Sperm diluent, method for preparing mammalian sperm, artificial insemination method, and in vitro fertilization method

Abstract

To provide a sperm diluent capable of obtaining a sperm population rich in X-chromosome-bearing sperm with acrosome retention by a simple method, to provide a method for preparing mammalian sperm, to provide an artificial insemination method, and to provide an in vitro fertilization method.SOLUTION: A sperm diluent induces an acrosome reaction in Y chromosome-bearing sperm to obtain a sperm population rich in X chromosome-bearing sperm that retain an acrosome and contains a TLR7 ligand and has a citric acid content of 4 mM or less. A method for preparing mammalian sperm comprises inducing an acrosome reaction in Y chromosome-bearing sperm by culturing with the sperm diluent to obtain a sperm population rich in X chromosome-bearing sperm that retain an acrosome.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a sperm diluent, a method for preparing mammalian sperm, an artificial insemination method, and an in vitro fertilization method. [Background technology]

[0002] The technology to selectively produce male and female livestock contributes greatly to the efficient production of livestock. For example, in the case of dairy cows, successor dairy cows can be obtained by selectively producing females in a planned manner. Furthermore, dairy cows need to be kept pregnant in order to produce milk, but after a successor dairy cow has been obtained, the dairy farmer's business can be greatly improved by implanting the fertilized eggs of Japanese black cattle, which have a high calf price, into the dairy cow and giving birth to a Japanese black cattle.

[0003] In pig farming, castrated males grow faster and require fewer days of fattening to reach shipping weight compared to females, so the birth of males in pork pig production not only improves the management of pig farmers, but also contributes to increasing global food production by increasing the amount of pork produced per feed. On the other hand, castration in pig production was performed without anesthesia for a long time, but in recent years, castration without anesthesia has been banned in Europe and Canada from the perspective of animal welfare, and an increase in production costs is unavoidable in these countries. Since the meat of uncastrated male pigs is low in fat and emits a foul odor called boar stench, its value is extremely low, so selecting and birthing females is expected to have the effect of mitigating the increase in production costs in overseas countries where castration without anesthesia is prohibited.

[0004] Due to these circumstances, there is a high demand for sex selection technology in the livestock industry, and techniques such as those described in Patent Document 1 are known as such technology.

[0005] In Patent Document 1, focusing on the fact that Toll-like receptors 7 and 8 (TLR7 / 8) are expressed only in X chromosome-bearing sperm, sperm with reduced motility are separated from Y chromosome-bearing sperm with good motility by culturing sperm in a medium containing a drug that activates TLR7 / 8. Then, male and female offspring are selectively born using the separated Y chromosome-bearing sperm and X chromosome-bearing sperm, respectively. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2019-10094 A Summary of the Invention [Problem to be solved by the invention]

[0007] In Patent Document 1, the X chromosome-bearing sperm can be efficiently separated from the Y chromosome-bearing sperm. However, when attempting to use the X chromosome-bearing sperm in artificial insemination or in vitro fertilization, the motility of the X chromosome-bearing sperm is reduced with a drug. Therefore, after isolating the X chromosome-bearing sperm, steps such as multiple centrifugation washings and additional culture are required to restore motility, which is cumbersome for livestock farming.

[0008] The present invention has been made in consideration of the above-mentioned circumstances, and an object of the present invention is to provide a sperm diluent, a method for preparing mammalian sperm, an artificial insemination method, and an in vitro fertilization method, which are capable of obtaining a sperm population rich in X chromosome-bearing sperm having acrosome by a simple method. [Means for solving the problem]

[0009] The sperm diluent according to the first aspect of the present invention comprises: A sperm dilution solution for inducing an acrosome reaction in Y chromosome-bearing sperm to obtain a sperm population rich in X chromosome-bearing sperm retaining an acrosome, comprising: Contains a TLR7 ligand and has a citric acid content of 4 mM or less. It is characterized by:

[0010] It is also preferable that the composition does not contain citric acid.

[0011] It is preferred that it contains no animal protein.

[0012] Moreover, the TLR7 ligand is preferably one or more selected from the group consisting of resiquimod, imiquimod, gardikimod, and loxoribine.

[0013] A method for preparing mammalian sperm according to a second aspect of the present invention comprises: Cultivating mammalian sperm in the sperm diluent according to the first aspect of the present invention to induce an acrosome reaction in Y chromosome-bearing sperm, thereby obtaining a sperm population enriched in X chromosome-bearing sperm retaining an acrosome; It is characterized by:

[0014] It is also preferable to incubate for longer than 30 minutes and shorter than 90 minutes.

