PV0212 Blanchard

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1/8Copyright 2012 Vetstreet Inc. This document is for internal purposes only. Reprinting or posting on an external website without written permission from Vetlearn is a violation of copyright lawsVetlearn.com |February 2012 | Compendium: Continuing Education for Veterinarians

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Terry L. Blanchard, DVM, MS, DACTDickson D. Varner, DVM, MS, DACT

Steven P. Brinsko, DVM, MS, PhD, DACTCharles C. Love, DVM, PhD, DACTTexas A&M University

Abstract:Determining the cause of failure to ejaculate sperm can be a diagnostic dilemma. The first diagnostic step is to ascertain whether

the stallion is ejaculating. If the stallion appears to ejaculate, but there is azoospermia (absence of sperm in the seminal fluid), testing

alkaline phosphatase (ALP) activity in seminal plasma can determine whether testicular and epididymal fluids are present. If ALP activity

is low, the possibility of either blockage to sperm outflow in the excurrent duct system or retrograde ejaculation should be pursued

diagnostically. If ALP activity is high, the possibility of a testicular defect should be pursued diagnostically. In some cases (notably plugged

ampullae or transient, thermally induced testicular degeneration), treatment or the passage of time may restore a stallions fertility.

Azoospermia in Stallions:Determining the Cause

In a review o ejaculatory dysunction, McDonnell1reported that

approximately one-ourth o stallions reerred to a ertility clinic

had evidence o ejaculatory problems. Most o these cases were

associated with anejaculation (ejaculatory ailure). Less than 1%

o the horses were truly azoospermic. Azoospermia can be difficult

to accurately diagnose and to correct.2,3Once anejaculation is ruled out, diagnostic efforts can be directed

at determining the cause o azoospermia (the absence o sperm

in seminal fluid). Some disorders, notably ampullary obstruction

o sperm outflow (plugged ampullae), can be corrected, restoring

a stallions ertility.4 Likewise, some disorders (e.g., transient,

thermally induced testicular degeneration) that result in ailure

o spermatogenesis (sperm production in the testes) sel-correct

with time, resulting in restoration o sperm output and thereore

ertility.5Some disorders resulting in obstruction o sperm outflow

(e.g., chronic epididymitis with blockage o tubules) or ailure o

spermatogenesis (e.g., age-related testicular degeneration) are

not correctable, causing permanent inertility and necessitating

retirement o the affected breeding stallion.

Tis article discusses diagnostic modalities or some o the

causes o azoospermia in stallions (FIGURE 1). Case summaries

urther illustrate diagnostic approaches.

Confirming EjaculationClinical evaluation o stallions that seem to be inertile should

begin with determining whether ejaculation is occurring. Lack

o secondary signs o ejaculation (i.e., flagging the tail, treading

on hind eet, and strong urethral pulsations, usually ollowed by

dismount with the glans penis still ully or partially engorged), in

conjunction with azoospermia, suggests that the stallion did not

ejaculate.3Several articles13describe therapies or anejaculation,

which are beyond the scope o this article. Tese therapies include

the ollowing (FIGURE 1):

Breeding and/or pharmacologic management to increase

sexual stimulation beore and during breeding

reatment and/or breeding management to minimize poten-

tial musculoskeletal pain that could interrupt emission and

ejaculation

Pharmacologic manipulation to lower the threshold to

emission and ejaculation

echniques or managing repeated anejaculation can be arduous

and time-consuming.3

When breeding behavior and ejaculation appear to be normal,

but sperm are not present in seminal fluid, azoospermia should

be suspected. Secondary signs o ejaculation during collection

may convince a clinician that ejaculation occurred. However,

these signs can occasionally occur without seminal emission

(i.e., semen does not move into the urethra beore ejaculation).

Confirming that ejaculation did occur by demonstrating the

presence o gel (which is produced in the seminal vesicles and

appears near the end o the ejaculatory process) and a high alkaline

phosphatase (ALP) concentration in seminal fluid devoid o sperm

indicates ailure o spermatogenesis in testes. Similar findings

with a low ALP level (i.e., values below serum concentration and

similar to the pre-ejaculatory fluid concentration) in seminal


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Azoospermia in Stallions: Determining the Caus

fluid suggests obstruction o sperm outflow rom the testes and

epididymides.4ALP levels o 6913 to 22,180 U/L are ound in

ejaculates o clinically normal stallions.6In our laboratory, values


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According to unpublished observations, administration o gonad-

otropin-releasing hormone may ail to elicit a normal increase in

circulating testosterone concentration in some cases o advanced

age-related testicular degeneration.

