Effect of ACTH (tetracosactide) on steroid hormone levels in ...pub.epsilon.slu.se/1275/1/YHAfin3.pdfAnimal Reproduction Science xxx (2006) xxx–xxx Effect of ACTH (tetracosactide)

Animal Reproduction Science xxx (2006) xxx–xxx

Effect of ACTH (tetracosactide) on steroidhormone levels in the mare

Part B: Effect in ovariectomized mares(including estrous behavior)

Y. Hedberg a,∗, A.-M. Dalin a, M. Forsberg b, N. Lundeheim c,G. Sandh d, B. Hoffmann e, C. Ludwig e, H. Kindahl a

a Division of Comparative Reproduction, Obstetrics and Udder Health, Department of Clinical Sciences,P.O. Box 7054, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden

b Division of Diagnostic Imaging and Clinical Pathology, Department of Biomedical Sciences and Veterinary PublicHealth, P.O. Box 7009, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden

c Division of Pig Breeding, Department of Animal Breeding and Genetics, P.O. Box 7023,Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden

d Division of Large Animal Surgery, Department of Clinical Sciences, P.O. Box 7018,Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden

e Klinik fur Geburtshilfe, Gynakologie und Andrologie der Groß-und Kleintiere mit Tierarztlicher Ambulanz,Justus-Liebig-Universitat, D-35392 Giessen, Germany

Received 7 February 2006; received in revised form 12 May 2006; accepted 12 June 2006

Abstract

The mare is the only non-primate species known to display estrous signs after ovariectomy and adrenalhormones have been implicated as a possible cause. Moreover, in several species, estradiol seems to havea stimulatory effect on the hypothalamic–pituitary–adrenal axis. The aim of the present study was to com-pare the effect of ACTH (tetracosactide) on pertinent hormones [cortisol, progesterone, androstenedione,testosterone (intact and ovariectomized mares) and estradiol (ovariectomized mares only)] in intact maresin estrus with the same mares after ovariectomy (n = 5). Blood samples were collected hourly from 12:00until 14:00 h the following day (half-hourly between 14:00 and 17:00 h) on two occasions, with saline orACTH treatment at 14:00 h (saline treatment day or ACTH treatment day). The mares, both when intact andafter ovariectomy, showed a significant increase in all measured hormones, except estradiol (not measured inintact mares), after ACTH treatment, lasting at least 3 h post-treatment (P < 0.001). On the saline treatment

∗ Corresponding author. Tel.: +46 18 671154; fax: +46 18 673545.E-mail address: [email protected] (Y. Hedberg).

0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.anireprosci.2006.06.007

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2 Y. Hedberg et al. / Animal Reproduction Science xxx (2006) xxx–xxx

day, cortisol levels in ovariectomized mares were lower than in intact mares in the evening (18:00–23:00 h),but higher at night (24:00–05:00 h). No differences in cortisol response between mares, when intact and afterovariectomy, were found after ACTH treatment (P = 0.3). Androstenedione levels were lower (P < 0.001)and increased less after ACTH treatment in ovariectomized mares, as compared to when intact (P < 0.05).Progesterone concentrations were lower in the ovariectomized mares at night (24:00–05:00 h) on the salinetreatment day and at all times on the ACTH treatment day (P < 0.05). Testosterone concentrations were lowerin ovariectomized mares on both treatment days, as compared to when intact (P < 0.001). It was concludedthat ovariectomy affected basal cortisol pattern. Ovarian androstenedione and testosterone contributed to thebasal circulating levels and, in the case of androstenedione, was stimulated by ACTH. Endogenous estradioldid not act stimulatory on adrenal gland hormone production in the mare.© 2006 Elsevier B.V. All rights reserved.

