Abstract Birth of offspring of a pre-determined sex using flow cytometrically sorted fresh spermatozoa was first achieved in rabbits by Johnson et al. (1989). Since then offspring have been produced using sex-sorted spermatozoa from several different species (reviewed by Johnson, 2000). Initially, efficiency of the sex-sorting technology was poor with only low numbers of spermatozoa sorted per hour. Thus, the offspring derived from flow cytometrically sorted spermatozoa were produced with the use of artificial reproductive technologies (ART) such as in vitro fertilisation (IVF) and culture (IVC), intracytoplasmic sperm injection (ICSI) and deep artificial insemination (AI) which facilitated low dose insemination of potentially compromised spermatozoa. More recently, the development of high-speed sorters (Johnson and Welch, 1999) has facilitated the production of offspring using conventional AI techniques with low dose inseminates (Seidel et al., 1999) and successful cryopreservation of sorted spermatozoa (Schenk et al., 1999; Johnson et al., 2000; Lindsey et al., 2002; Schenk and DeGrofft, 2003). Increased efficiency of sorting bull spermatozoa has evolved through significant instrumentation and biological developments which have enabled the commercialization of the sperm sexing technology in the dairy industry, although conception rates in cows after low dose AI with sexed frozen-thawed spermatozoa are still lower than after standard frozen semen AI (Seidel et al., 1999). Subsequently, over 20 000 calves of pre-determined sex have been produced from commercially available sex-sorted frozen-thawed bull spermatozoa (Seidel, 2003). However, similar developments have not been made in the sheep industry and were examined in this thesis. In this study, successful cryopreservation of sex-sorted ram spermatozoa and production of offspring of the pre-determined sex (X: 94.4 %; Y: 100 %) was achieved after low dose (2-4 x 106 total) insemination using conventional laparoscopic intrauterine (IU) AI. However, the overall pregnancy rate for ewes inseminated with sex-sorted frozen-thawed spermatozoa was low (25 %) compared to ewes inseminated with a commercial dose (140 x 106 total) of non-sorted frozen-thawed spermatozoa (54 %). Cryopreservation has been found to not only reduce the proportion of motile spermatozoa, but cause the remaining spermatozoa to undergo changes that advance membrane maturation thereby shortening their lifespan, especially after in vivo fertilisation (Gillan and Maxwell, 1999). It was found that sorting prior to cryopreservation accelerated the maturation of sperm membranes and after co-incubation with oviducal cells in vitro, sorted frozen-thawed spermatozoa were released more rapidly than non-sorted (control) frozen-thawed spermatozoa. The potentially reduced lifespan of sorted frozen-thawed spermatozoa, and practical constraints on the number of spermatozoa that can be sorted for an insemination dose, makes insemination close to the site of fertilisation and time of ovulation critical for successful fertilisation. After treatment of ewes with GnRH to increase the precision of insemination in respect to the time of ovulation, there was no difference in pregnancy rate between ewes inseminated before, during or after the assumed time of ovulation. Furthermore, there was no difference in pregnancy rate after IU AI with similar doses of sorted frozen-thawed and non-sorted frozen-thawed spermatozoa in GnRH-treated ewes. The minimum dose of sorted frozen-thawed spermatozoa required for commercially acceptable pregnancy rates determined after IU AI was high (20 x 106 motile). Consequently, alternative methods for efficiently producing large numbers of offspring of a pre-determined sex using flow cytometrically sorted ram spermatozoa were investigated. Ram spermatozoa can be stored for short periods of time in a chilled state (liquid storage) or for an indefinite period of time in a frozen state (frozen storage; Salamon and Maxwell, 2000). The fixed location of the sperm sorter requires the need for transport of semen from the point of collection to the site of sorting and processing, but also from the sperm sorter site to the recipient females under artificial conditions. In this study, ram spermatozoa liquid stored for 24 h prior to sorting were efficiently sorted, frozen, thawed and after in vitro fertilisation and culture produced a high proportion of grade 1 blastocysts. Similarly, spermatozoa stored at reduced temperatures after sorting maintained high sperm quality for up to 6 days. Furthermore, frozen-thawed spermatozoa from rams and some non-human primates were successfully prepared for sorting and efficiently sorted producing spermatozoa with high quality in vitro parameters. The quality of frozen-thawed ram spermatozoa after sorting was such that successful re-cryopreservation after sorting was possible. Low numbers of frozen-thawed sorted and re-frozen and thawed spermatozoa were optimal for IVF and a high proportion of grade 1 in vitro embryos of a pre-determined sex were produced. These embryos were either transferred immediately or vitrified prior to transfer, extending the application of the sperm sexing technology further. The birth of lambs of pre-determined sex after transfer of both fresh and vitrified embryos derived from frozen-thawed sorted spermatozoa was achieved. The findings in this thesis suggest that sorted frozen-thawed ram spermatozoa may have more advanced membrane maturation state than non-sorted frozen-thawed spermatozoa, resulting in a decreased fertilizing lifespan in the female reproductive tract. Despite this, the use of sexed ram spermatozoa in a number of physiological states (fresh, liquid, frozen) with several different ARTs is possible in producing significant numbers of offspring of a pre-determined sex. Improved efficiency in both sperm sexing and associated reproductive technologies is required for commercialization to be achieved in the sheep industry.

