Chapter 8 Richard M. Hopper and E. Heath King Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Starkville, Mississippi, USA An evaluation of semen is performed for several reasons, most commonly following the physical examination as part of a standard bull breeding soundness examination (BBSE). The BBSE in turn is performed for pre-purchase, annually or semi-annually prior to breeding season turnout, or for diagnostic purposes. A semen evaluation alone can be performed for prognostic reasons following an illness or injury to the reproductive system or in the case of an older or injured bull of high genetic merit that might be a candidate for use by artificial insemination. This chapter primarily deals with microscopic examination of semen as a part of a BBSE. A basic assessment of semen consists of the microscopic evaluation of motility and morphology. An assessment of sperm numbers or sperm production is determined by measurement of the scrotal circumference (covered in Chapter 7). The assessment of morphology has been shown to be of critical importance and therefore the proper evaluation of sperm morphology, as well as the significance of certain sperm abnormalities, will be emphasized. Spermatogenesis is a dynamic process and so dramatic changes in the quality of semen produced by bulls can occur within days. This basic fact renders the results of this examination historic in nature within days. Only after successfully meeting the standards of the physical examination is a bull subjected to collection and evaluation of his semen. To proceed with the evaluation only negates the all-important minimum standard placed on scrotal circumference or the importance of structural soundness. In order to evaluate the semen a sample must be collected in a safe and efficient manner. Semen motility should be assessed immediately but a slide for examination of cell morphology can be prepared and analyzed immediately or at a later date. Semen can be collected by electroejaculation, manual (per rectal) stimulation of the internal genitalia, or with an artificial vagina utilizing a jump animal. Additionally, an internal artificial vagina placed within the vagina of an estrual female can be utilized.1,2 Collection via manual stimulation provides samples of inconsistent quality among bulls,3 but can be useful in young bulls that respond negatively to electroejaculation. Use of an artificial vagina and jump animal is typically used only at bull studs for collection of semen slated for freezing. The internally placed artificial vagina provides the advantage of allowing the assessment of libido. However, of these, EE is the most consistent and reliable method of obtaining a diagnostic sample. Observation of bulls and specifically the vocalization of some bulls during electroejaculation have led to concerns that this procedure may be painful or at least stressful. A study was performed in which serum cortisol and progesterone levels, relative aversion, and the degree of vocalization were all quantified.4 While the results of the aversion metric appeared to be equivocal, there were increases in serum cortisol and progesterone, along with increased incidence of vocalization, tending to substantiate these concerns to some degree. However, with the recent elucidation of substance P as a neuropeptide involved in the integration of pain as well as stress and anxiety, it has been shown that electroejaculation does not specifically result in pain.5 All the currently used electroejaculator models are similar in function, although there are differences in operation and in bull response. Some models utilize a rheostat that allows manual increases in voltage, others a preprogrammed increase, and at least one model allows the selection of either. Thus the type of model selected is typically based on personal preference (Figure 8.1). Figure 8.1 Pulsator IV Electroejaculator and probe. Courtesy of Lane Manufacturing, Inc. From a technical standpoint little has changed in the voltage delivery of an ejaculator in the more than 50 years since the first patent was issued (Marden-US #2,808,834). However, continual progress has been made in probe design, with respect to electrode placement, size, and the yoke. In general the largest diameter probe that can easily be placed in the rectum of a bull should be the size used and heavier probes seem to ensure better contact than lighter probes. A general guideline for probe size is the use of a 7.5-cm diameter probe for bulls weighing 544–907 kg and a 9-cm probe for larger as well as older bulls.6 The larger (9 cm) also seems to work best in Brahman (Figure 8.2) and Brahman cross bulls. Smaller probes (6–6.5 cm) are available and are often necessary for yearling bulls. The probe yoke is the U-shaped extension on the back of the probe which fits around the tail and serves to prevent probe displacement from the rectum and maintain proper electrode alignment. A yoke that is oriented upward is preferred as a “butt bar” is often used in the chute to facilitate restraint. If a bull attempts to lie down in the