Abstract
Assessment of sperm vitality is an important component of semen analysis. It helps to distinguish spermatozoa that are alive and immotile from those that are dead. Sperm vitality can be assessed routinely on all semen samples by assessing the membrane integrity of the cell by identifying the spermatozoa with an intact cell membrane. This can be done by using 1) the dye exclusion test or 2) the hypotonic or hypoosmotic swelling test. Sperm vitality can therefore provide a good comparison with the motility of the sample. Eosin is used as a marker for dead cells because eosin can penetrate the cells when the membrane is damaged, while cells that have an intact membrane remain unstained. Nigrosin is a background stain that increases the contrast to the otherwise faintly stained cells [1, 2, 3, 4]. Both the single step and two-step staining using eosin and nigrosin have been used to assess sperm vitality. Both the wet preparation and the air-dried methods have been compared to study the correlation with motility [5, 6, 7]. The wet preparations evaluated by using either positive or negative phase-contrast microscopy consistently showed higher percentage of nonviable cells compared to the air-dried eosin-nigrosin smears. The air-dried smears have consistently shown that the sum of the motile (viable) and stained (presumed dead) preparations never exceeded 100 percent indicating that the air-dried method is the method of choice for determining vitality. In this chapter, we describe the staining protocols for vitality, the cut-off of motility when vitality must be tested, indications for poor motility and quality control recommended for performing sperm vitality in conjunction with basic semen analysis.
6.1 Introduction/ Background
Assessment of sperm vitality is an important component of semen analysis. It helps to distinguish spermatozoa that are alive and immotile from those that are dead. Sperm vitality can be assessed routinely on all semen samples by assessing the membrane integrity of the cell by identifying the spermatozoa with an intact cell membrane. This can be done by using 1) the dye exclusion test or 2) the hypotonic or hypoosmotic swelling test. Sperm vitality can therefore provide a good comparison with the motility of the sample. Eosin is used as a marker for dead cells because eosin can penetrate the cells when the membrane is damaged, while cells that have an intact membrane remain unstained. Nigrosin is a background stain that increases the contrast to the otherwise faintly stained cells [1, 2, 3, 4]. Both the single step and two-step staining using eosin and nigrosin have been used to assess sperm vitality. Both the wet preparation and the air-dried methods have been compared to study the correlation with motility [5, 6, 7]. The wet preparations evaluated by using either positive or negative phase-contrast microscopy consistently showed higher percentage of nonviable cells compared to the air-dried eosin-nigrosin smears. The air-dried smears have consistently shown that the sum of the motile (viable) and stained (presumed dead) preparations never exceeded 100 percent indicating that the air-dried method is the method of choice for determining vitality. In this chapter, we describe the staining protocols for vitality, the cut-off of motility when vitality must be tested, indications for poor motility and quality control recommended for performing sperm vitality in conjunction with basic semen analysis.
6.2 Principle/Mechanism of Eosin-Nigrosin Test
The dye exclusion or the eosin-nigrosin test or the vitality test is based on the dye exclusion method as a result of structural damage in the sperm plasma membrane. A damaged sperm plasma membrane as in the case of dead cells looses its semi-permeability and forms tiny pores that allow normally membrane-impermeant stains to enter the cell, whereas an intact sperm membrane does not allow the stain to enter [8].
6.2.1 One-Step versus Two-Step Eosin-Nigrosin Test
The one-step eosin-nigrosin stain contains a mixture of eosin (0.67 percent) and nigrosin (10 percent) dissolved in water [3]. This technique was later modified by including 0.9 percent NaCl in distilled water [9]. Equal volumes of liquefied semen and eosin-nigrosin solution are mixed for 30 seconds at room temperature and smears are prepared for assessing vitality.
The two-step eosin-nigrosin technique was introduced by Eliasson in 1971 [13]. In this case, the solution contains 1 percent eosin Y and 10 percent nigrosin. Semen samples are incubated with eosin for 15 seconds and nigrosin for ≤15 seconds. This technique was later modified by Dougherty et al. [14] by incubating 5 percent eosin Y for 15 seconds and nigrosin for ≤15 seconds. Björndahl et al. compared the modified one-step technique with motility and found it to be reliable, easy and simple compared to the one-step eosin alone irrespective of the number of dead cells [9].
6.3 Preparation of Stains for One- and Two-Step Eosin-Nigrosin Technique
Sperm vitality can be measured using the eosin staining either alone or in combination with nigrosin.
6.3.1 One-Step Eosin-Nigrosin Technique
This technique uses nigrosin to increase the contrast between the background and the sperm heads making it easier to visualize [8, 9].
6.3.2 Preparation of the Reagents
6.3.2.1 Eosin Y
1. Dissolve eosin Y (0.67 g; color index 45380) and 0.9 g of sodium chloride in 100 mL of purified water with gentle heating.
2. Eosin-nigrosin solution: add 10 g of nigrosin (color index 50420) to the 100 mL of eosin Y solution.
3. Boil the suspension, allow it to cool to room temperature.
4. Filter the solution through filter paper (90g/m2) to remove coarse and gelatinous precipitates.
5. Store in sealed dark-glass bottles.
6. After complete liquefaction, mix the semen sample thoroughly.
7. Mix equal volumes of the sample (50 µL) and eosin-nigrosin solution in a porcelain spot plate. Wait for 30 seconds.
8. Mix the sample again before removing another replicate aliquot and repeat step 7 above.
9. Make a smear on a glass slide and allow to air dry.
10. Mount with a cover slip and observe each slide with brightfield optics at 1000× magnification and oil immersion.
11. Evaluate at least 200 spermatozoa in replicate.
12. Calculate and report the average and percentage of vital cells from the replicate slides.
13. Report the percentage of vital spermatozoa to the nearest whole number.
6.3.3 Staining Using Eosin Alone
1. NaCl 0.9 percent (w/v): prepare by dissolving 0.9 g of NaCl in 100 mL of purified water.
2. Eosin Y: 0.5 percent (w/v): prepare by dissolving 0.5 g of eosin Y (color index 45380) in 100 mL of 0.9 percent NaCl.
6.3.4 Procedure for Staining
1. Mix the liquefied semen sample.
2. Mix one volume of the sample with equal volume of eosin Y solution on the microscope slide. Mix well by swirling the sample on the slide.
3. Cover with a 22 mm × 22 mm coverslip and leave for 30 seconds. Remix the sample and remove an aliquot, mix with eosin and repeat steps 2 and 3.
4. Examine each slide under a negative phase contrast optics at 200× or 400× magnification.
5. Evaluate at least 200 spermatozoa in duplicate.
6. Calculate and report the average and percentage of vital cells from the replicate slides.
7. Report the percentage of vital spermatozoa to the nearest whole number.
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