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Effect of vitamin D and sunlight on growth and bone development of young ponies

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Title:
Effect of vitamin D and sunlight on growth and bone development of young ponies
Creator:
El Shorafa, Waleed M., 1948-
Publication Date:
Language:
English
Physical Description:
xi, 101 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Ashes ( jstor )
Bones ( jstor )
Calcium ( jstor )
Horses ( jstor )
Phosphatases ( jstor )
Phosphorus ( jstor )
Plasmas ( jstor )
Rats ( jstor )
Rickets ( jstor )
Vitamin D ( jstor )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Horses -- Growth ( lcsh )
Rickets ( lcsh )
Vitamin D in the body ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 93-100.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Waleed M. El Shorafa.

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EFFECT OF VITAMIN D AND SUNLIGHT ON GROWTH AND BONE
DEVELOPMENT OF YOUNG PONIES










By

WALEED M. EL SHORAFA


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY













UNIVERSITY OF FLORIDA


1978




































This dissertation is dedicated to my wife, Annalee, to my daughter, Rhonda, for their love, understanding, and patience during the course of endeavor, and to my mother and father for their faith in my potential.

















ACKNOVIEDGEMENTS


The author owes a most sincere debt of gratitude to Dr. J.P. Feaster, who served as chairman of the supervisory committee, for his patience and understanding during the course of this endeavor. Dr. E.A. Ott was most helpful in his guidance and instruction through the research phase conducted at the Horse Research Center, Ocala. Special thanks to Dr. R.L. Shirley, Dr. C.M. Allen, Jr., and Dr. D.C. Sharp III as members of this graduate committee and for their guidance to make this study possible. The author also thanks C. Albiol, manager, as well as all other employees of the Horse Research Center who helped to make the work less of a chore and more of a learning experience. Special thanks to Dr. Asquith for this sincere help in the radiographs carried out for this project. The thanks extend to Dr. Littell, W. Offen, M. Vernon, and C.R. Smith for their aid in statistical and laboratory analysis. Special thanks to Dr. A.E. Green and his assistants for their aid in determination of ultraviolet light.


i iii
















TABLE OF CONTENTS


ACKNOWLEDGEMENTS. . . . . LIST OF TABLES. . . . . . LIST OF FIGURES...... ABSTRACT. . . . . . . . . CHAPTER

I INTRODUCTION . . .

II LITERATURE REVIEW.

Introduction . . .

Rickets in Rats. .

General Remarks on Rickets in Poultry Rickets in Swine .

Rickets in Cattle.

Rickets in Humans.

Rickets in Horses.


Page

iii


. . . . . . . . . . . . . . . . . . v i

. . . . . . . . . . . . . . . . . v i i i

. . . . . . . . . . . . . . . . . . ix


Rickets


in


Rats


1

3

3

4

11 11 15 18 19 25 27 27

29 30 30 31 31 31 31 31


III MATERIAL AND METHODS .


Conditions and Experimental Design Sample and Measurements. . . . . . Laboratory Analysis. . . . . . . .


Blood plasma calcium and magnesium determination. Blood plasma phosphorus determination . . . . . . Blood plasma alkline phosphatase determination. . Plasma 25-hydroxy vitamin D determination . . . . Bone ash and cortex aria. . . . . . . . . . . . .
Ca, P, and M" bone ash. . . . . . . . . . . . . .


.











Page

Bone breaking strength. . . . . . . . . . . . . . . . 32
Radiographic classification . . . . . . . . . . . . . 32
Kidney and liver analysis . . . . . . . . . . . . . . 34

Statistical Analysis . . . . . . . . . . . . . . . . . . . 34

IV RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . 36

External Appearance. . . . . . . . . . . . . . . . . . . . 36

Rate of Growth and Feed Efficiency . . . . . . . . . . . . 38

Blood Analysis . . . . . . . . . . . . . . . . . . . . . . 42

Plasma calcium (Ca), phosphorus (P), and
magnesium (Mg) levels . . . . . . . . . . . . . . . 42
Plasma alkaline phosphatase . . . . . . . . . . . . . 54
Plasma 25-hydroxy vitamin D . . . . . . . . . . . . . 56

Bone Analysis. . . . . . . . . . . . . . . . . . . . . . . 59

Water concentration of bone . . . . . . . . . . . . . 59
Bone ash (percent of dry, fat-free bone).. . . . . . . 62
Bohe calcium, phosphorus, and magnesium (% of ash). . 62
Bone cortex area. . . . . . . . . . . . . . . . . . . 63
Bone density. . . . . . . . . . . . . . . . . . . . . 65
Bone breaking strength. . . . . . . . . . . . . . . . 65
Bone epiphyseal closure . . . . . . . . . . . . . . . 66

Liver Analysis (Water, Ash, Calcium, Phosphorus, and
Magnesium Concentration) . . . . . . . . . . . . . . . . . 70

Kidney Analysis (Water, Ash, Calcium, Phosphorus, and
Magnesium Concentration) . . . . . . . . . . . . . . . . . 72

V SUMMARY AND CONCLUSION . . . . . . . . . . . . . . . . . . 73

APPENDIX ANALYSIS OF VARIANCE TABLES . . . . . . . . . . . . . 80

LITERATURE CITED. . . . . . . . . . . . . . . . . . . . . . . . . 93


BIOGRAPHICAL SKETCH . . . . . . . . . . . . . . . . . . . . .1


101


















LIST OF TABLES


Table Page

1. COMPOSITION OF THE VITAMIN D-DEFICIENT DIET . . . . . . . . 28

2. EFFECT OF VITAMIN D AND SUNLIGHT ON RATE OF GAIN AND
FEED EFFICIENCY OF PONIES . . . . . . . . . . . . . . . . . 39

3. AVERAGE WEEKLY PLASMA CALCIUM LEVELS DURING 27 WEEKS OF EXPERIMENTAL PERIOD. . . . . . . . . . . . . . . . . . . 43

4. AVERAGE PLASMA PHOSPHORUS LEVELS DURING 27 WEEKS OF EXPERIMENTAL PERIOD . . . . . . . . . . . . . . . . . . . . 47

5. AVERAGE PLASMA MAGNESIUM LEVELS DURING 27 WEEKS OF EXPERIMENTAL PERIOD . . . . . . . . . . . . . . . . . . . . 50

6. PLASMA ALKALINE PHOSPHATASE AT 0, 4, 12, 20, and 27 WEEK PERIOD . . . . . . . . . . . . . . . . . . . . . . . . 55

7. INDIVIDUAL AND AVERAGE VALUES OF PLASMA 25-HYDROXY VITAMIN D OF THE TWELVE PONIES. . . . . . . . . . . . . . . 58

8. SELECTED COMPOSITION AND PHYSICAL CHARACTERISTICS OF RIGHT METACARPAL BONE . . . . . . . . . . . . . . . . . . . 60

9. SELECTED COMPOSITION AND PHYSICAL CHARACTERISTICS OF LEFT METACARPAL BONE. . . . . . . . . . . . . . . . . . . . 61

10. EPIPHYSEAL CLOSURE OF THE DISTAL OF THE THIRD METACARPAL
(MD), THE PROXIMAL OF THE FIRST PHALANX (PIP), AND THE
SECOND PHALANX (P2P) OF BONES OF GROUP I PONIES . . . . . . 67 11. AVERAGE WATER, ASH, CALCIUM, PHOSPHORUS, AND MAGNESIUM
CONCENTRATIONS OF LIVER AND KIDNEY. . . . . . . . . . . . . 71

12. ANALYSIS OF VARIANCE OF FEED EFFICIENCY . . . . . . . . . . 80

13. WEEKLY PLASMA CALCIUM LEVELS FOR INDIVIDUAL PONIES. . . . . 81 14. ANALYSIS OF VARIANCE FOR PLASMA CALCIUM LEVELS. . . . . . . 82 15. WEEKLY PLASMA PHOSPHORUS LEVELS FOR INDIVIDUAL PONIES . . . 83 16. WEEKLY PLASMA MAGNESIUM LEVELS FOR INDIVIDUAL PONIES. . . . 84












Table Page

17. ANALYSIS OF VARIANCE FOR PLASMA ALKALINE PHOSPHATASE . . . . 85 18. ANALYSIS OF VARIANCE FOR PLASMA 25-OH VITAMIN D. . . . . . . . 86 19. ANALYSIS OF VARIANCE FOR SELECTED COMPOSITION OF BONES . . . 87 20. ANALYSIS OF VARIANCE FOR SELECTED PHYSICAL CHARACTERISTICS
OF BONES . . . . . . . . . - . . . . . . . . . . . . . . . . 88

21. WATER, ASH, CALCIUM, PHOSPHORUS, AND MAGNESIUM CONCENTRATION OF LIVER AND KIDNEY FOR INDIVIDUAL PONIES. . . . . . . 89 22. ANALYSIS OF VARIANCE FOR LIVER, WATER, ASH, CALCIUM,
PHOSPHORUS, AND MAGNESIUM CONCENTRATIONS . . . . . . . . . . 91 23. ANALYSIS OF VARIANCE FOR KIDNEY WATER, ASH, CALCIUM,
PHOSPHORUS, AND MAGNESIUM CONCENTRATIONS . . . . . . . . . . 92


i 11


















LIST OF FIGURES


Figure Page

1. INSTRON TENSIL STRENGTH TESTING APPARATUS FOR DETERMINING BREAKING STRENGTH OF BONES . . . . . . . . . . . . . 33

2. EFFECTS OF TREATMENT ON EXTERNAL APPEARANCE OF 3 OF
THE PONIES IN GROUP I . . . . . . . . . . . . . . . . . . . 37

3. WEEKLY BODY WEIGHT OF GROUP I PONIES.. . . . . . . . . . . . 40

4. WEEKLY BODY WEIGHT OF GROUP II PONIES . . . . . . . . . . . 41

5. AVERAGE WEEKLY PLASMA CALCIUM OF GROUP I. . . . . . . . . . 45

6. AVERAGE WEEKLY PLASMA CALCIUM OF GROUP II . . . . . . . . . 46 7. AVERAGE WEEKLY PLASMA PHOSPHORUS IN GROUP I . . . . . . . . 48 8. AVERAGE WEEKLY PLASMA PHOSPHORUS IN GROUP II. . . . . . . . 49 9. AVERAGE PLASMA MAGNESIUM LEVELS IN GROUP I. . . . . . . . . 52 10. AVERAGE PLASMA MAGENSIUM LEVELS IN GROUP II . . . . . . . . 53 11. STANDARD CURVE FOR THE COMPETITIVE BINDING ASSAY ON
25-OH VITAMIN D 3. .. ..... ..... ....... 57

12. EFFECT OF TREATMENTS ON METACARPAL BONES OF THREE PONIES
OF GROUP I. . . . . . . . . . . . . . . . . . . . . . . . . 64

13. EFFECT OF TREATMENTS ON THE EPIPHYSEAL CLOSURE OF
GROUP I . . . . . . . . . . . . . . . . . . . . . . . . . . 68

14. EFFECT OF TREATMENTS ON EPIPHYSEAL CLOSURE OF GROUP II. . . 69


vi i i














Abstract of Dissertation Presented to the Graduate Council,
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECT OF VITAMIN D AND SUNLIGHT ON GROWTH AND BONE
DEVELOPMENT OF YOUNG PONIES

By

Waleed M. El Shorafa

March, 1978

Chairman: Dr. J.P. Feaster
Major Department: Animal Science

A 2 x 3 factorial design experiment was carried out to study the

effect of vitamin D and sunlight on growth and bone development of young ponies. Two groups of ponies (G = two months of age and G2 = eight months of age) were assigned to three treatments (T = no sunlight without vitamin D supplement, T = no sunlight with a daily supplement of 1,000 I.U. of vitamin D, T = sunlight without vitamin D supplement). All ponies were put in a windowless barn with very minimum ultraviolet light exposure for one month (to deplete their body stores of vitamin D) and then were assigned to one of the above treatments. A vitamin Ddeficient diet, adequate in all other nutrients required for optimum growth, was fed to the ponies three times daily (ad libitum) for five months.

Weekly blood samples were obtained to study the effect of treatments and age groups on plasma calcium (Ca), phosphorus (P), magnesium (Mg), alkaline phosphatase (Alk), and 25-hydroxy vitamin D (25-OH Vit. D) levels. Monthly X-rays were taken to study the effect of treatments

and groups on the development of epiphyseal plates at the end of the











long bones of the front legs. All ponies were killed at the termination of the experiment. The two front metacarpal bones, liver, and kidney were obtained for determination of water, ash, Ca, P, and Mg concentration. One of the metacarpal bones was used to determine bone cortex area and the other was used to determine bone breaking strength and bone density.

The data were analyzed factorially by statistical analysis. In case of interaction effect, a Duncan's multiple range test was done to show the effect of treatments within the groups.

Loss of appetite, difficulty in standing, and slightly lower feed efficiency were found in T1, compared to T2 and T but the actual ex33
ternal appearance of rickets (bowed legs and inability to stand) did not occur.

Plasma Ca, P, and Mg were within the normal range and there were no significant differences among treatments. The results indicated higher plasma Ca levels (p < .01) of group II than group I (G = 10.5-13.5 vs. G2 = 12.6-15.2 mg/100 ml). Plasma alkaline phosphatase levels varied and were not affected by age or treatments. Plasma 25-hydroxy vitamin D levels were normal for all the ponies, and there were no significant differences among treatments. Average plasma 25-OH vitamin D for T, T 2 and T3 were 63.4, 64.1, and 61.2 ng/ml. Ponies in group II (G ) had significantly (p < .01) higher plasma 24-OH vitamin D than ponies in group I. For G and G2 the levels were 67.8 and 57.9 ng/ml.

Bone water concentrations were higher in TI (p < .05) than T2 and T 3 and in G1 (p < .05) than G2 ponies. Ponies on treatment 1 (T ) had lower bone ash concentration (p , .01) than ponies on T2 and I 3 of dry fat-free bone, but there were no sign icant differences between











groups. Calcium, P, and Mg as percent of bone ash were not affected by either treatments or groups. Bone cortex area of ponies in T was lower (p < .07) than T2 and T3 and group 11 had a higher cortex area (p < .05) than group I. Bones in T appear to have low bone breaking strength
2 ?2
(1308.8 kg/cm ) than T2 (1583.1 kg/cm2) or T3 (1583.1 kg/cm ). Bone breaking strength was higher (p , .03) in G2 than G . The epiphyseal plates (at the distal end of the metacarpal bone, proximal end of the first phalanx and the proximal end of the second phalanx) of T within group I were irregular, wider, poorly defined, and late in closing, compared to T2 and T 3

Factorial analysis indicated that ash concentration of liver and

kidney were not affected by treatments or age groups. Water concentration of liver and kidney were lower (p < .02) in G2 than G . Ponies in group 1 had higher Ca (p < .01) and P (p < .01) liver concentration than ponies in group II. There was an interaction effect concerning liver Mg concentration. Duncan's multiple range test indicated that magnesium level under T3 was significantly lower (p < .05) from TI or T2 within group I. Calcium, P, and Mg kidney concentrations were not affected by either age groups or treatments.

In conclusion, the ponies in T (especially those in the younger age) suffered from early symptoms of rickets compared to T2 and T3 ponies. The symptoms were loss of appetite, low feed efficiency, and low bone ash and breaking strength with epiphyseal plates of the long bones which were wider, irregular, lacking definition, and delayed in closure.


Xi


















CHAPTER I

INTRODUCTION



For several decades, scientists have recognized the significance of an agent that prevents rickets in young animals. This agent has been identified as vitamin D and its active form is the hormone 1,25 dihydroxy vitamin D. Animals obtain their needed vitamin D either from exposure of their skin to ultraviolet light or from diet. The study of vitamin D and its relation to rickets needs further investigation, especially after the recent increase in the knowledge of vitamin D metabolism and mechanism of action. Rickets is of interest to ecologists, since it is probably the first disease which could be classified as a result of air pollution, and to anthropologists, who have postulated that the effect of ultraviolet light on vitamin D metabolism in the skin was an important factor in the development of races with pigmented and non-pigmented skin

and in their geographical distribution. The disease is of interest to physiologists, since it is an example of a deficience of a single element, and to biochemists, because recent data on vitamin D metabolism not only explain the rachitic syndrome, but may uncover many of the mysteries of mineral regulatory mechanisms. It is, however, for the nutritionist that rickets holds the greatest interest.

Park (1923) stated that rickets is a common disturbance among puppies, pigs, lambs, poultry, and kids, but occ urs less frequently among colts, calves, and rabbits. It is easily produced in most domestic


I-








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animals by simply omitting vitamin D from the diet and preventing exposure to sunlight or other forms of ultraviolet radiation: In rats, however, lowered phosphorus intake also is required to produce rickets.

Techniques for producing and diagnosing rickets were developed

mostly in rats, dogs, poultry, and humans. There is a marked difference in species responses to vitamin D. It has been assumed that horses, like other animals, suffer adverse effects from a lack of vitamin D intake and the absence of ultraviolet light. This assumption is based on results with other animals, but does the young horse develop rickets? Occasionally, the colt, less than one year old, experiences a period of pain and shows an altered gait. The colt in most instances is not yet weaned and is receiving the major portion of its nutrition from the mare's milk. Regular radiography of the joint shows enlargements similar to those of rickets, but is it rickets? Much of the accepted knowledge of vitamin D and its importance in horse nutrition has been largely borrowed from other species. Currently there is a large volume of research to evaluate the actual nutritional requirements of horses. The horse industry has become a billion dollar twentieth century phenomenon. The

horse population is increasing in number and quality at a rapid pace. Universities, scientific foundations, and government facilities have begun allocating time and funds to equine research. Since horses have been bred for the purpose of racing, showing, and pleasure, bone development in young horses is an important factor.

The present study was carried out to determine if horses need

vitamin D or if sunlight is adequate to bring about optimum bone development.


















CHAPTER Ii

LITERATURE REVIEW



Introduction


The discovery of the cause and cure of rickets is one of the great triumphs of biochemical medicine. Rickets is known as a bone disease which occurs in young animals. As a result of poorly calcified bone, supporting weight is painful and results in lameness or disinclination to move. A clear description of the clinical picture of rickets was published in the 17th century, although mention of various aspects of

the syndrome goes back even further.

The first successful attempt to induce rickets experimentally in

animals was made at the University of Glasgow in 1908 by Leonard Findlay, who published conclusive pictures of puppies that had been confined in cages and developed rickets. Melanby (1919) also used pups in his early work on rickets and concluded that rickets was due to a deficiency of a specific dietary factor. These results aroused considerable interest and led to a large number of investigations in this field during the following decade. Dogs, hens, and rats have been used most for experimental studies of rickets. Accumulated knowledge on rickets shows that there are biochemical changes in bone, kidney, liver, and blood and definite external manifestations of the disease in the animals. In studies of the biochemical changes in blood in rickets, most research

has dealt with the levels of calcium, phosphorus, magnesium, 25-OH








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vitamin D, and alkaline phosphatase. Calcium, P, Mg, ash content, organic matter, water content, breaking strength, and cortex area of the bone have also been investigated. Radiographic study of the epiphyseal closure provides further evidence in diagnosis of rickets. In addition to these, the external appearance, growth, and feed efficiency have been measured to show rickets. Since liver and kidney are very much involved in vitamin D metabolism and its relation to Ca, P, and Mg regulation in the body, the level of these minerals in the liver and kidney has been studied. The findings of research which has shown the biochemical, radiographic, and clinical abnormalities of rickets in different species of animals are described below in some detail.



Rickets in Rats


Rats are not as susceptible to rickets as are the higher mammals

and poultry. Typical rickets in this type of animal can be produced only if the diet is abnormal with respect to calcium or phosphorus as well as deficient in vitamin D. The earliest diets of this type were developed by McCollum et al. (1922). The diet was composed principally of cereals and is high in calcium and moderately low in phosphorus. McCollum et al.'s experiment demonstrated clearly the existence of a growth factor or vitamin in the diet which regulates bone metabolism.

Steenbock and Nelson (1923) laid the foundation for an experimental method in which, by the use of ultraviolet light, growth can be used as a measure of the comparative amount of vitamin D occurring in food.

Steenbock and Black (unpublished data) showed no significant difference in growth of irradiated or non-irradiated rats. Shortly after this,







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Hume and Smith (1923) showed an increase in growth of irradiated rats compared with non-irradiated ones.

In 1922, it was found that X-ray images of the bones could provide visual evidence of richets. Pappenheimer et al. (1922) presented X-rays of the hind leg of a rachitic rat which showed a lack of calcification and a wide area of uncalcified cartilage at the junction of the diaphyses and the epiphyses.

Dutcher and Rothrock (1925) made detailed studies of the changes of the bone ash of rachitic rats. They reported a bone ash of 62% in dyr, fat-free bones from normal rats; in rachitic rats it was 26.5%.

Steenbock and Black (1924) reported that by irradiation with a

quartz mercury vapor lamp, rat rations could be activated, making them growth-promoting and bone-calcifying to the same degree as when the rats were irradiated directly. Also, their results indicated that liver taken from irradiated rats was growth-promoting while liver from nonirradiated rats was inactive. The same was found true of lung and muscle tissue. Inactive muscles, exposed after removal from the body to the radiations of the lamp, were found to have become activated, being both growth-promoting and bone-calcifying. Liver treated the same way also promoted bone calcification.

Again Steenbock and Black (1925) studied the induction of growthpromoting and calcifying properties in fats and their unsaponifiable constituents by exposure to light. Their results gave clear evidence of the antirachitic activity of irradiation of various fats to promote growth and increase calcium content of rat bones.

Dodds and Camerson (1943) studied the relation of rickets to growth with special reference to the bones. Their studies were based on 135








6-


albino rats, in most of which rickets was produced by the SteenbockBlack diet 2965. They traced the development and healing of rickets and growth of the bones by weekly roentgenograms. Graphic methods were used to show rickets, healing, and growth. The rachitic rats were sub-normal in weight, but their growth did not follow any single pattern. The growth of the leg bones and of the vertebral columns of the rachitic rats was greatly retarded. The retardation of the vertebral columns was relatively greater than of the leg bones. The epiphyseal cartilage of the tibia (typical for all long bones), during the first week or two on the rachitogenic diet, continued to make its contribution to the length of the shaft of Lh bone, but in decreasing amount. After about the third week the shaft ceased to elongate, and the pathologic thickening of the epiphyseal cartilage and the elongation of the bone became equal and identical.

Bethke et al. (1923-24) showed that with diets in which the Ca/P ratio was very high vitamin D did not induce growth.

Nicolaysen and Jansen (1939) compared the bones of vitamin Ddeficient and vitamin D-treated rats. Their results indicate no difference in the percentage of ash in the bones between the two groups, but the anatomical findings indicate a failure of newly formed matrix to calcify in the bones of the vitamin D-deficient animals. Unfortunately, Nicolaysen and Jansen did not give any data on the serum Ca and serum P.

Carlsson (1952) studied the effect of vitamin D on the skeletal

metabolism of calcium and phosphorus in rats. He concluded that vitamin D favored the removal of lime salts from the bones. The other effects of the vitamin observed in his experiment (increased serum Ca level,








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increased growth, and calcification of the skeleton) were regarded as secondary effects.

Carlsson (1954) studied the cause of hypophosphatemia and hypocalcemia in vitamin D-deficient rats. He stated that in rats on a P-free diet deficient vitamin D produced its typical effects on the level of serum inorganic P and on the uptake of P in the skeleton. These effects could not be explained as consequences of increased absorption or decreased excretion of P, since the element was retained almost completely even in the absence of vitamin D. He showed also that vitamin D-deficient rats were unable to utilize their bone store for maintaining a normal serum Ca, even if the bone stores had been well filled by feeding a diet with a good Ca/P ratio. In conculsion, he proved that the essential cause of hypophosphatemia and hypocalcemia in vitamin D deficiency is an insufficient utilization of stored bone salt.

Bellin et al. (1954) obtained considerable growth in rats by the

addition of vitamin D to a diet containing 0.62% and as little as 0.034% Ca.

Steenbock and Herting (1955) studied vitamin D and growth in rats. In a series of experiments with young rats, they found that a low Ca diet adequately supplied with phosphorus and other dietary essentials presented optimum conditions for eliciting the maximum growth differential which can be obtained with vitamin D. This effect of the vitamin was accompanied by a decrease in serum inorganic P, an increase in serum Ca, a decrease in the percentage of bone ash, an increase in the organic matrix of bone, and slight increase in the width of the cartilagenous

metaphyses. Vitamin D always tended to bring the serum P to a normal level. On the other hand, it-; only rf fect on the level of serum Ca was








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to increase it. They concluded that weanling rats require vitamin D for optimum performance.

Harrison et al. (1958) fed 3 week old rats a vitamin D-deficient diet adequate in calcium and phosphorus and the rats showed biochemical evidence of vitamin D-deficiency without the characteristic bone changes of rickets. The significant findings were hypocalcemia with normal serum phosphorus levels. Body weight gain increments were reduced in the vitamin D-deficient rat. One hundred units of vitamin D increased serum calcium.

Dixit (1967) studied the influence of vitamin D and starvation on serum calcium and phosphorus. He showed no significant changes in the serum calcium level in rachitic rats following vitamin D administration. The serum inorganic phosphorus of rats administered vitamin D 48 hours earlier was 5.84 mg/100 ml compared with 4.04 mg in the untreated control; this difference was not statistically significant. The product of calcium and phosphorus (mg/100 ml) in the serum of untreated rachitic control was 41.6, whereas at 24, 48, 72, and 96 hours after vitamin D treatment the values were 46.1, 60.7, 60.8, 57.1, respectively. The Bon Kossa silver staining and radiological examination of the metatarsals indicate that the earliest signs of "healing" were evident at or after 48 hours of vitamin D treatment. Perhaps the healing is initiated when the product of calcium and phosphorus rises to above 60.

Deluca and Steenbock (1956) reported that the alkaline phosphatase in the plasma of rats on various semisynthetic, vitamin D-free rations was higher than that of animals on a vitamin D-containing stock diet.

The administration of vitamin D reduced the values to approximately those found in stock rats.








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Hurwitz et al. (1969) investigated the role of vitamin D in plasma calcium regulation. They observed slower growth and low bone ash, bone Ca, and bone P in rachitogenic rats compared to the controls. A slightly elevated plasma P was observed in the rachitogenic rats.

Baylink et al. (1970) studied formation, mineralization, and resorption of bone in vitamin D-deficient rats. They obtained a lower growth, tibia length, and serum calcium, and higher serum phosphorus in vitamin D-deficient rats than in rats provided adequate vitamin D.

Simmons and Kunin (1970) studied the development and healing of rickets in rats. Weanling rats were rendered rachitic by maintenance on a low phosphate, vitamin D-free diet. The results indicated decreased growth, greater width of the proximal tibial epiphyseal cartilage, reduction of voluntary food intake, lower bone percent bone ash, and lower feed efficiency of a group of rats maintained on a rachitic diet, low in phosphorus and deficient in vitamin D, compared with a control group.

Al-Ganhari et al. (1973) studied vitamin D-deficiency in rats. Their results indicated that vitamin D-deficient rats gained significantly less weight than control rats. Also, as a result of vitamin D deficiency, the liver weight decreased compared with that of the controls. The rachitogenic rats developed symptoms of rickets which were shown by a distinctive gait and enlarged joints.

Omdahl and Deluca (1973) demonstrated that physiologic doses of vitamin D3 must be metabolized in the liver to 25-hydroxy vitamin D3 (25-OH D 3) and subsequently in the kidney to 1,25-dihydroxy vitamin D3 (1,25-(OH) 2 3) before it can function.








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Clark et al. (1973) showed that in vitamin D-deficient rats hypocalcemia, stunted growth, and soft bones occurred some weeks prior to total disappearance of 25-OH vitamin D from serum.

In studying the role of 1,25-dihydroxy vitamin D3 in maintaining serum phosphorus and curing rickets, Tanaka and Deluca (1974) showed that the intravenous injection of a single dose of 650 p moles of 1,25dihydroxy vitamin D3 to rats fed a vitamin D-deficient, low-phosphorus diet, caused an elevation of serum phosphorus within 5 hours which reached a maximum in about 10-12 hours and returned to deficiency levels 2-3 days later. On the other hand, a single injection of 650 p moles of 25-hydroxy vitamin D3 produced a significant rise in phosphorus at 12 hours which reached a maximum in 24 to 36 hours and was maintained for at least 7 days. The single dose of 1,25-dihydroxy vitamin D3 supported little calcification of bone, whereas the 25-hydroxy vitamin D3 produced marked calcification. The vitamin D-deficient rats showed low serum phosphorus, normal serum calcium, and decreased bone ash.

Yoshiki and Uanayisawa (1974) investigated the role of vitamin D in the mineralization of dentine in rats made rachitic by a diet low in calcium and deficient in vitamin D. After two weeks, weight gains were only 20-30 g as compared with 80-90 g in the control rats. The serum inorganic phosphorus and calcium levels dropped to 4.8 mg/100 ml and

8.5 mg/100 ml, respectively. Physiological amounts of vitamin D, given orally to rachitic rats, increased their serum phosphorus from 4.8 0.5 mg/100 ml to 7.5 0.4 mg/100 ml.








-11-


General Remarks on Rickets in Rats


Studies of experimental vitamin D deficiency in the rat are complicated by the fact that this species develops rachitic changes only when the Ca/P ratio of the diet is also modified. Coleman et al. (1950) reported that a disorder simulating rickets con be produced in growing rats by a pure deficiency of phosphorus even in the presence of adequate amounts of vitamin D. McClendon and Blanstein (1965) reported that young rats develop skeletal changes typical of rickets upon specific calcium deficiency. These and other observations have cast doubts on the importance of vitamin D in mineral metabolism of the rat. Generally speaking, calcium and phosphorus content of the diet must be taken into account in any experiment involving vitamin D deficiency in the rat.



Rickets in Poultry


Normal chicks, experimentally raised without sunlight or vitamin D, regularly develop rickets, called leg weakness by poultrymen. In birds, unlike rats, leg weakness can be produced by the absence of the vitamin alone, regardless of the calcium and phosphorus content of the diet.

The first experimental production of rickets in poultry was by

Hart et al. (1922). Since that time many investigations have been done in poultry to determine the effect of vitamin 1 on growth and other parameters involved.

Hart et al. (1923-24) demonstrated the striking effect of sunlight

on the growth of chicks on a synthetic diet which contained ample calcium and phosphorus but was low in vitamin D. One group was given the basal ration without sunlight and the other received the same diet but was








-12-


exposed to summer sunlight 30 minutes each day. At the end of 6 weeks on the experimental regimes, the two remaining rachitic fowls (no sunlight) weighed 80 and 90 grams, respectively, and the two controls (irradiated) weighed 145 and 180 gm. Bethke et al. (1928-29) observed marked increases in the growth rate of chicks when cod liver oil (1%) was added to a diet low in vitamin D. Steenbock et al. (1923-24) found low phosphorus in the serum of rachitic chicks. Common (1936) found high serum alkaline phosphatase during rickets in chicks. Hart et al. (192324) published X-rays of the complete boney structure of a rachitic chick and of a normal control. In the rachitic animal there was very little differentiation between cortex and marrow cavity and the whole skeleton was almost devoid of dense bone.

Steenbock et al. (1923) used blood inorganic phosphorus and calcium as criteria in the demonstration of the existence of a specific antirachitic vitamin in chickens. They showed that by the administration of cod liver oil freed from vitamin A, the inorganic phosphate and calcium of the blood were restored to normal, and the ash content of the bones was increased.

Massengale and Nussmeir (1930) studied the action of activated

ergosterol in the chicken. Their results indicate that activated ergosterol brings serum calcium and phosphorus to a normal level in rachitic chickens. Serum calcium was normal or slightly below normal, but serum phosphorus was always below normal in rachitic chickens.

McGinnis and Evans (1946) studied the response of turkey poults to vitamin D. They supplied broad breasted bronze turkey poults with graded chick unit levels of vitamin 1). They found that a level of 80 chick units from all sources (cod liver oil, salmon oil, corn oil,








-13-


solution of irradiated 7-dehydrocholesterol and corn oil solution of activated ergosterol) except the activated ergosterol gave maximum bone ash and growth. The mortality caused by rickets in the D-deficient group was 100%.

