Monitoring of the reproductive cycle in the female dog is necessary in order to determine the stage of the oestrus cycle; this is fundamental for the management of breeding strategies and particularly for the management of artificial insemination, to identify reproductive disorders, to have an indication on the whelping date, to choose the best timing for elective ovariectomy and, in general, for the optimal management of dog breeding farms.
The oestrus cycle can beassessed by evaluating the female dog’s behaviour, especially when in the presence of the male, by analysing the modification of reproductive organs (via colposcopy, ultrasonography, colpocytology) and via the determination (RIA, ELISA) of circulating hormones, particularly of progesterone (P4) and luteinizing hormone (LH).
OESTRUS CYCLE
The duration of the inter-oestral interval, i.e. the period between one cycle and the next, is on average of seven months, with inter and intra-individual variations ranging from 3.5 to 13 months, partly due to environmental factors. Although seasonality is accentuated in certain breeds, such as in the Basenji, in which females show oestrus only in autumn, in the majority of dog breeds a clear influence of the season on the oestrus cycle has not been established and oestrus can be present throughout the year; a tendency in favour of a higher frequency during the late winter-early spring and again in late summer-early autumn has however been observed by some authors.
The oestrous cycle of the bitch is normally divided into four stages: proestrus, oestrus (follicular phase), dioestrus (luteal phase) and anoestrus, depending on the blood concentration of oestrogens, LH and progesterone.
The bitch ovulates spontaneously towards the end of the follicular phase, the mean duration of which is of around 3 weeks. In parallel with follicular development there is a progressive increase in circulating oestrogens, which starting from baseline levels of 2-10 pg/ml then reacha peak of 50-120 pg/ml; a concomitant progressive rise of testosterone and androstenedione is also present. Increased oestrogen production is responsible forthe physical signs of proestrus: bloody vaginal discharge, vulvar and perineal hyperaemiaand oedema, hypertrophy of the mucosa of the caudal portion of the vagina, elongation of the uterine horns, the formation of endometrial folds and thickening of the cervix. High oestrogen concentrations also promote the secretion of pheromones, with the consequent attraction of males, even if the bitch is still not sexually receptive. The behaviour associated with proestrus usually lasts 5 to 10 days, but can be limited to 2-3 days or extended up to 3-4 weeks. The transition between proestrus and oestrus is characterized by the start of sexual receptivity, with the bitch acquiescently accepting the male for mating. In most cases this takes place in association with maximum follicular development, the decline in circulating oestrogens, the rapid increase of follicular luteinization (which had started previously and which is related to an increase in progesteronaemia) and with the preovulatory release of pituitary LH, followed, 2 days later, by ovulation. The change in behaviour (rejection/acceptance) during the proestrus/oestrus transition phase can be rapid (8-12 hours) or slower (1-3 days). The beginning of oestrus is usually synchronous with the LH ovulatory surge; however, it may at times precede (2-3 days) or follow (4-5 days) LH release.
The most striking feature of proestrus consists in vulvar hypertrophy and oedema and blood discharge resulting from uterine diapedesis; these signs may appear simultaneously or progressively in 1-4 days. Proestrus usually lasts from 5 to 9 days, but can be reduced to 2-3 days or extended up to 3-4 weeks. The expanding vulvar oedema reaches its peak in late proestrus, coinciding with the oestrogen peak. During this phase the bitch, while still attracting males in view of the production of one or more pheromones (methylhydroxybenzoate), is not sexually receptive and tends to retract, snarl and fight when males try to mount it. The increasing oestrogen levels progressively predispose to a more playful attitude towards the male, but the bitch still remains passive and indifferent to mounting attempts, and does not allow copulation. During late proestrus the bitch may acquire a masculine attitude (mounting of other dogs with coital-like pelvic movements), due to increasing concentrations of androgens (testosterone and androstenedione). With the rising levels of oestrogens colposcopy shows a pinkish vaginal mucosa with oedematous, rounded and turgid mucosal folds.
During proestrus, colpocytology is characterized by a progressive reduction in parabasal and small intermediate cells and by an increase in large intermediate and superficial cells, which, in the proximity of the LH surge, make up almost the totality of vaginal epithelial cells. Rare neutrophils and abundant red blood cells are also present, although they tend to progressively decrease; the presence of mucoproteins and cellular debris, as well as of bacteria, may complete the colpocytological pattern (Figs. 1 and 2).
