THE RESULTS reported in Part 1

Transcription

1 Studies of Variation in Initial Quality of Chicken Eggs 1 2. CHEMICAL PROPERTIES OF THE ALBUMEN J. H. SKALA 2 AND MILO H. SWANSON Department of Poultry Husbandry, University of Minnesota, St. Paul 1, Minnesota THE RESULTS reported in Part 1 (Skala and Swanson, 1962) of this series do not preclude the possibility that the distribution, concentration, or environment of specific constituents of egg white may influence the broken-out appearance of the albumen through an apparent effect on amount or condition of the middle thick layer of white. Conrad and Scott (1942) compared relatively high and low quality eggs with regard to albumen ph and content of ovomucin, calcium, magnesium, potassium, and sodium. Ovomucin was determined by the method of Conrad and Phillips (1938) as well as by histological techniques. These workers concluded that observed variations in sodium and potassium contents related to quality (Haugh units) were too variable to be the determining factors. Differences in ph, ovomucin, calcium, and magnesium contents were not significant. The ovomucin and tryptophan content of the middle thick and combined thin white layers of high and low quality eggs was determined by Hill et al. (1949). Results indicated that "apparent" ovomucin was present in larger amounts in higher quality middle thick white, while no difference was noticed in the pooled thin white samples. Observed tryptophan concentrations suggested that composition of the ovomucin 1 Published as Paper No. 4788, Scientific Journal Series of the Minnesota Agricultural Experiment Station. 3 Present address: University of Wisconsin, Madison, Wisconsin. (Received for publication February 22, 1962) 1537 precipitates differed with respect to quality of the eggs. Charkey et al. (1947) determined total solids by drying, Kjeldahl total nitrogen, and tryptophan in samples of yolk, middle thick white, and combined inner and outer thin white of high and low quality eggs laid by New Hampshire hens. No difference was noted with respect to quality in the concentration of these constituents in yolk samples. Both egg white samples of higher quality eggs were higher in total solids concentration. The total nitrogen concentration (percent on dry weight basis) was higher in the high quality middle thick white samples, but not in the pooled thin white samples. While the tryptophan content of the higher quality egg white layers appeared to be higher, no interpretation was attempted. In conclusion it was suggested that the type or composition of the whole white protein pattern varies with quality, particularly in the thick white layer, and that this results in "a reduced water holding capacity in high quality eggs." Refractive indices of the various layers of the white of different quality eggs produced by White Leghorn hens of different ages were measured by Sturkie and Polin (1954). No differences in the apparent water content of these samples and samples of oviducal eggs were discovered. It was concluded that albumen quality declines after the egg leaves the magnum and enters the uterus without change in the solids concentration. It was also sug-

