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Plasma Alkaline Phosphatase in the Fowl: Changes with Starvation
Plasma Alkaline Phosphatase in the Fowl: Changes with Starvation R. W. BIDE AND W. J. DOEWARD
Animal Pathology Division, Health of Animals Branch, Canada Department of Agriculture, Animal Diseases Research Institute (Western), P.O. Box 640, Lethbridge, Alberta, Canada (Received for publication November 27, 1969)
INTRODUCTION
F
MATERIALS AND METHODS
All chickens used in the study were White Leghorns of East Lansing Strain 15 I. The birds were raised to 6 weeks of age on Ottawa Starter Ration and then maintained on Ottawa Intermediate Rearing Ration custom made by Master Feeds Division of Maple Leaf Milling Company, Calgary, Alberta. Plasma was prepared from blood (1 ml.) drawn into heparinized syringes from wing veins. The plasma alkaline phosphatase levels
1 The 3 day gap is present because the laboratory works a 5-day week.
708
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 20, 2015
OR many years the changes in the normal plasma alkaline phosphatase which occur during growth and at the onset of lay, have been associated with bone formation and calcification (Common, 1936; Bell, 1959; Kuan, et al. 1966; Amador and Wacker, 1965; Yong, 1967; Hill and Sammons, 1967; Fishman and Gosh, 1967). However, when more direct methods were applied to the identification of the plasma isoenzymes (Bide, 1969), the plasma alkaline phosphatase was shown to be of intestinal and liver origin. Stutts et al. (1957), reported earlier that the plasma alkaline phosphatase levels were reduced by starvation. This observation and the apparent intestinal origin of the part of the plasma enzyme, would indicate that the plasma alkaline phosphatase levels may be related to the intestinal activity. This report describes the changes in the plasma alkaline phosphatases of the chicken that are induced by starvation.
and the percentage of the activity which was unchanged after 15 min. incubation with 4M urea (% urea-stable enzyme) were estimated using an automated system (Technicon AutoAnalyzer) described by Bide (1969), which is a modification of the Technicon N-6a phenyl phosphate method. The percentage urea-stable enzyme is a rough measure of the tissue source of the alkaline phosphatase isoenzymes as the bone, liver and intestinal isoenzymes are inactivated 100, 60 and 0% respectively by the urea treatment (Bide, 1969). Electrophoresis on vertical starch gels was used to check the isoenzyme differentiation data (Smithies 1959; Bide, 1969). The groups of experiments were performed using 10- and 15-week birds. In each experiment, on the first day, the birds were grouped 5 to a cage in large cages with ample trough space to avoid feeding problems arising from pecking order. Blood samples were taken that day, and on the third and fifth days. On the eighth day1 the feed was taken away from 25 birds and 10 birds were given feed ad libitum to act as controls. Blood samples were taken at that time and in the early morning of the ninth and tenth days. On the tenth day, after the blood samples were taken, the feed was replaced and blood samples were taken each hour for 6 hours. The feed consumption over the period was recorded. The total phosphatase and the % urea-stable isoenzyme were estimated on each blood sam-
PLASMA PHOSPHATASE AND STARVATION
pie. As the experiments progressed, groups of 5 birds were sacrificed for post-mortem examination. RESULTS
Effects of Starvation and Feeding on the
Plasma Alkaline Phosphatase. Experiment with 15-Week Birds. There was no significant change P = 0.10) in the alkaline phosphatase level and the proportion of urea-stable isoenzyme in these birds, either in the control groups maintained throughout the experiment, or in the experimental birds during the week prior to the experimental period (Table 1). The mean level of 1.11 ± 0.65 I.U.2/ml. was essentially the same as the values reported for this age group in the 15 I strain birds (Bide, 1969). Starvation caused a 48% decrease in the plasma alkaline phosphatase; the majority of the change occurred within 24 hours (Fig. 1). A change in the proportion of "intestinal" isoenzyme occurred at the same time as indicated by a significant drop (P = 0.01) in the % urea-stable isoenzyme (Table 1, Fig. 2). Electrophoresis of the plasmas indicated that the major isoenzyme remaining was of liver origin. When the starved birds were fed, the plasma alkaline phosphatase levels rose rapidly, in two hours the average value was 28% higher than the normal levels before starvation (Fig. 1). After 5 hours the level was again essentially normal. The proportion of the intestinal isoenzyme, however, did not alter significantly during the period. Thus, the increases observed were probably due to release of liver isoenzyme into the plasma. Experiments with 10-week birds. The plasma alkaline phosphatase levels in birds of this age group indicated that they were in a transition state between the high levels and the lower, constant levels in the 5-week and 15-week birds respectively (Bide, 1969; cf. also Common, 1936; Bell, 1959; Stutts et al. 1957). Thus the control values in these birds showed a rapid decline with fluctuations over the experimental period 2
lMmole/min. @37°C.
