Orientador: Laura Satiko Okada Nakaghi / Banca: Hugo Pereira Godinho / Banca: Alexandre Ninhaus Silveira / Resumo: O Astronotus ocellatus é originário da Bacia Amazônica, climatizado e disseminado, desde 1938, em açudes e rios do Nordeste brasileiro, pelo Departamento Nacional de Obras Contra as Secas. Trata-se de um peixe de grande potencial, devido a características adequadas para pesca esportiva, cultivado como ornamental e com uma carne saborosa e apreciada pela população do Norte e Nordeste do Brasil. Existem na literatura atual poucos estudos a respeito da biologia reprodutiva dessa espécie. Embora seja de desova parcelada e se reproduza bem em cativeiro, estudos de reprodução artificial têm suma importância para diversas pesquisas de biologia e desenvolvimento embrionário, cujos eventos devem ser acompanhados numa faixa de tempo conhecida e assim contribuir para uma melhor compreensão do cultivo, manejo e controle da espécie. O presente trabalho teve por objetivo avaliar a resposta do Astronotus ocellatus a aplicação de dois hormônios estimuladores de reprodução (gonadodropina coriônica humana - HCG e extrato de hipófise de carpa - EHC) e estudar a estrutura, ultraestrutura e morfometria de seus ovos e larvas. Foram utilizados 16 casais de reprodutores pertencentes ao Centro de Aqüicultura da Universidade Estadual Paulista, Campus de Jaboticabal (SP) e quatro desovas ocorridas naturalmente nos viveiros. Observouse que cinco das dezesseis fêmeas responderam ao estímulo hormonal, sendo três com HCG e duas com EHC. Dos machos, apenas um respondeu positivamente ao HCG. O acará-açu possui ovos demersais, adesivos, pouco resistentes ao tato, de formato ovóide pouco acentuado. "In vivo" apresentaram coloração amarelada, quando fertilizados, e branca opaca, quando não fertilizados, com grande esfera vitelina e pequeno espaço perivitelino. As desovas apresentaram ovos com valores médios variando de 1,75±0,056 a ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Astronotus ocellatus is a fish from the Amazon Basin, adapted and widely reared since 1938 in ponds and rivers of Brazilian northeast region by the Departamento Nacional de Obras Contra as Secas. This fish has a great potential due to appropriate characteristics for sporting fishing, reared as ornamental and owning a very tasty meat being appreciate by the people in north and northeast regions of Brazil. Despite having all these adjectives for aquaculture there are a few studies about reproductive biology of this species. Although spawning does not occur at once and reproduction in captivity is well successful, studies about artificial reproduction are extremely important for several researches in biology and early embryonic development of which events must be followed in a known lack of time and like this to contribute for a better understanding of rear conditions, management and control of the species. Therefore, this present research aimed to assess the Astronotus ocellatus responses to administration of two kinds of reproductive hormones (Human Chorionic Gonadotropin - HCG and carp hypophysis extract - CHE) and studying the structure, ultrastructure and morphometry of its eggs and larvae. Sixteen couples from Centro de Aqüicultura of Universidade Estadual Paulista, Campus of Jaboticabal (SP) and four spawnings occurred naturally in ponds were also catched. Five of sixteen females responded to hormonal stimuli, three of them with HCG and two with CHE. In relation to males, only one responded favorably to HCG. Acará-açu females have demersal and adhesive eggs, somewhat resistant to the touch, discreet egg shape-like, yellowish colour "in vivo" and when fertilized and white opalescent without fertilization, with a large vitelline sphere and a small space between yolk and chorion. The spawnings presented eggs with average values of 1,75±0,056 ...(Complete abstract click electronic access below) / Mestre

Sistemas para a Transposição de Peixes são implantados para atenuar os efeitos negativos dos barramentos sobre os peixes migradores ou de piracema. No Brasil, como na maioria dos paises subdesenvolvidos, devido a fatores históricos, financeiros, políticos, institucionais e culturais desfavoráveis, há uma defasagem tecnológica quanto aos Sistemas para a Transposição de Peixes, pelo menos secular, com conseqüências imensuráveis ao ambiente e ao universo dos peixes. A importância dos nossos peixes fluviais, onde se destacam os migradores como os de maior interesse comercial e esportivo, é indiscutível. Assim, esta dissertação propõe-se a inventariar o "estado da arte" dos Sistemas para a Transposição de Peixes, isto é, conhecer as necessidades dos nossos peixes, estudar alternativas hidráulico-estruturais de transposição e, também, vencer as correntes retrógradas. Os estudos e projetos internacionais não devem ser aplicados indiscriminadamente para a realidade nacional, pois diferem quanto à diversidade dos nossos peixes, sob a pena de incorrer nos insucessos como os australianos africanos e os brasileiros. Nesta dissertação, após à introdução, há um breve relato sobre a importância entre as relações ecológicas e ambientais; relações entre os elementos vivos e o meio aquático e as interferências humanas; conhecimentos físicos e biológicos sobre os peixes; história, conceitos fundamentais e legislação sobre os STP's; estudos para viabilização de estruturas-tipo, em modelo reduzido e estudos futuros. / Fish Transponding System are usually adopted to mitigate negative effects of dam and reservoir construction on migratory fishes. In Brazil, as with most undeveloped countries, owing to historical, political, economical and cultural adverse factors, there is a centenary technological gap relating Fish Transponding System, which has caused unbearable consequences to environment and to the fish realm. The importance of our fluvial fishes, among which the migratory ones represent major commercial and sportive interest, is unquestionable. This dissertation intends to furnish information on the State of the Art in Fish Transponding System, to present native fish habits and needs, to compare hydraulic and structural transponding system types and, last but not least, to introduct a new way of facing and dealing with fish transponding problems, in a contribution to breaking the prevailing retrograde vision. This dissertation shows that international current projects and practices should not be adopted widespread to national reality, as they usually don’t match our fishes habits and needs; the adoption of those practices could lead to a project breakdown, according to some recorded Australian, African, and even Brazilian experiences. The work also presents a brief explanation on: ecological and environmental relations; living specimens, aquatic media and human actions; Fish physical and biological knowledge; history, basic concepts and legislation about FTS; considerations aiming to make feasible standard structures, reduced scale models and further investigations.

