Effect of Dietary 1,25-Dihydroxycholecalciferol Level on Broiler Performance1

Effect of Dietary 1,25-Dihydroxycholecalciferol Level on Broiler Performance1 KEVIN D. ROBERSON and HARDY M. EDWARDS, JR.2 Department of Poultry Science, Livestock-Poultry Building, The University of Georgia, Athens, Georgia 30602 ABSTRACT Studies were conducted to evaluate the level of dietary 1,25-dihydroxycholecalciferol [1,25(OH)2D3] required to decrease the incidence of tibial dyschondroplasia (TD) in male broilers at 3 and 5 wk of age. The birds were reared in floor pens with wood shavings and fed a corn-soybean meal diet supplemented with 0, 3, 6, or 9 MgAg l,25-(OH)2D3. The diet contained, by averaged analyses, 0.73% calcium, 0.74% total phosphorus, and 0.22% phytate phosphorus. There was no treatment effect on body weight or gain: feed at either age. The incidence and severity of TD and

the percentage of severe lesions were decreased and bone ash was increased by 6 ^g/kg l,25-(OH)2D3 at 3 wk of age. At 5 wk of age, the incidences of TD and severe lesions were decreased when 6 ng/kg l,25-(OH)2D3 was fed. Bone ash was increased by this level in one of the two experiments. Plasma calcium was increased at 5 wk when 9 /ig/kg l,25-(OH)2D3 was fed, but there was no treatment effect on plasma dialyzable phosphorus or l,25-(OH)2D3. The results indicate that 6 ^g/kg l,25-(OH)2D3 is effective for decreasing TD under practical rearing conditions.

(Key words: 1,25-dihydroxycholecalciferol, bone ash, broiler, plasma 1,25-dihydroxycholecalciferol, tibial dyschondroplasia) 1996 Poultry Science 75:90-94 TD in broilers reared under practical conditions and fed a diet marginal in calcium according to the NRC (1984).

INTRODUCTION The addition of 10 /^g/kg 1,25-dihydroxycholecalciferol [l,25-(OH)2D3] to a low calcium, high phosphorus broiler diet has been reported to decrease the incidence and severity of tibial dyschondroplasia (TD) and increase bone ash (Edwards, 1989, 1990; Edwards et ah, 1992). However, in some cases, feed efficiency (gain: feed) was decreased when 10 Mg/kg was fed. A recent report utilizing histological techniques to diagnose TD confirmed the finding of Edwards (1990) that 5 ^g/kg may be effective in decreasing TD in broilers (Rennie et al, 1993). Whitehead (1992) reported that 5 /xg/kg l,25-(OH)2D3 will also prevent TD in broilers fed a diet balanced for calcium and phosphorus. In all of these studies, experiments were conducted in electrically heated battery brooders. The level of l,25-(OH)2D3 required to decrease the incidence of TD in broilers housed similar to a commercial setting has not been evaluated. The objective of this study is to estimate the requirement of l,25-(OH)2D3 for the prevention of

MATERIALS AND METHODS Animals and Diets Two identical experiments were conducted utilizing day-old Peterson x Arbor Acres broiler cockerels. The birds were placed in 24 floor pens with wood shavings at 24 birds per pen. Each of the four treatments was replicated six times. The chicks were reared on a continuous light schedule using incandescent bulbs. Feed and water were provided for ad libitum consumption throughout the 5-wk experimental period. The basal diet used was a TD-inducing diet (Edwards and Veltmann, 1983), which is low in calcium and high in phosphorus (Table 1). Levels of l,25-(OH)2D33 were added at 3, 6, or 9 Mg/kg to the basal diet in liquid form using propylene glycol as a carrier. The same diets were fed for the entire 5 wk. Natural zeolite was added at 0.20% as an indigestible marker for the analysis of calcium and phosphorus retentions. However, due to technical difficulties, these parameters were not evaluated. The diets were calculated to contain 22% crude protein, 3,232 kcal/kg metabolizable energy, 0.65% calcium, 0.72% total phosphorus, and 0.50% available phosphorus and were analyzed after each mixing. The analyzed values for

Received for publication May 4, 1995. Accepted for publication August 29, 1995. Supported by state and Hatch funds allocated to the Georgia Agricultural Experiment Stations of The University of Georgia. 2 To whom correspondence should be addressed. 3 Hoffmann-LaRoche Inc., Nutley, NJ 07110.

