Guppy Nutrition - Part 2.


Before I continue I have to fix two errors in Table 2 at Part 1. I wrongly typed that a proximate analysis of microworm resulted in 79% crude protein in the dry matter...The right  score is 49 % crude protein in dry matter. Second I didn't say that levels reported at Table 3 are on feed basis instead dry matter basis.

Proteins and Amino Acids (continued).

I was not able to get an analysis for the amino acid (AA) profile of my fishes so the only way is to figure a recommendation for AA levels in guppy diet based in data available for another fish species.

Based on the idea that AAs content in whole body and the dietary AAs requirements should be correlated some researchers recommend use body tissue analysis to estimate essential amino acid (EAA) requirements of those species for what requirements were not yet determined by the classical dose response curves. This concept is called "ideal protein balance". Anyway this methodology is not the best available to quantify amino aids needs because it assumes that all ingested AAs are deposited in body tissues and  we know that is not true.

Oohara et. al (no date, pp85) already compared the EAAs requirements and their profiles in the body of several fish species based on data available in literature using an index called A:E ratio. This index is given by: [(each EAA content ÷ total AA content including cystine and tyrosine) x 1000]. This methodology was used to overcome the variations due to the different protein levels and AA profiles of test diets observed when absolute values are compared. They found that (1) species belonging to the same family have similar AAs requirements; and (2)  A:E ratios of whole body tissues of different species shown lower variation  than A:E ratios of dietary requirements.

Table 4 - Comparison Between A:E Ratios of Whole Body Tissues of Different Fish Species and that of Swordtail.

I included Table 4 in this piece in order to compare whole body AA profiles of all fish species for what I got data to the whole body AAs profile of swordtails that is the guppy's closest species for what I found data. Kruger et. al. (2201) were not able to quantify the levels of cystine, tyrosine and tryptophan in swordtail tissues. Then is clear that A:E ratios of  methionine + cystine and phenylalanine + tyrosine were underestimated and that A:E ratios of left AAs were overestimated. Taking this methodological particularity into account I see that swordtails have an AA profile that matches that ones of most fishes and for analogy I assume the same for guppies. 

Like (1) guppies should have a whole body AA profile similar to species for what we know AAs requirements and (2) crude protein requirement of guppies is in the same range like the requirements of fish species for what we know AA requirements (30-40% crude protein) I feel comfortable to recommend that guppy diet be planned to fit something in between the average and maximum recommended levels of AAs already determined for food fish species for what I found recommendations (Table 5) perhaps using data from Table 6 or whatever you get on your hands.       

Table 5 - Essential Amino Acid plus Cystine and Tyrosine Requirements of Some Food Fish Species (% dry matter). 

Table 6 - Essential Amino Acid plus Cystine and Tyrosine Content of Some Guppy Feeds (% dry matter).

The explanation for different colors of types is the same I used in Part 1, values in black exceeds the maximum recommendations in Table 5; values in blue are between the average and the maximum values; and values in red do not even fit the average recommended levels. The recommend levels for cystine and tyrosine alone were determined comparing levels of methionine + cystine and phenylalanine + tyrosine to the recommend levels of methionine and phenylalanine, respectively.

The point that caught my eyes in this table is that sulfur AAs (methionine and cystine) looks to be the first limiting ones in live feeds. Since main protein sources of pastes (beef liver and beef heart) and a dry feed formulated with a good quality should be relatively rich in sulfur AAs is clear the importance of a diversified diet to meet guppies nutritional requirements. 

Luckily we bred an specie that readily accept inert food since it was a newborn so we can, "we must" I should say, combine different feeds, balance their whole diet, in order to meet their dietary requirements for maximum performance. At the same time we should try to save the energy that would be expended in the excretion of exceeding nutrients and to avoid worsen water quality due useless waste  load into the environment. Also researchers already observed that "Some adverse interactions may occur between AAs that are structurally related when their concentrations in the diet are imbalanced." NRC (1993c).

I do not mean you must see guppies diet as an exercise of mathematics, actively that is impossible, but just try balance their diet. 

One last point about AAs... From NRC (1993d): "The primary modes of feed detection by fish are through olfaction or sight, but the taste of the item is the key factor in determining whether the item is swallowed or rejected (Adron and Mackie, 1978). There appears to be a well defined and species-specific tuning of the taste receptors of fish for the particular cues present in their feed items (Goh and Tamura, 1980). ...  In general, carnivores show the greatest positive response to alkaline and neutral substances, such as glycine, proline, taurine, valine and betaine, while herbivores respond more to acid substances, such as aspartic acid and glutamic acid. The pattern is reasonable considering the chemical characteristics of the feed items the fish would seek in the natural environment (Mackie, 1982; Adams and Johnsen, 1986a)."  All but one of the substances cited above as feeding stimulants are AAs... Kasumyan and Nikolaeva (1997) tested the taste preferences of male guppies for L-stereoisomers of 21 AAs and  4 other classical taste substances. They observed that 5 AAs (glycine, glutamic acid, glutamine, lysine and cystine) plus citric acid and sucrose have feeding stimulant effects. Glycine, a non EAA shown the highest response, comparable to extracts of mosquito larvae and bloodworm; and that guppies of different origins shown the same response to all substances except sucrose.  For this reason I thought would be interesting park here a last table reporting the levels of non essential AAs that could boost feeding behavior of guppies. 

