EVSSAR symposium Milan 01march2001 part1 · Session 4: Pregnancy and neonatology S. Romagnoli (Pis a Italy) Pregnancy disease and pregnancy failures in the bitch 109 M. Lennoz (Lyon

2.

I

3.

The president words

President Introduction.

European Veterinary Society For

Small Animal Reproduction

/\ Ii(

* Milan 1 March 2001

Dear EVSSAR Member

I am happy to welcome you to this 4

th Meeting of the EVSSAR, in Milan in beautiful

Italy. This will be an exciting meeting with some excellent clinical papers as well as interesting poster presentations, and as all this is matched with the Italian hospitality and cuisine I am sure the meeting 'will be a success like the previous ones in Barcelona in 1998, Lyon in 1999 and Oslo in 2000.

The Society has moved from strength to strength in the year 2000, with the very successful and enjoyable meeting in Oslo as the highlight, and at the moment there are well over 200 EVSSAR members in 30 countries, also several from outside Europe. This coming year will be both stimulating and exciting for Small Animal Reproduction.

The Annual General Meeting of the Society will be held after the general program on March 1s

t, at around 18.30 hrs, and I hope that as many of our members as possible will make the effort to attend. At the meeting we will be holding the elections for new Members of the Society as well as new officers to

Page 3 of 160

EVSSAR SYMPOSIUM Milano 1st March 2001

The president words

replace those resigning from the Board, and we need your ideas about the future activities of the Society!

I will also take this opportunity to remind you to make a note in your calendar that the next EVSSAR congress will be held in Liege, Belgium in May 2002, and am happy to inform you that there are plans to organize the first examination to become Diplomate of the ECAR (European College of Animal Reproduction) in connection with this meeting. You will soon find more information about this in our Newsletter and on our Website

I wish you all a stimulating and enjoyable meeting!

Catharina Linde-Forsberg

Acting President

I would also like to take this opportunity to ask you to check the status of your membership fees, and to complete a new Credit Card Payment Form if your card has expired. Would you please be kind enough to send this information directly and urgently to John Verstegen either by fax (+ 32436 642 31) or by email: ([email protected]) so that they can be processed immediately.

Your fees are essential to allow the work of the EVSSAR to continue.

Page 4 of 160

EVSSAR SYMPOSIUM Milano 15t March 2001

Table of content

Table of contents Program of the symposium

7

The EVSSAR bylaws 9

The 1997 - 2001 EVSSAR board 19

Abstracts of the lecture 21

Session 1: Follicular growth and cycle induction

Mss Luvoni (Milano) Follicular growth and cycle regulation in bitches and queen 21 Eva Axner (Uppsala Sweden) : Oestrus induction in the queen 25 S. Romagnoli (Pisa Italy): Oestrus induction in the bitch 31

Session 2: Semen preservation J. Verstegen (Liege Belgium) Chilled Semen preservation 41 A. Rota (Pisa Italy) Frozen semen preservation 69

Session 3: Artificial Insemination

K. Onclin (Liege Belgium) Breeding management and timing of insemination 83

C. Linde-Forsberg (Upssala Sweden) Semen shipping and banking

for artificial insemination in the bitch 91

Session 4: Pregnancy and neonatology

S. Romagnoli (Pis a Italy) Pregnancy disease and pregnancy failures in the bitch 109

M. Lennoz (Lyon France) Morbidity and mortality in neonate puppies. 115

Abstract of the Poster 129

Information and EVSSAR Registration form 159

EVSSAR Continuous Credit Card authority 160

Page 5 of 160

EVSSAR SYMPOSIUM Milano 1 st March 2001

EVSSAR symposium Milano Program of the day

1 st of March 2001

PROGRAM Fiera Milano

Registration from 8:15

Programme Scientifique Scientific program


9-9:45: Talk I: 40 minutes + 5 min discussion: Mss Luvoni (Milano)

Follicular growth and cycle regulation in bitches and queen

9:45 - 10:15: Talk 2: 25 + 5 min= Eva Axner (Uppsala Sweden) :

Oestrus induction in the queen 10: 15-10:45: Talk 3: 25 + 5 : S. Romagnoli (Pisa Italy): Oestrus

induction in the bitch

10:45-11:30 45 minutes coffee break

Session 2: Semen preservation

11:30-12: 15 Talk 4: 40 + 5 min: J. Verstegen (Liege Belgium)

Chilled Semen preservation:

12:15 - 13:00 Talk 5: 40 + 5 min: A. Rota (Pisa Italy) Frozen semen

preservation

13:00 - 14:15 Lunch

Session 3: Artificial Insemination

14:15-15:00 Talk 6: 40 + 5 min: K. Onclin (Liege Belgium) Breeding

management and timing of insemination

15:00-15:45 Talk 7: 40 + 5 min: C. Linde-Forsberg (Upssala Sweden)

Semen shipping and banking for artificial insemination in the bitch

15:45-16:15: Coffee Break


16:15 - 17:00: Talk 8: 40 + 5 min: S. Romagnoli (Pisa Italy)

Pregnancy disease and pregnancy failures in the bitch

17:00 - 17:45: Talk 9: 40 + 5 min: M. Lennoz (Lyon France)

Morbidity and mortality in neonate puppies.

Session 5: Poster

17:45 - 18: 30 Poster discussion, Poster Award &end ash;

Conclusions and general discussion

EVSSARAnnual general Assembly: 18:30

Page 7 of 160


EVSSAR symposium Milano Program of the day

151 of March 2001

PROGRAM

Fiera Milano

Registration from 8:15

Programme Scientifique Scientific program


9-9:45: Talk I: 40 minutes + 5 min discussion: Mss Luvoni (Milano)

Follicular growth and cycle regulation in bitches and queen

9A5 - 10:15: Talk 2: 25 + 5 min= Eva Axner (Uppsala Sweden)

: Oestrus induction in the queen

10:15-10:45: Talk 3: 25 + 5 : S. Romagnoli (Pis a Italy): Oestrus

induction in the bitch

10:45-11:3° 45 minutes coffee break

Session 2: Semen preservation 11:30-12:15 Talk 4: 40 + 5 mm: J. Verstegen (Liege Belgium)

Chilled Semen preservation:

12:15 - 13:00 Talk 5: 40 + 5 min: A. Rota (Pisa Italy) Frozen semen

preservation

13:00 - 14:15 Lunch

Session 3: Artificial Insemination 14:15-15:00 Talk 6: 40 + 5 min: K. Onclin (Liege Belgium) Breeding

management and timing of insemination

15:00-15:45 Talk 7: 40 + 5 min: C. Linde-Forsberg (Upssala Sweden)

Semen shipping and banking for artificial insemination in the bitch

15:45-16:15: Coffee Break


16: 15 - 17:00: Talk 8: 40 + 5 min: S. Romagnoli (Pisa Italy)

Pregnancy disease and pregnancy failures in the bitch

17:00 - 17:45: Talk 9: 40 + 5 min: M. Lennoz (Lyon France)

Morbidity and mortality in neonate puppies.