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1,25-DIHYDROXYCHOLECALCIFEROL FOR BROILERS TABLE 1. Composition of the basal diet Ingredients Ground yellow corn Soybean meal (dehulled) Poultry fat (stabilized) Iodized sodium chloride Dicalcium phosphate (feed grade) Limestone Vitamin premix1 DL-methionine (98%) Trace mineral premix2 Se (0.02% from sodium selenite) Zeolite Calculated analyses Crude protein ME, kcal/kg Calcium Phosphorus, total Phosphorus, available

Amount (%) 56.61 35.00 5.00 0.45 1.86 0.28 0.25 0.20 0.10 0.05 0.20 22.10 3,232 0.65 0.72 0.50

Supplied in milligrams per kilogram of diet: (except as noted): vitamin A (as all-frans-retinyl acetate), 5,500 IU; cholecalciferol, 1,100 IU (27.5 ng/kg); vitamin E (all-rac-a-tocopherol acetate), 1.1 IU; riboflavin, 4.4; calcium pantothenate, 12; nicotinic acid, 44; choline CI, 220; vitamin B12,9 /ig; vitamin B6,3.0; menadione (as menadione sodium bisulfite), 1.1 thiamin (as thiamin mononitrate), 2.2; folic acid, 3; biotin, 0.3; and ethoxyquin, 125. 2 Supplied in milligrams per kilogram of diet: Mn0 2 , 222; ZnO, 150; FeS04-7H2O, 200; FeC03, 83; CuS04, 29; and Ca(103)2, 15.

Experiment 1 for the first 3 wk are 0.80% calcium, 0.77% total phosphorus, and 0.24% phytate phosphorus. At 5 wk, the values are 0.71% calcium, 0.77% total phosphorus, and 0.23% phytate phosphorus. In Experiment 2, the values at 3 wk are 0.75% calcium, 0.74% total phosphorus, and 0.18% phytate phosphorus. At 5 wk, the analyzed values are 0.67% calcium, 0.70% total phosphorus, and 0.22% phytate phosphorus. The high values for calcium may have been due to the possible incorporation of limestone into the soybean meal to make the meal a better free-flowing feed ingredient.

Blood At 3 and 5 wk of age, one bird per pen was chosen randomly and a blood sample was obtained by cardiac puncture. Plasma calcium (Section N-31) 4 and dialyzable phosphorus (Section 7 N-46) 4 were measured each time birds were bled. Plasma l,25-(OH)2D3 was measured at 3 wk of age in Experiment 1. The receptor for the l,25-(OH)2D3 assay was obtained from the thymus gland of a 5-mo-old calf and the cytosol protein was extracted (Reinhardt and Hollis, 1986).

Bone At 3 wk of age, 12 birds per pen were killed by asphyxiation and inspected for incidence and severity of

4 Technicon Autoanalyzer Methodology, Technicon Corp., Tarrytown, NY 10591.

91

TD (Edwards and Veltmann, 1983). The left tibia of five birds per pen was removed for bone ash determination on a dry, fat-free basis (Association of Official Agricultural Chemists, 1955). The remaining birds were killed at 5 wk of age and the procedure was repeated as before.

Statistical

Analysis

Data were analyzed together for the two experiments using the General Linear Models (GLM) procedure for ANOVA and regression analyses. Treatment mean differences for the combined experiments were separated by orthogonal contrasts at P < 0.05. The data for incidence of TD and severe lesions were subjected to nonlinear regression and the breakpoint between the descending line and the plateau was determined using the DUD method of the NLIN procedure (Helwig and Council, 1979). All statistics were conducted on the basis of pen means where pen is the experimental unit.