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 20, 2015
Clinical Observations. There were no changes in the actions or appearance of either the 10- or 15-week birds during the period of starvation even though the feces of all birds became watery and yellow probably due to the absence of solid materials. After the feed was replaced all of the birds ate immediately. By the end of the 6hour observation period most had taken two feedings. Postmortem examination of birds fasted for 48 hours showed that only remnants of the feed found in the gastrointestinal tract were some fibrous materials in the gizzard. One hour after feeding resumed the duodenum in most of the birds was full. After two hours the food had reached the ileum and in some cases the rectum, but the contents of the tract showed little evidence of digestion. After 3 hours the entire intestinal tract was full and digestion was in progress, as indicated by the presence of fecal material. However, the duodenum and jejunem were not distended as had been the case at two hours post-feeding. After four hours the entire tract was again distended with food and the contents were similar in appearance to those found in a normal chicken. Thus it would appear from gross examination of the alimentary tract that intestinal activity was restored three hours after the birds were fed following starvation. Histological examination of intestinal segments showed no pathological changes in the tissues and no signs of parasitemia were found. The feed consumption over the 6-hour period was 40 gm. and 59 gm./bird for the 10-week and 15-week birds respectively.
709
710
R. W. BIDE AND W. J. DORWARD TABLE 1.—Effects
of starvation and feeding on chicken plasma alkaline phosphatase Plasma alkaline phosphatase
Treatment
No of birds***
15-week birds
10-week birds
I.U./ml. I S t . dev.
% Urea-stable 1 St. dev.
I.U./ml. I S t . dev.
% Urea-stable 1 St. dev. 84.1111.2 77.2112.1 8 2 . 2 1 9.6 78.4110.3
50 50 50 50
1.11 + 0.62
6 4 . 7 1 9.1
1.24+0.32 1.09 + 0.50
62.4110.3 64.2 + 12.5
4.2214.0 2.5211.47 1.8210.81 2.0411.92
Starved 1 day Control
50 20
0.7110.54* 1.09 + 0.53
48.8+13.0* 63 7 1 1 5 . 0
2.1611.32* 3.1811.63
76.3115.4 80.7113.9
Starved 2 days Control
45 20
0.6910.30* 1.0810.38
4 9 . 2 1 9.6* 7 1 . 6 1 9.7
1.3411.18* 2.4911.60
67.3113.0* 8 5 . 0 1 7.2
Feeding trial Control
20
1.0810.42
7 1 . 5 1 9.6
1.5510.82
8 6 . 0 1 8.14
After food replaced 1 hour 2 hour 3 hour 4 hour 5 hour 6 hour
40 35 30 25 20 15
0.8310.21* 1.3911.49 1.1810.30 1.21 + 0.46 1.0510.27
52.2110.3* 47.6113.5* 45.9111.6* 4 8 . 3 1 7.5* 4 9 . 3 1 8.6*
1.0810.24* 1.1110.23* 1.2210.62 1.1810.48** 1.0410.16* 0.9810.17*
72.5112.5* 71.8+13.0* 76.6+11.7 6 7 . 0 1 8.6* 64.6+ 6.4* 66.415.6*
* Significant difference from control **5% * 1 % . The data are the average of two experiments. *** No. of birds was the same in experiments with both age groups.
