Artificial insemination technique for dogs. Artificial mating of dogs of different breeds. Evaluation of fresh semen

artificial insemination- a gynecological operation, which consists in obtaining sperm from a male, its evaluation, dilution and introduction into the genitals of a female using special tools.

Benefits of artificial insemination

With the help of artificial insemination, a number of problems in dog breeding can be solved.

when using frozen sperm, it is possible to accumulate it from outstanding producers and store it in a cryobank for many years, for future use;

the use of cryopreserved semen allows its international exchange without the need to transport the dogs themselves. At the same time, it becomes possible to order insemination of bitches with sperm from any manufacturer;

easier to comply with quarantine restrictions that prevent the movement of animals from one country to another;

artificial insemination helps to prevent the occurrence of many sexually transmitted diseases, since antibiotics are necessarily introduced into the composition of synthetic semen diluents, and sexual contact of animals is also excluded, most microbes and viruses are not transmitted during artificial insemination.

In addition, artificial insemination may also be recommended in a number of cases:

the impossibility of natural mating due to the narrow and short vagina in bitches;

excessively aggressive behavior of animals during mating;

low efficiency of natural mating due to certain breed characteristics of dogs (very large and heavy breeds, etc.);

the presence of certain diseases in a male or female that do not allow a full-fledged mating (disease of the limbs, hips, backs, etc.).

6. Methods for obtaining sperm

In males, sperm is obtained by masturbation, on an artificial vagina and with the help of electroejaculation.

Obtaining sperm by masturbation

Sperm from dogs can be obtained by masturbation. To do this, after a light massage through the prepuce, the penis is brought out beyond the cavernous body, wrapped around the fingers under the cavernous body in a ring-like manner and rhythmically squeezed. After an erection, the male can perform energetic search and friction movements with his pelvis. After the onset of a full erection, the male begins to roll over, which corresponds to the mating phase, which should last K)-15 minutes. Bonding is also imitated by the fact that one of the pelvic limbs is raised onto the hand that conducts masturbation.

The assistant is usually located to the right of the dog (if he is right-handed). In hunting, the bitch is fixed by the neck and head. When a dog tries to mount, the assistant fixes the priest's member with the right hand through the prepuce (foreskin). The hand moves the foreskin back and forth, then, when an erection occurs, the prepuce is pulled to the base of the penis so that the bulbs of the penis appear from the prepuce. Then the body of the penis is clamped at the base of the bulbs of the penis, after which ejaculation begins and the sperm is collected in prepared containers (sperm receptacles).

The method is considered less physiological than getting on an artificial vagina.

Before carrying out artificial insemination (AI) - in case of probable problems during mating - both the owner of the animal and the veterinarian must take into account the psychological and physiological mechanisms of normal mating. Moreover, not all breeders understand the psychology of relationships during mating. For mating, the bitch is delivered to the manufacturer, and not vice versa; compliance with this condition is especially important when it comes to the first mating of a young male. It is desirable that a young male is paired with an experienced female, and vice versa - a mature male was chosen as a partner for an inexperienced female.

A certain relationship develops between a male and a female, sympathy arises or, conversely, hostility, which can lead to the fact that one of the partners refuses to coitus. The social status of the dog or bitch and the dominance factor can also create problems. A young male, who occupies a subordinate position, experiences timidity in the company of a dominant bitch, which turns into difficulties when trying to mount or intromission. The dominant bitch, even during the period of estrus, can suppress such a partner, forcing him to refuse mating, but she will willingly mate with a male that occupies a dominant position. Since there are problems whose causes are beyond the scope of physiology, it is advisable to consider some psychological factors.

Often, signs of approaching estrus become noticeable a few weeks before the onset of proestrus. The bitch flirts or mounts other dogs, both males and females. Her appetite decreases or, on the contrary, increases, she begins to urinate more often, shows disobedience, behaves worse during training. For an experienced breeder, this behavior is a signal that estrus is approaching.

During proestrus, the bitch has an increased interest in socializing with other dogs. But she reacts passively to the approach of a male, she can even bite and drive him away if he tries to sniff her. At the end of proestrus, behavior changes. The bitch becomes playful, takes the initiative in communicating with males, allows them to sniff her. The onset of oestrus is marked by characteristic behavior, such as the bitch standing wagging her tail and moving it aside. Some females, when sniffed by a male, raise their croup, showing their vulva. When a male puts his paw on the bitch's back, she bends (lordosis) and can flirt with him herself (push with her nose, put her paw on her back, imitate a cage) if he shows timidity for some reason. During mating, the female stands calmly, her hind legs wide apart, her croup raised and her tail set aside.

Young males show cage from a very early age (from 4-5 weeks), this is considered the norm. Such activity is important for the male and is the training of sexual behavior. In addition, mounting other dogs or inanimate objects promotes puberty. However, such behavior must be strongly suppressed if it is directed at people, and especially at children.

The pheromones contained in the vaginal secretions and urine of an estrus bitch attract males even at a considerable distance and encourage them to follow her. When kept together, a male often even refuses food and water if the bitch is in heat. At this time, it is better to remove it. Some males show a propensity for vocal expression of excitement and howl tirelessly for days on end. Others try to escape from the owners by knocking out a door or a window for this purpose, making a tunnel or jumping over a fence; to stop such attempts, they often respond with destructive behavior. In males with prostatic disease (eg, cystic hyperplasia), bloody discharge from the penis is observed, due to rupture due to excitement, blood-filled cysts.

