Method for processing semen

Abstract

To provide a method for restoring sperm motility that has been reduced by diluting collected semen with an aqueous solution containing a chelating agent.SOLUTION: Provided is a method for processing semen in which collected semen is diluted with an aqueous solution containing calcium ions and a chelating agent, and then hydrostatic pressure of 5 to 40 MPa is applied for 0.1 to 15 minutes, after which the pressure is released. Preferably, a content of the calcium ions is 0.1 to 10 mM and a content of the chelating agent is 0.1 to 50 mM, with ethylene diamine tetraacetic acid or glycol ether diamine tetraacetic acid being also preferred as the chelating agent.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for treating semen, and particularly to a method for treating semen for artificial insemination, in vitro fertilization or intracytoplasmic sperm injection. The method for treating semen of the present invention can also be used for human assisted reproductive medicine and for livestock breeding.

Background Art

[0002] In human infertility treatment, methods such as artificial insemination, in vitro fertilization, and intracytoplasmic sperm injection are adopted, and ensuring the motility of the collected sperm is important in any method. This is the same in artificial insemination for livestock breeding. On the other hand, whether it is a human or a livestock, it is inevitable that a certain amount of time is required from semen collection to use in artificial insemination or the like. Therefore, various methods for treating and storing semen have been proposed to improve sperm motility or suppress the decrease in sperm motility. For example, a method of devising a diluent for diluting semen has been proposed (for example, Patent Documents 1 to 4).

[0003] On the other hand, measures to improve sperm motility by physical treatment have also been studied, and it has been proposed to apply hydrostatic pressure to sperm. Patent Document 5 shows that when bovine sperm were subjected to a hydrostatic pressure of 30 to 90 MPa for 30 to 90 minutes and then frozen and then thawed to examine the motility of the sperm, the motility of the sperm was higher than that of the specimen frozen without prior pressure treatment. In particular, it was shown that applying a hydrostatic pressure of 30 MPa for 90 minutes was effective. Patent Document 6 shows that when porcine sperm were subjected to a hydrostatic pressure of 20 to 80 MPa for 40 to 120 minutes and then maintained in a cold environment for 5 hours, the motility of the sperm was higher than that of the specimen maintained in a cold environment without prior pressure treatment.

[0004] Non-Patent Document 1, which includes some of the inventors of Patent Documents 5 and 6 as authors, reports that when hydrostatic pressure is applied to sperm before freezing or refrigeration, the motility of the sperm is improved compared to when freezing or refrigeration is performed without applying hydrostatic pressure. It is shown that by applying hydrostatic pressure in advance, the protein profile after freezing or refrigeration changes, and it is suggested that this change in protein affects the motility of sperm.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] In order to be used for artificial insemination, in vitro fertilization, micro-insemination, etc., semen after collection is often diluted with an aqueous solution containing a chelating agent, and may be further frozen or refrigerated as necessary. In such cases, a problem is that the motility of sperm decreases.

[0008] The present invention has been made to solve the above problems, and an object thereof is to provide a method for recovering the motility of spermatozoa reduced by diluting semen after collection with the above aqueous solution.

Means for Solving the Problems

[0009] The present inventors have intensively studied to solve the above problems and completed the present invention. That is, the present invention is as follows [1] to [5]. [1] A method for treating semen, comprising diluting the collected semen with an aqueous solution containing calcium ions and a chelating agent, applying a hydrostatic pressure of 5 to 40 MPa for 0.1 to 15 minutes, and then releasing the pressure. [2] The method for treating semen according to [1], wherein the content of calcium ions in the aqueous solution is 0.1 to 10 mM, and the content of the chelating agent is 0.1 to 50 mM. [3] The method for treating semen according to [1] or [2], wherein the chelating agent is ethylenediaminetetraacetic acid or glycol ether diamine tetraacetic acid. [4] The method for treating semen according to any one of [1] to [3], comprising diluting with the aqueous solution, cryopreserving and then thawing, and then applying the hydrostatic pressure and releasing the pressure. [5] The method for treating semen according to any one of [1] to [4] for artificial insemination, in vitro fertilization or intracytoplasmic sperm injection.

