Fluorine Deposition in Bone as Related to Physiological State1 J. NORBERTO MICHEL,2 J. W. SUTTIE, 3 and M. L. SUNDE2'4 University of Wisconsin-Madison, Madison, Wisconsin 53706 (Received for publication July 14, 1983)
1984 Poultry Science 63:1407-1411 INTRODUCTION
Approximately 99% of the fluorine (F) retained in the normal body is stored in the bone. The sequestering of F by the skeleton is influenced by 1) previous F exposure, 2) skeletal F concentration, and 3) age of the individual (National Research Council, 1974). The F deposition in the bone is a function of age as well as level of dietary F (Pool et al., 1965). Many suggestions have been made in the literature to explain the relationship of high F diets to retarded growth of chickens. Phillips et al. (1935) reported that F caused a growth inhibition by the restriction of feed consumption. Intraperitoneal injection of F also restricted feed consumption. The restriction of
1
Research supported by the Government of Mexico through a grant to the senior author and by the College of Agriculture and Life Sciences, University of Wisconsin-Madison, Madison, WI 53706. 2 Department of Poultry Science. 3 Department of Biochemistry. "To whom correspondence should be addressed.
feed intake by both methods indicated that the action of fluoride was systemic in nature and independent of any action in the digestive tract. It has been known that the bones of old hens contain more fluoride than those of old stags, but the reasons for this have not been determined (Murphy et al., 1979). Almost all experiments on F have been done on a short-time basis and using birds that have been grown on a diet different from the one being tested. A 2-year experiment was completed in which day-old chicks were grown on two dietary F levels to observe the effects of time on bone fluoride deposition, which could be obscured by starting with partially or fullgrown animals. A second experiment was then done to try to determine whether purely sex differences or physiological factors associated with egg production were responsible for the difference in bone F deposition. MATERIALS AND METHODS
Two hundred day-old New Hampshire X Single Comb White Leghorn unsexed chicks
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ABSTRACT Chicks were fed either a corn-soy basal or the same diet with 252 ppm added fluorine (F) from raw rock phosphate. These diets were fed continuously from hatching to 104 weeks of age to both males and females. Birds were sacrificed at 4, 13, 27, 39, 52, 78, and 104 weeks of age, and their tibiae were analyzed for F on an ash basis. At 4 and 13 weeks there were no significant differences in bone fluoride between males and females fed either the basal diet or between the sexes of those fed the added F. However, there were 3 to 10 times greater levels of bone fluoride in the birds receiving added F. At 27 weeks of age, males and females had bone contents of 767 ppm F and 1,066 ppm F, respectively, when fed the basal diet. These values for the F-added groups were 7,000 and 7,900, respectively. The values at 39 weeks of age were 904, 1,530, 8,000 and 9,187, respectively, and for 52 weeks 1,055, 1,906, 8,675, and 9,125, respectively. The values at 78 weeks of age were 1,487, 3,057, 10,300, and 11,975, respectively, and for 104 weeks 1,251, 3,550, 10,895, and 12,454, respectively. This suggests that females concentrate the F in the bones after sexual maturity. The experimental procedure described was also conducted with restricted ovulator birds, but they were sacrificed at 52 weeks of age. This experiment was conducted to determine if the sex or actual egg production was the important factor. The F content of the tibiae were 972, 814, 6,800, and 5,500 ppm F for the male and female without and with F, respectively. These results showed that there is no difference between males and sexually inactive females in bone F deposition but that physiological factors associated with egg production are responsible for the increased deposition. (Key words: fluorine, tibiae, sex difference, restricted ovulator)
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MICHEL ET AL.
just 3 to 5 small, odd eggs, but otherwise they show all the physical appearances of a normal layer. They also have an extremely high level of lipids in their blood. Because the RO birds do not lay eggs, this would help us determine if the physiological factors associated with egg production were responsible for the difference in bone fluoride. At 7 weeks, the birds were culled and 10 males and 10 females were retained for each group. At 19 weeks, the females were placed into individual laying cages to determine the true RO birds. At 52 weeks, 5 males and 5 true RO females from each group were sacrificed, their tibiae removed, and the procedure followed as previously described. Data were subjected to analysis of variance and statistical significance between means (BMDP, Statistical Software Department of Biomathematics, 1981). RESULTS AND DISCUSSION
The average feed consumption during the first 4 weeks of age was slightly higher for the birds fed the added F than those on the control
TABLE 1. Experimental
diets + F
Control Ingredients
0—8 w e e k s
0—8 weeks
8—104 weeks
8 - 1 0 4 weeks
(g/kg) Ground corn Ground oats Wheat middlings Alfalfa meal (17% CP') Meat scraps (50% CP 1 ) Soybean meal (44% CP 1 ) Ground limestone Dicalcium phosphate Raw rock phosphate Iodized salt Vitamin-mineral premix 2
530 100 20 40 280 10 10
570 70 100 40 190 10 10
5 5
5 5
20.9 1728 1.12 .80 27
16.5 2791 .73 .58 27
530 100 20 40 280 10 5 7 5 5
570 70 100 40 190 10 5 7 5 5
Calculated analysis Percent protein Metabolizable energy, kcal/kg Percent calcium Percent phosphorus Total fluorine, ppm 1 2
20.9 2728 1.12 .81 280
16.5 2792 .73 .59 280
Crude protein.
