N&I

ELSEVIER

EFFECT OF STAGE OF ANESTRUS ON THE INDUCTION OF ESTRUS BY THE DOPAMINE AGONIST CABERGOLINE IN DOGS J.P. Verstegen, 1 K. Onclin, 1 L.D.M. Silva 1 and P.W. Concannon2 1Department of Small Animal Reproduction, College of Veterinary Medicine 2 University of Liege, 4000 Liege Belgium Department of Physiology, College of Veterinary Medicine Comell University, Ithaca, NY 14853 USA Received for publication: 7 A p r i l 1998 Accepted:24 August 1998 ABSTRACT Beagle bitches were administered the dopamine D 2 receptor agonist cabergoline in 3 groups of 5 animals each, starting on known days of the estrous cycle. Cabergoline treatment was started in either early anestrus (Days 93 to 108), mid-anestrus (Days 123 to 156), or late anestrus (Days 161 to 192) at doses of 5 ug/kg/d, per os, and was continued until the confirmation of induced proestrus or for 40 d. Reproductive parameters were compared with those in 5 control anestrous bitches (Days 90 to 150). In control bitches, the mean ( SEM) interval to the next proestrus (73 + 11 d) resulted in an interestrus interval (192 -4-9 d) similar to that of the previous cycles (196 + 11 d). In 14 of the 15 cabergoline-treated bitches, the next proestrus occurred within 4 to 30 d, was premature in early and mid-anestrous bitches and developed with low variability within groups. The resulting intervals to proestrus in bitches treated with cabergoline in early anestrus (20 2 d), mid-anestrus (14 3 d) and late anestrus (6 + 1 d) resulted in interestrus intervals in those groups of 131 5, 166 7 and 196 + 2 d, respectively. In response to treatment, interestrus intervals were reduced (P<0.05) and more synchronous (P<0.05) in early and mid-anestrus bitches, and were more synchronous (P<0.05) in late-anestrous bitches compared with those of control bitches or those of the previous cycle. Perovulatory estradiol and progesterone profiles of induced cycles in treated bitches were similar to those of spontaneous cycles in control bitches. Four of 5 control bitches and 12 of the 14 responding cabergoline-treated bitches became pregnant and produced normal litters. Plasma prolactin concentrations at Days 2 and 5 of treatment (0.3 0.1 ng/mL) and at the onset of proestrus shortly before the end of treatment (0.4 0.1 ng/mL) were lower (P<0.05) than those present in anestrus prior to treatment (1.7 0.6 ng/mL) or in control bitches. Prolactin was also low at the onset ofproestrus in control bitches (0.5 0.2 ng/mL). The results demonstrate that prolactinlowering doses of the dopamine agonist cabergoline can terminate the normal obligate anestrus in dogs, and that the effect occurs more slowly in early anestrus than in mid or late anestrus.
1999 by ElsevierScience Inc.

Key words: ovary, estrus, prolactin, dopamine

INTRODUCTION The ovarian cycle of the dog is unique among those of domestic animals in that there is an obligatory anestrus following the termination of the luteal phase. A similar anestrus also occurs following withdrawal of exogenous progestin (5,10). Canine anestrus varies in duration from 3 to
Theriogenology 51:597-611, 1999 1999 by Elsevier Science inc. 0093-691X/99/$-see front matter PII S0093-691X(99)00013-8

598

Theriogenology

10 mo among bitches, and can vary slightly or greatly among cycles within the same bitch (1,36). The luteal phase lasts about 2 mo. Normal interestrus intervals range from 5 to 12 mo and average about 7 mo in both fertile and nonbred cycles (8,34). Except in 1 or 2 breeds there is no evidence of pronounced seasonality, and bitches of most breeds may cycle any month of the year (1,3,5,6,36). Anestrus in the bitch is characterized by apparent ovarian inactivity, low levels of estrogen and elevated levels of FSH (6,16). The termination of anestrus appears to be associated with increased secretion of gonadotropin (6) and/or increased sensitivity to GnRH (39), but the mechanisms responsible for such changes are not known. The mechanisms controlling the reinitiation of folliculogenesis and its timing in dogs may differ from those proposed for other species, since initiation of the follicular phase is temporally dissociated from the preceding luteal phase. However, progesterone withdrawal is more than just permissive in dogs, in that prostaglandin-induced or dopamine agonist-induced premature luteolysis results in shortened interestrus intervals and possibly a shortened duration of anestrus (27,28,32). Elevations in prolactin are associated with seasonal anestrus in some species, and hyperprolactinemia is a cause ofamenorrhea in women (33). Administration ofprolactin-lowering doses of the dopamine agonist bromocriptine throughout the luteal phase and anestrus reduced the duration of both the luteal phase and of anestrus (25). Bromocriptine administered beginning in anestrus terminated anestrus within 50 d in one study (38) and within 28 d in another (6). Another ergot alkaloid, metergoline, reduced the interestrus interval when administered repeatedly in anestrus in one study (15) but not in another (24). However, both bromocriptine and metergoline are not selective dopamine agonists. Bromocriptine is a dopamine D 2 receptor agonist, but it also affects serotonergic and adrenergic receptors (22). Metergoline is primarily a serotonin receptor antagonist which has dopaminergic activity only at doses much higher than those required for antiserotonergic effects (19,22). Furthermore, both readily cross the blood-brain barrier. Therefore, studies using those compounds leave it unclear whether their ability to terminate anestrus in dogs is limited to action in the median eminence and pituitary, or involves higher centers, and whether the actions on neuroendocrine elements involve receptors other than dopamine receptors. The ergot alkaloid and dopamine agonist cabergoline has a bio-potency and D 2 receptor affinity greater that ofbromocriptine, a lower affinity for serotonergic 5 HT-2 receptors and a longer duration ofaction (11,12,22,31). Furthermore, cabergoline has fewer central nervous system effects than bromocriptine. Side effects of dopamine agonist therapy in dogs and humans include nausea and emesis, apparently secondary to central nervous system effects. At doses which terminate clinical pseudopregnancy in dogs, cabergoline has fewer and milder side-effects than bromocriptine (17,27,30). Moreover, in vivo, cabergoline does not affect striatal dopaminergic activity whereas bromocriptine does (11). Administration ofcabergoline was reported to terminate anestrus in dogs with an abnormally prolonged anestrus but to have no effect on cycle duration when administered during anestrus in normal cycles (17). Such observations suggested that additional studies were warranted to confirm and evaluate the potential role of dopaminergic pathways in the regulation of obligate anestrus in normal ovarian cycles of dogs. To better understand the process of obligate anestrus in dogs, it would also be helpful to know whether stage of anestrus affects response to dopamine agonist treatment. An effect of stage would suggest that in normal anestrus there are changes in sensitivity to endogenous dopamine or dopamine-regulated elements. Prior reports on termination of anestrus with ergot alkaloids in dogs have varied in the time course of the response and have not examined the possible effect of the stage of anestrus on the response (6,17,25).

