. W w u ~u—nu 'v “M, . ‘ J v... . . . . . ,. . . ;.‘. . . ‘ . _ V _ . . . A , . . . ‘ . i. . . .. .. A D.- v eer‘of .Ph {AL NUTR‘EENT _ IEN Degr Yhesis for the 0 9.. An 00 RH. EL EL K m N" . MICHEGAN STATE UN-lVERSiTYf £9.70 muses: HENRY WELLENREITER- : . . ‘ u o v . . , ;:i: .: -1 .; ., ‘ 4ithé, 1.x :2 ‘11 3 .hi‘ This is to certify that the thesis entitled NICKEL AS A POTENTIAL NUTRIENT presented by Rodger Henry Wellenreiter has been accepted towards fulfillment of the requirements for L I B H A P 1’ Michigan State University Ph. D. degree ianusbandry and Institute of Nutrition ma. Major professor Date MA? /3/_/770 0-169 gill fr”..¥§.i;. pl... 3?? . agar... NICKEL AS A POTENTIAL NUTRIENT By Rodger Henry Nellenreiter A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry and Institute of Nutrition 1970 ( -ézjfi'él/ Ices-70 NICKEL AS A POTENTIAL NUTRIENT BY Rodger Henry Wellenreiter Ten experiments were conducted to investigate the possible involvement of nickel in the nutrition of the Japanese quail (Coturnix coturnix japonica). The experiments consisted of a generation study conducted in an attempt to deplete successive generations of quail of any possible body stores of nickel and, in turn, produce symptoms of a nickel deficiency. A second series of experiments, conducted simultaneously with the generation study, dealt with a postulated involvement of nickel in the activation of the enzyme, arginase, in which attempts were made to induce at least a partial in vivo activation of the enzyme by nickel. This was accomplished by altering the dietary levels of arginine and manganese in an effort to increase the demand for an active arginase while at the same time removing its normal in 3113 activator, manganese. Appropriate nickel-supplemented control quail were also carried through each of the 10 experiments. To prevent exposure of the quail to airborne nickel contaminants, they were housed in rigid plexiglass and/or balloon type plastic isolators. originally designed for gnotobiotio research, equipped with glass media air filters. H. H. Wellenreiter Feeders and waterers were made of plexiglass or plastic which contained no detectable nickel. A highly purified diet was developed which supported normal growth, egg production, fertility. hatchability and chick weight. Casein was used as the protein source in the diet, and in the course of development of the diet, it was shown that arginine is the first limiting amino acid in casein for the Japanese quail as it is for the chick. Supplementa- tion of a 35 percent casein diet with 1.13 percent of L-arginine hydrochloride alleviated the growth limiting effects of the casein diet and increased plasma arginine, indicating that the arginine requirement had been met. Dietary vitamin levels recommended for the chick proved to be inadequate for the quail. The high vitamin requirement was not due to the stress of being housed in isolators since quail housed under con- ventional conditions exhibited the same high vitamin require- ments when fed the purified diet. Of course, this does not exclude the purified diet, itself, as a contributing factor. In three of the experiments, the quail were raised to adults and used to produce eggs from which a succeeding generation was hatched. Two other experiments, involving fourth generation birds. were terminated before egg produc- tion began. Supplemental arginine was added to all of the diets used in these experiments, so the only dietary variable was the nickel concentration. The basal diet was H. H. Nellenreiter assayed to contain 74 ppb of nickel, and the nickel supple- mented diet was assayed to contain a final concentration of 1780 ppb of nickel. The dietary nickel concentration had no significant effect on weight gain in four of the five experi- ments and no apparent effect on reproductive performance in any of the three trials in which egg production occurred. Weight gain was significantly increased by supplemental nickel in one experiment involving fourth generation quail. An attempt to repeat this performance in another group of fourth generation birds was unsuccessful. At the conclusion of each experiment, the birds were killed by decapitation and blood collected for plasma amino acid analysis. The lungs, liver and pancreas were collected for tissue nickel assays. and the kidneys were collected for kidney arginase assays. Due to the very small size of the organs, an accurate quantitation of the tissue nickel concen- tration was not possible by the methods used. Increased arginase activity was not consistently associated with the higher dietary nickel concentration and no conclusions can be drawn as to the in 3139 activation of arginase by nickel. Hematocrit. hemoglobin and total plasma protein determinations revealed no consistent correlation between the values obtained and the nickel concentration of the diet. The other five trials were devoted to a study of the H. H. Wellenreiter possible role of nickel as an activator of arginase. Two levels of nickel and two levels of arginine were included in the diets in a 2 x 2 factorial design. The first level of supplemental arginine used, 0.88 percent of the diet, proved to be somewhat inadequate and the supplemental level was in- creased to 1.13 percent. Significant increases in weight gain were produced by supplemental arginine in all five experiments. There was some indication that the addition of nickel to an arginine deficient diet stimulated growth. Plasma amino acid analyses revealed a lower essential to non- essential amino acid ratio in the nickel supplemented, argi- nine deficient quail. Thus, nickel may increase the efficiency of utilization of arginine when arginine is rela- tively deficient. Dietary nickel concentration had no significant effect on growth of quail given diets adequate in their arginine content. The dietary concentration of nickel had no effect on arginase activity, but the addition of arginine to the diet stimulated arginase activity in diets otherwise adequate in all the other known nutrients. With respect to the last statement, two experiments were conducted in which the protein content of the diet was de- creased by 8 percent and further imbalanced by the addition Of one percentcn?tyrosina,The addition of tyrosine had been reported to increase arginase activity, but no increase was R. H. Nellenreiter observed, possibly due to the low level added. Decreased weight gain occurred on all treatments, as compared with previous trials, and the decrease was attributed principally to an inadequate protein level. An attempt was made to lower the nickel concentration of the basal diet used in these two trials by washing of the calcium, magnesium and phosphorus sources with dimethylglyoxime. The nickel content of each of the minerals was considerably lowered but a net decrease of only 3 ppb, from 7b to 71 ppb, occurred in the mixed basal diet. A decreased dietary manganese concentration was also employed in an attempt to deprive arginase of its normal activating ion and, in turn, induce nickel to act as an in £132 activator. The nickel concentration of the supplemented diets was increased to 4660 ppb for these two trials. The results obtained from the arginase assays were variable and inconclusive. The dietary concentration of nickel employed had no apparent effect on hematocrit, hemoglobin or total plasma protein values. The results obtained suggest that the nickel requirement of the Japanese quail fed an otherwise adequate diet is less than 7h ppb, if indeed there is a bona fide need for nickel in the diet. ACKNOWLEDGEMENTS The author wishes to express appreciation to Dr. D. a. Ullrey and Dr. E. R. Miller for guidance in conducting this research and for their constructive reading of this manu- script. The opportunity to work with such qualified and well known scientists has indeed been a valuable and pleasurable experience. Appreciation is also extended to Drs. S. R. Heisey and R. w. Luecke for their helpful guidance and interest during the author's graduate program, and to Dr. A. L. Trapp who kindly performed the post-mortem examinations on the experimental subjects. Appreciation is extended to the Department of Animal Husbandry and the Institute of Nutrition for financial support throughout the graduate program. Special thanks are due Mrs. Joice Adams and Mrs. Kathryn Ide for their efficient and skillful typing of this manuscript. Appreciation is also extended to fellow graduate students. laboratory personnel and department secretaries for their assistance during the author's stay at Michigan State. Above all, the author is indebted to his wife, Jackie, and children, Janeen and Darren, whose love and encourage- ment have made these years of study tolerable and worthwhile. ii Rodger Henry Nellenreiter Candidate for the degree of Doctor of Philosophy DISSERTATION: Nickel as a Potential Nutrient OUTLINE OF STUDIES: Rain Area: Animal Husbandry and Institute of Nutrition Minor Areas: Biochemistry and Physiology BIOGRAPHICAL ITEMS: Born: October 23, 1942, Bloomington, Illinois Undergraduate Studies: Illinois State University, 1960-19o4 Graduate Studies: Michigan State University, 1964-1970 Experience: Graduate Assistant, 1964-1969 NIH Trainee, 1969-1970 KEMBER: Society of Sigma Xi iii TABLE OF C NTENTS I. IN'PRODUCTIOAJ O O C O O O O O O O O O O C II. REVIEW OF LITERATURE . . . . . . . . . . Nickel . . . . . . . . . . . . Nickel as a toxic element Nickel as a carcinogen . . Nickel in soil and plants. Nickel in animal tissues . Excretion of nickel . . . . Evidence of a physiological role nickel . . . . . . . . . . . Analytical techniques fo nickel cor-43000000 O The Experimental Use of Japanese Quail ISOlatorS O 0 O O O O O O O O O O O Arginase and Arginine... . . . . . . . Plasma Amino Acids . . . . . . . . . . III. EXPERIMENTAL PROCEDURE . . . . . . . . . Introduction . . . . . . . . . . . . . General Conduct of Experiments . . . . Analytical Procedures . . . . . . . . Statistical Analyses . . . . . . . . . IV. RESULTS AND DISCUSSION . . . . . . . . . Experiment I . . . . . . . . . . . . . Experiment II . . . . . . . . . . . . Experiment III . . . . . . . . . . . . Experiment IV . . . . . . . . . . . . Experiment V O O O O O O O O O O O O 0 iv TABLE OF CONTENTS (Continued) Page Experiment VI . . . . . . . . . . . . . . . 