Thesis (Ph.D.). / Written for the Dept. of Neurology and Neurosurgery. Title from title page of PDF (viewed 2008/07/24). Includes bibliographical references.

Thesis (M.Sc.). / Written for the Dept. of Neurology and Neurosurgery. Title from title page of PDF (viewed 2009/06/18). Includes bibliographical references.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The presence of drugs in aquatic environments has been studied for decades and their actions have as consequence changes of several physiological mechanisms. It is known that there is a complex chemical communication between the species that inhabit this environment, but the perception of intra and interspecific communication substances is unknown. The studies developed aim to evaluate if the fish are able to identify different stress situations and drugs, analyzing the neuroendocrine, behavioral and osmoregulatory effects. Seven studies were elaborated: 1) acute stress test and osmoregulatory evaluation with fluoxetine; 2) test of the action of fluoxetine in different responses of stressors (physical or chemical); 3) individual perception test to different conspecific situations; 4) testing preference at different concentrations of drugs and detection by olfaction; 5) experimental anosmia test in type-anxiety behavior; 6) experimental anosmia test, by ZnSO4, in behavioral and physiological responses; 7) test of the effects of acute stress on social behavior and "anxiety" in jundias (Rhamdia quelen). Based on the results it was verified that the acute exposure to fluoxetine is able to inhibit osmoregulatory changes caused by stress. Fluoxetine attenuates the cortisol response to physical stressor stimulus, but not to chemical stressor stimulus. Zebrafish can perceive and trigger aversive behaviors when in contact with conditioned waters of physical, chemical, and food stress (acute fasting). Zebrafish is attracted to psychotropic drugs such as diazepam, fluoxetine, risperidone and buspirone, which are probably detected by olfaction. Temporary experimental anosmia (by lidocaine and ZnSO4) modulates anxiety behaviors in adult zebrafish. Jundia can be used for behavioral studies of "anxiety" and social interaction. In general, these results contribute to a better understanding of the effects and perceptions of pharmacological action and chemical communication in fish. / A presença de fármacos em ambientes aquáticos tem sido estudada há décadas e suas ações têm como consequências alterações de diversos mecanismos fisiológicos. Sabe-se que existe uma complexa comunicação química entre as espécies que habitam esse ambiente, mas a percepção de substâncias das comunicações intra e interespecíficas é desconhecida. Os estudos desenvolvidos objetivam avaliar se os peixes são capazes de identificar diferentes situações estressoras e fármacos, analisando os efeitos neuroendócrinos, comportamentais e osmorregulatórios. Foram elaborados sete estudos: 1) teste de estresse agudo e avaliação osmorregulatória com fluoxetina; 2) teste da ação da fluoxetina em diferentes respostas de estressores (físicos ou químicos); 3) teste de percepção individual a diferentes situações de coespecíficos; 4) teste de preferência a diferentes concentrações de fármacos e detecção pela via olfatória; 5) teste de anosmia experimental em comportamento tipo-ansiedade; 6) teste de anosmia experimental, por ZnSO4, em respostas comportamentais e fisiológicas; 7) teste dos efeitos do estresse agudo sobre comportamento social e "ansiedade" em jundiás (Rhamdia quelen). Com base nos resultados, verificou se que a exposição aguda de fluoxetina é capaz de inibir as alterações osmorregulatórias causadas pelo estresse. A fluoxetina atenua a resposta de cortisol a estímulo estressor físico, mas não a estímulo estressor químico. O zebrafish pode perceber e desencadear comportamentos aversivos quando em contato com águas condicionadas de estresse físico, químico e alimentar (jejum agudo). O zebrafish apresenta atração por psicofármacos como diazepam, fluoxetina, risperidona e buspirona, os quais provavelmente são detectados pela via olfatória. A anosmia experimental temporária (por lidocaína e ZnSO4) modula comportamentos de ansiedade em zebrafish adulto. O jundiá pode ser utilizado para estudos comportamentais de "ansiedade" e interação social. De modo geral, esses resultados contribuem para um melhor entendimento dos efeitos e percepções da ação farmacológica e de comunicação química em peixes.

