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Research Note: Responses of Laying Hens on Saline Drinking Water to Dietary Supplementation with Various Zinc Compounds
Research Note: Responses of Laying Hens on Saline Drinking Water to Dietary Supplementation with Various Zinc Compounds D. BALNAVE and D. ZHANG Department of Animal Science, University of Sydney, Werotnbi Road, Camden, New South Wales, 2570, Australia
1993 Poultry Science 72:603-606
INTRODUCTION Recently, Moreng et al. (1992) reported that the adverse effects of saline drinking water on the incidence of eggshell defects and eggshell quality can be alleviated by the simultaneous supplementation of the diet with a metal-amino acid complex, Znmethionine (Zinpro-200).1 These beneficial responses may be related to the Zn content of Zn-methionine. Yoselewitz and Balnave (1989) had previously reported a reduced activity of carbonic anhydrase (CA), a Zn-dependent enzyme, in the shell
Received for publication September 14, 1992. Accepted for publication November 19, 1992. iZinpro Corp., Edina, MN 55439-2441.
gland mucosa of laying hens receiving saline drinking water. There is other evidence suggesting that the supply of carbonate ion to the lumen of the shell gland may be a major factor limiting eggshell quality under these circumstances (Balnave et al, 1989). Because Zn is an integral component of CA, it is possible that supplying Zn-methionine in the diet may have had a direct effect on eggshell quality through an increased activity of this enzyme. Moreng et al. (1992) reported no improvements in eggshell defects or in eggshell quality when Zn sulfate (ZnSGv^O) was provided in the diet to approximate the same dietary Zn concentration as the Zn-methionine supplement (.1 g/kg). It is possible that the lack of
603
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on April 13, 2015
ABSTRACT Production variables, eggshell defects, eggshell quality, the concentration of calcium-binding protein (CaBP), and the activity of carbonic anhydrase (CA) in the shell gland mucosa were determined in hens receiving town water (10 mg Na/L), or town water supplemented with 2 g NaCl/L (796 mg Na/L). Five treatments were examined. Control hens received town water and a proprietary layer mash containing .17% Na. The remaining four treatments received the water containing NaCl (2 g/L). Hens of Treatment 2 were fed the proprietary layer mash and those of Treatments 3, 4, and 5 received, respectively, the layer mash containing supplements of Zn-methionine (Zinpro200; .5 g/kg), Zn sulfate (ZnS04-7H20; .46 g/kg), or chelated Zn-EDTA (.54 g/ kg) to supply the same concentration of Zn (.1 g/kg). The treatments were applied for 6 wk. Hens receiving the saline drinking water without any dietary Zn supplement produced significantly (P < .05) more eggs with shell defects than hens receiving the town water. This increase in the incidence of eggshell defects was associated with significant reductions in eggshell breaking strength, the concentration of CaBP, and the activity of CA. Supplementing the saline drinking water with any of the three Zn compounds significantly reduced the incidence of eggshell defects and in some cases improved shell breaking strength, the concentration of CaBP, and the activity of CA. (Key words: laying hens, shell defects, shell quality, calcium-binding protein, carbonic anhydrase)
604
BALNAVE AND ZHANG
response to Zn sulfate may have reflected the differences in Zn availability known to exist when these compounds are fed to chickens (Wedekind et al, 1990). Accordingly, in the present study a Zn-EDTA chelate was used for comparison with Znmethionine and Zn sulfate. MATERIALS AND METHODS
Ingredients and analysis Sorghum Wheat middlings Meat and bone meal (50% CP) Sunflower meal (35% CP) Cottonseed meal (38% CP) Beef tallow Limestone Bentonite Sodium chloride Sodium bicarbonate L-lysine Liquid methionine (40%) Layer premix 1 Calculated analysis ME, kcal/kg CP TSAA Methionine Calcium Available phosphorus Sodium, g / k g Chlorine, g / k g Zinc, g / k g
Percentage 43.73 25.00 8.50 7.00 2.00 4.00 7.50 1.25 .08 .07 .10 .27 .50 2,600 16 .53 .35 3.80 .56 1.7 1.6 .12
Provided the following per kilogram of diet: vitamin A, 8,000 IU; cholecalciferol, 1,600 IU; vitamin E, 10 mg; menadione, .9 mg; riboflavin, 4 mg; calcium pantothenate, 3.5 mg; thiamin, .4 mg; niacin, 20 mg; biotin, 10 jtg; vitamin Bx2, 10 /tg; choline, 100 mg; copper, 5.2 mg; iron, 45 mg; manganese, 66 mg; zinc, 50 mg; iodine, 1.3 mg; molybdenum, .4 mg; ethoxyquin (antioxidant), 100 mg; carophyl (yolk pigment), 55 mg.
