La recoleccion del semen canino y el manejo de la infertilidad en el macho

Transcription

1 La recoleccion del semen canino y el manejo de la infertilidad en el macho Canine semen collection and management of male infertility Michelle A. Kutzler Oregon State University Department of Animal Science-Companion Animal Industries 312 Withycombe Hall Corvallis, OR Telephone: Fax: michelle.kutzler@oregonstate.edu

2 INTRODUCTION Semen is routinely collected from male dogs for the purposes of artificial insemination. Occasionally, when both the male and female dogs are present and the female dog (bitch) is at the proper stage of the estrous cycle to accept breeding (estrus), artificial insemination is still requested due to presence of vaginal anomalies in the bitch (e.g. narrow vagina (maiden), vaginal-vestibular stricture, vaginal septum, vaginal hyperplasia) or a behavioral incompatibility between the male and female dogs. More commonly though, the male and female dogs are located in distant locations and transportation of the semen is less expensive and less stressful that shipping either the male or female dog. Only the first (pre-sperm) and second (sperm-rich) fractions of the ejaculate are needed for semen used for artificial insemination. However, semen should be collected until ejaculation of the third (prostatic) fraction is initiated because it is important that a complete ejaculation is obtained. This can be determined by measuring seminal plasma alkaline phosphatase in the combined first and second fractions. A seminal plasma alkaline phosphatase >10,000 U/ml is consisted with complete ejaculation. 1 SEMEN COLLECTION Semen can be easily collected from the dog using digital manipulation. The materials needed for semen collection in the male depend on the collector s level of expertise with this procedure. At minimum, two 50 ml sterile centrifuge tubes or specimen cups can be used to collect semen as it is ejaculated (one for the combined first and second fractions and the other for the third fraction). For beginners, using a semen collection cone with a 12 ml sterile centrifuge tube attached minimizes chances that precious few drops of sperm-rich (second) fraction will be lost during the thrusting phase of semen collection. Lane manufacturing sells these disposable semen collection cones for approximately $1.00 each ( Latex semen collection cones (artificial vaginas) can also be used with digital manipulation for semen collection in dogs. However, latex semen collection cones need to be carefully cleaned between males including removal of all disinfectant residues and water prior to the next use. In addition, Althouse and colleagues (1991) found that contact of dog semen with latex gloves for 1 minute has a detrimental effect on sperm motility. Contact with vinyl gloves or talcum powder had minimal effect on sperm motility. 2 Under ideal conditions, canine semen collection is performed in the presence of an estrous bitch. The presence of a teaser bitch in estrus will increase the probability that nervous or virgin males will ejaculate when semen collection is attempted. However, the presence of a submissive bitch at any stage of the estrous cycle or even a castrated male can be useful. Estrous bitch synthetic pheromones are commercially available (methyl p-hydroxybenzoate; Aldrich Chemical, Milwaukee, WI) or estrous vaginal secretions from healthy, Brucella canis-negative bitches can be preserved within gauze sponges from earlier examinations in a regular freezer (-20 C) for future use. These pheromones can either be presented on a gauze sponge to the male or better when rubbed over the base of the tail of the teaser animal. It is important to note that the majority of dogs from which this author has collected semen from have not been in the presence an estrous bitch. The lack of availability of an estrous bitch should not be a reason to not attempt to collect semen from a dog. However, it is imperative that any distractions or procedures that would induce anxiety be eliminated or minimized. Fear and pain will prohibit a dog from attaining a complete erection and ejaculating. For excessively timid males, allowing the male to play with the teaser or owner prior to collection may improve the quality of the ejaculate. Initially, the dog s penis is vigorously massaged through the prepuce at the level of the bulbous glandis (caudal-most aspect of the prepuce) until a partial erection develops (initial engorgement of the bulbous

