阅读说明：本技术 一种小鼠体外受精试剂及其制备方法和体外受精方法 (Mouse in-vitro fertilization reagent, preparation method thereof and in-vitro fertilization method ) 是由 李斌 陈新 于 2019-11-12 设计创作，主要内容包括：本发明涉及小鼠培育技术领域,且公开了一种小鼠体外受精试剂及其制备方法和体外受精方法,包括NACL5.8～6.0g、KCL0.4～0.5g、KH2PO4 0.038～0.045g、MgSO4.7H2O 0.088～0.095g、丙酮酸钠0.03～0.04g、葡萄糖0.55～0.6g、NaCO3 2.1～2.15g、乳酸钠4.19～4.641g、CaCL2.2H2O 0.3～0.35g、BSA4～5g、牛磺酸0.30～0.312g和双抗(100x)5.4～5.6ML,还包括GlutaMAX和EDTA(5%)。该小鼠体外受精试剂及其制备方法和体外受精方法,结合现有的受精试剂的基料配方,进行改进,能使小鼠的受精效率大大提高,具有很好的应用前景,解决了现有的受精试剂在应用于小鼠测验时,国内的试剂受精效率较低,会耗费大量相关试剂,而国外试剂价格较为昂贵,并不适用于对小鼠的大量培育测验的问题。(The invention relates to the technical field of mouse cultivation, and discloses a mouse in-vitro fertilization reagent, a preparation method thereof and an in-vitro fertilization method, wherein the reagent comprises 5.8-6.0 g of NACL, 0.4-0.5 g of KCL, KH2PO40.038-0.045 g of MgSO4.7H2O, 0.088-0.095 g of sodium pyruvate, 0.03-0.04 g of glucose, 0.55-0.6 g of NaCO32.1-2.15 g of sodium lactate, 4.19-4.641 g of CaCL2.2H2O, 0.3-0.35 g of BSA 4-5 g of taurine, 0.30-0.312 g of double antibody (100 x) and 5.4-5.6 ML of double antibody, and further comprises GlutaMAX and EDTA (5%). The mouse in-vitro fertilization reagent, the preparation method thereof and the in-vitro fertilization method are combined with the existing base material formula of the fertilization reagent, are improved, can greatly improve the fertilization efficiency of mice, have good application prospects, and solve the problems that when the existing fertilization reagent is applied to mouse tests, the fertilization efficiency of the domestic reagent is low, a large amount of related reagents are consumed, the price of foreign reagents is high, and the existing fertilization reagent is not suitable for a large amount of breeding tests of the mice.)

1. A mouse in-vitro fertilization reagent is characterized by comprising 5.8-6.0 g of NACL, 0.4-0.5 g of KCL, KH2PO40.038-0.045 g, 0.088-0.095 g of MgSO4.7H2O, 0.03-0.04 g of sodium pyruvate, 0.55-0.6 g of glucose, 0.1-2.15 g of NaCO32, 4.19-4.641 g of sodium lactate, 0.3-0.35 g of CaCL2.2H2O, 26-5 g of BSA4, 0.30-0.312 g of taurine and 5.4-5.6 ML of diabase (100 x).

2. The reagent for mouse in vitro fertilization according to claim 1, further comprising GlutaMAX and EDTA (5%).

3. A method for preparing a mouse in vitro fertilization reagent, wherein the fertilization reagent of claim 1 or 2 is used, comprising the steps of:

1) taking out a glass dish with the volume of 1L, weighing 800ML hydrogen peroxide, putting the hydrogen peroxide into the 1L glass dish, sequentially weighing 5.8-6.0 g of NACL, 0.4-0.5 g of KCL, KH2PO40.038-0.045 g of MgSO4.7H2O, 0.088-0.095 g of sodium pyruvate, 0.03-0.04 g of sodium pyruvate, 0.55-0.6 g of glucose, 0.1-2.15 g of NaCO32, 4.19-4.641 g of sodium lactate, 0.3-0.35 g of CaCL2.2H2O, 4-5 g of BSA and 0.30-0.312 g of taurine, and putting the glass dish into the glass dish for dissolving;

2) and then weighing 5.4-5.6 ML (100 x) of double antibody, GlutaMAX and EDTA (5%) and sequentially putting the double antibody, the GlutaMAX and the EDTA into a glass dish for dissolving, after dissolving for 3 hours, filtering by using a 0.22um filter, putting the filtered solution into a new glass dish to obtain the HTF fertilization reagent, and putting the reagent into a temperature of-20 ℃ for storage.

