Blood tests for progesterone in a Siberian Husky. Laboratory diagnostics

Progesterone is a female sex hormone. Its main task is to maintain pregnancy in dogs.

When are progesterone levels prescribed?

assessment of aspects of ovarian function in bitches and cats;
determination of the time of ovulation to determine the time of mating (in bitches);
predicting the date of birth;
confirmation of the presence of a remnant of ovarian tissue;
assessment of the function of the corpus luteum in cases of abortion;
detection of asymptomatic estrus;
detection of the presence of luteal cysts, etc.

In veterinary practice, progesterone levels are most often examined to determine the optimal mating time. This is extremely important when artificial insemination with frozen or chilled semen, or when the male is at a great distance and you need to know the exact mating date in order to bring the bitch or male.

How to prepare an animal for research?

There is no need for special preparation. Blood sampling is usually done in the morning on an empty stomach. The main condition is that the animal should not take drugs that affect the level of progesterone.

When is the test to be carried out?

A study of the level of progesterone can be carried out every 2-3 days, starting from 3-5 days from the onset of estrus. Usually, the analysis is ready within 24 hours. The average analysis time is up to 4 hours.

What is the research method?

The study is carried out by the method enzyme immunoassay(IFA).

This is a quantitative method for determining the level of progesterone in the blood serum. The study is performed in the laboratory using an appropriate enzyme immunoassay analyzer using special reagents.

The principle of determining progesterone is based on the use of a competitive ELISA method. Mouse monoclonal antibodies to progesterone are immobilized on the inner surface of the wells of the tablet. The test sample's progesterone competes with conjugated progesterone for binding to antibodies on the surface of the well. The result is a plastic-bound "sandwich" containing peroxidase. During incubation with the tetramethylbenzidine substrate solution, staining of the solutions in the wells occurs. The intensity of the color, which is determined on the analyzer, is inversely proportional to the concentration of progesterone in the test sample.

How to interpret the results of the analysis?

The quantitative value of progesterone in the blood is determined in the pre- and postovulatory periods. The level of progesterone in the blood serum, both in different dogs and in the same individual (from cycle to cycle) can change quite quickly.

Averages are presented in table.

Results in different laboratories may vary. This depends on the settings of the apparatus and reagents. An accurate interpretation of the results should be carried out by a veterinarian who has the appropriate qualifications and experience.

Optimal mating time:

When the progesterone level reaches 15.9 nmol / l (5 ng / ml), mating is carried out after 24-48 hours.

Artificial insemination with chilled semen is carried out 4 days after reaching a progesterone level of 7.95 nmol/l (2.5 ng/ml) or 48 hours after reaching 15.9 nmol/l (5 ng/ml).

AI with frozen semen is performed 5 days after 7.95 nmol/L (2.5 ng/mL) or 72 hours after 15.9 nmol/L (5 ng/mL)

What is the benefit of this study?

Determining the timing of ovulation allows you to increase not only the percentage of successful matings or artificial insemination, but also fertility. BUT additional methods studies based on the use of vaginal swabs and

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Platonova N.P., Ph.D. sciences, senior researcher,
Chernushenko O.V., doctor veterinary medicine, Veteko LLC
Satska L.V., student of NUBiP of Ukraine
The article was published in the journal "Modern Veterinary Medicine" No. 3, 2013

Female mammals produce progesterone corpus luteum(VT) of the ovaries during the luteal phase of the sexual cycle and persists with the onset of pregnancy, inhibits the formation of follicles and maintains pregnancy until the placenta is fully formed, which takes part in the development of the fetus, and therefore the production of progesterone VT gradually stops. Males do not produce this hormone. Progesterone is a steroid hormone. Progesterone and its synthetic analogues used in humanitarian and veterinary medicine under common name progestins, or gestagens, are a powerful tool for correcting the reproductive function of both productive animals and hobby class animals.

Progesterone inhibits the activity of the myometrium and stimulates the development of the endometrium of the uterus, it regulates the development of the mammary glands during the luteal phase of the sexual cycle. Progesterone preparations suppress the secretion of gonadotropic hormones, and, consequently, the follicular phase of the bitch's sexual cycle. High doses of progesterone have a sedating and stabilizing effect on nervous system due to the fact that it is a precursor of the neurosteroid aallopregnanolone, which has a pronounced antidepressant effect and is used in humanitarian medicine to correct neurological disorders.

