Establishment Of The Freeze-drying Sperm Techniques To Preserve Boar Spermatozoa And Fertilization By Intracytoplasmic Sperm Injection

Freeze-dried boar spermatozoa were stored for long time in a refrigerator at 4℃. Successful offspring production affter intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock. Freeze-drying for spermatozoa was also thought as hot topic in the field of cryopreservation, and relevant reports about boar spermatozoa was rarely found compared with other preservation methods.In the present study, the extenrnal form and ultrastructure of freeze-dried sperm was observed by staining and electron microscopy. A modified version of the neutral comet assay was employed to evaluate the effect of the freeze-drying process on boar sperm DNA integrity. The effect of different cryoprotectants (trehalose and EDTA) and Storage temperature (room temperature and -20℃) et al. on freeze-drying boar spermatozoa was tested. We wished that the results could provides a theoretical and practical basis for the development of freeze-drying mammal spermatozoa. Results show that:The protective effect of trehalose is better than EDTA for boar sperm in the freeze-drying process. Different storage times(2,4 and 6 months) of freeze-drying boar sperm of trehalose group had not obvious effect on its sperm morphology under 4℃or-20℃.In the ultrastructure of freeze-dried boar spermatozoa, main cryogenic damage in freeze-dried boar spermatozoa was swelling, damage or rupture in the sperm acrosome, neck and tail in trehalose and EDTA group. The ultrastructure of freeze-dried boar spermatozoa was better than the control group. No significant difference was found in the percentage of sperm with damaged DNA between fresh and freeze-dried boar sperm(P>0.05). The result indicated that cryopreservation can caouse boar sperm NDA damage and compared with the freeze-dried sperm it had remarkable difference (P<0.05).Those results indicated that the efficiencies of ICSI of the porcine oocytes which were activated by 1.0 kV/cm,30μs and 10μg/mL CHX was highest among other different activated protocols. The rate of male pronuclear formation (68.52%), cleavage (59.17%) and blastocyst formation (19.16%) of 0.2 mol/L trehalose groups was higher than those of 50 mmol/L EDTA group (64.59%,56.26% and 15.62%)(P<0.05). The rate of male pronuclear formation and blastocyst formation of trehalose group was significantly than control group (35.36%,52.33% and 8.60%)(P<0.01). No significant difference in the rate of male pronuclear formation, cleavage and blastocyst formation by ICSI with freeze-dried spermatozoa stored with different times at 4℃(P>0.05). The male pronuclear, cleavage and blastocyst formation of freeze-dried spermatozoa incubated for 1h was significantly than that incubated for 1-2 h in the 0.2 mol/L trehalose (P<0.01).Therefore, under the electron micoscopy, the sperm morphology was best in trehalose group. No significant difference was found in the percentage of sperm with damaged DNA between fresh and freeze-dried boar sperm(P>0.05). The efficiencies of ICSI of the porcine oocytes which were activated by 1.0 kV/cm,30μs and 10μg/mL CHX was highest among other different activated protocols.