Boisen* - Jaroslav Heger* - Amino Acids in Animal Nutrition

Jaroslav Heger*

S. Boisen*

Department of Animal Nutrition and Physiology, Research Centre Foulum, Tjele, Denmark

Introduction

The optimal dietary supply of amino acids to farm animals has been intensively studied for many years. Most studies have focused on the establishment of the requirement for one single amino acid, in particular, for one of those essential amino acids that are most commonly limiting in practical diets (for references see NRC, 1988, 1998).

Lysine is generally the first-limiting amino acid in practical cereal-based pig diets and has also been the most investigated. Thus, in a sur- vey of studies on lysine requirements published in international journals from 1985 to 1995, NRC (1998) refer to estimations of lysine requirement for piglets up to about 20 kg live weight from 18 publications, for growing pigs in the live-weight range 20–50 kg from 23 publications, and for finishing pigs in live-weight range 50–100 kg from 26 publications.

Nevertheless, a general agreement on the amino acid requirements for pigs has not yet been established. The major reason for this is probably a number of different factors that may influence the actual requirement of each individual amino acid and, consequently, the results obtained in the specific study.

Obviously, if these factors are not understood or properly considered the obtained results will not have general validity.

Among these factors, the dietary supply of the other amino acids, in particular all the essential amino acids, is of significant importance. In fact, the requirement for one specific amino acid can only be established if the dietary supply of all other essential amino acids, as well as crude protein, is adequate, i.e.

is not limiting responses of variable supply of the investigated amino acid in the actual study.

It follows that when the requirement for one single amino acid is determined all other essential amino acids need to be considered.

Consequently, some knowledge of the ideal profile of all essential amino acids other than the one investigated is necessary in order to secure proper experimental diets.

The concept of ideal protein and proposals for its composition were introduced more than 20 years ago (ARC, 1981). The continu- ous research on amino acid requirements has led to several proposals for modified ideal proteins for growing pigs as reviewed by, for example, Cole and Van Lunen (1994) and Boisen (1997). Furthermore, the officially rec- ommended dietary amino acid profile used in different countries is still relatively variable.

The aims of this chapter are: (i) to discuss the expression and establishment of the ideal dietary amino acid profile for pigs; (ii) to demonstrate how this profile can be utilized for characterizing the protein quality in feeds,

2nd edition (ed. J.P.F. D’Mello) 157

*E-mail address: [email protected]

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and finally (iii) to discuss how experimental conditions may be optimized and standardized for obtaining a generally agreed ideal dietary amino acid profile for pigs. Because the ideal protein for sows is discussed in Chapter 12, the present chapter will focus on growing pigs from weaning to slaughter.

Expression of the Ideal Dietary Amino Acid Profile

Essential and non-essential amino acids

The twenty amino acids commonly occurring in proteins are given in Fig. 9.1. Together with the different properties (chemically reactive groups), by which the different amino acids can be characterized, Fig. 9.1 also illustrates the non-essential amino acids that can be syn- thesized by simple transamination, or in some cases more complex reactions, of metabolites from the oxidation products of glucose or, as for arginine, from the urea cycle in mammals.

In piglets, the synthesis rate of arginine,

and probably also of proline that include several synthetic steps, may not be sufficiently high to fully satisfy the requirements for these amino acids during the first rapid growth phase (Fuller, 1994). On the other hand, the dietary supply of these two amino acids appear always to be in surplus in relevant practical diets for piglets.

Therefore, this possible insufficiency will not be considered as a matter of practical relevance.

Figure 9.1 also illustrates that the two amino acids, cysteine and tyrosine can be syn- thesized from the essential amino acids, methionine and phenylalanine, respectively. Thus, an undersupply of cysteine and tyrosine can be compensated by an oversupply of methionine and phenylalanine, respectively. On the other hand, cysteine and tyrosine cannot compensate for undersupply of methionine and phenylalanine, respectively. Consequently, a complete composition of ideal protein for pigs includes also the sum of sulphur amino acids (methionine + cysteine) and aromatic amino acids (phenylalanine + tyrosine) together with methionine and phenylalanine, respectively.

