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Effect of Vitamin C, Environmental Temperature, Chlortetracycline, and Vitamin D3 on the Development of Tibial Dyschondroplasia in Chickens1
Effect of Vitamin C, Environmental Temperature, Chlortetracycline, and Vitamin D3 on the Development of Tibial Dyschondroplasia in Chickens1 HARDY M. EDWARDS, JR. Department of Poultry Science, Livestock-Poultry Building, University of Georgia, Athens, Georgia 30602 (Received for publication May 4, 1988)
1989 Poultry Science 68:1527-1534 INTRODUCTION
The addition of ascorbic acid to a diet that induced a lesion resembling tibial dyschondroplasia in willow ptarmigans prevented the birds from developing the abnormal cartilage condition (Hanssen et al., 1979). Scorbutic guinea pigs have lesions (Murray and Kodicek, 1949) that resemble those described by Hanssen et al. (1979) in willow ptarmigans and tibial dyschondroplasia in chickens (Leach and Nesheim, 1965). A recent report indicates that supplemental dietary ascorbic acid in two experiments with two different diets had no effect on the development of tibial dyschondroplasia in broiler type chickens (Leach and Burdette, 1985). In the present report, the significant reduction in the incidence of tibial dyschondroplasia resulting from ascorbic acid supplementation in an initial experiment was not repeated in two subsequent experiments. Therefore, several experiments were carried out to see if factors previously reported to influence ascorbic acid responses in poultry, such as temperature (Thornton et al., 1959), microbial infection, and antibiotic treatment (Hill and Garren, 1955, 1958) or vitamin D3
^Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia.
status (Thornton et al., 1959; Thornton and Brownrigg, 1961; Ramp and Thornton, 1966; Thornton and Omdahl, 1969) may be influencing the effects of ascorbic acid supplementation on the development of tibial dyschondroplasia. MATERIALS AND METHODS
General Procedures. Newly hatched male broiler chickens were used in all experiments. All experiments were conducted in electrically heated battery brooders with wire mesh floors, with feed and water always available. The basal diet shown in Table 1 was used with slight modifications in the various experiments. Materials were added to the basal diet at the expense of ground com. Experiments 1 to 6 were conducted for 20 days, and Experiment 7 was conducted for 16 days. At the termination of the experiments, birds were weighed by pens and their feed consumption recorded. They were then killed and examined at random for tibial dyschondroplasia; a longitudinal cut was made across the right tibia, which was scored for incidence and severity (Edwards and Veltmann, 1983). A photograph illustrating the scoring system used to judge degree of tibial dyschondroplasia in 3-wk-old chickens has been published (Edwards and Veltmann, 1983). The system
1527
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ABSTRACT Seven experiments were conducted to test the influence of dietary supplementary ascorbic acid on the development of tibial dyschondroplasia in broiler chickens. Ascorbic acid supplementation significantly reduced the incidence and number of birds with a large mass of cartilage in the tibia in the first experiment but not in the two subsequent experiments. Because environmental temperature, microbial infection, and vitamin D 3 status had been reported in the literature to influence ascorbic acid metabolism in the chicken, experiments were conducted to see if these variables could influence supplemental ascorbic acid effects on development of tibial dyschondroplasia. Results of the experiments indicated that none of these factors influenced the effect of ascorbic acid on the development of tibial dyschondroplasia. The presence of vitamin D3 in the diet significantly influences the incidence of this disorder. (Key words: ascorbic acid, tibial dyschondroplasia, temperature, chlortetracycline, chicken)
1528
EDWARDS, JR. TABLE 1. Composition of the basal diet
Ingredients
Amounts
Ground yellow corn Denuded soybean meal Poultry fat (stabilized) Iodized salt DL-Methionine (feed grade) Vitamin premix1 Trace mineral premix2 Selenium concentrate (.02% from sodium selenite) Dicalcium phosphate (feed grade) Ground limestone
56.81 35.00 5.00 .45 .20 .25 .10 .05 1.86 .28
assigns scores from 0 to 3: 0) normal cartilage, narrow with little irregularities; 1) cartilage is thickened or shows considerable irregularities; 2) cartilage is thickened with evidence of persisting prehypertrophic cartilage that is not calcified and has not been invaded by vessels from the metaphysis; deep irregularities of such cartilage are apparent; and 3) large mass of cartilage in the proximal end of the tibia. The left tibia was removed for bone ash determination on the fat-free dry basis (Association of Official Agricultural Chemists, 1955). The analysis of variance and regression analysis were computed using the general linear model procedure (Helwig and Council, 1979). Where appropriate, main effect means were separated by Duncan's multiple range test. Ascorbic Acid Supplementation (Experiments 1 to 3). These experiments were conducted to determine the effect of ascorbic acid supplementation on the incidence and severity of tibial dyschondroplasia in broilers fed a diet low in calcium (.65%) and containing adequate dietary levels of phosphorus (.71%), chloride (.32%), and vitamin D 3 (1,100 ICU/kg) and known to induce a high incidence of the disorder (Edwards and Veltmann, 1983). In these three experiments, six pens of ten cockerels were fed each dietary treatment. The cockerels were obtained from a
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1 Vitamin premix provides in milligrams/kilogram of diet (except as noted): vitamin A (as all-tra/ts-retinyl acetate), 5,500 IU; cholecalciferol, 1,100 ICU; vitamin E (all-rac-atocopheryl acetate), 11 IU; riboflavin, 4.4; d-calcium pantothenate, 12; nicotinic acid, 44; choline chloride, 220; vitamin Bj2, 6.6 fig; vitamin Bg, 2.2; menadione (as menadione sodium bisulfite), 1.1; thiamin (as thiamin mononitrate), 2.2; and ethoxyquin, 125. ^ r a c e mineral premix provides in milligrams per kilogram of diet: Mn0 2 , 222; ZnO, 150; FeS0 4 -7H 2 0, 200; FeC0 3 , 83; CuS0 4 , 29; and Ca (I0 3 ) 2 , 15.