142 Experiment VII . . . . . . . . . . . . . . 145 Experiment VIII . . . . . . . . . . . . . . 1A9 Experiment IX . . . . . . . . . . . . . . . 153 Experiment X . . . . . . . . . . . . . . . 155 General Discussion . . . . . . . . . . . . 156 v. CONCLUSIONS . . . . . . . . . . . . . . . . . 173 VI. BIBLIOGRAPHY . . . . . . . . . . . . . . . . 175 VII. APPENDIX . . . . . . . . . . . . . . . . . . 199 Table \OG)\10\\J\ 10 11 12 13 14 15 LIST OF TABLES Organ concentrations of nickel in mice . . . . Occurrence of nickel in RNA isolated from various sources . . . . . . . . . . . . . . Approximate nutrient requirements of Japanese quail . . . . . . . . . . . . . . . Levels of minerals and vitamins adequate for growth of Japanese quail . . . . . . . . . . Percent amino acid composition of casein . . . Composition of basal diet . . . . . . . . . . Mineral composition of basal diet . . . . . . Vitamin composition of basal diet . . . . . . Weights and analytical data of Japanese quail fed 2 levels of nickel - experiment I. . . . Reproductive data of Japanese quail fed 2 levels of nickel - experiment I . . . . . . Weights of Japanese quail fed 2 levels of nickel and 2 levels of arginine - experiment II . . . . . . . . . .. . . . . . Weights and analytical data of Japanese quail fed 2 levels of nickel and 2 levels of arginine - experiment III . . . . . .. . Weights and analytical data of Japanese quail fed 2 levels of nickel - experiment 17 Reproductive data of Japanese quail fed 2 levels of nickel - experiment IV . . . . . . Nickel content of reagent grade minerals before and after washing with dimethylglyoxime vi Page 14 21 39 61 99 100 101 116 123 124 128 134-135 136 139 LIST OF TABLES (Continued) Table 16 17 18 19 20 21 22 23 24 25 26 2? Weights and analytical data of Japanese quail fed 2 levels of nickel and 2 levels of arginine - experiment V . . . . . . . . . Weights and analytical data of Japanese quail fed 2 levels of nickel and 2 levels of arginine - experiment VI . . . . . . . . Weights and feed use data of Japanese quail fed 2 levels of nickel - experiment VIII . . Reproductive data of Japanese quail fed 2 levels of nickel - experiment VII . . . . . Weights and analytical data of Japanese quail fed 2 levels of nickel and 2 levels of arginine - experiment VIII . . . . . . . Weights and analytical data of Japanese quail fed 2 levels of nickel - experiment IX Weights and feed use data of Japanese quail fed 2 levels of nickel - experiment X . . . Patterns of free amino acids in blood plasma of Japanese quail fed a commercial diet and of chicks fed a crystalline amino acid reference diet . . . . . . . . . . . . . . . Pattern of free amino acids in blood plasma of Japanese quail fed 2 levels of nickel - experiments I and IV . . . . . . . . . . . . Pattern of free amino acids in blood plasma of Japanese quail fed 2 levels of nickel and 2 levels of arginine - experiment II . . Pattern of free amino acids in blood plasma of Japanese quail fed 2 levels of nickel and 2 levels of arginine - experiment III. . Pattern of free amino acids in blood plasma of Japanese quail fed 2 levels of nickel, 2 levels of arginine and 1% of supplemental tyrosine - experiment V . . . . . . . . . . vii Page 140 143 146 147 150 154 156 159 160 161 162 LIST OF TABLES (Continued) Table 28 29 30 Pattern of free amino acids in blood plasma of Japanese quail fed 2 levels of nickel, 2 levels of arginine and 1% of supplemental tyrosine - experiment VI . . . Pattern of free amino acids in blood plasma of Japanese quail fed 2 levels of nickel and 2 levels of arginine - experiment \‘IIII O O O O O O O O O O O O 0 Analytical data on purified diet viii Page 163 164 172 Figure 1 LISP OF FIGURES Isolator set up as used in the experiments ix Table c- k0 m LIST OF APPENDIX Composition of mineral premix Composition of vitamin premix Preparation of nickel standards Solutions for arginase assay TABLES 3515: 199 200 201 202 I. INTRODUCTION The relatively recent discoveries of the importance of the trace elements selenium (Schwarz and Foltz, 1957) and crawl-fl 1" " : chromium (Schwarz and hertz, 1959) in nutrition has accentuated the probability that essential metabolic func- tions for other minerals remain to be elucidated. Analyses of human tissues have revealed that there are at least 15 trace elements consistently present for which no metabolic function is yet known (Tipton, 1960). Although some of the 15 may be accumulated with age as inert con- taminants with no biological function, others very possibly are essential for life with their function awaiting discovery by some enterprising scientist. A quantity of indirect evidence indicating that nickel may exert a biological function is accumulating. For example, nickel is consistently present in animal tissue in- cluding the newborn human (Schroeder 32 21., 1962); it activates various enzyme systems in vitrg, including arginase (Hellerman and Perkins, 1935); and nickel is present in ribonucleic acids from various sources (Wacker and Vallee, 1959a; 1959b). Nickel is also reported to be greatly elevated in the serum of patients suffering from myocardial infarction (D'Alonzo and Pell, 1963). However, a nickel de- ficiency in animals or man has not yet been reported. For an element to be considered essential, Underwood (1962) has suggested that the following criteria must be met: (1) Lack of the element results in a state of deficiency. (2) Supplementation of the element results in a reversal of the deficiency which may be accompanied by a growth response. (3) A correlation between the deficiency symptoms and subnormal levels in blood or tissues in some instances. At present several trace elements are classified as toxic and have not yet been demonstrated to be essential. However, as was the case with selenium, the toxic properties of an element are often recognized prior to proof of its essentiality. Based on the indirect evidence of a physiological func- tion for nickel, research concerned with elucidating such a function was considered worthy of pursuit. The Japanese quail was chosen as the experimental animal, and attempts were made to produce a state of deficiency and to demonstrate a role for nickel as an in vivo enzyme activator. i If "“" — .‘nflnv A ”-4- mb. _ u II. REVIEW OF LITERATURE Nickel Nickel as a toxic element Long before selenium was found to be an essential element in nutrition, the adverse effects of ingestion of excess selenium had been reported (Potter and Elvehjem, 1937). Although fluorine has not been definitely established as an essential nutrient, fluorosis was recognized as a problem before observations were reported that fluorine, at least under certain conditions, also exhibits beneficial effects (Cass, 1961; Davis, 1961). So it must be with nickel. At present no biological function has been demonstrated for nickel. However, the toxic effects of excess nickel have been recognized at least since the reports of Laborde and Riche (1888) and of Lehmann (1908). These workers reported that oral ingestion Of inorganic nickel salts over long periods of time by guinea pigs, rabbits, dogs and cats did not produce toxic effects, while subcutaneous or intravenous injections pro- duced toxic manifestations. More recently, Arnold (1939) found no evidence of nickel toxicity when young rats orally ingested amounts of nickel pectinate varying from 98.4 to 1256.0 mg per kg of body weight over an 8 week experimental period. No detectable differences in weight gain between the nickel pectinate treated group and the controls were observed, and no pathological lesions were found at necrOpsy. Nickel, at the level of 5 ppm in the drinking water, was found to have no effect on the growth and survival of mice up to 18 months of age (Schroeder at 31., 1963). In a follow-up article, the same authors (Schroeder 33 al.. 1964) reported on the effects of the continued ingestion of 5 ppm of nickel in the drinking water of the same mice until all had died. The total elapsed time was 36 months. They re- ported that nickel was not carcinogenic at the level used and, in fact, reduced the number of tumors observed in 473 nickel-supplemented mice as compared with controls. However, they reported a decreased survival rate beyond 18 months of age for the group given nickel and concluded that nickel seems to have an innate toxicity of an undefined nature, eXpressed by lessened male survival. No accumulation of nickel was observed in the tissues of the mice over their life time. Using weanling mice, fed much higher levels of nickel as the acetate salt for a period of 4 weeks, Weber and Reid (1969) found that 1600 ppm of supplemental nickel in the diet significantly reduced growth rate of male mice; but no -_-T! " - ..I- . ——‘ 3'.