- Email: [email protected]
Amino Acid Fortification of a Low-Protein Corn and Soybean Meal Diet for Chicks
METABOLISM AND NUTRITION Amino Acid Fortification of a Low-Protein Corn and Soybean Meal Diet for Chicks YANMING HAN, HIROYUKI SUZUKI,1 CARL M. PARSONS, and DAVID H. BAKER2 Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801 ABSTRACT Seven experiments were conducted to investigate whether a 19% CP corn and soybean meal (CS) diet could be fortified properly with amino acids (AA) to produce performance in 1- to 3-wk-old chicks equal to that obtained with a 23% CP, CS diet supplemented with Met. In one experiment, the assay was carried out to 6 wk of age. During 4- to 6-wk posthatching, the positive control was a 20% CP, CS diet with added Met and the low-protein diet was a 16% CP, CS diet fortified with limiting AA. The two most limiting AA in the low-protein diet (19% CP) were found to be Met and Lys. Arginine, Val, and Thr were observed to be limiting as well. Weight gain and feed efficiency were substantially increased and body fat content decreased when the low-protein diet was supplemented with the five limiting AA and amino nitrogen in the form of Glu. Addition of potassium had no effect on performance of chicks fed the AA-fortified, low-protein diet. With all trials considered together, chicks fed the low-protein diet fortified with the five limiting AA and Glu gained at the same rate with similar feed efficiency and had estimated body fat levels comparable to birds fed the 23% CP, positive control diet. From 3 to 6 wk of age, chicks fed the AA-fortified, 16% CP diet had growth performance similar to chicks fed the 20% CP, positive control diet. {Key words: low-protein diets, amino acids, broilers, growth performance, potassium) 1992 Poultry Science 71:1168-1178
INTRODUCTION Dietary amino acids (AA) in excess of the needs of chicks impair feed intake and growth rate (Waldroup et al., 1976). In practice it is very difficult, if not impossible, to formulate diets with natural feed ingredients that will provide all the AA needed by chicks in adequate quantities and also to maintain an optimal AA balance with minimal excesses. Recent advances in commercial production of crystalline AA make several indispensable AA (IAA) more economically feasible for
Received for publication September 13, 1991. Accepted for publication March 6, 1992. Visiting scientist, Ajinomoto Co., Inc., Technology Commercialization Laboratory, 2004 South Wright Street, Urbana, IL 61801. To whom correspondence should be addressed.
use in animal feeds. Addition of IAA to diets permits a reduction in dietary protein content and at the same time provides the needs of all IAA by chicks with an optimal AA profile. It is known that broiler chicks fed diets marginal in protein but fortified with Met or Met and Lys will perform as well as those fed a diet higher in protein (Jensen, 1991). There are great discrepancies, however, in attempts that have been made to feed broiler chicks lowprotein diets that are supplemented with several crystalline IAA. Bornstein and Lipstein (1975) compared a 19.7% CP diet (supplemented with Met and Lys) to a 23.1% CP diet supplemented with Met and found that chick growth and feed efficiency were equal for these two diets. Waldroup et al. (1976) reported that maximal growth of young chicks could be obtained with a 19% CP diet supple-
1168
AMINO ACID SUPPLEMENTATION
mented with crystalline AA. Protein utilization efficiency was improved as a consequence of mirtimizing excess levels of IAA in the diets. When feeding young chicks a 16% CP diet supplemented with all IAA equivalent to the levels present in a 20% CP diet, Schutte (1987) observed equal weight gain and feed efficiency during the period 7- to 21-days posthatching. More recently, Parr and Summers (1991) reported that chicks fed low-protein diets (ranging from 21 to 16.5% CP) supplemented with IAA had similar growth rates, feed efficiencies, and total carcass protein contents as those fed a 23% CP diet. Conversely, University of Georgia researchers (Fancher and Jensen, 1989a,b,c; Pinchasov et al, 1990; Colnago et al, 1991) concluded that optimal performance of starter and grower chicks could not be achieved with low-protein diets supplemented with crystalline AA. Similar findings were observed by Edmonds et al. (1985) when the protein level of corn and soybean meal (CS) diets was reduced from 24 to 16%. The reasons for these different findings among investigators are not readily apparent. The objective of the studies reported here was to approach this problem systematically in the hope of answering the following questions: 1) Can a lowprotein diet be properly fortified with crystalline AA so that performance will equal that of the birds fed a higher protein diet? 2) Which AA are limiting in the lowprotein diet and what quantities of the AA are needed? 3) Is dispensable AA N necessary for optimal chick performance and body composition? and 4) Is reduced K level in the low-protein (low soybean meal) diet a factor in utilization of the supplemental AA? MATERIALS AND METHODS One-week-old male chicks resulting from the cross of New Hampshire males and Columbian Plymouth Rock females were used in all chick assays, except for one assay wherein male Hubbard chicks (Hubbard x Hubbard) were used. The chicks were fed a 24% CP, CS pretest diet during the 1st wk posthatching. Following an overnight fast, the chicks were weighed
1169
and wing-banded and then allotted to dietary treatments as described by Sasse and Baker (1974). Four (in Experiments 1 to 5) or five (in Experiments 6 and 7) groups of five chicks were assigned to each dietary treatment. The chicks were housed in thermostatically controlled starter batteries with raised wire floors in an environmentally controlled room. Feed and water were supplied for ad libitum intake. Light was provided 24 h daily. Seven experiments were conducted, and all but Experiment 6 involved the 14-day feeding period 8- to 22-days posthatching. Birds in Experiment 6 were fed experimental diets from Day 8 to Day 42 posthatching. At Day 22, they were moved to finishing batteries. Weight gain was calculated by subtracting final from initial body weight. Feed efficiency was calculated as gain:feed. Experimental diets contained corn and soybean meal as the only sources of intact protein. Prior to diet formulation, the CP contents of corn and soybean meal were analyzed (Association of Official Analytical Chemists, 1980). Amino acid contents were then calculated from the analyzed CP values and the ratios of AA to CP listed by the National Research Council (NRC, 1984). The NRC (1984) recommended 23% CP level was used as a positive control and a 19% CP level was chosen as a low-protein negative control diet (Table 1). These diets contained 23 and 19% intact protein, respectively. All crystalline AA used were L-isomers except Met, which was provided as the DLisomer. Also, all AA were provided in free base forms except Lys, which was provided as Lys-HCl. Addition of AA to diets was made at the expense of cornstarch. At the end of Experiments 1, 2, and 3, all the birds from selected dietary treatments were killed by cervical dislocation and freeze-dried to constant weight. A regression equation (Velu et al., 1972) was used to estimate whole body fat from whole body water. Experiment 1 This preliminary assay was conducted for three purposes: 1) to determine which AA are needed in the low-protein diet; 2) to
HAN ET AL.
1170 TABLE 1. Composition of 23% and 19% CP diets 1 Ingredients and composition Corn (9.1% CP) Dehulled soybean meal (48.4% CP) Cornstarch Corn oil Dicalrium phosphate Limestone Nad Trace-mineral mix 2 Vitamin mix 3 Choline-a DL-Met Sand Calculated composition CP ME n/ kcal/kg K Na CI Ca P (available) Arg His lie Leu
Lys Met + Cys
Met Phe Tyr Thr Trp Val
23% CP diet
19% CP diet
51.02
52.58
37.93 1.00 6.00 2.20 1.00 .40 .05 .10 .10 20
29.37 5.30 6.00 220 1.00 .40 .05 .10 .10 .20 2.70
23.0 3,200.0 .92 .18 28 .99 .55 1.65 .61 1.17 2.02 133 .94 .58 1.05 1.00 .92 30 1.30
19.0 3,200.0 .76 .18 .28 .97 .54 1.35 .50 .96 1.72 1.07 .82 .52 .88 .84 .77 25 1.08
1
The composition was the same in Experiments 1, 2, and 3 except that sand was added only in Experiment 1. For the remaining experiments, the composition varied slightly due to variation of CP level in corn and soybean meal. Provided the following amounts per kilogram of diet: manganese, 75 mg; iron, 75 mg; zinc, 75 mg; copper, 5 mg; iodine, .75 mg; selenium, .1 mg. Supplied the following amounts per kilogram of diet: vitamin A, 4,400 IU; cholecakiferol, 1,000 ICU; vitamin E, 11 IU; vitamin Bj2, -011 mg; riboflavin, 4.4 mg; d-pantothenic acid, 10 mg; niacin, 22 mg; menadione sodium bisulfite complex, 2.33 mg.
ascertain whether dispensable AA N (as Glu) is necessary in the low-protein diet; and 3) to determine whether the reduced K level associated with the reduction of soybean meal in the low-protein diet is a factor in the efficacy of the low-protein diet. Treatments 1 and 2 were the high protein (23%) positive control and the low-protein
(19%) negative control diet (Table 1). Based upon NRC (1984) recommended requirements, Met, Lys, Arg, and Thr were calculated to be deficient, but Val was adequate. Evidence from Edmonds et al. (1985), however, indicated all five of these AA were limiting in a 16% CP, CS diet. Therefore in Treatment 3, the five suspect limiting AA (Met, Lys, Arg, Thr, and Val) were supplemented to the low-protein diet so that the total concentration of each of these AA was equal to that present in the 23% CP diet. In Treatment 4, in addition to the five suspect limiting IAA, six additional IAA, i.e., His, He, Leu, Phe, Tyr, and Trp, were supplemented to the low-protein diet to make levels of these AA equal to those in Treatment 1. To study the effect of nonspecific amino N on performance of chicks fed the low-protein diet supplemented with IAA, Glu was added to Diets 5 and 6 in addition to the supplemented 5 or 11 IAA to make the diets isonitrogenous to the 23% CP diet. In Treatments 7 to 11, K 2 C0 3 was supplemented to the Diets (as Treatments 2 to 6 plus K2CO3) to make dietary K levels equal to the level present in the 23% CP diet. Finally, ground sand was used in this experiment to make all diets isocaloric.
