Artificial insemination of sows

The present invention relates to a process for, the preservation of the sperm of the sow by low freezing and then thawing and the introduction into the uterus of the sow - for artificial fertilization, for the purpose! obtaining 5 of a solution of sperm of egg sperm mobility of at least 50%.

in the breeding of animals, from economic considerations, artificial fertilization is progressing more and more. while at the bo¹⁰important successes have already been achieved, in the case of sows, artificial fertilization or the help of frozen sperm below has only happened by chance. The reason is considered to be due to the fact that bull sperm is less sensitive to preservation through low freezing than sperm, since a much higher percentage of bull sperm survives conservation. 20 In the practice so far, for the conditioning of the sperm of semen proceed as follows: the ejaculate, about 200 to 500 ml, is centrifuged, the liquid above 'settles, the sediment is' ^3Sdilute, with a diluent containing glucose, sodium encrypted, egg yolk, EDTA, distilled water, milk, antibiotics, diluted sperm freeze, ie bring to a temperature of. about -180 to 30 -200 ° C and kept at this temperature until sperm is needed. For use, the sperm is thawed according to different methods, for example by introducing frozen sperm in both 40 ° C diluent and resulting in a solution with a sperm dilution that ensures a good distribution of sperm in the uterus and this solution is then directly introduced in the womb.

This way of conditioning spermatozoa in pigs leads to reduced sperm fertility and motility.

The present invention removes the disadvantages shown by the fact that, in order to maintain in the frozen sperm a mobility of 50% and to present in solution at 39'C for at least 4 hours a good advancement movement, subject the ejaculate before of centrifugation inactivation by adding an aqueous solution containing glucose and a buffer system, after centrifugation the sediment is subjected to predilution with a solution that allows low freezing, freezing is gradually keeping semen partially diluted at low freezing for 1 ~ Ll ⁷ hours at 5C and peak. on dry ice by CO₂, so as to form small pellets, which are then covered with liquid nitrogen, and thawing is done with a solution of mobi53562

LEI PRICE 2.20

5S562 sperm lysis, at a temperature of 60 ... 70 ° C in the amount needed to achieve the dilution sought.

Two examples of embodiments of the invention are given below.

Example 1. The ejaculate is diluted, immediately after sampling, with an inactivation solution of about 3 (c in a ratio of 1: 3, with the following composition: 87.5 g of d-glucose, 5.4 g of ethylenediftityrolothetracetic acid-dihydrate, 5, 4 g sodium cipher, 7.5 g sodium bicarbonate, 1000 ml bidistilled water.

Diluted semen se. pass into centrifuge tubes and allow to stand for] - 2 hours at room temperature. During this time, the temperature and the room temperature decrease and the sperm immobilization decreases. After that the centrifuge tubes are carefully inserted into a centrifuge and centrifuged for 7 minutes at about 2500 rpm, After removing the tubes from the centrifuge, the liquid above is completely decanted by reversing the tubes. The sediment is then diluted with a low freezing diluent at a ratio of 1: 3, with the following composition: 6.06 g tristhydromethylamine, 2.50 g fructose, 3.46 g citric acid, 10.00 ml glycerin, 25.00 ml egg yolk, 200 ml bidislilaf water.

The sperm and the diluent mix well with careful stirring with a glass rod. The diluted semen is cooled, then in the refrigerator for 1 - up to a maximum of 2 hours at about 5C. After that, the diluted semen is brought, with the aid of a pipette, onto dry CO2 ice in the form of droplets, which freeze for 5 minutes as small pellets. These pellets are then passed into liquid nitrogen.

When using sperm, it dissolves according to the present invention, by introducing, respectively, 20 to 30 pellets, that is, the amount required for fertilization, in 20, respectively, 30 ml (for 1 tablet 1 ml) of inactivation solution of 60 almost 70 ° C. All pellets for each fertilization dose are suddenly added to the inactivation solution. The tablets dissolve immediately by gently shaking the vessel.

The pre-diluted defrosted semen is then inseminated, that is, it is introduced into the uterus, after which 75 ml of a mobilizing solution for sperm is introduced and which has also been heated to a temperature of about 40C and has the following composition: 24, 3 g of citrate. sodium 2H₃O; 2.0 g sodium bicarbonate; 0.4 potassium chloride; 3.0 g glucose; 3.0 g sulfanifamide per 1000 ml bidistilled water.

After combining the residual components, the solution is heated for 10 minutes at 90 ° C. (In sterile packaging, the solution can be stored for a long time and can be used when needed.)

The three solutions used in the process according to the present invention may differ in their composition, but it is important that the inactivation solution contains an adequate buffer system and d-glucose; at the low freezing diluent it is important that the egg yolk in quantity is not overtaken. The mobilizing action of the third solution is determined by the increased amount of sodium salt. The solution must also contain a buffer system and, conveniently, a sulfonamide is added to ensure the absence of germs.

The sperm solution should be inserted into the s uterus with a temperature of about 30 to 39 ° C. The semen in the solution obtained after the procedure described above has a vitality of about 2 hours at this temperature. This time, however, is usually too short for practice, because the sperm requires a longer period until it reaches the tube. between clogging and failure in the fallopian tube may sometimes pass 4, in the most favorable cases up to 7 hours.

Example 2. In another form of exe-. The invention is simplified, the process simplified, i-nocution and mobilization done with the same solution and low freezing with a solution which, apart from antifreeze agents, does not contain any additional components.

