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Feeding 5 per cent l -phenylalanine to chicks of pullets fed same levels of l -phenylalanine
Comp. Biochem. Physiol., 1968, Vol. 24, pp. 417 to 425. Pergamon Press. Printed in Great Britain
F E E D I N G 5 PER CENT L-PHENYLALANINE TO CHICKS OF P U L L E T S FED SAME LEVELS OF L-PHENYLALANINE H.S. TAMIMIE Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Illinois
(Received 26 April 1967) A b s t r a c t - - 1 . Feeding 5 per cent L-phenylalanine depressed the growth rate, weight gain and feed conversion in chicks hatched from pullets fed basal diet alone. The reverse occurred with chicks hatched from pullets fed basal + 5 per cent L-phenylalanine. 2. A maternal as well as dietary effect of feeding the 5 per cent L-phenylalanine-supplemented diet was noted with chicks hatched from pullets fed the same level of L-pheny!a!anine. 3. Regardless of the maternal dietary regimen, chicks fed the 5 per cent L-phenylalanine-supplemented diet had elevated levels of plasma phenylalanlne and reduced concentration of brain serotonln. Levels of norepinephrlne were not affected.
INTRODUCTION IN nMPLOYINGcommercial, day-old and 4-weekold, crossbred, male chicks in the study of experimental phenylketonuria, Tamimie & Pscheidt (1966a) found that feeding a diet containing 5 per cent L-phenylalanine for a period of 4 weeks caused the following conditions: depressed growth rate, lower feed conversion, poor motor co-ordination, reduced hemoglobin content of the blood, swollen hock, curled toes, enlarged crop, rough feather development and darker-color shank, face and eyelids. Similar physical alterations proportional to the amount of L-phenylalanine in the supplemented diets were seen in another commercial strain of crossbred males and inbred White Leghorn female chicks (Tamimie & Pscheidt, 1966b). The inbred females were more affected than the crossbred males. The serotonin concentration of the brain of chicks fed 5 per cent L-phenylalanine was reduced while the level of norepinephrine was unaffected (Pscheidt & Tamimie, 1966). This depression in serotonin, caused by the feeding of diets rich in phenylalanine, was also found in rats (Wang et al., 1962). Similarly, a growth depression was observed in rats (Harper et al., 1955) and in chicks (Jones, 1964; Tamimie & Pscheidt, 1966a, b) fed diets supplemented with high levels of amino acids. In contrast, the feeding of amino acids in excess amounts led to increased concentration of these amino acids in the blood of dog (Hier, 1947), rat (Kumta & Harper, 1962), monkey (Waisman et al., 1959) and chicks (Dean & Scott, 1966). Furthermore, the influence of high levels of L-phenylalanine included not only the aforementioned parameters, but also the reproductive performance of pregnant
418
H.S. TAMIMIE
and lactating rats (Boggs & Waisman, 1964). Also, in crossbred pullets, Tamimie & Pscheidt (1966c) noted that feeding a diet supplemented with 5 per cent L-phenylalanine decreased the egg weight and the percentage of hen day production, fertility, hatchability and hatching chick weight without, however, producing deformed viable chicks. The chicks obtained from these pullets were used in this study to determine the effect of maternal feeding on the growth performance, concentration of serotonin and norepinephrine, level of plasma phenylalanine and external defects of these chicks fed the same level (5 per cent) of L-phenylalanine as their maternal parents. Because of (a) the importance of norepinephrine and serotonin in the normal physiological processes of the brain, (b) the concentration of these two biogenic amines known more in adult chickens than in young chicks (Pscheidt & Haber, 1965), (c) the report of Pscheidt & Himwich (1965) that, in contrast to other species, adult chicken brain contains relatively large amounts of norepinephrine and (d) the depressive effect of feeding high levels of phenylalanine on the serotonin, the serotonin and norepinephrine contents of the brain in chicks fed the normal "basal" ration or that basal supplemented with 5 per cent L-phenylalanine were determined. Similarly, in view of the previously mentioned reports showing the influence of dietary amino acid supplementation on the plasma free amino acids of rats and chicks, the phenylalanine content of the blood of these two kinds of chicks was also determined. MATERIALS AND METHODS Four sub-groups of unsexed, day-old, crossbred chicks (sixty-four chicks total), hatched from pullets fed one of two kinds of diets, were used in this study. Three of these groups (BI, B2 and B3) were derived from pullets fed daily a limited amount (100 g/bird/20 hr) of a commercial 16 per cent protein, corn-soy basal diet for three consecutive 10-day periods. The fourth group (B4) were from pullets fed ad lib. for an additional 10 days. Chicks of the other four groups (LP1, LP~, LP3 and LP4) were obtained from pullets fed in similar pattern a basal diet +5 per cent L-phenylalanine for the first three 10-day periods and then fed the basal diet during the fourth 10-day period. In order to control the intake of an expensive L-phenylalanine-supplemented diet by the pullet, and in the meantime prevent the males from consuming such a diet without affecting the egg fertility, the pullets which produced the LP chicks were fed in a special manner. A simplified procedure was therefore developed in which the male was left with the pullets for a period of 4 hr (8 a.m.-12 noon) during which time the feed troughs were removed from the pens. On the basis of 120/g/pullet/24 hr, age 5-8 months, the aforementioned amount was calculated to be 100 g/puUet/20 hr. Equal numbers of chicks (four chicks per group) from each of the four hatches were selected, weighed, banded and distributed into four lots. Chicks from the basal-fed pullets (B) were fed either a commercial 19 per cent corn-soy starter ration, or the same starter supplemented with 5 per cent L-phenylalanine for a period of 3 weeks. Similarly, the chicks from pullets fed the 5 per cent L-phenylalanine-supplemented diets were also subdivided and fed one of the two diets for
