Different methods for determining ovulation in dogs. Determination of ovulation in dogs by vaginal smears and saliva crystallization. Cytological studies from personal experience

Ovulation is the process of rupture of a follicle - a vesicle that, under the influence of pituitary hormones, matures in the dog's ovary during each estrus. Determining ovulation is often of fundamental importance, since according to statistics, 80% of dogs that remain infertile after mating were bred at the wrong time.

Additional difficulties arise for owners of dogs with hidden (anemic or bloodless estrus), a significant amount of fur and low libido. In this case, sometimes it is necessary to determine not only the time of ovulation, but also whether the dog is in heat in general.

ovulation begins

The egg leaving the follicle matures for fertilization in 2 days, so the optimal time for mating is 1-2 days after the follicle ruptures. In most cases, it is customary to breed dogs on the 12-14th day of estrus, however, ovulation in different dogs and even in the same dog in different cycles can occur from the 3rd to the 23rd day of estrus.

To determine the moment of ovulation, there are quite a few methods of varying degrees of complexity, both home and medical. An indirect sign of ovulation can be an increase in the bitch's libido, an increase in her attractiveness to males, and a lighter color of discharge from the loop. Many breeders use measurement of glucose levels in vaginal mucus, as well as special devices for determining the electrical conductivity of this mucus.

Methods for determining the onset of ovulation in a dog

If we talk about more scientific methods, there are three of them: vaginal cytology, ultrasound of the ovaries and blood progesterone analysis. All methods have their pros and cons, their limitations.

Vaginal cytology: a smear taken with a sterile cotton swab from the dorsal wall of the vagina. The essence of the method is that under the influence of hormones secreted by the “lid” of the follicle (these hormones are called estrogens), the vaginal epithelium turns from 4 layers to 40 layers, accordingly, a significantly larger number of surface cells are exfoliated, which we find in the smear, having previously colored it .

The advantage of the method is its accessibility and speed - the analysis takes from 5 minutes, but all we can say from this analysis is that the follicle is maturing/ripe. We cannot judge its rupture and release of the egg.

When a follicle ruptures, its bottom begins to turn into the corpus luteum of pregnancy. This process always normally occurs after ovulation, regardless of the fact of mating of the bitch. The corpus luteum of pregnancy secretes the hormone that supports pregnancy - progesterone. In the blood after ovulation, we observe a rapid jump in the level of this hormone - in two days the level of progesterone can rise from 3 to 45 nmol. The advantage of the analysis is high accuracy. If the progesterone level corresponds to the postovulation level, it means the follicle has definitely matured and has definitely ruptured. The difficulty is that taking a blood test for progesterone every day is, to put it mildly, not very convenient.

On an ultrasound of the ovaries, we can see the follicles, approximately count them, observe their rupture, that is, observe ovulation “online”. But this method also has limitations: the ovaries of an overly “rounded” or very large dog (for example, a well-fed Alabai) are not easy to examine; gases that are often found in the dog’s intestines and features of the anatomical structure can also pose difficulties for visualization (in flat and long dogs the ovaries are covered last ribs).

Large breed dogs take longer to develop physically than other breeds. Therefore, the first heat often occurs no earlier than 8 months, and sometimes at 1-1.5 years.

Female domestic dogs usually have two such sexual cycles per year. Often two cycles occur within an interval of 1.5 years. And sometimes bitches shed once every 10-11 months, and environmental factors, daylight hours, temperature, and the like affect only those dogs that are kept free-range. For most dogs living in city apartments, the cyclicity depends more on the characteristics of a particular organism, which can be influenced by a variety of external conditions (rearing, feeding, illness, etc.)

There are four phases of the dog cycle: pre-estrus, heat, post-estrus and rest. What we in practice call estrus actually involves three phases, which are accompanied by bloody-mucous vaginal discharge, as well as characteristic behavior. Fertilization occurs only in the second phase.

Precursor (proestrus) lasts 7-10 days. At this time, the eggs are actively maturing. Thanks to the hormone estrogen, the body actively prepares for mating. The loop in this period is enlarged, bloody discharge is abundant and bright. Despite the fact that the bitch arouses interest in males, and even responds to advances, she does not let the male near and does not allow mounting, demonstratively showing aggression. Bitches with increased sexual excitability can let a male dog in, but due to the fact that the vagina is not yet ready for copulation, both partners usually quickly lose interest in mating.

Hunting (estrus) is the period when the bitch is ready for mating, as the maturation of the reproductive cells begins (ovulation). It usually lasts 3-5 days. However, it can either be reduced to one day or last up to 10 days. During ovulation, an egg is released from the follicle. The egg remains in the ovary for several hours, then enters the reproductive tract.