Motzok and Wynne (1950) pointed out the increase in blood alkaline phosphatase activity of rachitic chicks. They concluded that the blood level of alkaline phosphatase could be used to determine antirachitic activity of different sources of vitamin D, instead of bone ash. The potencies obtained by the phosphatase method differed from the value given by the bone ash method by amounts varying from 25-40%.

Spinka (1960) studied the relative effectiveness of vitamin D2 and D3 in a bone test on chickens. The results showed that weight gain, length, weight, and mineral content of femur, serum Ca, and total minerals in the blood were better in chickens given vitamin D3 than those

which were given vitamin D In the experiment, all chickens were sheltered from sunlight.

Chen and Bosmann (1963) investigated the effect of vitamin D and D3 on serum calcium and phosphorus in rachitic chicks. The results showed that chicks fed the rachitogenic diet exhibited low serum calcium concentrations and percentage bone ash, but had significantly higher serum phosphorus levels than chicks fed a standard chick diet or treated adequately with either form of vitamin D. The vitamin D3 to D2 efficiency ratio was estimated at about 8:1 to 11:1.

Waldroup et al. (1963) studied the effect of various levels of

vitamin D3 on phosphorus utilization by broiler-type chicks. They found that increased level of vitamin D3 up to 360 international chick units (ICU)/pound resulted in increased body weight and percent bone ash.








-14-


However, the response to increased levels of the vitamin became less as the calcium and phosphorus levels more closely approached the optimum.

Waldroup et al. (1964) studied the vitamin D3 requirement of the

broiler chick. Their results showed that 90 ICU of vitamin D per pound as suggested by N.R.C. are adequate to support maximum growth and bone ash at the calcium and phosphorus levels recommended by this group (1.0% Ca and 0.6% P).

Canas et al. (1969) studied the effect of vitamin D3 on cortical

bone of rachitic chicks. They found that when expressed on a volume basis the cortical bone from rachitic chicks had decreased levels of both ash and organic material as compared with normal controls. Upon treatment with 8 IU/day of vitamin D3 normal bone composition is restored within 7-8 days.

Thornton (1970) investigated the skeletal and plasma calcium changes in chicks during recovery from vitamin D deficiency with normal calcium intakes. In his experiment, hypocalcemia was shown in vitamin deficient chicks with normal calcium intake. This was corrected within 48 hours by vitamin dosage.

McNutt and Haussler (1973) measured the nutritional effectiveness of 1,25-dihydroxycholecalciferol in preventing rickets in chicks, and found that its effectiveness was similar to that of 25-hydroxycholecalciferol, both metabolites being 1.5 to 2.2 times as active as cholecalciferol with respect to stimulation of weight gain and maintenance of plasma calcium levels.

Yang et al. (1973) evaluated the effect of different forms of vitamin D in preventing rickets in turkeys. The results indicated
t 3
tlhat growth, femur beoiie ash, bone length , breaiking, strength, pl asma







-15-


alkaline phosphatase and inroganic phosphorus correlated well with vitamin D status.

Cork et al. (1973) studied the effectiveness of 1,25 dihydroxy vitamin D3 in preventing rickets in the chick. In their experiment, 1,12 di-OH-D3 was 5.1, 3.4, and 3.7 times more potent than D3 in stimulating weight gain, maintenance of plasma calcium, and promotion of increased percent bone ash, respectively.

Wong and Norman (1974) studied the mechanism of action of calciferol in white leghorn cockerels. In the experiment, all vitamin Ddeficient chicks had slow growth, low serum calcium (6.3 mg/100 ml serum), and a low bone ash of 25Z. Cholecalciferol restored these to normal levels.

Crenshaw et al. (1974) studied the effects of dietary vitamin D

levels on the in-vivo mineralization of chicks' metaphyses. The chicks fed a rachitogenic diet became hypocalcemic and formed hypomineralized bones compared with chicks fed a control diet.

Gonnerman et al. (1975) studied the effect of vitamin D, dietary calcium, and parathyroid hormone in chicks. Compared to chicks on control diet, chicks on the D-deficient diet had significantly decreased plasma Ca levels at 2 and 4 weeks and increased plasma P at 17 and 21 days.



Rickets in Swine


Vitamin D has for several decades been termed a nutrient required for optimum gain and skeletal development of swine. Investigations have shown that a low calcium intake plus vitamin D deficiency will cause rickets in swine. Loeffel et al. (1931) observed low calcium and low








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inorganic phosphorus in the sera and a reduction on the percentage of ash in long bones of rachitic pigs. Elliot et al. (1922) indicated that there is no need, as measured by growth response, for supplemental vitamin D in rations for growing pigs confined in the absence of sunlight. Dunlop (1935) and Braude et al. (1943) concluded that pigs do not need vitamin D.

Johnson and Palmer (1938) studied individual and breed variations in pigs diets devoid of vitamin D. They indicated that pigs need supplements of vitamin D in the diet to obtain maximum growth, and that the addition of irradiated yeast to the ration caused an improvement in appetite. Bethke et al. (1946) studied the comparative efficacy of vitamin D from irradiated yeast and cod liver oil for growing pigs, with observations on their vitamin D requirement. The results indicated that the minimum practical vitamin D requirement of growing pigs fed a ration containing 0.6% calcium and 0.45% phosphorus is approximately 90 U.S.P.

units per pound of ration.

Sinclair (1929) studied the influence of ultraviolet rays and

vitamin D on bone ash of fall farrowed pigs. He did not find any difference in bone ash percentage in treated pigs or a control group.

Wahlstrom and Stolte (1958) studied the effect of supplemental vitamin D in rations for pigs fed in the absence of direct sunlight. They concluded that the addition of 90 U.S.P. units of vitamin D per pound to a mixed ration complete in other known dietary factors resulted in little difference in the rate of gain, calcium, and inorganic phosphorus content of the blood, or calcium, phosphorus and total ash content of femurs.








-17-


Johnson and Palmer (1941) observed that vitamin D supplied in the ration of weanling pigs by sun-cured alfalfa hay at levels of about 72 IU/kg was sufficient to cure gross symptoms of rickets and to correct serum calcium and phosphorus values.

Miller et al. (1964) studied vitamin D2 requirements of baby pigs (1-2 days old). The baby pigs sheltered away from sunlight were given a purified diet containing 0.8% Ca, 0.6% P, and 350 ppm Mg for five weeks. The effects of different levels of vitamin D2 from zero to 10,000 IU/kg of diet were studied. All pigs receiving no dietary vitamin D and no sunlight showed rickets pathology. The pigs which survived had low serum calcium (6.1 mg/100 ml) and higher serum alkaline phosphatase (39.1 Bessey-Lowry unit). Bone analyses in these pigs showed lower ash content, Ca, phosphorus, and breaking strength than the pigs supplied with vitamin D. All pigs receiving 100 IU or more of vitamin D2/kg diet exhibited optimal rates of growth and economy of diet utilization together with normal levels of serum Ca, P, Mg, and alkaline phosphatase and adequate skeletal development with an absence of rachitic pathology.

To evaluate vitamin D requirements and study possible metabolic roles, Combs et al. (1966a) added various levels of vitamin D to the ration of 115 pigs weaned at 2 weeks of age. All animals were housed in the absence of sunlight from birth until termination of the experiments. The results indicated no significant difference in bone ash percent or rachitic symptoms among the treatments nor were the quantities of calcium and phosphorus in the bone ash significantly different. Serum calcium of the unsupplemented pigs was significantly higher than in those given 110 IU of vitamin D, but all treatment groups exhibited








-18-


satisfactory serum calcium level. No significant differences in either serum phosphorus or magnesium were found among treatment groups.

Combs et al. (1966b) studied levels and sources of vitamin D for pigs fed diets containing varying quantities of calcium. The results indicated that average daily gain, feed intake, and bone ash were not significantly influenced by vitamin D treatment.



Rickets in Cattle


Park (1923) stated that rickets occurs less frequently among calves than other animals. The signs of rickets in the calf have been described by Bechtel et al. (1936:150) as "the skeletal changes including bowing of the forelegs either forward or to the side, swelling of the knee and hock joints, straightening of the pasterns, and humping of the back." Other symptoms frequently observed were stiffness of gait, dragging of the rear feet, standing with the rear legs crossed, irritability, tetany, rapid respiration, bloat, anorexia for grain and roughages but not for milk, weakness and inability to stand for any length of time, and finally the retardation or complete cessation of growth. He also reported that the first detectable signs of rickets in calves are a decrease in the level of inorganic phosphorus in the serum, low serum calcium, and in some cases tetany. X-rays of rachitic calves showed that the junction of the diaphysis and cartilage was irregular and indefinite and in places showed areas of incomplete calcification.

Rupel et al. (1933) observed high levels of serum alkaline phosphatase and a reduction in the percentage of ash in long bones of

rachitic calves.








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Rickets has been observed in calves by several early writers. In 1901, Law listed it as a disease affecting cattle. Hutyra and Mark (1914) have a splendid photograph of a rachitic calf. In 1920, Becker observed rickets in calves fed whole milk and grain. Rachitic calves were produced experimentally by Reed and Huffman (1926) in connection with the heavy feeding of concentrates without the proper quality of roughage. Olson (1929) reported that rickets occurred among calves allowed free choice of feeds. The rachitic symptoms were associated with low hay intakes. Huffman et al. (1930) reported a case of rickets in a young animal which was fed a ration containing a limited amount of wheat straw. Hill (1930) reported that calves fed a rickets-producing ration and exposed to ultraviolet rays showed improved calcification of the bones. Huffman and Duncan (1935) studied the antirachitic value of hay in the ration of dairy cattle. The rachitic calves showed anorexia, low growth, low serum calcium, low serum phosphorus, low bone ash content, and low bone mineral content.

Colovos et al. (1951) studied the effect of vitamin D on calves. The results indicated blood Ca and inorganic P were lowered by the deficiency, while the alkaline phosphatase activity was increased. The deficiency slowed gain in body weight and produced the usual symptoms of rickets, such as arched back, large knees, and soreness of joints.



Rickets in Humans


The signs and diagnosis of rickets in humans are not particularly different from those in animals.

-Howland and Kramer (1921) discuss calcium and phosphorus in the serum in relation to rickets in children. They conclude that in








-20-


non-rachitic infants and young children, the concentration of calcium is from 10-11 mg/100 ml of serum, and the inorganic phosphorus is definitely reduced and sometimes extremely low. They observed that cod liver oil corrects these abnormalities. All the children under 2-1/2 years of age, in whom they found an inorganic phosphorus content of the serum of

3.0 mg or less, were suffering from active rickets. Tetany sometimes was associated with rickets.

Orr et al. (1923) studied calcium and phosphorus metabolism in

rickets, with special reference to ultraviolet ray therapy. They showed that ultraviolet radiation caused an increase in serum calcium and phosphorus in rachitic children. Jaffe (1934) studied rickets in children. He stated that serum calcium level is not a reliable criterion of the severity of rickets, but low serum inorganic phosphorus and high serum alkaline phosphatase are better for diagnosing rickets. Serum alkaline phosphatase, the normal range of which in children may be stated as between 5 and 15 King-Armstrong (KA) units/100 cc, rises in mild cases

of rickets to about 20 or 30 units, in marked cases to about 60 units, and in very severe cases over 60 units.

Thomas et al. (1959) measured antirachitic activity in normal children. Results indicated that the mean antirachitic activity of sera from 18 normal subjects was equivalent to 2 IU of vitamin D per ml.

Dunnigan et al. (1962) made a survey of the existence of rickets in 5-10 year old Pakistani children. Compared with normal children, rachitic children had low serum calcium, low serum phosphorus, high serum alkaline phosphatase, and enlargement of the ends of the knee and wrist bones. They indicated that the reason for these abnormal it ies is deficiency of dietary vitamin D and not lack of exposure to sunlight.







-21-


Richards et al. (1968) studied the infantile rickets in Glasgow. They showed that serum alkaline phosphatase of 25 KA units/100 ml or above plus loss of definition of metaphyseal line at the end of the radius and ulna and broad bands of increased density replacing the sharp metaphyseal lines, are reliable methods of diagnosing rickets in children.

Lipson (1970) studied nutritional rickets in Sydney. He obtained a low serum calcium (6.0 mg/100 ml), low serum phosphorus (2.1 mg/lOG ml), and high serum alkaline phosphatase (71 KA units) in rachitic children compared with the normal children (serum calcium 9-11 mg/100 ml, serum phosphorus 4-6 mg/100 ml, serum alkaline phosphatase 15-35 KA units). The X-rays of rachitic children showed the features of rachitic bones. He related these abnormalities to limited exposure of the children to sunlight. Treatment with 5,000 IU of vitamin D/day corrected the serum and bone abnormalities in the rachitic children.

Stephen and Stephenson (1971) measured plasma alkaline phosphatase from children receiving vitamin D supplements in the London area and from children exposed to sunlight in the West indies. The distribution of values showed that there was no precise upper limit which would be used in the diagnosis of subclinical vitamin D deficiency.

In the diagnosis of rickets in immigrants, Wills et al. (1972)

measured plasma calcium, plasma phosphorus, plasma alkaline phosphatase levels, and X-rays of the radius. They showed that rachitic children had low plasma calcium (6.0 mg/100 ml), low plasma phosphorus (3.9 mg/ 100 ml), high plasma alkaline phosphatase (50 KA units per 100 ml), and widening of the epiphyseal plates of the radius.

Balsan and Garabedian (1972) studied the effect of 25-hydroxycholecalciferol in curing rickets in children. ft was shown that after








-22-


8 days of 16,000 IU of 25-hydroxy vitamin D3 the treated children had normal serum calcium (raised from 7.4 mg/100 ml to normal 10.0 mg/100 ml), and alkaline phosphatase (reduced from 40 Bodansky units to normal 20 Bodansky units).

Ford et al. (1972) made a survey of the occurrence of rickets in the Glasgow Pakistani community. Serum calcium below 8.3 mg/100 ml, serum inorganic phosphorus below 3.0 mg/100 ml, serum alkaline phosphatase above 30 KA units/100 ml, and widening of the epiphyseal plate of the long bone were taken as evidence of rickets. All children with these abnormalities were treated with 3,000 IU of calciferol daily and brought up to the normal level.

Holmes et al. (1972) investigated rickets among the Asian immigrant population. In the study the children who had rickets had loss of definition of the metaphyseal lines of the radius and ulna, serum calcium below 8.0 mg/100 ml, serum phosphorus below 3.0 mg/100 ml, serum magnesium below 1.9 mg/100 ml, and serum alkaline phosphatase 45 KA units per 100 ml of the serum. The external appearance of rickets included bowed legs and muscular weakness. Vitamin D treatment increased serum calcium to 9.8 mg/100 ml, serum phosphorus to 4.2 mg/100 ml, and reduced serum alkaline phosphatase to 31 KA units.

Revusova et al. (1972) studied the effect of vitamin D on serum

magnesium. The results indicated that vitamin D in high doses increases intestinal magnesium absorption and serum magnesium concentration.

In studying the natural and synthetic sources of circulating 25-hydroxy vitamin D in man, Haddad and Hahn (1973) concluded that approximately 90% of serum 25-hydroxy vitamin D, was derived from








-23-


irradiation of 7-dehydrocholestrol of the skin. The range of serum value obtained was 16 to 41 ng/ml.

Cooke et al. (1973) investigated serum alkaline phosphatase and rickets in urban school children. Among 569 school children (386 boys and 183 girls) aged 14-17 years, 233 had serum alkaline phosphatase values of 30 KA units or greater. There was no significant difference in the levels in Asian, white, or VWest Indian children. The mean values were significantly greater in boys than girls and both showed a fall in mean value with increasing age. The investigation suggested that most children with alkaline phosphatase levels above 30 KA units have rickets.

Preece et al. (1973) tried to use serum level of 25-hydroxy vitamin D in the diagnosis of rickets. in rachitic children, the mean values of serum 25-hydroxy vitamin D, calcium, phosphorus, and alkaline phosphatase were 0.8 ng/ml or undetectable, 8.66 mg/100 ml, 3.0 mg/100 ml, and 101.8 KA units/100 ml, respectively. In the normal group the serum values were 25-hydroxy vitamin D 12.0 ng/ml, calcium 9.19 mg/100 ml, and inorganic phosphorus 3.8 mg/100 ml. It was concluded that the measurement of circulating 25-hydroxy vitamin D by competitive proteinbinding provides a more sensitive method than serum alkaline phosphatase to diagnose rickets in children.

Mankodi et al. (1973) studied rickets in pre-school age children in and around Bombay. They concluded that serum alkaline phosphatase above 30 KA units and X-rays of long bones were good evidence of rickets in children. Arneil (1973) reported cases of rickets in children. He concluded that rachitic children had bowed legs or knock-knees and serum alkaline phosphatase values above 25 KA units/100 ml. The








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condition resolved rapidly when 2,000 IU (international unit) of calciferol were given daily.

Gupta et al. (1974) showed the spontaneous cure of vitamin D

deficiency in Asian children during summer in Britain. They measured plasma levels of calcium, phosphorus, alkaline phosphatase, and 25-hydroxy vitamin D in groups of healthy Asian children in early spring and again in the late summer. In summer, mean plasma calcium rose from 8.8 to

9.25 mg/100 ml and 25-OH vitamin D from 9.9 to 14.7 ng/ml. Plasma phosphorus and alkaline phosphatase did not change. There was a highly significant correlation between plasma calcium and 25-OH vitamin D. These results emphasize the importance of summer sunlight in the maintenance of vitamin D nutrition and the prevention of rickets.

Hojer and Gebre-Medin (1975) studied rickets and exposure to sunlight. They showed low serum calcium, phosphorus, and high alkaline phosphatase in rachitic children which were not exposed to the sunlight. These children were exposed to sunshine for 30 minutes daily. Clinical improvement, normalization of serum biochemical values, and radiographical signs of healing were observed after three weeks of treatment.

Miller and Chutkan (1976) investigated vitamin D deficiency rickets in Jamaican children. The rachitic children had slightly low serum calcium (8.0 mg/100 ml), low serum phosphorus (1.7 mg/100 ml), higher serum alkaline phosphatase (130 KA units), and radiological signs of rickets. It was indicated that limited exposure to sunlight and lack of vitamin D in the diet are the reasons for these radiological and biochemical abnormalities. Treatment with vitamin D or sunlight or both corrected the abnormalities.








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Rickets in Horses


While significant advances have been made in studying vitamin D and its relation to bone and mineral metabolism in other species, information regarding rickets in horses is not extensive. There appears to be no reliable information available on the effects of vitamin D deficiency in the horse.

Nieberle and Chors (1954) denied the existence of true rickets in the horse. Park (1923) stated that rickets occurs less frequently in horses than in other animals. Smith and Jones (1957) assumed that rickets occurs in horses in a form similar to that in animals and man. Adams (1974) stated that signs of, rickets occur in horses up to three years of age; however, foals between six months and one year are most commonly affected. He also defined rickets in horses as a disease of the epiphysis rather than of the joint itself.

At the present time, diagnosis of rickets in horses rests heavily on radiological interpretation; however, external evidence may be present.

Manning (1962) discussed rickets in horses, including radiography of possible rickets cases. He concluded that extrapolation of information data from man to the horse is not a valid procedure.

Sippel et a I. (1964) determined the normal values of plasma calcium, phosphorus, and magnesium in horses. Average values for plasma calcium, phosphorus, and magnesium were 11.0 mg/100 ml, 7.5 mg/100 ml, and 2.4 mg/100 ml, respectively.

Myers and Emmerson (1966) studied the age and manner of epiphyseal

closure in the forelegs of two Arabian foals which were maintained under








-26-


ideal conditions. All of the epiphyseal lines below the carpus were closed before the end of the ninth month. The distal epiphyses of the radius and ulna had completely joined before the end of the ninth month.

Monfort (1967) made a radiographic survey of epiphyseal maturity in thoroughbred foals from birth to three years of age. fie suggested that the distal epiphysis of the third metacarpal is an ideal segment for the estimation of bone maturity. He pointed out that this closed completely at one year. Coffman (1969) studied bone maturation in horses. Findings indicated that the growth plate in the canon bone is normally closed at 12 months, while the radius closes between 24 and 33 months. Haugh et al. (1971) studied the breaking strength of the metacarpal bones of normal Shetland ponies at two years of age. The average failure stress they obtained was 35,000 pounds per square inch of the

third metacarpal bone.


















CHAPTER III

MATERIAL AND METHODS



Conditions and Experimental Design


A 2 x 3 factorial design experiment using 12 ponies (males and females, Shetland and Welsh ponies) was carried out to determine the effect of vitamin D and sunlight on growth and bone development of young ponies. The experiment consisted of two age groups and three levels of vitamin D treatments. The two age groups were 2 months and 8 months of age. The three treatment levels were as follows: 1) Treatment 1 (T ) with no vitamin D supplement and protected from ultraviolet (uv) light; 2) Treatment 2 (T 2), 1,000 IU of vitamin D supplement daily and protected from uv light; and 3) Treatment 3 (T 3), no vitamin D supplement and kept outdoors. Vitamin D was given orally (appropriate amount of vitamin D was dissolved in water and mixed with the diet).

The diet was formulated to meet all nutrient requirements according to the Nutrient Requirements Council (N.R.C., 1973), but without vitamin D. Table 1 shows that the diet consisted of Coastal Bermuda hay pellets (dehydrated, not sun-cured) 40.0%, corn 35.75%, soy bean meal 16.0%, molasses 7.0%, calcium carbonate 0.025%, salt 0.5%, vitamins (except vitamin D), and minerals. Laboratory analysis of the diet indicated

it to have 16.68% protein, 3.85% fat, 0.55% calcium, and 0.42% phosphorus. Ponies were fed three times daily (ad libitum). The dietary ingredients had minimum or no vitamin ). To ascertain this, the diet was tested by


1 7








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a
TABLE 1. COMPOSITION OF THE VITAMIN D-DEFICIENT DIET .


Ingredient


Coastal Bermuda Hay (Dehydrated, not sun-cured) Corn

Soybean Meal Molasses Calcium Carbonate NaCl (iodized) Trace Mineral Mix Vitamin A, Ec


% of Diet


40.00


35. 75 16.00 7.00 0.25 0.50 0.50


Adequate in all other nutrients required for optimum growth. bProvided the following (mg/kg diet): Fe 20, Mn 8.8, Ze 20, Co 0.1, I 0.1, Cu 2.8.
c
Provided 4400 IU Vit. A, 11 IU Vit. E per kilogram of diet.








-29-


the line test on rats (A.O.A.C., 1973). Rats 20 days old were put on a rachitic diet for 25 days, then after they showed rachitic symptoms they were divided into three groups: Group I was given the vitamin Ddeficient pony diet, Group II was given a synthetic complete diet, and Group III was kept on a synthetic rachitic diet. The three groups were kept on these diets for 10 days. Since the pony diet did not heal the rickets symptoms (A.O.A.C. line test procedure), it was concluded that the pony diet did not have vitamin D. The rats with the complete diet recovered from rickets symptoms, while the rats in Group III had severe rickets.

At the beginning of the experiment, all ponies were sheltered away from the uv light for one month before being divided into groups for treatment, in order to deplete them of stored body vitamin D. The treatment period was five months (from the end of the depletion period to the termination of the experiment). All the ponies were killed at the end of the experiment. At the beginning and at the end of the trial the intensity of uv light in the barn where the ponies were sheltered was determined by the use of a IP28 RCA photomultiplier with a 334 mm interference filter. The intensity of light relative to outside radiation (1/Io) was found to be 3 x 10- 5, indicating a bare minimum or none, compared with outdoors.



Sample and Measurements


Blood samples (20 ml) were obtained weekly from the jugular vein. The samples were centrifuged and plasma was obtained and frozen for laboratory analysis. Ponies were weighed weekly and feed consumption was recorded.








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Anteroposterior (A.P.) and Lateriomedial (L.M.) radiographs were taken of front feet, 1st phalanx, 2nd phalanx, and the metacarpal bones of the 2.0 month old group of ponies. Since the epiphyseal areas of these bones had already closed in the older ponies group (8 months old), A.P. and L.M. radiographs of the front feet (the metacarpal, the carpus, and the radius and ulna) were taken. Radiographs were made at approximately one-month intervals.

Radiographs were obtained using a small compact portable X-ray unit (Picker X-ray Corp., Cleveland, Ohio). DuPont Cornex 4 industrial X-ray film (E.l., DuPont De Nemours and Co., Inc.) in an X-ray cassette (Wafer rigidform cassette 10 x 12, Hansley X-ray Product, Brooklyn, New York) was used. Radiographic settings were 15 mA, 80 kvp, and 0.75 sec exposure. The distance between the X-ray unit and the leg was 26 inches with the film pack placed directly against the extremity. Film was developed in Kodak developer.

At the termination of the experiment all ponies were killed and

liver, kidney, and the two metacarpal bones of the front legs were taken for laboratory analysis. The samples were put in plastic bags and stored in the freezer. All ponies were photographed at the beginning and at the end of the experiment to show any signs of leg abnormalities (rickets).



Laboratory Analysis


Blood plasma calcium and magnesium determination. To determine

plasma calcium and magnesium, I ml of plasma was diluted with 9 ml 10% (w/v) trichloracetic acid (TCA) for precipiLation of protein. One ml








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of the supernatent was diluted with 4 ml 1% LaCl3 and it was assayed using an atomic absorption spectrophotometer (Perkin-Elmer, Model. 306).

Blood plasma phosphorus determination. Using the Fiske and Subbarow (1925) method, the lanthanum solution prepared for determination of calcium was used for the colorimetric determination of plasma phosphorus.

Blood plasma alkaline phosphatase determination. Using the modified Bessey-Lowry-Brock method and with reagents obtained from Dade (American Hospital Supply Corporation, Miami, Fla.), plasma alkaline phosphatase was measured at 0, 4, 12, 20, and 27 weeks of the experiment.

Plasma 25-hydroxy vitamin D determination. The method of Hollis and Conrad (1976) was used to measure plasma 25-hydroxy vitamin D levels at 0, 4, 12, 20, and 27 weeks of the experiment. The binding protein was obtained from normal rat serum.

Bone ash and cortex area. One of the metacarpal bones was used to determine bone chemical composition and cortex area. It was cleaned of adhering tissue, and length and fresh weights were obtained. The bone was cut transversely at the longitudinal center with a saw and using a planimeter the total area (AT) and the marrow area (AM) were measured. The cortex area (AC) was equal to AT - AM. The bones were then dried to constant weight at 105 0C, defatted by ether extraction in a Soxhlet apparatus. Dry bone weights were recorded and bone ash weights were obtained after ashing at 650 C overnight. Contents were expressed as percentage of dry, defatted bone. Bone density was equal to fresh bone weight divided by bone volume, determined by submerging the bone in water

in a graduated cylinder.

Ca, P, and Mg bone ash. The ashed bone was ground with mortar and pestle. One gram of bone ash was put into a tared crucible and acid







-32-


hydrolysis was carried out. The solution was evaporated to about 5 ml and the solution was transferred quantitatively to a 50 ml volumetric flask for analysis of calcium, phosphorus, and magnesium. Atomic absorption spectrophotometry, as previously described, was used for calcium and magnesium determinations. The Fiske and Subbarrow colorimetric method also described previously was used to determine bone ash phosphorus.

Bone breaking strength. An Instron (TT-c English units tensil

machine with CF load cell, Fig. 1) and a technique similar to that used by Haugh et al. (1971) was used to measure the breaking strength of the metacarpal bones from each pony. A simple three-point flexural loading technique was used for testing. 'the bone was positioned on two fixed

supports with its center lines placed an equal distance apart on the bone. The load was applied to the bone midway between the two support points with a single fixture identical to the supports. The load was applied by the downward movement of the crosshead of the testing machine to which the single fixture was attached. The bone was loaded to failure. The force was applied gradually and slowly. Failure stress was calculated as follows: S = x where S = failure stress
3.14 x C3
(kg/cm2 ), F = maximum force (kg), 1 = length of bone between supports (in cm), C = distance from centroidal axis to edge of bone (in cm).

Radiographic classification. The stage of maturity or the degree of epiphyseal closure was classified on the basis of "A+" = complete epiphyseal closure; "A-" = 3/4 epiphyseal closure; "B+" = 1/2 epiphyseal closure; "B-" = 1/4 epiphyseal closure, and "C" = complete open epiphysis.





-33-


I
-1 U
~~ p0~


FIGUnE 1. I4ST=O1 TENSILE ST EHGT2 TESTING APPAPATUS FOR DETEPINING
BLEAICING ST21ENGTH1 OF BCNES.








- 34-


Kidney and liver analysis. Twenty-five grams of the right lobule of fresh liver (or of a consistant part of the kidney) were dried in an oven at 100 C for 16 hours. Water content was calculated as 25 grams minus dry weight. The dried tissue (liver or kidney, approximately 5 g) was digested on a hot plate using concentrated nitric acid. It was then ashed in a muffle furnace at 5000C for 24 hours. The ashed liver (or kidney) sample was placed on a hot plate for acid hydrolysis. Five ml of hydrochloric acid and deionized water were used for acid hydrolysis of the sample. With 10% hydrochloric acid the tissue solution was transferred to a 25 ml volumetric flask and made to volume with deionized water. One ml of this was used to determine calcium, magnesium, and phosphorus levels as previously described.



Statistical Analysis


The effect of three treatments (in the two age groups) on blood,

kidney, and liver, and feed efficiency and bone were analyzed factorially (Steel and Torrie, 1960). The Statistical Analysis System (Barr et al., 1976) was used in the processing of the data. When there was an interaction effect, a Duncan's multiple range test was done to show differences within a particular treatment or group.

In case of bone, liver,and kidney analysis, the following model was used: Y = A + T + AT + E , where Y = water, ash, Ca, P, and Mg concentration of the liver or kidney or bone, A = age groups effect, T = treatments effect, AT = interaction of age groups and treatments effect, Ea = error (a). In case of plasma Ca, P, Mg, 25-OH vitamin D, and alkaline phosphatase levels, the following model was used:








-35


Y = A + T + AT + Ea + W + AW + TW + ATW + E where Y, = Ca, P, Mg, 25-OH vitamin D, and alkaline phosphatase, W = weeks effect (27 weeks), and AT, AW, TW, ATW are the interaction effect with Eb = error (b).

















CHAPTER IV

RESULTS AND DISCUSSION



External Appearance


Loss of appetite and difficulty in standing occurred in T ponies, but the actual external appearance of rickets (bowed legs and inability to stand) did not occur. Figure 2 shows photographs of ponies at the beginning and at the end of the experiment.

These findings agree with those of other investigators. Park (1923) stated that rickets occurs less frequently among horses than puppies, pigs, lambs, and kids. Results of Harrison et al. (1958) showed no external appearance of rickets in vitamin D-deficient rats when he fed them a diet adequate in both calcium and phosphorus. Dunlop (1935) and Braude et al. (1943) concluded from their study that pigs do not need

vitamin D; they were not able to produce rickets. Nieberle and Chors (1954) denied the existence of true rickets in the horse. Manning (1962) surveyed rickets in horses and did not believe that rickets exists in the horse. He concluded that extrapolation of information data from man to the horse is not a valid procedure. Combs et al. (1966a) failed to produce rickets in pigs deprived of sunlight and vitamin D supplement. On the other hand, many other extensive experiments showed very clearly rickets in puppies (Findlay, 1908), rats (Al-Ganhari et al., 1973), poultry (Hart et at_., 1922), swine (Miller et al., 1964), cattle (Bechtel et al., 1936), iind humans (Holmes et nI., 1972). Gneral ly,





-37-


L-....-..-..-TL4


/


~ I


L. ~&h~


T2
2


I;, ~ 4


V4-


T


T1


I -j
L
C

L

k~Ai
jJ*


ii'


FIGURE 2-


EFFECTS OF TREATMENT ON EXTERNAL /APPEARANCE OF THREE OF THE PONIES IN GROUP I.


AAL start of experImnt. BAt ind, 6 moi lmC ltV.


A


B








- 38-


the external indication of rickets were joint enlargement, distinctive gait, bowing of the forelegs, swelling of the knees, dragging of the rear feet, standing with the rear legs crossed, irritability, rapid respiration, anorexia, and finally retardation or complete cessation of growth.



Rate of Growth and Feed Efficiency


Growth (gain/day) and feed efficiency for the twelve ponies are presented in Table 2. Figures 3and 4 present weekly body weight for group I and group II ponies. It appears that ponies of T had slower growth curves than ponies in T2 and T . Group I (ponies started the experiment at two months of age and were terminated after six months) had slightly lower gains per day and feed efficiencies than Group II (ponies started the experiment at eight months of age and were terminated after six months). Feed efficiency was essentially the same for all treatments (T = ponies deprived of exposure to ultraviolet light and vitamin D supplement; T2 = ponies supplied with adequate vitamin D, but deprived of ultraviolet light exposure; T3 = ponies exposed to sunlight, but without vitamin D supplement). Feed efficiency data (grams intake per grams gain) were analyzed by factorial analysis. There was no significant difference in feed efficiency between either of the two groups or among the three treatments. Table 12 (Appendix) presents the analysis of variance for feed efficiency.

The effect of vitamin D supplement and/or sunlight on animals' growth has been reported in other results in other species. For example, Steenbock and Black (unpublished data) showed no significant











TABLE 2. EFFECT OF VITAMIN D AND SUNLIGHT ON RATE OF GAIN AND FEED EFFICIENCY OF PONIES.


Treatment


T1
(no vit. D no uv)

T9
(+ vit. D no uv)

T3
(no vit. D + uv)



Ti
(no vit. D no uv)

T2
(+ vit. D no uv)

T3
(no vit D + uv)


Initial wt. kga


20.4 18.2


20.4 27.9


40.4 51.3


38.6
89.4


103.5
85.4


83.5 90.3


Final wt. kga


Total gain kga


Feed intake kga


Expt. period daya


42.2 22.2 176.2 193 36.8 17.4 142.7 180


36.3 15.9 179.6 193 65.4 37.5 237.5 180


70.4 30.0 294.6 193 89.4 38.1 258.6 180


40.9 140.3


2.3 114.5 180 50.9 475.7 180


156.6 53.1 435.7 180 118.9 33.5 367.7 180


131.2 47.7 480.9 180 145.7 55.4 534.4 180


Weight gain g/daya



112.9
103.4


82.3 208.1


155.3 211.9


282.5


295.1
186.6


287.5 307.7


Avg.


108.2




145. 2


183.6


282.5




240. 9


297.6


Grams intake per
grams
gain


8.1 7 .7


11.3 6.3


9.8 6.8


9.4


8.2 11.0 9.3 9.6


Avg.


7.9 8.8


8.3


9.4 9.6


9.5


aEach value represents one pony.


Group


I

(Started exp. at
2 months of age)


II

(Started exp. at 8 months of age)









-40-


T,..o.*. No Vit. D, No uv light

12 + Vit. D, No uv light

. No Vit. 1, + uv Iight


am 00


00 100 00 go 10


- ---U'

- /I00~
/
/


/


44
0 ME=- f


-. ...


.

.....
'*.. ..., ,.--


I I I I I I I I i I I I i


2 6 H 10 1i 1 4 H IS


W E E K


) ) )6 '?8


FIGURE 3. m1IlIiKIY BoDY gEIGH l GROUP I PONIES.
(St.r ted 1 two uOmit hs of ;g )


() o


0 /


/
/
/


L i C)
0









m


4 f) 40


3'


30 25


20


15


to


03


7








--41


180 TJi ** ... No Vit. 1, No Liv light

1-+ Vit. D, No uv I ight T 3 .. No Vit. D, + uv I ht


9 in









IS0
6000
- 00


g0 so 0 z ] -0

dot- ~ ~ ~ ~ ~ 0 lplp




S i i I i I
1190




8 0 :

70)


O 60







0 / 4 8 In I I I 18 .)L.EE0EE....E.EI...



W E E K

FIGURE 4. WEEKLY BODY WEIGHT OF GROUP II PONIES.
(Started at eight months of age)








-42-


difference in growth of irradiated and non-irradiated rats. Bethke et al. (1923-24) showed that with a diet in which the Ca/P ratio was very high vitamin D did not induce growth. Hart and Steenbock (1922) indicated that there is no need, as measured by growth response, for supplemental vitamin D in rations for growing pigs confined in the absence of sunlight. Wahlstrom and Stolte (1958) showed that the addition of 90 U.S.P. units of vitamin D per pound to a mixed ration complete in other known dietary factors resulted in little difference in the rate of gain of pigs. Combs et al. (1966a) indicated that average daily gain and feed intake were not significantly influenced by supplying pigs with vitamin D. In other experiments there were positive responses of growth to vitamin D and sunlight supplements in rats (Carlsson, 1952; Bellin

et al. , 1954; Simmons and Kunin, 1970), poultry (Wong and Norman, 1974), swine (Miller et al., 1964), and cattle (Colovos et al., 1951).

In the present study there were some individual ponies (Table 2)

which showed a reduction in growth, but the difference in the means was not significant. The author believes that if the ponies were put into the experiment at one day of age, they would have shown a better response to treatments. It is possible that the ponies had enough vitamin D storage in their bodies before they started the experiment to get along for six months without dietary vitamin D.



Blood Analysis


Plasma calcium (Ca), phosphorus (P), and magnesium (Mg) levels.

Table 3 presents the average levels of plasma Ca of the twelve ponies in twenty-seven weeks of the experiment. Table 13 presents weekly plasma








-43


TABLE 3. AVERAGE WEEKLY PLASMA CALCIUM LEVELS (mg/100 ml) DURING 27
WEEKS OF EXPERIMENTAL PERIOD.a


Group I (Started Ex at 2 Months of Age)

No Vit. D + Vit. D No uv No uv
(T ) 2 )


11 .1
12.3 11.0
11.2 11.2 11.3 11.6 10.7
10.2 11.4 11.9
11.4 11.8
11.9
12.0 11.7 11.8
12.0
11.8 11.6 11.5
12.0 11.4 11.2 11.6
11.4 11.9


10.7 10.5 11.9 11.5 10.5 11.5 11.6
12.3 10.8
12.1
12.6 10.9 11.5 11.7 11.6
12.3 11.8 11.3 11.5
11.2 10.8 10.7 10.8 10.8 10.6 10.6 11.0


periment


Week


Group II (Started Experiment at 8 Months of Age)b


No Vit. D No uv Or


No Vit. D
+ uv (13)


11.3
11.4 11.9 11.5
12.0
11.8 11.5 11.6
11.1 11.5
12.1 11.5 11.7
11.7 11.9
12.1 11.8 11.8 11.8 11.5 11.8 11.9 11.8 11.8
12.1 11.5 11.0


+ Vit. D No Vit. D No uv + uv (2( 3)


13.5
13.4 14.5 13.3
14.1 13.8 13.6 13.9 13.2 13.3 13.7 13.7 13.7 13.2
14.1 14.1 13.6 13.6
14.0 13.6 13.8
13.4 14.1 13.7 13.6
14.1 14.0


13.2 13.5 13.6 13.7 13.7 13.7 13.7 13.3
14.0 14.0 13.2 13.6 13.7 13.7 13.1
13.4 13.4 13.9 13.7 15.2 13.3 13.9
14.2 13.7 13.7
14.1 13.5


Each value represents average for two ponies. bAll values significantly (p < .01) higher than for Group 1.


13.4 12.6 13.2 13.3 13.7 13.5
13.4 14.0 14.0 13.2
14.0 12.8
13.4 13.2 13.2 13.2 13.8 13. 2 13.3 13.2
12.1 14.0 13.0 13.3 12.9 13.5 13.1


1
2
3
4
5
6
7
8
9
10 11
12 13
14 15 16 17 18 19
20 21 22 23
24 25 26 27








-44


Ca levels for individual ponies. All ponies, during the experimental period, indicated plasma Ca levels within the normal range. Significantly (p < .01) higher plasma Ca levels were found in Group II than in Group I. The normal plasma Ca levels in all ponies led to the conclusion that plasma Ca levels were not appreciably affected by treatment. Statistical analysis showed no significant difference among T , T2, and T3. Figures 5 and 6 show the average values of plasma Ca for the three treatments in Group I and II. Average plasma Ca of Group I ranged from 10.5-13.5 mg/100 ml, while Group II ranged from 12.6-15.2 mg/100 ml. Ranges for T, T and T3 were 10.9-13.9, 10.5-14.0, and 11.1-14.0 mg/100 ml, respectively. Analysis of variance for plasma Ca levels is presented in Table 14 (Appendix).

Within the normal plasma P range (4.0-8.0 mg/100 ml), ponies in

Group I had slightly higher values than ponies in Group II. The values within the groups were varied and did not follow a consistent pattern (increase or decrease). Table 4 presents the average weekly plasma P levels for the two ponies on each treatment throughout the 27-week period of study. Table 15 (Appendix) presents individual values of plasma P levels for ponies in each treatment. Average plasma P levels ranged

between 4.6-7.7 mg/100 ml and 3.4-6.5 for Group I and Group II. Average plasma P levels for T1, T and T3 ranged from 4.7-7.7, 4.7-7.7, and

4.1-7.6 mg/100 ml. Figures 7 and 8 present the average values of plasma P levels of Group I and Group I.

The average plasma Mg levels are given in Table 5. Observations on plasma Mg levels pointed out that the levels did not affect from lack of vitamin D or sunlight or both. The averages of plasma Mg levels were within the normal range, at 1.1-1.9 mg/100 ml for Group I and 1.8-2.5








-45-


i L *SS*OO*** No Vit. D, No uv T2 + Vit. D, No uv


13.. No Vit. 0, +u



.
* eS


*- : - I.

- e e - .


: Iij k u1I : 4

* .- 06 0 0 I ' '










U * 0


..
U.


I I I I I I I I I h I I - U


2 6 8 10 1'2 1 10 18 2



W E E K


FIGURE 5. AVERAGE WEEKLY PLASMA CALCIUM OF GROUP I.
(SLarted experiment at 2 months of age)


-j


0


12.2


12 n 11.&





11.1?


10.8 10.6 10. Z


10 .2 -


26 6 28


v


...









-'+6-


- Ti:J *....*..q** .


T ': -- --- -


No Vit. 1), No uv + Vit. D, No uv No Vit. D, + uv


14. l


- .(i


1 3 . ,


0

0


13 .


12.8 12. 4


12.2


I
I
I
I
I
I
I
I


~


:
50 I
~,* i I.
V* I.

I E .~ '~ 44/..
I'. 0
0
** * v~ * 4
S 0
0 Se
- 0
0 0@
00


- 0* .06


I
I
I
I
I
'A

IAI

LX'\\' ;1

fe:: ~: '~


~LE1
0@I

~ I
I
* 0 *0 0
* 0.0 0 *
0 00 S
b 0 00 0
0 ** "'SI
0
* 0 0 0
S
* 0 0
0
* 0
* 0 , S
* 0
* 0 0@ 00
* 6
*5
*0 00 S.
*0 00 S.
~0


I I I I I I I _ I I I I I I I


2 6 6 8 1 1 22 24 . 28


W E E K



FIGURE 6. AVERAGE WEEKLY PLASMA CALCIUM OF GROUP ii.
(Started experiment at 8 months of age)


'5.S


1,
Ii








4~7


TABLE 4. AVERAGE PLASMA PHOSPHORUS LEVELS (mg/100 ml) DURING 27 WEEKS
OF EXPERIMENTAL PERIOD.a


Group I (Started Experiment at 2 Months of Age)

Np Vit. D + Vit. D No Vit. D No uv No uv + uv
(TI ) (T2 (T3)


7.7 6.0 5.7
4.8 4.5 4.9 4.8 4.7 5.3
4.8 5.2
4.8 5.3 6.6 6.2 6.3
4.9 5.8 5.9
4.9 6.5 6.5 6.3 5.6 6.5
7.4 6.3


7.0 6.6
5.4 5.2 4.7
6.0 6.3
6.4 7.6 7.0 6.2 6.3
6.9
6.4 5.9 6.8
4.9 5.7
4.9 5.9 5.2 4.7
5.1 5.5
4.5 5.5
4.9


7.6 6.8 6.3
5.8
5.4 6.8
5.4 6.1
6.4 5.2 5.9 5.5 5.3 5.2
5.4 5.8 5.5 5.9 5.9 5.9 6.1
5.5 5.9 6.0 6.0 5.7
6.2


Group II (Started Experiment at 8 Months of Age)

No Vit. D + Vit. D No Vit. D No uv No uv + uv
(T ) (T2 (T _


5.1
6.4 5.8 6.2
4.8 5.7
4.5 4.9 4.8 4.2 5.0 5.6 5.2 4.7 5.3
4.8 5.1
4.8 4.4 4.5 5.3
4.5 5.2
4.3 4.5 4.4 4.7


3.4 4.4 5.9
4.9 4.1 5.0 3.5
4.4 4.5 3.4 4.5 4.1
5.3
4.1
4.4 4.7 4.9 4.7
4.3 3.9
4.9 4.7
4.6 4.9 4.3
4.0 5.4


4.4 5.7 5.8 6.0
4.8 6.0
4.6 4.0 4.9 4.5 5.1 6.5
5.4 5.1
4.9 6.0 6.0
4.8 5.3
4.8 4.2 4.9 4.1
4.5 4.3 4.1 5.7


Each value represents average for two ponies.


Week


1
2
3
4
5
6
7
8
9
10 11
12
13
14 15 16 17 18 19
20
21 22 23
24 25 26 27










-48-


7 . (I


Sc......e.. No Vit. D, No uv 1 2 * - + Vit. D, No uv


7.


/ I


0


5.2 ,5.()



04







4.8 4 .


2 4 6 8 10 1 '0 2 ' 21 88



W E E K




FIGURE 7. AVERAGE PLASMA PHOSPHORUS LEVEI S IN GROUP I.
(Started experiment at 2 months of age)


5
0
0
0 0




I'


C

) 4







4'


0~
* 0
* I
0



0
0I
n
SI
I

0

0~l ~

0







:4

0 0
- 0
0 0
U;






I I


I I I I I I I I L I I


7. 2



7 .02


I


. .. . . I


4r I3e~~No Vit. U, + tiv




0S
00
00
00
0
00
* S
* 0
* 0
* 0
* 0
* 0 0
* 00e
0 0
* S
Mg 0~ 0 0
Mg 0 t;**~ 0 0
* 0 0
* 0
11/ ,e~ 0~ ~
Mg. 00
Il 0 0 0
~ 0 0 0
* k 0 * 0 g
Is I 0 0 * I
0 0 5" 0

Egg ~ * 0 *~~j~I' A~\:
Mg, 0
0

I, ~ ~ I:": : ~

II ~i :. *
t~ I, 4 '1
11 1~
~II t* . . 0
0 00 ~ * 0
~lII1 lo 0
I

( ::~~ :~' 0
00 00 0 00
* 0 00 0 00
* 0 0 0 0 40 00
* 0 0 0 0 00 00
.~ SO
Z .~ *. C
*o * [1 r





F1*




















6.6


6.


Oe




. .
* 0 * 0







- a


- ~ :a







-l


D, No uv , No uv D, + uv


I I I I I I I I I I I I I I


2 ( 8 10 1 1 18 26 28


W E E K

F'l(41RE 8. AVERAGE PLASM PHOSPHORUS 1,EVILS IN GROUP If.
(Stl.(l d Cxperiumnil :1[ 8 Tlm)[ths ()I age)


-j e e - 0 0 0 No Vi t.

T+ Vit. D .. "- No Vit.

lji i'I





*- i -i*











. 0 . I
.: I a
g f
- el


6.0


3.8


5 .0C


..8


0,,.




6-2


S 4.0




3.6


3. 2


5 . 'i








-50-


TABLE 5. AVERAGE PLASMA MAGNESIUM LEVELS (mg/100 ml) DURING 27 WEEKS
OF EXPERIMENTAL PERIOD.a



Group I (Started Experiment Group II (Started Experiment
at 2 Months of Age) at 8 Months of Age)b
Week
No Vit. D + Vit. D No Vit. D No Vit. D + Vit. D No Vit. D
No uv No uv + uv No uv No uv + uv
(T1) (T2) (T3 ) (TI) (T2) (T3)_


1
2
3
4
5
6
7
8
9
10 11
12 13
14 15 16 17 18 19
20
21 22 23
24 25 26 27


1.6 1.8 1.5 1.5 1.5 1.6 1.5 1.5 1.6 1.5 1.7 1.5 1.6 1.9 1.7 1.7
1.6 1.5 1.5 1.6 1.5 1.7 1.6 1.6 1.8 1.8 1.9


1.5 1.0 1.3 1.5 1.5 1.6
1.6
1.8 1.6 1.7
1.4 1.3 1.3 1.3
1.4 1.5 1.3 1.3 1.3 1.1
1.2
1.3
1.4 1.4 1.4 1.4 1.6


1.7
1.2
1.5 1.6
1.6 1.8
1.5
1.5
1.6 1.4 1.7
1.2 1.5
1.7 1.6
1.7 1.7 1.7 1.7
1.6 1.5
1.5 1.7
1.5 1.5
1.6 1.6


2.1
1.8
2.0 2.3 2.5
2.4 2.3
2.2 2.4 2.2 2.5 2.3 2.3
2.1
2.2 2.3
2.4 2.3
2.4 2.2 1.8 2.3
2.2 2.2 2.3
2.2 2.2


2.0 1.8
2.2 2.2 2.2 2.3
2.1 2.2
2.0 2.0 2.0 2.1 2.3
2.0 2.0 2.0 2.0 2.2 2.3
2.3
2.4 2.5 2.3
2.2 2.2 2.1 2.1


2.0 1.8
2.1 2.4 2.3 2.3
2.1
'2.1
2.3
2.1 1.9
2.1 2.2 2.2 1.9
2.0 2.1 2.1 2.0 2.1 2.2 2.5
2.4 2.5
2.4 2.3
2.4


aEach value represents average for two ponies. bAll values significantly (p < .01) higher than for Group I.








-51-


mg/100 ml for Group II. Group II had higher (p < .01) plasma Mg than Group I. Ranges for TI, T and T3 were 1.5-2.5, 1.2-2.5, 1.2-2.5 mg/100 ml. Factorial analysis indicated no significant differences between treatments. Figures 9 and 10 show the average plasma Mg of Group I and Group II. Table 16 (Appendix) presented individual values of plasma Mg levels for all ponies.

Plasma Ca, P, and Mg levels (Tables 3, 4, 5 and Figures 5, 6, 7,

8, 9, 10) were not affected significantly by the treatments. The values were variable and did not follow particular patterns. Some of these values were toward the upper or lower end of the normal range, but generally did not indicate the existence of vitamin D deficiency. The data pointed out slight decreases (but within the normal range) in the levels of P and Mg in the first six weeks of the treatment, especially in Group I, and thereafter an increase is observed. This could be explained as follows. When plasma Ca, P, and Mg levels are low, bone is readily mobilized to raise them to normal levels. The significant differences (p < .01) between treatments in bone ash (later in this paper) indicate that the ponies kept their plasma Ca, P, and Mg within normal levels by mobilizing bone. In addition, supplying the ponies with a diet adequate in Ca, P, and Mg, and exposing them to sunlight (for two

months for Group I and eight months for Group II) before they were put on the treatments were probably other factors which kept plasma Ca, P, and Mg within the normal range in the vitamin D-deficient ponies.

Harrison et al. (1958) showed normal plasma Ca and low plasma P in vitamin D-deficient rats supplied with a diet adequate in Ca and P. Yoshiki and Uanayisawa (1974) found hypocal cemia and hypophosphotemia in vitamin D-deficient rats which were suppLi ed with a diet deficient








-52-


Tj . .. eNo Vit. D, No uv T2*4----- + Vit. D, No uv T s - . No Vit. D, + uv



























2 '4 6 8 10 0'l I 1 ' 20 26 28

W E E K




FIGURE 9. AVERAGE PLASMA MAGNESIUM LEVELS IN GROUP 1.
('started experiment at 2 months of age)






-53-


Ti o......... No Vit. D, No uv T2* + Vit. D, No uv
T3..,.., No Vit. D, + uv




-~g :-. .0 \0
.. g gI 0. g






I i * * ** *









W E E K



FIGURE 10. AVERAGE PLASMA MAGENSIUM LEVELS IN GROUP 11.
(started experiment at 8 months of age)











in Ca and adequate P. Coleman et al. (1950) and McLendon and Bainstein (1965) produced rickets in rats by a diet adequate in vitamin D but deficient in either Ca of P. Gonnerman et al. (1975) showed that vitamin D-fed chicks provided with 1.4% Ca had hypocalcemia and hypophosphotemia, while vitamin D-deficient chicks provided with 2.4% Ca had near normal plasma Ca and P levels. Wahlstrom and Stolte (1958) found little improvement in plasma Ca and P from adding supplemental vitamin D in rations of pigs fed in the absence of sunlight, while Miller et al. (1964) showed low plasma Ca and P in vitamin D-deficient pigs deprived of sunlight. Colovos et al. (1951) showed low plasma Ca and P in vitamin D-deficient calves. In humans the data showed markedly low plasma Ca, P, and Mg in vitamin D-deficient children (Lipson, 1970; Ford et al., 1972; Miller and Chutkan, 1976). The average levels of plasma Ca, P, and Mg in the ponies in this experiment are within the normal ranges reported by Sippel et al. (1964) in horses.

Plasma alkaline phosphatase. The individual and average plasma

alkaline phosphatase levels after 0, 4, 12, 20, and 27 weeks on experiment are given in Table 6. In Group II there was a tendency for levels to decrease as the ponies aged. In Group I a decrease in plasma alkaline phosphatase occurred but it was not regular. The overall picture of the data indicates a difference between individuals but not between treatments. Factorial analysis was done to determine the effect of treatments and groups. There were no significant differences between treatments or groups. Table 17 (Appendix) presents the analysis of variance of plasma alkaline phosphatase.

The present study did not demonstrate any consistant increase in

plasma alkaline phosphatase with continuing vitamin D deficiency as shown








-55-


TABLE 6. PLASMA ALKALiNE PHOSPHATASE AT 0, 4, 12, 20, AND 27 WEEK
PERIOD.


Group


Treatment Week T0


No
Vit. D No uv


T 2
+
Vit. D No uv


4
12 20 27


0
4
12 20 27


I (Started Experiment at 2 Months of Age)


Individual
IU


130 187 59
43 67


124 187 62
64 46


105 79 52 59 53


99
115 89 78
74


Avg.


118 133 56 51 60


112
151 76 71 60


T3 0 79 99 89
No 4 65 106 86
Vit. D 12 70 124 97
+ uv 20 96 73 85
27 79 92 86


Group Average 88.0


11 (Started Experiment at 8 Months of Age)

Individual


I t


130 55 36 98 98


Avg.


62 92
122 163


130 59 65
110 131


I - f


187 101 52 96
113


146 127 75
113 95


69
113
122
146


187 85 83
109 103


-- 146
72 100
75 75
93 103
103 99


102.0


Treatment Average


89.0


101.0


93.0







-56-


in some of the results with rats (Deluca and Steenbock, 1956), poultry (Yang et al., 1973), swine (Miller et al., 1964), cattle (Colovos et al., 1951), and humans (Lipson, 1970) by others. Some experiments have shown no significant change in plasma alkaline phosphatase in vitamin D-deficient children (Stephen and Stephenson, 1971: Gupta et al., 1974).

Plasma 25-hydroxy vitamin D. Since plasma 25-OH vitamin D level is felt to be an important indicator of the vitamin D status of an animal, the levels of plasma 25-OH vitamin D were determined by a competitive protein binding procedure developed by Hollis and Conrad (1976). Normal rat serum was used as source of binding protein. Three dilutions of rat serum with lipoprotein barbital-acetate buffer, 1:5000, 1:8000, and 1:10,000 were made to obtain a standard curve which showed maximum binding efficiency. Figure 11 presents the standard curves with the three different dilutions. The 1:10,000 dilution was chosen for this analysis.

The individual and the average values of plasma 25-hydroxy vitamin D at 0, 4, 12, 20, and 27 weeks for all ponies are given in Table 7. Since this probably represents the first attempt to determine plasma 25-hydroxy vitamin D in ponies and no values are available in the literature, comparison was made between the results in this paper and values reported in the bovine by Hollis and Conrad (1976). It appears that all the values obtained in the present study are normal. Statistical analysis showed no significant difference among treatments in plasma 25-OH vitamin D levels. The average 25-hydroxy vitamin D of Ti, T and T3 were 63.4, 64.1, and 61.2 ng/ml. Group I ponies had higher (p < .01) plasma 25-OH vitamin D than Group IL. In Groups I and II, the averages were 67.8 and 57.9 ng/ml. 'Fable 18 (Appendix) presents the analysis of


















L-D
=3





H


I


U


U


0.25

NG 25-


0 50 V.75

OH VITAMIN N 0DITUBE


FIGURE 11. STANDARD CURVE FOR THE COMPETITIVE BINDING ASSAY OF 25-011 D3*.


~c


C=
L
0


-.. 1 :5000 *-----e. i:s,oco ~~*.


0..





*-..


S.00


I I I


~-I1







-58-


TABLE 7. INDIVIDUAL AND AVERAGE VALUES OF PLASMA 25-HYDROXY VITAMIN D
OF THE TWELVE PONIES AT 0, 4, 12, 20, AND 27 WEEK PERIOD OF
THE EXPERIMENT.


Group


Treatment


Week


I (Started Experiment at 2 Months of Age)

Individual
ng/ml value Avg.


Ti 0 -- 73 73.0 53
No 4 73 67 67.0 57
Vit. D 12 68 58 63.0 52
No uv 20 69 79 74.0 53
27 74 64 69.0 61


T2 0 -- 64 64.0 61
+ 4 71 65 68.0 62
Vit. D 12 71 75 73.5 63
No uv 20 80 69 74.5 61
27 78 65 71.5 62


T 3 No
Vit. D + uv


0
4
12 20 27


-t -


60 61 60 62


62 67 66 67 66


62.0 63.5 63.5 63.5
64.0


58 55 57
64 57


II (Started Experiment at 8 Months of Age)

Individual
ng/ml value Avg.


61 58 58 50 56


57.0 57.5 55.0 56.5 58.5


Treatment Average




63.4


54 57.5
54 58.0
56 59.5 64.1
54 57.5
51 56.5


58 60 60 59 62


58.0 57.5 58.5 61 .5
59.5


61.2


67.8


57.9


Group Average







-59-


variance for 25-OH vitamin D. The values of ponies in T3 are similar to the values obtained in normal cow plasma (Koshy and VanDerslik, 1976; Hollis and Conrad, 1976) and normal human plasma (Haddad and Hahn, 1973). On the other hand, the values for vitamin D-deficient ponies (treatment 1) are normal, in contrast to the abnormal values (lower than normal) in vitamin D-deficient humans (Preece et al., 1973; Gupta et al., 1974).

Since ponies in T are in the early stage of rickets, the reduction of 25-OH vitamin D level, which had been shown in other results, might have been seen at a later stage. This conclusion is compatible with the findings of Clark et al. (1973) in rats, who noted the decrease of 25-OH vitamin D levels at advanced stage of rickets.



Bone Analysis


Tables 8 and 9 summarize the bone analysis of the right and the left metacarpal bones. All data were analyzed by factorial analysis (Tables 19 and 20, Appendix).

Water concentration of bone. Water concentration was lower in G

(p < .04) and T (p < .05) than G2 and TI or T. respectively. The means of TI, T and T3 were 31%, 26%, and 27% for right metacarpal and 28%, 25%, and 26% for the left metacarpal bones. The means of G and G2 were 30% and 27% for right metacarpal and 28% and 24% for the left metacarpal bones. There was no interaction effect between treatments and groups.

Because ponies of Group II were older than those of Group I, higher bone water concentration in G than G2 was expected. It has been











TABLE 8. SELECTED COMPOSITION AND PHYSICAL CHARACTERISTICS OF RIGHT METACARPAL BONE.a


Ash (Dry Ca P Mg Density Strength
Group Treatment H 20% Fat-Free) Percent Percent Percent 3 2 2
% of Ash of Ash of Ash g/cm kg/cm (lb/in )


(TI)
No Vit. D 32.1 60.2 35.8 16.1 .4843 1.3 796.2 (11326)
I No uv 28.9 61.4 37.1 14.7 .4711 1.3 -- -(Started
Experiment (T2)
at 2 + Vit. D 30.3 60.4 36.3 15.6 .4828 1.3 1160.8 (16512)
Months No uv 27.9 62.9 34.9 15.3 .4311 1.4 1435.9 (20425)
of Age)
(T3)
No Vit. D 29.5 62.6 37.8 16.6 .4867 1.4 1618.0 (23015)
+ uv 24.1 62.0 37.8 16.3 .4911 1.4 -- -(T1)
No Vit. D 26.9 62.7 36.3 15.9 .4635 1.4 1516.2 (21568)
II No uv 27.3 62.6 36.8 16.4 .5065 1.4 1613.9 (22957)
(Started
Experiment (T2)
at 8 + Vit. D 22.3 64.0 37.2 16.1 .4570 1.4 1694.2 (24100)
Months No uv 21.2 61.2 36.3 15.4 .4208 1.5 2041.7 (29042)
of Age)
(T3)
No Vit. D 25.2 60.8 37.3 16.1 .4729 1.5 - -+ uv 24.9 62.3 36.3 16.1 .5115 1.5 1523.6 (21673)


aEach value represents bone from one pony.


I










TABLE 9. SELECTED COMPOSITION AND PHYSICAL CHARACTERISTICS OF LEFT METACARPAL BONE .a


Ash (Dry Ca P Mg Density Cortex
Group Treatment H 0% Fat-Free) Percent Percent Percent Area
% of Ash of Ash of Ash g/cm cm2


(T1)
No Vit. D 34.8 57.3 38.0 16.2 .5229 1.2 1.3
I No uv 30.7 59.2 37.0 14.8 .4858 1.3 1.4
(Started
Experiment (T?)
at 2 + Vit. D 29.5 59.0 37.4 15.6 .5116 1.4 1.7
Months No uv 30.4 57.5 36.6 15.6 .4536 1.4 1.5
of Age)
(T3)
No Vit. D 30.0 61.0 36.0 15.1 .4741 1.4 2.9
+ uv 26.6 61.6 37.9 16.6 .4866 1.4 1.8


(Ti)
No Vit. D 32.3 61.1 35.3 15.5 .4529 1.3 1.7
II No uv 28.0 59.3 36.2 15.8 .5118 1.4 2.9
(Started
Experiment (T2)
at 8 + Vit. I) 24.5 60.5 36.5 15.9 .4361 1.5 2.9
Months No un 23.0 59.8 37.3 15.6 .4584 1.5 2.5
of Age)
(T3)
No Vit. 1) 28.8 62.1 37.3 15.5 .4934 1.5 3.1
+ uv 25.2 61.3 34.7 14.8 .4537 1.5 3.1


Each value represents bone from one pony.


1>








-62-


indicated that as the rat matures, there is a progressive replacement of water by mineral, with the organic fraction remaining relatively constant (Hammett, 1925).

Bone ash (percent of dry, fat-free bone). The percentage of bone

ash of the right and left metacarpal bones respectively are presented in Tables 8 and 9. The average ash concentration of both right and left leg were analyzed statistically to find the main effects (treatments and groups) and whether there is interaction between groups and treatments*. There was no significant difference between groups and no interaction effect. The ash means (percent of dry, fat-free bone) for Group I was 60.4 and for Group II was 60.8. Ponies in T had significantly low bone ash (p < .01) than ponies in T2 or T . The means for Tl, T2 and T3 were respectively 59.9, 60.1, and 61.7% of dry, fat-free bones. Te

The finding of this difference between treatments agrees with many other experiments in other species. Dutcher and Rothrock (1925) reported a bone ash of 62% in dry, fat-free bones from normal rats; in rickets it was 26.5%. Other results which showed low bone ash in vitamin D deficiency are in rats (Simmons and Kunin, 1970), poultry (Yang et al., 1973), swine (Miller et al., 1964), and cattle (Huffman and Duncan, 1935).

The low ash concentration of vitamin D-deficient ponies results from the low efficiency of these ponies in absorbing the mineral from the intestine and the use of the bone mineral to keep plasma mineral within the normal level.

