Methods for freezing rat sperm and in vitro fertilization

Abstract

The purpose of the present invention is to provide a rat sperm freezing method and an in vitro fertilization method that uses frozen rat sperm. The present invention provides a method for preparing frozen rat sperm. The method is characterized by including the following steps. Step a: a preparation step for collecting rat sperm from rat cauda epididymides and preparing a sperm suspension; step b: a cooling step for cooling the sperm suspension to approximately 1°C or below; and step c: a freezing step for freezing the rat sperm suspension that has been cooled to approximately 1°C or below.

Description

[Technical Field]

[0001] The present invention relates to a method for cryopreserving rat sperm, and also to an in vitro fertilization method using rat sperm cryopreserved using said method. [Background technology]

[0002] Over the past decade, transgenic rats with gene deletions or introductions and point mutation rats with base substitutions have been produced in large numbers worldwide. Genome editing technology has also become increasingly common in rat research. Therefore, it is predicted that many more genetically modified rats will be produced using genome editing in the near future. However, because rats are approximately 10 times larger than mice, there are limitations on the number of rats that can be raised in laboratory animal facilities. As a result, many genetically modified rat strains have been preserved as gametes or cryopreserved embryos in rat resource centers (Rat Banks). Representative rat banks include the National BioResource Project - Rat at Kyoto University in Japan, the Rat Resource & Research Center at the University of Missouri in the United States, and the Gene Editing Rat Resource Center at the Medical College of Wisconsin in the United States.

[0003] One method for preserving rat strains is the cryopreservation of rat embryos. This method requires the collection of embryos from the oviducts of 30–50 mated females, since 400–500 embryos per strain are stored. Because the number of females that can be mated at one time is limited, and males must be mated multiple times, it takes 2–4 months to obtain a sufficient number of embryos for cryopreservation when using males with several genetic modifications. In contrast, the cryopreservation of rat sperm allows for the cryopreservation of a large number of sperm immediately after collection from the male's epididymis. Therefore, for the preservation of genetically modified rat strains, sperm cryopreservation is far simpler, more efficient, and more cost-effective than embryo cryopreservation.

[0004] In vitro fertilization using fresh rat sperm was demonstrated by Toyoda et al. in 1974, but for quite some time thereafter, no successful cases of in vitro fertilization using frozen rat sperm were reported. Regarding the cryopreservation of rat sperm, Nakatsukasa et al. first reported it in 2001 (Nakatsukasa et al., Reproduction 122, 463-467 (2001)). Although many reports have been published since then, Seita et al. is the only one to report the production of offspring from embryos obtained by in vitro fertilization using frozen rat sperm (Non-Patent Document 2: Seita et al., Biol. Reprod. 80, 503-510 (2009)). According to their report, when frozen / thawed sperm treated with IBMX were used for in vitro fertilization, the rates of pronuclear formation and blastocyst formation were significantly higher (pronuclear formation rate: 50%; blastocyst formation rate: 20%; live birth rate: 49%) compared to when frozen / thawed sperm not treated with IBMX were used; however, the low fertilization rate made this technique unfeasible. Since this study, there have been no reports demonstrating that frozen sperm retain sufficient motility to produce live offspring using methods such as in vitro fertilization with frozen sperm and embryo transfer of fertilized eggs. Therefore, a satisfactory protocol for freezing rat sperm and a protocol for in vitro fertilization using such a protocol have not yet been established. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Nakatsukasa et al., Reproduction 122, 463-467 (2001) [Non-patent document 2] Seita et al., Biol. Reprod. 80, 503-510 (2009) [Non-patent document 3] Nakatsukasa et al., Comp. Med. 53, 639-641 (2003) Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for cryopreserving rat sperm and an in vitro fertilization method using cryopreserved rat sperm. [Means for solving the problem]

[0007] Rat sperm are known to be highly sensitive to environmental changes, such as the viscosity of the solution in which they are placed and osmotic stress. The commonly used rat sperm cryopreservation solution (CPA) has a higher viscosity and osmotic pressure (370-380 mOsm) than the diluent mTHF (300-310 mOsm). Therefore, the inventors suspected that diluting a CPA containing sperm with a diluent immediately after thawing could damage the cryopreserved sperm. To address this issue, the inventors conducted extensive research into methods to mitigate environmental changes in rat sperm. As a result, they discovered that freezing and / or thawing rat sperm using a specific process can achieve higher fertilization rates and offspring development potential in in vitro fertilization compared to conventional methods, leading to the completion of the present invention.

[0008] The present invention includes the following. (Freezing preservation method) [1] A method for preparing cryopreserved rat sperm, comprising the steps of: Step a: A preparation step of collecting rat sperm from the rat cauda epididymis and preparing a sperm suspension; Step b: A cooling step of cooling the sperm suspension to about 1°C or less (preferably about 0.5°C or less, more preferably about 0°C), and Step c: a freezing step of freezing the rat sperm suspension cooled to about 1°C or below; A method comprising: [2] The method according to [1] above, wherein the cooling step in step b is carried out to substantially reduce the motility of rat sperm. [3] The method according to [1] or [2] above, wherein step b comprises two steps: (b-1) cooling the rat sperm suspension to about 4°C to about 6°C (preferably about 4°C to about 5°C) (hereinafter, sometimes referred to as the first cooling of step b), and (b-2) further cooling the first-cooled sperm suspension to about 1°C or less (preferably about 0.5°C or less, more preferably about 0°C) (hereinafter, sometimes referred to as the second cooling of step b). [4] The method according to the above [3], wherein at least the step (b-2) is carried out in a cryopreservation container (preferably a straw-shaped cryopreservation container) for cryopreserving sperm. [5] The method according to [3] or [4] above, wherein step (b-2) is carried out by placing the cryopreservation container containing the sperm suspension on ice for about 15 to about 45 minutes (preferably about 25 to about 35 minutes, more preferably about 30 minutes). [6] The method according to any one of [1] to [5] above, wherein in step b, the motility of the sperm after cooling is 20% or less (preferably 15% or less, more preferably 10% or less, even more preferably 5% or less, and even more preferably 2% or less) of the motility of the sperm before cooling. [7] The method according to any one of [1] to [5] above, wherein in step b, the motility of sperm after cooling is 50% or less (preferably 40% or less, more preferably 30% or less, and even more preferably 20% or less) compared to the motility before cooling. [8] The method according to any one of the above [1] to [7], wherein the rat cauda epididymis is refrigerated and stored. [9] The method according to any one of [1] to [8] above, wherein the rat sperm is sperm derived from a genetically modified rat.

[10] Use of cryopreserved rat sperm prepared by the method according to any one of [1] to [9] above for in vitro fertilization.

[0009] (Freezing and thawing method)

[11] A method for preparing rat sperm for use in in vitro fertilization, comprising the steps of: Step A: a thawing step of warming a cryopreservation solution containing frozen rat sperm to a temperature of about 35°C to about 37.5°C (preferably about 36°C to about 37.5°C, more preferably about 37°C) to prepare a thawed rat sperm suspension; Step B: A swim-up step in which the thawed rat sperm suspension is placed at the bottom of a medium contained in a container and allowed to stand, allowing the sperm to swim up; Step C: a first recovery step of recovering sperm; and Step D: A second recovery step of transferring the recovered sperm to a sperm culture medium and recovering highly motile sperm; A method comprising:

[12] The method according to the above

[11] , wherein the lower part of the container in step B is a tube having a tapered or conical tip.

[13] The method according to

[11] or

[12] above, wherein the volume of the medium in step B is about 5 to about 20 times the volume of the sperm suspension.

[14] The method according to any one of the above

[11] to

[13] , wherein the standing in step B is carried out for about 20 to about 40 minutes.

[15] The method according to any one of

[11] to

[14] above, wherein step C is a step of mixing the thawed rat sperm suspension with a culture medium in a container, followed by centrifuging at low speed to collect the precipitate, thereby recovering the sperm.

[16] Use of rat sperm prepared by the method according to any one of

[11] to

[15] above for in vitro fertilization.

[0010] (In vitro fertilization method)

[17] The process of: (i) thawing the cryopreserved rat sperm prepared by the method described in any one of [1] to [9] above using the method described in any one of

[11] to

[15] above to prepare them for in vitro fertilization, and then pre-culturing the prepared rat sperm in a sperm pre-culture medium (wherein the sperm pre-culture medium is also the medium used for in vitro fertilization in step (iii)); (ii) administering equine chorionic gonadotropin (eCG) to female rats, then administering human chorionic gonadotropin (hCG) to the rats, and then collecting unfertilized eggs to prepare unfertilized eggs; (iii) a step of mixing the sperm pre-cultured in step (i) with the unfertilized eggs prepared in step (ii) to perform insemination; IVF methods including:

[18] The in vitro fertilization method described in

[17] above, wherein step (ii) further comprises a cumulus removal step of removing the cumulus from the prepared unfertilized eggs.

[19] The in vitro fertilization method described in

[17] or

[18] above, wherein the sperm preculture medium in step (i) is mHTF medium containing about 1 to about 80 mg / mL (preferably about 4 to about 80 mg / mL, more preferably about 10 to about 60 mg / mL, even more preferably about 20 to about 60 mg / mL, and even more preferably about 30 to about 50 mg / mL) of bovine serum albumin.

[0011] (Sperm pre-incubation medium)

[20] A rat sperm preculture medium, which is mHTF medium or TYH medium containing about 20 mg / mL to about 80 mg / mL (preferably about 20 to about 60 mg / mL, more preferably about 30 to about 50 mg / mL) of bovine serum albumin, used for preculture of rat sperm in in vitro fertilization using cryopreserved rat sperm. (Fertilization medium)

[21] A rat in vitro fertilization medium for use in in vitro fertilization using cryopreserved rat sperm, which is mHTF medium or TYH medium containing about 20 to about 80 mg / mL (preferably about 20 to about 60 mg / mL, more preferably about 30 to about 50 mg / mL) of bovine serum albumin.

[0012] One aspect of the present invention relates to the cryopreservation of rat sperm, and provides a method for freezing rat sperm that can achieve a better fertilization rate and / or offspring development ability than conventional techniques when in vitro fertilization is performed using frozen-thawed rat sperm. Another aspect of the present invention relates to thawing frozen rat sperm, and provides a method for thawing frozen rat sperm that can achieve a better fertilization rate and / or offspring development ability than conventional techniques when in vitro fertilization is performed using the thawed rat sperm. Another aspect of the present invention relates to in vitro fertilization using frozen rat sperm, and provides an in vitro fertilization method for rat sperm that can achieve a better fertilization rate and / or offspring development ability than conventional techniques by combining the method for freezing rat sperm of the present invention with the method for thawing frozen rat sperm. [Effects of the Invention]

[0013] The method for cryopreserving rat sperm and / or the method for thawing frozen rat sperm of the present invention provide in vitro fertilization using frozen rat sperm, which can achieve a better fertilization rate and / or offspring development ability than conventional techniques. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram of a method for cryopreserving rat sperm, which is one embodiment of the present invention. [Figure 2] FIG. 1 is a schematic diagram of a method for thawing frozen rat sperm, which is one embodiment of the present invention. [Figure 3] 1 is a diagram showing one embodiment of an instrument that can be used in the rat sperm freezing method of the present invention, the figure showing the instrument as seen from diagonally above. [Figure 4]FIG. 4 is a view of the cryopreservation container placement stand used in the freezing assisting device shown in FIG. 3, seen obliquely from above. [Figure 5] FIG. 4 is a top view of the cryopreservation container placement stand used in the freezing assisting device shown in FIG. 3. [Figure 6] FIG. 4 is a side view of the cryopreservation container placement stand used in the freezing aid of FIG. 3. [Figure 7] FIG. 10 is a diagram showing another embodiment of an instrument that can be used in the rat sperm freezing method of the present invention. [Figure 8] FIG. 8 is a view of the cryopreservation container placement stand used in the freezing assisting device shown in FIG. 7, seen obliquely from above. [Figure 9] FIG. 8 is a top view of the cryopreservation container placement stand used in the freezing assist device shown in FIG. 7. [Figure 10] FIG. 8 is a side view of the cryopreservation container placement stand used in the freezing assist device shown in FIG. 7. [Figure 11] This figure shows the temperature change of the sperm suspension during the process of freezing rat sperm using the rat sperm freezing method of the present invention. Each operational step is indicated with the corresponding step number shown in Figures 1 and 2. A indicates the start of cooling the sperm suspension on ice (C in Figure 1), B indicates the start of filling the straw with the sperm suspension (D in Figure 1), C indicates the start of cooling the straw on ice (F in Figure 1), and D indicates the start of freezing the straw in liquid nitrogen (G in Figure 1). [Figure 12] This is a microscopic photograph of a blastocyst-stage embryo obtained by in vitro fertilization using rat sperm that had been cryopreserved and thawed using the method of the present invention. The green embryos (which appear pale in black and white) are embryos that are emitting a GFP signal. [Figure 13] This is a photograph of offspring born after embryo transfer into a rat from fertilized eggs obtained by in vitro fertilization using rat sperm that had been cryopreserved and thawed using the method of the present invention. The green offspring (which appear white in black and white) are those that emit GFP signals. [Figure 14]The results show that sperm motility was confirmed after preserving rat cauda epididymis in Lifor® refrigerated preservation solution containing various concentrations of dimethyl sulfoxide (DMSO) and quercetin (Q). The left graph shows the percentage of motile sperm 2 hours after sperm suspension preparation, and the right graph shows the percentage of motile sperm 6 hours after preparation. [Figure 15] These are photographs of eggs prepared using conventional methods (untreated eggs: left image) and eggs that have also had the cumulus removed (cumulus-removed eggs: eggs with the surrounding cumulus cells removed: right image). DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described in detail below by way of illustrative embodiments, but the present invention is not limited to the embodiments described below. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In addition, any materials and methods equivalent or similar to those described herein can be used in the practice of the present invention. In addition, all publications and patents cited herein that relate to the inventions described herein are incorporated by reference, for example, to describe methodologies, materials, or the like that might be used in the present invention.

[0016] When the expression "X to Y" is used in this specification, it means that X is the lower limit and Y is the upper limit, or that Y is the lower limit and X is the upper limit. In this specification, the term "about" is used to mean that ±10% is acceptable.

[0017] By using the method of the present invention described below, the present inventors have succeeded in cryopreserving transgenic rat sperm and in in vitro fertilization of eggs with the cryopreserved sperm. Normal offspring were also produced from the in vitro fertilized eggs.

[0018] [Method for freezing and preserving rat sperm] One aspect of the present invention is a method for cryopreserving rat sperm, comprising the following steps: Step a: A preparation step of collecting rat sperm from the rat cauda epididymis and preparing a sperm suspension; Step b: A cooling step of cooling the sperm suspension to about 1°C or less; and Step c: A freezing step of freezing the rat sperm suspension cooled to about 1°C or below. An outline of the method for cryopreserving rat sperm is shown in Figure 1A to 1H.

[0019] (1) A step of preparing a sperm suspension In the method for cryopreserving rat sperm of the present invention, a rat sperm suspension is first prepared. Rat sperm are collected from the cauda epididymis of a rat according to standard methods and suspended in a cryopreservation solution to prepare a sperm suspension. The cryopreservation solution (CPA) can be any cryopreservation solution that can be used for preserving sperm from animals, preferably small animals, with appropriate modifications as necessary. Alternatively, commercially available cryopreservation solutions may be used.

[0020] Although the method for preparing a rat sperm suspension is not limited to this, it can be preferably performed, for example, as follows. The cauda epididymis is collected from a euthanized adult male rat. The collected cauda epididymis is placed in a CPA drop (1 cauda epididymis / 1.5 mL) prepared in the center of a petri dish at room temperature (20-25°C), and the cauda epididymis is incised multiple times, preferably about ten times, using scissors or a scalpel to prepare a sperm suspension. The petri dish is then placed on a metal plate (e.g., a tin plate) with excellent thermal conductivity placed on crushed ice, and left to cool for preferably about 10 minutes or longer to cool the sperm suspension. This allows the sperm suspension to cool to about 4-6°C.

[0021] Sperm-containing cauda epididymis collected from rats and then refrigerated can be used. For example, cauda epididymis can be collected from euthanized adult male rats, placed in a refrigerated preservation solution, and stored refrigerated (e.g., at approximately 4°C) until preparation of the sperm suspension. Examples of preservation solutions include, but are not limited to, Lifor® preservation solution (Lifeblood Medical Inc.), a refrigerated preservation solution for human organs. Lifor preservation solution is an artificial solution containing nutrients, growth factors, and non-protein enzymes and nutrient carriers. Preferably, Lifor is supplemented with dimethyl sulfoxide (DMSO) and / or quercetin. The concentration of dimethyl sulfoxide added to the preservation solution is typically 1-30%, preferably 5-25%, and more preferably 5-20%. The concentration of quercetin added to the preservation solution is usually 10 to 1000 μg / mL, preferably 50 to 250 μg / mL, and more preferably 50 to 200 μg / mL. The refrigerated storage period is usually within 7 days, preferably within 5 days, and more preferably within 3 days. Furthermore, since refrigerated storage is possible, transportation in a refrigerated state is also possible.

[0022] (2) A cooling step in which the rat sperm suspension is cooled to below approximately 1°C. The method for cryopreserving rat sperm of the present invention is characterized by including a cooling step in which a rat sperm suspension is cooled to approximately 1°C or below before freezing (but without freezing the rat sperm). This cooling step cools the rat sperm suspension to approximately 1°C or below, preferably approximately 0.5°C or below, and more preferably approximately 0°C, a temperature at which the rat sperm will not freeze. This cooling step substantially reduces the motility of the rat sperm in the suspension. Substantially reducing the motility of the rat sperm reduces damage to the rat sperm during the subsequent cryopreservation step, including the freezing step, and allows the preparation of a satisfactory cryopreserved rat sperm suspension. When subsequently thawed and used for in vitro fertilization, the frozen rat sperm prepared in this manner achieves an improved fertilization rate compared to sperm stored by a conventional method, i.e., by cooling the rat sperm in a cryopreservation solution on a petri dish on ice, transferring them to a cryopreservation container, and immersing them in liquid nitrogen for freezing.

[0023] The step of cooling the rat sperm suspension to about 1°C or below is preferably carried out by cooling the sperm suspension to about 4°C to about 6°C (preferably about 4°C to about 5°C) (hereinafter sometimes referred to as "primary cooling"), followed by further cooling to about 1°C or below (hereinafter sometimes referred to as "secondary cooling"). However, cooling to about 1°C or below continuously without dividing the cooling into two stages is also included in the method of the present invention. Methods for primary cooling of the sperm suspension include, but are not limited to, placing a petri dish containing the sperm suspension on ice, preferably placing a thermally conductive metal plate, such as a tin plate, on ice so that the bottom of the petri dish is cooled evenly. Alternatively, cooling can be performed by immersing a container (e.g., a tube) containing the sperm suspension in a solution cooled to about 4°C, or by placing a tube or petri dish containing the sperm suspension in a constant-temperature cooling bath or cooling box set to about 4°C.

[0024] An example of a method for cooling the primarily cooled sperm suspension to approximately 1° C. or below is to transfer the primarily cooled sperm suspension to a cryopreservation container, and then cool the sperm suspension in the cryopreservation container to approximately 1° C. or below. There are no particular limitations on the cryopreservation container as long as it is a container that can be used for cryopreserving rat sperm, and examples include straw-shaped containers and tube-shaped containers.

[0025] When using refrigerated cauda epididymis, a sperm suspension may be prepared and cooled before the above steps. Alternatively, the entire procedure for preparing a sperm suspension can be performed at a low temperature (e.g., about 4°C). For example, a petri dish can be placed on a heat-conductive metal plate (e.g., a tin plate) placed on crushed ice. The cauda epididymis (1 cauda epididymis / 1.5 mL) can be placed in a cooled CPA drop (e.g., cooled to about 4°C) in the center of the dish, and the sperm suspension can be prepared by making multiple cuts, preferably about ten times, in the cauda epididymis using scissors or a scalpel. When refrigerated cauda epididymis is used and the entire procedure for preparing a sperm suspension is performed at a low temperature, the above-mentioned primary cooling step can be omitted.

[0026] Although not limited to this, a preferred method for secondary cooling is to transfer the primarily cooled sperm suspension to a straw-shaped cryopreservation container and place it again on ice, preferably on a heat-conductive metal plate (e.g., a tin plate) placed on ice, to cool it to approximately 1°C or below. The time for placing it on ice or on a metal plate is not particularly limited as long as the sperm suspension cools to approximately 1°C or below. For example, when using a straw-shaped container, the time is approximately 15 to 45 minutes, preferably approximately 20 to 40 minutes, more preferably approximately 25 to 35 minutes, and even more preferably approximately 30 minutes. Here, "on ice" or "on a metal plate" means placing the cryopreservation container (e.g., a straw-shaped container) directly on ice or a metal plate. This allows the sperm suspension to cool more uniformly.

[0027] Secondary cooling of the sperm suspension can also be performed by placing the container containing the sperm suspension in ice. Alternatively, secondary cooling of the sperm suspension can be performed by placing the cryopreservation container containing the sperm suspension in a constant temperature cooling box set to approximately 0°C and cooling it. When using a programmable cooling box, the cryopreservation container containing the sperm suspension can be placed in the cooling box and the primary and secondary cooling can be performed consecutively.

[0028] Any of the methods described above can be used for step b, but a preferred method is to place the petri dish containing the sperm suspension on a metal plate placed on ice and cool it to about 4°C to about 6°C, then transfer the sperm suspension to a straw-shaped cryopreservation container and cool it again on the metal plate on ice to about 1°C or below. The sperm suspension can be inserted into the straw-shaped cryopreservation container using a known method. For example, a straw-shaped container with a major axis of 1.9 mm (inner diameter 1.6 mm, length 133 mm) can be used. 30 μL of mHTF solution is carefully aspirated into the straw along with 10 mm of air. 150 μL of sperm suspension is then aspirated. The syringe plunger is then pulled up until the 30 μL of mHTF solution reaches the cotton plug of the straw. Finally, the tip of the straw is sealed with an impulse sealer.

