EFFECTS OF suscumnsous msEcnoNs . . , - 0F RAW EGG'WHHE UPON PERFORMANCE ; 0F GROWING swms AND cmcxms ; - Thesis #0:: the Degree of M. S. MBCHEGM SYATE UNWERSETY BENG TAU OH 1975 IIIII J’HESIS . .. ., - ‘5‘: ‘ .4: <1 “.1.“- n’bék“ In: .1 f! S’} ‘ x 6. ‘6‘}... \f' 'menctwa-.z , . o I 5- amoma av V ”055ml 3“? a: 3523K 1"'YUU’Y THC. __ . M ABSTRACT EFFECTS or SUBCUTANEOUS INJECTIONS OF RAW EGG-WHITE upon PERFORMANCE OF GROWING SWINE AND CHICKENS By BENG TATT OH Attempts to stimulate the growth rate of swine by using chicken egg-white injections were unsuccessful. A total of 66 pigs divided into three trials were used and the pigs were injected subcutaneously with the following dosages of chicken egg albumen: 15 ml, 20 ml, 25 ml, 30 ml, 40 ml, 50 m1, and 55 m1. Injectins were made at weekly intervals for four weeks with increased dosage each week. Injected pigs failed to show any improved body weight gain. In the second trial the treated pigs which received 15 ml, 20 ml, 25 m1, and 30 ml of chicken egg-white injections had better feed efficiency than did controls. The feed conversion for the treated group was 3.3 kg of feed to 1 kg gain in weight and for the control h.2 kg of feed to 1 kg gain in weight. No noticeable toxicity symptom was found in the pigs. When the dosages were increased to ho ml, 50 ml, or 55 ml in Trial III depressed appetite, vomiting and loose stool were noticed in the treated pigs. Therefore, dosage above 40 m1 may produce some toxicity which also reduce the rate of growth. Weekly subcutaneous injection of chicken egg albumen at 5 m1, 1 ml, 1% ml, 2 m1 levels per week for four weeks to 92 growing chicks did not produce any significant gain in body weight. Female chickesn showed slight response to the chicken albumen injection but statistically the gain in weight was not significant. A total of 40 hens, divided into four treatments, namely quail egg albumen (QEA), chicken egg albumen (CEA), Ringer's solution (RS), and control (CONT) were conducted. During 107 days of egg production QEA treated hens produced 906 eggs, control hens 803 eggs, CEA treated hens 797 eggs, and RS treated hens 779 eggs. Hen housed production percentage was 84.7%, 75.1%, 7h.5% and 72.8% for QEA, CONT., CEA, AND RS, respectively. The OEA treated hens had a significantly higher (P 0.33502 e£=ez> 0533 52:20 201:0.— uo 30.032 3.2;? Eleven—v.0 3:: es. 512...... 10 Composition of Egg Albumen The egg albumen consists shiefly of water, of which it is the developing embryo's principal reservoir. The albumen's main organic constituent is protein; other components are small amounts of carbohydrates and minerals and trace amounts of lipids. These amounts are shown in Table 6. TABLE 6 COMPOSITION OF EGG ALBUMEN Total Egg Albumen Amounts (gram) Total 32.9 Water 28.9 Solids 4.0 Organic Matter 3.8 Proteins 3.5 Lipids trace Carbohydrates 0.3 Inorganic Matter 0.2 Source: Romanoff & Romanoff (1949) The whites of eggs laid by a single bird are usually fairly uniform in their proportions of water and solids. On the other hand, the average percentages of the various 11 constituents of the albumen are much the same in the eggs of all species of precocial birds. Table 7 shows the proportional amounts of major chemical constituents in the egg albumen of various birds. TABLE 7 PROPORTIONAL AMOUNT OF MAJOR CHEMICAL CONSTITUENTS IN EGG ALBUMEN OF VARIOUS BIRDS Chemical Chicken Turkey .Guinea Fowl Duck Goose Constituent (32.9 gm)* (44.2 gm)* (19.9 gm)* (40.4 gm)* (110.2 gm)* - (%) (%) (% (%) ) U Water 87.8 86.5 86.6 86.8 86.7 Solids 12.1 13.5 13.4 13.2 13.3 Organic Matter 11.5 12.8 12.6 12.4 12.5 Proteins 10.6 11.5 11.6 11.3 11.3 Fats (Lipids) 0.03 0.03 0.03 0.08 0.04 Carbohydrates 0.9 1.3 1.0 1.0 1.2 Inorganic Matter 0.6 0.7 , 0.8 0.8 0.8 *Indicate the average weight of egg albumen of each species. Source: Romanoff & Romanoff (1949) 12 As indicated in the table above there is a great similarity in albumen from eggs of the Species shown and this may explain why it is possible, in the laboratory, to substitute duck for chicken albumen without seriously inter- fcrring with early embryonic develOpment (Loisel, 1900; Romanoff and Romanoff, 1949). Various Layers of Albumen Egg-white is essentially an aqueous solution of proteins. Four distinct layers can be recognized. (l) a fluid "outer thin" layer (Outer Liquid) (2) a firm "thick" layer (Middle Dense) (3) another fluid layer (Inner Liquid) (4) a shallow dense layer (Chalaziferous) The percentage of water in the four layers varies in different species as shown in Table 8. These layers differ in chemical and physical properties; in particular, the concentration of ovomucin is much greater in the "thick" layer than in the "thin" ones. The proportions of the different layers vary widely, but it has been established that the percentage of the thick white is characteristic of the individual (Parkinson, 1966), (see Table 9). Table 10 shows the concentration of the major elements in albumen of egg. Earlier work on egg proteins has been comprehensively reviewed by Fevold (1951) and by Warner (1954) and later work 13 TABLE 8 PERCENTAGE OF WATER IN THE FOUR LAYERS OF ALBUMEN IN VARIOUS BIRDS Species Outer Liquid Middle Dense Inner Liquid Chalaziferous Chicken 88.8 87.6 86.4 84.3 Pheasant 89.0 88.0 86.3 85.3 Quail 88.1 87.2 85.8 84.9 Duck 87.4 86.7 85.8 84.3 Average 88.3 87.4 86.1 84.7 _L ( )Investigators: Romanoff (1929, 1943), Almquist and Lorenz 1933 TABLE 9 PROPORTION AND SOLID (PROTEIN) CONTENT OF THE VARIOUS COMPONENTS OF CHICKEN EGG ALBUMEN Component Weight (g) Percentage Percentage of Total Solids in Albumen Each Component Outer Thin Layer 7.7 23.0 11.2 Thick Layer 18.9 57.0 12.4 Inner Thin Layer 5.6 17.0 13.6 Chalaziferous Layer 0.9 0.2 15.7 Chalaza 1 0.9 0.2 15.5 Chalaza 2 0.9 0.8 15.5 Fro the data of Almquist and Lorenz (1933). Romanoff and Romanoff ?1949) and Besch and Sluka (1966). 14 TABLE 10 CONCENTRATION OF THE MAJOR ELEMENTS IN ALBUMEN OF THE LAID EGG (Calculgted on the basis of the weight of the albumen being 33.2 g. Element mg/total albumen Sodium 48.8 Potassium 46.5 Calcium 4.3 Magnesium . 3.3 Iron .003 Sulphur 65.0 Chloride 42.2 Phosphate (as P) 3.7 Recalculated from the collated data of Shenstone (1968). has been summarized by Feeney (1964), Parkinson (1966), Baker (1968) and Feeney and Allison (1969); a collated summary of their data is given in Table 11. 15 TABLE 11 REPRESENTATIVE COMPOSITION OF EGG-WHITE SOLIDS (Calculated on the basis of the egg weighing 58 g.) Ovalbumen Ovotransferrin ( conalbumen) Ovomucoid Ovomucin Lysozyme (G1 - globulin) G2 - globulin G - globulin 3 Ovomacroglobulin Ovoglycoprotein Flavoprotein - apoprotein Ovoinhibitor Avidin Percentage of Total Solids 54.0 13.0 11.0 1.5 - 2.9 3.5 4.0 4.0 0.5 0.5 - 1.0 0.8 0.1 — 1.5 0.05 Nutritive Value of Eggs Eggs are designed by nature to supply all of the nutrients needed in the development of a healthy, sturdy chick. They are a rich source of such high quality protein 16 that experimental nutritionists often use them as a standard 'for measuring the quality of other food proteins. Eggs are also an important source of unsaturated fatty acids (mainly oleic), iron, phosphorus, trace minerals, Vitamins A, E, and K and the B Vitamins, including B 12. As a natural source of Vitamin D, eggs rank second only to fish liver oils. In Table 12 are listed the values of the important nutrients of a fresh egg without shell. The listed value in the table for a specific nutrient in a whole raw egg may vary from the combined values for a raw white and yolk because of differences in analytical technique and source of data. Many studies have shown that the nutritive value of eggs can vary with the hen's feed and with storage time and conditions. Reports vary as to the effect of diet of the laying bird on cemposition of eggs produced. Pollard and Carr (1924) and Gerber and Carr (1930) presented evidence indicating that the percentage of protein of pigeon eggs varies with the type of grain fed. Significant differences in protein percentage of eggs of hens on different diets have been reported by Titus, Byerly and Ellis (1933). Csonka (1950) found that hens on a high protein diet produced eggs with a greater percentage of protein than those on a low protein diet. In contrast to the above reports several workers (Szorenyl and Ossezehas, 1941; McFarlane 23, 31., 1930; Calvery and Titus, 1934; and Radar, 1939), have found that 17 protein content of eggs was not influenced by the diet of the layer. The general consensus of available reports is that actual grams of protein per egg do not decrease on aging, although evaporation of water and shifts of water from white to yolk sometimes cause changes in percentage of protein (Jenkins 2}, 31., 1920; Mitchell, 1932; Reder, 1939; Romanoff, 1940; Silva, 1947; and Evans and Davidson, 1953). Significant, but small, genetic differences in nitrogen content of eggs were demonstrated by Arroyave gt.‘gl. (1957). Cotterill and Winter (1954) found significant differences among strains in nitrogen content of egg whites from hens of different lineage. Although the extent that season, environmental tempera- ture, management practices, nutrition, genetics, age of layer and others affect egg quality have been extensively studied, however, satisfactory explanation for these quality variations in relation to differences in composition which affect physical and performance characteristics associated with quality have not been elucidated. Smith.g§.‘§l. (1954) found that the composition of eggs produced by a hen was an individual characteristic and that changes in egg composition could be associated with laying rate, environmental tempera- ture and age of bird. They reported that higher temperature increased the dry matter, phosphorus, sodium and potassium levels of egg-white. 18 Cunningham gt. 21. (1960) indicated that season was highly significant in its effect on the variation of sodium, calcium, and chlorine in egg-white but had little or no effect on potassium, phosphorus, and protein content. They also reported that age of bird was highly significant in its effect on the variation of phosphorus, chlorine, and protein in egg-white; affected the calcium content to a lesser degree; and had no effect on the sodium and potassium content. May and Stadelman (1960) found that the strain of hen signifi- cantly influenced percentage of moisture, protein of fresh eggs and protein of dried egg. They also reported that age of ‘hen and season significantly influenced egg contents, weight, albumen height, Haugh units, grams of protein per egg and in one case, percentage moisture and percentage protein of dried egg. The values of the composition of fresh shell eggs reported in Table 12 and Table 13 were obtained directly from Dr. Owen J. Cotterill of the University of Missouri- Columbia, Mo. , who had kindly granted the author the permission to use them here. Biochemistzy of Egg-White Proteins Egg white has been shown to contain about 40 individual proteins. Of these 24 or so are minor proteins which have been found on starch-gel electrophoretograms. In the main, these minor components of egg—white remain uncharacterized, 19 TABLE 12 COMPOSITION OF FRESH SHELL EGGS (100 g. Liquid Basis) GROSS AMINO FATTY ACIDS° (Goldfisch: COMPOSITION ProteiB (N x 6.25) - 8. Lipids (total) - g. Saturated MOnounsaturated Pglyunsaturated Ash (total) - g. AC 113'sb Alanine - g. Arginine - g. Aspartic acid - g. Glutamic acid - g. Glycine - g. Histidine - g. Isoleucine - g. Leucine - g. Lysine - g. Methionine - g. Phenylalanine - g. Proline - g. Serine - g. Threonine - g. e Tryptophane - g. Tyrosine - g. valine - g. Caprylic (8:0) - g. Capric (10:0; - g. Lauric 12:0 - g. Myristic (14:0) - g. Myristoleic (14:1) - g. Whole White Yolk 25.28 10.72 50.78 12.03 10.07 16.16 12.31 - 34.10 5057 " 13035 7e03 " 17.02 2019 " 5023 .98 .69 1.65 .644 .580 .793 .771 .560 1.137 1.197 1.090 1.302 .274 .298 .262 1.487 1.320 1.817 .393 .330 .479 0279 0212 0396 .600 .510 .808 .998 .828 1.384 .851 .660 1.204 .388 .388 .376 .572 .550 .618 .548 .450 .643 .921 .636 1.297 .597 .422 .808 .173 .148 .237 .528 .408 .726 .781 .665 .942 .056 .014 .009 .053 .018 chloroform-—methanol extraction) .152 .022 .019 .160 .045 20 TABLE 12 (Con't.) Whole White Yolk Palmitoleic (16:1) - g. .625 - 1.432 Oleic (18: 1) - g. 6.388 - 15.541 Linoleic (18:2) - g. 2.056 - 4.908 Linolenic 218: .3 - g. .050 - .106 Arachidic 20 0 - g. .075 - .167 Arachidonic (20: 4) - g. .083 - .220 Behenic (22:0) - g. .222 - .615 VITAMINS Retinal - ug. 124.0 - 228.2 D - m 3.62 - l6e98 E - mg. 1.014 - 3.92 B1 - mcg. 1.08 .02 3 l6 Otin - mcg. 18.3 5.1 40. 