Theriogenology

599

The present study was conducted 1) to determine the effects on interestrus intervals of prolactin-lowering doses of cabergoline administered at known days of the cycle during early, middle, or late anestrus in beagle bitches; 2) to evaluate the resulting proestrus and estrus in terms of timing, behavior, vaginal cytology, fertility and peripheral concentrations of estradiol and progesterone; and 3) to confirm prolactin suppression in relation to prolactin levels present in anestrus. MATERIALS AND METHODS Animals The study utilized 20 mature beagle bitches, 3 to 8 yr of age. All the animals were bom, housed and provided routine veterinary examinations in animal facilities at the University of Liege. All animal experimentation was conducted in accordance with the Guide for the Care and Use of Laboratory Animals promulgated by the US National Research Cotmcil. Health examinations were provided every 1 to 2 mo, and immediately prior to treatment. Bitches were housed in groups of 2 to 5 in indoor-outdoor runs (3.5 x 10 m), exposed to natural light (50.50 N latitude), provided water ad llbltmn and fed a commercial dry food once daily in amounts sufficient to maintain body weight. For each bitch, the cycle of treatment and the previous cycle had been characterized in terms of proestrous and estrous vaginal cytology, the day ofpreovulatory rise in progesterone, and plasma progesterone concentrations during the luteal phase, including those on the first day of metestrus. The days of the cycle were designated relative to the estimated day of the preovulatory LH surge, i.e., the day on which plasma progesterone increased rapidly above 1.0 to 2.0 ng/mL from relatively unchanged concentrations of 0.4 to 0.9 ng/mL observed on the previous 2 to 3 d (9,27). The interestrus interval was calculated as the period between the estimated days of preovulatory LH surges of successive cycles.
. . a

Treatments Three groups of 5 bitches each were administered cabergoline starting at known days after the day of the preovulatory LH surge. Treatment was initiated in either early anestrus (Days 93 to 108), mid-anestrus (Days 123 to 156) or late-anestrus (Days 161 to 192). The mean day of treatment initiation in these groups was 102 + 6, 142 + 13 and 188 -4-5 d of the cycle, respectively. The treated bitches and the 5 untreated bitches designated as the controls were monitored daily for signs of proestrus. The control bitches at the start of study ranged from 90 to 150 d post estrus. The first day of treatment was considered to be treatment Day 0. Treatment for all dogs was initiated in the same week in November of the same year to preclude potential effects of season among groups. Bitches were assigned to treatment and control groups semi-randomly to ensure that there were no differences between groups in mean age, body weight or body condition.

aHill's Canine Maintenance Scientific Diet, Hill's Animal Feeds, Brussels, Belgium

