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Dietary Protein Effects on Urinary Nitrogen Components of the Hen
1260
B . W . BlERER AND T . H . ELEAZER
T h e r e w a s n o clinical evidence t h a t t h e a t t e n u a t e d vaccine a d v e r s e l y affected t h e turkeys. REFERENCES Bain, R. V. S., 1963. Hemorrhagic septicemia. Food and Agriculture Organization of the United Nations, Agricultural Studies No. 62, p. 46. Birdsall, J. J., 1967. Official communication, Wisconsin Alumni Research Foundation, Dec. 13,
1967. Harschfield, G. S., 1965. Fowl cholera in Diseases of Poultry, Biester and Schwarte Ed., Iowa State University Press, Ames, Iowa, Fifth Ed., p. 367. Sinha, S. K., T. F. Tachibana and R. Fagan, 1957. A quantitative evaluation of immune response in mice following intraperitoneal inoculation of killed strains and an avirulent strain of Pasteuretta multocida (type I I ) . Cornell Veterinarian, 47: 281-291.
Dietary Protein Effects on Urinary Nitrogen Components of the Hen R. A. TEEKELL, C. E. RICHARDSON* AND A. B. WATTS Department of Poultry Science, Louisiana State University and A & M College, Baton Rouge, Louisiana 70803 (Received for publication December 27, 1967)
I
T HAD been shown that dietary energy level had an effect on hen's urinary nitrogen components (Richardson et al., 1968). It was of interest to study possible effects of dietary protein on certain nitrogenous components of the urine, i.e. uric acid, ammonia, urea, creatine and amino acid nitrogen. Folin's laws (Folin, 1905) of human urine composition were based on such a study. Rather than switch from a high-protein to a non-protein diet as Folin, it was decided to conduct a protein depletion type study. It was reasoned that by following the daily urinary nitrogen components, these might indicate the protein level necessary for maintenance. It was hypothesized that as long as the nitrogen requirement was met or exceeded, the urinary nitrogen excretion would depend on the intake, but when the maintenance requirement was not met, the hen would mobilize body stores and the relative amounts of * Present address: Hoffman-LaRoche, Inc., Technical Services, Ames, Iowa.
urinary nitrogen components would then change. MATERIALS AND METHODS
Eighteen week-old White Leghorn hens were surgically modified to have exteriorized recta according to the procedure of Dixon (1958) and modified by Richardson et al. (1960). Fifteen hens were modified; however, only five were chosen for this experiment. Mortality, location of anal opening and failure to consume sufficient ration accounted for the remainder. When birds were selected for this study, they had been surgically modified from 2 to 6 months and were laying regularly. Birds were kept in large cages and fecal and urinary collections were made in the manner described by Richardson et al. (1960). Unconsumed feed was weighed and discarded at midnight each night, the next day's ration was allocated and the lights were turned off. Fecal and urinary collections for a given day were made at 9:30 o'clock the following morning. This was
1261
URINARY NITROGEN
done in order for the hens to pass the undigested material from the previous day's feeding. The hens had to eat the purified diet in order to have a fecal excretion on a given day and it was reasoned they must eat a portion of the next days ration to clean the digestive tracts of the previous day's ration. The formula of the diet used are shown in Table 1 and Table 2. The non protein basal ration was assayed and found to contain 1.0 percent crude protein (N X 6.2S). It is believed this amount can be attributed to the vitamin premix and possibly corn starch. Table 2 presents the amount of extracted egg added to the non-protein basal diet, the percent protein of the ration and the ration allowance for each day. Dried whole egg (Anheuser Busch, St. Louis, Mo.) was extracted in our laboratory, a 24-hour extraction was made with a 3:2 (V/V) mixture of acetone and ethanol. This was followed by a 24-hour extraction with diethyl ether. The pinkish-white pow-
TABLE 1.—Non-protein basal ration Ingredient Corn Starch Cerelose Vegetable Oil Agar Methyl Cellulose Vitamin Mix* Jones Foster Salt Mixture Oyster Shell Dicalcium Phosphate Al Si 0 2 Micro Mineral Mix** NaCl Chromic oxide
Percent 50.00 27.50 5.00 2.50 1.25 1.25 5.625 2.50 2.50 0.62 . 13 gm. .625 .25
* The vitamin supplement furnished the following per 454gm. feed: thiamine HC1,11.3 mg.; riboflavin, 7.26 mg.; Ca pantothenate, 9.08 gm.; vitamin B12, 9 mg.; pyridoxine HC1, 2.72 gm; biotin, 0.27 gm.; folic acid, 1.82 gm.; inositol, 45.4 gm.; 2 methyl napthaquinone, 2.27 mg.; niacin, 68.8 gm.; choline chloride, 800 mg.; vitamin A 4000,1.U.; vitamin D 3 , 750 I.G.U.; vitamin E 10 I.U. ** Supplied the following trace elements: Mo 10 p.p.m.; Bo 1.5 p.p.m.; Br 8.0 p.p.m. and Se
0.1 p.p.m.
