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Analysis of Liver Glycogen in Chicks
PHYSIOLOGY AND REPRODUCTION Analysis of Liver Glycogen in Chicks P. C. ALLEN and M. D. RUFF US Department ofAgriculture, Science and Education Administration, Agricultural Research, Animal Parasitology Institute, Poultry Parasitic Diseases Laboratory, Beltsville, Maryland 20705 (Received for publication December 29, 1980) ABSTRACT The glycogen levels in homogenates from livers from 3- to 4-week-old broilers were measured using alkaline digestion and ethanol precipitation followed by direct hydrolysis with amyloglucosidase. Glycogen was determined with glucose oxidase. Glycogen levels were not affected by rapid freezing in liquid nitrogen and storage at —IOC. Glycogen was stabile at 4 C for at least 1 hr; however, at room temperature, over 50% of the glycogen was lost within 1 hr. Homogenization with .05 M ethylenediaminetetraacetate (EDTA) prevented this loss. Liver glycogen decreased rapidly (within 1 hr) after feed withdrawal and was sometimes undetectable within 2 hr. There was considerable variation in glycogen levels among individual birds. Liver glycogen levels were up to 50% higher at 1100 than at 0800 hr. (Key words: chickens, liver glycogen) 1981 Poultry Science 60:2697-2700
INTRODUCTION Studies at our laboratory on the physiological changes in p o u l t r y caused by coccidiosis required t h e m e a s u r e m e n t of liver glycogen levels. It soon b e c a m e a p p a r e n t t h a t sample handling techniques and t h e n u t r i e n t status of t h e bird affected t h e glycogen levels measured in uninoculated controls. N u m e r o u s m e t h o d s for assaying liver glycogen in m a m m a l s have been described (van der Vies, 1 9 5 4 ; Pfleiderer, 1 9 6 3 ; J o h n s o n and Fusaro, 1 9 6 6 ; Orrell and Bueding, 1 9 6 4 ; Keppler and Decker, 1 9 7 4 ) . Reports on avian species are limited and only a few papers discuss factors such as tissue processing and bird m a n a g e m e n t t h a t affect levels of chick liver glycogen (Cleland and Bannister, 1 9 7 9 ; Huff et al, 1979). This r e p o r t describes effects of various sample handling and storage treatm e n t s o n glycogen values obtained and d e m o n strates t h e i m p o r t a n c e of maintaining chicks on feed up until t i m e of slaughter. MATERIALS AND METHODS Animals and Care. Chicks used in these experiments were 3- to 4-week-old broilers. T h e y were housed 5 t o 10 per cage on constant lighting, fed a standard starter ration, and given water ad libitum. In some experiments feed was removed for periods u p t o 3 hr before slaughter. F o u r t o 5 broilers composed each treatm e n t group.
Tissue Processing. Chicks were killed by cervical dislocation and liver tissue removed within 30 sec after d e a t h . Tissue was either quickly frozen in liquid N 2 or immediately homogenized (1 g in 15 ml liquid) with a Brinkm a n p o l y t r o n homogenizer in ice-cold H 2 0 or .05 M e t h y l e n e d i a m i n e t e t r a a c e t a t e (EDTA) (pH 5.9). Frozen tissues were stored at —10 C for 1 week t h e n homogenized in either H 2 0 or .05 M E D T A while still in t h e frozen state and aliquots analyzed immediately for glycogen. Homogenates of fresh tissue were analyzed for glycogen immediately or quick frozen on dry ice and stored at —10 C for a week. F r o z e n h o m o g e n a t e s were t h a w e d rapidly (within 3 min) and aliquots analyzed immediately for glycogen. Glycogen Analysis. H o m o g e n a t e aliquots (1 ml) were digested with 30% K O H at 100 C for 20 min and glycogen was isolated b y ethanol precipitation (Pfleiderer, 1 9 6 3 ) . T h e glycogen was suspended in 2 ml of .1 M acetate buffer pH 4 . 0 , and .75 ml aliquots reacted with .05 ml amyloglucosidase (Boehringer-Manheim Co., E.C. 3.2.1.3) (10 mg/ml ice-cold distilled H 2 0 ) for 1 hr (adapted from Keppler and Decker, 1 9 7 4 ) . T h e liberated glucose was assayed with glucose oxidase (Sigma Chemical Co., E.C. 1.1.3.4) according to M a t t e n h e i m e r ( 1 9 7 0 ) . Homogenates were analyzed for p r o t e i n according t o Lowry et al. ( 1 9 5 1 ) , and values for glycogen were expressed as micrograms per milligram protein.