Ultrasonography shows increased endometrial thickness, as a function of possible embryo implantation (Fig. 3); the ovaries appear as isoechogenic oval formations (1.5-2 cm) and the follicles as rounded formations, which are totally anechogenic during the development phase (from <5 mm to 6-9 mm) (Fig. 4).
During oestrus the bitch, while maintaining a playful behaviour, tries to attract the interest of the male, accepts courtship (sniffing and licking of the vulva), mating and penetration. Some bitches show a preference for males with a clear willingness to mount, while sexually dominant females may reject males considered as hierarchically inferior. The onset of oestrus occurs rapidly, from 8 to 32 hours after the LH surge; it can however be asynchronous: from 3 days before (transient decline of oestrogen or rise of progesterone before the final oestrogen peak) to 4-6 days after the LH surge (relative insensitivity of the nerve centres to the initial decline in the oestrogen to progesterone ratio). In a typical cycle, the oestrous behaviour lasts from 6 to 12 days; this can at times be reduced to only 2-3 days, when the beginning of oestrus is delayed with respect to the LH surge and ovulation, or protracted to up to 3 weeks.
Just prior to the LH surge the rounded and smooth summit of the vaginal folds becomes slightly crumpled, sacculated or plicated, acquiring a more wrinkled, pointed and jagged appearance; these changes probably reflect the sudden fall in oestrogen-dependent fluid retention.
The complete, or nearly-complete keratinization (95-100%) of superficial cells, the concomitant reduction in cells of the non-superficial epithelial layers of the vaginal mucosa and the disappearance of erythrocytes and leukocytes (Fig. 5) signify that follicular maturation and oestrogen secretion have reached a level capable of stimulatingthe LH surge, with the subsequent consequent ovulation. Vaginal smears performed in conjunction with the ovulatory LH surge are characterized by their sharpness, probably as a result of a reduction in vaginal mucoprotein content (Fig. 6).
Ultrasonography at the time of ovulation is characterized by the disappearance of the follicles, while the ovaries become difficult to locate; however, just before ovulation, a thickening of the follicular wall may be present, due to preovulatory luteinisation, together with the presence, a few hours after ovulation, of a tiny accumulation of periovarian fluid (Fig. 7).
The duration of metoestrus/dioestrus, which typically lasts 2-3 months, may vary depending on the criteria used (end ofmale acceptance, termination of the keratinization process and the presence of leukocytes at colpocytology). Progesterone secretion continues to rise, reaching maximum levels 20-30 days after the LH surge, to then decrease in the remaining 30-100 days. Clinically, no evident physical signs are present; the vulva returns (size and turgur) to its initial state before oestrogen secretion; mating is rejected and the male stops showing interest for the female. During the regression phase of progesteronaemia mammary hypertrophy becomesevident, starting from around 35 days after the LH surge, even in the absence of a pseudopregnancy. Mammary hypertrophy and uterine discharge, characteristic of the second half of the luteal phase, may result from a modest increase in prolactinaemia. In fact, prolactin receptors have been identified at mammary and endometrial level which increase in proestrus, decrease during the initial stages of dioestrus and then subsequently rise again, apparently depending on the oestrogen to progesterone ratio. Colposcopy is characterized by the presence of flattened and flaccid vaginal folds. Colpocytology, performed 7-8 (6-10) days after the LH surge, reveals a sudden decrease in superficial cells, which are replaced by intermediate and parabasal cells, indicating the beginning of dioestrus (Fig. 8); leukocytes are also present (Figs. 8 and 9). Typical of this phase of the cycle is the presence of metoestral cells (parabasal cells that have phagocytosed a neutrophil) and foam cells (parabasal cells with vacuolated nuclei).
Ultrasonography of the ovaries allows the detection of the corpora lutea: roundish, hypoechoic, slightly dishomogeneous formations, 6-9 mm in diameter (Fig. 10).