The egg is viable for 8 days, but after 5-6 days its ability to fertilize sharply decreases, the maturation of the eggs does not occur simultaneously, and therefore ovulation occurs with an interval of approximately 3 hours. The discharge during this period is almost colorless or faintly colored, the cervical canal is open, the labia are very swollen, although Hilary Harmar believes that during the period of ovulation, the labia sharply fall and become flaccid, which indicates the onset of ovulation. When you touch the croup, the bitch moves her tail to the side and spreads her hind legs, taking a stable position. If at this moment you touch the lower part of the loop, where the clitoris is located, the bitch becomes very excited and twitches the loop. During this period, the bitch willingly accepts a male.

Post-estrus is the stage preceding the dormant period if fertilization has not occurred. Its duration is 30-60 days. Progesterone released by the body suppresses the ovulation process, and the reproductive tract returns to its normal state. At the beginning of the period, vaginal discharge changes sharply, the loop “falls off”. The bitch's sexual desire decreases and she snaps at the males.

If fertilization is accomplished, then the return to the original state begins after childbirth and the end of feeding the cubs with milk.

The resting period - for most breeds lasts 90-130 days, for Dogues de Bordeaux it can be longer. Towards the end of the period, hormones stimulate new maturation of eggs and a new cycle begins.

Note:

The sexual cycle of a female dog, despite general trends, is purely individual and within normal limits. There are at least 7 options for the second phase of the sexual cycle - hunting. Researcher Kovalenko E.E. (1993) presents a range of seven options:

1) hunting lasts 3-5 days and 1-3 days after that the discharge ends; in the middle of the period the bitch is as ready as possible for mating;

2) the hunt lasts 5-8 days, the readiness for mating is the same, it is lower than normal, the transition to after-estrus is slow, 3-5 or more days;

3) hunting lasts 1-2 days with maximum readiness, after which the discharge stops and post-estrus occurs;

4) the hunt lasts 6-10 or more days, readiness initially increases, then falls and rises again (2 peaks) towards the end of the period; the transition to after-flow can be both slow and fast; readiness during peaks is the same, but not maximum;

5) the option is similar to the previous one, but the readiness during one of the peaks is much greater than during the other;

6) the option is similar to the previous one, but the interval between peaks of readiness can reach a week or more, during the interval the activity of the gonads is significantly reduced;

7) intermittent estrus: in young bitches, before normal estrus, sometimes there is a peculiar period when bright bloody discharge appears, making one assume that estrus has begun. After 3-7 days, the discharge completely stops. After another 3-7 or more days, and in some bitches almost a month later, estrus begins again. The pre-temperature stage in this embodiment can be very short or normal.

E. Kovalenko also observed an additional pregnancy, when the bitch had two heats, separated by a short interval, and fertilization occurred in each of them. Normal puppies were born within the prescribed period from the first mating, labor ended and the bitch carried the rest to term. The researcher recommends in such cases not to artificially stimulate the continuation of labor, since premature rejection of the remaining fetuses may occur.

In the case of a prolonged estrus, when the time between ovulations is increased, there are few eggs in the female’s body at any given time. Conversely, an active and rapid ovulation process leads to multiple births.

Extended ovulation is typical for bitches with metabolic disorders (obesity) and for old bitches. To obtain the maximum number of puppies in these cases, a larger number of matings are required at intervals of 1.5-2 days and, in addition, the use of males with very active and viable sperm.

In some cases, prolonged ovulation is associated with a decrease in the activity of the gonads. In this case, it is necessary to use methods to increase the activity of the reproductive system: increase physical activity, walking at least 5-6 km with the bitch, and carry out vitamin therapy. An obese dog should reduce the amount of food and the number of feedings. You can arrange fasting days (1-2 per week).

Significant deviations in the sexual cycle, even if they do not lead to infertility, mean hormonal disorders and require specialist intervention. In particular, prolonged estrus may be associated with ovarian hyperfunction, which may be accompanied by changes in the reproductive tract under the influence of excessive amounts of estrogens.

Mild estrus is also a pathology. This estrus occurs at normal times, but the bitch does not show any desire to mate. The cause is hormonal deficiency.

Such unusual heats are probably associated with the dog’s disrupted hormonal levels, which, however, does not always affect fertility and childbearing. However, it is worth paying attention to this circumstance, and if fertility decreases or if the period between estrus is too long, it is recommended to carry out hormonal correction with the help of drugs, be sure to consult a veterinarian.

To eliminate the above-described deviations in the course of the reproductive cycle, a complete biochemical examination of the dog is necessary. The use of hormonal drugs for treatment is always associated with the risk of serious hormonal disorders, therefore the dosage of hormonal drugs should be carried out by a doctor who monitors the dog throughout the course of treatment.

A prolonged absence of estrus may be due to a lack of pituitary and thyroid hormones, as well as estrogen deficiency (weak ovarian function).