Progesterone preparations are used in bitches:

to prevent estrus by subcutaneous or oral administration during anestrus and by subcutaneous or oral administration during proestrus;
for treatment clinical signs false pregnancy(due to the suppression of prolactin secretion);
for the treatment of estrogen-dependent tumors of the mammary glands;
for the prevention of miscarriages, however, in this case it is necessary to correlate the possible positive and negative effects of such prevention.

Progesterone preparations are used in males:

to suppress aggressive behavior;
to reduce sexual activity;
for the treatment of neoplasia and benign prostatic hyperplasia (alone or in combination with estrogens, gonadotropin-releasing hormone analogues and antiandrogens);
for contraception;
for the prevention of epileptic disorders.

The negative effects of progesterone administration vary in type and intensity, depending on the drug used. The most common are:

the production of growth hormones, which leads to an increase in appetite, an increase in body weight; change in temperament and increased drowsiness; the development of insulin antagonism and the development diabetes type 2 due to the resistance of peripheral insulin receptors;
breast enlargement and lactation, the appearance of breast neoplasia;
change in coat (may cause hair discoloration and hair loss at the injection site);
blistering endometrial hyperplasia and pyometra (this pathology often occurs as a result of long-term use of progesterone (or the use of prolonged-acting progesterone), especially against the background of an increased concentration of estrogens - during estrus)). Some synthetic analogues of progesterone, such as proligeston (Neonidan, Delvosterone, Depopromon, Covinan) or delmadinone acetate, are largely devoid of the above disadvantages, but there are no drugs that are recommended for suppressing estrus in immature bitches;
the use of progesterone drugs during pregnancy can lead to inhibition of labor (especially when using prolonged-release drugs) and an increase in the number of cases of cryptorchidism in puppies;

In males, the administration of progesterone preparations can cause changes in sperm quality and temporary or prolonged infertility. However, as a rule, short-term therapy with progesterone preparations at recommended doses does not significantly change the quality of sperm and fertility in male dogs.

Many owners of hobby-class animals turn to veterinarians with a request to prescribe a drug that will suppress estrus in sexually mature bitches, since castration is an inhumane method for them. But, as mentioned above, synthetic analogues of progesterone are recommended for short-term use, and their long-term use entails a number of negative consequences.

Rice. 1. Bubble hyperplasia of the endometrium of the female uterus

Rice. 2. Closed pyometra

Rice. 3. Open pyometra

Rice. 4. Bubble hyperplasia of the endometrium of the female uterus

Laetitia Barlerin

By controlling the reproduction of dogs, for a long time they were content with suppressing estrus in bitches. The development of purebred dog breeding led to the birth of a real canine gynecology, developed in particular detail by Dr. Foitbonnet, a participant in the last CNVSPA congress.

Consultations about infertility in females received for last years widespread in the treatment of dogs. Currently, the combination of methods of radioimmunoassay for determining the level of progesterone in the blood (which is now possible in the clinic thanks to small kits that have become commercial) and taking vaginal swabs allows you to accurately determine the optimal moment of mating and fertilization. This level of diagnostics allows you to solve most of the problems associated with the fertility of the producer. Indeed, 50-80% of these problems are the result of an incorrectly determined mating moment! There are cases of infertility where simply tracking the onset of ovulation is not enough: a number of studies have to be carried out, even of a preventive nature (for the fastest possible response without waiting for the next sexual cycle) during the entire period of estrus and pregnancy. Splen Fontbonnet (ENVL * Ecole Nationale Veterinaire a "Lyon) at the last CNVSPA congress in Lyon in December 1996 outlined the main features of in-depth observation of the bitch, the course of diagnosis, as well as possible therapy.

indications for in-depth observation

repeated infertility.

If after several attempts, despite the reliable determination of the optimal moment of mating carried out by classical methods, the bitch still became sallow, it is necessary to conduct a more complete cycle of observations of the stage of maturation of the follicles, as well as a possible pregnancy.

atypical and abnormal estrus

Atypical estrus: repetitive (sometimes monthly), abnormal volume spotting(too much or too little); weak attraction of males; interrupted estrus (observed in flocks in young females at the time of the onset of oestrus in the dominant female; estrus of normal duration resumes after about a month).

