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Abortifacient and endocrine effects of metergoline in beagle bitches during the second half of gestation
Theriogenology 59 (2003) 1929±1940
Abortifacient and endocrine effects of metergoline in beagle bitches during the second half of gestation Johan O. NoÈthlinga,*, David Gerbera, Cornelia Gerstenberga, C. Kaiserb, M. DoÈbelib a
Department of Production Animal Studies, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, Republic of South Africa b Clinic for Andrology and Gynaecology, Faculty of Veterinary Science, University of Zurich, Zurich, Switzerland Received 20 July 2001; accepted 15 July 2002
Abstract The purpose of this study was to investigate the abortifacient effects of high doses of metergoline when administered to pregnant beagle bitches during the second half of gestation and to de®ne the endocrine effects of this treatment as represented by plasma progesterone and estradiol concentrations. Previously, metergoline had been shown to be incompletely luteolytic and induced abortion in only one of eight pregnant bitches when 0.4±0.5 mg/kg were administered twice daily for 5 days from Days 18 to 20 of diestrus. Nine pregnancies in six beagle bitches were used for the present study. Three bitches were treated in each of two consecutive pregnant cycles. Metergoline was administered at a dose of 0.6 mg/kg per os twice daily, starting on Day 28 after the cytological onset of diestrus. Abortion was induced in eight of the nine treated pregnancies and started after 3±23 days of treatment (mean 12.5 days, S.D. 6.4 days). The abortions were completed within 0.5±8 days (mean 2.2 days, S.D. 2.7 days). There were no side effects associated with metergoline treatment and none of the abortions was associated with complications that required intervention. In the single bitch that did not abort, an ovarian granulosa cell tumor was discovered when the single fetus had to be removed surgically at term. Plasma progesterone concentrations declined after the start of metergoline administration in all pregnancies but levels below 4.8 nmol/l were required for successful abortions. Plasma estradiol concentrations showed a tendency to decline and ¯uctuate concurrently with the plasma progesterone levels. However, suppression of plasma estradiol concentrations by metergoline was not as complete as the suppression of progesterone and did not seem a prerequisite for abortion. The hormone pro®les and treatment period required for abortion tended to be similar for both cycles of the three bitches that were treated during two consecutive pregnancies. This suggests a bitch effect on the factors that determine the ef®cacy of metergoline to induce abortion. The large variation and length of the treatment period that was * Corresponding author. Tel.: 27-12-5298218; fax: 27-12-5298314. E-mail address: [email protected] (J.O. NoÈthling).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0093-691X(02)01292-X
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required until abortion commenced was probably related to the relatively early start of treatment compared to other studies. The results of this investigation suggest that, similar to other prolactin suppressing ergot derivatives, metergoline causes complete luteolysis and can be used to reliably induce abortion only during the last 3 weeks of gestation. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Metergoline; Abortion; Bitch; Progesterone; Estradiol
1. Introduction Ovariohysterectomy as a method of terminating an unwanted pregnancy is not an option if the breeding potential of the bitch is to be preserved and several different approaches have been adopted under these circumstances [1]. Estrogens can be used to prevent implantation but commonly cause side effects that compromise fertility or even endanger the life of the bitch [2,3]. These risks, together with the fact that estrogens have to be administered before the necessity of intervention can be ascertained by pregnancy diagnosis, make this option unacceptable to most clinicians [1,2,4]. Currently, PGF2a and its synthetic derivatives are the most commonly used abortifacient drugs for canine pregnancies [4±6]. However, PGF2a treatment regimes can be problematic and usually necessitate hospitalization for prolonged periods. At high dose rates, administration twice daily for a minimum of 4 days of PGF2a inevitably causes unpleasant side effects [6±8], while low dose rates that are better tolerated by the patient, require three to four administrations daily for up to 11 days [9]. Therefore, a substantial number of studies have been conducted to ®nd alternative pharmacological agents that can be administered orally and cause fewer side effects. Progesterone blocking agents, the socalled antigestagens like mifepristone [10±12] and aglepristone [13], are described as promising abortifacient drugs that elicit virtually no side effects in bitches. However, they tend to be expensive and are not registered in many countries due to ethical considerations regarding the legality of pregnancy termination in women. Certain ergot derivatives that suppress prolactin secretion in dogs are being investigated as another alternative for inducing abortion in bitches [14±16]. Luteal activity during the second half of canine gestation is dependent on hypophyseal prolactin [17,18] and active corpora lutea are the sole source of progesterone for the maintenance of pregnancy in the bitch [19,20]. Prolactin suppression during the second half of gestation, therefore, leads to a decrease in plasma progesterone concentrations that can be suf®cient to induce abortion [15,16,21]. While levels below 8 nmol/l [22] or 6.4 nmol/l [23] for prolonged periods may lead to termination of pregnancy, most studies conclude that plasma progesterone concentrations have to fall below 4.8 nmol/l for bitches to abort [8,21,24]. Most of the studies on the use of ergot derivatives have concentrated on bromocryptine [8,17,25] and cabergoline [15,16,26,27]. The former is generally unacceptable because of a high level of gastrointestinal side effects [8,17] and cabergoline is unavailable in many countries. Metergoline is another ergot derivative that has been shown to inhibit prolactin release in bitches [28,29]. Like cabergoline and bromocryptine, it has a dopaminergic effect, but seems to act primarily by blocking serotonin (5-hydroxytryptamine) receptors in