600

Theriogenology

Cabergolineb was administered daily at a dose of 5 ug/kg, per os, by application of the solution (50 ug/mL) directly into the mouth using a medicine dropper. Cabergoline administration was continued until 3 to 8 d after the onset of the first signs of proestrus and until the progress of proestrus was confirmed by both external signs (i.e., vulval swelling and serosanguinous discharge) and vaginal cytology (i.e., > 50% intermediate or superficial type epithelial cells), or until 40 d without signs ofproestrus. Observations Examinations, including evaluation of vulval swelling, vaginal discharge and collection of vaginal smears, were conducted twice weekly before treatment and daily throughout the period of cabergoline treatment in all bitches, including the untreated controls. Smears were obtained daily until the .onset . of metestrus and then every 3 Cto 4 d until after parturition. Vaginal smears were . . . . stained with a modified Wnghts-Glemsa stain and were evaluated for cell types and approximate percentages of epithelial cells, as previously described (7). The first day of induced proestrus was defined as the first day with evidence of vulval swelling or serosanguinous discharge, and vaginal smear containing erythrocytes and intermediate or superficial cells which represented >30% of epithelial cells present in increased numbers. The first day of estrus and first day of the cycle (Cycle Day 0) was defined endocrinologically as the estimated day of the preovulatory LH surge based on an abrupt increase in plasma progesterone above 1 ng/mL. The first day ofmetestrus (diestrus) was the day characterized by an abrupt 10 to 60% decrease in superficial cells and the reappearance of intermediate and parabasal cells in vaginal smears collected at the end of estrus (7). For the bitches in the 3 treatment groups and the control group the previous cycle and interestrus interval was well characterized, and parameters did not differ among groups. The preceding mean interestrus interval was 194 5 d; mean proestrus length was 8.4 0.5 d; and estrus length was 7.6 a: 0.2 d. Plasma Estrogen and Progesterone Blood samples (5 to 7 mL) were obtained by jugular venipuncture into heparinized tubes, centrifuged at 1000 x g for 15 min within 30 min of collection, and the multiple plasma aliquots (500 uL) stored at -20C until assay. Plasma samples were obtained from all animals once a week for 1 or more cycles prior to study. With few exceptions, samples were obtained from cabergolinetreated bitches every 2 to 4 d for 1 to 3 wk prior to treatment, and daily from start of treatment until 1 week after the onset ofmetestrus. Samples were obtained from control animals every 3 to 4 d or more frequently during anestrus, and daily during proestrus and estrus. Most of the samples were assayed for both estradiol and progesterone content, with attention to estradiol during treatment and to progesterone in late proestrus and estrus. In some instances, estradiol and progesterone were assayed in alternating samples in order to conserve samples. Values were aligned to Day 0 of

bGalastop, Vetem-Centralvet s.p.a., Milan, Italy. CHemacolor; Merck, Darmstadt, Germany.

Theriogenology

601

treatment and, retrospectively, also to the day of the ovulatory LH surge of the subsequent cycle. Mean values were determined for each day with values available for > 3 animals. Values for days with _<2 animals represented were included with values for the next day. Each mean included only a single value per animal. Progesterone concentrations were measured by radioimmunoassay using a commercial kitd as previously described (28) and validated for dog plasma (37). The sensitivity of the progesterone assay at 95% binding was 0.1 ng/mL, with inter- and intra-assay coefficients of variation of 5.1 and 8.8%, respectively. Concentrations of estradiol were determined using commercial assay reagents and methodology was validated for use with dog plasma (Verstegen, unpublisheddata). Validation was based on detection of physiological changes, which were determined in the present study and which were similar to those reported by others during canine proestrus and estrus (8,23,26), and on evaluation for parallelism and recovery of added estradiol. Serial dilutions of estradiol dissolved in human calibrator serum, phosphate buffer, estradiol-free canine plasma and serum were assayed along with serial dilutions of normal canine plasma. All curves were parallel (P>0.82) but the estradiol dilution curves in canine plasma and serum were displaced from that of the human calibrator standard. Therefore, samples were assayed as recommended by the manufacturer but using estradiol standard curves prepared by dilution of estradiol in estradiol-free canine plasma. In addition, in each assay, estradiol-free aliquots of each sample were also assayed as sample blanks to correct for any sample-specific plasma effect. Estradiol-free plasma was prepared by mixing 1 mL plasma with 1 mL Dextran-T70-coated charcoal solution (1 gm Norit A charcoal, 0.25 gm Dextran T70, 70 mL of 0.05 M phosphate buffered saline, pH 7.6). After incubation for 20 min at 4C and centrifugation at 3000 x g for 20 min at 4C, the supernatant was collected and used for assay. Assay steps included incubation of 200-uL sample with 100 uL of antiserum for 2 h at room temperature, addition of 100 uL 1-125 estradiol conjugate and incubation for 1 h, addition of 1 mL precipitation solution and, after 10 min, centrifugation at 3000 x g, followed by decantation of supematant and counting of precipitate in a gamma spectrometer. The sample blank if different from the standard blank value (range of difference, 1 to 19 pg) was subtracted from the sample value extrapolated from the standard curve. The addition of 20 and 50 pg ofestradiol to each of 3 plasma samples (4, 22, and 45 pg/mL) resulted in an assayed recovery of 18 4 and 46 6 pg/mL, respectively. The sensitivity of the estradiol assay at 95% binding was 2 pg/mL, with intra- and inter-assay coefficients of variation of 4 and 9%, respectively. Plasma Prolactin Prolactin content was assayed in the plasma samples obtained at Days 0, 2 and 5 of treatment and at the onset ofproestrus in the treated bitches, and in samples obtained at Days 0, 2, 5 and 21 of study and at the onset of proestrus in control bitches. This was done to confirm prolactin suppression in treated bitches and to compare concentrations during anestrus and early proestrus.
e

dDPC Progesterone-Coat-a-Count Diagnostic Product Inc., Humbeek, Belgium. eDPC Double Antibody Estradiol Radioimmunoassay, DPC, Humbeek, Belgium.

