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Changes in Plasma Calcium Concentration During Egg Formation
ADVANCES IN PHYSIOLOGY
Poultry Sci. 31: 227. Weiss, H. S., R. K. Ringer and P. D. Sturkie, 1957. Development of the sex difference in blood pressure of the chick. Am. J. Physiol. 188: 383. Weiss, H. S., and M. Sheahan, 1958. The influence of maturity and sex on the blood pressure of the turkey. Am. J. Vet. Res. 19: 209. Winget, C. M., and A. H. Smith, 1957. Blood calcium and shell formation in the laying hen. Poultry Sci., 36: 1169. Wingstrand, K. G., 1951. The Structure and Development of the Avian Pituitary. C. W. K. Gleerup/Lund, Sweden.
Changes in Plasma Calcium Concentration During Egg Formation CHARLES M. WINGET AND ARTHUR H. SMITH Department of Poultry Husbandry, University of California, Davis (Received for publication October 7. 1957)
D
URING egg formation, large quantities of calcium are deposited as carbonate on the developing shell. The site of shell formation, the oviduct shell gland, is not a calcium storage organ; consequently, calcium must be mobilized rapidly from body stores to meet the periodic requirements of shell formation. Since the body calcium is located almost entirely in osseous tissue, it must pass through the circulatory system in rather large quantities during each period of active shell formation (about 14 hours; Bradfield, 1951). There is also an increase in calcium absorption from the gut during shell formation (Buckner et al., 1930), which contributes to the fluctuation in blood calcium concentration. The literature contains several reports of the influence of shell formation on blood calcium. Charles and Hogben (1933) found that plasma calcium concentration during egg formation was about 20% higher than during non-laying periods. However, their data for hens with a form-
ing egg present in the oviduct varied widely. Diurnal variations in the blood calcium level of laying hens were found by Rochlina (1934). The birds were bled three times daily, morning, midday, and evening. The maximum blood calcium values (about 35% greater than the minimum) were found in the midday samples. Low levels in the plasma calcium of laying hens, associated with shell mineralization, and relatively higher levels when the shell gland was not active in shell formation have also been reported (Kno wles etal., 1935). Recently, Polin and Sturkie (1957) studied the concentrations of total and diffusible plasma calcium and related them to the condition of eggs present in the oviduct. Only small differences were noted between hens containing no egg and those with a soft- or hard-shelled egg in the oviduct. The experiments reported herein are concerned with changes in the plasma
Downloaded from http://ps.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 9, 2015
tionship between individual laying hens. J. Agr. Sci. 48: 171. Vanstone, W. E., W. A. Maw and R. H. Common, 1955. Levels and partition of the fowl's serum proteins in relation to age and egg production. Canad. J. Biochem. Physiol. 33: 891. Vanstone, W. E., D. G. Dale, W. F. Oliver and R. H. Common, 1957. Sites of formation of plasma phosphoprotein and phospholipid in the estrogenized cockerel. Canad. J. Biochem. Physiol. 35: 659. Weiss, H. S., and P. D. Sturkie, 1952. Time of oviposition as affected by neuromimetric drugs.
509
510
C. M . WlNGET AND A . H . SMITH
calcium concentration at various times in the laying cycle. METHOD
RESULTS
The concentrations of total diffusible and nondiffusible plasma calcium at various stages of egg formation are given in Table 1. The total plasma calcium remains fairly constant from the time of oviposition until onset of active shell forma-
TABLE 1.—Plasma calcium concentration in laying hens at specific times after oviposit Time since last laying; • position of egg 0-1 hr. No egg 4hr. Isth.