After ovulation of the egg, a corpus luteum forms at the site of the ruptured follicle. Corpus luteum hormones play a major role in maintaining pregnancy. In the absence of fertilization, the corpus luteum resolves at the end of the reproductive cycle and, even if the female is not fertilized, sometimes creates a hormonal background corresponding to pregnancy. This state of the bitch is called false pregnancy.

False puppiness, if it is strongly expressed, is accompanied by mental and physiological disorders in the dog’s body.

Sometimes the infertility of a bitch is associated with an incorrectly calculated mating period.

True infertility can be caused by one or a number of different reasons, which can be read about in the specialized literature on dog diseases.

Artificially inducing estrus

In large nurseries where one-time production of offspring is required (for example, if it is desirable to produce puppies only in the spring-summer period), a synchronous onset of estrus in all bitches is possible due to the administration of hormonal drugs. For this procedure, the following drugs are used: gonadotropin-releasing hormone, pregnant mare serum gonadotropin, human chorionic gonadotropin.

Synthetic analogues of the hormone GnRH help increase the number of ovulating eggs if it is administered to female dogs during mating. It is administered to low-fertility bitches to increase the number of puppies born. The treatment regimen, dose and mode of administration of hormones should be developed by a specialist.

The introduction of the hormone oxytocin ensures the synchronous onset of labor. This procedure does not negatively affect the mother's health, and in addition reduces the number of protracted labors requiring the intervention of an obstetrician. It should be noted, however, that an overdose of oxytocin and its analogues can cause excessive contraction of the uterus and rupture of its walls, therefore, when using such drugs, it is recommended to first consult a veterinarian.

The simultaneous mating of bitches and their whelping ensures the raising of age-matched young animals in the most optimal conditions.

Fertilization process

The process of fertilization in dogs is preceded by insemination. During the process of copulation, semen containing spermatozoa is injected into the female genital tract. In dogs, the process of insemination and ovulation are combined in time, thus, fertilization can only be carried out if the bitch’s reproductive tract is completely ready.

At the moment of copulation, the sperm and secretions of the male’s gonads are introduced into the female’s genital tract: first, a clear mucous liquid without sperm is ejected. It acts as a lubricant. A few seconds after the first, even before the formation of a complete lock, a thick whitish liquid with sperm is thrown in.

First, sperm moves due to contractions of the walls of the ducts and cilia on their walls. But as a result of the release of secretions from the prostate gland, sperm acquire the ability to move independently. At the same time, the walls and cilia help them move directionally.

Fertilization does not depend on the time of mating, since sperm is released in excess quantities by the male dog at the very beginning of sexual intercourse. All three stages of ejaculation occur in 1-3 minutes, although a bitch can hold a male for an average of 15-30 minutes, and in some cases a minimum of 5, a maximum of 40-50 minutes or more. Thus, a lock lasting 5 minutes is quite enough for fertilization.

The absence of mating in most cases indicates that the bitch is not ready. However, some bitches are always bred without a lock.

After successful mating, the bitch’s activity usually decreases for some time, so it is not advisable to carry out control mating in less than a day.

Determining the optimal readiness of bitches

Since the size of the litter largely depends on the bitch, it is desirable, if possible, to more accurately determine the period of ovulation of the female. There are several methods:

1) analysis for hormonal readiness;

2) vaginal smear method;

3) indicator method of testing for glucose;

4) visual method.

The first method is relatively labor-intensive and is not yet accessible to our dog breeders. It consists of taking a blood sample from the bitch and, after some manipulations, the color of the sample is compared with the color of control solutions. If the color of the sample is less intense or equal to the lower limit, this means that ovulation has occurred. If the color is between control samples, it will occur within the next 48 hours. This method is quite accurate and it is a pity that we have not put it into practice.

The second method is also effective - the method of vaginal smears, proposed by Candidate of Sciences E.E. Kovalenko and her colleagues, employees of the Department of Vertebrate Zoology of St. Petersburg State University.

This method is well known in Western countries. It is based on the fact that the mucous membrane of the genital tract changes in direct connection with changes in the ovaries during the development and maturation of germ cells. Thanks to samples taken from the vagina of bitches, it is possible to observe the entire process of the sexual cycle.

To obtain samples, a cotton swab on a stick is used to collect mucus in the vagina and this smear is transferred to a glass slide. Samples are taken once a day. Depending on whether we want to get a complete picture of the cycle or determine the days of maximum ovulation, different numbers of samples are taken.

For a complete examination and scheduling, 20-25 samples are needed, starting from the 3-5th day of emptying. If there is a schedule, samples are taken only selectively - 5-8 or one-time - 2-3 to clarify the timing of mating.

