Method for artificial insemination of ICR strain mice

Abstract

The invention discloses a method for artificial insemination of ICR strain mice, and belongs to the technical field of mouse reproduction, the artificial insemination AI technology is one of the most important reproduction means in the assisted reproduction technology ART, and the mouse AI plays an important role in experimental animal reproduction; rodents such as mice are used as conventional experimental mode organisms, and proper tools and methods provide reliable guarantee for feasibility and high efficiency of artificial insemination operation; according to the artificial insemination method for the ICR strain mice, assistance can be provided for experimental animals difficult to propagate and experimental problems needing artificial insemination operation, the insemination operation can be carried out on the experimental mice through a simple device, high operability and lower experimental cost without assistance of other special instruments and equipment, and the artificial insemination method for the ICR strain mice is high in practicability and high in practicability. And the fertilization rate of the in-vivo ova of the normal insemination female rats is equivalent to that of natural mating.

Description

Technical Field

[0001] The present invention belongs to the technical field of mouse breeding, and particularly relates to a method for artificial insemination of ICR strain mice. Background Art

[0002] Artificial Insemination (AI) technology is one of the most important reproductive means in Assisted Reproductive Technology (ART). Due to its relatively simple operation, less impact on recipient females, and lower cost, it has achieved great development in the livestock industry. AI is an assisted reproductive technology widely used in medium and large animals, but it is not common in small rodents such as mice and rats. Although in small rodents, the intraoviductal / intrauterine transfer of in vitro fertilized embryos is widely used, AI is a simple and potentially powerful tool for producing offspring.

[0003] In small rodents, there are various AI methods. The sperm insemination methods that require surgical procedures include insemination near the fimbria of the oviduct between the ovary and ovarian bursa, insemination in the ampulla of the oviduct, and uterine insemination, etc. surgical methods. In addition, there is also non-surgical artificial insemination by the transcervical method. Surgical AI can be performed with fewer sperm numbers than non-surgical AI and is effective for animals with difficult sperm collection (such as genetically modified mice), but requires advanced surgical techniques. On the other hand, the non-surgical AI (insemination by the transcervical method) requires relatively more sperm, but avoids the stress and physical damage caused by surgery, and has the advantages of simple operation and high feasibility. In addition, non-surgical AI enables repeated sperm insemination and offspring production in the same female animal. Therefore, non-surgical AI is not only effective for wild species and rare species with insufficient population numbers, but also of great significance for laboratory animals, meeting the 3R principles (replacement, reduction, and refinement).

[0004] In summary, non-surgical AI in mice plays an important role in the reproduction of experimental animals, and appropriate tools and methods are required during the sperm insemination process of rodents such as mice to ensure the feasibility and efficiency of the insemination operation. Summary of the Invention

[0005] Technical problems to be solved:

[0006] The present application aims at the deficiencies in the prior art, such as the difficulty in reproduction expansion and the need for advanced surgical techniques during the reproduction process of experimental animals, and provides a method for artificial insemination of ICR strain mice, which is simple and reasonable in design, simple in operation, convenient to use, low in cost, high in feasibility, and does not require auxiliary equipment of redundant instruments, improving the insemination efficiency.

[0007] Technical solutions:

[0008] To achieve the above object, the present application is implemented through the following technical solutions:

[0009] A method for artificial insemination of ICR strain mice, the specific steps are as follows:

[0010] First step, prepare the insemination tool: Connect an ophthalmic irrigation needle to the syringe barrel of a 1 mL syringe;

[0011] Second step, prepare a mouse vaginal speculum: The mouse vaginal speculum is prepared from a 200 μL pipette tip, and a length of 0.8 - 1 cm is intercepted from a distance of 1.3 - 1.7 cm from the tip outlet;

[0012] Third step, hormone treatment of female mice: Inject 10 IU of PMSG at 17:00 - 18:00 on the first day afternoon; Inject 10 IU of hCG at 17:00 - 18:00 on the third day afternoon;

[0013] Fourth step, prepare the mouse sperm in vitro capacitation solution HTF: Take 593.75 mg of NaCl, 34.96 mg of KCl, 4.93 mg of MgSO4·7H2O, 30 mg of CaCl2·2H2O, 50 mg of Glucose, 2398.9 mg of Sodium Lactate (60%), 3.65 mg of Sodium pyruvate, 5.04 mg of KH2PO4, 210 mg of NaHCO3, 400 mg of BSA according to the mass - volume ratio and dissolve them in 100 mL of sterile water, and adjust the pH to 7.2 - 7.4;

