ISSN 0008-5286 THE CANADIAN VETERINARY LA REVUE VETERINAIRE JOURNAL CANADIENNE Volume 24 December-decembre 1983 No. 12

Effects of Ethinylestradiol and on Embryo Survival and Implantation in Mated Beagle Bitches

W.T.K. BOSU AND R.O. WAELCHLI-SUTER Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario Nl G 2 WI

SUMMARY oestradiol et du danazol sur la nida- compounds such as diethylstilbestrol Eleven mature Beagle bitches were tion, la survie des embryons et le main- or menstranol have been used with a artificially inseminated and then used tien de la gestation. Trois chiennes high degree of success to terminate to determine the effects of ethinyles- servirent de temoins, quatre recurent potential in bitches, pro- tradiol or danazol on nidation, de l'ethinyloestradiol et les quatre vided the treatment is administered embryo survival and maintenance of autres, du danazol. L'administration within a day or two after mating . The dogs were grouped so de ces produits se fit par la voie buc- (3,4,5). However, the use of estrogens that three were kept as controls, four cale, a divers intervalles ulterieurs a and diethylstilbesterol for this purpose others were treated with ethinylestra- l'insemination. On en determina les has been reported to lead to bone mar- diol and the remaining four were effets d'apres la condition des ovaires row toxicity or pyometra in some cases treated with danazol. The compounds et de l'uterus, au moyen de la lapara- (5,6,7,8). Furthermore, once preg- were administered orally at varying tomie, et les resultats de la parturition. nancy is confirmed at four weeks or intervals after insemination. The L'ethinyloestradiol exerca tres peu more, there appears to be no reliable effects of the compounds were d'influence sur le developpement et or safe method for induction of abor- deduced from the condition of the l'implantation des embryons, puisque tion in the bitch. In this regard, reports and uterus examined at lapa- trois des quatre chiennes qui en concerning the results obtained after rotomy, and the results of whelping. avaient recu mirent bas. Le danazol the use of prostaglandins in pregnant Ethinylestradiol had minimal effect on donna des resultats analogues. 11 sem- bitches are conflicting (9,10,11,12) and embryo development and implanta- ble par consequent que ces deux pro- the recommended doses and regimens tion. Three offour bitches treated with duits, d'apres l'importance et le nom- seem dangerous. Clearly an urgent ethinylestradiol whelped. Similarly, bre de doses utilisees au cours de cette need for a safe and effective method danazol treatment had little or no experience, n'entravent pas de fa9on for the treatment of misalliance exists. effect on embryonic survival and significative la survie ou l'implanta- The present investigation, which implantation. It is concluded that, tion des embryons, chez des chiennes formed part of a wider search in our ethinylestradiol or danazol at the dos- Beagle gravides. Clinic for a reliable and efficient ages and schedules used in this study method of canine population control, did not significantly interfere with I N T R O D U C T O N including a treatment for misalliance embryonic survival or implantation in The increase in the number of in bitches and queens, was initiated to pregnant Beagle bitches. unwanted dogs and cats is a serious investigate the potential antinidatory problem in many countries. The signif- effects of two compounds, ethinyles- R SU Mg: icance of the problem and its eco- tradiol (EE) and danazol in the mated Effets de l'ethinyloestradiol et du nomic, sociological and public health bitch. Ethinylestradiol is the biologi- danazol sur la survie et l'implantation implications have been the subject of cally active principle in menstranol des embryons, chez des chiennes Bea- several reviews (1,2,3). One of the (I 7-ethinylestradiol-2-methylether) a gle inseminees major sources of unwanted pets is mis- compound that has been reported to Cette experience consistait 'a effectuer alliance. Apart from spaying, the interfere with embryo survival in l'insemination artificielle de 11 treatment of choice for misalliance is mated bitches (13,14). Danazol, (17a- chiennes Beagle et a les utiliser ensuite prevention of nidation or induction of pregn{2,3-d] isoxazol-17-ol) is a syn- pour determiner les effets de l'ethinyl- abortion. Estrogens and estrogenic thetic analogue of ethinyl testosterone

Present address of senior author: School of Veterinary Medicine, Department of Medical Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706.

