Livestock Research for Rural Development 14 (6) 2002

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Methods and techniques for the determination of amino acid digestibility
A Review

*Khieu Borin, Brian Ogle and Jan Erik Lindberg

Department of Animal Nutrition and Management, Swedish University of Agriculture Sciences
* Home institution: Department of Animal Production and Health, Ministry of Agriculture Forestry and Fisheries, Cambodia 
 

Abstract

Methods to estimate the digestibility of nitrogenous components and amino acids in chickens and ducks have been developed and tested by numerous authors. The discrepancies between the findings of different researchers make it very difficult to make clear and definite conclusions as to which method is most scientifically appropriate and relevant, and can be used as a standard method for the determination of digestibility of nutrients and amino acid retention. Besides the scientific aspects, the appropriateness of the methods to be used must be based also on the local context and available facilities and be economically viable. 

The researchers of the studies reviewed in this article on the digestibility of nutrients and amino acids with birds (chickens and ducks) have chosen three different approaches that the authors could use for the evaluation of cassava leaf meal as a replacement for fish meal in poultry diets: (i) analysis of excreta – ie. total tract digestibility; (ii) analysis of ileal digesta  and (iii) growth performance.

Keywords: Digestibility; Nutrients; Amino acids; Nitrogen; Ileal digesta; Excreta; Chickens; Ducks; Intact birds; Caecectomised birds


Introduction
 

Several factors stimulate researchers and producers to carefully study potential ingredients for feed formulation. However, the appropriate quantity and quality of a formulated feed that results in an optimum economic return is the main objective of those involved in poultry-related businesses. All dietary components, such as amino acids, vitamins and minerals, are important when formulating feeds for poultry, but critical attention should be given to the dietary amino acids in the form of protein, as approximately 25% of the cost of practical poultry diets can be accounted for by amino acids (McNab 1994). A deficiency of an essential amino acid will result in a reduction in performance (Buttery et al 1994). In addition, excess of amino acids in the diet above the requirements, are excreted, and can be a source of pollution as well as resulting in animal welfare problems, for example carcass defects, breast blisters and hock burn (McNab 1994). The usual method to quantify and qualify dietary feed is to conduct digestibility studies and growth performance trials with birds. 

Methods to estimate the digestibility of nitrogenous components and amino acids in chickens and ducks have been developed and tested by numerous authors (Payne et al 1971; Nordheim and Coon 1984; Sibbald 1986; Green et al 1987a; Revington et al 1991; Adeola et al 1997; King et al 1997; Ravindran et al 1999; Ragland et al 1999; Short et al 1999; van Leeuwen et al 2000; Hong et al 2001). However there is still debate as to the accuracy and suitability of different methods used by individuals or group of scientists. The central issue in the debate is the role of the lower part of the digestive tract including the ileal-caecal-rectal junction due to the metabolic faecal component (secretions, abraded cells, mucus, bile) and the endogenous faecal fraction (bacteria and bacterial debris) (Sibbald 1987).  

It is also important to point out that earlier work on the digestibility of nutrients, including amino acids, had been mainly carried out using hybrid birds and conventional feeds. The objective of this review is to look for more appropriate methods (economically and with respect to the facilities available at the research site) that can be used in studies with both local and exotic chickens and ducks for the evaluation of the protein in available feed resources. 


Amino acid availability and digestibility 

Sibbald (1987) in his extensive review on the estimation of bioavailable amino acids (AA) in different feedstuffs for poultry and pigs indicated that the amino acid concentrations vary widely among feeding stuffs due to many different factors, such as species, including genotype, plant fraction, stage of maturity, location of growth, soil fertility, season and year of growth and the situation is further complicated because amino acid digestibility, measured either in vitro or in vivo (with pigs and with birds) also varies among plant species. 

The total and available amino acid concentrations in feedingstuffs may be altered if heating occurs during the processing (soybean meal) and as a result of chemical changes affecting amino acids during storage due to temperature, moisture and time of storage. 

