Endocrine Abnormalities and Hormonal Therapy

Endocrine Abnormalities and Hormonal Therapy

STALLION MANAGEMENT 0749-0739/92 $0.00 + .20 ENDOCRINE ABNORMALITIES AND HORMONAL THERAPY R.H. Douglas, PhD, and Norman Umphenour, DVM Very little...

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STALLION MANAGEMENT

0749-0739/92 $0.00

+ .20

ENDOCRINE ABNORMALITIES AND HORMONAL THERAPY R.H. Douglas, PhD, and Norman Umphenour, DVM

Very little information is available regarding endocrine dysfunction in stallions, and even less is known about therapeutic uses of hormones to improve reproductive performance. Such a paucity in information is especially disgruntling to owners and managers of valuable breeding stallions (some of which are worth millions of dollars). One of the main obstacles in this regard is the difficulty of assaying glycoprotein hormones, such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH), in stallions. To date, no commercial assay kits are available for measuring these two hormones: Virtually all data available were generated using heterologous radioimmunoassays, which vary from laboratory to laboratory. Moreover, there has been no standardization among laboratories as to methodology or establishment of "normal" values for fertile stallions. The practicing veterinarian therefore has not relied heavily on endocrinologic data in decision-making for evaluation and management of breeding stallions. During the last decade, we have been collecting fertility and endocrinology data on stallions and have found that a limited number of hormonal changes may be associated with a decrease in fertility. At present, it is possible to hormonally identify stallions that are undergoing testicular degeneration leading to sub fertility, and, often, sterility; however, it is not possible to predictably treat such stallions with any hope for either restoring fertility or preventing the anticipated rapid decline in fertility. The hope for the future rests in the following: (1) greater use of routine endocrine assessment of stallions (i.e., to establish characteristic From BET Reproductive Laboratories, Inc. (RHD), and Gainesway Farm (NU), Lexington, Kentucky

VETERINARY CLINICS OF NORTH AMERICA: EQUINE PRACTICE VOLUME 8 • N UMBER 1 • APRIL 1992

237

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DOUGLAS & UMPHENOUR

patterns for each stallion while he is fertile); (2) improved availability and quality of therapeutic agents for treating endocrine-compromised stallions. For example, our results with therapeutic use of gonadotropinreleasing hormone (GnRH) in subfertile stallions would probably be greatly improved if we had access to a sustained release system such as microsphere-loaded native GnRH; (3) knowledge of treatment effects (i.e., dosage, frequency of administration, long term effects, etc.) on endocrine function with respect to season; (4) development of diagnostic endocrine criteria that indicate accurately when to intervene with therapy in advance of a decline in fertility; and (5) a working knowledge of endocrine function and dysfunction in the stallion by the veterinary practitioner. HORMONES ASSAYED

Our studies and those of others have focused on one or more of the following hormones: testosterone, estrogens, FSH, LH, thyroxine (T4), cortisol and, more recently, insulin. Table 1 provides test protocols for assessing endocrine function in stallions. References as to normal or abnormal values are those used by BET laboratories, where all the radioimmunoassays (RIA) were performed. RELATIONSHIP OF HORMONES TO FERTILITY

By 1974, a high blood concentration of FSH was considered diagnostic of irreversible damage to the seminiferous epithelium of men. 5 Circulating concentrations of FSH were above normal in oligospermic (low sperm numbers in ejaculates) men, whereas LH, prolactin, testosterone, and estradiol were within normal limits.!2 In serum of azoospermic (ejaculates devoid of sperm) men, elevated serum concentrations of FSH, LH, and estrogen, a slight elevation in 17ahydroxyprogesterone, and decreased concentrations of testosterone and dihydrotestosterone were reported. 6 In 1984, the first association between elevated plasma concentrations of FSH and subfertility was made in stallions.! Testosterone and LH concentrations were within normal limits for stallions in that study.! The authors have since evaluated endocrine profiles on over 300 different stallions, of which 82 had some degree of fertility loss . Blood samples for endocrine analyses were collected from the jugular vein during different times of the day. Although variations in hormone concentrations occur throughout the day (i.e., diurnal variations), interpretations can be made regarding altered hormonal concentrations (Table 2). Seasonal effects on plasma hormone concentrations are more pronounced (Table 3). Blood LH concentrations parallel those for testosterone; concentrations in the spring and summer are higher than concentrations in winter. 3 Concentrations of FSH appear to be less