[0014] Fifth step: Place the mouse semen in the mouse sperm in vitro capacitation solution HTF at 6:00 in the morning on the fourth day and incubate at 37 °C for 1 hour;

[0015] Sixth step: Dip a cotton swab in physiological saline to moisten the vagina of the female mouse, help open the vagina of the female mouse through the mouse vaginal speculum, install the ophthalmic irrigation needle on the syringe barrel of a 1 mL syringe, and according to the insemination volume of 90 μL per female mouse, suck the incubated semen into the syringe, and deliver the semen to the uterus of the female mouse through the ophthalmic irrigation needle passing through the cervix.

[0016] Further, in the first step, the material of the ophthalmic irrigation needle is a metal material, and the needle tip is a straight tip.

[0017] Further, in the first step, the needle tip model of the ophthalmic irrigation needle is #5, and the needle tip length is 3 cm.

[0018] Further, in the second step, the mouse vaginal speculum is trumpet - shaped and can be inserted into the vagina of the mouse from the place with a smaller opening.

[0019] Furthermore, in the second step, the mouse vaginal opener needs to be heated on an alcohol lamp before use to passivate the thinner port to prevent scratching the mouse vagina.

[0020] Beneficial effects:

[0021] The present application provides a method for artificial insemination of ICR strain mice, which has the following beneficial effects compared with the prior art:

[0022] 1. The present invention provides a method for artificial insemination of ICR strain mice, which does not require the use of a fiber cold light source to find the cervical opening. Even a novice can quickly find the cervical opening under the guidance of a vaginal opener and easily perform artificial insemination;

[0023] 2. The ophthalmic flushing needle used in the present invention is made of metal, which can be repeatedly sterilized by high pressure, has a longer service life, and can also avoid the need to clear the cervix in advance when using a soft insemination tip to a certain extent;

[0024] 3. The artificial insemination method proposed in the present invention can more easily realize artificial insemination of mice through simple equipment and high operability without the assistance of other special instruments and equipment such as fiber cold light sources;

[0025] 4. The vaginal opener used in the present invention is a modified pipette tip, which has low cost, high feasibility, and can be reused;

[0026] 5. The non-surgical artificial insemination used in the present invention can make the two-cell development rate of fertilization in mice reach 92.58%, which is higher than the two-cell rate of 83.9% of surgical artificial insemination in mice reported in existing research. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagrams of the preparation of the insemination tool and the mouse vaginal vaginal opening device of the present application, wherein the left figure is a schematic diagram of the preparation of the insemination tool, and the right figure is a schematic diagram of the preparation of the mouse vaginal vaginal opening device;

[0028] Figure 2 This is a schematic diagram of the situation in which sperm enters the fallopian tube in Example 1 of the present application;

[0029] Figure 3 is a schematic diagram of the in vivo fertilized egg obtained in Example 2 of the present application;

[0030] Figure 4 This is a schematic diagram of the fertilized egg in the Lypd4 knockout mouse obtained in Example 3 of the present application. DETAILED DESCRIPTION

[0031] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but should not be used to limit the protection scope of the present invention.

[0032] Example 1

[0033] A method for artificial insemination of ICR strain mice, the specific steps are as follows:

[0034] First step, prepare the insemination tool: an ophthalmic irrigation needle is connected to the syringe barrel of a 1 mL syringe. The ophthalmic irrigation needle is made of metal, the needle tip is straight, the needle tip model is #5, and the needle tip length is 3 cm;

[0035] Second step, prepare the mouse vaginal speculum: The mouse vaginal speculum is prepared from a 200 μL pipette tip. Cut a length of 0.8 - 1 cm from 1.3 - 1.7 cm away from the tip outlet of the pipette tip. The mouse vaginal speculum is trumpet-shaped and can be inserted into the mouse vagina from the place with a smaller opening. The mouse vaginal speculum needs to be heated on an alcohol lamp before use to blunt the port of the thinner part to prevent scratching the mouse vagina;

[0036] Third step, hormone treatment of female mice: Inject 10 IU of PMSG at 17:00 - 18:00 on the first afternoon; Inject 10 IU of hCG at 17:00 - 18:00 on the third afternoon;