[0029] In step b, rat sperm are cooled to about 1°C or below to substantially reduce sperm motility. In this step, "substantially reducing sperm motility" refers to the motility of sperm after cooling being reduced to about 20% or less, preferably about 15% or less, more preferably about 10% or less, even more preferably about 5% or less, even more preferably about 2% or less, and most preferably about 1% or less, of the motility of sperm before cooling. Here, sperm motility refers to the ability of sperm to move. Examples of motile sperm include, but are not limited to, (1) sperm that move quickly and in a straight line, (2) sperm that move slowly but in a straight line, (3) sperm that move quickly but not in a straight line, (4) sperm that rotate in place, and (5) sperm that show movement of the head or tail but do not show whole-body movement. Therefore, comparing motility can be done, for example, by checking the number of sperm (1) to (5) above using the sperm suspension before the cooling process, and by checking the number of sperm (1) to (5) above using the sperm suspension after cooling to approximately 1°C or below, and comparing the two.

[0030] The above criteria (1) to (5) indicating the state of motile sperm can be arbitrarily selected and combined. For example, since cooled rat sperm mainly contain sperm classified as (4) or (5), the percentage of sperm classified as (1) to (3) ([number of sperm classified as (1) to (3)] / [number of sperm classified as (1) to (5)] x 100%) can be determined using a sperm suspension before the cooling step and a sperm suspension after cooling to approximately 1°C or below, and then compared. Therefore, "substantially reducing sperm motility" means that the percentage of sperm classified as (1) to (3) after cooling is approximately 20% or less, preferably approximately 15% or less, more preferably approximately 10% or less, even more preferably approximately 5% or less, even more preferably approximately 2% or less, and most preferably approximately 1% or less, compared to before cooling. When rat sperm are cooled using conventional methods, i.e., when a sperm suspension placed in a tube is cooled on ice, the motility of rat sperm, measured as the ratio of the number of sperm in (1) to (3) above (number of sperm in [(1) to (3)] / number of sperm in [(1) to (5)] x 100%), is only reduced to about 20% to about 30% compared to before cooling. However, in the method of the present invention, rat sperm are cooled to about 1°C or below, so that the motility of rat sperm is further suppressed, to about 20% or less compared to before cooling.

[0031] In the present invention, "substantially reducing sperm motility" refers, as another indicator, to a state in which the sperm motility rate after cooling is about 50% or less, preferably about 40% or less, more preferably about 30% or less, even more preferably about 20% or less, and most preferably about 10% or less, compared to the motility rate before cooling. Sperm motility is the ratio of the number of motile sperm to the total number of sperm, and is, for example, but not limited to, the ratio of sperm that fall into the above categories (1) to (5) to the total number of sperm. Therefore, this refers to a state in which the number of motile sperm after cooling is about 50% or less, preferably about 40% or less, more preferably about 30% or less, even more preferably about 20% or less, and most preferably 10% or less, compared to the motility rate before cooling. When rat sperm are cooled using conventional methods, i.e., when a sperm suspension placed in a tube is cooled on ice, the rat sperm motility, as measured by the ratio of motile sperm to the total number of sperm, is reduced to only about 50% to about 60% of the value before cooling. However, in the method of the present invention, rat sperm are cooled to about 1°C or below, further reducing the motility of rat sperm to about 50% or less of the value before cooling. The motility of rat sperm before cooling is typically about 60% to about 70%. Therefore, "substantially reducing sperm motility" means that the sperm motility after cooling (the ratio of motile sperm to the total number of sperm) is reduced to about 35% or less, preferably about 30% or less, more preferably about 20% or less, even more preferably about 15% or less, and most preferably about 10% or less.

[0032] (3) A freezing step for freezing the rat sperm suspension The freezing process can be performed by immersing a cryopreservation container containing a sperm suspension cooled to approximately 1°C or below in liquid nitrogen. While known freezing methods can be used, the method of the present invention more preferably involves placing a freezing container containing the sperm suspension, preferably a straw-shaped freezing container, on a polystyrene foam frame, immediately floating it on liquid nitrogen in a polystyrene foam box, and leaving it for a desired period of time, such as approximately 10 minutes or more, to freeze the sperm. The freezing container is then transferred to a dedicated cassette and immersed in liquid nitrogen in a tank for storage until use. The rat sperm thus cryopreserved are thawed before use for in vitro fertilization.

[0033] An apparatus that can be preferably used in the freezing step of the method of the present invention will now be described. One embodiment of a freezing auxiliary apparatus that can be used in the method of freezing rat sperm of the present invention is shown in Figure 3. The freezing aid will be described below with reference to Figures 3 to 7. The illustrated device is a freezing aid 100 for freezing a rat sperm suspension contained in a straw-shaped cryopreservation container 15. The freezing aid comprises a cryopreservation container placement stand 1 and a float 20 onto which the cryopreservation container placement stand can be fixed. The cryopreservation container placement stand 1 has storage container holders 11 on the left and right sides of a main base 10 for placing straw-shaped storage containers (hereinafter simply referred to as "storage containers") 15. The main base 10 has a space to allow efficient passage of cold air from liquid nitrogen, a cooling source located below the main base. The main base may be made of any material, but is preferably made of a material with good thermal conductivity, such as metal. In the figures, the main base is made of multiple metal rods, but there is no limit to the number of rods as long as storage container holders can be placed thereon; for example, it may be made of only a circumferential portion. There are no particular limitations on the storage container holder 11, as long as it can hold the storage container and maintain an appropriate distance between the liquid nitrogen cooling source and the storage container. While Figure 3 shows a coiled member with a rod 12 disposed within it, this is not a limitation. The rod may have any shape, but is preferably a round or square rod. There are no particular limitations on the material of the coiled member and the rod. When a storage container is placed on it, it is held in place by the coil and the rod disposed within it, maintaining an appropriate distance between the storage container and the cooling source. After cooling, the storage container is transferred to a dedicated cassette and frozen more slowly than if it were immersed in liquid nitrogen in a tank. This reduces damage to the sperm.

[0034] As shown in Figure 3, the cryopreservation container placement stand, on which storage container holders for holding straw-shaped cryopreservation containers are arranged on the left and right, is fixed on a float part for floating on liquid nitrogen. The straw-shaped cryopreservation container containing the sperm suspension is placed on the storage container holder of the freezing instrument storage stand, which is fixed on the float part, so that it does not move on its own even when the freezing aid is floating on the liquid nitrogen. The cryopreservation aid is floated on the liquid nitrogen and the sperm suspension is frozen. The frozen sperm suspension is then transferred to a tank of liquid nitrogen and stored.

[0035] Figure 4 is a view from diagonally above of the cryopreservation container placement stand used in the freezing aid shown in Figure 3. Figure 5 is a view from above of the cryopreservation container placement stand used in the freezing aid shown in Figure 3. Figure 6 is a view from the side of the cryopreservation container placement stand used in the freezing aid shown in Figure 3.

[0036] Figures 7 to 10 are views showing another embodiment of an instrument that can be used in the rat sperm freezing method of the present invention. In Figure 7, the storage container holding part 13 is composed of a long, thin, pedestal-shaped member, and the pedestal has a small notch 14 to prevent the storage container from rolling. Figure 8 is a view from diagonally above of the cryopreservation container placement table used in the freezing aid shown in Figure 7. Figure 9 is a view from above of the cryopreservation container placement table used in the freezing aid shown in Figure 7. Figure 10 is a view from the side of the cryopreservation container placement table used in the freezing aid shown in Figure 7.

