Method for preparing mammalian sperm, artificial insemination method, in vitro fertilization method, and culture medium

Abstract

This mammalian sperm preparation method includes: culturing of mammalian sperm in a medium that contains TLR7 ligand and that has a pH of 7.7-7.9; inducing the acrosomal reaction of Y chromosome-containing sperm; and obtaining a sperm group that is rich with X chromosome-containing sperm and that retains acrosome. This sperm group is used for artificial insemination and in-vitro fertilization for selective male production.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing mammalian sperm and an artificial insemination method. 、 IVF method and culture medium Regarding. [Background technology]

[0002] The technology to selectively breed livestock makes a significant contribution to the efficient production of livestock. For example, in the case of dairy cows, successor dairy cows can be obtained by selectively breeding females. Furthermore, dairy cows need to be kept pregnant in order to produce milk, but once 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] Furthermore, in pig farming, castrated males grow faster and require fewer fattening days to reach shipping weight compared to females. Therefore, the birth of male pigs in pork production not only improves the management of pig farmers, but also contributes to increasing global food production by increasing pork production per feed. Meanwhile, castration in pig production was long performed without anesthesia, but in recent years, castration without anesthesia has been banned in Europe and Canada from an animal welfare perspective, resulting in an unavoidable increase in production costs in these countries. Since the meat of uncastrated male pigs is low in fat and emits a foul odor known as boar taint, its value is extremely low. Therefore, selectively breeding female pigs is expected to mitigate the increase in production costs in overseas countries where castration without anesthesia is prohibited.

[0004] As such, 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 sex selection 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, the researchers cultured sperm in a medium containing a drug that activates TLR7 / 8, thereby separating X chromosome-bearing sperm with reduced motility from Y chromosome-bearing sperm with good motility. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 2019-10094 Summary of the Invention [Problem to be solved by the invention]

[0007] While Patent Document 1 enables efficient separation of X-chromosome-bearing sperm and Y-chromosome-bearing sperm, when applying X-chromosome-bearing sperm to artificial insemination or in vitro fertilization, the motility of the X-chromosome-bearing sperm is reduced with a drug. Therefore, after separating the X-chromosome-bearing sperm, steps such as multiple centrifugation washes and additional culture are required to restore motility, which is cumbersome for livestock farming.

[0008] The present invention has been made in view of the above circumstances, and its object is to provide a method for preparing mammalian sperm, an artificial insemination method, and a method for producing a sperm population enriched in acrosome-retaining X chromosome-bearing sperm by a simple method. 、 IVF method and culture medium The purpose is to provide [Means for solving the problem]

[0009] A method for preparing mammalian sperm according to a first aspect of the present invention comprises: TLR7 Ligand , glucose and albumin Contains Contains no pyruvic acid or lactic acid Mammalian spermatozoa were cultured in a medium with a pH of 7.7 to 7.9. More than 30 minutes and less than 90 minutes Cultivate the spermatozoa and induce the acrosome reaction in the Y chromosome-bearing spermatozoa, thereby obtaining a spermatozoon rich in X chromosome-bearing spermatozoa that retain the acrosome. It is characterized by:

[0013] Furthermore, the TLR7 ligand is preferably one or more selected from the group consisting of resiquimod, imiquimod, gardikimod, and loxoribine.

[0014] The artificial insemination method according to the second aspect of the present invention comprises: artificial insemination of a non-human mammal using sperm of a non-human mammal prepared by the method for preparing mammalian sperm according to the first aspect of the present invention; It is characterized by:

[0015] The in vitro fertilization method according to the third 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 first aspect of the present invention; It is characterized by: A culture medium according to a fourth aspect of the present invention comprises: Mammalian sperm More than 30 minutes and less than 90 minutes A medium for culturing spermatozoa containing a Y chromosome to induce the acrosome reaction of the spermatozoa containing a Y chromosome and to obtain a spermatozoa rich in spermatozoa containing an X chromosome, The medium contains a TLR7 ligand , glucose and albumin Contains Contains no pyruvic acid or lactic acid pH is 7.7 to 7.9, It is characterized by: [Effects of the Invention]