EFFECTS OF FEEDING 5 PER CENT L - P H E N Y L A L A N I N E TO CHICKS
419
the same period of time. The chicks were reared in standard battery brooders with raised wire floors. Their weights were taken at weekly intervals. At 3 weeks of age, the study of each group was terminated, at which time the final weight was taken, and external defects were checked for swollen hock, curled toes and poor feather development. The coloration of shank, face and eyelid as compared to the control chicks was checked too. These defects were scored by an arbitrary score of 1-3 ; 1 = mild, 2 = medium and 3 ~- medium-severe. Feed conversions were computed from the grams of gain per grams of feed intake. Blood was withdrawn by heart puncture. The chicks were then weighed and decapitated, and the heart, liver, spleen, gizzard and intestine were weighed and examined for abnormal appearance. The serotonin and norepinephrine contents of the brain were determined by the method of Shore & Olin (1958) as modified by Mead & Finger (1961). The phenylalanine content of the blood was determined by the fluorometric procedure of McCaman & Robins (1962). Data analyses were computed by Mann-Whitney U- non-parametric statistic (1956), and "Student's" t-test (Table 5). RESULTS
As early as 1 week and continuing through the third week of experiment, it was found that feeding a commercial corn-soy basal diet supplemented with 5 per cent L-phenylalanine (LP) resulted in poorer growth than feeding the same basal diet alone (B) (Table 1). An improvement in third-week weight, weight gain and feed conversion was obtained by feeding the basal (B) as compared with the supplemented diet (LP) for the four hatching groups. This improvement held for chicks hatched from pullets fed a basal diet and with chicks hatched from pullets fed a supplemented diet. The third-week weight in the basal fed groups was significantly (P<0.001) greater than that of the L-phenylalanine supplemented groups. This finding was not only true for chicks hatched from basal fed pullets but for chicks hatched from pullets fed basal+5 per cent L-phenylalanine, during the three hatchings. Similar significant (P<0.001) improvement in third-week weight was observed in the fourth hatch. In studying the maternal effect, however, it was found that feeding the same chick's basal to the two types of chicks reveals that chicks hatched from pullets fed basal had a significantly (P<0.001) better third-week weight than that of chicks hatched from pullets fed basal + 5 per cent L-phenylalanine (Table 2). Such an observation was not only true for the first three hatches but for the fourth one too. The contrast occurred with the feeding of L-phenylalaninesupplemented diet. In this case chicks hatched from pullets fed the L-phenylalanine-supplemented diet were more benefited (P< 0.001) by feeding the chicks' L-phenylalanine-supplemented diet than chicks hatched from pullets fed basal alone. The latter observation reversed itself in the fourth hatch.
4-20
H . S . TAMIMIE
TABLE I p E ~ c T
OF 5 PER CENT L - P H E N Y L A L A N I N E ON THE GROWTH PERFORMANCE OF CHICKS HATCHED FROM PULLETS FED LIMITED AND a d l i b ~ / L m AMOUNTS OF DIET/DAY Basal (19 per cent protein) (B) No. of hatch
First three hatchest Fourth hatcht
B-groups*
LP-groups
Av. 3rdwk wt. (g)
Av. wt. gain (g)
Av. feed conversion
Av. 3rdwk wt. (g)
Av. wt. gain (g)
Av. feed convero sion
302 338
267 295
2"2 2"3
228 321
194 284
2"7 2"6
Basal + 5 per cent L-phenylalanine (LP) No. of hatch
First three hatches t Fourth hatch t
B-groups
LP-groups
Av. 3rdwk wt. (g)
Av. wt. gain (g)
Av. feed conversion
Av. 3rdwk wt. (g)
Av. wt. gain (g)
Av. feed conversion
178 202
130 159
3"2 3"0
196 191
162 154
3"0 3"0
* (B) and (LP) groups represent the chicks hatched from pullets fed basal or basal + 5 per cent L-phenylalanine respectively. Half of the chicks from each of the four hatches were placed on one of these dietary treatments. t See text (Materials and Methods).
Regardless of the maternal origin, the third-week weight in the fourth hatch as compared to the first three hatches showed (with the exception where T.-phenylalanine-supplemented diet was fed to the pullets and their chicks, L P - L P ) a significant ( P < 0-001) growth improvement in all the other groups. T h e reverse occurred in the exception group (Table 2). By the end of the third week a n u m b e r of chicks in each of the four groups fed the 5 per cent L-phenylalanine showed a n u m b e r of external defects, such as swelling of the hock, poor feather development and, in comparison to the control chicks, a darker-color shank, face and eyelid were observed (Table 3). In contrast, none of the chicks in the four groups fed the basal diet exhibited such conditions. T h i s was true whether the chicks were hatched from pullets fed basal or basal + 5 per cent L-phenylalanine. N o gross pathological conditions were observed in any of the internal organs of the chicks. However, the gizzards of the 5 per cent L-phenylalanine fed groups were dark green in color and had slight erosions. A very slight difference in the organ weights was observed among the four basal-fed or the 5 per cent L-phenylalanine-fed groups (Table 4).
EFFECTS
OF FEEDING
5 PER
CENT
L-PHENYLALANINE
421
TO CHICKS
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422
TABI~ 3 ~ T O T A L MALFORMED EXTERNAL SCORE OF CHICKS FED 5 PER L - P ~ A L A N I N E DIET
Hatching type
Poor feather development
B-LPt L P - L P ~:
14 22 §
Swollen hock
Discolored* face and eyelid
Discolored* shank
Total
10 14
8 12
50 70
18 20 [[
* T h e discoloration of face, eyelid and shank refers to darker color as compared to the same criteria in the control chicks. t B - L P corresponds to chicks hatched from pullets fed basal (B). These chicks were fed basal + 5 per cent L-phenylalanine. L P - L P corresponds to chicks fed similarly to * but were hatched from pullets fed basal + 5 per cent L-phenylalanine (LP). § Each chick in each group was scored individually (see text). [[ Each number indicates the total defects computed from scoring the four hatches of the two types of chicks. TABLE 4---THE THIRD-VCEEKORGAN~VEIGHTO F CHICKS* FED BASALOR BASAL ~-5 PER CENT L-PHENYLALANINE Brain
Heart
Liver
Wt. (g)
% of body wt.
Wt. (g)
% of body wt.
Wt. (g)
% of body wt.
1"70t 1"44~
0"56 0"75
2"03 1"52
0"70 0"78
7"60 4'67
0-25 0"24
Spleen
Gizzard
Wt. (g)
% of body wt.
Wt. (g)
0"38t 0"31.*
0"11 0"15
10"4 7"22
% of body wt.
Intestine Wt. (g)
% of body wt.
0-35
19"8
0"67
0"37
13"7
0"71
* Chicks hatched from pullets fed basal or basal + 5 per cent L-phenylalanine in limited and ad libitum amounts of diet/day. t Corresponds to chicks fed basal alone. Corresponds to chicks fed basal + 5 per cent L-phenylalanine. C h e m i c a l analyses s h o w e d that the chicks receiving the L-phenylalanine diet h a d elevated levels of this a m i n o acid in their plasma ( T a b l e 5). H o w e v e r , there was an extreme variability in the a m o u n t s f o u n d in different animals. F e e d i n g 5 per cent L-phenylalanine r e d u c e d the brain concentration of serotonin w i t h o u t h a v i n g a n y effect o n the c o n t e n t of n o r e p i n e p h r i n e ( T a b l e 5).
423
EFFECTS OF FEEDING 5 PER CENT L-PHENYLALANINETO CHICKS
TABLE 5--AWaAGE PLASMAPHENYLALANINELEVELSAND
CONCENTRATIONSOF BIOGENIC
AMINES I N BRAINS OF CHICKS FED A BASAL DIET COMPARED TO THOSE FED 5 PER CENT L-PHENYLALANINE
Group and diet
No. of animals
Plasma phenylalanine (mg/lO0 ml)
B-B + LP-B* basal B-LP + LP-LP t 5 per cent L-phcnylalanine Significance of difference between m e a n s
32 32
2.6 + 0.6 52+45 P<0.01
Serotonin (/zg/g) 0.99 + 0.23 0.62_+0.19 P<0.01
Norepinephrine (brg/g) 0.54 + 0.17 0.55_+0.15 N.S.
* B-B and LP-B correspond to half of chicks hatched from pullets fed basal (B) or basal + 5 per cent L-phenylalanine (LP). These two kinds of chicks were fed basal. Corresponds to the other half of chicks as in * but were fed basal + 5 per cent L-phenylalanine.
DISCUSSION This study confirms a previous report of Tamimie & Pseheidt (1966a) which showedthat feedingday-oldchicksa commercialcorn-soybasal diet supplemented with 5 per cent L-phenylalanine resulted in poor growth and poor feed conversion. A significant ( P < 0.001) difference in the third-week weight of chicks fed basal as compared to the feeding of basal + 5 per cent L-phenylalanine was obtained in the present study. This poor performance by feeding excess levels oi L-phenylalanine might have been caused by amino acid imbalance or by the toxicity of L-phenylalanine and/or its metabolites, or by the impairment of the diet palatability. Phenylalanine is known to be bitter. The reduction in total energy, protein and other micronutrients might have entered the picture of this study. Dietary additions of large amounts of individual amino acids caused growth depression in rats (Harper et al., 1955) and in chicks (Jones, 1964; Tamimie & Pscheidt, 1966a, b). This investigation reveals also that a significant ( P < 0.001) difference due to maternal feeding was observed not only in chicks fed basal diet, but in chicks ied basal + 5 per cent L-phenylalanine. In this case, chicks hatched from basal-fed pullets were more benefited by feeding chick's basal than chicks hatched from pullets fed basal + 5 per cent L-phenylalanine. Tamimie & Pscheidt (1966c) noted, along with numerous detrimental effects of feeding 5 per cent L-phenylalanine-supplemented diet to laying pullets, a reduction of 4-8 g. in the hatching weight of chicks obtained from these pullets in four consecutive incubations. Thus, it is possible that such effect of feeding the L-phenylalanine-supplemented diet might have extended its influence beyond the first day of life, by showing that chicks hatched from pullets fed the L-phenylalanine-supplemented diet had a lower third-week weight upon the feeding of chicks' basal than chicks hatched from puUets fed basal alone. However, and for unknown reason(s), the
424
H.S. TAMIMIE
