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THE desirability of detecting incipient decomposition in shell eggs by chemical means led a number of years ago to a study of their ammonia nitrogen concentration (Jenkins, 1919; Jenkins and Hendrickson, 1918; Lythgoe, 1927; Pennington, 1912; Pennington et al., 1914 and 1916). These investigations showed that this constituent increased progressively in mixed whole eggs during cold storage. It seemed to the authors that information concerning possible variations in the concentration of this constituent in yolks and whites of individual eggs produced under controlled conditions might contribute to a better understanding of the deterioration of cottonseed eggs during cold storage. Consequently a study was made of the influence of such factors as feed, breed, fertility, and preliminary holding periods before storage upon the ammonia nitrogen content of non-cottonseed and cottonseed eggs. EXPERIMENTAL In our earlier work, the ammonia nitrogen was determined by an adaptation of Hendrickson and Swan’s (1918) aeration method . . .

THE vitamin B12 content of fresh and stored shell eggs has been determined as part of a study of changes in composition occurring when shell eggs are kept in cold storage. Losses of nutrients during holding and storage of shell eggs are of importance, because large numbers of eggs are stored for extended times and because fresh eggs are usually several days old before they are eaten. According to previous work from this laboratory, eggs stored for twelve months lost 18 percent of the niacin, 51 percent of the vitamin B6, 14 percent of the riboflavin, 8 percent of the pantothenic acid, and 27 percent of the folic acid (Evans et al., 1951b, c, 1952a, b, 1953a) but did not lose choline nor biotin (Evans et al., 1951a, 1953b). Data are herein presented which show a loss of vitamin B12 from shell eggs that had been stored for twelve months.

Little or no destruction of threonine, glycine, aspartic acid, and glutamic acid occurred when soybean oil meal, soybean protein, or the protein plus sucrose, glucose, dextrin, agar-agar, gum arabic, or soybean oil was autoclaved for 4 hr. at 15 lb. pressure. A portion of each of these amino acids was “inactivated” or tied up in such a manner that the biologically active amino acid could be liberated by acid but not by in vitro trypsin and erepsin digestion. Studies with soybean protein and mixtures of soybean protein and other constituents of soybean oil meal indicated that two types of inactivation occurred during autoclaving. Protein-bound threonine, glycine, and glutamic acid reacted with sucrose or glucose (glycine to some extent with dextrin or agar-agar) to form a linkage resistant to enzymatic but not to acid digestion. Protein-bound aspartic and glutamic acids reacted with some other constituent of the protein to form a linkage resistant to trypsin and erepsin digestion in vitro but hydrolyzed by acid digestion. Part of the second carboxyl groups of protein-bound aspartic and glutamic acids are combined with ammonia as the amide and part are free. It was postulated that the free carboxyl groups react with the free amino groups of lysine and arginine and with the imidazolyl group of histidine to form a peptide-type linkage resistant to enzymatic cleavage.

ALMQUIST (1952), after reviewing the literature, proposed that chicks fed a diet containing 20 percent protein require 0.45 percent methionine, and 0.35 percent cystine, or 0.80 percent methionine plus cystine in that diet for optimal growth, which is lower than the 0.54 percent methionine and 1.10 percent methionine plus cystine earlier proposed by Grau and Almquist (1943). McGinnis and Evans (1947) found that New Hampshire chicks fed soybean oil meal, which had been steamed for 30 minutes at 100°C., as the only source of protein grew very well. Growth was not improved by the addition of methionine, cystine, or methionine plus cystine to the diet. The diet contained 22.4 percent protein, 0.26 percent methionine and 0.46 percent cystine, or 0.72 percent methionine plus cystine. Methionine and cystine were determined by the differential oxidation procedure (Evans, 1945), and the values are, therefore, maximum ones, because they were determined from the total …

THE liquid phase of the intestinal contents is important nutritionally because it is the fraction from which absorption takes place. Since this is in intimate contact with a solid phase composed of undigested feed residues, the question arose as to what effect an excess of mineral might have upon its normal composition. A study was made, therefore, of the partition of Ca and P between the liquid and solid phases of the intestinal contents of chicks fed rations adequate in Ca and P and those containing an excess of oyster shell or bone meal. Preliminary experiments were also conducted in vitro with simple systems to ascertain the nature of the effects produced. EXPERIMENTS IN VITRO Procedure Varying amounts of oyster shell, c.p. CaCO3, bone meal, or c.p. Ca3(PO4)2 (all through 300 mesh sieve) were shaken for three hours at 38°C. with a solution of Ca and P made by dissolving . . .

