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Methionine and Cystine Requirements of Growing Turkeys1,2
Methionine and Cystine Requirements of Growing Turkeys1 ' 2 B. R. BEHRENDS 3 and P. E. WAIBEL Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108 (Received for publication January 17, 1979)
INTRODUCTION
The metabolic relationship of methionine to cysteine has long been known. Methionine is an indispensable amino acid and must be provided by the diet. Cysteine can be synthesized from dietary methionine and, therefore, is dispensable. The reverse of the pathway does not occur (Rose and Rice, 1939). That this relationship exists for turkeys was confirmed by Kratzer et al. (1949). Practical turkey rations contain both methionine and cysteine (in the form of cystine) in protein. Cystine fulfills the physiological requirement for cysteine and has a "sparing" effect on methionine, i.e., the methionine requirement is dependent on the amount of cystine in the diet. A major problem in interpreting research on methionine requirements of turkeys is the different cystine levels in the diets used by various investigators. A remedy to this problem has been to evaluate the dietary cystine content (assuming it is not in excess), add it to the
1
Based, in part, on the senior author's Ph.D. thesis. Scientific Journal Series Paper No. 10,623, Minnesota Agricultural Experiment Station. 'Present address: Sparboe Agricultural Corp., 125 East Commercial St., Litchfield, MN 55355. 2
dietary methionine level found to be optimum, and report a requirement for "total sulfur amino acids" (TSAA) or "methionine plus cystine" (M + C). This practice does not consider the maximum amount of cystine that can be utilized or the effect of cystine levels on TSAA requirements. The maximum proportion of the methionine requirement that can be replaced by cystine (cystine replacement value) is not well defined for turkeys. A single study (Kratzer et al., 1949) determined the maximum cystine requirement with methionine adjusted to the minimal level and reported a requirement of .30% for starting poults. The methionine requirement (determined with excess cystine) was .50%, resulting in a TSAA requirement of .80%. The resultant cystine replacement value of 37% (.3%/.8%) is lower than recently reported values for other species. Graber et al. (1971) reported cystine replacement values of 56, 65, and 67% for broiler chicks during the 2 n d , 5 t h , and 8 t h week of life, respectively. Values of 56 and 60% have been reported for the growing pig and the growing rat, respectively (Baker et al., 1969;Stockland
849
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ABSTRACT Sulfur amino acid requirements of starting, growing, finishing male Large White turkeys were investigated in three experiments using 3000 turkeys. Requirements were determined by least squares analyses of response curves obtained by supplementing methionine and cystine deficient basal diets with graded levels of DL-methionine and/or L-cystine. Experiments were conducted from 1 to 4, 8 to 12, and 16 to 20 weeks of age. Diets included starch, soybean meal, faba beans, and field peas. Requirements for total sulfur amino acids (TSAA) determined with diets marginally deficient in cystine were .95 to 1.01, .70 to .71, and .43 to .48% of the diet for starting (1 to 4 weeks), growing (8 to 12 weeks), finishing (16 to 20 weeks) turkeys, respectively, in different experiments. On the basis of dietary metabolizable energy, the respective TSAA requirement values (in %/therm) were .298 to .332, .205 to .221, and .128 to .134. Minimum methionine requirements (determined with excess dietary cystine) were .46, .30, and .19% of the diet for starting, growing, and finishing turkeys, respectively. Cystine replacement values, calculated by dividing the maximum usable levels of cystine (TSAA requirements minus methionine requirements) by TSAA requirements were 55, 58, and 57% of the TSAA requirement for starting, growing, and finishing turkeys, respectively. 1980 Poultry Science 59:849-859
850
BEHRENDS AND WAIBEL
MATERIALS AND METHODS All of the studies utilized Large White turkeys (Nicholas strain, commercial males). The birds were housed in floor pens (1.82 x 2.43m) on wood shavings in a windowless house. Brooding was accomplished with infrared heating units. An intermittent lighting program was employed after the brooding period; with it, lights were on during 0800 to 1100 hr, 1400 to 1700 hr, 2000 to 2200 hr, and 0200 to 0400 hr.
Experimental diets containing graded levels of methionine and/or cystine were fed from 1 to 4, 8 to 12, and 16 to 20 weeks of age. The birds were fed corn-soy turkey rations, appropriate for the age period, during the first week of life, from 4 to 8 weeks, and from 12 to 16 weeks of age. Poults were sorted into stratified weight groups and assigned randomly to pens in order to assure equivalent weight distribution in each pen. At 1 week of age, there were 20 birds/pen. Upon termination of the first experimental period, the birds from the more deficient treatments and very small and large birds were discarded. The remaining birds were reweighed in the 7 t h week and assigned to pens (10 birds/pen), again from stratified weight groups, at 8 weeks of age for the second experimental period. A similar technique was used to make up pens (6 birds/pen) for the third experimental period (16 to 20 weeks). This procedure was used to insure that birds in the latter age groups had received adequate quantities of methionine and cystine throughout their lives. The experimental design and levels of amino acid additions are presented in Table 1. The composition and calculated analysis of the basal diets are given in Table 2. The major consideration in formulation was to develop diets deficient in methionine and cystine but adequate in all other nutrients for growing turkeys specific to a given age period. Basal diet series 1 contained dehulled soybean meal, corn starch, animal fat, corn oil, minerals, and vitamins. Basal diet series 2 was similar except faba beans (Vicia faba L.) and field peas (Pisum sativum) were used to provide a portion of the protein. The diets were not pelleted. The methionine and cystine contents of each basal diet were calculated from analytical values obtained for the protein-containing ingredients (Table 3) using the performic acid-oxidation method of Schram et al. (1954). These values represent the average of analyses conducted for each shipment of ingredients. Birds were weighed at the initiation and termination of each experiment. Feed consumption records were kept. Birds with obvious physical trauma, wrong sex birds, and obvious outliers were eliminated from the average daily gain calculations. All weight gains were included in the feed efficiency calculations. Pen means were considered as the experimental unit in all studies. Analysis of variance and linear regression were conducted as outlined by Snedecor and
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and Murillo and Jensen (1976a) have reported requirements in excess of 1% while Kummero et al. (1971) and D'Mello (1976) have suggested requirements of .82 and .83%, respectively. The lack of agreement on requirements for starting poults and the low cystine replacment value for turkeys (compared to other species) may be due to problems with analytical techniques for methionine and cystine. Research on analytical procedures by Schram et al. (1954) has demonstrated that cystine is partially destroyed by acid hydrolysis unless precautions are taken. This has not always been considered when using analytical values to determine dietary sulfur amino acid levels and may be responsible for low requirements. A recalculation of the data of Kummero et al. (1971) by Murillo and Jensen (1976a) using currently published methionine and cystine values for dietary ingredients revealed a requirement of 1.08% compared to the reported requirement of .82% based on analytical values. Considerably less data exist on methionine and cystine needs of turkeys older than 4 weeks and requirements are based on data obtained from studies of methionine supplementation. A single study (Murrillo and Jensen, 1976b) of TSAA requirements of growing turkeys (8 to 12 weeks) reported requirements of .77 to .88% of the diet for both summer and winter conditions. In order to gain much needed information on methionine and cystine requirements, especially for turkeys older than 4 weeks, three experiments were conducted. The objectives were a) to determine TSAA requirements, b) to determine minimum methionine requirements, and c) to determine maximum usable levels of cystine with methionine adjusted to minimal requirement levels. Each experiment was conducted with starting (1 to 4 weeks), growing (8 to 12 weeks), and finishing (16 to 20 weeks) turkeys.