eggs from 3 consecutive days to accumulate on the cage fronts. Eggs were collected and inspected manually. One egg from each hen was collected during the final 2 days of the experiment and was used for shell quality measurements as described previously (Balnave et al, 1991). Feed and water intakes were measured over the complete experiment. The feed and water treatments were continued for 3 days beyond the 6-wk experimental period. On each of these days two hens from each treatment were killed between 0900 and 1200 h. Each hen was killed by cervical dislocation; then the shell gland was rapidly removed and placed on ice. It was cut open immediately and the mucosa removed by scraping gently with the edge of a microscope slide. The tissue was weighed and suspended in 10 vol of deionized water (CA) or 5 vol of Tris buffer (CaBP). Calcium-binding protein
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on April 13, 2015
The experiment was carried out using 300 71-wk-old White Leghorn x New Hampshire hens that, during lay, had been given free access to municipal town drinking water and a commercial layer mash (Table 1). The mash was calculated to contain 2,600 kcal ME/kg, 16% CP, .35% methionine, .53% TSAA, 3.8% Ca, and .56% available P. The drinking water contained 10 mg Na, 41 mg CI, and 1 mg Zn/L and the corresponding concentrations in the diet were 1.7 g Na, 1.6 g CI, and .12 g Zn/kg, respectively. During the experiment two replicates of 30, individually caged hens were allocated at random to each of five treatments. Hens of Treatment 1 received the municipal town water (10 mg Na/L) and the proprietary layer mash. Hens of Treatment 2 received the town water containing 2 g NaCl/L (796 mg Na/L) and the proprietary layer mash. Hens of Treatments 3, 4, and 5 were also given the drinking water containing 2 g NaCl/L and the proprietary layer mash to which had been added, respectively, .5 g Zn-methionine/kg diet from Zinpro-200 (Treatment 3), .46 g ZnS0 4 -7H 2 0/kg (Treatment 4), or .54 g Zn-EDTA/kg (Treatment 5). The ZnSC>4-7H20 and the EDTA were reagent grade, and all dietary supplements supplied the same amount of Zn (.1 g/kg) when allowance was made for differences in dry matter content. Hens were given free access to feed and drinking water at all times and light was provided daily between 0430 and 2030 h throughout the 6-wk experiment. Each treatment replicate was treated as an experimental unit with egg production, feed intake, and water intake being recorded for the complete group. The numbers of eggshell defects (i.e., soft, broken, cracked, or deformed shells) were determined once weekly after allowing the
TABLE 1. Composition of layer mash
RESEARCH NOTE
was measured by the Chelex-100 assay described by Corradino et al. (1968) and CA as described by Yoselewitz and Balnave (1989). The method used for the assay of CA was shown previously to minimize contamination of the enzyme extracts with blood (Yoselewitz and Balnave, 1989). The data were analyzed by one-way ANOVA and means were compared by least significant difference (Steel and Torrie, 1982). Time was used as a repeat measure in the analysis of egg production and eggshell defects.
The results are shown in Table 2. N o significant treatment effect was observed for feed consumption, water intake, egg production, egg weight, shell thickness, shell weight:egg weight (x 100), or shell weight per unit surface area. The incidence of eggshell defects from hens receiving the saline drinking water and the unsupplemented layer mash was significantly greater than for any other treatment. These shell defects consisted of 76% cracked shells, 14% soft shells, and 10% broken shells. The increased incidence of eggshell defects in hens receiving the saline drinking water was reflected in a significant reduction in eggshell breaking strength. However, the fact that none of the other eggshell quality measures showed a significant response to the saline water contrasts with the results of previous studies (Balnave and Yoselewitz, 1987; Balnave et al, 1989, 1991), in which all shell quality measures varied in a concerted fashion. The decrease in shell breaking strength in the absence of any reduction in the amount of shell deposited may indicate that, in this particular study, the NaCl was influencing the structural organization of the shell (Bain, 1992). The incidence of eggshell defects was reduced significantly by the Znmethionine (P < .01), Zn-EDTA (P < .01), and ZnSC>4 (P < .05) supplements to values approximating that obtained for hens on the municipal town water. The beneficial response to the ZnSO^TF^O observed in the present study was greater than that noted previously with
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RESULTS AND DISCUSSION
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ACKNOWLEDGMENTS
The technical assistance of M. Zimmerman is acknowledged. This work was supported by the Egg Industry Research and Development Council and the Poultry Research Foundation, University of Sydney.