THE vitamin A content of fresh and stored shell eggs has been determined as part of a study of changes in composition occurring when shell eggs are kept in cold storage. Egg yolk is one of the better food sources of vitamin A, one egg furnishing 4 to 16 percent of the daily requirement (Boucher, 1941). Any losses of vitamin A from such a rich dietary source is of importance nutritionally. Large numbers of eggs are stored for extended times, and so-called fresh eggs are usually several days old before they are eaten. According to previous work from this laboratory, eggs stored for 12 months lost 18 percent of the niacin (Evans et al., 1951a), 51 percent of the vitamin B6 (Evans et al., 1951b), 14 percent of the riboflavin (Evans et al., 1952a), 8 percent of the pantothenic acid (Evans et al., 1952b), 27 percent of the folic acid . . .

A FRESHLY laid egg has a white which is firm and stands up well. After the shell egg has been kept for a few days at room temperature or for a longer period in cold storage, the broken out egg white contains less firm white (Almquist and Lorenz, 1932; Lorenz and Almquist, 1936) and becomes less viscous and spreads out much more (Evans and Carver, 1942b; Evans, 1943). The whites of these older eggs do not stand up well and are considered to be of poorer quality (Wilhelm and Heiman, 1938; Evans and Carver, 1942a). Evidently some changes in the protein structure of the white must occur during storage of shell eggs, because egg white is composed primarily of water and protein. Evans and Bandemer (1956) developed a method for the separation of egg white proteins and their quantitative estimation by filter paper electrophoresis in order to make possible a . . .

Low-density lipoprotein isolated from the yolk of hen's eggs contained 86% lipid. Approx. 19% of the total lipids were extracted by diethyl ether from a solution of native low-density lipoprotein in 1.0 M NaCl solution. Treatment with urea, guanidine hydrochloride, sodium thioglycolate, or non-ionic detergents did not increase the amount of lipid extracted, but treatment with sodium dodecylsulfate or sodium deoxycholate increased lipid extracted by ether to 50–64%. Digestion of low-density lipoprotein with phospholipase C increased lipid extracted to 74%, but phospholipase D digestion had no effect. Ether extracted 98% of the lipid from trypsin-digested low-density lipoprotein, and this lipid was composed of 73% glycerides, 7% sterols, and 21% phosphatides compared to 36% glycerides, 9% sterols, and 52% phosphatides in the residue lipid. Trypsin released 84% of the protein as soluble peptides. Further digestion of the residue with papain released lipids and peptides, and digestion of that residue with pronase also released lipids and peptides. The residue left after pronase digestion contained 27% protein, 39% lipid, 1% carbohydrate, and 33% of unknown material. The data obtained support the view that the low-density lipoprotein is a sphere of lipid surrounded by a layer of protein and phospholipid with hydrophobic groups on the inside and hydrophylic groups oriented to the outside making it very soluble in water and insoluble in organic solvents.

ROBERTS and Rice (1890) first described the small, dark colored yolks found in eggs produced by hens fed cottonseed meal. The yolks turned olive or chocolate brown in color after the eggs were stored (New Mexico, 1927). Schaible, Moore and Moore (1934) found gossypol to be the causative agent in cottonseed meal, and Swensen et al. (1942) showed the characteristic olive color of the yolks to be due to a chemical combination of the gossypol from the cottonseed meal with ferric iron released from the yolk proteins. Sherwood (1928, 1931) first described the type of discoloration in which the yolks of eggs from hens fed cottonseed meal turned salmon in color and the egg whites turned pink during cold storage. He observed that “cottonseed eggs” had more yolk and less white than normal stored eggs and that the yolk contained less fat and more water than normal egg yolk. He . . .

