DISCRIMINATION BETWEEN CARBOHYDRATES Molybdenum Supplementation of Chick Diets

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1 DISCRIMINATION BETWEEN CARBOHYDRATES 1127 Perry, J. W., A. E. Moore, D. A. Thomas and F. J. R. Hird, 196. Galactose intolerance observations on an experimental animal. Acta. Paediatrica, 4: Richter, C. P., and K. H. Cambell, 194. Taste thresholds and preferences of rats for five common sugars. J. Nutrition, 2: Schutz, H. G., and F. J. Pilgrim, 197. Sweetness of various compounds and its measurement. Food Research, 22: Supplementation of Chick Diets SINCE it appeared that tungsten supplementation of breeder hen rations produced chicks deficient in molybdenum (Teekell and Watts, 199), it was decided to use these chicks in evaluating the effect of adding various levels of molybdenum to the chicks' diet. The anomalous response of the chicks from breeder hens fed supplemental tungsten and from nonsupplemented hens which had been previously observed, raised some question as to the nature of the condition produced by the tungsten supplementation. The purpose of this study was to test the effect of molybdenum supplementation at various levels in a purified diet and a practical corn-soybean diet. Chicks from tungsten-supplemented and non-supplemented (control) were used in this study. EXPERIMENTAL The purified ration portion of this study involved 96 chicks in a series of 1 trials from the tungsten-supplemented hens and 84 chicks from non-tungsten supplemented hens. A purified cerelose-drackett C-l assay protein ration was formulated and was calculated to contain less than ROGER A. TEEKELL* AND A. B. WATTS Louisiana State University, Baton Rouge, Louisiana (Received for publication March 16, 199) * Present Address: UT-AEC Agricultural Research Laboratory, P.O. Box 142, Oak Ridge, Tenone ppm. molybdenum (Table 1). supplementations (as sodium molybdate) of, 1, 2,,, 1,, 1,, 1, and 2, ppm. were tested. A practical corn-soybean ration (Table 2) was fed to over 4 chicks in a second experiment. All chicks were raised in electric starter-type battery brooders with raised wire screen floors. The purified diet and practical broiler ration were chemically analyzed for molybdenum, tungsten, copper, sulfur, phosphorus and potassium. was determined as a dithiol (4-methyl-l, 2- dimercaptobenzene) complex developed in w-amyl acetate. Tungsten from the same sample was reduced by stannous TABLE 1.Purified chick ration Ingredient Drackett C-l assay protein Cerelose Jones-Foster salt mix no Alpha Cel (cellulose) Crisco Wesson oil Cod liver oil 2 Dl^glycine Di^methionine Creatine hydrate Percent The following materials were added as grams per pound: Gm./lb. Gm./lb. Alpha Tocopherol. Riboflavin.7 Choline chloride.9 Biotin.27 Inositol.4 Folic acid.2 Para-aminobenzoic acid.2 Thiamine HC1.14 Calcium pantothenate 1.1 Menadione.4 Niacin.1 Vitamin B12.1 Pyridoxine. DPPD.1 Vitamin Ds.7 1 Nutritional Biochemicals Inc. s 2,2 USPA and I.C.U. Di/lb. Downloaded from at Penn State University (Paterno Lib) on May 12, 216

2 1128 R. A. TEEKELL AND A. B. WATTS TABLE 2.Practical type chick ration Ingredient Percent Yellow corn meal 8. Soybean oil meal (% protein) 27.4 Menhaden fish meal. Alfalfa leaf meal. Dicalcium phosphate 2. Oyster shell flour 1. Animal fat (stabilized) 4. Sodium chloride. Vitamin premix*.6 Total 1. * Supplies the following per pound: Riboflavin 2 mgm.; calcium pantothenate 4 mgm.; niacin 9 mgm.; choline chloride mgm.; Vitamin D 64 I.C.U.; Vitamin A, I.U.; Vitamin E 2.4 I.U.; Vitamin B12 6 mcgm.; manganese 4 ppm.; and bacitracin mgm. chloride and titanium III and was determined as a tungsten-dithiol compound also developed in»-amyl acetate (a modification of the method of Greensberg, 197). Copper was determined by a diethyldithiocarbamate method and sulfur by a gravimetric method as barium sulfate (Parks et al., 194). Phosphorus was determined photometrically using vanadomolybdate (Epps, 19; and Austin et al., 19). Potassium was determined by the use of a Beckman DU Flame Spectrophotometer in the manner described by Austin et al. (19). RESULTS AND DISCUSSION TABLE.Mineral content of rations Element Purified diet Practical ration Tungsten Copper Sulfur Phosphorus Potassium ,649.,988. 4, ,6.,419. 