chute, the horizontally oriented yoke will hang on butt bars forcing the anterior aspect of the probe down. While this rarely results in serious damage to the rectum, it should be avoided (Figure 8.3). Figure 8.2 Electroejaculator probes (60, 75, and 90 mm). Utilization of the largest diameter probe that can easily be inserted facilitates semen collection. Note that the two larger-diameter models have an upright yoke and are “weighted” (heavier than standard models). Figure 8.3 Adequate restraint for a bull to be examined for soundness and utilization of upward oriented yoke. Efficient collection of semen from a bull by electroejaculation can be facilitated by a couple of techniques employed during the physical examination. During per rectal palpation of the bull’s internal genitalia, spend a few extra minutes massaging the ampullae and applying digital pressure to the urethralis muscle and bulbourethral gland. This will stimulate relaxation of the penis and often the bull will begin to dribble pre-seminal and seminal fluid. Next, before introducing the electroejaculator probe, close your hand into a loose fist and thrust your arm back and forth, fatiguing and relaxing the bull’s rectum. This action facilitates entry of the probe. The aforementioned massage and fatiguing of the rectum take only a few minutes and seem to both decrease the bull’s reaction to electrostimulation and speed the process of collection. Then, as seamlessly as possible, insert the probe into the rectum as the arm is being removed. After the probe has been inserted into the rectum, either a preprogramed cycle of stimulation can be used or a manually controlled stimulation cycle. The preprogramed cycle has the advantage of freeing up the operator to perform other tasks, and also provides stimulation in a controlled cyclical frequency that through experience has been shown to be effective in a majority of bulls. Manual control provides the operator with the flexibility to provide less stimulation if the bull seems sensitive or, conversely, to both speed up the frequency and increase the intensity of stimulation if necessary. Again, selection of a certain model of ejaculator is largely based on personal preference. To summarize, when utilizing manual control, provide the first two to three rounds of stimulus slowly, carefully observing the bull. Depending on his response, increase the amount of stimulus with each successive attempt, pausing for a “thousand-one, thousand-two, thousand-three count” at the peak of stimulus, then turning stimulus down for a one-count rest and repeat. Do not collect the first emissions; wait until the seminal fluid becomes cloudy. Since the penis should be extended at this time, it is also a good time to evaluate the penis for defects. Likewise the penis can be grasped with a gauze pad as this will allow better visualization, ensure a cleaner semen sample, and prevent retraction (Figure 8.4). Figure 8.4 Bull with penis extended allowing proper visualization. Occasionally, ejaculation will not have occurred even with the full utilization of electrical stimulation. At this point stop stimulation and allow a rest period. Usually within 2–3 min of rest, semen will spontaneously dribble from the penis. If semen is not obtained following this “rest” period, reinitiate stimulation with two to four rapidly increasing cycles. In general bulls that have been out with cows and actively breeding within the last 24 hours will be harder to collect. Also, nervous and fractious bulls are often harder to collect. If sedation is necessary, xylazine (20–50 mg i.v.) can be used. Acepromazine should not be used as it hampers collection and increases the incidence of urine contamination (urospermia). Semen can be collected into a cone attached to a collection handle and this in turn can be enclosed within a warm water “bath” or likewise insulated. Alternatively, semen can be collected into a Styrofoam coffee cup. These cups seem to adequately protect the sample from moderate temperature stress and as these cups are used in the food service industry, cleanliness (sterility?) is assumed. After collection the semen should first be observed grossly. A rough estimation of the concentration can be made based on the opacity (or lack of) and the color of the semen. Very concentrated samples look like heavy cream while very dilute samples have the appearance of watered-down skim milk. Yellow-tinted semen can result from urine contamination and this can be substantiated by smell or by use of a blood urea nitrogen test strip. Additionally, semen contaminated with urine will have rapidly declining or often no motility when examined microscopically. Conversely, a light-yellow or gold appearance is also associated with very highly concentrated semen and the presence of riboflavin,7 which is a common finding in many Jersey and some Angus bulls. Red- or brown-colored semen is due to the presence of blood or blood pigments and the source of this contamination must be determined. After quickly evaluating the semen grossly, a small “standing” drop is placed