Die Osteoglossomorpha stellen eine basale Gruppe der Teleostei dar mit einer Mischung von plesiomorphen und apomorphen Merkmalen bezogen auf Reproduktion und ontogenetische Entwicklung. Bezüglich reproduktiver Gilden und ontogenetischer Entwicklung gibt es immer noch nur begrenzte Daten zu dieser Gruppe. Der größte Teil tiefer gehender Studien bezieht sich auf Mormyriden, detaillierte Beschreibungen und experimentelle Daten sind kaum vorhanden bei den anderen Gruppen sowie bei Notopterus notopterus und Osteoglossum bicirrhosum. Im Rahmen dieser Untersuchung wird zum ersten Mal eine detaillierte Beschreibung der ontogenetischen Entwicklung dieser beiden Arten auf der Basis von Laborzuchten vorgestellt. Nur bei N. notopterus gelangen Zuchtexperimente unter Laborbedingungen. Die experimentellen Daten zeigten, dass die Umweltparameter abnehmende Leitfähigkeit, Erhöhung des Wasserstandes und leichte Temperaturvariation keinen Einfluss auf Gonadenentwicklung oder Balzverhalten bei N. notopterus hatten. Die frisch abgelegten, klebrigen Eier von 3.8-4 mm Größe werden vom Männchen bis zum Schlupf bewacht. Die Eihülle besitzt äußere Rillen, die ringförmig um die Mikropyle herum angeordnet sind. Das Schlüpfen erfolgt im Alter von 168-204 Stunden. In dieser Studie wird zum ersten Mal eine Methode zur erfolgreichen Aufzucht von O. bicirrhosum unter Laborbedingungen bei 28 °C vorgestellt. Die nicht-klebrigen Eier von 12 mm Größe besitzen eine transparente Eihülle. Das Schlüpfen erfolgt im Alter von 162-166 Stunden und die geschlüpften Embryonen haben eine Länge von 16 mm. Bei O. bicirrhosum findet sich keine Larval-Periode; auf die Embryonal-Periode folgt sofort die Juvenil-Periode: Die Ontogenese kann somit als direkte Entwicklung klassifiziert werden. N. notopterus hingegen ist gekennzeichnet durch eine intermediäre Entwicklung unter Bezug auf Reproduktionsstrategien da sie eine höhere Anzahl von mittelgroßen Eiern produzieren bei gleichzeitiger Brutpflege. / The Osteoglossomorpha represent a basal group of teleostean fish comprising taxa with a mixture of both plesiomorphic and apomorphic characters of reproduction and ontogenetic development. Concerning reproductive styles and ontogenetic development of this group, there are still very limited data available so far. Most in depth studies are available for mormyrids, but detailed descriptions and experimental data remain scarce in the other groups as in Notopterus notopterus and Osteoglossum bicirrhosum. This study will describe in detail for the first time the ontogenetic development of these two species in laboratory-reared specimens. Only N. notopterus succeeded in the breeding experiment. Experimental data demonstrated that the environmental factors decreasing conductivity, slight variation of temperature, and water level have no influence on gonad development or courtship behaviour in N. notopterus. Newly spawned 3.8–4 mm adhesive eggs are guarded by the male until hatching. The egg envelope has external ridges, which are centred around the micropyle. Hatching occurs within 168–204 hours. For the first time, this study describes a method of successfully raising O. bicirrhosum at 28 °C under laboratory conditions. The non-adhesive eggs measure 12 mm with a transparent egg envelope. Hatching occurs around 162–166 hours and newly hatched embryos measure 16 mm. Actually there is no larval period found in O. bicirrhosum. The embryonic period is directly followed by the juvenile period and ontogeny can be characterized as direct development. N. notopterus is classified as intermediate species in an interpretation at reproductive strategies since they produce a higher number of medium-sized eggs and show parental care.

Arapaima gigas is the largest scaled freshwater fish in the world reaching over 250 kg. With growth rates of 10 kg+ within 12 months, A. gigas is considered as a promising candidate species for aquaculture development in South America. However, the lack of reproductive control in captivity is hindering the industry expansion. The work carried out in this doctoral thesis therefore aimed to better understand the species’ reproductive physiology, develop tools to identify gender and monitor gonad development, test hormonal therapies to induce ovulation and spawning and characterise the cephalic secretion for its potential roles in pheromone release and during parental care. Initially, a genomic study investigated the overall extent of polymorphism in A. gigas, which was found to be surprisingly low, with only 2.3 % of identified RAD-tags (135 bases long) containing SNPs. Then, a panel with 293 single nucleotide polymorphism (SNP) was used to characterise the genetic diversity and structure of a range of Amazon populations. Results revealed populations from the Amazon and Solimões appeared to be genetically different from the Araguaia population, while Tocantins population comprised individuals from both stocks. This data provided a tool for broodstock identification and future management. The PhD then aimed to evaluate the effects of slow-release mGnRH implants and different broodstock size pairings on maturation and spawning. Results showed that the implants stimulated the brain-pituitary-gonad axis resulting in increased plasma levels of testosterone (females) and 11-ketotestosterone in males, respectively regardless of pairing sizes. However, no spawning was observed. Results also showed the release of sex steroids with potential pheromonal action through the cephalic secretion, a biological fluid released from the adult head along the reproductive period. Thereafter, a non-surgical field endoscopy method was developed and validated for ovarian assessment and gender identification. The method was then used to describe the female gonopore and obtain biopsy of the ovary through cannulation which allowed the description of oogenesis in A. gigas. Importantly, oocytes obtained by cannulation confirmed that adult females under investigation were maturing with oocytes in final maturation stage but failed to ovulate/spawn. Another hormonal induction trial was therefore performed in which a combination of GnRHa (mGnRHa/sGnRHa) was used by injection to induce ovulation and spawning in selected maturing females with effects on oocyte maturation monitored post-induction through biopsy. However, this trial appeared to not be successful at inducing ovulation or spawning. Finally, the peptidome and proteome of the cephalic secretion was further characterised through the comparison between parental and non-parental fish. Results highlighted the complex role of this biological fluid including potential roles on the developing offspring during the parental care period. Overall, this doctoral thesis provided new basic and applied data on A. gigas reproduction and tools that can be used in future studies to better understand the environmental and hormonal control of oogenesis and spawning.