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the central nervous system [30,31]. Several studies have shown metergoline to be an effective agent for the suppression of lactation and the symptoms of pseudopregnancy in bitches [28,32,33]. In a previous study, Gerstenberg and NoÈthling [34] showed that metergoline suppressed luteal function in pregnant bitches. When metergoline was administered for 5 days at 0.4±0.5 mg/kg twice daily per os to eight pregnant beagle bitches from Days 18 to 20 of diestrus, it proved to be incompletely luteolytic and plasma progesterone concentrations recovered in a subsequent 5-day period without treatment. After a further 3 days of pretreatment with metergoline, a short course of twice daily PGF2a treatment (mean 4.8 treatments) was suf®cient to reliably induce abortion. One bitch aborted due to metergoline treatment alone on Day 32 of diestrus. The other ergot derivatives, cabergoline and bromocryptine, have been found to be fully luteolytic and reliably induced abortion only if administered at high doses during the last third of gestation [15,16, 25±27]. This study, therefore, set out to investigate the endocrine and abortifacient effects of a slightly higher dose of oral metergoline (0.6 mg/kg per os, twice daily) administered to beagle bitches for prolonged periods at a later stage of pregnancy (after Day 28 of diestrus). 2. Materials and methods 2.1. Animals Six healthy beagle bitches were used in the study (Bitches No. 1±6). Three of the bitches (Bitches No. 1±3) were treated twice during each of two consecutive pregnant cycles. The bitches were hospitalized for estrus observation and breeding and throughout the whole treatment period until all sampling had been completed. While in the hospital, the bitches were housed in large cages. A commercial canine diet, consisting of dry pellets, was fed once daily and water was provided ad libitum. 2.2. Estrous periods All estrous cycles occurred spontaneously. Starting at the beginning of the pro-estrous period, each bitch was monitored daily by vaginoscopy and vaginal cytology [35±37]. The bitches were bred naturally or by arti®cial insemination of fresh semen from a single beagle male of known fertility. Day 1 of diestrus was determined by means of vaginal cytology [35±37]. 2.3. Pregnancy monitoring by ultrasound Pregnancy was con®rmed by B-mode ultrasonography (5 MHz convex linear, phase array transducer) between Days 18 and 26 after the onset of diestrus [38]. From the ®rst day of treatment (Day 28 of diestrus), the bitches were monitored twice daily by ultrasound scanning until abortion was complete [39]. During these examinations, the total number of conceptuses and the number of conceptuses with visible heartbeats were recorded as well
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as any other indications of fetal viability (for example, the size of a conceptus). Abortion was deemed to have started during the last 12 h preceding an ultrasound scan, if the number of fetuses was reduced compared to the previous examination. 2.4. Monitoring of bitches Bitches were monitored twice daily starting on Day 26 or 27 after the onset of diestrus until at least 7 days after the abortion was complete. A record sheet was completed at 12-h intervals. Any changes in habit, behavior, temperament, appetite, nature of feces, evidence of vomiting, presence and nature of vaginal discharge, and any other observations that could have been ascribed to the loss of pregnancy or side effects of the treatment were recorded [28,32]. 2.5. Metergoline treatment Every bitch was treated twice daily with 0.6 mg/kg per os metergoline1 at 12-h intervals. Treatment started on Day 28 after the onset of diestrus and continued until abortion was complete. 2.6. Blood sampling Daily blood samples were obtained from all treated bitches starting on Day 27 after the onset of diestrus and continuing until the abortion was completed. Thereafter, at least one additional blood sample was obtained 7 days later. The blood samples were taken at 08:00 to avoid any diurnal variations of plasma progesterone concentrations [40]. Cephalic vein puncture was used to collect the samples into heparinized blood tubes that were centrifuged within 30 min. The supernatant plasma was decanted into sterile cryotubes2 and duplicate samples were stored at 18 8C until the hormone assays were performed. 2.7. Hormone assays 2.7.1. Progesterone assay Plasma progesterone concentrations were determined with an RIA using a double antibody technique and dextran-coated charcoal separation [41]. The cross-reactions of the progesterone and plasma progesterone assay were reported as follows: progesterone 1.0; 5pregnene-3b-ol-20-one 0.005; 11b-hydroxyprogesterone 0.29; 11a-hydroxyprogesterone 0.054; 17-hydroxyprogesterone 0.013; testosterone 0.0004; 4-androstene-3,17-dione <0.0001; estrone, 17b-estradiol, estriol <0.0001; cholesterol <0.0001. The detection limit of the assay was 0.24 nmol/l. The intra-assay coef®cients of variation for progesterone concentrations above and below 10 nmol/l were 12.7 and 9.4%, respectively. The interassay coef®cient of variation was 14.3%. 1 2
Liserdol, Pharmacia Ltd., Johannesburg, South Africa. InterMed, Denmark.
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2.8. Estradiol assay Plasma estradiol concentrations were measured with a commercially available extraction RIA3. The cross-reactions of the estradiol assay were reported as follows: estradiol 1.0; estrone 0.02; estriol 0.02; ethinyl-estradiol <0.001; progesterone <0.001; testosterone, androstenedione <0.001. The detection limit of the assay was 9.2 pmol/l. The intra- and inter-assay coef®cients of variation were 4.8 and 13.2%, respectively. 3. Results At the start of treatment, ultrasonographic scanning revealed that the bitches carried a range of one to seven fetuses (mean 4, S.D. 1.7, n 9). The minimum and maximum of one and seven fetuses were seen only in one of the bitches (Bitch No. 6 and 5), respectively. Before the treatment with metergoline was started, all bitches were diagnosed pregnant on ultrasound examination with conceptuses that exhibited the typical features as expected at the relevant stage of pregnancy [38,39]. Abortion was induced in eight of the nine treated pregnancies (Fig. 1). The bitches started aborting after 3 to 23 days of treatment (mean 12.5 days, S.D. 6.4 days), which corresponded to between Days 31 and 51 after the onset of diestrus (mean 40.5 days, S.D. 6.4 days). The abortions were completed within 0.5 to 8 days (mean 2.2 days, S.D. 2.7 days) and all fetuses had been expelled by Day 35 to 52 of diestrus (Fig. 1). Bitch No. 1 showed a reduction in plasma progesterone concentrations to basal levels within 2 to 6 days after the start of treatment, and aborted before Day 43 after the onset of diestrus in each of her two consecutive treatment cycles. On both occasions all fetuses were expelled within 1 day after the start of abortion. Bitch No. 2 displayed a delay in the decline of plasma progesterone concentrations to basal levels on each of her consecutive treatment cycles. On both occasions, Bitch No. 2 started aborting after Day 43 of diestrus and completed the expulsion of all fetuses within 2 days. Bitch No. 3 showed a rapid and persistent decline of plasma progesterone concentrations, similar to Bitch No. 1, when she was treated on two consecutive pregnant cycles. She started aborting relatively soon after the onset of treatment (5 and 3 days) but required a relatively long period for all fetuses to be expelled (8 and 4 days) on both occasions. (Fig. 1) The bitch that did not abort was the one that carried only a single fetus at the start of treatment (Bitch No. 6). Metergoline therapy was discontinued on Day 56 after the onset of diestrus. The bitch showed evidence of mammary milk production on Day 60 and went into partus a day later. A hysterotomy was required to remove the single malpresented fetus which died intra-operatively. During surgery an abnormal mass was discovered on the right ovary. Histopathological examination con®rmed the diagnosis of a granulosa cell tumor. After the start of metergoline therapy, plasma progesterone concentrations decreased in all bitches. Plasma estradiol concentrations showed a tendency to decline and ¯uctuate together with the progesterone concentrations but they were not consistently reduced to baseline levels. The decline of plasma progesterone and estradiol concentrations after the 3
I-125 Oestradiol Extraction Kit, Sodiag SA, Switzerland.
