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Once the filtered sample is in the measuring cylinder, its colour and consistency can be assessed. This is normally done by eye but requires microscopic examination for confirmation. A good sample should appear milky white in colour, though it may range from watery to creamy, depending upon the spermatozoan concentration within the sample (Kuklin, 1983). The sample should also have a neutral smell and be evenly turbid with no evidence of clots, and should be the consistency of thin single cream (Kuklin, 1983; Frayrer-Hosken and Caudle, 1989).

Factors affecting seminal appearance

Abnormal colours may indicate contamination with urine, blood or pus. Significant increases in the volume of such samples will also be an indication of the extent of the problem.

UROSPERMIA

Urospermia is the term given to the condition resulting in contamination of a semen sample with urine, for which there are several causes (Voss and Pickett, 1975; Slusher, 1997).

Causes. One of the reasons for the condition of urospermia is a disruption of the neural mechanisms involved in ejaculation, which can lead to accidental bladder contraction and hence urine leakage during ejaculation (Rasbech, 1975; Leendertse et al., 1990; Mayhew, 1990). Control of the bladder sphincter and seminal emission is via the a-adrenergic sympathetic nervous system. Hence any interference with, or disruption of, this pathway might be involved in the condition (Voss and McKinnon, 1993). It has also been reported that the condition may be associated with self-mutilation in stallions (Samper, 1995a). In addition, neuropathies which cause bladder paralysis may also result in urospermia. This may be associated with equine herpes virus type I, as a secondary effect, or a result from poisoning with sorghum grass or due to cauda equina neuritis (Varner and Schumacher, 1991; Voss and McKinnon, 1993). Stallions suffering from urospermia may appear normal, with no neurological defects and with adequate libido and mating ability. The condition may be continuous or intermittent in appearance and its occurrence may be unpredictable. Contamination may occur at any time during ejaculation and may be as little as 1 ml or as much as 250 ml or more in volume (Varner and Schmacher, 1991). Evidence suggests that contamination is not likely to be due to just leakage but to an all-or-nothing effect (Nash et al., 1980).

Diagnosis. Gross contamination of a sample is apparent from the semen's yellow to amber colour and characteristic odour of urine. A sediment may also be evident. Microscopically, crystals associated with urine may be observed. In order to diagnose slight contamination, concentrations of urea nitrogen or creatinine may be measured (Danek et al., 1994a), values > 25-30 mg dl-1 and > 2 mg dl-1, respectively, being diagnostic of the condition (Hurtgen, 1987; Varner and Schumacher, 1991). Field tests are now available which enable urine contamination of 10% or greater to be diagnosed. Field tests using test strips are also available for urea nitrogen, nitrates and creatinine (Althouse et al., 1989). The test strips diagnose the presence of urine by changing colour (Voss and McKinnon, 1993). Urea nitrogen strips (Azostix, Miles, Inc., Ag-Vet, Shawnee, Kansas) turn from yellow to green to indicate a positive result. Nitrate reagent strips (Multistix, Miles, Inc., Ag-Vet, Shawnee, Kansas) turn from yellow to radiant orange when positive (Voss and McKinnon, 1993). Elevated osmolarity (> 350 mOsm) may also be indicative of urospermia.

The effect of urine contamination on spermatozoan viability. Urine contamination within a semen sample adversely affects the motility of spermatozoa and their capacity to fertilize an ovum (Hurtgen, 1987; Varner and Schumacher, 1991). Urine contamination may cause an elevation in sample pH which reduces spermatozoan viability primarily via an effect on plasma membrane structure. Finally, changes in osmotic pressure are seen due to the introduction of hyperosmotic urine. This will cause water to pass out of the spermatozoa and down the osmotic gradient to the seminal plasma, resulting in desiccation of the spermatozoa and a reduction in viability (Hurtgen, 1987; Samper, 1995a). The severity of the above problems seems to be dose dependent. It is apparent that stallion spermatozoa can tolerate minute amounts of urine without deterioration but the threshold level for contamination has not yet been determined and is unlikely to be a simple single value.

Treatment Samples with minor urine contamination may be diluted in an appropriate extender immediately upon collection, to try to counteract, or protect the spermatozoa from, the potential urine-related damage. The sample may also be centrifuged after dilution in an appropriate extender; the supernatant is then decanted off, and the urine along with it, and the spermatozoan pellet is re-suspended in urine-free extender prior to insemination (Varner and Schumacher, 1991).