602

Theriogenology

Prolactin was measured using ~acommercial canine prolactin immunoenzymometric assay and as described by the manufacturer-and recently validated (29). The sensitivity of the prolactin assay at 95% binding was 0.25 ng/mL. The intra- and inter-assay coefficients of variation were 5 and 8%, respectively. Mating and Pregnancy Bitches in estrus were mated naturally on the first day of acceptance of the male and every other day thereafter until metestrus, except for 2 control bitches. The latter were inseminated with fresh or frozen semen at 3 and 5 d after the estimated day of the preovulatory LH peak, and both became pregnant. Pregnancy was detected and monitored by ultra-sonographyg at 20, 30 and 40 d after the last mating as previously described (40). All means are reported + standard error of the mean (SEM). Differences in ovarian cycle parameters within groups were determined by paired or unpaired t-tests or by Chi-square tests of equality of variance, and those between treatment groups were determined by one-way analysis of variance and LSD (least significant difference) tests or by Chi-square tests of variance (20,35). RESULTS The onset of proestrus and of estrus in cabergoline-treated bitches occurred earlier during study than in control bitches, with the exception of 1 experimental bitch. The onset ofproestrus in the cabergoline-treated bitches occurred within 4 to 30 d compared with 48 to 105 d in control bitches. In each treatment group, the interval from start of treatment to onset of proestrus was shorter (P<0.05) and less variable (P<0.05) than in the control group (Table 1). In early and mid anestrous bitches, cabergoline treatment resulted in interestrus intervals which were shorter (P<0.05) than in control bitches or in previous cycles. In late-anestrous bitches, cabergoline treatment resulted in interestrus intervals that were less variable (P<0.05) but not shorter than in control bitches or in previous cycles (Table 1). The 1 cabergoline-treated early-anestrous bitch which did not respond to treatment was repeatedly observed to vomit following administration of treatment. Treatment was stopped after 40 d and the results for this animal were not included in mean results for that group. No other side effects were observed, except for apparent apathy and reduced food intake in 2 bitches during the first 4 days of treatment. In each of the 14 cabergoline-treated bitches which responded to treatment, erythrocytes were observed in vaginal smears beginning at 4 to 7 d of treatment, were variable in amounts, and were either present continuously (n=9) or intermittently (n=5). Early proestrus smears in treated bitches typically had more debris and leucocytes than those in control bitches but were otherwise similar to those in controls, as regards erythrocytes and epithelial cell changes. Differences in vaginal cytology were not quantified, and were assumed to be due to the earlier than normal occurrence of proestrus. Among cabergoline-treated bitches, the interval to onset ofproestrus based on vulval swelling and comification of vaginal smears varied with the stage of the cycle at the start of treatment

fDPC, Canine Prolactin Immulite, DPC, Humbeek, Belgium. gModel 480 Scanner B, Pie Medical, Maastricht, Holland.

Theriogenology
Table 1.

603

Mean (+ SEM) intervals and durations of reproductive responses and resulting interestrus intervals associated with termination of anestrus by oral administration of cabergoline (5 ug/kg/d) in beagle bitches Cabergoline-Treated Bitches Control Anestrous bitches n=5 90-150 NA 73 11 c NA 81 26 d 8.4 0.9 9.0 0.8 22 2 4/5 4.8 0.4

Parameters Dogs treated Day of cycle (range) Dogs responding (n) Treatment onset to proestrus (days) Duration of treatment (days) Treatment onset to progesterone rise (days) Duration of proestrus (days) Duration of estrus (days) Metestrus progesterone (ng/mL) Dogs pregnant (n) Litter size Interestrus intervals Prior cycle Treatment cycle

Early anestrus n=5 93-108 4/5 20 3 a 26 3 a 29 2 a 8.5 0.6 8.3 0.8 20 2 3/4 f 4.5 0.6

Middle anestrus n=5 123-156 5/5 14 3 a 18 3 b 21 3 b 7.3 + 0.9 9.0 0.8 21 2 5/5 5.3 1.0

Late anestrus n=5 161-192 5/5 6 1b 10 2 c 10 2 c 6.6 0.8 8.0 0.7 20 2 4/5 3.8 0.5

192 13 131 5 a

195 15 166 7 b

191 8 196 2 c'e

196 11 192 9 d'e

NA = Does not apply abcd Means with different superscripts are different (P<0.05) based on LSD test, Dunnet's test, or Chi-square test of variance.

Chl-square test of variance. fA fourth bitch developed pyometra with no evidence of pregnancy.