Bone calcium, phosphorus, and magnesium (% of ash). Tables 8 and 9 show percent of Ca, P, and Mg of bone ash for right and left metacarpal bones. Factorial analysis showed no significant difference between







-63-


treatments or groups regarding percent of Ca, P, and Mg of bone ash. The means of bone ash Ca were 37% and 36% of ash for G1 and G and 36%, 36%, and 36% for T1, T and T Phosphorus means were 15% for both GI and G2 and for TI, T and T , 15% for. all. For Mg the means of G and G2 were .49 and .47 and Ti, T2' 3 were .49, .47, and .48.

Since the bone crystals need certain minimum levels of Ca, P,

Mg, and other minerals to form the total bone crystal, bone ash was expected to be low in vitamin D-deficient ponies, but the percentage of Ca, P, Mg in the bone ash was expected to be the same for all treatments. Hurwitz et al. (1969) showed low percent of Ca and Mg in bone ash of rats, while Wahlstrom and Stolte (1958) and Combs et al. (1966a) showed little difference in percent of Ca and P of bone ash of pigs supplied with, or without, vitamin D.

Bone cortex area. Table 9 shows the value of the cortex area of the left metacarpal bone. Ponies on TI appear to have low bone cortex area (p < .07) than ponies in T2 or T3. Group II ponies had significantly (p < .05) higher cortex area than ponies in Group I. Figure 12 presents the cortex areas of three ponies in Group I, in which lower cortex area in TI than T2 and T3 appears. Cortex area averages of G, G2 were

1.7 and 2.7, and of T, T2, and T3 were 1.2, 2.1, and 2.7 cm , respectively.

As the young animal grows there is an increase of its bone cortex area, but the bone ash deposit will be less in vitamin deficient animals than the normal, with no difference in their bone cortex areas. This is what has been found in this experiment. There was no significant difference between treatments but there was a significant difference between age groups.





-64-


T2 T3



























FIGURE 12. EFFECT OF TREATMENT ON METACARPAL BONES OF THREE PONIES 02 CYIOUP I. aOverall appearance. bCross-section showing cortex area at midpoint. Designations Ti, T2 and T3 indicate respective treatments.







-65-


Bone density. Group I ponies had significantly lower (p <.01) bone density than ponies in Group II, and ponies in T had significantly (p < .01) lower bone density than ponies in T2 or T There were no interactions between groups and treatments. The means of bone density (g/cm 3) were 1.3 and 1.4 for GI and G2 and 1.32, 1.41, and 1.45 for T1, T2, and T . Tables 8 and 9 show the individual values of bone density for the twelve ponies.

Bone breaking strength. Table 8 shows the breaking strength of right metacarpal bones of the twelve ponies. Some of the values were lost through use of a defective testing apparatus in early determinations. Ponies in T appear to have lower bone breaking strength than ponies' bones in T2 or T but this difference was not significant. Group I ponies had lower (p < .05) bone breaking strength than ponies in Group II. The means of bone breaking strength were 1252.7 and 1677.9
2 2
kg/cm for G and G2, and 1308.8, 1583.1, 1570.8 kg/cm for TI, T2' and T3'

Since ponies in T had lower ash content than ponies in T2 and T3' it was expected to find low density and breaking strength in the bones of the vitamin D-deprived ponies in T The results in this experiment did indeed show lower bone density and breaking strength for T than T2 and T3*

Miller et al. (1964) indicated low bone breaking strength of

vitamin D-deficient pigs. The values of breaking strength of ponies in T3 was similar to values which were obtained by Haugh et al. (1971) in normal Shetland ponies.







-66-


Bone epiphyseal closure. Table 10 shows the findings of the epiphyseal closure measurements in Group I, employing the following grading system: "A+" = epiphyseal plate completely closed, "A-" = epiphyseal plate 3/4 closed, "B+" = epiphyseal plate 1/2 closed, "B-" = epiphyseal plate 1/4 closed, "C" = completely open epiphyseal plate. At the beginning (3 months of age), the distal end of the first phalanx (P 1D) and the distal end of the second phalanx (P2D) plates were closed (A+), but the distal end of the third metacarpal (MD), the proximal of the first and the second phalanges (P1P and P2P) were open (C). The last three epiphyseal plates were used to study treatment differences. The radiographs made at the termination of the treatments (Table 10) indicated that the MD, P IP, and P2P graded B-, B+, and B- for ponies in T . In T2 they were B+, A-, and B+. Radiographs in T3 indicated A+, A-, and A-. These observations demonstrate a difference in the epiphyseal closure between T, T and T . The radiographs of the left leg showed similar differences among treatments.

In addition to delay of epiphyseal closure of the vitamin Ddeficient ponies in T (Group I), lack of calcification, thickening, widening, and irregularity of the epiphyseal cartilage (epiphyseal plate) at the junction of the diaphyses and the epiphyses were observed.

Since MD, P P, PID, P2P, and P2D plates were closed in Group II (started at 8.0 months old) the distal end of the radius (RD), which develops later, was examined and comparison was made between treatments. At the end of the experiment there were no differences between treatments as to RD epiphyseal plate closure. Figures 13 and 14 present the effect of treatments on ponies of T and T3 of G and G2,







-67-


TABLE 10. EPIPHYSEAL CLOSURE OF THE DISTAL OF THE THIRD METACARPAL (MD), THE PROXIMAL OF THE FIRST PHALANX (PIP), AND THE SECOND
PHALANX (P2P) OF BONES OF GROUP I PONIES.a


Treat- Right Leg Left Leg
Age ment MD P 1p P 2P MD P IP P 2P
Ag1mnt2 1 2

c b
T C C C C C C b
3T 2d C C C C C C C C C C C C

Months 2e
T C C C B- C C C C B- - C C

T C - B+ - C - - B- - B- - C

T C B- B- B+ C B- - B-
Months 2
T3 B- B- B+ B+ B- B- B- B- B+ B+ B-BT C B- B+ B+ C B- B- - B+- C
T C - B- - B - C B- A- B- C BMonths
T3 B- B- A- B+ B- B- B- - A- - B+


6Ti - B- - B+ - B- - - - - -

Months T C - B+ - C -C- - - - - -
T B- - A+ - B+ - B- - A+ - B-

T B- B- B+ C B- B- B- B+ B+ B+ B- B+

T2 C B- A-- B+ C B- C B+ A- B- C BMonths 2
T3 - B+ - A- - A- B+ B+ A+ A+ B- AT B+ B- A- B+ B- B- B+ B+ A- B+ B-BT B- B+ A- A- B- B+ B- A- A- A- B- B+
T3 A+- - A- - A- - A+ A+ A- A+ A- A+


Each letter represents grade of pony (started at 2 months old).
A= completely closed B+ = 1/2 closed C = completely open
A- = 3/4 closed B- = 1/4 closed

Ra~diograph not clear enough to grade.

cT1 = No vitamin D, no uv light.

T2 = + vitamin D, no uv light.

eT3 = No vitamin D, + uv light.






-68-


A
















T3



C











FIGURE 13.


T,





B
















T3















EFFECT OF TREATMENTS ON THE EPIPHYSEAL CLOSURE OF GROUP I. Lateromedial (LM) radiograph of the right front foot, showing the delay of the epiphyseal closure of the pony in Group I on T1 (B) compared with the pony on T3 (D) in the same group. A, C = 3 months old; B, D = 8 months old Group I = started expt. at 2 months of age T = No Vit. D, no uv; T3 = No Vit. D, + uv






-69-


A 13
















FIGURE 14.


T

B


T3


EFFECT OF TREATMENTS ON EPIPHYSEAL CLOSURE OF GROUP II. Anteroposterior (AP) radiograph of the distal end of the radius showing similar appearance of the epiphyseal closure in Group II ponies on T, and T3 (B and D). A, C = 9 months old; B, D = 14 months old Group II = started expt. at 8 months of age T, = No Vit. D, no uv; T3 = No Vit. D, + uv







-70-


These abnormalities in development of the epiphyseal plate (the

junction of the diaphysis) which were shown in the ponies in T of this experiment have been shown in results with many other species. For example, these symptoms of vitamin D deficiency were shown in rats (Simmons and Kunin, 1970), cattle (Bechtel et al., 1936), and humans (Richards et al., 1968; Wills et al., 1972; and Holmes et al., 1972). The normal epiphyseal closure of ponies in T3 in this experiment is similar to that in X-rays shown by Myers and Emmerson (1966), Monfort (1967), and Coffman (1969) in horses.



Liver Analysis (Water, Ash, Calcium, Phosphorus, and Magnesium Concentration)


Table 11 presents the average of liver water, ash, Ca, P, and Mg concentrations (on dry basis), and Table 20 (Appendix) presents liver composition of individual ponies. Analysis of variance for liver compositions and the effect of treatments and age on it is presented in Table 22 (Appendix).

Ponies in Group II had lower (p < .07) concentration of liver water than Group I. There were no significant differences for liver ash concentration among groups or treatments. Average of liver (%) water concentration of G and G2 were 73.5, 72.0, and for T., T2, T3 were 73.0, 72.7, and 72.7. Average liver ash (%) for G G2 and TI, T and T3 were 4.3, 4.2, and 4.1, 4.3, 4.3.

Ponies in Group I had higher (p < .05) liver Ca (ppm) than Group II ponies, and ponies on T2 had higher (p < .01) liver Ca (ppm) than T1 or T 3 Liver phosphorus (ppm) was lower in Group II ponies than Group I.







-71-


TABLE 11. AVERAGE ASH (%), WATER (%), CALCIUM, PHOSPHORUS, AND
MAGNESIUM CONCENTRATION (ppm) OF LIVER AND KIDNEY.a
(Dry weight basis)

Mineral Organ Liver Kidney

Group I II I II
Start Start Start Start
at 2 at 8 at 2 at 8
Treat- Months Months Avg. Months Months Avg.
ment of Age of Age of Age of Age

T1b 224.4 190.5 207.5 1437.0 1286.0 1361.5

Ca 2 265.0 233.0 249.0 1805.0 2870.0 2337.8
T3 d 206.0 208.0 207.0 579.0 1289.5 934.3
Avg. 231.8 210.5 1273.8 1815.2

T 9971.0 79490.0 8980.5 11215.0 10700.0 10958.0
T2 10334.5 8323.0 9328.8 11203.0 11307.0 11255.3
T3 9282.5 8795.5 9021.0 10244.0 10574.0 10409.3
Avg. 9862.7 8357.5 10887.7 10860.7

T 572.5 503.0 537.8 575.5 690.0 723.8

Mg T2 567.0 500.0 533.5 763.0 745.0 754.3
T3 497.5 520.5 509.0 642.0 742.0 692.0
Avg. 545.7 507.8 720.8 725.8

T 4.4 3.9 4.2 7.1 6.4 6.8

Ash T2 4.4 4.4 4.4 6.7 6.9 6.8
% T3 4.2 4.4 4.3 6.6 6.5 6.5
Avg. 4.3 4.2 6.8 6.6

T 74.0 72.0 73.0 82.5 82.0 82.3

Water 2 73.0 72.5 72.8 82.5 81.5 82.0
% T3 73.5 72.0 72.8 82.5 80.5 81.5
Avg. 73.5 72.2 82.5 81.3

aEach value represents average of two ponies. bNo Vit. D, no uv.

N Vit. D, no uv.

dNo Vit. D, + uv.







-72-


An interaction effect of groups and treatments was found in liver magnesium concentration. Duncan's multiple range test for liver magnesium indicated that T3 was significantly (p < .05) higher than T and T2 in Group II, but not in Group I.

Liver water, ash, calcium, phosphorus, and magnesium concentration values obtained in this study indicated normal values as reported in normal horse liver (Schryver et al., 1974), and the differences were due to individuals but not to treatments. Al-Ganhari et al. (1973) reported lower liver weight in vitamin D-deficient rats compared to the control group.



Kidney Analysis (Water, Ash, Calcium, Phosphorus, and Magnesium Concentration)


Table 11, previously noted, presents the average and the individual ponies kidney composition. The analysis of these compositions and its

effect by age and treatments are presented in Table 23 (Appendix).

Kidney water, ash, calcium, phosphorus, and magnesium concentrations in all ponies were normal and were not affected by age groups and treatments. There were no significant differences for kidney compositions among groups or treatments.

















CHAPTER V

SUMMARY AND CONCLUSION



A 2 x 3 factorial design was carried out to study the effect of

vitmain D and sunlight on the growth and bone development of young ponies. Two groups of ponies (G two months of age, G2 eight months of age) were assigned to three treatments (T no sunlight without vitamin D supplement, T2 no sunlight with daily 1,000 IU of vitamin D supplement, and T3 sunlight without vitamin D supplement). All ponies were put in a windowless barn with very minimum ultraviolet light exposure for one month (to deplete the ponies of possible vitamin D body stores) and then they were assigned to the treatments for five months. A diet deficient in vitamin D (lack of vitamin D was confirmed by testing the diet on rats) and adequate in all other nutrients was fed to the ponies three times daily (ad libitum).

Weekly blood samples were obtained from the jugular vein to study the difference within and among treatments and groups regarding plasma calcium (Ca), phosphorus (P), magnesium (Mg), alkaline phosphatase (Alk), and 25-hydroxy vitamin D (25-OH Vit. D) levels. Monthly X-rays were taken to study the development of the metacarpus and phalanx junction in Group I, and the radius and metacarpus junction in Group II. The stage of maturity or the degree of epiphyseal closure was classified on the basis of "A+" = complete epiphyseal closure; "A-" = 3/4 epiphyseal closure; "B+" = 1/2 epiphyseal closure, "B-" = 1/4 epiphyseal closure,


-7.3-







-74-


and "C" = completely open epiphysis. At the termination of the experiment all ponies were killed; liver, kidney, and the two metacarpal bones were taken for laboratory analysis. Water, ash, Ca, P, and Mg contents of liver and kidney were determined. One of the metacarpal bones was used to determine cortex area while the other was used to determine bone breaking strength. Water ash (dry, fat-free basis), Ca (%), P (%), and Mg (%) (of bone ash), and bone bensity were determined in the bone.

The data were analyzed factorially and a statistical analysis system was used. In the case of interaction effects, a Duncan's multiple range test was done to show the effect of treatments within the group.

Symptoms of an early stage of rickets (difficulty in standing, loss of appetite, and low feed efficiency) occurred in ponies of T (ponies deprived from uv light and vitamin D supplement), but the bowed legs and inability to stand (typical characteristics of advanced stage of rickets in other species) did not appear in any of the ponies.

Group I had sli'ghtly lower feed intake and feed efficiency than Group II. Factorial analysis indicated no significant differences of feed efficiency among groups or treatments. Averages of feed efficiency for TI, T and T3 in Group I were 7.9, 8.8, 8.3 grams intake/gram gain and for T, T2' 3 in Group II were 9.4, 9.6, 9.5 grams intake/gram gain.

Plasma Ca levels were within the normal range. There was no significant difference among treatments, but Group II had higher (p < .01) plasma Ca level than Group I. Plasma calcium of Group II ranged from 12.6-15.2 mg/100 ml, while Group I was 10.5-13.5 mg/100 ml. Plasma calcium levels ranged in T , T and T3 were 10.9-13.9, 10.5-14.0, and 11.1-14.0 mg/100 mi.







-75-


Although all plasma P values were within the normal range, ponies in Group I appear to have higher levels than Group II. There were no significant differences among groups or treatments. Plasma P levels ranged from 4.6-7.7 mg/100 ml for Group I and 3.4-6.5 mg/100 ml for Group II. The plasma P ranges for TI, T2, and 13 were 4.2-7.7, 3.4-7.7, and 4.1-7.6 mg/100 ml, respectively.

Observations on plasma Mg levels led to the conclusion that the level was not affected from lack of vitamin D or sunlight. All plasma Mg levels were within the normal range. The ranges for Group I and Group II were 1.1-1.9 and 1.8-2.5 mg/100 ml. Ranges for T, T and

T3 were 1.5-2.5, 1.2-2.5, and 1.2-2.5 mg/100 ml. Factorial analysis indicated no significant differences between groups or treatments.

The results of other studies (in other species) on the effect of

vitmain D deficiency on the levels of plasma Ca, P, and Mg are not consistent, but all researchers indicated that vitamin D deficient animals mobilized their bone to keep their plasma Ca, P, and Mg levels within the normal range. The plasma mineral levels of vitamin D-deficient animals will drop slightly at the beginning of the deficiency, and thereafter the bone is mobilized to raise these levels to the normal ranges. This was shown, in the vitamin D-deficient ponies, to be generally true.

Plasma alkaline phosphatase levels were not affected by either

treatments or age groups. The values were varied and did not follow a particular pattern. No significant differences between treatments or groups were found. Average plasma alkaline phosphatase for C. G2 and TT T 3 were 88.0, 102.0 and 89.0, 101.0, 93.0 International Unit

(IU), respectively. Most results reported for other species indicated







-76-


an increase in plasma alkaline phosphatase with vitamin D-deficient animals. This is established in humans.

Plasma 25-OH vitamin D levels were normal and approximately the same for all ponies. The ranges of plasma 25-OH vitamin D for G , 2 and TI, T2, T3 were 58-80, 50-63 and 50-79, 51-80, 57-67 ng/ml, respectively. There is no information on the effect of vitamin D deficiency on the level of plasma 25-OH vitamin D in horses, but it is believed, in other species, that the decrease and the disappearance of 25-OH vitamin D occurred at later stage of rickets.

Bone water concentration was about the same for treatments and

groups. Average bone water content for G,, G2 and T, T2, T3 were 30%, 27% and 31%, 26%, 27%, respectively. There were no significant differences in bone water concentration among groups or treatments.

Bone ash concentration was lower in T (p < .01) than T2 or T3 ponies, and was lower (p < .05) in Group I ponies than Group II. Ash concentration (% of dry, fat-free bone) for G and G, were 60.4% and 60.8%, and T, T2, and T3 were 59.9, 60.1, and 61.7, respectively. Calcium, P, and Mg, as percent of bone ash, were not affected by age or treatment. Bone cortex areas of ponies deprived of sunliTht and vitamin D supplement (T ) were slightly lower than T2 and T The older group (G2) had higher (p .05) bone cortex area than group I (younger ponies). Bone cortex area of G and G2 ranges were 1.3-2.9 and 1.7-3.1
2
cm . The ranges for T, T2, and T were 1.3-2.9, 1.7-2.9, and 1.8-3.1
- 3
2
cm.

The foals on T appear to have lower bone breaking strength than foals of T and T but differences were not significant. The means of bone







-77-


breaking strength were 1252.7, 1677.9 kg/cm2 for Group I and Group II,
2
and 1308.8, 1583.1, 1570.8kg/cm for TI, T , and T .
1' 2' 3
Ponies in Group I had lower bone density (p < .01) than ponies in Group II. Also, ponies in T had lower bone density (p < .05) than ponies in T2 and T . The means of bone density were (g/cm 3) 1.3 and

1.4 for G and G? and 1.3, 1.4, and 1.5 for T, T and T3'

The epiphyseal plates (at the distal end of the metacarpal, proximal end of the first phalanx and the proximal end of the second phalanx) of T in Group I showed delayed closing, were irregular and wide and instead of having clear, sharp lines, and poor definition. Bone analyses which indicated low bone ash (% of dry, fat-free basis), breaking strength, density,and abnormality of epiphyseal closure of vitamin Ddeficient ponies are similar to reports in other vitamin D-deficient animals (other species).

Liver and kidney water and ash concentrations (on dry basis) were not significantly affected by age or treatment as tested by factorial analysis. Average liver water contents for G, G2 were 73.4 and 72.0, and for T, T2' T3 were 73.0, 72.7, and 72.7 percent of liver. Percent liver ash contents were 4.3, 4.2 and 4.1, 4.3, 4.3 for GI, G and Ti, T2, T . Kidney water content (%) for G, G were 82.5 and 81.3 and for Tl, T2' T3 were 82.3, 82.0, and 81.5. Kidney ash content for G, G2 and T, T2, T3 (%) were 6.7, 6.6 and 6.8, 6.8, 6.6.

There were significant differences (p < .05) between groups (GI

higher than G2) and highly significant differences among treatments (T ) (T2

higher than T and T 3) in liver Ca levels. For liver P content, a highly significant difference (p < .01) among groups (G > G2) occurred. In the case of Mg, interaction of groups and treatments occurred.







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Duncan's multiple range test showed T3 significantly lower (p < .05) from T and T2 in Group I but not in Group II. Kidney Ca, P, and Mg concentrations were not affected by either age or treatment. The mean values (ppm) of Ca, P, and Mg contents of liver and kidney are shown in Table 11.

In conclusion, what could be considered an early symptom of rickets occurred in ponies of TI (deprived of uv light and vitamin D supplement),

especially those of the younger group (started the experiment at 2 months of age). These ponies lost their appetite, had low feed efficiency, and difficulty in standing compared with ponies in T, (deprived of uv light and supplied with daily 1000 IU of vitamin D), and 13 (exposed to sunlight and deprived of vitamin D supplement). In addition to these symptoms, a slight drop in plasma P and Mg was observed at the early period of the experiment, and after which mobilization of these minerals from bone may have provided amounts sufficient to maintain their levels within the normal range. This mobilization resulted in lowering the ash content of the bone. The lack of adequate Ca, P, and Mg for proper bone mineralization resulted in delayed closing, irregularity, widening and loss of definition in the epiphyseal plates at the end of the long bones

in the ponies of T in Group I compared to T2 and T Since ponies in Group II (started experiment at 8 months of age) were exposed to sunlight for a longer period before being placed on experiment than those of Group I, they did not show very clearly these early symptoms of

rickets.

The author believes that for any further study of the importance

of vitamin D in pony nutrition, the age of the ponies and the intake of minerals should be evaluated.


































APPENDIX

ANALYSIS OF VARIANCE TABLES











TABLE 12. ANALYSIS OF VARIANCE FOR FEED EFFICIENCY.


Source Degree
Variable of of
Variation Freedom


Feed Age 1
efficiency (A)


Treatment
(T)


A x T


2


2


Sum of Squares PR > F



3.2634 0.4189a


0.6492


0.9269a


0.1990


0.9767a


aNon significant.


-80-









TABLE 13. WEEKLY PLASMA CALCIUM (mg/100 ml) LEVELS FOR INDIVIDUAL PONIES.


Group I (Started Experiment at 2 Months of Age)


No Vit. D No uv
(T I)


+ Vit. D No uv
(TO)


No Vit. D + uv
3


1
2
3
4
5
6
7
8
9
10 11
12
13
14 15 16
17 18 19
20 21
22 23
24 25 26 27


Group II (Started Experiment at 8 Months of Age)


No Vit. D No uv
- (T I)


+ Vit. D No uv (T2 )


2 3


11.1 10.7 -- -- 11.3
12.3 10.5 -- -- 11.4
11.0 12.1 10.7 11.7 12.1 11.6 11.7 11.2 11.2 11.9 11.5 11.5 9.5 11.8 12.2 11.8 11.8 11.1 11.2 12.3 11.8 12.5 10.7 11.9 11.2 10.9 12.5 12.1 11.1 12.1 10.6 10.8 10.8 10.4 11.7 11.4 11.3 12.8 10.6 12.4 11.7 11.6 13.5 12.2 12.0 11.8 11.3 10.5 -- 11.5 11.1 11.1 11.8 11.8 11.6
12.0 12.1 11.3 11.4 11.9 12.0 12.3 10.8 11.7 12.2 11.5 12.0 12.3 11.8 12.4 12.0 12.4 11.2 12.2 11.5 12.0 12.3 10.3 11.3 12.2 11.9 11.9 11.0 11.9 11.7 11.6 11.5 10.8 11.1 12.0 11.6 11.5 10.1 11.3 12.3 12.3 11.2 10.1 11.9 11.9 11.4 11.1 10.4 11.9 12.6 11.0 11.8 9.8 12.9 10.6 11.6 11.4 9.8 12.1 12.0 11.9 11.4 9.7 11.5 11.4 12.3 11.8 10.2 10.6 11.4


_______________________________


Week


No Vit. D + uv (T 3)


12.3 14.4 12.3 11.8 13.3 13.2 12.1 14.3 12.9 12.7 13.8 12.7 13.5 13.9 13.9 12.8 14.2 13.4 12.5 14.3 13.4 13.2 14.7 13.1 13.0 14.9 12.9 11.8 14.5 13.4 13.0 14.9 13.4 12.8 -- 13.4 12.4 14.3 13.0 13.0 13.4 12.6
12.0 14.4 13.9 12.0 14.3 13.8 12.7 14.8 13.6 12.0 14.3 13.5 12.0 14.6 13.8 11.9 14.4 13.5
12.4 11.8 13.2 13.6 14.3 13.4 12.1 13.8 14.3 12.3 14.2 13.5 12.2 13.6 13.3 12.8 14.2 13.7 12.0 14.1 13.2


10.9 10.7 10.9 10.7 11.3 10.5 9.8
11.4 12.0 11.2 12.4 11.7
12.0 11.9 11.6 11.9 11. 7 11.6 11.3 11.6
11.4 11.4 11.5 10.9
11.4


14.6 13.5 16.0 13.9
14.2 14.2 13.7
14.6
13.5
14.2 14.0 14.0 14.3 13.8
14.3 14.3 13.5 13.9
14.2 13.9
14.3 13.4 13.8 13.9 13.9
14.5 14.7


13.8

13.5 13.9
13.4 13.8 13.9 13.2
14.5

13.6
14.0 13.8 13.8 13.1 12.7 12.6 13.5 13.6 13.7 13.2 13.9 13.9 13.9 13.6
14.4 14.2


12.5 13.5 13.6
13.4 14.0 13.6 13.5
13.4 13.5
14.0 12.7 13.1 13.6 13.5 13.0
14.1 14.1 14.2 13.7 16.6 13.2 13.8
14.4 13.4 13.8 13.8 12. 7,







-82-


TABLE 14. ANALYSIS OF VARIANCE FOR PLASMA CALCIUM LEVELS.



Source Degree
of of Sum of Squares F Value
Variation Freedom


Age 1 13.0175 37.7428a
(A)

Treatment 2 4.8900 6.9440b
(T)

A x T 2 5.8400 8.4640b

Week 26 7.6748 0.6683b
(W)

W x T 54 14.9500 0.8143b

1 x A 26 13.0175 1.4516b

A x T x 1 50 17.3400 0.9310b


aHighly significant (p < .01).

b
Non significant.









TABLE 15. WEEKLY PLASMA PHOSPHORUS (mg/100 ml) LEVELS FOR INDIVIDUAL PONIES.


Group I (Started Experiment at


No Vit. D No uv
(TI)


7.7 6.0
4.7 2.8
4.4 4.5 3.4 4.7 3.9
4.4 3.4 3.6 3.9
4.7 5.5 5.7 3.8 5.2
5.4 4.4 5.4 6.1
6.5 6.3 6.1 5.7


6.7 6.7
4.5 5.3 6.1
4.7 6.6 5.2 7.0 6.0 6.7
8.4 6.8 6.9 5.9 6.3
6.4 5.4 7.6 6.9 6.0
4.9 6.8 9.1


+ Vit. D No uv
(T,)


7.0 6.6 5.2
4.9 4.6 5.5 5.9 6.3 6.6 8.1 5.7 7.0 7.6 6.8 5.5 7.7 5.0 6.6
4.9 6.5 6.5 5.7 6.1 6.3 5.7 5.5


5.6
5.4 4.7 6.6 6.6 6.5 8.6 5.9 6.7 5.5 6.2 6.0 6.2 6.0
4.8 4.8 4.8 5.2 3.9 3.6
4.0 4.6 3.3
5.4


27 5.7 6.9 5.7 4.0


2 Months of Age)


No Vit. + uv
(T )


6.5 6.8 5.3 7.2 6.6 6.7 7.2 5.2
6.4 4.7 5.0 5.7 5.7 5.6 5.7 7.2 5.8 6.6 7.7
6.4 5.7 6.2
5.4 5.8


D


7.6 6.8 6.0
4.7 5.4 6.3
4.2 5.4 5.5 5.2
5.4 6.3 5.5
4.7 5.0 5.9 5.2
4.5 6.0 5.2
4.5 4.5 6.1 5.7 6.6 5.5


6.8 5.5


Group 11 (Started Experiment at 8 Months of Age)


Week


D


+ Vit. D No uv (T 9)


No Vit. No uv
(T )

5.7 6.9 5.0 5.7 3.9
4.8 3.4 4.2 3.5 2.8
3.4 3.8
4.2 3.4
4.4 3.1 3.7 3.2 3.7 3.6
4.1 3.4 3.4 2.9
3.4 2.9 2.8


3.8 5.0 5.9 5.7
4.7 5.0

5.1 5.1 3.3 5.1 5.0 5.6
4.6 4.7 5.0 5.2 5.2
4.8 4.2 5.9 5.8
4.9 5.6
4.6 4.1 5.7


3.0 3.8

4.0 3.4 5.0 3.5 3.6 3.9
3.4 3.9 3.1 5.0 3.6
4.1 4.4 4.6 4.2 3.8 3.6 3.9 3.6
4.2 4.2 3.9 3.9


No Vit. + uv (T3

4.8

6.4 6.2
4.1 5.5
4.5 3.6
4.9

5.0
6.4 5.6 5.0 5.2 6.7
6.4 3.8
4.9 4.0 3.1 5.0 3.6
3.4 3.6 3.6


D


3.9 5.7 5.2 5.8 5.5
6.4 4.7 4.3 4.8 4.5 5.1 6.5 5.1 5.2
4.6 5.2 5.5 5.5 5.6 5.6 5.2
4.8 4.6 5.5
4.9 4.5


U0)


4.4 5.8 6.6 6.6 5.7 6.6 5.6 5.5 6.0 5.6 6.6 7.3 6.2 6.0 6.2
6.4 6.4 6.3 5.1
5.4 6.4 5.5 7.0 5.6 5.6 5.8 6.6


5.1 4.8 6.5


1
2
3
4
5
6
7
8
9
10 11
12 13
14 15 16
17 18 19
20 21 22 23
24 25 26










TABLE 16. WEEKLY PLASMA MAGNESIUM LEVELS (mg/100 ml) FOR INDIVIDUAL PONIES.


Group I (Started Experiment at 2 Months of Age) Group II (Started Experiment at 8 Months of Age)

Week No Vit. D + Vit. D No Vit. D No Vit. D + Vit. D No Vit. D
No uv No uv + uv No uv No uv + uv
(T ) (T) 2 (T
(T) T)3 2 3


1.8 1.9 1.5
1.6 1.5 1.6 1.9 1.7
2.0 1.8 1.9
2.2 1.8 1.6 1.5
1.4 1.4 1.5 1.5 1.7 1.3
1.4 1.7 1.9


1.5 1.0 1.0
1.2 1.2 1.4
1.5 1.5
1.2 1.7
1.2 1.0 1.3
1.4 1.6 1.7 1.3
1.4
1.2 1 .1
1.0 1.0 1.3
1.4
1.5
1.4


27 2.0 1.7 1.6


1.6
1.8 1.8 1.8 1.6
2.0 1.9
1.7 1.6 1.6
1.3
1.2 1.2
1.3 1.3
1.2 1.3 1.1
1.2 1.3
1.3
1.2 1.3
1.4


1.6 1.7 1.5 1.7 1.7 1.6 1.8 1.5
2.0

1.9
1.9 1.8 1.9 1.8
2.0 1.9 1 .7 1.7
1.7 1.8 1.6 1.6 1.8


1.7 1.2
1.4 1.4 1.6
2.0 1.3
1.4 1.3
1.3
1.3
1.2 1.1 1.5
1.4 1.4 1.5 1 .4
1.4 1.4
1.2 1.3 1.6
1.4 1.4 1.3


1.5 1.7 1.4


2.1 1.8 1.9
2.0 2.5
2.2 2.1 2.1
2.3 1.9
2.4 2.3
2.2
2.2
2.1 2.0 2.1 2.0 2.0 2.0 2.0 2.1 2.0 2.0 2.0 1.9
2.0


2.0 1.7
2.0 2.5
2.4 2.6 2.5 2.3
2.2 2.4 2.5

2.5
2.0 2.3 2.5 2.7 2.6 2.7 2.3 1.6
2.4 2.4 2.4 2.5 2.5 2.3


1.7
1.7
2.2 2.1 2.2 2.0 2.0 2.1 2.0 1.9 1.9 1.9 2.2
1.9
2.0 2.0 1.9
2.0 2.2 2.2 2.4 ').5
2.3
2.1 2.1 1.9


2.3 1.8
2.1 2.3
2.2 2.5
2.2 2.2 2.0 3.1
2.1 2.3
2.4
2.1 2.0 2.0 2.0 2.3
2.3
2.4 2.4 2.4 2.3 2.3 2.3
2.2


2.0 2.2


2.0

2.1 2.4 2.2 2.2 2.1 2.1 2.4

1.9
2.1 2.2 2.2 1.8
1.8 1.8 1.7 1.8
2.0 1.9
2.4 2.4 2.4 2.4 2.5


1.9 1.8
2.1 2.4 2.4 2.3
2.1 2.0
2.1 2.1 1.8
2.1
2.1 2.2 1.9
2.1 2.3
2.4 2.1 2.2 2.4 2.5
2.4 2.5 2.4
2.1


02 4>


2.3 2.4


1
2
3
4
5
6
7
8
9
10 11
12 13
14 15 16 17 18 19
20 21 22 23
24 25 26


1.6 1.8 1.1 1.1
1.4 1.5
1.4 1.4 1.3 1.3 1.3
1.2 1.2 1.6 1.6 1.7 1.7 1.5
1.5
1.6 1.5 1.7 1.8 1.7
1.9 1.6








-85-


TABLE 17. ANALYSIS OF VARIANCE FOR PLASMA ALKALINE PHOSPHATASE.