Experiments 2 and 3 Results of Experiment 1 indicated that chicks fed the low-protein diet supplemented with the five suspect limiting IAA (Met, Lys, Arg, Thr, and Val) together with Glu performed at a level equal to those fed the high-protein diet. Therefore, Experiment 2 was conducted to determine whether all five of the suspect IAA were limiting in the low-protein diet and the order in which they were limiting. The five AA were added alone or in different combinations to the low-protein diet (containing 4.62% of supplemental Glu) to provide levels of the five IAA to equal the levels present in the 23% CP diet. The 23% CP diet was fed as a positive control. N o attempt was made to equalize MEn of the diets fed in this experiment. Experiment 3 was essentially the same as Experiment 2 except that an additional treatment was added. In Experiment 2, chicks fed the 19% CP diet supplemented with Met, Lys, and Arg did not respond to Thr but did respond
1171
AMINO ACID SUPPLEMENTATION
TABLE 2. Supplemental levels of indispensable amino acids added to the 19% CP diet in Experiment 4 Level 1
100% 75% 50%
DL-Met
L-Lys
L-Arg
L-Val
L-Thr
.33 .25 .17
27 21 .14
.31 23 .14
22
.16
.11
!()8
amino adds added at this level brought the levels of the five deficient indispensable amino acids to the same levels present in the 23% CP, positive control diet (Table 1).
to Thr and Val added together. Therefore, the purpose of Experiment 3 was to confirm the results of Experiment 2 and to study whether Val was more limiting than or equally limiting with Thr.
Experiment 4 This assay was conducted to determine the optimal levels of the limiting AA needed in the 19% CP diet. In all previous experiments, the AA were added to equal the levels to those present in the positive control diet (23% CP). It is well known that AA in crystalline form are highly bioavailable to poultry (Izquierdo et ah, 1988; Han et al, 1990; Chung and Baker, 1991). Therefore, it was believed that it should be possible to lower the supplementation levels (Table 2) and at the same time maintain similar chick growth performance. The level of Glu in the AA-fortified, low-protein diets was maintained at 4.62%.
Experiment 5 This assay was done to determine an adequate level of nonspecific AA N (as Glu) needed in the low-protein diet containing reduced levels of the five LAA established in Experiment 4. Glutamic acid levels were 0, 25,50,75, and 100% of the 4.62% level used in the previous experiments.
Experiment 6 This w a s a long-term s t u d y (1- to 6-wk posthatching). During Weeks 1 to 3, dietary treatments consisted of the 23% CP, CS diet supplemented with Met, a 19% CP, CS diet, and a 19% CP, CS diet fortified with the AA package developed in Experiments 4 and 5. During Weeks 4 to 6, the positive
control was a 20% CP (NRC, 1984), CS diet supplemented with .2% DL-Met and .06% L-Lys to meet the requirements (NRC, 1984). A 16% CP, CS diet was arbitrarily selected as the low-protein diet. The AA package developed for Weeks 1 to 3, with slight modifications (Lys and Thr increased slightly considering higher corn:soybean meal ratio in the 16% CP diet), was added to the 16% CP diet. At the end of this experiment, birds were euthanatized by cervical dislocation and the abdominal fat pad was removed and weighed.
Experiment 7 Male commercial Hubbard (Hubbard x Hubbard) broiler chicks were used in Experiment 7 to establish whether a fastgrowing commercial strain would respond in the same manner as the slower growing crossbred strain used in Experiments 1 to 6. Dietary treatments were the same as those in Experiment 6 during 1- to 3-wk posthatching.
Statistical Analysis Data from all chick experiments were subjected to analysis of variance procedures appropriate for completely randomized designs (Steel and Torrie, 1980). Differences among treatment means within experiment were assessed by the least significant difference pairwise multiple-comparison procedure of Carmer and Walker (1985), and in some cases, by the single df contrast procedure (Steel and Torrie, 1980). Data from Experiments 2 and 3 were also pooled together and analyzed as a randomized complete-block design (Steel and Torrie, 1980) with experiments as the blocks. In Experiment 6, growth performance data for the period of 3 to 6 wk of age were analyzed with analysis of covariance with body
1172
HAN ET AL. TABLE 3. Performance of chicks fed a 19% CP diet supplemented with amino acids (AA) (Experiment 1)
Treatment 1. 23% CP 2. 19% CP 3. As 2 + 4. As 3 + 5. As 3 + 6. As 4 + 7. As 2 + 8. As 3 + 9. As 4 + 10. As 5 + 11. As 6 + Pooled SEM
+ .2% DL-Met + .2% DL-Met AA mix l 2 AA mix 2 s 4.62% L-Glu 3.36% L-Glu 28% K 2 C 0 3 .28% K 2 C 0 3 .28% K 2 C 0 3 .28% K 2 C 0 3 .28% K 2 C 0 3
Weight gain
Gain:feed
Body fat
(g)
(%)
290 s 271 c 291 a 2ggab 298 a 298 a 274 bc 291 a 288 ab 292 a 298 a 5
8.9 s 11.5C 10.3 b 9.0 3 9.0* 8.7 s
.3
a-e
Means within columns with no common superscripts differ significantly (P < .05). ^Data are means of four groups of five male crossbred chicks from 8 to 22 days posthatching; average initial weight was 96 g. Supplied .12% DL-Met, .27% L-Lys, .31% L-Arg, .22% L-Val, and .16% L-Thr. Supplied .11% L-His, .21% L-Ile, .30% L-Leu, .17% L-Phe, .16% L-Tyr, and .05% L-Trp.
weights at the end of 1- to 3-wk period as covariate.
RESULTS Experiment 1 Weight gain and feed efficiency of chicks fed the 19% CP diet, even with added Met, was lower (P < .05) and body fat content was higher (P < .05) compared with those of chicks fed the 23% CP positive control diet (Table 3). Supplementation of the five suspect limiting AA (Met, Lys, Arg, Thr, and Val) to the low-protein diet improved (P < .05) weight gain and feed efficiency and decreased (P < .05) body fat. Weight gain and gain:feed values of chicks fed the IAAfortified, low-protein diet were similar to those of chicks fed the positive control, 23% CP diet; body fat, however, was still higher (P < .05) in chicks fed the IAA-fortified, lowprotein diet. Addition of all 11 IAA decreased body fat to a level not different from that present in birds fed the positive control diet. Adding the five IAA along w i t h Glu t o t h e 19% p r o t e i n d i e t (isonitrogenous to the 23% CP diet) resulted in chick performance and body fat that were similar to those observed in birds fed the positive-control diet. Adding K to the lowprotein diets elicited no response. Single df comparisons indicated that adding Glu to
the IAA-fortified low-protein diets improved (P < .05) feed efficiency (Diets 5, 6, 10, and 11 versus Diets 3, 4, 8, and 9) and reduced body fat concentration (Diets 5 and 6 versus Diets 3 and 4).
Experiments 2 and 3 Feeding the low-protein diet without AA fortification decreased weight gain and feed efficiency and increased body fat concentration compared with feeding the 23% protein diet supplemented with Met (Tables 4 and 5). Addition of Glu alone to the low-protein diet did not yield any response in growth performance. Chicks fed the low-protein diet responded to the addition of Met, Met and Lys, and Met, Lys, and Arg. There was no response to the addition of Thr on top of the first three Umiting AA. Feed efficiency, however, responded slightly to the addition of Val in Experiment 3 (Table 5). With Thr and Val together, there was a consistent, though small, improvement in feed efficiency; in Experiment 3 the response to Val plus Thr over the diet containing Glu, Met, Lys, and Arg was significant (P < .05). As observed previously, the low-protein diet supplemented with IAA plus Glu yielded weight gain and body fat concentrations equal to those obtained with the positive control diet (Tables 4 and 5). Analysis of pooled data from Experiments 2
1173
AMINO ACID SUPPLEMENTATION TABLE 4. Determination of limiting amino acids in the 19% CP corn and soybean meal diet (Experiment 2) 1 Treatment 2 1. 2. 3. 4. 5. 6. 7. 8. 9.
23% CP 19% CP As 2 + As 3 + As 3 + As 3 + As 3 + As 7 + As 7 +
+ .2% DL-Met 4.62% L-Glu DL-Met DL-Met + L-Lys DL-Met + L-Arg DL-Met + L-Lys + L-Arg L-Thr L-Thr + L-Val
Pooled SEM
Weight gain
Gain:feed
Body fat
(g)
(g*g) 671ab 565 d 550 d 630° 656 b 632 c 671ab 670 a b 687 s
(%)
287 a b 241 d 240 d 273 c 281 b c 282 a b c 287 a b 284 a b c 295 a
5
7
7.8 b 10.8 a
8.3D
8.4 b S2h
a
Means within columns with no common superscripts differ significantly (P < .05). *Data are means of four groups of five male crossbred chicks from 8 to 22 days posthatching; average initial weight was 86 g. 2 Levels of indispensable amino acids added were .33% DL-Met, .27% L-Lys, .31% L-Arg, .22% L-Val, and .16% L-Thr.
and 3 indicated a significant (P < .01) response in feed efficiency to the addition of Val and Thr. The data suggested that Val and Thr were limiting in the low-protein diet.
