Artificial insemination method of animal having ability of multiple pregnancy

Abstract

To provide an artificial insemination method of an animal having an ability of multiple pregnancy, the method being capable of improving a male childbirth ratio without requiring a separation process.SOLUTION: Sperm of an animal having an ability of multiple pregnancy is incubated in a medium containing a TLR7 ligand and having a pH of 7.7 to 7.9, and the artificial insemination method of an animal having an ability of multiple pregnancy is performed in artificial insemination of the animal having an ability of multiple pregnancy within 12 hours after the incubation.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for artificial insemination of multiparous animals. [Background technology]

[0002] The technology of sex selection for livestock greatly contributes to efficient livestock production. For example, in pig farming, males grow faster and require fewer days of fattening to reach shipping weight compared to females, so it is sometimes desirable to produce male offspring for meat pig production. On the other hand, the meat of intact males tends to be less valuable than females because it has a low fat content and emits a foul odor called boar taint, so it is sometimes desirable to select females and produce them.

[0003] Thus, there is a high demand for sex selection technology in the livestock industry, and known sex selection techniques include those described in Patent Document 1. Patent Document 1 focuses on the fact that Toll-like receptors 7 and 8 (TLR7 / 8) are expressed only in X chromosome-bearing sperm, and separates X chromosome-bearing sperm with reduced motility from Y chromosome-bearing sperm with good motility by culturing sperm in a medium containing a drug that activates TLR7 / 8. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2019-10094 A Summary of the Invention [Problem to be solved by the invention]

[0005] In Patent Document 1, the Y chromosome-carrying sperm in the upper layer and the X chromosome-carrying sperm in the lower layer are separated and used for artificial insemination, so a separation step is necessary.

[0006] The present invention has been made in consideration of the above-mentioned circumstances, and an object of the present invention is to provide a method for artificial insemination of multiparous animals which is capable of increasing the ratio of male offspring without requiring a separation step. [Means for solving the problem]

[0007] The method for artificial insemination of a multiparous animal according to the present invention comprises the steps of: Cultivating sperm from multiple animals in a medium containing a TLR7 ligand and having a pH of 7.7 to 7.9; After culturing, the animals are subjected to artificial insemination of multiple live births within 12 hours. It is characterized by:

[0008] It is also preferable to use the medium containing albumin.

[0009] It is also preferable to use the above medium which does not contain pyruvic acid or lactic acid.

[0010] It is also preferable to incubate for longer than 30 minutes and shorter than 90 minutes.

[0011] Moreover, the TLR7 ligand is preferably one or more selected from the group consisting of resiquimod, imiquimod, gardikimod, and loxoribine.

[0012] The multiparous animal may also be a pig. Effect of the Invention

[0013] According to the present invention, there is provided a method for artificial insemination of multiparous animals that can increase the ratio of male offspring without requiring a separation step. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The method for artificial insemination of multi-birth animals according to the present embodiment can increase the ratio of male offspring among offspring born by artificially inseminating spermatozoa of multi-birth animals cultured in a medium containing a TLR7 ligand at a pH of 7.7 to 7.9 within 12 hours after the culture. Examples of multi-birth animals include pigs, sheep, goats, etc., and the method can be applied to these multi-birth animals.

[0015] In this specification, sperm carrying an X chromosome are referred to as "X sperm" and sperm carrying a Y chromosome as "Y sperm." When an X sperm penetrates an egg and fertilization is completed, it becomes a fertilized egg with XX chromosomes, and a female offspring is born. On the other hand, when a Y sperm penetrates an egg and fertilization is completed, it becomes an XY fertilized egg, and a male offspring is born.

[0016] Cultivating sperm in a medium having a pH of 7.7 to 7.9 and containing a TLR7 ligand increases the sperm metabolism of Y sperm, whereas the sperm metabolism of X sperm is suppressed in the above medium.

[0017] If the sperm are cultured in the above medium and then artificially inseminated within 12 hours, the Y sperm, which have high sperm metabolism, will preferentially penetrate the egg and become more likely to fertilize it. This will increase the proportion of XY fertilized eggs, and the proportion of male offspring will be higher among the offspring that are born. However, if the time between sperm culture and artificial insemination is too long, this will lead to acrosome defects in the Y sperm, which have high sperm metabolism. As a result, the Y sperm will lose their ability to fertilize, and the proportion of male offspring will decrease.

