Method and device for increasing sperm motility

Abstract

To provide methods and apparatuses for more efficiently increasing the motility of animal sperms.SOLUTION: A method for treating sperms includes: irradiating the sperms of a non-human animal with light having wavelengths in a range of 390-420 nm at a dosage effective to increase the motility of the sperms by 75% or more.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method and apparatus for increasing sperm motility, and more particularly to a method and apparatus for increasing the motility of animal sperm using light in a specific wavelength range. [Background technology]

[0002] Artificial insemination is used to breed a variety of animals, including livestock. However, the conception rate of cattle, for example, is declining year by year, and there is a demand for development of technology to improve the conception rate, and various research efforts are being conducted. For example, a technique has been developed that aims to increase the conception rate by culturing fertilized eggs in a specific medium in vitro (Patent Document 1).

[0003] On the other hand, it is known that sperm with higher motility have a higher conception rate, and so research has been conducted focusing on sperm motility. For example, there are methods for increasing sperm motility using chemical substances (Patent Documents 2 and 3) and methods for selecting sperm with high motility (Non-Patent Document 1). However, there are safety concerns regarding the use of chemical substances, and complicated methods are time-consuming and costly. It has been reported that, particularly in cattle, the addition of chemical substances to the components of the medium used to handle sperm and fertilized eggs has a detrimental effect on conception (Non-Patent Document 2). As a simple technique for enhancing the motility of sperm, a method using light irradiation has been developed (Patent Documents 4 and 5). Patent Document 4 discloses a method of irradiating sperm with light in a wide visible wavelength range, and Patent Document 5 discloses a method of intermittently irradiating sperm with red light (620 to 630 nm). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. WO2015 / 056727 [Patent Document 2] U.S. Pat. No. 5,453,354 [Patent Document 3] U.S. Patent No. 5,780,230 [Patent Document 4] U.S. Patent No. 6,379,939 [Patent Document 5] Special Publication No. 2017-529085 [Non-patent literature]

[0005] [Non-Patent Document 1] Maria Portia B. Nagata et al., PNAS, 2018, 115(14), E3087-3096 [Non-Patent Document 2] Hoshi Hoshi, http: / / group.lin.gr.jp / 2008 / siryo / hoshi_sro.pdf Summary of the Invention [Problem to be solved by the invention]

[0006] Patent Document 4 describes that the method described therein increased sperm motility by about 50%. The document does not describe or suggest that light of a specific wavelength can efficiently increase sperm motility. Patent Document 3 describes that the method described therein significantly improved motility, but does not specifically show the degree of increase. The document does not describe or suggest that light of a specific wavelength other than the red light used can efficiently increase sperm motility. There remains a need for methods and devices that more efficiently increase the motility of animal sperm. [Means for solving the problem]

[0007] According to the present invention, there is provided a method for processing sperm, which comprises irradiating non-human animal sperm with light in the wavelength range of 390 nm or more and 420 nm or less at an amount effective for increasing the motility of the sperm by 75% or more. The present invention also provides a method for producing sperm with increased motility, which comprises treating sperm of a non-human animal with the above-mentioned treatment method.

[0008] Furthermore, according to the present invention, there is provided an apparatus for processing animal sperm to increase their motility, characterized in that it comprises a holding section for holding a sperm storage container or a mounting section for placing a sperm storage container, an irradiation section that can irradiate the sperm storage container with light in the wavelength range of 390 nm or more and 420 nm or less when the sperm storage container is held by the holding section or when the sperm storage container is placed on the mounting section, and a control section for controlling the irradiation section. Effect of the Invention

[0009] According to the present invention, the motility of animal sperm can be efficiently increased. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing an embodiment of an apparatus of the present invention. [Diagram 2] 1 shows the emission spectrum of the light source used in the experiments described herein. [Diagram 3] This shows that exposure to light in a specific wavelength range significantly increases sperm motility. [Figure 4] 1 shows the effect of relatively high doses of light on sperm motility. [Diagram 5] The effect of irradiation time on sperm motility is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In this specification, the numerical range "a to b" (where a and b are specific numerical values) means a range including the values ​​"a" and "b" at both ends. In other words, "a to b" is synonymous with "a or more and b or less."

[0012] <How to dispose of sperm> In one aspect, the present invention provides a method for treating sperm, more specifically, a method for treating animal sperm to improve their motility. The animal sperm processing method of the present invention (hereinafter also referred to as the "processing method of the present invention") is characterized by irradiating non-human animal sperm with light in the wavelength range of 390 nm or more and 420 nm or less, at an irradiation amount effective for increasing the motility of the sperm by 75% or more. As shown in the examples below, the present invention is based on the new finding that when animal sperm are irradiated with light in the wavelength range of 390 nm to 420 nm, their motility can be significantly increased compared to when they are irradiated with light in other wavelength ranges. Therefore, the treatment method of the present invention can efficiently increase the motility of animal sperm.

[0013] In the present specification, "motility" can be evaluated using at least one parameter selected from sperm motility rate, motile sperm concentration, straight-line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and beat-cross frequency (BCF). These parameters can be measured, for example, using a computer-aided sperm analyzer (CASA). An example of a CASA is a sperm motility analysis system (SMAS). Motility can be evaluated using at least one parameter selected from linear velocity, curvilinear velocity, average velocity, head amplitude, and head frequency, more preferably linear velocity, curvilinear velocity, and average velocity. In some embodiments, motility is evaluated using average velocity (VAP). The average of values ​​for multiple fields (e.g., three fields) can be used for evaluation.

