Improving the method of cryopreservation and thawing of bovine in vitro fertilized blastocysts
A bovine body, in vitro mature technology, applied in biochemical equipment and methods, preservation of human or animal bodies, embryo cells, etc., can solve problems such as reduced embryo survival rate
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Embodiment 1
[0067] Example 1: Cultivation method of bovine in vitro fertilized embryos (collected in vitro)
[0068] In this example, in step (1), the collected oocytes were placed in a transport medium containing HEPES at 38.8° C., without carbon dioxide supply, and transported back to the laboratory within 24 hours.
[0069] 1. Reagents
[0070] In the specific test of the present invention, unless otherwise stated, the relevant reagents used are described in detail as follows:
[0071]The transport medium containing HEPES used in this example contains: glycine 50.0 mg / L, L-alanine 25.0 mg / L, L-arginine hydrochloride 70.0 mg / L, L-aspartic acid 30.0mg / L, L-cystine dihydrochloride 26.0mg / L, L-glutamic acid 75.0mg / L, L-glutamine 100.0mg / L, L-histidine hydrochloride monohydrate 21.88mg / L, L-hydroxyproline 10.0mg / L, L-isoleucine 40.0mg / L, L-leucine 60.0mg / L, L-lysine hydrochloride 70.0mg / L, L-Methionine 15.0mg / L, L-Phenylalanine 25.0mg / L, L-Proline 40.0mg / L, L-Serine 25.0mg / L, L-Threon...
Embodiment 1A
[0116] Example 1A: Cultivation method of bovine in vitro fertilized embryos (collected in vitro)
[0117] With reference to the method of Example 1, tests were carried out on three types of cattle, namely Holstein (dairy cattle breed), Simmental cattle (beef cattle breed), and Chinese buffalo (draft breed), and the results showed that the cleavage rates were all in the range of 85 to 90%. The morula rate was in the range of 62-67%, the blastocyst rate was in the range of 50-55%, the apoptosis rate was in the range of 4-6%, and the hatching rate of blastocyst on the 9th day was in the range of 72-55%. Within the range of 77%; the maturation rates of in vitro maturation of oocytes from three types of cattle, Holstein, Simmental, and Chinese buffalo, were 83.3%, 80.6%, and 75.6%, respectively.
[0118] In the experiments of Example 1 and Example 1A on four types of cattle, in step (3), the in vitro culture, preservation and thawing of embryos were performed using a freezing sol...
Embodiment 1B
[0122] Example 1B: Cultivation method of bovine in vitro fertilized embryos (collected in vitro)
[0123] With reference to the method of Example 1, tests were carried out for four kinds of cattle of Chinese yellow cattle (Nanyang cattle, draught type), Holstein cattle (dairy cattle species), Simmental cattle (beef cattle species), Chinese buffalo (draft cattle species), However, the recipes of fertilization medium and semen preparation medium were modified respectively, and the other test solutions and operations remained unchanged. The fertilization medium formula was changed to: 112.0 mM sodium chloride, 4.02 mM potassium chloride, 2.25 mM calcium chloride dihydrate, 0.52 mM magnesium chloride hexahydrate, 0.83 mM potassium dihydrogen phosphate, 37.0 mM sodium bicarbonate, 1.25 mM mM sodium pyruvate, 10μg / ml heparin, 4mg / ml bovine serum albumin (BSA), 100U / ml penicillin, 100μg / ml streptomycin, 10umoL / L α-lipoic acid, 25μg / ml folic acid, 3mg / ml glutamine Aqueous solution ...
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