Source of
Variation


Degree of
Freedom


Age
(A)

Treatment 2
(T)

A x T 2

Week 4
(W)

A x W 4

T x W 8

A x T x W 8



aI\on significant. bHighly significant (p < .01).


Sum of Squares


PR > F


215.8


1026.3


2493.6 1434.3 19221.2 3748.5 9038.4


0.4879a 0.1892 a 0.0046b 0.0010 b 0.7055 a 0.1743 a







-86-


TABLE 18. ANALYSIS OF VARIANCE FOR 25-OH VITAMIN D.


Source Degree
of of Sum of Squares PR > F
Variation Freedom


Age 1 1430.60 0.0026a
(A)

Treatment 2 109.00 0.4468b
(T)

A x T 2 296.80 0.1609b

Week 4 21.31 0.8485b
(W)

A x W 4 46.74 0.5732b

T x W 8 119.57 0.4980b

A x T x W 8 135.43 0.4155b


aHighly significant (p

b
Non significant.


< .01).







-87-


TABLE 19. ANALYSIS OF VARIANCE FOR SELECTED COMPOSITION OF BONES.


Source Degree
Variable of of Sum of Squares PR > F
Variation Freedom

Age (A) 1 33.3333 0.0441a
Water Treatment (T) 2 50.1667 0.0558b


A x T 2 8.1667 0.4958b


Age (A) 1 0.9600 0.3839b
Ash c
% Treatment (T) 2 14.6425 0.0101

A x T 2 1.1575 0.6259a


Age (A) 1 2.4300 0.1834b
Cab % Treatment (T) 2 0.5267 0.7902

of ash A x T 2 1.2800 0.5810b


P

of ash


Age (A) 1

Treatment (T) 2

A x T 2


Age (A) 1

Treatment (T) 2

A x T 2


Mg

of ash


0.0533 0.0617 0.4817


0.0014 0.0016

0.0004


0.7297b 0.9279 b 0.5825b 0.2559b

0.4428 0.7978b


aSignificant (p < .05).

b
Non significant. cHighly significant (p < .01).







-88-


TABLE 20. ANALYSIS OF VARIANCE FOR SELECTED PHYSICAL CHARACTERISTICS
OF BONES.



Source Degree
Variable of of Sum of Squares PR > F
Variation Freedom


Age (A) 1 0.0396 0.0033a

Density Treatment (T) 2 0.0362 0.0120b

A x T 2 0.0006 0.8496c


Age (A) 1 100039387.4 0.032 b
Breaking Treatment (T) 2 48337245.7 0.1653
Strength

A x T 2 46249622.3 0.1731c


Age (A) 1 2.6508 0.0131b
Cortex Treatment (T) 2 1.7580 0.0780c
Area
Ax T 2 0.0582 087


aHighly significant (p < .01).

b
Significant (p < .05). cNon significant.







-89-


TABLE 21. WATER, ASH, CALCIUM, PHOSPHORUS, AND MAGNESIUM CONCENTRATION
OF LIVER AND KIDNEY FOR INDIVISUAL PONIES.a


Organ Liver Kidney
Content b
TGb G2 G 1 G2

T 73.5 72.5 83.1 81.8
1 74.4 71.2 81.9 81.5

Water T2 e 73.5 72.5 82.6 82.2
% 72.2 71.6 82.4 81.2
f
T3 73.5 70.8 82.6 80.5
73.3 73.0 81.6 80.0


T 4.3 4.0 7.2 6.1
4.5 3.8 6.9 6.7

Ash TI) 4.4 4.8 7.4 7.4
% 4.4 3.9 6.0 6.3

T3 4.0 4.5 6.6 6.7
4.2 4.2 6.5 6.3


T 237.6 188.0 2159.3 775.2
211.0 193.8 715.2 1997.3

Calcium T2 266.8 229.0 3059.2 3892.8
ppm 263.4 237.0 552.0 1847.8

T3 208.6 195.6 557.7 636.4
203.2 219.6 580.6 1943.3


T 9406.0 8192.0 11683.8 10672.4
10536.0 7787.8 10746.8 10729.0

Phosphorus T 9989.2 8273.0 12298.8 11895.8
ppm 10680.0 8372.0 10107.2 10719.6

T3 9121.4 8439.0 10020.0 10599.8
9444.0 9079.7 10469.2 10549.7




Full Text

PAGE 1

EFFECT OF VITAMIN D AND SUNLIGHT ON GROWTH AND BONE DEVELOPMENT OF YOUNG PONIES By WALEED M. EL SHORAFA A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA

PAGE 2

This dissertation is dedicated to my wife, Annalee, to my daughter, Rhonda, for their love, understanding, and patience during the course of endeavor, and to my mother and father for their faith in my potential.

PAGE 3

ACKNOWLEDGEMENTS The author owes a most sincere debt of gratitude to Dr. J.P. Feaster, who served as chairman of the supervisory committee, for his patience and understanding during the course of this endeavor. Dr. E.A. Ott was most helpful in his guidance and instruction through the research phase conducted at the Horse Research Center, Ocala. Special thanks to Dr. R.L . Shirley, Dr. C.M. Allen, Jr., and Dr. D.C. Sharp III as members of this graduate committee and for their guidance to make this study possible. The author also thanks C. Albiol, manager, as well as all other employees of the Horse Research Center who helped to make the work less of a chore and more of a learning experience. Special thanks to Dr. Asquith for this sincere help in the radiographs carried out for this project. The thanks extend to Dr. Littell, W. Offen, M. Vernon, and C.R. Smith for their aid in statistical and laboratory analysis. Special thanks to Dr. A.E. Green and his assistants for their aid in determination of ultraviolet light. iii

PAGE 4

TABLE OF CONTENTS Page ACKNOWLEDGEMENTS m LIST OF TABLES vi LIST OF FIGURES viii ABSTRACT ix CHAPTER I INTRODUCTION 1 II LITERATURE REVIEW 3 Introduction 3 Rickets in Rats 4 General Remarks on Rickets in Rats 11 Rickets in Poultry H Rickets in Swine 15 Rickets in Cattle 18 Rickets in Humans 19 Rickets in Horses 25 III MATERIAL AND METHODS 27 Conditions and Experimental Design 27 Sample and Measurements 29 Laboratory Analysis 30 Blood plasma calcium and magnesium determination. . . 30 Blood plasma phosphorus determination 31 Blood plasma alkline phosphatase determination. ... 31 Plasma 25 -hydroxy vitamin D determination 31 Bone ash and cortex area 31 Ca, P, and Mg bone asli 31

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Page Bone breaking strength 32 Radiographic classification 32 Kidney and liver analysis . . 34 Statistical Analysis 34 IV RESULTS AND DISCUSSION 36 External Appearance 36 Rate of Growth and Feed Efficiency 38 Blood Analysis 42 Plasma calcium (Ca) , phosphorus (P) , and magnesium (Mg) levels 42 Plasma alkaline phosphatase 54 Plasma 25-hydroxy vitamin D 56 Bone Analysis 59 Water concentration of bone 59 Bone ash (percent of dry, fat-free bone) 62 Bone calcium, phosphorus., and magnesium (% of ash) . . 62 Bone cortex area 63 Bone density 65 Bone breaking strength 65 Bone epiphyseal closure 66 Liver Analysis (Water, Ash, Calcium, Phosphorus, and Magnesium Concentration) 70 Kidney Analysis (Water, Ash, Calcium, Phosphorus, and Magnesium Concentration) 72 V SUMMARY AND CONCLUSION 73 APPENDIX ANALYSIS OF VARIANCE TABLES 80 LITERATURE CITED 93 BIOGRAPHICAL SKETCH . 101

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LIST OF TABLES Table Page 1. COMPOSITION OF THE VITAMIN D-DEFICIENT DIET 28 2. EFFECT OF VITAMIN D AND SUNLIGHT ON RATE OF GAIN AND FEED EFFICIENCY OF PONIES 39 3. AVERAGE WEEKLY PLASMA CALCIUM LEVELS DURING 27 WEEKS OF EXPERIMENTAL PERIOD 43 4. AVERAGE PLASMA PHOSPHORUS LEVELS DURING 27 WEEKS OF EXPERIMENTAL PERIOD 47 5. AVERAGE PLASMA MAGNESIUM LEVELS DURING 27 WEEKS OF EXPERIMENTAL PERIOD 50 6 . PLASMA ALKALINE PHOSPHATASE AT 0, 4, 12, 20, and 27 WEEK PERIOD 55 7. INDIVIDUAL AND AVERAGE VALUES OF PLASMA 25-HYDROXY VITAMIN D OF THE TWELVE PONIES 58 8 . SELECTED COMPOSITION AND PHYSICAL CHARACTERISTICS OF RIGHT METACARPAL BONE 60 9. SELECTED COMPOSITION AND PHYSICAL CHARACTERISTICS OF LEFT METACARPAL BONE 53 10. EPIPHYSEAL CLOSURE OF THE DISTAL OF THE THIRD METACARPAL (MD), THE PROXIMAL OF THE FIRST PHALANX (P.P), AND THE SECOND PHALANX (P P) OF BONES OF GROUP I PONIES 67 11. AVERAGE WATER, ASH, CALCIUM, PHOSPHORUS, AND MAGNESIUM CONCENTRATIONS OF LIVER AND KIDNEY 71 12. ANALYSIS OF VARIANCE OF FEED EFFICIENCY 80 13. WEEKLY PLASMA CALCIUM LEVELS FOR INDIVIDUAL PONIES 81 14. ANALYSIS OF VARIANCE FOR PLASMA CALCIUM LEVELS 82 15. WEEKLY PLASMA PHOSPHORUS LEVELS FOR INDIVIDUAL PONIES ... 83 16. ' WEEKLY PLASMA MAGNESIUM LEVELS FOR INDIVIDUAL PONIES. ... 84

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Table Page 17. ANALYSIS OF VARIANCE FOR PLASMA ALKALINE PHOSPHATASE ..... 85 18. ANALYSIS OF VARIANCE FOR PLASMA 25-OH VITAMIN D 86 19. ANALYSIS OF VARIANCE FOR SELECTED COMPOSITION OF BONES ... 87 20. ANALYSIS OF VARIANCE FOR SELECTED PHYSICAL CHARACTERISTICS OF BONES 88 21. WATER, ASH, CALCIUM, PHOSPHORUS, AND MAGNESIUM CONCENTRATION OF LIVER AND KIDNEY FOR INDIVIDUAL PONIES 89 22. ANALYSIS OF VARIANCE FOR LIVER, WATER, ASH, CALCIUM, PHOSPHORUS, AND MAGNESIUM CONCENTRATIONS 91 23. ANALYSIS OF VARIANCE FOR KIDNEY WATER, ASH, CALCIUM, PHOSPHORUS, AND MAGNESIUM CONCENTRATIONS 92

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LIST OF FIGURES Figure Page 1. INSTRON TENSIL STRENGTH TESTING APPARATUS FOR DETERMINING BREAKING STRENGTH OF BONES 33 2. EFFECTS OF TREATMENT ON EXTERNAL APPEARANCE OF 3 OF THE PONIES IN GROUP I 37 3. WEEKLY BODY WEIGHT OF GROUP I PONIES 40 4. WEEKLY BODY WEIGHT OF GROUP II PONIES 41 5. AVERAGE WEEKLY PLASMA CALCIUM OF GROUP 1 45 6 . AVERAGE WEEKLY PLASMA CALCIUM OF GROUP II 46 7. AVERAGE WEEKLY PLASMA PHOSPHORUS IN GROUP I 48 8 . AVERAGE WEEKLY PLASMA PHOSPHORUS IN GROUP II 49 9. AVERAGE PLASMA MAGNESIUM LEVELS IN GROUP 1 52 10. AVERAGE PLASMA MAGENSIUM LEVELS IN GROUP II 53 11. STANDARD CURVE FOR THE COMPETITIVE BINDING ASSAY ON 25-OH VITAMIN D 3 57 12. EFFECT OF TREATMENTS ON METACARPAL BONES OF THREE PONIES OF GROUP I 64 13. EFFECT OF TREATMENTS ON THE EPIPHYSEAL CLOSURE OF GROUP I 68 14. EFFECT OF TREATMENTS ON EPIPHYSEAL CLOSURE OF GROUP II. 69 vi i i

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Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECT OF VITAMIN D AND SUNLIGHT ON GROWTH AND BONE DEVELOPMENT OF YOUNG PONIES By Waleed M. El Shorafa March, 1978 Chairman: Dr. J.P. Feaster Major Department: Animal Science A 2 x 3 factorial design experiment was carried out to study the effect of vitamin D and sunlight on growth and bone development of young ponies. Two groups orf ponies (G^ = two months of age and G ? = eight months of age) were assigned to three treatments (T = no sunlight without vitamin D supplement, T^ = no sunlight with a daily supplement of 1,000 I.U. of vitamin D, T^ = sunlight without vitamin D supplement). All ponies were put in a windowless barn with very minimum ultraviolet light exposure for one month (to deplete their body stores of vitamin D) and then were assigned to one of the above treatments. A vitamin Ddeficient diet, adequate in all other nutrients required for optimum growth, was fed to the ponies three times daily (ad libitum) for five months . Weekly blood samples were obtained to study the effect of treatments and age groups on plasma calcium (Ca) , phosphorus (P), magnesium (Mg), alkaline phosphatase (Aik), and 25-hydroxy vitamin D (25-OH Vit. D) levels. Monthly X-rays were taken to study the effect of treatments and groups on the development of epiphyseal p]ates at the end of the

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long bones of the front legs. All ponies were killed at the termination of the experiment. The two front metacarpal bones, liver, and kidney were obtained for determination of water, ash, Ca, P, and Mg concentration. One of the metacarpal bones was used to determine bone cortex area and the other was used to determine bone breaking strength and bone density. The data were analyzed factorially by statistical analysis. In case of interaction effect, a Duncan's multiple range test was done to show the effect of treatments within the groups. Loss of appetite, difficulty in standing, and slightly lower feed efficiency were found in T^ , compared to T 0 and T , but the actual external appearance of rickets (bowed legs and inability to stand) did not occur . Plasma Ca , P, and Mg were within the normal range and there were no significant differences among treatments. The results indicated higher plasma Ca levels (p < .01) of group II than group I (G = 10.5-13.5 vs. = 12.6-15.2 mg/100 ml). Plasma alkaline phosphatase levels varied and were not affected by age or treatments. Plasma 25-hydroxy vitamin D levels were normal for all the ponies, and there were no significant differences among treatments. Average plasma 25-OH vitamin D for T , T^ , and T^ were 63.4, 64.1, and 61.2 ng/ml. Ponies in group II (G ? ) had significantly (p < .01) higher plasma 24-OH vitamin D than ponies in group I. For G^ and the levels were 67.8 and 57.9 ng/ml. Bone water concentrations were higher in T^ (p < .05) than T 2 and T^, and in G^ (p < .05) than G^ ponies. Ponies on treatment 1 (T ) had lower bone ash concentration (p < .01) than ponies on T^ and T , % of dry fat-free bone, but there were no significant differences between

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groups. Calcium, P, and Mg as percent of bone ash were not affected by either treatments or groups. Bone cortex area of ponies in was lower (p < .07) than T ^ and and group II had a higher cortex area (p < .05) than group I. Bones in appear to have low bone breaking strength (1308.8 kg/cm 2 ) than 1' 2 (1583.1 kg/cm 2 ) or T 3 (1583.1 kg/cm 2 ). Bone breaking strength was higher (p < .03) in G^ than G^ . The epiphyseal plates (at the distal end of the metacarpal bone, proximal end of the first phalanx and the proximal end of the second phalanx) of within group I were irregular, wider, poorly defined, and late in closing, compared to T 2 and T^. Factorial analysis indicated that ash concentration of liver and kidney were not affected by treatments or age groups. Water concentration of liver and kidney were lower (p < .02) in G ? than G x . Ponies in group I had higher Ca (p < .01) and P (p < .01) liver concentration than ponies in group II. There was an interaction effect concerning liver Mg concentration. Duncan's multiple range test indicated that magnesium level under T^ was significantly lower (p < .05) from T^ or t 2 within group I. Calcium, P, and Mg kidney concentrations were not affected by either age groups or treatments. In conclusion, the ponies in T (especially those in the younger age) suffered from early symptoms of rickets compared to and T^ ponies. The symptoms were loss of appetite, low feed efficiency, and low bone ash and breaking strength with epiphyseal plates of the long bones which were wider, irregular, lacking definition, and delayed in closure . xi

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CHAPTER I INTRODUCTION For several decades, scientists have recognized the significance of an agent that prevents rickets in young animals. This agent has been identified as vitamin D and its active form is the hormone 1,25 dihydroxy vitamin D. Animals obtain their needed vitamin D either from exposure of their skin to ultraviolet light or from diet. The study of vitamin D and its relation to rickets needs further investigation, especially after the recent increase in the knowledge of vitamin D metabolism and mechanism of action. Rickets is of interest to ecologists, since it is probably the first disease which could be classified as a result of air pollution, and to anthropologists, who have postulated that the effect of ultraviolet light on vitamin D metabolism in the skin was an important factor in the development of races with pigmented and non-pigmented skin and in their geographical distribution. The disease is of interest to physiologists, since it is an example of a deficience of a single element, and to biochemists, because recent data on vitamin D metabolism not only explain the rachitic syndrome, but may uncover many of the mysteries of mineral regulatory mechanisms. It is, however, for the nutritionist that rickets holds the greatest interest. Park (1923) stated that rickets is a common disturbance among puppies, pigs, lambs, poultry, and kids, but occurs less frequently among colts, calves, and rabbits. It is easily produced in most domestic

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2 animals by simply omitting vitamin D from the diet and preventing exposure to sunlight or other forms of ultraviolet radiation: In rats, however, lowered phosphorus intake also is required to produce rickets. Techniques for producing and diagnosing rickets were developed mostly in rats, dogs, poultry, and humans. There is a marked difference in species responses to vitamin D. It has been assumed that horses, like other animals, suffer adverse effects from a lack of vitamin D intake and the absence of ultraviolet light. This assumption is based on results with other animals, but does the young horse develop rickets? Occasionally, the colt, less than one year old, experiences a period of pain and shows an altered gait. The colt in most instances is not yet weaned and is receiving the major portion of its nutrition from the mare's milk. Regular radiography of the joint shows enlargements similar to those of rickets, but is it rickets? Much of the accepted knowledge of vitamin D and its importance in horse nutrition has been largely borrowed from other species. Currently there is a large volume of research to evaluate the actual nutritional requirements of horses. The horse industry has become a billion dollar twentieth century phenomenon. The horse population is increasing in number and quality at a rapid pace. Universities, scientific foundations, and government facilities have begun allocating time and funds to equine research. Since horses have been bred for the purpose of racing, showing, and pleasure, bone development in young horses is an important factor. The present study was carried out to determine if horses need vitamin D or if sunlight is adequate to bring about optimum bone development .

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CHAPTER II LITERATURE REVIEW Introduction The discovery of the cause and cure of rickets is one of the great triumphs of biochemical medicine. Rickets is known as a bone disease which occurs in young animals. As a result of poorly calcified bone, supporting weight is painful and results in lameness or disinclination to move. A clear description of the clinical picture of rickets was published in the 17th century, although mention of various aspects of the syndrome goes back even further . The first successful attempt to induce rickets experimentally in animals was made at the University of Glasgow in 1908 by Leonard Findlay, who published conclusive pictures of puppies that had been confined in cages and developed rickets. Melanby (1919) also used pups in his early work on rickets and concluded that rickets was due to a deficiency of a specific dietary factor. These results aroused considerable interest and led to a large number of investigations in this field during the following decade. Dogs, hens, and rats have been used most for experimental studies of rickets. Accumulated knowledge on rickets shows that there are biochemical changes in bone, kidney, liver, and blood and definite external manifestations of the disease in the animals. In studies of the biochemical changes in blood in rickets, most research has dealt with the levels of calcium, phosphorus, magnesium, 25-OH

PAGE 15

-4vitamin D, and alkaline phosphatase. Calcium, P, Mg, ash content, organic matter, water content, breaking strength, and cortex area of the bone have also been investigated. Radiographic study of the epiphyseal closure provides further evidence in diagnosis of rickets. In addition to these, the external appearance, growth, and feed efficiency have been measured to show rickets. Since liver and kidney are very much involved in vitamin D metabolism and its relation to Ca, P, and Mg regulation in the body, the level of these minerals in the liver and kidney has been studied. The findings of research which has shown the biochemical, radiographic, and clinical abnormalities of rickets in different species of animals are described below in some detail. Rickets in Rats Rats are not as susceptible to rickets as are the higher mammals and poultry. Typical rickets in this type of animal can be produced only if the diet is abnormal with respect to calcium or phosphorus as well as deficient in vitamin D. The earliest diets of this type were developed by McCollum e^t aJ. (1922) . The diet was composed principally of cereals and is high in calcium and moderately low in phosphorus. McCollum et al . ' s experiment demonstrated clearly the existence of a growth factor or vitamin in the diet which regulates bone metabolism. Steenbock and Nelson (1923) laid the foundation for an experimental method in which, by the use of ultraviolet light, growth can be used as a measure of the comparative amount of vitamin D occurring in food. Steenbock and Black (unpublished data) showed no significant difference in growth of irradiated or non-i rradiated rats. Shortly after this,

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-5Hume and Smith (1923) showed an increase in growth of irradiated rats compared with non-irradiated ones. In 1922, it was found that X— ray images of the bones could provide visual evidence of richets. Pappenheimer et al . (1922) presented X-rays of the hind leg of a rachitic rat which showed a lack of calcification and a wide area of uncalcified cartilage at the junction of the diaphyses and the epiphyses. Dutcher and Rothrock (1925) made detailed studies of the changes of the bone ash of rachitic rats. They reported a bone ash of 62% in dyr, fat-free bones from normal rats; in rachitic rats it was 26.5%. Steenbock and Black (1924) reported that by irradiation with a quartz mercury vapor lamp, rat rations could be activated, making them growth-promoting and bone-calcifying to the same degree as when the rats were irradiated directly. Also, their results indicated that liver taken from irradiated rats was growth-promoting while liver from nonirradiated rats was inactive. The same was found true of lung and muscle tissue. Inactive muscles, exposed after removal from the body to the radiations of the lamp, were found to have become activated, being both growth-promoting and bone-calcifying. Liver treated the same way also promoted bone calcification. Again Steenbock and Black (1925) studied the induction of growthpromoting and calcifying properties in fats and their unsaponif iable constituents by exposure to light. Their results gave clear evidence of the antirachitic activity of irradiation of various fats to promote growth and increase calcium content of rat bones. Dodds and Camerson (1943) studied the relation of rickets to growth with special reference to the bones. Their studies were based on 135

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6 albino rats, in most of which rickets was produced by the SteenbockBlack diet 2965. They traced the development and healing of rickets and growth of the bones by weekly roentgenograms. Graphic methods were used to show rickets, healing, and growth. The rachitic rats were sub-normal in weight, but their growth did not follow any single pattern. The growth of the leg bones and of the vertebral columns of the rachitic rats was greatly retarded. The retardation of the vertebral columns was relatively greater than of the leg bones. The epiphyseal cartilage of the tibia (typical for all long bones), during the first week or two on the rachitogenic diet, continued to make its contribution to the length of the shaft of the bone, but in decreasing amount. After about the third week the shaft ceased to elongate, and the pathologic thickening of the epiphyseal cartilage and the elongation of the bone became equal and identical. Bethke ££ _al. (1923-24) showed that with diets in which the Ca/P ratio was very high vitamin D did not induce growth. Nicolaysen and Jansen (1939) compared the bones of vitamin Ddeficient and vitamin D-treated rats. Their results indicate no difference in the percentage of ash in the bones between the two groups, but the anatomical findings indicate a failure of newly formed matrix to calcify in the bones of the vitamin D-deficient animals. Unfortunately, Nicolaysen and Jansen did not give any data on the serum Ca and serum P. Carlsson (1952) studied the effect of vitamin D on the skeletal metabolism of calcium and phosphorus in rats. He concluded that vitamin D favored the removal of lime salts from the bones. The other effects of the vitamin observed in his experiment (increased serum Ca level,

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-7increased growth, and calcification of the skeleton) were regarded as secondary effects. Carlsson (1954) studied the cause of hypophosphatemia and hypocalcemia in vitamin D-deficient rats. He stated that in rats on a P-free diet deficient vitamin D produced its typical effects on the level of 32 serum anorganic P and on the uptake of P in the skeleton. These effects could not be explained as consequences of increased absorption or decreased excretion of P, since the element was retained almost completely even in the absence of vitamin D. He showed also that vitamin D-deficient rats were unable to utilize their bone store for maintaining a normal serum Ca, even if the bone stores had been well filled by feeding a diet with a good Ca/P ratio. In conculsion, he proved that the essential cause of hypophosphatemia and hypocalcemia in vitamin D deficiency is an insufficient utilization of stored bone salt. Beilin ejt al. (1954) obtained considerable growth in rats by the addition of vitamin D to a diet containing 0.62% and as little as 0.034% Ca. Steenbock and Herting (1955) studied vitamin D and growth in rats. In a series of experiments with young rats, they found that a low Ca diet adequately supplied with phosphorus and other dietary essentials presented optimum conditions for eliciting the maximum growth differential which can be obtained with vitamin D. This effect of the vitamin was accompanied by a decrease in serum inorganic P, an increase in serum Ca , a decrease in the percentage of bone ash, an increase in the organic matrix of bone, and slight increase in the width of the car tilagenous metaphyses. Vitamin D always tended to bring the serum P to a normal level. On the other hand, its only effect on the level of serum Ca was

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8 to increase it. They concluded that weanling rats require vitamin D for optimum performance. Harrison e_t al_. (1958) fed 3 week old rats a vitamin D-deficient diet adequate in calcium and phosphorus and the rats showed biochemical evidence of vitamin D-deficiency without the characteristic bone changes °f rickets. The significant findings were hypocalcemia with normal serum phosphorus levels. Body weight gain increments were reduced in the vitamin D-deficient rat. One hundred units of vitamin D increased serum calcium. Dixit (1967) studied the influence of vitamin D and starvation on serum calcium and phosphorus. He showed no significant changes in the serum calcium level in rachitic rats following vitamin D administration. The serum inorganic phosphorus of rats administered vitamin D 48 hours earlier was 5.84 mg/100 ml compared with 4.04 mg in the untreated control; this difference was not statistically significant. The product of calcium and phosphorus (mg/100 ml) in the serum of untreated rachitic control was 41.6, whereas at 24, 48, 72, and 96 hours after vitamin D treatment the values were 46.1, 60.7, 60.8, 57.1, respectively. The Bon Kossa silver staining and radiological examination of the metatarsals indicate that the earliest signs of "healing" were evident at or after 48 hours of vitamin D treatment. Perhaps the healing is initiated when the product of calcium and phosphorus rises to above 60. Deluca and Steenbock (1956) reported that the alkaline phosphatase in the plasma of rats on various semisynthetic, vitamin D-free rations was higher than that of animals on a vitamin D-containing stock diet. The administration of vitamin D reduced the values to approximately those found in stock rats.

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-9Hurwitz et. al. (1969) investigated the role of vitamin D in plasma calcium regulation. They observed slower growth and low bone ash, bone Ca, and bone P in rachitogenic rats compared to the controls. A slightly elevated plasma P was observed in the rachitogenic rats. Baylink ej^ a^. (1970) studied formation, mineralization, and resorption of bone in vitamin D-deficient rats. They obtained a lower growth, tibia length, and serum calcium, and higher serum phosphorus in vitamin D-deficient rats than in rats provided adequate vitamin D. Simmons and Kunin (1970) studied the development and healing of rickets in rats. Weanling rats were rendered rachitic by maintenance on a low phosphate, vitamin D— free diet. The results indicated decreased growth, greater width of the proximal tibial epiphyseal cartilage, reduction of voluntary food intake, lower bone percent bone ash, and lower feed efficiency of a group of rats maintained on a rachitic diet, low in phosphorus and deficient in vitamin D, compared with a control group. Al-Ganhari e_t a^. (1973) studied vitamin D-deficiency in rats. Their results indicated that vitamin D-deficient rats gained significantly less w£^i§ht than control rats. Also, as a result of vitamin D deficiency, the liver weight decreased compared with that of the controls. The rachitogenic rats developed symptoms of rickets which were shown by a distinctive gait and enlarged joints. Omdahl and Deluca (1973) demonstrated that physiologic doses of vitamin D^ must be metabolized in the liver to 25-hydroxy vitamin (25-OH D ) and subsequently in the kidney to 1,25-dihydroxy vitamin D^ (1, 25(OH) 2 D ) before it can function.

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10 Clark e t al . (1973) showed that in vitamin D-deficient rats hypocalcemia, stunted growth, and soft bones occurred some weeks prior to total disappearance of 25-OH vitamin D from serum. In studying the role of 1,25-dihydroxy vitamin D in maintaining serum phosphorus and curing rickets, Tanaka and Deluca (1974) showed that the intravenous injection of a single dose of 650 p moles of 1,25dihydroxy vitamin to rats fed a vitamin D— deficient, low— phosphorus diet, caused an elevation of serum phosphorus within 5 hours which reached a maximum in about 10-12 hours and returned to deficiency levels 2-3 days later. On the other hand, a single injection of 650 p moles of 25-hydroxy vitamin produced a significant rise in phosphorus at 12 hours which reached a maximum in 24 to 36 hours and was maintained for at least 7 days. The single dose of 1,25-dihydroxy vitamin supported little calcification of bone, whereas the 25-hydroxy vitamin produced marked calcification. The vitamin D-deficient rats showed low serum phosphorus, normal serum calcium, and decreased bone ash. Yoshiki and Uanayisawa (1974) investigated the role of vitamin D in the mineralization of dentine in rats made rachitic by a diet low in calcium and deficient in vitamin D. After two weeks, weight gains were only 20-30 g as compared with 80-90 g in the control rats. The serum inorganic phosphorus and calcium levels dropped to 4.8 mg/100 ml and 8.5 mg/100 ml, respectively. Physiological amounts of vitamin D, given ora lly 1° rachitic rats, increased their serum phosphorus from 4.8 ± 0.5 mg/100 ml to 7.5 ± 0.4 mg/100 ml.