-. it" '13 um such reduction was observed at a level of 1100 ppm. A significant growth reduction at both levels was noted in female mice. The amount of nickel consumed by the female mice was slightly less at the 1100 ppm level and about equal at the 1600 ppm level to the amount consumed by the males. TL These results suggest that growing female mice have less tolerance to high levels of nickel than do males. Nickel 1 supplementation resulted in no marked changes in apparent digestibility of either energy, fat or protein and no apparent changes in bone metabolism as measured by bone calcium, phosphorus and citrate levels. Enzyme activity studies indicated that nickel exerts its influence in the kidney and liver where the element is known to accumulate, and, while nickel did not appear to affect the activity of any given enzyme, it did decrease the overall activity of both the Kreb's cycle and the electron transport system. The same authors also reported that the high levels of nickel had no effect on mature body weights or on conception rate. The average number of pups born was not significantly decreased with increasing dietary levels of nickel. However, the average number of pups weaned was significantly decreased in animals receiving 1600 ppm of dietary nickel as compared to those receiving 1100 ppm of dietary nickel or a basal diet containing no additional nickel. Nickel toxicity in the growing chick was demonstrated by Weber and Reid (1968) by including from 0 to 1300 ppm of ‘V 1‘4 0 supplemental nickel as the acetate or sulfate salt. differences in growth rates were reported in chicks fed the two forms of nickel. As the birds ingested increasing r amounts of nickel, weight gain decreased as dietary nickel increased, and this effect of nickel was thought to be in add ition to its effect in reducing feed consumption. How- ever , when feed intake was equalized in a subsequent study by paired feeding, no significant effect of dietary nickel on the growth rate of chicks to 4 weeks of age was observed. The two highest levels of nickel supplementation decreased nitI‘Ogen retention in both studies. Nickel toxicity has also been reported in the bovine ( 0' Dell §_t_ El” 1970). Supplemental nickel as the carbonate, at levels of 0, 62.5, 250 and 1000 ppm was given in the I‘ O o 1 I r atlon of male calves for a perioc; of 8 weeks - from 12 to 20 weeks of age. Growth rate was not affected by the 62.5 ppm leVel while slight growth retardation occurred at the 250 ppm level. At the 1000 ppm level, growth was arrested and f eed consumption markedly decreased. However, the animals W high received this higher level did not appear starved nor We be they lethargic. The general appearance was that of a 3’0 LIrigger animal. Histopathological examination of selected t i S SUtes revealed no abnormality associated with the feeding of nickel. When supplemental nickel was removed from the diet , growth rates were comparable for those formerly given O and 1000 ppm nickel indicating the transitory nature of the growth retardation seen with high levels of nickel T3 su pplementat ion. -v - The toxic effects of excess nickel and cobalt were ob- Jar-W served by Griffith gt a}: (1942) to be less severe as the prOtein content of the diet was increased from 10 to 25 percent - “or..- m0 - 31173 were less severe on the low protein diet if supplemental Sillfur amino acids were also added. Cysteine was much more effective than cystine or methionine. The authors postulated the formation of a detoxification complex between the metals and sulfur containing; compounds such as cysteine, glutathione and homocystine in the body. They postulated that the main effect of cobalt poisoning; is the result of binding of Sulfhydryl compounds in the tissues and interference with cellular oxidation due to the formation of such complexes, with glutathione for instance, may be the stimulus to the hematopoietic system which causes cobalt polycythemia. Pre— SLllllably the same type of mechanism could be responsible for the adverse effects seen when high levels of nickel are ingeSted. Nickel as a carcinogen Closely related to the toxic effects of nickel is its ‘ A _. n A _ va...‘.. lo - , . .q ~.. 4’ ‘ x Q...‘ .~ ‘“‘r~-v- .4 ~"0-AV' \..__ > s . I u—-.._. .A I . vs. . ~ R. e...~ .- ~QI‘J V’vl “’-.. .~ ~ . x .v ‘ .Pk 4 “.«\ .‘R apparent ability to cause carcinoma of the respiratory tract. Th is is especially prevalent in miners who are in constant contact with air containing nickel and nickel compounds. Among workers in the nickel refineries of South Wales prior to 1921+, the incidence of lung cancer was reported to be five times, and cancer of the nose to be 150 times, the normal rate (Doll, 1958). The causative factor was thought to be nickel carbonyl (Ni(CO)u). Carcinoma of the respiratory passages was observed after approximately 25 years of exposure to nickel in nickel miners in England (kincaid, 1956). Nickel carbonyl has been implicated as a pulmonary care inogen in tobacco smoke. In 1957, Sunderman gt a__l_. demon- strated that rats inhaling small amounts of nickel carbonyl, equ ivalent to 1930 mg of nickel, develOped extensive Squamous metaplasia of the bronchial epithelium. Six brands of Cigarettes analyzed by Sunderman and Sunderman (1961) were found to contain an average of 1.99 pg of nickel per cigarette. They analyzed the smoke given off by a burning cigarette for Carbon monoxide and found the smoke to contain five to seven percent carbon monoxide. Carbon monoxide and nickel or nickel containing compounds will readily unite at the tempe‘I‘C—lture of a burning cigarette to form nickel carbonyl. Based On the nickel content of cigarettes and the amount of carbon monoxide formed in the burning of the tobacco, these wor kerS estimated that a person smoking two to three packs "“"E -. . ., ‘ _. IA r1: _ r «.4.,". ‘ ~ \ h. _ \Axu“‘_‘n U'V' v“. o ‘ . n~ ,. r ,A V. A "-..5 4‘ P . 5“ (\- 4-4,”, | n “;V\\," .‘L-._. ,5, ‘n '4 ,r‘,‘ H... . ..“_1 a At. ~. of cigarettes per day would inhale approximately 5400 pg of assuming that all of the smoke was in- nickel in one year, hal ed. This amount of nickel would be about three times the observed carcinogenic level for rats. Cigarette smoke con- ta ins about 140 ppb of nickel carbonyl while the maximum amount considered safe in air has been estimated by the American Conference of Governmental Industrial Hygienists (1957) to be one ppb. In further studies on the carcinogenic effects of nickel carbonyl, Hackett and Sunderman (1967) parenterally administered nickel carbonyl. Quantitative doses could easily be given by injection and this method was less They reported L050 values for more hazardous to the researchers. rats of 2.2 i" 0.1, 2.1 i 0.4 and 1.3 i 0.1 lug of nickel per 100 g of body weight for intravenous, subcutaneous and intra- peritoneal injections, respectively, and that the pulmonary parehohyma appeared to be the target tissue of nickel carbonyl regardless of the route of administration. Increased mitotic act 1V1ty of the alveolar lining cells was noted- It has been suggested that the toxicity of nickel cax‘bonyl does not indicate toxicity of nickel pg; g3. as other metal carbonyls, including iron carbonyl, are toxic (Schroeder _e_t_ gin 1962)~ In contrast to the increased mitotic activity observed b - y Ha‘lett and Sunderman (1967) . Swierenga and Basrur (1968) 10 have reported a marked decrease in the mitotic index of rat embryo muscle cells cultured on a medium with a nickel con- centration of 1 gig of nickel per ml of culture medium. In addition to a decreased mitotic index, cultures of all ages when exposed to a nickel-containing medium exhibitied a large percentage of pyknotic nuclei, with a majority of such affected cells at some stage of mitosis. They concluded that the occurrence of a percentage of abnormal mitotic figures exhibiting multipolar spindles, altered polarity and unequal segregation of nuclear and cytoplasmic material suggests that the carcinogenic effect of nickel may be involved with the spindle mechanisms within the cell. The exact mechanism by which nickel produces cancer has not been elucidated, but Neisburger 13.32 §_l_. (1963) believes that the presence of large amounts of ions capable of being chelated leads to an imbalance in some crucial process. ggickel in soil and plants CDhe presence of nickel in the earth's crust was dis- Covered by Mokragnatz (1922). Since this discovery, numerous Cfittempts have been made to determine the nickel concentration of the soil and of the plants grown thereon. A nickel concentration of 3.9 ppm was found in 3011 from Ifentucky by IvchaI‘g’ue (1925) and a nickel concentration of 3- 9 ppm was found in soybeans grown on the soil. A soil 11 concentration of 22 to 66 ppm of nickel has been reported by Hunter and Vergnano (1952) for soils in Scotland. An average concentration of 20 ppm of nickel was found by Painter _e_t_ al. (1953) for soils of New Jersey. The same workers reported higher nickel concentrations in coastal .—_._‘.L_.r. ’ soils indicating that the nickel apparently was carried down- I‘M” ward by drainage waters, or inward from the sea by the winds. Thus , it is quite evident that the nickel content of the Soil varies from point to point on the earth's surface. The nickel content of plants is dependent upon the Spec 1es of the plant, part of the plant, stage of maturity, nickel content of the soil and soil acidity (Iéitchell, 19’45). Plants normally have a higher nickel concentration than animals (Schroeder, 1956). Concentrations of nickel in Various plant species as reported by Schroeder gt a_l_. (1962) include: oats, 2.6 ppm; corn, 0.70 ppm; rice, 0.70 ppm; raw 1 potatoes, 0.56 ppm; cocoa, 5.00 ppm and tea, 7.60 ppm. Tne leaVes of plants contain more nickel than the stems, the younger parts of the plant more than the older parts, and the grain contains more nickel than the straw (Hunter and vergl’lano, 1952). A decrease in the nickel content of grasses with ad- vancing maturity has been reported by Kirchgessner (1965). He I”s-‘E30rted a decrease of the nickel concentration to only abo ut 20 percent of the level present at the time the first _..x.~—..—— Hon...- 12 shoots appeared. Numerous reports have appeared indicating that available, or exchangeable, nickel in the soil is favored by a low pH and. that liming of the soil, with the resultant increase in pH. decreases the amount of exchangeable nickel (Hunter and . ‘ Vergnano, 1952). There may be little correlation between 8011 levels of nickel and nickel levels of plants grown there- on if the pH is alkaline (Painter _e_t_ §_l_.. 1953). The same authors indicate that a concentration of #0 to 60 ppm of nickel in certain plants is toxic to the plants. An inhibition Of plant growth by high concentrations of available nickel in the soil was suggested by Eviitchell (1945), and he indicated that the problem could be alleviated by liming of the soil. Iiickel in animal tissues The relative lack of data available prior to 1960 on tis Sue concentration of nickel, prompted Herring Q 2.1; (1950) to clonclude that not enough data were available to be able to State "normal" values with certainty. In fact, the failure of Herring fl al. (1960) to find nickel in the plasma and erythrocytes from all subjects was regarded by them as eviderice against the existence of a physiological function for 131118 element. If an element has a physiological function, it stands to reason that it should be present in the newborn. The flr St report of nickel in the newborn was made in 1938 by 13 Rusoff and Gaddum who detected the presence of nickel in the newborn rat. Since then, Leonov (1960) and Schroeder _e_t_ al. (1962) have detected nickel in human fetuses and newborn. The distribution of nickel in the various tissues of the animal body has received considerable attention. Using radioactive bBIIiClZ dissolved in saline and injected intra- per itoneally, wase gt _a__l;. (195M) studied the distribution of 63101 in the tissues of the mouse. They related that the (/ OBNi was achieved in two to twelve ; I———_—:.