Resumo
Nos últimos anos, Pimelodus maculatus vem sendo utilizado como espécie modelo para o desenvolvimento de técnicas de biotecnologia em Siluriformes. Neste estudo foram abordados aspectos da micromanipulação de embriões e obtenção de espermatozoides de P. maculatus. Assim, o objetivo do artigo 1 foi identificar e rastrear a rota migratória das Células Germinativa Primordiais (PGCs) em P. maculatus. Inicialmente, foram testadas soluções para incubação de embriões decorionados, e a solução de Characin apresentou resultados satisfatórios. Para identificar as PGCs, os embriões decorionados foram microinjetados com mRNA 3'UTR GFP-nos1, e o desenvolvimento foi monitorado em estereomicroscópio de fluorescência. Inicialmente, as PGCs foram visualizadas no estágio 6-10 somitos na região medial do embrião. Posteriormente, as PGCs migraram gradativamente na direção anteroposterior e, no estágio 20-24 somitos, localizaram-se próximo à extremidade posterior da região de extensão do vitelo. Nas larvas recém-eclodidas, as PGCs foram encontradas nas cristas genitais. No artigo 2, foi estabelecida uma metodologia não letal para obtenção de espermatozoides de P. maculatus, e também descritos aspectos da morfologia dos espermatozoides e da histologia dos testículos. No experimento 1, avaliamos os parâmetros espermáticos de amostras obtidas de peixes submetidos a diferentes protocolos de indução: 1) Solução Fisiológica Salina (controle), 2) Extrato Bruto de Hipófise de Carpa (CCPE) 10 mg kg-1 e 3) CCPE 10 mg kg-1 + ocitocina 5 UI kg-1 . O protocolo que apresentou melhores resultados foi a indução com CCPE 10 mg kg-1 , e no experimento 2 foi comparado ao procedimento de indução hormonal seguido de maceração dos testículos. Neste experimento, não houve diferença entre esses procedimentos quanto aos parâmetros avaliados, o que indicou que a indução com 10 mg kg-1 de CCPE pode ser satisfatoriamente utilizada para a obtenção do sêmen de P. maculatus sem a necessidade de sacrificar os machos para remoção dos testículos. Com relação à morfologia, P. maculatus exibiu espermatozóides do tipo aquasperms, com características típicas de espécies com fertilização externa. A análise histológica dos testículos revelou diferenças entre as regiões anterior e posterior, onde a primeira mostrou atividade espermatogênica, enquanto a segunda apresentou atividade secretora.
In the last few years, Pimelodus maculatus has been used as a model species for the development of biotecnology techniques in Siluriformes. In this study were covered aspects of embryo micromanipulation and obtainment of sperm cells of P. maculatus. Thus, the aim of article 1 was to identify and trace the migratory route of Primordial Germ Cells (PGCs) in P. maculatus. Initialy, it was made a screening of solutions for incubation of dechorionated embryos, and Characin solution showed satisfactory results. To identify the PGCs, dechorionated embryos were microinjected with GFPnos1 3'UTR mRNA, and development was monitored in fluorescence stereomicroscope. Initially, PGCs were visualized at 6-10 somites stage in the medial region of the embryo. Later, PGCs migrated gradually in anteroposterior direction, and in the 20-24 somites stage, were located near the posterior extremity of the yolk extension region. In newly hatched larvae, PGCs were found in the genital ridges. In article 2, we have established a non-lethal methodology to obtain sperm cells from P. maculatus, and have also described aspects of spermatozoa morphology and histology of testes. In experiment 1, we assessed the sperm parameters of samples obtained from fish submitted to different induction protocols: 1) Physiological Saline (control), 2) Crude Carp Pituitary Extract (CCPE) 10 mg kg-1 and 3) CCPE 10 mg kg-1 + Oxytocin 5 UI kg-1 . The protocol that showed the best results was induction with CCPE 10 mg kg-1 , and in experiment 2 it was compared to the procedure of hormonal induction followed by testes maceration. In this experiment, there was no difference between such procedures concerning the parameters evaluated, which indicated that induction with CCPE 10 mg kg-1 may be satisfactorily used to obtain semen from P. maculatus without having to kill the fish for testes removal. With regard to its morphology, P. maculatus exhibited aquasperm spermatozoa, with some typical characteristics of species with external fertilization. Histological analysis of the testes revealed differences between the anterior and posterior regions, where the former showed spermatogenic activity, while the latter showed secretory activity.