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11 General Remarks on Rickets in Rats Studies of experimental vitamin D deficiency in the rat are complicated by the fact that this species develops rachitic changes only when the Ca/P ratio of the diet is also modified. Coleman e_t al. (1950) ported that a disorder simulating rickets can be produced in growing rats by a pure deficiency of phosphorus even in the presence of adequate amounts of vitamin D. McClendon and Blanstein (1965) reported that young rats develop skeletal changes typical of rickets upon specific calcium deficiency. These and other observations have cast doubts on the importance of vitamin D in mineral metabolism of the rat. Generally speaking, calcium and phosphorus content of the diet must be taken into account in any experiment involving vitamin D deficiency in the rat. Rickets in Poultry Normal chicks, experimentally raised without sunlight or vitamin D, regularly develop rickets, called leg weakness by poultrymen. In birds, unlike rats, leg weakness can be produced by the absence of the vitamin alone, regardless of the calcium and phosphorus content of the diet. The first experimental production of rickets in poultry was by Hart et al. (1922). Since that time many investigations have been done in poultry to determine the effect of vitamin D on growth and other parameters involved. Hart e_t al. (1923-24) demonstrated the striking effect of sunlight on the growth of chicks on a synthetic diet which contained ample calcium and phosphorus but was low in vitamin D. One group was given the basal ration without sunlight and the other received the same diet but was re-

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12 exposed to summer sunlight 30 minutes each day. At the end of 6 weeks on the experimental regimes, the two remaining rachitic fowls (no sunlight) weighed 80 and 90 grams, respectively, and the two controls (irradiated) weighed 145 and 180 gm. Bethke e_t al. (1928-29) observed marked increases in the growth rate of chicks when cod liver oil (1%) was added to a diet low in vitamin D. Steenbock ejt al. (1923-24) found low phosphorus in the serum of rachitic chicks. Common (1936) found high serum alkaline phosphatase during rickets in chicks. Hart et al. (192324) published X-rays of the complete boney structure of a rachitic chick and of a normal control. In the rachitic animal there was very little differentiation between cortex and marrow cavity and the whole skeleton was almost devoid of dense bone. Steenbock et al. (1923) used blood inorganic phosphorus and calcium as criteria in the demonstration of the existence of a specific antirachitic vitamin in chickens. They showed that by the administration of cod liver oil freed from vitamin A, the inorganic phosphate and calcium of the blood were restored to normal, and the ash content of the bones was increased. Massengale and Nussmeir (1930) studied the action of activated ergosterol in the chicken. Their results indicate that activated ergosterol brings serum calcium and phosphorus to a normal level in rachitic chickens. Serum calcium was normal or slightly below normal, but serum phosphorus was always below normal in rachitic chickens. McGinnis and Evans (1946) studied the response of turkey poults to vitamin D. They supplied broad breasted bronze turkey poults with graded chick unit levels of vitamin I). They found that a level of 80 chick units from all sources (cod liver oil, salmon oil, corn oil.

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-13solution of irradiated 7-dehydrocholesterol and corn oil solution of activated ergosterol) except the activated ergosterol gave maximum bone ash and growth. The mortality caused by rickets in the D-deficient group was 100%. Motzok and Wynne (1950) pointed out the increase in blood alkaline phosphatase activity of rachitic chicks. They concluded that the blood level of alkaline phosphatase could be used to determine antirachitic activity of different sources of vitamin D, instead of bone ash. The potencies obtained by the phosphatase method differed from the value given by the bone ash method by amounts varying from 25-40%. Spinka (1960) studied the relative effectiveness of vitamin D ? and in a bone test on chickens. The results showed that weight gain, length, weight, and mineral content of femur, serum Ca, and total minerals in the blood were better in chickens given vitamin than those which were given vitamin . In the experiment, all chickens were sheltered from sunlight. Chen and Bosmann (1963) investigated the effect of vitamin and on serum calcium and phosphorus in rachitic chicks. The results showed that chicks fed the rachitogenic diet exhibited low serum calcium concentrations and percentage bone ash, but had significantly higher serum phosphorus levels than chicks fed a standard chick diet or treated adequately with either form of vitamin D. The vitamin to efficiency ratio was estimated at about 8:1 to 11:1. Waldroup e_t a]^. (1963) studied the effect of various levels of vitamin on phosphorus utilization by broiler-type chicks. They found that increased level of vitamin up to 360 international chick units (ICU)/pound resulted in increased body weight and percent bone ash.

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-14However , the response to increased levels of the vitamin became less as the calcium and phosphorus levels more closely approached the optimum. Waldroup e t al . (1964) studied the vitamin requirement of the broiler chick. Their results showed that 90 ICU of vitamin per pound as suggested by N.R.C. are adequate to support maximum growth and bone ash at the calcium and phosphorus levels recommended by this group (1.0% Ca and 0.6% P) . Canas e^ £kl. (1969) studied the effect of vitamin on cortical bone of rachitic chicks. They found that when expressed on a volume basis the cortical bone from rachitic chicks had decreased levels of both ash and organic material as compared with normal controls. Upon treatment with 8 IU/day of vitamin normal bone composition is restored within 7-8 days. Thornton (1970) investigated the skeletal and plasma calcium changes in chicks during recovery from vitamin D deficiency with normal calcium intakes. In his experiment, hypocalcemia was shown in vitamin deficient chicks with normal calcium intake. This was corrected within 48 hours by vitamin dosage. McNutt and Haussler (1973) measured the nutritional effectiveness °f 1 , 25-dihydroxycholecalciferol in preventing rickets in chicks, and found that its effectiveness was similar to that of 25-hydroxycholecalciferol, both metabolites being 1.5 to 2.2 times as active as cholecalciferol with respect to stimulation of weight gain and maintenance of plasma calcium levels. Yang e^t al_. (1973) evaluated the effect of different forms of vitamin in preventing rickets in turkeys. The results Indicated that growth, femur bone ash, bone length, breaking strength, plasma

PAGE 26

-15alkaline phosphatase and inroganic phosphorus correlated well with vitamin D status. Cork et al. (1973) studied the effectiveness of 1,25 dihydroxy vitamin in preventing rickets in the chick. In their experiment, 1,12 di-OH-D^ was 5.1, 3.4, and 3.7 times more potent than D in stimulating weight gain, maintenance of plasma calcium, and promotion of increased percent bone ash, respectively. Wong and Norman (1974) studied the mechanism of action of calciferol in white leghorn cockerels. In the experiment, all vitamin Ddeficient chicks had slow growth, low serum calcium (6.3 mg/100 ml serum), and a low bone ash of 25%. Cholecalciferol restored these to normal levels . Crenshaw et_ al . (1974) studied the effects of dietary vitamin D levels on the in-vivo mineralization of chicks' metaphyses. The chicks fed a rachitogenic diet became hypocalcemic and formed hypomineralized bones compared with chicks fed a control diet. Gonnerman e_t al. (1975) studied the effect of vitamin D, dietary calcium, and parathyroid hormone in chicks. Compared to chicks on control diet, chicks on the D— deficient diet had significantly decreased plasma Ca levels at 2 and 4 weeks and increased plasma P at 17 and 21 days . Rickets in Swine Vitamin D has for several decades been termed a nutrient required for optimum gain and skeletal development of swine. Investigations have shown that a low calcium intake plus vitamin D deficiency will cause rickets in swine. Loeffe] al . (1931) observed low calcium and low

PAGE 27

-16inorganic phosphorus in the sera and a reduction on the percentage of ash in long bones of rachitic pigs. Elliot al. (1922) indicated that there is no need, as measured by growth response, for supplemental vitamin D in rations for growing pigs confined in the absence of sunlight. Dunlop (1935) and Braude et al. (1943) concluded that pigs do not need vitamin D. Johnson and Palmer (1938) studied individual and breed variations in pigs diets devoid of vitamin D. They indicated that pigs need supplements of vitamin D in the diet to obtain maximum growth, and that the addition of irradiated yeast to the ration caused an improvement in appetite. Bethke et_ aJL . (1946) studied the comparative efficacy of vitamin D from irradiated yeast and cod liver oil for growing pigs, with observations on their vitamin D requirement. The results indicated that the minimum practical vitamin D requirement of growing pigs fed a ration containing 0.6% calcium and 0.45% phosphorus is approximately 90 U.S.P. units per pound of ration. Sinclair (1929) studied the influence of ultraviolet rays and vitamin D on bone ash of fall farrowed pigs. He did not find any difference in bone ash percentage in treated pigs or a control group. Wahlstrom and Stolte (1958) studied the effect of supplemental vitamin D in rations for pigs fed in the absence of direct sunlight. They concluded that the addition of 90 U.S.P. units of vitamin D per pound to a mixed ration complete in other known dietary factors resulted in little difference in the rate of gain, calcium, and inorganic phosphorus content of the blood, or calcium, phosphorus and total ash content of femurs.

PAGE 28

-17Johnson and Palmer (1941) observed that vitamin D supplied in the ration of weanling pigs by sun-cured alfalfa hay at levels of about 72 IU/kg was sufficient to cure gross symptoms of rickets and to correct serum calcium and phosphorus values. Miller e_t jrL. (1964) studied vitamin requirements of baby pigs (1-2 days old). The baby pigs sheltered away from sunlight were given a purified diet containing 0.8% Ca, 0.6% P, and 350 ppm Mg for five weeks. The effects of different levels of vitamin D ? from zero to 10,000 IU/kg of diet were studied. All pigs receiving no dietary vitamin D and no sunlight showed rickets pathology. The pigs which survived had low serum calcium (6.1 mg/100 ml) and higher serum alkaline phosphatase (39.1 Bessey-Lowry unit). Bone analyses in these pigs showed lower ash content, Ca , phosphorus, and breaking strength than the pigs supplied with vitamin D. All pigs receiving 100 IU or more of vitamin D^/kg diet exhibited optimal rates of growth and economy of diet utilization together with normal levels of serum Ca, P, Mg, and alkaline phosphatase and adequate skeletal development with an absence of rachitic pathology. To evaluate vitamin D requirements and study possible metabolic roles. Combs e_t a_l. (1966a) added various levels of vitamin D to the ration of 115 pigs weaned at 2 weeks of age. All animals were housed in the absence of sunlight from birth until termination of the experiments. The results indicated no significant difference in bone ash percent or rachitic symptoms among the treatments nor were the quantities of calcium and phosphorus in the bone ash significantly different. Serum calcium of the unsupplemented pigs was significantly higher than in those given 110 IU of vitamin D, but all treatment groups exhibited

PAGE 29

-18satisfactory serum calcium level. No significant differences in either serum phosphorus or magnesium were found among treatment groups. Combs et al . (1966b) studied levels and sources of vitamin D for pigs fed diets containing varying quantities of calcium. The results indicated that average daily gain, feed intake, and bone ash were not significantly influenced by vitamin D treatment. Rickets in Cattle Park (1923) stated that rickets occurs less frequently among calves than other animals. The signs of rickets in the calf have been described by Bechtel et al. (1936:150) as "the skeletal changes including bowing of the forelegs either forward or to the side, swelling of the knee and hock joints, straightening of the pasterns, and humping of the back." Other symptoms frequently observed were stiffness of gait, dragging of the rear feet, standing with the rear legs crossed, irritability, tetany, rapid respiration, bloat, anorexia for grain and roughages but not for milk, weakness and inability to stand for any length of time, and finally the retardation or complete cessation of growth. He also reported that the first detectable signs of rickets in calves are a decrease in the level of inorganic phosphorus in the serum, low serum calcium, and in some cases tetany. X-rays of rachitic calves showed that the junction of the diaphysis and cartilage was irregular and indefinite and in places showed areas of incomplete calcification. Rupel £t _al. (1933) observed high levels of serum alkaline phosphatase and a reduction in the percentage of ash in long bones of rachitic calves.

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-19Rickets has been observed in calves by several early writers. In 1901, Law listed it as a disease affecting cattle. Hutyra and Mark (1914) have a splendid photograph of a rachitic calf. In 1920, Becker observed rickets in calves fed whole milk and grain. Rachitic calves were produced experimentally by Reed and Huffman (1926) in connection with the heavy feeding of concentrates without the proper quality of roughage. Olson (1929) reported that rickets occurred among calves allowed free choice of feeds. The rachitic symptoms were associated with low hay intakes. Huffman e t al . (1930) reported a case of rickets in a young animal which was fed a ration containing a limited amount of wheat straw. Hill (1930) reported that calves fed a rickets-producing ration and exposed to ultraviolet rays showed improved calcification of the bones. Huffman and Duncan (1935) studied the antirachitic value of hay in the ration of dairy cattle. The rachitic calves showed anorexia, low growth, low serum calcium, low serum phosphorus, low bone ash content, and low bone mineral content. Colovos et_ cLU (1951) studied the effect of vitamin D on calves. The results indicated blood Ca and inorganic P were lowered by the deficiency, while the alkaline phosphatase activity was increased. The deficiency slowed gain in body weight and produced the usual symptoms of rickets, such as arched back, large knees, and soreness of joints. Rickets in Humans The signs and diagnosis of rickets in humans are not particularly different from those in animals. Howland and Kramer (1921) discuss calcium and phosphorus in the serum in relation to rickets in children. They conclude that in

PAGE 31

20 non-rachitic infants and young children, the concentration of calcium is from 10-11 mg/100 ml of serum, and the inorganic phosphorus is definitely reduced and sometimes extremely low. They observed that cod liver oil corrects these abnormalities. All the children under 2-1/2 years of age, in whom they found an inorganic phosphorus content of the serum of 3.0 mg or less, were suffering from active rickets. Tetany sometimes was associated with rickets. Orr ert al . (1923) studied calcium and phosphorus metabolism in rickets, with special reference to ultraviolet ray therapy. They showed that ultraviolet radiation caused an increase in serum calcium and phosphorus in rachitic children. Jaffe (1934) studied rickets in children. He stated that serum calcium level is not a reliable criterion of the severity of rickets, but low serum inorganic phosphorus and high serum alkaline phosphatase are better for diagnosing rickets. Serum alkaline phosphatase, the normal range of which in children may be stated as between 5 and 15 King-Armstrong (KA) units/100 cc, rises in mild cases of rickets to about 20 or 30 units, in marked cases to about 60 units, and in very severe cases over 60 units. Thomas e_t al_. (1959) measured antirachitic activity in normal children. Results indicated that the mean antirachitic activity of sera from 18 normal subjects was equivalent to 2 IU of vitamin D per ml. Dunnigan et al . (1962) made a survey of the existence of rickets in 5-10 year old Pakistani children. Compared with normal children, rachitic children had low serum calcium, low serum phosphorus, high serum alkaline phosphatase, and enlargement of the ends of the knee and wrist bones. They indicated that the reason for these abnormalities is deficiency of dietary vitamin I) and not lack of exposure to sunlight.

PAGE 32

21 Richards e_t _al. (1968) studied the infantile rickets in Glasgow. They showed that serum alkaline phosphatase of 25 KA units/100 ml or above plus loss of definition of metaphyseal line at the end of the radius and ulna and broad bands of increased density replacing the sharp metaphyseal lines, are reliable methods of diagnosing rickets in children. Lipson (1970) studied nutritional rickets in Sydney. He obtained a low serum calcium (6.0 mg/100 ml), low serum phosphorus (2.1 mg/100 ml), and high serum alkaline phosphatase (71 KA units) in rachitic children compared with the normal children (serum calcium 9-11 mg/100 ml, serum phosphorus 4-6 mg/100 ml, serum alkaline phosphatase 15-35 KA units). The X-rays of rachitic children showed the features of rachitic bones. He related these abnormalities to limited exposure of the children to sunlight. Treatment with 5,000 IU of vitamin D/day corrected the serum and bone abnormalities in the rachitic children. Stephen and Stephenson (1971) measured plasma alkaline phosphatase from children receiving vitamin D supplements in the London area and from children exposed to sunlight in the West Indies. The distribution of values showed that there was no precise upper limit which would be used in the diagnosis of subclinical vitamin D deficiency. In the diagnosis of rickets in immigrants. Wills et al. (1972) measured plasma calcium, plasma phosphorus, plasma alkaline phosphatase levels, and X-rays of the radius. They showed that rachitic children had low plasma calcium (6.0 mg/100 ml), low plasma phosphorus (3.9 mg/ 100 ml) , high plasma alkaline phosphatase (50 KA units per 100 ml) , and widening of the epiphyseal plates of the radius. Balsan and Garabedian (1972) studied the effect of 25-hydroxycholecalciferol in curing rickets in children. It was shown that after

PAGE 33

22 8 days of 16,000 IU of 25-hydroxy vitamin the treated children had normal serum calcium (raised from 7.4 mg/100 ml to normal 10.0 mg/100 ml), and alkaline phosphatase (reduced from 40 Bodansky units to normal 20 Bodansky units) . Ford e_t (1972) made a survey of the occurrence of rickets in the Glasgow Pakistani community. Serum calcium below 8.3 mg/100 ml, serum inorganic phosphorus below 3.0 mg/100 ml, serum alkaline phosphatase above 30 KA units/100 ml, and widening of the epiphyseal plate of the long bone were taken as evidence of rickets. All children with these abnormalities were treated with 3,000 IU of calciferol daily and brought up to the normal level. Holmes e^ al. (1972) investigated rickets among the Asian immigrant population. In the study the children who had rickets had loss of definition of the metaphyseal lines of the radius and ulna, serum calcium below 8.0 mg/100 ml, serum phosphorus below 3.0 mg/100 ml, serum magnesium below 1.9 mg/100 ml, and serum alkaline phosphatase 45 KA units per 100 ml of the serum. The external appearance of rickets included bowed legs and muscular weakness. Vitamin D treatment increased serum calcium to 9.8 mg/100 ml, serum phosphorus to 4.2 mg/100 ml, and reduced serum alkaline phosphatase to 31 KA units. Revusova et. al. (1972) studied the effect of vitamin D on serum magnesium. The results indicated that vitamin D in high doses increases intestinal magnesium absorption and serum magnesium concentration. In studying the natural and synthetic sources of circulating 25-hydroxy vitamin D in man, Haddad and Hahn (1973) concluded that approximately 90% of serum 25-hydroxy vitamin D,^ was derived from

PAGE 34

-23irradiation of 7-dehydrocholestrol of the skin. The range of serum value obtained was 16 to 41 ng/ml. Cooke ejt a_l. (1973) investigated serum alkaline phosphatase and rickets in urban school children. Among 569 school children (386 boys and 183 girls) aged 14-17 years, 233 had serum alkaline phosphatase values of 30 KA units or greater. There was no significant difference in the levels in Asian, white, or West Indian children. The mean values were significantly greater in boys than girls and both showed a fall in mean value with increasing age. The investigation suggested that most children with alkaline phosphatase levels above 30 KA units have rickets. Preece e^ a_l. (1973) tried to use serum level of 25-hydroxy vitamin D in the diagnosis of rickets. In rachitic children, the mean values of serum 25-hydroxy vitamin D, calcium, phosphorus, and alkaline phosphatase were 0.8 ng/ml or undetectable, 8.66 mg/100 ml, 3.0 mg/100 ml, and 101.8 KA units/100 ml, respectively. In the normal group the serum values were 25-hydroxy vitamin D 12.0 ng/ml, calcium 9.19 mg/100 ml, and inorganic phosphorus 3.8 mg/100 ml. It was concluded that the measurement of circulating 25-hydroxy vitamin D by competitive proteinbinding provides a more sensitive method than serum alkaline phosphatase to diagnose rickets in children. Mankodi e_t_ aT . (1973) studied rickets in pre-school age children in and around Bombay. They concluded that serum alkaline phosphatase above 30 KA units and X-rays of long bones were good evidence of rickets in children. Arneil (1973) reported cases of rickets in children. He concluded that rachitic children had bowed legs or knock-knees and serum alkaline phosphatase values above 25 KA units/100 ml. The

PAGE 35

-24condition resolved rapidly when 2,000 IU (international unit) of calciferol were given daily. Gupta e_t^ al. (1974) showed the spontaneous cure of vitamin D deficiency in Asian children during summer in Britain. They measured plasma levels of calcium, phosphorus, alkaline phosphatase, and 25-hydroxy vitamin D in groups of healthy Asian children in early spring and again in the late summer. In summer, mean plasma calcium rose from 8.8 to 9.25 mg/100 ml and 25-OH vitamin D from 9.9 to 14.7 ng/ml. Plasma phosphorus and alkaline phosphatase did not change. There was a highly significant correlation between plasma calcium and 25-OH vitamin D. These results emphasize the importance of summer sunlight in the maintenance of vitamin D nutrition and the prevention of rickets. Hojer and Gebre-Medin (1975) studied rickets and exposure to sunlight. They showed low serum calcium, phosphorus, and high alkaline phosphatase in rachitic children which were not exposed to the sunlight. These children were exposed to sunshine for 30 minutes daily. Clinical improvement, normalization of serum biochemical values, and radiographical signs of healing were observed after three weeks of treatment. Miller and Chutkan (1976) investigated vitamin D deficiency rickets in Jamaican children. The rachitic children had slightly low serum calcium (8.0 mg/100 ml), low serum phosphorus (1.7 mg/100 ml), higher serum alkaline phosphatase (130 KA units), and radiological signs of rickets. It was indicated that limited exposure to sunlight and lack of vitamin D in the diet are the reasons for these radiological and biochemical abnormalities. Treatment with vitamin D or sunlight or both corrected the abnormalities.

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-25Rickets in Horses While significant advances have been made in studying vitamin D and its relation to bone and mineral metabolism in other species, information regarding rickets in horses is not extensive. There appears to be no reliable information available on the effects of vitamin D deficiency in the horse. Nieberle and Chors (1954) denied the existence of true rickets in the horse. Park (1923) stated that rickets occurs less frequently in horses than in other animals. Smith and Jones (1957) assumed that rickets occurs in horses in a form similar to that in animals and man. Adams (1974) stated that signs of> rickets occur in horses up to three years of age; however, foals between six months and one year are most commonly affected. He also defined rickets in horses as a disease of the epiphysis rather than of the joint itself. At the present time, diagnosis of rickets in horses rests heavily on radiological interpretation; however, external evidence may be present . Manning (1962) discussed rickets in horses, including radiography of possible rickets cases. He concluded that extrapolation of information data from man to the horse is not a valid procedure. Sippel et al. (1964) determined the normal values of plasma calcium, phosphorus, and magnesium in horses. Average values for plasma calcium, phosphorus, and magnesium were 11.0 mg/100 ml, 7.5 mg/100 ml, and 2.4 mg/ 100 ml, respectively. Myers and Emmerson (1966) studied the age and manner of epiphyseal closure in the forelegs of two Arabian foals which were maintained under

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-26ideal conditions. All of the epiphyseal lines below the carpus were closed before the end of the ninth month. The distal epiphyses of the radius and ulna had completely joined before the end of the ninth month. Monfort (1967) made a radiographic survey of epiphyseal maturity in thoroughbred foals from birth to three years of age. He suggested that the distal epiphysis of the third metacarpal is an ideal segment for the estimation of bone maturity. He pointed out that this closed completely at one year. Coffman (1969) studied bone maturation in horses. Findings indicated that the growth plate in the canon bone is normally closed at 12 months, while the radius closesbetween 24 and 33 months. Haugh et_ a_l_ . (1971) studied the breaking strength of the metacarpal bones of normal Shetland ponies at two years of age. The average failure stress they obtained was 35,000 pounds per square inch of the third metacarpal bone.

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CHAPTER III MATERIAL AND METHODS Conditions and Experimental Design A 2 x 3 factorial design experiment using 12 ponies (males and females, Shetland and Welsh ponies) was carried out to determine the effect of vitamin D and sunlight on growth and bone development of young ponies. The experiment consisted of two age groups and three levels of vitamin D treatments. The two age groups were 2 months and 8 months of age. The three treatment levels were as follows: 1) Treatment 1 (T ) with no vitamin D supplement and protected from ultraviolet (uv) light; 2) Treatment 2 (T 0 ) , 1,000 IU of vitamin D supplement daily and protected from uv light; and 3) Treatment 3 (T ) , no vitamin D supplement and kept outdoors. Vitamin D was given orally (appropriate amount of vitamin D was dissolved in water and mixed with the diet). The diet was formulated to meet all nutrient requirements according to the Nutrient Requirements Council (N.R.C., 1973), but without vitamin D. Table 1 shows that the diet consisted of Coastal Bermuda hay pellets (dehydrated, not sun-cured) 40.0%, corn 35.75%, soy bean meal 16.0%, molasses 7.0%, calcium carbonate 0.025%, salt 0.5%, vitamins (except vitamin D) , and minerals. Laboratory analysis of the diet indicated it to have 16.68% protein, 3.85% fat, 0.55% calcium, and 0.42% phosphorus. Ponies were fed three times daily ( ad 1 i bi turn) . The dietary ingredients had minimum or no vitamin D. To ascertain this, the diet was tested by

PAGE 39

-28TABLE 1. COMPOSITION OF THE VITAMIN D-DEFICIENT DIET^ Ingredient % of Diet Coastal Bermuda Hay (Dehydrated, not sun-cured) 40.00 Corn 35.75 Soybean Meal 16.00 Molasses 7.00 Calcium Carbonate 0.25 NaCl (iodized) 0.50 Trace Mineral Mix^ 0.50 c Vitamin A, E + Adequate in all other nutrients required for optimum growth. ^Provided the following (mg/kg diet): Fe 20, Mn 8.8, Ze 20, Co 0.1, I 0.1, Cu 2.8. Provided 4400 IU Vit. A, 11 IU Vit. E per kilogram of diet.

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-29the line test on rats (A.O.A.C., 1973). Rats 20 days old were put on a rachitic diet for 25 days, then after they showed rachitic symptoms they were divided into three groups: Group I was given the vitamin Ddeficient pony diet, Group II was given a synthetic complete diet, and Group III was kept on a synthetic rachitic diet. The three groups were kept on these diets for 10 days. Since the pony diet did not heal the rickets symptoms (A.O.A.C. line test procedure), it was concluded that the pony diet did not have vitamin D. The rats with the complete diet recovered from rickets symptoms, while the rats in Group III had severe rickets . At the beginning of the experiment, all ponies were sheltered away from the uv light for one month before being divided into groups for treatment, in order to deplete them of stored body vitamin D. The treatment period was five months (from the end of the depletion period to the termination of the experiment) . All the ponies were killed at the end of the experiment. At the beginning and at the end of the trial the intensity of uv light in the barn where the ponies were sheltered was determined by the use of a 1P28 RCA photomultiplier with a 334 mm interference filter. The intensity of light relative to outside radiation (I/Io) was found to be 3 x 10 , indicating a bare minimum or none, compared with outdoors. Sample and Measurements Blood samples (20 ml) were obtained weekly from the jugular vein. The samples were centrifuged and plasma was obtained and frozen for laboratory analysis. Ponies were weighed weekly and feed consumption was recorded.

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-30Anteroposterior (A.P.) and Lateriomedial (L . M. ) radiographs were taken of front feet, 1st phalanx, 2nd phalanx, and the metacarpal bones of the 2,0 month old group of ponies. Since the epiphyseal areas of these bones had already closed in the older ponies group (8 months old), A.P. and L.M. radiographs of the front feet (the metacarpal, the carpus, and the radius and ulna) were taken. Radiographs were made at approximately one-month intervals. Radiographs were obtained using a small compact portable X-ray unit (Picker X-ray Corp., Cleveland, Ohio). DuPont Cornex 4 industrial X-ray film (E.I., DuPont De Nemours and Co., Inc.) in an X-ray cassette (Wafer rigidform cassette 10 x 12, Hansley X-ray Product, Brooklyn, New York) was used. Radiographic settings were 15 mA, 80 kvp , and 0.75 sec exposure. The distance between the X-ray unit and the leg was 26 inches with the film pack placed directly against the extremity. Film was developed in Kodak developer. At the termination of the experiment all ponies were killed and liver, kidney, and the two metacarpal bones of the front legs were taken for laboratory analysis. The samples were put in plastic bags and stored in the freezer. All ponies were photographed at the beginning and at the end of the experiment to show any signs of leg abnormalities (rickets) . Laboratory Analysis Blood plasma calcium and magnesium determination . To determine plasma calcium and magnesium, 1 ml of plasma was diluted with 9 ml 10% (w/v) trichloracetic acid (TCA) for precipitation of protein. One ml

PAGE 42

-31of the supernatent was diluted with 4 ml 1% LaCl^ and it was assayed using an atomic absorption spectrophotometer (Perkin-Elmer , Model. 306). Blood plasma phosphorus determination . Using the Fiske and Subbarow (1925) method, the lanthanum solution prepared for determination of calcium was used for the colorimetric determination of plasma phosphorus. Blood plasma alkaline phosphatase determination . Using the modified Bessey-Lowry-Brock method and with reagents obtained from Dade (American Hospital Supply Corporation, Miami, Fla.), plasma alkaline phosphatase was measured at 0, 4, 12, 20, and 27 weeks of the experiment. Plasma 25-hydroxy vitamin D determination . The method of Hollis and Conrad (1976) was used to measure plasma 25-hydroxy vitamin D levels at 0, 4, 12, 20, and 27 weeks of the experiment. The binding protein was obtained from normal rat serum. Bone ash and cortex area . One of the metacarpal bones was used to determine bone chemical composition and cortex area. It was cleaned of adhering tissue, and length and fresh weights were obtained. The bone was cut transversely at the longitudinal center with a saw and using a planimeter the total area (AT) and the marrow area (AM) were measured. The cortex area (AC) was equal to AT AM. The bones were then dried to constant weight at 105°C, defatted by ether extraction in a Soxhlet apparatus. Dry bone weights were recorded and bone ash weights were obtained after ashing at 650 C overnight. Contents were expressed as percentage of dry, defatted bone. Bone density was equal to fresh bone weight divided by bone volume, determined by submerging the bone in water in a graduated cylinder. Ca , P, and Mg bone ash . The ashed bone was ground with mortar and pestle. One gram of bone ash was put into a tared crucible and acid

PAGE 43

32 hydrolysis was carried out. The solution was evaporated to about 5 ml and the solution was transferred quantitatively to a 50 ml volumetric flask for analysis of calcium, phosphorus, and magnesium. Atomic absorption spectrophotometry, as previously described, was used for calcium and magnesium determinations. The Fiske and Subbarrow colorimetric method also described previously was used to determine bone ash phosphorus . Bone breaking strength . An Instron (TT-c English units tensil machine with CF load cell, Fig. 1) and a technique similar to that used by Haugh e_t a_l. (1971) was used to measure the breaking strength of the metacarpal bones from each pony. A simple three-point flexural loading technique was used for testing. The bone was positioned on two fixed supports with its center lines placed an equal distance apart on the bone. The load was applied to the bone midway between the two support points with a single fixture identical to the supports. The load was applied by the downward movement of the crosshead of the testing machine to which the single fixture was attached. The bone was loaded to failure. The force was applied gradually and slowly. Failure stress 8 LF was calculated as follows: S = — — r x — ~ where S = failure stress 3.14 q 3 2 (kg/cm ), F = maximum force (kg), L = length of bone between supports (in cm), C = distance from centroidal axis to edge of bone (in cm). Radiographic classification . The stage of maturity or the degree of epiphyseal closure was classified on the basis of "A+" = complete epiphyseal closure; "A-" = 3/4 epiphyseal closure; "B+" = 1/2 epiphyseal closure; "B-" = 1/4 epiphyseal closure, and "C" = complete open epiphysis .

