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The Effect of Leucine on Egg Production and Hatchability*
T h e Effect of Leucine on Egg Production and Hatchability* W.W.
CRAVENS
Poultry Department, University of Wisconsin, Madison, Wisconsin (Received for publication February 20, 1948)
D
URING the course of studies designed to determine the effect of choline on egg production and hatchability, a purified ration was formulated which was inadequate for laying hens. The ration was designed to be low in choline and methionine. However, the addition of these two factors failed to complete the ration for laying hens. The ration used was identical in composition to ration B21 (Table 1) except that it contained no choline and corn oil was used in place of soybean oil. Inasmuch as choline or methionine alone or in combination were ineffective supplements, experiments were undertaken to determine what other deficiencies existed in the low choline ration.
batteries and given feed and water ad libitum. They were inseminated weekly with pooled semen collected from a group of New Hampshire cockerels. All eggs were pedigree marked, weighed and incubated at weekly intervals. Records of egg production and feed consumption were kept. All egg production data will be expressed on a percentage basis. All birds were weighed weekly, at which time observations on molting were made since in preliminary experiments molting was observed. The composition of the diets used in these studies is given below: TABLE 1.—Composition of rations
The studies to be reported herein demonstrate that leucine is an important amino acid for laying hens and further that either or both tryptophane and methionine are essential. A preliminary report on part of this work was given by Cravens and Halpin (1946).
dextrin casein (ale. ext'd.) gelatin salts IV soybean oil liver fraction L fish oil (400D-2,000A) chlorine chloride % riboflavin thiamin HC1 calcium pantothenate nicotinic acid pyridoxin HC1 biotin a tocopherol 2-methyl 1-4 napthoquinone
EXPERIMENTAL AND RESULTS
Single Comb White Leghorn pullets were used in all experiments. The birds were housed in individual cage laying
oyster shell * Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from The Borden Company, New York, N. Y. We are indebted to Merck & Company, Rahway for the crystalline vitamins and to The Wilson Laboratories, Chicago, Illinois, for the Liver fraction L.
B20
B 21
64 18 S 5 3 3 2 0.2 6 4 15 100 4 0.2 3 0.5
71 9 7 5 3 3 2 0.2 6 4 15 100 4 0.2 3 0.5
Ad Libitum
All vitamins, except choline, added as indicated in milligrams per kilogram of ration.
Experiment 1.—Four groups of three birds each which had been receiving the choline low basal diet supplemented with 562
EFFECT OF LEUCINE ON REPRODUCTION
choline, methionine and cystine or some combination of these were redistributed into two groups of six birds each. Since rate of egg production was at a very low level, it was decided to feed group 1 diet B21 which was identical to the low choline diet except for the modifications pointed out previously. Group 2 was fed diet B20 which had proven satisfactory in previous experiments on the effect of various members of the vitamin B complex on egg production and hatchability.
>n
563
1-5) shows that egg production for the two groups did not differ significantly at the start of the experiment. However, there was a rapid increase in rate of egg production of the birds of group 2 which were fed ration B20. The rate increased from approximately 30 percent to about 75 percent in 4 weeks. Group 1 which was fed ration B21 continued to lay at about the same rate as during the pre-experimental period. The reversal of the rations fed the 2
B20- casein l g ; gelatin 5 B21- casein 9; gelatin 7
so 70 6o 50 to 30 20
Group 1
Group 2
' Rations reversed
10
2
3
H
5
6
7 Week
8
5
10
11
12
1}
14
15
FIG. 1. Production of groups fed rations B 2 0 and B 2 1 .
This experiment was designed to obtain information on two points: (1) the time required for the laying bird to respond to a "complete" ration after being fed the "deficient" ration, or the time necessary for a measurable response after shifting from the complete to the deficient ration, (2) to gain evidence that ration B21 is incomplete insofar as protein is concerned. The results of experiment 1 are given in Figure 1. The pre-experimental period (weeks
groups after 5 weeks shows definitely that ration B21 is deficient in some factor or factors essential for the laying hen. Egg production of group 2 which was shifted to ration B21 dropped from approximately 75 percent to 30 percent in four weeks. On the other hand, egg production of group 1 which was shifted to ration B20 increased from about 30 percent to 80 percent in 5 weeks. The results of this experiment indicate that a measurable response could be ob-
564
W. W. CRAVENS
tained in from 3-5 weeks and further that we were dealing with a deficiency related to the protein makeup of the ration. While this study was in progress Krehl, Teply, Sarma and Elvehjem (1945) reported the growth inhibition of rats as a result of feeding large amounts of corn grits and the counteraction of this effect by adding either tryptophane or nicotinic acid. Later these same workers Krehl, Teply, Sarma and Elvehjem (1946) demonstrated that similar results were obtained with diets containing a protein supply very similar to that of diet B21. Although diet B21 contained 100 mg. of nicotinic acid per kg., it seemed advisable to test the effect of tryptophane additions. Experiment 2 was designed to test this amino acid. Experiment 2.—Eighteen pullets were equally divided into 6 groups. It was decided to conduct a curative trial and the ration changes were made after a 3 weeks "depletion" period. The experimental plan and egg production data are presented in Table 2. The test period was of 8 weeks duration. TABLE 2.—Effect
of tryptophane and other modi-
fications of ration B21 on egg production Experiment 2 Egg Ration "deple- production tion" perperiod cent B20 B20 B21 B21 B21
69.8 74.6 52.4 52.4 46.0
B21
34.9
Ration test period B20 B21 B21 B20 B21 less gelatin B21+0.5% DL
Egg production percent
tryptophane
52.4 42.9 16.7 54.8 16.7 8.9
The results of experiment 2 given Table 2 indicate that the addition tryptophane to ration B21 was of value in promoting egg production.
