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Effects of Vitamin D3 Deficiency on Adenosine Triphosphatase Activity of Jejunums from White Leghorn Hens1
Effects of Vitamin D 3 Deficiency on Adenosine Triphosphatase Activity of Jejunums from White Leghorn Hens1 A. A. GRUNDER and C. P. W. TSANG Animal Research Centre, Agriculture Canada, Ottawa, Ontario K1A 0C6 (Received for publication August 29, 1984)
1984 Poultry Science 63:1073-1075
INTRODUCTION
Vitamin D in combination with other factors influences the transport of calcium (Ca) across the intestinal wall. Wasserman and Taylor (1966) described a Ca-binding protein of the chick duodenum and found this protein to be dependent on vitamin D 3 (D 3 ). Haussler et al. (1970) observed that D 3 induced adenosine triphosphatase (ATPase) in the brush border of chick intestines. The time course of the appearance of this enzyme in D3-deficient chicks, following a D 3 dose, was correlated with Ca absorption. They also presented evidence that Mg2+-ATPase, Ca2+-ATPase, and alkaline phosphatase assays measure activity of a single enzyme. The present study was initiated to determine whether D 3 deficiency would influence jejunal ATPase activity of laying hens. The jejunum was chosen for study over other portions of the small intestine because Hurwitz et al. (1973) showed that the upper jejunum was the major site of Ca absorption. MATERIALS AND METHODS
The study involved 30 Strain 1 Single Comb White Leghorn hens that had been selected over several years for multiple traits with special
1 Contribution Number 1177 from the Animal Research Centre.
emphasis on egg production (Gowe and Fairfull, 1980). Prior to dietary treatment, the birds were provided a laying mash diet containing 3.1% Ca and 1100 ICU added D 3 /kg of diet. Beginning at 227 days of age, an experimental group of 15 received the diet without supplemental D 3 , until all hens were killed 30 to 37 days later, while a control group of 15 hens continued to receive the D 3 -supplemented diet. Egg traits and plasma Ca were measured. Four days before and 14 days after dietary treatment, the following egg measurements were taken-, egg weight, egg specific gravity (SG) by Archimedes' principle, dried shell plus membrane weight (Hamilton, 1978), shell plus membrane weight as a percent of egg weight and shell thickness (Voisey and Hunt, 1974). Plasma Ca was assayed on 5 ml blood samples obtained from the brachial vein using the anticoagulant heparin. Blood was taken from 8 to 10 birds from each group 28 days before, all birds 14 days after, and from all birds killed 30 to 37 days after the start of dietary treatment. Plasma was stored at —20 C until assayed for total Ca by atomic absorption spectrophotometry. Activity of ATPase was measured on jejunums collected from birds 30 to 37 days after start of the dietary treatment. After being killed by electrocution and exsanguination to minimize blood content of the intestinal wall, jejunums were removed and contents were washed out with .9% NaCl. Portions of jejunum
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Downloaded from http://ps.oxfordjournals.org/ by guest on April 16, 2015
ABSTRACT Supplemental vitamin D 3 (D 3 ) was removed from the diet given to an experimental group of White Leghorn hens, at 227 days of age, while a control group continued to receive a supplemental diet. By 14 days after D 3 withdrawal, egg weight, egg specific gravity, shell weight, percent shell, shell thickness, and plasma calcium were lower (P<.05) in the experimental compared to the control group. At 30 to 37 days after D 3 withdrawal, experimental hens had less (P<.05) jejunal adenosine triphosphatase (ATPase) activity than the control hens. The study indicated that lack of D 3 supplementation in laying diets reduced jejunal ATPase activity as well as egg shell quality. (Key words: vitamin D 3 , jejunum, adenosine triphosphatase, laying hens)
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were weighed a n d placed in a centrifuge t u b e with .25 M sucrose solution, 4 ml/g of tissue. T u b e and c o n t e n t s were frozen o n d r y ice and assayed for ATPase t h e following d a y as described b y G r u n d e r ( 1 9 8 3 ) . Various combinations of cations used in t h e assay are n o t e d in Table 1. Differences b e t w e e n c o n t r o l a n d experimental hens for egg a n d shell traits a n d plasma Ca were analyzed b y S t u d e n t ' s t test. A two-way, least squares m o d e l for t h e analysis of variance, with a linear contrasts p r o c e d u r e , was used t o assess effect of t r e a t m e n t a n d cation c o n t e n t of assay m i x t u r e on jejunal ATPase activity.