The behavior of a male and the postures he takes when meeting a female in heat usually indicate a playful mood with an admixture of fear, the latter directly depends on the social status of the male. He approaches the bitch, wagging his tail, pricking up his ears, avoiding looking at her intently. If she allows him to approach, the male licks her ears and muzzle, sniffs her, puts her paw or head on her back. If the bitch does not show friendliness, the male may start the game in order to lull her vigilance - he falls to the ground or offers the bitch to catch up with him.

Consists of 6 stages:

2. The male makes active movements and intromission - the initial erection.

3. Erection - swelling of the glans penis - separation of the first fraction of the ejaculate (transparent secret of the prostate gland).

4. Ejaculation - separation of the second fraction (white liquid containing sperm) - rotation.

5. Lock - promotion of sperm - separation of the third fraction (transparent secret of the prostate gland).

6. Rupture of the lock and the end of knitting.

If the bitch lets the male in (Fig. 9.1), he grabs her croup with his front paws and makes characteristic movements. An inexperienced male usually controls his movements, trying to orient his penis and locate the female's vulva. The initial erection provides intromission with the help of the penile bone. Upon contact with the vulva, the movements of the male are activated.

sadka

After the introduction of the penis into the vulva, the movements of the male become more active and energetic. He steps from foot to foot (dances), slightly rising above the bitch.

During this stage, a full erection is achieved. There is an elongation of the glans penis, the bulb remains attached to the bone of the penis, while pars longa glandis moves forward over the bone of the penis. The bulb swells, securing the penis to the bitch's vagina (lock). The sounds emitted by a bitch in heat and her smell stimulate an erection, which occurs due to impulses coming from excitatory (pelvic splanchnic) nerves, consisting of parasympathetic fibers of the pelvis and sacral nerves. Nerve impulses provide expansion of the external and internal pudendal arteries leading to the cavernous bodies of the penis; contraction of the sciatic-urethral muscles prevents the outflow of venous blood. Blood lingers in the sinuses of the cavernous tissue of the bulb, causing it to swell. The contraction of the ischiocavernosus and bulbospongius muscles, as well as the constriction of the vulva during the lock, maintain the intensity of the erection. During this phase, the first fraction of the ejaculate (1–2 ml of clear prostate secretion) is released.

Upon reaching a full erection, the second fraction containing spermatozoa (1–2 ml) is separated. This process takes 1-2 minutes. Ejaculation occurs as a result of stimulation of the nerve endings of the penis. Sperm with the secretion of the prostate gland is ejected due to contractions of the muscles surrounding the urethra, in particular, the bulbocavernosus and ischiocavernosus muscles. As a rule, at this stage, the male stops active movements, which coincides with the onset of ejaculation.

The male raises one paw and turns around its axis, taking the position "tail to tail" (Fig. 9.2), while the head of the penis remains in its original position. The elastic section of the penis, located behind the head, provides a 180-degree turn. The reversal does not cause discomfort to the animal, since the penile bone prevents obstruction of the urethra during erection and reversal. According to the existing assumption, rotation causes occlusion of the main vein of the penis and thereby prevents the cessation of erection. Some males do not turn around, others remain in the cage position, which can cause some inconvenience to the bitch, especially if the male is large; in such cases, it is carefully lifted, facilitating the position of the female.

rotation

During the lock, the male usually stands quietly in a tail-to-tail position (Fig. 9.3). At this stage, ejaculation of the third fraction of seminal fluid (5–20 ml of prostate secretion) occurs. Simultaneously with the movements of the tail, rhythmic contractions of the perineal muscles occur.

The castle lasts from 10 to 45 minutes, but the author of the article observed a castle that lasted at least an hour. As far as can be judged, the duration of the castle does not affect the results of mating. Fertilization also occurs in the absence of a lock, after ejaculation of the second fraction of the ejaculate containing spermatozoa, i.e., after the male has completed jerky movements. There is an assumption that the absence of a castle affects the number of litters, however, according to the author's observations, this opinion is erroneous.

During mating (cage, intromission and lock) experienced bitches stand still. In some cases, the bitch may try to leave, dragging the dog with her. Usually this behavior does not harm the animals, but it is wiser to hold the bitch by the collar, preventing such an attempt.

Before separation, the erection of the glans penis subsides and the partners separate. Usually the male licks the penis, which is then retracted into the prepuce. If there is hair between the opening of the prepuce and the penis, or the skin of the outer layer of the prepuce has got there, causing pain, the male is helped. Complete retraction of the penis is achieved by pressing on the prepuce 2–3 cm behind the orifice of entry, causing the opening to open.

Lock

After mating, the bitch usually vigorously licks the vagina, removing the leaked semen. Some females may show arousal, jump and play, or roll on the ground. Stopping the animal or not allowing it to urinate is impractical, since during the castle, most of the ejaculate has already entered the uterus and fallopian tubes.

Despite the fact that the bitch lets the male in and allows him to mount, if the mating time is not chosen correctly, there may be difficulties due to significant vaginal edema. The male is not able to enter the penis deep enough, as a result, it is not possible to reach the castle. Often a second attempt the next day is quite successful. If the animals are young and inexperienced, then the dog should be helped: supporting him by the croup, direct the penis into the vagina.

Some bitches, usually calm and peaceful, may panic during intromission, show aggression towards the male, and even try to bite the owner. In some cases, it may be appropriate to use tranquilizers or artificial insemination, although in the latter case, the Kennel Club's rules for registering puppies born as a result of AI should be taken into account.

Constriction of the annular muscles of the vagina, caused by hymenal rigidity or stricture, prevents complete intromission. The retention of the hymen is characteristic of young females; the defect is repaired by inserting a gloved finger into the vagina. In some cases, it may be necessary to surgically remove the hymen, but the formation of scar tissue can be a source of problems during childbirth. Artificial insemination is the best alternative for vaginal stricture or hymen retention, if the available lumen allows the insertion of appropriate instruments. The author's experience indicates that this type of vaginal stricture does not cause dystocia during labor.