851
METHIONINE AND CYSTINE FOR TURKEYS
T A B L E 1. Experiments,
Experiment
age periods,
Age period (weeks)
Basal diets
1-4
l a (soy-starch) 2a (pea-bean)
8-12
l b (soy-starch)
16-20
l c (soy-starch) 2c (pea-bean)
1-4 8-12 16-20 1-4 8-12 16-20
2a + .10% cystine 2a + .36% cystine 2b + .08% cystine 2 b + .18% cystine 2c + .06% cystine 2c + .14% cystine 2a + . 1 2 2 % m e t h i o n i n e 2a + . 1 7 2 % m e t h i o n i n e 2 b + .033% methionine 2b + . 0 6 3 % m e t h i o n i n e 2b + . 0 9 3 % m e t h i o n i n e 2 c + .048% methionine 2c + . 0 6 8 % m e t h i o n i n e 2c + . 0 8 8 % m e t h i o n i n e
Cochran (1967); means were separated, when appropriate, using the least significant difference test as outlined by Snedecor and Cochran (1967). Methionine and/or cystine requirements and their standard deviations were determined by least squares analysis of response curves utilizing the two phase regression model described by Hinkley (1971). The intersection of the response and plateau lines represented the requirement. Sloping response lines were determined by finding the line of best fit to the data points to the left of the intersection. Plateau lines were determined by averaging the points to the right of the intersection or by regression analysis where appropriate. Average daily gain (ADG) and gain/feed (G/F) were the response criteria. Experiment I. Experiment 1 included TSAA requirement studies for starting, growing, and finishing turkeys. The two basal diets for each age period were supplemented with graded levels of DL-methionine to obtain five dietary TSAA levels for each age period and basal diet combination. The levels of supplementation were chosen to provide dietary TSAA levels higher and lower than published requirements.
acids
Levels of a m i n o acids a d d e d .20, .30, .40% m e t h i o n i n e ; .10% m e t h i o n i n e .10% cystine .20, .30, .40% m e t h i o n i n e ; .10% m e t h i o n i n e .10% cystine 0, . 0 8 , .16, .24, .32% m e t h i o n i n e ; .08% m e t h i o n i n e .08% cystine 0, .08, .16, .24, .32% m e t h i o n i n e ; .08% m e t h i o n i n e .08% cystine o, .06, .12, . 1 8 , .24% m e t h i o n i n e ; .06% m e t h i o n i n e .06% cystine o, .06, .12, . 1 8 , .24% m e t h i o n i n e ; .06% m e t h i o n i n e .06% cystine 0, . 0 5 , .10, . 1 5 , . 2 0 , . 2 5 , .30, . 3 5 % m e t h i o n i n e 0, . 0 5 , . 1 0 , .15, .20, . 2 5 , .30, . 3 5 % m e t h i o n i n e 0, • 0 3 , .06, .09, .12, . 15, .18, . 2 1 % m e t h i o n i n e o, . 0 3 , .06, •09, . 1 2 , . 15, .18, . 2 1 % m e t h i o n i n e o, .02, .04, .06, . 0 8 , . 10, . 1 4 , .22% m e t h i o n i n e 0, .02, .04, .06, . 0 8 , . 10, .14, .22% m e t h i o n i n e o, .05, .10, . 1 5 , .20, . 2 7 5 , . 3 7 5 % cystine o, .05, • 10, . 1 5 , .20, . 2 7 5 , . 3 7 5 % cystine 0, . 0 3 , .06, .09, .12, . 15, . 1 9 5 , . 2 5 5 % cystine 0, . 0 3 , .06, . 0 9 , .12, . 15, . 1 9 5 , . 2 5 5 % cystine 0, . 0 3 , .06, .09, .12, . 15, . 1 9 5 , . 2 5 5 % cystine o, .02, .04, .06, . 0 8 , . 10, . 1 3 , .17% cystine 0, .02, •04, .06, . 0 8 , . 10, . 1 3 , .17% cystine 0, .02, .04, .06, . 0 8 , . 10, . 1 3 , .17% cystine
o, .10, o, .10,
DL-Methionine was added in increments of .10, .08, and .06% of the diet for the 1 to 4, 8 to 12, and 16 to 20 week periods, respectively. In addition, a DL-methionine, L-cystine combination was included with each basal diet-age group combination to determine if the diets were deficient in cystine. Each of the dietary treatments was fed to four pens of turkeys in each age period. Experiment 2. Experiment 2 evaluated methionine requirements at two dietary cystine levels for the 3 ages. Dietary cystine levels were adjusted to "adequate" or "excessive" levels by addition of L-cystine to basal diet series 2. The adequate levels were equal to the levels of cystine supplementation used in Experiment 1 for the three age groups. The excessive levels were arrived at by adding enough L-cystine to the basal diets to provide dietary sulfur amino acid levels equal to the TSAA requirements determined in Experiment 1. Two levels of cystine were used to insure that the physiological requirement would be met from dietary supplies. Eight methionine levels were used with each cystine level. The DL-methionine was added in
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2b (pea-bean)
and levels of added sulfur amino
BEHRENDS AND WAIBEL
852
TABLE 2. Composition of basal diets
Basal diet:
2c
lc
(%) 64.86 25.66
2.74
2.47
2.13
1.84
.41 .55 .13
.45 .67 .13
.39 .49 .11
.44 .67 .10
.28
.27
.20
.19
26.27 3057 1.34
20.05 3405 1.16
19.88 3201 1.09
12.69 3649
12.56 3370
.67
.58
.54
.91 .44
.84 .40
.335 .359 1.804
.272 .270 1.464
.236 .271 1.377
.171 .170 .967
.125 .173 .893
49.04 40.68
3.28
3.06
.50 .67 .14 .28
.52 .72 .14 .27
4.00 2.00
4.00 2.00
4.00 2.00 .15
.15
.15
Calculated analysis Protein, % 26.28 Metabolizable energy, kcal/kg 3195 Calcium, % 1.41 Phosphorus, inorganic, % .70 Methionine, % .357 Cystine, % .354 Lysine, % 1.885
43.65 7.10 20.00 20.00 4.00 2.00
33.33 26.68 15.00 15.00 4.00 2.00
24.73 44.56 10.00 10.00 4.00 2.00
35.57 53.40
Trace mineral mixture MN-74 was formulated to contain 2% iron (from ferrous sulfate), .2% copper (from copper sulfate), 6% manganese (from manganese sulfate), 6% zinc (from zinc oxide), .12% iodine (from ethylene diamine dihydroiodide), and .02% cobalt (from cobalt carbonate). Vitamin mixture MTS-74 supplied (per kilogram of mixture) 4,400,000 IU vitamin A acetate; 1,650,000 ICU vitamin D 3 ; 5,500 IU vitamin E acetate; 1.1 g menadione dimethylpyrimidinol bisulfite; 2.65 g riboflavin; 4.0 g d-calcium pantothenate; 26.5 g niacin; 198.4 g choline chloride; 4 mg vitamin B 1 2 ; .22 g folic acid; .55g pyridoxine; and 22 mg biotin. c Vitamin mixture MTG-74 supplied (per kilogram of mixture) 3,300,000 IU vitamin A acetate; 1,200,000 ICU vitamin D3 ; 3,300 IU vitamin E acetate; .77 g menadione dimethylpyrimidinol bisulfite; 1.98 g riboflavin; 2.6 g d-calcium pantothenate; 20 g niacin; 110.2 g choline chloride; and 2.76 mg vitamin B 1 2 .
increments of .05, .03, and .02% of the diet for the 1 to 4, 8 to 12, 16 to 20 week periods, respectively. In the 16 to 20 week period, the highest level of DL-methionine was increased to insure levels in excess of the requirement. Three replicate pens of birds were fed each methioninecystine combination. Experiment 3. Experiment 3 evaluated cystine requirements with dietary methionine adjusted to be less than, equal to, or greater
than the minimum methionine requirements determined in Experiment 2. The deficient and highest levels were included to verify the minimum methionine requirements determined in Experiment 2. The three levels of dietary methionine were combined with 8 levels of dietary cystine in a 3 X 8 factorial design for each age period. Basal diet series 2 was used for the experiment. The 1 to 4 week study was repeated as a
TABLE 3. Methionine and cystine contents of protein-containing dietary ingredients Material
Methionine (%)
Cystine (%)
Dehulled soybean meal Faba beans (Vicia faba L) Field peas (Pisum sativum)
.668 ± .032" .182 ± .006 .208 ± .006
.664 ± .026 .310 ± .008 .318+ .014
Mean ± standard deviation.
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Ingredient Starch, corn Soybean meal, dehulled Faba beans Field peas Animal fat, stabilized Corn oil L-lysine (78.4%) Defluorinated phosphate Calcium carbonate Salt (iodized) Trace mineral mix MN-74 Vitamin mix MTS-74 Vitamin mix MTG-74 0
2b
lb
2a
la
1 6 - 2 0 wetiks
8 - 1 2 weeks
1 —4 weeks
Age period:
METHIONINE AND CYSTINE FOR TURKEYS
853
consequence of an error in diet mixing. The facilities employed allowed the use of only one pen of 70 birds for each treatment. Two replicate pens were used per treatment for the 8 to 12 and 16 to 20 week age periods.
