Appendix 8: Determining Sperm Concentration and Sperm:Egg Ratio

For experimental studies of fertilization, it is frequently essential to ascertain the sperm concentration or the sperm:egg ratio. The following procedure works well for a 1% (v/v) sperm suspension:

  1. Dilute a small amount of a 1% sperm suspension by a factor of 200, and add a small amount of fixative to the diluted sperm to immobilize them.
  2. Add a small amount of the well-mixed sperm suspension to a hemacytometer slide and examine under a 43X objective lens.
  3. Count the number of sperm over 5 separate groups of 16 of the small squares and calculate the mean.
  4. The number of sperm/ml of the original 1% suspension will be: (sperm/group) / (2x104 ml).

A 1% sperm suspension is 1-2 x 108 sperm/ml.

The sperm:egg ratio is calculated as follows: (sperm/ml) x (volume of sperm) ÷ (eggs/ml) x (volume of eggs).

Sperm:egg ratios of 100-500:1 usually give rapid, synchronous monospermic fertilization. Significantly higher ratios yield progressively higher percentages of polyspermic eggs; significantly lower ratios may result in asynchronous fertilization and fertilization of less than 100% of the eggs.