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A Bioassay for Available Amino Acids and True Metabolizable Energy in Feedingstuffs1
A Bioassay for Available Amino Acids and True Metabolizable Energy in Feedingstuffs1 I. R. SIBBALD
Animal Research Institute, Central Experimental Farm, Ottawa, Ontario, Canada, K1A 0C6 (Received for publication July 19, 1978)
1979 Poultry Science 58:668-673 INTRODUCTION The amino acids (AA) contained in feedingstuffs are not necessarily available to the animal which consumes them. However, the establishment of available AA (AAA) requirement data and the formulation of diets based on the AAA in feed ingredients has been thwarted by the lack of a simple, rapid assay for AAA in feedingstuffs. A simple rapid bioassay for true metabolizable energy (TME) in feedingstuffs was developed by Sibbald (1976). The possibility of applying the basic methodology to the measurement of AAA is the subject of this report. Recently Likuski and Dorrell (1978) reported the AAA values for corn and soybean meal obtained by an adaption of the TME procedure. However, it has not been established that the relationship between AA output in excreta and AA input as feed is linear. Unless linearity exists, the methodology of the TME procedure will not be directly applicable. At best it will be necessary to evaluate each feedingstuff at several levels of input. MATERIALS AND METHODS
The birds were adult, male, Single Comb White Leghorns of the Kentville Control Strain housed in individual wire cages in a windowless room where they received 12 hr of light each day. Ninety-six birds were selected and starved for 24 hr to ensure that no feed residues re-
' Contribution No. 771. Animal Research Institute.
mained in their alimentary canals. The birds were then force-fed the appropriate dietary treatment and the excreta voided during the subsequent 24 hr was collected quantitatively, frozen, freeze-dried, and weighed. The treatments are described in Table 1. Treatment 1 was a negative control included to permit the measurement of the combined metabolic and endogenous excretion. It may be argued that a nitrogen-free diet should be administered to the negative control birds but this introduces the problem of what diet and in what quantity. Further, the use of such a diet would preclude the simultaneous measurement of TME. Treatments 2 to 6 involved feeding graded levels of glucose monohydrate which served as an AA-free source of energy. The purpose of these treatments was to measure the effect of energy input on AA excretion. Treatments 7 to 11 involved feeding graded levels of soybean meal (49% protein) to measure the effect of protein input on AA excretion. Treatments 12 to 15 involved an input of 30g of feed, consisting of mixtures of soybean meal and glucose monohydrate, to determine the effect of supplementary energy on the excretion of AA from soybean meal. The soybean meal and glucose were pelleted in a cold pellet mill. Samples of the soybean meal, glucose, and excreta (lg) were hydrolyzed with 6N HC1 as described by Smith et al. (1965) and AA concentrations were measured in a Beckman, Model 121M, automatic AA analyzer. The feeds and excreta, if sufficient, were assayed for gross energy content in an adiabatic oxygen bomb calorimeter. The procedures used to interpret 668
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ABSTRACT An experiment was made to determine if the basic methodology of the bioassay for true metabolizable energy (TME) can be applied to the measurement of available amino acids (AAA) in feedingstuffs. Administration of graded levels of glucose (0—30 g) had no effect on the excretion of 13 AA by adult roosters. Feeding graded levels of soybean meal, alone or in combination with glucose, caused linear increases in AA excretion. Methods of measuring AAA are described and the importance of correcting for metabolic and endogenous AA excretion is discussed. A bioassay for AAA which may be combined with the measurement of TME is described.
AMINO ACID-ENERGY BIOASSAY TABLE 1. The experimental treatments3Soybean meal input (g)
Glucose input (g)
0
6
12
0 6 12 18 24 30
1 2 3 4 5 6
7
8
... 12
13
24
30
nt number 9 10 15 14
11
18
the resulting data are described in the following section. RESULTS AND DISCUSSION One bird fed 30g of glucose monohydrate regurgitated and had to be discarded. In addition one bird on treatment 2 and another on treatment 6 provided insufficient excreta for gross energy determinations. The first phase of the experiment involved measuring the effect of glucose input on AA excretion. The AA output expressed as mg per bird (Y) was regressed on glucose input (g) (X). The results obtained for 13 AA, based on data of treatments 1 to 6, are summarized in Table
2. Most of the correlation coefficients were negative and none was significant. The intercepts of the regression equations ranged from 10.2 for histidine to 30.9 for aspartic acid. More important are the regression coefficients, none of which was significant. Consequently, the best description of each Y is its mean with the appropriate standard error. The lack of significance among the regression coefficients indicates that the AA excretion of the birds in this experiment was independent of glucose input. This is important because practical feedingstuffs contain energy sources other than proteins and AA. If energy input affected amino acid output, then the development of a bioassay for AAA based on the TME methodology would be exceedingly difficult. The second phase of the experiment involved measuring the effect of soybean meal input on AA excretion. The AA output was regressed on soybean meal input using data from treatments 1 and 7 to 11. The results are summarized in Table 3. The correlation coefficients indicate that the relationships between the two variables were linear. When correlations were measured using mean values for each treatment, the r values were much larger, ranging from .937 for tyrosine to .981 for alanine. The intercepts of the linear regressions provide estimates of the AA excretions of birds receiving no soybean meal; that is, they are indicative of the combined metabolic and endogenous excre-
TABLE 2. The relationships between amino acid excretion (Ymg) and glucose input (Xg)a
Amino acid
Correlation coefficient (r)
Intercept (a)
Regression coefficient (b)
Mean of Y
SEM
Alanine Arginine Aspartic acid Histidine Isoleucine Leucine Lysine Phenylalanine Proline Serine Threonine Tyrosine Valine
-.011 -.064 -.069 -.258 -.023 -.073 -.138 -.030 + .016 -.018 -.133 + .004 -.004
16.0 17.4 30.9 10.2 13.9 27.4 14.1 11.2 18.2 22.3 19.8 12.2 15.7
-.005 -.025 -.05 3 -.065 -.008 -.044 -.067 -.009 + .006 -.007 -.057 + .002 -.002
16.1 17.1 30.1 9.2 13.8 26.7 13.1 11.1 18.3 22.2 19.0 12.2 15.7
.8 .6 1.2 .4 .5 1.0 .8 .5 .6 .7 .7 .6 .7
a
There were 40 pairs of observations for each AA. None of the coefficients were significant at the 5% level of probability.
