Submitted by admin tede (tede@pucgoias.edu.br) on 2017-11-10T16:06:34Z No. of bitstreams: 1 KELLER GABRIEL MARTINS.pdf: 1151760 bytes, checksum: 3a640f73b5ee5ea85d89f69f6ccd9aa9 (MD5) / Made available in DSpace on 2017-11-10T16:06:34Z (GMT). No. of bitstreams: 1 KELLER GABRIEL MARTINS.pdf: 1151760 bytes, checksum: 3a640f73b5ee5ea85d89f69f6ccd9aa9 (MD5) Previous issue date: 2017-08-04 / The determination of fetal sex using maternal plasma is a noninvasive prenatal diagnostic test (NIPDT), offered to pregnant women, especially those with an increased risk of having children with conditions X-linked inheritance. Because it is a non-invasive technique, it presents a greater advantage over other methods of invasive prenatal diagnosis such as amniocentesis, cordocentesis and biopsy of chorionic villi. The use of cell free fetal DNA (cffDNA) in maternal plasma has become a promising alternative for the diagnosis of noninvasive and early sex determination. The porpuse this study is to conduct a qualitative test in pregnant women with gestational age ranging from 6 to 20 weeks using the noninvasive prenatal test for the early sex determination of fetal by the real time PCR technique. Twenty-one healthy pregnant women, over 18 years of age, single gestation and attended at the Gynecology and Obstetrics Clinic of the Unigen Laboratory belonging to the Private Health Care Network of the City of Goiânia-GO were selected. After venous blood collection, plasma was separated and frozen (-20 ° C). The material to be analyzed followed the laboratory of the company Qiagen® located in São Paulo, SP, where DNA extraction and purification were performed using fully automated equipment (QIAcube®, with the QIAamp® DNA Micro kit, using the protocol "Purification of viral nucleic acids from large body-fluid samples", according to the manufacturer's specifications), and then the quantitative PCR technique (Rotor-Gene® Qiagen) was run for specific Y-sequence amplification. Of the 21 pregnant women selected, two participants aborted and were excluded from the study. The results showed that, in the 19 cases analyzed, comparing the results of qPCR with fetal sex determined by ultrasonography (USG), 7/19 (36.8%) pregnancies with positive results for the Y chromosome (determining the male sex) and 11/19 (57.9%) pregnancies with a negative result for the Y chromosome (determining the female sex) and only one gestation (5.3%) presented false-negative results for males. The analysis of the concordance index, between the results of the qPCR and the USG, found a concordance of 0.89. These results confirmed a good sensitivity and specificity of the method for the gestation period studied (mean of 12 weeks), indicating that this procedure should be used in the medical routine as an auxiliary tool in cases where fetal sexing becomes necessary for health fetal and/or decreased parents anxiety. / A determinação do sexo fetal utilizando o plasma materno é um teste de diagnóstico pré-natal não invasivo (DPIN), oferecido as gestantes, principalmente para aquelas com risco aumentado de terem crianças com doenças ligadas ao sexo. Por se tratar de uma técnica não invasiva, apresenta-se com maior vantagem sobre outros métodos de diagnóstico pré-natal invasivos como a amniocentese, cordocentese e a biópsia de vilosidades coriônicas. O diagnóstico pré-natal (DPN) tem sido importante no acompanhamento de gestações com anormalidades fetais, além de permitir um planejamento mais adequado para o parto e de cuidados neonatais específicos. Dessa forma, o DPN tem sido estabelecido na prática obstétrica moderna e integra um conjunto de procedimentos para identificar uma anormalidade no feto durante a gravidez. Diversas pesquisas têm buscado a utilização de novas tecnologias para o diagnóstico pré-natal não invasivo (DPNI). O uso de DNA fetal livre (cffDNA) no plasma materno passou a ser uma alternativa promissora para diagnóstico do sexo não invasivo e precoce. O objetivo do presente trabalho é realizar um estudo qualitativo em pacientes gestantes, com idade gestacional variando de 6 a 20 semanas utilizando o teste pré-natal não invasivo para a determinação precoce do sexo fetal pela técnica de qPCR. Foram selecionadas 21 gestantes saudáveis, maiores de 18 anos e gestação única e atendidas em clínica de ginecologia e obstetrícia do Centro de Diagnóstico Clínico UNIGEN pertencente à Rede Privada de Atendimento à Saúde da Cidade de Goiânia-GO. Após a coleta de sangue venoso, houve a separação e o congelamento do plasma (-20°C). O material a ser analisado seguiu para o laboratório da empresa Qiagen® localizado