Composition for freezing canine sperm

FIELD: veterinary science.

SUBSTANCE: the suggested composition contains based upon 100 ml distilled water: from 0.25 to 4.5 g sodium citrate, from 0.45 to 4.0 g dextrose, from 0.01 to 0.3 g penicillin, from 50 000 to 500 000IU streptomycin, from 0.25 to 0.45 g catalase, from 0.045 to 0.25 g yolk, from 1.5 to 15 ml glycerol and from 0.02 to 0.1 mcg canine blood serum. The innovation keeps canine sperm for prolonged period of time, maintains viability and mobility of spermatozoa and provides high spermatic fertility after defrosting.

EFFECT: higher efficiency.

2 ex

 

Technical area

The present invention relates to compositions for freezing dog sperm, to a method for freezing canine semen with the use of these compositions and to the method of artificial insemination using frozen dog semen. More specifically, the present invention relates to compositions for freezing canine semen in which sperm count, living in the sperm, as well as their shape and vitality saved when thawing sperm within a certain period of time after freezing, to a method for freezing canine semen with the use of these compositions and to the method of artificial insemination, including the thawing of the frozen dog sperm in the right place and at the right time and inserting it into the vagina of the dog is female.

Prerequisites to the creation of inventions

In accordance with the recent industrial development of genetic engineering has become an important area of research. Genetic engineering is necessary for the preservation and development of valuable species of animals and increase the duration of human life, and to date, it has achieved a high level compared with the initial stage.

In the case of livestock or Pets artificial OEM is out with the use of liquid allows sperm to fertilize several females animals by mixing sperm taken from the male animal, with a dilution liquid to increase the amount of fluid available for injection. Thus, when performing artificial insemination collect superior or pure sperm, and you can fertilize several females animals simultaneously, which contributes to the improvement of breeds and preservation of pure breeds.

In order to achieve successful artificial insemination, has several advantages described above, an important issue is the development of an appropriate dilution of liquids. Because search dilution liquids began a few decades ago, were developed and introduced into practice of diluting liquid, suitable for pigs, horses and cattle. However, the development of diluting liquids, suitable for dog semen is not yet completed.

A way to preserve the sperm of animals over a long period of time without loss of freshness will need to use excellent sperm for fertilization of the female animal that will make a great contribution to the development of animal husbandry and breeding of pet animals.

The method of freezing is the most convenient to save the sperm for a long period of time. However, the method for freezing dog sperm has not yet developed completely. Even n the absence of clear wording, the specific cause of lack of development, perhaps, is that the reagents for freezing semen of dog spermatozoa which have some special features, as well as methods of freezing with the use of these reagents are not yet sufficiently developed.

Description of the invention

The first object of the present invention is to provide compositions for freezing dog sperm, through which the dog's sperm with special sperm can be frozen for storage.

The second object of the present invention is to provide a method for freezing canine semen collected from male dogs, with the use of these compositions with which the sperm can be stored for a long time and is easy to transport and with which the frozen dog semen can be thawed at a time suitable for artificial insemination.

The third object of the present invention is a method of artificial insemination using frozen sperm based on the method of freezing, which can be cultivated superior animals, and which can be widely applied.

For the implementation of the first aspect of the developed composition for freezing dog sperm, comprising, based on 100 ml of distilled water, 0.25 to 4.5 g of citrate n is sodium, from 0.45 to 4.0 g of dextrose, from 0.01 to 0.3 g of penicillin, from 50 000 to 500 000 IU of streptomycin, 0.25 to 0.45 g of catalase from 0,045 to 0.25 g of yolk, from 1.5 to 15 ml of glycerol and from 0.02 to 0.1 μg of the blood serum of the dog.

For the implementation of the second aspect, a method of freezing canine semen using a given composition, including the stage of (a) the taking of sperm in male dogs by way of massage or electric shock, (b) centrifugation of the collected semen for separation and removal of the supernatant liquid, leaving only the lower layers containing sperm, (C) mixing equal volumes of the lower strata, containing sperm, and composition, and primary cooling the mixture, (d) further adding the composition to the mixture obtained in stage (C), to reduce the volumetric ratio of sperm and composition to 1:2, and the secondary cooling the resulting mixture, and (e) adding liquid nitrogen to the mixture obtained in stage (d), and freezing the resulting mixture.

