- Email: [email protected]
Lysine and Total Sulfur Amino Acid Requirements of Turkey Poults, One Day to Three Weeks
Lysine and Total Sulfur Amino Acid Requirements of Turkey Poults, One Day to Three Weeks V. E. KUMMEEO,1 J. E. JONES AND C. B. LOADHOLT2 Clemson University, Clemson, South Carolina 29631 (Received for publication October 9, 1970)
T
752
Downloaded from http://ps.oxfordjournals.org/ by guest on May 7, 2015
HE lysine and total sulfur amino acid the lysine and T.S.A.A. requirements of requirements of turkey poults have growing turkey poults from one day to been discussed by a number of workers. three weeks of age. In view of the relationGrau et al. (1946) suggested a lysine re- ship of amino acid requirements to energy quirement of 1.3% of the diet at protein level of the diet, these requirements are exlevels of 20 and 24% when sesame meal pressed on the basis of a percent per megawas the protein source. German et al. calorie metabolizable energy per kilogram (1949) using feather pigmentation as a cri- (kg.). terion, suggested the requirement for lysine Two experiments were conducted to dewas 1.1 to 1.2% of the diet. Gartley et al. termine the levels of lysine and T.S.A.A. (1950) using sunflower seed meal as a di- necessary for optimum growth during the etary protein source, suggested the lysine first three weeks after hatching for the requirement was 1.2% of the diet from Nicholas strain of Large White turkeys. day-one to four weeks of age. Kratzer et al. (1949) used DL-methionine Experiment 1. A representative sample was as a dietary supplement to study the total taken from each of the raw feed ingredients sulfur amino acid requirements of poults. set aside for this experiment: soybean Their work indicated a need for 0.5% meal, Peruvian fish meal, and ground corn. methionine and 0.3% cystine for a total of Part of each sample was subjected to pro0.8% sulfur amino acids in the diet. Alm- tein and fat analysis by the method of quist (1952), in his review, listed the lysine A.O.A.C. (1965) and fiber analysis by the requirement for starting poults as 1.3% method of Van Soest and Wine (1967). of the diet, the methionine requirement Another part was analyzed for amino acid as 0.45% of the diet and the cystine re- content by the methods of Spackman et al. quirement as 0.3% of the diet, or the total (1958) and Moore et al. (1958). sulfur amino acid (T.S.A.A.) requirement From the selected raw feed ingredients, as 0.75% of the diet. The National Re- twenty-five diets were formulated using the search Council (N.R.C.) (1966) require- values resulting from the analyses. The ments for starting turkey poults for lysine diets were made up of all possible combinaand T.S.A.A. are respectively 1.5 and 0.87% tions of five levels each of lysine and (methionine 0.52% minimum) when the T.S.A.A. These diets had a final calculated diet contains 28% protein. protein range of from 28.38 to 28.32% and were isocaloric, containing 2,908 kilocaloThis study was undertaken to establish ries metabolizable energy per kilogram (kcal. M.E. per kg.). Lysine was added in Published with the approval of the Director of the Clemson Agricultural Experiment Station as increments of 0.034% from the basal level Technical Contribution No. 884. of 1.63% up to 1.76% of the diet and DLP r e s e n t Address: P. O. Box 5735, Athens, Ga. methionine in increments of 0.075% from 2 Present Address: Dept. of Biometry, Med. the basal level of 0.67% T.S.A.A. up to Univ. of S. C , Charleston, S. C.
753
LYSINE AND SULFUR AMINO ACID REQUIREMENTS
Experiment 2. Lots of yellow corn, 49% soybean meal, ground coastal Bermudagrass, sesame meal, and Peruvian fish meal were set aside; samples were taken and analyzed as previously described in experiment 1; and formulas were calculated from these analyses. The basal diet is shown in Table 1 with additional lysine and T.S.A.A. levels achieved by substituting 50% lysine and DL-methionine for sand. The sixteen diets were considered isocaloric at 2,858 kcal. M.E./kg. The diets were formulated with all possi-
TABLE 1.—Composition o} basal experimental diet Experiment 1
2
%
%
1.03 50.38 0.0 36.40 0.00 5.17 0.17 0.52 4.13 0.00 0.65 1.552
2.00 31.00 1.00 41.00 20.00 1.00 0.00 0.25 1.25 0.75 1.25 0.50'
100.00
100.00
Calculated analysis of diet, % Protein 28.38 Fat 9.26 Lysine 1.63 1 T.S.A.A. 0.67 Calcium 1.47 Phosphorus 0.90 M.E. (kcal./kg.) 2904
28.25 5.40 1.38 0.72 1.25 0.88 2858
Fish meal (Peruvian) Soybean meal (49% protein) Coastal Bermudagrass Ground yellow corn Sesame Meal Fat (Blended) DL-methionine Salt Defluorinated rock phosphate Limestone Sand Premix
1
T.S.A.A. =Total sulfur amino acid. Adds per kg. of finished diet: vit. A, 5159 I.U Vit. D 3 , 1549 I.U.; vit. B12, 3.1 meg.; Folic acid .90 mg.; Menadione, 2.0 mg.; Biotin, 4.4 meg. MnSO t , 199.76 mg., ZnO, 90 mg.; Choline chloride 1.89 gm.; Niacin, 66.11 mg.; vit. E, 22.68 I.U Riboflavin, 5.5 mg.; d-Pantothenic acid, 11.0 mg. 3 Same vitamin and mineral content/kg. as in 2 above except ZnO increased to 103 mg. 2
ble combinations of 4 levels of lysine and 4 levels of T.S.A.A. Lysine levels were 0.482%, 0.527%, 0.573% and 0.618% per megacalorie per kg. T.S.A.A. levels were 0.250%), 0.295%, 0.341%, and 0.386% per megacalorie per kg. Feed and water were fed ad libitum. Sexed, day-old, Large White turkey poults were randomly allotted into 32 groups of 5 males and 5 females each, then wing banded, weighed and placed in 32 pens of heated battery brooders. After 2 weeks, the heat was removed and the battery room was maintained at temperatures comparable to experiment 1. Body weight and feed consumption were measured weekly by pens. Body weight sains and feed conversions were calculated
Downloaded from http://ps.oxfordjournals.org/ by guest on May 7, 2015
0.97% of the diet (Table 1). Cystine was at a level of .15% of the diet. All diets were maintained isocaloric. Lysine and DLmethionine were substituted for sand. Replicate diets were obtained by splitting the initially prepared mixes. Feed and water were supplied ad libitum to each group of ten poults in electric brooders with raised wire floors. Twenty-five diets were randomly assigned to fifty pens. Five-hundred straight-run, day-old poults from a commercial strain of Large White turkeys were randomly selected, wing banded, individually weighed and assigned to pens. The heated area of the brooder was maintained at 36.7°C. for two weeks. The ambient room temperature was maintained at 26.1-26.7°C. throughout the duration of the experiment. At weekly intervals, feed consumption was determined and poults were individually weighed. The experiment was terminated after three weeks. Feed consumption, for a period in which a bird in a pen died, was calculated by computing the fraction (bird days attributable to the survivors of the period divided by the total bird days had all birds lived) and multiplying this by the observed feed consumption. This corrected feed consumption to exclude the feed eaten by birds that died during the weekly period.