Keywords: Mare; Ovariectomy; Adrenal gland; Sex steroid hormones; ACTH; Estrous behavior

1. Introduction

We have previously demonstrated that increases in cortisol, progesterone, androstenedioneand testosterone occur in the intact mare when treated with exogenous adrenocorticotrophichormone (ACTH, Synachten) (Hedberg et al., 2006; accompanying paper). Similarly, otherprevious studies have shown that the mare adrenal gland produces androgens both in vivo(Watson and Hinrichs, 1989) and in vitro (Silberzahn et al., 1984) although no adrenal pro-gesterone production was found (in vivo). Hormones of adrenal origin have been suggested asa cause of estrous signs in ovariectomized mares and rhesus monkeys, since dexamethasonetreatment (which suppresses the hypothalamic–pituitary–adrenal axis (HPA)) reduced sexualbehavior in both species (Everitt and Herbert, 1971; Asa et al., 1980b). Estrous behaviorin the mare having no ovarian estrogen is most likely not due to previously learned behav-ior, since it has been demonstrated that fillies ovariectomized before the onset of pubertymay still show estrous signs at the time puberty would have set in (Wesson and Ginther,1981).

Further, in several species, a gonadal–adrenal interaction has been shown, where ovarianhormones modulate the HPA axis at several sites. For example, in the rat, estradiol intensifiesthe corticosterone response to stress or ACTH (Kitay, 1963; Viau and Meaney, 1991; Burgessand Handa, 1992). Sex-related factors also appear to stimulate the HPA axis in ewes, at leastunder some stressful conditions (Canny et al., 1999; Turner et al., 2002; Van Lier et al., 2003).An effect of estradiol on the HPA axis at both the hypothalamic–pituitary and adrenal levelhas been described in rats and sheep (Burgess and Handa, 1992; Handa et al., 1994 (review);Nowak et al., 1995; Van Lier, 2003) and estradiol receptors have been demonstrated in the adrenalgland of rats, sheep and rhesus monkeys (Cutler et al., 1978; Hirst et al., 1992; Van Lier, 2003).However, to our knowledge, estradiol effect on adrenal activity has not yet been investigated inthe mare.

The aim of the present study was to compare the effect of ACTH on hormone concen-trations of cortisol, progesterone, androstenedione and testosterone in intact mares at estruswith the effect on the same mares after ovariectomy, i.e. to investigate whether endogenousestradiol (during estrus) has an effect upon the quantity of adrenal hormone produced. Theeffect of ACTH on estradiol levels in ovariectomized mares was also examined. An addi-tional aim was to study estrous behavior of non-ovarian origin, using two different teasingmethods.

Y. Hedberg et al. / Animal Reproduction Science xxx (2006) xxx–xxx 3

2. Materials and methods

All procedures were approved by the Ethical Committee for Experimentation with Animals,Sweden.

2.1. Animals, management and housing

Five intact mares used in the earlier part of the study (March–July 2003) (Hedberg etal., 2006; accompanying paper; i.e. mares at estrus) were ovariectomized (OVX) at the Divi-sion of Large Animal Surgery, at least 1 month prior to the experimental part performed inJune–November 2004. Ovariectomy was performed in the mare standing and under sedation withdetomidine (Domosedan, 10 mg/ml, Orion Pharma AB, Animal Health) and butorphanol (Torbu-gesic, 10 mg/ml) using flank incision. The mares had an age span from 6 to 12 years and weighedbetween 400 and 550 kg. During both years, the mares were housed, managed and fed in the samemanner as described earlier (Hedberg et al., 2006; accompanying paper).

