Novas investigações sobre o desempenho reprodutivo de pacu (Piaractus mesopotamicus) envolvendo variações no uso de prostaglandina F2α exógena e no horário de indução hormonal
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Data
2023-02-28
Autores
Sato, Rafael Tomoda [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Ao longo da última década desenvolvemos um protocolo de hipofisação associado a prostaglandina F2α (PGF2α) que aumentou a previsibilidade na desova do pacu, mas ainda assim, falhas na ovulação persistem em algumas fêmeas tratadas. Por isso, esta tese teve como objetivo principal aprimorar este protocolo, investigando dois novos aspectos: a) a associação entre ovulação bem-sucedida com o momento de aplicação e a concentração de PGF2α sintética (manuscrito 1); e b) a associação entre ovulação bem-sucedida com o horário de aplicação da segunda dose hormonal, considerando especialmente os ritmos circadianos de melatonina (MTN) na espécie (manuscrito 2). Os manuscritos foram compostos por dois experimentos cada; e testes - piloto não relatados. No experimento 1 (manuscrito 1), 24 fêmeas foram hipofisadas e receberam 2 mL/peixe de PGF2α sintética no momento da segunda dose (2D); ou cinco horas (5H) ou sete horas (7H) após a segunda dose da hipofisação. No experimento 2 (manuscrito 1), as fêmeas foram induzidas com cinco doses de PGF2α variando de 1,0 a 7,0 mL/kg aplicadas no momento da segunda dose da hipofisação. O manuscrito 2, sobre ritmos circadianos de MTN, teve o experimento 1 composto por três grupos: controle (sem indução); controle salino (indução com solução salina); e hipofisação. Neste experimento, os animais tiveram o sangue coletado a cada quatro horas, ao longo de 24 horas antes de ovulação (7h dia um até 7h dia 2), para quantificação dos níveis plasmáticos de MTN. No experimento 2 (manuscrito 2), com base nos níveis circadianos de MTN, o desempenho reprodutivo com indução hormonal iniciada às 19 horas (19H) e 0 horas (0H) foi comparado. Observamos no primeiro manuscrito, sobre variações no momento de aplicação da PGF2α exógena, uma taxa de desova de 66,7% nos grupos 2D e 5H e de 50% no grupo 7H. Além disso, apenas o grupo 2D não apresentou desovas ruins (fecundidade <35.000 ovócitos/kg peixe). No experimento 2, sobre doses de PGF2α exógena, as doses mais elevadas (5,5 e 7,0 mL/kg) foram as únicas que propiciaram 100% de desova, porém a viabilidade embrionária nestes grupos não foi satisfatória. No segundo manuscrito, sobre MTN, seus níveis foram similares ao longo de 24 horas entre os grupos controle e hipofisados. Quando analisados independentemente do tratamento, observamos que os níveis de MTN aumentam no início da noite às 19 horas do dia 1 e diminuem às 15 horas do dia 2. No experimento 2, as taxas de ovulação foram de 75% e 50% para os grupos 19H e 0H, respectivamente. O grupo 19H não apresentou desovas ruins. Além disso, o grupo (19H) obteve uma produção total de larvas (números absolutos) de 737.567, enquanto o grupo (0H) obteve 250.762. Portanto, nossas observações com o primeiro manuscrito não confirmam a especulação teórica que, aplicando PGF2α mais próximo da ovulação e/ou elevando sua dose, mitigariam as falhas de desova ainda recorrentes com o protocolo em uso (estabelecido por Criscuolo-Urbinati et al., (2012)). Porém, em ambos experimentos, as fortes correlações positivas entre níveis de PGF2α no momento da ovulação e fecundidade indicam que investigações neste sentido devem ser continuadas. No segundo manuscrito, apesar do desempenho reprodutivo similar entre os grupos, a produção de larvas superior do grupo 19H indica que fêmeas submetidas a um maior período escuro pós-segunda dose possam apresentar maior sucesso na ovulação e na viabilidade embrionária. Portanto, este estudo consolida o uso da PGF2α exógena como ferramenta indutora da ovulação do pacu e traz fortes indícios iniciais, que precisam ser aprofundados, mostrando que a MTN tem associação importante com a ovulação da espécie e o início do período noturno é o mais adequado para indução hormonal, entre os períodos por nós testados.