commercial broiler hatchery and were Ross x Arbor Acre for Experiment 1 and Petersen x Arbor Acre for Experiments 2 and 3. In Experiments 1 and 2, the dietary treatments were the basal diet (Table 1) and this diet supplemented with 500 mg/kg of ascorbic acid. The basal diet and a series of diets containing 250, 500, and 1,000 mg/kg of ascorbic acid were fed in Experiment 3. Environmental Temperature (Experiments 4 to 5). These experiments were conducted to determine if environmental temperature could be a variable in determining when an effect was obtained from ascorbic acid supplementation on the incidence of tibial dyschondroplasia. The experiments were conducted in three rooms where the environmental temperatures were maintained at approximately 18 C, 24 C, and 30 C. The brooder temperatures were maintained at between 32 to 35 C during the course of the experiment. In each room, six pens of ten birds each were fed die basal diet or this diet supplemented with 500 mg/kg of ascorbic acid. Room Fumigation and Antibiotic Feeding (Experiment 6). This experiment was conducted in two Petersime (Petersime Incubator Co., Gettysburg, OH) battery brooders, located in identical rooms (6.2 m wide x 7.4 m long x 2.5 m high). One room received normal cleaning of the battery and room with water and detergent before the start of the experiment. The otfier room and battery were cleaned in die same manner; the room was then sealed witii paper and tape and fumigated with 500 g KMn04 and 900 mL formaldehyde overnight. Three dietary treatments in each room were basal diet (Table 1), basal + 500 mg/kg ascorbic acid, and the basal + chlortetracycline concentrate to give 100 mg chlortetracycline/ kg of diet. Four pens of 10 Petersen x Arbor Acre cockerels were fed each dietary treatment in each room. Vitamin D3 Deficiency (Experiment 7). This experiment was conducted to determine if ascorbic acid supplementation of the diet would influence the development of tibial dyschondroplasia in the vitamin D3-deficient chicken. The vitamin mixture for this study supplied the same amounts of each vitamin as the one shown in Table 1 except it contained no cholecalciferol and it supplied a slighdy higher level of vitamin B12 to the diet (9 |0.g/ kg). The treatments were: vitamin D3-deficient basal diet, the basal diet plus 1,100 ICU/kg of
VITAMIN C AND ENVIRONMENT ON TIBIAL DYSCHONDROPLASIA
vitamin D3, the basal diet plus 500 mg/kg of ascorbic acid, and the basal diet plus 1,100 ICU/kg of vitamin D3 plus 500 mg/kg of ascorbic acid. Six pens of ten Petersen x Arbor Acre cockerels were fed each dietary treatment. The fluorescent lights in the battery brooder were turned off, the fluorescent lights in the room were covered with plastic, and the blind for the outside window was drawn during this experiment. RESULTS
Room Fumigation and Antibiotic Feeding (Experiment 6). Fumigation of the room did
not have a significant effect on any of the criteria measured (Table 4). The addition of chlortetracycline to the diet caused a significant increase in the 20-day weights and gain: feed ratio but had no effect on any of the other criteria studied. Vitamin D3 Deficiency (Experiment 7). Addition of ascorbic acid to the vitamin D3deficient or supplemented diet caused a significant decrease in bone ash and an increase in the incidence of tibial dyschondroplasia (Table 5). The addition of vitamin D3 to the diets, with or without ascorbic acid, caused significant increases in 16-day weight and bone ash, while causing a significant decrease in the incidence and number of Number 3 scores for tibial dyschondroplasia. The results and statistical analysis do not indicate any vitamin D3 x ascorbic acid interaction. DISCUSSION
Results obtained in these studies confirm the report of Leach and Burdette (1985), which indicated that dietary ascorbic acid was not involved in the development of tibial dyschondroplasia in young broiler chickens. The effects of ascorbic acid in all seven experiments may be summarized as follows: positive effects were decreased incidence and number of Number 3 scores of tibial dyschondroplasia in Experiment 1 and increased growth rate in Experiment 2; negative effects were decreased bone ash in Experiments 4, 5, and 7 and increased incidence of tibial dyschondroplasia in Experiment 7. Only the effect of ascorbic acid in decreasing bone ash would appear to be consistent enough to conclude that it may be a true effect and not just a chance occurrence. The other possibility of course still exists that some factor that influences the effect that ascorbic acid has on the development of tibial dyschondroplasia is not under control in these experiments and the effect of ascorbic acid as seen in Experiment 1 would be repeatable under more controlled experimental conditions. The chickens used in Experiment 1 were of different breeding (Ross x Arbor Acre) than those used in the other six experiments. A previous report from this laboratory (Edwards, 1984) has shown a difference in the commercial strains of broilers in their susceptibility to developing tibial dyschondroplasia. However, in those studies there were no significant differences in susceptibility of the two particular strains used in the present studies (Ross x Arbor Acre and Petersen x Arbor Acre). The
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Ascorbic Acid Supplementation (Experiments 1 to 3). Results of the experiments on the effect of ascorbic acid supplementation on tibial dyschondroplasia are presented in Table 2. Ascorbic acid supplementation caused a significant decrease in the incidence of tibial dyschondroplasia in Experiment 1 but had no effect in Experiments 2 and 3. Ascorbic acid supplementation also caused a significant increase in growth rate in Experiment 2. In both Experiments 1 and 2, the number of birds scored as having Number 3 lesions was reduced by ascorbic acid supplementation, but there was no significant effect on the overall score. Environmental Temperature (Experiments 4 to 5). Results of the experiments to determine if environmental temperature influences the effect of ascorbic acid supplementation on the incidence of tibial dyschondroplasia are presented in Table 3. Significant differences between experiments were found for gain:feed and bone ash; however, for both of these criteria, no significant interactions of experiment and ascorbic acid or experiment and temperature were found; therefore, analysis of the combined experiments is presented. Temperature had a significant effect on all of the criteria measured: the high temperature (30 C) caused lower 20-day weights, gainrfeed ratio, bone ash, and incidence and severity of tibial dyschondroplasia than the low temperature (18 C). The addition of ascorbic acid to the diet significantly decreased bone ash but had no significant effect on any of the other criteria measured. However, in the case of body weights at 20 days of age and incidence and severity of tibial dyschondroplasia, there was a significant temperature x experiment interaction, indicating some experimental betweenexperiments variable caused different responses to temperature in the two experiments.