Anomalous heat:

By duration of more than 25 or less than 7 days. "Short" estrus, however, are often the result of an incorrect determination by the owner of her first day; similarly, some bitches are capable of fertilization after the 25th day of their cycle (Berthe Alleman).

By the absence of ovulation diagnosed as a result of observations of previous estrus.

anomalies of interestrus

In the case of a very short interval between successive prolonged estrus, hyperestria should be suspected due to an ovarian tumor or follicular cysts. In the case of too long interestrus between prolonged estrus, one can think of a syndrome of hypogonadism (low hormonal activity of the gonads), associated with insufficient maturation of the follicles.

abortion and preterm birth

Before 40-45 days of gestation, loss of embryos or fetuses is not always noticeable due to their intrauterine resorption: monitoring of pregnancy should include serological studies on Herpes virus.

deep surveillance deployment

estrus monitoring

During the observation period, various studies are carried out.

Vaginal swabs

The analysis is simple, but gives little for the forecast. It is carried out to determine (by repeated, multiple samples) the rate of keratinization of vaginal cells, which indicates the effect of estradiol produced by ovarian follicles. Low rate keratinization (less than 50%) during proestrus and even estrus (when the female accepts a male) or, conversely, rapidly growing from the beginning of proestrus and after the end of estrus is a sign of an anomaly that requires study and, possibly, hormonal correction.

Vaginal swab

Quantification of hormone content

The method for determining the concentration of estradiol (in practice - estradiol 17) is original in relation to the classical methods of monitoring ovulation. Apply in the stage of proestrus or during the maturation of the follicles. On the chart 1: Normally, plasma estradiol concentration progresses during proestrus and reaches a peak (about 80-120 pmol / 1 * according to the author's and laboratory data) approximately 24 hours before the peak of secretion of luteinizing hormone LH (Prodan B). It then gradually declines and settles at a low level during estrus. With a "fine-toothed" nature of the curve, blood tests are resumed every 48 hours from the 3rd day of estrus.

The course of some curves indicates anomalies in the maturation of the follicles, causing the absence of ovulation: a rapid increase in estradiol secretion to abnormally high values \u200b\u200b(more than 400 pmol / l) indicates estrogenization of the body. On the contrary, an abnormally low curve (less than 50-60 pmol / l) is a sign of hypoestrogenism associated with insufficient maturation of the follicles.

Estradiol is an unstable hormone, it is stored poorly. A blood sample on an anticoagulant (eg, heparin) should be centrifuged within the next half hour; the plasma is then cooled; its further movements are made only in thermal containers. It is recommended that sample vials be sent only to laboratories specializing in the determination of hormones in dogs, as they have lower estradiol concentrations than humans and are difficult to detect in a "classical" laboratory. Determination of the content of testerone is carried out during the period of estrus, since it reveals random anomalies of ovulation. On the chart 1: In a normal cycle, progesterone levels, which are low during proestrus, increase immediately after LH reaches its peak and set at a high level. During metaestrus, it decreases very slowly (regardless of the presence of "a" or the absence of pregnancy "b") and reaches its minimum level around the 60th day after ovulation. Determination of progesterone content is a classic method of monitoring ovulation to determine the optimal moment of mating. At the end of estrus (determined by vaginal smears), a final blood test is done to check the activity of the corpus luteum at this stage. A decrease in the level of progesterone is proof of the end of ovulation or premature insufficiency of the functions of the corpus luteum.

Determination of hormone content thyroid gland T4 is indicated in cases of hyperthyroidism leading to abnormal ovulation (probably through hyperlactinemia), preterm labor or stillbirth.

Serology for Herpes Virus.

It is important to conduct research during proestrus, as during this period, herpes-infected bitches may experience virus reactivation with seroconversion. In the case of a positive result, a re-analysis must be done after 15 days to judge the dynamics of antibody development.

Sonography (ultrasound) of the ovaries.

Performed at the end of proestrus to detect follicular cysts.

in terms of practice

To conduct an in-depth observation, it is necessary to take a series of blood samples from the onset of proestrus. The plasma is cooled and sent to the laboratory only if the bitch does not conceive within a month: thus avoiding waiting for a new estrus and the need to investigate new random problems. The scheme recommended by Dr. Fontbonne:

Proestrus: blood samples taken on days 3,5,7,9 and 11, cool and prepare samples 7 or 9 for serology for Herpes virus.

Estrus: Prepare some progesterone samples.