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TABLE 1. Glycogen concentration (lig/mg protein) in homogenate after incubation at37C
TABLE 2. Glycogen concentration (lig/mg protein) in chick livers homogenized with distilled water and .05 M EDTA
Homogenate Time (hr)
H20 n=5
0 1 2 3
59.5 16.5 4.2 0
Homogenate
.05 M EDTA n =5 ± 11.0 a ± 7.4 b ± 1.9b ± Ob
103.5 89.0 94.3 78.7
± 46.2 a ± 26.5a ± 23.2 a ± 12.8 a
a' bValues within a column with the same letter are not significantly different (P<.05).
Data were evaluated by analysis of variance and the significance of differences between means determined using Duncan's multiple range test (P<.05). In this paper, trial refers to the repeat of the particular assay technique. Trials of different techniques were made on different birds and are not comparable. RESULTS AND DISCUSSION General Considerations. The methods employed in this study to isolate and hydrolyze glycogen are widely used and are ammenable to the processing of large numbers of samples. The use of homogenates is advantageous because analyses for protein, nucleic acids, lipid, dry weight, and some enzymes can be done on the same sample. The reliability of the procedures was checked three different ways. First, in five determinations of glycogen from a single chicken liver, the mean value was 78.4 ± 1.39 jug/mg protein with a coefficient of variation of 3.9%. Second, recovery of commercially available glycogen processed by the above procedure was 85.7 ± 3.0% (6 replicates). Third, amylogluco-
Trial
H2 O n=5
1 2
106.2 ± 19.6 a 185.0 ± 57.0 a
.05 M EDTA n=5 116.7 ± 5.1 a 153.4 ± 44.0 a
values withlin a row with the same letter are not
significantly different (P<.05).
sidase was added directly to the homogenate without alkaline digestion and the amount of glycogen compared with the amount recovered with alkaline digestion; recovery was 96.4 ± 5.8% (6 replicates). Preparation of Tissue. Liver glycogen in fresh H 2 0 homogenates was stabile for up to 1 hr when homogenates were held in ice. However, at 25 C the glycogen degraded rapidly. In fact, in one experiment, glycogen in 6 birds decreased an average of 54% in 20 min. It was hypothesized that activation of phosphorylases in homogenates caused the glycogen degradation and that EDTA would inhibit the degradation since EDTA binds Mg + + , an essential ion for activation of phosphorylase. Glycogen in H^O homogenates degraded rapidly at 37 C, whereas glycogen in EDTA homogenates remained stabile with a slight decrease after 3 hr (Table 1). The mean glycogen content of fresh H 2 0 homogenates was not significantly different from that of fresh homogenates containing .05 M EDTA (Table 2). Glycogen remained stable in H2O or EDTA homogenates stored at —10 C for a week and in liver tissue stored at —10 C
TABLE 3,, Effect of EDTA and storage conditions on liver glycogen content (lig/mg protein) Trial 1
Trial 2
Treatment
H,0 n=4
.05 M EDTA n=4
H20 n=5
.05 M EDTA n =5
Fresh homogenate Frozen homogenate Frozen tissue
162.7 ± 12.3 a 125.7 ± 15.9a 148.5 ± 12.7a
146.9 ± 13.7a
106.2 ± 19.6 a 123.7 +25.7a 106.1 + 11.4a
116.7 ± 5.1 a 131.6 ± 7 . 5 a 109.3 + 8.8 a
Values within a row with the same letter are not significantly different (P<.05).
LIVER GLYCOGEN IN CHICKS
2699
TABLE 4. Effects of short term feed withdrawal on glycogen concentrations in chick livers Glycogen (/ug/mg protein)
Time postprandial (hr)
Trial 1 n= 5
Trial 2 n=5
138.5 + 87.6 ± 36.0 ± 23.6 ±
13.6a 16.9b 9.5C 6.6C
139.0 73.5 37.0 9.0
Trial 3 n =5
± ± ± ±
17.0 a 17.6 b 7.8bc 2.9 C
124.6 40.9 0 0
+18.03 ± 24.0 b ± 0C ± oc
a,b,c Values within a column with the same letter are not significantly different (P<.05).