TABLE 2.—Varying protein levels fed in trial NonRation Protein Extracted Egg Basal 15 13 11 9 7 6 5 4 3 2 1 0
Gm. 80 80 80 80 80 80 80 80 80 80 80 80
Gm. 19.2 16.6 14.1 11.5 9.0 7.7 6.4 5.1 3.8 2.6 1.3 0.0
Crude Protein
%
14.5 13.5 12.2 10.5 8.2 7.2 6.7 5.4 4.2 3.2 2.5 1.0
Egg Protein
%
13.7 12.7 11.3 9.6 7.3 6.3 5.8 4.5 3.2 2.2 1.5 0.0
Total Ration Allowance Gm.
99.2 96.6 94.1 91.5 89.0 87.7 86.4 85.1 83.8 82.6 81.3 80.0
der had a protein content of 79 percent (N X 6.25). The extracted egg was quite digestible as will be seen later. In certain cases when a bird did not eat over 25 percent of the ration on a given day, the data from that bird were not used in computing the daily averages. The ration was pelleted before feeding. The ration allowance was calculated to represent the maximum amount of the nonprotein basal the hens had consumed during a one week period a month prior to the beginning of the experiment. At the time this value was determined the hens received a 15 percent protein ration formulated from the non-protein basal plus zein, Drachett protein and dried whole eggs. The experimental plan was to reduce the protein intake each day in such a manner that on the twelfth day and for two succeeding days the birds received no protein. On the fifteenth day, the hens received no feed. Water was supplied ad libitum during the fifteen day trial period. Total nitrogen in the feed, feces and urine was determined using the Kjeldahl method. Uric acid was determined by the method of Richardson (1959). Ammonia, urea, creatine and creatinine was determined on each individual urine collection (Hawk et al., 1954). Amino acid nitrogen was determined on a composite sample of the urine collected for a given day. Urea, ammonia were removed in a manner similar
1262
R. A. TEEKELL, C. E. RICHARDSON AND A. B. WATTS
1401 '
I'ay lr
i 2 3 13.7 12.7 11.3
4 9.6
5 7.3
fa 7 6 . 3 5.B
8 4.5
9 3.2
10 2.2
II 1.5
12 0
13 0
14 0
15 W
FIG. 1. Dietary protein intake vs. absorbed nitrogen.
to the method of Awapara and Sato (1956). The urine was then desalted (Dowex—2 resin, Na form). Unknown urinary components were taken as total nitrogen minus nitrogen entities determined. In addition, carbohydrates were determined using the An throne method (Loewus, 19S2).
creases in nitrogen excretion preceding and followed by lower nitrogen excretions. In general, there is an overall decrease in the urinary nitrogen components as the protein levels of the ration decreased. It is interesting to note the increased urinary nitrogen between the fourteenth and fifteenth days. On the fifteenth day, the hens received no feed. This would indicate that the energy intake on the thirteenth and fourteenth days spared body protein. A comparison of the values obtained for the third day (Figure 1 and Figure 2A) indicates that ration protein level has more effect on the urinary nitrogen excretion than the amount of absorbed nitrogen. Uric acid excretion as affected by protein intake is shown in Figure 2B. The relationship between the protein level and uric acid excretion is almost linear between the 12.7 and 6.3 percent supplementary
RESULTS AND DISCUSSION
The relationship between the supplementary protein and absorbed nitrogen is presented in Figure 1. With the exception of one point (day three) the relationship is linear. On the third day, the feed consumption dropped and the nitrogen absorption acted accordingly. The plateau between the sixth and seventh day might have been due to the small difference in protein levels between the two rations. The effect of protein level on the urinary nitrogen excretion (Figure 2A) shows a linear relationship from the highest protein level to the 6.3 percent protein intake. When protein supplementation was decreased beyond this point there were in-
FIG. 2A. Dietary protein intake vs. urinary nitrogen excretion. FIG. 2B. Dietary protein intake vs. uric acid excretion.
URINARY NITROGEN
protein levels. The values obtained from the 6.3 and 5.8 percent protein are very similar. Beyond the seventh day the uric acid excretion is quite erratic. In general, there is an overall decrease in uric acid excretion from the first through the fourteenth day. The ammonia nitrogen excretion (Figure 3A) follows a very similar pattern to that of uric acid excretion except that the values are considerably more erratic. If the source of urinary ammonia is the oxidative deamination of circulating amino acids (Lotspeich and Pitts, 1947), then it would appear likely that such a drop, as that observed between the fifth and sixth days, must reflect a change in the level of circulating amino acids. These data indicate that as the dietary protein level decreases the urinary ammonia will decrease accordingly. According to the current theory, the urinary amino nitrogen is a "spill-over" from the blood and not reabsorbed in the kidney
FIG. 3A. Ammonia nitrogen excretion as affected by protein intake. FIG. 3B. Dietary protein intake and amino acid nitrogen.