Premix contained 2200 IU vitamin A, 881 ICU vitamin D 3 , 8 mg riboflavin, .01 mg, vitamin B 1 2 , 136 mg manganese oxide, 450 mg DL-methionine, 18 mg. Procaine penicillin per kilogram of diet. Wheat middlings were added to make 5 g.
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were fed two corn-soy diets, one of which had 252 ppm F added in the form of raw rock phosphate; calcium and phosphorus levels were kept constant (Table 1). Birds were wing-banded and reared in battery brooders with raised wire floors. Feed and water were supplied ad libitum. Birds were moved to larger cages and separated by sex at 7 weeks. The diets were modified at 8 weeks (Table 1). Body weight and feed consumption were determined for the different periods (Table 2, Figs. 1 and 2). Oyster shell was offered ad libitum to the females after 20 weeks of age. Five males and five females from each group were killed at 4, 13, 27, 39, 52, 78, and 104 weeks of age and their tibiae removed, cleaned of all flesh, and one of them prepared for F analysis using the methods of Cralley et al. (1969) and Fry and Taves (1970). At the same time, 75 day-old Leghorn-type strain-cross restricted ovulatory (RO) (Toda et al., 1980) chicks were separated in two groups. Each group was fed either one of the two diets previously described (Table 1). The RO defines a heterozygous gene. True RO females lay no eggs throughout their life, or
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FLUORIDE LEVELS IN THE BONE TABLE 2. Average daily feed consumption per period Period
Control
+F
(g/day/bird) —
(weeks) 0-4 4-8 8-13 13 - 19 1 9 - 27 2 7 - 39 3 9 - 52 52 - 78 78 - 104
24.4 48.7 62.7 79.7 80.4 91.8 88.7 96.7 93.5
2.5 2.4
24.2 23.8
Average fluorine/day (mg) Males Females
1 Mean values within a row are not significantly different (P<.05).
diet (Table 2). After 8 weeks, feed consumption for the birds on the control diet was slightly higher than for those on the treatment diet, and this continued for most of the experiment, but the values were not significantly different (P>.05). Males consumed about 13% more feed than females during the period
*
• • • •
CONTROL NORMAL +F NORMAL CONTROL R.O. +F R.C.
4 13 27 39 52
78
WEEKS OF AGE FIG. 1. Average body weight of males.
104
• CONTROL NORMAL • +F NORMAL • CONTROL R.O. • +F R.O.
4 13 27 39 52
78
WEEKS OF AGE FIG. 2. Average body weight of females.
104
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21.4 1 47.6 64.7 82.6 92.0 94.4 92.5 99.5 88.3
preceding sexual maturity, which would explain the slightly higher F content of their bones as compared to those of the females during this period (Table 3). Males consumed an average of 2.45 mg F/day and females 2.42 mg F/day on the basal diet, and on the treatment diets the consumptions were 24.2 and 23.8 mg F/day for males and females, respectively. The level of F in the bone appears to be directly related to the total F consumed per day. The length of time for which this consumption continues also influences the total bone F. Males and females were fed from the same container. Therefore, feed consumption data were pooled for analysis. Hens eat approximately the same amount of feed as males of a similar age (Adkins et al., 1957). In fact, Vo et al. (1978) also reported that at 32 weeks in two trials at 21.1 C the two sexes ate approximately the same amount of feed per week. The rate of growth was not affected by the addition of 252 ppm F, regardless of the sex. The body weights of males as well as of females were very similar for both diets throughout the experiment (Figs. 1 and 2); at 104 weeks the control males had a higher, but not significantly (P>.05), different body weight than the Fsupplemented birds. These weights are typical for males of this cross at this stage. There was a highly significant (P<.01) difference between the F content of the bones of birds on the control diet and those on the treatment diet regardless of the sex. Birds fed the added F (Figure 3) had a F content in the
MICHEL ET AL.
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TABLE 3. Fluorine deposition in bone tissue (ppm, ash basis) Restricted ovulator
Normal
Control males Control females + Fluorine males + Fluorine females 1
52 weeks
4 weeks
13 weeks
52 weeks
702 ± 38' 705 ± 56 6008 ± 1026 5087 ± 1448
616± 97 580+ 71 6450± 1632 5575 ± 1107
1055 ± 112 1906 ± 529 8675 ± 1523 9125 ± 3371
972 ± 49 814± 171 6800 ± 277 5500±1591
Standard deviation.
relates closely to bone F unless the birds are laying eggs. There was a significant difference (P<.002) between normal and RO females in their F content of the bone for both diets, which shows that the physiological factors associated to egg production account for the difference in the F level of the bone of normal adult males and females (Table 3). The reason that the level of F increased in the bones of laying females was not determined. The RO females had approximately the same levels of F in the bone as males. It could be postulated that the greater F level found in the bones of females than in those of males was caused by the more active mineral metabolism in bones of females producing eggs. When calcium is removed from the bones for eggshell
12000 • 11000 • 10000 • 9000 • 8000 7000 • 6000 • 5000 - mr
/\ 1000 •
•--•"
,-• /
, * • " '
•---•'.
