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Effects of Vitamin D3 Metabolites on Physiological Traits of White Leghorn Hens1
PHYSIOLOGY AND REPRODUCTION Effects of Vitamin D 3 Metabolites on Physiological Traits of White Leghorn Hens1 A. A. GRUNDER and C.P.W. TSANG Animal Research Centre, Agriculture Canada, Ottawa, Ontario, Canada, K1A 0C6 R. NARBATIZ Department of Anatomy, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Received for publication June 14, 1989)
1990 Poultry Science 69:1204-1208 INTRODUCTION
Although the importance of cholecalciferol, or vitamin D3 (D3), for the production of good eggshells has been known for some time, there is recent evidence that the active metabolite is 1,25-dihydroxycholecalciferol [1,25(OH)2D3], (Soares, 1984). Tsang et al. (1990b) observed that feeding l,25(OH)2D3 at 5 Jig per kg ration to hens produced better eggshells than did control hens fed D3. Another study has indicated that this level of l,25(OH)2D3 is optimal for producing strong eggshells (Tsang et al, 1990a). Some effort has been made to study the effect of D3 on certain physiological traits believed to play a role in eggshell deposition. Wasserman and Taylor (1966) found that Cabinding protein (CaBP) of the chick duodenum was dependent on D3. Haussler et al. (1970) observed that D3 induced adenosine triphosphatase (ATPase) in the chick intestines and Ca absorption from the intestines. Vitamin
D3 also induces Ca-dependent ATPase in reproductive organs of quail (Regal, 1983). Grander and Tsang (1984) have shown that layer diets without supplemental D3 reduced the level of jejunal ATPase. In a physiological study of l,25(OH)2D3, Nys and DeLaage (1984a) found that injections of l,25(OH)2D3 did not alter CaBP or ATPase levels in uteri of hens induced to lay shell-less eggs, but did influence alkaline phosphatase activity in duodenal mucosa and restored CaBP levels in all sections of the intestines (Nys and DeLaage, 1984b). The purpose of the present study was to confirm the effect of deleting D3 from the diet and to determine the effect of replacement with 1,25(011)^3 or 24,25(OH)2D3 on uterine ATPase and carbonic anhydrase, jejunal ATPase, plasma Ca, BW, and uterine weight of laying hens. MATERIALS AND METHODS
Birds Contribution Number 1617 from the Animal Research Centre.
Single Comb White Leghorn hens were produced from nonpedigree matings of 60 Strain
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ABSTRACT This study was conducted to determine the effect of feeding vitamin 03(03) metabolites on BW of hen, weight of uterus, plasma Ca, jejunal and uterine adenosine triphosphatase (ATPase), and carbonic anhydrase. At 416 days of age each of 7 groups of laying hens was fed the basal ration supplemented with one of 7 concentrations (ng per kg) of D3 or its metabolites as treatments: 0 jig of D3; 27.5 ug of D3; 3,5, or 7 ug of l,25(OH)2D3; 5 |ig of 24,25(OH)2D3; and 5 ug of 24,25(OH)2D3 plus 5 Jig of l ^ O H ) - ^ . Treatment effects were compared at various periods after the start of the study. Hens fed the unsupplemented ration had lower (P<05) values for all traits man hens fed the D3supplemented ration by 162 days after the start of treatment In a comparison of all dietary treatments except the one involving 0 ug D3, from 154 to 161 days after the start of the experiment, treatment effects were significant (P£.05) for BW, uterine ATPase, and carbonic anhydrase; hens fed 5 Ug of 24^5(OH)2D3 per kg of ration ranked the lowest of all treatment groups for these traits. Hens fed 27.5 ug of D3 and those fed 5 Ug of l,25(OH)2D3 per kg of ration did not differ (P>.05) for any traits studied. The results suggest that 5 ug of 1^5(OH)2D3 per kg of ration can replace 27.5 ug of D3 per kg of ration but that 5 fig of 24,25(OH)2I>3 per kg of ration tends to have a negative effect on physiological systems of the hen. (Key words: vitamin D metabolites, body weight, laying hen, adenosine triphosphatase, carbonic anhydrase)
VITAMIN D METABOLITES AND PHYSIOLOGICAL TRAITS
1 males to 460 Strain 2 females. These experimental strains had been selected for multiple traits with emphasis on egg production (Fairfull et al, 1983). After rearing to 18 wk of age, birds were caged individually and fed a balanced laying ration containing 3.1% Ca and supplemented with 27.5 ug of D3 per kg of ration. Light was gradually increased from 8 h at 133 days of age by .5 h per week to 16 h per day in a windowless house.
