CN105861424B - A kind of rats in vitro fertilization nutrient solution and its application - Google Patents
A kind of rats in vitro fertilization nutrient solution and its application Download PDFInfo
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Abstract
The invention discloses a kind of rats in vitro fertilization nutrient solution RTF (Rat Tube Fluid) and its applications in culture rat fertilized eggs.Special role of the present invention according to sodium ion and calcium ion in animal sperm ovum binding proposes a kind of rats in vitro fertilization nutrient solution in conjunction with the specific demand of rat sperm ovum binding.Rats in vitro fertilization nutrient solution of the invention carries out the human assistance reproduction of rat, and can be realized realizes microbiological purification while saving lines progenies again.
Description
Technical field
The invention belongs to Experiment of Zoology zoology general fields, and in particular to rat spermatozoa and ovum nutrient solution in vitro fertilization
(RTF) it and its applies.
Background technique
Rat is very important experimental animal, and usage amount is only second to mouse, but due to the particularity of itself, rat
It is in vitro fertilization relatively difficult always, for biological reagent in the market also without the dedicated liquid in vitro fertilization of rat, people are commonly external
The liquid in vitro fertilization such as IVF-20 or IVF-30 of people are generally by sperm, due to the otherness of people and rat, user's is external
By sperm carry out rat it is in vitro fertilization, often rate of fertilization and developmental rate are lower, can not obtain sufficient amount of rat embryo into
Row transfer.The liquid in vitro fertilization of people is formulated on the basis of human tubal fluid (HTF) ingredient, composition proportion see
Table 1.
Table 1HTF culture solution ingredient (Human Tube Fluid)
| Ingredient | Concentration (mM/l | Concentration (grams per liter |
| Sodium chloride | 101.6 | |
| Potassium chloride | 4.69 | |
| Potassium dihydrogen phosphate | 0.37 | |
| Magnesium sulfate | 0.2 | |
| Sodium lactate | 21.4 | |
| Sodium Pyruvate | 0.33 | |
| Glucose | 2.78 | |
| Sodium bicarbonate | 25.0 | |
| Calcium chloride | 2.04 | |
| Bovine serum albumin(BSA) | 4.0 |
At present because the RTF of market supply, many laboratories cannot be rapidly not numerous after the preparation of big mouse disease model
Grow and carry out scientific research in groups, once reproductive organs obstacle occurs for transgenosis or knockout rat, can not using in vitro by
The mode of essence retains offspring, can only be allowed to extinction, germ plasm resource can not save.When the rat of rare strain faces dead or life
When growing organ defect, people often sue and labour to it, it is desirable in the case where saving its life, restore its reproductive function, so
It carries out caesarean birth afterwards or embryo transfer carries out saving the strain, but sue and labour in the irreclaimable situation of unsuccessful or reproductive function,
Can only leave strain model rat loss.
Summary of the invention
In order to overcome the above-mentioned defects in the prior art, the invention proposes a kind of rats in vitro fertilization nutrient solution RTF
(Rat Tube Fluid) and its application in culture rat fertilized eggs.The present invention is according to sodium ion and calcium ion in animal essence
Special role in ovum combination proposes a kind of rats in vitro fertilization nutrient solution in conjunction with the specific demand of rat sperm ovum binding.
It include: sodium chloride 101.6mmol/L, potassium chloride 4.69 the present invention provides a kind of rats in vitro fertilization nutrient solution
Mmol/L, potassium dihydrogen phosphate 0.37mmol/L, magnesium sulfate 0.2mmol/L, sodium lactate 15-19.5mmol/L, Sodium Pyruvate
0.335-0.345mmol/L, glucose 2.78mmol/L, sodium bicarbonate 25.0mmol/L, calcium chloride 3.0-6.0mmol/L, ox
Seralbumin 4.0g/L.Solvent is distilled water.
Preferably, rats in vitro of the invention fertilization nutrient solution includes: sodium chloride 101.6mmol/L, potassium chloride 4.69
Mmol/L, potassium dihydrogen phosphate 0.37mmol/L, magnesium sulfate 0.2mmol/L, sodium lactate 17-19mmol/L, Sodium Pyruvate 0.34-
0.345mmol/L, glucose 2.78mmol/L, sodium bicarbonate 25.0mmol/L, calcium chloride 4.0-6.0mmol/L, ox blood are pure
Albumen 4.0g/L.Solvent is distilled water.