3 glandis). The step is not necessary for males that present with a partial erection. If the collector is righthanded, it is least awkward to collect the semen from the dog s left side, holding the dog s penis with the right hand and the collection container in the left hand. If a teaser bitch is present, the dog may be allowed to mount the teaser bitch. The prepuce is quickly retracted past the bulbous glandis and firm constant pressure is applied to the penis behind the bulbous glandis by squeezing the penis between index finger and thumb. Back and forth manipulation of the penis at this stage is not necessary and may result in the loss of the erection (detumescence). If the bulbous glandis is too engorged, the preputial opening will not be large enough to allow the prepuce to pass by the bulbous glandis. Should this occur, the procedure should be discontinued until the bulbous glandis engorgement subsides (within a few minutes). Distracting the dog by offering him food or taking him for a brief walk will hasten the detumescence of the bulbous glandis. Pelvic thrusting (minimal to exaggerated) may occur following application of pressure behind the bulbous glandis during the development a full erection. Erection is due to impulses from the nervi erigentes composed of parasympathetic fibers from the pelvic and sacral nerves. These nerve impulses lead to dilation of the external and internal pudendal arteries to the cavernous body of the penis. Due to contraction of the ischiourethral muscles, venous return from the cavernous body of the penis is prevented. Blood retained in the sinuses of the cavernous tissue causing the bulbus glandis to swell, glans penis to elongate and the pars longa glandis to slide forward over the os penis. Following the development of a complete erection, the male may attempt to tie as demonstrated by lifting his leg over the collector s right arm (if the collector is right-handed). The erect penis is rotated 180 caudally maintaining the dorsum of the penis dorsally. The collector should continue applying firm pressure behind the bulbous glandis as well as gently pull the penis caudally away from the dog. The 180 turn is made possible by a twist of the penis just behind the bulbus. The penis is very elastic in this area, and the twisting does not seem to cause any discomfort. 3 The penile bone of the dog prevents occlusion of the urethral opening during the erection and twist. It has been suggested that the rotation causes occlusion of the emissary vein of the glans, thus preventing detumescence. Ejaculation will begin immediately following the placement of pressure behind the bulbous glandis. Ejaculation is caused by stimulation of the sympathetic nerves of the penis. The semen and the prostatic fluid are expelled by peristaltic contractions in the muscles surrounding the urethra, particularly the bulbocavernosus and ischiocavernosus muscles. The ejaculate is composed of three fractions: first (presperm), second (sperm-rich) and third (prostatic fluid). The characteristics of the ejaculate should be monitored (dripping; cloudy secretion = first and second fraction, spurting; clear secretion third fraction). The release of the sperm-rich fraction takes 1 2 minutes. The sperm-rich fraction should be thick and white, with average volume of ml, depending on the size of the dog. Collection of the third fraction is only performed to insure complete collection of the first two fractions. A new collection container should be used for the collection of the third fraction. However, it is important to note that addition of prostatic fluid does not appear to affect the motility and viability of chilled spermatozoa. 4 The semen should be protected from abrupt changes in temperature, undue agitation, water, germicides, and detergents. Canine sperm is not as easily cold shocked as are sperm from other species (e.g. bovine, equine). However, canine sperm can be heat shocked very easily. To prevent harming the sperm, the semen should be maintained between body temperature (37 C) and room temperature (20 C). The semen should be evaluated for color, volume, motility, concentration, morphology, etc. However, it is outside the scope of this review to discuss evaluation of the canine ejaculate. If the ejaculate contains fewer than 200 million progressively motile spermatozoa, then the male may be collected immediately following the initial collection to increase the total sperm number shipped by approximately 70% when