Total calcium
Diffusible calcium As % of total calcium
Nondiffusible calcium1 mg. %
.1
32.3 + 1.5
22.5 + 2.7
(8)
10.4+ .2
38.7 + 1.3
16.3 + 1.0
No. hens
mg-%
No. hens
(9)
26.4 + 1.02
(7)
26.1±
.6
(12)
mg. % 8.7+
10 hr. No egg
(4)
28.1 + 1.5
(3)
13.2+ .3
48.3+4.4
14.5 + 2.2
10 hr. Ut. S.S.3
(5)
26.6 + 1.4
(3)
11.8+ .3
47.5 + 4.6
13.5 + 2.3
16 hr. Ut. egg
(7)
24.2 + 1.5
(4)
12.4+1.4
54.2 + 8.7
11.0 + 2.5
20 hr. Ut. egg
(8)
22.1+
.8
(6)
11.1+
47.9±2.5
11.6 + 0.7
20 hr. No egg
(3)
21.5+
.5
(2)
10.9 + 0
51.6 + 1.6
10.3 + 0.6
1 2 3
.4
Determined as difference between total calcium and diffusible. Mean + standard error. Eggs in which calcification was insufficient to give the shell rigidity.
Downloaded from http://ps.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 9, 2015
S. C. White Leghorn hens from the departmental flock were kept in individual cages under normal husbandry conditions for this experiment. Times of oviposition were observed closely, and blood samples were drawn at specific times thereafter. The birds were sacrificed immediately after bleeding, and the condition of the oviduct was examined. A description of the experimental animals and the weights of their reproductive organs have been reported previously (Smith el al., 1957). A schedule of the bleeding times and the number and status of birds involved are given in Table 1. Blood was obtained by the anterior heart-puncture method (Blalock, 1956). The syringes and collecting bottles were heparinized to prevent clotting of the blood. The plasma was prepared from the
blood samples in a refrigerated centrifuge at a temperature of 5°C. immediately after collection, and then stored frozen until the analyses were carried out. Plasma ultrafiltrates containing the ionic calcium were obtained by placing 5 cc. of plasma in bags of cellulose casing ("Visking" 0.008 inch wall thickness) which had been dried previously. These bags were placed in bacterial separation cups and centrifuged at 2,300 rpm. for 8 hours at 10°C. The ultrafiltrate obtained was about a third of the original plasma volume. All calcium determinations were made by the method of Fales (1953).
BLOOD CALCIUM AND LAYING
511
and proceeds at a constant rate (Burmester, 1941; Bradfield, 1951). The principal change in total plasma calcium concentration is a decrease which coincides with this period of shell gland activity. However, the similarity between the plasma calcium concentrations in hens with an egg forming in their oviducts and those without one indicates that the fluctuations associated with shell mineralization may not be a direct result of that process. Egg formation is a complex process, involving many organs and under elaborate endocrine control (described in detail by Sturkie, 1954), and it is quite likely that the dynamics of the calcium metabolism of the laying hen are regulated by some process more general than shell mineralization.
DISCUSSION
The total calcium in the plasma of a laying hen appears to fluctuate diurnally, depending on shell gland activity. The similarity of calcium concentration changes in hens with active and with inactive oviducts indicates that these
The developing egg remains in the shell gland of the oviduct approximately 20 hours During the early parts of this period water is transferred to the egg; after 5 hours shell mineralization starts
Although the sampling times used in this experiment represented most of the normal period of egg formation, the data do not indicate a "cyclic" picture. Very rapid changes in plasma calcium concentration must proceed oviposition to complete the cyclic nature of the calcium metabolism. The decrease in plasma calcium, presumably the source of shell calcium, is the organically-bound (nondiffusible) form. It does not seem likely that the shell calcium leaves the circulation in this form. Probably the inorganic fraction is transferred by the shell gland, and the concentration of this form is maintained by a rapid release of the organically bound calcium. SUMMARY
Downloaded from http://ps.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 9, 2015
tion, when it begins to decrease. When shell formation is essentially complete (about 20 hours after oviposition), total plasma calcium has decreased about 15% (statistically highly significant). The diffusible (inorganic) and nondiffusible (organically bound) components of the plasma calcium appear to change independently through the period of egg formation. The organically-bound plasma calcium concentration is greatest soon after oviposition, and decreases almost linearly for 20 hours thereafter (Table 1). The inorganic plasma calcium concentration is lowest soon after oviposition; it increases up to the onset of shell mineral deposition (about 10 hours after oviposition) and remains fairly constant through this period. The partition of plasma calcium (inorganic as percent of total; Table 1) changes considerably during egg formation. Soon after oviposition, about one third of the plasma calcium is inorganic. At the onset of shell mineralization, about half the plasma calcium is inorganic, and this ratio is maintained through the period of shell formation. Similar changes in the concentration of total, diffusible, and non-diffusible plasma calcium were evident in hens which did not ovulate after oviposition (i.e., those which did not contain developing eggs at the time of bleeding). Although the concentrations of some plasma calcium fractions were different at some times between hens with active and inactive oviducts, none of these variations were statistically significant.
