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The establishment of a dietary interaction between molybdenum and selenium based on weight gain and feed… Weisstock, Silvia Rita 1980

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THE ESTABLISHMENT OF A DIETARY INTERACTION BETWEEN MOLYBDENUM AND SELENIUM BASED ON WEIGHT GAIN AND FEED CONSUMPTION IN BROILERS B.Sc, The University of Brit ish Columbia, 1977 A Thesis Submitted in Partial Fulfilment of the Requirements for the Degree of Master of Science in The Faculty of Graduate Studies (Department of Poultry Sciency) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMIBA August, 1980 (c) S i lv ia Weisstock, 1980 S i lv ia Rita Weisstock by In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i i ABSTRACT A series of three experiments were carried out in order to de-monstrate an interaction between molybdenum and selenium in broilers. Trai l I investigated the interaction of selenium (0, 0.2, 0.4, and 0.8 ppm) with various toxic and subtoxic dietary levels of molyb-denum (0, 0.5, 100, and 330 ppm), supplemented to a wheat based diet to broilers from one to four weeks of age. Results indicated that at 300 ppm molybdenum, increasing selenium levels resulted in progress-ive decline in weight gain, compared to a non-significant decline across these selenium levels when no molybdenum was supplemented. At these levels of molybdenum, selenium appeared to be acting antagonisti-cal ly with molybdenum. At lower molybdenum levels, selenium exerted no apparent effects on weight gain. Selenium at toxic levels responded different from selenium at subtoxic dietary levels over molybdenum levels. Tria l II, used 480 broiler chicks, assigned in a randomized block (RB) experimental de-sign and 12 treatment combinations of selenium and molybdenum. Although the overall interaction effect was non-significant for weeks 1 to 4 i n -clusive, there were some definite interaction trends. Results indicated that at either basal or 3 ppm Se over basal 100, 200, or 300 ppm Mo, a non-significant difference in weight gain and feed consumption occurred. Selenium and molybdenum appeared to be interacting reciprocally. At 6 ppm dietary supplementation of selenium, however, combining increas-ing levels of molybdenum appeared to result in an independent toxic effect on weight gain which was additive for the two mineral tox ic i t ies , and not interactive. i i i Using 480 broilers chicks assigned to a 3 x 4 x 3 x 3 multi-factorial arrangement of 12 treatments an experiment was performed to investigate the effect on weight gain and feed consumption upon feeding toxic levels of molybdenum and selenium. Selenium levies ranged from basal, to 6, 12, and 18 ppm and molybdenum levels from basal, 400, and 800 ppm. Treatments were arranged in a RB experi-mental design. Results indicated that combining toxic dietary levels of selenium and molybdenum resulted in a measurable interaction in birds based on weight gain to feed consumption from one to four weeks of the experimental period. As the toxic dietary levels of selenium increased from basal to 6, 12, and 18 ppm the adverse effect of molybdenum at basal, 400 and 800 ppm became progressively reduced. At 18 ppm selenium, weight gain and feed consumption were the same irrespective of whether basal, 400, 800 ppm Mo was supplemented to the diet. The presence of toxic levels of selenium appeared to either reduce toxicity of molybdenum, or induce an increased tolerance for increasingly toxic levels of molybdenum. The nature of the interaction between selenium and molybdenum is discussed. Dr. D. B. Bragg (Thesis Supervisor) Professor, Chairman Department of Poultry Science University of Brit ish Columbia Vancouver, Brit ish Columbia iv TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES vi LIST OF FIGURES ix LIST OF APPENDIX FIGURES xi ACKNOWLEDGEMENTS x i i INTRODUCTION 1 Trace Elements 1 Mineral Interactions 2 LITERATURE REVIEW 6 Selenium 6 Molybdenum 7 Toxicity and Mineral Interactions 7 EXPERIMENTAL PROCEDURES 16 Trial I - The Effect of Various Dietary Levels of Selenium With Various Toxic and Subtoxic Levels of Molybdenum on Weight Gain and Feed Consumption in Broilers. 16 Trial II- The Effect of Toxic and Subtoxic Dietary Levels of Selenium and Molybdenum on Weight Gain and Feed Consumption in Broilers. 18 Trial III- The Dietary Interaction Between Various Toxic Levels of Selenium and Molybdenum Based on Weight Gain and Feed Consumption in Broilers. 19 TABLE OF CONTENTS RESULTS AND DISCUSSION Tria l I Trial II Trial III General Discussion SUMMARY AND CONCLUSIONS BIBLIOGRAPHY APPENDIX vi LIST OF TABLES Page Table 1 Composition of the Basal Ration 15 Table 2 Trial I, Analysis of Variance Summary Table for Weight Gain During Weeks 1, 2, 3, and 4 of the Experimental Period. 21 Table 3 Trial I, Effect of Various Dietary Levels of Selenium with Various Toxic and Subtoxic Levels of Molybdenum, Based on Weight Gain in Broilers at 1 Week of Age. 2 6 Table 4 Trial I, Effect of Various Dietary Levels of Selenium with Various Toxic and Subtoxic Levels of Molybdenum, Based on Weight Gain in Broilers at 2 Weeks of Age. 27 Table 5 Tr ia l I, Effect of Various Dietary Levels of Selenium with Various Toxic and Subtoxic Levels of Molybdenum, Based on Weight Gain in Broilers at 3 Weeks of Age. Table 6 Trial I, Effect of Various Dietary Levels of Selenium with Various Toxic and Subtoxic Levels of Molybdenum, Based on Weight Gain in Broilers at 4 Weeks of Age. 28 29 Table 7 Trial I, Analysis of Variance Summary Table for Feed Consumption During Weeks 1, 2, 3, and 4 of the Experimental Period. 3 3 Table 8 Trial II, Analysis of Variance Summary Table for Weight Gain for Weeks 1, 2, 3, and 4 of the Experimental Period. 3 ^ Table 9 Trial II, Effect of Varioua Toxic and Sub-toxic Dietary Levels of Selenium and Molyb-denum on Weight Gain in Broilers at 1 Week of Age. 4 3 Table 10 Trial II, Effect of Various Toxic and Sub-toxic Dietary Levels of Selenium and Molyb-denum on Weight Gain in Broilers at 2 Weeks of Age. 4 4 0 V l l LIST OF TABLES Page Table 11 Trial II, Effect of Various Toxic and Sub-toxic Dietary Levels of Selenium and Molyb-denum on Weight Gain in Broilers at 3 Weeks of Age. 4 5 Table 12 Trial II, Effect of Various Toxic and Sub-toxic Dietary Levels of Selenium and Molyb-denum on Weight Gain in Broilers at 4 Weeks of Age. 4 6 Table 13 Trial II, Analysis of Variance Summary Table for Feed Consumption During Weeks 1, 2, 3, and 4 of the Experimental Period. 52 Table 14 Trial II, Effect of Various Toxic and Sub-toxic Levels of Selenium and Molybdenum on Feed Consumption in Broilers at 4 Weeks of Age. 5 3 Table 15 Trial III, Analysis of Variance Summary Table for Weight Gain During Weeks 1, 2, 3, and 4 of the Experimental Period. 55 Table 16 Trial III, Effect of Various Toxic and Dietary Levels of Selenium and Molybdenum Based on Weight Gain in Broilers at 1 Week of Age. 6 3 Table 17 Trial III, Effect of Various Toxic and Dietary Levels of Selenium and Molybdenum Baseid on Weight Gain in Broilers at 2 Weeks of Age. 64 Table 18 Trial III, Effect of Various Toxic and Dietary Levels of Selenium and Molybdenum Based on Weight Gain in Broilers at 3 Weeks of Age. 6 5 Table 19 Trial III, Effect of Various Toxic and Dietary Levels of Selenium and Molybdenum Based on Weight Gain in Broilers at 4 Weeks of Age. 6 6 VI11 LIST OF TABLES Table 20 Trial III, Analysis of Variance Summary Table for Feed Consumption During Weeks 1, 2, 3, and 4 of the Experimental Period. Table 21 Trial III, Effect of Various Toxic Levels of Selenium and Molybdenum on Feed Consumption in Broilers at 4 Weeks of Age. ix LIST OF FIGURES Page Figure 1 The Effect of Various Subtoxic Dietary Levels of Selenium in Broilers at 3 Weeks of Age. 2 2 Figure 2 The Effect of Various Toxic and Subtoxic Dietary Levels of Molybdenum in Broilers at 3 Weeks of Age. 2 4 Figure 3 The Effect of Various Dietary Levels of Selenium with Various Toxic and Subtoxic Levels of Molybdenum Based on Weight Gain in Broilers at 4 Weeks of Age. 3 u Figure 4 Effect of Various Toxic Levels of Selenium on Three Week Weight Gain in Broilers. 37 Figure 5 Effect of Various Toxic and Subtoxic Molybdenum Levels on Two Week Gain in Broilers. 3 9 Figure 6 Effect of Various Toxic and Subtoxic Molybdenum Levels on Three Week Weight Gain in Broilers. 4 0 Figure 7 Effect of Various Toxic and Subtoxic Levels of Molybdenum on Four Week Weight Gain in Broilers. 4 2 Figure 8 Effect of Various of Toxic and Subtoxic Levels of Selenium and Molybdenum on Weight Gain in Broilers at Four Weeks of Age. 4 7 Figure 9 Effect of Various Toxic and Subtoxic Levels of Selenium and Molybdenum on Weight Gain in Broilers at Three Weeks of Age. 4 8 Figure 10 Effect of Various Toxic and Levels of Molybdenum on Weight Gain in Broilers at Three Weeks of Age. 56 Figure 11 Effect of Various Toxic Levels of Selenium on Weight Gain in Broilers at Four Weeks of Age. 5 8 X LIST OF FIGURES Figure 12 Effect of Various Toxic Levels of Selenium and Molybdenum on Weight Gain in Broilers at Four Weeks of Age. Figure 13 Reverse Axis of Figure 12. Showing Selenium's Effect on Molybdenum in Broilers at Four Weeks of Age. Figure 14 Effect of Various Toxic Dietary Levels of Molybdenum on Weight Gain in Broilers at Four Weeks of Age. Figure 15 Effect of Various Toxic Dietary Levels of Selenium and Molybdenum on Weight Gain in Broilers at Three Weeks of Age. Figure 16 Effect of Various Dietary Levels of Molybdenum in Broilers Fed Diets Con-taining 18 ppm Selenium, from One to Four Weeks of Age. Figure 17 Theoretical Three-Way Interaction Between Selenium, Molybdenum, and Copper (Dotted Line), based on known Interactions of Selenium with Copper with Selenium and Molybdenum with Copper (Solid Lines). xi LIST OF APPENDIX FIGURES Appendix Figure 1 Diagrammatic Representation of the Experimental Room for Tr ia l I. Appendix Figure 2 Trial I Multifactorial Arrange-ment of Four Levels of Selenium and Three Levels of Molybdenum, over Two Blocks and the Two Sexes. Appendix Figure 3 Trial II Diagrammatic Representa-tion of the Experimental Room for Trials I and II. Appendix Figure 4 Tria l II Multifactorial Arrange-ment of Three Selenium Levels and Four Molybdenum Levels, over Four Blocks and the Two Sexes. Appendix Figure 5 Trial III Multifactorial Arrange-ment of the Four Selenium Levels and Three Molybdenum Levels over Four Blocks and the Two Sexes. xi i ACKNOWLEDGEMENTS The author wishes to acknowledge her advisor, Dr. B. D. Bragg, Chairman and Professor, Department of Poultry Science, for his helpful advise throughout this thesis. The author also wishes to acknowledge her committee members: Dr. C. Krishnamurti, Professor, Department of of Animal Science; Dr. J . Sim, Instructor, Department of Poultry Science; and Dr. R. Fitzsimmons, Professor, Department of Poultry Science, for their constructive cr i t ic ism throughout the writing of this thesis. Their guidance was truly appreciated. The author also wishes to acknowledge the late, Dr. C. W. Roberts for this s tat i s t ica l expertice during the i n i t i a l stages of her thesis experimentation. 1 INTRODUCTION Trace Elements Originally many of the elements which occurred in l iv ing tissues were described as "trace elements" because they were present in such small amounts that methods of detection and measurement were unavail-able to quantitate them precisely. With the advent of modern methods of analysis, levels of trace elements were much more clearly and accurately established but the descriptive heading "trace elements" has been retained. In the past 35 years, many elements have been added to the l i s t of trace elements which now includes: iron, zinc, copper, manganese, nickel, cobalt, molybdenum, selenium, chromium, iodine, f luorine, t i n , s i l i con , vanadium, boron, cadmium, mercury, and arsenic. A feature, common to a l l the above elements, is their apparent essentially for normal body function. Cotzias (1967) defines an essential trace element by the follow-ing c r i t e r i a : (1) i ts presence in a l l healthy tissues in a l l l iv ing things; (2) i ts concentration from in different animals is f a i r l y constant; (3) i ts withdrawal from the body induces reproducible physiological and structural abnormalities regardless of the species studied; (4) its addition either reverses or prevents those ab-normalities; (5) the abnormalities induced by a deficiency of the element are always accompanied by pertinent, specific biochemical changes; and, (6) these biochemical changes can be prevented or cured when the deficiency is prevented or cured. 