and subsequently homogenized with either distilled H 2 0 or EDTA (Table 3). Thus, liver homogenates may be stored frozen for later analyses provided they are thawed rapidly and analyzed immediately. Our findings for frozen tissue are similar to those of Cleland and Bannister (1979) who found no glycogen decrease after storage at —21 and —70 C. The range of liver glycogen levels reported here are similar to those found by Cleland and Bannister (1979), Seaton et al. (1978), Raheja and Linsheer (1978), and Huff et al. (1979). As noted by others (Cleland and Bannister, 1979), a large bird to bird variation in glycogen content was found within treatment groups. For example, in one group of 6 chicks glycogen values ranged from 67.2 to 181.9 Mg/mg protein. Nutrient State of the Birds. In preliminary unreported studies, chicks were removed from pens before slaughter, sometimes for periods of up to 2 hr. This procedure appeared to cause a loss of liver glycogen. In subsequent controlled studies on feed withdrawal, liver glycogen decreased when chicks were off feed for as
TABLE 5. Effect of time of day on levels of liver glycogen (ug/mg protein) in broilers Time Trial
0800 hr n=5
1 2
109.6 ± 7.9 a 123.7 ± 25.7 a
1100 hr n= 5 186.8 + 45.3 b 199.3 ± 44.3 b
ab ' Values within a row with the same letter are not significantly different (P*S.05).
short a time as 1 hr and glycogen was sometimes undetectable after 2 and 3 hr (Table 4). Therefore, to insure optimal levels of liver glycogen it appears critical to keep chicks on feed up until the time of slaughter. Such a procedure would be particularly important in the study of pathological conditions that tend to cause decreases in liver glycogen. Glycogen content of chick liver is known to vary considerably with diet (Seaton et al., 1978; Raheja and Linsheer, 1979; Rosebrough et al., 1978). In caged chicks where pecking orders have been established, the variation in rates of feeding may cause variations in levels of liver glycogen. Our findings that time of day influenced glycogen levels (Table 5) is in agreement with reports of others (Hazelwood, 1976; Twiest and Smith, 1970; Smith, 1972) that liver glycogen levels are affected by circadian rhythms in the chick. In summary, to assure optimal liver glycogen levels, chicks should be kept on feed up until the time of slaughter which clearly should occur from midmorning to midafternoon. If, because of large numbers of samples, analyses cannot be done at the time of slaughter, liver tissue or liver homogenates may be quickly frozen and stored at —10 C for later analysis. Use of .05 M EDTA in the homogenization medium is an effective measure against glycogen degradation by endogenous phosphorylases. ACKNOWLEDGMENTS The authors thank Lyndon Owens, Pamela Hawkins, and Constance Swann for able technical assistance. REFERENCES Cleland, M. E., and D. W. Bannister, 1979. Effects of
2700
ALLEN AND RUFF
storage conditions in vitro on the assay of chick liver glycogen. Brit. Poultry Sci. 20: 307-310. Hazelwood, R. L., 1976. Carbohydrate metabolism. In Avian physiology. 3rd ed. D. D. Sturkie, ed. Springer-Verlag, New York, NY. Huff, W. E., J. A. Doerr, and P. B. Hamilton, 1979. Decreased glycogen mobilization during ochratoxicosis in broiler chickens. Appl. Environ. Microbiol. 37:122-126. Johnson, J. A., and R. M. Fusaro, 1966. The quantitative enzymatic determination of animal liver glycogen. Anal. Biochem. 15:140—149. Keppler, D., and K. Decker, 1974. Glycogen. Pages 1171—1176 in Methoden des Enzymatischen Analyse. 2nd ed. Vol. II. H. U. Bergmeyer, ed. Verlan Chemie, Weinheim. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265275. Mattenheimer, H., 1970. Pages 107-109 in Micromethods for the clinical and biochemical laboratory. Ann Arbor Sci. Publ., Ann Arbor, MI. Orrell, S. A., and E. Bueding, 1964. A comparison of the products obtained by various procedures used
for the extraction of glycogen. J. Biol. Chem. 239:4021-4026. Pfleiderer, G., 1963. Glycogen. Pages 5 9 - 6 2 in Methods of enzymatic analysis. H. U. Bergmeyer, ed. Academic Press, New York, NY. Raheja, K. L., and W. G. Linsheer, 1978. Effect of dietary composition on liver glycogen accumulation and lipid metabolism in the hypothyroid chick (Gallus domesticus). Comp. Biochem. Physiol. 61A:31-34. Rosebrough, R. W., E. Geis, K. Henderson, and L. T. Frobish, 1978. Glycogen depletion and repletion in the chick. Poultry Sci. 57:1460-1462. Seaton, K. W., O. P. Thomas, R. M. Gous, and E. H. Bossard, 1978. The effect of diet on liver glycogen and body composition in the chick. Poultry Sci. 57:692-698. Smith, C.J.V., 1972. Blood glucose levels in young chickens: the influence of light regimes. Poultry Sci. 51:268-273. Twiest, G., and C. J. Smith, 1970. Circadian rhythm on blood glucose levels of chickens. Comp. Biochem. Physiol. 32:371-375. van der Vies, J., 1954. Two methods for the determination of glycogen in liver. Biochem. J. 57: 410-416.