Employees of the Department of Obstetrics, Gynecology and Artificial Insemination of Animals of the St. Petersburg Academy of Veterinary Medicine V.U. Davydov, G.S. Stepanov and N.B. Bazhenov improved the cytodiagnostic technique in veterinary gynecology. They argue that throughout the animal's life, the vaginal mucosa is capable of reflecting fluctuations in the level of endogenous hormones, as well as the introduction of exogenous hormones into the body. They have developed a device for obtaining fingerprint smears from the vaginal mucosa. The test material is taken from the cranial third of the upper fornix using a vaginal speculum. The location of the collection was determined due to the fact that this particular part of the vagina is particularly sensitive to hormonal stimulation in mammals. When taking material from the lower vaginal vault, the smears, as a rule, contain cells that were rejected much earlier.

The preovulation period is characterized by the appearance of a large number of parabasal and basal cells in smears. The cells of the superficial layers are few in number, located in isolation, their edges are straightened with large nuclei.

After ovulation, the smear is dominated by superficial cells with pyknotic nuclei. During this period, the cells become “crumpled” and their edges become twisted, and the cell boundaries are unclear. During the heat period and after ovulation, the appearance of secretory cells in smears is observed, their number increases significantly by the middle of the cycle and decreases again in the proestrum phase.

Throughout the entire cycle, neutrophils were detected in smears, the number of which increases significantly after ovulation.

Ideally, the picture of vaginal smears coincides with the analysis of hormonal activity. This indicates the exact time of ovulation and allows you to determine the best time for mating.

The procedure for obtaining a vaginal smear is simple and accessible to every dog ​​owner. In order to receive a vaginal smear preparation, you must have:
1. Distilled water 2-3 liters.
2. Several eye droppers with rounded ends.
3. Glass slides (30 pieces).
4. Petri dishes or Koch dishes (you can also use small saucers).
5. “Sleds” - 2 glass tubes with a diameter of 3-4 mm, a length of 6-7 cm, connected at both ends by rubber tubes, with a diameter that allows them to fit tightly onto glass tubes, 4-6 cm long. The sled is placed on the bottom Petri (or Koch) dishes or saucers. The slide can be successfully replaced with two matches placed on the bottom of cups or saucers parallel to each other. Slides are placed on sleds or matches.
6. Filter paper.
7. A glass (as high as the length of the slide) for rinsing the colored preparation.
8. Dye.
a. Eosinic acid methylene blue according to May-Grunwald or Leishman (ready solution).
b. Giemsa solution (methylene blue and eosin).
Ready-made solutions of these dyes are available in laboratories: medical or biological, where blood smears or vaginal smears are dyed.
9. Glass vessels (small bottles or cups) for dye and distilled water.

Technique for obtaining and staining smears.
1. In a Petri dish (or Koch dish) or at the bottom of a saucer, on a “sled” or on matches, place a clean, washed and degreased dry glass slide.
2. Fill a clean pipette with of its volume of distilled water.
3. Insert a pipette into the bitch’s vagina (if possible without touching its outer edge), release the water and immediately draw liquid from the vagina into the pipette (“rinse” the vagina).
4. Immediately apply the contents of the pipette to the middle of the prepared glass slide.
5. Dry the smear in air, covering the Petri (or Koch) dish or saucer with filter paper from dust.
6. Having collected the dye into a separate pipette, apply 5-6 drops to the dried smear and leave for 3-5 minutes.
7. Apply 5-6 drops of distilled water to the smear so that the water completely covers the smear, leave for another 5-10 minutes, then drain the solution and immerse the glass with the colored smear for a very short time (up to 1-1.5 minutes). into a glass filled to the top with distilled water.
Dry gently between sheets of filter paper.
8. Label the smear. On the edge (any edge, since there is a smear in the middle) of the slide, write: drug number, dog’s name, date and ordinal day of emptying. It is better to write the inscription in ink and fill it with BF glue.
Coloring works better on very fresh smears, but coloring smears that are 2-4 days old is quite acceptable.
The preparation is ready; it should be examined under a microscope at 10-40x magnification.
On a stained preparation (according to May-Grunwald or Leishman), the “scales” are colored reddish, the cytoplasm of epithelial cells is bluish, and the nuclei are blue. Leukocytes are stained blue, almost violet.
When stained by Giemsa: “scales” are dark blue, the cytoplasm is blue, the nuclei are blue. Leukocytes are dark blue.
The intensity of coloring of cellular elements may depend on the freshness of the dye.
Let me briefly dwell on the differences in the cytological picture at different stages of the reproductive cycle.
As mentioned above, the entire cycle consists of four stages or phases, of which we are interested in only three - pre-estrus, estrus and post-estrus (proestrus, estrus and metaestrus), the fourth stage - rest - is the interval between two estrus and is characterized by complete inactivity of the reproductive system.
1. The pre-estrus phase, or preparatory phase, lasts from 9 to 11 days and is characterized by bloody discharge, sometimes profuse; at this time, intensive development of follicles occurs in the dog’s ovaries. The dog is playful, carefully examines all the dogs’ urinary marks, constantly sits down, leaving literally droplets of urine containing a specific smell that attracts males. The concentration of estrogen in the blood increases rapidly and reaches a peak 1-2 days before the end of proestrus. The picture of a vaginal smear at the beginning of the phase is characterized by a large number of round-shaped epithelial cells with rather large nuclei. At the end of the phase (9-10 days from the start of emptying with a normal sexual cycle), individual keratinized, anucleate polygonal cells, the so-called “scales,” appear in the smear.
2. The estrus phase itself lasts from 2 to 10 days and is characterized by almost colorless or pale pink vaginal discharge. In this phase, mature follicles rupture and eggs are released, which after a few hours enter the oviducts, where they remain for up to 7 - 8 days; This is where the meeting with sperm (in case of mating) and fertilization takes place. Eggs have the highest ability to fertilize in the first 5-6 days. The level of the hormone estradiol in the blood sharply increases, which leads to an increase in the number of completely keratinized epithelial cells in the vagina - “scales”. The optimal time for mating should be considered the day when the vast majority of cellular elements on a vaginal smear preparation are represented by scales.
3. The post-estrus or inhibition phase takes about 10-14 days in the sexual cycle and gradually passes into the stage of complete generative rest.
During this phase, vaginal discharge may still occur; it is usually colorless, but can also be pale pink; the bitch becomes calm and aggressive towards male dogs. In place of the burst follicles, corpus luteum forms, producing the hormone progesterone, which suppresses ovulation. Unfertilized eggs are destroyed and die. A vaginal smear shows a small amount of “scales” and an abundance of young epithelial cells, rounded with large nuclei. A good indicator of this phase is the increasing number of leukocytes.
Thus, the picture of a vaginal smear quite clearly characterizes and reflects the physiological state of the bitch and her readiness for mating. It also allows you to evaluate, based on a number of characteristics, the characteristics of the reproductive cycles of families, and the possibility of conception, to identify the causes of “false pregnancy”, low fertility, etc.