2.2. Blood sampling

The mares, when intact (Part A), were sampled during two estrous periods, when a folliclesize of 3 cm or larger was detected and the mare showed estrous signs and/or had edema in theuterus (Hedberg et al., 2006; accompanying paper). After ovariectomy, frequent blood samplingswere performed 1 week apart (June 23 and 30, 2004). The mares were randomly assigned to atest order in a crossover design with three mares receiving saline treatment and two mares, ACTHtreatment (tetracosactide, Synachten, 0.5 mg) on the first sampling occasion and on the second, thereverse. Blood sampling and ACTH treatment were performed as described in Part A (Hedberg etal., 2006; accompanying paper). Briefly, samples were collected for a 26-h period on each occa-sion; hourly 12:00–14:00 h (pre-treatment samples), half-hourly 14:30–17:00 h and again hourly17:00–14:00 h the following day through a permanent catheter inserted in the jugular vein. All sam-ples (30 samples/mare/occasion) were analyzed for cortisol, progesterone and androstenedionecontent. Pre-treatment samples and samples obtained during the first 8-h period post-treatmentwere analyzed for testosterone content (14 samples/mare/occasion). One pre-treatment sampleand samples taken during the first 5 h post-treatment were analyzed for estradiol content in OVXmares (10 samples/mare/occasion). To confirm high endogenous plasma levels of estradiol inintact mares at estrus, two samples from each of the five intact mares during both frequent sam-pling periods in Part A (samples at 14:00 h prior to ACTH/saline treatment and at 02:00 h aftertreatment) were also analyzed for estradiol content.

2.3. Behavioral testing

All of the mares were teased twice daily when intact (Part A, 2003) and once daily afterovariectomy (June–September 2004) using conventional teasing with a stallion. On both treatmentdays, mares were also teased in the afternoon, during time period two, when (on the ACTHtreatment day) maximum adrenal hormone concentrations were expected (Hedberg et al., 2006;accompanying paper). Estrous signs were scored as follows: +1 for standing in a straddled posture,+1 for flashing clitoris/urinating, +1 for raising tail, −1 for kicking, −1 for switching tail and −1for ears back. The mare was considered to be in full estrus if she had at least two positive scores.Teasing of OVX mares was performed from 2 weeks prior to the first frequent sampling day in


June and until the end of September 2004. In July, the mares were, in addition to the conventionalteasing, teased in a paddock (24 m × 24 m) together with the stallion. This ‘paddock teasing’involved the mare and stallion being let loose in the paddock for 10 min and their behavior videorecorded. Each mare was teased in this manner three times during 3 weeks, with 4–7 days betweenteasing occasions. ‘Paddock teasing’ was again performed in November, but on this occasion allOVX mares were teased for 10 min each and only for 1 day. Again, the behavior of mare andstallion was video recorded. Full and weak estrus was defined as described by Asa et al. (1980a),although they used 20-min long teasing periods. Definitions of full and weak estrus were thus asfollows:

Full estrus:(1) ≥20 s of tail raise near the stallion (TRNS) and >2 urinations.Weak estrus:(1) ≥20 s of TRNS and one occurrence of urination or(2) ≥2 urinations without TRNS or(3) ≥60 s of TRNS without urination.

2.4. Gynecological examination

Intact mares (March–July 2003) were rectally examined every second to every fourth daythroughout the study period with ultrasonography (6 MHz linear-array scanner, 485 Anser, PieMedical, Netherlands) to determine follicle size, presence of edema in the uterus or presence of acorpus luteum. Ovariectomized mares were rectally examined using ultrasonography to determinethe presence of fluid and/or edema in the uterus for 3 consecutive days following paddock teasingin July and on five occasions in the beginning of November. Edema in the uterus was graded asdescribed in Part A.

2.5. Hormone assays

The concentrations of cortisol, progesterone, androstenedione and testosterone were analyzedas described in Part A (Hedberg et al., 2006; accompanying paper). The analyses for 17-�-estradiol were performed at the Department of Biomedical Sciences and Veterinary Public Health,Swedish University of Agricultural Sciences, Uppsala, Sweden. 17-�-Estradiol concentration wasdetermined by radioimmunoassay using a DPC kit (Diagnostic Product Co., Los Angeles, CA,USA), as reported for use in bovine plasma (Sirois and Fortune, 1990). The method has previouslybeen validated (Meikle et al., 1997). All samples were run in duplicates. The inter- and intra-assaycoefficients of variation were as follows:

20.0 and 42.5% at 3.2 pmol/l; 7.7 and 5.0% at 46.5 pmol/l; and 12.0 and 6.2% at 123.2 pmol/l.The minimal detectable concentration of 17-�-estradiol was 2.1 pmol/l.