Made available in DSpace on 2016-09-27T13:39:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-03-17. Added 1 bitstream(s) on 2016-09-27T13:45:06Z : No. of bitstreams: 1 000864722.pdf: 2629115 bytes, checksum: 66c11f771d4567d46705749086273afb (MD5) / Os procedimentos durante a sexagem pelo citômetro de fluxo garantem a acuidade de 85%, mas causam danos na viabilidade espermática que levam a baixa taxa de prenhez após os 90 dias. Os objetivos dessa Dissertação foram: 1) caracterizar a expressão gênica diferencial em transcriptoma de espermatozoides congelados pelo método convencional, sexados por centrifugação em gradiente de densidade ou sexados por citometria de fluxo; 2) comparar as taxas de clivagem e de blastocistos produzidos in vitro de bovinos utilizando sêmen convencional, sexado por citometria de fluxo ou por centrifugação em gradiente de densidade. Os grupos experimentais para sêmen e embriões foram: 1) grupo controle: sêmen convencional submetido ao gradiente de PercollTM 45/90% e respectivos embriões produzidos com esse sêmen; 2) grupo gradiente: centrifugação em gradiente de densidade e respectivos embriões produzidos com esse sêmen; 3) grupo citometria: sêmen sexado pelo citometro de fluxo, submetido ao mini gradiente de PercollTM 45/90% e respectivos embriões produzidos com esse sêmen. O RNA total foi extraído dos espermatozoides (20 x 10 6 células) obtendo-se 400 ng RNA/ 20 x 10 6 células/ animal, ou seja, 20 fg RNA por espermatozoide. Entretanto, não foi posível a construção da biblioteca de cDNA, provavelmente, devido ao alto grau de degradação do RNA. Os Complexos cumulus oócito (COCs) classificados como graus 1, foram aspirados de folículos antrais de 3 a 8 mm de diâmetro a partir de ovários de abatedouros e maturados durante 18 horas em estufa a 38,5°C, 100% de umidade e atmosfera de 5% de CO2 em ar. Os oócitos maturados serão colocados em contato com os espermatozoides previamente preparados para fecundação e incubados por 20 horas em 5% de CO2, na temperatura de 38,5°C. Os prováveis zigotos serão lavados por três vezes em meio SOF (meio sintético de fluido de oviduto) sem SFB e sem glicose e cultivados e transferidos... / The procedures for sexing by flow cytometry guarantee the accuracy of 85%, but cause damage in sperm viability leading to lower pregnancy rate after 90 days of pregnancy. The objectives of this study were: 1) to characterize differential gene expression in transcriptome of spermatozoa frozen by conventional method, sexed by density gradient centrifugation, by flow cytometry. 2) Compare blastocyst and cleavageratesproduced in vitro by conventional semen sexed by flow cytometry and by density gradient centrifugation. The experimental groups for semen and embryos were: 1) control group: conventional semen subjected to PercollTM gradient of 45/90% and their embryos produced with this semen, 2) gradientgroup: centrifugation in density gradient and their embryos produced with this semen, 3) cytometry group: semen sexed by flow cytometer, subjected to PercollTMmini gradient 45/90% and their embryos produced with this semen. Total RNA was extracted from sperm (20 x 10 6 cells) and it was possible to extract 400 ngRNA/ 20x10 6 cells/animal or it means, 20 fg of RNA per spermatozoa. However cDNA libraries were not built, probably due a high RNA degradation. The cumulus oocyte complexes (COCs) were classified as grade 1, aspirated from antral follicles with 3-8 mm in diameter, ovariesfromslaughterhouse were used and matured for 18 hours in an incubator at 38.5°C, 100% humidity and atmosphere of 5% of CO2 in air. The matured oocytes are going to be placed in contact with the prepared spermatozoa for fertilization and incubated for 20 hours in 5% CO2 at a temperature of 38.5 °C. Presumptive zygotes are going to be washed three times in SOF (half synthetic oviduct fluid) without FBS and without glucose, cultivated and transferred to plates with four wells containing 500 ul of the same medium used for washing zygotes after fertilization. Embryonic development was evaluated 7-8 days after fertilization. The cleavage and blastocysts rates were ...

