'THE ROLE OR ZINC IN POULTRY NUTRITION By ROBERT HARVEY ROBERSON AN a b s t r a c t Submitted to the School of Advanced Graduate Studies at Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OP PHILOSOPHY Department of Poultry Science 1959 Approved ABSTRACT Experiments were conducted on the growing chick with the use of semipurified diets and a relatilvey zinc free environment to determine a) if the chick requires zinc, (2) the quantitative requirement of zinc for growth (3) the relative availability for the chick of zinc from the chloride, sulfate, carbonate and oxide compounds, and (A) the effect of excessive amounts of calcium and phosphorus on the amount of zinc re­ quired. In experiments with feeds composed of natural feedstuffs, studies were made on (5) the effect of high levels of zinc on growth and feed efficiency in young chicks, (6) the effect of supplemental zinc and calcium, alont or in combination, on egg production, hatchability, laying-house mortality, egg weight, Haugh score, and eggshell thickness in the laying hen. Zinc was found to be an essential nutrient for the young chick for growth, feather development, efficient feed utilization, bone growth, and maintenance of healthy skin. A deficiency of zinc caused retarded growth rate, poor feather development, retention of body down, poor feed utilization, enlarged hocks, and dermatitis of tops of feet and foot pads. The zinc requirement was twenty ppm of available zinc or less. Zinc in the form of the chloride, sulfate, oxide, and carbonate was equally utilized by the chick. Excessive amounts of calcium in the diet aggravated the zinc-deficiency symptoms and increased the zinc requirement. With the use of practical diets, zinc levels of 1,000 ppm or less, as the sulfate, oxide,and carbonate, were well tolerated. Above this level the compounds depressed growth and feed efficiency in the following increasing order: oxide, sulfate, and carbonate. The addition of zinc and calcium, alone or in combination, to a laying ration, did not affect egg production, hatchability, layinghouse mortality, egg weight, and Haugh score. Eggshell thickness was increased in cool but not warm weather by the combination of zinc and calcium, but not by calcium or zinc alone. the; r o le or zinc in poultry nutrition By ROBERT HARVEY ROBERSON A THESIS Submitted to the School of Advanced Graduate Studies at Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OR PHILOSOPHY Department of Poultry Science 1959 ProQ uest Number: 10008541 All rights reserved INFO RM ATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete m anuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQ uest 10008541 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 A ckn owledge ment s The author wishes to express his sincere thanks to Dr. Philip J. Schaible for his constant personal interest, motivation and continual guidance throughout this work. The writer is greatly indebted to Dr. Erwin J. Benne and Dr. Eustace K. Johnson for assistance in the chemical analyses and also for their interest and many helpful suggestions. The author wishes to express his appreciation to Dr. Howard C. Zindel for the use of the facilities of the Department of Poultry Science, the many courtesies which he extended, and for the constructive review of this thesis. Sincere thanks are due Michigan State University for financial support through an Assistantship and to Hardy Salt Company for financial support of part of the work through a grant. The writer is indebted to Dr. Robert K. Ringer and Dr. Richard U. Byerrum for their helpful suggestions, and constructive review of this thesis. The writer is indebted to his fellow graduate students, Charles W. Pope, Hugh p. Travis, William K. Warden and Simon T. L. Tsang for their assistance and many helpful suggestions* Above all, the author is indebted to his wife, Billie, for her constant interest and encouragement during the preparation of this thesis. Jinally, the author is greatful to his two sons Dennis and Tom and to his daughter Janet for their perseverance. TABLE OF CONTENTS Pages INTRODUCTION . ............................................... 1 REVIEW O F 'L I T E R A T U R E ........................................ 2 Zinc, an essential n u t r i e n t ............... . .......... 2 Zinc deficiency symptoms ................................ 4 Environmental effects on the development of a zinc deficiency in chicks ......................... 5 The physiological role of z i n c ......................... 9 Zinc and h o r m o n e s .................................. 9 Zinc and p o r p h y r i n s ....................... 10 Zinc containing e n z y m e s ............ 10 The interrelationships of zinc with other m i n e r a l s .................... 12 Calcium and phosphorus Copper 12 . . . . . 14 The possible relationship between zinc and unidentified growth f a c t o r s .................. 14 P R O C E D U R E ............................. 16 ZINC, AN ESSENTIAL MINERAL IDE THE GROWING- C H I C K .......... 18 EXPERIMENT I ............................................ 18 general experimental Experimental P r o c e d u r e ............................. Results and D i s c u s s i o n ......................... . 18 18 P&ges EXPERIMENT I I ............ 21 Experimental Procedure .............. ............ 21 Results and Discussion . . . . . . . . . . 21 ZINC DEFICIENCY SYMPTOMS OF THE C H I C K .......................... 25 THE ZINC CONTENT OF THE YOLK-SACOF ONE-DAY-OLD CHICKS . . . 31 EXPERIMENT I I I ........................................ 31 Experimental Procedure ............................. 31 Results and Discussion . . . . . . . . . . . . . . . 31 THE ZINC REQUIREMENT OF THE C H I C K ......................... 33 EXPERIMENT I V .......................................... 33 Experimental Procedure ............................. 33 Results and D i s c u s s i o n ............................. 33 THE TOLERANCE OF GROWING CHICKSFOR Z I N C .................. EXPERIMENT V .......................................... Experimental Procedure ...................... . . . . 37 37 37 Results and D i s c u s s i o n ............................. 37 EXPERIMENT V I .......................................... 41 Experimental Procedure ......................... 41 Results and Discussion . . . . . . . ........ . . . . 41 EXPERIMENT V I I .......................................... 46 Experimental Procedure ............................... 46 Results and Discussion 46 the availability of zinc ....................... from different compounds TO THE C H I C K ...................................... 50 Pages EXPERIMENT V I I I ................................... 50 Experimental Procedure .................................. 50 Results and D i s c u s s i o n .............. 50 EXPERIMENT I X ............................................ 53 Experimental Procedure .................................. 53 Results and D i s c u s s i o n .................................. 53 THE INTERRELATIONSHIP OF ZINC WITH CALCIUM AND PHOSPHORUS . . . 57 EXPERIMENT X .............................................. 57 Experimental Procedure .................................. 57 Results and D i s c u s s i o n .................................. 57 EXPERIMENT X I .............................................. 61 Experimental P r o c e d u r e ...................................... 6l Results and Discussion ........................... . . 6 1 EXPERIMENT X I I ............................................ 65 Experimental P r o c e d u r e ................ 65 Results and D i s c u s s i o n .................. 65 EXPERIMENT X I I I ............................................ 67 Experimental P r o c e d u r e .............. Results and Discussion ....................... 67 . . . . . 67 THE EFFECT OF SUPPLEMENTAL ZINC IN ABSENCE AND PRESENCE OF ELEVATED CALCIUM LEVEL ON THE p e r f o r m a n c e OF THE LAYING H E N ........................................ 69 EXPERIMENT X I V ............................................ 69 Experimental Procedure .................................. 69 Results end D i s c u s s i o n .................................. 70 GENERAL D I S C U S S I O N .............................................. 80 pages C O N C L U S I O N S ...................................................... 86 0 REFERENCES C I T E D .................................... APPENDIX . ..................................................... 93 LIST or t a b l e s Table Page 1 COMPOSITION OP BASAL DIET USED IN EXPERIMENT I ........... 2 THE EPPECT OP SUPPLEMENTAL ZINC ON CHICK GROWTH TO POUR WEEKS OP A C E .................................... 20 THE EPPECT OP SUPPLEMENTAL ZINC ON CHICK GROWTH, LIVABILITY a n d P E E D ...................................... 23 3 A analysis op variance op 19 chick w e ights a t p o u r WEEKS (Experiment I I ) .................................... 5 6 7 24 THE ZINC CONTENT OP YOLK-SACS OP ONE-lAY-OLD WHITE LEGHORN MALE CHICKS . ........................... CHICK GROWTH RESPONSE TO GRADES LEVELS OP ZINC, AS THE SULPATE IN THE DIET TO POUR WEEKS OP A (S3 . . . . o p v a r i a n c e o p b o d y ,w e i g e t s o p p o u r week-old CHICKS (Experiment I V ) ........................ 32 35 analysis 36 8 COMPOSITION OP THE BASAL DIET USED IN EXPERIMENT V ...............................................39 9 THE EPPECT OP VERY HIGH LEVELS OP ZINC ON THE GROWTH RATE AND PEED CONVERSION OP CROSS-BRED WHITE LEGHORN COCKERELS; ANALYSIS OP VARIANCE OP 5-WEEK CHICK WEI (SITS (Experiment V ) ................................. 40 10 COMPOSITION OP BASAL DIET USED IN EXPERIMENT 11 EPPECT OP THREE LEVELS OP DIFFERENT SALTS OP ZINC UPON GROWTH, LIVABILITY AND PEED EPPICIENCY OP CHICKS TO POUR WEEKS OP A 0 3 ............................... 44 12 V I ............. A3 Analysis op variance op chick weicuts (Experiment V i ) . . ..................................... 45 13 THE EPPECT OP AN EXCESS OP ZINC ON CHICK PERPOBMANCS TO 4 WEEKS OP A 0 0 ............................ 48 14 ANALYSIS OP VARIANCE OP POUR-WEEK CHICK WEI GETS (Experiment V I I ) ......................... 15 THE AVAILABILITY OP ZINC PROM TWO COMPOUNDS TO THE CHICK TO POUR WEEKS OP A G E .................... G9 51 16 ANALYSIS OF VARIANCE OP THE WEI GHT 05' POURWEEK-OLD CHICKS (Experiment VIII) ................... 17 THE AVAILABILITY OP ZINC PROM THESE COMPOUNDS TO THE CHICK TO POUR WEEKS OP A G E ................. 18 analysis 19 THE EPPECT OP ELEVATED LEVELS OP DIETARY CALCIUM ON THE CHICKS TO POUR WEEKS OP AGE ........ 20 ANALYSIS OP VARIANCE OP CHICK WEIGHTS TO POUR WEEKS OP ACS (Experiment X ) ....................... 21 THE EPPECT OP ELEVATED CALCIUM LEVELS ON THE ZINC REQUIREMENT OP THE GROWING CHICK TO POUR WEEKS OP A ® . . . ......................... 22 ANALYSIS OP VARIANCE OP POUR-WEEK WEIGHTS (Experiment XI) .................................... 23 THE EP5ECT 05' A HI GH LEVEL OP DIETARY PHOSPHORUS ON THE CHICK REQUIREMENT POR ZINC TO POUR WEEKS GP A G S ...................................... 24 ANALYSIS OP VARIANCE OP CHICK WEI (SITS AT POUR WEEKS OP A (3) (Experiment XII) ..................... 25 THE in vitro REMOVAL OP ZINC PROM SOLUTION BY MINERAL SUPPLEMENTS........................... 26 DIETS POR REARING PULLETS USED IN EXPERIMENT XIV . . . 27 BASAL DIET POR EXPERIMENT X I V ................ 28 ^HE EPPECT OP ZINC IN THE PRESENCE AND ABSENCE OP ADDITIONAL CALCIUM ON THE PERFORMANCE OP THE LAYING HEN ............................... 