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Toxicity of a Vitamin D Steroid to Laying Hens1
ENVIRONMENT AND HEALTH Toxicity of a Vitamin D Steroid to Laying Hens1 J. H. SOARES, JR., 2 D. M. KAETZEL, 3 J. T. ALLEN, and M. R. SWERDEL4 Department of Poultry Science, University of Maryland, College Park, Maryland 20742 (Received for publication February 8, 1982)
198"! Poultry Science 6 2 : 2 4 - 2 9 steroids caused laying hens t o p r o d u c e egg shells of significantly altered quality. In addition, data were sought using Single C o m b White Leghorn (SCWL) hens t h a t corroborated previous observations (Kaetzel and Soares, 1979) with quail t h a t indicated there was a relatively low t o x i c level for l a - O H - D 3 .
INTRODUCTION A continuing concern of t h e p o u l t r y industry is t h e high incidence (6 t o 12%) of egg losses in t h e laying house due t o p o o r egg shell quality (Cox, 1 9 6 7 ; R o l a n d , 1 9 7 6 ) . Often these losses are enhanced b y increasing age and h o t environmental temperatures. Because calcium homeostasis is a key factor in egg shell f o r m a t i o n and vitamin D is a major c o n t r o l p o i n t for calcification, this research is concerned w i t h t h e role of vitamin D 3 , its m e t a b o l i t e s , and its h o r m o n e form in egg and egg shell p r o d u c t i o n . In an earlier r e p o r t McLoughlin and Soares ( 1 9 7 6 ) found improved egg shell quality in aged hens w h e n 25 hydroxycholecalciferol (25-OHD 3 ) was fed with oyster shell and, t o a lesser e x t e n t , limestone. Dose level of t h e vitamin D ; metabolites has proven t o be a critical factor, because s o m e researchers (Roland and Harms, 1 9 7 6 ; Waldroup, 1977) have n o t been able to o b t a i n similar results even w h e n m u c h higher levels of these steroids were fed. T h e objectives of t h e following e x p e r i m e n t s were t o d e t e r m i n e if feeding several levels of vitamin D
MATERIALS AND METHODS Experiment 1. In this e x p e r i m e n t , 56-weekold SCWL hens were selected for a 22 week, summer-fall s t u d y . T e n hens were allocated t o each t r e a t m e n t and h o u s e d in individual cages. Crystalline vitamin D 3 , 2 5 - O H - D 3 , and t h e vitamin D h o r m o n e analogue la-hydroxycholecalciferol ( l a - O H - D 3 ) were added t o a low vitamin D corn-soybean m e a l basal (Table 1) such t h a t diets contained one of these vitamin D 3 steroids at a level equivalent t o 6 0 0 IU D 3 / k g diet. This a m o u n t e d t o 15, 6, and 3.41 /ig/kg diet of cholecalciferol ( D 3 ) , 25-OHD 3 , and l a - O H - D 3 , respectively. After 12 weeks on these diets each t r e a t m e n t group was further divided into t w o groups (5 hens each). Five of t h e birds remained o n t h e respective diets described and t h e o t h e r 5 were given diets containing twice as m u c h of t h e particular steroid for 10 additional weeks.
'Scientific Article No. A-3103 Contribution No. 6168 of the Maryland Agriculture Experiment Station (Department of Poultry Science). 2 Reprint requests. 3 Veterans Administration Hospital, Cleveland, OH. 4 Department of Nutrition and Food Science, Rutgers University, Newark, NJ.
Egg shell thickness, specific gravity (using salt solutions), and breaking strength, based o n all eggs laid in a 3-day period, as well as egg p r o d u c t i o n and feed c o n s u m p t i o n , were determ i n e d weekly. In addition, t o e ash, serum
24
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ABSTRACT Two experiments were conducted with 56-week-old or 104-week-old Leghorn hens to determine if feeding vitamin D steroids in excess of requirement levels caused any marked affects on eggshell quality. In_the first experiment caged hens had reduced feed consumption, egg shelLqualitv. and egg production as early as 6 weeks after initially_cj)nsuming_a basaldiet supplemented with 6.8 Mg la-hydroxvcholecalciferol (la-5H-D,)/kg. The second experiment confirmed the previous results and showed that extensive weight loss occurred with continued feeding of 10 or 15 Mg la-OH-D,/kg diet. No adverse affects were observed in either experiment when the level of la-OH-D 3 supplementation was 5.0jig/kg diet or less. No toxic effects were observed when the hormone precurser 25-OH-D3 was supplemented to diets at 6 or 12 Mg/kg. It is suggested that the pathological effects observed are related to the potent calcium homeostatic properties of laOH-D3 that at elevated levels may cause aberrations in circulating calcium. (Key words: vitamin D steroids, toxicity, hens) ^
VITAMIN D STEROID TOXICITY TABLE 1. Laying hen diet for Vitamin D steroid studies
(%) Corn, yellow Soybean meal (49%) Limestone Defluorinated phosphate Corn oil NaCl Vitamin premix 1 Mineral mix' DL-Methionine 1
70.618 19.070 6.500 2.270 .900 .500 .050 .050 .042
As described by Soares et al, 1978.