Swelling of the vaginal mucosa, observed during the estrus stage, can lead to its excessive swelling and prolapse. Hyperplasia of the mucosa prevents coition, and often the introduction of instruments for artificial insemination. For treatment, astringents and disinfectants are prescribed; in case of severe lesions, surgical removal of hypertrophied tissue is recommended. Uncomplicated hyperplasia of the vaginal mucosa usually regresses after the end of estrus, but these animals tend to relapse into the next estrus. For this reason, bitches prone to hyperplasia of the vaginal mucosa are not used in breeding.

One of the most common problems that cause difficulties during mating is the inexperience of the male. In such cases, the male often tries to intromission in the anus or below the vulva. Young males often experience overexcitation leading to premature erection of the head of the penis, preventing intromission. At the stage of initial erection, ejaculation of the prostate secretion occurs. Ejaculation of the second fraction occurs later. It is necessary to temporarily separate the male from the partner and let him calm down, and after 30 minutes make a second attempt. In case of failure, the best way out is artificial insemination.

Rarely, young males are unable to coitus due to phimosis caused by adhesion of the penis and prepuce or stricture of the prepuce orifice. In both cases, surgery is necessary.

The lack of libido in a male can be due to a number of reasons, which include dislike for a sexual partner, an error in choosing the timing of mating, testicular hypoplasia, prostate disease, urinary calculi (rarely), balanoposthitis, epididymitis, vertebral arthrosis (often found in dogs of such breeds such as the Danish Hound and the Pekingese), diseases of the lower spine (spondylosis in boxers) and hind limbs (hip dysplasia). Clinical examination is performed to identify the cause. If libido is reduced due to psychological problems, changing a partner can correct the situation. While aroused, the male mates with another female or collects sperm for artificial insemination. In cases where diseases of the prostate gland, urinary calculi, epididymitis and balanoposthitis are detected, appropriate treatment is carried out. If problems with libido are explained by hereditary diseases, it is necessary to discuss the ethical side of the issue with the owner and explain why animals with genetic defects are not used in breeding.

Based on my experience, the author believes that the conditions for semen collection include: the presence of an irritant - usually a female in estrus (preferably at the peak of estrus, but if the bitch behaves calmly, any stage will do); a separate room and access to the site, in case the male prefers to play with the female beforehand; the minimum number of people. These conditions have a positive effect on sperm parameters and greatly facilitate the procedure.

The dog is given the opportunity to make a landing. When pushing the penis out of the prepuce, the operator pulls the latter back and squeezes the penis with his fingers caudally from the head (Fig. 9.4).

Sperm collection is not possible until a full erection is achieved and the male's jerky movements stop. The author uses a disposable funnel-shaped plastic cup to collect semen. When carrying out the manipulation, it is necessary to exclude the possibility of injury to the penis on the edge of the vessel. Such an injury can lead to rupture of the small vessels of the head of the penis and the ingress of blood into the ejaculate. Older dogs are especially prone to bleeding. It is not known how the presence of blood affects semen parameters, but its presence makes analysis difficult. Ideally, the sample should consist of only the second fraction (containing spermatozoa), especially if the sperm is to be frozen. For immediate use for artificial insemination, 1-2 ml of prostate secretion is added to the semen tube and used without additional solvents.

Semen collection using a female in heat as a stimulus. The operator retracts the prepuce, grasps the bulbs in the palm of his hand, and with his fingers squeezes the base of the penis behind the bulbs. Sperm is collected directly into a plastic tube

During the analysis, the color of the seminal fluid is determined. Shades from milky to slightly creamy are considered normal, depending on the density; a yellow tint is unacceptable and indicates urine ingress or an inflammatory process; red indicates the presence of blood. The reason for the deviation of the color of the ejaculate from the norm is revealed during a microscopic examination. Semen containing leukocytes, erythrocytes, or urine is centrifuged at 300–1000 rpm for 5 minutes in TRIS diluent (Table 9.1) or antibiotic solution, the supernatant is removed, and the pellet is reconstituted in fresh diluent containing antibiotics, and used for artificial insemination.

Buffer base contains antibiotics

Trishydroxymethylaminomethane - 6.056 g;

Citric acid - 3.400 g;

Fructose - 2.500 g;

Distilled water of double purification - 200 ml;

Crystalline penicillin - 200,000 IU;

Dihydrostreptomycin - 0.2 g;

Egg yolk is injected immediately before adding water (20% by volume) 2 ml of yolk per 8 ml of TRIS base + antibiotics.

Evaluation of sperm density, i.e. the concentration of spermatozoa per unit volume, is carried out using a counting chamber and a microscope or a photometric sperm counter (Fig. 9.5). The number of spermatozoa in the ejaculate is calculated based on their number per unit volume and total volume. The total number of spermatozoa in the semen of dogs varies from 200 to 1200 x 10 6 , the content in the second fraction is 100–700 x 10 6 /ml.

Spermacue™ photometric calibrator for measuring the density (sperm count per ml) of freshly obtained undiluted dog and fox semen

Progressive mobility (PP) is assessed visually under a microscope at 100x magnification. The indicator of progressive motility (the number of spermatozoa with a pronounced forward movement) can minimally be up to 5% at a rate of 75–90% in undiluted freshly collected ejaculate. In frozen semen, if it is to be used for artificial insemination, spermatozoa motility after thawing must be at least 50%. Sperm activity (progressive speed) is also an important indicator, which is subjectively assigned a value from 0 to 5, where 0 means no progressive movement (necrospermia) and 5 is the highest score. Normal fertile sperm usually have a score of 3–5; semen to be frozen should have a high reading before freezing, preferably 5. Low temperature reduces sperm motility, therefore it is recommended to heat the glass slide for analysis, especially when working with thawed semen. Saline solutions, especially those containing glycerol, also temporarily reduce sperm activity, but on a heated glass slide it usually recovers after 2-3 minutes.