[0016] According to the present invention, a method for preparing mammalian sperm and an artificial insemination method are provided that can easily obtain a population of sperm rich in X chromosome-bearing sperm that retain the acrosome. 、 IVF method and culture medium can be provided. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is a graph showing the acrosome retention rate in Experiment 1. [Figure 2] 10 is a graph showing the acrosome retention rate in Experiment 2. [Figure 3] 10 is a graph showing the acrosome retention rate in Experiment 3. [Figure 4] 10 is a graph showing the acrosome retention rate in Experiment 4. [Figure 5] 10 is a graph showing the percentage of X sperm in Experiment 5. [Figure 6]10 is a graph showing the acrosome retention rate in Experiment 7. [Figure 7] 10 is a graph showing the acrosome retention rate in Experiment 9. [Figure 8] 10 is a graph showing the proportions of X sperm and Y sperm in Experiment 9. [Figure 9] 10 is a graph showing the proportions of XX embryos and XY embryos in Experiment 10. DETAILED DESCRIPTION OF THE INVENTION

[0018] The method for preparing mammalian sperm according to this embodiment involves culturing mammalian sperm in a medium containing a TLR7 ligand and having a pH of 7.7 to 7.9, inducing the acrosome reaction in Y chromosome-bearing sperm, and obtaining a population of sperm enriched in X chromosome-bearing sperm that retain acrosomes. 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."

[0019] After being ejaculated into the female accessory reproductive organs, sperm swim depending on the surrounding environment until they reach the fallopian tube, where fertilization occurs. When sperm encounter an egg in the fallopian tube, the acrosome at 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 fallopian tube; sperm that undergo the acrosome reaction early are phagocytosed in the uterus and are unable to reach the fallopian tube. In other words, to complete fertilization, it is essential that the sperm retain their acrosome until they reach the fallopian tube, and that the acrosome reaction occur at the appropriate time.

[0020] When an X sperm penetrates an egg and fertilization is complete, a female embryo is produced and a female offspring is born. On the other hand, when a Y sperm penetrates an egg and fertilization is complete, a male embryo is produced and a male offspring is born.

[0021] In this embodiment, by culturing sperm in a medium having a pH of 7.7 to 7.9 and containing a TLR7 ligand, sperm metabolism of Y sperm is increased and the acrosome reaction is induced. The Y sperm then lose their acrosome and lose their fertilization ability. On the other hand, sperm metabolism of X sperm is suppressed in the above medium, so the acrosome is maintained and fertilization ability is ensured. Therefore, in the sperm population after culturing, most of the sperm that retain the acrosome become X sperm. Therefore, a sperm population rich in X sperm that retain the acrosome is obtained.

[0022] A medium containing a TLR7 ligand can be prepared by adding a TLR7 ligand to a basal medium. A medium typically used for culturing mammalian spermatozoa can be used as the basal medium. Suitable basal media include embryo culture media such as HTF (Human Tubal Fluid) medium (composition: NaCl, KCl, KH2PO4, MgSO4·7H2O, CaCl2·2H2O, NaHCO3, glucose, sodium pyruvate, sodium lactate, gentamicin sulfate salt, and phenol red).

[0023] The pH of the medium is 7.7 to 7.9, and if it is outside this range, the significant difference in the acrosome retention rate between X sperm and Y sperm becomes smaller, as shown in the Examples below. The pH can be adjusted by adding a pH adjuster such as NaOH to the medium.

[0024] Furthermore, the medium preferably contains albumin. When sperm are cultured in a medium that does not contain albumin, sperm metabolism is suppressed regardless of whether they are X sperm or Y sperm. Therefore, when sperm are cultured in a medium that does not contain albumin but contains a TLR7 ligand, sperm metabolism of Y sperm is also suppressed, resulting in impaired acrosome loss, making it difficult to obtain a sperm population rich in X sperm that retain acrosomes. For example, bovine serum albumin (BSA) can be used as albumin. The albumin content in the medium is preferably about 2 to 8 g / L, and more preferably about 5 g / L.

[0025] Furthermore, it is preferable that the medium does not contain pyruvic acid or lactic acid. When the basal medium does not contain pyruvic acid or lactic acid, the retention rate of the acrosome of X sperm can be further improved.

[0026] Examples of TLR7 ligands to be added include resiquimod, imiquimod, gardiquimod, and loxoribine. These may be used alone or in combination of two or more.

