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Immunological Reactivity of Fowl Ferritin
1140
RESEAKCH NOTES
TABLE 2.—Performance of birds on constant and variable dietary protein regimens, compared Protein H e n " d a y '-I potion Constant Variable
75.9 76.5
M
Feed °rtailty ^ « c y % 17.8 21.4
kg./doz. 2.4 2.4
Av. egg si J" gm. 61.8 62.0
REFERENCES Jennings, R. C , and T. R. Morris, 1965. The recovery of laying birds from a period of low protein feeding. Brit. Poultry Sci. 6: 321-326. Waggle, D. H., and C. W. Deyoe, 1966. Relationship between protein level and amino acid composition of sorghum grain. Feedstuffs, 38: 18, 19. Waggle, D. H., C. W. Deyoe and P. E. Sanford, 1967. Relationship of protein level of sorghum grain to its nutritive value as measured by chick performance and amino acid composition. Poultry Sci. 46: 655-659. Waldroup, P. W., and R. H. Harms, 1964. The effect of dietary protein restriction of laying hens on subsequent performance. Poultry Sci. 4 3 : 792794.
IMMUNOLOGICAL REACTIVITY OF FOWL FERRITIN MOHENDRA J . G. S. MERRIMAN AND C. LE Q. DARCEL Animal Pathology Division, Health oj Animals Branch, Canada Department of Agriculture, Animal Diseases Research Institute (Western), Box 640, Lethbridge, Alberta, Canada (Received for publication May 25, 1970)
Ferritin appears in the plasma of man and animal in certain diseases, where it can be recognized by immunological procedures (Aungst, 1968). In avian erythroblastosis (Eb) there is elevation of plasma iron (Darcel and Bather, 1964) which it was suspected might be due to the presence of ferritin and it was thought that an immunological method might be required for its recognition. To this end, we have isolated fowl liver ferritin (Darcel, 1961) from normal East Lansing, Line 15 I, White Leghorns inoculated subcutaneously with 0.5 ml. iron-dextran1 24-48 hours before col1
Imferon, from Stevenson, Turner and Boyce, London, Ontario.
lecting the liver and compared it with liver ferritin from Eb birds. Rabbit antisera were prepared against fowl ferritin and against a purified preparation of horse spleen ferritin2 with the aid of Freund's' incomplete adjuvant.3 Standard immunodiffusion and immunoelectrophoretic techniques were followed using both agar gel and cellulose acetate as supporting media. Barbiturate buffer, pH 8.6, 0.075 ionic strength was employed. Immunoelectrophoresis in cellulose acetate was carried out with a
"Nutritional Biochemicals Corp., Cleveland, Ohio. "Difco Laboratories, Detroit, Michigan.
Downloaded from http://ps.oxfordjournals.org/ at Dalhousie University on May 19, 2015
no time dropped below 17 gm.; that could explain the lack of an effect of variable treatment on egg production. Waldroup and Harms (1964) and Jennings and Morris (1965) reported that layers responded immediately to an adequate level of protein after being on low-protein diets. Mortality caused generally by leukosis was 3.6% higher for birds on variable protein than for birds on constant protein (Table 2). No relationship between treatments and average egg size and between
treatments and feed efficiency were observed (Table 2). In this study frequent, short-term dietary protein variations had little effect on rate of lay, mortality, average egg size and feed efficiency.
1141
RESEARCH NOTES
:
I
>
FF
FF
FIG. 1. Immunoelectrophoretic analysis of fowl ferritin (FF) and horse ferritin (HF) run against fowl ferritin antiserum (FFA) and horse ferritin antiserum (HFA) in cellulose acetate. Arrow indicates point of origin. The electrophoresis band on the extreme right shows the migration of the fowl ferritin (FF). Staining was by Perls' method.
FIG. 2. Agar gel immunodiffusion of fowl ferritin (FF) and horse ferritin (HF) against fowl ferritin antiserum (FFA) and horse ferritin antiserum (HFA). Staining was by Perls' method.
