Measurement of Spermatozoal Concentration

Measurement of the concentration of spermatozoa in ejaculates is a fundamental and extremely important step in the evaluation and then in the processing of ejaculates for artificial insemination.

The simplest and most inaccurate method to estimate spermatozoal concentration is by means of visual inspection. This conservative processing technique is used effectively when, for example, a minimum number of insemination doses must be produced and when semen availability is not limited. In the case of liquid boar semen, for example, a fresh ejaculate that has the appearance similar to whole milk, i.e. creamy white, might be extended to yield 6–8 insemination doses; this would compare with 20–24 doses if actual concentration measurements were performed. In contrast, a semiclear, non-fat or skimmed milk-like appearance of an ejaculate might be extended to yield only 2–3 doses.

Suggested method for the determination of the concentration of spermatozoa in semen using an Improved Neubauer Haemocytometer

1. Prepare suitable diluted subsamples of the semen. The dilution used is somewhat dependent upon the species being studied. For example, for rams or bulls, ejaculates are typically very concentrated, so a 1:201 dilution could be used. Boar ejaculates are less concentrated, so a 1:101 dilution could be used.

Notes:

(i) It is important that dilutions are properly prepared. The outside surface of sample pipette tips should be carefully wiped to remove adherent cells. Tips should be wiped from the top towards the open end. The open end of the tip should not be touched, as this will result in sample loss by wicking action from inside the tip.

(ii) It is important that dilutions are properly prepared. For example, 0.01 µl (10 µl) sample plus 1.0 µl diluent results in a 1:101 dilution; 0.01ml (10 µl) sample plus 2.0 µl diluent results in a 1:201 dilution.

(iii) For accurate counts, a diluent that results in immotile spermatozoa should be used, e.g. 10% formaldehyde in saline (10 µl of 40% formaldehyde in saline).

(iv) Prepare a dilution that results in a count of approximately 150 spermatozoa in 5 large squares of the counting chamber.

Note: Overfilling or underfilling of the chamber will result in inaccuracy of concentration measurements.

3. Wait approximately 5–10 min for the sperm to settle to the bottom of the haemocytometer. Do not allow the haemocytometer to dry out before counting. In order to be sure that the haemocytometer does not dry out, the following procedure can be followed. Place a paper towel large enough to hold the haemocytometer in the bottom of a dish and wet the towel. Then place two microscope slides on the wet paper towel as a base, positioned so that the haemocytometer can be placed on the slides above the wet towel. The slides prevent the bottom of the haemocytometer from getting wet, and avoid any need to manually dry the chamber, as doing so may dislodge the haemocytometer coverslip. Cover the dish and allow the necessary time for the cells to settle.

4. The haemocytometer consists of a grid system. Five large squares should be counted – either each of the corners and the centre square, or from corner to corner diagonally (see Figs 6.4 and 6.5).

Counting suggestions:

(i) Count the sperm heads only, ignore the sperm tails.

(ii) Establish a pattern to use every time.

(iii) Count the sperm heads touching two of the sides of the large squares, e.g. left and bottom. Do not count the sperm heads touching the remaining two sides, e.g. right and top.

(iv) Total counts for the two sides of each individual chamber should be within 10% of each other. If not, these counts should be discarded and another chamber prepared and counted.

5. Add the counts from all five squares of one side of the haemocytometer as one total count, and then count the other side. It is recommended to count at a minimum four sides, using two haemocytometers for each semen sample under evaluation.

6. Calculate the total number and concentration of spermatozoa as follows (steps A–D):

A. Average the haemocytometer counts.

B. Multiply the average of the counts by the dilution factor.

C. Multiply the result of step ‘B’ by 50,000 (5 × 10⁴). This result is the number of cells/ml ejaculate. (Note: the factor 50,000 is used only if five large squares of the chamber are counted.)

D. Multiply the result of step ‘C’ by the volume of the ejaculate. This result is the total number of sperm in the ejaculate.

Example:

Ejaculate volume = 10 µl

Total counts in each of the five large squares are: 161, 168, 164, 167 after a dilution of 101 (counts from two chambers, two sides each).