Session 5: Poster

17:45 - 18: 30 Poster discussion, Poster Award &end ash;

Conclusions and general discussion

EVSSAR Annual general Assembly: 18:30

Page 7 of 160

EVSSAR SYMPOSIUM Milano 151

March 2001

EVSSAR Board Members 1997-2001

EVSSAR BOARD MEMBERS 1997-2001

President:

M. Lennoz, DVM

Clinique Veterinaire Les Roches Rue Marchandes F 8090 Villefontaine France Tel:

+ 33 4 74 962550

Junior President and President at Interim

C. Linde-Forsberg, DVM, PhD Swedish University of Agricultural Science Department of Obstetrics and Gynaecology Box 7039 S 75007 Uppsala Sweden Tel: + 46 18 672163 Fax: + 46 18 673545

E-mail: [email protected]

Past-President:

J. Verstegen, DVM, Msc, Agrege Enseignement Superieur. Service d'Obstetrique et des troubles de la Reproduction Universite de Liege Bd Colonster 20, Bt 44B 4000 Liege Belgique Tel: + 32 43 664236 Fax: + 3243664231

E-Mail: [email protected]

Secretary

Matteo Spalarossa Via quinot 8R 16166 Genova Italy

[email protected]

Treasurer:

G. England, BVetMed PhD FRCVS MIBiol CertVA DVR DVReprod DiplomateACT The Royal

Veterinary College University of London Department of Large Animal Surgery and medicine

Hawksheadlane Hatfield North Mymms AL9 7TA Herts United Kingdom

Tel: + 49 707 666 236 Fax: + 49 707660

671 E-mail: [email protected]

EVSSAR Newsletter Editor

W. Farstad, DVM, PhD Norwegian College of Veterinary Medicine Department of Reproduction and Forensic Animals Po Box: 8146 N 0033 Oslo Norway

Tel: + 472964855 Fax: + 472565704 E-mail: [email protected]

Board members

A. Prat, DVM Centro Veterinario El Masnou San Miguel 9 08320 El Masnou Barcelona Spain

Tel: + 93 555 73 52 Fax: + 93 555 73 52

K. Lange, DVM Buelowstr. 54, D-26384 Wilhelmshaven, Germany

Tel: + 49-4421-31911. Fax: + 49-4421-42911

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Follicular Growth C. Luvoni Milano Italy

FOLLICULAR GROWfH AND CYCLE REGULATION IN

BITCHES AND QUEENS

Gaia Cecilia Luvoni

Istituto di Clinica Ostetrica e Ginecologica, Facolta di Medicina Veterinaria, U

niversita degli Studi, Milano

Follicular growth and cycle regulation in domestic carnivores have been studied extensively and have led to a number of clinical applications. A different and more ample perspective on the issue has been introduced by the recent development of advanced technologies and biotechnologies. In fact indepth knowledge on follicular growth and cycle regulation in bitches and queens is needed to approach two problems of great topical interest: conservation of biodiversity and overpopulation control.

These are contrasting aspects of research, since the conservation of biodiversity is based on the improvement of reproductive performances, whereas overpopulation control needs an efficient contraception.

The most important tools for the improvement of reproductive performances are assisted reproductive technologies (ART) such as artificial insemination (AI), embryo transfer (ET) and in vitro fertilization (IVF). On the other hand overpopulation control includes surgery, hormonal therapy and more recently immunological control (specifically zona pellucida immunocontraception), which represents a valuable alternative to the previous options.

Thus, in Carnivores, ART are indicated when bitches and queens are valuable pets or when they are considered as a comparative model for Canine and Feline endangered species, whereas zona pellucida contraception has an important role with stray animals.

It is important to stress that AI and ET are successfully applied if cycle regulation is well known, In fact the chance to obtain a pregnancy is strictly related to the optimal time for performing insemination or transfer.

Among domestic animals the dog has a peculiar oestrous cycle. It is well known that the bitch is monoestrous and that the period of fertility begins with a decline in oestrogen and a rise in progesterone associated with the LH surge. Moreover the hormone profiles observed during pregnancy are not very different from those observed in absence of pregnancy since the cyclic corpus luteum lasts longer after ovulation (see Concannon et al., 1989).

Reproductive physiology of the domestic cat is characterized by induced ovulation. Ovulation requires the release of LH determined by copulation, even if recent studies have demonstrated that ovulation can also occur in group-housed females in the absence of mating (Lawler et al., 1993).

In vitro fertilization is still under investigation. The results are encouraging in cats, while in dogs they are still limited, although many studies aimed at the investigation of mechanisms involved in reproductive physiology in Carnivores have been published recently. These studies have been

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performed mainly in vitro and peculiar characteristics of folliculogenesis and gamete

interaction during fertilization have been recognized. In canine species the female gamete has unique characteristics compared to

oocytes of many other domestic mammals. The main differences are represented both by the follicular environment and the oocyte meiotic stage at ovulation. In fact, in most mammalian species, oestrogen dominates the preovulatory follicular environment and the meiotic division is resumed shortly before ovulation as a consequence of the preovulatory LH -surge. Ovulated oocytes are in metaphase II of the meiotic division and are ready for fertilization.

In dogs, ovarian follicles luteinize prior to ovulation, exposing oocytes to high concentrations of progesterone. After the LH -surge, the oocytes are ovulated spontaneously as primary oocytes, at the beginning of the first meiotic division. Subsequent stages of the meiotic maturation are resumed in the oviduct and take 2-3 days to complete (Holst and Phemister, 197

1). Moreover the canine oocyte is very

rich in lipids (Guraya, 1965) and the cumulus cell mass around the oocyte is tight and multilayered and remains attached to the gamete longer after fertilization (Renton et al., 1991). The presence of primary oocytes in the oviducts increases the chance of the oocyte meeting spermatozoa before or during maturation and it has been shown that canine sperm can penetrate homologous immature oocytes (Mahi and Yanagimachi, 1976). Significant differences in the glycosylation of the dog zona pellucida glycoproteins compared to other species have also been . investigated (Barber et al., 1999).

