Indução à maturação final e ovulação de lambari (Astyanax altiparanae) com análogos do hormônio liberador de gonadotropinas (GnRH)
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2020-08-05
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Universidade Estadual Paulista (Unesp)
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A presente tese foi dividida em três capítulos, no primeiro é apresentada uma revisão bibliográfica dos temas relacionados à tese, os outros dois capítulos são manuscritos gerados a partir dos experimentos realizados durante o doutorado. No manuscrito I (capítulo II) avaliamos o desempenho reprodutivo de fêmeas de lambari (Astyanax altiparanae) usando diferentes protocolos de reprodução em cativeiro, como a administração de análogo do hormônio liberador de gonadotrofina de salmão (sGnRHa) e extrato de pituitária de carpa (CPE), bem como sem o uso de terapia hormonal. Foram realizados dois experimentos consecutivos com machos e fêmeas maduros de lambari (seis meses de idade) usando dose única (Exp. 1) ou fracionada (10% + 90% - intervalo de 12h) (Exp. 2). No Exp. 1, seis doses de sGnRHa (variando de 1 a 160 µg kg-1) foram aplicadas em três repetições (cinco fêmeas e 10 machos cada); no Exp. 2, três doses de sGnRHa (10, 100 e 1000 µg kg-1) foram aplicadas em quatro repetições (cinco fêmeas e 10 machos cada). Em ambos os experimentos, 6 mg de CPE kg-1 e solução salina foram usados como controles positivo e negativo, respectivamente. A quantificação de 17α-20β-dihidroxi-4-pregnen-3-ona (17,20β-P) e níveis plasmáticos de prostaglandina F2α e avaliações histológicas ovarianas foram introduzidas no Exp. 2 separando fêmeas desovadas e não desovadas. Usando uma única dose, a taxa de desova foi baixa e semelhante entre todos os grupos (de 0 a 20%). Usando doses fracionadas, a taxa de desova de CPE (60%) foi maior do que todos os outros grupos (de 10 a 25%), com exceção da dose mais alta de sGnRHa (40%). A desova e elevação dos níveis de 17,20β-P ocorreram em algumas fêmeas do controle negativo (Exp. 2). Portanto, a terapia com doses fracionadas de CPE melhorou a ovulação de lambari, mas não foi condicional para a obtenção da ovulação. Apenas as fêmeas não desovadas induzidas com doses fracionadas de CPE, mas não com sGnRHa e nem no controle negativo, atingiram a maturação final. Além disso, a desova nos grupos com sGnRHa ocorreu em uma extensão semelhante à do controle negativo e os grupos tratados com sGnRHa não mostraram qualquer resposta dose-dependente considerando todas variáveis avaliadas. Portanto, é possível que a desova nos grupos sGnRHa se deva apenas ao agrupamento de peixes, semelhante ao controle negativo. Em conclusão, a dose fracionada de CPE foi o único tratamento que provocou maturação final em todas as fêmeas tratadas e um aumento significativo no desempenho reprodutivo em comparação com todos os outros tratamentos. No manuscrito II (capítulo III) avaliamos o efeito do análogo do hormônio liberador do hormônio luteinizante (LHRHa) com e sem domperidona no desempenho reprodutivo de lambari e na liberação de 17,20β-P e PGF2α, bem como avaliação histológica das estruturas ovarianas em dois experimentos independentes e consecutivos. No primeiro experimento cinco doses de LHRHa (20, 60, 180, 540 e 1620 μg kg-1) foram testadas com três repetições cada, enquanto no segundo experimento a dose de 180 μg kg-1 foi testada em combinação com 20 mg kg-1 de domperidona (D) com quatro repetições. Em ambos os experimentos utilizamos solução salina 0,9% como controle negativo e 6 mg de CPE kg-1 como controle positivo e as fêmeas de todos os grupos receberam duas injeções (10% + 90% - intervalo de 12h). As doses testadas de LHRHa não apresentram resultados superiores aos do controle negativo. Associando o LHRHa com D não foram obtidos resultados superiores ao LHRHa sem associação, sugerindo que a dopamina parece não apresentar um papel no controle neuroendócrino da reprodução dessa espécie ou que este antidopaminérgico não é o mais indicado para o lambari. O grupo tratado com CPE foi o único com melhor desempenho que o controle negativo, indicando que o CPE foi a melhor terapia hormonal para induzir a desova em lambari (entre os testados por nós). Houve uma diminuição nas taxas de fertilização e eclosão que coincidiram com as duas doses mais altas de LHRH (540 e 1620 μg kg-1), sugerindo que essas doses podem ter causado uma superestimulação da hipófise na produção de LH, a qual pode ter afetado a qualidade dos ovos.