2.6. Statistical analyses

In the statistical calculations, the five OVX mares were compared with the results obtainedfrom when they were intact. Statistical analyses were carried out using the SAS software (Ver. 8;SAS Institute Inc., Cary, NC, USA). Repeated measurement analyses of variance were performedusing the MIXED procedure. The observations were divided into five time periods: 12:00–14:00 h


Table 3.1Number of days per month (June–September) with estrous behavior in both intact and OVX mares

Mare Reproductive state June July August September

An Intact 6 0* 0* –OVX 0 0 3 0

M Intact 14 2 5# 0OVX 0 6 21 12

B Intact 6 6 – –OVX 0 19 31 28

Ae Intact 1 1 1# –OVX 0 0 2 0

Z Intact 2 0 0 0#OVX 0 16 3 11

(*) prolonged luteal phase; (#) teased 2–3 weeks only; (–) not applicable.

(period one; pre-treatment samples); 14:30–17:00 h (period two); 18:00–23:00 h (period three);24:00–05:00 h (period four); 06:00–14:00 h (period five). The analyses were performed for eachtreatment (saline or ACTH), as well as for the difference between treatments (ACTH minus saline,within mare and sampling time). The fixed effects included in the statistical models were groupof mares (intact or OVX), time period (five periods), sampling time nested within time period,and the interaction between group of mares and time period. The time periods were chosen basedon results from a pilot study performed at the Division of Comparative Reproduction, Obstetricsand Udder Health, where the same dose of ACTH as used in the present study caused an increasein cortisol level for at least 3 h (period two in the present study) (Dalin et al., 2002). Period fourwas defined because of negative feedback (overcompensation by the HPA axis for cortisol) duringthis time period found in the pilot study. Least-squares means were estimated, and pair-wise testsof significance of the difference between Least-squares means were performed.

3. Results

3.1. Estrous detection scores (daily teasing)

The number of days per month each mare (intact in 2003 and OVX in 2004) showed estrus(i.e. at least two positive scores) is shown in Table 3.1. In OVX mares, the number of days ofestrus/mare varied during the period July–September (2, 3, 30, 39 and 78 days, respectively).None of the OVX mares showed estrous behavior in June. The increase in adrenal hormonesduring time period two on the ACTH treatment day (i.e. on June 23 or 30) had no effect onestrous behavior. Four of five OVX mares began to show estrous signs after the paddock teasingstarted in July. In general, OVX mares showed more days of estrus compared with when they wereintact, the year before (44 days of estrus for all intact mares compared to 152 days for all OVXmares during June–September). However, complete comparison between years is not possiblesince daily teasing was for technical reasons not performed on all days of the experimental periodin all mares (see Table 3.1). Estrous behavior after ovariectomy did not follow the normal cyclicpattern seen in intact mares; for example, one OVX mare (mare B) showed 78 consecutive daysof estrus.


3.2. Paddock teasing (OVX mares only)

In July, two mares showed full estrus on all three teasing occasions (mares B and Z). Oneof these mares allowed mounting and ejaculation by the stallion (mare B). One mare showed noestrus the first time she was teased, but consequently showed full estrus during the next two teasingoccasions (mare M). She did not, however, allow the stallion to mount. Two of the mares showed nosymptoms of estrus (mares An and A). In November, only one mare showed full estrous signs (mareZ). She did not allow the stallion to mount. The other four mares showed no symptoms of estrus.