604

Theriogenology

(Table 1). Onset ofproestrus in bitches treated during late anestrus occurred within 3 to 9 d, and earlier (P<0.05) during treatment than in bitches treated during mid-anestrus (6 1 versus 14 4- 3 d). In bitches treated during early anestrus, onset of the induced proestrus occurred within 12 to 24 d and tended to occur the latest in the course of treatment (20 + 3 d), albeit not significantly later (P= 0.14) than the 6 to 20 d in bitches treated in mid-anestrus. However, the resulting number of days of cabergoline treatment until confirmed proestrus differed (P<0.05) among the 3 treatment groups (Table 1). There were no differences between cabergoline-treated animals and controls in the durations ofproestrus or estrus, in plasma progesterone at the start ofmetestrus, or in fertility (Table 1). Each of the 14 responding cabergoline-treated bitches ovulated, based on plasma progesterone results, and was mated; 12 of the 14 became pregnant and produced normal litters of 4 to 8 pups each. In the early-anestrus treatment group, 1 bitch which ovulated developed a pyometra and was subsequently ovariectomized. There was no evidence of pregnancy at surgery. Mating or insemination resulted in pregnancy in 4 of the 5 control bitches. Mean concentrations of estradiol and progesterone were evaluated in relation to the day of cabergoline treatment (Figure 1). Concentrations of both hormones were low at the start of treatment in early, mid, and late anestrous bitches. Estradiol increased in each group, but the time of the increase appeared to vary depending on the stage ofanestrus at treatment. Estradiol increased above pretreatment values within 1 to 3 d after the start of treatment in late-anestrous bitches but not until 6 to 12 d in mid-anestrous bitches, and 12 to 18 d in early anestrous bitches. Changes in mean concentrations of estradiol and of progesterone, examined in relation to the day of the subsequent estimated day of the LH surge, were similar in cabergoline-treated and control groups (Figure 2). The increase in estradiol occurred shortly before the onset ofproestrus in treated and control bitches. In each group there was typically a low and sometimes transient elevation in estradiol concentration 1 to 4 d prior to the observed onset of proestrus. The pre-proestrus elevation in estradiol in individual bitches averaged 14.2 4- 1.5 pg/mL and was significant (P<0.05) in comparison with pretreatment values of 2.3 4- 0.6 pg/mL. Preovulatory peak concentrations ofestradiol ranged from 28 to 47 pg/mL in control animals and from 27 to 62 pg/mL in the 14 cabergoline-treated bitches in which anestrus was prematurely terminated, and did not differ among groups. Mean concentrations of progesterone at the onset of metestrus were similar in treated and control animals (Table 1) and were similar to those of previous cycles (data not shown). Progesterone concentrations between Days 10 and 20 of the cycle varied extensively within and between bitches in each group. Peak levels.of progesterone ranged from 22 to 60 ng/mL in control bitches, and 27 to 58 ng/mL in the 14 responding cabergoline-treated bitches. There were no differences among treated or control groups in mean peak estradiol or progesterone concentrations. Plasma prolactin concentrations at the start of treatment were similar in early, mid and late anestrous bitches (1.8 + 0.9, 1.6 4- 0.7, and 1.6 + 0.8 ng/mL, respectively) and did not differ from those of control anestrous bitches at the start of the study (1.7 + 0.1 ng/mL) or at Days 2, 5 and 21 (1.8 4- 0.7, 2.0 + 0.6, 1.9 4- 0.2 ng/mL, respectively). In all 14 bitches which tolerated cabergoline treatment, prolactin concentrations at Day 2 of the study (0.4 4- 0.1 ng/mL) and Day 5 of the study (0.3 4- 0.1 ng/mL) were subsequently reduced (P<0.05) compared with the pretreatment values, and were lower (P<0.05) than in control bitches on those days. Prolactin concentrations shortly before

Theriogenology
60-

605
-40

{,
3o" 20-

-3o

- 10

o
IO

O 20 30 40 50

6OCAB Mid AE
=

-40

5040302010OI0 0

T .~
20

T Tv T~w TT

-30

- 20

~L'
40

&

- I0

30

50

60-] CAB l ~ t e A E 5o-

-40

l
/ ~ IT T T
I0

-30

302010 o 0 i
20

-20

0
30

50

Days
Figure 1. Mean (4- SEM, n = 3 to 5) plasma concentrations of estradiol (o) and progesterone (e) in groups of beagle bitches administered cabergoline (5 ug/kg/day, po) beginning in early anestrus (n = 4), mid-anestrus (n = 5), or late anestrus (n = 5). Error bar not shown when smaller than symbol size.

606 ESTRADIOL
6080-]CAB early AE
~ 40"
50

Theriogenology PROGESTERONE ~ 4oE CAB early A / ~ "] _ ~ 1

201

) 20"L
O. . . . . .. . . . .

T T'--I

~ !.0t
O__ --. , . I

-40
80

-30

-20

40

l0

-40 -30 -20 40

lO
.,.

20

1cAB~
,

]i:tcAB~.~
~,o1
~" o |

~
o

-'~
-30

,"~--

-40

-30

-20

-10

10

-40

-20

-10

10

20

C~a~

~=~ C ~ ~ate~

~o1
f ,01
0 10 -40 -30 -20 -10 0 10 20

~ 2o~
-40 -30 -20 10

~6o
4

,o7 Controls
1 0 ~
20
O

=,oq Controls 40
T =o 30 ~ 20t
~ ,o
0 Z= , --I II I / I

-40

-30

-20

-I0

10

-40

-30

-20

-10

10

20

Days
Figure 2.

Days

Mean (4- SEM, n = 3 to 5) plasma concentrations of estradiol (left panels) and progesterone (right panels), relative to the estimated day of the subsequent preovulatory LH surge (Day 0), in groups of 4 to 5 beagle bitches administered cabergoline (5 ug/kg/d, po) beginning in early anestrus (upper panels), mid-anestrus (second panels), or late anestrus (third panels), and in 5 untreated control bitches (bottom panels). The mean (4- SEM) time of the initiation of treatment in treated bitches was, respectively, at 294- 2, 214- 3 and 104- 2 days prior to Day O. The day of LH surge was estimated as the time of the initial increase in progesterone above 1 ng/mL. Error bar not shown when smaller than symbol size.