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Ascorbic acid supplementation
1
.77 .91
.40
.68
.38
38.1 37.3 37.9 37.5 ±.3
(%) 38.2 38.3 ±.2 39.2 39.2 ±.4
Bone ash
.92 .20
(g/g) .664 .665 ±.010 .667 .676 ±.005 .635 .650 .654 .647 ±.016
Gain:feed ratio
.32 .03
(g) 559 569 ±9 508 530* ±6 555 537 551 539 ±9
BW, 20 days of age
*P<05.
Number of birds that scored Number 3/number of birds examined at the end of the experiment.
(mg/kg) None 500 Pooled SE 2 None 500 Pooled SE 3 None 250 500 1,000 Pooled SE ANOVA, probabilities Treatments, Experiment 1 (df, 1) Treatments, Experiment 2 (df, 1) Regression analysis Experiment 3 Ascorbic acid, linear (df 1)
Experiment
.60
.03 .19
(%) 59 34* ±7 68 58 ±5 38 51 43 46 ±7
Incidence
TABLE 2. Effect of dietary ascorbic acid supplementation on tibial dyschondroplasia (Exp
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'Number of birds that scored Number 3/number of birds examined at the end of the experiment.
.0 .2 .0 .0 .0 .4 .6
30 35
40.8" 40.3 b
<001 .001 .044 <001 .339 .309 .471
36a 37a 23 b
<*) 33 31 9 31 52 14 ±6 48 27 30 34 38 39 ±8
Inci
40.7" 40.8* 40.2b
(%) 41.2 41.6 40.9 41.3 40.7 40.7 ± .2 40.3 40.8 40.0 40.2 40.0 39.2 ± .2
Bone ash
'""'Values of the same variable with no common superscripts are significantly different (P<.05).
<.001 .944 .785 <.001 .164 .630 .493
.695 .695
601 595
<001 .318 .519 .734 <.001 .471 .036
.700" .715" .670b
.670 .700 .652 ±.012 .721 .741 .674 .739 .729 .680 ±.020
(g/g) .672 .690 .674
Gain:feed ratio
593b 627a 573 c
565 646 601 567 618 596 ±10
(g) 635 616 543 605 630 552 ±10
(mg/kg) None None None 500 500 500
(C) 18 24 30 18 24 30 Pooled SE 18 24 30 18 24 30 Pooled SE
None None None 500 500 500
BW, 20 days of age
Ascorbic acid supplementation
Room temperature
ANOVA probabilities (Experiments 4 + 5 combined) Sources of variation, df Temperature (T), 2 Ascorbic acid (AA), 1 T x AA, 2 Experiment (EXP), 1 T x EXP, 2 AA x EXP, 1 T x AA x EXP, 2
Main effect means Temperature 18 24 30 Ascorbic acid None 500 mg/kg
5
4
Experiment
TABLE 3. Effect of environmental temperature and ascorbic acid on tibial dyschonaropla
om http://ps.oxfordjournals.org/ at Michigan State University on December 24, 2014
.261 .758 .334
'Number of birds that scored Number 3/mimber of birds examined at the end of the experiment.
' Values of the same variable with no common superscripts are significantly different (P<05).
ab
.003 .070 .082
39 37
38.2 38.6
.646 .673
523 535
.038 .393 .672
44 36 33
38.4 37.3 39.5
.628b .653b .696"
.447 .775 .925
±8
38.3 36.2 40.3 38.5 38.5 38.7 ±1.3
505 b 529 ab 553"
Pooled SE Main effect means Diet supplements None Ascorbic acid Chlortetracycline Fumigated room Yes No ANOVA, probabilities Sources of variation, df Diet, 2 Fumigated room, 1 Diet x fumigated room, 2
No
(%) 43 38 35 45 33 32
(%)
(g/g) .592 .658 .680 .662 .645 .709 ±.017
None Ascorbic acid Chlortetracycline None Ascorbic acid Chlortetracycline
Yes
Incidence
Bone ash
Gain:feed ratio
BW, 20 days of age
Diet supplementation
Fumigated room
TABLE 4. Effect of ascorbic acid, chlortetracycline, and room fiimigation on the development of tibi
om http://ps.oxfordjournals.org/ at Michigan State University on December 24, 2014
.241 .001 .724
.236 .146 .821
.708 .673
30.1 b 31.2a
.679 .705
330 344
367" 306b
± .5
.698 ±.021
359 ±11
'Number of birds that scored Number 3/number of birds examined at the end of the experiment.
