Selection of the Most Appropriate Medium for Assessing Motility and DNA Uptake of Bovine Spermatozoa

Abstract

Objective: The aim of this study was to select the best medium to maintain spermmotility during sperm-DNA incubation and assess the DNA uptake by spermatozoaof Iranian Holstein bulls and its effects on sperm motility.Materials and Methods: frozen sperms from an Iranian Holstein bull were thawedand centrifuged. Motile sperms were separated through Puresperm gradient(40/80%) followed by two times washing in SP-TALP medium. Then, sperms werewashed once (PBS, Opti-MEM and SP-TALP) and incubated with DNA in each mediafollowed by sperm motility estimation. The plasmid pEGFP-C1 was linearizedand incubated with sperms at 37°C for 1 hour. Sperm-DNA mixture was treated withDNase I and the sperm pellet was washed with PBS.DNA extraction from sperms and supernatants from the last washing were used astemplate for PCR. Data was analyzed using SAS package and mean comparisonsbetween sperm motility in different media were performed.Results: Sperm motility after incubation in PBS, Opti-MEM and SP-TALP were40(±2.89), 2(±1.53) and 54(±4.41) percent, respectively. PCR results from transfectedsperms indicated that EGFP transgene internalized into the bovine spermsand DNaseI treatment could not eliminate it.Conclusion: In conclusion the best medium for sperm and DNA incubation was SPTALP.The DNA not only could attach to the post acrosomal region of spermatozoabut also could integrate into it. So bovine spermatozoa can be used as transgenecarrier into oocyte.

Keywords

• Transfection
• Sperm Motility
• Transgene
• EGFP
• PCR