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No. of bitstreams: 2 Dissertação - Fabiana Dias Lopes - 2018.PDF: 5144650 bytes, checksum: e26937e82c98a8ad97b968f19bfaccef (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-09-22 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Brazil has been occupying a prominent position in agribusiness since 2008, considered one of the largest consumers of agrochemicals in the world. Among these, herbicides based on glyphosate (HGLY) have been the most used herbicides. H is able to inhibit the enzyme 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS), an enzyme present in plants, but absent in animals. 5-enolpyruvyl-xykimate-3-phosphate synthase (Gly) and RTR are quite toxic to animals, but some studies suggest that the toxicity is related to the surfactants, as well as the POEA-polyoxyethyleneamine present in the commercial formulation of RTR In this study we evaluated the toxic effect of the herbicide Gly, the POEA adjuvant and the RTR, through the acute exposure on D. rerio embryos. The embryos were exposed and evaluated in a 24-well plate for up to 96 hours observing their vital and developmental parameters through the Leica AZ microscope. The embryotoxic activity on D. rerio of the compounds (RTR, Gly and POEA) is of the concentration-time dependent type. The RTR was the most embryotoxic, presenting LC50 / 24h were 44.6 times higher than Gly and 6 times higher than POEA. All compounds led to changes in eye formation and presence of pericardial edema at concentrations chosen as sub-lethal; 3.75, 30 and 25 μg / mL, respectively, for RTR, Gly and POEA for up to 96 hours. Heart rate analysis (beats per minute - bpm) showed embryotoxicity for the compounds tested. Exposure to RTR showed a decrease of bpm at the 24 and 48 h times for the concentrations of 7.5 and 3.75 μg / mL. At 96 h the concentrations of 3.75 and 1.875 μg / mL were equally significant. Gly had an effect on bpms with difference only at 24 and 48h times, most evident at concentrations of 120 and 60 μg / mL. The POEA, unlike Gly, presented a change in bpm at 48 and 72 h for the concentrations of 50, 25 and 12.5 μg / mL. The morphological changes observed were pericardial edema, spinal curvature, which, with advancing exposure, especially in sub-lethal concentrations, intensified; and associations such as hatching delay and death arose. Finally, the results showed high toxicity of RTR compared to Gly and POEA and a great concern for human health, using the experimental model D. rerio. / O Brasil vem ocupando uma posição de destaque no agronegócio desde 2008, considerado um dos maiores consumidores de agrotóxicos do mundo. Dentre estes, os herbicidas a base de Gly (HGLY) tem sido os herbicidas mais utilizados. O HGLY é capaz de inibir a enzima 5-enolpiruvoil-xikimato-3-fosfato sintase (EPSPS), enzima presente nas plantas, porém ausente nos animais. O HGLY Roundup Transorb® (RTR) vem apresentando grandes efeitos tóxicos e sua ação sobre diferentes organismos apontam para a inibição da enzima acetilcolinesterase em animais. Estudos sugerem que o Gly (Gly) e RTR são bastante tóxicos para os animais, porém alguns estudos sugerem que a toxidade esteja relacionada com os surfactantes, tal como o POEA – poli-oxietilenoamina, presentes na formulação comercial de RTR. Neste estudo avaliamos o efeito tóxico do herbicida Gly, do surfactante POEA e de RTR, através do teste de embriotoxidade com o Zebrafish, Danio rerio. Os embriões foram expostos e avaliados em placa de 24 poços por um período de até 96 horas observando seus parâmetros vitais e de desenvolvimento através do microscópio Leica AZ. A atividade embriotóxica observada sobre D. rerio dos compostos (RTR, Gly e POEA) demonstrou ser concentração e tempo dependente. O RTR foi o mais embriotóxico apresentando CL50 24h 44,6 vezes maior que o Gly e 6 vezes maior que POEA. Todos os compostos levaram a alterações na formação dos olhos e presença de edema de pericárdio, nas concentrações eleitas como sub-letais; 3,75, 30 e 25 µg/mL, para respectivamente RTR, Gly e POEA, em período de até 96h. A análise da taxa de batimentos cardíacos (batimentos por minuto - bpm) evidenciou embriotoxidade para os compostos expostos. A exposição ao RTR apresentou diminuição dos bpm nos tempos de 24 e 48 h para as concentrações de 7,5 e 3,75 µg/mL. No tempo de 96h as concentrações de 3,75 e 1,87 µg/mL, foram igualmente significativas. O Gly apresentou efeito sobre os bpms com diferença somente nos tempos de 24 e 48h, de modo mais evidente nas concentrações de 120 e 60 µg/mL. O POEA, diferentemente do Gly, apresentou alteração no bpm nos tempos de 48 e 72 h para as concentrações de 50, 25 e 12,5 µg/mL. As alterações morfológicas observadas foram edemas de pericárdio, curvatura espinhal, que com o avançar da exposição, em especial em concentrações sub-letais, se intensificaram; com o surgimento de associações como retardo de eclosão e morte. É possível que os resultados obtidos quanto a taxa de batimentos cardíacos e mortalidade de embriões e larvas de D. rerio com RTR sejam o resultado sinérgico de Gly e POEA, demonstrando grande preocupação para a saúde humana e ambiental, utilizando o modelo experimental D. rerio.