.028 .001 .607
34.2* 27.2 b
33.5
27.6 26.8 34.9
(%)
(g/g) .692 .651 .719
(g) 311 302 376
Bone ash
Gain:feed ratio
BW, 16 days of age
"'Values of the same variable with no common superscript are significantly different (P<.05).
ANOVA, probabilities Sources of variation, df Ascorbic acid, 1 Vitamin D3, 1 Ascorbic x vitamin D3, 1
Vitamin D3 1,100 ICU/kg None
None Ascorbic acid, 500 mg/kg Vitamin D 3 , 1,100 ICU/kg Ascorbic acid, 500 mg/kg plus vitamin D 3 , 1,100 ICU/kg Pooled SE Main effect means Ascorbic acid 500 mg/kg None
Supplements to basal diet
TABLE S. Effect of dietary ascorbic supplementation on the development of tibial dyschondroplasia by ch in vitamin D3 (Experiment 7)
om http://ps.oxfordjournals.org/ at Michigan State University on December 24, 2014
1534
EDWARDS, JR.
ACKNOWLEDGMENTS
The author wishes to thank Frances Denman for her technical assistance. The advice of Glenn Ware regarding the statistical analysis is appreciated. REFERENCES Association of Official Agricultural Chemists, 1955. Official Methods of Analysis. 8th ed. Assoc. Offic. Agric.
Chem., Washington, DC. Edwards, H. M., Jr., 1984. Studies on the etiology of tibial dyschondroplasia in chickens. J. Nutr. 114: 1001-1013. 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. Hanssen, I., H. J. Grav, J. B. Steen, and H. Lysnes, 1979. Vitamin C deficiency in growing willow ptarmigan (Lagopus lagopus lagopus). J. Nutr. 109:2260-2276. Helwig, J. T., and K. A. Council, eds., 1979. SAS User's Guide. SAS Inst. Inc., Raleigh, NC. Hill, C. H., and H. W. Garren, 1955. The effect of high levels of vitamins on the resistance of chicks to fowl typhoid. Ann. N.Y. Acad. Sci. 63:186-194. Hill, C. H., and H. W. Garren, 1958. Plasma ascorbic acid levels of chicks with fowl typhoid. Poultry Sci. 37: 236-237. Horio, F., K. Ozaki, A. Yoshida, S. Makino, and Y. Hayashi, 1985. Requirement for ascorbic acid in a rat mutant unable to synthesize ascorbic acid. J. Nutr. 115: 1630-1640. Leach, R. M., Jr., and J. H. Burdette, 1985. The influence of ascorbic acid on the occurrence of tibial dyschondroplasia in young broiler chickens. Poultry Sci. 64: 1188-1191. Leach, R. M., Jr., and M. C. Nesheim, 1965. Nutritional, genetic and morphological studies of an abnormal cartilage formation in young chicks. J. Nutr. 86: 236-244. Makino, S., and K. Katagiri, 1980. Osteogenic disorder rat. Exp. Anim. (Tokyo) 29:374-375. Murray, P.D.F., and E. Kodicek, 1949. Bones, muscles and vitamin C. III. Repair of the effects of total deprivation of vitamin C at the proximal ends of the tibia and fibula in guinea pigs. J. Anat. 83:285-293. Ramp, W. K., and P. A. Thornton, 1966. Skeletal response to exogenous ascorbic acid by vitamin D3 deficient chicks. Proc. Soc. Exp. Biol. Med. 121:1248-1250. Thornton, P. A., and D. Brownrigg, 1961. Calcium utilization and skeletal development in chicks as influenced by parental dietary ascorbic acid. J. Nutr. 75:354-360. Thornton, P. A., and J. L. Omdahl, 1969. Further evidence of skeletal response to exogenous ascorbic acid. Proc. Soc. Exp. Biol. Med. 132:618-621. Thornton, P. A., C. W. Weber, and R. E. Moreng, 1959. The effect of ascorbic acid in the diet of adult chickens on calcium utilization by progeny. J. Nutr. 69:33-38.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on December 24, 2014
possibility that genetic differences in the ability of broiler chickens to synthesize ascorbic acid might cause deficiencies in some chickens of certain strains still exists. The production of a chicken mutant that cannot convert gulonolactone to ascorbic acid and the subsequent study of ascorbic acid deficiency in chickens is desirable. A mutant rat strain that cannot convert gulonolactone to ascorbic acid has recently been isolated (Makino and Katagiri, 1980; Horio et al., 1985). High environmental temperature significantly reduced the incidence and score of tibial dyschondroplasia, and this can be partially accounted for by the reduction in growth rate that resulted from the high temperature. The small but significant lowering of the percentage of bone ash in broilers reared at the highest temperature (30 C) is unusual in view of the effects on growth and incidence of tibial dyschondroplasia. The effects of vitamin D3 deficiency produced in Experiment 7 was not as severe on growth rate as expected after the precautions had been taken to reduce the ultraviolet light on the chickens. This probably indicates that substantial vitamin D3 reserves from the dams were present in the day-old chicks.
