Sample washing :
Washing is oldest method described. The aim is to remove only the seminal plasma, and not the cellular debris, bacteria or non-motile spermatozoa.
After liquefaction, the semen is mixed with culture medium (1:1) and centrifuged at 300-400 g for 10 minutes. The supernatant is discarded and pellet is suspended in 2 ml of culture medium. This is centrifuged again at 300-400 g for 5-10 minutes and supernatant against discarded. The final pellet is suspended in 0.4-0.5 ml of medium and immediately used for insemination. This is especially useful when sperm density is very low and you are doing an IUI.
Swim – up form Pellet
This is the most widely used technique for separation of most sperms from non-motile sperms and cellular debris. It is used with normal semen samples and is based on principle active self-migration.
After liquefaction, the ejaculate is mixed with culture medium and then centrifuged at 300-400 g for 10 minutes. The supernatant is discarded and the sperm pellet is gently over lay with 1 ml of culture medium and stored inside incubator at 37°C for 30-45 minutes. The 0.4-0.5 ml of the supernatant is then gently aspirated and used for insemination.
Swim-up from Ejaculate :
This is the ideal method because it obviates the needs for centrifugation . There are some workers who still prefer to centrifuge the recovered sperms.
Discontinuous Gradient Centrifugation :
The layers are made with 1-2 ml of gradient solution (80% lower and 40% upper layer) 2 ml of semen is then carefully layered on top of 40% and centrifuged at 400-600 g for 15-20 min. cell debris, immobile an abnormal sperm all accumulate at interfaces and a soft pellet is formed at the bottom of the tube. This pellet is aspirated and suspended in 2 ml of culture medium (Depending on sperm density either simple washing or wash with swim-up technique is used). This is centrifuged at 300 g for 5-10 min. the pellet is now suspended in 0.3-0.5 ml of culture medium and used for insemination.
The final wash with culture medium to remove the gradient is drawback of this method.
Sedimentation Method / Layering under paraffin.
Only method to succeed if the number and motility of sperm is very low and is now usually used only in patients undergoing IVF with ICSI. It is very effective in removing cells and debris. The disadvantage is that the preparation takes a long time.
The initial steps are similar to the simple washing procedure. Suspend final pellet in the form of a single droplet and put it under paraffin oil in a petridish at 37°C in 5 percent Co2 incubator. Leave it for 1-24 hours depending on progression of sperms. All the cellular debris will settle at the bottom of droplet and using a fine drawn pipette under dissecting microscope one can ensure that only the upper part of droplet is picked up. Especially useful for very low counts during ICSI.
Sperm Incubation with Pentoxifylline :
It is well known that adding pentoxyfylline to the semen will enhance the motility of the spermatozoa and improved pregnancy rates.
Equal volumes of pentoxifylline solution is added to semen and centrifuged at 200 g for 5 minutes. The pellet is suspended in culture medium and centrifuge is repeated. The final pellet is suspended in 0.3-0.5 ml of culture medium and used for insemination immediately.