EMMA SOPs - Spermfreezing

 

Cryopreservation of Mouse Spermatozoa in 1.8ml cryotubes

Equipment

Method

Place 9ml Sigma H2O (W-1503) in a screw top 15ml Falcon tube (2097) and equilibrate to 60oC in a water bath. Add 1.8g raffinose and dissolve by gentle inversion. Add 0.3g skim milk and dissolve by gentle inversion. Make up to 10ml if necessary. Aliquot into microfuge tubes and centrifuge at 14,000 rpm for 10 min. Collect supernatant and filter the solution using a 0.45µm syringe end filter. Place 1.1ml aliquots into cryotubes and store at -20oC.

Cryopreservation method

To prepare the cooling apparatus place a platform (e.g. the insert from a Gilson yellow tip box) into the polystyrene box. This acts as a support for the cryotube rack. Carefully pour liquid nitrogen into the polystyrene box to just cover the platform. Place a cryotube rack on top of the platform so that it is suspended in liquid nitrogen vapour. Replace the lid on the polystyrene box and allow it to fill with vapour. Replenish the liquid nitrogen as necessary during the freezing session, but do not allow the level to rise above the platform.

Thaw one 1.1ml aliquot of cryoprotectant solution for each male mouse and bring to 37oC in the incubator or hot block. Mix by inversion if there is any precipitation. Pipette 1.0ml CPA into a small culture dish and place on the hot block at 37oC. Dissect the vasa deferentia and cauda epididymides from the mouse and clean off all fat and blood. This is easily achieved by placing the organs on a paper tissue and examining them under a microscope. Transfer the cleaned organs to the dish of CPA and using watchmaker's forceps mince the epididymis and squeeze sperm gently out of the vas deferens. To disperse the sperm, gently shake the culture dish for ~30 seconds. Place the dishes lid on a shelf in the CO2 incubator, then rest the base of the dish, containing the sperm, at an angle on the lid. Incubate for sperm preparation for 10 minutes at 37oC.

Keeping the culture dish at an angle, remove the animal tissues from the suspension by scraping them to one side of the culture dish with a pipette tip. Place 100µl aliquots into each of 8 cryotubes and tighten the screw cap to seal the cryotube. Place the cryotubes in a pre-cooled rack, supported in the liquid nitrogen vapour phase of the freezing apparatus and leave for 10 minutes. Remove the cryotubes from the freezing apparatus and plunge them into liquid nitrogen. The sperm samples can now be stored in a liquid nitrogen refrigerator until required.

Thawing mouse spermatozoa frozen in cryotubes

Equipment

Method

Note: Take special care when following this protocol that the cryotubes are not filled with liquid nitrogen before plunging them into the water bath, such tubes may explode. If liquid nitrogen is present in the cryotube, wait for it to evaporate and escape before plunging the cryotube into the water bath.

Using forceps, hold the cryotube in air for 30 seconds and then thaw rapidly by plunging it into a 37oC water bath. When the sample has thawed, gently agitate the cryotube to ensure the sperm sample is evenly mixed. Then gently pipette 5µl of the sperm suspension directly into your in vitro fertilization drop. Sperm motility will initially appear low but within 10 minutes sperm quality and motility can be assessed. If necessary add a second aliquot of sperm to the in vitro fertilization drop. The in vitro fertilization drops are now ready to accept the cumulus-oocyte masses.

 

Cryopreservation of Mouse Spermatozoa in French straws

Equipment

Method

To prepare the freezing canister: insert a piece of styrofoam tightly into the bottom of a 50 ml-syringe, heat seal the outlet of the syringe, and fix the syringe to the acrylic bar. Thaw an aliquot of CPA in the incubator. Fill one well of the 4-well dish with 200 µl 0.9% NaCl, another well with 160 µl CPA.

Sacrifice the male mouse by cervical dislocation and dissect the two caudae epididymides. Place the epididymides into 0.9% NaCl of the 4-well dish (on ice, alternatively at 37oC). Under a microscope remove all remaining fat and big blood vessels with a watchmaker forceps and a spring scissor. Place both caudae epididymides into the CPA in the well of the 4-well dish (on ice). Cut them several times with a spring scissor and let the spermatozoa swim out for 3-5 minutes (on ice or at 37oC). Shake the dish carefully until the suspension is homogenous.

Pipette 10 x 15 µl-aliquots on the lid of a dish. Connect the 1 ml syringe with a 0.25 ml straw, aspirate successively 100 µl HTF medium, an air bubble, one 15 µl-drop, another air bubble. Heat seal both ends of the straw. Repeat for all drops. Place samples in the freezing canister and put it in the liquid nitrogen vapour phase of the flask and leave it there for 10 minutes then plunge the freezing canister directly in liquid nitrogen (-196oC).

Place both caudae epididymides into the CPA in the well of the 4-well dish

Place both caudae epididymides into the CPA in the well of the 4-well dish.

Thawing of mouse spermatozoa frozen in French straws

Equipment

Method