Honored Expert of Russia Candidate of Biological Sciences
A.E. Hayrapetyan

The third method is based on the fact that during ovulation, vaginal mucus contains glucose, which can be detected using indicator tapes used by diabetics. A strip of this tape is inserted into the vagina, where it is moistened with secretions. Within 1 minute it turns green. In this case, we can talk about the bitch’s maximum readiness in the next 24 hours. In infantile bitches, the concentration of secretion is lower, so the sensitivity of the indicator tape may be insufficient.

The fourth is the visual method. Our breeders who are not familiar with the methods described above, as a rule, use the visual method. It is the most accessible, but not the most reliable, since the nature of the discharge may not adequately reflect the condition of the bitch.

The recommended average mating period for Dogue de Bordeaux is 12-13 days of estrus, some bitches even later. With each subsequent whelping, the days of ovulation may shift upward. For example, if you first mated your bitch on days 11 and 13, the next mating it is better to do this on days 12 and 14, or even on days 13 and 15. However, each owner should focus on the behavior of his bitch, since the dog itself has a good sense of its physiological state. Usually, to determine whether a bitch is ready for mating, the owner places one hand on the standing bitch's croup, and with a finger of the other hand touches the loop or slightly higher. The ready bitch begins to twitch the loop upward, raise and move her tail, while spreading her hind legs wide. This method allows you to determine the readiness of the bitch for mating in the next 48 hours.

The condition of the bitch's loop naturally changes at different stages of estrus. It gradually swells to its maximum size, but remains hard to the touch. As soon as the loop becomes soft, flaccid, and sometimes hangs like a bag, this means that ovulation has occurred and the bitch is ready for mating.

Male reproductive cycle

It takes several days for sperm to mature. The development of a new batch begins when the previous one is consumed. It follows that the sexual cycle of a male dog depends on the regularity of mating.

It is important to know that in one mating, a male does not consume the entire supply of sperm, even from one testis, and can successfully, with an interval of several hours, fertilize several females. In this case, time is needed only to restore sexual arousal. How many matings a male dog can produce depends on his individual characteristics, namely age, the number of cells in the gonads, and the number of cells ejected during each fertilization. Theoretically, the gonads of a male dog can work continuously as semen is consumed, provided that in the intervals between matings the sperm supply has time to be restored.

In practice, the processes of sperm maturation, the formation and release of semen, and mating itself are a big burden for the male dog and require energy and nervous costs.

Therefore, prolonged intensive use of a male dog depletes his nervous system, negatively affects his health, leads to loss of interest in mating, premature aging and decrepitude of the male dog.
. Factors influencing efficient reproduction in dogs.