Submitted by BRUNA CRISTINA JERONIMO null (bruna.jer@gmail.com) on 2016-09-27T23:44:59Z No. of bitstreams: 1 Dissertação Bruna - Correções Adriane versão final para imprimir.pdf: 2531830 bytes, checksum: 468a11d8d93e4d6cdb8212717e577790 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-09-29T18:29:14Z (GMT) No. of bitstreams: 1 jeronimo_bc_me_bot.pdf: 2531830 bytes, checksum: 468a11d8d93e4d6cdb8212717e577790 (MD5) / Made available in DSpace on 2016-09-29T18:29:14Z (GMT). No. of bitstreams: 1 jeronimo_bc_me_bot.pdf: 2531830 bytes, checksum: 468a11d8d93e4d6cdb8212717e577790 (MD5) Previous issue date: 2016-06-21 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Em contraste ao padrão de cromossomos sexuais de mamíferos (XX/XY), as aves apresentam um sistema de determinação sexual em que os machos representam o sexo homogamético (ZZ) e as fêmeas constituem o sexo heterogamético (ZW). Adicionalmente, embora mamíferos apresentem um mecanismo de compensação de dose, a inativação completa de um dos cromossomos Z não é observada em machos de aves e, portanto, estes possuem um maior nível de expressão de vários genes presentes nesse cromossomo. A despeito disso, um mecanismo ainda não completamente esclarecido de compensação de dose parcial em aves resulta em expressão equivalente entre os sexos para alguns genes do cromossomo Z. A região MHM (Male Hypermethylated), localizada no cromossomo Z de Galliformes, está associada a um padrão de hipermetilação em machos e hipometilação em fêmeas, levando à síntese de um RNA não-codificante longo (lncRNA) somente em fêmeas. A presença deste lncRNA é associada ao aumento da expressão de genes próximos à região MHM em fêmeas, o que parece resultar em uma compensação de dose local entre os sexos. Dado que, até o momento, segmentos MHM foram somente identificados em Galiformes e Anseriformes, o presente estudo visou isolar e caracterizar esta região em Galliformes (galinha doméstica, codorna européia, peru) e também em Struthioniformes (avestruz), Strigiformes (coruja-orelhuda, corujinha-do-mato, coruja-da-igreja, coruja-buraqueira), Piciformes (tucanuçu), Psittaciformes (arara-azul-grande) e Apodiformes (beija-flor-da-banda-branca, beija-flor-tesoura, beija-flor-preto). Indivíduos adultos e embriões com seis dias de desenvolvimento foram sexados com base em caracteres morfológicos e moleculares - por meio de PCR (Polymerase Chain Reaction) para amplificação de uma região intrônica dos genes CHD1-Z e CHD1-W, seguida de eletroforese em gel de agarose e poliacrilamida, análise SSCP e análise automatizada de fragmentos de DNA. Métodos de sexagem molecular mostraram-se adequados para identificação de machos e fêmeas de galinha doméstica, codorna européia, peru, tucanuçu, arara-azul-grande, coruja-orelhuda, corujinha-do-mato, coruja-buraqueira, beija-flor-da-banda-branca, beija-flor-tesoura e beija-flor-preto. Entretanto, as técnicas moleculares utilizadas não permitiram identificar diferenças entre machos e fêmeas de avestruz. Análises in silico da região MHM de galinha doméstica mostraram que esta se encontra localizada no braço curto do cromossomo Z, sendo constituída por 260Kb (chrZ:27,000,000-27,260,000) e alto conteúdo de CG. Esta região é delimitada por duas LINES (Long Interspersed Nuclear Elements) e possui múltiplos elementos repetitivos da classe LTR (Long Terminal Repeat), especialmente os pertencentes à família EVRL (Endogenous Retrovirus), denominados GGLTR5A. Com base em sua composição genômica, a região MHM de galinha doméstica foi subdividida em sub-regiões - denominadas de 1 (chrZ:27,176,712-27,260,282), 2 (chrZ:27,132,044-27,174,901), 3a (chrZ:27,094,512-27,132,043), 4 (chrZ:27,036,950-27,094,511) e 3b (chrZ:27,000,000-27,036,949) - que apresentam-se compostas por três unidades de repetições diferentes (denominadas de repeats 1, 2 e 3) flanqueadas por LTRs específicas. PCR multiplex em amostras de galinha doméstica levou à amplificação de dois fragmentos de DNA de aproximadamente 240 e 750 pares de bases, sendo o fragmento maior correspondente à repeat 1 da região MHM. Assim como observado para galinha doméstica, também foram gerados, via PCR, dois fragmentos de DNA de diferentes tamanhos associados à região MHM para as outras espécies de aves estudadas. Um maior nível de identidade (80-97%) foi observado entre a região MHM de galinha doméstica e as sequências nucleotídicas obtidas de codorna européia, peru e avestruz, o que demonstra que a presença de segmentos MHM não se restringe ao genoma de Galliformes e Anseriformes. Ensaios de digestão enzimática em DNA genômico de galinha doméstica, codorna européia e peru, por meio do uso de endonucleases de restrição sensíveis à metilação e dependentes de metilação (MspI, HpaII e McrBC), seguidos de amplificação de um fragmento de DNA associado à sub-região 1 MHM, evidenciaram padrões diferenciais de metilação dessa região entre os sexos, sendo hipometilada em fêmeas e hipermetilada em machos. Tais padrões diferenciais mostram-se potencialmente adequados para aplicação em testes de sexagem molecular em espécies de aves. / In contrast to the sexual chromosomes pattern found in mammals (XX/XY), birds present a sex determination system in which males represent the homogametic sex (ZZ) and females correspond to the heterogametic sex (ZW). Furthermore, although mammals present a dosage compensation mechanism, the