Beckman Microzone apparatus1 (Merriman and Darcel, 1967). Protein arcs and zones were stained with Nigrosin. Perls' method was used to detect iron (Lillie, 1947). Study of our fowl ferritin preparation by electron microscope confirmed it to be composed of ferritin molecules (Yamamoto, 1969). The precipitin reactions produced by cellulose Immunoelectrophoresis and agar gel 4
Beckman Instruments Inc., Spinco Division, Palo Alto, California.
ACKNOWLEDGMENTS Our thanks are due to Mrs. Barbara Jones for the immunoelectrophoretic and immunodiffusion studies and to Miss Vivienne Smith and Mrs. Norma Dow for assistance with the preparation of ferritin. REFERENCES Aungst, C. W., 1968. Ferritin in body fluids. J. Lab. Clin. Med. 71: 517-522. Darcel, C. le Q., 1961. Avian erythroblastosis. IV.
Downloaded from http://ps.oxfordjournals.org/ at Dalhousie University on May 19, 2015
HF
Immunoelectrophoresis and immunodiffusion were identical. The results are illustrated in Figs. 1 and 2. It is clear that antiserum to horse ferritin does not react with fowl ferritin and vice versa in both immunodiffusion and immunoelectrophoretic procedures. When horse ferritin reacted with its homologous antiserum, only a single immunoprecipitin arc was produced. In both techniques the reaction between fowl ferritin and its corresponding antiserum produced three immunoprecipitin arcs which stained both for iron and protein even though the fowl ferritin moved as a single band in electrophoresis. It is possible that with the appropriate electrophoretic conditions one may be able to separate fowl ferritin into distinct electrophoretic bands since different forms of ferritin have already been found in man, rats, and horses (Zamari and Mason, 1968). Fowl ferritin obtained from Eb birds was indistinguishable by these techniques from ferritin prepared from normal livers. Ferritin was recoverable from the livers of Eb birds which had not been inoculated with iron-dextran but normal birds required prior inoculation with iron-dextran. The use of this anti-fowl ferritin rabbit serum in an attempt to identify fowl ferritin in the plasma of birds with Eb will be described in another paper (Darcel and Merriman, 1970).
1142
RESEARCH NOTES
A note on the separation of fowl hemoglobin, hematin and "ferritin" by paper electrophoresis. Can. J. Comp. Med. 25: 129-131. Darcel, C. le Q., and R. Bather, 1964. Iron metabolism in avian erythroblastosis. International Conference on Avian Tumor Viruses. National Cancer Institute Monograph, 17: 735-746. Darcel, C. le Q., and M. Merriman, 1970. Ferritin in the plasma of birds with erythroblastosis. In preparation.
Lillie, R. D., 1947. Histological Techniques and Practical Histochemistry. Blakiston Division, McGraw-Hill Book Co., New York. p. 244. Merriman, M., and C. le Q. Darcel, 1967. Electrophoretic and immunological studies of plasma a-lipoproteins in avian erythroblastosis. Can. J. Biochem. 45: 1468-1470. Yamamoto, T., 1969. Personal communication. Zamiri, I., and J. Mason, 1968. Electrophoresis of ferritins. Nature, 217: 2 58-259.
T. SELVARAJAH,* F. N. JEROME, J. D. SUMMERS AND B. S. REINHART Department of Poultry Science, University of Guelph, Guelph, Ontario, Canada (Received for publication June 10, 1970)
When dwarf hens reputed to lay normalsized eggs first appeared on the scene in the United States some twenty years ago, there was considerable speculation that dwarf hens, like dwarf maize, might play an important role in an increasingly efficient agricultural industry. Such speculation was heightened with enthusiasm when it was established that the type of dwarfing observed followed a Mendelian pattern of inheritance which was due to a single recessive sex-linked mutant gene (Hutt, 1949). Even though early reports were not consistent about the economic future of these dwarfs (Hutt, 19S9; and Bernier and Arscott, 1960, 1966) recent interest has been shown in these birds as a potential source of layers. Consequently, it was felt that some critical studies should be undertaken to evaluate the economic merit of the dwarfs from a genetic and nutritional standpoint. Hence, two experiments were conducted to investigate the effects of recessive sex-linked dwarfism in White Leghorn fowls. Experiment 1 was carried out to investigate the effect of two suitably chosen diets (19% protein and high energy * Present address: Poultry Research and Breeding Station, Johore Bahru, Malaysia.