-‘A4“o-n M..-__ .7.“ .- . maximum uptake of injected hours and that the kidney, lung and plasma contained high concentrations of 631x11, with brain and muscle containing the 63 least. They also noted the rapid disappearance of Hi from all tissues except the lungs and brain. No values were given as to the absolute concentration of 631in in the tissues. The relative absence of nickel in most animal products as Compared to vegetable products was reported by Schroeder 533—; a. (1962). Tissue values for nickel concentration in mice a after their entire life had been spent exposed to drink ing water containing either 5 ppm nickel or no additional nickel , revealed that nickel has a predilection for spleen and heart, with little tendency to accumulate in other Organs in amounts much greater than the controls (Schroeder a ~8&- , 1964). The tissue values obtained are shown in tabl e 1 11+ TABLE I. ORGAN COl-ECZNTU’XTIOI‘IS F NICKEL Ii: i-‘lICE (pg 9; Net '.-'«"C.) Organ 4.3181 +Z'I'ib’c Kidney 0.50 0.99 Liver 0.“? 0.77 Heart 0.30 0.90 lhlns oJu o.&3 Spleen 0.39 3.39 A“ a?! ' aBeatsal diet contained 0.40 ppm nickel. ’05 ppm of nickel supplied in the water as nickelous acetate. o‘m—‘m-K- M». The predilection of nickel for the spleen and heart was also found in the rat where generally higher concentrations were found than in mice receiving identical concentrations of nickel in the diet and water supply (Schroeder e_t_ é‘l" 1967). The nickel concentration of the various tissues has been highly correlated with the blood volume perfusing the tissues at O - 25, 2, 6 and 16 hours following a single injection of 63511 (Smith and Hackley, 1968). The kidney was the only Organ with significant amounts of 63M 72 hours following an ”'2 O’Ni. All intravenous injection of either 2.5 01‘ 5-0 )1C 0f 01° the 631“ activity in the blood could be accounted for by the activity in the plasma. A direct count of the washed erythrocytes showed no detectable 631%. This is in contrast to the data of Wase e_t_ 31; (1954) who found an increasing pe I“heritage of the 63:61 activity of the blood of mice in the 31‘ Ythl‘ocytes with increasing post-injection time. However, 7' 19 63 Ni did not accumulate in the blood cells as has been re- ported for zinc (Robertson and Burns, 1963), selenium (Burke SE Ell-- 1967) and chromium (Gray and Sterling, 1950). Nickel activity was found in the erythrocytes of rat blood following an L050 dose injection of radioactive nickel ‘ , 1" carbonyl into the saphenous vein or administration by inhala- tion of a LDSO dose (Sunderman and Selin, 1968). The LD50 dose amounted to 2.2 mg of nickel per 100 g of body weight for 'trie injected dose and 0.20 mg nickel per liter of air when 1NIua]_ed.for a period of 15 minutes. No absolute values for nioliefil concentration of tissues were given but muscle plus fat; (zontained 7.3, bone plus connective tissue 5.5, viscera 91118 blood 4.7 and brain plus spinal cord 0.2 percent of the injeoted dose after 24 hours. The remainder of the dose was excxrueted. The 63Ni activity present in the erythrocytes one hOLLI‘ post-injection accounted for 48.1 percent 0f the actiixrity of the whole blood. After six hours the activity of ‘tlie erythrocytes accounted for only 8.4 percent, with the tradislocation of 63Ni from the erythrocytes to the plasma COI‘I‘elated with the disappearance of nickel carbonyl from the blo0d. Measurements were made of the distribution of the injected dose in the serum six hours after injection, and 80-5 percent of the total serum 63Ni was bound to serum protein. Of that bound to the serum proteins, 88.1 percent 16 Evas; bound to albumin and the remainder (11.9 percent) was prwesumed to be bound to the globulins. The latter figure was; believed to be in error due to the tailing of the altnimun fraction in the electrophoretic separation. In an earlier report, Sunderman (1964) reported that 72 perwoeent of the nickel remaining in the lungs following in- haleat310n of nickel carbonyl was bound to macromolecules and could not be separated by dialysis. (Ether reports concerned principally with the concentra- tiori <>f nickel in the blood have appeared. A range of 5.3 to ‘5..2 pg of nickel per 100 ml of human blood was reported by lBtrtt gt El' (1962). and an average value of 3.0 pg of niCikxel per 100 ml of human blood was given by Imbus gt 3;. (1396C3). A positive correlation between increased serum niiliieil concentration and the occurrence of myocardial in- faJTCrtion.has been observed (D'Alonzo and Pell, 1963)- They suésénest that high serum nickel levels are associated with lnfetrotion but not with other manifestations of coronary heaJ?t disease, but they could not determine if the high levEsls of serum nickel were a consequence of infarction or whee‘ther they were involved in the processes that led to infarction. If the latter is found to be true, they suggest that increased serum nickel can be used as a diagnostic indicator of acute myocardial infarction, and if nickel is .. ~>- -— - T u a ’Zfi; .."AA~¢ 17 the cause, treatment with a chelating agent to reduce serum levels micht be feasible. The fulfillment of any potential requirement for nickel by the newborn mammal would depend upon a body reserve I already present at birth or upon the nickel content of milk. A Cow' 8 milk was found to be free of nickel, even when 145 mg 0f elemental nickel were fed per day for two months (Archi- bald, 1949), and Archibald concluded that any nickel found in milk would be due to contamination from equipment used in handling the milk. Recent workers have shown that nickel is pres ent in detectable amounts in milk from humans and cows (Leonov, 1958; Narano and Rainone, 1959; Tokovoi and Lapshina, 1962 ; Stoobun e_t_ El.” 1962). The amount of nickel found was higl‘lest in the colostrum and gradually decreased as lactation proStressed; however, no concentrations were given. Excretion of nickel In one of the earliest reports on nickel excretion, Fq~lrrn and Imouge (1928) determined that 98.5 to 99 percent of tbs? nickel consumed in foods was excreted in the feces. calljolle (1937) found significant amounts of nickel in the bj~1e of dOgs following an injection of nickel chloride. FeCal nickel excretion was found to be maximal in the first eight hours and urinary excretion maximal during the first four hours after an intraperitoneal injection of 1 p0 of 63N1012, dissolved in saline (wase e_t_ 2}.” 1954). Total fecal ex- cretion greatly exceeded total urinary excretion. Excretion studies on growing pigs indicated that 94 to 96 percent of an oral dose of nickel was excreted in the feces and 4 to 6 percent in the urine (liirchgessner, 1965). Following an '7‘ / \ intxuavenous injection of 03N1C1,, over 60 percent of the in- 380136xi dose was excreted in the urine within 72 hours and less; ‘than 6 percent excreted in the feces during the same DEIViCNi (Smith and Hackley, 1968). Urinary excretion peaked twc> kuours and fecal excretion eight hours following the in- JeCt ion. 'Ehe excretion of 63Ni following an intravenous injection Of é>31\Ii((3(3)4 was studied by Sunderman and Selin (1968). Four da3’81 after the injection, urinary excretion amounted to 31.2 peITCwent and fecal excretion 2.4 percent of the injected dose. Bil—leery excretion during the first six hours after injection aVWEIuaged only 0.2 percent; no further measurements were made On‘ tdie biliary route. The major excretory route for in- jecrted 63Ni(Co)4 was found to be through the lungs. During theAfirst six hours after the injection, 38.4 percent of the T51 was exhaled. N0 63N1 was detected in the breath after six hours. Nickel carbonyl was determined to be the exhaled fOI‘m. The same workers recovered 81.4 percent of an injected dose of 03N1012 in the urine and 3 percent in the feces within four days following the injection. 19 From the results reported, it is apparent that the rcnite of nickel excretion is dependent upon the route of ad- miriistration and, to some extent, upon the form of nickel adniiIUstered. The majority of orally administered nickel appears in the feces as it is mostly unabsorbed; the major POI“t110n of an injected dose appears in the urine. When nicl:e].is administered as the carbonyl, the lungs can be eXINeczted to play a major role in the excretion of nickel. The: lgarge percentage of an oral dose that is excreted in the fecies; and the much higher oral intakes required to produce a nicikxel toxicity imply that there is a mechanism limiting 1milemstinal absorption as postulated by Schroeder (1962). Evidence of a physiological role for nickel. 