was similar t o t h a t used in E x p e r i m e n t 1. Egg shell quality (breaking strength, specific gravity, and p e r c e n t shell), were d e t e r m i n e d for all hens o n 3 consecutive days every 4 w e e k s . Plasma Ca, P, and l , 2 5 ( O H ) 2 D 3 and tibia ash m e a s u r e m e n t s were also m a d e . T o t a l plasma Ca and P were d e t e r m i n e d before t h e e x p e r i m e n t began a n d u p o n t e r m i n a t i o n of t h e e x p e r i m e n t . However, l , 2 5 - ( O H ) 2 D 3 and b o n e ash were d e t e r m i n e d only on t h e last day of t h e experim e n t . Egg shell quality m e a s u r e m e n t s were d e t e r m i n e d as described b y Kaetzel and Soares ( 1 9 7 9 ) . Plasma calcium was d e t e r m i n e d b y a t o m i c absorption spectrophotometry and serum p h o s p h o r u s analysis was b y t h e phosp h o m o l y b d i c acid m e t h o d modified for a u t o analysis ( A n o n y m o u s , 1 9 6 4 ) . Plasma 1,25( O H ) 2 D 3 was d e t e r m i n e d b y t h e m e t h o d s described by L a m b e r t et al. ( 1 9 7 9 ) , Hollis et al. ( 1 9 8 1 ) , and Eisman et al. ( 1 9 7 6 ) . Tibias were removed after sacrifice, cleaned of all flesh, dried in a forced air oven overnight a t 9 0 C, and t h e n ashed in a muffle furnace a t 5 3 0 C for 2 0 hr. All d a t a were subjected t o an analysis of variance according t o the p r o c e d u r e s of Snedecor and Cochran ( 1 9 6 7 ) .
TABLE 2. Egg shell quality of hens fed D3,, 25-OH-D3, or la-OH-D3forup
Treatment
Specific gravity
Breaking strength (kg)
to 22 weeks (Experiment 1) Eggshell thickness (mm)
Phase I 15' 25-OH-D3 40-Q la-OH-D 3 3.41
1.077
± .007 2
1.17 ± .24
.33 ± .03
1.075
± .007
1.06 ± .20
.31 ± .03
1.076
± .007
1.20 ± .16
.33 ± .02
Phase:II 15 30 25-OH-D3 6 12 la-OH-D 3 3.41 6.82
1.079 a ± .003 1.074 a ± .005
1.43a ± .35 1.56 a + .26
,39a + .07 ,36a ± .01
1.077a + .005 1.076 a ± .006
1.52 a + .06 1.48 a + .18
,35a ± .06 ,39 a ± .01
1.075a + .057 1.067b ± .005
1.37a ± .15 1.12 b ± .25
,35a ± .06 27b + .04
a ' b Means bearing different superscripts are significantly different (P<.05). 1
Micrograms per kilogram of diet.
2
Mean ± SE.
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calcium, and serum p h o s p h o r u s were m e a s u r e d in all h e n s a t t h e end of t h e e x p e r i m e n t (for m e t h o d s see E x p e r i m e n t 2 ) . Experiment 2. Two-year-old SCWL hens of a commercial strain were fed four dietary treatm e n t s for 8 weeks in t h e fall of t h e year. Each diet was fed t o 8 individually fed and caged hens. T h e e x p e r i m e n t a l diets were t h e basal s u p p l e m e n t e d with 15 /ig D 3 / k g (control) 5, 10, or 15 £tg l a - O H - D 3 / k g diet. T h e basal diet
25
SOARES, JR., ETAL.
26 RESULTS AND DISCUSSION
T h e results of E x p e r i m e n t 1 show t h a t during t h e first 12 weeks of feeding, n o differences in egg shell quality occurred b e t w e e n t h e three g r o u p s (Table 2).
Experiment 2. T h e results of b o n e calcification m e a s u r e m e n t s (Table 5) s h o w little or n o differences b e t w e e n t h e various t r e a t m e n t groups. With t h e possible e x c e p t i o n of t h e 5 Mg/kg level of d i e t a r y i n c o r p o r a t i o n , l a - O H D 3 appears t o have had little or n o short-term depleting effect on skeletal calcium. Egg shell quality d a t a (Table 6) were similar, b u t egg prod u c t i o n was again m a r k e d l y reduced with levels of l a - O H - D 3 s u p p l e m e n t a t i o n above 5 jUg/kg. Blood Ca and P c o n c e n t r a t i o n s (Table 7) det e r m i n e d before and after t h e 8-week experimental period were lower ( P < . 0 5 ) after vitamin D steroid t r e a t m e n t in all groups except those given 5 //g/kg l a - O H - D 3 . A t t h e latter level of s u p p l e m e n t a t i o n , l a - O H - D 3 maintained plasma
TABLE 3. Egg production and average feed consumption of hens fed two levels of three vitamin D3 steroids (Experiment 1, Phase II)
HDP2
Feed consumption (g/day)
.453 ± .097^ .529 ± .068 a
88.3 3 100.5
25-OH-D3 6 12
.486 ± .069 a .436 ± .071*
91.9 90.1
la-OH-D 3 3.41 JL&2_
.598 ± .048 a .281 ± .090 b
83.0 70.9
Treatment
15' 30
a' b Means bearing different superscripts are significantly different (P<.05). ' Micrograms per kilogram diet. 2
Hen-day production.