Availability is a function of two processes: digestion and metabolism. There are a number of biological tests, which have commonly been used to measure available protein. Most of these, such as nitrogen balance, biological value, net protein utilization and protein efficiency ratio have no value for the formulation of mixed diets (Johnson 1992). The potential value of a food for supplying a particular nutrient can be determined by chemical analysis, but the actual value of the food to the animal can be arrived at only after making allowances for the inevitable losses that occur during the digestion, absorption and metabolism (McDonald et al 1998). 

Ideally, the bioavailability of a nutrient should be a characteristic of the feedstuff in which it is contained and independent of the animal to which it is fed (Sibbald 1987). Quantitatively, the requirement for an amino acid is driven by the sum of three processes: (i) the rate at which amino acids are needed for functional processes such as protein accretion, or as precursors for other metabolites; (ii) the rate of endogenous loss of protein and amino acids – these occur mostly from the digestive tract; and  (iii) the rate at which amino acids are lost to oxidation or other metabolic pathways (Klasing 1998).  


Methods used in poultry digestibility studies 

Three types of methods can be used to estimate the bioavailable AA: in vitro methods, indirect and direct in vivo methods. However, only the direct in vivo method will be discussed here, as it was pointed out (Sibbald 1987) that in vitro and in vivo indirect methods appear to have several disadvantages for the estimation of bioavailable amino acids. For example, AA may not be bioavailable through enzymatic digestion, the absolute values are not constantly reliable but best apply to quality control (chemical assay), data lack accuracy but are suitable for comparative purposes (microbiological assay), they serve a rather specialized requirement of cereal breeders and offer little potential for evaluating animal feedingstuffs (insect assays). Plasma-free AA concentrations can be used to identify the limiting bioavailable AA in feeding stuffs but cannot yet describe the bioavailable AA in absolute amounts and prediction of AA availability from nitrogen digestion might be useful for ranking many samples of a particular feeding stuff but lack the accuracy and precision required for most practical purposes. However, some of these methods could be useful for ranking many samples of a particular feedingstuff. 

The direct in vivo methods of estimation of bioavailable amino acids focus on three techniques:  the growth assay, balance trial and nylon-bag techniques. However, only the growth and balance trials are selected here for further review. 

Growth assays 

The common criteria used in the growth assay technique are animal body weight gain, body weight gain as a function of live body weight, gain:feed or feed:gain ratio, and nitrogen retention. Independent variables used to establish response relationships include dietary AA concentration and AA intake (Sibbald 1987). Further the same author indicated that the basic growth assay for an available AA involves several steps:  a basal diet, deficient in the AA of interest, is supplemented with one or more levels of that AA and fed to animals to establish a relationship between the response and the AA level. Simultaneously, the same basal diet, supplemented with one or more levels of the material to be assayed, is fed to comparable animals under the same conditions. The performance of the latter group is compared with the response, and the AA relationship and the available AA concentration in the test material are calculated. 

In this technique, the selection of birds and of basal diets demands careful attention, but perhaps the most important variable is experimental design. Further independent and dependent variables are used. In this case, AA concentration in the diet is preferred as independent and body weight gain as dependent variables. Intuitively, nitrogen retention is preferable because AA at sub-optimal intakes, should affect tissue protein synthesis and weight gain, appears to give comparable results and is much easier to measure. 

Balance experiments 

A balance experiment measures the difference between the input and output of a material. The difference between nutrient intake and nutrient output may be an estimate of balance but it is not a satisfactory estimate of bioavailability (Sibbald 1987). The feedingstuff may be offered alone or as part of a multi-ingredient mixture and reference materials are given to control animals and markers and indicators may be included. It is also fundamentally important to make a proper design, which includes species, sex, age, housing in a specific environment and the number of animal cages, their distribution according to treatments and replicates. 

When describing AA in terms of their availability for birds for their different functions, it is important to understand the process where the AA contained in the dietary protein are absorbed from the gastrointestinal tract during the digestion, and which are expressed as digestibility coefficients. The measurement, which has been used for more than 100 years and contributing to nutritional knowledge, is expressed in terms of the difference between the intake and that excreted as faeces as a proportion of amount consumed (McNab 1994).

