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The nutritive value of barley for broilers Chan, Robert D. Y. 1983-04-19

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THE NUTRITIVE VALUE OF BARLEY FOR BROILERS by ROBERT D.Y. CHAN B.Sc, University of British Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Poultry Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1983 (c) Robert D.Y. Chan , 1983 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Robert D.Y. Chan Department of Poultry Science The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date October 14, 1983 DE-6 (3/81) ii. ABSTRACT Availability of Ca, P, Mg, Mn, Cu and Zn were determined from six barley samples using broiler chicks. Each of the barley samples was compared with corn in respect to growth performance and feed conversion. Metabolizable energy of barley and corn samples were evaluated by using a recently developed method. The results of this study indicate that six minerals were highly available from barley sampies with values of 93.9%, 85.2%, 81.4%, 70.4%, 71.3% and 76.5% for Ca, P, Mg, Mn, Zn and Cu, respect ively. Variations in availability of minerals among some of the barley samples were observed. Supplying each barley sample in the control diet at approximately 20% or 40% of the total cereal grains at the expense of corn resulted in no alteration in growth performance, feed con sumption and feed conversion to the control except for one barley sample which differed only in feed conversion at 40% composition of cereal grains. Metabolizable energy of barley and corn samples were comparable. These values correlate with the performance of the broiler chicks. The results of this study suggest that the availability values from feedstuffs should be considered in formulating diets and that the values from N.R.C. publications may not accurately represent the nutritive quality of a feedstuff. iii. TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iii LIST OF TABLES v LIST OF APPENDIX TABLES vi ACKNOWLEDGEMENTS viINTRODUCTION 1 LITERATURE REVIEW 2 Nutritional value of various cereal grains 2 Absorption and regulation of various minerals 6 Effect on mineral availability by various factors 8 Availability of minerals from various cereal grains 17 Methods of determining mineral availability 19 Methods of determining metabolizable energy of feed ingredients 22 Metabolizable energy from various cereal grains 25 Amino acid availability of various cereal grains 26 CHEMICAL ANALYSIS OF BARLEY 30 EXPERIMENT 1. Availability of minerals in six barley samples to broiler chicks 32 EXPERIMENTAL PROCEDURE 3RESULTS 35 CalciumPhosphorus 35 Magnesium 8 Manganese 3iv. Page Zinc 38 CopperDISCUSSION 39 EXPERIMENT 2. Effect of barley on growth performance and feed conversion of broiler chicks using practical diets 43 EXPERIMENTAL PROCEDUREDIETARY TREATMENTS 43 RESULTS AND DISCUSSIONEXPERIMENT 3. Metabolizable energy of six barley samples and one corn sample 49 EXPERIMENTAL PROCEDURERESULTS AND DISCUSSION 49 SUMMARY AND CONCLUSIONS 53 REFERENCES 55 APPENDIX 66 LIST OF TABLES Table 1 Chemical analysis of six barley samples and one corn sample 2 Composition of synthetic diet 3 Content of minerals in six barley samples ( dry matter basis ) 4 Availability of minerals from six barley samples ( dry matter basis ) 5 Composition of experimental diets 6 Feed consumption, body weight gain and feed conversion per chick fed different composition of barley samples 7 Metabolizable energy of barley and corn samples ( dry matter basis ) 8 Recalculated metabolizable energy values LIST OF APPENDIX TABLES Table 1 Analysis of variance for mineral availability from barley 2 Analysis of variance for body weight gain at four weeks of age 3 Analysis of variance for cumulative feed consumption at four weeks of age 4 Analysis of variance for feed conversion at four weeks of age ACKNOWLEDGEMENTS I am deeply grateful to my research supervisor, Dr. D.B. Bragg, Professor and Chairman, Department of Poultry Science, for his patience, advice and encouragement, during the course of the study. I also like to thank the other members of my graduate committee: Dr. R.M. Beames, Associate Professor, Department of Animal Science; Dr. R.C. Fitzsimmons, Associate Professor, Department of Poultry Science; Dr. J.S. Sim, Assistant Professor, Department of Poultry Science, for their helpful suggestions in this work. The author wishes to express his sincere thanks to Dr. Al Leslie, Ritchie Smith Feed Ltd., for supplying the barley samples and providing the necessary information that made this study possible. Finally, I like to extend my gratitude to the following people for their assistance, encouragement and advice: Mel Hudson, Ray Soong, Carol McMillan, Fred Ming, Matt Wolde-Tsadick, Grant Schierman, Betty Carlson, Dean Crick, Roland Low, Kamily Cheung, Lai-mon Aw-yong and Stanley Chu. Robert D.Y. Chan 1. INTRODUCTION Cereal grains contribute the major part of the farm ani mal 's diet. Grains are used principally to provide the energy and protein required for body growth, maintenance and productivity of the animal. The values used in feed formulation are based on the con tent of various nutrients in feedstuffs and not on the availability of those nutrients. The nutritive value of a feedstuff is deter mined principally by the degree of availability of its nutrients which may not be accurately represented by its contents. The Na tional Research Council stated minimum requirement levels of various nutrients in various poultry diets. Due to insufficient knowledge in regard to nutrient availability of feedstuffs and interactions among different nutrients, minimum recommended requirement levels are subject to criticism. There is limited research on nutrient availability of ce real grains, although more work has been completed on energy and ami no acid availability than on mineral availability. The purpose of this study was to determine the availability of various minerals in barley. Also, metabolizable energy was meas ured in barley and corn by a recently developed method. Practical balanced diets, composed of different combinations of barley and corn were fed to broiler chicks to assess the nutritional value of each cereal grain based on growth performance and feed conversion. 2. LITERATURE REVIEW NUTRITIONAL VALUE OF VARIOUS CEREAL GRAINS Cereal grains are a major source of food nutrients in many parts of the world. Many countries consume from 100 to 500 pounds of wheat/capita/year and consequently, obtain from 19 to 62 grams of protein/capita/day from wheat and wheat products ( Anonymous, 1981 ). In Nigeria, sorghum and pearl millet are staple foods for more than 60 percent of the population ( Olatunji et al_., 1982 ). Sorghum and millet comprised more than 80 percent of the cereals grown in Nigeria. Cereal grains contribute about 42 percent of the total daily calories and 49 percent of total daily protein to the Nigerian diet. About 80 percent of the calories and 64 percent of the protein from cereals are from sorghum and millet ( Olatunji et aj_., 1982 ). Production trends for the period 1968-1978 indicate that per capita production of cereal grains such as wheat, rice, maize and sorghum in economically developing countries has increased ( Anonymous, 1981 ). Cereal grains are consumed mainly by humans in economically developing countries with 59 to 98 percent consumed by humans and 7 to 41 percent consumed by animals. In the developed coun tries, however, animals consumed 58 to 86 percent of the total cereal grains and humans 14 to 42 percent ( Anonymous, 1980 ). Corn is the most commonly used cereal grain as a source of car bohydrate in commercial poultry rations. Several investigators have 3. tested barley as a substitute for corn in poultry diets but it is dif ficult to make valid comparisons because of experimental variables. In some studies, the substitutions were made on a weight for weight ba sis rather than on a protein and energy basis so that there were varia tions in energy, protein, mineral and vitamin contents among treatments. Arscott ( 1963 ) found that replacing corn for barley as a sole cereal grain at 67 percent of the diet improves body weights and feed conversion. Also, a decrease in sticky droppings was noted. Sibbald and Slinger ( 1963 ) observed that body weight gains of white leghorn chicks fed wheat, barley or oats at different levels of 20, 40 and 60 percent of the diet, were greater for the wheat diet followed by the barley diet with the oats diet being the lowest. Feed efficiency were also better for wheat followed by barley and then oats. Various researchers have tried different methods to improve the utilization of barley by poultry. Leong et al_. ( 1960 ) found that the addition of a fungal enzyme or treatment with water improved the utilization of barley 18 and 25 percent, respectively. Arscott et al. ( 1960 ) also presented similar results and theorized that barley treat ed with the methods probably remove an inhibitor(s) or inhibiting act-ion(s). Lucas et al_. ( 1973 ) found that acid autoclaving of barley significantly improves the nutritional value of barley but the sticky feces condition was not improved when the chick diets contained 55 per cent of the test grain. The low nutritive value of barley was suggested to be princi pally due to the highly viscous condition in the alimentary tract of chicks caused by B-D-glucan ( Burnett, 1966 ). Burnett ( 1966 ) pointed out that barley with varying levels of B-D-glucan caused different degrees of 4. viscosity. Barleys with high nutritive values were those which con tain high levels of B-D-glucanase. This enzyme decreased the highly viscous material in the intestine to improve digestibility of protein and carbohydrate. Willingham et al_. ( 1960 ) noted that barley grown in western parts of the U.S. had higher levels of B-D-glucan than eastern U.S. barley and therefore, lower nutritive value. Burnett ( 1966 ) also showed that Australian barley was more viscous than Irish barley because of the higher level of B-D-glucan. Prentice et al. ( 1980 ) analyzed