characterized by high values of standard deviation (Table 1). On the other hand, the value of % urea-stable enzyme varied little from the average value of 80.1 ± 11.0% for the period. Starvation reduced the levels of alkaline phosphatase to 47 and 45% on the first and second day in these birds (Table 1, Fig. 1). However, the levels of urea-stable enzyme showed a mathematically significant change (P = 0.01) only after 48 hours of starvation (Table 1, Fig. 2), although a definite shift in the distribution of values had occurred after 24 hours as seen from the higher standard deviation of the data (Table 1). Three hours after the birds were fed the levels of the total and urea-stable enzymes had risen so that they were not significantly different from the control values (P = 0.10). However, one hour later both levels
had again fallen and continued significantly (P = 0.01) lower to the end of the experimental period. The distribution of the proportion of the urea-stable enzyme altered over the feeding period (Fig. 3) so that the value of the standard deviation changed from 17% of the values the first hour after feeding, to 8.5% of the values after 6 hours. DISCUSSION
The levels of plasma alkaline phosphatase in the chicken have been known for their high variations for some time. The presence of a large proportion of "intestinal" isoenzyme in the plasma and the connection observed here between starvation, feeding i.e. intestinal activity and the plasma alkaline phosphatase levels may account for a great deal of the variation (Table 1, Bell, 1959). Starvation caused marked changes in both the total enzyme
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 20, 2015
Before starving 7 days 5 days 3 days 0 days
711
PLASMA PHOSPHATASE AND STARVATION
Hours After Feeding
FIG. 1. Changes in the plasma alkaline phosphatase levels upon starvation and recovery. The birds were starved for 2 days and then fed for recovery. The lines show the response in (O) 10week and ( • ) lS-feed birds.
Hours After Feeding
FIG. 2. Changes in the proportion of urea-stable isoenzyme upon starvation and recovery. The birds were starved for 2 days and then fed. Fig. 2a shows the response found in 10-week birds and Fig. 2b the response in 15-week birds. Closed symbols represent control values, open symbols the treated birds and the terminated lines the standard deviation of the values.
sumed. In addition, there was a wide variation in the response of this parameter to feeding. In both age groups the variation in values was markedly increased immediately after feeding. In the 10-week birds only there was a definite increase in the level of the urea-stable "intestinal" isoenzyme in some of the birds, so that after 3 hours, two distinct populations could be mathematically defined (Fig. 3; P = 0.01). After 6 hours, however, this distinction was no longer present so that in both age groups there was only one population, that was much more uniform than any of the other groups described. There was no apparent reason for this segregation as it did not correspond to any of the usual biological parameters such as sex, size, etc. The Line IS I Leghorns used in this
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 20, 2015
levels and in the proportion of urea-stable "intestinal" isoenzymes. When feeding was resumed, further changes occurred in the plasma alkaline phosphatase levels over the 6 hour observation period. The increase observed after feeding did, however, coincide with the filling of the gastrointestinal tract and roughly with the initiation of digestion, as evidenced by the condition of the intestinal contents. The response of the two age groups after feeding was not the same (Fig. 1, 3). These differences in the recovery sequence may be attributed to the observation that the alkaline phosphatase levels in the 10-week birds were in a transition stage. The final level attained in the 10-week birds was essentially the same as the "normal" level in the 15-week birds and the starvation cycle may have simply accelerated the transition which was in progress. In both age groups, the proportion of urea-stable isoenzymes did not return to the original levels after feeding was re-
712
R. W. BIDE AND W. J. DORWARD
experiments are being contemplated. Similarly in disease studies, if the feed intake of the birds is reduced, there may be changes in the plasma alkaline phosphatase levels related to anorexia rather than to the direct action of the disease upon the body organs. SUMMARY
80
60
40
20
100
80
60
40
80
FIG. 3. The distribution of values of urea-stable isoenzyme. The graphs on the left show the response in 15-week birds; those on the right 10week birds, (a) shows the distribution in the "normal" population, (b) values after 48 hours starvation, (c) the values 2 hours after feeding, (d) 3 hours after feeding, (e) 6 hours after feeding (f) the distribution of values found with tissue extracts (from Bide, 1969).
study are a selected line which has both a higher level of plasma alkaline phosphatases and a higher proportion of the ureastable "intestinal" isoenzyme than other strains of Leghorns (Bide, 1969). Because of this difference, the results presented here may not be typical of other strains or breeds of chickens. The work described here leaves many questions unanswered. Although these problems, and the interesting aspects regarding the effects of diets on the levels of both the liver and intestinal alkaline phosphatase in the plasma could not be studied extensively in this pathology laboratory, several points previously mentioned should be stressed. The large proportion of the intestinal alkaline phosphatase in the plasma must be borne in mind when feeding or diet
ACKNOWLEDGMENTS
The authors wish to thank Mr. H. Klassen and Mr. H. Boumans for their technical assistance in this study and Dr. R. McKay of Animal Diseases Research Institute (Eastern), Hull, P.Q., for the histological examination carried out. Thansk are also due to Dr. S. E. Magwood for his assistance with the manuscript. REFERENCES Amador, E., and W. E. C. Wacker, 1965. Enzymatic methods used for diagnosis. Meth. Biochem. Anal. 13: 265-356.