Total tract AA digestibility =

AA consumed – AA in faeces

AA consumed

Birds excrete faeces including a metabolic faecal component and an endogenous faecal fraction, but McNab (1994) grouped these together as “endogenous faecal materials” which are included in the following equation: 

True AA digestibility =

AAC – (AAF + EAAF)
AAC

AAC = (amino acid consumed); AAF = (amino acid in faeces);
EAAF = (endogenous amino acid in faeces); 

Inert external markers such as chromic oxide (Cr2O3), titanium oxide (TiO2) (Short et al 1996) and ferric oxide (Fe2O3) (Kuiken and Lyman 1948; Elwell and Soares 1975) as well as internal marker acid insoluble ash (AIA) have been extensively used in animal digestibility studies to estimate quantitatively feed intake and excreta output. The same technique can be applied to measure the digestibility of amino acids at the ileum and then the apparent digestibility coefficient is expressed as:

Apparent AA digestibility  =

AA in feed

AA in ileum

marker in feed

marker in ileum

 

AA in feed

 

 

marker in feed 

 
             

McDonald et al (1998) reported that chromic oxide is the most common indigestible marker used for digestibility studies and it seems unlikely to be present as a major constituent of foods. The same authors also suggested using titanium dioxide as indicator for non-ruminants but more studies have used chromic oxide as indicator (Summers and Robblee 1985; Atteh and Leeson 1985; Jamroz et al 2001). Three to five grams of chromic oxide are added per kilogram of feed according to Kadim and Moughan (1997) and Jamroz et al (2002). The use of indicators in balance trials introduces at least three assumptions: the indicator is dispersed uniformly throughout the alimentary canal at the same rate as the residues containing AA of interest and the indicator is inert and unabsorbed (Sibbald 1987). 


Techniques used in balance trials
 

Methods to determine available amino acids are numerous and include both in vivo and in vitro approaches. Some authors use only the digestibility values, while others utilize both digestibility and growth performance tests with animals to evaluate the quality of the dietary feed. Dalibard and Paillard (1995) studied the determination of the requirement for lysine and sulphur amino acids of broilers and proposed a combination of digestibility and growth tests, which is applicable to any set of recommendations for feed formulation. 

The balance experiment methodology, a technique in which the birds are given free access to the diet, still seems to have the widest acceptance and is subjected to the least criticism. However, great care must be taken to avoid food losses, to recover lost food (including any in the drinker), to avoid separation of food components, to monitor dry matter changes and to take representative samples for analysis (McNab 1994). These aspects are just part of the care and management of the experiment related to feed. Although several techniques have been developed to estimate precisely the nutritive value of the feed, such as using fasted birds and force or tube feeding using caecectomised or intact birds, none of these techniques has become a universal standard method or technique for digestibility studies in poultry. 


Surgical techniques 

The aim of the caecectomised birds is to avoid the intense microbial activity that may synthesize AA from other nitrogenous compounds and degrade AA, which are not available to the host animals. The caecectomy technique was first used with chickens (Payne et al 1971; Parsons et al 1983; Parsons 1984; Raharjo and Farrel 1984; Green et al 1987a, b) and was later modified for use with ducks (Ragland et al 1999). Adult birds of different ages are used, for example: 24 weeks old male broilers (Payne et al 1971); 50 weeks old White Leghorn hens (Parsons 1983); Warren cockerels at 3 kg live weight (Green et al 1987a); and 32-34 weeks old male White Pekin ducks (Ragland et al 1999). 

The cockerels are deprived of solid food for 24 h before surgery, then anaesthetized and the caeca are removed (Green et al 1987a). After the operation, solid food is withheld for 24h, but water is supplied ad libitum. After 10 days the skin sutures are removed. Cockerels are allocated to test 4 weeks after surgery. The caecectomy technique with ducks (Ragland et al 1999) has been adapted from the technique used for cockerels, as described by Payne et al (1971).  Another technique for ducks is to fit them with an excreta collection apparatus (Whirl-Paktm bag) (Adeola et al 1997; King et al 1997 and 2000; Hong et al 2001). Adult male ducks of approximately 16 weeks old and 3.8 kg are surgically fitted with modified plastic retainer rings. During excreta collection, Whirl-Paktm bags are placed through the bore of a bottle, which is then screwed onto the modified retainer ring attached to the bird, with the threads of the ring and bottle securing the bag in place. 