ENDOCRINE ABNORMALITIES AND HORMONAL THERAPY

239

Table 1. TEST PROTOCOLS FOR ASSESSING ENDOCRINE FUNCTION IN STALLIONS· Test

Dosage

Time for Collection of Blood Samples

Hormones Assayed

ACTH Stimulation

100lU/IV

1) 0 2) 2 hr

Cortisol

Dexamethasone suppression

10 mg/lM

1) 2) 3) 4) 5)

Cortisol, insulin

Dexamethasone suppressionl ACTH Stimulation

10 mg/lM (DEX) 100lU/IV (ACTH)

1) 2) 3) 4)

TRH response

1 mg/lV

1) 2) 3) 4) 5) 6)

0 15 30 1 2 3

min min hr hr hr

1) 2) 3) 4) 5) 6)

0 1 2 4 8 24

hr hr hr hr hr

GnRH response

HCG Stimulation

1 mg/lV

6000lU/IM

0 2 6 12 24

hr hr hr hr

0 3 hr after 3 hr sample 2 hr post ACTH

Cortisol

Cortisol, insulin, T4

1) 0 2) 1 hr 3) 2 hr

LH, FSH, testosterone, estrogens

Testosterone, estrogens

*0 time samples are always taken prior to administration of test drug; either serum or plasma may be assayed.

variable throughout the year. Additionally, application of an artificial photoperiod primarily affects concentrations of testosterone and LH without profoundly affecting concentrations of FSH.3 Age affects gonadotropin concentrations (i.e., older stallions have higher mean concentrations of LH and FSH than do younger stallions), whereas testosterone and estrogen concentrations are less correlated with age in stallions, except that testosterone concentrations tend to decrease somewhat in aged stallions. Hormone concentrations in an individual stallion therefore must be evaluated in relation to the stallion's age, type of photoperiod in which he is maintained, and the time of year. Table 4 summarizes some relationships between hormonal concentrations and stallion fertility. Data were obtained from evaluation of differing levels of fertility in stallions in a natural-cover breeding management program. All data are from stallions on the same farm. Values shown for FSH and total estrogen concentrations were obtained from blood samples taken in October, a month when low hormonal

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DOUGLA S & UMPHENOUR

Table 2. CONCENTRATIONS OF TESTOSTERONE AND ESTROGENS THROUGHOUT THE DAY IN A SUBFERTILE STALLlON* Time

Testosterone (pg/mL)

Total estrogens (pg/mL)

0800 0830 0900 0930 1000 1030 1100 1130 1200 1230 1300 1330 1400 1430 1500 1530 1600

1203 1134 1062 1157 1040 938 913 969 932 940 1066 1032 923 1029 1026 1087 975

46 55 45 56 53 46 45 53 54 49 52 60 60 55 44 99 45

"April

concentrations and seminal characteristics would be obtained. Stallions were evaluated for hormone concentrations on at least three other occasions during each year; usually winter, spring, and summer. Our conclusions were not altered by the season of sample collection. Only data for estrogens and FSH are shown because these two hormones were more consistently associated with changes in fertility and thus may be good markers to predict future changes in fertility. Table 3. SEASONAL CHANGES IN MEAN TESTICULAR DIAMETER AND BLOOD CONCENTRATIONS OF TESTOSTERONE,TOTALESTROGENS, AND FSH (N = 46 STALLIONS) Month January February March April May June July August September October November December

Scrotal Width

Testosterone (pg/mL)