8 Choline - mg. 824.1 1.25 1109.1 Folic acid - mg. .293 .001.673 Inositol - mg. 15.46 3.73 33. 97 Niacin - mg. .089 .094 .059 Pantothenic acid - mg. 1.376 .127 4. 904 Pyridoxine - mg. .137 .005 349 Riboflavin - mg. .320 .253 .457 Thiamine - mg. .089 .003 .253 MINERALS Calcium - mg. 58.45 8.61 136.39 Chlorine - mg. 172.1 175.5 165.5 Copper - mg. .062 .023 .132 Iodine - mg. .072 .0074 .167 Iron - mg. 2.25 .011 5 92 Magnesium - mg. 12.41 12.44 12.35 Manganese - mg. .0413 .0057 .1132 Phosphorus - mg. 237.94 14.26 607 34 Potassium - mg. 138.0 147.2 110.1 Sodium - mg. 139.14 183.44 60.73 Sulfur - mg. 165.3 158.4 165.5 Zinc - mg. 1.50 .01 3.76 Source: 0. J. Cotterill 23. 21., 1973. 21 TABLE 13 CONVERSION FACTORS FOR EGG SAMPLES . Whole Egg White Yolk 3121.4: Based on total egg* 87.2% 54.8% 32.4% Based on liquid only - 62.9% 37.1% Amount/egg (g.) 53.0 33.3 19.7** Solids Level: Liquid ’25.28% 10.72% 50.78% Freeze-dried 97.75% 94.67% 98.66% *Total egg weight was 60.76 g. **Includes adhering albumen. Source: 0. J. Cotterill 23. 51,, 1973. and not all are always present (Gilbert, 1971). From starch-gel electrophoresis, Lush (1961) distinguished 19 minor components of fowl's egg-white, some undoubtedly represent slight genetical and/or chemical heterogeneity of the 12 major types. These are, in the order of concentrations in the albumen as in Table 14; which shows a summary of characteristics of egg-white proteins from the hen (data were taken from Baker, 1968, and Feeney and Allison, 1969. 22 Several of the egg-white proteins are known in homo— geneous form (Warner, 1954); recent work has extended knowledge of their chemical and physical properties (see Table 14 and 15). Egg:flhite Proteins Avian egg-white is the type of material which is primarily a solution of proteins with a relatively small amount of sugar and salts. This makes the job of protein separation and purification much easier than with many other biological materials. Indeed, egg-white has been the source of several of the standard proteins for biochemists. Chicken egg-white has been examined by many different electrOphoretic techanues. ElectrOphoretic studies have shown some of the egg- white proteins to exist in different forms. The first exten- sive study by this method was that of Longsworth gt. a;. (1940) using a standard Tiselius type of apparatus and covering the pH range 3.9 - 7.8. They identified eight proteins, including two ovalbumins (A1 and A2) and three globulins (61’ 62, and GB) as well as conalbumin, ovomucoid and ovomucin; there was evidence that conalbumin could also exist in two forms (C1 and C2). Bain and Deutsch (1947), published electrophoretic patterns at pH 8.6 for the egg-white 23 000.000 op daasaoao 000.000 0 0.0 : m.0 0.0 u ohoweo>0 .0 000.00 o# sw>0amopaa moaam 000.Nm 0H H.0 u m.n 0.0 caopopaopwam .0 coapwcavsHmmmamwn Hwhfi>avn< 0 0H 0 m.N a n.H Gaosao>o .0 mahmpomn momha "mmcacfiawmoosaw 000.0H - a A0uav m o» 60000000 000000 0000.00 0 0.00 n 0.00 0.0 00006004 .0 000.00 a 0.0 0.0 00 mafiasaoac N 000.0m 0 m.m 0.0 o .0 0000060 mpapancH 000.00 mm 0.0 . 0.m 0.Ha caoosao>0 .0 00000600 0000000 008 000.00 “0:00 .mmoammcda op 000066 .0000 00:00 000.00 0 0.0 u 00.0 0.00 canuounnagpo>0 .0 000.00 0 0.0 - 0.0 0.00 caasnaaso .0 uoappoamum vnmwoz *chovnoo . Hwoamoaoam amazooaoz opuuvknomuao ma oappoodmonH a .xopn a. L zmonmo NEH 20mm mZHmaomm mBHmkloom mo mUHHmHmmHoo .OH 000.00 00 0.0 0.0 - 0.0 00opouaooh0mo>0 .0 000Puomopm pnmwoz X vaovcou 000000000m 00030000: 09000>nonhmo ma oauvooauomH R .xoangm A»P.COUM‘¢H mamgfi 25 00.0 00.0 00.0 00.0 00.0 00 00.0 00.0 00.0 000000 00.0 00.0 00.0 00.0 00.0 0 00.0 00.0 00.0 0000000 00.0 00.0 00.0 00.0 00.0 0 00.0 00.0 00.0 0000000000000 00.0 00.0 00.0 00.0 00.0 0 00. 00.0 00.0 0000000002 00.0 00.0 00.0 00.0 00.0 0 00.0 00.0 00.0 000000 . 00.0 00.0 00.0 00.0 00.0 0 00.0 00.0 00.0 0000000 00.0 00.0 00.0 00.0 00.0 0 00.0 00.0 00.0 0000000000 00.0 00.0 00.0 00.0 00.0 0 00.0 00.0 00.0 000000000 00.0 00.0 00.0 00.0 00.0 00 00.0 00.0 00.0 0000000 00.0 00.0 00.00 00.00 . 00.00 00 00.0 00.00 00.00 000000000 00.0 00.0 00.0 00.0 00.0 0 00.0 00.0 00.0 0000000 00.00 00.0 00.0 00.0 00.0 000 00.00 00.00 00.0 000000000 00.0 00.0 00.0 00.0 00.0 00 00.0 00.0 00.0 0000000< 00.0. 0.0 00.0 00.0 00.0 00 00.0 00.0 00.0 0000004 GOHsnoao GaopOLAOhn<\ wwwmmqa cahuow a000>< Acu0QHnu0o>0 nououao>0 c0opOHAo>uah c0osao>o mahuoahn caoosao>o Innuavobo caaSDHm>o mZHMHomm NHHmbnuom £004: mma mo onaHmomzco H mum0m 00 0000800 0000 00080 000 czocx 00 0030 0am 00805000 0000 00080 009 .08000000 0cm vnmoxo 0000o0n m 000 009 0000000809000 00V U000 08080 050000n80 Ho 800m 00 80>0m 000 00500» HH< .08080950m no 00500000 m 0005008Hp .0800000900 no 00500000 n0 000500800 .Ammmfiv 8000HH< 080 008000 "ooh5om I I I I 00.0 I I I I 0008000000000 #q.n Oh.n ON.m oo.m mm.m I wo.NH No.0 nm.H 080800005Ho NN.0 00.0 00.0 om.m 00.0 I 00.m No.0 00.0 mmOXom I I . no.0 I 00.0 I Nm.o 00.0 00.0 vwo< oaHuum 00.0 mm.m 0m.m 00.0 00.0 0 00.0 m0.0 00.0 00000> 00.0 00.0 00.0 00.0 00.0 0 00.0 00.0 00.0 00000000 00.0 00.0 00.0 00.0 00.0 0. 00.0 00.0 00.0 0000000000 mm.NH wN.m 00.0 Om.N mh.¢ b HN.m ow.¢ no.n 080800089 00059000 80vvounopg<\ wwwmmca 800000 8000>< 000020580o>o Io0omao>o 80cpouno>0nh 800580>o 08000000 00005ao>o I080000>o 8085nao>o A.P.coov ma m4m<9 27 proteins of thirteen different species of birds. For chicken egg-white their diagrams were similar to those obtained by Longsworth 23, 31., at pH 7.8, but showed two distinct conalbumin fractions; globulins and ovomucoid were not easily distinguished and formed one heterogeneous region. Forsythe andFoster'(l9h9)used this technique to examine egg-white proteins, other than ovomucin, and found no marked differences in the electrophoretic patterns of outer thin, middle, thick and inner thin layers. The same authors (1950) demonstrated slight but statistically significant differences in the composition of egg-white protein in six genetic strains of chickens. Gordon‘gt.'gl. (1949), introduced 1% agar gel as a medium for electrophoresis of egg—white proteins at pH values of 6.8 and 8.0, and followed the protein movement by measuring the ultraviolet absorption at 280 mu of l-cm sections. Their patterns had a broad similarity to the Schbieren diagrams of Longsworth 9;. El- Using starch-gel electrophoresis, Lush (1961) established consistent differences between the patterns from eggs from individual hens of different breeds. There appeared to be nineteen separate constituents on some of the electrophero- grams. Stevens (1962) obtained evidence of sixteen consti- tuents in a similar study and stated that the protein composi- tion of the thick and the inner and outer thin layers of egg-white appeared to be the same. Feeney and co-workers (1963), using the same technique, confirmed the existence of 28 several uncharacterized minor protein constituents, one of which was isolated by a combination of precipitation and ion-exchange chromatography and characterized as a globulin. Evans andSandemer (1956') , separated egg-white proteins by paper electrophoresis, distinguishing the three forms of ovalbumin. Rhodes 23, 31. (1958) obtained the different proteins by ion-exchange chromatography on carboxymethyl-cellulose. Proteins of high purity were obtained in high yields and the method appeared to have several advantages over electro- phoresis and the more conventional methods. Mandeles (1960) separated egg-white protein into eleven fractions on a column of diethylaminoethyl cellulose. Ovalbumin Ovalbumin can be obtained in crystalline form and with the possible exception of lysozyme, is the most studied egg- white protein. Despite this, it has no known biological function, although it contains all the "essentialf'amino acids; whether this is important in embryonic nutrition is not known. Ovalbumin, the most abundant protein in egg- white comprises about 5A% of the total (see Table 14). Ovalbumin is the primary protein of chicken egg-white present at four to five times the concentration of the secondary constituents, ovotransferrin and ovomucoid. The properties of egg-white are primarily those of the ovalbumin 29 with the other constituents contributing mainly to the bio- logical properties of the egg-white. An obvious exception to this is the ovomucin which appears responsible for the high viscosity of thick egg-white. Ovalbumin is a glyco- protein. .It has a molecular weight of about 46,000 and an isoelectric pH between 4.5 and 4.8. Other variants of ovalbumin are S-ovalbumin and plakalbumin. S-ovalbumin is a more stable form of ovalbumin and increases during storage of eggs; its properties are similar to ovalbumin, but it possibly arises through alteration of one disulphide bridge (Smith, 1964; Smith and Back, 1965). Plakalbumin has properties very similar to those of ovalbumin (Linderstrom - Lang and Ottesen, 1947; Perlmann, 1952; Lush, 1964; Gournaris and Ottesen, 1965). It is formed by removal of a heptapep— tidase by subtilopeptidase A (see Ottesen, 1958) and has a slightly lower molecular weight (43,000) (Linderstrom - Lang and Ottesen, 1947). It migrates more slowly in Tiselius electrOphoresis (Lush, 1964). The finding of Yamafuji (1972) that the capacity of egg ovalbumin to bind with and break DNA isolated from spleen may contribute to investigations on the mechanism of antibody formation. Conalbumin(pvotransferrin) Although conalbumin was only identified as the iron- binding protein and anti-bacterial agent in the middle 1940's (Alderton 33, 31., 1946), it is receiving a relatively 30 large amount of attention from chemists. Probably the principal reason for the current interest in conalbumin is the fact that it is a homologue of its counterpart in blood serum, serum transferrin (Warner and Weber, 1953). Feeney (1964) referred to avian conalbumin as ovotransferrin. Ovotransferrin comprises about 13 percent of the total protein of egg-white but its precise function is not clear. Its metal-binding properties seem unimportant since less than 1% of its binding potential is used. For embryonic develop- ment it is probable that sufficient iron is bound to yolk transferrin although the ability of ovotransferrin in binding copper could be important to the embryo. In the event of bacterial contamination it could compete for metallic ions with the bacterial enzyme systems and thus act as an anti- bacterial agent. Since it binds copper, its presence may help to preserve the bactericidal properties of lysozyme, which is inactivated by this metal. Ovotransferrin, a glyc0protein with a molecular weight between 80,000 and 86,000 (Fuller and Briggs, 1956) has an isoelectric pH of about 6.0. It is a single chain (Bezkorevainy 23, $1., 1968; Feeney and Allison, 1969) with alanine as the N-terminal residue (Williams, 1967). Ovotransferrin can bind other polyvalent metallic ions; in decending order, cobalt - iron - manganese — copper - zinc (Fraenkel — Conrat and Feeney, 1950: Warner and Weber, 1953; Baker, 1967). 31 The homogeneity of ovotransferrin has been in question (see Stratil, 1967; Baker, 1968; Feeney and Allison, 1969). As Baker (1968) points out, the amount of iron bound (one or more atoms) will affect the physical properties of the complex and thus could lead to resolution of a variety of artefactual "ovotransferrins." Thus in vivo, care must be taken to distinguish between genetical variants of the protein itself and chemical variants resulting from metal-binding. However, Rhodes 23, 91, (1958) positively identified two forms of ovotransferrin and Lush (1961) and Feeney 35. 