on a prewarmed slide and evaluated under low-power microscopy (40–200×) for gross motility. Thick, dark, rapidly oscillating swirls are indicative of excellent motility (defined as high-velocity or high-speed motility), a high percentage of sperm that are progressively motile, and a sample of high concentration. This type of sample would typically be classified as “very good.” A sample that displays slower moving swirls is classified as “good.” A “fair” sample displays no swirls, but significant individual sperm movement. A “poor” sample has no or very little movement/oscillation. Because the concentration of a sample impacts the gross motility designation, individual motility should be assessed if there is any question about the validity of a motility rating based on gross motility (i.e., low-power evaluation of the standing drop). Individual motility can be assessed using 200–400× microscopy and, depending on the concentration, a coverslip over either the previously examined droplet or a diluted droplet (diluted with warmed sodium citrate solution). Individual motility is classed “very good” if greater than 70%, “good” if 50–69%, “fair if 30–49%, and “poor” if less than 30%.8 Based on the current standards set forth by the Society for Theriogenology (SFT), bulls must have a minimum of “fair” sperm motility based on either individual or gross assessment.9 While this may seem to be low, it is a minimum threshold and while there is a positive correlation between motility and fertility with the use of artificial insemination, this does not appear to be the case with natural service bull fertility.10 Accurate evaluation of sperm morphology begins with the preparation of a stained sample of diagnostic quality and the use of a bright-field microscope that has at least 100× oil immersion objective (1000× magnification) or the use of a high-quality phase-contrast microscope. Additionally, one must make a commitment to the careful examination of at least 100 sperm cells. Although, strictly speaking, one who could differentiate normal from abnormal sperm could perform this procedure adequately, the ability to identify the various commonly occurring sperm abnormalities and an understanding of their significance is important. Evaluation of sperm morphology depends on the preparation of a good semen smear. Begin with a clean warm slide, realizing that some “new” slides may be contaminated with detergents, etc. that interfere with staining. For years India ink was utilized as a semen stain. It does not actually stain the sperm cells, but provides a dark background to the white unstained cells. A vital stain, eosin–nigrosin, is currently recommended for field use, due to its ease and consistent staining properties. The eosin portion will penetrate dead sperm cells, staining them pink (red is dead) and leaving live cells unstained (white) against the dark background provided by the nigrosin component. A smear stained with Diff Quik will allow better visualization of white cells and is also an adequate stain for visualizing sperm morphology. Another useful staining procedure is the Feulgen staining method. This technique begins with preparation of a smear and then allowing it to dry for an hour. Next, place the slide in 5 N HCl for 30 min. Wash the slide by running water into the corner of a staining dish containing the slide for 2 min. Then place the slide in Schiff’s reagent for 30 min. Wash again as before and air dry. The Feulgen technique is superior for identifying the nuclear vacuole (crater) defect and because the process removes fat globules, it is an excellent staining technique for smears from extended semen. To stain a semen sample, first place a small droplet of stain on the slide, then add a drop of semen and mix. Placing the semen drop first followed by the stain can result in contamination of the stain solution if the tip of the bottle or dropper inadvertently touches the semen. Once the stain and semen are mixed, a second slide is used to push (spread) the semen across the slide in the same manner as a blood smear is prepared. Because the feathered edge is the best area for evaluation, an alternative method is to create several thickness gradients by stopping and starting as the mixture is spread. It is also often a good idea to make a second slide at the same time, as it is faster to make two than to come back later and make another slide if it turns out that the first slide was not of diagnostic quality (Figure 8.5). Figure 8.5 Two techniques for preparing a semen smear. The stop/start smear provides multiple thickness gradients. Once the slide is dry, the smear can be examined either with phase-contrast or bright-field microscopy at 1000× (i.e., the oil immersion objective for the bright-field microscope). In an attempt to better quantify sperm abnormalities, several classification systems have been developed.6,11 The commonly employed classifications are “primary/secondary,” “major/minor,” and “compensable/uncompensable.” Additionally, abnormalities can simply be counted by each specific defect, which is