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Foetal development in rats FED AIN-76A diets supplemented with excess calcium
Fd Chem. Toxic. Vol. 31, No. 12, pp. 953-961, 1993 Printed in Great Britain
0278-6915/93 $6.00+ 0.00 Pergamon Press Ltd
FOETAL DEVELOPMENT IN RATS FED AIN-76A DIETS SUPPLEMENTED WITH EXCESS CALCIUM M. E. SHACKELFORD,T. F. X. COLLINS,J, J. WELSH, T. N. BLACK, M. J. AMES, R. K. CHI and M. W. O'DONNELL Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington, DC 20204, USA (Accepted 12 July 1993) Abstract--This study was designed to evaluate the developmental effects of moderate dietary calcium increases in rats fed nutritionally adequate diets. Female Charles River CD/VAF Plus rats were given 0.50 (control), 0.75, 1.00 or 1.25% dietary calcium as calcium carbonate in AIN-76A diets for 6 wk before mating, during mating and for 20 days of gestation. On gestation day 20, the animals were killed and caesarean sections were performed. Both the non-pregnant and pregnant rats in the 0.75, 1.00 and 1.25% groups ate slightly more than did the control group during most of the intervals measured, but not all the increases were statistically significant. There was no consistent pattern of increase or decrease in weight gain. No dose-related changes were found in maternal clinical findings, the average number of implantations, resorptions and viable foetuses, or foetal length or weight. Under the conditions of the study, there were no statistically significant increases as compared with the control group in the litter incidence regarding specific external, visceral or skeletal variations of the foetuses. Dietary calcium was neither foetotoxic nor teratogenic at the concentrations used.
INTRODUCTION The 1984 NIH Consensus Development Conference Panel on Osteoporosis (1986) recommended that premenopausal women and women who are pregnant or nursing increase their daily calcium consumption through the use of calcium-rich foods and calcium supplements. The recommendations represented an approximate 1.2-fold increase over the 1980 recommended dietary allowances (RDA). Calcium supplement sales increased from $46 million in 1983 to $200 million in 1987 (Consumer Reports, 1988). Adverse reproductive and haematological effects have been reported from ingestion of increased dietary calcium during pregnancy and lactation (Richards and Greig, 1952; Greig, 1952). Adult female Swiss mice were fed experimental diets for four reproductive cycles. These diets were adjusted with calcium carbonate to 0.54, 0.73 and 1.11% calcium and maintained constant in phosphorus at 0.48%. For all the litters evaluated, there was no significant effect on the total number of animals or litters born. There was a significant decrease in the total weight and number of animals weaned on diets that contained the two-fold increase in dietary calcium (1.11% calcium). Calcium supplementation resulted in hypertrophy of the heart and anaemia in adult and weanling mice. Iron supplementation of the
Abbreviations: ANOVA -- analysis of variance; ANOCOVA = analysis of covariance; LSD = least significant difference; MAS = milk-alkali syndrome; RDA = recommended dietary allowances.
diet diminished the anaemia and the effects on the heart. However, anaemia was already established in the litters from the control groups when they were compared with litters from animals fed several other stock diets. Analysis of the diet formulations revealed possible dietary inadequacies. These diets, which consisted of whole wheat and dried whole milk, were not supplemented with vitamins and minerals (O'Dell et al., 1961). Nutritional inadequacies in addition to excess calcium in the diets may have contributed to the reproductive and haematological results observed. Lai et al. (1984) fed rats a semi-purified diet that contained 0.6 or 1.0% calcium, 20% soya-protein isolate and a constant (0.45%) level of phosphorus. The study reported hypophagia near term and a significant decrease in the total feed intake and total body weight gain of the pregnant female rats fed the 1.67-fold increase in dietary calcium during the 22 days of gestation. However, foetal weight and litter size were not affected. The nutrient interaction between phytate in the protein source, the inadequate amount of zinc (4.8 mg/kg diet) in the Harper's mineral mix and excess calcium in the diet could have reduced zinc bioavailability and affected feed consumption and body weight gain (Davies and Nightingale, 1975; Davies and Olpin, 1979, Davies and Reid, 1979; Harper, 1959). Because of these problems, the effects attributable to dietary calcium could not be assessed. We therefore designed a study to evaluate the developmental effects of excess dietary calcium in rats that were fed nutritionally adequate diets. Female
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M. E. SHACKELFORDet al.
954
Charles River C D / V A F Plus rats were given 0.50, 0.75, 1.00 or 1.25% dietary calcium as calcium carbonate for 6 wk before mating, during mating and for 20 days o f gestation. The effects o f multiple dietary factors were eliminated by feeding the rats A I N - 7 6 A diet with casein, which lacks dietary phytate, as the protein source. MATERIALS
AND
METHODS
Test material. US Pharmacopeia-grade calcium carbonate was obtained from Pfizer (Adams, MA, USA). The chemical was 98.62% pure, and only one lot was used. The test material was incorporated into a modified A I N - 7 6 A diet that was prepared according to our specifications by B I O - S E R V (Frenchtown, N J, USA). Diets. The formulation o f p o w d e r e d AIN-76A diet (Table 1) is r e c o m m e n d e d as nutritionally adequate for rats by the American Institute o f Nutrition (1977 and 1980). During the duration o f the study, the diets were stored in a walk-in refrigerator ( - 1 to - 6 ° C ) to maintain nutrient stability (Fullerton and Greenman, 1982). The mineral mix o f the AIN-76a diet was slightly modified. The copper content was increased from 6 to 9 mg/kg diet (Spoerl and Kirchgessner, 1976) by a separate addition o f cupric carbonate. Calcium p h o s p h a t e and calcium carbonate were added separately to adjust the diets to specified levels o f calcium and p h o s p h o r u s . Calcium p h o s p h a t e was the primary source o f calcium and p h o s p h o r u s . Dietary calcium was adjusted to 0.50, 0.75, 1.00 and 1.25% with calcium carbonate, and dietary phosphorus was maintained at approximately 0.40%. At
the time of preparation, a separate lot was prepared for each o f the specified dose levels o f calcium. The diets were assayed for homogeneity and concentrations o f calcium, p h o s p h o r u s , magnesium, manganese, zinc, iron and copper by the New Jersey Feed Laboratory, Inc. (Trenton, N J, USA), using the m e t h o d s of the Association o f Official Analytical Chemists (Padmore, 1990). The diets at 1.0 and 1.25% calcium were slightly higher in iron than the other diets used in this study because o f contributions from calcium carbonate. Animals. 156 male and 291 female C D / V A F Plus Charles River rats were obtained from Charles River Laboratories, Inc. (Wilmington, MA, USA); when received, the females were 52 days old and the males were 44 days old. At the end of the 6-day quarantine, 276 female animals were assigned to the diets by a stratified r a n d o m m e t h o d according to body weight. F o u r groups received 0.50 (control), 0.75, 1.00 or 1.25% calcium in powdered AIN-76A diet for 6 wk before mating, t h r o u g h o u t mating and for 20 days o f gestation. O f the 291 female animals used in the study, 135 were randomly selected for the determination o f tissue mineral levels at three times during the study: 15 animals before assignment o f the remaining 276 to diet groups; 60 non-pregnant animals (15 per group) after 6 w k on the diets; and 60 pregnant animals (15 per group) at the time o f caesarean section on day 20 of gestation. F r o m these 15 pregnant females, two foetuses from each o f 12 litters were also used for whole-body mineral determinations. The mineral data are presented in a separate paper (Shackelford et al., 1994). Female animals were weighed and feed c o n s u m p t i o n was measured every
Table 1. Composition of the purified diets supplementedwith excesscalcium fed to female CD/VAF Plus rats Concentration of calcium in the diet (%) Component* (%) 0.50 0.75 1.00 1.25 Casein 20.0 20.0 20.0 20.0 Choline bitartrate 0.2 0.2 0.2 0.2 DL-Methionine 0.3 0.3 0.3 0.3 Corn starch 21.7 21.7 21.7 21.7 Sucrose 20.0 19.4 18.8 18.I Dextrose 21.7 21.7 21.7 21.7 Cellulose 5.0 5.0 5.0 5.0 Corn oil 5.0 5.0 5.0 5.0 Vitamin mix 1.0 1.0 1.0 1.0 Mineral mix 3.5 3,5 3.5 3.5 Cupric carbonate 0.05 0.05 0.05 0.05 Calcium phosphate 1.49 1.49 1.49 1.49 Calcium carbonate 0.15 0.77 1.40 2.03 Minerals Ca 0.46+0.01 0.71 _+0.02 0.92+_0.01 1.20-+0.02 P 0.45 + 0.01 0.46 +, 0.03 0.46 +-0.02 0.46 +, 0.02 Mg (mg/kg diet) 464 +, 15 458 _+9 503 +_33 497 +, 60 Mn (mg/kg diet) 53 -+ 3 55 +_2 56 +_2 52 + 2 Fe (mg/kg diet) 42 +, 9 42 -+2 53 -+2 51 _-4-2 Zn (mg/kg diet) 32 +, 2 29 -+ 2 34 + 1 34 -+ 1 Cu (mg/kg diet) 8 +, I 8 -+ I 9 +, I 9_+ 1 *Components are in percentagesof the diet unless otherwiseindicated. The vitamin mix of the purified diets was the same as that specified for diet AIN-76A (American Institute of Nutrition, 1977 and 1980). The mineral mix was modified as described in Materials and Methods. The mineral composition of the diets was determined by analysis. Results are means +- SD of duplicate samples from the top, middle and bottom of each lot.