OSBORNE and Campbell (1900) gave the name lecithovitellin to the lipoprotein that they isolated from egg yolks. Alderton and Fevold (1945) and Fevold and Lausten (1946) separated lecithovitellin into two lipoproteins, lipovitellin and lipovitellenin. Recent investigations by Vandegaer et al., (1956), Martin et al. (1957), Martin and Cook (1958), Joubert and Cook (1958a, b), Turner and Cook (1958), Smith and Turner (1958), Martin et al. (1959), and Sugano (1957, 1958a, b, 1959) have shown the classical lipovitellin and lipovitellenin to be mixtures of proteins. Evans and Bandemer (1957) isolated the lipovitellenin and lipovitellin complexes from eggs without ether treatment and observed that each migrated on paper electrophoresis strips as distinct bands. Extraction of lipovitellenin with ether changed its electrophoretic behavior. Lipids removed from egg lipoprotein by ether extraction have not been considered to be part of the lipoprotein. However, Vandegaer et al. (1956) observed that the stability and solubility of…

IN RECENT years, it has been shown that certain phenylarsonic acid compounds increase growth rate in young chickens and turkeys (Frost 1953; Morehouse 1949). Sometimes the growth response has been in addition to that provided by antibiotics and sometimes it has not (Bird, 1952a). The exact mode of action of these compounds in promoting growth is unknown but indications are that the beneficial effects result from their action upon the intestinal microflora —as is the case with antibiotics. In applying these findings to practical feeds two arsenicals have been incorporated generally in commercial broiler feeds —3-nitro-4-hydroxyphenylarsonic acid (“3-nitro”) and p-amino phenylarsonic acid (arsanilic acid). Although these chemicals would do the same for flock-replacement pullets as for broilers, they have not been used much for the growing flock because there is less interest in fast, early growth and improved pigmentation in birds intended for egg production. Very little research has been …

SEVERAL years ago studies were initiated in this laboratory to isolate the compounds in crude cottonseed oil which cause pink-colored whites and large salmon-colored yolks in stored eggs produced by hens fed the oil. Since this work was started Masson et al. (1957) reported that sterculic acid from Sterculia foetida seeds caused pink white discoloration, and Faure (1956) showed that sterculic acid gave a positive Halphen reaction. Lorenz (1939) believed the substance in crude cottonseed oil that causes “pink-white” egg discoloration is the same as the one that gives the Halphen reaction or something very closely associated with it. Shenstone et al. (1965) isolated sterculic acid from cottonseed oil by reversed phase chromatography. Shenstone and Vickery (1956) obtained from cottonseed oil a fatty acid fraction that gave a positive Halphen reaction and caused “pink-white” discoloration of stored eggs. Macfarlane et al. (1957) isolated from this fraction a fatty acid, which …

SINCE certain yolk ingredients have been shown to diffuse into the whites during cold storage of cottonseed oil eggs, the question arose as to whether the pink color of the whites of such eggs could be (1) diffused normal yolk pigments, (2) the natural pigment of whites, riboflavin, modified by diffused yolk constituents, or, (3) some cottonseed oil pigment, deposited in the yolk as a result of its consumption in feed and later diffused into the whites during storage. To investigate these possibilities the following experiments were conducted. EXPERIMENTAL Absorption spectra of a normal white from a fresh egg and a normal-appearing white and pink white from stored cottonseed oil eggs are presented in Figure 1. The Beckman quartz spectrophotometer and the Cenco-Sheard Spectrophotelometer were used for the determinations. The untreated whites were filtered clear and diluted with 0.9 percent NaCl solution to obtain the proper concentrations for specific wave . . .

HEN’S eggs are a rich source of lipides. An average size 51.6 gm. egg contains 6.1 gm. of lipide (Romanoff and Romanoff, 1949). Any changes in composition of the egg yolk fatty acids resulting from changes in the hen's diet are important in human nutrition. Cruickshank (1934) used iodine and thiocyanogen values; and Reiser (1950 and Reiser (1951), Fisher and Leveille (1957), and Feigenbaum and Fisher (1959) used alkali isomeration to determine differences in amounts of saturated and unsaturated acids in eggs from hens fed fats of widely differing fatty acid compositions. The development of gas-liquid chromatographic methods for separation of the methyl esters of fatty acids gives a tool for the direct determination of fatty acids in egg lipides. In this study, hens were fed a basal ration containing 4 percent lipide plus smaller amounts of crude cottonseed oil or various fractions of the mixed fatty acids from the oil and …

BECAUSE it is alleged that MnSO4 causes a loss of iodine from rations, the more insoluble MnCO3 has recently found its way into feeding trade channels. The product being distributed is the finely ground natural ore, rhodochrosite, which preliminary work at this institution (Schaible, Bandemer, and Davidson, 1938) had shown was ineffective in preventing perosis in chickens. In view of the beneficial results obtained with precipitated MnCO3, this problem was investigated more thoroughly in order to learn the reason for the divergent effects of different forms of the same substance. Since rhodochrosite ground to pass a 40-mesh sieve had been used, there was a question whether its unavailability was caused by lack of surface or the presence of deleterious impurities. To settle this point, feeding experiments were conducted using a perotic ration containing rhodochrosite at various levels to determine the effects of particle size, impurities, and additional treatment of the . . .

