How to Investigate Azoospermia in Stallions

Home , Azoospermia, Retrograde ejaculation

NON-PREGNANT MARE AND STALLION

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

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 tubule 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 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. Testicular Biopsy Testicular biopsy may be indicated to assess status of spermatogenesis.13 To perform testicular biopsy, 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 adhesions present that prevented manual displacement during palpation (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 of more advanced germ cells, and the presence or PAS-Toluidine Blue. To the trained observer, of numerous basilar vacuoles within the seminifer- examination of testicular parenchyma under light ous epithelium. Reduced size and number of Ley- microscopy will reveal individual cell types and dig 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 Fig. 10. Ultrasonographic appearance (longitudinal image) of a normal egg shape, did not move freely within the scrotum, and dilated ampulla caused by obstruction (sperm stasis) of the am- was quite ﬁrm and irregular on palpation. pullary lumen.

334 2009 ր Vol. 55 ր AAEP PROCEEDINGS NON-PREGNANT MARE AND STALLION pelvic vas deferens or ampullae,b16 and prostatic adenocarcinoma resulting in blockage of excurrent duct openings into the pelvic urethra have also been identiﬁed.

References and Footnotes 1. McDonnell SM. Ejaculation: physiology and dysfunction. Vet Clin North Am [Equine Pract] 1992;8:57–70. 2. McDonnell SM. Normal and abnormal sexual behavior. Vet Clin North Am [Equine Pract] 1992;8:71–89. 3. Varner DD, Schumacher J, Blanchard TL, et al. Diseases and management of breeding stallions. Goleta, CA: Ameri- can Veterinary Publications, 1991. 4. Turner RM. Alkaline phosphatase activity in stallion semen: characterization and clinical implications, in Proceed- ings. Annual Meeting of the Society for Theriogenology 1996;284–293. 5. Love CC, Riera FL, Oristaglio Turner RM. Sperm occluded (plugged) ampullae in the stallion, in Proceedings. Annual Meeting of the Society for Theriogenology 1992;117–125. Fig. 11. Ultrasonographic appearance (cross-sectional image) of 6. Brinsko SP. Retrograde ejaculation in a stallion. JAmVet a dilated ampulla caused by obstruction (sperm stasis) of the Med Assoc 2001;218:551–553. ampullary lumen. 7. Love CC. Ultrasonographic evaluation of the testis, epididymis, and spermatic cord of the stallion. Vet Clin North Am [Equine Pract] 1992;8:167–182. 8. Ladds PW. The male genital system. In: Jubb KVF, seminiferous tubules are hallmarks of advanced tes- Kennedy PC, eds. Pathology of domestic animals, 3rd ticular degeneration.8 ed. Orlando, FL: Academic Press Inc., 1985;420–424. 9. McEntee K. The male genital system. In: Jubb KVF, 6. Examination for Evidence of Obstruction to Sperm Kennedy PC, eds. Pathology of domestic animals, 2nd ed. Outflow New York: Academic Press Inc., 1970;447–449. 10. Blanchard TL, Woods JA, Brinsko SP. Theriogenology ques- Physical examination of scrotal contents and inter- tion of the month: Epididymal hypoplasia in a stallion. J nal genitalia are required to diagnose the location of Am Vet Med Assoc 2000;217:825–826. an obstruction to sperm outflow.3,5 Thorough pal- 11. Roser JF. Endocrine profiles in fertile, subfertile, and infertile stallions: testicular response to human chroionic gonad- pation and ultrasonographic examination can reveal otropin in infertile stallions. Bio Reprod 1995;1:661–669. a number of abnormalities that may contribute to 12. Douglas RH, Umphenour N. Endocrine abnormalities and the obstruction of sperm outflow. The presence of hormonal therapy. Vet Clin North Am [Equine Pract] 1992; space-occupying lesions within testicular or epidid- 8:237–250. 13. Blanchard TL, Varner DD, Bretzlaff KN, et al. Testicular ymal tissue (such as tumors or extensive fibrosis, degeneration in large animals: identification and treat- sometimes with calcification) has been ob- ment. Vet Med 1991;86:537–542. served.6,7,13 Firm, enlarged epididymides, some- 14. Swierstra EE, Pickett BW, Gebauer MR. Spermatogenesis times with dilated ducts as with chronic obstructive and duration of transit of spermatozoa through the excurrent epididymitis, can be adhered to the testicular tu- ducts of stallions. J Reprod Fertil 1975;23(Suppl):53–57. 13,15 15. Blanchard TL, Varner DD, Schumacher J, et al. Manual of nics. Extensive adhesions between vaginal equine reproduction, 2nd ed. St Louis, MO: Mosby, 2003. and parietal tunics can result from hematocele, or- 16. Estrada A, Samper JC, Lillich JD, et al. Azoospermia asso- chitis, periorchitis, or testicular rupture (Figs. ciated with bilateral segmental aplasia of the ductus deferens 6–9).7,13,15 Ultrasonographic examination may re- in a stallions. J Am Vet Med Assoc 2003;222:1740–1742. veal dilated ampullae, vas deferens, or ducts of the a cauda epididymides with sperm stasis or ampullar Bard Monopty Biopsy Instrument, C. R. Bard, Inc., Covington, 7 GA 30015. blockage (Figs. 10 and 11). Although rare, stal- bVarner DD, et al. Unpublished observations, 2002. lions with congenital aplasia of the epididymides,10 cVarner DD, et al. Unpublished observations, 2000.

AAEP PROCEEDINGS ր Vol. 55 ր 2009 335