1263
FIG. 4A. Urea nitrogen excretion as affected by protein intake. FIG. 4B. Creatine nitrogen excretion as affected by protein intake.
tubules. The data from this experiment do not necessarily support this view unless oxidative deamination of the amino acids in kidneys is reduced during protein depletion (Figure 3B). As may be seen from this Figure, the amino acid nitrogen excretion remained quite constant throughout this trial. As may be seen from Figure 4A, urea nitrogen was very variable from day to day. The energy level of the ration did not necessarily favor urea excretion, however, there tended to be an overall decrease in excretion levels of urea throughout this trial. Attention is called to the break in the overall pattern which occurred after the seventh day. If urea is derived solely from arginine, there was considerable mobilization of this amino acid on the eighth day. The creatine excretion (Figure 4B) appears to indicate some increase in arginine catabolism did occur during this period. These data represent creatine and creatinine excretion. The fluctuations in creatine
1264
R. A. TEEKELL, C. E. RICHARDSON AND A. B. WATTS
1
I ». I a Day W
. i 2 3 J 3 . 7 12.7 n . 3
4 9.6
5 7.3
6 7 6 . 3 5-8
8 4.5
9 3.2
10 11 2.2 1.5
12 0
13 0
14 0
15 H
FIG. 5A. Dietary protein effect on unknown urinary nitrogen components. FIG. SB. Urinary carbohydrate excretion as influenced by dietary protein.
excretion seem to reflect an inate ability of the hen to control nitrogen catabolism within certain limits. The erratic appearing data in this figure do not appear great when compared to the total urinary nitrogen excretion; however, the lowest value represents a decrease of about 33 percent from the first day of the experiment. It is interesting to note the excretion pattern of the unknown nitrogen components (Figure 5A). These data tended to vary in a manner similar to that of creatine. If the complete pattern is considered there is a tendency for this excretion to remain somewhat constant. The data presented in Figure 5B were unexpected. It was not suspected that the protein level of the ration would affect carbohydrate excretion. From these data, there is little doubt that there was indeed a direct or indirect relationship. This rise in
carbohydrate excretion became very pronounced between the seventh and thirteenth day. At this same time, there was a very erratic excretion of the nitrogen components on a day to day basis, especially in the urea, creatine and uric acid excretions. Amino nitrogen showed no marked change during this period; however, variance was noted in the excretion pattern of ammonia and the unknown components but not to the extent of the forementioned nitrogenous components. These data suggest the hen may have an ability to adjust the nitrogen metabolism in accordance with the requirements established by the protein intake. The data further indicate the maintenance requirement for the birds was met at about 6.3 to 5.8 percent supplemental egg protein. Body weight showed that prior to the seventh day the weight gain or loss of the individual hens depended more upon the feed intake than upon the protein level. After the seventh day the hens consistently lost body weight, except in a few instances. The heavier birds began losing weight sooner than did their lighter sisters. Ten of the fifteen eggs laid during the trial were collected prior to the seventh day. The average gain of the bird during this period was a minus 16 grams; after the seventh day the birds lost on an average of 87 grams on the day an egg was laid. At the beginning of the trial there was no effort made to relate the level of protein to egg production. The fact that such weight losses of birds after egg production on the lower protein ration showed that at least 5.8% protein is indeed required for maintenance of normal body functions. This corresponds to about 3.8 grams of egg protein based on the average feed consumption on the seventh day. In a previous maintenance protein level study, Ariyoshi (1957) stated the mainte-
1265
URINARY NITROGEN
nance may be lower than 1.8 gm. of whole egg protein per day. In these trials, birds were depleted for 42 days in order to determine the endogenous nitrogen excretion. The birds were then allowed 22 days to regain some of their lost weight and again placed on a depletion ration for 14 days (one gm. of dried egg per day as protein source). After this time he fed birds either 3 or S percent whole egg for 9 days. A close examination of the weights of these birds indicates that some adapted to the low-protein rations while others did not. This might have affected the determinations of the endogenous nitrogen excretion. In this study there were two plateaus in the urinary nitrogen excretion (Figure 2A). The nitrogen excretion at the maintenance protein level (day 7) was 304 milligrams and on the non-protein ration (days 13 and 14) the urinary nitrogen excretion was 180 to 186 milligrams. The urinary nitrogen excretion at the maintenance level was about 100 milligrams higher than obtained by Ariyoshi on heavier birds; however, he used cocks and capons and the sex difference might have influenced the urinary nitrogen excretion at maintenance protein levels. The urinary nitrogen excretion from the non-protein diets in this study was about 42 milligrams lower than obtained by Ariyoshi (185 vs. 227 mg.). SUMMARY