100.0
.434
1 1 90.0
i > Basal diet la Basal diet 2a
A
/
.490
,i Z~)—Y
y
• 144.17X + 24.13
- Y • 353.33X - 57.24
— •
*— - • »
-3 70.0 •
Basal diet lc Basal diet 2c
50X -25.99 25X - 28.26
& 25.0-
60.0'
.210. 50.0
680-
/'
f /
X
#L_
A
40.0
15.0
/A
1896X +
0.M5
Y -
600 • A
s^--Y
^ AS
' G.7562X - 0 . 0 6 8 3
.6167X -
.1089
.0626
.140-
Y • 0.8612X - 0.M79
/?
500 •
.100' 420'
\
.280 .800
.900
1.000
1.100
.400
.500
.600
Total sulfur amino acids K of diet)
Tobl sulfur amino acids ft of diet)
FIG. 1. Effect of dietary total sulfur amino acid levels on average daily gain and gain/feed. Experiment 1, 1 to 4 week period.
FIG. 3. Effect of dietary total sulfur amino acid levels on average daily and gain/feed. Experiment 1, 16 to 20 week period.
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RESULTS AND DISCUSSION Experiment 1. The results of Experiment 1 are presented in Figures 1, 2, and 3. Both average daily gain (ADG) and gain/feed ratio (G/F) increased with increasing levels of methionine supplementation and then plateaued as requirements were reached. Regression coefficients of the response lines were highly significant in all cases. The plateau lines were determined by averaging the response points to the right of the intersection. The responses to added dietary cystine in TSAA responsive diets are presented in Table 4. The ADG responses to L-cystine (average of 6 comparisons, 15.5%) were similar (6 of 6 comparisons, P<.05) to those from equivalent .600 .700 .800 .900 DL-methionine (average of 6 comparisons, FIG. 2. Effect of dietary total sulfur amino acid 17.8%). These data indicate that both diet series were deficient in cystine as well as levels on average daily gain and gain/feed. Experiment 1, 8 to 12 week period. methionine. Total sulfur amino acid requirements and their standard errors as determined in Experi-
BEHRENDS AND WAIBEL
854
TABLE 4. Effect of methionine and cystine supplementation of basal diets on average daily gain and gain/feed ratio of Large White turkey males (Experiment 1) Age period (weeks)
Basal diet
Methionine
1 1 1 2 2 2 1 1 1 2 2 2 1 1 1 2 2 2
.457 .457 .557 .435 .435 .535 .352 .352 .432 .316 .316 .396 .231 .231 .291 .185 .185 .245
Cystine
Average daily gain (g/day)
Gain/ feed
.354 .454 .354 .359 .459 .359 .270 .350 .270 .271 .351 .271 .170 .230 .170 .173 .233 .173
23.0* 26.4" 27.8° 24.4 a 26.9" 28.3° 84.0* 93.2" 95.6" 82.2* 93.l" 93.8° 80.7* 88.8° 91.2 b 70.0* 93.5" 90.l"
.554 .598 .616 .563* .603" .608" .325 .362 .361 .331 .349 .347 .186 .211 .196 .154* .193" .198°
s«V
1-4
16-20
ab ' Means with different superscripts are significantly different (P<.05) within an age period-diet combination.
m e n t 1 are presented in Table 5. Requirem e n t s agreed well b e t w e e n diets and response criteria for t h e 1 t o 4 and 8 t o 12 week age periods. For t h e 16 t o 20 week age period average T S A A r e q u i r e m e n t s of .481 and .430 were o b t a i n e d for basal diets 1 and 2, respectively. T h e slopes of t h e response lines were markedly different for t h e t w o basal diets and this m a y have c o n t r i b u t e d t o t h e difference in
requirements for this age period. Because m o r e accurate requirements with lower standard errors o f intersection points (Table 5) are o b t a i n e d with steep response curves, basal diet series 2 was used in E x p e r i m e n t s 2 and 3. In retrospect, t h e reason for t h e shallow slope with basal diet 1 is n o t certain; however, t h e higher basal c o n t e n t s of T S A A and biological variation m a y be c o n t r i b u t i n g factors.
TABLE 5. Total sulfur amino acid requirements (% of diet) of Large White turkey males as determined in Experiment 1 Response criteria:
Average daily gain
Gain/feed
Mean'a
(% of diet) Basal diet 1 1 —4 weeks 8 - 1 2 weeks 1 6 - 2 0 weeks
.960 u ± .022 .676 ± .036 .490 + .065
.945 ± .040 .718+ .051 .472 ± .103
.952 .697 .481
Basal diet 2 1 —4 weeks 8 - 1 2 weeks 1 6 - 2 0 weeks
.945 ± .035 .694 ± .037 .434 + .045
.945 ± .020 .720 ±.058 .425 ± .042
.945 .707 .430
Average of requirements determined by average daily gain and gain/feed. Intersection of slope line and plateau line. Standard deviation of intersection point.
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8-12
855
METHIONINE AND CYSTINE FOR TURKEYS
Experiment 2. The results of Experiment 2 are presented in Figures 4, 5, and 6. The regression coefficients of the sloping lines were highly significant in all cases.
•8 |
70.0-
< - • Dietary cystine • . 3 5 1 * 60.0-
-* Dietary cystine - . 4 5 1 *
50.0
£ c
.300-
-Y • .5211X • .1530
3
-Y - .S444X + .0776
.236
.266
296
.326
.356
.386
.416
.446
Methionine (* of diet)
FIG. 5. Effect of dietary methionine and cystine levels on average daily gain and gain/feed. Experiment 2, 8 to 12 week period.
100.0 -
5 90.0 -
>.80.0
60.0 55.0 -
£
. 150.
y
.125
.165
705
.265
Methionine 1% of diet)
Methionine I* of diet) FIG. 4. Effect of dietary methionine and cystine' levels on average daily gain and gain/feed. Experiment 2, 1 to 4 week period.
FIG. 6. Effect of dietary methionine and cystine levels on average daily gain and gain/feed. Experiment 2, 16 to 20 week period.
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Methionine requirements as determined in Experiment 2 are presented in Table 6. For each age period-response criterion combination, methionine requirements were lower when determined with the diets containing the higher levels of cystine. This would indicate that the lower levels of cystine were not adequate and methionine was being converted to cystine. Because cystine was not in excess with these diets, TSAA requirements were estimated by adding the methionine requirement to the dietary cystine level. These requirements are summarized in Table 7 along with requirements determined in Experiment 1. Experiment 3. The results of Experiment 3 are presented in Figures 7, 8, and 9. Valid intersection points could be established only for data in the 8 to 12 week period. Cystine levels were apparently not deficient enough to allow accurate determination of intersection points in all cases. For the 8 to 12 week period, it was possible to determine cystine requirements for the intermediate methionine level using the ADG response and for the high methionine level using both ADG and G/F as response criteria.
100.0-
856
BEHRENDS AND WAIBEL
TABLE 6. Methionine requirements (% of diet) of Large White turkey males as determined in Experiment 2 1—4 weeks
Age period: Dietary cystine:
.459%
.719%
8—12 weeks .351%
1 6 - 2 0 weeks
.451%
.233%
.313%
.216 ± .015 .219 + .023 .218
.192 ± .020 .192 ± .035 .192
(% of diet) Response criteria: Average daily gain .505 a ± .024 Gain/feed .605 ±.045 MeanC .555
.455 + .014 .464 ± .052 .460
.341 + .027 .368 ± .024 .354
.302 ± .009 .296 ± .020 .299
Standard deviation of intersection point. Average of requirements determined by average daily gain and gain/feed.
Regression coefficients of the sloping lines were highly significant for these cases. Regression analyses were conducted for the remaining age period-methionine level combinations. First order and higher level equations are presented in the figures where significant, Because intersection points could not be obtained, thereby providing direct cystine requirement data, maximum usable levels of cystine were determined indirectly by calculation from the results of Experiments 1 and 2. Total Sulfur Amino Acid Requirements, Total sulfur amino acid requirements, expressed as percent of the diets, and per therm of dietary metabolizable energy, as determined in
Experiments 1 and 2, are presented in Table 7. These are the means for requirements determined by ADG and G/F for each age period and diet combination. The TSAA requirements agreed well between experiments for the 1 to 4 and 8 to 12 week age periods. For the 16 to 20 week period requirement estimates ranged from .430 to .481% of the diet. The experimentally determined TSAA requirements for 1- to 4-week-old poults of .95 to 1.01% of the diet are higher than the requirements of .82 and .83 reported by Kummero «( al. (1971) and D'Mello (1976), respectively, and somewhat lower than the requirements of 1.05 and 1.03 reported by Murillo and Jensen
TABLE 7. Summary of total sulfur amino acid requirements of Large White turkey males as determined in Experiments 1 and 2 Age period, weeks Experiment 1 (Diet series 1) Diet energy, kcal ME/kg TSAA requirement, % of diet TSAA requirement, % per therm ME Methionine level, % Cystine level, % Experiment 1 (Diet series 2) Diet energy, kcal ME/kg TSAA requirement, % of diet TSAA requirement, % per therm ME Methionine level, % Cystine level, % Experiment 2 (Diet series 2) TSAA requirement, % of diet TSAA requirement, % per therm ME Methionine level, % Cystine level, %
1-4
8-12
3,195
16-20
3,405
3,057
3,649 .481 .132 .311 .170
.697 .205 .427 .270
.952 .298 .598 .354
3,370
3,201 .945 .309 .586 .359
.707 .221 .435 .271
.430 .128 .257 .173
1.014 .332 .555 .459
.705 .220 .354 .351
.451 .134 .218 .233
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Intersection of slope line and plateau line.
