Animal Reproduction Science 101 (2007) 134–144

The effects of a low dose of cabergoline on inductionof estrus and pregnancy rates in anestrous bitches�

Umut Cirit a,∗, Suleyman Bacinoglu a, I. Taci Cangul b,Huriye Horoz Kaya c, Muzaffer Tas d, Kemal Ak a

a Istanbul University, Faculty of Veterinary Medicine, Department of Reproduction andArtificial Insemination, 34320 Avcilar, Istanbul, Turkey

b Uludag University, Faculty of Veterinary Medicine, Department of Pathology, 16059 Bursa, Turkeyc Istanbul University, Faculty of Veterinary Medicine, Department of Obstetrics and Gynaecology,

34320 Avcilar, Istanbul, Turkeyd Dicle University, Faculty of Veterinary Medicine, Department of

Obstetrics and Reproduction, 21280 Diyarbakir, Turkey

Received 2 May 2006; received in revised form 11 August 2006; accepted 5 September 2006Available online 9 September 2006

Abstract

This is the first report of successful induction of normal estrus and ovulation in breeder bitches with as a lowdose as 0.6 �g/kg/day of cabergoline formulation marketed for use in women. Sixty-one pure breed bitchesfrom various breeds were used in the study at their already determined periods of anestrus. Twenty-fourdogs formed the control group, while 37 bitches were administered with two different doses of cabergoline(recommended dose group, n = 10, 5 �g/kg/day and low dose group, n = 27, 0.6 �g/kg/day). Induced estrusrates and mean treatment and proestrus durations of dogs in these two dose groups were compared.

At the second phase of the study, the effects of 500 IU human chorionic gonadotropin (hCG) administeredon days 1 and 3 of estrus induced by the low dose of cabergoline, on the duration of behavioral estrus,ovulation rates, pregnancy rates and the number of offspring were investigated. For this purpose, the dogswith signs of proestrus (22/27) following the treatment in the low dose group were assigned into twosubgroups. Five hundred IU of hCG (Pregnyl, Organon, Turkey) was intramuscularly administered to eightof these dogs [low dose (hCG+) group] on days 1 and 3 of estrus. The remaining 14 dogs were not treatedwith hCG [low dose (hCG−) group]. An aqueous solution of cabergoline (Dostinex, Pharmacia, Italy) wasorally administered until 2 day after the onset of proestrus or for a maximum of 42 days. Blood sampleswere taken daily from all treatment and 11 control bitches during the first five days of behavioral estrus tomeasure progesterone concentrations. In the recommended dose and low dose groups, estrus was induced

� The present work was supported by the Research Fund of Istanbul University. Project No. 183/15012004.∗ Corresponding author. Tel.: +90 212 473 70 70x17264; fax: +90 212 473 72 41.

E-mail addresses: ucirit@istanbul.edu.tr, ucirit@gmail.com (U. Cirit).

0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.anireprosci.2006.09.005

U. Cirit et al. / Animal Reproduction Science 101 (2007) 134–144 135

between days 8–45 and 4–48 (mean: 23.63 ± 14.33 and 24.41 ± 14.31 days), in the ratio of 80.0 and 81.5%,respectively (p > 0.05). In both dose groups, post-treatment interestrous intervals were significantly shorterthan both those of the control group and their own pre-treatment interestrous intervals (p < 0.05).

Ovulation rates, pregnancy rates and mean number of offspring delivered by the dogs in the recommendeddose, low dose (hCG−), low dose (hCG+) and control groups were found to be similar (p > 0.05). However,the mean duration of behavioral estrus of the dogs in the low dose (hCG+) group was found to be significantlylonger compared to dogs in all other groups (p < 0.05). In both dose groups, no correlation could be foundbetween the anestrus stages and treatment durations (p > 0.05).

Shortly, it has been concluded from the study that (1) normal and fertile estrus can be induced moreeconomically in bitches during different stages of anestrus using as a low dose of 0.6 �g/kg of cabergolineformulation marketed for use in women, and that (2) hCG injections on days 1 and 3 of the estrus inducedby this method has no positive effects on the ovulation rates, pregnancy rates and the number of offspringper pregnancy.© 2006 Elsevier B.V. All rights reserved.