A. (161 + 168 + 164 + 167) ÷ 4 = 660 ÷ 4 = 165

B. 165 × 101 = 16665 = 16.7 × 10³

C. (16.7 × 10³) × 50,000 = (16.7 × 10³) × (5 × 10⁴) = 8.4 × 10⁸ = no. cells/ml

D. 8.4 × 10⁸ × 10 µl = 8.4 × 10⁹ = 8.4 billion sperm total in the ejaculate.

7. Clean the haemocytometer and coverslip carefully.

Note: These are coverslips unique to the haemocytometer and cannot be replaced with an ‘ordinary’ microscope coverslip.

Improved Neubauer Haemocytometer maths

• Counting chamber depth: 0.100 mm.

• Ruling pattern: 1/400 square mm.

• Rulings cover 9 mm² (9 squares of 1 × 1mm).

• The centre square is subdivided into 25 large squares separated by triple lines.

• Each large square is subdivided into 16 small squares of 1/400 mm².

• The chamber height is 1/10mm, so the volume over each small square is 1/4000 mm³ or 0.00025 mm³.

• If ‘N’ is the number of cells counted in five large (80 small) squares:

• Then: N = no. of cells in 80/4000 mm³.

• So in 1 mm³, there are 4000 (N)/80 cells.

• Therefore, in 1 µl, there are 4,000,000 (N)/80 cells.

• If ‘D’ is the dilution factor, then the number of cells/ml is: 4,000,000 (N) (D)/80 or (5 × 10⁴) (N) (D).

Lorton (1986) described a technique for evaluating initially extended semen using a spectrophotometer. Photometric estimations of sperm concentration typically are simple and relatively quick (30–60 s). Photometers/spectrophotometers must be calibrated, i.e. to give OD versus sperm concentration, when purchased, as well as by periodic checks and after changing the light source. Calibrations change over time, especially in machines equipped with incandescent light sources, although they vary to a lesser degree in machines with light emitting diodes (LED) as the light source. Foote et al. (1978) reviewed calibration techniques when using haemocytometer counts. Calibrations must be made for each individual species of sperm under study. The NucleoCounter^® SP-100^™ (see below) provides more repeatable and more accurate data than haemocytometers and can be used for calibrations instead. The electronic function of photometers/spectrophotometers should be evaluated frequently (daily, weekly) with turbidity standards and/or filters.

Evaluation of concentrations can be made using the flow cytometer (Evenson et al., 1993). Within these machines, individual cells pass in a liquid fluid stream through a laser and are electronically counted. ‘Gates’ based upon cell size can be established in order to reduce error from debris and cells other than sperm (COTS), and extremely accurate data can be obtained. However, flow cytometers are expensive and relatively difficult to maintain and operate. Few semen production laboratories utilize them for concentration measurements alone, though they are used for more complex morphological and functional analyses (see below).

Anzar et al. (2009) concluded that the Nucleocounter^® SP-100^™ and the flow cytometer could be used with equal confidence for the measurement of sperm concentration. These authors also provided evidence that 13% additional bull artificial insemination doses could be produced when using the SP-100^™ rather than haemocytometers for the calibration of spectrophotometers. The SP-100^™ has become the instrument of choice for evaluating sperm concentration in neat and extended semen from several species, including stallions (Comerford et al., 2008; Morrell et al., 2010), bulls (DeJarnette and Lefevre, 2008; S. Lorton, unpublished), pigs, dogs, sheep, deer, wolves (S. Lorton, unpublished) and trout (Nynca and Ciereszko, 2009). Owing to the accuracy and repeatability of the Nucleocounter^® SP-100^™, it should be considered as the ‘new gold standard’ for the measurement of sperm concentration.

A later study (S.P. Lorton and M.M. Pace, unpublished) involving approximately 20,000 first inseminations also verified the model and provided additional evidence that the asymptotic value for individual bulls differed significantly. However, it was shown that, across bulls, approximately 10–12 × 10⁶ motile spermatozoa per insemination dose resulted in the asymptotic level of fertility (non-return rate). Den Daas et al. (1998) provided similar results and also discussed the variability between bulls. Foote and Kaproth (1997) presented evidence that the total sperm number per artificial insemination dose could be reduced to 10 × 10⁶ with only a one percentage point reduction in non-return rate. Further, Chenoweth et al. (2010) described data showing a strong correlation between the number of progressively motile spermatozoa per insemination dose (1.5, 3.5 and 7.0 × 10⁶) and palpated pregnancy rate. A specialized process developed in New Zealand using Caprogen extender and sperm storage at ambient (15–23°C) temperatures results in optimal fertility at levels with approximately 1.5 × 10⁶ total (1.0–1.2 × 10⁶ motile) spermatozoa per insemination (Shannon et al., 1984). The number of motile spermatozoa inseminated is, therefore, a compensable semen trait. (See Saacke et al., 1991 for a review of compensable and noncompensable semen traits.)