Estrogens (pg/mL)

FSH (ng/mL)

109 113 118 114 112 116 117 118 107 109 104 113

1243 1794 1660 1769 1627 1306 1056 956 1155 659 615 950

330 309 391 308 317 258 242 218 192 203 253 269

8.8 8.3 9.1 8. 1 8.3 8.1 7.5 7.4 6.3 5.9 7.4 8.0

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ENDOCRINE ABNORMALITIES AND HORMONAL THERAPY

Table 4. RELATIONSHIP BETWEEN FERTILITY AND BLOOD CONCENTRATIONS OF ESTROGENS AND FSH IN THOROUGHBRED STALLIONS Average No. of Breedings/ Pregnancy

Season Pregnancy Rate (%)

Serum Total Estrogen Concentration

Serum FSH Concentration

Age

Year

No. of Mares Bred

21 22 25 26 27

84 85 88 89 90

48 47 44 34 16

1.98 2.40 2.09 1.76 2.06

90 83 66 74 75

214 192 394 404 478

7.2 4.6 3.3 7.8 9.0

2 2 2 2 2

13 14 17 18 19

84 85 88 89 90

41 47 49 51 50

1.85 1.89 1.82 1.67 1.70

94 94 94 94 94

321 288 345 206 270

3.1 4.6 3.0 8.2 2.7

3 3 3 3 3

5 6 9 10 11

84 85 88 89 90

46 61 44 52 56

1.68 1.98 1.95 1.90 1.84

96 87 82 79 · 88 .

240 110 239 303 317

5.3 6.5 2.7 10.6 2.4

4 4 4 4 4

10 11 14 15 16

84 85 88 89 90

54 56 55 56 30

1.83 1.96 2.31 3.20 2.97

89 84 91 63 43

310 364 118 212 137

7.3 8.1 10.4 19.4 30.3

5 5 5 5 5

13 14 17 18 19

84 85 88 89 90

60 57 54 62 29

2.10 2.40 2.88 2.42 2.45

85 79 72 56 52

198 78 240 124 100

6.7 8.2 7.3 28.1 25.0

6 6 6 6 6

11 12 15 18 19

84 85 88 89 90

47 60 55 51 36

2.11 2.38 2.33 2.08 2.44

81 67 75 80 69

156 48 409 328 255

3.3 5.1 3.5 10.9 2.8

7 7 7 7 7

15 16 19 20 21

84 85 88 89 90

76 56 57 47 49

2.26 2.68 2.75 2.66 2.41

87

94

77

77

79 85 78

120 192 125

4.5 4.2 6.0 13.3 4.3

8 8 8 8

15 16 19 20

84 85 88 89

76 68 24 21

1.99 2.24 4.75 3.00

80 88 42 14

105 18 112 8

8.2 10.1 23.1 40.9

9 9 9 9

19 20 23 24

84 85 88 89

45 45 46 30

1.93 2.51 2.20 1.60

93 80 78 57

128 25 134 163

4.7 8.9 14.5 16.5

Stallion

The key clinical points regarding the relationship between fertility and blood concentrations of total estrogen and FSH are as follow: 1. Low total estrogen concentration frequently is present in stallions with a high average breeding per pregnancy rate. 2. Low total estrogen concentration mayor may not be present in stallions with inadequate libido.