31. (1963a) showed these were in the ratio of 4 to 1. Further work, showing that at least three co-dominant alleles are involved in the coding of ovotransferrin and transferrin (Ogden 23. 31., 1962; Baker, 1967; Stratil, 1967), also indicated that ovotransferrin is not homogeneous. Ovomucoid Ovomucoid was identified as the trypsin inhibitor in egg-white by Lineweaver and Murray (1947). This protein is distinguished by its high content of carbohydrate and its relatively high heat stability. It is a non-heat coagulable glycoprotein. Lewis 33, 21, (1950) determined its amino acid composition by microbiological methods and drew attention to the absence of free sulphydryl groups (Table 18). Longsworth gt, 2;, (1940) and Frederica.and Deutsch (1949b); concluded independently that at least two 32 electrophoretically distinct forms exist. Rhodes 33, 31. (1958) at first found that the ovomucoid fraction obtained from egg-white by chromatography on carboxymethyl-cellulose appeared to be homogeneous when examined by paper electro- phoresis. Later, however, refractionation of the preparation of ovomucoid by eluting with buffers of different pH values gave three separate components differing in their contents of sialic acid (Rhodes 33, 31., 1960). Feeney and Allison (1969) could not find homogeneous ovomucoid. Feeney gt. 31, (1967) noticed that in all animals, the characteristic of this fraction is its heterogeneity. The major biochemical importance of ovomucoid lies in its power to inhibit proteases. Three proteolytic inhibitors are known in egg-white; the others are ovoinhibitor and a papain-ficin inhibitor. Fowl ovomucoid (also geese and quail) inhibits only trypsin, whereas ovomucoid from other species (turkey, game pheasant, guinea fowl and duck) inhibits both trypsin and chymotrypsin; ovomucoid from yet other birds inhibits only chymotrypsin (Lineweaver and Murray, 1947; Rhodes 33, 31,, 1960; Feeney, 1964). The mechanisms of its action are not understood. However, during the complexing of ovomucoid with trypsin it is possible that important aspects are the electrostatic charges involved, covalent bond(s) and the epsilon groups of the ovomucoid lysine residues. For a full discussion of this subject see Simlot and Feeney (1966) and Feeney and Allison (1969). 33 Globulins, 02 and G1 Longsworth 22°.él- (1940) showed in egg-white the pres- ence of three globulins termed 01' 02 and G}; Alderton 33. 31. (1945) subsequently identified G1 and lysozyme. 02 and G3 were further studied by Kaminski (1954) who showed them to be distinct from lysozyme, ovomucoid, ovotransferrin and ovalbumin. Baker and Manwell (1962) identified them on starch-gel electrophoretograms and Feeney 33. 31. (1963b) were able partially to characterize each fraction. G2 and G3 appear in egg-white in concentrations each of about 4%. Gz-globulin, isolated by a combination of chromatography on DEAE and CM-celluloses and precipitation with ammonium sulphate, was found to have a molecular weight greater than 36,000 but less than 45,000. Isoelectric pH's were estimated as 5.5 for 02 and 5.8 for G}. Little other information is available and their functions remain unknown. Recent biological-genetical work has centered on these proteins. It is known that Gz-globulins are coded or controlled at two alleles (Lush, 1961, 1964; Baker and Manwell, 1962; Cochrane and Annau, 1962; Feeney 33. 31., 1963b) but further variants of 02 have been reported (Baker, 1964, 1968). G3 is controlled at a single locus with 10 dominant alleles (Lush 1961, 1964; Baker and Manwell, 1962); subse— quent investigations have indicated further variants (Baker, 1964, 1968). Baker (1968) suggests that up to 8 variants of 34 G3 are present in different strains of hens. The phylogenet- ical importance of these globulins has been reCOgnized and considerable investigations reported (see Baker, 1968). Lysozyme (Globulin 01) The globulin of_egg—white termed G1 by Longsworth 33. 31. (1940) was characterized by these workers chiefly by its electrophoretic behavior on examination of diluted egg~white. Further characterization, as well as isolation of 01’ has resulted from research work designed to isolate and characterize a bacteriolytic agent of egg-white. This factor was called lysozyme byFleming (1922), and has been shown to be identical with G1 (Alderton 33. 31., 1945). (a) Bacteriolytic Properties Fleming (1922) published a paper entitled, "On A Remarkable Bacteriolytic Agent Occurring in Tissues and Secretions," reporting the detection of lytic agents in tears, sputum, saliva, serum, plasma, leucocytes, eggs and organs of animal body, such as heart, spleen, liver, lungs, and kidneys. The lytic agent was termed lysozyme (lytic enzyme). The highest concentration was found in egg-white, which was active against test organisms in a dilution of l to 50,000. That lysozyme is in fact an enzyme, was later demonstrated by Meyer 33. 31. (1936) and by Epstein and Chain (1940) who showed that it disintegrates the cell walls 35 of susceptible microorganisms by acting on a specific carbo- hydrate component of the cell walls. The specific substrate was shown to be present in the cell walls of all susceptible organisms which were investigated but not in those which were resistant to lysozyme (Epstein and Chain, 1940). The susceptibility of various microorganisms to lysozyme has been reported by Fleming (1922), and Waksmann and Woodruff (1942). (b) Basic Properties Abraham (1939) described lysozyme as a basic protein containing approximately twenty-two titratable basic groups per mole (m.w. 18,000) as measured by histidine, arginine, and lysine content, and by acid binding at pH 3.0. This value is in good agreement with the data of Fraenkel-Conrat and Cooper (1944) who reported 12.5 basic groups per 104 grams. Alderton 33. 31. (1945) found that on electrodialysis of lysozyme solutions the pH gradually increased to 9.5, and a partial precipitation of lysozyme resulted. 0n subse— quent adjustment of the pH to 11.0 re-solution was apparent. As has already been mentioned, Longsworth 33. 31. (1940) found on electrophoretic examination of diluted egg- white that the component G1 maintained a positive charge over the entire range studied (pH 3.9 - 7.8). Alderton 33. 31. (1945), in studying the electrophoretic behavior of purified lysozyme, found good agreement with the mobilities 36 reported for 61‘ (c) Composition Lysozyme has been characterized quite completely as regards amino acid composition by the work of two groups of investigators (Fromegeot and de Garilhe, 1949, 1950; Lewis 31. 31., 1950; Canfield 1963). Prior to amino acid analysis, Fromegeot and de Garilhe fractionated the amino acids of HCL hydrolyzates by chromatographic methods into four groups; namely, the basic, neutral aromatic, acidic and the neutral non-aromatic groups, while Lewis 33. 31. (1950) carried out microbiological analyses directly on the hydrolyzates. The data from the two laboratories agree fairly well for most amino acids. Fromageot and de Carilhe (1950) did not originally find proline or methionine in their fractionated hydrolyzates, but later verified the data of Lewis 33. 31. (1950) who found both of these amino acids to be present. Later, the complete amino acid sequence was independently determined by Canfield (1963). The most interesting biological property of lysozyme is its lytic activity against bacterial cell walls during which D—glucosamine, muramic acid and reducing groups are liberated. Because the structure of egg-white lysozyme is known, considerable attention has been directed towards an understanding not only of its enzymatic properties, but also of the physical relationship between the enzyme and 37 its substrate. Much attention has been given to the action mechanism of lysozyme: early work pointed to the importance of the tertiary protein structure (the three-dimensional structure was determined by X-ray analysis by Blake 33. 31., 1965, 1967a, b), the disulphide bonds, the E-amino groups (from lysine) and the carboxyl groups. Current evidence supports the view that the disulphide bridges are important (Fraenkel- Conrat 33. 31., 1951; Goldberger and Epstein, 1963) but some of these can be reduced without affecting enzymic activity (Isemura 33. 31., 1961; Caputo and Zito, 1961). It is interesting that Manwell (1967), concerned with "molecular palaeogenetics," claims that lysozyme is closely related to ribonuclease and may have evolved from a common protogene. The widespread distribution of lysozymes through- out the animal and vegetable kingdoms and in the bacterio- phage suggests that it may well have evolved early in the formation of living systems. In the hen, qualitative genetic differences in lysozyme have been found (Baker, 1968); it has been suggested that the locus has two co-dominant alleles. Quantitative differ- ences are also inherited (Wilcox and Cole, 1957). (d) Stability, Inactivation, and Denaturation Meyer 33.131. (1936) reported that lysozyme was very stable to heat and cold, but unstable to alkali. Solution 38 in 2% acetic acid lost no activity when held at 100°C for 45 minutes. Neutral solution similarly treated lost all activity, while the same result was achieved in 5 minutes at pH 9.0. Alderton 3E. 31. (1946) found no loss in activity at pH 9.0 or below, over a period of three months at 23°C and only a 25% loss took place at pH 11.0. At pH 12.0, however, 60% loss took place in 7 days. The same investi- gators found that in HCL solution, pH 3.0, at a temperature of 96°C., 9, 21, and 40% of the activity was lost after 10, 25, and 50 minutes, respectively. The isolated lysozyme was found to be resistant to enzymic hydrolysis by trypsin, papain, mild proteinase, and bacterial proteinase (Alderton 33. 31., 1945). Pepsin hydrolyzed the material at a slow, but measurable rate. After heating in acid solution, however, the material, while still active, was markedly more susceptible to hydrolysis by the enzyme; trypsin, for example, inactivating 66% of the material, whereas before heating and under the same condi- tions no measurable destruction had resulted. Heat may, therefore, alter the lysozyme molecule, sensitizing it to enzyme action without altering the biological activity (Fevold, 1951). Ovomucin Ovomucin contributes about 2% to the total protein of albumen (Baker, 1968, 1.5%; Feeney and Allison, 1969, 2.9%). 39 It is an insoluble, fibrous, acidic glycoprotein with high carbohydrate content of 18.6% (Donovan_e_t_. 31,, 1970). Feeney'3g. 31. (1960) and Donovan 33. 31. (1970) reported a sialic acid content of between 2.5 and 4% but Odin (1951) gave values as high as 8%. The hexosamine content is between 7 and 12%»(0din, 1951; Brooks and Hale, 1961; Robinson and Mbnsey, 1964; Donovan 33. 31., 1970). Donovan 33. 31. (1970) also reported the presence of about 7% hexose and about 2 and 14% respectively of sulphur and total nitrogen. Ovomucin is insoluble because it is a large molecule and is fibrous. Its electrophoretic characters have not been resolved, nor have estimates been made of its molecular weight or isoelectric pH. However, Sharp 33. 31. (1951) did resolve it into three fractions on moving-boundry electrophoresis. Ovomucin's natural function in egg-white is not known, but it inhibits viral haemagglutination (Lanni 3:. 31., 1949); despite numerous suggestions to the contrary, its sialic component is probably not involved in this activity. Other suggestions have ascribed to ovomucin the origin of the gel-like qualities of egg-white, particularly since it appears in greater concentration in the Chalaziferous region and the thick white. Moreover, much appears to be present as a complex with lysozyme (Hawthorne, 1950; Feeney and Nagy, 1952; Cotterill and Winter, 1954; Sugihara 33. 