The following article is re-printed from the Australian Association of Cattle Veterinarians “Veterinarians Examination of Bulls” handbook; edited by M McGowan, D Galloway, E Taylor, K Entwistle and P Johnston.

INITIAL EXAMINATION OF SEMEN

EVALUATION OF DENSITY

The following density scale may be used:

Table 3. Bull semen density scale

SCALE	GROSS APPEARANCE	APPROXIMATE SPERM CONCENTRATION 10 SPERM/ML)
0	clear to cloudy	0 to 200
1	cloudy to milky	200 to 400
2	milky	400 to 800
3	thick milky	800 to 1200
4	creamy	1200 to 1800
5	thick creamy	1800+

EVALUATION OF MOTILITY

Prewarmed (37°C) microscope slides, glass coverslips, disposable glass or plastic pipettes and a vial of phosphate buffered saline are required. As the relationship between sperm motility and fertility has been established under ideal laboratory conditions, it is vital that when examining semen samples in the field, good laboratory techniques are practiced. Semen samples exposed to sudden decreases in temperature (cold shock) will have significantly lowered motility. A good quality binocular microscope (preferably with phase contrast) is essential. Further, the use of a temperature controlled portable microscope stage warmer will greatly reduce the risk of cold shock. Urine contamination will also significantly reduce sperm motility.

Mass activity or wave motion is determined by placing a drop of semen on a prewarmed microscope slide and examining the edge of the drop (x40 or x 100 power). A description of the mass activity rating recommended is presented below (Table 4). Satisfactory quality raw semen has a mass activity of at least 3 i.e. a distinct slow swirl is seen. However some semen samples are too dilute because of the method of collection (or response of the bull) to show wave motion. With these samples only the percentage progressively motile sperm can be assessed.

Table 4. Bull semen mass activity rating system

RATING	MICROSCOPIC APPEARANCE
0	no swirl – nil or sporadic oscillation of individual sperm
1	no swirl – generalized oscillation of individual sperm only
2	very slow distinct swirl
3	slow distinct swirl
4	moderately fast distinct swirl
5	fast distinct swirl – appearance of good quality ram semen

The percentage of progressively motile sperm is determined by examining a small drop under a coverslide. Dense semen may be diluted by mixing a small drop of semen with a drop of prewarmed phosphate buffered saline or buffered 2.9% sodium citrate diluent on a microscope slide and then applying a coverslip. This examination is best done using phase contrast microscopy (x 400 power) but can be done with a good quality light microscope (rack down the condenser and reduce the light). Satisfactory quality raw semen has at least 50% progressively motile sperm. In situations where only a clear to cloudy sample of semen is collected (e.g. partial ejaculate only collected due to poor response of bull to electroejaculation), then assessment of percentage progressively motile sperm will be the only motility assessment that can be applied and the semen is usually not diluted.

It may be also useful to score the rate (speed) of progression of the spermatozoa as follows:

Table 5. Scoring system for the rate of progression of bovine spermatozoa

0	no sperm movement
1	slight tail undulation without forward motion
2	slow tail undulation with slow or stop and start forward motion
3	forward progression at a moderate speed
4	rapid forward progression
5	very rapid progression in which cells are difficult to follow visually

The following are some guidelines to assist in interpretation of semen samples:

• A bull with well developed testicles and good testicular consistency usually produces satisfactory quality semen. Failure to obtain a sample of satisfactory quality suggests inadequate collection technique. Very few bulls have “no spermatozoa”.
• If the semen is of good density, wave motion and motility on initial examination and does not contain pus, a representative sample has been obtained and the bull is capable of producing high quality semen. These findings reflect normal testicular function.
• If a massaged or electroejaculated semen sample is of low concentration but of high motility this also suggests normal function of the reproductive organs. However, use of the technique has resulted in failure to obtain a representative sample with respect to sperm numbers. Scrotal circumference provides information about the quantitative output of the testicles.
• If a sample is of low concentration and has low motility, then the interpretation is more difficult. Another sample should be taken immediately to see if motility is improved. Avoid cold chock and take care when handling the sample. Over 50% motility is generally regarded as reflecting normal testicular and epididymal function. If a sample of semen showing 50% motility or more cannot be obtained then the results have to be interpreted in the light of the difficulties of obtaining the sample, morphology of the spermatozoa, the size and consistency of the testicles, the condition of the epididymal tails and the history, age, body condition and health of the bull.