2 1538 J. H. SKALA AND M. H. SWANSON gested that the amount of ovomucin secreted in the magnum might account for differences in the quality of laid eggs. Cotterill and Winter (1954) investigated the relationship between relative quality of eggs and activity of lysozyme, anticipating that there might be a difference in lysozyme activity affecting its association with ovomucin [explored previously by Hawthorne (1950) and Feeney et al. (1952)]. No appreciable variation with respect to quality was observed. Wilcox (1955) also studied the relation between egg quality and lysozyme content of eggs from lines within each of two strains of White Leghorn hens bred for, high and low lysozyme level. A positive and significant regression within strains for albumen quality on lysozyme concentration was reported. From data presented it is not possible to determine whether or. not lysozyme content variation was related to variation in concentration of total solids or total nitrogen. Although considerable information is available concerning variations in chemical constituents of egg white with differences in egg quality, the problem of determining a cause for quality variation is not yet resolved. Accordingly it was decided to reinvestigate some of the earlier work to clarify the apparent disagreements over the concentration of egg white solids and to continue the study of egg white constituents as yet unexplored in an attempt to clarify interpretation of the results of Part 1 of this series. EXPERIMENTAL Three eggs were obtained from each of two hens from each group of the 60 White Leghorn hens managed as described in Part 1 of this series. The hens were chosen on the basis of albumen quality characterization so as to provide the maximum possible spread in Haugh unit scores. The 3 whole whites, including chalazae, were composited and homogenized in a blendor at a low speed to minimize denaturation of the proteins, providing one composite sample per bird. Samples were stored in sealed containers under refrigeration for a short time until analyses could be performed. Layers of white were obtained for analysis as outlined in Part 1 of this series by using a screening or sieve technique. Three eggs were obtained from each of 3 hens in each group. The 9 individual similar layers of white (e.g., inner thin white) were composited and homogenized to provide a group composite sample of the particular albumen layer. Thus there were 3 samples available per group for chemical analysis, representing the middle thick, the inner thin, and the outer thin layers of albumen. These samples were also stored a short time until analyses could be completed. Sampling was conducted on a staggered schedule so as to prevent back-logging and possible sample deterioration. Both the whole white and white layer samples were subjected to certain chemical analyses as outlined below. Total Solids. Five gram aliquots were dried in a vacuum oven for 5 hours at 100 C. and inches of vacuum. Total Ask. The dry residues from the determination of total solids in whole white composite samples were pulverized, redried for a short while, and weighed into porcelain crucibles. It was observed that pulverizing the dry white solids prior to ignition for total ash determination reduced the usual foaming appreciably. Total ash was determined by a method adapted from techniques applied to cereal products (summarized by Bailey, 1937). To the dried sample in each crucible were added 2 milliliters of magnesium acetate-methanol solution (15 grams of Mg(C 2 H ) 3

3 ALBUMEN COMPOSITION AND EGG QUALITY H 2 0 to 1 liter with methanol); the methanol was then evaporated on a steam bath. First proposed for use in cereal product ashing by Spalding (1930), the magnesium acetate serves to reduce volatilization of ash constituents. The crucibles were placed in a cool muffle furnace with the door open, and the samples were slowly charred until the furnace approached dull red heat. After removal from the furnace, 5 drops of concentrated nitric acid were added to each sample to hasten and insure oxidation. The crucibles were then evaporated to dryness on a steam bath and returned to the hot muffle furnace. Ignition was carried out at a temperature not exceeding 500 C. (to avoid fusion and possible carbon occlusion) until constant weights were attained, usually within hours. Final ash weights were corrected for blank determinations on 2 milliliters of the magnesium acetatemethanol solution. Sodium. The ash residue from the preceeding step was dissolved in concentrated hydrochloric acid and diluted solutions were analyzed for sodium using a flame photometer with internal standards. Total Nitrogen. A semi-micro Kjeldahl technique was used to analyze duplicate aliquots. Ovomucin. The technique of Balls and Hoover (1940) was applied to duplicate aliquots. It is generally acknowledged that this provides only the roughest indication of true ovomucin content; however, the North Central Marketing Regional Project, NCM-7, Subcommittee on Chemical Methods (1957) suggested that it should be useful in comparative studies. Paper Electrophoresis. Bandemer and Evans (1956) have described a method for determining egg white proteins by paper electrophoresis in a Durrum ridgepole type cell. Best results were obtained after modifying the aforementioned method somewhat for use with the horizontal open strip type cell used in this study. Six 1-inch wide strips of Whatman No. 3MM paper were placed on the carrier and moistened with 0.1 ionic strength barbital-acetate buffer of ph 8.6. The cell was assembled and allowed to equilibrate under current for 2 hours. A 10 microliter sample of a oneplus-one dilution of egg white with buffer was applied across the strip. Zone development was carried out by passing a constant current of 0.8 milliamperes per strip through the cell for a 16 hour period. The strip carrier was then removed from the cell and dried at 125 C. for 30 minutes. The strips were stained for 16 hours in a 5% acetic acid solution containing 0.1 gram of bromphenol blue plus 50 grams of zinc sulfate heptahydrate. The strips were then rinsed in two successive baths of 5% acetic acid for 6 minutes each, fixed in a solution of 3.0 grams of sodium acetate trihydrate made up to a liter with 5% acetic acid, blotted and then dried for 15 minutes at 125 C. The strips were cut into zones and the fractions were eluted by shaking gently with 25 ml. of 0.01 N sodium hydroxide for 15 minutes. The absorbance of each eluate was determined at 590 millimicrons wavelength with a Coleman Model 14 Spectrophotometer. The percent of each protein of total protein on the strip was calculated from the fraction each zone absorbance was of the total absorbance (Bandemer and Evans, 1956). The method provided fairly well defined separation of lysozyme, nonmobile protein, conalbumin, and ovalbumin. The separation of ovomucoid plus ovoglobulin was less definite. In addition to the above procedures, the ph of the thick white of one fresh egg from each of the 60 hens was determined. A section of the thick white was manipulated away from the other layers of white