2 Venchikov (1974) applied Bertrand's Law (1912) of dose dependent response in the form of a curve with two plateaus. The f i r s t part of the curve, showing an increasing effect with increasing concentra-tions until a plateau is reached expresses the biological action of the element. The plateau breadth expresses optimal supplementation and normal function (the requirement range). With further increase in dose the element enters a phase of i r r i ta t ion and stimulation of some func-tion, expressing the pharmacological action. S t i l l higher doses of the elements result in the appearance of signs of tox ic i ty, expressing a toxicological action of the element. The snap of the plateau which represents the requirement range can fluctuate and is dependent on many factors. These factors include: age, species, developmental stage, protein and energy content of the diet, and relative amounts of other elements in the diet. This latter factor, introduced the concept of mineral interactions. Mineral Interaction The term interaction can be defined either physiologically or s ta t i s t i ca l l y . Physiologically, interaction means to act reciprocally or to act on each other. Thus, a mineral interaction refers to the action (which maybe uptake or ut i l izat ion) of one mineral on another. The interaction may take the form of either a mutual stimulation or antagonism between mineral elements. S tat i s t i ca l ly , an interaction is a measurable effect apparent i;f the minerals have a combined effect which is different from the sum of the effects were the minerals applied separately. The individual mineral effects are not additive 3 in their parameters of measure therefore, one mineral is dependent or interrelated to another for the observed overall effect of the combined minerals. Researchers have employed various forms of measure in an attempt to demonstrate or detect an interaction between two (or more) minerals. This may range from the specif ic measures of enzyme act iv i ty and pro-tein binding a f f i n i t i e s , to the more general measures such as weight gain and mortality. Generally, a mineral interaction cannot be un-covered without some type of observable change in overall performance in l iv ing tissues, organisms, or in a population of animals which can be demonstrated or repeated. Moxon and DuBois (1939) showed that 5 ppm cadmium, molybdenum, or zinc.(along with other minerals) in the drinking water as soluble salts fed to rats in a basal diet containing 11 ppm selenium as seleniferous wheat caused increased mortality when compared with unsupplemented controls. H i l l (1974), on the other hand, demonstrated that chick mortality due to selenosis was not increased through supplementation of the diet with cadmium. The finding that cadmium part ia l ly counteracted the toxic effects of selenosis in poultry is contradictory to the report of Moxon and DuBois work on rats. Chicks fed either 57 ppm cadmium or 40 ppm selenium (in this case, Se fed as sodium selenite) exhibited mortality s ignif icantly greater than controls but i t was not further increased when both cadmium and selenium were added to the diet together. The cadmium-selenium interaction has been repeatedly demonstrated in other experiments of this nature (Underwood, 1977),thus raising question to the early findings of Moxon and DuBois (1939). 4 In l ight of these contradictory findings of Moxon and DuBois1 work on the cadmium-selenium interaction, a l iterature review was under-taken for the molybdenum-selenium interaction this search indicates that the original work on molybdenum and selenium had not been thoroughly investigated. A study undertaken by the author, at the University of Brit ish Columbia poultry experimental farm indicated that selenium supple-mented to the basal diet at 0, 0.2, 0.4, 0.6, and 0.8 ppm resulted in a non-significant difference in weight gain in birds reared to four weeks of age. Four thousand chicks were assigned to 10 pens, with 5 pens per block. Commercial poultry farmers frequently supplement the diet with levels of selenium similar to those in the above study. In some cases (water supplementation), even higher levels are ingested for short periods of time. This is done (successfully) in an attempt to al leviate leg problems and increase weight gain in broi lers. The idea was presented that various levels of other minerals may interact and/or alter the level of selenium available to the birds and may be responsible for determining whether or not a beneficial response to selenium supplementation would occur. Due to scant information on selenium response to molybdenum in broi lers, an experiment was set forth to investigate this response. The objectives of the present study were to demonstrate a selenium -molybdenum interaction in poultry. A hypothesis was set forth that 5 selenium and molybdenum interact with each other and result in a measurable effect on weight gain on feed consumption. The interaction is based on 'stat ist ica l analysis of the weight gain and feed con-sumption in broilers. 6 LITERATURE REVIEW Selenium The nutritional significance of selenium was discovered in the 1950's. This lead., to the establishment of selenium requirement-levels in many species of animals. Scott et al_. (1955) showed that brewer's yeast contained a factor which replaced vitamin E for prevention of exudative diathesis. Schwartz and Foltz (1957) then discovered that the unknown factor in yeast was a selenium compound and that inorganic selenium (sodium selenite) was as effective as brewer's yeast in pre-venting nutritional l i ver necrosis in rats. In poultry the minimum requirement for selenium was established in a number of studies by Scott and associates. A dietary deficiency of selenium in chicks results in poor feed eff ic iency, poor feathering, and growth retardation, marked by poor growth of muscles, l i ver , pancreas, and ultimate death. Chicks suffering from acute vitamin E and selenium deficiencies may show one or more of the three deficiency diseases; exudative diathesis, encephalomalacia, and/or nutritional muscular dystrophy. Neischeim and Scott (1958) found that a Torula Yeast diet con-taining 0.056 ppm Se and 100 I.U. of vitamin E per pound did not support growth in chicks unless supplemented with 0.04 ppm selenium as selenite. 01dfield (1963) also established a requirement level of selenium in sheep by demonstrating that in lambs a minimum require-ment of 0.06 ppm selenium was necessary for the prevention of white muscle disease (WMD). •7 Molybdenum Molybdenum was discovered as an essential element, independent-ly by two groups of workers (Renzo et al_., 1953 and Richert et a l . , 1935) who showed that the flavoprotein enzyme zanthine oxidase is an molybdenum-containing metalloenzyme which is dependent for its act iv i ty on the presence of this element. The rat and chick have an extremely low molybdenum requirement. In l ight of this, the re-quirement level of molybdenum is not clearly established in poultry (Higgins et al_. 1956). Molybdenum requirement levels are also dependent on the relative amounts of other minerals such as copper (Underwood, 1977). Toxicity and Mineral Interactions Prior to the establishment of a selenium requirement, i n i t i a l attention was devoted to selenium toxicity. Franke (1934) demonstrated that a high selenium content in plant foodstuffs of South Dakota was responsible for 'a lkal i disease' in cattle and other livestock. Cases of selenium toxicity were f i r s t demonstrated by Moxon (1937) and re-viewed by Rosenfeld and Beath (1945). There are two types of selenium toxicity or selenosis: (1) Alkal i disease, or (2) Blind staggers. Alkal i disease is a condition arising from chronic toxicity which occurs when animals consume seleniferous plants containing 3-30 ppm selenium over a prolonged period (Oldfield, 1971). Conversely, the acute form of selenosis, blind staggers, resulted from the consumption usually in a single feeding, a suff ic ient quantity of highly seleniferous weeds which produces severe symptions. According to Rosenfeld and 8 Beath (1964) selenium is among the few elements which are capable of being accumulated by certain forage plants in suff ic ient amounts to create toxicity hazards in animals. High concentrations of selenium accumulate in these plant species when grown on seleni-ferous so i l s . Martin and Gerlach (1972) state that other than hydrogen selenide, selenite is generally regarded as the most toxic chemical form of selenium. Irrespective of the mode of administration selenite rapidly finds i t s way into the general circulation where i t rapidly combines loosely with plasma albumin. Prior to becoming more firmly attached to the various globular fractions, i t has the opportunity to detach i t se l f from the albumin and penetrate the cel l s of the body. Once in the c e l l , i t exerts i ts toxic effect by catalyzing the oxida-tion of important sulfhydryl metabolities, thus rendering them in -active. Once many of the acute disorders were brought under control and/or prevented, i t became apparent that a series a milder maladies involving the trace elements also existed which had less specif ic manifestations and affected more animals and greater areas than the acute conditions which prompted the original investigations. The deficiency or toxicity states were often found to be ameliorated or exacerbated, by the extent to which other elements or compounds were present or absent from the environment. Furthermore, the tolerance or 'safe' dietary levels of potentially toxic trace elements exist. The range of the dietary requirement is dependent on the extent to which other elements are present in the diet. For example, a copper 9 deficiency may be induced in sheep i f the diet is supplemented with excess levels of the copper antagonists, molybdenum or sulfur. This was indicated clearly by Dick (1954) who showed a 3-way interaction between copper, molybdenum, and inorganic sulfur. The ab i l i ty of molybdenum to l imit copper retention in the animal can only be expressed in the presence of adequate sulfate. In fact, i t is d i f f i cu l t to prescribe- a copper or molybdenum requirement in poultry without considering levels of both elements in the diet (Underwood, 1977). In the event that the copper;molybdenum ratio is not ideal (ranging from 2:1 to 6:1) excess molybdenum may induce a copper deficiency or visa versa. The molybdenum-copper-sulfate interaction occurs in the gut (Mil ls, 1961). Mi l ls contends that molybdate and sulfate restr ic t copper ut i l izat ion in sheep by depressing copper so lubi l i ty in the digestive tract through the precipitation of insoluble cupric su l -f ide. Copper becomes unavailable by 2 mechanisms.':'.|1.);with molybdate to form a biological ly unavailable Cu-Mo complex called cupric molybdate, and (2) by the formation of insoluble copper sulfide in the rumen, intestine, or tissues (Underwood, 1977). Furthermore, higher than normal sulfur induced a copper deficiency. An antagonism exists between copper and molybdenum plus sulfur. Kratzer (1952) provides support for a metabolic difference between species of animals by the following observation: catt le and sheep fa i l to form hair pigmentation when fed high molybdenum diets (dark coloured animals become l ight coloured). In turkey poults, however, molybdenum had no effect on pigmentation for a test period 10 of over 4 weeks. He suggest that either: (1) the mechanism of pig-ment formation in turkeys differs from that in ruminants, or (2,) the level of copper while adequate in poult rations may have been marginal in these experiments dealing with ruminants. Furthermore, Comar et al_. (1949) suggest that the growth depressing effect of molybdenum is due to the presence of the element in the tissues. Evidence of a copper-selenium interaction was presented in a paper by Jensen (1975a). Adding high levels of copper to a chick diet marginal in selenium resulted in a selenium deficiency in chicks at 3 weeks. Chicks receiving 0.2 ppm Se (as sodium selenite) supple-mented with 800 or 1600 ppm copper resulted in increased mortality and exudative diathesis, and nutritional muscular dystrophy when compared with control diets which did not exhibit these maladies and grew well. Evidence was presented for the copper induced selenium deficiency by the finding that adding 0.5 ppm more selenium to the diet completely prevented the deficiency symptoms and reduced the mortality. In this study Jensen noted that a similar selenium de-ficiency prof i le was observed in chicks fed 0.2 ppm selenium diet supplemented with 2,100-4,100 ppm z:inc-'. Again selenium deficiency symptoms were completely prevented and mortality reduced by feed an additional 0.5 ppm selenium to the diet. In other words, the selenium requirement of the chick was raised from 0.2 to 0.7 ppm by the treat-ments imposed. The establishment of a selenium requirement and its dependency on relative amounts of other minerals in the diet not only includes copper and zinc but also arsenic, cadmium, s i lver , mercury, and sulfur. Moxon (1938) demonstrated the effect of arsenic on selenium toxic ity. Five ppm arsenic as arsenite in the drinking water pre-vented a l l signs of selenosis is rats. Moxon and DuBois (1938) then performed a series of experiments to investigate the effect of com-bining various elements with high levels of selenium. Their para-meters of measure were weight gain and mortality. They concluded that 5 ppm of either tungsten, f luoring, molybdenum, chromium, vanadium, cadmium, zinc, cobalt, n ickel , or uranium given in water as soluble salts to rats fed a basal diet containing 11 ppm selenium as seleniferous wheat resulted in increased mortality. More import-ant, however, was the finding that 5 ppm arsenic in the drinking water completely prevented the symptoms of selenosis. Arsenic has since been used to al leviate selenium poisoning in pigs, dogs, chicks and catt le. The protection afforded by arsenic is due, at least in part, to increased b i l ia ry (Lavander and Bauman, 1966) and pulmonary (Kamstra and Bonhorst, 1953) selenium excretion. Some years later Ganther et al_. (1972), introduced the idea that mineral interactions might take place between selenium and other elements. He demonstrated that selenosis could by counteracted in chicks by feeding mercury. But i t wasn't until a study by H i l l (1974) that mineral interaction studies were undertaken. Using chicks he showed that selenium toxicity could be reversed by using either mercury, copper, or cadmium. Adding 500 ppm of mercury in the chick diet containing 40 ppm of selenium completely al leviated the toxic effects of selenium when compared to mercury-free diets. Furthermore, the most effective ratio of mercury to selenium was a 1:1 molar ratio. At a comparable ratio copper was as effective as mercury in reducing the mortality due to selenosis but did not prevent the growth in -hibition as did mercury. Hi l l (1974) suggested that mercury, copper, and cadmium exert protection against selenosis by reacting with the selenium probably within the intestinal tract to form relat ively innocuous compounds. Starcher (1969) discovered a plausible mechanism to explain the interaction or close relationship by identif ication of a single metal-binding protein in the duodenum and demonstrating that this protein in the duodenum would bind completely with either copper, cadmium, or zinc. Dietary copper at 500 ppm was capable of markedly reducing selenosis mortality but only part ia l ly counteracted growth depression. However, using 32 ppm copper in the diet combined with 40 ppm selenium was extremely effective in reducing the mortality due to selenosis (H i l l , 1974). He concluded with the following four points extrapolated from this study: (1) the interaction between copper and selenium was evident; (2) copper is as effective as mercury in al leviat ing the mortality induced by selenosis; (3) selenium toxicity is alleviated by toxic levels of copper but in doing so l ike ly resulted in a copper related mineral imbalance, which would account for the incomplete recovery of weight gain; and, (4) although copper alleviated selenium induced mortality there is questionable differences between the beneficial effects of feeding copper at 500 ppm or 32 ppm, indicating that there may be an 'optimal' range for copper supplementation at various levels of selenium in the toxic regions. Jensen (1975b) provided evidence that elements which induce a 13 selenium deficiency should also counteract a selenium toxicity. This experiment employed various toxic levels of selenium ranging from 0.5, 10, 20, 40, to 80 ppm. Se supplemented with 1000 ppm copper. Addi-tion of copper resulted in weight gain that was similar across a l l selenium levels. Combining copper and selenium at these levels pro-duced gains that were not additive in their independent tox ic i t ies , as one might expect i f copper and selenium acted independently. Copper appeared to have l i t t l e effect on selenium absorption. Copper did, however, cause a marked accumulation of selenium in the l i ver . Cadmium reduced (as does copper) the toxic effects of high dietary levels of selenium. As further evidence to"this, selenium has a protective effect against the carcinogenic (Gunn et_ al_. 1963), teratogenic (Holmberg and Ferm, 1969), and hyppertensive (Perry and Erlauger, 1974) effects of cadmium. Jensen (1975b) also showed that a marked protective effect against both mortality and growth retardation induced by dietary levels of selenium ranging from 0, 5, 10, 20, 40, and 80 ppm Se resulted from chicks fed s i lver, as s i lver nitrate, at 1000 ppm Ag. Comparative weight gains were higher (1000 ppm Ag) than with copper (1000 ppm Cu) over the selenium levels, and mortality was somewhat lower with 1000 ppm Ag than with 1000 ppm Cu supplementation. Silver appeared to interfere with selenium absorption or accumulation, as shown by the reduced levels of hepatic absorption in chicks fed s i lver . Furthermore, the metabolism of the portion of selenium which is absorbed appears to be affected also by the s i lver ions, as heptic selenium accumulated in comparison with those chicks fed basal diets. The accumulated selenium is apparently in a non-deleterious form, as chicks 14 containing high levels of selenium in the l iver lived and grew well. Final ly, increasing the levels of sulfate up to 0.87% of a sulfate-free diet progressively relieves the growth inhibition i n -duced in young rats by feeding 10 ppm selenium as selenite or selenate (Halverson, 1960). In summary, i t appears that many trace minerals both essential and non-essential have been shown to experimentally interact with selenium. These elements include: arsenic, mercury, copper, zinc, s i l ver , cadmium, iron, and sulfur. An inspection of the periodic table reveals a striking s imilar ity between these minerals. H i l l et aj_ (1970) and Matrone (1974) hypothesized that the electron configuration of the various elements is the major factor in deter-mining the apparent nature of theobserved interactions between the elements. For example, selenium and copper interact. Their electron configuration is similar as i t is with zinc, iron, cobalt, and arsenic. Al l these elements interact with selenium and have a similar argon core base of electrons. It should also be kept in mind that the chemical property of elements is to a large extent determined by the electron configuration in its outermost shel l . Likewise, s i lver , cadmium, and mercury interact with selenium and these elements have a similar krypton electron base core. 15 Table 1 Composition of the Basal Ration Ingredients % % C P . % M.I Wheat, hard 57.5 7.18 782.0 2 Soybean, meal 29.5 14.13 339.3 Meat, meal 2.0 1.08 18.2 A l fa l f a , meal 1.0 .17 5.0 Animal tallow 7.0 - 245.0 Limestone 1.5 - -Calcium Phosphate 0.5 - -* Premix 1.0 _ 100.0 22.75 1389.5 * Premix ingredients/kg: vitamin A (500,000 IU/gm), 8800IU; vitamin D3 (200,000 ICU/gm), 880 ICU; vitamin E (227,000 IU/lb), 22 IU; vitamin B-j2 (60 mg/lg), 13.3 meg; r ibof lavin, 6.6 mg; calcium pantothenate, 8.8 mg; niacin, 22 mg; choline chloride (50%), 220 mg; amprol 25%, 124.9 mg; baciferin (50 gm/lb), 9.7 mg; methionine (0.1%), 999 mg; MnS04, 100 mg; ZnO, 48.4 mg; CUSO4, 7.8 mg; iodized salt , 4.6 gm. 1 Lysine = 1.18%; Methionine = 0.3523%; Calcium = 1.099%; Phosphorus = 0.5679%; Selenium content, based on fluorometric analysis = 0".6 ppm (Tiral I); 0.5 ppm (Trials II & III). 2 Crude Protein Analysis (CP.) = 46.2'3%. 3, Metabolic energy (M.E.) 16 EXPERIMENTAL PROCEDURES Trial I - The Effect of Various Dietary Levels of Selenium with Various Toxic and Subtoxic Levels of Molybdenum on Weight Gain and Feed Consumptions in Broilers Trial I was designed to test the hypothesis that molybdenum interacts with selenium to segregate an otherwise non-significant difference in weight gain and feed consumption across various selenium levels. Four levels of molybdenum (0, 0.5, 100, and 300 ppm) were added to diets containing 0, 0.2, 0.4, or 0.8 ppm supplemented selenium as a 4 x 4 factorial arrangement of treatments. This study was based in part, on the work of Moxon and DuBois (1939) who showed that 5 ppm molybdenum increased mortality in rats fed 11 ppm selenium of seleni-ferous wheat when compared to selenium-free controls. Experimental: Three hundred and twenty commercial broi ler chicks (one day of age) were assigned in a randomized block (RB) experimental design to four battery brooders. Two battery brooders represent one block containing 16 pens, with each representing one treatment (Appendix Figure 1). Al l chicks were individually weighed and wing-banded according to sex which had been previously determined at the hatchery. Five male and five female chicks were randomly distributed to each of 16 pens in each block. The 16 treatments were than assigned to each block at random. Each week (seven days) during a four week experimental period individual bird weights were recorded. Feed con-sumption was also calculated each week on a per pen basis. Diet: The 16 treatments consisted of four levels of selenium and four levels of molybdenum added to the basal diet (T:able 1) including 17 a l l combinations. The experimental design took the form of a 4 x 4 factorial arrangement of treatments with two blocks and the two sexes per treatment (Appendix Figure 2). Molybdenum was added to the basal diet as the anhydrous acid, molybdate, and selenium was added to the basal diet as sodium selenite (in water). Minerals added to the basal diet were mixed for 15 minutes in a horizontal mixer. The following levels of each trace mineral were used: (1) molybdenum control (basal), molybdenum 0.5 ppm, molybdenum 100 ppm, and molybdenum 300 ppm; and, selenium control (basal), selenium 0.2 ppm, selenium 0.4 ppm, and selenium 0.8 ppm. Feed and water were available ad libitum over the four week experimental period. Analysis: The analysis of the 4 x 4 x 2 x 2 multifactorial arrangement of the data in the RB experimental design follows the model: Y. .. = ti + S. + M. + B. + Z, + MS. . + SB.. + MB.. + MZ., + BZ. , + l jk i j k 1 i j ik jk j l kl SMB... + SMZ.., + SBZ.,, + MBZ. ( 1 + SMBZ. .. , + e . . . , . Ij k i j l ikl j kl i j kl i j kl where, 1 = 1, ...4 selenium levels j = 1, ...4 molybdenum levels k = 1, ...2 blocks 1 = 1, ...2 sexes and, e. . . , = the random environmental effect that varied among individuals irrespective of treatment, block, or sex effect. Sa t i s t i -cal analysis of the 16 above variables were performed each week based on comparative weight gain and feed consumption of chicks. Data was subject to analysis of variance (AN0VA) by computer multiple regression package, BMD:10V. Mean comparisons were also performed on this data 18 by computer by Student-Newman-Keuls (SNK) test. Tria l III - The Effect of Various Toxic and Subtoxic Dietary Levels of Selenium and Molybdenum on Weight Gain and Feed Consumption in Broilers  The objectives of this t r i a l were to study the combined effect of various toxic and subtoxic dietary levels of selenium on various toxic and subtoxic dietary levels of molybdenum. Results to Trial I indicate a selenium-molybdenum interaction become apparent at the higher levels of selenium. A hypothesis was set forth that at subtoxic and toxic levels selenium and molybdenum interact with each other in the diet and this interaction results in a measurable change in weight gain and feed consumption which can be quantiated. Experimental: Four hundred and eighty commercial broi ler chicks (one day of age) were assigned in a randomized block (RB) experimental design to four Petersime battery brooders. Each battery brookder re-presents one block containing 12 pens in which each represents one treatment (Appendix Figure 3). Chicks were individually weighed and wingbanded according to sex which had been previously determined at the hatchery. Five male and f ive female chicks were randomly distributed to each of the 12 pens in each block. The 12 treatments were then assigned to each block at random. Each week (seven days) during the four week experimental period individual bird weights and feed con-sumption were recorded. Diet: 12 treatments consisted of theee levels of selenium and four levels of molybdenum added to the basal diet (Table 1), includ-ing combinations. The RB experimental design takes the form of a 4 x 3 factorial arrangement of treatments with four blocks and the two sexes per treatment (Appendix Figure 4). Refer to Tria l I for diet 19 preparations. The following levels of each trace mineral were used: (1) molybdenum control (basal), molybdenum 100 ppm, molybdenum 200 ppm, and molybdenum 300 ppm; and, (2) selenium control (basal), selenium 3 ppm, and selenoum 61 ppm. Feed and water were available ad libitum over the four week experimental period. Analysis: Analysis of the 4 x 3 x 4 x 2 multifactorial arrange-ment of the data