Mouse IVF - (Sep/30/2010 )
I have kind of a problem..
For over a month I've been trying to do in vitro fertilization using Balb/c mice but embryos never get past the 1-cell phase.
I follow a standard protocol; I do a PMS injection, 48 hrs later an hCG injection and 13 hrs post hCG I collect the mature oocytes (around 30 from each mice).
First problem, mature oocytes should be by that time in the oviducts, however I always find them inside the ovaries. I thought that something is wrong with the hormones I'm injecting so I ordered new ones but they haven't arrived yet.
After collecting mature oocytes I incubate for 30 min (37 C, 5% CO2). At this point I have groups of 10 oocytes in 1ml M2 medium.
At the same time, I collect sperm from the epididymis (males are absent for more than 3 days, but no more than a week) and incubate for 1h 30min in 500μl M2 medium (sperm capacitation).
I do the insemination adding 100μl of sperm into each group of oocytes and incubate for 4 hrs.
Finally, after the 4 hour incubation, I move the fertilized oocytes into 20μl drops of M16 medium (3 oocytes max per drop) covered with mineral oil and incubate.
And here is the major problem... my embryos stay at the 1-cell phase and I have no idea what's wrong with them.
I could really use some advice or anything, I 'm getting desperate here!
Thank you so much for reading all these
1. if you find your oocytes in ovary 13 hrs post hCG, you can collect them like 17hrs after hcg injection