[0015] The artificial insemination method according to the third aspect of the present invention comprises: performing artificial insemination of a non-human mammal using sperm of the non-human mammal prepared by the method for preparing mammalian sperm according to the second aspect of the present invention; It is characterized by:

[0016] The in vitro fertilization method according to the fourth aspect of the present invention comprises: performing in vitro fertilization of a non-human mammal using sperm of the non-human mammal prepared by the method for preparing mammalian sperm according to the second aspect of the present invention; It is characterized by: Effect of the Invention

[0017] According to the present invention, it is possible to provide a sperm diluent, a method for preparing mammalian sperm, an artificial insemination method, and an in vitro fertilization method, which are capable of obtaining a sperm population rich in X chromosome-bearing sperm having acrosome by a simple method. [Brief description of the drawings]

[0018] [Figure 1] 1 is a graph showing the rate of acrosome normality in experiment 1. [Diagram 2] 13 is a graph showing the normal acrosome rate in experiment 2. [Diagram 3] 13 is a graph showing the percentage of Y sperm in experiment 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The sperm diluent according to the present embodiment is a sperm diluent for inducing an acrosome reaction in Y chromosome-bearing sperm to cause acrosome loss, thereby obtaining a sperm group rich in X chromosome-bearing sperm that retain the acrosome. In this specification, X chromosome-bearing sperm will be referred to as "X sperm" and Y chromosome-bearing sperm will be referred to as "Y sperm".

[0020] Normally, after being ejaculated into the female accessory reproductive organs, sperm swim in a manner dependent on the surrounding environment until they reach the oviduct, where fertilization occurs. When sperm encounter an egg in the oviduct, the acrosome present in the sperm head cleaves and secretes an enzyme. This acrosome reaction is essential for the sperm to penetrate the egg and complete fertilization. This acrosome reaction can also occur spontaneously outside the oviduct, and sperm that undergo the acrosome reaction early are phagocytosed in the uterus and are unable to reach the oviduct. In other words, in order to complete fertilization, it is essential that the sperm retain the acrosome until they reach the oviduct, and that the acrosome reaction occurs at the appropriate time.

[0021] When the X sperm penetrates the egg and fertilization is complete, a female embryo is produced and a female offspring is born. On the other hand, when the Y sperm penetrates the egg and fertilization is complete, a male embryo is produced and a male offspring is born.

[0022] The sperm diluent according to this embodiment contains a TLR7 ligand and has a citric acid content of 4 mM or less.

[0023] Citric acid promotes sperm metabolism and induces the acrosome reaction in both X and Y sperm. On the other hand, TLR7 ligands act sex-specifically on sperm. When sperm are cultured in a sperm diluent containing TLR7 ligands, sperm metabolism of X sperm is suppressed and the acrosome is preserved. This ensures the fertilization ability of X sperm.

[0024] That is, in a sperm diluent containing a TLR7 ligand and a citric acid content of 4 mM or less, the sperm metabolism of Y sperm is increased by citric acid, and the acrosome reaction is induced. On the other hand, the sperm metabolism of X sperm is suppressed by the TLR7 ligand. Therefore, after culture, most of the sperm that retain the acrosome become X sperm. Therefore, the proportion of X sperm is high in the sperm group that retains the acrosome.

[0025] Examples of TLR7 ligands include resiquimod, imiquimod, gardiquimod, and loxoribine. These may be contained alone or in combination of two or more.

[0026] The content of the TLR7 ligand is appropriately determined depending on the type of TLR7 ligand and the animal species of the sperm used, and is preferably, for example, 3 nM to 3 μM.

[0027] The sperm diluent can be obtained by adding a TLR7 ligand to a basal diluent. As the basal diluent, a commercially available embryo culture medium or the like can be used as long as the citric acid concentration is 4 mM or less.

[0028] Examples of the components of the basal diluent include glucose, lactose, sodium bicarbonate, EDTA·2Na2 hydrate, and tris(hydroxymethyl)aminomethane (hereinafter, Tris). The contents of these components are, for example, glucose: 20-40 mM, lactose: 100-150 mM, sodium bicarbonate: 8-15 mM, EDTA·2Na2 hydrate: 3-10 mM, and Tris: 40-60 mM.

[0029] In addition, Patent Document 1 discloses a medium in which bovine serum albumin (BSA) and a TLR7 ligand are added to a human tubular fluid (HTF) medium as a basal medium. If BSA is not added to the HTF medium, sperm metabolism is suppressed in both X sperm and Y sperm, and the acrosome reaction is not induced in the Y sperm. For this reason, Patent Document 1 adds BSA to the HTF medium.

