Application of octanoylated ghrelin in promoting bovine oocyte maturation in vitro
An oocyte and in vitro maturation technology, applied in the field of livestock gamete embryo engineering, can solve problems such as uncertain effects, achieve the effect of less toxic effect, promote cleavage rate and blastocyst rate, and promote the effect of synchronous maturation
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Embodiment 1
[0025] Embodiment 1 oocyte maturation culture
[0026] (1) Oocyte collection
[0027] Use a 10ml syringe containing 2ml egg extraction solution (containing 25mM hepes M199+1%PVA+200uM IBMX+1% double antibody) to extract follicles with a diameter of 3-8mm from the surface of bovine ovaries obtained from the slaughterhouse. Put the egg-absorbing liquid after absorbing the oocytes into the 7cm 3 In domestic petri dishes, cumulus-oocyte omplexes (COCs) with uniform cytoplasm and three or more layers of dense cumulus granulosa cells were selected under a stereo microscope for pre-maturation in vitro to cultivate.
[0028] (2) Oocyte pre-maturation treatment
[0029] The selected COCs were washed 3 times in the egg washing solution (containing hepes M199+1%PVA+200uM IBMX+1% double antibody), and washed in the pre-maturation solution containing octanoylated Ghrelin (adding 1-1000ng / ml concentration of octane Acylated Ghrelin's M199 culture medium) was washed 3 times, and then put...
Embodiment 2
[0044] Example 2 In vitro fertilization and in vitro embryo production
[0045] Further experiments were carried out with the oocytes matured and cultured in Example 1.
[0046] (1) In vitro fertilization
[0047] Using the petri dish micro-drop method, first wash the mature oocytes in fertilization fluid (BO solution + 20mg / ml BSA + 20mg / mL heparin sodium + 100mg / mL streptomycin + 100IU / mL penicillin) for 2-3 times Put it into the balanced fertilization fluid (the amount of 60 μl of fertilization fluid, 20 oocytes), and the oocytes cultured in the pre-maturation fluid without octanoylated Ghrelin as the control; then use the flotation method to process the frozen semen , the sperm floated up in 2ml of semen washing solution (BO solution + 3.38mg / mL caffeine) for 30min, then took 600-800μl of the supernatant and put it into a 1.5ml centrifuge tube for centrifugation (1800 rpm, 5min) twice, after centrifugation, remove the supernatant and add The final volume of fertilized fl...
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