29 ANALYSIS OP VARIANCE OP E G G PRODUCTION (Experiment XIV) ................. o p v a r i a n c e o p p o u r -w e e k - o l d c h i c k V.EICHTS (Experiment I X ) ........................... . . . . T&Lle Page 30 analysis op variance op e g o weiget (pebruary); ANALYSIS OP VARIANCE OP E G G VOSI GHT (JUNE ) (Experiment X I V ) ............................................ 76 31 ANALYSIS OP VARIANCE OP E G G SHELL THICKNESS (PEBRllARY); ANALYSIS OP VARIANCE OP EGG SHELL THICKNESS (JUNE) (Experiment X I V ) ....................... 32 ANALYSIS OP VARIANCE OP EAUCH SCORE (PEBRUARY) ANYLSIS OP VARIANCE OP HAUCR SCORE (JUNE) (Experiment X I V ) ............................ 77 LIST OF FIGURES Figure 1. 2. 3. 4. Page THE NORMAL CHICKEN (TO?) VS THE ZINC-DEFICIENT CHICKEN (BOTTOM)..................... 27 THE FOOT OF A NORMAL'CHICKEN (TOP) VS THE FOOT OF A ZINC-DEFICIENT CHICKEN ( B O T T O M ) ................. 28 THE FOOTPAD OF A NORMAL CHICKEN (TOP) VS THE NECROTIC FOOTPAD OF A ZINC-DEFICIENT C H I C K E N ........ 29 THE HOCK OF A NORMAL CHICKEN (TOP) VS THE ENLARGED HOCK OF A ZINC-DEFICIENT CHICKEN ( B O T T O M ) ........... 30 Introduction Por many years poultrymen have been concerned with the proper feeding of farm poultry. As the science of poultry nutrition has advanced, the demand for more qualitative knowledge for all the nutrients has developed and assumed great importance. Probably in no area of animal nutrition has the qualitative and quantitative determination of essential nutrients received greater impetus than in the growing chick. In recent years, the nutrient requirements of the laying hen have also been intensively investigated. In view of the report of Tucker and Salmon (1955) that swine fed practical rations required additional zinc, it seemed desirable to determine if supplemental zinc is needed in the ration of chickens. The research described in this thesis is concerned with the study of zinc in the nutrition of poultry. Qualitative and quantitative studies were made to determine if the growing chick requires zinc. Toxicity studies were undertaken to determine the tolerance level of zinc for the growing chick and the interrelationships of zinc with calcium and phosphorus were also studied. With the laying hen, the effect of zinc on egg production, egg weight, egg shell thickness, and embryonic development was studied. 2 REVIEW OF LITERATURE Zinc, an essential nutrient The presence of zinc in plant and animal tissues (Lechartier, 1877) and in the muscle and liver of man (Raoult, 1877) was first established in 1877 * This followed by several years the work of Raulin (1869) » a pupil of Pasteur, who determined that zinc was needed for growth of the mold Aspergilus niger. Much-later Tsui (19^8) provided a possible explanation for the role of zinc in plants. Zinc is an essential nutrient for groxsrth because the tryptophane content is lowered in zinc-deficient plants. Since indoleacetic acid, a plant growth hoimone, is produced from tryptophane, a deficiency of this amino acid causes a deficiency of this hormone and results in a lowered growth rate. During the 1920*s, several investigators (McHargue, 1926; Hubbell and Mendel, 1927) made attempts to determine whether zinc performed an essential function in animal nutrition but met with limited success. The diets employed were so deficient in essential nutrients, especially vitamins, that even with added zinc the experimental rats and mice survived only a short time. Todd, Elvehjem and Hart (193*0 were more successful - by using purified diets they demonstrated that zinc is essential for the growth of rats. Diets adequate in the other nutrients but low in zinc were subsequently used by the Wisconsin workers and others. These studies disclosed unquestionably the importance of zinc in the nutrition of mice as well as rats (Day and Skidmore, 19**7; Edwards et a l ., 1958). 3 Uishimura (1952) found, that newborn mice, which were removed from their mother before receiving colostrum and placed with foster mothers in later stages of lactation, developed disorders similar to those report­ ed for zinc deficiency, The administration of colostrum or zinc salts at birth prevented the development of the condition. In early attempts to investigate the essentiality of zinc for rats, exhaustive purifi cat ions of the casein in the diet was necessary to produce a deficiency. Recently Edwards et al. (1958) easily produced a zinc de­ ficiency in rats by the use of isolated soybean protein. Tucker and Salmon (1955) aad Luecke e_t al. (195&) found that zinc prevented parakeratosis and thus was an essential mineral for the pig. Titus and Ginn (1931) noted that rice bran was very effective in the prevention of perosis in chicks. In the following year, the zinc content of rice bran was found to be higher than in most feedstuffs and an experiment was initiated to determine if this mineral had any effect on perosis. The perosis preventative effect of rice bran was not found to be due to its zinc content. This constituted the first investigation to determine if the chick benefits from zinc. Insko ejfc al. (1938) also reported that zinc was ineffective in preventing perosis. However, Wilgus et_ a l . (1937) concluded that zinc decreased the incidence of perosis but was less effective than manganese. 0*liell and Savage (195?) reported that the addition of 6.6 ppm and 56 ppm of zinc, or five percent of distillers solubles promoted the growth of chicks. The chicks were maintained in galvanized (zinc-coated) batteries, and fed a Drackett Assay C-l p rotein - Cerelose type diet which contained 1.72 percent calcium, 0.6 percent phosphorus and 52 ppm 4 of zinc. Roberson and Schaible (1958) used plastic-coated or glass equipment, purified rations that contained exceedingly low contents of zinc and distilled drinking water and thus proved that the chick requires zinc for growth, feather development and maintenance of normal skin condition. Edwards et. al. (1958) produced a zinc deficiency in the chick using plastic equipment or epoxy resin-coated batteries; they did not produce the condition in regular galvanized batteries. Uorris et al . (1958) and Morrison and Sarett (1958) also reported that the growing chick requires zinc. Supplee ejb al. (1958) reported that the growing turkey requires zinc for rapid normal growth, feather development and prevention of a non­ rachitic hock disorder which denoted abnormal bone development. Zinc deficiency symptoms Eollis et al.(l9hl) described zinc deficiency symptoms in the rat. They were extreme parakeratosis of the esophagus with a thick layer of partially keratinized cells, lesions of the buccal cavity and in some cases, corneal changes of the eyes. The skin showed hyperkeratinization with loss of hair follicles and hair. Kernkamp and Eerrin (1953) described a condition in swine, which they termed parakeratosis, characterized by retarded growth, diarrhea, vomiting, anorexia, severe dermatitis and finally death in acute cases. Tucker and Salmon (1955) obtained parakeratosis in practical swine rations containing 3h - hh ppm zinc and found that high levels of calcium supplements aggravated the condition. Symptoms similar to those reported by Kernkamp and Eerrin (1953) were observed. C^Dell and Savage (1957) described symptoms caused by a slight deficiency of zinc in the chick as depressed growth ana impaired bone 5 development. According to Roberson and Schaible (1958a, 1958b) conditions causing an acute zinc deficiency in the growing chick produced depressed growth, poor utilization of feed, failure to feather normally with retention of body down and a ragged condition of feathers and dermatitis of the tops of feet and on footpads which caused a highstepping walk, Norris et_ al. (1958) observed similar deficiency symptoms and, in addition, found a decrease in bone ash* Symptoms were produced when plastic-coated equipment liras used but were not produced when chicks were maintained in galvanized batteries. In turkey poults, Supple© e_fc ad. (1958) noted that a zinc defic­ iency caused depressed growth, poor feathering, and impaired bone dev­ elopment which resulted in a high incidence of hock disorders. Symptoms attributable to a zinc deficiency have not been reported for cattle, sheep, dogs, cats, ducks, geese, pheasants or other poultry* However, in view of the essentiality of zinc for the rat, pig, chick, and turkey, it seems likely that zinc is an essential nutrient for other farm animals and pets. Environmental effects on the development of a zinc deficiency in the chick Since the zinc coating has been noted to erode quite rapidly on starting batteries, especially the waterers and feeders, only materials preventing the exposure of chicks to extraneous zinc were employed when the research reported in this thesis was initiated. Mehring (1956) observed that enough zinc is dissolved from the gal­ vanized water pans to add 3?pm to the drinking water* If it is assumed that the bird consumes two to three times as much water as feed, an additional 6 to 19 ppm would be added to the level provided 6 in the feed* In the preliminary experiments, plastic cages, glass feeders and waterers, and reagent grade minerals were used. Subsequent experiments were conducted in Jamesway Batteries with all exposed galvanized parts coated with epoxy resin. The galvanized waterers were replaced with one- gallon glass baby chick founts with plastic bases. Previous research on unidentified minerals, reported by Morrison e~k al, (I95 5. 1956) w&s conducted in galvanized Petersime Batteries. In the early experiments, the galvanized waterers were not coated; in later work, they were sprayed with plastic varnish. In proving the adequacy of their diet, graded levels of zinc ( 5 to 100 mg/kg) were added to the basal diet which contained h.8 ppra added zinc without producing a sig­ nificant growth response in chicks reared in zinc-coated batteries with zinc-coated waterers. 0*Bell and Savage (1957) obtained a chick growth response when a semi-purified diet was supplemented with graded levels of zinc, but as stated previously the work was conducted in galvanized batteries. The basal diet contained 1.75 percent calcium and 0.6 percent phosphorus. In view of the experimental work reviewed on the calcium-zinc inter­ relationship, the zinc deficiency they obtained could have resulted from the high level of calcium. Deficiency symptoms were not nearly as severe as those on extremely low levels of zinc and normal levels of calcium reported by Roberson and Schaible (1958)* la other laboratories in which plastic-coated equipment was .sed, researchers (Eorris et a l . . 