It consists in determining the number of sperm per unit volume. This determination can be performed by different methods, the most common are the photocolorimeter and the Bürker chamber.
COUNT WITH PHOTOCOLORIMETER
The determination of the concentration by spectrophotometer or photocolorimeter is a method widely used in major A.I. centers. This method is based on the correlation between the number of sperms per volume unit with the opacity of the semen. However, this method has the disadvantage of presenting important errors in the determination of the concentration of sperm produced by the unpredictable opalescence of seminal plasma and because the concentration of proteins present in the said plasma is highly variable. The photocolorimeter must be calibrated regularly, to avoid mismatches of the appliance.
The photocolorimeter contains a photodetector that reacts to the light pass through the colorimetric tubes, measuring the different optical density of semen samples. This allows us to construct a curve pattern in which there is a correspondence between the values of absorbance / transmittance with different sperm concentrations. Absorbance readings at the colorimeter, of different dilutions of semen which concentration known means are carried Bürker chamber.
Once calibrated accurately, photocolorimeter provides a fast method of determining the sperm concentration.
The colorimetric method for assessing the concentration of sperm cells is performed by reading the absorbance of diluted semen sample against a solution of sodium citrate.
- Sodium citrate: 3.46 g
- Distilled water: 100 ml
Select the 560 nm wavelength filter (between 540 and 570 nm). The tube with the calibration solution is placed, and the instrument is set to 0 in absorbance.
The pattern curve is obtained by performing serial dilutions of ejaculates, with the starting concentration point measured by Bürker chamber. The different absorbance readings are made, and with them we will build the curve pattern.
Another way to construct the curve, is measuring the absorbance of 15 or 20 different ejaculates in the in photocolorimeter, of which concentrations are known with Bürker chamber, thus drawing the calibration curve.
COUNT WITH BÜRKER CHAMBER
The counting WITH Bürker chamber is the used method in farm AI centers for its simplicity and afordability.
The procedure is as follows:
- Mix the ejaculate well before taking 1 ml of pure semen with a pipette.
- Dilute 1: 100 in saline formalin 3%, using a 100 cc volumetric flask.
- Mix the solution gently, and fill with a drop of semen the Bürker chamber using the following method:
- Set the coverslip well over the Bürker chamber.
- Place the Pasteur pipette between the cover and the chamber, allowing the semen fill the reticle by capillarity.
- Observe in a microscope with 40x magnification.
- Perform the count of sperm present in 40 squares.
We will count those sperm whose heads are located within the small boxes on the grid, and those whose head hits the top and right side, and top or bottom corner of the box. Always the same two sides and two corners of the box.
The concentration of sperm per mm3 (C) is equal to the sum of sperm counted (A) X 10,000 = (C)