1989 Poultry Science 68:1527-1534 INTRODUCTION
The addition of ascorbic acid to a diet that induced a lesion resembling tibial dyschondroplasia in willow ptarmigans prevented the birds from developing the abnormal cartilage condition (Hanssen et al., 1979). Scorbutic guinea pigs have lesions (Murray and Kodicek, 1949) that resemble those described by Hanssen et al. (1979) in willow ptarmigans and tibial dyschondroplasia in chickens (Leach and Nesheim, 1965). A recent report indicates that supplemental dietary ascorbic acid in two experiments with two different diets had no effect on the development of tibial dyschondroplasia in broiler type chickens (Leach and Burdette, 1985). In the present report, the significant reduction in the incidence of tibial dyschondroplasia resulting from ascorbic acid supplementation in an initial experiment was not repeated in two subsequent experiments. Therefore, several experiments were carried out to see if factors previously reported to influence ascorbic acid responses in poultry, such as temperature (Thornton et al., 1959), microbial infection, and antibiotic treatment (Hill and Garren, 1955, 1958) or vitamin D3
^Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia.
status (Thornton et al., 1959; Thornton and Brownrigg, 1961; Ramp and Thornton, 1966; Thornton and Omdahl, 1969) may be influencing the effects of ascorbic acid supplementation on the development of tibial dyschondroplasia. MATERIALS AND METHODS
General Procedures. Newly hatched male broiler chickens were used in all experiments. All experiments were conducted in electrically heated battery brooders with wire mesh floors, with feed and water always available. The basal diet shown in Table 1 was used with slight modifications in the various experiments. Materials were added to the basal diet at the expense of ground com. Experiments 1 to 6 were conducted for 20 days, and Experiment 7 was conducted for 16 days. At the termination of the experiments, birds were weighed by pens and their feed consumption recorded. They were then killed and examined at random for tibial dyschondroplasia; a longitudinal cut was made across the right tibia, which was scored for incidence and severity (Edwards and Veltmann, 1983). A photograph illustrating the scoring system used to judge degree of tibial dyschondroplasia in 3-wk-old chickens has been published (Edwards and Veltmann, 1983). The system
1527
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ABSTRACT Seven experiments were conducted to test the influence of dietary supplementary ascorbic acid on the development of tibial dyschondroplasia in broiler chickens. Ascorbic acid supplementation significantly reduced the incidence and number of birds with a large mass of cartilage in the tibia in the first experiment but not in the two subsequent experiments. Because environmental temperature, microbial infection, and vitamin D 3 status had been reported in the literature to influence ascorbic acid metabolism in the chicken, experiments were conducted to see if these variables could influence supplemental ascorbic acid effects on development of tibial dyschondroplasia. Results of the experiments indicated that none of these factors influenced the effect of ascorbic acid on the development of tibial dyschondroplasia. The presence of vitamin D3 in the diet significantly influences the incidence of this disorder. (Key words: ascorbic acid, tibial dyschondroplasia, temperature, chlortetracycline, chicken)
1528
EDWARDS, JR. TABLE 1. Composition of the basal diet
Ingredients
Amounts
Ground yellow corn Denuded soybean meal Poultry fat (stabilized) Iodized salt DL-Methionine (feed grade) Vitamin premix1 Trace mineral premix2 Selenium concentrate (.02% from sodium selenite) Dicalcium phosphate (feed grade) Ground limestone
56.81 35.00 5.00 .45 .20 .25 .10 .05 1.86 .28
assigns scores from 0 to 3: 0) normal cartilage, narrow with little irregularities; 1) cartilage is thickened or shows considerable irregularities; 2) cartilage is thickened with evidence of persisting prehypertrophic cartilage that is not calcified and has not been invaded by vessels from the metaphysis; deep irregularities of such cartilage are apparent; and 3) large mass of cartilage in the proximal end of the tibia. The left tibia was removed for bone ash determination on the fat-free dry basis (Association of Official Agricultural Chemists, 1955). The analysis of variance and regression analysis were computed using the general linear model procedure (Helwig and Council, 1979). Where appropriate, main effect means were separated by Duncan's multiple range test. Ascorbic Acid Supplementation (Experiments 1 to 3). These experiments were conducted to determine the effect of ascorbic acid supplementation on the incidence and severity of tibial dyschondroplasia in broilers fed a diet low in calcium (.65%) and containing adequate dietary levels of phosphorus (.71%), chloride (.32%), and vitamin D 3 (1,100 ICU/kg) and known to induce a high incidence of the disorder (Edwards and Veltmann, 1983). In these three experiments, six pens of ten cockerels were fed each dietary treatment. The cockerels were obtained from a
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on December 24, 2014
1 Vitamin premix provides in milligrams/kilogram of diet (except as noted): vitamin A (as all-tra/ts-retinyl acetate), 5,500 IU; cholecalciferol, 1,100 ICU; vitamin E (all-rac-atocopheryl acetate), 11 IU; riboflavin, 4.4; d-calcium pantothenate, 12; nicotinic acid, 44; choline chloride, 220; vitamin Bj2, 6.6 fig; vitamin Bg, 2.2; menadione (as menadione sodium bisulfite), 1.1; thiamin (as thiamin mononitrate), 2.2; and ethoxyquin, 125. ^ r a c e mineral premix provides in milligrams per kilogram of diet: Mn0 2 , 222; ZnO, 150; FeS0 4 -7H 2 0, 200; FeC0 3 , 83; CuS0 4 , 29; and Ca (I0 3 ) 2 , 15.