reverse occurred by feeding the same types of chicks the L-phenylalanine-supplemented diet. Similarly, and for unknown reason(s), the latter observation reversed itself with chicks hatched (fourth hatch) from pullets fed basal in ad libitum amounts. The possibility(ies) that chicks hatched from pullets fed only basal might be more sensitive to the feeding of L-phenylalanine-supplemented diet than chicks hatched from pullets fed basal+ L-phenylalanine might be considered. Also the reverse occurrence of such observation between the first three hatches and the fourth hatch might have been caused by the pullet's ad libitum feeding as compared to limited feeding. Further investigation in this aspect is required. The 30 per cent reduction of the brain serotonin level confirms a previous finding of Pscheidt & Tamimie (1966) and is in agreement with that reported for rat (Wang et aL, 1962) where a 5 per cent L-phenylalanine-supplemented diet was fed. However, in the work of Pscheidt & Tamimie, a decreased level of serotonin in other strains of chicks fed 2, 4 and 8 per cent L-phenylalanine-supplemented diets was not obtained. This failure might indicate that the breed of chick employed plays an important part. The increased level of plasma phenylalanine of chicks fed a diet supplemented with 5 per cent L-phenylalanine confirms those findings obtained with dog (Hier, 1947), rat (Kumta & Harper, 1962), monkey (Waisman et al., 1959) and chicks (Dean & Scott, 1966). Finally, it can be concluded from the growth data that there was a maternal as well as dietary effect of feeding the 5 per cent z-phenylalanine-supplemented diet to chicks hatched from pullets fed the same level of L-phenylalanine. This effect shows that chicks hatched from pullets fed the L-phenylalanine-supplemented diet were more benefited by feeding the chicks' L-phenylalanine-supplemented diet than chicks hatched from pullets fed basal diets alone. In contrast, chicks hatched from pullets fed basal diet alone were more benefited by feeding the chicks' basal than chicks hatched from pullets fed basal + L-phenylalanine. In considering only the dietary effect the growth data reveal also that feeding 5 per cent L-phenylalanine-supplemented diet was more detrimental to the growth of chicks not only with chicks hatched from pullets fed limited amounts of diet but with chicks hatched from pullets fed ad lib. amounts of diet. REFERENCES BoGcs D. E. & WAISMANH. A. (1964) Influences of excess dietary phenylalanine on pregnant rats and their fetuses. Proc. Soc. exp. Biol. Med. 115, 407-410. DEAN W. F. & SCOTTH. M. (1966) Use of free amino acid concentrations in blood plasma of chicks to detect deficiencies and excesses of dietary amino acids. J. Nutr. 88, 75-83. HARPERA. E., BENTOND. A. & ELVEHJEMC. A. (1955) L-Leucine and isoleucine antagonist in the rat. Archs Biochem. Biophys. 57, 1-10. HIER S. W. (1947) Influence of ingestion of single amino acids on the blood level of free amino acids. J. blol. Chem. 171, 813-820. JONESJ. D. (1964) Lyslne-arginine antagonism in the chick. J. Nutr. 84, 313-321. K~MTA U. S. & HARPERA. E. (1962) Amino acid balance and imbalance---IX. Effect of amino acid imbalance on blood amino acid pattern. Proc. Soc. expt. Biol. Med. 110, 512-517.
EFFECTS OF FEEDING 5 PER CENT L-PHENYLALANINE TO CHICKS
425
McCAMAN M. W. & ROBINS E. (1962) Fluorometric method for the determination of phenylalanine in serum. 07. lab. din. Med. 59, 885-890. MEAD J. A. R. & FINGER K. F. (1961) A single extraction method for the determination of both norepinephrine and serotonin. Biochem. Pharmac. 6, 52-53. PSCHEIDT G. R. & HABERB. (1965) Regional distribution of dehydroxyphenylalanine and 5-hydroxytryptophan decarboxylase and of biogenic amines in the chicken central nervous system. 07. Neurochem. 12, 613-618. PSCHEIDTG. R. & HIMWlCHH. E. (1965) Chicken brain amines: Normal levels and effects of reserpine and monoamine oxidase inhibitors. Progress in Brain Research (Edited by HIMWICHW. A. & SCHAD~J. P.) Vol. 16, pp. 235-249. Elsevier, Amsterdam. PSCHEIDTG. R. & TAMIMIES . S. (1966) Brain amines and brain weights in growing chicks: Some normal values and effects of feeding excess dietary L-phenylalanine. Biochem. Pharmac. 15, 1629-1632. SHORE P. A. Sg OLIN J. S. (1958) Identification and chemical assay of norepinephrine in brain and other tissues, o7. Pharmacol. expt. Ther. 122, 295-300. SIEGEL S. (1956) Nonparametric Statistics for the Behavioral Sciences. pp. 116-127. McGraw-Hill, New York. TAMIMm H. S. & PSCHEIBTG. R. (1966a) Effect of feeding 5 per cent L-phenylalanine to young chicks. Comp. Biochem. Physiol. 17, 609-622. TAMIMIE H. S. & PSCHEIDTG. R. (1966b) Effect of feeding high levels of L-phenylalanine to day-old chicks and its relationship to sex and breed. Comp. Biochem. Physiol. 17, 1159-1166. TAMIMIE H. S. & PSCHEIDTG. R. (1966c) Influence of feeding 5 per cent L-phenylalanine on reproductive performance of pullets. Am. 07. Physiol. 211, 955-958. WAISMANH. A., WANGH. L., HARLOWH. & SPONHOLZR. R. (1959) Experimental phenylketonuria in the monkey. Proc. Soc. exp. Biol. Med. 101, 864--865. WANGH. L., HARWALIamV. H. & WAISMANH. A. (1962) Effect of dietary phenylalanine and tryptophan on brain serotonin. Archs Biochem. Biophys. 97, 181-184.