often done when a BBSE is performed for diagnostic reasons. Quantifying each defect can be cumbersome as there are at least 25 currently recognized sperm defects.12 The primary/secondary sperm classification system, which is still utilized by the SFT, categorizes an abnormality based on the suspected origin of the defect. Primary abnormalities are testicular in origin or, more specifically, occur during spermatogenesis,13 whereas secondary abnormalities are epididymal in origin. However, as advances are made abnormalities are continually reclassified and in fact some abnormalities can be caused during either phase.6 There is also a tendency to ascribe more importance or significance to those abnormalities designated as primary and this may not be appropriate for each individual abnormality (Table 8.1). Table 8.1 Sperm abnormalities as categorized by Society for Theriogenology. The major/minor sperm classification system is based on the suspected significance on fertility.14 Major defects are obviously those whose presence in the ejaculate decreases fertility. Minor defects, which at the time of the original work were not thought to be especially significant, have been subsequently shown to decrease fertility but only when in significant numbers. Interestingly, the primary/secondary categories that came along later and were designated based on presumed origin differ only slightly from the major/minor categories defined by Blom. Categorizing semen defects that result in reduced pregnancy rates into a classification of compensable/uncompensable was introduced by Saacke et al.15 Simply defined, compensable defects in an ejaculate, or more specifically an insemination dose, are those that will not result in a lowered fertility rate if there are adequate additional normal sperm. Thus these defects can be “compensated for” in natural breeding and also in artificial insemination providing additional normal sperm are present in adequate numbers. Examples of these are defects where motility or the ability to traverse the barriers of the female genital tract is compromised, rendering the sperm unable to reach the ovum or able to reach the ovum but unable to initiate the fertilization process.16 Conversely, additional normal sperm will not compensate for the presence of some abnormalities and these defects are categorized as uncompensable. Sperm with these defects can reach the ovum and initiate the fertilization process but are unable to sustain it or result in poor embryonic viability.15,16 These uncompensable sperm compete with and often preempt a normal sperm’s opportunity to fertilize an ovum. Thus, an uncompensable defect will theoretically decrease fertility by the percentage rate that it is present in an ejaculate or insemination dose. Complicating the designation of sperm defects as compensable/uncompensable is the fact that, like the other classification schemes, new advances are likely to result in changes to a category of defects and the complex nature of male infertility in general. However, approaching evaluation of abnormalities with this approach is of tremendous value in helping the veterinarian determine the prognostic implications of a prevalent defect in an ejaculate. Further complicating the issue of evaluation of sperm morphology is the reality that within a specific ejaculate there may be morphologically normal sperm that have chromatin damage. Sperm with chromatin damage are uncompensable in that they can achieve fertilization but the resulting embryo is often of poor quality. However, chromatin-damaged but normally appearing sperm can likely be predicted by the predominant presence of morphologically abnormal cells in an ejaculate. This has been demonstrated by scrotal insulation studies which revealed that the resultant manifestation of defects is dependent on the stage of spermatogenesis at the time of an insult17 and that both the number and type of defects seen reflect a larger number of defective but normally appearing sperm.15 The best approach therefore is to realize that abnormal sperm in an ejaculate are either a reflection of testicular health as it relates to spermatogenesis or a genetic etiology. Carefully evaluate a minimum of 100 sperm at 1000× and “pass” only those bulls that have a minimum of 70% normal. Note prevalent or predominant sperm abnormalities, since the occurrence of certain defects make the presence of “normally appearing” but defective sperm more likely. Bulls with over 70% normal but with 20–25% nuclear or other uncompensable defects should be closely scrutinized. Thus, it is prudent when evaluating the bull with a borderline spermiogram to consider the type of defects and their prevalence and perhaps err on the side of caution. Since there may be some confusion in the categorization of abnormal sperm by veterinarians performing a basic BBSE, the ability to accurately ascertain the percent normal may be the most important aspect of evaluating the spermiogram. In fact some veterinary andrologists have suggested that classification of abnormal sperm as part of the routine