Nutrition as a variable in European research Robert Geisler

Nutrition as a variable in European research Robert Geisler

October 15, 2019 0 By Ewald Bahringer


ROBERT GEISLER: Thanks a lot for inviting
me. So, I was invited to talk about zebrafish
nutrition variable in Europe. I wasn’t actually sure what to report on. So, Stephen and Zoltan encouraged me to do
a small survey of … through the mailing list of the European Society for Fish Models
and Biomeds—EuFishBioMed—and maybe I should introduce that. It is a regional society of zebrafish labs
that corresponds to the international zebrafish—IZTS, IZFS—International Federation of Zebrafish
Scientists but at a regional level. And this complements the study that Zoltan
will report on later on in the lunch session. So, we mailed approximately 500 labs. The mailing list of EuFishBioMed contains,
more or less, all zebrafish labs in Europe. We received only a small number of replies,
it was also only just a short time. In total, we had 27 replies, so obviously
that’s the highest self-selected sample of laboratories that are interested in this
issue. Also, not from all European countries. There were no respondents from Britain. I’m not sure why that is so, but I still
I still think there are some … ROBERT GEISLER: I’m sorry? UNIDENTIFIED MALE: It’s not in Europe anymore!
(laughter) ROBERT GEISLER: Oh well, but Turkey is not
in the EU and Israel is not in the EU. Maybe so. But I think there are some interesting answers,
which I would like to share with you. So, what feeds do people use? A wide-range of dry feeds is used, and all
of these are commercial. None of the labs that responded made their
own food, and they include major manufactures of pet store commercial foods, like JBL, Sera,
Tetra, and laboratory suppliers. The only thing that was clear is that GEMMA
is spreading, it is the most popular, which was used by about half … excuse me, about
half of the labs, and also very popular is SPAROS. I think we have people from SPAROS listening
in … listening in to the meeting? And then there are quite a number of other
supplies, some of which are very popular in the U.S. as well—I don’t know about that. And interestingly, 33 percent of the labs,
one-third of the labs, combine multiple brands of dry food. That is presumably to even out differences
… perceived differences, between foods from … feeds from individual manufacturers. In fact, we use feed from two manufacturers
as well at the EZRC. Most labs supplement dry feed with Artemia
with … and other live feeds, but mostly Artemia, which is used by about three-quarters
of the labs, which is also recommended by EuFishBioMed. Rotifers are … we are in the process of
testing rotifers now. So, some labs use rotifers as well as paramecia,
flatworms (i.e. Chironomus larvae), I use (indiscernible),
spirulina, tetrahymena. Just as an example, instead of what that’s
… what we do with this protocol, also published in the husbandry special issue of Zebrafish
that appeared a couple of years ago. So, this is just an example of the very traditional
protocol that we use successfully vis a vis the EZRC and have not changed because it works
well, which is based on caviar, a commercial food. And this protocol was originally imported—as
an example of how this happened—it was originally imported to our lab from the Strasburg Fish
Facility, and we continued to use it because it worked well. So, different sizes of caviar are used and
supplemented with Artemia and (indiscernible) with TetraMin flakes, so part of what we feed
is flake food. And of course, this is a rather complex protocol. There are quite a number of factors that could
be optimized if one wanted to optimize it systematically. So, this was all done by trial and error,
and while some aspects have been tested, and it works quite well, and so we use it at the
moment. Now, do labs think that feed variability is
an issue? And it turned out that most people do not
… who responded do not see that as an issue for their own research. Either they don’t see it at all as an issue
for research but … Is there a pointer here? Ah, this doesn’t work. I cannot find the mouse … Anyways. I see it. So, the labs in gray says no, not an issue
at all for the kinds of experiments they are doing. Then there are two labs that said that it
is not an issue for us because we have established an internal standard for our lab, which of
course makes it difficult to compare to what other people are doing, to help with sensibility. Then there are the people that … people
who say that yes, feed variability is an issue, or they say that it could in principle be
an issue for zebrafish research, just not what we are doing at the moment. That category again would include us at the
EZRC because we’re very interested in growing up and producing fish, but obviously it can
be an issue for things like screening as is … that we also use. And among the responses, two Italian labs
wanted to point out that it would be helpful to use feeding devices, such as the BlowFish
device—which they codeveloped—which is a feeding device for flake food that’s pneumatically
operated, and it can help to feed consistent amounts. Of course, this is just an example. There are similarly robotic devices and others,
but this is just to point out that feeding a consistent amount is a major issue for zebrafish,
especially since zebrafish tend to fatten and get obese and sterile if they are fed
a little bit too much in contrast to other organisms … animal organisms. So, big question: What do people think about
open-formulation, chemically defined food? And there are multiple people who think that
this would be useful and at some point would be specifically useful for experiments involving
obviously metabolism, metabolomics, toxicology, ecotoxicology, and so on. And a number of arguments were in favor of
chemically defined food … with having food with known and, if possible, low pollutant
content. I must point out that commercial food—one
person believes it’s generally too high in protein—that it would be useful to be
able to boost specific nutrients for in order to produce, for example, better quality oocytes,
by changing nutrients … the conditions on top of the standard concentration that would
be given in the defined food and also, obviously, to improve reproducibility. There were also a number of arguments against. There is a general feeling that live food
is crucial for the health of zebrafish, and the feeling is that chemically defined food
could be potentially expensive. And, of course, as one person pointed out,
never change a running system. So, there will be some resistance as well. We also asked labs whether they would be willing
to test such a food if it became available. Sixteen of the labs said they would be willing
to test a chemically defined feed. That even included one of the labs that said
they didn’t see a reason to have it in the first place, but they would test it if it
became available. And some pointed out the lack of capacity
for setting up and controlling the experiment, such as setting up (indiscernible). Finally—and its already the end of this
survey—since this was organized by EuFishBioMed, we asked whether the society should get in
involved in an effort to standardize feeds. For example, it could include a white paper,
and there was overwhelming support for that, with only one person being against it. That person cautioned—and I put this at
the bottom of the slide: “Before standardizing, you would have to make quantitative comparisons
of different feeding protocols that are being used in different places to see what really
works best. To do this properly would be a huge effort,
and I am not sure this is worth it, except for labs working specifically on metabolomics
or related issues.” So obviously it would require a large amount
of experiments, as we have seen this morning, and I think EuFishBioMed could be involved
in popularizing chemically defined diets and making them available to labs and creating
a feeling in the community that such … that such feeds … such diets are needed. EuFishBioMed probably does not have the resources
to do a lot in experimentally evaluating and helping to develop such diets. I should also point out that EuFishBioMed
has published a white paper, that’s now also published as a paper, on zebrafish husbandry,
which should help regulatory authorities in Europe, as well as labs … especially labs
starting to work on zebrafish. And these guidelines do contain a paragraph
on feeding, and this is very vague, and in part this is purposely very vague because
other people working on these guidelines … others saw a problem in being too restrictive because
European authorities—several different authorities in several countries—would perhaps take
the opportunity to take away freedom from laboratories. If the guidelines say you have to feed whatever
three times a day, then everybody would be compelled to do that. One shouldn’t be too specific in formulating
such guidelines, because thinking of the potential regulatory ramifications—and these are very
good reasons to formulate the guidelines in that way—one has to be very careful in that
respect and work to propose and how to write up guidelines for (indiscernible). They will also apply to chemically defined
feeds, which are probably not good for everyone. Okay, that’s the end of what I was going
to say already. I would like to thank people from EuFishBioMed
who helped with the survey and of the ITG, EZRC Fish Facility, and Uwe Strähle, the
head of EuFishBioMed. Thanks. (applause)
MODERATOR: There is time for questions. UNIDENTIFIED MALE: You were saying the open-formula,
chemically defined diet. A chemically defined diet is not an actual
ingredient type. Did you mean, you know, a diet made from,
you know, the basic nutrients components, like fatty acids and … some of the fatty
acids and amino acids? You’re not talking about a semi-purified
diet, or are you talking about a natural … when you say chemically defined diet, do you mean
that nutrients are defined? ROBERT GEISLER: The way the survey was put
… I think chemically defined diet, as we heard today, one where the individual … a
purified diet, where the individual components are purified. But, you know, obviously, when you try to
standardize feed, I think that is something that should be done definitely, seeing how
far backwards we are in the zebrafish field compared to aquaculture. It was pointed out today that one should have
both. There are works to be done there. Zoltan? ZOLTAN VARGA: So, I think you hit the nail
on the head, as we would say in Germany. When we engage in a project that could last
20 years, I think, and different communities come together—obviously the language we
speak is not the same—and I think it is really, really important that there is some
sort of effort put into a common language that we understand, whether we really mean
chemically defined or laboratory-defined or whatever we do. So, for the later discussion this afternoon,
this should be something we should put into the pathway for developing a standardized
food. How, you know, with the nutrition community
and the zebrafish community, you know, exchange information and agree on terms. UNIDENTIFIED MALE: I think some of the results
… some of the responses to the questionnaire really highlight some of the challenges we
are going to face. And speaking from my perspective, in our facility,
we’ve got, maybe, 15 labs that use zebrafish, and I am probably the only one that does anything
past 5 days. So, you know, any analysis done when they
are independently fed, you know, one of these diets for the experimental fish. And I should imagine there is going to be
a lot of resistance to changing things, because I would imagine other … if you’re looking
at 1-day-old embryos, the majority of people would be like, if the fish are growing, you
know, at a reasonable rate and producing enough embryos—don’t change anything. So I think that’s going to be, for me and
my facility, that’s going to be a big problem implementing some of this. ROBERT GEISLER: Yeah, I think probably you
can’t … you can’t force zebrafish labs to use something specific and, at one point,
to be forced by authorities either. That’s referred to. But still, I think that the survey results
are encouraging in that a lot of people do see in Europe … at least do see a need to
standardize feed in some way, and that will probably not be chemically defined feed. But to have guidelines—especially if you
start a new lab, you want some sort of guide, you don’t want to start from the beginning
by trial and error, and so there is a need for some help to be given to labs. And some would adopt the guidelines, and some
wouldn’t. UNIDENTIFIED MALE: I think it also highlights
the need for education, because we are scientists, and many of our colleagues that are developmental
biologists—which kind of founded the system—don’t get, or aren’t aware of, the body of literature
on the subsequent affects downstream. For example, there’s tons of developmental
biologists working on beta cell development and the beta cell number. Now we have data to say we know epigenetic
changes that occur, and maybe their own research, whatever sets the number of beta cells, could
have … be affected by the kinds of diets that the mother or the parents had. So, I think that there is a lot of biology
that suggests that maybe there are things that people are studying that they think are
independent are actually affected by these husbandry questions. I think this is an education … so I think
our colleagues … if we communicated the importance and what we know about how the
microbiome changes and what we know … that may open up more possibilities, because many
of folks just think, this embryo and that yolk are just the same in every embryo, not
the idea that the yolk lipids make up the lipids that (indiscernible) and changing those
things change their embryo. So, you know, I guess I’m just arguing there
is an education component to all the ways that these things affect what the developmental
biologists do not perceive right now as important. MODERATOR: Okay, with that, we’ll …