Keywords: Bitch; Dog; Estrus induction; Anestrus; Cabergoline; hCG

1. Introduction

Dogs are monestrus and experience an obligate anestrus of 2–10 months following the lutealphase having an average duration of about 75 days (Bouchard et al., 1991b). Endocrine changesleading to the termination of anestrus and the start of a new follicular phase in the bitch arestill not completely understood. Owners of pure bred bitches with normally long interestrousintervals often request to shorten interestrous intervals, so the number of litters per year can beincreased (Gobello and Corrada, 2001). Several protocols with exogenous gonadotropins, syn-thetic estrogens, GnRH agonists and dopamine agonists (DA) have been tried for induction ofestrus in anestrus bitches (Bouchard et al., 1991a; Inaba et al., 1998; Horoz et al., 2000; Gunayet al., 2004). Use of gonadotropins for induction of estrus can result in low fertility and abnormalluteal function (Horoz et al., 2000; Concannon, 2002). The use of DA to prematurely termi-nate anestrus typically results in a physiological estrus and spontaneous ovulation (Verstegenet al., 1999; Gobello et al., 2002). Moreover, satisfactory results could not be obtained withtreatments based on PMSG (eCG), GnRH, diethylstilbestrol (DES) and gonadotropin agonistadministrations when compared to cabergoline in terms of practical application and adverseeffects for stimulating estrus and ovulation in bitches (Gunay and Soylu, 2001; Rota et al.,2003).

Dopamine agonists such as cabergoline, bromocriptine or metergoline are ergot derivated alka-loids that exert an anti-prolactinergic effect. However, both bromocriptine and metergoline arenot selective DA. Metergoline is primarily a serotonin receptor antagonist, which has dopamin-ergic activity and shortens interestrous interval only at doses much higher than those requiredfor antiserotonergic effects (Gobello and Corrada, 2001). Bromocriptine often causes emesis inearly stages of treatment, can cause anorexia and is not approved for veterinary use (Concannon,2002). Cabergoline has a high specificity for D2 receptors, long specific activity on pituitary lac-totroph cells and has fewer central nervous system effects than bromocriptine (Rains et al., 1995).Cabergoline (Galastop, Vetem, Milan, Italy), marketed as a veterinary drug in some Europeancountries, successfully induce fertile estrus in most bitches but can be cost prohibitive in theother countries, where these drugs are not readily available for veterinary use (Kutzler, 2005).Cabergoline (Dostinex, Pharmacia) is also used in the treatment of hyperprolactinemia in women

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and can be purchased easily. However, its use for the induction of estrus in heavy breed dogs maynot be cost effective.

No widely used, effective, easily applicable and cost effective protocol has been found yet forthe induction of estrus in bitches. In addition, as far as we know, there are no comparative studiesin which different doses of cabergoline have been used for induction of estrus. Five �g/kg/daydose of cabergoline has been reported to have mild side effects in dogs (Gobello et al., 2001).Therefore, the aim of this field study was to investigate the estrus-inducing effect of a quite lowerdose (0.6 �g/kg/day) of aqueous cabergoline solution in bitches. In addition, the effects of humanchorionic gonadotropin (hCG) administered on days 1 and 3 of the estrus induced by low dosesof cabergoline on the ovulation rates, pregnancy rates and the number of offspring per pregnancywere investigated.

2. Materials and methods

2.1. Animals and experimental design

The study was carried out between June and October of the same year in a breeder kennel inorder to minimize the effects of feeding, management and environmental conditions. Sixty-onepure breed bitches of various breeds were used in the study. The animals were 2–8-year-old,weighing 15–45 kg, and were at 97–280 days after the beginning of their previous proestrus. Theday of anestrus was determined based on the breeding records. All bitches were clinically examinedat the beginning of the study and anestrus condition was confirmed based on vulvar observationand on the absence of intermediate or superficial cells in vaginal smears. Bitches were housed inindividual indoor–outdoor runs, exposed to natural light, provided with water ad libitum and feda commercial dry food once daily in amounts sufficient to maintain body weight. The first day oftreatment was considered to be treatment day 0. Treatment for all dogs was initiated in the sameweek in June to preclude potential effects of season among groups. Bitches were assigned into thetreatment and control groups in a manner not to have any difference between their mean anestrusdurations. Dogs from different breeds were distributed to groups as proportionally as possible tothe dog numbers (Table 1). The interestrous interval was defined as the number of days betweenproestrus onsets. Pre-treatment interestrous intervals were calculated from the breeding records.

Thirty-seven dogs on days 104–280 (mean: 182.2 ± 51.53 day) after the beginning of their lastproestrus formed the treatment group. In order to determine the effect of low dose of cabergolineon the induction of estrus, in the first phase dogs in the treatment group were distributed intotwo dose groups. Dogs in the recommended dose group (n = 10), which were on days 104–267of anestrus (mean: 186.9 ± 57.35 day), were treated with cabergoline (Dostinex, Pharmacia) atdose of 5 �g/kg/day. Dogs in the low dose group (n = 27), which were on days 110–280 (mean:180.4 ± 50.27 day) of anestrus, were treated with 0.6 �g/kg/day cabergoline. The rates of inducedestrus and the mean time interval between the beginning of treatment and proestrus of the dogs inthese two dose groups were compared. In the second phase, the effects of 500 IU hCG (Pregnyl,Organon) administered on the first and third days of estrus induced by the low dose of cabergolineon the ovulation rates, pregnancy rates and the number of offspring were investigated. For thispurpose, the dogs with signs of proestrus (22/27) following the treatment in the low dose groupwere assigned into two subgroups. Five hundred IU of hCG was administered intramuscularly toeight of these dogs [low dose (hCG+) group] on days 1 and 3 of estrus. The remaining 14 dogswere not treated with hCG [low dose (hCG−) group]. Twenty-four bitches on days 97–272 (mean:194.7 ± 46.58 day) of their anestrus formed the control group and were kept untreated. The average