4. A method for in vitro fertilization of a mouse using the fertilization reagent of claim 3, comprising the steps of:

1) taking a plurality of culture dishes, respectively dripping 1 piece of HTF reagent solution with the volume of 0.2ml and 2 pieces of HTF reagent solution with the volume of 0.1ml into the sperm culture dish and the fertilization culture dish, then covering the sperm culture dish and the fertilization culture dish by mineral oil or paraffin oil, and placing the culture dishes into an incubator for culture at the temperature of 37 ℃, under the conditions of 5% CO2 and 95% humidity;

2) placing the sperm of the mouse into a balanced sperm culture dish for culture capacitation;

3) putting the ovum of the mouse into the balanced fertilization culture dish, and then taking the sperm which can be obtained in the step (2) by using a micropipettor and putting the sperm into the fertilization culture dish;

4) the fertilization dishes were incubated in an incubator containing 5% CO2 at 37 ℃.

5. The method for in vitro fertilization according to claim 4, wherein the sperm capacitation culture dish is placed in an incubator containing 5% CO2 at 37 ℃ for a sperm capacitation time of 60 minutes.

6. The method of in vitro fertilization of a mouse according to claim 4, wherein the method of ova retrieval comprises the steps of:

1) the mice are superexcreted by injecting PMSG and HCG according to the dose of 5IU per mouse, and the interval time is twenty-four hours;

2) the superovulated donor mother mouse is sacrificed about 15-17 hours after the injection of HCG, the oviduct is fixed with fine forceps and the expanded portion of the oviduct is punctured with a dissecting needle, bringing out the oocyte pellet to a fertilization culture dish.

Technical Field

The invention relates to the technical field of biology, in particular to a mouse in-vitro fertilization reagent, a preparation method thereof and an in-vitro fertilization method.

Background

In vitro fertilization refers to a technique in which sperm and ovum of a mammal complete a fertilization process in an environment artificially controlled in vitro, and is abbreviated as IVF in English, and since it is inseparable from an embryo transfer technique (ET), and is abbreviated as IVF-ET, in biology, an animal obtained after an in vitro fertilized embryo is transferred to a mother body is called a tube animal.

At present, both domestic and overseas relate to relevant reagents for mouse in-vitro fertilization, and the reagents have effects on mouse fertilization, but when the domestic fertilization reagents are applied to mice, the fertilization efficiency is low, a large amount of relevant reagents are consumed, and the reagents for mouse fertilization selected from the foreign reagents are expensive in price and high in cost, so that the reagents are not suitable for a large amount of tests on the mice.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides a mouse in-vitro fertilization reagent, a preparation method thereof and an in-vitro fertilization method, which have the advantages of low cost, good fertilization effect and the like, and solve the problems that when the existing fertilization reagent is applied to mouse tests, the fertilization efficiency of the domestic reagent is low, a large amount of related reagents are consumed, the price of foreign reagents is high, and the existing fertilization reagent is not suitable for a large amount of breeding tests of mice.

(II) technical scheme

In order to achieve the purposes of low cost and good fertilization effect, the invention provides the following technical scheme: a mouse in-vitro fertilization reagent comprises 5.8-6.0 g of NACL, 0.4-0.5 g of KCL, KH2PO40.038-0.045 g of MgSO4.7H2O, 0.088-0.095 g of sodium pyruvate, 0.03-0.04 g of glucose, 0.55-0.6 g of NaCO32.1-2.15 g of sodium lactate, 4.19-4.641 g of CaCL2.2H2O, 0.3-0.35 g of BSA 4-5 g of taurine, 0.30-0.312 g of bis-antibiotics (100 x) and 5.4-5.6 ML.