857
METHIONINE AND CYSTINE FOR TURKEYS
~V • 1LUX » 24.27 • 37.97 X • 23.45X2 • 17.83
.507*
<8 Average
•—• Dietary methionine • *—* Dietary methionine •
1
,457*
iy
^~~Y
s I 25.0/
XI. •Y • 38.75X * 58.96 70.
1
.5690
.340-
3
.320-
.SO'
.300.359
.409
.459
.509
.559
.634
S
.360-
2.546SX « 5.5365X2 • 3 . M 1 S ( ' • 1.9315
560'
.734
.280-
Cystine H of dletl
Dietary methionine • 0.269%
»•-»
Dietary methionine • 0.299*
-o
Dietary methionine • 0.329*
0.380
.380. 08325X «
•—•
J*v»
'f ^ ^
• *
_,.,•'•'"
A
°
- • —o—'
-—-*•"
^
^P^ ^
°
\ • ^— \~ \
•
o
Y • 0.4367X + 0.2065 Y -0.194IX • 0.2788 Y • 0.2189X • 0.2041
,'t
FIG. 7. Effect of dietary methionine and cystine levels on average daily gain and gain/feed. Experiment 3, 1 to 4 week period.
(1976a) and Potter and Shelton (1974), respectively. The TSAA requirements for growing turkeys (8 to 12 weeks) determined herein of .70 to .71 are somewhat lower than the requirement of .77 to .82% reported by Murillo and Jensen (1976b). The TSAA requirements for finishing turkeys of .43 to .48 are lower than the requirement (.55%) reported by the National Research Council (NRC, 1977). Methionine and Cystine Requirements. Because dietary methionine is used to synthesize cysteine when dietary cystine is deficient, the physiological requirement for methionine must be determined with dietary cystine at or in excess of its maximum usable level. The minimum methionine requirement plus the maximum usable level of cystine will theoretically represent the minimum requirement for TSAA's. The maximum percentage of the TSAA requirement that can be provided by cystine is termed the "cystine replacement value". Requirements for methionine were determined in Experiment 2 with dietary cystine adjusted to adequate and excessive levels. In all cases, methionine requirements (Table 6) were lower when dietary cystine was at the higher (excessive) level. The higher levels provided enough cystine to fulfill the TSAA requirements determined in Experiment 1 when
.331
.391
.466
Cystine 1% of diet)
FIG. 8. Effect of dietary methionine and cystine levels on average daily gain and gain/feed. Experiment 3, 8 to 12 week period.
-8
- - \
Y - 65.70X* 87.64
90.0 86.0• Dietary methionine - . 173* *—-•* Dietary methionine • . 1 9 3 * ° Dietary methionine - . 2 1 3 * .230 .220 .210 .200'
. wo.180-
.213
.253
.303
.343
Cystine I * of diet)
FIG. 9. Effect of dietary methionine and cystine levels on average daily gain and gain/feed. Experiment 3, 16 to 20 week period.
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60.
BEHRENDS AND WAIBEL
858
TABLE 8. Methionine and cystine requirements of Large White turkey males Age period, weeks:
S-12
1-4
16-20
.705
.451
.555 .459
.354 .351
.218 .233
Minimum methionine requirements (Experiment 2)
.460
.299
.192
Maximum usable level of cystine (TSAA requirementsminimum methionine requirements)
.554
.406
.259
Cystine replacement value (Maximum usable level of cystine/TSAA requirements) X 100
55%
combined with the M + C contained in the basal diet. This would insure that cystine was in excess and would verify that the methionine requirements determined with these diets are independent of needs for cystine. Because useful intersections were not obtained in Experiment 3, maximum usable cystine was calculated as the difference between the minimum methionine requirements and the TSAA requirements. The TSAA requirements used were those determined in Experiment 2 with the low cystine diets. The minimum methionine requirements were determined with the high cystine levels. This type of calculation is subject to the nonequivalent conversion in mass of methionine to cystine (Creek, 1968), but it was felt that this effect would be negligible with the level of cystine contained in the diets. A summary of these calculations and the resultant requirements is presented in Table 8. The cystine replacement values of 5 5, 5 8, and 57% for starting, growing, and finishing turkeys, respectively, are higher than the 37% replacement value reported by Kratzer et al. (1949) and agree well with values reported for other species. Values of 53, 56, and 60% have been reported for chicks, pigs, and rats, respectively (Sasse and Baker, 1974; Baker et al, 1969; Stockland et al, 1973). There did not appear to be an effect of age on cystine replacement value as shown by Graber et al. (1971). Although cystine replacement values were determined indirectly in these studies, based on minimum methionine requirements, it seems safe to
58%
57%
assume that at least 50% of the turkey's TSAA requirement can come from cystine.
REFERENCES Baker, D. H., W. W. Clausing, B. G. Harmon, A. H. Jensen, and D. E. Becker, 1969. Replacement value of cystine for methionine for the young pig. J. Anim. Sci. 29:581-584. Creek, R. D. 1968. Non equivalence in mass in the conversion of phenylalanine to tyrosine and methionine to cystine. Poultry Sci. 47:1385— 1386. D'Mello, J.P.F., 1976. Requirements of the young turkey for sulfur amino acids and threonine: comparison with other species. Brit. Poultry Sci. 17:157-162. Graber, G., H. M. Scott, and D. H. Baker, 1971. Sulfur amino acid nutrition of the growing chick: Effect of age on the capacity of cystine to spare dietary methionine. Poultry Sci. 50:1450-1455. Hinkley, D. V., 1971. Inference in two-phase regression. J. Amer. Statist. Ass. 66:737-743. Kratzer, F. H., D. E. Williams, and B. Marshall, 1949. The sulfur amino acid requirements of turkey poults. J. Nutr. 37:377- 383. Kummero, V. E., J. E. Jones, and C. B. Loadholt, 1971. Lysine and total sulfur amino acid requirements of turkey poults, one day to three weeks. Poultry Sci. 50:752-758. Murillo, M. G., and L. S. Jensen, 1976a. Sulfur amino acid requirement and foot pad dermatitis in turkey poults. Poultry Sci. 55:554-562. Murillo, M. G., and L. S. Jensen, 1976b. Methionine requirement of developing turkeys from 8—12 weeks of age. Poultry Sci. 55:1414-1418. National Research Council, 1977. Nutrient requirements of poultry. Nat. Acad. Sci., Washington, DC.
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1.014
TSAA requirements (Experiment 2) Methionine level Cystine level
METHIONINE AND CYSTINE FOR TURKEYS Potter, L. M., and J. R. Shelton, 1974. Methionine and protein requirements of young turkeys. Poultry Sci. 53:1967-1968. Rose, W. C , and E. C. Rice, 1939. The utilization of certain sulfur-containing compounds for growth purposes. J. Biol. Chem. 130:305-323. Sasse, C. E., and D. H. Baker, 1974. Factors affecting sulfate-sulfur utilization by the young chick. Poultry Sci. 5 3:652-662. Schram, E., S. Moore, and E. J. Bigwood, 1954. Chromatographic determination of cystine as
859
cysteic acid. Biochem. J. 57:3 3—37. Snedecor, G. W., and W. G. Cochran, 1967. Statistical methods. Iowa State University Press, Ames, IA. Stockland, W. L., R. J. Meade, D. F. Wass, and J. E. Sowers, 1973. Influence of levels of methionine and cystine on the total sulfur amino acid requirement of the growing rat. J. Anim. Sci. 36:526— 530. Warnick, R. E., and J. O. Anderson, 1973. Essential amino acid levels for starting poults. Poultry Sci. 52:445-452..