T h a t hens lacking D 3 s u p p l e m e n t a t i o n h a d lower plasma Ca levels and lower jejunal ATPase activities t h a n hens fed supplemental D 3 , provides evidence t h a t ATPase is involved in t h e intestinal a b s o r p t i o n of Ca in laying hens. This e n z y m e also influences a b s o r p t i o n of Ca in t h e chick intestine (Haussler et al, 1970).
TABLE 1. Means of jejunal adenosine triphosphatase (ATPase) activity of hens fed diets with (+) and without (—) vitamin D 3 and effects of various cations on ATPase activity ATPase activity Cations added
+ (9)'
Na+ 45 m/W; K+ 50 mAf; Mg2+ 2.5 mM Mg2+ 2.5 mM Ca2+ 30 mM Mg 2+ 2.5m/W;Ca 2+ 30 mM Nil
28.1 28.4 16.8 29.0 8.1
(8) 1 (Mg P/mg protein/5 min)
+ 3.60 + 1.20 ± 1.26 + 2.35 + 1.58
12.3 ± 1.27**** 15.5 ± 1.70**** 10.0+ 1.10* 13.1 ± 1.56**** 3.9 ± 1.25
1 Each mean ± standard error of 9(+) or 8(—) hens with an egg undergoing early calcification in the uterus at death.
*P<.05. ****P<.0001 compared with +D 3
Downloaded from http://ps.oxfordjournals.org/ by guest on April 16, 2015
RESULTS AND DISCUSSION Effect of Vitamin D 3 Deficiency on Egg Shell Quality and Calcium in Plasma. No significant differences existed b e t w e e n control a n d experimental hens for plasma Ca 2 8 days before and for egg a n d shell m e a s u r e m e n t s 4 days before dietary t r e a t m e n t . During t h e second week after D 3 withdrawal, eggs from t h e experimental g r o u p were often b r o k e n and partially shelled. By 14 days after D 3 withdrawal, egg and shell parameters and plasma Ca were all lower ( P < . 0 5 ) for t h e D 3 -deficient hens t h a n for t h e c o n t r o l g r o u p . F r o m 1 4 days t o t i m e of kill at 30 t o 37 days after d i e t a r y t r e a t m e n t , egg quality a n d p r o d u c t i o n of t h e e x p e r i m e n t a l hens were t o o erratic for m e a s u r e m e n t of shell quality. F o r plasma Ca, t h e difference was still present ( P = . 0 6 ) at t h e t i m e of kill 30 t o 37 days after t h e start of t h e d i e t a r y t r e a t m e n t , an
observation consistent with t h a t of Haussler et al. ( 1 9 7 0 ) in rachitic chicks. Effect of Vitamin D3 Deficiency on Jejunal Adenosine Triphosphatase. Jejunal ATPase activity in control and experimental hens is summarized in Table 1 o n l y for birds with a developing egg in utero at t i m e of kill. Analysis of variance indicated t h a t t h e r e were differences ( P < . 0 0 0 1 ) b e t w e e n diets and a m o n g cation sources a n d t h a t t h e r e was an interaction ( P < . 0 1 ) b e t w e e n t h e t w o sources of variation. Linear contrasts revealed t h a t there was greater ( P < . 0 5 ) jejunal ATPase activity for t h e c o n t r o l compared t o t h e e x p e r i m e n t a l group, regardless of t h e cation added t o t h e assay m i x t u r e , b u t n o significant differences were observed w h e n n o cations were added (Table 1). T h e r e was a b o u t twice t h e jejunal ATPase activity in t h e control compared to the experimental group, regardless of t y p e or presence of a d d e d cations in t h e assay m i x t u r e . In b o t h c o n t r o l a n d treatm e n t groups, jejunal ATPase activity was less ( P < . 0 0 1 ) w i t h o u t added cations than with a n y or all of t h e o t h e r cations added. F o r t h e control g r o u p o n l y , t h e activity was less when only Ca 2 + was added ( P < . 0 5 ) t h a n when Mg 2 + alone or in c o m b i n a t i o n with o t h e r cations was added.
