RESUMO
In the present study, we examined various freezing protocols, effects of controlled seeding, and changes in cooling rate and determined the endpoint (temperature at which sample could be plugged into liquid nitrogen (LN) without visible effect on survival rate after thawing) to reveal the relative importance of each different stage of cooling on freezing success during cryobanking of carp sperm. Sperm samples from different individual carp males were frozen in 0.5 mL straws by conventional freezing. Cooling rates were determined by monitoring the sample's internal temperature. We compared four freezing protocols, which involved placing sperm samples at various levels (1, 3, 6, and 9 cm) above the LN surface (corresponding to -190°C, -150°C, -110°C, and -70°C, respectively) for 20 minutes followed by transferring the samples into LN. Freezing at 3 cm above the LN surface resulted in the highest motility (33% ± 8%) and velocity (118 ± 9 µm/s) of spermatozoa after thawing and diluting in swimming medium. We determined that -90°C is an optimal temperature at which immersing the samples in LN does not affect sperm motility after thawing and shorten the process of freezing for around three times. Motility of spermatozoa cryopreserved with or without a seeding procedure was not significantly different after thawing. Therefore, we hypothesize that supercooling the sample during the conventional freezing procedure is not the main damaging factor during carp spermatozoa cryopreservation. However, the cooling rate itself is important, because it determines the ability of the sperm to dehydrate and survive cryopreservation.