REFERENCES Bain, M. M, 1992. Eggshell strength: a relationship between the mechanism of failure and the ultrastructural organisation of the mammillary layer. Br. Poult. Sci. 33:303-319. Balnave, D., and I. Yoselewitz, 1987. The relation between sodium chloride concentration in drinking water and egg shell damage. Br. J. Nutr. 58:503-509. Balnave, D., I. Yoselewitz, and R. J. Dixon, 1989. Physiological changes associated with the production of defective egg-shells by hens receiving sodium chloride in the drinking water. Br. J. Nutr. 61:35-43. Balnave, D„ D. Zhang, and R. E. Moreng, 1991. Use of ascorbic acid to prevent the decline in eggshell quality observed with saline drinking water. Poultry Sci. 70:848-852. Corradino, R. A., R. H. Wasserman, M. H. Pubols, and S. I. Chang, 1968. Vitamin D3 induction of a calcium-binding protein in the uterus of the laying hen. Arch. Biochem. Biophys. 125: 378-380. Eastin, W. C, and E. Spaziani, 1978. On the mechanism of calcium secretion in the avian shell gland (uterus). Biol. Reprod. 19:505-518. Moreng, R. E., D. Balnave, and D. Zhang, 1992. Dietary zinc methionine effect on eggshell quality of hens drinking saline water. Poultry Sci. 71:1163-1167. Pearson, T. W., T. J. Pryor, and A. M. Goldner, 1977. Calcium transport across avian uterus. HI. Comparison of laying and nonlaying birds. Am. J. Physiol. 232:E437-E443. Steel, R.G.D., and J. H. Torrie, 1982. Principles and Procedures of Statistics. A Biometrical Approach. 2nd ed. McGraw-Hill, Kogakusha Ltd., Tokyo, Japan. Wedekind, K. J., A. E. Hortin, and D. H. Baker, 1990. Bioavailability of zinc in a zinc methionine chelate. J. Anim. Sci. 68(Suppl. l):394.(Abstr.) Yoselewitz, I., and D. Balnave, 1989. The influence of saline drinking water on the activity of carbonic anhydrase in the shell gland of laying hens. Aust. J. Agric. Res. 40:1111-1115.
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on April 13, 2015
ZnS0 4 H 2 0 (Moreng et al., 1992). This may reflect a reduced availability of Zn from the monohydrate salt. The shell breaking strength of eggs from hens receiving saline water was improved by all three Zn supplements although only the Zn-EDTA response was significant (P < .05) and approached the value for the hens receiving the town water. These results suggest that the Zn component of the Znmethionine was the factor responsible for the reduction in eggshell defects observed previously when Zn-methionine was added to the diet of hens on saline drinking water (Moreng et al, 1992). This is not unexpected because the concentrations of Zn-methionine used in the present and previous work (.2 to .5 g/kg) only supplied .008 to .02% methionine to the diet. The significantly lower shell gland CaBP concentration and CA activity observed in hens receiving the saline water and laying eggs with defective shells in the present study can be associated with the significantly lower Ca and bicarbonate ion concentrations observed by Balnave et al. (1989) in the shell gland fluid of similarly treated hens. The concentration of CaBP was significantly (P < .05) increased by the Zn-methionine and ZnSC>4 supplements, and the CA activity was increased (P < .01) by the Zn-EDTA supplement, the increase with Znmethionine just failing to attain statistical significance (P < .06). Improvements of between 30 and 78% in CA activity and between 10 and 17% in CaBP were obtained with the Zn supplements. It is likely that the supply of Ca and carbonate ions to the lumen of the shell gland are interdependent. In this regard, Pearson et al. (1977) suggested that Ca transport across the avian shell gland is related to the activity of CA in this tissue, and Eastin and Spaziani (1978) have reported a dependency of Ca secretion on lumen bicarbonate concentration and bicarbonate production.