The ileostomy technique has been used with adult cockerels of 2.8-3.0 kg by Schutte et al (1991) and van Leeuwen et al (2000) for the determination of ileal digestibility of protein and amino acids in the diet, and involves the surgical fitting of a cannula for collection of ileal digesta. 


Intact birds
 

Kuiken and Lyman (1948) developed a technique based on excreta analysis to evaluate the foodstuff using rats for the digestibility studies. Although there is still controversy around digestibility studies with intact birds using this technique most of the available published data have been derived from excreta analysis (Sibbald and Morse 1982a, b; Dale et al 1985; Sibbald and Wolynetz 1985; Nordheim and Coon 1984; Summers and Robblee 1985; Short et al 1999; Ravindran et al 1999; Svihus and Hetland 2001; Jamroz et al 2002). This technique with intact birds can be used for total excreta or digesta, or both excreta and digesta (from same birds) collection. Poultry void faeces and urine via a common cloaca as a single excretum and this natural mixture of faeces and urine  the term “Excreta” is used.

Excreta collection is usually done once daily in the morning during the last 4 days of the assay, and care must be taken to avoid contamination from feathers, scales and debris (Ravindran et al 1999). Most authors have used forced feeding of birds fasted for 24-48 hours before the assay and then 36 hours (Parsons 1984) or 48 hours (Green et al 1987a) post feeding for excreta collection. For the collection of ileal digesta birds are normally killed 2 hours after forced feeding (Short et al 1996; Jamroz et al 2002). However, Kadim and Moughan (1997) found that the maximum amount of digesta was collected 4 hours after feeding and that the mean apparent ileal nitrogen digestibility had the lowest variation after 4 hours. 

The most suitable site for ileal digesta collection was reported to be 15 cm of the terminal ileum (Kadim and Moughan 1997). The complete contents of small intestine, starting from the vitelline diverticum to a point 40 mm proximal to the ileo-caecal junction, have also been collected by gently flushing out the digesta with distilled water. However, other authors have collected digesta from the lower gastro-intestinal tract between Meckel’s diverticulum and ileo-caecal-colonic junction. 

After the collection excreta are dried for 24 hours at 80C in a forced-air oven or freeze dried. Dried excreta are then ground and stored in airtight plastic containers at –4C for subsequent analyses (Kadim and Moughan 1997; Ravindran et al 1999). 


Discussion
 

It is recognized that amino acid digestibility is a sensitive indicator of amino acid availability in dietary ingredients for poultry. Several methods to estimate the digestibility of nitrogen and amino acids have been developed and tested by numerous authors (Kuiken and Lyman 1948; Payne et al 1971; Nordheim and Coon 1984; Sibbald 1986; Adeola et al 1997; King et al 1997; Ravindran et al 1999; Ragland et al 1999; and Hong et al 2001).  

Due to the fact that birds excrete faeces and urine plus the endogenous materials together as excreta earlier studies therefore suggested different methods to determine the digestibility of different feedstuffs in order to evaluate the bioavailability of nutrients (amino acids), of a single ingredient or synergism of a mixture. Numerous methods have been developed and used to estimate the bioavailable amino acids, either through in vitro methods and indirect or direct in vivo methods. The direct in vivo method has been widely used for both studies on energy and amino acids digestibilities. Three techniques, ie. growth assays, balance trials and nylon-bags are commonly used for studies on monogastric animals. In balance trials with poultry, workers have developed different ways to treat birds, such as caecectomy (Payne et al 1971), later adapted for use with ducks (Ragland et al 1999), collection of ileal digesta (Ravindran et al 1999) and ileostomy (van Leeuwen et al 2000), in order to avoid mixture of faeces and urine. 