3.3. Gynecological findings

Gynecological findings of the intact mares were described previously (Hedberg et al., 2006;accompanying paper). All OVX mares had edema in the uterus when the ultrasound examinationwas performed in July, the day after the last “paddock teasing” was carried out, regardless ofthe mare having been mounted by the stallion or not. The average edema score was 1.8 (±S.D.0.3). A small amount of fluid was found in the mare that had been mounted by the stallion. Sinceedema was found in the uterus of all mares, ultrasound examination of the uterus was performedfor an additional 2 days. The edema decreased over the 2 days to 1.7 (±S.D. 0.3) (day 2 afterpaddock teasing) and 1.2 (±S.D. 0.4) (day 3 after paddock teasing). No fluid was found in anyof the mares during the last two examinations. In October–November, the mares had no edemain the uterus and the uterus of all mares was thin and flaccid.

3.4. Estradiol levels during frequent sampling (intact and OVX)

The mean (±S.D.) estradiol concentrations in the five intact mares during the frequent samplingperiods in estrus were 46.2 (±13.1) and 31.6 (±18.4) pmol/l on the saline treatment day and ACTHtreatment day, respectively. Estradiol concentrations (pre-treatment sample and samples 5 h post-saline/ACTH injection) in the OVX mares were close to the assay’s detection limit, with a meanestradiol level below the minimal detection concentration of the assay.

3.5. Effect of ACTH on hormone concentrations

3.5.1. CortisolOn the saline treatment day, there was a significant effect of time period, showing a diurnal

rhythm (P < 0.001). No overall difference between mare groups (intact and after OVX) was noted.However, a significant interaction between mare group (intact and after OVX) and time periodwas found, i.e. a difference in cortisol pattern on the saline treatment day between mare groups(P < 0.05). OVX mares had significantly lower cortisol levels in time period three (18:00–23:00 h)(P < 0.001), but higher cortisol levels in time period four (P < 0.05), compared with when intact(Fig. 3.1a). In the mares (intact and after OVX), cortisol values increased significantly as a resultof the ACTH injection in time periods two (P < 0.001) and three (P < 0.01), as compared withthe saline treatment day (i.e. difference between treatment days). Peak values (±S.D.) werereached in time period two in both mare groups (intact and after OVX). The highest averagelevels reached were 428 (±121) nmol/l when the mares were intact (2.5 h post-injection) and446 (±111) nmol/l after ovariectomy (2 h post-injection). There was no overall significant effectof mare group (intact and after OVX) in cortisol response to ACTH (i.e. difference betweentreatment days). In the ACTH cycle, no significant difference between mare groups (intact andafter OVX) was found (Fig. 3.1b).


Fig. 3.1. (a) Mean cortisol values on the saline treatment day in intact mares at estrus and after ovariectomy, accordingto sampling time. ***Significant at P < 0.001 and *significant at P < 0.05 (between mare groups). (b) Mean cortisol valueson the ACTH treatment day in intact mares at estrus and after ovariectomy, according to sampling time.

3.5.2. ProgesteroneOn the saline treatment day, an overall significant effect of mare group (intact and after OVX)

was found (P < 0.001), with OVX mares having significantly lower progesterone levels in timeperiod four than when intact (24:00–05:00 h) (P < 0.05) (Fig. 3.2a). As a result of the ACTH


Fig. 3.2. (a) Mean progesterone values on the saline treatment day in intact mares at estrus and after ovariectomy, accordingto sampling time. *Significant at P < 0.05 (between mare groups). (b) Mean progesterone values on the ACTH treatmentday in intact mares at estrus and after ovariectomy, according to sampling time. *Significant at P < 0.05 (between maregroups).

treatment, progesterone levels in both groups (intact and after OVX) increased significantly intime periods two (P < 0.001) and three (P < 0.05). Peak values (±S.D.) were reached in timeperiod two in both groups (intact and after OVX). The highest average levels reached were 1.2(±0.7) nmol/l when the mares were intact and 0.4 (±0.3) nmol/l after ovariectomy (1.5 h post-


injection). In the ACTH samples, OVX mares had significantly lower progesterone values at alltime periods as compared with when being intact (P < 0.05) (Fig. 3.2b). There was also an overallsignificant effect of mare group (intact and after OVX) in the difference between treatment days(saline versus ACTH) (P < 0.001), with intact mares showing a greater difference between salineand ACTH samples in time periods one, two, three and five (P < 0.01), as compared with afterovariectomy.