LABORATORY EXAMINATION OF SEMEN

Laboratory examination of the semen can be helpful in reaching a diagnosis when there is testicular and/or duct system dysfunction. It adds information and therefore confidence in reporting that the reproductive system is functioning normally. Some diagnostic semen laboratories send out, on request, a kit for preparing samples for laboratory examination. This kit may contain slides for smears for assessment of head morphology and cellular elements other than spermatozoa: nigrosin-eosin for assessment of % alive and sperm morphology, buffered formol saline for assessment of % abnormalities of the head (including acrosome) midpiece and tail, a slide carrier and an empty vial for some of the samples for concentration assessment. In addition there are directions for making a kit up and a form on which to record details of the bull and the result of clinical examinations and initial examination of the semen. The report obtained from the laboratory contains the results sent in of the veterinarian’s examination on the complete picture with respect to normality or a likely diagnosis of abnormality.

EXAMINATION OF SPERM MORPHOLOGY

The morphology of individual sperm is determined by either examining an eosin-nigrosin or other appropriately stained semen smear under oil immersion (x 1000 power) or by examining using phase contrast microscopy (x 400) a thin coverslip preparation of semen preserved in 0.2% buffered glutaraldehyde or buffered formol saline. The latter technique has the advantage that acrosomal abnormalities and nuclear vacuoles can more readily be detected. A good quality microscope is essential for the study of sperm morphology.

The recommended technique for the preparation of an eosin-nigrosin stained semen smear is as follows:

• Place a 4 to 5mm drop of warm stain near the end of a warm microscope slide
• Place a drop of semen near the stain and mix the two on the slide using either a pasteur pipette or applicator slide. The size of the drop of semen varies with the density of the semen sample. For very concentrated semen use a drop 2mm in diameter. For dilute semen use a larger drop, up to 6mm in diameter.
• To make the smear, a second slide held at a 30° to 40° angle is pushed against the drop of stained semen and pulled back slowly across the slide.

(From Barth, A. & Oko, R., Abnormal Morphology of Bovine Spermatozoa, 1989).

When using the eosin-nigrosin stain, the stain should be prewarmed prior to use and stained smears should be rapidly dried (for optimal definition of sperm cell morphology) preferably on a temperature controlled slide warmer or microscope warm stage. Unstained smears can be made and stained in the laboratory with carbol fuchsin eosin (Williams) stain.

Plate 25 (page 56) illustrates sperm cell abnormalities commonly observed in bull semen. Table 6 (page 56) summarises the effects of specific sperm cell abnormalities on bull fertility. Usually 200 sperm cells are counted and the total percentage morphologically normal sperm recorded. Usually abnormal sperm are categorized and percentages determined using a standard haematology cell counter. For a detailed description of sperm morphology practitioners should consult the following reference text : A.D. Barth and R.J. Oko, Abnormal Morphology of Bovine Spermatozoa. It is recommended that morphological abnormalities be reported by their descriptive name (see Plate 25). Artificial classifications and groupings such as “major and minor” or “primary and secondary” should not be used.

Satisfactory quality semen has at least 70% morphologically normal sperm. Increased percentages of morphological abnormalities are indicative of degenerative changes or hypoplasia of the testis and/or epididymis. Frequently, following examination of a single semen sample, it is difficult to determine whether the increased proportion of sperm abnormalities are indicative of permanent or transient testicular/epididymal dysfunction. Approximately 61 days are required for spermatogenesis plus an additional 10 to 12 days for passage of the sperm through the epididymis.