various cereal grains for B-D-glucan content and the results were as follows: oat 6.6%; wheat 1.4%; rye 2.9%; triticale 1.2%; sorghum 1.0%; and barley ( malting ) 4.6-8.2%. Many of the early studies with barley did not consider equal izing either the protein or carbohydrate in diets when different cereal grains were compared for nutritive value. Fry et al_. ( 1956 ) reported that substitution of barley for corn on a weight by weight basis depress ed growth and feed efficiency of chicks. However, the addition of tallow to barley diets to equalize the energy content resulted in growth approx imately equal to corn. The results of their studies indicate that dif ference in available energy content of corn and barley accounted largely for the difference in feeding value of these grains. Arscott et al . ( 1957 ) observed that 50 percent dietary corn can be replaced by Hann-chin barley provided three percent fat was included in the diet. Fry et al. ( 1957 ) showed that a combination of pearled barley and corn at a ratio of 50:50 gave comparable results to those of corn alone at similar protein level with or without adding tallow. Both growth and feed effi ciency of chicks at four weeks of age were similar. Arscott et al. 5. ( 1955 ) showed that use of barley up to 25 percent of the cereal grain component ( or 15.25 percent of total diet ) in a high effi ciency broiler ration exerted no significant adverse effect on growth of chicks while growth depression was observed when 50 per cent and 100 percent of the grain was barley. In a study on two types of barley ( Hiproly and Hiproly normal ) in which they were compared with wheat in diets either low ( 13% and 16% ) or high ( 16% and 20% ) protein during the starter and growing periods for broiler chicks, Moss et a]_. ( 1975 ) showed in the low protein diets that both types of barley produced a better performance than wheat. Birds fed the two types of barley had simi lar body weight gain, however, feed conversion was the best for Hi proly barley followed by wheat and Hiproly normal barley, both of which were not significantly different. Chicks on the high protein level diet showed the highest body weight gain and best feed conver sion for Hiproly barley, followed by Hiproly normal barley and fin ally wheat. Fernandez et ajk ( 1974 ) studied various cereal grains ( rye, triticale trail blazer, yellow corn, opaque-2 corn, gaines wheat, high protein wheat, Piroline barley and Hiproly barley ) in diets of one protein level . They found that chicks fed Hiproly bar ley and opaque-2 corn diets had the best body weight gains among the cereal grains tested. Although body weight gains were not significant ly different, Hiproly barley was observed to be slightly better than opaque-2 corn. For the PER ( protein efficient ratio: chick growth/ protein intake ), opaque-2 corn was significantly better than Hiproly barley, followed by other cereal grains. There were no significant differences in body weight gain among the other grains tested. ABSORPTION AND REGULATION OF VARIOUS MINERALS 6. It has been shown with the everted gut sac technique in rats that active transport of Ca is highly developed in the duodenum ( Schac-ter et al_., 1960 ). Harmeyer and DeLuca ( 1969 ) showed that in chicks the main site of active transport was also in the duodenum. At any giv en concentration of Ca, the duodenum has greater ability to absorb Ca per unit length than the jejunum or ileum when measured by the everted gut sac technique. The mechanics of the Ca uptake have been analyzed in inverted sacs of the rat small intestine which indicated that Ca first accumulates within the mucosal surface and then is transported to the serosal surface. It was shown by Wasserman and Taylor ( 1973 ) with studies 32 using the ligated loop technique in chicks that P was rapidly trans located across all segments of the small intestine. When the values 32 were expressed per cm. of intestine, the duodenum absorbed more P than the jejunum or ileum. In an in vivo study in the chick, different 32 results were obtained in which most of the P was absorbed at the upper jejunum. Magnesium is generally absorbed throughout the digestive tract. Guenter and Sell ( 1973 ) observed that the majority of dietary Mg was taken up by the duodenum, followed by the ileum, colon and jejunum. The only single study located in the literature on the absorp tion sites of zinc in chickens was done by Miller and Jensen ( 1966 ). The purpose of their study was to observe the differences in the effects of dietary protein on absorption and excretion of zinc in different sec tions of the digestive tract of the chick. Their findings showed that 65 the majority of the Zn was absorbed by the proventriculus and the small 7. intestine. There was more information on Zn absorption in rat studies. Van Campen and Mitchell ( 1965 ) measured uptake of 65Zn by selected tissues three hours after injection of the isotope into ligated intes tinal segments of adult rats. They showed that the duodenum had the highest rate of absorption followed by the ileum and jejunum with al most no Zn absorbed from the stomach. The results of Van Campen and Mitchell ( 1965 ) have been confirmed by Methfessel and Spencer ( 1966 ) 65 who reported that the absorption of Zn was 40 percent of the dose for the ligated duodenum, 15 percent for the mid-jejunum and ileal loops and only one to two percent for the stomach and colon. No information was located on absorption sites for copper in the chicken. From studies on the rat, Cu was absorbed from the stomach and upper gut ( Van Campen and Mitchell, 1965 ). Two separate mechanisms were described for Cu absorption ( Marceau ejt a]_., 1970; Crampton et aj_., 1 965 ). The first involves the transport of a copper-amino acid complex to the mu cosa of the small intestine and the second involves passive diffusion which accounts for the majority of the ionic Cu absorbed. Evans and Le-Blanc ( 1976 ) isolated and characterized a copper-binding protein from the rat intestine which was different from the conventional metallothio-nein. It was suggested that this intestinal copper-binding protein plays a role in copper absorption by regulating the passage of Cu from the mu cosa to the blood. There was no information observed in the literature on manganese absorption. Apparently, dietary Mn is generally accepted to be poorly ab sorbed by the chicken ( Scott et aj_., 1976 ). From rat studies, it Was suggested that variable excretion of Mn rather than absorption probably plays an efficient homeostatic regulatory mechanism for maintaining ba-8. lanced Mn tissue concentrations ( Abrams et al_., 1976 ). The area of Mn absorption is one aspect of Mn metabolism which needs to be studied. EFFECT ON MINERAL AVAILABILITY BY VARIOUS FACTORS Minerals play obscure but important roles in animal metabol ism. A deficiency or excess of some minerals result in metabolic dis turbances in the animal. Interaction between two or more minerals or between minerals and organic compounds such as vitamins and amino acids, may improve or impair efficiency of absorption or metabolic function. High levels of Ca or P in the diet prevent the deposition of Mg in the bone. Increasing Mg in the diet caused the bone Mg to increase ( Nugara and Edwards, 1963 ). It appears that the high Mg-low Ca diet may have caused Mg to replace Ca by deposition in the bone thereby re sulting in the higher Mg content of bone. Feeding of Zn was also assoc iated with an increase in the excretion of Ca and P. On the other hand, added dietary supplements of Ca and P can facilitate the removal of ex cess Zn from the animal body ( Stewart and Magee, 1964 ). Olson et aK ( 1972 ) demonstrated that, at low doses in the isolated vascularly perfused rat small intestine, calcitonin markedly inhibited Ca absorption whereas large doses of calcitonin markedly in creased Ca absorption. This effect remains unexplained. The authors suggested that calcitonin may play a direct role in preventing hyper calcemia resulting from excessive Ca absorption. An effect of calciton in was also noted by Swaminathan et al_. ( 1974 ) and Fox et al_. ( 1974 ) who found in sheep and pigs that large doses of calcitonin caused an ini tial increase in Ca absorption whereas small doses of calcitonin ultimately 9. caused a significant reduction in Ca absorption. In everted intestinal loops of the rat, cortisone has been shown to reduce active Ca transport and was thought to antagonize the effect of vitamin D on Ca diffusion ( Harrison and Harrison, 1960 ). This decrease in.active Ca transport has been confirmed by other work ers ( Kimberg et al_., 1971; Favus ejt al_., 1973 ). Another type of cor ticosteroid, prednisolone, also have shown to reduce active Ca transport ( Lukert et al_., 1973 ). Kimberg et a]_. ( 1971 ) showed that 25-OHD3 did not reverse the cortisone block of active Ca transport and that there were normal amounts of Ca binding protein in the intestinal mucosa of cortisone-treated rats. Favus et al_. ( 1973 ) showed that 1,25-(0H)2D3 failed to restore Ca absorption to control levels in cortisone-treated rats and that the cortisone effect was not mediated via the intermediate metabol ism of vitamin D. They proposed that the antagonism between glucocorti coids and vitamin D may be due to a steroid hormone-related alteration in end-organ functions that are independent of any direct interaction between the hormone and vitamin D or its metabolites. Wasserman and Carridino ( 1973 ) have confirmed that the administration of cortisone to rachitic animals does not inhibit calcium binding protein synthesis in response to vitamin D but does inhibit vitamin D stimulation of Ca absorption. It has been demonstrated that certain amino acids can increase Ca absorption. Wasserman et al_. ( 1956 ) found that L-lysine and L-argin-ine were particularly effective in increasing Ca absorption. Several other amino acids had this property to a lesser extent such as L-leucine, L-tryp-tophan and L-methionine but glycine, L-valine and L-phenylalanine were re latively ineffective. It had been suggested