3.5.3. AndrostenedioneLevels of androstenedione in both the saline and ACTH sampling periods were significantly

higher in intact mares as compared to after ovariectomy at all time periods (P < 0.001) (Fig. 3.3aand b). Androstenedione concentrations increased significantly as a result of the ACTH treatmentin time period two in both intact mares and after ovariectomy (P < 0.001). Highest average level(±S.D.) reached was 783 (±330) pmol/l in intact mares (half an hour post-treatment). In OVXmares, high androstenedione levels were observed from 30 min to 2.5 h post-treatment, withthe highest average peak level (±S.D.) at 2 h post-treatment [461 (±273) pmol/l]. After ACTHtreatment, androstenedione values increased significantly more in time period two in intact marescompared to after ovariectomy (difference between treatment days; P < 0.05).

3.5.4. TestosteroneLevels of testosterone in both the saline and ACTH sampling periods were significantly higher

in intact mares as compared to after ovariectomy at all time periods (one to three) (P < 0.001)(Fig. 3.4a and b). Testosterone concentrations increased significantly as a result of the ACTHtreatment in time period two (P < 0.001). Highest average levels (±S.D.) were 200 (±78) pmol/l inintact mares (2 h post-treatment) and 154 (±51) pmol/l) after ovariectomy (2.5 h post-treatment).

3.5.5. EstradiolThe concentration of estradiol was only measured in selected samples from the OVX mares

(one pre-treatment sample and samples up to and including 5 h post-treatment). In the controlsamples of these mares, estradiol values ranged from <1 to 3 pmol/l, with the majority of sampleshaving a concentration of 1 pmol/l. Since the minimal detection limit of the assay was 2.1 pmol/land the intra-assay variation at 3.2 pmol/l was very high (45%), all samples were considered equalto null. In the ACTH samples, estradiol values ranged from <1 to 6 pmol/l in all samples analyzed,with the majority of samples having a concentration of 1–3 pmol/l. As for the control samples,the concentration of estradiol was below or near the detection limit of the assay.

4. Discussion

All of the OVX mares in the present study showed estrous behavior during the conventionalteasing procedure. This is in accordance with earlier studies (Asa et al., 1980a; Wesson andGinther, 1981; Hooper et al., 1993). As expected, the mares in the present study lost the normalpattern of estrous cyclicity after ovariectomy. In most species, estradiol is needed for estrousbehavior. However, in mares this is not the case (see Section 1). Progesterone produced by thecorpus luteum during the luteal phase in mares is considered to have an inhibitory effect on estrousbehavior, with estrous signs resulting if progesterone levels are low (Munro et al., 1979; Asa etal., 1980a, 1984).

The possible effect of adrenal steroid hormones on estrous behavior could be due to either adirect effect of androgens or alternatively as a result of a peripheral conversion into estrogens, since


Fig. 3.3. (a) Mean androstenedione values on the saline treatment day in intact mares at estrus and after ovariectomy,according to sampling time. ***Significant at P < 0.001 (between mare groups). (b) Mean androstenedione values onthe ACTH treatment day in intact mares at estrus and after ovariectomy, according to sampling time. ***Significant atP < 0.001 (between mare groups).

androgens are converted to estrogens by the enzyme complex aromatase (cytochrome P450 andNADPH-cytochrome reductase) (Conley and Hinshelwood, 2001). Watson and Hinrichs (1989)found no increase in estradiol with adrenal stimulation. The same result was found in the presentstudy, i.e. no increase in estradiol concentrations in the OVX mares after ACTH treatment. Fur-