Testicular Biopsyesticular biopsy may be indicated to assess status o spermato-

genesis.15 o perorm testicular biopsy, the stallion is sedated

(e.g., 8 to 10 g/kg IV o detomidine hydrochloride and 0.01 to

0.02 mg/kg IV o butorphanol tartrate) and the scrotal skin is

scrubbed and disinected. Sterile gloves are donned, and the tes-

tis is grasped and stabilized ventrally in the scrotum. I desired,

local anesthetic (e.g., lidocaine, mepivacaine) can be injected

subcutaneously at the intended biopsy site, but we do not usually

use local anesthesia. A sterile, spring-loaded biopsy instrument

is pushed laterally to medially through the scrotal skin, tunica

dartos, testicular tunics, and tunica albuginea into the cranial

mid-testis. Tis 14-gauge instrument is 16 cm in length, with a

22-mm penetration depth and a 1.7-cm sample notch. Te biopsy

punch is triggered to procure the specimen, then the instrument

is removed rom the testis and scrotum. Digital pressure is main-

tained or 1 to 2 minutes on the tunica albuginea and scrotal skin

over the biopsy site to control hemorrhage. Te testicular paren-

chyma is gently removed rom the exposed notch o the biopsy

instrument and is transerred to Bouin solution or 4% paraor-

maldehyde or 24 hours. Te fixed tissue is then transerred to

alcohol and submitted to a histology laboratory or processing

and mounting. Preerred stains are periodic acid-Schiff (PAS)

hematoxylin or PAStoluidine blue. o the trained observer, exami-

nation o testicular parenchyma under light microscopy reveals

individual cell types and whether spermatogenesis is proceeding

to completion. Although the amount o tissue is insufficient to

determine accurate percentages o tubules within each o eight

stages, the presence o several stage VIII tubules (FIGURE 2)reveals

that spermatogenesis is proceeding to completion and sperm are

being released into the seminierous tubule lumina.16I straight

tubules (which connect seminierous tubules with the rete testis)are present in the biopsy specimen, the presence o released

sperm within the lumina can be assessed. A diagnosis o testicular

degeneration or hypoplasia can be made i high numbers o de-

generating germ cells (sometimes sloughed into the tubule lumina)

are present, more advanced germ cells are absent, and numerous

basilar vacuoles are present within the seminierous epithelium.

Reduced size and number o Leydig cells in interstitial tissue and

Sertoli cell only seminierous tubules are hallmarks o advanced

testicular degeneration.5,10

Examination for Evidence of Obstruction of Sperm OutflowPhysical examination o scrotal contents and internal genitalia is

required to determine the location o an obstruction o sperm

outflow.3,4 Torough palpation and ultrasonographic examina-

tion can reveal a number o abnormalities that may contribute to

obstruction o sperm outflow. Space-occupying lesions (e.g., tumors

or extensive fibrosis, sometimes with calcification) have been

observed within testicular or epididymal tissue.5,8Firm, enlarged

epididymides, sometimes with dilated ducts due to chronic ob-

structive epididymitis, can adhere to the testicular tunics.5,17Exten-

sive adhesions between vaginal and parietal tunics can result

rom hematocele, orchitis, periorchitis, or testicular rupture5,8,17

Table 1. Resting Hormone Concentrations

Resting Hormone Concentrations (Obtained From Pooled Samples Taken at HourlyIntervals During a 4-Hour Period) in a 24-Year-Old Thoroughbred Stallion With SevereOligospermia.While this stallion was not truly azoospermic, very few sperm werepresent in ejaculates (despite a high seminal plasma alkaline phosphatase level),

and the stallion failed to produce any pregnancies between February and April.Testicular size had decreased from 220 cc in January 2007 to 91 cc in March 2009.

The following values were obtained in April.