PAGE 44

33 FIGURE 1. INSTRON TENSILE STRENGTH TESTING APPARATUS FOR DETERMINING BREAKING STRENGTH OF BONES.

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-34Kidney and liver analysis . Twenty-five grams of the right lobule of fresh liver (or of a consistant part of the kidney) were dried in an oven at 100 C for 16 hours. Water content was calculated as 25 grams minus dry weight. The dried tissue (liver or kidney, approximately 5 g) was digested on a hot plate using concentrated nitric acid. It was then ashed in a muffle furnace at 500°C for 24 hours. The ashed liver (or kidney) sample was placed on a hot plate for acid hydrolysis. Five ml of hydrochloric acid and deionized water were used for acid hydrolysis of the sample. With 10% hydrochloric acid the tissue solution was transferred to a 25 ml volumetric flask and made to volume with deionized water. One ml of this was used to determine calcium, magnesium, and phosphorus levels as previously described. Statistical Analysis The effect of three treatments (in the two age groups) on blood, kidney, and liver, and feed efficiency and bone were analyzed factorially (Steel and Torrie, 1960). The Statistical Analysis System (Barr et al., 1976) was used in the processing of the data. When there was an interaction effect, a Duncan's multiple range test was done to show differences within a particular treatment or group. In case of bone, liver, and kidney analysis, the following model was used: Y = A + T + AT + E , where Y = water, ash, Ca , P, and Mg concentration of the liver or kidney or bone, A = age groups effect, T = treatments effect, AT = interaction of age groups and treatments effect, E a = error • In case of plasma Ca , P, Mg, 25-OH vitamin D, and alkaline phosphatase levels, the following model was used:

PAGE 46

-35Y 1 »A+T+AT+Ea+W+AW+TW+ ATW + E fc , where Y = Ca, P, Mg, 25-OH vitamin D, and alkaline phosphatase, W = weeks effect (27 weeks), and AT, AW, TW, ATW are the interaction effect with E. = error (b) . b

PAGE 47

CHAPTER IV RESULTS AND DISCUSSION External Appearance Loss of appetite and difficulty in standing occurred in T^ ponies, but the actual external appearance of rickets (bowed legs and inability to stand) did not occur. Figure 2 shows photographs of ponies at the beginning and at the end of the experiment. These findings agree with those of other investigators. Park (1923) stated that rickets occurs less frequently among horses than puppies, pigs, lambs, and kids. Results of Harrison ej^ jal. (1958) showed no external appearance of rickets in vitamin D-deficient rats when he fed them a diet adequate in both calcium and phosphorus. Dunlop (1935) and Braude al. (1943) concluded from their study that pigs do not need vitamin D; they were not able to produce rickets. Nieberle and Chors (1954) denied the existence of true rickets in the horse. Manning (1962) surveyed rickets in horses and did not believe that rickets exists in the horse. He concluded that extrapolation of information data from man to the horse is not a valid procedure. Combs ej: al. (1966a) failed to produce rickets in pigs deprived of sunlight and vitamin D supplement. On the other hand, many other extensive experiments showed very clearly rickets in puppies (Findlay, 1908), rats (Al-Ganhari et al . , 1973), poultry (Hart et al., 1922), swine (Miller et al. , 1964), cattle (Bechtel e^ aj.. , 1936), and humans (Holmes et al . , 1972). Generally, ) £

PAGE 48

’ 37 FIGURE 2 • EFFECTS OF TREATMENT ON EXTERNAL APPEARANCE OF THREE OF THE PONIES IN GROUP I. AAt start of experiment. ^At end, 6 months later.

PAGE 49

-38the external indication of rickets were joint enlargement, distinctive gait, bowing of the forelegs, swelling of the knees, dragging of the rear feet, standing with the rear legs crossed, irritability, rapid respiration, anorexia, and finally retardation or complete cessation of growth. Rate of Growth and Feed Efficiency Growth (gain/day) and feed efficiency for the twelve ponies are presented in Table 2. Figures 3 and 4 present weekly body weight for group I and group II ponies. It appears that ponies of T had slower growth curves than ponies in and T 3 Group I (ponies started the experiment at two months of age and were terminated after six months) had slightly lower gains per day and feed efficiencies than Group II (ponies started the experiment at eight months of age and were terminated after six months). Feed efficiency was essentially the same for all treatments (T = ponies deprived of exposure to ultraviolet light and vitamin D supplement; = ponies supplied with adequate vitamin D, but deprived of ultraviolet light exposure; = ponies exposed to sunlight, but without vitamin D supplement). Feed efficiency data (grams intake per grams gain) were analyzed by factorial analysis. There was no significant difference in feed efficiency between either of the two groups or among the three treatments. Table 12 (Appendix) presents the analysis of variance for feed efficiency. The effect of vitamin D supplement and/or sunlight on animals' growth has been reported in other results in other species. For example, Steenbock and Black (unpublished data) showed no significant

PAGE 50

TABLE 2. EFFECT OF VITAMIN D AND SUNLIGHT ON RATE OF GAIN AND FEED EFFICIENCY OF PONIES. -39tO > < c •H CU CO a £ c CO to •"-I £ (U H CU cU fcC u u cu o 4-1 co > < 4-> CU rC c CO *H CU •H CU T) » o O H cU .X 00 CN rH rH m m i — 1 CN 00 m 4-> > •H rH > G CN -H G m > > H H > H G 0 O O o G G G c + Q Q Q 4-1 • ^ 4-» •H > H > •rH rH 0* G CN -H G cn > > H H > H G O O O O G v-/ G + c G + CL G O H O CO XI 4-> rC /«~N
PAGE 51

body WEIGHT kg 40 2 4 6 8 10 12 14 18 18 20 22 24 26 28 WEEK FIGURE 3. WEEKLY BODY W'EIGHt OF GROUP 1 PONIES. (SLarted at two months of age)

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BODY WEIGHT KG -41WEEK FIGURE 4. WEEKLY BODY WEIGHT OF GROUP II PONIES. (Started at eight months of age)

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-42difference in growth of irradiated and non-irradiated rats. Bethke et_ ahL. (1923-24) showed that with a diet in which the Ca/P ratio was very high vitamin D did not induce growth. Hart and Steenbock (1922) indicated that there is no need, as measured by growth response, for supplemental vitamin D in rations for growing pigs confined in the absence of sunlight. Wahlstrom and Stolte (1958) showed that the addition of 90 U.S.P. units of vitamin D per pound to a mixed ration complete in other known dietary factors resulted in little difference in the rate of gain of pigs. Combs e_t a_l. (1966a) indicated that average daily gain and feed intake were not significantly influenced by supplying pigs with vitamin D. In other experiments there were positive responses of growth to vitamin D and sunlight supplements in rats (Carlsson, 1952; Beilin et al., 1954; Simmons and Kunin, 1970), poultry (Wong and Norman, 1974), swine (Miller et al . , 1964), and cattle (Colovos et al., 1951). In the present study there were some individual ponies (Table 2) which showed a reduction in growth, but the difference in the means was not significant. The author believes that if the ponies were put into the experiment at one day of age, they would have shown a better response to treatments. It is possible that the ponies had enough vitamin D storage in their bodies before they started the experiment to get along for six months without dietary vitamin D. Blood Analysis Plasma calcium (Ca) , phosphorus (P) , and magnesium (Mg) levels. Table 3 presents the average levels of plasma Ca of the twelve ponies in twenty-seven weeks of the experiment. Table 13 presents weekly plasma

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-43TABLE 3. AVERAGE WEEKLY PLASMA CALCIUM LEVELS (mg/100 ml) DURING 27 WEEKS OF EXPERIMENTAL PERIOD. a Week Group I (Started Experiment at 2 Months of Age) Group II (Started Experiment at 8 Months of Age)^ No V i t . D No uv (T x ) + Vit. D No uv (t 2 ) No Vit. D + uv (t 3 ) No Vit. D No uv (T]_) + Vit. D No uv (t 2 ) No Vit. D + uv (t 3 ) 1 11.1 10.7 11.3 13.4 13.5 13.2 2 12.3 10.5 11.4 12.6 13.4 13.5 3 11.0 11.9 11.9 13.2 14.5 13.6 4 11.2 11.5 11.5 13.3 13.3 13.7 5 11.2 10.5 12.0 13.7 14.1 13. 7 6 11.3 11.5 11.8 13.5 13.8 13.7 7 11.6 11.6 11.5 13.4 13.6 13.7 8 10.7 12.3 11.6 14.0 13.9 13.3 9 10.2 10.8 11.1 14.0 13.2 14.0 10 11.4 12.1 11.5 13.2 13.3 14.0 11 11.9 12.6 12.1 14.0 13.7 13.2 12 11.4 10.9 11.5 12.8 13.7 13.6 13 11.8 11.5 11.7 13.4 13.7 13.7 14 11.9 11.7 11.7 13.2 13.2 13.7 15 12.0 11.6 11.9 13.2 14.1 13.1 16 11.7 12.3 12.1 13.2 14.1 13.4 17 11.8 11.8 11.8 13.8 13.6 13.4 18 12.0 11.3 11.8 13.2 13.6 13.9 19 11.8 11.5 11.8 13.3 14.0 13.7 20 11.6 11.2 11.5 13.2 13.6 15.2 21 11.5 10.8 11.8 12.1 13.8 13.3 22 12.0 10.7 11.9 14.0 13.4 13.9 23 11.4 10.8 11.8 13.0 14.1 14.2 24 11.2 10.8 11.8 13.3 13.7 13.7 25 11.6 10.6 12.1 12.9 13.6 13.7 26 11.4 10.6 11.5 13.5 14.1 14.1 27 11.9 11.0 11.0 13.1 14.0 13.5 Each value represents average for two ponies. All values significantly (p < .01) higher than for Group 1.

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-44Ca levels for individual ponies. All ponies, during the experimental period, indicated plasma Ca levels within the normal range. Significantly (p < .01) higher plasma Ca levels were found in Group II than in Group I. The normal plasma Ca levels in all ponies led to the conclusion that plasma Ca levels were not appreciably affected by treatment. Statistical analysis showed no significant difference among , T 9 , and T . Figures 5 and 6 show the average values of plasma Ca for the three treatments in Group I and II. Average plasma Ca of Group I ranged from 10.5-13.5 mg/100 ml, while Group II ranged from 12.6-15.2 mg/100 ml. Ranges for T-j^, T 2 , and T 3 were 10.9-13.9, 10.5-14.0, and 11.1-14.0 mg/100 ml, respectively. Analysis of variance for plasma Ca levels is presented in Table 14 (Appendix) . Within the normal plasma P range (4. 0-8.0 mg/100 ml), ponies in Group I had slightly higher values than ponies in Group II. The values within the groups were varied and did not follow a consistent pattern (increase or decrease) . Table 4 presents the average weekly plasma P levels for the two ponies on each treatment throughout the 27-week period of study. Table 15 (Appendix) presents individual values of plasma P levels for ponies in each treatment. Average plasma P levels ranged between 4.6-7. 7 mg/100 ml and 3.4-6. 5 for Group I and Group II. Average plasma P levels for ^ , T 2 , and T 3 ranged from 4. 7-7. 7, 4.7-7. 7, and 4. 1-7. 6 mg/100 ml. Figures 7 and 8 present the average values of plasma P levels of Group I and Group II. The average plasma Mg levels are given in Table 5. Observations on plasma Mg levels pointed out that the levels did not affect from lack of vitamin D or sunlight or both. The averages of plasma Mg levels were within the normal range, at 1.1-1. 9 mg/100 ml for Group I and 1.8-2. 5

PAGE 56

-45WEEK FIGURE 5. AVERAGE WEEKLY PLASMA CALCIUM OF GROUP I. (Started experiment at 2 months of age)

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46 FIGURE 6. AVERAGE WEEKLY PLASMA CALCIUM OF GROUP II. (Started experiment at 8 months of age)

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-4 7TABLE 4. AVERAGE PLASMA PHOSPHORUS LEVELS (mg/100 ml) DURING 27 WEEKS OF EXPERIMENTAL PERIOD. 3 Week Group I (Started Experiment at 2 Months of Age) Group II (Started Experiment at 8 Months of Age) N[p Vit. D No uv (T x ) + Vit. D No uv (t 2 ) No Vit. D + uv (t 3 ) No Vit. D No uv (T x ) + Vit. D No uv (t 2 ) No Vit. D + uv (t 3 ) 1 7.7 7.0 7.6 5.1 3.4 4.4 2 6.0 6.6 6.8 6.4 4.4 5.7 3 5.7 5.4 6.3 5.8 5.9 5.8 4 4.8 5.2 5.8 6.2 4.9 6.0 5 4.5 4.7 5.4 4.8 4.1 4.8 6 4.9 6.0 6.8 5.7 5.0 6.0 7 4.8 6.3 5.4 4.5 3.5 4.6 8 4.7 6.4 6.1 4.9 4.4 4.0 9 5.3 7.6 6.4 4.8 4.5 4.9 10 4.8 7.0 5.2 4.2 3.4 4.5 11 5.2 6.2 5.9 5.0 4.5 5.1 12 4.8 6.3 5.5 5.6 4.1 6.5 13 5.3 6.9 5.3 5.2 5.3 5.4 14 6.6 6.4 5.2 4.7 4.1 5.1 15 6.2 5.9 5.4 5.3 4.4 4.9 16 6.3 6.8 5.8 4.8 4.7 6.0 17 4.9 4.9 5.5 5.1 4.9 6.0 18 5.8 5.7 5.9 oo 4.7 4.8 19 5.9 4.9 5.9 4.4 4.3 5.3 20 4.9 5.9 5.9 4.5 3.9 4.8 21 6.5 5.2 6.1 5.3 4.9 4.2 22 6.5 4.7 5.5 4.5 4. 7 4.9 23 6.3 5.1 5.9 5.2 4.6 4.1 24 5.6 5.5 6.0 4.3 4.9 4.5 25 6.5 4.5 6.0 4.5 4. 3 4.3 26 7.4 5.5 5.7 4.4 4.0 4.1 27 6.3 4.9 6.2 4.7 5.4 5.7 Each value represents average for two ponies.

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-48WEEK FIGURE 7. AVERAGE PLASMA PHOSPHORUS LEVELS IN GROUP I. (Started experiment at 2 months of age)

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49 WEEK FIGURE 8. AVERAGE PLASMA PHOSPHORUS LEVELS IN GROUP II. (St .1 r ted oxpor i men L at 8 moiiLlis of a^e) , No uv No uv , + uv

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-50TABLE 5. AVERAGE PLASMA MAGNESIUM LEVELS (mg/100 ml) DURING 27 WEEKS OF EXPERIMENTAL PERIOD. 3 Group I (Started Experiment Group II (Started Experiment at 2 Months of Age) at 8 Months of Age) b Week No Vit. D + Vit. D No Vit. D No Vit. D + Vit. D No Vit. D No uv No uv + uv No uv No uv + uv (T x ) (t 2 ) (t 3 ) (T 1 ) (t 2 ) (t 3 ) 1 1.6 1.5 1.7 2.1 2.0 2.0 2 1.8 1.0 1.2 1.8 1.8 1.8 3 1.5 1.3 1.5 2.0 2.2 2.1 4 1.5 1.5 1.6 2.3 2.2 2.4 5 1.5 1.5 1.6 2.5 2.2 2.3 6 1.6 1.6 1.8 2.4 2.3 2.3 7 1.5 1.6 1.5 2.3 2.1 2.1 8 1.5 1.8 1.5 2.2 2.2 '2.1 9 1.6 1.6 1.6 2.4 2.0 2.3 10 1.5 1.7 1.4 2.2 2.0 2.1 11 1.7 1.4 1.7 2.5 2.0 1.9 12 1.5 1.3 1.2 2.3 2.1 2.1 13 1.6 1.3 1.5 2.3 2.3 2.2 14 1.9 1.3 1.7 2.1 2.0 2.2 15 1.7 1.4 1.6 2.2 2.0 1.9 16 1.7 1.5 1.7 2.3 2.0 2.0 17 1.6 1.3 1.7 2.4 2.0 2.1 18 1.5 1.3 1.7 2.3 2.2 2.1 19 1.5 1.3 1.7 2.4 2.3 2.0 20 1.6 1.1 1.6 2.2 2.3 2.1 21 1.5 1.2 1.5 1.8 2.4 2.2 22 1.7 1.3 1.5 2.3 2.5 2.5 23 1.6 1.4 1.7 2.2 2.3 2.4 24 1.6 1.4 1.5 2.2 2.2 2.5 25 1.8 1.4 1.5 2.3 2.2 2.4 26 1.8 1.4 1.6 2.2 2.1 2.3 27 1.9 1.6 1.6 2.2 2.1 2.4 Each value represents average for two ponies. All values significantly (p < .01) higher than for Group I.

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-51mg/100 ml for Group II. Group II had higher (p < .01) plasma Mg than Group I. Ranges for T^ , T^, and were 1.5-2. 5, 1.2-2. 5, 1.2-2. 5 mg/100 ml. Factorial analysis indicated no significant differences between treatments. Figures 9 and 10 show the average plasma Mg of Group I and Group II. Table 16 (Appendix) presented individual values of plasma Mg levels for all ponies. Plasma Ca, P, and Mg levels (Tables 3, 4, 5 and Figures 5, 6, 7, 8, 9, 10) were not affected significantly by the treatments. The values were variable and did not follow particular patterns. Some of these values were toward the upper or lower end of the normal range, but generally did not indicate the existence of vitamin D deficiency. The data pointed out slight decreases (but within the normal range) in the levels of P and Mg in the first six weeks of the treatment, especially in Group I, and thereafter an increase is observed. This could be explained as follows. When plasma Ca, P, and Mg levels are low, bone is readily mobilized to raise them to normal levels. The significant differences (p < .01) between treatments in bone ash (later in this paper) indicate that the ponies kept their plasma Ca, P, and Mg within normal levels by mobilizing bone. In addition, supplying the ponies with a diet adequate in Ca , P, and Mg, and exposing them to sunlight (for two months for Group I and eight months for Group II) before they were put on the treatments were probably other factors which kept plasma Ca, P, and Mg within the normal range in the vitamin D-deficient ponies. Harrison et al . (1958) showed normal plasma Ca and low plasma P in vitamin D-deficient rats supplied with a diet adequate in Ca and P. Yoshiki and Uanayisawa (1974) found hypocalcemia and hypophosphotemia in vitamin D-deficient rats which were supplied with a diet deficient

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-52FIGURE 9. AVERAGE PLASMA MAGNESIUM LEVELS IN GROUP I. (Started experiment at 2 months of age)

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-53T,-. T2*= No Vit. D, No uv + Vit. D, No uv WEEK FIGURE 10. AVERAGE PLASMA MAGENSIUM LEVELS IN GROUP II, (Started experiment at 8 months of age)

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-54in Ca and adequate P. Coleman e_t al. (1950) and McLendon and Balnstein (1965) produced rickets in rats by a diet adequate in vitamin D but deficient in either Ca of P. Gonnerman et_ aT. (1975) showed that vitamin D-fed chicks provided with 1.4% Ca had hypocalcemia and hypophospho temia , while vitamin D-deficient chicks provided with 2.4% Ca had near normal plasma Ca and P levels. Wahlstrom and Stolte (1958) found little improvement in plasma Ca and P from adding supplemental vitamin D in rations of pigs fed in the absence of sunlight, while Miller et cLL. (1964) showed low plasma Ca and P in vitamin D-deficient pigs deprived of sunlight. Colovos et al. (1951) showed low plasma Ca and P in vitamin D-deficient calves. In humans the data showed markedly low plasma Ca, P, and Mg in vitamin D-deficient children (Lipson, 1970; Ford et_ al. , 1972; Miller and Chutkan, 1976) . The average levels of plasma Ca, P, and Mg in the ponies in this experiment are within the normal ranges reported by Sippel _et al. (1964) in horses. Plasma alkaline phosphatase . The individual and average plasma alkaline phosphatase levels after 0, 4, 12, 20, and 27 weeks on experiment are given in Table 6. In Group II there was a tendency for levels to decrease as the ponies aged. In Group I a decrease in plasma alkaline phosphatase occurred but it was not regular. The overall picture of the data indicates a difference between individuals but not between treatments. Factorial analysis was done to determine the effect of treatments and groups. There were no significant differences between treatments or groups. Table 17 (Appendix) presents the analysis of variance of plasma alkaline phosphatase. The present study did not demonstrate any consistant increase in plasma alkaline phosphatase with continuing vitamin D deficiency as shown

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-55TABLE 6. PLASMA ALKALINE PHOSPHATASE AT 0 , 4, 12, 20, AND 27 WEEK PERIOD. Treatment Group Week I (Started Experiment at 2 Months of Age) II (Started Experiment at 8 Months of Age) Individual IU Avg. Individual IU Avg. Treatment Average T 1 0 130 105 118 130 130 No 4 187 79 133 55 62 59 Vit. D 12 59 52 56 36 92 65 89.0 No uv 20 43 59 51 98 122 110 27 67 53 60 98 163 131 T 2 0 124 99 112 187 187 + 4 187 115 151 101 69 85 Vit. D 12 62 89 76 52 113 83 101.0 No uv 20 64 78 71 96 122 109 27 46 74 60 113 146 103 T 3 0 79 99 89 146 146 No 4 65 106 86 127 72 100 Vit. D 12 70 124 97 75 75 75 93.0 + uv 20 96 73 85 113 93 103 27 79 92 86 95 103 99 Group Average 88.0 102.0

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-56in some of the results with rats (Deluca and Steenbock, 1956) , poultry (Yang ej^ a_l. , 1973) , swine (Miller ej: aM . , 1964) , cattle (Colovos ej; a_L . , 1951), and humans (Lipson, 1970) by others. Some experiments have shown no significant change in plasma alkaline phosphatase in vitamin D-deficient children (Stephen and Stephenson, 1971: Gupta et al., 1974). Plasma 25-hydroxy vitamin D . Since plasma 25-OH vitamin D level is felt to be an important indicator of the vitamin D status of an animal, the levels of plasma 25-OH vitamin D were determined by a competitive protein binding procedure developed by Hollis and Conrad (1976) . Normal rat serum was used as source of binding protein. Three dilutions of rat serum with lipoprotein barbital-acetate buffer, 1:5000, 1:8000, and 1:10,000 were made to obtain a standard curve which showed maximum binding efficiency. Figure 11 presents the standard curves with the three different dilutions. The 1:10,000 dilution was chosen for this analysis . The individual and the average values of plasma 25-hydroxy vitamin D at 0, 4, 12, 20, and 27 weeks for all ponies are given in Table 7. Since this probably represents the first attempt to determine plasma 25-hydroxy vitamin D in ponies and no values are available in the literature, comparison was made between the results in this paper and values reported in the bovine by Hollis and Conrad (1976). It appears that all the values obtained in the present study are normal. Statistical analysis showed no significant difference among treatments in plasma 25-OH vitamin D levels. The average 25-hydroxy vitamin D of T^ , T^ , and T^ were 63.4, 64.1, and 61.2 ng/ml. Group I ponies had higher (p < .01) plasma 25-OH vitamin D than Group II. In Groups I and II, the averages were 67.8 and 57.9 ng/ml. Table 18 (Appendix) presents the analysis of

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-57FIGURE 11. STANDARD CURVE FOR THE COMPETITIVE BINDING ASSAY OF 25-OH D

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-58TABLE 7. INDIVIDUAL AND AVERAGE VALUES OF PLASMA 25-HYDROXY VITAMIN D OF THE TWELVE PONIES AT 0 , 4, 12, 20, AND 27 WEEK PERIOD OF THE EXPERIMENT. Group I (Started Experiment II (Started Experiment at 2 Months of Age) at 8 Months o f Age) TreatIndividual Individual Treatment ment Week ng/ml value Avg. ng/ml value Avg. Average T 1 0 73 73.0 53 61 57.0 No 4 73 67 67.0 57 58 57.5 Vit. D 12 68 58 63.0 52 58 55.0 63.4 No uv 20 69 79 74.0 53 50 56.5 27 74 64 69.0 61 56 58.5 T 2 0 64 64.0 61 54 57.5 + 4 71 65 68.0 62 54 58.0 Vit. D 12 71 75 73.5 63 56 59.5 64.1 No uv 20 80 69 74.5 61 54 57.5 27 78 65 71.5 62 51 56.5 T 3 0 ____ 62 62.0 58 58 58.0 No 4 60 67 63.5 55 60 57.5 Vit. D 12 61 66 63.5 57 60 58.5 61.2 + uv 20 60 67 63.5 64 59 61.5 27 62 66 64.0 57 62 59.5 Group Average 67.8 57.9

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-59variance for 25-OH vitamin D. The values of ponies in are similar to the values obtained in normal cow plasma (Koshy and VanDerslik, 1976; Hollis and Conrad, 1976) and normal human plasma (Haddad and Hahn, 1973). On the other hand, the values for vitamin D-deficient ponies (treatment 1) are normal, in contrast to the abnormal values (lower than normal) in vitamin D-deficient humans (Preece et al, , 1973; Gupta et al . , 1974) . Since ponies in are in the early stage of rickets, the reduction of 25-OH vitamin D level, which had been shown in other results, might have been seen at a later stage. This conclusion is compatible with the findings of Clark e^ al. (1973) in rats, who noted the decrease of 25-OH vitamin D levels at advanced stage of rickets. Bone Analysis Tables 8 and 9 summarize the bone analysis of the right and the left metacarpal bones. All data were analyzed by factorial analysis (Tables 19 and 20, Appendix). Water concentration of bone. Water concentration was lower in G (p < .04) and T^ (p < .05) than G 9 and T^ or T 9 respectively. The 1 means of T^ , T^, and T were 31%, 26%, and 27% for right metacarpal and 28%, 25%, and 26% for the left metacarpal bones. The means of G^ and G were 30% and 27% for right metacarpal and 28% and 24% for the left metacarpal bones. There was no interaction effect between treatments and groups. Because ponies of Group II were older than those of Group I, higher bone. water concentration in G^ than G was expected. It has been

PAGE 71

TABLE 8. SELECTED COMPOSITION AND PHYSICAL CHARACTERISTICS OF RIGHT METACARPAL BONE. 60 CM Z“N — n s "s /— \ /— "s /-N c M5 cm o LO oo o CM co rC •H CM 1 rH CM rH | M0 o O NT 1 4-» \ CO | to - • 1 MO 1 O O • 1 CO I MO CO 'M' rH 1 I CO 00 Ob MO CO rH i — 1 rH Ob c co i— i 00 rH rrH in m o oo Ob LO 0) CO NT rH CM rH M0 rH CO MO ro CM rH 00 O < oo r*. cc co oo Ob M0 o LO CM i — 1 LO MO mo m MO MO cu 4-1 rH rH rH rH rH rH rH rH rH rH rH rH pc, O 4-1 c rC cu CO 00 i-H CO Ob 00 oo CO co CM CO CO CO n3 u < • • • . . . • • u u to mo or^ MO MO mo r^MO (U 4-i co co co co CO CO CO CO co co co CO PH o >> cu U rn rrt MO M0 M0 MO MO MO MO MO MO M0 MO MO < fti 6^ rH Q> CO Ob LO rH Ob CO CO CM OJ Ob O • • • • • • * • • . • CM CM 00 O r** Ob NT mo r-. CM rH LO CN • > CO 4-1 1 — HI > CM • > CO 4-» 4-i H •H 0 H 4-» 0 H •H > H •H 0 H 4-1 0 H •H > co > •s •H > 3 '*_X > w •H > a> O > O O > O u O 2 O + O Z ZZ o + H 53 + Z JZJ + z 4-1 4-1 TD a TO c a) CU w cu CU cu W cu Cl 4-i B cm m: oc 4-i B CO MO GC P M U •H 4-i < M U •H 4-» < o
PAGE 72

TABLE 9. SELECTED COMPOSITION AND PHYSICAL CHARACTERISTICS OF LEFT METACARPAL BONE. -61X d) 4-1 QJCM CO > 4-1 co •H £ CM CO co CM • > CO 4-» i — 1 4-1 > CM • G CO 4-1 H •H G H 4-1 G H *H > H •H G H 4-1 G H •H > CD V > •H ' — ' > G ' — x > v-/ •H > G CD O > G O > O Jh O P P 0 + O p G + H z + z P + 2 4-1 4-> X) G /-N X G a CD CD co CD G
PAGE 73

-62indicated that as the rat matures, there is a progressive replacement of water by mineral, with the organic fraction remaining relatively constant (Hammett, 1925) . Bone ash (percent of dry, fat-free bone) . The percentage of bone ash of the right and left metacarpal bones respectively are presented in Tables 8 and 9. The average ash concentration of both right and left leg were analyzed statistically to find the main effects (treatments and groups) and whether there is interaction between groups and treatments. There was no significant difference between groups and no interaction effect. The ash means (percent of dry, fat-free bone) for Group I was 60.4 and for Group II was 60.8. Ponies in T^ had significantly low bone ash (p < .01) than ponies in T^ or T^. The means for T^ , T^, and T^ were respectively 59.9, 60.1, and 61.7% of dry, fat-free bones. . The finding of this difference between treatments agrees with many other experiments in other species. Dutcher and Rothrock (1925) reported a bone ash of 62% in dry, fat-free bones from normal rats; in rickets it was 26.5%. Other results which showed low bone ash in vitamin D deficiency are in rats (Simmons and Kunin, 1970) , poultry (Yang e_t jil. , 1973), swine (Miller ej^ al. , 1964), and cattle (Huffman and Duncan, 1935) . The low ash concentration of vitamin D-deficient ponies results * from the low efficiency of these ponies in absorbing the mineral from the intestine and the use of the bone mineral to keep plasma mineral within the normal level. Bone calcium, phosphorus, and magnesium (% of ash) . Tables 8 and 9 show percent of Ca, P, and Mg of bone ash for right and left metacarpal bones. Factorial analysis showed no significant difference between

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-63treatments or groups regarding percent of Ca, P, and Mg of bone ash. The means of bone ash Ca were 37% and 36% of ash for and G^, and 36%, 36%, and 36% for T^, , and T^. Phosphorus means were 15% for both G^ and G^ and for T^, , and T^, 15% for. all. For Mg the means of G^ and G^ were .49 and .47 and , T^, were .49, .47, and .48. Since the bone crystals need certain minimum levels of Ca, P, Mg, and other minerals to form the total bone crystal, bone ash was expected to be low in vitamin D-deficient ponies, but the percentage of Ca, P, Mg in the bone ash was expected to be the same for all treatments. Hurwitz e_t al. (1969) showed low percent of Ca and Mg in bone ash of rats, while Wahlstrom and Stolte (1958) and Combs et a_l. (1966a) showed little difference in percent of Ca and P of bone ash of pigs supplied with, or without, vitamin D. Bone cortex area . Table 9 shows the value of the cortex area of the left metacarpal bone. Ponies on T^ appear to have low bone cortex area (p < .07) than ponies in T 2 or T . Group II ponies had significantly (p < .05) higher cortex area than ponies in Group I. Figure 12 presents the cortex areas of three ponies in Group I, in which lower cortex area in T^ than T^ and T^ appears. Cortex area averages of G^ , G^ were 1.7 and 2.7, and of T^ T 2 , and T 3 were 1.2, 2.1, and 2.7 cm 2 , respectively . As the young animal grows there is an increase of its bone cortex area, but the bone ash deposit will be less in vitamin deficient animals than the normal, with no difference in their bone cortex areas. This is what has been found in this experiment. There was no significant difference between treatments but there was a significant difference between age groups.

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-64FIGURE 12. EFFECT OF TREATMENT ON METACARPAL BONES OF THREE PONIES OF CROUP I. a Overall appearance. Cross-section showing cortex area at midpoint. Designations T T and T indicate respective treatments. 1Â’ 2 3

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-65Bone density . Group I ponies had significantly lower (p < .01) bone density than ponies in Group II, and ponies in had significantly (p < .01) lower bone density than ponies in or . There were no interactions between groups and treatments. The means of bone density 3 (g/cm ) were 1.3 and 1.4 for G x and G 2 and 1.32, 1.41, and 1.45 for T , T 2 > and T^. Tables 8 and 9 show the individual values of bone density for the twelve ponies. Bone breaking strength . Table 8 shows the breaking strength of right metacarpal bones of the twelve ponies. Some of the values were lost through use of a defective testing apparatus in early determinations. Ponies in T^ appear to have lower bone breaking strength than ponies' bones in T^ or T^, but this difference was not significant. Group I ponies had lower (p < .05) bone breaking strength than ponies in Group II. The means of bone breaking strength were 1252.7 and 1677.9 2 ? kg/cm for G 1 and G^, and 1308.8, 1583.1, 1570.8 kg/cm for T , , and T^. it of did Since ponies in T^ had lower ash content than ponies in T^ and T^, was expected to find low density and breaking strength in the bones the vitamin D-deprived ponies in T^ . The results in this experiment indeed show lower bone density and breaking strength for T^ than and T^ . Miller et al . (1964) indicated low bone breaking strength of vitamin D-deficient pigs. The values of breaking strength of ponies in T^ was similar to values which were obtained by Haugh et al. (1971) in normal Shetland ponies.