in the RB experimental design follows the model presented in Trial I: where, i = 1, ...3 selenium levels j = 1, ...4 molybdenum levels k = 1, ...4 blocks 1 = 1, ...2 sexes Analysis of weight gain and feed consumption follows those undertaken in Trial I. Tr ia l III - The Apparent Dietary Interaction Between Various Toxic Levels of Selenium and Molybdenum Based on Weight Gain and Feed Consumption in Broilers Objectives of this study were to determine the effect on weight gain, feed consumption and mortality in broilers fed different toxic levels of selenium and molybdenum in a wheat based starter diet. A hypothesis was set forth that selenium and molybdenum (both at toxic levels) interact with each other and result in a measurable change in performance which can be quantitated. Experimental: Four hundred and eighty commercial broi ler chicks (one day of age) averaging 42.5 gm were assignedHn a randomized block (RB) experimental design to four Petersime battery brooders. The experimental set up was similar to that in Trial I and II. 20 Diet: The 12 treatments consisted of three levels of molybdenum and four levels of selenium supplemented to the basal diet (Table 1), including combinations. Treatments were assigned in a 3 x 4 factorial arrangement with four blocks and the two sexes per treatment (Appendix Figure 5). The following levels of each trace minerals were used: (1) molybdenum control (basal), molybdenum 400 ppm, and molybdenum 800 ppm; and, (2),selenium control (basal), selenium 6 ppm, selenium 12 ppm, and selenium 18 ppm. Feed and water were available ad libitum throughout the experimental period. Diet preparations follow the outline presented in Trial I and II. Analysis: Follows the model given in Trials I and II. where, i = 1, ...4 selenium levels j = 1, ...3 molybdenum levels k = 1, ...4 blocks 1 = 1, ...2 sexes Analysis of the feed consumption and weight gain is described in Trials I and II. Table 2. Trial I, Analysis of Variance Summary Table for Weight Gain for Weeks 1, 2, 3, and 4 of the Experimental Period. Source of Variation D. F. Mean Square 2 Week 1 J Week 2 Week 3 Week 4 Molybdenum, Mo 3 0.02405900* 0.01920700* 0.02050000* 0.02210600* Selenium, Se 3 0.03867800* 0.01656600* 0.00861090* 0.00111550 Block, B 1 0.00942990 0.00254580 0.00000788 0.01171400* Sex, Z 1 0.00154020 0.03134500* 0.09353800* 0.15919000* Mo X Se 9 0.01823200* 0.00683830* 0.00269300 0.00508210 Mo X B 3 0.03364600* 0.00423530 0.00045621 0.00054998 Se X B 3 0.03245400* 0.01277100* 0.00614960 0.01456000* Mo X Z 3 0.00638850 0.00311700 0.00161100 0.00205670 Se X Z 3 0.00129410 0.00086645 0.00069280 0.00172760 B X Z 1 0.00077147 0.00001434 0.00000367 0.00005338 Mo X Se X B 9 0.00919630 0.00412360 0.00426590 0.00690360 Mo X Se X Z 9 0.01198600 0.00756100* 0.00724880* 0.00502380 Mo X B X Z 3 0.00159920 0.00330830 0.00518620 0.00191310 Se X B X Z 3 0.00244520 0.00264600 0.00353210 0.00110880 Mo X Se X B X Z 9 0.00327780 0.00200550 0.00146950 0.00237850 Error 208 0.00560880 0.00288830 0.00241250 0.00332020 1. Multifactorial Analysis, Sokol, R. R. and F. J . Rolf, 1969. Biometry. W. H. Freedman and Company, U.S.A. pps.346-362. 2. Indicates level of significance, 5% ( ). 3. AN0VA based on l og , n values of weight gain from weeks 1 to 4. mo 4 X 490 Vb > 4So 1 ° BASAL O.Z O.if SELEA//UM LEVELS (ppm) FIGURE /. EFFECTS OF VARIOUS DlETffRY LEVELS OF SELENIUM ON WEIGHT GG/At IN BROILERS AT THR££ WEEKS OF A&E . 23 RESULTS AND DISCUSSION Trial I Weight Gain Stat ist ica l analysis of the selenium main effects indicated a signif icant difference in weight gain which was apparent from week 1 to week 3 (Table 2). Graphical representation of SNK comparisons of the various selenium levels i l lustrated the nature of the significance (Figure 1). Selenium levels of 0.2 or 0.4 ppm did not s ignif icantly a lter weight gain from those birds fed basal (unsupplemented) diets. In fact, there was a sl ight but non-significant increase in weight gain in birds receiving 0.2 or 0.4 ppm Se in their diets. Supplement-ing the diet with 0.8 ppm selenium, however, resulted in a signif icant decline in weight gain when compared to control gains. This effect was evident in week 1, 2, and 3. During week 4, weight gain across a l l selenium levels (0, 0.2, 0.4, and 0.8 ppm) were similar. Results of four weeks in this t r i a l were consistent with the results obtained in a preliminary study conducted by the author. In this study, using 4,000 broilers raised to four weeks of] age on diets supplemented with either 0, 0.2, 0.4, or 0.8 ppm selenium, results indicated a non-signif icant difference in weight gain. The observed difference in weight gain, in the present study during weeks 1, 2, and 3 weeks, among birds fed similar selenium levels may have its basis in the inter-action between selenium and molybdenum-discussed later. A significant difference in weight gain was observed upon supplementing the diet with molybdenum at various levels (0, 0.5, 100, Soo-^ 48o\ V5 470 > to 450 J BASAL 0.5 100 300 MOLYBDENUM LEVELS FIGURE Z. EFFECT OF VARIOUS TOXIC AND SUBTOXIC DIETARY LEVELS OF M0LV60EMUM ON WEIGHT GRIN IN BROILERS AT THREE WEEKS OF AGE. 25 and 300 ppm). This effect was apparent from week 1 to week 4, i n -clusive. Table 2 summarizes these results. Figure 2 indicated this trend in birds at three weeks of age. Molybdenum at levels of basal, 0.5, or 100 ppm resulted in weight gain that was not s ignif icantly different from the f i r s t through to the fourth week of experimentation. Dietary levels of 300 ppm molybdenum, however, resulted in a 12% drop in weight gain that was s ignif icantly different from the weight gain at basal, 0.5, or 100 ppm Mo. This trend held true in birds from one to four weeks of age. During week 1 i t appeared that supplementing the diet with 0.5 ppm Mo resulted in reduced weight gain when compared to the basal diet weight gains. This event was also noted in week 2 but was not signif icant (based on SNK test). The indifferent gains observed when molybdenum levels ranged from basal to 100 ppm Mo is in agreement with previous l iterature (Kratzer, 1952). He showed that weight gain only appeared to commence declining after 200 ppm or more molybdenum was added to the diet. The observed decline in weight gain upon supplementing the diet with 0.5 ppm Mo is questionable as i t was only marginally apparent during week 1 and 2. Experimental/environmental error or variation cannot be ruled out here. There was a s ignif icant interaction between selenium and molyb-denum during weeks 1 and 2 (Table 2). The interaction was not signif icant during weeks 3 and 4, although there were definite inter-action trends during this time that were similar to those in weeks 1 and 2. Tables 3-6 examine the interaction subsets of the various 26 Table 3. Trial I,. Effects of Various Dietary Levels of Selenium with Various Toxic and Subtoxic Levels of Molybdenum Based on Weight Gain in Broilers at One Week of Age. 2 Means Diet Basal Selenium +0.2 p.p.m. Selenium +0.4 p.p.m. Selenium +0.8 p.p.m. Selenium Basal Molybdenum 88.52 b c d ' 90.58 b c d 95.62 d 87.30 b c d +0.5 p.p.m. Molybdenum 74.95 a b cd 91.85 C Q 96.18 d 77.80 a b c +100 p.p.m. Molybdenum 92.38 C d 82 22 ^^""^ 94.00 d 84.15 a b c d +300 p.p.m. Molybdenum 90.15 b c d 87.30 b c d 80.92 a b c d 71.68 a 1. Weight gain values which do not have the same subscript are s ignif icantly different, p _<0.05, based on Student-Newman-Keuls test. 2. Based on 2 observations per treatment. 27 Table 4. Tria l I, Effects of Various Dietary Levels of Selenium with Various Toxic and Subtoxic Levels of Molybdenum Based on Weight Gain in Broilers at Two Weeks. 2 Means Basal +0.2 p.p.m. +0.4 p.p.m. +0.8 p.p.m. Diet Selenium Selenium Selenium Selenium Basal 3 1 Molybdenum 263.2 a 267.3 3 280.4 a 251.5 a +0.5 p.p.m. Molybdenum 241.5 a 263.0 a 263.5 a 245.6 6 +100 p.p.m. Molybdenum 271.5 9 251.3 a 272.3 a 251.3 3 +300 p.p.m. Molybdenum 260.7 a 252.2 a 242.3 3 216.5 b 1. Weight gain values which do not have the same subscript are s ignif icantly different, p _< 0.05, based on Student-•Newman-Keuls test. 2. Based on 2 observations per treatment. 28 Table 5. Trial I, Effects of Various Dietary Levels of Selenium with Various Toxic and Subtoxic Levels of Molybdenum Based on Weight Gain in Broilers at Three Weeks. Means Diet Basal Selenium +0.2 p.p.m. Selenium +0.4 p.p.m. Selenium +0.8 p.p.m. Selenium Basal Molybdenum 524.8 b " 500.0 b 470.3 b 496.5 a b +0.5 p.p.m. Molybdenum 482.0 a b 507.0 b 508.2 b 481.0 a b +100 p.p.m. Molybdenum 490.0 b 486.6 a b 506.0 b 488.5 a b +300 p.p.m. Molybdenum 483.1 a b 483.2 a b 464.2 a b 429.5 a b 1. Weight gain values which do not have the same subscript are s ignif icantly different, p 0.05, based on Student-Newman-Keuls test. 2. Based on 2 observations per treatment. 29 Table 6. Trial I, Effects of Various Dietary Levels of Selenium with Various Toxic and Subtoxic Levels of Molybdenum Based on Weight Gain in Broilers at Four Weeks. 2 Means Diet Basal Selenium +0.2 p.p.m. Selenium +0.4 p.p.m. Selenium +0.08 p.p.m. Selenium Basal Molybdenum 798.0 b " 758.5 a b 790.5 b 787.0 b +0.5 p.p.m. Molybdenum 700.0 a b 757.0 a b 780.0 a b 776.5 a b +100 p.p.m. Molybdenum 763.8 a b 756.9 a b 772.8 a b 758.8 a b +300 p.p.m. Molybdenum 741.5 a b 751.5 a b 712.8 a b 660.8 a 1. Weight gain values which do not have the same subscript are s ignif icantly different, p _<0.05, based on Student-Newman-Keuls test. 2. Based on 2 observations per treatment. ioo 4 -1Q0 -7bO 730 W." 7oc 6<to o ***** / LEGEND / / BASAL AJo / / / / / 0,S" ppm Mo • • • • 100 pp>m ,t<tp xxxx 300 pfi* Mo / / / BASffL 0.1 <3.V 0.9 SELENIUM LEVELS FIGURE 3. EFFECT OF VARIOUS DIETR&Y LEVELS OF SELENIUM WITH VARIOUS TOXIC RNQ SUBTOXIC LEVELS OF MOLSB0ENOM BRSEO OA/ WEIGHT GRIN M &R01LERS AT FOUR WEEKS OF AGE. Co o 31 selenium and molybdenum combinations on weight gain. Over molybdenum basal, 0.5, and 100 ppm levels, selenium does not appear to exert a signif icant influence on weight gain. At molybdenum basal, 0.5,and 100 ppm levels, selenium does not appear to have a signif icant in -fluence on weight gain. Molybdenum basal, 0.5, and 100 ppm were v irtual ly non-significant from weeks 1 to 4 of the experimental period. Weight gain for the range of selenium (0, 0.2, 0.4, or 0.8 ppm) and molybdenum levels (0, 0.5, or 100 ppm) resulted in no significant difference in weight gain when compared to each other at these levels. Birds fed either basal, 0.5, or 100 ppm Mo followed the same weight gain pattern as the level of selenium increased. Molybdenum and selenium at these levels could have been compensated for by birds metabolism such that the overall effect on weight gain remains unchanged. At dietary levels of 300 ppm Mo, however, as the selenium levels i n -creased from basal, to 0.2, 0.4, and 0.8 ppm, the weight gain pro-gressively decreased. At this level of molybdenum, selenium appeared to be acting antagonistically with the effect of molybdenum, resulting in a net adverse effect on weight gain. This effect was observed from weeks 1 to 4 inclusive. Figure 3 i l lustrates this effect at 4 weeks. The detrimental effects of feeding toxic levels of molybdenum appear to be compounded by increasing levels of selenium. Furthermore, selenium now appears to be exerting a toxic effect where i t previously had had non effect on weight gain. The presence of molybdenum at 300 ppm resulted in a lowered threshold for selenium toxicity. Levels of 0.4 - 0.8 ppm selenium were now toxic. Toxicity normally arises at levels of selenium beyond 4 ppm in the diet (Puis, 1978). 