commercial broiler hatchery and were Ross x Arbor Acre for Experiment 1 and Petersen x Arbor Acre for Experiments 2 and 3. In Experiments 1 and 2, the dietary treatments were the basal diet (Table 1) and this diet supplemented with 500 mg/kg of ascorbic acid. The basal diet and a series of diets containing 250, 500, and 1,000 mg/kg of ascorbic acid were fed in Experiment 3. Environmental Temperature (Experiments 4 to 5). These experiments were conducted to determine if environmental temperature could be a variable in determining when an effect was obtained from ascorbic acid supplementation on the incidence of tibial dyschondroplasia. The experiments were conducted in three rooms where the environmental temperatures were maintained at approximately 18 C, 24 C, and 30 C. The brooder temperatures were maintained at between 32 to 35 C during the course of the experiment. In each room, six pens of ten birds each were fed die basal diet or this diet supplemented with 500 mg/kg of ascorbic acid. Room Fumigation and Antibiotic Feeding (Experiment 6). This experiment was conducted in two Petersime (Petersime Incubator Co., Gettysburg, OH) battery brooders, located in identical rooms (6.2 m wide x 7.4 m long x 2.5 m high). One room received normal cleaning of the battery and room with water and detergent before the start of the experiment. The otfier room and battery were cleaned in die same manner; the room was then sealed witii paper and tape and fumigated with 500 g KMn04 and 900 mL formaldehyde overnight. Three dietary treatments in each room were basal diet (Table 1), basal + 500 mg/kg ascorbic acid, and the basal + chlortetracycline concentrate to give 100 mg chlortetracycline/ kg of diet. Four pens of 10 Petersen x Arbor Acre cockerels were fed each dietary treatment in each room. Vitamin D3 Deficiency (Experiment 7). This experiment was conducted to determine if ascorbic acid supplementation of the diet would influence the development of tibial dyschondroplasia in the vitamin D3-deficient chicken. The vitamin mixture for this study supplied the same amounts of each vitamin as the one shown in Table 1 except it contained no cholecalciferol and it supplied a slighdy higher level of vitamin B12 to the diet (9 |0.g/ kg). The treatments were: vitamin D3-deficient basal diet, the basal diet plus 1,100 ICU/kg of
VITAMIN C AND ENVIRONMENT ON TIBIAL DYSCHONDROPLASIA
vitamin D3, the basal diet plus 500 mg/kg of ascorbic acid, and the basal diet plus 1,100 ICU/kg of vitamin D3 plus 500 mg/kg of ascorbic acid. Six pens of ten Petersen x Arbor Acre cockerels were fed each dietary treatment. The fluorescent lights in the battery brooder were turned off, the fluorescent lights in the room were covered with plastic, and the blind for the outside window was drawn during this experiment. RESULTS
Room Fumigation and Antibiotic Feeding (Experiment 6). Fumigation of the room did
not have a significant effect on any of the criteria measured (Table 4). The addition of chlortetracycline to the diet caused a significant increase in the 20-day weights and gain: feed ratio but had no effect on any of the other criteria studied. Vitamin D3 Deficiency (Experiment 7). Addition of ascorbic acid to the vitamin D3deficient or supplemented diet caused a significant decrease in bone ash and an increase in the incidence of tibial dyschondroplasia (Table 5). The addition of vitamin D3 to the diets, with or without ascorbic acid, caused significant increases in 16-day weight and bone ash, while causing a significant decrease in the incidence and number of Number 3 scores for tibial dyschondroplasia. The results and statistical analysis do not indicate any vitamin D3 x ascorbic acid interaction. DISCUSSION
Results obtained in these studies confirm the report of Leach and Burdette (1985), which indicated that dietary ascorbic acid was not involved in the development of tibial dyschondroplasia in young broiler chickens. The effects of ascorbic acid in all seven experiments may be summarized as follows: positive effects were decreased incidence and number of Number 3 scores of tibial dyschondroplasia in Experiment 1 and increased growth rate in Experiment 2; negative effects were decreased bone ash in Experiments 4, 5, and 7 and increased incidence of tibial dyschondroplasia in Experiment 7. Only the effect of ascorbic acid in decreasing bone ash would appear to be consistent enough to conclude that it may be a true effect and not just a chance occurrence. The other possibility of course still exists that some factor that influences the effect that ascorbic acid has on the development of tibial dyschondroplasia is not under control in these experiments and the effect of ascorbic acid as seen in Experiment 1 would be repeatable under more controlled experimental conditions. The chickens used in Experiment 1 were of different breeding (Ross x Arbor Acre) than those used in the other six experiments. A previous report from this laboratory (Edwards, 1984) has shown a difference in the commercial strains of broilers in their susceptibility to developing tibial dyschondroplasia. However, in those studies there were no significant differences in susceptibility of the two particular strains used in the present studies (Ross x Arbor Acre and Petersen x Arbor Acre). The
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Ascorbic Acid Supplementation (Experiments 1 to 3). Results of the experiments on the effect of ascorbic acid supplementation on tibial dyschondroplasia are presented in Table 2. Ascorbic acid supplementation caused a significant decrease in the incidence of tibial dyschondroplasia in Experiment 1 but had no effect in Experiments 2 and 3. Ascorbic acid supplementation also caused a significant increase in growth rate in Experiment 2. In both Experiments 1 and 2, the number of birds scored as having Number 3 lesions was reduced by ascorbic acid supplementation, but there was no significant effect on the overall score. Environmental Temperature (Experiments 4 to 5). Results of the experiments to determine if environmental temperature influences the effect of ascorbic acid supplementation on the incidence of tibial dyschondroplasia are presented in Table 3. Significant differences between experiments were found for gain:feed and bone ash; however, for both of these criteria, no significant interactions of experiment and ascorbic acid or experiment and temperature were found; therefore, analysis of the combined experiments is presented. Temperature had a significant effect on all of the criteria measured: the high temperature (30 C) caused lower 20-day weights, gainrfeed ratio, bone ash, and incidence and severity of tibial dyschondroplasia than the low temperature (18 C). The addition of ascorbic acid to the diet significantly decreased bone ash but had no significant effect on any of the other criteria measured. However, in the case of body weights at 20 days of age and incidence and severity of tibial dyschondroplasia, there was a significant temperature x experiment interaction, indicating some experimental betweenexperiments variable caused different responses to temperature in the two experiments.