A 14 day protein depletion study was made using surgically modified birds. Supplemental egg protein level of the rations was decreased from 13.7 percent to zero protein level in twelve days, a non-protein ration fed for two days and feed was withdrawn on the fifteenth day. Daily individual fecal and urine collections were made. Total nitrogen of feed, feces and urine was determined. Individual determinations, of uric acid, ammonia, urea and creatine nitrogen were made of the urine in addition
to the daily composite determination of amino acid nitrogen. Findings from this experiment were that as dietary protein decreased, ammonia and uric acid nitrogen decreased. Urea nitrogen excretion varied considerably on a daily basis; however, there was an overall decrease as dietary protein decreased. Creatine nitrogen was quite variable on a daily basis with a tendency to maintain a constant level for the duration of this trial. Amino acid nitrogen excretion remained rather constant regardless of the protein intake. The unknown nitrogen excretion of the urine did not appear to be related to the dietary treatment, as protein level decreased throughout this trial, there was a marked increase in urinary carbohydrate excretion, as determined by the Anthrone method. From these data it is concluded that the hen can adapt to decreases in ration protein until a level below her maintenance needs is attained. Below this maintenance level the hen loses weight as she attempts to adapt herself to the lowered protein level. REFERENCES Ariyoshi S., 1957. Studies on the nitrogen metabolism of the fowl, 2. Protein requirement and amino acid balance for maintenance. Bull. Natl. Inst. Agri. Sci. G13 : 93-104. Awapara, J., and Y. Sato, 1956. Paper chromatography of urinary amino acids. Clin. Chem. Acta, 1: 75-86. Dixon, J. M., 1958. Investigation of urinary water reabsorption in the cloaca and rectum of the hen. Poultry Sci. 37 : 410-414. Folin, O., 1905. Laws governing the chemical composition of urine. Am. J. Physiol. 13: 66-116. Hawk, P. B., B. L. Oser and W. H. Summerson, 1954. Practical Physiological Chemistry, 13th edition. The Blakiston Co., Inc., N.Y., N.Y. Loewus, F. A., 1952. Improvement in anthrone method for determination of carbohydrates. Analytical Chem. 22: 219-220. Lotspeich, W. D., and R. F. Pitts, 1947. Role of
1266
R. A. TEEKELL, C. E. RICHARDSON AND A. B. WATTS
amino acids in the renal tubular secretion of ammonia. J. Biol. Chem. 168: 611-622. Meyer, H., 1957. Ninhydrin and its analytical applications. Biochem. J. 67: 333-340. Richardson, C. E., 1959. The effect of dietary protein and energy level upon the nitrogen components in the urine of the domestic hen. Dissertation, Louisiana State University, Baton
Rouge, La. Richardson, C. E., A. B. Watts, W. S. Wilkinson and J. M. Dixon, 1960. Techniques used in metabolism studies with surgically modified hens. Poultry Sci. 39:432-440. Richardson, C. E., R. A. Teekell and A. B. Watts, 1968. The effects of energy levels on nitrogen components of urine. Poul. Sci. 47:288-292.
Safflower Meal—Utilization as a Protein Source for Broiler Rations D. D. KUZMICKY AND G. O. KOHLER Western Regional Research Laboratory, Agricultural Research Service, U. S. Department of Agriculture, Albany, California 94710 (Received for publication December 27, 1967)
P
ARTIALLY decorticated safflower meal, except for deficiencies in lysine and possibly methionine, has been reported to be a good protein source for chicks (Kratzer and Williams, 1947, 1951; Peterson et al., 1957; Halloran, 1961; Young and Halloran, 1962; Valadez et al., 1965; Kohler et al., 1966). When the safflower meal is adequately supplemented it was shown to promote a growth response comparable to that of soybean meal. The commercially available partially decorticated meal contains about 42% protein and 12-16% fiber. This high fiber content is due mainly to the safflower hulls remaining in the meal. Consequently, the metabolizable energy of the meal is relatively low and this presents a problem for its use in high energy broiler rations. (Zablan et al., 1963; Kohler et al, 1966; Shoji et al, 1966). Our earlier results showed that the chick growth rate from lysine-supplemented safflower rations exceeded that from soy rations (Kohler et al, 1966). This present research was conducted to further investigate these results in chick experiments
where rations were varied in protein and energy content. EXPERIMENTAL
Nine chick experiments were conducted utilizing safflower and soybean meals as the main protein source. Table 1 shows the ingredients which were TABLE 1.—Ration ingredients common to all soy and sajftower rations Ingredient
% of ration
Dehydrated alfalfa meal (20% protein) Dried fish solubles Vitamin mix WU-24G1 Mineral mix MK-5 2 CaC0 3 DL-Methionine
3.00 1.00 1.00 3.00 1.50 0.20
1 Supplies the following per kg. of ration: 10,600 LIT. vitamin A, 1,660 I.C.U. vitamin D, 20 I.U. vitamin E, 2.0 g. choline chloride, 53 mg. niacin, 18.5 mg. calcium pantothenate, 5.74 mg. riboflavin, 5.74 mg. pyridoxine, 3.54 mg. thiamine, 2.2 mg. folic acid, .18 mg. biotin, 17.6 meg. vitamin B12, .98 mg. menadione, 50 mg. inositol, 7,215 U.procainepenicillin G and 3.43 g. corn starch as a carrier. 2 Supplies the following per kg. of ration: 8.02 g. CaCOs, 9.05 g. feed grade CaHP0 4 -2H 2 0, 4.5 g. K2HPO4, 3.48 g. NaCl, 3.65 g. Na 2 HP0 4 , 0.86 g. MgCOs, 0.20 g. ferric citrate, 0.16 g. MnS0 4 -H 2 0. 20 mg. KI, 50 mg. ZnC0 3 , 5 mg. CuS0 4 , 3.6 mg. CoCl 2 -6H 2 0, 1.2 mg. Na 2 Mo0 4 -2H 2 0 and .15 mg, Na 2 Se0 3 .
