Aim: The C57BL/6 mouse strain is now commonly used for producing transgenic/knockout strains. However, the fertilizing ability of these spermatozoa decreases as a result of cryopreservaion. Although the micromanipulation technique has been established to increase their fertilizing ability, it requires a considerable degree of technical skill. In the present report, we investigate the simple microdissection of zona pellucida by laser to increase the fertilizing ability of cryopreserved spermatozoa. Methods: C57BL/6J spermatozoa were cryopreserved using a solution consisting of 18% raffinose/3% skim milk. Oocytes of the same strain were placed in PB1 medium containing 0, 0.25, 0.50 or 0.75 mol sucrose. The zona pellucida of oocytes was microdissected by laser with different pulse lengths selected from 0.45 to 0.65 ms. Microdissected oocytes were then fertilized with cryopreserved spermatozoa, and the subsequent development of embryos was assessed. Results: When oocytes were microdissected in PB1 medium without sucrose, 81.5% of the oocytes were fertilized. The fertilization rates increased significantly as the pulse length was lengthened when compared with oocytes with intact zona pellucida. Furthermore, normal offspring were obtained in all experiments. Conclusion: The fertilizing ability of cryopreserved spermatozoa is improved when oocytes with their zona pellucida microdissected by laser were used. (Reprod Med Biol 2006; 5: 249-253).
Aim: The C57BL/6 mouse strain is now commonly used for producing transgenic/knockout strains. However, the fertilizing ability of these spermatozoa decreases as a result of cryopreservaion. Although the micromanipulation technique has been established to increase their fertilizing ability, it requires a considerable degree of technical skill. In the present report, we investigate the simple microdissection of zona pellucida by laser to increase the fertilizing ability of cryopreserved spermatozoa. Methods: C57BL/6J spermatozoa were cryopreserved using a solution consisting of 18% raffinose/3% skim milk. Oocytes of the same strain were placed in PB1 medium containing 0, 0.25, 0.50 or 0.75 mol sucrose. The zona pellucida of oocytes was microdissected by laser with different pulse lengths selected from 0.45 to 0.65 ms. Microdissected oocytes were then fertilized with cryopreserved spermatozoa, and the subsequent development of embryos was assessed. Results: When oocytes were microdissected in PB1 medium without sucrose, 81.5% of the oocytes were fertilized. The fertilization rates increased significantly as the pulse length was lengthened when compared with oocytes with intact zona pellucida. Furthermore, normal offspring were obtained in all experiments. Conclusion: The fertilizing ability of cryopreserved spermatozoa is improved when oocytes with their zona pellucida microdissected by laser were used. (Reprod Med Biol 2006; 5: 249-253).
Entities:
Keywords:
C57BL/6 mouse; cryopreservation; in vitro fertilization; laser‐microdissection; spermatozoa