During vertebrate gastrulation, a well-orchestrated series of cell movements leads to the formation of the three germ layers: ectoderm, mesoderm and endoderm. In zebrafish, a model organism for vertebrate development, the mesendodermal progenitor cells separate from the ectodermal cells and migrate towards the animal pole. To identify proteins controlling these processes, I used a comparative proteomic approach following two alternative strategies: (1) Based on the notion that Wnt11 regulates cell movement and morphology during gastrulation independent of transcriptional regulation, I performed a screen aimed at the identification of proteins phosphorylated upon Wnt11 signalling. To regulate Wnt11 expression tightly, I engineered a transgenic slb/wnt11-/- fish line expressing wnt11 under the control of a heat shock promoter. Using this line, I performed a quantitative comparison of protein phosphorylation with or without Wnt11 pathway activation by analysing 32P-labelled embryo extracts on 2D gels. (2) Since these experiments did not reveal any Wnt11 targets, I addressed, in the second approach, proteomic differences causal for the changes in cell adhesion and motility observed in mesendodermal cells upon involution. Quantitative 2D gel analysis comparing ectodermal and mesendodermal cells revealed 37 significantly regulated spots, 36 of which I identified by mass spectrometry. Interestingly, the majority of these proteins were not regulated on a transcriptional level as determined by an accompanying microarray analysis confirming the complementary nature of proteomics and transcriptomics. Among the identified targets, several proteins, including Ezrin2, had previously been assigned a cytoskeleton-related function. I characterised Ezrin2 in more detail showing that Ezrin2 is specifically activated by phosphorylation in mesendodermal cells and that it is required for proper gastrulation movements. In the course of this study, I developed techniques for proteomic analysis of early zebrafish embryos, including a protocol to remove the yolk. I identified several cytoskeleton-related proteins in a comparative proteomic screen for regulators of gastrulation movements. The subsequent characterisation of Ezrin2 confirmed the power of proteomics for the analysis of developmental processes. In conclusion, this work provides a foundation to study developmental and cell biological questions in early zebrafish embryos using proteomics.

Les bactéries appartenant au complexe Burkholderia cepacia (Bcc) sont des pathogènes opportunistes intracellulaires qui causent des infections pulmonaires chez les patients atteints de mucoviscidose, aggravant leur pronostic clinique. Ces infections pulmonaires sont caractérisées par des périodes chroniques avec des exacerbations intermittentes détériorant la fonction pulmonaire, et pouvant causer des nécroses broncho-pulmonaires et septicémies fatales reconnues sous le nom du “Syndrome Cepacia”. Ces bactéries intrinsèquement multi-résistantes aux antibiotiques sont aussi responsables de sérieuses infections émergentes dans des contextes hors mucoviscidose, à la fois dans des conditions intra et extra-hospitalières. Burkholderia cenocepacia, l’une des espèces les plus répandues et isolées chez les patients, est capable d’échapper à la dégradation par les macrophages de l’hôte en bloquant la maturation des (auto)phagosomes. Nous avons récemment démontré que les macrophages servent de niche essentielle pour la réplication intracellulaire de B. cenocepacia K56-2, et sont nécessaires pour le développement d’une réponse pro-inflammatoire aiguë et fatale dans des larves de poisson zèbre. Cette étude exploite d’autant plus le modèle du poisson zèbre pour mieux comprendre quels sont les facteurs bactériens et de l’hôte qui sont impliqués dans la différence entre infection aiguë et persistante, et dans la transition entre ces deux phases infectieuses.ShvR est un