Treatment of the condition is not simple and is often unsuccessful. Treatment may be by alteration in management techniques - for example, by delaying covering or collection until immediately after natural urination. Alternatively, urination may be stimulated by the administration of diuretics, such as frusemide or ephedrine sulphate, immediately prior to covering or collection (Voss and McKinnon, 1993). The effectiveness of such management practices in reducing the problem is disputed (Hurtgen, 1987). Urination may also be encouraged by the presence of faeces, from another stallion or a mare in oestrus, so inducing the stallion's natural territory-marking activity. Some stallions can be trained to urinate upon command, while others urinate in response to feeding, fresh bedding or exercise. As a more extreme measure, bladder catheterization has been used to evacuate urine prior to collection, but repeated use of this technique is not advised due to the risk of urethritis or cystitis (Voss and McKinnon, 1993; Varner and Schumacher, 1991). Pharmacological therapy has been used primarily to close the neck of the bladder, using a-blockers. This treatment apparently works in men suffering from retrograde ejaculation, but this condition is not reported as a significant cause of urospermia in horses and so the effectiveness of the treatment is limited (Varner and Schumacher, 1991; Voss and McKinnon, 1993; Samper, 1995a). Other alternatives, including the use of oxytocin to induce contraction of the bladder sphincter, or bethanecol chloride or flavoxate hydrogen chloride, have been tried but with only limited success (Varner and Schumacher, 1991; Voss and McKinnon, 1993). Significant work is still required before the major causes of the condition can be ascertained and feasible treatments and regimes devised. One of the biggest problems is the unpredictability of the condition, with many stallions showing the condition only intermittently. In general, however, urine is passed during the latter stages of ejaculation. An open-ended AV (section 5.2.4) may be used to identify and collect the various fractions of the ejaculate, so allowing the later jets to be discarded.

HAEMOSPERMIA

Haemospermia is the term given to contamination of a semen sample by blood.

Causes. The causes of haemospermia are varied and more commonly may include lacerations of the exterior of the penis caused, for example, by a tail hair from the mare entering the vagina at covering, or other accidental or puncture wounds of the glans penis area. The condition may also be caused by urethral defects (Schumacher et al., 1995), the damaged areas tending to haemorrhage at erection and ejaculation due to the increased blood pressure.

If it is not treated immediately, the condition is made worse and may predispose to infections or, more specifically, to surface infestation with Habronema or Druschia larvae (summer sore) (Hurtgen, 1987; Varner and Schumacher, 1991; Voss and McKinnon, 1993). Habronemiasis on the glans penis area may itself also cause haemorrhage. The larvae are carried by house and stable flies, which are attracted to the relatively humid environment of the genital area. The larvae are then deposited on the penis when the flies feed, particularly in spring and summer (Varner and Schumacher, 1991). The presence of these larvae induces rapid production of eosinophilic granulation tissue, which surrounds the larvae. As a result this whole area becomes friable and tends to haemorrhage at erection and ejaculation. Particular problems arise if significant infestation of the urethral process occurs (Hurtgen, 1987; Varner and Schumacher, 1991; McKinnon and Voss, 1993). Good hygiene and fly control help to control the condition and organophosphates and ivermectins are used to treat it (Bowen, 1986; S. Revell, Wales, 1998, personal communication).

Urethritis, usually due to bacterial infection of the urethra, is a further common cause of haemospermia. The bacteria most commonly associated with the condition are Pseudomonas aeruginosa, Streptococcus species and Escherichia coli (Blanchard et al., 1987; Samper, 1995a). Stallions with a history of infection with Pseudomonas aeruginosa have an increased chance of suffering from haemospermia (McKinnon et al., 1988). Viral urethritis causes inflammation and vesiculation which may result in haemorrhage at erection and ejaculation (Voss and Pickett, 1975). Seminal vesiculitis, or infection of other accessory glands, may also be a rare cause of haemorrhaging (Blanchard et al., 1987). Blood contamination of semen associated with migration, through the seminal vesicles, of the parasite Strongylus endenatus has been reported (Pickett et al., 1981).

Ulceration of the urethra and rupture of the urethral subepithelial blood vessels have been reported as causes of haemospermia (Schumacher et al., 1995). The latter is reported to be associated with the use of stallion rings to discourage masturbation (Voss and McKinnon, 1993). The use of these rings should be discouraged.