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 20, 2015
100
The plasma alkaline phosphatase levels and the proportion of urea-stable "intestinal" isoenzyme in the plasma were followed in 10- and 15-week Leghorns of Strain IS I through a cycle of 48 hours starvation and subsequent recovery. Starvation reduced the plasma alkaline phosphatase levels by 45% and 48% respectively in the two groups and the proportion of urea-stable isoenzyme was significantly reduced. During recovery, the plasma alkaline phosphatase levels were restored only in the 15week age group. The levels of urea-stable isoenzyme did not return to the original or the control values during recovery. The changes noted, as well as the fact that there is a large proportion of the urea-stable "intestinal" isoenzyme in the plasma of these birds must be recognized and considered, whenever attempts are made to correlate the plasma alkaline phosphatase levels with pathology in disease states or with the results of nutrition experiments. •
PLASMA PHOSPHATASE AND STARVATION Bell, D. J., 1960. Tissue components of the domestic fowl 4. Plasma-alkaline phosphatase activity. Biochem. J. 75: 224-229. Bide, R. W., 1969. Plasma Alkaline Phosphatase in the Fowl: Differentiation of Tissue Isoenzyme by Urea. Technicon Symposia. Common, R. H., 1936. Serum phosphatase in the domestic fowl. J. Agric. Sci. 26: 492-508. Fishman, W. H., and N. K. Ghosh, 1967. Isoenzymes of human alkaline phosphatase. Adv. Clin. Chem. 10: 255-370. Hill, P. G., and R. C. Sammons, 1967. An interpretation of the elevation of serum alkaline phosphatase in disease. J. Clin. Path. 20: 654659.
713
Kuan, S. S., W. G. Martin and H. Patrick, 1966. Alkaline phosphatases of the chick. Partial characterization of the tissue isozymes. Proc. Soc. Exptl. Biol. Med. 122: 172-177. Saini, P. K., and S. Posen, 1969. The origin of serum alkaline phosphase in the rat. Biochem. Biophys. Acta, 177 : 42-49. Smithies, O., 1959. Zone electrophoresis in starch gels and its application to studies of serum proteins. Advances in Protein Chem. 14: 65-113. Stutts, E. C , W. E. Briles and H. O. Kunkel, 1957. Plasma alkaline phosphatase activity in mature inbred chickens. Poultry Sci. 36: 249-276. Yong, J. M., 1967. Origins of serum alkaline phosphatase. J. Clin. Path. 20: 647-653.
Department
A. E. WOODARD, H. ABPLANALP AND W. O. WILSON of Avian Sciences, University of California, Davis, California 95616 (Received for publication November 27, 1969)
R
EPRODUCTION in most wild passerine and gallinaceous birds is much the same in that most are seasonal layers and produce their young in the spring of the year, when favorable light and temperature conditions prevail. Under domestication, chukars (Alectoris graeca chukar) and pheasants (Phasianus colchicus) can be induced to lay in midwinter, rather than in the spring, provided the birds are preconditioned by short day lengths before being exposed to long daily photoperiods (Funk et al., 1942). Rowan (1929) demonstrated that repeated cycles of gonadal development could be induced in slate-colored juncos (Junco hyemalis) by subjecting these birds to alternating periods of short and long days. His results were substantiated by Wolfson (1954), who reported that multiple cycles of gonadal development, fat deposition, and periods of molt could be induced in juncos and whitecrowned sparrows (Zonotrichia leucophrys leucophrys) by subjecting these species to
alternating periods of short (9L:15D) and long (20L:4D) days. A lighting program which would induce chukars to lay more frequently than once a year would be highly acceptable to the game bird producer. It is conceivable that chukars may be used as an experimental animal in studying the effect of agricultural pesticides on reproduction in game birds. The purpose of the present study was to determine if chukar partridge, through manipulation of day length, could be induced to lay more frequently than once a year. PROCEDURE
Chukar chicks hatched in April, 1967, were brooded in a five-deck, commercial game-bird battery to 6 weeks of age. They were then moved to floor pens (6.2 m. X 10.0 m.) and reared under natural daylength to 28 weeks of age. Thereafter, all birds were maintained in modified chicken cages, one, two or three birds to a cage, depending upon the mating ratio. They were
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 20, 2015
Induced Cycles of Egg Production in the Chukar Partridge
Animal Pathology Division, Health of Animals Branch, Canada Department of Agriculture, Animal Diseases Research Institute (Western), P.O. Box 640, Lethbridge, Alberta, Canada (Received for publication November 27, 1969)
INTRODUCTION
F
MATERIALS AND METHODS
All chickens used in the study were White Leghorns of East Lansing Strain 15 I. The birds were raised to 6 weeks of age on Ottawa Starter Ration and then maintained on Ottawa Intermediate Rearing Ration custom made by Master Feeds Division of Maple Leaf Milling Company, Calgary, Alberta. Plasma was prepared from blood (1 ml.) drawn into heparinized syringes from wing veins. The plasma alkaline phosphatase levels