Sistemas para a Transposição de Peixes são implantados para atenuar os efeitos negativos dos barramentos sobre os peixes migradores ou de piracema. No Brasil, como na maioria dos paises subdesenvolvidos, devido a fatores históricos, financeiros, políticos, institucionais e culturais desfavoráveis, há uma defasagem tecnológica quanto aos Sistemas para a Transposição de Peixes, pelo menos secular, com conseqüências imensuráveis ao ambiente e ao universo dos peixes. A importância dos nossos peixes fluviais, onde se destacam os migradores como os de maior interesse comercial e esportivo, é indiscutível. Assim, esta dissertação propõe-se a inventariar o "estado da arte" dos Sistemas para a Transposição de Peixes, isto é, conhecer as necessidades dos nossos peixes, estudar alternativas hidráulico-estruturais de transposição e, também, vencer as correntes retrógradas. Os estudos e projetos internacionais não devem ser aplicados indiscriminadamente para a realidade nacional, pois diferem quanto à diversidade dos nossos peixes, sob a pena de incorrer nos insucessos como os australianos africanos e os brasileiros. Nesta dissertação, após à introdução, há um breve relato sobre a importância entre as relações ecológicas e ambientais; relações entre os elementos vivos e o meio aquático e as interferências humanas; conhecimentos físicos e biológicos sobre os peixes; história, conceitos fundamentais e legislação sobre os STP's; estudos para viabilização de estruturas-tipo, em modelo reduzido e estudos futuros. / Fish Transponding System are usually adopted to mitigate negative effects of dam and reservoir construction on migratory fishes. In Brazil, as with most undeveloped countries, owing to historical, political, economical and cultural adverse factors, there is a centenary technological gap relating Fish Transponding System, which has caused unbearable consequences to environment and to the fish realm. The importance of our fluvial fishes, among which the migratory ones represent major commercial and sportive interest, is unquestionable. This dissertation intends to furnish information on the State of the Art in Fish Transponding System, to present native fish habits and needs, to compare hydraulic and structural transponding system types and, last but not least, to introduct a new way of facing and dealing with fish transponding problems, in a contribution to breaking the prevailing retrograde vision. This dissertation shows that international current projects and practices should not be adopted widespread to national reality, as they usually don’t match our fishes habits and needs; the adoption of those practices could lead to a project breakdown, according to some recorded Australian, African, and even Brazilian experiences. The work also presents a brief explanation on: ecological and environmental relations; living specimens, aquatic media and human actions; Fish physical and biological knowledge; history, basic concepts and legislation about FTS; considerations aiming to make feasible standard structures, reduced scale models and further investigations.

Tesis por compendio / [ES] La criopreservación de semen de peces es una técnica que puede aumentar la eficiencia de la reproducción en cautiverio de especies de peces de agua dulce y marinas. A lo largo de las últimas décadas, se han establecido protocolos para criopreservación de semen de diversas especies de peces. Sin embargo, el foco principal de los pescadores ha sido en tener éxito en el congelamiento y descongelamiento de los espermatozoides, no llevando en cuenta el período en que los gametos se quedan expuestos a la solución crioprotectora en un momento previo a la fertilización. Esta exposición de los espermatozoides a las soluciones crioprotectoras después del descongelamiento puede ser perjudicial a la calidad de los gametos, ya que pueden ser tóxicos. La mayoría de los protocolos establecidos utilizan recipientes de plástico ultrarresistentes para almacenar el semen durante el proceso de criopreservación. Estos recipientes normalmente no se reutilizan, generando residuos altamente contaminantes al medio ambiente. Así, el objetivo principal de la tesis fue crear y probar métodos de bajo costo que potencialicen el uso de los espermatozoides descongelados de peces y torne el proceso de criopreservación de semen menos contaminante al medio ambiente. Los experimentos de los capítulos 1 y 2 se llevaron a cabo en Brasil. Utilizamos el jundiá gris Rhamdia quelen, especie considerada modelo experimental para peces nativos de América del Sur. En el capítulo 1, probamos el uso de la dilución de semen descongelado para disminuir la toxicidad de la solución crioprotectora. La técnica se utiliza comúnmente en protocolos de criopreservación de semen de mamíferos, pero nunca antes se había aplicado al semen descongelado de peces suramericanos. Muestras de semen descongelado de R. quelen se diluyeron en un diluyente salino (NaCl al 1,1% - 325 mOsm kg-1; pH 7,6). Después, observamos que los espermatozoides de muestras diluidas mostraron mayores velocidades, rectitud, progresión y frecuencia de batido flagelar que las muestras no diluidas. La dilución del semen descongelado también proporcionó mayores tasas de fertilización y eclosión que el grupo no diluido. De esta manera, la dilución de semen descongelado de R. quelen resultó ser una metodología sencilla, económica y eficiente que debe incluirse en el protocolo de criopreservación del semen de la especie. En los capítulos 2 y 3 desarrollamos y probamos la metodología para el uso de cápsulas de gelatina biodegradables (colágeno) y cápsulas de hipromelosa biodegradables (HPMC) como recipiente alternativo al uso de pajuelas de plástico en la criopreservación de semen de peces. En el segundo capítulo observamos que las cápsulas biodegradables mantuvieron los parámetros cinéticos y la capacidad reproductiva del esperma de R. quelen así como las pajuelas de plástico.Los procedimientos experimentales del capítulo 3 se llevaron a cabo en España. En este capítulo aplicamos la metodología desarrollada en el capítulo 2 para la criopreservación del semen de anguila europea Anguilla anguilla, dorada Sparus aurata y lubina Dicentrarchus labrax. En estas tres especies, las cápsulas biodegradables conservaron los parámetros cinéticos y la integridad de la membrana de los espermatozoides, así como las pajuelas de plástico. Además, observamos que el daño al ADN en muestras de semen de anguila europea y lubina europea criopreservadas en cápsulas y pajuelas no difirió. Sin embargo, las muestras de semen de dorada mostraron mayor daño en el ADN que las criopreservadas en pajuelas. Aunque, el nivel de daño que observamos en las muestras almacenadas en las cápsulas se considera bajo, por lo que pueden no comprometer el desarrollo embrionario. Evaluamos los resultados y concluimos que las cápsulas de gelatina biodegradables y las cápsulas de HPMC biodegradables pueden utilizarse como recipientes alternativos al uso de pajuelas de plástico para la criopreservación de semen de las cuatro especies de peces. / [CA] La criopreservació de l'esperma de peixos és una tècnica que pot augmentar l'eficiència de la reproducció en captivitat d'espècies de peixos d'aigua dolça i marins. Al llarg de les dècades passades, s'han establert protocols per a la criopreservació de l'esperma de diverses espècies de peixos. No obstant això, el focus principal dels pescadors ha estat tenir èxit en la congelació i descongelació dels espermatozoides, sense tenir en compte el temps en què els gamets queden exposats a la solució crioprotectora abans de la fecundació. Aquesta exposició dels espermatozoides a les solucions crioprotectores després de la descongelació pot ser perjudicial per a la qualitat dels gàmetes, ja que poden ser tòxics. La majoria dels protocols establerts utilitzen recipients de plàstic ultrarresistents per emmagatzemar l'esperma durant el procés de criopreservació. Aquests recipients normalment no es reutilitzen, generant residus altament contaminants per al medi ambient. Així, l'objectiu principal de la tesi va ser criar i provar mètodes de baix cost que potenciïn l'ús dels espermatozoides descongelats de peixos i facin que el procés de criopreservació de l'esperma sigui menys contaminant per al medi ambient. Els experiments dels capítols 1 i 2 es van dur a terme a Brasil. Vam utilitzar el jundia gris Rhamdia quelen, una espècie considerada com a model experimental per a peixos natius d'Amèrica del Sud. En el capítol 1, vam provar l'ús de la dilució de l'esperma descongelat per reduir la toxicitat de la solució crioprotectora. Aquesta tècnica s'utilitza comúment en protocols de criopreservació de l'esperma de mamífers, però mai abans s'havia aplicat a l'esperma descongelat de peixos sud-americans. Mostres d'esperma descongelat de R. quelen es van diluir en un diluent salí (NaCl al 1,1% - 325 mOsm kg-1; pH 7,6). Després, vam observar que els espermatozoides de mostres diluïdes mostraven majors velocitats, rectitud, progressió i freqüència de batuda flagel·lar que les mostres no diluïdes. La dilució de l'esperma descongelat també va proporcionar majors taxes de fecundació i eclosió que el grup no diluït. D'aquesta manera, la dilució de l'esperma descongelat de R. quelen va resultar ser una metodologia senzilla, econòmica i eficient que ha d'incloure's en el protocol de criopreservació de l'esperma de l'espècie. En els capítols 2 i 3 vam desenvolupar i provar la metodologia per a l'ús de càpsules de gelatina biodegradables (col·lagen) i càpsules d'hipromelosa biodegradables (HPMC) com a recipient alternatiu a l'ús de canuts de plàstic en la criopreservació de l'esperma de peixos. En el segon capítol vam observar que les càpsules biodegradables mantenien els paràmetres cinètics i la capacitat reproductiva de l'esperma de R. quelen així com els canuts de plàstic. Els procediments experimentals del capítol 3 es van dur a terme a Espanya. En aquest capítol vam aplicar la metodologia desenvolupada al capítol 2 per a la criopreservació de l'esperma d'anguila europea Anguilla anguilla, daurada Sparus aurata i llobarro Dicentrarchus labrax. En aquestes tres espècies, les càpsules biodegradables van conservar els paràmetres cinètics i la integritat de la membrana dels espermatozoides, així com els canuts de plàstic. A més, vam observar que el dany a l'ADN en mostres d'esperma d'anguila europea i llobarro europeu criopreservades en càpsules i canuts no es va diferir. No obstant això, les mostres d'esperma de daurada van mostrar més dany a l'ADN que les criopreservades en canuts. Tot i això, el nivell de dany que vam observar a les mostres emmagatzemades en càpsules es considera baix, pel que poden no comprometre el desenvolupament embrionari. Vam avaluar els resultats i vam concloure que les càpsules de gelatina biodegradables i les càpsules d'HPMC biodegradables es poden utilitzar com a recipients alternatius a l'ús de canuts de plàstic per a la criopreservació de l'esperma de les quatre espècies de peixos. / [EN] Fish sperm cryopreservation is a technique that can increase the reproduction in captive efficiency of freshwater and marine fishes.Over the last few decades, protocols for sperm cryopreservation from many fishes have been established. However, the researchers' main focus was successfully freezing and thawing sperm, neglecting the period in which the gametes remain in contact with the cryoprotective solution until fertilization. Exposure of sperm to cryoprotectant solutions after thawing can be harmful to the quality of the gametes since they can be toxic. The majority of the established protocols use ultra-resistant plastic containers to store sperm during the cryopreservation process. These containers usually are not reused, generating highly polluting waste for the environment. Furthermore, in some countries, the containers usually used are sold by a few industries, which makes acquisition difficult and increases the product's price. Thus, the main objective of the thesis was to create and test low-cost methodologies that enhance the use of fish post-thaw