Mouse sperm freezing protecting agent
A cryoprotectant and mouse technology, applied in the field of bioengineering, can solve the problem of ineffective cryoprotection of C57BL/6J and achieve the effect of improving the fertilization rate
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Embodiment 1
[0027] Example 1 , Preparation of cryoprotectant
[0028] 1.1. Configuration of R18S3 cryoprotectant: Weigh 3g of skimmed milk (Becton, Kickinson and Company), put it into 90ml of three-distilled water, and dissolve it in a water bath at 60°C for 2 hours. After the milk powder particles are completely dissolved, weigh 18g of Raffinose (produced by Sigma Company) was put into it, and triple distilled water was added to 100ml, and continued to dissolve in a 60°C water bath until the raffinose particles were completely dissolved. The suspension was centrifuged under the condition of 15000g / min for 60 minutes, the supernatant was taken out, and put into a clean glass bottle for subsequent use, the cryoprotectant prepared in this way contained 2.4% skimmed milk and 14.4% raffinose (volume ratio) .
[0029] 1.2. Preparation of SPF egg yolk: Carry out the following operations in an ultra-clean workbench: take an SPF egg, carefully break the eggshell at the end of the egg with air c...
Embodiment 2
[0031] Example 2 , the use of cryoprotectants
[0032] 2.1. Take adult male mice of each strain listed in Table 1-Table 8, kill them by cervical dislocation, disinfect the abdomen with 75% alcohol, cut the skin and muscles, carefully pull out the testes, expose the epididymis, and use ophthalmic scissors Separate tissues such as fat, blood vessels, and ligaments, cut off the epididymis, clean the blood, body fluids, and fat on sterilized filter paper, put them into 120 μl of each cryoprotectant prepared in step 1.3, cut the epididymis with scissors, and wait for the sperm After the natural outflow, the tissue can be removed, the sperm and the cryoprotectant are gently mixed, and then put into a 0.25ml straw (purchased from IMV Company, France).
[0033] Among them, the method of loading the sperm suspension into the straw is as follows:
[0034] First, use a syringe to inhale about 100 μl of HTF culture solution; then inhale 10 mm of air; then inhale about 10 μl of sperm su...
Embodiment 3
[0043] Example 3 , In vitro fertilization with frozen-thawed sperm
[0044] 3.1. Collection of oocytes
[0045] 1) About 15-17 hours after the injection of hCG (human chorionic gonadotropin, a conventional sex hormone, produced by Ningbo Second Hormone Factory), the donor female mice (8-12 weeks old) who had undergone superovulation treatment were killed;
[0046] 2) After wiping the abdomen of the mouse with alcohol cotton, open the abdominal cavity of the mouse, and use tweezers to move the internal organs of the abdominal cavity to expose the uterus, fallopian tubes and ovaries, etc., take out the fallopian tubes, and reduce blood, interstitial fluid and fat as much as possible;
[0047] 3) Put the fallopian tube on the sterilized filter paper, wipe off the blood and interstitial fluid, etc.;
[0048] 4) place the oviduct in mineral oil in a Petri dish containing the droplets of fertilization solution HTF;
[0049] 5) Fix the fallopian tube with a pair of fine forceps, ...
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