1958; Pensack and Klussendorf, 1958) substantiated this interpretation. Edwards ejt al. (1958) also studied the effect of enviroment on 2inc deficiency in the chick. Chicks housed in galvanized batteries 7 with galvanized waterers did. not respond to supplemental zinc added to diets apparently deficient in zinc. Chicks, however, developed severe zinc deficiency symptoms when housed in plastic end stainless steel ca-ges and were given distilled water in glass waterers. A less severe, hut quite variable, zinc deficiency was obtained with chicks housed in galvanized batteries and given distilled drinking water. When chicks were fed a zinc—deficient diet, in open wooden troughs, growth was increased, but when the troughs were covered to prevent fecal contam­ ination, no growth increase occurred. When the galvanized batteries were coated with epoxy resin, a very severe zinc deficiency, similar to that in plastic cages occurred. Morrison and Sarett (1958) obtained a growth response in chicks from graded levels of zinc (0 ,5 *25, 100 ppm) added to a semi-purified diet (Morrison et al.1956). The birds were maintained in galvanized batteries and given distilled water in glass jars. A zinc deficiency in this study consisted of a growth depression, and an enlargement and elongation of the tibiotarsal-tarsometatarsal (hock) joint. was not affected. Bone ash The deficiency symptoms were much less severe than those in which a similar diet was used but the birds were reared in plastic-coated batteries. Water contaminated with zinc has been another source of experimental error in nutritional research. Edwards ejb al. (1958) found that the tap water he used contained 1.0 ppm zinc and that this gave a chick grov/th response over distilled water when a semi-purified diet was used. Romanoff end Romanoff (19^9) state that almost all the zinc in the egg is in the yolk. In work reported in this thesis, it is concluded that ^he quantity of zinc in the yolk sac of day-old chicks is too small to cause significant experimental error in growth, experiments. In agreement with this conclusion, deutectomized chicks grow at the same rate as normal chicks when fed a zinc-deficient, semi-purified diet (Edwards £t al. 1958). The source of protein in experimental rations has also affected the results obtained in zinc studies. O'Dell and Savage (1957) obtained a growth response in chicks when supplemental zinc was added to a semipurified diet containing Drackett Assay- C-l Protein but failed when casein or alpha— protein replaced the soybean x>rotein* The soybean protein, casein and alpha-protein contain similar quantities of zinc* Norris ejb al. (1958) found that a total zinc content of 20 mg/kg was required by the chick when a casein-type diet containing 5«2 mg/kg was fed. On the other hand, -when a Drackett Assay C-l Protein diet containing nine ppm zinc was used a total of thirty ppm zinc was required. They concluded that the zinc in the soybean, protein was not utilizable by the chick. In studies with poults, Supplee e_t al. (1958) rarely observed abnor­ mal hocks and poor feathering when an autoclaved soybean protein— starch type diet was fed; whereas, a high incidence of hock disorder and poor feathering was observed on & soybean protein-sucrose-type diet. Possibly the improvement was due to the release of the zinc from the soybean protein during eutoclaving so that it became more available to the poults. However, it must not be overlooked that corn starch contains 73 ppm zinc; whereas, sucrose contains very little zinc. Consequently, starch, when used as a primary energy source in a diet could contribute a considerable quantity of zinc. Edv/ards et al. (1958) produced without great difficulty a zinc 9 deficiency in rate vjhen soybean protein was used. This is in contrast with early attempts to produce a zinc deficiency in rats with a caseintype diet. Exhaustive purification of the casein was necessary to produce a zinc deficiency in the rats. Although Morrison and Sarett (1953) obtained a substantial grov/th response from zinc when soybean protein was used, they obtained only a slight response when casein or gelatin was used as the protein source. Both type diets contained approximately 30 ppm zinc. The addition of excess calcium to the soybean protein diet depressed weight gains and feed efficiency. Supplemental zinc counteracted these effects. The addition of excess calcium to the casein-and gelatin-type diet failed to depress growth or produce other symptoms indicative of a zinc deficiency. M s h i m u r a (1952) pointed out that the level of zinc in mouse colostrum was great enough to protect newborn mice from a zinc deficiency; whereas, the zinc content of mouse milk was inadequate for normal performance of baby mice. The physiological role of zinc Zinc and Hormones The crystallization of insulin as a zinc salt by Eisher and Scott (1935) l©d to the misconception that zinc was an integral part of the insulin molecule. This thought persisted in spite of the fact that other metals, namely nickel, cobalt, and cadmium, can be used in the crystallization. Amorphous insulin is as active physiologically as crystallin insulin, and there is little convincing evidence that zinc and insulin must combine in vivo to form an active compound (Vallee, 1957)* There is far more zinc in the pancreas than would be necessary for insulin activation and some of it is now accounted for by its presence in carboxypeptidase ( Vallee and Neuroth, 1955). Zinc and Porphyrins It appears certain that a zinc uroporphyrin exists in higher animals as well as in mollusk ( snails, etc. ). The zinc complex of the Waldenstrom uroporphyrin (1937) has been shown to be a constant consti­ tuent of the urine and feces in cases of intermittent, acute porphyria. In congenital cases of this condition, the porphyrin is excreted in the free state and zinc uroporphyrin is found in the liver. A zinc cop­ roporphyrin has been found in cases of lead poisoning and in the urine of victims of acute rheumatic fever ( Vallee, 1957). Zinc-Containing Enzymes In contrast to the highly-colored iron and copper proteins and enzymes, the zinc proteins and enzymes are colorless. This fact has probably been responsible for the slow recognition of the importance of zinc in enzymes. Keilin and Mann (19^0) first observed that the carbonic anhydrase ( an enzyme which catalyzes the reaction HgO + C O ) of the blood cells of oxen contained 0*33 percent zinc which does not exchange freely with ionic zinc. Zinc is an integral part of the molecule of carbonic anhydrase and the removal of the metal results in irreversible inactivation. Carbonic anhydrase is found in almost all tissue but erythrocytes contain the greatest quantity. Practically all the zinc of erythrocytes is present in their carbonic anhydrase (Vallee, 1957). Wachtel et al, (19A1) studied the carbonic anhydrase activity in rats deficient in zinc. Althou^a. a slight anemia was observed, there was no lowering of the carbonic anhydrase activity of the erythrocytes. Carboxypeptidase of bovine pancreatic juice was shown by Vallee and Bfeurath (1955) to be a zinc-containing enzyme. This exopeptidase splits terminal amino acids from peptides having a free alpha carboxyl group adjacent to the peptide bond. The enzyme contains one atom of zinc per molecule and the zinc is indispensible for its enzymatic activity. A number of zinc-containing dehydrogenases ( enzymes which remove hydrogen from a substrate in biological oxidation) have been reported and zinc is necessary for their activity. All of the enzymes are dependent upon diphosohopyridine nucleotide (DPR) for their activity. For example, Vallee and Hoch (1955) stated that yeast alcohol dehydrogenase contained, as an integral part, four atoms of zinc per molecule of enzyme. Theorell _et al. (1956) found that horse liver alcohol dehydrogenase contained two atoms of zinc per molecule of enzyme and that zinc is essential for enzymatic activity. Glutamic dehydrogenase of beef liver and lactic dehydrogenase of rabbit skeletal muscle were found to contain zinc as an integral part of the enzyme system (Vallee et. al. (1955* 1956). The zinc in these enzymes explains the hij^a content of this mineral in the liver and retina of the eye. The metabolic role of zinc in the development of bone, skin and feathers is unknown at the present time* O ’Dell and Savage (1957) obtained impaired bone development in zinc-deficient chicks while Horris e,t al. (1958) found a decrease in bone ash. Roberson and Schaible (1958) described dermatitis of the feet ana poor feather development in zinc-deficient chicks. As yet, no specific role of zinc in metabolism of these tissues has been reported. Mawson and Fisher (1951) found high concentrations of zinc in the prostate of the rat, and Mawson (1952) noted a similar condition 12 in the rabbit and man. Gunn et_ al. (1956) reported that Zn^5 was selectively taken up by the dorsolateral prostate of the ratspecifically by the cells of the lateral acini ( Gunn and GodLd, 1956, 1957) and elaborated in the glandular secretion ( Gunn and Gould, 1956). ejacula.tion. The Zn65 was found throughout the uterine tract after However, the removal of large amounts of the zinc from the eja-culate did not alter fertility or fecundity ( Gunn and Gould, 1958 ). U lus , the zinc in the prostake gland does not appear to play any particular role in reproduction. Zinc is, however, found in the sperm (Mawson, 1953) aad doubtlessly plays an indispensable part in its production or metabolism. Serum inorganic phosphorus levels were depressed in pigs with parakeratosis but serum calcium, magnesium and alkaline phosphatase were not appreciably affected ( Stevenson and Earle, 1956). Significant increases in the zinc concentration of blood plasma, liver and kidney followed supplementation of a ration with 50 PP™ zinc (Hoekstra e_t al. 1956). The zinc concentration of muscle, spleen, lungs and skin were not affected by zinc supplementation. The addition of two percent bone meal to a non— supplemented diet did not affect the zinc concentration of organs. However, the addition of the two percent bone meal to a diet supplemented with fifty ppm zinc resulted in decreased zinc concentration of the liver and kidneys but did not affect that in the other organs. Morrison and Sarett (1958) reported that zinc deficiency in the chick has no effect on blood-sugar, serum phosphatase, lactic acid and alkaline phosphatase levels, but results in depressed intestinal alkaline phosphatase. Zinc deficiency did not affect the percentage 13 of ash in the tibiotarsal bones or on the composition of the liver or carcass. The interrelationship of zinc with other minerals Calcium and 'phosphorus Tucker and Salmon (1955) reported that the level of calcium and/or phosphorus affected the incidence and severity of parakeratosis. Increasing the calcium level of the ration decreased weight gains and hastened the onset of parakeratosis (Lewis ejfc al. , 1956). Increasing the phosphorus level of the diet by the addition of monosodium phosphate had no effect on weight gains but decreased the skin lesions when compared with the results obtained with a basal diet containing 0.47 percent phosphorus and 0.82 percent calcium. One hundred ppm supplement­ al zinc were necessary to completely prevent parakeratosis. Luecke et^ a l , (1956) produced a 100 percent incidence of par­ akeratosis in swine by feeding 1.50 percent calcium and 0.80 percent phosphorus in a ration containing 31 ppm zinc. Twenty ppm supplemental zinc prevented parakeratosis in nine of ten pigs. When the calcium level was reduced to 0.98 percent of the diet and phosphorus to 0.70 percent, the incidence of parakeratosis was lowered to three of ten pigs. Supplementation of this ration with twenty ppm zinc completely pre­ vented parakeratosis. The experience of many workers suggests that the interaction between calcium and zinc may be within the intestinal tract. However, the results obtained at the hands of different investigators suggest that other factors may be involved in this interaction. For example, recently