Recently, a test for membrane integrity and viability of sperm (hypoosmotic test) has been widely used. The test involves incubating sperm in a hypoosmotic environment that causes the tail of the sperm to swell. This test correlates well with sperm motility and viability, but not with sperm concentration.

Morphological analysis (MA) is used to determine the percentage of spermatozoa with a normal appearance, either using phase-contrast microscopy at 200x magnification, or using a less powerful microscope, but then with nigrosine-eosin staining (see Chapter 8). Morphological parameters are considered normal if the percentage of morphologically unchanged spermatozoa ranges from 65 to 90%. The minimum percentage of morphologically normal sperm at which normal fertility is maintained has not been established in dogs, but values ​​below 60% usually indicate testicular or epididymal dysfunction. In males during puberty, up to 40% of spermatozoa may have proximal cytoplasmic inclusions. Sperm intended for freezing must contain at least 75% of morphologically normal spermatozoa.

The indicator of acrosome integrity is especially important for the morphological evaluation of sperm after thawing. Spermac™ staining is used in the author's laboratory to count spermatozoa with an intact acrosome.

For artificial insemination with freshly collected semen, it is not necessary to dilute it. However, if semen is to be transported and used after 2-3 hours, it is diluted with special diluents and transported at 4°C, since such conditions help maintain the viability of spermatozoa. For the transport of freshly collected semen, the author successfully uses two types of extenders (Tables 9.1 and 9.2).

In an experiment, diluents containing egg yolk, egg yolk in combination with sour cream and egg yolk in combination with milk were compared. TRIS extender is best for storing semen at 4°C. In all solutions, the yolk protects the sperm membrane from shock caused by exposure to low temperature. Semen dilution is usually carried out in a ratio of 1:6, depending on the initial concentration. It is important that the volume of the solution exceeds the volume of semen, and if the semen was not divided into fractions during collection, it is centrifuged and, after removing excess prostate secretion, mixed with the solution.

Pasteurized cream 12% fat - 8 ml;

Egg yolk 20% by volume - 2 ml.

Transport refrigeration is provided by placing the sample in a thermos partially filled with crushed ice. Use plastic centrifuge tubes (eg, 10 mL cell culture screw cap tube) to transport the sample.

The sample tube is placed in an insulated flask, the flask is placed in a thermos over a layer of ice and wrapped in paper. Before insemination, the sperm is gradually warmed up to 30–35 °C.

Numerous protocols for diluting, freezing, and thawing semen are described in detail in the literature. The author of this chapter prefers to work with TRIS extender, which provides good storage results for dog and fox semen. The success rate of subsequent insemination is 67-80% with intrauterine insemination using semen with a concentration of 50-150 x 10 6 spermatozoa per semen dose and insemination in two steps with an interval of 24 hours.

For freezing, glycerol and antibiotics are added to the TRIS stock solution (Table 9.1). The volume of added glycerin is from 12 to 16 ml (6 and 8% of the total volume, respectively) depending on the freezing technique: 6% glycerol is added for automatic freezing, 8% for manual freezing. Distilled water is added to the solution (188 or 184 ml instead of the standard 200 ml).

Add 20% egg yolk to the TRIS stock solution with glycerin. Egg yolk is obtained from hens tested for the absence of pathogenic factors (destined for export or domestic). The yolk is separated from the protein, spread on a sheet of clean paper, carefully pierced and poured into a funnel and vigorously whipped with a glass spatula, crushing its particles as much as possible - this reduces the binding of sperm heads by yolk particles, which makes it difficult to analyze the sperm. Finally, the yolk is mixed with TRIS buffer preheated to 30°C. The mixture is shaken well and heated to 35 °C. The pre-mixed diluent can be frozen and stored as such for up to 2 months. Before use, it is thawed in a water bath at a temperature of 35 °C.

A microscopic examination of the sperm is carried out for compliance with the criteria listed above, then a diluent heated to 35 ° C is added dropwise to the second fraction of the ejaculate until the required concentration is reached. In the author's laboratory, samples with a total spermatozoa concentration of 100 x 10 6 /ml are usually used for freezing. However, with high sperm quality, the concentration can be reduced by half.

After dilution, a few drops of the sample are examined under a microscope, and the diluted semen is poured into plastic centrifuge tubes, which are placed in a vessel with water heated to 35 °C. Then the vessel is transferred to the refrigerator and left for 2 hours at a temperature of 4 °C. During this time, the sperm is cooled to 4-5 ° C, and glycerol, which acts as a protective factor, penetrates the sperm membrane.

The semen is removed from the refrigerator, gently shaken and immediately placed in plastic straws (straws) with a capacity of 0.5 ml. To perform the procedure, a special suction is used or sucked by mouth through a latex tube. When distributing semen, care should be taken to ensure that the powder between the ends of the straw filters is saturated with liquid and hardens. First, the straws are filled halfway, after which, leaving an air bubble, they are topped up, not bringing 1 cm to the top. The air bubble prevents the transfusion of sperm (due to pressure changes during defrosting). After that, the straw is sealed.