U. Cirit et al. / Animal Reproduction Science 101 (2007) 134–144 137

Table 1Distribution of breeds in groups and induced estrus and pregnancy rates of dogs in the treatment groups (lowdose + recommended dose groups) according to breeds

Breed Dog numbers Treatment groups(low dose + rec. dose)

Rec. dose Low dose(hCG−)

Low dose(hCG+)

Con. Inducedestrus

Pregnancy

German Shepherd dog 3 6 2 7 10/11 8/10Labrador Retriever 2 5 2 6 8/9 5/8Irish Setter 2 1 1 2 2/4 2/2Kangal dog 1 1 1 2 3/3 3/3Rottweiler 1 2 0 2 1/3 1/1Akbash dog 1 1 0 2 2/2 1/2Border Collie 0 1 1 1 1/2 1/1German Shorthaired Pointer 0 1 1 1 2/2 2/2Belgian Shepherd Malinois 0 1 0 1 1/1 0/1

Rec., recommended; Con., control.

anestrus periods at the beginning of the study were 179.5 ± 47.91, 182.5 ± 58.95, 186.9 ± 57.35and 194.7 ± 46.58 days (p > 0.7) for low dose (hCG−), low dose (hCG+), recommended doseand control groups, respectively.

Chemical structure of cabergoline is N-[3-(dimethylamino)propyl]-N-[(ethylamino) carbonyl]-6-(2-propenyl)ergoline-8�-carboxamide (Persiani et al., 1994), so it has free amine groups in themolecule. Cabergoline is freely soluble in alcohol, slightly soluble in 0.1 M hydrochloric acidand not practically soluble in water. But, in our pilot trial, we observed that Dostinex tabletstotally and easily dissolved in distilled water at room temperature and pH value of this aqueoussolution (10 �g cabergoline/mL) was 5.5–6.0. Dostinex tablets used in this study contain 0.5 mgof cabergoline and inactive ingredients consist of leucine and lactose. As known, organic com-pounds with free amine groups become soluble in water by the quaternization of the molecules.Solubility of Dostinex tablet is probably due to the either quaternized cabergoline in the tabletor the interaction of leucine molecule as inactive ingredient, which contains polar free aminegroups and its isoelectric point pH 5.98, with cabergoline to form the highest hydrogen bindingcapacity.

Persiani et al. (1994) found that relative bioavailability of tablets versus aqueous solution ofcabergoline was 99% (geometric mean with the 90% confidence intervals of 68–144%). Theirresults indicated that the pharmacodynamics and relative bioavailability of cabergoline are notinfluenced by formulation, as tablet or solution (Persiani et al., 1994). As it would be difficult toadjust the proper dosage in tablet form, we dissolved the cabergoline tablets in distilled water.The bitches were weighed at the beginning of the trial. The total cabergoline amount to beused and number of Dostinex tablets to be dissolved were calculated daily. All the tablets werepowdered in a glass mortar, were dissolved in warm (37 ◦C) distilled water to make a solutioncontaining 10 �g active substance per milliliter and homogenized by using a magnetic stirrer. Thissolution was prepared fresh and administered orally using a medical dropper within 15 min afterpreparation.

Cabergoline administration was continued until 2 day after the onset of the first signs ofproestrus, where the progress of proestrus was confirmed by both external signs (i.e., vul-var swelling and serosanguinous discharge) and vaginal cytology (i.e., >50% intermediate or

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superficial type epithelial cells), or until 42 day without signs of proestrus. The time intervalbetween the beginning of drug applications and the first day of proestrus was determined as thetreatment interval.