Preferably, GlutaMAX and EDTA (5%) are also included.

Another technical problem to be solved by the present invention is to provide a method for preparing a mouse in vitro fertilization reagent, comprising the following steps:

1) taking out a glass dish with the volume of 1L, weighing 800ML hydrogen peroxide, putting the hydrogen peroxide into the 1L glass dish, sequentially weighing 5.8-6.0 g of NACL, 0.4-0.5 g of KCL, KH2PO40.038-0.045 g of MgSO4.7H2O, 0.088-0.095 g of sodium pyruvate, 0.03-0.04 g of sodium pyruvate, 0.55-0.6 g of glucose, 0.1-2.15 g of NaCO32, 4.19-4.641 g of sodium lactate, 0.3-0.35 g of CaCL2.2H2O, 4-5 g of BSA and 0.30-0.312 g of taurine, and putting the glass dish into the glass dish for dissolving;

2) and then weighing 5.4-5.6 ML (100 x) of double antibody, GlutaMAX and EDTA (5%) and sequentially putting the double antibody, the GlutaMAX and the EDTA into a glass dish for dissolving, after dissolving for 3 hours, filtering by using a 0.22um filter, putting the filtered solution into a new glass dish to obtain the HTF fertilization reagent, and putting the reagent into a temperature of-20 ℃ for storage.

Another technical problem to be solved by the present invention is to provide a method for in vitro fertilization of a mouse, comprising the following steps:

1) taking a plurality of culture dishes, respectively dripping 1 piece of HTF reagent solution with the volume of 0.2ml and 2 pieces of HTF reagent solution with the volume of 0.1ml into the sperm culture dish and the fertilization culture dish, then covering the sperm culture dish and the fertilization culture dish by mineral oil or paraffin oil, and placing the culture dishes into an incubator for culture at the temperature of 37 ℃, under the conditions of 5% CO2 and 95% humidity;

2) placing the sperm of the mouse into a balanced sperm culture dish for culture capacitation;

3) putting the ovum of the mouse into the balanced fertilization culture dish, and then taking the sperm which can be obtained in the step (2) by using a micropipettor and putting the sperm into the fertilization culture dish;

4) the fertilization dishes were incubated in an incubator containing 5% CO2 at 37 ℃.

Preferably, the sperm capacitation culture dish is placed in an incubator containing 5% CO2 at 37 ℃ for 60 minutes.

Preferably, the method for taking the ovum comprises the following steps:

1) the mice are superexcreted by injecting PMSG and HCG according to the dose of 5IU per mouse, and the interval time is twenty-four hours;

2) the superovulated donor mother mouse is sacrificed about 15-17 hours after the injection of HCG, the oviduct is fixed with fine forceps and the expanded portion of the oviduct is punctured with a dissecting needle, bringing out the oocyte pellet to a fertilization culture dish.

(III) advantageous effects

Compared with the prior art, the invention provides a mouse in-vitro fertilization reagent and a preparation method thereof, and the mouse in-vitro fertilization reagent has the following beneficial effects:

according to the mouse in-vitro fertilization reagent, the preparation method and the in-vitro fertilization method, when the mouse fertilization reagent is prepared, besides the mouse fertilization reagent raw materials are basically prepared, the EDTA and the GlutaMAX are added, so that the stability of the reagent can be enhanced when the fertilization reagent is prepared, the fertilization efficiency of the fertilization reagent can be enhanced, the success rate of the fertilization reagent used for cultivating mice is higher by combining the operation of the mouse in-vitro fertilization method, the base material cost of the fertilization reagent is not high, and the reagent can be suitable for being used for a large number of tests of the mice.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

A mouse in-vitro fertilization reagent is characterized by comprising 5.8-6.0 g of NACL, 0.4-0.5 g of KCL, KH2PO40.038-0.045 g, 0.088-0.095 g of MgSO4.7H2O, 0.03-0.04 g of sodium pyruvate, 0.55-0.6 g of glucose, 0.1-2.15 g of NaCO32, 4.19-4.641 g of sodium lactate, 0.3-0.35 g of CaCL2.2H2O, 4-5 g of BSA, 0.30-0.312 g of taurine, 5.4-5.6 g of diabase (100 x), GlutaMAX and EDTA (5%).