sperm and make the sperm cryopreservation process more environmentally friendly. The experiments in the Chapters 1 and 2 were developed in Brazil. We used the South American silver catfish Rhamdia quelen, a species considered an experimental model for native South American fishes. In Chapter 1, we tested the use of post-thawing dilution to reduce the toxicity of the cryoprotectant solution. This technique is commonly used in mammalian sperm cryopreservation protocols but has never before been applied to post-thaw sperm of South American fishes. South American silver catfish post-thaw sperm samples were diluted in a saline extender (1.1% NaCl - 325 mOsm kg-1; pH 7.6). The post-thaw sperm diluted samples showed higher velocities, straightness, progression, and flagellar beat frequency than the cells of undiluted samples (control). The post-thawing dilution also provided higher fertilization and hatching rates than the control group. Thus, the post-thawing sperm dilution proved to be a simple, cheap, and efficient methodology that should be included in the silver catfish sperm cryopreservation protocol. In Chapters 2 and 3, we developed, tested, and described the methodology for using biodegradable gelatin (collagen) and hypromellose (HPMC) capsules as an alternative container to plastic straws in the fish sperm cryopreservation. In the second chapter, we observed that the biodegradable capsules maintained the kinetic parameters and reproductive capacity of South American silver catfish sperm just as effectively as plastic straws. The experimental procedures in Chapter 3 were carried out in Spain. We apply the methodology developed in Chapter 2 to the cryopreservation of sperm from European eel Anguilla anguilla, gilthead seabream Sparus aurata, and European sea bass Dicentrarchus labrax. In these three species, biodegradable capsules preserved the sperm kinetic parameters and membrane integrity just as effectively plastic straws. We observed that DNA damage in European eel and European sea bass sperm samples cryopreserved in capsules and straws did not differ. On the other hand, gilthead seabream sperm samples showed higher DNA damage than those cryopreserved in straws. However, the damage level observed in samples stored in capsules is considered low, thus, may not compromise embryonic development. We observed the results and concluded that biodegradable gelatin and HPMC capsules could be used as alternative containers to plastic straws for sperm cryopreservation from the tfour aquaculture fishes. / This study was supported by MICINN with funding from European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana (THINKINAZUL/2021/012;THINKINAZUL/2021/024;THINKINAZUL/2021/042) including the contract of FF-G.WAG-L has a Margarita Salas postdoctoral contract (RD 289/2021. UAB) by the Spanish Ministry of Universities. LF has a PhD contract from Generalitat Valenciana (GRISOLIAP/2020/063). TSF (141717/2019-0 and 200285/2021-1) and MPS (200452/2022-3) have fellowships from Brazilian National Council for Scientific and Technological Development (CNPq). / De Souza França, T. (2024). Development and Application of Low-Cost and Environment-Friendly Techniques for Fish Sperm Cryopreservation [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/204486 / Compendio

Made available in DSpace on 2016-06-02T19:32:06Z (GMT). No. of bitstreams: 1 DissMGV.pdf: 1081292 bytes, checksum: 700b8d44e1688d266b85b0a69648d0f3 (MD5) Previous issue date: 2003-03-13 / Financiadora de Estudos e Projetos / The majority of information related to reproductive strategy of Erythrinidae species has its origin from observations on natural environment. In this context, several tactics are not effectively evaluated and precious collection of information relevant to commercial fishing culture or even to effort of fauna reconstitution are simply lost. Studies on induced reproduction of Hoplerythrinus unitaeniatus Hoplias malabaricus and Hoplias lacerdae were performed at the Fish culture and Hydrobiology Station of the CODEVASF-Três Marias-MG, from September to December of the years 2000 and 2001 with the objective of search its reproductive characteristics. The Hoplerythrinus unitaeniatus presented to be the most fecund of the three studied species with smaller eggs and larvae sizes, followed by the Hoplias malabaricus presenting intermediate values between Hoplerythrinus unitaeniatus and Hoplias lacerdae, the least fecund one but with bigger eggs and larvae than the other two species. The larvae of the two species of Hoplias did not presented the vertical movement on the water column observed on Hoplerythrinus unitaeniatus. This behavior is considered of extreme importance to the larvae dispersion in different environment. The Erythrinidae eggs are in general big with yellow color and opaque, presenting good adhesive property and light hydration. Comparing this group with others Characiformes from São Francisco river basin it was observed that these three species present as major peculiarities, the low fecundity and enhanced parental care, evidenced by the eggs and larvae characteristics, representing a high unit value of reproductive investment, placing the Erythrinidae here studied next to the strategic group of k-selected equilibrium proposed by Winemiller in 1989 and in the nest spawner guild proposed by BALON in 1985. At the present work we found out the importance of knowing a few breeding tactics/strategies that results in benefits that certainly yield specialization on handling reproductors, eggs and larvae. / A maioria das informações sobre as estratégias reprodutivas das espécies de Erythrinidae estudadas têm origem a partir de observações em ambiente natural e, dentro deste contexto, várias táticas não foram efetivamente avaliadas podendo constituir informações importantes para o aproveitamento de representantes do grupo na piscicultura comercial ou mesmo em esforços de reconstituição ictiofaunística. No presente trabalho, estudos de reprodução induzida de Hoplerythrinus unitaeniatus, Hoplias