All bitches were examined twice daily (09:00 a.m. and 04:30 p.m.) for the presence of vulvarswelling and serosanguinous vaginal discharges until the onset of proestrus. Vaginal smears wereobtained every fourth day until the onset of proestrus and daily during proestrus and estrusuntil the second day of metestrus. Vaginal smears were stained with Giemsa (Merck, Darmstadt,Germany) and were evaluated for cell types and approximate percentages of epithelial cells,as previously described (Concannon and DiGregorio, 1986). Female dogs were placed togetherwith an active male dog every second day from day 4 of proestrus and every day from the daythey showed interest in male dogs, and their behaviors were observed. A bitch was consideredto be in estrus when showing tail deflection, allowing the male to mount and initiating coitus.Estrus was confirmed by epithelial exfoliative cytology (>90% cornification) also. The first day ofmetestrus (diestrus) was the day characterized by refusal of male and was confirmed by an abrupt10–60% decrease in superficial cells and the reappearance of intermediate and parabasal cells invaginal smears collected at the end of estrus (Verstegen et al., 1999). For the determination of thebehavioral estrus period, bitches were mated naturally on the first day of acceptance of the maleand every day thereafter until refusal. Breeder male dogs with proven fertility, pre-determinedby the company, were used in all matings. One male reserved for breeding in the low dosagegroup and one male reserved for the recommended dose group could not mate with their partners,although they mounted on females. As the breeding program of the kennel was already designed,no other males could be used and therefore artificial insemination was performed by fresh sementaken from these males. In both cases the bitches became pregnant. According to the matingschedule of the company, four control bitches were not mated and excluded from the statisticalanalysis regarding the pregnancy rates.

Blood samples were taken from all animals in the treatment group and 11 control bitches dailyduring the first five days of estrus. Blood samples (7–10 mL) were obtained by jugular venipunc-ture, centrifuged at 3000 × g for 15 min within 30 min of collection, and multiple plasma aliquots(500 �L) were stored at −20 ◦C until assay. Progesterone (P4) concentrations were measured byradioimmunoassay using a commercial kit (DSL-3900, DSL, Texas, USA). The sensitivity of theassay was 0.12 ng/mL. Ovulation was considered to have occurred if P4 concentrations ≥5 ng/mLwere observed or if pregnancy occurred (Wildt et al., 1979). Following the mating, the dogs wereobserved until delivery and the litter size was determined. Pregnancy rate was determined as theratio of delivered bitches to those showing behavioral estrus signs and mated.

2.2. Statistical analysis

Pregnancy, ovulation and induced estrus rates were analyzed by the Chi-square test. Themean proestrus and behavioral estrus durations, the time interval between the beginning oftreatment and proestrus, the interestrous intervals and the serum P4 levels were analyzed bymeans of one-way ANOVA and significance controls between the groups were carried out bythe Duncan-test. The pre- and post-treatment interestrous intervals of bitches in the low dose,recommended dose and control groups were compared with independent samples t-test. The cor-relation between the anestrus stage and treatment duration in the low dose and recommendeddose groups were analyzed by Pearson Correlation Test. All statistical analyses were carried outusing the SPSS program V. 10.0 for Windows. Results are presented as mean ± standard deviation(mean ± S.D.).

U. Cirit et al. / Animal Reproduction Science 101 (2007) 134–144 139

Table 2Comparison of data obtained from dogs in the low dose (0.6 �g/kg/day), recommended dose (5 �g/kg/day) and controlgroups

Groups n Duration of proestrus (day) Experiment onset toproestrus (day)

Dogs responding

Low dose 27 7.9 ± 2.34a 24.4 ± 14.31a 81.5% (22/27)a

Recommended dose 10 7.4 ± 1.85a 23.6 ± 14.33a 80.0% (8/10)a

Control 24 8.8 ± 2.24a 49.5 ± 25.14b NA

Within columns, means with no common letters (a and b) are statistically different (p < 0.001). NA, does not apply.

3. Results

Twenty-two of 27 dogs (81.5%) treated with low dose of cabergoline showed proestrus betweendays 4 and 48 (mean: 24.4 ± 14.31 days) from the beginning of the experiment. Proestrus wasdetermined in 12 of these dogs between days 4 and 22, and in the remaining 10 dogs betweendays 30 and 48. Proestrus started in three dogs following the end of 42 days of cabergolinetreatment (at days 45, 46 and 48). Eight of 10 dogs (80.0%) treated with recommended dose ofcabergoline showed estrus signs between days 8 and 45 (mean: 23.6 ± 14.33 days). Proestrussigns were observed in six of these eight dogs between days 8 and 26, while on the third dayfrom the end of treatment in the remaining two (day 45). Signs of proestrus were seen in 16–113(49.5 ± 25.14) days after the initiation of treatment in the control group. Induced estrus rates andmean time interval between treatment and proestrus in dogs treated with low and normal dose ofcabergoline were found to be very similar (Table 2, p > 0.05). In all cabergoline responded bitches,proestrus progressed to behavioral estrus. No statistically significant difference was found amongthe mean proestrus durations of dogs in the low dose, the recommended dose and the controlgroups (Table 2, p > 0.05). The mean time intervals between the beginning of the treatment andproestrus of dogs in the treatment groups were found to be significantly shorter compared to thosein the control group (Table 2, p < 0.001). The post-treatment interestrous intervals of dogs in thetreatment group were significantly shorter than in the control group (p < 0.05). While the averagepre- and post-treatment interestrous intervals in the control group were similar (p > 0.05), in bothtreatment groups, post-treatment estrus intervals were significantly shorter than the pre-treatmentestrus intervals (Table 3, p < 0.05). Ovulation rates, pregnancy rates and the mean number ofoffspring of bitches in the recommended dose, low dose (hCG−), low dose (hCG+) and thecontrol groups were found to be similar (p > 0.05, Table 4). The mean behavioral estrus durationsof dogs in the low dose (hCG+) group were seen to be significantly longer (p < 0.05), and theirmean serum P4 concentrations on day 5 of estrus were found to be numerically lower (p > 0.05),

