Motile sperm sorting device and method for sorting motile sperm

Abstract

To provide a motile sperm sorting device that can easily and efficiently sort motile sperm and suppress DNA damage to motile sperm, and to provide a method for sorting motile sperm.SOLUTION: The present invention relates to a motile sperm sorting device that includes a supply tank 4 that contains a sperm-containing liquid that contains sperm, a collection tank 5 that contains a liquid medium, and multiple flow paths 6 that connect the supply tank 4 and the collection tank 5 to each other. By generating a flow of medium from the collection tank 5 to the supply tank 4 in the multiple flow paths 6, motile sperm contained in the sperm are made to migrate upstream from the supply tank 4 to the collection tank 5 through the multiple flow paths 6, and the motile sperm are sorted.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a motile sperm sorting device and a method for sorting motile sperm.

Background Art

[0002] In in vitro fertilization, in order to efficiently create fertilized eggs, it is desirable to use sperm with good motility (hereinafter referred to as motile sperm). As a method for sorting motile sperm, there are methods such as the Percoll method that performs centrifugation. However, such methods may damage the DNA of sperm by centrifugation. In addition, a skilled technician is required for the operation, and there is a problem that it takes time to collect sperm. Therefore, there is a need for a method that can easily sort motile sperm and suppress damage to the DNA of motile sperm. In addition, there is a need for a method that can efficiently sort motile sperm.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] The present invention provides a motile sperm sorting device that can easily and efficiently sort motile sperm and suppress damage to the DNA of motile sperm. In addition, the present invention provides a method for sorting motile sperm that can easily and efficiently sort motile sperm and suppress damage to the DNA of motile sperm.

Means for Solving the Problems

[0005] The motility sperm sorting device according to an embodiment of the present invention includes a supply tank that stores a sperm-containing liquid containing sperm, a recovery tank that stores a liquid medium, and a plurality of flow paths that communicate the supply tank and the recovery tank with each other. By generating a flow of the medium from the recovery tank toward the supply tank in the plurality of flow paths, the motile sperm contained in the sperm are caused to swim upstream from the supply tank to the recovery tank through the plurality of flow paths, and the motile sperm are sorted.

[0006] Further, the method for sorting motile sperm according to an embodiment of the present invention includes a supply tank that stores a sperm-containing liquid containing sperm, a recovery tank that stores a liquid medium, and a plurality of flow paths that communicate the supply tank and the recovery tank with each other. By generating a flow of the medium from the recovery tank toward the supply tank in the plurality of flow paths, the motile sperm contained in the sperm are caused to swim upstream from the supply tank to the recovery tank through the plurality of flow paths, and the motile sperm are sorted. The method includes the steps of preparing a motility sperm sorting device, injecting a medium into the recovery tank, injecting a sperm-containing liquid into the supply tank, and generating a flow of the medium from the recovery tank toward the supply tank in the plurality of flow paths.

Advantages of the Invention

[0007] According to the present invention, it is possible to provide a motility sperm sorting device that can easily and efficiently sort motile sperm and suppress damage to the DNA of motile sperm. Further, the present invention can provide a method for sorting motile sperm that can easily and efficiently sort motile sperm and suppress damage to the DNA of motile sperm.

Brief Description of the Drawings

[0008]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7A

Figure 7B

DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, this embodiment will be described with reference to the drawings. Note that the disclosure is merely an example, and for those that can be easily conceived by those skilled in the art with appropriate modifications while maintaining the gist of the invention, they are naturally included in the scope of the present invention. Also, for the sake of clearer explanation, the drawings may schematically represent the width, thickness, shape, etc. of each part compared to the actual aspect, but this is merely an example and does not limit the interpretation of the present invention. Further, in this specification and each figure, components that are the same as or have similar functions to those described above with respect to the previously shown figures may be given the same reference numerals, and detailed descriptions that are redundant may be omitted as appropriate.

[0010] <First Embodiment> The motility sperm sorting device according to the first embodiment includes a supply tank that stores a sperm-containing liquid containing sperm, a recovery tank that stores a liquid medium, and a plurality of flow paths that communicate the supply tank and the recovery tank with each other. By generating a flow of the medium from the recovery tank toward the supply tank in the plurality of flow paths, the motile sperm contained in the sperm are caused to swim upstream from the supply tank to the recovery tank through the plurality of flow paths, thereby sorting the motile sperm.

[0011] In this specification, motile sperm are sperm with high curvilinear velocity, linearity, head amplitude, and head frequency measured using a sperm motility analysis device (SMAS). The motility sperm sorting device according to the first embodiment can easily and efficiently sort such motile sperm and can suppress damage to the DNA of the motile sperm.

[0012] Hereinafter, the motility sperm sorting device according to the first embodiment will be described with reference to FIGS. 1 to 3.

[0013] As shown in FIGS. 1 to 3, the motile sperm sorting device 1 includes a substrate 2 and a chip 3. As shown in the drawing, an X-axis, a Y-axis, and a Z-axis that are orthogonal to each other are described. The direction along the X-axis is referred to as the X direction, the direction along the Y-axis is referred to as the Y direction, and the direction along the Z-axis is referred to as the Z direction. The X direction corresponds to the width direction of the motile sperm sorting device 1. The Y direction corresponds to the depth direction of the motile sperm sorting device 1. The Z direction corresponds to the thickness direction of the motile sperm sorting device 1. Looking at the X-Y plane defined by the X-axis and the Y-axis is referred to as a plan view.