31., 1955; Brooks and Hale, 1959, 1961; Oades and Brown, 1965). 4O Flavoprotein Rhodes 33. 31. (1958) first reported riboflavin in egg-white as a flavoprotein complex. Rhodes 31. 31. (1959) showed that albumin contained about 0.8 to 1% (see Table 14) of equal amounts of flavoprotein and its apoprotein. Subsequent work (see Baker, 1968; Feeney and Allison, 1969) verified these findings and showed that the apOprotein had a molecular weight of between about 32,000 and 36,000 and air isoelectric pH between 3.9 and 4.1. Its amino acid composition (Feeney and Allison, 1969) (see Table 15) differs only slightly from that reported by Farrel 33. 31. (1969). There appear to be no free sulphydryl groups, although the protein is highly crossed-linked by eight disulphide bridges (Farrel 31. 31., 1969). It contains 14% of carbohydrate derived from mahnose, galactose and glucosamine (Farrel _e_t. 31 ., 1969). The protein contains 0.7% Or 08%. phosphorus; there are two forms, one containing seven phosphate groups and the other eight (Rhodes 33. 31., 1959). On starch-gel and paper electrophoresis it moves ahead of ovalbumin components (Rhodes 33. 31., 1959; Baker and Manwell, 1962; Baker 33. 31., 1966). Its most obvious characteristic is its ability to bind the vitamin riboflavin (in 1:1 molar ratio), hence all riboflavin in egg-white is held in an extremely stable complex (Winter 3:. 31., 1967); this is unusual in that the flavin moiety is riboflavin and not the customary flavin 41 mononucleotide or flavinadeninedinucleotide. It has been suggested that the form found in egg-white is identical with the plasma flavoprotein complex (Ostrowski 31. 31., 1962). Clearly a possible function for ovoflavoprotein is to transfer riboflavin to the embryo, since riboflavin injected into riboflavin-deficient eggs will allow hatching (Hawes and Buss, 1965). However, the generally accepted relation- ship between riboflavin deficiency, "clubbed down: and poor hatchability (Lepkovsky 33. 31., 1938; Coles and Cumber, 1955) is in some doubt, particularly since riboflavin- deficient eggs do not necessarily lead to chicks with "clubbed down" (Hawes and Buss,1965). Ovomacroglobulin Ovomacroglobulin forms about 0.5% of egg-white. It was first seen by Lush (1961) on starch-gel electrophoreto- grams and was described in more detail by Feeney 33. 31. (1963a). It is the heaviest egg-white protein, with possible exception of ovomucin, having a molecular weight of around 800,000; its isoelectric pH is between 4.5 and 4.7 (see Table 14). Feeney and Allison (1969) state that it has no special noteworthy features as a protein (see Table 15), but little is known of its major characteristics. It is absent in the turkey and many genetic lines of Japanese quail. Its main interest lies in that it is the only component 42 of egg-white with a wide spectrum of immunological cross- reactivity (Miller and Feeney, 1964, 1966); it is strongly immunogenic. Further information on this aspect is to be found in Feeney and Allison (1969). Ovoglycoprotein Little is known of this acidic glycoprotein. It was first noted by Ketterer (1962, 1965). It has a molecular weight of 24,400 and an isoelectric pH of 3.9 and contains about 13.6% of hexose, 13.8% glucosamine and 3.0% sialic acid. Feeney and Allison-(1969) suggest that it might be identical with an acidic fraction isolated by Rhodes 33. 31. (1960), which contained approximately 4% sialic acid. Montreuil 33. 31. (1965) also found a similar protein. It possibly forms the most advanced band on starch—gel and may be heterogeneous. It has no antihaemagglutination activity and inhibits neither trypsin nor chymotrypsin. Ovoinhibitor Matsushima (1958) first described a protein in chicken egg-white which inhibited bovine chymotrypsin (Rhodes 33. 31., 1960; Feeney 33. 31., 1963a). Further characterization (Rhodes 33. 31., 1960: Tomimatsu 33. 31., 1966) established that it would inhibit both trypsin and chymotrypsin in the ratio of two molecules of each proteinase to each molecule of protein; hence four molecules of enzyme could be inhibited 43 by each molecule of ovoinhibitor. Ovoinhibitor occurs in albumen at concentrations between about 0.1% (Baker, 1968) and 1% (Feeney and Allison, 1969). The isoelectric pH is about 5.2 (see Table 14). It is composed of a single polypeptide chain (Davis 3;. 31., 1969) of the amino acid composition given in Table 15. Ovoinhibitor is a glycoprotein containing about 3.5% hexose and 2.7% hexosamine, with little sialic acid (Rhodes 33. 31., 1960). Ovoinhibitor migrates on starch-gel just ahead of the ovotransferrin bands (Tomimatsu 33. 31., 1966). It is not homogeneous and Tomimatsu et. a1. (1966) distinguished three forms whilst Davis 33. 31. (1969) described five which differed only with respect to the carbohydrate moiety. Avidin This is a minor component of egg-white (0.05%). It binds biotin and was first described by Eakin 31. 31. (1940, 1941). Early physico-chemical studies gave a molecular weight of 48,000 to 66,000 and an isoelectric pH of 9.5; it was thought to consist of three peptide chains, each capable of binding one mole of biotin (Melamed and Green, 1963). Recent work suggests that it is composed of four chains and has a molecular weight nearer 70,000 (Green, 1964). Its amino acid composition has been determined (Table 15) (Melamed and Green, 1963). Avidin may occur in three forms (Melamed and Green, 44 1963), two of which differ in composition whilst the third is complexed with another substance. Fraenkel-Conrat 33. 31. (1952) have previously described three forms composed of avidin plus nucleic acid, avidin-A formed from this complex and avidin complexed with an acidic glycoprotein. As it has not been possible unequivically to demonstrate avidin on starch-gels (Baker and Manwell, 1962; Lush, 1964), it has not been established whether the different forms represent genetical variants. The importance of avidin lies in its capacity to bind three moles of biotin to about 64,000 g of protein as an extremely stable complex (Feeney and Allison, 1969) and so render biotin unavailable as a vitamin or coenzyme. In this way it can act as an antibacterial agent. Minor Proteins Several acidic proteins containing sulphydryl groups have been reported in egg-whites of different birds (Feeney 33. 31., 1960, 1966). The amounts present are extremely small, and little is known about their properties. Feeney 3'3. 31. (1966) and Baker andManwell (1967) suggest that some cathodallymigrating proteins may be present. Enzyme Inhibitors The following were found to be enzyme inhibitors: (1) Ovomucoid (see page 31) 45 (II) Ovoinhibitor (see page 42) (III) Papain Inhibitor (IV) Trypsin Inhibitor Papain Inhibitor Fossum and Whitaker (1968) have partially characterized a protein, comprising about 0.1% of egg-white, which inhibits both papain and ficin. Its molecular weight may be 12,700; apparently it is devoid of carbohydrate. Trypsin Inhibitor Kanamori and Kawabata (1964) identified trace amounts of an acidic protein thought to contain flavin; they suggested that it had antitryptic properties. Enzymatic Proteins The enzymatic proteins of egg-white are of the following: (a) lysozyme (see page 34) (b) glycosidases (c) catalase activity (d) peptidase (e) esterases Glycosidases Lush and Conchie (1966), in hen's egg-white, discovered two enzymes, Lemannosidase and B-N-acetylglucosaminidase 46 (E.C. 3.2.1.30). The latter splits aryl-N-acetyl glucos- aminides and more correctly it should be called B-2- acetamido-2-deoxy—D—glucosidase . Catalase Activity Lineweaver 33. 31. (1948) reported that egg-white had catalase activity as well as very slight esterase and peptidase activities. However, as pointed out by Baker (1968), care must be taken to distinguish between catalase activity (decomposition of H202) and the substance catalase itself. On starch-gels, catalase activity seems to be associated with the ovalbumin and transferrin region in the fowl (Baker and Manwell, 1962; Corbin and Brush, 1966) and in other birds (Baker, 1965; Baker 32. 31., 1966; Baker and Manwell, 1967), but this activity may be artefactual (Baker and Manwell, 1962; Corbin and Brush, 1966). True catalase has not been found in the fowl but Lush (1966) reported its presence in the quail; this was confirmed by Baker and Manwell (1967). Its migration on starch-gel is consistent with a molecular weight of at least 200,000. Further characterization has not been carried out. Catalase is a heam-protein, the function in quail egg albumen is not known. Dawson 9;. _a1. (1959) list H202 as an inhibitor of lysozyme, which suggests that albumen catalase may fill a protective role but Lush (1966) viewed it unlikely to have any important physiological function because it is absent 47 from chicken egg albumen. Peptidases Despite the original caution regarding peptidases in egg-white, Manwell 33. 31. (1967) reported that chicken egg- whites gives two zones on starch-gels which have been shown to be true peptidases. Esterases Egg-white of many birds shows esterase activity, but there is considerable inter- and intra-generic and specific variation (Baker and Manwell, 1962, 1967; Baker, 1965; Baker 33. 31., 1966). Moors and Stockx (1966, 1968) have reported the presence of alkaline and acid phosphomonoesterases and phosphodiesterases. Japanese Quail Egg (Coturnix coturnix japonica Egg) Interest in the quail as a domestic bird has been renewed and it is kept for meat and egg production and as a laboratory animal. It is now possible to resolve Coturnix egg-white into at least twelve distinct proteins and the results of two- dimensional electrophoresis suggest that there are more (Baker and Manwell, 1967). The literature cited and informa- tion reported here are mainly of Baker and Manwell (1967), as the author cannot find any other material related to the 48 same subject. lungs; of Coturnix Egg-White When whole Coturnix eggs or separated egg-whites have been stored at 0-4°C the electrophoretic resolution of proteins deteriorates noticeably. At higher temperature, the deterior- ation starts sooner and is more rapid. Ovalbumin is one of the soonest to deteriorate (Baker and Manwell, 1967). Identif1gation of Igg1yidual Proteins of Coturnix Egg-White Figure 2 shows the composite diagram of coturnix egg- white proteins observed in several different buffers. The diagram has been expanded to include all the observed phenotypes. The trace proteins are only visible in pH 5.7 acetate gels under particularly favorable conditions. Catalase As mentioned before true catalatic activity is found only in Japanese quail and not in chicken. Baker and Manwell (1967) confirmed the report by Lush and Conchie (1966). Esterase Esterase is found in the Coturnix egg-white. The esterase does not stain well in the acid phosphate gels, in. contrast to many other esterases (Baker and Manwell (1967). Z: } Prealbumln: Albumlnt Postzlbumlns - < Conalbumin 5 SH F M '—"" Slow u-llke protein Simple-Insertion slovI it: - _ — — — — “X" P’°“"‘ — _ — m: SM 5r 5 M F _ — — — }"Y" proteln ‘ _ — S M ‘ MF SH SF ...... < Trace preteln — '— V Lyioqme . — .... F SF 5 < Trace protein FIG-a. Composite diagram of Commie egg-white proteins observed in several dilicrent liuil’ers. The diagram has been expanded to include all the observvd Phenotypes. The trace proteins are only visible in pll 5'7 acetate gels under parti- cularly favourable conditions. 49 50 Peptidase Some of the peptidases located with napthyl conjugates are very active. Four zones of "peptidase" are observed after electrophoresis of Coturnix egg-white, using three substratea(fibnmell 33. 