How to Investigate Azoospermia in Stallions

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1 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 ; gmail.com AAEP. 1. Introduction In a review of ejaculatory dysfunction, McDonnell 1 reported that 25% of stallions referred to a fertility clinic had evidence of ejaculatory problems. The vast majority of cases were anejaculatory (failure to ejaculate). Less than 1% of horses in that survey were truly azoospermic (i.e., ejaculated seminal fluids devoid of sperm). Failure to ejaculate sperm can be a troublesome problem that requires accurate diagnosis, determination of prognosis for correction (sometimes necessitating retirement as a breeding stallion), and arduous treatment and/or breeding management to correct. 2,3 Figure 1 represents an attempt at a schematic overview of an approach to diagnosis of lack of sperm in ejaculates. 2. Confirming Ejaculation Clinical evaluation of stallions failing to produce sperm in ejaculates should begin with determining whether or not ejaculation is occurring. Lack of secondary signs of ejaculation (i.e., flagging of the tail, treading on hindlimbs, and presence of strong urethral pulsations) followed by dismount with the glans penis still partially engorged and an absence of sperm in seminal fluids suggest that the stallion did not ejaculate. 3 A number of reports describe therapy indicated for ejaculation failure, but they are not the subject of this report. Briefly, they include breeding and/or pharmacological management to increase sexual stimulation before and during the breeding process, treatment and/or breeding management to minimize potential musculoskeletal pain that could interrupt the emission and ejaculatory process, and pharmacologic manipulation to lower the threshold to emission and ejaculation. 1 3 Techniques used to manage repeated ejaculatory failure can be arduous and time consuming, and they are reviewed by Varner et al. 3 When breeding behavior and apparent ejaculation seems to be normal but no sperm are present in the seminal fluids collected, azoospermia should be suspected. Secondary signs of ejaculation (e.g., flagging the tail, urethral pulsations, etc.) that occur during a collection process may convince the clinician that ejaculation occurred. However, these signs can occur without seminal emission (i.e., semen does not move into the urethra before ejaculation). Confirming that ejaculation did occur by showing the presence of gel (i.e., gel is produced in the seminal vesicles and appears near the end of the NOTES AAEP PROCEEDINGS Vol

2 Lack of Sperm in Ejaculates Appears not to Ejaculate Appears to Ejaculate Increase Sexual Simulation High Alkaline Phosphatase Tease to several mares Provide fence-line access to mares Adjust phantom/smaller mount mare height Ensure solid footing Ground ejaculation Low Alkaline Phosphatase Evaluate testes, epididymides, spermatic cords Size, texture, adhesions Ultrasonography Evaluate Artificial Vagina -model, pressure, temperature, position Palpation Ultrasonography Biopsy Degeneration Hypoplasia Cessation of spermatogenesis Hot towel compress Evaluate penile sensation/filling defects Evaluate internal and external genitalia for evidence of semen outflow obstruction Rule-out Retrograde Ejaculation Examine Urine Midstream free-catch Bladder catherization Hormonal Characteristics Baseline LH, FHS, E2, T GnRH response hcg response Pharmacotherapy 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 phosphatase concentration in seminal fluids devoid of sperm indicates failure of sperm production in testes. Alkaline phosphatase levels of ,180 units/l are found in ejaculates of clinically normal stallions.4 The same findings with low levels of alkaline phosphatase (i.e., below serum value and similar to pre-ejaculatory fluid) in seminal fluids suggest that obstruction to sperm outflow from the testes and epididymides is present.5 Retrograde ejaculation into the urinary bladder should also be ruled out for stallions that exhibit behavioral signs of ejaculation but yield ejaculates that are of low volume and devoid of sperm.6 Collecting and microscopically examining centrifuged urine sediment immediately after ejaculation will confirm whether or not retrograde ejaculation occurred Relationship to Hormone Concentrations If an adequate population of Leydig cells exists in the interstitium, resting and stimulated (i.e., after administration of gonadotropin releasing hormone (GnRH) or human chorionic gonadotropin (hcg)) concentrations of testosterone may be within normal limits. However, in advanced cases of testicular degeneration, resting concentrations of testosterone and estrogen in the circulation will be lower than normal (sometimes similar to those of geldings). 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 have advanced degeneration. However, testicular 332 degeneration or hypoplasia also occur in testes of normal size.9, Ⲑ Vol. 55 Ⲑ AAEP PROCEEDINGS Fig. 2. Bard monopty biopsy punch used in procuring testicular biopsy.