1 The 3 day gap is present because the laboratory works a 5-day week.
708
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 20, 2015
OR many years the changes in the normal plasma alkaline phosphatase which occur during growth and at the onset of lay, have been associated with bone formation and calcification (Common, 1936; Bell, 1959; Kuan, et al. 1966; Amador and Wacker, 1965; Yong, 1967; Hill and Sammons, 1967; Fishman and Gosh, 1967). However, when more direct methods were applied to the identification of the plasma isoenzymes (Bide, 1969), the plasma alkaline phosphatase was shown to be of intestinal and liver origin. Stutts et al. (1957), reported earlier that the plasma alkaline phosphatase levels were reduced by starvation. This observation and the apparent intestinal origin of the part of the plasma enzyme, would indicate that the plasma alkaline phosphatase levels may be related to the intestinal activity. This report describes the changes in the plasma alkaline phosphatases of the chicken that are induced by starvation.
and the percentage of the activity which was unchanged after 15 min. incubation with 4M urea (% urea-stable enzyme) were estimated using an automated system (Technicon AutoAnalyzer) described by Bide (1969), which is a modification of the Technicon N-6a phenyl phosphate method. The percentage urea-stable enzyme is a rough measure of the tissue source of the alkaline phosphatase isoenzymes as the bone, liver and intestinal isoenzymes are inactivated 100, 60 and 0% respectively by the urea treatment (Bide, 1969). Electrophoresis on vertical starch gels was used to check the isoenzyme differentiation data (Smithies 1959; Bide, 1969). The groups of experiments were performed using 10- and 15-week birds. In each experiment, on the first day, the birds were grouped 5 to a cage in large cages with ample trough space to avoid feeding problems arising from pecking order. Blood samples were taken that day, and on the third and fifth days. On the eighth day1 the feed was taken away from 25 birds and 10 birds were given feed ad libitum to act as controls. Blood samples were taken at that time and in the early morning of the ninth and tenth days. On the tenth day, after the blood samples were taken, the feed was replaced and blood samples were taken each hour for 6 hours. The feed consumption over the period was recorded. The total phosphatase and the % urea-stable isoenzyme were estimated on each blood sam-
PLASMA PHOSPHATASE AND STARVATION
pie. As the experiments progressed, groups of 5 birds were sacrificed for post-mortem examination. RESULTS
Effects of Starvation and Feeding on the
Plasma Alkaline Phosphatase. Experiment with 15-Week Birds. There was no significant change P = 0.10) in the alkaline phosphatase level and the proportion of urea-stable isoenzyme in these birds, either in the control groups maintained throughout the experiment, or in the experimental birds during the week prior to the experimental period (Table 1). The mean level of 1.11 ± 0.65 I.U.2/ml. was essentially the same as the values reported for this age group in the 15 I strain birds (Bide, 1969). Starvation caused a 48% decrease in the plasma alkaline phosphatase; the majority of the change occurred within 24 hours (Fig. 1). A change in the proportion of "intestinal" isoenzyme occurred at the same time as indicated by a significant drop (P = 0.01) in the % urea-stable isoenzyme (Table 1, Fig. 2). Electrophoresis of the plasmas indicated that the major isoenzyme remaining was of liver origin. When the starved birds were fed, the plasma alkaline phosphatase levels rose rapidly, in two hours the average value was 28% higher than the normal levels before starvation (Fig. 1). After 5 hours the level was again essentially normal. The proportion of the intestinal isoenzyme, however, did not alter significantly during the period. Thus, the increases observed were probably due to release of liver isoenzyme into the plasma. Experiments with 10-week birds. The plasma alkaline phosphatase levels in birds of this age group indicated that they were in a transition state between the high levels and the lower, constant levels in the 5-week and 15-week birds respectively (Bide, 1969; cf. also Common, 1936; Bell, 1959; Stutts et al. 1957). Thus the control values in these birds showed a rapid decline with fluctuations over the experimental period 2
lMmole/min. @37°C.