Hoefer at ad. (1958) reported tha.t copper would prevent parake ratosis. Ik Copper Smith and Larson. (1946) depressed growth and produced anemia in rats by feeding a diet excessively high in zinc* The addition of c0PPer to the diet corrected the anemia but did not restore normal growth* Similarly, Gray and Ellis (1950) produced anemia in rats by feeding a high level of zinc but growth was not depressed. Supplemental copper likewise corrected the anemia. The possible relationship between zinc and unidentified factors Accoring to Dannenburg jst al. (1955) the ash of distillers dried solubles produced a growth stimulation which was equivalent to approximately one-half that obtained from the unashed material. The addition of 0*5 percent calcium to the basal diet, which already con­ tained 1.08 percent calcium, depressed growth. Addition of three percent distillers dried solubles restored normal growth. O ’Dell and Savage (1957) ascertained that zinc would replace part, if not all, of the unidentified minerals supplied by distillers dried solubles. Morrison and Sarett (1958) found that the addition of 2.5 percent of dried fish solubles to an isolated soybean-protein-cerelose type diet (protein level kept constant) produced a ten percent growth response; whereas, no growth response was obtained in the presence of ten ppm of added zinc. Furthermore, fish solubles corrected the depression in growth brought about by the addition of 0.5 percent excess calcium. Kratzer e_t al. (1958) produced a hock disorder and depressed growth in turkeys by feeding an isolated soybean protein basal diet containing 1.88 percent calcium, 0.75 percent phosphorus and 25 p ,-m zinc. The addition of 38 PP® of supplemental zinc to the diet or replacement of the isola.ted soybean meal with regular soybean meal containing 66 ppm of zinc was required to give optimum growth and have a maxium effect in reducing the incidence of enlarged hock. He concluded that the zinc in the soybean meal corrected the depressed growth and enlarged hock. Soybean meal is a source of an unidentified factor. IS GENERAL EXPERIMENTAL PROCEDURE Similar procedures for the different experiments are outlined in this section; departures therefrom, are described under the individual experiments. The randomization of chicks to pens or replicates was made in the following manner: one-day— old chicks received from a commercial hatchery or hatched at Michigan State University were weighed and distributed into consecutive weight groups, such as 36- 38 , 39-41, etc. each having a weight range of three grams. weights were discarded. The groups with the lowest and highest An equal number of chicks from each weight group was randomized to each pen. Thus, this method minimized the effect of differences in initial weights of the chicks on the final results. Lots were randomly assigned to replicates. In experiments with practical diets, each lot appeared in each battery, therefore, each battery held one replicate of all treatments. This method was used to prevent differences due to position of the batteries in the room. Birds were maintained in electrically-heated, starting batteries with raised wire floors. Where purified diets were used, all battery parts were coated with epoxy-resin, except the wire floors and dropping pans which were coated with shellac or clear metal primer. The gal­ vanized (zinc plated) water troughs were replaced with one-gallon, glass, baby chick founts with plastic bases. The batteries were kept in a special room which was relatively free of dust. Thus, precautions were taken not only to reduce the amount of zinc in the test ration but also to prevent accidental zinc contamination from equip ~ ment end environment. Where practical diets were used, galvanized batteries (uncoated) were utilized. Group weights of the birds were recorded at the end of two weeks c-nd individual weights at the end of the experiment. Feed consumption was obtained by lot in experiments with purified diets and by replicate with practical diets. In preparation for sn experiment, the heaters on the batteries were turned on and regulated at least one day prior to the arrival of chicks. Feed was placed in the troughs and, at the start, on paper which was laid on the wire floors. Experimental diets and water were supplied ad libitum. In experiments with purified diets, distilled water was used; with prac­ tical diets, tap water was employed. Hardware cloth (uncoated) was placed on the feed in the troughs to minimize feed wastage when practical diets were utilized. With purified diets, this was not practical because of the type of battery used. Purified diets were mixed in a plastic, twin-shell blender or in a small, steel concrete mixer. Other diets were mixed in a one— half ton upright mixer or in a small concrete mixer. Feeds prior to feeding were handled as follows: Purified diets were placed in plastic or paper bags and stored in a refrigerator at 35° F. Practical diets were stored in galvanized cans in the starting battery room at room temperature ( approximately 60° F). Experiment I In an exploratory experiment, two groups of five White Leghorn cockerels, hatched at Michigan State University, were allocated to two pens having plastic mesh sides and rubber mesh floors. One group of chicks was fed a basal diet which was a slight modification of that used by Morrison et_ al. (1955) Table 1, but not including the supplemental zinc. of zinc. The basal diet contained 16 ppm The second group was supplied the basal diet supplemented with 100 ppm of zinc as the sulfate. were added to the diets. Analytical reagent grade minerals Distilled water was supplied for drinking and both feed and water were provided ad libitum in glass feeders and waterers. The experiment was terminated when the birds were four weeks of sge. Results and Discussion: The results are shown in Table 2. The group receiving the basal diet grew poorly, developed dermatitis of the feet, failed to feather properly and developed a high-stepping walk. Symptoms began to appear about the 14th and were severe by the 21st day. The group fed the zinc-supplemented diet grew satisfactorily during the four-week test period and did not exhibit any of the symptoms noted in the zincdeficient group. 19 TABLE 1 COMPOSITION OF BASAL DIET USED IN EXPERIMENT I Ingredients Grlucose^ Isolated soybean protein^ Corn oil Ground cellulose^ DL-Methionine Glycine CaHPOj, CaCOo KH NaCl Mgso ^ FeSO.*?H 20 MnSQ^.HoO KI CuSO^ *5 ^ 0 C 0CI 2 '6H 2O Na^MoOj^.2HoO Choline chloride Inositol Niacin Ca Pantothenate a-Toeopherol acetate Thiamin HC1 Riboflavin Pyridoxine HC1 Folic acid Menadione Biotin Vitamin A Vitamin D~ Vitamin B^g Amt./lOO gins 61.55 gra 25.57 3.00 3.00 0.70 0.30 2.151 1.492 0.867 0.600 0.25 it 0.0333 0.0333 Tl nig 0.26 mg 1.67 mg 0.17 mg 0.83 150.00 mg 25.00 mg 5.00 mg 2.00 mg 2.00 mg mg 1.00 mg 1.00 0.45 mg 0.40 mg mg 0.05 mg 0.02 500 IU 37.5 icu 2.00 meg 1- Cerelose, C o m Products Sales Co., 440 New Center Bldg., Detroit, Michigan 2- Drackett Assay Protein C-l, The Drackett Products Co., Cincinnati 32* Ohio 3_ Solka Floe, The Brown Co., Berlin, New Hampshire 20 TABLE 2 THE EFFECT OF SUPPLEMENTAL ZINC ON CHICK GROWTH TO FOUR WEEKS OF AGE Lot Zinc Additions to the basal (ppm) Chick weights at k weeks (gm) 1 None 100 2 100 226 Experiment II In view of the surprising results obtained in the preliminary experiment, a more comprehensive experiment was designed to substantiate the preliminary experimental findings. In this experiment the isolated soybean protein was washed with an HC1 solution at pH 4,6 in an attempt to remove its residual zinc. The protein was dried for 36 hours at a temperature of approximately 100° C. Two basal diets were used, one contained unv/ashed isolated soybean protein and the other washed isolated soybean protein. They contained 19 ®-nd 7 ppm zinc, respectively, and were derived from the formula show in Table 1. To each basal ration was added 100 ppm of zinc. White Meateor X White Rock, one-day-old, sexed chicks were allotted as previously described into 12 pens of five males end five females each. birds. E©-ch treatment was applied to three replicates of The chicks were maintained in a special laboratory which was kept relatively free from dust m d at a temperature of 75° P. Feed consumption was recorded for each treatment* Results and Discussion: The results are presented in Table 3. An analysis of variance of the data (Table 4) revealed a significant difference (P among lo^s and a significant difference (p responses of the two sexes. .01) .05 ) between the Comparisons between treatments were made by Duncanfs method (1955). With both basal rations, the addition of zinc to the diets increased growth significantly (P .01). Extracting the protein in the diet depressed growth significantly (P .01) in the absence of 22 supplemental zinc and ( P ^ . 05 ) in the presence of supplemental zinc. Extracted protein in the presence of supplemental zinc did not depress growth significantly at the one percent level of probability. As in Experiment I, the unsupplemented basal diet produced poor growth, retention of body down with poor feather growth, ragged feathers, enlarged hocks, a severe foot dermatitis and a high— stepping walk. The birds huddled near the source of heat, were inactive, but never emaciated. first week. Retardation of growth was apparent at the end of the Dermatitis began to appear at the second week and was extremely severe by the end of the third week. The addition of zinc to the diet produced normal growth and prevented all the deficiency symptoms. The livability of chicks receiving supplemental zinc was better than that of chicks receiving no supplemental zinc. Usually, chicks with the more severe symptoms died first. Feed utilization was improved by supplements! zinc both in the presence of washed and unwashed protein. Washing the protein decreased the efficiency of feed utilization. It is obvious that extracting the isolated soybean protein is unnecessary to produce a zinc deficiency if other possible zinc con­ taminants are avoided. The zinc content of the natural protein is low enough, or perhaps not available so that an a cute zinc deficiency can be produced in the chick. 23 TABLE 3 THE EFFECT OF SUPPLEMENTAL ZINC CN CHICK GROWTH, LTV ABILITY A m FEED EFFICIENCY TO FOUR WEEKS OF AGE Lot Treatment Average chick at 4 wks. (gin) Surviving chicks of 30 started Gain/feed 1 Basal I (unwashed protein) 183 27 0.45 2 Basal I + 100 ppm zinc 363 29 0.69 3 Basal II (washed protein) 101 28 0.35 4 Basal II + 100 ppm zinc 333 29 0.62 24 TABLE 4 ANALYSIS OF VARIANCE OF CH.ICK WEIGHTS AT FOUR WEEKS (Experiment II) Total Subclass Degrees of freedom Mean square F values Calculated vr\ O• II Ph Source of variation P = .01 112 23 56,609 30.09** 2.11 Lot 3 too, 752 213.39** 4.04 Replicate 2 3.334 Sex 1 to, 758 T X R 6 615 0.32 2.21 T X S 3 4,412 2.34 2.71 R X S 2 5,634 3.00 3*10 T X R X S 6 3.354 1.78 2.20 89 1,878 Error 3.10 1.77 23.80** ** Significant (P<*.01) Comparisons among lots at 5 percent level of probability Lot No* 4 wk. wt. 3 1 101 183 4 2 333 363 4 2 At 1 percent level of probability Lot No. 4 wk. w t . 