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There were 7 individual birds assigned to treatments 2 to 6 and 6 birds assigned to each of the other treatments.
669
670
SIBBALD TABLE 3. The relationships between amino acid excretion (Ymg) and soybean meal input (Xg)*
Intercept a
Regression coefficient b
(mg/g)
(%)
Alanine Arginine Aspartic acid Histidine Isoleucine Leucine Lysine Phenylalanine Proline Serine Threonine Tyrosine Valine
.904 .921 .918 .833 .903 .897 .800 .868 .841 .900 .907 .832 .840
13.7 14.2 28.7 9.7 13.0 24.9 12.0 10.3 17.0 21.2 18.4 11.0 14.3
1.69 3.32 3.96 .86 1.58 3.00 .99 1.45 1.39 1.84 1.78 .90 1.52
16.7 29.1 47.6 10.9 16.1 31.0 26.2 20.4 21.1 20.8 15.5 13.6 16.5
90.8 89.7 92.3 92.7 91.1 91.2 96.4 93.4 93.8 91.8 89.7 93.8 91.6
There were 36 pairs of observations for each AA.
tions. As such they may be compared with the mean values of Y in Table 2. The regression coefficients are estimates of the increase in the amount of AA excreted for each gram of soybean meal input. Subtracting the regression coefficient from the concentration of the AA in the soybean meal yields an AAA value which can be expressed either as a weight or percentage. Using this procedure the availabilities of the AA in the sample of soybean meal ranged from 89.7 to 96.4%.
The third phase of the experiment involved measuring the effect of soybean meal to glucose mixtures on A A excretion. The AA ouput was regressed on soybean meal input using data from treatments 6 and treatments 11 to 15. The results are summarized in Table 4. The results obtained are very similar to those obtained with soybean meal alone (Table 3) confirming the earlier observation that glucose input did not affect the AA availabilities. The data reported demonstrate the relation-
TABLE 4. The relationship between amino acid excretion (Ymg) and the input of soybean meal (Xg) made to 30 g with glucose*
Amino acid
Correlation coefficient (r)
Intercept a
Regression coefficient b
(mg/g)
(%)
Alanine Arginine Aspartic acid Histidine Isoleucine Leucine Lysine Phenylalanine Proline Serine Threonine Tyrosine Valine
.913 .901 .916 .772 .907 .912 .730 .908 .855 .890 .912 .837 .880
13.4 7.0 28.2 9.0 12.8 24.7 14.2 10.1 17.5 21.1 18.1 11.1 14.1
1.59 3.12 3.67 .82 1.46 2.74 .85 1.31 1.27 1.66 1.64 .81 1.38
16.6 29.3 47.9 11.0 16.2 31.2 26.4 20.5 21.2 20.9 15.6 13.7 16.6
90.2 90.4 92.9 93.2 91.7 91.9 97.0 94.0 94.3 92.6 90.5 94.4 92.3
a
There were 36 pairs of observations for each AA.
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Amino acid
Correlation coefficient (r)
AMINO ACID-ENERGY BIOASSAY
AAA (mg/g) =
AA input — AA output ; boy input
where: AA input and AA output are expressed as mg/bird and soy input is g/bird. Simultaneously AAA corrected for the mean metabolic and endogenous AA excretions of birds on treatment 1 were calculated: AAinput—(AA output F—AA output C) AAA c (mg/g)= Soy input
where: AA output F is the AA excretion of the fed birds, and AA output C is the AA excretion of the negative control birds. The corrected data are comparable to the AAA values of Tables 3 and 4 which were obtained by linear regression analysis. The AAA and AAAC data, calculated as above, are displayed in Table 5. The AAA values tend to increase in a curvilinear manner with the increase in soybean input. This is expected because the output of endogenous and metabolic AA is charged against an increasing input and therefore has a diminishing effect. This has been illustrated experimentally for energy in an earlier report (Sibbald, 1975). The AAAC values also display some variation but it tends to be independent of the soybean input. The final two columns of data in Table 5 show that AAA values are lower than AAAC values and tend to be more variable when there is a range of inputs. The correction, by the method of calculation, must increase the AAA value.