B . W . BlERER AND T . H . ELEAZER
T h e r e w a s n o clinical evidence t h a t t h e a t t e n u a t e d vaccine a d v e r s e l y affected t h e turkeys. REFERENCES Bain, R. V. S., 1963. Hemorrhagic septicemia. Food and Agriculture Organization of the United Nations, Agricultural Studies No. 62, p. 46. Birdsall, J. J., 1967. Official communication, Wisconsin Alumni Research Foundation, Dec. 13,
1967. Harschfield, G. S., 1965. Fowl cholera in Diseases of Poultry, Biester and Schwarte Ed., Iowa State University Press, Ames, Iowa, Fifth Ed., p. 367. Sinha, S. K., T. F. Tachibana and R. Fagan, 1957. A quantitative evaluation of immune response in mice following intraperitoneal inoculation of killed strains and an avirulent strain of Pasteuretta multocida (type I I ) . Cornell Veterinarian, 47: 281-291.
Dietary Protein Effects on Urinary Nitrogen Components of the Hen R. A. TEEKELL, C. E. RICHARDSON* AND A. B. WATTS Department of Poultry Science, Louisiana State University and A & M College, Baton Rouge, Louisiana 70803 (Received for publication December 27, 1967)
I
T HAD been shown that dietary energy level had an effect on hen's urinary nitrogen components (Richardson et al., 1968). It was of interest to study possible effects of dietary protein on certain nitrogenous components of the urine, i.e. uric acid, ammonia, urea, creatine and amino acid nitrogen. Folin's laws (Folin, 1905) of human urine composition were based on such a study. Rather than switch from a high-protein to a non-protein diet as Folin, it was decided to conduct a protein depletion type study. It was reasoned that by following the daily urinary nitrogen components, these might indicate the protein level necessary for maintenance. It was hypothesized that as long as the nitrogen requirement was met or exceeded, the urinary nitrogen excretion would depend on the intake, but when the maintenance requirement was not met, the hen would mobilize body stores and the relative amounts of * Present address: Hoffman-LaRoche, Inc., Technical Services, Ames, Iowa.
urinary nitrogen components would then change. MATERIALS AND METHODS
Eighteen week-old White Leghorn hens were surgically modified to have exteriorized recta according to the procedure of Dixon (1958) and modified by Richardson et al. (1960). Fifteen hens were modified; however, only five were chosen for this experiment. Mortality, location of anal opening and failure to consume sufficient ration accounted for the remainder. When birds were selected for this study, they had been surgically modified from 2 to 6 months and were laying regularly. Birds were kept in large cages and fecal and urinary collections were made in the manner described by Richardson et al. (1960). Unconsumed feed was weighed and discarded at midnight each night, the next day's ration was allocated and the lights were turned off. Fecal and urinary collections for a given day were made at 9:30 o'clock the following morning. This was
1261
URINARY NITROGEN
done in order for the hens to pass the undigested material from the previous day's feeding. The hens had to eat the purified diet in order to have a fecal excretion on a given day and it was reasoned they must eat a portion of the next days ration to clean the digestive tracts of the previous day's ration. The formula of the diet used are shown in Table 1 and Table 2. The non protein basal ration was assayed and found to contain 1.0 percent crude protein (N X 6.2S). It is believed this amount can be attributed to the vitamin premix and possibly corn starch. Table 2 presents the amount of extracted egg added to the non-protein basal diet, the percent protein of the ration and the ration allowance for each day. Dried whole egg (Anheuser Busch, St. Louis, Mo.) was extracted in our laboratory, a 24-hour extraction was made with a 3:2 (V/V) mixture of acetone and ethanol. This was followed by a 24-hour extraction with diethyl ether. The pinkish-white pow-
TABLE 1.—Non-protein basal ration Ingredient Corn Starch Cerelose Vegetable Oil Agar Methyl Cellulose Vitamin Mix* Jones Foster Salt Mixture Oyster Shell Dicalcium Phosphate Al Si 0 2 Micro Mineral Mix** NaCl Chromic oxide
Percent 50.00 27.50 5.00 2.50 1.25 1.25 5.625 2.50 2.50 0.62 . 13 gm. .625 .25
* The vitamin supplement furnished the following per 454gm. feed: thiamine HC1,11.3 mg.; riboflavin, 7.26 mg.; Ca pantothenate, 9.08 gm.; vitamin B12, 9 mg.; pyridoxine HC1, 2.72 gm; biotin, 0.27 gm.; folic acid, 1.82 gm.; inositol, 45.4 gm.; 2 methyl napthaquinone, 2.27 mg.; niacin, 68.8 gm.; choline chloride, 800 mg.; vitamin A 4000,1.U.; vitamin D 3 , 750 I.G.U.; vitamin E 10 I.U. ** Supplied the following trace elements: Mo 10 p.p.m.; Bo 1.5 p.p.m.; Br 8.0 p.p.m. and Se
0.1 p.p.m.