[0014] In the present invention, sperm are irradiated with light in a wavelength range (also referred to as a "specific wavelength range" in this specification) that is effective in efficiently increasing the motility of the sperm, i.e., light in the range of 390 nm to 420 nm, more specifically, light in the range of 395 nm to 415 nm, and even more specifically, light in the range of 400 nm to 410 nm. The amount of light in a specific wavelength range irradiated onto sperm is not particularly limited as long as it is an amount effective for increasing the motility of the sperm by 75% or more, preferably 90% or more, more preferably 100% or more, more preferably 125% or more, more preferably 150% or more, and more preferably 175% or more. The specific amount of irradiation may vary depending on the sperm used. In the case of bovine sperm, the specific amount of irradiation is, for example, 450 mJ / cm. 2 More than 7000 mJ / cm 2 Below, 450 mJ / cm 2 More than 6500 mJ / cm 2 Less than or equal to 450 mJ / cm 2 More than 6000 mJ / cm 2 Less than or equal to 500 mJ / cm 2 More than 6000 mJ / cm 2 Less than or equal to 500 mJ / cm 2 More than 5500 mJ / cm 2 Less than or equal to 500 mJ / cm 2 More than 5000 mJ / cm 2 Less than or equal to 500 mJ / cm 2 More than 4500 mJ / cm 2 Less than or equal to 550 mJ / cm 2 More than 4500 mJ / cm 2 Less than or equal to 550 mJ / cm 2 More than 4000 mJ / cm 2 Less than or equal to 550 mJ / cm 2 More than 3500 mJ / cm 2 Less than or equal to 550 mJ / cm 2 More than 3000 mJ / cm 2 Less than or equal to 600 mJ / cm 2 More than 3000 mJ / cm 2 Less than or equal to 600 mJ / cm2 More than 2500 mJ / cm 2 Less than or equal to 600 mJ / cm 2 More than 2000 mJ / cm 2 Less than or equal to 600 mJ / cm 2 More than 1500 mJ / cm 2 Less than or equal to 650 mJ / cm 2 More than 1500 mJ / cm 2 Less than or equal to 650 mJ / cm 2 More than 1000 mJ / cm 2 In the present invention, the "irradiation amount" refers to the amount of irradiation on the light-receiving surface of sperm or a suspension containing sperm. The amount of irradiation can be substituted with a value measured in an area corresponding to the light-receiving surface on the outer surface of a container (sperm container) that contains sperm.

[0015] The illuminance of the light of a particular wavelength is not particularly limited as long as it is an amount effective for bringing about the above-mentioned predetermined increase in the motility of the irradiated spermatozoa. For example, the illuminance may be 0.1 mW / cm 2 More than 200 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 150 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 100 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 50 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 20 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 10 mW / cm 2 Less than or equal to 0.1 mW / m 2 More than 5 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 2 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 1 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 0.8 mW / m2 Less than or equal to 0.1 mW / cm 2 More than 0.5 mW / m 2 The illuminance can be less than 0.1 mW / cm 2 or less than 200 mW / cm 2 If the exposure exceeds 100%, the increase in sperm motility may not be efficiently achieved. In the present invention, the "illuminance" refers to the illuminance at the light-receiving surface of the sperm or the suspension containing the sperm. The illuminance may be substituted with a value measured in a region corresponding to the light-receiving surface on the outer surface of the sperm storage container.

[0016] The irradiation time of the light of the specific wavelength range is not particularly limited as long as it is an amount effective for bringing about the above-mentioned predetermined increase in the motility of the irradiated sperm, but is, for example, from 1 minute to 90 minutes, preferably from 1 minute to 60 minutes, more preferably from 2 minutes to 60 minutes, more preferably from 5 minutes to 60 minutes, more preferably from 5 minutes to 30 minutes, more preferably from 10 minutes to 30 minutes. If the irradiation time is less than 1 minute or exceeds 90 minutes, the increase in sperm motility may not be efficiently achieved.

[0017] The light in the specific wavelength range may be irradiated as continuous light or as intermittent light (e.g., pulsed light). When intermittent light is used, it is possible to avoid or reduce the temperature rise of the irradiated sperm and / or the light source emitting the light in the specific wavelength range. The pulsed light may have a pulse width of, for example, 100 ms or less, more specifically 50 ms or less, more specifically 20 ms or less, more specifically 10 ms or less, more specifically 5 ms or less. The pulsed light may also have a duty ratio of, for example, 50% or less, more specifically 40% or less, more specifically 30% or less, more specifically 20% or less, more specifically 10% or less, more specifically 5% or less.

[0018] The light in the specific wavelength range may be irradiated alone or in combination with light in a wavelength range other than the specific wavelength range. When the light in the specific wavelength range is irradiated in combination with light in a wavelength range other than the specific wavelength range, the light may be irradiated to the sperm simultaneously (as a composite light or a mixed light) or alternately. As an example of light that can be combined with light in a specific wavelength range, light in a wavelength range of 615 nm to 635 nm can be given. As shown in the examples described below, when light in a wavelength range of 615 nm to 635 nm is irradiated to sperm, it can effectively increase the motility of the sperm, although it is not as strong as light in a specific wavelength range. Therefore, when light in a specific wavelength range and light in a wavelength range of 615 nm to 635 nm are irradiated to sperm in combination, the motility of the sperm can be increased more efficiently than when light in a specific wavelength range is irradiated alone.

