A feeding trial of 5-week duration was performed to assess the response of broiler chicks to diet... more A feeding trial of 5-week duration was performed to assess the response of broiler chicks to dietary supplementation with different doses of myco-fabricated zinc oxide nanoparticles (ZONPs) on blood indices, physiological, immunological response, antioxidant status, intestinal microbial count, and histological changes in immune organs. A total of 162 3-day-old Ross 308 broiler chicks were weighed individually and distributed equally into 3 dietary treatments with 6 replicate of 9 chicks in each in a completely randomized design. Chicks were fed ad libitum a basal ration prepared as starter, grower, and finisher supplemented with 0 (T1, control), 40 (T2), and 60 (T3) mg zinc oxide nanoparticles (ZONPs)/kg feed. Results showed that supplementing with ZONPs at both studied levels increased the relative weights of the spleen, bursa, thymus, and liver and decreased the relative weight of the kidney, gizzard, and intestine. A significant increase in the concentrations of hemoglobin (Hb), ...
The chorioallantoic membrane (CAM) of birds is an epithelial tissue that actively transports larg... more The chorioallantoic membrane (CAM) of birds is an epithelial tissue that actively transports large amounts of Ca during embryonic development. In this study the effect of vitamins D and K on Ca uptake by the CAM was studied. Four dietary treatments were used to produce eggs that are the following: deficient in vitamins D and K (-D/-K), sufficient in both (+D/+K), or deficient in one and sufficient in the other (-D/+K or +D/-K). Vitamin D-deficient (-D) Japanese quail embryos (from hens fed 1,25-dihydroxyvitamin D3) do not hatch because of severe Ca deficiency resulting from their inability to obtain Ca from shell, whereas vitamin K deficiency results in only 14% reduction in hatchability. The results demonstrate that Ca uptake by CAM is vitamin D dependent and only slightly vitamin K dependent. Ca-binding activity of CAM extracts was unchanged by vitamin K deficiency, and only a small increase was provided by vitamin D treatment. Vitamin D stimulated both Ca entry and exist from the...
Effects of vitamin D deficiency (-D) on mineral homeostasis were investigated in Japanese quail e... more Effects of vitamin D deficiency (-D) on mineral homeostasis were investigated in Japanese quail embryos. The -D embryos from 1,25(OH)2D3-fed hens became progressively calcium deficient, as documented by hypocalcemia and reduced calcium accumulation by the skeleton, yolk sac, and allantoic fluid. Plasma phosphate was progressively elevated between days 11 and 15. Increased calcium accumulation by the skeleton, yolk sac, and allantoic fluid occurred between days 12 and 15 in +D embryos. Phosphate and adenosine 3',5'-cyclic monophosphate (cAMP) concentrations of allantoic fluid increased in +D embryos during the period of shell calcium mobilization. Further increases in phosphate and cAMP excretion into allantoic fluid occurred in -D embryos, although no calcium was absorbed from the shell. Renal 25(OH)D-1-hydroxylase activity increased between days 11 and 13, whereas the adenylate cyclase response to parathyroid hormone was lost in -D embryos by day 14. These changes in renal function are indicative of secondary hyperparathyroidism in the -D embryos. Differentiation of villus cavity and capillary covering cells occurred in the chorionic epithelium of -D embryos, but eggshell calcium was apparently not absorbed. In contrast, 75% of the total body calcium of newly hatched (+D) chicks was obtained from the eggshell. Thus the dissolution and/or transport of eggshell calcium is dependent on vitamin D in quail embryos.
An experiment was carried out to investigate the effects of age of laying hens (young = 22 wk vs ... more An experiment was carried out to investigate the effects of age of laying hens (young = 22 wk vs old = 120 wk) in maintaining Ca homeostasis during periods of Ca depletion then repletion with Ca. Plasma Ca and P, tibia breaking strength and percentage ash, renal 25-hydroxycholecalciferol-1-hydroxylase (1 alpha-hydroxylase), and parathyroid hormone (PTH)-stimulated adenylate cyclase activities were studied during 28 d of Ca depletion on a .08% Ca diet (LC) and 28 d of Ca repletion on a 3.75% Ca diet (HC). When laying hens on a HC diet were placed on a LC diet, plasma Ca and P, tibia breaking strength and ash percentage, and renal PTH-dependent adenylate cyclase activity were significantly depressed, but renal 1 alpha-hydroxylase activity was significantly stimulated. These changes were greater in the young hens than in the older hens; therefore an interaction between age and dietary Ca was found. These changes were of a lesser magnitude at 28 d of Ca depletion, probably due to the cessation of egg laying and to the desensitization of hormone-mediated function. 1 alpha-Hydroxylase activity was significantly less during the repletion period. The age effect was most pronounced for 1 alpha-hydroxylase, with the younger birds expressing significantly higher activity and ability to respond to hypocalcemia. There was a significant increase in kidney weights in Ca-deficient groups at 14 and 28 d of Ca depletion. It is concluded that younger hens have greater adaptive responses to Ca restriction than do older hens.
Avian kidney function adapts during reproduction to provide the calcium required for eggshell for... more Avian kidney function adapts during reproduction to provide the calcium required for eggshell formation. Adaptive changes in kidney function are 1) increased parathyroid hormone (PTH)-dependent adenylate cyclase activity; 2) elevated numbers of PTH receptors; and 3) increased synthesis of 1,25-dihydroxycholecalciferol. Because exogenous estrogen mimics these changes, this study explored the physiological role of estrogen in the regulation of kidney function by altering egg-laying status or levels of estradiol. In hens, treatment with the coccidiostatic drug, nicarbazin, led to cessation of egg laying with maintenance of the reproductive tract and of plasma estradiol and calcium. The PTH-dependent adenylate cyclase activity remained elevated (upregulated). However, when molting was induced by altering the photoperiod and diet, plasma estradiol, plasma calcium, and renal PTH-dependent adenylate cyclase activity all decreased. The depressed responsiveness to PTH was restored by administration of estradiol either during the molt or upon return to egg laying following the molt. When the estrogen antagonist, tamoxifen, was administered to laying hens, reproduction ceased and the PTH-dependent adenylate cyclase activity of renal membranes was decreased. In all three groups of nonlaying birds, the activity of kidney 25-hydroxycholecalciferol-1-hydroxylase was markedly decreased relative to that of laying hens irrespective of the amount of plasma estradiol. It was concluded that estrogen regulates the PTH-dependent adenylate cyclase system of avian kidney, whereas the activity of the 25-hydroxycholecalciferol-1-hydroxylase of kidney and thus, the synthesis of 1,25-hydroxycholecalciferol may be governed at least in part by the regulation of renal receptors for PTH by estrogen.
Vitamin D-deficient (-D) Japanese quail embryos [from hens fed 1,25-dihydroxycholecalciferol (1,2... more Vitamin D-deficient (-D) Japanese quail embryos [from hens fed 1,25-dihydroxycholecalciferol (1,25-(OH)2D3)] die at Day 15 of incubation from severe calcium deficiency. Single doses of 125 ng cholecalciferol, 600 ng 24,25-dihydroxycholecalciferol [24,25-(OH)2D3], or 100 ng 1,25-(OH)2D3 were found to increase hatchability when injected into eggs prior to incubation. Cholecalciferol could be used from 125 to 1,250 ng per egg with no detrimental effects on hatchability, whereas single doses of 1,25-(OH)2D3 lower or higher than 100 ng per egg reduced hatchability. Injection of 125 ng cholecalciferol per egg supported the hatching of -D embryos when eggs were treated as late as 10 days of incubation. Sharply reduced hatchability occurred when cholecalciferol was injected at Day 11 or 12 of incubation. Experiments designed to evaluate the physiological state of 1-day-old quail treated with a single dose of cholecalciferol metabolites in ovo prior to incubation revealed that chicks had hypocalcemia, reduced total calcium content, and a six- to sevenfold increase in renal 25-hydroxycholecalciferol-1-hydroxylase activity. On the other hand, chicks from eggs treated with cholecalciferol were relatively normal. It appears that cholecalciferol administered in ovo is the compound of choice for supporting sustained development of the skeleton, mobilization of shell calcium, and prevention of hypocalcemia, probably because cholecalciferol is utilized slowly as needed to support development of the chick skeleton.
