we work in IVF of bovine oocytes .
1. After aspiration when we collect COCs (cumulus oocyte Complex) to washing media it sometimes forms a jelly like structure . (I cannot use it further)
2. Sometimes they do not form jelly but just get stick to each other. although I am getting embryos in this case after IVF.
If you know reason behind these one reason I am thinking is due to aspiration from bad follicle. still need your valuable suggestions . please let me know how could I avoid them.
Thanks
Which size of follicles do you aspire? The jelly structure happened to me when I accidentally aspire big follicles ( > 8 mm). Normally we work with small follicles but it is normal that they stick to each other so don't worry.
thanks Angela next time i will use smaller follicle. for second part as they stick, And I require 15 COCs/Experiment so they all form a single bunch of COC and after that You cannot count . They are almost impossible to separate from each other.even after 20 hour post IVF they are needed to be separated mechanically by use of needle. and this is major problem with my sample i want to avoid this but not able to point out reason for that..
You have to collect oocyte before putting in the maturation media. Create a group of 15 and put together in the ivm drop or petri dish so that in future you don't have to separate anything.
thanks a lot Navid ... what could be reason behind this means clumping of oocytes ?
Hi Prakash,
The clumping of oocyte is due to formation of fibrin, a thread like structure of blood protein fibrinogen. Fibrin and platelets in the blood make a clump. If this happens, it is not possible to separate oocytes except needles. The more you pipette, the clump will become stronger. The presence of blood in the aspirated follicular fluid is cause of this. I am not sure if this protein is present in the bovine follicular fluid also. As suggested by Navidad, heparin will prevent the coagulation. Collect the oocytes in larger amount of wash medium so that blood is diluted. Try to prevent addition of blood. Also washing the COCs multiple times in the wash medium to remove presence of any blood will prevent this coagulation. I am afraid, if you collect COCs from very small follicles, fertilization and embryo development will be reduced. Good luck!
thank you Murid .. this explanation was helpful I tried heparin and that worked... and Navid was asking about collection of oocytes from smaller folliclicular size equal to or less than 8mm diameter. earlier I use to aspirate from larger ones also ... that may have created problem... thanks for your valuable comments...
Thank you B Sanaei ... I always keep expanded/Nude COC's out of my collection Drop... ...
Dear Chandra,
I agree with Navid and Murid. Nevertheless, I want to add my opinions. Size or diameter of follicles you carry out aspiration can be important factor causing jelly or sticky formations. If you aspirate larger follicles i.e. 8 mm or higher these follicles may contains matured oocytes or expanded cumulus oocyte complexes. In this situation, some proteins produced by cumulus cells cause sticky and jelly forms. Please prefer only 2-8 mm follicules for aspiration.
You can use heparin to prevent jelly formation. And also you can use BSA or PVA against stickiness of oocytes. Also you have to use these additives in your collection medium. You can increase dosage of heparin and PVA a little bit if situation continue.
Briefly,
1. Prefer only 2-8 mm follicles for COCs collection,
2. Use enough doses of heparine in collection medium,
3. Use BSA or PVA,
4. Use suitable needle and vacuum for collection,
My best wishes,
Umut
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