Relation between storage temperature and fertilizing ability of freeze-dried mouse spermatozoa.
The advantage of freeze-dried mouse spermatozoa is that samples can be stored in the refrigerator (+4 degrees C). Moreover, the storage of freeze-dried spermatozoa at ambient temperature would permit spermatozoa to be shipped easily and at low cost around the world. To examine the influence of the storage temperature on freeze-dried spermatozoa, we assessed the fertilizing ability of spermatozoa stored at different temperatures. Cauda epididymal spermatozoa were freeze-dried in buffer consisting of 50 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 50 mM NaCl, and 10 mM Tris-HCl (pH 8.0). Samples of freeze-dried spermatozoa were stored at -70, -20, +4, or +24 degrees C for periods of 1 week and 1, 3, and 5 months. Sperm chromosomes were maintained well at -70, -20, and + 4 degrees C for 5 months, and oocytes fertilized with these spermatozoa developed to normal offspring. Moreover, the chromosomal integrity of spermatozoa stored at -20 or + 4 degrees C did not decrease even after 17 months. In contrast, the chromosomes of spermatozoa stored at +24 degrees C were maintained well for 1 month but became considerably degraded after 3 months. In addition, to investigate the cause of deterioration of sperm chromosomes during storage at +24 degrees C, spermatozoa were freeze-dried in buffer containing DNase I. The chromosomes of spermatozoa freeze-dried with 1 or 0.2 units/ml of DNase I, 100% or 72%, respectively, exhibited chromosomal abnormalities. Our findings suggest that freeze-dried spermatozoa can be stored long-term with stability at +4 degrees C, and the suppression of nucleases present in the buffer or spermatozoa during storage led to the achievement of long-term storage of freeze-dried spermatozoa.
Kaneko
Takehito
T
Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto 860-0811, Japan.
Nakagata
Naomi
N
D000822:Animal Husbandry / Q000379:methods*; D000818:Animals; D002869:Chromosome Aberrations; D003080:Cold Temperature; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D004624:Embryo Transfer / Q000662:veterinary; D047108:Embryonic Development; D005260:Female; D005306:Fertilization / Q000502:physiology*; D047109:Fetal Development; D008297:Male; D051379:Mice; D011247:Pregnancy; D011256:Pregnancy Outcome; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D020554:Sperm Injections, Intracytoplasmic / Q000662:veterinary; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology*