S. Prawirodigdo
Assessment Institute for Agricultural Technology Central Java, Ungaran 50501
ABSTRACT
This article discusses aspect of availability assessment in amino acids of feed for pigs. The usefulness of amino acids profile and character of feedstuff for formulating diet of monogastrict animals has received more attention than the protein content. It has been accepted that ileal digestibilities of amino acids in some feedstuffs reflected their availabilities. On the other hand, digestibility as a synonym of availability is inappropriate in some cases such as amino acids from heat-processed meals, where some bound amino acids are absorbed but not available for utilization. There are sixt techniques to approach the utilization of feed protein in pigs. Nevertheless, each technique requires specific precaution due to several disadvantages in it that may provide inaccurate estimate. Overall, it is concluded that the apparent ileal digestible amino acids balance assay is a rapid and inexpensive technique which may be more reliable to estimate amino acids utilization of feeds than other techniques.
Keywords : availability, apparent, ileal digestible amino acids, nitrogen balance, feed, pig
ABSTRAK
Makalah ini mendiskusikan aspek pengukuran ketersediaan asam amino pakan untuk babi. Kegunaan nilai profil dan karakter asam amino bahan pakan untuk formulasi pakan terrnak berperut tunggal lebih mendapat perhatian daripada data kandungan protein. Disatu sisi telah diterima bahwa dayacerna ileum asam amino beberapa macam bahan pakan mereflesikan dayamanfaatnya. Tetapi di sisi lain, rumusan dayacerna sebagai sinonim daya manfaat tidak terapan untuk asam amino pada bahan pakan yang mengalami pemanasan dalam proses pembuatannya. Dalam pengukuran dayaguna protein pakan untuk babi dikenal enam teknik. Namun demikian, masing-masing teknik memerlukan penanganan khusus karena memiliki kelemahan yang dapat mengakibatkan estimasi yang tidak akurat. Secara keseluruhan dapat disimpulkan bahwa pengukuran keseimbangan asam amino pakan berdasarkan dayacerna ileum merupakan teknik yang lebih dapat dipercaya untuk estimasi nilai dayaguna asam amino dari pada teknik-teknik lainnya.
Kata kunci : ketersediaan, semu, ileal, asam amino tercerna, neraca nitrogen, pakan, babi
INTRODUCTION
In the formulation of diets for monogastric animals, there has been a change in focus to the amino acids rather than the usefulness of protein content (Baker, 1993}. Despite the formulation of diets based on amino acid utilization being simple and straightforward (Baker, 1993), there is no guarantee that formulation of diet based on protein only will satisfy the amino acid requirements of the pigs (Prawirodigdo, 1999)..
104 J.Indon.Trop.Anim.Agric.28(2) June 2003

To a large extent, the terms availability and digestibility have the same meaning (Batterham,
1992). This is based on the fact that almost all the digested amino acids have potential for use in protein synthesis (Erbersdobler, 1976). Several investigators
(Batterham et al ., 1990 a; Moughan et al , 1991;
Leibholz, 1992) have shown that digestibilities of amino acids in some feedstuffs (e.g. soybean meal,
SBM) reflected their availabilities. However, digestibility as a synon ym of availability is inappropriate in some cases such as amino acids from heat-processed meals (e.g. cottonseed meal,
CSM), where some bound amino acids are absorbed but not available for utilization (Batterham et al .,
1990 a, b ; Moughan et al ., 1991; Wiseman et al ., 1991).
Thus, Batterham (1992) suggested that for amino acids, availability should be defin ed as th e proportion of digestible amino acids in a feed that is absorbed in a form suitable for protein synthesis.
The present study is addressed to discuss th e tech niques for deter min ing amino acids availability of feed in pigs.
TECHNIQUES OF ASSESSMENT
Van Barneveld (1993 b ) has discussed five existing techniques to rapidly assess amino acid availability in feedstuffs, including microbiological techniques, simulation models, chemical approaches, ileal digestibility measurements and growth assays.
These tehcniques are discussed below.
Microbiological Simulation Technique
An example of the application of the microbiological ( Tetrahymena pyriformis ) simulation technique for estimating amino acid availability in feed in pigs was performed by Batterham (1973). It was found that the pigs fed heat-treated soybean exh ibited low per forman ce of gr owth r ate.
Furthermore, when the effect of inclusion of free amino acids (lysine, methionine and tryptophan) was examined using Tetrahymena pyriformis, the availability estimate was only 43%. The results lead
Batterham to the conclusion that the Tetrahymena estimate was not a reliable technique to predict the availability of heat-treated meal in pigs. Varnish and
Carpenter (1975) also provided evidence that microbiological estimates of total amino acid content were not in close agreement with the chemical estimates for the heated protein.