31., 1967). The real nature and significance of these enzymes are unknown, but they appear to be widely distributed. Lysozyme (G1 Globulin; Muramidase) Lysozyme is the most cathodal of the major proteins of coturnix egg-white. There are two types of coturnix lysozyme, "fast" and "slow." The genetic interpretation of these data is that the "fast" and "slow" only are homozygotes and the two-banded pattern of "fast" and "slow" represents the heterozygote. Preliminary studies on the activity of the three lysozyme phenotypes on bacterial cell wall substrate did not reveal any marked differences in kinetic properties. §19w -o§-;Like Protein This protein corresponds electrophoretically to a post-conalbumin slow -cK - like protein in the domestic fowl and pheasant (Baker‘ahdManwell, 1962; Baker 33;. 31. , 1966). It is probable that these slow -c£ - like proteins also correspond to "band 18" or ovomacroglobulin, a high molecular weight protein (Miller and Feeney, 1966). 51 X - Protein This protein is called "X" temporarily because it has not yet been possible to find any clear similarity between it and any of the characterized chicken egg-white proteins. Y - Protein (Ovomucoid) The coturnix egg-white protein referred to as "Y" has some of the characteristic prOperties of ovomucoid. It can be obtained by methods used to purify ovomucoid from the egg-white of chicken (Lineweaver and Murray, 1947; Warner, 1954) and other species (Stevens and Feeney, 1963); "Y"-protein also has strong trypsin-inhibiting activity as reported by Feeney (1964). This report was supported and confirmed by Baker and Manwell (1967). From the information concerning the migration of chicken ovomucoid and ovoinhibitor in starch-gel (Tomimatsu 33.,31., 1966), Y-protein would seem more likely to be ovoinhibitor and postalbumin to be ovomucoid. Postalbumin Baker andManwell (1967) found that postalbumin protein is also present in TCA-acetone-precipitated "ovomucoid." They reported that it was possible that a true ovomucoid or ovoinhibitor may be in this region. 52 Ovalbumin Attempts to purify Coturnix ovalbumin by "salting out" with ammonium sulphate were unsuccessful (Baker and Manwell, 1967). Feeney 33. 31. (1966) also failed to crystallize ovalbumin by this method from the Adelic penguin egg-white. Individual variation in Coturnix ovalbumin was reported by Baker and Manwell (1962), Whether the variation represents actual molecular mutation in the ovalbumin, or in the enzymes involved in phosphorylation during ovalbumin synthesis in the oviduct, or in any enzyme capable of dephosphorylating the finished ovalbumin in egg-white is not known (Baker and Manwell, 1967). Prealbumins The prealbumins have not been studied extensively as the optimum conditions for their resolution are suboptimal for most of the other egg-white proteins. Four prealbumins can be resolved and some variation among individuals in the presence or absence of one band has been noted. Other Interesting_033ervations Amos (1966) used Japanese quail to test the effect of DDT on the hatching of osprey eggs. There were no differences in mortality and hatchability between control quail and the groups fed varying levels of DDE and DDD up to 50 ppm. This finding suggests that Coturnix are unsuitable for testing the 53 toxicity of pesticides. The results may give a false idea that certain compounds are relatively harmless. It is also of interest that Ames (1966) DDE and DDD fed quail deposited large amounts of these substances in their eggs. In this connection it is worth noting that the Greeks and Romans were aware that Coturnix could COpe with a diet including natural substances poisonous to man and for this reason, did not eat the birds (Zeuner, 1963). The author had read in Chinese literature that in the olden daya Japanese quail eggs were served only to the Kings and that there were instances of therapeutic effects of quail eggs. For these reasons quail egg albumin was used in the observation later by the author. Physiologic Availag111ty of Nutrients in Eggs The classic work of Atwater and Bryant (1899) concerned with the availability of proteins and fats of eggs revealed that approximately 97% of the proteins and 95% of the total fat were absorbed by healthy human subjects. 0f many items tested, no other food was found to be superior in these respects. Research dealing with the utilization of amino acids indicates that the amino acids of eggs are readily available to the consumer. Guthneck, 33. 31. (1953) observed that 98% of the lysine of dried whole egg was utilized to support weight gain in protein-depleted rats. Murlin 33. 31. (1938) 54 demonstrated the excellent performance of animals receiving egg proteins. 0n the other hand feeding experiments con- ducted by Boas (1927) and Parsons (1931) demonstrated that high levels of unheated egg-white produced a detrimental effect on test animals. Following numerous studies of the "toxic" effect of a substance occurring in egg-white, Parsons and Kelly (1933) concluded that the active material was proteinaceou's in‘nature. The harmful effect disappeared when egg-white was heated. A crystalline material isolated from egg-white and capable of producing lesions similar to those described by Boas and Parsons was announced by Pennington 23. 31. (1942). This substance, named avidin was proved to combine with a vitamin (biotin) (see page 43), rendering the essential factor unavailable to test animals. Raw egg-white was, therefore, found to contain a physiologically active substance which combines with biotin to form a complex 9 which the host cannot absorb. Boyd 33. 31. (1966) fed raw egg-white powder to rats at a dose of 50 gm/Kg of diet/day, produced inhibition of growth, anorexia, diuresis, glycosuria, proteinuria, diarrhea, a decrease in weight of most organs except brain, gastrointestinal tract, salivary glands and kidneys and dehydration of several organs. Peters (1967) produced a toxic syndrome by giving levels of 20%, 40%, 60%, 80%, or 100% of raw egg-white to young adult female albino rats. The toxic signs caused by 55 increasing intake of dried raw egg-white powder were: decreased food intake, weight loss, soft stool, diarrhea, glycosuria and death. Water intake and urine output rose with increasing raw egg-white powder in the diet. In all groups the urine was alkaline and the urinary output of protein increased. At autopsy there was a decrease in the absolute weight and in the water content of most body organs with increasing amounts of raw egg-white powder in the diet. The toxicity syndrome was not prevented by a biotin supplement, but was largely prevented by heat denaturation of the egg-white powder; 80% of denatured egg-white was well tolerated, as was 80% of casein in the diet. This indicated that the syndrome was due to the direct toxic effects of large amounts of dietary raw egg-white powder and not to biotin deficiency. The results of this study showed that inclusion of as little as 20% raw egg-white powder, but particularly of 80% or more, in the diet produced measurable signs of acute toxicity in adult female and young male rats. Bateman's (1916) original description of reduced food intake, weight loss, some development of tolerance after 7-10 days, soft stool and diarrhea was confirmed in the study by Peters (1967) and extended to include other clinical and pathological measurements. The results indicated that the toxicity syndrome originally reported by Bateman (1916) was due to biotin deficiency, and also to the direct toxicity of raw egg-white powder. 56 Embgyological Functions of Egg-White There is very little information on the function or importance of egg-white proteins in embryological development. Unproven but possible functions of the egg—white proteins during the development of the embryo are the following: (1) A protective aqueous physical environment for the developing embryo on the surface of the egg yolk. (2) A source of water and protein to the developing embryo. The embryo apparently "drinks" the egg-white during develOpment. (3) A source of certain particular constituents, such as the ovotransferrin, for transfer to the blood. (4) A direct functional activity of a nutritional nature. One of these may be transport of calcium. (5) An antimicrobial barrier and protection for the embryo. (6) Buffer. Direct evidence for any of these functions is apparently available. Studies have involved a removal of the egg-white during incubation of the embryo and its replacement by various substances. A preliminary interpretation of this data is that the egg-white may prove to be not particularly important physiologically but may be utilized for its nutritional value. As early as 1890, Wurtz noted that various organisms, such as typhoid bacilli andpythogenic cocci,could not survive in egg albumen. Albumen, when left in an Open, sterile dish, 57 may remain free of bacteria growth for as long as two months (Laschtschenko, 1909, quoted by Romanoff and Romanoff, 1949). The egg's chemical defenses against bacteria are contained in the albumen. The ability of the albumen to protect the yolk from contamination by the microorganisms of the external environment is clearly shown in figure 3. Bacteria, after penetrating through the shell and membranes, multiply in the albumen for a short time and then rapidly decrease in number. Contamination of the yolk is delayed and comparatively light. It has been recognized for many years that the failure of bacteria to grow in egg albumen is due chiefly to the presence there of a substance, or substances, possessing germicidal activity (Laschtschenko, 1909; Rettger and Sperry, 1912). The Romans reputedly recommended egg-white for the treatment of eye infections. The most interesting discovery was made by Fleming (1922) (see lysozyme). Since then many other egg-white proteins which show antimicrobial or anti- enzyme properties could be considered as potential microbial antagonists. These include lysozyme, ovoflavoprotein, avidin, ovotransferrin and the inhibitors of proteolytic enzymes, ovomucoid, and ovoinhibitor. Lysozyme has received the most notoriety and indeed is bacteriacidal to a limited number of species. Until fifteen years ago there was only a slow acquisition of data concerning the action of this enzyme (Thompson, 1940, 1941; Salton, 1957) but it was established 4000 r- A / SHELL ucusemc u' U \ F g 3000 " o 8 S a: bi t) 2000 __ .... f ALBUMCN 4 ‘s (D , '\ u I O 'I 0 (I u IOOO " '0' m r " I 5 : Z a‘ I a ‘O‘ .... 0 I0 20 30 40 50 60 70 80 INCUBATION TIME AT 25° T0 26°C. (nouns) Fm. 3:5. The extent of contamination in shell membrane, albumen, and yolk observed during 80 hours' incubation of eggs smeared externally with a culture of Pseudmnouas acruginosa. (After Stuart and McNally, 1943.) 