Abstract
In spotted wolffish Anarhichas minor aquaculture, cryopreservation is used to secure sperm availability throughout the entire spawning season. Under current protocols, sperm is cryopreserved in 0.5-mL straws. This implies thawing a considerable number of straws for insemination with cryopreserved sperm. In this work, we scale up the spotted wolffish sperm cryopreservation procedure through the development of a protocol for sperm cryopreservation in 5-mL cryovials. Different freezing (distances from the liquid nitrogen surface) and thawing rates were tested. The best results were obtained with cryovials frozen at a distance of 1.5 cm from the liquid nitrogen surface and thawed either at 15 or 10 °C for 4 and 6 min, respectively. Under these conditions, similar percentage of motile cells, sperm velocity and percentage of viable cells were obtained in comparison with the sperm cryopreserved in the traditional 0.5-mL straws. This protocol will facilitate the process of insemination with cryopreserved sperm in the spotted wolffish hatcheries.
Highlights
In order to scale up the spotted wolffish sperm cryopreservation procedure and facilitate handling of cryopreserved sperm, in this study, we developed a protocol for sperm cryopreservation in 5-mL cryovials
The spotted wolffish sperm could be preserved in large storage volumes without a decrease in quality compared with small volumes
The lowest percentage of motile cells and sperm velocity were observed in samples cryopreserved at 4.5 cm distance from the LN2. For both sperm motility parameters, there was no difference between subsamples cryopreserved in the straws and in the cryotubes at 1.5 cm distance from the LN2, regardless of the thawing rate
Summary
For the VCL (μm/s), the Bartlett test indicated that there was no homogeneity of variances (p = 0.0003672), most likely because of the variability observed for the sperm cryopreserved in cryotubes using a freezing height of 4.5 cm. A significant difference in sperm velocity between the sperm cryopreserved in cryotubes using a freezing height of 4.5 cm and the fresh control was found (Fig. 1b).
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