[0027] The amount of TLR7 ligand added to the basal medium may be appropriately determined depending on the type of TLR7 ligand used and the animal species of the sperm used, and is preferably 3 nM to 3 μM, for example.

[0028] The incubation time is preferably long enough to activate the Y sperm, for example, longer than 30 minutes. Furthermore, 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.

[0029] In this embodiment, as described above, sperm are cultured in a medium containing a TLR7 ligand, and therefore a large amount of sperm can be processed.

[0030] As mentioned above, the cultured sperm population is enriched in X sperm that have an X chromosome and retain the acrosome. Therefore, when this sperm population is used for artificial insemination or in vitro fertilization of mammals, the X sperm fertilize the eggs, resulting in zygotes with XX chromosomes, allowing selective female offspring production. Artificial insemination and in vitro fertilization can be performed using conventional methods.

[0031] The cultured sperm population may or may not be separated into X and Y sperm. Since many of the Y sperm contained in the cultured sperm population lack acrosomes and have lost their fertilization ability, if the cultured sperm population 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.

[0032] As described above, the method for preparing mammalian sperm according to the present embodiment can obtain a sperm population rich in X sperm in a simple manner, and does not require any complicated steps such as additional culture, so it can be easily carried out in livestock farming settings. [Example]

[0033] (Experiment 1) Verification of the effect of albumin The diluted boar semen collected the day after collection was centrifuged (300×G, 3 minutes). In addition, a modified HTF medium was prepared by adding BSA to the HTF medium. The composition of the modified HTF medium is shown in Table 1.

[0034] [Table 1]

[0035] Boar semen was added to HTF medium and modified HTF medium, and the medium was incubated in an incubator at 37°C for 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. After washing by centrifugation, the sperm were subjected to flow cytometry to calculate the ratio of PNA- / PI- sperm (viable sperm with acrosome retention) and determine the acrosome retention rate.

[0036] The results are shown in Figure 1. The percentage of sperm with acrosome retention was significantly lower in the modified HTF medium than in the HTF medium. We confirmed that culturing boar sperm in a medium containing albumin reduced the acrosome retention rate.

[0037] (Experiment 2) Verification of the effects of medium pH and R848 on acrosome retention in boar sperm The modified HTF medium was adjusted to various pH levels by adding NaOH, and Resiquimod R-848 (hereinafter referred to as R848) (3 nM) was added as a TLR7 ligand.

[0038] Using each modified HTF medium, boar sperm were cultured and the acrosome retention rate was determined in the same manner as in Experiment 1. For comparison, the same procedure was performed using a medium without R848 (non-additive group).

[0039] The results are shown in Figure 2. In the pH range of 7.7 to 7.9, the percentage of sperm with retained acrosomes was significantly higher in the R848-added group compared to the control group. Note that the time point for 0 minutes was determined by adding PNA-FITC and PI to diluted semen and leaving it to stand for 30 minutes at 15°C.

[0040] (Experiment 3) Verification of the effect of incubation time on acrosome retention in boar sperm Boar sperm were cultured in modified HTF medium adjusted to pH 7.8 and supplemented with R848 (3 nM) in the same manner as in Experiment 1, except for varying the incubation time, and the acrosome retention rate was determined (R848-supplemented group). For comparison, the same experiment was also performed using medium without R848 (non-supplemented group). The incubation times were 0, 30, 60, and 90 minutes.

[0041] The results are shown in Figure 3. The effect of R848-added sperm in significantly increasing the proportion of sperm with normal acrosomes compared to the control group was not observed after 30 minutes, but was significantly observed after 60 minutes. However, after 90 minutes of incubation, the acrosome reaction occurred even in the R848-added group, and the retention rate was the same as in the control group. Therefore, it is clear that an incubation time of more than 30 minutes and less than 90 minutes is preferable, with 60 minutes being more preferable.

[0042] (Experiment 4) Verification of the effect of energy substrates in the medium on acrosome retention in boar sperm The modified HTF medium contains energy substrates other than glucose, such as pyruvate and lactate, so we investigated the effects of these energy substrates.

[0043] Boar sperm were cultured in the same manner as in Experiment 1, except that the pH was adjusted to 7.8, R848 (3 nM) was added, and different energy substrates were used. The acrosome retention rate was determined (R848-added group). For comparison, the same procedure was performed using modified HTF medium without R848 (non-added group).

