Breeding Soundness Examination of Bulls
In bulls, assessment of libido is often not possible during a routine breeding soundness examination (BSE). However, if possible, the bull should be observed serving cows to allow assessment of his desire to breed, ease of mounting, ability to achieve erection and extend the penis, and presence of penile deviation or other abnormalities that may prevent successful service. Libido and serving capacity tests (scoring the number of services achieved during a set time that a bull is in a pen with a restrained cow) have been devised but are time consuming and difficult to standardize under field conditions. In addition, the results are difficult to interpret in light of the variety of stocking conditions used, eg, single versus multiple bulls or small paddocks versus large ranges.
BSE forms have been developed and should be used to ensure the systematic completion of the examination and accurate reporting of results. The bull should be restrained in a chute for the examination. Body condition should be scored, and a general physical examination conducted with special attention paid to the feet, legs, eyes, and sheath. The inguinal rings and internal genitalia should be palpated per rectum to detect any abnormalities, eg, seminal vesiculitis. The scrotum should be palpated to evaluate the testes, epididymides, spermatic cords, and scrotal skin. The testes should be symmetrical in size, smooth, resilient, and freely movable within the scrotum. Cryptorchidism is considered an undesirable heritable trait and renders a bull unsatisfactory for breeding even though his semen quality might be acceptable. In ruminants, the epididymides run caudomedially along the testes in a dorsoventral orientation, with the tail most ventrad. The epididymis should have no palpable masses. The measurement of scrotal circumference (SC) is considered a reliable predictor of paired testis weight, which in turn provides an accurate estimate of daily sperm production and quality. The SC should be measured at its maximal diameter, and size depends greatly on bull age and breed. As a general rule, SC should be ≥30 cm for bulls <15 mo old, 31 cm for bulls 16–18 mo, 32 cm for bulls 19–21 mo, 33 cm for bulls 22–23 mo, and 34 cm for bulls ≥2 yr old. However, readers should consult the latest published breed-specific average SC measures.
Although not used routinely in practice, additional techniques to evaluate the scrotal contents include thermography and ultrasonography. Bull testes must be 2°–6°C (35.6°–42.8°F) cooler than core body temperature for optimal sperm production, and increases in scrotal temperature negatively affect semen quality. Infrared thermography can be used to evaluate scrotal temperature, which should yield a left to right symmetry with a 6° to 4°C (42.8° to 39.2°F) temperature gradient from top to bottom. Abnormal thermograms are almost always associated with poor sperm quality and have been correlated with decreased pregnancy rates. Conversely, evaluation of the testes by ultrasonography does not appear to improve the predictive value of standard evaluation techniques (physical examination, palpation, SC measurement, sperm evaluation). Therefore, it appears that the value of ultrasonography as a tool in BSE in bulls rests on assessment and characterization of grossly detectable lesions of the testes.
For the purpose of a routine BSE, semen is most often collected via electroejaculation; semen can be collected with an artificial vagina (AV) in bulls trained to use one (eg, those in bull studs). The penis usually extends during electroejaculation and should be examined at that time for any abnormalities. If it is not extended during electroejaculation, it should be gently exteriorized for examination by grasping the glans with a cotton gauze and, if necessary, by putting pressure on the sigmoid flexure immediately caudal to the scrotum. Preputial wash samples may be taken for isolation and culture of Campylobacter fetus venerealis (using Clark’s medium) or Tritrichomonas foetus (using Diamond’s medium or a commercially available diagnostic medium kit), in particular when investigating subfertility in bulls ≥4 yr old.
The electroejaculator consists of a rectal probe that has a series of linear banded electrodes connected to a variable current and voltage source. The bull is restrained in a chute, the rectum is emptied, and the entire lubricated probe is inserted rectally with the electrodes oriented ventrally. A hand-operated rheostat permits intermittent pulses of current to be given as the voltage is gradually increased. The response varies considerably, but it is common to use 2- to 4-sec pulses repeated at 5- to 7-sec intervals. After a variable number of such stimulations, erection and protrusion of the penis may be seen, followed by a flow of seminal fluid, or the bull may ejaculate into the sheath without protruding the penis. The semen may be collected by any convenient method; typically a rubber AV cone inserted within a plastic cylinder attached to an 18-in. handle, with a test tube attached to the cone, is used. In some bulls, ejaculation is seen only after a final series of momentary pulses at 1- to 2-sec intervals. Older bulls usually require a higher voltage for ejaculation. In some large bulls, the probe may not reach the correct areas for stimulation; having two or more probe sizes is recommended if BSEs are to be done on a variety of sizes of bulls. The semen collected via electroejaculation is not representative of a full ejaculate in regard to volume and concentration, and thus the sperm production potential of the bull is based on the evaluation of SC. Conversely, sperm quality (motility and morphology) does not differ from that in an ejaculate collected with an AV.
