e estatisticamente significativos (≤ 0,01), encontramos dois segmentos de CNV significativamente associados ao peso do peito, um ao rendimento de peso de peito, seis ao peso de carne de peito, 18 ao rendimento de carne de peito, quatro ao peso de filé de peito e dois ao rendimento do filé de peito. Esses segmentos de CNV significativamente associados estão sobrepostos com 181 genes codificadores de proteínas. O CNVseg 300, que foi associado a todas as características e abrange seis CNVRs, foram sobrepostos a um total de 26 genes codificadores de proteínas. Entre estes genes, o gene MYL1 (Myosin Light Chain 1) é expresso nas fibras rápidas do músculo esquelético, e os genes MLPH (Melanophilin), PRLH (Prolactin Releasing Hormone) e RAB17 (Member RAS Oncogene Family), que foram anteiromente associados ao fenótipo de cor azul acinzentado de penas e à regulação da homeotermia e do metabolismo. O presente estudo melhora o conhecimento sobre CNVs no genoma de frango, especialmente sobre a distribuição de CNV herdadas, não herdadas e de novo, em duas populações experimentais de frango. Além disso, a associação genômica foi a primeira realizada na população de frangos de corte com características do músculo do peito, que são muito importantes para a avicultura. Os resultados do GWAS nos permitem compreender a provável relação entre alguns genes e CNVRs que foram significativamente associados às características do músculo do peito.

The study of the formation of new cardiac muscle cells during postnatal development is a relatively new field. During fetal development, new cells are formed as the heart grows. However, the proliferative ability of postnatal cardiomyocytes is still debated. While several studies have been made on mammals, less is known about the chicken cardiac cells and their postnatal proliferation. As almost all previous studies have used microscopy-based cell counting methods, there has been some limitations on accuracy and amounts of cells that could be counted. The aim of this study is to develop a method for using flow cytometry to analyze proliferative ability of chicken cardiomyocytes and to investigate if any postnatal proliferation exists. For this study, 4 weeks old Red Junglefowl (Gallus gallus) chickens were used for isolating cardiomyocytes. In addition, 19 days old Red Junglefowl embryos were used to asses if a longer incubation time would yield a higher number of proliferative cells. Cells were stained using a commercial EdU imaging kit and analyzed using flow cytometry and imaging flow cytometry. The produced results could not be used for determining the proliferative ability of the cardiomyocytes, but provides crucial information for possible method improvements. In conclusion, this study has laid important groundwork for future studies on the proliferative ability of chicken cardiomyocytes.

Cognition (i.e. how individuals perceive, process and react to environmental cues) is fundamental to all animals’ life. Despite this, what explains variation in cognitive abilities is still mainly unclear. Environment is assumed to influences cognitive variation, but the mechanisms for this are still unknown. According to the social intelligence hypothesis, living in a group with a rich social environment, generate challenges that can enhance cognitive abilities. Impulsivity (to not be able to inhibit impulses), one aspect of cognition, may be influenced by the social environment, however this has not yet been experimentally tested. Impulsivity can complicate life, both for humans and animals. In humans, high levels of impulsivity and lack of self-control are associated with addictions and psychiatric disorders, thus is considered to be maladaptive. In animals, impulsivity correlates with stereotypies. To improve our understanding of impulsivity, I experimentally investigated how early social environment affects individual variation in impulsivity. To test this, red junglefowl chicks were used because their group living nature, and our accumulated knowledge on their cognition and behaviour. To manipulate the social environment, chicks either grew up in larger groups (with 17 individuals) or smaller groups (with 7 individuals). During the chicks’ first five weeks of life, three aspects of impulsivity were tested; impulsive action, persistence (in a detour reaching test) and routine formation (in a reversal learning test). Chicks that grew up in larger groups tended to perform less impulsive actions, while social environment did not explain variation in persistence. Chicks from larger groups had less strong routine formation compared to chicks raised in smaller groups. This partially supports the social intelligence hypothesis, and suggest that early social life can affect cognitive traits and explain individual variation in such.