4 1540 J. H. SKALA AND M. H. SWANSON TABLE 1. Chemical composition oj whole whites of high and low quality fresh eggs Determination Haugh units % Total Solids % Total Nitrogen of total white % Total Nitrogen of total solids % Total Ash of total white % Total Ash of total solids Sodium (mg./gm. white) Sodium (mg./gm. solids) %Ovomucin of total white % Ovomucin of total protein High quality (N = 12) Low quality (N = ll) Observed t * 4.055* 4.770* 3.667* * * 2.868* * Denotes significance at 5% level or better; expected t at 0.05 probability for 21 d.f. is and transferred to a small beaker for measurement using a glass electrode system without further stirring or homogenization. RESULTS AND DISCUSSION The composition of the whole whites of these relatively high (98.4 Haugh units) and low (76.5 Haugh units) quality fresh eggs in terms of several chemical constituents is shown in Tables 1 and 2. One low quality composite sample was lost in handling; therefore, statistical analysis was accomplished using a form of Student's t-test modified for unpaired data. The higher quality whole whites con- TABLE 2. Composition of the total protein of whole whites of high and, low quality fresh eggs {horizontal open strip paper electrophoresis) Percent Each Protein of Total Protein Protein High Low fraction quality quality nht. prvp,i ( Observed Haugh Haugh units) units) % % 1) Ovalbumin ) Ovomucoid +Ovoglobulin ) Conalbumin )+3) ) Non-mobile ) Lysozyme (Expected value for t at 0.05 probability for 21 d.f. is ) tained a higher concentration of total solids (12.54%) and of total nitrogen (1.72%) than the lower quality whites (11.28% and 1.50%, respectively). The higher quality whites contained 10.75% protein (N X 6.25) and the lower quality whites 9.38% protein. These results might be predicted from the work of Charkey et al. (1947). Calculation of the concentration of total nitrogen on a dry basis indicates that the higher quality whites contained a higher concentration, 13.68% vs %. This could reflect either a difference in the composition of the total protein pattern or in the pattern of total solids. The latter possibility is strongly favored by subsequent analytical data. Total ash by ignition was present in the whites of both quality levels in equal concentrations. As would be expected, then, the ash of lower quality whites comprised a greater proportion of the total solids (4.94%) compared with the higher quality whites (4.54%). Such a difference in the pattern of the total solids might be expected based on the findings reported in Part 1 of this series of papers. In that phase of the study it was observed that lower quality whites contained a larger proportion of outer thin white. The origin of this layer is believed to be the chiefly non-nitrogenous inorganic secretion of the uterus, contrasted with the chiefly nitrogenous secretion of the magnum from which the middle thick white and inner thin white layers are ultimately thought to be formed. Thus with variation in amounts and concentration of either or both secretions, there would appear to be ample opportunity for differences in the composition of the total solids pattern. Sodium concentrations were the same in the whites of both quality levels. This does not confirm the significant although variable difference reported by Conrad and