[0019] When the light of the specific wavelength range is irradiated to the sperm as a composite light or a mixed light with light of a wavelength other than the specific wavelength range, the illuminance of the light of the specific wavelength range is preferably higher than the illuminance of the light of the wavelength other than the specific wavelength range. Thus, in some preferred embodiments, the illuminance of the light of the specific wavelength range is 50% or more (more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 100%) of the illuminance of the light of the entire wavelength range irradiated to the sperm. According to this embodiment, the energy intensity is increased, and the motility of the sperm can be increased more efficiently.

[0020] The light in the specific wavelength range preferably has a maximum peak wavelength in the wavelength range of 390 nm to 420 nm, more preferably in the wavelength range of 395 nm to 415 nm, and more preferably in the wavelength range of 400 nm to 410 nm. The use of such light is preferable in that the light in the specific wavelength range, which is effective in efficiently increasing the motility of sperm, can be efficiently irradiated onto sperm. In some preferred embodiments, the light in the specific wavelength range is light having a peak wavelength of 405±15 nm (more preferably 405±10 nm) and a wavelength spectrum with a half-width of 0.1 nm to 50 nm (more preferably 0.1 nm to 20 nm). This embodiment increases energy intensity and can more efficiently increase sperm motility.

[0021] The light in a specific wavelength range may be light extracted from a light source that emits light having a broad wavelength spectrum, such as a halogen lamp, by using an optical filter. The light in the specific wavelength range may include light emitted from a light emitting diode (LED) or a laser diode (LD) as a light source, or may consist of such light alone. The use of LEDs or LDs is preferable from the standpoint of energy efficiency and economy due to their energy intensiveness, low heat generation, low power consumption, and long life. In addition, it becomes easier to control or manage the amount of irradiation.

[0022] As used herein, an animal may be, for example, a vertebrate, preferably selected from mammals, birds and fish, more preferably from mammals and birds, more preferably from mammals. The animal may be a human or a non-human animal. The non-human animal may be a farm animal, a race horse, a pet animal, an animal kept in a zoo, an endangered animal, etc. Examples of mammals include humans, non-human primates, cows, horses, pigs, sheep, goats, rabbits, dogs, cats, etc. Examples of birds include chickens, ducks, wild ducks, turkeys, guinea fowl, geese, ostriches, etc. Examples of fish include salmon, trout, sturgeon, tuna, goldfish, koi carp, zebrafish, killifish, etc.

[0023] The sperm may be from any time immediately after collection until immediately before artificial insemination, for example, between collection and loading into a sperm storage container (e.g., a straw tube), between being frozen and thawing, or between thawing and immediately before artificial insemination. During the light irradiation, the sperm may be contained in a container that does not prevent the light (including light in a specific wavelength range) from being irradiated to the sperm placed therein. The container may be, for example, a petri dish, a beaker, a test tube, a microtube, a straw tube, etc. The material of the container is not particularly limited, but at least a part of the container is made of a material that is transparent to the light (particularly light in a specific wavelength range) irradiated to the sperm according to the processing method of the present invention, so that the light irradiated from the outside of the container must penetrate to the inside of the container.

[0024] The sperm concentration during light irradiation is not particularly limited, but may be, for example, 100,000 / mL to 10,00,000 / mL, more specifically 500,000 / mL to 10,00,000 / mL, more specifically 1,000,000 / mL to 10,00,000 / mL, and more specifically 3,000,000 / mL to 6,000,000 / mL. During the light irradiation, the sperm may be present in a physiological buffer, a sperm pre-culture medium, or a sperm storage medium. As the medium, a medium that can be generally used for animal sperm may be used, for example, TCM-199, BO medium, CR1 medium, SOF medium, NCSU medium, PZM-5 medium, HTF medium, TALP medium, BWW medium, TYH medium, DMEM medium, Ham-F10 medium, Whitten medium, Whittingham medium, M16 medium, and modified media thereof, but are not limited thereto. The medium may contain additives generally used in the art, if necessary. The additives also include substances used to increase the survival rate of sperm or to induce fertilization capacitation. Examples of additives include, but are not limited to, serum (FCS), serum albumin (HSA, BSA, etc.), pyruvic acid, calcium, cyclodextrin or derivatives thereof, and the like. The cryopreservation medium may further contain chicken egg yolk, glycerin (glycerol), ethylene glycol, dimethyl sulfoxide (DMSO), and the like.

[0025] The sperm may be agitated during irradiation. Agitation can be achieved using a shaker or stirrer. During the light irradiation, the sperm may be heated and / or maintained at a temperature close to the body temperature of the animal (other than fish) from which the sperm originates or at a temperature close to the water temperature during the spawning season of the fish from which the sperm originates. The body temperature of the animal (other than fish) may vary depending on the species, but may be, for example, around 35°C to 45°C. The water temperature suitable for spawning of fish may also vary depending on the species, but may be, for example, 15°C to 30°C. The sperm treated by the method of the present invention may be used for artificial insemination, in vitro fertilization, or intracytoplasmic sperm injection. Alternatively, the sperm treated by the method of the present invention may be cryopreserved until it is used for artificial insemination, in vitro fertilization, or intracytoplasmic sperm injection.

[0026] <Sperm manufacturing method> From another aspect, the present invention provides a method for producing sperm with increased motility. The method for producing sperm of the present invention (hereinafter also referred to as "the production method of the present invention") is characterized by processing sperm of a non-human animal by any of the processing methods of the present invention described above in <Sperm processing method>. According to the production method of the present invention, sperm of non-human animals having increased motility (and therefore increased fertilization ability) can be provided efficiently (e.g., in terms of time and / or cost).

[0027] The non-human animals used in the production method of the present invention are the same as those explained in the above <Method of processing sperm>. The sperm used in the production method of the present invention may be unfrozen, frozen, or thawed from a frozen state. When frozen sperm are used, sperm during thawing from a frozen state and / or sperm after thawing can be treated by the treatment method of the present invention.

[0028] In the manufacturing method of the present invention, the sperm may be packed into a suitable transport or storage container before or after the treatment by any of the processing methods of the present invention. Thus, in some embodiments, the manufacturing method of the present invention further comprises a step of packing the sperm into a suitable transport or storage container before or after the treatment by any of the processing methods of the present invention. The transport or storage container may be a petri dish, a beaker, a test tube, a microtube, a straw tube, etc. The processed sperm may then be frozen (after packaging, if the sperm are packaged in a storage container after processing). Thus, in some embodiments, the manufacturing method of the present invention further comprises the step of freezing the processed sperm (after packaging, if applicable).

[0029] <Sperm treatment device> Another aspect of the present invention provides an apparatus for processing animal sperm so as to increase motility. The animal sperm treatment device of the present invention (also referred to as the "device of the present invention" in this specification) is A holding part for holding a sperm storage container or a placing part for placing a sperm storage container; an irradiation unit capable of irradiating the sperm storage container with light having a wavelength range of 390 nm or more and 420 nm or less when the sperm storage container is held in the holding unit or when the sperm storage container is placed in the placement unit; A control unit for controlling the irradiation unit; The present invention is characterized by comprising: The inventive apparatus is suitable for carrying out the inventive treatment method or the inventive production method. With respect to the device of the present invention, the animal is an animal as explained in the above <Method of processing sperm>, and may be a human.

[0030] Hereinafter, the apparatus of the present invention will be described with reference to FIG. 1, which is a schematic diagram showing an embodiment of the apparatus of the present invention. Some embodiments of the device of the present invention (Figure 1A) include a holding section 12 for holding a sperm storage container 11 (shown by a dashed line in the figure), an irradiation section 14 that can irradiate the container 11 with light in the wavelength range of 390 nm or more and 420 nm or less when the container is held in the holding section, and a control section 15 for controlling the irradiation section. Some other embodiments of the device of the present invention (Figures 1B to 1D) include a mounting section 23, 33, 43 for mounting a sperm storage container 21, 31, 41 (shown by dashed lines in the figures), an irradiation section 24, 34, 44 that can irradiate the container 21, 31, 41 with light in the wavelength range of 390 nm or more and 420 nm or less when the container is mounted on the mounting section, and a control section 25, 35, 45 for controlling the irradiation section.

[0031] The device of the present invention has at least one holding part (12) or placing part (23, 33, 43) for holding or placing a container (11, 21, 31, 41). The holding part or placing part can be designed appropriately depending on the sperm storage container to be used. The sperm storage container (11, 21, 31, 41) used with the device of the present invention can be, for example, a petri dish, a dish, a beaker, a sample cup, a test tube, a microtube, a straw tube, etc. The holding part (12) holds the container (11) in a detachable manner. The holding part is not particularly limited as long as it has a structure and size that can hold the sperm storage container to be used in a predetermined position or area. The holding part is not limited to one that holds the sperm storage container in an upright or erect position, but may also hold the container in a tilted, tipped, or inverted position, provided that the sperm inside do not leak out. The holder may have a structure for holding the sperm container by accommodating at least a part of the sperm container in its internal space. In this case, the holder may have, for example, a cylindrical member and hold the sperm container by accommodating the bottom or the bottom and side of the sperm container in its internal space. The holder may hold the sperm container by supporting at least a part of the sperm container. The supporting part (which may be a point, a line, or a surface) may be any part of the sperm container, for example, the bottom, the side, or the top, or two or more of these.

[0032] In some specific embodiments, the holding portion has a set of two clamping members or a set of three gripping members, one or more of which are movable, and each clamping member or gripping member is arranged so that it can simultaneously abut against the side of the sperm storage container, and the container is held in a clamped or gripped state by the clamping members or gripping members. In some other specific embodiments, the holding portion has one or more grooves having a square, triangular or arc-shaped cross-section, and holds the sperm container by abutting the inner wall of the groove against the side of the sperm container (e.g., the outer side of a straw). In some other specific embodiments, the holding part has a first member that supports one end of the sperm storage container, a second member that supports the other end of the container, and / or a third member that supports the middle part. Each member can be appropriately designed according to the shape of the sperm storage container to be used. For example, the first member has a plate-like structure, and its upper surface is a member that supports the lower end of the sperm storage container in the holding state, and the second and third members have openings and are members that support the side of the container inserted into the openings.

[0033] The holding member may be made of, for example, metal (e.g., stainless steel, brass, etc.), glass, or plastic (e.g., acrylic resin, polycarbonate, polyvinyl chloride, etc.), but if at least a part of the holding member may be located on the optical path of the light irradiated from the irradiation unit to the sperm storage container, it is preferable that at least a part of the holding member is made of a material that is transparent to the light (including light in the specific wavelength range).