58 59 that under normal conditions gram-positive bacteria are on the whole more sensitive to lysozyme than are gram-negative organisms. The factor almost entirely overlooked, however, is the antibacterial activity of the high pH of egg-white during the first few days of incubation of the egg. Ovotransferrin has been directly proven to be an important antimicrobial substance in a rather unique manner. When eggs are washed for commercial sales, there was a large difference in the rate of spoilage which could not be explained on the basis of sanitation and source of the eggs (Garibaldi and Bayne, 1962). Most people will not buy eggs if the shell has dirt and feces on them. The washing process, while removing the dirt, will, if not properly done aid in transporting microorganisms through the thousands of pores of the egg shell. After a long period of research it was shown that the reason for the difference in subsequent spoilage of the eggs was directly related to the iron content of the water used for washing. The higher the iron content, the more the subsequent spoilage and growth of microorganisms in the egg-white. The conclusion was that the small amounts of iron gaining entrance into the egg-white or in the pores of the shell was sufficient for microorganisms to initiate growth even in the presence of the high concen- tration of the iron chelating agent, ovotransferrin. The biological properties of the proteins of the albumen are given in Table 16. These data have provided the 60 TABLE 16 THE BIOLOGICAL PROPERTIES OF COMPONENTS OF THE ALBUMEN OF THE CHICKEN'S EGG Component Action Investigator 1. Lysozyme (a) Lysis of cell Laschtschenko (1909) walls of cer- tain bacteria (b) Flocculation Fleming (1922) of bacterial cells , Freidberger and Hoder (c) Hydrolysis of (1932) B-l, 4-g1ycosi- . dic Berger and Weiser (1957) 2. Conalbumen Chelation of iron, Shade and Caroline {(1944) (Ovotransferrin) zinc and copper Alderton, Ward.and Fevold (1946) 3. Ovomucoid Inhibition of Balls and Swenson (1934) trypsin Lineweaver and Murray (1947) 4. Avidin Combination with Eakin Snell and Williams biotin (1940) Woolley'and~Longsworth ' (1942) Baumgartner (1957) 5. Riboflavin Chelation of Feeney and Nagy (1952) Cations 6. Uncharacterized (a) Inhibition of Rhodes, BennettHand trypsin and Feeney (1960) chemotrypsin (b) Inhibition of Matsushima (1958) fungal protease (c) Combination Rhodes, Bennett and with ribo- Feeney (1959) flavin 61 TABLE 16 (Con't.) Component Action Investigator (d) Combination Evans, Butts and with vitamin Davidson (1951) Be (e) Chelation of Abels (1936) calcium Source: Board, 1968. basis for the currently accepted concept of the antimicrobial defense of the albumen, namely that it is a medium unsuitable for microbial growth (Board, 1968). Contamination of the Egg Research in the field of microbial deterioration of shell eggs still remains a problem to the poultry industry. The available literature found for marshalling evidence has been attempted in four publications only (Haines, 1939; Romanoff & Romanoff, 1949; Brooks and Taylor, 1955; Board, 1966). In spite of the numerous microorganisms on the surface of the shell, bacteria are not usually found in the yolk in more than 10 percent of all fresh eggs or in albumen in more than 3 percent (Haines, 1939; Romanoff’and-Romanoff, 1949) 62 although higher percentages of contamination have been reported by some workers. Many workers have claimed that the shell is easily invaded following laying (Ferdinadov, 1944; Lorenz 33. 31., 1952; Graves & MacLaury, 1962) and that degree of contamination of the contents of fresh eggs is related directly to the porosity of the shell (Kraft 3:. 31., 1958). When reviewing this facet of egg micro- biology, Brooks and Taylor (1955) were forced to generalize that "roughly" 90% of newly laid eggs are free from micro- organisms and the true value may be even higher. The commonest contaminants of the contents of fresh eggs are micrococci which grow poorly, if at all, at the body temperature of the hen (Hadley and Caldwell,'1916'; Haines, 1938; Miller and Crawford, 1953). Micrococci have been recovered also from ova taken from hens which had been killed and dissected in the laboratory (Harry, 1963). Harry (1963) favored the view that blood-borne organisms are primarily responsible for contamination of the ova. There is ample evidence (Rettger, 1913; May, 1924; Buxton.and Gordon, 1947; Gordon and Tucker, 1965) that pathogens such as salmonella species pass from the alimentary canal via the blood to the ovaries, but there is no conclusive evidence of such migration by organisms capable of rotting eggs. Brooks and‘Taylor'(1955) summarized in their observations that rot producing organisms are primarily of extragenital origin and that less than 1% of naturally clean eggs rot during prolonged storage. 63 When eggs were collected aseptically at the time of oviposition, Stuart and McNally (1943) recovered organisms from the shells of two eggs only. This suggests that the shell of a few eggs are contaminated when passing through the cloaca but.that the main contamination occurs after lay- ing. Fertile and infertile eggs ordinarily are contaminated to an approximately equal extent (Bushnell and Maurer, 1914; Hadly and Caldwell, 1916 )n. The slightly lower incidence of contamination found in fertile eggs in one study (Rettger, 1913) is possibly without significance. Gramppositive bacteria are numerically dominant on clean or slightly soiled shells whereas gram-negative ones can be dominant on badly soiled eggs. These contaminants are almost certainly derived from dust, soil, and faeces (Haines, 1939; Zagaevsky and Lutikova,'19'4'4; Boards}. 31., 1964). Boardand Board (1968) characterized and identified the commonest contaminants as members of the genera Alcali- genes, Achromobacter, Pseudomonas, Serratia, Cloaca, Hafnia, Citrobacter, Proteus and Aeromonas. Viruses such as Lymphoid Leukosis Virus (LLV), Infections Bronchitis Virus (IBM), Avian.Encephalcmyelitis Virus (AEV), Newcastle Disease Virus (NDV), and other viruses of an infected hen may transfer the infection to the egg in the oviduct at some stage between development of the ovum and completion of the egg in the oviduct or 64 infection can also be due to extragenital contamination by the virus. The occurrence of egg transmission of LLV was firmly established by the work of Burmester (1962), Rubin 3t. 31. (1961), and Rubin 33. 31. (1962). Infection by IBV is widespread in commercial flocks. Fabricant and Levine (1951) showed that IBV could be isolated from the eggs from infected hens up to 36 days after infection. Egg transmission of ARV is an important natural route of spread (Galnek 33. 31. (1960). Transmission occurs when the ovulating hen becomes infected and ceases when immunity develops. Egg transmission of NDV appears to be rare. DeLay (1947) and Hofstad (1949) isolated NDV from dead embryos and infertile eggs from infected stock, and DeLay also isolated the virus from hatched chicks. Hofstad found that egg transmission ceased as egg production recovered following outbreaks of the disease. In addition to the disease-associated viruses mentioned above other viruses of little or no pathogenicity occur in the fowl and may be transmitted to the embryo. Note that most of the viruses are found in the egg yolk. Mycoplasma gallisepticum is widespread in the field where it is responsible for respiratory infections of chickens and turkeys. In addition to spread by contact, egg trans- mission occurs (Van Roekel 33. 31., 1952; Fahey'and Crawley, 1954). Yoder and Hofstad (1964) reported isolation of the organisms from ovary, oviduct and semen of infected birds. 65 Immunological Comparisons Egg-whites and, in particular, chicken albumin have been used in many immunological studies. In 1938 Lansteiner 25.,31. showed the immunological relationships of several avian species. Landsteiner's experiments are now classical and used both ovalbumin and hemoglobulins of several species. More extensive data on egg-whites using three different proteins, ovalbumin, ovotransferrins, and lysozyme came from Deutsch's laboratory at Wisconsin in the early 1950's (Wetter,3§. 31., 1953). Turkey, guinea hen, pheasant, duck, and goose whites were analyzed with antibodies to chicken proteins and were found to cross-react with each other. These investigators also obtained evidence indicating that a major portion of the antibody response (immunogenicity) of rabbit to chicken egg-white was directly against minor unidentified protein components. These minor components were present in other egg-whites examined and cross-reacted strongly. Ovalbumins of different species have been compared immunologically by many workers (Kaminski, 1962; Jennings and Kaplan, 1962; Fothergill and Perrie, 1966). Wilson and co-werkers (1968) studied the complement fixation by antiserum against chicken ovalbumin with a large number of different egg-whites in twenty-two taxonomic groups. This type of study has been extended to lysozyme by Arnheim and Wilson (1967). 66 Miller and Feeney (1964) have employed immunoelectro- phoresis and immunodiffusion for comparative immunological studies. .Antibodies were prepared in the rabbit against chicken, Japanese quail, duck, and cassowary egg-whites as well as purified chicken ovalbumin and ovotransferrin and cassowary ovotransferrin. They confirmed the observations of wetter 33. 31. (1952).that a minor component was found to be strongly antigenic and showed extensive cross-reactions between all species examined. This component was identified as component 18 named ovomacroglobulin noted on starch-gel electrophoresis by Lush (1961) and Feeney et. al. (1963b). It was found widely distributed in the various flocks of chickens at the University of California at Davis. It was absent from many genetic lines of Japanese quail and not found in turkey egg-white selected to obtain a population sample (Feeney and Allison, 1969). The physical and chemical properties of an immunologi- <=ally cross-reacting macroglobulin in avian egg-whites were studied by Miller and Feeney (1966). They reported that nmsacroglobulin has comparatively high antigenicity in rabbits and cows and shows extensive immunological cross-reactivity ‘vrlnen the proteins prepared from the egg-whites of different 8IDecies are tested against antibodies to chicken protein. Orr (1971) and Smith (1972) found high titers of reagins "“flr11ch.were obtained in rats by initial immunization with egg albumen; Varies and Harink (1973) reported that about 50 67 percent of guinea-pigs immunized with egg albumen possess strong antibody activity in an immunoglobulin class with antigenic properties different from those of the well-known guinea-pig immunoglobulins. This immunoglobulin is present in normal guinea-pig serum in very small amounts and its concentration rises during immunization with egg albumen. Immunization with bovine insulin or bovine X—globulin does not stimulate increased synthesis of this immunoglobulin class. Antibodies usually inhibit enzymes, though in some cases they cause activation (Pollock, 1964; Suzuki 33. 31. 1969). In addition, their efficiency of neutralization of enzymic activities varies depending on their specificities (Imanishi et. a1., 1969). Since different parts of a protein molecule play different roles in maintaining the function and conformation of the protein, the most probable explana- tion for the diverse functions of antibodies is that they differ in antibody specificity (Fujio et. a1., 1971). Now detailed information on antigenicity of lysozyme has been ¢1t>tained from many laboratories (Arnon and Sela, 1969; Habeeb 3‘5. 31., 1969; Bonavida 33. 31., 1969; Strosberg and Kanarek, 1970; Young and Leung, 1970; Maron 33. 31., 1971; Percht 31;. 31., 1971). Many immunological studies have been Carried out on hen egg-white lysozyme because its primary Etr1¢1 tertiary structures have been fully characterized. KnOwledge of this enzyme's antigenic determination has been derived from studies of the immunological behavior of peptide 68 fragments by many investigators mentioned above. Data have become available for the complete amino acid sequence of human leukemia lysozyme (Canfield 31. 31., 1971). In addition, preliminary X-ray crystallographic data on this lysozyme at a 6 3. resolution have indicated that the three- dimensional structure of the two lysozymes (HEL and human leukemic lysozyme) appears to be the same (Blake and Swan, 1971). Lysozyme is an enzyme known to be widely distributed in vertibrates. If the structures responsible for the enzymic activity of the enzyme from different sources are exactly the same, it is unlikely that it will be possible to make an antibody which is specific for the active center of enzyme from all these sources. However, Canfield 33. 