Developmental effects of excess dietary calcium 3 days for 6 wk before mating and during gestation, but not during mating. Feed consumption and body weights were not measured for male rats. Male rats were fed pelleted Purina Rodent Chow 5002 (Purina Mills, Inc., Richmond, IN, USA) except during mating, when they also received one of the four experimental diets. Animals, identified by numbered metal ear tags, were housed in stainless-steel cages, with feed and deionized water supplied ad lib. The study room was environmentally monitored with a hy'grothermograph for temperature (18-27°C) and humidity (25-72%). There was an automatic 12-hr light/dark cycle. Exposure to extraneous sources of zinc was minimized by the use of stainless-steel animal housing, Aqua Cool deionized water (Ionics, Inc., Watertown, MA, USA) and polyethylene waterbottle stoppers. Powder-free, polyvinyl chloride gloves (VWR Scientific Co., Baltimore, MD, USA) were used to minimize contamination of the animals. Procedures. After 6 wk on the control and test diets, the female rats were mated to the males in a 2: I ratio, with cohabitation beginning at approximately 4.30 pm. The following morning, a vaginal smear was made for each female to determine copulation. The day on which sperm was found in the vaginal smear was considered to be day 0 of gestation. The spermpositive females were continued on the respective control or test diets during pregnancy. The dams were killed by carbon dioxide asphyxiation on day 20 of gestation. A laparotomy was then performed on each dam, and the major organs were examined. In each case, the uterus was opened and examined in situ for the presence and position of resorption sites and foetuses (dead or alive) and the number of implantation sites. Deciduomas (brownish implantation sites without placentas) were considered early deaths; implantation sites with placentas and complete but non-viable foetuses that were of subnormal size, showed retarded development or were in a macerated condition were classified as late deaths, according to the terminology of Bateman and Epstein (1971) and the MARTA (Mid-Atlantic Reproduction and Teratology Association) Committee on Terminology (1969). The uteri of animals that did not appear to be pregnant were stained with sodium sulfide to enhance resorption sites. Foetuses were examined individually for gross abnormalities. The uterine position, sex, weight and crown-rump length of each foetus were recorded. Corpora lutea were counted under a lighted magnifying lens. Any foetus that weighed less than 70% of the average weight of the concurrent male or female controls was considered to be a runt (Leuschner and Czok, 1973). Foetuses were assigned to skeletal or visceral analysis on an alternate basis following the sequence of foetal numbers. From 12 litters in each group, two foetuses (one from each uterine horn) were randomly selected and used for whole-body mineral determinations. Approximately half of the foetuses were fixed in alcohol, macerated in potassium hydroxide, stained with Alizarin Red S
955
and examined microscopically for skeletal variations (Dawson, 1926). The remaining half were fixed in Bouin's solution, sectioned by razor blade and microscopically examined for internal variations of the soft tissues (Wilson and Warkany, 1965; Wilson, 1973). Each sperm-positive female was given a blind-test number, which was carried over as a litter number. To preclude the possibility of bias, we evaluated the dams and foetuses during caesarean sections and the foetuses during skeletal or visceral analyses without knowing the dose levels. Statistical analysis. All statistical analyses were performed by the Division of Mathematics, US FDA. The incidence of clinical observations was analysed by comparing treatment groups with the control group by using Fisher's exact test. Data on feed consumption were submitted to an analysis of variance (ANOVA) and a protected least-significant difference (LSD) test (two-tailed). Data on weight gain were submitted to an analysis of covariance (ANOCOVA), after adjusting for the initial body weight, and an LSD test (two-tailed). Trend analysis was done on feed consumption. It was not done on body weight data, because we analysed these data by covariance. A trend is present if the linearity P value is below 0.05 (borderline trend if below 0.10) and, at the same time, the departure P value is above 0.10. The number of corpora lutea, the average number of implantations, the average number of viable foetuses for each litter and the average number of male or female foetuses for each litter were analysed by ANOVA and an LSD test (one-tailed). Data on implantation efficiency, early deaths, late deaths and total resorptions (early and late deaths) were analysed by using the Freeman-Tukey arcsine transformation for binomial proportions (Freeman-Tukey, 1950). We then analysed transformed data for each litter by ANOVA and an LSD test (one-tailed), comparing the control group with experimental groups. With Fisher's exact test, we analysed data on litters with runts and litters totally resorbed, comparing the control group with treated groups. Foetal body weights and crown-rump lengths were analysed by a nested ANOVA (Sokal and Rohlf, 1981) and an LSD test (one-tailed). We analysed data on the specific litter incidence of sternebral, skeletal and visceral variations by using Fisher's exact test to compare the control group with treatment groups. Proportions of litters with foetuses showing external variations were analysed by using Fisher's exact test. The average number of foetuses with variations per litter was analysed by using the Freeman-Tukey arcsine transformation and then an ANOVA and a protected LSD test (one-tailed). Litters that had foetuses with one or more sternebral, skeletal and visceral variations were analysed with Fisher's exact test. We did trend analysis on the litter data for external variations, using the Cox exact one-tailed test for unadjusted positive trend. A trend is significant if the P value is below 0.05. Trend analysis was not done on data that were
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M.E. SHACKELFORDet al.
analysed by using the Freeman-Tukey arcsine transformation for binomial proportions.
Table 2. Mean daily feed consumption (g/animal/day) of rats given excess dietary calcium
Concentration of calcium in the diet (%) Days
RESULTS
Clinical findings included the following: alopecia, exudate around the eyes, exudate around the nose, bent teeth, lesion, lump in the left flank or leg and mammary lump. None of the above findings was dose related. The number of animals with alopecia was significantly increased in animals fed 1.00% calcium (8.7 v. 0% in the control) during the 6wk before mating and in the groups fed 0.75 and 1.00% calcium (10 and 16.3%, respectively, v. 0% in the control) during gestation. One non-pregnant animal was observed to have diarrhoea on day 4 after assignment to the 1.25% calcium diet. One pregnant female fed 1.00% dietary calcium developed bleeding from the vagina on days 15 and 16 of gestation. At caesarean section on day 20, this animal had 17 implants with 13 resorptions and three living pups. The incidence of diarrhoea and bleeding from the vagina was not considered to be related to the dietary calcium. Three females died during the study, but the deaths were not attributable to excess calcium in the diet. One animal in the 0.75% calcium group was killed on day 40 because mechanical trauma to the jaw prevented the animal from eating properly. One animal in the control group died because of mechanical injury during smearing. A second animal in the 0.75% calcium group was found moribund and bleeding from the urethra on day 3 of gestation. At autopsy, the animal was found to have bladder calculi. The final diagnosis was acute renal failure and obstructive uropathy. The condition was considered to be a random occurrence and was not thought to be caused by the 0.75% level of calcium in the diet. Values for mean daily feed consumption per female given excess dietary calcium for 6 wk before mating and during gestation are shown in Table 2. The 0.75, 1.00 and 1.25% groups ate slightly more than the controls during most of the intervals measured, but not all the increases were statistically significant. The trend analysis for feed consumption for non-pregnant rats showed a significant (P < 0.05) positive trend during most intervals measured, but the trend was borderline positive (P =0.0514) during days (~12. There was no linear trend during days 12-18 and 18-20 of gestation, but there was a significant positive trend (P < 0.05) during the other intervals measured. Values for mean body weight gain for animals given excess dietary calcium for 6 wk before mating and gestation are given in Table 3. Initial body weight on day 0 for non-pregnant rats and sperm-positive females was similar for all groups. Although some differences in weight gain between control and treated groups were significant, there was no consistent pattern of increase or decrease in the body weight gain. Weight gain increased during some intervals measured but decreased for other intervals.
0.50
0.75
1.00
1.25
Non-pregnant rats 6 wk before mating 0-6 6-12 12-18 18-24 24-30 30-36 3~42 ff42
(69) 15.2_+0.4 17.4_+0.3 17.0,+0.3 17.9,+0.3 17.6-+0.3 17.8-+0.3 17.8_+0.3 17.3,+0.2
(69) 15.7,+0.3 17.7_+0.3 17,6_+0.3 18.1_+0.3 17.9-+0.3 17.7_+0.3 17.8_+0.3" 17.5_+0,2"
(69) 15.8_+0.3 17.8,+0.3 18.1 +0.3 b 18.8+0.3 ~ 18.8_+0.3 b 18.6_+0.3 d 18.6_+0.3 a 18.1_+0.2 ~
(69) 16.4_+0,4 18.2_+0.3 18.4_+0.3 ¢ 18.9+0.3 b 18.6+0.3 a 18.4-+0.2 18.5_+0.3 d 18.2_+0.2 b
0~ 6-12 12-18 18-20 0-20
Pregnant rats from days 0-20 (45) (47) (44) 19.6_+0.4 20.4,+0.4 20.8,+0.4" 22.8,+0.4 23.3_+0.3 23.9_+0.4 d 23.5,+0.4 24.3-+0.3 24.1 -+0.5 23.7 _+0.5 24.0 _+0.4 23.7 _+ 0.6 22.2_+0.4 22.8_+0.3 23.0_+0.4
(48) 21.0+0.3 b 24.2+0.4 a 24.2-+0.3 23.0 _+ 0.5 23.1 _+0.3
*Because one animal was autopsied in extremis on day 40 in the 0.75% group, the number of rats was reduced to 68 for the data for days 36-42 and 0-42. Numbers of animals are indicated in parentheses. Values are means _+ SEM; superscripts indicate differences between control and test values (aP~<0.05; bp~<0.01; cP~<0.00h a0.05 < P < 0.10).
Maternal reproductive parameters are listed in Table 4. There were no adverse reproductive effects associated with ingestion of excess dietary calcium. Significant increases were observed in implantation efficiency at 1.25% dietary calcium and in the average number of viable female foetuses at 0.75 and 1.25% dietary calcium. These increases were not dose related and were not considered to be caused by dietary calcium. Table 3. Mean body weight gains and initial body weights of rats given excess dietary calcium Concentration of calcium in the diet (%) Days
0.50
0,75
(69)
(69)
Body weight gain (g) of non-pregnant (~6 6-12 12-18 18 24 24-30 30-36 36-42 0~.2 0
1.00
1.25
(69) (69) rats 6 wk before mating 9.1_+1.1 7.7+1.4 21.7_+1.1 20.1+1.1 16.1 _+ 1.0~ 16.3 _+0.T 15.7_+0.9 14.1 +0.7" 11.9 _+0.9 a 10.6 + 0.7 10.1 + 1.0 9.6_+0.8 4.9 _+ 0.8 6.4 _+ 0.6 89.5_+3.0 84.7+2.6
8.1+1.4 9.9-+1.2 22,1 -+ 1.1 21.1_+0.9 12.1 -+0.8 14.7+0.9 a 16.3_+0.8 14.0+0.7 a 9.0 _+ 0.6 9.8 _+0.8 11.0_+0.9 9.2_+0.8 5.3 + 0.7 5.8 _+ 0.9* 83.9_+2.9 85.1-+3.1" Initial body weight (g) of non-pregnant rats 217.7-+1.0 217.6_+1.0 217.4_+1.0 217.7_+1.0 (45)
(47)
(44)
(48)
Body weight gain (g) of pregnant rats 0~ 6-12 12-18 18-20 0-20 0
29.8 _4- 1.2 29.7 + 1.3 29.8 + 1.1 29.4 + t.0 30.4+ 1.0 32.0_+0.8 30.2_+0.7 31.9_+0.9 45.2 _+ 1.8 51.1 ± 1.4' 48.0 -+ 2.6 48.9 -+ 1.5 25.8_+ 1.1 27.9_+0.9 26.2_+1.1 25.1 _+ 1.0 131.2_+3.1 140.7+2.6 ~ 134.2_+3.9 135.3_+3.0 Initial body weight (g) of pregnant rats 297.6 _+ 3.6 300.8 + 3.7 306.5 _+4.8 300.0 -+ 3.8
*Because one animal was autopsied in extremis on day 40 in the 0.75% group, the number of rats was reduced to 68 for days 36-42 and 0-42. Numbers of animals are indicated in parentheses. Values are means _+ SEM; superscripts indicate differences between control and test values (ap ~<0.05; ~P ~<0.0h eP ~<0.00k d0.05 < P < 0.10).