RESEARCH NOTE ACKNOWLEDGMENTS T h e a u t h o r s wish t o t h a n k J. W. Dickie for technical assistance.
REFERENCES Gowe, R. S., and R. W. Fairfull, 1980. Performance of six long-term multitrait Leghorn strains and three control strains, and a single cross evaluation of the selected strains. Pages 141—162 in Proc. S. Pacific Poult. Sci. Conv., Auckland, New Zealand. Grander, A. A., 1983. Uterine adenosine triphosphatase in relation to shell quality. Poultry Sci. 62: 512-518. Hamilton, R.M.G., 1978. The effects of dietary phos-
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phorus, vitamin D 3 , and 25-hydroxy vitamin D 3 levels on feed intake, productive performance, and egg and shell quality in two strains of forcemolted White Leghorns. Poultry Sci. 59:598— 604. Haussler, M. R., L. A. Nagode, and H. Rasmussen, 1970. Induction of intestinal brush broiler alkaline phosphatase by vitamin D and identity with Ca-ATPase. Nature 228:1199-1201. Hurwitz, S., A. Bar, and I. Cohen, 1973. Regulation of calcium absorption by fowl intestine. Am. J. Physiol. 225:150-154. Voisey, P. W., and J. R. Hunt, 1974. Measurement of eggshell strength. J. Texture Stud. 5:135—182. Wasserman, R. H., and A. N. Taylor, 1966. Vitamin D 3 -induced calcium-binding protein in chick intestinal mucosa. Science 152:791—793. Downloaded from http://ps.oxfordjournals.org/ by guest on April 16, 2015
1984 Poultry Science 63:1073-1075
INTRODUCTION
Vitamin D in combination with other factors influences the transport of calcium (Ca) across the intestinal wall. Wasserman and Taylor (1966) described a Ca-binding protein of the chick duodenum and found this protein to be dependent on vitamin D 3 (D 3 ). Haussler et al. (1970) observed that D 3 induced adenosine triphosphatase (ATPase) in the brush border of chick intestines. The time course of the appearance of this enzyme in D3-deficient chicks, following a D 3 dose, was correlated with Ca absorption. They also presented evidence that Mg2+-ATPase, Ca2+-ATPase, and alkaline phosphatase assays measure activity of a single enzyme. The present study was initiated to determine whether D 3 deficiency would influence jejunal ATPase activity of laying hens. The jejunum was chosen for study over other portions of the small intestine because Hurwitz et al. (1973) showed that the upper jejunum was the major site of Ca absorption. MATERIALS AND METHODS
The study involved 30 Strain 1 Single Comb White Leghorn hens that had been selected over several years for multiple traits with special