em São Paulo-SP, onde foram realizadas a extração e purificação do DNA utilizando equipamento automatizado (QIAcube®, com o kit QIAamp® DNA Micro, empregando-se o protocolo “Purification of viral nucleic acids from large body-fluid samples”, conforme especificações do fabricante), e em seguida foi ralizada a técnica de PCR quantitativa (Rotor-Gene® Qiagen) para amplificação de sequência Y específica. Das 21 gestantes selecionadas, duas participantes abortaram, sendo dessa forma excluídas do estudo. Os resultados revelaram que dos 19 casos analisados, ao comparar os resultados da qPCR com o sexo fetal determinado pela ultrassonografia (USG), 7/19 (36,8%) gestações com resultados positivos para o cromossomo Y (determinando o sexo Masculino) e 11/19 (57,9%) gestações com resultado negativo para o cromossomo Y (determinando o sexo Feminino) e apenas uma gestação (5,3%) apresentou resultado falso-negativo para o sexo masculino. A análise do índice de concordância, entre os resultados da qPCR com a USG foi de 0,89. Esses resultados confirmaram uma boa sensibilidade e especificidade do método para o período de gestação estudado (média de 12 semanas), indicando que este procedimento deve ser utilizado na rotina médica como ferramenta auxiliar nos casos onde a sexagem fetal torna-se necessária para tratamento fetal e/ou diminuição da ansiedade dos pais.

<p>In this thesis, genetic methods for individual identification and sex determination of wolverines from non-invasive samples were developed and applied in genetic monitoring of Scandinavian wolverine populations. Paternity and mating system of wolverines were studied by combining genetic analysis with telemetry data. Moreover, the possibility to obtain DNA from claws left on tanned carnivore hides was investigated.</p><p>Non-invasive genetic sampling was effective in revealing important population parameters. For the subpopulation in southern Norway, a population size of approximately 90 individuals, an equal sex ratio and similar levels of genetic diversity as in the main Scandinavian population were revealed. Genetic erosion in this small population has likely been counteracted by immigration of individuals from the main population since its re-establishment around 1970.</p><p>During the 1990s, two areas in east-central Sweden were colonised by wolverines. In a survey comprising 400 non-invasive samples collected during five winters, a total of 22 wolverines were detected. Genetic data suggest that inbreeding has occurred in both areas and that the two populations were founded by as few as 2-4 individuals. These findings suggest that gene flow from the main population is crucial for their survival even in a short time perspective. The detection of occasional stray individuals from the main population shows that this is indeed feasible. </p><p>Paternity analysis of 145 wolverine offspring in northern Sweden and southern Norway confirmed a polygamous mating system in wolverines. Breeding pair formation was generally consistent with the territories held by males and females, i.e. breeding pairs had overlapping territories. In the majority of litters, siblings were assigned the same father, thus indicating that multiple paternity is rare. </p><p>Tanning is a common form of preservation of mammalian specimens that normally precludes genetic analysis. Nevertheless, I demonstrate the possibility to successfully extract and amplify DNA from claws left on tanned carnivore hides.</p>

In this thesis, genetic methods for individual identification and sex determination of wolverines from non-invasive samples were developed and applied in genetic monitoring of Scandinavian wolverine populations. Paternity and mating system of wolverines were studied by combining genetic analysis with telemetry data. Moreover, the possibility to obtain DNA from claws left on tanned carnivore hides was investigated. Non-invasive genetic sampling was effective in revealing important population parameters. For the subpopulation in southern Norway, a population size of approximately 90 individuals, an equal sex ratio and similar levels of genetic diversity as in the main Scandinavian population were revealed. Genetic erosion in this small population has likely been counteracted by immigration of individuals from the main population since its re-establishment around 1970. During the 1990s, two areas in east-central Sweden were colonised by wolverines. In a survey comprising 400 non-invasive samples collected during five winters, a total of 22 wolverines were detected. Genetic data suggest that inbreeding has occurred in both areas and