[0030] On the other hand, the sperm diluent according to the present embodiment does not contain animal proteins mainly composed of albumin such as BSA. Even though the sperm diluent according to the present embodiment does not contain animal proteins such as BSA, the citric acid concentration is set to 4 mM or less, and as described above, the acrosome reaction of Y sperm is promoted. Furthermore, while BSA is expensive, the sperm diluent does not require animal proteins such as BSA, so the manufacturing cost can be reduced.

[0031] In addition, HTF medium containing BSA cannot be stored because precipitation occurs when stored in a cool place. On the other hand, the sperm diluent according to the present embodiment does not have such a disadvantage, and has the advantage that it can be stored for a long period of time, for example, for more than one month, even at room temperature.

[0032] The method for preparing mammalian sperm according to the present embodiment involves culturing mammalian sperm in the sperm diluent described above, inducing the acrosome reaction of Y sperm, and preparing a sperm population enriched in X sperm with retained acrosomes.

[0033] In this embodiment, by culturing sperm in the above-mentioned sperm dilution solution, sperm metabolism of Y sperm is increased and the acrosome reaction is induced. Then, the Y sperm lose the acrosome and lose fertilization ability. On the other hand, the sperm metabolism of X sperm is suppressed in the above-mentioned sperm dilution solution, so the acrosome is maintained and fertilization ability is guaranteed. Therefore, in the sperm group after cultivation, most of the sperm that retain the acrosome become X sperm. Therefore, a sperm group rich in X sperm that retain the acrosome is obtained.

[0034] The incubation time is preferably long enough to activate the Y sperm, for example, longer than 30 minutes. If the incubation time is too long, the loss of the acrosome of the X sperm will progress, so the incubation time is preferably shorter than 90 minutes. The incubation time is more preferably 60 minutes.

[0035] In this embodiment, as described above, sperm are cultured in a sperm diluent, so that a large amount of sperm can be processed.

[0036] As mentioned above, the sperm population after the culture is rich in X sperm that have an X chromosome and retain the acrosome. Therefore, when artificial insemination or in vitro fertilization of mammals is performed using this sperm population, the X sperm fertilize the egg and become a fertilized egg with an XX chromosome, so that it is possible to selectively produce female offspring. Artificial insemination and in vitro fertilization can be performed by the usual method.

[0037] It is not necessary to separate the X and Y sperm from the cultured sperm group. Since most of the Y sperm contained in the cultured sperm group lack acrosomes and have lost the ability to fertilize, if the cultured sperm group is subjected to artificial insemination or in vitro fertilization without separating the X and Y sperm, female offspring can be produced at a high rate.

[0038] As described above, the method for preparing mammalian sperm according to the present embodiment can easily obtain a sperm population rich in X-sperm, and can be easily carried out in livestock farming facilities since it does not require any complicated steps such as additional culture. EXAMPLES

[0039] (Experiment 1) Verification of the effect of citric acid concentration in sperm diluent on acrosome retention of boar sperm A sperm diluent (pH 7.0) was prepared by mixing glucose, lactose, sodium bicarbonate, EDTA·2Na2 hydrate, and Tris. Citric acid was then added to this sperm diluent to prepare sperm diluents with citric acid concentrations of 40 mM, 20 mM, 4 mM, and 0 mM (hereinafter referred to as sperm diluent A, sperm diluent B, sperm diluent C, and sperm diluent D). The compositions of sperm diluent A, sperm diluent B, sperm diluent C, and sperm diluent D are shown in Table 1.

[0040] [Table 1]

[0041] The diluted boar semen on the day after collection was centrifuged (300×G, 3 minutes) to separate sperm from the semen. The separated sperm were added to each sperm dilution solution and incubated in an incubator (37°C, 60 minutes). The sperm were stained with PNA-FITC (peanut agglutinin fluorescein isothiocyanate), an acrosome retention marker, and PI (podium iodide), a cell viability marker, washed by centrifugation, and then subjected to flow cytometry to calculate the percentage of PNA- / PI- sperm (sperm that were viable and had retained their acrosome) and determine the acrosome normality rate.

[0042] The results are shown in Figure 1. As the citric acid concentration in the sperm diluent decreases, the rate of normal sperm acrosomes decreases. It can be seen that when the citric acid concentration is low, the acrosome reaction is induced and acrosome defects occur. There was also no significant difference between 4 mM and 0 mM citric acid concentrations, indicating that a citric acid concentration of 4 mM or less can promote acrosome defects in sperm.