EGGS laid by hens fed rations containing either crude cottonseed oil or Sterculia foetida seeds contained higher levels of stearic acid and lower levels of oleic acid than eggs produced by hens fed a normal laying ration (Evans et al., 1961). Livers, blood plasma, and ovaries of these hens also contained more stearic acid and less oleic acid than those of normal hens, but only slight changes occurred in fatty acid distribution of heart lipides or of depot fat (Evans et al., 1962). Both crude cottonseed oil and S. foetida seed oil contain some materials that interfere with fatty acid metabolism in the hen. The interference causes the hen to produce more saturated fatty acids and less monoenoic fatty acids than are produced by hens not fed these oils rather than causing the hen to deposit more stearic acid and less oleic acid in the eggs (Evans et al., 1962).

CONSIDERABLE difficulty was experienced in ashing the whites and yolks of individual eggs preliminary to the determination of their iron content. This was especially true with the whites where it was necessary to use the entire amount of each egg in order to obtain sufficient iron for analysis. Ashing this quantity in the muffle furnace was impossible as the material foamed out of the crucibles. Drying the egg whites did not prevent the foaming. The following experiments were conducted, therefore, to overcome this difficulty and, at the same time, obtain an acidsoluble ash from which the iron could be easily and completely recovered. EXPERIMENTAL Egg White—After much trial and error, it was found that the maximum amount of egg white from individual eggs (38 ml.) could be kept within the confines of No. 3 (75 ml.) crucibles if the egg whites were heated rapidly over Meeker burners until the . . .

IT HAS been recognized for some time that poultry develops perosis if the ration is deficient in Mn or contains an excess of mineral supplements, such as bone meal or oyster shell (Wilgus, Norris, and Heuser, 1937; Schaible, Bandemer, and Davidson, 1938). It has also been shown (Bandemer and Schaible, 1942) that the concentration of insoluble Ca and P is considerably greater in the intestinal contents when chicks are fed high-mineral rations. The present study was made to determine if such insoluble material reduces the solubility of Mn and, in this manner, decreases its availability to the chick. If so, the similar effects of high-mineral and Mn-deficient rations would be explained. EXPERIMENTAL Effect of Mineral Supplements upon Mn in Solution To simplify matters, experiments were first carried out in vitro under conditions roughly approximating those in the gastrointestinal tract. Varying amounts of bone meal, c.p. Ca3(PO4)2, or oyster shell were . . .

EGGS are known to be one of the best sources of iron in the human dietary. Whether or not the iron content can be increased by feeding hens iron supplements is in dispute, according to Sherman (1935). The purpose of the following experiments, therefore, was to ascertain if any of several different iron compounds in the ration of the hen would increase the iron content of the white or yolk of her eggs. In making a critical study, it was deemed necessary to find out if eggs of individual birds or specific breeds of poultry had characteristic levels of iron and if the distribution of iron between the yolk and white was affected by storage of the eggs before analysis. PROCEDURE Analyses were made upon the separated yolks and whites of individual eggs laid before and after the feeding of various iron supplements. Except as otherwise mentioned, all eggs showing . . .

STUDIES of changes in the chemical composition of shell eggs during cold storage were started at the Michigan Agricultural Experiment Station nearly 20 years ago. At the time the investigations were started the objectives were to see if there were changes in egg composition that would affect the nutritive value of stored eggs and to study changes in the composition that could be related to changes in the interior quality of the eggs, such as the loss of viscosity of the egg white. Studies of changes in vitamin content of eggs during storage showed that eggs stored for 12 months lost 18 percent of the niacin (Evans et al., 1951a), 51 percent of the vitamin B1 (Evans et al., 1951b), 14 percent of the riboflavin (Evans et al., 1952a), 8 percent of the panthothenic acid (Evans et al., 1952b), 27 percent of the folic acid (Evans et al., 1953a), 33 …