The present thesis was divided into three chapters, in the first one a review of the themes related to the thesis is presented, the other two chapters are manuscripts prepared from the experiments carried out during the doctorate. In the manuscript I (chapter II) we evaluated lambari (Astyanax altiparanae) reproductive performance using different protocols of reproduction in captivity, such as the administration of analogue of salmon gonadotropin-releasing hormone (sGnRHa) and carp pituitary extract (CPE) as well as without the use of hormonal therapy. We performed two consecutive experiments with mature lambari males and females (six months old) using single (Exp. 1) or fractioned doses (10% + 90% - 12h interval) (Exp. 2). In Exp. 1, six sGnRHa doses (ranging from 1 to 160 µg kg-1) were applied in three replicates (five females and 10 males each); in Exp. 2, three sGnRHa doses (10, 100 and 1000 µg kg-1) were applied in four replicates (five females and 10 males each). In both experiments 6 mg of CPE kg-1 and saline solution were used as positive and negative controls, respectively. Quantification of 17α-20β-dihydroxy-4-pregnen-3-one (17,20β-P) and prostaglandin F2α plasma levels and ovarian histological evaluations were introduced in Exp. 2 via the separation of spawned and unspawned females. Using a single dose, the spawning rate was low and similar between all groups (from 0 to 20%). Using fractioned doses, the spawning rate of CPE (60%) was higher than all other groups (from 10 to 25%), with the exception of the highest sGnRHa dose (40%). Spawning and elevation of 17,20β-P levels occurred in some females of the negative control (Exp. 2). Therefore, only CPE fractioned doses therapy improved lambari ovulation but were not conditional for obtaining ovulation. Only unspawned females treated with fractioned CPE doses, but neither sGnRHa nor negative control, attained final oocyte maturation.In addition, spawning in sGnRHa groups occurred in a similar extent as in negative control and sGnRHa-treated groups did not show any dose dependent response considering all variables measured. Therefore, it was possible that spawning in sGnRHa groups were just due to grouping of fish, similar to negative control. In conclusion, the fractioned dose of CPE was the only treatment that provoked final oocyte maturation in all treated females and a significant enhance in reproductive performance comparing to all other treatments. In the manuscript II (chapter III) we evaluated the effect of luteinizing hormone-releasing hormone ethylamide analogue (LHRHa) with and without domperidone on reproductive performance of lambari and the quantification of plasma levels of 17,20β-P and PGF2α, as well as histological evaluation of ovarian structures in two independent and consecutive experiments. In the first, five doses of LHRHa (20, 60, 180, 540 and 1620 μg kg-1) were tested with three replicates each, while in the second the dose of 180 μg kg-1 was tested in combination with 20 mg kg-1 domperidone (D) with four replicates. In both experiments, we used 0.9% saline as a negative control and 6 mg CPE kg-1 as a positive control and the females of all groups received two injections (10%; and 90% after 12 h). The doses we tested of LHRHa did not show results superior to those of the negative control. When associating LHRHa with D, we did not obtain results superior to LHRHa without association, suggesting that dopamine does not seem to play a role in the neuroendocrine control of the reproduction of this species or that this antidopaminergic is not the most suitable for lambari. The group treated with CPE was the only one with better performance than the negative control, indicating that CPE was the best hormonal therapy to induce spawning in lambari (among those tested by us). There was a decrease in fertilization and hatching rates that coincided with the two highest doses of LHRH (540 and 1620 μg kg-1), suggesting that these doses may have caused an overstimulation of the pituitary in the production of LH, which may have affected egg quality.