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jejunum was opened longitudinally and washed with .9% Nad. Two sections of approximately 1 g each were cut from the anterior end of the jejunum, weighed, and placed in a polypropylene tube containing a .25 M sucrose solution (4 mL per g of tissue), frozen on dry ice, and stored at -20 C. Assays
Plasma Ca was assayed using an atomicabsorption spectrophotometer. Jejunal and uterine ATPase were assayed as described by At 416 days of age hens were randomly Grander (1983) and Grander and Tsang (1984). assigned to one of seven dietary treatment However, each homogenate of jejunum or groups consisting of 28 or 56 hens. Each group uterine mucosa was centrifuged at 1,200 x g for was fed the same basal ration supplemented with 25 min instead of 12,000 x g (Grander, 1983). different concentrations (ug per kg ration) of D3 Assay of uterine carbonic anhydrase activity in or its metabolites as follows: 0 |xg of D3; 27.5 ug these preparations that were diluted one in five of D 3 (control); 3,5, or 7 ug of l,25(OH)2D3; 5 with water was based on the method of Wilbur ug of 24,25(OH)2D3; 5 ug each of l,25(OH)2D3 and Anderson (1948). All reaction mixtures and 24,25(OH)2D3. were cooled by ice-cold water. Three mL of H 2 0 Hens were killed by cervical dislocation to saturated with CQj was added to .60 mL of H 2 0 obtain samples and to make specific compar- and 2.4 mL of veronal buffer (.044 M sodium isons at various periods after the start of barbital and .032 M barbituric acid) at a pH of treatment. For comparing the 0 ug of D 3 to Jhe 8.15. The pH was recorded every 10 s and was control treatment, 7 and 6 hens, respectively, plotted on semi-log paper. The time it took for were killed at either 56 or 57 days; 6 hens were the pH to dropfrom8.1 to 6.4 was recorded (To) killed per treatment at either 98 or 100 days; 12 and the average of four such observations was hens were killed per treatment over six dates at utilized in subsequent calculations. The time it 154, 155, 157, 158, 161, and 162 days. For took for a similar change in pH when .02 mL of comparisons among all treatments excluding the the enzyme preparation replaced the same 0 ug of D 3 per kg of ration, data were obtained volume of H 2 0 in the reaction mixture was also from 10 of 12 hens in the control group and 5 recorded (Ts). The assay was repeated until two hens killed between 154 and 161 days (five observations of Ts were within 5% of each other. dates) from each of the other treatment groups. A value was recorded as the mean of these In order to enhance and focus on the comparison observations. Carbonic anhydrase activity was between the control versus the diet supple- expressed as (To - Ts)/(To+Ts) units per mg of mented with 5 ug of l,25(OH)2D3 per kg of protein (Lowry et al., 1951) in the enzyme ration, the latter being superior to the former for preparation. All treatments were represented as eggshell quality (Tsang et al., 1990a,b), an much as possible on each day of assay to additional five hens from each of these two minimize the influence of storage on treatment treatments were killed at 199 days, and the data comparisons. Nevertheless, the authors have not obtainedfromthese hens were pooled with those observed significant changes in ATPase activity of hens killed at 154 to 161 days. Within each of tissue stored at -20 C over several months or comparison, all treatment groups were repreof enzyme preparations for assay of ATPase or sented on a kill date. carbonic anhydrase stored at -20 C over a few On a given day, hens were sampled only if a days. palpable, developing egg was present in the uterus. The hens were weighed, bled from the Statistical Analysis brachial vein into heparinized vacuum tubes, and killed by cervical dislocation. Blood was The data were analyzed by three specific centrifuged and the plasma stored at -20 C. The comparisons. In the first comparison, the control uterus was weighed, placed in a plastic bag, was compared to the 0 ug of D 3 per kg of ration frozen on dry ice, and stored at -20 C. The at three different times from the start of Treatments
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GRUNDER ET AL. TABLE 1. Effect of omitting vitamin D3 from a ration for various periods of time on weight and physiological traits (x ± SE)
Source of variation
n
Plasma Ca
Jejunal ATPase1
(kg)
(g)
(mg %)
- (figP/mg protein/5 min) -
Uterine ATPase
Uterine carbonic anhydrase (units/mg protein)
24 25
1.94 ± .04" 19.3 ± .7a 1.65 ± .04b 14.4 ± .6b
22.3 ± l . l a 51.8 ± 1.9a 58.9 ± 2.1* 15.1 ± 1.0b 46.4 ± 1.9b 45.6 ± 2.1 b
277 ± 20 s 142 ± 20 b
13 12 24
1.79 ± .06" 15.3 ± .9a 1.86 ± .06a 18.0 ± .9a 1.75 ± .04a 17.2 ± .6a
19.8 ± 1.4a 53.9 ± 2.5 a 49.0 ± 2.8b 18.4 ± 1.4a 41.8 ± 2.6 b 49.6 ± 2.9b 17.8 ± 1.0s 51.5 ± 1.8a 58.0 ± 2.0"
169 ± 26 b 196 ± 27 ab 263 ± 19 s
"••Treatment means for each source of variation within a column with no common superscripts are significantly different (P5.05) according to Duncan's multiple range test. 'ATPase = adenosine triphosphatase.
treatment using an ANOVA based on a factorial design where sources of variation included treatment, time, and their interaction. In the second comparison, six treatments were compared having omitted that with 0 (Xg of D3 per kg. Data collected between 154 to 161 days after the start of treatment were subjected to an analysis of covariance where time after the onset of treatment was the covariate and treatment was the source of variation. For the third comparison, the control treatment and 5 jig of l,25(OH)2D3 per kg of ration were evaluated using analysis of covariance where the covariate was time after the onset of treatment (154 to 161 days and 199 days). Duncan's multiple range test was used to compare means. Statistical programs were those of the Statistical Analysis System (SAS Institute, 1985). RESULTS AND DISCUSSION
A comparison of traits of hens that received the control ration with those that received the D3-deficient ration for up to 162 days is shown in Table 1. All values are greater (P£.05) for the control than for the D3-deficient ration. There were differences (P<.05) among the three sampling periods (Table 1) for jejunal and uterine enzyme activities, but there were no interactions between treatment and duration (data not shown). The negative effect on jejunal ATPase caused by omitting D3 from the ration confirms an earlier observation of Grander and Tsang (1984). However, the levels of ATPase of the present study were approximately twice those of the earlier study.