512
C. M. WINGET AND A. H. SMITH
changes may not be a direct result of shell mineralization. The decrease in circulating calcium during egg formation occurs mainly in the bound form (nondiffusible). The ionic plasma calcium increases relative to total calcium while the shell gland is actively forming shell mineral.
A Case of a Hen Yolk with Seven Blastodiscs JAROSLAV KRIZENECKY, JOSEPH SAJNER AND RUZENA J. VANCIKOVA Laboratory for Physiology of Domestic Animals, Slovahian Academy of Sciences, Ivdnka on Danube (Received for publication July 4, 1957)
I
T IS known that yolks with two blastodiscs occur, and numerous reports of this phenomenon have been published. Romanoff and Romanoff (1949) included 10 references to such cases. Levi (quoted by Romanoff and Romanoff, 1949) described a case of twin pigeons from an egg containing only one yolk. Therefore this yolk must have had two blastodiscs. Curtis (also quoted by Romanoff and Romanoff, 1949) described an abnormally large yolk and believed that the presence of two blastodiscs was the result of the fusion of two yolks. The existence of a thin white line on the surface of the yolk, separating the blastodiscs from each other, confirmed this opinion. Romanoff and Romanoff (1949) share this view. Ac-
cording to Curtis the origin of two blastodiscs from division of one oocyte is very doubtful. During our research on many thousands of eggs we have observed several yolks with two discs. They were located near each other (in one case only 5 mm. apart), or near the poles of a plane vertical to an axis between the chalazae. Since similar observations have been reported they were not recorded in detail. We cannot, therefore, furnish exact morphological and numerical data. According to their external appearance the discs were fertilized in all cases. In experiments on the formation of sccalled vacuoles in unfertilized eggs we were greatly surprised to find a yolk
Downloaded from http://ps.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 9, 2015
REFERENCES Blalock, H. G., Jr., 1956. Hematology as an aid in the diagnosis of poultry diseases. J. Am. Vet. Med. Assn. 128: 547-549. Bradfield, J. R. G., 1951. Radiographic studies on the formation of the hen's egg shell. J. Exp. Biol. 28:125-140. Buckner, G. D., J. H. Martin and W. N. Insko, 1930. The blood calcium of laying hens varied by the calcium intake. Am. J. Physiol. 94: 692-695. Burmester, B. R., 1941. Experiments on the physiology of egg white secretion. Poultry Sci. 20: 224-226.
Charles, E., and L. Hogben, 1933. The serum calcium and magnesium level in the ovarian cycle of the laying hen. Quant. J. Exp. Physiol. 23: 343-349. Fales, F. W., 1953. A micromethod for the determination of serum calcium. J. Biol. Chem. 204: 577-585. Knowles, H. R., E. B. Hart and J. G. Halpin, 1935. The variation in the calcium level of the blood of the domestic fowl. Poultry Sci. 14: 83-89. Polin, D., and P. D. Sturkie, 1957. The influence of the parathyroids on blood calcium and shell deposition in laying hens. Endocrinology, 60: 778-784. Rochlina, M., 1934. Les taux des differents elements dans le sang des poules en liaison avec la ponte. Bull. Soc. Chim. Biol. 16: 1652-1662. Smith, A. H., G. N. Hoover, J. O. Nordstrom and C. M. Winget, 1957. Quantitative changes in the hen's oviduct associated with egg formation. Poultry Sci. 36: 353^357. Sturkie, P. D., 1954. Avian Physiology, Comstock Publishing Association, Ithaca, New York.