F E E D I N G 5 PER CENT L-PHENYLALANINE TO CHICKS OF P U L L E T S FED SAME LEVELS OF L-PHENYLALANINE H.S. TAMIMIE Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Illinois
(Received 26 April 1967) A b s t r a c t - - 1 . Feeding 5 per cent L-phenylalanine depressed the growth rate, weight gain and feed conversion in chicks hatched from pullets fed basal diet alone. The reverse occurred with chicks hatched from pullets fed basal + 5 per cent L-phenylalanine. 2. A maternal as well as dietary effect of feeding the 5 per cent L-phenylalanine-supplemented diet was noted with chicks hatched from pullets fed the same level of L-pheny!a!anine. 3. Regardless of the maternal dietary regimen, chicks fed the 5 per cent L-phenylalanine-supplemented diet had elevated levels of plasma phenylalanlne and reduced concentration of brain serotonln. Levels of norepinephrlne were not affected.
INTRODUCTION IN nMPLOYINGcommercial, day-old and 4-weekold, crossbred, male chicks in the study of experimental phenylketonuria, Tamimie & Pscheidt (1966a) found that feeding a diet containing 5 per cent L-phenylalanine for a period of 4 weeks caused the following conditions: depressed growth rate, lower feed conversion, poor motor co-ordination, reduced hemoglobin content of the blood, swollen hock, curled toes, enlarged crop, rough feather development and darker-color shank, face and eyelids. Similar physical alterations proportional to the amount of L-phenylalanine in the supplemented diets were seen in another commercial strain of crossbred males and inbred White Leghorn female chicks (Tamimie & Pscheidt, 1966b). The inbred females were more affected than the crossbred males. The serotonin concentration of the brain of chicks fed 5 per cent L-phenylalanine was reduced while the level of norepinephrine was unaffected (Pscheidt & Tamimie, 1966). This depression in serotonin, caused by the feeding of diets rich in phenylalanine, was also found in rats (Wang et al., 1962). Similarly, a growth depression was observed in rats (Harper et al., 1955) and in chicks (Jones, 1964; Tamimie & Pscheidt, 1966a, b) fed diets supplemented with high levels of amino acids. In contrast, the feeding of amino acids in excess amounts led to increased concentration of these amino acids in the blood of dog (Hier, 1947), rat (Kumta & Harper, 1962), monkey (Waisman et al., 1959) and chicks (Dean & Scott, 1966). Furthermore, the influence of high levels of L-phenylalanine included not only the aforementioned parameters, but also the reproductive performance of pregnant
418
H.S. TAMIMIE
and lactating rats (Boggs & Waisman, 1964). Also, in crossbred pullets, Tamimie & Pscheidt (1966c) noted that feeding a diet supplemented with 5 per cent L-phenylalanine decreased the egg weight and the percentage of hen day production, fertility, hatchability and hatching chick weight without, however, producing deformed viable chicks. The chicks obtained from these pullets were used in this study to determine the effect of maternal feeding on the growth performance, concentration of serotonin and norepinephrine, level of plasma phenylalanine and external defects of these chicks fed the same level (5 per cent) of L-phenylalanine as their maternal parents. Because of (a) the importance of norepinephrine and serotonin in the normal physiological processes of the brain, (b) the concentration of these two biogenic amines known more in adult chickens than in young chicks (Pscheidt & Haber, 1965), (c) the report of Pscheidt & Himwich (1965) that, in contrast to other species, adult chicken brain contains relatively large amounts of norepinephrine and (d) the depressive effect of feeding high levels of phenylalanine on the serotonin, the serotonin and norepinephrine contents of the brain in chicks fed the normal "basal" ration or that basal supplemented with 5 per cent L-phenylalanine were determined. Similarly, in view of the previously mentioned reports showing the influence of dietary amino acid supplementation on the plasma free amino acids of rats and chicks, the phenylalanine content of the blood of these two kinds of chicks was also determined. MATERIALS AND METHODS Four sub-groups of unsexed, day-old, crossbred chicks (sixty-four chicks total), hatched from pullets fed one of two kinds of diets, were used in this study. Three of these groups (BI, B2 and B3) were derived from pullets fed daily a limited amount (100 g/bird/20 hr) of a commercial 16 per cent protein, corn-soy basal diet for three consecutive 10-day periods. The fourth group (B4) were from pullets fed ad lib. for an additional 10 days. Chicks of the other four groups (LP1, LP~, LP3 and LP4) were obtained from pullets fed in similar pattern a basal diet +5 per cent L-phenylalanine for the first three 10-day periods and then fed the basal diet during the fourth 10-day period. In order to control the intake of an expensive L-phenylalanine-supplemented diet by the pullet, and in the meantime prevent the males from consuming such a diet without affecting the egg fertility, the pullets which produced the LP chicks were fed in a special manner. A simplified procedure was therefore developed in which the male was left with the pullets for a period of 4 hr (8 a.m.-12 noon) during which time the feed troughs were removed from the pens. On the basis of 120/g/pullet/24 hr, age 5-8 months, the aforementioned amount was calculated to be 100 g/puUet/20 hr. Equal numbers of chicks (four chicks per group) from each of the four hatches were selected, weighed, banded and distributed into four lots. Chicks from the basal-fed pullets (B) were fed either a commercial 19 per cent corn-soy starter ration, or the same starter supplemented with 5 per cent L-phenylalanine for a period of 3 weeks. Similarly, the chicks from pullets fed the 5 per cent L-phenylalanine-supplemented diets were also subdivided and fed one of the two diets for