malabaricus e Hoplias lacerdae foram realizados na Estação de Hidrobiologia e Piscicultura da CODEVASF-Três Marias-MG, de setembro a dezembro de 2000 e de 2001, para avaliação de suas características reprodutivas. O jejú, Hoplerythrinus unitaeniatus, foi caracterizada como a de maior fecundidade entre as três espécies, porém com ovos e larvas de menores dimensões comparativamente à traíra Hoplias malabaricus, que por sua vez, apresentou valores intermediários entre Hoplerythrinus unitaeniatus e o trairão Hoplias lacerdae considerada a de menor fecundidade entre as três espécies, porém com ovos e larvas de maiores dimensões. As larvas das duas espécies de Hoplias não apresentaram movimento vertical na coluna d água, como o observado para Hoplerythrinus unitaeniatus. Este comportamento é considerado de extrema importância para a dispersão das larvas em diferentes ambientes. Os ovos destes eritrinídeos apresentaram-se, de maneira geral, grandes e opacos, adesivos, de coloração amarela e relativamente pouco hidratáveis. Comparando-se o grupo das traíras com outros Characiformes da bacia do São Francisco para quais já foram realizados estudos de indução reprodutiva, observou-se que este grupo apresenta como peculiaridades principais a relativa baixa fecundidade, o período de embriogênese longo e o cuidado parental acentuado, confirmado pelas características de ovos e larvas que representam um alto valor unitário de investimento reprodutivo, colocando os eritrinídeos aqui estudados próximos ao grupo dos estrategistas de equilíbrio kselecionados, de acordo com a proposta de Winemiller em 1989 e na guilda dos guardadores, segundo a proposta de Balon em 1985. No presente trabalho verificou-se que a importância do conhecimento de algumas táticas/estratégias reprodutivas reside na contribuição que elas seguramente propiciarão ao aprimoramento do manejo tanto dos reprodutores, quanto dos ovos e das larvas.

Made available in DSpace on 2014-12-17T15:36:43Z (GMT). No. of bitstreams: 1 WallaceSN_TESE.pdf: 8655030 bytes, checksum: d55633f6c1ca42d23115efea83e7b07c (MD5) Previous issue date: 2014-06-04 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / This work deals with the life strategy of an endangered annual fish, Hypsolebias antenori (Cyprinodontiformes: Rivulidae), of the Brazilian semiarid region. The eggs of these fish hatch at the onset of the rainy season, grows rapidly and they reproduce during the rainy season. When the water puddles dry out, the entire population dies. The resistant eggs which are buried in the bottom of the dried pools go through diapause stages, during which time the embryonic development becomes temporarily arrested. With the onset of the next rainy season, the eggs hatch and a new generation is formed. Specimens of H. antenori were captured during 2011 and 2013, in temporary water pools located in the hydrographic basin of river Jaguaribe in Cear?, Brazil. Sex ratio, the length-weight relationship, the growth type, first sexual maturity, anatomy and histology of the digestive tract, development of gonads, reproductive strategy, karyotypic pattern of the species, and the conservation status of H. antenori were investigated. The results of this study are presented in the form of eight articles. The first article is about the fish faunal composition of the hydrographic basins of Rio Grande do Norte, Brazil, wherein the record of H. antenori is included. The second article deals with the sex ratio, secondary sexual characteristics of males, the length-weight relationship and the type of growth. Males show a pattern of intense coloration with well developed fins. The sex ratio showed a significant predominance of females (1M:1.7 F). Males were larger in length and weight. The equation of weight and total length relationship was Wt=0.0271Lt3,8937, showing a positively allometric growth, indicating greater increase in weight than in length. The third article discusses the anatomy and histology of the digestive tract of H. antenori. It is considered as a generalist feeder with characteristics of omnivore, which utilizes different food sources. The fourth article discusses the stages and phases of gonad development and type of spawning of H. antenori. The fifth article is about the r reproductive strategy adopted by H. antenori which helps in successful reproduction over a short period of life. The sixth article deals karyotypic pattern of the species, constituting the first cytogenetic contribution to the genus. The seventh article discusses about the risk of extinction of this species which suffers a series of threats, such as, habitat loss through land use, deforestation, construction of reservoirs, pollution due to domestic and industrial sewage, besides pesticides and agrochemicals. Furthermore, decreasing rainfall and intensification of aridity due to global climate changes, interferes with the reproductive cycle. The eighth article deals with aggressive behavior adopted between males and among females during reproduction. All temporary water pools sampled during this study were in high degree of degradation, mainly due to human action. There is a great need for conservation measures to protect the populations of annual fish, including the creation of protected areas in the semiarid ephemeral aquatic environments of Brazil / Este trabalho trata sobre a estrat?gia da vida de um peixe anual em perigo Hypsolebias antenori (Cyprinodontiformes: Rivulidae), do semi?rido brasileiro. Os ovos dessa esp?cie nascem em po?as tempor?rias, crescem rapidamente, reproduzem e no per?odo de estiagem quando a po?a seca, toda a popula??o morre. Os ovos resistentes depositados no substrato da po?a s?o capazes de passar por um est?gio de diapausa, quando seu est?gio de desenvolvimento embrion?rio ? estacionado. Com a chegada do per?odo de chuvas, os ovos eclodem e uma nova gera??o ? formada. Os exemplares de H. antenori foram capturados durante 2011 e 2013, nas po?as tempor?rias localizadas na bacia do rio Jaguaribe no Cear?, Brasil. Foram investigadas a propor??o