JP2896184B2 - Concentrated freezing method of swine semen - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for concentrating and freezing pig semen.

[Prior Art] Artificial insemination of pigs has been widely performed for the purpose of efficiently producing high-quality pigs. In this case, pig semen is used in terms of long-term storage, easy availability and easy use. Has been attempted to freeze. However, when the frozen pig semen is thawed, the number of high-vigor spermatozoa generally decreases compared to that of raw pig semen, and the number of high-vigor spermatozoa decreases remarkably for some pigs. Impossible. Since such a decrease in the number of high-vigor spermatozoa due to the freezing treatment significantly lowers the fertilization rate, improvement thereof has been required.

[Contents of the Invention] The present inventors have continued research to solve the above-mentioned drawbacks. As a result, the pig semen was divided into a sperm category with high vitality and a sperm category with low vitality, and the sperm category with high vitality was divided. The present invention has been completed by finding that a frozen pig semen having a high activity and a small decrease in the number of sperm can be obtained by collecting and freezing the concentrated semen and freezing.

That is, the present invention is a method for concentrating and freezing swine semen, which comprises separating and recovering a highly viable sperm fraction from concentrated semen as a concentrated semen using the difference in density, and then freezing.

As a method for separating and recovering swine semen into a sperm fraction having a high vitality and a sperm fraction having a low vitality, polyvinylpyrrolidone-coated colloidal silica gel (a typical example is polyvinylpyrrolidone sold by Pharmacia under the trade name of Percoll) A sucrose / epichlorohydrin copolymer aqueous solution (a typical example of which is Ficoll (trade name) sold by Pharmacia), a sucrose solution ,
A method in which swine semen is put into a solution such as a polyvinylpyrrolidone solution, and the semen with a high vitality sperm count collected at the bottom by centrifugation is separated and collected as a concentrated semen. A method of placing pig semen on the top and stirring it up and down with a stirring bar or the like and separating and collecting semen collected at the bottom as concentrated semen (stirring density gradient method) may be considered for the time being.

Among the above-mentioned separation and recovery methods, the present invention employs a method in which swine sperm is placed in a solution of polyvinylpyrrolidone-coated colloidal silica gel (hereinafter referred to as “modified colloidal silica gel”) and centrifuged, and the modified colloidal silica gel is used. The solution is made into a three-layer solution having different concentrations, and pig semen is placed on the solution and centrifuged. At this time, concentration 40
50% by volume modified colloidal silica gel first solution, concentration
The modified colloidal silica gel second solution of 50 to 60% by volume and the modified colloidal silica gel third solution of the concentration of 60 to 70% by volume are composed of three layers, and the concentration difference between the second solution and the first solution is 10 to 20. A three-layer solution having a volume percentage of 10 to 20% by volume and a concentration difference between the third solution and the second solution is formed, pig semen is placed on top of the solution, centrifuged, and then pigs collected at the bottom Separating and collecting semen is more effective for separating and collecting high-vigor sperm fractions.

The modified colloidal silica gel in that case is a liquid composed of colloidal silica gel having a coating of polyvinylpyrrolidone, and usually has a density of about 1.130 ± 0.005. Representative examples of such modified colloidal silica gels include Barcol manufactured by Pharmacia described above. However, in the present invention, any of modified colloidal silica gels having the same physical properties as Percoll can be used, and is not limited to Percoll.

Then, the first, second and third solutions of the modified colloidal silica gel having the above specific concentration forming the three-layer solution are prepared by converting the modified colloidal silica gel solution into a physiological salt solution or other diluting solution. It can be prepared by diluting to each of the above concentrations. Examples of the physiological salt solution and the diluting solution at this time include physiological saline (0.15 M), sucrose solution (0.25 M), bicarbonate buffer-based culture solution [eg, Ham F12 manufactured by Nissui Pharmaceutical Co., Ltd.] (Trade name)] can be used, and Ham F12 is particularly preferable because it maintains sperm vitality.

The first, second and third solutions of the modified colloidal silica gel are desirably in the above concentration ranges, and the difference in concentration between the second solution and the first solution is 10 to 20% by volume (hereinafter, the volume% is simply referred to as the volume%). %) And the concentration difference between the third solution and the second solution
Desirably, it is 10 to 20%. The density difference in this case is
A value (%) obtained by subtracting the concentration of the first solution from the concentration of the second solution or a value (%) obtained by subtracting the concentration of the second solution from the concentration of the third solution. For example, when the concentration of the second solution is 55%. When the concentration of the first solution is 45%, the concentration difference becomes 10%. When the concentration of each solution is out of the above range or the concentration difference is out of the above range, separation of high-activity swine semen and low-activity swine semen or seminal plasma may not be performed smoothly.