3
Feed consumption based on total feed consumed minus total weight of feed.
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During t h e second period of this e x p e r i m e n t , however, egg p r o d u c t i o n , feed c o n s u m p t i o n , and egg shell quality began t o decline a r o u n d 6 weeks i n t o t h e t r e a t m e n t period and hens exhibited m a r k e d l y reduced egg p r o d u c t i o n and shell q u a l i t y b y 8 weeks when given l a - O H - D 3 as a vitamin D source. Both vitamin D 3 levels as well as t h e lower levels of 2 5 - O H - D 3 and l a - O H - D 3 s u p p o r t e d equally good p e r f o r m a n c e t h r o u g h o u t t h e e x p e r i m e n t . However, m a r k e d l y poorer egg p r o d u c t i o n and feed c o n s u m p t i o n were observed in those hens given t h e higher level of l a - O H - D 3 (Table 3). These d a t a d e m o n s t r a t e t h e critical nature of t h e level of t h e l a - O H - D 3 c o m p o u n d in t h e diet and m a y explain w h y published results conflict. It is clear t h a t levels of l a - O H - D 3 equivalent t o 1 2 0 0 IU D 3 / k g diet (7.0 Mg/kg of this steroid) result in toxicity. In contrast, vitamin D 3 fed at this level did n o t have any adverse effect on t h e hens. It is possible t h a t t h e greater biological activity of l a - O H - D 3 stimulates intestinal Ca absorption or b o n e resorption so effectively as t o raise b l o o d Ca with coincidental reduced a p p e t i t e . This would b e consistent with t h e t h e o r y t h a t t h e laying hens a p p e t i t e can be modified b y calcium i n t a k e (Mueller et al,
1978) such t h a t high Ca levels reduce a p p e t i t e . It appears t h a t a p p e t i t e was suppressed w h e n l a - O H - D 3 c o n s u m p t i o n is high (Table 3). However, we did n o t d e t e c t elevated b l o o d Ca (Table 4). In addition t o a depression in a p p e t i t e and egg p r o d u c t i o n , egg shell quality fell sharply w h e n 6.82 fig l a - O H - D 3 / k g diet was fed t o laying hens. This can be seen in Table 2 where egg specific gravity, shell breaking strength, and shell thickness are all signifiantly depressed at this level of l a - O H - D 3 s u p p l e m e n t a t i o n . N o t r e a t m e n t effect, however, can b e seen in plasma p h o s p h o r u s and calcium or t o e ash (Table 4), indicating t h a t t h e anorexia is induced before severe drain of b o d y calcium takes place.
VITAMIN D STEROID TOXICITY
27
TABLE 4. Serum calcium and phosphorus of hens fed vitamin D steroids (Experiment I, Phase II) Serum Treatment
P
Ca
Toe ash
(mg/dl)1
(%) 27.4 ± 2.78 31.3 ± 6.63
18.1 ± 1.17 19.4 ± .28
25-OH-D3 6 12
2.2 ± .43 2.7 ± 1.21
34.5 ± 5.21 31.1 ± 19.88
17.3 ± 2.00 18.1 ± 1.08
la-OH-D 3 3.41 6.82
3.1 ± .70 2.1 + .90
33.3 : 7.66 29.2: 17.89
19.3 ± 1.70 18.8 ± 2.02
1
Micrograms per kilogram diet.
TABLE 5. Tibial calcification in hens fed D3 or various levels of 1 a-OH-D3 for 8 weeks (Experiment 2) Tibia parameters Treatment
15' la-OH-D 3 5 10 15 1 2
Ash
Breaking strength
Cortical thickness
(%)
(kg)
(mm)
61.7 ± 3.50 2
7.6 ± 3.47
.90 ± .048
62.8 ±4.02 57.9 ± 3.71 61.8 ±4.60
8.1 ± 1.08 7.0 ± .76 8.8 ± 1.25
.71 ± .049 .83 ± .063 .83 ± .150
Micrograms per kilogram diet. Mean ± SE.
TABLE 6. Egg production and eggshell characteristics of hens fed D j or three levels of la-OH-D3 (Experiment 2)
Treatment
(%)
Egg weight (g)
% Shell
Specific gravity
D3 15 1
47 ± 5.9 a
63.3 ± 1.52
8.7 ± .10
1.077 ± .0009
49 ± 6.6 a 39 ± 8.5* b 17 + 8 . 1 b
64.7 ± 1.15 65.4 ± 2.64 61.2 ± 1.62
8.8 ± .27 8.7 ± .30 9.0+ .38
1.080 ± .0077 1.079 ± .0019 1.075 ± .0031
HDP
la-OH-D 3 5 10 15
1
' Means ± SE bearing different superscripts are significantly different (P<.05). Micrograms per kilogram diet.