BBSE should, or could, be discontinued (6). Most of our clients and virtually all veterinary students when looking at a magnified display of a stained population of sperm can intuitively identify a normal sperm cell. However, subtle abnormalities, or what we as veterinarians would consider not so subtle such as the nuclear vacuole defect, will be easily missed by the novice or casual observer. Thus a complete description of the normal is warranted. Basically, the normal sperm cell (Figure 8.6) can be divided simply into the head and tail for the purposes of description and also as a way to better define the location of defects. The head, which is 8–10 µm long, 4–4.5 µm wide, and 1–1.5 µm thick,7 is composed of nuclear material and the acrosome. The tail, which is approximately 40–45 µm in length, can be further divided into the neck, midpiece, principal piece, and endpiece. Figure 8.6 (a) Normal sperm; (b) distal droplet defect; (c) bent tail defect; (d) Dag defect. As previously stated, when stained with a vital stain such as eosin–nigrosin, dead cells will take up the stain and appear pink, with the acrosome staining much lighter and thus differentiating the head. As the head of the sperm is virtually completely composed of chromatin, the nuclear shape is correlated to fertility18 and indeed the sperm heads of highly fertile bulls are very consistent in size.13 A BBSE is in its essence a prognostic evaluation. Therefore an understanding of the etiology and significance of sperm abnormalities is useful to the veterinarian performing the examination. The goal of this section is to provide a reference for each of the more common sperm defects with respect to classification, etiology, and prognostic significance. Small numbers of these defects may be found in the ejaculate of normal bulls and especially peripubertal bulls. Normally, droplets should be “shed” during epididymal transit. Proximally located cytoplasmic droplets or simply proximal droplets are due to abnormal spermiogenesis,13 are testicular in origin, and appear to be a manifestation of sperm incompetence. Indeed, in men decreased fertility due to the increased presence of cytoplasmic droplets has been shown to result from higher levels of sperm DNA denaturation.11 Distally located cytoplasmic droplets (distal droplets) are not believed to be of much significance and are thought to be epididymal in origin. Thus the proximal droplet is classified as either a primary or major defect, while the distal droplet is classified as either a secondary or minor defect. The distal droplet defect is believed to be compensable and the proximal droplet defect uncompensable.6,13 However, the possibility that the proximal droplet defect is compensable may be supported by work that used ejaculates of either high or low numbers of sperm with this defect for in vitro fertilization.19 In this study, fertilization was decreased as the percentage of proximal droplets in an ejaculate increased, but of the ova that were fertilized, cleavage rates were similar (note that in this study there were normal sperm in each population of sperm cells exposed to the ova). This is a good example of the complexity of categorizing sperm defects. Using the basic metric of a defect that lowers fertility at the level of its expression in the ejaculate, then the proximal droplet defect is clearly uncompensable. However, another standard definition for an uncompensable defect is one that can initiate fertilization but which does not lead to normal embryonic development. Thus the placement of the proximal droplet defect in the uncompensable category is problematic, since the aforementioned study19 validated the literature13 in asserting that a sperm with a proximal droplet is unable to fertilize an ovum (Figure 8.7). Figure 8.7 Proximal droplet defect. Perhaps the best approach is to treat this defect (proximal droplet) as a clinical sign or “marker” for abnormal spermiogenesis, with the potential underlying cause either immaturity or conversely testicular degeneration. So young bulls can be placed in the deferred category and retested since improvement of the spermiogram is likely, but older bulls with this defect have a more guarded to poor prognosis for return to fertility. The distal midpiece reflex (DMR) is the most common abnormality of the sperm tail.13 Considered to be epididymal in origin and therefore a secondary defect, it is also categorized as a minor defect and compensable. This defect is compensable due to the lack of forward progressive motility. Evidence for its origin is based on its rapid appearance in the ejaculate of bulls within a few days of a thermal insult. This defect appears as a sharp hairpin bend at the distal midpiece,20 with a cytoplasmic droplet within the bend. If there is no droplet present, it is likely that the “bend” is due to contact with a hypotonic solution, presumably the stain that was used. It can also be identified during the evaluation of motility as these sperm will be swimming backwards (Figure 8.8). Figure 8.8 Distal midpiece reflex defect. The