in of no It
may appear that egg production was sustained at a moderate rate when ration B21 was fed during the "depletion" period. However it should be pointed out that the average production for the period does not show the rapid drop in production which actually occurred. For groups 3, 4, 5 and 6 during the third week of the "depletion" period percent production was 33.8, 28.6, 23.8 and 9.5 respectively. The low rate of production for birds fed ration B21 for an extended period is reflected in the production rate of group 3 during the test period. The data are not presented graphically since negative results were obtained with tryptophane. Experiment 3.—-Inasmuch as some of the birds exhibited pronounced molting during experiment 2, it was thought that perhaps a prophylatic trial might better test the value of supplements to ration B21. Thus an experiment to further test tryptophane was conducted. In addition to tryptophane, methionine and cystine additions to ration B21 were made since this ration had been designed to be low in these amino acids. Additional casein was also added to the diet in order to determine if we could gain further evidence that we were dealing with an amino acid deficiency. Eighteen birds were divided equally among 6 groups. This experiment was of 6 weeks duration. The experimental plan and egg production data are given in Table 3. The results of this experiment (Table 3) show that some deficiency other than possible deficiencies of tryptophane and/or the sulfur containing amino acids exist in ration B21. That the deficiency is an amino acid is suggested by the results obtained by adding 4 percent additional casein (group 5). Egg production of this group was 64.8 percent while that for the group receiving B21 was 34.3 percent
E F F E C T O F L E U C I N E ON R E P R O D U C T I O N
565
able forms of amino acids were used. T h e forms and levels used in grams percent were: DL methionine 0.20, L arginine monohydrochloride 0.2, glycine 0.025, L lysine monohydrochloride. H2O 0.33, DL t r y p t o p h a n e 0.10, DL threonine 0.20, DL
(group 1) a n d the control (group 6) was 77.8 percent. Since essentially negative results h a d been obtained thus far in elucidating the specific deficiency or deficiencies of ration B21 i t was decided t o supplement the PERCENT PRODUCTION
B21 / 5% CASEIN
B21 / U AMINO ACIDS
2
3
U
WEEK FIG. 2. Effect of rations on egg production.
diet with the essential amino acids that would be supplied by 5 percent casein. Experiment 4 was designed to test this treatment. Experiment 4.—Sixteen pullets were divided into equal groups and fed the rations as follows: Group 1—B20; 2— B21; 3—B21+5 percent purified casein; 4—B21+essential amino acids equivalent to 5 percent casein. Commercially avail-
valine 0.35, L histidine monohydrochloride H 2 0 0.15, L leucine 0.65, DL isoleucine 0.30, DL phenylalanine 0.25. The experiment was terminated at four weeks. The results of experiment 4 are given in Figure 2. The results of experiment 4 shown in figure 2 indicate that ration B21 is deficient in one or more essential amino acids for laying hens. The addition of the
566
W. W. CRAVENS
11 essential amino acids in the quantities that would be supplied by 5 percent casein was effective in maintaining egg production. It may be observed, however that the group receiving the amino acids additions showed a gradual decline in rate of egg production. When compared with the group receiving B21 alone, it is obvious that the amino acid supplemented group TABLE 3.—Effect of supplements to ration B21 on egg production Experiment 3
group 4 and thus the results are based on only 3 birds.) Group 1. 2. 3. 4.
