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.
Classification and Evaluation of Sperm Morphology
Differential counts of sperm morphology are done with oil immersion, bright field microscopy at 1000× magnification. Always use immersion oil labeled for microscopy. Other oils, e.g. mineral oil, have a different refractive index and may penetrate the objective and effectively ruin it. Blood cell counters relabeled for use as sperm cell counters are an invaluable tool for the practitioner who evaluates more than a few bulls each year. At the Western College of Veterinary Medicine we label keys by the location of the defect on the sperm as shown in Table 9.1.
Table 9.1 Layout of an example label for keys used by the Western College of Veterinary Medicine, Saskatoon.
| Head | Midpiece | Principal piece | Detached (loose) abnormal | Detached (loose) normal | Proximal droplet | Acrosome (other) | Normal |
An image of a labeled sperm cell counter is shown in Figure 9.6. “Detached” or “loose” refers to detached heads. Detached heads with abnormalities, for example, the pyriform shape or nuclear vacuoles, should be counted as “detached abnormal.” Heads without abnormalities are counted as “detached normal.” Every sperm within a field should be counted to avoid bias. Exceptions will be if clumps of sperm are present obscuring other sperm, or if there are simply too many sperm preventing the complete inspection of individual cells. Fields should be inspected in systemic fashion – left to right, top to bottom has been an effective method for the author.
Figure 9.6 Cell counter with keys labeled for sperm cell morphology.
Differential counts are reported as “defects per 100 cells.” Each time a key is pushed a cell is counted and added to the total. In cases where a sperm cell has more than one defect the respective keys should be depressed simultaneously. The two defects are counted, yet only one cell is added to the total. When the counter reaches 100 cells a bell is sounded. Counting just 100 sperm cells will be sufficiently representative if just a few abnormalities are recorded. When many abnormalities are encountered it is advised to count at least 300 cells to improve the reliability of the morphology assessment. Reliable counts should not differ by more than 10%, or, in other words, 10 cells. When reviewing my counts, I generally disregard any outliers in favor of completing another 100‐cell count. Once satisfied that my counts are representative I will report the average of the suitable counts.
When sperm morphology is examined, several questions may arise about the specific type of defects observed:
Is it an aberration or a significant abnormality?
What would be the effect on fertilizing ability (sperm transport, binding to the oocyte, oocyte penetration, zygote formation)?
What would be the tolerable level for bulls in natural service, or semen used in AI?
What are the implications for the bull (cause, prognosis)?
Readers of this chapter are encouraged to review sperm structure and spermatogenesis to develop a more complete understanding of sperm morphology (see Chapter 3). Classification systems have been developed to try to simplify semen evaluation; however, there appears to be quite a lot of misunderstanding of them. The earliest system used was the primary/secondary sperm defect system. By definition, a primary defect is one that originates during spermatogenesis – within the testicle – and a secondary defect is one that originates within the epididymis [28]. All head defects, such as knobbed acrosomes (KAs), microcephalic sperm, pyriform heads, and nuclear vacuoles, would thus be primary defects. Some tail defects would be primary, but others, notably the Dag defect and the distal midpiece reflex defect, develop in the epididymis and would thus be secondary defects. However, if these defects developed in the epididymis because of a weakness in structure that occurred in spermatogenesis they might be defined as primary defects. Following this line of reasoning there would then really be very few secondary defects. Distal midpiece reflexes and proximal droplets appear to develop in sperm that were structurally normal upon entering the epididymis. However, it could be argued that sperm with malformed heads or tails are more likely to retain a cytoplasmic droplet in the proximal position. The supposed origin of a defect bears little relevance to the effect of the defect on fertility and probably is only useful for determining when a defect might appear in the spermiogram following an insult to spermatogenesis.
The “major and minor” system of sperm morphology classification was created to remove some of the confusion associated with the primary/secondary sperm defect system. In this system a major defect was a sperm aberration which had been associated with infertility. Minor defects were those that had not been shown to be associated with infertility [29, 30]. Over time, more evidence has been gathered, including the reporting of defects not included in the original list. Sperm aberrations that were categorized as minor have been found to have a significant effect on fertility. In defense of the early work, it would seem that at that time very few bulls had been found that produced high numbers of some of these defects. It is notable that two sperm aberrations that were listed as minor defects, abaxial tails and distal droplets, have been shown to have no effect on fertility and should be disregarded when classifying sperm as normal or abnormal.
Use of the primary/secondary and major/minor sperm defect classification systems has been replaced by the differential count of sperm defects system described previously, where the predominant defects are listed as a percentage and the effect on fertility is determined based on the current knowledge of each type of defect.
A system of classifying sperm defects that has proven useful in predicting the effect on fertility is the compensable/uncompensable system. Based on the understanding that a certain population of live, motile sperm must travel to the site of fertilization and that some abnormal sperm can penetrate the ovum while others cannot, the compensable/uncompensable system has been particularly useful for determining sperm concentrations per insemination dose for the artificial insemination industry. Sperm with impaired