The urinary AA contribution to poultry excreta is small and its amino acids concentration is unaffected by the nature of the diet (McNab 1994; Mosenthin et al 2000). However, McNab (1994) recommended critical examination of the technique, particularly when comparisons are being drawn from data derived from ileal and excreta balance studies. Ragland et al (1999), in agreement with Johnson (1992), suggested the use of caecectomized poultry for amino acid digestibility studies. Studies by Green et al (1987a, b) on the digestibilities of amino acids in maize, wheat and barley meals found no differences between intact and caecectomised cockerels with respect to the quantities of nitrogen excreted and the true digestibility of nitrogen and amino acids. However, there were significant differences for some amino acids between intact and caecectomised cockerels in studies on soybean, sunflower and groundnut meals and it has been stated that caecectomy may encourage proliferation of microorganisms in other regions of the hindgut (Raharjo and Farrell 1984). 

Some amino acids in maize husks seem to have been digested, degraded or retained in both caecectomised and intact birds (Green 1988), and caecectomy can only reduce a proportion of the microbial activity in the intestinal tract, so in addition to the bird’s enzymic digestion, some degradation of dietary amino acids by microbes may still be occurring in caecectomised birds. In addition, the removal of the caeca may influence digestion in the gastrointestinal tract anterior to the caeca (Sakata 1987). Ragland et al (1999) found consistent results as described for caecetomised chickens by various authors and no differences were found between intact and caecectomised birds, but this is dependent on the feedstuff under consideration (Ragland et al 1999), the type of dietary protein (Parsons 1982a) and that the general effects of caecectomy are similar for ducks and chickens. 

McNab (1994) reviewed the studies of O’Dell et al (1960) and Teekell et al (1968) and concluded that only about 2% of the nitrogen in chicken urine is in the form of amino acids and colostomised birds excreted greater quantities of endogenous amino acids compared to intact birds (Bragg et al 1969). However, Parsons (1984) in studies on dietary fibre or starch, suggested that microbial metabolism in the caeca significantly affects the excretion of amino acids by poultry, and therefore microbial protein had a larger effect on the excreta from intact hens than on that from caecectomised hens. Approximately 25% of the excreta nitrogen in poultry was reported to be from microbial origin (Parsons et al 1982b) when birds were fed a high fibre diet. The increased bacterial synthesis of amino acids in the large intestine and caeca partially accounted for the increased amino acid excretion (Parsons et al 1983), but no effect was found on the digestibility of amino acids when corn starch and cellulose were fed to intact fasting roosters (Parsons et al 1982a). The bacteria in the caeca may also modify the amino acid composition of the endogenous component of the digesta (Salter and Fulford 1974), and this could have a significant effect on digestibility values calculated from excreta analysis (Johnson 1992). 

When carbohydrate is limiting, such as with fasted birds or birds given highly-digestible diets, amino acid excretion is probably reduced due to bacterial deamination (Parsons et al 1983) that varies according to the type of ingredient and the individual amino acid (Ravindran et al 1999; Ragland et al 1999). The quantity of nitrogen (N) and amino acids excreted after feeding the protein-free diet did not differ between intact and caecectomised birds, and neither did the true digestibility coefficient of N and amino acids. However, the differences between intact and caecectomised birds were significant only for threonine, which was excreted to a lesser extent from intact cockerels (Green et al 1987a). Microbes in the small intestine had a tendency to act preferentially on endogenous muco-proteins and enzymes rather than on undigested protein residues in the hindgut (Salter and Fulford 1974). 

Determination of apparent digestibility through analysis of excreta samples has been criticized because this approach fails to distinguish amino acids voided which are not of direct dietary origin (endogenous excretory losses, EEL) (Short et al 1999). Bragg et al (1969) found that individual chicks in surgically modified groups sometimes exhibited amino acid digestibility values greater than 100%. Salter and Fulford, (1974) using germ-free and conventional chicks in their studies, concluded that gut microflora had little influence on the digestion of protein in the diets but may serve an important role in the degradation of endogenous proteins and the recycling of N. 

Raharjo and Farrell (1984) and Ravindran et al (1999) reported that amino acid metabolism by the hindgut microflora in chickens may be substantial, and that digestibilities measured in the terminal ileum are more accurate measures of amino acid availability than those measured in the excreta. A review by McNab (1994) on different methods used for determination of amino acid digestibility concluded though that the digestibility values based on the droppings from normal intact birds have the decided advantage of simplicity over those taken from caecectomized birds or of values based on ileal concentrations. 