complete inactivation of one Z chromosome is not observed in male birds and, therefore, they have a higher expression level of several genes that are found in this chromosome. Despite this, a mechanism of partial dosage compensation that was not clearly explained so far for birds results on an equivalent expression between sexes for some of the genes found at the Z chromosome. The MHM region (Male Hypermethylated), localized at the Z chromosome of Galliformes, is associated to a hypermethylation pattern in males and hypomethylation in females, which leads to the synthesis of a long non-coding RNA (lncRNA) only in females. The presence of this lncRNA is associated with a higher expression of genes that are located near to the MHM region in females, which seems to result in a local dosage compensation between sexes. As MHM segments were so far identified only in Galliformes and Anseriformes, the present study aimed to isolate and characterize this region on Galliformes (chicken, European quail, turkey) and also on Struthioniformes (ostrich), Strigiformes (striped owl, tropical screech-owl, barn owl, burrowing owl), Piciformes (toco toucan), Psittaciformes (hyacinth macaw), and Apodiformes (versicolored emerald, swallow-tailed hummingbird, black jacobin). Adult individuals and six-day embryos were sexed based on morphological and molecular characters - throughout PCR (Polymerase Chain Reaction) to amplify an intronic region of the CHD1-Z e CHD1-W genes, followed by agarose and polyacrylamide electrophoresis, SSCP analysis and automated fragment DNA analysis. Molecular sexing methodologies were useful for the identification of males and females of chicken, European quail, turkey, toco toucan, hyacinth macaw, striped owl, tropical screech-owl, burrowing owl, versicolored emerald, swallow-tailed hummingbird, and black jacobin. However, the applied techniques were not effective to identify differences between male and female ostriches. In silico analyses of the chicken MHM region showed that it is localized at the short arm of the Z chromosome and is constituted by 260Kb (chrZ:27,000,000-27,260,000) and a high CG content. This region is delimited by two LINES (Long Interspersed Nuclear Elements) and presents multiple repetitive elements of the LTR (Long Terminal Repeat) class, especially those of the EVRL (Endogenous Retrovirus) family, denominated GGLTR5A. Based on its genomic composition, the MHM region was subdivided into sub regions – denominated as 1 (chrZ:27,176,712-27,260,282), 2 (chrZ:27,132,044-27,174,901), 3a (chrZ:27,094,512-27,132,043), 4 (chrZ:27,036,950-27,094,511), and 3b (chrZ:27,000,000-27,036,949) - that are composed by three different repeat units (denominated as repeats 1, 2 e 3) flanked by specific LTRs. Multiplex PCR on chicken samples resulted in the amplification of two different size DNA fragments of around 240 and 750 base pairs, and the larger fragment corresponds to the repeat 1 of the MHM region. As observed for chicken, two different DNA fragments associated to the MHM region were also generated, by PCR, for the other studied species. A higher identity index (80-97%) was recognized between the chicken MHM region and the obtained nucleotide sequences of European quail, turkey and ostrich, which evidences that the presence of MHM segments is not restricted to the Galliformes and Anseriformes genomes. Enzymatic digestion assays in genomic DNA samples of chicken, European quail and turkey, through the use of methylation sensitive and methylation dependent restriction endonucleases (MspI, HpaII e McrBC), followed by the amplification of a DNA fragment associated to the sub region 1 MHM, showed differential methylation patterns between sexes - hypomethylated in females and hypermethylated in males. These differential patterns are potentially applicable for molecular sexing tests in bird species. / CNPq: 131152/2014-9

Gender of animal offspring is of great interest to farmers where gender selection is achieved via the separation of male-bearing from female-bearing sperms prior to performing artificial insemination. A start-up company (Ab Biotech Inc.) has developed a technique for gender selection based on the production of an intracellular single-domain antibody (sdAb) using the bacterium Escherichia coli capable of sexing bovine sperm. The purpose of this research was to provide a recommendation to Ab Biotech Inc. for the lysis of E. coli. An efficient lysis technique was required in order to release the intracellular sdAb. In the dairy industry, sexing for female calves is preferred since male calves are not useful for the purpose of milk production. Multiple lysis techniques were tested in order to provide a feasible recommendation for Ab Biotech Inc. These techniques included high pressure homogenization, sonication, bead milling and enzymatic/chemical lysis using lysozymes and Triton X-100. Required lysis time, extent of lysis and potential operating costs were contributing factors for determining an optimal technique. The extent of lysis was determined by quantifying the total amount of released protein using SDS-PAGE densitometry. It was recommended to choose bead milling for potential process upscaling since a large amount of fractional lysis (0.70) was obtained over a short amount of lysis time (3 min) with an inexpensive ($9.50/kg) 0.3 mm mixture of glass beads.