Developmental effects of excess dietary calcium
957
Table 4. Maternal and reproductive outcome at autopsy and foetal data from rats given excess dietary calcium Concentration of calcium in the diet (%) Parameter No. of pregnant females No. of corpora lutea/female* Implantation e f f i c i e n c y (%/female)*? No. of implants/female* No. of viable foetuses/female* Viable male foetuses/litter* Viable female foetuses/litter* Resorptions/female (mean %)*{ No. of early deaths/litter? No. of late deaths/littert No. of early and late deaths/litter?~ No. of litters totally resorbed Body weight (g) Males Females C r o w n - r u m p length (era) Males Females No. of runts~ Males Females *Means
0.50
0.75
1.00
1.25
45 16.27 + 0.41
47 16.83 + 0.40
44 16.52 _-4-0.27
48 15.98 + 0.32
82.01 + 3.73 13.58 _+ 0.68 11.82+0.66 6.29 _+ 0.43 5.53 _+ 0.39 12.50_+ 1.98 1.71 0.04 1.76 0
90.29 + 1.89 15.26 _ 0.43 13.53+0.46 6.49 _+ 0.33 7.04 _+ 0.38 b 11.15_+ 1.73 1.70 0.02 1.72 0
86.51 + 3.46 14.25 + 0.60 12.82_+0.65 6.95 _+ 0.49 5.86 5:0.37 12.92_+3.19 1.41 0.02 1.43 1
93.41 + 2.28 b 15.15 ___0.50 13.545:0.58 6.83 _+ 0.37 6.71 _+ 0.43" 13.81 _+3.09 1.56 0.04 1.60 2
3.84 3.61
3.90 3.69
3.83 3.66
3.79 3.58
4.0 3.9
4.0 3.9
4.0 3.9
4.0 3.9
5 (4) 4 (4)
5 (2) 4 (1)
3 (3) 1 (1)
3 (3) 3 (2)
+ SEM.
)'Statistical analysis was performed on these data after application of the Freeman-Tukey arcsine transformation. :~tThe number of resorptions includes early deaths and late deaths per Litter. §The number of litters is indicated in parentheses. Values marked with superscripts indicate significant differences between control and test values (ap ~<0.05; bp ~<0.01; cp ~<0.001; d0.05 < P < 0.10).
There were no adverse effects to the foetuses caused by feeding excess dietary calcium to the dams (Table 4). Foetal body weights and c r o w n - r u m p lengths of male and female animals given excess dietary calcium were similar to those of the control groups. Feeding excess dietary calcium had no effect on the number of litters with male or female runts. The incidences of specific external variations are shown in Table 5. In the control group, one foetus did not possess a m o u t h (astomia) and a lower jaw (agnathia). At the 1.25% dietary calcium level, one
Table 5. Incidence of specific external variations in foetuses from rats given excess dietary calcium Concentration of calcium in the diet (%) Parameter
0.50
0.75
1.00
1.25
No. foetuses (litters examined 532 (45) 636 (47) 564 (43) 650 (46) No. of foetuses (litters) with variations 2 (2) 2 (2) l (l) 8 (6) Haemorrhages, external I (1) -l (I) 2 (2) Cleft palate l (1) -2 (2) Agnathia I (1) --l(1) Astomia 1 (1) Thread-like tail 1(1) -1(1) Kinked tail --2 (2) Oedema --3 (2) Malformed head --(1) Beaked snout --(1) Protruding tongue --(1) Open eye --(l) Diaphragmatic cleft* --(1) Omphalocele --(1) _ _ (]) Club foot Scoliosis --(1) Imperforate anus --2 (2) The number of litters is indicated in parentheses. *This parameter includes exposed heart and lungs.
foetus had multiple anomalies: agnathia, cleft palate, protruding tongue, beak-like snout, malformed head, open eye on the fight side, oedema of the trunk, omphalocele, exposed heart and lungs (diaphragmatic cleft), club foot on the left front paw, scoliosis, imperforate anus and kinked tail. The occurrence of this foetus with multiple anomalies was not thought to be related to feeding the rats excess dietary calcium before and during pregnancy. The data in Table 5 were analysed by trend analysis and Fisher's exact test. The Cox exact trend test showed no significant positive trends in the litter incidence of individual or combined variations. When analysed by Fisher's exact test, there were no significant differences between treated and control groups in the litter incidence of these same parameters. Under the conditions of the study, there were no significant increases in the litter incidence of specific or combined external variations. The numbers of foetuses with sternebral ossification deficiencies and the litter incidences are summarized in Table 6. A comparison of the proportion of litters with foetuses having sternebral variations showed no significant differences among the groups given excess calcium and the control group. The increase in the number of litters with foetuses with bipartite sternebrae in the 1.25% group was not significant (P = 0.0854). The incidences of sternebral variations are presented in Table 7. The variations counted are based on the classification of the types (e.g. missing or bipartite) and not on the individual sternebrae affected. The average number of foetuses per litter with one or more sternebral variations increased in
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M.E. SHACKELFORDet al.
Table 6. Incidence o f specific sternebral variations in foetuses o f rats given excess dietary calcium
Table 8. Incidence o f specific skeletal variations in foetuses o f rats given excess dietary calcium
Concentration o f calcium in the diet ( % ) Parameter
0.50
No. o f foetuses (litters) examined 251 (44) No. of foetuses with specific sternebral variations Incomplete ossification 74 (31) Bipartite 4 (4) Unossified 50 (27) Malaligned 8 (7) Fused 1 (1)
0.75
1.00
305 (47)
Concentration of calcium in the diet ( % ) 1.25
273 (43) 313 (46)
84 (35) 3 (3) 49 (27) 2 (2) 0(0)
88 (36) 4 (3) 40 (18) 10 (9) 0(0)
The n u m b e r o f litters is indicated in parenthesis. Supercript indicates difference between control (a0.05 < P < 0.10).
95 (35) 10 (10) d 54 (31) I 1 (11) 1 (1)
and test values
Parameter
0.50
No. o f foetuses (litters) examined 251 (44) Ribs, fused -Ribs, wavy -14th rib (C7) 1 (1) 14th rib (L1) 22(12) 13th rib, short 4(3) Centra, missing 6 (5) Centra, fused . . Centra, bipartite 1 (1) Dorsal arches, extra centre of ossification -Last caudal ossification* -T y m p a n i c arch, red. oss. 1 (1) Interparietal, red. oss. 3 (2) Parietal, red. oss. 1 (1) Frontal, red. oss. 1 (I) Supra occipital, red. oss. I (I) Supra occipital, bipartite 1 ( 1) Hyoid, red. oss. I (1) Nasal, red. oss. 41 (22) Mandible, red. oss. 1 (I) Pubis, red. oss. 2 (2) Metacarpals, red. oss. 1 (I) Metatarsals, red. oss. 5 (4) Maxilla, red. oss. --
0.75
1.00
1.25
305 (47) 273 (43) 313 (46) I (I) --I (I) . . . . ---17(9) 12(12) 13(12) 5(3) 6(5) 4(2) 7 (7) I I (9) 9 (8) . . . . . . . . . 1 (1) 4 ( 1)
----.
2 (1) I (I) --
the 0.75, 1.00 and 1.25% groups, but the differences were not significant. There was no significant increase in the average number of foetuses per litter with at least two or three sternebral variations in the groups fed excess dietary calcium. Statistical analysis of the proportion of litters that had foetuses with variations (at least one, two or three) showed no significant increase between the excess calcium and the control groups. The incidences of specific skeletal variations, excluding sternebrae, were not dose related, and there were no significant differences among any of the values (Table 8). The average number of sacral and caudal vertebrae was not affected by dietary calcium (data not shown). The average number of foetuses per litter with at least one skeletal variation was lower in the 0.75, 1.00 and 1.25% dietary calcium groups than in the control (Table 9). However, the A N O V A of the F r e e m a n Tukey arcsine-transformed data showed no differences in the average number of foetuses with variations (P > 0.10). The proportion of litters that had foetuses with variations (at least one, two or three)
showed no dose-related or significant differences between any of the values obtained in this study. There was no dose-related effect on the incidence of any type of soft-tissue variation (Table 10). The proportion of litters with hydrocephalus was greater in the control group than in the treated groups, but the differences were not significant. The incidence of visceral problems by foetus and by litter is shown in Table 11. The average number of foetuses with at least one, two or three soft-tissue variations was similar in all groups. Although the
Table 7. Incidence o f sternebral variations in foetuses of rats given excess dietary calcium
Table 9. Incidence o f skeletal variations in foetuses of rats given excess dietary calcium
Parameter
0.50
0.75
1.00
1.25
142 3.30
171 3.72
118 2.51 38.69
112 2.60 41.03
138 3.00 44.09
42 89.36
38 88.37
39 84.78
273 43
4 (2) 6 (3)
I ( I) -4 (4) I (1)
Concentration of calcium in the diet ( % )
138 2.94
305 47
36(20) -1 ( 1) -2 (2) --
I ( 1) -41 (17)
red. oss. = reduced ossification *Absence of caudal ossification. The number o f litters is indicated in parentheses.
Concentration o f calcium in the diet ( % )
Sternebral variations* Total no. 137 M e a n no./litter 3.11 Foetuses with one or more sternebral variations Total no. 105 M e a n no./littert 2.39 Foetuses affected ( % ) 41.83 Litters with foetuses with one or more variations Total no. 37 Litters affected ( % ) 84.09 No. o f foetuses examined 251 No. o f litters examined 44
1 (1) -41 (21)
313 46
*Statistical analysis was not performed on this parameter. tStatistical analysis was performed on these data after application of the F r e e m a n - T a k e y arcsine transformation.