'Nickel has been implicated in a number of physiological I'01-'38 throughout the years. Some of the postulated roles haW’EB been definitely disproven and some have not been StuClied adequately enough for any definite conclusions. One of the first reports of a possible role for nickel 111 the animal body was published by Bertrand (1926). In his St'L’Ldies he found appreciable amounts of nickel in insulin arki suggested that a proinsulin may be converted to insulin by nickel. In the 1930's, several reports appeared which attempted to implicate nickel as an element necessary for the preven- tion of anemia. Nickel was reported to have a marked a :— m‘nm‘h’ - f erg/thropoietic action when fed in daily doses of 0.05 mg to raizs receiving diets adequate in iron content (Myers and Beaurdq 1931). Copper was found to be more effective, but Dmrezss and Beard concluded that cOpper was not specific as a Sugipfilement to iron for the prevention and cure of nutritional anemiiad Reports by Keil and Nelson (1931) and by Underhill 22 gig:. (1931) established that there was a specific need for COEUDexr in the prevention of anemia and that this need could not toe met by nickel. iNickel has been reported to preserve the activity of the a0CHSJ_erator-globulin involved in the blood clotting mechanism (LEBiliin and Bessman, 1956), and Herring g: §l° (1960) re- pOI“t€md that nickel may affect the clotting mechanism by Stéikbilizing the labile factor. One of the most potentially significant roles for nickel aI‘iESes from the work of Nacker and Vallee (1959a, 1959b) who det352rmined that nickel, along with several other elements, Vfiis found in all samples of ribonucleic acid (RNA) analyzed. A QOnstant ratio of the sum of all the metals which were foltnd in RNA to the number of phosphate groups of the RNA's fIWDm the various sources was found to be 2.2 x 10"2 gram atoms of total metals per mole of RNA phosphorus. Therefore, not all phosphate groups are saturated. The ratio was found to be somewhat higher in rabbit reticulocyte RNA and somewhat 21 lower in calf thymus RNA. Deoxyribonucleic acid (DNA) from the same sources contained lesser amounts of nickel. The nickel content of RNA isolated from several sources is given in table 2. TAEHLEE 2. OCCURRENCE OF N CKEL IN RNA ISOLATED FROM VARIOUS SOURCES Source Nickel (mg/g RNA) Calf pancreas 130 (sRN-A) calf pancreasa 18 Calf thymus 74 Horse kidney ML Rabbit reticulocyte 51 Eiuézlena gracilis 60 Rat liver 64 r‘ SCDluble RNA isolated from supernatant of pancreas. The metals found in RNA were firmly bound, as dialysis algallist metal-free water did not remove the metals. Some of the metals were partially or completely removed by dialysis aga inst various chelating agents. The authors concluded that metals may play a role in the maintenance of configuration of the RNA molecule, perhaps linking purine or pyrimidine bases, 01‘ both, through covalent bonds possibly involving nitrogen atOms or 17 electrons of the bases. As they play a role in RNA, metals may bear a functional relationship to protein 7—.-. "“‘—‘—~—*» ="T.. a 2rd .21“ 22 S3FnthBSiS and the transmission of genetic information. In an airtempt to determine the effect of nickel on the incorpora- tixln of amino acids into protein in the lens of the eye, DeVri and Banerjie (1965) found protein synthesis to be comuoletely inhibited by nickel. Concentrations of 48,ng of nicliel per gram of rat lung RNA and 29,ug of nickel per gram CM‘ I13t liver RNA have been reported by Sunderman (1965). Both of these values are well within the range of values reENDITted by Hacker and Vallee (1959b). The binding of 63Ni t0 IRIIA in rat liver and lung following an injection of 63IJi.(CKDu,has been observed (Sunderman and Selin, 1968). .A more recent study conducted by Smith and Hackley (1968) reV'Etaled no significant correlation between the RNA content Of‘ Eselected tissues and the distribution of 63Ni in these tiissfues. This does not conflict with the work of Hacker and Vallee (1959a, 1959b) as they gave no definite ratio between n1431§el and RNA, but rather a definite and constant ratio betWeen the sum of all the metals present and RNA. Nickel has been shown to strongly inhibit ribonuclease CLjJA and Wang, 1964) and Kaindl and Altmann (1964) have Srflggested that at least one of the possible functions of trace elements in RNA is to stabilize it against the action or ribonuclease. Nickel carbonyl has been found to be an 1nhibitor of DNA dependent RNA polymerase activity in hepatic nuclei following an intravenous injection of 2.2 mg D' 9.. A -lfl.‘A-.T1 -" - I 23 of nickel (as the carbonyl) per 100 g of body weight (Sunderman and Esfahani, 1968). The inhibition of the in- corporation of 14C-orotic acid into RNA of rat liver follow- ing an injection of nickel carbonyl was reported by Reach and Sunderman (1969). A possible role for nickel in main- taining the conformation of ribosomes has been reported by Tal (1969). The implication of such a finding with respect to protein synthesis is quite evident. One of the first reports of what was thought to be a response to nickel supplementation in an intact animal appeared in 1937 (Dixon, 1937). He found that dietary nickel (0.16 mg per week) plus dietary cobalt supplementation to sheep gave a greater growth response than cobalt alone (0.8 mg cobalt per week). The nickel source was stated to be cobalt-free so that the response supposedly was not due to additional cobalt supplied by the nickel supplement. Cobalt-free nickel is difficult to prepare, and quite possibly the nickel source was contaminated with cobalt. Io(1945) concluded that nickel has a physiological role in promoting both carotene and citrin formation in plant metabolism. The first report indicating that nickel might be of importance in increasing crop yield was published by Roach and Barclay (1946) who found an increase in yield of wheat, potatoes and broad beans which had been sprayed with a 24 nickel solution. The nickel concentration of the solution was not given. One of the most interesting roles postulated for nickel is its possible involvement in the pigmentation process. The in_zlt£g ability of nickel to replace copper in the enzyme polyphenol oxidase (tyrosinase) has been demonstrated (Kertesz, 1951). On the basis of the strong affinities of melanin and its precursors for metals, Kikkawa at al. (1955) proposed that color depends upon specific genes which control the metals found in melanin. They reported that black rabbit hair contained cobalt, copper and nickel; yellow hair con- tained titanium, molybdenum and nickel; and white hair con- tained only nickel. Furthermore, the addition of titanium to melanin precursors produced yellow pigment; the addition of molybdenum produced red; and the addition of nickel pro- duced white pigment. Similar relationships were also ob- served in analyses of fish skin, bird feathers, moth wings, guinea pig hair and human hair. This possible relationship between nickel and hair color has been carried a step further by Abe (1956) who reported that the absorption of metals in various organs was found to depend on the hair colors of the mice used. He reported that more cobalt was taken into the bodies of black mice than of white mice, and that more nickel was absorbed by the white mouse. Future work with this possible role for nickel 25 should prove quite interesting. The possibility that nickel, in addition to iodine, is necessary for the prevention of endemic goiter has been re- ported by Lyaonau (1960). Nickel, tOgether with copper, cobalt, iron and manganese, is believed to contribute to the assimilation of iodine (Savchenko, 1964). A possible involvement of nickel as a causative factor of bloat in ruminants has been reported by Harris and Sebba (1965). In their studies on the in 21:33 foam-forming tendencies of lucerne, they observed a greater stability of the foam when nickel was added. The foam-forming tendencies of aged lucerne solutions decreased, and Harris and Sebba concluded that the nickel in fresh lucerne appeared to be available for attachment to the protein while in aged lucerne, although nickel was still present, it was no longer in an available form. The significance of nickel, or other metals, in the cause of bloat in 3333 has not been determined. Nickel has been postulated to interfere with the active transport of specific sugars into yeast cells (Rothstein and Steveninck, 1966). They believe the transport to occur by the interaction of the sugar with the phosphoryl sites of the membrane carrier. This complex (carrier-phosphoryl sugar) is then translocated to the site of the glycolytic system where the phosphoryl group is transferred to the sugar 2O phosphate, which proceeds through the glycolytic scheme ultimately to carbon dioxide and alcohol. The carrier is released to the outer surface and the phosphoryl sites are regenerated presumably by the adenosine triphosphate (ATP) produced in the glycolytic chain. The nickelous ion is be- lieved to interfere with the active transport by combining with the phosphoryl sites and thereby reducing or virtually eliminating the transfer of phosphate to the carrier. No phosphoryl sites can be regenerated, and the phosphoryl sites already present are used to transport glucose and to phos- phorylate glucose. Once all of the phosphoryl sites have been used in the transport process, the active transport must stop. Hence, the presence of nickel ultimately results in the stoppage of active transport of glucose by the yeast cell. Nickel, at the level of 5 ppm in the drinking water for rats, has been reported to affect the serum cholesterol level (Schroeder, 1968). Nickel nonsignificantly decreased serum cholesterol levels in males and significantly decreased serum cholesterol levels in female rats. Chromium and niobium had similar effects to those of nickel while increased serum cholesterol levels were associated with the 10 other metals tested. Nickel has been shown to activate a number of enzymes, to have no effect on some and to inhibit others when added to in vitro enzyme systems. Since most of the biologically active metals usually exert their effects through enzyme systems (Herring 22 21., 1960), any study concerned with es- tablishing a physiological role for a metal should at least consider the role of that element as a potential enzyme activator. In this respect, Hellerman and Perkins (1935) determined that nickel, cobalt, manganese and iron (ferrous) could act as in zitrg activators for the urea cycle enzyme, arginase. They observed that an arginase preparation would deteriorate with age if unactivated, but would remain active for months if activated with metal ions, and they suggested that a coordination complex between the metal and enzyme may be responsible for the metal ion activation of arginase. Nickel has also been shown to activate trypsin (Sugai, 1944), oxalacetic carboxylase (Speck, 1949), enolase (Mold and Ballou, 1957), carboxypeptidase (Coleman and Vallee, 1960), wheat root phosphodiesterase (Ibuki §t_al., 1964) and acetyl coenzyme A synthetase (Webster, 1965). The latter enzyme appeared to have a double requirement for activating, divalent cations. A specific block in the alcohol dehydro- genase reaction leading to a partial inhibition of fermenta- tion was observed in yeast cells exposed to a nickel contain- ing medium (Fuhrmann and Rothstein, 1968). As pointed out earlier, nickel was shown by Liu and Wang (1964) to inhibit ribonuclease. 