TABLE 2.—Varying protein levels fed in trial NonRation Protein Extracted Egg Basal 15 13 11 9 7 6 5 4 3 2 1 0
Gm. 80 80 80 80 80 80 80 80 80 80 80 80
Gm. 19.2 16.6 14.1 11.5 9.0 7.7 6.4 5.1 3.8 2.6 1.3 0.0
Crude Protein
%
14.5 13.5 12.2 10.5 8.2 7.2 6.7 5.4 4.2 3.2 2.5 1.0
Egg Protein
%
13.7 12.7 11.3 9.6 7.3 6.3 5.8 4.5 3.2 2.2 1.5 0.0
Total Ration Allowance Gm.
99.2 96.6 94.1 91.5 89.0 87.7 86.4 85.1 83.8 82.6 81.3 80.0
der had a protein content of 79 percent (N X 6.25). The extracted egg was quite digestible as will be seen later. In certain cases when a bird did not eat over 25 percent of the ration on a given day, the data from that bird were not used in computing the daily averages. The ration was pelleted before feeding. The ration allowance was calculated to represent the maximum amount of the nonprotein basal the hens had consumed during a one week period a month prior to the beginning of the experiment. At the time this value was determined the hens received a 15 percent protein ration formulated from the non-protein basal plus zein, Drachett protein and dried whole eggs. The experimental plan was to reduce the protein intake each day in such a manner that on the twelfth day and for two succeeding days the birds received no protein. On the fifteenth day, the hens received no feed. Water was supplied ad libitum during the fifteen day trial period. Total nitrogen in the feed, feces and urine was determined using the Kjeldahl method. Uric acid was determined by the method of Richardson (1959). Ammonia, urea, creatine and creatinine was determined on each individual urine collection (Hawk et al., 1954). Amino acid nitrogen was determined on a composite sample of the urine collected for a given day. Urea, ammonia were removed in a manner similar
1262
R. A. TEEKELL, C. E. RICHARDSON AND A. B. WATTS
1401 '
I'ay lr
i 2 3 13.7 12.7 11.3
4 9.6
5 7.3
fa 7 6 . 3 5.B
8 4.5
9 3.2
10 2.2
II 1.5
12 0
13 0
14 0
15 W
FIG. 1. Dietary protein intake vs. absorbed nitrogen.
to the method of Awapara and Sato (1956). The urine was then desalted (Dowex—2 resin, Na form). Unknown urinary components were taken as total nitrogen minus nitrogen entities determined. In addition, carbohydrates were determined using the An throne method (Loewus, 19S2).
creases in nitrogen excretion preceding and followed by lower nitrogen excretions. In general, there is an overall decrease in the urinary nitrogen components as the protein levels of the ration decreased. It is interesting to note the increased urinary nitrogen between the fourteenth and fifteenth days. On the fifteenth day, the hens received no feed. This would indicate that the energy intake on the thirteenth and fourteenth days spared body protein. A comparison of the values obtained for the third day (Figure 1 and Figure 2A) indicates that ration protein level has more effect on the urinary nitrogen excretion than the amount of absorbed nitrogen. Uric acid excretion as affected by protein intake is shown in Figure 2B. The relationship between the protein level and uric acid excretion is almost linear between the 12.7 and 6.3 percent supplementary
RESULTS AND DISCUSSION
The relationship between the supplementary protein and absorbed nitrogen is presented in Figure 1. With the exception of one point (day three) the relationship is linear. On the third day, the feed consumption dropped and the nitrogen absorption acted accordingly. The plateau between the sixth and seventh day might have been due to the small difference in protein levels between the two rations. The effect of protein level on the urinary nitrogen excretion (Figure 2A) shows a linear relationship from the highest protein level to the 6.3 percent protein intake. When protein supplementation was decreased beyond this point there were in-
FIG. 2A. Dietary protein intake vs. urinary nitrogen excretion. FIG. 2B. Dietary protein intake vs. uric acid excretion.
URINARY NITROGEN
protein levels. The values obtained from the 6.3 and 5.8 percent protein are very similar. Beyond the seventh day the uric acid excretion is quite erratic. In general, there is an overall decrease in uric acid excretion from the first through the fourteenth day. The ammonia nitrogen excretion (Figure 3A) follows a very similar pattern to that of uric acid excretion except that the values are considerably more erratic. If the source of urinary ammonia is the oxidative deamination of circulating amino acids (Lotspeich and Pitts, 1947), then it would appear likely that such a drop, as that observed between the fifth and sixth days, must reflect a change in the level of circulating amino acids. These data indicate that as the dietary protein level decreases the urinary ammonia will decrease accordingly. According to the current theory, the urinary amino nitrogen is a "spill-over" from the blood and not reabsorbed in the kidney
FIG. 3A. Ammonia nitrogen excretion as affected by protein intake. FIG. 3B. Dietary protein intake and amino acid nitrogen.