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2.4 ± .56 1.8 ± .90
15 30
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SOARES, JR., ETAL. TABLE 7. Serum levels of calcium,1 phosphorus,1 and l,25(OH)2D3 in laying hens given vitamin D steroids (Experiment 2) Initial
(pg/ml)
8 Weeks
Treatment
P
Ca
P
Ca
l,25-(OH) 2 D 3
D3 15 2
3.2 ± .12
29.2 ± 2.44 a
3.1 + .21
22.8 ± 2 . 7 1 b
308 ± 62.6
la-OH-D 3 5 10 15
3.0 ± .11 3.6 ± .33 3.7 + .35
28.8 ± 3.43 29.3 ± 1.98 a 31.6±2.50a
3.4 ± .23 2.7 ± .27 3.3 ±.33
30.3 ± 1.51 22.8 ± 2.85 b 26.6 ± 2.19 b
263 ± 19.4 278 ± 58.6 250 ± 49.2
a
calcium at n o r m a l levels. These d a t a are consist e n t with o u r observations t h a t ovulation was inhibited w h e n l a - O H - D 3 was fed at 10 or 15 Mg/ kg feed and t h e hens subsequently b e c a m e anorexic, resulting in emaciation. T h e percentage of hens showing severe emaciation u p o n postm o r t e m e x a m i n a t i o n wereO, 0, 1 2 . 5 , and 50 for t h e controls ( D 3 ) and 5, 10, and 15 jug/kg l a OH-D3, respectively. This effect does n o t appear t o be d u e t o gross skeletal changes and m a y be t h e result of neurological changes induced b y s o m e undisclosed a b n o r m a l i t y in calcium homeostasis. However, because only gross observations of k i d n e y and o t h e r organs were undert a k e n , n o definitive s t a t e m e n t t o this effect can b e m a d e at this t i m e . It appears t h a t t h e order of events is such t h a t appetite is reduced and, c o n s e q u e n t l y , egg p r o d u c t i o n stops, b u t egg shell quality m a y or m a y n o t be significantly affected. Plasma l , 2 5 ( O H ) 2 - D 3 levels (Table 7) were n o t different a m o n g t h e four t r e a t m e n t groups. This was true even t h o u g h t h e 10 and 15 £tg/kg l a - O H - D 3 fed groups contained m o r e nonlayers. It remains t o be d e t e r m i n e d w h e t h e r or n o t a t r u e elevation of plasma l , 2 5 ( O H ) 2 D 3 occurs early o n before egg p r o d u c t i o n , feed c o n s u m p tion and weight gains are decreased. F u r t h e r investigations are needed t o ascertain if transit o r y changes in plasma associated with high l a O H - D 3 c o n s u m p t i o n cause a t o x i c effect t o laying hens. ACKNOWLEDGMENTS T h e a u t h o r s wish t o t h a n k HoffmannL a R o c h e , Inc., N u t l e y , and T h e U p j o h n Co., K a l a m o z o o , MI, for t h e gifts of 2 5 - O H - D 3 and l a - O H - D 3 , respectively.
REFERENCES Anonymous, 1964. Technicon Auto Analyzer Methodology N-4c-Inorganic Phosphate (Modified from Fiske and SubbaRow, 1925. J. Biol. Chem. 66:375.) Techicon Instruments Corp. Tarrytown, NY. Cox, J., 1967. How much is egg breakage costing you? Poultry Trib. 10:46. Eisman, J. A., A. J. Hamstra, B. E. Kream, and H. F. DeLuca, 1976. A sensitive, precise and convenient method for determination of 1,25-dihydroxyvitamin D in human plasma. Arch. Biochem. Biophys. 176:235-243. Hollis, B. W., B. A. Roos, H. H. Draper, and P. W. Lambert, 1981. Vitamin D in human and bovine milk. J. Nutr. 111:1240-1248. Kaetzel, D. M., Jr., and J. H. Soares, Jr., 1979. Effects of cholecalciferol steroids on bone and egg shell calcification in Japanese quail. J. Nutr. 109:1601-1608. Lambert, P. W., D. O. Toft, S. F. Hodgson, E. A. Lindmark, B. J. Witrak, and B. A. Roos, 1979. An improved method for the measurement of l,25-(OH) 2 D 3 in human plasma. Endocrinol. Res. Comm. 5:293-310. Mueller, W. S., A. Pro-Martinex, I. G. Joshua, and B. Lobaugh, 1978. Calcium appetite in chickens. Pages 82—87 in Proc. Maryland Nutr. Conf. McLoughlin, C. P., and J. H. Soares, Jr., 1976. A study of the effects of 25-hydroxycholecalciferol and calcium source on egg shell quality. Poultry Sci. 55:1400-1410. Roland, D. A., Sr., 1976. Recent advances in egg shell quality. Pages 19—22 in Proc. Maryland Nutr. Conf. Roland, D. A., Sr., and R. H. Harms, 1976. The lack of response of 25-hydroxyvitamin D 3 on egg shell quality or other criteria in laying hens. Poultry Sci. 44:1983-1985. Snedecor, G. W., and W. G. Cochran, 1967. Analysis of variance. Pages 273-275 in Statistical Methods. 6th ed. Iowa State Univ. Press, Ames, IA. Soares, J. H. Jr., M. R. Swerdel, and E. H. Bossard,
Downloaded from http://ps.oxfordjournals.org/ at East Carolina University on June 28, 2015
' Means within the same line bearing different superscripts are significantly different (P<.05). Milligrams per decaliter. 2 Micrograms per kilogram of diet. 1
VITAMIN D STEROID TOXICITY 1978. Vitamin D metabolites and phosphorus utilization. Poultry Sci. 57:1305—1312. Waldroup, P. W., W. C. Bussell, and A. R. Cobb, 1977.