The concentrations of the modified colloidal silica gel first, second and third solutions are each within the above range, and the second solution and the first solution
When the concentration difference between the solutions and the concentration difference between the third solution and the second solution are within the range of the present invention, the third solution is located at the lowermost position, and the second solution and the A three-layer solution in which one solution is layered is formed. The shape of the three-layer solution is determined by using a test tube for centrifugation, an elongated container made of plastic or glass, or the like, and storing the third solution and the second solution in the container.
Preferably, the solution and the first solution are added sequentially. The amount of the solution used is 1: about 1 by the volume ratio of the first solution: the second solution: the third solution.
1: About 1 is recommended.

Then, the semen collected from the boar is placed on the uppermost portion of the above three-layer solution (that is, on the first solution) using a dropper or the like. In this case, about 1/4 to 1/2 volume part, preferably about 1/3 volume part of pig semen is put on 1 volume part of the first, second and third solutions in total, which is vitality. It is desirable in terms of efficiently separating and collecting high sperm. At that time, the temperature of the three-layer solution and the swine semen is preferably kept at about 20 to 37 ° C.
The pig semen to be placed on the three-layer solution may be as collected from boars, or may be obtained by previously diluting the collected pig semen with a physiological salt solution or another diluent storage solution for pigs. Alternatively, fine sediment such as cytoplasmic droplets, epidermal cells, cell debris, and cell granules may be contained in swine semen, which may be obtained by previously purifying raw semen to remove or reduce impurities contained therein. Since it is mixed, it is preferable that the semen be preliminarily treated by centrifugation or the like to remove or reduce impurities. When purifying by centrifugation, raise the temperature of
It is preferable to centrifuge at about 37 ° C. and about 50 × 200 × G for about 3 to 10 minutes.

Next, the unpurified or purified raw pig semen is placed on the uppermost layer of the modified three-layered colloidal silica gel solution, and then centrifuged to separate a highly viable sperm fraction as concentrated semen. For the centrifugation, any of the methods and apparatuses generally used for centrifugation of semen can be employed. Especially using a centrifugal separator consisting of a swingout rotor about 150-300x
It is preferable to centrifuge at a centrifugal force of G for about 10 to 20 minutes. Due to the difference in specific gravity after completion of centrifugation, the desired vigorous porcine sperm gathers at the bottom of the container, while the first solution and the second
Porcine semen consisting of inactive sperm gathers at the boundary with the solution, and the seminal plasma component separates on the first solution,
Separation and collection of highly active porcine sperm collected at the bottom as highly active concentrated semen. The separation and recovery can be performed by any method, for example, by decantation, suction, or the like.

Next, in the present invention, the collected highly active concentrated pig semen is frozen. Freezing can be performed by any of the methods commonly used for freezing semen and the like, and can be performed by, for example, a tableting freezing method (pellet method), a straw method, an aluminum pack method, or the like. In particular, in the case of the tableting freezing method, about 300 to 500 concentrated swine semen tablets can be conveniently prepared from swine semen for one ejaculation. When freezing swine semen, using a cryoprotectant such as glycerol, usually about -80 ℃ ~ about-
It is good to freeze at 200 ℃. Depending on the type of freezing method, it is convenient to freeze using about 0.1 to 5 ml of semen per frozen product during artificial fertilization. Concentrated and frozen swine semen can be stored semipermanently without loss of activity when stored in liquid nitrogen (-196 ° C), and can be used at any time.

When artificial insemination is performed using the concentrated and frozen pig semen, it is heated and melted at about 30 to 50 ° C. before use. The artificial insemination may be performed by any method employed in artificial insemination of pigs, such as an intratubal injection method by laparotomy or an intracervical injection method without surgical operation. The concentrated frozen swine semen frozen by the tableting freezing method has a small amount of semen per tablet (usually about 0.1 to 0.2 ml). Performing artificial insemination is desirable because a sow can be fertilized at a high rate with 1/2 to 1 tablet semen per insemination, and the concentrated frozen semen can be used efficiently.

[Effect of the Invention] Since the swine semen obtained by the concentration and recovery method of the present invention contains high-vigority swine sperm in a concentrated state at a high density, it is thawed compared to the case where raw swine semen is frozen and stored as it is. There is little decrease in vitality.

The concentrated frozen swine semen obtained by the method of the present invention can be stored for a long period of time (semi-permanently) while maintaining the fertilizing ability, and can be easily used when necessary and convenient.

According to the method of the present invention, the semen can be cryopreserved for a long time without losing the fertilizing ability even if the semen of the individual is resistant to cryopreservation.

ADVANTAGE OF THE INVENTION According to this invention, the highly active concentrated swine semen containing a large amount of highly active porcine spermatozoa can be separated and collected with high efficiency.

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto.

Example 30 ml of fresh semen collected from a boar (Large Yorkshire species: December age: 200 kg) was placed in a test tube, heated to about 37 ° C., and then centrifuged (CT5L: manufactured by Hitachi, Ltd.). , 150 × G
For 5 minutes. The supernatant consisting of seminal plasma and the contaminants precipitated at the bottom were separated and removed, and the intermediate solution was collected as purified raw pig semen (about 20 ml).