In the present study, centrifugation of the tissue homogenates was purposely done at a low force of 1,200 x g compared to 12,000 x g in the earlier study (Grander and Tsang, 1984) so that enzyme activity of the mitochondrial and microsomal fractions would be retained. Therefore, the greater amount of enzyme activity could have been due to greater retention of mitochondrial or other material and greater duration of treatment in the present study as compared with the study of Grander and Tsang (1984). In addition to confirming the negative effect of D3-deficient rations on plasma Ca (Tsang et al., 1990a,b) and jejunal ATPase (Grander and Tsang, 1984), this study showed the negative effect of this treatment on uterine ATPase and carbonic anhydrase as well as on BW and uterine weight. Lowered feed consumption previously observed (Tsang et al., 1990a) would account for low BW. This result was consistent with the positive effect of D3 on Cadependent ATPase in the reproductive organs of quail (Regal, 1983). The enzymes of the present study have been implicated in the control of eggshell quality. Gutowska and Mitchell (1945) found higher uterine carbonic anhydrase activity among hard-shelled than soft-shelled layers or nonlayers. Nelson et al. (1979) also reported that duodenal and uterine CaBP protein and uterine carbonic anhydrase activity were greater for a group of hens chosen for high compared with a group chosen for low shell quality. In contrast, several studies indicated that uterine carbonic anhydrase is not related to shell quality (Mueller, 1962; Heald et al, 1968; Grander
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Treatment 27.5 (lg D3/kg OugDs/kg Duration of feeding 56, 57 days 98, 100 days 154 to 162 days
BW
Uterine weight
VITAMIN D METABOLITES AND PHYSIOLOGICAL TRAITS
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TABLE 2. Effect of various dietary treatments involving vitamin D3 and its metabolites on weight and physiological traits (x ± SE) at 154 to 161 days of consumption
Dietary treatment1
BW
10 5 5 5 5
(kg) 1.86 1.89 1.75 1.93 1.53
5
± ± ± ± ±
(g) .06" 18.8 .08* 20.3 .08 al'20.9 .08" 19.0 .08b 15.8
± ± ± ± ±
.9* 12* 12* 12* 12*
Plasma Ca
Jejunal ATPase2
Uterine ATPase
(mg %) 23.4 ± 1.3a 20.6 ± 1.8a 23.1 ± 1.8" 22.7 ± 1.8a 18.9 ± 1.8"
- QlgP/mg protein/5 nrin) 55.4 ± 2.4 a 65.0 ± 2.9° 53.5 ± 3.4 a 61.6 ± 4 . 1 * 63.2 ± 3.4 a 66.1 ± 4.1 a 64.4 ± 3.4 a 57.4 ± 4 . 1 * 54.8 ± 3.4 a 50.6 ± 4.1 b
1.87 ± .08* 18.8 ± 12* 20.6 ± 1.8a 56.5 ± 3.4" 49.9 ± 4.1 b
Uterine carbonic anhydrase (units/mg protein) 340 ± 3 0 * 271 ± 43 a b c 293 ± 43*° 352 ± 43 a 198 ± 43 c 217 ± 43 b c
"""Treatment means within a column with no common superscripts are significantly different (PS.05) according to Duncan's multiple range test Vitamin D 3 or metabolite in basal ration. 2 ATPase = adenosine triphosphatase.
and Hollands, 1976; Klingensmith and Hester, 1985). Several factors such as different methods of assessing shell quality, presence of an egg in the uterus, and time after oviposition or method for measuring carbonic anhydrase could explain why these authors did not observe a positive relationship between this enzyme and shell quality. Eastin and Spaziani (1978) concluded that secretion of Ca is dependent, at least in part, on uterine ATPase which supplies energy for active transport. Grander (1983) did not observe a relationship between uterine ATPase and shell deposition 12 h after oviposition. Watanabe et al. (1989) observed significantly more uterine Ca ATPase activity in their high compared with low shell strength line at 22 but not at 8 or 15 h after oviposition; no significant differences between lines were noted for this enzyme in the duodenum. Haussler et al. (1970) observed in chicks that D3 induced intestinal ATPase activity and Ca absorption. The results of the present study, in which all hens had a developing egg in utero, suggest that D3 does influence jejunal and uterine ATPase and uterine carbonic anhydrase and support the data implicating these enzymes in the control of shell quality. This is because hens without supplemental D 3 had low shell quality (Tsang et al., 1990a,b) in addition to low enzyme activity (Table 1). Six dietary treatments, assessed from 154 to 161 days after initiation, were tested for their effects on the same traits discussed above. Significant differences (P£.05) were observed
for BW, uterine ATPase, and carbonic anhydrase (Table 2). Hens that received a ration supplemented with 5 ug of 24,25(OH)2D3 per kg ranked within the lowest grouping of means among all treatment means for these traits. Inclusion of 5 ug of l,25(OH)2D3 per kg along with 5 ug of 24,25(OH)2D3 per kg of ration was only partially effective in restoring the level of these traits to that of the control ration. Body weight was the only one of these three traits which was significantly restored. Numbers of hens per treatment were small and, hence, there is a risk of accepting or rejecting the null hypothesis erroneously. Because hens were killed only if they had a developing egg in the uterus, physiological traits would tend to approach values for birds fed the control diet regardless of treatment, thereby making detection of differences difficult. Soares (1984) reviewed evidence which suggests that 24,25(OH)2D3 plays a relatively minor role in stimulating eggshell calcification. This metabolite seems to have much less potency than D 3 in maintaining uterine ATPase or carbonic anhydrase (Table 2). Furthermore, 24,25(OH)2D3 suppresses the action of l,25(OH)2D3 as far as eggshell quality is concerned (Tsang et al., 1990b). In the present study, 24,25(OH)2D3 in combination with l,25(OH)2D3 nullified the action of the latter on uterine ATPase. A comparison between hens fed the control ration and hens fed the ration supplemented with 5 ug of l,25(OH)2D3 per kg did not reveal a significant difference for BW or
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27.5 ug D3/kg 3 Jig l,25(OH)2D3/kg 5 jig l,25(OH)2D3/kg 7 fig l,25(OH)2D3/kS 5 fig 24,25(OH)2D3/kg 5 ug U5(OH) 2 D 3 + 5 jig 24,25(OH)2D3/kg
n
Uterine weight
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GRUNDER ET AL.