[0034] The placement section (23, 33, 43) is not particularly limited as long as it has a structure and size that allows the container (21, 31, 41) to be used to be placed on its upper surface. The placement section is not limited to one on which the sperm storage container is placed in an upright or erect position, but may be one on which the container is placed in a sideways or inverted position, provided that the sperm inside do not leak out. The mounting section may be composed of, for example, the upper surface of a shelf or stand, the inner bottom surface of a box, tray or basket, or at least a part of the inner bottom surface of a housing. The mounting surface is not limited to one continuous surface, but may be composed of multiple separate surfaces, or may be a virtual surface such as the upper surface of a mesh-like or lattice-like shelf. The member constituting the mounting surface may be made of, for example, metal (e.g., iron, stainless steel, aluminum, brass, etc.), glass, or plastic (e.g., acrylic resin, polycarbonate, polyvinyl chloride, etc.), but when the device of the present invention is configured so that light from the irradiation section is irradiated through the mounting surface to the sperm storage container placed on the mounting surface, it is preferable that at least a part of the mounting surface is made of a material transparent to the light (including light in the specific wavelength range).

[0035] The mounting part may have a recess or a protrusion that defines an area (mounting surface) for mounting the sperm container or an area for receiving light from the irradiation part. In the case of a recess, the sperm container may be placed on the bottom surface of the recess. In the case of a protrusion, the sperm container may be placed on the top surface of one protrusion, or may be placed within an area defined by multiple protrusions. The shape of the placement portion when viewed from above may be, for example, circular or rectangular. The mounting portion may have an anti-slip member or a member for preventing slippage. If the mounting portion has an anti-slip member, the mounting surface may be the upper surface of the anti-slip member. The anti-slip member may be made of any material that generates a large frictional force against the part of the container that it comes into contact with.

[0036] The holding unit or the placing unit may be provided with a rotating or shaking mechanism for rotating or shaking the sperm storage container. The shaking mechanism may be of a reciprocating or rotating type. By providing the rotating or shaking mechanism, it becomes easier to more uniformly irradiate the sperm in the sperm storage container with light of a specific wavelength range. In addition, if applicable, by providing the shaking mechanism, it becomes possible to agitate the sperm in the physiological buffer, sperm pre-incubation medium, or sperm storage medium at the same time as irradiating the sperm in the sperm storage container with light of a specific wavelength range. In a specific embodiment, the placing unit is composed of a turntable.

[0037] The device of the present invention includes at least one irradiation unit (14, 24, 34, 44). The irradiation unit (14, 24, 34, 44) is arranged so that the light (including light in a specific wavelength range) emitted from the irradiation unit can be irradiated to the container (11, 21, 31, 41) held in the holding unit 12 or placed on the placement unit (23, 33, 43) (and therefore can be irradiated to the sperm contained in the container when the device of the present invention is used). The irradiation unit can be arranged so that the light can be irradiated from at least one of the above (e.g., directly above), below (e.g., directly below), and side (e.g., directly to the side) of the sperm storage container.

[0038] The irradiation unit includes at least one light source that emits light in at least a specific wavelength range (i.e., light in the range of 390 nm to 420 nm, more specifically, light in the range of 395 nm to 415 nm, more specifically, light in the range of 400 nm to 410 nm). As such a light source, for example, a light-emitting diode (LED), a laser diode (LD), a sodium lamp, a xenon lamp, a fluorescent lamp, an incandescent lamp, a white lamp, a metal halide lamp, a high-pressure mercury lamp, or the like can be used. When the ratio of the component in the specific wavelength range in the light emitted by the light source itself to be used (the ratio of illuminance to the light in the entire wavelength range) is relatively low (for example, when it is less than 30%, more specifically, 40%, or more specifically, 50%), it is preferable to increase the ratio by using a filter whose transmittance for the light in the specific wavelength range is higher than the transmittance for the light other than the specific wavelength range.

[0039] From the standpoint of energy efficiency, the light source that emits light in a specific wavelength range is preferably a light source that emits mainly light in the wavelength range of 390 nm or more and 420 nm or less, and more preferably a light source that emits substantially exclusively light in the wavelength range of 390 nm or more and 420 nm or less. In this specification, "a light source that mainly emits light in the wavelength range of 390 nm to 420 nm" refers to a light source in which the amount of radiation of emitted light in the wavelength range of 390 nm to 420 nm is 50% or more, more specifically 55% or more, more specifically 60% or more, more specifically 65% ​​or more, more specifically 70% or more, more specifically 75% or more, more specifically 80% or more, more specifically 85% or more, more specifically 90% or more of the total amount of emitted light in the entire wavelength range. In this specification, "a light source that substantially exclusively emits light in the wavelength range of 390 nm to 420 nm" refers to a light source in which the amount of radiation of emitted light in the wavelength range of 390 nm to 420 nm is 95% or more, more specifically 98% or more, more specifically 99% or more of the total amount of emitted light in the entire wavelength range.

