1. New insight in The Ovarian cycle

The more we study the bitch hormonal cycle, the more it appears complex. Indeed, the interest for the bitch reproductive cycle was up to some years ago relatively reduced and in fact often associated with extrapolations from studies in other species. Since 10-20 years, progressively, more and more input has been given in the study of this species and the peculiarity of canine among the vast majority of other mammalians slowly arises. Nowadays, dogs are studied per se but also as models for other species of carnivore particularly endangered species of wild carnivores. This allows for an emerging field of discovery relative to physiology of reproduction.

We will have an overview of the most recent observations and of their consequences in clinical practice and breeding management. Among these, the biology of pregnancy has not been investigated in dogs as vigorously as it has been in other species of common domestic and laboratory animals.

2. The bitch cycle

Variability is certainly the main characteristic of the bitch reproductive cycle and this term can be apply to any part of the bitch reproductive life: urge differences in the hormonal profiles, in the ovarian events, in reproductive behaviour.....

All this makes the clinical work of the practitioner often relatively complex and the one of the breeder even more difficult!

2.1. The hormonal profile of the bitch

In dogs, initiation of oestrus behaviour may be as early as 5 days before ovulation or as late as 3 days after ovulation. Ovulation occurs spontaneously about 2 days after the preovulatory LH surge. Oocytes are ovulated as primary oocytes which undergo maturation slowly, over 2 or 3 days in the oviduct. Following oocyte maturation, many bitches remain fertile for 3 days or longer Dog sperm may survive in the female tract for up to 8 days and still retain motility. In some cases, sperm can survive in the bitch for 5 days before ovulation and still result in fertilization and pregnancy.

The lengthy fertile life spans of the spermatozoa and maybe the mature oocytes may explain the high fertility rates (>90%) often obtained in commercial dog breeding facilities. Ovulation is timed clinically by using serum progesterone assays to detect rapid preovulatory luteinization which occurs during the preovulatory LH surge, or by monitoring the vulval detumescence and vaginal wrinkling that accompany the preovulatory decline in estradiol. Ovulation has also been timed using ultrasound to detect the loss of the anechoic appearance of preovulatory follicular fluid. Ovulation can be timed retrospectively as having occurred 5-7 days before the decline in vaginal cornification at the end of oestrus.

Ovarian and plasma progesterone in this species begins to increase from around the same period estradiol begins to decrease. This happens approximately 24-48 hours before the LH surge. In fact this increase of progesterone appears to be required for the LH surge to occur. Concannon demonstrated that if progesterone does not increase and then the progesterone/estradiol ratio does not change the LH surge may not be observed and ovulation may not occur. This is sometime observed in clinical conditions where prolonged pro-oestrus and follicular cysts are present.

Following this preovulatory luteinization progesterone continues to increase to reach maximum values around days 20-30 and then slowly decreases to reach basal values sometime during anoestrus. In fact progesterone decrease is slow and if values below 1 ng are observed from around days 60-70 after the LH surge (defining this way the end of diestrus), a deep look into serum progesterone concentration allows for the observation that the lowest plasmatic levels of progesterone (around 10-50 pg/ml) are only observed in mid- to late anoestrus. This observation has been confirmed simultaneously by Jeffcoate and our laboratory examining ovarian progesterone contents in anoestrus. It appears clearly that ovarian progesterone content is never basal before days 130-160 of the cycle. The role of this progesterone continuous production by the corpus luteum with regard to oestrous cycle regulation remains to be determined.

The corpus luteum is an ephemeral endocrine gland appearing after rapid growth and differentiation of both theca and granulosa cells. To survive, the luteal cells are dependent of different substances called "luteotropins." The luteal function is regulated by endocrine, paracrine or autocrine luteotropins. The main luteotropins are: LH and prolactin arriving from the pituitary. Indeed, it was demonstrated that hypophysectomy induced immediate abortion in pregnant animals and in non pregnant animal a dramatic drop of plasmatic progesterone (Concannon, 1980, Okkens et al., 1986). The reality of a dynamic luteal regulation in bitch has not yet been established nor studied. However, the relative dependency of the corpus luteum to LH and prolactin seems to be different during the different period of pregnancy. Indeed, the corpus luteum seems to be more independent during the first third of pregnancy and the closest to the LH surge you are the more independent the corpus luteum seems to be. Before day 10 , hypophysectomy is unable to induce corpus luteum functional arrest (Concannon, 1980, Okkens et al., 1986), similarly corpus luteum remains resistant to several indirect luteolytic agents like dopaminergic agonists (bromocriptine Concannon et al., 1987) or GnRH antagonists (Vickery et al., 1987, 1988), and direct like prostaglandin F2a (Concannon et Hansel, 1977). The reasons for this relative and apparent independence of CL during the first third and may be half of pregnancy are still not yet elucidated. This appear to be independent of LH or prolactin receptors variations as Fernandes et al. (1987) observed a constant expression of both receptors during luteal phases in non pregnant animals.

Recently it was demonstrated that if progesterone concentration appears similar in blood from pregnant and non-pregnant diestrus animals, plasma progesterone concentration does not mimic corpus luteum progesterone production. Progesterone production is indeed significantly increase in pregnant animals comparatively to non-pregnant animals. However, metabolism and consumption of progesterone is significantly higher reflected as demonstrated by Concannon and co-workers by a highly significantly increase plasma or faeces concentration of progesterone metabolites.