The relationships between AA inputs and outputs appear to parallel the relationships observed previously for energy (Sibbald, 1975, 1976); therefore it is reasonable to assume that the basic methodology of the TME assay can be applied successfully to the measurement of AAA in feedingstuffs. The assay is summarized: 1) Adult male SCWL birds are starved for 24 hr to empty their alimentary canals. 2) A bird is selected and force-fed a known quantity of the feedingstuff to be assayed, placed in a cage over a plastic tray, and the time is recorded. 3) Twenty-four hours later the tray is removed, the excreta are collected quantitatively, frozen, freeze-dried, and weighed. 4) A similar starved bird serves as a negative control and is given no feed before placed in a cage over a tray at a recorded time. The excreta voided in 24 hr are treated as in step 3.5) Samples of the feed and excreta are assayed for AA content and the AAC values of the feedingstuff are calculated as described above. Details of the TME methodology, including the force-feeding procedure, have been published elsewhere (Sibbald, 1977a). The possibility of microbial action in the alimentary canal affecting AAA was discussed by Likuski and Dorrell (1978). Erbersdobler and Riedel (1972) found no differences in AAA values measured with germfree, monoinfected, and conventional chickens. Salter and Fulford (1974) concluded that the gut microflora of chicks had little influence on the digestion of dietary proteins but may serve an important role in the degradation of endogenous proteins. In the present experiment the feeding of glucose alone or with soybean meal did not change AA availability nor did the changes in soybean input. These findings support the view that microbial action probably had little effect under the conditions of the assay. This requires further investigation as do other aspects of the procedure, but it appears that the basic methodology is applicable to available AA measurement. The experiment provided an opportunity to obtain additional information about the TME procedure and to demonstrate the possibility of combining TME and AAA bioassays. The results of the TME bioassay are contained in Table 6. Although the mean value for glucose at the 6g level of input (3.36 kcal/g) was a little lower than the values obtained at higher inputs it was not significantly (P>.05) different. Similarly, the mean value for soybean meal at the same level of input appeared to be slightly out
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ship between AA excretion and the input of a source of AA, such as soybean meal, is linear for at least 13 AA. Thus it is feasible to describe a bioassay for AAA in poultry feedingstuffs. However, before progressing to this step it is necessary to stress that the estimates of AA availability contained in Tables 3 and 4 were based on multiple input levels. This is expensive and time consuming. The TME bioassay is based on a single input level for each feedingstuff with a correction being made for the combined metabolic and endogenous energy excretion of negative control birds. The results of this experiment provide an opportunity to determine if a similar procedure is feasible in the assessment of A A availability. The mean values for the AA outputs on each soybean treatment (7 to 15) were used to calculate the AAA/g of soybean meal:
671
SIBBALD
672
TABLE 5. Effect of the correction for combined metabolic and endogenous amino acid excretion on the observed available amino acids in soybean meal* Soybean glucose nput/bird (g) 6:0
12:0
18:0
24:0
30:0
6:24
12:18
18:12
24:6
Mean
SEM
Alanine A A A AAAC Arginine A A A AAA C Aspartic acid AAA AAA C Histidine A A A AAA C Isoleucine A A A AAAC Leucine A A A AAA C Lysine A A A AAAC Phenylalanine A A A AAAC Proline A A A AAAC Serine A A A AAAC Threonine A A A AAAC Tyrosine AAA AAA C Valine AAA AAA C
14.3 16.6 27.5 30.1 42.9 47.6 9.3 11.2 14.0 16.2 27.2 31.4 24.2 26.6 18.7 20.4 18.6 21.5 17.5 21.0 12.6 15.8 11.8 13.6 14.1 16.4
15.9 17.0 28.4 29.7 45.8 48.2 10.4 11.4 15.3 16.4 29.4 31.5 25.5 26.7 19.8 20.6 19.8 21.2 19.3 21.0 14.3 15.9 12.8 13.6 15.5 16.6