Effects of the Invention

[0010] According to the method for treating semen of the present invention, the motility of spermatozoa reduced by diluting semen after collection with an aqueous solution containing a chelating agent can be recovered.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Mode for Carrying Out the Invention

[0012] The present invention is a method for treating semen, which comprises diluting the collected semen with an aqueous solution containing calcium ions and a chelating agent, then applying a hydrostatic pressure of 5 to 40 MPa for 0.1 to 15 minutes and then releasing the pressure.

[0013] Generally, semen collected for use in artificial insemination, in vitro fertilization, intracytoplasmic sperm injection, etc. is subjected to treatments such as dilution, washing, centrifugation, freezing, etc. as appropriate, and then used for artificial insemination, etc. The diluent used at this time has a composition considering the preservability of sperm and contains various electrolytes. Among them, calcium ions are considered to affect the motility of sperm.

[0014] On the one hand, when the diluent contains a chelating agent typified by ethylenediaminetetraacetic acid (EDTA), polyvalent cations such as calcium ions contained in the diluent are captured. Thus, by controlling the free calcium ion concentration in the diluent, it may be possible to suppress sperm death during long-term storage or cryopreservation, and it is widely practiced to include a chelating agent in the diluent.

[0015] However, if the free calcium ion concentration decreases too much, sperm motility will decrease. Therefore, there has been a demand for a method that can ensure sperm motility while improving preservation properties by incorporating a chelating agent.

[0016] In Patent Documents 5 and 6 and Non-Patent Document 1, it has been proposed to apply hydrostatic pressure to diluted semen, and experimental examples in which a hydrostatic pressure of 20 to 90 MPa is applied for 30 to 120 minutes are described. It shows that by applying hydrostatic pressure in advance before freezing or refrigeration, the protein profile after freezing or refrigeration has changed, and it is suggested that this change affects sperm motility. In this example, hydrostatic pressure is applied for a long time of 30 minutes or more to change the protein profile.

[0017] In contrast, the present inventor has found that after diluting the collected semen with a diluent containing a chelating agent, applying hydrostatic pressure for a short time can restore the sperm motility that has decreased due to the influence of the chelating agent. The reason why sperm motility that has once decreased can be restored by applying hydrostatic pressure for a short time is not necessarily clear, but it is presumed that by applying hydrostatic pressure, calcium ions are taken into sperm cells even in the presence of a chelating agent, and sperm motility is restored. Since it only requires the movement of ions without the movement of high-molecular substances such as protein profile changes, it is considered that the effect is manifested in a short time. Thus, by simply applying hydrostatic pressure for a short time, it is possible to improve sperm motility decreased by a chelating agent, which is easily applicable in the fields of medicine and livestock production and is of great significance.

[0018] Hereinafter, the present invention will be specifically described.

[0019] The semen used in the present invention is not particularly limited. It may be human semen or semen of other animals. As other animals, mammals are preferred, and in particular, pigs, cows, horses, sheep, goats, dogs, cats, etc. are exemplified as suitable ones.

[0020] The semen collected from these animals is used for reproduction, and specific methods thereof include artificial insemination, in vitro fertilization, intracytoplasmic sperm injection, etc. Artificial insemination is a method of injecting sperm into the uterine cavity. In vitro fertilization is a method of fertilizing eggs taken out of the body before ovulation by an egg collection operation with sperm outside the body. Intracytoplasmic sperm injection is a method of injecting sperm into the taken-out eggs using a glass needle under a microscope in the same manner as in vitro fertilization. In any method, sperm that has been washed and diluted in advance is used, and in that case, a diluent containing a chelating agent is often used, which is a suitable application of the treatment method of the present invention.

[0021] In the present invention, the collected semen is diluted with an aqueous solution containing calcium ions and a chelating agent. Prior to dilution, the semen may be washed with a washing solution or centrifuged to remove sperm.