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Fig. 1. Plasma progesterone concentrations (nmol/l) and estradiol concentrations (pmol/l) of pregnant beagle bitches treated with metergoline (0.6 mg/kg per os, twice daily) from Day 28 after the onset of diestrus. Three of the bitches (Bitches No. 1, 2, and 3) were treated twice on two consecutive pregnant cycles (a and b). (~), plasma estradiol concentrations. (*), plasma progesterone concentrations. (&), period of abortion process.
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Fig. 1. (Continued ).
start of treatment was least obvious and convincing in Bitch No. 6, which did not abort. (Fig. 1) The pattern of the decline of progesterone concentrations varied considerably. In cycles in which abortion failed to occur or was delayed beyond Day 43 after the onset of diestrus, the plasma progesterone concentrations either did not decline rapidly (Bitch No. 6) or, after an initial decrease, the progesterone levels displayed some degree of temporary resurgence (Bitch No. 2 (a and b), Bitch No. 5). In six of the eight treatment cycles that resulted in termination of pregnancy, the plasma progesterone concentrations were below 8 nmol/l for 1 to 11 days and below 4.8 nmol/l for 1 to 10 days before abortion commenced. Only Bitch No. 3 started aborting at higher plasma progesterone concentrations in both of her treatment cycles, but the levels had declined to below 4.8 nmol/l for several days by the time that all fetuses had been expelled (Fig. 1). There was no clinical evidence of side effects due to the metergoline therapy in any of the bitches. None of the abortions was associated with complications that required
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intervention. None of the bitches that aborted showed mammary development and no bitch lactated after an abortion. All conceptuses remained viable on ultrasound examination until the abortion process started. Occasionally, fetuses without a detectable heartbeat were seen in the uterus within 12 to 24 h before they were expelled. One aborted fetus was seen of Bitch No. 5 and ®ve aborted fetuses, of which one was alive, were observed in the ®rst pregnancy of Bitch No. 2. The bitches had probably eaten all the other fetuses as soon as they were expelled. 4. Discussion In this study, metergoline was shown to be fully luteolytic in eight of nine cases, if administered to pregnant beagle bitches at a dose of 0.6 mg/kg per os bid from Day 28 after the onset of diestrus. Persistence of the corpus luteum after abortion is undesirable as the prolonged diestrous phase may predispose the bitch to uterine pathology [5]. Abortion was induced successfully in eight of the nine treated pregnancies but there was a large variation in the period of treatment required until abortion commenced, from as short as 3 days to as long as 23 days (mean 12.5 days, S.D. 6.4 days, n 8). Most bitches completed the abortion relatively rapidly within 0.5 to 2 days. Treatment failure in the one bitch that did not abort may have been due to abnormal control of hormone production by the granulosa cell tumor present on one ovary [42]. The tumor cells may have been able to continue producing progesterone and estrogen despite a decline of plasma prolactin concentrations. One bitch, treated in two consecutive pregnant cycles, required the shortest duration of treatment until abortion commenced but the longest period for all fetuses to be expelled on both occasions. Furthermore, there was a tendency for the hormone pro®les and treatment period required for abortion to be similar for both cycles of the other two bitches that were treated in two consecutive pregnancies. It, therefore, cannot be excluded that there is an individual bitch effect on the factors that determine the ef®cacy of metergoline to induce abortion. Both cabergoline and bromocryptine have been shown to be fully luteolytic and reliably induced abortion only if the treatment was started more than 40 days after the ®rst mating [15,16,25±27] or more than 42 days after the LH surge [17]. At this stage of gestation, most animals had to be treated for less than 6 days until abortion [15±17,25,27] but in most of these studies it is unclear when the expulsion of all fetuses was completed. However, a larger study, using similar doses, recorded that 4 to 10 days of treatment were required for abortion to commence (mean 6.1 days) if cabergoline was administered from Days 40 to 45 after mating. The treatment failed in 1 of 12 bitches treated at this late stage of gestation and in all 3 bitches that were treated at a slightly earlier stage from Days 35 to 37 after mating [26]. Estrous behavior is associated with the progesterone rise around the LH peak [43] and it may be assumed that the ®rst mating occurs on average 3 days after the LH peak [16]. The LH peak occurs approximately 8 days before Day 1 of cytological diestrus [35,44]. In the present study, abortion commenced around a mean of 40.5 days after the onset of diestrus, which is not dissimilar to previous studies using ergot derivatives. The prolonged period of metergoline administration that was required for termination of pregnancy, therefore, might have been a result of the early start of treatment on Day
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28 of pregnant diestrus rather than due to the relative inef®cacy of metergoline as compared to cabergoline and bromocryptine. As suggested by Gerstenberg and NoÈthling [34], it may be possible to shorten this period by additional PGF2a administration during the ®nal phase of metergoline treatment. This study con®rms previous ®ndings which indicate that the corpus luteum is either only partially dependent on prolactin as a luteotrophic agent during early to mid-gestation [14,18,21], or that it becomes more sensitive to reduced luteotrophic support in late gestation [16]. Induction of abortion in the last 2 weeks of gestation can be problematic because owners may be distressed if they ®nd the expelled fetuses. Furthermore, the ethics of inducing abortions at such a late stage of pregnancy could be questioned. This prompted the emergence of protocols that combine various pharmaceutical agents with antiluteotrophic and ecbolic actions in order to induce abortion in bitches at an earlier stage during mid-gestation [34,45,46]. In addition, both PGF2a [47] and the progesterone receptor blocking agents [10±13] show considerable promise for being able to ef®ciently prevent implantation or terminate early pregnancies without undue side effects. Plasma concentrations