Role of the Shell Gland in Determination of Albumen Quality

Role of the Shell Gland in Determination of Albumen Quality R. E . AUSTIC

Department of Poultry Science, Cornell University, Ithaca, New York 14853 (Received for publication June 9, 1976)

POULTRY SCIENCE 56: 202-210, 1977

A

LBUMEN firmness is an important trait affecting the market value of eggs. However, little is known about the biochemical basis for albumen structure and much less about the basis for alteration in structure as affected by heredity, aging, nutrition or environment. The gel structure of albumen appears to be related to the ovomucin content of eggs. This relationship was originally reported by Almquist and Lorenz (1932), and has been observed by several other investigators (McNally, 1933; Balls and Hoover, 1940; Brooks and Hale, 1961; Skala and Swanson, 1962; Kato et al., 1970a; Baliga et al., 1971; Robinson and Monsey, 1971). Thick albumen contains the highest amount of ovomucin, the concentration of which declines in association with the thinning of egg white during storage (Conrad and Scott, 1939; Brooks and Hale, 1961; Kato et al., 1970b; Baliga et al, 1970; Donovan et al., 1972; Robinson and Monsey, 1972; Sauveur, 1973a). Skala and Swanson (1962) observed that fresh eggs which differed

in albumen firmness also differed in ovomucin content. Those which had the thickest albumen contained the most ovomucin. Although the important role of ovomucin in the gel structure of egg white appears well established, the processes by which gel firmness becomes differentiated during egg formation have not been investigated. This study was conducted to determine the relative contributions of shell gland and more proximal portions of the oviduct to variations in the ovomucin content of eggs. METHODS Hens from unselected populations, fed the Cornel] breeder diet, were identified on the basis of Haugh unit score (Haugh, 1937) for use in each experiment. Eggs were collected daily over a period of several successive days, held overnight at 55° C. and 65% relative humidity, and broken the day following collection for determination of albumen height. In each experiment several hens were identi202

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ABSTRACT Experiments were conducted to determine the crude ovomucin contents of eggs at various stages of egg formation. The total ovomucin content was greatest in eggs obtained from the isthmus four and one-half hours after oviposition of the previous egg. It was unchanged at six hours, after the egg had entered the shell gland, but declined precipitously during the first few hours in the shell gland, remaining relatively constant thereafter until oviposition. The rapid decline in ovomucin content of eggs in shell gland corresponded to the period of rapid egg white plumping. Fresh eggs which differed in albumen quality also differed in crude ovomucin content. Hens which were selected for differences in albumen quality of fresh eggs did not differ in the total ovomucin content of eggs obtained from the isthmus. However, the differences in ovomucin content associated with egg quality were apparent in eggs obtained from the shell gland within eight hours after oviposition of the previous egg. Hens fed a diet supplemented with 2% ammonium chloride in an attempt to increase albumen quality also did not differ in ovomucin content of isthmal eggs. Ovomucin was markedly increased in eggs of this group after oviposition; however, variations in ovomucin content among hens selected for differences in egg quality were not due to differences in the dilution of egg white by plumping fluid in the shell gland. High and low quality groups contained comparable amounts of albumen in eggs at all stages of formation. Thus, variations in albumen quality among hens within a strain become differentiated during the first few hours in the shell gland and are probably related to chemical interactions between plumping fluid and preformed albumen.

OVOMUCIN AND ALBUMEN QUALITY

TABLE 1.—Basal diet Percent Ingredients of diet Ground yellow corn 64.20 Soybean meal (50% protein) 17.00 Vegetable oil 2.50 Corn distillers dried solubles 2.50 Alfalfa meal 2.50 Limestone 8.20 Dicalcium phosphate 2.30 Vitamin-mineral premix' 0.50 Salt, iodized 0.25 DL-Methionine 0.05 'Provided 2200 I.U. vitamin A palmitate, 1100 I.U. vitamin D 3 , 2.2 mg. menadione sodium bisulfite, 3.3 mg. riboflavin, 110 mg. ZnO and 220 mg. MnS04 • H2 O per kg. of diet.