INTRODUCTION Studies at our laboratory on the physiological changes in p o u l t r y caused by coccidiosis required t h e m e a s u r e m e n t of liver glycogen levels. It soon b e c a m e a p p a r e n t t h a t sample handling techniques and t h e n u t r i e n t status of t h e bird affected t h e glycogen levels measured in uninoculated controls. N u m e r o u s m e t h o d s for assaying liver glycogen in m a m m a l s have been described (van der Vies, 1 9 5 4 ; Pfleiderer, 1 9 6 3 ; J o h n s o n and Fusaro, 1 9 6 6 ; Orrell and Bueding, 1 9 6 4 ; Keppler and Decker, 1 9 7 4 ) . Reports on avian species are limited and only a few papers discuss factors such as tissue processing and bird m a n a g e m e n t t h a t affect levels of chick liver glycogen (Cleland and Bannister, 1 9 7 9 ; Huff et al, 1979). This r e p o r t describes effects of various sample handling and storage treatm e n t s o n glycogen values obtained and d e m o n strates t h e i m p o r t a n c e of maintaining chicks on feed up until t i m e of slaughter. MATERIALS AND METHODS Animals and Care. Chicks used in these experiments were 3- to 4-week-old broilers. T h e y were housed 5 t o 10 per cage on constant lighting, fed a standard starter ration, and given water ad libitum. In some experiments feed was removed for periods u p t o 3 hr before slaughter. F o u r t o 5 broilers composed each treatm e n t group.
Tissue Processing. Chicks were killed by cervical dislocation and liver tissue removed within 30 sec after d e a t h . Tissue was either quickly frozen in liquid N 2 or immediately homogenized (1 g in 15 ml liquid) with a Brinkm a n p o l y t r o n homogenizer in ice-cold H 2 0 or .05 M e t h y l e n e d i a m i n e t e t r a a c e t a t e (EDTA) (pH 5.9). Frozen tissues were stored at —10 C for 1 week t h e n homogenized in either H 2 0 or .05 M E D T A while still in t h e frozen state and aliquots analyzed immediately for glycogen. Homogenates of fresh tissue were analyzed for glycogen immediately or quick frozen on dry ice and stored at —10 C for a week. F r o z e n h o m o g e n a t e s were t h a w e d rapidly (within 3 min) and aliquots analyzed immediately for glycogen. Glycogen Analysis. H o m o g e n a t e aliquots (1 ml) were digested with 30% K O H at 100 C for 20 min and glycogen was isolated b y ethanol precipitation (Pfleiderer, 1 9 6 3 ) . T h e glycogen was suspended in 2 ml of .1 M acetate buffer pH 4 . 0 , and .75 ml aliquots reacted with .05 ml amyloglucosidase (Boehringer-Manheim Co., E.C. 3.2.1.3) (10 mg/ml ice-cold distilled H 2 0 ) for 1 hr (adapted from Keppler and Decker, 1 9 7 4 ) . T h e liberated glucose was assayed with glucose oxidase (Sigma Chemical Co., E.C. 1.1.3.4) according to M a t t e n h e i m e r ( 1 9 7 0 ) . Homogenates were analyzed for p r o t e i n according t o Lowry et al. ( 1 9 5 1 ) , and values for glycogen were expressed as micrograms per milligram protein.
2697
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ALLEN AND RUFF
TABLE 1. Glycogen concentration (lig/mg protein) in homogenate after incubation at37C
TABLE 2. Glycogen concentration (lig/mg protein) in chick livers homogenized with distilled water and .05 M EDTA
Homogenate Time (hr)
H20 n=5
0 1 2 3
59.5 16.5 4.2 0
Homogenate
.05 M EDTA n =5 ± 11.0 a ± 7.4 b ± 1.9b ± Ob
103.5 89.0 94.3 78.7
± 46.2 a ± 26.5a ± 23.2 a ± 12.8 a
a' bValues within a column with the same letter are not significantly different (P<.05).