Hormone Concentration

Laboratory-ReportedNormal Ranges

EndocrineLaboratory

TAMUResearch

Testosterone 334 pg/mL 8002000 pg/mL 1020 11 pg/mL

Estradiol-17 28.0 pg/mL 48 4 pg/mL

LH 13.12 ng/mL 110 ng/mL 6.77 1.69 ng/mL

FSH 23.40 ng/mL 212 ng/mL 4.91 1.69 ng/mL

Inhibin 1.77 ng/mL 2.23.4 ng/mL 3.48 0.55 ng/mL

Interpretation: The hormone concentrations are consistent with a substantial degree

of testicular degeneration. Leydig and Sertoli cells are experiencing some dysfunction

as indicated by lower-than-normal levels of testosterone, estradiol, and inhibin. The

pituitary is compensating for testicular dysfunction by increasing secretion of

gonadotropins (luteinizing hormone [LH] and follicle-stimulating hormone [FSH]).

Figure 2.Histologic appearance of a stage VIII seminiferous tubule of a stallion.

Elongated spermatids line the lumen in preparation for release into the tubule.

(magnification: 450)


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(FIGURE 3; FIGURE 4; FIGURE 5; FIGURE 6). Ultrasonographic examina-

tion may reveal dilated ampullae, vas deerens, or ducts o the cauda

epididymides with sperm stasis or ampullar blockage4,8(FIGURE 7).

Although rare, congenital aplasia/hypoplasia o the epididymides,11

pelvic ductus deerens or ampullae,18,19or prostatic neoplasia as an

extension o an ampullary adenocarcinoma20can block the excur-

rent duct system, preventing sperm rom entering the ejaculate.

Te ollowing case summaries illustrate procedures and find-

ings or determining the cause o azoospermia in stallions.

Case 1A 33-month-old, 450-kg (990-lb) Paint stallion was examined

because o azoospermia. Historical review indicated that the stallion

had bred two mares by natural mating the previous summer, but

neither mare became pregnant. wo weeks beore admission to

our clinic, semen had been collected and examined by a reerring

veterinarian. Sperm were not detected in the ejaculate.

Afer arrival at our clinic, two ejaculates were collected rom

the stallion, using a Missouri-model artificial vagina and an

ovariectomized mount mare. Te stallion displayed normal libido

and breeding behavior and appeared to ejaculate on both collec-

tions. Indirect evidence or ejaculation included engorgement o the

glans penis, urethral pulsations (our or five pronounced pulsations

o the urethra palpable on the ventral aspect o the base o the

penis), flagging o the tail, and the presence o gel (60 cc in the

Figure 5. Thick, encompassing adhesions surrounding a stallions testis. Someepididymal tissue is evident. The testis had lost its normal egg shape, was not

freely moveable within the scrotum, and was firm and irregular on palpation.

Figure 6. A firm, thickened epididymis. Some fibrinous adhesions prevented thebody of the epididymis from being displaced dorsally during palpation. Both

epididymides were involved, and complete obstruction of sperm outflow resulted

in azoospermia.Figure 3. Ultrasonogram of a testiculartumor in a stallion. Most of the

parenchyma has been obliterated by

the tumor.

Figure 4. Ultrasonogram of a testis andan epididymis surrounded by fibrous

tissue. Extensive fibrous tissue

deposition not only insulates the testis,

causing degeneration, but also may

constrict ductile lumina, preventing

movement of sperm into the ductus

deferens.

Figure 7. Cross-sectional ultrasonogram of a dilated ampulla due to obstruction(sperm stasis) of the ampullary lumen.


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first ejaculate; 23 cc in the second ejaculate). Afer removal o the

gel rom each ejaculate,


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embedding, sectioning, and staining with PAS-hematoxylin. Exami-

nation o the biopsy specimens revealed only a ew seminierous

tubules containing round or elongated spermatids (stages V to

VII, with elongated spermatid heads embedded deep within the

Sertoli cells). No stage VIII tubules were noted in either biopsy

specimen. Numerous degenerating spermatocytes were evident

in many tubules, some with pronounced epithelial vacuolation

(FIGURE 9). A diagnosis o severe testicular degeneration was

made. Because o the longstanding history o the condition, theowners elected to castrate the stallion.