There are two alternative freezing methods - manual (static protocol) and automatic (dynamic protocol). The proportion of glycerol in the diluent depends on the method used. For manual freezing, use a container (30 x 40 x 30 cm) made of polystyrene with a removable metal shelf placed 10 cm below the edge. The container is filled with liquid nitrogen to a level 4 cm below the shelf.

The filled straws (8–10 maximum) are grasped with tweezers, placed horizontally on a shelf and left in evaporating nitrogen for 8 minutes. Then the tweezers are cooled in liquid nitrogen, and the straws are lifted and rotated with it, one by one, to make sure that their contents are completely crystallized. After that, the payettes are immersed in liquid nitrogen. Such a procedure is called static due to the presence of a constant uncontrolled flow of evaporating nitrogen during the period of cooling and freezing.

Automatic freezing involves the use of a machine. This method is called dynamic, since the evaporating nitrogen enters the freezer at different rates depending on the set program, which allows you to adjust the freezing rate. The freezing program is carried out by the Planer 10™ machine developed by Hofmo (1988) for freezing fox semen. This instrument is currently in use in the author's laboratory, which has an extensive stock of semen samples. Paillettes are frozen in a horizontal position on a shelf with a removable lid, and there can be several such shelves in the freezer. The freezing program includes the following modes:

2 ?С/min. from +4 °C to -7 °C;

50 ?С/min. -7 °C to -100 °C;

25 ?С/min. from -100 ?С to -180 °C.

after the end of the procedure, the shelf is placed directly in liquid nitrogen.

Payeggs are thawed in a water bath (in a thermos) at a temperature of 70 ° C for 8 seconds. After defrosting, they are placed vertically with the filter down, the lid is opened, shaken so that the air bubble can rise up. The straw is opened and a few drops of the sample are placed on a heated glass slide to assess the parameters of the sperm after thawing. Before the restoration of sperm motility takes some time, usually 2 minutes is enough. Due to the fact that different laboratories use different freezing techniques, when thawing a semen sample obtained from a particular laboratory, all recommendations received from there must be strictly followed, since the freezing and thawing processes are closely related.

It is practiced in cases where natural mating is impossible for a number of reasons: male and female live quite far from each other; the breeder intends to inseminate several bitches or is afraid of direct contact of the bitch with the sire due to the prevalence of infectious diseases.

This is usually done if the producer is far away, and it is not possible to send fresh sperm, either due to the ban on bringing fresh sperm across the border, or in the event of the death of the producer. In Europe and the USA, there are sperm banks in national dog clubs, scientific institutes and some private companies.

In the presence of fresh sperm, the insemination procedure is carried out on the day of ovulation, and then repeated after 2 days. When using frozen sperm, which is characterized by reduced viability, since the freezing and thawing processes have a negative effect on acrosomes and sperm membranes, insemination is carried out 1–2 days after ovulation, and the second procedure is carried out 24 hours after the first. Serum progesterone tests are a good indicator of ovulation. Progesterone concentration, determined by radioimmunoassay, should be maintained at 30 nmol/l (10 ng/ml) on the first day and between 55 and 75 nmol/l (18–25 ng/ml) on the second day of insemination. Rapid enzyme immunoassay (ELISA) determination is also suitable for assessing the qualitative and quantitative content of progesterone. In foxes, artificial insemination allows you to achieve an increase in the number of litters, and with the right choice of date, fertilization occurs as a result of one procedure.

Fresh semen is introduced into the vagina using a rigid plastic catheter used for insemination of cows, but shortened to the desired size. The catheter is equipped with a 2–5 ml syringe with a rubber plunger (Fig. 9.6). It must be borne in mind that not every syringe can be used, since some types of rubber have a toxic effect on spermatozoa.

The injected volume should not exceed 3 ml for large breeds and 2 ml for small breeds, otherwise sperm may leak from the female genital tract. The vulva is raised with the index finger and a plastic catheter is gradually inserted into the vagina. After insertion, the caudal end of the catheter is raised and guided along the dorsal wall of the vagina to the vaginal fornix in order to avoid the catheter entering the bladder, since the opening of the urethra is located on the ventral wall of the vagina. With the second hand, the operator palpates the cervix through the abdominal wall, correcting the advancement of the catheter. Sperm is injected slowly, after which the female is kept for 10 minutes in a position with a raised croup. Stroking the vulva causes uterine contractions, which promote the movement of sperm.

For insemination with fresh and frozen-thawed sperm, the author's laboratory developed a special intrauterine catheter (Fig. 9.6), consisting of a nylon guide and a metal catheter with an attached syringe. The technique consists in inserting a guide tube into the vagina, with a metal catheter inside; after the tube touches the vaginal fornix, the metal catheter is advanced and inserted into the cervix, holding it with the thumb and forefinger through the abdominal wall (Fig. 9.7). The cervical canal is straight with slight folds and is almost completely dilated during estrus, so insertion of the catheter is usually straightforward. To perform the procedure, the animal is raised to the table, sedation is usually not required (Fig. 9.8). The manipulation requires the presence of certain skills, takes no more than 1–2 minutes from an experienced operator and does not cause discomfort to the animal. After performing intrauterine insemination, it is not necessary to lift the pelvic region of the bitch.

Three different types of nylon-guided catheters used for transcervical intrauterine artificial insemination in dogs. Below is a conventional plastic bovine catheter shortened to the required length for intravaginal insemination in dogs.

Artificial insemination of a dog with an intrauterine catheter. A plastic tube is put over the catheter to prevent contamination by vaginal microflora or trauma to the vaginal mucosa with a metal catheter

Transcervical artificial insemination. The animal is on the table. Sedation is usually not required and the procedure takes less than 5 minutes.