29
From Leidahl R. 1977 Test: No thicker shells with new vitamin D methobolites. Feedstuffs 49(40): 15.
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198"! Poultry Science 6 2 : 2 4 - 2 9 steroids caused laying hens t o p r o d u c e egg shells of significantly altered quality. In addition, data were sought using Single C o m b White Leghorn (SCWL) hens t h a t corroborated previous observations (Kaetzel and Soares, 1979) with quail t h a t indicated there was a relatively low t o x i c level for l a - O H - D 3 .
INTRODUCTION A continuing concern of t h e p o u l t r y industry is t h e high incidence (6 t o 12%) of egg losses in t h e laying house due t o p o o r egg shell quality (Cox, 1 9 6 7 ; R o l a n d , 1 9 7 6 ) . Often these losses are enhanced b y increasing age and h o t environmental temperatures. Because calcium homeostasis is a key factor in egg shell f o r m a t i o n and vitamin D is a major c o n t r o l p o i n t for calcification, this research is concerned w i t h t h e role of vitamin D 3 , its m e t a b o l i t e s , and its h o r m o n e form in egg and egg shell p r o d u c t i o n . In an earlier r e p o r t McLoughlin and Soares ( 1 9 7 6 ) found improved egg shell quality in aged hens w h e n 25 hydroxycholecalciferol (25-OHD 3 ) was fed with oyster shell and, t o a lesser e x t e n t , limestone. Dose level of t h e vitamin D ; metabolites has proven t o be a critical factor, because s o m e researchers (Roland and Harms, 1 9 7 6 ; Waldroup, 1977) have n o t been able to o b t a i n similar results even w h e n m u c h higher levels of these steroids were fed. T h e objectives of t h e following e x p e r i m e n t s were t o d e t e r m i n e if feeding several levels of vitamin D
MATERIALS AND METHODS Experiment 1. In this e x p e r i m e n t , 56-weekold SCWL hens were selected for a 22 week, summer-fall s t u d y . T e n hens were allocated t o each t r e a t m e n t and h o u s e d in individual cages. Crystalline vitamin D 3 , 2 5 - O H - D 3 , and t h e vitamin D h o r m o n e analogue la-hydroxycholecalciferol ( l a - O H - D 3 ) were added t o a low vitamin D corn-soybean m e a l basal (Table 1) such t h a t diets contained one of these vitamin D 3 steroids at a level equivalent t o 6 0 0 IU D 3 / k g diet. This a m o u n t e d t o 15, 6, and 3.41 /ig/kg diet of cholecalciferol ( D 3 ) , 25-OHD 3 , and l a - O H - D 3 , respectively. After 12 weeks on these diets each t r e a t m e n t group was further divided into t w o groups (5 hens each). Five of t h e birds remained o n t h e respective diets described and t h e o t h e r 5 were given diets containing twice as m u c h of t h e particular steroid for 10 additional weeks.
'Scientific Article No. A-3103 Contribution No. 6168 of the Maryland Agriculture Experiment Station (Department of Poultry Science). 2 Reprint requests. 3 Veterans Administration Hospital, Cleveland, OH. 4 Department of Nutrition and Food Science, Rutgers University, Newark, NJ.
Egg shell thickness, specific gravity (using salt solutions), and breaking strength, based o n all eggs laid in a 3-day period, as well as egg p r o d u c t i o n and feed c o n s u m p t i o n , were determ i n e d weekly. In addition, t o e ash, serum
24
Downloaded from http://ps.oxfordjournals.org/ at East Carolina University on June 28, 2015
ABSTRACT Two experiments were conducted with 56-week-old or 104-week-old Leghorn hens to determine if feeding vitamin D steroids in excess of requirement levels caused any marked affects on eggshell quality. In_the first experiment caged hens had reduced feed consumption, egg shelLqualitv. and egg production as early as 6 weeks after initially_cj)nsuming_a basaldiet supplemented with 6.8 Mg la-hydroxvcholecalciferol (la-5H-D,)/kg. The second experiment confirmed the previous results and showed that extensive weight loss occurred with continued feeding of 10 or 15 Mg la-OH-D,/kg diet. No adverse affects were observed in either experiment when the level of la-OH-D 3 supplementation was 5.0jig/kg diet or less. No toxic effects were observed when the hormone precurser 25-OH-D3 was supplemented to diets at 6 or 12 Mg/kg. It is suggested that the pathological effects observed are related to the potent calcium homeostatic properties of laOH-D3 that at elevated levels may cause aberrations in circulating calcium. (Key words: vitamin D steroids, toxicity, hens) ^
VITAMIN D STEROID TOXICITY TABLE 1. Laying hen diet for Vitamin D steroid studies
(%) Corn, yellow Soybean meal (49%) Limestone Defluorinated phosphate Corn oil NaCl Vitamin premix 1 Mineral mix' DL-Methionine 1
70.618 19.070 6.500 2.270 .900 .500 .050 .050 .042
As described by Soares et al, 1978.
