semen on a microscope slide which is then viewed using bright field microscopy at 40–125× magnification [2]. Vigorous swirls and eddies are indicative of a concentrated semen with a very high proportion of vigorous motile sperm. A depression affecting one or more of the three factors will, in turn, have a negative effect on gross motility. For example, sperm motility may be very high in a poorly concentrated semen sample yet the gross motility appears poor. Another common occurrence is when a highly concentrated sample becomes chilled, negatively affecting sperm vigor. As a result, gross motility should be interpreted carefully, leaving individual motility as a more reliable method of assessing sperm motility.
Individual Motility
Individual motility or more precisely percentage of progressively motile sperm is determined by placing a 2‐ to 4‐mm drop of semen on a clean microscope slide over which a cover slip is dropped in place. The seminal fluid should spread just to the edge of the cover slip to form a thin layer in which the sperm can be viewed within the same focal plane [2]. Too much semen and the cover slip will float, making visualization of individual sperm more difficult, and very concentrated samples may be impossible to evaluate without dilution. Although readily available, the low pH of physiologic saline can diminish sperm motility. Warmed sodium citrate solution [17], commercial semen extender, or even fresh sperm‐free seminal fluid are better choices to use as a diluent [2]. Individual motility is recorded as a percentage, with minimum acceptable proportions of 30% [1] and 60% [2] being used by the respective SFT and Western Canadian Association of Bovine Practitioners' guidelines. Several fields should be examined at 400× magnification, observing the proportion moving forward versus those that are immotile, or barely motile, and an average of the percentage motile sperm can be estimated, usually rounded to the nearest multiple of 10. Individual sperm motion is most effectively observed using phase contrast microscopy. Most modern microscopes can be fitted with phase contrast for a modest cost. Sperm motility can be easily affected by heating, chilling, and contamination with urine or other fluids, soaps, etc. A finding of poor motility should be substantiated by sperm morphology and vitality (percentage staining live) before it is considered reflective of fertility. If a finding of poor motility cannot be substantiated then a second sample should be evaluated.
Sperm Morphology
An Assessment of sperm morphology should always be performed. In most cases, irregularities noted with one or more of the other parameters will be evident in the sperm morphology assessment. The minimum percentage of morphologically normal sperm is 70.
Percentage Staining Alive
Percentage staining alive requires the use of a vital stain (see section “Preparation of Semen Smears”) wherein eosin will cause the dead sperm to stain pink. The percentage staining alive is a good indicator of sperm vitality which will be positively correlated with progressive motility when semen handling and slide preparation have been performed correctly. Example reasons that the percentage staining alive may be high but motility is poor could occur with midpiece/principal piece defects or if there has been urine or other chemical contamination of the semen sample.
Preparation of Semen Smears
Evaluation of sperm morphology begins with a well‐prepared smear. Eosin‐nigrosin is the most recommended and widely used stain in use for evaluation of bull sperm. Eosin penetrates damaged sperm membranes to stain dead and non‐viable cells pink, hence it is referred to as a vital, or vitality, stain. Nigrosin provides a dark, purple background, enhancing the appearance of both live and non‐viable cells. Eosin‐aniline blue is another vital stain that is less popular nowadays. Aniline‐blue (blue color) takes the place of nigrosin (purple color) as the background stain. Live sperm do not take up the eosin stain and appear white; half‐stained sperm are protected by the acrosomal membrane covering the anterior end, but eosin is able to penetrate the non‐acrosomal membrane distal to the equatorial region. These half‐stained sperm are more common in semen smears prepared under less than ideal conditions such as cold ambient temperatures. They are non‐viable, but in the opinion of the author they are iatrogenic in origin versus the entirely pink sperm that was non‐viable or dead for a sufficient period of time before staining to lose or suffer damage to the acrosomal membrane. When semen samples are handled correctly and there are no morphologic defects affecting sperm motility, the proportion of sperm staining live and the percentage of progressively motile sperm should be close to the percentage of live sperm, being approximately 5–10% higher.
Non‐vital stains such as modified Wright Giemsa (Diff Quik) and India ink provide a dark background, but do not have a cell wall‐penetrating component. They may be used to evaluate sperm morphology but should be used only if eosin‐nigrosin is not available. Diff Quik is a superior stain for definitively identifying white blood cells, displaying the characteristic bean‐shaped nucleus of neutrophils very well (Figure 9.3). Neutrophils appear as large, white, circular structures, often one and a half to two times the size of a sperm head when viewed on eosin‐nigrosin stained smears (Figure 9.4). Diff Quik is a superior stain for definitively identifying white blood cells. A distinct advantage associated with the use of eosin‐nigrosin is the rapidity with which slides can be prepared and evaluated. Stains that require long periods of air drying and several steps not only are time consuming but also can result in the loss of cells. Unstained wet mounts should never be the sole means of evaluating sperm morphology – the ability to identify many sperm defects is inadequate. Wet mounts are useful for observing the unusual motility patterns of live sperm with midpiece defects and for verifying whether a perceived staining artifact is real or not.
Figure 9.3 White blood cells (neutrophils) on a Diff Quik stained smear. Note the faintly stained sperm.
Figure 9.4 White blood cells (wbc), a speroid cell (sc), detached head (dh), a shed droplet (sd), and distal midpiece reflexes (dmr) on an eosin‐nigrosin stained smear.
Prior to beginning the evaluation of a semen sample, clean slides should be warmed on a warming stage for several minutes to prevent any artifactual, cold‐related changes. To prepare a semen smear, place a 4‐ to 5‐mm drop of stain at one end of the slide, adjacent to the frosted end if applicable. Frosted slides are ideal for labeling, which will facilitate the filing of semen smears as part of the medical record. Stain should always be placed first to limit exposure of the sperm to the damaging effects of chilling and desiccation before fixing with stain. Place a slightly smaller drop of semen adjacent to the stain and immediately mix the two together. Lifting the slide off of the warm stage with one hand and mixing with the other hand is far less awkward then leaving the slide on a flat surface. Wooden applicator sticks (Applicators, Puritan Medical Products Company LLC, Guilford, ME, USA) work well for obtaining droplets of semen and mixing. Using the stir stick, spread the mixture down the length of the slide in a stopping and starting fashion which will effectively create thick and thin areas of stain (Figure 9.5). This will help to provide the ideal area to count approximately 10–20 sperm per microscope field once the slide is dry. Another way to prepare a smear is to first mix the semen and stain as described above then use a second microscope slide to make the smear,