Data were evaluated by analysis of variance and the significance of differences between means determined using Duncan's multiple range test (P<.05). In this paper, trial refers to the repeat of the particular assay technique. Trials of different techniques were made on different birds and are not comparable. RESULTS AND DISCUSSION General Considerations. The methods employed in this study to isolate and hydrolyze glycogen are widely used and are ammenable to the processing of large numbers of samples. The use of homogenates is advantageous because analyses for protein, nucleic acids, lipid, dry weight, and some enzymes can be done on the same sample. The reliability of the procedures was checked three different ways. First, in five determinations of glycogen from a single chicken liver, the mean value was 78.4 ± 1.39 jug/mg protein with a coefficient of variation of 3.9%. Second, recovery of commercially available glycogen processed by the above procedure was 85.7 ± 3.0% (6 replicates). Third, amylogluco-
Trial
H2 O n=5
1 2
106.2 ± 19.6 a 185.0 ± 57.0 a
.05 M EDTA n=5 116.7 ± 5.1 a 153.4 ± 44.0 a
values withlin a row with the same letter are not
significantly different (P<.05).
sidase was added directly to the homogenate without alkaline digestion and the amount of glycogen compared with the amount recovered with alkaline digestion; recovery was 96.4 ± 5.8% (6 replicates). Preparation of Tissue. Liver glycogen in fresh H 2 0 homogenates was stabile for up to 1 hr when homogenates were held in ice. However, at 25 C the glycogen degraded rapidly. In fact, in one experiment, glycogen in 6 birds decreased an average of 54% in 20 min. It was hypothesized that activation of phosphorylases in homogenates caused the glycogen degradation and that EDTA would inhibit the degradation since EDTA binds Mg + + , an essential ion for activation of phosphorylase. Glycogen in H^O homogenates degraded rapidly at 37 C, whereas glycogen in EDTA homogenates remained stabile with a slight decrease after 3 hr (Table 1). The mean glycogen content of fresh H 2 0 homogenates was not significantly different from that of fresh homogenates containing .05 M EDTA (Table 2). Glycogen remained stable in H2O or EDTA homogenates stored at —10 C for a week and in liver tissue stored at —10 C
TABLE 3,, Effect of EDTA and storage conditions on liver glycogen content (lig/mg protein) Trial 1
Trial 2
Treatment
H,0 n=4
.05 M EDTA n=4
H20 n=5
.05 M EDTA n =5
Fresh homogenate Frozen homogenate Frozen tissue
162.7 ± 12.3 a 125.7 ± 15.9a 148.5 ± 12.7a
146.9 ± 13.7a
106.2 ± 19.6 a 123.7 +25.7a 106.1 + 11.4a
116.7 ± 5.1 a 131.6 ± 7 . 5 a 109.3 + 8.8 a
Values within a row with the same letter are not significantly different (P<.05).
LIVER GLYCOGEN IN CHICKS
2699
TABLE 4. Effects of short term feed withdrawal on glycogen concentrations in chick livers Glycogen (/ug/mg protein)
Time postprandial (hr)
Trial 1 n= 5
Trial 2 n=5
138.5 + 87.6 ± 36.0 ± 23.6 ±
13.6a 16.9b 9.5C 6.6C
139.0 73.5 37.0 9.0
Trial 3 n =5
± ± ± ±
17.0 a 17.6 b 7.8bc 2.9 C
124.6 40.9 0 0
+18.03 ± 24.0 b ± 0C ± oc
a,b,c Values within a column with the same letter are not significantly different (P<.05).
and subsequently homogenized with either distilled H 2 0 or EDTA (Table 3). Thus, liver homogenates may be stored frozen for later analyses provided they are thawed rapidly and analyzed immediately. Our findings for frozen tissue are similar to those of Cleland and Bannister (1979) who found no glycogen decrease after storage at —21 and —70 C. The range of liver glycogen levels reported here are similar to those found by Cleland and Bannister (1979), Seaton et al. (1978), Raheja and Linsheer (1978), and Huff et al. (1979). As noted by others (Cleland and Bannister, 1979), a large bird to bird variation in glycogen content was found within treatment groups. For example, in one group of 6 chicks glycogen values ranged from 67.2 to 181.9 Mg/mg protein. Nutrient State of the Birds. In preliminary unreported studies, chicks were removed from pens before slaughter, sometimes for periods of up to 2 hr. This procedure appeared to cause a loss of liver glycogen. In subsequent controlled studies on feed withdrawal, liver glycogen decreased when chicks were off feed for as
TABLE 5. Effect of time of day on levels of liver glycogen (ug/mg protein) in broilers Time Trial
0800 hr n=5
1 2
109.6 ± 7.9 a 123.7 ± 25.7 a
1100 hr n= 5 186.8 + 45.3 b 199.3 ± 44.3 b
ab ' Values within a row with the same letter are not significantly different (P*S.05).