ACKNOWLEDGMENTS
The authors wish to thank A. R. Morrison and staff for looking after the birds; J. Stefancsisc and J. W. Dickie for collecting data and performing assays, respectively; and M. E. Reid for computer analysis of the data. REFERENCES Eastin, W. C , Jr., and E. Spaziani, 1978. On the mechanism of calcium secretion in the avian shell gland (uterus). Biol. Reprod. 19:505-518. Fairfull, R. W., R. S. Gowe, and JAJB. Emsley, 1983. Diallel cross of six long-term selected Leghorn strains with emphasis on heterosis and reciprocal effects. Br. Poult. Sci. 24:133-158. Grunder, A. A., 1983. Uterine adenosine triphosphatase in relation to shell quality. Poultry Sci. 62:512-513. Grunder, A. A., and K. G. Hollands, 1976. Carbonic anhydrase isozymes of blood and oviductal tissues of Leghorn hens. Poultry Sci. 55:2255-2261. Grunder, A. A., and C.P.W. Tsang, 1984. Effects of vitamin D3 deficiency on adenosine triphosphatase activity of
jejunums from White Leghorn hens. Poultry Sci. 63: 1073-1075. Gutowska, M. S., and C. A. Mitchell, 1945. Carbonic anhydrase in the calcification of the egg shell. Poultry Sci. 24:159-167. Haussler, M. R., L. A. Nagode, and H. Rasmussen, 1970. Induction of intestinal brush border alkaline phosphatase by vitamin D and identity with Ca-ATPase. Nature 228:1199-1201. Heald, P. J., D. Pohlman, and E. G. Martin, 1968. Shell strength and carbonic anhydrase activity of the shell gland of the domestic fowl. Poultry Sci. 47:858-862. Klingensmith, P. M., and P. Y. Hester, 1985. Phosphorus phase feeding and uterine and isthmus mucosal enzymes and minerals in relation to soft-shelled and shell-less egg production. Poultry Sci. 64:2180-2188. Lowry, O. H., N. J. Rosenbough, A. L. Farr, and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Mueller, W. J., 1962. Carbonic anhydrase, diuretics and egg shell formation. Poultry Sci. 41:1792-1796. Nelson, R. A., C. W. Kang, O. E. Olson, and C. W. Carlson, 1979. Calcium-binding protein and carbonic anhydrase activity in aged hens with high and low quality egg shells. Fed. Proc. Fed. Am. Soc. Exp. Biol. 38:766. (Abstr.) Nys, Y., and X. DeLaage, 1984a. Effects of suppression of eggshell calcification and of 1^5(OH)2D3 on Mg 2+ , C ? + and Mg24HC03 ATPase, alkaline phosphatase, carbonic anhydrase and CaBP levels. I. The laying hen uterus. Comp. Biochem. Physiol. 78A:833-838. Nys, Y., and X. DeLaage, 1984b. Effects of suppression of eggshell calcification and of l,25(OH>2 D3 on Mgr+, Ca24" and M^+HCOj ATPase, alkaline phosphatase, carbonic anhydrase and CaBP levels. H. The laying hen intestine. Comp. Biochem. Physiol. 78A:839-844. Regal, D. S., 1983. Experimental studies of extraosseous calcification in hypervitaminosis D3. 3. Effect of goldhafer on formation on proteoglycans and on calcium dependent ATPase in the egg laving organs of quails. Zentralbl. Veterinaermed. Reihe A 30:93-113. SAS Institute, 1985. SAS® User's Guide: Statistics, Version 5 Edition. SAS Institute Inc., Cary, NC. Soares, J. H., Jr., 1984. Calcium metabolism and its control-a review. Poultry Sci. 63:2075-2083. Tsang, C.P.W., A. A. Grunder, and R. Narbaitz, 1990a. Optimal dietary level of la,25-dihydroxycholecalciferol for eggshell quality in laying hens. Poultry Sci. 69:(Suppl. l):135.(Abstr.) Tsang, C.P.W., A. A. Grunder, J. H. Soares, Jr., and R. Narbaitz, 1990b. The effect of loC25-dihydroxycholecalciferol on egg shell quality and egg production. Br. Poult. Sci. 31:241-247. Wasserman, R. H., and A. N. Taylor, 1966. Vitamin D 3 induced calcium-binding protein in chick intestinal mucosa. Science 152:791-793. Watanabe, E. S., S. Kobayashi, Y. Terashima, and H. Itoh, 1989. Adenosine triphosphatase in the uterus and duodenum of chicken hens during egg shell formation. Poultry Sci. 68:564-568. Wilbur, K. M., and N. G. Anderson, 1948. Electronic and colorimetric determination of carbonic anhydrase. J. Biol. Chem. 176:147-154.
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physiological traits after 154 to 161 days of consumption (Table 2). A further analysis of these data pooled with data obtained from 10 hens killed at 199 days after start of treatment also did not result in significant differences (data not shown). Tsang et al. (1990a,b) have demonstrated that 5 |ig of l,25(OH>2D3 per kg ration resulted in better shell quality than the control ration consisting of 27.5 u.g of D3 per kg. It would seem that too few hens were studied to detect significant differences in the physiological traits listed in Table 2 or that l,25(OH)2D3 has a positive effect on shell quality by influencing other physiological traits. This study has confirmed the negative effect of omitting D3 from laying rations on jejunal ATPase and plasma Ca; it has revealed negative effects on BW, uterine ATPase, and carbonic anhydrase, and uterine weight. The study also showed that for some traits, substitution of D3 with 3, 5, or 7 |Xg of l,25(OH)2D3 per kg of ration resulted in values similar to those of hens fed the control ration of 27.5 ug of D3 per kg. Replacement of D 3 with 24i25(OH)2D3 resulted in lower (P<.05) values for BW and uterine ATPase and carbonic anhydrase in those hens as compared with hens fed the control ration.