1529
Ascorbic acid supplementation
1
.77 .91
.40
.68
.38
38.1 37.3 37.9 37.5 ±.3
(%) 38.2 38.3 ±.2 39.2 39.2 ±.4
Bone ash
.92 .20
(g/g) .664 .665 ±.010 .667 .676 ±.005 .635 .650 .654 .647 ±.016
Gain:feed ratio
.32 .03
(g) 559 569 ±9 508 530* ±6 555 537 551 539 ±9
BW, 20 days of age
*P<05.
Number of birds that scored Number 3/number of birds examined at the end of the experiment.
(mg/kg) None 500 Pooled SE 2 None 500 Pooled SE 3 None 250 500 1,000 Pooled SE ANOVA, probabilities Treatments, Experiment 1 (df, 1) Treatments, Experiment 2 (df, 1) Regression analysis Experiment 3 Ascorbic acid, linear (df 1)
Experiment
.60
.03 .19
(%) 59 34* ±7 68 58 ±5 38 51 43 46 ±7
Incidence
TABLE 2. Effect of dietary ascorbic acid supplementation on tibial dyschondroplasia (Exp
om http://ps.oxfordjournals.org/ at Michigan State University on December 24, 2014
'Number of birds that scored Number 3/number of birds examined at the end of the experiment.
.0 .2 .0 .0 .0 .4 .6
30 35
40.8" 40.3 b
<001 .001 .044 <001 .339 .309 .471
36a 37a 23 b
<*) 33 31 9 31 52 14 ±6 48 27 30 34 38 39 ±8
Inci
40.7" 40.8* 40.2b
(%) 41.2 41.6 40.9 41.3 40.7 40.7 ± .2 40.3 40.8 40.0 40.2 40.0 39.2 ± .2
Bone ash
'""'Values of the same variable with no common superscripts are significantly different (P<.05).
<.001 .944 .785 <.001 .164 .630 .493
.695 .695
601 595
<001 .318 .519 .734 <.001 .471 .036
.700" .715" .670b
.670 .700 .652 ±.012 .721 .741 .674 .739 .729 .680 ±.020
(g/g) .672 .690 .674
Gain:feed ratio
593b 627a 573 c
565 646 601 567 618 596 ±10
(g) 635 616 543 605 630 552 ±10
(mg/kg) None None None 500 500 500
(C) 18 24 30 18 24 30 Pooled SE 18 24 30 18 24 30 Pooled SE
None None None 500 500 500
BW, 20 days of age
Ascorbic acid supplementation
Room temperature
ANOVA probabilities (Experiments 4 + 5 combined) Sources of variation, df Temperature (T), 2 Ascorbic acid (AA), 1 T x AA, 2 Experiment (EXP), 1 T x EXP, 2 AA x EXP, 1 T x AA x EXP, 2
Main effect means Temperature 18 24 30 Ascorbic acid None 500 mg/kg
5
4
Experiment
TABLE 3. Effect of environmental temperature and ascorbic acid on tibial dyschonaropla
om http://ps.oxfordjournals.org/ at Michigan State University on December 24, 2014
.261 .758 .334
'Number of birds that scored Number 3/mimber of birds examined at the end of the experiment.
' Values of the same variable with no common superscripts are significantly different (P<05).
ab
.003 .070 .082
39 37
38.2 38.6
.646 .673
523 535
.038 .393 .672
44 36 33
38.4 37.3 39.5
.628b .653b .696"
.447 .775 .925
±8
38.3 36.2 40.3 38.5 38.5 38.7 ±1.3
505 b 529 ab 553"
Pooled SE Main effect means Diet supplements None Ascorbic acid Chlortetracycline Fumigated room Yes No ANOVA, probabilities Sources of variation, df Diet, 2 Fumigated room, 1 Diet x fumigated room, 2
No
(%) 43 38 35 45 33 32
(%)
(g/g) .592 .658 .680 .662 .645 .709 ±.017
None Ascorbic acid Chlortetracycline None Ascorbic acid Chlortetracycline
Yes
Incidence
Bone ash
Gain:feed ratio
BW, 20 days of age
Diet supplementation
Fumigated room
TABLE 4. Effect of ascorbic acid, chlortetracycline, and room fiimigation on the development of tibi
om http://ps.oxfordjournals.org/ at Michigan State University on December 24, 2014
.241 .001 .724
.236 .146 .821
.708 .673
30.1 b 31.2a
.679 .705
330 344
367" 306b
± .5
.698 ±.021
359 ±11
'Number of birds that scored Number 3/number of birds examined at the end of the experiment.