4 both ejaculates are combined. 5 Two hundred million motile sperm cells is commonly recommended as the minimum number to use per artificial insemination in dogs. 6-8 It is also important to note that the administration of oxytocin (10 IU IM) or prostaglandin F-2α (2.5 mg IM) ten minutes before semen collection does not increase the number of spermatozoa in the ejaculate. 9 Occasionally, hairs from the abdomen and prepuce can be trapped between the penis and the preputial orifice during the retraction (detumescence), or the skin on the outer layer of the prepuce may be rolled inward (inverted). This may cause pain to the dog, and he may then require some assistance. Application of a non-spermicidal, water-soluble lubricant (e.g. Priority Care ) to the penile mucosa especially behind the bulbous glandis around the opening of the prepuce following semen collection will prevent the dry mucosa of the penis from sticking to the prepuce and becoming inverted. SEMEN EXAMINATION There are four basic components of canine semen evaluation: determination of ejaculate volume, percentage of progressively motile sperm, number of spermatozoa per ejaculate and percentage of morphologically normal spermatozoa. In evaluation for infertility, additional tests may include determination of seminal plasma alkaline phosphatase concentration, semen culture and sensitivity, prostatic fluid (3 rd fraction) cytology, ultrasound examination and biopsy of the prostate and testes, and endocrinology. VOLUME & COLOR Volume alone is not correlated with fertility, but an accurate determination of volume of the 1 st /2 nd fraction is important for calculating total sperm count. Total ejaculate volume varies from ml of 1 st /2 nd fraction and 1-20 ml of 3 rd fraction depending on the amount of prostatic fluid collected. Normal canine ejaculate is the color of skim milk. The etiology of clear or discolored semen should be determined. Yellow normally indicates contamination with urine (urospermia), which can be spermicidal. Blood (hemospermia) may be observed as a red tinge to the semen and the origin (preputial, penile, urethral, prostatic) should be determined. Green or yellowish-green semen denotes the presence of neutrophils and is associated with an infection. Coffee ground-colored semen often denotes a prostatitis and concurrent infection. MOTILITY Progressively forward motility is best evaluated by examining an undiluted sample of the 1 st /2 nd fraction on a cover-slipped microscope slide with the 40X objective of a phase contrast microscope equipped with a stage warmer (<37 C) assisted by computerized linear imaging. If sperm are too concentrated to easily observe, the sample can be diluted with appropriately warmed physiologic saline (0.9% without preservatives), or 2.9% sodium citrate solution. In private practice, undiluted semen can be evaluated at room temperature with light microscopy as rapidly after collection as possible. The number of sperm moving rapidly and progressively forward in a straight line should be estimated. There is great variability among trained observers in estimating of percentage motility, even when the same sample is being evaluated. Nevertheless, most observers can discriminate between very poor motility and very good motility. A rough rule of thumb is that good motility allows the observer to just determine the sperm cell s shape as the cell transverses the 40X field. Fresh canine semen should have progressive motility 70%. Decreased motility may be a response to elevated body temperature,

5 infection or trauma. Contaminated collection containers or incomplete ejaculation should be ruled out as other likely causes for low motility. CONCENTRATION It is essential to determine the total number of sperm within an ejaculate. A WBC Unopette chamber (lavender color code) and a 20 l pipette (1:100 dilution) can be used to dilute a 20 l sample of 1 st /2 nd fraction. The diluted sample within the Unopette should be gently mixed, the first few drops discarded and then both chambers of a hemacytometer slide should be charged. Using a 10X objective, the number of sperm within the center large square that contains 25 small squares should be counted. The counts between