1263
FIG. 4A. Urea nitrogen excretion as affected by protein intake. FIG. 4B. Creatine nitrogen excretion as affected by protein intake.
tubules. The data from this experiment do not necessarily support this view unless oxidative deamination of the amino acids in kidneys is reduced during protein depletion (Figure 3B). As may be seen from this Figure, the amino acid nitrogen excretion remained quite constant throughout this trial. As may be seen from Figure 4A, urea nitrogen was very variable from day to day. The energy level of the ration did not necessarily favor urea excretion, however, there tended to be an overall decrease in excretion levels of urea throughout this trial. Attention is called to the break in the overall pattern which occurred after the seventh day. If urea is derived solely from arginine, there was considerable mobilization of this amino acid on the eighth day. The creatine excretion (Figure 4B) appears to indicate some increase in arginine catabolism did occur during this period. These data represent creatine and creatinine excretion. The fluctuations in creatine
1264
R. A. TEEKELL, C. E. RICHARDSON AND A. B. WATTS
1
I ». I a Day W
. i 2 3 J 3 . 7 12.7 n . 3
4 9.6
5 7.3
6 7 6 . 3 5-8
8 4.5
9 3.2
10 11 2.2 1.5
12 0
13 0
14 0
15 H
FIG. 5A. Dietary protein effect on unknown urinary nitrogen components. FIG. SB. Urinary carbohydrate excretion as influenced by dietary protein.
excretion seem to reflect an inate ability of the hen to control nitrogen catabolism within certain limits. The erratic appearing data in this figure do not appear great when compared to the total urinary nitrogen excretion; however, the lowest value represents a decrease of about 33 percent from the first day of the experiment. It is interesting to note the excretion pattern of the unknown nitrogen components (Figure 5A). These data tended to vary in a manner similar to that of creatine. If the complete pattern is considered there is a tendency for this excretion to remain somewhat constant. The data presented in Figure 5B were unexpected. It was not suspected that the protein level of the ration would affect carbohydrate excretion. From these data, there is little doubt that there was indeed a direct or indirect relationship. This rise in
carbohydrate excretion became very pronounced between the seventh and thirteenth day. At this same time, there was a very erratic excretion of the nitrogen components on a day to day basis, especially in the urea, creatine and uric acid excretions. Amino nitrogen showed no marked change during this period; however, variance was noted in the excretion pattern of ammonia and the unknown components but not to the extent of the forementioned nitrogenous components. These data suggest the hen may have an ability to adjust the nitrogen metabolism in accordance with the requirements established by the protein intake. The data further indicate the maintenance requirement for the birds was met at about 6.3 to 5.8 percent supplemental egg protein. Body weight showed that prior to the seventh day the weight gain or loss of the individual hens depended more upon the feed intake than upon the protein level. After the seventh day the hens consistently lost body weight, except in a few instances. The heavier birds began losing weight sooner than did their lighter sisters. Ten of the fifteen eggs laid during the trial were collected prior to the seventh day. The average gain of the bird during this period was a minus 16 grams; after the seventh day the birds lost on an average of 87 grams on the day an egg was laid. At the beginning of the trial there was no effort made to relate the level of protein to egg production. The fact that such weight losses of birds after egg production on the lower protein ration showed that at least 5.8% protein is indeed required for maintenance of normal body functions. This corresponds to about 3.8 grams of egg protein based on the average feed consumption on the seventh day. In a previous maintenance protein level study, Ariyoshi (1957) stated the mainte-
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nance may be lower than 1.8 gm. of whole egg protein per day. In these trials, birds were depleted for 42 days in order to determine the endogenous nitrogen excretion. The birds were then allowed 22 days to regain some of their lost weight and again placed on a depletion ration for 14 days (one gm. of dried egg per day as protein source). After this time he fed birds either 3 or S percent whole egg for 9 days. A close examination of the weights of these birds indicates that some adapted to the low-protein rations while others did not. This might have affected the determinations of the endogenous nitrogen excretion. In this study there were two plateaus in the urinary nitrogen excretion (Figure 2A). The nitrogen excretion at the maintenance protein level (day 7) was 304 milligrams and on the non-protein ration (days 13 and 14) the urinary nitrogen excretion was 180 to 186 milligrams. The urinary nitrogen excretion at the maintenance level was about 100 milligrams higher than obtained by Ariyoshi on heavier birds; however, he used cocks and capons and the sex difference might have influenced the urinary nitrogen excretion at maintenance protein levels. The urinary nitrogen excretion from the non-protein diets in this study was about 42 milligrams lower than obtained by Ariyoshi (185 vs. 227 mg.). SUMMARY