1. High quality refers to eggs with firm albumen gels and low quality refers to eggs having weaker gel structure as indicated by the height of the thick albumen in the broken-out egg. 2. A commercial preparation of pentobarbital and chloral hydrate. Jensen-Salisbury Laboratories, Kansas City, MO 64141.

oviduct, they were transferred to a plate glass surface where the shell membranes were removed and the albumen plus chalazae were separated from the yolk using blunt curved forceps. Albumen, including chalazae, was scraped off the plate into sample jars, sealed and stored at 0° C. Albumen from oviposited eggs was collected in a similar manner. A modification of the technique of Brooks and Hale (1961) was used for preparation of ovomucin. Albumen was blended using a teflon Potter-Elvehjem homogenizer, and one gramaliquots were transferred to test tubes. Four milliliters of water were added and the samples were mixed, causing the crude ovomucin to precipitate. The samples were centrifuged and the water fraction discarded. The crude ovomucin precipitate was washed three times with 2% K G solution and then three times with water. The residue was dissolved in 0.5N NaOH and analyzed for protein content according to the method of Lowry et al. (1951). Sialic acid was assayed in ovomucin preparations according to the method of Svennerholm (1957). The ovomucin content of eggs during the course of egg formation was investigated in the second experiment. Pullets which produced eggs averaging 78-85 Haugh units over a 5-day period immediately preceding the experiment were used. On the day of experiment, the time of oviposition was noted for each hen. Eggs were obtained at oviposition: from the isthmus 4 hours and from the shell gland 6, 8, 11 and 15 hours after oviposition. Albumen was collected from three hens per time period as described previously. Albumen samples were held on ice overnight and analyzed for crude ovomucin content the following day. The total sample of each egg was transferred to 250 ml. glass centrifuge bottles, diluted four-fold (w./v.) with water and stirred periodically during a 20-30 minute interval using a stainless steel spatula. After centrifugation, the supernatant was removed by aspiration and discarded.

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fied as consistent producers of the highest and lowest, or intermediate quality eggs,' within the population. The first experiment was conducted to determine the ovomucin content of eggs from two groups of hens which produced eggs differing in albumen firmness (quality). These hens were selected from a single strain based on the quality of eggs produced over a five day period immediately prior to experiment. The higher quality group and one-half of the lower quality group were fed a practical laying diet (Table 1). The remaining hens in the lower quality group were fed the same diet supplemented with 2% NH 4 C1 in an attempt to increase albumen quality (Hall and Helbacka, 1959; Hunt, 1964). The diets were fed for one month, after which isthmal and oviposited eggs were obtained for analysis. Hens were killed four and one-half hours after oviposition by intracardial injection of 7 ml. equithesan. 2 After eggs were removed from the

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RESULTS Preliminary experiments to test the correlation between crude ovomucin content of egg albumen and albumen quality indicated a direct relationship between the two parameters. A typical example is shown in Table 2. Although the range in albumen quality as measured in Haugh units was relatively narrow, significant differences in crude ovomucin were observed. The ovomucin content of isthmal and oviposited eggs from the first experiment is shown in Table 3. Fresh eggs from the high and low quality groups averaged 85 and 75 Haugh units with the low quality group fed NH 4 C1 similar in quality to the high quality

TABLE 2.—Ovomucin content of fresh eggs

Haugh units

Number of eggs analyzed

Ovomucin M-g-/galbumen 71 ± 1 5 943 ± 115a1 78 ± 1 5 1287 ± 200aa 85 ± 1 6 2155 ± 272bb 1 Mean ± S.E. Means with same superscript are not significantly different (P > .05.)