of progesterone and estradiol in mid-gestation, before the start of metergoline treatment, were similar to those reported in previous studies [40,48,49]. In this regard, it is interesting to note that although the plasma estrogen concentrations were relatively low and varied considerably between the different bitches, they showed an obvious tendency to decrease and ¯uctuate in parallel with plasma progesterone concentrations after the start of metergoline treatment. There have been different opinions about whether the ovaries or the placenta are the most likely source of estrogens during gestation in the bitch [19,21,48,49]. The parallel hormone pro®les in the present study suggest that the corpora lutea of pregnancy are at least partially responsible for the plasma estrogen levels during the second half of gestation in the bitch. However, suppression of estrogen production by metergoline was not as complete as the suppression of progesterone and did not seem to be a prerequisite for abortion. Additional production of estradiol by miscellaneous follicles may have contributed to the substantial variation of estrogen concentrations between bitches, especially after plasma progesterone concentrations had declined to basal levels. The results of Chakraborty [48] suggest that the majority of estradiol-17b is converted to estrone during pregnancy. Due to possible cross-reactions in the assay system used in this study, it remains unclear how much of the measured estradiol concentrations are attributable to estrone, a problem that was pointed out previously by Concannon et al. [49]. An interesting aspect is the prolonged period of low plasma progesterone concentrations in some bitches before the onset of abortion, which is similar to the observations made by the authors during a previous study [34]. Repeated, twice daily, ultrasound scanning of all fetuses showed that they only started to die shortly before or at the point of expulsion. It might, therefore, be assumed that the abortifacient effect of low progesterone levels is not so much due to a direct effect on placental ef®ciency and fetal well-being but rather because one of the main protective functions of progesterone in late pregnancy is to ensure the quiescence of the myometrium [50,51]. Therefore, the presence or absence of miscellaneous ecbolic stimuli may be partially responsible for the large variation between bitches in the time period of treatment until the start of abortion. Differences of local endometrial hormone metabolism between individual bitches may contribute to this
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variation. However, the demonstrated concurrent decline of plasma estrogen and progesterone concentrations make it unlikely that a possible sensitization of the myometrium by an increase of estradiol levels during the latter parts of gestation could play a role in this regard [34,51]. Previous studies have shown moderate to severe behavioral abnormalities in pseudopregnant bitches treated with metergoline at low doses (0.1±0.2 mg/kg twice daily) [28,32,33]. The authors did not observe any of these side effects in the present investigation or in a previous study [34] that used similarly high doses of metergoline (0.4±0.6 mg/kg twice daily). It is a matter for speculation whether this is due to the drug being used in pregnancy as opposed to pseudopregnancy, whether it indicates a lower sensitivity of the beagle breed to the behavioral side effects of metergoline, or whether the incidence of side effects is dependent on different formulations of metergoline used in the various studies. References [1] Shille VM. Mismating and termination of pregnancy. Vet Clin North Am Small Anim Pract 1982;12:99±106. [2] Bowen RA, Olson PN, Behrendt MD, Wheeler SL, Husted PW, Nett TM. Ef®cacy and toxicity of estrogens commonly used to terminate canine pregnancy. J Am Vet Med Assoc 1985;186:783±8. [3] Schalm OW. Exogenous estrogen toxicity in the dog. Canine Pract 1978;5:57±61. [4] Olson PN, Johnston SD, Root MV, Hegstad RL. Terminating pregnancy in dogs and cats. Anim Reprod Sci 1992;28:399±406. [5] Feldman EC, Nelson RW. Canine female reproduction. Canine and feline endocrinology and reproduction. Philadelphia: Saunders; 1996. [6] Fieni F, Dumon C, Tainturier D, Bruyas JF. Clinical protocol for pregnancy termination in bitches using prostaglandin F2a. J Reprod Fertil Suppl 1997;51:245±50. [7] Sciorsci RL, Lacalandra GM, Lograno MD, Valentini L, Daniele E, Minola P. Reduction in the bitch of prostaglandin side-effects by atropine, anti-H1 and anti-H2 drugs. In vivo and in vitro observations. Twelfth Int Cong Anim Reprod. The Hague; 1992. p. 1817±9. [8] Wichtel JJ, Whitacre MD, Yates DJ, van Camp SD. Comparison of the effects of PGF2a and bromocryptine in pregnant beagle bitches. Theriogenology 1990;4:829±36. [9] Hubler M, Arnold S, Casal M, FluÈckinger M, Hauser B, Corboz L, et al. Anwendung von niedrig dosiertem Prostaglandin F2a bei HuÈndinnen. Schweiz Arch Tierheilk 1991;133:323±9. [10] Concannon PW, Yeager A, Frank D, Iyampillai A. Termination of pregnancy and induction of premature luteolysis by the antiprogestagen, mifepristone, in dogs. J Reprod Fertil 1990;88:99±104. [11] Linde-Forsberg C, Kindahl H, Madej A. Termination of mid-term pregnancy in the dog with oral RU486. J Small Anim Pract 1992;33:331±6. [12] Sankai T, Endo T, Kanayama K, Sakuma Y, Umezu M, Masaki J. Antiprogesterone compound, RU486 administration to terminate pregnancy in dogs and cats. J Vet Med Sci 1991;53:1069±70. [13] Fieni F, Tainturier D, Bruyas JF, Badinand F, Bertholet X, Ronsin P, et al. Etude clinique d'une antihormone pour provoquer l'avortement chez la chienne: l'agleÂpristone. ReÂc MeÂd VeÂt 1996;172:359±67. [14] JoÈchle W. Prolactin in canine and feline reproduction. Reprod Domestic Anim 1997;32:183±93. [15] JoÈchle W, Arbeiter K, Post K, Ballabio R, D'Ver AS. Effects of pseudopregnancy, pregnancy and interoestrous intervals on pharmacological suppression of prolactin secretion in female dogs and cats. J Reprod Fertil Suppl 1989;39:199±207. [16] Onclin K, Silva LDM, Donnay I, Verstegen JP. Luteotrophic action of prolactin in dogs and the effects of a dopamine agonist, cabergoline. J Reprod Fertil Suppl 1993;47:403±9. [17] Concannon PW, Weinstein P, Whaley S, Frank D. Suppression of luteal function in dogs by luteinizing hormone antiserum and by bromocriptine. J Reprod Fertil 1987;81:175±80. [18] Okkens AC, Bevers MM, Dieleman SJ, Willemse AH. Evidence for prolactin as the main luteotrophic factor in the cyclic dog. Vet Q 1990;12:193±201.