Can Vet J 1983; 24: 371-374. 371 that has been shown to inhibit pitui- TABLE I tary gonadotrophin secretion, ovarian CORPORA LU1EA. IMPLANTATION AND WHELPING DATA IN UNTREAIED BITCHES steroidogenesis and cause atrophy of the in women (15,16, Findings at Laparotomy 17,18), It was theorized that if danazol No. of displayed the same effects in the mated Bitch No. Days of Corpora No of bitch, the end result would be failure of & Wt. (kg) Pregnancy Lutea Implantations Remarks implantation. 1, 16kg 41 RO 3 RH 3 Aborted day42 LO 3 LH 3 MATERIALS AND METHODS 2, 20 kg 30 RO 5 RH 5 Never whelped Eleven conditioned purebred Beagle LO 5 LH 5 bitches, weighing 9 to 20 kg and 3, 12 kg 30 RO 3 RH 3 Whelped 4 puppies, between three and five years of age, LO 3 LH 3 65 days gestation were used. They were housed in indi- vidual cages in an indoor facility, fed Vaginal swabs obtained from each 11 inclusive (Table 111). once a day and allowed free access to bitch following insemination were An exploratory laparotomy was water. The bitches were examined examined for cytological evidence of performed on each bitch between day daily to determine the onset of proes- ovulation which included reappear- 25 and 41 after insemination (Tables trus which was marked by swelling of ance of leukocytes, parabasal cells, 1-111). Each bitch was anesthetized and the vulva and serosanguineous dis- and the detection of metestrous cells in prepared in the routine manner for charge at the vulvar lips. Starting from the smears (18,19,20). surgery. Following premedication the onset of proestrus, daily vaginal The eleven bitches were randomly with a mixture of atropine-demerol swabs were obtained from each bitch. assigned for treatment as follows: and acepromazine anesthesia was Two smears were made from each three bitches in group I and four induced with thiopental 10-12 mg/kg harvest, processed and examined bitches each in groups II and 111. The intravenously. Anesthesia was main- using techniques previously described bitches in the control group, group 1, tained with 2-2.5% halothane and (18,19). In addition a vaginal swab were inseminated but received no oxygen at a flow rate of 1 to 1.5 liters from each bitch obtained at the onset treatment (Table 1). The group 11 per minute. A midline incision was of proestrus was submitted for bacte- bitches were treated with ethinylestra- made to expose the reproductive tract. rial culture. At the onset of proestrus, diol after mating. Two animals Each uterine horn was examined to the bitches were introduced daily to a (bitches 4 and 5) received single oral count the number of conceptual swel- male dog in order to determine the day doses of ethinylestradioll on day 6 at lings. The ovaries were palpated and of onset of standing estrus. Each bitch the rate of 5 mg/ kg bodyweight. The the number of corpora lutea present was then artificially inseminated on remaining two (bitches 6 and 7) was recorded. Ovariohysterectomies the second day of standing estrus, received single oral doses of ethinyles- were performed in all of the Group III using 3 to 6 mL of semen collected tradiol on day 10 at the rate of 10 bitches following this examination from a proven and fertile Beagle dog. mg/kg body weight (Table 11). The and the reproductive tracts were fixed The day of insemination was desig- group IlI bitches were treated with 400 in Bouin's3 solution immediately after nated day 1 of presumptive pregnancy. mg danazol2 orally, from day 2 to day surgery. Sections across and between

TABLE 11 EFFECTS OF EE ON IMPLANTATION AND MAINTENANCE OF PREGNANCY IN BITCHES

EE Treatment Findings at Laparotomy Bitch No. Days of Dose Days of No. of No. of Wt. (kg) Pregnancy (mg) Pregnancy Corpora Lutea Implantations Remarks 4, 10.6 6 5/kg 22 RO 2 RH I Aborted day 23 gestation LO I LH 0 5, 10 6 5/kg 28 RO 5 RH 3 Dystocia 63 days gestation LO 5 LH 5 C-Section / 7 puppies, 5 alive All had short tails, one had a genesis of distal forelimb 6, 17 10 10mg/kg 33 RO 0 RH 3 Dystocia 61 days gestation LO 0 LH 5 C-Section / 7 puppies, 4 alive 7, 15 10 10mg/kg 37 RO 0 RH 4 Whelped 60 days gestation LO I LH 3 6 live puppies (I small)

'Linoral Forte, N.V. Organon, OSS, Holland. 2Cyclomen, Winthrop Laboratories, Aurora, Ontario. 3BDH Chemicals, Toronto, Ontario.