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INTRODUCTION
The metabolic relationship of methionine to cysteine has long been known. Methionine is an indispensable amino acid and must be provided by the diet. Cysteine can be synthesized from dietary methionine and, therefore, is dispensable. The reverse of the pathway does not occur (Rose and Rice, 1939). That this relationship exists for turkeys was confirmed by Kratzer et al. (1949). Practical turkey rations contain both methionine and cysteine (in the form of cystine) in protein. Cystine fulfills the physiological requirement for cysteine and has a "sparing" effect on methionine, i.e., the methionine requirement is dependent on the amount of cystine in the diet. A major problem in interpreting research on methionine requirements of turkeys is the different cystine levels in the diets used by various investigators. A remedy to this problem has been to evaluate the dietary cystine content (assuming it is not in excess), add it to the
1
Based, in part, on the senior author's Ph.D. thesis. Scientific Journal Series Paper No. 10,623, Minnesota Agricultural Experiment Station. 'Present address: Sparboe Agricultural Corp., 125 East Commercial St., Litchfield, MN 55355. 2
dietary methionine level found to be optimum, and report a requirement for "total sulfur amino acids" (TSAA) or "methionine plus cystine" (M + C). This practice does not consider the maximum amount of cystine that can be utilized or the effect of cystine levels on TSAA requirements. The maximum proportion of the methionine requirement that can be replaced by cystine (cystine replacement value) is not well defined for turkeys. A single study (Kratzer et al., 1949) determined the maximum cystine requirement with methionine adjusted to the minimal level and reported a requirement of .30% for starting poults. The methionine requirement (determined with excess cystine) was .50%, resulting in a TSAA requirement of .80%. The resultant cystine replacement value of 37% (.3%/.8%) is lower than recently reported values for other species. Graber et al. (1971) reported cystine replacement values of 56, 65, and 67% for broiler chicks during the 2 n d , 5 t h , and 8 t h week of life, respectively. Values of 56 and 60% have been reported for the growing pig and the growing rat, respectively (Baker et al., 1969;Stockland
849
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ABSTRACT Sulfur amino acid requirements of starting, growing, finishing male Large White turkeys were investigated in three experiments using 3000 turkeys. Requirements were determined by least squares analyses of response curves obtained by supplementing methionine and cystine deficient basal diets with graded levels of DL-methionine and/or L-cystine. Experiments were conducted from 1 to 4, 8 to 12, and 16 to 20 weeks of age. Diets included starch, soybean meal, faba beans, and field peas. Requirements for total sulfur amino acids (TSAA) determined with diets marginally deficient in cystine were .95 to 1.01, .70 to .71, and .43 to .48% of the diet for starting (1 to 4 weeks), growing (8 to 12 weeks), finishing (16 to 20 weeks) turkeys, respectively, in different experiments. On the basis of dietary metabolizable energy, the respective TSAA requirement values (in %/therm) were .298 to .332, .205 to .221, and .128 to .134. Minimum methionine requirements (determined with excess dietary cystine) were .46, .30, and .19% of the diet for starting, growing, and finishing turkeys, respectively. Cystine replacement values, calculated by dividing the maximum usable levels of cystine (TSAA requirements minus methionine requirements) by TSAA requirements were 55, 58, and 57% of the TSAA requirement for starting, growing, and finishing turkeys, respectively. 1980 Poultry Science 59:849-859
850
BEHRENDS AND WAIBEL
MATERIALS AND METHODS All of the studies utilized Large White turkeys (Nicholas strain, commercial males). The birds were housed in floor pens (1.82 x 2.43m) on wood shavings in a windowless house. Brooding was accomplished with infrared heating units. An intermittent lighting program was employed after the brooding period; with it, lights were on during 0800 to 1100 hr, 1400 to 1700 hr, 2000 to 2200 hr, and 0200 to 0400 hr.
Experimental diets containing graded levels of methionine and/or cystine were fed from 1 to 4, 8 to 12, and 16 to 20 weeks of age. The birds were fed corn-soy turkey rations, appropriate for the age period, during the first week of life, from 4 to 8 weeks, and from 12 to 16 weeks of age. Poults were sorted into stratified weight groups and assigned randomly to pens in order to assure equivalent weight distribution in each pen. At 1 week of age, there were 20 birds/pen. Upon termination of the first experimental period, the birds from the more deficient treatments and very small and large birds were discarded. The remaining birds were reweighed in the 7 t h week and assigned to pens (10 birds/pen), again from stratified weight groups, at 8 weeks of age for the second experimental period. A similar technique was used to make up pens (6 birds/pen) for the third experimental period (16 to 20 weeks). This procedure was used to insure that birds in the latter age groups had received adequate quantities of methionine and cystine throughout their lives. The experimental design and levels of amino acid additions are presented in Table 1. The composition and calculated analysis of the basal diets are given in Table 2. The major consideration in formulation was to develop diets deficient in methionine and cystine but adequate in all other nutrients for growing turkeys specific to a given age period. Basal diet series 1 contained dehulled soybean meal, corn starch, animal fat, corn oil, minerals, and vitamins. Basal diet series 2 was similar except faba beans (Vicia faba L.) and field peas (Pisum sativum) were used to provide a portion of the protein. The diets were not pelleted. The methionine and cystine contents of each basal diet were calculated from analytical values obtained for the protein-containing ingredients (Table 3) using the performic acid-oxidation method of Schram et al. (1954). These values represent the average of analyses conducted for each shipment of ingredients. Birds were weighed at the initiation and termination of each experiment. Feed consumption records were kept. Birds with obvious physical trauma, wrong sex birds, and obvious outliers were eliminated from the average daily gain calculations. All weight gains were included in the feed efficiency calculations. Pen means were considered as the experimental unit in all studies. Analysis of variance and linear regression were conducted as outlined by Snedecor and
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and Murillo and Jensen (1976a) have reported requirements in excess of 1% while Kummero et al. (1971) and D'Mello (1976) have suggested requirements of .82 and .83%, respectively. The lack of agreement on requirements for starting poults and the low cystine replacment value for turkeys (compared to other species) may be due to problems with analytical techniques for methionine and cystine. Research on analytical procedures by Schram et al. (1954) has demonstrated that cystine is partially destroyed by acid hydrolysis unless precautions are taken. This has not always been considered when using analytical values to determine dietary sulfur amino acid levels and may be responsible for low requirements. A recalculation of the data of Kummero et al. (1971) by Murillo and Jensen (1976a) using currently published methionine and cystine values for dietary ingredients revealed a requirement of 1.08% compared to the reported requirement of .82% based on analytical values. Considerably less data exist on methionine and cystine needs of turkeys older than 4 weeks and requirements are based on data obtained from studies of methionine supplementation. A single study (Murrillo and Jensen, 1976b) of TSAA requirements of growing turkeys (8 to 12 weeks) reported requirements of .77 to .88% of the diet for both summer and winter conditions. In order to gain much needed information on methionine and cystine requirements, especially for turkeys older than 4 weeks, three experiments were conducted. The objectives were a) to determine TSAA requirements, b) to determine minimum methionine requirements, and c) to determine maximum usable levels of cystine with methionine adjusted to minimal requirement levels. Each experiment was conducted with starting (1 to 4 weeks), growing (8 to 12 weeks), and finishing (16 to 20 weeks) turkeys.