In the ideal amino acid profile suggested by

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Glutamine

Isoleucine Threonine

Histidine Methionine

Valine Leucine

Lysine Arginine

Alanine Serine

Proline Glutamate

Glycine

Aspartate Asparagine

Cysteine

AMINO ACIDS

Semi-essential: Essential:

3-P-Glycerate Pyruvate Acetyl-CoA

OAA+

α-KGA TCA cycle

Urea cycle Arginine

Ornithine Citrulline

Hydroxy amino acids

Acidic Amino acids

Urea Glucose

Tryptophan Phenylalanine Tyrosine

CO₂

Non-essential:

CO₂

NH₄⁺

NH₄⁺ NH₄⁺ NH₄⁺

NH₄⁺

Sulphur amino acids Branched- chain amino acids

Aromatic amino acids

Basic amino acids NH₄⁺

Fig. 9.1. Essential amino acids and synthesis routes of semi-essential and non-essential amino acids.

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the ARC (1981) lysine was used as reference for the other amino acids. Tryptophan has also been suggested as a reference for the other essential amino acids (NRC, 1988). However, it is now generally accepted that lysine is most suitable (ARC, 1991; NRC, 1998). Some very good reasons for using lysine as reference are:

(i) lysine is commonly the first limiting amino acid in practical diets, (ii) lysine is the most investigated amino acid with respect to requirement; (iii) the requirement for lysine is, together with leucine, higher than the other amino acids;

and (iv) lysine does not contribute quantitatively to other specific functions than protein synthesis.

Order of essential amino acids according to their degree of limitation

The order of essential amino acids describing the ideal amino acid profile in relation to their general order of limitation in common cereal- based diets is given together with their specific properties in Table 9.1. Furthermore, the essential amino acids can be separated into two groups according to their general degree of limitation.

1. A primary limiting group which includes lysine, threonine, methionine (and sulphur amino acids) and tryptophan. The requirements for these amino acids can generally be met after supplementation of industrial products of lysine, threonine, methionine and tryp-

tophan. These amino acids are generally used for improving the protein quality in the diets in practical pig feed production.

2. A secondary limiting group which includes: (i) isoleucine, leucine and valine (all branched-chain amino acids); (ii) histidine, and (iii) phenylalanine (and aromatic amino acids).

Each of these amino acids can become limiting when the diets are supplemented with amino acids from the primary limiting group.

However, at present the amino acids from the secondary limiting group are not commercially available at reasonable prices. Furthermore, due to the present insufficient knowledge on their precise requirements the benefit for a further reduction in the dietary protein level by their supplementation is difficult to assess.

Definition of the ideal dietary amino acid profile

The first proposals for ideal amino acid profiles referred directly to the concentration of amino acids in the diet. However, due to vari- ations in the availability of the individual amino acids in dietary proteins the profile should rather be related to the available amino acids in the diet. Generally, the ileal digestibility is considered to be the most correct mea- sure for availability, although the availability of some amino acids, e.g. lysine, may be over- estimated by this method, in particular in heat treated feedstuffs (Batterham, 1994, Moughan and Rutherfurd, 1996).

Ideal Dietary Amino Acid Profiles for Pigs 159

Table 9.1. Essential amino acids according to their general order of limitation in common practical diets for growing pigs.

Essential amino acid Chemical property Order of limitation

Lysine Basic amino acid Primary

Threonine Hydroxy amino acid

Methionine Sulphur amino acid

Methionine + cystine Sulphur amino acid Tryptophan Indole amino acid

Isoleucine Branched-chain amino acid Secondary

Leucine Branched-chain amino acid

Valine Branched-chain amino acid

Histidine Imidazole amino acid

Phenylalanine Aromatic amino acid Phenylalanine + tyrosine Aromatic amino acid Amino Acids - Chap 09 12/3/03 12:25 pm Page 159

Several tables of apparent ileal digestibility of amino acids were published about 10 years ago and ideal amino acid profiles have been suggested on this basis. However, as discussed by, for example, Boisen and Moughan (1996a,b) and Mosenthin et al.(2000) values of apparent digestibility are influenced by the total, i.e. basal plus extra feed specific, endogenous protein losses during the digestion processes and, therefore, are influenced by the protein level in the experimental diets used for their determination. On the other hand, values of standardized (or true) digestibility only include the feed-specific losses which are correctly debited on the feed- stuff itself. Therefore, for obtaining maximal accuracy (and general validity) in the ideal dietary amino acid profile, it should be related to values of standardized digestibility of the individual amino acids.

It is now generally accepted that values of standardized ileal digestibility of amino acids are those to be used in practical feed evaluation. Recently, several tables on true or standardized ileal digestibility of amino acids in common feedstuffs for pigs have been published (NRC, 1998; Rademacher et al., 1999; CVB, 1999; AmiPig, 2000; Pedersen and Boisen, 2002).