3 1 101 183 322____262 6.93 Zinc deficient chicks are compared with normal chicks in the following figures. deficiency. Figurel shows the stooped posture typical of zinc Wing feathers grew to a limited extent but became rough- due to the absence of barbules. The weight of the normal chick was 363 grams as compared to 183 grams for the zinc—deficient chick, thus growth is severely impaired in a zinc deficiency. Figure II shows the dermatitis which is concentrated mainly between the toes and on the skin covering the junction of the Tarsometatarsus with the phalanges. The footpads of a normal and a zinc-deficient chick are shown in Figure III. The dermatitis of the footpads becomes extremely severe, necrotic, and often bleeds. These conditions cause the high— stepping walk characteristic of a deficiency. In Figure IV the hocks of normal and zinc-deficient chicks are shown. The enlarged hocks resemble those of a manganese deficiency but there is no slipping of the tendon. As mentioned before, these symptoms can be overcome by supplementation of the ration with zinc. Other researchers have also described zinc deficiency symptoms. O'Dell and Savage (1957) ahd Morrison and S&rett (1958) reported that symptoms of a zinc deficiency in chicks included depressed growth, shorter and thicker long bones and decreased ash content of the leg bones. Since their experiments were conducted in galvanized batteries with substantial amounts of zinc in the basal diet, it is considered that the symptoms given represent a mild, rather than a severe, zinc deficiency. Pensack and Klussendorf (1957) a^d Norris et al. (1958) found zinc deficiency symptoms exactly as described by this author. Norris et al (1958) also made histological examinations of the enlarged bone, which, revealed a derangement of the cartilage cells in the zone of cal cification similar to that found in manganese and choline deficiencies These symptoms were also reported by Edwards e_t al. (1958). Zinc deficiency symptoms of the growing chick as noted by this author, and other workers are depressed growth, impaired bone devel­ opment causing enlarged hocks and shortened long bones, failure to feather properly, severe dermatitis of tops of feet and between toes and on the footpads, and poor utilization of feed. to the diet prevents all the deficiency symptoms. Zinc additions 2Z FIGURE I. THE NORMAL CHICKEN (TO?) VS THE ZINC-DEFICIENT CHICKEN (BOTTOM) 28 f i-ier FIGUEE 2. THE FOOT OF A NOKM&L CHICKEN (TOP) VS THE FOOT Off A ZINC DEFICIENT CHICKEN (BOTTOM) r%'m' 'aC $}nm «r ty ^ •V 29 FIGURE 3. THE FOOTPAD OF A EGBMAL CHICICEH (TOP) VS THE E35CROTIC FOOTPAD OF A ZIHC-EEFICIENT CHICKEE (BOTTOM) 30 / PI GOES A. THE HOCK OP A NORMAL CHICKEN (TOP) VS THE ENLARGED HOCK OP A ZINO-DEPICIENT CHICKEN (BOTTOM) *: , I ^ ' \ v **f'^' ? ~ >v • :,‘,'■.'/A * T V.- . • ? .*•’ • "<* ’ ,.v . • " '"'K J'X : r ' y 's & V '- f : . . . *•-•>■ - -s’ - * V .&• A H A A * ■ ,;v- *1* * - ''• '"'>■£ • ■•*■. '*' * / ■ •:: *•*’• * : f ~ $<♦": 31 EXPERIMENT III This work was conducted to determine if the yolk sac of day-old chicks contained enough zinc to induce a sizable error into experimental growth data. The yolk-sacs from one-day— old. White Leghorn, male chicks were carefully removed, weighed, and placed in separate 200 ml Erlenmeyer flasks. To each flask was added 25 ml of concentrated HNO^. • The contents were digested overnight on a steam bath* To each flask containing the clear digested liquid was added eight ml of perchloric acid. The contents in the flasks were heated on a hot plate until dry then taken up in fifty ml of 0*1 N HCL. Aliquots in duplicate from each flask were ahalyzed for zinc (Benne, 1955)* The results were combined into four lots (Table 5) according to the yolk sac weights. Results and Discussion The zinc content of the yolk sac seems to be quite small. Only two grams of feed containing twenty ppm of zinc would equal the zinc content of the entire yolk sac. Consequently, differences in zinc content of retained yolk sacs would not introduce experimental error into the results previously obtained. This conclusion was substant­ iated by Ed.wards et_ al. (1958) who found that chicks with the yolk sacs removed did not respond differently than normal chicks to zinc deficient diets. 32 o o O W i —I o CO -p CO ->— . •p fiin m O O >* v r\ 0^ o°v £N-3- c *• rH O • i—1 r—1 ON VO• C^\ <*i cm o o 10 O c *H +5 CO < tsa c w G H • < 3* ° o • C\2 a w g p ps m c L w 4^*. -Pc? y-. o r-H o a, rH (0 •a cO 3 m E 1 hD -P C- i —I -3CM C"\ * CM p~\ -=h VO C'-N 00 -3- H <>sj r O m • -p ps Pt- o CO cO 2 CO g q 1 o •H pp •H E rH O X! O C~\ ho CO • M t s ~ PC s hD w Ch o • O , > 1 o • -H o X! S VO VO O CM 33 EXPERIMENT IV Since the previous experiments proved that zinc is an essential mineral for chick growth, an experiment was designed to determine its requirement* One-day-old White Meateor X White Rock,chicks, obtained from a commercial hatchery, were allotted as previously described Into five lots, each containing three replicates of ten chicks each. The birds were reared in a plastic-coated battery in a room relatively free of dust at a temperature approximately 75° The basal diet was that shown in Table 1, except that Ami coy, another isolated soybean protein, was used in place of Drackett protein* Added minerals were analytical reagent grade, except the calcium carbonate and dicalcium phosphate which were USP grade. analyzed ten ppm zinc (Benne, 1955)* The basal diet To the basal diet 0, 10 , 20, AO and 80 ppm of zinc, respectively, were added as the sulfate. Experimental diets were fed ad libitum and distilled water was furnished in glass, baby chick founts with plastic bases, growth rate, feed consumption, mortality, and deficiency symptoms were observed and recorded. Results and Discussion The experimental design and results are shown in Table 6 . An analysis of variance of the data is given in Table 7. The addition of zinc at all levels improved growth over that of thecontrol,lot 1. The addition of ten ppm gave significant (P increased growth over the basal diet diet and the basal plus ten ppm zinc. ci-S *01) did twenty ppm over the basal The growth of chicks receiving suoolemental levels of 20 , A 0 and 80 ppm were not significantly 3A different but all were better (P *01 ) than the 0 and 10 ppm levels. Peed utilization by the chick was improved at all levels of supplemental zinc. However, levels above ten ppm made no further improvement. Other research concerning the quantitative requirement of zinc by the chick has been reported since the completion of that conducted by the author. Horris at al. (1958) stated that the chick required no more than a total of twenty ppm of zinc when a diet containing casein was used; whereas, a total of 27 ppm (20 ppm of added zinc) was needed when isolated-soybean protein was used. Since the zinc in the isolated soybean protein was found previously to be unavailable to the chick, they c on eluded that the chick's requirement for this mineral was no more than twenty ppm. Young jet al. (1958) added graded levels of 0 , 10 , 20 , A 0 , 60 and 80 ppm to a, diet containing isolated soybean protein and found that the chick required no more than a A 0 ppm supplement. When the data were plotted on semi-log paper, the intersec­ tion of the sub-maximal and maximal growth lines Indicated a zinc re­ quirement of 25 to JO ppm. Moeller and Scott (1958) reported that the chick required 20 ppm supplemental zinc when isolated soybean protein was used in a diet containing 13 ppm zinc. These recent reports confirm the findings of the author that the growing chick requires no more then twenty ppm of available zinc. If isolated soybean protein is used in the diet, the total zinc needed is about thirty ppm because the zinc in this product is bound so that it is not utilizable by the chick. 35 TABLE 6 CHICK GROWTH RESPONSE TO GRADED LEVELS OF ZINC, AS THE SULFATE IN THE DIET TC FOUR WEEKS OF AGE Lot Zinc additions to the basal (ppm) Zinc by analysis (ppm) Average chick weights at 4 weeks (gm) Surviving chicks of 30 started Gain/feed 1 None 10.0 126 29 0.41 2 10 17.6 327 29 0.63 3 20 30.4 390 28 0.64 4 40 52.5 407 27 0.67 5 80 107.6 396 28 0.65 36 TABLE 7 ANALYSIS OF VARIANCE OF BCDY WEIGHTS OF FOUR-MEEK OLD CHICKS (Experiment XV) Source of variation Total Subclass Degrees of freedom Mean square F Calculated Values P = .05 P = .01 140 14 1X4,776 57.64** 2.23 Lot 4 397,463 H 9 .62** 3.47 Replicate 2 1,029 0,51 3.07 L X R 8 1,869 0.83 2.01 126 1,991 Error ** Significant at P < . 0 1 Comparisons among lots at 1 percent level of probability Lot 4 wk. wt. 1 2 3 5 4 126 327 390 396 407 at 5 percent level of probability Lot 4 wk. wt. 1 126 2 327 3 3 4 390 396 407 37 THE TOLERANCE OF THE GROWING CHICK FOB ZINC. As information was being acquired concerning the chick*s specific need for zinc, it was felt desirable to determine if higher levels were well tolerated or toxic. This knowledge is important because practical supplementation of rations requires a reasonable margin of safety. Furthermore, if a mistake were made in the amount of zinc added to practical rations, then the toxic symptoms and the level at which they occur would be important. Hence, experiments xvere conducted to determine the zinc tolerance level of growing chicks. EXPERIMENT V Male chicks, hatched at Michigan State University from White Leghorn males X strain-cross White Leghorn females were reared on a basal diet (Table 7) until one week of age. At this time they were randomized on the basis of weight Into fifty pens of ten chicks each. Five replicates were run on each of nine high levels of zinc oxide in addition to the control lot ( Table 9). Ten pens in each of five batteries were used for the experiment in such a way that a portion of each lot appeared once in each battery; thus, a battery constituted a replicate. Feed and tap water were supplied to the chicks ad libitum throughout the experimental period and feed consumption was recorded. When the birds were five weeks of age, group weights were taken. Results and Discussion The results end an analysis of variance of the data are shown in Table 9. Zinc was tolerated by the chicks over ^he entire range of levels tested without significantly depressing growth. significant differences (P position. However, .05 ) occurred among replicates due to battery Since all lots were allotted to each battery, this did not 38 invalidate the effect of the different levels of zinc. Feed conversion was also not affected by the addition of the high levels of zinc to the ration. 39 TABLE 8 COMPOSITION OF BASAL DIET USED IB EXPERIMENT V Percent Ingredients A5.0 5.0 5.0 Corn, grd. yellow Oats, ground Wheat middlings, flour "Wheat bran Alfalfa meal, dehyd. 1?% prot. Meat and bone meal, 50$ prot. 5.0 5.0 5.0 20.0 Soybean meal, prot. solvent extracted Fish m e a l , red Whey, dried cheese 2.5 2.5 Xeast, dried brewers Limestone, grd. Bone meal, steamed Salt, iodized Vitamin A and D feeding oil (2250A, 300 Manganese sulfate, 70$ feeding grade Choline chloride, 25$ dry form Pro-pen with 3^2* 2.5 1.5 1.0 0.30 ) 0.25 0.02 0.15 0.05 * Contains: 2 gm procaine penicillin and 3 mg vitamin 3 ^ per pound. 