9,. TABLE 4. and tungsten levels in day-old-chicks Dam treatment Non-supplemented Non-supplemented Non-supplemented Chick tissue Whole carcass Liver Kidney Whole carcass Liver Kidney The mineral determinations are shown in Table. and tungsten determinations were also made on whole carcasses, livers, and kidneys (wet-weight basis) of day-old chicks from all treat Tungsten ments. Five replicate determinations were made from each tissue sample (Table 4). The data in Table 4 show that there was an apparent carry-over of the tungsten from the to chicks. This is particularly evident in the case of kidney and liver. The 2-day gain and feed data from the purified ration study are shown in Table. A statistical analysis of variance (Snedecor, 196) indicated that the addition of 1, 2,, and 1 ppm. supplemental molybdenum gave a statistically significant gain increase, while 1 and ppm. gave a highly significant increase in the 2-day gain in chicks from tungsten-supplemented. An apparent molybdenum "toxicity" level appeared to lie between 1 and ppm. as the higher level of supplementation TABLE.Gain and feed of chicks fed purified diet with added molybdenum Added , 2, day gain Control Downloaded from at Penn State University (Paterno Lib) on May 12, 216

3 MOLYBDENUM IN CHICK DIETS 1129 resulted in a highly significant growth depression. Higher levels of molybdenum supplementation (1, and 2, ppm) gave even greater depressions in 2-day growth rate. Statistical analyses revealed that the lower levels of supplementation (i.e. 1, 2 and ppm.) had no effect on the feed rates of chicks from tungstensupplemented. The addition of, and 1 ppm. molybdenum gave significant improvements while 1 ppm. molybdenum supplement gave a highly significant improvement in the feed s when compared to the non-molybdenum supplemented ration. High levels of molybdenum (1, and 2, ppm.) gave highly significant depressions in the feed rates. It is believed that these higher levels of supplementation were definitely toxic to the chicks, although no gross clinical symptoms or lesions were observed. In the case of chicks from non-tungsten supplemented, supplemental levels of 1, 2,,, 1,, 1 and ppm. molybdenum had no significant effect on the rate of gain or feed rate of the 2-day-old chicks. It should be noted that ppm. molybdenum caused a significant growth depression in the chicks from the tungsten-supplemented. The molybdenum toxicity level of the chicks from non-supplemented appeared to be considerably higher as 1, ppm. molybdenum gave a significant depression in growth rate but a highly significant growth depression was found only when the level of molybdenum was 2, ppm. There were no statistically significant differences between the feed rates until the two highest levels of molybdenum were reached. Both (1, and 2, ppm.) gave a significant depression in the feed rates. In order to determine if the treatment of the dam exhibited an effect on the chicks fed the purified rations, an analysis was made comparing the data obtained from all chicks of each dam treatment. It was found that the chicks from the nontungsten supplemented grew at a rate which was highly significantly superior to the chicks from the tungsten supplemented. This suggests that the tungsten treatment of the caused a statistically significant depression in the rate of gain for a period of at least 2 days. Although these differences between dam treatments were found to exist, attention is called to the fact that the chicks (from the tungsten-supplemented ) when fed the purified diet containing 1 or ppm. molybdenum, showed a greater increase in the rate of gain than did chicks from the control, when supplemented with molybdenum at any level. This suggests a synergistic relationship between tungsten and molybdenum. The fact that the number of chicks is rather large (an average of 2 groups of 1 chicks each per treatment) adds confidence that this difference in the rates of gain is a real difference. It was also desirable to test the effects of the molybdenum supplementation on chicks fed a practical ration. The 2-day gain and feed rate from chicks from each hen treatment are shown in Table 6. Supplemental molybdenum levels of 1, and 1 ppm. gave statistically significant increases in the growth rates of chicks from the tungsten supplemented. However, no beneficial effects on the rate of gain was observed in the chicks (from that had not received tungsten supplementation) from any level of added molybdenum. Higher levels of molybdenum supplementation to this practical diet (i.e. and 1, ppm. molybdenum) appeared to have a toxic effect as evidenced by the rates of gain of the chicks Downloaded from at Penn State University (Paterno Lib) on May 12, 216