Table 3Comparison of pre- and post-treatment interestrous intervals within each group (rows) and comparison of pre- and post-treatment interestrous intervals between groups (columns)

Groups Interestrous interval (day)

Pre-treatment Post-treatment

Low dose 254.8 ± 95.04a 203.3 ± 59.09b

Recommended dose 262.3 ± 54.64a 200.8 ± 56.80b

Control 250.1 ± 60.55a 244.2 ± 60.90a

Within rows and columns, means with no common letters (a and b) are statistically different (p < 0.05).

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Table 4Some reproductive results of bitches showing symptoms of behavioral estrus in recommended dose, low dose (hCG−),low dose (hCG+) and control groups (mean ± S.D.)

Groups

Recommended dose Low dose (hCG−) Low dose (hCG+) Control

n = 8 n = 14 n = 8 n = 24

Duration of proestrus (day) 7.4 ± 1.85a 8.1 ± 2.14a 7.4 ± 2.72a 8.8 ± 2.24a

Duration of behavoiralestrus (day)

12.9 ± 2.03a 11.2 ± 4.79a 17.5 ± 5.24b 10.5 ± 2.43a

Plasma P4 at the fifth day ofestrus (ng/mL)

11.1 ± 5.75a 11.7 ± 6.78a 7.7 ± 2.62a 13.6 ± 7.29a*

Litter size 5.8 ± 1.94a 5.9 ± 2.17a 5.5 ± 2.59a 5.2 ± 2.75a

Bitches ovulated 87.5% (7/8)a 92.9% (13/14)a 100.0% (8/8)a 100.0% (11/11)a*

Bitches pregnant 75.0% (6/8)a 78.6% (11/14)a 75.0% (6/8)a 85.0% (17/20)a

Within rows, means with no common letters (a and b) are statistically different (p < 0.05).* Data of 11 dogs from the control group are included in the analysis.

compared to dogs in all other groups (Table 4). Two of eight bitches responding to cabergolinein the low dose (hCG+) group exhibited an estrus longer than 21 day, and one of them did notovulate and conceive.

In order to investigate the role of anestrus period on treatment duration, dogs in the low dosegroup were classified retrospectively according to their anestrus stages. The average treatmentdurations in the early (days 110–120; n = 3), middle (days 123–159; n = 5), late (days 164–198;n = 5) and very late (days 207–280; n = 9) phases of anestrus were 31.7 ± 12.58, 20.8 ± 11.78,16.2 ± 6.76 and 28.6 ± 13.93 days, respectively (p > 0.05). In the recommended dose group, asthe number of dogs in each anestrus stage was limited, statistical analysis could not be performed.Both in the low dose and the recommended dose groups, no correlation could be found betweenthe anestrus stage and treatment duration (r = −0.408 and −0.425, respectively; p > 0.05). Nodetectable side effects such as vomiting were found in any of the dogs both treated with recom-mended dose and low dose of cabergoline.

4. Discussion

Although DA can provide a successful physiological tool to improve canine reproductionperformance, the precise mechanism of action of DA in induction of estrus is not yet completelyunderstood. Formerly, it was believed that the induction of estrus in bitches by DA was the result ofthe suppression of prolactin (PRL) secretion. Contrarily, treatment with the serotonin antagonistmetergoline did not result in shortening of the interestrous interval, although there was a decrease inplasma PRL concentration (Okkens et al., 1997). These findings have raised the question whether areduction in plasma PRL concentration or some other dopamine-agonistic influence is the criticalfactor in the shortening of the estrus cycle. Recently, it has been shown in bitches receiving a lowdose of bromocriptine that there was significant shortening of the interestrous interval without adecrease in plasma PRL concentration (Beijerink et al., 2003). Serum follicle stimulating hormone(FSH) concentrations are increased throughout much of canine anestrus while luteinizing hormone(LH) concentrations are low except near the end of anestrus (Concannon, 1993). However, bothLH and FSH appear to be follicotropic in the dog as administration of pharmacologic doses

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of either LH or FSH alone induces estrus (Shille et al., 1984). It has been demonstrated thatshortening of the interestrous interval by bromocriptine in a dose that also lowers plasma PRLconcentration is associated with an increase in plasma FSH concentration without a concomitantincrease in plasma LH concentration (Kooistra et al., 1999).