group. Despite the differences in albumen quality, no differences in concentration of crude ovomucin of isthmal eggs were observed. The crude ovomucin content of oviposited eggs was similar for the high and low quality unsupplemented groups but markedly higher in the eggs from hens fed the NH 4 C1 supplemented diets. Albumen samples from this experiment were stored at -20° C. and analyzed later for sialic acid content of ovomucin (Table 4). Less ovomucin was obtained in this analysis but the relative amounts among treatments were similar to those of the earlier assay. The sialic acid of isthmal ovomucin was determined. The mean value for all experimental groups was 3.6% of ovomucin, a concentration similar to the value reported by Donovan et al. (1970) for highly purified ovomucin from oviposited eggs. Since the ovomucin content of isthmal and oviposited eggs differed markedly, the second experiment was conducted to determine the ovomucin content of eggs intercepted at various times during the course of egg formation. Ovomucin content was highest in eggs obtained from the isthmus (4 hr.), slightly lower at 6 hours (shell gland) and reduced markedly by 8 hours post-oviposition, remaining constant thereafter (Fig. 1). Albumen weight was lowest at 4 hours. At 6 hours it had increased slightly but not significantly. By 8 hours, however, it had increased 64% and by 10 hours had nearly plateaued at twice the original amount. The pronounced reduction in ovomucin content between 6 and 8 hours coincided with the most rapid increase in albumen volume. Six experiments were conducted to determine the ovomucin content of oviducal eggs from hens which laid "high" or " l o w " quality eggs. Five hens were utilized for each sampling period. One such experiment is shown in Figure 2. No numerical difference in ovomucin content of isthmal eggs was observed. Although the high and low quality

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The insoluble residue was washed three times with 150 ml. of 2% KC1 followed by a double wash with 150 ml. water and dissolved in 0.5N NaOH for protein analysis according to the method of Lowry et al. (1951). Several subsequent experiments were performed to determine the relative importance of the shell gland and proximal portion of the oviduct in differentiating albumen quality among hens. In six experiments hens were identified as "high" or " l o w " quality by screening for albumen thickness for several days prior to experiment. Albumen was analyzed for ovomucin as described for the previous experiment, except that ovomucin was determined gravimetrically in three experiments.

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OVOMUCIN AND ALBUMEN QUALITY

groups appeared to differ in ovomucin content of oviposited and shell gland (8 hr.) eggs, the variability was sufficiently high that the differences were not statistically significant (P > .05). When the data from the six experiments, all similar in design, were pooled for statistical analysis, the differences in ovomucin content between high and low quality groups were significant (P < .01) for oviposited eggs and eggs from the shell gland but not significant (P > .05) for eggs obtained from the isthmus. The data are depicted graphically in Figure 3. The total ovomucin recovery was higher on the average than for the previous experiments. This probably can be attributed to less successful purification of ovomucin in three of the six experiments. The reason for variation between experiments is not known. The analytical methods were similar for all experiments; however, the study spanned a period of three years during which successive generations of the same strain of hens were used.

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The mean albumen contents of eggs from the latter experiments are shown in Table 5. Despite the significant differences in ovomucin contents of eggs from the two quality groups (Fig. 3), no differences in albumen weight were observed at any stage of egg formation. The pH of albumen was determined in one experiment. The mean values for five samples at 4.5, 8 hours and at oviposition were 7.0 ± 0 . 1 , 7.0 ± 0.1 and 8.2 ± 0.1, respectively for the high quality group and 7.1 ± 0.2, 7.0 ± 0.2 and 8.5 ± 0.1 for the low quality group. The two groups appeared to differ only at oviposition (P < .05).