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[44] Bouchard GF, Solorzano N, Concannon PW, Youngquist RS, Bierschwal CJ. Determination of ovulation time in bitches based on teasing, vaginal cytology, and ELISA for progesterone. Theriogenology 1991;35: 603±11. [45] 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±22. [46] Onclin K, Verstegen JP. Practical use of a dopamine agonist and a synthetic prostaglandin analogue to terminate unwanted pregnancy in dogs. J Small Anim Pract 1996;37:211±6. [47] Romagnoli SE, Camillo F, Cela M, Johnston SD, Grassi F, Ferdeghini M, et al. Clinical use of prostaglandin F2a to induce early abortion in bitches: serum progesterone, treatment outcome and interval to subsequent oestrus. J Reprod Fertil Suppl 1993;47:42±431. [48] Chakraborty PK. Reproductive hormone concentrations during estrus, pregnancy, and pseudopregnancy in the labrador bitch. Theriogenology 1987;27:827±40. [49] Concannon PW, McCann JP, Temple M. Biology and endocrinology of ovulation, pregnancy and parturition in the dog. J Reprod Fertil Suppl 1989;39:3±25. [50] Heap RB, Flint APF. Pregnancy. In: Austin CR, Short RV, editors. Reproduction in Mammals. Book 3: hormonal control of reproduction. Cambridge: Cambridge University Press; 1984. p. 153 94. [51] van der Weyden GC, Taverne MAM, Dieleman SJ, Wurth Y, Bevers MM, van Oord HA. Physiological aspects of pregnancy and parturition in dogs. J Reprod Fertil Suppl 1989;39:211±24.
Abortifacient and endocrine effects of metergoline in beagle bitches during the second half of gestation Johan O. NoÈthlinga,*, David Gerbera, Cornelia Gerstenberga, C. Kaiserb, M. DoÈbelib a
Department of Production Animal Studies, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, Republic of South Africa b Clinic for Andrology and Gynaecology, Faculty of Veterinary Science, University of Zurich, Zurich, Switzerland Received 20 July 2001; accepted 15 July 2002
Abstract The purpose of this study was to investigate the abortifacient effects of high doses of metergoline when administered to pregnant beagle bitches during the second half of gestation and to de®ne the endocrine effects of this treatment as represented by plasma progesterone and estradiol concentrations. Previously, metergoline had been shown to be incompletely luteolytic and induced abortion in only one of eight pregnant bitches when 0.4±0.5 mg/kg were administered twice daily for 5 days from Days 18 to 20 of diestrus. Nine pregnancies in six beagle bitches were used for the present study. Three bitches were treated in each of two consecutive pregnant cycles. Metergoline was administered at a dose of 0.6 mg/kg per os twice daily, starting on Day 28 after the cytological onset of diestrus. Abortion was induced in eight of the nine treated pregnancies and started after 3±23 days of treatment (mean 12.5 days, S.D. 6.4 days). The abortions were completed within 0.5±8 days (mean 2.2 days, S.D. 2.7 days). There were no side effects associated with metergoline treatment and none of the abortions was associated with complications that required intervention. In the single bitch that did not abort, an ovarian granulosa cell tumor was discovered when the single fetus had to be removed surgically at term. Plasma progesterone concentrations declined after the start of metergoline administration in all pregnancies but levels below 4.8 nmol/l were required for successful abortions. Plasma estradiol concentrations showed a tendency to decline and ¯uctuate concurrently with the plasma progesterone levels. However, suppression of plasma estradiol concentrations by metergoline was not as complete as the suppression of progesterone and did not seem a prerequisite for abortion. The hormone pro®les and treatment period required for abortion tended to be similar for both cycles of the three bitches that were treated during two consecutive pregnancies. This suggests a bitch effect on the factors that determine the ef®cacy of metergoline to induce abortion. The large variation and length of the treatment period that was * Corresponding author. Tel.: 27-12-5298218; fax: 27-12-5298314. E-mail address: [email protected] (J.O. NoÈthling).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0093-691X(02)01292-X
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required until abortion commenced was probably related to the relatively early start of treatment compared to other studies. The results of this investigation suggest that, similar to other prolactin suppressing ergot derivatives, metergoline causes complete luteolysis and can be used to reliably induce abortion only during the last 3 weeks of gestation. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Metergoline; Abortion; Bitch; Progesterone; Estradiol
1. Introduction Ovariohysterectomy as a method of terminating an unwanted pregnancy is not an option if the breeding potential of the bitch is to be preserved and several different approaches have been adopted under these circumstances [1]. Estrogens can be used to prevent implantation but commonly cause side effects that compromise fertility or even endanger the life of the bitch [2,3]. These risks, together with the fact that estrogens have to be administered before the necessity of intervention can be ascertained by pregnancy diagnosis, make this option unacceptable to most clinicians [1,2,4]. Currently, PGF2a and its synthetic derivatives are the most commonly used abortifacient drugs for canine pregnancies [4±6]. However, PGF2a treatment regimes can be problematic and usually necessitate hospitalization for prolonged periods. At high dose rates, administration twice daily for a minimum of 4 days of PGF2a inevitably causes unpleasant side effects [6±8], while low dose rates that are better tolerated by the patient, require three to four administrations daily for up to 11 days [9]. Therefore, a substantial number of studies have been conducted to ®nd alternative pharmacological agents that can be administered orally and cause fewer side effects. Progesterone blocking agents, the socalled antigestagens like mifepristone [10±12] and aglepristone [13], are described as promising abortifacient drugs that elicit virtually no side effects in bitches. However, they tend to be expensive and are not registered in many countries due to ethical considerations regarding the legality of pregnancy termination in women. Certain ergot derivatives that suppress prolactin secretion in dogs are being investigated as another alternative for inducing abortion in bitches [14±16]. Luteal activity during the second half of canine gestation is dependent on hypophyseal prolactin [17,18] and active corpora lutea are the sole source of progesterone for the maintenance of pregnancy in the bitch [19,20]. Prolactin suppression during the second half of gestation, therefore, leads to a decrease in plasma progesterone concentrations that can be suf®cient to induce abortion [15,16,21]. While levels below 8 nmol/l [22] or 6.4 nmol/l [23] for prolonged periods may lead to termination of pregnancy, most studies conclude that plasma progesterone concentrations have to fall below 4.8 nmol/l for bitches to abort [8,21,24]. Most of the studies on the use of ergot derivatives have concentrated on bromocryptine [8,17,25] and cabergoline [15,16,26,27]. The former is generally unacceptable because of a high level of gastrointestinal side effects [8,17] and cabergoline is unavailable in many countries. Metergoline is another ergot derivative that has been shown to inhibit prolactin release in bitches [28,29]. Like cabergoline and bromocryptine, it has a dopaminergic effect, but seems to act primarily by blocking serotonin (5-hydroxytryptamine) receptors in