Parameter
0.50
Skeletal variations* Total no. 93 Mean no./litter 2.11 Foetuses with one or more skeletal variationst Total no. 69 Mean no#litter 1.57 Foetuses affected ( % ) 27.49 Litters with foetuses with one or more skeletal variations Total no. 31 Litters affected ( % ) 70.45 No. o f foetuses examined 251 No. o f litters examined 44
0.75
1.00
1.25
88 1.87
68 1.58
77 1.67
72 1.53 23.61
60 1.40 21.98
67 1.46 21.41
32 68.09
32 74.42
30 65.22
305 47
273 43
313 46
*Statistical analysis was not performed on this parameter. "['Statistical analysis was performed on these data after application o f the F r e e m a n - T u k e y arcsine transformation.
Developmental effects of excess dietary calcium Table I0. Incidence of specific soft-tissue variations in foetuses of rats given excess dietary calcium Concentration o f calcium in the diet (%)
Parameter
0,50
0.75
1.00
1.25
No. of foetuses (litters)
examined
257 (43) 307 (47) 267 (42) 313 (46) 5 (5) 5 (2) -3 (2) Haemorrhages, internal 2 (2) 1 (1) -2 (2) Kidney, ectopic ---1 (1) Hydro-ureter, moderate 41 (25) 51 (25) 38 (20) 37 (20) Hydro-ureter, severe 6 (5) 9 (8) 11 (8) 10 (6) Enlarged renal pelvis, moderate -2 (2) 1 (1) 3 (1) Enlarged renal pelvis, severe --1 (1) -Enlarged uretal kidney, moderate 15 (12) 27 (17) 13 (11) 18 (9) Enlarged uretal kidney, severe 2 (2) 1 (1) 5 (4) 3 (2) Thyroid, brown -1 (1) --Uterus, swollen ---1 (I) Hydrocephalus
The n u m b e r of litters is indicated in parentheses.
average number of foetuses with at least one variation was lower in the 1.00 and 1.25% groups, the ANOVA did not indicate a significant decrease compared with the controls. Although there was a dose-dependent decrease in the proportion of litters with foetuses with at least one variation, Fisher's exact test (two-tailed) did not show any significant difference. Thus, the proportion of litters with foetuses with at least one, two or three soft-tissue variations was similar in all groups. DISCUSSION
The 1984 NIH Consensus Development Conference Panel on Osteoporosis (1986) recommended increases in the daily calcium consumption that represented an approximate 1.2-fold increase above the 1980 RDA. However, there is inadequate information on the reproductive and developmental effects of moderate increases in dietary calcium. This study was designed to evaluate the effects of 1.5-, 2.0- and Table 11. Incidence of soft-tissue variations in foetuses of rats given excess dietary calcium Concentration of calcium in the diet (%) Parameter
0.50
Soft-tissue variations* Total no. 71 Mean no./litter 1.65 Foetuses with one or more soft-tissue variationst Total no. 57 Mean no./litter 1.33 Foetuses affected (%) 22.18 Litters with foetuses with one or more variations Total no. 29 Litters affected (%) 67.44 No. of foetuses examined 257 No. of litters examined 43
0.75
1.00
1.25
97 2.06
69 1.64
78 1.70
68 1.45 22.15
49 1.17 18.35
52 I. 13 16.61
31 65.96
23 54.76
22 47.83
307 47
267 42
313 46
*Statistical analysis was not performed on this parameter. $Statistical analysis was performed on these data after application of the F r c e m a n - T u k e y arcsine transformation.
959
2.5-fold increases in dietary calcium on reproductive performance and foetal development in rats fed diets that were nutritionally adequate. The differences between our results and those obtained in the study by Lai et al. (1984) can be attributed to dietary composition. Rogers et al. (1985) studied the sensitivity of Long-Evans hooded rats to maternal zinc deficiency during pregnancy, using purified diets. The source of protein was egg white, and the levels of zinc were I00 mg/kg diet for the control group and 9.0, 4.5 or 0.5 mg/kg diet for the treatment groups. Long-Evans dams fed 0.5, 4.5 or 9.0 mg zinc/kg diet ate less than did animals fed 100 mg zinc/kg diet during the last 3 days of pregnancy. Rats fed 0.5 or 4.5 mg zinc/kg diet gained significantly less weight than did those fed 9.0 or 100mg zinc/kg diet; weight gains did not differ between rats fed 9.0 mg zinc/kg diet and those fed 100 mg zinc/kg diet. The decreases in feed consumption and body weight gain and the increased hypophagia near term observed in the study by Lai et al. (1984) are consistent with reduced zinc bioavailabilty, which was probably caused by the phytate-zinc-calcium interaction rather than by the increase in dietary calcium (Davies and Olpin, 1979). The absence of a similar effect in the present study can be attributed to the use of casein as the protein source and a level of zinc reported to be adequate for gestation according to the American Institute of Nutrition (1977 and 1980). One useful approach to the study of the reproductive effects of excess dietary calcium is to induce hypercalcaemia during pregnancy and/or lactation by feeding rodents excess calcium in the diet and drinking water. This technique was introduced by Fairney and Weir (1970), who produced hypercalcaemic female rats by feeding them 3% calcium as calcium carbonate in powdered Oxoid Breeding Diet and 4 g calcium lactate per 100 ml drinking (tap) water. The hypercalcaemic parental female rats had litters that were small, weighed less than normal at birth and grew poorly. Liebgott and Srebrolow (1989) studied the effects of experimentally induced maternal hypercalcaemia on foetal development in CDI mice. The method of inducing hypercalcaemia was similar to that used by Fairney and Weir (1970), except that calcium carbonate was added to Purina Rodent Chow 500, which contains excess nutrients. The excess calcium was given during gestation, and caesarean sections were performed on day 18. Foetal skeletons were doublestained for cartilage and bone with alcian blue and Alizarin Red S. When the data were analysed per litter, the mean number of resorptions and foetal deaths were higher than those of the control group, but the differences were not significant. Excessively high calcium intake in mice during gestation significantly decreased foetal weight and retarded foetal skeletal and dental development. No gross abnormalities were observed in the control or experimental
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M.E. SHACKELFORDet al.
groups. The authors recommended that excessive calcium intake should be avoided during pregnancy to prevent hypercalcaemia and that plasma calcium should be monitored during pregnancy. Unlike Fairney and Weir (1970) and Liebgott and Srebrolow (1989), in the present study moderate increases in dietary calcium were used to examine the developmental effects of this element. The concentrations used in our study resemble more closely the increases recommended by the 1984 N I H Consensus Development Conference Panel on Osteoporosis (1986). Plasma calcium levels were not measured, but were not expected to increase with the levels of dietary calcium used. Liebgott and Srebrolow (1989) hypothesized that the observed delayed skeletal and dental calcification might have been due to decreased foetal parathyroid hormone levels and vitamin D metabolites subsequent to the maternal hypercalcaemia. Under the dietary conditions of the present study, there was no observed dose-related effect on the average number of implantations, resorptions and viable foetuses, or on the length and weight of the foetuses. N o effects on foetal visceral and skeletal development were seen. O f what relevance are the studies with excess dietary calcium to human pregnancy? The first study on the ingestion of large amounts of calcium carbonate and calcium-containing food during pregnancy was reported by Ullian and Linas (1988). The patient had the milk-alkali syndrome, which is the cause of hypercalcaemia in ulcer patients who consume large amounts of milk and antacid products (Federal Register, 1979). A patient was hospitalized in wk 23 of pregnancy with complications of hypercalcaemia, dehydration, renal insufficiency and pancreatitis. At wk 37 of gestation, a stillborn foetus was delivered. The foetus had short limbs, low ears and normal chromosome analysis. According to the authors, the relationship between M A S and congenital abnormalities was not apparent. The present study used nutritionally adequate diets to study the effects of supplemental dietary calcium on maintenance of pregnancy and foetal development. This study did not address issues raised by Lai et al. (1984) and Richards and Greig (1952) concerning the reproductive and developmental effects of excess dietary calcium and dietary deficiencies of zinc and iron. At the concentrations used here, dietary calcium was neither foetotoxic nor teratogenic. Acknowledgements--We express our appreciation to George Gray, Randolph Jackson, Robert Jones, Dorothy Quander, James Rorie and Michael Scott for their excellent technical support. We thank the Technical Editing Branch, Food and Drug Administration, for assistance in preparing the manuscript. REFERENCES
American Institute of Nutrition (1977) Report of the American Institute of Nutrition ad hoc committee on standards
for nutritional studies. Journal of Nutrition 107, 1340-1348. American Institute of Nutrition (1980) Second report of the ad hoe committee on standards for nutritional studies. Journal of Nutrition 110, 1726. Bateman A. J. and Epstein S. S. (1971) Dominant lethal mutations in mammals. In Chemical Mutagens. Principles and Methods for Their Detection. Edited by A. Hollaender. Vol. 2. p. 541. Plenum Press, New York. Consumer Reports (1988) The truth about calcium. Consumer Reports May 1988, 288-291. Davies N. T. and Nightingale R. 0975) The effects of phytate on intestinal absorption and secretion of zinc, and whole-body retention of zinc, copper, iron, and manganese in rat. British Journal of Nutrition 34, 243-258. Davies N. T. and Olpin S. E. (1979) Studies on the phytate: zinc molar contents in diets as a determinant of Zn availability to young rats. British Journal of Nutrition 41, 591~03. Davies N. T. and Reid H. (1979) An evaluation of the phytate, zinc, copper, iron and manganese of, and Zn availability from, soya-based textured-vegetable-protein meat-substitutes or meat-extenders. British Journal of Nutrition 41, 579-589. Dawson A. B. (1926) A note on the staining of the skeleton of cleared specimens with Alizarin Red S. Stain Technology 1, 123-124. Fairney A. and Weir A. A. (1970) The effect of abnormal maternal plasma calcium levels on the offspring of rats. Journal of Endocrinology 48, 337 345. Federal Register (1979) Vitamin and mineral drug products for over-the-counter human use. Federal Register 44, 16175-16178. Freeman M. F. and Tukey J. W. (1950) Transformation related to the angular and the square root. Annals of Mathematical Statistics 21, 607q511. Fullerton F. and Greenman D. L. (1982) Effects of storage conditions on general deterioration and stability of nutrients in experimental diets. National Center for Toxicological Research (NCTR) Final Report for Experiment 336. Food and Drug Administration, Washington, DC. Greig W. A. (1952) The effects of additions of calcium carbonate to the diet of breeding mice. 2. Haematology and histopathology. British Journal of Nutrition 6, 280-294. Harper A. E. (1959) Amino acid balance and imbalance. 1. Dietary level of protein and amino acid imbalance. Journal of Nutrition 68, 405-418. Lai A., Kiyomi-Ito M., Komatsu K. and Niiyama Y. (1984) Effects of various levels of dietary calcium during pregnancy on maternal calcium utilization and fetal growth in rats. Journal of Nutrition, Science and Vitaminology 30, 285-295. Leuschner F. and Czok G. (1973) Reversibility of prenatal injuries induced by caffeine in rats. Comptes Rendus du Colloque International sur la Chimie des CafOs 5, 388--391. Liebgott B. and Srebrolow G. (1989) Fetal toxicity caused by excessive maternal dietary calcium. Journal o f the Canadian Dental Association 55, 129-133. MARTA (Mid-Atlantic Reproduction and Teratology Association) Committee on Terminology (1969) Teratology and Reproduction Glossary. p. 71. NIH Consensus Development Conference Panel on Osteoporosis (1986) Osteoporosis: Cause, Treatment, Prevention. NIH publication no. 862226. National Institutes of Health, Bethesda, MD. O'Dell B. L., Hardwick B. C. and Reynolds G. (1961) Mineral deficiencies of milk and congenital malformations in the rat. Journal of Nutrition 73, 151-157.
