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Relation between storage temperature and fertilizing ability of freeze-dried mouse spermatozoa
Takehito Kaneko1, Naomi Nakagata
1Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto 860-0811, Japan.
Abstract:
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.
Insights
Freeze-dried mouse spermatozoa can be stored stably at +4°C for long-term use. Suppressing nucleases in the storage buffer is key to maintaining sperm chromosomal integrity and fertilizing ability.
Area of Science:
- Reproductive Biology
- Cryobiology
- Genetics
Background:
- Spermatozoa preservation is crucial for assisted reproduction and genetic resource banking.
- Freeze-drying offers potential for cost-effective, long-term storage and global transport of spermatozoa.
- Optimal storage conditions for freeze-dried spermatozoa require further investigation to ensure viability and genetic integrity.
Purpose of the Study:
- To evaluate the impact of different storage temperatures on the fertilizing ability and chromosomal integrity of freeze-dried mouse spermatozoa.
- To identify the optimal storage temperature for long-term preservation of freeze-dried spermatozoa.
- To investigate the role of nucleases in the degradation of freeze-dried spermatozoa during storage.
Main Methods:
- Cauda epididymal mouse spermatozoa were freeze-dried using a specific buffer solution.
- Freeze-dried spermatozoa samples were stored at various temperatures (-70°C, -20°C, +4°C, +24°C) for extended periods (up to 5 months).
- Fertilizing ability was assessed by oocyte fertilization and subsequent offspring development. Chromosomal integrity was analyzed over time, with additional experiments involving DNase I to assess nuclease activity.
Main Results:
- Spermatozoa stored at -70°C, -20°C, and +4°C maintained chromosomal integrity and produced normal offspring after 5 months.
- Chromosomal integrity remained stable for up to 17 months at -20°C and +4°C.
- Spermatozoa stored at +24°C showed significant chromosomal degradation after 3 months, linked to nuclease activity.
Conclusions:
- Freeze-dried mouse spermatozoa can be reliably stored long-term at +4°C, preserving both fertilizing ability and chromosomal integrity.
- Storage at ambient temperatures (+24°C) leads to rapid deterioration due to enzymatic activity.
- Inhibiting nuclease activity during the freeze-drying and storage process is essential for successful long-term preservation of spermatozoa.