Poultry Sci. 31: 227. Weiss, H. S., R. K. Ringer and P. D. Sturkie, 1957. Development of the sex difference in blood pressure of the chick. Am. J. Physiol. 188: 383. Weiss, H. S., and M. Sheahan, 1958. The influence of maturity and sex on the blood pressure of the turkey. Am. J. Vet. Res. 19: 209. Winget, C. M., and A. H. Smith, 1957. Blood calcium and shell formation in the laying hen. Poultry Sci., 36: 1169. Wingstrand, K. G., 1951. The Structure and Development of the Avian Pituitary. C. W. K. Gleerup/Lund, Sweden.
Changes in Plasma Calcium Concentration During Egg Formation CHARLES M. WINGET AND ARTHUR H. SMITH Department of Poultry Husbandry, University of California, Davis (Received for publication October 7. 1957)
D
URING egg formation, large quantities of calcium are deposited as carbonate on the developing shell. The site of shell formation, the oviduct shell gland, is not a calcium storage organ; consequently, calcium must be mobilized rapidly from body stores to meet the periodic requirements of shell formation. Since the body calcium is located almost entirely in osseous tissue, it must pass through the circulatory system in rather large quantities during each period of active shell formation (about 14 hours; Bradfield, 1951). There is also an increase in calcium absorption from the gut during shell formation (Buckner et al., 1930), which contributes to the fluctuation in blood calcium concentration. The literature contains several reports of the influence of shell formation on blood calcium. Charles and Hogben (1933) found that plasma calcium concentration during egg formation was about 20% higher than during non-laying periods. However, their data for hens with a form-
ing egg present in the oviduct varied widely. Diurnal variations in the blood calcium level of laying hens were found by Rochlina (1934). The birds were bled three times daily, morning, midday, and evening. The maximum blood calcium values (about 35% greater than the minimum) were found in the midday samples. Low levels in the plasma calcium of laying hens, associated with shell mineralization, and relatively higher levels when the shell gland was not active in shell formation have also been reported (Kno wles etal., 1935). Recently, Polin and Sturkie (1957) studied the concentrations of total and diffusible plasma calcium and related them to the condition of eggs present in the oviduct. Only small differences were noted between hens containing no egg and those with a soft- or hard-shelled egg in the oviduct. The experiments reported herein are concerned with changes in the plasma
Downloaded from http://ps.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 9, 2015
tionship between individual laying hens. J. Agr. Sci. 48: 171. Vanstone, W. E., W. A. Maw and R. H. Common, 1955. Levels and partition of the fowl's serum proteins in relation to age and egg production. Canad. J. Biochem. Physiol. 33: 891. Vanstone, W. E., D. G. Dale, W. F. Oliver and R. H. Common, 1957. Sites of formation of plasma phosphoprotein and phospholipid in the estrogenized cockerel. Canad. J. Biochem. Physiol. 35: 659. Weiss, H. S., and P. D. Sturkie, 1952. Time of oviposition as affected by neuromimetric drugs.
509
510
C. M . WlNGET AND A . H . SMITH
calcium concentration at various times in the laying cycle. METHOD
RESULTS
The concentrations of total diffusible and nondiffusible plasma calcium at various stages of egg formation are given in Table 1. The total plasma calcium remains fairly constant from the time of oviposition until onset of active shell forma-
TABLE 1.—Plasma calcium concentration in laying hens at specific times after oviposit Time since last laying; • position of egg 0-1 hr. No egg 4hr. Isth.