1990 Poultry Science 69:1204-1208 INTRODUCTION
Although the importance of cholecalciferol, or vitamin D3 (D3), for the production of good eggshells has been known for some time, there is recent evidence that the active metabolite is 1,25-dihydroxycholecalciferol [1,25(OH)2D3], (Soares, 1984). Tsang et al. (1990b) observed that feeding l,25(OH)2D3 at 5 Jig per kg ration to hens produced better eggshells than did control hens fed D3. Another study has indicated that this level of l,25(OH)2D3 is optimal for producing strong eggshells (Tsang et al, 1990a). Some effort has been made to study the effect of D3 on certain physiological traits believed to play a role in eggshell deposition. Wasserman and Taylor (1966) found that Cabinding protein (CaBP) of the chick duodenum was dependent on D3. Haussler et al. (1970) observed that D3 induced adenosine triphosphatase (ATPase) in the chick intestines and Ca absorption from the intestines. Vitamin
D3 also induces Ca-dependent ATPase in reproductive organs of quail (Regal, 1983). Grander and Tsang (1984) have shown that layer diets without supplemental D3 reduced the level of jejunal ATPase. In a physiological study of l,25(OH)2D3, Nys and DeLaage (1984a) found that injections of l,25(OH)2D3 did not alter CaBP or ATPase levels in uteri of hens induced to lay shell-less eggs, but did influence alkaline phosphatase activity in duodenal mucosa and restored CaBP levels in all sections of the intestines (Nys and DeLaage, 1984b). The purpose of the present study was to confirm the effect of deleting D3 from the diet and to determine the effect of replacement with 1,25(011)^3 or 24,25(OH)2D3 on uterine ATPase and carbonic anhydrase, jejunal ATPase, plasma Ca, BW, and uterine weight of laying hens. MATERIALS AND METHODS
Birds Contribution Number 1617 from the Animal Research Centre.
Single Comb White Leghorn hens were produced from nonpedigree matings of 60 Strain
1204
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 7, 2015
ABSTRACT This study was conducted to determine the effect of feeding vitamin 03(03) metabolites on BW of hen, weight of uterus, plasma Ca, jejunal and uterine adenosine triphosphatase (ATPase), and carbonic anhydrase. At 416 days of age each of 7 groups of laying hens was fed the basal ration supplemented with one of 7 concentrations (ng per kg) of D3 or its metabolites as treatments: 0 jig of D3; 27.5 ug of D3; 3,5, or 7 ug of l,25(OH)2D3; 5 |ig of 24,25(OH)2D3; and 5 ug of 24,25(OH)2D3 plus 5 Jig of l ^ O H ) - ^ . Treatment effects were compared at various periods after the start of the study. Hens fed the unsupplemented ration had lower (P<05) values for all traits man hens fed the D3supplemented ration by 162 days after the start of treatment In a comparison of all dietary treatments except the one involving 0 ug D3, from 154 to 161 days after the start of the experiment, treatment effects were significant (P£.05) for BW, uterine ATPase, and carbonic anhydrase; hens fed 5 Ug of 24^5(OH)2D3 per kg of ration ranked the lowest of all treatment groups for these traits. Hens fed 27.5 ug of D3 and those fed 5 Ug of l,25(OH)2D3 per kg of ration did not differ (P>.05) for any traits studied. The results suggest that 5 ug of 1^5(OH)2D3 per kg of ration can replace 27.5 ug of D3 per kg of ration but that 5 fig of 24,25(OH)2I>3 per kg of ration tends to have a negative effect on physiological systems of the hen. (Key words: vitamin D metabolites, body weight, laying hen, adenosine triphosphatase, carbonic anhydrase)
VITAMIN D METABOLITES AND PHYSIOLOGICAL TRAITS
1 males to 460 Strain 2 females. These experimental strains had been selected for multiple traits with emphasis on egg production (Fairfull et al, 1983). After rearing to 18 wk of age, birds were caged individually and fed a balanced laying ration containing 3.1% Ca and supplemented with 27.5 ug of D3 per kg of ration. Light was gradually increased from 8 h at 133 days of age by .5 h per week to 16 h per day in a windowless house.
