10. Cytochemical Reaction for the Determination of Endogenic Reductase

What is the objective of the cytochemical reaction for the determination of endogenic reductase?

The objective of the cytochemical reaction is to evaluate the vitality of individual sperm cells by determining the activity of the enzyme endogenic dehydrogenase located in the mitochondria of the sperm neck. This assessment provides information about the motility and vitality of the sperm cells.

What is the principle behind the cytochemical reaction for the determination of endogenic reductase?

The principle is based on the fact that the mitochondria in the neck of sperm cells produce the energy necessary for sperm motility. The enzyme endogenic dehydrogenase plays a vital role in this energy production. As the sperm cells age, the activity of this enzyme gradually decreases along with their motility. Therefore, the presence or absence of enzyme activity can be used to determine the vitality and motility of the sperm cells.

How is the cytochemical reaction for the determination of endogenic reductase performed?

The procedure involves mixing a drop of semen with an indicator (nitro VT) and incubating it at 37°C for 30 minutes. Smears are then prepared and dried in an incubator. The smears are fixed in a 10% formol solution, rinsed with water, and dried. They are subsequently stained with a 0.5% eosin solution for 5 minutes, rinsed, and dried. Macroscopic and microscopic evaluations are performed to assess the activity of endogenic reductase in the sperm cells.

How is the evaluation of the cytochemical reaction performed?

* Macroscopic evaluation: degree of purple-tinge of sample. Colour intensity relate to activity of sperm cells, their density and time elapsed since incubation
* Microscopic evaluation: activity of endogenic reductase is determined by amount, size and tinge of granules in the mitochondrial part of the sperm cells. The higher enzymatic activity, the bigger and more tinge granules appear.
* The activity of at least 400 sperm cells is determined, and the cells are divided into four groups
* 1. High activity spermatozoa: intense staining granules in whole length of mitochondrial part
* 2. Middle active spermatozoa: large and distinct granules in m-part. Some parts are pale.
* 3. Low activity spermatozoa: pale granules with low reaction in m-part.
* 4. Spermatozoa without activity of endogenic reductase