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DOUGLAS & UMPHENOUR

3. Decreased total estrogen concentration sometimes precedes a decrease in stallion fertility. 4. High FSH concentration almost always occurs concurrently with a low concentration of total estrogen. 5. High concentration of FSH concurrent with low concentrations of total estrogen is associated with low fertility, expressed as an increased number of breedings per pregnancy. Stallions over 20 years of age with concentrations of total estrogen greater than 200 pg/mL and FSH concentrations less than 15 ngl mL generally achieve seasonal pregnancy rates greater than 75% with two or fewer covers per pregnancy. Stallions of any age that consistently have total estrogen concentrations less than 125 pg/mL and FSH concentrations greater than 25 ng/mL generally achieve seasonal pregnancy rates less than 75 % and require more than two covers per pregnancy. 6. Stallions with a low concentration of total estrogen and a high concentration of FSH tend to have decreased testicular diameter with palpable evidence of testicular degeneration (i.e., small, soft testes with prominent wrinkling of the tunica albuginea). Ejaculates from such stallions usually have a low concentration of sperm «80 x 106 /mL) and poor sperm motility «50% progressive sperm motility) . Preliminary observations also suggest sperm chromatin content may be reduced.

It is important to remember that there are numerous causes of stallion infertility and subfertility. All stallions with low fertility do not have obvious endocrine anomalies, and the cause of poor fertility frequently remains undetermined.

ENDOCRINE MECHANISMS OF TESTICULAR DEGENERATION

Evaluation of data in Table 5 and from other sources suggests that stallions are similar to men in regard to changes in FSH concentrations that are associated with damage to the seminiferous epithelium. Whether high FSH concentrations are a result of, or a cause of, testicular degeneration, oligospermia, and reduced fertility remains unknown. If the anterior pituitary gland secretes a polymorphic form of FSH that Table 5. RELATIVE POTENCY OF GnRH AND GnRH ANALOGUES Compound

Relative Potency

GnRH GnRH (amino acid substituted at 10 position) GnRH (amino acid substituted at 6 position) GnRH (amino acid substituted at 6 and 10 position) Leuprolide Buserelin Deslorelin Histrelin

1 4 4 4 15 20 144 210

Data from Conn PM, Crowley WF: Gonadotropin·releasing hormone and its analogues. N Engl J Med 324:93-103, 1991

ENDOCRINE ABNORMALITIES AND HORMONAL THERAPY

243

contains predominantly biologically inactive moieties, testicular tissue may lack adequate endocrine support for normal function. Resulting testicular degeneration could lead to decreased testicular production of estrogen and inhibin and increased production of biologically inactive FSH. Radioimmunoassay techniques measure only immuno-active FSH. No data exist on the ratio of biologically active to biologically inactive FSH in the blood of stallions. In contrast, if seminiferous epithelial damage occurs first, the normal inhibitory feedback that moderates FSH secretion (testis to the hypothalamus/anterior pituitary gland) would be lost. If loss of negative feedback results in increased FSH production and secretion from the anterior pituitary gland, an increased blood FSH concentration, perhaps with altered bioactivity, would follow. The important clinical concept is that for any therapy to be effective, it must be initiated before irreversible damage occurs either at the level of the testis or the hypothalamus/anterior pituitary gland. Obtaining routine seasonal endocrine profiles for individual stallions may become important in predicting when degeneration is starting to occur. Combining endocrine data with seminal parameters and indices of fertility calculated monthly may facilitate development of an accurate model for predicting endocrine mediated decreases in fertility. Another condition that may playa role in testicular degeneration is pituitary tumors. Their incidence in stallions is not well documented. Endocrine alterations are present long before classic signs of hirsutism, polyuria, polydipsia, weight loss, and visual deficits are manifest in affected animals. These include consistently elevated blood concentrations of insulin and, sometimes, glucose. The stallion usually fails to have a normal response to either a dexamethasone suppression test or thyrotropic-releasing hormone (TRH) response test (see Table 1). Cortisone is not suppressed in response to dexamethasone administration and is abnormally elevated in response to TRH administration. We have identified this condition in a 17-year-old stallion that had a rapid decline in fertility and became oligospermic. More research obviously is needed in this area. Although the impact of thyroid dysfunction on fertility in stallions is less clear than in mares, anecdotal data suggest that blood concentrations of total thyroxine (T4) consistently less than 6.0 ng/mL may be associated with decreased libido. Young stallions retired from racing are likely to have low T4 concentrations due to injuries or other stress. Auto-immune thyroiditis is probably uncommon in the stallion; however, no studies of thyroid-stimulating hormone (TSH) concentrations concomitant with T4 evaluations have been done in stallions. Testosterone concentrations are seldom altered in subfertile stallions younger than 15 years of age but are sometimes low in subfertile, aged stallions. Serum concentrations of LH (LH stimulates testicular Leydig cells to produce testosterone) often are low in stallions with poor libido of organic origin. Conversely, serum LH concentrations are high in about 50% of stallions with oligospermia and reduced fertility.