Diagnosis. The extent of the haemorrhage is reflected in the colour of the semen sample, which varies from red to pale pink. However, if no coloration is evident, blood contamination may be shown to be present by microscopic examination for erythrocytes. External haemorrhage is evident from examination of the genitalia for wounds, lacerations, etc. Urethritis, ulceration of the urethra or rupture of the subepithelial vessels around the urethra may be diagnosed by the use of penile and pelvic urethral endoscopy performed on a standing tranquillized stallion. Air or saline may be used to dilate the urethra and aid viewing (Hurtgen, 1987; Voss and McKinnon, 1993). This method may also be used to assist in the diagnosis of accessory gland infection, by examination of the ducts during massage per rectum, and examination of the resulting extruded fluid. Biopsies may be taken of suspect tissue. Positive contrast and double contrast radiographic studies, using barium sulphate solution infused into the urethra, may be used to identify occlusions, prolapses, adhesions, strictures, etc. (Walker and Vaughan, 1980; Voss and McKinnon, 1993). The use of ultrasonography allows more detailed examination of accessory gland structure and function, and may be used as an aid to diagnosis (Varner and Schumacher, 1991). Smears of cell debris left on the urethral process immediately post coitum can be used, after fixation, for the identification of viruses and neoplastic or other abnormal cells.

The effect of blood contamination on spermatozoa viability. The mechanism by which blood contamination affects spermatozoa quality is unclear. It is thought to be an effect modulated by the erythrocytes, white blood cells or platelets, rather than via the serum (Voss et al., 1976; McKinnon et al., 1988; Varner and Schumacher, 1991). Experimental contamination of semen for AI with serum or whole blood resulted in significantly different pregnancy rates: mares inseminated with semen contaminated only with serum showed significantly higher pregnancy rates than those inseminated with semen contaminated with whole blood (Voss et al., 1976).

Treatment. External lesions can be treated in the normal fashion with topical antibiotics to prevent infection and aid the healing process. Cessation of mating until complete healing is achieved is essential to prevent recurrence. Early treatment of Habronema lesions with anti-inflammatory agents (for example, with corticosteroids) may be successful (Hurtgen, 1987).

Subischial urethrostomy has been reported to be successful. The operation involves longitudinal incision of the urethral wall and the enveloping carvenous tissue, in the perineal area. The resulting wound is left to heal naturally (Varner and Schumacher, 1991). This technique was originally developed to investigate the cause of haemospermia prior to the advent of the endoscope. Post urethrostomy and sexual rest stallions who had normal fertility rates prior to the advent of haemospermia are reported to return to their previous breeding ability. However, those with long-term infertility problems before the advent of haemospermia showed no improvement in fertility rates (Sullins et al., 1988). While the reasons for the reported success of this technique are unclear, Voss and McKinnon (1993) have suggested that it may be due to a counter-irritant effect or the temporary diversion of urine.

Bacterial urethritis may be treated, after identification of the bacterium, by using an appropriate specific antibiotic. This is not always successful, due to the relative inaccessibility of the internal genital tract. If persistent genital tract infection is evident, but no pus or haemorrhage is seen, then antibiotic seminal extenders may be used either with AI or by infusion into the mare after natural service (Hurtgen, 1987). Stallions with early habronemiasis lesions or inflammation of the distal urethra may be collected from using an open-ended AV. This allows the blood-free jets to be collected and only these used for insemination (Hurtgen, 1987). Centrifugation of contaminated semen with subsequent resuspension in extenders may also be used (Samper,

1995a). Many conditions leading to urethritis are associated with damp, infected bedding; general improvement in hygiene may have a beneficial effect (Bowen, 1986).

Accumulation of pus or pyospermia is an important indication of infection. This will be discussed in further detail later in this chapter when the bacteriological evaluation of semen is considered. Regardless of the cause, the presence of a significant amount of pus excludes a sample from use in AI. Natural covering should also be avoided until full recovery is evident (Samper, 1995a).

EJACULATION OF A SMALL VOLUME

Samples with significantly smaller volumes than expected may be obtained consistently from some stallions. These are often the result of incomplete ejaculation (Fayrer-Hosken and Caudle, 1989; Samper, 1995a) and a second ejaculation should be taken to confirm this. It must be remembered that there is considerable variation in the volume of semen produced normally by different stallions. Some stallions consistently produce small volume samples (even as low as 15-20 ml) but these will often have higher than average spermatozoan concentrations.

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