For the implementation of the third aspect, a method of artificial insemination of dogs, which includes stages: (a) survey female dogs on the subject of appropriate ovulatory phase to improve the fertility rate, (b) add frozen sperm, obtained by the method stated in paragraph 2, to a mixture of 0.3 to 1.5 wt.% solution of sodium chloride and blood serum of the dog hot on the temperature from 30 to 40° With and thawing, (C) study the motility of thawed sperm in the semen under the microscope, and (d) injection thawed sperm in the vagina of the female dog and massaging of the clitoris in female dogs.

Extensive investigations have been made to develop a method for freezing dog sperm and compositions for use in the specified way and it was found that the dog's sperm can be frozen for storage by combining small amounts of blood serum of a dog with a composition similar to the usual dilution of fluid used for cattle or pigs, to complete the present invention.

The present invention will be described hereinafter in more detail. First will be described composition for freezing dog sperm.

Composition for freezing dog sperm of the present invention is made homogeneous adulteration to 100 ml of distilled water, 0.25 to 4.5 g of sodium citrate to control the buffer function, acidity (pH) and osmotic pressure of sperm, from 0.45 to 4.0 g of dextrose as a source of metabolism and energy, from 0.01 to 0.3 g of penicillin as an antibiotic to prevent infection, from 50 000 to 500 000 IU of streptomycin, 0.25 to 0.45 g of catalase from 0,045 to 0.25 g of yolk, from 1.5 to 15 ml of glycerol and from 0.02 to 0.1 µg serum blood FDS is key. Made so the composition is frozen in units of volume of about 2 ml, designed to thaw before using them at any time.

The composition according to the present invention stores the sperm in for a longer period of time, maintaining the viability and motility of spermatozoa contained in the frozen sperm, and contains the blood serum of dogs, showing normal factors of fertility and childbirth after thawing.

Next will be described a method of freezing canine semen using the said composition.

For freezing dog sperm first, you need to collect. As a method of semen collection can be used in this area ways without limitation, preferably, the method of massage or electric shock. Hereinafter will be briefly described by way of massage, which is used most widely.

Because when mixed with urine sperm die quickly, it is preferable that the male dog before taking his sperm piss. Then the sexual organ of the male dog massage using tubes inserted in her artificial vagina to get a male dog to ejaculate in a test tube. To accelerate the capture of the sperm in this way the female dog during this period of time came the heat, allow nah is occurring close to male dogs. Another way is that when collecting the sperm of the male dogs give to smell the cotton or sponge containing vaginal discharge in female dog that first freeze, and before use, thaw.

In the case of taking sperm by using method of massage cum divided into three samples, collected in different tubes. In this case, the sample of semen, containing sperm, represents a second sample. Each sample collected during a time interval of about 10-20 seconds. However, in some cases, the interval between the first and second samples is too short to be visible. In the present invention preferably uses a second sample. However, in the case where it is impossible to distinguish between the first and second samples, all three samples are collected in one tube, which is designed for use.

Then collected in this way the sperm is separated by centrifugation, and the supernatant liquid is poured, after which remains the lowest layer of deposited sperm. Centrifugation carry about 500-1100 rpm for 6 minutes.

Equal volumes of lower layer deposited sperm and composition, prepared as described above, are mixed at room temperature and then the mixture is initially cooled. In this case, the cooling time is approximately 10 to 20 minutes, and temperaturesare is approximately 5° C. a Mixture of sperm and composition in a volumetric ratio of 1:2 then mixed with the primary chilled mixture at room temperature, and then again cooled at the same temperature and for the same time period. In further stages of primary and secondary cooling sperm and the composition is homogeneous mix.