1. A trial was conducted to examine the effects of dietary vitamin E content, age and sex on haem... more 1. A trial was conducted to examine the effects of dietary vitamin E content, age and sex on haematological indices and liver enzymes of Japanese quails. A total of 800 1-d-old quail chicks were assigned at random into 4 equal groups and fed on starter and layer diets containing 0, 1, 5 or 10 times the NRC recommended supplements of vitamin E. No selenium was added to the basal deficient diets; the other diets were supplied with 0.2 mg selenium/kg diet. 2. The investigation covered the age span of 3 to 12 weeks in female and male birds. Blood samples were collected at 3-week intervals and tested for haematological indices (erythrocyte count; leucocyte count; susceptibility of erythrocytes to haemolysis; haemoglobin concentration (Hb); packed cell volume (PCV); and mean corpuscular volume (MCV)) and liver enzymes (aspartate transaminase, AST, and glutathione peroxidase, GSH-Px). 3. The significant differences between the 4 dietary treatments indicated that as the levels of selenium and/or vitamin E increased, the percentage of erythrocytes haemolysed and AST activity decreased, whereas Hb and GSH-Px concentrations increased. 4. Differences between age groups showed that older quails had higher erythrocyte susceptibility to haemolysis, higher AST levels and but lower erythrocyte count and PCV. 5. Females had lower erythrocyte haemolysis and higher Hb concentrations than males. 6. The interaction between dietary groups and age groups revealed that the differences between age groups were reduced as the level of selenium and/or vitamin E increased, leading to similar group means over the age period of study. 7. In conclusion, NRC recommended supplements of vitamin E (12 and 25 mg/kg diet) were not adequate. Doses equal to, at least, 5 times that recommended were advised to improve GSH-Px (index of antioxidant status) and Hb concentrations.
This study describes the toxicity signs that developed when the diet of male broiler chickens was... more This study describes the toxicity signs that developed when the diet of male broiler chickens was artificially contaminated with different levels of the mycotoxin ochratoxin A (OTA). Chicks were assigned randomly to three groups of 80 chicks that were fed a diet containing 0 parts per billion (ppb) (control, group 1), 400 ppb (group 2) or 800 ppb (group 3) OTA from day 1 to 5 weeks of age. Signs of ochratoxicosis were assessed on the basis of changes in the following criteria: body weight, relative weights of two representative internal organs (gizzard and thymus), feed consumption, feed conversion ratio, mortality, thyroid activity, blood profile, humoral and cell mediated immunity. Feeding OTA at levels of 400 and 800 ppb (groups 2 and 3) significantly decreased the body weight, thymus weight, feed consumption, feed conversion ratio and thyroxine concentration (P < 0.05). The OTA groups developed anaemia manifested by a significant decrease in the red blood cell count, packed cell volume percentage and haemoglobin concentration (P < 0.05). By the end of the experiment both groups that received OTA showed a 37% reduction in red blood cell count compared with the control group. Furthermore, a significant decrease in the white blood cell count, humoral immune response and cell-mediated immunity was found in both groups fed ochratoxin compared with the control group (P < 0.05). The reduction in the above parameters was more noticeable with time and was proportional to the level of OTA exposure. A significant increase in relative gizzard weight, cumulative mortality and triiodothyronine concentration was found in OTA-fed chicks (P < 0.05). These data provide a description of ochratoxicosis in broilers that should be useful in diagnosis and in improved understanding of the practical implications on broiler performance and health, a problem that can threaten the poultry industry.
1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is essential for the transport of eggshell calcium to the... more 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is essential for the transport of eggshell calcium to the embryo across the chorioallantoic membrane (CAM). CAM contains the vitamin D receptor that increases following 1,25-(OH)2D3 injection into embryos at day 10 of incubation. Further, a single injection of 100 ng of 1,25-(OH)2D3 into vitamin D-deficient quail eggs at day 10 of incubation resulted in a significant increase in both body and yolk calcium. This is accompanied by an increase in carbonic anhydrase from low levels in deficiency to normal levels. Acetazolamide (AZ), a specific carbonic anhydrase inhibitor injected into the quail embryos, caused hypocalcemia and hyperphosphatemia. This is similar to the hypocalcemia and hyperphosphatemia found in vitamin D-deficient embryos. These results suggest that one mechanism of action of vitamin D in the mobilization of eggshell calcium is the activation of carbonic anhydrase that acidifies the calcium carbonate shell.