End of heat: blood sampling.

pregnancy monitoring

It is necessary, since pregnant females may not bring it to a natural end.

Determining the level of progesterone

1-2 samples per week during the first month of pregnancy will reveal the insufficiency of the corpus luteum, leading to fetal resorption or abortion, and ensure timely treatment.

abdominal ultrasound

Carried out on the 18-20th day of pregnancy and repeated weekly to detect various pathologies: death of the litter without clinical signs, abnormal sizes of the fetus and fruit membranes, gladulocystic hyperplasia, etc.

ovarian cysts

Serological studies

In case of fetal resorption or abortion, the presence of infection should be checked as possible cause(Herpes virus, Brucella).

Note: During estrus and pregnancy, in the presence of suspicious vaginal discharge, a bacteriological analysis of cervical smears is performed.

treatment

hormone therapy

Insufficient maturation of follicles (hypoestrogenism) is a consequence of the impaired action of the pituitary follicle-stimulating hormone FSH (Prolan A) on the development of follicles. This is corrected with serum gonadotropin obtained from the blood of a foal mare (FFK, Folligon nd). Intramuscularly at 30 mg / kg per day from the onset of proestrus for 3-7 days - until 60% of vaginal smears with signs of keratinization are obtained.

The drug of "classical" medicine is used - Menotropin (Humegon ND) or hMG (Inductor ND) - a drug with a dominant effect of FSH and residual LH. The current trend is towards the use of highly purified FSH (eg, Metrodin ND, not yet tested in dogs), as in women, pure FSH gives better results than mixed FSH. Therefore, Dr. Fontbonnet does not recommend the use of hypothalamic releasing factor analogs (Receptal ND) in bitches, as they stimulate the dual release of Prolans A and B, which prevents the maturation of follicles. During this treatment, it is not necessary to stimulate ovulation with hCG, as normally mature follicles ovulate on their own.

Hyperestrogenism and lack of ovulation, not amenable to classical methods of treatment

Abnormally early and excessively abundant secretion of estrogens by the follicles causes blockade at the level of the hypothalamus. In human medicine (in this case and with reference to previously unsuccessful treatment) antiestrogens are used, for example Clomiphene (Clomid ND). Its action, unfortunately, has not yet been tested on dogs.

Lack of ovulation without follicular deficiency or prolonged estrus

Ovulation is stimulated by the use of drugs with the action of LH (Gonadotrophie chorionique Endo nd, Chomlon ND) - three intramuscular injections of 50 IU / kg with an interval of 48 hours.

Attention: The immunosensitizing effect of this glycoprotein in women and mares is well known! It is also capable of provoking ovulation disorders by blocking the peak of endogenous LH values, therefore, unless absolutely necessary, it is better not to use it.

Functional insufficiency of corpus luteum

Violation of the secretion of progesterone by the corpus luteum is corrected by the use of this hormone:

Oral: Utrogestan nd, effective for bitches at a dose of 1-2 capsules in the morning and evening until the 58th day of pregnancy

Intramuscularly 1-2 times a week Tocogestan nd, Progest 500 nd. Since the metabolism of progesterone is different in different bitches, a 2-week monitoring of its level is recommended.

Note: In some bitches, stopping progesterone makes normal delivery impossible and requires C-section. Good to warn the owner!

antibiotic treatment

Quinolones (Entrofloxacine) are considered the most effective in mycoplasmosis.

The use of antibiotics for preventive purposes should not be systematic, but should be used in cases of true infertility of bacterial origin, as indicated by the antibiogram.

surgery

Ovariectomy of one or both ovaries in breeding bitches is indicated for suspected ovarian tumor or follicular cysts.

conclusion

Thanks to the medicinal arsenal that a modern veterinarian has, he can really offer dog breeding research that is no less profound than in human medicine, the correct diagnosis and treatment of gynecological diseases.

Gynecology "Veterinarian" - № 0 1997

Progesterone is a hormone produced in females during estrus and pregnancy and related to estrogen. This is one of the most important components of successful fertilization and subsequent childbirth, since the state of the uterus, the name system, the mammary glands and much more depends on the level of progesterone.

With a high amount of the hormone, it allows the embryos in the uterus to attach without problems, suppresses immune system, preventing her from reacting to neoplasms as a danger and in the future is responsible for successful lactation. Low level during estrus or pregnancy will lead to the inability to conceive and bear healthy viable offspring.