Insemination season.
According to some authors (S. Guerra, S. Petit, R. Badino), a decrease in fertility occurs when matings or artificial insemination are carried out towards the end of the summer period, which coincides with a decrease in the quality of sperm in producers. But these studies cannot carry a large share of probability, since a seasonal decrease in productivity is not always observed at this time of year and depends on many factors. In any case, it would be preferable to avoid this period and reproduce animals with a low probability of fertilization in winter to increase the chance of success.

Today, a competent, science-based approach to dog breeding comes to the fore. The high breeding value and uniqueness of some individuals is a prerequisite for a thorough examination of the reproductive system of dogs, not only to exclude infectious diseases, but also to identify the timing of ovulation. This examination is especially relevant for animals traveling for mating to other regions of the country and the world.

Many breeders understand the need to diagnose estrus, however, the lack of information, the fragmentation of data on this issue, as well as the lack of specialists in some cases leads to the receipt of unreliable data or their incorrect interpretation. Thus, the presence of great demand for diagnostics of the reproductive cycle in females was the reason for the development of an algorithm for its study with the derivation of standard indicators and determination of ovulation parameters.

First of all, it is necessary to give a brief description of the reproductive cycle of bitches. The dog is a monocyclic animal, the cycle frequency ranges from 4 to 12 months. The following stages are distinguished: proestrus, estrus, metestrus and anestrus. The onset of estrus, or proetrus, is manifested by corresponding changes in the reproductive apparatus: there is a growth of follicles and an increase in the number of FSH receptors in them. Clinically, this stage is characterized by hyperemia of the vaginal mucosa, swelling of the vulva, and the appearance of bloody discharge. Under the influence of follitropin, together with various growth factors, the development of primordial follicles is stimulated, as well as the differentiation and proliferation of granulosa cells. Of the many follicles that begin to grow, only a few mature. The estrus stage corresponds to heat and ovulation, and it is during this period that mating should be carried out. It is difficult to discern a clear boundary between the stages, however, during estrus there is a softening of the vulva, a change in the nature of vaginal discharge, and the animal’s readiness to mate. Then, at the site of the burst follicle, a cavity is formed, which is quickly filled with granulosa cells filled with the yellow pigment lutein. The development of the corpus luteum occurs during the metestrus stage, regardless of whether fertilization has occurred or not. As you know, pregnancy in dogs lasts 58-65 days, and after its resolution, the stage of anestrus, or sexual rest, begins.

It becomes clear that preparation for ovulation occurs under the influence of a complex of factors, the most important role of which is played by hormones. It is known that estrogens and gestagens regulate proliferative processes in the vaginal mucosa, which consists of multilayered squamous epithelium. Depending on the degree of maturity, the vaginal epithelium can be represented by parabasal, intermediate, superficial intermediate and superficial cells. Morphologically, they differ as follows: parabasal cells are the smallest, round in shape, the nucleus occupies most of the cell. Intermediate cells are approximately twice as large due to the cytoplasm; superficial intermediate cells become thinner and acquire a somewhat angular shape. Superficial cells are the largest, often anucleate, containing keratohyalin granules. In the anestrus phase, the vaginal epithelium is thin and pale, mainly represented by parabasal cells. The ovulatory peak is characterized by the highest degree of maturity of the vaginal epithelium, i.e. detection of more than 90% of superficial cells in a vaginal smear with the highest percentage of karyopyknosis (more than 80%) and high sensitivity to the dye. The development of the corpus luteum leads to the appearance in the smear of a large number of horny scales, leukocytes and intermediate cells.

In our study we set the following tasks:

To identify the dynamics of changes in estradiol and progesterone during the period of proestrus and estrus and compare it with the results of a cytological examination of vaginal smears
analyze data on the timing of mating and fertilization in connection with the endocrine status and degree of maturity of the vaginal epithelium in order to determine the optimal parameters for mating

The study involved 22 dogs aged from 1.5 to 7 years during various periods of proestrus and estrus. The concentration of progesterone and estradiol in the blood serum was determined by the enzyme immunoassay method using a vertical scanning analyzer and special diagnostic kits at the clinical and biochemical laboratory of St. Petersburg State Medical University. Smears were taken with sterile cotton swabs from the lateral vaginal vault; the preparations were fixed with a special May-Grunwald dye and stained with Romanovsky-Giemsa dye. In the smears, the percentage of parabasal, intermediate, superficial-intermediate and superficial cells out of 100 cells was calculated.