both chambers is averaged and this number is the number of sperm X 10 6 /ml of 1 st /2 nd fraction. Multiply this number by the volume of the 1 st /2 nd fraction to give you the total number of sperm in the ejaculate. The total number of sperm/ejaculate should be >200 X 10 6, but this will vary with size of dog and ejaculation frequency (Table 1). Table 1. Body weight (BW), total scrotal width (TSW), daily sperm output (DSO) and resting sperm output (RSO) in normal dogs (modified from Amann, 1991) 10 ; *95% confidence interval. BW (kg) TSW (mm)* DSO (X 10 6 ) RSO (X 10 6 ) For artificial insemination of fresh or chilled semen, vaginal placement of a minimum of 400 X 10 6 progressively motile, morphologically normal sperm cells are needed to obtain pregnancy. For artificial insemination of frozen semen, uterine placement of a minimum of 100 X 10 6 progressively motile, morphologically normal sperm cells needed to obtain. MORPHOLOGY This is an integral, albeit time consuming, part of a breeding soundness examination. Prepare a smear of a sample of 1 st /2 nd fraction on a slide, allow the slide to air dry and stain the slide with any stain that will clearly show morphology of an entire sperm. Morphology is best evaluated using a vital dye exclusion stain, such as eosin-nigrosin. The eosin is a vital dye and is excluded from live cells with undamaged plasma membranes. In the case of cells with damaged cell membranes (dead or dying cells), these cells stain red. For private practice, Diff Quik stain is useful, but slides must be immersed in each solution for 5 minutes (15 minutes total). A Diff Quik stained slide using routine staining techniques (30 seconds per solution) is helpful for identifying leukocytes and bacteria for diagnosing inflammatory conditions arising from the reproductive tract. Sperm cell morphology must be evaluated with an oil emersion lens and at least 100 sperm cells must be counted. Through use of the morphology evaluation, a veterinarian may determine the cause of an apparent infertility be it the rest of defective sperm development (testes) or maturation (epididymides). A normal ejaculate has >80% morphologically normal sperm. SEMINAL ALKALINE PHOSPHATASE (ALKP)

6 The testes and epididymides secrete a unique isoform of ALKP, which is present in the highest concentration in the cauda epididymis. Because the sperm-rich (2 nd ) fraction also originates from the cauda epididymis, high concentrations (>10,000 IU/ml) of ALKP in the 2 nd fraction can differentiate between aspermia (incomplete ejaculation with few or no sperm cell present) and azoospermia (complete ejaculation with few or no sperm cells present). The 2 nd fraction should be centrifuged to pellet any cellular material and the seminal plasma can be measured in a standard serum chemistry instrument used commonly in private practice. For a complete ejaculation, serial dilutions will need to be made to make an accurate measurement of the ALKP concentration. CULTURE & SENSITIVITY Sperm-rich (1 st /2 nd fraction) and prostatic fluid (3 rd fraction) samples as well as a sterile cystocentesis may be cultured for aerobes, anaerobes, Mycoplasma sp and Ureaplasma sp bacteria if cytologic examination of the ejaculate indicates an inflammatory process is present. Samples should be submitted in Amies media with charcoal and cultured under optimal environmental conditions or useful information will not be gained. ULTRASOUND EXAMINATION & BIOPSY OF THE PROSTATE & TESTES Examination of the prostate gland by rectal digital palpation is limited to the caudal dorsal aspect and can be especially unrewarding if the gland enlarges sufficiently to fall cranially out of the pelvis. For this reason and when scrotal palpation or the spermiogram reveal testicular abnormalities, ultrasound examination of the reproductive tract is essential. Prostatic enlargement identified during a breeding soundness evaluation is typically asymptomatic or may involve either some degree of blood dripping from the prepuce, hematuria or hemospermia. Ruleouts should include benign prostatic hyperplasia, chronic prostatitis or prostatic cysts and these can be differentiated on the basis of echotexture and prostatic fluid (3 rd fraction) cytology. Prostatic biopsies are rarely necessary for obtaining a definitive diagnosis during a breeding soundness evaluation and can result in serious intra-abdominal