that the amino acids were able to solubilize Ca phosphates and hence render them more available for 10. absorption. However, it was found that lysine and glycine solubilized Ca phosphate to about the same degree whereas lysine was more than twice as effective as glycine in enhancing Ca absorption. Also, lysine was found to inhibit Ca absorption in the chick and this species difference cannot be explained if the mechanism depends primarily upon an interacts ion between amino acids and Ca ( Wasserman and Taylor, 1969 ). Raven et al . ( 1960 ) found that the action of lysine was greater in the ileum and the amino acid must be present in the same segment as Ca for absorption to be enhanced. The mechanism of this phenomenon remains unexplained. Condon e_t al_. ( 1970 ) have shown by balance studies that in corporation of lactose into the diet increased the absorption and retent ion of Ca in man. Sugars which are apparently inactive^such as glucose, are those that are rapidly absorbed by the upper small intestine but even glucose increases Ca absorption when injected straight into the lumen of the rat ileum ( Vaughan and Filer, 1960 ). The mechanism of lactose enhance ment of Ca absorption is not known but several theories have been proposed. One suggestion was that the sugars chelate or complex Ca and another that they exert a non-specific inhibitory effect on the energy-producing mech anisms of the intestinal cell which in turn leads to an increased permea bility to Ca ( Wasserman and Taylor, 1969 ). The vitamin D metabolite 1,25-dihydroxy-cholecalciferol is re cognized as the most potent naturally occurring derivative of the parent vitamin ( Henry et al_., 1976 ). There is no evidence which indicates that vitamin D plays a direct role in bone calcification ( Thornton, 1970 ). Nevertheless, it is generally accepted that the vitamin enhances the in testinal absorption of Ca and probably P, thereby assuring an adequate mineral supply for this process. The bulk of Ca and P absorption occurred 11. in the proximal small intestine with both hens and chicks ( Hurwitz and Bar. 1971 ). The pH in the intestinal tract has shown to be a factor in Ca availability ( Hurwitz and Bar, 1971 ). Almost all Ca flowing into the duodenum was in a soluble form at a pH of one to two. The increase of pH in the duodenum to about 6.5 resulted in a massive precipitation of Ca and P in this segment as evidenced by the appearance of a large non-ultra-filterable fraction. Morgan ( 1969 ) showed that vitamin D enhanced P absorption in the presence of Ca in the everted chick intestine but obtained no evidence that vitamin D had directly affected P transport. Similarly, Sal lis and Holdsworth ( 1962 ) using perfused duodenal loops of rachitic chicks in vitro found no evidence that vitamin D affected the intestinal absorption 32 of P. Furthermore, Neville and Holdsworth ( 1968 ) showed that only in the presence of Ca did vitamin D increase phosphate translocation in the rachitic chick intestine. In contrast, evidence has been reported that vitamin D directly affects P absorption. Kowarski and Schacter ( 1969 ) concluded that vitamin D increased phosphate transport selectively in the direction of mucosa to serosa by in vitro gut segments and that this was independent of the Ca transport mechanism. Hurwitz and Bar ( 1972 ) were unable to demonstrate any correla tion in the chick between Ca and P absorption. They found that the maxi mum effect of vitamin D was in the jejunum for phosphate absorption and in the duodenum for Ca absorption, thus indicating separate identities for 32 the two processes. Wasserman and Taylor ( 1973 ) found that P was not absorbed as a co-ion to calcium. Taylor ( 1974 ) showed that ouabain re-32 duced P transfer in vitro in the chick ileum whereas it did not affect 12. Ca transport. Chen et al_. ( 1974 ) showed that vitamin D stimulated phos phate transport by everted sacs of rat intestine. In the duodenum, Ca greatly increased phosphate transport but had no effect on phosphate transport in the jejunum. These workers concluded that vitamin D stim ulation of phosphate in the jejunum was not related to the Ca transport system of the intestine. Chen et al_. ( 1974 ) also showed that in the jejunum, vitamin D, 25-OHg or 1,25-(0H)2D3 stimulated phosphate trans port but 24,25-(0H)2D3 was not effective. Failure of 24,25-(0H)2D3 to activate phosphate transport at 48 hours was observed whereas it clearly activated Ca transport by this time ( Boyle et al_., 1973 ) by conversion to 1 ,24,25-(0H)3D3. Thus, it was suggested that 1 ,24,25-(0H)3D3 is a form of vitamin D3 which can specifically activate intestinal Ca trans port without activating phosphate transport. These results provided further evidence for two separate systems for Ca and P transport in the intestine. Parathyroid hormone is primarily concerned with the regulation of Ca homeostasis but is not part of a homeostatic mechanism for regula ting plasma phosphate. Sherwood et aj_. ( 1968 ) have shown with cows and Reiss et al_. ( 1970 ) in man that plasma phosphate does not regulate parathyroid hormone secretion directly but can do so by its effect on plasma Ca. Evidence for an interaction between calcitonin and phosphate comes from mammalian studies. When the diet of rats is low in P but ade quate in Ca, hypocalcemic responses to injected calcitonin are abolished. However, adequate P, in combination with low Ca in the diet, does not in hibit the hormone response ( Massry et al_., 1977 ). 13. Controversial reports have appeared concerning local effects of Ca on Mg absorption and vice versa. It has been reported that Mg de presses intestinal Ca absorption in experimental animals ( Alcock and Maclntyre, 1962 ) and also that Mg augments intestinal Ca absorption ( Clark, 1968 ). Excess Ca seems to be absorbed in dietary Mg deficiency and excess Mg absorbed in dietary Ca deficiency ( Alcock and Maclntyre, 1962 ). The explanation for the discrepancy could partly lie in the relationship out lined by Ebel and Comar ( 1968 ). At extremely low dietary Mg levels ( too low for normal growth ) Ca retention is high. With increasing diet ary levels of Mg, Ca retention declines, until the level of dietary Mg necessary for normal growth is reached. The Ca retention then rises abruptly, stays unaltered at even higher Mg levels and rises again at very high dietary Mg levels. The mode of action by which Mg exerts its influ ence on Ca metabolism may be in its effect on the parathyroid gland ( Chou et al., 1979 ). There is some evidence that Mg concentration affects parathyroid hormone secretion and that parathyroid hormone affects Mg homeostasis but the results were not conclusive. Maclntyre and Davidsson ( 1958 ) showed that Mg deficiency in rats produced hypercalcemia and nephrocalcinosis and Eliel et al_. ( 1969 ) subsequently showed that this response could be pre vented by parathyroidectomy. This suggests that Mg depletion causes para thyroid stimulation. Studies also have shown that low Mg concentrations stimulate ( Sherwood et aj_., 1971 ) and high concentrations suppress ( Git-elman et al_., 1968 ) parathyroid secretion. Mg has also been shown to in crease calcitonin secretion but only in high concentrations ( Littledike and Arnaud, 1971 ). In studies on Mg levels with broiler chicks, Lee and Britton ( 1980 ) observed that excess amounts of Mg caused diarrhea, lower 14. 28-day body weights, increased mortality and leg problems. Increasing dietary P significantly lessened the detrimental effects of Mg toxicity. The authors suggested that the presence of this element in excess may be a reason why there is controversy as to the P requirements of chickens. Several suggestions have been made concerning the mechanism whereby Ca exerts its antagonistic effect on Zn absorption ( Heth and Hoekstra, 1965; Hoekstra, 1964 ) but most evidence indicates that this effect is mediated by phytic acid ( Likuski and Forbes, 1965; Oberleas et al_., 1966 ). This viewpoint was supported by in vitro experiments which have shown that phytate forms a stable complex with Zn at pH 7.4 ( Vohra et al_., 1965 ). Precipitation of Zn from solution is depend ent upon the presence of both Ca and phytic acid ( Byrd and Matrone, 65 1965 ). The uptake of Zn by the mucosal cells of rat intestinal strips decreased progressively as the ratio of Ca:phytate in the medium increased while control strips with Ca alone showed similar effect ( Oberleas et aj_., 1966 ). These interactions assume that when insol uble Ca phytate is formed in the presence of adequate dietary Ca and phy tic acid, Zn was removed from solution by binding with Ca-phytate and therefore was rendered unavailable for absorption ( Byrd and Matrone, 1965 ). Because phytin ( Ca-Mg phytate ) comprises 60-80 percent of the total P in cereals and other plant seeds, the problem of unavailability of minerals bound to phytate was a significant one, especially for animals which are normally fed cereal grains and for humans in those countries where cereals and seed products make up a large portion of the diet ( Byrd and Matrone, 1965 ). It appears that a Ca depression influence on Zn absorption can occur in the presence of either a high level of phytic acid or inorganic phosphate, although phytic acid may be more effective 15 because of its greater insolubility and its poorer absorption. However, studies with semi-purified diets devoid of phytic acid demonstrated the antagonistic effect of Ca ( Heth et al_., 1966 ). This effect was found to be dependent upon the level of dietary inorganic phosphorus. Ca 65 significantly depressed Zn absorption from diets containing about one percent P but not when P level was 0.3 to 0.5 percent. Dietary phytate not only reduced the availability of Ca and Zn but also Mg, Fe, Mn and Cu ( Davies and Nightingale, 1975 ). A dietary phytate-Zn molar ratio of 6:1 supplies adequate Zn to maintain serum levels in humans subjects ( Harland and Prosky, 1979 ). Davies and Olpin ( 1979 ) have shown that phytate:Zn values exceeding 15-20 resulted in low hair and plasma Zn concentrations and low