Developmental effects of excess dietary calcium Padmore J. M. (1990) Animal feed. In Official Methods of
Analysis of the Association of Official Analytical Chemists. Edited by K. Helrich. 15th Ed. pp. 84~8. AOAC, Arlington, VA. Richards M. B. and Greig W. A. (1952) The effects of additions of calcium carbonate to the diet of breeding mice. 1. Effects on reproduction and on the heart and thymus weights of the weanlings. British Journal of Nutrition 6, 265-280. Rogers J. M., Keen C. L. and Hurley L. S. (1985) Zinc deficiency in pregnant Long-Evans hooded rats: teratogenicity and tissue trace elements. Teratology 31, 89-100. Shackelford M. E., Collins T. F. X., Welsh J. J., Black T. N , Ames M. J., Chi R. K. and O'Donnell M. W. (1994) Foetal development in rats fed AIN-76A diets
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supplemented with excess calcium. Food and Chemical Toxicology 32. In press. Sokal R. R. and Rohlf F. J. (1981) Biometry. pp. 293-308. W. H. Freeman and Company, San Francisco. Spoerl R. and Kirchgessner M. (1976) Studies on the copper requirements of pregnant and lactating rats. Archives Tierern~hrung 26, 25-34. Ullian M. E. and Linas S. L. (1988) The milk-alkali syndrome in pregnancy. Mineral and Electrolyte Metabolism 14, 208-210. Wilson J. G. (1973) Environment and Birth Defects. pp. 227-232. Academic Press, New York. Wilson J. G. and Warkany J. (1965) Teratology Principles and Techniques pp. 262-277. University of Chicago Press, Chicago.
0278-6915/93 $6.00+ 0.00 Pergamon Press Ltd
FOETAL DEVELOPMENT IN RATS FED AIN-76A DIETS SUPPLEMENTED WITH EXCESS CALCIUM M. E. SHACKELFORD,T. F. X. COLLINS,J, J. WELSH, T. N. BLACK, M. J. AMES, R. K. CHI and M. W. O'DONNELL Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington, DC 20204, USA (Accepted 12 July 1993) Abstract--This study was designed to evaluate the developmental effects of moderate dietary calcium increases in rats fed nutritionally adequate diets. Female Charles River CD/VAF Plus rats were given 0.50 (control), 0.75, 1.00 or 1.25% dietary calcium as calcium carbonate in AIN-76A diets for 6 wk before mating, during mating and for 20 days of gestation. On gestation day 20, the animals were killed and caesarean sections were performed. Both the non-pregnant and pregnant rats in the 0.75, 1.00 and 1.25% groups ate slightly more than did the control group during most of the intervals measured, but not all the increases were statistically significant. There was no consistent pattern of increase or decrease in weight gain. No dose-related changes were found in maternal clinical findings, the average number of implantations, resorptions and viable foetuses, or foetal length or weight. Under the conditions of the study, there were no statistically significant increases as compared with the control group in the litter incidence regarding specific external, visceral or skeletal variations of the foetuses. Dietary calcium was neither foetotoxic nor teratogenic at the concentrations used.
INTRODUCTION The 1984 NIH Consensus Development Conference Panel on Osteoporosis (1986) recommended that premenopausal women and women who are pregnant or nursing increase their daily calcium consumption through the use of calcium-rich foods and calcium supplements. The recommendations represented an approximate 1.2-fold increase over the 1980 recommended dietary allowances (RDA). Calcium supplement sales increased from $46 million in 1983 to $200 million in 1987 (Consumer Reports, 1988). Adverse reproductive and haematological effects have been reported from ingestion of increased dietary calcium during pregnancy and lactation (Richards and Greig, 1952; Greig, 1952). Adult female Swiss mice were fed experimental diets for four reproductive cycles. These diets were adjusted with calcium carbonate to 0.54, 0.73 and 1.11% calcium and maintained constant in phosphorus at 0.48%. For all the litters evaluated, there was no significant effect on the total number of animals or litters born. There was a significant decrease in the total weight and number of animals weaned on diets that contained the two-fold increase in dietary calcium (1.11% calcium). Calcium supplementation resulted in hypertrophy of the heart and anaemia in adult and weanling mice. Iron supplementation of the
Abbreviations: ANOVA -- analysis of variance; ANOCOVA = analysis of covariance; LSD = least significant difference; MAS = milk-alkali syndrome; RDA = recommended dietary allowances.
diet diminished the anaemia and the effects on the heart. However, anaemia was already established in the litters from the control groups when they were compared with litters from animals fed several other stock diets. Analysis of the diet formulations revealed possible dietary inadequacies. These diets, which consisted of whole wheat and dried whole milk, were not supplemented with vitamins and minerals (O'Dell et al., 1961). Nutritional inadequacies in addition to excess calcium in the diets may have contributed to the reproductive and haematological results observed. Lai et al. (1984) fed rats a semi-purified diet that contained 0.6 or 1.0% calcium, 20% soya-protein isolate and a constant (0.45%) level of phosphorus. The study reported hypophagia near term and a significant decrease in the total feed intake and total body weight gain of the pregnant female rats fed the 1.67-fold increase in dietary calcium during the 22 days of gestation. However, foetal weight and litter size were not affected. The nutrient interaction between phytate in the protein source, the inadequate amount of zinc (4.8 mg/kg diet) in the Harper's mineral mix and excess calcium in the diet could have reduced zinc bioavailability and affected feed consumption and body weight gain (Davies and Nightingale, 1975; Davies and Olpin, 1979, Davies and Reid, 1979; Harper, 1959). Because of these problems, the effects attributable to dietary calcium could not be assessed. We therefore designed a study to evaluate the developmental effects of excess dietary calcium in rats that were fed nutritionally adequate diets. Female
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M. E. SHACKELFORDet al.
954
Charles River C D / V A F Plus rats were given 0.50, 0.75, 1.00 or 1.25% dietary calcium as calcium carbonate for 6 wk before mating, during mating and for 20 days o f gestation. The effects o f multiple dietary factors were eliminated by feeding the rats A I N - 7 6 A diet with casein, which lacks dietary phytate, as the protein source. MATERIALS
AND
METHODS
Test material. US Pharmacopeia-grade calcium carbonate was obtained from Pfizer (Adams, MA, USA). The chemical was 98.62% pure, and only one lot was used. The test material was incorporated into a modified A I N - 7 6 A diet that was prepared according to our specifications by B I O - S E R V (Frenchtown, N J, USA). Diets. The formulation o f p o w d e r e d AIN-76A diet (Table 1) is r e c o m m e n d e d as nutritionally adequate for rats by the American Institute o f Nutrition (1977 and 1980). During the duration o f the study, the diets were stored in a walk-in refrigerator ( - 1 to - 6 ° C ) to maintain nutrient stability (Fullerton and Greenman, 1982). The mineral mix o f the AIN-76a diet was slightly modified. The copper content was increased from 6 to 9 mg/kg diet (Spoerl and Kirchgessner, 1976) by a separate addition o f cupric carbonate. Calcium p h o s p h a t e and calcium carbonate were added separately to adjust the diets to specified levels o f calcium and p h o s p h o r u s . Calcium p h o s p h a t e was the primary source o f calcium and p h o s p h o r u s . Dietary calcium was adjusted to 0.50, 0.75, 1.00 and 1.25% with calcium carbonate, and dietary phosphorus was maintained at approximately 0.40%. At
the time of preparation, a separate lot was prepared for each o f the specified dose levels o f calcium. The diets were assayed for homogeneity and concentrations o f calcium, p h o s p h o r u s , magnesium, manganese, zinc, iron and copper by the New Jersey Feed Laboratory, Inc. (Trenton, N J, USA), using the m e t h o d s of the Association o f Official Analytical Chemists (Padmore, 1990). The diets at 1.0 and 1.25% calcium were slightly higher in iron than the other diets used in this study because o f contributions from calcium carbonate. Animals. 156 male and 291 female C D / V A F Plus Charles River rats were obtained from Charles River Laboratories, Inc. (Wilmington, MA, USA); when received, the females were 52 days old and the males were 44 days old. At the end of the 6-day quarantine, 276 female animals were assigned to the diets by a stratified r a n d o m m e t h o d according to body weight. F o u r groups received 0.50 (control), 0.75, 1.00 or 1.25% calcium in powdered AIN-76A diet for 6 wk before mating, t h r o u g h o u t mating and for 20 days o f gestation. O f the 291 female animals used in the study, 135 were randomly selected for the determination o f tissue mineral levels at three times during the study: 15 animals before assignment o f the remaining 276 to diet groups; 60 non-pregnant animals (15 per group) after 6 w k on the diets; and 60 pregnant animals (15 per group) at the time o f caesarean section on day 20 of gestation. F r o m these 15 pregnant females, two foetuses from each o f 12 litters were also used for whole-body mineral determinations. The mineral data are presented in a separate paper (Shackelford et al., 1994). Female animals were weighed and feed c o n s u m p t i o n was measured every
Table 1. Composition of the purified diets supplementedwith excesscalcium fed to female CD/VAF Plus rats Concentration of calcium in the diet (%) Component* (%) 0.50 0.75 1.00 1.25 Casein 20.0 20.0 20.0 20.0 Choline bitartrate 0.2 0.2 0.2 0.2 DL-Methionine 0.3 0.3 0.3 0.3 Corn starch 21.7 21.7 21.7 21.7 Sucrose 20.0 19.4 18.8 18.I Dextrose 21.7 21.7 21.7 21.7 Cellulose 5.0 5.0 5.0 5.0 Corn oil 5.0 5.0 5.0 5.0 Vitamin mix 1.0 1.0 1.0 1.0 Mineral mix 3.5 3,5 3.5 3.5 Cupric carbonate 0.05 0.05 0.05 0.05 Calcium phosphate 1.49 1.49 1.49 1.49 Calcium carbonate 0.15 0.77 1.40 2.03 Minerals Ca 0.46+0.01 0.71 _+0.02 0.92+_0.01 1.20-+0.02 P 0.45 + 0.01 0.46 +, 0.03 0.46 +-0.02 0.46 +, 0.02 Mg (mg/kg diet) 464 +, 15 458 _+9 503 +_33 497 +, 60 Mn (mg/kg diet) 53 -+ 3 55 +_2 56 +_2 52 + 2 Fe (mg/kg diet) 42 +, 9 42 -+2 53 -+2 51 _-4-2 Zn (mg/kg diet) 32 +, 2 29 -+ 2 34 + 1 34 -+ 1 Cu (mg/kg diet) 8 +, I 8 -+ I 9 +, I 9_+ 1 *Components are in percentagesof the diet unless otherwiseindicated. The vitamin mix of the purified diets was the same as that specified for diet AIN-76A (American Institute of Nutrition, 1977 and 1980). The mineral mix was modified as described in Materials and Methods. The mineral composition of the diets was determined by analysis. Results are means +- SD of duplicate samples from the top, middle and bottom of each lot.