16.0 16.8 27.4 28.3 45.6 47.2 10.4 11.0 15.3 16.0 29.3 30.7 25.7 26.5 19.7 20.3 19.9 20.9 19.3 20.4 14.4 15.4 12.8 13.4 15.6 16.3
15.9 16.5 28.4 29.1 45.7 46.9 10.5 11.0 15.3 15.8 29.4 30.4 25.4 26.0 19.6 20.0 20.0 20.7 20.1 21.0 14.4 15.2 12.9 13.3 15.5 16.0
16.4 16.9 28.8 29.3 47.1 48.0 10.6 11.0 15.8 16.3 30.5 31.3 25.9 26.4 20.2 20.6 20.8 21.3 20.3 21.0 15.1 15.7 13.4 13.7 16.3 16.7
14.9 17.2 28.9 31.5 44.2 49.0 9.9 11.9 14.5 16.7 28.1 32.4 24.6 27.0 19.2 20.9 19.0 21.8 18.0 21.6 13.3 16.6 12.2 13.9 14.8 17.0
15.6 16.8 29.0 30.3 45.2 47.5 10.3 11.3 15.0 16.1 28.8 31.0 24.7 25.9 19.5 20.3 19.4 20.8 18.9 20.7 14.0 15.6 12.5 13.4 15.2 16.3
16.0 16.8 29.2 30.0 45.9 47.5 10.4 11.0 15.4 16.2 29.7 31.1 25.6 26.4 19.9 20.5 20.0 21.0 19.6 20.8 14.5 15.5 13.0 13.6 15.7 16.5
16.3 16.9 29.0 29.6 46.7 47.9 10.8 11.3 15.7 16.2 30.2 31.2 25.8 26.4 20.1 20.5 20.3 21.0 20.1 21.0 14.9 15.7 13.1 13.6 16.0 16.5
15.7 16.8 28.5 29.8 45.4 47.8 10.3 11.2 15.1 16.2 29.2 31.2 25.3 26.4 19.6 20.4 19.8 21.1 19.2 20.9 14.2 15.7 12.7 13.6 15.4 16.5
.23 .07 .22 .30 .42 .20 .15 .10 .19 .08 .34 .19 .20 .11 .15 .08 .22 .12 .32 .11 .26 .13 .16 .06 .22 .09
Data expressed as mg/g of soybean meal containing 91.0% of dry matter. AAA available amino acid calculated from the mean input and output data; AAAC corrected for the mean amino acid excretion of the negative control birds.
TABLE 6. The TME values of glucose monohydrate and soybean meal at several levels of input Input (g) Glucose
Soybean meal
6 12 18 24 30 Mean 6 12 18 24 30 Mean 24 18 12 6
6 12 18 24
No. of observations
TME kcal/g air dry Mean
SEM
6 7 7 7 5
3.36 3.55 3.51 3.50 3.54
.07 .03 .04 .02 .02
32
3.49
.02
6 6 6 6 6
3.14 2.92 2.81 2.82 2.91
.17 .07 .06 .03 .03
30
2.92
.04
6 6 6
3.46 3.21 3.17 3.07
.01 .04 .04 .08
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Amino acid b
AMINO ACID-ENERGY BIOASSAY of line. However, t h e standard errors of these t w o means were also relatively large t h u s confirming the finding of Sibbald ( 1 9 7 7 b ) t h a t t h e sensitivity of t h e assay t e n d s t o increase with feed i n p u t u p t o levels at which regurgitation m a y occur.
ACKNOWLEDGMENTS T h e a u t h o r wishes t o t h a n k S. T o b i n and D . T u t t e for their able technical assistance. T h e a m i n o acid analyses were performed b y t h e Chemical and Biological Research Institute of Agriculture Canada.
REFERENCES Erbersdobler, H., and G. Riedel, 1972. Bestimmung der Aminosaurenverdaulichkeit bei keimfrei und konventionell gehaltenen Kuken. 1. Mitteilung. Archiv fur Geflugelkunde 36:218-222. Likuski, H. J. A., and H. G. Dorrell, 1978. A bioassay for rapid determinations of amino acid availability values. Poultry Sci. 57:1658-1660. Salter, D. N., and R. J. Fulford, 1974. The influence of the gut microflora on the digestion of dietary and endogenous proteins: studies of the amino acid composition of the excreta of germ-free and conventional chicks. Brit. J. Nutr. 32:625-637. Sibbald, I. R., 1975. The effect of level of feed intake on metabolizable energy values measured with adult roosters. Poultry Sci. 54:1990-1997. Sibbald, I. R., 1976. A bioassay for true metabolizable energy in feedingstuffs. Poultry Sci. 55:303—308. Sibbald, I. R., 1977a. The true metabolizable energy system. Part 1. Feedstuffs 49(42):21-22. Sibbald, I. R., 1977b. The effect of level of feed input on true metabolizable energy values. Poultry Sci. 56:1662-1663. Smith, P., M. E. Ambrose, and G. M. Knobl, 1965. Possible interference of fats, carbohydrates, and salts in amino acid determinations in fish meals, fish protein concentrates, and mixed animal feeds. J. Agr. Food Chem. 13:266-268.
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It is a p p a r e n t t h a t t h e TME and A A A bioassays m a y be combined t h u s reducing t h e t i m e and cost of obtaining t h e t w o sets of d a t a for a feedingstuff. T h e time r e q u i r e m e n t for t h e c o m b i n e d assay d e p e n d s u p o n t h e analytical techniques e m p l o y e d but it should be possible t o c o m p l e t e t h e w o r k in less t h a n a week.
