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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, similar to making blood smear. A better result will be achieved if the second slide is pulled back slowly, not rapidly as is the recommended practice when preparing a blood smear. Smears should be dried as quickly as possible to prevent the occurrence of bowed tails, an artifactual occurrence due to the hypotonic nature of eosin‐nigrosin. This may be accomplished by returning the slide to the slide warmer and by blowing on the slide to speed drying.

Figure 9.5 Semen smear preparation.

The Feulgen staining technique is a multistep procedure that begins with an air‐dried smear. The exposure of the dried sperm cells to hydrochloric acid exposes aldehyde groups in the DNA which then bind with the Schiff's reagent, resulting in magenta staining of the DNA [27]. Feulgen staining is an excellent way to augment eosin‐nigrosin stained smears, particularly when abnormal DNA condensation is suspected or to get a more accurate differential count of the number and type of nuclear vacuoles.