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the central nervous system [30,31]. Several studies have shown metergoline to be an effective agent for the suppression of lactation and the symptoms of pseudopregnancy in bitches [28,32,33]. In a previous study, Gerstenberg and NoÈthling [34] showed that metergoline suppressed luteal function in pregnant bitches. When metergoline was administered for 5 days at 0.4±0.5 mg/kg twice daily per os to eight pregnant beagle bitches from Days 18 to 20 of diestrus, it proved to be incompletely luteolytic and plasma progesterone concentrations recovered in a subsequent 5-day period without treatment. After a further 3 days of pretreatment with metergoline, a short course of twice daily PGF2a treatment (mean 4.8 treatments) was suf®cient to reliably induce abortion. One bitch aborted due to metergoline treatment alone on Day 32 of diestrus. The other ergot derivatives, cabergoline and bromocryptine, have been found to be fully luteolytic and reliably induced abortion only if administered at high doses during the last third of gestation [15,16, 25±27]. This study, therefore, set out to investigate the endocrine and abortifacient effects of a slightly higher dose of oral metergoline (0.6 mg/kg per os, twice daily) administered to beagle bitches for prolonged periods at a later stage of pregnancy (after Day 28 of diestrus). 2. Materials and methods 2.1. Animals Six healthy beagle bitches were used in the study (Bitches No. 1±6). Three of the bitches (Bitches No. 1±3) were treated twice during each of two consecutive pregnant cycles. The bitches were hospitalized for estrus observation and breeding and throughout the whole treatment period until all sampling had been completed. While in the hospital, the bitches were housed in large cages. A commercial canine diet, consisting of dry pellets, was fed once daily and water was provided ad libitum. 2.2. Estrous periods All estrous cycles occurred spontaneously. Starting at the beginning of the pro-estrous period, each bitch was monitored daily by vaginoscopy and vaginal cytology [35±37]. The bitches were bred naturally or by arti®cial insemination of fresh semen from a single beagle male of known fertility. Day 1 of diestrus was determined by means of vaginal cytology [35±37]. 2.3. Pregnancy monitoring by ultrasound Pregnancy was con®rmed by B-mode ultrasonography (5 MHz convex linear, phase array transducer) between Days 18 and 26 after the onset of diestrus [38]. From the ®rst day of treatment (Day 28 of diestrus), the bitches were monitored twice daily by ultrasound scanning until abortion was complete [39]. During these examinations, the total number of conceptuses and the number of conceptuses with visible heartbeats were recorded as well
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as any other indications of fetal viability (for example, the size of a conceptus). Abortion was deemed to have started during the last 12 h preceding an ultrasound scan, if the number of fetuses was reduced compared to the previous examination. 2.4. Monitoring of bitches Bitches were monitored twice daily starting on Day 26 or 27 after the onset of diestrus until at least 7 days after the abortion was complete. A record sheet was completed at 12-h intervals. Any changes in habit, behavior, temperament, appetite, nature of feces, evidence of vomiting, presence and nature of vaginal discharge, and any other observations that could have been ascribed to the loss of pregnancy or side effects of the treatment were recorded [28,32]. 2.5. Metergoline treatment Every bitch was treated twice daily with 0.6 mg/kg per os metergoline1 at 12-h intervals. Treatment started on Day 28 after the onset of diestrus and continued until abortion was complete. 2.6. Blood sampling Daily blood samples were obtained from all treated bitches starting on Day 27 after the onset of diestrus and continuing until the abortion was completed. Thereafter, at least one additional blood sample was obtained 7 days later. The blood samples were taken at 08:00 to avoid any diurnal variations of plasma progesterone concentrations [40]. Cephalic vein puncture was used to collect the samples into heparinized blood tubes that were centrifuged within 30 min. The supernatant plasma was decanted into sterile cryotubes2 and duplicate samples were stored at 18 8C until the hormone assays were performed. 2.7. Hormone assays 2.7.1. Progesterone assay Plasma progesterone concentrations were determined with an RIA using a double antibody technique and dextran-coated charcoal separation [41]. The cross-reactions of the progesterone and plasma progesterone assay were reported as follows: progesterone 1.0; 5pregnene-3b-ol-20-one 0.005; 11b-hydroxyprogesterone 0.29; 11a-hydroxyprogesterone 0.054; 17-hydroxyprogesterone 0.013; testosterone 0.0004; 4-androstene-3,17-dione <0.0001; estrone, 17b-estradiol, estriol <0.0001; cholesterol <0.0001. The detection limit of the assay was 0.24 nmol/l. The intra-assay coef®cients of variation for progesterone concentrations above and below 10 nmol/l were 12.7 and 9.4%, respectively. The interassay coef®cient of variation was 14.3%. 1 2
Liserdol, Pharmacia Ltd., Johannesburg, South Africa. InterMed, Denmark.
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2.8. Estradiol assay Plasma estradiol concentrations were measured with a commercially available extraction RIA3. The cross-reactions of the estradiol assay were reported as follows: estradiol 1.0; estrone 0.02; estriol 0.02; ethinyl-estradiol <0.001; progesterone <0.001; testosterone, androstenedione <0.001. The detection limit of the assay was 9.2 pmol/l. The intra- and inter-assay coef®cients of variation were 4.8 and 13.2%, respectively. 3. Results At the start of treatment, ultrasonographic scanning revealed that the bitches carried a range of one to seven fetuses (mean 4, S.D. 1.7, n 9). The minimum and maximum of one and seven fetuses were seen only in one of the bitches (Bitch No. 6 and 5), respectively. Before the treatment with metergoline was started, all bitches were diagnosed pregnant on ultrasound examination with conceptuses that exhibited the typical features as expected at the relevant stage of pregnancy [38,39]. Abortion was induced in eight of the nine treated pregnancies (Fig. 1). The bitches started aborting after 3 to 23 days of treatment (mean 12.5 days, S.D. 6.4 days), which corresponded to between Days 31 and 51 after the onset of diestrus (mean 40.5 days, S.D. 6.4 days). The abortions were completed within 0.5 to 8 days (mean 2.2 days, S.D. 2.7 days) and all fetuses had been expelled by Day 35 to 52 of diestrus (Fig. 1). Bitch No. 1 showed a reduction in plasma progesterone concentrations to basal levels within 2 to 6 days after the start of treatment, and aborted before Day 43 after the onset of diestrus in each of her two consecutive treatment cycles. On both occasions all fetuses were expelled within 1 day after the start of abortion. Bitch No. 2 displayed a delay in the decline of plasma progesterone concentrations to basal levels on each of her consecutive treatment cycles. On both occasions, Bitch No. 2 started aborting after Day 43 of diestrus and completed the expulsion of all fetuses within 2 days. Bitch No. 3 showed a rapid and persistent decline of plasma progesterone concentrations, similar to Bitch No. 1, when she was treated on two consecutive pregnant cycles. She started aborting relatively soon after the onset of treatment (5 and 3 days) but required a relatively long period for all fetuses to be expelled (8 and 4 days) on both occasions. (Fig. 1) The bitch that did not abort was the one that carried only a single fetus at the start of treatment (Bitch No. 6). Metergoline therapy was discontinued on Day 56 after the onset of diestrus. The bitch showed evidence of mammary milk production on Day 60 and went into partus a day later. A hysterotomy was required to remove the single malpresented fetus which died intra-operatively. During surgery an abnormal mass was discovered on the right ovary. Histopathological examination con®rmed the diagnosis of a granulosa cell tumor. After the start of metergoline therapy, plasma progesterone concentrations decreased in all bitches. Plasma estradiol concentrations showed a tendency to decline and ¯uctuate together with the progesterone concentrations but they were not consistently reduced to baseline levels. The decline of plasma progesterone and estradiol concentrations after the 3
I-125 Oestradiol Extraction Kit, Sodiag SA, Switzerland.
