**3.4. Sperm motility and mobility**

Another concern when using any semen evaluation method is variation in the operator's techniques. Consistency is the key to repeatable data. The technical staff all must follow the same standard operating procedures (SOPs). For example, when counting sperm with a hemocytometer, all individuals in a lab should following the same SOP for how long the sperm are permitted to settle on the grid and which sperm to count or omit from the count. Also, is the photometer zeroed with the same buffer? If a procedure calls for an incubation period, such as in a live-dead stain, are the samples being incubated for the same duration each time using the same stain concentrations? A lack of consistency in following the SOPs within a laboratory will lead to unwarranted variation and non-reproducible and inaccurate data.

186 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

In the context of semen evaluation, reference to 'viable' sperm simply implies that such sperm possess an intact plasmalemma and are assumed to be functional. Plasmalemma in‐ tegrity is frequently determined using either a dead-cell or a live-cell stain alone or simulta‐ neously. The dead-cell stains are excluded by sperm with an intact plasmalemma but stain dead sperm possessing a permeable plasmalemma. Live-cell stains permeate the intact sperm plasmalemma and become visible only after reacting with cytosolic enzymes or inter‐ acting with sperm nuclear proteins. Both eosin and propidium iodide are popular dead-cell stains while calcein AM and SYBR-14 are frequently used live-cell stains (see [86] for extend‐ ed discussion and availability for the live-cell probes). On a commercial breeder farm, the nigrosin/eosin (N/E) technique is most likely the procedure to be used to determine sperm viability [4]. Briefly, sperm are stained with N/E and a smear of the stained sperm is made on a slide (Figure 7). Under a bright field microscope the viable sperm remain pearly white, while eosin will stain non-viable sperm a pink to magenta color. The nigrosin serves as a background to enhance differentiation between the non-viable and viable sperm. In contrast to the N/E technique, a more sophisticated laboratory may use flow cytometry that sorts via‐ ble from non-viable sperm after staining with calcein AM or SYBR-14 and propidium iodide.

**Figure 7.** The left panel shows a nigrosin eosin preparation of turkey sperm with nearly 100% viable sperm (un‐ stained) white nuclei and midpieces. The sperm head is clearly visible as the white arcing segment; the acrosome and midpiece are difficult to differentiate from the nucleus. The upper right panel reveals a normal sperm and a second sperm with an abnormally curved and swollen midpiece. Observed in the lower right panel is a nonviable sperm stained with eosin throughout the nucleus and midpiece. Barely visible at the anterior end of the nucleus is the un‐

**3.3. Sperm viability**

stained, conical shaped acrosome.

Sperm motility can be progressive (forward direction) or non-progressive (random movement or oscillations) movement. Generally, progressive motility is determined subjectively at ambient temperature using a microscope at low magnification (hanging-drop technique) or objectively using a computer-assisted semen analysis system. These techniques are reviewed by Bakst and Long [4]. Motility evaluated by microscopy has been shown to have little correlation with fertility and simply reveals that the sperm are motile. First described by Froman and McLean [87] and further elaborated for commercial use by Froman [88], the sperm mobility assay has gained popularity as a measure of an individual male's ability to produce highly mobile sperm [mobility defines the ability of sperm to move progressively against a viscous medium (Accudenz) at 41° C] that are more likely to fertilize an ovum than males producing less mobile sperm. While the sperm mobility assay is a powerful tool for the selection of the most fecund males to be used in AI, it necessitates attention to details and accurate and consistent preparation of the reagents.