B 21 B 20 B 21+0.65% L leucine B 21+0.65% L leucine+ 0.1% DL tryptophane+0.2% DL methionine 5. B 2 1 + 5 % purified casein
Tryptophane and methionine were added for reasons stated earlier in this paper. Ration B21 was suspected of being Egg low in leucine because of comparisons Group Ration and supplement production made of its calculated amino acid makeup percent with that of certain simplified laying 1 B21 34.3 rations composed of crude ingredients and 2 B21+0.5%DL tryptophane 31.5 which had proven satisfactory for laying hens. 3 B21+0.5%DL tryptophane 38.1 0.3% DL methionine The results of experiment 5 are given in 0.4% L cystine Figure 3. 4 B21+0.3%DL methionine 47.2 The data in Figure 3 show that the addition of leucine alone was ineffective in 0.4% L cystine supplementing ration B21 for laying hens. 5 B21+4% purified casein 64.8 The addition of leucine, methionine and 6 B20 77.8 tryptophane to ration B21 resulted in egg produced at a higher rate. The group of production comparable to the control birds receiving B21 alone entirely ceased ration B20 or to ration B21 supplemented to lay during the second week of the ex- with 5 percent casein. Thus it is indicated periment and produced at a very low rate that diet B21 is deficient in leucine. It is for the balance of the experiment. Weekly impossible to determine from this experivariations in rate of production are quite ment whether tryptophane or methionine marked due to the small numbers of birds or both are deficient in ration B21. All possible combinations of these amino involved. The results of this experiment demon- acids were not tested because of the exstrated clearly that an amino acid de- pense involved. Methionine and tryptoficiency was responsible for the poor phane had proven ineffective in experiproduction which resulted when ration ment 3 and thus this treatment was not B21 was fed to laying hens. The problem repeated. The data indicate, however, thus became one of determining which of that one or both of these amino acids is the 11 essential amino acids was deficient. limiting. Experiment 5 was designed to obtain Hatchability Results.—Due to the small evidence on this point. number of eggs produced by the pullets Experiment 5.—Twenty pullets were fed ration B 21, composite hatchability data equally divided into 5 groups and fed the from all experiments are presented. The rations listed below: (one bird died in data for all eggs produced by the birds
EFFECT OF LEUCINE ON REPRODUCTION
TABLE 4.—Effect of an amino acid deficiency on hatchability
Egg set Eggs fertile No. chicks Percent fertile Percent hatchability
Ration B20
Ration B21
521 457 378 87.7 82.7
329 292 236 88.8 80.8
receiving rations B20 and B21 for experiments 2-5 are given in Table 4. The data in Table 4 indicate that the amino acid deficiencies of ration B21 did not affect hatchability. The percent hatchability of eggs from the birds fed the control ration (B20) was 82.7 while that
567
of the eggs from the amino acid deficient group was 80.8. Effect of an amino acid deficiency on the
egg laying cycle.—In order to present an accurate picture of the effect of an amino acid deficiency of the hen on the egg production cycle, the complete daily egg production record of all birds of experiment 5 are given in Figure 4. In addition, observations on molting of the birds are listed on the egg record sheet. The birds were frequently handled and observations recorded as to the laying condition of the bird. The following system was followed in recording information on molting and laying condition:
PERCENT PRODUCTION
B21 / 5* CASEIN B21 / B20
'""'"-
/'LEUCINE < TRYPTOPHANE ^METHIONINE
B21 / LEUCINE
2
3
4
I
WEEK FIG. 3. Effect of rations on egg production.
568
W. W. CRAVENS
(1) obvious pin feathers on neck and perhaps body (2) molting heavily (3) laying condition as judged by vent and pubic bone spread (4) not in laying condition It is obvious from Figure 4 that the amino acid deficiency of ration B21 caused
the diets used contained adequate amino acids for maintenance but an insufficient amount for the additional strain of egg production. The birds in group 3, receiving leucine, continued to lay but at a much reduced rate. One bird of this group showed evidence of a neck molt. To obtain evidence as to why the birds
FIG. 4. Effect of an amino acid deficiency on the egg laying cycle. (Experiment 5.)
a marked reduction in the egg laying cycle. It is of interest however that 2 of the 4 birds receiving the deficient ration continued to lay an occasional egg. It thus appears that the rate of production can be influenced by an amino acid deficiency without complete cessation of laying. Three of the birds of group 1 were showing some degree of molting at the end of the experiment. Occasionally a bird fed the deficient ration would go into a complete body molt and lose weight. No marked weight changes were observed which were unattended by heavy molting. Apparently
continued to lay at a reduced rate, or why pauses were so common among birds fed ration B21, two birds from each group were killed at the conclusion of the experiment and the ovaries examined. Observations were made on the length and weight of the oviduct and the number of ova falling within 5 mm. size classes. The ova were measured parallel to the suture line with a vernier caliper. These data are given in Table 5. The data in Table 5 show that the birds in group 2-5 contained ova in nearly every size class. It is of considerable inter-
EFFECT OF LEUCINE ON REPRODUCTION
est that abnormal ova were observed in the birds in groups 1, 3 and 4. These ova appeared to have developed to a certain size, then cease to grow and degenerate or be reabsorbed. The ova appeared irregular in outline and flabby rather than round and turgid. It may be seen that several were observed in the birds of group 1 and one of the birds of group 3. Such "resorp-
569
sumption may or may not be valid. In fact one is led to theorize that it would not be valid in view of the rather high content in egg protein of certain amino acids. Further the present study indicates that the laying hen has a leucine requirement (1.35-2.0 percent) not less than that reported by Almquist (1947) as being required by the growing chick.