[0018] The medium containing the TLR7 ligand may be prepared by adding the TLR7 ligand to a basal medium. As the basal medium, a medium that can be generally used for culturing mammalian spermatozoa can be used. As the basal medium, for example, HTF (Human Tubal Fluid) medium (composition: NaCl, KCl, KH 2 PO 4 , MgSO 4 7H 2 O, CaCl 2 2H 2 O, NaHCO 3Embryo culture media such as medium containing 100% concentrated culture medium such as 100% concentrated culture medium containing ... containing 100% concentrated culture medium containing 100% concentrated culture medium such as 100% concentrated culture medium containing 100% concentrated culture

[0019] Examples of TLR7 ligands to be added to the medium include resiquimod, imiquimod, gardiquimod, and loxoribine. These may be contained alone or in combination of two or more.

[0020] 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, for example, 3 nM to 3 μM.

[0021] The pH of the medium is 7.7 to 7.9, and the pH can be adjusted by adding a pH adjuster such as NaOH to the medium.

[0022] In addition, it is preferable that the medium does not contain pyruvic acid or lactic acid.

[0023] The incubation time is preferably sufficient to activate the Y sperm, for example, 30 to 90 minutes.

[0024] Furthermore, in this embodiment, as described above, sperm are cultured in the above-mentioned medium, so that a large amount of sperm can be processed.

[0025] In this way, the artificial insemination method for multiple animals according to the present embodiment does not require a process for separating X sperm from Y sperm, and the semen for artificial insemination prepared by culturing is used for artificial insemination as is. Since the semen for artificial insemination can be used for artificial insemination within 12 hours after culturing without a process for separating X sperm from Y sperm, the method can be easily carried out even in livestock farming. EXAMPLES

[0026] As described below, semen for artificial insemination of pigs was prepared, and artificial insemination was performed by inseminating pigs with the semen, and the sex of the offspring born was verified.

[0027] (Preparation of medium) A modified HTF medium having the composition shown in Table 1 was prepared.

[0028] [Table 1]

[0029] (Preparation of semen for artificial insemination) Boar semen diluted to 5 billion / 100 mL with HIRO-SWINE B solution (manufactured by Hiroshima Cryopreservation Service Co., Ltd.; HIRO-SWINE is a registered trademark) 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, R848 added (3 nM)) as the supernatant, and cultured in an incubator (60 min, 37°C). After the culture, the mixture was centrifuged (500 G, 5 minutes, room temperature) to remove the supernatant, and then suspended in a diluent for artificial insemination to prepare semen for artificial insemination.

[0030] (artificial insemination) Sows were inseminated in two farms (Farm A and Farm B) in the following two ways. (A) Same-day insemination (artificial insemination within 12 hours after preparation) The above-mentioned semen for artificial insemination was prepared in the morning, and the first insemination was carried out in the afternoon of the same day, and the second insemination was carried out half a day after the first insemination.

[0031] (B) Insemination the next day (artificial insemination 12 hours after preparation) The above-mentioned semen for artificial insemination was prepared in the morning, the first insemination was carried out in the morning of the following day, and the second insemination was carried out half a day after the first insemination.

[0032] At Farm A, 3 pigs were inseminated on the same day and 7 pigs were inseminated the next day. At Farm B, 3 pigs were inseminated on the same day and 11 pigs were inseminated the next day.

[0033] The male ratio of piglets born is shown in Table 2.

[0034] [Table 2]

[0035] On Farm A, the percentage of male offspring was 57% for same-day breeding and 50% for next-day breeding, while on Farm B, the percentage of male offspring was 53% for same-day breeding and 41% for next-day breeding.

[0036] In both farms, the percentage of male offspring born in same-day insemination exceeded 50%. The percentage of male offspring born in same-day insemination was higher than that born the next day. As described above, it was proven that it is possible to increase the percentage of male offspring in artificial insemination of pigs. [Industrial Applicability]

[0037] This can increase the ratio of male offspring for multiparous animals such as pigs, and can be used in the livestock industry, etc.

Claims

1. Cultivating sperm from a multifetal animal in a medium containing a TLR7 ligand and having a pH of 7.7 to 7.9; After culturing, the animals are subjected to artificial insemination of multiple liveborn animals within 12 hours. A method for artificial insemination of multi-birth animals.

2. The medium containing albumin is used.

2. The method for artificial insemination of a multiparous animal according to claim 1.

3. The medium does not contain pyruvate or lactate.

2. The method for artificial insemination of a multiparous animal according to claim 1.

4. Incubate for more than 30 minutes but less than 90 minutes; 2. The method for artificial insemination of a multiparous animal according to claim 1.

5. The TLR7 ligand is one or more selected from the group consisting of resiquimod, imiquimod, gardikimod, and loxoribine.

2. The method for artificial insemination of a multiparous animal according to claim 1.

6. The multiparous animal is a pig.

6. The method for artificial insemination of a multiparous animal according to claim 1 .

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