sexual, a rela??o peso-comprimento, o tipo de crescimento, a primeira matura??o sexual, anatomia e histologia do trato digest?rio, desenvolvimento das g?nadas, estrat?gia reprodutiva, dados cariot?picos da esp?cie, e o estado de conserva??o de H. antenori. Os resultados desse trabalho est?o apresentados sob a forma de oito artigos. O primeiro artigo ? sobre a composi??o da ictiofauna das bacias hidrogr?ficas do Rio Grande do Norte, Brasil, incluindo o registro de H. antenori. O segundo artigo trata sobre a propor??o sexual, caracter?sticas secund?rios sexuais dos machos, a rela??o pesocomprimento e o tipo de crescimento. Os machos apresentam um padr?o de colora??o intensa com nadadeiras mais desenvolvidas. A propor??o sexual apresentou uma predomin?ncia significativa de f?meas (1M: 1,7F). Os machos foram maiores em comprimento e em peso. A equa??o originada da rela??o peso total e comprimento total foi Wt = 0,0271Lt3,8937, apresentando um crescimento alom?trico positivo, indicando maior incremento no peso do que em rela??o ao comprimento. O terceiro artigo trata sobre a anatomia e histologia do trato digest?rio de H. antenori. ? considerado como um generalista com as caracter?sticas de on?voria, que aproveita fontes alimentares diferentes. O quarto artigo trata sobre os est?dios e fases de desenvolvimento das g?nadas e o tipo de desova de H. antenori. O quinto artigo trata sobre a estrat?gia reprodutiva r adotada pela H. antenori que auxilia na reprodu??o bem sucedida durante um curto per?odo de vida. O sexto artigo trata sobre os dados cariot?picos da esp?cie, constituindo a primeira contribui??o citogen?tica para o g?nero. O s?timo artigo trata sobre o risco de extin??o dessa esp?cie, que v?m sofrendo uma s?rie de amea?as, tais como, a perda de habitats pela ocupa??o do solo, desmatamento, constru??o dos reservat?rios, polui??o devido aos efluentes dom?sticos, industriais, pesticidas e agrot?xicos. Al?m disso, a diminui??o da pluviosidade e intensifica??o da aridez em decorr?ncia das mudan?as clim?ticas globais vem interferindo em seu ciclo reprodutivo. O oitavo trabalho trata sobre o comportamento agressivo adotado entre os machos e entre as f?meas durante a reprodu??o. Todas as po?as tempor?rias amostradas durante este trabalho estavam em um alto grau de degrada??o, principalmente devido ? a??o antr?pica. H? uma grande necessidade de medidas de conserva??o para proteger as popula??es de peixes anuais, entre elas especialmente a cria??o de ?reas de prote??o nos ambientes aqu?ticos ef?meros do semi?rido brasileiro

Tesis por compendio / [ES] Los tiburones y rayas aparecieron hace 420 millones de años, conformando el antiguo y ecológicamente diverso, grupo de vertebrados acuáticos conocido como elasmobranquios. Este variado grupo posee unas estrategias vitales que los hace muy vulnerables a los cambios rápidos del entorno, como los derivados de la acción antrópica. Pese a ser elementos clave en la regulación de los ecosistemas en los que habitan, en la actualidad, se trata de uno de los grupos vertebrados más amenazados del planeta. Paralelamente a la conservación in situ, los programas de conservación ex situ se pueden utilizar para mejorar la situación de algunas especies sensibles. Entre estos programas, los planes de cría en cautividad aumentarían la sostenibilidad de acuarios públicos y centros de investigación, además de permitir el desarrollo de estrategias de conservación en estado salvaje. Sin embargo, para que sean efectivos, estos planes deberían incluir el uso de técnicas de reproducción asistida. Desafortunadamente, estas técnicas apenas se han desarrollado en el pasado, por lo que su utilidad no se ha demostrado adecuadamente. Esta tesis aborda el uso de técnicas de reproducción en elasmobranquios, con énfasis en la obtención y preservación del esperma. Inicialmente, se centró en dos especies modelo, Scyliorhinus canicula y Raja radula, pero luego se incluyeron otras especies. Esto reveló variabilidad en las estructuras reproductivas, lo que puede afectar la eficacia y calidad de las técnicas. Los capítulos 1 y 2 se enfocaron en catalogar y describir estas estructuras en diversas especies, destacando las mejores prácticas de obtención de gametos e inseminación. La conservación a corto, medio y largo plazo del esperma es esencial para planes ex situ, reduciendo el transporte de machos, conflictos durante el cortejo y endogamia. El Capítulo 3 detalla fórmulas para mantener el esperma fresco y un medio para su supervivencia por más de 30 días. Luego, se describe la criopreservación del esperma de varias especies de rayas y tiburones mediante crioprotectores. El Capítulo 4 aborda desafíos en la manipulación de espermatozoides elasmobranquios debido a su morfología helicoidal y la formación de agregaciones llamadas espermatozeugma. Se resalta la importancia de la viscosidad en las técnicas de reproducción asistida. En la Discusión, se compara la eficacia de los métodos y se exploran nuevas oportunidades al tener acceso a diversas especies. Esto incluye descripciones detalladas con análisis de imagen, evaluación de viscosidad y agregados en la preservación, y la consideración de inseminaciones artificiales. / [CA] Els taurons i les ratjades van aparèixer fa 420 milions d'anys, formant el grup conegut com a elasmobranquis. Aquest grup divers posseeix estratègies vitals que els fan molt vulnerables als canvis ràpids de l'entorn. Malgrat ser elements clau en la regulació dels ecosistemes on habiten, actualment són un dels grups més amençats del planeta. Els programes de conservació ex situ es poden utilitzar per millorar la situació d'algunes espècies. Entre aquests programes, els plans de cria en captivitat augmentarien la sostenibilitat dels aquaris públics i els centres de recerca, a més de permetre el desenvolupament d'estratègies de conservació a l'estat salvatge. No obstant això, aquests plans haurien d'incloure l'ús de tècniques de reproducció assistida. Desafortunadament, aquestes tècniques gairebé no s'han desenvolupat en el passat. Davant d'aquesta