31. (1971) compared the amino acid sequences of human lysozyme and hen egg lysozyme (HEL) and found that the amino acid moieties differed at a considerable number of points near the catalytic site. Therefore, it may be possible to make an antibody which is specific for the region close to the catalytic site of enzyme. Hill and Sercarz (1974) studied the cellular and humoral antibody response in C57BL/6 mice to egg-white lysozyme (HEL) and six other immunochemically related lysozymes: bob-white (BBL), guinea hen (NEL), Japanese quail (JEL), pea-fowl (PEL), ringed-neck pheasant (REL) and turkey (TEL). Ninety percent 0f the animals are completely unresponsive to HEL, BEL, NEL, 69 and PEL, while the remaining 10% respond at a very low level and exhibit relatively restricted patterns by isoelectric focusing. All C57BL/6 mice are strong responders to JEL, and REL, and moderate responders to TEL. Egg-White Protein and Poultry Breeding The discovery of genetic polymorphism of egg-white raised hopes that it would provide information of use in breeding practice (Baker, 1960; Lernernand Donald, 1966). Egg-white protein polymorphism combines the advantages of discrete genetic characters and gene expression is the object of economic interest. Several of the larger poultry firms have typed their stocks for variants but the results have not been publicized. A measure of the success of egg-white polymorphism as a breeding tool is the fact that a number of poultry breeding firms have discontinued their typing; other firms only continue to type as a check on pedigree records (Baker, 1968). A check on the accuracy of pedigrees is a useful asset for which protein polymorphisms are idealy suited (Baker andManw'ell,.l962, 1967). Dried Egg-White Dehydration is a successful way of preserving eggs. Research has played a major role in solving problems which involve stability, functional properties and quality of dried egg products. Dried eggs have the following advantages: 70 (a) They can be stored at low cost under dry storage or refrigeration with reduced space requirement. (b) Transportation cost is low because water has been removed. (c) They are easy and clean to use. (d) They can be used in, and are necessary for, many new convenience foods. Eggs can be divided into two basic categories when considering their drying characteristics: (a) Egg-white products (b) Whole egg and yolk products. Egg-white products are virtually fat free, while whole egg and yolk products contain the highly emulsified lipids which are closely associated with the proteins and other components of the yolk. Almost all dried-egg products are uncooked. For eggs to be useful, the native characteristics of the raw egg must be preserved (Bergquist, 1973). The density of egg products is not affected by dehydration. Since water is an integral part of the protein molecule, its removal may cause certain changes to occur in the properties of egg-white. Egg-white is pro-treated in several different ways before drying. The primary purpose of pre-treatment is to remove the glucose. Acids are added to egg-white primarily for-the purpose of pH adjustment. Various methods of treating egg-white before drying have been reported (Epstein, 71 1933; Balls and Swenson, 1936; Littlefield, 1938; Mulvany, 1941). Some of these methods involve the use of acids and/or enzymes. Whipping aids are added to egg-white products to give them more uniformity and to compensate for any changes that might occur during processing and drying. The most common whipping aids used commercially and approved by FDA include sodium lauryl sulfate (Mink, 1939), triethyl citrate (Kothe, 1953), sodium desoxycholate (Kline and Singleton, 1959), and triacetin (Maturi 33. 31., 1960). These additives are used at about 0.1% base on egg-white solids and depending on type of additive. There are also other additives which have been reported to improve whipping properties, including acetyle methyl carbinol (Jensen and Hale, 1954), polyphos- phates (mecane and Mitchell, 1954), salts of :‘capri‘c' acid (Silliker 33. 31., 1959), and calcium stearyl-Z lactylate (Carmen and Keith, 1960). Injections of Egg Albumen to Animals Very few articles relating to the subject of injecting animals with egg albumen can be found. Most of the animals used in the literature cited, were laboratory animals. When animals are injected with egg albumen, large absorption droplets form in proximal tubule cells of the Imunmaliam kidney which contain mitochondrial material and ingested albumen (Oliver, 1948; Oliver 33. 31., 1954; Rhodin, 72 1954). The NADH-D activity of the mitichondria that form the bulk of the droplets is rapidly lost and the number of mito- chondria decreases simultaneously with droplet formation (Pugh, 1972). The large lysozymes gradually disappear and the affected cells can renew their mitochondria within a few days. The normal pattern of mitochondria distribution is. soon restored in proximal kidney tubules after damage caused by administration of egg albumen. This restoration which is facilitated by the ability of kidney cells to elaborate mitochondria from other cell organelles in addition to the division of pre-existing mitochondria was reported by Pugh (1972). The rats were injected intraperitoneally with 100 mgegg albumen in 1 ml of saline. Coe (1971) found that when hamsters were inoculated with soluble hen egg albumen (BEA) synthesis of antibody to HEA was restricted to one of the FS immunoglobulins (FSy- globulin). This selective induction occurred regardless of the amount of the BEA-saline injection (.001-5.0 mg) or the route of inoculation. In contrast, HEA in Freund's adjuvants induced synthesis of anti-REA in both the 78y1 and 7Sy2- globulins even when as little as 0.1 ug of HEA was used. Repeated inoculations of hamsters with BEA-saline increased or maintained 78y1 antibody but did not induce HEA synthesis in the 7Sy2-globulins. IV. EXPERIMENTAL PROCEDURES A. Growing Swine General A total of 66 pigs were used in three trials. The treated groups were injected subcutaneously with egg albumen weekly for four weeks and compared to control groups injected with .85% physiological saline solution. In Trial III, one group was fed 1% egg-white powder in the basal ration and compared to control groups not injected with physiological saline solution. The pigs were maintained at MSU Swine Research and Teaching Center in pens with slotted concrete floors and had free access to water and feed at all times. Feed and growth data were collected at weekly intervals. Trial I was started on September 29, 1973, and completed on October 29, 1973. Trial II was started on April 1, 1974, and ended on April 29, 1974. Trial III was started on August 5, l97h, and completed on September 9, 1974. Trial I Sixteen pigs were randomly allotted into two lots of eigfifl:each with sex and weight balanced. After lotting them, one barrow died from injuries sustained on the second day due to fighting. This lot served as control. The average 73 74 weight of the fifteen pigs was 37.27 Kg. (82 lbs). The eight pigs in the treated group were injected subcutaneously with the following volume of egg albumen: week 1 - 15 ml; week 2 - 20 ml; week 3 - 25 ml; and week 4 - 30 ml. The control group was injected with the same volume of .85% physiological saline solution. There was no noticeable side effects due to introduction of foreign protein. The feed used in this trial was "MSU 16 Percent Ration" the composition of which is shown in Table 17. TABLE 1 COMPOSITION OF RATION - TRIAL I Ingredients % Corn 79.0 Soybean Meal (49%) 18.0 Dicalcium Phosphate 1.0 Limestone 1.0 Salt .5 m Premix (MSU)a ____,_5_ Total 100.0 8Composition of MSU vitamin and trace mineral premix is Shown in Table 18. 75 Trial II Heavier pigs were used in this trial. Twelve pigs with an average of 79.9 K3 (17h lbs) were randomly divided by weight into two equal groups. One pig in the control group was found to have one lame hind leg and was removed from the trial. The volume of egg albumen and saline solution were given the same as in Trial I- MSU 12 Percent Ration was fed free choice throughout the trial. The sex was disregarded in this trial. TABLE 18 MSU VITAMIN AND TRACE MINERAL PREMIX Amount in 4.55 KB Ingredients (10 lbs.) of premix Vitamin A, million 3.0 1.0. Vitamin D2, million 0.6 1.0. Vitamin E, thousand 10.0 1.0. Riboflavin 3.0 gm Nicotinic Acid 16.0 gm JD-pantothenic Acid 12.0 gm Choline Chloride 100.0 gm Vitamin B12 18.0 mg Zinc 68.0 gm Manganese 34.0 gm 76 TABLE 18 (Con't.) Amount in 4.55 KB (10 lbs.) of premix Iodine 2.5 gm Copper 9.0 gm Iron 54.0 gm Antioxidant (Ethoxyquin) 45.0 gm Carrier (Ground Yellow Corn) to bring total to 4.55 Kg The compostiion of ration is shown in Table 19. TABLE 12 COMPOSITION OF THE RATION IN TRIAL II Ingredients: % Corn 89.2 Soybean Meal (49) 8.0 .Dicalcium Phosphate 1.0 Limestone .8 Stilt .5 FESU VTM Premix (see Table 18) .5 Total 100.0 77 Trial III Forty pigs with an average weight of 68.3 Kg (150.25 lbs.) were randomly allotted into four equal lots with weight balanced. They were treated as outlined in Table 20. TABLE 20 OUTLINE OF EXPERIMENT IN TRIAL III Lot No. Sex No. of Pigs Treatments 1 6F, 4M 10 Basal feed 10% protein 2 BF, 7M 10 Basal feed (10% protein) plus 1% egg-white powder 3 6F, 4M 10 Basal feed (10% protein) plus egg albumen injection weekly 4 4F, 6M 10 Finisher feed 13% In this trial only Lot No. 3 pigs were injected with egg albumen subcutaneously. Data' on weekly feed consumption and weight gain were collected as in the other two trials. 131a levels of albumen used in this trial were much higher 78 than in the first two trials. Injections of weekly dosages of 40 ml, 50 ml, 55 m1, and 55 ml, respectively, were made. On the third week of injection it was found that too high a dosage of egg albumen injected subcutaneously behind the ear caused some stress and irritation at the site of the injection, therefore, the dosages for the next two weeks were kept at 55 ml per week. The composition of the rations used is shown in Table 21. At the end of the fifth week, blood samples from five pigs of each lot were taken. The blood samples were obtained from the anterior vena cave of all animals by the technique described by Carla and Dewhirst (1942). Blood samples were taken again from the same five pigs in Lot 3, 24 hours after the injection of egg albumen. Fourteen ml of blood was obtained from each pig. An additional 2 m1 of blood was placed in a heparinized vial to be used for hematocrit and hemoglobin determinations. All tubes for containing the blood samples were tightly corked except when they were being sampled. Serum samples were "rimmed" in the tube before putting them in the centrifuge. Separation of serum from cells was accomplished with the use of an international centrifuge, size 2, Model V, at 2000 x g for 25 minutes. Serum was removed, placed in vials and chilled 79 TABLE 21 COMPOSITION OF RATIONS IN TRIAL III Ingredients Basal 10% (Kg) Basal 10% + Finisher Egg-White (Kg) Ration 13% (%) (%) (%) Egg-White Powder 0 1.0 0 Corn #2 91.5 90.5 85.0 Soybean Meal (49%) 5.0 5.0 11.5 Dicalcium Phosphate 1.0 1.0 1.0 Limestone 1.0 1.0 1.0 Salt .5 .5 .5 MSU VTM Premix .5 .5 .5 VIT E-Se Premix .5 .5 .5 ' Total . 100 100 100 80 in a cold room at 4°C for determination of ammonia N, urea N, and agarbgel electrOphoresis. Later the serum was frozen for use in total serum protein determination. Determinations of hematocrit and hemoglobin were made within 2 to 3 hours after the samples were collected. Hematocrit values were determined by the procedure outlined by MbGovern £3. 31. (1955). Hemoglobin was determ- ined by the cyanmethemoglobin method described by Crosby 23. ‘21. (1954). Conway's (1957) micro diffusion technique was used for serum urea N and serum ammonia N. Total serum protein was determined according to the Lawry's method mod- ified and described by Waddel (1956). The serum protein fractions were separated on a spinco, Mbdel R, agar-gel electrophoresis system. The technique used by Cawley and Eberhardt (1962) was followed very closely. The relative intensities of the separated proteins were determined by scanning with a Spinco Model RB Analytrol equipped with two 500 millimicron filters and a B-5 cam. The rations in Trial III were analyzed for their energy values by using oxygen bomb calorimetry. The Kjeldahl‘tech-e nique was used to determine the nitrogen content of the feed. Table 22 shows the protein and energy values. 