All biological and non-biological particles in nature are known to have an electrostatic potential. During sperm maturation in the epididymis, negative charged glycoproteins are added to the sperm membrane (Veres, 1968), which provides the sperm its characteristic negative membrane potential (Bedford, 1983). The sperm negative membrane charge was termed the zeta potential or electro-kinetic potential by Ishijima etal. (1991). In the Zeta sperm selection method, this negative membrane charge is utilized as a biomarker to select mature sperm. Later, Chan et al (2006) developed the Zeta test to select sperm according to its electrostatic potential.
The zeta sperm selection method is performed on density gradient centrifugation selected sperm. A new centrifuge 15 mL tube is used as a platform to isolate highly negatively charged sperm. The electrostatic charge of an untouched centrifuge tube is positively and will attract negatively charged sperm. Touching the tube without the use of glove will ground the tube, resulting in the loss of electrostatic potential. Extreme care should be taken to place the tube inside a latex glove up to the cap and hold the cap of the tube at all time. DGC washed sperm (0.1 mL) is diluted with 5 mL of serum-free HEPES - HTF medium and gently pipetted in the tube. The tube with the sperm sample should be rotated two or three turns clockwise and incubated at room temperature (23° C) for 1 min to allow adherence of the charged sperm to the wall of the centrifuge tube. Following incubation, the tube is centrifuged at 200 X g for 5 min and the tube is slowly inverted to drain out all non-adhering sperm and other contaminant cells. The excess liquid at the mouth of the tube is removed by placing the tube upside down on a tissue paper. Three percent serum supplemented with HEPES-HTF medium (0.2 mL) is pipetted into the tube, by allowing the medium to trickle down the side of the tube wall. This process helps to neutralize
Figure 1.4 Diagram of sperm selection using the Zeta test. Negatively charged mature sperm is adhered to the positively charged tube surface, while immature sperm remain suspended in the media. (See color plate section forthe color representation of this figure.)
the positive charge of the tube and detach the adhering sperm from the wall (Figure 1.4). The collected medium at the bottom of the tube is re-pipetted and used to rinse the wall of the same tube several times to increase the concentration of recovered sperm (Chan et al., 2006; Kam etal., 2007; Khajavi etal., 2009).