673
Animal Research Institute, Central Experimental Farm, Ottawa, Ontario, Canada, K1A 0C6 (Received for publication July 19, 1978)
1979 Poultry Science 58:668-673 INTRODUCTION The amino acids (AA) contained in feedingstuffs are not necessarily available to the animal which consumes them. However, the establishment of available AA (AAA) requirement data and the formulation of diets based on the AAA in feed ingredients has been thwarted by the lack of a simple, rapid assay for AAA in feedingstuffs. A simple rapid bioassay for true metabolizable energy (TME) in feedingstuffs was developed by Sibbald (1976). The possibility of applying the basic methodology to the measurement of AAA is the subject of this report. Recently Likuski and Dorrell (1978) reported the AAA values for corn and soybean meal obtained by an adaption of the TME procedure. However, it has not been established that the relationship between AA output in excreta and AA input as feed is linear. Unless linearity exists, the methodology of the TME procedure will not be directly applicable. At best it will be necessary to evaluate each feedingstuff at several levels of input. MATERIALS AND METHODS
The birds were adult, male, Single Comb White Leghorns of the Kentville Control Strain housed in individual wire cages in a windowless room where they received 12 hr of light each day. Ninety-six birds were selected and starved for 24 hr to ensure that no feed residues re-
' Contribution No. 771. Animal Research Institute.
mained in their alimentary canals. The birds were then force-fed the appropriate dietary treatment and the excreta voided during the subsequent 24 hr was collected quantitatively, frozen, freeze-dried, and weighed. The treatments are described in Table 1. Treatment 1 was a negative control included to permit the measurement of the combined metabolic and endogenous excretion. It may be argued that a nitrogen-free diet should be administered to the negative control birds but this introduces the problem of what diet and in what quantity. Further, the use of such a diet would preclude the simultaneous measurement of TME. Treatments 2 to 6 involved feeding graded levels of glucose monohydrate which served as an AA-free source of energy. The purpose of these treatments was to measure the effect of energy input on AA excretion. Treatments 7 to 11 involved feeding graded levels of soybean meal (49% protein) to measure the effect of protein input on AA excretion. Treatments 12 to 15 involved an input of 30g of feed, consisting of mixtures of soybean meal and glucose monohydrate, to determine the effect of supplementary energy on the excretion of AA from soybean meal. The soybean meal and glucose were pelleted in a cold pellet mill. Samples of the soybean meal, glucose, and excreta (lg) were hydrolyzed with 6N HC1 as described by Smith et al. (1965) and AA concentrations were measured in a Beckman, Model 121M, automatic AA analyzer. The feeds and excreta, if sufficient, were assayed for gross energy content in an adiabatic oxygen bomb calorimeter. The procedures used to interpret 668
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ABSTRACT An experiment was made to determine if the basic methodology of the bioassay for true metabolizable energy (TME) can be applied to the measurement of available amino acids (AAA) in feedingstuffs. Administration of graded levels of glucose (0—30 g) had no effect on the excretion of 13 AA by adult roosters. Feeding graded levels of soybean meal, alone or in combination with glucose, caused linear increases in AA excretion. Methods of measuring AAA are described and the importance of correcting for metabolic and endogenous AA excretion is discussed. A bioassay for AAA which may be combined with the measurement of TME is described.
AMINO ACID-ENERGY BIOASSAY TABLE 1. The experimental treatments3Soybean meal input (g)
Glucose input (g)
0
6
12
0 6 12 18 24 30
1 2 3 4 5 6
7
8
... 12
13
24
30
nt number 9 10 15 14
11
18
the resulting data are described in the following section. RESULTS AND DISCUSSION One bird fed 30g of glucose monohydrate regurgitated and had to be discarded. In addition one bird on treatment 2 and another on treatment 6 provided insufficient excreta for gross energy determinations. The first phase of the experiment involved measuring the effect of glucose input on AA excretion. The AA output expressed as mg per bird (Y) was regressed on glucose input (g) (X). The results obtained for 13 AA, based on data of treatments 1 to 6, are summarized in Table
2. Most of the correlation coefficients were negative and none was significant. The intercepts of the regression equations ranged from 10.2 for histidine to 30.9 for aspartic acid. More important are the regression coefficients, none of which was significant. Consequently, the best description of each Y is its mean with the appropriate standard error. The lack of significance among the regression coefficients indicates that the AA excretion of the birds in this experiment was independent of glucose input. This is important because practical feedingstuffs contain energy sources other than proteins and AA. If energy input affected amino acid output, then the development of a bioassay for AAA based on the TME methodology would be exceedingly difficult. The second phase of the experiment involved measuring the effect of soybean meal input on AA excretion. The AA output was regressed on soybean meal input using data from treatments 1 and 7 to 11. The results are summarized in Table 3. The correlation coefficients indicate that the relationships between the two variables were linear. When correlations were measured using mean values for each treatment, the r values were much larger, ranging from .937 for tyrosine to .981 for alanine. The intercepts of the linear regressions provide estimates of the AA excretions of birds receiving no soybean meal; that is, they are indicative of the combined metabolic and endogenous excre-
TABLE 2. The relationships between amino acid excretion (Ymg) and glucose input (Xg)a
Amino acid
Correlation coefficient (r)
Intercept (a)
Regression coefficient (b)
Mean of Y
SEM
Alanine Arginine Aspartic acid Histidine Isoleucine Leucine Lysine Phenylalanine Proline Serine Threonine Tyrosine Valine
-.011 -.064 -.069 -.258 -.023 -.073 -.138 -.030 + .016 -.018 -.133 + .004 -.004
16.0 17.4 30.9 10.2 13.9 27.4 14.1 11.2 18.2 22.3 19.8 12.2 15.7
-.005 -.025 -.05 3 -.065 -.008 -.044 -.067 -.009 + .006 -.007 -.057 + .002 -.002
16.1 17.1 30.1 9.2 13.8 26.7 13.1 11.1 18.3 22.2 19.0 12.2 15.7
.8 .6 1.2 .4 .5 1.0 .8 .5 .6 .7 .7 .6 .7
a
There were 40 pairs of observations for each AA. None of the coefficients were significant at the 5% level of probability.