-'
*'.*'' ,'-•' %
•
•
•
•
-•
•
m
CONTROL MALES CONTROL FEMALES +F MALES +F FEMALES
3000 . 2000 • 1000 • f 13 27 39 52
78
101
FIG. 3. Fluorine deposition in bone tissue.
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tibia 3 to 10 times that of birds on the basal diet. This agrees with the data of Pool et al. (1965), which showed that the F content of the bone is also a function of dietary fluoride. As the birds grew older the F content of their bones increased regardless of the sex, which shows that the bone F content is also a function of age and, as shown later, egg shell formation. There were no significant differences (P> .05) in the F content of the bones between males and females, regardless of the diet, up to 13 weeks of age. However, a significant difference (P<.05) was observed in the bone F content between males and females in both diets after egg production started (Fig. 3). As birds grew older the F in the bone continued to rise for both diets and for both sexes. The F deposition curves (Fig. 3) are greatly different, but the slopes for both males and females are similar for the two levels of F. For the RO birds, there was no significant difference (P>.05) in growth rates between chickens fed the two diets, and there was a significant (P<.05) effect of the two diets on the F content of the tibia (Table 3, Figs. 1 and 2). There was no significant difference (P>.05) in bone F content between males and females within each diet; in fact, in each group the bones from the females had a lower F content than the males (Table 3). The RO females at 52 weeks showed values for bone F somewhat lower (Table 3) than the RO males, which could be due to a lower feed consumption by the females. Although these values were not significantly different they were consistent for the control diet and the fluoride treatment. Because the RO females did not lay eggs, their feed consumption was less than for those hens laying eggs. Therefore, again, fluoride intake
FLUORIDE LEVELS IN THE BONE
ACKNOWLEDGMENTS We wish to express our deepest gratitude to Arnold B. Clay for his invaluable help in conducting the fluorine analyses.
REFERENCES Adkins, J. S., H. R. Bird, and M. L. Sunde, 1957. A study of the feed consumption of breeder males. Poultry Sci. 36:1095-1096.
BMDP Statistical Software Department of Biomathematics, 1981. Univ. California, Los Angeles. Univ. California Press. Cralley, L. V., L. V. Haff, A. W. Hook, E. J. Schneider, J. D. Strauther, C. R. Thompson, and L. H. Weinstein, 1969. Tentative method of analysis for fluoride content of the atmosphere and plant tissues (semi-automated). Health Lab. Sci. 6:84-101. Fry, B. W., and D. R. Taves, 1970. Serum fluoride analysis with the fluoride electrode. J. Lab. Clin. Med. 75:1020-1025. Murphy, E. W., C. R. Brewington, B. W. Willis, and M. A. Nelson, 1979. Health and Safety Aspects of the Use of Mechanically Deboned Poultry. Food Safety Qual. Serv. US Dept. Agric., Washington, DC. National Research Council, 1974. Effects of Fluorides in Animals. 4th ed. Natl. Acad. Sci., Washington, DC. Phillips, P. H., H. E. English, and E. B. Hart, 1935. Augmentation of the toxicity of fluorosis in chicks by feeding dessicated thyroid. J. Nutr. 10:399-407. Pool, M. F., W. J. Tango, and A. A. Klose. 1965. The fluoride content of commercial broiler backs and necks. Poultry Sci. 44:1545-1550. Toda, T., D. Leszczynski, W. H. McGibbon, and F. A. Kummerow, 1980. Coronary arterial lesions in sexually mature non-layers, layers, and roosters. Virchows Arch. A. Path. Anat. Histol. 3 8 8 : 1 2 3 135. Vo, K. V., M. A. Boone, and W. E. Johnston, 1978. Effect of three lifetime ambient temperatures on growth, feed and water consumption and various blood components in male and female Leghorn chickens. Poultry Sci. 57:798-803.
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formation, absorption of calcium increases and an increased F absorption may accompany this. Probably F moves with calcium into the bone, but when calcium is needed for eggshell formation F does not come out of the bone but becomes more concentrated. The data show that the physiological factors associated with egg production may be the primary factor causing the increase in F in the bones. When a hen deposits calcium in the bone, F is incorporated in the same manner. The input and output of calcium is a daily occurrence over the period of months of egg production. Apparently, the calcium is removed efficiently but portions of the F remain and a concentration of F occurs over a period of months. The reason that this occurs is not known at the present time.
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