In addition, the canine embryos require a long period for the passage through the oviduct and enter the uterus 7-9 days after ovulation as an embryo of 16 cells or more (Holst and Phemister, 1971). Thus, the oviduct supports long term survival of oocytes that in this tract complete maturation, undergo fertilization, and develop up to the morula-blastocyst stage.

In cats the ovulated oocyte is in metaphase II of meiosis and it also is very dark in appearance because of a high intracellular concentration of lipid (Guraya, 1965). The cat zona pellucida is bilayered and the inner zona layer appears to function as a partial barrier to sperm penetration affecting the number and kind of sperm entering the oocyte (Andrews et al., 1992).

As far as zona pellucida immunocontraception is concerned some attempts have been performed to establish a vaccination with purified zona pellucida glycoproteins and,

although the preliminary results are promising,further studies are needed to allow this technology to be efficiently and safely applied in practice.

From what has been mentionned above, it is evident that Carnivores represent an interesting model for the studies concerning physiological mechanisms of reproduction of which some aspects have been already clarified, but we are still far from a thorough

understanding of the phenomena.

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References.

- Andrews J.C., Howard J.G., Bavister B.D., Wildt D.E. (1992), Sperm capacitation in the domestic cat (Felis catus) and leopard cat (Felis bengalensis) as studied with a salt-stored zona pellucida penetration assay, Mol. Reprod. Dev. 31, 200-207. - Barber M.R., Merkle R.K., Fayrer-Hosken R.A. (1999), Evaluation of carbohydrates of the dog, cat and elephant zona pellucida using lectins. Theriogenology 51, 278. - Concannon P.W., McCann J.P., Temple M. (1989), Biology and endocrinology of ovulation, pregnancy and parturition in the dog. J. Reprod. Fertil. Suppl. 39, 3-25.

- Guraya S.S. (1965), A histochemical analysis of lipid yolk deposition in the oocytes

of cat and dog, J. Exp. Zool. 160, 123-136.

- Holst P.A., Phernister R.D. (1971), The prenatal development of the dog.

Preimplantation events, Biol, Reprod. 5, 771-779. - Lawler D.F., Johnston S.D., Hegstad R.L., Keltner D.G., Owens S.F. (1993), Ovulation without cervical stimulation in domestic cats, J. Reprod. Fertil. SuPp

I47,57-

61.

- Mahi C.A., Yanagirnachi R. (1976), Maturation and sperm penetration of canine ovarian oocytes in vitro, J. Exp. Zool. 196, 189-196. - Renton J.P., Boyd J.S., Eckersall P.D., Ferguson J.M., Harvey M.J.A., Mullaney J., Perry B. (1991), Ovulation, fertilization and early embryonic development in the bitch (Canis familiaris l.,l. Reprod. Fertil. 93, 221-231.

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Oestrus induction in the queen A. Axner Uppsala Sweden

OESTRUS INDUCTION IN THE QUEEN

Eva Axner-

Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden

BACKGROUND

In natural conditions the female domestic cat is seasonally polyoestrus, with a long seasonal anoestrus from September/October to January in the northern hemisphere. The cat is a long photoperiod breeder and decreasing hours of daylight induces the anoestrus period (12). Household cats kept under the influence of artificial light may be non-seasonal breeders and it is possible to produce kittens throughout the year in a cat colony kept under a controlled light program (10, 13). It is more common for cats that are allowed outdoors to have a non-breeding season than for cats confined indoors (10). Genetic factors probably influence the individual female's sensitivity to changes in daylight, since Jemmet and Evans (10) found that a period of anoestrus was more common for longhaired than for shorthaired breeds.

Methods for artificially inducing oestrus are of particular value for embryo transfer and in vivo fertilisation protocols. Oestrus induction may also be of value for breeders wishing to breed their queen during the natural anoestrus period or in cases of delayed puberty or prolonged anoestrus. Natural methods of oestrus induction such as social stimuli or light stimuli are harmless in these cases, but careful consideration should be given, and a thorough investigation be performed before using exogenous hormones, since there are potential side effects related to the use of these hormones.

METHODS FOR INDUCTION OF OESTRUS

Social stimuli and light manipulation The most natural methods to induce oestrus in the anoestrus female are social stimuli and light stimuli. Placing the queen with other cycling females or with a male cat may shorten the period to oestrus (12). Since the cat is a long-day breeder it is possible to induce oestrus by increasing the day length. The exact mechanism that controls the seasonal cyclicity in the female cat has not been studied but it is believed that changes in light affects the reproductive process via the pineal gland and the hormone melatonin (11). The concentrations of melatonin and prolactin are higher under a day length regimen with 8 hours of light in 24 hours than with 14 hours of light. The concentrations of both these hormones are lower during oestrus than during interoestrus. Keeping queens in a photoperiod of 8 hours of light in 24 hours will suppress oestrus (11) while cats maintained with a minimum of 10 hours of light may cycle year-round (15). A light flash for one hour during the dark period shortens the

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EVSSAR SYMPOSIUM Milano 15t March 200 1

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time to oestrus (12). In a home environment where females experience a change in the length of natural light through windows, response to artificial light stimuli is more unpredictable than in a colony of research cats. The change in day length outdoors can be a stronger stimulus than the artificial light indoors (Hakan Andersson, personal communication).

FSH-P

FSH is secreted by anterior pituitary cells. It stimulates follicular growth and oestrogen production. Repeated injections are required due to the relatively short half-life. FSH preparations also have inherent LH activity, which may vary from lot to lot (1, 19). Different regimens have been used to induce oestrus in the anoestrus queen. Doses of 2.0 mgjday until oestrus gave a pregnancy result of 71% (5 of 7 queens) after natural matings with a mean litter size of 5.8 kittens. Oestrus was seen after an average of 4.6 days from the first injection. It was recommended that this treatment should not be continued for more than 5 days due to the hyperstimulative effect of this hormone (19). Dresser et al. (6) found that a lower dose of 0.75 mgjday FSH for 5 days and 0.25 mg day 6 followed by 375 IU hCG given day 6 and 7 was efficient for embryo recovery. Treatment of prepubertal cats results in high numbers of unovulated follicles (I8). It seems as if FSH-P treatment may compromise embryo quality and fertilization rates in IVF (7). Formation of ancillary follicles is seen after FSH - P treatment, probably as a result of the residual effect of this hormone (19).

