Sperm concentration and motility

The sperm concentration and motility/kinematic parameters were determined with Computer Aided Sperm Analysis [CASA] (Sperm Class Analyzer version 5.4 - SCA®, Microptic, S.L., Barcelona, Spain) with a disposable eight-cell chamber Leja slide (LJ; 20-μm depth; Leja® Products B. V., Nieuw-Vennep, the Netherlands) at 37°C. The SCA® is equipped with a Basler A312fc digital colour camera (Microptic, S.L., Barcelona, Spain), mounted on a Nikon E200 Microscope (IMP, Cape Town, South Africa) and a stage warmer (Omron™, Kyoto, Japan) which was heated to 37°C. The camera settings for the SCA® system are listed in Table 3.1.

Table 3. 1: Camera settings for the SCA® system

Parameter Setting

Acquisition control

Acquisition mode Timed

Exposure time 19900

Time base 20 µs

Exposure time 995

Acquisition frame rate 50 FBS

Counter and timer controls Time duration Raw 4095

Gain 435

Analog controls Black level 168

Balance ration 77

Balance ration 1.20313

FBS = frame per second.

In accordance with the manufacturer’s guidelines, 2 μl of the pre-incubated semen (37°C, 5 % CO2) was loaded into a single chamber of the Leja slide using an Eppendorf micropipette. If bubbles were found, another chamber was loaded. The slide was then allowed to rest on the warm stage (37°C, 30sec) to avoid liquid flow in the chamber. All the preparations were made with pre-warmed (37°C) slides and pipette tips. A minimum of 1000 spermatozoa per semen sample were analysed in several systematically selected areas within the central part of each chamber. The CASA parameters assessed are listed in Table 3.2. As displayed by the SCA® system, various motility colour tracks are presented in Figure 3.2.

Figure 3. 2: SCA® analysis displaying the different colour paths for motility rating. Red (Type A); green (type B); blue (Type C) and yellow (type D)

The following SCA® settings were used; green filter; Ph1 condenser; positive phase contrast observation setting; brightness ± 400; contrast ± 100; objective, 10x; eyepiece, 10x; capture, 50 images per second; chamber, Leja 20; scale, 10x; 2µ²˂ particle area ˂50µ²; VCL, 25μm/s<slow<40μm/s, 40μm/s<medium<50μm/s, 50μm/s<rapid; progressivity, >80 % of STR;

circular, <50 % LIN; connectivity, 12; VAP points, 5μm/s; filter, on; temperature, 37°C. WHO reference values adopted in this study are as follows: sperm concentration, ≥15 10⁶/mL; total motility

>40 %; progressive motility >32 %.

Table 3. 2: SCA® concentration, motility and kinematic parameters

WHO = World Health Organization, T.S.C. = total sperm count, VCL = Curvilinear velocity, VSL = Straight-line velocity, VAP = Average path velocity, LIN = Linearity, WOB = Wobble, ALH = Amplitude of lateral head displacement, BCF = Beat-cross frequency, µm = micrometre, s = second, Hz = hertz.

3.2.7 Sperm morphology

For sperm morphology assessment, approximately 10 μl of semen (≥20x10⁶ spermatozoa/mL), was placed near one end of a labelled microscope slide (76 x 26mm). A second slide was placed onto the drop at a 45° angle and allowed to spread along its back edge, then moved forward spreading the semen drop over the surface of the first slide to make an even smear. The slide was left on the lab bench overnight to air dry at room temperature. Following the air drying period, the slide was fixed and stained according to the manufacture’s guidelines (Van der Horst and Maree, 2009). The slide was immersed into a Coplin jar containing SpermBlue® fixative (SpermBlue® fixative, Microptic, S.L., Barcelona. Spain), and left undisturbed for 10 minutes. The slide was removed carefully from the Coplin jar and left upright at an angle of about 70^◦, allowing contact with a paper towel to drain the excess fixative. The slide was then dipped into a Coplin jar containing SpermBlue® staining solution (SpermBlue® stain, Microptic, S.L., Barcelona. Spain) and left for a 15 minutes period, after which it was gently immersed in distilled water for few seconds to remove the excess stain. The slide was then left to drain and air-dried at room temperature as described above. Following completion of the staining procedures, the slide was permanently mounted by applying a drop of non-aqueous DPX mounting medium (Dako, CA, USA) and a cover slip to the slide. Two morphology slides were prepared from each sample.

Morphological characteristics of the stained spermatozoa were evaluated by Computer Aided Sperm Morphology Analysis (CASMA) using the SCA® module. (blue filter, 100x oil immersion objective, 10x eyepiece, brightness±435, contrast=100). The SCA® system equipped with a Basler A312fc digital colour camera (Microptic, S.L., Barcelona, Spain), mounted on a Nikon Eclipse 200 Microscope (IMP, Cape Town, South Africa). As shown in Figure 3.3, a total of 100 spermatozoa per semen sample from various systematically selected microscopic fields were analysed according to WHO criteria (WHO, 2010). Overlapping or clumping spermatozoa and those with heads obscured by tail or debris were excluded, as they were not possible to be analysed adequately.

Figure 3. 3: SCA morphology analysis of spermatozoa stained with SpermBlue. Acrosome region stains yellow, postacrosomal region of the head dark blue, and the midpiece green.

3.2.8 Sperm viability

The integrity of the sperm plasma membrane was estimated by means of a dye exclusion technique (WHO, 2010), using the membrane-impermeant Eosin-Nigrosin stain (Sigma-Aldrich, St Louis, MO, USA), which penetrates only the compromised plasma membrane of dead and dying cells. A 10 μl of diluted semen adjusted to a concentration of 5x10⁶ spermatozoa/mL was added and mixed with 20 μl Eosin and 30 μl Nigrosin. A drop of the mixture was spread across the length of a labelled microscope slide using another slide to create a uniformly spread smear. After air drying, the slide was mounted using a DPX mounting medium (Dako, CA, USA) and covered with a cover slip. Two viability slides were made from each sample. Viability status of spermatozoa was assessed using CASA (Sperm Class Analyzer version 5.4 - SCA®, Microptic, S.L., Barcelona, Spain) equipped with a Basler A312fc digital colour camera (Microptic, S.L., Barcelona, Spain), mounted on a Nikon Eclipse 200 Microscope (IMP, Cape Town, South Africa), with bright field optics. From each sample, the viability

of 100 spermatozoa was evaluated using the counter module of the SCA^® system (positive phase contrast observation setting; 20x objective lens; 10x eyepiece and a blue filter). The number of pink-stained (non-viable) spermatozoa and those unpink-stained (viable) were counted in randomly selected fields. Results were reported as percentage of viable spermatozoa (Figure 3.4).

Figure 3. 4: Viability assessment of spermatozoa stained with dye exclusion, using the counter module of the SCA® system. Unstained cells (1) are viable; Stained cells (2) are non-viable.