There have been similar, but less extensive, sperm number titration studies in the pig. Baker et al. (1968) demonstrated an interaction between the number of sperm inseminated and the volume inseminated. By examining the number of spermatozoa recovered from oviducts of gilts and the number of spermatozoa bound to the zonae pellucidae of ova, these authors concluded that 5–10 × 10⁹ total spermatozoa in a volume of approximately 100 µl resulted in optimum fertility. Johnson et al. (1981, 1982) demonstrated significant differences among boars in farrowing rate after insemination with 3 × 10⁹ total sperm. In contrast, Xu et al. (1998) were unable to demonstrate a difference in farrowing rate, while there was a significant difference in total live-born, when the insemination dose was reduced from 3 × 10⁹ to 2 × 10⁹ total sperm. Flowers (2002) reported a linear relationship of litter size among some boars (32 of 200 boars) versus an asymptotic relationship among most boars (133 of 200 boars) when the number of sperm inseminated was increased from 1 × 10⁹ to 9 × 10⁹. The authors commented that if additional motile spermatozoa were inseminated, a plateau effect may also have been detected for the animals showing the linear relationship. In contrast, Alm et al. (2006) reported a significant decrease in 60-day non-return rate and litter size when the insemination dose decreased from 3 × 10⁹ to 2 × 10⁹ total sperm. Reicks et al. (2008) reported a decrease in litter size when sperm number inseminated was reduced from 4 × 10⁹ to 2.5 × 10⁹ total sperm, while farrowing rate did not differ. These studies were all performed using ‘conventional’ cervical artificial insemination (AI) with chilled semen.

More recently, post-cervical and deep uterine surgical insemination studies in pigs have demonstrated that both insemination volume and number of sperm can be reduced without a reduction in pregnancy rate or litter size, e.g. from 3–3.5 × 10⁹ total sperm in 75–80 µl to approximately 1–1.5 × 10⁹ total sperm in a volume of 35–40 µl (post-cervical insemination), or even 0.5 µl (deep uterine insemination, using fresh or frozen–thawed semen) (Krueger and Rath, 2000; Rozeboom et al., 2004; Wongtawan et al., 2006; Vazquez et al., 2008 (review); Sonderman et al., 2011).

Evaluation of Spermatozoal Morphology

5. Loosen the knurled condenser locking screw on the side of the condenser mounting ring (see Figs 6.10 and 6.11).

6. Insert the phase condenser into its mounting ring. The top sleeve of the condenser should be inserted completely ‘up’ into the mounting ring.

7. Tighten the knurled locking screw to secure the condenser in position.

8. Place a specimen drop on a microscope slide and cover with a coverslip.

9. Place the slide on the microscope stage and adjust the stage so that the edge of cover glass is under the 10× lens.

10. Rotate the phase adjustment control of the condenser so that the bright-field (BF) setting is seen at the front.

18. While looking into the centring telescope, push inwards with each hand the two condenser centring screws that extend outwards from each side of the phase condenser. When completely pushed in, these screws engage centring screws within the condenser assembly.

20. Disengage the two condenser centring screws by allowing/pulling both to retract to their ‘out’ position. Do not attempt to rotate the condenser adjustment control while the condenser centring screws are in their ‘in’ position.

21. Rotate the condenser control to position ‘20‘ and then rotate the 20× objective to the ‘use‘ position. Repeat the phase rings centring procedure (steps 18–20), if necessary.

22. Repeat the centring steps for the 40× (condenser position ‘40‘) and 100× (condenser position ‘100‘) objectives, if necessary.

23. Remove the centring telescope and replace with the eyepiece. The microscope is now ready for use.

Note: When the condenser is in the phase positions (10, 20, 40 or 100), the condenser diaphragm should be in the completely open position. When using bright-field microscopy, e.g. with stained specimens, rotate the condenser control to position ‘BF’ and adjust the diaphragm as desired.

Specimen observation

1. Prepare a slide of the semen sample with a coverslip and place on the microscope stage.

2. Position the slide such that the edge of the coverslip is centred under the 10× objective, with the condenser control positioned at ‘10’.