Experiment 4 As observed in all the previous experiments, chicks fed the low-protein diet supplemented with the five IAA at levels to
equal those in the 23% CP diet (Treatment 6) produced weight gain and gauvfeed results equal to those of chicks fed the positive control diet (Table 6). In the low-protein diets containing 4.62% Glu, the first three limiting AA could be reduced to 75% (Treatment 4), or all five IAA reduced to 50% (Treatment 5), of the initial supplementation levels to produce a growth performance equal to that of the positive control diet. However, feed efficiency was inferior
TABLE 5. Determination of limiting amino acids in the 19% CP corn and soybean meal diet (Experiment 3) 1 Treatment 2 1. 23% CP 2. 19% CP 3. As 2 + 4. As 3 + 5. As 3 + 6. As 3 + 7. As 3 + 8. As 7 + 9. As 7 + 10. As 7 + Pooled SEM a_e
+ 2% DL-Met 4.62% L-Glu DL-Met DL-Met + L-Lys DL-Met + L-Arg DL-Met + L-Lys + L-Arg L-Thr L-Val L-Thr + L-Val
Weight gain
Gain:feed
Body fat
(g)
276 a 226 d 213 d 252 c 264 a b c 251 c 272ak 261 b c
682 a 529 e 538 e 597 d 653*0 604 d 641 c 642 c 671ab 680 s
(%) 9i ba c
onnab
278 a
5
8
13.5
9.8**= 9.4 b c 8.8C 10.1 b
.4
Means within columns with no common superscripts differ significantly (P < .05). 1 Data are means of four groups of five male crossbred chicks from 8 to 22 days posthatching; average initial weight was 82 g. 2 Levels of indispensable amino acids added were .33% DL-Met, .27% L-Lys, .31% L-Arg, .22% L-Val, and .16% L-Thr.
1174
HAN ET AL. TABLE 6. Performance of chicks fed 19% CP diet supplemented -with varying levels of supplemental amino acids (Experiment 4) 1
Treatment + .2% DL-Met 4.62% Glu + [Met-Lys-Arg] (100%) 4.62% Glu + [Met-Lys-Arg] (75%) 4.62% Glu + [Met-Lys-Arg] (50%) [Val-Thr] (100%) [Val-Thr] (100%) [Val-Thr] (100%) [Val-Thr] (50%) [Val-Thr] (50%) [Val-Thr] (50%)
Gain:feed
(g) 270*
(g*g> 675 abc 580e
III
1. 23% CP 2. 19% CP 3. As 2 + 4. As 2 + 5. As 2 + 6. As 3 + 7. As 4 + 8. As 5 + 9. As 3 + 10. As 4 + 11. As 5 + Pooled SEM
Weight gain
252c 271* 263 bc 262bc 270* 279* 271*
667 abcd
644d 696a 669abcd 654cd 682 abc 688* 652"1 11
a
~*Means within columns with no common superscripts differ significantly (P < .05). *Data are means of four groups of five male crossbred chicks from 8 to 22 days posthatching; average initial weight was 77 g.
(P < .05) to that produced by the fully fortified diet (Treatment 6). With Lys, Met, and Arg at 75%, and Thr and Val at 50% of the initial supplementation levels (Treatment 10), growth performance was equal to that of the positive control. Results from this experiment indicated that the supplemental levels of Met, Lys, and Arg could be reduced to at least 75% and Val and Thr to at least 50% of the initial levels. Single df comparison (Treatments 3 and 4 versus Treatments 6, 7, 9, and 10) indicated a significant (P < .01) improvement in feed efficiency by Val and Thr.
Experiment 5 As expected, chicks fed the low-protein diet without AA fortification exhibited decreased weight gain and feed efficiency (Table 7). Fortification of the low-protein diet with the lowered levels of the five IAA brought growth performance back to that observed with the positive control. The lack of response to the addition of 1.16% Glu was mainly due to the poor performance of one group of birds in that treatment. The response in feed efficiency was significant (P < .05) for 2.31% Glu. Single df comparisons (Diets 5, 6, and 7 versus Diet 3) indicated that adding 2.31 % or more Glu to the low-protein diet containing optimal levels of the five IAA improved feed efficiency (P < .05).
Experiment 6 During 1 to 3 wk of age, chicks fed the low-protein diet gained less weight (P < .05) and utilized feed less efficiently (P < .05) than those fed the positive control diet (Table 8). Supplementing the low-protein diet with IAA plus 2.31% Glu, the amount established as being the optimal efficacious level (Tables 6 and 7), resulted in growth performance equal to that obtained with the 23% CP, positive control diet. During the period of 3 to 6 wk of age wherein the positive control was a Met-fortified 20% CP diet and the negative control was a 16% CP diet, addition of Glu plus IAA to the lowprotein diet brought growth and feed efficiency u p to the values not different from those recorded for the positive control diet. Overall, from 1 to 6 wk of age, chicks fed the low-protein, AA-fortified diet performed at levels equal to those obtained with 20% CP diet. Abdominal fat was reduced markedly (P < .05) in birds fed the AA-fortified, low-protein diet compared with that in birds fed the unsupplemented, low-protein diet, but abdominal fat in birds fed the positive-control diet was lower still (albeit slightly) (P < .05).
Experiment 7 Fast-growing Hubbard chicks fed the low-protein diet with AA supplementation
1175
AMINO ACID SUPPLEMENTATION
TABLE 7. Effects of glutamic acid level on performance of chicks fed the 19% CP diet fortified with amino acids (Experiment 5) 1 Treatment
Weight gain
Gain:f
(g) 1. 23% CP + 2% DL-Met 2. 19% CP 3. As 2 + IAA mix3 4. As 3 + 1.16% L-Glu 5. As 3 + 2.31% L-Glu 6. As 3 + 3.47% L-Glu 7. As 3 + 4.62% L-Glu Pooled SEM
268* 227b 264* 258* 269* 264* 269* 6
675*b 587= 661 b 663*b 692* 687*b 682*b 10
a_c
Means within columns with no common superscripts differ significantly (P < .05). ^Data are means of four groups of five male crossbred chicks from 8 to 22 days posthatching; average initial weight was 76 g. Diets 5, 6, and 7 (pooled observation) are significantly different (P < .05) from Diet 3. 3 Indispensable amino acid (IAA) mix provided .25% DL-Met, .21 % L-Lys, .23% L-Arg, .11% L-Val, and .08% LThr. These levels were those determined to be adequate in Experiment 4.
gained as rapidly as those fed the Metfortified, 23% CP, positive control diet during the period 8- to 22-days posthatching (Table 9). Feed efficiency also did not differ between the two groups. DISCUSSION The limiting AA in the 19% CP corn and soybean diet for 1- to 3-wk-old chicks were Met, Lys, Arg, Val, Thr, and amino
N (as Glu). Although Met was clearly firstlimiting and Lys second-limiting, the exact order of limitation for the remaining AA could not be accurately predicted from the experiments done herein. Nonetheless, these limitations resemble those found in a previous study by Edmonds et ah (1985) in which a 16% CP, CS diet for young chicks was found deficient in Met (first-limiting), Lys and Arg (equally second-limiting), and Val and Thr (equally tbird-limiting).
TABLE 8. Performance of chicks fed low-protein diets fortified with amino acids during 1 to 6 wk of age (Experiment 6) 1
Treatment
1 to 3 wk Weight Gain: gain feed
1. Positive control2 2. Low CP diet2 3. As 2 + amino acids3 Pooled SEM
(g) 267* 229b 270* 4
a_c
(g*g) 697* 580b 694* 6
3 to 6 wk Weight Gain: gain feed
(g:kg) 656* 528b 633* 8
Weight gain
1 to 6 wk Gain: Abdominal feed fat
(g) 970* 768b 971* 9
(g*g> 666* 543b 649* 7
(% BW) .5C 1.4* .7 b .05
Means within columns with no common superscripts differ significantly (P < .05). 'Data are means of five groups of five male crossbred chicks from 8 to 22, 22 to 42, and 8 to 42 days posthatching; average initial weight was 76 g. %ee Table 1 for dietary composition during 1 to 3 wk of age. During 4 to 6 wk of age, the positive control was a 20% CP diet (59.8% corn and 31.1% soybean meal) fortified with .2% DL-Met and .06% L-Lys, and the low-protein diet was 16% CP (66.0% corn and 21.6% soybean meal). 3 Quantities supplied during 1 to 3 wk of age were .25% DL-Met, .21% L-Lys, .23% L-Arg, .11% L-Val, .08% LThr, and 2.31% L-Glu. During 3 to 6 wk of age, quantities supplied were .25% DL-Met, .25% L-Lys, .23% L-Arg, .11% L-Val, .13% L-Thr, and 2.31% LrGla.
1176
HAN ET AL.
TABLE 9. Performance of commercial Hubbard chicks fed the 19% CF diet supplemented with amino acids (Experiment 7) Treatment
Weight gain
Gain: feed
(g) (g*g) 636* 776* b 546 680b 629* 771* 16 16 *^Means within columns with no common superscripts differ significantly (P < .05). 1 Data are means of five groups of five male Hubbard chicks from 8 to 22 days posthatching; average initial weight was 121 g. 2 Indispensable amino acid (IAA) mix provided .25% DL-Met, .21% L-Lys, .23% L-Arg, .11% L-Val, and .08% L-Thr. These levels were those determined to be adequate in Experiment 4.