Then a certain number of secondary chilled mixture is taken with a pipette and drip 2-3 drops on the surface of dry ice. Since the surface temperature of dry ice is approximately -79°C, the mixture throughout freezes. The frozen mixture is separated from the dry ice and placed in a container with liquid nitrogen with a temperature of about -196°S, which is then hermetically closed, completing thus the process of freezing dog sperm. This method is called "method of pellets", because the frozen mixture in liquid nitrogen is in the form of granules.

In addition to the above method of pellets, dog sperm can be frozen using the method, which uses a straw, and which hereinafter will be briefly described.

Secondary chilled mixture is again divided into samples of approximately 0.5 ml and placed in straws. Every 20-30 straws group and immersed in a container of liquid nitrogen. When the straw is immersed in a container for freezing, the POG is ujaut gradually, instead of immediately immersed at the bottom of the container. Their first immersed to the depth of the top of the container and left it for a certain time, then they are lowered to the depth of the middle of the container and left it for a certain time, and then to the bottom of the container.

Frozen in this way, the sperm can be transferred and stored for a long time and use if necessary for artificial insemination after thawing.

Then will be described by way of artificial insemination using frozen sperm.

First, for the implementation of artificial insemination test is done to determine whether female dog ovulatory phase. Test ovulatory phase performed by the methods known in the art to which the present invention is intended, such as the test cells of the vagina or hormonal test.

Then the frozen semen was thawed by adding frozen sperm to a mixture of 0.3 to 1.5 wt.% solution of sodium chloride and a small quantity of dog sperm heated to a temperature of from 30 to 40°that is conducive to maintaining the viability and shape of sperm. Also, although the mixture can be heated directly, because it is quite difficult to properly control the temperature, it is preferable to place a hermetically sealed container, aderrasi mixture solution of sodium chloride and dog sperm in hot water with a temperature of about 50°and heat.

Before using thawed using this method of sperm for artificial insemination study the number, shape and motility of live sperm under a microscope. Successful artificial insemination depends on the number of sperm that are moving forward. Spermatozoa are classified according to four types based on their mobility.

Class 0 refers to the case where the sperm move without tails forward, class 1 refers to the case where less than 20% of the sperm are moving forward, class 2 refers to the case where from 20 to 50% of the sperm are moving forward, class 3 refers to the case where 50 to 80% of the sperm are moving forward, and class 4 refers to the case where more than 80% of the sperm are moving forward. When thawed semen was placed on a glass slide and examined under a microscope, the sperm with class mobility 2 and above you can use for artificial insemination.

Then thawed sperm injected into the vagina of the female dog that is in phase, ovulation, and massage the clitoris of the female dog to complete artificial insemination.

In addition to the above method of artificial insemination, the thawed sperm can be injected into the uterus of the female dogs surgically. In other the words, the lower part of the navel cut about 2 cm to lift the uterus and the sperm of the male dog is injected directly into the uterus of a female dog with a syringe for injection. Then sew the peritoneum, the abdominal muscle and skin of the female dog.

The best option is the implementation of the present invention

The present invention is further illustrated by the following examples which should not be construed as limiting the scope of the present invention.

Example 1

Preparation of a composition for freezing dog sperm

A mixture having the following composition is homogeneous mixed using a mixer, to obtain a composition for freezing dog sperm, based on 100 ml of distilled water.

Sodium citrate0.35 g
Dextrose2.5 g
Penicillin0.02 g
Streptomycin100,000 IU
Catalase0.35 g
Yolk0.05 g
Glycerin2 ml
The serum of the dog0.05 mg

The method of freezing dog sperm

Sperm was good for a 6-year-old shepherd mix weighing 43 kg method of electric shock. The amount collected the semen was 6.2 ml Then the collected sperm were separated by centrifugation to remove the supernatant liquid and leaving only the bottom sediment. Centrifugation was performed at 500-1100 rpm for 6 minutes.

Equal volumes separated in this way, the lower layer of the sperm and composition, prepared as described above were mixed at room temperature, and then subjected to primary cooling. In this case, the cooling time was 10 to 20 minutes, and the cooling temperature was about 5°C. the Composition was additionally added to the initially cooled mixture, so that the volume ratio of the sperm and the composition was equal to 1:2. This resulting mixture is then subjected to secondary cooling at the same temperature and for the same period of time.