eCG

Equine chorionic gonadotropin is secreted by foetal trophoblastic cells of the endometrial cups in the horse. eCG principally exerts FSH-like activity but it also has LH-like bioactivity (1). It has a long half-life, estimated to be between 39 and 55 hours (17), and therefore a single injection is enough to induce follicular growth, which is an advantage over FSH-P. Repeated injections with eCG can lead to the formation of gonadotropin-neutralizing immunoglobulins and a decreased effect of this treatment. An interval of at least 4 months between treatments is therefore recommended to avoid ovarian refractoriness (16). Treatment with eCG is usually combined with hCG treatment to induce ovulation for artificial insemination or IVF. Treatment with hCG should probably be avoided in cases of natural mating where the mating stimuli will induce ovulation (14). Doses of !OO or 150 IU eCG i.m. followed by 75 or 100 IU ~G 84 hours later is recommended (4, 8, 9). Using this regimen Howard et al. (9) Obtained a 50% pregnancy result in 12 females laparoscopically inseminated with fresh semen after ovulation. Higher doses of hCG should not be used since it results in more degenerated oocytes and lower fertilisation rates in vitro (8). The oocytes are sensitive to the interval between the two stimuli. If hCG is given too early after the eCG treatment it appears as if final oocyte maturation is impaired (8). An interval between eCG and hCG that exceeds 88 hours on the other hand gives more degenerated oocytes (4). Females treated with eCG do not always display overt oestrus behaviour but usually have follicles with mature oocytes (5). Queens have been naturally mated with pregnancy results of 78% (7 of 9 queens) after injection of lQQJY eCG day 1 followed by 50 IU hCG day 7, the same day as mating. New data ~.__ '':;c:~

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suggests that mating 80 to 88 hours after eCG might be better (4, 8, 14). Increasing the dose of eCG compromises pregnancy results (2). Wildt et al. (19) using total doses of 500 to 1000 IV eCG found that some queens ovulated before mating, probably as a result of the LH component in the eCG preparation. As with FSH-P, formation of ancillary follicles is seen after injection with eCG (9, 19), which may be attributed to persistence of eCG (17). Young females (<1 year) that are treated with eCG tend to produce cystic follicles that do not ovulate (3). Treatment with eCG does not seem to compromise egg or embryo quality (5, 14).

Table 1.

Oestrus induction Ovulation induction Referenc

es eCG 100 to 150 IV hCG 75 to 100 IV 80 to 88 hours after 4,8,9,14

injection of eCG eCG 100 IV day 1 Mating and 50 IV hCG day 7 . 2

eCG 100 IV Mating 80 hours after eCG 14

FSH-P 2.0 mgjday for 5 Mating each day in oestrus 19

davs FSH-P 0.75 mgjdays 1 to 5 hCG 375 IV day 6 and 7 and mating from 6

days 0.25 mg day 6 day 6

SUMMARY

Induction of oestrus can easily be achieved with the use of exogenous hormones in the queen. There are, however, potential side effects related to the use of these hormones. It is easy to over stimulate the ovaries. After injection with FSH - P or eCG there is a delayed secondary follicle development and CL formation after ovulation (8, 19). Exogenous hormones should not be used to induce oestrus in prepubertal females since it results in formation of large numbers of unovulating follicles (2, 18). Natural methods can be considered harmless and should always be the first choice. Social stimuli may, however, not always be possible or efficient, and an efficient light stimulus is not always practical in a home environment.

REFERENCES

1. Carruthers TD. Principles of hormone therapy in theriogenology. In:

Morrow DA ed. Current Therapy in Theriogenology. 2nd ed. Philadelphia. WB

Saunders, 1986: 3-13.

5. Cline EM, Jennings LL, Sojka NJ. Breeding Laboratory cats during artificially induced estrus. Lab Anim Sci 1980;30:1003-1005.

3. Colby ED. Induced estrus and timed pregnancies in cats. Lab Anim Care

1970;20:1075-1080.

4. Donoghue AM, Johnston LA, Munson L, Brown JL, Wildt DE. Influence of

gonadotropin treatment interval on follicular maturation, in vitro

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fertilisation, circulating steroid concentrations, and subsequent luteal function in

the domestic cat. Biol Reprod 1992; 46:972-980.

5. Donoghue AM, Johnston LA, Goodrowe KL, O'Brien SJ, Wildt DE.

Influence of day of oestrus on egg viability and comparative efficiency of in vitro

fertilization in domestic cats in natural or gonadotrophin-induced oestrus. J Reprod Fert 1993;98:85-90.

6. Dresser BL, Sehlhorst CS, Wachs KB, Keller GL, Gelwicks EJ, Turner JL.

Hormonal stimulation and embryo collection in the domestic cat (Felis catus).

Theriogenology 1987;28: 915-927.

7.Goodrowe KL, Howard JG, Wildt DE. Comparison of embryo recovery,

embryo quality, oestradiol-t zf and progesterone profiles in domestic cats (Felis

catus) at natural or induced oestrus. J Reprod Fert 1988a;82:553561.

8. Goodrowe KL, Wall RJ, O'Brien SJO, Schmidt PM, Wildt DE.

Developmental competence of domestic cat follicular oocytes after fertilisation in

vitro. Biol Reprod 1988b;

39:355-372.

9. Howard JG, Barone MA, Donoghue AM, Wildt DE. The effect of preovulatory anaesthesia on ovulation in laparoscopically inseminated domestic cats. ,J Reprod Fert 1992;96:175-186.

10. Jemmet JE, Evans JM. A survey of sexual behaviour and reproduction of female

cats. J small Anim Pract 1977;18: 31-37.

11. Leyva H, Madley T & Stabenfeldt GH. Effect of light manipulation on ovarian

activity and melatonin and prolactin secretion in the domestic cat. J Reprod Fert.

SUppI1989;39: 125-133.

12. Michel C. Induction of oestrus in cats by photoperiodic manipulations and social

stimuli. Lab Anim 1993;27:278-280.

13. Robinson R, Cox HW. Reproductive performance in a cat colony over a 10year

period. Lab Anim Sci 1970;4:99-112.

14. Roth TL, Wolfe BA, Long JA, Howard JG, Wildt DE. Effects of equine chorionic

gonadotropin, human chorionic gonadotropin, and laparoscopic artificial

insemination on embryo, endocrine, and luteal characteristics in the domestic cat.

Biol Reprod 1997;57:165-171.