THE cyclopropenoid ring containing fatty acids, sterculic and malvalic, have been shown to give positive Halphen reactions (Macfarlane et al., 1957; Faure, 1956) and to cause pink-white discoloration in stored eggs from hens fed these compounds (Shenstone and Vickery, 1959; Masson et al., 1957). Evans et al. (1962) isolated from 4,000 grams of crude cottonseed oil, a 45 gram fraction of fatty acids which gave a strong Halphen reaction and caused pink-white discolorations in stored eggs from hens fed it. Treatment with urea separated the concentrate into two fractions, a urea insoluble one that gave a strong Halphen color reaction and variable degrees of pink-white discoloration and a urea soluble fraction which gave a negative Halphen reaction but gave consistent pink-white discoloration. More than 90% of the methyl esters of the urea soluble fatty acids had gas-liquid chromatographic retention times similar to methyl linoleate. No methyl esters were detected in…

Some eggs that had been in cold storage for several years, when opened, were observed to contain curdy masses just beneath the yolk membrane. Mold was suspected although the eggs did not smell moldy nor show mold growth inside the shell. Microscopic examination of the curds disclosed that they were not molds but crystals in the form of rosettes. Further examination showed that the yolk mass contained similar rosettes or single crystals whereas the egg white had an apparently different crystal formation. A series of eggs from freshly laid ones to some that had been in cold storage for various periods up to three years were examined. Crystals similar to those observed in the whites of the original eggs were found in the firm whites of freshly laid eggs and appeared in increasing amounts in the thin whites as the age of the eggs increased. No crystals were observed in . . .

Difficulty was encountered in the determination of cysteic acid with ninhydrin after elution from a resin column, using a Time-Flow fraction-collector. Since the trouble arose from the variable pH obtained on the addition of sodium hydroxide to the eluate, a buffered solution of 2 N sodium hydroxide saturated with sodium citrate was used to produce the pH 5.0 required for maximum color development. The calibration for 10 ml of the tubes collecting the eluate, and the use of a standard cysteic acid curve facilitated the calculation of the cysteic acid concentration in the sample.

Egg yolk very low density lipoprotein contained on the average 75% of lipid which could be extracted by ether and 25% of a residual lipoprotein, the classical lipovitellenin. The ether-extracted lipid was composed of 75% triglycerides, 7% sterols, 2% mono- and diglycerides, and 16% phospholipids. Lipovitellenin contained 48% lipid composed of 87% phospholipids, 11% triglycerides, and 2% sterols. The protein vitellenin was composed for the most part of units of 74,000 and 270,000 daltons molecular weight. Egg yolk very low density lipoprotein is polydisperse. Preparative ultracentrifugation separated it into six fractions of different average molecular size, and gel chromatography separated it into five. The fractions of larger molecular size contained more lipid and triglyceride than did the fractions of smaller molecular size. The proteins of the fractions appeared to be similar. Egg yolk very low density lipoproteins appear to be a series of molecules composed of cores of lipid of varying sizes with each core surrounded by a layer of lipovitellenin, which is composed principally of glycoprotein and phospholipid.

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Chicks fed rations containing high levels of uncooked egg white develop a dermatitis of a pellagrous character after three or four weeks (Ringrose, Norris and Heuser, 1931). A similar condition is observed if the rations are deficient in biotin (Hegsted et al. 1940; Ansbacher and Landy, 1941). Egg white contains avidin which fixes the biotin of the ration into a complex which cannot be absorbed from the intestine. This explains the similarity of results produced by the two types of rations. As the deficiency disease progresses, the skin on the feet becomes rough and calloused and exhibits numerous hemorrhagic cracks and a dry scaliness. The corners of the mouth develop crusty scabs which spread to the area around the beak. The eyelids may become red, swollen and stuck together. In addition to these symptoms which have been previously reported, we have observed that chicks develop abnormal beaks. At first, upper . . .

Hens fed rations containing crude cottonseed oil produce eggs that develop viscous pink whites and large salmon colored yolks after six months or more of cold storage (Sherwood, 1928). Lorenz and Almquist (1934) observed that crude kapok oil, the seeds of cheeseweed (buttonweed or mallow), and other plants of the family Malvacea also caused “pink-white” storage discoloration in eggs when fed to laying hens, but that coconut, peanut, sesame, linseed, and soybean oils did not. Thompson (1934) observed color changes in stored eggs from hens fed cottonseed, peanut, soybean, or linseed meals. Because of the increasing use of fats and oils in high energy poultry rations and the scarcity of information on the production of “pink-white” eggs by feeding oils other than cottonseed to laying hens, it was decided to study a number of the more common crude vegetable oils. Each group consisted of twelve S.C. White Leghorn pullets housed . . .