Case 4A 6-year-old Toroughbred stallion had no sperm in dismount

samples collected afer mating and examined as wet mounts under

a microscope. Several semen collection attempts were made using

a Missouri-model artificial vagina and an ovariectomized mount

mare. All ejaculates were devoid o sperm despite the presence o

strong urethral pulsations, flagging o the tail, and gel in the ejac-

ulates. Seminal plasma rom several ejaculates was assayed or

ALP activity, yielding values


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Case 5A 4-year-old Quarter horse stallion presented with a history o

ailing to establish pregnancy in six mares bred by natural service

at pasture. Several semen collection attempts were made using a

Missouri-model artificial vagina and an ovariectomized mount

mare. Although the stallion appeared to ejaculate, the ejaculatory

fluids were clear and devoid o sperm. Te urinary bladder was

catheterized afer semen collection was attempted, and the urine

was centriuged. Examination o urine sediment ailed to reveal

the presence o sperm. Palpation and ultrasonography o the scrotal

contents revealed normal-sized testicles with enlarged cauda

epididymides. Te ducts o the cauda epididymides were greatly

distended (FIGURE 11). ransrectal palpation and ultrasonographicexamination revealed flattened, hypoplastic or aplastic ductus

deerens at the junction o the ampullae, and the ductus deerens

proximal to this junction were grossly distended. Numerous attempts

to collect semen, which were preceded by administration o either

oxytocin (20 U IV) or cloprostenol (125 g IM) and vigorous

massage o the ampullae and pelvic ductus deerens, ailed to

produce sperm in ejaculates. Exploratory laparoscopic surgery

confirmed aplastic segments o the terminal ductus deerens where

it coursed over the undus o the bladder (FIGURE 12), whereas the

ductus deerens was distended ventrally to the internal inguinal

ring. With the stallion under general anesthesia, needle aspiration

o the distended ductus deerens yielded thick, creamy concen-

trated semen with sperm clumping, and virtually all sperm heads

were detached. Attempts to pass cannulas through the terminal

ductus deerens into the ampullary lumina were unsuccessul.

Te diagnosis was bilateral segmental aplasia o the ductus deerens,

and the stallion was removed rom stud service.

Case 6A 9-year-old Quarter horse stallion presented to our clinic with a

history o neutrophils in ejaculates. Te stallion had previously

been bred or 2 years to approximately 12 mares per year by natural

service, resulting in 90% or better seasonal pregnancy rates. Tree

months previously, the stallion had been moved to another arm,

and the arm manager had noted fluctuation in the stallions scrotal

size. Te ollowing month, the stallion developed a ever o 105F

with prominent swelling o the testes. Culture o semen collected

in an artificial vagina yielded Klebsiella pneumoniae, and the stallion

was treated by the reerring veterinarian, who administered systemic

antimicrobials, which were not identified in the history. While

the scrotal swelling decreased, neutrophils remained in the ejaculate.

Te stallion was then bred to five mares, producing one pregnancythat was subsequently lost.

On arrival at our clinic, the stallion was teased to erection and

the penis washed and dried. wo ejaculates were collected in an

artificial vagina. Neither ejaculate contained sperm, but both

contained numerous degenerating neutrophils. For bacteriologic

culture, the urethra was swabbed beore, and immediately afer,

ejaculation and the filtered ejaculatory fluid was swabbed. Heavy

growth o both Pseudomonas aeruginosaand Klebsiella pneumoniae

was recovered rom the ejaculatory fluid and postejaculatory

urethral swabbings. Palpation and ultrasonographic examination

o the scrotal contents revealed firm, small testes with large, firm

epididymides. Te epididymides could not be moved rom the

surace o the testes, suggesting adhesion had occurred. Te stallion

was sedated, and endoscopic examination o the pelvic urethra,

seminal colliculus, and seminal vesicles was perormed. Prostatic

and urethral gland secretions expressed by per-rectumpalpation

during the examination were clear. Te endoscope was passed into

the seminal vesicles, which contained thick, mucopurulent debris.

Because o the extent o inectionand epididymitis that ap-

parently blocked sperm outflow into the ductus deerensthe

owners elected to castrate the horse and retire it rom stud service.

Afer castration, the seminal vesicles were flushed with sterile saline

Figure 11. Ultrasonogram of distended epididymal tubules in the cauda epididymisof a stallion with bilateral segmental aplasia of the pelvic ductus deferens.

Figure 12. Laparoscopic view of an aplastic segment of the terminal ductusdeferens as it courses over the fundus of the bladder.


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and inused with 1 g o amikacin every other day or three treat-

ments, and the horse was discharged rom the hospital.