.028 .001 .607
34.2* 27.2 b
33.5
27.6 26.8 34.9
(%)
(g/g) .692 .651 .719
(g) 311 302 376
Bone ash
Gain:feed ratio
BW, 16 days of age
"'Values of the same variable with no common superscript are significantly different (P<.05).
ANOVA, probabilities Sources of variation, df Ascorbic acid, 1 Vitamin D3, 1 Ascorbic x vitamin D3, 1
Vitamin D3 1,100 ICU/kg None
None Ascorbic acid, 500 mg/kg Vitamin D 3 , 1,100 ICU/kg Ascorbic acid, 500 mg/kg plus vitamin D 3 , 1,100 ICU/kg Pooled SE Main effect means Ascorbic acid 500 mg/kg None
Supplements to basal diet
TABLE S. Effect of dietary ascorbic supplementation on the development of tibial dyschondroplasia by ch in vitamin D3 (Experiment 7)
om http://ps.oxfordjournals.org/ at Michigan State University on December 24, 2014
1534
EDWARDS, JR.
ACKNOWLEDGMENTS
The author wishes to thank Frances Denman for her technical assistance. The advice of Glenn Ware regarding the statistical analysis is appreciated. REFERENCES Association of Official Agricultural Chemists, 1955. Official Methods of Analysis. 8th ed. Assoc. Offic. Agric.
Chem., Washington, DC. Edwards, H. M., Jr., 1984. Studies on the etiology of tibial dyschondroplasia in chickens. J. Nutr. 114: 1001-1013. 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. Hanssen, I., H. J. Grav, J. B. Steen, and H. Lysnes, 1979. Vitamin C deficiency in growing willow ptarmigan (Lagopus lagopus lagopus). J. Nutr. 109:2260-2276. Helwig, J. T., and K. A. Council, eds., 1979. SAS User's Guide. SAS Inst. Inc., Raleigh, NC. Hill, C. H., and H. W. Garren, 1955. The effect of high levels of vitamins on the resistance of chicks to fowl typhoid. Ann. N.Y. Acad. Sci. 63:186-194. Hill, C. H., and H. W. Garren, 1958. Plasma ascorbic acid levels of chicks with fowl typhoid. Poultry Sci. 37: 236-237. Horio, F., K. Ozaki, A. Yoshida, S. Makino, and Y. Hayashi, 1985. Requirement for ascorbic acid in a rat mutant unable to synthesize ascorbic acid. J. Nutr. 115: 1630-1640. Leach, R. M., Jr., and J. H. Burdette, 1985. The influence of ascorbic acid on the occurrence of tibial dyschondroplasia in young broiler chickens. Poultry Sci. 64: 1188-1191. Leach, R. M., Jr., and M. C. Nesheim, 1965. Nutritional, genetic and morphological studies of an abnormal cartilage formation in young chicks. J. Nutr. 86: 236-244. Makino, S., and K. Katagiri, 1980. Osteogenic disorder rat. Exp. Anim. (Tokyo) 29:374-375. Murray, P.D.F., and E. Kodicek, 1949. Bones, muscles and vitamin C. III. Repair of the effects of total deprivation of vitamin C at the proximal ends of the tibia and fibula in guinea pigs. J. Anat. 83:285-293. Ramp, W. K., and P. A. Thornton, 1966. Skeletal response to exogenous ascorbic acid by vitamin D3 deficient chicks. Proc. Soc. Exp. Biol. Med. 121:1248-1250. Thornton, P. A., and D. Brownrigg, 1961. Calcium utilization and skeletal development in chicks as influenced by parental dietary ascorbic acid. J. Nutr. 75:354-360. Thornton, P. A., and J. L. Omdahl, 1969. Further evidence of skeletal response to exogenous ascorbic acid. Proc. Soc. Exp. Biol. Med. 132:618-621. Thornton, P. A., C. W. Weber, and R. E. Moreng, 1959. The effect of ascorbic acid in the diet of adult chickens on calcium utilization by progeny. J. Nutr. 69:33-38.
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possibility that genetic differences in the ability of broiler chickens to synthesize ascorbic acid might cause deficiencies in some chickens of certain strains still exists. The production of a chicken mutant that cannot convert gulonolactone to ascorbic acid and the subsequent study of ascorbic acid deficiency in chickens is desirable. A mutant rat strain that cannot convert gulonolactone to ascorbic acid has recently been isolated (Makino and Katagiri, 1980; Horio et al., 1985). High environmental temperature significantly reduced the incidence and score of tibial dyschondroplasia, and this can be partially accounted for by the reduction in growth rate that resulted from the high temperature. The small but significant lowering of the percentage of bone ash in broilers reared at the highest temperature (30 C) is unusual in view of the effects on growth and incidence of tibial dyschondroplasia. The effects of vitamin D3 deficiency produced in Experiment 7 was not as severe on growth rate as expected after the precautions had been taken to reduce the ultraviolet light on the chickens. This probably indicates that substantial vitamin D3 reserves from the dams were present in the day-old chicks.