1. 23% CP + DL-Met 2. 19% CP 3. As 2 + IAA mix2 Pooled SEM
That Met, Lys, Arg, and (possibly) Thr might be deficient in lower CP (lower soybean meal) diets for broiler chicks is not surprising. Indications that Val was deficient, however, are surprising because relative to the NRC (1984) Val requirement estimate, the level of this AA in the low-protein diet used herein is calculated to be in considerable excess. There appears to be no plausible explanation for the Val response observed here or in the authors' previous work (Edmonds et al, 1985), other than that the NRC table overestimated Val content of corn and soybean meal 0ensen and Colnago, 1991). Glutamic acid supplementation of the low-protein diet containing the five limiting IAA improved feed efficiency and reduced body fat (Table 3). The improved leanfat ratio of chick carcasses was as great with Glu as was the case when six additional IAA were added to the diet already containing the five limiting IAA. This supports the view that after Met, Lys, Arg, Val, and Thr were supplemented, no other IAA were deficient in the lowprotein diet. In fact, the data indicate that amino N (for dispensable AA biosynthesis) was also a limiting factor in the lowprotein diet. The low-protein diet required smaller quantities of the five limiting AA than that present in the 23% CP positive control
diet. This is not surprising because 1) crystalline AA are more bioavailable than intact, protein-bound AA (Izquierdo et al, 1988; Han et al, 1990; Chung and Baker, 1991); 2) lowering CP of a diet results in a reduction in AA requirements (Griminger et al, 1956; Rosenberg and Baldini, 1957; Nelson et al, 1960; Boomgaardt and Baker, 1971; Abebe and Morris, 1990); and 3) the 23% CP diet contained levels of some of the AA in excess of the requirements. Downward revision in AA levels in the IAA package cannot be interpreted as supplemental minimum levels. It is very likely that some of the IAA levels could be reduced further. Although the low-protein diet used here contained 19% CP from corn and soybean meal (16% in the case of the study done with 3- to 6-wk-old birds), after adding the determined optimal levels of the five IAA plus 2.31% Glu, the lowprotein diets actually contained 21.4% CP (18.5% CP for the older birds), using the factor N x 6.25 to account for the added AA N. This factor may be important in attempts to interpret the literature on the subject of low-protein, AA-fortified diets for broiler chicks. Thus, several workers have reported that performance of chicks is as good with a low-protein, AA-fortified diet as with a diet containing a conventional protein level (Bornstein and Lipstein, 1975; Waldroup et al, 1976; Uzu, 1982, 1986; Summers and Leeson, 1985; Schutte, 1987; Stillborn and Waldroup, 1989; Parr and Summers, 1991). However, recent work from the University of Georgia has indicated that chick performance is inferior on low-protein, CS diets, regardless of AA supplementation (Fancher and Jensen, 1989a,b,c; Pinchasov et al, 1990; Colnago et al, 1991). In the Georgia studies, the low-protein diets were formulated such that the AA N was accounted for, and this effectively lowered the soybean meal (at the expense of corn) more than was the case in the current studies. Unpublished data from another research project in the authors' laboratory wherein a true 19% CP AA-fortified diet was fed resulted in weight gains that were the same as those obtained with a Metfortified 23% CP diet. Feed efficiency, however, was reduced in birds fed the
AMINO ACID SUPPLEMENTATION
19% CP diet. The low-CP diet in this case contained no added Glu, and it was slightly lower in soybean meal. Chicks fed 19% CP diets with supplemental AA (21.4% total CP) during Weeks 1 to 3 posthatching, and those fed AAfortified 16% CP diets (18.5% total CP) during Weeks 3 to 6 posthatching gained as fast, converted feed to gain as efficiently, and had similar body fat levels as those fed conventional protein levels of 23% or 20%, respectively, for the early and later growing periods. Feed efficiency of birds during the early growing period, in fact, tended to be superior for the low-protein, AA-fortified diet compared with the positive control, Met-fortified, 23% CP diet. When data for these two diets were pooled across experiments (Treatments 1 and 5 of Experiment 1; Treatments 1 and 9 of Experiment 2; Treatments 1 and 10 of Experiment 3; and Treatments 1 and 6 of Experiment 4), gain:feed was, in fact, greater (P < .05) for the low-protein diet containing the five IAA plus Glu than it was for the positive control diet. Because the laboratory broiler strain (New Hampshire x Columbian) grows only about one-half as fast as commercial broiler strains, Hubbard chicks were tested in the last experiment. These chicks also performed as well on the low-protein, AA-fortified diet as they did on the 23% CP, positive control diet. This might have been expected because AA requirements expressed in terms of dietary concentration are virtually the same for the fastgrowing Hubbard chicks as they are for the slower growing laboratory strain (Robbins and Baker, 1980; Han and Baker, 1991). REFERENCES Abebe, S., and T. R. Morris, 1990. Effects of protein concentration on responses to dietary tryptophan by chicks. Br. Poult. Sci. 31:267-272. Association of Official Analytical Chemists, 1980. Official Methods of Analysis. 13th ed., Association of Official Analytical Chemists, Washington, DC. Boomgaardt, J., and D. H. Baker, 1971. Tryptophan requirement of growing chicks as affected by dietary protein level. J. Anim. Sci. 33:595-599. Bornstein, S., and B. Lipstein, 1975. The replacement of some of the soybean meal by the first limiting amino acids in practical broiler diets. I. The
1177
value of special supplementation of chick diets with methionine and lysine. Br. Poult. Sci. 16: 177-188. Carmer, S. G., and W. M. Walker, 1985. Pairwise multiple comparisons of treatment means in agronomic research. J. Agron. Ed. 14:19-26. Chung, T. K., and D. H. Baker, 1991. Apparent and true digestibility of amino acids in casein and in a complete amino acid mixture: comparison of pig ileal digestibility with the cecectomized cockerel assay. J. Anim. Sci. 69(Suppl. 1): 38.(Abstr.) Colnago, L., A. M. Penz, Jr., and L. S. Jensen, 1991. Effect of response of starting broiler chicks to incremental reduction in intact protein on performance during the grower phase. Poultry Sci. 70(Suppl. l):153.(Abstr.) Edmonds, M. S., C. M. Parsons, and D. H. Baker, 1985. Limiting amino acids in low-protein cornsoybean meal diets fed to growing chicks. Poultry Sci. 64:1519-1526. Fancher, B. I., and L. S. Jensen, 1989a. Male broiler performance during the starting and growing periods as affected by dietary protein, essential amino acids, and potassium levels. Poultry Sci. 68:1385-1395. Fancher, B. I., and L. S. Jensen, 1989b. Influence on performance of three to six-week-old broilers of varying dietary protein contents with supplementation of essential amino acid requirements. Poultry Sci. 68:113-123. Fancher, B. I., and L. S. Jensen, 1989c. Dietary protein level and essential amino acid content: influence upon female broiler performance during the grower period. Poultry Sci. 68:897-908. Griminger, P., H. M. Scott, and R. M. Forbes, 1956. The effect of protein level on the tryptophan requirement of the growing chick. J. Nutr. 59: 67-76. Han, Y., and D. H. Baker, 1991. Lysine requirements of fast- and slow-growing broiler chicks. Poultry Sci. 70:2108-2114. Han, Y., F. Castanon, C. M. Parsons, and D. H. Baker, 1990. Intestinal absorption and bioavailability of DL-methionine hydroxy analog compared to DL-methionine. Poultry Sci. 69:281-287. Izquierdo, O. A., C. M. Parsons, and D. H. Baker, 1988. Bioavailability of lysine in L-lysineHCl. J. Anim. Sci. 663590-2597. Jensen, L. S., 1991. Are peptides needed for optimum nutrition? Feed Management 42, No. 8:37-40. Jensen, L. S., and G. L. Colnago, 1991. Amino acid and protein for broilers and laying hens. Pages 29-36 in: Proceedings Maryland Nutrition Conference for Feed Manufacturers. Baltimore, MD. National Research Council, 1984. Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. Nelson, T. S., R. J. Young, R. B. Bradfield, J. B. Anderson, L. C. Norris, F. W. Hill, and M. L. Scott, 1960. Studies on the sulfur amino acid requirement of the chick. Poultry Sci. 39: 308-314. Parr, J. F., and J. D. Summers, 1991. The effects of minimizing amino acid excesses in broiler diets. Poultry Sci. 70:1540-1549.
1178
HAN ET AL.
Pinchasov, Y., C. X. Mendonca, and L. S. Jensen, 1990. Broiler chick response to diets supplemented with synthetic amino acids. Poultry Sci. 69:1950-1955. Robbins, K. R., and D. H. Baker, 1980. Effect of sulfur amino acid level and source on the performance of chicks fed high levels of copper. Poultry Sci. 59:1246-1253. Rosenberg, H. R., and J. R. Baldini, 1957. Effect of dietary protein level on the methionine-energy relationship in broiler diets. Poultry Sci. 36: 247-252. Sasse, C. E., and D. H. Baker, 1974. Factors affecting sulfate-sulfur utilization by the young chicks. Poultry Sci. 53:652-662. Schutte, J. B., 1987. Utilization of synthetic amino acids in poultry. Pages RT11-12 in: World's Poultry Science Association 6th European Symposium on Poultry Nutrition. Konigslutter, Germany. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics. A Biometrical Ap-
proach. 2nd ed. McGraw-Hill Book Co., New York, NY. Stillborn, H. L., and P. W. Waldroup, 1989. Utilization of low-protein grower diets for broiler chickens. Poultry Sci. 68(Suppl. l):142.(Abstr.) Summers, J. D., and S. Leeson, 1985. Broiler carcass composition as affected by amino acid supplementation. Can. J. Arum. Sci. 65:717-723. Uzu, G., 1982. Limit of reduction of the protein level in broiler feeds. Poultry Sci. 61(Suppl. 1): 1557-1558.(Abstr.) Uzu, G., 1986. Threonine requirement in broilers. Alimentation Equilibre Commentry, Document No. 242. Commentary, France. Velu, J. G., D. H. Baker, and H. M. Scott, 1972. Regression equations for determining body composition of young chicks. Poultry Sci. 51: 698-699. Waldroup, P. W., R. J. Mitchell, J. R. Payne, and K. R. Hazen, 1976. Performance of chicks fed diets formulated to minimize excess levels of essential amino acids. Poultry Sci. 55:243-253.