Downloaded from http://ps.oxfordjournals.org/ at Albert R. Mann Library on December 15, 2014
There were 7 individual birds assigned to treatments 2 to 6 and 6 birds assigned to each of the other treatments.
669
670
SIBBALD TABLE 3. The relationships between amino acid excretion (Ymg) and soybean meal input (Xg)*
Intercept a
Regression coefficient b
(mg/g)
(%)
Alanine Arginine Aspartic acid Histidine Isoleucine Leucine Lysine Phenylalanine Proline Serine Threonine Tyrosine Valine
.904 .921 .918 .833 .903 .897 .800 .868 .841 .900 .907 .832 .840
13.7 14.2 28.7 9.7 13.0 24.9 12.0 10.3 17.0 21.2 18.4 11.0 14.3
1.69 3.32 3.96 .86 1.58 3.00 .99 1.45 1.39 1.84 1.78 .90 1.52
16.7 29.1 47.6 10.9 16.1 31.0 26.2 20.4 21.1 20.8 15.5 13.6 16.5
90.8 89.7 92.3 92.7 91.1 91.2 96.4 93.4 93.8 91.8 89.7 93.8 91.6
There were 36 pairs of observations for each AA.
tions. As such they may be compared with the mean values of Y in Table 2. The regression coefficients are estimates of the increase in the amount of AA excreted for each gram of soybean meal input. Subtracting the regression coefficient from the concentration of the AA in the soybean meal yields an AAA value which can be expressed either as a weight or percentage. Using this procedure the availabilities of the AA in the sample of soybean meal ranged from 89.7 to 96.4%.
The third phase of the experiment involved measuring the effect of soybean meal to glucose mixtures on A A excretion. The AA ouput was regressed on soybean meal input using data from treatments 6 and treatments 11 to 15. The results are summarized in Table 4. The results obtained are very similar to those obtained with soybean meal alone (Table 3) confirming the earlier observation that glucose input did not affect the AA availabilities. The data reported demonstrate the relation-
TABLE 4. The relationship between amino acid excretion (Ymg) and the input of soybean meal (Xg) made to 30 g with glucose*
Amino acid
Correlation coefficient (r)
Intercept a
Regression coefficient b
(mg/g)
(%)
Alanine Arginine Aspartic acid Histidine Isoleucine Leucine Lysine Phenylalanine Proline Serine Threonine Tyrosine Valine
.913 .901 .916 .772 .907 .912 .730 .908 .855 .890 .912 .837 .880
13.4 7.0 28.2 9.0 12.8 24.7 14.2 10.1 17.5 21.1 18.1 11.1 14.1
1.59 3.12 3.67 .82 1.46 2.74 .85 1.31 1.27 1.66 1.64 .81 1.38
16.6 29.3 47.9 11.0 16.2 31.2 26.4 20.5 21.2 20.9 15.6 13.7 16.6
90.2 90.4 92.9 93.2 91.7 91.9 97.0 94.0 94.3 92.6 90.5 94.4 92.3
a
There were 36 pairs of observations for each AA.
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Amino acid
Correlation coefficient (r)
AMINO ACID-ENERGY BIOASSAY
AAA (mg/g) =
AA input — AA output ; boy input
where: AA input and AA output are expressed as mg/bird and soy input is g/bird. Simultaneously AAA corrected for the mean metabolic and endogenous AA excretions of birds on treatment 1 were calculated: AAinput—(AA output F—AA output C) AAA c (mg/g)= Soy input
where: AA output F is the AA excretion of the fed birds, and AA output C is the AA excretion of the negative control birds. The corrected data are comparable to the AAA values of Tables 3 and 4 which were obtained by linear regression analysis. The AAA and AAAC data, calculated as above, are displayed in Table 5. The AAA values tend to increase in a curvilinear manner with the increase in soybean input. This is expected because the output of endogenous and metabolic AA is charged against an increasing input and therefore has a diminishing effect. This has been illustrated experimentally for energy in an earlier report (Sibbald, 1975). The AAAC values also display some variation but it tends to be independent of the soybean input. The final two columns of data in Table 5 show that AAA values are lower than AAAC values and tend to be more variable when there is a range of inputs. The correction, by the method of calculation, must increase the AAA value.
