Magister Scientiae (Medical Bioscience) - MSc(MBS) / A functional sperm is critical for successful fertilization in order to deliver an intact genome to the site of fertilization. It is often characterized by high motility and normal morphology. Moreover, sperm hyperactivated motility is imperative for both detachment from the oviductal wall and for penetration into the zona pellucida, subsequently resulting in fertilization. Several semen parameters such as volume, colour, sperm morphology and sperm concentration are used to clinically discriminate between fertile and sub-fertile males. Additionally, several sperm functional tests assess sperm function and a male’s fertility potential. A sperm feature that is not currently assessed clinically, but could possibly discriminate between fertility and infertility, is hyperactivation. The aim of this project was to investigate motility degrees (good, medium and poor) of sperm subpopulations and induce hyperactivation in each subpopulation, as well as to sperm in semen, by addition of caffeine and procaine. This was achieved by separating three sperm subpopulations from a semen sample using the Puresperm density gradient separating technique. Sperm subpopulations were exposed to 5mM caffeine and 2 mM procaine respectively for 15, 30, 60, 90 and 120 minutes. Sperm in semen was exposed to caffeine and procaine using a flush technique and analysed at 0, 5, 15, 30, 45 and 60 minutes. Sperm displaying hyperactivation was determined using cut-offs for curvilinear velocity, linearity and amplitude of lateral head displacement. The results indicate significant differences in overall percentage motility, sperm kinematic parameters and hyperactivation among the three subpopulations (p<0.05). Procaine and caffeine both induced hyperactivation in subpopulations, although the most pronounced effect of procaine was evident after 15-30 minutes compared to caffeine (60-90 minutes) in subpopulations. Maximum hyperactivation of sperm in semen was seen after 15- 30 minutes in both procaine and caffeine. Moreover, caffeine had significantly higher stimulating effect than procaine. The results suggest that the existence and distinct motility characteristics of subpopulations should be considered in future during clinical assessment of male fertility, especially when assessing hyperactivation. The immediate and higher stimulation response of sperm with the flush technique indicates that the technique may be an ideal sperm functional test compared to the separation technique. The separation technique may be used to categorize sperm subpopulation of a patient in terms of motility (high motile or low motile) and to stimulate such subpopulations with chemicals for use in assisted reproduction technologies.