1 Contribution Number 1177 from the Animal Research Centre.
emphasis on egg production (Gowe and Fairfull, 1980). Prior to dietary treatment, the birds were provided a laying mash diet containing 3.1% Ca and 1100 ICU added D 3 /kg of diet. Beginning at 227 days of age, an experimental group of 15 received the diet without supplemental D 3 , until all hens were killed 30 to 37 days later, while a control group of 15 hens continued to receive the D 3 -supplemented diet. Egg traits and plasma Ca were measured. Four days before and 14 days after dietary treatment, the following egg measurements were taken-, egg weight, egg specific gravity (SG) by Archimedes' principle, dried shell plus membrane weight (Hamilton, 1978), shell plus membrane weight as a percent of egg weight and shell thickness (Voisey and Hunt, 1974). Plasma Ca was assayed on 5 ml blood samples obtained from the brachial vein using the anticoagulant heparin. Blood was taken from 8 to 10 birds from each group 28 days before, all birds 14 days after, and from all birds killed 30 to 37 days after the start of dietary treatment. Plasma was stored at —20 C until assayed for total Ca by atomic absorption spectrophotometry. Activity of ATPase was measured on jejunums collected from birds 30 to 37 days after start of the dietary treatment. After being killed by electrocution and exsanguination to minimize blood content of the intestinal wall, jejunums were removed and contents were washed out with .9% NaCl. Portions of jejunum
1073
Downloaded from http://ps.oxfordjournals.org/ by guest on April 16, 2015
ABSTRACT Supplemental vitamin D 3 (D 3 ) was removed from the diet given to an experimental group of White Leghorn hens, at 227 days of age, while a control group continued to receive a supplemental diet. By 14 days after D 3 withdrawal, egg weight, egg specific gravity, shell weight, percent shell, shell thickness, and plasma calcium were lower (P<.05) in the experimental compared to the control group. At 30 to 37 days after D 3 withdrawal, experimental hens had less (P<.05) jejunal adenosine triphosphatase (ATPase) activity than the control hens. The study indicated that lack of D 3 supplementation in laying diets reduced jejunal ATPase activity as well as egg shell quality. (Key words: vitamin D 3 , jejunum, adenosine triphosphatase, laying hens)
1074
GRUNDER AND TSANG
were weighed a n d placed in a centrifuge t u b e with .25 M sucrose solution, 4 ml/g of tissue. T u b e and c o n t e n t s were frozen o n d r y ice and assayed for ATPase t h e following d a y as described b y G r u n d e r ( 1 9 8 3 ) . Various combinations of cations used in t h e assay are n o t e d in Table 1. Differences b e t w e e n c o n t r o l a n d experimental hens for egg a n d shell traits a n d plasma Ca were analyzed b y S t u d e n t ' s t test. A two-way, least squares m o d e l for t h e analysis of variance, with a linear contrasts p r o c e d u r e , was used t o assess effect of t r e a t m e n t a n d cation c o n t e n t of assay m i x t u r e on jejunal ATPase activity.
T h a t hens lacking D 3 s u p p l e m e n t a t i o n h a d lower plasma Ca levels and lower jejunal ATPase activities t h a n hens fed supplemental D 3 , provides evidence t h a t ATPase is involved in t h e intestinal a b s o r p t i o n of Ca in laying hens. This e n z y m e also influences a b s o r p t i o n of Ca in t h e chick intestine (Haussler et al, 1970).
TABLE 1. Means of jejunal adenosine triphosphatase (ATPase) activity of hens fed diets with (+) and without (—) vitamin D 3 and effects of various cations on ATPase activity ATPase activity Cations added
+ (9)'
Na+ 45 m/W; K+ 50 mAf; Mg2+ 2.5 mM Mg2+ 2.5 mM Ca2+ 30 mM Mg 2+ 2.5m/W;Ca 2+ 30 mM Nil
28.1 28.4 16.8 29.0 8.1
(8) 1 (Mg P/mg protein/5 min)