Chemical Approach
The use of chemical analysis to assess amino acid availability of a feed has been reviewed extensively by van Barneveld (1993 b ). Generally, techniques are used to predict lysine availability are based on binding the free Î-amino group of lysine with specific sensitiveness or dye (van Barneveld,
1993 b ). The use of several dye-binding procedures for the assessment of heat-damaged pr otein availability (heated plasma albumin, blood meal, fish meal, peanut meal and milk powder) have been examin ed usin g azo dyes with th e dir ect fluorodinitrobenzene (FDNB) analysis (Hurrell and
Carpenter, 1975). Hurrel and Carpenter (1975) stated that dye-binding procedures are rapid, inexpensive and suitable for estimating the amount of free lysine, however, the cresol red procedure appeared to be rarely applicable. Nevertheless, van Barneveld
(1993 b ) concluded that the dye binding procedures overestimate biological availability, except for estimating lysine availability, and is dangerous due to the use of highly toxic chemicals.
Recently, the reactive lysine analysis method was claimed to be the new method reliable for predicting lysine availability for the heatprocessed meal (Moughan and Rutherfurd, 1996;
Rutherfurd and Moughan, 1997). Unfortunately, the results of the experiments to confirm the new method were inconsistent. Moughan and Rutherfurd (1996) were not certain whether the use of their method underestimated or overestimated lysine availability of heat-treated feeds (Table 1), while Batterham et al . (1990 b ) documen ted th at appar en t ileal digestibility of heat processed meal overestimated availability. Table 1 shows the distinctions between digestible lysine of heated lactose casein (Moughan and Rutherfurd, 1996), glucose casein (Moughan et al.
, 1996) and peas (Rutherfurd and Moughan, 1997) in the rat determined using conventional amino acid analysis and reactive lysine analysis.
Previously, Rutherfurd et al. (1995) used the rat as a model for the pig for determining true ileal reactive lysine digestibility, and found that rats
Assessment of Amino Acids Availability in Feed for Pigs (Prawirodigdo) 105

Protein intake
Muscle
Kidney
Stomach
Small intestine
Large
Intestine Urinary nitrogen
(Bladder)
Figure 1.
Schematic Diagram of the Pathway of Nitrogen Losses via Urine (Adapted from: Prawirodigdo, 1999) and pigs showed similar performance in a true lysine digestibility when the experimental animals were fed with a heated lactose casein. The results guided
Rutherfurd et al .
(1995) to the conclusion that the rat is a dependable model for digestibility studies in the pig. However, Rutherfurd and Moughan (1995) found that the true reactive ileal digestible lysine
(predicted available lysine) of CSM fed to rats was
10.3 g/kg. This result (Rutherfurd and Moughan,
1995) overestimates the available lysine of CSM (3.78
to 5.2 g/kg) in pigs determined using the carcass analysis method (Baterham et al., 1990 b ). It seems that the rat is not a reliable model for determining ileal digestibility of lysine of CSM in pig. Batterham et al . (1979) compared the results of the slope-ratio assays with pigs and rats indicated considerable discr epan cies between these an imals in th e availability of lysine from the same dietary protein sources. Other researchers (Huisman et al ., 1991;
Donkoh et al ., 1994) also noted that the rat may not be a useful model for the pig in the digestibility studies for all feedstuffs.
Isotope Tracer Technique
The use of the isotope tracer technique to trace the final destiny of ingested amino acids and to evaluate amino acid availability in feed for pigs was proposed by Black and Batterham (1987). In
14 C was expected to be a tracer suitable for labelling carbon product from protein oxidation.
The hypothesis was that, the 14 C labelled amino acid included in the diet will mix with the free amino acid pool and move along the alimentary canal and enter the various metabolic routes in the same proportion as the unlabelled amino acid entering that pool (Black and Batterham, 1987). As the supply of an amino acid for protein synthesis changes from a deficit to an excess, there will be a major increase in its oxidation and thus in the proportion of the labelled dose appearing as 14 CO
2
. Black and Batterham (1987) assumed that the difference between 14 CO
2
excretion and amino acid intake will depend on the availability of the amino acids. For example, the increase in 14 CO
2 excretion occurring at a higher feed will be associated with low amino acid availability. It was suggested
(Black and Batterham, 1987) that (1) the amino acid has to be supplied in excess of the requirement, (2) the amino acid profile in the free pool and the proportion of them following metabolic routes do n ot ch an ge substan tially with in cr ease in concentration of the amino acid, and (3) the feeding
106 J.Indon.Trop.Anim.Agric.28(2) June 2003

Table 1.