[0037] Therefore, the following instruments can be used in the freezing step of the cryopreservation method of the present invention. (1) A sperm freezing aid (sperm freezing aid) used for freezing sperm, which comprises a float part 20 for floating on liquid nitrogen, and a cryopreservation container placement stand 1 placed or fixed on the float part, and the cryopreservation container placement stand has a main body base part 10 that is approximately square or approximately rectangular (here, the main body base part has a space part that is at least partially penetrated), and at least two storage container holding parts 11 placed on the main body base part (here, the storage container holding parts have a mechanism for holding straw-shaped storage containers and restricting their free movement). (2) The sperm freezing aid according to (1) above, wherein the main body base is composed of a plurality of metal rods. (3) A sperm freezing aid described in (1) or (2) above, wherein the space of the base portion constitutes more than half of the base portion, preferably more than 70%, more preferably more than 80%, and even more preferably more than 90%. (4) The sperm freezing aid according to any one of (1) to (3) above, wherein the two storage container holding parts are arranged facing each other near the outer periphery of the main body base part. (5) A sperm freezing aid according to any one of (1) to (4) above, wherein the storage container holding part is made of a coil-shaped member and a square or round rod disposed therein. (6) The sperm freezing aid according to any one of (1) to (4) above, wherein the storage container holding part is made of a long, thin, pedestal-shaped member having a notch on part of its surface.

[0038] [Method for thawing frozen rat sperm] Another aspect of the present invention is a method for thawing frozen rat sperm, which comprises the following steps: The thawed rat sperm can then be used for in vitro fertilization. The outline of the method for thawing frozen rat sperm is shown in Figures 2 I to M. Step A: A thawing step of preparing a thawed rat sperm suspension by heating a frozen storage solution containing rat sperm to a temperature of about 35°C to about 37.5°C; Step B: A swim-up step in which the thawed rat sperm suspension is placed at the bottom of a medium contained in a container and allowed to stand, allowing the sperm to swim up; Step C: a first recovery step of recovering sperm; and Step D: The second recovery step involves transferring the recovered sperm to a culture medium and recovering sperm with high motility.

[0039] <1> Preparing a thawed rat sperm suspension In the method for thawing frozen rat sperm of the present invention, frozen rat sperm are first thawed to prepare a thawed rat sperm suspension. The frozen storage solution containing rat sperm is thawed according to standard methods to prepare a rat sperm suspension. To thaw frozen rat sperm, the frozen sperm suspension is heated to a temperature of about 35°C to about 37.5°C, preferably about 36°C to about 37.5°C, and more preferably about 37°C. A container containing the frozen sperm suspension, preferably a straw-shaped container, is placed in a 37°C water bath and pre-incubated to thaw the sperm suspension. The incubation time in the water bath is not particularly limited as long as the sperm are thawed, but is, for example, about 5 to about 30 minutes, preferably about 10 to about 20 minutes, and more preferably about 15 minutes.

[0040] <2> Sperm swim-up process The method for thawing frozen rat sperm of the present invention is characterized by including a swim-up step in which thawed rat sperm suspension is placed at the bottom of a culture medium and the sperm swim up. Placing at the bottom of a culture medium here refers to placing the sperm suspension at a location below or below the culture medium. The method for placing the sperm suspension at the bottom of the culture medium is not particularly limited. For example, but not limited to, placing an appropriate amount of culture medium in a tube, preferably a tube with a tapered or conical tip (e.g., an Eppendorf tube or a culture tube), and then placing the thawed sperm suspension at the bottom of the tube using a capillary or tip. Alternatively, the sperm suspension can be placed at the bottom of the culture medium by placing the sperm suspension at the bottom of the tube and slowly layering the culture medium on top. A preferred method is to place the sperm suspension at the bottom of a tube containing culture medium. Because the sperm suspension has a higher density than the culture medium, it remains at the bottom of the tube. As a result, an environment is formed in which the sperm suspension and the culture medium are in contact with each other at an interface, allowing healthy sperm to swim up from the suspension into the culture medium. The ratio of the volume of thawed sperm suspension to the volume of medium is not particularly limited as long as an environment in which sperm can swim up is formed. However, the volume of medium relative to the volume of sperm suspension is preferably about 5 to 20 times, more preferably about 5 to 15 times, and even more preferably about 5 to 10 times. By employing such a swim-up process, sperm can be gradually transferred from the cryopreservation solution environment to the culture medium environment for in vitro fertilization. As a result, the degree of environmental change is significantly reduced compared to the conventional method of adding a thawed sperm suspension to a culture medium and mixing it. The time for allowing sperm to swim up is not particularly limited, but can be, for example, about 10 to 60 minutes, preferably about 20 to 40 minutes, and more preferably about 25 to 35 minutes.

[0041] The medium for causing sperm to swim up from the sperm suspension is not particularly limited as long as it is a medium that can be used for culturing sperm (sperm culture medium), and examples thereof include mHTF medium and TYH medium, and commercially available media can be used without limitation. Preferably, bovine serum albumin (BSA) is added to the medium, preferably at about 1 to about 80 mg / mL, more preferably at about 4 to about 80 mg / mL. The addition of BSA can increase the fertilization rate.

[0042] <3> Sperm collection process (first collection process) The sperm are allowed to swim up to the culture medium, after which the sperm are collected. Preferably, the sperm suspension and culture medium are mixed before collecting the sperm, preferably slowly to ensure homogeneity. For example, this can be done by slowly inverting the tube several times or slowly inverting the tube. The method for collecting sperm from the culture medium is not particularly limited. For example, sperm can be collected by centrifugation at a low speed (e.g., about 200 to about 500 g, preferably about 200 to about 400 g) for about 30 seconds to about several minutes, preferably about 60 seconds. After centrifugation, the suspension containing the sperm pellet collected at the bottom of the tube can be collected using, for example, a pipette with a wide-bore pipette tip (large orifice tip).

[0043] <4> Sperm collection process (second collection process) The sperm collected in the first collection step are transferred back to a sperm culture medium (sperm preculture medium), and motile sperm are collected according to standard methods. For example, a suspension containing the sperm pellet collected in the first collection step is transferred to a drop of sperm preculture medium covered with paraffin liquid placed in a petri dish, and the sperm are cultured. Sperm with high motility swim around the periphery of the drop, while sperm with low motility gather in the center of the drop. The volume of the culture medium drop is not particularly limited as long as it can culture the sperm and separate highly and less motile sperm, but is, for example, about 100 μL to about 300 μL, preferably about 150 μL to about 250 μL, and more preferably about 200 μL. The medium used is not particularly limited as long as it is a medium (sperm culture medium) that can be used to culture sperm, and examples thereof include mHTF medium and TYH medium, and commercially available media can be used without restriction. Preferably, bovine serum albumin (BSA) is added to the medium at a concentration of about 1 to about 80 mg / mL, preferably about 4 to about 80 mg / mL, more preferably about 10 to about 60 mg / mL, even more preferably about 20 to about 60 mg / mL, and even more preferably about 30 to about 50 mg / mL. The addition of BSA can increase the fertilization rate. After culturing (allowing) the sperm to swim in the drop for about 20 to about 40 minutes, preferably about 25 to about 35 minutes, and more preferably about 30 minutes, highly motile sperm are collected. Highly motile sperm may be collected by collecting highly motile sperm swimming around the drop with a pipette or the like, or by removing less motile sperm with a pipette or the like. The sperm collected in the second collection step are then pre-incubated in the medium and then inseminated with eggs.

[0044] [In vitro fertilization method] Another aspect of the present invention is a method of in vitro fertilization using sperm cryopreserved by the sperm cryopreservation method of the present invention. Another aspect of the present invention is a method of in vitro fertilization using sperm thawed by the frozen sperm thawing method of the present invention. Another aspect of the present invention is a method of in vitro fertilization using sperm cryopreserved by the sperm cryopreservation method of the present invention and further thawed by the frozen sperm thawing method of the present invention. In vitro fertilization can be performed by referring to known reports, such as the report by Nakagata et al. (Jikken Dobutsu. 41, 443-447 (1992)) and the report by Anzai et al. (Jikken Dobutsu. 43, 445-448 (1994)). For example, the method can be performed by the following steps. (i) pre-incubating rat sperm in a sperm pre-incubation medium; (ii) administering equine chorionic gonadotropin (eCG) to female rats, then administering human chorionic gonadotropin (hCG) to the rats, and then collecting unfertilized eggs to prepare unfertilized eggs; (iii) A step of mixing the sperm pre-cultured in step (i) with the unfertilized eggs prepared in step (ii) in a medium to perform insemination.