A 14 day protein depletion study was made using surgically modified birds. Supplemental egg protein level of the rations was decreased from 13.7 percent to zero protein level in twelve days, a non-protein ration fed for two days and feed was withdrawn on the fifteenth day. Daily individual fecal and urine collections were made. Total nitrogen of feed, feces and urine was determined. Individual determinations, of uric acid, ammonia, urea and creatine nitrogen were made of the urine in addition
to the daily composite determination of amino acid nitrogen. Findings from this experiment were that as dietary protein decreased, ammonia and uric acid nitrogen decreased. Urea nitrogen excretion varied considerably on a daily basis; however, there was an overall decrease as dietary protein decreased. Creatine nitrogen was quite variable on a daily basis with a tendency to maintain a constant level for the duration of this trial. Amino acid nitrogen excretion remained rather constant regardless of the protein intake. The unknown nitrogen excretion of the urine did not appear to be related to the dietary treatment, as protein level decreased throughout this trial, there was a marked increase in urinary carbohydrate excretion, as determined by the Anthrone method. From these data it is concluded that the hen can adapt to decreases in ration protein until a level below her maintenance needs is attained. Below this maintenance level the hen loses weight as she attempts to adapt herself to the lowered protein level. REFERENCES Ariyoshi S., 1957. Studies on the nitrogen metabolism of the fowl, 2. Protein requirement and amino acid balance for maintenance. Bull. Natl. Inst. Agri. Sci. G13 : 93-104. Awapara, J., and Y. Sato, 1956. Paper chromatography of urinary amino acids. Clin. Chem. Acta, 1: 75-86. Dixon, J. M., 1958. Investigation of urinary water reabsorption in the cloaca and rectum of the hen. Poultry Sci. 37 : 410-414. Folin, O., 1905. Laws governing the chemical composition of urine. Am. J. Physiol. 13: 66-116. Hawk, P. B., B. L. Oser and W. H. Summerson, 1954. Practical Physiological Chemistry, 13th edition. The Blakiston Co., Inc., N.Y., N.Y. Loewus, F. A., 1952. Improvement in anthrone method for determination of carbohydrates. Analytical Chem. 22: 219-220. Lotspeich, W. D., and R. F. Pitts, 1947. Role of
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amino acids in the renal tubular secretion of ammonia. J. Biol. Chem. 168: 611-622. Meyer, H., 1957. Ninhydrin and its analytical applications. Biochem. J. 67: 333-340. Richardson, C. E., 1959. The effect of dietary protein and energy level upon the nitrogen components in the urine of the domestic hen. Dissertation, Louisiana State University, Baton
Rouge, La. Richardson, C. E., A. B. Watts, W. S. Wilkinson and J. M. Dixon, 1960. Techniques used in metabolism studies with surgically modified hens. Poultry Sci. 39:432-440. Richardson, C. E., R. A. Teekell and A. B. Watts, 1968. The effects of energy levels on nitrogen components of urine. Poul. Sci. 47:288-292.
Safflower Meal—Utilization as a Protein Source for Broiler Rations D. D. KUZMICKY AND G. O. KOHLER Western Regional Research Laboratory, Agricultural Research Service, U. S. Department of Agriculture, Albany, California 94710 (Received for publication December 27, 1967)
P
ARTIALLY decorticated safflower meal, except for deficiencies in lysine and possibly methionine, has been reported to be a good protein source for chicks (Kratzer and Williams, 1947, 1951; Peterson et al., 1957; Halloran, 1961; Young and Halloran, 1962; Valadez et al., 1965; Kohler et al., 1966). When the safflower meal is adequately supplemented it was shown to promote a growth response comparable to that of soybean meal. The commercially available partially decorticated meal contains about 42% protein and 12-16% fiber. This high fiber content is due mainly to the safflower hulls remaining in the meal. Consequently, the metabolizable energy of the meal is relatively low and this presents a problem for its use in high energy broiler rations. (Zablan et al., 1963; Kohler et al, 1966; Shoji et al, 1966). Our earlier results showed that the chick growth rate from lysine-supplemented safflower rations exceeded that from soy rations (Kohler et al, 1966). This present research was conducted to further investigate these results in chick experiments
where rations were varied in protein and energy content. EXPERIMENTAL
Nine chick experiments were conducted utilizing safflower and soybean meals as the main protein source. Table 1 shows the ingredients which were TABLE 1.—Ration ingredients common to all soy and sajftower rations Ingredient
% of ration
Dehydrated alfalfa meal (20% protein) Dried fish solubles Vitamin mix WU-24G1 Mineral mix MK-5 2 CaC0 3 DL-Methionine
3.00 1.00 1.00 3.00 1.50 0.20
1 Supplies the following per kg. of ration: 10,600 LIT. vitamin A, 1,660 I.C.U. vitamin D, 20 I.U. vitamin E, 2.0 g. choline chloride, 53 mg. niacin, 18.5 mg. calcium pantothenate, 5.74 mg. riboflavin, 5.74 mg. pyridoxine, 3.54 mg. thiamine, 2.2 mg. folic acid, .18 mg. biotin, 17.6 meg. vitamin B12, .98 mg. menadione, 50 mg. inositol, 7,215 U.procainepenicillin G and 3.43 g. corn starch as a carrier. 2 Supplies the following per kg. of ration: 8.02 g. CaCOs, 9.05 g. feed grade CaHP0 4 -2H 2 0, 4.5 g. K2HPO4, 3.48 g. NaCl, 3.65 g. Na 2 HP0 4 , 0.86 g. MgCOs, 0.20 g. ferric citrate, 0.16 g. MnS0 4 -H 2 0. 20 mg. KI, 50 mg. ZnC0 3 , 5 mg. CuS0 4 , 3.6 mg. CoCl 2 -6H 2 0, 1.2 mg. Na 2 Mo0 4 -2H 2 0 and .15 mg, Na 2 Se0 3 .