that the two populations were founded by as few as 2-4 individuals. These findings suggest that gene flow from the main population is crucial for their survival even in a short time perspective. The detection of occasional stray individuals from the main population shows that this is indeed feasible. Paternity analysis of 145 wolverine offspring in northern Sweden and southern Norway confirmed a polygamous mating system in wolverines. Breeding pair formation was generally consistent with the territories held by males and females, i.e. breeding pairs had overlapping territories. In the majority of litters, siblings were assigned the same father, thus indicating that multiple paternity is rare. Tanning is a common form of preservation of mammalian specimens that normally precludes genetic analysis. Nevertheless, I demonstrate the possibility to successfully extract and amplify DNA from claws left on tanned carnivore hides.

The PhD research studied two aspects in tilapia, firstly the analysis of sex determination in Nile tilapia (evidence of complex sex-determining systems) and secondly the genetic management of the tilapia species, using different genomic analysis approaches. This research started with the development of two techniques: minimally invasive DNA sampling from fish mucus, which was found to be suitable for standard genotyping and double-digest restriction-site associated DNA sequencing – ddRADseq; and pre-extraction pooling of tissue samples for ddRADseq (BSA-ddRADseq), which was found to be suitable for identifying a locus linked to a trait of interest (sex in this case). The first molecular evidence concerning the sex determination in genetically improved farmed tilapia (GIFT) was described using BSA-ddRADseq. Given the multiple stock origin of GIFT, surprisingly only a single locus (in linkage group 23) was found to be associated with the phenotypic sex across the population. The first evidence of LG23 influence on phenotypic sex in the Stirling population of Nile tilapia was also found. Different combinations of estrogen hormones and high temperature were tested for feminising Nile tilapia: a combined treatment of estrogen hormone and high temperature was found to be more efficient in feminising Nile tilapia than the estrogen alone. A set of species-diagnostic SNP markers were tested which were found to be suitable to distinguish pure species (O. niloticus, O. mossambicus and O. aureus), and these were used to analyse species contribution to GIFT and a selected tilapia hybrid strain. The results of the current research added novel information to our understanding of sex determination in Nile tilapia, which will be helpful in the development of marker-assisted selection in GIFT and other Nile tilapia strains towards the production of all male offspring. The methods developed also have broader applicability in genetic and genomics research.

No description available.

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.

The aim of the current thesis was the analysis of the genetics of sex determination of farmed fish with sexual dimorphism, using Next Generation Sequencing. Three different species of farmed fish with sex-determining systems of varying complexity were studied. Both full-sibs and more distantly related specimens of Atlantic halibut (Hippoglossus hippoglossus), Nile tilapia (Oreochromis niloticus) and European sea bass (Dicentrarchus labrax) were used for this study. Application of Restriction-site Associated DNA sequencing (RAD-seq) and double digest Restriction-site Associated DNA sequencing (ddRAD-seq), two related techniques based on next generation sequencing, allowed the identification of thousands of Single Nucleotide Polymorphisms (SNPs; &gt; 3,000) for each of the above species. The first SNP-based genetic maps for the above species were constructed during the current study. The first evidence concerning the location of the sex-determining region of Atlantic halibut is provided in this study. In the case of Nile tilapia both novel sex-determining regions and fine mapping of the major sex-determining region are presented. In the study of European sea bass evidence concerning the absence of a major sex-determining gene was provided. Indications of putative sex-determining regions in this species are also provided. The results of the current thesis help to broaden current knowledge concerning sex determination in three important farmed fish. In addition the results of the current thesis have practical applications as well, towards the production of mono-sex stocks of those species for the aquaculture industry.

