Rhamdia quelen's semen pre-cryopreservation processing and sperm viability time after thawing / Processo de pré-criopreservação de sêmen de Rhamdia quelen e tempo de viabilidade do esperma após o descongelamento

Ricardo Andrei Krause, Giovano Neumann, Ahiana Cássia de Oliveira Pedreira, Mirna Adriane Syperreck, Amanda Moreira Malacarne, Milene Neves Ferreira Braga, Sara Ugulino Cardoso, Robie Allan Bombardelli

Abstract


The study’s aim was to evaluate the sperm movement in the semen of Rhamdia quelen, cryopreserved at different times of exposure to nitrogen vapor, besides different equilibration times and with different concentrations of methanol, and in thawed semen kept at 25ºC for different periods of time. Three distinct experiments were carried out. In the first experiment, fresh semen aliquots were diluted in a solution containing 5% D-Fructose, 5% milk powder (Nestle®, Ninho Fortificado®) and 10% methanol, filled into 0.25 mL straws and immediately kept in nitrogen vapor by 0,5; 1.0; 4.0; 8.0; 12.0 and 18.0h. After these times, the straws were transferred to the liquid nitrogen (-196°C). After 72 h, the semen was thawed by immersing the straws in water at (25 ° C) for 10 seconds. The sperm activation was evaluated by diluting the thawed semen in distilled water in the proportion of 1:250 (semen:activating solution; v/v). Motility and spermatic velocity in thawed semen were measured using the CASA system. In the second experiment, the fresh semen was diluted in solutions with glucose and powdered milk (Nestle®, Ninho Fortificado®) and 5.0; 7.5; 10.0; 12.5% methanol. Immediately after, it was filled into 0.25mL straws and kept at 25ºC by equilibrium times corresponding to 0, 20, 40 and 60 minutes. Subsequently, part of the straws was used for spermatic evaluation before cryopreservation and others were submitted to cryopreservation in nitrogen vapor for 30 minutes and after transferred to liquid nitrogen (-196°C). The processes of thawing and sperm evaluation were performed as described above. In the third experiment, the cryopreserved semen was thawed and kept at 25°C for 0.0; 7,5; 15,0; 22.5 and 30.0 minutes. After each time, the spermatic movement was evaluated as described above. The exposure time of straws to nitrogen vapor and the kept time of the thawed semen at 25ºC did not affect (p > 0.05) the sperm movement. On the other hand, methanol concentrations and equilibrium time (p < 0.05) influenced the spermatic movement interactively both, before and after cryopreservation. The best results were achieved when the semen was cryopreserved immediately after dilution. The cryopreservation of Rhamdia quelen semen can be successfully performed when the semen is diluted in a solution containing 5% D-Fructose, 5% milk powder and 11.66% of methanol and immediately filled into 0.25 mL straws and kept in nitrogen vapor by 30 min before being transferred to liquid nitrogen. In addition, after thawing, the semen can be kept at 25ºC for up to 30 minutes without damage to spermatic movement.


Keywords


CASA, equilibrium time, motility, nitrogen vapor, spermatic velocity

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References


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DOI: https://doi.org/10.34117/bjdv7n4-669

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