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DOUGLAS & UMPHENOUR

HORMONAL THERAPY

Evaluation of response to hormonal therapy regarding improvement or correction of reproductive function in males of any species is limited by deficiencies in both the size of the data base and in the small arsenal of drugs available to the veterinarian. The hormone that has received the most attention for treatment of subfertility in men and stallions has been GnRH. This hormone is a decapeptide produced by the hypothalamus. Synthesized GnRH is secreted directly into the hypophysioportal circulation with some probably diffusing into cerebrospinal fluid. At the level of the anterior pituitary gland, GnRH stimulates production and release of LH and FSH. Gonadotropinreleasing hormone also stimulates secretion of free a-subunits of these two glycoproteins, which are normally made up of a- and f3-subunits. 4 Both LH and FSH are thought to stimulate testicular steroidogenesis and gametogenesis. In vivo, GnRH secretion is pulsatile . Prolonged treatment with continuously administered GnRH in some species desensitizes the anterior pituitary gland, resulting in suppression of FSH and LH production (i.e., down-regulation), culminating in gonadal dysfunction. 4 However, this effect is dependent on the form of GnRH used and the frequency of its administration. For example, when oligospermic men with elevated serum FSH concentrations were treated with 5 /-Lg GnRH every 2 hours for 6 weeks, concentrations of FSH were decreased, concentrations of estradiol and testosterone were increased, and concentrations of LH remained unaltered by the end of treatment. 12 When the same treatment protocol was administered for 1 instead of 6 weeks, no adverse effects on gonadotropin or testicular steroid concentrations were detected, but administration of 5 /-Lg GnRH every 30 minutes resulted in decreased serum FSH concentrations in 1 week. l l Some researchers have postulated that if high FSH concentration is the cause rather than the result of spermatogenic failure it may be possible to improve sperm production by regulating FSH production by exogenous GnRH therapy.12 It should be noted that formulations of GnRH have variable potencies (Table 5) . The relative potencies are determined by the sequence of amino acids or the substitution of the heterologous amino acids at certain positions within the ten amino acid chain and by the specific amino acid that is substituted in that position. In general, GnRH is used for two basic reasons in men. One use is to restore fertility in men thought to be deficient in GnRH (although GnRH cannot be measured in such patients) by administering natural GnRH via external pumps. Usually, the natural GnRH decapeptide is pulsed every 2 hours. In hypogonadotropic (low FSH and LH concentrations, presumably caused by a GnRH deficiency) men, several months of pulsatile GnRH therapy results in growth of the testes and induction of spermatogenesis by restoring episodic secretion of gonadotropins and normal concentrations of sex steroids.13, 15 Another use of GnRH