32-These results of Trial I are somewhat consistent with those of Moxon and DuBois (1939). These researchers noted that feeding 11 ppm selen-ium in the diet as seleniferous wheat when combined with 5 ppm molybdenum resulted in increased mortality when compared to controls. Selenium toxicity was enhanced by feeding molybdenum at otherwise non-toxic levels. There was a block effect during week 4 of the experimental period. Birds in Block B gained 2.6% more than birds in Block A. This block effect could have occurred for one or more of the follow-ing reasons: (1) the water troughs may contributed by contamination to other minerals, such as iron. Iron may indirectly a lter the requirement for selenium or molybdenum; and (2) the experiment was performed in February, which was during cold weather. Block A was located near the outside vent which received more cold a i r . When birds are chi l led they tend to huddle under the heaters. This could result in two things: i) they don't eat when under the heaters, and i i ) they expand more energy in keeping warm, therefore lessening the available energy for growth. This block effect resulted in Treatment X Block two-way inter-actions in week 4 which could be accounted for by the previously described events but was not compounded by treatment level and type. During weeks 1, 2, and 3 the block effect was not signif icant (Table 2). From weeks 1 to 4 male birds gained more than the female birds under equivalent treatments. Male birds normally gain more than female birds (Sturke, 1976). Table 7. Trial I, Analysis of Variance Summary Table for Feed Consumption During Weeks 1, 2, 3, and 4 of the Experimental Period. Source of Mean c 2 Square Variation D.F. Week 1 Week 2 Week 3 Week 4 Molybdenum, Mo 3 73.70 683.86 3,444.10 7,908.80 Selenium, Se 3 162.85 390.22 946.23 577.05 Se X Mo 9 303.50 385.87 697.22 3,373.80 Error 16 244.60 271.51 814.55 4,296.20 1. Two Factor ANOVA, G. W. Iowa. pps. 338-343. Snedecor, Statist ical Methods. Iowa State College Press, Ames, 2. Indicates level of significance, 5% ( ). ,34 Feed Consumption Increasing dietary levels of selenium from basal to 0.2, 0.4 and 0.8 ppm Se resulted in no signif icant difference in feed con-sumption among birds in these treatments over the entire experimental period (Table 7)... Conversely, weight gain analysis showed a s i g n i f i -cant difference in weight gain as the levels of selenium increased, especially selenium levels beyond 0.8 ppm. At these levels of selenium i t appeared that birds are able to metabolize the varying levels of selenium and maintain optimal weight gain without altering feed intake. Stat ist ical analysis of the feed consumption data indicated a non-significant difference in intakes as the level of molybdenum increased from basal to 0.5, 100, and 300 ppm Mo during weeks 1, 2, and 4 (Table .7). During week 3 there was a signif icant difference in feed consumption. Birds fed 300 ppm molybdenum ate s ignif icantly less than control birds or birds fed 100 ppm Mo, based on SNK com-parisons. This trend was also observed during weeks 1, 2, and 4 but was not s ignif icant. Week 3 results for feed consumption were consistent with those of weight gain results. Here as molybdenum levels increased beyond 100 ppm weight gain progressively decreased (Figure T). In an effort to cope with toxic levels of molybdenum birds reduce their feed consumption (Ewing, 1947). The interaction between selenium and molybdenum based on feed consumption was not s ignif icantly different. Birds fed these dietary treatment of molybdenum and selenium were able to maintain normal (basal) diet feed consumption. They were not as ef f ic ient in . 3 6 maintaining weight gain. Weight gain analysis indicates a signif icant interaction between selenium and molybdenum during weeks 1 and 2. Thus i t appears that the birds ab i l i t y to optimalty u t i l i ze the diet was affected by high dietary levels of molybdenum and selenium as feed consumption is not s ignif icantly reduced by the diet imposed. Furthermore, any feed tending to upset normal digestion, thereby putting the bird out of condition cannot r ightful ly be c lass i f ied as unpalatable, since the lower feed consumption is a direct result of the effect on the bird rather than the palatabi l i ty or lack of palatabi l i ty of the feed (Ewing, 1947). Table 8. Tria l II, Analysis of Variance Summary Table for Weight Gain for Weeks, 1, 2, 3, and 4 of the Experimental Period. Source of Variation D.F. Mean Square 2 Week 1 Week 2 Week 3 Week 4 Selenium, Se 2 139.39 13,471.00* 48,979.00* 403,609.* Molybdenum, Mo 3 1,637.30* 9,060.80* 75,125.00* 478,290.* Block, B 3 257.34 649.18 564.45 18,811. Sex, Z 1 2,275.80* 29,296.00* 198,950.00* 1,271.900.* Se X Mo 6 227.16 731.73 4,769.20 107,300. Se X B 6 120.50 1,684.40 2,447.80 96,493. Se X Z 9 261.39* 1,056.80 1,154.50 89,379. Mo X B 2 197.64 39.16 848.89 137,760. Mo X Z 3 101.94 154.22 829.95 121,450. B X Z 3 200.86 2,308.40 1,016.10 77,088. Se X Mo X B 18 179.11 1,258.80 5,034.00* 97,162. Se X Mo X Z 6 83.37 732.20 4,787.70 124,390. Se X B X Z 6 140.90 2,500.10* 3,632.10 100,130. Mo X B X Z 9 140.38 1,382.90 1 ,780.10 93,438. Se X Mo X B X Z 18 127.66 986.88 2,458.60 95,088. Error 320 134.77 1 ,032.40 3,032.40 92,747. 1. Multifactorial Analysis, Sokal, R. R. and F. J . Rolf, 1969. Biometry. W. H. Freedman and Company, U.S.A. pps. 346-362. 2. Indicates level of significance, 5% ( ). 440 43o + 420 4 4io 4oo oo J i — , — I i — , — I 1 — . — 1 o BAS0L 3 4> SELENIUM LEVELS C ppm) FIGURE H. EFFECT OF VARIOUS TOXIC LEVELS OF SELENIUM ON WEIGHT GAIN jN BIRDS AT THKEE WEEKS OF AGE. 38 Trial II Weight Gain Table 8. summarizes the effects of selenium,molybdenum, block, and sex and a l l the interaction components from one to four weeks of the experimental period. Selenium at supplemented, dietary levels of 0, 3, and 6 ppm resulted in weight gain among birds in these treatments that was not signficantly different during the f i r s t week of analysis. During the f i r s t few days of chick development post-hatch the yolk sac is being reabsorbed and consistutes a non-treatment source of nutrients. It seems plausible therefore, that the diet imposed during this period would not constitute the sole influence on one week weight gain. Weight gain was s ignif icantly different by the second week. Figure 4\ i l lustrates that at 6 ppm selenium weight gain was less than/that at 3 ppm and this inturn was less than weight gain at basal levels of selenium. Selenium at basal and 3 ppm supplemented selenium showed no difference in weight gain^based on SNK comparisons. This same trend in weight gain was again noted at 3 weeks in the experimental period. Prior to week 4 weight gain analysis, birds fed diets containing 3 ppm supplemented selenium gained the same as birds fed basal diets. During week 4, birds fed the 3 ppm Se diet appeared to gain more than birds on the basal diet. Munsell et al_. (1936) suggested that selenium levels greater than 3-4 ppm Se in the diet are considered to have an adverse effect on weight gain. Thus a progressive decline in weight gain was expected as the selenium level in the diet increased from basal to 3 and 6 ppm 39 o o "0 Co O 5 §: _• UJ s CO o I T . 8 C5 V6 4W4 4i°l o 4(0 4 o /OO S 4 0 L i & D E N V M 2oo Levees Cppm) FIG Ode 6 , EFFECT OF VARIOUS TOXIC AND SUBTOXIC LEVELS ON WE I GMT 6f)/N IN 6&0ILFR.S THREE WEEKS OF A&E. 300 MOLYBPEAJOM AT 41 Se. There are several explanations for the observations made in this t r i a l : (1) the amount of exogenous selenium required to reduce a selenium toxicity or meet a selenium requirement is dependent on the endogenous amount of selenium; (2) requirement and toxicity levels are also dependent on other mineral profi les in the diet. Mineral imbalances can lead to increased requirements (Jensen, 1975b); (3) broiler diets are protein rich formulations which may affect selenium requirements in two ways: i) high protein reduces selenium toxicity (Smith, 1939); and, i i ) the birds grow faster and therefore have an increased need for selenium; and, (4) experimental error in the data analysis cannot be ruled out. Figure 8 indicates this trend for increased need as time progress in the experimental period. Broiler chicks fed molybdenum supplemented diets ranging in level from basal, 100, 200 to 300 ppm Mo resulted in weight gain that progressively decreased as molybdenum levels increased during weeks 1 and 2. Figure 5 i l lustrates this trend. Results are consistent with those obtained by Kratzer (1952) who showed that as molybdenum levels increased beyond 200 ppm dietary Mo, weight gain progressively de-creased. At week 3 the drop in weight gain became more pronounced as molybdenum levels increased. Figure 6 i l lustrates this trend. Chicks on the basal and 100 ppm molybdenum diets gained 458.9 gm and 432.1 gm respectively, while birds on 200 ppm and 300 ppm molybdenum diets gained at a reduced level (401.7 gm and 400.2 gm, respectively). The latter two gains were not s ignif icantly different during weeks 2 and 3. 42 o tn o o o g — t — e 3 43 Table 9. Trial II, Effect of Various Toxic and Subtoxic Dietary Levels of Selenium and Molybdenum on Weight Gain in Broilers at One Week of Age. Means Diet Basal Selenium +3 p.p.m. Selenium + 6 p.p.m. Selenium Basal Molybdenum 88.85+13 .23 c 82.08+11. 5 g abc 84.74+9.68bc +100 p.p.m. Molybdenum 82.34+10 . 8 5 a b c 84.34+10. 97 b c 79.48+11.80a +200 p.p.m. Molybdenum 77.73+15 .74 a b 79.86+12. n a b 78.38+11.16ab +300 p.p.m. Molybdenum 74.20+13 .55 a 75.75+12. 25 a b 75.51+10.97ab 1 . Weight gain values which do not have the same subscript are signif icantly different, p. _<0.05, based on Student-Newman-Keuls test. 2. Based on a minimum of 36 observations per treatment. 44 Table 10. Trial II, Effect of Various Toxic and Subtoxic Dietary Levels of Selenium and Molybdenum on Weight Gain in Broilers at Two Weeks. 2 Means Diet Basal Selenium +3 p.p.m. Selenium +6 p.p.m. Selenium Basal Molybdenum 251.1+32. c1 ,c 239.2+31. Qbc 233.2+25. t-bc 0 +100 p.p.m. Molybdenum 240.7+24. gbc 239.2+25. gbc 222.5+33. 5 a b +200 p.p.m. Molybdenum 224.9+32. 7abc 232.7+26. gbC 213.4+34. 2 a +300 p.p.m. Molybdenum 228.6+62. 4abc 221.5+31. Qabc 207.5+27. 3 a 1. Weight gain values which do not have the same subscript are s ignif icantly different, p. _< 0.05, based on Student-Newman-Keuls test. 2. Based on a minimum of 36 observations per treatment. 45 Table 11. Trial II, Effect of Various Toxic and Subtoxic Dietary Levels of Selenium and Molybdenum on Weight Gain in Broilers at Three Weeks of Age. Means Diet Basal Selenium +3 p.p.m. Selenium +6 p.p.m. Selenium Basal Molybdenum 483.2+55. y 457.3+48. 6 C 437.1+50. 2bc +100 p.p.m. Molybdenum 442.1+46. 437.8+72. ^bc 415.0+66. 4ab +200 p.p.m. Molybdenum 405.4+55, 2ab 423.4+54. ^ bc 376.9+69. 4 a -+300 p.p.m. Molybdenum 407.0+62 . 3 a b 407.8+60. 9 4abc 378.3+57. ,2 a 1. Weight gain values which do not have the same subscript are s ignif icantly different, p. j<0.05, based on Student-Newman-Keuls test. 2. Based on a minimum of 36 observations per treatment. -46 Table 12. Trial II, Effect of Various Toxic and Subtoxic Dietary Levels of Selenium and Molybdenum on Weight Gain in Broilers at Four Weeks of Age. 2 Means Diet Basal Selenium +3 p.p.m. Selenium +6 p.p.m. Selenium Basal Molybdenum 750.6+85.6 a t ) 1 ; 706.5+82.l a b 685.1+79.5ab +100 p.p.m. Molybdenum 702.5+81.8ab 901.1+1056.7b 654.8+110.7ab +200 p.p.m. Molybdenum 615.9+91.8a 665.2+84.7ab 558.7+120.5a +300 p.p.m. Molybdenum 626.7+101.3a 631.9+100.5ab 557.2+99.4a 1. Weight gain values which do not have the same subscript are s ignif icantly different, p _< 0.05, based on Student-Newman-Keuls test. 2. Based on a minimum of 36 observations per treatment. 3 ppi* Se . . . . . 6 ppm Se — i — loo — f FIQURE 8. 200 30O MOLYBDENUM LEVELS Cpfm) EFFECT OF VAKlOOS TOXIC AND SUBTOXIC LEVELS OF SELENIUM I9AIO MOLYBDENUM ON lAl£l6HT 6A/M /N B201LERS AT FOVtt WEEKS OF A&E. 48 -t 1 1 1 1 1 \ k 1 1 1 1 r+ 49 By week 4 (Figure 7) weight gain at 100 ppm molybdenum was greater than basal weight gain (751.3 gm and 712.0 gm, respectively), there-fore feeding chicks 100 ppm molybdenum to four weeks of age resulted in greater weight gain ppm molybdenum produced beneficial effect by increasing weight gain. Stat ist ical analysis of the interaction between molybdenum and selenium indicates a non-significant interaction source of variation from week 1 through to week 4 (Table 8). Upon closer analysis of the interaction data, results demonstrated selenium-molybdenum interaction trends based on SNK analysis. Tables 9-12 presented these trends. Graphing the interaction data allows for visual observation of these interaction trends. For example, Figure 8 i l lustrates that birds fed 3 ppm dietary selenium and 100 ppm molybdenum to 4 weeks resulted in an increase in weight gain which exceeded that of basal Se-Mo weight gain. The observed effect for 3 ppm Se-100 ppm Mo represented a marked interaction that may s i gn i f i -cantly a lter the main effect weight gain for selenium or molybdenum, and provided an explanation for the observed beneficial effect on weight gain upon feeding Se or Mo at these levels. The poss ibi l i ty also exists that this observed interaction trend in Figure 8 is an anomally as i t was not observed during weeks 1, 2, or 3 (Tables 9, 10, or 11, respectively). Figure 9 shows the interaction trends at week 3 of the experi-mental period, week 1 and 2 trends are similar. During this period the results for the interaction between selenium and molydbenum indicated 50 a non-significant difference in weight gain upon feeding basal or 3 ppm selenium over the molybdenum levels ranging from basal to 300 ppm. At these levels, molybdenum and selenium appeared to be interacting reciprocally, resulting in similar weight gains. Molybdenum appeared to be altering the effects of selenium. With no selenium present weight gain appeared to decline in a l inear fashion as molybdenum levels increased. When 3 ppm selenium was added to the diet, weight gain was reduced to less than selenium basal gains but as molybdenum levels increased the adverse effect of 3 ppm selenium in the diet was nu l l i f i ed . Weight gain at 3 ppm Se became the same as basal Se weight gains over molybdenum levels ranging from 100, 200, to 300 ppm. At 6 ppm selenium in the diet, combining this level of selenium with increasing levels of molybdenum resulted in an independent toxic effect of one mineral which was additive, and therefore not inter-active with the other mineral. Molybdenum over these levels does not appear to be influencing selenium weight gains, or visa versa. Stat ist ical analysis of the sex effect during the experimental period showed that males gained s ignif icantly more weight than females over a given time period. During week 1 males gained 7% more than females, during week 2, 8% more, during week 3, 11% more, and during week 4, 16% more. The block effect was non-significantly during the experimental period, indicating that weight gain for treatment and sex in one block did not d i f fer from that observed in another block. Two- and three-way interaction sources of variation were not s ignif icantly different for each of the four weeks of study with the 51 exception of a selenium X block X sex e f fect in week 2 and a selenium X molybdenum X block e f fect in week 3. Refer to Table 8 for s ign i f icance leve l s . Table 13. Trial II, Analysis of Variance Summary Table for Feed Consumption During Weeks 1, 2, 3 and 4 of the Experimental Period. Source of Mean Square' c Variation D.F. Week 1 Week 2 Week 3 Week 4 Selenium, Se 2 20.9 1,989.5* 13,198.0* 60,590.0* Molybdenum, Mo 3 * 197.6 2,663.8* 9,444.1* 26,231.0* Sex, Mo 6 51.3 287.4 682.8 20,403.0* Error 36 54.4 351.7 1 ,482.1 3,122.4* 1. Two Factor AN0VA, G. W. Snedecor, Statist ical Methods. Iowa State College. Press, Ames, Iowa, pps. 338-343. * 2. Indicates level of significance, 5% ( ). 