[0014] The substrate 2 has an upper surface 2a and a lower surface 2b. As shown in FIG. 1(a), the substrate 2 has a plate-like shape in which the upper surface 2a and the lower surface 2b are flat and the upper surface 2a and the lower surface 2b are parallel. The shape of the substrate 2 is not limited to a plate-like shape, and may be, for example, a cube, a rectangular parallelepiped, a cylindrical shape, or the like. The material of the substrate 2 is not particularly limited, but is preferably glass.

[0015] The chip 3 has an upper surface 3a and a lower surface 3b. As shown in FIG. 1(a), the chip 3 has a substantially rectangular parallelepiped shape. The shape of the chip 3 is not limited to a substantially rectangular parallelepiped, and may be, for example, a substantially cubic shape, a cylindrical shape, or the like. Further, the chip 3 is formed with a hole 7 for the supply tank 4 and a hole 8 for the recovery tank 5 that penetrate the upper surface 3a and the lower surface 3b. Further, on the lower surface 3b side of the chip 3, a plurality of grooves 9 for a plurality of flow paths 6 that communicate the hole 7 for the supply tank 4 and the hole 8 for the recovery tank 5 are formed. The material of the chip 3 is not particularly limited, but is preferably a resin, and more preferably polydimethylsiloxane (hereinafter referred to as PDMS).

[0016] As shown in FIG. 1(a), by adhering the upper surface 2a of the substrate 2 and the lower surface 3b of the chip 3, a supply tank 4, a recovery tank 5, and a plurality of flow paths 6 that communicate the supply tank 4 and the recovery tank 5 with each other are formed.

[0017] As shown in FIG. 1(a), one side surface LS4 of the supply tank 4 and one side surface LS5 of the recovery tank 5 are parallel to each other in the Y direction and face each other in the X direction. Also, as shown in FIG. 1(a), the plurality of flow paths 6 are connected to the opposing surfaces LS4 and LS5. Further, as shown in FIGS. 1(a) and 1(b), the plurality of flow paths 6 have a linear shape extending in a direction (X direction) orthogonal to the opposing surfaces LS4 and LS5. More specifically, the plurality of flow paths 6 have a rectangular parallelepiped shape in which the cross-section in the direction (Y direction) parallel to the opposing surfaces LS4 and LS5 is rectangular. By configuring the supply tank 4, the recovery tank 5, and the plurality of flow paths 6 in this way, the lengths L3 of all the plurality of flow paths 6 become the same length. Thereby, in the method for selecting motile sperm described later, the amount of motile sperm ascending each flow path 6 and the height of the motility of the motile sperm can be made uniform. Also, since the cross-section of the plurality of flow paths 6 in the direction (Y direction) parallel to the opposing surfaces LS4 and LS5 is rectangular, they have the width W3, height H3, and length L3 shown in FIG. 1(b). Thereby, the average flow velocity u M ) of the medium 11 in the plurality of flow paths 6 described later M can be easily designed.

[0018] As shown in FIGS. 1 and 2, the supply tank 4 and the recovery tank 5 have a rectangular parallelepiped shape. The shapes of the supply tank 4 and the recovery tank 5 are not limited to rectangular parallelepipeds, and may be cubes, cylindrical shapes, etc. The area of the bottom surfaces of the supply tank 4 and the recovery tank 5 is, for example, 20 mm 2 or more and 64 mm 2 or less.

[0019] As shown in FIGS. 1 and 2, when the supply tank 4 and the recovery tank 5 have a rectangular parallelepiped shape, it is preferable that the values of the width W1, depth D1 of the supply tank 4 shown in FIG. 1(a), and the width W2, depth D2 of the recovery tank 5 are combinations that satisfy the range of the area of the bottom surface. For example, the widths W1 and W2 are each 1.6 mm or more and 7.0 mm or less, and the depths D1 and D2 are each 4.0 mm or more and 40 mm or less.

[0020] As shown in FIGS. 1 and 2, the motile sperm sorting device 1 includes a plurality of flow paths 6 that communicate the supply tank 4 and the recovery tank 5 with each other. Preferably, the motile sperm sorting device 1 includes the flow paths 6 at a density of 10 to 50 per 1 mm with respect to the lateral length per 1 mm of the side surface LS4 of the supply tank 4. By the motile sperm sorting device 1 including a plurality of flow paths 6, when motile sperm swim upstream through the flow paths 6, the surface that the motile sperm contacts increases, so that it becomes easier to induce the rheotactic property of the motile sperm. Therefore, by utilizing the rheotactic property of the motile sperm, more motile sperm can be moved to the recovery tank 5, and the motile sperm can be sorted efficiently. In FIGS. 1 to 3, a motile sperm sorting device 1 including six flow paths 6 is schematically shown, but the number of flow paths is not limited to six.

[0021] The width W3 of the flow path 6 is preferably as small as possible within the range of 10 μm or more. Thereby, since motile sperm are more likely to contact the inner surfaces LS61 and LS62 of the flow path 6, the rheotactic property of the motile sperm can be induced more, and the motile sperm can be sorted more efficiently. If the width W3 is less than 10 μm, there is a possibility that it becomes difficult for motile sperm to swim upstream through the flow path 6. Further, it is preferable that the height H3 (H3 / W3) with respect to the width W3 of the flow path 6 satisfies 0.1 < H3 / W3 < 10. If H3 / W3 is 0.1 or less, when the chip 3 is formed of PDMS, the upper surface of the flow path 6 may bend and contact the lower surface of the flow path 6 (that is, the upper surface 2a of the substrate 2), and there is a possibility of blocking at least a part of the flow path 6. Also, if H3 / W3 is 10 or more, when the chip 3 is formed of PDMS, the side surface of the flow path 6 may bend and the inner surfaces LS61 and LS62 may contact each other, and there is a possibility of blocking at least a part of the flow path 6.

[0022] As shown in FIGS. 3(a) and (b), the bottom surfaces of the supply tank 4, the recovery tank 5, and the plurality of flow paths 6 are preferably located on the same plane. Thereby, it becomes easy to control the average flow velocity u M of the medium flow F(u M ) in the plurality of flow paths 6 described later.

[0023] The motility sperm sorting device 1 according to the first embodiment causes a flow F(u M ) of a medium from the recovery tank 5 toward the supply tank 4 in a plurality of flow paths 6, so that the motile sperm contained in the sperm are caused to ascend from the supply tank 4 to the recovery tank 5 through the plurality of flow paths 6, and the motile sperm are sorted. Thereby, motile sperm can be easily and efficiently sorted, and damage to the DNA of the motile sperm can be suppressed. The method of causing the flow (u M ) of the medium is not particularly limited. For example, the liquid level of the medium stored in the recovery tank 5 may be made higher than the liquid level of the sperm-containing liquid stored in the supply tank 4, thereby causing the flow (u M ) of the medium. Thereby, the flow (u M ) of the medium can be caused by an easy operation of making the liquid level of the medium higher than the liquid level of the sperm-containing liquid. Further, the pressure in the recovery tank 5 may be made higher than the pressure in the supply tank 4, thereby causing the flow (u M ) of the medium.

[0024] (Manufacturing method of the motility sperm sorting device according to the first embodiment) Next, a manufacturing method of the motility sperm sorting device according to the first embodiment will be described. Here, the manufacturing method of the motility sperm sorting device 1 shown in FIGS. 1 to 3 will be described. The manufacturing method of the motility sperm sorting device 1 according to the first embodiment includes a step of preparing a substrate 2, a step of preparing a chip 3 in which a hole 7 for the supply tank 4, a hole 8 for the recovery tank 5, and a plurality of grooves 9 for the plurality of flow paths 6 are formed, and an adhesion step of adhering the upper surface 2a of the substrate 2 and the lower surface 3b of the chip 3.

[0025] The step of preparing the chip 3 can be performed by a known method. For example, the holes 7, 8 and grooves 9 may be formed in the chip 3 before the holes 7, 8 and grooves 9 are formed by cutting, photolithography or the like to produce the chip 3. Further, a mold provided with the shapes of the holes 7, 8 and grooves 9 may be produced from metal, photoresist, silicon or the like, and the chip 3 may be produced by performing molding such as injection molding using the mold. Further, the chip 3 may be produced by electroforming using nickel or the like.

[0026] In the subsequent process, it is preferable to bond the upper surface 2a of the substrate 2 and the lower surface 3b of the chip 3 by plasma treatment or the like. Bonding using an adhesive is not preferable because, in the method for selecting motile sperm described later, the components contained in the adhesive may dissolve in the medium and adversely affect the motility of the sperm.

[0027] By the method described above, the motile sperm selection device 1 shown in FIGS. 1 to 3 can be manufactured. According to such a manufacturing method, a plurality of flow paths 6 can be easily formed by processing a plurality of grooves on the lower surface 3b of the chip 3.

[0028] (Method for Selecting Motile Sperm Using the Motile Sperm Selection Device According to the First Embodiment) Next, a method for selecting motile sperm using the motile sperm selection device according to the first embodiment will be described.

[0029] The method for selecting motile sperm includes a supply tank that stores a sperm-containing liquid containing sperm, a recovery tank that stores a liquid medium, and a plurality of flow paths that communicate the supply tank and the recovery tank with each other. By generating a flow of the medium from the recovery tank toward the supply tank in the plurality of flow paths, the motile sperm contained in the sperm are caused to swim upstream from the supply tank to the recovery tank through the plurality of flow paths, and a motile sperm selection device (that is, the motile sperm selection device according to the first embodiment) is prepared in step (S1). In addition, step (S2) of injecting the medium into the recovery tank, injecting the sperm-containing liquid into the supply tank, and generating a flow of the medium from the recovery tank toward the supply tank in the plurality of flow paths is included. Further, the method for selecting motile sperm may further include step (S3) of recovering the motile sperm that have moved into the recovery tank.

[0030] Hereinafter, a method for selecting motile sperm using the motile sperm selection device 1 shown in FIGS. 1 to 3 will be described.

[0031] First, in the step of preparing the motile sperm selection device 1 (S1), the motile sperm selection device 1 is placed on a horizontal surface. The motile sperm selection device 1 described in the first embodiment can be used.

[0032] Next, in the step of generating a flow of the medium (S2), the medium is injected into the recovery tank 5, the sperm-containing liquid is injected into the supply tank 4, and a flow F(u M ) of the medium from the recovery tank 5 toward the supply tank 4 is generated in the plurality of flow paths 6.

[0033] The medium is not particularly limited as long as it is a liquid substance capable of causing motile sperm contained in the sperm to flow back to the recovery tank 5 when a flow F(u M ) of the medium from the recovery tank 5 toward the supply tank 4 is generated in the plurality of flow paths 6. The density of the medium is preferably about the same as that of water. The viscosity of the medium is, for example, 1×10 -3 Pa·s or more and 1×10 2 Pa·s or less. Also, the viscosity of the medium is preferably about the same as that of water. As such a medium, one selected from the group consisting of medium solutions such as IFP963B (manufactured by Functional Peptide Laboratories Co., Ltd.), culture solutions for in vitro fertilization, buffer solutions, physiological saline, and other culture solutions, or a mixture of two or more thereof, in which albumin such as bovine serum albumin (BSA) is dissolved, is preferred. When using a mixture of two or more, their combinations and ratios can be arbitrarily selected according to the purpose.

[0034] The method of injecting the medium into the recovery tank 5 is not particularly limited, and for example, it may be performed by injecting the medium into the recovery tank 5 using a micropipette, a syringe, or the like.

[0035] The sperm-containing liquid is not particularly limited as long as it is a liquid containing sperm, but is preferably semen, a semen suspension, or the like. The semen suspension is obtained by suspending semen in at least one or a mixture of two or more selected from the group consisting of a sperm cryopreservation solution, a culture solution, a buffer solution, and a physiological saline solution. When using a mixture of two or more, their combination and ratio can be arbitrarily selected according to the purpose. The sperm can be sperm of any animal, but is preferably sperm of mammalian animals such as cows, pigs, and humans. The sperm may be in the state as collected from the above-described animals, or may be in the state of being cryopreserved after collection and then thawed.

[0036] The method of injecting the sperm-containing liquid into the supply tank 4 is not particularly limited, and for example, it may be performed by injecting the sperm-containing liquid into the supply tank 4 using a micropipette, a syringe, or the like.

[0037] In the step (S2) of generating the flow of the medium, a flow F(u M ) of the medium from the recovery tank 5 to the supply tank 4 is generated in the plurality of flow paths 6. The motile sperm swim upstream from the supply tank 4 to the recovery tank 5 through the plurality of flow paths 6 against the flow F(u M ) by the rheotaxis and thigmotaxis of the motile sperm and move to the recovery tank 5. On the other hand, sperm with low motility remain in the supply tank 4 because it is difficult for them to swim upstream against the flow F(u M ). Thus, the motile sperm sorting device 1 according to the first embodiment can sort motile sperm and sperm with low motility by utilizing the rheotaxis and thigmotaxis of the motile sperm. In addition, since motile sperm can be sorted without using a method that may damage the DNA of sperm such as centrifugation, damage to the DNA of motile sperm can be suppressed.

[0038] The average flow velocity u M of the flow F(u M ) of the medium from the recovery tank 5 to the supply tank 4 generated in the plurality of flow paths 6 is preferably 10 μm / s or more and 100 μm / s or less, and more preferably 20 μm / s or more and 50 μm / s or less. The flow F(u MThe average flow velocity u M is in the range of 10 μm / s or more and 100 μm / s or less, motile sperm can swim upstream against the flow F(u M ) and move to the recovery tank 5. On the other hand, sperm with low motility will have difficulty swimming upstream against the flow F(u M ). Therefore, motile sperm and sperm with low motility can be accurately sorted. Also, when the average flow velocity u M is 20 μm / s or more, it becomes even more difficult for sperm with low motility to swim upstream against the flow F(u M ), so motile sperm and sperm with low motility can be sorted more accurately. When the average flow velocity u M is less than 10 μm / s, there is a risk that the flow of the medium F(u M ) will be unstable or the medium will flow backward. When the average flow velocity u M exceeds 100 μm / s, there is a risk that motile sperm will have difficulty swimming upstream against the flow F(u M ) and moving to the recovery tank 5.

[0039] The method of generating the flow of the medium is not particularly limited. For example, by making the liquid level of the medium contained in the recovery tank 5 higher than the liquid level of the sperm-containing liquid contained in the supply tank 4, the flow F(u M ) of the medium may be generated. Also, by using a syringe pump or the like to make the pressure in the recovery tank 5 higher than the pressure in the supply tank 4, the flow F(u M ) of the medium may be generated.

[0040] Hereinafter, the method of generating the flow F(u M ) of the medium by making the liquid level of the medium contained in the recovery tank 5 higher than the liquid level of the sperm-containing liquid contained in the supply tank 4 will be described with reference to FIGS. 4(a) to (e).

[0041] First, as shown in FIGS. 4(a) and (b), the medium 11 is injected into the recovery tank 5, and the medium 11 is caused to flow from the recovery tank 5 into the supply tank 4 through a plurality of flow paths 6.

[0042] The medium 11 can be made to flow from the recovery tank 5 into the supply tank 4 through a plurality of flow paths 6, and in a subsequent step, a sufficient amount of the medium 11 is injected into the recovery tank 5 so that the liquid level FL 11 of the medium 11 is higher than the liquid level FL 10 of the sperm-containing liquid 10. By injecting a sufficient amount of the medium 11 into the recovery tank 5, the medium 11 flows from the recovery tank 5 into the supply tank 4 through a plurality of flow paths 6.

[0043] After injecting the medium 11 into the recovery tank 5, it is preferable to confirm that the medium 11 has flowed from the recovery tank 5 into the supply tank 4. Whether the medium 11 has flowed from the recovery tank 5 into the supply tank 4 can be determined, for example, by visually checking the state where the medium 11 has flowed into the supply tank 4 from the opening of the supply tank 4 as shown in FIG. 4(b). Thereby, it can be confirmed that the plurality of flow paths 6 communicate the recovery tank 5 and the supply tank 4 with each other.

[0044] Next, as shown in FIG. 4(c), the sperm-containing liquid 10 is injected into the supply tank 4 so that the liquid level FL 11 of the medium 11 is higher than the liquid level FL 10 of the sperm-containing liquid 10. The difference ΔFL (i.e., ΔFL = FL 11 - FL 10 ) between the height FL 11 of the liquid level of the medium 11 and the height FL 10 of the liquid level of the sperm-containing liquid 10 (hereinafter referred to as "liquid level difference ΔFL") can be set to any value within the range where the average flow velocity uM of the flow F(uM) of the medium is 10 μm / s or more and 100 μm / s or less in the formula (1) described later. However, if the liquid level difference ΔFL is too small, there is a risk that the medium 11 will flow backward due to an impact or the like. On the other hand, if the liquid level difference ΔFL is too large, it is necessary to increase the length L3 of the flow path 6 in order to make the average flow velocity uM of the flow F(uM) of the medium fall within the above range. The liquid level difference ΔFL can be, for example, 0.7 mm or more and 10 mm or less.

[0045] The liquid level FL 11 of the medium 11 is higher than the liquid level FL 10By injecting the sperm-containing liquid 10 into the supply tank 4 so as to be higher, the hydraulic pressure P of the medium 11 in the recovery tank 5 11 becomes greater than the hydraulic pressure P of the sperm-containing liquid 10 in the supply tank 4. 10 Therefore, even after injecting the sperm-containing liquid 10 into the supply tank 4, as shown in FIGS. 4(c) to (e), the medium 11 flows from the recovery tank 5 into the supply tank 4 through the plurality of flow paths 6. At this time, in the plurality of flow paths 6, there is a flow F(u M ) of the medium 11 in the direction from the recovery tank 5 to the supply tank 4 with an average flow velocity u. M As shown in FIGS. 4(d) and (e), the motile sperm 12a, due to the rheotaxis and rheotactility possessed by the motile sperm 12a, goes upstream from the supply tank 4 to the recovery tank 5 against the flow F(u M ) through the plurality of flow paths 6 and moves to the recovery tank 5. On the other hand, the sperm 12b with low motility remains in the supply tank 4 because it is difficult to go upstream against the flow F(u M ).

[0046] Hereinafter, the relationship between the average flow velocity u M of the flow F(u M ) of the medium 11 in the plurality of flow paths 6, the shape of the flow path 6, and the liquid level difference ΔFL will be described. The average flow velocity u M of the flow F(u M ) can be expressed by the following formula using the width W3, height H3, and length L3 of the flow path 6 shown in FIG. 1(b), and the liquid level difference ΔFL. In the following formula, g is the acceleration due to gravity, ρ is the density of the medium 11, and μ is the viscosity of the medium 11.

[0047]

Equation

[0048] The liquid level difference ΔFL, and the width W3, height H3, and length L3 of the flow path 6 are, in the above formula, the average flow velocity u MIt can be set to any value within the range of 10 μm / s or more and 100 μm / s or less. For example, when the density ρ and viscosity μ of the medium 11 are about the same as those of water, the liquid level difference ΔFL can be set to 0.7 mm or more and 10 mm or less, the width W3 of the flow path 6 can be set to 10 μm or more and 60 μm or less, the height H3 can be set to 10 μm or more and 60 μm or less, and the length L3 can be set to 1 mm or more and 80 mm or less.

[0049] The time from injecting the sperm-containing liquid 10 into the supply tank 4 until the motile sperm 12a is moved into the recovery tank 5 is not particularly limited, but for example, it is 15 minutes or more and 60 minutes or less.

[0050] As described above, the step (S2) of generating the flow of the medium may be performed. Thereby, the liquid level FL of the medium 11 11 is made higher than the liquid level FL of the sperm-containing liquid 10 10 and the flow of the medium can be generated by an easy operation.

[0051] In the steps after the step (S2) of generating the flow of the medium, it is preferable to keep the sperm-containing liquid and the medium in the motile sperm sorting device 1 at a temperature suitable for the sperm to move. The appropriate temperature varies depending on the type of animal of the sperm used. For example, in the case of humans, it is 32 °C or more and 37 °C or less, and in the case of cows, it is 37 °C or more and 39 °C or less. The method of keeping the sperm-containing liquid and the medium at an appropriate temperature is not particularly limited, but the motile sperm sorting device 1 may be placed in an incubator and the temperature may be controlled by the incubator.

[0052] After the step (S2) of generating the flow of the medium, the step (S3) of collecting the motile sperm that has moved into the recovery tank 5 may be performed. The method of collecting the motile sperm is not particularly limited, but the motile sperm may be collected together with the medium remaining in the recovery tank 5 using a micropipette, a syringe, or the like.

[0053] The recovered motile sperm may be stored, for example, together with a medium in a microtube or the like, or the microtube or the like may be frozen and stored. The recovered and stored motile sperm can be used for in vitro fertilization. The motile sperm selected using the motile sperm sorting device according to the first embodiment has less DNA damage compared to sperm selected by a conventional sorting method such as the Percoll method that involves centrifugation. Therefore, by using the motile sperm selected using the motile sperm sorting device according to the first embodiment for in vitro fertilization, the cleavage rate, blastocyst rate, and cryopreservable embryo rate of in vitro fertilization can be increased.

[0054] Hereinafter, the effects of the first embodiment will be described. The motile sperm sorting device according to the first embodiment causes a flow of a medium from a recovery tank to a supply tank in a plurality of flow paths, thereby causing the motile sperm contained in the sperm to ascend from the supply tank to the recovery tank via the plurality of flow paths and sorting the motile sperm. In this way, the motile sperm sorting device can sort motile sperm by an easy operation of causing a flow of a medium from a recovery tank to a supply tank in a plurality of flow paths. Further, since the motile sperm is sorted by utilizing the rheotaxis and thigmotaxis of the motile sperm without using a method that may damage the DNA of the motile sperm such as centrifugation, damage to the DNA of the motile sperm can be suppressed. In addition, the motile sperm sorting device according to the first embodiment includes a plurality of flow paths that communicate the supply tank and the recovery tank with each other. As a result, when the motile sperm ascends through the flow path, the number of surfaces that the motile sperm contacts increases, so it becomes easier to induce the thigmotaxis of the motile sperm. Therefore, since more motile sperm can be moved to the recovery tank by utilizing the thigmotaxis of the motile sperm, the motile sperm can be sorted efficiently.

[0055] (Example) Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

[0056] <Example 1> (Preparation of Motile Sperm Sorting Device) A device similar to the motile sperm sorting device 1 shown in FIGS. 1 to 3 was prepared. The supply tank 4 and the recovery tank 5 are rectangular parallelepipeds of substantially the same shape, with the widths W1 and W2 shown in FIG. 1(a) being 3.5 mm, the depths D1 and D2 being 8.2 mm, and the heights H1 and H2 being 5 mm. Also, as shown in FIGS. 1(a) and (b), the plurality of flow paths 6 are rectangular parallelepipeds with a rectangular cross-section in the Y direction, having a width W3 of 50 μm, a height H3 of 10 μm, and a length L3 of 5 mm. Further, the motile sperm sorting device 1 was provided with 80 of these flow paths 6 arranged in parallel in the Y direction. The motile sperm sorting device 1 was installed on a horizontal surface.

[0057] (Preparation of sperm-containing liquid and medium) Frozen bovine semen in a bovine frozen semen straw (sire name: Black Wagyu "Katsumi Sakura", Morioka Sire Center, Livestock Improvement Association of Japan) was thawed in a 38.5°C water bath to prepare bovine semen as the sperm-containing liquid. Also, bovine serum albumin (albumin, derived from bovine serum (BSA), pH 7.0, produced in New Zealand, manufactured by Fujifilm Wako Pure Chemical Corporation) (hereinafter referred to as BSA) was dissolved in the basic medium for sperm IFP963B (manufactured by Functional Peptide Laboratories Co., Ltd.) (hereinafter referred to as BO solution) to prepare a BO solution with a BSA concentration of 0.2% by mass (hereinafter referred to as 0.2% BSA-added BO solution) as the medium.

[0058] (Sorting of motile sperm) 100 μL of the prepared medium was injected into the recovery tank 5 using a micropipette. It was visually confirmed from the opening of the supply tank 4 that the medium had flowed from the recovery tank 5 into the supply tank 4 through the plurality of flow paths. Next, 80 μL of semen was injected into the supply tank 4. At this time, the liquid level of the medium was higher than the liquid level of the sperm, and the difference ΔFL between the height of the liquid level of the medium and the height of the liquid level of the semen was 0.7 mm or more and 10 mm or less. Also, after injecting the semen into the supply tank 4, the average flow velocity u of the medium flow F(u M ) in the plurality of flow paths 6 Mwas 14.8 μm / s. Next, the motility sperm sorting device 1 was placed in a CO2 incubator (MCO-170AICUV-PJ, manufactured by PHC Corporation), and the temperature of the medium and semen was maintained at 38.5 degrees and left for 30 minutes. During this period, a plurality of flow channels 6 were observed with a microscope from above, and the behavior of motile sperm moving upstream from the supply tank 4 to the recovery tank 5 through the plurality of flow channels 6 was observed. Microscopic photographs taken at that time are shown in FIGS. 5(a) to 5(c). FIG. 5(a) is a microscopic photograph showing a state in which motile sperm are moving upstream in the flow channel 6. FIG. 5(b) is a microscopic photograph showing the state about 1.4 seconds after the state of FIG. 5(a), and FIG. 5(c) is a microscopic photograph showing the state about 2.8 seconds after the state of FIG. 5(a). In FIGS. 5(a) to 5(c), one of the plurality of flow channels 6 is indicated by reference numeral 6, its inner surfaces are indicated by reference numerals LS61 and LS62, and one of the motile sperm is indicated by reference numeral 12a. As is clear from the microscopic photographs shown in FIGS. 5(a) to 5(c), it can be seen that a plurality of motile sperm are moving upstream against the flow F(u M ) of the medium in the plurality of flow channels 6 due to their rheotaxis. Also, since a large number of motile sperm are present along the inner surfaces LS61 and LS62 of the flow channel 6 and are moving along the inner surfaces LS61 and LS62, it can be seen that the motile sperm are also moving upstream in the plurality of flow channels 6 due to their thigmotaxis.

[0059] After leaving for 30 minutes, a mixture of the motile sperm that had moved to the recovery tank 5 and the medium remaining in the recovery tank 5 was aspirated from the recovery tank 5 using a micropipette, placed in a microtube, and recovered. Also, a mixture of the semen remaining in the supply tank 4 and the medium that had flowed from the recovery tank 5 into the supply tank 4 was aspirated from the supply tank 4 using a micropipette, placed in a microtube, and recovered. Then, using a sperm motility analysis device (SMAS, manufactured by Detect Co., Ltd.), the curvilinear velocity, linearity, head amplitude, and head frequency of the sperm contained in each mixture recovered from the recovery tank 5 and the supply tank 4 were measured respectively, and the motility of the sperm was evaluated. The results are shown in FIG. 6. In FIG. 6, the error bars indicate the standard error, the symbol "*" indicates that the p-value is less than 0.05 (p < 0.05), and the symbol "n.s." indicates that it is not statistically significant.