Theriogenology

607

the end of treatment, i.e., at the onset ofproestrus (0.3 + 0.1 ng/mL), were likewise lower (P<0.05) than those of control bitches around that time, i.e., at Day 21 of the study. Prolactin at the onset of proestrus in control bitches (0.5 -4-0.3 ng/mL) was also reduced (P<0.05) in comparison with that present previously during anestrus. DISCUSSION The present results confirm and extend observations that daily administration of prolactinlowering doses of a dopamine agonist can hasten the termination of anestrus in dogs as reported previously with administration of bromocriptine (6,25,38). These results also demonstrate the efficacy of the dopamine agonist cabergoline in this regard. The rapid, 3- to 9-d response of bitches treated with cabergoline in late anestrus is more rapid than previously reported but is similar to the observation that cabergoline administration for 5 to 15 d resulted in proestrus in bitches treated clinically for abnormal and prolonged anestrus at 7 to 19 mo after the last estrus (17). The termination of anestrus in bitches treated in early and mid anestrus in our present study is in contrast with the previously reported absence of treatment effect for beagle bitches treated with similar doses for 14 d beginning at 4 to 6 mo after estrus (17). The difference in results could be due to differences in formulation, mode of oral administration, or duration of administration. The treatment period required to induce proestrus in many of the early and mid-anestrous bitches in the present study was longer than 14 d. The cycles resulting from cabergoline-induced termination of anestrus were normal in most respects, since the resulting proestrus and estrus parameters, fertility and serum hormone profiles were very similar to those of control bitches. The increased debris and leucocytes in vaginal smears obtained during early proestrus in the cabergoline-treated bitches may have been due to earlier than normal proestrous endocrine changes. It is not uncommon to find leucocytes in vaginal smears in early proestrus of some normal cycles (7). The rapid initial appearance of erythrocytes in vaginal smears of cabergoline-treated bitches possibly represents uterine diapedesis in response to early increases in estradiol, and may be similar to that which occurs in early proestrus of normal cycles (7). If so, then increases in estradiol below the level of detection in the early and mid anestrous bitches may be physiologically important. The efficacy of dopamine agonists in terminating anestrus in the bitch may involve suppression ofprolactin secretion. When bromocriptine administration induced premature proestrus in 4 of 5 anestrous bitches, only in the 1 nonresponding bitch did the treatment fail to suppress prolactin secretion (6). The time course ofprolactin suppression was not fully characterized in the present study. However, the data obtained demonstrate reduced prolactin concentrations within 2 d of treatment and at the onset of proestrus shortly before the end of treatment, whereas prolactin was not reduced at comparable times in the untreated controls. The reduced prolactin concentrations during treatment confirm that the doses of cabergoline used in this study suppress circulating concentrations ofprolactin in anestrous bitches, as previously reported for pregnant and for diestrous bitches (29,30). The findings of reduced prolactin concentrations observed at the onset ofproestrus in control bitches suggests that in normal cycles prolactin concentrations decline near the end of
anestrus.

608

Theriogenology

The termination of anestrus in this study is presumed to be due to effects of cabergoline on dopaminergic receptors. Cabergoline is considered to function as a D2-dopamine agonist, possessing little effect on other neuroendocrine receptors (22), and to have a lower affinity for serotonin and adrenergic receptors than bromocriptine (31). It seems unlikely that the effect of cabergoline in affecting the canine estrous cycle is directly on the GnRH neurons. In rats, dopamine administered into the third ventricle inhibited LH release in one study (14), and cabergoline had no effect on LH secretion in vivo or in vitro in others (11-13). In dogs, cabergoline administered during the luteal phase had no effect on LH (29). Because of the D2-receptor specificity of cabergoline, the results suggest that the efficacy of the other ergot-alkaloids in terminating anestrus also involves effects on dopaminergic neurons or dopaminergic receptors. That is presumably the case with bromocriptine (6,25). Metergoline is primarily an anti-serotonin agent at low doses but can have direct dopaminergic effects at high doses (22). Metergoline treatment induced premature proestrus in dogs in one study (15) but not in another (24). In instances where metergoline appears to have been effective (15), the effect may have been indirect and mediated by dopaminergic or other neurons, as observed in the serotonergic control ofprolactin secretion in rats (18,19). Or, since high doses were used, it may have involved a direct dopaminergic effect. In the present study, the observed prolactin suppression by cabergoline was presumably due to a direct effect on lactotroph D2 receptors (31 ). Effects proximal to the pituitary and median eminence, perhaps at the hypothalamic level, cannot be discounted, although in vivo studies in rats suggest a limited ability of cabergoline to cross the blood-brain barrier and bind to striatal D 2 receptors (11). Whether the mechanism mediating induction of proestrus-like follicle growth by dopamine agonists involves a change in gonadotropin secretion or efficacy remains to be determined. Whether the concurrent declines in prolactin secretion or in plasma concentrations ofprolactin are required for the response is also not known. The suppression ofprolactin alone is reportedly not sufficient to terminate anestrus in bitches. The administration of the serotonin antagonist metergoline to anestrous bitches suppressed prolactin to levels observed during bromocriptine treatment but, unlike bromocriptine, failed to shorten anestrus (24). It is possible that the effect of dopamine agonists in anestrous bitches involves both prolactin suppression and effects on additional dopaminergic pathways. Or, perhaps the extent and duration of prolactin are critical and have varied among studies. If the termination of anestrus is dependent on prolactin suppression, it is unrelated to the role ofprolactin as a luteotropin, since progesterone levels in all bitches were already low at the start of treatment. The anti-fertility effects ofprolactin in humans with hyperprolactinemia have been considered to possibly involve inhibitionof ovarian aromatase, antagonism of FSH action on follicle cells (21), and suppression of hypothalamic GnRH pulsatility and LH pulse amplitude (4). There were few apparent side effects except for the vomiting response in 1 dog. This is similar to the low (4 to 25%) incidence of vomiting observed in bitches administered similar oral doses for treatment of pseudopregnancy (2). The greater potency and fewer side effects of cabergoline compared to bromocriptine in dogs are possibly related to the fact that cabergoline has a greater affinity and specificity for the D 2 dopamine receptor (22) and more limited access to the central nervous system (11).