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jejunum was opened longitudinally and washed with .9% Nad. Two sections of approximately 1 g each were cut from the anterior end of the jejunum, weighed, and placed in a polypropylene tube containing a .25 M sucrose solution (4 mL per g of tissue), frozen on dry ice, and stored at -20 C. Assays
Plasma Ca was assayed using an atomicabsorption spectrophotometer. Jejunal and uterine ATPase were assayed as described by At 416 days of age hens were randomly Grander (1983) and Grander and Tsang (1984). assigned to one of seven dietary treatment However, each homogenate of jejunum or groups consisting of 28 or 56 hens. Each group uterine mucosa was centrifuged at 1,200 x g for was fed the same basal ration supplemented with 25 min instead of 12,000 x g (Grander, 1983). different concentrations (ug per kg ration) of D3 Assay of uterine carbonic anhydrase activity in or its metabolites as follows: 0 |xg of D3; 27.5 ug these preparations that were diluted one in five of D 3 (control); 3,5, or 7 ug of l,25(OH)2D3; 5 with water was based on the method of Wilbur ug of 24,25(OH)2D3; 5 ug each of l,25(OH)2D3 and Anderson (1948). All reaction mixtures and 24,25(OH)2D3. were cooled by ice-cold water. Three mL of H 2 0 Hens were killed by cervical dislocation to saturated with CQj was added to .60 mL of H 2 0 obtain samples and to make specific compar- and 2.4 mL of veronal buffer (.044 M sodium isons at various periods after the start of barbital and .032 M barbituric acid) at a pH of treatment. For comparing the 0 ug of D 3 to Jhe 8.15. The pH was recorded every 10 s and was control treatment, 7 and 6 hens, respectively, plotted on semi-log paper. The time it took for were killed at either 56 or 57 days; 6 hens were the pH to dropfrom8.1 to 6.4 was recorded (To) killed per treatment at either 98 or 100 days; 12 and the average of four such observations was hens were killed per treatment over six dates at utilized in subsequent calculations. The time it 154, 155, 157, 158, 161, and 162 days. For took for a similar change in pH when .02 mL of comparisons among all treatments excluding the the enzyme preparation replaced the same 0 ug of D 3 per kg of ration, data were obtained volume of H 2 0 in the reaction mixture was also from 10 of 12 hens in the control group and 5 recorded (Ts). The assay was repeated until two hens killed between 154 and 161 days (five observations of Ts were within 5% of each other. dates) from each of the other treatment groups. A value was recorded as the mean of these In order to enhance and focus on the comparison observations. Carbonic anhydrase activity was between the control versus the diet supple- expressed as (To - Ts)/(To+Ts) units per mg of mented with 5 ug of l,25(OH)2D3 per kg of protein (Lowry et al., 1951) in the enzyme ration, the latter being superior to the former for preparation. All treatments were represented as eggshell quality (Tsang et al., 1990a,b), an much as possible on each day of assay to additional five hens from each of these two minimize the influence of storage on treatment treatments were killed at 199 days, and the data comparisons. Nevertheless, the authors have not obtainedfromthese hens were pooled with those observed significant changes in ATPase activity of hens killed at 154 to 161 days. Within each of tissue stored at -20 C over several months or comparison, all treatment groups were repreof enzyme preparations for assay of ATPase or sented on a kill date. carbonic anhydrase stored at -20 C over a few On a given day, hens were sampled only if a days. palpable, developing egg was present in the uterus. The hens were weighed, bled from the Statistical Analysis brachial vein into heparinized vacuum tubes, and killed by cervical dislocation. Blood was The data were analyzed by three specific centrifuged and the plasma stored at -20 C. The comparisons. In the first comparison, the control uterus was weighed, placed in a plastic bag, was compared to the 0 ug of D 3 per kg of ration frozen on dry ice, and stored at -20 C. The at three different times from the start of Treatments
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1206
GRUNDER ET AL. TABLE 1. Effect of omitting vitamin D3 from a ration for various periods of time on weight and physiological traits (x ± SE)
Source of variation
n
Plasma Ca
Jejunal ATPase1
(kg)
(g)
(mg %)
- (figP/mg protein/5 min) -
Uterine ATPase
Uterine carbonic anhydrase (units/mg protein)
24 25
1.94 ± .04" 19.3 ± .7a 1.65 ± .04b 14.4 ± .6b
22.3 ± l . l a 51.8 ± 1.9a 58.9 ± 2.1* 15.1 ± 1.0b 46.4 ± 1.9b 45.6 ± 2.1 b
277 ± 20 s 142 ± 20 b
13 12 24
1.79 ± .06" 15.3 ± .9a 1.86 ± .06a 18.0 ± .9a 1.75 ± .04a 17.2 ± .6a
19.8 ± 1.4a 53.9 ± 2.5 a 49.0 ± 2.8b 18.4 ± 1.4a 41.8 ± 2.6 b 49.6 ± 2.9b 17.8 ± 1.0s 51.5 ± 1.8a 58.0 ± 2.0"
169 ± 26 b 196 ± 27 ab 263 ± 19 s
"••Treatment means for each source of variation within a column with no common superscripts are significantly different (P5.05) according to Duncan's multiple range test. 'ATPase = adenosine triphosphatase.
treatment using an ANOVA based on a factorial design where sources of variation included treatment, time, and their interaction. In the second comparison, six treatments were compared having omitted that with 0 (Xg of D3 per kg. Data collected between 154 to 161 days after the start of treatment were subjected to an analysis of covariance where time after the onset of treatment was the covariate and treatment was the source of variation. For the third comparison, the control treatment and 5 jig of l,25(OH)2D3 per kg of ration were evaluated using analysis of covariance where the covariate was time after the onset of treatment (154 to 161 days and 199 days). Duncan's multiple range test was used to compare means. Statistical programs were those of the Statistical Analysis System (SAS Institute, 1985). RESULTS AND DISCUSSION
A comparison of traits of hens that received the control ration with those that received the D3-deficient ration for up to 162 days is shown in Table 1. All values are greater (P£.05) for the control than for the D3-deficient ration. There were differences (P<.05) among the three sampling periods (Table 1) for jejunal and uterine enzyme activities, but there were no interactions between treatment and duration (data not shown). The negative effect on jejunal ATPase caused by omitting D3 from the ration confirms an earlier observation of Grander and Tsang (1984). However, the levels of ATPase of the present study were approximately twice those of the earlier study.