It is further preferred that rats in vitro fertilization nutrient solution of the invention includes: sodium chloride 101.6mmol/L, potassium chloride
4.69 mmol/L, potassium dihydrogen phosphate 0.37mmol/L, magnesium sulfate 0.2mmol/L, sodium lactate 18mmol/L, Sodium Pyruvate 0.34
Mmol/L, glucose 2.78mmol/L, sodium bicarbonate 25.0mmol/L, calcium chloride 5.0mmol/L, 4.0 g/ of bovine serum albumin(BSA)
L.Solvent is distilled water.
The invention also provides the applications for the culture that rats in vitro fertilization nutrient solution is used for rats in vitro fertilized eggs.
The beneficial effects of the present invention are: due to important function of the rat in human disease model's preparation, the people of rat
The human disease model population that work assisted reproductive technology becomes rat expands numerous and Germ-plasma resources protection bottleneck, and in vitro fertilization
The break-through point of human assistance reproduction, the application of rats in vitro fertilization nutrient solution can make rat it is in vitro fertilization be able to it is suitable
Benefit carries out, in the case where natural mating can not obtain rat resource or natural propagation group expanding speed is too low, so that it may utilize body
Outer fertilization technique completes the combination of sperm and ovum in RTF culture solution, sterilized rat embryo is obtained, after embryo transfer
Establish SPF progeny population.Break through the bottleneck of male mouse, female mice natural mating.It is provided safeguard for rat Germ-plasma resources protection.
Rats in vitro fertilization nutrient solution of the invention carries out the human assistance reproduction of rat, by using external
Just dead male rat is dissected, takes its epididymis and fallopian tubal, discharge sperm therein, after treatment by fertilization technique
Carry out it is in vitro fertilization after, then carry out embryo transfer, the final offspring for saving the strain realizes what microbiology purified again
Purpose.
Specific embodiment
In conjunction with following specific embodiments, the present invention is described in further detail.Implement process of the invention, condition,
Experimental method etc. is among the general principles and common general knowledge in the art in addition to what is specifically mentioned below, and the present invention does not have
There is especially limitation content.
It is basic liquid with distilled water, prepares rats in vitro fertilization nutrient solution, comprising: sodium chloride 101.6mmol/L, potassium chloride
4.69mmol/L, potassium dihydrogen phosphate 0.37mmol/L, magnesium sulfate 0.2mmol/L, sodium lactate 15-19.5mmol/L, Sodium Pyruvate
0.335-0.345mmol/L, glucose 2.78mmol/L, sodium bicarbonate 25.0mmol/L, calcium chloride 3.0-6.0mmol/L, ox
Seralbumin 4.0g/L.Solvent is distilled water, after mixing well, is filtered with 0.22 micron of virus filters, 0.5 milliliter of packing
It is spare afterwards, it is labeled as RTF.
Using: (1) acquisition of sperm: adult 12-14 week old male SD rat cervical dislocation is put to death, sterile taking-up epididymis
Tail cuts 3-4 aperture with eye scissors on cauda epididymidis, immerses in 37 DEG C of liquid in vitro fertilization of 200ul-400ul preheating, uses
Mineral oil covering, is placed in 37 DEG C, in the incubator of carbon dioxide 5%, swims out of from cauda epididymidis to sperm spare.By what is swum out of
Sperm is transferred in 37 DEG C of liquid in vitro fertilization of 100ul preheating, and adjustment quantity is 106-7It is covered with mineral oil, is placed in 37 DEG C, two
In the incubator of carbonoxide 5%, place 1-1.5 hours.This is capacitation process.