Over the last decade we have developed a pituitary protocol associated with prostaglandin F2α (PGF2α) that increased the predictability of pacu spawning, but even so, failures in ovulation persist in some treated females. Therefore, this thesis had as main objective to improve this protocol, investigating two new aspects: a) the association between successful ovulation with the moment of application and the concentration of synthetic PGF2α (manuscript 1); and b) the association between successful ovulation and the time of application of the second hormonal dose, especially considering the melatonin circadian rhythms (MTN) in the species (manuscript 2). The manuscripts were composed of two experiments each; and tests - pilot not reported. In experiment 1 (manuscript 1), 24 females were hypophysed and received 2 mL/fish of synthetic PGF2α at the time of the second dose (2D); either five hours (5H) or seven hours (7H) after the resolving dose of hypophysation. In experiment 2 (manuscript 1), females were induced with five doses of PGF2α ranging from 1.0 to 7.0 mL/kg applied at the time of the resolving dose of hypophysation. Manuscript 2, about circadian rhythms of MTN, had experiment 1 composed of three groups: control (without induction); saline control (induction with saline solution); and hypophysation. In this experiment, the animals had their blood collected every four hours, over the 24 hours before ovulation (7h day one to 7h day 2), to quantify the plasma levels of MTN. In experiment 2 (manuscript 2), based on circadian levels of MTN, reproductive performance with hormone induction starting at 7 pm (19H) and midnight (0H) was compared. In the first manuscript, on variations in the moment of application of exogenous PGF2α, a spawning rate of 66.7% in the 2D and 5H groups and of 50% in the 7H group. Furthermore, only the 2D group did not have poor spawning (fecundity <35,000 oocytes/kg fish). In experiment 2, on exogenous PGF2α doses, the highest doses (5.5 and 7.0 mL/kg) were the only ones that provided 100% spawning, but embryonic viability in these groups was not satisfactory. In the second manuscript, on MTN, its levels were similar over 24 hours between the control and pituitary groups. When analyzed independently of treatment, we observed that MTN levels increase in the early evening at 7 pm on day 1 and decrease at 3 pm on day 2. In experiment 2, ovulation rates were 75% and 50% for groups 19H and 0H, respectively. The 19H group did not present bad spawns. Furthermore, the group (19H) obtained a total production of larvae (absolute numbers) of 737,567, while the group (0H) obtained 250,762. Therefore, our observations with the first manuscript do not confirm the theoretical speculation that, applying PGF2α closer to ovulation and/or increasing its dose, would mitigate the still recurrent spawning failures with the protocol in use (established by Criscuolo-Urbinati et al., (2012)). However, in both experiments, the strong positive correlations between PGF2α levels at the time of ovulation and fertility indicate that investigations in this direction should be continued. In the second manuscript, despite the similar reproductive performance between the groups, the higher larval production of the 19H group indicates that females subjected to a longer post-resolving dose dark period may be more successful in ovulation and embryonic viability. Therefore, this study consolidates the use of exogenous PGF2α as a tool to induce pacu ovulation and provides strong initial evidence, which needs to be deepened, showing that MTN has an important association with the ovulation of the species and the beginning of the nocturnal period is the most appropriate for hormonal induction, between the periods we tested.
Over the last decade we have developed a pituitary protocol associated with prostaglandin F2α (PGF2α) that increased the predictability of pacu spawning, but even so, failures in ovulation persist in some treated females. Therefore, this thesis had as main objective to improve this protocol, investigating two new aspects: a) the association between successful ovulation with the moment of application and the concentration of synthetic PGF2α (manuscript 1); and b) the association between successful ovulation and the time of application of the second hormonal dose, especially considering the melatonin circadian rhythms (MTN) in the species (manuscript 2). The manuscripts were composed of two experiments each; and tests - pilot not reported. In experiment 1 (manuscript 1), 24 females were hypophysed and received 2 mL/fish of synthetic PGF2α at the time of the second dose (2D); either five hours (5H) or seven hours (7H) after the resolving dose of hypophysation. In experiment 2 (manuscript 1), females were induced with five doses of PGF2α ranging from 1.0 to 7.0 mL/kg applied at the time of the resolving dose of hypophysation. Manuscript 2, about circadian rhythms of MTN, had experiment 1 composed of three groups: control (without induction); saline control (induction with saline solution); and hypophysation. In this experiment, the animals had their blood collected every four hours, over the 24 hours before ovulation (7h day one to 7h day 2), to quantify the plasma levels of MTN. In experiment 2 (manuscript 2), based on circadian levels of MTN, reproductive performance with hormone induction starting at 7 pm (19H) and midnight (0H) was compared. In the first manuscript, on variations in the moment of application of exogenous PGF2α, a spawning rate of 66.7% in the 2D and 5H groups and of 50% in the 7H group. Furthermore, only the 2D group did not have poor spawning (fecundity <35,000 oocytes/kg fish). In experiment 2, on exogenous PGF2α doses, the highest doses (5.5 and 7.0 mL/kg) were the only ones that provided 100% spawning, but embryonic viability in these groups was not satisfactory. In the second manuscript, on MTN, its levels were similar over 24 hours between the control and pituitary groups. When analyzed independently of treatment, we observed that MTN levels increase in the early evening at 7 pm on day 1 and decrease at 3 pm on day 2. In experiment 2, ovulation rates were 75% and 50% for groups 19H and 0H, respectively. The 19H group did not present bad spawns. Furthermore, the group (19H) obtained a total production of larvae (absolute numbers) of 737,567, while the group (0H) obtained 250,762. Therefore, our observations with the first manuscript do not confirm the theoretical speculation that, applying PGF2α closer to ovulation and/or increasing its dose, would mitigate the still recurrent spawning failures with the protocol in use (established by Criscuolo-Urbinati et al., (2012)). However, in both experiments, the strong positive correlations between PGF2α levels at the time of ovulation and fertility indicate that investigations in this direction should be continued. In the second manuscript, despite the similar reproductive performance between the groups, the higher larval production of the 19H group indicates that females subjected to a longer post-resolving dose dark period may be more successful in ovulation and embryonic viability. Therefore, this study consolidates the use of exogenous PGF2α as a tool to induce pacu ovulation and provides strong initial evidence, which needs to be deepened, showing that MTN has an important association with the ovulation of the species and the beginning of the nocturnal period is the most appropriate for hormonal induction, between the periods we tested.
Descrição
Palavras-chave
Prostaglandinas, Melatonina, Peixe desova artificial, Ovulação induzida, Fotoperiodismo