primary etiology is a negative effect on epididymal function due to depressed testosterone levels, which can in turn be caused by stress, thermal stress (either high or low), exogenous estrogen, or induced hypothyroidism, although normal fertile bulls can have up to 25% of this defect in an ejaculate13 due presumably to its compensable nature. However, when this defect is present at a prevalence of 20–25% in the ejaculate of a bull that meets the standards (>70% normal) when tested at a time of moderate weather and absence of stress, it must be realized that although the bull is fertile now levels of this defect can increase dramatically during times of environmental temperature extremes. Additionally, it has been shown to be heritable in Jersey bulls, some of which would have up to 100% DMR defective sperm in an ejaculate,6 and the possibility of this defect being heritable in other breeds is something that should be considered. This category of defects includes the “pseudodroplet” defect, the mitochondrial sheath defect, and segmental aplasia. Additionally, the midpiece may appear swollen, “corkscrew,” bent, or asymmetric (Figure 8.9). Compensable because of the impact this defect has on motility, it is classified as a primary defect in the SFT system. Since the development of this sperm region occurs almost completely during spermiogenesis,13 the specific origin for most of these defects is undoubtedly testicular. It has been shown that some forms of this group of defects can be caused by increased levels of gossypol,21 a compound found in the cotton plant, and specifically cottonseed, in the diet of bulls. Bulls fed diets high in gossypol appear to be especially sensitive to this compound during puberty.22 The etiology of defects caused by gossypol appears to result from damage to sperm structure during spermiogenesis with further damage occurring during epididymal transit.21 Figure 8.9 Pseudodroplet defect. Also referred to as the “Dag” defect, this is also an abnormality of the midpiece (Figure 8.10). Although small numbers of this defect appear in the ejaculate of normal bulls, it can be found in high numbers (>50%) in some individuals. This defect has been classified as primary and of epididymal origin,23 specifically the cauda epididymis. Named for the Jersey bull from whose ejaculate this defect was first identified, it was immediately believed to have a genetic basis because the same defect was identified in a full brother.24 A breeding trial utilizing a normal bull that had produced offspring with the defect confirmed this suspicion when he was bred to 120 of his daughters and produced sons with the defect. At least in the Danish Jersey, the Dag defect can be transmitted to offspring as a recessive trait.25 Figure 8.10 Dag defect. Additionally, there are increased zinc levels in the plasma and sperm of bulls affected with this defect (bulls with >50% defects). This could presumably be due to either dietary imbalance or a genetically predisposed sensitivity and thus it is not known whether zinc plays a causal or contributory role.25 The abaxial placement sperm type is listed as a secondary defect in the SFT classification scheme. In a series of breeding trials consisting of superovulated heifers that were slaughtered 7 days after breeding for embryo recovery, the artificial breeding of synchronized heifers, and finally a competitive breeding situation, the presence of high numbers (50, 88, and 100%) of abaxial sperm did not negatively impact fertility by either decreasing conception or embryonic survivability.26 Thus abaxial implantation should be considered as simply a variant of the normal as it is in horses and swine. The knobbed acrosome defect can be identified as an apical swelling that may protrude from, or fold over, the head27 and appears most often as a flattening or indention of the apex.13 Early on this defect was identified as having a genetic etiology, specifically an autosomal sex-linked recessive trait in the Friesian breed.24,27 A genetic etiology should be considered when this defect is prominent in the ejaculate over time, but when identified with several other defects an environmental (temperature-related) cause is likely.13,17,28 It is considered to be both a major and primary defect, and the best current evidence is that it is uncompensable.28 The uncompensable nature of this defect is not straightforward as it actually appears to be compensable based on the fact that sperm with this defect do not traverse the reproductive tract of cows efficiently,29 and those that do are unable to penetrate the zona pellucida.28 However, this defect is an example of those imperfections whose presence denotes the occurrence of normal-appearing but defective cohorts.17,28,30 These defective but morphologically normal cells, though able to penetrate ova, yielded lower rates of fertilization and reduced cleavage by zygotes.28,30 Therefore from a practical perspective this defect should be deemed uncompensable. When this defect is encountered in large numbers with other head defects, it is prudent to