The present thesis was divided into three chapters, in the first one a review of the themes related to the thesis is presented, the other two chapters are manuscripts prepared from the experiments carried out during the doctorate. In the manuscript I (chapter II) we evaluated lambari (Astyanax altiparanae) reproductive performance using different protocols of reproduction in captivity, such as the administration of analogue of salmon gonadotropin-releasing hormone (sGnRHa) and carp pituitary extract (CPE) as well as without the use of hormonal therapy. We performed two consecutive experiments with mature lambari males and females (six months old) using single (Exp. 1) or fractioned doses (10% + 90% - 12h interval) (Exp. 2). In Exp. 1, six sGnRHa doses (ranging from 1 to 160 µg kg-1) were applied in three replicates (five females and 10 males each); in Exp. 2, three sGnRHa doses (10, 100 and 1000 µg kg-1) were applied in four replicates (five females and 10 males each). In both experiments 6 mg of CPE kg-1 and saline solution were used as positive and negative controls, respectively. Quantification of 17α-20β-dihydroxy-4-pregnen-3-one (17,20β-P) and prostaglandin F2α plasma levels and ovarian histological evaluations were introduced in Exp. 2 via the separation of spawned and unspawned females. Using a single dose, the spawning rate was low and similar between all groups (from 0 to 20%). Using fractioned doses, the spawning rate of CPE (60%) was higher than all other groups (from 10 to 25%), with the exception of the highest sGnRHa dose (40%). Spawning and elevation of 17,20β-P levels occurred in some females of the negative control (Exp. 2). Therefore, only CPE fractioned doses therapy improved lambari ovulation but were not conditional for obtaining ovulation. Only unspawned females treated with fractioned CPE doses, but neither sGnRHa nor negative control, attained final oocyte maturation.In addition, spawning in sGnRHa groups occurred in a similar extent as in negative control and sGnRHa-treated groups did not show any dose dependent response considering all variables measured. Therefore, it was possible that spawning in sGnRHa groups were just due to grouping of fish, similar to negative control. In conclusion, the fractioned dose of CPE was the only treatment that provoked final oocyte maturation in all treated females and a significant enhance in reproductive performance comparing to all other treatments. In the manuscript II (chapter III) we evaluated the effect of luteinizing hormone-releasing hormone ethylamide analogue (LHRHa) with and without domperidone on reproductive performance of lambari and the quantification of plasma levels of 17,20β-P and PGF2α, as well as histological evaluation of ovarian structures in two independent and consecutive experiments. In the first, five doses of LHRHa (20, 60, 180, 540 and 1620 μg kg-1) were tested with three replicates each, while in the second the dose of 180 μg kg-1 was tested in combination with 20 mg kg-1 domperidone (D) with four replicates. In both experiments, we used 0.9% saline as a negative control and 6 mg CPE kg-1 as a positive control and the females of all groups received two injections (10%; and 90% after 12 h). The doses we tested of LHRHa did not show results superior to those of the negative control. When associating LHRHa with D, we did not obtain results superior to LHRHa without association, suggesting that dopamine does not seem to play a role in the neuroendocrine control of the reproduction of this species or that this antidopaminergic is not the most suitable for lambari. The group treated with CPE was the only one with better performance than the negative control, indicating that CPE was the best hormonal therapy to induce spawning in lambari (among those tested by us). There was a decrease in fertilization and hatching rates that coincided with the two highest doses of LHRH (540 and 1620 μg kg-1), suggesting that these doses may have caused an overstimulation of the pituitary in the production of LH, which may have affected egg quality.
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Tratamento com hormônios exógenos, Hormônios esteroides sexuais, Reprodução induzida, Desenvolvimento ovariano, Maturação e ovulação de oócitos, Prostaglandina, Manejo de reprodutores, Exogenous hormone treatment, Sex steroids hormones, Controlled reproduction, Ovarian development, Oocyte maturation and ovulation, Prostaglandin, Broodstock management