vs. 16% protein and low energy) on the performance of dwarf and non-dwarf halfsibs and in Experiment 2 the comparative performance of dwarf and non-dwarf fullsibs sampled on the same environment. The results indicated that the dwarfs matured significantly later (8 days and 14 days later in Experiment 1 and 2 respectively) and were significantly smaller at 19 and 44 weeks of age. For the 24-week period, feed intake of the dwarfs was only 77 and 69% that of the non-dwarf in experiment 1 and 2 respectively, the differences between genotypes being highly significant. Average egg weight of the dwarf was 89 and 88% that of the non-dwarf in Experiment 1 and 2 respectively, the differences between genotypes being highly significant. Percent egg production (rate of lay on hen-day basis) was similar for both genotypes in experiment 1 (50.5 vs. 51.0% for the nondwarfs) but was significantly different in experiment 2 (43.5 vs. 58.1% for the nondwarfs). The dwarfs were more efficient in experiment 1 with feed per dozen eggs and feed mass/egg mass being significantly greater in the non-dwarfs (1.40 and 2.33 kg. vs. 2.09 and 3.13 kg. for the nondwarfs), while there were no significant differences in experiment 2 (1.54 and 2.62 kg.
Downloaded from http://ps.oxfordjournals.org/ at Dalhousie University on May 19, 2015
SOME EFFECTS OF SEX-LINKED DWARFISM IN LAYER-TYPE FOWLS
RESEAKCH NOTES
TABLE 2.—Performance of birds on constant and variable dietary protein regimens, compared Protein H e n " d a y '-I potion Constant Variable
75.9 76.5
M
Feed °rtailty ^ « c y % 17.8 21.4
kg./doz. 2.4 2.4
Av. egg si J" gm. 61.8 62.0
REFERENCES Jennings, R. C , and T. R. Morris, 1965. The recovery of laying birds from a period of low protein feeding. Brit. Poultry Sci. 6: 321-326. Waggle, D. H., and C. W. Deyoe, 1966. Relationship between protein level and amino acid composition of sorghum grain. Feedstuffs, 38: 18, 19. Waggle, D. H., C. W. Deyoe and P. E. Sanford, 1967. Relationship of protein level of sorghum grain to its nutritive value as measured by chick performance and amino acid composition. Poultry Sci. 46: 655-659. Waldroup, P. W., and R. H. Harms, 1964. The effect of dietary protein restriction of laying hens on subsequent performance. Poultry Sci. 4 3 : 792794.
IMMUNOLOGICAL REACTIVITY OF FOWL FERRITIN MOHENDRA J . G. S. MERRIMAN AND C. LE Q. DARCEL Animal Pathology Division, Health oj Animals Branch, Canada Department of Agriculture, Animal Diseases Research Institute (Western), Box 640, Lethbridge, Alberta, Canada (Received for publication May 25, 1970)
Ferritin appears in the plasma of man and animal in certain diseases, where it can be recognized by immunological procedures (Aungst, 1968). In avian erythroblastosis (Eb) there is elevation of plasma iron (Darcel and Bather, 1964) which it was suspected might be due to the presence of ferritin and it was thought that an immunological method might be required for its recognition. To this end, we have isolated fowl liver ferritin (Darcel, 1961) from normal East Lansing, Line 15 I, White Leghorns inoculated subcutaneously with 0.5 ml. iron-dextran1 24-48 hours before col1
Imferon, from Stevenson, Turner and Boyce, London, Ontario.