During pregnant diestrus a concomitant or closely related increase of relaxin and prolactin is observed. The role of relaxin, essentially placenta produced in this species, is not yet clear. Relaxin may play a major role in furthering directly at the corpus luteum levels or indirectly through prolactin, progesterone secretion by the luteal cells of pregnant animals. Indeed, in non-pregnant animals, no increase of prolactin nor relaxin nor progesterone synthesis and metabolism are observed.

2.2. The main questions related to the bitch hormonal cycle and their consequences for breeding management

Many questions remain to be elucidated regarding bitch cycle and regulation. However some are of major importance and either deserve studies or have been studied recently.

 Termination of anoestrus. The bitch is characterised by a long and obligate anoestrus. The factors regulating termination of anoestrus and reinitiation of the next oestrus cycle are not yet clear

 Regulation of corpus luteum during pregnant and non-pregnant diestrus. The most recent studies depict a time related and dose dependent corpus luteum hormono-dependancy with prolactin being the major luteotropin. LH appearing more as a facultative luteotropin enhancing directly or indirectly progesterone secretion.

 Ovulation, maturation, and fecundation of the oocytes in bitches. Recent data suggest that ovulation is probably synchronous for all follicles or supposed to occur on a very short period of time.

3. Control of the reproductive cycle

Methods for contraception, post-coital contraception and induction of abortion in dogs are necessary to prevent the oestrous cycle or the birth of undesirable litters. There are numerous reasons to justify this need: pet overpopulation, preservation of the reproductive potential in animals devoted to breeding, travels, unwanted matings, first estrous matings in young bitches, reproduction management, current health problems or expected problems during labor due to pelvis abnormalities or disproportion between the male and female.

Due to social, cultural, financial or ethical differences, great variations exist between countries in the management of these problems. In North America, the majority of bitches are surgically sterilized and contraception is rarely used. Furthermore the products available to control reproduction are few. In the Scandinavian countries, surgical sterilization being ethically controversial, medical contraception is the main technique for population control. Europe appears to sit in between these 2 extreme positions regarding surgery and is characterized by a particularly large number of commercialized drugs available for reproductive purposes. Similarly, out label use of authorized drugs for humans or other species is relatively common in this part of the world.

The techniques for contraception have not dramatically changed during the last two decades but new methods and development (vaccination, implants, slow release, gene targeting) will be warranted in a close future.

3.1 Cycle control by steroid hormones

Many different steroid hormones have been used with success to control reproductive behavior and cycle in the bitch and queen. No real discovery has occurred in this field of medical contraception so far, however, things have to change and will change in a near future regarding the methods of administrations of drugs already known and on the other hand regarding new drugs or new modes of administration. However, contraceptive steroids derivative of the female pathway, as currently used in large dosages and long-acting formula, are known or supposed to promote acromegaly, insulin resistance, diabetes mellitus, or the development of the cystic endometrial hyperplasia/pyometra complex, mammary development and tumors whereas androgens derivatives may induce external genitalia masculinization. Obesity and increased body weight have also often been described as possible side effects. Therefore, the use of contraceptive steroids is of limited importance and particular attention is given on the route of administration, the dose, the duration of treatment and the stage of the cycle at initiation of the treatment. Recent data have suggested that many of the side effects may be related to the misuse of these agents ( high doses) rather than to the drugs per se. Indeed, for the vast majority of these "old" drugs, no data are available regarding the correct dose, as dose titration studies have not been performed to demonstrate clearly the minimal amount of product needed to prevent the onset of estrous cycle. These studies are warranted and further results will probably demonstrate that a correct use at an appropriate dosage of these drugs may not be associated with significant side effects or even more be beneficial . Misdorp^, in studies on Progestogens and mammary tumors in dogs and cats, pointed out a possible dose-related tumorigenic effect of progestins with the moderate doses being found to be associated with a slight increased risk for benign tumors but no carcinogenic effect and the low doses being associated with a protective effect possibly resulting from the suppression of ovarian function leaving the animals at a level of exposure to sex steroids that is less than under physiological conditions. Further studies are needed before condemning definitively the use of progestins as contraceptive agents in dogs but it is correct to say that their current use is far from being the safest way to control reproduction in dogs and cats.

3.2 Non-steroidal control of cycle

Devices

Vaginal devices have been developed but with non constant or with equivocal results in bitches. Inflammatory reactions or device expulsion result in an unacceptable failure rate. Intrauterine devices are so far not available due to the difficulty in cannulating the canine cervix (reserved to specialists).

Vaccines

Anti-fertility vaccines have been studied in dogs and in other species but with poor or non consistent results^. The problems with GnRH, LH and FSH immunization seem to be related to a poor consistency in response. ZP immunization appears to be irreversible but is still under investigation and development. The main concerns are related to the immunization process (irritation, skin lesions), to the vaccine adjuvant and to the duration of immunity as well as its efficacy. To date, no contraceptive immunization protocol for dogs has been submitted to large-scale clinical trials. However, this is certainly one route for future development.

3.3 Treatments with GnRH agonists or antagonists

GnRH antagonists, if available, would certainly represent one of the main interesting ways of controlling reproduction in dogs. By continuously blocking GnRH receptors at the pituitary level, these antagonists block the synthesis and release of the major portion of LH and FSH. Cyclic control is inhibited whereas modification of the tonic constant release is questionable. Questions on the use of GnRH agonists or antagonists will have to be analyzed by taking into account the new development regarding gonadotrophin secretion and regulation.

References

References are available on request.