Analyzing the results obtained, we identified a general pattern of changes in hormone dynamics for all studied animals and identified the following phases of proestrus:

1. Relative rest phase. Continues from the beginning of emptying for 5-15 days. Despite the pronounced changes in the condition of the external genitalia, the level of progesterone and estradiol is at a relatively low level, not exceeding the normative values ​​corresponding to the stage of anestrus. It is believed that during this period the development of primordial follicles is stimulated, the proliferation of granulosa cells under the influence of FSH.
2. The phase of the beginning of the growth of estradiol. Within 2 - 3 days after the first phase, a slight increase in the concentration of estradiol is detected. During this period, the follicles continue to mature and synthesize a sufficient amount of estrogens, which, in turn, stimulate the release of luteinizing hormone.
3. The phase of the beginning of progesterone growth. Under the influence of luteotropin, steroidogenesis in the follicles switches to the production of progesterone. In the future, the increase in progesterone will continue, bypassing ovulation, up to the metestrus stage. However, one more phase stands out:
4. Estradiol peak phase. The duration of this phase is 1 - 3 days. A sharp release of estrogen is caused by the death of a large number of follicles that have not reached final maturity with the release of a large amount of follicular fluid. Peak concentrations of estradiol, determined in the blood, lead to a powerful release of GnRH by the hypothalamus, and consequently to the secretion of gonadotropic hormones, most notably LH. The preovulatory release of luteotropin promotes a further increase in progesterone levels. The latter, together with FSH and LH, induces the synthesis of proteolytic enzymes in the ovaries, and a complex of factors (natriuretic peptide, angiotensin II, prolactin, neuropeptide Y, vasopressin, bradykinin) increase hydrostatic pressure in the follicular cavity.

Thus, under the influence of the above reasons, the follicular wall ruptures and ovulation occurs. According to various researchers, ovulation corresponds to a progesterone concentration in the range of 35 - 55 nmol/l. In our studies, we found that when progesterone increased to 32 - 43 nmol/l, there was a rather sharp drop in estradiol levels.

The research results are presented in the table.

DYNAMICS OF CHANGES IN THE CONCENTRATION OF PROGESTERONE AND ESTRADIOL DURING THE STAGES OF PROESTRUS AND ESTRUS IN BITCHES

It should be noted that there is a clear relationship between the hormonal status during a specific emptying period and the cytological picture of vaginal smears. Thus, smears taken in the 1st phase of proestrus were characterized by a large number of erythrocytes, the epithelium was represented mainly by parabasal cells (67.4 + 4.3%) and intermediate (30.5 + 2.8%). Superficial intermediate cells were rare (3.1+0.4%). In the second phase, the ratio of cells changed slightly: the content of intermediate cells increased to 47.3+3.7% and superficial intermediate cells to 15.6±2.1%, while the percentage of parabasal cells decreased to 37.1+1.2%. Further estrogenization leads to proliferation of the vaginal epithelium, as evidenced by a change in the cytological profile at stage 3 of proestrus towards an increase in the percentage of superficial intermediate cells (32.4±2.0%) and the appearance of single superficial cells (4.1±0.32%) . The proportion of intermediate cells decreased to 42.0+2.6%, and parabasal cells - to 21.5+1.8%. Quite dramatic cytological changes occurred in phase 4 - peak concentrations of estradiol led to a significant increase in the most mature cells. The percentage of superficial epithelial cells during this period was about 76.3+4.9%, many of which were anucleate. The proportion of superficial-intermediate and intermediate was 20.4±1.6% and 3.3+0.3%, respectively . It should be noted that many cells changed their sensitivity to the dye, becoming zosinophilic. And finally, during estrus, almost all vaginal epithelial cells in the smear were superficial (91.3+ 3.2%), most of them lacked a nucleus. And only 8.7+0.34% were superficial intermediate cells.

We examined 17 bitches to determine the optimal breeding day. The presence of latent infections was previously excluded using PCR diagnostics. During the emptying period, the levels of progesterone and estradiol were determined in each individual, as well as a cytological examination of vaginal smears. We recommended planning mating at a progesterone level of 30-35 nmol/l and again after 48 hours. The fertilization rate was 100%.

Progesterone concentration is a key criterion for determining the timing of ovulation, but determination of estradiol levels and cytological examination of vaginal smears provide valuable clarifying data. When making your first visit during pregnancy, we recommend doing the first test on days 3-5 of proestrus. At low concentrations of sex hormones and the absence of proliferative changes in the vaginal epithelium, repeated tests should be taken after 3-4 days. It should be noted that when progesterone begins to rise, it is possible to predict the dynamics of its changes by doing at least two studies with an interval of 48 hours. According to our research, on average, the level of this hormone in the blood increases by 6.52 nmol/l per day, however, individual schedules should be followed. We recommend using the data we obtained for the practical work of veterinary specialists and dog handlers.

Vasilyeva S.V., Vasiliev R.M.
St. Petersburg State Academy of Veterinary Medicine

Ovulation is the process of rupture of a follicle - a vesicle that, under the influence of pituitary hormones, matures in the dog's ovary during each estrus. Determining ovulation is often of fundamental importance, since according to statistics, 80% of dogs that remain infertile after mating were bred at the wrong time.