851
METHIONINE AND CYSTINE FOR TURKEYS
T A B L E 1. Experiments,
Experiment
age periods,
Age period (weeks)
Basal diets
1-4
l a (soy-starch) 2a (pea-bean)
8-12
l b (soy-starch)
16-20
l c (soy-starch) 2c (pea-bean)
1-4 8-12 16-20 1-4 8-12 16-20
2a + .10% cystine 2a + .36% cystine 2b + .08% cystine 2 b + .18% cystine 2c + .06% cystine 2c + .14% cystine 2a + . 1 2 2 % m e t h i o n i n e 2a + . 1 7 2 % m e t h i o n i n e 2 b + .033% methionine 2b + . 0 6 3 % m e t h i o n i n e 2b + . 0 9 3 % m e t h i o n i n e 2 c + .048% methionine 2c + . 0 6 8 % m e t h i o n i n e 2c + . 0 8 8 % m e t h i o n i n e
Cochran (1967); means were separated, when appropriate, using the least significant difference test as outlined by Snedecor and Cochran (1967). Methionine and/or cystine requirements and their standard deviations were determined by least squares analysis of response curves utilizing the two phase regression model described by Hinkley (1971). The intersection of the response and plateau lines represented the requirement. Sloping response lines were determined by finding the line of best fit to the data points to the left of the intersection. Plateau lines were determined by averaging the points to the right of the intersection or by regression analysis where appropriate. Average daily gain (ADG) and gain/feed (G/F) were the response criteria. Experiment I. Experiment 1 included TSAA requirement studies for starting, growing, and finishing turkeys. The two basal diets for each age period were supplemented with graded levels of DL-methionine to obtain five dietary TSAA levels for each age period and basal diet combination. The levels of supplementation were chosen to provide dietary TSAA levels higher and lower than published requirements.
acids
Levels of a m i n o acids a d d e d .20, .30, .40% m e t h i o n i n e ; .10% m e t h i o n i n e .10% cystine .20, .30, .40% m e t h i o n i n e ; .10% m e t h i o n i n e .10% cystine 0, . 0 8 , .16, .24, .32% m e t h i o n i n e ; .08% m e t h i o n i n e .08% cystine 0, .08, .16, .24, .32% m e t h i o n i n e ; .08% m e t h i o n i n e .08% cystine o, .06, .12, . 1 8 , .24% m e t h i o n i n e ; .06% m e t h i o n i n e .06% cystine o, .06, .12, . 1 8 , .24% m e t h i o n i n e ; .06% m e t h i o n i n e .06% cystine 0, . 0 5 , .10, . 1 5 , . 2 0 , . 2 5 , .30, . 3 5 % m e t h i o n i n e 0, . 0 5 , . 1 0 , .15, .20, . 2 5 , .30, . 3 5 % m e t h i o n i n e 0, • 0 3 , .06, .09, .12, . 15, .18, . 2 1 % m e t h i o n i n e o, . 0 3 , .06, •09, . 1 2 , . 15, .18, . 2 1 % m e t h i o n i n e o, .02, .04, .06, . 0 8 , . 10, . 1 4 , .22% m e t h i o n i n e 0, .02, .04, .06, . 0 8 , . 10, .14, .22% m e t h i o n i n e o, .05, .10, . 1 5 , .20, . 2 7 5 , . 3 7 5 % cystine o, .05, • 10, . 1 5 , .20, . 2 7 5 , . 3 7 5 % cystine 0, . 0 3 , .06, .09, .12, . 15, . 1 9 5 , . 2 5 5 % cystine 0, . 0 3 , .06, . 0 9 , .12, . 15, . 1 9 5 , . 2 5 5 % cystine 0, . 0 3 , .06, .09, .12, . 15, . 1 9 5 , . 2 5 5 % cystine o, .02, .04, .06, . 0 8 , . 10, . 1 3 , .17% cystine 0, .02, •04, .06, . 0 8 , . 10, . 1 3 , .17% cystine 0, .02, .04, .06, . 0 8 , . 10, . 1 3 , .17% cystine
o, .10, o, .10,
DL-Methionine was added in increments of .10, .08, and .06% of the diet for the 1 to 4, 8 to 12, and 16 to 20 week periods, respectively. In addition, a DL-methionine, L-cystine combination was included with each basal diet-age group combination to determine if the diets were deficient in cystine. Each of the dietary treatments was fed to four pens of turkeys in each age period. Experiment 2. Experiment 2 evaluated methionine requirements at two dietary cystine levels for the 3 ages. Dietary cystine levels were adjusted to "adequate" or "excessive" levels by addition of L-cystine to basal diet series 2. The adequate levels were equal to the levels of cystine supplementation used in Experiment 1 for the three age groups. The excessive levels were arrived at by adding enough L-cystine to the basal diets to provide dietary sulfur amino acid levels equal to the TSAA requirements determined in Experiment 1. Two levels of cystine were used to insure that the physiological requirement would be met from dietary supplies. Eight methionine levels were used with each cystine level. The DL-methionine was added in
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2b (pea-bean)
and levels of added sulfur amino
BEHRENDS AND WAIBEL
852
TABLE 2. Composition of basal diets
Basal diet:
2c
lc
(%) 64.86 25.66
2.74
2.47
2.13
1.84
.41 .55 .13
.45 .67 .13
.39 .49 .11
.44 .67 .10
.28
.27
.20
.19
26.27 3057 1.34
20.05 3405 1.16
19.88 3201 1.09
12.69 3649
12.56 3370
.67
.58
.54
.91 .44
.84 .40
.335 .359 1.804
.272 .270 1.464
.236 .271 1.377
.171 .170 .967
.125 .173 .893
49.04 40.68
3.28
3.06
.50 .67 .14 .28
.52 .72 .14 .27
4.00 2.00
4.00 2.00
4.00 2.00 .15
.15
.15
Calculated analysis Protein, % 26.28 Metabolizable energy, kcal/kg 3195 Calcium, % 1.41 Phosphorus, inorganic, % .70 Methionine, % .357 Cystine, % .354 Lysine, % 1.885
43.65 7.10 20.00 20.00 4.00 2.00
33.33 26.68 15.00 15.00 4.00 2.00
24.73 44.56 10.00 10.00 4.00 2.00
35.57 53.40
Trace mineral mixture MN-74 was formulated to contain 2% iron (from ferrous sulfate), .2% copper (from copper sulfate), 6% manganese (from manganese sulfate), 6% zinc (from zinc oxide), .12% iodine (from ethylene diamine dihydroiodide), and .02% cobalt (from cobalt carbonate). Vitamin mixture MTS-74 supplied (per kilogram of mixture) 4,400,000 IU vitamin A acetate; 1,650,000 ICU vitamin D 3 ; 5,500 IU vitamin E acetate; 1.1 g menadione dimethylpyrimidinol bisulfite; 2.65 g riboflavin; 4.0 g d-calcium pantothenate; 26.5 g niacin; 198.4 g choline chloride; 4 mg vitamin B 1 2 ; .22 g folic acid; .55g pyridoxine; and 22 mg biotin. c Vitamin mixture MTG-74 supplied (per kilogram of mixture) 3,300,000 IU vitamin A acetate; 1,200,000 ICU vitamin D3 ; 3,300 IU vitamin E acetate; .77 g menadione dimethylpyrimidinol bisulfite; 1.98 g riboflavin; 2.6 g d-calcium pantothenate; 20 g niacin; 110.2 g choline chloride; and 2.76 mg vitamin B 1 2 .
increments of .05, .03, and .02% of the diet for the 1 to 4, 8 to 12, 16 to 20 week periods, respectively. In the 16 to 20 week period, the highest level of DL-methionine was increased to insure levels in excess of the requirement. Three replicate pens of birds were fed each methioninecystine combination. Experiment 3. Experiment 3 evaluated cystine requirements with dietary methionine adjusted to be less than, equal to, or greater
than the minimum methionine requirements determined in Experiment 2. The deficient and highest levels were included to verify the minimum methionine requirements determined in Experiment 2. The three levels of dietary methionine were combined with 8 levels of dietary cystine in a 3 X 8 factorial design for each age period. Basal diet series 2 was used for the experiment. The 1 to 4 week study was repeated as a
TABLE 3. Methionine and cystine contents of protein-containing dietary ingredients Material
Methionine (%)
Cystine (%)
Dehulled soybean meal Faba beans (Vicia faba L) Field peas (Pisum sativum)
.668 ± .032" .182 ± .006 .208 ± .006
.664 ± .026 .310 ± .008 .318+ .014
Mean ± standard deviation.
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Ingredient Starch, corn Soybean meal, dehulled Faba beans Field peas Animal fat, stabilized Corn oil L-lysine (78.4%) Defluorinated phosphate Calcium carbonate Salt (iodized) Trace mineral mix MN-74 Vitamin mix MTS-74 Vitamin mix MTG-74 0
2b
lb
2a
la
1 6 - 2 0 wetiks
8 - 1 2 weeks
1 —4 weeks
Age period:
METHIONINE AND CYSTINE FOR TURKEYS
853
consequence of an error in diet mixing. The facilities employed allowed the use of only one pen of 70 birds for each treatment. Two replicate pens were used per treatment for the 8 to 12 and 16 to 20 week age periods.