TABLE 5.—Eject of an amino acid deficiency on development of ova
Group
1 2 3 4 5
Hen No.
25 28 30 31 33 34 37 39 41 45
No. of ova by 5 mm. classes
Oviduct Layingf condition Length Weight
6-10
11-15
16-20
21-25
cm. 21 23 24 24 25 32 24 29 27 29
9* 10* 3 5 7* 4 6 5 11 6
1 1 1 1
2*
2* 3*
— +?
+ + _? + + + + +
gms. 28 26 40 39 26 62 43 40 46 51
26-30
Egg in 31-35 Oviduct
1
1 1
1 1 1 1
1 1 1 1 2
3* 1 1 1 1
1* 1*
— — —
+ — + + + + +
* One or more ova showing evidence of "resorption." Size of such ova only aproximate. t As judged by handling.
tion" of ova might explain the erratic production of the birds in these groups. DISCUSSION OF RESULTS The data presented in this paper show that a deficiency of leucine results in an abrupt cessation of egg production. The data do not show which of the other two amino acids, tryptophane or methionine, are deficient in the basal diet used. A response to tryptophane or methionine, alone or in combination was not obtained, probably because of the leucine deficiency. By calculation one might predict that methionine is limiting but such calculations would be based on the supposition that the amino acid needs of the laying hen are the same as those of the growing chick, taking into account the difference in total protein requirements. Such an as-
The data presented in Table 4 indicate that a deficiency of leucine and either or both tryptophane and methionine does not affect hatchability. Rather the data show that the hen ceases laying. Such data suggest that the amino acid makeup of the egg is not influenced by the diet fed the hen or that the change in egg composition is insufficient to result in failure of embryonic development. Recently Csonka, Denton and Ringel (1947) have shown that the quantity of the sulfur containing amino acids in eggs may be modified by dietary means. Earlier work by McFarlane, Fulmer and Jukes (1930) had indicated no changes in the amino acid content of eggs due to dietary modifications while Gerber and Carr (1930) presented data indicating that egg protein might be' modified by the diet fed the bird. Unfor-
570
W. W. CRAVENS
tunately no amino acid analyses of eggs were made in the present investigation. The egg laying cycle of the amino acid deficient birds are of particular interest. From Figure 4 it may be seen that some hens will apparently stay in laying condition as determined by external appearances and even produce an occasional egg. Thus the egg laying cycle may definitely be influenced by the amino acid makeup of the diet and in cases of an apparent borderline deficiency egg production with a marked reduction in intensity or rate will occur. By correlating the egg laying cycle with the development of ova in the ovary one is led to suggest that an amino acid deficiency may lead to a resorption of yolk material from certain ova, whereas others continue to develop until normal ovulation occurs. The possibility must not be overlooked, however, that the rate of growth of ova is actually modified by the amino acid deficient diets. The tendency to pause was observed in all birds fed diet B21 but considerable variation was observed, with some birds ceasing to lay and starting a general molt. SUMMARY
Experiments are described which indicate that leucine is an important amino acid for laying hens. The requirement for the laying bird appears to be not less than
1.35 percent of the ration. The data also indicate that either tryptophane or methionine or both are essential for the laying hen. Data are presented which show the effect of an amino acid deficiency on intensity of egg production and on development of egg yolks in the ovary of the hen. REFERENCES
Almquist, H. J., 1947. Evaluation of amino acid requirements by observations on the chick. J. Nutrition 34: 543-563. Cravens, W. W. and J. G. Halpin, 1946. Studies on the protein nutrition of breeding hens. Poultry Sci. 25: 400. Csonka, F. A., C. A. Denton and S. J. Ringel, 1947. The methionine and cystine content of hen's eggs. J. Biol. Chem. 169: 259-265. Gerber, L. and R. H. Carr, 1930. A chemical and immunological study of egg protein obtained under restricted diets. J. Nutrition 3: 245-256. Krehl, W. A., L. J. Teply, P. S. Sanaa and C. A. Elvehjem, 1945. Growth retarding effect of corn in nicotinic acid-low rations and its counteraction by tryptophane. Science 101: 489-490. Krehl, W. A., P. S. Sarma, L. J. Teply and C. A. Elvehjem, 1946. Factors affecting the dietary niacin and tryptophan requirement of the growing rat. J. Nutrition 31: 85-106. McFarlane, W. D., H. L. Fulmer and T. H. Jukes, 1930. Studies in embryonic mortality in the chick I. The effect of diet upon the nitrogen, amino-nitrogen, tyrosine, tryptophan, cystine and iron content of the proteins and on the total copper of the hen's egg. Biochem. J. 24: 16111631.