In the present study, centrifugation of the tissue homogenates was purposely done at a low force of 1,200 x g compared to 12,000 x g in the earlier study (Grander and Tsang, 1984) so that enzyme activity of the mitochondrial and microsomal fractions would be retained. Therefore, the greater amount of enzyme activity could have been due to greater retention of mitochondrial or other material and greater duration of treatment in the present study as compared with the study of Grander and Tsang (1984). In addition to confirming the negative effect of D3-deficient rations on plasma Ca (Tsang et al., 1990a,b) and jejunal ATPase (Grander and Tsang, 1984), this study showed the negative effect of this treatment on uterine ATPase and carbonic anhydrase as well as on BW and uterine weight. Lowered feed consumption previously observed (Tsang et al., 1990a) would account for low BW. This result was consistent with the positive effect of D3 on Cadependent ATPase in the reproductive organs of quail (Regal, 1983). The enzymes of the present study have been implicated in the control of eggshell quality. Gutowska and Mitchell (1945) found higher uterine carbonic anhydrase activity among hard-shelled than soft-shelled layers or nonlayers. Nelson et al. (1979) also reported that duodenal and uterine CaBP protein and uterine carbonic anhydrase activity were greater for a group of hens chosen for high compared with a group chosen for low shell quality. In contrast, several studies indicated that uterine carbonic anhydrase is not related to shell quality (Mueller, 1962; Heald et al, 1968; Grander
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Treatment 27.5 (lg D3/kg OugDs/kg Duration of feeding 56, 57 days 98, 100 days 154 to 162 days
BW
Uterine weight
VITAMIN D METABOLITES AND PHYSIOLOGICAL TRAITS
1207
TABLE 2. Effect of various dietary treatments involving vitamin D3 and its metabolites on weight and physiological traits (x ± SE) at 154 to 161 days of consumption
Dietary treatment1
BW
10 5 5 5 5
(kg) 1.86 1.89 1.75 1.93 1.53
5
± ± ± ± ±
(g) .06" 18.8 .08* 20.3 .08 al'20.9 .08" 19.0 .08b 15.8
± ± ± ± ±
.9* 12* 12* 12* 12*
Plasma Ca
Jejunal ATPase2
Uterine ATPase
(mg %) 23.4 ± 1.3a 20.6 ± 1.8a 23.1 ± 1.8" 22.7 ± 1.8a 18.9 ± 1.8"
- QlgP/mg protein/5 nrin) 55.4 ± 2.4 a 65.0 ± 2.9° 53.5 ± 3.4 a 61.6 ± 4 . 1 * 63.2 ± 3.4 a 66.1 ± 4.1 a 64.4 ± 3.4 a 57.4 ± 4 . 1 * 54.8 ± 3.4 a 50.6 ± 4.1 b
1.87 ± .08* 18.8 ± 12* 20.6 ± 1.8a 56.5 ± 3.4" 49.9 ± 4.1 b
Uterine carbonic anhydrase (units/mg protein) 340 ± 3 0 * 271 ± 43 a b c 293 ± 43*° 352 ± 43 a 198 ± 43 c 217 ± 43 b c
"""Treatment means within a column with no common superscripts are significantly different (PS.05) according to Duncan's multiple range test Vitamin D 3 or metabolite in basal ration. 2 ATPase = adenosine triphosphatase.
and Hollands, 1976; Klingensmith and Hester, 1985). Several factors such as different methods of assessing shell quality, presence of an egg in the uterus, and time after oviposition or method for measuring carbonic anhydrase could explain why these authors did not observe a positive relationship between this enzyme and shell quality. Eastin and Spaziani (1978) concluded that secretion of Ca is dependent, at least in part, on uterine ATPase which supplies energy for active transport. Grander (1983) did not observe a relationship between uterine ATPase and shell deposition 12 h after oviposition. Watanabe et al. (1989) observed significantly more uterine Ca ATPase activity in their high compared with low shell strength line at 22 but not at 8 or 15 h after oviposition; no significant differences between lines were noted for this enzyme in the duodenum. Haussler et al. (1970) observed in chicks that D3 induced intestinal ATPase activity and Ca absorption. The results of the present study, in which all hens had a developing egg in utero, suggest that D3 does influence jejunal and uterine ATPase and uterine carbonic anhydrase and support the data implicating these enzymes in the control of shell quality. This is because hens without supplemental D 3 had low shell quality (Tsang et al., 1990a,b) in addition to low enzyme activity (Table 1). Six dietary treatments, assessed from 154 to 161 days after initiation, were tested for their effects on the same traits discussed above. Significant differences (P£.05) were observed
for BW, uterine ATPase, and carbonic anhydrase (Table 2). Hens that received a ration supplemented with 5 ug of 24,25(OH)2D3 per kg ranked within the lowest grouping of means among all treatment means for these traits. Inclusion of 5 ug of l,25(OH)2D3 per kg along with 5 ug of 24,25(OH)2D3 per kg of ration was only partially effective in restoring the level of these traits to that of the control ration. Body weight was the only one of these three traits which was significantly restored. Numbers of hens per treatment were small and, hence, there is a risk of accepting or rejecting the null hypothesis erroneously. Because hens were killed only if they had a developing egg in the uterus, physiological traits would tend to approach values for birds fed the control diet regardless of treatment, thereby making detection of differences difficult. Soares (1984) reviewed evidence which suggests that 24,25(OH)2D3 plays a relatively minor role in stimulating eggshell calcification. This metabolite seems to have much less potency than D 3 in maintaining uterine ATPase or carbonic anhydrase (Table 2). Furthermore, 24,25(OH)2D3 suppresses the action of l,25(OH)2D3 as far as eggshell quality is concerned (Tsang et al., 1990b). In the present study, 24,25(OH)2D3 in combination with l,25(OH)2D3 nullified the action of the latter on uterine ATPase. A comparison between hens fed the control ration and hens fed the ration supplemented with 5 ug of l,25(OH)2D3 per kg did not reveal a significant difference for BW or
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27.5 ug D3/kg 3 Jig l,25(OH)2D3/kg 5 jig l,25(OH)2D3/kg 7 fig l,25(OH)2D3/kS 5 fig 24,25(OH)2D3/kg 5 ug U5(OH) 2 D 3 + 5 jig 24,25(OH)2D3/kg
n
Uterine weight
1208
GRUNDER ET AL.