Although four approaches: (i) use of germ-free animals; (ii) the feeding of antibiotics; (iii) sampling ileal contents either after slaughter or from birds fitted with appropriate cannulae; and (iv) sampling droppings from birds whose caeca have been surgically removed, have been adopted to avoid the influence of the intestinal bacteria (McNab 1994), there is still no clear consensus, as to which can be used as a standard model. The important statement made again by McNab (1994) is that bacterial proteases may release amino acids that are absorbed and ultimately benefit the host animals, or amino acids may be converted into bacterial protein and consequently lost to the host.  

Assays based on ileal digesta also have the added advantage of not being influenced by urinary amino acids, which may confound digestibility estimates based on excreta (Ravindran et al 1999). In addition, the digesta analysis has a further advantage over excreta due to the contamination of excreta with scurf and feathers. 

Several studies have suggested that due to the highly liquid excreta of ducks, which can be subjected to contamination with feed, or scales, ducks surgically fitted with retainer rings and collection bags could be used (Adeola et al 1997; King et al 1997; Hong et al 2001). Lists of useful precautions to be taken include: more frequent collection of excreta (12 hourly) and continuous mechanical blowing to remove scurf are devices, which might be judged beneficial (Sibbald 1986). Jamroz et al (2001) found considerable AA synthesis by microbes in the caeca-colon of chickens, ducks and especially geese, and these AA are not absorbed and utilized in the body but excreted in the faeces, therefore he concluded that ileal digestibility would be a better estimate of available amino acids than excreta. 

Ragland et al (1999) in a study on metabolizable energy and amino acid digestibility with ducks found similar results to those reported by several other researchers in studies conducted with caecectomised chickens. These results are also in agreement with Parsons, (1985) who found that caecectomised roosters excreted higher amounts of energy and amino acids and tended to have lower amino acid digestibility coefficients than intact chickens. However, these arguments are not conclusive due to reasons, which are explained earlier in relation to caecectomised cockerels (O’Dell et al 1960; Teekell et al 1968; Bragg et al 1969; Salter and Fulford 1974; Parsons 1984; Raharjo and Farrell 1984; Sakata 1987; Green et al 1987a; Green 1988; McNab 1994; Mosenthin et al 2000). 

Neither the extensive reviews by Sibbald (1987) nor McNab (1994) have reached any firm conclusions on the methodology, which is most appropriate to apply to a single or all species when studying digestibility in poultry. Sibbald (1987) stated that although techniques for measuring the bioavailabilities of AA in feedingstuffs are still being developed, substantial progress has been made, particularly during the last 10 years. McNab (1994) suggested more detailed studies are required to determine precisely what factors affect endogenous losses and whether the use of caecectomised birds results in the derivation of significantly different and more meaningful coefficients. Other interesting factors, which so far lack supporting data, are the effects of age within breed and whether the digestibility coefficients derived with hybrid chickens or ducks are applicable to local chickens and ducks, or whether studies with chickens are applicable to other poultry species.  


Conclusions
 

The discrepancies between the findings of different researchers make it very difficult to make clear and definite conclusions as to which method is most scientifically appropriate and relevant, and can be used as a standard method for the determination of digestibility of nutrients and amino acid retention. Besides the scientific aspects, the appropriateness of the methods to be used must be based also on the local context and available facilities and be economically viable. 

The researchers of the studies reviewed in this article on the digestibility of nutrients and amino acids with birds (chickens and ducks) have chosen three different approaches that the authors could use for the evaluation of cassava leaf meal as a replacement for fish meal in poultry diets: (i) analysis of excreta – ie. total tract digestibility (Kuiken and Lyman 1948); (ii) analysis of ileal digesta (Ravindran et al 1999; Short et al 1999) and (iii) growth performance (Sibbald 1987). In addition, chromium oxide can be added as an indigestible maker, and the time and site of sampling used could be based on the results of Kadim and Moughan (1997).  


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Received 30 April 2002

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