Comparison between D igestible Lysine Content of Heated-feedstuffs (g/kg) Determined Using the
Cconventional Amino Acid and Reactive Lysine Analyses
Items Methods of amino acid analysis Significance
Lactose casein
1
Lysine content
Ileal lysine digestibility
Digestible lysine
Glucose casein
2
Lysine content
Ileal lysine digestibility
Digestible lysine
Peas
3
Conventional
38.0
0.705
26.8
31.6
0.54.
17.1
Guanidination
33.0
0.859
28.4
25.0
0.59
14.8
N. M.
***
N. M.
N. M.
N. M.
N. M.
Ileal lysine digestibility
Heat treatment : 110 o
C
135
o
C
150
o
C
13.2
12.6
13.3
12.6
N. S.
N. S.
9.5 8.8 ***
165
o
C 3.7 3.6 N. S
Adapted from:
1
, Moughan and Rutherfurd (1996);
2
Moughan et al . (1996);
3
, Rutherfurd and Moughan (1997); N. M., Not mentioned;
N. S., Not significant; ***, P<0.001.
regime is provides the free amino acids which are completely available. Such a hypothesis was examined by Ball et al . (1995), however, the results were not in close agreement with the hypothesis.
Thus, it was suggested that the isotope labelling technique was not a reliable to estimate amino acid availability in feed. Ball et al . (1995) found that the lysine pool was smaller in pigs given CSM diet than in pigs receiving SBM diet. The pattern of plasma lysine profile in pigs fed a CSM diet was peculiar, being low, constant, and unaffected by the meal.
Ball et al . (1995) suggested that lysine availability in intact-protein sources is a function of both ileal digestibility and lysine catabolism.
the ileal digestibility values have been used routinely to formulate diets (Batterham, 1992).
However, Batterham (1992) concluded that the ileal digestibility values are only appropriate to estimate availability of amino acids in feed where the protein nature is not damaged by heat treatment. In the other words, the ileal digestibility assay cannot be used to estimate amino acid availability in heatprocessed meals (Batterham et al ., 1990 a, b ;
Moughan et al ., 1991; Wiseman et al ., 1991).
Ileal Digestibility Assay
The use of the ileal digestibility assay to estimate the availability of amino acids has been accepted widely. Batterham (1992) explained that the application of the ileal digestibility assay for estimating availability was based on the assumption that amino acids which are not recovered in the small intestine are available for protein synthesis. Thus,
Growth Assay
The growth assay has been used in pigs to evaluate lysine in meat and bone meal (Batterham et al ., 1986 a, b ) and tryptophan availability in some feedstuffs (Sato et al ., 1987). According to Batterham
(1992), the growth assay is the growth response of pigs to the diets containing the amino acid being examined, as a proportion of the slope of the response to the free amino acid (as a standard). The inclusion of some points is necessary to ensure that the response to the specific amino acid is linear. The hypothesis is proposed that the slope of the response
Assessment of Amino Acids Availability in Feed for Pigs (Prawirodigdo) 107

Figure 2. Schematic diagram of bladder catheter placement (adapted from Prawirodigdo, 1999). 2.1.1, positioning of the catheter, 2.1.2, inflation of the catheter balloon; R, Rectum; C, Catheter; Blf, Blind fold; Bld,
Bladder; V, Vagina; Bln, Balloon; U, Urine.
108 J.Indon.Trop.Anim.Agric.28(2) June 2003

to the inclusion of the particular amino acid is due to the availability of the test amino acid and is not affected by other nutrients contributed by the protein source (Batterham, 1992).
In order to reach a suitable criterion of response, the use of carcass base for measuring feed conversion ratio in pig is preferable to using liveweight data. The requirement of condition to obtain an accurate result was clearly detailed by
Batterham (1992). The slope assay has been considered an accurate technique to evaluate amino acid availability of feedstuffs, and is recommended for use as a standard technique for validating other techniques (van Barneveld, 1993 b ). Unfortunately, the slope ratio assay is costly, labor intensive and only one amino acid can be examined at one measurement.
Alternate Techniques to the Existing Techniques for Predicting Availability
There are three kinds of technique that may be suitable for predicting amino acid availability in heat-processed meals. These techniques are the near infra red (NIR) spectrophotometry (Batterham, 1992; van Barneveld, 1993)., the portal absorption technique (Dunshea, 1998) and the ileal digestible N balance technique (Prawirodigdo et al . 1997).