Ο Ευρωπαϊκός Θαλασσοκόρακας (Phalacrocorax aristotelis) περιλαμβάνει τρία υποείδη που διαχωρίζονται με βάση μορφολογικές και συμπεριφορικές διαφορές και εξαπλώνονται σε διαφορετικές γεωγραφικές περιοχές. Το ατλαντικό υποείδος, P. a. aristotelis, κατά την αναπαραγωγική του περίοδο, εξαπλώνεται στον Ατλαντικό από τη Β. Ρωσσία μέχρι τις ατλαντικές ακτές της Ιβηρικής χερσονήσου, το υποείδος P. a. riggenbachi διαβιεί στις ακτές της Β. Αφρικής και το μεσογειακό (Phalacrocorax aristotelis desmarestii) θεωρείται ενδημικό υποείδος της Μεσογείου και της Μαύρης Θάλασσας. Η βιολογία και η οικολογία του μεσογειακού θαλασσοκόρακα δεν είναι μελετημένη, ιδιαίτερα στις ανατολικές περιοχές της εξάπλωσής του, παρότι η περιοχή του βόρειου Αιγαίου περιλαμβάνεται στις σημαντικότερες περιοχές αναπαραγωγής του είδους. Η παρούσα διδακτορική διατριβή, αποτελεί την πρώτη μελέτη σχετικά με την οικολογία της αναπαραγωγής και την ανάλυση της γενετικής δομής του μεσογειακού θαλασσοκόρακα σε αποικίες του Αιγαίου. Συγκεκριμένα, μελετώνται τέσσερα θέματα της βιολογίας του υποείδους: (1) η αναπαραγωγική επιτυχία και οι πιθανοί περιβαλλοντικοί παράγοντες που ενδέχεται να την επηρεάζουν, (2) η αναλογία του φύλου των νεοσσών, (3) οι διατροφικές συνήθειες κατά την αναπαραγωγική περίοδο, και (4) η γενετική δομή αναπαραγωγικών πληθυσμών του Αιγαίου, καθώς και οι φυλογεωγραφικές σχέσεις μεταξύ τους και με άλλους μη ελληνικούς πληθυσμούς. / The European Shag (Phalacrocorax aristotelis) is currently divided in three subspecies based on plumage differences, non-overlapping distributions and phenology. The nominate subspecies, P. a. aristotelis, has a breeding distribution from northern Russia to the Atlantic coast of Iberia, P. a. riggenbachi is found along the northern African coasts and the Mediterranean Shag (Phalacrocorax aristotelis desmarestii) is considered an endemic subspecies of the Mediterranean and the Black Seas. The Mediterranean subspecies’ biology and ecology are poorly studied, especially in the eastern part of its distribution, despite the fact that North Aegean Sea (Greece) is considered one of the most important regions for its reproduction. This study presents the first results regarding the study of its reproduction ecology and genetic structure in colonies from the Aegean Sea region. Specifically, four aspects of the its biology are addressed here: (1) breeding success and the possible ecological factors that may affect it, (2) the sex ratio of fledglings, (3) feeding habits during reproduction, and (4) the genetic structure of breeding populations in the Aegean and their phylogeographic relationships with other non-Greek populations.