3. Focus on the edge of the coverslip.

4. Using the stage movement controls, move the slide so that the centre of the coverslip is approximately under the objective lens.

5. Focus the image, primarily using the ‘fine’ focus knob.

6. Change the magnification by turning the nosepiece to the desired objective. Set the condenser control to the setting matching the objective.

7. When using phase contrast lenses, the green filter may be fitted on to the recess of the light housing or in a filter holder under the condenser. A blue filter is often employed when using bright-field microscopy, but this may also be used for alternative effects during phase microscopy.

Differential interference contrast (DIC) microscopy is another technique used to enhance contrast of objects using a light microscope. DIC provides an appearance of the 3D structure of viewed objects. The physics of this technique has also been reviewed by Abramowitz (1987). The techniques to set up DIC differ by microscope design and model, are quite complicated, and therefore will not be summarized here. The reader is referred to documentation accompanying individual microscopes.

Wet smears using phase or DIC microscopy may be performed on spermatozoa that are unfixed (Saacke and Marshall, 1968) or fixed with gluteraldehyde or formaldehyde (Hancock, 1957; Pursel and Johnson, 1974; Johnson et al., 1976; Barth and Oko, 1989; Richardson et al., 1992), or on spermatozoa that have had their motility inhibited with sodium fluoride (Pursel et al., 1972, 1974). Potential artefacts associated with the staining and preparation of smears are avoided by the use of wet mounts.

Morphological evaluations are performed at 400–1000× magnification, e.g. 40× or 100× (oil) objectives and 10× oculars. Several morphological classification systems have been used. In some laboratories, morphological abnormalities are classified as being ‘primary’ (originating in the testes), ‘secondary’ (originating during epidydimal passage) and ‘tertiary’ (occurring post ejaculation). Blom (1972), as referenced by Barth and Oko (1989), suggested a ‘major’ and ‘minor’ classification scheme, according to the importance of the defect with respect to fertility. With these schemes, however, it is often difficult or impossible to classify a particular defect. Often, the origin of the defect is unknown, its importance is unclear, and/or spermatozoa may exhibit more than one defect. A simpler classification may then be used, as follows: abnormal head, abnormal acrosome, abnormal tail, proximal cytoplasmic droplets, distal cytoplasmic droplets and, of course, ‘normal’. COTS, debris, etc., should also be noted.

SEM and TEM can also be used to identify and study specific sperm abnormalities. These techniques require specific and meticulous sample preparation. Individual microscopes are expensive. Samples are often transferred to specific laboratories specializing in these techniques.

Irrespective of the classification scheme used, in general a maximum level of 15–20% total abnormal sperm is often considered acceptable. However, whenever performing analyses, it is critical to understand that some abnormalities, e.g. nuclear vacuoles, are extremely significant with respect to fertility. Also, observed abnormalities may be indicative of abnormalities that cannot be observed, e.g. DNA defects. In these cases, the general consideration of 15–20% as acceptable must be reconsidered. In essence, this issue is the basis of the ‘major’ and ‘minor’ classification scheme proposed by Blom (1972).

It must also be realized that a morphological evaluation is merely a snapshot of the ability of the male to produce ‘normal’ spermatozoa. The analysis is performed on a sample collected on a particular day and is indicative only for that particular collection. If the male is young, has been ill, or has been exposed to environmental conditions detrimental to sperm production, a satisfactory semen analysis may indicate adequate/acceptable semen production. Likewise, an unsatisfactory analysis only indicates that on that particular day, the collection consisted of abnormal cells. In both cases, repeated analyses must be done to clearly elucidate the seminal status.

After several satisfactory analyses, repeated in-depth analyses must be done on a periodic basis, depending upon the frequency of semen collection. In between in-depth analyses, laboratory personnel should be aware of abnormal sperm during routine microscopic semen evaluations, e.g. for motility. Salisbury and Mercier (1945) provided evidence that data obtained by examination of 100 spermatozoa on one slide was as reliable as the examination of multiple slides and the examination of up to 500 cells, although differences between technicians may account for greater variation (Root Kustritz et al., 1998; Brito et al., 2011). The proportion of cells examined is, then, extremely low (100–500) compared with the total cells (billions) in the ejaculate. Kuster et al. (2004b) discussed the statistical implications of sample size when performing morphological analyses.