[0040] A light source that emits light mainly or substantially exclusively in the wavelength range of 390 nm to 420 nm may be a light source that emits light having a maximum peak wavelength in the wavelength range of 390 nm to 420 nm, preferably in the wavelength range of 395 nm to 415 nm, more preferably in the wavelength range of 400 nm to 410 nm. More specifically, such a light source may be a light source that emits light having a peak wavelength of 405±15 nm (preferably 405±10 nm) and a wavelength spectrum of 0.1 nm to 50 nm (preferably 0.1 nm to 50 nm) (particularly, one having a single peak). Specific examples of such light sources are light-emitting diodes (LEDs) or laser diodes (LDs). LDs or LEDs may be provided as arrays or clusters. The use of LEDs or LDs is also preferable from the viewpoint of energy efficiency and economy due to their energy intensiveness, low heat generation, low power consumption and long life. In addition, the illuminance or irradiance can be easily controlled or managed.

[0041] The irradiation unit may include a light source that emits light mainly or substantially exclusively in the wavelength range of 390 nm to 420 nm, and a light source that emits light outside the specific wavelength range. The light source that emits light outside the specific wavelength range may be a light source that emits light in the wavelength range of 615 nm to 635 nm. The form of the light source may be any form, and may be appropriately designed according to the shape of the sperm container to be used and / or the arrangement of the container and the light source, etc. Specific examples of the light source may be a line light source and a panel light source.

[0042] The device of the present invention includes a control unit (15, 25, 35, 45) that controls the irradiation unit (14, 24, 34, 44). The control unit may control the irradiation unit so that the light emitted from the irradiation unit is continuous light, intermittent light, or a combination of both. When the light is intermittent light, it is possible to avoid or reduce a temperature rise of the sperm and / or the light source that emits light in a specific wavelength range. The control unit may control the irradiation unit so that the light emitted from the irradiation unit is pulsed light having, for example, a pulse width of 100 ms or less, more specifically 50 ms or less, more specifically 20 ms or less, more specifically 10 ms or less, or more specifically 5 ms or less, and a duty ratio of 50% or less, more specifically 40% or less, more specifically 30% or less, more specifically 20% or less, more specifically 10% or less, or more specifically 5% or less.

[0043] The control unit may also control the irradiation unit so that the illuminance of the light of a specific wavelength range emitted from the irradiation unit is within a predetermined range. In the present invention, the "illuminance" refers to the illuminance on the inner surface or the outer surface (preferably the outer surface) of the sperm storage container 11. The predetermined range may vary depending on the sperm used, but is, for example, 0.1 mW / cm 2 More than 200 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 150 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 100 mW / cm2 Less than or equal to 0.1 mW / cm 2 More than 50 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 20 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 10 mW / cm 2 Less than or equal to 0.1 mW / m 2 More than 5 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 2 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 1 mW / cm 2 Less than or equal to 0.1 mW / cm 2 More than 0.8 mW / m 2 Less than or equal to 0.1 mW / cm 2 More than 0.5 mW / m 2 By controlling the light source of the irradiation unit 14 within the above range, it is possible to provide the sperm in the container 11 with an illuminance or amount of irradiation that is effective in increasing their motility. 2 or less than 200 mW / cm 2 If the concentration exceeds 100 ppm, the increase in sperm motility may not be achieved efficiently. A control for these purposes may be, for example, a pulse width modulation circuit.

[0044] The control unit may also control the irradiation unit so that the irradiation time of the light in the specific wavelength range emitted from the irradiation unit falls within a predetermined range. The specified time may be, for example, 1 minute or more and 90 minutes or less, preferably 1 minute or more and 60 minutes or less, more preferably 2 minutes or more and 60 minutes or less, more preferably 5 minutes or more and 60 minutes or less, more preferably 5 minutes or more and 30 minutes or less, and more preferably 10 minutes or more and 30 minutes or less. A control for this purpose can be, for example, a timer.

[0045] In a specific embodiment, the control unit controls the illuminance and irradiation time (or irradiation amount) of the light in a specific wavelength range emitted from the irradiation unit. In this case, the control unit may be composed of, for example, a pulse width modulation circuit and a timer. In a more specific embodiment, the control unit controls the amount of light irradiated from the irradiation unit to the sperm storage container to be, for example, 450 mJ / cm 2 More than 7000 mJ / cm 2 Below, 450 mJ / cm 2 More than 6500 mJ / cm 2 Less than or equal to 450 mJ / cm 2 More than 6000 mJ / cm 2 Less than or equal to 500 mJ / cm 2 More than 6000 mJ / cm 2 Less than or equal to 500 mJ / cm 2 More than 5500 mJ / cm 2 Less than or equal to 500 mJ / cm 2 More than 5000 mJ / cm 2 Less than or equal to 500 mJ / cm 2 More than 4500 mJ / cm 2 Less than or equal to 550 mJ / cm 2 More than 4500 mJ / cm 2 Less than or equal to 550 mJ / cm 2 More than 4000 mJ / cm 2 Less than or equal to 550 mJ / cm 2 More than 3500 mJ / cm 2 Less than or equal to 550 mJ / cm 2 More than 3000 mJ / cm 2 Less than or equal to 600 mJ / cm 2 More than 3000 mJ / cm 2 Less than or equal to 600 mJ / cm 2 More than 2500 mJ / cm 2 Less than or equal to 600 mJ / cm 2 More than 2000 mJ / cm 2 Less than or equal to 600 mJ / cm 2 More than 1500 mJ / cm 2Less than or equal to 650 mJ / cm 2 More than 1500 mJ / cm 2 Less than or equal to 650 mJ / cm 2 More than 1000 mJ / cm 2 The irradiation unit may be controlled so as to fall within the following ranges: In the present invention, the "irradiation amount" refers to the amount of irradiation on the inner or outer surface (preferably the outer surface) of the sperm storage container.