Aquaculture is an essential developing sector for world food production, however the attainment of sexual maturity during commercial on-growing is a major bottleneck to industry expansion. Sexual maturation brings a commercial loss due to reduced growth performance as well as reduced immune function. Furthermore, serious concerns exist over potential genetic interaction with native stocks through broadcast spawning or spawning interaction by escapees. In the north Atlantic region, the Atlantic cod (Gadus morhua) and Atlantic halibut (Hippoglossus hippoglossus) are key aquaculture species in which industry expansion is limited by pre-harvest sexual maturation. However, through a species specific combination of modern technologies and refinement in management practices it is possible that this sexual maturation can be controlled and on-growing potential enhanced. Thus the overall aim of this thesis was to conduct novel research that will improve our understanding of the underlying mechanisms that regulate sexual maturation, whilst also advancing the optimisation of technologies for the management of maturation in cod and halibut. In Atlantic cod, owing to the inconsistent inhibition of maturation in commercial conditions, ever increasing intensities of light and in some cases narrow spectrum technologies are being used to try to combat this problem. Firstly, this PhD project investigated the potential welfare impacts of high intensity artificial lighting which have not been studied to date (Chapter 2). The work specifically investigated the effect of traditional metal halide and novel green cathode lighting on the stress response, innate immunity, retina structure, feeding activity and light perception of Atlantic cod. Results indicated that although acute responses to light were observed, there were no clear significant long term effects of any of the lighting treatments on these parameters. Regarding light perception, interestingly even when subjected to high intensity constant lighting (metal halide mean tank intensity: 16.6 watts m-2), cod still demonstrated a day/night rhythm in melatonin release which suggests perception of the overlying ambient photoperiod. The second trial of this PhD project investigated the efficacy of shading of ambient photoperiod in addition to constant lighting to inhibit maturation of cod outdoors (Chapter 3). This aimed at improving the performance of artificial lighting regimes in the open cage system during commercial on-growing by reducing the relative difference between day/night light intensities. The trial was conducted over a one year period where a low and high shade treatment were tested in outdoor tanks. Shading increased the relative night time illumination to 6.6% and 31.3% of daytime levels respectively, compared to <2% in an unshaded set-up. Both shading treatments were effective at suppressing sexual development in cod as confirmed through measurements of gonadosomatic index, histological analysis of gonadal development, oocyte diameter measurements and sex steroid profiles as well as measurements of growth. In addition to research at the applied level in Atlantic cod, this thesis has also extended to the fundamental level and explored one of the potential mechanisms relaying photoperiod signal to the endogenous regulation of sexual maturation in cod, namely the kisspeptin system (Chapter 4). Partial sequences for the signal peptide Kiss2 and its receptor Kissr4 were isolated and described showing similarity to other teleost species such as the medaka, Oryzias latipes and stickleback, Danio rerio. Novel molecular qPCR assays were designed and developed to measure the expression of both genes in male and female cod over a maturation cycle and compared to cod under constant lighting which remained immature. Interestingly, expression patterns of kiss2 and kissr4 did not reveal any clear association with season or photoperiod treatment. However, pituitary expression of gonadotropins (FSH, follicle stimulating hormone; LH, luteinising hormone) did show a differential expression in relation to treatment from early winter approximately 4-6 months after the photoperiod change. These new results are in contradiction with the hypothesis that the kisspeptin system would be involved in the initiation of gametogenesis, as shown in mammals. However, the FSH/LH data defines a window during which time kisspeptin or another GnRH stimulating mechanism must be active, this compels the need further investigation. In Atlantic halibut farming, all-female production removes the concerns of production losses through sexual maturation. Accordingly, this thesis investigated the potential/feasibility of generating monosex populations by FACS (fluorescence activated cell sorting) semen sexing based on cellular DNA content, as proven in terrestrial agriculture. Results however did not show any clear differences between the DNA of sperm in a range of species tested (Atlantic halibut, cod, sea bass, perch) suggesting that this technique may not be applicable in such species. The project also focussed on the production of a population of sex reversed halibut broodstock (neomales) that will generate, in the long term, a basis for traditional monosex population generation in the UK. Two in feed MDHT (17α-methyldihydrotestosterone) treatments were tested with the aim to reduce the use of hormone. Results were very successful with a hormone treatment of 5ppm MDHT generating a 97% phenotypic male population thus suggesting the presence of sex-reversed halibut which can be used for future monosex production. Overall, this work aimed to develop and/or refine potential remediation techniques for sexual maturation in two key commercially important farmed marine fish species, cod and halibut, as well as further our understanding on the regulation of puberty. The knowledge gained from this work provides a means to optimise the techniques employed in the industry and has the potential to increase production and profitability without compromising farmed animal welfare, thus ultimately promoting the sustainable expansion of the Atlantic cod and halibut aquaculture.