15. Shille VM, Sojka NJ. Feline reproduction. In: Ettinger SJ, Feldman EC eds.

Textbook of Veterinary Internal Medicine. Philadelphia. WB Saunders,

1995;1690-1698.

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16. Swanson WF, Roth TL, Graham K, Horohov DW, Godke RA. Kinetics of the

humoral immune response to multiple treatments with exogenous gonadotropins and relation to ovarian responsiveness in domestic cats. AJVR 1996a;ST 302-307.

17. Swanson WF, Graham K, Horohov DW, Thompson DL, Godke RA.

Ancillary follicle development and secondary corpora lutea formation following

exogenous gonadotropin treatment in the domestic cat and effect of passive

transfer of gonadotropin-neutralizing antisera. Theriogenology 1996b;4S:S61-S72.

18. Verstegen JP, Onclin K, Silva LD, Donnay I, Mettens P, Ectors F.

Superovulation and embryo culture in vitro following treatment with ultrapure

follicle-stimulating hormone in cats. J Reprod Fert Suppl 1993;47:209-218.

19. Wildt DE, Kinney GM, Seager SWJ. Gonadotrophin induced reproductive

cyclicity in the domestic cat. Lab Anim Sci 1978;28: 301-307.

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Oestrus induction in the bitch. S. Romagnoli Padova Italy

Clinical considerations on oestrus induction in the bitch

Stefano Romagnoli, DVM, MS

Istituto di Patologia e Igiene Veterinaria, Facolta di Medicina Veterinaria

University of Padova, Italy - if +39-049-827.2948, fax 827.2602, JeJ [email protected]

INTRODUCTION

Canine anoestrus lasts 2-10 months. During this stage, FSH is present in relatively high concentrations while LH is low (Olson et al., 1982). An increase in sensitivity to GnRH followed by release of FSH and LH and by an increase in oestradiol concentrations have always been known to be the main events occurring prior to the onset of proestrus. Therefore, oestrus induction in the bitch has been achieved in the past mostly with estrogens and/or gonadotropins, two classes of drugs, which anticipate the onset of heat, by acting at the ovarian and at the pituitary level.

Over the last decade a major advance in this field has been the discovery of the role of prolactin (PRL) in regulating length of anoestrus in the canine. It is now well established that lowering PRL concentrations in the bitch is a key endocrine feature characterizing the transition from anoestrus to proestrus. This led to a number of experimental and clinical trials on oestrus induction using antiprolactinics, a class of compounds which inhibit PRL secretion by stimulating dopamine (a PRL inhibiting factor) or by decreasing serotonine (a PRL stimulating factor). Currently, anti-PRL drugs are considered by many clinicians among the most reliable compounds to induce fertile oestrus in the bitch, although GnRH as well as other gonadotropins are still considered fairly effective treatments. This paper will:

a) review oestrus induction protocols in the canine considering

products which are commercially available and clinically useful

such as estrogens, gonadotropins and antiprolactinics;

b) present clinical considerations on patient selection and choice of drug;

c)provide criteria for a critical evaluation of papers on oestrus induction in the

bitch.

A REVIEW OF CANINE OESTRUS INDUCTION PROTOCOLS

Oestrogens - Folliculogenesis is initiated and guided by rising levels of estrogens, which enable FSH to induce LH receptors on granulosa cells (Richards, 1980) thereby increasing responsiveness to basal LH concentrations and causing further follicular growth and oestradiol production. Treatment of anoestrous bitches with 200 Ilg oestradiol (once every other day for 3 days) consistently causes proestrus-like vaginal discharge 4-7 days later, persisting for up to 7 days (Chakraborty et al., 1982). This indicates that exogenous oestradiol is capable to start follicular growth: the resulting priming of the pituitary by endogenous oestradiol causes the LH

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surge and ovulation. Using 3 injections of increasing doses of stilbestrol dipropionate (from o.o5 to 1.0 mg) Renton et al. (1981) obtained normal oestrus behaviour in 4/4 and pregnancy in 0/4 bitches, respectively. Moses and Shille (1988) obtained 100% of pregnancies in bitches treated with diethylstilbestrol (DES) protocol combined with porcine LH and FSH given around ovulation time. Using a similar DES protocol at the dose of 5 mg /dog/day orally for 6-9 days combined with 3 injections of FSH-P administered around the expected ovulation time, Bouchard et al. (1991) obtained oestrus in 6/8 and pregnancy in 3/8 bitches treated in late anoestrus, respectively. Using DES alone the same authors obtained oestrus and pregnancy in 5/5 treated dogs (Bouchard et al., 1993). Oestrogen pretreatment has improved efficacy of some gonadotropin treatments; for instance, when pre-treated with 100-300 Ilg/ day of oestrone for 5-6 days anoestrous bitches showed vaginal bleeding; when further treated with 200400 IU PMSG and 1000 IU HCG once on the day of withdrawal of bleeding and on the day of oestrus, 6/7 bitches conceived and delivered normal litters (Takeishi et al., 1976). Potential side effects of estrogens (pyometra, bone marrow aplasia - not investigated with these protocols) as well as anecdotal evidence of a relatively low efficacy (Verstegen, 2001; Root-Kustritz, 2001) undoubtedly make estrogens not the most popular choice among small animal clinicians for canine oestrus induction. However, DES is still favoured by some authors, who report a satisfactory efficacy when acting fairly close to the expected date of proestrus onset (Bouchard, 2001).

Gonadotropins - PMSG is the drug most intensively investigated in experimental protocols for oestrus induction in the bitch. Its combined FSHlike and LH-like activity provides an excellent ovarian stimulation. It has been used at doses of 5OO-1000 IU/dog or 20 IU/kg/dog for 5-10 days (Wright, 1980; 1982). Pregnancy rates vary between 0 and 25% when used alone and between 0 and 5o% when used combined with HCG (Thun, 1977; Renton et al., 1981; Allen, 1982; Arnold et al., 1989). The most common problem when using PMSG is the unpredictability of the bitch's response with regard to onset of oestrus and number of developing follicles. This is due to the fact that potency of PMSG can vary considerably from one lot to another, resulting in various degrees of ovarian stimulation, which are quite difficult to predict. Also, PMSG is a large molecule and may cause an immunologic or refractory response to repeated injections. Because of variations in potency of different lots as well as its inherent LH-like activity, PMSG can result in ovarian hyperstimulation if used carelessly. PMSG treatments longer than 5 days have been associated with:

a) increased incidence of hyperoestrogenism with peak oestradiol values of

300-500 pg/ml (normal is 5O-100 pg/ml in late proestrus) leading to

abnormal vaginal bleeding, thrombocytopenia and aplastic anaemia (Arnold et al., 1989);

b) reduced fertility probably due to an inhibitory effect on implantation (Arnold et al., 1989).

