spermNMR Methods #1: Sperm washing by density gradient centrifugation

12 February 2019

When couples go for Assisted Reproduction Techniques (ART) one of the common methods for preparing sperm is called density gradient centrifugation (DGC). This is used to separate the sperm from the rest of the seminal fluid so that the sperm are ready to be used in procedures like IVF (in vitro fertilisation).

We use the separation technique DGC to prepare samples for our experiments as it ensures

  • that we are able to look at the metabolites only in the sperm themselves, rather than in the seminal fluid

  • we filter out any other cells present in semen, eg immune and skin cells, which can affect our results

  • it separates sperm into two populations of higher and lower motility which means one population of sperm can swim well and the other swims less well. We can analyse these two samples to compare differences in metabolism between the good swimmers and the poorer ones.

Below is a simple flow diagram of the steps taken to prepare sperm for our experiments.

Density gradient centrifugation method

Step one

The semen is collected in a pot by masturbation and contains seminal fluid and sperm. Freshly ejaculated semen starts very thick and viscous. Enzymes breakdown proteins in the semen. After about 30 minutes it turns to a much more watery solution and allows the sperm to swim out of the liquid so it is ready for us to process it.

Step two

The density gradient centrifugation process involves two layers of silica beads suspended in a simple fluid, such as phosphate buffered saline (PBS). Further PBS is used to form two higher and lower density concentrations (we use 40% and 80% bead solutions).

It is possible to carefully layer one solution on top of the other so that we end up with two layers; the 80% bead layer at the bottom and a 40% bead layer on top (a clear line can be seen where the refractive index of the two solutions changes). We can then carefully add the semen on top of the two bead layers.

The whole preparation is then put in a centrifuge and spun at 300 times gravitation force so that the denser sperm are pulled down through the bead layers, whilst the lighter cells and seminal fluid remain floating on top as separate layers.

Step three

After centrifugation, there are three distinct layers in the tube which provide us with different samples. At the very top is the seminal fluid and other cells. In the middle (purple box) are lower motility sperm and at the very bottom (green box) are higher motility sperm.

Step four

Each of the three layers is very carefully removed in turn (seminal fluid, lower motility sperm and higher motility sperm) so as not to cross contaminate them. A few extra washing steps are performed before being used in our experiments.

The above are only very brief details of the experiments. The full scientific protocol is provided in our publications on sperm techniques for 1H-MRS and 13C-MRS.


  1. S Reynolds, S J Calvert, M N Paley, A A Pacey; 1H Magnetic Resonance Spectroscopy of live human sperm, MHR: Basic science of reproductive medicine, Volume 23, Issue 7, 1 July 2017, Pages 441–451,

  2. S J Calvert, S Reynolds, M N Paley, S J Walters, A A Pacey; Probing human sperm metabolism using 13C-magnetic resonance spectroscopy, MHR: Basic science of reproductive medicine, Volume 25, Issue 1, 1 January 2019, Pages 30–41,