ENDOCRINE ABNORMALITIES AND HORMONAL THERAPY

245

results in "biochemical castration" and is achieved by reversible suppression of the pituitary-gonadal axis. This requires continuous administration of high doses of GnRH or long-acting GnRH agonists. Clinically, this is now accomplished by using depot delivery formulations containing potent GnRH analogues such as goserelin acetate implants (Zoladex, U.S. and Canada). 7 These implants contain 3.6 mg goserelin base dispersed in a biogradable and biocompatible matrix of D,L,-lactic and glycolic acids copolymer (13.3-14.3 mg per dose) containing approximately 2.1 mg GnRH analogue. 7 In men with Zoladex implants, peak concentrations of 2.5 ng/mL goserelin is achieved in 12 to 15 days; the implant lasts 28 days. Testosterone secretion is stimulated during the first week of therapy but declines to castrate levels within 2 to 4 weeks and remains low for the duration of therapy. Unfortunately, hypogonadotropic subfertile stallions have not been identified; therefore, pulsatile administration of natural GnRH probably would be of no benefit. Clinically, this has been found to be the case in the small number of stallions treated (Tables 6-8). The stallion represented in Table 8 had no evide'n ce of testicular degeneration but finished the year with a 36% seasonal pregnancy rate. Typically, restoration of low estrogen concentrations to within normal limits was inconsistent. Even in cases in which high FSH concentrations were decreased, no significant improvement in fertility ensued. A recent Table 6. EFFECTS OF PULSATILE ADMINISTRATION OF GnRH AT A RATE OF 10 f-L9 EVERY 120 MINUTES IN A SUBFERTILE STALLION WITH LOW CONCENTRATIONS OF TOTAL ESTROGENS AND HIGH CONCENTRATIONS OF FSH Hormone Concentrations

Date No GnRH April 15 GnRH pump in April 17 April 28 June 1 June 8 No GnRH August 10 September 10 GnRH pump in October 17 November 11 Next year

°

Pregnancy Testosterone Rate (pg/mL)

Total Estrogen LH (pg/mL) (ng/mL)

FSH (ng/mL)

33%

1088

35

20.0

22.0

73%

1203 991 1007

46 124 148

25.0 8.9 10.4

25.0 32.7 24.3

390 411

297 63

7.6 0.6

50.0 50.0

426 300

204 50

1.0 25.0

51.0 65.0

0%

Notes: Total estrogen concentrations are decreased compared to normal stallions except on Aug. 1 and Nov. 11. Testosterone concentrations are similar to normal stallions except on Aug. 10, Sep. 10, and Nov. 11 . Concentrations of FSH are increased in all samples compared to normal stallions.

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DOUGLAS & UMPH ENOUR

Table 7. EFFECTS OF DAILY INJECTIONS OF GnRH IN A STALLION WITH DECLINING FERTILITY AND LOW TESTOSTERONE AND TOTAL ESTROGEN CONCENTRATIONS AND HIGH FSH CONCENTRATIONS Pregnancy Rate Month

Year 1 Year 2

February March April May June July

27 42 43 31 54 50

12 29 34 33 34 40

Total

81

71

-Hormone Concentration Year 2

Year 3

Year 4

FSH Estrogens (pg/mL) (ng/mL)

Testosterone (pg/mL)

82 170 28 53 261 61

876 938 857 802 453 636 42

13.0 18.0 4.9 12.5 24.1 35.0

14

' 500 flog of GnRH given intramuscularly daily from March 31 to July 31 . Administration of GnRH did not appear to affect hormone concentrations or fertility.

report indicated that either pulsatile (10 fLg every 2 hrs) or constan t (120 fLg/day) administration of natural GnRH for 20 weeks did not elevate secretion of testosterone, enhance growth of testes, or alter sperm output in reproductively sound or unsound stallions. 14 Effects of higher doses of GnRH are unknown in such stallions. Table 8. EFFECTS OF PULSATILE ADMINISTRATION OF 10 1L9 GnRH EVERY 2 HOURS ON HORMONE CONCENTRATIONS IN A SUBFERTILE STALLION Hormone Concentrations Date November 1 March 19 April 16 April 21 May 5 May 13 May 19 May 20 May 22 May 27 May 29 June 1 June 5 June 24 July 4 July 6 July 9 October 5

Testosterone (pg/mL) 216 502 GnRH 748 969 557 1500 GnRH GnRH 207 GnRH GnRH 1124 185 GnRH 467 469 285

Estrogens (pg/mL)

T4 (ng/mL)

LH (ng/mL)

158 369

8.6 3.3

1.6 8.7

3.1 3.9

305 446 190 445

9.2 8.8' 20.0 20.1

17.0 20.0 23.0 20.0

3.4 4.6 5.1 4.5

237

9.4

16.4

3.5

489 335

19.0 13.7

8.5 10.6

7.6 5.0

212 242 152

10.1 9.9 14.0

4.3 7.6 14.0

5.7 0.4 0.8

FSH (ng/mL)

pump in

pump out pump in pump out pump in pump out

' Thyroxine treatment started .