The recommended dose for intrauterine insemination with frozen-thawed sperm is 100 x 10 6 spermatozoa per insemination, however, fertilization is also ensured by a smaller number of spermatozoa - 35–40 x 10 6 per insemination, provided that the date of the procedure is correctly chosen and the technology of sperm thawing is observed. The dose of 100 x 10 6 /ml is based on the results of the author's experiments with fox sperm, during which the size of the litter was an important parameter. Insemination during laparotomy can be limited to even less sperm, however, to the best of the author's knowledge, such experiments have not been carried out.

Despite the possibility of intravaginal insemination when using frozen-thawed semen, intrauterine insemination provides the best results, which also makes it possible to reduce the dose of semen.

If catheterization is not possible or the operator is unfamiliar with the technique, artificial insemination by laparoscopy or endoscopy is recommended. In the United States and Canada, surgical methods of artificial insemination are the most common. However, in most European countries, intravaginal insemination, as well as catheter and endoscope techniques, are considered more ethical.

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SIA "Dzīvnieku veselības centrs"

veterinarian Marina Khitrova

In artificial insemination of dogs, sperm is introduced into the reproductive tract of the bitch with the help of special devices. The method of insemination also depends on how the sperm is prepared.

It is quite natural that this method is used only in the breeding of purebred animals and responsible and experienced breeders are careful in choosing suitable individuals for breeding. There are many situations where this method is best suited to achieve a particular goal. Let's look at some situations where this method can be used.

• Animals are far from each other, in different countries. Preparing for the trip, buying tickets at the last moment, because the date of the trip is determined by the readiness of the bitch for mating, finding a hotel where the dog would be allowed to stay ... This is a huge expense and stress not only for the owners, but also for the animal. The animal, often as a parcel, is sent to the owners of the dog, and in what conditions and how everything happened, the owner of the bitch will not know. And if you think about what an animal experiences in such situations, how scary and lonely he has to be far from home?! In addition, flights, stress and long trips can block the ovulation process, progesterone levels will drop and mating will be pointless.
• Another situation is when the animals do not approve of your choice, they show aggression towards each other and mating becomes very problematic.
• There are situations when, due to anatomical reasons, mating becomes difficult. For example, animals differ greatly from each other in size or, due to injury or problems with the musculoskeletal system, the male cannot land or turn around.
• Often, it is necessary to check the semen quality before mating, and the artificial insemination method is the best choice.
• Artificial insemination completely protects the male from sexually transmitted infections

SOME FACTS ABOUT THE ANATOMY OF THE REPRODUCTIVE TRACT OF THE BITCH

• Outside, the female reproductive tract consists of a vulva (loop), consisting of the labia,
• Further, on the lower wall is the clitoris and the fossa of the clitoris.
• The external genitalia pass into the vestibule of the vagina (vestibulum) - a hollow, tube-like organ with smooth walls that narrows at the border with the vagina.
• On the border between the vestibule and the vagina, on the lower wall, there is an urethra.
• Then the vagina begins - a very long hollow muscular tube with longitudinal folds, which increase many times during estrus. In large females, the length of the vagina can reach 30-40 cm! Therefore, it is impossible to see the cervix and simply introduce something into the uterus! The anterior part of the vagina is very narrow and only a very thin instrument can penetrate the cervix.
• The vagina ends with a vaginal fornix (fornix vaginae)
• Next comes the cervix. In dogs, the cervix is ​​quite difficult to penetrate, since it is located on top of the "ceiling" of the vagina and the opening of the cervical canal is directed downward.
• After the cervix follows the uterus (uterus), like the body of the uterus - a short segment, which is divided into the right and left horns of the uterus. The horns of the uterus run towards the right and left kidneys, where they pass into the very thin fallopian tubes. At the ends of the uterine horns are the ovaries.
• The ovaries are sex glands that secrete various sex hormones, the main ones being progesterone and estrogenic hormones. The ovaries contain follicles - vesicles, each of which contains one egg. During estrus, a group of follicles matures, they greatly increase in size and, at a certain moment, burst - ovulation occurs.

TO GET A GOOD RESULT WITH ARTIFICIAL INSEMINATION, YOU NEED:

• properly prepared sperm of good quality;
• correct determination of the fertile period of the bitch;
• good fertility (fertility) of the bitch;
• correct insemination technique;
• correct choice of materials.

TYPES OF SEMEN FOR ARTIFICIAL INSEMINATION

Sperm used for artificial insemination can be:

• fresh (fresh semen);
• chilled (chilled semen);
• frozen (frozen semen).

INSEMINATION WITH FRESH SEMEN

fresh cum- this is a product that has just been obtained, which does not need to be specially prepared, cooled.

Such sperm in the reproductive tract of the bitch has the greatest viability, and as you know, good quality sperm retains its qualities for up to 7 days. The only thing is that such semen must be used immediately after collection, it cannot be stored.

Fresh semen can be used for all types of insemination.

• intravaginal– when semen is introduced into the anterior part of the vagina using a long catheter. Intravaginal AI requires a larger volume of semen, 3-10 ml, depending on the size of the dog

After the introduction of sperm into the vagina, the bitch is lifted by the hind legs for 10 minutes + a massage of the vaginal fornix is ​​desirable.

• Intrauterine insemination- the introduction of sperm directly into the uterine cavity. For this, a Scandinavian probe is used, which consists of a plastic conductor and a metal catheter, and the endoscopic method is widely used when a thin catheter is inserted into the uterus using a special endoscopic probe. The required amount of sperm is very small, only 1-3 ml.

Using these techniques does not require the use of general anesthesia. Sometimes, if the dog is too restless, light sedation can be used.