Our results confirm and extend observations that daily administration of a prolactin-loweringdose (5 �g/kg/day) of cabergoline (Post et al., 1988) induces normal and fertile estrus in dogs asreported previously (Verstegen et al., 1999; Gobello et al., 2002; Gunay et al., 2004). Our dataalso demonstrate the efficacy of a quite lower dose (0.6 �g/kg/day) than recommended dose ofcabergoline in this regard. Induced estrus rates and mean treatment days in both dose groups werevery similar to those reported for Beagle bitches (83.3% and 23.5 ± 3.2 day, respectively) treatedwith 5 �g/kg/day cabergoline (Galastop) on the 120–480 (mean 291 ± 45) days of their anestrus(Rota et al., 2003). In both dose groups, post-treatment interestrous intervals were significantlyshorter than both those of the control group and their own pre-treatment interestrous intervals(p < 0.05).

Since lactation in the bitch is under prolactin control, suppression of lactation has been used as abioassay for dose determinations with prolactin inhibitors (Mayer and Schutze, 1973; Jochle et al.,1987; Post et al., 1988). Jochle et al. (1987) showed that 1.25 �g/kg/day oral dose of cabergolinewas ineffective in suppressing or terminating lactation in bitches. In our study, PRL levels werenot measured. Therefore, it is difficult to conclude whether 0.6 �g/kg/day cabergoline inducedestrus by lowering the PRL levels or by another mechanism.

Cabergoline (Galastop) is only marketed in some European countries as a veterinary drug fortreatment of pseudopregnancy in dogs (Concannon, 2002). Therefore, in most countries, provisionof this product can be money or time consuming. On the other hand, cabergoline (Dostinex) whichis used in the treatment of hyperprolactinemia in women can be purchased easily. Results of ourstudy clearly showed that fertile and normal estrus could be induced in dogs at a lower cost byusing 0.6 �g/kg/day of cabergoline formulation marketed for use in women. Induced estrus rates,mean response times and fertilities of dogs in both of the low and recommended dose groupswere very similar. The only difference determined with respect to the treatment efficacy was thatthe estrus in the recommended dose group seemed somewhat more synchronized. Proestrus signswere seen between 8 and 26 day post-treatment in six of eight dogs in the recommended dose groupthat responded to cabergoline. On the other hand, proestrus was seen in 12/22 bitches betweendays 4 and 22 in the low dose group, and between day 30 and 48 in the remaining 10 dogs. Thisdata suggested that there would be a relation, even not induced estrus rate, between cabergolinedose and response time. Results of Gunay et al. (2004) also support this hypothesis. In their study,they treated the dogs on determined days of anestrus with cabergoline at 6 �g/kg/day orally andachieved proestrus in 12 of 13 dogs within 14 days. A disadvantage of induction of estrus usingcabergoline is that, this method of induction of estrus may require over 30 days of treatmentbefore the onset of proestrus (Kutzler, 2005) depending on the stage of anestrus (Verstegen et al.,1999; Gunay et al., 2004). It is likely to shorten the treatment period by using higher doses ofcabergoline than recommended doses. However, in order to demonstrate this, comparative studiesassessing the side effects also are needed. In studies with cabergoline, some side effects such asvomiting have been reported in the rate of 0–25% in dogs (Arbeiter et al., 1988; Gobello et al.,2001; Gunay et al., 2004). No vomiting or other side effects have been determined in any of thedogs in both dose groups in our study.

It has been suggested that treatment duration of cabergoline necessary to terminate anestrusis shorter in late anestrus period than early anestrus period (Verstegen et al., 1999; Gunay et al.,2004). In the present study, a similar tendency was observed in the low dose group, but it could

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not be statistically proven (31.7 ± 12.58 and 16.2 ± 6.76 days for early and late anestrus stages,p > 0.05). The cause may be the difference in dog numbers per anestrus period or the high standarddeviations. Unexpectedly, the average treatment period in the very late anestrus (days 207–280)was slightly longer than of dogs in the late anestrus period (days 164–198) (16.2 ± 6.76 versus28.6 ± 13.93; respectively, p > 0.05).

Provoked estrus in bitches with the administration of gonadotropin and estrogen preparationsfailed to result in reliable and reproducible data (Van Haften et al., 1988). In a study usinghuman menopausal gonadotropin (hMG), proestrus signs were not marked in 30% of the dogsand lasted only 2–3 days (Wanke et al., 1997). Similarly, compared to spontaneous estrus, 18%of bromocriptine-induced estruses have been characterized by shorter duration, less pronouncedvaginal bleeding and clinically fewer expressed estrus symptoms (Zoldag et al., 2001). We havefound that the cycles resulting from cabergoline-induced termination of anestrus were normal inboth dose groups, since the resulting proestrus and estrus parameters, serum progesterone levelsat the fifth day of estrus, fertility and litter sizes were very similar to those of control bitches.

