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Volume 61, issue 3 | Copyright
Arch. Anim. Breed., 61, 351-358, 2018
© Author(s) 2018. This work is distributed under
the Creative Commons Attribution 4.0 License.

Original study 03 Sep 2018

Original study | 03 Sep 2018

Evaluating bovine sperm transfection using a high-performance polymer reagent and assessing the fertilizing capacity of transfected spermatozoa using an in vitro fertilization technique

Ali Jafarnejad1, Mohammad Zandi2, Mehdi Aminafshar1, Mohammad Reza Sanjabi2, and Naser Emamjomeh Kashan1 Ali Jafarnejad et al.
  • 1Department of Animal Science, Faculty of Agricultural Sciences and Food Industries, Science and Research Branch, Islamic Azad University, Tehran, Iran
  • 2Department of Agriculture, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran

Abstract. Sperm-mediated gene transfer (SMGT) has been considered as an innovative device for transgenesis on a mass scale by taking advantage of live spermatozoa to transfer exogenous DNA. However, the fertilizing ability of transfected sperm cells and the poor reproducibility of this method are still matters of controversy. Hence, the current study was conducted to evaluate transfecting the enhanced green fluorescent protein (EGFP) as the source of exogenous DNA into bovine spermatozoa using a high-performance polymer reagent as well as assessing the fertilizing capacity of transfected sperm cells by in vitro fertilization (IVF). In the first experiment, three different concentrations of rhodamine-labeled DNA and high-performance polymer transfection reagent, X-tremeGENE HP, were used to transfect bovine spermatozoa. In the second experiment, IVF and fluorescence microscopy methods were utilized to assess the fertilizing capacity of sperm cells carrying exogenous DNA when X-tremeGENE HP was used either alone or with dimethyl sulfoxide (DMSO) treatment. Findings revealed that at 1µL X-tremeGENE HP and 1µg of DNA concentration, approximately one-third of total spermatozoa were transfected. However, following IVF and fluorescence microscopy, no EGFP expression was detected in zygotes and morula-stage embryos. Results of this study showed that, although X-tremeGENE HP could transfer EGFP to bovine spermatozoa, transfected sperm cells were unable to transfer foreign DNA to matured bovine oocytes. Under our experimental conditions, we hypothesized that the absence of the EGFP fluorescence signal in embryos could be due to the detrimental effects of transfection treatments on sperm cells' fertility performance as well as incompetency of IVF to produce transgenic embryos using transfected sperm cells.

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In our study, we attempted to introduce a new device for optimizing sperm-mediated gene transfer. We utilized X-tremeGENE™ HP reagent from the Roche company in bovines, and one-third of total spermatozoa were transfected. We also examined the effect of DMSO on increasing the efficiency of the sperm/DNA transfer to oocytes via IVF. We examined these two reagents in bovine spermatozoa for the first time, which can be considered as the novelty of our work together with its originality.
In our study, we attempted to introduce a new device for optimizing sperm-mediated gene...