EFFECTS OF FEEDING 5 PER CENT L - P H E N Y L A L A N I N E TO CHICKS
419
the same period of time. The chicks were reared in standard battery brooders with raised wire floors. Their weights were taken at weekly intervals. At 3 weeks of age, the study of each group was terminated, at which time the final weight was taken, and external defects were checked for swollen hock, curled toes and poor feather development. The coloration of shank, face and eyelid as compared to the control chicks was checked too. These defects were scored by an arbitrary score of 1-3 ; 1 = mild, 2 = medium and 3 ~- medium-severe. Feed conversions were computed from the grams of gain per grams of feed intake. Blood was withdrawn by heart puncture. The chicks were then weighed and decapitated, and the heart, liver, spleen, gizzard and intestine were weighed and examined for abnormal appearance. The serotonin and norepinephrine contents of the brain were determined by the method of Shore & Olin (1958) as modified by Mead & Finger (1961). The phenylalanine content of the blood was determined by the fluorometric procedure of McCaman & Robins (1962). Data analyses were computed by Mann-Whitney U- non-parametric statistic (1956), and "Student's" t-test (Table 5). RESULTS
As early as 1 week and continuing through the third week of experiment, it was found that feeding a commercial corn-soy basal diet supplemented with 5 per cent L-phenylalanine (LP) resulted in poorer growth than feeding the same basal diet alone (B) (Table 1). An improvement in third-week weight, weight gain and feed conversion was obtained by feeding the basal (B) as compared with the supplemented diet (LP) for the four hatching groups. This improvement held for chicks hatched from pullets fed a basal diet and with chicks hatched from pullets fed a supplemented diet. The third-week weight in the basal fed groups was significantly (P<0.001) greater than that of the L-phenylalanine supplemented groups. This finding was not only true for chicks hatched from basal fed pullets but for chicks hatched from pullets fed basal+5 per cent L-phenylalanine, during the three hatchings. Similar significant (P<0.001) improvement in third-week weight was observed in the fourth hatch. In studying the maternal effect, however, it was found that feeding the same chick's basal to the two types of chicks reveals that chicks hatched from pullets fed basal had a significantly (P<0.001) better third-week weight than that of chicks hatched from pullets fed basal + 5 per cent L-phenylalanine (Table 2). Such an observation was not only true for the first three hatches but for the fourth one too. The contrast occurred with the feeding of L-phenylalaninesupplemented diet. In this case chicks hatched from pullets fed the L-phenylalanine-supplemented diet were more benefited (P< 0.001) by feeding the chicks' L-phenylalanine-supplemented diet than chicks hatched from pullets fed basal alone. The latter observation reversed itself in the fourth hatch.
4-20
H . S . TAMIMIE
TABLE I p E ~ c T
OF 5 PER CENT L - P H E N Y L A L A N I N E ON THE GROWTH PERFORMANCE OF CHICKS HATCHED FROM PULLETS FED LIMITED AND a d l i b ~ / L m AMOUNTS OF DIET/DAY Basal (19 per cent protein) (B) No. of hatch
First three hatchest Fourth hatcht
B-groups*
LP-groups
Av. 3rdwk wt. (g)
Av. wt. gain (g)
Av. feed conversion
Av. 3rdwk wt. (g)
Av. wt. gain (g)
Av. feed convero sion
302 338
267 295
2"2 2"3
228 321
194 284
2"7 2"6
Basal + 5 per cent L-phenylalanine (LP) No. of hatch
First three hatches t Fourth hatch t
B-groups
LP-groups
Av. 3rdwk wt. (g)
Av. wt. gain (g)
Av. feed conversion
Av. 3rdwk wt. (g)
Av. wt. gain (g)
Av. feed conversion
178 202
130 159
3"2 3"0
196 191
162 154
3"0 3"0
* (B) and (LP) groups represent the chicks hatched from pullets fed basal or basal + 5 per cent L-phenylalanine respectively. Half of the chicks from each of the four hatches were placed on one of these dietary treatments. t See text (Materials and Methods).
Regardless of the maternal origin, the third-week weight in the fourth hatch as compared to the first three hatches showed (with the exception where T.-phenylalanine-supplemented diet was fed to the pullets and their chicks, L P - L P ) a significant ( P < 0-001) growth improvement in all the other groups. T h e reverse occurred in the exception group (Table 2). By the end of the third week a n u m b e r of chicks in each of the four groups fed the 5 per cent L-phenylalanine showed a n u m b e r of external defects, such as swelling of the hock, poor feather development and, in comparison to the control chicks, a darker-color shank, face and eyelid were observed (Table 3). In contrast, none of the chicks in the four groups fed the basal diet exhibited such conditions. T h i s was true whether the chicks were hatched from pullets fed basal or basal + 5 per cent L-phenylalanine. N o gross pathological conditions were observed in any of the internal organs of the chicks. However, the gizzards of the 5 per cent L-phenylalanine fed groups were dark green in color and had slight erosions. A very slight difference in the organ weights was observed among the four basal-fed or the 5 per cent L-phenylalanine-fed groups (Table 4).