ACKNOWLEDGMENTS
The authors wish to thank A. R. Morrison and staff for looking after the birds; J. Stefancsisc and J. W. Dickie for collecting data and performing assays, respectively; and M. E. Reid for computer analysis of the data. REFERENCES Eastin, W. C , Jr., and E. Spaziani, 1978. On the mechanism of calcium secretion in the avian shell gland (uterus). Biol. Reprod. 19:505-518. Fairfull, R. W., R. S. Gowe, and JAJB. Emsley, 1983. Diallel cross of six long-term selected Leghorn strains with emphasis on heterosis and reciprocal effects. Br. Poult. Sci. 24:133-158. Grunder, A. A., 1983. Uterine adenosine triphosphatase in relation to shell quality. Poultry Sci. 62:512-513. Grunder, A. A., and K. G. Hollands, 1976. Carbonic anhydrase isozymes of blood and oviductal tissues of Leghorn hens. Poultry Sci. 55:2255-2261. Grunder, A. A., and C.P.W. Tsang, 1984. Effects of vitamin D3 deficiency on adenosine triphosphatase activity of
jejunums from White Leghorn hens. Poultry Sci. 63: 1073-1075. Gutowska, M. S., and C. A. Mitchell, 1945. Carbonic anhydrase in the calcification of the egg shell. Poultry Sci. 24:159-167. Haussler, M. R., L. A. Nagode, and H. Rasmussen, 1970. Induction of intestinal brush border alkaline phosphatase by vitamin D and identity with Ca-ATPase. Nature 228:1199-1201. Heald, P. J., D. Pohlman, and E. G. Martin, 1968. Shell strength and carbonic anhydrase activity of the shell gland of the domestic fowl. Poultry Sci. 47:858-862. Klingensmith, P. M., and P. Y. Hester, 1985. Phosphorus phase feeding and uterine and isthmus mucosal enzymes and minerals in relation to soft-shelled and shell-less egg production. Poultry Sci. 64:2180-2188. Lowry, O. H., N. J. Rosenbough, A. L. Farr, and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Mueller, W. J., 1962. Carbonic anhydrase, diuretics and egg shell formation. Poultry Sci. 41:1792-1796. Nelson, R. A., C. W. Kang, O. E. Olson, and C. W. Carlson, 1979. Calcium-binding protein and carbonic anhydrase activity in aged hens with high and low quality egg shells. Fed. Proc. Fed. Am. Soc. Exp. Biol. 38:766. (Abstr.) Nys, Y., and X. DeLaage, 1984a. Effects of suppression of eggshell calcification and of 1^5(OH)2D3 on Mg 2+ , C ? + and Mg24HC03 ATPase, alkaline phosphatase, carbonic anhydrase and CaBP levels. I. The laying hen uterus. Comp. Biochem. Physiol. 78A:833-838. Nys, Y., and X. DeLaage, 1984b. Effects of suppression of eggshell calcification and of l,25(OH>2 D3 on Mgr+, Ca24" and M^+HCOj ATPase, alkaline phosphatase, carbonic anhydrase and CaBP levels. H. The laying hen intestine. Comp. Biochem. Physiol. 78A:839-844. Regal, D. S., 1983. Experimental studies of extraosseous calcification in hypervitaminosis D3. 3. Effect of goldhafer on formation on proteoglycans and on calcium dependent ATPase in the egg laving organs of quails. Zentralbl. Veterinaermed. Reihe A 30:93-113. SAS Institute, 1985. SAS® User's Guide: Statistics, Version 5 Edition. SAS Institute Inc., Cary, NC. Soares, J. H., Jr., 1984. Calcium metabolism and its control-a review. Poultry Sci. 63:2075-2083. Tsang, C.P.W., A. A. Grunder, and R. Narbaitz, 1990a. Optimal dietary level of la,25-dihydroxycholecalciferol for eggshell quality in laying hens. Poultry Sci. 69:(Suppl. l):135.(Abstr.) Tsang, C.P.W., A. A. Grunder, J. H. Soares, Jr., and R. Narbaitz, 1990b. The effect of loC25-dihydroxycholecalciferol on egg shell quality and egg production. Br. Poult. Sci. 31:241-247. Wasserman, R. H., and A. N. Taylor, 1966. Vitamin D 3 induced calcium-binding protein in chick intestinal mucosa. Science 152:791-793. Watanabe, E. S., S. Kobayashi, Y. Terashima, and H. Itoh, 1989. Adenosine triphosphatase in the uterus and duodenum of chicken hens during egg shell formation. Poultry Sci. 68:564-568. Wilbur, K. M., and N. G. Anderson, 1948. Electronic and colorimetric determination of carbonic anhydrase. J. Biol. Chem. 176:147-154.
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physiological traits after 154 to 161 days of consumption (Table 2). A further analysis of these data pooled with data obtained from 10 hens killed at 199 days after start of treatment also did not result in significant differences (data not shown). Tsang et al. (1990a,b) have demonstrated that 5 |ig of l,25(OH>2D3 per kg ration resulted in better shell quality than the control ration consisting of 27.5 u.g of D3 per kg. It would seem that too few hens were studied to detect significant differences in the physiological traits listed in Table 2 or that l,25(OH)2D3 has a positive effect on shell quality by influencing other physiological traits. This study has confirmed the negative effect of omitting D3 from laying rations on jejunal ATPase and plasma Ca; it has revealed negative effects on BW, uterine ATPase, and carbonic anhydrase, and uterine weight. The study also showed that for some traits, substitution of D3 with 3, 5, or 7 |Xg of l,25(OH)2D3 per kg of ration resulted in values similar to those of hens fed the control ration of 27.5 ug of D3 per kg. Replacement of D 3 with 24i25(OH)2D3 resulted in lower (P<.05) values for BW and uterine ATPase and carbonic anhydrase in those hens as compared with hens fed the control ration.