sperm freezing

[May-Su YOU]

I am Maysu You, now working at IMCB (Institute of Molecular and Cell  
Biology) in Singapore. We are now working on sperm  
squeezing,freezing and in vitro fertilization of Zebrafish. We got around  
90% of IVF successful rate when we used fresh sperm (from both  
squeezed sperm and dissected testes) . So we moved to freezing step.  
We tried two different cryoprotectants (methanol and BSMIS: Buffered  
sperm motality inhibitor solution). After thawing, we cann't get any IVF  
embryo. Therefore, we doubt about the freezing procedure which we  
used at the moment. We also tried to observe the sperm, but even with  
trypan blue, we are not sure wheather the sperm are live or dead.  
According to the results,I think there are some questions to be  
answered by whom has experties in this work   

1.Maybe our freezing protocal is not totally correct (is there any critical  
point that we didn't catch?)  

2.How can we check the viability of the sperm? (we observed sperm  
under 400 time magnification)  

3. What will be the best way to fertilize eggs with frozen sperm. Your  
help will give us a big improvement of our work.Thank you in advance.   

Best regards 

Maysu 


************************************************ 
Dr. May-Su YOU 
Research Officer 
Institute of Molecular and Cell Biology, 
A*STAR building, 1 Research Link, 
The National University of Singapore, 
117604 Singapore 
Republic of Singapore 

Tel: (65) 68727092 (lab) 
Fax: (65) 68727007 
e-mail: youms at imcb.a-star.edu.sg 

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