Fish Physiology - Science topic

Hello

I advise you to consult this technical document

Dear kindly search for the areas of research you what to cover.

Hello

Kindly look at PDF files will help you

No feeding during the experiment will surely affect your results. To feed fish daily is as important as you also enjoy food everyday!

You can add this herbicide in fish feed formulation and just compare your results with control and treated. 7 day trial is too short for the true results, It should be of 90 days at least. 

Good Luck!

You can find 61 species' TL-gape relationships in my publication, as well as general ones based on species' functional trophic groups:

KARACHLE PK & STERGIOU KI. 2011. Mouth allometry and feeding habits in fishes. – Acta Ichthyologica et Piscatoria, 41 (4): 255-275. (attached)

To my knowledge there is no such general relationship .

Regards

Voula

The International Society for Fish Endocrinology has now been established, see https://isfendo.com/

As the question is no longer valid, I tried to delete it, but found out that it's not possible to delete RG questions which have already received answer(s).

Higgs 2004 "Neuroethology and sensory ecology of teleost ultrasound detection" I supose best answer is in this paper.

Dear Paraskevi,

Please check the following resources.

Regards

SM Najim

Nitrogen Free Extract (NFE)

NFE is determined by mathematical calculation. It is obtained by subtracting the sum of

percentages of all the nutrients already determined from 100.

%NFE = 100-(%moisture + %CF + %CP + %EE + %Ash)

NFE represents soluble carbohydrates and other digestible and easily utilizable non-nitrogenous substances in feed.Total carbohydrate component was crud fiber and soluble carbohydrate (NFE), NFE was the source of energy since most fishes can not digest fiber.

A.Y.Al-Dubakel

Hi Adel, may you dissolve vitamin A in oil and spray over pellets?

Hi Björn,

I used the Distell Fish Fatmeter to evaluate small pelagic fish lipid content (mainly anchovy, sardine and sprat). It works quite well, we compared Fatmeter estimations with lipid extraction by Folch method and it correlated well.

I confirm the fact that "the standardized measurement of well-defined body zones that need to be strictly the same for all fish if we want to be able to compare them" as Nicolas said.

In the different studies, the lowest fat content that I measured was 1.8%.

Pablo

Hello, 

You can also use this illustrated database: http://fishbone.nottingham.ac.uk/

Silurus is among the entries. 

Radu V. (2005)

Dear all,

for sure, dissection will bring a final result for this! But, as everyone knows it is an invasive and destructive method. Testing if echographia could be applied here could be interesting. At least, it will preserve animals.

This technique may provide results for mature animals, but for juveniles and immatures, I guess that dissection remains the sole applicable method.

Reasons will be related to light penetration on clear waters as hill streams. Fish skin have melanophores that, stimulated by sunlight can produce different colour  as we see in the Pantanal. The same fish, if it is in clear water,  is more colourfull than in  lowland water.

My experience is that with the injection of hormone (Ex: Ovaprim) as a releasing agent, eggs final maturation occur and if they do not spawn naturally or by striping septicemic condition might occur and brood fish might die in due course. we should ensure water hygienic condition. Most of the time overripe eggs may biologically absorbed and refurbish at the next cycle.

Hi Wolfgang,

Thank you for your response. Admittedly there are a number of histopathology handbooks that state the maxim of "20:1 volume to volume ratio', however my statement of using 10:1 is not merely based on my own professional experience but is also in the literature.

If you refer to the highly regarded histological book "Theory and Practice of Histological Techniques" 6th Ed, edited by Bancroft and Gamble, you will find the comment "fixative volume should be at least ten times the  volume of the tissue specimen...".

There are even papers that suggest as little volume as 2:1 is adequate (under controlled temperature and pressure variables). Though this isn't a ratio that I have come across in practice.

Using lower volumes of formalin is more than an 'economic factor' in any laboratory where health and safety is a consideration. 

I would also suggest that some of the tissue specimens you mention that have suffered from inadequate fixation may have suffered in delay from theatre to the lab, or whether the specimen was 'opened up' in a timely manner to allow inner tissue penetration.

I think we can probably agree that understanding fixation and its principles is more useful than sticking to a one rule fits all ratio.

Kind regards,

Sarah

Dear Afaf Mohamed ,

I usually use the Bouin's fluid (Aquous solution of picric acid = 75ml + formaldehide PA = 25ml + 05 ml de Acetic Acid) for animal tissues in general, for traditional histology with hematoxylin-eosin .

This is because it does not deform the histological parts.

It causes immediate cristalization of proteins, which prevents the muscle fibers to contract with the fixation .

It also allows a brighter color, leaving the affinity heads anilines more receptive to the stainings.

It is a faster fixator and the histological fragments should have a maximum of 1 cubic centimeter. Use of 8 to 20 times the volume of the histological fragment by up to 3 days.

Empty Bouin liquid and pass the pieces to 70 % alcohol.

But if you are studying glycogen storage , I indicate the alcoholic Bouin ( Gendre ) , because the alcohol will not allow the muscle or liver glycogen dissolves and colorings with Periodic Acid Schiff occur properly .

Kind regards.

It looks like subcutaneous lesions - possibly of parasitic origin. I would try to dissect into one of the spots and examine microscopically to see what's there.

Yes - the best reference would be:

Peterson et al. 2008.  When eradication is not an option: modeling strategies for electrofishing suppression of nonnative brook trout to foster persistence of sympatric native cutthroat trout in small streams.  North American Journal of Fisheries Management 28:1847-1867

But the following would also be good to read to help provide context:

Peterson and Fausch 2003 Testing population-level mechanisms of invasion by a mobile vertebrate: a simple conceptual framework for salmonids in streams.  Biological Invasions 5:239-259.

Peterson et al. 2004.  Population ecology of an invasion: effects of brook trout on native cutthroat trout.  Ecological Applications 14:754-772

These papers studied wild fish, but ratios should be analogous...

If you're looking for simple procedures, you should homogenize the fish tissue in an adequate buffer (either using an ultra-sounds probe or a mechanical homogenization approach), centrifuge the debris and use the supernatant in the quantification kit.

This wont render a protein extract neither clean enough for further applications, nor 100% efficient in the extraction of protein.

Another (the most important) consideration to make is the choice of buffer. Standard protocol involves using PBS, which I recomend for its simplicity and compatibility with most quantification kits. However, some other buffers might be more efficient.

As to this point, if you want further help, it would be nice for you to say what protein quantification kit you have available... when extraction is made with the sole purpose of quantifying protein, it is better to design homogenization having the kit in consideration.

·         Weighted the organs i.e liver.

·         Added phosphate buffer (pH 7.4) 4 times greater than the weight of organ i.e 1:4.

·         Homogenize it for 15 minutes with the help of pestle and mortar or with the help of homogenizer with short intermissions.

·         Passed the homogenized tissues through muslin cloth to remove the biomass.

·         Filtered the liquid obtained from muslin cloth with the help of whatman filter paper no. 1.

·         Centrifuged the filtrate obtained above step in refrigerator centrifugal machine at 10, 000 rotates per minutes for 15 minutes.

·         Both sediments and supernatants separated for further analysis.

·         All the enzyme isolation steps will be carried out at 4°C.

The supernatants and sediments were stored at 4°C until further analysis.

0.371 g NaH₂PO₄ and 0.270 g Na₂HPO₄ was taken in a flask and added distilled water to make volume upto 100 mL and pH was adjusted 7.0.

Hi Afaf,

the following link provides the desired buffer table

Best regards,

Chris

Hi Michael,

what are you trying to get with the ELISA?

For simple and easy method use Turk's solution. You can see the WBC as colored by blue or violet under microscope.

Hg.and Pb-  

Hg because form dimethyl compound or methylmercury cation

Pb because i have been found that from concentracion of 1  ppm changes the behaviour of fishes,

Besten Dank! Werde nachfragen, alles Gute!

First of all it depends on age of fish.If you want to get  slides of good quality you have to decalcify Gills of adult fish

Dear Christopher, thank you for your kindness reply.

It is easy to perform procedures for sex inversion (sex reversal) using enclosed facilities. The procedures for the sex reversal of open water fish depends on a lot of factors. What species of fish will you be using and at what stage of the fish will you administer sex reversal? For species like tilapia, sex reversal is performed by feeding them with hormone-treated feeds for 17-21 days (at fry stage) . There are also research conducted by immersing eggs in hormone solution for 24-96 hours. You may try immersing the fry / egg first in a hormone solution. 

Thanks for the articles. I have got the 1st one. Please send me the 2nd article.

Fixation in buffered formalin may have a small effect on gonad weight, however it shouldn't be large as the fixative is intended to avoid significant distortion of tissues. I have provided a link to a study done on spanish mackerel, which are superficially similar to sablefish, and provides conversion equations which may be useful.  

You cannot use whole body values instead of blood values for many hormones, since their targets in the body can be widely varying in the tissues, and because blood volume is only 3-4 % of the total fish volume. So, even if you in large fish, where blood sampling succeeds easily, see a significant change in a hormone concentration of blood, if hormone binding occurs only in a small percentage of the rest of the body, any change may remain undetected in whole body measurements because of limits of resolution of the methods. Where the hormones are secreted to water (only some of them are), the water measurements can be a useful measure (of changes).

Maybe this review is helpful

Yes indeed, the clock is temperature compensated.

From my experience - I mean in vitro experiments using pike or white sucker pineal glands-  temperature cycles can synchronize the melatonin secretion rhythm under LD. Temperature cycles can also synchronize the rhythm under DD, i.e., it can synchronize the pineal clocks. However, a return to constant conditions (temperature & DD) results in an loss of the endogenous melatonin rhythm. This suggests to me that temperature cycles cannot entrain the pineal clocks. Another indication of this is that the melatonin rhythm  is of high amplitude providing the temperature applied during the night (or subjective night) is as closed as possible to the preferred fish temperature. Whether this applies to other clocks in the fish organism, I don't know. I can send you some refs later if you want to.

Melatonin is one output of the pineal clocks. How does temperature impact the clock genes ? I cannot tell. I suspect it may have to do with the fish thermal preferences and aerobic scope.

Accumulation of copper and zinc in higher concentrations in comparison with other organs is characteristic of the liver, which is associated with exchange - depositing liver function. Gill epithelia in comparison with other integuments of fish has a substantially larger surface area and actively interacts with the external environment, so gills virtually have no protection from the effects of different substances present in water including metals.

Stressed fish generally grow more slowly than non-stressed fish. Stress elevates cortisol which has a direct suppressive effect on growth. Also, energy that would otherwise be directed to somatic growth is used to combat the effects of stressors. I've attached an old article of ours with some useful citations.

You can use endogenous or internal markers which are naturally available in the feed such as: ash, crude fiber, cellulose and hydrolysis resistant organic matter. You only need to determine one of these internal indicators in the feed and fecal, then use the formula according to Cho and Slinger (1979) for calculating ADC. For more information please find attached file.

ADC (%) = 100 – [100 × (marker in diet / marker in faeces)] × [(% nutrient in faeces / % nutrient in diet)]. 

Cho, C.Y., Slinger, S.J., 1979. Apparent digestibility measurements in feed stuffs for rainbow       trout. In: Halver, J.E., Tiews, K. (Eds.), Finfish Nutrition and Fish feed Technology, Vol.     2. Heinemann, Berlin, Germany, pp. 239–247.

Best regards

Mansour

Hi Marvin, was increase in protein content correlated with decrease in fat content? If yes, it might be related to differences in water temperature due to oceanographic (climate) changes or sample bias. In warmer waters expenses on metabolism are higher so less of fat to deposit.

Hi Pierre,

The logics in your supposition is an obvious, and based on the following strong experimental and epidemiologic data. Thus, in Stefanelli et al. paper entitled “Organochlorine compounds in tissues of swordfish (Xiphias gladius) from Mediterranean Sea and Azores island” (Mar Pollut Bull. 49(11-12):938-50, 2004), to develop sensitive biomarkers for the evaluation of toxicological risk, 34 congeners of PCB were measured in swordfishes (Xiphias gladius) taken from the Mediterranean Sea along the Sicilian coast (Strait of Messina, Italy) and in the PCB-clear Atlantic Ocean along the Azores Islands. In the tissues of Mediterranean swordfishes the sum of the determined PCBs congeners ranged from 4.61 to 4651.17 ng g(-1) on fresh tissue basis. In the liver of Azores Island swordfishes lower levels of summation PCBs (8.43-294.17 ng/g w.w.). Thereafter, Maria Fossi et al continue that approach by publishing a paper entitled “Endocrine Disruptors in Mediterranean Top Marine Predators” (ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH 13(3):204-7, 2006), where they investigated P4501A1 activity (namely, ethoxyresorufin-o-deethylase) in the pelagic fishes live in neither close to the bottom nor near the shore, such as the swordfish and also in tuna fish from the same Strait of Messina. A 4-year survey revealed almost 3 times higher P4501A1 activity in the liver of that species, comparing to that ones from Atlantic Ocean along the Azores Islands. The role of increased bottom level of PCBs along the Strait of Messina statistically correlated with the above induction of ethoxyresorufin-o-deethylase in the livers from swordfish and tuna fish.

Best wishes,

Ilya

We just conducted a study investigating a growth-promoting chemical with yellow perch (Perca flavescens) reared at 24°C. The fish were fed a high quality diet to satiation twice daily. The controls had specific growth rates of around 3-4. The experimental fish had SGRs from 5-8. In the wild SGRs for this species are in the 1-2 range. 

Thank you all. You will see my paper very soon.

None of these  species occurs in Mediterranean.

In histological sections through the intestine image, can be observed some goblet cells (large white dots). Other dots (smaller) are vesicle for endocytosis - pinocytosis.

Yes indeed. Try this document by Walt Golet.

Golet WJ, Galuardi B, Cooper AB, Lutcavage ME (2013) Changes in the Distribution of Atlantic Bluefin Tuna (Thunnus thynnus) in the Gulf of Maine 1979-2005. PLoS ONE 8(9): e75480. doi:10.1371/journal.pone.0075480

I would suggest to consult the following references:

Capapé, C. and M. Desoutter 1979. Nouvelle description de Aetomylaeus nichofi (Bloch et Schneider, 1801) (Pisces, Myliobatidae). Premières observations biologiques. Cahiers de l'Indo-Pacifique v. 4: 305-322.

Ruocco, N. L. , L. O. Lucifora, J. M. Díaz de Astarloa, E. Mabragaña, and S. M. Delpiani 2012. Morphology and DNA barcoding reveal a new species of eagle ray from the southwestern Atlantic: Myliobatis ridens sp. nov. (Chondrichthyes: Myliobatiformes: Myliobatidae). Zoological Studies v. 51 (no. 6): 862-873.

White, W. T. 2014. A revised generic arrangement for the eagle ray family Myliobatidae, with definitions for the valid genera. Zootaxa 3860 (no. 2): 149-166.

White, W. T. , J. Kawauchi, S. Corrigan, E. Rochel and G. J. P. Naylor 2015. Redescrition of the eagle rays Myliobatis hamlyni Ogilby, 1911 and M. tobijei Bleeker, 1854 (Myliobatiformes: Myliobatidae) from the east Indo-West Pacific. Zootaxa 3948 (no. 3): 521-528.

White, W. T. , P. R. Last and L. Baje 2015. Aetomylaeus caeruleofasciatus, a new species of eagle ray (Myliobatiformes: Myliobatidae) from northern Australia and New Guinea. Ichthyological Research: [1-16].

White, W. T. , P. R. Last, G. J. P. Naylor, K. Jensen and J. N. Caira 2010. Clarification of Aetobatus ocellatus (Kuhl, 1823) as a valid species, and a comparison with Aetobatus narinari (Euphrasen, 1790) (Rajiformes: Myliobatidae). In: Descriptions of new sharks and rays from Borneo. CSIRO Marine and Atmospheric Research Paper No. 032: 141-164.

White, W. T. and A. B. M. Moore 2013. Redescription of Aetobatus flagellum (Bloch & Schneider, 1801), an endangered eagle ray (Myliobatoidea: Myliobatidae) from the Indo-West Pacific. Zootaxa 3752 (no. 1): 199-213.

Hello Ahasan,

I suggest that you start simple - no transformation yet. Using residuals of fish lengths and weights, check if assumptions (eg normality, etc) of your analysis are satisfied. I suggest that you do data transformations only if you can extract additional information that is not available from the simple analysis.

You mentioned reason of performing log-transformation: to remove the size-dependent variation. Again, ensure that you can communicate what resulting log transformed coefficients mean.

hi, you can use one of microscopic lamel after anesthesia of fish. for collection of mucus pull the lamel on the gill filaments, gently.

EE2 is a well known endocrine disruptor, entering aquatic environments mostly due to it being a major, stable component of contraceptive pills. It's an estradiol-mimicker, stimulating cellular processes through its stimulation of the estrogen receptor, which is found in both male and female fish. One of the earliest signs on EE2 exposure will be the appearance of vitellogenin in the circulation of the male fish, which of course is abnormal. So it really depends on your definition of "safe" when you ask if it is safe for the fingerlings..

There is a huge amount of literature on the disruptive effects of EE2 in fish which you'll easily find by simple data search.

I'm wondering what the purpose of the research is?  You haven't explained that. Is it to study endocrine disruption - in that case you should use levels similar as are found in contaminated waters.

You seem to be proposing to expose your fish to EE2 for an extended length of time, from "30 days post-hatch until the bass becomes sexually mature". This must be years rather than months, which means that you have to add a lot of EE2 to the water the fish are being raised in. I hope you have an aquarium system that will not release this EE2 contaminated water into the environment as it's really harmful, and also that you also make very sure that none of the exposed fish goes to human consumption!

In the Type image databae of the California Academy of Sciences: Ichthyology, there is an x-ray of the head and the caudal fin of the holotype of Carcharias azureus Gilbert & Starks 1904 (CAS-SU 11890), which is a junior synonym of Carcharhinus leucas (Müller & Henle 1839) -- see the link below.

Malcolm's idea of making a video is quite good enough.I would like to add one point.Activity of a fish as observed is through movement by fins & its eyes. So number of times a particular fin makes a movement as well as eye ball movement can be measured from both the sides.  

Sometimes this can be dietary derived, for example the deposition of pigments from corn (maize) that has been included in the feed

This was previously not uncommon in channel catfish farming in USA

Soon, I'II have image with opercular bones of Danube salmon (Hucho hucho). Probably, in a month. For now, we process them (should be dried). 

I agree with Ramon, in red muscle tissues; in some species of fish one can find more red muscle tissue below the lateral line, especially near the tail-end of the fish. 

The pituitary is usually attached to the brain ventrally, in the midline between the inferior lobes, anterior to the saccus vasculosus.

For illustrations of the pituitary in several teleost families see the following paper:

Honma, Y. 1957. On the pituitary gland of some Japanese teleosts. Japanese Journal of Ichthyology, v. 5 (nos 3-6): 175-181.

Wstern blotting is OK, on the other hand one does not determine protein with qPCR, only the template so one does not know anything about protein level without previous inormation about mRNA protein correspondence

Please refer to follow book.

Methods for the estimation of production of aquatic animals.  by G.G. Winberg

I think this book will be good for you

Water Pollution and Fish Physiology

Hi Andrey,

This is a fascinating topic - The pattern of absences and presences strongly suggests that convergent evolutions of a gallbladder are at least possible. And certainly it is often lost (as in many bird groups). This is a secondary source, but Kardong's textbook (Vertebrates) states that the gallbladder is lacking in "cyclostomes" (518). This might also be loss; people tend to forget that hagfish and lampreys are highly specialized animals. 

If you intend to pursue this further, the next step might be to rework a Gorham and Ivy -style diagram, but with modern relationships. Since their paper, taxonomic opinions and cladistics in particular have changed much of this phylogeny. I see you have added Afrotheria, which brings out the pattern of loss in Elephants. Contra the figure sharks certainly have a gall bladder, by the median lobe of the liver in dogfish for example.

I can't agree more that there are some fascinating lacunae left behind in modern science - ample work for us to pursue. 

Some times government quality control officer visited our fish processing industry once or twice in a month; they usually defrost our frozen processing sea-food. Defrost, frost in once, it might be OK to avoid shrinkage; but more than once it might be caused for shrinkage. Controlling of temperature during transportation is the helpful for avoiding shrinkage. I am referring the HACCP manual which means Hazard Analysis Critical Control Point. 

Using either gamma counter of Elisa technique.You are able to perform with either of them.Ask more question if you need.

Ah. Sympathies! I hate when that happens. A few things to add to the good advice already given:

Check that the light cycle timers are working - ours was once set for 14/10 but stuck "on", and that devastated breeding cycles for a while. Same can happen if someone's there at 'night' opening the door and disrupting the dark period.

Another thing you could try could be to tilt the mating tank inserts so that the grating lies at an angle - the zebrafish like to spawn on the shallow "beach" end, almost out of the water. Sometimes that helps when mine won't lay. You could also try autoclaved plastic plants or marbles to give the mating tanks some more natural features, and make sure there are plastic plants in the adult aquariums if there are only a few adults, because they're sometimes stressed/bullied by dominant in that situation.

I don't know how chubby the females are, but if they haven't laid it a while you could try gently squeezing them, perhaps they're eggbound? Also, how old are the fish you're using?

Goodluck!

There are many reasons for use juvenile:

- It’s a critical life-stage for the survival of a population

- juveniles are more sensitive to toxic

- adults may have been subjected to selection pressure : juveniles represent all the genetic variability

- there are more confounding factor with adults : stage of maturity, sex (M/F), etc. could modulate the response to toxic

- juvenile are more small and more practical for laboratory experiment with many individuals

- …

Dear Plante,

It is hard to accept the retention time of a certain compound differs in different medium (acetonitril and plasma). specially when fluorescent detection method is applied. However, I faced same problem in my assay of amino acids (gradient mobile phase). By changing the percentage of mobile phases, my problem was solved.

Hope it helps.

Hi Nitin, in addition to the pCO2, you are going to need either the total alkalinity or the total CO2 (the dissolved inorganic carbon). 

Assuming you have either of those, you can download the Excel-based CO2sys ocean carbonate calculator, available at http://cdiac.esd.ornl.gov/oceans/co2rprt.html

If you are familiar with the r-project, there is a very nice library called seacarb (http://www.obs-vlfr.fr/~gattuso/seacarb.php).  

Resting plasma glucose levels (mg/dL)

Dear David,

I think you should use a camera to avoid the stress caused by your presence.

The whole experiment could be filmed which would be better than visual observation because you can watch the film as many times as you want.

Cheers, Roland

Thanks for the suggestion. My original thought was to compare growth parameters between regions. Additionally determine if differences in growth parameters are due to tissue contaminate concentrations.

Below may helpful for you.

I tried several time for preparing primary cell line from some fish, it is a bit difficult but you should attention about the degree around trypsinized tissue

Dear Ardavan

i think you can be use follow article for you research

Blood glucose in crayfish—II. Variations induced by artificial stress

Physiological Responses to Acid Stress in Crayfish (Orconectes): Haemolymph Ions, Acid–Base Status, and Exchanges with the Environment

Neuroendocrine and metabolic responses of Pacific whiteleg shrimp Litopenaeus vannamei exposed to acute handling stress

Stress reduces hemolymph ecdysteroid levels in the crab: mediation by the eyestalks‏

i hope usefull

One need to be very careful to interpret the relative amount of nutrients. If lipids are reduce according to protein, then the protein will increase relative to the lipids. This does not mean that protein increases itself. One could for instance try to manage net changes. If you have the initial weight from the animals with their proximate composition in absolute numbers, you can compare to those data after starvation. By analysing their reduction in weight in absolute number an related to the proximate composition.

while acknowledging the fact that there are many studies on lipid profiling (fatty acid, cholesterol, oxysterol, phytosterol and phospholipid content) in response to toxicants, heavy metals, etc., you could expand your idea on testing antioxidant enzymes, which are directly associated with lipid profiling. Some pesticides such as dicofol increased oxidative stress by elevation of lipid peroxidation index associated with depletion in glutathione level. Cu intake at high concentration induced adverse effects on lipid profile, associated with oxidative stress and diminished activities of antioxidant enzymes.

Björn, cortisone and corticosterone only differ by a keto group on C11 in cortisone (substituted by a hydroxyl group in corticosterone), but perhaps the papers by Franci Weyts on cortisone and cortisol in carp are interesting. She uses an antibody raised against a cortisol-BSA-adduct with <10% cross-reactivity to cortisone.

She also measures an increase in plasma levels of both cortisol and cortisone in response to a stressor.

I believe that the metals are the cause of steatosis that you have found. In human pathology is well known that one of the causes of non-alcoholic steatohepatitis are heavy metals (even if the root cause is metabolic and metals probably interfere with the metabolism). I beg to enclose an article that perhaps it will be useful (Advances in metal-induced oxidative stress and human disease). good work

I suggest that you make a frequency distribution of all your resting/routine determination and the fit them to the sum of two normal distributions - as described in "Steffensen, J. F., Bushnell, P. G. & Schurmann, H. (1994). Metabolic rate of 4 Arctic species of teleosts from Greenland. Polar Biology, 14; 49-54" that can be downloaded from here: http://www.mbl.ku.dk/jfsteffensen/Publications/PolarBiol%201993%20JFS.pdf or from Research Gate.

The normal distribution to the left represent resting or standard metabolic rate - the normal distribution to the left routine metabolism.

This method requires that you have quite a few metabolic rate determinations. This can be obtained by intermittent respirometry as described in: " Steffensen, J.F. (1989). Some errors in respirometry of aquatic breathers: how to avoid and correct for them. Fish. Physiol. Biochem. 6; 49-59" that can be downloaded from here"http://www.mbl.ku.dk/jfsteffensen/Publications/FishPhysiolBiochem%201989%20JFS.pdf or from Research Gate.

Software (free-ware and open-source) for automated respirometry can be found at http://www.AquaResp.com - a new version written in Phyton will be available soon. This version will be able to run on PCs, Macs as well as Raspberrry Pi

John Fleng Steffensen

Hi Javed

To clearly answer your question, I think you should search on google to find out the related studies. I recommend you to read one of my paper and find the paper's references and I think that you will answer your question. Good luck with your studies.

Le et al, 2010. Distribution of Trace Metals and Methylmercury in Soft Tissues of the Freshwater Eel Anguilla marmorata in Vietnam. Arch Environ Contam Toxicol