INTRODUCTION Dietary amino acids (AA) in excess of the needs of chicks impair feed intake and growth rate (Waldroup et al., 1976). In practice it is very difficult, if not impossible, to formulate diets with natural feed ingredients that will provide all the AA needed by chicks in adequate quantities and also to maintain an optimal AA balance with minimal excesses. Recent advances in commercial production of crystalline AA make several indispensable AA (IAA) more economically feasible for
Received for publication September 13, 1991. Accepted for publication March 6, 1992. Visiting scientist, Ajinomoto Co., Inc., Technology Commercialization Laboratory, 2004 South Wright Street, Urbana, IL 61801. To whom correspondence should be addressed.
use in animal feeds. Addition of IAA to diets permits a reduction in dietary protein content and at the same time provides the needs of all IAA by chicks with an optimal AA profile. It is known that broiler chicks fed diets marginal in protein but fortified with Met or Met and Lys will perform as well as those fed a diet higher in protein (Jensen, 1991). There are great discrepancies, however, in attempts that have been made to feed broiler chicks lowprotein diets that are supplemented with several crystalline IAA. Bornstein and Lipstein (1975) compared a 19.7% CP diet (supplemented with Met and Lys) to a 23.1% CP diet supplemented with Met and found that chick growth and feed efficiency were equal for these two diets. Waldroup et al. (1976) reported that maximal growth of young chicks could be obtained with a 19% CP diet supple-
1168
AMINO ACID SUPPLEMENTATION
mented with crystalline AA. Protein utilization efficiency was improved as a consequence of mirtimizing excess levels of IAA in the diets. When feeding young chicks a 16% CP diet supplemented with all IAA equivalent to the levels present in a 20% CP diet, Schutte (1987) observed equal weight gain and feed efficiency during the period 7- to 21-days posthatching. More recently, Parr and Summers (1991) reported that chicks fed low-protein diets (ranging from 21 to 16.5% CP) supplemented with IAA had similar growth rates, feed efficiencies, and total carcass protein contents as those fed a 23% CP diet. Conversely, University of Georgia researchers (Fancher and Jensen, 1989a,b,c; Pinchasov et al, 1990; Colnago et al, 1991) concluded that optimal performance of starter and grower chicks could not be achieved with low-protein diets supplemented with crystalline AA. Similar findings were observed by Edmonds et al. (1985) when the protein level of corn and soybean meal (CS) diets was reduced from 24 to 16%. The reasons for these different findings among investigators are not readily apparent. The objective of the studies reported here was to approach this problem systematically in the hope of answering the following questions: 1) Can a lowprotein diet be properly fortified with crystalline AA so that performance will equal that of the birds fed a higher protein diet? 2) Which AA are limiting in the lowprotein diet and what quantities of the AA are needed? 3) Is dispensable AA N necessary for optimal chick performance and body composition? and 4) Is reduced K level in the low-protein (low soybean meal) diet a factor in utilization of the supplemental AA? MATERIALS AND METHODS One-week-old male chicks resulting from the cross of New Hampshire males and Columbian Plymouth Rock females were used in all chick assays, except for one assay wherein male Hubbard chicks (Hubbard x Hubbard) were used. The chicks were fed a 24% CP, CS pretest diet during the 1st wk posthatching. Following an overnight fast, the chicks were weighed
1169
and wing-banded and then allotted to dietary treatments as described by Sasse and Baker (1974). Four (in Experiments 1 to 5) or five (in Experiments 6 and 7) groups of five chicks were assigned to each dietary treatment. The chicks were housed in thermostatically controlled starter batteries with raised wire floors in an environmentally controlled room. Feed and water were supplied for ad libitum intake. Light was provided 24 h daily. Seven experiments were conducted, and all but Experiment 6 involved the 14-day feeding period 8- to 22-days posthatching. Birds in Experiment 6 were fed experimental diets from Day 8 to Day 42 posthatching. At Day 22, they were moved to finishing batteries. Weight gain was calculated by subtracting final from initial body weight. Feed efficiency was calculated as gain:feed. Experimental diets contained corn and soybean meal as the only sources of intact protein. Prior to diet formulation, the CP contents of corn and soybean meal were analyzed (Association of Official Analytical Chemists, 1980). Amino acid contents were then calculated from the analyzed CP values and the ratios of AA to CP listed by the National Research Council (NRC, 1984). The NRC (1984) recommended 23% CP level was used as a positive control and a 19% CP level was chosen as a low-protein negative control diet (Table 1). These diets contained 23 and 19% intact protein, respectively. All crystalline AA used were L-isomers except Met, which was provided as the DLisomer. Also, all AA were provided in free base forms except Lys, which was provided as Lys-HCl. Addition of AA to diets was made at the expense of cornstarch. At the end of Experiments 1, 2, and 3, all the birds from selected dietary treatments were killed by cervical dislocation and freeze-dried to constant weight. A regression equation (Velu et al., 1972) was used to estimate whole body fat from whole body water. Experiment 1 This preliminary assay was conducted for three purposes: 1) to determine which AA are needed in the low-protein diet; 2) to
HAN ET AL.
1170 TABLE 1. Composition of 23% and 19% CP diets 1 Ingredients and composition Corn (9.1% CP) Dehulled soybean meal (48.4% CP) Cornstarch Corn oil Dicalrium phosphate Limestone Nad Trace-mineral mix 2 Vitamin mix 3 Choline-a DL-Met Sand Calculated composition CP ME n/ kcal/kg K Na CI Ca P (available) Arg His lie Leu
Lys Met + Cys
Met Phe Tyr Thr Trp Val
23% CP diet
19% CP diet
51.02
52.58
37.93 1.00 6.00 2.20 1.00 .40 .05 .10 .10 20
29.37 5.30 6.00 220 1.00 .40 .05 .10 .10 .20 2.70
23.0 3,200.0 .92 .18 28 .99 .55 1.65 .61 1.17 2.02 133 .94 .58 1.05 1.00 .92 30 1.30
19.0 3,200.0 .76 .18 .28 .97 .54 1.35 .50 .96 1.72 1.07 .82 .52 .88 .84 .77 25 1.08
1
The composition was the same in Experiments 1, 2, and 3 except that sand was added only in Experiment 1. For the remaining experiments, the composition varied slightly due to variation of CP level in corn and soybean meal. Provided the following amounts per kilogram of diet: manganese, 75 mg; iron, 75 mg; zinc, 75 mg; copper, 5 mg; iodine, .75 mg; selenium, .1 mg. Supplied the following amounts per kilogram of diet: vitamin A, 4,400 IU; cholecakiferol, 1,000 ICU; vitamin E, 11 IU; vitamin Bj2, -011 mg; riboflavin, 4.4 mg; d-pantothenic acid, 10 mg; niacin, 22 mg; menadione sodium bisulfite complex, 2.33 mg.
ascertain whether dispensable AA N (as Glu) is necessary in the low-protein diet; and 3) to determine whether the reduced K level associated with the reduction of soybean meal in the low-protein diet is a factor in the efficacy of the low-protein diet. Treatments 1 and 2 were the high protein (23%) positive control and the low-protein
(19%) negative control diet (Table 1). Based upon NRC (1984) recommended requirements, Met, Lys, Arg, and Thr were calculated to be deficient, but Val was adequate. Evidence from Edmonds et al. (1985), however, indicated all five of these AA were limiting in a 16% CP, CS diet. Therefore in Treatment 3, the five suspect limiting AA (Met, Lys, Arg, Thr, and Val) were supplemented to the low-protein diet so that the total concentration of each of these AA was equal to that present in the 23% CP diet. In Treatment 4, in addition to the five suspect limiting IAA, six additional IAA, i.e., His, He, Leu, Phe, Tyr, and Trp, were supplemented to the low-protein diet to make levels of these AA equal to those in Treatment 1. To study the effect of nonspecific amino N on performance of chicks fed the low-protein diet supplemented with IAA, Glu was added to Diets 5 and 6 in addition to the supplemented 5 or 11 IAA to make the diets isonitrogenous to the 23% CP diet. In Treatments 7 to 11, K 2 C0 3 was supplemented to the Diets (as Treatments 2 to 6 plus K2CO3) to make dietary K levels equal to the level present in the 23% CP diet. Finally, ground sand was used in this experiment to make all diets isocaloric.