was similar t o t h a t used in E x p e r i m e n t 1. Egg shell quality (breaking strength, specific gravity, and p e r c e n t shell), were d e t e r m i n e d for all hens o n 3 consecutive days every 4 w e e k s . Plasma Ca, P, and l , 2 5 ( O H ) 2 D 3 and tibia ash m e a s u r e m e n t s were also m a d e . T o t a l plasma Ca and P were d e t e r m i n e d before t h e e x p e r i m e n t began a n d u p o n t e r m i n a t i o n of t h e e x p e r i m e n t . However, l , 2 5 - ( O H ) 2 D 3 and b o n e ash were d e t e r m i n e d only on t h e last day of t h e experim e n t . Egg shell quality m e a s u r e m e n t s were d e t e r m i n e d as described b y Kaetzel and Soares ( 1 9 7 9 ) . Plasma calcium was d e t e r m i n e d b y a t o m i c absorption spectrophotometry and serum p h o s p h o r u s analysis was b y t h e phosp h o m o l y b d i c acid m e t h o d modified for a u t o analysis ( A n o n y m o u s , 1 9 6 4 ) . Plasma 1,25( O H ) 2 D 3 was d e t e r m i n e d b y t h e m e t h o d s described by L a m b e r t et al. ( 1 9 7 9 ) , Hollis et al. ( 1 9 8 1 ) , and Eisman et al. ( 1 9 7 6 ) . Tibias were removed after sacrifice, cleaned of all flesh, dried in a forced air oven overnight a t 9 0 C, and t h e n ashed in a muffle furnace a t 5 3 0 C for 2 0 hr. All d a t a were subjected t o an analysis of variance according t o the p r o c e d u r e s of Snedecor and Cochran ( 1 9 6 7 ) .
TABLE 2. Egg shell quality of hens fed D3,, 25-OH-D3, or la-OH-D3forup
Treatment
Specific gravity
Breaking strength (kg)
to 22 weeks (Experiment 1) Eggshell thickness (mm)
Phase I 15' 25-OH-D3 40-Q la-OH-D 3 3.41
1.077
± .007 2
1.17 ± .24
.33 ± .03
1.075
± .007
1.06 ± .20
.31 ± .03
1.076
± .007
1.20 ± .16
.33 ± .02
Phase:II 15 30 25-OH-D3 6 12 la-OH-D 3 3.41 6.82
1.079 a ± .003 1.074 a ± .005
1.43a ± .35 1.56 a + .26
,39a + .07 ,36a ± .01
1.077a + .005 1.076 a ± .006
1.52 a + .06 1.48 a + .18
,35a ± .06 ,39 a ± .01
1.075a + .057 1.067b ± .005
1.37a ± .15 1.12 b ± .25
,35a ± .06 27b + .04
a ' b Means bearing different superscripts are significantly different (P<.05). 1
Micrograms per kilogram of diet.
2
Mean ± SE.
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calcium, and serum p h o s p h o r u s were m e a s u r e d in all h e n s a t t h e end of t h e e x p e r i m e n t (for m e t h o d s see E x p e r i m e n t 2 ) . Experiment 2. Two-year-old SCWL hens of a commercial strain were fed four dietary treatm e n t s for 8 weeks in t h e fall of t h e year. Each diet was fed t o 8 individually fed and caged hens. T h e e x p e r i m e n t a l diets were t h e basal s u p p l e m e n t e d with 15 /ig D 3 / k g (control) 5, 10, or 15 £tg l a - O H - D 3 / k g diet. T h e basal diet
25
SOARES, JR., ETAL.
26 RESULTS AND DISCUSSION
T h e results of E x p e r i m e n t 1 show t h a t during t h e first 12 weeks of feeding, n o differences in egg shell quality occurred b e t w e e n t h e three g r o u p s (Table 2).
Experiment 2. T h e results of b o n e calcification m e a s u r e m e n t s (Table 5) s h o w little or n o differences b e t w e e n t h e various t r e a t m e n t groups. With t h e possible e x c e p t i o n of t h e 5 Mg/kg level of d i e t a r y i n c o r p o r a t i o n , l a - O H D 3 appears t o have had little or n o short-term depleting effect on skeletal calcium. Egg shell quality d a t a (Table 6) were similar, b u t egg prod u c t i o n was again m a r k e d l y reduced with levels of l a - O H - D 3 s u p p l e m e n t a t i o n above 5 jUg/kg. Blood Ca and P c o n c e n t r a t i o n s (Table 7) det e r m i n e d before and after t h e 8-week experimental period were lower ( P < . 0 5 ) after vitamin D steroid t r e a t m e n t in all groups except those given 5 //g/kg l a - O H - D 3 . A t t h e latter level of s u p p l e m e n t a t i o n , l a - O H - D 3 maintained plasma
TABLE 3. Egg production and average feed consumption of hens fed two levels of three vitamin D3 steroids (Experiment 1, Phase II)
HDP2
Feed consumption (g/day)
.453 ± .097^ .529 ± .068 a
88.3 3 100.5
25-OH-D3 6 12
.486 ± .069 a .436 ± .071*
91.9 90.1
la-OH-D 3 3.41 JL&2_
.598 ± .048 a .281 ± .090 b
83.0 70.9
Treatment
15' 30
a' b Means bearing different superscripts are significantly different (P<.05). ' Micrograms per kilogram diet. 2
Hen-day production.
3
Feed consumption based on total feed consumed minus total weight of feed.