CRAVENS
Poultry Department, University of Wisconsin, Madison, Wisconsin (Received for publication February 20, 1948)
D
URING the course of studies designed to determine the effect of choline on egg production and hatchability, a purified ration was formulated which was inadequate for laying hens. The ration was designed to be low in choline and methionine. However, the addition of these two factors failed to complete the ration for laying hens. The ration used was identical in composition to ration B21 (Table 1) except that it contained no choline and corn oil was used in place of soybean oil. Inasmuch as choline or methionine alone or in combination were ineffective supplements, experiments were undertaken to determine what other deficiencies existed in the low choline ration.
batteries and given feed and water ad libitum. They were inseminated weekly with pooled semen collected from a group of New Hampshire cockerels. All eggs were pedigree marked, weighed and incubated at weekly intervals. Records of egg production and feed consumption were kept. All egg production data will be expressed on a percentage basis. All birds were weighed weekly, at which time observations on molting were made since in preliminary experiments molting was observed. The composition of the diets used in these studies is given below: TABLE 1.—Composition of rations
The studies to be reported herein demonstrate that leucine is an important amino acid for laying hens and further that either or both tryptophane and methionine are essential. A preliminary report on part of this work was given by Cravens and Halpin (1946).
dextrin casein (ale. ext'd.) gelatin salts IV soybean oil liver fraction L fish oil (400D-2,000A) chlorine chloride % riboflavin thiamin HC1 calcium pantothenate nicotinic acid pyridoxin HC1 biotin a tocopherol 2-methyl 1-4 napthoquinone
EXPERIMENTAL AND RESULTS
Single Comb White Leghorn pullets were used in all experiments. The birds were housed in individual cage laying
oyster shell * Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from The Borden Company, New York, N. Y. We are indebted to Merck & Company, Rahway for the crystalline vitamins and to The Wilson Laboratories, Chicago, Illinois, for the Liver fraction L.
B20
B 21
64 18 S 5 3 3 2 0.2 6 4 15 100 4 0.2 3 0.5
71 9 7 5 3 3 2 0.2 6 4 15 100 4 0.2 3 0.5
Ad Libitum
All vitamins, except choline, added as indicated in milligrams per kilogram of ration.
Experiment 1.—Four groups of three birds each which had been receiving the choline low basal diet supplemented with 562
EFFECT OF LEUCINE ON REPRODUCTION
choline, methionine and cystine or some combination of these were redistributed into two groups of six birds each. Since rate of egg production was at a very low level, it was decided to feed group 1 diet B21 which was identical to the low choline diet except for the modifications pointed out previously. Group 2 was fed diet B20 which had proven satisfactory in previous experiments on the effect of various members of the vitamin B complex on egg production and hatchability.
>n
563
1-5) shows that egg production for the two groups did not differ significantly at the start of the experiment. However, there was a rapid increase in rate of egg production of the birds of group 2 which were fed ration B20. The rate increased from approximately 30 percent to about 75 percent in 4 weeks. Group 1 which was fed ration B21 continued to lay at about the same rate as during the pre-experimental period. The reversal of the rations fed the 2
B20- casein l g ; gelatin 5 B21- casein 9; gelatin 7
so 70 6o 50 to 30 20
Group 1
Group 2
' Rations reversed
10
2
3
H
5
6
7 Week
8
5
10
11
12
1}
14
15
FIG. 1. Production of groups fed rations B 2 0 and B 2 1 .
This experiment was designed to obtain information on two points: (1) the time required for the laying bird to respond to a "complete" ration after being fed the "deficient" ration, or the time necessary for a measurable response after shifting from the complete to the deficient ration, (2) to gain evidence that ration B21 is incomplete insofar as protein is concerned. The results of experiment 1 are given in Figure 1. The pre-experimental period (weeks
groups after 5 weeks shows definitely that ration B21 is deficient in some factor or factors essential for the laying hen. Egg production of group 2 which was shifted to ration B21 dropped from approximately 75 percent to 30 percent in four weeks. On the other hand, egg production of group 1 which was shifted to ration B20 increased from about 30 percent to 80 percent in 5 weeks. The results of this experiment indicate that a measurable response could be ob-
564
W. W. CRAVENS
tained in from 3-5 weeks and further that we were dealing with a deficiency related to the protein makeup of the ration. While this study was in progress Krehl, Teply, Sarma and Elvehjem (1945) reported the growth inhibition of rats as a result of feeding large amounts of corn grits and the counteraction of this effect by adding either tryptophane or nicotinic acid. Later these same workers Krehl, Teply, Sarma and Elvehjem (1946) demonstrated that similar results were obtained with diets containing a protein supply very similar to that of diet B21. Although diet B21 contained 100 mg. of nicotinic acid per kg., it seemed advisable to test the effect of tryptophane additions. Experiment 2 was designed to test this amino acid. Experiment 2.—Eighteen pullets were equally divided into 6 groups. It was decided to conduct a curative trial and the ration changes were made after a 3 weeks "depletion" period. The experimental plan and egg production data are presented in Table 2. The test period was of 8 weeks duration. TABLE 2.—Effect
of tryptophane and other modi-
fications of ration B21 on egg production Experiment 2 Egg Ration "deple- production tion" perperiod cent B20 B20 B21 B21 B21
69.8 74.6 52.4 52.4 46.0
B21
34.9
Ration test period B20 B21 B21 B20 B21 less gelatin B21+0.5% DL
Egg production percent
tryptophane
52.4 42.9 16.7 54.8 16.7 8.9
The results of experiment 2 given Table 2 indicate that the addition tryptophane to ration B21 was of value in promoting egg production.
