P-85: Epididymal Sperm Parameters following Sperm Mediated Gene Transfer of EDFP-pIRES Vector in Rat

Background: Sperm mediated gene transfer was approved for producing transgenic animals. There are many studies which reported different success rate by this procedure. Failure in this project mostly is returned to transfection and stability of transferred genes. The other problem that may contribute in project failure may return to the testicular damages which occur at the time of transfection, that have impact on male fertility. In this part of the study we followed the impact of gene injection on epididymal sperm parameters in rat. Materials and Methods: Total numbers of 55 head Rats were assigned in 5 groups as follow; Control (n=10) were the animals which did not receive any treatment, DOTAP (n=15) received intratesticular 100 μL of cationic DOTAP solution, D-Plasmid (n=15) that received 50 μg of EGFP-pIRES gene plasmid in 100μL DOTAP solution, Plasmid (n=15) that received 50 μg of EGFP-pIRES gene plasmid. Days 2, 5, 10, 30 and 60 after gene injection the rats were euthanized with ethical considerations and their cauda epididymis was separated and minced in TALP medium for 15 min. Then sperm parameters include sperm motility, viability and any morphological abnormalities were analyzed using conventional procedures which approved by WHO. Data were analysed using GLM procedure in SAS. Results: The results showed that sperm count and sperm motility were not affected by testicular microinjection (p>0.05). There was significant changes in percentages of sperm abnormalities and sperm viability following injection of different Materials (p<0.05). Sperm abnormalities increased at days 10 and 30 in DOTAP group (p<0.05). Injection of Plasmid with or without DOTAP cause to significant increase in the percentage of sperm abnormalities at days 2, 5 and 10 (p<0.05), then decrease at days 30 to 60. Sperm viability significantly decreased after injection of plasmid with or without DOTAP at day 2 of sperm collection (p<0.05). However, the values of sperm viability returned to normal condition at day 10 and over. Conclusion: Some sperm fertility potentials following intra-testicular microinjection are affected and may contribute in failure of intra-testicular gene transfer procedure. Optimizing the favorite time for breeding following intra-testicular microinjection may improve the procedure outcome.