If I'm not mistaken, probiotics are different from immunostimulants or vaccines. The main aim of using probiotic bacteria is to make them colonize the fish intestine to (i) prevent the development of pathogens and (ii) make them metabolize more uptakable nutrient for the fish. Thus, it is not rare that probiotics enhance growth rate in vertebrates. These are even suspected of playing a role in human obesity by some researchers (yogourt probiotics).

Therefore, intuitively, the preference order would be: feed > water >> injection. Taking into account that marine fish drinks much more than freshwater fish. Still intuitively, when you inject, you may also elicit an undesirable infection and immune response. What do you think ?

If I'm not mistaken, probiotics are different from immunostimulants or vaccines. The main aim of using probiotic bacteria is to make them colonize the fish intestine to (i) prevent the development of pathogens and (ii) make them metabolize more uptakable nutrient for the fish. Thus, it is not rare that probiotics enhance growth rate in vertebrates. These are even suspected of playing a role in human obesity by some researchers (yogourt probiotics).

Therefore, intuitively, the preference order would be: feed > water >> injection. Taking into account that marine fish drinks much more than freshwater fish. Still intuitively, when you inject, you may also elicit an undesirable infection and immune response. What do you think ?

In my opinion feeding treatment with probiotics is more effective than other ways, because the aim of using probiotics is change of microbial colony of fish gut. If you work on fish larvae you can also enrich live food or mix probiotic in water of culture system.

Depending on the fish species of interest, some species are more hardy than others and it will be unlikely to inject a fish without causing it stress. Thus infection via feed is the preferred option of infection and a more natural approach.

I fully agree with earlier comments, that addition of probiotic bacteria to food is preferred. You may also try the route via water, but injection will probably evoke undesired immune responses in fish.

Aliyu you have some questions there. However I think it may be better to introduce the probiotic via feed. Besides I think the best word to use is not infection since probiotics are modulators and enhancers. The fish are small and can as well receive the probiotics through diets if the diets are well prepared. I am presently involved in collaborative research involving feeding of smaller fish. Alternatively mini piping can be used in introducing the probiotics. This may be more direct and specific method ensuring absolute result.

yes it is recommended that probiotics mixed in feeds and given to the fish.

From my researrch, i found injection of fish intraperitoneally very convenient for them. They had feed conversion rates of 0.55, which was most desirable compared with other treatments. Although after administration, they showed signs of morbidity, they recovered with a bang and outgrew my control samples in multiple folds. Yes, intraperitoneal administration might evoke some undesired immune response like Willem B. Van Muiswinkel said, yet i find intraperitoneal administration fast and reliable. Incorporation in feed is most convenient and less vigorous, but may not reach the desired sites and in enough concentration as to effect the desired result. So, you could incorporate it into the feed for convenience sake, but at a higher concentration. Hope this helps

Fundamentally

-What is the mechanism of action you are after with your probiotic?

-What site are you trying to get the probiotic to?

If it’s the conventional site in the intestine, feed is ok, fat rich feeds even better since the fat will protect the bacteria from the stomach acid. Even better still is a capsule that only breaks down in the intestine or a cannula (i.e. a direct feeding tube) into the intestine so you can be sure what is delivered, note this may not be practical in fish (It works very well on large animals like sheep or cows!). Furthermore if your work is applied (so creating a product), you want to create a system that works industrially-a formulated feed is one of the few ways you can achieve a result.

However if you are after an immune-modulation effect, you might get away with injection (as alluded to above). The science of this is that you target the TLR9 receptor that recognises a DNA motif found in bacteria (particularly in the lactobacilli). The activation of TLR9 seems to modulate the immune system and reduce inflammation. The science of this is particularly well studied since it’s the basis some asthma therapies that use synthetic DNA and it appears to be the mechanical link between the hygiene hypothesis and asthma.

These are given through feed.

I reckon it is better to mixed in feeds. But, I I feel if you will also test them through culture water or injected IP, them you can compare it with the one which is recommended. Difference between good researcher and extra ordinary researcher is that latter keeps eye to everything and does not rule out any possibility.

..the probiotic bacteria are required to line the gut of the animal, and be situated in the crevices and folds of the epithelial cells creating the required surface area and spread, and this efficient spread will only be best achieved via feeds.

Moreso the retention of undigested food in the hind-gut of the fish will increase the probability of the tissue surfaces in the hind-gut picking up and retaining these probiotic organisms.

Feed it is!

Thanks everyone for the interesting contributions.

However, when we mix probiotics in feed, the application becomes continuous. Meanwhile, I am rather aiming for pulse application, where the effects of application could be measured within that specific period, as continuous application has been shown to elicit negative effects when applied in some species. In any case, the current trial is aimed to evaluate immune response (via ELISA and genomic assays) following the feeding of an immuno-stimulant in the diet, in the presence or absence of probiotic bacteria. Since the immuno stimulant is already included in the diet, the attempt is to verify if adding the probiotics in the culture water would be effective. In this case therefore, Grant Bennett's questions add some interesting 'spices' to the discussion, exposing us to other perspectives. Considering dietary route alone as the most effective in this case becomes, what? Redundant, lazy or narrowing possibilities?

What would anyone recommend in this scenario? Remember, immune response (especially if one is targeting to measure pro-inflammatory cytokines like TNF alpha, IL-1 etc.) is time bound.

The problem I see coming if you add probiotics into the water is that if you have no probiotic effects, you will never know if it is because the probiotic has no effect or because the probiotic did not enter into the host. If I were you, I would definitely add the probiotic to the feed to get rid of this possible situation. Moreover, when you add probiotic to the water, you don't know its behaviour. Its concentration may vary with time from tank to tank causing higher variability in the biological response. However, these are only thoughts. I have only work once on this. What do you think ?

Hi Neil, the immuno-stimulant we are testing acts potentially as a prebiotic. In vitro, we have been able to prove that this prebiotic promotes the proliferation of the probiotics bacteria as a preferred carbon source. Our current objective is to prove that the probiotic bacteria are NATURALLY attracted to this prebiotic in the fish gut (in vivo), the reason why we included it in the diet. Therefore, to put the probiotic bacteria in the culture tanks is supposed to be our 2nd treatment. Thus the experimental design would be;

- Control- Feed fish a diet without the prebiotic

- Treatment 1- Feed fish a diet with the prebiotic (qty previously established)

- Treatment 2- Feed same diet as Treatment 1 but with probiotic bacteria in culture water

After feeding for 4-6 weeks;

1. Challenge (dip) all surviving fish from all treatments with a vibrio (the pathogen we are testing) and monitor mortality

2. Take tissues (liver, intestine & kidney) for ELISA (cytokines) and qPCR(genomic analysis for selected enzymes; iNOS, COX-2)

Our reasoning are that;

*Probiotics in culture water mimics the mode by which the bacteria naturally scout for nutrients and when they do encounter their preferred choice, they proliferate. Adding the probiotics in the feed thus seem artificial?

*Dip challenge mimics natural mode of fish infection, compared to IP injection

In any case, we always check the fish gut to find out if the probiotics are there.

What are your thoughts?

Dear Mohammed, first of all, it is a pleasure to discuss with you. Your experiment sounds really great ! Plus, investigating the fish gut to see if probiotics are established is a very good idea. I didn't know this could be done. This way, you can indeed test the probiotic in the water without the difficulty of interpreting a no-effect result.

However, will you work on freshwater or marine fish ? I ask this because it seems that freshwater fish don't drink much, contrary to marine fish that drink a lot (osmoregulation). I suppose that the main route leading to fish gut is drinking? So, even if it is not more than a hypothesis, one can expect that adding probiotic to the water is much more efficient with marine fish than with freshwater fish ? Someone could confirm or infirm that ?

You can check this paper: we worked on Eurasian perch larvae (freshwater fish) and tested a probiotic on growth and survival. The only treatment that gave no effect was when the probiotic was only added to the water.

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2109.2010.02744.x/abstract

Besides, I don't think that adding to the feed would be superficial. In the wild, I suppose that fish can also be contaminated by what they eat.

Could you tell me what is your idea of the mechanisms by which the bacteria in the water would be naturally attracted to the prebiotic that is within the fish gut ?

Hi, best method for infect fish with probiotics in 2gr is mixed to food.

It is a good topic. The probiotic or prebiotic applications (as a food additives for fish) are popular. The beneficial effects are still studied with respect to the well-being and health of fish. Future Expectations: probiotics significantly reduce antibiotic usage in aquaculture.

The good method to infect fish with probiotic bacteria is to give in the feed is a good approach to control the disease in the fish and also in the water than with injection. with injection we have to catch every fish and make it stressful and many times they wont recover and die with infections .If there is a disease in the culture water we have to mix probitoics in water or in sand , after that we have to go for feed probiotics.

The pathogenic bacteria will be more in water and than in the guts. probiotics are very useful to enhance the health of fish and also water system i suggest both are very important either the water probiotic or the feed probiotics.

IP injection will stress the fish,predispose them to infection which may eventually lead to mortality. Addition of probiotic bacteria to the water and the feed will be very effective.

I think water is the efficient way of introduction of probiotic preparation for Aquaculture

You can try with Bio-encapsulation technique through Artemia. This would be an efficient technique without any loss of prebiotic and probiotic.

Hi everyone, inline with this interesting discussion i was wondering in which "carrier substance"would you add the probiotics into feed pellets?

Dear Mohammed Aliyu-Paiko

How did you proceed with this in your study?

Since you have mentioned about pulse application, why don't you try with oral drops. Hope this will give the intended result than feed trials. IP injection is not suitable for this kind of study.