40 TABLE 9 THE EFFECT OF VERY HIGH LEVELS OF ZINC ON THE GROWTH RATE AND FEED CONVERSION OF CROSSBRED WHITE LEGHORN COCKERELS TO FIVE WEEKS OF AGE Lot Zinc additions to the basal as the oxide (ppm) Average chick "wts at 5 wks (grn) ♦ Gain/feed 1 None 485 0*35 2 200 492 0.35 3 300 ' 496 0.35 400 476 0.34 5 500 494 0.36 6 600 488 0.35 7 700 471 0.35 8 800 474 0.34 9 900 478 0.35 10 1,000 473 0.34 4 Analysis of variance of 5-'w,eek ■weights: Source of variation Degrees of freedom Mean square Experiment V F values Calculated. P = .05 Total 48 Lot 9 354 1.55 2.16 Replicate 4 685 2.99* 2.64- 35 229 Error * Significant ( P ^ .05) P = .01 3-91 41 EXPERIMENT VI The preceding experiment proved that Leghorn chickens tolerate zinc oxide at very high levels in the ration. It was thought desirable to extend this study to broiler-type chickens and zinc In the form of the sulfate, carbonate, as well as the oxide. The latter two compounds are inexpensive forms of zinc and are used in the supplementation of practical swine rations. One-day-old Cobb's strain of White Rock, male chicks obtained from a commercial hatchery were randomized into thirty pens of ten chicks each in the manner previously described so as to minimize differences in initial weights. The various lots were assigned to three batteries in such a manner that each one appeared once in each battery; thus, each battery had a complete series of lots* The basal diet of practical ingredients is given in Table 8 and the experimental outline is in Table 9* Three high levels of zinc (1 ,000 , 2 ,000 , and 3*000 ppm) in the forms of the oxide, sulfate and carbonate were added to the basal diet at the expense of c o m . birds were reared as previously described. The Group weights were taken at the end of two weeks, individual weights at the end of four weeks of age. Experimental diets and tap water were supplied ad libitum throughout the experimental period. Feed consumption and mortality were recorded. Results and Discussion The results are given in Table y.ind the data are evaluated statistically in Table 9* Zinc at 1,000 ppm in the forms of oxide sulfate, or carbonate were equally well tolerated and did not depress growth as compared to 42 the basal diet. There was no significant difference among the average weights of lots of birds fed 2,000 ppm of the three compounds, however, when compared with the basal lot growth was depressed at this level of the sulfate and carbonate but not the oxide. Three thousand ppm of the three compounds depressed growth as compared to both the lower levels and the basal. The efficiency of feed utilization was lowered with each increase in the zinc level. This was true with all three of the compounds. Only 3*000 ppm of zinc, as the carbonate, caused rather severe mortality. The other forms of zinc at this level or below did not adversely affect livability. At higher levels the zinc compounds were tolerated in the following decreasing order: zinc oxide, zinc sulfate, and zinc carbonate. 43 TABLE 10 CQMPCSITIOM OF BASAL DIET USED IN EXPERIMENT VI Ingredients Corn, Grd. yellow Soybean oil meal, 50$ prot. Menhaden fish meal, 60$ prot. Amount in 100 lbs. of feed 50.8 32.7 2.0 lb n ti Pleat and bone scraps, 50$ prot. Alfalfa leaf meal, 20$ prot. Whey product, delactosed 2.0 2.0 0.5 II it tt Teast, dried brewers Yellow grease, stabilized Dicalcium phosphate 0.5 6.5 1.0 it tf Limest one, g r d . Salt, iodized 1.0 0.5 it tt 11 DL-methionine Trace mineral mix* Vitamin Suppl. 249C 45.4 gm 45.4 tt 11 34.0 Vitamin A (10,000 IU/gm) Vitamin D 3 (1,500 ICU/gm) Choline chloride, 25$ dry form 19.0 10.0 30.0 it tt it Vitamin B 12 suppl. (6 mg/lb) 45.0 ti 1- Delamix: at 0.1 percent of diet it supplies in mg per lb. of feed: manganese 2?, iodine 0*54, iron 9 » copper 0.9 and cobalt 0 .09 * 2 Contains per lb: 2 grains riboflavin, 4 grams pantothenic acid, 9 grams niacin and 10 grams choline TABLE 11 EFFECT OF THREE LEVELS OF DIFFERENT SALTS OF ZINC UPON GROWTH, LIVABILITY AND FEED EFFICIENCY OF THE CHICK TO 4 WEEKS OF AGE Lot Zinc additions to the basal (ppm) 1 None 2 1,000 3 Source of zinc Av. chick ■wts • at 4 wks. (gm) Surviving chicks of 30 started Gain/feed U93 29 0.5^ ZnO 472 29 0*58 2,000 ti 441 30 0-53 4 3,000 it 337 29 0*44 5 1,000 464 30 0*59 6 2,000 II 419 28 0*52 7 3,000 It 282 29 0.41 8 1,000 483 30 0.58 9 2,000 it U07 29 0.48 10 3,000 it 214 23 0.29 ZnSO^ ZnC03 45 TABLE 12 ANALYSIS OF VARIANCE OF CHICK WEIGHTS (Experiment VI) Source of variation Degrees of freedom Total Mean square Calculated F values P = .05 P = .01 28 5 Subclass 29 77,246 17.40** 1.79 Lot 9 233,499 54.90** 2.50 Replicate 2 9,557 2.24 3.02 18 6,807 1.59 1.65 266 4,256 L X R Error ** Significant (P<^*01) Comparisons among lots at 5 percent level of probability Lot 10 7 4 9 4 w k . wt. 21A 282 337 6 407 3 419 5 1 8 2 441 *j64 476 463 488 at 1 percent level of probability Lot 10 4 wk. wt. 214 7 4 9 6 3 5 1 8 2 282 337 407 419 441 464 476 483 488 EXPERIMENT VII This experiment- was designed to study farther the tolerance of the growing chick for high levels of zinc. Cobb's strain, White Rock, male chicks obtained from a commercial hatchery were randomized into 32 pens of ten chicks each, as described in the previous experiment. Four lots, each consisting of four replicates, were used as shown in Table 11. The basal diet was the same as that used in Experiment VI. Results and Discussion The results are reported in Table 12 and the data are evaluated in Table 12. The addition of fifty ppm of zinc as the sulfate and 1,000 ppm in the form of sulfate, oxide, or carbonate did not affect growth but 1,500 ppm depressed growth significantly (P< 0.01). At this higher level zinc oxide was less toxic than zinc sulfate or carbonate. The efficiency of feed utilization was not affected by the lower levels of zinc but the 1,500 ppm level of all forms affected it adversely. The livability of chicks was good in all lots. Certain research reports appearing recently have also dealt with this problems with chicks. Mehring et al. (1956) found that zinc levels up to 814 ppm did not adversely affect growth and feed efficiency Klussendorf and Pensack (1957 observed that a level of 1,3^7 ppm of zinc as the proteinate, did not depress growth; whereas, 1,823 ppm did depres growth, the chloride being more drastic. They found zinc proteinate, carbonate or acetate at the 2,000 ppm level equal in this regard, but compared to the non—supplemented basal diet these levels depressed growth. The difference between them was not significant but a trend was apparent. They concluded that zinc in excess of 1,000 ppm may be ^7 detrimental. Norris et al. (1958) observed that 1,000 ppm zinc caused poorer growth when a semi-purified, isolated soybean-protein-type diet was used but a level of 500 ppm did not. The form in which the zinc was added was not disclosed. These recent reports confirm the results of the present study that the growing chick can tolerate 1,000 ppm or less of zinc in the ration and that the oxide form is tolerated to a greater degree than the sulfate and chloride. Since the requirement of the growing chick is not greater than twenty ppm of available zinc, reasonable levels of supplementation of practical rations can be made with assurance that they will be well tolerated. 48 TABLE 13 THE EFFECT OF AH EXCESS OF ZINC ON CHICK PERFORMANCE TO 4 WEEKS OF AGE Lot Zinc additions to the basal (ppm) 1 None 2 50 3 Source of zinc A v . chick wts. at 4 wks. (gm) Surviving chicks of 30 started Gain/feed ^91 38 0 .6 2 ZnSO^ 500 40 0 .5 7 1 ,0 0 0 ZnO 497 38 0 .6 0 4 1 ,0 0 0 ZnSO^ k6o 39 0 .5 9 5 1 ,0 0 0 ZnCO-j 490 bo 0 .5 6 6 1 ,5 0 0 ZnO 455 38 0 .5 4 7 1 ,5 0 0 ZnSO^ 4D2 39 0 .5 1 8 1 ,5 0 0 ZnCO^ 390 40 0 .5 0 49 TABLE 14 AMLYSIS OF VARIANCE OF FOUR-WEEK WEIGHTS (Experiment VII) Source of variation Degrees of freedom Mean square Calculated 31 19.992 6•08** 1.79 Lot 7 73.778 21.53** 2.73 Replicate 3 7,990 2.43 2.62 21 3,777 1.14 1.60 280 3,287 Total Subclass ** Significant (P<.01) Comparisons among lots 4- wk. wt 00 at 1 percent level of probability Lot 390 402 6 4 5 455 470 490 3 1 2 491 .. 4 g z . 50.0 at 5 percent of probability Lot A wk. wt, P = .01 311 L X R Error F values P = .05 8 390 7 6 402 455 4 5 470 1 490 3 491 2 497 500 50 the availability to the chick of zinc from DIFFERENT COMPOUNDS A g*oup of experiments was conducted to study the availability of zinc in different zinc compounds. experiment VIII This experiment compared the availability of zinc as the sulfate and chloride with respect to rate of chick growth and feed efficiency* White Meateor X White Rock, male chicks from a commercial hatchery were allotted to six pens of nine chicks each* Duplicate lots were fed the basal ration, the basal containing zinc sulfate and the basal with zinc chloride (Table 15)* The birds were maintained in a manner previously described for purified d-iet studies. Results and Discussion Zinc in the form of both the sulfate and the chloride at the 100 ppm level produced increased growth as compared to the basal lot. There was no difference in performance of chicks between the two compounds. Thus, the zinc In the compounds was available and adequate for normal growth at the level used. Feed utilization was improved at a comparable rate by both the zinc compounds. Livability of the chicks was good In all lots. 51 TABLE 15 THE AVAILABILITY OF ZINC FROM TWO COMPOUNDS TO THE CHICK TO FOUR WEEKS OF AGE Lot Zinc additions to the basal (ppm) 1 None 2 100 3 100 Zinc compound None M 1 2 ZnSO^ Surviving chicks of 30 started Gain/feed 112 18 0.33 398 18 0.65 itoo 17 0.62 Average chick wts. at U weeks (g*0 52 TABLE 16 ANALYSIS OF VARIANCE OF THE WEIGHT OF FOUR-WEEK-OLD CHICKS (Experiment VIII) Source of variation Degrees of freedom Total Mean square Calculated F values P = .05 P - *01 51 Subclass 5 189,512 145.11** 3.44 Lot 2 468,795 358.95** 5.10 Replicate 1 2,646 2,03 4.05 L X R 2 3,663 2.80 3.20 46 1,306 Error ** Significant (P<.0l) Comparisons among lots at 1 percent level of probability Lot 4 wk. wt. 1 112 2 3 398______ 400 at' 5 percent level of probability Lot 4 wk. wt. 1 112 2 3 398______ 400 53 EXPERIMENT IX In this experiment, zinc in the form of the oxide and carbonate was compared with the sulfate. The three compounds were included at levels of ten and twenty ppm of zinc. Since the zinc requirement had been established at twenty ppm of available zinc, the lower level provided a more critical evaluation of the availability of the zinc. White Rock, male chicks from a commercial hatchery were randomized to three replicates of ten birds for each lot and reared as previously described for purified diet studies. The experimental design is given in Table 17. Results and Discussion The results are given in Table 17 and the data statistically evaluated in Table 18. The addition of both levels of supplemental zinc increased growth in every case, but deficiency symptoms were prevented only at the higher level* At each level of zinc, the sulfate, oxide, and the carbonate proved to be about equal, but growth was greater at the higher level. Comparison of twenty ppm of the sulfate and ten ppm of the oxide at the five percent level of probablility revealed a. significant weight decrease with this level of oxide, but no difference at the one percent level. The efficiency of feed utilization was increased in each inst­ ance in which supplemental zinc was added to the diet. The higher level of zinc improved feed efficiency to a greater extent than did the lower. Thus, the zinc in the three compounds is equally available to 54 the chick and can "be used interchangeably in supplementation of practical poultry rations. This supports the conclusion of Pensack at al. (1958) who showed that zinc from the carbonate, oxide, chloride and proteinate was equally available when 6, 20 and 40 ppm of each compound were added to a