[0045] The in vitro fertilization medium (sperm pre-culture medium) used in step (iii) can be, for example, mHTF medium, and any general or commercially available fertilization medium can be used without limitation. Preferably, bovine serum albumin (BSA) is added to the medium at a concentration of about 1 to about 80 mg / mL, preferably about 4 to about 80 mg / mL, more preferably about 10 to about 60 mg / mL, even more preferably about 20 to about 60 mg / mL, and most preferably about 30 to about 50 mg / mL. The addition of BSA can increase the fertilization rate.

[0046] The unfertilized eggs prepared in step (ii) above may also be used after removing the cumulus. Cumulus removal can be performed by appropriately referring to known methods. For example, removal can be achieved by enzymatic treatment using 0.05 to 0.5% hyaluronidase. By using eggs from which the cumulus has been removed, the fertilization rate can be improved in in vitro fertilization of rat strains with low fertility rates, such as Long-Evans, BN, and F344 rat strains. [Example]

[0047] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

[0048] 1. Materials and Methods (1)Animals Sperm donors were 11-12 week-old male transgenic rats (SD-Tg(CAG-EGFP)4Osb) purchased from Slc Japan (Shizuoka, Japan) (http: / / www.anim.med.kyoto-u.ac.jp / NBR / strains / Strains_d.aspx?StrainID=559&s_geneAffected=GFP&s_References=GFP&s_livingAnimals=1). Egg donors were 4-5 week-old SD female rats purchased from Slc Japan. 10-15 week-old Crl:CD (SD) female rats were purchased from Charles River Japan (Kanagawa, Japan) as recipients. All animals were housed under a 12-hour light / dark cycle (light period: 7:00 AM to 7:00 PM) at a room temperature of 22 ± 1°C, with food and water available ad libitum. All animal experiments were conducted with the approval of the Kumamoto University School of Medicine Experimental Animal Committee.

[0049] To investigate the difference in fertility among various strains of rats, we purchased and used Wister, Long-Evans, BN, and F344 rats from Charles River Japan (Kanagawa, Japan) in addition to SD rats. In the following examples, SD rats were used unless otherwise specified.

[0050] (2) Sperm cryopreservation solution (Sperm Cycroprotective Agent: Sperm CPA) Sperm cryopreservation solution (CPA) was prepared essentially according to the method reported by Nakatsukasa et al. (Non-Patent Document 1: Nakatsukasa et al., Reproduction 122, 463-467 (2001); Non-Patent Document 3: Nakatsukasa et al., Comp. Med. 53, 639-641 (2003)). A solution of 8% (w / v) lactose and 23% (w / v) chicken egg yolk, supplemented with 1,000 units / mL penicillin G and 1 mg / mL streptomycin sulfate, was mixed with distilled water. The pH of the mixture was adjusted to 7.4 with 10% trisaminomethane solution, and the solution was centrifuged at 1,600 G for 15 minutes, and the upper layer was collected. Next, 0.7% Equex™ (ES: Nova Chemical Sales, Inc., USA) and 0.1% ATP (Adenosine 5'-triphosphate disodium salt hydrate) were added to the solution and mixed on a stir plate. Finally, the CPA was dispensed into aliquots and stored at -20°C until use.

[0051] (3) Sperm cryopreservation One transgenic rat was used for each experiment to cryopreserve sperm. The cryopreservation process is outlined in Figures 1A-H. The cryopreservation process using fresh cauda epididymis was carried out as follows. 1. Male rats were euthanized by cervical dislocation, and then the two cauda epididymides were aseptically removed and transferred to a 300 μL drop of CPA placed in a 35 mm dish at room temperature (Figure 1A). 2. Under a microscope, 10 to 12 deep incisions were made in the epididymis using scissors (Figure 1B). 3. The dish containing the CPA drop was placed on a tin plate placed on crushed ice and left to stand for 10 minutes (Figure 1C). 4. While the sperm were equilibrating in the CPA, the connector at the tip of a 1 mL syringe was attached to the end of a straw (Figure 1D), which was then placed on the tin plate described above to cool. For each of the two cauda epididymides of one male, 15 straws were prepared. At 5.0°C, 30 μL of mHTF (Kyudo Co., Ltd., Japan) was carefully aspirated into a straw with 10 mm of air. Then, 150 μL of sperm suspension was aspirated, and the syringe plunger was pulled up until 30 μL of mHTF reached the cotton plug of the straw. The tip of the straw was then sealed using an impulse sealer (Figure 1E). 6. The 15 sealed straws were placed on a tin plate placed on crushed ice and allowed to stand for 30 minutes (Figure 1F). 7. The straws were transferred to a float (a polystyrene foam frame), and the polystyrene foam frame holding the straws was then immediately floated on liquid nitrogen in a polystyrene foam box and left there for 10 minutes (Figure 1G). After 10 minutes, the straws were immersed in liquid nitrogen. They were then transferred to a conical cassette and stored in the tank for 4–8 weeks (Figure 1H).

[0052] (4) Cryopreservation of sperm using refrigerated cauda epididymis For sperm cryopreservation, one transgenic rat was used per experiment. First, male rats were euthanized by cervical dislocation. Then, two cauda epididymis were aseptically removed and transferred to a tube containing a refrigerated preservation solution (Lifor® refrigerated preservation solution supplemented with dimethyl sulfoxide and quercetin). The tube was then cooled to approximately 4°C. The tube was then stored in a refrigerated state for a period of time (1 to 11 days). The cauda epididymis was then removed from the tube and transferred to a 300 μL CPA drop placed in a 35 mm Petri dish. The frozen sperm were then prepared by repeating steps 2 to 8 above.

[0053] (5) Thawing frozen sperm For each experiment, 3 to 4 straws out of 15 frozen straws were used for the sperm thawing experiment. The thawing process is outlined in Figure 2, I to M. The thawing process was carried out as follows. 1.1 mL of mHTF was placed in a 1.5 mL tube, and the medium was equilibrated in an incubator (37°C, 5% CO2) for 30 minutes before use. 2. The frozen straws were removed from the liquid nitrogen tank, placed in a floating container, and then pre-incubated in a 37°C water bath for 15 minutes to thaw (Figure 2I). 4. The straws were removed from the water bath and the water on the surface of the straws was wiped off using a paper towel. 5. The straw was cut at the area between the sperm suspension and the seal and placed in the tube containing the mHTF prepared in step 1. Next, after cutting the end of the cotton plug, the straw was inserted into the straw connector and the sperm suspension was transferred to the bottom of the tube by depressing the plunger of a 1 mL syringe. The tube was then placed on its side and kept in a humidified incubator (37°C, 5% CO2) for 30 minutes (Figure 2J). After 30 minutes, the tube was gently inverted 2-3 times to mix and then centrifuged at 300 g for 60 seconds (Figure 2K). Next, using a 200 μL pipette with a large orifice tip (catalog number 4290-00S, Funakoshi Co., Ltd., Japan), 50 μL of the sediment containing the sperm pellet was aspirated from the bottom of the tube. The sediment was transferred to a 200 μL mHTF drop containing BSA (1-80 mg / mL) covered with paraffin liquid in a Petri dish (Figure 2L). After 7.30 min, 125 μL of medium containing dead sperm was removed from the mHTF drop (Figure 2M), and the sperm suspension was preincubated for 2 h before insemination.