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 20, 2015
Clinical Observations. There were no changes in the actions or appearance of either the 10- or 15-week birds during the period of starvation even though the feces of all birds became watery and yellow probably due to the absence of solid materials. After the feed was replaced all of the birds ate immediately. By the end of the 6hour observation period most had taken two feedings. Postmortem examination of birds fasted for 48 hours showed that only remnants of the feed found in the gastrointestinal tract were some fibrous materials in the gizzard. One hour after feeding resumed the duodenum in most of the birds was full. After two hours the food had reached the ileum and in some cases the rectum, but the contents of the tract showed little evidence of digestion. After 3 hours the entire intestinal tract was full and digestion was in progress, as indicated by the presence of fecal material. However, the duodenum and jejunem were not distended as had been the case at two hours post-feeding. After four hours the entire tract was again distended with food and the contents were similar in appearance to those found in a normal chicken. Thus it would appear from gross examination of the alimentary tract that intestinal activity was restored three hours after the birds were fed following starvation. Histological examination of intestinal segments showed no pathological changes in the tissues and no signs of parasitemia were found. The feed consumption over the 6-hour period was 40 gm. and 59 gm./bird for the 10-week and 15-week birds respectively.
709
710
R. W. BIDE AND W. J. DORWARD TABLE 1.—Effects
of starvation and feeding on chicken plasma alkaline phosphatase Plasma alkaline phosphatase
Treatment
No of birds***
15-week birds
10-week birds
I.U./ml. I S t . dev.
% Urea-stable 1 St. dev.
I.U./ml. I S t . dev.
% Urea-stable 1 St. dev. 84.1111.2 77.2112.1 8 2 . 2 1 9.6 78.4110.3
50 50 50 50
1.11 + 0.62
6 4 . 7 1 9.1
1.24+0.32 1.09 + 0.50
62.4110.3 64.2 + 12.5
4.2214.0 2.5211.47 1.8210.81 2.0411.92
Starved 1 day Control
50 20
0.7110.54* 1.09 + 0.53
48.8+13.0* 63 7 1 1 5 . 0
2.1611.32* 3.1811.63
76.3115.4 80.7113.9
Starved 2 days Control
45 20
0.6910.30* 1.0810.38
4 9 . 2 1 9.6* 7 1 . 6 1 9.7
1.3411.18* 2.4911.60
67.3113.0* 8 5 . 0 1 7.2
Feeding trial Control
20
1.0810.42
7 1 . 5 1 9.6
1.5510.82
8 6 . 0 1 8.14
After food replaced 1 hour 2 hour 3 hour 4 hour 5 hour 6 hour
40 35 30 25 20 15
0.8310.21* 1.3911.49 1.1810.30 1.21 + 0.46 1.0510.27
52.2110.3* 47.6113.5* 45.9111.6* 4 8 . 3 1 7.5* 4 9 . 3 1 8.6*
1.0810.24* 1.1110.23* 1.2210.62 1.1810.48** 1.0410.16* 0.9810.17*
72.5112.5* 71.8+13.0* 76.6+11.7 6 7 . 0 1 8.6* 64.6+ 6.4* 66.415.6*
* Significant difference from control **5% * 1 % . The data are the average of two experiments. *** No. of birds was the same in experiments with both age groups.