[0046] In an embodiment in which the irradiation unit includes a light source that emits light in a specific wavelength range and a light source that emits light outside the specific wavelength range, the control unit may control the irradiation unit so that the illuminance of the light from the light source that emits light in the specific wavelength range is 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more of the illuminance of the light in the entire wavelength range emitted from the irradiation unit, and / or may control the irradiation unit so that the light in the specific wavelength range and the light outside the specific wavelength range are emitted simultaneously or alternately from the irradiation unit. According to this embodiment, energy intensity is increased, and a more efficient increase in sperm motility can be provided.

[0047] The device of the present invention may further include a housing (36, 46) having at least a holding part or a placing part and an irradiation part therein. In this embodiment, the control part may be disposed outside the housing or may be built into the housing. The housing may have an opening on one surface (preferably the top surface or the side surface, more preferably the side surface) and may be provided with a door for opening and closing the opening. Alternatively, the housing may include a main body having an opening on the upper side, and a lid body attached to the main body for opening and closing the opening. The shape of the housing may be, for example, a cube, a rectangular parallelepiped, or a cylinder. The housing may be made of, for example, metal (e.g., iron, stainless steel, aluminum, brass, etc.), glass, or plastic (e.g., acrylic resin, polycarbonate, polyvinyl chloride, etc.). The internal space of the housing may be surrounded by a thermal insulating material.

[0048] The device of the present invention may further include a temperature control mechanism for heating and / or maintaining the sperm container at a predetermined temperature. According to this embodiment, the temperature of the sperm in the sperm container can be maintained at an appropriate temperature, so that it is possible to easily reduce the influence of temperature changes before, during and / or after irradiation on the survival, fertilization and / or motility of the sperm. The temperature control mechanism can be capable of heating and / or maintaining the temperature within a range of, for example, 15° C. to 50° C. (specifically, 35° C. to 45° C. or 15° C. to 30° C.) depending on the sperm used. The temperature adjustment mechanism may be composed of, for example, a heating element (e.g., a heater, a Peltier element, etc.) and / or a heat medium (e.g., water, oil, metal, etc.) and a temperature control unit 47 that controls the temperature of the heating element and / or the heat medium. One example of the temperature adjustment mechanism is a heating table. In this case, the upper surface of the heating table may constitute the mounting portion. Another example of the temperature adjustment mechanism is a thermostatic bath or a thermostatic chamber. In this case, the inside of the housing may function as a thermostatic chamber or a thermostatic bath. When a thermostatic bath is used as the temperature adjustment mechanism, when a predetermined amount of water is contained in the thermostatic chamber, the holding portion or the mounting portion is arranged so as to hold the sperm storage container in a state where it is located in the water, and the irradiation portion may or may not be submerged in the water. EXAMPLES

[0049] <Experiment 1> method (Sperm preparation) The spermatozoa were straw-packed frozen semen from dairy cows. The frozen semen was thawed by immersing it in 37°C warm water for 1 minute, and the semen was taken out into a microtube and centrifuged (3000 rpm x 5 minutes). The medium was removed and the spermatozoa were collected. The collected sperm were diluted with SP-TALP medium (Caisson Labs, model IVL03-100ML) and placed in a sterile disposable petri dish.

[0050] (Light irradiation) Light irradiation was performed from above the sterile disposable petri dish with the lid removed. The following was used as the light source for irradiation. "290LED" and "340LED": Nichia Corporation's NCSU234B series "365LED", "385LED" and "405LED": Nichia Chemical Industries NVSU233B series "430LED": OSA Opto OCU-440 series "450LED", "540LED" and "620LED": NCS manufactured by Nichia Corporation * 119B-V1 Series "700LED", "765LED", "810LED" and "880LED": EPIGAP Ceramic SMD 3838 series Halogen lamp with IR cut: An EMINENT MAIN halogen lamp (model number GEC10-P) was fitted with heat absorbing glass (model number KG3 50MM) made by Edmund Optics. The spectrum of the light source used is shown in Figure 2. The illumination intensity on the semen surface was 0.2 mW / cm 2 , irradiation time was 60 min, and the dose was 720 mJ / cm 2 The illuminance was measured using a combination of an OPHIR VEGA display and a PD300-UV sensor. In order to achieve uniform illuminance across the entire sterile disposable petri dish, a highly reflective aluminum plate (ALANOD MIRO series) was used to homogenize the dish.

[0051] (Evaluation of athletic ability) After light irradiation under the above conditions, a portion of the semen was transferred to a disposable sperm counting chamber (Kitazato; code SP-ACE P) and sperm motility analysis was performed using a sperm motility analysis system (SMAS, manufactured by Detect Co., Ltd.). Sperm motility was evaluated based on the average velocity (VAP [μm / sec]). In addition, considering the variation in observation position, the average value of three fields of view was used for evaluation. As a control, the same procedures were carried out except for the light irradiation.

[0052] result The results are shown in Figure 3. Figure 3 shows the changes due to each light irradiation, with the average speed of the control taken as the standard (100%). As is clear from the figure, irradiation with light of wavelengths around 405 nm significantly increased the motility of sperm. In addition, irradiation with light of wavelengths around 620 nm, which has been known in the past, also increased the motility of sperm compared to irradiation with light of other wavelengths, but the increase rate was higher than that of light of wavelengths around 405 nm. It was about half the rate.