The details of the morphologic evaluation of the semen (spermiogram) can be used as evidence of normal function or dysfunction in the reproductive tract and they assist in making a diagnosis.

Morphologically abnormal heads reflect a disturbance of the spermatogenic epithelium. Normal bulls have <20% of these abnormalities and over this figure the disturbance of function is expected to influence fertility. Abnormal sperm heads are increased in testicular degeneration and sometimes in testicular hypoplasia. The higher the percentage the more severe the disturbance. Changes over time in cases of testicular degeneration are helpful in determining a prognosis.

Spermatozoa with attached proximal cytoplasmic droplets should be lower than 4% of spermatozoa in the ejaculate. Higher figures suggest immaturity of testicular function, inability of the spermatozoa leaving the spermatogenic epithelium to mature properly in the epididymis or a dysfunction of the epididymis with respect to maturation of sperm atozoa.

Increased percentages of distal cytoplasmic droplets (should be <4%) can also be a result of dysfunction of the testicle or the epididymis.

Tailless heads (should be <15%) are a prominent feature of testicular degeneration but may also be present in testicular hypoplasia and disturbances of the duct system (e.g. epididymitis, ampullitis).

Distal reflex defect or singly bent tails (should be <8%) are present in dysfunctions of the testicle and the epididymis but may also appear as an artifact (simple bent tail, no retained distal droplet) if the semen was cold chocked or subjected to an abnormal osmotic environment, e.g. water in the collection tube or a leaking artificial vagina. Singly bent tails are often the main abnormality in frozen semen and result from damage in the freezing and thawing process.

The ‘Dag’-like defect or doubly bent tails (should be <4%) can reflect a disturbance in the testicle or epididymis.

Coiled tails (should be <3%) are interpreted similarly to the singly bent tails.

The pattern of abnormalities and the proportion of spermatozoa affected help to diagnose disturbance of function and its severity. In some samples of semen single characteristics mildly increased above the normal range may reflect a temporary fluctuation in that characteristic emanating from an otherwise normal reproductive tract and requires careful interpretation in the light of all findings in the bull. Change in the spermiogram over time in serial samples help to establish a prognosis.

The findings from the microscopic examination of semen must always be interpreted in light of the findings from the history, general clinical examination and physical examination of the reproductive organs. 

Plate 25. Common sperm abnormalities.

A. Knobbed acrosome (common form)
B. Knobbed acrosome (beaded form)
C. Pyriform head (severe)
D. Pyriform head (moderate)
E. Pyriform head (slight)
F. Nuclear vacuoles
G. Diadem defects
H. Detached head
I. Distal reflex
J. Dag-like defect (broken midpiece)
K. Dag-like defect (severely bent midpiece)
L. Proximal droplet
M. Distal droplet
N. Teratoid (severe)
O. Teratoid (moderate)
P. Normal spermatozoa

Table 6. Effect of specific sperm abnormalities on the reproductive performance of bulls

(modified from Barth & Oko, 1986)

