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How to Investigate Azoospermia in Stallions

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How to Investigate Azoospermia in Stallions NON-PREGNANT MARE AND STALLION How to Investigate Azoospermia in Stallions Terry L. Blanchard, DVM, MS, Diplomate ACT; Steven P. Brinsko, DVM, MS, PhD, Diplomate ACT; Dickson D. Varner, DVM, MS, Diplomate ACT; and Charles C. Love, DVM, PhD, Diplomate ACT Authors’ address: Department of Large Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843-4475; e-mail: stalliondoc@ gmail.com. © 2009 AAEP. 1. Introduction did not ejaculate.3 A number of reports describe In a review of ejaculatory dysfunction, McDonnell1 therapy indicated for ejaculation failure, but they reported that ϳ25% of stallions referred to a fertility are not the subject of this report. Briefly, they in- clinic had evidence of ejaculatory problems. The clude breeding and/or pharmacological management vast majority of cases were anejaculatory (failure to to increase sexual stimulation before and during the ejaculate). Less than 1% of horses in that survey breeding process, treatment and/or breeding man- were truly azoospermic (i.e., ejaculated seminal flu- agement to minimize potential musculoskeletal pain ids devoid of sperm). Failure to ejaculate sperm that could interrupt the emission and ejaculatory can be a troublesome problem that requires accurate process, and pharmacologic manipulation to lower diagnosis, determination of prognosis for correction the threshold to emission and ejaculation.1–3 Tech- (sometimes necessitating retirement as a breeding niques used to manage repeated ejaculatory failure stallion), and arduous treatment and/or breeding can be arduous and time consuming, and they are management to correct.2,3 Figure 1 represents an reviewed by Varner et al.3 attempt at a schematic overview of an approach to When breeding behavior and apparent ejaculation diagnosis of lack of sperm in ejaculates. seems to be normal but no sperm are present in the seminal fluids collected, azoospermia should be sus- 2. Confirming Ejaculation pected. Secondary signs of ejaculation (e.g., flag- Clinical evaluation of stallions failing to produce ging the tail, urethral pulsations, etc.) that occur sperm in ejaculates should begin with determining during a collection process may convince the clini- whether or not ejaculation is occurring. Lack of cian that ejaculation occurred. However, these secondary signs of ejaculation (i.e., flagging of the signs can occur without seminal emission (i.e., se- tail, treading on hindlimbs, and presence of strong men does not move into the urethra before ejacula- urethral pulsations) followed by dismount with the tion). Confirming that ejaculation did occur by glans penis still partially engorged and an absence showing the presence of gel (i.e., gel is produced in of sperm in seminal fluids suggest that the stallion the seminal vesicles and appears near the end of the NOTES AAEP PROCEEDINGS ր Vol. 55 ր 2009 331 NON-PREGNANT MARE AND STALLION Lack of Sperm in Ejaculates Appears not to Ejaculate Appears to Ejaculate Increase Sexual Simulation High Alkaline Phosphatase Low Alkaline Phosphatase Tease to several mares Provide fence-line access to mares Adjust phantom/smaller mount mare height Ensure solid footing Evaluate testes, epididymides, spermatic cords Evaluate internal and external genitalia for evidence of semen outflow obstruction Ground ejaculation Size, texture, adhesions Ultrasonography Palpation Evaluate Artificial Vagina Ultrasonography Biopsy -model, pressure, temperature, position Degeneration Rule-out Retrograde Ejaculation Hypoplasia Examine Urine Hot towel compress Cessation of spermatogenesis Midstream free-catch Bladder catherization Evaluate penile sensation/filling defects Hormonal Characteristics Baseline LH, FHS, E2, T Pharmacotherapy GnRH response hCG response GnRH Imipramine Short-term testosterone Xylazine or detomidine Oxytocin or prostaglandin Fig. 1. Schematic representation of an approach to diagnosis of cause of lack of sperm in stallion ejaculates. ejaculatory process) and a high alkaline phospha- degeneration or hypoplasia also occur in testes of tase concentration in seminal fluids devoid of sperm normal size.9,10 indicates failure of sperm production in testes. Alkaline phosphatase levels of 6913–22,180 units/l 4. Relationship to Hormone Concentrations are found in ejaculates of clinically normal stal- If an adequate population of Leydig cells exists in lions.4 The same findings with low levels of alka- the interstitium, resting and stimulated (i.e., after line phosphatase (i.e., below serum value and administration of gonadotropin releasing hormone similar to pre-ejaculatory fluid) in seminal fluids (GnRH) or human chorionic gonadotropin (hCG)) suggest that obstruction to sperm outflow from the concentrations of testosterone may be within normal testes and epididymides is present.5 Retrograde limits. However, in advanced cases of testicular ejaculation into the urinary bladder should also be degeneration, resting concentrations of testosterone ruled out for stallions that exhibit behavioral signs and estrogen in the circulation will be lower than of ejaculation but yield ejaculates that are of low normal (sometimes similar to those of geldings). volume and devoid of sperm.6 Collecting and mi- croscopically examining centrifuged urine sediment immediately after ejaculation will confirm whether or not retrograde ejaculation occurred. 