I am trying to cryopreserve immature bovine oocytes in .25 ml french straws by vitrification but i am facing problems:
1. I have used almost all vitrification media composition and protocols available online for cryopreservation of immature bovine (Bos indicus) but still not getting good results. sometimes i get maturation after thawing but rarely get embryos after IVF.what could be explainable reason for this type of results?
2. Any special step or ingredient that you could suggest? ( I have used DMSO and/or EG with sucrose or glucose)
3. What precautions I must take during vitrificatio?( i use fresh vitrification media)
Any help would be appreciated . Thank you for your time
You probably have a specific reason to cryoprerserve immature oocytes as mature oocytes should survive vitrification much better. One thing to look into is the cumulus cells. To what degree do you denude the oocytes before vitrification and how do the cumulus cells look like after thawing?
Dear Peter thank you for attention . I am vitrifying with cumulus cells . cumulus cells sometimes get scattered/ loosen but not always. i know that they are live because they gets converted to fibroblasts after maturation.
The exposure to cryoprotectants is then obviously sufficient for keeping membranes intact. Nevertheless, the exposure time may have affected cytoskeletal components resulting in maturation failure. Maybe you could try to shorten the last incubation step(s) before vitrification to avoid intracellular damage. EG might be the better choice as a cryoprotectant because of its better permeability.
I have tried:
Equilibration time: 45 sec, 1 min , 2 min, 2.5 min , 3 min , 5 min , 6min
and vitrification: 30 sec, 45 sec , 60 sec, 1 min ,2 min
vitrification without Equilibration
and I mostly used EG in higher ratio with or without DMSO.
What kind of medium and temperature you have used for equilibration, usually for the first equilibration is in the medium with more low cryoprotectant concentration and in the presence of sucrose.
The temperature at moment of exposure of oocyte to vitrification medium is also important factor, some people do this step with vitrification medium keep on the ice. The combination of permeable cryoprotectant ( PROH, EG, ..) with sucrose, trealose is better for vitrification and rapid thawing.
There is also the influence of the type of straw (size, normal or open pulled straw) or the way of exposure to liquid nitrogen (in straw or with microdrop).
Dear Nguyen,
I keep low concentration in equilibration but without sucrose because i did not found any literature that suggest to use sucrose while equilibration. if you have any research article please send me link.
people suggest EG is better as compare to others . I do equilibration at room temperature not on ice.
I am using french straws (.25 ml) .
please suggest about :
how much concentration of EG or DMSO or Combination I use for equilibration and exposure time in both equilibration as well as vitrification media. for hand about way to exposure . if you could suggest any way because I directly plunge straw into liquid Nitrogen .
thank you
Prakash Chandra
Dear P Chandra,
Enclose din attachement you find please my papers on embryo rapid freezing and vitrification. Just to have idea how to made the combination of sucrose, CR or ficol.
The selection of type of CR, concentration is different following the species , stage of oocyte or embryos.
Dear Nguyen
Thanks a lot . will let you know my results.
regards
Prakash
yes vitrification of oocyte is less successful in comparison to cleavage stage embryos in human. the reason may be physical reasons. the standing principle is to prepare
1. Equilibration solution should be half of vitrification solution
2. 0.5/1M sucrose is an ideal candidate as non permeating CPA.
3. Mixture of CPAs should be standardised by testing the toxicity. (10% of 4CPAs or 15% of 3 CPA).
4. Work on vitrification kit made by SAGE, USA.
Dear Dr.Chandra
I think the paper ( Two-step freezing of two-cell rabbit embryos after
partial dehydration at room temperatureli) by J.-P. Renard, Bui-Xuan-Nguyen and V. Garnier puplished in Journal of Reproduction and Fertilization(1984) 71:573-580
To Mr. Prakash Chandra.
I present you some suggestion :
1. use the vitrification tool ( cryotop) instead of strow .
2. reduce the time of exposed the gametes to the cryoprotectant such as DMSO or EG below 1 minute .
3. this is very important requirements to success vitrification. thank you for your question
can anyone please share the step - wise protocol for vitrification of oocyte. Also suggest me wheather is it possible to vitrify the oocyte at the field or slaughter house itself.
@ M. Das Gupta .. as per my experience ...preserving immature oocytes is really difficult task .. i cannot disclose you my procedure .. but you can have many protocols online.. and you can also buy vitrification kits.
I guess vitrification at field/slaughter house is not possible due to risk of contamination and also it requires steps like equilibration some protocols require pre equilibration than equilibration and finally vitrification .
hope this will help you
@Prakash Chandra ....can you please tell me the price of vitrification kit
@M.Das Gupta Sorry we don't use vitrification kit..but the price varies from company to company.
Hello . Mr. chandra . the 0.25 straw is not a good tool to appliance the vitrification of oocyte or embryo you can use the cryotop its successful tools . Thank you.
using of straw in the vitrification protocol is very bad choice . to make
successful vitrification you can use the device named cryotop.
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