The near infra red spetrophotometry technique.The application of NIR spectrophotometry has been suggested for predicting amino acid availability (Batterham, 1992; van Barneveld, 1993 b ).
The advantage of the NIR is that it is rapid and inexpensive (Batterham, 1992). Baterham (1992) hypothetized if the changes in the amino acid profile due to heat treatment are the reflections of the protein qualities, then the NIR is suitable for predicting availability. However, Batterham (1992) reported that the need for an accurate standard for calibrating the equipment was a major limitation for the development of NIR analyses.
The portal absorption technique. The use of the portal absorption technique allows the measurement of absorption and hepatic exchanges of nutrients between the alimentary tract and liver
(Dunshea, 1998). Dunshea (1998) reported this technique has been used for measuring the effect of por cine somatotr opin (PST) on amino acid metabolism in pigs, and suggested that amino acid absorption and blood flow were more stable in pigs fed more frequently than once a day. However, this technique involves the employment of sophisticated surgical practice to enable implantation of blood catheters.
Ileal digestible n itr ogen balan ce.
Theoretically, it is well understood that the residue of ingested dietary amino acids and N is excreted through the final pathways in various forms such as urine, faeces and sweat (Manatt and Garcia, 1992).
According to Moughan (1993), urinary N is the major route of elimination of excess N. The following figure
(Figure 1, Prawirodigdo, 1999) displays the schematic diagram pathway of the major excreted amino acids and N in the pig. Figure 1 shows that ingested proteins are absorbed from the small intestine through the liver and passed to the muscle. Then, the unutilized amino acids and N are transferred into the kidney and excreted as the urinary N via the bladder. In addition, the absorbed N from the large intestine is transported to the liver as ammonia and directly transferred to the kidney and finally released into the bladder and voided via urine (Zebrowska,
1975). Thus, significant information on the absorption characteristics of amino acids in different dietary protein sources could be obtained by N balance studies which are relatively easy to conduct
(Gatel and Grosjean, 1992) .
The N balance technique has been used for a long time for predicting amino acids utilization of feeds in humans or animals. Manatt and Garcia
(1992) reviewed that during the 1940’S-1950’S the N balance technique was used for determining the indispensable amino acids of food for humans, particularly to show that N equilibrium will not be obtained or maintained when any indispensable amino acid is ingested in insufficient quantity.
Basically, the N balance technique involves the accurate measurement of the weights of both the N ingested and excreted N. The N balance technique has been used intensively for determining protein availability of several feedstuffs in pigs
(Zebrowska, 1975; Fuller et al ., 1979; Just et al ., 1982;
Gatel and Grosjean, 1992; Jongbloed and Lenis, 1992; van Barneveld, 1993 a ; Marty and Chavez, 1995; Grala et al ., 1997; Prawirodigdo et al ., 1997).
Assessment of Amino Acids Availability in Feed for Pigs (Prawirodigdo) 109

Table 2. Nitrogen (N) balance in pigs fed diets containing cottonseed meal (CSM) or soybean meal (SBM) *
Diets SED P =
Nitrogen intake (g/day)
IDN intake (g/day)
Faecal N (g/day)
Apparent absorbed N (g/day)
Urinary N (g/day)
CSM
45.4
35.2
8.5
36.9
19.6
SBM
41.8
35.8
2.0
39.8
16.5
0.96
0.22
0.54
1.26
0.56
0.01
NS
+
<0.001
0.061
0.001
(% N intake)
(% absorbed N)
(%IDN intake)
43.5
55.3
55.8
39.4
41.4
44.0
1.85
2.69
2.97
0.084
0.004
0.011
Retained N (g/day)
(% N intake)
17.3
38.0
23.3
55.8
1.47
2.43
0.007
<0.001
(% absorbed N) 46.7 58.6 2.69 0.004
(% IDN intake) 44.2 55.2 2.97 0.011
*, Adapted from Prawirodigdo et al . (1997); NS, Not significant,
+
, P<0.10; SED, Standard error of the differences;
IDN, Ileal digestible nitrogen.
Eggum (1989) stated that the N balance technique is an accurate approach for estimating N availability of feeds. Furthermore, Eggum (1989) argued that, theoretically amino acid and N retention in animals should be identical, when determined using the N balance compared to the chemical analysis of gross N balance method. Unfortunately such agreement was difficult to achieve because of many sources of errors e.g accuracy of weighing, collection of faeces and urine, chemical analysis
(Eggum, 1989) and measurement of feed intake.