régulateur transcriptionnel appartenant aux LTTRs (“LysR-Type Transcriptional Regulators”) chez B. cenocepacia K56-2. Il a été démontré dans le modèle d’infection pulmonaire chez le rat, que ShvR a un rôle important dans l’induction de la réponse pro-inflammatoire, mais pas dans les infections persistantes. Pour cette étude nous utilisons une approche bioinformatique, le modèle du poisson zèbre et des études transcriptomiques afin d’obtenir plus d’informations sur le rôle de ShvR dans la virulence et dans la transition entre infection persistante et réponses pro-inflammatoires. Nos données bioinformatiques suggèrent que le gène shvR s’est adapté par évolution divergente, et a été perdu dans une sous-classe du Bcc. Grâce au modèle du poisson zèbre, nous avons démontré que ShvR n’est pas essentiel pour les stades intra-macrophagiques, mais qu’il est requis pour la dissémination de B. cenocepacia K56-2 des macrophages et pour le développement d’une réponse pro-inflammatoire fatale. Le profile persistant de l’infection a été confirmé par l’analyse du transcriptome de l’hôte, donnant plus d’informations sur les différentes réponses de l’hôte envers les infections par des mutants comparées à la souche sauvage. Le travail de cette thèse a contribué non seulement à une meilleure compréhension du rôle de ShvR et aux gènes cibles régulés par ce dernier qui joue un rôle important dans les infections aiguës, mais également à établir de nouvelles pistes pour développer de nouvelles stratégies thérapeutiques luttant contre les infections par les bactéries appartenant au complexe Bcc. / Bacteria belonging to the Burkholderia cepacia complex (Bcc) are opportunistic pathogens with an intracellular life style. Pulmonary infections with these bacteria significantly worsen clinical outcome for cystic fibrosis (CF) patients. Chronic infections with recurrent acute exacerbations deteriorate lung function with sometimes fatal necrotizing pneumonia and septicaemia (Cepacia Syndrome). These intrinsically multi resistant bacteria are also emerging as the culprit of serious infections in non-CF settings, both in- and outside the hospital. B. cenocepacia, one of the more prevalent species in the complex, is able to avoid degradation by host macrophages by arresting (auto)phagosome maturation. We have recently shown that macrophages provide a critical site for intracellular replication of B. cenocepacia K56-2 and development of acute fatal pro-inflammatory infection in zebrafish larvae. This study further explores the zebrafish infection model to better understand bacterial and host factors involved in the difference between persistent and acute infection, and the transition between these stages.ShvR, a LysR-type transcriptional regulator of B. cenocepacia K56-2, has been shown to play an important role in the induction of pro-inflammatory responses in a rat lung infection model, but not in persistent infection. We used bioinformatics, the zebrafish infection model, and host transcriptome profiling to gain more insight into the role of ShvR in virulence, and in transition between persistent and pro-inflammatory responses. Our bioinformatics study suggests that shvR has adapted by divergent evolution, and has been lost in a subclade of the Bcc. Using the zebrafish embryo model, we demonstrate that ShvR is not important for intramacrophage stages, but is required for dissemination of B. cenocepacia K56-2 from infected macrophages and the development of pro-inflammatory fatal disease. The persistent character of the infection was confirmed by host transcriptomic analysis, giving insight into the differential host response towards the mutant compared to wildtype infection. This thesis contributes to a better understanding of the role of ShvR and its possible target genes that play an important role in acute infection and to future perspectives of development of new targets for the treatment of Bcc infections.

