[Zbrafish] embryo contamination

David and Don,

Inside the chorion? I've seen this before, and it is almost always
associated with low quality eggs, usually from low quality fish. It is
likely some opportunistic fungal contaminant, although I am not sure that it
is the same organism that commonly infests the outside of the chorion. You
could try to put a band-aid on it by using methylene blue in your embryo
medium, but this won't work if it is within the chorion, and it doesn't
address the cause of the problem.

My guess, without knowing really anything about your situation, is that your
fish are producing poor quality embryos. The reason for this? Could be one
or more of many.

Let's start with the water quality. It probably ISN'T water quality if your
parameters are STABLE and within the general range conducive to freshwater
tropical fish culture. so pH 6.8 - 8.0. conductivity 300-2000uS, hardness
50-200 ppm, TAN 0, NO2 0, NO3 0-80. The actual values, as long as you're
within this range are not as important as the stability. If you're not
within this range, or if you swing wildly, then this could be part of the
problem. I believe that one under appreciated chronic stressor on zebrafish
in culture is wide daily swings in pH produced when systems with moderate to
high loads of fish are buffered solely by addition of sodium bicarbonate.
There are also other water quality issues to consider including presence of
chlorines, chloramines, copper, etc. This will depend on source water and
chemical filtration. Needless to say, these values should be zero. Also
goes without saying that water quality of water embryos are placed in after
collection is very important.

If you can eliminate water quality, then look into DIET. This is probably
where the issue lies. Low quality diet will result in low quality embryos
and low quality fish (not necessarily in that order). Because zebrafish are
tough and adaptable, the effects of dietary deficiencies may not be
manifested right away and often intensify over time, especially as fish age
and if they are spawned intensively. Rapidity of drop-off in
quality/viability will be further influenced by overall quality of fish
stock. Bad stock will have more pronounced, abrupt drop-offs due to dietary
deficiencies. What are you feeding? Diet should be balanced and ideally
you should be feeding more than one type of feed. Are you storing it
properly? Dry feeds like flakes and pellets have short shelf lives (~3
months if kept dry at 4C). Are you feeding it properly? Water soluble
nutrients in formulated feeds leach out into water upon hydration very
rapidly, so it is best to deliver them dry. Basic question is are you
getting proper balance of nutrients to animals? Probably not if you are
feeding only one kind of feed, and certainly not if you're not handling or
delivering it properly. Varied diets of mixed feed types (with live
zooplankton as predominant) are best for long-term health and productivity
of fish. If this is the issue and the fish you're observing problems with
are not too far gone, then a correction in diet will result in a pronounced
improvement in the quality of eggs (and decrease in the contaminant you
see).

As I've mentioned several times above, the quality of the fish themselves is
another major factor. The maintenance of laboratory strains of zebrafish
is an issue that should be taken seriously, because generation by necessity
involves inbreeding and bottlenecking. If these things are not considered
during the propagation of lines (i.e. you don't think much about making a
new generation with a pair cross or continuously incross homozygous
transgenic or viable mutant fish) the quality of the fish goes downhill. A
great example of this is what happens when you work with a strain like SJD,
which is nearly isogenic. Maintaining SJD populations by normal inbreeding
of small populations (in which bottlenecking and inbreeding effects are even
more pronounced) result in the production of embryos of extremely poor
quality that often do not hatch, and are highly susceptible to ciliate and
fungal infestations (the first example that comes to mind relative to the
symptoms you observe is what I see with SJD). Outcross them, though, and
the eggs are fine, presumably from infusion of heterozygosity. Age and
stress (from bad husbandry, excessive handling, etc.) will also affect the
quality of fish. Even high quality fish will break down eventually,
inevitably from age, but sooner if stressed. Poor quality fish will break
down much faster. You can offset the effects of aging to some degree and
even poor quality (in some cases, such as SJD, you have little control over
the matter) by keeping husbandry conditions favorable and by feeding the
fish an excellent diet, one high in certain nutrients like Vitamin C (a
known antioxidant).


So. this is clearly a wide net that I've cast here, and I'm making lots of
pessimistic (but not ad hominem) assumptions about your practices, but
hopefully this will allow you to narrow down the source of the problem.

Lastly, seems to me that bleaching within 20 minutes after fertilization is
a bit early. Any particular reason why you're treating them so quickly?
Don't know if it could be a problem or not, but this is much earlier than
what I am familiar with (usually between 10 -24 hpf).


Good luck, and naturally feel free to follow up with me off-list.


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-----
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Sent: Wednesday, April 04, 2007 12:19 PM
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Subject: [Zbrafish] embryo contamination

embryos. A 10% system water change occurs 3-4X per week and water quality
tests show normal ranges. The fertilized eggs are washed with .003% bleach
within 20 minutes of fertilization. Our breeders are about 1 year old. The
embryo media is changed every 24 hours.
increases over time until the embryo dies. Does anybody have any
suggestions as to how we may combat this issue.

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