754
V. E.
KUMMERO, J. E. JONES AND C. B. LOADHOLT
TABLE 2.—Split plot in time analysis, Experiment 1 Source A, A2 A1XA2 Error (a) Time TiXAi TiXA. T1XA1XA2 Error (b) Total
MS 4 4 16 25 2 8 8 32 50
325 1,497 3,402 3,345 501,190 245 526 2,243 3,205
81.2 374.3 212.6 133.8 250,594.9 30.6 65.8 70.1 64.1
.607 2.798* 1.589 3,909.498" .477 1.026 1.094
149
* Denotes significance at 5% level. ** Denotes significance at 1% level. Ai Denotes Lysine. A2 Denotes total sulfur amino acids. Time denotes one week.
Source
df
SS
T.S.A.A. Lineal Quadratic Cubic Quartic Lysine Linear Quadratic Cubic Quartic T.S.A.A. XLysine Error
4
4489 1697 1909 778 105 957 858 97 14
16 25
10,205 10,028
MS 1697 1909 778 105
4.231 4.759 1.940 .262
858 97 14
2.139 .242 .035
637. 81 401. 12
1.590
* Denotes significance at 5% level. ** Denotes significance at 15% level.
kg. A second order response function equation: Y = 437.395 - 2.679X, + 21.559X2 - 0.834X,2 - 3.700X22 + 1.585XiX2
was also derived where X x = the level of lysine, X 2 = the level of total sulfur amino acids and Y = the predicted weight gain. Solving this equation using the suggested optimums gave a maximum predicted RESULTS AND DISCUSSION weight gain of 470 grams. The highest obExperiment 1. The split plot in time analy- served weight in this experiment was 491.5 sis (Table 2) showed that the levels of grams (Table 4). Figure 1 is a graphic repT.S.A.A. significantly affected weight gain resentation of the prediction formula. The (P < 0.0S). There was no interaction be- curves are contours of constant response for tween amino acid levels and time. Orthogo- weight gains. For any predicted weight gain nal polynomials were calculated to deter- in the area covered by the curves, a determine the nature of the response for the en- mination can be made of levels of lysine and tire three-week period (Table 3). This T.S.A.A. necessary to produce that preanalysis demonstrated both a linear and a dicted response. If visualized as a contour quadratic relationship between the levels of map, the curves of the graph will represent T.S.A.A. and weight gains at the 5% level levels of predicted weight gains. In interof significance. This analysis also suggested preting the closeness of the curves to mean that the lysine content of the diet affected a rather steep depression in rate of weight weight gains at the 15% level of signifi- gain and the large central area as a plateau cance. A second order response surface of constant weight gain rates, it was conanalysis was used to determine the opti- cluded that the levels of dietary lysine and mum combinations of lysine and total sul- T.S.A.A. within the area represented by the fur amino acids. This analysis suggested central plateau were optimum for maximum optimum values for lysine as 0.564% and weight gains. for T.S.A.A. as 0.282% per megacalorie per The variables in this experiment were
Downloaded from http://ps.oxfordjournals.org/ by guest on May 7, 2015
on a weekly basis for the 3-week trial period. When a poult died, the feed and poults of the pen were weighed immediately and average weight gain and feed conversion were calculated on a poult day basis. The data were analyzed by using a split plot in time analysis, orthogonal polynomials (Steele and Torrie, 1960) and response surface analysis (Cochran and Cox, 1964). Time was designated by three "weekly intervals.
TABLE 3.—Orthogonal polynomials. Experiment 1
LYSINE AND SULFUR AMINO ACID REQUIREMENTS
755
TABLE 4.—Average weight gains of poults in Experiment 1 (Grams) Lysine % Per Megacalorie Per Kg.
Total Sulfur Amino Acid % Per Megacalorie % Per Kg.
0.232 0.259 0.282 0.309 0.336
Treatment Means
0.SS9
0.573
0.582
0.595
0.609
450.6 476.1 463.6 472.4 448.4
427.9 491.5 481.0 460.5 456.1
445.3 449.7 470.9 468.0 463.9
423.0 465.9 469.6 432.5 489.1
455.5 427.1 456.0 462.4 455.2
462.2
463.4
459.5
456.0
451.2
amino acid imbalance was seen at the higher levels of lysine in Trial 1. As the lysine content was increased past the second lowest level, a trend toward depressed weight gains was noted. Gardiner and Agudu (1968) found that 0.35% added methionine reversed the depression of weight gains caused by 0.15% lysine added to the diet. This finding helps to explain why a relatively large depression in weight gains was not noted at the highest levels of lysine studied. Experiment 2. Feed conversion was based on pen data eliminating the sex variable. Therefore, a split plot in time analysis was
/ / \^-^Z \
g
.60 •
/
/ / ^ ^ \ .