[0037] Fourth step, prepare the mouse sperm in vitro capacitation solution HTF: Take 593.75 mg of NaCl, 34.96 mg of KCl, 4.93 mg of MgSO4·7H2O, 30 mg of CaCl2·2H2O, 50 mg of Glucose, 2398.9 mg of Sodium Lactate (60%), 3.65 mg of Sodium pyruvate, 5.04 mg of KH2PO4, 210 mg of NaHCO3, 400 mg of BSA and dissolve them in 100 mL of sterile water, and adjust the PH to 7.2 - 7.4;

[0038] Fifth step: Place the mouse semen in the mouse sperm in vitro capacitation solution HTF at 6:00 in the morning on the fourth day and incubate at 37 °C for 1 hour;

[0039] Sixth step: Dip a cotton swab in physiological saline to moisten the vagina of the female mouse. Help open the vagina of the female mouse through the mouse vaginal speculum. Install the ophthalmic irrigation needle on the syringe barrel of a 1 mL syringe. According to the insemination volume of 90 μL / only for the female mouse, suck the incubated semen into the syringe, and deliver the semen to the uterus of the female mouse through the ophthalmic irrigation needle passing through the cervix.

[0040] Before the insemination operation, in order to obtain more estrous female mice suitable for insemination, the mice were first synchronized in estrus through hormone treatment. The specific operation plan is as follows: Inject 10 IU of PMSG at 17:00 - 18:00 on the first day afternoon; Inject 10 IU of hCG at 17:00 - 18:00 on the third day afternoon; Decapitate 2 male mice at 8:00 in the morning on the fourth day. Take a 1.5 mL centrifuge tube and add 0.5 mL of mouse sperm in vitro capacitation solution HTF which has been preheated at 37°C. Place the epididymal tails and vas deferens of the male mice in the 1.5 mL centrifuge tube and cut them several times, then incubate in a 37°C cell culture incubator for 1 h to capacitate the sperm. After staining the capacitated sperm with 2 μM Hoechst33342 for 5 min, perform insemination; Inject 80 - 100 μL of capacitated sperm into each female mouse. After 3 - 3.5 h of insemination, decapitate the female mouse and take the oviduct. Observe the movement of sperm in the oviduct under a fluorescence microscope.

[0041] Example 2

[0042] Select 6 ICR female mice at 6 - 8 weeks old. Synchronize the estrus of the mice through hormone treatment. The specific plan is as follows: Inject 10 IU of PMSG at 17:00 - 18:00 on the first day afternoon; Inject 10 IU of hCG at 17:00 - 18:00 on the third day afternoon; Decapitate 3 male mice at 6:00 in the morning on the fourth day. Take the epididymal tails and vas deferens and place them in a 1.5 mL EP tube, add 0.7 mL of mouse sperm in vitro capacitation solution HTF, then cut them several times in the capacitation solution, and incubate the sperm in a 37°C cell culture incubator for 1 h to capacitate the sperm. After the sperm capacitation is completed, anesthetize the female mouse, then lift the mouse's tail with one hand to fix it on the cage box with the tail upward and the head downward. Then moisten the self-made speculum with HTF and open the vagina of the mouse, and perform insemination with the capacitated sperm, 100 μL for each mouse. About 22 h after insemination, decapitate the female mouse and take the oviduct of the female mouse. Prick the ampulla of the oviduct with a 1 mL syringe needle under a microscope to release the fertilized eggs in the ampulla of the oviduct. Observe the fertilization of the eggs in the oviduct and find that through the insemination tool and method of the present invention, the eggs in the oviduct can be fertilized, and the fertilization rate is close to 100%.

