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This study was conducted to establish a freezing method of miniature pig spermatozoa. The semen ejaculated from PWG M-type miniature pig was collected by gloved-hand method. The semen was diluted with same volume extender (m-Modena B). The frozen solution used frozen solution of four different (LEY, TCG, BF-5 and m-Modena+egg yolk) for find optimal frozen solution in miniature pig sperm. The diluted semen for frozen rate assay was added to LEY solution (solution Ⅰ: 11% lactose+egg yolk; solution Ⅱ: solutionⅠ+glycerol+OEP), and frozen depending on freezing rate by the three different freezing methods (A: until 5℃ for 1 hrs, holding at —102℃ for 10 min; B: until 5℃ for 2 hrs, holding at —102℃ for 10 min; C: until 5℃ for 3 hrs, holding at —80 and —102℃ for 10 min). Semen cooled until 5℃ was added with glycerol 1, 3 and 5%, and take a equilibrium time for 0, 10 and 30min. Frozen-thawed sperm were evaluated for viability, acrosomal status and morphological abnormality. The results of frozen-thawed sperm ability by frozen solution, viability was higher in LEY solution compared to other three different frozen solution. AR pattern of LEY solution were lower than other three different frozen solution. The results of freezing rate, viability was higher in B method compared to other methods (p<0.05). Acrosomal statute was intacted in A and B methods than C method. The experiment for glycerol condition was showed that sperm viability was higher in extender with 1% and 3% glycerol and equilibrium time of 0 min. The acrosome damage was lower in extender with 1% glycerol and equilibrium time of 10 min than other conditions. In conclusion, the optimal conditions for cryopreservation of miniature pig spermatozoa obtained in LEY frozen solution, cooling rate of 1～2 hrs, 1～3% glycerol concentrations and glycerol equilibrium time of 0～10 min.