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66 Bone epiphyseal closure . Table 10 shows the findings of the epiphyseal closure measurements in Group I, employing the following grading system: "A+" = epiphyseal plate completely closed, "A-" = epiphyseal plate 3/4 closed, "B+" = epiphyseal plate 1/2 closed, "B-" = epiphyseal plate 1/4 closed, "C" = completely open epiphyseal plate. At the beginning (3 months of age), the distal end of the first phalanx (P^D) and the distal end of the second phalanx (P 2 D) plates were closed (A+) , but the distal end of the third metacarpal (MD) , the proximal of the first and the second phalanges (P^P and P 2 P) were open (C) . The last three epiphyseal plates were used to study treatment differences. The radiographs made at the termination of the treatments (Table 10) indicated that the MD, P^P, and P^P graded B-, B+, and Bfor ponies in T^ . In T^ they were B+, A-, and B+. Radiographs in T^ indicated A+, A-, and A. These observations demonstrate a difference in the epiphyseal closure between T^ , T^, and T^. The radiographs of the left leg showed similar differences among treatments. In addition to delay of epiphyseal closure of the vitamin Ddeficient ponies in T^ (Group I), lack of calcification, thickening, widening, and irregularity of the epiphyseal cartilage (epiphyseal plate) at the junction of the diaphyses and the epiphyses were observed. Since MD, P^P, P-^D, P 2 ?> anc ^ ^2° P^ ates were closed in Group II (started at 8.0 months old) the distal end of the radius (RD) , which develops later, was examined and comparison was made between treatments. At the end of the experiment there were no differences between treatments as to RD epiphyseal plate closure. Figures 13 and 14 present the effect of treatments on ponies of T^ and T^ of G^ and .

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-67TABLE 10. EPIPHYSEAL CLOSURE OF THE DISTAL OF THE THIRD METACARPAL (MD) , THE PROXIMAL OF THE FIRST PHALANX (PjP) , AND THE SECOND PHALANX (P 2 P) OF BONES OF GROUP I PONIES. 3 TreatRight Leg Left Leg Age ment MD P 1 P P 2 P MD P 1 P P 2 P T h C C C C C C b _ _ 3 T C C C C C c C C C C C C Months „ 6 T 3 c C c Bc c C C BC C 4 T 1 c B+ c BBc T 9 c BBB+ c BMonths Z BBB+ T 3 B+ BBBBB+ B+ BB5 T 1 C BB+ B+ C BBB+ C T 2 C BB C BABC BMonths T 3 BBAB+ BBB~ A— B+ ~ 6 T 1 BB+ BT 9 c B+ C — — _ _ _ Months T 3 BA+ B+ BA+ ~ B— 7 T 1 BBB+ C BBBB+ B+ B+ BB+ T 2 C BAB+ C BC B+ ABC BMonths T 3 B+ " AAB+ B+ A+ A+ BA8 T 1 B+ BAB+ BBB+ B+ AB+ BBT 2 BB+ AABB+ BAAABB+ Months T 3 A+ AA' A+ A+ AA+ AA+ Each letter represents grade of pony (started at 2 months old). A+ = completely closed B+ = 1/2 closed C = completely open A= 3/4 closed B= 1/4 closed Radiograph not clear enough to grade, c T^ = No vitamin D, no uv light. ^T^ = + vitamin D, no uv light. 0 T^ = No vitamin D, + uv light.

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68 Tl 8 •Si FIGURE 13. EFFECT OF TREATMENTS ON THE EPIPHYSEAL CLOSURE OF GROUP I. Lateromedial (LM) radiograph of the right front foot, showing the delay of the epiphyseal closure of the pony in Group I on Ti (B) compared with the pony on T 3 (D) in the same group. A, C = 3 months old; B, D = 8 months old Group I = started expt. at 2 months of age T^ = No Vit. D, no uv; T^ = No Vit. D, + uv

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-69\ FIGURE 14. EFFECT OF TREATMENTS ON EPIPHYSEAL CLOSURE OF GROUP II. Anteroposterior (AP) radiograph of the distal end of the radius showing similar appearance of the epiphyseal closure in Group II ponies on T^ and T 3 (B and D) . A, C = 9 months old; B, D = 14 months old Group II = started expt. at 8 months of age T^ = No Vit. D, no uv; T^ = No Vit. D, + uv

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-70These abnormalities in development of the epiphyseal plate (the junction of the diaphysis) which were shown in the ponies in of this experiment have been shown in results with many other species. For example, these symptoms of vitamin D deficiency were shown in rats (Simmons and Kunin, 1970), cattle (Bechtel ejt _al . , 1936), and humans (Richards et al. , 1968; Wills _et aX . , 1972; and Holmes et al., 1972). The normal epiphyseal closure of ponies in in this experiment is similar to that in X-rays shown by Myers and Emmerson (1966) , Monfort (1967) , and Coffman (1969) in horses. Liver Analysis (Water, Ash, Calcium, Phosphorus, and Magnesium Concentration) Table 11 presents the average of liver water, ash, Ca, P, and Mg concentrations (on dry basis) , and Table 20 (Appendix) presents liver composition of individual ponies. Analysis of variance for liver compositions and the effect of treatments and age on it is presented in Table 22 (Appendix) . Ponies in Group II had lower (p < .07) concentration of liver water than Group I. There were no significant differences for liver ash concentration among groups or treatments. Average of liver (%) water concentration of C,^ and G^ were 73.5, 72.0, and for T^ , T ? , T^ were 73.0, 72.7, and 72.7. Average liver ash (%) for G^, G^ and T^ , T^ , and T^ were 4.3, 4.2, and 4.1, 4.3, 4.3. Ponies in Group I had higher (p < .05) liver Ca (ppm) than Group II ponies, and ponies on T^ had higher (p < .01) liver Ca (ppm) than T^ or Ty Liver phosphorus (ppm) was lower in Group II ponies than Group I.

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-71TABLE 11. AVERAGE ASH (%) , WATER (%) , CALCIUM, PHOSPHORUS, AND MAGNESIUM CONCENTRATION (ppm) OF LIVER AND KIDNEY. 3 (Dry weight basis) Mineral Organ Liver Kidney \ Group I II I II Start Start Start Start at 2 at 8 at 2 at 8 TreatMonths Months Avg. Months Months Avg. ment \ of Age of Age of Age of Age T l b 224.4 190.5 207.5 1437.0 1286.0 1361.5 Ca 265.0 233.0 249.0 1805.0 2870.0 2337.8 T d 3 206.0 208.0 207.0 579.0 1289.5 934.3 Avg. 231.8 210.5 1273.8 1815.2 T 1 9971.0 79490.0 8980.5 11215.0 10700.0 10958.0 T 2 10334.5 8323.0 9328.8 11203.0 11307.0 11255.3 P T 3 9282.5 8795.5 9021.0 10244.0 10574.0 10409.3 Avg. 9862.7 8357.5 10887.7 10860.7 T 1 572.5 503.0 537.8 575.5 690.0 723.8 Mg T 2 567.0 500.0 533.5 763.0 745.0 754.3 T 3 497.5 520.5 509.0 642.0 742.0 692.0 Avg. 545.7 507.8 720.8 725.8 T 1 4.4 3.9 4.2 7.1 6.4 6.8 Ash T 2 4.4 4.4 4.4 6.7 6.9 6.8 % T 3 4.2 4.4 4.3 6.6 6.5 6.5 Avg. 4.3 4.2 6.8 6 . 6 T 1 74.0 72.0 73.0 82.5 82.0 82.3 Water T 2 73.0 72.5 72.8 82.5 81.5 82.0 % T 3 73.5 72.0 72.8 82.5 80.5 81.5 Avg. 73.5 72.2 82.5 81.3 Each value represents average of two ponies. ^No Vit. D, no uv. c + Vit. D, no uv. ^No Vit. D, + uv.

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-72An interaction effect of groups and treatments was found in liver magnesium concentration. Duncan's multiple range test for liver magnesium indicated that was significantly (p < .05) higher than T and in Group II, but not in Group I. Liver water, ash, calcium, phosphorus, and magnesium concentration values obtained in this study indicated normal values as reported in normal horse liver (Schryver e_t al. , 1974) , and the differences were due to individuals but not to treatments. Al-Ganhari et_ aJL. (1973) reported lower liver weight in vitamin D-deficient rats compared to the control group. Kidney Analysis (Water, Ash, Calcium, Phosphorus, and Magnesium Concentration) Table 11, previously noted, presents the average and the individual ponies kidney composition. The analysis of these compositions and its effect by age and treatments are presented in Table 23 (Appendix) . Kidney water, ash, calcium, phosphorus, and magnesium concentrations in all ponies were normal and were not affected by age groups and treatments. There were no significant differences for kidney compositions among groups or treatments.

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CHAPTER V SUMMARY AND CONCLUSION A 2 x 3 factorial design was carried out to study the effect of vitmain D and sunlight on the growth and bone development of young ponies Two groups of ponies (G^ two months of age, eight months of age) were assigned to three treatments (T no sunlight without vitamin D supplement T^ no sunlight with daily 1,000 IU of vitamin D supplement, and T^ sunlight without vitamin D supplement) . All ponies were put in a windowless barn with very minimum ultraviolet light exposure for one month (to deplete the ponies of possible vitamin D body stores) and then they were assigned to the treatments for five months. A diet deficient in vitamin D (lack of vitamin D was confirmed by testing the diet on rats) and adequate in all other nutrients was fed to the ponies three times daily ( ad libitum ) . Weekly blood samples were obtained from the jugular vein to study the difference within and among treatments and groups regarding plasma calcium (Ca) , phosphorus (P) , magnesium (Mg), alkaline phosphatase (Aik), and 25-hydroxy vitamin D (25-OH Vit. D) levels. Monthly X-rays were taken to study the development of the metacarpus and phalanx junction in Group I, and the radius and metacarpus junction in Group II. The stage of maturity or the degree of epiphyseal closure was classified on the basis of "A+" = complete epiphyseal closure; "A-" = 3/4 epiphyseal closure; "B+" =1/2 epiphyseal closure, "B-" =1/4 epiphyseal closure, 73 -

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-74and "C" = completely open epiphysis. At the termination of the experiment all ponies were killed; liver, kidney, and the two metacarpal bones were taken for laboratory analysis. Water, ash, Ca, P, and Mg contents of liver and kidney were determined. One of the metacarpal bones was used to determine cortex area while the other was used to determine bone breaking strength. Water ash (dry, fat-free basis), Ca (%) , P (%), and Mg (%) (of bone ash), and bone bensity were determined in the bone. The data were analyzed factorially and a statistical analysis system was used. In the case of interaction effects, a Duncan's multiple range test was done to show the effect of treatments within the group. Symptoms of an early stage of rickets (difficulty in standing, loss of appetite, and low feed efficiency) occurred in ponies of (ponies deprived from uv light and vitamin D supplement) , but the bowed legs and inability to stand (typical characteristics of advanced stage of rickets in other species) did not appear in any of the ponies. Group I had slightly lower feed intake and feed efficiency than Group II. Factorial analysis indicated no significant differences of feed efficiency among groups or treatments. Averages of feed efficiency for T^ , T^, and in Group I were 7.9, 8.8, 8.3 grams intake/gram gain and for , T , in Group II were 9.4, 9.6, 9.5 grams intake/gram gain. Plasma Ca levels were within the normal range. There was no significant difference among treatments, but Group II had higher (p < .01) plasma Ca level than Group I. Plasma calcium of Group II ranged from 12.6-15.2 mg/100 ml, while Group I was 10.5-13.5 mg/100 ml. Plasma calcium levels ranged in T^ , 1 ^, and T^ were 10.9-13.9, 10.5-14.0, and 11.1-14.0 mg/100 ml.

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-75A1 though all plasma P values were within the normal range, ponies in Group I appear to have higher levels than Group II. There were no significant differences among groups or treatments. Plasma P levels ranged from 4.6-7. 7 mg/100 ml for Group I and 3. 4-6. 5 mg/100 ml for Group II. The plasma P ranges for T^ , T ? , and T^ were 4. 2-7. 7, 3.4-7. 7, and 4. 1-7. 6 mg/100 ml, respectively. Observations on plasma Mg levels led to the conclusion that the level was not affected from lack of vitamin D or sunlight. All plasma Mg levels were within the normal range. The ranges for Group I and Group II were 1.1-1. 9 and 1.8-2. 5 mg/100 ml. Ranges for T , T ? , and T^ were 1.5-2. 5, 1. 2-2.5, and 1.2-2. 5 mg/100 ml. Factorial analysis indicated no significant differences between groups or treatments. The results of other studies (in other species) on the effect of vitmain D deficiency on the levels of plasma Ca, P, and Mg are not consistent, but all researchers indicated that vitamin D deficient animals mobilized their bone to keep their plasma Ca, P, and Mg levels within the normal range. The plasma mineral levels of vitamin D-deficient animals will drop slightly at the beginning of the deficiency, and thereafter the bone is mobilized to raise these levels to the normal ranges. This was shown, in the vitamin D-deficient ponies, to be generally true. Plasma alkaline phosphatase levels were not affected by either treatments or age groups. The values were varied and did not follow a particular pattern. No significant differences between treatments or groups were found. Average plasma alkaline phosphatase for G^, G^ and Tf , T^ , T^ were 88.0, 102.0 and 89.0, 101.0, 93.0 International Unit (IU), respectively. Most results reported for other species indicated

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-76an increase in plasma alkaline phosphatase with vitamin D-deficient animals. This is established in humans. Plasma 25-OH vitamin D levels were normal and approximately the same for all ponies. The ranges of plasma 25-OH vitamin D for G , G ? and T , T , T 3 were 58-80, 50-63 and 50-79, 51-80, 57-67 ng/ml, respectively. There is no information on the effect of vitamin D deficiency on the level of plasma 25-OH vitamin D in horses, but it is believed, in other species, that the decrease and the disappearance of 25-OH vitamin D occurred at later stage of rickets. Bone water concentration was about the same for treatments and groups. Average bone water content for G^ , G^ and T^ , T ? , T 3 were 30%, 27% and 31%, 26%, 27%, respectively. There were no significant differences in bone water concentration among groups or treatments. Bone ash concentration was lower in T^ (p < .01) than T^ or T 3 ponies, and was lower (p < .05) in Group I ponies than Group II. Ash concentration (% of dry, fat-free bone) for G and G 9 were 60.4% and 60.8%, and T^ , T 9 , and T 3 were 59.9, 60.1, and 61.7, respectively. Calcium, P, and Mg, as percent of bone ash, were not affected by age or treatment. Bone cortex areas of ponies deprived of sunlight and vitamin D supplement (T^) were slightly lower than T ? and T^ The older group (G^) had higher (p < .05) bone cortex area than group 1 (younger ponies). Bone cortex area of G^ and G ^ ranges were 1.3-2. 9 and 1. 7-3.1 2 cm . The ranges for T^ T 2 , and T 3 were 1.3-2. 9, 1. 7-2.9, and 1. 8-3.1 2 cm . The foals on T^ appear to have lower bone breaking strength than foals °f T^ and T 3> but differences were not significant. The means of bone

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-772 breaking strength were 1252.7, 1677.9 kg/cm for Group I and Group II, and 1308.8, 1583.1, 1570.8kg/cm 2 for 1^, and T . Ponies in Group I had lower bone density (p < .01) than ponies in Group II. Also, ponies in T had lower bone density (p < .05) than ponies in and T^. The means of bone density were (g/cm^) 1.3 and 1.4 for G^ and G ^ and 1.3, 1.4, and 1.5 for , and T . The epiphyseal plates (at the distal end of the metacarpal, proximal end of the first phalanx and the proximal end of the second phalanx) of T^ in Group I showed delayed closing, were irregular and wide and instead of having clear, sharp lines, and poor definition. Bone analyses which indicated low bone ash (% of dry, fat-free basis), breaking strength, density, and abnormality of epiphyseal closure of vitamin Ddeficient ponies are similar to reports in other vitamin D-deficient animals (other species). Liver and kidney water and ash concentrations (on dry basis) were not significantly affected by age or treatment as tested by factorial analysis. Average liver water contents for G , G^ were 73.4 and 72.0, and for T^ , , T^ were 73.0, 72.7, and 72.7 percent of liver. Percent liver ash contents were 4.3, 4.2 and 4.1, 4.3, 4.3 for G^ , G ? and T , T 2 , T 3 . Kidney water content (%) for G , G ? were 82.5 and 81.3 and for T 1 Â’ T 2Â’ T 3 were 82.3, 82.0, and 81.5. Kidney ash content for G^ , G ^ and T , T^ (%) were 6.7, 6.6 and 6.8, 6.8, 6.6. There were significant differences (p < .05) between groups (G^ higher than G^) and highly significant differences among treatments (T higher than T^ and T ) in liver Ca levels. For liver P content, a highly significant difference (p < .01) among groups (G^ > G ? ) occurred. In the case of Mg, interaction of groups and treatments occurred.

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-78Duncan's multiple range test showed significantly lower (p < .05) from and T ? in Group I but not in Group II. Kidney Ca, P, and Mg concentrations were not affected by either age or treatment. The mean values (ppm) of Ca, P, and Mg contents of liver and kidney are shown in Table 11. In conclusion, what could be considered an early symptom of rickets occurred in ponies of T^ (deprived of uv light and vitamin D supplement) , especially those of the younger group (started the experiment at 2 months of age). These ponies lost their appetite, had low feed efficiency, and difficulty in standing compared with ponies in T. ? (deprived of uv light and supplied with daily 1000 IU of vitamin D) , and T^ (exposed to sunlight and deprived of vitamin D supplement) . In addition to these symptoms, a slight drop in plasma P and Mg was observed at the early period of the experiment, and after which mobilization of these minerals from bone may have provided amounts sufficient to maintain their levels within the normal range. This mobilization resulted in lowering the ash content of the bone. The lack of adequate Ca, P, and Mg for proper bone mineralization resulted in delayed closing, irregularity, widening and loss of definition in the epiphyseal plates at the end of the long bones in the ponies of T ^ in Group I compared to T^ and T^. Since ponies in Group II (started experiment at 8 months of age) were exposed to sunlight for a longer period before being placed on experiment than those of Group I, they did not show very clearly these early symptoms of rickets . The author believes that for any further study of the importance °f vitamin D in pony nutrition, the age of the ponies and the intake of minerals should be evaluated.

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APPENDIX ANALYSIS OF VARIANCE TABLES

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TABLE 12. ANALYSIS OF VARIANCE FOR FEED EFFICIENCY. Variable Source of Variation Degree of Freedom Sum of Squares PR > F Feed Age 1 3.2634 0.4189 3 efficiency (A) Treatment 2 0.6492 0.9269 3 (T) A x T 2 0.1990 0.9767 3 a Non significant . 80 -

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TABLE 13. WEEKLY PLASMA CALCIUM (mg/100 ml) LEVELS FOR INDIVIDUAL PONIES. -810) oc < 4-1 mm'0 H co in sr co n m iDoooooH^^Dmior'iNiMriouDstN + ^ • i 1 oo o on i n tn n tn tn n cNcNr^vOLncNOOmoocoroLno^cN(^cOvcrcoc^a>mr^ e a •U 3 O'! X •H H w > O ^ fnNo\NffiNt ^ 3 •H D i — I o > H rocoi—tn'LncomtNocoocO'crooor^'OOaN^rvo— imcNooo o ^ o o ^ NHlNM(niMMtntnH< T l(Nn)nNCNMM(NHNniNCM(MC'lnl 1 if — It — It — It — IrHt — It — It — It — It — It — ItHt — It — It — It — It — It — It — ItHt — IrHt — It — 1 rH t — 1 CD oc < f r lstH(^CMfnNHN ^ i~; •H D ro 4J > H -fw rvcMooNaiHNfiotN cO'jr'mmcnaiHtnoioioir1 m \o o O 1 i H H t 1 rH i 1 t — 1 O O CN | t — 1 t — | t — | rH CN , — | , — | , — | , — | | t — | CNJ CNJ tH O s 2 1 — IHt — It — It — It — It — i rH t — 1 1 — It — It — It — 1| — It — 1 i — It — It — It — It — |t — It — It — It — | CN 4-1 cd NCMinHNHcocointncorocomrMnocoHHsrcocoNCN 4-1 i i o i i o'* i i o cnj o on o i — i i — i o cn i — i o i — i o o o o cjn ct^ o c Q Ht — 1 i — It — It — IHHt — IHHHHHt — IHt — IrHHHH H E • > ^ •H u 3 cn •H H OJ a. > o ^ r^miHr^LTiooLrimooco^ocOr-it— i /-n *H D i — 1 a > H O ^ (7\NC^NrOi/)CGNj-004sf|NOO>vDa\rNvOnvDN]-vJinO>sJo o 2: i 1 OOOOHOCTtt — 1 CN t — | (NJ t — 1 C\J t — It — IHHt — It — It — IHHHOH O ' — i ' — 1' — It — It — It — | t — IrHt — It — It — It — It — It — It — It — It — It — It — |t — J , — |t — 1| — | t — | rX 0) 1 — |MCf)stinvDNCOC^Oi — ICNCO-H’LOvDr^'OOCTiOt — 1 M n sf LO v£) N jl) ' — 1' — 1' — It — ItHHt — It — 1 i — It — 1 CN CN CN CN CN CNJ CN CN ‘ —

PAGE 93

-82TABLE 14. ANALYSIS OF VARIANCE FOR PLASMA CALCIUM LEVELS. Source of Variation Degree of Freedom Sum of Squares F Value Age 1 13.0175 37. 7428 3 (A) Treatment 2 4.8900 6.9440 b (T) > X H 2 5.8400 8.4640 b Week 26 7.6748 0.6683 b (w) W x T 54 14.9500 0.8143 b W x A 26 13.0175 1.4516 b A x T x W 50 17.3400 0.9310 b a Highly significant (p < .01). b Non significant.

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TABLE 15. WEEKLY PLASMA PHOSPHORUS (mg/100 ml) LEVELS FOR INDIVIDUAL PONIES. -83M < o w rC 4-J a o CO 4-1 C cu e •H d> CX X w dJ 4-J P on CN 00 LO •nT oCO oc LO 1 — 1 LO i — i CN vO CN LO LO vO NO CN oo NO LO ON LO LO Q CO lO LO LO LO VO ^ •H P CO > H + oo 1 CN rH LO LO vO On 1 o vO O CN o* CC ON o pH o vO CO CO CO LO CO co CO co CO CO LO CO + o w 53 00 o ON O' o 1 1 — 1 r— i co pH o vD vO O' O CN CN co 04 ON co ON NO vO rH co LO LO LO •H P r> H O w O 2 55 4-J •H > O 2; > /P CO H + ^ iN^orNO\cosj-fNmco o v^ a X w + o vO CN ON NO LO ON co vO pH oo VO 00 LO O'. o vO ON LO LO • — 1 co LO ovO LO or * N •H 0 1 1 a > H P o o 00
PAGE 95

TABLE 16. WEEKLY PLASMA MAGNESIUM LEVELS (mg/100 ml) FOR INDIVIDUAL PONIES. -84-

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-85TABLE 17. ANALYSIS OF VARIANCE FOR PLASMA ALKALINE PHOSPHATASE. Source of Variation Degree of Freedom Sum of Squares PR > F Age (A) 1 215.8 0.5822 a Treatment (T) 2 1026.3 0.4879 a A x T 2 2493.6 0.1892 a Week (W) 4 1434.3 0.0046 b A x W 4 19221.2 o.ooio b T x W 8 3748.5 0.7055 3 A x T x W 8 9038.4 0.1743 3 a Mon significant. ^Highly significant (p < .01).

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86 TABLE 18. ANALYSIS OF VARIANCE FOR 25-OH VITAMIN D. Source of Variation Degree of Freedom Sum of Squares PR > F Age 1 1430.60 0.0026 a (A) Treatment 2 109.00 0.4468 b (T) A x T 2 296.80 0.1609 b Week 4 21.31 0.8485 b (W) A x W 4 46.74 0.5732 b T x W 8 119.57 0.4980 b A x T x W 8 135.43 0.4155 b a Highly significant (p < .01). Non significant.

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-87TABLE 19. ANALYSIS OF VARIANCE FOR SELECTED COMPOSITION OF BONES. Source Degree Variable of of Sum of Squares PR > F Variation Freedom Age (A) 1 33.3333 0.0441 a Water % Treatment (T) 2 50.1667 0.0558 b A x T 2 8.1667 0.4958 b Age (A) 1 0.9600 0.3839 b Ash % Treatment (T) 2 14.6425 0.0101 C A x T 2 1.1575 0.6259 a Age (A) 1 2.4300 0.1834 b Ca % Treatment (T) 2 0.5267 0. 7902 b of ash A x T 2 1.2800 0.5810 b P Age (A) 1 0.0533 0.7297 b % Treatment (T) 2 0.0617 0.9279 b of ash A x T 2 0.4817 0.5825 Age (A) 1 0.0014 0.2559 b Mg % Treatment (T) 2 0.0016 0.4428 b of ash A x T 2 0.0004 0. 7978 b . . . . Significant (p < .05). Non significant, c Highly significant (p < .01).

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88 TABLE 20. ANALYSIS OF VARIANCE FOR SELECTED PHYSICAL CHARACTERISTICS OF BONES. Variable Source of Variation Degree of Freedom Sum of Squares PR > F Age (A) 1 0.0396 0.0033 a Density Treatment (T) 2 0.0362 0.0120 b A x T 2 0.0006 0.8496 C Age (A) 1 100039387.4 0.0321 b Breaking Strength Treatment (T) 2 48337245.7 0.1653 C A x T 2 46249622.3 0.1731 C Age (A) 1 2.6508 0.0131 b Cortex Area Treatment (T) 2 1.7580 0.0780 C A x T 2 0.0582 0.8778° a Highly significant (p < .01). ^Significant (p < .05). C Non significant.

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-89TABLE 21. WATER, ASH, CALCIUM, PHOSPHORUS, AND MAGNESIUM CONCENTRATION OF LIVER AND KIDNEY FOR INDIVISUAL PONIES . a Content Organ Liver Kidney "\G Y b <4 C G 1 G 2 h d 73.5 72.5 83.1 81.8 74.4 71.2 81.9 81.5 Water h" 73.5 72.5 82.6 82.2 % 72.2 71.6 82.4 81.2 T f 73.5 70.8 82.6 80.5 73.3 73.0 81.6 80.0 T i 4.3 4.0 7.2 6.1 4.5 3.8 6.9 6.7 Ash T 9 4.4 4.8 7.4 7.4 % 4.4 3.9 6.0 6.3 T 4.0 4.5 6.6 6.7 4.2 4.2 6.5 6.3 T i 237.6 188.0 2159.3 775.2 211.0 193.8 715.2 1997.3 Calcium T 9 266.8 229.0 3059.2 3892.8 ppm 263.4 237.0 552.0 1847.8 T 208.6 195.6 557.7 636.4 203.2 219.6 580.6 1943.3 T i 9406.0 8192.0 11683.8 10672.4 10536.0 7787.8 10746.8 10729.0 Phosphorus T 9 9989.2 8273.0 12298.8 11895.8 ppm 10680.0 8372.0 10107.2 10719.6 T 3 9121.4 8439.0 10020.0 10599.8 9444.0 9079.7 10469.2 10549.7

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-90TABLE 21. (continued) Content Organ Liver Kidney T <5° G 1 G 2 T i d 554.6 518.8 833.2 665.8 590.0 487.0 682.0 715.0 Magnesium L e 568.2 489.4 870.0 765.0 ppm 566.0 511.4 656.0 725.0 T 3 f 499.8 496.8 659.0 689.0 496.0 543.7 625.0 795.0 a Each value represents one pony. b = Ponies started experiment at 2 months of , age. c = Ponies started experiment at 8 months of ; age. d = No Vit. D, no uv light = + Vit. D, no uv light. = No Vit. D, + uv light.

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-91TABLE 22. ANALYSIS OF VARIANCE FOR LIVER WATER, ASH, CALCIUM, PHOSPHORUS, AND MAGNESIUM CONCENTRATIONS. Variable Source of Degree of Sum of Squares PR > F Variation Freedom Age (A) 1 5. 3334 0.0764 3 Water 7 Treatment (T) 2 0.1667 0.9318 b A x T 2 1.1667 0.6297 b Age (A) 1 0.0408 0.5081 b Ash 7 Treatment (T) 2 0.1017 0.5710 b /o A x T 2 0.2517 0. 1914 b Age (A) 1 1365.3 0.0151 3 Ca Treatment (T) 2 4648.7 0.0024 C ppm A x T 2 818.7 0.1031 b Age (A) 1 6796580.0 0.0012 C P Treatment (T) 2 290171.2 0.5276 b ppm A x T 2 1447442.2 0.0959 a Age (A) 1 4294.1 0.0180 3 Mg Treatment (T) 2 1926.5 0.1780 b ppm A x T 2 5554.2 0.0294 3 a Signif icant (p < .05). Non significant, c Highly significant (p < .01).

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-92TABLE 23. ANALYSIS OF VARIANCE FOR KIDNEY WATER, ASH, CALCIUM, PHOSPHORUS, MAGNESIUM CONCENTRATIONS. Variable Source of Variation Degree of Freedom Sum of Squares PR > F Age (A) 1 4.0833 0.0203 a Water 7 Treatment (T) 2 1.1667 0.2170 b /o A x T 2 1.1667 0. 3170 b Age (A) 1 0.1200 0 . 5577 ^ Ash 7 Treatment (T) 2 0.1517 0. 7914 b /o A x T 2 0.3950 0.5628 b Age (A) 1 879125.3 0 . 4382 b Ca Treatment (T) 2 4140558.5 0.2733 b ppm A x T 2 781647.2 0.7470 Age (A) 1 2187.0 0.9541 b P Treatment (T) 2 1473600.2 0. 3605 b ppm A x T 2 382528.5 0.7409 Age (A) 1 75.0000 0.9214 b Mg Treatment (T) 2 7751.1 0.6059 b ppm A x T 2 14787.5 0.4083 Significant (p < .05). Non significant.

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100 Thomas, W.C., H.G. Morgan, T.B. Conners, L. Haddock, C.E. Bills, and J.E. Howard. 1959. Antirichitic activity in human serum. J. Clin. Invest. 38:1078. Thornton, P.A. 1970. Skeletal and plasma changes in chicks during recovery from rickets. J. Nutr. 100:1187. Wahlstrom, R.C. and D.E. Stolte. 1958. The effect of supplemental vitamin D in rations for pigs fed in the absence of direct sunlight. J. Animal Sci. 17:699. Waldroup, P.W., C.B. Ammerman, and R.H . Harms. 1963. Effect of vitamin D 2 levels on phosphorus utilization. Poultry Science 42:982. Waldroup, P.W., J.E. Stearns, C.B. Ammerman, and R.H. Harms. 1964. Studies on the vitamin D 3 requirement of the broiler chick. Poultry Science 44:543. Wills, M.R., J.B. Phillips, R.G. Day, and E.C. Bateman. 1972. Phytic acid and nutritional rickets in immigrants. Lancet 1:771. Wong, R.G. and A.W. Norman. 1974. Studies on the mechanism of action of calciferol. J. Biol. Chem. 7:2411. Yang, H.S., P.E. Waibel, and J. Brenes. 1973. Evaluation of vitamin D 3 supplements by biological assay using the turkey. J. Nutr. 103:1187. Yoshiki , S. and T. Uanayisawa. 1974. The role of vitamin D in the mineralization of dentin in rats made rachitic by a diet low in calcium and deficient in vitamin D. Calc. Tiss. Res. 15:295.

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BIOGRAPHICAL SKETCH Waleed El Shorafa was born May 28, 1946, in Beer Sheba, Palestine. In June, 1963, he graduated from Cairo High School in Egypt. In June, 1968, he graduated from Ean-Shams University in Cairo, Egypt. From June, 1968, to June, 1970, he worked at the Agricultural Experiment Station in Cairo, Egypt. In June, 1970, he immigrated to the United States. In the fall of 1973, he enrolled in the University of Florida. In April, 1974, he obtained his Master of Science in Agriculture from the University of Florida. At present, he is a candidate for the Ph.D. degree in animal science. He is married to the former Annalee Ruprecht and they have a daughter, Rhonda. 101 -

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. 'J.P. Feaster, Chairman Professor of Animal Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. E . A. Ott Associate Professor of Animal Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. C.M. Allen, Jr. Associate Professor of Biochemistry

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. R.L . Shirley Professor of Animal Science f I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. / D.C. Sharp, III J Assistant Professor of Animal Science This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. March, 1978 Dean, Graduate School