The solution used for inactivation and mobilization preferably contains glucose, sodium bicarbonate sodium citrate, antibiotics and dilution of centrifuged sperm before freezing is made with a low freezing diluent, containing skimmed milk powder, glucose, egg yolk, egg yolk antibiotics ethylenediaminetetraacetic acid and potassium chloride,

By this, the whole process is greatly simplified. Only one solution must be prepared and kept in reserve. Confusions of solutions are excluded, in addition to the semen of the boar, after it is mobilized, it is resistant for a short time to 4 hours at 39 ° C. In practice, the solutions of sperm obtained according to the present invention were kept for more than 6 hours at 39'C and then showed an additional mobility of about 70 ° C.

The exact composition of the solution used as an inactivator and mobilizer, hereinafter referred to as diluent], as well as the solution used, preferably for low freezing, referred to below diiuiuent 2, is presented as follows:

Dj'Iuen / î:

- 100 ... 140 g, preferably 120 g, gin · COZ;

- 10 ... 15 g, preferably 12 g, sodium cipher;

- 1 ... 3 g, preferably 2 g, sulEani'lamide;

- 0.5 ... 3 g, preferably 1.5 g, potassium chloride, · - 1.5 ... 4 g, preferably 2.5 g, ptilendi'amlntetraacetic acid;

- 1,5 ... 4 g, preferably 2.5 g, sodium bicarbonate and antibiotics.

Mortar 1 is prepared as follows: 2 liters of bidistilled water are boiled, the chemicals are added, allowed to cool briefly and allowed to cool, after which 1.7 million international units of penicillin G sodium and 1.7 are added. million international units dihydrostreptomycin base and the pH is raised to 6.8 with 4% NaOH. The solution obtained is filtered.

Thinner 2:

- 100 ... 120 g, preferably 100 g, skimmed milk powder;

- 20, 30 g, preferably 25 g, glucose;

- 0,5., 3 g, preferably 1 g, ethyldetylnidylaminjteJbraiaoetiic acid;

- 0.5 ... 1.5 g, preferably 0.8 g, potassium chloride, antibiotics, egg yolk, glycerin.

Diluent 2 is prepared as follows: 1 liter of bidistilled water is boiled, the chemicals are added, briefly boiled and allowed to cool. To the solution with room temperature add 1 million international units penicillin G sodium and 1 million international units dihydrosfreptomycin base. From this solution, take 100 ml and add 18 ml of egg yolk and 6.0 ml of glycerine.

For the execution of this preferred form of the present invention, the way of working is as follows: ejaculate obtained from a boar is treated with diluent 1 in relation to 1 part ejaculated to 3 parts diluenrt. After that, the diluted ejaculate is passed into centrifuge tubes, is 80 ml and allowed to settle for 24 hours at 15 ° C. The sedimented ejaculate is centrifuged for 5 minutes at 1700 rpm. This speed of centrifugation and the duration of time are not required. Of course, it can be centrifuged faster and slower, naturally, at lower centrifugation speeds and the duration should be properly extended. After centrifugation, the centrifuge is decanted and the sediment from each vial6 centrifuge rod is added double to triple diluent 2 and mixed with the seed. The seed thus diluted is cooled in a tube with a water level of 160 ml for 1V2 hours at 5 ° C in the cooler. The semen thus diluted and cooled freezes down on dry CO ice₂ after the pelletization process. The freeze time is 5 minutes. The pellets are inserted into transparent plastic tubes and stored in liquid nitrogen.

When using sperm, for defrosting, the entire contents of a plastic tube are placed in 30 ml of diluent 1 heated to 50X1, and, by vigorous shaking, defrost and dilute at a rate of seconds. After that, thawed sperm together with diluent 1 to "the same temperature is filled to 100 ml. The sperm solution is transported in a vessel to the place where it is needed. At fertilization, with the aid of a catertw, in the oeirvix and in the uterus, approximately 50 ml of diluent 1 is infused first, then the sperm solution is introduced and then 20 ml of diluent 1 is infused.

The sperm solution - obtained according to this preferred embodiment of the process according to the present invention can be kept, as already mentioned, for a longer time at 39 ° C. This temperature is set in the uterus, after the frozen pellets below have been thawed in 50 ° C diluent and together have taken 1 egg diluent of a temperature slightly higher than that of the thawed sperm solution and brought to 100 ml. It has been observed that the sperm solutions thus prepared present after 6, even after 8 hours, a sperm mobility of over 50%. The solutions gave birth to 10 to 15 piglets.

Low frozen sperm pellets obtained according to the invention are characterized by a quiet, mobilizable sperm content contained in a mass of frozen water containing small amounts of glycerin, sodium citrate, albuminous substances and antibiotics. These pellets can be stored anytime. liquid nitrogen. After thawing the appropriate number of pellets with diluent 1 at 50 ° C, a solution of sperm with a mobility of sperm of at least 50% was reached, which is characterized by an average amount of sperm needed for a fertilization in 100 ml bidistilled water and small quantities of albuminoid substance, glucose, sodium citrate, sodium bicarbonate., ethylenediaminetetraacertic acid, potassium chloride, antibiotics and sodium bicarbonate.

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Process according to the invention has the following advantages:

- a sperm is obtained which, after motorization, is resistant to 39 ° C at least 4 hours, and an egg with a mobility of approx. ^£70% ;

- makes it possible to economically fertilize pigs;

- at thawing with diluent a product is obtained at body temperature; ^1t - .to establish the final concentration ou. The solution of mobilization only in the uterus the sperm is maintained until the moment of use in a less mobile state, being thus protected;

- the sperm solutions, prepared according to the invention, gave birth to

10 ... 15 holes.

- the process is easy to do.