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ENDOCRINE ABNORMALITIES AND HORMONAL THERAPY

Table 9. GUIDE FOR HORMONAL EVALUATION IN STALLIONS Problem Libido Sperm concentration Hair coat Testicle size Polyuria/polydipsia Excess gel Motility Fertility Weight loss p s

T4

Testosterone

Estrogens

Insulin

FSH

LH

Cortisol

P

P

P

s

s

s

s

p p p p p

s

p p p

p s p p s s p p

p

p

s

p

p

s p

p

p s p

P

p p

p p s p

p

P p

P

p

P P

s p

P

= primary = secondary

We have implanted two stallions with Zoladex. One stallion was sterile and had previously been found to be unresponsive to GnRH (see Table 1). The second stallion was oligospermic, had a seasonal pregnancy rate of 43% with 2.97 breedings per pregnancy in the year immediately preceding implant injection, and was responsive to the GnRH. Implants were injected in December. The first stallion showed virtually no response in testosterone, estrogen, LH, and FSH concentrations even after two impl<;lnts at 28-day intervals. The second stallion received three implants at 28-day intervals. Testosterone, estrogens, LH, and FSH concentrations changed following insertion of the second implant, but down-regulation had occurred by insertion of the third implant, and testosterone and estrogen declined to extremely low concentrations. Concentrations of testosterone and estrogens were normal within 30 days after the 28 day life-span of the third implant. The second stallion failed to impregnate a single mare during the treatment period. Thus, even this relatively low dose (approximately three times lower than the dosage for humans) of a potent GnRH analogue was able to cause down-regulation of the pituitary gland. Whether a microsphere implant of natural GnRH would induce downregulation is unknown. Most stallions we studied were treated with GnRH after testicular degeneration had developed. If any form of GnRH therapy is to be beneficial in stallions, it will likely have to be initiated prior to decline in serum estrogen concentrations below 125 pg/mL. Extremely low estrogen concentrations probably reflect irreversible damage to the germinal epithelium. More studies that address various dosages and frequencies of administration must be conducted before we give up completely on GnRH therapy in subfertile stallions. A futuristic concept lies in the transplantation of fetal GnRH neurons into the cerebral ventricles of adults. This has been done in sterile hypogonadal mice.8-l0 After deposition into the floor of the third ventricle, the neurons migrate into the arcuate nuclei, extend nerve terminals into the median eminence, and release GnRH into the

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DOUGLAS & UMPHENOUR

hypophysioportal circulatory system. Reproductive function is restored, and females are then capable of mating and becoming pregnant. One potential hormonal therapy that has been overlooked in stallions is the use of equine gonadotropins. Injection of 500 FevoldHisaw units of equine pituitary extract in stallions resulted in significant increases of LH (1.6 times pretreatment concentrations) similar to that achieved in the same stallions with a single injection of 400 j-lg natural GnRH. Unfortunately, a commercially available source of equine gonadotropins does not exist. Human gonadotropins have been shown to be effective in hypogonadotropic men, but their use has not been investigated in stallions. Table 9 gives an overview for use of endocrine assays to screen stallions for specific endocrine problems. When endocrine anomalies, presumably involving pituitary tumors, occurred, therapy with perogolide (Permax) at a dosage of 3 to 5 mg orally per day reduced insulin concentrations, improved hair coat, and corrected polydipsia/polyuria, but effects on fertility usually were not altered.