1993 Poultry Science 72:603-606
INTRODUCTION Recently, Moreng et al. (1992) reported that the adverse effects of saline drinking water on the incidence of eggshell defects and eggshell quality can be alleviated by the simultaneous supplementation of the diet with a metal-amino acid complex, Znmethionine (Zinpro-200).1 These beneficial responses may be related to the Zn content of Zn-methionine. Yoselewitz and Balnave (1989) had previously reported a reduced activity of carbonic anhydrase (CA), a Zn-dependent enzyme, in the shell
Received for publication September 14, 1992. Accepted for publication November 19, 1992. iZinpro Corp., Edina, MN 55439-2441.
gland mucosa of laying hens receiving saline drinking water. There is other evidence suggesting that the supply of carbonate ion to the lumen of the shell gland may be a major factor limiting eggshell quality under these circumstances (Balnave et al, 1989). Because Zn is an integral component of CA, it is possible that supplying Zn-methionine in the diet may have had a direct effect on eggshell quality through an increased activity of this enzyme. Moreng et al. (1992) reported no improvements in eggshell defects or in eggshell quality when Zn sulfate (ZnSGv^O) was provided in the diet to approximate the same dietary Zn concentration as the Zn-methionine supplement (.1 g/kg). It is possible that the lack of
603
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on April 13, 2015
ABSTRACT Production variables, eggshell defects, eggshell quality, the concentration of calcium-binding protein (CaBP), and the activity of carbonic anhydrase (CA) in the shell gland mucosa were determined in hens receiving town water (10 mg Na/L), or town water supplemented with 2 g NaCl/L (796 mg Na/L). Five treatments were examined. Control hens received town water and a proprietary layer mash containing .17% Na. The remaining four treatments received the water containing NaCl (2 g/L). Hens of Treatment 2 were fed the proprietary layer mash and those of Treatments 3, 4, and 5 received, respectively, the layer mash containing supplements of Zn-methionine (Zinpro200; .5 g/kg), Zn sulfate (ZnS04-7H20; .46 g/kg), or chelated Zn-EDTA (.54 g/ kg) to supply the same concentration of Zn (.1 g/kg). The treatments were applied for 6 wk. Hens receiving the saline drinking water without any dietary Zn supplement produced significantly (P < .05) more eggs with shell defects than hens receiving the town water. This increase in the incidence of eggshell defects was associated with significant reductions in eggshell breaking strength, the concentration of CaBP, and the activity of CA. Supplementing the saline drinking water with any of the three Zn compounds significantly reduced the incidence of eggshell defects and in some cases improved shell breaking strength, the concentration of CaBP, and the activity of CA. (Key words: laying hens, shell defects, shell quality, calcium-binding protein, carbonic anhydrase)
604
BALNAVE AND ZHANG
response to Zn sulfate may have reflected the differences in Zn availability known to exist when these compounds are fed to chickens (Wedekind et al, 1990). Accordingly, in the present study a Zn-EDTA chelate was used for comparison with Znmethionine and Zn sulfate. MATERIALS AND METHODS
Ingredients and analysis Sorghum Wheat middlings Meat and bone meal (50% CP) Sunflower meal (35% CP) Cottonseed meal (38% CP) Beef tallow Limestone Bentonite Sodium chloride Sodium bicarbonate L-lysine Liquid methionine (40%) Layer premix 1 Calculated analysis ME, kcal/kg CP TSAA Methionine Calcium Available phosphorus Sodium, g / k g Chlorine, g / k g Zinc, g / k g
Percentage 43.73 25.00 8.50 7.00 2.00 4.00 7.50 1.25 .08 .07 .10 .27 .50 2,600 16 .53 .35 3.80 .56 1.7 1.6 .12
Provided the following per kilogram of diet: vitamin A, 8,000 IU; cholecalciferol, 1,600 IU; vitamin E, 10 mg; menadione, .9 mg; riboflavin, 4 mg; calcium pantothenate, 3.5 mg; thiamin, .4 mg; niacin, 20 mg; biotin, 10 jtg; vitamin Bx2, 10 /tg; choline, 100 mg; copper, 5.2 mg; iron, 45 mg; manganese, 66 mg; zinc, 50 mg; iodine, 1.3 mg; molybdenum, .4 mg; ethoxyquin (antioxidant), 100 mg; carophyl (yolk pigment), 55 mg.