A feeding trial of 5-week duration was performed to assess the response of broiler chicks to diet... more A feeding trial of 5-week duration was performed to assess the response of broiler chicks to dietary supplementation with different doses of myco-fabricated zinc oxide nanoparticles (ZONPs) on blood indices, physiological, immunological response, antioxidant status, intestinal microbial count, and histological changes in immune organs. A total of 162 3-day-old Ross 308 broiler chicks were weighed individually and distributed equally into 3 dietary treatments with 6 replicate of 9 chicks in each in a completely randomized design. Chicks were fed ad libitum a basal ration prepared as starter, grower, and finisher supplemented with 0 (T1, control), 40 (T2), and 60 (T3) mg zinc oxide nanoparticles (ZONPs)/kg feed. Results showed that supplementing with ZONPs at both studied levels increased the relative weights of the spleen, bursa, thymus, and liver and decreased the relative weight of the kidney, gizzard, and intestine. A significant increase in the concentrations of hemoglobin (Hb), ...
The chorioallantoic membrane (CAM) of birds is an epithelial tissue that actively transports larg... more The chorioallantoic membrane (CAM) of birds is an epithelial tissue that actively transports large amounts of Ca during embryonic development. In this study the effect of vitamins D and K on Ca uptake by the CAM was studied. Four dietary treatments were used to produce eggs that are the following: deficient in vitamins D and K (-D/-K), sufficient in both (+D/+K), or deficient in one and sufficient in the other (-D/+K or +D/-K). Vitamin D-deficient (-D) Japanese quail embryos (from hens fed 1,25-dihydroxyvitamin D3) do not hatch because of severe Ca deficiency resulting from their inability to obtain Ca from shell, whereas vitamin K deficiency results in only 14% reduction in hatchability. The results demonstrate that Ca uptake by CAM is vitamin D dependent and only slightly vitamin K dependent. Ca-binding activity of CAM extracts was unchanged by vitamin K deficiency, and only a small increase was provided by vitamin D treatment. Vitamin D stimulated both Ca entry and exist from the...
Effects of vitamin D deficiency (-D) on mineral homeostasis were investigated in Japanese quail e... more Effects of vitamin D deficiency (-D) on mineral homeostasis were investigated in Japanese quail embryos. The -D embryos from 1,25(OH)2D3-fed hens became progressively calcium deficient, as documented by hypocalcemia and reduced calcium accumulation by the skeleton, yolk sac, and allantoic fluid. Plasma phosphate was progressively elevated between days 11 and 15. Increased calcium accumulation by the skeleton, yolk sac, and allantoic fluid occurred between days 12 and 15 in +D embryos. Phosphate and adenosine 3',5'-cyclic monophosphate (cAMP) concentrations of allantoic fluid increased in +D embryos during the period of shell calcium mobilization. Further increases in phosphate and cAMP excretion into allantoic fluid occurred in -D embryos, although no calcium was absorbed from the shell. Renal 25(OH)D-1-hydroxylase activity increased between days 11 and 13, whereas the adenylate cyclase response to parathyroid hormone was lost in -D embryos by day 14. These changes in renal function are indicative of secondary hyperparathyroidism in the -D embryos. Differentiation of villus cavity and capillary covering cells occurred in the chorionic epithelium of -D embryos, but eggshell calcium was apparently not absorbed. In contrast, 75% of the total body calcium of newly hatched (+D) chicks was obtained from the eggshell. Thus the dissolution and/or transport of eggshell calcium is dependent on vitamin D in quail embryos.