A method for preparing a mouse in vitro fertilization reagent, wherein the fertilization reagent of claim 1 or 2 is used, comprising the steps of:

1) taking out a glass dish with the volume of 1L, weighing 800ML hydrogen peroxide, putting the hydrogen peroxide into the 1L glass dish, sequentially weighing 5.8-6.0 g of NACL, 0.4-0.5 g of KCL, KH2PO40.038-0.045 g of MgSO4.7H2O, 0.088-0.095 g of sodium pyruvate, 0.03-0.04 g of sodium pyruvate, 0.55-0.6 g of glucose, 0.1-2.15 g of NaCO32, 4.19-4.641 g of sodium lactate, 0.3-0.35 g of CaCL2.2H2O, 4-5 g of BSA and 0.30-0.312 g of taurine, and putting the glass dish into the glass dish for dissolving;

2) and then weighing 5.4-5.6 ML of double antibody (100 x), 3.0-3.2 ML of GlutaMAX and 0.15-0.25ML of EDTA (5%) in turn, putting the materials into a glass dish for dissolving, filtering by using a 0.22um filter after dissolving for 3 hours, putting the filtered solution into a new glass dish to obtain the HTF fertilization reagent, and putting the reagent at the temperature of minus 20 ℃ for storage.

A method for in vitro fertilization of a mouse using the fertilization reagent of claim 3, comprising the steps of:

1) taking a plurality of mice, injecting PMSG according to 5IU dose per mouse, injecting HCG with 5IU per mouse on the first day of 17:00, on the third day of 17:00, and starting to take eggs on the fourth day of 9: 00;

2) taking a plurality of culture dishes, respectively dripping 1 piece of HTF reagent solution with the volume of 0.2ml and 2 pieces of HTF reagent solution with the volume of 0.1ml into the sperm culture dish and the fertilization culture dish, then covering the sperm culture dish and the fertilization culture dish by mineral oil or paraffin oil, and placing the culture dishes into an incubator for culture, wherein the temperature is 37 ℃, and the culture dish contains 5% CO2 and 95% humidity;

3) taking 2-3 mature male mice (3-6 months old), taking the tail of the epididymis after sacrifice, and removing fat, blood, interstitial fluid and the like around the male mice as far as possible; placing the tail part of the epididymis on sterile filter paper, removing blood and tissue fluid of the epididymis, moving the tail part of the epididymis into a sperm culture dish, clamping the tail part of the epididymis by using fine forceps, then taking a dissecting needle, alternately extruding forwards to extrude a sperm cluster at the tail part of the epididymis, introducing the extruded sperm cluster into an overnight HTF culture solution by using the dissecting needle, and placing the culture dish into a culture box with 37 ℃ and 5% CO2 to culture for 60 minutes to obtain energy;

4) killing donor mice subjected to superovulation treatment about 15-17 hours after HCG injection, opening the abdominal cavity, taking out the oviduct, reducing blood, tissue fluid, fat and the like as much as possible, placing the oviduct on a sterilization filter paper, wiping off blood stains, tissue fluid and the like, placing the oviduct in a fertilization culture dish, fixing the oviduct by using a fine forceps, piercing the expansion part of the oviduct by using a dissecting needle, carrying out oocyte group to HTF culture solution, and then taking sperm subjected to energy harvesting in the step (2) by using a micropipettor and placing the sperm in the fertilization culture dish;

5) culturing the fertilization culture dish in an incubator containing 5% CO2 at 37 deg.C, fertilizing for 5-6 hr, washing with fresh M2 culture solution for 3 times, and observing embryo;

6) after 18-22 hours at 37 ℃ in an incubator containing 5% CO2, the split eggs are counted and may be transplanted or embryos frozen.