hemorrhage (hyperplasia) and peritonitis (prostatitis) regardless of the method they are obtained. Painful conditions involving the prostate such as acute bacterial infections, abscesses, and neoplasia are diagnosed as emergency situations or during extensive work-ups. Ultrasound examination of the testes and epididymides is an invaluable method for identifying the presence neoplasia or ductal blockage. However, ultrasound examination can not reveal information regarding the spermatogenic capacity of the testes. In dogs that are producing no (azoospermia) or very few (oligospermia) sperm cells with seminal ALKP concentrations >10,000 IU/ml, a testicular biopsy is necessary to obtain a definitive diagnosis of testicular degeneration. If you plan to invade the testicular parenchyma, do a biopsy and not a needle aspirate. Testicular biopsy procedures include general anesthesia and must be an extremely sterile surgery. As with castration, the skin is incised cranial to the scrotum and slightly lateral to the midline. The testes and epididymis are exteriorized and carefully examined. A small, 1 to 2 cm, incision is made in the tunica and a simple interrupted suture is placed in one end of the incision as a means of manipulating the incision line without the use of forceps. A stab incision is made through the tunica albuginea testis. The seminiferous tissue will bulge through the stab wound. The tissue protruding is shaved off with a sterile thin razor blade. The tissue is carefully but rapidly placed in Bouin's fixative, which will not distort the seminiferous tubule morphology. The tunica albuginea is not sutured and the tunica vaginalis is closed with horizontal mattress sutures. The skin is

7 closed with single interrupted sutures. ENDOCRINOLOGY Serum testosterone concentrations can be measured accurately by radioimmunoassay and range from ng/ml (mean: 1 4 ng/ml) in intact male dogs. However, the interpretation of the baseline values is complicated by the daily variation in testosterone secretion, fluctuating as much as 2.5 ng/ml in a 24- hour period. The testosterone producing capacity of the testes may be tested with an injection of human chorionic gonadotropin (hcg) at a dosage of IU/dog. HCG administration produces a rapid (3-4 hour) and greatly elevated (6-fold) secretion of testosterone. However, FSH levels are a better indicator of testicular function. Excessively high FSH levels (5-10 times normal base line) are indicative of significant Sertoli cell damage. Veterinary diagnostic laboratories occasionally contract with pharmaceutical companies to run canine FSH radioimmunassays and can run clinically obtained samples at these times. Radioimmunoassays derived against non-canine FSH are not useful for diagnosing infertility in male dogs. Hypothyroidism is a highly contentious as source of infertility in male dogs. To reliably diagnose thyroid hormone dysfunction, it is essential to evaluate both basal hormone levels as well as the ability of the thyroid to respond to thyroid stimulating hormone. Thyroid replacement therapy has not been proven to be beneficial in the male dog. However, many breeders will prevail upon you to put their male dog, with or without an apparent infertility, on thyroid replacement therapy to improve or maintain their fertile state. I urge you as a practicing clinician to not take the path of least resistance and refrain from placing client s breeding dogs on thyroid replacement therapy when it is truly not indicated. It is also important to remember that hypothyroidism may be a familial trait and as with other genetic disorders, it behooves you as the veterinarian to advise the clients against breeding affected animals and perpetuating the trait. MANAGEMENT OF STUD DOG INFERTILITY Stud dogs with normal mating ability and normal semen parameters are rarely infertile. When faced with a case of male infertility, a careful history, physical examination and initial laboratory results should be collected. It is useful to tentatively define the problem using these criteria: Is ejaculatory ability or behavior normal? Abnormal ejaculatory ability or behavior could represent a systemic, neurologic, or behavioral problem. Is the semen evaluation normal? Is the infertility congenital or acquired? NORMAL SEMEN EVALUATION Following a normal semen evaluation, the next step is to investigate the fertility of the bitches that