53 Table 14. Trial II, Effect of Various Toxic Dietary Levels of Selenium and Molybdenum on Feed Consumption in Broilers at Four Weeks. Mean ± Standard Deviation 2 i Diet Basal Selenium +3 p.p.m. Selenium +6 p.p.m. Selenium Basal Molybdenum 1331.0+52.0cd 1189.0+57 .4 b 1263.0+56 .obcd +100 p.p.m. Molybdenum 1277.0+97.4b c d 1350.0+39 .9 d 1215.0+38 .obc +200 p.p.m. Molybdenum 1221.0+47.9bc 1317.+77. 9cd 1101.0+42 .9 a +300 p.p.m. Molybdenum 1249.0+30.3b c d 1213.0+53 . 6 b c 1069.0+42 .5 a 1. Feed consumption values which do not have the same subscript as s ignif icantly different, p _<0.05, based on Student-Newman-Keuls test. 2. Based on 4 observations per treatment. 54 Table 13 summarizes the effect of selenium and molybdenum on feed consumption in Trial II. During weeks 2, 3, and 4 the selenium effect was s ignif icant. As the selenium level increased from basal to 3 and 6 ppm, feed consumption progressively decreased. During week 4, feed consumption at basal silenium levels equalled that at 3 ppm Se levels. Stat ist ical analysis of the molybdenum treatments indicated that as molybdenum levels from basal, to 100, 200,and 300 ppm resulted in a s ignif icant difference in feed consumption from 1 to 4 weeks of the experimental period. Closer observation of the molybdenum trends were performed by SNK comparisons. This revealed that feed consumption from basal to 200 ppm were not s ignif icantly different during weeks 1 and 2. By week 3, basal and 100 ppm Mo fed birds exhibited a feed consumption that was the same. At week 4, 100 ppm Mo feed consumption actually exceeded basal, although this was not s ignif icantly different. During weeks 3 and 4, levels of 200 and 300 ppm Mo resulted in progressively reduced feed consumptions when compared to basal and 100 ppm diets. The selenium-molybdenum interaction effects on feed consumption were only s ignif icantly different during week 4 of the experimental period. (Table 14). These results indicate that at 3 ppm selenium feed con-sumption was reduced s ignif icantly from those on basal diets. Supple-menting these diets with 100 ppm Mo, however, resulted in increased feed consumption which was not s ignif icantly different from birds on basal Mo and Se diets. Results for selenium, molybdenum, and selenium-molyb-denum interaction effects follow the same trends as those for the equivalent weight gain responsed discussed in Tria l II. Table 15. Trial III, Analysis of Variance Summary Table for Weight Gain for Weeks, 1, 2, 3, and 4 of the Experimental Period. Source of Variation D.F. 2 Mean Square Week 1 Week 2 Week 3 Week 4 Molybdenum, Mo 2 12,493.00* 94,339.00* 384,470.00* 1,059,300.00* Selenium, Se 3 29,430.00* 406,000.00* 1,668,400.00* 4,139,000.00* Block, B 3 348.73 702.54 909.48 1,178.30 Sex, Z 1 367.41* 3,849.00* 29,547.00* 48,367.00* Mo X Se * * * * 6 2,207.10 17,619.00 76,966.00 185,680.00 Mo X B 6 277.26 1,311.70 1,812.20 1,638.60 Mo X Z 2 488.86* 3,462.40* 9,305.20* 10,464.00 Se X B 9 154.86 743.03 1,704.20 7,354.00 Se X Z 3 68.35 918.00 4,601.90 12,154.00 B X Z 3 178.76 620.43 1,848.00 5,696.40 Mo X Se X B 18 161.66 1,002.50 2,552.30 7,073.80 Mo X Se X Z 6 60.10 544.49 1 ,520.80 2,806.40 Mo X B X Z 6 128.06 732.81 2,898.10 6,259.60 Se X B X Z 9 114.53 930.05 1,682.00 4,647.70 Mo X Se X B X Z 18 92.39 762.45 2,288.80 6,064.70 Error 362 138.24 970.51 2,776.60 6,870.80 1. Multifactorial Analysis, Sokal, R. R. and F. J . Rolf, 1969. Biometry. W. H. Freedman and Company, U.S.A. pps. 346-362. 2. Indicates level of significance, 5% ( ). _ si — 3oo + 2oo 4 loo-\ 4-BrV?HL FIGURE 10. 4oo MOLS SOENUM 8oo LEVELS Cppm') EFFECT OF VARIOUS TOYIC LEVELS OF MOLSBOENUM ON WEIGHT GRIN IN BROILERS AT TARES WEEUS OF A&E. 57 Trial III Weight Gain Table 15 summarizes the effects of selenium, molybdenum, block, and sex and a l l their interaction components from one to four weeks of the experimental period. Stat ist ical analysis of the data indicates a significant difference in weight gain in chicks upon feeding different levels of molybdenum ranging from basal, to 400 ppm and 800 ppm. Figure 10 i l lustrates these effects at three weeks. As the molybdenum levels increased weight gain decreased in a l inear fashion. These results are consistent with those obtained by Kratzer (1952) who noted a 25% reduction in weight gain in chicks, started on treat-ment at one week of age, subject to 300 ppm dietary molybdenum during the second and third weeks. In this experiment a 19.7% drop in weight gain was observed at 400 ppm molybdenum and a 36.7% reduction in weight gain at 800 ppm molybdenum supplemented in the diet to birds from hatch to four weeks of age when compared to control birds. Intakes of at least 2,000 ppm molybdenum are needed to induce severe growth depression in chicks and this is accompanied by anemia when dietary levels are raised to 4,000 ppm Mo (Arthur et al_. 1953, and Davis, et al_. 1960). Results of this study show that birds on the 800 ppm Mo diet were poorly developed and growth was retarded when compared to control birds. Physically, four week old birds looked similar to normal birds which were two,and a half weeks old. Other than retarded develop-ment, birds on high dietary levels of molybdenum displayed no other notable 58 4-w CM _ 5 CD 5 8 o SWV&S> HI Mb/9 'J.M 9AV 59 physical abnormalities. Increasing selenium in the control diet from basal to 6, 12sand 18 ppm Se resulted in a signif icant difference in weight gain evident during weeks, 1, 2, 3, and 4 (Table 15). Figure 11 shows this effect at 4 weeks. Increasing the dietary supplementation of selenium result-ed in a progressive decline in weight gain at 1, 2, 3, and 4 weeks. Munsell et al_. (1936) concluded that toxicity signs would ultimately develop at dietary selenium levels of 3-4 ppm. Puis (1978) in a l iterature review concluded that diets containing greater than 3 ppm selenium were considered toxic for poultry. Jensen (1975b) induced 7-10% mortality in broi lers, at 2 weeks of age, by feeding them diets containing 20 ppm selenium (fed as sodium selenite), and a 40% mortability by feeding birds 40 ppm dietary selenium. Mortality in the control birds of this experiment was 3%. Feeding 10 ppm selenium in this experiment resulted in 5% mortality which was not s i gn i f i -cantly different from basal or 20 ppm Se diets. Based on Jensen's results (1975b) i t was assumed in the present study, that supplementing the diet with 18 ppm selenium would be sublethal. It was hypothesized that by subjecting birds to sublethal levels of selenium and toxic levels of molybdenum addit ivity of the two mineral toxic it ies would result in increased mortality. Addit i -vity of the two mineral toxicit ies would indicate an independent behavior of the selenium or molybdenum effects. The corollary, non-addit iv ity between the effects of the two minerals, would indicate an interaction. Analysis of variance (Table 15) indicate that a significant 60 8 | | | S SHVdO Nl WW UA1 '9W 61 62. o o EG o o ft. _ -a CO > o -J CO O VO > >1 u g to >. o a >-51 4 63 Table 16. Tria l III, Effects of Toxic Dietary Levels of Selenium and Molybdenum Based on Weight Gain in Broilers at One Week of Age. Means Diet Basal Molybdenum +400 p.p.m. Molybdenum +800 p.p.m. Molybdenum Basal Selenium 82.59+9.96T 64.98+16.01de 43.99+11.40b +6 p.p.m. Selenium 69.26+16.97e 60.51+12.87d 50.80+12.70C +12 p.p.m. Selenium 49.85+9.72° 40.47+12.03b 37.74+10.41b +18 p.p.m. Selenium 31.47+8.923 28.26+8.27a 27.60+9.79a 1. Weight gain values which do not have the same subscript are s ignif icantly different, p. < 0.05, based on Student-Newman-Keuls test. 2. Based on a minimum of 36 observations per treatment. 64 Table 17. Trial III, Effects of Toxic Dietary Levels of Selenium and Molybdenum Based on Weight Gain in Broilers at Two Weeks of Age. 2 Means Diet Basal Molybdenum +400 p.p.m. Molybdenum +800 p.p.m. Molybdenum Basal Selenium f1 250.9+23.9 204.6+39.9d 141.6+33.6c +6 p.p.m. Selenium 198.5+39.2d 191.3+35.8d 152.6+35.2° +12 p.p.m. Selenium 127.6+33.0e 110.2+27.5b 96.6+27.8b +18 p.p.m. Selenium 80.0+22.2a 60.7+20.la 64.7+25.03 1. Weight gain values which do not have the same subscript are s ignif icantly different, p < 0.05, based on Student-Newman-Keuls test. 2. Based on a minimum of 36 observations per treatment. 65 Table 18. Trial III, Effects of Toxic Dietary Levels of Selenium and Molybdenum Based on Weight Gain in Broilers at Three Weeks of Age. Means Diet Basal Molybdenum +400 p.p.m. Molybdenum +800 p.p.m. Molybdenum Basal Selenium 495.0+44.8""1 394.8+72.4d 274.4+58.7° +6 p.p.m. Selenium 378.5+67.ld 314.4+53.5h 270.0+52.5° +12 p.p.m. Selenium 225.4+51.19 200.9+40.8f 174.1+51.5e +18 p.p.m. Selenium 137.9+44.lb 99.71+35.5a 114.4+45.9ab 1. Weight gain values which do not have the same subscript are s ignif icantly different, p _< 0.05, based on Student-Newman-Keuls test. 2. Based on a minimum of 36 observations per treatment. 66 Table 19. Trial III, Effect of Toxic Dietary Levels of Selenium and Molybdenum Based.on Weight Gain in Broilers at Four Weeks of Age. Basal +400 pTp.m. +800 p.p.m. Diet Molybdenum Molybdenum Molybdenum Basal Selenium 770.7+65.X 601.6+114. l f 440.5+91.l d e +6 p.p.m. Selenium 623.0+95.8f 458.7+96. l e 403.6+87.2d +12 p.p.m. —i Selenium 348.9+79.1° 301.1+73.8° 267.9+75.19 +18 p.p.m. Selenium 206.2+66.4b 150.5+58.9a 175.7+66.8ab 1. Weight gain values which do not have the same subscript are s ignif icantly different, p. _< 0.05, based on Student-Newman-Keuls test. 2. Based on a minimum of 36 observations per treatment. 67 interaction exists between molydbenum and selenium from one through to the four week experimental period based on comparative weight gain. Plotting the mean values of the interaction subsets, in graphical form fac i l i ta tes visual observation of the trends which make up the overall interaction effect. For example, at any part i -cular selenium level one may observe the interaction effect due to molybdenum (Figure 12), or visa versa (Figure 13). Student-Newman-Keuls multiple comparison test of the interaction substantiates their significance (Tables 16-19). The selenium-molybdenum interaction is present in birds by the f i r s t week of the experimental period. By the fourth week the following interactions between selenium and molybdenum were observed. Table 19 results of weight gain for selenium basal over various molybdenum levels coraborates with molybdenum main effects noted in Figure 14. In this case, as molybdenum levels increase the basal to 400 and 800 ppm Mo over selenium basal, weight gain progressively decreased. This event is consistent from one to four weeks of analysis. S imi lar i ly, at 6 ppm selenium, weight gain declined as molybdenum levels increased from basal, 400 and 800 ppm Mo over a l l weeks, but to a lesser extent than at selenium control over these molybdenum levels. At 6 ppm Se the rate of decline is reduced as the molybdenum levels increased. At 6 ppm Se-800 ppm Mo weight gain.was not s ignif icantly different from Se basal-800 ppm Mo at 2, 3, and 4 weeks (Tables 17, 18, and 19). And, at one week of age (Table 16) chicks fed 6 ppm Se-800 ppm Mo actually gained more than Se basal-800 ppm Mo birds. 