[0054] (6) Sperm pre-incubation medium (in vitro fertilization medium) The sperm pre-incubation medium (IVF medium) used was modified human tubal fluid (mHTF) containing BSA (4 mg / mL or 40 mg / mL). The prepared medium was sterilized by filtration (0.22 μm) and stored at 4°C.

[0055] (7) In vitro fertilization and embryo development In each experiment, IVF was performed using cryopreserved and fresh sperm collected from four male rats. For cryopreserved sperm, sperm suspensions with a total motility of 30% and a progressive motility of 10% or more were used for IVF, as measured by computer-assisted sperm analysis (Integrated Visual Optical System, Hamilton Thorn Inc., USA) before insemination.

[0056] In vitro fertilization was performed according to the procedures described by Nakagata et al. (Jikken Dobutsu. 41, 443-447 (1992)) and Anzai et al. (Jikken Dobutsu. 43, 445-448 (1994)). Briefly, the procedure is as follows. Immature female rats were ovulated by administering 30 IU equine chorionic gonadotropin (eCG, Aska Pharmaceutical) and 30 IU human chorionic gonadotropin (hCG, Aska Pharmaceutical) 54–56 h apart. 15–16 h after hCG administration, the rats were euthanized by cervical dislocation, and the oviducts were rapidly harvested. All intact cumulus-oocyte complexes were isolated from the harvested oviducts and placed in a preincubated sperm suspension (sperm concentration: 500–1200 cells / μL) for insemination. As a control, superovulated oocytes obtained using the same method were transferred to a fresh sperm suspension (sperm concentration: 500 cells / μL) that had been incubated for 2 h. Twenty hours after insemination, the eggs were observed under an inverted microscope, and the fertilization rate was calculated by dividing the number of fertilized eggs (those in which two pronuclei were visible in the cytoplasm or where two pronuclei had fused and only the sperm tail was visible) by the total number of eggs mixed with sperm and multiplying the result by 100.

[0057] In experiments using cumulus-free oocytes, intact cumulus-oocyte complexes were isolated from the oviduct, transferred to a medium containing 0.1% hyaluronidase, and treated with the enzyme for 1-3 minutes, followed by washing with the medium. The oocytes were then mixed with a pre-incubated sperm suspension (sperm concentration: 500-1200 cells / μL) in IVF medium (insemination). Fertilization rates were calculated as described above.

[0058] For each experiment, 20 fertilized eggs were transferred into the oviducts of female Crl:CD(SD) rats on the day a vaginal plug was observed (day 1 of pseudopregnancy) by transmural embryo transfer, with 10 embryos per oviduct. The number of offspring was recorded 22–23 days later. The remaining fertilized eggs were cultured for an additional 8 hours, and 100 fertilized eggs developed to the 2-cell stage were cultured in mR1ECM until the blastocyst stage. GFP signals within the developed blastocysts were observed under a fluorescent microscope.

[0059] (8) GFP gene introduction To construct the transgene, the pCAGGS vector, developed by Niwa et al. (1991, 108, 193-9) based on the pUC13 plasmid, was inserted with the chicken beta-actin promoter, cytomegalovirus enhancer, enhanced green fluorescent protein (EGFP), and rabbit beta globin polyA. Linear DNA was then prepared by digestion with BamH I and Sal I. The resulting linear DNA fragment was injected into the pronuclei of fertilized eggs from SD rats and implanted into the oviducts of pseudopregnant female rats. The offspring obtained by this procedure were confirmed to have green fluorescence by UV irradiation, confirming that the GFP gene had been introduced. GFP rats were crossed with SD rats to generate heterozygous rats, and the strain was maintained by mating heterozygous male rats with SD female rats.

[0060] (9) Statistical analysis Statistical analysis was performed using Prism version 5.0 (GraphPad). Results are expressed as mean ± standard deviation (SD). Group results were compared using analysis of variance after arcsine transformation of percentages. p < 0.05 was considered statistically significant.

[0061] (10) Evaluation of sperm motility The pre-cultured sperm were diluted with mHTF and introduced into a dedicated chamber (Hamilton Thorne, Inc.). The sperm were then placed in an HTM-IVOS (Hamilton Thorne, Inc.) and analyzed for sperm motility. The percentage of motile sperm can be expressed, for example, as the percentage of sperm that moved 5 μm or more per second out of the total number of sperm. The percentage of progressively motile sperm can be expressed, for example, as the percentage of sperm with an average sperm path velocity (path velocity) of 50 μm / s or more and a straightness of 50% or more out of the total number of sperm.

[0062] 2. Experimental Example Example 1 Rat sperm were frozen using the sperm cryopreservation method (3) described above. The temperature change of the sperm suspension was measured from the start of cooling until freezing. The results are shown in Figure 11. It can be seen that the conventional method of cooling in a petri dish on ice can only cool to about 5°C. On the other hand, it was confirmed that the method of the present invention further cooled the sperm to about 1°C or less.

[0063] Example 2: In vitro fertilization of fresh or cryopreserved rat sperm Using the method described above, in vitro fertilization was performed using fresh and cryopreserved sperm. High fertilization rates were achieved with both sperm. The results are shown in Table 1. However, the fertilization rate of cryopreserved sperm was slightly lower than that of fresh sperm.

[0064] [Table 1]

[0065] (Example 3) In vitro developmental ability of fertilized rat eggs Using the method described above, fertilized eggs were cultured and developed in vitro. Nearly all fertilized eggs developed into two-cell embryos 28 hours after fertilization. Whether derived from cryopreserved or fresh sperm, more than 60% of the two-cell embryos developed into blastocysts. Half of these blastocysts had green fluorescent protein (GFP) signals. The results are shown in Figure 12. The development rates of in vitro 2-cell stage embryos to blastocysts are shown in Table 2 below.

[0066] [Table 2]

[0067] (Example 3) In vivo developmental ability of fertilized rat eggs Fertilized eggs were developed in vivo using the method described above. A total of 46 normal offspring (26 offspring with GFP signal: 57%) were obtained from cryopreserved sperm, and a total of 48 normal offspring (24 offspring with GFP signal: 50%) were obtained from fresh sperm. The results are shown in Figure 13. The in vivo development rates of fertilized eggs are shown in Table 3 below.

[0068] [Table 3]

[0069] (Example 4) Refrigerated storage of the cauda epididymis After removing the rat cauda epididymis, the specimens were refrigerated at 4°C for three days in a refrigerated storage solution containing various concentrations of dimethyl sulfoxide and quercetin. A sperm suspension was then prepared from the cauda epididymis, and the percentage of motile sperm in the suspension was determined. The results are shown in Figure 14. It was found that rat sperm could be refrigerated and preserved by adding a combination of dimethyl sulfoxide and quercetin. Particularly favorable results were obtained by combining 5-25% dimethyl sulfoxide with 50-250 μg / mL quercetin.

[0070] After refrigerating rat cauda epididymis for 1-3 days in Lifor refrigerated preservation solution containing 5% dimethyl sulfoxide and 100 μg / mL quercetin, sperm suspensions were prepared and IVF was performed. The IVF media used were mHTF medium and medium supplemented with 40 mg / mL BSA (referred to as Rat IVF medium). The fertilization rate results are shown in the table below.

[0071] [Table 4] Fertilization medium supplemented with BSA (Rat IVF medium) provided good fertilization rates even after extended refrigeration periods.