were bred and the breeding management used. If these cannot be ruled out as causes of infertility by history, a planned breeding using excellent breeding management and a bitch of proven fertility should be planned. If problems with intromission or the tie are suspected, check the bitch for vaginal obstructions and check the bulbus glandis for engorgement prior to full intromission. This would result in an outside tie or no tie at all. The os penis should be palpated. When the penis is flaccid, the os penis should extend almost to the tip of the glans penis. If it is too short, the end of the glans will not be directed forward or the penis will not be rigid enough to successfully enter the vagina. The os penis should also be checked for a fracture. The presence of a persistent penile frenulum should be ruled-out. A frenulum

8 is a thin strand of tissue attaching the tip of the penis to the inner preputial mucosa. Normal males, as well as intersex dogs may have a persistent frenulum. Some dogs that have apparently normal semen will have sperm counts change significantly with frequent ejaculation. The daily sperm output (DSO) can be determined with daily ejaculation for 7 days. Sperm counts on days 6 & 7 should represent the DSO. The DSO can then be compared to that of normal dogs of similar body weight. 10 Frequent ejaculation of normal dogs will not change daily sperm output by the testes, but will reduce sperm reserves in the epididymides, resulting in a decrease in number of sperm/ejaculate after several ejaculations. In normal dogs, the sperm count should stabilize within the normal range for DSO, which is adequate for fertility. There are some dogs, old ones in particular, in which sperm counts fall below normal and out of the fertile range by the second or third ejaculate. Breeding management with these males requires initial sexual rest and then use of only one or two ejaculates at the precise time. Abnormal sperm morphology should be ruled-out. Sperm with normal appearing morphology still may not be normal. Numerous ultrastructural and biochemical defects which inhibit normal sperm function have been reported in horse and bovine sperm. These have not been well researched in the dog. However, an acrosome-nuclear abnormality was found by electron microscopy which was not apparent by light microscopy 11 and a midpiece abnormality (appeared to be droplets, but were not) associated with infertility has also been reported NORMAL SEMEN EVALUATION WITH EITHER ABNORMAL SEMINAL FLUID APPEARANCE, PH, OR CYTOLOGY; HIGH WBC COUNT; POSITIVE CULTURE; SPERM CELL AGGLUTINATION The significance of an abnormal appearing seminal fluid in the dog is unknown, although urine and blood in ejaculates from other species is known to decrease sperm viability. Normal prostatic fluid (3 rd fraction) is clear and acellular. Yellow fluid maybe due to urine in the ejaculate and should warrant evaluation of bladder sphincter tone. Green exudate may coincide with bacterial infection and should be examined by cytology. Red or brown fluid may be due to hemoglobin. In the absence of trauma during semen collection, further testing to rule-out prostatic hyperplasia, neoplasia or infection should be done. Normal prostate fluid should have a ph of The ph can deviate from this with bacterial prostatitis. WBC s may be present in cytology preparations with hyperplasia or infection, although degenerative WBC should be only be present with infection. An elevated WBC count in the 1 st and 2 nd fractions (>2000 cells/ l) indicates that cytology evaluation and culture for aerobic, anaerobic, Mycoplasma sp. or Ureaplasma sp. bacteria should be performed. However, positive semen cultures need to be interpreted in relationship to clinical signs. Agglutination of sperm cells has been reported with B. canis infection in the dog. Antibodies to sperm cells can cause agglutination and infertility in man, however, the role of anti-sperm antibodies in the dog is not clear. TERATOZOOSPERMIA WITH NORMAL SPERM CELL COUNT AND MOTILITY The presence of >10% primary sperm abnormalities or >20% secondary sperm abnormalities is abnormal. The first collection of a young dog (just after puberty) will often have abnormal morphology. The semen should be rechecked at 60-day intervals. In adult dogs, testicular disorders such as orchitis, toxin exposure, hormonal or chemotherapeutic agents, should be investigated. The effects of abnormal morphology may be transient, depending upon the cause. Congenital defects in spermatogenesis should be considered if the semen has always had a high percentage of abnormal sperm. If there is a high percentage of secondary defects, make sure collection and preparation of sample was not traumatic to sperm. When abnormal sperm morphology is the only abnormality and the number of morphologically