4 11 R. A. TEEKELL AND A. B. WATTS TABLE 6.Gain and feed of chicks fed practical ration with added molybdenum ,. 1 2 day ; gain Control regardless of the dam treatment. No clinical lesions were observed, however. In work reported previously (Teekell and Watts, 199) considerable variation was encountered in the xanthine oxidase (dehydrogenase) determinations as measured by the Warburg Constant Volume Respirometer. In an attempt to secure less variable results for this enzyme determination, it was decided to measure uric acid synthesis of liver and kidney tissues for use as an indicator of xanthine dehydrogenase activity. The tissues of each organ were homogenized with Michaelis' Universal buffer ph 7.2 and xanthine was used as the substrate. Colorimetric determinations were made at 89 m/j, using a Beckman Model DU Spectrophotometer. Two chicks from purified ration groups fed,,, 1 and 2, ppm. supplemental molybdenum, and practical ration groups fed,, 1, and 2, ppm. molybdenum were sacrificed and uric acid synthesis was determined in liver and kidney tissues. The results of these analyses are shown in Table 7. As may be seen, the various levels of added molybdenum appeared to have no effect on uric acid synthesis in either of the organs. It was then decided to make other physiological tests. A difference in alkaline phosphatase activity had been observed (Canatarow and Trumper, 19) during certain hepatic and renal malfunctions. Since high levels of tungsten were found to be contained in kidneys of chicks from tungstate supplemented, it was decided to determine if correlations existed between the activity of this enzyme and kidney tungsten level. Chicks fed the purified diet were selected from various groups, slaughtered and alkaline phosphatase activities were made on liver and kidney tissues from chicks from tungsten supplemented and on kidneys in chicks from that had received no additional tungsten. The results are shown in Table 8. These values may not be absolute, but are relative since all determinations within a series were made at the same time. Good agreement was noted between replicate determinations. There did not appear to be TABLE 7.Uric acid values* of chick tissues Added molybdenum 1 1, 2, 1 1, 2, Practical ration Purified ration Liver ^ Kidney^ Control Purified ration * Uric acid expressed as milligrams uric acid per gram tissue (wet weight). f Net enzymatic oxidation (gross oxidation minus endogenous oxidation). Downloaded from at Penn State University (Paterno Lib) on May 12, 216

5 MOLYBDENUM IN CHICK DIETS 111 TABLE 8.Alkaline phosphatase levels of chicks fed purified diets Added molybdenum 2 1 1, 2,, 2 1 1, 2,, From tungsten- From non-tungfed sten-fed (net yield)* (net yield)* (mg. P)f Liver Kidney (mg. P)f * Net yield = Incubated sample minus control sample. f Values expressed as milligrams phosphorus per gram tissue (wet weight). any definite trend in the liver alkaline phosphatase values until the suspected toxic levels were reached. The alkaline phosphatase values for the "toxic" levels (, 1,, 2, and, ppm. molybdenum) were from 1. to 2. times the values obtained with the chicks which received no molybdenum supplementation. These enzyme values from the kidney tissues appeared to become successively higher as the level of molybdenum was increased. Since high alkaline phosphatase levels may be associated with certain bone malfunctions, bone ash determinations were made in the manner prescribed by A.O.A.C. (19). Two chicks from groups fed levels of molybdenum were selected (chicks from tungsten-supplemented fed purified diets), slaughtered and bone ash was determined on the left tibia. The results were: 2 ppm. molybdenum,.94 percent; ppm. molybdenum.2 percent; and, ppm. molybdenum.2 percent bone ash. A statistical analysis of variance did not reveal any real differences in the percent bone ash of the 2-day old chicks. Therefore molybdenum did not appear to affect bone formation in these growing chicks. SUMMARY It was found that there was a carryover of tungsten from the dam (fed supplemental tungsten) to the chicks and that the kidney contained large amounts of