Table 7 - Content of Non Essential Amino Acids with Feeding Stimulant Properties in Some Guppy Feeds (% dry matter).

Lipids.

Lipids comprise several groups of compounds that share the property of being soluble in fat. For now I'll keep my focus on the fats and oils, phospholipids (PLs) and sterols. 

Fats and oils are made up by fatty acids (FAs) and glycerol. They differ at the point that fats are solid at room temperature while oils are liquid. Triglycerides are the most common compounds of fats and oils and the free FAs from triglycerides are most important aerobic fuel source for fish muscle, energy.   

PLs are also made up by fatty acids (FAs) and glycerol but added of phosphatidic acid or phosphoric acid and a nitrogenous base. PLs play their role on the constitution, maintenance and function of cellular and intracellular membranes, transport of fats within the body and emulsification of fats during digestion.

Among steroids there is the cholesterol that together with PLs and proteins constitute the cellular membranes, that is evolved in fat absorption, that is a precursor of another sterols like bile acids (evolved in fats digestion),  steroid hormones (androgens and estrogens,  related to reproductive processes, and corticosteroids that fight against inflammations and allergies) and vitamin D3. 

A key point among these lipids is that they enclose FAs in their structure. References to fatty acids are made using a notation made up by 3 numbers, like 18:2(n-6), where: "18" is the number of carbons atoms; "2" is the number of double bonds; "n" is a nick for Greek letter omega (w); and "6" is the number of carbon atoms between the methyl terminal and the fist double bond. Polyunsaturated fatty acid (PUFA) are fatty acids equal or above 18:2 and highly unsaturated fatty acids (HUFA) are fatty acids equal or above 20:2. 

Until today it was observed that five FAs can be essential fatty acids (EFAs) for fishes: 18:3(n-3) - linolenic acid - LNA; 18:2(n-6) - linoleic acid  -  LLA; 20:5(n-3) - eicosapentaenoic acid - EPA; 22:6(n-3) - docosahexaenoic acid - DHA and 20:4(n-6) - arachidonic acid - ADA. 

Although like most warm  freshwater fishes can desaturate and elongate 18 carbons EFAs to their 20 and 22 carbons relatives and like none vertebrate can synthesize LLA and LNA, these two later ones are sometimes taken as the truly EFAs, I mean the only which should really be present in the diet, for warm freshwater fishes despite it was observed  several times that the HUFAs of n-3 and n-6 series  have stronger biological activity and that they could alone fulfill the whole requirement of EFAs with superior performance than their 18 carbon respective relatives.  

The FA profile of fish tissues, and I suppose their requirements in some extent, are effected by the environment, diet and season accordingly Halver (1980) besides Tacon (1987c) only cites: "On a general basis, the dietary EFA requirement of fish have been found to increase with increasing dietary lipid level and/or with decreasing water temperature (Castell et. al., 1986).". Tacon's statement is clear to me in the sense that a given ratio between FAs must be kept in the lipid fraction of the diet and that a "winter diet" should be richer in lipids than a "summer diet". Fishes raised at lower temperatures eat less, have relatively increased energy needs because grow slowly consequently demanding less protein per unit of energy and because lipids carry more energy than any other feed fraction.

Halver (1980) cites salinity and temperature as environmental factors that effect FA profile of fish tissues but I would like include water hardness among them. The backbone behind this truth is that n-3 FAs allow  greater membrane fluidity, flexibility and permeability than  n-6 FAs. This way fishes kept at lower temperatures, higher salinity and higher water hardness would require more n-3 FAs than fishes kept in inverse conditions. These changes are more clear in the FAs content of PLs since they are straight evolved with cellular and intracellular walls and in a lesser extent in the FAs content of triglycerides that is an energy storage form.  The role of temperature is specially clear when you compare the content of n-3 EFAs in the tissues of guppies fed Artemia nauplii  at Table 8. 

Table 8 also illustrate the role of diet on the FAs profile of guppies. In short, they have a tendency to mirror the FA profile of their diet.  In general guppies fed Artemia related feeds have more n-3 FAs, that fed Moina have equal amounts of n-3 FAs and n-6 FAs and those fed trout pellets have more n-6 FAs following the characteristic FAs patterns found in marine, freshwater and land environments, respectively. On the other hand they look be able to favor in some extent the deposition of n-3 FAs instead n-6 FAs when needed. Halver (1980)  cited that the trout pellet fed to guppies had a n-6/n-3 ratio of 2.75 while they retained FAs in pattern of 2.06 and 1.3 in cold and warm water, respectively. Sounds to me that the guppies fed these trout pellets retained all n-3 they could get from the diet and complemented their needs with the n-6 FAs available. From NRC (1993e): "In fish species that can further desaturate and chain elongate 18:2(n-6) or 18:3(n-3), an absence of either of these fatty acids in the diet leads to the desaturation and chain elongation of oleic acid, 18:1(n-9), to 20:3(n-9), which is characteristic of an EFA deficiency in many terrestrial animals. Thus when EFAs are deficient, increased concentrations of 20:3(n-9) are incorporated into tissue polar lipids in place of 20:4(n-6), 20:5(n-3), or 22:6(n-3).". Unfortunately there was no data on the content of 20:3(n-9) in guppies but they clearly deposited much less EPA and DHA than guppies fed Artemia (Table 8).

 Only take care in order do not straightly compare the data got from the difference sources cited because their methodology for FAs determination possibly is not the same.  

Table 8 - Fatty Acid Profiles of Guppies Fed Different Feeds and Raised at Different Temperatures (mg/g dry weight).

Note:FAs in red are EFAs.

Personally I think that talk about seasonal changes caused by seasonal variations in feeds availability do not make sense when we are thinking in fancy guppies, this way I'll limit myself to look for a relationship between the feeds recommended for the different life phases of guppies and their fatty acid profile.  

As far I know there are two articles comparing the growth of guppies and another one comparing their  reproductive performance when fed different diets that I can take into account in order to try establish a suggestion for EFAs levels in guppy diet:

Lim et al. 2002  compared the performance of guppies fed brine preserved decapsulated artemia cysts, dried decapsulated artemia cysts, artemia nauplii and moina. In general lines they observed that guppies fed Artemia related feeds performed significantly better than those fed Moina and a tendency , that was most evident in fries (1 to 30 days old) although present in adults (2 to 3 months old) too, of fishes fed dried cysts perform better than those fed brine cysts and nauplii in decreasing order. See this table and this other table. The EFA content of experimental feeds is reported in Table 9 under reference #2. These results show a straight positive correlation between LNA, DHA, Total PUFA in EFA and Total n-3 in EFA content of the diets and in some extent to the EPA content too, while n-6/n3 Ratio show negative correlation (inversely proportional). In short, to me sounds that moinas shortage in LNA harm the performance of fishes fed on it. 

Shim and Bajrai (1982) compared the performance of guppies fed tubifex, bloodworms, moina and Aquavite (a brand of dry food). Despite the lacking of significance fishes fed tubifex always shown better performance followed by those fed Aquavite, bloodworm and moina, respectively. Fishes fed Aquavite did a little better than those fed tubifex during a large part of the experiment but they somewhat suddenly stop to grow like they had reached sexual maturity. These results are inversely proportional to the lipid content of the diet but in this case sound to me that EPA content of tubifex made THE difference.

So THE classical Larr et a. (no date).  They observed that guppies fed earthworms, beef heart and tubifex, in decreasing order, produced the largest offsprings among tested feeds. These results are negatively correlated with total amount of EFAs in the diet (Total PUFA in EFA). Here the trick sounds be to limit the ingestion of n-3 EFAs while increasing or keeping n-6 EFAs levels .  

Table 9 - Essential Fatty Acids Content of Some Guppy Feeds (% dry matter).

Actually I see that most important than simply recommend dietary levels of EFAs is to understand the interactions that occur among them. FAs share the same metabolic pathways (see Figure 2) in a way they show a behavior of competitive inhibition between FAs of different series. The n-3 FAs are the most potent inhibitors followed by n-6 and n-9 FAs in decreasing order of power. 

Figure 2 - Pathways for the biosynthesis of C20 and C22 PUFA from 18:3(n-3) and 18:2(m-6) showing the two possible routes for the production of 22:6(n-3) (and 22:5(n-6) from 20:4(n-6)). D6, D5 and D4 represent microssomal fatty acyl desaturase activities; E1, E2 and E3 denote microssomal fatty acyl elongase activities; and CS denotes peroxisomal chain shortening. The dotted lines indicate pathways for which there is no direct evidence in fish. 

From Tocher et al. (2003).

All said I fell free to suggest always to use lowest lipid level possible. Fries sound to be able to manage larger amounts of lipids mainly during their early growth phase, lets say until they are 1 month, but lipids level must be decreased from there on.  See lipids levels recommended by Tacon (1987a) and Tacon (1987b). For the growth phase I suggest a heavy supply of n-3, much better if used n-3 HUFA for maximum performance, do not matter n-6 level since n-3 will inhibit n-6 metabolism, see tubifex case where high levels of LLA do not impair guppy growth in the presence of adequate levels of EPA, and since HUFA have stronger biological activity than shorter chain FAs. DHA:EPA ratio don't sound be a matter of concern for guppies, tubifex is completely lacking in DHA, anyway it matters for marine fishes (Copeman et al. 2002)  and  guppies deposit more DHA than EPA (Table 8)... The main sources of n-3 HUFA we have easily available are live feeds and marine fish related feedstuffs. Personally I think that most dry fish food factories are always saving in marine fish related feedstuffs because they are relatively expensive so if you are that kind of breeder who do not want, or can not, hassles with n-3 rich live feeds you have as option to include LNA in the diet of your fishes using linseed and flaxseed and/or n-3 HUFA using cod liver oil. About n-6 EFAs my suggestion is to keep their level as low as possible in order to try delay sexual maturation of guppies that could have undesirable effects on their growth, although short generation intervals could be interesting to whose play with genetics and genetic improvement. Besides n-6 metabolism is inhibited by n-3 levels I think it is better option to avoid guppies expend energy managing unnecessary fat. In short I suggest try 0.5-1% LLA (linoleic acid - 18:2(n-6)), <0.5% ADA (arachidonic acid - 20:4(n-6)); 1-2% LNA (linolenic acid - 18:3(n-3)); 0.5-0.75% EPA (eicosapentaenoic acid - 20:5(n-3)) and 0.5% DHA (docosahexaenoic acid - 22:6(n-3)). always as percentage of dry matter. 

About lipids level in breeders diet sounds that it is a case where less is better. On EFAs levels in diets of broodstock I think you first have to decide if do you want a relatively large number of small fries or a few larger ones (Shikano and Taniguchi, 2005).  Homogamma-linolenic acid (20:3(n-6)), ADA and EPA are precursors of the so called eicosanoids (Seppanen-Laakso, 2004 pp 22)  which are partially responsible by the effects of FAs on reproduction (Izquierdo et al., 2001) and immunity  (Lall, 2000 pp 15) of fishes. Frankly speaking I am not sure that ADA and its derived eicosanoids have nothing but beneficial effects on guppy reproduction (Venkatesh et al. 1992) but analyzing data from  Larr et a. (no date) and from Table 9 becomes clear to me that decreasing the amount of n-3 and LLA in diet until ADA equals total n-3 EFAs, lets say at 0.25-0.5% of dry matter, is better than keep n-3 and LLA levels and increase ADA amount instead both approaches can increase the number of fries produced per brood. Anyway as far as I know the only pure source of ADA is an stuff called ARASCO®  . In short I suggest try decrease amount of fat in the diet of  broodstock and replace heavy feeding with artemia related feeds by a pinch of claroderans and/or worms and/or beef heart or liver.

Another point I would like discuss here but I have no tolls to do it deeply for now is the role of FAs on (1) the resistance of guppies to transport; may be fishes heavily fed on lipids before transport could resist better to this starvation period due increased energy reserves in fatty tissues; (2) adaptation to different water conditions; cholesterol, PLs and FAs are important for the proper function of the membranes which will work hard during adaptation of the fish to new water conditions;  and (3) to diseases they were never exposed to; eicosanoids and corticosteroids related stuff.    

"A beneficial effect of dietary phospholipid (PL) supplementation in purified diets in terms of survival, growth, resistance to stress tests, and occurrence of deformities has been demonstrated in larval and juvenile stages of various species of fish and crustaceans." Coutteau et a. (1997). See also Geurden at al. (1998)  and Wet (2003). Perhaps it is an issue that do not matter that much for guppies since artemia nauplii, that is usually taken as "The food" for guppy fries, is relatively poor in PLs (Shields et al., 1999), although dry feeds always contain soy lecithin,  and reports on the benefits of PL supplementation are mostly related to fish larvae that, at least theoretically, have a less developed digestive tube than guppy fries. 

Last, it is not an endorsement of any product neither I can be sure that things in real world will work like in literature, biology is not maths..., but feel free to experiment based on my thoughts because I did my best gathering the best informations available, anyway I do not want any lawyer kicking my door, take your decisions for yourself, analyze pros and cons, and feel free to share your opinion in our publication or in our forum!!!

References:

Kasumyan, A.O. and E. V.; Nikolaeva. 1997.  Taste preferences of the guppy Poecilia reticulata (Cyprinodontiformes). Voprosy-Ikhtiologii. 37(5):696-703.

Shim, K. F. and J. R. Bajrai. 1982. Growth rates and food conversion in young guppy (Poecilia reticulata Peters) fed on natural and artificial foods. Singapore Journal of  Primary  Industries 10(1):26-38.