Theriogenology

609

The effect of the stage of cycle on the duration of cabergoline treatment required to cause an increase in estradiol and terminate anestrus suggests that during normal anestrus there are progressive changes in hypothalamic, pituitary or gonadal function that affect the response to dopamine agonist treatment. Relevant in that regard is the observation that pituitary sensitivity to GnRH increases during the course of anestrus in dogs (39). More complete evaluation of bitches administered dopamine agonist in early vs late anestrus is needed to elucidate the mechanism of action and the endogenous factors that affect the response. In summary, the present study demonstrates that treatment with the dopamine agonist cabergoline can be used to shorten interestrus intervals in dogs, that it results in normal estrus and fertile ovulation, and that the duration of treatment required depends on the stage ofanestrus. However, the mechanism of action and the role prolactin suppression in the response have not been determined. REFERENCES 1. 2. 3. 4. Andersen AC, Simpson ME. The Ovary And Reproductive Cycle of the Dog (Beagle). Los Altos, CA: Geron-X Press, 1973. Arbeiter K, Brass W, Ballabio R, Jochle W. Treatment ofpseudopregnancy in the bitch with cabergoline, an ergoline derivative. J Small Anim Pract 1988;29:781-788. Christie DW, Bell ET, Horth CE, Palmer RF. Peripheral plasma progesterone levels during the canine oestrous cycle. Acta Endocrinol (Copenh) 1971;68:543-550. Cohen-Becker JR, Selmanoff M, Wise PM. Hyperprolactinemia alters the frequency and amplitude of pulsatile luteinizing hormone secretion in the ovariectomized rat. Neuroendocrinology 1986;42:328-333. Concannon PW. Reproduction in the dog and cat. In: Cupps PT (ed), Reproduction in Domestic Animals. New York: Academic Press Inc, 1991;517-554. Concannon PW. Biology ofgonadotrophin secretion in adult and prepubertal female dogs. J Reprod Fertil 1993;47:3-27. Concannon PW, DiGregorio GB. Canine vaginal cytology. In: Burke T (ed), Small Animal Reproduction and Infertility. Philadelphia: Lea and Febiger, 1986;96-111. Concannon PW, Hansel W, Visek W. The ovarian cycle of the bitch: plasma estrogen, LH and progesterone. Biol Reprod 1975;13:112-121. Concannon PW, McCann JP, Temple M. Biology and endocrinology of ovulation, pregnancy and parturition in the dog, J Reprod Fertil 1989;39(Suppl):3-25. Concannon PW, Meyers-Wallen VN. Current and proposed methods for contraception and termination of pregnancy in dogs and cats. J Am Vet Med Assoc 1991;198:1214-1225. Di Salle E, Giudici D, Omati G, Carfagna N, Caccia C, Moretti A, Pegrassi L, Rossi A. Prolactin lowering and central dopaminergic activities of the new ergoline derivative FCE 21336 in vivo and in vitro in the rat. Proc Symp Euro Neuroendo Assn (Basel) 1984; 122 abstr. Di Salle E, Ornati G, Giudici D. A comparison of the in vivo and in vitro duration ofprolactin lowering effect in rats of FCE 21336, pergolide and bromocriptine. J Endocrinol Invest 1984;7(Suppl 1):30-32. Di Salle E, Ornati G, Giudici E, Ragazzi P. Effect of the new ergoline derivative FCE 21336 on prolactin and LH secretion in the rat. J Endocrinol Invest 1983;6(Suppl 1):6 abstr. Gallo R, Drouva S. Effect of intraventricular infusion of catecholamines on luteinizing hormone release in ovariectomized rats. Neuroendocrinology 1979;29:14 abstr.

5. 6. 7. 8. 9. 10. 11.

12.

13. 14.

610
15.