Then a certain number of secondary chilled mixture was collected by pipette and 2-3 drops dripped on the surface of dry ice. Since the surface temperature of dry ice is approximately -79°C, the mixture throughout froze. The frozen mixture was separated from the dry ice and placed in a container with liquid nitrogen with a temperature of about -196°S, which is then hermetically closed, completing thus the process of freezing dog sperm.

Artificial insemination using frozen sperm

For the art of the con insemination used three 3-year-old shepherd female weighing 30 kg To determine the phase of ovulation in female dogs used test of vaginal cells.

Then it was necessary to thaw sperm, frozen as described above. Thawing was carried out as follows: frozen sperm was added to a mixture of 0.3 to 1.5 wt.% solution of sodium chloride and a small quantity of dog sperm; the mixture was heated to a temperature of 38°and then left to thaw for 5 hours.

Then, before using thawed sperm for artificial insemination, investigated the mobility of live sperm by placing the thawed semen on a glass slide and examine it under the microscope. Successful artificial insemination depends on the number of sperm that are moving forward. The percentage of sperm that are moving forward was approximately 85%, which is suitable for artificial insemination.

Then the thawed semen was divided into three portions and were injected with the vagina of the female dogs that were in the phase of ovulation. Then each of the clitoris in females dogs massaged for 5 minutes to complete artificial insemination.

A pregnancy test was performed 1 month after the implementation of artificial insemination, and all three female dogs were pregnant. Later the three female dogs were born 4, 6, and 9 healthy pups, respectively.

Example 2

In this example, the composition for freezing dog sperm was prepared as described in example 1, except that used was 0.02 µg serum of the dog.

Subsequently, the freezing of dog semen and artificial insemination with the use of the above compositions was carried out as described in example 1.

In this embodiment, using a 5-year-old male dog weighing 40 kg and three 3-year-old female dog weighing 25 kg, 30 kg and 28 kg

Test on the motility of thawed sperm in the semen showed that the percentage of sperm that are moving forward was 60%, which is a suitable condition for artificial insemination.

A pregnancy test was performed 1 month after the implementation of artificial insemination, and two female dogs were pregnant. Later two female dogs were born 5 and 6 healthy pups, respectively.

Comparative example 1

In this comparative example, a composition for freezing dog sperm was prepared as described in example 1, and the sperm was frozen as described in example except that the volumetric ratio of the composition and dog sperm was 1:1. Dog sperm were collected as described in example 1, the same male dogs.

Frozen semen was thawed as described in example 1, and studied the mobility of spermat is Saidov in the thawed semen. The test showed that the percentage of sperm moving forward, was less than 20%, which is not an appropriate condition for artificial insemination.

Comparative example 2

In this comparative example, a composition for freezing dog sperm was prepared as described in example 1, except that the composition was not the blood serum of the dog. Methods of freezing and thawing were the same as in example 1, and canine semen was collected as described in example 1, the same male dogs.

Test on sperm motility in frozen and thawed sperm, according to this comparative example, showed that sperm was observed only movement tails, without moving forward, that is not an appropriate condition for artificial insemination.

As described above, the present invention relates to a method for freezing dog sperm, which is difficult to freeze to store because of the peculiarities inherent in the sperm dog sperm, and to compositions for use in the specified way. Thus, dog sperm can be stored in a frozen state, which contributes to the preservation of good dogs breed lines. Frozen dog semen can be used for artificial insemination at the right time and the right place that helps is to improving fertility.

Composition for freezing dog sperm, comprising, based on 100 ml of distilled water and 0.25÷4.5 g of sodium citrate, 0,45÷4.0 g of dextrose, 0.01 to÷0.3 g of penicillin, 50 000÷500 000 IU of streptomycin, and 0.25÷0.45 g of catalase, 0,045÷0.25 g of yolk, 1,5÷15 ml of glycerol and 0.02÷0.1 ág serum of the dog.



 

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