• Intrauterine insemination during surgery- when the abdominal cavity opens, the surgeon exposes the uterus and injects the sperm directly into the uterus through an intravenous catheter.

When using the surgical method, keep in mind:

• ethical aspect;
• it is an invasive method;
• general anesthesia required, risk;
• only 1 IO is possible;
• the possibility of postoperative complications.

INSEMINATION WITH COLD SEMEN

chilled cum is a specially prepared product, cooled to a temperature of +4 °C. The duration of storage of such sperm is from several days to a week, and will depend both on the quality of the male's sperm and on the solvents used - extenders. Only extenders intended for dogs can be used. Such sperm is prepared in a certain way. Use only sperm-rich fraction of sperm, free from prostatic fluid. Often, the semen has to be centrifuged to separate the prostatic fluid. Then the extender is added drop by drop, and it is important to ensure that there is no temperature difference, then the prepared sperm is slowly cooled to +4 °C. Such semen is prepared for transportation. Use special containers with cooling elements. For example, Minitube containers are able to provide the required temperature for 48 hours.

All materials used for collecting and preparing semen must be non-spermicidal!
When inseminating with chilled sperm, all methods of insemination can be used, but it must be taken into account that such sperm no longer has such vitality as fresh, it survives in the genital tract for about 24-72 hours, therefore, it is necessary to determine the favorable period for mating, that is, determining the moment ovulation.

INSEMINATION WITH FROZEN SEMEN

Such sperm can be stored for many years in sperm banks and stored in special containers with liquid nitrogen at a temperature of -196°C.

Shipping such semen is quite expensive and requires a transport container for liquid nitrogen, the so-called dry-shipper.

It is important that the receiving party also has containers for semen storage (sperm bank), as transport containers are only able to maintain a low temperature for a few days. After thawing, the viability of such sperm is much lower - in the genital tract, it survives only max 12 hours! After defrosting, it must be used without delay!
Therefore, an accurate determination of the moment of ovulation is required and sperm is injected at the most favorable moment.
Only intrauterine sperm injection is used!

SEMEN REQUIREMENTS:

• recommended sperm count from 150-200 x 10⁶ progressively motile spermatozoa;
• mobility >70%;
• defects< 40 %;
• preferably for two IOs.

WHEN TO DO MATE OR ARTIFICIAL INSEMINATION?

• Natural mating or AI - artificial insemination with fresh semen:
  • if 2 matings are ideal, on the 1st and 3rd day after ovulation;
  • if mating is 1 time - ideally, on the 2nd or 3rd day after ovulation;
• AI with chilled or frozen semen:
  • various protocols;
  • ideally 2-3 days after ovulation.

SENDING SEMEN

Often, transferring the semen is the most difficult part of using artificial insemination. You can use express mail companies such as DHL, FedEx or use the services of cargo companies to send sperm. The shipment is carried out within 1-2 days. Just keep in mind that companies do not carry out transportation on weekends and holidays. That is, if the AI ​​on Friday, Saturday, Sunday or Monday sperm must be sent

• Thursday - 1 day delivery
• Wednesday - 2 days delivery

Semen should be kept refrigerated and temperature fluctuations should be avoided.

MINIMUM DOCUMENTATION:

• Veterinary health certificate for semen export
• Sperm Quality Certificate
• Frozen semen: recommendations for thawing, number of straws per AI
• Dog's name as in official documents
• A package of copies of documents

On these official websites on the Internet, you can get acquainted with the requirements of different countries for the export / import of semen, find out which veterinarians deal with insemination and reproduction problems in different countries and other valuable information:

www.evssar.org
www.canirep.com
www.canine-semenbanks-europe.com

RGAU-MSHA them. K. A. Timiryazeva

Department of Obstetrics, Animal Hygiene and Veterinary.

Essay on obstetrics on the topic:

"Mating and artificial insemination of dogs".

Moscow 2006

INTRODUCTION

Inspection of bitches and males should be carried out 3 weeks before mating. A male from which healthy puppies were born in the previous mating does not need confirmation of fertility. However, if more than a year has passed since the last fertile mating or the age of the sire is more than 7 years, it is still recommended to examine him. The clinical examination should include assessment of general health, semen analysis, examination of the penis and prepuce, and palpation of the prostate, testes, and epididymis. The bitches are assessed for their general health, making sure that vaccinations and deworming have been carried out. Examination of the vulva includes inspection of the vagina and vestibule for stricture, stenosis, or retention of the hymen. To calculate the optimal timing of mating, a study of vaginal discharge and / or a test for the concentration of progesterone is carried out.

Psychological aspects of mating

Before carrying out artificial insemination - in case of probable problems during mating - both the owner of the animal and the veterinarian must take into account the psychological and physiological mechanisms of normal mating. Moreover, not all breeders understand the psychology of relationships during mating. On the day of mating, the bitch is delivered to the manufacturer, and not vice versa; compliance with this condition is especially important when it comes to the first mating of a young male. It is desirable that a young male is paired with an experienced female, and vice versa - a mature male was chosen as partners for an inexperienced female.

A certain relationship develops between a male and a female, sympathy arises or, conversely, hostility, which can lead to the fact that one of the partners refuses to coitus. The social status of the dog or bitch and the dominance factor can also create problems. A young male, who occupies a subordinate position, experiences timidity in the company of a dominant bitch, which turns into difficulties when trying to mount or intromission. The dominant bitch, even during the period of estrus, can suppress such a partner, forcing him to refuse mating, but she will readily mate with a dominant male. Since there are problems whose causes are beyond the scope of physiology, it is advisable to consider some psychological factors.