Another aim of our study was the determination of the effects of 500 IU hCG administered ondays 1 and 3 of estrus induced by low dose of cabergoline. Ovulation rates, pregnancy rates andthe mean litter size in hCG receiving and not receiving bitches were similar. However, the meanbehavioral estrus duration of dogs in the low dose (hCG+) group was found to be significantlylonger (p < 0.05), and furthermore, their mean serum P4 concentrations on day 5 of estrus werefound to be numerically lower (p > 0.05), compared to dogs in all other groups. Two of eightbitches responding to cabergoline in the low dose (hCG+) group exhibited an estrus longer than21 day. The average proestrus duration in the low dose (hCG+) group were very similar to othergroups. Therefore, it can be concluded that in our study hCG had an effect by extending estrusinto diestrus rather than shortening proestrus. Even if the two dogs that had prolonged estrusare not included in the statistics, the average estrus duration in the low dose (hCG+) group waslonger than in the low dose (hCG−) and control groups (p < 0.05). These data prove that theeffect of hCG on estrus duration is not coincidental. It has been reported that two of eight bitchesreceiving 500 IU hCG in late metergoline induced proestrus exhibited a prolonged proestrusafter injection of hCG. In the remaining six bitches, the average duration of proestrus and estruswas 4.8 ± 1.7 and 6.5 ± 2.8 days, respectively, which was significantly shorter (p < 0.05) than inbitches not given hCG (Kusuma and Tainturier, 1993). These data show that when administeredto dogs responding DA during late proestrus or early estrus, hCG affects the proestrus and estrusduration (by prolonging or shortening), and such effects can show variation according to the timeof administration.

As some of the breeds used in the study were limited in number, it has been not possible toshow whether the estrus inducing effect of cabergoline has variation according to the breeds ornot. Nonetheless, when breeds with three or higher number of dogs are assessed regardless ofthe dose, it can be suggested that German Shepherd, Labrador Retriever and Kangal breeds werevery responsive to cabergoline (Table 1).

5. Conclusion

Shortly, it has been concluded from the study that (1) normal and fertile estrus can be inducedmore economically in bitches during different stages of anestrus, using a low dose as 0.6 �g/kg/dayof cabergoline marketed for use in women, and (2) hCG injections on days 1 and 3 of the estrusinduced by this method has no positive effects on the ovulation rates, pregnancy rates and thenumber of offspring per pregnancy.

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References

Arbeiter, K., Brass, W., Ballabio, R., Jochle, W., 1988. Treatment of pseudopregnancy in the bitch with cabergoline, anergoline derivative. J. Small Anim. Pract. 29, 781–788.

Beijerink, N.J., Dieleman, S.J., Kooistra, H.S., Okkens, A.C., 2003. Low doses of bromocriptine shorten the interestrousinterval in the bitch without lowering plasma prolactin concentration. Theriogenology 60, 1379–1386.

Bouchard, G., Youngquist, R.S., Clark, B., Concannon, P.W., Braun, W.F., 1991a. Estrus induction in the bitch using acombination diethylstilbestrol and FSH-P. Theriogenology 36, 51–65.

Bouchard, G., Youngquist, R.S., Vaillancourt, D., Krause, G.F., Guay, P., Paradis, M., 1991b. Seasonality and variabilityof the interestrous interval in the bitch. Theriogenology 36, 41–50.

Concannon, P.W., 1993. Biology of gonadotrophin secretion in adult and prepubertal female dogs. J. Reprod. Fertil. Suppl.47, 3–27.

Concannon, P.W., 2002. Methods for induction of estrus in dogs using gonadotropins. GNRH or dopamine ago-nists. In: 27th Wsava Congress, Granada, Spain, October 3–6, http://www.vin.com/proceedings/Proceedings.plx?CID=WSAVA2002&PID=2680&Category=425.

Concannon, P.W., DiGregorio, G.B., 1986. Canine vaginal cytology. In: Burke, T. (Ed.), Small Animal Reproduction andInfertility. Lea and Febiger, Philadelphia, pp. 96–111.

Gobello, C., Corrada, Y., 2001. Estrus induction with dopaminergic agonists in the bitch: a review. Commun. Theriogenol.1 (1) (Doc 2).

Gobello, C., Castex, G., Corrada, Y., 2002. Use of cabergoline to treat primary and secondary anestrus in dogs. J. Am.Vet. Med. Assoc. 220, 1653–1654.

Gobello, C., de la Sota, R.L., Goya, R.G., 2001. Study of the change of prolactin and progesterone during dopaminergicagonist treatments in pseudopregnant bitches. Anim. Reprod. Sci. 66, 257–267.

Gunay, A., Gunay, U., Soylu, M.K., 2004. Cabergoline applications in early and late anoestrus periods on German Shepherddogs. Rev. Med. Vet. 155, 557–560.