Anoestrus is the transition phase from an oestrous cycle to the next; it is characterized by the presence of circulating levels of progesterone <1 ng/ml and its duration ranges from 1 to 6 months. Occasional rises of FSH and oestrogen have been reported during anoestrus but with no physical signs of oestrogenic activity present, probably due to the sporadic nature of these increases. During this phase of sexual rest the uterus resumes its normal size: the uterine horns shorten, the utero-tubal junction is reconnected and the endometrial glands regress.
The colpocytological pattern is characterized by low cellularity, with the presence of mostly parabasal and intermediate cells, and, frequently, with few, often aggregated leukocytes (Fig. 11). Visualization of the ovaries byultrasonography is difficult as the ovaries are isoechoic with the adipose tissue of the ovarian bursa.
MONITORING OF OVULATION
Given that neither the clinical evaluation of the genital organs nor the animal’s sexual behaviour are sufficient to predict with satisfactory reliability the day of ovulation, additional investigations are necessary. Colpocytology is only of retrospective value, as the sudden appearance of intermediate and parabasal cells is detected on the fifth post-ovulatory day. Vaginal endoscopy, although capable of indicating the fertile period, does not allow an accurate prediction of the day of ovulation.
Hormonal assays are instead of considerable support. Identification of the ovulatory LH surge is the ideal method, however LH plasma concentration assays are rather time consuming (2 samples per day) and expensive. Progesterone assays seem sufficiently reliable in indicating the day of ovulation, which is characterized by plasma levels of at least 11 ng/ml.
ENDOCRINE CONTROL OF THE OESTRUS CYCLE
The adenohypophysis is involved in the reproductive process via the secretion of luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin and growth hormone (GH). As the name implies, FSH promotes the growth and development of the antral follicles of the ovary and has also a role in the synthesis of oestrogens. LH is also involved in normal follicular development and maturation, as well as in steroidogenesis, being responsible for the conversion of cholesterol into progesterone, the precursor of androgens and oestrogens.LH is considered the ovulation hormone (mean ovulatory surge 8-15 ng/ml); during this process, the follicles are luteinized and transformed into corpora lutea, which secrete progesterone, the activity of which is supported by both LH and prolactin (luteotropic activity). In the dog, as in other species, the LH release is pulsatile, with a time interval between pulses ranging from 1.5 to over 7 hours. During anoestrus the circulating levels of LH are low (~ 1 ng/ml), with occasional pulses (2-25 ng/ml) at intervals of 4-24 hours. In the week preceding proestrus the interval between pulses (> 3 ng/ml) is usually of around 1-2 hours, probably as a stimulus for the development of the relevant ovulatory wave; during proestrus, levels decrease because of the negative feedback of oestrogens. LH concentrations reach their peak level (8-15 ng/ml on average) 1-3 days after the oestrogen peak. The LH (and FSH) pituitary release mechanism is stimulated by gonadotropin-releasing hormone (GnRH), a decapeptide of hypothalamic origin, and is inhibited by oestrogens. In fact, LH levels are chronically elevated in ovariectomized bitches and may be suppressed with the administration of oestrogens. During the normal transition from proestrus to oestrus the reduction in the oestrogen-progesterone ratio, which is registered with follicular maturation, is the triggering factor for the LH surge, with ovulation within 36-50 hours. The basal levels of LH during dioestrus have a luteotropic effect for the functioning of the corpora lutea. In contrast to oestrogens, progesterone does not appear to have a negative feedback effect on the release of LH; in fact, in ovariectomized bitches the administration of high doses of progestogens do not reduce LH levels.
Basal FSH levels tend to increase throughout anoestrus, with no signs of oestrogenic activity present; the role or the bioactivity of FSH during this phase of the cycle of the bitch remains therefore unclear, as well as the timing of FSH-induced aromatase activity or the possible variation in the levels of FSH receptors. Furthermore, it is also unclear whether the increase in oestrogen secretion at the beginning of proestrus is caused by an increase in aromatase synthesis or if it is the result of LH-induced androgen synthesis already available for aromatase. In the dog, as assumed for the pig, the significant autocrine and paracrine activity might perhaps be responsible for the development of dominant follicles as well as for the acquisition of receptors for LH and for the expression of insulin-like growth factor-binding protein 4. Indeed, it is interesting to note that both the dominant follicles in the late follicular phase of the pig and the follicles in mid-proestrus of the dog exhibit clear morphological signs of luteinization.