8 58' III/
5 -
46s
\ \
\
»
•" n ////i
4 7 0
s H I III .23 TSAA
.25
.27
/////
{//III .30
.33
(% PER MEGACALORIE PER KG)
FIG. 1. Contours of constant growth response in grams for Experiment 1.
Downloaded from http://ps.oxfordjournals.org/ by guest on May 7, 2015
classified as quantitative and continuous. Heretofore, with this type of data, the least significant difference test would have been used to determine the optimum among treatments tested. However, since a response surface analysis is a valid test of continuous data (Loadholt, 1969), this analysis was employed to determine the combination of the variables which would produce an optimum response in weight gain. A response surface analysis should be used only after determining if there were significant effects for the data collected. The differences among weight gains attributable to the levels of T.S.A.A. in the diets were significant at the 5% level of probability. Data of Table 4 shows the treatment means and the average weight gains for the twenty-five diet combinations. The treatment means for T.S.A.A. suggested the existence of a curve with a definite peak response. The treatment means for lysine suggested the existence of a linear relation ship between dietary lysine content and growth rate. These results may have several interpretations. The range of T.S.A.A. chosen encompassed the desired levels for maximum growth. The levels of lysine existing in a practical corn-soy type diet are at or near the level which will produce maximum growth of turkey poults. Imbalances of both lysine and total sulfur amino acids depress weight gains (Boorman and Fisher, 1966). The most dramatic effect of this
440.4 462.1 468.2 459.1 462.5
756
V. E. KUMMERO, J. E. JONES AND C. B. LOADHOLT TABLE 5.—Analysis of weight gain Source
Lysine T.S.A.A. LXT.S.A.A. Error (a) Sex SXL SXT.S.A.A. SXLXT.S.A.A. Error (b) Weeks LXW T.S.A.A. X W SXW LXT.S.A.A. X W LXSXW T.S.A.A. X S X W LXT.S.A.A. X S X W Error (c)
df
SS
F
MS
3 1,355.2 4,065.6 6.699"* 3 1,727.8 575.9 2.847* 9 1,100.9 122.3 .605 16 3,236.2 202.3 1 6,836.7 6,836.7 56.455*** 3 816.8 2.249 272.3 3 168.2 56.1 .463 9 996.9 110.8 .915 16 1,937.0 121.1 2 589,933.5 294,966.8 4859.4*** 6 88.4 2.247* 136.4 6 1,374.8 229.1 3.774"* 2 1,878.3 939.1 15.471*** 18 2,000.7 111.2 1.831** 6 218.2 36.4 .600 6 69.9 11.6 .191 18 477.7 26.5 .437 64 3,883.6 60.7
calculated. As in experiment 1, no effect of varying levels of lysine or T.S.A.A. was detected on feed conversion. A split plot in space and time analysis (Steele and Torrie, 1960) was calculated for weight gain on a weekly basis for 3 weeks. This analysis is presented in Table 5 where the sex variable accounts for the split plot in space and the week variable the split plot in time. Average weight gains for the poults fed the 16 diet combinations (Table 6) indicates lower gains for the poults in experiment 2 than in experiment 1. The diets fed in experiment 2 contained less energy, less added fat and different ingredient composition as compared to those fed in experiment 1 (Table 1) which might account for part of the difference in gain. For weight gain, there was an overall effect of lysine (P < .01) and T.S.A.A. (P < .10) which was the same for both sexes. It is also apparent from Table 5 that the TABLE 6.—Average weight gains of poults in Experiment 2 (Grams) Lysine % Per Megacalorie Per Kg.
Total Sulfur Amino Acid Per Megacalorie Per Kg.
.250 .295 .341 .386
0.482
0.527
0.573 0.618
380.4 370.4 358.9 360.4
394.5 391.2 360.6 368.9
388.1 416.4 379.5 406.2
402.7 409.9 393.0 399.8
Y = 388.00342 + 10.00671XL - 18.70500XT - .36237XL2 - .51081X T 2 + 1.52180XLXT + .15545X L X T 2 + 1.60057XL2XT + .10537XL2 - .56008XL3 + 1.99442XT3 + .11724X L X T 3 - .20434X L 2 X T 3 - .17182Xr,3XT -
.01281XL3XT2 -
.00406XL3XT3
Y denotes the estimated weight gain, X L denotes the level of lysine and X T denotes the level of T.S.A.A. Using this estimated equation, contours of constant response were calculated for weight gain and are presented in Figure 2.
Downloaded from http://ps.oxfordjournals.org/ by guest on May 7, 2015
* Denotes significance at 10% level. ** Denotes significance at 5% level. ** Denotes significance at 1% level.
protein requirements for starting poults changed during the first 3 weeks. However, since 3 weeks is a common time for changing to a lower protein diet in commercial practice, weekly intervals were not considered in further analyses. A covariance analysis (Steele and Torrie, 1960) was then calculated using the initial weight of the bird as the covariate. Since the covariate, i.e., initial weight of the bird, was significant (P < .05), the observed weight gains for the three week period were adjusted to a common initial weight. A response surface analysis (Cochran and Cox, 1964) was then calculated for weight gain over time ignoring sex since no interactions of sex with amino acids was detected (Table 5). For experiment 1, a second order regression equation was estimated as the first step in the response surface analysis. Since in experiment 2 the ranges of both amino acids were greater than in experiment 1, it was necessary to estimate a third order regression equation. By coding the four levels of each amino acid successively from the lowest to the highest level, as —3, — 1 , 1 and 3, the estimated regression equations for weight gain is as follows:
LYSINE AND SULFUR AMINO ACID REQUIREMENTS
.30
.34
.39
TSAA (% PER MEGACALORIE PER.KG)
FIG. 2. Contours of constant growth response in grams for Experiment 2.