[0044] The modified HTF medium used was as follows: Free medium: A medium that does not contain glucose (D-glucose), pyruvic acid, or lactic acid (Na-lactate) among the ingredients listed in Table 1. Glu medium: A medium that does not contain pyruvic acid or lactic acid (Na-lactate) among the ingredients listed in Table 1. Pyr medium: A medium that does not contain glucose (D-glucose) or lactic acid (Na-lactate) among the ingredients listed in Table 1. Lac medium: A medium that does not contain glucose (D-glucose) or pyruvic acid among the ingredients listed in Table 1. All medium: A medium containing all the ingredients listed in Table 1

[0045] The results are shown in Figure 4. The acrosome retention effect of R848 was observed in Glu medium. On the other hand, the acrosome retention effect of R848 was not observed in Free medium, Pyr medium, or Lac medium. Therefore, it can be seen that the acrosome retention effect is enhanced in a medium containing R848 but not containing pyruvate or lactate.

[0046] (Experiment 5) Separation of X sperm Acrosome-deficient sperm exhibit a circular motility pattern and are difficult to swim up. Taking advantage of this, we attempted to separate X sperm by swim-up.

[0047] Boar sperm were cultured (1 hour at 37°C) in modified HTF medium (Glu medium, pH 7.8) supplemented with R848 using the same method as in Experiment 1. After incubation, the reacted sperm were suspended in viscous 2% PVP (Polyvinylpyrrolidone) (1 mL) and then placed in the lower layer of 2 mL of modified HTF medium. After 30 minutes, sperm swimming up into the modified HTF medium were collected.

[0048] The collected sperm were then subjected to PNA staining and their fluorescence intensity was quantified by flow cytometry. The fluorescence intensity of the sperm remaining in the lower layer was also quantified in the same manner.

[0049] As a result, it was confirmed that most of the sperm that swam to the upper layer retained their acrosomes, just like the sperm before culture, while the acrosomes of the sperm that remained in the lower layer were damaged.

[0050] The percentage of X-sperm was measured using real-time PCR for the sperm collected from the upper layer (R848-added group). For comparison, the same procedure was performed on the modified HTF medium without R848, and the percentage of X-sperm was measured (no-added group).

[0051] The results are shown in Figure 5. In the R848-added group, the rate of X-sperm among upward-facing sperm was significantly higher than in the control group. Therefore, it was confirmed that most of the sperm with retained acrosomes were X-sperm.

[0052] (Experiment 6) Artificial insemination of pigs Boar semen diluted to 5 billion / 100 mL with Hiloswine solution B was centrifuged (500 G, 5 minutes, room temperature) and the supernatant was removed. Boar sperm were suspended in the same volume of modified HTF medium (pH 7.8, supplemented with 3 nM R848) and cultured in an incubator (60 minutes, 37°C). After culture, the supernatant was removed by centrifugation (500 G, 5 minutes, room temperature), and the sperm were suspended in artificial insemination diluent and used for artificial insemination.

[0053] The results are shown in Table 2. In the untreated group, 25 out of 50 were male and 25 were female, for a male-female ratio of 50:50, whereas artificial insemination using semen treated with culture medium containing R848 resulted in 13 out of 36 males and 23 females, for a female ratio of 64%.

[0054] [Table 2]

[0055] (Experiment 7) Verification of mouse sperm Sperm were collected from the cauda epididymis of 12-week-old adult male mice and cultured in modified HTF medium (Glu medium, pH 7.8, supplemented with 3 nM R848) for 60 minutes at 37°C.

[0056] After incubation, mouse sperm were fixed and stained with PNA-FITC, and the acrosome retention rate was measured by flow cytometry (R848-added group).For comparison, the acrosome retention rate was measured in the same manner as above, except that the medium was not supplemented with R848 (no addition group).

[0057] The results are shown in Figure 6. For mouse sperm, the acrosome retention rate was also significantly higher in the R848-added group than in the non-added group.

[0058] (Experiment 8) In vitro fertilization and artificial insemination of mice In vitro fertilization was performed using sperm cultured in the same manner as in Experiment 7. Five days later, embryos that had reached the blastocyst stage were collected, and the ratio of X sperm to Y sperm was calculated using real-time PCR.