An experiment was carried out to investigate the effects of age of laying hens (young = 22 wk vs ... more An experiment was carried out to investigate the effects of age of laying hens (young = 22 wk vs old = 120 wk) in maintaining Ca homeostasis during periods of Ca depletion then repletion with Ca. Plasma Ca and P, tibia breaking strength and percentage ash, renal 25-hydroxycholecalciferol-1-hydroxylase (1 alpha-hydroxylase), and parathyroid hormone (PTH)-stimulated adenylate cyclase activities were studied during 28 d of Ca depletion on a .08% Ca diet (LC) and 28 d of Ca repletion on a 3.75% Ca diet (HC). When laying hens on a HC diet were placed on a LC diet, plasma Ca and P, tibia breaking strength and ash percentage, and renal PTH-dependent adenylate cyclase activity were significantly depressed, but renal 1 alpha-hydroxylase activity was significantly stimulated. These changes were greater in the young hens than in the older hens; therefore an interaction between age and dietary Ca was found. These changes were of a lesser magnitude at 28 d of Ca depletion, probably due to the cessation of egg laying and to the desensitization of hormone-mediated function. 1 alpha-Hydroxylase activity was significantly less during the repletion period. The age effect was most pronounced for 1 alpha-hydroxylase, with the younger birds expressing significantly higher activity and ability to respond to hypocalcemia. There was a significant increase in kidney weights in Ca-deficient groups at 14 and 28 d of Ca depletion. It is concluded that younger hens have greater adaptive responses to Ca restriction than do older hens.
Avian kidney function adapts during reproduction to provide the calcium required for eggshell for... more Avian kidney function adapts during reproduction to provide the calcium required for eggshell formation. Adaptive changes in kidney function are 1) increased parathyroid hormone (PTH)-dependent adenylate cyclase activity; 2) elevated numbers of PTH receptors; and 3) increased synthesis of 1,25-dihydroxycholecalciferol. Because exogenous estrogen mimics these changes, this study explored the physiological role of estrogen in the regulation of kidney function by altering egg-laying status or levels of estradiol. In hens, treatment with the coccidiostatic drug, nicarbazin, led to cessation of egg laying with maintenance of the reproductive tract and of plasma estradiol and calcium. The PTH-dependent adenylate cyclase activity remained elevated (upregulated). However, when molting was induced by altering the photoperiod and diet, plasma estradiol, plasma calcium, and renal PTH-dependent adenylate cyclase activity all decreased. The depressed responsiveness to PTH was restored by administration of estradiol either during the molt or upon return to egg laying following the molt. When the estrogen antagonist, tamoxifen, was administered to laying hens, reproduction ceased and the PTH-dependent adenylate cyclase activity of renal membranes was decreased. In all three groups of nonlaying birds, the activity of kidney 25-hydroxycholecalciferol-1-hydroxylase was markedly decreased relative to that of laying hens irrespective of the amount of plasma estradiol. It was concluded that estrogen regulates the PTH-dependent adenylate cyclase system of avian kidney, whereas the activity of the 25-hydroxycholecalciferol-1-hydroxylase of kidney and thus, the synthesis of 1,25-hydroxycholecalciferol may be governed at least in part by the regulation of renal receptors for PTH by estrogen.
Vitamin D-deficient (-D) Japanese quail embryos [from hens fed 1,25-dihydroxycholecalciferol (1,2... more Vitamin D-deficient (-D) Japanese quail embryos [from hens fed 1,25-dihydroxycholecalciferol (1,25-(OH)2D3)] die at Day 15 of incubation from severe calcium deficiency. Single doses of 125 ng cholecalciferol, 600 ng 24,25-dihydroxycholecalciferol [24,25-(OH)2D3], or 100 ng 1,25-(OH)2D3 were found to increase hatchability when injected into eggs prior to incubation. Cholecalciferol could be used from 125 to 1,250 ng per egg with no detrimental effects on hatchability, whereas single doses of 1,25-(OH)2D3 lower or higher than 100 ng per egg reduced hatchability. Injection of 125 ng cholecalciferol per egg supported the hatching of -D embryos when eggs were treated as late as 10 days of incubation. Sharply reduced hatchability occurred when cholecalciferol was injected at Day 11 or 12 of incubation. Experiments designed to evaluate the physiological state of 1-day-old quail treated with a single dose of cholecalciferol metabolites in ovo prior to incubation revealed that chicks had hypocalcemia, reduced total calcium content, and a six- to sevenfold increase in renal 25-hydroxycholecalciferol-1-hydroxylase activity. On the other hand, chicks from eggs treated with cholecalciferol were relatively normal. It appears that cholecalciferol administered in ovo is the compound of choice for supporting sustained development of the skeleton, mobilization of shell calcium, and prevention of hypocalcemia, probably because cholecalciferol is utilized slowly as needed to support development of the chick skeleton.