characterized by high values of standard deviation (Table 1). On the other hand, the value of % urea-stable enzyme varied little from the average value of 80.1 ± 11.0% for the period. Starvation reduced the levels of alkaline phosphatase to 47 and 45% on the first and second day in these birds (Table 1, Fig. 1). However, the levels of urea-stable enzyme showed a mathematically significant change (P = 0.01) only after 48 hours of starvation (Table 1, Fig. 2), although a definite shift in the distribution of values had occurred after 24 hours as seen from the higher standard deviation of the data (Table 1). Three hours after the birds were fed the levels of the total and urea-stable enzymes had risen so that they were not significantly different from the control values (P = 0.10). However, one hour later both levels
had again fallen and continued significantly (P = 0.01) lower to the end of the experimental period. The distribution of the proportion of the urea-stable enzyme altered over the feeding period (Fig. 3) so that the value of the standard deviation changed from 17% of the values the first hour after feeding, to 8.5% of the values after 6 hours. DISCUSSION
The levels of plasma alkaline phosphatase in the chicken have been known for their high variations for some time. The presence of a large proportion of "intestinal" isoenzyme in the plasma and the connection observed here between starvation, feeding i.e. intestinal activity and the plasma alkaline phosphatase levels may account for a great deal of the variation (Table 1, Bell, 1959). Starvation caused marked changes in both the total enzyme
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 20, 2015
Before starving 7 days 5 days 3 days 0 days
711
PLASMA PHOSPHATASE AND STARVATION
Hours After Feeding
FIG. 1. Changes in the plasma alkaline phosphatase levels upon starvation and recovery. The birds were starved for 2 days and then fed for recovery. The lines show the response in (O) 10week and ( • ) lS-feed birds.
Hours After Feeding
FIG. 2. Changes in the proportion of urea-stable isoenzyme upon starvation and recovery. The birds were starved for 2 days and then fed. Fig. 2a shows the response found in 10-week birds and Fig. 2b the response in 15-week birds. Closed symbols represent control values, open symbols the treated birds and the terminated lines the standard deviation of the values.
sumed. In addition, there was a wide variation in the response of this parameter to feeding. In both age groups the variation in values was markedly increased immediately after feeding. In the 10-week birds only there was a definite increase in the level of the urea-stable "intestinal" isoenzyme in some of the birds, so that after 3 hours, two distinct populations could be mathematically defined (Fig. 3; P = 0.01). After 6 hours, however, this distinction was no longer present so that in both age groups there was only one population, that was much more uniform than any of the other groups described. There was no apparent reason for this segregation as it did not correspond to any of the usual biological parameters such as sex, size, etc. The Line IS I Leghorns used in this
Downloaded from http://ps.oxfordjournals.org/ at UPVA on April 20, 2015
levels and in the proportion of urea-stable "intestinal" isoenzymes. When feeding was resumed, further changes occurred in the plasma alkaline phosphatase levels over the 6 hour observation period. The increase observed after feeding did, however, coincide with the filling of the gastrointestinal tract and roughly with the initiation of digestion, as evidenced by the condition of the intestinal contents. The response of the two age groups after feeding was not the same (Fig. 1, 3). These differences in the recovery sequence may be attributed to the observation that the alkaline phosphatase levels in the 10-week birds were in a transition stage. The final level attained in the 10-week birds was essentially the same as the "normal" level in the 15-week birds and the starvation cycle may have simply accelerated the transition which was in progress. In both age groups, the proportion of urea-stable isoenzymes did not return to the original levels after feeding was re-
712
R. W. BIDE AND W. J. DORWARD
experiments are being contemplated. Similarly in disease studies, if the feed intake of the birds is reduced, there may be changes in the plasma alkaline phosphatase levels related to anorexia rather than to the direct action of the disease upon the body organs. SUMMARY
80
60
40
20
100
80
60
40
80
FIG. 3. The distribution of values of urea-stable isoenzyme. The graphs on the left show the response in 15-week birds; those on the right 10week birds, (a) shows the distribution in the "normal" population, (b) values after 48 hours starvation, (c) the values 2 hours after feeding, (d) 3 hours after feeding, (e) 6 hours after feeding (f) the distribution of values found with tissue extracts (from Bide, 1969).
study are a selected line which has both a higher level of plasma alkaline phosphatases and a higher proportion of the ureastable "intestinal" isoenzyme than other strains of Leghorns (Bide, 1969). Because of this difference, the results presented here may not be typical of other strains or breeds of chickens. The work described here leaves many questions unanswered. Although these problems, and the interesting aspects regarding the effects of diets on the levels of both the liver and intestinal alkaline phosphatase in the plasma could not be studied extensively in this pathology laboratory, several points previously mentioned should be stressed. The large proportion of the intestinal alkaline phosphatase in the plasma must be borne in mind when feeding or diet
ACKNOWLEDGMENTS
The authors wish to thank Mr. H. Klassen and Mr. H. Boumans for their technical assistance in this study and Dr. R. McKay of Animal Diseases Research Institute (Eastern), Hull, P.Q., for the histological examination carried out. Thansk are also due to Dr. S. E. Magwood for his assistance with the manuscript. REFERENCES Amador, E., and W. E. C. Wacker, 1965. Enzymatic methods used for diagnosis. Meth. Biochem. Anal. 13: 265-356.