Gunay, A., Soylu, M.K., February 2001. A study of induction of oestrus and ovulation in bitches in anoestrus period. In:34. Jahrestagung Physiologie und Pathologie der Fortplanzung und 26. Veterinar-Humanmedizinische Gemeinschaft-stagung, Gie�en, p. 127.

Horoz, H., Sonmez, C., Baran, A., Ak, K., 2000. Effect of serum gonadotrophin (PMSG) administration on oestrus andovulation in anestrous bitches. J. Fac. Vet. Med. Istanbul Univ. 26, 49–59.

Inaba, T., Tani, H., Gonda, M., Nakagawa, A., 0hmura, M., Mori, J., Torii, R., Hamada, H., Sawada, T., 1998. Induction offertile estrus in bitches using a sustained-release formulation of a GnRH agonist (Leuprolide Acetate). Theriogenology49, 975–982.

Jochle, W., Ballabio, R., di Salle, E., 1987. Inhibition of lactation in the beagle bitch with the prolactin inhibitor cabergoline(FCE 21336): dose response and aspects of long-term safety. Theriogenology 27, 799–810.

Kooistra, H.S., Okkens, A.C., Bevers, M.M., Popp-Snijders, C., van Haaften, B., Dieleman, S.J., Schoemaker, J., 1999.Bromocriptine-induced premature oestrus is associated with changes in pulsatile secretion pattern of FSH in beaglebitches. J. Reprod. Fertil. 117, 387–393.

Kusuma, P.S., Tainturier, D., 1993. Comparison of induction of oestrus in dogs using metergoline, metergoline plushuman chorionic gonadotrophin, or pregnant mares’ serum gonadotrophin. J. Reprod. Fertil. Suppl. 47, 363–370.

Kutzler, M.A., 2005. Induction and synchronization of estrus in dogs. Theriogenology 64, 766–775.Mayer, P., Schutze, E., 1973. Effect of 2-Br-�-ergokryptine (CB 154) on lactation in the bitch. Experimentia 29, 484–

485.Okkens, A.C., Kooistra, H.S., Dieleman, S.J., Bevers, M.M., 1997. Dopamine agonistic effects as opposed to prolactin

concentrations in plasma as the influencing factor on duration of anoestrus in bitches. J. Reprod. Fertil. Suppl. 51,55–58.

Persiani, S., Sassolas, G., Piscitelli, G., Bizollon, C.A., Poggesi, I., Pianezzola, E., Edwards, D.M., Strolin Benedetti,M., 1994. Pharmacodynamics and relative bioavailability of cabergoline tablets vs. solution in healthy volunteers. J.Pharm. Sci. 83 (10), 1421–1424.

Post, K., Evans, L.E., Jochle, W., 1988. Effects of prolactin suppression with cabergoline on the pregnancy of the bitch.Theriogenology 29, 1233–1243.

Rains, C.P., Bryson, H.M., Fitton, A., 1995. Cabergoline. A review of its pharmacological properties and therapeuticpotential in the treatment of hyperprolactinaemia and inhibition of lactation. Drugs 49, 255–279.

Rota, A., Mollo, A., Marinelli, L., Gabai, G., Vincenti, L., 2003. Evaluation of cabergoline and buserelin efficacy foroestrous induction in the bitch. Reprod. Domest. Anim. 38, 440–443.

144 U. Cirit et al. / Animal Reproduction Science 101 (2007) 134–144

Shille, V.M., Thatcher, M.J., Simmons, K.J., 1984. Efforts to induce estrus in the bitch, using pituitary gonadotropins. J.Am. Vet. Med. Assoc. 184, 1469–1473.

Van Haften, B., Dieleman, S.J., Okkens, A.C., Bevers, M.M., Willemse, A.H., 1988. Induction of a fertile oestrus inbeagle bitches with PMSG and bromocriptine. In: 11th International Congress on Animal Reproduction and ArtificialInsemination, Dublin, June 26–30, p. 463.

Verstegen, J.P., Onclin, K., Silva, L.D.M., Concannon, P.W., 1999. Effect of stage of anestrus on the induction of estrusby the dopamine agonist cabergoline in dogs. Theriogenology 51, 597–611.

Wanke, M.M., Farina, J., Loza, M.H., Rebuelto, M., Concannon, P.W., 1997. Induction of estrus in bitches with normaland persistent anestrus using human menopausal gonadotropin (hMG). Theriogenology 47, 935–942.

Wildt, D.E., Panko, W.B., Chakraborty, P.K., Seager, S.W., 1979. Relationship of serum estrone, estradiol-17beta andprogesterone to LH, sexual behavior and time of ovulation in the bitch. Biol. Reprod. 20, 648–658.

Zoldag, L., Fekete, S., Csaky, I., Bersenyi, A., 2001. Fertile estrus induced in bitches by bromocryptine, a dopamineantagonist: a clinical trial. Theriogenology 55, 1657–1666.