81 TABLE 22 PROTEIN AND ENERGY ANALYSIS OF FEED Lot 1 Lot 2 Lot 3 Lot 4 Basal Ration Basal Ration Basal + 1% Egg-White Basal + 1% Egg-White Basal Ration Basal Ration Finisher Ration Finisher Ration Energy Protein 3890.0 cal/g 10.8% 3916.9"ca1/g 11.3% .3973 .o cal/'3 11.9% 3913.67 cal/g ‘ 12.0% 3885.1” cal/g. 12.3% 3872 ..9 cal/g 12.4% 3860.4 cal/g, 12.6% 3889.47 cal/g 13.1% pigs of each lot. and Discussion." Carcass evaluation was also made and it was from five slaughter was 91.76 kilograms. The results are shown under "Results The average weight of pigs at the time of 82 B. Chickens General Three trials were conducted and each trial was composed of different types of chickens and at a different time and place. Trial I was started on May 5, 1973. and completed on May 29, 1973, with Barred Plymouth Rock. Trial 11 was started on July 27, 1973, and completed on August 24, 1973. with SCWL. Barred Plymouth Rocks were used in the same trial which was started on July 30, 1973, and completed on August 27, 1973. Trial III was started on October 29, 1974, and completed on April 2, 1975. Trial I Twelve Barred Plymouth Rock.fema1es, 7 weeks old, were used in this trial. They were divided into two lots of six birds each. One was used as control and the others were treated with egg albumen. Three m1 of chicken egg albumen were injected subcutaneously at weekly intervals for 4 weeks and the growth data were collected. These birds were kept in battery cages at one bird ‘per cage in the cage room in the basement of Anthony Hall. Commercial grower mash was used. 83 Trial II Forty SCWL chicks, three-weeks old, and 40 Barred Plymouth Rock chicks, three-weeks old, were used in this trial. They were sexed and then divided into eight lots as outlined in Table 23. TABLE 22 OUTLINE OF AN EXPERIMENT FOR TRIAL II Type of Chicks Control Treated SCWL 0" 10 10 SCWL 3 10 lo Barred Plymouth Rock 0" 9 9 Barred Plymouth Rock 4’. _2_l_1_ }_1_._ Total 40 4O All the chicks were fed with regular starter ration and were weighed at weekly intervals. The treated group ‘was injected subcutaneously with chicken egg albumen at 3% m1, 1 ml, 15 m1, and 2 ml each week, respectively, for 84 four weeks. Growth data were collected weekly. The birds were kept in a brooder battery in the cage room in the basement of Anthony Hall. Feed consumption data were not taken. Trial III Initially, 80 pullets were housed in House No. 5. pen A at the MSU Poultry Science Research and Teaching Center. Half of the pullets were hatched on April 23, 1974, and the other half on May 7, 1974. The birds were individ- ually kept in battery cages. The birds were observed for two weeks and then they were allotted into lots A, B, C, and D with ten birds per lot. The age of the chicks in each group was equally balanced, i.e., five pullets hatched on April 23, 1974, and five pullets hatched on May 7, 1974, were put in each lot. Selection was also made so that the egg production of the four lots was about the same based on egg production obtained during the observation period. The rest of the birds were assigned to lots in E, F, G, and H. They were without age balance and were used for observation. The outline of the experiment is shown in Table 24. 85 TABLE 24 OUTLINE OF EXPERIMENT IN TRIAL III (a) Lots Number of Birds Treatments A 10 Control, no treatments B 10 Injected 4 m1 chicken albumen C 10 Injected 4 ml quail albumen D 10 Injected 4 m1 Ringer's solution The treatments were started on November'l3, 1975. The birds were injected with 4 m1 of chicken albumen, quail albu- men, or Ringer's solution each week for five weeks. Individ- ual production data were collected during the period of treatment. On February 18, 1975, lots E, F, G, and H; were treated as outlined in Table 25, because lot C hens treated with quail albumenwere showing some positive results. After one week pre—treatment observation the birds ‘were injected subcutaneously with 4 ml of quail egg albumen per week for five weeks. Individual egg production data were collected. 86 TABLE 25 OUTLINE OF REPLICATIONS OF QUAIL ALBUMEN TREATMENTS (Trial IIIb) Lots Number of Birds Treatments E 9 Injected 4 m1 quail albumen F 10 Control, no treatment G 9 Injected 4 ml quail albumen H 10 Control, no treatment V. RESULTS AND DISCUSSION A. Growing Swine Trial I A summary of the data obtained in this trial is presented in Table 26 and AppendixTables 40 and 41. Pigs in each treatment were group-fed; therefore, a statistical analysis could not be made on feed consumption and feed efficiency data. The rate of growth was approximately the same for the treated and control groups as can be seen in Table 26. As mentioned in the introduction, the author had made some observations in Malaysia and there was a significant increase in growth rate of pigs injected with egg albumen as compared to that of controls. In comparing this trial with the trial in Malaysia it could possibly have been due to any or all of the following: MSU Malaysia Breed: l. Undoubtedly the pigs were Poor breed of a superior breed Age: 2. Used younger pigs (2% Used older pigs (5} months) months) 87 TABLE 26 SUMMARY OF DATA IN TRIAL I Number of Pigs Average Initial Weight (Kg)a Average final weight (Kg)b Average Daily Gain (Kg) Average Daily Feed (Kg) Feed/Gain (Kg) Feed Cost/Kg - Gain (12.l¢/Kg) Gain in weight/Pig (Kg) Feed Cost During Period Extra Labor Charge for Treatment Cost of Feed and Treatment Control Treated 7 8 37.41 37.22 58.18 57.33 0.74 0.72 1.78 1.74 2.40 2.42 29.04 29.28 20.77 20.11 3 6.03 S 5.89 - _1_..29. S 6.03 3 6.89 aStandard errOr for control 0.96 and treated 0:89. b Standard error for control 1:32 and treated 1.29. MSU Malaysia Diet: 3. High quality balanced diet Poor quality diet 4. Dry and free choice feed Slop-feeding twice a day Housing: 5. Controlled.environmenta1 Pigs on concrete floor housing with low walls and a roof Treatment: 6. Without bathing Bathing the pigs twice a day to keep them cool From this comparison it is obvious that an important factor has been overlooked-—the age of pigs. The author recalled that he had tried the injection of egg albumen on younger pigs in Malaysia but with no response. A second trial was proposed and was approved. Trial 11 In Table 27 and AppendixTablesi42 and 43 are shown a summary of the data obtained in Trial II. No statistical analysis could be made on feed consumption and feed efficiency data because pigs in each treatment were group fed. Weight gain between the mo groupsw'as about the same. There was a fairly large difference in the feed efficiency which, however, ' could not be tested Statistically. The treated group required only 3.3 Kg of feed per kilogram of body weight gain, whereas the control group required 4.1 Kg of feed. There was TABLE 22 SUMMARY OF DATA IN TRIAL II Control Treated Number of Pigs 5 6 Average Initial Weight (Kg)a 79.46? 79.09‘ Average Final Weight (Kg)b 98.64- 98.185 Average Daily Gain (Kg) 0.6947 0.69- Average Daily Feed 2.81 2.26 Feed/Gain (Kg) 4.10 3.30. .Feed Cost/Kg Gain (11.66¢/Kg) 47.81 38.48 Gain in‘Weight/Pig 19.18 19.09 Feed Cost/Pig During Period 8 9.17 8 7.35 Extra Labor Charge for Treatment/Pig - __;;QQ Cost of Feed and Treatment/Pig S 9.17 3 8.35 aS.E. for control = 2.69 treated = 2.32 bS.E. for control = 3.12 treated 2.70 91 considerable saving of feed during the period of observation. Even though extra labor was charged for handling and treat- ment of pigs, 8 savings of 82 cents could be realized on the cost of feeding each pig (see Table 27). The authoris hypothesis is that pigs eat more than the body can absorb when feed is given free choice and the excess food that is not digested is excreted and wasted. Egg albumen depresses the appetite and thus controls the food intake. MSU pigs had. reached their fullest potential for weight gain and 'egg albumen treatment did not stimulate any higher rate of growth. Malaysian pigs were given poorer types of feed and rate of grow'thw'as below its own potential, thus egg albumen helped to stimulate growth. The author did not keep records on feed intake in Malaysia because the treated and the control pigs were kept in the same pen. Carcass evaluation was also taken but it was found that there was no significant difference in the dressing-out percentage or the percent of loin between the two lots. Trial III Table 28 and Appendix Tables 44, 45, 46 and 47 show the summary of performance data of Trial III. A higher level of egg albumen injection (40 m1 and above) was found to have some harmful effects though not very obvious. Two pigs were found to have soft stools and.one was vomiting during the time of weighing. Two pigs developed abscesses at the sites 92 of injection. This may be due to injecting the high dosage of egg albumen at one time on the same spot subcutaneously or due to contamination which causes the abscesses. The abscesses were healed within a week but during that period there was no noticeable gain in weight. Although Lot No. 1. which was on basal diet showed a better gain in weight over 0 Lot No..3 which was treated with egg albumen injection, statistically there was no significant difference. In comparing Lot No. 3 with Lot No. 4 which was fed with 13% finisher ration, gain in weight was significantly (P4 0.01) greater in Lot No. 4. Also, in Lot No. 4 body weight gain was significantly (P<0.05) greater than Lot No. 1, Since Lot No. 4 was the positive control, this result was expected. The failure to improve weight and feed efficiency by injecting might be due to the harmful effect of too high a dosage. Lot No. 4 had the best utilization of feed gain among the four lots. Lot No. l was the poorest feed converter. Lots No. 2 and No. 3 had the same feed efficiency. The poor performance of pigs injected with egg albumen might be due to some low level of toxicity such as that described by Harper 22. 31. (1955), Boyd gt. 31. (1966), Peters (1967), and Pugh (1972). The injected pigs had depressed appetites as can be seen in Table 28. The average daily feed intake was the lowest of all in Lot 3. Vomiting and loose stools may also indicate some toxicity. 93 TABLE 28 SUMMARY OF PERFORMANCE DATA IN TRIAL III Lots Treatments Number of Pigs Average initial weight (Kg) Average final weight (Kg) Average daily gain (Kg) Average daily feed (Kg) Feed/gain (Kg) Feed cost/Kg gain (¢) Gain in weight/pig (Kg) 1 Basal Feed 10% 10 ' 68.14: 86.45: 0.53* 2.34 2.00 27.8 18.27“ 2 Basal Feed + 1% egg white 10 69.321 88.41: 0.59- 2.45 1.89 33.26 20.32 3 Basal Feed + egg al- bumen injec- tion 10 68.23: 84.18: o.51** 2.12 1.89 26.27 17.as~~ h Finisher Feed 13% 10 67.591 91.683 0.679 2.44 1.66 23.74 24.09 *Significant difference (P<0.05) as compared to lot 4. ”‘Highly significant difference (P<0.0l) as compared to lot 4. 94 The average values for hematocrit, hemoglobin, serum protein, urea N, ammonia N, electrophoretic pattern of growing pigs are shown in Table 29. ‘ Analysis of hematocrit and hemoglobin data indicated no significant differences among treatments. Total serum protein was not significantly different between lots. There was a slight increase of .20 gm/lOO ml of total serum protein level in the pigs 24 hours after injection of egg albumen. The total serum protein level for pigs in Lot 1 and Lot 3, seven days after albumen injection, was the same (7.41 gm/lOO m1). This could also indicate that the effect of egg albumen was nil on the 7th day. Lot 4 (13% finisher) had the highest total serum protein level (7.87 gm/lOO ml) and Lot 2 (Basal + 1% egg-white was second (7.60 gm/lOO m1). Duncan's multiple—range test was used and no statis- tically significant difference was found between treatments in the plasma ammonia N, plasma urea N, and in the electro- phoretic patterns of the plasma proteins. Table 30 shows a summary of the carcass evaluation. Although there is no statistically significant difference among the groups it is interesting to note that Lot 2 and Lot 3, both of which had been treated with egg-white, were 1.5% higher than Lot 1 in dressing-out percent and 1.1-1.6% higher in the percent of ham and loin. 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