No / Sex assessment is key when investigating human remains either from medicolegal contexts or archaeological sites. Sex is usually assessed by examination of the skull and pelvis, but this may not always be possible if skeletal material is fragmented or incomplete. The present study investigated the potential for using carpals to assess sex, utilizing one hundred individuals of known-sex from the Christ Church, Spitalfields Collection, curated at the Natural History Museum (London). A series of newly-defined measurements are applied to all eight carpals. Inter- and intra- observer error tests show that all measurements are satisfactorily reproduced by the first author and another observer. Paired t-tests to investigate side asymmetry of the carpals reveal that some, but not all, measurements are consistently larger on the right hand side than the left. Independent t-tests confirm that all carpals are sexually dimorphic. Univariate measurements produce accuracy levels that range from 64.6 to 84.7%. Stepwise discriminant function analysis, devised separately for left and right sides, provides reliable methods for assessing sex from single and multiple carpals, with an accuracy range of 71.7 to 88.6%. All functions derived are tested for accuracy on a sample of twenty additional individuals from the Christ Church, Spitalfields Collection.

The mosquito Aedes aegypti is responsible for 50 million dengue fever infections in humans each year. A novel control strategy, named RIDL (Release of Insects carrying a Dominant Lethal) relies on releasing large numbers of genetically sterile male insects in order to control pest populations. This thesis describes the development of new tools to improve the efficiency of RIDL against Ae. aegypti and assessment of candidate strains for field release. <strong>Chapter 3</strong> describes a new selection system for Ae. aegypti based on ethanol susceptibility conferred by the alcohol dehydrogenase gene (Adh) from Drosophila melanogaster. I observed that the susceptibility of Ae. aegypti larvae to ethanol can be triggered by expression of Adh in larvae. <strong>Chapters 4</strong> and <strong>5</strong> focus on RIDL strains with a genetic sexing mechanism, for easy and stringent selection for males before mass-releases, eliminating disease- transmitting females. In <strong>Chapter 4</strong>, I describe the creation of a late-acting sexing strain of Ae. aegypti based on the Ae. aegypti doublesex (Aedsx) alternative splicing system. In <strong>Chapter 5</strong>, I describe an attempt to create an early-acting sexing system. Killing the females of the release generation early would free space and resources for the production of males. This was done by combining the Adh gene and the Aedsx alternative splicing system described in <strong>Chapters 3</strong> and <strong>4</strong>. <strong>Chapter 6</strong> reports the results of a comparison, in terms of quality and productivity, between an existing Ae. aegypti RIDL strain and a wild-type control. Results showed equivalent female quality and productivity between the two strains, while RIDL males were less fertile in comparison with wild-type males. RIDL eggs also seemed more susceptible to long- term storage. The results of this work show promise for development of novel RIDL strains that may be used in the field to control disease-transmitting mosquitoes.

Orientador: Adriane Pinto Wasko / Banca: Carlos Roberto Teixeira / Banca: Renata Cristina Batista Fonseca / Resumo: Entre os animais silvestres envolvidos em tráfico e comércio ilegal no Brasil, as aves compreendem um dos grupos mais atingidos, especialmente devido a características como canto e colorido das penas. Atualmente, análises genéticas representam uma das formas mais eficazes de gerar dados para solucionar e minimizar os resultados de crimes ambientais e comércio ilegal de animais silvestres. Desta forma, este trabalho teve como objetivo realizar análises genéticas de sexagem em diversas aves, incluindo espécies comumente associadas ao tráfico de animais e apreendidas pela Polícia Ambiental e pelo IBAMA (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis), e gerar um material de divulgação científica sobre tráfico e conservação deste grupo de vertebrados. Amostras de DNA foram obtidas de penas e sangue de 124 exemplares de 53 espécies das famílias Accipitridae, Cacatuidae, Cardinalidae, Cariamidae, Columbidae, Cuculidae, Emberezidae, Falconidae, Icteridae, Musophagidae, Psittacidae, Ramphastidae, Sturnidae, Thraupidae, Tinamidae, Trochilidae e Turdidae, mantidas junto a um Centro de Recepção, Triagem e Reabilitação de Animais Silvestres - CETAS (ONG Instituto Floravida, Botucatu, SP), a um Centro de Medicina e Pesquisa em Animais Silvestres - CEMPAS (Faculdade de Medicina Veterinária e Zootecnia, UNESP, Botucatu, SP) e a dois criadouros científicos de fauna silvestre para fins de conservação (Criadouro C.A., Itatiba, SP e Criadouro Poços de Caldas, Poços de Caldas, MG). Perfis genéticos sexo-específicos foram gerados por meio da amplificação de segmentos de DNA dos cromossomos Z e W. Um conjunto de primers que se anelam a regiões de éxons dos genes CHD-Z e CHD-W (chromo helicase-DNA binding) e que amplificam uma região de íntron que difere em tamanho entre os dois genes foi utilizado em PCR (Polymerase Chain Reaction) e os produtos de amplificação foram visualizados em gel de agarose 2%. ... / Abstract: Birds represent a large part of the animals associated to illegal trade and commerce in Brazil, mainly due to some characteristics as song and feathers colors. Nowadays, genetic analyses comprehend one of the most efficient approaches to generate data in order to solve and minimize the results of environmental crimes and illegal