tungsten compared to chicks from nontungsten supplemented. When chicks from the tungsten supplemented hens were fed either purified or practicaltype broiler ration, they grew at a significantly slower rate than did chicks from non-supplemented. When molybdenum was added to the ration in supplemental levels up to 1 ppm. there was no effect on growth or feed in the case of chicks from the control ; however, the chicks from tungsten supplemented made significantly greater gains over those obtained on the respective basal rations when supplemented with molybdenum up to 1 ppm. It was of interest to find that these chicks made greater gains when fed the basal rations with added molybdenum (1 and ppm. molybdenum in purified diets) than did chicks from the control regardless of the levels of molybdenum supplementation. This suggested a synergistic effect between molybdenum and tungsten for growing chicks. The addition of ppm. molybdenum to either the purified or practical ration resulted in a significant depression in the rates of gain and feed s of chicks from the tungsten supplemented. This apparent "toxic level" was not reached in Downloaded from at Penn State University (Paterno Lib) on May 12, 216

6 112 R. A. TEEKEIX AND A. B. WATTS chicks from the control until the molybdenum supplementation reached 1, ppm. in the purified diet; however, ppm. molybdenum appeared to be toxic in the practical ration. Results obtained from measurements of uric acid synthesis in the liver and kidney did not reflect any difference in feed supplementation levels of molybdenum used. Alkaline phosphatase activities appeared to increase with the increasing levels of molybdenum supplementation. When the so-called "toxic levels" were reached, as measured by rates of gain and feed s, there were marked increases in the levels of alkaline phosphatase activity. REFERENCES Association of Official Agricultural Chemists, 19. Book of Methods, 6th Edition. HEN studies conducted at this station, Berg et al. (196), have shown that, under the climatic conditions of western Washington, hens will lay equally well on corn and barley diets. However, barley was found to be only 82.7 percent as efficient as corn in the production of eggs. It was also noted than hens fed barley rations voided a larger volume of wetter droppings than did hens fed corn diets. Fry et al. (197) found that water treating ground barley markedly improved the nutritional value of barley for the 1 Scientific Paper No. 1826, Washington Agricultural Experiment Stations, Pullman. Project No Austin, H. C, Jr., W. P. Denson and E. A. Epps, Jr., 19. Instrumental methods for the determination of available phosphoric acid and potash in fertilizer. J. Assoc. Off. Agric. Chem. 6 (): Cantarow, A., and M. Trumper, 19. Clinical Biochemistry, Fifth Edition, W. B. Saunders Co., Philadelphia, Penn. Epps, E. A., Jr., 19. Photometric determination of available phosphorus pentoxide in fertilizers. Anal. Chem. 22: Greensberg, P., 197. Spectrophotometric determination of tungsten in tantalum, titanium, and zirconium using dithiol. Anal. Chem. 29: Parks, R. W., S. L. Hood, C. Hurwitz and G. H. Ellis, 194. Quantitative chemical microdetermination of twelve elements in plant tissue. Ind. Eng. Chem. 1: 27-. Snedecor, G. W., 196. Statistical Methods, Fifth Edition. Iowa State College Press, Ames, Iowa. Teekell, R. A., and A. B. Watts, 199. Tungsten supplementation of breeder hens. Poultry Sci. 8: Enzyme Supplementation of Barley Diets for Laying Hens 1 LAWRENCE R. BERG State College of Washington, Western Washington Experiment Station, Puyallup, (Received for publication March 17, 199) Washington chick. The growth rate and feed efficiency with treated barley were practically equal to those obtained with corn. Subsequently, in the same laboratory, Jensen et al. (197) showed that the addition of a fungal enzyme preparation to a barley ration would also improve the nutritional value of barley for chicks. Unpublished data from our laboratory have confirmed these findings and extended them to show that enzymes of bacterial origin and those present in a certain malt barley were also effective in increasing growth rate and feed efficiency in chicks when added to barley diets. In view of the marked responses obtained in chicks from water treating bar- Downloaded from at Penn State University (Paterno Lib) on May 12, 216