Zebrafish thrombocytes exhibit characteristics of human platelets and megakaryocytes, making them valuable for studying megakaryopoiesis and thrombopoiesis. Using single-cell RNA sequencing, we analyzed gene expression in young and mature zebrafish thrombocytes. We identified 394 protein-coding genes unique to young thrombocytes, many corresponding with human orthologs, suggesting shared regulatory mechanisms in zebrafish and humans. We hypothesized knocking down these 394 genes should identify the novel regulatory genes that control thrombocyte maturation. To address this, we used the piggyback knockdown method to knock down these genes to study their biological functions in zebrafish thrombopoiesis. We first found the knockdown of nfe2, nfe2l1a, and nfe2l3 reduced both young and mature thrombocyte counts, confirming their role in thrombopoiesis. A comprehensive knockdown screening of the uniquely expressed genes in young thrombocytes identified 7 candidate genes associated with thrombopoiesis. We selected the spi1b gene for further mutant characterization, which revealed its critical role in young thrombocyte development, with homozygous mutations leading to embryonic lethality. Considering megakaryocyte properties in thrombocytes, we studied the potential for polyploidization in zebrafish thrombocytes. The inhibition of AURKA led to the development of polyploid thrombocytes resembling mammalian megakaryocytes, suggesting the retention of genetic programs for megakaryocyte development in zebrafish thrombocytes and providing insights into the evolutionary basis of thrombopoiesis. Thus, our study reveals critical gene expression patterns and regulatory factors in zebrafish thrombocyte development, offering insights into conserved mechanisms relevant to developmental biology and research in thrombosis and hemostasis disorder.

As a functional unit involved in both maintaining endocrine homeostasis and also producing mature eggs, the ovary plays a central role in female reproduction. The development and function of the ovarian follicles are controlled by gonadotropins released from the pituitary. It is widely accepted that the action of gonadotropins on ovarian follicles is mediated by paracrine/autocrine factors produced by the somatic cells surrounding the oocyte. Increasing evidence indicates that the Igf system is involved in mediating the action of gonadotropins in the ovary. Previously, we identified a gonad-specific Igf subtype (Igf3) distinct from Igf1 and Igf2. This fmding further highlights the importance of the Igf system in the fish ovary. In this thesis, efforts were made to understand the role of the Igf system in ovary using zebrafish as the model organism, and attention was focused on Igf3. / Because the expression of Igf3 is correlated with the LH receptor in zebrafish follicles, the regulation of igf3 by gonadotropins was subsequently studied in the ovary. The expression of igf3 was significantly up-regulated in both ovarian fragments and isolated follicles upon treatment with hCG in dose- and time-dependent manners. Treatment with 8-Br-cAMP or IBMX mimicked the effects of hCG on the expression of igf3 in follicles of different stages. / Four Igfs are present in zebrafish, and our results show that all four igfs are expressed in the ovary of zebrafish and exhibit the differential expression profiles during folliculogenesis. Using a primary culture of zebrafish follicle cells, we demonstrated that hCG stimulated igf2b and igf3 expression but suppressed igf2a expression. Moreover, the effect of gonadotropin could be mimicked by IBMX, which increased the intracellular levels of cAMP, suggesting the possible involvement of cAMP in the gonadotropin-based regulation and differential expression of igf2a, igf2b and igf3. These results also show that the Igf3 is the Igf subtype most sensitive to gonadatropin and cAMP. / In addition, the expression patterns of igf1, igf2a, igf2b, igf3, igf1ra and igf1rb were also studied during zebrafish embryogenesis. The unique temporal and spatial expression patterns of igf1, igf2a, igf2b, igf3, igf1ra and igf1rb were revealed by both real-time PCR and whole mount in situ hybridization, the results suggest divergent functions for these Igfs in early zebrafish development. / Taken together, the present studies provide substantial information about the Igf system, especially that of Igf3 in the zebrafish ovary. Data were gathered regarding Igf3 expression, regulation and functions, which is not only helpful for the understanding of the role of the Igf system in fish reproduction, but also contributes toward uncovering the ovarian signaling network involved in oocyte maturation across vertebrates. This study of igfs gene expression provides direct information to the study of Igf signaling in zebrafish. / To study the function of Igf3, bioactive recombinant Igf3 proteins were prepared using a bacterial expression system. Incubation of follicles with recombinant zebrafish Igf3 significantly enhanced oocyte maturation in time-, dose- and stage-dependent manners. The potential mechanisms of Igf3-induced oocyte maturation were then investigated. Igf3 stimulated oocyte maturation via a steroid-independent manner. Igf3 induced oocyte maturation through Igf1rs and the