RESULTS There were no treatment effects on body weight or feed efficiency at 3 or 5 wk of age. Plasma calcium, dialyzable phosphorus, and l,25-(OH)2D3 were not affected at 3 wk. However, there was a linear increase in plasma calcium at 5 wk. Plasma calcium was significantly higher in the birds consuming 9 /xg/kg l,25-(OH) 2 D3 than the birds eating the basal diet. Supplementation of the diet with l,25-(OH)2D3 resulted in linear decreases in TD incidence (P < 0.065), TD severity, and the percentage of severe lesions at 3 wk of age (Table 2). At 5 wk, TD incidence decreased linearly (Table 3). There was a treatment by experiment interaction for percentage number 3 scores. The percentage of severe lesions was decreased linearly (P < 0.068) in Experiment 1. However, a quadratic response was observed in Experiment 2. The effective dosage of dietary l,25-(OH)2D3 to decrease incidence and severity of TD in this study was 6 ng/kg. Bone ash was increased at 3 wk of age when 6 jtg/kg l,25-(OH)2D3 was added to the diet. However, there was a treatment by experiment interaction for bone ash at 5 wk (Table 3). Bone ash continued to be higher when at least 6 /xg/kg l/25-(OH)2D3 was fed in Experiment 1. There was no effect of l,25-(OH)2D3 on bone ash at 5 wk in Experiment 2. Because 3 ptg/kg l,25-(OH)2D3 was generally not effective in decreasing the incidence of TD in these studies and 9 ^ g / k g was not any more beneficial than 6 /ig/kg, the data were contrasted between 0 and 3 ^ g / k g vs 6 and 9 Mg/kg l,25-(OH) 2 D 3 . At 3 wk of age, there was a decrease in incidence of TD (P < 0.009), severity of TD (P < 0.021), percentage of severe lesions (P < 0.001), and an increase in bone ash (P < 0.001) when 6 /xg/kg or more l,25-(OH) 2 D 3 was fed (Table 2). At 5 wk, there was a decrease in incidence of TD (P < 0.005) and percentage of severe lesions (P < 0.006), and increases in bone ash (P < 0.046) and plasma calcium (P < 0.015) (Table 3). The

92

ROBERSON AND EDWARDS, JR. TABLE 2. Effect of dietary 1,25-dihydroxycholecalciferol [l,25-(OH) 2 D 3 ] on growth, plasma minerals, bone ash, and the development of tibial dyschondroplasia in broiler chickens at 3 wk of age, Experiments 1 and 2 Plasma

1,25-•(OH) 2 D 3

BW

(fg/kg) (g) Experiment 1 763 0 758 3 778 6 742 9 Experiment 2 719 0 728 3 739 6 725 9 Pooled SE, combined experiment 14 Combined analysis of both experiments ANOVA summary Source of variation df Experiment (Ex) 1 0.002 l,25-(OH) 2 D 3 3 0.348 Ex x l,25-(OH) 2 D 3 3 0.770 Regression analysis l,25-(OH) 2 D 3 Linear 1 0.909 Quadratic 1 0.213 Overall effects Experiment 1 760* 2 728b l,25-(OH) 2 D 3 0 741 3 743 6 759 9 734

Gain: feed

Calcium

Dialyzable phosphorus

(mg/dL)

feg) 0.726 0.719 0.715 0.713

11.25 11.42 11.39 11.16

8.24 8.32 8.47 7.99

0.727 0.737 0.735 0.732

10.91 11.07 11.07 11.01

7.08 7.25 7.23 6.64

0.006

0.32

0.41

0.003 0.825 0.392

0.207 0.933 0.988

50.001 0.562 0.989

0.467 0.646

0.989 0.504

0.505 0.316

Tibial dyschondroplasia

1,25(OH) 2 D 3

Bone ash

Incidence

(pg/mL)

(%)

(%)

288 295 267 277

35.9 36.2 37.5 37.3

59 58 44 42

2.69 2.49 2.35 2.48

49 36 21 24

40.3 39.8 41.4 40.3

37 39 24 36

2.67 2.53 2.25 2.22

28 24 15 16

33 — Probabilities NA 0.938 NA 0.675 0.998

0.4

Score

No. 3

(%)

0.16

50.001 0.002 0.371

50.001 0.052 0.492

0.443 0.087 0.791

50.001 50.001 0.300

0.210 0.686

0.065 0.574

0.017 0.314

50.001 0.175

0.718" 0.733"

11.30 11.01

8.26= 7.05"

36.7" 40.4"

51" 34"

2.50 2.42

33" 21b

0.726 0.729 0.725 0.722

11.08 11.24 11.21 11.08

7.66 7.79 7.85 7.32

38.1b 38.0b 39.4" 38.8"b

48" 48" 34b 39"b

2.68" 2.51* 2.30b 2.35*

38" 30" 18b 20b

"•bMeans within a < umn with no common superscript differ significantly (P 5 0.05).