+ 3.60 + 1.20 ± 1.26 + 2.35 + 1.58
12.3 ± 1.27**** 15.5 ± 1.70**** 10.0+ 1.10* 13.1 ± 1.56**** 3.9 ± 1.25
1 Each mean ± standard error of 9(+) or 8(—) hens with an egg undergoing early calcification in the uterus at death.
*P<.05. ****P<.0001 compared with +D 3
Downloaded from http://ps.oxfordjournals.org/ by guest on April 16, 2015
RESULTS AND DISCUSSION Effect of Vitamin D 3 Deficiency on Egg Shell Quality and Calcium in Plasma. No significant differences existed b e t w e e n control a n d experimental hens for plasma Ca 2 8 days before and for egg a n d shell m e a s u r e m e n t s 4 days before dietary t r e a t m e n t . During t h e second week after D 3 withdrawal, eggs from t h e experimental g r o u p were often b r o k e n and partially shelled. By 14 days after D 3 withdrawal, egg and shell parameters and plasma Ca were all lower ( P < . 0 5 ) for t h e D 3 -deficient hens t h a n for t h e c o n t r o l g r o u p . F r o m 1 4 days t o t i m e of kill at 30 t o 37 days after d i e t a r y t r e a t m e n t , egg quality a n d p r o d u c t i o n of t h e e x p e r i m e n t a l hens were t o o erratic for m e a s u r e m e n t of shell quality. F o r plasma Ca, t h e difference was still present ( P = . 0 6 ) at t h e t i m e of kill 30 t o 37 days after t h e start of t h e d i e t a r y t r e a t m e n t , an
observation consistent with t h a t of Haussler et al. ( 1 9 7 0 ) in rachitic chicks. Effect of Vitamin D3 Deficiency on Jejunal Adenosine Triphosphatase. Jejunal ATPase activity in control and experimental hens is summarized in Table 1 o n l y for birds with a developing egg in utero at t i m e of kill. Analysis of variance indicated t h a t t h e r e were differences ( P < . 0 0 0 1 ) b e t w e e n diets and a m o n g cation sources a n d t h a t t h e r e was an interaction ( P < . 0 1 ) b e t w e e n t h e t w o sources of variation. Linear contrasts revealed t h a t there was greater ( P < . 0 5 ) jejunal ATPase activity for t h e c o n t r o l compared t o t h e e x p e r i m e n t a l group, regardless of t h e cation added t o t h e assay m i x t u r e , b u t n o significant differences were observed w h e n n o cations were added (Table 1). T h e r e was a b o u t twice t h e jejunal ATPase activity in t h e control compared to the experimental group, regardless of t y p e or presence of a d d e d cations in t h e assay m i x t u r e . In b o t h c o n t r o l a n d treatm e n t groups, jejunal ATPase activity was less ( P < . 0 0 1 ) w i t h o u t added cations than with a n y or all of t h e o t h e r cations added. F o r t h e control g r o u p o n l y , t h e activity was less when only Ca 2 + was added ( P < . 0 5 ) t h a n when Mg 2 + alone or in c o m b i n a t i o n with o t h e r cations was added.
RESEARCH NOTE ACKNOWLEDGMENTS T h e a u t h o r s wish t o t h a n k J. W. Dickie for technical assistance.
REFERENCES Gowe, R. S., and R. W. Fairfull, 1980. Performance of six long-term multitrait Leghorn strains and three control strains, and a single cross evaluation of the selected strains. Pages 141—162 in Proc. S. Pacific Poult. Sci. Conv., Auckland, New Zealand. Grander, A. A., 1983. Uterine adenosine triphosphatase in relation to shell quality. Poultry Sci. 62: 512-518. Hamilton, R.M.G., 1978. The effects of dietary phos-
1075
phorus, vitamin D 3 , and 25-hydroxy vitamin D 3 levels on feed intake, productive performance, and egg and shell quality in two strains of forcemolted White Leghorns. Poultry Sci. 59:598— 604. Haussler, M. R., L. A. Nagode, and H. Rasmussen, 1970. Induction of intestinal brush broiler alkaline phosphatase by vitamin D and identity with Ca-ATPase. Nature 228:1199-1201. Hurwitz, S., A. Bar, and I. Cohen, 1973. Regulation of calcium absorption by fowl intestine. Am. J. Physiol. 225:150-154. Voisey, P. W., and J. R. Hunt, 1974. Measurement of eggshell strength. J. Texture Stud. 5:135—182. Wasserman, R. H., and A. N. Taylor, 1966. Vitamin D 3 -induced calcium-binding protein in chick intestinal mucosa. Science 152:791—793. Downloaded from http://ps.oxfordjournals.org/ by guest on April 16, 2015