When 5-day and 10-day 20 IU/kg PMSG treatments (combined with 5OO IU HCG on the last day) were compared, pregnancy rates were 5o% and 0%,

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respectively, and incidence of hyperoestrogenism was 0% and 29%, respectively (Arnold et al., 1989). When deciding length of a PMSG treatment in order to maximize its efficacy, one should keep in mind that two split doses of 500-1000 IU of PMSG given 6 days apart or 20-50 IU/kg/day of PMSG given for 9 days appear equally effective in inducing follicular development and oestrous behaviour (Chakraborty et al., 1982). This means that PMSG protocols should be carefully monitored in order to avoid hyperstimulation. The 5-day 20 IU/kg/day protocol (combined with 500 IU HCG on the last day) has proven effective and safe (Arnold et al., 1989; England and Allen, 1991)

Variability of response to PMSG could be due to relative amounts of FSH-like activity of different lots. Concentrations of endogenous FSH are high during anoestrus, while LH is low. The use of FSH alone is often not capable of inducing follicular growth and signs of oestrus even when used at high, repeated doses (Chakraborty et al., 1982; Shille et al., 1984), while LH alone seems to be effective: Verstegen et al. (1997) treated anoestrous bitches 3 times daily for 7 days with 0.1 IU /kg of a purified porcine LH preparation: 12/16 treated bitches showed heat within 16+3 days and 7/16 conceived and whelped. Therefore, in the anoestrous bitch LH alone is capable of causing resumption of follicular growth in the bitch leading to ovulation.

Antiprolactinics - Currently, there are 3 PRL-Iowering drugs which are commercially available: cabergoline (Galastop, a veterinary compound) and bromocryptine (Parlodel, a human compound) which are dopamine agonists (they increase the concentration of dopamine, a PRL-inhibiting factor); and metergoline (Contralac TM, a veterinary compound) a serotonineantagonist (it lowers the concentration of serotonine, a PRL-stimulating factor). All these 3 products have been used for oestrus induction in the bitch. Cabergoline and bromocryptine have consistently given positive results, while metergoline's results have been more variable depending on dosage. An 1M formulation of metergoline administered at the dose of 12.5 mg/bitch every 3 days until the first signs of vulvar swelling and discharge, induced oestrus and ovulation within 40 days of treatment in 10/12 bitches and pregnancy in 9/12 bitches (Handaja Kusuma and Tainturier, 1993). Using a much lower dose (0.1 mg/kg BID) of the commercial oral preparation of metergoline administered from 100 days after ovulation until the following proestrus, Okkens et aI. (1997) were not able to shorten the interoestrous interval significantly. The administration of bromocryptine in anoestrus will induce oestrus within 2850 days (Concannon, 1993; van Haaften et al., 1993). We have used bromocryptine at the dose of 10-25 ).lg/kg in 5 bitches with prolonged anoestrus: 4/5 came in oestrus within 13-28 days, and all 4 conceived and whelped (Sbragia et al., 1999). Using cabergoline (5 ).lg/kg, once daily for up to 28 days) or natural PGF2alpha (100 ug/kg SC, BID for 5 days starting on cytological dioestrus day 10) we achieved an interoestrous interval of 6 months in 6 treated bitches as opposed to an interval of 9 months in 9 control bitches (Rota and Romagnoli, 2000). We have also used cabergoline (5 ug/kg, once daily for up to 28 days) in 9 bitches (7 Rough Collies, 1 Shetland sheepdog and 1 English Setter) for a total of 11 cycles: fertile oestrus was induced in 10/11 cycles in 24±11 days with a reduction of the interoestrous

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interval of 1.8+0.2 months (Rota et al., unpublished). In our experience, the clinical use of antiprolactinics to induce oestrus has proven to be safe and highly effective. Side effects are minimal (particularly with cabergoline), being mostly related to the gastrointestinal tract (nausea, rare vomiting) with no other reproductive effect (unlike estrogens or PMSG which can both cause oestrogen toxicity).

CONSIDERATIONS :FOR A CLINICAL APPROACH TO CANINE OESTRUS INDUCTION

Presence of functioning corpora lutea - It is of utmost importance to confirm that dioestrus is finished. The bitch has an unusually long luteal phase which spans over the first 2 months after ovulation with serumjplasma progesterone (P4) concentrations consistently >2.0 ngjml. In pregnant bitches, P4 drops rapidly (within 24-48 hours) to ~ 2.0 ngjml prior to parturition, while in the non-pregnant female such a drop occurs during several days starting about 65-70 days after ovulation. During dioestrus, when P 4 is ~2.0 ngjml, bitches tend to respond poorly, if at all, to oestrus induction and induced oestruses are generally not fertile (Verstegen et al., 1999). The luteal phase is followed by another 2-3 months (or less if anoestrus is short) during which P4 concentrations gradually go from 1.S±o.S ngjml down to 0.5-0.0 ngjml. During these first 2-3 months of what is technically considered anoestrus (but which could as well be considered a prolongation of dioestrus), the bitch's responsiveness to oestrus induction protocols is low at the beginning and then gradually increases. However, a P4 concentration < 2.0 ngjml does not necessarily mean that the bitch is deeply into anoestrus and that a fertile oestrus can be induced. Therefore, it is probably not adequate to try to discriminate between bitches that are ready for oestrus induction vs. those that are not using a P4 concentrations of 2.0 ngjml as a threshold. It is probably wiser to state that the lower the P 4 concentration, the higher the efficacy of oestrus induction treatments. Drawing a blood sample to assay P4 should be done whenever history is unavailable and or the owner is not sure about the date of onset of the previous oestrous cycle. Although any type of P 4 assay will be better than no P4 assay, semi-quantitative assays generally discriminate only between> or < 2.0 ng/ml, while a quantitative assay (such as a radioimmunoassay) will enable clinicians to understand how deep the bitch is into her anoestrus.

