[Zbrafish] fertilization

christian lawrence clawrence at rics.bwh.harvard.edu
Mon Aug 21 11:05:33 EST 2006

Hi ---  Lots of potential things going on here.

1. Are you fertilizing with frozen sperm?  There is a big difference between
average fertilization rates between IVF using reconstituted thawed sperm and
freshly collected sperm. IVF with fresh sperm is much easier because sperm
can be easily damaged during freezing and thawing process.    You might want
to quality control by trying IVF with fresh, just to see if your technique
or egg quality aren't the problematic factors.

2. Sperm can be easily damaged by the freezing, storage, and thawing
processes, thereby decreasing the likelihood of successful fertilization
after reconstitution.  Very important to follow freezing and thawing
protocols EXACTLY, and also solutions should be remade regularly, etc.,  but
it equally if not more important that the quality of sperm being frozen is
high.  Factors contributing to this are age of the fish (younger the
better), diet (should be high in HUFA/PUFA and Vitamin C), holding
conditions (favorable, with routine exposure to females), and priming
(setting up in mock crosses with females and fresh water night the night
before is helpful).  In the field, reconstitution rates using frozen sperm
are typically low, but we have had fertilization rates with wt frozen sperm
>90% after 1 month of freezing.  It is not known how long sperm can be
stored for before it loses viability, but disparate data suggests that at
least some sperm remains viabile for up to 10 years.  This duration of this
period is probably impacted strongly by initial quality.

3. Fungus can be a problem, but should not be an overriding factor unless
the eggs are of low quality or you are not cleaning them at all, especially
when fertilization rates are low.  You can circumvent this by cleaning eggs
after day 0 and day 1, using methylene blue, and keeping them at low
densities in petri dishes (25 or less).

4. Are you sure that your eggs are fertilizing at all?  Zebrafish eggs are
activated by water, and not sperm.  If you're not carefully looking at them,
they can appear, at a quick careless glance (like my own) that they are
fertilized when in fact they are not.  There will be cell division, but it
will go awry or completely arrest. I'm not sure, but this could be due to
lack of fertilization altogether or perhaps incomplete fertilization by
damaged sperm.  

Typically, the problem with IVF from frozen sperm is due to problems with
fertilization, as it relates to sperm quality, although that may not always
be the case.

Good luck,

Christian Lawrence
Brigham and Women's Hospital
Karp Family Research Laboratories 06-004B
One Blackfan Circle
Boston, Massachusetts 02115
Tel: 617.355.9041
Fax: 617.355.9064

-----Original Message-----
From: zbrafish-bounces at oat.bio.indiana.edu
[mailto:zbrafish-bounces at oat.bio.indiana.edu] On Behalf Of jenny
Sent: Sunday, August 20, 2006 10:12 AM
To: bionet-organisms-zebrafish at moderators.isc.org
Subject: [Zbrafish] fertilization

     hi,everyone.Does anyone who is working on zebrafish sperm freezing and
IVF?I have some problems with the IVF. The hatching rate is very low by IVF,
only several embryos can survive from more than one hunderd fertilized
embryo.The most embryo can survive for 6-10h. after that, the embryo will
died gradually.but  I have control the
tempreture(25-30?) and fungus for the petri dish which were filled with
so my problem is that:
1. if the fugus exits, will all the embryo die?and how to kill the fugus, do
you have any methods more effectively?
2. My IVF method is following with  the zebrafish book, is there anyone who
make high hatching rate with this method?can you give me some
    Thank you for your help!

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