Owners of breeding bitches carefully monitor the level of this estrogen in the pet's blood during the breeding season, since it is by this level that readiness for conception is determined.

Modern veterinary medicine allows you to conduct tests not only in the laboratory, but also independently at home, significantly saving time and money for breeders.

Why is research needed?

An analysis for a hormone that shows the quality of the reproductive system and the condition of the bitch must be taken if you plan to have puppies or the animal has recently suffered diseases associated with the pelvic organs. This will allow you to accurately determine what is happening to the animal.

Most often, a blood sampling procedure is prescribed to determine the level of progesterone in dogs in the case of:

Definitions of the most auspicious days chutes for knitting. Precisely guessing the moment of ovulation is necessary, since it does not last long relative to the time of manifestation of sexual desire in dogs.
To evaluate ovarian function. This is a mandatory procedure for young females who are going to be introduced into breeding. It is also recommended to conduct it periodically to monitor the reproductive abilities of the dog. It is especially important not to forget to pass the test after infections and diseases of the genitourinary system.
To identify the state of the estrus. If the appointed time has come according to the calendar, and there are no visible signs, it is likely that this process is asymptomatic in the animal, but this does not mean that it will not be able to become pregnant.
If it is not possible to establish the exact date of mating, the level of the hormone in the blood will tell the exact date of the upcoming birth.
After an abortion, this procedure is necessary, as it allows you to evaluate the functions of the corpus luteum.
Also deviation from the norm content This type of estrogen in the blood will allow timely detection of certain diseases and neoplasms, such as luteal cysts.

For owners of titled bitches introduced into breeding, this analysis is extremely important, it helps to determine the exact time of ovulation, which is of great help when using the method of artificial insemination or in the case when the groom lives far enough away, and it is necessary to warn his owner about the visit in advance.

Methods

The only way to detect the level of the hormone is a specialized blood test, made using the enzyme immunoassay method.

Previously, it was possible to conduct it only in the laboratory with specialized equipment. The whole process from taking the material to obtaining the results took 24 hours, although 4 hours are enough to detect the reaction.

It is too slow and financially unprofitable if you need to conduct several studies in a short time. During the period of estrus and bearing offspring, especially if the bitches have any problems with the reproductive system, it is necessary to determine the growth of progesterone several times over a fairly short time.

However, in recent years, a portable tester has also been developed that allows you to make all measurements without leaving your home. It has become a real find for cattery owners who constantly need to conduct such analyzes.

The test strips are very easy to use:

All selections are removed from the outer part of the loop.
The tester is placed with the marked end close to the inside of the loop so that it comes into contact with it and is saturated with the released liquid.
After 10 seconds, the strip is pulled out and inspected for sufficient contact with the secretions.

The result will be visible immediately, but it is not very detailed, since the method is designed like a litmus test. The wetted field changes its color depending on the amount of hormones. The more of them, the darker the test becomes.

This method is ideal for use at home to determine ovulation and subsequent pregnancy, but it will not give accurate results that allow you to identify abnormalities and diseases.

Interpretation of results

It is very important to understand what the results of the blood test mean, because on the basis of them, vital issues for bitches are solved. And this is not so much about the moment of estrus, the duration of ovulation, which has come on successfully for mating, but rather about determining the causes of a pregnancy that did not occur or possible problems with ovarian health.

It is also worth remembering that the results may vary slightly depending on the characteristics of the laboratory equipment, age and breed of the bitch. Often in the same individual in the same period completely different indicators. The veterinarian who observes the dog on an ongoing basis should interpret the data obtained.

Table of indicators:

Phase	lower limit		Upper limit
Phase	nmol/l	ng/ml	nmol/l	ng/ml
Anaestrus
luteal phase
Ovulation
Pregnancy
Less than 2 days before delivery

The ideal mating diet is calculated taking into account the method of fertilization:

In a real meeting, you need to wait for an indicator of 15.5-16 nmol / l and mate within 48 hours.
With artificial insemination using chilled sperm, the same indicator is expected.
If pregnancy is induced by introducing a previously frozen material, then wait for a mark of 8 nmol / l and carry out the procedure for 5 days.

The benefits of laboratory tests for progesterone cannot be overestimated. After all, these studies not only help to track the most favorable period for conception, and hence obtaining high-quality offspring, but also helps to identify problems with conception and the functioning of the reproductive system.