Experiments 2 and 3 Results of Experiment 1 indicated that chicks fed the low-protein diet supplemented with the five suspect limiting IAA (Met, Lys, Arg, Thr, and Val) together with Glu performed at a level equal to those fed the high-protein diet. Therefore, Experiment 2 was conducted to determine whether all five of the suspect IAA were limiting in the low-protein diet and the order in which they were limiting. The five AA were added alone or in different combinations to the low-protein diet (containing 4.62% of supplemental Glu) to provide levels of the five IAA to equal the levels present in the 23% CP diet. The 23% CP diet was fed as a positive control. N o attempt was made to equalize MEn of the diets fed in this experiment. Experiment 3 was essentially the same as Experiment 2 except that an additional treatment was added. In Experiment 2, chicks fed the 19% CP diet supplemented with Met, Lys, and Arg did not respond to Thr but did respond
1171
AMINO ACID SUPPLEMENTATION
TABLE 2. Supplemental levels of indispensable amino acids added to the 19% CP diet in Experiment 4 Level 1
100% 75% 50%
DL-Met
L-Lys
L-Arg
L-Val
L-Thr
.33 .25 .17
27 21 .14
.31 23 .14
22
.16
.11
!()8
amino adds added at this level brought the levels of the five deficient indispensable amino acids to the same levels present in the 23% CP, positive control diet (Table 1).
to Thr and Val added together. Therefore, the purpose of Experiment 3 was to confirm the results of Experiment 2 and to study whether Val was more limiting than or equally limiting with Thr.
Experiment 4 This assay was conducted to determine the optimal levels of the limiting AA needed in the 19% CP diet. In all previous experiments, the AA were added to equal the levels to those present in the positive control diet (23% CP). It is well known that AA in crystalline form are highly bioavailable to poultry (Izquierdo et ah, 1988; Han et al, 1990; Chung and Baker, 1991). Therefore, it was believed that it should be possible to lower the supplementation levels (Table 2) and at the same time maintain similar chick growth performance. The level of Glu in the AA-fortified, low-protein diets was maintained at 4.62%.
Experiment 5 This assay was done to determine an adequate level of nonspecific AA N (as Glu) needed in the low-protein diet containing reduced levels of the five LAA established in Experiment 4. Glutamic acid levels were 0, 25,50,75, and 100% of the 4.62% level used in the previous experiments.
Experiment 6 This w a s a long-term s t u d y (1- to 6-wk posthatching). During Weeks 1 to 3, dietary treatments consisted of the 23% CP, CS diet supplemented with Met, a 19% CP, CS diet, and a 19% CP, CS diet fortified with the AA package developed in Experiments 4 and 5. During Weeks 4 to 6, the positive
control was a 20% CP (NRC, 1984), CS diet supplemented with .2% DL-Met and .06% L-Lys to meet the requirements (NRC, 1984). A 16% CP, CS diet was arbitrarily selected as the low-protein diet. The AA package developed for Weeks 1 to 3, with slight modifications (Lys and Thr increased slightly considering higher corn:soybean meal ratio in the 16% CP diet), was added to the 16% CP diet. At the end of this experiment, birds were euthanatized by cervical dislocation and the abdominal fat pad was removed and weighed.
Experiment 7 Male commercial Hubbard (Hubbard x Hubbard) broiler chicks were used in Experiment 7 to establish whether a fastgrowing commercial strain would respond in the same manner as the slower growing crossbred strain used in Experiments 1 to 6. Dietary treatments were the same as those in Experiment 6 during 1- to 3-wk posthatching.
Statistical Analysis Data from all chick experiments were subjected to analysis of variance procedures appropriate for completely randomized designs (Steel and Torrie, 1980). Differences among treatment means within experiment were assessed by the least significant difference pairwise multiple-comparison procedure of Carmer and Walker (1985), and in some cases, by the single df contrast procedure (Steel and Torrie, 1980). Data from Experiments 2 and 3 were also pooled together and analyzed as a randomized complete-block design (Steel and Torrie, 1980) with experiments as the blocks. In Experiment 6, growth performance data for the period of 3 to 6 wk of age were analyzed with analysis of covariance with body
1172
HAN ET AL. TABLE 3. Performance of chicks fed a 19% CP diet supplemented with amino acids (AA) (Experiment 1)
Treatment 1. 23% CP 2. 19% CP 3. As 2 + 4. As 3 + 5. As 3 + 6. As 4 + 7. As 2 + 8. As 3 + 9. As 4 + 10. As 5 + 11. As 6 + Pooled SEM
+ .2% DL-Met + .2% DL-Met AA mix l 2 AA mix 2 s 4.62% L-Glu 3.36% L-Glu 28% K 2 C 0 3 .28% K 2 C 0 3 .28% K 2 C 0 3 .28% K 2 C 0 3 .28% K 2 C 0 3
Weight gain
Gain:feed
Body fat
(g)
(%)
290 s 271 c 291 a 2ggab 298 a 298 a 274 bc 291 a 288 ab 292 a 298 a 5
8.9 s 11.5C 10.3 b 9.0 3 9.0* 8.7 s
.3
a-e
Means within columns with no common superscripts differ significantly (P < .05). ^Data are means of four groups of five male crossbred chicks from 8 to 22 days posthatching; average initial weight was 96 g. Supplied .12% DL-Met, .27% L-Lys, .31% L-Arg, .22% L-Val, and .16% L-Thr. Supplied .11% L-His, .21% L-Ile, .30% L-Leu, .17% L-Phe, .16% L-Tyr, and .05% L-Trp.
weights at the end of 1- to 3-wk period as covariate.
RESULTS Experiment 1 Weight gain and feed efficiency of chicks fed the 19% CP diet, even with added Met, was lower (P < .05) and body fat content was higher (P < .05) compared with those of chicks fed the 23% CP positive control diet (Table 3). Supplementation of the five suspect limiting AA (Met, Lys, Arg, Thr, and Val) to the low-protein diet improved (P < .05) weight gain and feed efficiency and decreased (P < .05) body fat. Weight gain and gain:feed values of chicks fed the IAAfortified, low-protein diet were similar to those of chicks fed the positive control, 23% CP diet; body fat, however, was still higher (P < .05) in chicks fed the IAA-fortified, lowprotein diet. Addition of all 11 IAA decreased body fat to a level not different from that present in birds fed the positive control diet. Adding the five IAA along w i t h Glu t o t h e 19% p r o t e i n d i e t (isonitrogenous to the 23% CP diet) resulted in chick performance and body fat that were similar to those observed in birds fed the positive-control diet. Adding K to the lowprotein diets elicited no response. Single df comparisons indicated that adding Glu to
the IAA-fortified low-protein diets improved (P < .05) feed efficiency (Diets 5, 6, 10, and 11 versus Diets 3, 4, 8, and 9) and reduced body fat concentration (Diets 5 and 6 versus Diets 3 and 4).
Experiments 2 and 3 Feeding the low-protein diet without AA fortification decreased weight gain and feed efficiency and increased body fat concentration compared with feeding the 23% protein diet supplemented with Met (Tables 4 and 5). Addition of Glu alone to the low-protein diet did not yield any response in growth performance. Chicks fed the low-protein diet responded to the addition of Met, Met and Lys, and Met, Lys, and Arg. There was no response to the addition of Thr on top of the first three Umiting AA. Feed efficiency, however, responded slightly to the addition of Val in Experiment 3 (Table 5). With Thr and Val together, there was a consistent, though small, improvement in feed efficiency; in Experiment 3 the response to Val plus Thr over the diet containing Glu, Met, Lys, and Arg was significant (P < .05). As observed previously, the low-protein diet supplemented with IAA plus Glu yielded weight gain and body fat concentrations equal to those obtained with the positive control diet (Tables 4 and 5). Analysis of pooled data from Experiments 2
1173
AMINO ACID SUPPLEMENTATION TABLE 4. Determination of limiting amino acids in the 19% CP corn and soybean meal diet (Experiment 2) 1 Treatment 2 1. 2. 3. 4. 5. 6. 7. 8. 9.
23% CP 19% CP As 2 + As 3 + As 3 + As 3 + As 3 + As 7 + As 7 +
+ .2% DL-Met 4.62% L-Glu DL-Met DL-Met + L-Lys DL-Met + L-Arg DL-Met + L-Lys + L-Arg L-Thr L-Thr + L-Val
Pooled SEM
Weight gain
Gain:feed
Body fat
(g)
(g*g) 671ab 565 d 550 d 630° 656 b 632 c 671ab 670 a b 687 s
(%)
287 a b 241 d 240 d 273 c 281 b c 282 a b c 287 a b 284 a b c 295 a
5
7
7.8 b 10.8 a
8.3D
8.4 b S2h
a
Means within columns with no common superscripts differ significantly (P < .05). *Data are means of four groups of five male crossbred chicks from 8 to 22 days posthatching; average initial weight was 86 g. 2 Levels of indispensable amino acids added were .33% DL-Met, .27% L-Lys, .31% L-Arg, .22% L-Val, and .16% L-Thr.
and 3 indicated a significant (P < .01) response in feed efficiency to the addition of Val and Thr. The data suggested that Val and Thr were limiting in the low-protein diet.