It is indicated in Figure 2 that the optimum weight gain of approximately 414 grams was achieved at a lysine level of 0.582% per megacalorie and 0.282% T.S.A.A. per megacalorie per kg. This is in very close agreement with the results of experiment 1. It is noted that at the highest T.S.A.A. level, there is an increasing response as the level of lysine is increased. There was an overall difference in body weight gain and feed conversion between the sexes with the females realizing less weight gain and better feed conversion. However, there was no interaction between sex and either amino acid indicating a common amino acid requirement by both sexes but on a different plane. SUMMARY
Two experiments were conducted to determine the lysine and total sulfur amino acid (T.S.A.A.) requirements of a commercial strain of large white turkey poults from 0-3 weeks of age. Natural feed ingredients were analyzed for amino acid content and diets were formulated based on these results. Five-hundred straight-run poults were fed 25 diets with 1
replication in experiment 1. Three-hundred and twenty sexed poults were fed 16 diets with 1 replication in experiment 2. The poults and feed were weighed weekly and body weight gain and feed conversion were calculated. A split plot in time was first calculated for both sets of data and then covariance analysis with initial body weight as the covariate. Response surface analysis was calculated for body weight gain in both experiments. The predicted optimum requirement of lysine was 0.564% per megacalorie per kg. in experiment 1 and 0.582% per megacalorie per kg. in experiment 2. The T.S.A.A. requirement was predicted to be 0.282% per megacalorie per kg. in both trials and for both sexes. ACKNOWLED GMENTS
The authors wish to acknowledge donations by Dow Chemical Company, Midland, Michigan, and Merck, Sharp & Dohme Co., Rahway, New Jersey, for DLMethionine and Lysine respectively, used in the diets. Appreciation is extended to Clemson Agricultural Chemical Services for the proximate analysis and to Dr. Lynn Crook of Clemson Food Science and Biochemistry for assistance in the amino acid analysis. REFERENCES Almquist, H. J., 1952. Amino acid requirements of chickens and turkeys—a review. Poultry Sci. 3 1 : 966-981. Association of Official Agriculture Chemists, 1965. Official Methods of Analysis, 10th ed. Washington, D.C. Boorman, K. N., and H. Fisher, 1966. The arginine-lysine interaction in the chick. Brit. Poultry Sci. 7: 39-44. Cochran, W. G., and G. M. Cox, 1964. Experimental Design, 2nd ed. John Wiley and Sons, Inc., New York. Gardiner, E. E., and E. W. Agudu, 1968. Effect of methionine on a growth depression induced by supplementing a corn-soybean meal diet with lysine. Poultry Sci. 47: 1631.
Downloaded from http://ps.oxfordjournals.org/ by guest on May 7, 2015
•25
757
758
V. E. KUMMERO, J. E. JONES AND C. B.
fonated polystyrene resins. Anal. Chem. 30(7) : 1185-1190. National Research Council, 1966. Nutrient requirements of domestic animals. 1. Nutrient requirements of poultry. Sth edition. National Academy of Sciences, National Research Council, Washington, D.C. Spackman, D. H., W. H. Stein and S. Moore, 1958. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30(7): 1190-1206. Steele, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc. New York. Van Soest, P. J., and R. H. Wine, 1967. Neutral detergent fiber. J. Assoc. Off. Agr. Chem. 50: 50-55.
Oxygen Uptake in the Shell Gland of Laying Hens G.
Central Institute
BEUVING
for Poultry Research "Het Spelderholt,"
Beekbergen,
The
Netherlands
(Received for publication October 13, 1970)
INTRODUCTION
MATERIALS AND METHODS
HE shell gland of laying hens has several functions. Beside the action of the muscles to move and expel the egg, it takes care of the secretion and possibly the synthesis of organic materials (shell matrix, the true cuticle and the ooporphyrins) and the secretion of inorganic materials for the plumping fluid and for the shell formation. The calcium content in the uterine fluids is higher than the calcium content of the blood (El Jack and Lake, 1967; Beadle et al., 1938), which suggests active transport of this ion. The oxygen uptake of the uterine tissue as determined by Brown and Badman (1962) and Misra and Kemeny (1964) was rather low for such an active tissue. Therefore, in this investigation the oxygen uptake in uterine tissue was studied again and was found much higher than those reported by the authors mentioned (Brown and Badman, 1962; Misra and Kemeny, 1964).