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DISCUSSION The gel structure of egg white is unknown. However, the studies of Kato et al. (1970a) and Robinson and Monsey (1971), Sleigh et al. (1973) and Smith et al. (1974) indicate

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TABLE 4.—Sialic acid content of ovomucin in eggs obtained from the isthmus Quality group'

Diet

Sialic acid

Ovomucin

H*-/galbumen HQ (8) 2810 ± 1302 LQ (6) 2750 ± 180 LQ (8) 3070 ± 90

Hours post-oviposition 2

Quality group

mg./g. ovomucin 36.7 ± 2.1 36.2 ± 2.9 35.6 ± 1.3

HQ LQ

4.5

8.0

Oviposited

g. albumen/egg 17.2 ± 0.4 3 28.4 ± 0.7 3 33.9 ± 0.6 3 17.9 ± 0.6 28.0 ± 0.8 34.5 ± 0.6

1 2

"HQ, high quality; LQ, low quality. Hens selected for each group during pre-experimental screening period. 2 Mean ± S.E.

HQ, high quality; LQ, low quality. Eggs obtained from the isthmus 4.5 hours and from the shell gland at 8 hours after oviposition of the previous egg. 'Mean ± S.E. for 29 eggs at 4.5 hours and at the time of oviposition, and for 14 eggs at 8 hours.

that ovomucin is a heterogeneous protein composed of two or more fractions which vary greatly in composition. One fraction contains approximately 58% carbohydrate

(w./w.) whereas another major fraction contains only about 15% (w./w.) carbohydrate (Robinson and Monsey, 1971). The former is particularly rich in sialic acid. Egg white

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FIG. 1. Changes in ovomucin content of eggs during egg formation. Ovomucin, thick line; albumen weight, thin line. Each point indicates mean for 3 samples. Vertical lines indicate standard error. Pooled standard error for albumen weight was 0.9 g. Four-hour samples obtained from isthmus; all others from shell gland except for oviposited (ovip.) eggs.

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Basal Basal + NH 4 C1

TABLE 5.—Albumen contents of eggs from hens differing in albumen quality

OVOMUCIN AND ALBUMEN QUALITY

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Time After Oviposition

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FIG. 2. Ovomucin content of eggs from hens laying "high" (open circles) or "low" (closed circles) quality eggs. Each point represents mean of four samples. Vertical lines indicate standard errors. Four and one-half and 8 hour samples obtained from isthmus and shell gland, respectively. Ovip. = oviposited.

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FIG. 3. Ovomucin content of eggs from hens laying "high" (open circles) or "low" (closed circles) quality eggs. Six experiments pooled. Each point at 4.5 hr. and at oviposition represents mean for 29 eggs. Each point at 8 hr. represents mean for 14 eggs. Vertical lines indicate standard errors. Four and one-half and 8 hour samples obtained from isthmus and shell gland, respectively. Ovip. = oviposited.

structure appears to be provided by a complex of ovomucin subunits, held together by disulfide bridges and in electrostatic association with lysozyme (Cotterill and Winter, 1955; Brooks and Hale, 1961; Kato et al., 1971, 1975). Thinning of egg white during storage appears to be due to the dissociation of the complex with loss of the carbohydrate-rich fraction of ovomucin to the soluble form (Kato et al., 1970b, 1972, 1975; Robinson and Monsey, 1972). It is difficult to obtain ovomucin of high purity. Brooks and Hale (1961) diluted egg white with four volumes of water with stirring, centrifuged the sample and washed the precipitate with the four volumes of water until the wash was free of protein. This was followed by successive washes with 2% KC1 until the wash was free of protein. Successive washes with water removed KC1 from the final product. Although the ovomucin preparations had been subjected to repeated washings they were contaminated with as much as 13.5% lysozyme. Donovan et al. (1970) obtained higher purification using prior precipitation of lysozyme from egg white, followed by dilution of egg white with water. The crude ovomucin which precipitated as a result of dilution was washed extensively atcontrolled pH with .01M KC1-.002M EDTA solution and finally with four successive washings with .5M KC1 solution. The product was relatively free of other protein contaminants. Because of the relatively large numbers of samples to be analyzed, the extensive purification procedures of Donovan et al. (1970), Kato et al. (1970) or Robinson and Monsey (1972), although ideal, did not appear feasible for this study. A modification of the technique of Brooks and Hale (1961) was used in these experiments. The washing cycles were reduced to two each of water, 2% KC1 and water respectively in an attempt to standardize the treatment for all samples, and to minimize the physical loss of ovomucin which