(2) preparation of ovum: taking SPF grade female mice of the 5-6 Zhou Yuxiong mouse with strain, tests preceding 3 days PMSG Injections (pregnant mother
Serum hormone) 30-50IU, after 48 hours HCG injection (human chorionic gonadotrophin) after 25-40IU, 14-16 hours, cervical vertebra
Dislocation is put to death, and abdominal cavity is opened, and separates fallopian tubal, and ovum group is chosen 37 DEG C of nutrient solutions in vitro fertilization into 100ul-200ul preheating
In, and wash 2-3 times, it is covered with mineral oil.37 DEG C are placed in, in the incubator of carbon dioxide 5%.
(3) fertilization process: in the ovum group for taking the sperm liquid 10ul of step (1) capacitation to be placed in (2), 37 DEG C are placed in, dioxy
Change and continue to cultivate in the incubator of carbon 5%, is fertilized.
(4) fertilized eggs are selected: after 6-7 hour, fertilization drop is placed under microscope and is selected, select dispersion ovum,
The 100ul for moving into mineral oil covering is preheated in 37 DEG C of RM1ECM culture solution, after continuing culture 20-22 hours, selects development
To 2-4 cell stage embryo, and calculate fertilization developmental rate.
Examples 1 to 4 configures different component rats in vitro and is fertilized nutrient solution, and it is spare to be respectively labeled as A, B, C, D, is shown in Table 1
It is shown,
The RTF culture solution ingredient of 1 Examples 1 to 4 of table
(1) acquisition of sperm: adult 12-14 week old male SD rat cervical dislocation is put to death, sterile taking-up cauda epididymidis, attached
3-4 aperture is cut with eye scissors on testis tail, immerses in 37 DEG C of liquid in vitro fertilization of 200ul-400ul preheating, is covered with mineral oil
Lid, is placed in 37 DEG C, in the incubator of carbon dioxide 5%, swims out of from cauda epididymidis to sperm spare.The sperm swum out of is shifted
In the 37 DEG C of liquid in vitro fertilization preheated to 100ul, adjustment quantity is 106-7It is covered with mineral oil, is placed in 37 DEG C, carbon dioxide
In 5% incubator, place 1-1.5 hours.This is capacitation process.
(2) preparation of ovum: taking SPF grade female mice of the 5-6 Zhou Yuxiong mouse with strain, and female mice is divided into 5 groups, and every group 5,
Test preceding 3 days PMSG Injections (pregnant mother's serum hormone) 30-50IU, HCG injection (human chorionic gonadotrophin) after 48 hours
After 25-40IU, 14-16 hours, cervical dislocation is put to death, and opens abdominal cavity, is separated fallopian tubal, ovum group is chosen pre- into 100ul-200ul
In 37 DEG C of liquid in vitro fertilization of heat, for control group using HTF by sperm, experimental group uses A, B, C, D by sperm respectively, and with respectively
From by semen washing 2-3 times, covered with mineral oil.37 DEG C are placed in, in the incubator of carbon dioxide 5%.
(3) it fertilization process: takes the sperm liquid 10ul of step (1) capacitation to be placed in the ovum group being respectively placed in (2), is placed in
37 DEG C, continues to cultivate in the incubator of carbon dioxide 5%, be fertilized.
(4) fertilized eggs are selected: after 6-7 hour, fertilization drop is placed under microscope and is selected, select dispersion ovum,
The 100ul for moving into mineral oil covering respectively is preheated in 37 DEG C of RM1ECM culture solution, continues to cultivate.
(6) it rate of fertilization: after 20-22 hours, selects and is developed to 2-4 cell stage embryo and calculates rate of fertilization.
Experimental result is see table 2
Fertilization situation of the 2. Examples 1 to 4 different component rats in vitro of table by sperm
Unit: %
The liquid IVF-30. in vitro fertilization (Sweden's Vitrolife product, article No. 10086) of control group user
| Control group | A group (embodiment 1) | B group (embodiment 2) | C group (embodiment 3) | D group (embodiment 4) |
| 2.5 | 4.2 | 23.7 | 86.5 | 24.4 |
It can be seen from the above result that the liquid in vitro fertilization of people is in vitro fertilization for rat progress, rate of fertilization is extremely low, tends not to
Sufficient amount of embryo is obtained for embryo transfer, is not suitable as the liquid in vitro fertilization of rat, rat fertilization process in vitro
In, fertilization process to by sperm sodium ion and calcium ion concentration it is sensitive, the variation of such small concentrations will bring rate of fertilization
Significant changes, for the concentration of calcium chloride in 3.0-6.0mmol, especially 4.0-6.0 can obtain effect of being preferably fertilized；?