3 Fig. 3. Procuring testicular biopsy. In response to the testicular defect culminating in low production of testicular steroids, circulating concentrations of gonadotropins (particularly follicle stimulating hormone (FSH)) can be higher than normal.11,12 In cases of advanced testicular degeneration, administration of GnRH or hcg may fail to elicit a normal increase in circulating testosterone concentration as well. 5. Fig. 5. Histologic appearance of a Stage VIII seminiferous tubule of a stallion. Elongated spermatids line the lumen in readiness for release into the tubule. Testicular Biopsy Testicular biopsy may be indicated to assess status of spermatogenesis.13 To perform testicular biopsy, the stallion is sedated (e.g., detomodine hydrochloride 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., lidocaine or carbocaine) bleb may be injected subcutaneously 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 16 cm in length with a 22-mm penetration depth and a 1.7-cm sample notch. The biopsy Fig. 4. Needle punch end of biopsy instrument with the testicular tissue visible in biopsy window. Fig. 6. Ultrasonographic appearance of testicular tumor in a stallion. Most of the parenchyma has been obliterated by the tumor. AAEP PROCEEDINGS Ⲑ Vol. 55 Ⲑ

4 Fig. 7. Ultrasonographic appearance of testis and epididymis surrounded by fibrous tissue. Extensive fibrous tissue deposition not only insulates the testis, which causes degeneration, but it also may constrict ductile lumina, which prevents sperm movement into the vas deferens. Fig. 9. Firm, thickened epididymis with some fibrinous adhesions present that prevented manual displacement during palpation (i.e., the epididymis could not be pushed dorsally away from the surface of the testis). Both epididymes were involved, and complete obstruction to sperm outflow resulted in azoospermia. punch is triggered, and the instrument is removed from the testis and scrotum. Digital pressure is maintained for 1 2 min on the tunica albuginea and scrotal skin over the biopsy site to control any hemorrhage. The testicular parenchyma is gently removed from the exposed notch of the biopsy instrument (Fig. 4), and it is transferred to Bouin s solution or 4% paraformaldehyde for 24 h. The fixed tissue is then transferred to alcohol and submitted to a histology laboratory for processing and mounting. Preferred stains are PAS-Hematoxylin or PAS-Toluidine Blue. To the trained observer, examination of testicular parenchyma under light microscopy will reveal individual cell types and whether or not spermatogenesis is proceeding to completion. Although insufficient tissue is present to determine accurate percentages of stages, the presence of several Stage VIII tubules (Fig. 5) reveals that sperm are being released into the seminiferous tuble lumina.13,14 If straight tubules (tubules connecting seminiferous tubules with the rete testis) are present in the biopsy, the presence or absence of released sperm within their lumina can be assessed. The diagnosis of testicular degeneration or hypoplasia can be made by the presence of high numbers of degenerating germ cells, the absence of more advanced germ cells, and the presence of numerous basilar vacuoles within the seminiferous epithelium. Reduced size and number of Leydig cells in interstitial tissue and Sertoli cell only Fig. 8. Thick, encompassing adhesions surrounding testis of a stallion. Some epididymal tissue is evident. The testis lost its normal egg shape, did not move freely within the scrotum, and was quite firm and irregular on palpation. Fig. 10. Ultrasonographic appearance (longitudinal image) of a dilated ampulla caused by obstruction (sperm stasis) of the ampullary lumen Ⲑ Vol. 55 Ⲑ AAEP PROCEEDINGS