All the birds used in the experiments were White Leghorn laying hens between 7 and 11 months old. The hens were decapitated and the shell glands excised and freed of fatty tissues. The gland was divided into three parts as shown in Figure 1. Each part was thoroughly minced with a pair of scissors in a watch glass on ice. From each minced portion three 75-150 mg. samples were transferred to 2 ml. incubation medium. A larger volume of incubation medium did not further enhance the oxygen uptake. The incubation medium was as described by Robinson (1949) but with the phosphate buffer concentration decreased from 3 mM. to 1.3 mM. and with a lOmM. collidine-HCl buffer pH 7.4 added. The ionic strength was readjusted to 0.158 by adding 1.0 ml. water per 100 ml. incubation medium. The gas uptake was measured at 41 °C. in a Gilson respirometer type nr G 14 (constant pressure type). The vessels
T
Downloaded from http://ps.oxfordjournals.org/ by guest on May 7, 2015
Gartley, K. M., S. J. Slinger and D. C. Hill, 19S0. Further observations of the use of sunflower seed oil meal in turkey starter rations. Poultry Sci. 29: 312-313. German, H. L., B. S. Schweigert, R. M. Sherwood and L. E. James, 1949. Further evidence of the role of lysine in the formation of normal Bronze turkey feathers. Poultry Sci. 28: 165167. Grau, C. R., F. H. Kratzer and V. S. Asmundson, 1946. The lysine requirements of poults and chicks. Poultry Sci. 25: 529-530. Kratzer, F. H., D. E. Williams and B. Marshall, 1949. The sulfur amino acid requirements of turkey poults. J. Nutr. 37: 377-383. Loadholt, C. B., 1969. Personal communication. Moore, S., D. H. Spackman and W. H. Stein, 1958. Chromatrography of amino acids on sul-
LOADHOLT
T
752
Downloaded from http://ps.oxfordjournals.org/ by guest on May 7, 2015
HE lysine and total sulfur amino acid the lysine and T.S.A.A. requirements of requirements of turkey poults have growing turkey poults from one day to been discussed by a number of workers. three weeks of age. In view of the relationGrau et al. (1946) suggested a lysine re- ship of amino acid requirements to energy quirement of 1.3% of the diet at protein level of the diet, these requirements are exlevels of 20 and 24% when sesame meal pressed on the basis of a percent per megawas the protein source. German et al. calorie metabolizable energy per kilogram (1949) using feather pigmentation as a cri- (kg.). terion, suggested the requirement for lysine Two experiments were conducted to dewas 1.1 to 1.2% of the diet. Gartley et al. termine the levels of lysine and T.S.A.A. (1950) using sunflower seed meal as a di- necessary for optimum growth during the etary protein source, suggested the lysine first three weeks after hatching for the requirement was 1.2% of the diet from Nicholas strain of Large White turkeys. day-one to four weeks of age. Kratzer et al. (1949) used DL-methionine Experiment 1. A representative sample was as a dietary supplement to study the total taken from each of the raw feed ingredients sulfur amino acid requirements of poults. set aside for this experiment: soybean Their work indicated a need for 0.5% meal, Peruvian fish meal, and ground corn. methionine and 0.3% cystine for a total of Part of each sample was subjected to pro0.8% sulfur amino acids in the diet. Alm- tein and fat analysis by the method of quist (1952), in his review, listed the lysine A.O.A.C. (1965) and fiber analysis by the requirement for starting poults as 1.3% method of Van Soest and Wine (1967). of the diet, the methionine requirement Another part was analyzed for amino acid as 0.45% of the diet and the cystine re- content by the methods of Spackman et al. quirement as 0.3% of the diet, or the total (1958) and Moore et al. (1958). sulfur amino acid (T.S.A.A.) requirement From the selected raw feed ingredients, as 0.75% of the diet. The National Re- twenty-five diets were formulated using the search Council (N.R.C.) (1966) require- values resulting from the analyses. The ments for starting turkey poults for lysine diets were made up of all possible combinaand T.S.A.A. are respectively 1.5 and 0.87% tions of five levels each of lysine and (methionine 0.52% minimum) when the T.S.A.A. These diets had a final calculated diet contains 28% protein. protein range of from 28.38 to 28.32% and were isocaloric, containing 2,908 kilocaloThis study was undertaken to establish ries metabolizable energy per kilogram (kcal. M.E. per kg.). Lysine was added in Published with the approval of the Director of the Clemson Agricultural Experiment Station as increments of 0.034% from the basal level Technical Contribution No. 884. of 1.63% up to 1.76% of the diet and DLP r e s e n t Address: P. O. Box 5735, Athens, Ga. methionine in increments of 0.075% from 2 Present Address: Dept. of Biometry, Med. the basal level of 0.67% T.S.A.A. up to Univ. of S. C , Charleston, S. C.
753
LYSINE AND SULFUR AMINO ACID REQUIREMENTS
Experiment 2. Lots of yellow corn, 49% soybean meal, ground coastal Bermudagrass, sesame meal, and Peruvian fish meal were set aside; samples were taken and analyzed as previously described in experiment 1; and formulas were calculated from these analyses. The basal diet is shown in Table 1 with additional lysine and T.S.A.A. levels achieved by substituting 50% lysine and DL-methionine for sand. The sixteen diets were considered isocaloric at 2,858 kcal. M.E./kg. The diets were formulated with all possi-
TABLE 1.—Composition o} basal experimental diet Experiment 1
2
%
%
1.03 50.38 0.0 36.40 0.00 5.17 0.17 0.52 4.13 0.00 0.65 1.552
2.00 31.00 1.00 41.00 20.00 1.00 0.00 0.25 1.25 0.75 1.25 0.50'
100.00
100.00
Calculated analysis of diet, % Protein 28.38 Fat 9.26 Lysine 1.63 1 T.S.A.A. 0.67 Calcium 1.47 Phosphorus 0.90 M.E. (kcal./kg.) 2904
28.25 5.40 1.38 0.72 1.25 0.88 2858
Fish meal (Peruvian) Soybean meal (49% protein) Coastal Bermudagrass Ground yellow corn Sesame Meal Fat (Blended) DL-methionine Salt Defluorinated rock phosphate Limestone Sand Premix
1
T.S.A.A. =Total sulfur amino acid. Adds per kg. of finished diet: vit. A, 5159 I.U Vit. D 3 , 1549 I.U.; vit. B12, 3.1 meg.; Folic acid .90 mg.; Menadione, 2.0 mg.; Biotin, 4.4 meg. MnSO t , 199.76 mg., ZnO, 90 mg.; Choline chloride 1.89 gm.; Niacin, 66.11 mg.; vit. E, 22.68 I.U Riboflavin, 5.5 mg.; d-Pantothenic acid, 11.0 mg. 3 Same vitamin and mineral content/kg. as in 2 above except ZnO increased to 103 mg. 2
ble combinations of 4 levels of lysine and 4 levels of T.S.A.A. Lysine levels were 0.482%, 0.527%, 0.573% and 0.618% per megacalorie per kg. T.S.A.A. levels were 0.250%), 0.295%, 0.341%, and 0.386% per megacalorie per kg. Feed and water were fed ad libitum. Sexed, day-old, Large White turkey poults were randomly allotted into 32 groups of 5 males and 5 females each, then wing banded, weighed and placed in 32 pens of heated battery brooders. After 2 weeks, the heat was removed and the battery room was maintained at temperatures comparable to experiment 1. Body weight and feed consumption were measured weekly by pens. Body weight sains and feed conversions were calculated
Downloaded from http://ps.oxfordjournals.org/ by guest on May 7, 2015
0.97% of the diet (Table 1). Cystine was at a level of .15% of the diet. All diets were maintained isocaloric. Lysine and DLmethionine were substituted for sand. Replicate diets were obtained by splitting the initially prepared mixes. Feed and water were supplied ad libitum to each group of ten poults in electric brooders with raised wire floors. Twenty-five diets were randomly assigned to fifty pens. Five-hundred straight-run, day-old poults from a commercial strain of Large White turkeys were randomly selected, wing banded, individually weighed and assigned to pens. The heated area of the brooder was maintained at 36.7°C. for two weeks. The ambient room temperature was maintained at 26.1-26.7°C. throughout the duration of the experiment. At weekly intervals, feed consumption was determined and poults were individually weighed. The experiment was terminated after three weeks. Feed consumption, for a period in which a bird in a pen died, was calculated by computing the fraction (bird days attributable to the survivors of the period divided by the total bird days had all birds lived) and multiplying this by the observed feed consumption. This corrected feed consumption to exclude the feed eaten by birds that died during the weekly period.
