associated with riboflavin in the diet [26] and is different from a yellow tinge that may be evident with urine contamination. A urine‐contaminated semen sample will usually be variably diluted and will have an odor of urine. (Editor's note: if unsure, utilization of a “BUN strip” [blood urea nitrogen] will confirm or rule out.) Preseminal fluid is clear, watery, and odorless. Semen samples diluted with preseminal fluid will not be opaque but rather have a degree of clarity on a spectrum, comparable with skim‐milk, watered‐down juice, or even clear fluid. Generally, preseminal fluid emission is noted during the EEJ process, but it is not collected in favor of waiting for the appearance of the sperm‐rich fraction. Especially in difficult collections, a variable amount of preseminal fluid will be collected, thereby diluting the sample. A pink or red hue or even streaks of red in the semen sample are usually indicative of blood contamination. The source of the blood should be addressed. Blood‐contaminated samples are common when semen collection follows preputial scraping for Tritrichomonas foetus and Campylobacter foetus, especially if penile protrusion did not occur during EEJ. Clumps of white cellular material in the vial are highly suggestive of a high neutrophil content.
Volume
Always note the volume as it is an indication that a representative sample was collected; however, volume is influenced by the collection process and is therefore not a relevant measure. In other words, the volume of an EEJ‐derived semen sample should not be compared with ejaculates from the same or different bulls. The one exception is in cases of sperm accumulation, the “rusty load phenomenon,” where 25–40 ml of concentrated semen may be collected easily during a single ejaculation [2]. A typical EEJ‐derived semen sample is 1–5 ml.
Concentration
A subjective evaluation of concentration should be made by examining the tube containing the semen to ensure that a representative sample has been obtained. A poorly concentrated sample will be watery and translucent containing <250 million sperm per milliliter, whereas a concentrated sample, typical of a mature bull, will appear creamy and grainy when examined against a dark background containing at least one billion sperm per milliliter. Very concentrated, high‐volume ejaculates occur in cases of sperm accumulation. A watery, poorly concentrated sample from a mature bull is typical in cases of testicular degeneration. Closely linked to volume, the concentration of a semen sample also varies with the collection technique. EEJ is not an appropriate method of determining the sperm‐producing capability of a bull.
Gross Motility
Gross motility or gross wave motion is influenced by the percentage of progressively motile sperm, the sperm concentration, and the vigor or rate of speed of the motile sperm. Determining gross motility is a fast and simple method requiring just a drop of 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