PI3 kinase, PDE3 and MAP kinase were necessary for Igf3-mediated oocyte maturation in zebrafish. / We first examined the gene expression patterns of Igf3 in the ovary. The igf3 gene in zebrafish was found to be alternatively spliced into two transcripts, with transcript variant 1 exclusively expressed in the gonads and transcript variant 2 only expressed during early development. Using specific antibodies developed for zebrafish Igf3, both the prepropeptide and the mature peptide forms of Igf3 were found to be predominantly expressed in the zebrafish ovary. Real-time PCR and in situ hybridization revealed that igf3 mRNA levels were relatively low in the early follicles but significantly increased after the mid vitellogenic stage (midstage III) and were high in the full grown follicles. In the full grown follicles, igf3 mRNA was detected primarily in the somatic follicular cells, with a low level of expression in the oocytes. Igf3 immunoreactivity was confined to the follicular cells alone. / Li, Jianzhen. / Advisers: Hui Zhao; Hon Ki Christopher Cheng. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 122-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Doctor of Philosophy / Department of Biology / Alexander E. Beeser / Multiple signaling pathways regulate development of the posterior zebrafish body, which is derived from a population of progenitor cells called the tailbud, a structure formed at the end of gastrulation. Fate specification and differentiation are closely linked with cell migration to ensure that, as some cells exit the tailbud and differentiate, other cells are retained in the tailbud as undifferentiated precursors to support later growth. The role of BMP signaling in specifying cell fate in the tailbud has been well-characterized. Among the lost ventral tissues like ventral tailfin and cloaca, embryos with compromised BMP signaling produce a curious phenotype-a ventrally located secondary tail containing both somitic muscle and notochord. This phenotype is proposed to be a fate-patterning defect when the BMP gradient lowered to a precise level. However, this morphogen mode is insufficient to explain secondary tail formation without considering BMP also regulates morphogenetic movements during gastrulation, promoting the convergence of lateral mesodermal cells towards the dorsal axis. In this study, we provide evidence that BMP signaling continues to mediate cell movements during tail development. Our data indicate that BMP signaling is activated in the ventroposterior tailbud to promote cell migration during tailbud protrusion, and that it is the defective migration of these cells which ultimately leads to bifurcation of the CNH domain, a presumptive stem cell pool in the tailbud, and formation of a secondary tail in BMP mutants. In parallel, the morphogenesis of tailbud cells is known to be under the control of noncanonical Wnt signaling, although the exact nature of the defect remains unclear. We find that inhibition of noncanonical Wnt signaling also leads to secondary tail formation. Additionally, we show that noncanonical Wnt signaling interacts with BMP signaling to maintain CNH integrity by affecting cadherin localization in CNH cells, possibly disrupting cell cohesion. We propose a model that BMP and a noncanonical Wnt pathway regulate tail morphogenesis by controlling cell migration and cell adhesion within the tailbud.

Cryopreservation of fish ovarian tissue fragments can be a viable alternative to cryopreservation of oocytes and embryos. The ability to cryopreserve both maternal and paternal gametes would provide a reliable source of fish genetic material for scientific and aquaculture purposes. The main aim of the present study was to develop an in-vitro culture protocol and cryopreservation protocol for zebrafish ovarian tissue fragments. In-vitro culture protocol for the tissue fragments containing stage I and stage II follicles were developed and the growth assessment of follicles were evaluated using biomarkers. To develop the cryopreservation protocol using control slow cooling method, the effect on freezing medium, cryoprotectants and cooling rate on the tissue fragments were investigated. The in-vitro culture experiments showed that L-15 medium (pH 9) containing 100mIU/ml FSH along with 20% FBS was effective for tissue fragments containing stage I and II follicles to grow in-vitro. The growth of the ovarian follicle stages was confirmed by the level of expression of p450aromA and vtg1 gene. The optimal cryopreservation protocol for the ovarian tissue fragments was found as 2M methanol+ 20%FBS in 90% L-15 medium with the cooling rate of 4°C/min. Although the highest survival rate obtained for stage II follicles within the fragments was 68.2±1.9% and stage I follicles within the fragments was 55.4±2.3% using TB staining, it showed a significant decrease in their ATP levels. This is the first study carried out on the zebrafish ovarian tissue fragments. Study on cryopreservation of the ovarian tissue fragments and development of the in-vitro culture protocol and use of biomarkers for the ovarian tissue fragments were reported here for the first time. The outcomes of this study have provided useful information for future cryopreservation protocol development.