1. A trial was conducted to examine the effects of dietary vitamin E content, age and sex on haem... more 1. A trial was conducted to examine the effects of dietary vitamin E content, age and sex on haematological indices and liver enzymes of Japanese quails. A total of 800 1-d-old quail chicks were assigned at random into 4 equal groups and fed on starter and layer diets containing 0, 1, 5 or 10 times the NRC recommended supplements of vitamin E. No selenium was added to the basal deficient diets; the other diets were supplied with 0.2 mg selenium/kg diet. 2. The investigation covered the age span of 3 to 12 weeks in female and male birds. Blood samples were collected at 3-week intervals and tested for haematological indices (erythrocyte count; leucocyte count; susceptibility of erythrocytes to haemolysis; haemoglobin concentration (Hb); packed cell volume (PCV); and mean corpuscular volume (MCV)) and liver enzymes (aspartate transaminase, AST, and glutathione peroxidase, GSH-Px). 3. The significant differences between the 4 dietary treatments indicated that as the levels of selenium and/or vitamin E increased, the percentage of erythrocytes haemolysed and AST activity decreased, whereas Hb and GSH-Px concentrations increased. 4. Differences between age groups showed that older quails had higher erythrocyte susceptibility to haemolysis, higher AST levels and but lower erythrocyte count and PCV. 5. Females had lower erythrocyte haemolysis and higher Hb concentrations than males. 6. The interaction between dietary groups and age groups revealed that the differences between age groups were reduced as the level of selenium and/or vitamin E increased, leading to similar group means over the age period of study. 7. In conclusion, NRC recommended supplements of vitamin E (12 and 25 mg/kg diet) were not adequate. Doses equal to, at least, 5 times that recommended were advised to improve GSH-Px (index of antioxidant status) and Hb concentrations.
This study describes the toxicity signs that developed when the diet of male broiler chickens was... more This study describes the toxicity signs that developed when the diet of male broiler chickens was artificially contaminated with different levels of the mycotoxin ochratoxin A (OTA). Chicks were assigned randomly to three groups of 80 chicks that were fed a diet containing 0 parts per billion (ppb) (control, group 1), 400 ppb (group 2) or 800 ppb (group 3) OTA from day 1 to 5 weeks of age. Signs of ochratoxicosis were assessed on the basis of changes in the following criteria: body weight, relative weights of two representative internal organs (gizzard and thymus), feed consumption, feed conversion ratio, mortality, thyroid activity, blood profile, humoral and cell mediated immunity. Feeding OTA at levels of 400 and 800 ppb (groups 2 and 3) significantly decreased the body weight, thymus weight, feed consumption, feed conversion ratio and thyroxine concentration (P < 0.05). The OTA groups developed anaemia manifested by a significant decrease in the red blood cell count, packed cell volume percentage and haemoglobin concentration (P < 0.05). By the end of the experiment both groups that received OTA showed a 37% reduction in red blood cell count compared with the control group. Furthermore, a significant decrease in the white blood cell count, humoral immune response and cell-mediated immunity was found in both groups fed ochratoxin compared with the control group (P < 0.05). The reduction in the above parameters was more noticeable with time and was proportional to the level of OTA exposure. A significant increase in relative gizzard weight, cumulative mortality and triiodothyronine concentration was found in OTA-fed chicks (P < 0.05). These data provide a description of ochratoxicosis in broilers that should be useful in diagnosis and in improved understanding of the practical implications on broiler performance and health, a problem that can threaten the poultry industry.
1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is essential for the transport of eggshell calcium to the... more 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is essential for the transport of eggshell calcium to the embryo across the chorioallantoic membrane (CAM). CAM contains the vitamin D receptor that increases following 1,25-(OH)2D3 injection into embryos at day 10 of incubation. Further, a single injection of 100 ng of 1,25-(OH)2D3 into vitamin D-deficient quail eggs at day 10 of incubation resulted in a significant increase in both body and yolk calcium. This is accompanied by an increase in carbonic anhydrase from low levels in deficiency to normal levels. Acetazolamide (AZ), a specific carbonic anhydrase inhibitor injected into the quail embryos, caused hypocalcemia and hyperphosphatemia. This is similar to the hypocalcemia and hyperphosphatemia found in vitamin D-deficient embryos. These results suggest that one mechanism of action of vitamin D in the mobilization of eggshell calcium is the activation of carbonic anhydrase that acidifies the calcium carbonate shell.
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