Effects Of Two Different Cryoprotectants On Fertilization Rate Of Frozen-Thawed Mouse Sperm

Introduction: The purpose of this research was to study the effects of two cryoprotectants on viability, motility, morphology and fertilization rate of mouse sperm after freezing-thawing.
Material and Methods: Spermatozoa were isolated from caudae epididymis of NMRI male mice (8 to 10 weeks-old) and divided to three groups of control, experimental 1 and 2. Cryoprotectant solution for the first experimental group was made of 18% raffinose (W/V) and 3% skim milk (W/V) in distilled water and for the second experimental group was made using 7% glycerol (V/V), 1% glucose (W/V) and 25% egg yolk (V/V) in PBS. They were added to sperm (1:1). After 2 minutes equilibration at room temperature, the samples were first cooled by introducing to nitrogen vapoure for 10 minutes and plunged directly into liquid nitrogen. For thawing, the samples were removed from nitrogen and diluted quickly. To remove the cryoprotectant from sperm, it was washed twice using T6 medium contaning 5% mg / ml BSA. After incubation, percentage of viability, motility and morphology of sperm at experimental groups examined and compared with control group. To evaluate the fertilization rate of frozen sperm, female NMRI mice, 6-10 weeks old, were superovulated using IP injection of 7.5 IU hMG and 7.5 IU hCG 48 hours later. Ovulated oocytes were collected from the ampullary portion of the oviducts at 12-13 hrs after hCG injection. After that oocytes were inseminated with epididymal sperm and examined pronucleous formation and development to two cell embryos.
Results: Results of this research showed that viability rate of spermatozoa at groups of control, experimental 1 and 2 were 80.33%, 35.8%, 20.5% respectively and the observed differences were significant. Their motility 75.4%, 31.8%, 18.6% and normal morphology were 57.2%, 43.2%, 37.8% respectively and again the differences were significant. Fertilization rate of sperm were in control and two experimental groups 87%, 31%, 20%. All differences were significant (between exp. and control groups).
Conclusion: Conservation of mouse sperm is still in research, however raffinose and skim milk is a suitable cryoprotectant and preserve sperm against freezing damages better than glycerol-glucose, but more studies to improve freezing techniques are required.