EFFECTS
OF FEEDING
5 PER
CENT
L-PHENYLALANINE
421
TO CHICKS
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422
TABI~ 3 ~ T O T A L MALFORMED EXTERNAL SCORE OF CHICKS FED 5 PER L - P ~ A L A N I N E DIET
Hatching type
Poor feather development
B-LPt L P - L P ~:
14 22 §
Swollen hock
Discolored* face and eyelid
Discolored* shank
Total
10 14
8 12
50 70
18 20 [[
* T h e discoloration of face, eyelid and shank refers to darker color as compared to the same criteria in the control chicks. t B - L P corresponds to chicks hatched from pullets fed basal (B). These chicks were fed basal + 5 per cent L-phenylalanine. L P - L P corresponds to chicks fed similarly to * but were hatched from pullets fed basal + 5 per cent L-phenylalanine (LP). § Each chick in each group was scored individually (see text). [[ Each number indicates the total defects computed from scoring the four hatches of the two types of chicks. TABLE 4---THE THIRD-VCEEKORGAN~VEIGHTO F CHICKS* FED BASALOR BASAL ~-5 PER CENT L-PHENYLALANINE Brain
Heart
Liver
Wt. (g)
% of body wt.
Wt. (g)
% of body wt.
Wt. (g)
% of body wt.
1"70t 1"44~
0"56 0"75
2"03 1"52
0"70 0"78
7"60 4'67
0-25 0"24
Spleen
Gizzard
Wt. (g)
% of body wt.
Wt. (g)
0"38t 0"31.*
0"11 0"15
10"4 7"22
% of body wt.
Intestine Wt. (g)
% of body wt.
0-35
19"8
0"67
0"37
13"7
0"71
* Chicks hatched from pullets fed basal or basal + 5 per cent L-phenylalanine in limited and ad libitum amounts of diet/day. t Corresponds to chicks fed basal alone. Corresponds to chicks fed basal + 5 per cent L-phenylalanine. C h e m i c a l analyses s h o w e d that the chicks receiving the L-phenylalanine diet h a d elevated levels of this a m i n o acid in their plasma ( T a b l e 5). H o w e v e r , there was an extreme variability in the a m o u n t s f o u n d in different animals. F e e d i n g 5 per cent L-phenylalanine r e d u c e d the brain concentration of serotonin w i t h o u t h a v i n g a n y effect o n the c o n t e n t of n o r e p i n e p h r i n e ( T a b l e 5).
423
EFFECTS OF FEEDING 5 PER CENT L-PHENYLALANINETO CHICKS
TABLE 5--AWaAGE PLASMAPHENYLALANINELEVELSAND
CONCENTRATIONSOF BIOGENIC
AMINES I N BRAINS OF CHICKS FED A BASAL DIET COMPARED TO THOSE FED 5 PER CENT L-PHENYLALANINE
Group and diet
No. of animals
Plasma phenylalanine (mg/lO0 ml)
B-B + LP-B* basal B-LP + LP-LP t 5 per cent L-phcnylalanine Significance of difference between m e a n s
32 32
2.6 + 0.6 52+45 P<0.01
Serotonin (/zg/g) 0.99 + 0.23 0.62_+0.19 P<0.01
Norepinephrine (brg/g) 0.54 + 0.17 0.55_+0.15 N.S.
* B-B and LP-B correspond to half of chicks hatched from pullets fed basal (B) or basal + 5 per cent L-phenylalanine (LP). These two kinds of chicks were fed basal. Corresponds to the other half of chicks as in * but were fed basal + 5 per cent L-phenylalanine.
DISCUSSION This study confirms a previous report of Tamimie & Pseheidt (1966a) which showedthat feedingday-oldchicksa commercialcorn-soybasal diet supplemented with 5 per cent L-phenylalanine resulted in poor growth and poor feed conversion. A significant ( P < 0.001) difference in the third-week weight of chicks fed basal as compared to the feeding of basal + 5 per cent L-phenylalanine was obtained in the present study. This poor performance by feeding excess levels oi L-phenylalanine might have been caused by amino acid imbalance or by the toxicity of L-phenylalanine and/or its metabolites, or by the impairment of the diet palatability. Phenylalanine is known to be bitter. The reduction in total energy, protein and other micronutrients might have entered the picture of this study. Dietary additions of large amounts of individual amino acids caused growth depression in rats (Harper et al., 1955) and in chicks (Jones, 1964; Tamimie & Pscheidt, 1966a, b). This investigation reveals also that a significant ( P < 0.001) difference due to maternal feeding was observed not only in chicks fed basal diet, but in chicks ied basal + 5 per cent L-phenylalanine. In this case, chicks hatched from basal-fed pullets were more benefited by feeding chick's basal than chicks hatched from pullets fed basal + 5 per cent L-phenylalanine. Tamimie & Pscheidt (1966c) noted, along with numerous detrimental effects of feeding 5 per cent L-phenylalanine-supplemented diet to laying pullets, a reduction of 4-8 g. in the hatching weight of chicks obtained from these pullets in four consecutive incubations. Thus, it is possible that such effect of feeding the L-phenylalanine-supplemented diet might have extended its influence beyond the first day of life, by showing that chicks hatched from pullets fed the L-phenylalanine-supplemented diet had a lower third-week weight upon the feeding of chicks' basal than chicks hatched from puUets fed basal alone. However, and for unknown reason(s), the
424
H.S. TAMIMIE
