Method for regulating and controlling calcium ion concentration of vitrification frozen bovine oocytes

A technology of vitrification and oocytes, which is applied in the field of regulating the concentration of calcium ions in vitrification frozen bovine oocytes, can solve the problems affecting the application range of vitrification oocytes, the decrease of fertilization rate, and the increase of concentration, and achieve The effect of protecting mitochondrial function and its developmental ability, low cost, and simple operation

Active Publication Date: 2017-08-18
INST OF ANIMAL SCI CAAS
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Problems solved by technology

The reason is that vitrification can cause Ca in oocytes 2+ Abnormally elevated concentrations, which in turn lead to premature release of cortical granules, hardening of the zona pellucida, and reduced fertilization rates (Larmanet a1.,2006,2007; Tian et al.,2007; Bogliolo et al.,2012; Nikiforaki et al.,2014) , seriously affected

Method used

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  • Method for regulating and controlling calcium ion concentration of vitrification frozen bovine oocytes
  • Method for regulating and controlling calcium ion concentration of vitrification frozen bovine oocytes
  • Method for regulating and controlling calcium ion concentration of vitrification frozen bovine oocytes

Examples

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[0039] Example 1 Effects of RR and BAPTA-AM treatment on the in vitro fertilization ability of vitrified bovine oocytes

[0040] 1. Oocyte collection and in vitro maturation

[0041] Bovine oocytes were collected from local slaughterhouses, stored in 35°C normal saline containing 75 μg / mL penicillin and 50 μg / mL streptomycin, and sent back to the laboratory within 2 hours, and cumulus was extracted from follicles with a diameter of 2-8mm- For the oocyte complex, the complex with at least 3 layers of dense cumulus cells is selected and washed for in vitro maturation. 50 cumulus-oocyte complexes are placed in a group containing 500 μL of in vitro maturation solution, and the surface is covered with 4-well plate with mineral oil at 38.5 °C 5% CO 2 Cultured in an incubator for 22-24 hours, then the cumulus cells were removed with hyaluronidase, and the MII stage oocytes were collected for subsequent experiments. .

[0042] In vitro maturation solution: M199 medium containing 10...

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Abstract

The invention provides a method for regulating and controlling calcium ion concentration of vitrification frozen bovine oocytes. The method comprises the following steps: placing bovine oocytes being mature in vitro for 22 hours in in-vitro mature liquid containing BAPTA-AM for incubating for 2 hours; then, freezing with a vitrification freezing solution containing 10muM BAPTA-AM and 1muM RR by adopting an OPS technology; unfreezing, and recovering for 30 minutes in in-vitro mature liquid containing RR in order to remarkably improve the cleavage rate, blastocyst rate, blastocyst cell number, in-vitro fertility and developmental capacity of the vitrification frozen bovine oocytes. The method is easy to operate, is low in cost, is nontoxic and harmless to the oocytes, can be used for effectively protecting the mitochondrial function and the developmental capacity of the vitrification frozen bovine oocytes, has important theoretical and practical significance to the expansion of the application range of the frozen bovine oocytes, and plays an important role in the field of ultralow-temperature freezing research of the bovine oocytes.

Description

technical field [0001] The invention relates to the technical field of ultra-low temperature freezing of bovine oocytes, in particular to a method for regulating and controlling the calcium ion concentration in vitrified frozen bovine oocytes. Background technique [0002] The cryopreservation of mammalian oocytes is an effective means to protect the diversity of species resources and save endangered animals. It can also provide abundant experimental materials for embryonic biotechnology such as in vitro fertilization, single sperm injection, and somatic cell nuclear transfer. Supply and use are not limited by time and space. Therefore, the cryopreservation of oocytes has very important theoretical significance and practical value, and has always been a research hotspot and focus in the field of cryobiology. At present, vitrification has become an effective method for oocyte cryopreservation due to its advantages of fast cooling speed, low freezing damage, and simple operat...

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 赵学明王娜朱化彬李崇阳赵亚涵郝海生
Owner INST OF ANIMAL SCI CAAS
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