Experiment 4 As observed in all the previous experiments, chicks fed the low-protein diet supplemented with the five IAA at levels to
equal those in the 23% CP diet (Treatment 6) produced weight gain and gauvfeed results equal to those of chicks fed the positive control diet (Table 6). In the low-protein diets containing 4.62% Glu, the first three limiting AA could be reduced to 75% (Treatment 4), or all five IAA reduced to 50% (Treatment 5), of the initial supplementation levels to produce a growth performance equal to that of the positive control diet. However, feed efficiency was inferior
TABLE 5. Determination of limiting amino acids in the 19% CP corn and soybean meal diet (Experiment 3) 1 Treatment 2 1. 23% CP 2. 19% CP 3. As 2 + 4. As 3 + 5. As 3 + 6. As 3 + 7. As 3 + 8. As 7 + 9. As 7 + 10. As 7 + Pooled SEM a_e
+ 2% DL-Met 4.62% L-Glu DL-Met DL-Met + L-Lys DL-Met + L-Arg DL-Met + L-Lys + L-Arg L-Thr L-Val L-Thr + L-Val
Weight gain
Gain:feed
Body fat
(g)
276 a 226 d 213 d 252 c 264 a b c 251 c 272ak 261 b c
682 a 529 e 538 e 597 d 653*0 604 d 641 c 642 c 671ab 680 s
(%) 9i ba c
onnab
278 a
5
8
13.5
9.8**= 9.4 b c 8.8C 10.1 b
.4
Means within columns with no common superscripts differ significantly (P < .05). 1 Data are means of four groups of five male crossbred chicks from 8 to 22 days posthatching; average initial weight was 82 g. 2 Levels of indispensable amino acids added were .33% DL-Met, .27% L-Lys, .31% L-Arg, .22% L-Val, and .16% L-Thr.
1174
HAN ET AL. TABLE 6. Performance of chicks fed 19% CP diet supplemented -with varying levels of supplemental amino acids (Experiment 4) 1
Treatment + .2% DL-Met 4.62% Glu + [Met-Lys-Arg] (100%) 4.62% Glu + [Met-Lys-Arg] (75%) 4.62% Glu + [Met-Lys-Arg] (50%) [Val-Thr] (100%) [Val-Thr] (100%) [Val-Thr] (100%) [Val-Thr] (50%) [Val-Thr] (50%) [Val-Thr] (50%)
Gain:feed
(g) 270*
(g*g> 675 abc 580e
III
1. 23% CP 2. 19% CP 3. As 2 + 4. As 2 + 5. As 2 + 6. As 3 + 7. As 4 + 8. As 5 + 9. As 3 + 10. As 4 + 11. As 5 + Pooled SEM
Weight gain
252c 271* 263 bc 262bc 270* 279* 271*
667 abcd
644d 696a 669abcd 654cd 682 abc 688* 652"1 11
a
~*Means within columns with no common superscripts differ significantly (P < .05). *Data are means of four groups of five male crossbred chicks from 8 to 22 days posthatching; average initial weight was 77 g.
(P < .05) to that produced by the fully fortified diet (Treatment 6). With Lys, Met, and Arg at 75%, and Thr and Val at 50% of the initial supplementation levels (Treatment 10), growth performance was equal to that of the positive control. Results from this experiment indicated that the supplemental levels of Met, Lys, and Arg could be reduced to at least 75% and Val and Thr to at least 50% of the initial levels. Single df comparison (Treatments 3 and 4 versus Treatments 6, 7, 9, and 10) indicated a significant (P < .01) improvement in feed efficiency by Val and Thr.
Experiment 5 As expected, chicks fed the low-protein diet without AA fortification exhibited decreased weight gain and feed efficiency (Table 7). Fortification of the low-protein diet with the lowered levels of the five IAA brought growth performance back to that observed with the positive control. The lack of response to the addition of 1.16% Glu was mainly due to the poor performance of one group of birds in that treatment. The response in feed efficiency was significant (P < .05) for 2.31% Glu. Single df comparisons (Diets 5, 6, and 7 versus Diet 3) indicated that adding 2.31 % or more Glu to the low-protein diet containing optimal levels of the five IAA improved feed efficiency (P < .05).
Experiment 6 During 1 to 3 wk of age, chicks fed the low-protein diet gained less weight (P < .05) and utilized feed less efficiently (P < .05) than those fed the positive control diet (Table 8). Supplementing the low-protein diet with IAA plus 2.31% Glu, the amount established as being the optimal efficacious level (Tables 6 and 7), resulted in growth performance equal to that obtained with the 23% CP, positive control diet. During the period of 3 to 6 wk of age wherein the positive control was a Met-fortified 20% CP diet and the negative control was a 16% CP diet, addition of Glu plus IAA to the lowprotein diet brought growth and feed efficiency u p to the values not different from those recorded for the positive control diet. Overall, from 1 to 6 wk of age, chicks fed the low-protein, AA-fortified diet performed at levels equal to those obtained with 20% CP diet. Abdominal fat was reduced markedly (P < .05) in birds fed the AA-fortified, low-protein diet compared with that in birds fed the unsupplemented, low-protein diet, but abdominal fat in birds fed the positive-control diet was lower still (albeit slightly) (P < .05).
Experiment 7 Fast-growing Hubbard chicks fed the low-protein diet with AA supplementation
1175
AMINO ACID SUPPLEMENTATION
TABLE 7. Effects of glutamic acid level on performance of chicks fed the 19% CP diet fortified with amino acids (Experiment 5) 1 Treatment
Weight gain
Gain:f
(g) 1. 23% CP + 2% DL-Met 2. 19% CP 3. As 2 + IAA mix3 4. As 3 + 1.16% L-Glu 5. As 3 + 2.31% L-Glu 6. As 3 + 3.47% L-Glu 7. As 3 + 4.62% L-Glu Pooled SEM
268* 227b 264* 258* 269* 264* 269* 6
a_c
Means within columns with no common superscripts differ significantly (P < .05). ^Data are means of four groups of five male crossbred chicks from 8 to 22 days posthatching; average initial weight was 76 g. Diets 5, 6, and 7 (pooled observation) are significantly different (P < .05) from Diet 3. 3 Indispensable amino acid (IAA) mix provided .25% DL-Met, .21 % L-Lys, .23% L-Arg, .11% L-Val, and .08% LThr. These levels were those determined to be adequate in Experiment 4.
gained as rapidly as those fed the Metfortified, 23% CP, positive control diet during the period 8- to 22-days posthatching (Table 9). Feed efficiency also did not differ between the two groups. DISCUSSION The limiting AA in the 19% CP corn and soybean diet for 1- to 3-wk-old chicks were Met, Lys, Arg, Val, Thr, and amino
N (as Glu). Although Met was clearly firstlimiting and Lys second-limiting, the exact order of limitation for the remaining AA could not be accurately predicted from the experiments done herein. Nonetheless, these limitations resemble those found in a previous study by Edmonds et ah (1985) in which a 16% CP, CS diet for young chicks was found deficient in Met (first-limiting), Lys and Arg (equally second-limiting), and Val and Thr (equally tbird-limiting).
TABLE 8. Performance of chicks fed low-protein diets fortified with amino acids during 1 to 6 wk of age (Experiment 6) 1
Treatment
1 to 3 wk Weight Gain: gain feed
1. Positive control2 2. Low CP diet2 3. As 2 + amino acids3 Pooled SEM
(g) 267* 229b 270* 4
a_c
(g*g) 697* 580b 694* 6
3 to 6 wk Weight Gain: gain feed
(g:kg) 656* 528b 633* 8
Weight gain
1 to 6 wk Gain: Abdominal feed fat
(g) 970* 768b 971* 9
(g*g> 666* 543b 649* 7
(% BW) .5C 1.4* .7 b .05
Means within columns with no common superscripts differ significantly (P < .05). 'Data are means of five groups of five male crossbred chicks from 8 to 22, 22 to 42, and 8 to 42 days posthatching; average initial weight was 76 g. %ee Table 1 for dietary composition during 1 to 3 wk of age. During 4 to 6 wk of age, the positive control was a 20% CP diet (59.8% corn and 31.1% soybean meal) fortified with .2% DL-Met and .06% L-Lys, and the low-protein diet was 16% CP (66.0% corn and 21.6% soybean meal). 3 Quantities supplied during 1 to 3 wk of age were .25% DL-Met, .21% L-Lys, .23% L-Arg, .11% L-Val, .08% LThr, and 2.31% L-Glu. During 3 to 6 wk of age, quantities supplied were .25% DL-Met, .25% L-Lys, .23% L-Arg, .11% L-Val, .13% L-Thr, and 2.31% LrGla.
1176
HAN ET AL.
TABLE 9. Performance of commercial Hubbard chicks fed the 19% CF diet supplemented with amino acids (Experiment 7) Treatment
Weight gain
Gain: feed
(g) (g*g) 636* 776* b 546 680b 629* 771* 16 16 *^Means within columns with no common superscripts differ significantly (P < .05). 1 Data are means of five groups of five male Hubbard chicks from 8 to 22 days posthatching; average initial weight was 121 g. 2 Indispensable amino acid (IAA) mix provided .25% DL-Met, .21% L-Lys, .23% L-Arg, .11% L-Val, and .08% L-Thr. These levels were those determined to be adequate in Experiment 4.