Oestrogens rise throughout the entire duration of proestrus, passing from baseline levels of 5-10 pg/ml to values of 45-120 pg/ml, usually 1-3 days before the LH ovulatory peak. The rapid decline of oestrogens after the ovulatory surge probably reflects an inhibitory effect of LH on the aromatase of mature follicles. As a result, with the exception of a slight rise during metoestrus, oestrogens remain at baseline levels throughout the entire luteal phase; oestrogens also rise during the last third of pregnancy, without however reaching proestral levels.
During proestrus the low basal levels of testosterone (around 0.1 ng/ml), typical of anoestrus, tend to gradually increase, similarly to oestrogens, reaching maximum values (0.3-1 ng/ml) immediately after the oestrogen peak and concomitantly with the LH ovulatory surge; they subsequently decrease (0.2 ng/ml) throughout the entire luteal phase. The evolution of androstenedione levels, which are low (0.2 ng/ml) during anoestrus, is partially similar to that of testosterone, but with higher values (0.6-2.3 ng/ml). In addition, androstenedione levels, which follow the evolution of progesterone, remain high during dioestrus, with or without pregnancy, with mean levels of 0.5-0.7 ng/ml. High androgen levels during proestrus reflect the production of follicular oestrogens, being a substrate for the enzymatic activity of aromatase.
Progesteronaemia, the lowest levels of which (0.2-0.4 ng/ml) are found during anoestrus (adrenal origin?), rises slowly during proestrus (0.6-1 ng/ml), reflecting partial follicular luteinization already 6 days before the LH surge. This rise, which could represent an excess of precursors for the synthesis of oestrogens, starts 1-2 weeks before proestrus.
The sudden and significant progesterone increase (2-3 ng/ml) is concomitant with the start of ovulatory LH secretion, to the point that the two events cannot be dissociated one from the other. Follicular luteinization during and immediately after the ovulatory surge seems to affect the morphology of granulosa and theca interna cells, suggesting a major contribution of these tothe formation of corpora lutea.
The gradual secretion of progesterone (15-80 ng/ml) requires, as luteotropic factors, both LH and prolactin, the luteotropic activity of which is evident starting from day 25, even if it probably already starts at day 12. During gestation the prolactin-induced luteal activity is significantly more pronounced compared to that of cyclic corpora lutea, even if progesteronaemia does not register significant differences, probably due to increased metabolism and the plasma dilution of progesterone. Starting from the third week of dioestrus, progesteronaemia undergoes a 30-50 days trend of slow decrease, down to values <1 ng/ml (0.6-0.3 ng/ml). It seems, therefore, that the corpora lutea of the bitch are ephemeral formations with the aim of supporting a possible gestation, without any apparent signs of luteolysis as in other species. The slow decline of progesterone in the non-gravid bitch does not seem to be caused by the action of prostaglandins, but rather by the progressive reduction in the expression of 3β-hydroxysteroid dehydrogenase, differently from what is recorded in the gravid bitch 12-24 hours before whelping with the PGF2α-induced fall in progesteronaemia (<1 ng/ml).
Suggested reading
- Concannon P.W.: Canine Physiology of Reproduction. In Burke T. “Small Animal Reproduction and Infertility. A Clinical Approach to Diagnosis and Treatment” Lea & Febiger, Philadelphia, 1986, pp: 23-77.
- Concannon P.W., Castracane V.D., Temple M., Montanez A. (2009). Endocrine control of ovarian function in dogs and other carnivores Animal Reproduction, 6 (1): 172-193.
- Feldman E.C., Nelson R.W.: Canine and Feline Endocrinology and Reproduction. Ed 2, Philadelphia, W.B. Saunders, 1996.
- Hașegan I., Șonea A., Matei M., Vintilă L., Ion C., Bîrțoiu A. (2012). Current Relevant Knowledge on Dog Reproductive Physiology – a Review Animal Science and Biotechnologies, 45 (1): 172-180.
- Wiebe V.J., Howard J.P. (2009) Pharmacologic Advances in Canine and Feline Reproduction. Topical Review,24 (2):71-99.