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Methodology Chapter (2) - Dissertation Example

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2. Methodology 2.1 Experimental Plan 2.1.1 In vitro effects of sildenafil on murine embryo development In order to assess the impacts of sildenafil on embryo development, the experiment required two cell embryos that were taken from naturally mated mice and grown to blastocyst stage in an environment supplemented with sildenafil (Figure 2.1)…
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Methodology Chapter (2)
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Download file to see previous pages Figure 2.1 In vitro effects of sildenafil on murine embryo development 2.1.2 In vivo effects of sildenafil on murine embryo development This part of the experiment was not conducted due to requirement for changes in the license. However, the in vivo effects on the development of oocyte would have been examined by injecting sildenafil on female mice for a period of four days with the following three doses: 0.5 mg/kg, 1.5mg/kg and 2.5mg/kg. Afterwards, the female mice would be mated naturally with males; then one-day old zygotes would be harvested from sacrificed females. The number of oocytes and corpora lutea recorded would be used to determine the rate of ovulation. Thereafter, the embryos would be grown to blastocysts again in a media not supplemented with sildenafil. The quality of development of the embryos would then be determined by examining their development rate, and assessing the numbers of the blastocyst cells (figure 2.2). Figure 2.2 In Vivo effects of sildenafil on murine embryo development 2.1.3 Expression of PDE5a in the mouse pre-implantation embryo The experiment was conducted by examining the presence of PDE5 mRNA in murine embryos at the successive stages of embryo development. The embryos used for study were obtained from mice that had been mated naturally. On the other hand, the blastocysts had been cultured in standard conditions. 2.2 Sildenafil Citrate purification from commercially available Viagra tablets In both the in vitro and in vivo experiments, sildenafil had been cleansed from commercially available Viagra tablets by leaving 20g sephadex G-25 overnight to swell in 100ml of distilled water. A column of 80ml was then applied with the sephadex gel and equilibrated in 100ml of distilled water at room temperature. A Viagra tablet of 38mg was then placed in 91.2ml of distilled water and slowly mixed with a magnetic stirrer at room temperature for a period of twenty minutes. It was then filtered for 20 minutes under a temperature of 4 degrees Celsius. The liquid in which sildenafil has been dissolved was then applied on the column. The column was rinsed with the Viagra solution just before it was flushed with 400ml of water to wash away any possible small molecules. This way, only sildenafil was left precipitated to resin. The column was then applied with 1% Formic acid to rinse the sildenafil off. The absorbance of the rinsed solution was then observed and according to Francis et al. (2003), the rinsed sildenafil had a sharp peak absorbance at 40ml (Figure 3.1). Figure 3.1 Elution of pure sildenafil from sephadex column After sildenafil was rinsed, the column was washed again with 160ml or distilled water to eliminate the formic acid. After the column was free of formic acid, it was then washed with 320ml of 0.2% sodium azide in order to preserve it for later use. The rinsed sildenafil was then frozen at temperatures of -80 degrees Celsius. It was then dried by freezing it in a high vacuum. This was done by first sublimating the contents for a period of 8 hours at 0.37 mbar and under a temperature of -53 degrees Celsius, then desorbing it at 0.001 mbar for 3 hours. The weight of the crystallized contents was determined by examining the rate of absorbance of sildenafil over the whole sildenafil that had been eluted, assuming that there had been a recovery rate of 60%. The eluted sildenafil was then dissolved in 0.1% formic acid in order to obtain the normal concentration of refined ...Download file to see next pagesRead More
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