The relationships between AA inputs and outputs appear to parallel the relationships observed previously for energy (Sibbald, 1975, 1976); therefore it is reasonable to assume that the basic methodology of the TME assay can be applied successfully to the measurement of AAA in feedingstuffs. The assay is summarized: 1) Adult male SCWL birds are starved for 24 hr to empty their alimentary canals. 2) A bird is selected and force-fed a known quantity of the feedingstuff to be assayed, placed in a cage over a plastic tray, and the time is recorded. 3) Twenty-four hours later the tray is removed, the excreta are collected quantitatively, frozen, freeze-dried, and weighed. 4) A similar starved bird serves as a negative control and is given no feed before placed in a cage over a tray at a recorded time. The excreta voided in 24 hr are treated as in step 3.5) Samples of the feed and excreta are assayed for AA content and the AAC values of the feedingstuff are calculated as described above. Details of the TME methodology, including the force-feeding procedure, have been published elsewhere (Sibbald, 1977a). The possibility of microbial action in the alimentary canal affecting AAA was discussed by Likuski and Dorrell (1978). Erbersdobler and Riedel (1972) found no differences in AAA values measured with germfree, monoinfected, and conventional chickens. Salter and Fulford (1974) concluded that the gut microflora of chicks had little influence on the digestion of dietary proteins but may serve an important role in the degradation of endogenous proteins. In the present experiment the feeding of glucose alone or with soybean meal did not change AA availability nor did the changes in soybean input. These findings support the view that microbial action probably had little effect under the conditions of the assay. This requires further investigation as do other aspects of the procedure, but it appears that the basic methodology is applicable to available AA measurement. The experiment provided an opportunity to obtain additional information about the TME procedure and to demonstrate the possibility of combining TME and AAA bioassays. The results of the TME bioassay are contained in Table 6. Although the mean value for glucose at the 6g level of input (3.36 kcal/g) was a little lower than the values obtained at higher inputs it was not significantly (P>.05) different. Similarly, the mean value for soybean meal at the same level of input appeared to be slightly out
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ship between AA excretion and the input of a source of AA, such as soybean meal, is linear for at least 13 AA. Thus it is feasible to describe a bioassay for AAA in poultry feedingstuffs. However, before progressing to this step it is necessary to stress that the estimates of AA availability contained in Tables 3 and 4 were based on multiple input levels. This is expensive and time consuming. The TME bioassay is based on a single input level for each feedingstuff with a correction being made for the combined metabolic and endogenous energy excretion of negative control birds. The results of this experiment provide an opportunity to determine if a similar procedure is feasible in the assessment of A A availability. The mean values for the AA outputs on each soybean treatment (7 to 15) were used to calculate the AAA/g of soybean meal:
671
SIBBALD
672
TABLE 5. Effect of the correction for combined metabolic and endogenous amino acid excretion on the observed available amino acids in soybean meal* Soybean glucose nput/bird (g) 6:0
12:0
18:0
24:0
30:0
6:24
12:18
18:12
24:6
Mean
SEM
Alanine A A A AAAC Arginine A A A AAA C Aspartic acid AAA AAA C Histidine A A A AAA C Isoleucine A A A AAAC Leucine A A A AAA C Lysine A A A AAAC Phenylalanine A A A AAAC Proline A A A AAAC Serine A A A AAAC Threonine A A A AAAC Tyrosine AAA AAA C Valine AAA AAA C
14.3 16.6 27.5 30.1 42.9 47.6 9.3 11.2 14.0 16.2 27.2 31.4 24.2 26.6 18.7 20.4 18.6 21.5 17.5 21.0 12.6 15.8 11.8 13.6 14.1 16.4
15.9 17.0 28.4 29.7 45.8 48.2 10.4 11.4 15.3 16.4 29.4 31.5 25.5 26.7 19.8 20.6 19.8 21.2 19.3 21.0 14.3 15.9 12.8 13.6 15.5 16.6