372 the implantation sites were later made the number of implantation sites this study did not always correlate in the routine manner for histological counted at surgery. In bitch 7, one of with the number of implantation sites, examination for evidence of progesta- the implantation sites appeared it was impossible to accurately deter- tional development of the endome- smaller than the rest at laparotomy. mine the level of preimplantation trium and placentation. The bitches in All seven puppies produced by bitch losses in the bitches. Our inability to groups I and 11 were allowed to carry 5 were abnormal, each having a short palpate corpora lutea in the ovaries of their pregnancies to term. tail while a single puppy had an addi- two bitches, 6 and 7, in which implan- tion, agenesis of both distal parts of tation sites were detected at surgery RESULTS the forelimbs. and which subsequently whelped was No pathogens were recovered on bac- The number of corpora lutea and noteworthy. Since the corpora lutea teriological cultures of the vaginal implantation sites observed in the are essential throughout gestation in swabs from the eleven bitches. There group III bitches are given in Table III. the bitch (23), it is reasonable to were no adverse reactions to the Conceptual swellings were evident in assume that corpora lutea were pre- treatments with either EE or danazol. three of the four bitches at laparot- sent in these ovaries, but were not The changes in the vaginal cytology omy. When the uterine sacculations grossly palpable. characteristic of ovulation were were opened fetal tissues and placenta In the present study, there were observed in all eleven bitches follow- were grossly visible in these three dogs, always fewer puppies whelped than the ing insemination. 8, 10 and 11. No conceptual swelling number of implantation sites counted The characteristics of the ovaries was observed in the fourth bitch, 9. at surgery. The fetal losses cannot be and the uteri in the lutea corresponded Examination of histological sections attributed to bacterial infections since with the number of conceptual swel- of implantation sites revealed normal no pathogens were recovered on cul- lings (implantation sites) present in progestational endometrium in all ture of prebreeding vaginal swabs. It is each bitch. Bitch I aborted a day after cases and the presence of fetal and pla- possible, however, that the abortions surgery and bitch 2 never whelped. cental tissues were observed in three and fetal losses could have resulted as The third bitch in the group whelped cases, 8, 10 and I 1. a consequence of anesthesia, laparot- four normal puppies after 65 days ges- omy and manipulation of the repro- tation. D I S C U S S IO N ductive tracts. In this regard, others Table I1 contains the laparotomy The conception rate in this study, have reported embryonic losses and findings in the bitches treated with EE. based on the number of inseminated abortions following laparotomy and Corpora lutea were not palpable in bitches with implantation sites was manipulation of the uterus in pregnant two bitches, 6 and 7, although several over 90%. However, only four of seven bitches (24). conceptual swellings were present in bitches (group I and group 11) which The failure of EE to prevent implan- each animal. In the other two bitches had implantation sites at laparotomy, tation in the present study contrasts to there were more corpora lutea than whelped. Conception rates for females the reported abortifacient effects of implantation sites. Bitch 4 exhibited a in established Beagle breeding colo- comparative or lower doses of men- dark bloody vaginal discharge several nies have been reported to be 80% or stranol in mated bitches (13,14). This hours after laparotomy, and was pre- more (21,22). The low whelping rate difference in results cannot be readily sumed to have aborted since she never (group I and group II) observed in the explained. It was our supposition at whelped. A culture of this discharge present study could be attributed to the onset of the study that, since the was negative for bacteria. Three abortion and fetal resorption. biological activity of menstranol bitches in this group, 5, 6 and 7 The degree of embryonic losses resides in its ethinylestradiol moiety, whelped after 63, 61 and 60 days' ges- reported by others for the Beagle breed the administration of this compound tation respectively. In each case, the was 1 1% (21). Since the number of should be equally effective. It is prob- number of puppies was one less than corpora lutea counted at surgery in able, however, that the different

TABLE III EFFECTlS OF DANAZOL ON IMPLANTAT1ION AND MAINTENANCE OF PREGNANCY IN BlTCHES

Danazol Treatment Findings at Laparotomy Bitch No. Days of Dose Days of No. of No. of Wt. (kg) Pregnancy (mg)/d Pregnancy Corpora Lutea- Implantations Remarks 8, 10 2-11 400 27 RO 3 RH 3 Histologically 9 CL in , LO 4 LH 6 all 9 embryos normal 9, 9 2-11 400 27 RO 4 RH 0 No evidence of placentation LO 4 LH 0 10, 11.5 2-11 400 25 RO 3 RH 2 Six normal embryos LO 4 LH 4 11, 10.2 2-11 400 30 RO 5 RH 4 Seven normal embryos LO 2 RH 3 RH - Right uterine horn LH - Left uterine horn

373 results obtained following the use of results obtained in the present study, 11. VICKERY B, McRAE G. Effects of synthetic menstranol in previous studies, as more studies are needed to determine prostaglandin analogue on pregnancy in compared to EE in the present study, the abortifacient effects of menstranol Beagle bitches. Biol Reprod 1980; 22: 438- may be due to differences in potency or ethinylestradiol in dogs. Such stu- 442. and bioavailability of these two com- dies should be aimed at determining 12. TSUTSUI T, TAKANTANI H, HIROSE 0, YAMAU- pounds in the bitch. the effective dose ranges, and periods CHI M. Effects of prostaglandin F2 on The deformity of pups produced by of treatment needed for these com- implantation and maintenance of preg- one bitch treated with ethinylestradiol pounds to consistently produce abor- nancy in the dog. Jap J Vet Sci 1982; 44: during pregnancy is of interest. This tifacient effects in mated bitches. 403-410. finding, although observed in only one 13. JOCHLE W, LAMOND DR, ANDERSEN AC. Men- ACKNOWLEDGMENTS stranol as an abortifacient in the bitch. The- litter, could be an indication that the riogenology 1975; 4: 1-9. compound may be teratogenic in this This study was supported by a grant 14. KENNELLY Ji. The effect of menstranol on species. Data on the teratogenecity of from the Research Advisory Board, canine reproduction. Biol Reprod 1969; 1: estrogens in pregnant dogs are scant University of Guelph. The technical 282-288. (26). assistance of Mr. J. Rahn is gratefully 15. POTTS GO, BEYLER AL, SCHANE HP. Pituitary Contrary to previous reports in acknowledged. gonadotrophin inhibitory activity of other species and humans (15,17), the danazol. Fertil Steril 1978; 25: 367-372. results obtained in this study indicated 16. BARBIERI RL. CANICK JA, MAKRIS A, TODD RB. REFERENCES DAVIES IJ. RYAN KJ. 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