Percoll was diluted using a physiological salt solution consisting of Ham F12 (pH 7.3 to 7.4: manufactured by Nissui Pharmaceutical Co., Ltd.) as a diluting solution to obtain a Percord first solution having a concentration of 45% and a Percoll first solution having a concentration of 55%. A second solution and a third solution of Percoll having a concentration of 65% were prepared.

To a test tube having a diameter of 3 cm and a depth of 11.5 cm, 7.5 ml of each of the above-prepared Percoll third solution, Percoll second solution and Percoll first solution were added in order of 7.5 ml to form a three-layer solution.

Next, 7.5 ml of the purified raw pig semen obtained above was added to the above-mentioned first solution in a test tube with a dropper (the temperature of the Percoll solution and semen was about 37 ° C.), and the same centrifugal machine as above was used. And centrifuged at 250 × G for 15 minutes. After completion of the centrifugation, the three kinds of Percoll solutions are again separated into three layers, semen sediments at the bottom of the test tube, and a part of semen gathers at the boundary between the Percoll first solution and the Percoll second solution, Further, seminal plasma separated at the top of the Percoll first solution.

Separating and removing seminal plasma and Percoll first, second and third solutions, semen at the boundary between Percoll first solution and Percoll second solution (boundary semen), and semen settled at the bottom of the test tube (sedimented semen) Were collected separately.

When the sperm vitality of the boundary semen and sedimented semen and the sperm vitality of the semen (raw semen) before Percoll solution treatment were measured by the following methods, the results shown in Table 1 were obtained.

[Measurement of sperm vitality] Three semen quality test boards were prepared, and
Put a drop (about 50μ), put it on a slide heating device and keep it at about 37 ° C, use a microscope to visually count the total number of spermatozoa in the field of view of the microscope, and the most active forward movement in the field of view The number of spermatozoa was calculated using the following formula.

From the results in Table 1 above, it can be seen that the concentrated semen (sedimented semen) having higher sperm vitality than the raw semen can be obtained by the concentration-freezing treatment of the present invention.

Next, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) 1.2
g, trishydroxymethylmethane (Tris) 0.2 g, glucose 3.2 g, egg yolk 20 ml and over es paste (OE
P) A primary diluent containing 0.5 ml was prepared, and a three-fold volume primary diluent was added to the sedimented semen obtained above, and gradually cooled to 5 ° C. over about 90 minutes. To the primary diluted semen cooled to 5 ° C., an equal volume of a secondary diluent (1 to 3% of glycerol added to the primary diluent) is added, and the mixture is thoroughly stirred. About 5
The mixture was dropped into a concave part of mm and rapidly frozen to obtain semen of a pellet.
Each of the pellet semen was individually placed in a plastic test tube, sealed and stored in liquid nitrogen for 30 days.

Remove the pellet semen put in a test tube from liquid nitrogen, immerse it in hot water at 40 ° C for 20 seconds, then
Was melted by adding 1 ml of the melt.

On the other hand, the above-mentioned raw semen not subjected to the concentration treatment with the Percoll solution was also subjected to the ligation and melting treatment in the same manner as described above.

The sperm vitality of the sedimented semen and the raw semen that were melted was measured in the same manner as described above, and the results are as shown in Table 2 below.

From the results in Table 2 above, in the case of using the concentrated freezing method of the present invention, a good concentrated frozen semen having a high fertilizing ability with a high percentage of sperm having high vitality can be obtained, while the raw semen is directly frozen as it is. In this case, it can be seen that the proportion of high-vigor spermatozoa is greatly reduced and the fertilizing ability is lost.

In addition, the lower abdomen of 6 artificially estrused female pigs (Large Yorkshire breed: 5 to 6 months old, weight: 90 to 110 kg / head) was incised and the melting obtained above in the fallopian tubes on the left and right. The sedimented semen was injected 0.1 ml each (number of spermatozoa 3 to 6 × 10 ⁷ ) and artificially inseminated. Five of the six were pregnant and four delivered, giving good results ( One is miscarriage).

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) A61D 19/02

Claims (2)

(57) [Claims] 1. A swine semen is placed on top of a polyvinylpyrrolidone-coated colloidal silica gel solution having different concentrations sequentially increasing in concentration from the uppermost layer, the middle layer, and the lowermost layer, and centrifuged. A method for concentrating and recovering swine semen, comprising separating and recovering a highly viable sperm fraction as concentrated semen from the sedimentation part of the above, and then freezing this. 2. The concentration of the three layers of polyvinylpyrrolidone-coated colloidal silica gel solution is 40-50% by volume for the top layer, 50-60% by volume for the middle layer and 60-70% by volume for the bottom layer,
2. The method of claim 1, wherein the concentration difference between the layers is at least 10%.