Fig. 3.4. (a) Mean testosterone values on the saline treatment day in intact mares at estrus and after ovariectomy, accordingto sampling time. ***Significant at P < 0.001 (between mare groups). (b) Mean testosterone values on the ACTH treatmentday in intact mares at estrus and after ovariectomy, according to sampling time. ***Significant at P < 0.001 (between maregroups).


thermore, the estrone level, measured in one of the OVX mares (mare Z) (unpublished results), didnot increase after ACTH treatment. Therefore, these results indicate that adrenal androgens mightbe directly involved in the estrous behavior shown by ovariectomized mares. In fact, androgens doaffect the sexual behavior of mares. For example, in mares, testosterone producing granulosa-thecacell tumors cause disturbed behavior varying from an absence of estrus, to nymphomania-likebehavior or stallion-like behavior (Meagher et al., 1978). Also, estrous behavior was inducedby testosterone propionate treatment (Thompson et al., 1983). Studies in humans and primatesindicate that androgens may act both directly in the brain through androgen receptors and also,after local aromatization, via estradiol receptors [Bancroft, 2005 (review)]. In the present study,there was no evidence of an immediate effect of the increase in androgen plasma level on estrousbehavior.

In the present study, most of the OVX mares started to show estrous behavior during conven-tional teasing after being in close contact with the stallion and not before. The paddock teasingresulted in a higher stimulatory effect on the mares than the conventional method and thus insome way initiated the estrous behavior. An unexpected observation was that all of the mares hadedema in the uterus after paddock teasing was performed. Ultrasound examination of the mares’uteri was not performed prior to paddock teasing, since, after OVX, we found no reason to dosuch an examination. Only one OVX mare was mated by the stallion, so this was not a reasonfor edema formation in the other four mares. A marked difference between the uterine edema inJuly and November was also found, with no edema and a flaccid, thin uterus in November. In theOVX mares, estradiol levels remained below the detection level after ACTH treatment. Therefore,adrenal estrogens as a cause of the edema are unlikely. The incidence of uterine edema in OVXmares is peculiar and difficult to explain.

In the present study, the frequent blood samplings in intact and later ovariectomized mares wereperformed during the same period of different years, i.e. during the ovulatory period (Ginther,1992), to exclude seasonal variation. Mares, intact and after ovariectomy, showed a diurnal rhythmof cortisol as shown by the significant effect of time period on the saline treatment day (P < 0.001).However, the significant interaction between mare group and time period suggests ovariectomyaffected the diurnal rhythm, since season of year was the same. After ovariectomy, mares hadsignificantly lower cortisol levels in the evening, the time of the expected nadir in the diurnalrhythm of cortisol (James et al., 1970; Larsson et al., 1979; Irvine and Alexander, 1994), butsignificantly higher levels at night time. Ovariectomy was shown to decrease basal corticosteronelevels in the rat (Kitay, 1963). In the rhesus monkey, ovariectomy significantly decreased bothmean and peak basal cortisol levels (Smith and Norman, 1987). These effects of ovariectomy wereattributed to the stimulatory effect of estradiol on the HPA axis. For instance, estradiol treatmentgiven to OVX rats stimulated adrenal gland corticosterone secretion (Kitay, 1963). Estrous cyclestage has also been shown to affect basal plasma corticosterone levels in the rat, with higher peakcorticosterone levels at pro-estrus, when endogenous estradiol levels are increased (Critchlowet al., 1963; Carey et al., 1995; Atkinson and Waddell, 1997). The results of the present studyindicate a different effect in the mare as no estradiol in the mare (due to ovariectomy) resultedin a decrease of the trough levels of cortisol, but not a decrease in peak levels as found in the ratand rhesus monkey (Smith and Norman, 1987; Carey et al., 1995; Atkinson and Waddell, 1997).In fact, when diurnal peak levels occurred (period five), mares had higher cortisol levels afterovariectomy than when intact, although this difference was not significant. Asa et al. (1983) haveshown that mares had higher cortisol levels in diestrus, when endogenous estradiol levels werelow, compared to in estrus. They suggested that low cortisol levels in the mare may be necessaryfor normal follicle growth and LH release, since dexamethasone treatment reduced both follicle