68 69 70 Birds fed a diet containing 12 ppm selenium gained less, overal l , than birds fed the basal or 6 ppm Se based diets, but at 12 ppm Se adding 400 or 800 ppm Mo resulted in only marginal reductions in weight gain. The same trend between selenium and molybdenum held true from one to four weeks of analysis. Final ly, with diets containing 18 ppm selenium, weight gain was the same across a l l molydbenum levels from weeks 1 to 4 inclusive (Tables 14-16). Selenium exerts an effect by altering the toxicity of molybdenum, this effect became more apparent as the level of selenium increased. At 18 ppm selenium, gains were the same irrespective of whether basal, 400 or 800 ppm Mo was superimposed. Figure 15 i l lustrates this. Molybdenum no longer exerted an in -dependent toxic effect of i ts own. Weight gain at 18 ppm Se-800 ppm Mo was in fact greater than 18 ppm Se-400 ppm Mo which were both less, but not s ignif icantly different than 18 ppm Se- Mo basal. In summary then, the toxic effects of molybdenum and selenium appeared to be non-additive. Although increasing molybdenum from control to 400 and 800 ppm Mo resulted in reduced gains, and likewise, increasing selenium from basal, to 6, 12, and 18 ppm resulted in reduced gains, a combination of the two minerals in the diet did not result in an additive decrease in weight gain. In fact, at 18 ppm selenium adding either control, 400 or 800 ppm molybdenum resulted in similar gains (Figure 16). At these levels, birds show indiscrimin-ate retarded growth and development. Results indicated that the interaction or interrelationship 71 between selenium and molybdenum is manifested on a physical basis by altering weight gain. Also, the presence of toxic levels of selenium reduced to nu l l i fy toxicity of molybdenum, or induced an increased tolerance for increasingly toxic levels of molybdenum. Selenium appears to be preferentially absorbed over molybdenum at these high levels (18 ppm Se). At lower levels of selenium (6 ppm Se) molybdenum appeared more active in the competition with selenium. In fact, weight gain at 6 ppm Se-800 ppm Mo was greater than basal Se-800 Mo during week 1, and not s ignif icantly different in weeks 2, 3, and 4. This effect was also somewhat evident at 6 ppm Se-400 ppm Mo and basal Se-400 ppm Mo. Figure 12 again i l lustrates this effect. The molybdenum-selenium interaction is dynamic - the observed influence of selenium upon molybdenum changes as the levels of these minerals changes. Molybdenum appeared more able to compete with selenium at low levels and have an ultimate joint influence with molybdenum on weight gain. Whereas, at higher selenium levels molybdenum's influence wavers, and at toxic selenium levels molyb-denum does not appear to exert a significant influence. The range of competition between molybdenum and selenium appears to be at a ratio of 80:1, Mo:Se. As this ratio becomes less, molybdenum is less effective on selenium. The concept of ratio was presented by Ganther e_t al_. (1972) who showed that a 1:1 molar ratio of mercury to selenium was most effective in preventing selenium toxic ity. H i l l (1974) also pro-vided evidence to explain the dynamics of mineral interactions by noting that both 500 and 32 ppm copper were effective in counter-72 acting selenosis, l ike ly resulted in a copper imbalance with other minerals and therefore reduced its beneficial effects. Based on the general nature of many minerals (copper, mercury, cadmium, or zinc) molybdenum appeared to interact within the in -testinal mucosa. This interaction was supported by - Starcher (1969) who discovered the mechanism of interaction between copper and cadmium by identifying a single metal-binding protein, metallothionine, which would bind with either copper, cadmium, or zinc and in so doing explained the competition for absorption. At present i t is unknown whether molybdenum competes for absorption at the same site on the binding protein for selenium. Support for this possible common selenium-molybdenum binding protein may rest in the s imilar ity of their electron configurations. H i l l et al_. (1979) and Matrone (1974) hypothesized that the electron con-figurations of elements is the major factor in determining the apparent nature of the interaction among minerals. For example, s i lver, cadmium, zinc, and iron a l l affect molybdenum and copper requirements (Underwood, 1977). Analysis of their electron configura-tions reveal that selenium, zinc, copper and iron a l l have a similar argon core of electrons. Furthermore, molybdenum, s i lver , cadmium, and mercury a l l have a similar krypton electron core base. It is known that selenium interacts with s i lver (Jensen, 1975b), cadmium (H i l l , 1974), and mercury (Ganther, 1972), and that zinc, copper, and cadmium have a single metal-binding protein (Starcher, 1969) in which they compete for absorption. Based on these chemical and bio-logical s imi lar i t ies i t seems plausible that molybdenum can interact Table 20. Trial III, Analysis of Variance Summary Table for Feed Consumption During Weeks 1, 2, 3, and 4 of the Experimental Period. Source of Mean Square 2 Variation D.F. Week 1 Week 2 Week 3 Week 4 Molybdenum, Mo 2 838.16* 18,795.0* 82,838.0* 210,200.8* Selenium, Se 3 3,285.50* 61,953.0* 321,090.0* 972,810.0* Mo X Se 6 220.16* 3,344.7* 17,776.0* 60,636.0* Error 36 43.27 661.1 2,664.4 3,643.8 1. Two Factor AN0VA, G. W. Senedecor, Statist ical Methods. Iowa State College Press. Ames, Iowa. pps. 338-343. * 2. Indicates level of significance, 5% ( ). 74 with selenium and compete for the single common metal - binding pro-tein. Feed Consumption Stat ist ical analysis of feed consumption data reveal a s i g n i f i -cant difference in feed consumption as selenium level increased. This event was observed in birds from one to four weeks of age Table 20). Birds fed basal (unsupplemented) levels of selenium ate more than birds fed 6 ppm.Se, which ate more than birds fed 12 ppm Se, which ate more than birds fed 18 ppm Se supplemented diets. This event was consistent with the weight gain results of Trai l III. In an effort for birds to compensate for these toxic dietary levels of selenium the net feed consumption was reduced to a level which allowed for metabolic turnover of the superfluous selenium. As molybdenum levels increased, feed consumption progressively decreased. This held true in birds from one to four weeks of age (Table 20). This trend was also noted in the weight gain analysis of Trial III. As dietary levels of molybdenum increased both feed consumption and weight gain decreased. Birds are less diet contain-ing toxic levels of molybdenum to avoid excess levels of molybdenum in their systems.and, in part, as a consequence of this they gain less weight. There exists a significant interaction between selenium and molybdenum for feed concumsed from week 1 to 4 inclusive. A summary table of the interaction subsets is presented in Table 21. Various molybdenum and selenium levels preferentially affect food consumption in a similar manner as to their affect on weight gain. That i s , as 75 Table 21. Effect of Various Toxic Dietary Levels of Selenium and Molybdenum on Feed Consumed by Broilers at Four Weeks. Study III. Mean + Standard Deviation Diet Basal Molybdenum +400 p.p.m Molybdenum +800 p.p.m. Molybdenum Basal Selenium 1,348.0+28.33e 1,115.0+46 .72d 826.7+36.90C +6 p.p.m. Selenium 1 ,157.0+46.46d 912.2+38 .16C 834.5+66.06° +12 p.p.m. Selenium 651.7+89.26b 642.1+81 .98 b 650.5+51.91b +18 p.p.m. Selenium 507.5+46.15a 447.7+60 .60 a . 433.2+79.18a 1. Feed consumption values which do not have the same subscript are s ignif icantly different, p j< 0.05, based on Student-Newman-Keuls test. 2. Based on 4 observations per treatment. 76 the level of selenium increased additional molybdenum becomes in -consequential in affecting food consumption. Birds ate the same at 12 ppm and 18 ppm selenium whether fed basal, 400 or 800 ppm molybdenum in the diet. This was noted at weeks!, 2, 3, and 4,also. At lower.levels of dietary selenium (basal and 6 ppm), i n -creasing molybdenum levels resulted in a decrease in feed consumed, from one to four weeks. It appeared that at higher levels of selenium (12 and 18 ppm) the birds were able to tolerate the higher levels of molybdenum or they could consume higher levels of molyb-denum and were not adversely affected by i t . These trends were con-sistent with those of weight gain. 77 General Discussion Franke (1934) demonstrated that high selenium content in plant foodstuffs of South Dakota was responsible for 'a lka l i disease' in cattle and other livestock. Alkali disease is a condition arising from chronic toxic ity which occurs when animals consume seleniferous plants containing 3-30 ppm selenium over a prolonged period of time (Oldfield, 1971). Early investigations of the 'a lkal i disease 1 re-vealed that arsenic, vanadium, molybdenum, and chromium as well as selenium were present in some so i l s , feeds, and waters from the toxic areas (Beath et a_K, 1937). The poss ib i l i ty that the combined effects of any of these other minerals would alter the selenium re-lated toxic i t ies was presented by Moxon and DuBois (1939). They sub-jected rats, f ive replicates per treatment, to dietary levels of 11 ppm selenium of selniferous wheat and combined with either 5 ppm of molybdenum, f luorine, chromium, vanadium, cadmium, zinc, cobalt, nickel, or uranium in the drinking water as water soluble salts. They noted that 5 ppm molybdenum with 11 ppm selenium resulted in increased mortality when compared with seleniferous-free wheat diets. They concluded that combining selenium and molybdenum resulted in increased mortality, but more important, that this effect may be an important factor in dealing with selenium related tox ic i t ies . The present studies also demonstrate a selenium-molybdenum interaction in poultry. Various levels of both selenium and molybdenum appeared to govern the type and extent of interaction observed;,, Tr ia l I results indicated that at dietary levels of 300 ppm Mo, weight gain progressively declined as the selenium levels increased 78 from basal to 0.2, 0.4, and 0.8 ppm. This is in contrast to a non-signif icant decline in weight gain across these selenium levels when no molybdenum was supplemented. This trend appeared to be similar to that observed by Moxon and DuBois (1939). At 300 ppm molybdenum, selenium appears to be acting antogonistically with molybdenum which resulted in a net adverse effect on weight gain. The detrimental effects of 300 ppm molybdenum are compounded by increasing levels of selenium. Furthermore, selenium now appears to be excerting a toxic effect where i t previously had no effect on weight gain. The presence of molybdenum appears to lower the threshold for the selenium toxicity. Levels of selenium ranging from 0.2 to 0.8 ppm dietary supple-mentation is quite common in present day feeding regimes. Poultrymen are now able to add up to 0.1 ppm Se in the feed. Furthermore, water supplementation with selenium is also common. Birds may ingest as much as 6 ppm dietary selenium via the drinking water. Thus, i f there was a molybdenum toxicity problem in the s o i l , feed, or water, detrimental effects oh weight gain and feed consumption in birds may be expected with these high selenium diets. Furthermore, this molyb-denum toxicity could be aggravated by low copper levels. Low copper could lead to an increase in available molybdenum. Trial II using basically the same molybdenum range as Trial I, investigated the effect of toxic and subtoxic levels of selenium over these molybdenum levels. Results indicated a dual response to selenium and molybdenum combinations. At either basal or 3 ppm selenium over molybdenum levels ranging from basal to 100, 200, or 79 300 ppm there was a non-significant difference in weight gain. At 6 ppm supplemented Se, however, combining increasing levels of molybdenum appears to result in an independent toxic effect on weight gain. This effect was additive for the two minerals, and therefore not interactive. At these levels of molybdenum and selen-ium at least three events may be taking place in the birds: (1) selenium and molybdenum were acting independent of each other, result-ing in an additive detrimental effect on weight gain; (2) at these mineral levels there was a tolerable balance between selenium and molybdenum uptake or u t i l i za t ion; and/or, (3) the minerals competed equally well for sites of absorption. Tr ia l III indicated that at extremely toxic levels of selenium, combining toxic dietary levels of molybdenum resulted in a measurable interaction based on weight gain and feed consumption. As the dietary selenium levels increased from 6, 12, to 18 ppm, the adverse