Resumo
Objetivou-se avaliar o efeito da manipulação de horas de luz e temperatura da água na produção de ovos de Astyanax altiparanae durante o inverno. O experimento foi realizado entre julho e agosto de 2013 (57 dias), utilizando-se dois grupos de 32 casais (G1 e G2). No G1 não foram controladas horas de luz e temperatura da água, enquanto que no G2 essas variáveis foram manipuladas de forma a apresentar valores próximos aos registrados na primavera (média de horas de luz e temperatura da água: 13,85 e 24,40°C, respectivamente). A cada 14 dias, 8 casais de cada grupo foram submetidos à indução hormonal com extrato bruto de hipófise de carpa, tendo-se acompanhado a liberação, número, fertilização e diâmetro dos ovos, a evolução dos ovários, e sobrevivência larval até o terceiro dia de vida. Posteriormente, foram estimados custos, receita e lucro da produção. A proporção de fêmeas do G2 que liberaram oócitos foi superior ao G1 (81,25 contra 9,38%; p=0,0000), assim como o número de ovos (g fêmea)-1 (G1: 710,95 ± 335,59; G2: 262,81 ± 179,42; p=0,0328). Os ovos do G1 apresentaram diâmetro médio superior aos do G2 (608,16 ± 22,57 contra 603,20 ± 24,34 m; p=0,0042). Não foi verificada diferença entre os grupos para os valores das taxas de fertilização e sobrevivência das larvas (p=0,0853 e p=0,2434, respectivamente). Nos ovários do G2 foi observada alta incidência de folículos pós-ovulatórios, oócitos primários e pré-vitelogênicos, enquanto que no G1 a maioria dos oócitos se encontrava em atresia. O lucro operacional da produção de ovos e larvas no regime de horas de luz e temperatura do G2 foi superior ao G1 (US$ 976,558 contra US$ -75,033), sendo o mesmo observado para a margem bruta (552,16% contra -77,74%). Em suma, os parâmetros reprodutivos verificados, juntamente com a análise econômica, indicam que a manipulação ambiental pode ser associada às atuais práticas de manejo reprodutivo estendendo a produção de ovos e larvas por maior período do ano.
The aim of this study was (1) to evaluate the effect of manipulating hours of light and temperature on the induced reproduction of Astyanax altiparanae during winter (out-of season), and (2) compare the reproductive parameters observed during winter and spring (natural breeding season). During the first phase of the experiment, accomplished during July and August 2013 (winter, 57 days), two groups of 32 couples were used. The first group was kept in natural environmental conditions of hours of light and temperature, on the other hand, on the second group these variables were manipulated in order to achieve similar conditions to those observed in spring. Each 14 days, eight couples of each group were hormonally induced with carp pituitary extract, and then eggs were collected after natural spawning. Amount of eggs, fertilization rates and diameter were measured from each spawn. Additionally, microscopiest changes in ovaries, larval survival (3th day) were evaluated. Costs, incoming and profit of the production were estimated. In the group that environmental conditions were controlled, the proportion of females that spawned was higher (81,25 vs.9,38%; p=0,0000), and the same was observed for the number of eggs (g female)-1 (710,95 ± 335,59 vs. 262,81 ± 179,42; p=0,0328). However, in this group, the diameter of the eggs was smaller (603,20 ± 24,34 vs. 608,16 ± 22,57 m). Values of fertilization and survival rates did not differ among the groups (p=0,0853 and p=0,2434, respectively). Most of the oocytes of fish kept in natural environmental conditions were atretic, in the other group were observed many postovulatory follicles, primary and previtellogenic oocytes. Operational profit from eggs and larvae produced by manipulation of hours of light and temperature was greater (US$ 976,558 vs. US$ -75,033), as well as the gross margin (552,16 vs. -77,74%). On the second phase, which occurred during October and November 2013, 32 couples were kept in a system were hours of light and water temperature were not controlled. Every 14 days the same procedures of data collection carried out on the first phase were performed. The reproductive parameters obtained were compared to those verified on the winter group which was kept in manipulated environmental conditions. No difference was observed on the proportion of females that released oocytes (p=0,2526), however, the number of eggs (g female)-1 on spring was greater than on winter (999,61 ± 367,53 vs. 710,95 ± 335,59; p=0,0066), as well as the diameter of the eggs produced (611,61 ± 31,54 vs. 603,20 ± 24,34 m; p=0,0000). The value of fertilization rate of winter group was greater than the value of the spring group (84,85 ± 18,35 vs. 71,70 ± 26,64%; p=0,0472), and no difference was observed for the survival rate (p=0,7955). Females from both groups that spawned had ovaries with many postovulatory follicles, and primary and previtellogenic oocytes, whereas in females that not spawned most of the oocytes were atretic. Operational profit from larvae production during spring was greater than during winter (US$ 1.498,542 vs. US$ 976,558), as well as the gross margin (1.552,64 vs. 552,16%). The results of both phases indicated that environmental manipulation was efficient on A. altiparanae out-of-season spawning and lead to responses that were comparable to those observed during natural breeding season. Thus, it is advantageous to adopt this strategy in order to enable the production of eggs and larvae during most of the year.