28 The possibility that nickel may be a biOIOgically important metal has attracted the attention of Smith (1968) and of Nielsen (1970). The former worker fed a purified diet consisting of dried skim milk, sucrose, corn oil and supplemental minerals and vitamins to growing rats housed in isolators. for a period of 55 days. The nickel content of the diet was assayed to be 0.08 ppm. No significant difference in growth rates were observed before or after intraperitoneal injections of 70,ug of nickel per day. Since environmental contamination could be virtually ruled out, Smith concluded that, if nickel is required by the rat, the dietary requirement is less than 80 ppb. The work reported by Nielson (1970) involved the feeding of purified diets to growing chickens. The chicks were maintained in an all-plastic, controlled environment and fed a diet containing 79 ppb nickel in 2 trials and a diet con- taining less than 40 ppb nickel in a third trial. Control chicks received the same diets supplemented with 3 to 5 ppm of nickel. In all trials, the chicks receiving the un- supplemented diets develOped a slight thickening of the long leg bone and a slightly enlarged back, and seemed to walk with an abnormal gait. The length to width ratio of the tibias from the unsupplemented group was significantly de- creased, and the leg color was bright orange-yellow instead 29 of pale yellow-brown. All of the observed changes were re- ported to be prevented by dietary nickel supplementation. Following oral administration of 25,uC of 63hi, an increased retention of the isotope after six hours was observed in the unsupplemented groups. The author concluded that nickel may have an important physiological role in the growing chicken. A requirement of 1 mg of nickel per day has been re- ported for a 40 kg castrated Kirgiz fine-wooled ram (Odynets and kambetov, 1962). No further explanation was given. Analytical techniques for nickel Atomic absorption spectrometry is currently the most sensitive method of determining nickel in biological materials (Sprague and Slavin, 1964; Sunderman, 1965; Takeucki EE.§£°' 1966; Rulford, 1966; Brooks §t_al., 1967; and Sachdev et al., 1967). In cases where the nickel concentration of samples is too low to permit a direct measurement of the nickel content, a concentrating procedure, solvent extraction, is used to enable an accurate assay (Lockyer 22 31., 1961; Sprague and Slavin, 1964; Takeucki e3 31., 1966; and Mulford, 1966). In solvent extraction, two immiscible liquids are brought into contact to effect the transfer of one or more elements from one liquid phase to the other. In the usual case, one phase is an aqueous solution of the sample, and the other is an \0 (D immiscible organic solvent. metal salts, being ionized, are usually much more soluble in aqueous media than in organic solvents, and to extract a metal into an organic solvent, the metal ion must be conver ed to an uncharged species. This is easy to accomplish as most metals fo~m stable, neutral and extractable complexes with one or more of a group of organic compounds known as chelating agents. The solvent used must completely dissolve the metal chelate and be immiscible with the sample solution, and, in addition, should burn with a clear, steady flame. For this reason, esters and ketones are extensively used as the organic phase. Solvent extraction can be used to eliminate interferences which might ordinarily arise from the sample matrix, and can be used when the sample size is too small for direct analysis. With solvent extraction an increased signal can be obtained due to: (1) concentration of the metal from the aqueous phase into a much smaller volume of organic phase, and (2) the higher volatility of the organic solvent, as compared to water, which results in a higher concentration of neutral atoms in the flame (Mulford, 1966). The experimental Use of Japanese Quail and Isolators If one hopes to establish an essential physiological role for a trace metal, such as nickel, in a living macro- ;\ ~x~ ».A . v g1 organism, the selection of that organism is very important. The potential of the Japanese quail (Coturnix coturnix 'aponic.) as a pilot animal for poultry research was first [:3 U reported by Padgett and Ivey (1939). They discussed th advantages of using quail in place of chickens. Among the advantages reported were rapid growth rate, early maturity and short incubation period which allow for the production of the next generation in about 10 weeks. From their experience with the Japanese quail, Eadgett and Ivey (1959) and Howes (1964) concluded that the coturnix nust have a high rate of metabolism due to the rapid rate of growth and the high rate of egg laying. Other advantages which the use of Japanese quail has over the use of chickens are of economic importance to the re- searcher with a limited budget, time and space (Wilson 33 31., 1959; Wilson 33 al.. 1961). Quail are much cheaper to main- tain than chickens and 20 to 30 birds can be kept for the same cost as one chicken (Howes and Ivey, 1961; Howes and Ivey, 1962). Because of the relatively small size of quail, space requirements are minimal which enables the quail to be easily adapted to germfree experimentation (Reyniers at 31., 1960; Beyniers and Sacksteder, 1960). A recent bulletin on the Japanese quail has been published by the National Academy of Sciences (1969). The publication includes information on breeding, care and management of b.) (‘v quail for research purposes. most of the recommendations included in the publication were derived from the work of the various scientists cited earlier. Careful management of the Japanese quail is very im- portant to insure success with the bird. eggs to be hatched should not be stored for longer than 14 days as the hatcha- bility decreases greatly if the eggs are held for a longer period of time (Padgett and Ivey, 1959; Howes, 1964). However, eggs can be stored for a somewhat longer time if stored in plastic bags (Becher et 31., 1963; Proudfoot, 1964a; Proud- foot, 1964b). Flushing of the plastic bag with nitrogen has been reported to increase storage time (Proudfoot, 1964b; Proudfoot, 1965). Storage of eggs at a temperature of 13 to 160 C is recommended (Howes, 1964). Incubation should be carried out at a temperature of 37 to 38° C, and the eggs should be turned every 3 to 4 hours (Howes, 1964). Fertility of the eggs collected at the start of egg production is low, but after 50 days of production may be as high as 85 to 90 percent (Padgett and Ivey, 1959; Noodard at al., 1969). The hatchability of fertile eggs is 60 to 70 percent (Padgett and lvey, 1959; doodard at al., 1969). The greatest single cause of poor hatchability of quail eggs has been reported to be dehydration as a result of microscopic cracks in the shells which could not be de- r... 14 tected by the naked eye (Howes, 1964). At hatching, the brooder temperature should be kept at 380 C for the first week and gradually lowered so that by four weeks of age the birds can be kept at room temperature, 24 to 25° C (National Academy of Sciences, 1969). The chicks weigh an average of 5 to 6 g at hatching (Reyniers and Sack- steder, 1960; Howes and Ivey, 1961) and it is necessary to place a small piece of screenwire or several small pebbles or marbles in the waterers for the first few days to prevent drowning (Wilson at al., 1961). Average feed consumption by growing Japanese quail, 20 to 35 days old, is about 11 g per day (Reyniers and Sacksteder, 1960). As sexual maturity approaches, a ratio of approximately one male to three females has been reported to give Optimal fertility (Padgett and Ivey, 1959; Woodard and Abplanalp, 1967). The density of the mature birds has been shown to be a factor in egg production and subsequent hatchability of the eggs (Ernst and Coleman, 1966). They report an increased number of cracked eggs at the higher densities studied (172 and 215 quail per square meter of floor space). However, they report that no delay in sexual maturity occurred as a result of rearing of the chicks at high density levels, since no difference in egg production was observed in full sibs which were raised at different densities and subsequently placed in individual cages during the laying period. An 34 earlier report by Wilson gt 31., (1961) indicated that de- velopment of quail was delayed one week by crowding, while Howes (1964) reported a depression of growth, increased mortality and cannabalism as a result of crowding. The National Academy of Sciences (1969) has recommended a minimum of 84 cm2 of floor space per bird (120 birds per m2) from 0 to 4 weeks of age and approximately 125 cm2 2). per adult quail (80 birds per m Male Japanese quail weigh approximately 90 to 100 g at 120 days and females 120 to 130 g (fieyniers and Sacksteder, 1960). Adult males average 100 to 1&0 g and adult females 110 to 160 g (Howes and Ivey, 1961). The heavier body weight of the female has been attributed to heavier gonads, liver and intestines (Wilson 33 al., 1961). The average egg weight has been reported to be about 9 g or approximately 7 percent of the body weight of the female, and Japanese quail require about 3 kg of feed to produce 1 kg of eggs (Wilson gt 31., 1961). The same authors report that under good en- vironmental conditions, quail may produce double the egg mass per unit of body weight as compared to good laying strains of chickens. Hence, the metabolic demands for egg laying should be exaggerated such as to allow the development of a nutri- tional deficiency which might not arise during growth of the ,quail, or might not arise at all in other species which have lower metabolic demands at all stages of the life cycle. 