754
V. E.
KUMMERO, J. E. JONES AND C. B. LOADHOLT
TABLE 2.—Split plot in time analysis, Experiment 1 Source A, A2 A1XA2 Error (a) Time TiXAi TiXA. T1XA1XA2 Error (b) Total
MS 4 4 16 25 2 8 8 32 50
325 1,497 3,402 3,345 501,190 245 526 2,243 3,205
81.2 374.3 212.6 133.8 250,594.9 30.6 65.8 70.1 64.1
.607 2.798* 1.589 3,909.498" .477 1.026 1.094
149
* Denotes significance at 5% level. ** Denotes significance at 1% level. Ai Denotes Lysine. A2 Denotes total sulfur amino acids. Time denotes one week.
Source
df
SS
T.S.A.A. Lineal Quadratic Cubic Quartic Lysine Linear Quadratic Cubic Quartic T.S.A.A. XLysine Error
4
4489 1697 1909 778 105 957 858 97 14
16 25
10,205 10,028
MS 1697 1909 778 105
4.231 4.759 1.940 .262
858 97 14
2.139 .242 .035
637. 81 401. 12
1.590
* Denotes significance at 5% level. ** Denotes significance at 15% level.
kg. A second order response function equation: Y = 437.395 - 2.679X, + 21.559X2 - 0.834X,2 - 3.700X22 + 1.585XiX2
was also derived where X x = the level of lysine, X 2 = the level of total sulfur amino acids and Y = the predicted weight gain. Solving this equation using the suggested optimums gave a maximum predicted RESULTS AND DISCUSSION weight gain of 470 grams. The highest obExperiment 1. The split plot in time analy- served weight in this experiment was 491.5 sis (Table 2) showed that the levels of grams (Table 4). Figure 1 is a graphic repT.S.A.A. significantly affected weight gain resentation of the prediction formula. The (P < 0.0S). There was no interaction be- curves are contours of constant response for tween amino acid levels and time. Orthogo- weight gains. For any predicted weight gain nal polynomials were calculated to deter- in the area covered by the curves, a determine the nature of the response for the en- mination can be made of levels of lysine and tire three-week period (Table 3). This T.S.A.A. necessary to produce that preanalysis demonstrated both a linear and a dicted response. If visualized as a contour quadratic relationship between the levels of map, the curves of the graph will represent T.S.A.A. and weight gains at the 5% level levels of predicted weight gains. In interof significance. This analysis also suggested preting the closeness of the curves to mean that the lysine content of the diet affected a rather steep depression in rate of weight weight gains at the 15% level of signifi- gain and the large central area as a plateau cance. A second order response surface of constant weight gain rates, it was conanalysis was used to determine the opti- cluded that the levels of dietary lysine and mum combinations of lysine and total sul- T.S.A.A. within the area represented by the fur amino acids. This analysis suggested central plateau were optimum for maximum optimum values for lysine as 0.564% and weight gains. for T.S.A.A. as 0.282% per megacalorie per The variables in this experiment were
Downloaded from http://ps.oxfordjournals.org/ by guest on May 7, 2015
on a weekly basis for the 3-week trial period. When a poult died, the feed and poults of the pen were weighed immediately and average weight gain and feed conversion were calculated on a poult day basis. The data were analyzed by using a split plot in time analysis, orthogonal polynomials (Steele and Torrie, 1960) and response surface analysis (Cochran and Cox, 1964). Time was designated by three "weekly intervals.
TABLE 3.—Orthogonal polynomials. Experiment 1
LYSINE AND SULFUR AMINO ACID REQUIREMENTS
755
TABLE 4.—Average weight gains of poults in Experiment 1 (Grams) Lysine % Per Megacalorie Per Kg.
Total Sulfur Amino Acid % Per Megacalorie % Per Kg.
0.232 0.259 0.282 0.309 0.336
Treatment Means
0.SS9
0.573
0.582
0.595
0.609
450.6 476.1 463.6 472.4 448.4
427.9 491.5 481.0 460.5 456.1
445.3 449.7 470.9 468.0 463.9
423.0 465.9 469.6 432.5 489.1
455.5 427.1 456.0 462.4 455.2
462.2
463.4
459.5
456.0
451.2
amino acid imbalance was seen at the higher levels of lysine in Trial 1. As the lysine content was increased past the second lowest level, a trend toward depressed weight gains was noted. Gardiner and Agudu (1968) found that 0.35% added methionine reversed the depression of weight gains caused by 0.15% lysine added to the diet. This finding helps to explain why a relatively large depression in weight gains was not noted at the highest levels of lysine studied. Experiment 2. Feed conversion was based on pen data eliminating the sex variable. Therefore, a split plot in time analysis was
/ / \^-^Z \
g
.60 •
/
/ / ^ ^ \ .