SUMMARY

Routine measurement of estrogens, testosterone, T4, insulin, FSH, and LH at least four times per year (e.g., during each of the four seasons) may improve the efficiency of stallion management. Benefits may not be realized in the short term but will provide valuable historical data on individual stallions that, when added to other data, will improve ability of management personnel to initiate early treatment and delay or slow declining fertility. This ability will be greatly improved as more data and products become available. There appears to be a relationship between low total estrogen concentration/high FSH concentration and subfertility. This condition is associated with high average breedings per pregnancy. A decrease in concentration of estrogen and an increase in FSH concentration often precede a decline in fertility associated with oligospermia. Hypogonadotropic stallions have not been reported. This condition is not likely to be a cause of declining fertility in stallions and greatly limits the potential efficacy of GnRH therapy in subfertile stallions. Much research must be done to elucidate the etiology of testicular degeneration associated with increased FSH concentrations and decreased estrogen concentrations in stallions. At present, no reliable hormonal therapeutic protocols exist that will improve fertility in subfertile stallions.

References 1. Burns PI, Douglas RH: Reproductive hormone concentrations in stallions with breeding problems: case studies. J Equine Vet Sci 5:40, 1984 2. Burns PI, Douglas RH: Effects of a single injection of GnRH or equine pituitary

ENDOCRINE ABNORMALITIES AND HORMONAL THERAPY

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

249

extract on plasma LH and FSH concentrations in stallions. J Equine Vet Sci 4:281, 1984 Burns PJ, Jawad MJ, Weld JM, et al: Effects of season, age, and increased photoperiod on reproductive hormone concentrations and testicular diameters in thoroughbred stallions. J Equine Vet Sci 4:202, 1984 Conn PM, Crowley WF Jr: Gonadotropin releasing hormone and its analogues. N Engl J Med 324:93, 1991 de Kretser DM, Burger HS, Hudson B: Relationship between germinal cells and serum FSH levels in males with infertility. J Clin Endocrinol Metab 38:787, 1974 Diez LCG, Buitrago JMG, Corrales J], et al: Hormone levels in serum and seminal plasma of men with different types of azoospermia. J Reprod Fertil 67:209, 1983 Drug information for the health care professional. USPDI 11:1397, 1991 Gibson MJ, Keiger DT, Charlton HM, et al: Mating and pregnancy can occur in genetically hypogonadal mice with preoptic area brain grafts. Science 225:949, 1984 Gibson MJ, Charlton HM, Perlow MJ, et al: Preoptic area brain grafts in hypogonadal female mice abolish effects of congenital hypothalamic gonadotropin-releasing hormone deficiency. Endocrinology 114:1938, 1984 Gibson MJ, Kokoris GJ, Silverman AJ: Positive feedback in hypogonadal mice with preoptic area brain transplants. Neuroendocrinology 48:112, 1988 Gross KM, Matsumots AM, Southworth MB, et al: Evidence for decreased luteinizing hormone releasing hormone pulse frequency in men with selective elevations of follicle stimulating hormone. J Clin Endocrinol Metab 60:197, 1985 Honigl W, Knuth UA, Nieschlag E: Selective reduction of elevated FSH levels in infertile men by pulsatile LHRH treatment. Clin Endocrinol 24:177, 1986 Santorn N, Filicori M, Crowley WF Jr: Hypogonadotropic disorders in men and women: diagnosis and therapy with pulsatile gonadotropic releasing hormone: Endocrinol Rev 7:11, 1986 Squires EL, Blue BJ, Nett TM, et al: Effect of pulsatile and continuous administration of GnRH on reproductive function of stallions. In Proceedings of the 5th International Symposium on Equine Reproduction, Neuville, France, 1990, p 14 Whitcomb RW, Crowley WF Jr: Diagnosis and treatment of isolated gonadotropic releasing hormone deficiency in men. J Clin Endocrinol Metab 70:3, 1990

Address reprint requests to R. H. Douglas, PhD BET Reproductive Laboratories, Inc. 6174 Jacks Creek Road Lexington, KY 40515