[0059] The results are shown in Table 3. In the untreated group, 8 out of 16 were male and 8 were female, for a male-female ratio of 50:50, whereas in the R848-added group, 5 out of 22 were male and 17 were female, for a female embryo yield rate of 77%.

[0060] [Table 3]

[0061] In addition, the sperm cultured in the same manner as in Experiment 7 were used for artificial insemination, and blastocyst-stage embryos were recovered, of which 83.3% (5 / 6) were female.

[0062] (Experiment 9) Verification of bovine sperm Frozen-thawed bovine spermatozoa were thawed and washed in modified HTF medium, then cultured in modified HTF medium (Glu medium, pH 7.7, supplemented with 3 nM R848) for 30 minutes at room temperature. After centrifugation and removal of the supernatant, the spermatozoa were resuspended in modified HTF medium containing 1% PVP and 3 nM R848 and placed in the lower layer of PVP-free modified HTF medium. After 15 minutes, 1.5 mL of the upper layer and 0.5 mL of the lower layer were collected. Acrosome staining was performed on each sample to determine the acrosome retention rate.

[0063] The results are shown in Figure 7. The acrosome retention rate of sperm in the upper layer was 70%, which was significantly higher than that of sperm in the lower layer.

[0064] In addition, the sperm collected from the lower layer were subjected to fluorescence in situ hybridization using a probe that recognizes the X chromosome, and the ratio of X sperm to Y sperm was determined.

[0065] The results are shown in Figure 8. 84% of the sperm in the lower layer were Y-sperm, which suggests that in bovine sperm, R848 also damages the acrosome of Y-sperm, while the acrosome of X-sperm is preserved.

[0066] (Experiment 10) In vitro fertilization of cattle Frozen-thawed bovine spermatozoa were thawed and washed in modified HTF medium, then cultured in modified HTF medium (pH 7.4, supplemented with 3 nM R848) for 30 minutes at room temperature. After centrifugation to remove the supernatant, the spermatozoa were resuspended in modified HTF medium containing 1% PVP and 3 nM R848 and placed in a lower layer of PVP-free modified HTF medium. After 15 minutes, 1.5 mL of the upper layer was collected, washed, and then used for in vitro fertilization. Blastocyst-stage embryos were sexed using real-time PCR.

[0067] The results are shown in Figure 9. As a result, 91% of the embryos were XX female embryos.

[0068] The present invention allows various embodiments and modifications without departing from the broad spirit and scope of the present invention. Furthermore, the above-described embodiments are intended to explain the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not the embodiments. Various modifications made within the scope of the claims and the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

[0069] This application is based on Japanese Patent Application No. 2022-9945 filed on January 26, 2022. The entire specification, claims, and drawings of Japanese Patent Application No. 2022-9945 are incorporated herein by reference. [Industrial Applicability]

[0070] The method for preparing mammalian sperm according to the present invention can obtain a sperm population rich in X-sperm with acrosome retention, and therefore, by subjecting it to artificial insemination or in vitro fertilization, it is possible to selectively produce male offspring, and the method can be used in the livestock industry, such as in the production of cattle and pigs.

Claims

1. Mammalian sperm are cultured in a medium containing a TLR7 ligand, glucose, and albumin, not containing pyruvate or lactic acid, and having a pH of 7.7 to 7.9, for a period of time longer than 30 minutes and shorter than 90 minutes, thereby inducing the acrosome reaction of Y chromosome-bearing sperm and obtaining a sperm population enriched in X chromosome-bearing sperm that retain an acrosome. A method for preparing mammalian sperm, comprising:

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

3. Artificial insemination of a non-human mammal is performed using sperm of a non-human mammal prepared by the method for preparing mammalian sperm according to claim 1 or 2. An artificial insemination method characterized by:

4. 3. In vitro fertilization of a non-human mammal is carried out using sperm of a non-human mammal prepared by the method for preparing mammalian sperm according to claim 1 or 2. An in vitro fertilization method characterized by:

5. A medium for culturing mammalian sperm for a period of time longer than 30 minutes and shorter than 90 minutes to induce the acrosome reaction of Y chromosome-bearing sperm and obtain a sperm population enriched in X chromosome-bearing sperm retaining acrosomes, comprising: The medium contains a TLR7 ligand, glucose, and albumin, is free of pyruvate and lactate, and has a pH of 7.7 to 7.

9. A culture medium characterized by:

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