Ovulation - Although the bitch is a spontaneous ovulator, most oestrous induction protocols use HCG (500-1000 IV per day for 1-2 days) or GnRH (one injection of 50 microgram/dog or two injections six hours apart) to ensure that ovulation occurs at the end of the induction treatment or after 2-4 days of oestrus behaviour (Chakraborty et al, 1982). It is generally accepted that these two drugs can have only a positive, if any, effect on the outcome of the induction treatment, due to the fact that their only action is to cause release of LH from the pituitary. This is particularly true for GnRH (whose molecular size makes it almost invisible to the immune system) while HCG can give rise to antibody formation, which might make this compound unsuitable if the induction treatment needs to be repeated at subsequent cycles. However, one

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Oestrus induction in the bitch. S. Romagnoli Pad ova Italy

should consider that also induction protocols which do not use HCG or GnRH have proved highly effective, which may question the rationale for trying to induce ovulation in the bitch. Also, in some cases where (both gonadotropin and antiprolatinic) treatments with or without HCG were compared, the use of H CG had a negative effect on follicular development and the hormonal milieu (Jones et al., 1973; Barta et al., 1982; Nakao et al., 1985; Handaja Kusuma and Tainturier.uqog}. A negative effect of HCG or GnRH could be due to a wrong timing of administration. Saturating ovarian LH receptors (with HCG) or depleting pituitary LH reservoirs (with GnRH) when follicles are still not ready to rupture might have a detrimental effect on ovulation, such as to cause follicles to luteinize without rupturing.

How late is "late anoestrus"? - Although there is not a universal agreement among researchers and clinicians about what is a late anoestrus, the last 4-6 weeks of reproductive quiescence can be assumed to be late anoestrus (Verstegen et al., 1999). Most bitches cycle every 6 months. However, starting an oestrus induction treatment 4.5 to 5 months after the previous oestrus may not necessarily be successful in the single bitch. Indeed, individual females or bloodlines of some breeds may have interoestrous intervals of 7-9 months, and bitches of breeds like the Basenji or the Collie often cycle once/year. Therefore, collecting an accurate history about onset of previous oestrous cycles is a key to success. Owners who cannot provide adequate historical information about the reproductive status of their bitches should be cautioned about the poor success rate of oestrus induction protocols.

When to treat during anoestrus: early, mid or late? - Efficacy of oestrus induction treatments in different periods of anoestrus can be summarized as follows: late anoestrus> mid anoestrus> early anoestrus. In general terms, a bitch treated in late anoestrus is much more likely to respond than a bitch treated in early anoestrus. However, variability in treatment length (interval from the start of treatment to onset of proestrus) has been assessed precisely only in a recent study on the effects of cabergoline in which duration of treatment in bitches treated in early, mid and late anoestrus was 26, 18 and 10 days, respectively (Verstegen et al., 1999). A complete set of experimental data such as the one from Verstegen et al. (1999) has not been produced yet for PMSG or any other compound available to induce oestrus in the dog. However, for practical purposes one can safely assume that even with PMSG oestrus induction will be more successful if treating late anoestrus as opposed to early anoestrus bitches. Nevertheless, the success of an oestrus induction treatment depends on the presenting complaint. A fertile oestrus occurring within a reasonable interval from treatment start can be acceptable when dealing with a case of prolonged anoestrus; however, this may not be considered satisfactory when the request is to reduce the interoestrous interval and/or to achieve more whelpings/year. For instance, the average interoestrous interval of 5 bitches treated with cabergoline in late anoestrus was not shorter than in control dogs (Verstegen et al., 1999), whereas shortening of the interoestrous interval was 61 and 29 days in bitches treated in early and mid-anoestrus, respectively (Verstegen et al., 1999). As a general rule, treatments in early anoestrus tend to be longer and less effective than

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treatments in late anoestrus; however, when opting for late anoestrus one must weigh a higher degree of success against a minimal or no difference in the

interoestrous interval. In order to obtain a more significant shortening of the

interoestrous interval, a shortening of dioestrus (using a luteolytic treatment) can

be combined with a shortening of anoestrus. Bitches can be treated with a 5-8 days course of prostaglandin Fzalpha (PGF) starting about 10-14 days after the

onset of cytological dioestrus or 16-20 days after ovulation. Natural PGF

compounds (100 IJ.g/kg SC BID) or synthetic analogues such as cloprostenol (1-

5 ug/kg SC BID) or alphaprostol (20 IJ.g/kg SC BID) can be used safely in the

bitch to cause luteolysis thereby shortening dioestrus. Both natural and synthetic

PGF can be used once daily and every other day (instead of daily BID) if the

bitch is being concurrently treated daily per os with cabergoline.

Abnormalities in the oestrous cycle - Incidence of split heat in naturally occurring canine oestrous cycles is thought to be around 1-2% (Arbeiter, 1989), although very little epidemiological data is available on this topic. Incidence of split heats in

induced canine oestrus is unknown. However, split heats are reported in induced

canine cycles (Verstegen et al., 1997). Also, as split heats are due to a relatively

abnormal hormonal milieu around ovulation (e.g. lack of LH), they are likely to be at least as (if not more) common when heat is induced. Lower degrees of vaginal

cornification as well as higher oestrogen and lower or shorter progesterone profiles

have been reported when inducing heat with gonadotropins (Jones et al., 1973; Barta

et al., 1982; England, 1991). Veterinarians should carefully monitor any induced cycle, making sure that ovulation occurs using at least vaginal cytology and P 4 assay.

How TO CRITICALLY READ A PAPER ON OESTRUS INDUCTION IN THE BITCH When

reading information on new drugs/protocols for canine oestrus induction,

clinicians should keep in mind a few key questions to be asked about each paper.

Table n" 1 shows key issues and the type of information which ideally papers

should convey on each issue.

KEy ISSUE THE IDEAL SITUATION

Was anoestrus History on previous interoestrous intervals as well as the date of las

clearly defined proestrus of treated dogs should be available. Serum P 4 should hav

and confirmed been measured at least once shortly prior to the onset of the stud

in treated with a quantitative assay.

bitches? Treating bitches in different stages of anoestrus produces differen

Were control results. The same protocol used in bitches in mid-anoestrus vs. lat

bitches in the anoestrus mayor may not shorten the interoestrous interva

same stage of (Verstegen et al., 1999). It is important that a range of anoestrus a

anoestrus? short as possible and as similar as possible be used both for treate

and control animals. An acceptable range should probably be 20-50 days depending on th

Did the length of anoestrus. If anoestrus lasts 7 months, each phase (earl)

treatment start mid and late) can be assumed to last about 9 weeks: therefore, i at the same time order to avoid overlapping treatments, each range should be kep

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or within an acceptable range in all

bitches?

Was pregnancy the end product

of the experiment?

Can different protocols be compared?

below 9 weeks. If average anoestrus of experimental animals is : months,

then each range should be no longer than 20 days.