Developmental effects of excess dietary calcium 3 days for 6 wk before mating and during gestation, but not during mating. Feed consumption and body weights were not measured for male rats. Male rats were fed pelleted Purina Rodent Chow 5002 (Purina Mills, Inc., Richmond, IN, USA) except during mating, when they also received one of the four experimental diets. Animals, identified by numbered metal ear tags, were housed in stainless-steel cages, with feed and deionized water supplied ad lib. The study room was environmentally monitored with a hy'grothermograph for temperature (18-27°C) and humidity (25-72%). There was an automatic 12-hr light/dark cycle. Exposure to extraneous sources of zinc was minimized by the use of stainless-steel animal housing, Aqua Cool deionized water (Ionics, Inc., Watertown, MA, USA) and polyethylene waterbottle stoppers. Powder-free, polyvinyl chloride gloves (VWR Scientific Co., Baltimore, MD, USA) were used to minimize contamination of the animals. Procedures. After 6 wk on the control and test diets, the female rats were mated to the males in a 2: I ratio, with cohabitation beginning at approximately 4.30 pm. The following morning, a vaginal smear was made for each female to determine copulation. The day on which sperm was found in the vaginal smear was considered to be day 0 of gestation. The spermpositive females were continued on the respective control or test diets during pregnancy. The dams were killed by carbon dioxide asphyxiation on day 20 of gestation. A laparotomy was then performed on each dam, and the major organs were examined. In each case, the uterus was opened and examined in situ for the presence and position of resorption sites and foetuses (dead or alive) and the number of implantation sites. Deciduomas (brownish implantation sites without placentas) were considered early deaths; implantation sites with placentas and complete but non-viable foetuses that were of subnormal size, showed retarded development or were in a macerated condition were classified as late deaths, according to the terminology of Bateman and Epstein (1971) and the MARTA (Mid-Atlantic Reproduction and Teratology Association) Committee on Terminology (1969). The uteri of animals that did not appear to be pregnant were stained with sodium sulfide to enhance resorption sites. Foetuses were examined individually for gross abnormalities. The uterine position, sex, weight and crown-rump length of each foetus were recorded. Corpora lutea were counted under a lighted magnifying lens. Any foetus that weighed less than 70% of the average weight of the concurrent male or female controls was considered to be a runt (Leuschner and Czok, 1973). Foetuses were assigned to skeletal or visceral analysis on an alternate basis following the sequence of foetal numbers. From 12 litters in each group, two foetuses (one from each uterine horn) were randomly selected and used for whole-body mineral determinations. Approximately half of the foetuses were fixed in alcohol, macerated in potassium hydroxide, stained with Alizarin Red S
955
and examined microscopically for skeletal variations (Dawson, 1926). The remaining half were fixed in Bouin's solution, sectioned by razor blade and microscopically examined for internal variations of the soft tissues (Wilson and Warkany, 1965; Wilson, 1973). Each sperm-positive female was given a blind-test number, which was carried over as a litter number. To preclude the possibility of bias, we evaluated the dams and foetuses during caesarean sections and the foetuses during skeletal or visceral analyses without knowing the dose levels. Statistical analysis. All statistical analyses were performed by the Division of Mathematics, US FDA. The incidence of clinical observations was analysed by comparing treatment groups with the control group by using Fisher's exact test. Data on feed consumption were submitted to an analysis of variance (ANOVA) and a protected least-significant difference (LSD) test (two-tailed). Data on weight gain were submitted to an analysis of covariance (ANOCOVA), after adjusting for the initial body weight, and an LSD test (two-tailed). Trend analysis was done on feed consumption. It was not done on body weight data, because we analysed these data by covariance. A trend is present if the linearity P value is below 0.05 (borderline trend if below 0.10) and, at the same time, the departure P value is above 0.10. The number of corpora lutea, the average number of implantations, the average number of viable foetuses for each litter and the average number of male or female foetuses for each litter were analysed by ANOVA and an LSD test (one-tailed). Data on implantation efficiency, early deaths, late deaths and total resorptions (early and late deaths) were analysed by using the Freeman-Tukey arcsine transformation for binomial proportions (Freeman-Tukey, 1950). We then analysed transformed data for each litter by ANOVA and an LSD test (one-tailed), comparing the control group with experimental groups. With Fisher's exact test, we analysed data on litters with runts and litters totally resorbed, comparing the control group with treated groups. Foetal body weights and crown-rump lengths were analysed by a nested ANOVA (Sokal and Rohlf, 1981) and an LSD test (one-tailed). We analysed data on the specific litter incidence of sternebral, skeletal and visceral variations by using Fisher's exact test to compare the control group with treatment groups. Proportions of litters with foetuses showing external variations were analysed by using Fisher's exact test. The average number of foetuses with variations per litter was analysed by using the Freeman-Tukey arcsine transformation and then an ANOVA and a protected LSD test (one-tailed). Litters that had foetuses with one or more sternebral, skeletal and visceral variations were analysed with Fisher's exact test. We did trend analysis on the litter data for external variations, using the Cox exact one-tailed test for unadjusted positive trend. A trend is significant if the P value is below 0.05. Trend analysis was not done on data that were
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M.E. SHACKELFORDet al.
analysed by using the Freeman-Tukey arcsine transformation for binomial proportions.
Table 2. Mean daily feed consumption (g/animal/day) of rats given excess dietary calcium
Concentration of calcium in the diet (%) Days
RESULTS
Clinical findings included the following: alopecia, exudate around the eyes, exudate around the nose, bent teeth, lesion, lump in the left flank or leg and mammary lump. None of the above findings was dose related. The number of animals with alopecia was significantly increased in animals fed 1.00% calcium (8.7 v. 0% in the control) during the 6wk before mating and in the groups fed 0.75 and 1.00% calcium (10 and 16.3%, respectively, v. 0% in the control) during gestation. One non-pregnant animal was observed to have diarrhoea on day 4 after assignment to the 1.25% calcium diet. One pregnant female fed 1.00% dietary calcium developed bleeding from the vagina on days 15 and 16 of gestation. At caesarean section on day 20, this animal had 17 implants with 13 resorptions and three living pups. The incidence of diarrhoea and bleeding from the vagina was not considered to be related to the dietary calcium. Three females died during the study, but the deaths were not attributable to excess calcium in the diet. One animal in the 0.75% calcium group was killed on day 40 because mechanical trauma to the jaw prevented the animal from eating properly. One animal in the control group died because of mechanical injury during smearing. A second animal in the 0.75% calcium group was found moribund and bleeding from the urethra on day 3 of gestation. At autopsy, the animal was found to have bladder calculi. The final diagnosis was acute renal failure and obstructive uropathy. The condition was considered to be a random occurrence and was not thought to be caused by the 0.75% level of calcium in the diet. Values for mean daily feed consumption per female given excess dietary calcium for 6 wk before mating and during gestation are shown in Table 2. The 0.75, 1.00 and 1.25% groups ate slightly more than the controls during most of the intervals measured, but not all the increases were statistically significant. The trend analysis for feed consumption for non-pregnant rats showed a significant (P < 0.05) positive trend during most intervals measured, but the trend was borderline positive (P =0.0514) during days (~12. There was no linear trend during days 12-18 and 18-20 of gestation, but there was a significant positive trend (P < 0.05) during the other intervals measured. Values for mean body weight gain for animals given excess dietary calcium for 6 wk before mating and gestation are given in Table 3. Initial body weight on day 0 for non-pregnant rats and sperm-positive females was similar for all groups. Although some differences in weight gain between control and treated groups were significant, there was no consistent pattern of increase or decrease in the body weight gain. Weight gain increased during some intervals measured but decreased for other intervals.
0.50
0.75
1.00
1.25
Non-pregnant rats 6 wk before mating 0-6 6-12 12-18 18-24 24-30 30-36 3~42 ff42
(69) 15.2_+0.4 17.4_+0.3 17.0,+0.3 17.9,+0.3 17.6-+0.3 17.8-+0.3 17.8_+0.3 17.3,+0.2
(69) 15.7,+0.3 17.7_+0.3 17,6_+0.3 18.1_+0.3 17.9-+0.3 17.7_+0.3 17.8_+0.3" 17.5_+0,2"
(69) 15.8_+0.3 17.8,+0.3 18.1 +0.3 b 18.8+0.3 ~ 18.8_+0.3 b 18.6_+0.3 d 18.6_+0.3 a 18.1_+0.2 ~
(69) 16.4_+0,4 18.2_+0.3 18.4_+0.3 ¢ 18.9+0.3 b 18.6+0.3 a 18.4-+0.2 18.5_+0.3 d 18.2_+0.2 b
0~ 6-12 12-18 18-20 0-20
Pregnant rats from days 0-20 (45) (47) (44) 19.6_+0.4 20.4,+0.4 20.8,+0.4" 22.8,+0.4 23.3_+0.3 23.9_+0.4 d 23.5,+0.4 24.3-+0.3 24.1 -+0.5 23.7 _+0.5 24.0 _+0.4 23.7 _+ 0.6 22.2_+0.4 22.8_+0.3 23.0_+0.4
(48) 21.0+0.3 b 24.2+0.4 a 24.2-+0.3 23.0 _+ 0.5 23.1 _+0.3
*Because one animal was autopsied in extremis on day 40 in the 0.75% group, the number of rats was reduced to 68 for the data for days 36-42 and 0-42. Numbers of animals are indicated in parentheses. Values are means _+ SEM; superscripts indicate differences between control and test values (aP~<0.05; bp~<0.01; cP~<0.00h a0.05 < P < 0.10).