increase in plasma Icium was actually caused by 9 jtg/ kg alone (P < 0.0c,. The treatment by experiment interactions for bone ash and percentage severe lesions were again evident when the data were contrasted separately (Table 4). Bone ash was increased (P < 0.001) and percentage severe lesions was decreased (P < 0.016) by 6 and 9 /ig/ kg l,25-(OH)2D3 in Experiment 1. However, there was no significant difference in bone ash (P < 0.456) or percentage severe lesions (P < 0.189) in Experiment 2. The decrease in severe lesions when 3 iig/kg l,25-(OH)2D3 was fed in Experiment 2 prevented a significant response. There was a decrease (P < 0.016) in percentage severe lesions in Experiment 2 when all three levels of l,25-(OH)2D3 supplementation were contrasted against the basal diet. The l,25-(OH)2D3 requirement to decrease TD in general and severe lesions was estimated by nonlinear regression to be 6 /xg/kg at 3 wk. After 5 wk, the requirement to decrease TD was still 6 Mg/kg. However, the requirement to prevent severe lesions was estimated to be 6.4 /xg/kg.

DISCUSSION The results of these studies confirm the previous reports that less than 10 iig/kg l,25-(OH)2D3 can decrease TD without affecting growth or feed efficiency (Edwards, 1990; Rennie et al, 1993). This report also demonstrates that l,25-(OH)2D3 can decrease the incidence of TD in broilers reared on litter. Tibial dyschondroplasia was induced by a low calcium, high phosphorus diet. The actual requirement for calcium for maximum bone ash when l,25-(OH)2D3 is fed is actually about two-thirds of the calcium requirement when l,25-(OH)2D3 is not in the diet (Edwards et al, 1992). The average level of calcium fed in the two experiments conducted was 0.73%. Hence, when an adequate level of l,25-(OH)2D3 was fed in these studies (6 iig/kg), the calcium level of the diet relative to the diet with no added l,25-(OH) 2 D 3 was about 1.10%. Therefore, the relative calcium to nonphytate phosphorus ratio was approximately 2.2:1. Even with the increase in phytate phosphorus utilization due to dietary l,25-(OH) 2 D 3 (Edwards, 1993), the relative calcium to available phosphorus ratio is similar to feed formula-

1,25-DIHYDROXYCHOLECALCIFEROL FOR BROILERS

93

TABLE 3. Effect of dietary 1,25-dihydroxycholecalciferol on growth, plasma minerals, and the development of tibial dyschondroplasia in broiler chickens at 5 wk of age, Experiments 1 and 2 Plasma

l,25-(OH) 2 D 3

BW

fog/kg) (8) Experiment 1 0 1,786 3 1,715 6 1,743 9 1,718 Experiment 2 0 1,698 3 1,730 6 1,697 9 1,691 Pooled SE, combined experiment 35 Combined analysis of both experiments ANOVA summary Source of variation df 0.147 1 Experiment (Ex) l,25-(OH) 2 D 3 3 0.759 Ex x l,25-(OH) 2 D 3 3 0.524 Regression analysis l,25-(OH) 2 D 3 linear 1 0.292 quadratic 1 0.951 Overall effects Experiment 1 1,740 2 1,704 l,25-(OH) 2 D 3 0 1,742 3 1,723 6 1,720 9 1,704

Gain: feed

Dialyzable phosphorus

Calcium

• (mg/dL)

teg)

-

Tibial dyschondrop!lasia

Bone ash

Incidence

(%)

(%)

Score

No. 3

(%)