short a time as 1 hr and glycogen was sometimes undetectable after 2 and 3 hr (Table 4). Therefore, to insure optimal levels of liver glycogen it appears critical to keep chicks on feed up until the time of slaughter. Such a procedure would be particularly important in the study of pathological conditions that tend to cause decreases in liver glycogen. Glycogen content of chick liver is known to vary considerably with diet (Seaton et al., 1978; Raheja and Linsheer, 1979; Rosebrough et al., 1978). In caged chicks where pecking orders have been established, the variation in rates of feeding may cause variations in levels of liver glycogen. Our findings that time of day influenced glycogen levels (Table 5) is in agreement with reports of others (Hazelwood, 1976; Twiest and Smith, 1970; Smith, 1972) that liver glycogen levels are affected by circadian rhythms in the chick. In summary, to assure optimal liver glycogen levels, chicks should be kept on feed up until the time of slaughter which clearly should occur from midmorning to midafternoon. If, because of large numbers of samples, analyses cannot be done at the time of slaughter, liver tissue or liver homogenates may be quickly frozen and stored at —10 C for later analysis. Use of .05 M EDTA in the homogenization medium is an effective measure against glycogen degradation by endogenous phosphorylases. ACKNOWLEDGMENTS The authors thank Lyndon Owens, Pamela Hawkins, and Constance Swann for able technical assistance. REFERENCES Cleland, M. E., and D. W. Bannister, 1979. Effects of
2700
ALLEN AND RUFF
storage conditions in vitro on the assay of chick liver glycogen. Brit. Poultry Sci. 20: 307-310. Hazelwood, R. L., 1976. Carbohydrate metabolism. In Avian physiology. 3rd ed. D. D. Sturkie, ed. Springer-Verlag, New York, NY. Huff, W. E., J. A. Doerr, and P. B. Hamilton, 1979. Decreased glycogen mobilization during ochratoxicosis in broiler chickens. Appl. Environ. Microbiol. 37:122-126. Johnson, J. A., and R. M. Fusaro, 1966. The quantitative enzymatic determination of animal liver glycogen. Anal. Biochem. 15:140—149. Keppler, D., and K. Decker, 1974. Glycogen. Pages 1171—1176 in Methoden des Enzymatischen Analyse. 2nd ed. Vol. II. H. U. Bergmeyer, ed. Verlan Chemie, Weinheim. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265275. Mattenheimer, H., 1970. Pages 107-109 in Micromethods for the clinical and biochemical laboratory. Ann Arbor Sci. Publ., Ann Arbor, MI. Orrell, S. A., and E. Bueding, 1964. A comparison of the products obtained by various procedures used
for the extraction of glycogen. J. Biol. Chem. 239:4021-4026. Pfleiderer, G., 1963. Glycogen. Pages 5 9 - 6 2 in Methods of enzymatic analysis. H. U. Bergmeyer, ed. Academic Press, New York, NY. Raheja, K. L., and W. G. Linsheer, 1978. Effect of dietary composition on liver glycogen accumulation and lipid metabolism in the hypothyroid chick (Gallus domesticus). Comp. Biochem. Physiol. 61A:31-34. Rosebrough, R. W., E. Geis, K. Henderson, and L. T. Frobish, 1978. Glycogen depletion and repletion in the chick. Poultry Sci. 57:1460-1462. Seaton, K. W., O. P. Thomas, R. M. Gous, and E. H. Bossard, 1978. The effect of diet on liver glycogen and body composition in the chick. Poultry Sci. 57:692-698. Smith, C.J.V., 1972. Blood glucose levels in young chickens: the influence of light regimes. Poultry Sci. 51:268-273. Twiest, G., and C. J. Smith, 1970. Circadian rhythm on blood glucose levels of chickens. Comp. Biochem. Physiol. 32:371-375. van der Vies, J., 1954. Two methods for the determination of glycogen in liver. Biochem. J. 57: 410-416.