Connaître le sexe d’un oiseau est important pour divers domaines notamment pour les vétérinaires, les écologistes ainsi que pour les éleveurs d’oiseaux qui veulent former des couples qui serviront à la reproduction. Plusieurs espèces d’oiseaux, juvéniles et adultes, n’ont pas de dimorphisme sexuel. L’utilisation de l’ADN est une façon rapide de déterminer le sexe à partir d’un échantillon de sang, de muscle, de plumes ou de fèces. Par contre, la méthode devrait être validée pour chaque espèce et idéalement, standardisée. Le premier objectif de cette étude est de développer une méthode de sexage par séquençage des oiseaux à partir des séquences du gène CHD, en utilisant les oiseaux de proie et les perroquets vus en clinique au Québec. Un deuxième objectif est de faire l’identification de l’espèce à sexer, à partir du gène mitochondrial COX-1 et aussi à partir des séquences CHD-Z et CHD-W, utilisés pour le sexage. Un troisième objectif est d’évaluer les séquences sorties (CHD-Z, CHD-W et COX-1) en vue d’une étude phylogénique. Une extraction d’ADN a été effectuée chez 27 espèces de perroquets, 34 espèces d’oiseaux de proie, une corneille (Corvus brachyrhynchos) et un poulet (Gallus gallus). Une amplification par PCR a été exécutée pour les exons partiels 23 et 24 du gène CHD. Le séquençage de cet amplicon permettait de savoir s’il s’agissait d’un mâle (séquence simple CHD-Z) ou d’une femelle (séquences CHD-Z et CHD-W qui se chevauchent). Afin d’avoir des séquences CHD-W distinctes, un sous-clonage a été fait chez les femelles de chaque espèce. De cette manière, les séquences partielles du gène CHD, Z et W, ont été trouvées pour les espèces échantillonnées. Une étude phylogénique a été effectuée avec les séquences de COX-1, CHD-Z et CHD-W grâce au site « Clustal-Omega ». La méthode de sexage des oiseaux par séquençage du gène CHD est standard et efficace. Le gène COX-1 permet une meilleure identification des espèces parentes et le gène CHD-Z est le plus utile pour étudier la phylogénie profonde. / Knowing the sex of a bird is important for many disciplines, notably for veterinary, ecological and evolutionary studies, not to mention for bird breeders who need to form pairs for reproduction. For many species of birds, juveniles and adults do not display a sexual dimorphism. The use of DNA, derived from a sample of blood, muscle, feathers or feces, is a rapid method to determine a bird’s sex. However, this method must be validated for each species, and ideally, standardised. The first objective of this work was to develop a method of sexing birds by sequencing portions of their CHD gene, for birds of prey and parrots seen in veterinary clinics in Quebec. A second objective was to identify the species being sexed, first of all using the mitochondrial gene COX-1, and second of all using the CHD-Z and CHD-W sequences used for sexing. The third objective of these studies was to evaluate the sequences obtained (CHD-Z, CHD-W and COX-1) for performing phylogenetic studies. DNA was extracted from 27 species of parrots, 34 species of birds of prey, from one species of crow (Corvus brachyrhynchos) and from the chicken (Gallus gallus). A PCR amplification was performed for partial exons 23 and 24 of the CHD gene. Sequencing this amplicon resulted in simple CHD-Z sequences for a male and overlapping CHD-Z and CHD-W sequences for a female, allowing sexing of the bird. In order to obtain distinct CHD-W sequences, a sub-cloning was performed for females of each species. In this fashion, partial sequences of the CHD gene, both -Z and –W, were generated for the species studied. A phylogenetic study was performed using COX-1, CHD-Z and CHD-W sequences and the site “Clustal-Omega”. The method of sexing birds by sequencing was found to be standard and efficient. The COX-1 gene permitted a better resolution of closely related species, while the CHD-Z gene was the most useful for estimating deep phylogenetic relationships.