Method for improving cultivation efficiency of bovine in vitro fertilization embryos
An embryo culture medium, bovine body technology, applied in embryo cells, culture process, tissue culture and other directions, can solve the problems of low efficiency, high cost of in vitro fertilization embryo production, and limited wide application.
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Embodiment 1
[0145] Embodiment 1: the culture method of bovine in vitro fertilization embryo
[0146] 1. Reagents
[0147] Mature medium: Containing 0.01IU / ml FSH, 10IU / ml LH, 1μg / ml estradiol, 100ng / m l TCM-199 culture solution of IGF, 50ng / ml EGF, 100U / ml penicillin, 100μg / ml streptomycin, 10% fetal bovine serum.
[0148] Wash semen: Containing 112.0mM NaCl, 4.02mM KCl, 2.25mM CaCl2 2H2O, 0.52mM MgCl2 6H2O, 0.83mM KH2PO3, 37.0mM NaHCO3, 1.25mM sodium pyruvate, 10μg / ml heparin, 4mg / ml of bovine serum albumin (Bovine serum albumin, BSA), 10 mM caffeine, 100 U / ml of penicillin, and 100 μg / ml of streptomycin in water.
[0149] Semen received: Containing 112.0mM NaCl, 4.02mM KCl, 2.25mM CaCl2 2H2O, 0.52mM MgCl2 6H2O, 0.83mM KH2PO3, 37.0mM NaHCO3, 1.25mM sodium pyruvate, 10μg / ml heparin, 4mg / ml of BSA, 100 U / ml of penicillin, and 100 μg / ml of streptomycin in water.
[0150] Pre-embryo culture medium: Contains 109.5mM NaCl, 3.1mM KCl, 26.2mM NaHCO3, 0.8mM MgCl2 6H2O, 1.19mM KH2...
Embodiment 2
[0201] Embodiment 2: the culture method of bovine in vitro fertilization embryo
[0202] With reference to Example 1, the only difference is that the composition of the embryonic late stage culture solution is changed to:
[0203] 109mM NaCl,
[0204] 3.1mM KCl,
[0205] 26.0mM NaHCO3,
[0206] 0.5mM MgCl2 6H2O,
[0207] 1.3mM KH2PO3,
[0208] 0.4mM sodium pyruvate,
[0209] 1.5mM glucose,
[0210] 5mM Calcium Galactonate,
[0211] 10v / v% fetal bovine serum,
[0212] 1mM L-Glutamine,
[0213] 2v / v% essential amino acids,
[0214] 1v / v% non-essential amino acids,
[0215] 3mM glutathione, and
[0216] Water as dosing solvent.
[0217] The test was carried out as in Example 1, and all the indicators of the results were basically the same as those of the 3mM glutathione group in Example 1. For example, in this example, the morula rate was 0.633, the blastocyst rate was 0.508, and the apoptosis rate was 0.050.
Embodiment 3
[0218] Embodiment 3: the culture method of bovine in vitro fertilization embryo
[0219] With reference to Example 1, the only difference is that the composition of the embryonic late stage culture solution is changed to:
[0220] 110mM NaCl,
[0221] 2.9mM KCl,
[0222] 26.5mM NaHCO3,
[0223] 1.0mM MgCl2 6H2O,
[0224] 1.0mM KH2PO3,
[0225] 0.4mM sodium pyruvate,
[0226] 1.5mM glucose,
[0227] 5mM Calcium Galactonate,
[0228] 10v / v% fetal bovine serum,
[0229] 1mM L-Glutamine,
[0230] 2v / v% essential amino acids,
[0231] 1v / v% non-essential amino acids,
[0232] 3mM glutathione, and
[0233] Water as dosing solvent.
[0234] The test was carried out as in Example 1, and all the indicators of the results were basically the same as those of the 3mM glutathione group in Example 1. For example, in this example, the morula rate was 0.636, the blastocyst rate was 0.521, and the apoptosis rate was 0.050.
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