Maternal reproductive parameters are listed in Table 4. There were no adverse reproductive effects associated with ingestion of excess dietary calcium. Significant increases were observed in implantation efficiency at 1.25% dietary calcium and in the average number of viable female foetuses at 0.75 and 1.25% dietary calcium. These increases were not dose related and were not considered to be caused by dietary calcium. Table 3. Mean body weight gains and initial body weights of rats given excess dietary calcium Concentration of calcium in the diet (%) Days
0.50
0,75
(69)
(69)
Body weight gain (g) of non-pregnant (~6 6-12 12-18 18 24 24-30 30-36 36-42 0~.2 0
1.00
1.25
(69) (69) rats 6 wk before mating 9.1_+1.1 7.7+1.4 21.7_+1.1 20.1+1.1 16.1 _+ 1.0~ 16.3 _+0.T 15.7_+0.9 14.1 +0.7" 11.9 _+0.9 a 10.6 + 0.7 10.1 + 1.0 9.6_+0.8 4.9 _+ 0.8 6.4 _+ 0.6 89.5_+3.0 84.7+2.6
8.1+1.4 9.9-+1.2 22,1 -+ 1.1 21.1_+0.9 12.1 -+0.8 14.7+0.9 a 16.3_+0.8 14.0+0.7 a 9.0 _+ 0.6 9.8 _+0.8 11.0_+0.9 9.2_+0.8 5.3 + 0.7 5.8 _+ 0.9* 83.9_+2.9 85.1-+3.1" Initial body weight (g) of non-pregnant rats 217.7-+1.0 217.6_+1.0 217.4_+1.0 217.7_+1.0 (45)
(47)
(44)
(48)
Body weight gain (g) of pregnant rats 0~ 6-12 12-18 18-20 0-20 0
29.8 _4- 1.2 29.7 + 1.3 29.8 + 1.1 29.4 + t.0 30.4+ 1.0 32.0_+0.8 30.2_+0.7 31.9_+0.9 45.2 _+ 1.8 51.1 ± 1.4' 48.0 -+ 2.6 48.9 -+ 1.5 25.8_+ 1.1 27.9_+0.9 26.2_+1.1 25.1 _+ 1.0 131.2_+3.1 140.7+2.6 ~ 134.2_+3.9 135.3_+3.0 Initial body weight (g) of pregnant rats 297.6 _+ 3.6 300.8 + 3.7 306.5 _+4.8 300.0 -+ 3.8
*Because one animal was autopsied in extremis on day 40 in the 0.75% group, the number of rats was reduced to 68 for days 36-42 and 0-42. Numbers of animals are indicated in parentheses. Values are means _+ SEM; superscripts indicate differences between control and test values (ap ~<0.05; ~P ~<0.0h eP ~<0.00k d0.05 < P < 0.10).
Developmental effects of excess dietary calcium
957
Table 4. Maternal and reproductive outcome at autopsy and foetal data from rats given excess dietary calcium Concentration of calcium in the diet (%) Parameter No. of pregnant females No. of corpora lutea/female* Implantation e f f i c i e n c y (%/female)*? No. of implants/female* No. of viable foetuses/female* Viable male foetuses/litter* Viable female foetuses/litter* Resorptions/female (mean %)*{ No. of early deaths/litter? No. of late deaths/littert No. of early and late deaths/litter?~ No. of litters totally resorbed Body weight (g) Males Females C r o w n - r u m p length (era) Males Females No. of runts~ Males Females *Means
0.50
0.75
1.00
1.25
45 16.27 + 0.41
47 16.83 + 0.40
44 16.52 _-4-0.27
48 15.98 + 0.32
82.01 + 3.73 13.58 _+ 0.68 11.82+0.66 6.29 _+ 0.43 5.53 _+ 0.39 12.50_+ 1.98 1.71 0.04 1.76 0
90.29 + 1.89 15.26 _ 0.43 13.53+0.46 6.49 _+ 0.33 7.04 _+ 0.38 b 11.15_+ 1.73 1.70 0.02 1.72 0
86.51 + 3.46 14.25 + 0.60 12.82_+0.65 6.95 _+ 0.49 5.86 5:0.37 12.92_+3.19 1.41 0.02 1.43 1
93.41 + 2.28 b 15.15 ___0.50 13.545:0.58 6.83 _+ 0.37 6.71 _+ 0.43" 13.81 _+3.09 1.56 0.04 1.60 2
3.84 3.61
3.90 3.69
3.83 3.66
3.79 3.58
4.0 3.9
4.0 3.9
4.0 3.9
4.0 3.9
5 (4) 4 (4)
5 (2) 4 (1)
3 (3) 1 (1)
3 (3) 3 (2)
+ SEM.
)'Statistical analysis was performed on these data after application of the Freeman-Tukey arcsine transformation. :~tThe number of resorptions includes early deaths and late deaths per Litter. §The number of litters is indicated in parentheses. Values marked with superscripts indicate significant differences between control and test values (ap ~<0.05; bp ~<0.01; cp ~<0.001; d0.05 < P < 0.10).
There were no adverse effects to the foetuses caused by feeding excess dietary calcium to the dams (Table 4). Foetal body weights and c r o w n - r u m p lengths of male and female animals given excess dietary calcium were similar to those of the control groups. Feeding excess dietary calcium had no effect on the number of litters with male or female runts. The incidences of specific external variations are shown in Table 5. In the control group, one foetus did not possess a m o u t h (astomia) and a lower jaw (agnathia). At the 1.25% dietary calcium level, one
Table 5. Incidence of specific external variations in foetuses from rats given excess dietary calcium Concentration of calcium in the diet (%) Parameter
0.50
0.75
1.00
1.25
No. foetuses (litters examined 532 (45) 636 (47) 564 (43) 650 (46) No. of foetuses (litters) with variations 2 (2) 2 (2) l (l) 8 (6) Haemorrhages, external I (1) -l (I) 2 (2) Cleft palate l (1) -2 (2) Agnathia I (1) --l(1) Astomia 1 (1) Thread-like tail 1(1) -1(1) Kinked tail --2 (2) Oedema --3 (2) Malformed head --(1) Beaked snout --(1) Protruding tongue --(1) Open eye --(l) Diaphragmatic cleft* --(1) Omphalocele --(1) _ _ (]) Club foot Scoliosis --(1) Imperforate anus --2 (2) The number of litters is indicated in parentheses. *This parameter includes exposed heart and lungs.
foetus had multiple anomalies: agnathia, cleft palate, protruding tongue, beak-like snout, malformed head, open eye on the fight side, oedema of the trunk, omphalocele, exposed heart and lungs (diaphragmatic cleft), club foot on the left front paw, scoliosis, imperforate anus and kinked tail. The occurrence of this foetus with multiple anomalies was not thought to be related to feeding the rats excess dietary calcium before and during pregnancy. The data in Table 5 were analysed by trend analysis and Fisher's exact test. The Cox exact trend test showed no significant positive trends in the litter incidence of individual or combined variations. When analysed by Fisher's exact test, there were no significant differences between treated and control groups in the litter incidence of these same parameters. Under the conditions of the study, there were no significant increases in the litter incidence of specific or combined external variations. The numbers of foetuses with sternebral ossification deficiencies and the litter incidences are summarized in Table 6. A comparison of the proportion of litters with foetuses having sternebral variations showed no significant differences among the groups given excess calcium and the control group. The increase in the number of litters with foetuses with bipartite sternebrae in the 1.25% group was not significant (P = 0.0854). The incidences of sternebral variations are presented in Table 7. The variations counted are based on the classification of the types (e.g. missing or bipartite) and not on the individual sternebrae affected. The average number of foetuses per litter with one or more sternebral variations increased in
958
M.E. SHACKELFORDet al.
Table 6. Incidence o f specific sternebral variations in foetuses o f rats given excess dietary calcium
Table 8. Incidence o f specific skeletal variations in foetuses o f rats given excess dietary calcium
Concentration o f calcium in the diet ( % ) Parameter
0.50
No. o f foetuses (litters) examined 251 (44) No. of foetuses with specific sternebral variations Incomplete ossification 74 (31) Bipartite 4 (4) Unossified 50 (27) Malaligned 8 (7) Fused 1 (1)
0.75
1.00
305 (47)
Concentration of calcium in the diet ( % ) 1.25
273 (43) 313 (46)
84 (35) 3 (3) 49 (27) 2 (2) 0(0)
88 (36) 4 (3) 40 (18) 10 (9) 0(0)
The n u m b e r o f litters is indicated in parenthesis. Supercript indicates difference between control (a0.05 < P < 0.10).
95 (35) 10 (10) d 54 (31) I 1 (11) 1 (1)
and test values
Parameter
0.50
No. o f foetuses (litters) examined 251 (44) Ribs, fused -Ribs, wavy -14th rib (C7) 1 (1) 14th rib (L1) 22(12) 13th rib, short 4(3) Centra, missing 6 (5) Centra, fused . . Centra, bipartite 1 (1) Dorsal arches, extra centre of ossification -Last caudal ossification* -T y m p a n i c arch, red. oss. 1 (1) Interparietal, red. oss. 3 (2) Parietal, red. oss. 1 (1) Frontal, red. oss. 1 (I) Supra occipital, red. oss. I (I) Supra occipital, bipartite 1 ( 1) Hyoid, red. oss. I (1) Nasal, red. oss. 41 (22) Mandible, red. oss. 1 (I) Pubis, red. oss. 2 (2) Metacarpals, red. oss. 1 (I) Metatarsals, red. oss. 5 (4) Maxilla, red. oss. --
0.75
1.00
1.25
305 (47) 273 (43) 313 (46) I (I) --I (I) . . . . ---17(9) 12(12) 13(12) 5(3) 6(5) 4(2) 7 (7) I I (9) 9 (8) . . . . . . . . . 1 (1) 4 ( 1)
----.
