Sperm DNA fragmentation index or Integrity test is the important tool for the male infertility. Now days there are many factors that involve the damaging of Sperm DNA. Our life style is responsible for the higher DNA integrity level of sperms. Damaged DNA is responsible for infertility, Impaired reproductive outcomes after any ART procedure, Increased risks for genetic/birth defects and rapid or continuous miscarriages.  Objective: To observe the best method for sperm preparation that results the healthier transmission of genetic information through sperms.  Material and Methods: Double Layer density gradient with Hepes buffered HTF medium, single layer density gradient with Hepes buffered HTF medium, Simple sperm wash with culture media contains Human serum albumin 5mg/ml, Endotoxin (LAL) <= 0.5 EU/ML, gentamicin sulphate 10 ug/ml, DNA fragmentation integrity kit.  Results: 50 semen samples were processed to check the sperm DNA integrity. Each semen sample was processed with all three methods. It was found that semen samples processed with Double Layer density gradient with Hepes buffered HTF medium having less Sperm DNA fragmentation index followed by the single layer density gradient with Hepes buffered HTF medium. The highest Sperm DNA fragmentation index was found with Simple sperm wash method, contains Human serum albumin 5mg/ml, Endotoxin (LAL) <= 0.5 EU/ML, gentamicin sulphate 10ug/ml.

Keywords : Endotoxin, HTF Medium, Human Serum Albumin, Male Infertility, Sperm DNA Integrity, Reactive Oxygen Species (ROS).