Bos taurus (Spermatogonial stem cells)

(Protocol for Cryopreservation of Bovine Spermatogonial Stem Cells in In Vitro Cultures)


For the cryopreservation of bovine spermatogonial stem cells after culture in vitro. This protocol utilizes bovine embryonic fibroblasts (BEF) as feeder cells in co-culture with bovine germ cells. The cryopreservation of in vitro cultured of sperm may be useful for research into male germ line fertility, regulatory factors of male fertility, as well as a means of preservation of a male germ line, or increased genetic gain within domestic herds. The addition of exogenous glial cell-derived neurotrophic factor (GDNF) allows for increased the maintenance of spermatogonial stem cells in vitro. The following protocol has been adapted from Oatley, et al (2004b).


• testicular parenchymal tissue
• 0.45µm pore size membrane
• Dulbecco modified Eagle medium
• Penicillin
• Streptomycin
• Liquid nitrogen
• -80C freezer
• 37C water bath
• Cryopreservation tubes
• Fetal bovine serum

For co-culture technique:
• Trypsin
• Hanks Balanced Salt Solution
• 25cm2 culture flasks
• Bovine embryonic fibroblast feeder cells
• Mitomycin-C


Basic cryopreservation:

1. Collect testicular parenchymal tissue by removing bull testes and culture on membrane (0.45µm pore size; Millipore) in 6-well culture plates (Oatley et al., 2004a).

2. Dilute cells to 1x107 cells / ml in Dulbecco modified Eagle medium (DMEM) + 30 mg/ml penicillin, and 50 mg/ml streptomycin + 10% dimethyl sulfoxide (DMSO).

3. Aliquot cells in cryopreservation tubes (approximately 1ml / tube) and store at -80°C for 24hr.

4. For long term storage, transfer vials to liquid nitrogen.

5. To thaw, incubate cells in a 37°C water bath.

6. Dilute cells in DMEM, centrifuge to pellet cells, then discard supernatant.

7. Resuspend cells in DMEM + 10% fetal bovine serum (FBS). Determine cell concentration and viability.

Embryonic Feeder cell co-culture with Bovine germ cells for cryopreservation:

Prepare bovine embryonic fibroblast (BEF) feeder cells:

1. Collect day 35 bovine embryo in saline solution, and chop with a sterile razor blade.

2. Add tissue pieces to 10ml Hanks Balanced Salt Solution (HBSS) + 0.05% trypsin + 1mM EDTA. Incubate at 37°C for 15mins.

3. Dissociate tissue by passing through a 14-gauge needle, and then allow cells to settle on ice for 2 mins.

4. Repeat steps 2 and 3.

5. Centrifuge cells at 300 x g. Discard supernatant. Resuspend cells in (DMEM) + 10% fetal bovine serum, 30 mg/ml penicillin, and 50 mg/ml streptomycin. Culture at 37°C.

Co-culture with BEF feeder cells:

6. Dilute bovine germ cells to 1 x 105 cells / ml in DMEM media.

7. Add diluted cells to a 25cm2 culture flask containing a monolayer of BEF feeder cells that have been mitotically-arrested by treatment with mitomycin-C.

8. Co-Culture cells DMEM + 10% fetal bovine serum, 30 mg/ml penicillin, and 50 mg/ml streptomycin for 1 week at 32°C in 5% CO2.

9. For cryopreservation protocol refer to the basic cryopreservation protocol above.


Oatley, J.M., D.M. de Avila, J.J. Reeves, and D.J. McLean. 2004a. Testis tissue explant culture supports survival and proliferation of bovine spermatogonial stem cells. Biol Reprod. 70:625-631.

Oatley, J.M., J.J. Reeves, and D.J. McLean. 2004b. Biological activity of cryopreserved bovine spermatogonial stem cells during in vitro culture. Biol Reprod. 71:942-947.

Added by: Carlton Hoyt on July 25, 2012

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