trade of wild animals. Therefore, this work intent to perform sex identification genetic analyses in several birds, including species that are commonly associated to illegal animal trade and apprehended by the Environmental Policy and by IBAMA (Brazilian Institute of Environment and Renewable Natural Resources), and generate a scientific broadcasting material about illegal trade and conservation of this vertebrate group. DNA samples were obtained from feathers and blood of 124 individuals that belong to 53 species of the families Accipitridae, Cacatuidae, Cardinalidae, Cariamidae, Columbidae, Cuculidae, Emberezidae, Falconidae, Icteridae, Musophagidae, Psittacidae, Ramphastidae, Sturnidae, Thraupidae, Tinamidae, Trochilidae, and Turdidae, maintained by a Wildlife Reception, Screening and Rehabilitation Center (Floravida Institute NGO), a Wildlife Animal Health and Research Center (Faculty of Veterinary Medicine and Zootechny, São Paulo State University Botucatu, SP) and to two scientific breeding grounds of wild fauna based on conservation purposes (Criadouro C.A., Itatiba, SP and Criadouro Poços de Caldas, Poços de Caldas, MG). Sex-specific genetic profiles were generated by the amplification of DNA segments of the Z and W chromosomes. A primer set that anneal to exon regions of the CHD-Z and CHD-W (chromo helicase-DNA binding) genes and that amplify an intron region that differ in size between the two genes was used in PCR (Polymerase Chain Reaction) and the amplification products were visualized in 2% agarose gel. Two different size DNA fragments were evidenced for females and a single fragment was ... / Mestre

<p>The white-tailed eagle is a formerly threatened raptor that is commonly used as a flagship and indicator species in conservation work. This thesis uses molecular genetic methods to study sex determination of nestlings, genetic variability, population structure and phylogeography of the white-tailed eagle.</p><p>Fourteen microsatellite markers were developed and tested for the white-tailed eagle.</p><p>A method to sex white-tailed eagle nestlings in the field is presented. The method is based on just one tarsus measure, and is suitable for situations where a single person is handling the nestlings alone in a treetop.</p><p>Most European white-tailed eagle populations underwent extreme declines during the 20th century. The results presented here show that bottlenecked populations have maintained significant levels of genetic diversity. Gene flow between regions is not a main explanation for this, as indicated by both genetic and ringing data. Instead, the long generation time of white-tailed eagles has acted as an intrinsic buffer against rapid loss of genetic diversity. Additionally, local conservation led to protection of more genetic diversity than if conservation had focused on the large remnant population in Norway.</p><p>Mitochondrial DNA of white-tailed eagles is structured in two main clades with a predominantly eastern and western Eurasian distribution. The clades likely correspond to separate Ice Age refugia but do not grant classification as evolutionary significant units given their current extensive overlap across large parts of Eurasia.</p><p>Microsatellite variation was studied in populations across Eurasia. Variability was rather constant across the continent, but clearly lower on Iceland and Greenland. This is best explained by founder effects during their colonisation, but only weak bottlenecks during colonisation of and persistence on the continent. Current population differentiation between Europe and eastern Eurasia is not compatible with a zero gene flow model but requires some amount of gene flow over evolutionary time scales.</p>

The white-tailed eagle is a formerly threatened raptor that is commonly used as a flagship and indicator species in conservation work. This thesis uses molecular genetic methods to study sex determination of nestlings, genetic variability, population structure and phylogeography of the white-tailed eagle. Fourteen microsatellite markers were developed and tested for the white-tailed eagle. A method to sex white-tailed eagle nestlings in the field is presented. The method is based on just one tarsus measure, and is suitable for situations where a single person is handling the nestlings alone in a treetop. Most European white-tailed eagle populations underwent extreme declines during the 20th century. The results presented here show that bottlenecked populations have maintained significant levels of genetic diversity. Gene flow between regions is not a main explanation for this, as indicated by both genetic and ringing data. Instead, the long generation time of white-tailed eagles has acted as an intrinsic buffer against rapid loss of genetic diversity. Additionally, local conservation led to protection of more genetic diversity than if conservation had focused on the large remnant population in Norway. Mitochondrial DNA of white-tailed eagles is structured in two main clades with a predominantly eastern and western Eurasian distribution. The clades likely correspond to separate Ice Age refugia but do not grant classification as evolutionary significant units given their current extensive overlap across large parts of Eurasia. Microsatellite variation was studied in populations across Eurasia. Variability was rather constant across the continent, but clearly lower on Iceland and Greenland. This is best explained by founder effects during their colonisation, but only weak bottlenecks during colonisation of and persistence on the continent. Current population differentiation between Europe and eastern Eurasia is not compatible with a zero gene flow model but requires some amount of gene flow over evolutionary time scales.