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Most of the protein of egg white is secreted in the regions of the oviduct proximal to the shell gland (Scott et al., 1937), and histological evidence supports the view that ovomucin is not secreted in the isthmus or shell gland (Cole, 1938; Solomon, 1971). The present results indicate that the thinning of egg white which occurs as the egg enters the shell gland (Sturkie and Polin, 1954) is due not only to dilution of albumen with plumping fluid, but also to a decrease in total water precipitable ovomucin. It is possible that plumping fluid may influence the physical and chemical properties of ovomucin, leading to weakened gel structure which, in turn, is reflected in lower recovery of ovomucin by egg white dilution. The nature of the changes in ovomucin which result in lower recovery have not been investigated. It would be worthwhile to determine whether they are similar to those which occur in oviposited eggs during egg white thinning (Kato, 1970b, 1971; Robinson and Monsey, 1972).

The present study attempted to determine when, during the course of egg formation, albumen thickness of the fresh egg is determined. Hens which produce fresh eggs differing in albumen thickness did not differ in ovomucin content of eggs obtained from the isthmus (Table 3, Figs. 2,3). This was a consistent observation in all but one experiment, the mean ovomucin content in six experiments (Fig. 3) being slightly but not significantly higher (P > .05) for the hens which produced thinner albumen. The amount of ovomucin obtained from eggs from the shell gland and after oviposition, in contrast, was less for these hens in all experiments (P < .01). Variations in albumen thickness of fresh eggs, therefore, appear to be determined at least in part in the shell gland. This happens during the first few hours after the egg enters the shell gland (Figs. 1,2,3), and coincides with the secretion of plumping fluid. Since no difference in ovomucin content of isthmal eggs was apparent when NH 4 C1 was fed (Table 1), it also appears that this compound exerted its effect in the shell gland. A marked effect of NH 4 C1 on albumen thickness was reported by Hall and Helbacka (1959), Hunt (1964) and Helbacka et al. (1968). The latter investigators also observed increased crude ovomucin contents of eggs from hens fed diets supplemented with NH 4 C1. Future studies on the basic processes of albumen quality in fresh eggs should focus on the events surrounding the plumping process in the shell gland, particularly the interaction between plumping fluid and albumen secreted by more proximal portions of the oviduct. Several investigators have noted differences in composition of eggs differing in albumen thickness. Sauveur (1970) observed increased calcium, potassium and chloride and decreased pH and sodium content of albumen from hens fed NH 4 C1, and has reported strain differences in these pa-

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might occur with multiple washing steps. In general, the ovomucin concentrations observed are similar to those reported by other investigators. The ovomucin content of eggs decreased precipitously during the first few hours in the shell gland. Although this decline was marked by 8 hours after oviposition of the previous egg in some experiments (i.e., Fig. 1), in others the decline was apparent but not as great (Figs. 3,4). The similar ovomucin contents of eggs at 4.5 hours (early isthmus) and 6 hours (early shell gland) suggest that the isthmus does not contribute to the thinning of the albumen. The present data do not exclude the possibility that some inherent differences in magnum or isthmal secretions are expressed after the egg enters the shell gland. In view of the similarity of ovomucin content of isthmal eggs (Table 3) and the sialic acid content of ovomucin (Table 4), however, this seems unlikely.

