A preliminary study of reactive oxygen species activity within motile Boer buck sperm using confocal laser scanning microscope (CLSM)

Objective. There is an association between reactive oxygen species (ROS) and DNA damage to sperm. Researchers believe that ROS is always present at the sperm's head. The variation of ROS concentration within the area has an impact on the integrity of the DNA. Materials and Methods. A study was conducted to confirm the location of ROS within a group of motile Boer buck sperm. A dual photon confocal laser scanning microscope (CLSM) with a 507 nm excitation and 529 nm emission was used on thawed cryopreserved sperms mixed with 10% polyvinyl pyrrolidone (PVP) and stained with 10 mmol/l dihydrorhodamine 123. ROS production was observed and captured under the CLSM at 400x magnification and a frame rate of 35 fps. All fluorescent images were then automatically overlaid with images obtained through Brightfield (BF) to the location of the sperm's membrane. A 3-D reconstruction of the sperm was also conducted to confirm the location of ROS activity. Results. ROS activity was present at the acrosome, midpiece one third of the sperm's tail and whole area of the sperm head. ROS in immotile sperms was also present at the acrosome, one third of sperm tail and the whole area of the head compartment. Relatively fluorescence intensity of ROS was found lower in motile Boer buck sperm as compared to the immotile Boer buck sperm. Conclusion. It is concluded that sperm viability largely depends on the ability of the sperm to confine ROS within the acrosome, midpiece and one third of the sperm's tail.