Additional difficulties arise for owners of dogs with hidden (anemic or bloodless estrus), a significant amount of fur and low libido. In this case, sometimes it is necessary to determine not only the time of ovulation, but also whether the dog is in heat in general.

ovulation begins

The egg leaving the follicle matures for fertilization in 2 days, so the optimal time for mating is 1-2 days after the follicle ruptures. In most cases, it is customary to breed dogs on the 12-14th day of estrus, however, ovulation in different dogs and even in the same dog in different cycles can occur from the 3rd to the 23rd day of estrus.

To determine the moment of ovulation, there are quite a few methods of varying degrees of complexity, both home and medical. An indirect sign of ovulation can be an increase in the bitch's libido, an increase in her attractiveness to males, and a lighter color of discharge from the loop. Many breeders use measurement of glucose levels in vaginal mucus, as well as special devices for determining the electrical conductivity of this mucus.

Methods for determining the onset of ovulation in a dog

If we talk about more scientific methods, there are three of them: vaginal cytology, ultrasound of the ovaries and blood progesterone analysis. All methods have their pros and cons, their limitations.

Vaginal cytology: a smear taken with a sterile cotton swab from the dorsal wall of the vagina. The essence of the method is that under the influence of hormones secreted by the “lid” of the follicle (these hormones are called estrogens), the vaginal epithelium turns from 4 layers to 40 layers, accordingly, a significantly larger number of surface cells are exfoliated, which we find in the smear, having previously colored it .

The advantage of the method is its accessibility and speed - the analysis takes from 5 minutes, but all we can say from this analysis is that the follicle is maturing/ripe. We cannot judge its rupture and release of the egg.

When a follicle ruptures, its bottom begins to turn into the corpus luteum of pregnancy. This process always normally occurs after ovulation, regardless of the fact of mating of the bitch. The corpus luteum of pregnancy secretes the hormone that supports pregnancy - progesterone. In the blood after ovulation, we observe a rapid jump in the level of this hormone - in two days the level of progesterone can rise from 3 to 45 nmol. The advantage of the analysis is high accuracy. If the progesterone level corresponds to the postovulation level, it means the follicle has definitely matured and has definitely ruptured. The difficulty is that taking a blood test for progesterone every day is, to put it mildly, not very convenient.

On an ultrasound of the ovaries, we can see the follicles, approximately count them, observe their rupture, that is, observe ovulation “online”. But this method also has limitations: the ovaries of an overly “rounded” or very large dog (for example, a well-fed Alabai) are not easy to examine; gases that are often found in the dog’s intestines and features of the anatomical structure can also pose difficulties for visualization (in flat and long dogs the ovaries are covered last ribs).

Operating principle: test - the strip changes color from pink to burgundy during the onset of ovulation. Testing can begin on the first day of the onset of estrus.

Step 1

Wipe your vulva clean and remove any blood.

Step 2

Open the vulva slightly and insert the test strip, the side with the pink indicator, 2-3 cm inward.

Step 3

Leave the test strip in this position for 10 seconds.

Step 4

Pull out the test strip and make sure it has been in contact with vaginal fluid.

Step 5

Read the results and compare them to the snapshot results below if you are unsure.

Negative indicator(ovulation has not occurred)- color test - stripes remain unchanged.

Positive indicator(ovulation has occurred) – a change in color of the test - stripes from burgundy and purple to black indicates the onset of ovulation. Results can vary from a small spot of color to staining the entire indicator surface of the test strip.

Attention! Do not touch the pink indicator surface of the test strip. This may lead to incorrect results.

Remember that ovulation in dogs can occur either 5 or 21 days after the start of estrus.

Dog mating

Dog breeders always need to determine the timing of mating. When should you breed a bitch?

The best time to coat a bitch is the day before ovulation and the first days of ovulation.

Ovulating dogs are the optimal days for mating a bitch. They may vary depending on the breed. They can also “shift” throughout life, even in the same dog. It is generally accepted that as the age of the bitch increases, mating must be carried out at a later date. In most cases this is true. However, there are cases when heat may occur earlier than in previous heats. Although, as a rule, hunting coincides with the period of ovulation. Also, in some dogs, heat may not coincide with the period of ovulation and may occur later than the days when they are most likely to become pregnant.

During dog ovulation, the discharge becomes more mucilaginous and lighter in color.

The color of the discharge, counting the days since the start of heat, the behavior of the bitch and the condition of the vulva are secondary signs that are caused by processes occurring in the body of the bitch. It is quite difficult to determine readiness for mating based on these signs.

Therefore, in order to be sure of the timing of mating, you can use a strip test. Using a test to determine the timing of ovulation in dogs is possible even at home.

For a more accurate result, the test for determining the mating of dogs must be used dynamically (from the beginning of estrus). The result can be compared in the photo above.