0.552 0.570 0.554 0.573

11.05 11.62 11.77 11.66

6.93 7.63 7.38 7.48

37.8 37.4 39.3 39.2

30 42 23 21

2.79 2.90 2.31 1.83

25 39 20 13

0.585 0.567 0.579 0.562

10.86 11.03 10.93 11.69

7.32 7.48 6.98 7.60

38.1 38.7 38.1 37.8

35 24 15 27

2.66 2.03 2.31 2.63

29 14 11 20

0.009

0.21

0.25

0.3

0.103 0.998 0.056

0.010 0.014 0.166

0.963 0.202 0.456

0.464 0.233 0.010

0.350 0.031 0.091

0.815 0.417 0.087

0.100 0.050 0.019

0.883 0.985

0.004 0.897

0.287 0.838

0.144 0.718

0.033 0.629

0.109 0.689

0.024 0.857

0.563 0.573

11.52" 11.13b

7.35 7.35

38.4 38.2

29 25

2.46 2.41

24 18

0.569 0.569 0.567 0.568

10.95" 11.32* 11.35* 11.68*

7.12 7.55 7.18 7.54

38.0 38.0 38.7 38.5

32" 33* 19b 24ab

2.73 2.47 2.31 2.23

27a 26 a 15 b 16 ab

0.32

• Probabilities

a b

- Means within a column with no common superscript differ significantly (P £ 0.05).

tions present in the industry. However, future studies with lower dietary phosphorus levels should be conducted to determine the benefits of l,25-(OH)2D3 on leg abnormalities in broiler chickens. The different responses to dietary l,25-(OH)2D3 for TD illustrates the variability involved when conducting research in this area, as the incidence of TD in the birds fed the basal diet was twice as high in Experiment 1 as in Experiment 2 at 3 wk of age. The different responses at various doses also reflects the variation inherent within treatments when only half the birds were examined for TD at 3 and 5 wk of age. The decrease in the development of TD was a smaller response than has been observed in experiments conducted previously in battery brooders (Edwards, 1990; Edwards et al, 1992; Rennie et al, 1993). The cholecalciferol requirement may be higher than 1,100 IU/kg under these growing conditions. Other factors such as mycotoxins may have influenced the results (Walsar et al, 1982) by affecting vitamin D metabolism. Although 6 Mg/kg l,25-(OH)2D3 clearly increased bone ash at 3 wk in both trials and at 5 wk in

Experiment 1, the lack of a response at 5 wk in Experiment 2 is surprising. Because only five bones per pen were used for bone ash analysis, random sampling and variation within pens of individual birds within each treatment may have resulted in lower ash levels than expected for the birds fed 6 or 9 jtg/kg l,25-(OH)2D3 in Experiment 2. The etiology of TD is complex. However, it is becoming more clear that a defect in vitamin D metabolism may be playing a major role. Xu et al. (1992) reported that the DNA binding domain of vitamin D receptors from chicks with TD is normal when measured in cultured growth plate chondrocytes. However, in recent in vivo studies, they found that chicks with TD had lower levels of calcium binding protein-28K and type X mRNA. 1,25-Dihydroxycholecalciferol is capable of upregulating its own receptor activity (Costa et al, 1985), which occurs at the messenger RNA level (Pike, 1991). Therefore, l,25-(OH)2D3 may autoinduce its own receptor protein. In the studies by Xu et al. (1993), chicks with TD did not have different levels of serum l,25-(OH)2D3 than

ROBERSON AND EDWARDS, JR. TABLE 4. Effect of dietary 1,25-dihydroxycholecalciferol [l,25-(OH) 2 D 3 ] on bone ash and severe tibial dyschondroplasia in broiler chickens at 5 wk of age Experiment 2

Experiment 1 1,25-•(OH)2D3

(Mg/kg) 0 3 6 9 Pooled SE ANOVA Source of variation l,25-(OH)2D3 Regression analysis Linear Quadratic

Bone ash

No. 3

37.8b 37.4b 39.3= 39.2» 0.3

25^ 39' 20b 13b 6

Bone ash

No. 3

38.1 38.7 38.1 37.8 0.5

29a 14b lib 20 ab 5

(%)

df 3

0.001

0.035

Probability 0.725

0.057

1 1

0.001 0.601

0.068 0.100

0.573 0.413

0.186 0.016

normal chicks. It was concluded that a vitamin D resistance was involved in the development of TD. Plasma l,25-(OH)2D3 was unaffected in these studies as well, despite the obvious effects dietary l,25-(OH)2D3 had on the development of TD. The absence of a relationship between TD and plasma l,25-(OH)2D3 has been reported previously (Newbrey et ah, 1988). Elliot and Edwards (1992) observed no correlation between the average TD lesion score and plasma l,25-(OH)2D3. It appears that the major cause of TD in the rapidly growing broiler may be a problem in vitamin D metabolism at the receptor level. This problem is overcome when any vitamin D metabolite hydroxylated at the 1 position is fed at an adequate amount (Edwards, 1990). The requirement for l,25-(OH) 2 D 3 in broilers to decrease the incidence of TD was estimated to be 6 /tg/ kg under practical conditions. However, dietary l,25-(OH)2D3 may not be required for the entire growing period.