[0022] In the present invention, the diluent used for diluting the collected semen is an aqueous solution containing calcium ions and a chelating agent. From the viewpoint of sperm motility, the calcium ion content is preferably 0.1 to 10 mM. The calcium ion content is more preferably 0.2 mM or more, and even more preferably 0.5 mM or more. Also, the calcium ion content is more preferably 8 mM or less, and even more preferably 5 mM or less.

[0023] In addition, the diluent used in the present invention contains a chelating agent in addition to calcium ions. The chelating agent used here may be any one that can capture polyvalent metal cations and is not particularly limited. Polyamines, polycarboxylic acids, etc. can be used. Among them, polyaminopolycarboxylic acids are preferred, and in particular, ethylenediaminetetraacetic acid (EDTA) and glycol ether diamine tetraacetic acid (EGTA) are exemplified as suitable ones. The content of the chelating agent is preferably 0.1 to 50 mM. The content of the chelating agent is more preferably 0.2 mM or more, and even more preferably 0.5 mM or more. Also, the content of the chelating agent is more preferably 40 mM or less, and even more preferably 30 mM or less.

[0024] The diluent may contain electrolytes other than calcium ions, and may contain cation species such as sodium ions, potassium ions, and magnesium ions, and anion species such as chloride ions, sulfate ions, phosphate ions, carbonate ions, bicarbonate ions, lactate ions, and pyruvate ions. Further, it may contain a nutrient source such as glucose, or may contain an antibiotic such as gentamicin to prevent bacterial contamination. In order to stabilize the pH of the diluent, it is preferably a buffer solution. Also, the pH of the diluent is preferably 6 to 8.

[0025] In the treatment method of the present invention, after diluting semen with a diluent, hydrostatic pressure is applied. By applying hydrostatic pressure to sperm in an aqueous solution containing calcium ions and a chelating agent, the motility of sperm that has been suppressed by the presence of the chelating agent can be restored. The hydrostatic pressure applied at this time is 5 to 40 MPa. If the hydrostatic pressure is too low, the restoration of sperm motility will be insufficient. The hydrostatic pressure is preferably 10 MPa or more. On the other hand, if the hydrostatic pressure is too high, there is a risk that the sperm will be damaged, and the pressurizing device may also become large-scale. The hydrostatic pressure is preferably 30 MPa or less.

[0026] The pressurization time when applying hydrostatic pressure is 0.1 to 15 minutes. If the pressurization time is too short, the recovery of sperm motility will be insufficient. The pressurization time is preferably 0.2 minutes or more, more preferably 0.5 minutes or more. On the other hand, if the pressurization time is too long, there is a risk that the sperm will be damaged. The pressurization time is preferably 10 minutes or less, more preferably 5 minutes or less, and even more preferably 3 minutes or less. Also, a short pressurization time is preferable from the viewpoint of workability, and it also facilitates sample management in infertility treatment clinics and the like. After pressurizing for a predetermined time, the pressure is released, and then it is used for artificial insemination or the like.

[0027] The method of applying hydrostatic pressure to the diluted semen is not particularly limited, and various general pumps, such as a hydraulic pump or a pneumatic pump, can be used. When using a hydraulic pump, since the working fluid is water, the sample can be put into the water in the pressure vessel and pressure can be applied. The pump can be driven manually or electrically. The container for putting the sample may be sealed and have flexibility to transmit external pressure. Examples of the container used include a plastic tube with a cap.

[0028] After diluting the semen with a diluent, it can be cryopreserved and then thawed, and then hydrostatic pressure can be applied and the pressure can be released. Thus, by applying hydrostatic pressure after freezing and thawing, it is also possible to recover the sperm motility reduced by the chelating agent. By freezing, it is possible to store sperm for a long period of time.

[0029] By treating the semen as described above, it is possible to recover the sperm motility reduced by the use of the chelating agent. The sperm treated in this way can be used for artificial insemination, in vitro fertilization, intracytoplasmic sperm injection, etc. in reproductive assisted medicine. It can also be used for artificial insemination for livestock breeding.