Manifestations of sexual behavior in bitches

Often, signs of approaching estrus become noticeable a few weeks before the onset of proestrus. The bitch flirts or mounts other dogs, both males and females. Her appetite decreases or, on the contrary, increases, she begins to urinate more often, shows disobedience, behaves worse during training. For an experienced breeder, this behavior is a signal that estrus is approaching.

During proestrus, the bitch has an increased interest in socializing with other dogs. But she reacts passively to the approach of a male, she can even bite and drive him away if he tries to sniff her. At the end of proestrus, behavior changes. The bitch becomes playful, takes the initiative in communicating with males, allows them to sniff her. The onset of oestrus is marked by characteristic behavior, such as the bitch standing wagging her tail and moving it aside. Some females, when sniffed by a male, raise their croup, showing their vulva. When a male puts his paw on the bitch's back, she bends (lordosis) and can flirt with him herself (push with her nose, put her paw on her back, imitate a cage) if he shows timidity for some reason. During mating, the female stands calmly, her hind legs wide apart, her croup raised and her tail set aside.

Manifestations of sexual behavior in males

Young males exhibit mounting from a very early age (from 4-5 weeks), this is considered the norm. Such activity is important for the male and is a training in sexual behavior. In addition, mounting other dogs or inanimate objects promotes puberty. However, such behavior must be strongly suppressed if it is directed at people, and especially at children.

The pheromones contained in the vaginal secretions and urine of the bitch in heat attract males even at a considerable distance and encourage them to follow her. When kept together, a male often even refuses food and water if the bitch is in heat. At this time, it is better to remove it. Some males show a propensity for vocal expression of excitement and howl tirelessly for days on end. Others try to escape from the owners by knocking out a door or a window for this purpose, making a tunnel or jumping over a fence; to stop such attempts, they often respond with destructive behavior. In males with prostatic disease (eg, cystic hyperplasia), bloody discharge from the penis is observed, due to rupture due to excitement, blood-filled cysts.

The behavior of a male dog and the postures he takes when he meets a bitch in heat usually indicate a playful mood with an admixture of fear, the latter directly depends on the social status of the male. He approaches the bitch, wagging his tail, pricking up his ears, avoiding looking at her intently. If she allows him to approach, the male licks her ears and muzzle, sniffs her, puts her paw or head on her back. If the bitch does not show friendliness, the male may start the game in order to lull her vigilance, he crouches down or invites the bitch to catch up with him.

NORMAL KNIT

Consists of 6 stages:

2. The male makes active movements and intromission - the initial erection.

3. Erection - swelling of the glans penis - separation of the first fraction of the ejaculate (transparent secret of the prostate gland).

4. Ejaculation - separation of the second fraction (white liquid containing sperm) - rotation.

5. Lock - promotion of sperm - separation of the third fraction (transparent secret of the prostate gland).

6. Rupture of the lock and the end of knitting.

sadka

If the bitch lets the male (Fig. 1). he grabs her croup with his front paws and makes characteristic movements. An inexperienced male usually controls his movements, trying to orient his penis and locate the female's vulva. The initial erection provides intromission with the help of the penile bone. Upon contact with the vulva, the movements of the male are activated.

Rice1 . sadka

intromission

After the introduction of the penis into the vulva, the movements of the male become more active and energetic. He steps from foot to foot (dances), slightly rising above the bitch.

Erection and swelling of the headsexualmember

During this stage, a full erection is achieved. There is an elongation of the glans penis, the bulb remains attached to the bone of the penis, while pars longa glandis moves forward over the bone of the penis. The bulb swells, securing the penis to the bitch's vagina (lock). The sounds made by the bitch in heat and her smell stimulate an erection, which occurs due to impulses coming from excitatory (pelvic splanchnic) nerves, consisting of parasympathetic fibers of the pelvis and sacral nerves. Nerve impulses provide expansion of the external and internal pudendal arteries leading to the cavernous bodies of the penis; contraction of the sedalis-urethral muscles prevents the outflow of venous blood. Blood lingers in the sinuses of the cavernous tissue of the bulb, causing it to swell. The contraction of the sciatic-cavernous and bulbous-spongy mice, as well as the constriction of the vulva during the lock, maintain the intensity of the erection. During this phase, the first fraction of the ejaculate (1-2 ml of clear prostate secretion) is released.

In this case, we are talking about artificial insemination, to which their owners gave the term "artificial mating of dogs."
This technique has been known since the eighteenth century. It began with the experiments of two Italians. A little later, the practice of artificial insemination was introduced in Russia, where Russian scientists continued to study the method and even developed methods for freezing and storing sperm. This made it possible in the middle of the twentieth century to carry out an insemination operation on a dog and get completely healthy puppies.

Artificial mating of dogs: why is it needed?

This question can arise in any person. But many breeders engaged in breeding purebred dogs have long appreciated the excellent possibilities of the technique. It allows you to use the semen of males with excellent performance, which are far from the bitch. For example, in another city or country.

The second "plus" of artificial mating can be considered the reduction to zero of the risk of diseases that can be transmitted from one individual to another through sexual contact. The fact is that synthetic semen thinners contain antibiotics that destroy the infection.
In addition, there are a number of indications when artificial mating of dogs is preferable to the usual one.

These include:
Physiological characteristics of the bitch, which has a narrow and short vagina;
exterior features that make mating difficult (heavy weight, large size, and so on);
The presence of non-infectious diseases that interfere with the mating of dogs (limbs or back);
unfriendly attitude of animals to each other.

In general, the technique of artificial knitting is well developed and gives positive results. However, dog owners should understand that such a procedure can only be carried out with the participation of highly qualified specialists with special equipment and materials. The health of dogs and their offspring depends on this.