Theriogenology
Handaja-Kusuma PS, Tainturier D. Comparison of induction of oestrus in dogs using metergoline, metergoline plus human chorionic gonadotrophin, or pregnant mares' serum gonadotrophin. J Reprod Fertil 1993;47(Suppl):363-370. Jeffcoate IA. Endocrinology of canine anoestrus. J Reprod Fertil 1993;47(Suppl):69-76. Jochle W, Arbeiter K, Post K, Ballabio R, D'Ver AS. Effects on pseudopregnancy, pregnancy and interoestrous intervals of pharmacological suppression of prolactin secretion in female dogs and cats. J Reprod Fertil 1989;39(Suppl): 199-207. Kordon C, Drouva S, Martinez-Escalara G, Weiner R. Role of classic and peptide neuromedicators in the neuroendocrine regulation of luteinizing hormone and prolactin. In: Knobil E, Neil J (eds), The Physiology of Reproduction. New York: Raven Press, 1995;1621-1681. Krulich L, McCann SM, Mayfield MA. On the mode of the prolactin release-inhibitingaction of the serotonin receptor blockers metergoline, methysergide, and cyproheptadine. Endocrinology 1981;108:1115-1124. Lentner G. Statistical methods. In: Anonymous, Introduction to Statistics, Statistical Tables and Mathematical Formulae. Basle: Ciba Geigy, 1982;199-203. Molitch MG. Disorders of the pituitary lactotroph. In: Adashi E, Rock JA, Rosinwelks Z (eds), Reproductive Endocrinology, Surgery and Technology, Vol. 2. New York: Lippincott-Raven Press, 1996;1303-1324. Muller EE, Nistico G. Neurotransmitter regulation of the anterior pituitary. In: Muller E, Nistico G (eds), Brain Messengers and the Pituitary. San Diego: Academic Press,

16. 17.

18.

19.

20. 21.

22.

1989;488-513.
23. Nett TM, Akbar AM, Phemister RD, Holst PA, Reichert LE Jr, Niswender GD. Levels of luteinizing hormone, estradiol and progesterone in serum during the estrous cycle and pregnancy in the Beagle bitch. Proc Soc Exp Biol Med 1975;148:134-139. Okkens A, Kooistra H, Dieleman S, Bevers M. Dopamine agonistic effects as opposed to prolactin concentrations in plasma as the influencing factor on the duration of anestrus in bitches. J Reprod Fertii 1997;5 l(Suppl):55-58. Okkens AC, Bevers M, Dieleman S, Willemse S. Shortening of the interestrus interval and lifespan of the corpus luteum of the cyclic dog by bromocriptine treatment. Vet Q 1985;7:173-176. Olson PN, Bowen RA, Behrendt M, Olson JD, Nett TM. Concentrations of reproductive hormones in canine serum throughout late anestrus, proestrus and estrus. Biol Reprod 1982;27:1196-1206. Onclin K, Silva LDM, Donnay I, Verstegen J. Luteotrophic action ofprolactin in dogs and the effects ofa dopamine agonist, eabergoline. J Reprod Fertil 1993;47(Suppl):403-409. Onclin K, Silva LDM, Verstegen JP. Termination of unwanted pregnancy in dogs with the dopamine agonist, cabergoline, in combination with a synthetic analog of PGF2alpha, either cloprostenol or alphaprostol. Theriogenology 1995;43:813-822. Onclin K, Verstegen JP. In vivo investigation of luteal function in dogs: effects on cabergoline, a dopamine agonist, and prolactin on progesterone secretion during mid-pregnancy and -diestrus. Dom Anita Endocrinol 1997; 14:25-38. Post K, Evans LE, Jochle W. Effects of prolactin suppression with cabergoline on the pregnancy of the bitch. Theriogenology 1988;29:1233-1243.

24.

25.

26.

27. 28.

29. 30.

Theriogenology
31.

611

32.

33.

34. 35. 36. 37.

38.

39.

40.

Rains CP, Bryson HM, Fitton A. Cabergoline: a review of its pharmacological properties and therapeutic potential in the treatment ofhyperprolactinemia and inhibition of lactation. Drugs 1995;49:255-280. Romagnoli SE, Camillo F, Cela M, Johnston SD, Grassi F, Ferdeghini M, Aria G. Clinical use of prostaglandin F2 alpha to induce early abortion in the bitch: serum progesterone, treatment outcome and interval to subsequent oestrus. J Reprod Fertil 1993;47(Suppl):433-438. Sauder SE. Abnormal patterns of pulsatile luteinizing hormone secretion in women with hyperprolactinemia and amenorrhea: responses to bromocriptine. J Clin Endocrinol Metab 1984;59:941-948. Shille VM, Stabenfeldt GH. Current concepts on reproduction of the dog and cat. Adv Vet Sci Comp Med 1980;24:211-243. Snedecor GW, Cochran WG. Statistical Methods. Ames IA: Iowa State University Press, 1980. Sokolowski JH, Stover DG, VanRavenswaay F. Seasonal incidence ofestrus and interestrous interval for bitches of 7 breeds. J Am Vet Med Assoc 1977;171:271-273. Srikandakumar A, Ingraham RH, Ellsworth M, Archbald LF, Liao A, Godke RA. Comparison of a solid-phase, no-extraction radioimmunoassay for progesterone with an extraction assay for monitoring luteal function in the mare, bitch and cow. Theriogenology 1986;26:779-793. van Haaften B, Dieleman SJ, Okkens AC, Bevers MM, Willemse AH. Induction ofoestrus and ovulation in dogs by treatment with PMSG and/or bromocriptine. J Reprod Fertil 1989;39(Suppl):330-331. van-Haaften B, Bevers MM, Van-Den-Brom WE, Okkens AC, Van-Sluijs- F J, Willemse AH, Dieleman SJ. Increasing sensitivity of the pituitary to gnrh from early to late anoestrus in the beagle bitch. J Reprod Fertil 1994; 101:221-225. Verstegen JP, Silva LDM, Onclin K, Donnay I. Echocardiographic study of heart rate in dog and cat fetuses in utero. J Reprod Fertil 1993;47(Suppl): 175-180.