Effects of sperm parameters and incubation conditions of sperms with foreign DNA in ovine sperm transfection

Document Type : Research Paper

Authors

1 Assistant Professor, Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran-Iran

2 Professor, Department of Theriogenology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

Abstract

    Sperm-mediated gene transfer (SMGT) based on the intrinsic ability of sperms to bind and take-up of exogenous DNA was introduced in 1989. From that time on, it has been a challenging topic. One of the serious challenges was the motility of transfected sperms and hence their ability to fertilize the oocyte. The present study aimed to determine the effects of media and incubation conditions and also sperm parameters on the transfection of ovine spermatozoa by foreign DNA. In this study, the effects of various incubation temperature (5, 20 and 37 ºC), three different media (PBS, TCM and DMEM), presence of FBS in medium, viability and motility of sperms and sperm capacitation in DNA absorption rate and intensity were assessed by using rhodamine-labeled EGFP plasmid. Results showed that incubation of spermatozoa with plasmid in 37ºC leads to more transfection rate but various incubation media and presence or absence of FBS had no significant effect on DNA uptake rate and intensity. Motility rate and capacitation of sperms had no significant effects too. However, in sperms with a damaged membrane, the DNA uptake rate increased significantly. All of the spermatozoa with true DNA absorption (post acrosome absorption) were immotile and none of the examined treatments in this study could produce motile transfected spermatozoa. Regarding the results of this study, it seems that membrane-damaged spermatozoa incubated with foreign DNA can be used for SMGT-ICSI to produce transgenic animals.

Keywords


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