[0043] (Experiment 2) Verification of the effect of no addition of citric acid and addition of TLR7 ligand on acrosome retention in boar sperm A sperm diluent (pH 7.0, hereinafter referred to as sperm diluent DR) was prepared by adding 3 mM resiquimod (R848) as a TLR7 ligand to the above-mentioned sperm diluent D. In addition, sperm diluent D and sperm diluent DR were also prepared by adding NaOH to them and adjusting the pH to 8.0. The sperm diluents used in Experiment 2 were sperm diluent A (pH 7.0), sperm diluent D (pH 7.0), sperm diluent DR (pH 7.0), sperm diluent D (pH 8.0), and sperm diluent DR (pH 8.0).

[0044] The diluted pig semen on the day after collection was centrifuged (300×G, 3 minutes). Boar sperm were incubated in each sperm dilution solution in an incubator (37°C, 60 minutes). After the culture, the supernatant was removed by centrifugation, and the cells were suspended in HIRO-SWINE B solution (manufactured by Hiroshima CryoPreservation Service Co., Ltd.; HIRO-SWINE is a registered trademark) containing seminal plasma and allowed to stand at 15° C. for one day. The sperm were stained with PNA-FITC (peanut agglutinin fluorescein isothiocyanate), an acrosome retention marker, and PI (podium iodide), a cell viability marker, washed by centrifugation, and then subjected to flow cytometry to calculate the percentage of PNA- / PI- sperm (sperm that were viable and had retained their acrosome) and determine the acrosome normality rate.

[0045] The results are shown in Figure 2. At either pH 7.0 or 8.0, when cultured in sperm diluent DR containing R848, the rate of acrosome normality was significantly higher than in sperm diluent D without R848.

[0046] (Experiment 3) Verification of the ratio of X sperm (Y sperm) In experiment 3, sperm diluent D (pH 7.0) and sperm diluent DR (pH 7.0) were used. The diluted pig semen on the day after collection was centrifuged (300×G, 3 minutes). Boar sperm were incubated in each sperm dilution solution in an incubator (37°C, 60 minutes). After the culture, the supernatant was removed by centrifugation, and the cells were suspended in HIRO-SWINE B solution (manufactured by Hiroshima CryoPreservation Service Co., Ltd.; HIRO-SWINE is a registered trademark) containing seminal plasma and allowed to stand at 15° C. for one day. Since sperm with normal acrosomes have good motility, motile sperm were collected by the swim-up method. The Y sperm rate was calculated by FISH (Fluorescence in situ hybridization) using a probe that recognizes the Y chromosome.

[0047] The results are shown in Figure 3. When cultured in sperm diluent DR containing R848, the proportion of Y sperm was about 30%. In other words, X sperm accounted for about 70%. The absence of citric acid in the sperm diluent induces the acrosome reaction in sperm, but R848 acts sex-specifically to inhibit the induction of the acrosome reaction in X sperm, resulting in a sperm population rich in X sperm. [Industrial Applicability]

[0048] The sperm dilution solution according to the present invention can obtain a sperm population rich in X-sperm with acrosome retention, and therefore can be used for artificial insemination or in vitro fertilization to selectively produce male offspring, and can be used in the livestock industry, such as for pigs.

Claims

1. A sperm dilution solution for inducing an acrosome reaction in Y chromosome-bearing sperm to obtain a sperm population rich in X chromosome-bearing sperm retaining an acrosome, comprising: Contains a TLR7 ligand and has a citric acid content of 4 mM or less; A sperm diluent characterized by the above.

2. Does not contain citric acid 2. The sperm diluent according to claim 1 .

3. Contains no animal protein 2. The sperm diluent according to claim 1 .

4. The TLR7 ligand is one or more selected from the group consisting of resiquimod, imiquimod, gardikimod and loxoribine; 2. The sperm diluent according to claim 1 .

5. 5. Incubating mammalian sperm with the sperm diluent according to any one of claims 1 to 4 to induce the acrosome reaction of Y chromosome-bearing sperm, thereby obtaining a sperm population rich in X chromosome-bearing sperm retaining the acrosome. A method for preparing mammalian sperm, comprising:

6. Incubate for more than 30 minutes but less than 90 minutes; 6. The method for preparing mammalian sperm according to claim 5.

7. Artificial insemination of a non-human mammal is performed using sperm of the non-human mammal prepared by the method for preparing mammalian sperm according to claim 5. A method for artificial insemination comprising the steps of:

8. 6. An in vitro fertilization of a non-human mammal is carried out using sperm of the non-human mammal prepared by the method for preparing mammalian sperm according to claim 5.

1. An in vitro fertilization method comprising:

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