3.3.2 Cryopreservation of Medaka Spermatozoa (trehalose buffer)†
Tokyo Institute of Technology
22.214.171.124 General information†
To stock many lines of medaka including mutagenesis screened, simple and inexpensive method for cryopresevation of spermatozoa is useful. This protocol is modified from the method previously described (Aoki et al., 1997).
126.96.36.199 Preparation for Freezing Spermatozoa†
- Prepare 1M trehalose in deionized distilled water and 100% FCS.
- Prepare serum tubes and a foam polystyrene box.
- Keep the male fish away from females at least 1 night before.
188.8.131.52 Freezing Spermatozoa†
- Dilute 1M trehalose by 1:1 with 100% FCS and chill this preservation solution on ice.
- Kill the adult male fish by cutting the medulla and extirpate its testis.
- Squeeze or shake gently the testis in the 30mm preservation solution using a pair of forceps.
- Chill on ice if necessary (in case for several samples).
- Get the spermatozoa solution(s) into the foam polystyrene box, put the top on the box, then store at -80ºC freezer for 1 over night.
- Transfer the frozen spermatozoa solution(s) from -80ºC freezer into a container filled with liquid nitrogen, then store .
184.108.40.206 Preparation for Fertilization†
- Prepare medaka ringer (110mM NaCl, 5mM KCl, 0.9mM CaCl2, 0.8mM MgSO4, 1.2mM NaHCO3).
- Turn on a water bath switch at 37ºC
- Kill the adult female fish kept away from males by cutting the medulla and extirpate its ovary and obtain unfertilized eggs.
- Put frozen sample tube into the 37ºC water bath shortly and melt spermatozoa solution partly.
- Immediately mix with 30microl of medaka ringer and add to unfertilized eggs.
- Transfer only fertilized eggs into fresh medaka ringer.
Medaka ringer is able to be altered to FCS.
Table1. Improvement of medaka spermatozoa cryopreservation
Aoki K, Okamoto Masanori, Tatsumi K, and Ishikawa Y. 1997. Cryopreservation of medaka spermatozoa. zool. sci. 14: 641-644.