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Fig. 1. Plasma progesterone concentrations (nmol/l) and estradiol concentrations (pmol/l) of pregnant beagle bitches treated with metergoline (0.6 mg/kg per os, twice daily) from Day 28 after the onset of diestrus. Three of the bitches (Bitches No. 1, 2, and 3) were treated twice on two consecutive pregnant cycles (a and b). (~), plasma estradiol concentrations. (*), plasma progesterone concentrations. (&), period of abortion process.
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Fig. 1. (Continued ).
start of treatment was least obvious and convincing in Bitch No. 6, which did not abort. (Fig. 1) The pattern of the decline of progesterone concentrations varied considerably. In cycles in which abortion failed to occur or was delayed beyond Day 43 after the onset of diestrus, the plasma progesterone concentrations either did not decline rapidly (Bitch No. 6) or, after an initial decrease, the progesterone levels displayed some degree of temporary resurgence (Bitch No. 2 (a and b), Bitch No. 5). In six of the eight treatment cycles that resulted in termination of pregnancy, the plasma progesterone concentrations were below 8 nmol/l for 1 to 11 days and below 4.8 nmol/l for 1 to 10 days before abortion commenced. Only Bitch No. 3 started aborting at higher plasma progesterone concentrations in both of her treatment cycles, but the levels had declined to below 4.8 nmol/l for several days by the time that all fetuses had been expelled (Fig. 1). There was no clinical evidence of side effects due to the metergoline therapy in any of the bitches. None of the abortions was associated with complications that required
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intervention. None of the bitches that aborted showed mammary development and no bitch lactated after an abortion. All conceptuses remained viable on ultrasound examination until the abortion process started. Occasionally, fetuses without a detectable heartbeat were seen in the uterus within 12 to 24 h before they were expelled. One aborted fetus was seen of Bitch No. 5 and ®ve aborted fetuses, of which one was alive, were observed in the ®rst pregnancy of Bitch No. 2. The bitches had probably eaten all the other fetuses as soon as they were expelled. 4. Discussion In this study, metergoline was shown to be fully luteolytic in eight of nine cases, if administered to pregnant beagle bitches at a dose of 0.6 mg/kg per os bid from Day 28 after the onset of diestrus. Persistence of the corpus luteum after abortion is undesirable as the prolonged diestrous phase may predispose the bitch to uterine pathology [5]. Abortion was induced successfully in eight of the nine treated pregnancies but there was a large variation in the period of treatment required until abortion commenced, from as short as 3 days to as long as 23 days (mean 12.5 days, S.D. 6.4 days, n 8). Most bitches completed the abortion relatively rapidly within 0.5 to 2 days. Treatment failure in the one bitch that did not abort may have been due to abnormal control of hormone production by the granulosa cell tumor present on one ovary [42]. The tumor cells may have been able to continue producing progesterone and estrogen despite a decline of plasma prolactin concentrations. One bitch, treated in two consecutive pregnant cycles, required the shortest duration of treatment until abortion commenced but the longest period for all fetuses to be expelled on both occasions. Furthermore, there was a tendency for the hormone pro®les and treatment period required for abortion to be similar for both cycles of the other two bitches that were treated in two consecutive pregnancies. It, therefore, cannot be excluded that there is an individual bitch effect on the factors that determine the ef®cacy of metergoline to induce abortion. Both cabergoline and bromocryptine have been shown to be fully luteolytic and reliably induced abortion only if the treatment was started more than 40 days after the ®rst mating [15,16,25±27] or more than 42 days after the LH surge [17]. At this stage of gestation, most animals had to be treated for less than 6 days until abortion [15±17,25,27] but in most of these studies it is unclear when the expulsion of all fetuses was completed. However, a larger study, using similar doses, recorded that 4 to 10 days of treatment were required for abortion to commence (mean 6.1 days) if cabergoline was administered from Days 40 to 45 after mating. The treatment failed in 1 of 12 bitches treated at this late stage of gestation and in all 3 bitches that were treated at a slightly earlier stage from Days 35 to 37 after mating [26]. Estrous behavior is associated with the progesterone rise around the LH peak [43] and it may be assumed that the ®rst mating occurs on average 3 days after the LH peak [16]. The LH peak occurs approximately 8 days before Day 1 of cytological diestrus [35,44]. In the present study, abortion commenced around a mean of 40.5 days after the onset of diestrus, which is not dissimilar to previous studies using ergot derivatives. The prolonged period of metergoline administration that was required for termination of pregnancy, therefore, might have been a result of the early start of treatment on Day
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28 of pregnant diestrus rather than due to the relative inef®cacy of metergoline as compared to cabergoline and bromocryptine. As suggested by Gerstenberg and NoÈthling [34], it may be possible to shorten this period by additional PGF2a administration during the ®nal phase of metergoline treatment. This study con®rms previous ®ndings which indicate that the corpus luteum is either only partially dependent on prolactin as a luteotrophic agent during early to mid-gestation [14,18,21], or that it becomes more sensitive to reduced luteotrophic support in late gestation [16]. Induction of abortion in the last 2 weeks of gestation can be problematic because owners may be distressed if they ®nd the expelled fetuses. Furthermore, the ethics of inducing abortions at such a late stage of pregnancy could be questioned. This prompted the emergence of protocols that combine various pharmaceutical agents with antiluteotrophic and ecbolic actions in order to induce abortion in bitches at an earlier stage during mid-gestation [34,45,46]. In addition, both PGF2a [47] and the progesterone receptor blocking agents [10±13] show considerable promise for being able to ef®ciently prevent implantation or terminate early pregnancies without undue side effects. Plasma concentrations of progesterone and