reverse occurred by feeding the same types of chicks the L-phenylalanine-supplemented diet. Similarly, and for unknown reason(s), the latter observation reversed itself with chicks hatched (fourth hatch) from pullets fed basal in ad libitum amounts. The possibility(ies) that chicks hatched from pullets fed only basal might be more sensitive to the feeding of L-phenylalanine-supplemented diet than chicks hatched from pullets fed basal+ L-phenylalanine might be considered. Also the reverse occurrence of such observation between the first three hatches and the fourth hatch might have been caused by the pullet's ad libitum feeding as compared to limited feeding. Further investigation in this aspect is required. The 30 per cent reduction of the brain serotonin level confirms a previous finding of Pscheidt & Tamimie (1966) and is in agreement with that reported for rat (Wang et aL, 1962) where a 5 per cent L-phenylalanine-supplemented diet was fed. However, in the work of Pscheidt & Tamimie, a decreased level of serotonin in other strains of chicks fed 2, 4 and 8 per cent L-phenylalanine-supplemented diets was not obtained. This failure might indicate that the breed of chick employed plays an important part. The increased level of plasma phenylalanine of chicks fed a diet supplemented with 5 per cent L-phenylalanine confirms those findings obtained with dog (Hier, 1947), rat (Kumta & Harper, 1962), monkey (Waisman et al., 1959) and chicks (Dean & Scott, 1966). Finally, it can be concluded from the growth data that there was a maternal as well as dietary effect of feeding the 5 per cent z-phenylalanine-supplemented diet to chicks hatched from pullets fed the same level of L-phenylalanine. This effect shows that chicks hatched from pullets fed the L-phenylalanine-supplemented diet were more benefited by feeding the chicks' L-phenylalanine-supplemented diet than chicks hatched from pullets fed basal diets alone. In contrast, chicks hatched from pullets fed basal diet alone were more benefited by feeding the chicks' basal than chicks hatched from pullets fed basal + L-phenylalanine. In considering only the dietary effect the growth data reveal also that feeding 5 per cent L-phenylalanine-supplemented diet was more detrimental to the growth of chicks not only with chicks hatched from pullets fed limited amounts of diet but with chicks hatched from pullets fed ad lib. amounts of diet. REFERENCES BoGcs D. E. & WAISMANH. A. (1964) Influences of excess dietary phenylalanine on pregnant rats and their fetuses. Proc. Soc. exp. Biol. Med. 115, 407-410. DEAN W. F. & SCOTTH. M. (1966) Use of free amino acid concentrations in blood plasma of chicks to detect deficiencies and excesses of dietary amino acids. J. Nutr. 88, 75-83. HARPERA. E., BENTOND. A. & ELVEHJEMC. A. (1955) L-Leucine and isoleucine antagonist in the rat. Archs Biochem. Biophys. 57, 1-10. HIER S. W. (1947) Influence of ingestion of single amino acids on the blood level of free amino acids. J. blol. Chem. 171, 813-820. JONESJ. D. (1964) Lyslne-arginine antagonism in the chick. J. Nutr. 84, 313-321. K~MTA U. S. & HARPERA. E. (1962) Amino acid balance and imbalance---IX. Effect of amino acid imbalance on blood amino acid pattern. Proc. Soc. expt. Biol. Med. 110, 512-517.
EFFECTS OF FEEDING 5 PER CENT L-PHENYLALANINE TO CHICKS
425
McCAMAN M. W. & ROBINS E. (1962) Fluorometric method for the determination of phenylalanine in serum. 07. lab. din. Med. 59, 885-890. MEAD J. A. R. & FINGER K. F. (1961) A single extraction method for the determination of both norepinephrine and serotonin. Biochem. Pharmac. 6, 52-53. PSCHEIDT G. R. & HABERB. (1965) Regional distribution of dehydroxyphenylalanine and 5-hydroxytryptophan decarboxylase and of biogenic amines in the chicken central nervous system. 07. Neurochem. 12, 613-618. PSCHEIDTG. R. & HIMWlCHH. E. (1965) Chicken brain amines: Normal levels and effects of reserpine and monoamine oxidase inhibitors. Progress in Brain Research (Edited by HIMWICHW. A. & SCHAD~J. P.) Vol. 16, pp. 235-249. Elsevier, Amsterdam. PSCHEIDTG. R. & TAMIMIES . S. (1966) Brain amines and brain weights in growing chicks: Some normal values and effects of feeding excess dietary L-phenylalanine. Biochem. Pharmac. 15, 1629-1632. SHORE P. A. Sg OLIN J. S. (1958) Identification and chemical assay of norepinephrine in brain and other tissues, o7. Pharmacol. expt. Ther. 122, 295-300. SIEGEL S. (1956) Nonparametric Statistics for the Behavioral Sciences. pp. 116-127. McGraw-Hill, New York. TAMIMm H. S. & PSCHEIBTG. R. (1966a) Effect of feeding 5 per cent L-phenylalanine to young chicks. Comp. Biochem. Physiol. 17, 609-622. TAMIMIE H. S. & PSCHEIDTG. R. (1966b) Effect of feeding high levels of L-phenylalanine to day-old chicks and its relationship to sex and breed. Comp. Biochem. Physiol. 17, 1159-1166. TAMIMIE H. S. & PSCHEIDTG. R. (1966c) Influence of feeding 5 per cent L-phenylalanine on reproductive performance of pullets. Am. 07. Physiol. 211, 955-958. WAISMANH. A., WANGH. L., HARLOWH. & SPONHOLZR. R. (1959) Experimental phenylketonuria in the monkey. Proc. Soc. exp. Biol. Med. 101, 864--865. WANGH. L., HARWALIamV. H. & WAISMANH. A. (1962) Effect of dietary phenylalanine and tryptophan on brain serotonin. Archs Biochem. Biophys. 97, 181-184.