Furthermore, in order to optimize effi- ciency, and also to reduce surplus-N, the optimal ratio between essential amino acids (EAA) and non-essential amino acids (NEAA) has been defined (Wang and Fuller, 1989; see also Chapter 6). On the other hand, experimentally determined EAA/NEAA ratios are influenced by the composition of the NEAA in the experimental diets, as well as of other available N-compounds, e.g. nucleic acids.

Therefore, a more precise characterization would be the ratio obtained from the amounts of standardized digestible EAA-N and total-N, respectively.

Alternatively, the ideal profile of essential amino acids can be directly expressed in relation to crude protein (g kg¹CP or g 160 g¹ N). This results in a simple and more informative definition of the dietary ideal amino acid profile. Furthermore, from these values the theoretical biological value (BV) of the diet, as well as the single protein sources, can be directly calculated as discussed later.

In conclusion, the ideal dietary amino acid profile refers to the optimal composition of the available amounts, i.e. ileal standardized digestible, of all the essential amino acids and N in the diet. Then, the dietary supply of all individual essential amino acids, as well as of N, should be equally limiting for covering the actual requirements for a certain production.

Establishment of the Ideal Dietary Amino Acid Profile for Pigs

Basically, it would be expected that sow’s milk, which can be considered to be optimized for suckling piglets during evolution, would also provide the weaned piglets with an ideal amino acid profile. This assumption is supported by the fact that the profile is very constant in sow’s milk and apparently not influenced by the dietary composition (Boisen, 1997).

Furthermore, this composition is very close to the amino acid composition in the body as well as in the deposited protein during growth (Table 9.2). Because the composition of deposited protein, during growth up to 100 kg live weight is relatively constant and, furthermore, the requirements for protein deposition account for a dominating portion of the total amino acid requirements, it would also be expected that the ideal dietary amino acid profile is relatively constant during this period. The residual amino acid requirements are related to maintenance requirement, which appear to be dominated by the ileal endogenous protein loss during the digestion processes (Fuller, 1991, 1994; Boisen and Moughan, 1996a).

Compared with sow’s milk ileal endogenous protein losses are low in most essential amino acids except threonine, cystine and tryptophan (Table 9.2). The concentration of cystine is furthermore very high in hair (Table 9.2) which is lost continuously during growth. Tryptophan and tyrosine are precursors for important hor- mones, i.e. serotonin, tyroxine and adrenaline.

After weaning, the requirements for these amino acids may increase due to extra losses of endogenous protein when dietary fibre and antinutritional factors in the feed are increased and a generally increased hormone production.

On the other hand, the higher concentration of histidine in deposited protein than in sow’s milk

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may be explained by its ability to be stored in carnosine when there is a dietary surplus (Fuller et al., 1989).

The amino acid profile (relative to lysine) of sow’s milk is compared with proposals for ideal protein from the literature in Table 9.3. Characteristically, amino acids in the primary limiting group are generally rep- resented at a higher ratio, whereas those in the secondary limiting group are often rep- resented at a lower ratio in the proposals than found in sow’s milk. As described in the introduction, all amino acids in the first limiting group have been intensively studied.

Nevertheless, there is still some disagree- ment about the relative requirements for these amino acids, in particular for the sulphur amino acids, which vary by up to 30%

between the different proposals.

The amino acids in the secondary limiting group are much less investigated. However, there is no obvious reason for a lower ratio of these amino acids in the ideal profile than found in sow’s milk.

It is generally believed that, due to the increasing contribution of maintenance requirements of protein, which is relatively

high in threonine, sulphur amino acids and tryptophan, the ideal dietary amino acid profile changes during growth (e.g. Rademacher et al., 1999). However, for fast-growing pigs this may be of little practical importance before 100 kg live weight (Boisen, 1997). A major portion of the extra maintenance amino acid requirements may derive from the feed-specific extra endogenous ileal protein losses which is compensated for when using values of standardized ileal digestible amino acids as discussed above. Thus, the optimal threonine:lysine ratio was not found to increase significantly up to 100 kg live weight in a recent study (Pedersen et al., 2003). On the other hand, the requirements of amino acids relative to energy appear to be generally reduced with almost 50% from the extrapolated value at zero to 100 kg live weight (for lysine and threonine from 10.5 and 6.8, respectively to 5.25 and 3.4, respectively) as shown in Fig. 9.2.

In conclusion, sow’s milk can be considered as a valuable guide for establishment of ideal dietary amino acid profile for growing pigs after weaning and until slaughter at 100 kg live weight in fast growing pigs.

