This method can help answer key questions in the field of sperm physiology, such as the chemical determinates of sperm quality. The main advantage of this technique is it's ability to separate large quantities of sample by quality, as well as adaptability when compared to alternative methods. Though this method can provide insight in the differences in sperm quality, it can also be applied to other samples such as mixed cell types or subcellular components with different densities.
To begin, make two 3 milliliter Percoll dilutions in two separate tubes. In a clean test tube, dilute 1 milliliter of semen sample with 2 milliliters of PBS. Pipe it gently to mix the solution.
Add 3 milliliters of the lower density Percoll sollution to a sterile conical tube. Then, carefully pipette 3 milliliters of the higher density Percoll solution beneath the lower density Percoll solution. While tilting the conical tube at a 45 degree angle, pipette 3 milliliters of the diluted semen sample on top of the Percoll density gradient.
After preparing a blank tube, centrifuge both tubes at 1500 Gs for 20 minutes. Insure that three distinct layers of semen have formed in the tub after centrifugation. Using a pipette collect the three layers of semen, starting with the top layer, followed by the middle layer, and ending with the hard pellet at the bottom of the tube.
Transfer each of the layers to a sterile micro centrifuge tube. Dilute each of the semen samples with 1.5 milliliters of PBS and centrifuge the samples at 1500 Gs for ten minutes. Finally, pour off the supernatant and reconstitute the pellets with motility buffer.
In this protocol, the Percoll density gradient centrifugation technique, or PDGC, was used to separate a semen sample into three distinct layers. Sperm separates into a high quality layer below the higher density solution, a medium quality layer between the higher and lower density solutions, and a low quality layer above the lower density solution. These differences in sperm quality are evidenced by differences in viability, mobility, and penetrability.
While attempting this procedure, it's important to remember that samples need to be brought to room temperature. The success of PDGC is reliant upon careful preparation of the gradient as well as careful collection of the isolated layers. After it's development, this technique paved the way for our research in the field of sperm perdiomics to explore bio markers of fertility in avian species.
