3.3.1 Cryo-preservation of Medaka sperm
Takao Sasado, Yasuko Kota, Makoto Furutani-Seiki, Hisato Kondoh
JST/SORST Kondoh Research Group, Kinki-chihou Hatsumei Center, Yoshida-Kawaracho 14, Sakyo-ku, Kyoto 606-8305, Japan
184.108.40.206 An overview
Freezing sperm coupled with artificial insemination provides an efficient means to preserve mutant or transgenic strains of model animals. With Medaka (Oryzias latipes), techniques for sperm freezing have developed to the level where its routine practice is reliable. We have been successful in using glass capillaries to hold sperm to freeze, and describe below the procedures.
The technique is based on a combination of two important elements, the freezing medium to suspend sperm developed by Aoki et al. (1997) and a simple freezing procedure using glass capillaries developed by Krone and Wittbrodt (1997). There are several advantages of using capillaries to hold sperm. First, sperm derived from a testis divided into multiple capillaries, which are separately thawed for use in artificial insemination. This feature is particularly advantageous in preserving and reviving precious fish lines. Second, it is space- saving if a large number of specimens are to be collected. Third, the air space between the glass capillaries and holding plastic cryo-tube provides an excellent damper for temperature change, allowing gradual yet rapid enough temperature decrease of the sperm suspension placed in a cryogen.
220.127.116.11 The procedure of cryo-preservation consists of the following steps:
(1) Preparation of sperm suspension on ice.
(2) Preparing 10 microL aliquots of the suspension using glass capillaries.
(3) Distributing the capillaries in plastic cryo-tubes.
(4) Placing the tube in a larger plastic tube embedded in crushed dry ice, allowing the suspension to freeze in 20 minutes.
(5) Putting the tube in liquid nitrogen, first in a Dewar flask and then in a large storage container.
(6) Examination of motility of the remaining sperm.
(1) Preparation of sperm suspension
• Sharp tweezers (Dumont No.5) X2
• Dissecting scissors
• 0.5 mL plastic centrifuge tubes (The lid part is used as a chamber to prepare sperm suspension)
• Large Styrofoam box containing ice (inner size 27X17X17 cm) for pre-cooling of tubes.
• Small Styrofoam box filled with ice (inner size 9X8X8 cm) for sperm preparation.
(2) Preparing 10 micro L aliquots of the suspension using glass capillaries.
• Glass capillaries (minicaps - disposable microcapillaries, 10 microL, Hirschmann laborgeraete #9000110)
• Mouth-pipeting assembly consisting of a capillary holder, connecting long tubing (1 m long) and mouthpiece.
(e.g. Drummond Scientific Company, Aspirator tube assembly / Clear, #2-040-000).
(3) Distributing the capillaries in plastic cryo-tubes.
• Cryo-tube (Nunc #377267,1.8 mL)
(4) (5) Freezing in dry ice and in liquid nitrogen.
• Large Styrofoam box containing dry ice (inner size 27X17X17 cm).
• Eight 15 ml centrifuge tubes.
• Dewar flask (1 L)
• Liquid nitrogen tank to store frozen sperm
• Four timers
(6) Examination of sperm motility
• Inverted phase contrast microscope
• Slide glass
(1) Reagents for freezing medium:
• Fetal calf serum (FCS) for tissue culture, heat-inactivated at 56 C for 30 min, cleared by centrifugation and frozen as 1.5 ml aliquots.
• High grade dimethylformamide
• Freezing medium: Mix FCS and dimethylformamide at 9:1 immediately before use, and keep the medium on ice.
66 microL of the mixture is used to make sperm suspension from a fish.
(2) Balanced salt solution (BSS) to examine sperm motility (Iwamatsu, 1983; Ando and Wakamatsu, 1995).
• 20x Solution A
Phenol Red 10mg
Adjust to 1 L with Milli Q water and autoclave.
• Solution B: 5% NaHCO3 in Milli Q water. Sterilize by filtration.
• BSS: Dilute 20X Solution A 20-fold with Milli Q water, autoclave, and adjust pH to 7.4 by addition of Solution B.
18.104.22.168 Detailed procedures.
 Sexually matured males. It does not matter whether the male has been isolated from the female, as whole testes are isolated to prepare sperm suspension.
 Pre-cooling of tubes before handling testes.