situació, aquesta tesi pretén omplir certes mancances en el coneixement sobre l'ús d'aquestes tècniques en diverses espècies d'elasmobranquis. La nostra investigació va començar centrant-se especialment en dues espècies model, el tauró gat Scyliorhinus canicula i la ratjada peluda Raja radula, però al llarg de l'estudi es va aconseguir tenir accés a altres espècies. Aquest accés ens va permetre ser conscients de la gran variabilitat en la disposició de les estructures del sistema reproductor. Malgrat que en la literatura hi ha descripcions generals i, en ocasions, detallades dels sistemes reproductors de certes espècies, aquestes descripcions mai s'han centrat en l'aplicació pràctica de les tècniques de reproducció assistida. Per aquest motiu, els capítols 1 i 2 es van centrar en la catalogació i descripció de les diferents estructures d'espècies que abasten un ampli espectre taxonòmic. La conservació de l'esperma juga un paper clau per poder desenvolupar plans de conservació en condicions ex situ. Disposar d'esperma de qualitat permet limitar el transport de mascles entre institucions, minimitzar problemes derivats de la manca de sincronia entre adults reproductors, reduir conflictes durant èpoques d'aparellament i reduir l'endogàmia. El tercer capítol es centra en com es van desenvolupar i provar diferents fórmules per aconseguir el manteniment en fresc de l'esperma, fins a aconseguir un medi on diluir l'esperma capaç de mantenir-lo amb vida durant més de 30 dies. Posteriorment, mitjançant l'addició de diversos crioprotectors (ou, DMSO i metanol) en diverses concentracions, s'explica com es va aconseguir la criopreservació de l'esperma de diverses espècies de ralles i, per primer cop, la criopreservació de l'esperma de diverses espècies de taurons. Un dels primers obstacles que es van detectar en treballar amb els espermatozoides es va deure a la morfologia d'aquestes cèl·lules en comparació amb les d'altres espècies. El cap dels espermatozoides presenta una forma helicoidal, amb un nombre variable de voltes en funció de l'espècie. A més, en moltes ocasions les cèl·lules no apareixen lliures en el fluid seminal, sinó que formen agregacions estructurades anomenades espermatotzeugma. Al llarg del capítol 4 s'explora com aquestes dues característiques sorgeixen com a resultat de la fecundació interna i les característiques mecàniques del medi en què han de realitzar la seva funció. Per primer cop, es pot observar com les cèl·lules reaccionen davant mitjans amb diferents propietats, posant de relleu la importància de la viscositat en l'aplicació de tècniques de reproducció assistida. Finalment, a la discussió, es compara l'eficàcia dels mètodes emprats i s'explora nous camins d'acció sorgits de tenir accés a més individus de diferents espècies. Això inclou la possibilitat de fer descripcions detallades mitjançant tècniques d'anàlisi d'imatge, avaluar la importància de la viscositat i els agregats en els processos de preservació i la possibilitat de realitzar inseminacions artificials. / [EN] Sharks and rays emerged 420 million years ago, forming the ancient and ecologically diverse group of aquatic vertebrates known as elasmobranchs. This diverse group possesses vital strategies that make them highly vulnerable to rapid environmental changes, such as those resulting from human activities. Despite being key elements in the regulation of the ecosystems they inhabit, they are currently one of the most threatened vertebrate groups on the planet. In parallel with in-situ conservation efforts, ex-situ conservation programs can be used to improve the status of some sensitive species. Among these programs, captive breeding plans would enhance the sustainability of public aquariums and research centers, as well as enable the development of conservation strategies in the wild. However, for these plans to be effective, they should include the use of assisted reproduction techniques. Unfortunately, these techniques have been scarcely developed in the past, and their utility has not been adequately demonstrated. This thesis addresses the use of reproduction techniques in elasmobranchs, with an emphasis on sperm retrieval and preservation. Initially, it focused on two model species, Scyliorhinus canicula and Raja radula, but later, other species were included. This revealed variability in reproductive structures, which can affect the effectiveness and quality of the techniques. Chapters 1 and 2 focused on cataloging and describing these structures in various species, highlighting best practices for gamete retrieval and insemination. The short, medium, and long-term conservation of sperm is essential for ex-situ plans, reducing the need for transporting males, conflicts during courtship, and inbreeding. Chapter 3 details formulas for maintaining sperm freshness and a medium for its survival for over 30 days. Subsequently, sperm cryopreservation in several species of rays and sharks is described using cryoprotectants. Chapter 4 addresses challenges in handling elasmobranch sperm due to its helical morphology and the formation of aggregates called spermatozeugma. The importance of viscosity in assisted reproduction techniques is emphasized. In the Discussion, the effectiveness of the methods is compared, and new opportunities are explored by having access to various species. This includes detailed descriptions with image analysis, viscosity assessment, and aggregate preservation, as well as the consideration of artificial inseminations. / This research was partially funded by the Fundación Biodiversidad ( ). PGS has a PhD contract from the European Union through the Operational Program of the European Social Fund (ESF) of the Comunitat Valenciana 2014–2020 ACIF 2018 (ACIF/2018/147). VG has a postdoc contract from the MICIU, Programa Juan de la Cierva-Incorporación (IJCI- 2017-34200). / García Salinas, P. (2023). Development and Application of Techniques for the Control of Captive Breeding in Elasmobranchs [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/201494 / Compendio