effect of molybdenum toxicity at 400 and 800 ppm Mo became progressively reduced. Although increasing molybdenum from basal, to 400 and 800 ppm resulted in reduced gain, and likewise increasing selenium from basal, to 6, 12, and 18 ppm resulted in reduced gain, a combination of the two minerals in the diet did not result in an additive decrease in weight gain. At 18 ppm selenium weight gains were the same irrespective of whether basal, 400 or 800 ppm Mo was supplemented to the diet. Molybdenum no longer exerts an independent toxic effect of its own. The toxic effects of molybdenum and selenium appear to be non-additive. The presence of toxic levels of selenium reduced or nu l l i f ied the toxicity of molybdenum or induced an increased 80 <' tolerance for the increasingly toxic levels of molybdenum. Further-more, selenium may be preferentially absorbed over molybdenum at these levels. Molybdenum appears more able to compete with selenium at low levels and have an ultimate joint influence on weight gain. Whereas, at higher levels of selenium, the effect of molybdenums' influence wavers. At toxic levels of selenium, molybdenum does not appear to exert a significant influence. The dual nature of the molybdenum-selenium interrelationship over the various dietary levels of these minerals may be due to a number of factors: (1) there may be a common metal-binding protein which is able to cope with absorption of both mineral at low levels of each but at high levels one mineral is preferentially absorbed or more competitive than the other mineral for both absorption and u t i l i za t ion ; (2) excess selenium may be making Mo unavailable for absorption. Increasing molybdenum excretion therefore, reducing the molybdenum available for absorption (3) extremely toxic levels at selenium and molybdenum more l ike ly results in gross mineral im-balances; and/or (4) Se and Mo may be affecting balances of other minerals (for example, copper). Excess molybdenum may induce a copper deficiency. This may be thought of as a primary mineral response. Also, excess selenium may induce a copper deficiency -another example of a primary mineral response. I t ' i s possible that a three-way interaction exists between copper, molybdenum, and selenium. Here, the selenium-molybdenum interaction may be a primary, direct mineral interaction response or a secondary, indirect mineral interaction response (Figure 17). This theory is based, 81 COPPER SELENIUM ---MOLYBDENUM I or 2 Figure 17. Theoretical three-way interaction between selenium, molybdenum, and copper (dotted l ine) , based on known interactions of selenium with copper and copper with molybdenum (solid l ine) . 82 in part, on the existing copper, molybdenum, sulfur three-way inter-action (Mil ls, 1961), and the fact that selenium is similar to sulfur (Berzelius, 1817). Furthermore, selenium and copper have the same argon core base of electrons. 83 SUMMARY AND CONCLUSIONS The objectives of the present study were to demonstrate a selenium-molybdenum interaction in poultry. A hypothesis was set forth that selenium and molybdenum interact with each other and result in a measurable effect on weight gain and feed consumption in poultry. Trial I investigated the interaction of subtoxic selenium (0, 0.2, 0.4, and 0.8 ppm) with various toxic and subtoxic dietary levels of molybdenum (0, 0.5, 100, and 300 ppm), supplemented to a wheat based diet to broilers from one to four weeks of age. Three hundred and twenty chicks were assigned in a randomized block (RB) experimental design to 16 treatments. Treatments were allocated in a 4 x 4 factorial among two blocks and each sex. Results indicated that a 300 ppm molybdenum, increasing selenium levels resulted in a progressive de-cl ine in weight gain, compared to a non-significant relat ively non-signif icant decline across these selenium levels when no molybdenum was supplemented. At these levels of molybdenum, selenium appeared to be acting antagonistically with molybdenum. Selenium appeared to be acting antagonistically with molybdenum. These results were consist-ent with those of Moxon and DuBois (1939) using rats fed 5 ppm Mo and 11 ppm Se (as seleniferous wheat). Selenium at subtoxic levels (as in the above experiment) res-ponded different from selenium at toxic dietary levels over similar molybdenum levels. Trial II, using 480 broi ler chicks, assigned in a RD experimental design and 12 treatment combinations of selenium and molybdenum. Results indicated that at either basal or 3 ppm Se over 84 basal, 100, 200, or 300 ppm Mo, a non-significant difference in weight gain and feed consumption occurred. Selenium and molybdenum appeared to be interacting reciprocally. At 6 ppm dietary supplementation of selenium, however, combining increasing levels of molybdenum appeared to result in an independent toxic effect on weight gain which was additive for the two tox ic i t ies , and not interactive. Using 480 broi ler chicks assigned to a 3 x 4 x 3 x 2 multi-factorial arrangement of 12 treatments, an experiment was performed to investigate the effect on weight gain and feed consumption upon feeding toxic levels of both selenium and molybdenum. Selenium ranged from basal to 6, 12, and 18 ppm, and molybdenum from basal, to 400 and 800 ppm, supplemented to the diet. Treatments were arranged in a RB experi-mental design. Results indicated that combining toxic dietary levels of selenium and molybdenum resulted in a measurable interaction based on weight gain and feed consumption in birds from one to four weeks of age. As the toxic dietary levels of selenium increased from basal to 6, 12, and 18 ppm the adverse effect of molybdenum at basal, 400, and 800 ppm became progressively reduced. At 18 ppm selenium, weight gain and feed consumption were the same irrespective of wheather basal, 400, or 800 ppm Mo was supplemented to the diet. The presence of toxic levels of selenium appeared to reduce toxicity of molybdenum, or less l i ke ly , induce an increased tolerance for i n -creasingly toxic levels of molybdenum. The nature of the interaction between selenium and molybdenum is discussed. 85 BIBLIOGRAPHY Arthur, D., Motzok, I., and H. D. Bram'on. 1958. Interaction of dietary copper and molybdenum in rations fed to poultry. Poultry Sc i . , 37:1181. (Abst.). Beath, 0. A., Eppson, H. F., and C. S. Gilbert. 1937. Selenium distribution in and seasonal variation of type vegetation occurring on seleniferous so i l s . J . Am. Pharm. Assoc. 26:394-405. Bertrand, G. 1912. Proc. Int. Congr. Appl. Chem. 8th, 28:30. Berzelius, J . J . 1817. Sur dex metaux nouveaus (litium and selenium). J . Schweigger, 21:1818-1823. Cotzias, G. C. 1967. Trace substances in environmental health. Proc. Univ. Mo. Annu. Conf., 1st, 5. Davis, R. E., Reid, B. L., Kurnich, A. A., and J . R. Couch. 1960. Sulfate on molybdenum toxicity in the chick. J . Nutri. 70:193-197. DeRenzo, E. C , Kaleita, E., Heyther, P., Oleson, J . J . , Hutchings, B. L., and J . H. Williams. 1953. Nature of the xanthine oxidase factor. J . Am. Chem. Soc. 75:753. Dick, A. T. 1954. Studies on the assimilation and storage of copper in crossbred sheep. Aust. J . Agri. Res. 5:511-544. Ewing, W. R. 1947. Poultry Nutrition, Third Edition. J . J . L i t t l e and Ives Co., New York. Franke, K. W. 1934. A new toxicant occurring naturally in certain samples of plant feedstuffs. 1. Results obtained in pre-liminary feed t r i a l s . J . Nutri. 8:597-608. Ganther, H. E., Goudie, C , Sunde, M. L., Kopecky, M. J . , Wagner, P., Oh, S. H., and W. G. Hoekstra. 1972. Selenium: relation to decreased toxicity of methylmercury added to diets contain-ing tune. Science 175:1122-1124. Gray, L. F., and L. J . Daniel. 1954. Some effects of excess molybdenum on the nutrition of the rat. J . Nurti. 53:43-51. Gunn, S. A., Gould, T. C , and W. A. Anderson. 1963. Cadnium-induced intestinal cess tumors in rats and mice and their prevention by zinc. J . Nutr. Cancer Inst. 31:745-753. 86 Halverson, A. W., and K. J . Monty. 1960. An effect of dietary sulfate on selenium poisoning in the rat. J . Nutri. 70:100-102. Hart, E. B., Steenbock, H., Waddell, J . , and C. A. Elvehjemc. 1928. Iron nutrit ion. VII. Copper as a supplement to iron for hemoglobin building in the rat. J . Biol . Chem. 77:797-812. H i l l , C. H. 1974. Reversal of selenium toxicity in chicks by mercury, copper, and cadmium. J . Nutri. 104:593-598. H i l l , C. H. and G. Matrone. 1970. Chemical parameters in the study of in vivo and in vitro interactions of transiton elements. Fed. Proc. Am. Soc. Exp. B io l . 29:1474-1481. Holmberg, R. E. and V. Ferm. 1969. Interrelationship of selenium, cadmium, and arsenic in mammalian teratogenesis. Arch. Environ. Health. 18:873-877. Jensen, L. 1975a. Precipitation of a selenium deficiency by high dietary levels of copper and zinc. Proc. Soc. Exp. Biol . Med. 149:113-116. Jensen, L. 1975b. Modification of a selenium toxicity in chicks by dietary s i lver and copper. J . Nutri. 105:769-775. Kamstra, L. D. and C. W. Bonhorst. 1953. Effect of arsenic on the expiration of volat i le selenium compounds by rats. Proc. S. D. Acad. Sc. 32:72-74. Kratzer, F. H. 1952. Effect of dietary molybdenum upon chick and poults. Proc. Soc. Exp. Biol . Med. 80:483-486. Lavander, 0. A. and C. A. Baumann. 1966. Selenium Metabolism. IV. Effects of arsenic on the excretion of selenium in the b i le . Toxicol. Appl. Pharmacol. 9:106-115. Martin, J . L. and M. L. Gerlach. 1972. Selenium metabolism in animals. Ann. N.Y. Acad. Sci . 192:193-199. Matrone, G. 1974. Chemical parameters in trace elements antagonism. In Hoekstra, W. G. "Trace element metabolism in animals". Vol. 2. Univ. Park Press., Baltimore, 91-104. Mi l l s , C. F. 1961. Rowett Res. Inst. Collect. Papers 17:71. In Underwood, E. J . 1977. "Trace elements in human and animal nutrit ion" Fourth Edition. Academic Press, New York, 90. Moxon, A. L. 1937. Alkali disease or selenium poisoning. South Dakota Agri. Exp. Sta. Bul l . #311:50. 87 Moxon, A. L. 1938. The effect of arsenic on the toxicity of seleniferous grains. Science. 88:81. Moxon, A. L. and K. P. DuBois. 1939. The influence of arsenic and certain other trace elements on the toxicity of seleniferous grains. J . Nutri. 18:447-457. Munsell, H. E., Devancy, G. M. and M. H. Kennedy. 1936. US. Dept. Agri. Tech. Bul l . 534. Neischeim, M. C. and M. L. Scott. 1958. Studies on the nutrit ive effects of selenium for chicks. J . Nutri. 65:601-618. Oldf ield, J . E., Schubert, J . R., and 0. H. Muth. 1963. Implications of selenium in large animal nutrit ion. J . Agri. Food. Chem. 11:388-390. Oldf ield, J . E. 1971. Selenium in nutrit ion. National Acad. Sci . Washington, D.C. Patterson, E. L., Milstrey, R., and E. L. R. Stokstad. 1957. Prevent-ing exudative diathesis in chicks. Proc. Soc. Exp. Biol . Med. 95:617-620. Perry, H. M. and M. W.Erlanger. 1974. Prevention of cadmium-induced hypertension by selenium. Fed. Proc. 33:357. (Abstr.). Puis, R. "Trace deficiency information". 1978. B. C. Ministry of Agriculture Publication. Richert, D. A. and W. W. Westerfeld. 1953. Isolation and ident i f ica-tion of the xanthine oxidase factor as molybdenum. J . Biol . Chem. 203:915-923. Rosenfeld, I. and D. A. Beath. 1946. The pathology of selenium poisoning. Wyo. Agr. Exp. Sta. Bul l . 275:1-27. Schwarz, K. and C. M. Foltz. 1957. Selenium as an integral part of factor 3 against dietary necrotic l iver degeneration. J . Am. Chem. Soc. 79:3292-3293. Scott, M. L., H i l l , F. W., Norris, L. C , Dobson, D. C , and T. S. Nelson. 1955. Studies on vitamin E in poultry nutrit ion. J . Nutri. 56:387-402. Smith, M. I. 1939. The influence of diet on the chronic toxicity of selenium. Public Health Repts. 54:1441-1453. 88 Starcher. B. C. 1969. Studies on the mechanism of copper absorpti in the chick. J . Nutri. 97:321. Sturkie, P. D. editor "Avian Physiology". Third Edition. Springer Verlag New York Inc, 1976. Underwood, E. J . 1977. editor "Trace elements in human and animal nutrit ion" Fourth Edition. Academic Press, New York. Venchikov, A. I., in "Trace element metabolism in animals" (W. G. Hoekstra, et al_. eds.) Vol. 2. Univ. Park Press, Baltimore, Maryland. 1974, 295. 89 APPENDIX 90 91 - J o Ul - J VO vV <*> ©V \> o o "b «*• •b I 3 o-V> tv ^ * 3 3 D ov V> N \> to ' I I 1 i 1 1 1 1 ( ^ i $ a eft «S u vb $ * 1 s 1 51 ^ 5 vu >4» 92 W E I 6 H T A B 1-6 le L 0 4 c K VfcH {TiUiATlo/J 7 2 X 3 4 /o ? li 6 n BLOCK I BLOCK 2 BLOCK- 3 APPENDIX FIGURE 3 . DIAGRAMMATIC REF>G£SEM7AriON OF RUdANGe/^iENT OF /3A77E£y BROOOBO.S \M\THlhJ ExPEGineMrAL. ROOM. TRIALS JT &L J J J . 93 I U> CO >> o 5: 5 , ^ on o X o o ns OH Ul Q CO > o 3 Q OH ri rf\ V/l Ul 5 m 3 «0 rrj b ri rjj a *3 3 vo Ul 3 Ul ft. 1 94 -k « 1 I o4 Hi N Uj 5: Ul -J Ul w 5 SO *5 ro ^ r i 03 rl 5 3 Ifs 9 5? * 3 * Ul k s; ui o VUj 0 K * o (8 CO I 00 DO 1 o I vj 0 -J I 

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