Homo sapiens (Spermatozoa (sperm))

(Short-Term Storage of Human Spermatozoa in Electrolyte-Free Medium Without Freezing)


This protocol is actually an alternative to cryopreservation that does not involve freezing and is suitable for short-term storage of human spermatozoa. The authors were attempting to minimize the loss of sperm motility and also minimize DNA fragmentation. The authors report that for short-term storage (1 week) this protocol performs significantly better than cryopreservation with regard to both metrics.


• Bovine Serum Albumin (BSA; Sigma-Aldrich, St. Louis, MO, cat # A7906)
• Percoll (Sigma Chemical, St. Louis, MO)
• Glucose (Sigma Chemical, St. Louis, MO)
• Human tubal fluid medium (HTFM; catalog no. ART-1006; Quinn’s Sperm Washing Media; Cooper Surgical Inc., Trumbull, CT)


1. Place 1 ml of semen on the top of a discontinuous Percoll gradient consisting of 1 ml of 66% (vol/vol) Percoll, 0.5% BSA, 0.33 M glucose; 4 ml of 44% (vol/vol) Percoll, 1.5% BSA, 0.33 M glucose; and 3 ml of 22% (vol/vol) Percoll, 2.5% BSA, and 0.33 M glucose.
2. Centrifuge the column for 20 min at 400 x g at room temperature.
3. Resuspend the pelleted sperm in 0.4 ml of Electrolyte-Free Medium (EFM; 0.33 M glucose, 3% BSA)
4. Divide the sperm suspension into 15 µl aliquots in 0.2 ml tubes and store at 4°C.
5. To revitalize the sperm after storage, add 15 µl of HTFM and incubate for 10 min at 37°C.


Riel JM, Yamauchi Y, Huang TT, Grove J, Ward MA. Short-term storage of human spermatozoa in electrolyte-free medium without freezing maintains sperm chromatin integrity better than cryopreservation. Biol Reprod. 2011 Sep;85(3):536-47.

Added by: Carlton Hoyt on July 25, 2012

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