In Deutschland werden jährlich über 40 Millionen männliche Eintagsküken aus Legelinien aufgrund vorrangig wirtschaftlicher Interessen getötet. Dies stellt sowohl ein ethisches als auch ein tierschutzrechtliches Problem dar (ANON. 2006, IDEL 2007). Gerade vor dem Hintergrund aktueller politischer Entscheidungen (MUNLV NRW 2013, NI MELV 2014) besteht ein Bedarf an Alternativen zur Tötung männlicher Eintagsküken. Verschiedene Lösungsansätze wie z. B. das Zweinutzungshuhn (ICKEN et al. 2013) oder aber die Mast männlicher Geschwisterhühner (KAUFMANN und ANDERSSON 2013) sind derzeit aus ökonomischen und ökologischen Gründen nicht flächendeckend realisierbar. Eine weitere Möglichkeit bietet die In-ovo-Geschlechtsbestimmung. Hierbei wird das embryonale Geschlecht bereits vor dem Schlupf identifiziert; nachfolgend können die Eier mit männlichen Embryonen aussortiert werden. Um sowohl ethischen als auch tierschutzrechtlichen Aspekten Genüge zu tun, sollte die Geschlechtsidentifikation dabei vor Einsetzen des embryonalen Schmerzempfindens stattfinden (Tag 10 + 12 h der Bebrütung; CLOSE et al. 1997). Ziel der vorliegenden Arbeit war die Entwicklung einer verlässlichen Methode zur In-ovo-Geschlechtsbestimmung anhand geschlechtsspezifischer Differenzen im Hormongehalt der Allantoisflüssigkeit sieben bis zehn Tage alter Hühnerembryonen. Nachfolgend wurde der Einfluss der Geschlechtsbestimmung auf die embryonale Entwicklung, Schlupferfolg, Aufzucht sowie die Leistungsparameter der adulten Tiere analysiert. Im Rahmen der ersten Teilstudie erfolgte die Beprobung von n = 750 Eiern des Braunlegehybrids Lohmann Brown (LB, Lohmann Tierzucht GmbH, Deutschland). Der minimalinvasiven Entnahme von Allantoisflüssigkeit folgte die Untersuchung auf 17β-Östradiol (E2), Östronsulfat (E1S) und Testosteron mittels an das Haushuhn angepassten Enzymimmunoassays (ELISA). Es konnten sowohl für E2 als auch für E1S signifikante (p < 0,01) geschlechtsspezifische Differenzen in der Allantoisflüssigeit von neun und zehn Tage alten Embryonen nachgewiesen werden. Die Testosteronkonzentration hingegen zeigte an keinem der untersuchten Tage geschlechtsabhängige Unterschiede und erwies sich somit für die In-ovo-Geschlechtsbestimmung als ungeeignet. Die statistische Auswertung ergab, dass die Bestimmung von E1S eine frühere und genauere Geschlechtsidentifikation ermöglicht als die von E2. Der für E1S festgelegte Grenzwert erreicht bei neun Tage alten Embryonen eine 86%ige Sensitivität und 83%ige Spezifität. In der zweiten Teilstudie wurde die zuvor etablierte Technik der Geschlechtsbestimmung mittels E1S an 8 + 4 h (n = 2420) und 9 + 4 h (n = 2850) Tage alten Embryonen der Herkunft LB sowie an n = 150 9 + 4 h alten Embryonen des Weißlegehybrids Lohmann Selected Leghorn (LSL, Lohmann Tierzucht GmbH, Deutschland) überprüft. Das Geschlecht der 8 + 4 h Tage alten Embryonen konnte zu 84 % korrekt identifiziert werden. Dieser Wert stieg bei 9 + 4 h Tage alten Embryonen auf 98 % (LB) bzw. 100 % (LSL) an. Im Vergleich zu einer unbehandelten Kontrollgruppe (n = 5258) wurde die Schlupfrate durch die Entnahme von Allantoisflüssigkeit um 1,4 - 3,5 (LB) bzw. 12,7 Prozentpunkte (LSL) reduziert. Nachfolgend wurden 150 Tiere der Versuchsgruppe und 80 Tiere der Kontrollgruppe für eine Aufzuchtperiode von 17 Wochen eingestallt. Hierbei zeigten sich hinsichtlich des Körpergewichtes signifikante (p < 0,05) Unterschiede zwischen Versuchs- und Kontrollgruppe in Woche 4 und 6, wobei die Zunahmen in der Versuchsgruppe geringer waren. Anschließend wurde die Leistung von 120 Tieren der Versuchsgruppe und 60 Tieren der Kontrollgruppe bis Lebenswoche 33 bezüglich Legeleistung, Eigewicht, Körpergewicht sowie Futterverbrauch analysiert. Bei keinem der untersuchten Parameter konnten signifikante Unterschiede zwischen den Gruppen festgestellt werden (p > 0,05). Die Resultate der vorliegenden Arbeit zeigen, dass eine verlässliche Geschlechtsbestimmung in ovo bei 9 + 4 h Tage alten Hühnerembryonen mithilfe einer Bestimmung der E1S-Konzentration in der Allantoisflüssigkeit möglich ist; zudem ist die beschriebene Methode bei verschiedenen Legelinien anwendbar. Die Entnahme von Allantoisflüssigkeit führt zwar zu einer minimalen Reduktion der Schlupfrate, bei adulten Legehennen kommt es jedoch zu keiner Beeinträchtigung der Produktionsleistung. Demnach erfüllt das etablierte Verfahren alle Grundvoraussetzungen für eine Anwendung in kommerziellen Brütereien. Da die Geschlechtsbestimmung