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Sperm freezing method and freezing liquid thereof

A freezing method and freezing liquid technology, applied in the field of sperm freezing method and freezing liquid, can solve the problems of frozen sperm survival and fertilization without significant improvement, achieve high fertilization ability, embryonic development rate, and simple operation

Inactive Publication Date: 2012-05-16
陈浩杰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, despite the increasing research on sperm freezing, the survival and fertilization of frozen sperm have not been significantly improved

Method used

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  • Sperm freezing method and freezing liquid thereof
  • Sperm freezing method and freezing liquid thereof
  • Sperm freezing method and freezing liquid thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 sperm preparation

[0027] 1. Fresh sperm:

[0028] Take rabbits (New Zealand rabbits aged 6-8 months, SPF grade, provided by Shanghai Slack Experimental Animal Co., Ltd., license number: SCXK2004-0005) fresh semen with D-PBS solution (commercially purchased) 2000r / min Centrifuge and wash three times, then slowly add D-PBS solution, and place at 38.5°C, 5% CO 2 , in an incubator at 100% humidity for 10 minutes.

[0029] 2. Freeze-thawed sperm:

[0030] Collect rabbit semen and freezing liquid (the formula of freezing liquid is: add 3.52g Tris HCL, 1.96g citric acid, and 1.59g glucose into 100ml deionized water and mix well as the base liquid; 100ml freezing liquid: add 20ml egg yolk, 4ml Glycerin, mix and pack, store in refrigerator at 4°C.) Dilute and mix at 1:1, slowly cool down at 4°C, seal with 0.25-0.30ml plastic thin tube after 3 hours, place on a floater (4cm away from the liquid nitrogen surface , -90--95°C), put into liquid nitrogen after 10 min...

Embodiment 2

[0031] Example 2 Oocyte preparation

[0032] 1. Fresh Oocytes

[0033] Donor female rabbits were primiparous New Zealand rabbits aged 6-8 months and weighing about 3kg. According to the method of Kennelly and Foote (1965), 2 times a day (12h interval) for 3 consecutive days, each subcutaneous injection of FSH50IU / only (Ningbo 12h after the last injection of FSH, inject 100 IU of HCG (manufactured by Ningbo Second Hormone Factory) into the ear vein and mate with ligated male rabbits. Oocyte retrieval was carried out 14, 16, and 18 hours after HCG injection. Cut off the fallopian tubes under sterile conditions, absorb 10ml of oviposit fluid, flush the eggs from the bell mouth to the junction of the uterine tube, use 5mL oviposit fluid on each side, put the cumulus oocyte complex in a container containing 0.2% In the Eppendorf tube of hyaluronidase D-PBS solution, shake and digest on a vortex mixer for 5 minutes to remove granule cells, pick out the enzyme-digested oocytes und...

Embodiment 3

[0040] Example 3 injecting sperm into oocytes

[0041] 1. Manufacture of operation panel

[0042] The design method of the operation plate is designed according to the style introduced in the third edition of the ICSI chapter of the "Mouse Embryo Operation Experiment Manual", that is, 4-5 rows of operation droplets are sequentially made on the cover of the 90mm cell culture dish, and each row of operation droplets is composed of Composed of three 3ul 10% PVP (polyvinylpyrrolidone) drops and three 5ul mTCM199 drops, absorb 2ul of floating upper layer sperm into the second PVP drop, place the eggs in the fourth mTCM199 drop, and the remaining small drops in the middle of the plate Both are used for cleaning the holding tube and the injection needle. After all the operation drops are done, cover the entire operation panel with 3-5ml paraffin oil. The design style of the operation panel operation drop is shown in the figure ( figure 1 ).

[0043] 2. Addition of spermatozoa in ...

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Abstract

The invention relates to a sperm freezing method which mainly comprises the following steps: 1) diluting and well mixing sperm and freezing liquid according to a ratio of 1:1; 2) slowly cooling at 4 DEG C, after 3 hours, individually adding the mixture in plastic tubules of 0.25-0.35 ml and sealing, putting on a suspended object with a distance from a liquid nitrogen surface of 4-6 cm at a temperature of from -90 to -95 DEG C; 3) putting the tubules into the liquid nitrogen after 10-15 minutes. The sperm freezing method provided by the invention has simple operations, and the frozen-thawed sperm still has high fertilization ability and an high embryonic development rate.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a freezing treatment method, in particular to a sperm freezing method and a freezing solution thereof. Background technique [0002] Intracytoplasmic sperm injection (ICSI), as a means of assisted fertilization, has developed rapidly in just a few decades. In terms of the nature and scope of ICSI applications, the current application of ICSI can be classified as There are two categories, one is that it can solve a series of infertility problems caused by factors such as sperm itself and blocked fallopian tubes, so it is widely used in the treatment of infertility in reproductive medicine; the other is that it is used as a biotechnology for biological In the field of engineering, the research on the basic theories of developmental biology such as animal husbandry production, wild animal protection and fertilization mechanism is of great significance. [0003] ICSI techniques usually i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 陈浩杰
Owner 陈浩杰
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