Diferentes técnicas e metodologias foram utilizadas no estudo do desenvolvimento do sistema nervoso, analisando como modelos experimentais, entre outros, o embrião do \"Gallus domesticus\" e embriões de diferentes anfíbios. Com o advento do microscópio eletrônico de varredura (M.E.V.) pode-se, com certas limitações, efetuar uma análise tridimensional de estruturas biológicas. Portanto, utilizando o M.E.V., neste trabalho apresentamos nossas observações sobre o desenvolvimento inicial do tecto óptico do embrião de \"Gallus domesticus. / Different techniques and methods were used in the study of the development of the nervous system, analyzing how experimental models, among others, the embryo of \"Gallus domesticus\" and embryos of different amphibians. With the advent of the scanning electron microscope (SEM) can be, with certain limitations, make an analysis of three-dimensional structures. Therefore, using the SEM, in this paper we present our observations on the initial development of the embryo optical roof of \"Gallus domesticus.

A avicultura brasileira tem alcançado altos índices de desempenho na produção de carne e ovos, como resultado da atualização constante de tecnologias no setor. A biotecnologia vem contribuindo nesse sentido, atuando especialmente em programas de seleção de animais com maior potencial de desenvolvimento e crescimento. Como toda a fisiologia animal é controlada direta e/ou indiretamente pela hipófise e hipotálamo, este trabalho propôs identificar e analisar genes expressos nestas estruturas de galinhas de duas linhagens divergentes quanto ao potencial de crescimento e avaliar a expressão dos genes correspondentes a transcritos cuja identidade não pôde ser revelada (sem similar nos bacos de dados), uma vez que estes podem representar possíveis genes novos. Para isto, foram construídas e analisadas bibliotecas a partir da hipófise e hipotálamo de aves de 21 dias de idade de uma linha macho de corte (TT) e uma linhagem de postura (CC), provenientes da Embrapa Suínos e Aves. Um total de 4.286 ESTs válidas foi obtido (no mínimo150 pb com qualidade PHRED acima de 20), correspondendo a 2.133 ESTs da biblioteca da linhagem TT e 2.153 ESTs da biblioteca da linhagem CC. O exercício de montagem, via programa Cap3, revelou 3.074 seqüências únicas, sendo 1.643 da biblioteca da linhagem TT e 1.649 da CC. Estas seqüências únicas foram automaticamente classificadas, de acordo com as categorias do GeneOntology, sendo que as seqüências de ambas as bibliotecas apresentaram uma distribuição similar entre os termos. Após montagem das ESTs e análise do padrão de expressão digital das seqüências constituintes, dos 389 contigs obtidos, 194 foram compostos por seqüências diferencialmente expressas entre as bibliotecas. Foram detectados 28 contigs contendo SNPs linhagem específicos, sendo 52 SNPs TT específicos e 25 CC específicos. Um total de 146 ESTs não pôde ter sua identidade revelada, porém destas, 133 puderam ser localizadas no genoma da galinha e apresentaram pelo menos uma fase de leitura aberta (ORF). Após análise de expressão gênica, os genes correspondentes a 78 destas ESTs sem identidade apresentaram-se diferencialmente expressos entre pelo menos dois dos tecidos de aves de uma linhagem comercial de corte de 21 dias de idade estudados: hipófise, hipotálamo, cérebro, fígado e músculo. Os resultados apresentados são promissores e merecem ser investigados em estudos futuros com o objetivo de identificar marcadores moleculares para programas de seleção e melhoramento animal. / The brazilian aviculture has reached a high development level in both meat and egg production as a result of constant technological atualization in the sector. Biotechnology can contribute in this way acting in selection programs of animals that show higher growth and development potential. The global animal physiology is direct or indirectly controlled by pituitary and hypothalamus. The aim of this work was to identify and analyse genes expressed in these structures of two divergent line chickens according to growth potential and evaluate the gene expression of corresponding transcripts classified as "no hit", once these ESTs could represent new genes. In this way, two pituitary and hypothalamus libraries of 21 days broiler (TT) and a layer (CC) chickens supplied by Embrapa Swine and Poultry National Research Center were constructed and analysed. A total of 4,286 valid ESTs was obtained (showing at least 150 bp with PHRED quality above 20) corresponding to 2,133 ESTs of TT line library and 2,153 ESTs of CC line library. The clustering process by Cap3 resulted in 3,074 unique sequences corresponding to 1,643 TT library sequences and 1,649 of CC sequences. These unique sequences were automatically annotated according GeneOntology categories and both library sequences showed a similar distribution between the terms. After ESTs clustering and northern digital analysis, 389 contigs were obtained – 194 of them showed differentially expressed sequences between the libraries. A total of 28 contigs showed line specific SNPs, corresponding to 52 TT specific SNPs and 25 CC specific SNPs. 146 ESTs were classified as "no hit" but 133 of these sequences were localized in chicken genome and showed open reading frame (ORF). After gene expression analysis, the genes of 78 "no hit" ESTs showed different expression level between the five tissues of commercial broilers with 21 days old: pituitary, hypothalamus, brain, liver and muscle. These results are promising and must be more investigated in future studies to identify molecular markers for use in animal selection and improvement programs.