1. 23% CP + DL-Met 2. 19% CP 3. As 2 + IAA mix2 Pooled SEM
That Met, Lys, Arg, and (possibly) Thr might be deficient in lower CP (lower soybean meal) diets for broiler chicks is not surprising. Indications that Val was deficient, however, are surprising because relative to the NRC (1984) Val requirement estimate, the level of this AA in the low-protein diet used herein is calculated to be in considerable excess. There appears to be no plausible explanation for the Val response observed here or in the authors' previous work (Edmonds et al, 1985), other than that the NRC table overestimated Val content of corn and soybean meal 0ensen and Colnago, 1991). Glutamic acid supplementation of the low-protein diet containing the five limiting IAA improved feed efficiency and reduced body fat (Table 3). The improved leanfat ratio of chick carcasses was as great with Glu as was the case when six additional IAA were added to the diet already containing the five limiting IAA. This supports the view that after Met, Lys, Arg, Val, and Thr were supplemented, no other IAA were deficient in the lowprotein diet. In fact, the data indicate that amino N (for dispensable AA biosynthesis) was also a limiting factor in the lowprotein diet. The low-protein diet required smaller quantities of the five limiting AA than that present in the 23% CP positive control
diet. This is not surprising because 1) crystalline AA are more bioavailable than intact, protein-bound AA (Izquierdo et al, 1988; Han et al, 1990; Chung and Baker, 1991); 2) lowering CP of a diet results in a reduction in AA requirements (Griminger et al, 1956; Rosenberg and Baldini, 1957; Nelson et al, 1960; Boomgaardt and Baker, 1971; Abebe and Morris, 1990); and 3) the 23% CP diet contained levels of some of the AA in excess of the requirements. Downward revision in AA levels in the IAA package cannot be interpreted as supplemental minimum levels. It is very likely that some of the IAA levels could be reduced further. Although the low-protein diet used here contained 19% CP from corn and soybean meal (16% in the case of the study done with 3- to 6-wk-old birds), after adding the determined optimal levels of the five IAA plus 2.31% Glu, the lowprotein diets actually contained 21.4% CP (18.5% CP for the older birds), using the factor N x 6.25 to account for the added AA N. This factor may be important in attempts to interpret the literature on the subject of low-protein, AA-fortified diets for broiler chicks. Thus, several workers have reported that performance of chicks is as good with a low-protein, AA-fortified diet as with a diet containing a conventional protein level (Bornstein and Lipstein, 1975; Waldroup et al, 1976; Uzu, 1982, 1986; Summers and Leeson, 1985; Schutte, 1987; Stillborn and Waldroup, 1989; Parr and Summers, 1991). However, recent work from the University of Georgia has indicated that chick performance is inferior on low-protein, CS diets, regardless of AA supplementation (Fancher and Jensen, 1989a,b,c; Pinchasov et al, 1990; Colnago et al, 1991). In the Georgia studies, the low-protein diets were formulated such that the AA N was accounted for, and this effectively lowered the soybean meal (at the expense of corn) more than was the case in the current studies. Unpublished data from another research project in the authors' laboratory wherein a true 19% CP AA-fortified diet was fed resulted in weight gains that were the same as those obtained with a Metfortified 23% CP diet. Feed efficiency, however, was reduced in birds fed the
AMINO ACID SUPPLEMENTATION
19% CP diet. The low-CP diet in this case contained no added Glu, and it was slightly lower in soybean meal. Chicks fed 19% CP diets with supplemental AA (21.4% total CP) during Weeks 1 to 3 posthatching, and those fed AAfortified 16% CP diets (18.5% total CP) during Weeks 3 to 6 posthatching gained as fast, converted feed to gain as efficiently, and had similar body fat levels as those fed conventional protein levels of 23% or 20%, respectively, for the early and later growing periods. Feed efficiency of birds during the early growing period, in fact, tended to be superior for the low-protein, AA-fortified diet compared with the positive control, Met-fortified, 23% CP diet. When data for these two diets were pooled across experiments (Treatments 1 and 5 of Experiment 1; Treatments 1 and 9 of Experiment 2; Treatments 1 and 10 of Experiment 3; and Treatments 1 and 6 of Experiment 4), gain:feed was, in fact, greater (P < .05) for the low-protein diet containing the five IAA plus Glu than it was for the positive control diet. Because the laboratory broiler strain (New Hampshire x Columbian) grows only about one-half as fast as commercial broiler strains, Hubbard chicks were tested in the last experiment. These chicks also performed as well on the low-protein, AA-fortified diet as they did on the 23% CP, positive control diet. This might have been expected because AA requirements expressed in terms of dietary concentration are virtually the same for the fastgrowing Hubbard chicks as they are for the slower growing laboratory strain (Robbins and Baker, 1980; Han and Baker, 1991). REFERENCES Abebe, S., and T. R. Morris, 1990. Effects of protein concentration on responses to dietary tryptophan by chicks. Br. Poult. Sci. 31:267-272. Association of Official Analytical Chemists, 1980. Official Methods of Analysis. 13th ed., Association of Official Analytical Chemists, Washington, DC. Boomgaardt, J., and D. H. Baker, 1971. Tryptophan requirement of growing chicks as affected by dietary protein level. J. Anim. Sci. 33:595-599. Bornstein, S., and B. Lipstein, 1975. The replacement of some of the soybean meal by the first limiting amino acids in practical broiler diets. I. The
1177
value of special supplementation of chick diets with methionine and lysine. Br. Poult. Sci. 16: 177-188. Carmer, S. G., and W. M. Walker, 1985. Pairwise multiple comparisons of treatment means in agronomic research. J. Agron. Ed. 14:19-26. Chung, T. K., and D. H. Baker, 1991. Apparent and true digestibility of amino acids in casein and in a complete amino acid mixture: comparison of pig ileal digestibility with the cecectomized cockerel assay. J. Anim. Sci. 69(Suppl. 1): 38.(Abstr.) Colnago, L., A. M. Penz, Jr., and L. S. Jensen, 1991. Effect of response of starting broiler chicks to incremental reduction in intact protein on performance during the grower phase. Poultry Sci. 70(Suppl. l):153.(Abstr.) Edmonds, M. S., C. M. Parsons, and D. H. Baker, 1985. Limiting amino acids in low-protein cornsoybean meal diets fed to growing chicks. Poultry Sci. 64:1519-1526. Fancher, B. I., and L. S. Jensen, 1989a. Male broiler performance during the starting and growing periods as affected by dietary protein, essential amino acids, and potassium levels. Poultry Sci. 68:1385-1395. Fancher, B. I., and L. S. Jensen, 1989b. Influence on performance of three to six-week-old broilers of varying dietary protein contents with supplementation of essential amino acid requirements. Poultry Sci. 68:113-123. Fancher, B. I., and L. S. Jensen, 1989c. Dietary protein level and essential amino acid content: influence upon female broiler performance during the grower period. Poultry Sci. 68:897-908. Griminger, P., H. M. Scott, and R. M. Forbes, 1956. The effect of protein level on the tryptophan requirement of the growing chick. J. Nutr. 59: 67-76. Han, Y., and D. H. Baker, 1991. Lysine requirements of fast- and slow-growing broiler chicks. Poultry Sci. 70:2108-2114. Han, Y., F. Castanon, C. M. Parsons, and D. H. Baker, 1990. Intestinal absorption and bioavailability of DL-methionine hydroxy analog compared to DL-methionine. Poultry Sci. 69:281-287. Izquierdo, O. A., C. M. Parsons, and D. H. Baker, 1988. Bioavailability of lysine in L-lysineHCl. J. Anim. Sci. 663590-2597. Jensen, L. S., 1991. Are peptides needed for optimum nutrition? Feed Management 42, No. 8:37-40. Jensen, L. S., and G. L. Colnago, 1991. Amino acid and protein for broilers and laying hens. Pages 29-36 in: Proceedings Maryland Nutrition Conference for Feed Manufacturers. Baltimore, MD. National Research Council, 1984. Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. Nelson, T. S., R. J. Young, R. B. Bradfield, J. B. Anderson, L. C. Norris, F. W. Hill, and M. L. Scott, 1960. Studies on the sulfur amino acid requirement of the chick. Poultry Sci. 39: 308-314. Parr, J. F., and J. D. Summers, 1991. The effects of minimizing amino acid excesses in broiler diets. Poultry Sci. 70:1540-1549.
1178
HAN ET AL.
Pinchasov, Y., C. X. Mendonca, and L. S. Jensen, 1990. Broiler chick response to diets supplemented with synthetic amino acids. Poultry Sci. 69:1950-1955. Robbins, K. R., and D. H. Baker, 1980. Effect of sulfur amino acid level and source on the performance of chicks fed high levels of copper. Poultry Sci. 59:1246-1253. Rosenberg, H. R., and J. R. Baldini, 1957. Effect of dietary protein level on the methionine-energy relationship in broiler diets. Poultry Sci. 36: 247-252. Sasse, C. E., and D. H. Baker, 1974. Factors affecting sulfate-sulfur utilization by the young chicks. Poultry Sci. 53:652-662. Schutte, J. B., 1987. Utilization of synthetic amino acids in poultry. Pages RT11-12 in: World's Poultry Science Association 6th European Symposium on Poultry Nutrition. Konigslutter, Germany. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics. A Biometrical Ap-
proach. 2nd ed. McGraw-Hill Book Co., New York, NY. Stillborn, H. L., and P. W. Waldroup, 1989. Utilization of low-protein grower diets for broiler chickens. Poultry Sci. 68(Suppl. l):142.(Abstr.) Summers, J. D., and S. Leeson, 1985. Broiler carcass composition as affected by amino acid supplementation. Can. J. Arum. Sci. 65:717-723. Uzu, G., 1982. Limit of reduction of the protein level in broiler feeds. Poultry Sci. 61(Suppl. 1): 1557-1558.(Abstr.) Uzu, G., 1986. Threonine requirement in broilers. Alimentation Equilibre Commentry, Document No. 242. Commentary, France. Velu, J. G., D. H. Baker, and H. M. Scott, 1972. Regression equations for determining body composition of young chicks. Poultry Sci. 51: 698-699. Waldroup, P. W., R. J. Mitchell, J. R. Payne, and K. R. Hazen, 1976. Performance of chicks fed diets formulated to minimize excess levels of essential amino acids. Poultry Sci. 55:243-253.