9 normal sperm is within the normal range for total sperm/ejaculate, offspring may be obtained with good breeding management. ASTHENOZOOSPERMIA WITH NORMAL SPERM COUNT Asthenozoospermia occurs when <25% of the sperm have progressively forward motility (the minimum percentage of motile sperm cells required for fertility in the dog is not known). When this is diagnosed, it is important to rule out the possibility of contaminated collection equipment. Collections made on different days will help to evaluate this. Lubricants, collection cups, funnels, artificial vaginas, slides, coverslips, and diluents should not contain water, urine, detergents, ethylene oxide, or formalin residues. Equipment should be rinsed several times in water only, and should be completely dry before use. If the equipment is not the problem, consider systemic disease, infection or toxin exposure. Semen alkaline phosphatase should be measured to be sure that a complete ejaculation has occurred. Congenital defects such as immotile cilia syndrome may be considered. In young dogs, motility may be abnormal initially and it should be rechecked at 60-day intervals. Ejaculates that repeatedly contain sperm with poor motility may indicate disturbances in sperm transport or storage, or defects in the sperm cell flagellum such as Kartagener's syndrome. OLIGOZOOSPERMIA Oligospermia is defined as sperm counts <200 X 10 6 sperm/ejaculate in dogs weighing >10 pounds. To place a dog in this category, you must be certain that the semen specimen collected was adequate and representative. This can be accomplished by repetitive collections under optimal conditions, ruling-out fear or discomfort problems interfering with collection. Sperm counts on ejaculates taken on different days are necessary to confirm consistently low count and DSO must be determined. If daily sperm output is abnormally low, testicular function is probably abnormal. 15 Systemic disease, infectious disease of the reproductive or urinary tracts, febrile conditions, drugs, toxins, steroids can all affect sperm production. Ultrasonography and semen cultures may be helpful in diagnosing the etiology. In the absence of an infection, gonadal steroids and gonadotropins before and after GnRH stimulation can be measured. Some dogs with counts in the range of X 10 6 sperm/ejaculate have been reported to produce offspring, but lowered fertility is expected (lowered conception % and smaller litter size). Breeding management of the bitch using only one or two ejaculates is best. This will require that ovulation timing in the bitch is precise. Retroejaculation or ejaculation of some portion of the 2 nd fraction into the urinary bladder has been hypothesized as a reason for oligozoospermia or azoospermia in the dog and probably occurs rarely. Low sperm count with evidence that some 2 nd fraction was ejaculated (borderline levels of alkaline phosphatase in the seminal plasma) may warrant investigation for retroejaculation. The normal route for elimination of sperm in the absence of ejaculation is from the tail of the epididymis to the vas deferens and then to the bladder, so presence of sperm in the bladder is not abnormal. When retroejaculation is suspected, the bladder is catheterized, all urine is removed, and a portion examined for spermatozoa. Then an ejaculate is collected, and the bladder is catheterized and the post-ejaculation urine is examined for spermatozoa. If retroejaculation is occurring, more sperm should be found in the post-ejaculation sample than the prior sample. Chronic treatment with - adrenergic agonists (phenylpropanolamine or ephedrine) has been described as successful. 16 AZOOSPERMIA Azoospermia occurs when there are no sperm in the ejaculate. This must be differentiated from an aspermic sample, which also is devoid of sperm but results from failure to ejaculate. To place a dog in this category, you must be sure that the semen specimen collected was adequate and representative.