glucose-casein, gelatin-type diet. 55 TABLE 17 THE AVAILABILITY OF ZINC FROM THREE COMPOUNDS TO THE CHICK TO FOUR ViEEKS OF AGE Average chick •weights at 4 weeks (gm) Lot Zinc addition to basal (ppm) Zinc compound 1 None None 163 0.36 2 10 ZnSO^ 293 0.50 3 20 ZnSO^ 343 0.53 A 10 ZnO 310 0.51 5 20 ZnO 352 0 ,5^ 6 10 ZnCO_ j 299 0.51 7 20 ZnCO^ 359 o.55 Gain/feed 56 TABLE 18 ANALYSIS OF VARIANCE OF FOUR-WEEK-OLD CHICK WEIGHTS (Experiment IX) Source of variation Degrees of freedom Total Mean square Calculated F values P = .05 F = -01 192 Subclass 20 39,329 15.10** 2.00 Lot 6 125,443 48.30** 2.92 Replicate 2 4,8^4 1.80 3.06 12 2,020 0.78 1.82 172 2,598 L X R Error ** Significant (P< .01) Comparisons among lots at 1 percent level of probability Lot 4 wk. wt. 1 2 6 4 3 5 163 293 299 310 .2£2l 352 ___359 5 7 352 352 7 at 5 percent level of probability Lot 4 wk. wt. 1 2 6 4 163 293 299 310 3 57 THE IHTERREIAT101TSHIP OF ZINC WITH CALCIUM AND PHOSPHORUS Since high levels of calcium in the ration aggravated parakeratosis, or zinc deficiency in swine, a series of experiments was conducted to study interrelationships between zinc and calcium or phosphorus. EXPERIMENT X Cobh's strain, White Rock, male chicks received from a commercial hatchery were randomized to 15 pens of ten chicks each. Each lot was replicated three times. The chicks were maintained in a plastic-coated battery under conditions previously described for purified diet studies. The basal diet conts.ined 1.23 percent calcium. A supplemental zinc level of eighty ppm was added to this diet in the absence and presence of one percent supplemental calcium (Table 19). Also, two levels of calcium (0.5 and 1.0 j)ercent) were added to the basal diet in the ebsence of additional zinc. Results and Discussion The chicks receiving the unsupplemented basal diet exhibited zinc deficiency symptoms and grew poorly. The addition of 0.5 percent calcium depressed growth only slightly but caused the incidence and severity of the symptoms to increase. The additions of one percent calcium definitely retarded growth, increased the incidence of symptoms, and made them more acute. Supplementation of the basal diet with zinc alone, or one percent extra calcium plus eighty ppm of zinc, produced norm?! growth end prevented all deficiency symptoms. At least, a part of the zinc in the "basal diet was available because the elevated calcium levels aggravated the deficiency condition. Since isolated soybean protein was the major source of zinc in the basal ration, part of its zinc must have been available to the chick. Thus it is quite evident that the adverse effect of calcium on growth and deficiency symptoms was produced through its antagonism with 59 TABLE 19 THE EFFECT OF ELEVATED LEVELS OF DIETARY CALCIUM GU THE PERFORMANCE OF CHICKS TO FOUR WEEKS OF AGE Lot Additions to the basal Calcium Zinc (ppm) Gf> Average chick weights at 4 weeks (gm) Surviving chicks of 30 started Gain/feed 1 — — 145 29 0.37 2 — 80 356 29 0.55 3 0.5 — 140 29 0.38 4 1.0 — 116 27 0.31 5 1.0 80 343 28 0.5^ 60 TABLE 20 ANALYSIS OF VARIANCE OF CHICK HEIGHTS AT FOUR WEEKS (Experiment X) Source of variation Degrees Of freedom Mean square Calculated 14 115,441 62.37** 5*56 Lot 4 400,267 216.25** 6.63 Replicate 2 152 L X R 8 1,851* Total Subclass Error F values P = .05 P = .01 141 127 0.08 19.37 865 * Significant ( P C . 05) interaction with which subclass, lot and replicate mean squares were compared. ** Significant (P^.Ol) Comparisons among lots at 1 percent level of probability Lot 4 wk. wt. A 3 1 116 140 145 5 2 343____ 356 at 5 percent level of probability Lot 4 wk. wt. A 3 1 5 2 116 140 145 343 353 61 EXPERIMENT XI In the previous experiment the retarded growth produced by elevated calcium levels was overcome "by zinc levels in excess of the actual requirement• This experiment was conducted to find out if the zinc requirement, as determined with a diet containing a normal level of calcium (1 .23 percent), was increased if higher levels of calcium were used. White Rock, male chicks from a commercial hatchery were allotted as previously described to 21 pens of ten birds each. replicated three times. The lots were The birds were maintained in a plastic-coated battery In the special laboratory under conditions as previously described for purified diet, stxidies. Two levels of calcium (0.5 and 1.0 percent) were added to the basal diet containing both twenty and eighty ppm of zinc (Table 21). The unsupplemented basal diet contained 1.23 percent cadcium. Results and Liscussion The chicks receiving the basal diet grew poorly and exhibited zinc deficiency symptoms similar to those previously described. The addition of 0.5 percent' calcium to the basal diet either in the presence of twenty or eighty ppm zinc produced normal growth and no deficiency symptoms. Similarly, normal performance was obtained following the addition of 1.0 percent calcium in the presence of eighty ppm of zinc. However, one percent of calcium in the presence of twenty ppm retarded growth but did not produce deficiency symptoms typical of acute cases. The efficiency of feed utilization was reduced in the lots receiving the basal diet and basal diet supplemented with 1.0 yerceni 62 calcium 20 ppm zinc "but was normal in the remaining lots. Livability was good in all lots. These results indicate that the requirement of the chick for twenty ppm of zinc is not increased if a slight (0.5 percent) excess of cal­ cium is added to the diet (1.73 percent total). However, twenty ppm of supplemental zinc is not sufficient if one percent calcium is added (2.23 percent total). Eighty ppm of zinc neutralizes the effect of one percent additions.! calcium* Therefore, 60 "ppm of zinc will over­ come the adverse effects of one percent of calcium. Thus, the zinc requirement is elevated with rather high levels of dietary calcium but not to the extent as in swine. Several other research workers have mentioned a zinc— calcium interrelationship. Morrison and S&rett (1958) found that the addition of 0.5 S-nd 1.0 percent calcium to an isolated-soybean-protein diet reduced 14— day gains and increased the incidence and severity of the hock disorder; bone ash percentage was not affected. of the diets with five ppm Supplemental:ion zinc only partially prevented the growth inhibition brought about by the extra calcium; whereas 25 ppm com­ pletely overcame the depression. in each case. Need efficiency paralleled growth Norris _et al. (1958) reported that the addition of one percent calcium to a diet containing 1.23 percent calcium caused deficiency symptoms to become more severe. Eifty ppm of zinc restored normal growth and alleviated all symptoms. Pensack et al. Conversely, (1958) observed that growth was not depressed with calcium—phosphorus ratios of l.O/O.o, 2.0/0.O ana 1.0/1.2 in the presence of o, 20, and 40 ppm of supplemental zinc. gelatin, however, were employed in ^heir diets. o<5-sexn a.nd 63 TABLE 21 THE EFFECT OF ELEVATED CALCIUK LEVELS ON THE ZINC REQUIREMENT OF THE GROWING CHICK TO FOUR WEEKS OF AOS Lot Additions to the basal Calcium Zinc (ppm) <*> Average chick Surviving weights chicks at 4 weeks 30 startedGain/feed (gm) 1 None None 153 30 0.39 2 0.5 20 377 30 0.58 3 0.5 80 368 28 0.56 k 1.0 20 310 29 0.50 5 1.0 80 382 28 0.57 64 TABLE 22 ANALYSIS OF VARIANCE OF FOUR WEEK WEIGHTS (Experiment XI) Source of variation Total Degrees of freedom Mean square F values Calculated P = .05 P = .01 144 14 80,757 43 *00** 2.23 Lot 4 280,618 150.00** 3.47 Replicate 2 701 0*30 3.07 L X R 8 841 0.40 2.01 130 1.871 Subclass Error ** Significant ( P C . 01) Comparisons among lots at 1 percent of probability Lot 1 4 w k . wt • 153 4 3 2 310 368 377 ,.J82 5 at 5 percent of probability Lot 4 w k . "wt • 1 4 3 2 5 153 310 368 377 382 65 EXPKKIKEHT XII The purpose of this experiment was to find out if a phosphorus level in excess of the requirement would affect the chicks quantitative need for zinc. White Rock, male chicks from a commercial hatchery were randomized to nine pens of nine chicks each. Each lot was replicated three times. The chicks were maintained under special conditions as previously described for purified diet studies. Sodium phosphate, at a level of 0.5 percent phosphorus was added to the basal diet, in the absence and presence of one hundred ppm of zinc (Table 23). The basal diet contained twenty ppm supplemental zinc and O .69 percent phosphorus. Results and Discussion The addition of 0.5 percent phosphorus to the basal diet did not increase the chick's zinc requirement. Peed efficiency, growth and feather condition were comparable regardless of the level of phos­ phorus or zinc. 66 TABLE 23 THE EFFECT OF A HIGH LEVEL OF DIETARY PHOSPHORUS OH THE CHICK REQUIREMENT FOR ZINC TO FOUR WEEKS OF AGE A v . chick w t . at 4 weeks (gm) Additions to the basal Lot Chicks surviving Gain/feed 27 started 1 None 389 27 0.57 2 O. 55S Phosphorus 371 26 0.58 3 0.5$ Phosphorus + 100 ppm zinc 384 26 0.56 TABLE 24 ANALYSIS OF VARIANCE OF CHICK WEIGHTS AT FOUR WEEKS OF AGE (Experiment XII) Source of variation Total Degrees of freedom Mean square Calculated F values P = .05 76 8 6,753 0.78 3.84 Lot 2 4,953 0.57 19.25 Replicate 2 4,695 0.54 19.25 L X R 4 8,681** Subclass Error 70 2,155 ** Significant interaction (P<*01) used as basis for comparisons with subclass, lot and replicate mean square. 67 EXPERIMENT XIII This experiment was conducted in vitro to find out if calcium and phosphorus supplements would remove zinc from solution. One gram samples of ground oyster shells, feed grade dicalcium phosphate, and "bone meal were weighed separately into duplicate flasks. To one flask of each miners! and a “blank were added forty ml of a solution containing fifty ppm of zinc, as the sulfate. To the other flasks were added forty ml of a solution containing one hundred ppm zinc, as the sulfate. The flasks were mechanically shaken at a temp­ erature of 37° C for 1.5 hours. In each flask a quantity of the mineral supplement remained in the solid phase. The contents of each flask were filtered and analyzed for zinc (Benne, 1955). Results and Discussion The results are given in Table 25- Ground oyster shells, dicalcium phosphate and bone meal removed practically all of the zinc from solution at both levels of zinc. Ground oyster shells and bone meal were more effective in this respect than dicalcium phosphate. These results indicate the manner by which high,or excess levels of mineral act in making zinc unutilizable to the cnick* 68 TABLE 25 THE IN VITRO REMOVAL OF ZINC FROM SOLUTION BY MINERAL SUPPLEMENTS Amount of mineral added (gm) ppm Zinc in ho ml of zinc sulfate solution Zinc solution after equilibrium (ppm) Lot Mineral 1 None — 50 h5 2 Ground oyster shells 1 50 <1 3 Dicalcium phosphate 1 50 5 h Bone meal 1 50 <1 5 None — 100 109 6 Ground oyster shells 1 100 < 1 7 Dicalcium phosphate 1 100 h 8 Bone meal 1 100 <1 69 EXPERIMENT XIV This experiment was conducted with laying hens to determine if supplemental zinc, in the presence or absence of one percent of additional calcium (as CaCO-j), affected egg production, hatchability and egg shell thickness. Spring-hatched, hybrid pullets from a commercial hatchery were reared in confinement on wood shavings for litter* A starting ration (Table 2&) containing approximately 35 ppm of zinc wag fed for the first eight weeks and a growing ration (Table 26) containing approx­ imately 36 ppm of