[Zbrafish] Re: Zebrafish sperm cryopreservation

Daniel Castranova via zbrafish%40net.bio.net (by castrand from mail.nih.gov)
Mon Oct 20 08:24:19 EST 2008


Hello Kevin and others,

I have been doing zebrafish sperm cryopreservation for a while and I  
agree, the 1-3ul of sperm described in many protocols isn't  
realistic.  I find that I get between 0.5 and 2ul with the average  
around 0.75 - 1ul.  I don't think that there is an extraction  
technique that will help increase your volume, but I don't think you  
should be that concerned about it.  I have fertilized over 800 eggs  
with 0.5ul of sperm (fresh not frozen) so I think there is enough  
sperm in small samples to get the job done, it is a matter of properly  
freezing and thawing the sample.  I have had the most success with a  
modified version of the protocol found in the zebrafish book.  I cut  
capillary tubes to 1" and freeze the tubes inside cryovials to save  
space in our N2 dewar.  I also use 10ul of freezing media.  If anyone  
would like a copy of our detailed protocol contact me off of the list  
and I will send it out.  I think the key to successful sperm freezing  
is practice.  It took me about a year before I was happy with my  
results and I spent a lot of time trying various protocols that I  
could never get to work.


I hope that helps,

Dan

Daniel Castranova
Aquatic Specialist
Charles River Laboratories - Contractor
Unit on Vertebrate Organogenesis
Laboratory of Molecular Genetics, NICHD, NIH
Building 6B, Room 322, 6 Center Drive
Bethesda, MD 20892
(301)594-0904
castrand from mail.nih.gov


On Oct 17, 2008, at 7:26 PM, finchg from ohsu.edu wrote:

> On Oct 16, 11:08 am, "Kotredes, Kevin"
> <Kevin_Kotre... from DFCI.HARVARD.EDU> wrote:
>> I'm interested in learning and practicing the procedure to prepare  
>> zebrafish
>> sperm for cryopreservation and later use for in vitro  
>> fertilization.  Does
>> anybody have a useful protocol they are willing to share or a  
>> resource that they
>> can suggest?  Thank you very much!
>>
>> Kevin P Kotredes
>> Research Technician
>> Look Zebrafish Lab
>> Jimmy Fund Building, RM G03
>> Dana-Farber Cancer Institute
>> Phone: 617-632-3545
>> Email: Kevin_Kotre... from dfci.harvard.edu
>>
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>
> Kevin,
> I'm trying to start up sperm cryopreservation at my lab as well.
> Multiple sperm freeze protocols can be found at the ZIRC site:
> http://zebrafish.org/zirc/documents/protocols.php
> Also, there is a protocol in the Christiane Nusslein-Volhard/Ralf Dahm
> Zfish book:
> http://books.google.com/books?id=zS-5CKOjCIgC&pg=PA30&dq=zebrafish+sperm+cryopreservation
> The Draper protocol appears to be the most widely used (e.g. at ZIRC,
> in TILLING at Moens Lab, etc.) and is well written/easy to follow.
>
> ....however, I am following it and thus far have been having a really
> difficult time getting the 1-3ul of sperm that the protocol
> recommends.  If anyone reading this can recommend or describe a
> squeezing technique that can produce larger volumes, please please
> take the time to post some advice.  I have tried everything I can
> think of, variety of forceps, fingers, variety of pressures, variety
> of anatomical regions, sexy music, etc.  Anyone?  Please........
>
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