8 58' III/
5 -
46s
\ \
\
»
•" n ////i
4 7 0
s H I III .23 TSAA
.25
.27
/////
{//III .30
.33
(% PER MEGACALORIE PER KG)
FIG. 1. Contours of constant growth response in grams for Experiment 1.
Downloaded from http://ps.oxfordjournals.org/ by guest on May 7, 2015
classified as quantitative and continuous. Heretofore, with this type of data, the least significant difference test would have been used to determine the optimum among treatments tested. However, since a response surface analysis is a valid test of continuous data (Loadholt, 1969), this analysis was employed to determine the combination of the variables which would produce an optimum response in weight gain. A response surface analysis should be used only after determining if there were significant effects for the data collected. The differences among weight gains attributable to the levels of T.S.A.A. in the diets were significant at the 5% level of probability. Data of Table 4 shows the treatment means and the average weight gains for the twenty-five diet combinations. The treatment means for T.S.A.A. suggested the existence of a curve with a definite peak response. The treatment means for lysine suggested the existence of a linear relation ship between dietary lysine content and growth rate. These results may have several interpretations. The range of T.S.A.A. chosen encompassed the desired levels for maximum growth. The levels of lysine existing in a practical corn-soy type diet are at or near the level which will produce maximum growth of turkey poults. Imbalances of both lysine and total sulfur amino acids depress weight gains (Boorman and Fisher, 1966). The most dramatic effect of this
440.4 462.1 468.2 459.1 462.5
756
V. E. KUMMERO, J. E. JONES AND C. B. LOADHOLT TABLE 5.—Analysis of weight gain Source
Lysine T.S.A.A. LXT.S.A.A. Error (a) Sex SXL SXT.S.A.A. SXLXT.S.A.A. Error (b) Weeks LXW T.S.A.A. X W SXW LXT.S.A.A. X W LXSXW T.S.A.A. X S X W LXT.S.A.A. X S X W Error (c)
df
SS
F
MS
3 1,355.2 4,065.6 6.699"* 3 1,727.8 575.9 2.847* 9 1,100.9 122.3 .605 16 3,236.2 202.3 1 6,836.7 6,836.7 56.455*** 3 816.8 2.249 272.3 3 168.2 56.1 .463 9 996.9 110.8 .915 16 1,937.0 121.1 2 589,933.5 294,966.8 4859.4*** 6 88.4 2.247* 136.4 6 1,374.8 229.1 3.774"* 2 1,878.3 939.1 15.471*** 18 2,000.7 111.2 1.831** 6 218.2 36.4 .600 6 69.9 11.6 .191 18 477.7 26.5 .437 64 3,883.6 60.7
calculated. As in experiment 1, no effect of varying levels of lysine or T.S.A.A. was detected on feed conversion. A split plot in space and time analysis (Steele and Torrie, 1960) was calculated for weight gain on a weekly basis for 3 weeks. This analysis is presented in Table 5 where the sex variable accounts for the split plot in space and the week variable the split plot in time. Average weight gains for the poults fed the 16 diet combinations (Table 6) indicates lower gains for the poults in experiment 2 than in experiment 1. The diets fed in experiment 2 contained less energy, less added fat and different ingredient composition as compared to those fed in experiment 1 (Table 1) which might account for part of the difference in gain. For weight gain, there was an overall effect of lysine (P < .01) and T.S.A.A. (P < .10) which was the same for both sexes. It is also apparent from Table 5 that the TABLE 6.—Average weight gains of poults in Experiment 2 (Grams) Lysine % Per Megacalorie Per Kg.
Total Sulfur Amino Acid Per Megacalorie Per Kg.
.250 .295 .341 .386
0.482
0.527
0.573 0.618
380.4 370.4 358.9 360.4
394.5 391.2 360.6 368.9
388.1 416.4 379.5 406.2
402.7 409.9 393.0 399.8
Y = 388.00342 + 10.00671XL - 18.70500XT - .36237XL2 - .51081X T 2 + 1.52180XLXT + .15545X L X T 2 + 1.60057XL2XT + .10537XL2 - .56008XL3 + 1.99442XT3 + .11724X L X T 3 - .20434X L 2 X T 3 - .17182Xr,3XT -
.01281XL3XT2 -
.00406XL3XT3
Y denotes the estimated weight gain, X L denotes the level of lysine and X T denotes the level of T.S.A.A. Using this estimated equation, contours of constant response were calculated for weight gain and are presented in Figure 2.
Downloaded from http://ps.oxfordjournals.org/ by guest on May 7, 2015
* Denotes significance at 10% level. ** Denotes significance at 5% level. ** Denotes significance at 1% level.
protein requirements for starting poults changed during the first 3 weeks. However, since 3 weeks is a common time for changing to a lower protein diet in commercial practice, weekly intervals were not considered in further analyses. A covariance analysis (Steele and Torrie, 1960) was then calculated using the initial weight of the bird as the covariate. Since the covariate, i.e., initial weight of the bird, was significant (P < .05), the observed weight gains for the three week period were adjusted to a common initial weight. A response surface analysis (Cochran and Cox, 1964) was then calculated for weight gain over time ignoring sex since no interactions of sex with amino acids was detected (Table 5). For experiment 1, a second order regression equation was estimated as the first step in the response surface analysis. Since in experiment 2 the ranges of both amino acids were greater than in experiment 1, it was necessary to estimate a third order regression equation. By coding the four levels of each amino acid successively from the lowest to the highest level, as —3, — 1 , 1 and 3, the estimated regression equations for weight gain is as follows:
LYSINE AND SULFUR AMINO ACID REQUIREMENTS
.30
.34
.39
TSAA (% PER MEGACALORIE PER.KG)
FIG. 2. Contours of constant growth response in grams for Experiment 2.