35 The amount and wavelength of light to which the develop- ing quail are exposed has an influence on the development of sexual maturity and on subsequent egg production. At least 12 to 14 hours of light per day are required to stimulate gonadal development (Padgett and Ivey, 1959; Wilson 33 31., 1962; Tanaka 33 31., 1965) with intermittent 12 hours of light more effective than continuous 12 hours of light (Wilson gt 31., 1962; Tanaka 33 31., 1965). Japanese quail will continue to lay under continuous light (Daniels, 1968). Growth of female quail is depressed by green or blue light as compared to red or white light and testicular weights were less for males kept under green or blue light (Woodard 32 31., 1969). The same authors report that females reached 50 percent lay two weeks earlier under red or white light than under green or blue light, and the fertility of eggs from quail kept under blue light was lower than for birds kept under green, red or white light. However, egg weight, feed conversion and hatchability of the fertile eggs were not affected by wavelength of light. In most trace element research, the use of purified or semipurified diets is usually dictated by the unavailability of natural feedstuffs low in the element in question. The first reported use of a purified diet for Japanese quail appeared in 1964 (Fox and Harrison, 1964), and was used for a study on zinc deficiency in quail. However, at least a 36 semipurified diet was used in a preliminary study with Japanese quail reported by Adkins 33 31. (1961). In the study, an attempt was made to replace the casein in the diet (20 percent) with an equivalent amount of crystalline amino aicds. The authors reported that this concentration of casein and the equivalent concentration of amino acids supported normal growth in the chicken, but depressed egg production and caused a loss of body weight when fed to mature quail over a period of four weeks. Low dietary pro- tein was cited as the probable cause of the poor performance of the quail. Purified diets were extensively used by Ketola (1967) in an effort to identify a growth factor in soy- beans for the Japanese quail, and to establish requirements for protein, energy, minerals and vitamins for the birds. Undesirable effects resulted from feeding a sucrose- base purified diet to day-old Japanese quail (Alford et al., 1967). Water containing small quantities of dissolved sucrose passed into the air passages of the baby quail, and upon dehydration sucrose crystals formed in the air passages causing asphyxia. This problem was not noted when the diet was offered to two to three week old quail, but poor feather development resulted which the authors attributed to a lack of fiber in the diet. The performance of Japanese quail fed purified or 37 commercial diets was studied by Gough et al. (1968) who re- ported, in general, rather poor performance of birds fed the purified diet. They observed a depression of growth rate, feather development and feed consumption, the incidence of a higher mortality during the first three weeks of life, de— creased egg production and egg size with many soft-shelled eggs, and lower fertility and hatchability for quail receiving the purified diet. The successful use of a purified diet for any species is dependent upon a knowledge of the nutrient requirements of that species. Very little work has been done on the nutrient requirements of the Japanese quail, and the poor growth and reproductive performance of quail fed purified diets is undoubtedly a reflection on the lack of knowledge in this area. The nutrient requirements of the Japanese quail as estimated by various workers are summarized in table 3. A complete listing of minerals and vitamins at levels which have been shown (Ketola, 1967) to be adequate for growth of Japanese quail is presented in table a. The levels of minerals included were assumed to be ade- quate as no growth response was observed when the levels were doubled. 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H 0 m . meHeHwQ 308330000 000H omaH 0a 00 n00.Hz to :oHpmectoeoe mHa+Hz+ waequ+ wH<+Hz- mHmq 038 QZd AQMOHZ mo m4fl>mq 038 Qmm QH £000 000 00050 go hmnasg psmwoaamh mommgpsoamm c0 mHmQESZN .Cmma oSp go 00000 flamesmpMR ( 0.H 00H0 0.H0 H0HV 0.00 000000 000 0 00.0 00H000.0 00H0HH.0 000H03 0000 0 Nu.o ***m©.m mmamamm 00.0 H0.H mmmawz 00.0 00H0H0.0 00Hv00.0 .00000 000H03 00>HH 0000 AmHvammom moamaom 0000 00 V0000H 000H0a HO0H 00 V 0H000 00H0H000H 00000>0000 0000 000 0 H000 000H0000N \00H0H .00HV 000H0 00Hv00mm0 0000>H00000 dmmmcamag 00.0 00.0 00000 00 000 0000\0H00 N.© 0.0 00000 0m cpvw .000 0009\0000 Scandaamgoo 000m 0.H 0*000000.00H 00H0000 0.0 00H00.00H 000H0a 0.0 H00 v0.0mH 00H v0.0mH 0000 0HH .0H000 0.0 000 00.00H 00H 0H.00H 0000 00 0.H 000 00.0HH 00H 00.0HH 0000 H0 0.H 0H0 00.00 00H 00.00 0000 00 0.H 0H0 00.00 00H 00.00 0000 00 0.0 0m0 v0.00 00H 00.00 0000 0w 0.0 000 00.00 000 00.00 0000 0H 0.0 000 00.00 000 00.00 0000 0 H.0 H0HH00.0 00HH00.0 H0H0H0H H.m.mH **N w .mPSmHGR 000H 00 000 .H000Hz 00000H0 .>H BZHEHmmmNfl I QHMOHZ mo mqm>mq ORB Oflm AH Hmsoa can» hmpmmhw 009:000002w0m00* .000. v my 05H0> 00300 saga nonwoaw 0Hp200000:w0m** .nmo. V my mdamb Hmsoa Sana nonwonw thQMOHmHQwam* .mmpssHa om pom N+gz Spas 00pmbfipo<0 .meofi: mo Hm>oa £000 pm 005H0> mo pom Uncomm How 00000 0000c0pmo .mosflp pgmam0000 03p pm 05000» 0800 so Uosampno 00000 How modam>w .Hsos\mdmmdp £0009 w\mmas mmaoa a0 .00.psoov .0H 04000 136 times higher than those obtained for the unactivated assay. As in experiment I, the arginase values for females tended to be higher than those of males. No treatment differences were observed in liver weights, but the livers from the females were significantly heavier (P < .005) than those from males. Some of the livers taken from the females were very oily. No hematocrit or hemoglobin determinations were made. Reproductive data from the second generation quail are presented in table 14. The birds were housed under the same conditions as described for experiment I so many eggs un- doubtedly were broken and therefore not recorded. Egg weights and eggs per female bird day were very similar to the figures presented in table 10 for experiment I. TABLE 14. REPRODUCTIVE DATA OF JAPANESE QUAIL FED TWO LEVELS OF NICKEL - EXPERIMENT IV. Dietary Nickel, ppb 74 1780 First egg, days 38 37 Total eggs laid 281 1290 Ave. wt, g 9.37 8.79 Eggs/female bird day .573 .648 No. eggs set 941 922 3641 0992 Net (uncracked) 89 77 282 390 No. fertile 62 47 188 247 % Hatch (of total set) 38.4 19.6 31.0 19.6 % Hatch ( of net) 40.5 23.4 40.1 25.1 % Fertile (of total) 66.0 51.1 51.6 49.5 % Fertile (of net) 69.7 61.0 66.7 63.3 % Hatch (of fertile) 58.1 38.3 60.1 39.7 1Eggs collected when birds were 10 to 12 weeks of age for pro- ,duction of third generation, experiment VII. Eggs collected when birds were 17 to 19 weeks of age for pro- duction of birds used in experiment VIII. 137 The experiment IV birds were used to provide eggs for hatching of birds used in two succeeding experiments, and, therefore, two sets of fertility and hatchability data are presented. The first set is similar to that observed in ex- periment I. In each case, eggs were collected for hatching when the birds were 10 to 12 weeks of age or in approximately the fourth to sixth weeks of egg production. The second set of data was compiled from eggs collected from the same birds but during the 17th to 19th weeks of egg production. Fertil- ity and hatchability of fertile eggs exhibited a definite decrease as the birds grew older. Experiment V This experiment was designed to stimulate arginase activity to such an extent that the quail receiving the arginine deficient diets would not grow as well as in previous experiments, and to determine if nickel could activate arginase lg 111g. If the latter were true, the group re- ceiving supplemental nickel but no supplemental arginine should not grow as well, and have higher arginase activities, than the group receiving neither supplemental nickel nor arginine. Growth on the diet supplemented with 1.13 percent of arginine and 4660 ppb. total nickel would be expected to decrease if nickel were capable of $2.21X2 activation of arginase and if the dietary arginine level were marginal or 138 deficient. If excess dietary arginine were present, its degradation by arginase would not be detrimental to the bird, and such birds would be expected to gain as rapidly as quail not receiving supplemental nickel. The attempt to stimulate arginase activity was carried out by reducing the casein content of all the diets from 35 to 25 percent and adding one percent of supplemental tyrosine to increase the tyrosine level from the original 1.36 percent in the 35 percent casein diet, to 1.98 percent in the 25 percent casein diet. Supplemental tyrosine has been reported to increase arginase activity two to four fold (Austic and Nesheim, 1969a; 1969b; 1969c). However, they added three percent tyrosine to chick diets. In addition to the attempt to stimulate arginase activity, an attempt was made to lower the nickel content of the basal diet. Analysis of each of the reagent grade minerals used in the basal diet revealed that the magnesium carbonate, calcium carbonate and calcium phosphate were relatively highly contam- inated with nickel, as shown in table 15. Nickel analysis of the same minerals, after washing with an alcoholic dimethyl- glyoxine solution revealed a considerably lowered nickel concentration in all three (table 15). The cellulose used in trials V and VI was acid washed and resulted in a reduc- tion of the nickel concentration from 97 to 90 ppb. However, 139 (D TA LE 15. NICKEL CONTEJT OF REAGENT GRADE MINERALS BEFORE AND AFTER WASHING WITH DINETHYLGLYOXINE. Nickel Concentration Mineral Before washing f After washing 4MgCOBD':g(OH)2°n(H20) 1300 i 301 388 i 38 021003 138 i 9 54 i 10 CaHPOa 395 i 25 224 i 26 1Standard error of the mean. the nickel concentration of the basal diet only decreased from 74 to 71 ppb, an insignificant change. When unwashed calcium carbonate was added to the basal diet to increase the calcium content for the laying birds in experiments I, IV and VII, the nickel content was increased from 74 to 101 ppb indicat- ing that the minerals, even though reagent grade, are con- taminated with nickel and can make a significant contribution to the total nickel content of a purified diet. The weights and analytical data