eggs from 3 consecutive days to accumulate on the cage fronts. Eggs were collected and inspected manually. One egg from each hen was collected during the final 2 days of the experiment and was used for shell quality measurements as described previously (Balnave et al, 1991). Feed and water intakes were measured over the complete experiment. The feed and water treatments were continued for 3 days beyond the 6-wk experimental period. On each of these days two hens from each treatment were killed between 0900 and 1200 h. Each hen was killed by cervical dislocation; then the shell gland was rapidly removed and placed on ice. It was cut open immediately and the mucosa removed by scraping gently with the edge of a microscope slide. The tissue was weighed and suspended in 10 vol of deionized water (CA) or 5 vol of Tris buffer (CaBP). Calcium-binding protein
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on April 13, 2015
The experiment was carried out using 300 71-wk-old White Leghorn x New Hampshire hens that, during lay, had been given free access to municipal town drinking water and a commercial layer mash (Table 1). The mash was calculated to contain 2,600 kcal ME/kg, 16% CP, .35% methionine, .53% TSAA, 3.8% Ca, and .56% available P. The drinking water contained 10 mg Na, 41 mg CI, and 1 mg Zn/L and the corresponding concentrations in the diet were 1.7 g Na, 1.6 g CI, and .12 g Zn/kg, respectively. During the experiment two replicates of 30, individually caged hens were allocated at random to each of five treatments. Hens of Treatment 1 received the municipal town water (10 mg Na/L) and the proprietary layer mash. Hens of Treatment 2 received the town water containing 2 g NaCl/L (796 mg Na/L) and the proprietary layer mash. Hens of Treatments 3, 4, and 5 were also given the drinking water containing 2 g NaCl/L and the proprietary layer mash to which had been added, respectively, .5 g Zn-methionine/kg diet from Zinpro-200 (Treatment 3), .46 g ZnS0 4 -7H 2 0/kg (Treatment 4), or .54 g Zn-EDTA/kg (Treatment 5). The ZnSC>4-7H20 and the EDTA were reagent grade, and all dietary supplements supplied the same amount of Zn (.1 g/kg) when allowance was made for differences in dry matter content. Hens were given free access to feed and drinking water at all times and light was provided daily between 0430 and 2030 h throughout the 6-wk experiment. Each treatment replicate was treated as an experimental unit with egg production, feed intake, and water intake being recorded for the complete group. The numbers of eggshell defects (i.e., soft, broken, cracked, or deformed shells) were determined once weekly after allowing the
TABLE 1. Composition of layer mash
RESEARCH NOTE
was measured by the Chelex-100 assay described by Corradino et al. (1968) and CA as described by Yoselewitz and Balnave (1989). The method used for the assay of CA was shown previously to minimize contamination of the enzyme extracts with blood (Yoselewitz and Balnave, 1989). The data were analyzed by one-way ANOVA and means were compared by least significant difference (Steel and Torrie, 1982). Time was used as a repeat measure in the analysis of egg production and eggshell defects.
The results are shown in Table 2. N o significant treatment effect was observed for feed consumption, water intake, egg production, egg weight, shell thickness, shell weight:egg weight (x 100), or shell weight per unit surface area. The incidence of eggshell defects from hens receiving the saline drinking water and the unsupplemented layer mash was significantly greater than for any other treatment. These shell defects consisted of 76% cracked shells, 14% soft shells, and 10% broken shells. The increased incidence of eggshell defects in hens receiving the saline drinking water was reflected in a significant reduction in eggshell breaking strength. However, the fact that none of the other eggshell quality measures showed a significant response to the saline water contrasts with the results of previous studies (Balnave and Yoselewitz, 1987; Balnave et al, 1989, 1991), in which all shell quality measures varied in a concerted fashion. The decrease in shell breaking strength in the absence of any reduction in the amount of shell deposited may indicate that, in this particular study, the NaCl was influencing the structural organization of the shell (Bain, 1992). The incidence of eggshell defects was reduced significantly by the Znmethionine (P < .01), Zn-EDTA (P < .01), and ZnSC>4 (P < .05) supplements to values approximating that obtained for hens on the municipal town water. The beneficial response to the ZnSO^TF^O observed in the present study was greater than that noted previously with
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RESULTS AND DISCUSSION
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606
BALNAVE AND ZHANG
ACKNOWLEDGMENTS
The technical assistance of M. Zimmerman is acknowledged. This work was supported by the Egg Industry Research and Development Council and the Poultry Research Foundation, University of Sydney.