It is indicated in Figure 2 that the optimum weight gain of approximately 414 grams was achieved at a lysine level of 0.582% per megacalorie and 0.282% T.S.A.A. per megacalorie per kg. This is in very close agreement with the results of experiment 1. It is noted that at the highest T.S.A.A. level, there is an increasing response as the level of lysine is increased. There was an overall difference in body weight gain and feed conversion between the sexes with the females realizing less weight gain and better feed conversion. However, there was no interaction between sex and either amino acid indicating a common amino acid requirement by both sexes but on a different plane. SUMMARY
Two experiments were conducted to determine the lysine and total sulfur amino acid (T.S.A.A.) requirements of a commercial strain of large white turkey poults from 0-3 weeks of age. Natural feed ingredients were analyzed for amino acid content and diets were formulated based on these results. Five-hundred straight-run poults were fed 25 diets with 1
replication in experiment 1. Three-hundred and twenty sexed poults were fed 16 diets with 1 replication in experiment 2. The poults and feed were weighed weekly and body weight gain and feed conversion were calculated. A split plot in time was first calculated for both sets of data and then covariance analysis with initial body weight as the covariate. Response surface analysis was calculated for body weight gain in both experiments. The predicted optimum requirement of lysine was 0.564% per megacalorie per kg. in experiment 1 and 0.582% per megacalorie per kg. in experiment 2. The T.S.A.A. requirement was predicted to be 0.282% per megacalorie per kg. in both trials and for both sexes. ACKNOWLED GMENTS
The authors wish to acknowledge donations by Dow Chemical Company, Midland, Michigan, and Merck, Sharp & Dohme Co., Rahway, New Jersey, for DLMethionine and Lysine respectively, used in the diets. Appreciation is extended to Clemson Agricultural Chemical Services for the proximate analysis and to Dr. Lynn Crook of Clemson Food Science and Biochemistry for assistance in the amino acid analysis. REFERENCES Almquist, H. J., 1952. Amino acid requirements of chickens and turkeys—a review. Poultry Sci. 3 1 : 966-981. Association of Official Agriculture Chemists, 1965. Official Methods of Analysis, 10th ed. Washington, D.C. Boorman, K. N., and H. Fisher, 1966. The arginine-lysine interaction in the chick. Brit. Poultry Sci. 7: 39-44. Cochran, W. G., and G. M. Cox, 1964. Experimental Design, 2nd ed. John Wiley and Sons, Inc., New York. Gardiner, E. E., and E. W. Agudu, 1968. Effect of methionine on a growth depression induced by supplementing a corn-soybean meal diet with lysine. Poultry Sci. 47: 1631.
Downloaded from http://ps.oxfordjournals.org/ by guest on May 7, 2015
•25
757
758
V. E. KUMMERO, J. E. JONES AND C. B.
fonated polystyrene resins. Anal. Chem. 30(7) : 1185-1190. National Research Council, 1966. Nutrient requirements of domestic animals. 1. Nutrient requirements of poultry. Sth edition. National Academy of Sciences, National Research Council, Washington, D.C. Spackman, D. H., W. H. Stein and S. Moore, 1958. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30(7): 1190-1206. Steele, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc. New York. Van Soest, P. J., and R. H. Wine, 1967. Neutral detergent fiber. J. Assoc. Off. Agr. Chem. 50: 50-55.
Oxygen Uptake in the Shell Gland of Laying Hens G.
Central Institute
BEUVING
for Poultry Research "Het Spelderholt,"
Beekbergen,
The
Netherlands
(Received for publication October 13, 1970)
INTRODUCTION
MATERIALS AND METHODS
HE shell gland of laying hens has several functions. Beside the action of the muscles to move and expel the egg, it takes care of the secretion and possibly the synthesis of organic materials (shell matrix, the true cuticle and the ooporphyrins) and the secretion of inorganic materials for the plumping fluid and for the shell formation. The calcium content in the uterine fluids is higher than the calcium content of the blood (El Jack and Lake, 1967; Beadle et al., 1938), which suggests active transport of this ion. The oxygen uptake of the uterine tissue as determined by Brown and Badman (1962) and Misra and Kemeny (1964) was rather low for such an active tissue. Therefore, in this investigation the oxygen uptake in uterine tissue was studied again and was found much higher than those reported by the authors mentioned (Brown and Badman, 1962; Misra and Kemeny, 1964).
All the birds used in the experiments were White Leghorn laying hens between 7 and 11 months old. The hens were decapitated and the shell glands excised and freed of fatty tissues. The gland was divided into three parts as shown in Figure 1. Each part was thoroughly minced with a pair of scissors in a watch glass on ice. From each minced portion three 75-150 mg. samples were transferred to 2 ml. incubation medium. A larger volume of incubation medium did not further enhance the oxygen uptake. The incubation medium was as described by Robinson (1949) but with the phosphate buffer concentration decreased from 3 mM. to 1.3 mM. and with a lOmM. collidine-HCl buffer pH 7.4 added. The ionic strength was readjusted to 0.158 by adding 1.0 ml. water per 100 ml. incubation medium. The gas uptake was measured at 41 °C. in a Gilson respirometer type nr G 14 (constant pressure type). The vessels
T
Downloaded from http://ps.oxfordjournals.org/ by guest on May 7, 2015
Gartley, K. M., S. J. Slinger and D. C. Hill, 19S0. Further observations of the use of sunflower seed oil meal in turkey starter rations. Poultry Sci. 29: 312-313. German, H. L., B. S. Schweigert, R. M. Sherwood and L. E. James, 1949. Further evidence of the role of lysine in the formation of normal Bronze turkey feathers. Poultry Sci. 28: 165167. Grau, C. R., F. H. Kratzer and V. S. Asmundson, 1946. The lysine requirements of poults and chicks. Poultry Sci. 25: 529-530. Kratzer, F. H., D. E. Williams and B. Marshall, 1949. The sulfur amino acid requirements of turkey poults. J. Nutr. 37: 377-383. Loadholt, C. B., 1969. Personal communication. Moore, S., D. H. Spackman and W. H. Stein, 1958. Chromatrography of amino acids on sul-
LOADHOLT