2 (1) I (I) --
the 0.75, 1.00 and 1.25% groups, but the differences were not significant. There was no significant increase in the average number of foetuses per litter with at least two or three sternebral variations in the groups fed excess dietary calcium. Statistical analysis of the proportion of litters that had foetuses with variations (at least one, two or three) showed no significant increase between the excess calcium and the control groups. The incidences of specific skeletal variations, excluding sternebrae, were not dose related, and there were no significant differences among any of the values (Table 8). The average number of sacral and caudal vertebrae was not affected by dietary calcium (data not shown). The average number of foetuses per litter with at least one skeletal variation was lower in the 0.75, 1.00 and 1.25% dietary calcium groups than in the control (Table 9). However, the A N O V A of the F r e e m a n Tukey arcsine-transformed data showed no differences in the average number of foetuses with variations (P > 0.10). The proportion of litters that had foetuses with variations (at least one, two or three)
showed no dose-related or significant differences between any of the values obtained in this study. There was no dose-related effect on the incidence of any type of soft-tissue variation (Table 10). The proportion of litters with hydrocephalus was greater in the control group than in the treated groups, but the differences were not significant. The incidence of visceral problems by foetus and by litter is shown in Table 11. The average number of foetuses with at least one, two or three soft-tissue variations was similar in all groups. Although the
Table 7. Incidence o f sternebral variations in foetuses of rats given excess dietary calcium
Table 9. Incidence o f skeletal variations in foetuses of rats given excess dietary calcium
Parameter
0.50
0.75
1.00
1.25
142 3.30
171 3.72
118 2.51 38.69
112 2.60 41.03
138 3.00 44.09
42 89.36
38 88.37
39 84.78
273 43
4 (2) 6 (3)
I ( I) -4 (4) I (1)
Concentration of calcium in the diet ( % )
138 2.94
305 47
36(20) -1 ( 1) -2 (2) --
I ( 1) -41 (17)
red. oss. = reduced ossification *Absence of caudal ossification. The number o f litters is indicated in parentheses.
Concentration o f calcium in the diet ( % )
Sternebral variations* Total no. 137 M e a n no./litter 3.11 Foetuses with one or more sternebral variations Total no. 105 M e a n no./littert 2.39 Foetuses affected ( % ) 41.83 Litters with foetuses with one or more variations Total no. 37 Litters affected ( % ) 84.09 No. o f foetuses examined 251 No. o f litters examined 44
1 (1) -41 (21)
313 46
*Statistical analysis was not performed on this parameter. tStatistical analysis was performed on these data after application of the F r e e m a n - T a k e y arcsine transformation.
Parameter
0.50
Skeletal variations* Total no. 93 Mean no./litter 2.11 Foetuses with one or more skeletal variationst Total no. 69 Mean no#litter 1.57 Foetuses affected ( % ) 27.49 Litters with foetuses with one or more skeletal variations Total no. 31 Litters affected ( % ) 70.45 No. o f foetuses examined 251 No. o f litters examined 44
0.75
1.00
1.25
88 1.87
68 1.58
77 1.67
72 1.53 23.61
60 1.40 21.98
67 1.46 21.41
32 68.09
32 74.42
30 65.22
305 47
273 43
313 46
*Statistical analysis was not performed on this parameter. "['Statistical analysis was performed on these data after application o f the F r e e m a n - T u k e y arcsine transformation.
Developmental effects of excess dietary calcium Table I0. Incidence of specific soft-tissue variations in foetuses of rats given excess dietary calcium Concentration o f calcium in the diet (%)
Parameter
0,50
0.75
1.00
1.25
No. of foetuses (litters)
examined
257 (43) 307 (47) 267 (42) 313 (46) 5 (5) 5 (2) -3 (2) Haemorrhages, internal 2 (2) 1 (1) -2 (2) Kidney, ectopic ---1 (1) Hydro-ureter, moderate 41 (25) 51 (25) 38 (20) 37 (20) Hydro-ureter, severe 6 (5) 9 (8) 11 (8) 10 (6) Enlarged renal pelvis, moderate -2 (2) 1 (1) 3 (1) Enlarged renal pelvis, severe --1 (1) -Enlarged uretal kidney, moderate 15 (12) 27 (17) 13 (11) 18 (9) Enlarged uretal kidney, severe 2 (2) 1 (1) 5 (4) 3 (2) Thyroid, brown -1 (1) --Uterus, swollen ---1 (I) Hydrocephalus
The n u m b e r of litters is indicated in parentheses.
average number of foetuses with at least one variation was lower in the 1.00 and 1.25% groups, the ANOVA did not indicate a significant decrease compared with the controls. Although there was a dose-dependent decrease in the proportion of litters with foetuses with at least one variation, Fisher's exact test (two-tailed) did not show any significant difference. Thus, the proportion of litters with foetuses with at least one, two or three soft-tissue variations was similar in all groups. DISCUSSION
The 1984 NIH Consensus Development Conference Panel on Osteoporosis (1986) recommended increases in the daily calcium consumption that represented an approximate 1.2-fold increase above the 1980 RDA. However, there is inadequate information on the reproductive and developmental effects of moderate increases in dietary calcium. This study was designed to evaluate the effects of 1.5-, 2.0- and Table 11. Incidence of soft-tissue variations in foetuses of rats given excess dietary calcium Concentration of calcium in the diet (%) Parameter
0.50
Soft-tissue variations* Total no. 71 Mean no./litter 1.65 Foetuses with one or more soft-tissue variationst Total no. 57 Mean no./litter 1.33 Foetuses affected (%) 22.18 Litters with foetuses with one or more variations Total no. 29 Litters affected (%) 67.44 No. of foetuses examined 257 No. of litters examined 43
0.75
1.00
1.25
97 2.06
69 1.64
78 1.70
68 1.45 22.15
49 1.17 18.35
52 I. 13 16.61
31 65.96
23 54.76
22 47.83
307 47
267 42
313 46
*Statistical analysis was not performed on this parameter. $Statistical analysis was performed on these data after application of the F r c e m a n - T u k e y arcsine transformation.
959
2.5-fold increases in dietary calcium on reproductive performance and foetal development in rats fed diets that were nutritionally adequate. The differences between our results and those obtained in the study by Lai et al. (1984) can be attributed to dietary composition. Rogers et al. (1985) studied the sensitivity of Long-Evans hooded rats to maternal zinc deficiency during pregnancy, using purified diets. The source of protein was egg white, and the levels of zinc were I00 mg/kg diet for the control group and 9.0, 4.5 or 0.5 mg/kg diet for the treatment groups. Long-Evans dams fed 0.5, 4.5 or 9.0 mg zinc/kg diet ate less than did animals fed 100 mg zinc/kg diet during the last 3 days of pregnancy. Rats fed 0.5 or 4.5 mg zinc/kg diet gained significantly less weight than did those fed 9.0 or 100mg zinc/kg diet; weight gains did not differ between rats fed 9.0 mg zinc/kg diet and those fed 100 mg zinc/kg diet. The decreases in feed consumption and body weight gain and the increased hypophagia near term observed in the study by Lai et al. (1984) are consistent with reduced zinc bioavailabilty, which was probably caused by the phytate-zinc-calcium interaction rather than by the increase in dietary calcium (Davies and Olpin, 1979). The absence of a similar effect in the present study can be attributed to the use of casein as the protein source and a level of zinc reported to be adequate for gestation according to the American Institute of Nutrition (1977 and 1980). One useful approach to the study of the reproductive effects of excess dietary calcium is to induce hypercalcaemia during pregnancy and/or lactation by feeding rodents excess calcium in the diet and drinking water. This technique was introduced by Fairney and Weir (1970), who produced hypercalcaemic female rats by feeding them 3% calcium as calcium carbonate in powdered Oxoid Breeding Diet and 4 g calcium lactate per 100 ml drinking (tap) water. The hypercalcaemic parental female rats had litters that were small, weighed less than normal at birth and grew poorly. Liebgott and Srebrolow (1989) studied the effects of experimentally induced maternal hypercalcaemia on foetal development in CDI mice. The method of inducing hypercalcaemia was similar to that used by Fairney and Weir (1970), except that calcium carbonate was added to Purina Rodent Chow 500, which contains excess nutrients. The excess calcium was given during gestation, and caesarean sections were performed on day 18. Foetal skeletons were doublestained for cartilage and bone with alcian blue and Alizarin Red S. When the data were analysed per litter, the mean number of resorptions and foetal deaths were higher than those of the control group, but the differences were not significant. Excessively high calcium intake in mice during gestation significantly decreased foetal weight and retarded foetal skeletal and dental development. No gross abnormalities were observed in the control or experimental
960
M.E. SHACKELFORDet al.
groups. The authors recommended that excessive calcium intake should be avoided during pregnancy to prevent hypercalcaemia and that plasma calcium should be monitored during pregnancy. Unlike Fairney and Weir (1970) and Liebgott and Srebrolow (1989), in the present study moderate increases in dietary calcium were used to examine the developmental effects of this element. The concentrations used in our study resemble more closely the increases recommended by the 1984 N I H Consensus Development Conference Panel on Osteoporosis (1986). Plasma calcium levels were not measured, but were not expected to increase with the levels of dietary calcium used. Liebgott and Srebrolow (1989) hypothesized that the observed delayed skeletal and dental calcification might have been due to decreased foetal parathyroid hormone levels and vitamin D metabolites subsequent to the maternal hypercalcaemia. Under the dietary conditions of the present study, there was no observed dose-related effect on the average number of implantations, resorptions and viable foetuses, or on the length and weight of the foetuses. N o effects on foetal visceral and skeletal development were seen. O f what relevance are the studies with excess dietary calcium to human pregnancy? The first study on the ingestion of large amounts of calcium carbonate and calcium-containing food during pregnancy was reported by Ullian and Linas (1988). The patient had the milk-alkali syndrome, which is the cause of hypercalcaemia in ulcer patients who consume large amounts of milk and antacid products (Federal Register, 1979). A patient was hospitalized in wk 23 of pregnancy with complications of hypercalcaemia, dehydration, renal insufficiency and pancreatitis. At wk 37 of gestation, a stillborn foetus was delivered. The foetus had short limbs, low ears and normal chromosome analysis. According to the authors, the relationship between M A S and congenital abnormalities was not apparent. The present study used nutritionally adequate diets to study the effects of supplemental dietary calcium on maintenance of pregnancy and foetal development. This study did not address issues raised by Lai et al. (1984) and Richards and Greig (1952) concerning the reproductive and developmental effects of excess dietary calcium and dietary deficiencies of zinc and iron. At the concentrations used here, dietary calcium was neither foetotoxic nor teratogenic. Acknowledgements--We express our appreciation to George Gray, Randolph Jackson, Robert Jones, Dorothy Quander, James Rorie and Michael Scott for their excellent technical support. We thank the Technical Editing Branch, Food and Drug Administration, for assistance in preparing the manuscript. REFERENCES
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