so far I have tried co-culture with oviductal cells in K-RVCl-50 media ( culture conditions are 38.5 ° C and 5% CO2 ) still we are reaching till morulla stage after that the embryos are degenerating . If you could provide a media composition and culture conditions so that we could get blastocysts.
Thank you
Dear Prakash,
Are you changing medium during 7 days culture, if not then change media after 48h. What percentage of embryos were cleaved, you need to take care of sperm processing to improve cleavage rate, very crucial step. Also used cumulus cells matrix for culture. RVCL is best suitable for buffalo cloned embryos, for cattle most workers used mSOF, try this media too. You need to continue the experiments many times to get reproducible results. Best Wishes_NLS
Have a look at the work that came from my late husband'd lab - Neal L. First. His efficiency was excellent! Changing media is critical, also the incubator should match that of bovine, not human. Using cumulus cells to co-culture is a good idea but also low O2 is critical.
Low O2 tension is critical for bovine blastocyst to develop in culture. I would recommend 5% O2.
I will agree with Marijo and Carolna as far as the O2 levels for bovine blastocyst, but I will encourage the presence of the cumulus cells and if it is possible to enrich your media with media of oviductal culture. Good luck.
we are doing oviductal co culture and I always take A-Grade oocytes ... thanks everyone ... I will try mSOF and regarding to oxygen I will try to do something... I will Update you .. after these experiment trials...
thanks again
Blastocyst stage is very critical point in embryonal development, there are some trouble treatment in this area. One of the most critical factors is also osmotic pressure of media. Please focus a little on this matter.Yours
In my opinion, the most important thing is related to the media. The basis of every IVC (whatever it is...) are the media. Even if you move the lab to another place, using the same techniques and the same equipment, the limiting factor is related to the media you need to use...Try to change the media and keep a single culture without cells as a control to compare after the IVC.
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