growth rates in rats. Franz et al_. ( 1980 ) indicated that the phytic acid content of various cereal grains and legume seeds was inversely related to Zn availability. However, phytic acid did not appear to hinder Zn availability in legumes as much as in cereals. The molar ratio of phytate:Zn has been suggested as a means to evaluate the availability to animals of Zn in various foodstuffs ( Davies and Olpin, 1979 ).. Like phytate, fiber also caused diminished bioavailability of minerals from plant sources. High fiber diets lead to increased fecal excretion of Ca, Zn, Mg and P and can therefore cause negative balances of these elements in the body ( Reinhold et aK, 1976 ). In studying the in fluence of dietary fiber on the mineral status in chicks, Thompson and Weber ( 1981 ) noted that dietary fiber sources ( wheat bran, corn bran, soy bran, oat hulls and cellulose ) showed no significant differences in body weights and feed intake with the exception of rice bran which caused depressed growth and feed consumption. Reinhold et al_. ( 1975 ) maintain that foodstuffs rich in both phytate and fiber ( whole meal bread and 16. bran-based products ) that fiber rather than phytate largely determines the degree to which polyvalent metals are absorbed through the intestinal walls. However, Davies et al_. ( 1977 ) reached the opposite conclusion from growth studies on rats in which phytate rather than fiber appeared to be the major determinant in respect to Zn availability. There are various minerals that show interrelationships with Cu. Both Zn and Cd are antagonistic to Cu utilization in the chick. When either element is added to Cu deficient diets, the severity of the manifestations of Cu deficiency is heightened. These effects were not observed when the diet is supplemented with Cu ( Hill et al_., 1963 ). Excessive dietary Mo produced a depression of growth, anemia and diarrhea in rats fed a low Cu diet. Supplementation of Cu prevented anemia and diarrhea but only when Cu was added with a high level of cysteine was growth brought back to normal using high Mo diets. Thompsett ( 1940 ) observed that less Cu was absorbed from a high Ca than from a low diet. Silver was also found to be antagonistic to Cu where Ag retard the growth of chicks in the absence of dietary Cu ( Hill et al_., 1964 ). It was demon strated that both Cu and Zn can induce a Se deficiency in chicks with relatively low dietary Se levels ( Jensen, 1975 ). Availability of Mn to chicks was decreased considerably with high levels of Ca and P in the diet ( Schaible and Bandemer, 1942 ). Mn is competitive with Fe and Co for common binding sites in the intestine ( Thomson et , 1971 ). Mn absorption may be influenced by ethanol in which ethanol enhances the transport of Mn two-fold in the intestine of the rat ( Schafer et al_., 1974 ). 17. AVAILABILITY OF MINERALS FROM VARIOUS CEREAL GRAINS Cereal grains constitute a major source of animal feed. Although they contribute most significantly as a source of carbohydrates and to a lesser extent as protein, their potential contribution of other nutrients including minerals is frequently overlooked. In nutritional evaluation of a feedstuff, one must consider the concentration of a particular nutrient as well as the biological availability to the animal. Few studies have been reported on the availability of minerals from cereal grains. In these studies various methods were used to deter mine the mineral availability. Therefore, the range in the estimated level of availability of a particular mineral is quite often different from one study to another. Using breaking strength as a criterion for measuring availability of P from various cereal grains, Hayes et aK ( 1979 ) compiled the follow ing results: corn 12%; hard red winter wheat 43%; soft red winter wheat 58% and barley 50%. The P contents in each of these cereal grains were: corn .27%; hard red winter wheat .37%; soft red winter wheat .30% and barley .44%. In respect to both content and availability, barley seemed to be the best grain as a source of P. Stober et al_. ( 1979 ) and Cromwell et al. ( 1974 ) showed that P in barley was more available than corn. Most of the P present in cereal grains is organically bound in the form of phytate ( Nelson ejt aK, 1968 ). According to McCance and Widdowson ( 1944 ) and Mollgaard ( 1946 ) cereal grains such as wheat and rye contain high levels of natural phytase with barley having intermediate levels and oats and corn having trace amounts of phytase. The biological Zn availabilities of various cereal grains obtain ed from a chick assay method ( O'Dell et al_., 1972 ) were: high lysine 18. corn ( .44% lysine; 10.2% protein ) 65%; control corn ( .30% lysine; 8.1% protein ) 63%; rice 62% and wheat 59%. Availability of Zn using a rat assay were: high lysine corn 55%; control corn 57%; rice 39% and wheat 38%. These results showed that the chick can utilize Zn from cereal grains better than the rat. Guenter and Sell ( 1974 ), using intramuscular injection of 28 radioactive Mg and assigning an availability index of 100 to MgSO^ in which the " true availability of Mg is 57.4% ", reported the following results from various cereal grains: oats 144; wheat 99; corn 97; bar ley 95 and rice 74. Assessing Mg bioavailability based on apparent absorption ( ie. feces collected quantitatively and on tissue concentra tions of Mg and Ca ) Ranhotra e_t al_. ( 1976 ) showed that about 70 per cent of the dietary Mg and Ca from wheat flour were absorbed by the rat. A more complete study on mineral availability of different cereal grains was done by Aw-Yong ( 1980 ) using the balance procedure of Nwokolo et al_. ( 1976 ) to determine availability in which retention of minerals was calculated as intake less fecal excretion corrected for endogenous components and expressed as percentage of intake. Aw-Yong ( 1980 ) determined Ca, P, Mg, Mn, Zn and Cu availability in wheat, bar ley, corn and triticale. His results, in percentages, are shown as follows: Ca P Mg Mn Zn Cu Wheat 69.50 67.50 53.11 47.85 48.89 77.47 Barley 68.90 68.80 54.90 54.90 49.10 77.50 Corn 70.00 60.90 51.00 60.00 57.60 87.20 Triticale 87.50 66.90 58.10 54.20 57.10 90.00 From the results of Aw-Yong ( 1980 ), wheat, barley and corn appear to be 19. quite similar in their mineral availabilities except for Mn, Zn and Cu in which corn was slightly better. Barley was similar to wheat in both Zn and Cu except for Mn in which barley was slightly better in availabil ity. Triticale, compared to the other three cereal grains, have the highest availability of Ca and Cu. The author concluded that minerals were highly available from cereal grains. METHODS OF DETERMINING MINERAL AVAILABILITY Various methods have been reported for determining the availabil ity of dietary minerals for animals. The following are some of the methods that have been used. 1) Isotope Dilution Method Radioisotopic procedures have been widely used for the determina tion of true digestibilities of dietary minerals, particularly Ca and P ( Evans et a]_., 1979 ). The isotope dilution method assumes (a) the plasma is uniformly labelled and in equilibrium with the tissues and (b) the endo genous excretion into the intestinal tract has the same specific activity as the plasma. The dilution of the total element in the feces by endogen ous excretion can be measured by difference in the plasma and fecal specific activities. 2) Comparative Balance Method This method estimates true digestibility directly, using radioiso tope balance data derived from pairs of animals, one dosed intravenously and the other orally with a single trace dose of radioactivity. The main assump-20. tion in the method is that the dietary source becomes fully labelled by the orally administered radioisotope for the true digestibility applies strictly to the administered radioisotope element. Aubert et al. ( 1963 ) proposed a modification of this method, eliminating the use of paired animals by injection of two different isotopes of the same element. 3) Apparent Digestibility Method This method measures the intake and total excretory output of test minerals ( Mitchell, 1964 ). Although this method is simple, it is very inaccurate in measuring digestibility of minerals because it does not take into account the endogenous losses from the animal. 4) Bone Ash Method This method involves the establishment of a standard response curve using a semi-purified basal diet and graded levels of an inorganic salt of the test element, assumed to be 100 percent utilized at low diet ary level. A straight line was obtained by plotting the percentage of bone ash of solvent extracted dry fat-free tibia of the chick and the logarithm of the percentage dietary mineral. The test ingredient was substituted for a small fraction of the basal diet. Biological avail ability was defined as the ratio, expressed as a percentage of the a-mount of the inorganic salt to the amount of test ingredient which pro duced the same bone ash when each was added to the basal diet. Nelson ( 1967 ) noted that bone ash was one of the most sensitive, practical criteria for evaluating the availability of dietary phosphorus. How ever, this method is limited to growing animals. 21. 5) Toe Ash Method This method is based on a linear relationship between diet ary phosphorus and toe ash content ( Yoshida and Hoshii, 1977 ). Avail ability of phosphorus was determined by using a slope ratio assay. The authors concluded that the toe ash method gave more accurate results than the bone ash method. 6) Carcass Analysis Method The method involves the use of litter mates, some of which were slaughtered at the beginning of the experiment to determine the ratios of body weight to the retention of test minerals ( Armstrong and Thomas, 1952 ). The other members of the litter were fed a controlled diet in which the test ingredient was the only source of the minerals under study. At end of the experimental period, the test animals are slaughtered, ashed and the mineral content determined. Minerals re tained, expressed as a fraction of mineral intake, is an indicator of availability. The disadvantage of this method is that it is limited to small animals. 