Step by step optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772)

Spotted wolffish (Anarhichas minor) sperm cryopreservation in 5-mL cryovials

“…Sperm cryopreserved in the 5-mL cryovials at 1.5 cm from the LN 2 and thawed either at 15 or 10°C presented similar percentage of motile cells, sperm velocity and percentage of viable cells in comparison with the sperm cryopreserved in the traditional 0.5-mL straws. In a previous work by Santana et al (2020), fertilizations rate above 80% were obtained with spotted wolffish sperm cryopreserved in 0.5-mL straws. We recommend for the cryovials the sperm to be thawed at 10°C for 6 min since this temperature is closer to the species normal temperature range.…”

“…The freezing and thawing paces were defined in a preliminary trial. A control subsample was cryopreserved in 0.5-mL straws at 4.5 cm distance from LN 2 and thawed at 5 °C for 1 min according to the protocol by Santana et al ( 2020 ). The cryotubes and control straws were stored in a LN 2 storage tank until thawing for analysis.…”

“…Spotted wolffish sperm is unique compared with sperm of other farmed species, because it is already motile at stripping, remains constantly motile for up to 2 days and loses its motility upon a contact with seawater (Kime and Tveiten 2002;González-López et al 2020). In this species, cryopreservation is used to secure sperm availability when eggs are spawned, because it is often impossible to find spermiating males (Le François et al 2008;Gunnarsson et al 2009;Santana et al 2020). Spotted wolffish spawn approximately 8000 to 30,000 large benthonic eggs (Falk-Petersen et al 1999;Falk-Petersen and Hansen 2003).…”

“…Currently, sperm is cryopreserved in 0.5-mL straws. In order to achieve good fertilization rates, depending on the sperm concentration, an approximate volume of 2 to 15 mL of fresh sperm and 2 h contact time (Beirão and Ottesen 2018 ) or 4 to 30 mL of cryopreserved sperm and 4 h contact time (Santana et al 2020 ) is recommended to inseminate 1 L of eggs (≈ 5000 eggs). This implies thawing a considerable number of straws for insemination with cryopreserved sperm.…”

Is it possible to store spotted wolffish (Anarhichas minor) sperm by refrigeration?

“…In addition, the sperm that is already motile at stripping remains motile from several hours up to 2 days, but is inactivated by contact with seawater (Kime and Tveiten 2002 ). Thus, sperm storage procedures, through cryopreservation, have been suggested as methods to ensure the availability of good quality sperm when required (Le François et al 2008 ; Gunnarsson et al 2009 ; Santana et al 2020 ).…”