<p>Domestication is a process in which animals become adapted to a life among humans by means of selection. A reduced fear of humans is probably one of the first aims of selection, intentionally or unintentionally. Animals that have undergone the process of domestication have a different appearance than animals in the wild (domestic phenotype) and behave in a different way towards humans. In this study I have looked at foraging and explorative behaviours in an unselected parental generation of red junglefowl and their offspring. The parental generation were bred in three lines, a high line, with birds displaying a strong fear of humans, an intermediate line, birds showing a modest fear, and one low line, with birds performing a more tame behaviour towards humans. I presented the birds with three different feeding alternatives, familiar chicken food, meal worms camouflaged with wood shavings and just wood shavings. I counted number of pecks in the different food options, number of changes between sites and how many sites a bird visited. The results show that females of both generations were more explorative than males, by pecking more in cups of meal worms hidden in wood shavings whereas the males pecked more in cups containing chicken food. Females also moved around more in the arena. Results from the first selected generation show significant differences between the selection lines among the females, with females from the high and low groups being the most explorative.</p>

Ett stort antal utrotningshotade djurarter ingår idag i bevarandeprogram världen över. Små populationer hålls då i skyddade miljöer, exempelvis i djurparker, och i många fall är målet att återintroducera djur till naturen. Dessvärre är det vanligt att det uppstår problem när djur återintroduceras vilket kan bero på beteendemässiga anpassningar som uppkommit under tiden i fångenskap. Syftet med den här studien var därför att undersöka beteendemässiga aspekter på bevarandeavel och försöka ta reda på om och hur djur påverkas beteendemässigt av att hållas i skyddade fångenskapsmiljöer. I projektet användes röda djungelhöns (Gallus gallus) som en fallstudie. En granskning av beteendevariation mellan olika populationer av röda djungelhöns i fångenskap konstaterade skillnader i antipredatorbeteende, socialt beteende och födosöksbeteende. Vid en genetisk studie av samma populationer upptäcktes dessutom ett samband mellan genetisk diversitet och beteendevariation som potentiellt kan vara intressant ur ett bevarandeperspektiv. Socialt beteende, födosöksbeteende och olika aspekter av rädsla studerades vidare i populationer med olika bakgrund som fick växa upp tillsammans i en grupp. Resultaten visade att populationerna bara skilde sig åt i rädslebeteenden vilket antyder att denna typ av beteende i större utsträckning påverkas av långvarig avel i en viss fångenskapsmiljö medan socialt beteende och födosöksbeteende istället kan bero på den omedelbara sociala eller fysiska miljön. Antipredatorbeteende studerades också i en longitudinell studie av två populationer över fyra generationer och det visade sig att populationerna blev mer lika varandra ju längre tiden gick då de hölls under likadana miljöförhållanden. Det verkar alltså som om antipredatorbeteende kan förändras av avel i en viss miljö efter bara ett fåtal generationer. Utöver detta studerades även dagliga beteendemönster i olika djurparksmiljöer samt dygnsrytm av galanden hos både vilda populationer och djurparkspopulationer inom artens naturliga utbredningsområde. Resultaten tyder på att sådana beteendemönster inte påverkas nämnvärt av att djur hålls i fångenskap. Fallstudien som presenteras här är ett av de första försöken att, ur ett bevarandeperspektiv, studera hur fångenskapsmiljöer kan påverka djurs beteende och resultaten talar för att dessa aspekter är viktiga att ta hänsyn till vid planering av bevarandeavel. / A number of endangered species are currently involved in conservation breeding programs worldwide. Conservation breeding deals with propagation of captive populations, often with the ultimate aim of releasing animals into the wild. However, an alarmingly high proportion of reintroductions have not been successful in establishing viable populations, possibly due to behavioural problems caused by genetic adaptation to captivity. The main aim of this thesis was to study behavioural aspects of conservation breeding and investigate whether, and how, maintenance