7) Body Weight Method The method involves standard response curves which are established by supplemental basal diets with graded levels of the test element in inor ganic form, assumed to be 100 percent available ( 01 Del 1 et aK , 1972 ). A plot of weight or weight gain versus the logarithm of the supplemental element gives a linear response curve at lower levels of supplementation. The test ingredients are analyzed and subsequently substituted for carbohy drate in the basal diet at low levels. The quantity of biologically avail-22. able mineral is estimated from the standard curve and divided by the content of the test mineral in the ingredient. The method has shown to give inaccurate measurement of available phosphorus and therefore did not correlate well with body weight ( Nelson, 1967 ). 8) True Digestibility Method This method ( Nwokolo et al_., 1976 ) is most economical in terms of labor spent and feed used when compared to the methods that have already been described. The method takes into account the endo genous mineral excretion besides minerals of feed origin. Estimation of endogenous mineral involves the use of purified mineral-free diets. Be cause endogenous minerals are taken into account in calculating true di gestibility, the results are usually higher than apparent digestibility results and are indicative of mineral availability from feed ingested. METHODS OF DETERMINING METABOLIZABLE ENERGY OF FEED INGREDIENTS Hill et al_. ( 1960 ) described an assay which involves feeding a reference diet containing 44.1 percent of glucose and a similar diet in which a portion of the glucose was replaced by the test material. This procedure has been Used extensively for determining the metabolizable ener gy of grain and grain products. The metabolizable energy ( ME ) values of the test material is calculated using a value of 15.2 KJ/g for glucose which was adopted by Anderson et al_. ( 1958 ). The disadvantage of this assay is that the ME value of glucose being used under different experi mental conditions was assumed to be constant and that all samples of glu cose are of identical purity. This constant value is open to critism be-23. cause Anderson et al_. ( 1958 ) reported that the ME values for glucose ranged from 14.64 to 15.43 KJ/g on a dry matter basis. Mcintosh et al_. ( 1962 ) measured the ME values of cereal grains fed alone or in combination with a basal diet. The overall mean values revealed no major differences between the two procedures but individual treatment comparisons showed some degree of variation which present doubts on the assay of grains alone. March and Biely ( 1973 ) described a ME bioassay in which three weeks old chicks are fasted for 24 hours and then offered either a reference diet or a test material reference diet mixture for a period of three days following which the birds were again fasted for 24 hours. Feed intake was measured and excreta collected from the end of the first fast until the end of the second fast. This assay tends to give low estimates of ME according to Sibbald( 1975 ) probably because the fecal metabolic energy and the endogenous urinary energy voided over a period of four days were against the feed consumed in three days. Sibbald and Slinger ( 1963 ) used reference diets composed of practical ingredients and prepared test diets by replacement of a portion of the reference diet. An important part of their procedure was to add the test material to the reference diet at various levels. This makes it possible to determine whether the ME value was constant irrespective of the level of inclusion and permits the estimation of values by regression analysis. A major advantage of this assay is that the reference diet serves as a standard and is assayed in every experiment. A disadvantage is that the ME value of a feedstuff may vary with the composition of the reference diet. 24. The following two methods, Sibbald's and Farrell's, have been recently developed in 1976 and 1978, respectively. Both methods consumed similar time to obtain results, use small amount of feed and are faster than the methods that are used in the past years. Some feed manufacturers are currently using either of these two methods for ME determinations of feedstuffs because of their economical application. The quick bioassay of Sibbald ( 1976 ) for the determination of true metabolizable energy involves force-feeding of a known quantity of feedstuff, usually 30 to 40 grams into the crop of young adult cockerels which were previously starved for 24 hours. The excrement were collected from these birds for 48 hours ( Sibbald, 1982 ). To obtain the endogen ous excrement, the procedure uses two or more birds which are to be unfed during the experimental period. The true metabolizable energy ( TME ) is calculated as: TME=IE-( FE+UE ) + ( F E+U E ) where FE and UE is the me energy voided by the fed bird and F^E and UgE is the average of the energy voided by the unfed birds. Sub-samples of feed and feces ( either of en dogenous origin or feed origin ) are determined for their gross energy on a gram dry matter basis. The rapid method of Farrell ( 1978 ) for ME, which have been revised by Farrell ( 1980 ), involves the use of young adult cockerels ( at least six months of age ) trained to consumed their daily feed allow ance ( about 80-110 grams ) within one hour. All feeds were cold-pelleted before feeding to the birds which were starved for 32 hours previously. The test material may be fed alone or as partial replacement for a refer ence diet. The total amount of feed consumed is noted and the excreta voided during the subsequent 32 hours ( Farrell, 1980 ) is collected quantitatively. Gross energy was determined on sub-samples of feed and 25. excreta on a gram dry matter basis. The advantage of Farrell's over Sibbald's method is that one person can carry out the experimental pro cedure whereas Sibbald's method requires two people. METABOLIZABLE ENERGY FROM VARIOUS CEREAL GRAINS Feed is the largest single cost in animal production and accounts for over 50 percent of the poultry production costs ( Sibbald, 1982 ). The bioavailable energy component of feed is about 70 percent of the feed cost ( Sibbald, 1982 ). It was suggested by Sibbald ( 1982 ) that reduction of bioavailability input costs, through the use of more accurate bioavailable energy values to estimate requirements and to form ulate rations, offers the greatest potential for increasing production efficiency. Various researchers reported different values for various types of cereal grains. McNab and Shannon ( 1974 ), utilizing colostomized laying hens, reported values of 2.66, 3.16, 2.64 and 2.91 Kcal/g ME for barley, maize, oat and wheat, respectively. Giurguis ( 1975 ) used a chick assay method to determine ME of various feedstuffs, with the test feedstuff replacing dextrose in basal diets. Among the feedstuffs tested were wheat, barley, corn and oat. The results that he obtained for the cereal grains were 3.37, 2.98, 3.45 and 2.86 Kcal/g respectively. Using the rapid method for measuring the ME of feedstuffs, Farrell ( 1980 ) reported values of 3.07, 3.68, 3.75, 3.56, 3.63, 3.68, 3.41 and 3.55 Kcal/g for barley, corn, sorghum #1, sorghum #2, triticale #1, triticale #2, wheat #1 and wheat #2, respectively when testing the cereal grains with young adult white leghorn roosters. Farrell ( 1981 ) 26. in another report, reported similar results compared to his previous study ( Farrell, 1980 ). In this study, he reported values of 3.22, 3.10, 3.75, 3.53 and 3.48 Kcal/g, respectively, for barley, oats, sorghum, triticale and wheat. Differences in ME values of various feedstuffs as influenced by age of chickens have been reported. Petersen e_t aj_. ( 1976 ), using a chick assay method, compared the energy utilization of feedstuffs be tween four weeks old broiler chicks and mature white leghorn hens. The ME for male broiler chicks were 3.11, 3.06 and 2.35 Kcal/g for corn, wheat and barley, respectively; for female broiler chicks, 3.16, 3.10 and 2.41 Kcal/g, respectively; and for hens, 3.49, 3.14 and 3.05 Kcal/g, respectively. They found that there were no significant differences between male and female chicks on ME utilization of different cereal grains. However, there were significant differences between the chicks and hens on ME utilization for corn and barley but not for wheat. Among those cereal grains with differences in ME utilization, the hens could utilize greater energy than chicks. March et aj_. ( 1973 ) also found that the laying pullet was more efficient than the growing chick when fed rapeseed meal diets. However, in contrast to the findings of Peter sen et aj_. ( 1976 ), Sibbald et aj_. ( 1960 ) observed no significant differences between ME values of corn determined with chicks and hens. AMINO ACID AVAILABILITY OF VARIOUS CEREAL GRAINS The amount of information on amino acid availability from cereal grains is relatively small. DeMuelenaere and Feldman ( 1960 ) reported availability values of 92.8, 89.5, 95.3 and 88.8 percent, respectively, 27. for isoleucine, lysine, methionine and threonine from corn when using the fecal analysis method on rats. DeMuelenaere et aj_. ( 1967 ) re ported lysine of corn and rice proteins to be highly available. Gupta et al_. ( 1958 ) have determined that lysine availability value was on ly 50 percent by using the growth assay method on weanling rats. Pick and Meade ( 1970 ) reported that lysine of opaque-2 corn is as avail able as that from a reference diet containing fish meal, soybean meal, dried skimmilk, fat and sugars. Using regression analysis ( weight gained versus lysine con sumed ) and applying a slope ratio technique in a growth assay method, Klein et_ al_. ( 1972 ) estimated that the average lysine availability values for normal, opaque-2 corn and opaque-2 were 70, 80 and 89 percent, respectively. Sasse and Baker ( 1973 ) used growth assays on eight days old chicks to estimate the availability of sulfur amino acids in corn gluten and corn protein by the