Example

[0030] [Example 1] A sample was prepared by diluting human semen (from 1 donor) collected manually 10-fold with a diluent. The diluent was human tubal fluid (HTF) manufactured by FUJIFILM Irvine Scientific and contained 2 mM Ca 2+ ions. Also, a sample was prepared by diluting 10-fold with a diluent in which ethylenediaminetetraacetic acid (EDTA) manufactured by Dojindo Laboratories was added to the HTF so that its concentration became 2 mM. After preparing these samples, they were immediately subjected to observation.

[0031] 5 μL of the diluent prepared in this way was dropped onto a slide glass (manufactured by Matsunami Glass Industry Co., Ltd.) to create a glass preparation. For observation, a high-speed camera "HAS-U2" manufactured by Detect Co., Ltd. was attached to a microscope "CX41" manufactured by Olympus Corporation, and using software "HAS-VIEWER" manufactured by Detect Co., Ltd., a video was taken at a frame rate of 200 fps (frames per second). Using the obtained video data, the movement of the sperm head was tracked for 0.1 seconds (20 frames), and the curved distance during that time was determined. This curved distance is the distance of the curved movement of the sperm head canceling the head shaking movement. Figure 1 shows the results of observing and analyzing each of the sample diluted only with HTF (number of specimens (number of sperm) 124) and the sample diluted by adding EDTA to HTF (number of specimens 135).

[0032] Next, a sample diluted by adding EDTA to HTF was prepared in the same manner as above, put into a PCR 8-tube, and a hydrostatic pressure of 20 MPa was applied for 1 minute using a hydraulic manual pump "WP-1B" manufactured by RIKEN KEIKI Co., Ltd. and then returned to atmospheric pressure, and then left standing at atmospheric pressure for 30 minutes. The results (number of specimens 90) of observing and analyzing this sample in the same manner as above are shown together with Figure 1. It was shown that the curved speed significantly decreased by adding EDTA, and also that the curved speed significantly recovered by applying hydrostatic pressure thereafter.

[0033] Figure 2 is an image obtained by superimposing the points of maximum luminance for 100 frames (0.5 seconds) in a sample diluted after adding EDTA to HTF, and the trajectory of sperm head movement during that time is observed. Further, Figure 3 is an image obtained in the same manner for a sample that was pressurized after being diluted by adding EDTA to HTF. By comparing these figures, it can be seen that the curvilinear velocity of the sperm head increases by applying hydrostatic pressure for a short time. The curvilinear velocity is obtained by measuring the length of the arrowed curve shown in Figure 4 from the image obtained in this way and dividing it by the measurement time.

[0034] Also, in the same manner as when obtaining the above curve distance, three types of samples were prepared, observed with the same apparatus, and the motility rate of sperm was determined. The number of specimens of the sample diluted only with HTF was 243, the number of specimens of the sample diluted by adding EDTA to HTF was 280, and the number of specimens of the sample pressurized after being diluted by adding EDTA to HTF was 259. These results are summarized in Figure 5. It was shown that the motility rate of sperm decreased by adding EDTA, and then recovered by applying hydrostatic pressure.