in of no It
may appear that egg production was sustained at a moderate rate when ration B21 was fed during the "depletion" period. However it should be pointed out that the average production for the period does not show the rapid drop in production which actually occurred. For groups 3, 4, 5 and 6 during the third week of the "depletion" period percent production was 33.8, 28.6, 23.8 and 9.5 respectively. The low rate of production for birds fed ration B21 for an extended period is reflected in the production rate of group 3 during the test period. The data are not presented graphically since negative results were obtained with tryptophane. Experiment 3.—-Inasmuch as some of the birds exhibited pronounced molting during experiment 2, it was thought that perhaps a prophylatic trial might better test the value of supplements to ration B21. Thus an experiment to further test tryptophane was conducted. In addition to tryptophane, methionine and cystine additions to ration B21 were made since this ration had been designed to be low in these amino acids. Additional casein was also added to the diet in order to determine if we could gain further evidence that we were dealing with an amino acid deficiency. Eighteen birds were divided equally among 6 groups. This experiment was of 6 weeks duration. The experimental plan and egg production data are given in Table 3. The results of this experiment (Table 3) show that some deficiency other than possible deficiencies of tryptophane and/or the sulfur containing amino acids exist in ration B21. That the deficiency is an amino acid is suggested by the results obtained by adding 4 percent additional casein (group 5). Egg production of this group was 64.8 percent while that for the group receiving B21 was 34.3 percent
E F F E C T O F L E U C I N E ON R E P R O D U C T I O N
565
able forms of amino acids were used. T h e forms and levels used in grams percent were: DL methionine 0.20, L arginine monohydrochloride 0.2, glycine 0.025, L lysine monohydrochloride. H2O 0.33, DL t r y p t o p h a n e 0.10, DL threonine 0.20, DL
(group 1) a n d the control (group 6) was 77.8 percent. Since essentially negative results h a d been obtained thus far in elucidating the specific deficiency or deficiencies of ration B21 i t was decided t o supplement the PERCENT PRODUCTION
B21 / 5% CASEIN
B21 / U AMINO ACIDS
2
3
U
WEEK FIG. 2. Effect of rations on egg production.
diet with the essential amino acids that would be supplied by 5 percent casein. Experiment 4 was designed to test this treatment. Experiment 4.—Sixteen pullets were divided into equal groups and fed the rations as follows: Group 1—B20; 2— B21; 3—B21+5 percent purified casein; 4—B21+essential amino acids equivalent to 5 percent casein. Commercially avail-
valine 0.35, L histidine monohydrochloride H 2 0 0.15, L leucine 0.65, DL isoleucine 0.30, DL phenylalanine 0.25. The experiment was terminated at four weeks. The results of experiment 4 are given in Figure 2. The results of experiment 4 shown in figure 2 indicate that ration B21 is deficient in one or more essential amino acids for laying hens. The addition of the
566
W. W. CRAVENS
11 essential amino acids in the quantities that would be supplied by 5 percent casein was effective in maintaining egg production. It may be observed, however that the group receiving the amino acids additions showed a gradual decline in rate of egg production. When compared with the group receiving B21 alone, it is obvious that the amino acid supplemented group TABLE 3.—Effect of supplements to ration B21 on egg production Experiment 3
group 4 and thus the results are based on only 3 birds.) Group 1. 2. 3. 4.
B 21 B 20 B 21+0.65% L leucine B 21+0.65% L leucine+ 0.1% DL tryptophane+0.2% DL methionine 5. B 2 1 + 5 % purified casein
Tryptophane and methionine were added for reasons stated earlier in this paper. Ration B21 was suspected of being Egg low in leucine because of comparisons Group Ration and supplement production made of its calculated amino acid makeup percent with that of certain simplified laying 1 B21 34.3 rations composed of crude ingredients and 2 B21+0.5%DL tryptophane 31.5 which had proven satisfactory for laying hens. 3 B21+0.5%DL tryptophane 38.1 0.3% DL methionine The results of experiment 5 are given in 0.4% L cystine Figure 3. 4 B21+0.3%DL methionine 47.2 The data in Figure 3 show that the addition of leucine alone was ineffective in 0.4% L cystine supplementing ration B21 for laying hens. 5 B21+4% purified casein 64.8 The addition of leucine, methionine and 6 B20 77.8 tryptophane to ration B21 resulted in egg produced at a higher rate. The group of production comparable to the control birds receiving B21 alone entirely ceased ration B20 or to ration B21 supplemented to lay during the second week of the ex- with 5 percent casein. Thus it is indicated periment and produced at a very low rate that diet B21 is deficient in leucine. It is for the balance of the experiment. Weekly impossible to determine from this experivariations in rate of production are quite ment whether tryptophane or methionine marked due to the small numbers of birds or both are deficient in ration B21. All possible combinations of these amino involved. The results of this experiment demon- acids were not tested because of the exstrated clearly that an amino acid de- pense involved. Methionine and tryptoficiency was responsible for the poor phane had proven ineffective in experiproduction which resulted when ration ment 3 and thus this treatment was not B21 was fed to laying hens. The problem repeated. The data indicate, however, thus became one of determining which of that one or both of these amino acids is the 11 essential amino acids was deficient. limiting. Experiment 5 was designed to obtain Hatchability Results.—Due to the small evidence on this point. number of eggs produced by the pullets Experiment 5.—Twenty pullets were fed ration B 21, composite hatchability data equally divided into 5 groups and fed the from all experiments are presented. The rations listed below: (one bird died in data for all eggs produced by the birds
EFFECT OF LEUCINE ON REPRODUCTION
TABLE 4.—Effect of an amino acid deficiency on hatchability
Egg set Eggs fertile No. chicks Percent fertile Percent hatchability
Ration B20
Ration B21
521 457 378 87.7 82.7
329 292 236 88.8 80.8
receiving rations B20 and B21 for experiments 2-5 are given in Table 4. The data in Table 4 indicate that the amino acid deficiencies of ration B21 did not affect hatchability. The percent hatchability of eggs from the birds fed the control ration (B20) was 82.7 while that
567
of the eggs from the amino acid deficient group was 80.8. Effect of an amino acid deficiency on the
egg laying cycle.—In order to present an accurate picture of the effect of an amino acid deficiency of the hen on the egg production cycle, the complete daily egg production record of all birds of experiment 5 are given in Figure 4. In addition, observations on molting of the birds are listed on the egg record sheet. The birds were frequently handled and observations recorded as to the laying condition of the bird. The following system was followed in recording information on molting and laying condition:
PERCENT PRODUCTION
B21 / 5* CASEIN B21 / B20
'""'"-
/'LEUCINE < TRYPTOPHANE ^METHIONINE
B21 / LEUCINE
2
3
4
I
WEEK FIG. 3. Effect of rations on egg production.
568
W. W. CRAVENS
(1) obvious pin feathers on neck and perhaps body (2) molting heavily (3) laying condition as judged by vent and pubic bone spread (4) not in laying condition It is obvious from Figure 4 that the amino acid deficiency of ration B21 caused
the diets used contained adequate amino acids for maintenance but an insufficient amount for the additional strain of egg production. The birds in group 3, receiving leucine, continued to lay but at a much reduced rate. One bird of this group showed evidence of a neck molt. To obtain evidence as to why the birds
FIG. 4. Effect of an amino acid deficiency on the egg laying cycle. (Experiment 5.)
a marked reduction in the egg laying cycle. It is of interest however that 2 of the 4 birds receiving the deficient ration continued to lay an occasional egg. It thus appears that the rate of production can be influenced by an amino acid deficiency without complete cessation of laying. Three of the birds of group 1 were showing some degree of molting at the end of the experiment. Occasionally a bird fed the deficient ration would go into a complete body molt and lose weight. No marked weight changes were observed which were unattended by heavy molting. Apparently
continued to lay at a reduced rate, or why pauses were so common among birds fed ration B21, two birds from each group were killed at the conclusion of the experiment and the ovaries examined. Observations were made on the length and weight of the oviduct and the number of ova falling within 5 mm. size classes. The ova were measured parallel to the suture line with a vernier caliper. These data are given in Table 5. The data in Table 5 show that the birds in group 2-5 contained ova in nearly every size class. It is of considerable inter-
EFFECT OF LEUCINE ON REPRODUCTION
est that abnormal ova were observed in the birds in groups 1, 3 and 4. These ova appeared to have developed to a certain size, then cease to grow and degenerate or be reabsorbed. The ova appeared irregular in outline and flabby rather than round and turgid. It may be seen that several were observed in the birds of group 1 and one of the birds of group 3. Such "resorp-
569
sumption may or may not be valid. In fact one is led to theorize that it would not be valid in view of the rather high content in egg protein of certain amino acids. Further the present study indicates that the laying hen has a leucine requirement (1.35-2.0 percent) not less than that reported by Almquist (1947) as being required by the growing chick.
