Improvement in the long-term stability of freeze-dried mouse spermatozoa by adding of a chelating agent.
This study demonstrates that a small amount of chelating agent in the freeze-drying solution is necessary to prevent the deterioration of spermatozoa during freeze-drying and subsequent preservation at 4 degrees C. We freeze-dried mouse epididymal spermatozoa in the solutions containing Tris-HCl and ethylenediaminetetraacetic acid (EDTA) as a chelating agent. Spermatozoa stored for various times up to 1 year at 4 degrees C were injected intracytoplasmically into individual oocytes, and the normality of chromosomes in fertilized oocytes was analyzed. In addition, embryos derived from freeze-dried spermatozoa were transferred into recipients to determine their developmental ability. Chromosomes were maintained well when spermatozoa were freeze-dried in a solution containing 10 mM Tris-HCl and 1mM EDTA (73%), and 57% of embryos developed to term. Of embryos derived from spermatozoa stored for 1 year, 65% developed into live offspring. On the other hand, when spermatozoa were freeze-dried in a solution containing 10 mM Tris-HCl and 0 or 50 mM EDTA, spermatozoa that maintained karyotypically normal chromosomes were 64% or 22%, and only 16% or 3% of embryos were developed to term, respectively. This finding suggested that mouse spermatozoa can be freeze-dried in a simple solution containing the same composition as that used to preserve extracted DNA.
Kaneko
Takehito
T
Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto 860-0811, Japan. take@kumamoto-u.ac.jp
Nakagata
Naomi
N
D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D005306:Fertilization; D005612:Freeze Drying; D006863:Hydrogen-Ion Concentration; D008297:Male; D051379:Mice; D012662:Semen Preservation / Q000379:methods*; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods; D013094:Spermatozoa / Q000378:metabolism*; D013696:Temperature
D002614:Chelating Agents; D004492:Edetic Acid