Please care about medium osmolarity and oocyte collection method. Especially i recommend that don’t use puncturation

Hi, teacher. The matured oocytes are grade 1, they demonstrate a good expansion of the cumulus, as well as the presence of 1 polar body. I believe something in between is being toxic, but besides not identifying, I've never seen this kind of problem. So maybe someone who may have been through this kind of problem could have some suggestion or solution.

Hello, Dear Leonardo,

I think you should verify time of activation or the concentration of the activator, specially if you use Ionomycin.

Best Regrads,

Leonardo, you can verify basic things like osmolarity and pH, if the gas mixture was changed recently, any expired reagent. If you use oil on your culture, this might also be a problem (we had problems with different lots of oil). During the years I experienced all kinds of problems that resulted in the embryos dying, from CO2 problems, expired reagents, external contamination with cleaning substances... If you can provide more information about any possible changes that occurred in your system, that might help to identify the problem.

I've had problems with 6DMAP too. If you use it on your protocol, prepare a new stock solution.

Replace one reagent/solution at a time, so you really identify what was harming your embryos.

So our system for producing embryos is not three gases, but we've ruled out that problem for now, as we were able to produce our embryos without all three gases previously. Today, we are running some other tests looking for a solution to the problem. Something we questioned a lot was our activators, but it's something we're still making changes to. Including preparing our stock solutions. Anyway, thank you so much for all the help!