[0060] As shown in Fig. 6(a), the motile sperm that moved to the recovery tank 5 had a higher curvilinear velocity than the sperm remaining in the supply tank 4. Also, as shown in Fig. 6(b), the motile sperm that moved to the recovery tank 5 had a higher linearity than the sperm remaining in the supply tank 4. Further, as shown in Fig. 6(c), the motile sperm that moved to the recovery tank 5 had a higher head amplitude than the sperm remaining in the supply tank 4, although it was not significant. Also, as shown in Fig. 6(d), the motile sperm that moved to the recovery tank 5 had a higher head frequency than the sperm remaining in the supply tank 4. Thus, it was found that the motile sperm that moved to the recovery tank 5 had higher motility than the sperm remaining in the supply tank 4. Therefore, by using the motile sperm sorting device according to the first embodiment, highly motile sperm could be easily and efficiently sorted.

[0061] (Example 2) Except that the number of flow paths 6 of the motile sperm sorting device 1 was 120, in the same manner as in Example 1, a mixture of the motile sperm that moved to the recovery tank 5 and the medium remaining in the recovery tank 5 was recovered from the recovery tank 5. The sperm concentration of the mixture was 500,000 sperm / mL.

[0062] (Example 3) Except that the length of the flow path of the motile sperm sorting device 1 was 3 mm, the number of flow paths was 120, and the average flow velocity u of the flow F(u M ) of the medium in the plurality of flow paths M was 24.8 μm / s, in the same manner as in Example 1, a mixture of the motile sperm that moved to the recovery tank 5 and the medium remaining in the recovery tank 5 was recovered from the recovery tank 5. The sperm concentration of the mixture was 500,000 sperm / mL.

[0063] (Comparative Example 1) First, 2 ml of 90% Percoll solution was placed into a centrifuge tube, and then 2 mL of 45% Percoll solution was layered on top of it. Bovine frozen semen straws (breeding bull name: Japanese Black "Katsumi Sakura", Morioka Breeding Bull Center of the Livestock Improvement Association of Japan) were thawed in warm water at 38.5°C, and the semen was layered on the surface of the 45% Percoll solution in the centrifuge tube. The centrifuge tube was centrifuged at a speed of 1000 rpm for 30 minutes to separate motile sperm from sperm with low motility. The supernatant was removed from the centrifuge tube, 1 mL of medium semen (IVF100, manufactured by Functional Peptide Research Institute Co., Ltd.) was added, and the mixture was pipetted to mix and prepare a sperm suspension. After counting the sperm concentration of the sperm suspension using SMAS, more medium semen (IVF100) was added to adjust the sperm concentration of the sperm suspension to 2.5 million cells / mL.

[0064] <In Vitro Fertilization> (Preparation of Oocytes) Cumulus-oocyte complexes were aspirated from the follicles of bovine ovaries together with follicular fluid using a syringe. Using a microscope, among the collected cumulus-oocyte complexes, those with three or more layers of cumulus cells attached to the oocytes were selected. The selected cumulus-oocyte complexes were introduced into a maturation medium (Medium199 (manufactured by Gibco) with 5% serum) in a petri dish and matured in an incubator at 38.5°C and 5% CO2 in air for 20 - 24 hours.

[0065] (Insemination and Embryo Culture) The matured oocytes were transferred to an insemination medium, and the mixture of motile sperm and medium recovered in Example 2 was added using a micropipette. Insemination was performed by culturing in an incubator at 38.5°C and 5% CO2 in air for 6 - 16 hours. The embryos after insemination were washed using phosphate-buffered saline (PBS) with 5% serum. Subsequently, the washed embryos were introduced into a development medium (BO-IVC (manufactured by IVF Bioscience)) and cultured for 192 hours. The mixture of motile sperm and medium recovered in Example 3, and the sperm suspension recovered in Comparative Example 1 were also subjected to the same insemination and embryo culture treatments.

[0066] <Measurement of cleavage rate, blastocyst rate, and cryopreservation-compatible embryo rate> After 48 hours from the start of development culture, the embryos were observed using a microscope, and the number of cleaved embryos was counted to obtain the cleavage number and cleavage rate (= cleavage number / number of test embryos × 100). Also, after 192 hours from the start of development culture, the embryos were observed using a microscope, and the number of developed blastocysts was counted to obtain the blastocyst number and blastocyst rate (= blastocyst number / number of test embryos × 100). Further, after 168 hours from the start of development culture, the embryos were observed using a microscope, and embryos with Code 2 or higher in the classification of the International Embryo Technology Society (IETS) were regarded as cryopreservation-compatible embryos, and the number of such cryopreservation-compatible embryos was counted to obtain the cryopreservation-compatible embryo number and cryopreservation-compatible embryo rate (= cryopreservation-compatible embryo number / number of test embryos × 100). The results are shown in Table 1.

[0067]

Table 1

[0068] As shown in Table 1, Examples 2 and 3 had a higher cleavage rate than Comparative Example 1. Also, Examples 2 and 3 had a higher blastocyst rate than Comparative Example 1. Further, Examples 2 and 3 had a higher cryopreservation-compatible embryo rate than Comparative Example 1. Thus, it was found that the motile sperm selected using the motile sperm selection device according to the first embodiment (Examples 2 and 3) had a higher cleavage rate, blastocyst rate, and cryopreservation-compatible embryo rate in in vitro fertilization than the motile sperm obtained by the conventional method (Percoll method) that performs centrifugation (Comparative Example 1). Therefore, by using the motile sperm selection device according to the first embodiment, it was possible to select motile sperm while suppressing damage to the DNA of the motile sperm more than the conventional method that performs centrifugation.