[0072] (Example 5) In vitro fertilization using frozen sperm prepared from refrigerated cauda epididymis Following the method described in (4) above, rat cauda epididymis was refrigerated for 2 or 3 days, after which frozen sperm were prepared. The refrigerated storage solution used was Lifor refrigerated storage solution supplemented with 5% dimethyl sulfoxide and 100 μg / mL quercetin. The frozen sperm were thawed according to the method described in (5) above. The percentage of motile sperm after thawing was approximately 1% to 2%. In vitro fertilization was then performed according to the method described in (6) above. The in vitro fertilization medium used was mHTF medium containing 40 mg / mL BSA, and the eggs used were those from which the cumulus had been removed by treatment with 0.1% hyaluronidase. Photographs of normal eggs (untreated eggs) and eggs from which the cumulus had been removed (cumulus-removed eggs) are shown in Figure 15. The fertilization rate (%) (number of eggs used for in vitro fertilization / number of fertilized eggs) was 47% (21 / 45) for sperm frozen and stored in the refrigerator for two days, and 32% (11 / 34) for sperm frozen and stored in the refrigerator for three days. This indicates that a good fertilization rate can be achieved by using the method of the present invention, even when using frozen sperm prepared from sperm stored in the refrigerator.

[0073] (Example 6) In vitro fertilization using frozen sperm from various strains of rats When in vitro fertilization was performed in the same manner as in Example 2 using F344 rats instead of SD rats, the fertilization rate (%) (number of eggs used in in vitro fertilization / number of fertilized eggs) was a very low 2.9% (2 / 70). Next, in vitro fertilization was performed in the same manner using mHTF medium (Rat IVF medium) supplemented with 40 mg / mL BSA instead of mHTF, and eggs from which the cumulus had been removed. The results for each condition are shown in the table below.

[0074] [Table 5] By using cumulus-free eggs and a medium containing BSA, a good fertilization rate was achieved even in F344 rats.

[0075] The results of in vitro fertilization using various strains of rats under the standard conditions described in Example 2 (untreated oocytes, mHTF medium) (condition A) and the conditions described in Example 6 (cumulus-removed oocytes, Rat IVF medium (mHTF medium containing 40 mg / mL BSA)) (condition B) are shown below. In the table, double circles indicate a fertilization rate of 50% or higher, triangles indicate a fertilization rate of 10-50%, and crosses indicate a fertilization rate of less than 10%.

[0076] [Table 6] By using cumulus-free eggs and a medium containing BSA, sufficient fertilization rates were achieved in all strains.

[0077] (Example 6) In vitro fertilization using frozen sperm from SD and Wistar rats Using the commonly used SD and Wistar rat strains, the fertilization rate was confirmed in in vitro fertilization using cumulus-removed oocytes in mHTF medium containing BSA, which is the preferred condition, according to the method of the present invention. The results are shown in the table below.

[0078] [Table 7] According to the method of the present invention, it was shown that in vitro fertilization using intact oocytes and cumulus-removed oocytes in mHTF medium containing BSA can achieve a fertilization rate of over 90%.

[0079] The above results show that fertilized eggs derived from either cryopreserved or fresh sperm developed similarly both in vivo and in vitro, demonstrating the effectiveness of the rat sperm cryopreservation method of the present invention.

[0080] The above description merely illustrates the purpose and scope of the present invention and is not intended to limit the scope of the appended claims. Various modifications and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings set forth herein, without departing from the scope of the appended claims. [Industrial Applicability]

[0081] The methods of the present invention provide a practically useful method for freezing and thawing rat sperm and a method for in vitro fertilization using cryopreserved rat sperm.

Claims

1. 1. A method for preparing cryopreserved rat sperm, comprising the steps of: Step a: A preparation step of collecting rat sperm from the rat cauda epididymis and preparing a sperm suspension; Step b: A cooling step of cooling the rat sperm suspension to a temperature below 1°C (not freezing) and maintaining the temperature for about 15 to about 40 minutes; and Step c: a freezing step of freezing the rat sperm suspension cooled to a non-freezing temperature of less than 1°C; A method comprising:

2. 2. The method of claim 1, wherein the non-freezing temperature of less than 1°C is a non-freezing temperature of about 0.5°C or less.

3. 3. The method according to claim 1 or 2, wherein step (b) is carried out by placing a straw-shaped cryopreservation container containing the rat sperm suspension on a tin plate placed on crushed ice.

4. 4. The method according to claim 1, wherein the cooling step in step b is carried out to substantially reduce the motility of rat sperm.

5. 3. The method according to claim 1 or 2, wherein step b comprises two steps: (b-1) cooling the rat sperm suspension to about 4°C to about 6°C; and (b-2) cooling the rat sperm suspension cooled to about 4°C to about 6°C to a temperature below 1°C where the suspension will not freeze, and maintaining the temperature for about 15 to about 40 minutes.

6. 5. The method according to claim 4, wherein the step of cooling the rat sperm suspension to a temperature below 1°C, where the temperature is not freezing, is carried out in a cryopreservation container for cryopreserving sperm.

7. The method according to any one of claims 1 to 6, wherein in step b, the motility of the sperm after cooling is 20% or less of the motility of the sperm before cooling.

8. 7. The method according to claim 1, wherein in step b, the motility of the sperm after cooling is 50% or less of the motility of the sperm before cooling.

9. The method according to any one of claims 1 to 8, wherein the rat cauda epididymis is refrigerated and stored.

10. The method according to any one of claims 1 to 9, wherein the rat sperm is sperm derived from a genetically modified rat.

11. 1. A method for preparing rat sperm for use in in vitro fertilization, comprising the steps of: Step A: A thawing step of preparing a thawed rat sperm suspension by heating a cryopreservation solution containing frozen rat sperm to a temperature of 35°C to 37.5°C; Step B: A swim-up step in which the thawed rat sperm suspension is placed at the bottom of a medium contained in a container and left to stand for about 20 to about 40 minutes to allow the sperm to swim up; Step C: a first recovery step of recovering sperm; and Step D: A second recovery step of transferring the recovered sperm to a sperm culture medium and recovering highly motile sperm; A method comprising:

12. The method according to claim 11, wherein in step D, the sperm culture medium is a sperm culture medium containing about 20 mg / mL to about 60 mg / mL of bovine serum albumin.

13. 13. The method according to claim 11 or 12, wherein the lower part of the container in step B is a tube having a tapered or conical tip.

14. The method according to any one of claims 11 to 13, wherein the volume of the medium in step B is about 5 to about 20 times the volume of the sperm suspension.

15. The method according to any one of claims 11 to 14, wherein step C is a step of recovering sperm by mixing the thawed rat sperm suspension with a medium in a container, followed by centrifugation at low speed to recover the precipitate.

16. The following steps: (i) thawing the cryopreserved rat sperm prepared by the method according to any one of claims 1 to 10 using the method according to any one of claims 11 to 15 to prepare them for in vitro fertilization, and then pre-incubating the prepared rat sperm in a sperm pre-incubation medium containing about 20 mg / mL to about 60 mg / mL of bovine serum albumin; (ii) administering equine chorionic gonadotropin (eCG) to female rats, then administering human chorionic gonadotropin (hCG), and then collecting unfertilized eggs to prepare unfertilized eggs; and (iii) adding the unfertilized eggs prepared in step (ii) to a medium containing the sperm pre-cultured in step (i) to carry out insemination; IVF methods including:

17. 17. The in vitro fertilization method according to claim 16, wherein step (ii) further comprises a cumulus removal step of removing the cumulus from the prepared unfertilized eggs.

18. A rat sperm pre-incubation medium, which is an mHTF medium or a TYH medium containing about 20 mg / mL to about 80 mg / mL of bovine serum albumin, used for pre-incubating rat sperm in in vitro fertilization using cryopreserved rat sperm.

19. A rat in vitro fertilization medium for use in in vitro fertilization using cryopreserved rat sperm, which is an mHTF medium or a TYH medium containing about 30 to about 50 mg / mL of bovine serum albumin.

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