estradiol in mid-gestation, before the start of metergoline treatment, were similar to those reported in previous studies [40,48,49]. In this regard, it is interesting to note that although the plasma estrogen concentrations were relatively low and varied considerably between the different bitches, they showed an obvious tendency to decrease and ¯uctuate in parallel with plasma progesterone concentrations after the start of metergoline treatment. There have been different opinions about whether the ovaries or the placenta are the most likely source of estrogens during gestation in the bitch [19,21,48,49]. The parallel hormone pro®les in the present study suggest that the corpora lutea of pregnancy are at least partially responsible for the plasma estrogen levels during the second half of gestation in the bitch. However, suppression of estrogen production by metergoline was not as complete as the suppression of progesterone and did not seem to be a prerequisite for abortion. Additional production of estradiol by miscellaneous follicles may have contributed to the substantial variation of estrogen concentrations between bitches, especially after plasma progesterone concentrations had declined to basal levels. The results of Chakraborty [48] suggest that the majority of estradiol-17b is converted to estrone during pregnancy. Due to possible cross-reactions in the assay system used in this study, it remains unclear how much of the measured estradiol concentrations are attributable to estrone, a problem that was pointed out previously by Concannon et al. [49]. An interesting aspect is the prolonged period of low plasma progesterone concentrations in some bitches before the onset of abortion, which is similar to the observations made by the authors during a previous study [34]. Repeated, twice daily, ultrasound scanning of all fetuses showed that they only started to die shortly before or at the point of expulsion. It might, therefore, be assumed that the abortifacient effect of low progesterone levels is not so much due to a direct effect on placental ef®ciency and fetal well-being but rather because one of the main protective functions of progesterone in late pregnancy is to ensure the quiescence of the myometrium [50,51]. Therefore, the presence or absence of miscellaneous ecbolic stimuli may be partially responsible for the large variation between bitches in the time period of treatment until the start of abortion. Differences of local endometrial hormone metabolism between individual bitches may contribute to this
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variation. However, the demonstrated concurrent decline of plasma estrogen and progesterone concentrations make it unlikely that a possible sensitization of the myometrium by an increase of estradiol levels during the latter parts of gestation could play a role in this regard [34,51]. Previous studies have shown moderate to severe behavioral abnormalities in pseudopregnant bitches treated with metergoline at low doses (0.1±0.2 mg/kg twice daily) [28,32,33]. The authors did not observe any of these side effects in the present investigation or in a previous study [34] that used similarly high doses of metergoline (0.4±0.6 mg/kg twice daily). It is a matter for speculation whether this is due to the drug being used in pregnancy as opposed to pseudopregnancy, whether it indicates a lower sensitivity of the beagle breed to the behavioral side effects of metergoline, or whether the incidence of side effects is dependent on different formulations of metergoline used in the various studies. References [1] Shille VM. Mismating and termination of pregnancy. Vet Clin North Am Small Anim Pract 1982;12:99±106. [2] Bowen RA, Olson PN, Behrendt MD, Wheeler SL, Husted PW, Nett TM. Ef®cacy and toxicity of estrogens commonly used to terminate canine pregnancy. J Am Vet Med Assoc 1985;186:783±8. [3] Schalm OW. Exogenous estrogen toxicity in the dog. Canine Pract 1978;5:57±61. [4] Olson PN, Johnston SD, Root MV, Hegstad RL. Terminating pregnancy in dogs and cats. Anim Reprod Sci 1992;28:399±406. [5] Feldman EC, Nelson RW. Canine female reproduction. Canine and feline endocrinology and reproduction. Philadelphia: Saunders; 1996. [6] Fieni F, Dumon C, Tainturier D, Bruyas JF. Clinical protocol for pregnancy termination in bitches using prostaglandin F2a. J Reprod Fertil Suppl 1997;51:245±50. [7] Sciorsci RL, Lacalandra GM, Lograno MD, Valentini L, Daniele E, Minola P. Reduction in the bitch of prostaglandin side-effects by atropine, anti-H1 and anti-H2 drugs. In vivo and in vitro observations. Twelfth Int Cong Anim Reprod. The Hague; 1992. p. 1817±9. [8] Wichtel JJ, Whitacre MD, Yates DJ, van Camp SD. Comparison of the effects of PGF2a and bromocryptine in pregnant beagle bitches. Theriogenology 1990;4:829±36. [9] Hubler M, Arnold S, Casal M, FluÈckinger M, Hauser B, Corboz L, et al. Anwendung von niedrig dosiertem Prostaglandin F2a bei HuÈndinnen. Schweiz Arch Tierheilk 1991;133:323±9. [10] Concannon PW, Yeager A, Frank D, Iyampillai A. Termination of pregnancy and induction of premature luteolysis by the antiprogestagen, mifepristone, in dogs. J Reprod Fertil 1990;88:99±104. [11] Linde-Forsberg C, Kindahl H, Madej A. Termination of mid-term pregnancy in the dog with oral RU486. J Small Anim Pract 1992;33:331±6. [12] Sankai T, Endo T, Kanayama K, Sakuma Y, Umezu M, Masaki J. Antiprogesterone compound, RU486 administration to terminate pregnancy in dogs and cats. J Vet Med Sci 1991;53:1069±70. [13] Fieni F, Tainturier D, Bruyas JF, Badinand F, Bertholet X, Ronsin P, et al. Etude clinique d'une antihormone pour provoquer l'avortement chez la chienne: l'agleÂpristone. ReÂc MeÂd VeÂt 1996;172:359±67. [14] JoÈchle W. Prolactin in canine and feline reproduction. Reprod Domestic Anim 1997;32:183±93. [15] JoÈchle W, Arbeiter K, Post K, Ballabio R, D'Ver AS. Effects of pseudopregnancy, pregnancy and interoestrous intervals on pharmacological suppression of prolactin secretion in female dogs and cats. J Reprod Fertil Suppl 1989;39:199±207. [16] Onclin K, Silva LDM, Donnay I, Verstegen JP. Luteotrophic action of prolactin in dogs and the effects of a dopamine agonist, cabergoline. J Reprod Fertil Suppl 1993;47:403±9. [17] Concannon PW, Weinstein P, Whaley S, Frank D. Suppression of luteal function in dogs by luteinizing hormone antiserum and by bromocriptine. J Reprod Fertil 1987;81:175±80. [18] Okkens AC, Bevers MM, Dieleman SJ, Willemse AH. Evidence for prolactin as the main luteotrophic factor in the cyclic dog. Vet Q 1990;12:193±201.
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