[0069] Also, as shown in Table 1, Example 3, in which the average flow velocity u M of the flow F(u M ) of the medium in a plurality of flow paths was in the range of 20 μm / s or more and 50 μm / s or less, had a higher cryopreservation-compatible embryo rate as a result compared to Example 2 in which the average flow velocity u M was less than 20 μm / s. Therefore, the average flow velocity u MBy setting it within the range of 20 μm / s or more and 50 μm / s or less, it was possible to further suppress the upstream movement of sperm with low motility to the collection tank and to select motile sperm with higher accuracy.

[0070] The above-described embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the gist of the invention. The embodiments and their modifications are included in the scope and gist of the invention, and are also included in the invention described in the claims and the equivalent scope thereof.

[0071] (Modification example of the motile sperm sorting device according to the first embodiment) Hereinafter, a modification example of the motile sperm sorting device 1 according to the first embodiment will be described with reference to FIGS. 7A and 7B. In FIGS. 7A(a) and (b), and FIGS. 7B(c) and (d), only the supply tank 4, the collection tank 5, and the plurality of flow paths 6 of the motile sperm sorting device 1 are extracted and illustrated respectively. Although only one flow path 6 is shown by omitting it, a plurality of flow paths are provided. The description of the same configuration as that of the above-described first embodiment is omitted by referring to the above description.

[0072] (First modification example) As shown in FIG. 7A(a), a collection tank 5 may be arranged between the two supply tanks 4, and the supply tank 4 and the collection tank 5 may be respectively communicated with each other by a plurality of flow paths 6 to constitute the motile sperm sorting device according to the first embodiment.

[0073] (Second modification example) As shown in FIG. 7A(b), two supply tanks 4 are arranged at both ends in the X direction, two supply tanks 4 are arranged at both ends in the Y direction, a collection tank 5 is arranged in the center of the four supply tanks 4, and each supply tank 4 and the collection tank 5 are respectively communicated with each other by a plurality of flow paths 6 to constitute the motile sperm sorting device according to the first embodiment.

[0074] (Third modification example) As shown in FIG. 7B(c), a plurality of supply tanks 4 and a plurality of recovery tanks 5 may be alternately arranged side by side in the X direction and the Y direction, and the supply tank 4 and the recovery tank 5 adjacent to each other in the X direction and the Y direction may be communicated with each other by a plurality of flow paths 6 to constitute a motility sperm sorting device according to the first embodiment.

[0075] (Fourth modification example) As shown in FIG. 7B(d), a cylindrical recovery tank 5 may be arranged at the center of a hollow cylindrical supply tank 4, and the side surface LS4 of the supply tank 4 and the side surface LS5 of the recovery tank 5 may be communicated with each other by a plurality of flow paths 6 to constitute a motility sperm sorting device according to the first embodiment. In the fourth modification example, the side surface LS4 of the supply tank 4 and the side surface LS5 of the recovery tank 5 are opposed to each other in parallel. Further, the plurality of flow paths 6 are radially arranged around the recovery tank 5, and each flow path 6 has a linear shape extending in a direction orthogonal to the side surface LS4 and the side surface LS5.

Explanation of reference numerals

[0076] 1... Motility sperm sorting device, 2... Substrate, 3... Chip, 4... Supply tank, 5... Recovery tank, 6... Flow path, 7... Hole for supply tank, 8... Hole for recovery tank, 9... Groove for flow path, 10... Sperm-containing liquid, 11... Medium, 12... Sperm.

Claims

1. A supply tank for containing a sperm-containing liquid containing sperm, A recovery tank for containing a liquid medium, and A plurality of flow paths communicating the supply tank and the recovery tank with each other, By generating a flow of the medium from the recovery tank toward the supply tank in the plurality of flow paths, motile sperm contained in the sperm are caused to swim upstream from the supply tank to the recovery tank through the plurality of flow paths, and a motile sperm sorting device for sorting the motile sperm.

2. The motile sperm sorting device according to claim 1, wherein a liquid level of the medium contained in the recovery tank is made higher than a liquid level of the sperm-containing liquid contained in the supply tank, thereby generating a flow of the medium from the recovery tank toward the supply tank in the plurality of flow paths.

3. The supply tank and the recovery tank have surfaces facing each other in parallel, The motile sperm sorting device according to claim 1, wherein the plurality of flow paths are connected to the facing surfaces and have a linear shape extending in a direction orthogonal to the facing surfaces.

4. The motile sperm sorting device according to claim 3, wherein the plurality of flow paths have a rectangular cross section parallel to the facing surfaces.

5. Comprising a substrate and a chip, The chip has an upper surface and a lower surface, A hole for the supply tank and a hole for the recovery tank, which penetrate the upper surface and the lower surface, are formed in the chip, and a plurality of grooves for the plurality of flow paths, which communicate the hole for the supply tank and the hole for the recovery tank, are formed on the lower surface side of the chip, The motile sperm sorting device according to claim 1, wherein the supply tank, the recovery tank, and the plurality of flow paths are formed by adhering the substrate and the lower surface of the chip.

6. A step of preparing the motile sperm sorting device according to claim 1, A method for selecting motile sperm, comprising the steps of injecting the medium into the recovery tank, injecting the sperm-containing liquid into the supply tank, and generating a flow of the medium from the recovery tank to the supply tank in a plurality of the flow paths.

7. The method for selecting motile sperm according to claim 6, wherein the sperm-containing liquid is injected into the supply tank such that the liquid level of the medium is higher than the liquid level of the sperm-containing liquid.

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