TABLE 5.—Eject of an amino acid deficiency on development of ova
Group
1 2 3 4 5
Hen No.
25 28 30 31 33 34 37 39 41 45
No. of ova by 5 mm. classes
Oviduct Layingf condition Length Weight
6-10
11-15
16-20
21-25
cm. 21 23 24 24 25 32 24 29 27 29
9* 10* 3 5 7* 4 6 5 11 6
1 1 1 1
2*
2* 3*
— +?
+ + _? + + + + +
gms. 28 26 40 39 26 62 43 40 46 51
26-30
Egg in 31-35 Oviduct
1
1 1
1 1 1 1
1 1 1 1 2
3* 1 1 1 1
1* 1*
— — —
+ — + + + + +
* One or more ova showing evidence of "resorption." Size of such ova only aproximate. t As judged by handling.
tion" of ova might explain the erratic production of the birds in these groups. DISCUSSION OF RESULTS The data presented in this paper show that a deficiency of leucine results in an abrupt cessation of egg production. The data do not show which of the other two amino acids, tryptophane or methionine, are deficient in the basal diet used. A response to tryptophane or methionine, alone or in combination was not obtained, probably because of the leucine deficiency. By calculation one might predict that methionine is limiting but such calculations would be based on the supposition that the amino acid needs of the laying hen are the same as those of the growing chick, taking into account the difference in total protein requirements. Such an as-
The data presented in Table 4 indicate that a deficiency of leucine and either or both tryptophane and methionine does not affect hatchability. Rather the data show that the hen ceases laying. Such data suggest that the amino acid makeup of the egg is not influenced by the diet fed the hen or that the change in egg composition is insufficient to result in failure of embryonic development. Recently Csonka, Denton and Ringel (1947) have shown that the quantity of the sulfur containing amino acids in eggs may be modified by dietary means. Earlier work by McFarlane, Fulmer and Jukes (1930) had indicated no changes in the amino acid content of eggs due to dietary modifications while Gerber and Carr (1930) presented data indicating that egg protein might be' modified by the diet fed the bird. Unfor-
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W. W. CRAVENS
tunately no amino acid analyses of eggs were made in the present investigation. The egg laying cycle of the amino acid deficient birds are of particular interest. From Figure 4 it may be seen that some hens will apparently stay in laying condition as determined by external appearances and even produce an occasional egg. Thus the egg laying cycle may definitely be influenced by the amino acid makeup of the diet and in cases of an apparent borderline deficiency egg production with a marked reduction in intensity or rate will occur. By correlating the egg laying cycle with the development of ova in the ovary one is led to suggest that an amino acid deficiency may lead to a resorption of yolk material from certain ova, whereas others continue to develop until normal ovulation occurs. The possibility must not be overlooked, however, that the rate of growth of ova is actually modified by the amino acid deficient diets. The tendency to pause was observed in all birds fed diet B21 but considerable variation was observed, with some birds ceasing to lay and starting a general molt. SUMMARY
Experiments are described which indicate that leucine is an important amino acid for laying hens. The requirement for the laying bird appears to be not less than
1.35 percent of the ration. The data also indicate that either tryptophane or methionine or both are essential for the laying hen. Data are presented which show the effect of an amino acid deficiency on intensity of egg production and on development of egg yolks in the ovary of the hen. REFERENCES
Almquist, H. J., 1947. Evaluation of amino acid requirements by observations on the chick. J. Nutrition 34: 543-563. Cravens, W. W. and J. G. Halpin, 1946. Studies on the protein nutrition of breeding hens. Poultry Sci. 25: 400. Csonka, F. A., C. A. Denton and S. J. Ringel, 1947. The methionine and cystine content of hen's eggs. J. Biol. Chem. 169: 259-265. Gerber, L. and R. H. Carr, 1930. A chemical and immunological study of egg protein obtained under restricted diets. J. Nutrition 3: 245-256. Krehl, W. A., L. J. Teply, P. S. Sanaa and C. A. Elvehjem, 1945. Growth retarding effect of corn in nicotinic acid-low rations and its counteraction by tryptophane. Science 101: 489-490. Krehl, W. A., P. S. Sarma, L. J. Teply and C. A. Elvehjem, 1946. Factors affecting the dietary niacin and tryptophan requirement of the growing rat. J. Nutrition 31: 85-106. McFarlane, W. D., H. L. Fulmer and T. H. Jukes, 1930. Studies in embryonic mortality in the chick I. The effect of diet upon the nitrogen, amino-nitrogen, tyrosine, tryptophan, cystine and iron content of the proteins and on the total copper of the hen's egg. Biochem. J. 24: 16111631.