lecting the liver and compared it with liver ferritin from Eb birds. Rabbit antisera were prepared against fowl ferritin and against a purified preparation of horse spleen ferritin2 with the aid of Freund's' incomplete adjuvant.3 Standard immunodiffusion and immunoelectrophoretic techniques were followed using both agar gel and cellulose acetate as supporting media. Barbiturate buffer, pH 8.6, 0.075 ionic strength was employed. Immunoelectrophoresis in cellulose acetate was carried out with a
"Nutritional Biochemicals Corp., Cleveland, Ohio. "Difco Laboratories, Detroit, Michigan.
Downloaded from http://ps.oxfordjournals.org/ at Dalhousie University on May 19, 2015
no time dropped below 17 gm.; that could explain the lack of an effect of variable treatment on egg production. Waldroup and Harms (1964) and Jennings and Morris (1965) reported that layers responded immediately to an adequate level of protein after being on low-protein diets. Mortality caused generally by leukosis was 3.6% higher for birds on variable protein than for birds on constant protein (Table 2). No relationship between treatments and average egg size and between
treatments and feed efficiency were observed (Table 2). In this study frequent, short-term dietary protein variations had little effect on rate of lay, mortality, average egg size and feed efficiency.
1141
RESEARCH NOTES
:
I
>
FF
FF
FIG. 1. Immunoelectrophoretic analysis of fowl ferritin (FF) and horse ferritin (HF) run against fowl ferritin antiserum (FFA) and horse ferritin antiserum (HFA) in cellulose acetate. Arrow indicates point of origin. The electrophoresis band on the extreme right shows the migration of the fowl ferritin (FF). Staining was by Perls' method.
FIG. 2. Agar gel immunodiffusion of fowl ferritin (FF) and horse ferritin (HF) against fowl ferritin antiserum (FFA) and horse ferritin antiserum (HFA). Staining was by Perls' method.
Beckman Microzone apparatus1 (Merriman and Darcel, 1967). Protein arcs and zones were stained with Nigrosin. Perls' method was used to detect iron (Lillie, 1947). Study of our fowl ferritin preparation by electron microscope confirmed it to be composed of ferritin molecules (Yamamoto, 1969). The precipitin reactions produced by cellulose Immunoelectrophoresis and agar gel 4
Beckman Instruments Inc., Spinco Division, Palo Alto, California.
ACKNOWLEDGMENTS Our thanks are due to Mrs. Barbara Jones for the immunoelectrophoretic and immunodiffusion studies and to Miss Vivienne Smith and Mrs. Norma Dow for assistance with the preparation of ferritin. REFERENCES Aungst, C. W., 1968. Ferritin in body fluids. J. Lab. Clin. Med. 71: 517-522. Darcel, C. le Q., 1961. Avian erythroblastosis. IV.
Downloaded from http://ps.oxfordjournals.org/ at Dalhousie University on May 19, 2015
HF
Immunoelectrophoresis and immunodiffusion were identical. The results are illustrated in Figs. 1 and 2. It is clear that antiserum to horse ferritin does not react with fowl ferritin and vice versa in both immunodiffusion and immunoelectrophoretic procedures. When horse ferritin reacted with its homologous antiserum, only a single immunoprecipitin arc was produced. In both techniques the reaction between fowl ferritin and its corresponding antiserum produced three immunoprecipitin arcs which stained both for iron and protein even though the fowl ferritin moved as a single band in electrophoresis. It is possible that with the appropriate electrophoretic conditions one may be able to separate fowl ferritin into distinct electrophoretic bands since different forms of ferritin have already been found in man, rats, and horses (Zamari and Mason, 1968). Fowl ferritin obtained from Eb birds was indistinguishable by these techniques from ferritin prepared from normal livers. Ferritin was recoverable from the livers of Eb birds which had not been inoculated with iron-dextran but normal birds required prior inoculation with iron-dextran. The use of this anti-fowl ferritin rabbit serum in an attempt to identify fowl ferritin in the plasma of birds with Eb will be described in another paper (Darcel and Merriman, 1970).