3. Relationship to Testicular Size and Texture If signs of ejaculation and high alkaline phosphatase levels are present in seminal fluids, examination of testes size, shape, and texture by palpation and ultrasonography is indicated.6,7 If testes are quite small and soft, advanced testicular degeneration or hypoplasia should be suspected.8,9 Young stallions in which testes size has never been large are most likely to be hypoplastic, whereas older stallions that previously had normal testes size are most likely to Fig. 2. Bard monopty biopsy punch used in procuring testicular have advanced degeneration. However, testicular biopsy. 332 2009 ր Vol. 55 ր AAEP PROCEEDINGS NON-PREGNANT MARE AND STALLION Fig. 3. Procuring testicular biopsy. Fig. 5. Histologic appearance of a Stage VIII seminiferous tu- bule of a stallion. Elongated spermatids line the lumen in readi- ness for release into the tubule. In response to the testicular defect culminating in low production of testicular steroids, circulating con- centrations of gonadotropins (particularly follicle stimulating hormone (FSH)) can be higher than nor- mal.11,12 In cases of advanced testicular degenera- tion, administration of GnRH or hCG may fail to elicit a normal increase in circulating testosterone concentration as well. 5. Testicular Biopsy Testicular biopsy may be indicated to assess status of spermatogenesis.13 To perform testicular bi- opsy, the stallion is sedated (e.g., detomodine hydro- chloride and butorphanol tartrate). After aseptic preparation of the scrotal skin and donning of sterile gloves, the testis is grasped and stabilized ventrally in the scrotum. If desired, an anesthetic (e.g., lido- caine or carbocaine) bleb may be injected subcuta- neously at the intended biopsy site, but the authors do not usually employ local anesthesia. A sterile, spring-loaded biopsy instrumenta (Fig. 2) is pushed laterally to medially through the scrotal skin, tunica dartos, testicular tunics, and tunica albuginea into the cranial mid-testis (Fig. 3). This instrument is 14 gauge ϫ 16 cm in length with a 22-mm penetra- tion depth and a 1.7-cm sample notch. The biopsy Fig. 6. Ultrasonographic appearance of testicular tumor in a Fig. 4. Needle punch end of biopsy instrument with the testic- stallion. Most of the parenchyma has been obliterated by the ular tissue visible in biopsy window. tumor. AAEP PROCEEDINGS ր Vol. 55 ր 2009 333 NON-PREGNANT MARE AND STALLION Fig. 9. Firm, thickened epididymis with some fibrinous adhe- sions present that prevented manual displacement during palpa- tion (i.e., the epididymis could not be pushed dorsally away from Fig. 7. Ultrasonographic appearance of testis and epididymis the surface of the testis). Both epididymes were involved, and surrounded by fibrous tissue. Extensive fibrous tissue deposi- complete obstruction to sperm outflow resulted in azoospermia. tion not only insulates the testis, which causes degeneration, but it also may constrict ductile lumina, which prevents sperm move- ment into the vas deferens. whether or not spermatogenesis is proceeding to completion. Although insufficient tissue is present punch is triggered, and the instrument is removed to determine accurate percentages of stages, the from the testis and scrotum. Digital pressure is presence of several Stage VIII tubules (Fig. 5) re- maintained for 1–2 min on the tunica albuginea and veals that sperm are being released into the semi- scrotal skin over the biopsy site to control any hem- niferous tuble lumina.13,14 If straight tubules orrhage. The testicular parenchyma is gently re- (tubules connecting seminiferous tubules with the moved from the exposed notch of the biopsy rete testis) are present in the biopsy, the presence or instrument (Fig. 4), and it is transferred to Bouin’s absence of released sperm within their lumina can solution or 4% paraformaldehyde for 24 h. The be assessed. The diagnosis of testicular degenera- fixed tissue is then transferred to alcohol and sub- tion or hypoplasia can be made by the presence of mitted to a histology laboratory for processing and high numbers of degenerating germ cells, the ab- mounting. Preferred stains are PAS-Hematoxylin sence
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