Sparus aurata (Spermatozoa / sperm)

(Cryopreservation of Gilthead Seabream (Sparus aurata) Sperm)

Intro/Description:

This protocol is taken from a paper in which the authors attempted to create an optimized cryopreservation protocol which minimized changes in plasma membrane lipid composition, as changes in plasma membrane lipid composition have been related to a decrease in sperm quality during cryopreservation. To achieve this, they use the antifreeze protein AFPIII.

Materials:

• Sodium chloride (NaCl)
• Dimethyl sulfoxide (DMSO)
• Antifreeze protein III (AFPIII; A/F Protein Canada, Inc.)
• 0.5 ml French straws (IMV Technologies)
• Liquid nitrogen
• Water bath

Protocol:

1. Dilute sperm 1 to 6 in extender solution (1% NaCl plus 5% DMSO with 1 µg / ml AFPIII).
2. Load sperm into 0.5 ml French straws
3. Allow filled straws to equilibrate at room temperature for 4 min.
4. Place the filled straws 2 cm from the liquid nitrogen surface for 10 min.
5. Plunge the straws directly into liquid nitrogen. They are now ready for long-term cryogenic storage.
6. To thaw, place the frozen straws in a 25°C water bath for 30 seconds.

References:

Beirão J, Zilli L, Vilella S, Cabrita E, Schiavone R, Herráez MP. Improving sperm cryopreservation with antifreeze proteins: effect on gilthead seabream (Sparus aurata) plasma membrane lipids. Biol Reprod. 2012 Feb 29;86(2):59.

Added by: Carlton Hoyt on July 25, 2012

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