1142
RESEARCH NOTES
A note on the separation of fowl hemoglobin, hematin and "ferritin" by paper electrophoresis. Can. J. Comp. Med. 25: 129-131. Darcel, C. le Q., and R. Bather, 1964. Iron metabolism in avian erythroblastosis. International Conference on Avian Tumor Viruses. National Cancer Institute Monograph, 17: 735-746. Darcel, C. le Q., and M. Merriman, 1970. Ferritin in the plasma of birds with erythroblastosis. In preparation.
Lillie, R. D., 1947. Histological Techniques and Practical Histochemistry. Blakiston Division, McGraw-Hill Book Co., New York. p. 244. Merriman, M., and C. le Q. Darcel, 1967. Electrophoretic and immunological studies of plasma a-lipoproteins in avian erythroblastosis. Can. J. Biochem. 45: 1468-1470. Yamamoto, T., 1969. Personal communication. Zamiri, I., and J. Mason, 1968. Electrophoresis of ferritins. Nature, 217: 2 58-259.
T. SELVARAJAH,* F. N. JEROME, J. D. SUMMERS AND B. S. REINHART Department of Poultry Science, University of Guelph, Guelph, Ontario, Canada (Received for publication June 10, 1970)
When dwarf hens reputed to lay normalsized eggs first appeared on the scene in the United States some twenty years ago, there was considerable speculation that dwarf hens, like dwarf maize, might play an important role in an increasingly efficient agricultural industry. Such speculation was heightened with enthusiasm when it was established that the type of dwarfing observed followed a Mendelian pattern of inheritance which was due to a single recessive sex-linked mutant gene (Hutt, 1949). Even though early reports were not consistent about the economic future of these dwarfs (Hutt, 19S9; and Bernier and Arscott, 1960, 1966) recent interest has been shown in these birds as a potential source of layers. Consequently, it was felt that some critical studies should be undertaken to evaluate the economic merit of the dwarfs from a genetic and nutritional standpoint. Hence, two experiments were conducted to investigate the effects of recessive sex-linked dwarfism in White Leghorn fowls. Experiment 1 was carried out to investigate the effect of two suitably chosen diets (19% protein and high energy * Present address: Poultry Research and Breeding Station, Johore Bahru, Malaysia.
vs. 16% protein and low energy) on the performance of dwarf and non-dwarf halfsibs and in Experiment 2 the comparative performance of dwarf and non-dwarf fullsibs sampled on the same environment. The results indicated that the dwarfs matured significantly later (8 days and 14 days later in Experiment 1 and 2 respectively) and were significantly smaller at 19 and 44 weeks of age. For the 24-week period, feed intake of the dwarfs was only 77 and 69% that of the non-dwarf in experiment 1 and 2 respectively, the differences between genotypes being highly significant. Average egg weight of the dwarf was 89 and 88% that of the non-dwarf in Experiment 1 and 2 respectively, the differences between genotypes being highly significant. Percent egg production (rate of lay on hen-day basis) was similar for both genotypes in experiment 1 (50.5 vs. 51.0% for the nondwarfs) but was significantly different in experiment 2 (43.5 vs. 58.1% for the nondwarfs). The dwarfs were more efficient in experiment 1 with feed per dozen eggs and feed mass/egg mass being significantly greater in the non-dwarfs (1.40 and 2.33 kg. vs. 2.09 and 3.13 kg. for the nondwarfs), while there were no significant differences in experiment 2 (1.54 and 2.62 kg.
Downloaded from http://ps.oxfordjournals.org/ at Dalhousie University on May 19, 2015
SOME EFFECTS OF SEX-LINKED DWARFISM IN LAYER-TYPE FOWLS