5 pelvic vas deferens or ampullae, b16 and prostatic adenocarcinoma resulting in blockage of excurrent duct openings into the pelvic urethra have also been identified. Fig. 11. Ultrasonographic appearance (cross-sectional image) of a dilated ampulla caused by obstruction (sperm stasis) of the ampullary lumen. seminiferous tubules are hallmarks of advanced testicular degeneration Examination for Evidence of Obstruction to Sperm Outflow Physical examination of scrotal contents and internal genitalia are required to diagnose the location of an obstruction to sperm outflow. 3,5 Thorough palpation and ultrasonographic examination can reveal a number of abnormalities that may contribute to the obstruction of sperm outflow. The presence of space-occupying lesions within testicular or epididymal tissue (such as tumors or extensive fibrosis, sometimes with calcification) has been observed. 6,7,13 Firm, enlarged epididymides, sometimes with dilated ducts as with chronic obstructive epididymitis, can be adhered to the testicular tunics. 13,15 Extensive adhesions between vaginal and parietal tunics can result from hematocele, orchitis, periorchitis, or testicular rupture (Figs. 6 9). 7,13,15 Ultrasonographic examination may reveal dilated ampullae, vas deferens, or ducts of the cauda epididymides with sperm stasis or ampullar blockage (Figs. 10 and 11). 7 Although rare, stallions with congenital aplasia of the epididymides, 10 References and Footnotes 1. McDonnell SM. Ejaculation: physiology and dysfunction. Vet Clin North Am [Equine Pract] 1992;8: McDonnell SM. Normal and abnormal sexual behavior. Vet Clin North Am [Equine Pract] 1992;8: Varner DD, Schumacher J, Blanchard TL, et al. Diseases and management of breeding stallions. Goleta, CA: American Veterinary Publications, Turner RM. Alkaline phosphatase activity in stallion semen: characterization and clinical implications, in Proceedings. Annual Meeting of the Society for Theriogenology 1996; Love CC, Riera FL, Oristaglio Turner RM. Sperm occluded (plugged) ampullae in the stallion, in Proceedings. Annual Meeting of the Society for Theriogenology 1992; Brinsko SP. Retrograde ejaculation in a stallion. JAmVet Med Assoc 2001;218: Love CC. Ultrasonographic evaluation of the testis, epididymis, and spermatic cord of the stallion. Vet Clin North Am [Equine Pract] 1992;8: Ladds PW. The male genital system. In: Jubb KVF, Kennedy PC, eds. Pathology of domestic animals, 3rd ed. Orlando, FL: Academic Press Inc., 1985; McEntee K. The male genital system. In: Jubb KVF, Kennedy PC, eds. Pathology of domestic animals, 2nd ed. New York: Academic Press Inc., 1970; Blanchard TL, Woods JA, Brinsko SP. Theriogenology question of the month: Epididymal hypoplasia in a stallion. J Am Vet Med Assoc 2000;217: Roser JF. Endocrine profiles in fertile, subfertile, and infertile stallions: testicular response to human chroionic gonadotropin in infertile stallions. Bio Reprod 1995;1: Douglas RH, Umphenour N. Endocrine abnormalities and hormonal therapy. Vet Clin North Am [Equine Pract] 1992; 8: Blanchard TL, Varner DD, Bretzlaff KN, et al. Testicular degeneration in large animals: identification and treatment. Vet Med 1991;86: Swierstra EE, Pickett BW, Gebauer MR. Spermatogenesis and duration of transit of spermatozoa through the excurrent ducts of stallions. J Reprod Fertil 1975;23(Suppl): Blanchard TL, Varner DD, Schumacher J, et al. Manual of equine reproduction, 2nd ed. St Louis, MO: Mosby, Estrada A, Samper JC, Lillich JD, et al. Azoospermia associated with bilateral segmental aplasia of the ductus deferens in a stallions. J Am Vet Med Assoc 2003;222: a Bard Monopty Biopsy Instrument, C. R. Bard, Inc., Covington, GA b Varner DD, et al. Unpublished observations, c Varner DD, et al. Unpublished observations, AAEP PROCEEDINGS Vol