of small populations in captivity cause behavioural modifications, which could affect the success of reintroductions. Throughout the project, the red junglefowl (Gallus gallus) was used as a case study, representing animals maintained in captive populations. A screening of behavioural variation revealed that captive populations differ in antipredator, social and exploratory behaviours, all of which are central components of life in the wild. A correlation was also found between genetic diversity and behavioural variation. This has not been reported before and may potentially have interesting implications for conservation breeding. When studying the behaviour of populations with different backgrounds being raised together as one group, the results suggested that fear-related behaviours may be more affected by long-term breeding in a certain captive environment than social and exploratory behaviours which seem to be more influenced by the immediate social or physical environment. A longitudinal study of antipredator behaviour in two populations across four generations revealed that the populations became more similar over time when maintained under identical conditions. This demonstrates that effects of a new environment can appear after only a few generations. Furthermore, daily behavioural routines in different captive environments as well as diurnal crowing rhythms in both wild and captive populations were studied in the species’ natural region of distribution and the results suggest that such behavioural patterns are not affected by the captive environments to any notable extent. The present case study is one of the first attempts to, from a conservation perspective, study how captive environments can affect behaviour and the results imply that these aspects are important to take into consideration in conservation breeding programs.

Tonic immobility, TI, is an innate fear response in many vertebrate animals, induced by brief physical restraint. It is a widespread theory that chickens (Gallus gallus) reared under stressful conditions react stronger in tonic immobility tests, i.e. stay still and lay still for a longer period of time, than individuals reared under more stress-free conditions. In our study we attempted to see if stressful conditions early in life (temporary isolation from the flock on a daily basis) had any effect on how the individuals handled the stressful and fear-evoking experience of tonic immobility tests. A total of 77 chickens of the HyLine strain of White Leghorn were used. Three sets of tests were performed; first at the hatchery facility at Linköpings universitet; second after a stressful experience – the moving to the Wood-Gush facility at Vreta jordbruksgymnasium; and a random sample with about half of the birds the third time, also at Vreta. The results were inconclusive, but pointed more in the direction of the early-in-life stress having no effect on TI tests rather than the other way around.

Domestication is a process in which animals become adapted to a life among humans by means of selection. A reduced fear of humans is probably one of the first aims of selection, intentionally or unintentionally. Animals that have undergone the process of domestication have a different appearance than animals in the wild (domestic phenotype) and behave in a different way towards humans. In this study I have looked at foraging and explorative behaviours in an unselected parental generation of red junglefowl and their offspring. The parental generation were bred in three lines, a high line, with birds displaying a strong fear of humans, an intermediate line, birds showing a modest fear, and one low line, with birds performing a more tame behaviour towards humans. I presented the birds with three different feeding alternatives, familiar chicken food, meal worms camouflaged with wood shavings and just wood shavings. I counted number of pecks in the different food options, number of changes between sites and how many sites a bird visited. The results show that females of both generations were more explorative than males, by pecking more in cups of meal worms hidden in wood shavings whereas the males pecked more in cups containing chicken food. Females also moved around more in the arena. Results from the first selected generation show significant differences between the selection lines among the females, with females from the high and low groups being the most explorative.