zinc was fed from eight to twenty weeks of age. The management practices employed were comparable to those used under practical conditions. Tap water and feed were given ad libitum . The pullets were housed at twenty weeks of age in floor pens with wood shavings for litter* Each pen contained 85 square feet of floor space, five trapnests, one hanging feeder, one automatic waterer, a-nd a h * X A' dropping pit. Ventilation was natural or forced draft depending on weather conditions but no supplemental heat was used. At 2k weeks of age the pullets were randomized on the basis of body weight, sexual maturity, and one-month's production into 12 pens of 21 pullets each. Each lot was replicated three times The basal laying ration in Table 27 contained two percent calcium. One hundred ppm of supplemental zinc as the sulfate was added to the basal diet, in the absence and presence of one percent calcium (Table 28). The hens were trapnested five days per week for 32 weeks beginning the first of November and continuing into June. In 70 February arid June, three eggs per hen were collected to determine egg weight, Haugh score and shell thickness. Efegs for February were collected during week days, stored in a refrigerator at 48° F, and measurements were made during the morning following the day the eggs were laid. E£g weights were taken in grams, albumen heights in millimeters, and eggshell thickness in thousandth's of an inch. From the egg weight and albumen height, t h e ‘Haugh score was derived (Haugh, 1937)* For hatchability data, one week's eggs of each hen were set for the months of February through June. The hatch during Kay was not included with final results because of incubator failure. Results and Discussion The results are given in T ahle 28 and the analyses of the data in Tables through 32. E&g production and hatchability were not affected by the addition of zinc to the basal ration, or the basal to which extra calcium was added. Also, extra calcium alone did not affect egg production and hatchability. There was no difference in egg weight or Haugh score among the treatments during either February or June. In February, eggshell thickness was increased (P< .05) by the addition of calcium and zinc to the ration compared to zinc alone. However, there was no increase in eggshell thickness when zinc or calcium alone were compared to zinc plus calcium. At the five percent level of probability, the addition of neither calcium nor zinc altered eggshell thickness as compared to the basal but the addition of calcium improved eggshell thickness compared to zinc alone. The addition of calcium plus zinc increased shell thickness when compared 71 to all other treatments. These results Indicated that neither calcium nor zinc alone had any significant effect on eggshell thickness, "but when both were added, the egg shell thickness was increased in cool weather. This combination of calcium and zinc did not increase eggshell thickness during warm weather. Apparently the eggshell thickness decreased with age of hen and environmental temperature, regardless of level of calcium or zinc used. 72 TABLE 26 DIETS FOR REARING PULLETS USED IN EXPERIMENT XII Ingredients Corn, ground yellow Oats, ground heavy Whe a t , ground Wheat bran Wheat, standard middlings Alfalfa leaf meal, dehyd. ITfo prot. Soybean oilraeal, solv. ext* ktyp prot. Meat and bone scraps Fish meal, red Whey, dried cheese least, dried brewers Limestone, ground Bone meal, steamed Salt, iodized Manganese sulfate, 70$> feed grade Vitamin A and D feeding oil (2250A, 300D) Choline chloride, 25f° dry mix Pro-pen1 Calcium pantothenate Amt. per 100 pounds Starter Grower (lbs') (lbs) 45.00 5.00 45.00 15.00 ... 10.00 5.00 5.00 5.00 5.00 15.00 2.50 1.25 1.25 5.00 5.00 20.00 5.00 2.50 2*50 2.50 1.50 1.00 0.3 0 0.02 0.25 0.15 0.005 1 An antibiotic supplement containing two grains of procaine penicillin and three grams of vitamin B ^2 per pound* — 1.25 1.50 0.50 0.013 0.150 — 0.10 200 mg 73 TABLE 27 BASAL DIET FOR EXPERIMENT XIV Ingredients Amt. per 100 lbs. C o m , yellow ground Soybean meal, 50% solv. extracted Wheat, flour middlings Meat and bone scraps, 50% prot. Fish meal Whey product, delactosed Alfalfa meal, 20% prot. Fat, No. 2 yellow grease Lime st one, g r ound Dicalcium phosphate Salt Trace mineral mix* Choline chloride, 25% dry mixture Vitamin B-j^ (6 mg/lb) Vitamin Do (3*000 ICU/gm) Vitamin A (10,000 IU/gm) Calcium pantothenate (32 mg/lb) Niacin, 100% 59.53 17.00 5.00 3.00 2.00 1.5 1.5 5.0 2.0 2.6 0.5 0.1 0.2 lbs n ti ti n it ti it it tt ii it , ti 23 grams 15 " " M 0.5 n 10 2 * Delamix - added at 0.1 percent level it provides in mg per pound of feed: manganese 25* iodine 0.5^» iron 9, copper 0.9 and cobalt 0 .09 * Calculated analysis: Ca 2.0 percent, P 1.0 percent, 2n 3^ ppm 74 TJ w bQ «H 0) IN- UN VO VO 00• -aCN CO• UN Ov IN- CM IN * CO W rQ O l—l TJ (N• vo ON I P *H x-x O rHN&p .H w cc* W CCS VO • CM H CO UN • CM 0> ^ ^.h rH i—i i—I rH ON ~3• CM rH VO• INCK UN UN ON ON !N - ON UN « CM rH • CM H o o cn o ON ON • ON *H hi).C £> -P S' CD O rH ON UN • ON i—1 rH iH -3- On • ON rH • ON i—1 00 IN - IN IN - IN­ IN - ON N- -d£N- VO IN - & vo un CM VO CM VO ON VO Ov UN 0O UN CO CO UN UN • ON VO • C5N VO UN • rH * VO vO s bO ,0 CD UN C o cu •H cd co -P P. P* bfl O P o !?•O§ cd > u *d P* un w I ps CO C rH O ctJ £ P* .H CO P o C'VO O O O o i—I rH cd •H ,0 *d O cd F ., 1877 • As quoted in Underwood, E . J ., 1956 • Trace element s in human and animal nutrition. Academic Press, Inc., New York, pp. 204-232. Roberson, R. H. and P. J. Schaible, 1958. SCIENCE, 127: 875-876. Roberson, R. chick. Zinc requirement of the chick. H. and P. J. Schaible, 1958. The zincrequirement Poultry Sci., 37s 1231 - 1323* Romanoff, A. L. and A. J. Romanoff, 19^9* and Sons, Inc., New York, pp. 358. TheAvian egg. of the John Wiley Schaible, P. J. and S. L. Bandemer, 1942. The effect of mineral supplements on the availability of manganese. Poultry Sci., 21: 8 - 14. Smith, S. R. and E. J. Larson, 1946. Zinc toxicity in rats: antagonistic effects of copper and liver. J. Biol. Chem., 163: 29- 38. Snedecor, G. W . , 1955* Ames, Iowa. Statistical methods. The Collegiate Press, Inc., Stevenson, J. W . , and I. P. Earle, 1956. Studies on parakeratosis in swine. J.Animal Sci., 15: 1036—104*5. Supplee, W. C., G. F. Combs and D. L. Blamberg, 1958. Zinc and potassium effects on bone formation, feathering and growth of poults. Poultry Sci., 37: 63-67* Supplee, W. C., D. L. Blamberg, O. D. Keene, G. F. Combs and G. L. Romoser, 1958. Observations on zinc supplementation of poultry rations. Poultry Sci., 37: 1245-1246. Theorell, H., A. P. Nygaard and R. K. Bonnischsen, 1955* Studies on liver alcohol dehydrogenase: III Influence of pH and some anions on reaction velocity constants. Acta Chem. Scandinav., 9: 1148-1165. Titus, H. W. and W. M. Ginn, 1931. Rice bran, a preventive of perosis (deforming leg weakness) in chickens. SCIENCE, 74: 249—250. Titus, H. W . , 1932. Perosis or deforming leg weakness in chickens. Poultry Sci., 11: 117-125* 92 Todd, W. E . , C. A. Elvehjem and E. B. Harte, 1934. Zinc in the nutrition of the rat. Am. J. Physiol., 107: 146-156. Tsui, C., 1948. The role of zinc in suxin synthesis in the tomato plant. An. J. Botany, 35: 172-178. Tucker, H. F. and W. D. Salmon, 1955- Parakeratosis or zinc deficiency in the pig. P r o c . Soc. Exp. Biol, and Med., 88 : 613-616. Underwood, E. J., 1956. Trace elements in human and animal nutrition. Academic Press Inc., Publishers, New York. Vallee, B. L., S. J. Adelstein and J. A. Olson, 1955* Glutamic dehydro­ genase of beef liver, a zinc metalloenzyme. J. An. Chem. Soc. 77: 5196. Vallee, B. L., and F. L. Hoch, 1955* Zinc, a component of yeast alcohol dehydrogenase. Proc. Nat. Acad. Sci. 41: 327-338. Vallee, B. L., and H. Neurath, 1955* Carboxypeptidase, a zinc metallo­ enzyme, J. Biol. Chem., 217: 253-261. Vallee, B. L* and F. L. Koch, 1956. Zinc, a component of alcohol dehydro­ genase of horse liver. Fed. Proc., 15: 619-620. Vallee, B. L . , 1957* Zinc and its biological significance. A.K.A. Arch, of Industrial Health, 16: 147-154. Vallee, B. L . , and J. H. R. Kagi, 1958. Zur bedeutung des zinks im stoffwechsel• Soderabdruck aus der Schweizerischen Medizinischen Wochenschrift. 88 : 1-10 . Waldenstrom, J., 1937* Studen uber porphyrie. Supp. 82, pp. 3— 254. Acta Med. Scandinav., Wachtel, L. W . , E. Hove, C. A. Elvehjem and E. B. Hart, 1941. Blood uric acid and liver uricase of zinc-deficient rats on various diets. J. Biol. Chem., 138: 361-368. Wilgus, H. S., Jr., L. C. Morris and G. F. Heuser. 1937- The role of manganese and certain other trace elements in the prevention of perosis. J. Nutrition, 14: 155-G67Young, R. J., H. K. Edwards, Jr., and M. B. Gillis, 1958. Studies on zinc in poultry nutrition. 2. Zinc requirement and deficiency symptoms of chicks. Poultry Sci., 1100—1107* APPENDIX EXPERIMENT I In a preliminary experiment a semi-purified diet was used to determine the effect of supplemental zinc on egg production, egg weight, Haugh score and eggshell thickness of eggs from laying hens. Twenty-eight White leghorn hens (strain cross), which had been in production for seven months, were divided into two pens of 14 hens each. The hens had previously received a corn-soy type diet containing approximately 34 ppm of zinc. The birds were housed in pens containing 74 sq. ft. of floor space a wooden slat floor, one wooden feeder, and two one-gallon baby chick founts for water. with tarpaper. The galvanized metal walls of the pens were covered All these precautions were taken to prevent the birds from obtaining extraneous zinc. The hens were placed on experimental diets on May 2, 1958, and trapnested for 43 days beginning May 5* Eggs were collected the last nine days of the period to determine eggshell thickness, Haugh score and egg weight. The composition of the basal diet is in Appendix Table II. Feed and distilled water were supplied ad libitum. Results and Discussion The results of the experiment are presented in Appendix Table I. There was no difference in egg production and egg weight between the two lots. The Haugh score of Lot 1 was significantly higher than Lot 2 The eggshell thickness ox Lot 2 receiving the supplemental zinc was significantly ihieker than Lot 1; however, the eggshell thickness was extremely poor in both lots* In this experiment the effects of warm 94 weather, age of hens and treatments are confounded and the differences noted are not necessarily due to the addition of zinc* APPENDIX TABLE I THE EFFECT OF ZINC SUPPLEMENTATION OF A SEMI-PURIBTED DIET ON ECO PRODUCTION, EGG WEIGHT, HAUGH SCORE AND EGGSHELL THICKNESS OF EGGS FROM LAYING HENS Lot Lot additions (ppm) Egg production (*) 1 None 67 64 83** 11.1 2 50 61 64 79 11.5** Significant (P ^ .01) Av. egg weight (gm)... . Haugh score Eggshell thickness (.001") 95 APPENDIX TABLE II BASAL DIET USED IN APPENDIX EXPERIMENT I Ingredients Percent 1 Glucose 59.72 2 Isolated soybean protein 18.00 Corn oil 10.00 5.00 Cellulose Butylated hydroxy toluene .02 DL-Me thi onine .2Ur CaHPOjj, 1.77 CaCO^ 3.88 k 2h p o ^ .50 NaCl .60 MgSO^ .25 3 Trace mineral mix 3 Vitamin mix 1. .0695 .193^ Cerelose, Corn Products Sales Co., Detroit, Michigan New Center Bldg., 2. Amisoy, The Glidden Company, Chicago, Illinois 3. Morrison et al. (1955) Calculated analyses: Protein Calcium Phosphorus Zinc 16.00 2.26 0.66 10 percent percent percent ppm