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During t h e second period of this e x p e r i m e n t , however, egg p r o d u c t i o n , feed c o n s u m p t i o n , and egg shell quality began t o decline a r o u n d 6 weeks i n t o t h e t r e a t m e n t period and hens exhibited m a r k e d l y reduced egg p r o d u c t i o n and shell q u a l i t y b y 8 weeks when given l a - O H - D 3 as a vitamin D source. Both vitamin D 3 levels as well as t h e lower levels of 2 5 - O H - D 3 and l a - O H - D 3 s u p p o r t e d equally good p e r f o r m a n c e t h r o u g h o u t t h e e x p e r i m e n t . However, m a r k e d l y poorer egg p r o d u c t i o n and feed c o n s u m p t i o n were observed in those hens given t h e higher level of l a - O H - D 3 (Table 3). These d a t a d e m o n s t r a t e t h e critical nature of t h e level of t h e l a - O H - D 3 c o m p o u n d in t h e diet and m a y explain w h y published results conflict. It is clear t h a t levels of l a - O H - D 3 equivalent t o 1 2 0 0 IU D 3 / k g diet (7.0 Mg/kg of this steroid) result in toxicity. In contrast, vitamin D 3 fed at this level did n o t have any adverse effect on t h e hens. It is possible t h a t t h e greater biological activity of l a - O H - D 3 stimulates intestinal Ca absorption or b o n e resorption so effectively as t o raise b l o o d Ca with coincidental reduced a p p e t i t e . This would b e consistent with t h e t h e o r y t h a t t h e laying hens a p p e t i t e can be modified b y calcium i n t a k e (Mueller et al,
1978) such t h a t high Ca levels reduce a p p e t i t e . It appears t h a t a p p e t i t e was suppressed w h e n l a - O H - D 3 c o n s u m p t i o n is high (Table 3). However, we did n o t d e t e c t elevated b l o o d Ca (Table 4). In addition t o a depression in a p p e t i t e and egg p r o d u c t i o n , egg shell quality fell sharply w h e n 6.82 fig l a - O H - D 3 / k g diet was fed t o laying hens. This can be seen in Table 2 where egg specific gravity, shell breaking strength, and shell thickness are all signifiantly depressed at this level of l a - O H - D 3 s u p p l e m e n t a t i o n . N o t r e a t m e n t effect, however, can b e seen in plasma p h o s p h o r u s and calcium or t o e ash (Table 4), indicating t h a t t h e anorexia is induced before severe drain of b o d y calcium takes place.
VITAMIN D STEROID TOXICITY
27
TABLE 4. Serum calcium and phosphorus of hens fed vitamin D steroids (Experiment I, Phase II) Serum Treatment
P
Ca
Toe ash
(mg/dl)1
(%) 27.4 ± 2.78 31.3 ± 6.63
18.1 ± 1.17 19.4 ± .28
25-OH-D3 6 12
2.2 ± .43 2.7 ± 1.21
34.5 ± 5.21 31.1 ± 19.88
17.3 ± 2.00 18.1 ± 1.08
la-OH-D 3 3.41 6.82
3.1 ± .70 2.1 + .90
33.3 : 7.66 29.2: 17.89
19.3 ± 1.70 18.8 ± 2.02
1
Micrograms per kilogram diet.
TABLE 5. Tibial calcification in hens fed D3 or various levels of 1 a-OH-D3 for 8 weeks (Experiment 2) Tibia parameters Treatment
15' la-OH-D 3 5 10 15 1 2
Ash
Breaking strength
Cortical thickness
(%)
(kg)
(mm)
61.7 ± 3.50 2
7.6 ± 3.47
.90 ± .048
62.8 ±4.02 57.9 ± 3.71 61.8 ±4.60
8.1 ± 1.08 7.0 ± .76 8.8 ± 1.25
.71 ± .049 .83 ± .063 .83 ± .150
Micrograms per kilogram diet. Mean ± SE.
TABLE 6. Egg production and eggshell characteristics of hens fed D j or three levels of la-OH-D3 (Experiment 2)
Treatment
(%)
Egg weight (g)
% Shell
Specific gravity
D3 15 1
47 ± 5.9 a
63.3 ± 1.52
8.7 ± .10
1.077 ± .0009
49 ± 6.6 a 39 ± 8.5* b 17 + 8 . 1 b
64.7 ± 1.15 65.4 ± 2.64 61.2 ± 1.62
8.8 ± .27 8.7 ± .30 9.0+ .38
1.080 ± .0077 1.079 ± .0019 1.075 ± .0031
HDP
la-OH-D 3 5 10 15
1
' Means ± SE bearing different superscripts are significantly different (P<.05). Micrograms per kilogram diet.
