EW floated but all sank after trying to remove top layer

Hi girls!

I am still on this LONG journey of trying to figure out whether I am brave enough to really go for it, but I am happy to say that I finally started experimenting for real this week.

I was so excited that the EW floated and stayed seperated for the hour in the incubator, but then sank when I put the pipette in to remove the top layer :((  Any ideas?

Also, I tried looking under the microscope at the mixture and saw NOTHING alive?? I think I saw brown spots---so they could have been dead.... At one point, the incubator went down to 96 or so but that must have only been for two-three minutes b/c I was watching them most of the time to adjust the temp. Could they have died in that short time?

TIA for your help!

I don't have any ideas for the EW because I'm still having problems with that myself.  As for the dead sperm, did you warm up the EW before putting it on top of the semen?  The first time we tried it we didn't and they all died.  The next time with the warm EW, they lived, but I had trouble with it floating.  The first time it just sank, but I accidently tilted the vial when I put it in, which I was told was a no-no.  The other time they were floating, but when we got them out of the incubator, then it was mixed.  They were all alive though.  Our incubator fluctuated a little bit with temp.  96 is still pretty warm so I would think they would be fine.

good job at floating!!

dead sperm are black

dont put the piplette into the sperm but barely touch the top then suck it up. You arent trying to push it into the sample or it will sink