defer and recheck the bull in 60–90 days. Also, scrutinize more closely those bulls whose ejaculate displays this defect predominantly in large numbers (>20%) as it is known it can have a genetic basis and expresses infertility at levels higher than its occurrence within an ejaculate. These include “pear-shaped” and tapered heads and are often referred to as such (Figure 8.11). This is the most common defect of the sperm head13 and is commonly found in low numbers in the ejaculates of fertile bulls.20 Because there are bulls of normal fertility that have narrowed sperm and because there appear to be variations in the range of “taperedness,” it can be hard to distinguish at what point a designation is made between normal and pyriform.13 In fact, in the human, sperm categorized as pyriform or pear-shaped are no longer considered abnormal.31 Figure 8.11 Pyriform defect. This abnormality is currently categorized as both a primary and major defect. The evidence for whether this abnormality is compensable is equivocal. In general, sperm with misshapen heads do not traverse the reproductive tract, but sperm with this defect apparently do,29 although this appears to depend on the level of deformity.32 The level of deformity also apparently impacts fertilization rates as trials evaluating this defect reveal decreased levels of zona penetration, fertilization, and cleavage rates.32,33 For example, the previously cited work revealed that semen containing this defect at high percentages (85% pyriform heads) had zona penetration at about half the rate of control (90% normal) semen. Considering that semen containing a high percentage of pyriform head defects still resulted in some, albeit much lower, fertilization, these authors came to the conclusion that this could be due to the presence of a small number of normal sperm as well as a percentage of less affected pyriform sperm that may be capable of successful fertilization, suggesting that this abnormality could be partially compensable.33 With respect to etiology, this defect is seen following environmental heat stress, validated by scrotal insulation studies,17 and also from bulls with testicular hypoplasia.13 In addition to environmental causes of heat stress, the scrotal insulation effects of fat deposition around the scrotum that results from heavy feeding during gain tests have the same deleterious effect. Bulls examined after recently coming off a gain test or experiencing adverse environmental extremes that have this abnormality in numbers that contribute to not meeting the metric for percent normal sperm should be deferred. In the case of bulls with testicular hypoplasia, they typically do not meet BBSE standards for scrotal circumference. Small (microcephalic) and giant (macrocephalic) heads are categorized as secondary and minor defects. They are commonly found in very small numbers in the ejaculates of bulls of normal fertility. These defects can be observed with a myriad of other defects (pyriform, vacuoles, etc.) following a disturbance in spermatogenesis, but still rarely exceed 5–7% of the ejaculate.13 Misshapen heads are generally excluded as these sperm traverse the reproductive tract and therefore would be considered compensable. Narrow heads, categorized as primary by the SFT classification and minor according to Blom, can be normal as there are fertile bulls that consistently produce sperm with a narrow head profile.13,32 However, in these bulls all the sperm heads in an ejaculate were very consistent in size. Therefore any sperm with a head size or profile that is observably narrower than the others should be counted as a defect. The nuclear vacuole defect is also termed a “crater” and includes the diadem defect, which is a string or line of vacuoles around the acrosome–nuclear cap junction34 (Figure 8.12). This abnormality is categorized as both a primary and major defect and is uncompensable. Figure 8.12 Nuclear vacuole defect. Courtesy of Dr Robert Carson.
Evaluation of Breeding Soundness: Basic Examination of the Semen
Introduction
Semen evaluation as part of a standard BBSE
Semen collection
Gross evaluation of semen
Evaluation of semen motility
Evaluation of sperm morphology
Preparation of a slide
Categorization of sperm abnormalities
Primary abnormalities
Underdeveloped
Double forms
Acrosome defect (knobbed acrosome)
Narrow head
Crater/diadem defect
Pear-shaped defect
Abnormal contour
Small abnormal head
Free abnormal head
Proximal droplet
Strongly folded or coiled tail (Dag)
Accessory tail
Secondary abnormalities
Small normal heads
Giant and short broad heads
Free normal heads
Detached, folded, loose acrosomal membranes
Abaxial implantation
Distal droplet
Simple bent tail
Terminally coiled tail
Normal sperm
Sperm abnormalities: classification, etiology and prognostic implications
Cytoplasmic droplet
Distal midpiece reflex
Abnormal midpiece
Strongly folded or coiled tail
Abaxial placement
Acrosome defect: knobbed acrosome
Pyriform-shaped heads
Other head size abnormalities
Nuclear vacuole defect
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