Sperm Freezing Protocol
- Anesthetize male in 2g/L MS222 at 20-24oC for 30 minutes.
- While anesthetizing the 1st male:
a. Thaw cryoprotectant aliquot on ice
b. Make L-15 (with glutamax)
i. 0.74 g L-15 in 50 mL sterile water, pH 7.5 + 0.1, filter sterilize into new 50 mL conical tube
- Rinse anesthetized frog in distilled water and dissect. The following steps should be completed in twenty minutes or less for the most efficient cryopreservation.
- Remove the heart into a petri dish; take 3 punches of tissue with a biopsy punch and place each in a labeled 1.5 mL cryotube. Place the tubes into liquid nitrogen.
- Remove testes and clean by rolling each on a paper towel with the blunt side of a scalpel blade.
- Aliquot 125 uL of L-15 into two 1.5 mL microfuge tubes. Place two additional aliquots of 125 uL of L-15 into a 60 x 15mm petri dish.
a. Make sure the aliquots in the petri dish are separate and not conjoined.
b. The purpose for separating the protocol into one designed for each testis (rather than a pair of testes) is because in our experience, many males may have abnormal testes, which include possessing only one testis. In this case it is much easier to treat individual testis, rather than trying to re-aliquot different volumes, as would be the case if the protocol was designed for a pair of testes.
- Rinse each testis in the aliquots of L-15 in the petri dish. Add one testis to each of the microfuge tubes containing L-15.
- Homogenize each testis using a sterile micropestle. Homogenize for 5-6 strokes with moderate pressure.
a. Before homogenizing, pull the testis up against the wall of the tube and press with the pestle to flatten. This prevents all of the testis from being forced into the conical bottom of the microfuge tube and ensure more complete homogenization.
- Add 125 uL of cryoprotectant to each homogenized testis and mix. Consolidate the volume of both tubes into one tube using a cut pipet tip and mix briefly.
a. If a non-cut pipet tip is used, the sperm will be sheared and result in little to no fertilization later.
- Aliquot 62 uL of sperm per 0.5 mL cryotube on ice using the cut pipet tip.
- Place all sperm aliquots in a styrofoam tube rack, cover with plastic wrap and place at –80oC.
a. The styrofoam racks we use are the packaging for 15 mL conical tubes. Generally the racks are cut in half to make two racks for sperm freezing.
- After at least one day in the –80oC freezer, an aliquot is tested for fertility with in vitro fertilization.
- When enough tubes have accumulated to fill a box, the sperm samples are moved from the foam rack to a liquid nitrogen freezer box and placed in the liquid nitrogen freezer.
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Last update: Feb. 13, 2008