By in the sodium salt component in sperm, the extracorporeal fertilization process of rat is particularly sensitive to the variation of organic acid sodium, the concentration of sodium lactate
Effect of being fertilized in 15-19.5mmol, especially 17-19mmol is preferable；The concentration of Sodium Pyruvate in 0.335-0.345mmol,
Effect of being preferably fertilized can be obtained when especially 0.34-0.345mmol.In above several embodiments, in embodiment 3, chlorine
When the matched proportion density for changing calcium, sodium lactate and Sodium Pyruvate is 5.0mmol, 18mmol and 0.34mmol, fertilization effect is best.
Protection content of the invention is not limited to above embodiments.Without departing from the spirit and scope of the invention, originally
Field technical staff it is conceivable that variation and advantage be all included in the present invention, and be with appended claims
Protection scope.
Claims (5)
- The nutrient solution 1. a kind of rats in vitro is fertilized, which is characterized in that the nutrient solution is by sodium chloride 101.6mmol/L, potassium chloride 4.69mmol/L, potassium dihydrogen phosphate 0.37mmol/L, magnesium sulfate 0.2mmol/L, sodium lactate 15-19mmol/L, Sodium Pyruvate 0.335-0.345mmol/L, glucose 2.78mmol/L, sodium bicarbonate 25.0mmol/L, calcium chloride 3.0-6.0mmol/L, ox Seralbumin 4.0g/L composition, solvent is distilled water.
- The nutrient solution 2. rats in vitro as described in claim 1 is fertilized, which is characterized in that the nutrient solution is by sodium chloride 101.6mmol/L, potassium chloride 4.69mmol/L, potassium dihydrogen phosphate 0.37mmol/L, magnesium sulfate 0.2mmol/L, sodium lactate 17- 19mmol/L, Sodium Pyruvate 0.34-0.345mmol/L, glucose 2.78mmol/L, sodium bicarbonate 25.0mmol/L, calcium chloride 4.0-6.0mmol/L, bovine serum albumin(BSA) 4.0g/L composition, solvent is distilled water.
- The nutrient solution 3. rats in vitro as claimed in claim 2 is fertilized, which is characterized in that the nutrient solution is by sodium chloride 101.6mmol/L, potassium chloride 4.69mmol/L, potassium dihydrogen phosphate 0.37mmol/L, magnesium sulfate 0.2mmol/L, sodium lactate 18mmol/L, Sodium Pyruvate 0.34mmol/L, glucose 2.78mmol/L, sodium bicarbonate 25.0mmol/L, calcium chloride 5.0mmol/L, bovine serum albumin(BSA) 4.0g/L composition, solvent is distilled water.
- 4. the people that the fertilization nutrient solution preparation of rats in vitro described in any one claim by claims 1 to 3 is used for rat The application of work supplementary reproduction reagent.
- 5. the fertilization nutrient solution preparation of rats in vitro described in any one claim by claims 1 to 3 is used for just dead Rat human assistance reproduction reagent application.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898048A (en) * | 2014-03-27 | 2014-07-02 | 安徽农业大学 | In vitro maturation culture method of denuded oocytes of mice |
| CN104164400A (en) * | 2014-08-12 | 2014-11-26 | 沈阳洁瑞生物技术有限公司 | In-vitro fertilization liquid and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898048A (en) * | 2014-03-27 | 2014-07-02 | 安徽农业大学 | In vitro maturation culture method of denuded oocytes of mice |
| CN104164400A (en) * | 2014-08-12 | 2014-11-26 | 沈阳洁瑞生物技术有限公司 | In-vitro fertilization liquid and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| "A protocol for rat in vitro fertilization during conventional";Toshihiro Aoto et al.,;《Transgenic Research》;20111231;第20卷(第6期);第1245-1252页 |
| "钙离子浓度对大鼠精子自发性顶体反应的影响";曾海涛等;《医学临床研究》;20030831;第20卷(第8期);第381页摘要部分 |
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