[0043] Example 3

[0044] Male mice with Lypd4 knockout are infertile because their sperm cannot pass through the uterotubal junction to fertilize eggs during normal mating. To explore whether this method can solve this problem, the specific operations are as follows: Before sperm injection, to obtain more estrous female mice suitable for sperm injection, the mice were first synchronized in estrus by hormone treatment. The specific protocol is as follows: Inject 10 IU of PMSG at 17:00 - 18:00 on the first afternoon; Inject 10 IU of hCG at 17:00 - 18:00 on the third afternoon; At 6:00 in the morning on the fourth day, 2 5 - 6 - month - old C57 male mice with Lypd4 knockout were sacrificed by cervical dislocation. The cauda epididymis and vas deferens were placed in a 1.5 mL centrifuge tube, and 0.4 mL of pre - warmed HTF was added to the centrifuge tube in advance. Then, after cutting several times in the capacitation medium with scissors, the sperm were incubated in a cell culture incubator for 1 h to capacitate the sperm. After sperm capacitation, the female mice were anesthetized. Then, one hand lifted the mouse's tail to fix it in an inverted position on the cage box. The self - made speculum was moistened with HTF and then used to open the vagina of the mouse. The capacitated sperm were injected into the uterus of the mouse, 100 μL for each mouse. About 22 h after sperm injection, the female mice were sacrificed by cervical dislocation. The oviducts of the female mice were taken. Under a stereomicroscope, the ampulla of the oviduct was punctured with a 1 mL syringe needle to release the fertilized eggs in the ampulla of the oviduct, and the fertilization of the oviduct eggs was observed and counted. The results showed that the sperm of male mice with Lypd4 knockout could fertilize some of the eggs in the oviduct through the sperm injection tool and method of the present invention, and the fertilization rate could reach 40%. While the in - vivo fertilization rate of male mice with Lypd4 knockout and female mice during normal mating was 0%. The application of this method achieved a breakthrough from scratch and could effectively help the rapid propagation of Lypd4 knockout mice.

[0045] Table 1 shows the statistics of the two - cell development in mice after sperm injection into female mice by the method provided in this application.

[0046] Table 1

[0047]

[0048] The selected embodiments in the above materials are for easy understanding and do not limit the process method. People in the same technical field can easily modify the process flow or transfer it to other cases without creative changes. If these changes also belong to the same type of claims or the same type of technology of the present invention, then the intention of the present invention also includes these changes.

Claims

1. A method for artificial insemination of ICR strain mice, characterized in that, The specific steps are as follows: Step 1: Prepare the insemination tool: Connect an ophthalmic irrigation needle to the syringe barrel of a 1 mL syringe; Step 2: Prepare a mouse vaginal speculum: The mouse vaginal speculum is made from a 200 μL pipette tip. Cut a length of 0.8 - 1 cm from a point 1.3 - 1.7 cm from the tip outlet; Step 3: Hormone treatment for female mice: Inject 10 IU of PMSG at 17:00 - 18:00 in the afternoon on the first day; Inject 10 IU of hCG at 17:00 - 18:00 in the afternoon on the third day; Step 4: Prepare the mouse sperm in vitro capacitation solution HTF: Take 593.75 mg of NaCl, 34.96 mg of KCl, 4.93 mg of MgSO4·7H2O, 30 mg of CaCl2·2H2O, 50 mg of Glucose, 2398.9 mg of Sodium Lactate (60%), 3.65 mg of Sodium pyruvate, 5.04 mg of KH2PO4, 210 mg of NaHCO3, 400 mg of BSA and dissolve them in 100 mL of sterile water, and adjust the pH to 7.2 - 7.4; Step 5: At 6:00 in the morning on the fourth day, place the mouse semen in the mouse sperm in vitro capacitation solution HTF and incubate at 37 °C for 1 hour; Step 6: Moisten the female mouse's vagina with a cotton swab dipped in physiological saline. Use the mouse vaginal speculum to help open the female mouse's vagina. Install the ophthalmic irrigation needle on the syringe barrel of a 1 mL syringe. According to the insemination volume of 90 μL per female mouse, suck the incubated semen into the syringe and deliver the semen to the female mouse's uterus through the ophthalmic irrigation needle passing through the cervix.

2. The method for artificial insemination of ICR strain mice according to claim 1, characterized in that: In the first step, the ophthalmic irrigation needle is made of metal material and the needle tip is straight.

3. The method for artificial insemination of ICR strain mice according to claim 1, wherein: In the first step, the model number of the ophthalmic irrigation needle tip is #5 and the needle tip length is 3 cm.

4. The method for artificial insemination of ICR strain mice according to claim 1, characterized in that: In the second step, the mouse vaginal speculum is trumpet-shaped and can be inserted into the mouse vagina from the place with a smaller opening.

5. The method for artificial insemination of ICR strain mice according to claim 1, characterized in that: In the second step, before using the mouse vaginal speculum, it needs to be heated on an alcohol lamp to blunt the port of the thinner part to prevent scratching the mouse vagina.

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