of the quail used in experiment V are presented in table 16. Weight gain was not significantly influenced by the nickel concentration of the diet but was significantly (P < .005) increased at all weigh periods, except the initial period, by 1.13 percent of supplemental arginine. Growth rates of the birds on all treatments were considerably less than the growth rates ob- served in the earlier experiments. This was probably due to .0500> 8000 000 00080 00 008888 080000000 00008080000 80 0008882 .00000Ha 00 000 0+0: 00H; 0000>H0000 .0808\0Smmap 80000 m\0008 00008 1W 140 .8008 080 00 00000 0000800mm 00Nv0m.0 00HO0H.0 000000.0 000000.0 Ha 00H\0 .8000000 080000 H0009 00 0H.0 00 V H.0H 00 0 0.0H H0 0 0.0 He 00H\0 .anchcs0m 00 0H.m0 HOH0 0.00 HOHv 0.00 00HV 0.H0 0 .0Haccp0smm 000000.0 000000.0 000000.N 000000.0 us 0060 0 00000H.m 000000.H 000000.H 000000.H 10 .00000 000H03 00>H0 N0NH 000000H0 000000H 0000000HH 00000000 0000>Hpc< 0000000H H0 0000H 00HV0H0H 0 00>Hpc0s0 M00080w0¢ 00.0 00.0 00.0 0000\2000 H.0 0.0 0.0 0 .000 00Hn\0000 80000880800 0000 00000.00 00000.00 00000.00 0000 0m 00000.00 00000.00 H000H.Hm 0000 00 00000.00 00000.00 00000.0H 0000 0H 00000.0H A0000.HH 00000.0 0000 0 00000.0 0H000.0 ~00H000.0 H0H0HsH . w .0000H00 00 oo om 800.88.00 8000000800800 0H.H 0H.H 0 0.00000000_H000000H0000 0000 H0 H0 000.Hz.0o :cH0000000000 000+Ha+ msanHz+ 000+qu 00 BzmszflmXfl I mszme< m0 mqm>mq 039 Dz< ammon mo mqm>mq 028 Gum QH¢DJ mmmz £000 000 00050 00 000858 080000000 00000080000 80 0009852N .8008 000 00 00000 00008000H 00.0 000000.: 00 000.0 000000.: 000000.0 00 000\0 .8000000 080000 00009 0.0 00000.00 00000.0 00000.0 00000.00 00 000\0 .0000000000 0.0 00000.00 00000.00 00000.00 00000.00 0 .0000000000 00.0 000000.0 000000.0 000000.: 000000.0 03 0000 0 00.0 000000.0 000000.o 000000.N 000000.o 0 .00000 000003 00>00 0000 000000000 00000000 000000000 00000000 00000>00o< 000 00000000 00000000 00000000 00000000 00000000000 M00080w0< 00.0 00.0 00.0 00.0 0000\0000 00.0 00.0 00.0 00.0 0 .000 0000\0000 80000850800 0000 0.0 00000.0: 00000.00 00000.0: 00000.00 0000 00 0.0 00000.00 00000.00 00000.00 00000.00 0000 00 0.0 00000.00 00000.00 00000.00 00000.00 0000 00 0.0 00000.00 00000.0 00000.00 00000.0 0000 0 0.0 00000.0 00000.0 00000.0 000000.0 0000000 0.0.0“ 0 .0080003 00 00 on 00 0800482 00 8000000800800 00.0 o 00.0 o 0.00000000 000000000000 0000 0000 00 00 naaflrfiz 00 0000000000000 00<+02+ 000-02+ 000+00- 000-00- 0000 .0> 0000000000 . 0200 000 00 000000 020 020 000000 00 000000 030 000 00000 00020000 00 0000 0000000000 020 0000002 .00 00000 144 and feed consumption data indicate that the quail receiving supplemental arginine utilized their feed more efficiently. Arginase activities were significantly (P < .005) in- creased by supplemental arginine, but the dietary level of nickel was without significant effect. Liver weights were significantly (P < .005) increased by supplemental dietary arginine but no significant difference was obtained when liver weight was expressed as a percent of body weight, indicating that the larger livers were simply due to larger birds. Significant (P < .01) increases in hematocrit due to nickel and a positive nickel-arginine interaflfion were ob- served. A significant (P < .05), positive nickel-arginine interaction was responsible for an increase in the hemo- globin levels. Arginine supplementation was without signi- ficant effect on either hematocrit or hemoglobin values but did significantly (P < .005) increase total plasma protein levels. In this experiment as in experiment V, the attempts to stimulate arginase were largely unsuccessful. An attempt was made to determine the nickel concentra- tion in the livers of the quail from experiment VI. Livers from 5 to 10 birds which had received the same dietary treatment were pooled such that the final weight of each l ! ‘ .IIIII" ll ‘III‘ ||l (I I ll- ‘ l \ | Ill-Ill I 145 pooled sample was 10 to 15 g. Nickel was determined by atomic absorption Spectroscopy following wet digestion of the livers, with nitric and perchloric acids, and following a solvent extraction procedure as described by Nulford (1966). The nickel concentration of the livers ranged between 0.055 and 0.3Q6 ppm on a fresh basis. No correlation was observed between the dietary nickel level and the concentra- tion of nickel in the liver. Unsuccessful attempts were made to determine the nickel concentration of the kidney homogenates used for the arginase assay and of blood plasma. The amount of sample available in each case was so small as to make a meaningful analysis for nickel impossible by the methods employed. Tissue analyses were not attempted for any of the other experiments. Experiment VII The quail used for this experiment consisted of the third generation of the nickel-low and nickel—supplemented birds and were offspring of the quail used in experiment IV. Weights and feed use data for experiment VII are pre- sented in table 18. The initial weights of the two groups were significantly (P < .05) different but hatching weight had not been depressed by the feeding of a purified diet for two generations. No other significant difference in weights 146 TABLE 18. HEIGHTS AND FEED USE DATA OF JAPANESE QUAIL FED TWO LEVELS OF NICKEL - EXPERIMENT VII. Dietary Nickel, ppb 74 1780 Weights, g ,* iS.E.1 Initial 6.6(47)3 6.3(78) 0.1 7 days 79(35) 17.6(67) 0.3 14 days 04(35) 38.9(65) 0.5 21 days 6h.1(34) 63.0(63) 0.8 28 days 8h. 2(28) 85.0(29) 1.1 35 days 97. 2(28) 97.2(29) 1.6 Adult, 190 days 126.8(27) 131.3(25) 2.3 Males3 118.0(17) 2.9 Females 133.9(35)*** 2.7 Feed Consumption Feed/bird day (to 35 days) 6.4 7.5 Gain/feed (to 35 days) 0.35 0.41 ;Standard error of the mean. Numbers in parentheses represent number of quail for each value. 3Statistical comparisons of males and females were conducted without regard to treatment. *Signifioantly greater than lower value (P < .05). ***Significantly greater than lower value (P < .005). was observed, except, once again, females were significantly (P < .005) heavier than males. At 28 days into the experiment, the number of birds was reduced to 28 or 29 per treatment with a ratio of one male to three females in each treatment. Feed consumption data indicate that the quail receiving supplemental nickel consumed slightly more feed per day and utilized it somewhat more efficiently. No tissues were taken when the birds were killed so no arginase or hematOIOgical values were obtained. 147 Eggs were collected from the birds for the production of the quail used in experimenuslx and X. A record of the reproductive performance of the third generation is pre- sented in table 19. Average egg weight was about the same TABLE 19. REPRODUCTIVE DATA OF JAPANESE QUAIL FED TWO LEVELS OF NICKEL - EXPERIMENT VII. Dietary Nickel, ppb 74 1780 First egg, days 43 40 Total eggs laid 1553 1278 Ave. wt, g 8.76 8.63 Eggs/female bird day 0.59 0.46 No. eggs set1 144 129 Net (uncracked) 114 82 No. fertile 103 72 % Hatch (of total set) 38.9 30.5 % Hatch (of net) 49.1 47.6 % Fertile (of total) 71.5 56.2 % Fertile (of net) 90.4 87.8 Z Hatch (of fertile) 54.4 54.2 1 Eggs collected when birds were 17 to 19 weeks of age for production of fourth generation, experiment IX. as for the second generation. The number of eggs per female bird day appeared to decrease in this trial, but most of this decrease can be attributed to the failure to collect the eggs soon after they were laid. Such a delay allowed for more breakage of eggs, and therefore, the record for eggs laid is rather inaccurate for this trial. 148 Fertility and hatchability data were collected from the eggs set for production of the quail to be used in experi- ment IX but not experiment X. Eggs were collected when the birds were 17 to 19 weeks of age. The fertility data ob- tained show that fertility of the eggs was higher than for the two previous generations. Hatchability of the fertile eggs was higher for the third than the second generation for eggs collected at comparable stages of production. No difference in fertility or hatchability was observed due to nickel treatment; both groups received 1.13 percent supple- mental arginine. The eggs collected for the production of the quail used for experiment X were collected when the experiment VII birds were 27 to 29 weeks of age. Although no record was made of the fertility and hatchability of these eggs, the hatchability of the eggs was very low. Of course, this could have been due to an excessive number of cracked eggs, but the age of the laying birds undoubtedly had some effect. Eggs from each of the first three generations were stored at least two weeks at 150 C before incubation. Eggs from the second generation were stored for approximately three weeks, due to the small number of surviving females in the low-nickel group. Hatchability of the eggs stored longer than two weeks was greatly reduced. l " llll" I‘llll' ‘l . II x. ‘1 II II, 149 Experiment VIII A 2 x 2 factorial design was used in this experiment as in experiments II, III. V and VI. The casein level was in- creased from the 25 percent level used in experiments V and VI to 35 percent, as used in experiments II and III. No supplemental tyrosine was included, arginine was supplemented at 1.13 percent of the diet and the nickel level of the supplemented diet was reduced back to the 1780 ppb level used in experiments II and III. Washing of the minerals and cellulose, as in experiments V and VI, was not carried out for the present experiment. The final diets were formulated to be identical to the diets used in experiment III, in an attempt to duplicate the feathering difference noted in that trial. The only difference in the two experiments involved the use of third generation quail (offspring of experiment IV) in the present trial. Weights and analytical data of the experiment VIII quail are presented in table 20. Arginine significantly (P < .005) increased the growth rate for all weigh periods subsequent to the initial weigh period. Supplemental nickel signifi- cantly (P < .05) increased growth rate for the six and 20 day weigh periods, and approached significance at the same level for the 13 day weight. 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