Ideal Dietary Amino Acid Profiles for Pigs 161

Table 9.2. Amino acid composition (g 160 g¹N) of sow’s milk compared with the composition in whole body and deposited protein during growth, ileal endogenous protein loss and hair, respectively.

Essential and semi- Endogenous

essential amino acids Sow’s milk^a Whole body^b Deposited^c protein^d Hair^c

Lysine 71 66 69 30 33

Threonine 39 39 38 45 59

Methionine 18 19 19 10 4

Cystine 13 11 10 16 134

Tryptophan 12 8 n.d.^e 12 n.d.

Isoleucine 41 35 40 25 35

Leucine 81 72 77 40 77

Valine 54 48 51 35 60

Histidine 25 29 32 15 11

Phenylalanine 39 39 37 30 23

Tyrosine 42 27 28 20 9

aMean of 32 samples (Boisen, 1997).

bDetermined at 20 kg live weight (Fuller, 1994).

cFrom 20 to 90 kg live weight (Jørgensen et al., 1988).

dMean of 36 determinations (Boisen and Moughan, 1996a).

en.d., not determined.

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Ideal Dietary Amino Acid Profile and Biological Value of Dietary Protein

Sources

The biological value (BV) of dietary protein is only related to the first limiting amino acid and can be determined experimentally in N-

balance studies in which N-intake and N- excretion with faeces and urine, respectively, are determined. This information is often relevant for complete diets, whereas for single protein sources traditional BV is of less use for feed evaluation and diet for- mulation.

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Table 9.3. Proposals for ideal dietary amino acid profile (relative to lysine) for growing pigs compared with the profile of sow’s milk.

A^a B C D E F G

Primary limiting amino acids

Lysine 100 100 100 100 100 100 100

Threonine 55 60 75 65 66 60 64

Methionine 25 26 27 31 – – 26

Met + Cys 44 50 59 60 50 55 52

Tryptophan 17 15 19 18 18 18 17

Secondary limiting amino acids

Isoleucine 58 55 61 60 50 54 57

Leucine 114 100 110 100 100 102 114

Valine 76 70 75 68 70 68 74

Histidine 35 33 32 32 33 32 35

Phenylalanine 55 49 59 51 – – 57

Phe + Tyr 114 96 122 95 100 93 114

Arginine 62 – – 42 – 48 –

aA: Sow’s milk (Table 9.2); B: ARC (1981); C: Fuller et al.(1989); D: Chung and Baker (1992); E: Cole and van Lunen (1994); F: NRC (1998); G: Boisen et al.(2000).

0 1 2 3 4 5 6 7 8 9 10 11 12

0 10 20 30 40 50 60 70 80 90 100

Threonine Lysine

Live weight (kg) Requirement (g FUp–1)

Fig. 9.2. Requirements for lysine and threonine (g per FUp (feed units for pigs)) relative to dietary energy of fast growing pigs until 100 kg live weight.

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Table 9.4 shows different proposals for the ideal profile of dietary amino acids in relation to crude protein. When using this profile (from column G in Table 9.4) relative to the composition of available amino acids

in single protein sources a detailed characterization of protein quality is obtained.

Table 9.5 describes the protein quality of two single feedstuffs and a simple diet, based only on those two protein sources.

Ideal Dietary Amino Acid Profiles for Pigs 163

Table 9.4. Proposals for amino acid composition (g 160 g¹N) of ideal protein for growing pigs.

A^a B C D E F G

Primary limiting amino acids

Lysine 70 65 59 81 (70)^b (70)^b 70

Threonine 42 47 44 53 46 42 45

Methionine 18 – 16 25 – – 18

Met + Cys 35 41 35 49 35 39 36

Tryptophan 10 12 11 15 13 13 12

Secondary limiting amino acids

Isoleucine 38 39 36 49 35 38 40

Leucine 70 72 65 81 70 71 80

Valine 49 49 44 55 49 48 52

Histidine 23 – – 26 23 22 25

Phenylalanine 34 – 35 41 – – 40

Phe + Tyr 67 78 72 77 70 65 80

aA: ARC (1981); B: Wang and Fuller (1989); C: Fuller et al.(1989); D: calculated from Chung and Baker (1992); E: Cole and van Lunen (1994); F: NRC (1998); G: Boisen et al.(2000).

bCalculated from Table 9.2 assuming lysine is 70 g 160 g¹N.

Table 9.5. Protein quality of protein sources and diets in relation to the ideal dietary amino acid pattern.