The use of the N balance technique for estimating the availability has been criticised by
et al . (1982) found that the N balance overestimated N retention when the technique was directly compared to the slaughter analysis, however, Just et al . (1982) calculated the retention based on the gross N balance data. Whereas, Zebrowska (1975) has proven that
N uptake in the hindgut has no biological value for the pig. Similarly Gatel and Grosjean (1992) also speculated that the N balance technique could lead to underestimation of N excretion due to N losses from evaporation of ammonia from urine and faeces.
Nevertheless, Gatel and Grosjean (1992) also used the gross N balance method. In addition, the technique used to collect urine in their study may be inappropriate and contributing a significant degree of error.
The use of the N balance technique for predicting N availability of feed may have to be conducted based on apparent ileal digestibility of N
(AIDN). The use of a urine catheter (Fuller et al .,
1979, Prawirodigdo et al ., 1997; Prawirodigdo, 1999) to allow direct urine collection into a urine container containing strong acid (e.g. H
2
SO
4
or HCl) may be necessary to minimize error due to urine spillage or
N evaporation during the measurement period.
Fuller (personal communication, 1993) demonstrated directly the placement of urine catheter in the bladder of conscious female pigs. The procedure for such urine catheter insertion in the bladder of con scious pig was descr ibed by
Prawirodigdo (1999, Figure 2).
Firstly, the female pig was kept in the metabolism cage and left undisturb for few days.
110 J.Indon.Trop.Anim.Agric.28(2) June 2003

Foley bladder catheter which was equipped with a nominal balloon capacity of 35 ml was inserted into con scious pig by fin ger guidance. Aseptic con dition s were maintained th rough out th e procedure which involved cleaning the exterior area of the gilt and the hands of the operator using
Betadine Antiseptic Solution and guiding the lubricated catheter tip along the floor of the vagina and into the urethra. The catheter tip was then manipulated gently until it cleared the diverticulum and entered the bladder. However, since placement of the urine catheter has been conducted on an unanaesthetised pig, the accuracy of this placement depended on the accuracy of the palpation of the orifice. Consequently, the operator could mislead the catheter into the blind fold (Blf, See 2.1.1., Figure
2). Therefore appearance of urine in the catheter was used for confirmation of positioning and 25 ml of sterile saline was used to inflate the balloon of th e catheter. An antibiotic (trimidin e) was administered orally (3.5 g/day) for three days to prevent urinary infections. Sterile polyvinyl tubings were attached to Foley catheters and placed into 10 litre buckets containing 50 ml of 5 M Hcl.
Nevertheless, the direct urine collection technique has been considered to be an accurate technique for estimating amino acids and N availability throughout the AID amino acids and N balance measurements. As a result, this technique has been adopted widely by some research institutes and pig industries in Australia.
Recently, Prawirodigdo et al . (1997) demonstrated the use of the IDN balance technique for measuring N retention of CSM and SBM in pigs.
The examined hypothesis was that IDN does not reflect availability of heat-processed feedstuffs.
They speculated that, if IDN reflects availability, pigs consumming either CSM or SBM diets of equal ID protein content should have similar urinary N levels.
If IDN over estimates availability then a greater portion of N will apear in the urine. It was documented that the pigs consuming CSM diet excreted larger amount (P<0.01) of urinary N than the pigs consuming SBM diet (See Table 2).
Results also suggested that IDN from CSM diet was retained by the pigs less efficiently (P<0.05) than IDN from SBM diet. It was possible that the Î amino acids in CSM was bound with sucrose during heat-process and not available for synthesis. Godin and Spensley (1971) stated that CSM used as the dietary protein source was subjected to severe heating (107-112 o C for 90-120 minutes) before mechanical breakdown and subsequent extraction.
Due to Maillard reaction, there may have been a large quantity of amino acids absorbed in unavailable form into the circulatory system of the pigs fed CSM diet.
Moreover, under these circumstances the pigs may not be able to metabolize nor utilize the amino acids of advan ced Maillar d pr oduct efficien tly.
Consequently, more N was wasted via urine of the pigs consuming CSM diet than of the pigs fed SBM diet.
The study proved the hypothesis that IDN value of CSM does not reflect N availability of such heat-processed protein meal. So that, the IDN measurement is not appropriate for detemining N availability of heat-processed protein meal such as
CSM.
In conclusion, the AID amino acids and N balance assay is a rapid and inexpensive technique that may be more reliable to estimate amino acids and N utilization of feedstuffs than other techniques.
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