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OVOMUCIN AND ALBUMEN QUALITY

Further studies concerning interaction of plumping fluid and isthmal albumen may provide a better understanding of the process of egg quality determination in the shell gland. Other studies are needed to determine whether the shell gland contributes to the changes in albumen thickness of fresh eggs

associated with age of the hen, housing temperature, or disease. REFERENCES Almquist, H. J., J. W. Givens and A. Klose, 1934. Transmission of light by egg albumen. Ind. Eng. Chem. 26: 847-848. Baliga, B. R., S. B. Kadkol and N. L. Lahiny, 1971. Thinning of thick albumen in shell eggs—changes in ovomucin. Poultry Sci. 50: 466-473. Balls, A. K., and S. R. Hoover, 1940. Behavior of ovomucin in the liquefaction of egg white. Ind. Eng. Chem. 32: 594-596. Brooks, J., and H. P. Hale, 1961. The mechanical properties of the thick white of the hen's egg. II. The relation between rigidity and composition. Biochim. Biophys. Acta, 46: 289-301. Cole, R. K., 1938. Histology of the oviduct of the fowl in relation to variations in the condition of the firm egg albumen. Anat. Rec. 71: 349-361. Conrad, R. M., and H. M. Scott, 1939. Changes in ovomucin during egg storage. Proc. 7th World's Poultry Congr., pp. 528-530. Cotterill, O. J., and A. R. Winter, 1955. Egg white lysozyme. 3. The effect of pH on the lysozymeovomucin interaction. Poultry Sci. 34: 679-686. Donovan, J. W., J. G. Davis and L. M. White, 1970. Chemical and physical characterization of ovomucin, a sulfated glycoprotein complex from chicken eggs. Biochim. Biophys. Acta, 207: 190-201. Donovan, J. W., J. G. Davis and M. B. Wiele, 1972. Viscosimetric studies of alkaline degradation of ovomucin. J. Agr. Food Chem. 20: 223-228. Draper, M. H., 1966. The accumulation of water and electrolytes in the egg of the hen. In: Physiology of the Domestic Fowl (Ed., C. Horton-Smith and E. C. Amoroso). Oliver and Boyd, Edinburgh, pp. 63-74. Hall, K. N., and N. V. Helbacka, 1959. Improving albumen quality. Poultry Sci. 38: 111-114. Haugh, R. R.. 1937. The Haugh unit for measuring egg quality. U.S. Egg Poultry Mag. 43: 552-555. Helbacka, N. V. L., C. T. Ragual and M. H. Taylor, 1968. Physical and chemical properties of eggs from hens fed NH4C1. Poultry Sci. 47: 904-911. Hunt, J. R., 1964. Electrolyte changes in albumen on feeding NH4C1. Poultry Sci. 43: 1331-1332. Kato,A.,T. Imotoand K. Yagishita, 1975. The binding groups inovomucin-lysozyme interaction. Agr. Biol. Chem. 39: 541-544. Kato, A., R. Nakamura and Y. Sato, 1970a. Studies on changes in stored shell eggs. Part V. The difference in the chemical and physiochemical proper-

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rameters (Sauveur, 1969). Draper (1966) observed higher calcium and lower sodium and potassium contents of albumen in one strain of chickens than in a second breed which produced thinner albumen. He also noted that within a strain the calcium content of oviposited eggs was lower and potassium higher than in eggs obtained from the isthmus. Whether the mineral content of plumping fluid may affect ovomucin from isthmal eggs is not clear. Robinson and Monsey (1972b) observed that small amounts of magnesium or sodium salts can influence the rate of albumen thinning. Sauveur (1973b) has demonstrated, using in vitro incubation of oviducal eggs with water or solutions of salts or glucose, that the height of albumen and viscosity was influenced by the solutions employed. An important factor affecting the properties of ovomucin is pH. A lower pH favors the maintenance of egg white gel structure. The decreased pH of eggs from hens fed NH 4 C1 may be expected to improve ovomucin recovery on this basis alone (Cotterill and Winter, 1955; Kato et al., 1975). Although pH did not differ significantly in oviducal eggs of high or low quality groups in one experiment where this paramater was measured in the present study, a slightly lower pH of oviposited eggs may have contributed to greater ovomucin recovery in the high quality group. The disulfide bridges between ovomucin subunits are susceptible to disruption by reducing agents (Robinson and Monsey, 1972; MacDonnell, 1951). Thus, variations in reducing compounds of plumping fluid also may cause variations in ovomucin content of eggs.