Total calcium
Diffusible calcium As % of total calcium
Nondiffusible calcium1 mg. %
.1
32.3 + 1.5
22.5 + 2.7
(8)
10.4+ .2
38.7 + 1.3
16.3 + 1.0
No. hens
mg-%
No. hens
(9)
26.4 + 1.02
(7)
26.1±
.6
(12)
mg. % 8.7+
10 hr. No egg
(4)
28.1 + 1.5
(3)
13.2+ .3
48.3+4.4
14.5 + 2.2
10 hr. Ut. S.S.3
(5)
26.6 + 1.4
(3)
11.8+ .3
47.5 + 4.6
13.5 + 2.3
16 hr. Ut. egg
(7)
24.2 + 1.5
(4)
12.4+1.4
54.2 + 8.7
11.0 + 2.5
20 hr. Ut. egg
(8)
22.1+
.8
(6)
11.1+
47.9±2.5
11.6 + 0.7
20 hr. No egg
(3)
21.5+
.5
(2)
10.9 + 0
51.6 + 1.6
10.3 + 0.6
1 2 3
.4
Determined as difference between total calcium and diffusible. Mean + standard error. Eggs in which calcification was insufficient to give the shell rigidity.
Downloaded from http://ps.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 9, 2015
S. C. White Leghorn hens from the departmental flock were kept in individual cages under normal husbandry conditions for this experiment. Times of oviposition were observed closely, and blood samples were drawn at specific times thereafter. The birds were sacrificed immediately after bleeding, and the condition of the oviduct was examined. A description of the experimental animals and the weights of their reproductive organs have been reported previously (Smith el al., 1957). A schedule of the bleeding times and the number and status of birds involved are given in Table 1. Blood was obtained by the anterior heart-puncture method (Blalock, 1956). The syringes and collecting bottles were heparinized to prevent clotting of the blood. The plasma was prepared from the
blood samples in a refrigerated centrifuge at a temperature of 5°C. immediately after collection, and then stored frozen until the analyses were carried out. Plasma ultrafiltrates containing the ionic calcium were obtained by placing 5 cc. of plasma in bags of cellulose casing ("Visking" 0.008 inch wall thickness) which had been dried previously. These bags were placed in bacterial separation cups and centrifuged at 2,300 rpm. for 8 hours at 10°C. The ultrafiltrate obtained was about a third of the original plasma volume. All calcium determinations were made by the method of Fales (1953).
BLOOD CALCIUM AND LAYING
511
and proceeds at a constant rate (Burmester, 1941; Bradfield, 1951). The principal change in total plasma calcium concentration is a decrease which coincides with this period of shell gland activity. However, the similarity between the plasma calcium concentrations in hens with an egg forming in their oviducts and those without one indicates that the fluctuations associated with shell mineralization may not be a direct result of that process. Egg formation is a complex process, involving many organs and under elaborate endocrine control (described in detail by Sturkie, 1954), and it is quite likely that the dynamics of the calcium metabolism of the laying hen are regulated by some process more general than shell mineralization.
DISCUSSION
The total calcium in the plasma of a laying hen appears to fluctuate diurnally, depending on shell gland activity. The similarity of calcium concentration changes in hens with active and with inactive oviducts indicates that these
The developing egg remains in the shell gland of the oviduct approximately 20 hours During the early parts of this period water is transferred to the egg; after 5 hours shell mineralization starts
Although the sampling times used in this experiment represented most of the normal period of egg formation, the data do not indicate a "cyclic" picture. Very rapid changes in plasma calcium concentration must proceed oviposition to complete the cyclic nature of the calcium metabolism. The decrease in plasma calcium, presumably the source of shell calcium, is the organically-bound (nondiffusible) form. It does not seem likely that the shell calcium leaves the circulation in this form. Probably the inorganic fraction is transferred by the shell gland, and the concentration of this form is maintained by a rapid release of the organically bound calcium. SUMMARY
Downloaded from http://ps.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 9, 2015
tion, when it begins to decrease. When shell formation is essentially complete (about 20 hours after oviposition), total plasma calcium has decreased about 15% (statistically highly significant). The diffusible (inorganic) and nondiffusible (organically bound) components of the plasma calcium appear to change independently through the period of egg formation. The organically-bound plasma calcium concentration is greatest soon after oviposition, and decreases almost linearly for 20 hours thereafter (Table 1). The inorganic plasma calcium concentration is lowest soon after oviposition; it increases up to the onset of shell mineral deposition (about 10 hours after oviposition) and remains fairly constant through this period. The partition of plasma calcium (inorganic as percent of total; Table 1) changes considerably during egg formation. Soon after oviposition, about one third of the plasma calcium is inorganic. At the onset of shell mineralization, about half the plasma calcium is inorganic, and this ratio is maintained through the period of shell formation. Similar changes in the concentration of total, diffusible, and non-diffusible plasma calcium were evident in hens which did not ovulate after oviposition (i.e., those which did not contain developing eggs at the time of bleeding). Although the concentrations of some plasma calcium fractions were different at some times between hens with active and inactive oviducts, none of these variations were statistically significant.