Barley Soybean meal Digestible (g kg¹) AA pattern^d SD SD

Total^a (%)^b Total¹ (%)^b Barley SBM Diet^c Barley SBM Diet Primary limiting amino acids

Lysine 3.8 75 28.6 92 2.9 26.3 5.1 48 92 64

Threonine 3.6 75 18.2 88 2.7 16.0 3.9 71 88 78

Methionine 1.8 84 6.6 93 1.5 6.1 1.9 99 83 94

Cystine 2.4 84 6.9 89 2.0 6.1 2.3 – – –

Met + Cys – – – – 3.5 12.2 4.2 116 83 105

Tryptophan 1.2 79 5.9 92 1.0 5.4 1.4 94 111 100

Secondary limiting amino acids

Isoleucine 3.7 81 20.8 91 3.0 18.9 4.4 89 116 99

Leucine 7.3 83 35.7 90 6.1 32.1 8.4 90 99 93

Valine 5.2 80 21.6 90 4.2 19.4 5.5 95 92 94

Histidine 2.4 81 12.5 92 1.9 11.5 2.8 92 113 100

Phenylalanine 5.5 84 23.7 91 4.6 21.6 6.1 137 133 136

Tyrosine 3.3 83 17.4 92 2.7 16.0 3.9 – – –

Phe + Tyr – – – – 7.3 37.6 10.0 109 116 112

Crude protein (N6.25) 105.5 80 456.4 89 84.4 406.2 112.3 – – –

aDegussa (1996).

bStandardized digestibility (Amipig, 2000).

c85% barley + 10% soybean meal + 5% non-protein ingredients.

dRelative to proposal for ideal protein (Table 9.4, column G).

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According to the amino acid profile of the diet only supplementation with lysine and threonine is needed to assure high dietary protein quality. Furthermore, these calcula- tions indicate that leucine and valine from the secondary limiting group are equally limiting with methionine from the first limiting group. Thus, a much more informative characterization of protein sources and diets is obtained by this simple method than the traditional BV.

Optimization and Standardization of Experimental Conditions

The accumulating knowledge from experimental determinations of requirements for amino acids has not yet led to a generally agreed standard ideal dietary amino acid profile. One reason for this is that a large number of factors influence amino acid requirements in practical pig production.

Obviously, many of these factors also influence experimental results when estimating requirements. Furthermore, the specific experimental basis may influence the results obtained and, consequently, the conclusions drawn. Therefore, all influencing factors should always be considered appropriately when estimating amino acid requirements.

Below, some generally influencing factors are discussed and possible ways of standardization of future studies on amino acid requirements are suggested.

Verification and control of available amino acids in the experimental diets

Firstly, the amounts of available amino acids, i.e. standardized digestible amino acids, in the actual batches of ingredients for the experimental diets should be known by using: (i) 100% digestible protein sources; (ii) table values of protein sources which are known to vary only little; (iii) direct in vivo digestibility measurements of the actual batches; or (iv) analyses for in vitro digestibility of protein from which the digestibility of the amino acids may be calculated with sufficient accuracy (Boisen, 2000).

Generally, supplementation of industrial amino acids is needed, in particular of the investigated amino acid because of the neces- sity of a series of experimental diets only varying in the composition of the investigated amino acid. These amino acids are generally expected to be 100% utilized. However, free amino acids may be more susceptible to reactions with other compounds in the feed than protein-bound ones. In particular, free methionine and tryptophan are known to be sensitive to destruction from free radicals pro- duced from oxidation of unsaturated fatty acids during storage.

The content of supplemented free amino acids should, therefore, generally be con- trolled throughout the experiment.

Utilization of free amino acids supplemented directly into the actual diet

Possible destruction of free amino acids can be avoided by direct supplementation to the diet immediately before feeding. However, because free amino acids may be quickly fermented in liquid feed, they would also be expected to be susceptible to the possible fermentation which may occur in the stomach before the digesta enter the small intestine. Furthermore, free amino acids may not be absorbed as efficiently as peptides actually generated after normal protein digestion. Thus, studies by van der Meulen et al. (1998) indicate that the effi- ciency in the recovery of free dietary amino acids in the portal vein may vary considerably, from 100% for isoleucine, to about 80% for lysine, threonine, methionine and tryptophan and only 60% for cystine.

The different possible influencing factors question the general expectation that industrial amino acids are 100% utilized and free amino acids may in some cases be utilized even less efficiently than protein-bound amino acids. Obviously, this possible overestimation of the actual concentration and utilization of supplemented industrial amino acids may result in overestimation of the amino acid requirements which are commonly based on experimental diets with increasing levels of the investigated amino acid supplemented in its free form.

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