Abstract
Aim: To investigate the influence of lipofection treatment and exogenous DNA uptake on the quality of sahiwal cattle spermatozoa. Materials and Methods: Semen collected from sahiwal bulls (n=7) were evaluated separately for color, volume, mass activity, concentration, motility and viability using standard procedures. Pooled sperm samples from selected bulls (n=3) were transfected with a model gene construct enhanced green fluorescent protein (p-EGFP) via lipofection method and confirmed the genome integration by PCR technique. Furthermore the effect of transfection on spermatozoa was assessed based on apotosis, viability and motility. Results: In the current investigation sahiwal bulls were selected based on their breeding records and better semen characteristics. Although the transfected sperm samples failed to show florescence under fluorescence microscope, PCR studies confirmed the successful uptake of the p-EGFP gene in to the host sperm cell genome. Moreover transfected samples showed a significant reduction in the viability and motility without causing any DNA damage induced apoptosis as demonstrated by DNA Ladder assay.
Highlights
Liposome mediated DNA delivery method i.e. lipofection is a technique used for introducing nucleic acids into cells and embryos
The capacity of sperm cells to capture foreign DNA had been reported by Brackett et al (1971) where, they observed that Simian virus 40 (SV40) adsorbs on surface of rabbit spermatozoa, but does not penetrate the cells [2]
For the present study sahiwal donor bulls were selected based on their semen qualities and thereby demon-strated that good quality sperm cells can be successfully transfected with pEGFP via lipofection without causing any apoptosis induced DNA fragmentation
Summary
Liposome mediated DNA delivery method i.e. lipofection is a technique used for introducing nucleic acids into cells and embryos. This report provided the first evidence that a heterologous genome can be incorporated into mammalian spermatozoa and subsequently carried into an ovum during the process of fertilization. This finding and its implications were ignored for around twenty years and rediscovered after the report by Lavitrano et al, in 1989 that mouse sperm cell can act as a vector for transferring foreign gene to the generation [3]. It has been reported in Sea urchin sperm cells. After this many success reports of in-vitro uptake of DNA constructs by animal sperm cells have been presented [3,5,6,7,8,9,10]
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