slope-ratio technique and also by a standard curve method. The availability estimates for corn gluten meal were 98.9% and 99.2% and for corn, 96.5% and 93.9% using the slope-ratio and standard curve methods, respectively. Using the true metabolizable energy bioassay for determining the apparent amino acid availability and true amino acid availability, Likuski and Dorrell ( 1978 ) had an average apparent amino acid avail ability of 82 percent and average true amino acid availability value of 97 percent for corn. True amino acid availability value was higher than apparent amino acid availability value because of the correction for meta bolic and endogenous amino acid excretion. For apparent amino acid avail ability, methionine and lysine values were 88 and 72 percent^espectively^ 28. In contrast, the true amino acid availability was 98 and 96 percent for methionine and lysine, respectively. Sibbald ( 1979 ) also used the true metabolizable energy assay procedure to measure the availability of amino acids of feedstuffs. When ten grams of each cereal grain was fed to adult cockerels, the true amino acid availability of lysine and methionine for corn was 95 and 93 percent, respectively; wheat 91 and 88 percent; Harmon oats 84 and 81 percent; Hinoat oats 96 and 97 percent; barley 81 and 68 percent. Sarwar and Bowland ( 1975 ) have determined availability of amino acids in different wheat cultivars ( Neepawa, Glenlea, Norquay and Purple ) for weanling rats. They reported that availability values of amino acids other than lysine and tyrosine were above 90 percent. The values for lysine in different wheat cultivars varied from 80 to 85 percent. Amino acid availabilities from triticale, wheat and barley for growing pigs weighing 10 and 30 kgs. were determined by the fecal analysis method ( Sauer et al_., 1974 ). In general, true amino acid availability of each essential amino acid decreased in the following order: triticale, wheat and barley. Lysine was found to be the." least'.available "essential amino acid from cereal grains. Availabilities of amino acid in Hiproly, Gait and three high-lysine experimental lines of barley were examined using rats ( Misir and Sauer, 1979 ). Availabilities of amino acids in the high lysine lines were equal to or greater than those of Gait barley and they ranged from 73 to 92 percent for the essential amino acids. Lysine availability was lowest in all the barley types. The lysine values were 72 and 64 percent for Gait and Hiproly, respectively, and 75, 73 and 75 percent for the three high lysine barleys. 29. Emebo and Roberson ( 1980 ) reported that the overall true amino acid availability of durham wheat, milo and corn were 96.6, 81.4 and 95.5 percent,respectively. The true lysine availability of wheat, milo and corn were 97.6, 73.1 and 94.6 percent,respectively. Bragg et al_. ( 1970 ) have shown that the average biological availability of amino acids in wheat was 92.1 percent. Stephenson et al. (1971 ) determined the amino acid content and availability of twenty-four grain sorghum hybrids and found that there were some variation in content and a large variation in availability. Four hybrids were found to be low in availability of all amino acids. There were also hybrids that were high in availability of all amino acids. Determinations of amino acid availability data for feedstuffs are needed for the most efficient use of feedstuffs. Elwell and Spares ( 1975 ) found that all diets utilizing available amino acids data re sulted in improved feed conversions. In general, amino acids of cereal grains are highly available. 30. CHEMICAL ANALYSIS OF SIX BARLEY SAMPLES AND ONE CORN SAMPLE Six barley samples from various locations in Alberta were received from Ritchie-Smith Feed Company in Abbots ford, B.C. All the barley samples were received by Ritchie-Smith Feed Company during the month of March, 1982 with the following description and location: SAMPLE DESCRIPTION LOCATION 1 Lambes Trucking Fort Saskatchewan Ln-5024 2 CN-427534 Bruderheim 3 ALPX-628376 Olds 4 CNWX-396939 Eckville 5 CPWX-601062 Didsbury 6 Unmarked Didsbury All the barley samples were of whole grain form. One ground corn sample from Surrey Co-op Feed Company was also analyzed. The bar ley and corn samples were all finely grounded in a Wiley Mill equipped with a one mm. screen in the U.B.C. Poultry Science laboratory before chemical analysis of the cereal grains was carried out. Chemical analy sis, using the methods described in A.O.A.C. ( 1965 ) was carried out for determining dry matter, crude protein ( N X 6.25 ), ether extract and ash content. The results are shown in Table 1. 31. Table 1. Chemical analysis of six barley samples and one corn sample ( dry matter basis ) Percent Sample Dry Matter Crude Ether Ash Protein Extract Lambes Trucking 85.9 14.4 2.5 2.5 CN-427534 85.6 11.5 3.8 2.9 ALPX-628376 85.7 11.2 3.4 3.0 CNWX-396939 86.5 13.7 3.1 3.3 CPWX-601062 87 .0 13.3 3.7 2.5 Unmarked 85.6 10.6 2.7 3.1 Corn 87.4 10.1 7.2 2.7 32. EXPERIMENT 1. AVAILABILITY OF MINERALS IN SIX BARLEY SAMPLES TO BROILER CHICKS EXPERIMENTAL PROCEDURE Day old broiler chicks from Horizon Hatchery in Abbotsford, B.C. were used to determine the availability of various minerals from the six barley samples. The chicks were raised in Petersime battery brooder cages to three weeks of age before the start of experiment. The chicks were fed a 20 percent crude protein commercial chick start er diet from Surrey Co-op Feed Company in Abbotsford, B.C. from day old to three weeks of age. Feed and water were supplied to the chicks ad 1ibitum. Chicks were weighed at three weeks of age and chicks of approximately similar body weights ( + 5 grams ) were used. Ninety-six birds were transferred to the stainless steel metabolism cages with four birds per cage. There were four cages of birds for each barley sample. The birds were given three days to acclimatize to the new environment before starting them on a balance procedure used for determining mineral availability ( Nwokol o et a]_., 1976 ). At 24 days of age, all birds were supplied with a starter diet containing 0.3 percent ferric oxide as a marker for four hours and fasted for 16 hours. Following the fasting period, birds were fed a synthetic diet ( Table 2 ) for four hours. Birds were then fasted for another hour before returning to the marker diet. Feces derived from the synthetic diet were collected ( starting from the end of the first batch of marked excreta and ending at the beginning of the second batch of marked excreted ). On the twenty-fifth day, the same procedure was repeated except the barley samples replaced the Table 2. Composition of synthetic diet Ingredients Percent Corn Starch 40.6 Sucrose 40.Alpha-cell 13.8 Corn Oil 5.0 100.0 34. synthetic diet. There were four replicates for each tested ingredient. Marked feed, including synthetic and barley diets and water, were offer ed ad libitum during the testing period. Limited water was given during the fasting period. Feed consumption of both synthetic and barley diets were recorded at the end of each period. Total marker-free feces were collected from each individual cage. The feces from each cage was oven dried after collection at 85 degrees celcius for 48 hours. The dry feces were weighed and finely ground in a Kurzzertbetrieb microgrinder to be analyzed for mineral content. Barley samples and feces from both synthetic and barley diets were wet ashed to determined the Ca, Mg, P, Mn, Zn and Cu contents by using the Jarrel Ash Atomic Absorption Spectrophotometer. Phosphorus was determined by a Unicam SP1800 Ultraviolet Spectrophotometer follow ing color development with ammonium molybdate. The formula used for calculating the percentage availability of minerals after analysis of ingredients and excreta was developed by Nwokolo et al_. ( 1976 ) as follows: Percent Mineral Availability = TMI ". TMI^ " EFME ^ X 100 where TMI = Total mineral intake from feed ingredient TFME = Total fecal mineral excreted EFME = Endogenous fecal mineral excreted The availability data was subjected to analysis of variance and the statistical comparison of mean by Duncan's Multiple Range Test ( Little and Hills, 1978 ). 35. RESULTS The mineral content of six barley samples is shown in Table 3. The average concentration of Ca, P, Mg, Zn, Mn and Cu were 686, 4397, 6619, 15, 70 and 21 ppm, respectively. The average mineral availa bility of the barley samples for Ca, P, Mg, Mn, Zn and Cu were 93.9%, 85.2%, 81.4%, 70.4%, 71.3% and 76.5%, respectively. These results are shown in Table 4. Calcium Results indicate that among the minerals analyzed, Ca in barley has the highest availability. There were significant differ ences among barley samples tested. The availability values of the various barleys ranged from 84.9% ( barley #3 ) to 98.7% ( barley #1 ). There were no significant differences among barleys #1, #4, #5 and #6 but barley #2 differed significantly from barleys #1, #3 and #4. Phosphorus Phosphorus was analyzed to have the second highest availa bility among the minerals. The average availability was 85.2%. The average values had a range from 78.1% ( barley #3 ) to 92.9% ( barley #1 ). There were no significant differences among barleys #2, #4, #5 and #6 as well as for barleys #1, #4, #5 and #6. There were no signi ficant differences among barleys #1, #2 and #3. Table 3. Content of minerals in six barley samples ( dry matter basis ) ppm Barley # Sample Ca P Mg Mn Zn Cu 1 lambes 668 41?0 67Q4 15 76 28 Trucking 2 CN-427534 98 2 468 0 7 1 54 1 5 8 1 22 3 ALPX-62837 6 7 9 4400 645 0 1 4 6 3 2 0 4 CNWX-396939 616 5200 6491 17 87 22 5 CPWX-601062 519 3730 6406 12 59 22 6 Unmarked 647 4200 6510 15 54 14 Average 686 4397 6619 15 70 21 Table 4. Availability of minerals from six barley samples ( dry matter basis ) Percent Barley # Sample Ca P Mg Mn Zn Cu 1 Lambes 98.7a 92.9a 90.7a 91 .la 93.7a 85.3al Trucking 2 CN-427534 92.3cd 82.3bc 79.4bc 78.3ab 78.9ab 68.5° 3 ALPX-628376 84.9e 78.1C 73.3C 46.8d 52. lc 68.lc 4 CNWX-396939 96.9ab 88.6ab 86.6ab 76.1ab 73.3b 87.7a 5 CPWX-601062 95.3abc 84.1abc 76.0C 57.4cd 56.1C 67.9° 6 Unmarked 95.2abcd 85.0abc 82.4abc 72.7bc 73.6b 81.3a Average 93.9 85.2 81.4 70.4 71.3 76.5 Means with different superscripts within a column are significantly different ( P f-0.05 ). 38. Magnesium Magnesium availability was highest in barley #1 ( 90.7% ) and lowest in barley #3 ( 73.3% ). There were no significant differ ences among barleys #1, #4 and #6; barleys #2, #4 and #6; and for barleys #2, #3, #5 and #6. Differences were noted between barley #1 and barleys #2, #3 and #5. Manganese No significant differences were noted among barleys #1, #2 and #4; barleys #2, #4 and #6; and barleys #3 and #5. Differences were noted among barleys #1, #3 and #6. Zinc Three barley samples had availability values in the 70's. These were barleys #2, #4 and #6 and were not significantly different. Two barley samples had values at the 50% level. The highest availabil ity value was found with barley #1 at the 90% level. Copper The availability of copper, like the other minerals was high with an average value of 76.5%. The availability values range from 67.9% ( barley #5 ) to 87.7% ( barley #4 ). There were no significant differences among barleys #1, #4 and #6 or for barleys #2, #3 and #5. Significant differences were noted between these two groups of barley. 39. DISCUSSION In general, all minerals were highly available from all barley samples. Mn and Zn in barleys #3 and #5 were somewhat low in comparison to other barley samples. Ca in these barley samples aver aged 93.9% in availability which is considerably higher than the 68.9% average value reported by Aw-Yong ( 1980 ). However, among the three barley samples analyzed by Aw-Yong ( 1980 ), the locations of two bar ley samples were known. The local barley from Chilliwack, B.C. has a Ca availability value of 51.9%; whereas, the barley from Alaska was de termined to have a considerably higher availability value of 80.5%. The barley samples from this report came from a small region in the province of Alberta and probably because of this, Ca availability of these barley samples did not differed as greatly as the barley samples reported by Aw-Yong ( 1980 ). The Ca availability of the barley samples from this report were comparable to the 80.5% availability value of the Alaska barley reported by Aw-Yong ( 1980 ). Other cereal grains can have high availability of Ca as shown for one wheat sample with availability value of 82.9% and triticale, with a value of 87.5% ( Aw-Yong, 1980 ). Furthermore, Armstrong and Thomas ( 1952 ) reported Ca availability from plant sources with values of 84.89%, 83.11% and 79.95% for lucerne,,red clever and wild white clover, respectively, using rats as the test animals. Armstrong et al. ( 1953 ) found that burnet, chicory and narrow-leaved plantain ( three herbs of grassland ) have availability values for Ca of 80.34%, 87.73% and 95.58%, respectively. Nwokolo e_t al_. ( 1976 ) reported that Ca was well utilized from soybean meal with an availability value of 85.6%. 40. Phosphorus availability of the barley samples in this re port was also quite high, having an average value of 85.2%. This was higher than the reported average value of 68.8% for P by Aw-Yong ( 1980 ). However, one barley sample he analyzed had a value of 73.2% compared to 66.0% value for the other two samples. Also, three wheat samples had P availability values ranging from 76% to 79%. Only one corn sample had a P availability value over 70% ( 72% ) with availability values of less than 60% for other corn samples. Nwokolo et al. ( 1976 ) reported P availability values of 89.3%, 76.9%, 74.8% and 70.8% for soybean meal, rapeseed meal, cottonseed meal and palm kernel meal, respectively. The results of this report and others indicate that high availability values for P from plant sources are not uncommon. This view is in contrast with that of the committee on animal nutrition ( NAS-NRC, 1960 ) in which only approximately 30% of the total P in plant materials is considered to be utilized by non-ruminants. Magnesium in barley samples was comparable to P in availability, having an average value of 81.4%. This value is considerably greater than the average value of Mg reported by Aw-Yong ( 1980 ) where three barley samples all had availability in the 50% range. One of the eleven wheat samples analyzed had a Mg availability value of 70.5% whereas other wheat samples were in the 40% or 50% range. These results suggest that the probable causes of different availability values from cereal grains may be due to the difference in geographical locations, genetical strains or other factor(s) that may influence mineral interactions and availabil ity. Nwokolo et al_. ( 1976 ) reported average Mg availability values of 74.6% and 77.8% for cottonseed meal and soybean meal samples, respectively. 41. Manganese availability, compared to Ca, P and Mg, is consid erably lower with an average value of 70.4%. Aw-Yong ( 1980 ) had one barley sample with an availability value of 65.8% whereas the other two samples had lower values of 42.8% and 56.2%. One wheat sample among the eleven tested, showed a high value of 64.3%. Other wheat samples have values between 30%, 40% or 50%. Two corn samples had values of 70% or greater ( 70.1% and 77.3% ). Nwokolo et al_. ( 1976 ) reported soybean meal and cottonseed meal to have average availability values of 76.1% and 76.3%, respectively. The average availability value for zinc was 71.3% compared to the report by Aw-Yong ( 1980 ) of 49.1% for three different barley samples. However, the average value was based on 39.3%, 37.5% and 70.6% for the three samples. The 70.6% value was from the Alaskan barley and is comparable to the value from this report. Aw-Yong ( 1980 ) also had two corn samples with availability values at the 70% level ( 72.5% and 77.1% ). Nwokolo It a]_. ( 1976 ) reported an availability value of 66.5% for soybean meal. 0'Dell et al_. ( 1972 ) reported a similar value for soybean meal ( 67% ). Barley samples, in this report, have an average availability value of 76.5% for Cu. This value is in agreement with Aw-Yong ( 1980 ) with the average value of 77.5%. This value was based on the range between 73.9% and 81.4% whereas the value in this report was based on a range of 68.1% to 87.7% for the six barley samples. Aw-Yong ( 1980 ) reported a corn sample average value of 87.2% based on high values rang ing from 81.5% to 94.5%. However, wheat samples had an average value of 78.5% which varied from 60.6% to 92.9%. Nwokolo and Bragg ( 1977 ) reported an average Cu availability value of 74.3% for rapeseed meal. In general, the results of this report showed that minerals in barley were highly available and were comparable to other cereal grains such as wheat and corn and protein sources such as soybean meal. 43. EXPERIMENT 2. EFFECT OF BARLEY ON GROWTH PERFORMANCE AND FEED CONVERSION OF BROILER CHICKS USING PRACTICAL DIETS EXPERIMENTAL PROCEDURE Three hundred and ninety broiler chicks, one day of age were obtained from a commercial hatchery for this experiment. The chicks were maintained in battery brooders during the four weeks experiment. Diets were prepared and supplied to chicks at one day of age. Feed and water were supplied ad libitum. Feed consumption, body weights and mortality were recorded on a weekly basis. There were 13 treatments with three replicates of ten chicks ( equal number of each sex ) placed on each treatment. DIETARY TREATMENTS A corn-soybean diet was used as the control treatment contain ing no barley ( Table 5 ). The control diet was calculated to supply approximately 23% protein with 3200 Kcal/Kg. Each barley sample was sup plied at approximately 20% or 40% of the total cereal grains at the ex pense of corn. All diets were calculated to have similar protein contents based on nutritional values of barley ( NRC, 1977 ). RESULTS AND DISCUSSION Body weight gain, feed conversion and feed consumption per chick are summarized in Table 6. Chicks fed.diets with barley containing 20% or 44. Table 5. Composition of experimental diets Percent Ingredients 0% a 20% b 40% c Soybean meal 33.75 32.90 32.00 Barley 0.00 11.00 23.00 Corn 55.25 45.10 34.00 Meat meal 4.00 4.00 4.00 Animal tallow 5.25 5.25 5.25 Dicalcium phosphate 0.75 0.70 0.65 Limestone 0.45 0.50 0.55 Iodized salt 0.25 0.25 0.25 D-L-Methionine 0.1217 0.1295 0.1379 Vitamin-mineral ** ** ** premi x Metabolizable Enerqy 3201 3123 3037 Kcal/Kg % Protein 23.34 23.34 23.34 % Calcium 0.9022 0.9051 0.9081 % Phosphorus 0.6974 0.6943 0.6918 % Lysine 1.3099 1.3024 1.2952 % Methionine 0.3795 0.3718 0.3635 Premix supplied per Kg diet: Mn, 38.98 mg; Se, 0.03 mg; Zn, 15.60 mg; nicotinic acid, 15.0 mg; calcium panto thenate, 12.0 mg; riboflavin, 4.0 mg; vitamin A, 4500 IU; Vitamin D3, 400 ICU; Vitamin E, 20 III; Vitamin K ( menadione ), 1.0 mg; biotin, 0.3 mg; Vitamin BJ2, 9.0 meg; santoquin, 125.0 mg. 45. OUTLINE OF EXPERIMENTAL TREATMENTS a) Diet #13 Diet with no barley ( ie. 0% of total cereal grain ) b) Diets #1 - #6 Diets containing each barley sample comprising 20% of total grain c) Diets #7 - #12 Diets containing each barley sample comprising 40% of total cereal grain Table 6. Feed consumption, body weight gain and feed conversion per chick fed different composition of barley samples. Grams Diet* Sample Composition of Average feed Average body Average feed cereal grain [%) consumption weight gain conversion 1 Lambes Trucking 20 1445.83 910.48 1.59b h 2 CN-427534 20 1515.29 940.67 1.61b 3 ALPX-628376 20 1425.13 899.01 1.59b 4 CNWX-396939 20 1421.58 900.44 1.58b 5 CPWX-601062 20 1413.22 878.77 1.61b 6 Unmarked 20 1407.10 873.64 1.61b 7 Lambes Trucking 40 1348.68 830.82 1.62b h 8 CN-427534 40 1363.59 848.18 • 1.61b 9 ALPX-628376 40 1437.31 889.76 1.62b 10 CNWX-396939 40 1406.66 834.14 1.69a 11 CPWX-601062 40 1414.10 876.33 1.61b 12 Unmarked 40 1457.26 896.05 1.63b 13 Corn 100 1403.52 884.03 1.59b Means with different superscripts within a column are significantly different 47. 