100.0
.434
1 1 90.0
i > Basal diet la Basal diet 2a
A
/
.490
,i Z~)—Y
y
• 144.17X + 24.13
- Y • 353.33X - 57.24
— •
*— - • »
-3 70.0 •
Basal diet lc Basal diet 2c
50X -25.99 25X - 28.26
& 25.0-
60.0'
.210. 50.0
680-
/'
f /
X
#L_
A
40.0
15.0
/A
1896X +
0.M5
Y -
600 • A
s^--Y
^ AS
' G.7562X - 0 . 0 6 8 3
.6167X -
.1089
.0626
.140-
Y • 0.8612X - 0.M79
/?
500 •
.100' 420'
\
.280 .800
.900
1.000
1.100
.400
.500
.600
Total sulfur amino acids K of diet)
Tobl sulfur amino acids ft of diet)
FIG. 1. Effect of dietary total sulfur amino acid levels on average daily gain and gain/feed. Experiment 1, 1 to 4 week period.
FIG. 3. Effect of dietary total sulfur amino acid levels on average daily and gain/feed. Experiment 1, 16 to 20 week period.
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RESULTS AND DISCUSSION Experiment 1. The results of Experiment 1 are presented in Figures 1, 2, and 3. Both average daily gain (ADG) and gain/feed ratio (G/F) increased with increasing levels of methionine supplementation and then plateaued as requirements were reached. Regression coefficients of the response lines were highly significant in all cases. The plateau lines were determined by averaging the response points to the right of the intersection. The responses to added dietary cystine in TSAA responsive diets are presented in Table 4. The ADG responses to L-cystine (average of 6 comparisons, 15.5%) were similar (6 of 6 comparisons, P<.05) to those from equivalent .600 .700 .800 .900 DL-methionine (average of 6 comparisons, FIG. 2. Effect of dietary total sulfur amino acid 17.8%). These data indicate that both diet series were deficient in cystine as well as levels on average daily gain and gain/feed. Experiment 1, 8 to 12 week period. methionine. Total sulfur amino acid requirements and their standard errors as determined in Experi-
BEHRENDS AND WAIBEL
854
TABLE 4. Effect of methionine and cystine supplementation of basal diets on average daily gain and gain/feed ratio of Large White turkey males (Experiment 1) Age period (weeks)
Basal diet
Methionine
1 1 1 2 2 2 1 1 1 2 2 2 1 1 1 2 2 2
.457 .457 .557 .435 .435 .535 .352 .352 .432 .316 .316 .396 .231 .231 .291 .185 .185 .245
Cystine
Average daily gain (g/day)
Gain/ feed
.354 .454 .354 .359 .459 .359 .270 .350 .270 .271 .351 .271 .170 .230 .170 .173 .233 .173
23.0* 26.4" 27.8° 24.4 a 26.9" 28.3° 84.0* 93.2" 95.6" 82.2* 93.l" 93.8° 80.7* 88.8° 91.2 b 70.0* 93.5" 90.l"
.554 .598 .616 .563* .603" .608" .325 .362 .361 .331 .349 .347 .186 .211 .196 .154* .193" .198°
s«V
1-4
16-20
ab ' Means with different superscripts are significantly different (P<.05) within an age period-diet combination.
m e n t 1 are presented in Table 5. Requirem e n t s agreed well b e t w e e n diets and response criteria for t h e 1 t o 4 and 8 t o 12 week age periods. For t h e 16 t o 20 week age period average T S A A r e q u i r e m e n t s of .481 and .430 were o b t a i n e d for basal diets 1 and 2, respectively. T h e slopes of t h e response lines were markedly different for t h e t w o basal diets and this m a y have c o n t r i b u t e d t o t h e difference in
requirements for this age period. Because m o r e accurate requirements with lower standard errors o f intersection points (Table 5) are o b t a i n e d with steep response curves, basal diet series 2 was used in E x p e r i m e n t s 2 and 3. In retrospect, t h e reason for t h e shallow slope with basal diet 1 is n o t certain; however, t h e higher basal c o n t e n t s of T S A A and biological variation m a y be c o n t r i b u t i n g factors.
TABLE 5. Total sulfur amino acid requirements (% of diet) of Large White turkey males as determined in Experiment 1 Response criteria:
Average daily gain
Gain/feed
Mean'a
(% of diet) Basal diet 1 1 —4 weeks 8 - 1 2 weeks 1 6 - 2 0 weeks
.960 u ± .022 .676 ± .036 .490 + .065
.945 ± .040 .718+ .051 .472 ± .103
.952 .697 .481
Basal diet 2 1 —4 weeks 8 - 1 2 weeks 1 6 - 2 0 weeks
.945 ± .035 .694 ± .037 .434 + .045
.945 ± .020 .720 ±.058 .425 ± .042
.945 .707 .430
Average of requirements determined by average daily gain and gain/feed. Intersection of slope line and plateau line. Standard deviation of intersection point.
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8-12
855
METHIONINE AND CYSTINE FOR TURKEYS
Experiment 2. The results of Experiment 2 are presented in Figures 4, 5, and 6. The regression coefficients of the sloping lines were highly significant in all cases.
•8 |
70.0-
< - • Dietary cystine • . 3 5 1 * 60.0-
-* Dietary cystine - . 4 5 1 *
50.0
£ c
.300-
-Y • .5211X • .1530
3
-Y - .S444X + .0776
.236
.266
296
.326
.356
.386
.416
.446
Methionine (* of diet)
FIG. 5. Effect of dietary methionine and cystine levels on average daily gain and gain/feed. Experiment 2, 8 to 12 week period.
100.0 -
5 90.0 -
>.80.0
60.0 55.0 -
£
. 150.
y
.125
.165
705
.265
Methionine 1% of diet)
Methionine I* of diet) FIG. 4. Effect of dietary methionine and cystine' levels on average daily gain and gain/feed. Experiment 2, 1 to 4 week period.
FIG. 6. Effect of dietary methionine and cystine levels on average daily gain and gain/feed. Experiment 2, 16 to 20 week period.
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Methionine requirements as determined in Experiment 2 are presented in Table 6. For each age period-response criterion combination, methionine requirements were lower when determined with the diets containing the higher levels of cystine. This would indicate that the lower levels of cystine were not adequate and methionine was being converted to cystine. Because cystine was not in excess with these diets, TSAA requirements were estimated by adding the methionine requirement to the dietary cystine level. These requirements are summarized in Table 7 along with requirements determined in Experiment 1. Experiment 3. The results of Experiment 3 are presented in Figures 7, 8, and 9. Valid intersection points could be established only for data in the 8 to 12 week period. Cystine levels were apparently not deficient enough to allow accurate determination of intersection points in all cases. For the 8 to 12 week period, it was possible to determine cystine requirements for the intermediate methionine level using the ADG response and for the high methionine level using both ADG and G/F as response criteria.
100.0-
856
BEHRENDS AND WAIBEL
TABLE 6. Methionine requirements (% of diet) of Large White turkey males as determined in Experiment 2 1—4 weeks
Age period: Dietary cystine:
.459%
.719%
8—12 weeks .351%
1 6 - 2 0 weeks
.451%
.233%
.313%
.216 ± .015 .219 + .023 .218
.192 ± .020 .192 ± .035 .192
(% of diet) Response criteria: Average daily gain .505 a ± .024 Gain/feed .605 ±.045 MeanC .555
.455 + .014 .464 ± .052 .460
.341 + .027 .368 ± .024 .354
.302 ± .009 .296 ± .020 .299
Standard deviation of intersection point. Average of requirements determined by average daily gain and gain/feed.
Regression coefficients of the sloping lines were highly significant for these cases. Regression analyses were conducted for the remaining age period-methionine level combinations. First order and higher level equations are presented in the figures where significant, Because intersection points could not be obtained, thereby providing direct cystine requirement data, maximum usable levels of cystine were determined indirectly by calculation from the results of Experiments 1 and 2. Total Sulfur Amino Acid Requirements, Total sulfur amino acid requirements, expressed as percent of the diets, and per therm of dietary metabolizable energy, as determined in
Experiments 1 and 2, are presented in Table 7. These are the means for requirements determined by ADG and G/F for each age period and diet combination. The TSAA requirements agreed well between experiments for the 1 to 4 and 8 to 12 week age periods. For the 16 to 20 week period requirement estimates ranged from .430 to .481% of the diet. The experimentally determined TSAA requirements for 1- to 4-week-old poults of .95 to 1.01% of the diet are higher than the requirements of .82 and .83 reported by Kummero «( al. (1971) and D'Mello (1976), respectively, and somewhat lower than the requirements of 1.05 and 1.03 reported by Murillo and Jensen
TABLE 7. Summary of total sulfur amino acid requirements of Large White turkey males as determined in Experiments 1 and 2 Age period, weeks Experiment 1 (Diet series 1) Diet energy, kcal ME/kg TSAA requirement, % of diet TSAA requirement, % per therm ME Methionine level, % Cystine level, % Experiment 1 (Diet series 2) Diet energy, kcal ME/kg TSAA requirement, % of diet TSAA requirement, % per therm ME Methionine level, % Cystine level, % Experiment 2 (Diet series 2) TSAA requirement, % of diet TSAA requirement, % per therm ME Methionine level, % Cystine level, %
1-4
8-12
3,195
16-20
3,405
3,057
3,649 .481 .132 .311 .170
.697 .205 .427 .270
.952 .298 .598 .354
3,370
3,201 .945 .309 .586 .359
.707 .221 .435 .271
.430 .128 .257 .173
1.014 .332 .555 .459
.705 .220 .354 .351
.451 .134 .218 .233
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Intersection of slope line and plateau line.