16.0 16.8 27.4 28.3 45.6 47.2 10.4 11.0 15.3 16.0 29.3 30.7 25.7 26.5 19.7 20.3 19.9 20.9 19.3 20.4 14.4 15.4 12.8 13.4 15.6 16.3
15.9 16.5 28.4 29.1 45.7 46.9 10.5 11.0 15.3 15.8 29.4 30.4 25.4 26.0 19.6 20.0 20.0 20.7 20.1 21.0 14.4 15.2 12.9 13.3 15.5 16.0
16.4 16.9 28.8 29.3 47.1 48.0 10.6 11.0 15.8 16.3 30.5 31.3 25.9 26.4 20.2 20.6 20.8 21.3 20.3 21.0 15.1 15.7 13.4 13.7 16.3 16.7
14.9 17.2 28.9 31.5 44.2 49.0 9.9 11.9 14.5 16.7 28.1 32.4 24.6 27.0 19.2 20.9 19.0 21.8 18.0 21.6 13.3 16.6 12.2 13.9 14.8 17.0
15.6 16.8 29.0 30.3 45.2 47.5 10.3 11.3 15.0 16.1 28.8 31.0 24.7 25.9 19.5 20.3 19.4 20.8 18.9 20.7 14.0 15.6 12.5 13.4 15.2 16.3
16.0 16.8 29.2 30.0 45.9 47.5 10.4 11.0 15.4 16.2 29.7 31.1 25.6 26.4 19.9 20.5 20.0 21.0 19.6 20.8 14.5 15.5 13.0 13.6 15.7 16.5
16.3 16.9 29.0 29.6 46.7 47.9 10.8 11.3 15.7 16.2 30.2 31.2 25.8 26.4 20.1 20.5 20.3 21.0 20.1 21.0 14.9 15.7 13.1 13.6 16.0 16.5
15.7 16.8 28.5 29.8 45.4 47.8 10.3 11.2 15.1 16.2 29.2 31.2 25.3 26.4 19.6 20.4 19.8 21.1 19.2 20.9 14.2 15.7 12.7 13.6 15.4 16.5
.23 .07 .22 .30 .42 .20 .15 .10 .19 .08 .34 .19 .20 .11 .15 .08 .22 .12 .32 .11 .26 .13 .16 .06 .22 .09
Data expressed as mg/g of soybean meal containing 91.0% of dry matter. AAA available amino acid calculated from the mean input and output data; AAAC corrected for the mean amino acid excretion of the negative control birds.
TABLE 6. The TME values of glucose monohydrate and soybean meal at several levels of input Input (g) Glucose
Soybean meal
6 12 18 24 30 Mean 6 12 18 24 30 Mean 24 18 12 6
6 12 18 24
No. of observations
TME kcal/g air dry Mean
SEM
6 7 7 7 5
3.36 3.55 3.51 3.50 3.54
.07 .03 .04 .02 .02
32
3.49
.02
6 6 6 6 6
3.14 2.92 2.81 2.82 2.91
.17 .07 .06 .03 .03
30
2.92
.04
6 6 6
3.46 3.21 3.17 3.07
.01 .04 .04 .08
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Amino acid b
AMINO ACID-ENERGY BIOASSAY of line. However, t h e standard errors of these t w o means were also relatively large t h u s confirming the finding of Sibbald ( 1 9 7 7 b ) t h a t t h e sensitivity of t h e assay t e n d s t o increase with feed i n p u t u p t o levels at which regurgitation m a y occur.
ACKNOWLEDGMENTS T h e a u t h o r wishes t o t h a n k S. T o b i n and D . T u t t e for their able technical assistance. T h e a m i n o acid analyses were performed b y t h e Chemical and Biological Research Institute of Agriculture Canada.
REFERENCES Erbersdobler, H., and G. Riedel, 1972. Bestimmung der Aminosaurenverdaulichkeit bei keimfrei und konventionell gehaltenen Kuken. 1. Mitteilung. Archiv fur Geflugelkunde 36:218-222. Likuski, H. J. A., and H. G. Dorrell, 1978. A bioassay for rapid determinations of amino acid availability values. Poultry Sci. 57:1658-1660. Salter, D. N., and R. J. Fulford, 1974. The influence of the gut microflora on the digestion of dietary and endogenous proteins: studies of the amino acid composition of the excreta of germ-free and conventional chicks. Brit. J. Nutr. 32:625-637. Sibbald, I. R., 1975. The effect of level of feed intake on metabolizable energy values measured with adult roosters. Poultry Sci. 54:1990-1997. Sibbald, I. R., 1976. A bioassay for true metabolizable energy in feedingstuffs. Poultry Sci. 55:303—308. Sibbald, I. R., 1977a. The true metabolizable energy system. Part 1. Feedstuffs 49(42):21-22. Sibbald, I. R., 1977b. The effect of level of feed input on true metabolizable energy values. Poultry Sci. 56:1662-1663. Smith, P., M. E. Ambrose, and G. M. Knobl, 1965. Possible interference of fats, carbohydrates, and salts in amino acid determinations in fish meals, fish protein concentrates, and mixed animal feeds. J. Agr. Food Chem. 13:266-268.
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It is a p p a r e n t t h a t t h e TME and A A A bioassays m a y be combined t h u s reducing t h e t i m e and cost of obtaining t h e t w o sets of d a t a for a feedingstuff. T h e time r e q u i r e m e n t for t h e c o m b i n e d assay d e p e n d s u p o n t h e analytical techniques e m p l o y e d but it should be possible t o c o m p l e t e t h e w o r k in less t h a n a week.
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