If an AV is to be used, bulls are induced to mount a teaser animal (eg, restrained steer, cow, or phantom), and the erect penis is directed into the AV by the collector as the bull mounts. In preparing the AV, the temperature, which is a critical factor in stimulating ejaculation, is maintained at 105°–107°F (40.5°–42°C). Temperatures up to 118°F (48°C) may assist collections in untrained bulls. The AV should be lubricated with nonspermicidal jelly. The typical volume of an ejaculate is 4–8 mL, and the concentration 1–1.5 billion sperm/mL. A semen sample can be collected from some bulls by transrectal massage of the accessory sex glands. With this technique, erection seldom occurs. After the rectum is completely emptied, the vesicular glands are massaged with a backward motion until a few mL of fluid drop from the sheath. The ampullae are then massaged, and an assistant collects the semen as for electroejaculation. This method is not always successful, and the quality of the semen emission is usually lower than the ejaculate collected by the other two methods.
Sperm concentration, ejaculate volume, and the total number of sperm in the ejaculate are evaluated only if the sample has been collected with an AV. The semen sample should be evaluated immediately for sperm motility. The materials that contact the sperm should be at the same temperature as the sperm (to avoid temperature shock), clean, dry, and nontoxic. Motility evaluation is best when the temperature of the semen is either maintained at ~37°C (~98.6°F) during the short time before it is evaluated, or when the semen is gently warmed to 37°C before evaluation. Both gross motility (“swirl pattern”) in the semen and motility of individual spermatozoa should be assessed; individual motility is the more accurate measure and should be used for classification of the bull.
Gross motility is a function of sperm concentration and individual sperm motility and is evaluated in an undiluted drop of semen placed on a slide without a coverslip and examined at low power (~100×). The intensity of wave motion may be classified into four categories: very good—intense swirling, rapid dark and light waves; good—slower swirling, waves not as intense; fair—slow movement with fewer waves; or poor—very little or no swirl activity. Individual motility is assessed in a sample diluted with warmed saline or semen extender. A drop of diluted sperm is placed on a slide, covered with a coverslip, and examined at 200–400×. The proportion of sperm that are moving progressively across the field of view is estimated by finding multiple groups of ~10 sperm and estimating how many sperm are progressive versus how many are not.
The environments in which BSE are done are variable, and there is a high chance of temperature shock to sperm before they may be evaluated. Therefore, >30% individual progressive motility, or fair gross motility, is considered acceptable. If the examination is performed under optimal conditions, some associations (eg, the Canadian Bovine Practitioners Association) recommend a minimum of 60% motility for a bull to be classified as a satisfactory potential breeder.
Visual estimates of sperm motility are considered appropriate for bull classification in field BSEs. However, in commercial artificial insemination centers, sperm motion evaluation is performed using computer-assisted sperm analysis (CASA), which provides more objective values as well as specific measures not only of percent motile sperm but also of velocities and the track followed by each sperm. For instance, the combination of measures such as beat cross frequency, linearity, average path velocity, straightness, and curvilinear velocity have been correlated with bull fertility. Moreover, some of the motion measures evaluated by CASA such as curvilinear velocity, straightness, and linearity correlate with sperm function such as the acquisition of hyperactivated motility.
The presence of cells other than spermatozoa in the sample should be investigated while estimating motility. RBCs, WBCs, and excess numbers of round epithelial cells and developing forms of spermatozoa may indicate a genital tract abnormality. Careful evaluation of the tract, especially the internal genitalia, may indicate the source of WBCs. The most common cause is seminal vesiculitis. Round, immature germ cells and spermatozoa with proximal cytoplasmic droplets may indicate immaturity or, alternatively, testicular degeneration. For a better assessment of the types of cells present in semen, staining an air-dried smear of fresh semen with Romanowski stain is recommended. This simple technique can provide critical information, in particular when cells other than mature sperm are present in the sample.
A sample of the semen should be fixed in a buffered formaldehyde saline solution to evaluate sperm morphology. This is best done with the fixed, unstained sperm examined under high power (1,000×) using a phase-contrast microscope. At least 100 sperm should be counted, and the proportion of sperm showing different types of abnormalities noted. However, the only number used in the evaluation of the bull is the proportion of normal sperm, which should be >70%. Sperm plasma integrity, which is required for sperm function, can be assessed with the use of a vital stain such as eosin. Eosin penetrates cells with a disrupted sperm membrane, which appear pink on evaluation under light microscopy, in contrast to live cells, which do not stain. Eosin is often combined with nigrosin, which provides a dark background that allows better observation of live/dead sperm cells. An eosin-nigrosin–stained smear can also be used to assess sperm morphology, although it is considered less reliable than using phase-contrast microscopy of an unstained wet preparation. Other, more sophisticated measures of sperm function are not used routinely in cattle BSEs, although they may become more common in referral and artificial insemination centers. Such tests may include combinations of fluorescent probes and flow cytometry to assess membrane integrity, mitochondrial function, or sperm chromatin integrity, and functional assays such as ability of sperm to undergo in vitro capacitation, zona binding, and/or homologous in vitro fertilization.
Bulls are classified as “satisfactory potential breeders” if they have no physical abnormalities that would prevent breeding and if they meet the minimal qualifications for SC, sperm motility, and sperm morphology. Bulls that do not meet these criteria are classified as “unsatisfactory potential breeders” or, if the results are marginal or questionable, they are considered “classification deferred” and a retest should be recommended.