[0035] [Example 2] A sample was prepared by diluting human semen (from 1 donor) collected manually 5-fold with a diluent. The diluent was the same HTF as in Example 1. Also, a sample diluted 5-fold with a diluent in which the same EDTA as in Example 1 was added to the HTF so that its concentration became 2 mM was prepared. Each sample was divided into 2 groups. In one group, the sample was placed in a PCR 8-tube, and a hydrostatic pressure of 20 MPa was applied for 1 minute using a hydraulic manual pump "WP-1B" manufactured by RIKEN KEIKI CO., LTD., and then returned to atmospheric pressure. For the 4 samples thus obtained, centrifugation (1000 rpm, 5 minutes) was performed to precipitate the sperm. After discarding the supernatant, a cryoprotectant manufactured by FUJIFILM Irvine Scientific was mixed with the precipitate. The resulting liquid was put into a straw and stored in a refrigerator at 4°C for 60 minutes. Next, the straw was exposed to the vapor of liquid nitrogen for 30 minutes and then placed in liquid nitrogen for freezing. After the next day, the frozen liquid in the straw was immersed in running water for thawing. Centrifugation (1000 rpm, 5 minutes) was performed on the thawed liquid to precipitate the sperm. After discarding the supernatant, HTF was mixed with the resulting precipitate to obtain an observation sample, which was observed and analyzed using a sperm motility analysis system "SMAS" manufactured by DETECT Co., Ltd. with a high-speed camera "HAS-U2" manufactured by DETECT Co., Ltd. attached to a microscope "TS100" manufactured by Nikon Corporation. Five locations were observed for each sample, and the average was calculated.

[0036] The above is the case where hydrostatic pressure was applied before freezing. However, in both the EDTA-added system and the non-added system, the tests when hydrostatic pressure was applied after freezing and the tests when no hydrostatic pressure was applied at all were conducted in the same manner. The results of a total of 6 tests are summarized in Table 1.

[0037]

Table 1

[0038] As shown in Table 1, it can be seen that by adding EDTA to the diluent, the number of motile sperm after freeze-thawing increases. Therefore, when performing cryopreservation, it is considered effective to capture metal ions with a chelating agent such as EDTA. And when hydrostatic pressure was applied after freezing in the case of adding EDTA, the straight-line velocity increased. The straight-line velocity is the straight-line distance between the first and last sperm heads within the measurement time and is a value that affects fertilization ability.

[0039] [Example 3] A sample was prepared by diluting 10-fold with a diluent in which EDTA was added to the same HTF as in Example 1 so that the concentration became 20 mM, using porcine semen (Duroc breed) purchased from Shimco Co., Ltd. After preparing the sample, it was immediately observed in the same manner as in Example 1. A video of sperm motility was taken at a frame rate of 200 fps, and a reconstructed image obtained by overlapping images for 0.1 seconds (20 frames) thereof is shown in FIG. 6. Further, after preparing the sample, a hydrostatic pressure of 20 MPa was applied for 1 minute in the same manner as in Example 1 and then returned to atmospheric pressure, and then left standing under atmospheric pressure for 10 minutes, and then a reconstructed image obtained by observing in the same manner is shown in FIG. 7.

[0040] When preparing the glass slide, since the heads of porcine sperm were bound to the glass, the motility of sperm was evaluated by the presence or absence of flagellar vibration. In FIGS. 6 and 7, sperm with vibrating flagella are circled in white. Before applying hydrostatic pressure, as shown in FIG. 6, the proportion of motile sperm was 4 / 34 = 0.12 (12%), but after applying hydrostatic pressure, as shown in FIG. 7, it increased to 39 / 111 = 0.35 (35%). Also in porcine sperm, as in human sperm, after dilution with an EDTA-containing diluent, the motility of sperm was improved by applying hydrostatic pressure.

Claims

1. A method for treating semen, comprising diluting the collected semen with an aqueous solution containing calcium ions and a chelating agent, applying a hydrostatic pressure of 5 to 40 MPa over 0.1 to 15 minutes, and then releasing the pressure.

2. The method for treating semen according to claim 1, wherein the content of calcium ions in the aqueous solution is 0.1 to 10 mM, and the content of the chelating agent is 0.1 to 50 mM.

3. The method for treating semen according to claim 1 or 2, wherein the chelating agent is ethylenediaminetetraacetic acid or glycol ether diamine tetraacetic acid.

4. The method for treating semen according to claim 1 or 2, comprising diluting with the aqueous solution, cryopreserving, thawing, applying the hydrostatic pressure, and then releasing the pressure.

5. The method for treating semen according to claim 1 or 2 for artificial insemination, in vitro fertilization, or microinjection.

Images