SPERM ABNORMALITY	PATHOGENESIS	EFFECT ON FERTILITY
Knobbed Acrosome	Abnormal pathogenesis due to disturbances in testis heat regulation e.g. systemic illness, toxicity, nutritional deficiencies, fat deposition around scrotum. Heritable.	Bull with 83-99% of spermatozoa affected was used for natural mating (single sire) – calving rate of cows mated was 9%. Sperm do not attach to zona pellucida.
Pyriform Head	Abnormal spermiogenesis due to disturbances of either heat regulation in testis of endocrine control of testicular function. Possible genetic predisposition.	Reduced fertility in superovulated heifers inseminated with semen containing a high proportion on affected cells.
Nuclear vacuoles (pouches, craters, diadem defect)	Unknown, possible stress induced. Diadem also thought to be associated with feeding high concentrate rations.	Bull with 80% diadems had a history of low fertility but produced apparently good quality semen (e.g normal post thaw motility). However, fertilization rate was reduced by 54%
Detached Heads	Testicular hypoplasia. Testicular degeneration, inflammation of ampullae, and epididymis. Appears to be a genetic predisposition.	Bull suffering from laminitis and toxaemia had 91% sperm cells affected. Four to 6 months later incidence decreased to 33%. Can be associated with sperm accumulation and cell death.
Distal Midpiece Reflex	Thermoregulation disturbances of the testis. Low testosterone levels. Can be induced when spermatozoa exposed to hypotonic solutions or cold shock. Genetic disposition.	High incidence may result in reduced fertility. Usually transient.
Dag-like Defect	Disturbance of spermiogenesis. Genetic disposition.	High incidence results in significant impairment of fertility.
Proximal droplet	Disturbance in later stages of spermiogenesis and in function of the caput epididymides. Immaturity of testicular function in young bulls.	Marked reduction in in vitro fertilization rate following use of semen containing >30% proximal droplets.
Teratoid (undeveloped)	Severe disturbance in spermatogenesis and spermiogenesis. Tubule degeneration and fibriosis.	High incidence results in significant impairment of fertility.

NB: When very high proportions of spermatozoa are affected by a specific abnormality for extended periods, in the absence of a significant representation of other abnormalities, the etiology may have a genetic basis. Alternatively, in the incidence of a range of sperm abnormalities is likely to be associated with testicular and epididymal pathology.

Bulls which produce semen which fails to meet the above minimum requirements, should be re-examined following an interval of 4 to 10 weeks to determine if there has been any significant change in semen quality. To provide an accurate prognosis for bulls with unacceptable quality semen, at least 2 semen collections (conducted 4 to 10 weeks apart) should be examined.

Samples of semen from normal bulls should contain only spermatozoa and occasional squamous epithelial cells from the penile and preputial epithelium. The presence of other cell types (e.g. leucocytes, germinal epithelial cells in the semen), is indicative of a pathologic process either in the testicles and duct systems or internal accessory sex organs. Specific identification of the cells present in a semen smear can be achieved by using a standard Diff Quik or haematoxylin eosin stain. Neutrophils are the most common inflammatory cells found in the semen of bulls with seminal vesiculitis. Neutrophils tend to be of similar size, have a characteristic nucleus and occur singly; they are seen in clumps if flakes of exudates are grossly present. Germinal cells are variable in size and their presence in a semen sample is indicative of testicular degeneration.

Difficulties of collecting a representative sample may be reflected in the same as adequate volume, low concentration, lowered initial motility and normal morphology. Collection of semen after a long period of sexual rest may be reflected in high density, lowered wave motion and motility with sometimes a mild increase in tailless heads. Improvement in successive samples suggests that sexual rest was the cause of poorer characteristics in earlier samples. In infertility which is the result of behavioral problems, the semen picture is often normal. A statement in the report can be made that if the bull were ejaculating this (normal) quality semen into normal cows then they should have become pregnant. This situation is found, for example, in many cases of corkscrew deviation of the penis.

Many semen characteristics may be affected when there is testicular dysfunction. Lowered concentration of spermatozoa and motility as well as increased abnormalities of the sperm herd, tailless heads and other midpiece and tail abnormalities are common. Specific to testicular problems are the reduced sperm output, sperm head abnormalities and structural abnormalities of the midpiece. In degenerative changes in the testicles the semen picture tends to change over time with increased severity in progressive degeneration or improvement when regeneration is taking place. Disturbance in the epididymis may be reflected in lowered motility and increases in the percentage of spermatozoa with various patterns of midpiece and tail abnormalities. Abnormality of the ampullae will often result in increased tailless heads. Inflammatory conditions of the duct system which is still patent will result in reduced motility and an increase in tailless heads accompanied by leucocytes and possibly epithelial cells from the damaged duct. In seminal vesiculitis leucocytes, epithelial cells and chromatin clumps may be found in the semen. Leucocytes and epithelial cells which have come from an inflamed part of the accessory genitalia will often be degenerate and they may be accompanied by some non specific cell debris.