size and inhibited ovulation (Asa and Ginther, 1982). The present results, with no difference inACTH response between mares, intact and OVX, do not support a stimulatory effect of estradiolon the HPA axis in the mare and contrasts with species such as the rat and sheep (Kitay et al.,1965; Viau and Meaney, 1991; Van Lier et al., 2003). Extrapolating data from other species isdifficult, however, due to differences in estrous cycles; e.g. the rat has approximately 30 h ofelevated estrogen levels compared with several days in the mare (Young, 1995).

Progesterone levels in intact mares compared to after ovariectomy were higher during nighttime on the saline treatment day and at all time periods on the ACTH treatment day (includingpre-treatment samples). Thus, some of the measured progesterone in the intact mares was mostlikely of ovarian origin. The progesterone levels in the ACTH cycle were higher in intact maresas compared to after ovariectomy already in the pre-treatment samples and ACTH did not have adirect effect on ovarian progesterone. In the mare, fluid of vascular follicles during estrus containsandrostenedione, epitestosterone and progesterone (Younglai, 1971).

Androstenedione concentrations were significantly higher in saline treated intact mares inestrus, compared to after ovariectomy, showing an ovarian contribution to androstenedione levelsduring estrus. Mares have been shown to have a pre-ovulatory rise in androstenedione (Younglai,1971; Munro et al., 1979). The theca interna cells in the ovary produce androgens (androstenedioneand testosterone) that are converted to estrogens (estrone and 17-�-estradiol) in the granulosa celllayer by the aromatase enzyme complex. The fact that androstenedione levels in the intact maresincreased significantly more as a result of the ACTH treatment than after ovariectomy, suggeststhat ACTH stimulated also the ovarian release of androstenedione. It has been demonstrated invitro that physiological concentrations of cortisol can directly increase the thecal cell productionof androstenedione in bovine ovary tissue (Spicer and Chamberlain, 1998). If this is true underin vivo conditions and in other species remains to be determined, but may give an explanation asto why, in the present study, androstenedione increased significantly more in the intact mares ascompared to after OVX.

Testosterone levels were also higher in intact mares at all time periods on both the saline andACTH treatment days, again showing an ovarian source of this hormone. Significant amountsof testosterone were measured in mares across the estrous cycle (estrus and luteal phase), withtestosterone peaks around ovulation and shortly before luteolysis (Silberzahn et al., 1978). Testos-terone was also found in mare follicular fluid at a concentration twice that found in peripheralplasma (Silberzahn et al., 1983).

5. Conclusion

The present study on intact and subsequently ovariectomized mares shows that basal pro-gesterone, androstenedione and testosterone levels in the mare are of both ovarian and adrenalorigin. Moreover, ACTH stimulated ovarian androstenedione production. Ovariectomy affectedthe basal cortisol pattern, by decreasing trough values, as opposed to peak values seen in otherspecies. Furthermore, endogenous estradiol levels at estrus did not have an enhancing effect onthe amount of cortisol produced upon ACTH stimulation, again contradictory to other species.Ovariectomized mares displayed estrous signs, without a normal cyclic pattern.

Acknowledgements

The authors wish to express their gratitude to Mari Wallbring, Carola Jansson and UlrikaMattsson for taking care of the mares, Asa Karlsson and Mari-Anne Carlsson for assistance with


the hormone analyses and to the Swedish Horse Racing and Totalisator Board (ATG) for financialsupport.

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