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2.4 ± .56 1.8 ± .90
15 30
28
SOARES, JR., ETAL. TABLE 7. Serum levels of calcium,1 phosphorus,1 and l,25(OH)2D3 in laying hens given vitamin D steroids (Experiment 2) Initial
(pg/ml)
8 Weeks
Treatment
P
Ca
P
Ca
l,25-(OH) 2 D 3
D3 15 2
3.2 ± .12
29.2 ± 2.44 a
3.1 + .21
22.8 ± 2 . 7 1 b
308 ± 62.6
la-OH-D 3 5 10 15
3.0 ± .11 3.6 ± .33 3.7 + .35
28.8 ± 3.43 29.3 ± 1.98 a 31.6±2.50a
3.4 ± .23 2.7 ± .27 3.3 ±.33
30.3 ± 1.51 22.8 ± 2.85 b 26.6 ± 2.19 b
263 ± 19.4 278 ± 58.6 250 ± 49.2
a
calcium at n o r m a l levels. These d a t a are consist e n t with o u r observations t h a t ovulation was inhibited w h e n l a - O H - D 3 was fed at 10 or 15 Mg/ kg feed and t h e hens subsequently b e c a m e anorexic, resulting in emaciation. T h e percentage of hens showing severe emaciation u p o n postm o r t e m e x a m i n a t i o n wereO, 0, 1 2 . 5 , and 50 for t h e controls ( D 3 ) and 5, 10, and 15 jug/kg l a OH-D3, respectively. This effect does n o t appear t o be d u e t o gross skeletal changes and m a y be t h e result of neurological changes induced b y s o m e undisclosed a b n o r m a l i t y in calcium homeostasis. However, because only gross observations of k i d n e y and o t h e r organs were undert a k e n , n o definitive s t a t e m e n t t o this effect can b e m a d e at this t i m e . It appears t h a t t h e order of events is such t h a t appetite is reduced and, c o n s e q u e n t l y , egg p r o d u c t i o n stops, b u t egg shell quality m a y or m a y n o t be significantly affected. Plasma l , 2 5 ( O H ) 2 - D 3 levels (Table 7) were n o t different a m o n g t h e four t r e a t m e n t groups. This was true even t h o u g h t h e 10 and 15 £tg/kg l a - O H - D 3 fed groups contained m o r e nonlayers. It remains t o be d e t e r m i n e d w h e t h e r or n o t a t r u e elevation of plasma l , 2 5 ( O H ) 2 D 3 occurs early o n before egg p r o d u c t i o n , feed c o n s u m p tion and weight gains are decreased. F u r t h e r investigations are needed t o ascertain if transit o r y changes in plasma associated with high l a O H - D 3 c o n s u m p t i o n cause a t o x i c effect t o laying hens. ACKNOWLEDGMENTS T h e a u t h o r s wish t o t h a n k HoffmannL a R o c h e , Inc., N u t l e y , and T h e U p j o h n Co., K a l a m o z o o , MI, for t h e gifts of 2 5 - O H - D 3 and l a - O H - D 3 , respectively.
REFERENCES Anonymous, 1964. Technicon Auto Analyzer Methodology N-4c-Inorganic Phosphate (Modified from Fiske and SubbaRow, 1925. J. Biol. Chem. 66:375.) Techicon Instruments Corp. Tarrytown, NY. Cox, J., 1967. How much is egg breakage costing you? Poultry Trib. 10:46. Eisman, J. A., A. J. Hamstra, B. E. Kream, and H. F. DeLuca, 1976. A sensitive, precise and convenient method for determination of 1,25-dihydroxyvitamin D in human plasma. Arch. Biochem. Biophys. 176:235-243. Hollis, B. W., B. A. Roos, H. H. Draper, and P. W. Lambert, 1981. Vitamin D in human and bovine milk. J. Nutr. 111:1240-1248. Kaetzel, D. M., Jr., and J. H. Soares, Jr., 1979. Effects of cholecalciferol steroids on bone and egg shell calcification in Japanese quail. J. Nutr. 109:1601-1608. Lambert, P. W., D. O. Toft, S. F. Hodgson, E. A. Lindmark, B. J. Witrak, and B. A. Roos, 1979. An improved method for the measurement of l,25-(OH) 2 D 3 in human plasma. Endocrinol. Res. Comm. 5:293-310. Mueller, W. S., A. Pro-Martinex, I. G. Joshua, and B. Lobaugh, 1978. Calcium appetite in chickens. Pages 82—87 in Proc. Maryland Nutr. Conf. McLoughlin, C. P., and J. H. Soares, Jr., 1976. A study of the effects of 25-hydroxycholecalciferol and calcium source on egg shell quality. Poultry Sci. 55:1400-1410. Roland, D. A., Sr., 1976. Recent advances in egg shell quality. Pages 19—22 in Proc. Maryland Nutr. Conf. Roland, D. A., Sr., and R. H. Harms, 1976. The lack of response of 25-hydroxyvitamin D 3 on egg shell quality or other criteria in laying hens. Poultry Sci. 44:1983-1985. Snedecor, G. W., and W. G. Cochran, 1967. Analysis of variance. Pages 273-275 in Statistical Methods. 6th ed. Iowa State Univ. Press, Ames, IA. Soares, J. H. Jr., M. R. Swerdel, and E. H. Bossard,
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' Means within the same line bearing different superscripts are significantly different (P<.05). Milligrams per decaliter. 2 Micrograms per kilogram of diet. 1
VITAMIN D STEROID TOXICITY 1978. Vitamin D metabolites and phosphorus utilization. Poultry Sci. 57:1305—1312. Waldroup, P. W., W. C. Bussell, and A. R. Cobb, 1977.
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From Leidahl R. 1977 Test: No thicker shells with new vitamin D methobolites. Feedstuffs 49(40): 15.
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