(1) Insert eight (4X2) 15 mL polypropylene centrifuge tubes in crashed dry ice in a large Styrofoam box. Insert tubes to the 10 mL line. Keep the tubes with lid closed for at least 30 min.
(2) Label cryo-tubes with information of the sperm-donor fish. Two tubes per individual fish. Cool the labeled tubes on ice in a large Styrofoam box.
(3) Insert 0.5 mL centrifugation tubes in crushed ice with the lid open and the lid bottom surface attached to the ice. The rim of the lid provides the well in which testes are suspended in freezing medium. Avoid water drops getting into the lid.
(4) (Glass capillaries are kept at ambient temperature to avoid development of moisture condensation).
 Before excision of testes from a male fish:
Move two labeled cryo-tubes and a 0.5 mL tube to a small ice-filled Styrofoam box. Dispense 66 microL of chilled freezing medium in the lid of the 0.5 mL tube.
 Isolation of a testis.
Pierce the brain and cut the spinal cord of the male fish with the dissecting scissors to instantaneously kill the animal under anesthesia achieved by keeping them in ice-cold water for ten seconds. Cut the abdominal wall from anus to gill along the dorsal peritoneal cavity with the scissors. Open the wall and the dorsal peritoneum using a pair of tweezers. Hold the testis by its anal side with tweezers, isolate from the animal, clean of the peritoneum and fat while still holding, and place the testis in the freezing medium dispensed in the well of the 0.5-mL centrifuge cap.
 Preparation of sperm suspension.
Quickly mince the testis with a pair of sharp tweezers for 1 min until no testis fragments remain identifiable.
 Freezing sperm suspension on dry ice.
(1) Suck sperm suspension into a glass capillary, insert the capillary in a cryo-tube held on ice, and repeat this process three times (three capillaries are held in a cryo-tube).
(2) To avoid breaking of capillaries while tightening the cap with inside screws, the following care should be taken. First, orient the cryo-tube in a slanted position with the opening side downward, to let the capillary tips slide into the concavity of the cap. Close the cap loosely and return the tube to an upright position. In this way, the capillaries are held inside the cap.
(3) Place the cryo-tube into a 15 mL centrifugation tube inserted in dry ice, close the lid of the dry ice box, and leave for 20 min (use a timer).
(4) Repeat this process for another set of three capillaries. Two cryo-tubes containing a total of six capillaries per male fish.
 Checking motility of sperms.
(1) Add 36 microL of balanced salt solution (BSS) to the rest of the sperm suspension in the lid of a 0.5 mL tube and mix gently using the yellow tip. This corresponds to ten-fold dilution of the sperm suspension, since remaining sperm suspension in the lid is usually ca. 4 microL.
(2) Drop diluted sperm suspension on a slide glass on the stage of an inverted phase contrast microscope, and check motility and the fraction of moving sperms. Only good specimens should be used for cryo-preservation.
 Freezing of the body.
If genomic DNA of the fish is to be examined later, place the remaining part of the fish body in a cryo-tube and freeze it in liquid nitrogen.
 After 20 min freezing in the dry-ice box, remove the cryo-tubes from the 15 mL tube, close caps tightly, and place them in liquid nitrogen in Dewar flasks.
As step  to  takes around five minutes, start the procedure for the next fish. To avoid contamination, replace the whole capillary holder set with new one. Also use a new set of scissors and tweezers, or at least clean them with 70% ethanol.
 Storage in liquid nitrogen.
Transfer cryo-tubes from Dewar flasks to a liquid nitrogen tank in storage boxes or on canes. To keep to a minimum increase in temperature of once frozen sperms, cryo-tubes should be handled on dry ice in a Styrofoam box.
Ando, S. and Wakamatsu, Y. (1995). Production of chimeric medaka (Oryzias latipes). Fish Biol. J. Medaka 7, 65-68.
Aoki, K., Okamoto, M., Tatsumi, K. and Ishikawa, Y. (1997). Cryopreservation of Medaka Spermatozoa. Zool. Sci. 14, 641-644.
Iwamatsu, T. (1983). A new technique for dechorination and observations on the development of the naked egg in Oryzias latipes. J. Exp. Zool. 228, 83-89.
Krone, A. and Wittbrodt, J. (1997). A simple and reliable protocol for cryopreservation of Medaka (Oryzias latipes) spermatozoa. Fish Biol. J. Medaka 9, 47-48.