[0053] <Experiment 2> method In experiment 2, the illuminance was increased by 10 times (2.0 mW / cm 2 )(Irradiance: 7200 mJ / cm 2 ) and did not irradiate with light at wavelengths around 290 nm, 340 nm, and 365 nm, but otherwise performed in the same manner as in Experiment 1. The reason for not using light at these three wavelengths was that there was a concern that increased irradiation dose could damage the cells.

[0054] result The results are shown in Figure 4. Figure 4 also shows the change due to each light irradiation, with the average speed of the control taken as the standard (100%). As is clear from the figure, irradiation with light with a wavelength of about 405 nm still increased sperm motility. However, this effect was reduced by an irradiation dose of 720 mJ / cm. 2 Irradiation with light with a wavelength of around 620 nm also increased sperm motility, but the effect was limited to an irradiation dose of 720 mJ / cm. 2 Therefore, it is understood that if the amount of irradiation is too high, the effect of increasing sperm motility by light irradiation decreases.

[0055] <Experiment 3> method The spermatozoa were prepared in the same manner as in Experiment 1 and were exposed to light of around 405 nm at an illuminance of 0.2 mW / cm 2 Sperm samples (irradiated samples) were collected 15, 30, 45, and 60 minutes after the start of irradiation. Meanwhile, a sample (unirradiated sample) was also taken from sperm that was similar to the above except that it was not irradiated with light. The motor abilities of the irradiated and unirradiated samples at each time point were evaluated in the same manner as in Experiment 1.

[0056] result No increase in locomotor capacity was observed in unirradiated samples. As for the irradiated samples, a significant increase in motor ability was observed in those irradiated for 45 minutes and 60 minutes. From this, in order to significantly increase sperm motility by irradiation with light around 405 nm, a certain amount of irradiation (at least about 300-400 mJ / cm2) is required. 2 ) is required.

[0057] <Test 4> method The ATP content was measured for the 60 min irradiated samples and the corresponding unirradiated samples obtained in Experiment 3. ATP was extracted from the samples using an ATP level test kit manufactured by AMERIC Co., Ltd. The amount of extracted ATP was quantified by measuring the amount of luminescence in a luciferase assay using a luminometer (Berthold Technologies Co., Ltd., Model: Lumat3 LB9508).

[0058] result The amount of ATP in the irradiated samples was approximately 2.4 times that in the unirradiated samples. This result, together with the fact that the energy source for sperm flagellar movement is ATP produced in mitochondria, Considering all of this, it can be inferred that irradiation with light around 405 nm promotes mitochondrial ATP production in sperm, thereby increasing their motility. More specifically, it is believed that light around 405 nm activates the electron transfer between heme a and heme a3-CuB in cytochrome C oxidase, which is the bottleneck in the mitochondrial electron transfer system. However, the present invention is not limited by the above theory. From the above, it is believed that light at around 405 nm can increase the motility of sperm from any animal that uses ATP as an energy source through a similar mechanism. [Explanation of symbols]

[0059] 11, 21, 31, 41 Sperm container 12 Holding part 23, 33, 43 Placement part 14, 24, 34, 44 Irradiation section 15, 25, 35, 45 Control section 36, 46 case 47 Temperature control unit

Claims

1. A holding part for holding a sperm storage container or a placing part for placing a sperm storage container; an irradiation unit capable of irradiating the sperm storage container with light having a wavelength range of 390 nm or more and 420 nm or less when the sperm storage container is held in the holding unit or when the sperm storage container is placed in the placement unit; A control unit for controlling the irradiation unit, The irradiation unit has a light-emitting diode or a laser diode that emits light having a peak wavelength of 405±15 nm and a wavelength spectrum with a half-width of 0.1 nm or more and 20 nm or less, 13. An apparatus for processing mammalian sperm to increase motility, further comprising a temperature control mechanism for heating and / or maintaining the sperm storage container at a predetermined temperature.

2. The device according to claim 1 , wherein the holding portion or the placing portion is provided with a rotating or shaking mechanism for rotating or shaking the sperm storage container.

3. A holding part for holding a sperm storage container or a placing part for placing a sperm storage container; an irradiation unit capable of irradiating the sperm storage container with light having a wavelength range of 390 nm or more and 420 nm or less when the sperm storage container is held in the holding unit or when the sperm storage container is placed in the placement unit; A control unit for controlling the irradiation unit, The irradiation unit has a light-emitting diode or a laser diode that emits light having a peak wavelength of 405±15 nm and a wavelength spectrum with a half-width of 0.1 nm or more and 20 nm or less, An apparatus for processing mammalian sperm to increase motility, characterized in that the holding unit or the placement unit is equipped with a rotating or shaking mechanism for rotating or shaking the sperm storage container.

4. The device according to any one of claims 1 to 3, wherein the control unit controls the irradiation unit as to whether the light emitted from the irradiation unit is continuous light, intermittent light, or a combination thereof.

5. 5. The device according to claim 1, wherein the control unit controls the irradiation unit so that the pulse width of the light emitted from the irradiation unit is 100 ms or less and the duty ratio is 50% or less.

6. The device according to any one of claims 1 to 5, wherein the control unit is a pulse width modulation circuit.

7. The device according to any one of claims 1 to 4, wherein the control unit is a timer.

8. The device according to any one of claims 1 to 7, further comprising a housing having the holding section or the placement section and the irradiation section therein.

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