Fertility of an induced oestrus should always be verified by mating th bitch to a fertile male and performing pregnancy diagnosis. A vas majority of oestrus induction protocols will cause proestrus and oestrus, many will cause a serum P 4 rise suggestive of ovulation, but only few will be followed by pregnancy. Too many things can go wrong when treating a bitch with hormones: she may show oestrus without ovulating; she may secrete progesterone as a consequence of luteinization of follicles not followed by ovulation; or she may conceive and implantation may not occur due to abnormally higl levels of estrogens.

Due to differences in stage of anoestrus between bitches in different papers, it is extremely difficult to compare protocols on oestrus induction. Even when dosages, time and route of administration are identical, the best-looking protocol may not necessarily be the most effective one, and a comparison may lead clinicians astray. Only when stage of anoestrus is clearly defined can comparative evaluations be safely made.

Table n? 1 - Key issues to be critically assessed when reading a paper on canine oestrus induction and the ideal experimental situation for each

one them.

REFERENCES

Allen WE - Attempted oestrus induction in four bitches using PMSG. J Small An

Pract 28: 233-231, 1982

Arbeiter K - Anovulatory ovarian cycles in dogs. J Reprod Fert SuPp147:453-

456,1989

Arnold S, Arnold P, Concannon PW, Weilenmann R, Hubler M, Casal M, Dobeli M,

Fairburn A, Eggenberger E, Rusch P - Effects of duration of PMSG treatment on

induction of oestrus, pregnancy rates and the complications of hyper-estrogenism in dogs. J Reprod Felt Suppl 39: 115122,1989

Barta M, Archbald LF, Godke RA - Luteal function of induced corpora lutea in the

bitch. Theriogenology 18: 541-549, 1982.

Bouchard G, Youngquist RS, Clark B, Concannon PW, Braun WF - Oestrus induction

in the bitch using a combination diethylstilbestrol and FSH - P. Theriogenology 36: 51-65, 1991

Bouchard G, Gross S, Ganjam VK, Youngquist RS, Concannon PW, Krause GF,

Reddy CS - Oestrus induction in the bitch with the synthetic estrogen

diethylstilbestrol. J Reprod Fert, SuPp147: 515-516, 1993

Bouchard G - Comment on the small animal reproduction discussion list

CAFEREPROD, February 6, 2001. (*)

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Concannon PW - Biology of gonadotrophin secretion in adult and prepubertal

female dogs. J Reprod Fert SuPp14T 3-27, 1993

England GCW, Allen WE - Repeatability of events during spontaneous and

gonadotrophin-induced oestrus in bitches. J Reprod Fert 93:443-448 (1991) J eukenne P, Verstegen J - Termination of dioestrus and induction of oestrus in

dioestrus nonpregnant bitches by the prolactin antagonist cabergoline. J

Reprod Fert Suppl 51:59-66, 1997

Jones GE, Boyns AR, Bell ET, Christie DW, Parkes MF - Immuno reactive luteinizing hormone and progesterone during pregnancy and following

gonadotropins administration in beagle bitches. Acta Endocr., Copenh 72: 573-

581, 1973

Moses DL, Shille VM - Induction of oestrus in Greyhound bitches with prolonged idiopathic anestrus or with suppression of oestrus after testosterone

administration. JA VMA 192:1541-1545 (1988)

Okkens AC, Kooistra HS, Dieleman SJ, MM Bevers - Dopamine agonistic effects

as opposed to prolactin concentrations in plasma as the influencing factor on the duration of anoestrus in bitches. J Reprod Fert Suppl 51, 5558,1997

Olson PN, Bowen RA, Behrendt M, Olson JD, Nett TM - Concentrations of

reproductive hormones in canine serum throughout late anoestrus, proestrus

and oestrus. Biol Reprod 27:1196-1206,1982

Renton JP, Munro CD, Heathcote H, Carmichael S - Some aspects of etiology,

diagnosis and treatment of infertility in the bitch. J Reprod Fert 61:289-

294,1981

Richards JS - Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation. Physiol Rev 60:51-

89,1980

Root-Kustritz - Comment on the small animal reproduction discussion list

CAFEREPROD, February 6, 2001. (*) Rota A, Romagnoli S - One year of breeding management in a dog kennel.

Proceedings 4th International Symposium on Dog and Cat Reproduction. Oslo, June 29-Aug 2,2000, pag 83

Sbragia G, Rota A, Vannozzi I, Ballabio R, Romagnoli S - Clinical use of

antiprolactinic drugs for oestrus induction in the bitch. Proceedings 24th

Congress World Small Animal Veterinary Association, Lyon, France, September 1999

Shille VM, Thatcher MJ, Simmons KJ: Efforts to induce oestrus in the bitch

using pituitary gonadotropins. JAVMA 184 (12): 1469-1473,1984

Takeishi M, Kodama Y, Mikami T - Studies on reproduction in the dog. XI:

Induction of oestrus by hormonal treatment and results of the following

insemination. Jap J Anim Reprod 22:71,1976

Thun R, Watson P, Jackson GL: Induction of oestrus and ovulation in the bitch

using exogenous gonadotropins. AJVR 38:483-486,1977

van Haaften B, Dieleman SJ, Okkens AC, Bevers MM, Willemse AH Induction of

oestrus and ovulation in dogs by treatment with PMSG and/or bromocriptine. J

Reprod Fert SuPp139: 330-331, 1989

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Verstegen J, Onclin K, Silva L, Concannon PW - Termination of obligate anoestrus

and induction of fertile ovarian cycles by administration of purified pig LH. J Reprod Fert 111:35-40, 1997

Verstegen JP, Onclin K, Silva LDM, Concannon PW - Effect of stage of anoestrus on

the induction of oestrus by the dopamine agonist cabergoline in dogs.

Theriogenology 51:597-611, 1999 Verstegen .J - Comment on the small animal reproduction discussion list

CAFEREPROD, February 6, 2001. (*) Wright PJ - The induction of oestrus in the bitch using daily injections of PMSG.

Australian Vet J, 59: 123-124,1982 Wright PJ - The induction of oestrus and ovulation in the bitch using PMSG and

HCG. Australian Vet J, 56: 137-140, 1980

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EVSSAR symposium Milan 01march2001 part1 · Session 4: Pregnancy and neonatology S. Romagnoli (Pis a Italy) Pregnancy disease and pregnancy failures in the bitch 109 M. Lennoz (Lyon

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