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The plasma alkaline phosphatase levels and the proportion of urea-stable "intestinal" isoenzyme in the plasma were followed in 10- and 15-week Leghorns of Strain IS I through a cycle of 48 hours starvation and subsequent recovery. Starvation reduced the plasma alkaline phosphatase levels by 45% and 48% respectively in the two groups and the proportion of urea-stable isoenzyme was significantly reduced. During recovery, the plasma alkaline phosphatase levels were restored only in the 15week age group. The levels of urea-stable isoenzyme did not return to the original or the control values during recovery. The changes noted, as well as the fact that there is a large proportion of the urea-stable "intestinal" isoenzyme in the plasma of these birds must be recognized and considered, whenever attempts are made to correlate the plasma alkaline phosphatase levels with pathology in disease states or with the results of nutrition experiments. •
PLASMA PHOSPHATASE AND STARVATION Bell, D. J., 1960. Tissue components of the domestic fowl 4. Plasma-alkaline phosphatase activity. Biochem. J. 75: 224-229. Bide, R. W., 1969. Plasma Alkaline Phosphatase in the Fowl: Differentiation of Tissue Isoenzyme by Urea. Technicon Symposia. Common, R. H., 1936. Serum phosphatase in the domestic fowl. J. Agric. Sci. 26: 492-508. Fishman, W. H., and N. K. Ghosh, 1967. Isoenzymes of human alkaline phosphatase. Adv. Clin. Chem. 10: 255-370. Hill, P. G., and R. C. Sammons, 1967. An interpretation of the elevation of serum alkaline phosphatase in disease. J. Clin. Path. 20: 654659.
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Kuan, S. S., W. G. Martin and H. Patrick, 1966. Alkaline phosphatases of the chick. Partial characterization of the tissue isozymes. Proc. Soc. Exptl. Biol. Med. 122: 172-177. Saini, P. K., and S. Posen, 1969. The origin of serum alkaline phosphase in the rat. Biochem. Biophys. Acta, 177 : 42-49. Smithies, O., 1959. Zone electrophoresis in starch gels and its application to studies of serum proteins. Advances in Protein Chem. 14: 65-113. Stutts, E. C , W. E. Briles and H. O. Kunkel, 1957. Plasma alkaline phosphatase activity in mature inbred chickens. Poultry Sci. 36: 249-276. Yong, J. M., 1967. Origins of serum alkaline phosphatase. J. Clin. Path. 20: 647-653.
Department
A. E. WOODARD, H. ABPLANALP AND W. O. WILSON of Avian Sciences, University of California, Davis, California 95616 (Received for publication November 27, 1969)
R
EPRODUCTION in most wild passerine and gallinaceous birds is much the same in that most are seasonal layers and produce their young in the spring of the year, when favorable light and temperature conditions prevail. Under domestication, chukars (Alectoris graeca chukar) and pheasants (Phasianus colchicus) can be induced to lay in midwinter, rather than in the spring, provided the birds are preconditioned by short day lengths before being exposed to long daily photoperiods (Funk et al., 1942). Rowan (1929) demonstrated that repeated cycles of gonadal development could be induced in slate-colored juncos (Junco hyemalis) by subjecting these birds to alternating periods of short and long days. His results were substantiated by Wolfson (1954), who reported that multiple cycles of gonadal development, fat deposition, and periods of molt could be induced in juncos and whitecrowned sparrows (Zonotrichia leucophrys leucophrys) by subjecting these species to
alternating periods of short (9L:15D) and long (20L:4D) days. A lighting program which would induce chukars to lay more frequently than once a year would be highly acceptable to the game bird producer. It is conceivable that chukars may be used as an experimental animal in studying the effect of agricultural pesticides on reproduction in game birds. The purpose of the present study was to determine if chukar partridge, through manipulation of day length, could be induced to lay more frequently than once a year. PROCEDURE
Chukar chicks hatched in April, 1967, were brooded in a five-deck, commercial game-bird battery to 6 weeks of age. They were then moved to floor pens (6.2 m. X 10.0 m.) and reared under natural daylength to 28 weeks of age. Thereafter, all birds were maintained in modified chicken cages, one, two or three birds to a cage, depending upon the mating ratio. They were
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Induced Cycles of Egg Production in the Chukar Partridge