REFERENCES Bain, M. M, 1992. Eggshell strength: a relationship between the mechanism of failure and the ultrastructural organisation of the mammillary layer. Br. Poult. Sci. 33:303-319. Balnave, D., and I. Yoselewitz, 1987. The relation between sodium chloride concentration in drinking water and egg shell damage. Br. J. Nutr. 58:503-509. Balnave, D., I. Yoselewitz, and R. J. Dixon, 1989. Physiological changes associated with the production of defective egg-shells by hens receiving sodium chloride in the drinking water. Br. J. Nutr. 61:35-43. Balnave, D„ D. Zhang, and R. E. Moreng, 1991. Use of ascorbic acid to prevent the decline in eggshell quality observed with saline drinking water. Poultry Sci. 70:848-852. Corradino, R. A., R. H. Wasserman, M. H. Pubols, and S. I. Chang, 1968. Vitamin D3 induction of a calcium-binding protein in the uterus of the laying hen. Arch. Biochem. Biophys. 125: 378-380. Eastin, W. C, and E. Spaziani, 1978. On the mechanism of calcium secretion in the avian shell gland (uterus). Biol. Reprod. 19:505-518. Moreng, R. E., D. Balnave, and D. Zhang, 1992. Dietary zinc methionine effect on eggshell quality of hens drinking saline water. Poultry Sci. 71:1163-1167. Pearson, T. W., T. J. Pryor, and A. M. Goldner, 1977. Calcium transport across avian uterus. HI. Comparison of laying and nonlaying birds. Am. J. Physiol. 232:E437-E443. Steel, R.G.D., and J. H. Torrie, 1982. Principles and Procedures of Statistics. A Biometrical Approach. 2nd ed. McGraw-Hill, Kogakusha Ltd., Tokyo, Japan. Wedekind, K. J., A. E. Hortin, and D. H. Baker, 1990. Bioavailability of zinc in a zinc methionine chelate. J. Anim. Sci. 68(Suppl. l):394.(Abstr.) Yoselewitz, I., and D. Balnave, 1989. The influence of saline drinking water on the activity of carbonic anhydrase in the shell gland of laying hens. Aust. J. Agric. Res. 40:1111-1115.
Downloaded from http://ps.oxfordjournals.org/ at Serials Acq (Law) on April 13, 2015
ZnS0 4 H 2 0 (Moreng et al., 1992). This may reflect a reduced availability of Zn from the monohydrate salt. The shell breaking strength of eggs from hens receiving saline water was improved by all three Zn supplements although only the Zn-EDTA response was significant (P < .05) and approached the value for the hens receiving the town water. These results suggest that the Zn component of the Znmethionine was the factor responsible for the reduction in eggshell defects observed previously when Zn-methionine was added to the diet of hens on saline drinking water (Moreng et al, 1992). This is not unexpected because the concentrations of Zn-methionine used in the present and previous work (.2 to .5 g/kg) only supplied .008 to .02% methionine to the diet. The significantly lower shell gland CaBP concentration and CA activity observed in hens receiving the saline water and laying eggs with defective shells in the present study can be associated with the significantly lower Ca and bicarbonate ion concentrations observed by Balnave et al. (1989) in the shell gland fluid of similarly treated hens. The concentration of CaBP was significantly (P < .05) increased by the Zn-methionine and ZnSC>4 supplements, and the CA activity was increased (P < .01) by the Zn-EDTA supplement, the increase with Znmethionine just failing to attain statistical significance (P < .06). Improvements of between 30 and 78% in CA activity and between 10 and 17% in CaBP were obtained with the Zn supplements. It is likely that the supply of Ca and carbonate ions to the lumen of the shell gland are interdependent. In this regard, Pearson et al. (1977) suggested that Ca transport across the avian shell gland is related to the activity of CA in this tissue, and Eastin and Spaziani (1978) have reported a dependency of Ca secretion on lumen bicarbonate concentration and bicarbonate production.