40% of the total cereal grain showed no significant differences in body weight gain and feed consumption when compared to chicks fed the diet containing corn without barley. It is interesting to note that all barley-containing diets were comparable to corn in feed consumption, body weight gain and feed conversion except for diet #10. Diet #10 showed a feed conver sion significantly higher from other treatments although there were no significant differences in feed consumption and body weight. This may indicate that there was a factor(s) in this barley sample which caused a slight decrease in absorption of a certain nutrient(s) as the chicks consuming this diet had a low body weight gain but with quite a high feed consumption. It appears that the amount of barley sample ( CNWX-396939 ) which could replace corn in the diet is limited for feed conversion because at 20% of total cereal grains there were no significant differences in feed conversion. Coon ejt al_. ( 1979 ) found that among the 22 varieties of bar ley tested, only two barley types were different from the other samples in feed efficiency when fed to chicks. Also, there were no significant differences in weight gains of the chicks. Wilson and McNab ( 1975 ) reported that no significant differences in feed efficiency ratios for the various barley samples tested when the diets were formulated similar in protein and energy contents or just protein content alone. Reasons for the findings of Coon et aK ( 1979 ) and the present report regarding variations in chick performance on different barley samples remains unexplained. It was suggested by Burnett ( 1966 ) that the probable cause of the low nutritive value of some barley vari eties may be due to their high B-D-glucan and low B-D-glucanase contents 48. because B-D-glucan can decrease the availability of protein and carbohydrate. They also noted that barleys with high levels of B-D-glucanase have greater nutritive value. Coon et al_. ( 1979 ) found that some barley types analyzed were low in either lysine or both lysine and threonine when compared to other types. The results in this report are in agreement with those of Arscott et al_. ( 1955 ) who showed that inclusion of barley up to 25% of the grain component in the diet exerted no significant effect on the growth of the chick. Arscott et al_. ( 1957 ) also reported that 50% of the corn in the diet can be replaced by Hannchin barley without growth depression. Fry et al. ( 1957 ) reported that a com bination of pearled barley and corn at a ratio of 50:50 gave comparable results to those of corn alone using similar protein levels with or without adding tallow. Both growth and feed efficiency of chicks at four weeks of age were similar. Fernandez et aj_. ( 1974 ) reported that Hiproly barley and opaque-2 corn gave the best body weight gains in chicks fed different cereal grains. Results of this report and earlier reports indicate that most barley varieties can replace a certain percentage of corn in chick diets. There are some reports showing that barley gives better performances than wheat ( Moss et al_., 1975; Fernandez et al_., 1974 ). Therefore, the general assumption in the past that barley is inferior to corn and wheat is open to criticism. Because the cost of barley is less than both corn and wheat, it would be profitable to use barley in poultry diets to replace corn and wheat at specified dietary levels. The levels of barley and the specific type of barley to replace corn and wheat should be further studied. 49. EXPERIMENT 3. METABOLIZABLE ENERGY OF SIX BARLEY SAMPLES AND ONE CORN SAMPLE EXPERIMENTAL PROCEDURE The rapid assay method of Farrell ( 1980 ) was used in this experiment with minor changes. Twenty months old white leghorn roosters were each placed in individual cages with an empty cage used for spacing. The birds were trained for three weeks to consume the daily feed allow ance in one hour. At the start of training, 16% protein commercial layer ration ( grounded ) was given to the roosters. The amount of feed con sumed within one hour ranged from 60-100 grams ( only birds consuming more than 60 grams were used in the study ). The birds were starved for 48 hours before given their tested barley or corn samples which were also grounded. There were seven out of twelve original roosters that consumed over 60 grams of feed and six were chosen for the experiment. Because body weights were different, the roosters were paired so that total body weight of the paired birds were similar. After the roosters had eaten their hourly allowance, a collection bag was placed under each rooster cage. Collection of excrement was for .48 hours during the fasting period. Excreta was removed and dried ( 85°C ) at the end of the 48 hour period. The oven-dried sample was then cooled to room temperature and grounded using the Kurzzertbetrieb microgrinder. Gross energy using the Parr oxygen bomb calorimeter was determined. RESULTS AND DISCUSSION The average ME values ( Table 7 ) in this report were considerably higher 50. Table 7. Metabolizable energy of barley and corn samples ( dry matter basis ) Kcal/gram Sample Gross energy Gross energy Metabolizable of grain of feces energy of grain Lambes Trucking 4.715 1.614 3.101 CN-427534 4.797 1.974 2.823 ALPX-628376 4.728 1.825 2.903 CNWX-396939 4.667 1.741 2.926 CPWX-601062 4.668 1.813 2.855 Unmarked 4.682 1.750 2.932 Corn 4.767 1.864 2.903 2.923 51. than the value stated in NRC ( 1977 ). Barley samples tested have an average value of 2.923 Kcal/g compared to 2.624 Kcal/g from NRC ( 1977 ). Although the value was higher than the NRC ( 1977 ) value, it was low er than the value reported by Farrell ( 1980 ) and Farrell ( 1981 ).. Farrell reported average values of 3.07 Kcal/g ( Farrell, 1980 ) and 3.22 Kcal/g ( Farrell, 1981 ) for his tested barleys. The ME value of corn in this report is lower than 3430 Kcal/g stated by NRC ( 1977 ). Farrell ( 1980 ) reported an average value of 3.68 Kcal/g. The differ ence between the results of this report and those of Farrell ( 1980, 1981 ) may be due to the difference in age and strain of the birds used. With these ME values, the total ME values of each experimental diet were recalculated. The recalculated ME values of each diet is shown in Table 8. The recalculated ME values of the diets containing different barley samples and corn are quite comparable to one another. The ME of the barley diets are either similar or slightly higher than the corn diet without barley. Also, the ME values of these diets are roughly 200 Kcal/Kg less than the original calculated ME values for these diets. From the new recalculated ME values, the difference between the diets with the lowest ( 2899.76 Kcal/Kg ) and highest ( 2963.70 Kcal/Kg ) values was 63.94 Kcal/Kg whereas the difference among the original ME values ( lowest, 3036.85 Kcal/Kg; highest, 3201.23 Kcal/Kg ) was 164.34 Kcal/Kg. There fore, the new recalculated values of ME for the experimental diets pro vide a better view of how the broiler chicks perform because chicks in all diets have similar performances in regard to body weight gain, feed consumption and feed conversion except for Diet #10 chicks which differ ed only in feed conversion. 52. Table 8. Recalculated metabolizable energy values Diet Composition of Sample Metabolizable cereal grain (%) energy ( Kcal/Kg ) 1 20 Lambes Trucking 2936 2 20 CN-427534 2905 3 20 ALPX-628376 2914 4 20 CNWX-396939 2917 5 20 CPWX-601062 2909 6 20 Unmarked 2917 7 40 Lambes Trucking 2964 8 40 CN-427534 2900 9 40 ALPX-628376 2918 10 40 CNWX-396939 2923 11 40 CPWX-601062 2907 12 40 Unmarked 2925 13 100 Corn 2910 53. SUMMARY AND CONCLUSIONS Broiler chicks were used to determine the mineral availability of Ca, P, Mg, Mn, Cu and Zn from six barley samples. Effects of partial replacement of corn for barley on growth performance and feed conversion of broiler chicks were examined. Metabolizable energy of the barley and corn samples were also evaluated using adult white leghorn roosters. The results of this study showed that six minerals were highly available from barley samples. The average availability for Ca, P, Mg, Mn, Zn and Cu were 93.9%, 85.2%, 81.4%, 70.4%, 71.3% and 76.5%,respect ively. Variations in mineral availability among some of the barley samples were observed. Partial replacement of corn by barley samples in the diet did not affect body weight gain or feed consumption among treatments with barley at 20% or 40% of the total cereal grains. Treatments show no significant differences in feed conversion except diet #10 consisting of barley CNWX-396939 at 40% replacement of dietary corn. This indicates that factors related to barley composition may decrease the availability of some nutrients in specific samples. Barley samples tested at two dietary levels were comparable to the corn diet in body weight gain, feed consumption and feed conversion except CNWX-396939 at the 40% replacement of dietary corn. Metabolizable energy values of the barley and corn samples were comparable. 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Analysis of variance for mineral availability from barley Mean Square Source of variance D.F. Ca P Mg Mn Zn Cu Treatment 5 0.00723 0.0105b 0.0170a0.01005a0.0933a0.0347a ReplicatesN'S" 3 0.0005 0.0006 0.0017 0.0021 0.00110.0023 Error 15 0.0006 0.0036 0.0034 0.0115 0.0098 0.0059 a Significant ( P £ 0.05 ) b Significant ( P £ 0.01 ). I S * ' Not significant Appendix Table 2. Analysis of variance for body weight gain at four weeks of age. Source of variance D.F. Sum of square Mean square Treatment Re pi ica.tes Error Total N-S' Not significant ( P £ 0.10 ) 12 0.0034 0.00028 2 0.0005 0.00025 24 0.0052 0.00022 38 0.0091 0.00024 Appendix Table 3. Analysis of variance for cumulative feed consumption at four weeks of age. Source of variance D.F. Sum of square Mean square Treatment Replicates Error Total N-S' Not significant ( P-f 0.10 ) 12 0.01 0.0008 2 0.01 0.0050 24 0.18 0.0075 38 0.20 ).0053 Treatment Replicates Error Total Appendix Table 4. Analysis of variance for feed conversion at four weeks of age. Sum of square 0.027 0.000 0.023 0.050 12 2 24 38 0.0023 0.000 0.00096 0.0013 N.S, N.S, Significant ( P -50.05 ) Not significant ( P £ 0.05 ) 01 VO 


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