vor Einsetzen des embryonalen Schmerzempfindens erfolgt, kann sie somit als Grundlage für eine ethisch vertretbare Alternative zum Töten männlicher Eintagsküken angesehen werden. / In Germany about 40 million day-old male chicks are culled each year predominantly because of economic reasons. From the animal welfare as well as the ethical point of view this is a problematic situation (ANON. 2006, IDEL 2007). Particularly with regard to current political decisions (MUNVL NRW, NI MELV 2014) alternatives to the culling of male day-old chicks are required. Different approaches such as a dual-purpose breed (ICKEN et al. 2013) or the fattening of male layer-hybrids (KAUFMANN and ANDERSSON 2013) are not ubiquitous marketable at present due to economic and ecological reasons. In ovo sexing represents another option; the embryonic gender is determined before hatch and the eggs containing male embryos can be eliminated subsequently. To comply with ethical and animal welfare aspects, the sexing should take place before the onset of embryonic pain perception (embryonic day 10 + 12 h; CLOSE et al. 1997). Aim of this thesis was the development of a reliable method for in ovo gender identification with the help of sex-specific differences in the hormone concentration of the allantoic fluid of seven to ten day old chick embryos. Subsequently, the influence of gender identification on embryonic development, hatching rate, rearing as well as production performance of the adult hens was analysed. Within the first study n = 750 eggs of the brown layer-hybrid Lohmann Brown (LB; Lohmann Tierzucht GmbH, Germany) were sampled for allantoic fluid. After the minimally invasive withdrawal the allantoic fluid was analysed via enzyme immunoassays (ELISA) adapted to domestic chicken for 17β-oestradiol (E2), oestrone sulphate (E1S) and testosterone. With regard to E2 and E1S, significant (P < 0.01) sex-specific differences were observed in the allantoic fluid of nine and ten day old embryos. Testosterone on the other hand displayed no gender-related variances on any of the analysed days. Therefore, it proved to be unsuitable for gender identification using the method applied in this study. Statistical analysis showed that the analysis of E1S allows an earlier and more accurate sexing than the E2-assay. The limit value determined for E1S has a sensitivity of 86 % and a specificity of 83 % for nine day old embryos. The previously established method for gender identification via E1S detection in the allantoic fluid was verified with a larger number of samples in the second study. The allantoic fluid of day 8 + 4 h (n = 2420) and day 9 + 4 h (n = 2850) old LB embryos as well as n = 150 day 9 + 4 h old embryos of the white layer-hybrid Lohmann Selected Leghorn (LSL; Lohmann Tierzucht GmbH, Germany) was analysed. For day 8 + 4 h old embryos the sex was correctly identified in 84 %. The accuracy of gender prediction increased for day 9 + 4 h old embryos up to 98 % (LB) and 100 % (LSL). Compared to an untreated control group (n = 5258) sampling of allantoic fluid reduced the hatching rate by 1.4 - 3.5 (LB) and 12.7 points of percentage (LSL). In the following, 150 animals of the experimental group and 80 animals of the control group were reared for a period of 17 weeks. With regard to the body weight significant differences (P < 0.05) were observed in weeks 4 and 6, with the animals of the experimental group having a lower body weight. Subsequently the production performance of 120 hens from the experimental and 60 hens from the control group was analysed up to an age of 33 weeks. With respect to egg production, egg weight, body weight and feed consumption no significant differences (P > 0.05) were observed between the groups. The results of this thesis demonstrate that a reliable in ovo sexing of day 9 + 4 h old chicken embryos is possible via the measurement of E1S in the allantoic fluid; additionally the method is not limited to a certain layer strain. The sampling of allantoic fluid reduces the hatching rate only marginally. The production performance of adult hens on the other hand is not affected. Therefore, the described technique fulfils all the basic requirements for an alternative method to the culling of day-old male layer chicks. Because gender identification takes place before the onset of embryonic pain perception it can serve as the basis for an ethical alternative to the culling of male day-old chicks from layer-hybrids.