857
METHIONINE AND CYSTINE FOR TURKEYS
~V • 1LUX » 24.27 • 37.97 X • 23.45X2 • 17.83
.507*
<8 Average
•—• Dietary methionine • *—* Dietary methionine •
1
,457*
iy
^~~Y
s I 25.0/
XI. •Y • 38.75X * 58.96 70.
1
.5690
.340-
3
.320-
.SO'
.300.359
.409
.459
.509
.559
.634
S
.360-
2.546SX « 5.5365X2 • 3 . M 1 S ( ' • 1.9315
560'
.734
.280-
Cystine H of dletl
Dietary methionine • 0.269%
»•-»
Dietary methionine • 0.299*
-o
Dietary methionine • 0.329*
0.380
.380. 08325X «
•—•
J*v»
'f ^ ^
• *
_,.,•'•'"
A
°
- • —o—'
-—-*•"
^
^P^ ^
°
\ • ^— \~ \
•
o
Y • 0.4367X + 0.2065 Y -0.194IX • 0.2788 Y • 0.2189X • 0.2041
,'t
FIG. 7. Effect of dietary methionine and cystine levels on average daily gain and gain/feed. Experiment 3, 1 to 4 week period.
(1976a) and Potter and Shelton (1974), respectively. The TSAA requirements for growing turkeys (8 to 12 weeks) determined herein of .70 to .71 are somewhat lower than the requirement of .77 to .82% reported by Murillo and Jensen (1976b). The TSAA requirements for finishing turkeys of .43 to .48 are lower than the requirement (.55%) reported by the National Research Council (NRC, 1977). Methionine and Cystine Requirements. Because dietary methionine is used to synthesize cysteine when dietary cystine is deficient, the physiological requirement for methionine must be determined with dietary cystine at or in excess of its maximum usable level. The minimum methionine requirement plus the maximum usable level of cystine will theoretically represent the minimum requirement for TSAA's. The maximum percentage of the TSAA requirement that can be provided by cystine is termed the "cystine replacement value". Requirements for methionine were determined in Experiment 2 with dietary cystine adjusted to adequate and excessive levels. In all cases, methionine requirements (Table 6) were lower when dietary cystine was at the higher (excessive) level. The higher levels provided enough cystine to fulfill the TSAA requirements determined in Experiment 1 when
.331
.391
.466
Cystine 1% of diet)
FIG. 8. Effect of dietary methionine and cystine levels on average daily gain and gain/feed. Experiment 3, 8 to 12 week period.
-8
- - \
Y - 65.70X* 87.64
90.0 86.0• Dietary methionine - . 173* *—-•* Dietary methionine • . 1 9 3 * ° Dietary methionine - . 2 1 3 * .230 .220 .210 .200'
. wo.180-
.213
.253
.303
.343
Cystine I * of diet)
FIG. 9. Effect of dietary methionine and cystine levels on average daily gain and gain/feed. Experiment 3, 16 to 20 week period.
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60.
BEHRENDS AND WAIBEL
858
TABLE 8. Methionine and cystine requirements of Large White turkey males Age period, weeks:
S-12
1-4
16-20
.705
.451
.555 .459
.354 .351
.218 .233
Minimum methionine requirements (Experiment 2)
.460
.299
.192
Maximum usable level of cystine (TSAA requirementsminimum methionine requirements)
.554
.406
.259
Cystine replacement value (Maximum usable level of cystine/TSAA requirements) X 100
55%
combined with the M + C contained in the basal diet. This would insure that cystine was in excess and would verify that the methionine requirements determined with these diets are independent of needs for cystine. Because useful intersections were not obtained in Experiment 3, maximum usable cystine was calculated as the difference between the minimum methionine requirements and the TSAA requirements. The TSAA requirements used were those determined in Experiment 2 with the low cystine diets. The minimum methionine requirements were determined with the high cystine levels. This type of calculation is subject to the nonequivalent conversion in mass of methionine to cystine (Creek, 1968), but it was felt that this effect would be negligible with the level of cystine contained in the diets. A summary of these calculations and the resultant requirements is presented in Table 8. The cystine replacement values of 5 5, 5 8, and 57% for starting, growing, and finishing turkeys, respectively, are higher than the 37% replacement value reported by Kratzer et al. (1949) and agree well with values reported for other species. Values of 53, 56, and 60% have been reported for chicks, pigs, and rats, respectively (Sasse and Baker, 1974; Baker et al, 1969; Stockland et al, 1973). There did not appear to be an effect of age on cystine replacement value as shown by Graber et al. (1971). Although cystine replacement values were determined indirectly in these studies, based on minimum methionine requirements, it seems safe to
58%
57%
assume that at least 50% of the turkey's TSAA requirement can come from cystine.
REFERENCES Baker, D. H., W. W. Clausing, B. G. Harmon, A. H. Jensen, and D. E. Becker, 1969. Replacement value of cystine for methionine for the young pig. J. Anim. Sci. 29:581-584. Creek, R. D. 1968. Non equivalence in mass in the conversion of phenylalanine to tyrosine and methionine to cystine. Poultry Sci. 47:1385— 1386. D'Mello, J.P.F., 1976. Requirements of the young turkey for sulfur amino acids and threonine: comparison with other species. Brit. Poultry Sci. 17:157-162. Graber, G., H. M. Scott, and D. H. Baker, 1971. Sulfur amino acid nutrition of the growing chick: Effect of age on the capacity of cystine to spare dietary methionine. Poultry Sci. 50:1450-1455. Hinkley, D. V., 1971. Inference in two-phase regression. J. Amer. Statist. Ass. 66:737-743. Kratzer, F. H., D. E. Williams, and B. Marshall, 1949. The sulfur amino acid requirements of turkey poults. J. Nutr. 37:377- 383. Kummero, V. E., J. E. Jones, and C. B. Loadholt, 1971. Lysine and total sulfur amino acid requirements of turkey poults, one day to three weeks. Poultry Sci. 50:752-758. Murillo, M. G., and L. S. Jensen, 1976a. Sulfur amino acid requirement and foot pad dermatitis in turkey poults. Poultry Sci. 55:554-562. Murillo, M. G., and L. S. Jensen, 1976b. Methionine requirement of developing turkeys from 8—12 weeks of age. Poultry Sci. 55:1414-1418. National Research Council, 1977. Nutrient requirements of poultry. Nat. Acad. Sci., Washington, DC.
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1.014
TSAA requirements (Experiment 2) Methionine level Cystine level
METHIONINE AND CYSTINE FOR TURKEYS Potter, L. M., and J. R. Shelton, 1974. Methionine and protein requirements of young turkeys. Poultry Sci. 53:1967-1968. Rose, W. C , and E. C. Rice, 1939. The utilization of certain sulfur-containing compounds for growth purposes. J. Biol. Chem. 130:305-323. Sasse, C. E., and D. H. Baker, 1974. Factors affecting sulfate-sulfur utilization by the young chick. Poultry Sci. 5 3:652-662. Schram, E., S. Moore, and E. J. Bigwood, 1954. Chromatographic determination of cystine as
859
cysteic acid. Biochem. J. 57:3 3—37. Snedecor, G. W., and W. G. Cochran, 1967. Statistical methods. Iowa State University Press, Ames, IA. Stockland, W. L., R. J. Meade, D. F. Wass, and J. E. Sowers, 1973. Influence of levels of methionine and cystine on the total sulfur amino acid requirement of the growing rat. J. Anim. Sci. 36:526— 530. Warnick, R. E., and J. O. Anderson, 1973. Essential amino acid levels for starting poults. Poultry Sci. 52:445-452..
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