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Sauveur, B., 1970. Acidoses metaboliques experimentales chez la poule pondeuse. II. action sur la composition minerale de 1'albumen de l'oeuf. Ann. Biol. Anim. Bioch. Biophys. 10: 81-100. Sauveur, B., 1973a. Relation entre les acides sialiques de l'ovomucine et la hauteur du gel d'albumen de l'oeuf. J. Recherches Avicole: 311-315. Sauveur, B., 1973b. Reconstitution in vitro du determinisme des proprietes physiques de 1'albumen de l'oeuf. J. Recherches Avicoles: 317-321. Scott, H. M., J. S. Hughes and D. C. Warren, 1937. Augmentation of nitrogen to the egg white after formation of the shell membranes in the fowl. Poultry Sci. 16: 53-61. Skala, J. H., and M. H. Swanson, 1962. Studies of variation in initial quality of chicken eggs. 2. Chemical properties of the albumen. Poultry Sci. 41: 1537-1545. Sleigh, R. W., G. J. H. Melrose and M. B. Smith, 1973. Isolation and characterization of hen egg white ovomucin. Biochim. Biophys. Acta, 310: 453-460. Smith, M. B., T. M. Reynolds, C. P. Buckingham and J. F. Back, 1974. Studies on the carbohydrate of egg-white ovomucin. Aust. J. Biol. Sci. 27: 349-360. Solomon, S. E., 1971. The characterization and distribution of acidic mucopolysaccharides in the oviduct of the domestic fowl. Res. Vet. Sci. 12: 225-227. Sturkie, P. D., and D. Polin, 1954. Role of the magnum and uterus in the determination of albumen quality of laid eggs. Poultry Sci. 33: 9-17. Svennerholm, L., 1957. Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method. Biochim. Biophys. Acta, 24: 604-611.

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ties of ovomucin (B) between the thick and thin white. Agr. Biol. Chem. 34: 854-859. Kato. A., R. Nakamura and Y. Sato, 1970b. Studies on changes in stored shell eggs. Part VI. Changes in the chemical composition of ovomucin during storage. Agr. Biol. Chem. 34: 1009-1013. Kato, A., R. Nakamura and Y. Sato, 1971. Studies on changes in stored shell eggs. Part VII. Changes in the physiochemical properties of ovomucin solubilized by treatment with mercaptoethanol during storage. Agr. Biol. Chem. 35: 351-356. Kato, A., and Y. Sato, 1972. The release of carbohydrate rich component from ovomucin gel during storage. Agr. Biol. Chem. 36: 831-836. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the phenol reagent. J. Biol. Chem. 193: 265-275. MacDonnell, L. R., H. Lineweaver and R. E. Feeney, 1951. Chemistry of shell egg deterioration: effect of reducing agents. Poultry Sci. 30: 856-863. McNally, E., 1933. Relative amount of mucin in thick and thin egg white. Proc. Soc. Exp. Biol. Med. 30: 1254-1255. Robinson, D. S., and J. B. Monsey, 1971. Studies on the composition of egg-white ovomucin. Biochem. J. 121: 537-547. Robinson, D. S., and J. B. Monsey, 1972a. Changes in the composition of ovomucin during liquefaction of thick egg white. J. Sci. Fd. Agric. 23: 29-38. Robinson, D. S., and J. B. Monsey, 1972b. Changes in the composition of ovomucin during liquefaction of thick egg white: The effect of ionic strength and magnesium slats. J. Sci. Fd. Agric. 23: 893-904. Sauveur, B., 1969. Etude de la composition electrolytique des differentes zones de 1'albumen de l'oeuf chez deux races de poule. Ann. Biol. Anim. Bioch. Biophys. 9: 563-573.