512
C. M. WINGET AND A. H. SMITH
changes may not be a direct result of shell mineralization. The decrease in circulating calcium during egg formation occurs mainly in the bound form (nondiffusible). The ionic plasma calcium increases relative to total calcium while the shell gland is actively forming shell mineral.
A Case of a Hen Yolk with Seven Blastodiscs JAROSLAV KRIZENECKY, JOSEPH SAJNER AND RUZENA J. VANCIKOVA Laboratory for Physiology of Domestic Animals, Slovahian Academy of Sciences, Ivdnka on Danube (Received for publication July 4, 1957)
I
T IS known that yolks with two blastodiscs occur, and numerous reports of this phenomenon have been published. Romanoff and Romanoff (1949) included 10 references to such cases. Levi (quoted by Romanoff and Romanoff, 1949) described a case of twin pigeons from an egg containing only one yolk. Therefore this yolk must have had two blastodiscs. Curtis (also quoted by Romanoff and Romanoff, 1949) described an abnormally large yolk and believed that the presence of two blastodiscs was the result of the fusion of two yolks. The existence of a thin white line on the surface of the yolk, separating the blastodiscs from each other, confirmed this opinion. Romanoff and Romanoff (1949) share this view. Ac-
cording to Curtis the origin of two blastodiscs from division of one oocyte is very doubtful. During our research on many thousands of eggs we have observed several yolks with two discs. They were located near each other (in one case only 5 mm. apart), or near the poles of a plane vertical to an axis between the chalazae. Since similar observations have been reported they were not recorded in detail. We cannot, therefore, furnish exact morphological and numerical data. According to their external appearance the discs were fertilized in all cases. In experiments on the formation of sccalled vacuoles in unfertilized eggs we were greatly surprised to find a yolk
Downloaded from http://ps.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 9, 2015
REFERENCES Blalock, H. G., Jr., 1956. Hematology as an aid in the diagnosis of poultry diseases. J. Am. Vet. Med. Assn. 128: 547-549. Bradfield, J. R. G., 1951. Radiographic studies on the formation of the hen's egg shell. J. Exp. Biol. 28:125-140. Buckner, G. D., J. H. Martin and W. N. Insko, 1930. The blood calcium of laying hens varied by the calcium intake. Am. J. Physiol. 94: 692-695. Burmester, B. R., 1941. Experiments on the physiology of egg white secretion. Poultry Sci. 20: 224-226.
Charles, E., and L. Hogben, 1933. The serum calcium and magnesium level in the ovarian cycle of the laying hen. Quant. J. Exp. Physiol. 23: 343-349. Fales, F. W., 1953. A micromethod for the determination of serum calcium. J. Biol. Chem. 204: 577-585. Knowles, H. R., E. B. Hart and J. G. Halpin, 1935. The variation in the calcium level of the blood of the domestic fowl. Poultry Sci. 14: 83-89. Polin, D., and P. D. Sturkie, 1957. The influence of the parathyroids on blood calcium and shell deposition in laying hens. Endocrinology, 60: 778-784. Rochlina, M., 1934. Les taux des differents elements dans le sang des poules en liaison avec la ponte. Bull. Soc. Chim. Biol. 16: 1652-1662. Smith, A. H., G. N. Hoover, J. O. Nordstrom and C. M. Winget, 1957. Quantitative changes in the hen's oviduct associated with egg formation. Poultry Sci. 36: 353^357. Sturkie, P. D., 1954. Avian Physiology, Comstock Publishing Association, Ithaca, New York.