10 Recommend repeat collections to verify, collect under optimal conditions, rule out fear or discomfort problems interfering with collection of a representative sample and determine alkaline phosphatase level of seminal plasma. Congenital causes of azoospermia include chromosomal abnormalities (e.g. XXY), endocrine abnormalities (1 or 2 hypogonadism), inborn errors of metabolism, gonadal abnormalities (e.g. testicular hypoplasia, Sertoli cell only syndrome, bilateral cryptorchidism, sperm cell formation defects), and ductal abnormalities (e.g. segmental aplasia anywhere from the rete testis to the urethra). 17 Two groups of azoospermic dogs have been identified. 18 The first group consists of dogs that have normal serum testosterone concentrations, normal or elevated serum LH concentrations, and elevated serum FSH concentrations, abnormal testes on physical examination, and abnormal spermatogenesis in testicular biopsy. The elevated FSH could be the result of pituitary malfunction. However, it is more likely that FSH elevation occurs as an appropriate response to decreased inhibin secretion following the decrease in spermatogenesis. No effective treatment regimen for this condition in the dog has been reported. The second group consists of dogs that have normal endocrinologic tests, normal testes and epididymides as determined by physical examination, normal spermatogenesis in testicular biopsies, and no sperm in smears of aspirates from the caudal epididymides. These are thought to have bilateral blockage of the epididymides, although this is difficult to document. Possible causes for this type of lesion include fibrosis secondary to infection. No treatment regimen has been reported for this condition. Diagnosis can be attempted with a karyotype, testicular biopsy, and gonadal steroid and gonadotropin concentrations. Acquired causes of azoospermia are not uncommon but there is little available data regarding this in dogs. The damage may be permanent or temporary, such that the semen should be rechecked every two months. It may be months before any improvement is seen. 19 If the sperm stem cells are permanently damaged, there is no effective treatment. REFERENCES 1. Kutzler MA, Solter PF, Hoffman WE, et al: Characterization and localization of alkaline phosphatase in canine seminal plasma and gonadal tissues. Theriogenology 2003;60(2): Althouse GC, Ko JCH, Hopkins SM, et al: Effect of latex and vinyl examination gloves on canine spermatozoa motility. J Am Vet Med Assoc 1991;199: Grandage J: The erect dog penis: a paradox of flexible rigidity. Vet Rec 1972;91: Sirivaidyapong S, Ursem P, Bevers MM, et al: Effect of prostatic fluid on motility, viability and acrosome integrity of chilled and frozen-thawed dog spermatozoa. J Reprod Fertil (Suppl) 2001;57: England GC: Semen quality in dogs and the influence of a short-interval second ejaculation. Theriogenology 1999;52(6): Gill HP, Kaufman CF, Foote RH, et al. Artificial insemination of beagle bitches with freshly collected, liquid stored, and frozen-stored semen. Am J Vet Res 1970;31: Mickelsen WD, Memon MA, Anderson PB, et al: The relationship of semen quality to pregnancy rate and litter size following artificial insemination in the bitch. Theriogenology 1993;39(2): Pinto CR, Eilts BE, Paccamonti DL: The effect of reducing hindquarter elevation time after artificial insemination in bitches. Theriogenology 1998;50(2):

11 9. Traas AM, Kustritz MV: Effect of administrating oxytocin or prostaglandin F2alpha on characteristics of the canine ejaculate. Can Vet J 2004;45(12): Amann RP Reproductive physiology and endocrinology. In Morrow DA (ed), Current Therapy in Theriogenology, 2nd ed. Philadelphia: WB Saunders, Hrudka F Acrosomo-nuclear syndrome in canine sperm. Andrologia 15: Aughey E, Renton JP The ultrastructure of abnormal spermatozoa in a stud dog. J Reprod Fertil 25: Renton JP, Aughey E Certain interesting aspects of infertility in two Shetland Collies. Vet Rec 88: Oettle EE, Soley JT Infertility in a Maltese poodle as a result of a sperm midpiece defect. J South Africa Vet Assoc 2: Olar TT, Amann RP, Pickett BW Relationships among testicular size, daily sperm production and output of spermatozoa, and extragonadal spermatozoal reserves of the dog. Biol Reprod 29: Feldman EC Infertility. In Ettinger SJ (ed), Textbook of Veterinary Internal Medicine. Philadelphia: WB Saunders, Majeed ZZ Segmental aplasia of the Wolffian duct; report of a case in a poodle. J Small Anim Pract 15: Freshman J, Amann RP, Soderberg SF, Olson PN Clinical evaluation of infertility in dogs. Comp Contin Educ 10: Evans J, Renton JP A case of azoospermia in a previously fertile dog with subsequent recovery. Vet Rec 92: