The use of N-alkanes as markers for detemination of intake and digestibility of fish feed

Author Publisher YearPlace
Ólafur Guđmundsson, Kristín HalldórsdóttirEIFAC1993Eichenau, Germany
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Conference
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    Introduction

    Measurements of  intake and digestibility are among the most critical estimates needed to determine the nutrient requirements and feed utilization in fish. Many methods have been devised to study these parameters, but all have their shortcomings and disadvantages, as direct measurements are not applicable. One of the most common methods used to measure intake of livestock is based on determining  faecal dry matter output and the respective dry matter digestibility of the diet. Intake is then calculated as:

                                                      Intake = (Faecal output)/(1- digestibility)                                                                                 (1)

    This method of measuring  intake has not gained  popularity in aquaculture. The main reason for this is because it is difficult to estimate the faecal output, as total collection of  faeces is hard to obtain. Total collection has been attempted by several researchers1,2,3,4, but the faecal output can also be estimated from the dilution in faeces of an indigestible marker (external marker) consumed by the fish in an exactly known quantity. Then the faecal output is calculated from the equation:

                              Faecal output = (External marker consumed)/(External marker in faeces)                                 (2)

    The most frequently used method of measuring diet digestibility, which is also required in equation 1, is determened  from the ratio of an indigestible marker (internal marker) in the diet and the respective faeces, from the following equation:

                                  Digestibility = 1-(Internal marker in diet)/(Internal marker in faeces)                                     (3)

    This should enable an estimate to be taken of intake and digestibility of fish feed, provided the daily consumption of external marker can be accurately defined and a representative sample of the faeces obtained. However, error in faecal output estimation causes equivalent error in intake, whereas error in digestibility  leads to proportionally higher error in indigestibility and consequently in intake. The ideal marker should  therefore be non toxic, readily analyzed, as well as being indigestible.

    In addition to the intake and digestibility it is important to know the preference of feedstuffs and digesta kinetics in fishes.  The easiest way to study this is by  using indirect marker substances. Therefore indirect marker-based approaches have become important in the study of  fish nutrition.

    Chromium sesquioxide (Cr2O3), and more recently celite, have been  popular markers to determine digestibility. Accurate measurements of faecal output, and therefore feed intake, has been harder to obtain mainly because of the difficulty to have the fish consume an exact daily amount of external marker and at the same time consume reliable amount of internal marker in the feed. Because of this, as well as other problems concerning the most common markers used today, the search for suitable markers has continued.

    Use of n-alkanes in measuring intake and digestibility in grazing animals has acquired some popularity 5,6,7, giving grounds for studying its use in fish as well.  The aim of this paper is to explore the potential use of n-alkanes in measuring intake and digestibility in fishes, with some reference to its use in studies of diet preference and digesta kinetics.


    Alkanes in feedstuff for fish

    Many feed ingredients contain n-alkanes. Some have only traces, while others, especially of plant origin, contain considerable amounts of cuticular wax components, which include n-alkanes.  The composition of the alkane fraction in several common ingredients used in fish feed is shown in table 1 (K. Halldorsdottir, unpublished data). Fish and soybean meal only contain minimal amounts of n-alkanes (less than 1 mg kg-1 DM), whereas wheat and some other grains contain somewhat higher amounts of odd-chain alkanes, but still less than 5 mg kg-1, giving a limited increase in the quantity of these in compound feeds for fishes. However, the amount of alkanes commonly used in feed as markers is approximately 400 mg kg-1. The low amount or lack of n-alkanes in the common  feedstuff for fish virtually eliminates the possibility of using n-alkane source from the feed as internal markers, which is the basis for its use in animal grazing experiments5; but it creates the possibility of using almost any long-chain alkanes as markers in the exact ratio or quantity desired in nutritional studies with fish. The n-alkanes most readily available at reasonable price are octacosane ( C28), dotriacontane (C32) and hexatriacontane (C36). 

    Sample preparation and analysis of alkanes

    In general, collection of faecal samples from fishes can be obtained by three methods: total collection, striping and settling. In total collection, no parts of the faeces should be lost, whereas the others are based on markers techniques where only samples of the faeces are required.  

    After obtaining the samples, they are freeze-dried, ground and directly saponificated in alcoholic KHO and the alkanes extracted in heptane, after which they are passed through silica gel making them ready for gas-liquid chromatographic determination (R.W. Mayes, personal communication). Quantitative identification of the alkanes has advanced with the development of more sophisticated gas-liquid chromatography, but the best results are obtained using capillary columns (R.W. Mayes, personal communication), although packed columns have been satisfactory5

    Alkanes as internal markers

    Long chain n-alkanes have been used for some years as markers in intake and digestibility studies with grazing animals5,8. This has been thoroughly reviewed by Dove and Mayes7.  It has been established that although recovery of  n-alkanes  from C28  to C36 is incomplete in ruminants, they can be used as internal markers as the recovery in faeces increases with the chain length5. This is probably also true in fishes, but direct measurements of faecal recovery of n-alkanes is difficult in fish, although faecal recovery relative to the longest chain alkane, such as C36, can easily be accomplished.

    Cravedi and associates9,10,11 studied the absorption of alkanes into fish tissue. They found that the alkanes are mainly accumulated in the liver and fat tissue. However, as the chain length increases from C14,  the absorption decreases and is virtually none above C20.

    When alkanes are used as markers, they are required in very low quantities and are easily mixed with the feedstuff;  but one of the great advantage of their use is that they do not necessarily have to be mixed in the feed but could be dissolved in fat and coated on the pellets.  They have an even flow through the digestive tract and are easy to measure, even in very low quantities as described before; and last but not least, they cause minimal or no pollution. However, as alkanes are water insoluble, they float in water like other lipids. If for some reason the fat becomes separated from the feed before consumption or from the faeces before collection, for example when using settling columns12,  it could cause an error.

    A study was conducted on farmed Arctic charr (Salvelinus alpinus) comparing  n-alkanes (C28 and C32) to Cr2O3  and celite as digestibility markers13,14. Faecal samples were collected by the Guelph method using settling columns12. Some of the results are shown in table 2. In general the digestibility was very high, which could be partly due to the method used for faecal collection. However, there was no significant difference (P>0.05) found in the digestibility between the two alkanes tested, nor was there a difference (P> 0.05) in digestibility of fats determined with n-alkanes and Cr2O3 or celite.  However, digestibility of dry matter and protein was for few feedstuffs lower (P< 0.05) when estimated with n-alkanes than with  Cr2O3 or celite.  It was concluded that the n-alkanes could be used for determination of digestibility of fish feed.

    Recently cholestane (cycloalkane) has also been studied as marker to determine digestibility of fat in salmon (Salmo salar)15, tilapia (Oreochromis niloticus) and fresh-water prawn (Macrobrachium rosenbergii)16 with good success.

    Alkanes as external markers

    In intake studies, the fish has to be dosed daily with an exact amount of external marker to determine the faecal output. This is difficult in fishes, but can be done, for example by mixing the external marker in a small amount of the experimental feedstuff to be fed in limited amount at the first feeding of each day, so as to know the exact consumption of the marker, followed by feeding the rest of the experimental feedstuff containing only the  internal marker. Faecal samples are then collected and feed and faeces analyzed for the internal and external markers. In this type of work, n-alkanes have great advantage above most other markers. Both the internal and external markers can be determined in the same analyses and it has been argued5 that incomplete recovery in the faeces will not matter provided the n-alkanes used as external markers to determine the faecal output (equation 2) are of similar chain length and therefore have the same recovery rate as the internal markers used to determine the digestibility (equation 3). When these are put into equation 1 they will cancel out errors associated with incomplete recoveries. This further assures correct results by using n-alkanes as markers, although as mentioned before, there is no evidence of incomplete recovery of n-alkanes above C20 in fishes.

    It has been suggested7 that unsaturated hydrocarbons (n-alkenes) and branched-chain alkanes (iso-alkanes and ante-iso-alkanes) could also be used as markers for determination of diet intake and composition.

    Alkanes to measure consumption of individual feeds from feed mixtures

    In addition to measuring the total intake, the n-alkanes can be used in estimating consumption of individual feeds from feed mixtures. This can be important when determining and comparing preference or palatability of feeds. Then a different alkane is included as an internal marker in each individual feed under testing. Theoretically the maximum number of different feeds that could be compared at each time is only limited to the numbers, or even ratios, of alkanes available as internal markers. If only internal alkanes are used, the proportions of different feeds can be determined without estimating the intake of each feed. If, however, both external and internal alkanes are used, the consumption of each feed can be determined. The total intake is then estimated using equation 1 and the intake of each feed quantified accordingly. The method can also be extended to studies where competition for different feeds between different species of fish, such as Salmon, Arctic charr and Trout, is compared. Further, it can probably have some role in studies of  behavior and learning.

    Alkanes to study digesta kinetics

    There are some questions concerning the use of n-alkanes in dealing with the measurement of digesta kinetics such as particle breakdown, retention times and digesta flow, as the affinity of alkanes for either the particulate or liquid phases of digesta in the fish is not known. However, this deserves a clarification, as, for example in ruminants, exogenous alkanes do not appear to have strong affinities for either one of these phases17.

    References
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    16 Ishikawa, M., Teshima, S., Kanazawa, A. and Koshio, S. 1993. Measurement of digestibilities of cholesterol and fatty acids using an indicator, 5a-Cholestane. Proc. EIFAC Workshop on Methodology for Determination of Nutrient Requirements in Fish, Eichenau, Germany, pp. 33.

    17 Mayes, R.W., Lamb, C.S. and Colgrove, P.M. 1988. Digestion and metabolism of dosed even-chain and herbage odd-chain alkanes in sheep. Proc. 12th General meeting, European Grassl. Fed., Dublin, Irealnd, pp. 159-163.