Bovine Reproduction. Группа авторов

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      Use of the primary/secondary and major/minor sperm defect classification systems has been replaced by the differential count of sperm defects system described previously, where the predominant defects are listed as a percentage and the effect on fertility is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 motility that are unable to travel to the site of fertilization, or that can travel but cannot penetrate the ovum, can be compensated for by motile sperm – compensable defects. Defective sperm, which are able to penetrate the zona pellucida and initiate the zona block are uncompensable [31, 32]. Noteworthy examples of uncompensable sperm are those with diadem vacuoles or abnormal DNA condensation. Although fertilization by a sperm with an uncompensable defect is equally as possible as a normal sperm, the risk of early embryonic loss is much greater with the uncompensable sperm defect.

As mentioned previously, differentially counting sperm defects to produce a spermiogram is a much less confusing approach to assessing sperm morphology. There are approximately 25 recognized sperm abnormalities – some are artifactual and some have no effect on fertility. Table 9.2 lists 19 defects; however, some defects have been grouped together. Examiners should regularly review abnormal sperm types. Comparing differential counts with colleagues and experts in sperm morphology are good ways to ensure that one is performing consistent, quality evaluations. Taking the time to compare a single sperm with a questionable appearance with images of known defects is an effective way to learn.

Table 9.2 Listing of recognized sperm defects.

Pyriform headsTapered headsMicrocephalic/macrocephalic headsVacuoles – diadem, single vacuoles, confluent vacuolesClumped DNADetached (loose) heads – normal, abnormalDecapitated defectRolled‐crested‐giant head syndromeTeratoidsKnobbed acrosome – beaded, flattenedRuffled and detached acrosomesDistal midpiece reflexMitochondrial sheath defectsDag defectStump tail defectCorkscrew defectPseudodroplet defectCoiled principal pieceProximal cytoplasmic droplets

Table 9.3 Abnormal sperm morphologies, artifacts, and other cells not included in a sperm morphology differential count.

Morphology	Artifact	Other cells
Abaxial tailsDistal droplets	Bowed midpieceSimple bent tail(hypotonic or cold shock)	White blood cellsMedusa cellsSpheroidsEpithelial cells

      A good quality, well‐maintained microscope and some knowledge on how to optimize image quality are important. Oil easily captures dust and debris, so it is good practice to clean the oil immersion objective regularly. A solution of 70% ethanol on a lens‐appropriate cloth works well.

      Sperm Abnormalities

      Several of the most common or noteworthy sperm defects are presented below.

      Normal Sperm

Nearly all novice examiners will be able to recognize a normal sperm at first glance, but if questioned they will be unsure. An effective technique is to develop a system so that each sperm is thoroughly examined. Normal sperm vary in shape both within a single smear and more so between bulls. Heads may appear short and wide or elongated and narrow. Sometimes a sperm head may look excessively narrow, but if there are no other, more obvious, disturbances in head shape and most of the sperm in the ejaculate appear similar then these sperm can be considered to be normal (Figure 9.7).

Figure 9.7 Normal sperm stained with eosin‐nigrosin. The dark staining sperm is dead.

      Sperm Morphology – Minimums and Maximums

      The widely accepted minimum proportion of morphologically normal sperm is 70%. Below this threshold it is expected that fertility will be impaired and a bull cannot be classified as a satisfactory potential breeder. Generally, bulls that are at or near this threshold have at least two or three sperm defects; however, as a precaution, it is recommended that the proportion of sperm with nuclear defects or proximal droplets should not exceed 20% and the proportion with acrosome or midpiece abnormalities should not exceed 25%.

      Artifacts and Debris

      Commonly found artifacts include bowed midpieces (also called bowed tails) and bent tails (also called simple bent tails and hypotonic shock). Neither of these anomalies should be counted as abnormal in the differential count. Bowed midpieces are easy to recognize as rainbow‐shaped, C‐shaped, or U‐shaped. A key characteristic is that the tails (midpiece and principal piece) do not fold as is the case with the distal midpiece reflex. The occurrence of bowed midpieces is associated with faulty slide preparation, most often when slides and the stain are cold. Examination of motile sperm on a wet mount can be used to confirm that this aberration is artifact [27].

Hypotonic shock is usually manifested as terminal whipping or looping of the principal piece and is caused by prolonged exposure to hypotonic stains (eosin‐nigrosin and eosin‐aniline blue) before drying. Drying is often prolonged when stains and slides are cold. The looping may appear similar to the distal midpiece defect (DMR) except that there will be no trapped droplet material. Closely examining the terminal portion of the