REFERENCES Association of Official Analytical Chemists, 1955. Official Methods of Analysis. 8th ed. Association of Official Analytical Chemists, Washington, DC. Costa, E. M, M. Hirst, and D. Feldman, 1985. Regulation of 1,25-dihydroxyvitamin D3 receptors by vitamin D analogs in cultured mammalian cells. Endocrinology 117: 2203-2210. Edwards, H. M, Jr., 1989. The effect of dietary cholecalciferol, 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol on the development of tibial dyschondroplasia in broiler chickens in the absence and presence of disulfiram. J. Nutr. 119:647-652. Edwards, H. M, Jr., 1990. Efficacy of several vitamin D compounds in the prevention of tibial dyschondroplasia in broiler chickens. J. Nutr. 120:1054-1061. Edwards, H. M., Jr., 1993. Dietary 1,25-dihydroxycholecalciferol supplementation increases natural phytate phosphorus utilization in chickens. J. Nutr. 123:567-577. Edwards, H. M, Jr., M. A. Elliot, and S. Sooncharernying, 1992. Effect of dietary calcium on tibial dyschondroplasia.

Interaction with light, cholecalciferol, 1,25-dihydroxycholecalciferol, protein, and synthetic zeolite. Poultry Sci. 71:2041-2055. Edwards, H. M., Jr., and J. R. Veltmann, Jr., 1983. The role of calcium and phosphorus in the etiology of tibial dyschondroplasia in young chicks. J. Nutr. 113:1568-1575. Elliot, M. A., and H. M. Edwards, Jr., 1992. Studies on the correlation between the severity of tibial dyschondroplasia, and weight gain, bone ash, plasma minerals, and plasma vitamin D3 metabolites in broilers. Poultry Sci. 71 (Suppl. 1):148. (Abstr.) Helwig, J. T., and K. A. Council, 1979. SAS® User's Guide. SAS Institute Inc., Cary, NC. National Research Council, 1984. Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. Newbrey, J. W., S. N. Baksi, A. S. Dhillon, N. G. Zimmerman, S. G. Truitt, and R. Riedinger, 1988. Histomorphometry and vitamin D metabolism of valgus-varus deformity in broiler chickens. Avian Dis. 32:704-712. Pike, J. V., 1991. Vitamin D3 receptors: Structure and function in transcription. Ann. Rev. Nutr. 11:189-216. Reinhardt, T. A., and B. W. Hollis, 1986. 1,25-Dihydroxyvitamin D microassay employing radioreceptor techniques. Meth. Enzymol. 123:176-185. Rennie, J. S., C. C. Whitehead, and B. H. Thorp, 1993. The effect of dietary 1,25-dihydroxycholecalciferol in preventing tibial dyschondroplasia in broilers fed on diets imbalanced in calcium and phosphorus. Br. J. Nutr. 69: 809-816. Walser, M. M., N. K. Allen, C. J. Mirocha, G. F. Hanlon, and J. A. Newman, 1982. Fusarium-induced osteochondrosis (tibial dyschondroplasia) in chickens. Vet. Pathol. 19: 554-560. Whitehead, C. C, 1992. Tibial dyschondroplasia in broilers and the role of vitamin D metabolites in its prevention. Pages 109-113 in: Proceedings of the Arkansas Nutrition Conference, Fayetteville, AR. Xu, T., J. H. Soares, Jr., and R. M. Leach, 1992. Alkaline phosphatase activity of cultured growth plate chondrocytes and affinity of vitamin D receptor for DNA in chicks with tibial dyschondroplasia. Poultry Sci. 71(Suppl. 1):103. (Abstr.) Xu, T., J. H. Soares, Jr., R. M. Leach, Jr., J. Kerr, and B. Hollis, 1993. Evidence of vitamin D resistance in chicks with tibial dyschondroplasia. Poultry Sci. 71(Suppl. 1):109. (Abstr.)