ConclusionWhen the cause o azoospermia in a stallion is investigated, the

first diagnostic step is to ascertain whether the stallion is ejaculating.I the stallion appears to ejaculate, a logical diagnostic examination

and testing approach can determine whether blockage o the ex-

current duct system or ailure o spermatogenesis is the cause o

azoospermia. Once a correct diagnosis is made, a prognosis or

return to ertility can be offered and treatment options explored.

References1. McDonnell SM. Ejaculation: physiology and dysfunction. Vet Clin North Am Equine

Pract 1992;8(1):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, Johnson L. Diseases and Management of

Breeding Stallions. Goleta, CA: American Veterinary Publications; 1991:159-174, 343.

4. Love CC, Riera FL, Oristaglio (Turner) RM. Sperm occluded (plugged) ampullae in thestallion. Proc Ann Mtg Soc Therio1992:117-125.

5. Blanchard TL, Varner DD, Bretzlaff KN, Elmore RG: Testicular degeneration in large

animals: II. Identication and treatment. Vet Med1991;86(5):537-542.

6. Turner RM. Alkaline phosphatase activity in stallion semen: characterization and clinical

implications.Proc Ann Mtg Soc Therio1996:284-293.

7. Brinsko SP. Retrograde ejaculation in a stallion. J Am Vet Med Assoc2001;218:551-553.

8. Love CC. Ultrasonographic evaluation of the testis, epididymis, and spermatic cord of

the stallion. Vet Clin North AmEquine Pract 1992;8:167-182.

9. McEntee K. The male genital system. In: Jubb KVF, Kennedy PC, eds. Pathology of

Domestic Animals.2nd ed. New York: Academic Press; 1970:447-449.

10. Ladds PW. The male genital system. In: Jubb KVF, Kennedy PC, eds. Pathology of

Domestic Animals.3rd ed. Orlando, FL: Academic Press; 1985:420-424.

11. Blanchard TL, Woods JA, Brinsko SP. Theriogenology question of the month: epididymal

hypoplasia in a stallion. J Am Vet Med Assoc2000;217(6):825-826.

12. Douglas RH, Umphenour N. Endocrine abnormalities and hormonal therapy. Vet Clin

North Am Equine Pract 1992;8(1):237-250.

13. Roser JF. Endocrine proles in fertile, subfertile, and infertile stallions: testicular

response to human chorionic gonadotropin in infertile stallions. Biol Reprod1995;suppl

1:661-669.

14. Blanchard T, Varner D, Miller C, Roser J. Recommendations for clinical GnRH challenge

testing of stallions. J Equine Vet Sci2000;20:678-682, 686, 731-737.

15. Blanchard TL. Testicular biopsy. In: McKinnon AO, Samper J, Pycock JC, Varner DD,

eds. Equine Reproduction.2nd ed. West Sussex, UK: Wiley-Blackwell; 2011:1517-1522.

16. Swierstra EE, Pickett BW, Gebauer MR. Spermatogenesis and duration of transit of

spermatozoa through the excurrent ducts of stallions. J Reprod Fertil Suppl1975;23:53-57.

17. Brinsko SP, Blanchard TL, Varner DD, et al. Manual of Equine Reproduction.3rd ed.

St Louis, MO: Mosby; 2011:176-206.

18. Estrada A, Samper JC, Lillich JD, et al. Azoospermia associated with bilateral segmental

aplasia of the ductus deferens in a stallion. J Am Vet Med Assoc 2003;222:1740-1742.

19. Varner DD. Congenital aplasia of the pelvic ductus deferens in a stallion. Unpublished

observations, 2002.20. Varner DD, Schumacher J. Abnormalities of the accessory sex glands. In: McKinnon

AO, Samper J, Pycock JC, Varner DD, eds. Equine Reproduction.2nd ed. West Sussex,

UK: Wiley-Blackwell; 2011:1113-1118.

21. Love CC, Garcia MC, Riera FR, Kenney RM. Evaluation of measures taken by ultra-

sonography and caliper to estimate testicular volume and predict daily sperm output in

the stallion. J Reprod FertilSuppl1991;44:99-105.

22. Johnson L, Varner DD, Thompson DL Jr. Effect of age and season on the establishment

of spermatogenesis in the horse. J Reprod Fertil Suppl1991;44:87-97.

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PV0212 Blanchard