5 ALBUMEN COMPOSITION AND EGG QUALITY 1541 Scott (1942). As in the case of the total ash, sodium constituted a higher proportion of the total solids of lower quality whites (12.64 mg./gm.) compared with the higher quality whites (11.60 mg./gm.). This indicates a fair degree of sensitivity for the ashing technique employed. Ovomucin, as determined in this study, was present in higher concentration in the higher quality whites (0.36%) than in the lower quality whites (0.31%). As indicated by calculation of the ovomucin on a total protein basis, ovomucin is present in the whites of both quality levels in direct relation to the amount of total protein present. No difference between qualities was noted in the concentration of five major protein fractions relative to total protein of whole whites (Table 2). The development of the zones was generally quite satisfactory with this electrophoretic method. However, the separation of the ovomucoid + ovoglobulin and the conalbumin fractions (items 2) and 3) in the table) was too indistinct to permit more than an arbitrary elution of the zones. Although they were eluted and reported separately, they were combined under item 2) + 3) for statistical precision. The inferences from these and the ovomucin results are that, within each quality level, each protein fraction is present in relation to the amount of total protein in the whole white, and that the composition of the protein pattern of different quality egg whites sampled as a whole unit does not differ. The composition of the three principal layers of white of relatively high (97.6 Haugh units) and low (78.4 Haugh units) quality fresh eggs in terms of several chemical constituents is shown in Tables 3 and 4. Statistical analyses were accomplished using a form of the t-test for paired data. TABLE 3. Chemical composition of the major layers of while from high and low quality fresh eggs Determination Haugh units % Total Solids % Total Nitrogen % Ovomucin of layer % Ovomucin of total protein in layer Layer of white High quality Low quality Observed t * 6.512* 4.686* * 5.303* * * * Denotes significance at 5% level or better; expected t at 0.05 probability for 10 d.f. is The concentration of total solids and total nitrogen was the same in the outer thin layers of white of eggs of both quality levels. The solids and nitrogen concentration increased toward the inner thin white layer of both quality levels, but at a higher rate in the higher quality eggs. Total solids concentrations of 14.60% and 12.69% were observed in the inner thin and middle thick white layers of higher TABLE 4. Composition of the total protein of the major layers of white from high and low quality fresh eggs (horizontal open strip paper electrophoresis) Protein fraction 1) Ovalbumin 2) Ovomucoid+Ovoglobulin 3) Conalbumin 2)+3) 4) Non-mobile 5) Lysozyme 1) Ovalbumin 2) Ovomucoid+OvogiobuUn 3) Conalbumin 2)+3) 4) Non-mobile 5) Lysozyme 1) Ovalbumin 2) Ovomucoid- -Ovoglobulin 3) Conalbumin 2)+3) 4) Non-mobile 5) Lysozyme Layer of white Trmpr Percent each protein of total protein High Low quality quality (97.6 Haugh (78.4 Haugh units) units) % % * 66.1± ± ± ± ± ± ± ± ± ± * Means are shown with their standard deviations ± ± ± ± ± ± ± ± ±Q.2

6 1542 J. H. SKALA AND M. H. SWANSON quality eggs, compared with concentrations of 12.87% and 11.70% in the lower quality eggs. Total nitrogen, as per cent of wet sample, followed a similar pattern (Table 3). This alignment of the solids of different quality whites has not previously been reported because other studies have employed different sampling procedures (Charkey et al., 1947). Ovomucin was present in higher concentration in the middle thick layer of higher quality whites (0.56% vs. 0.46%). Calculation of the concentration of ovomucin in relation to the total protein of the middle thick white layer again revealed a higher value favoring higher quality eggs (4.98% vs. 4.48%). Ovomucin appeared to constitute a smaller portion of the total protein of the inner thin white layer of higher quality eggs (3.37% vs. 3.76%), although these means were significant at a confidence level slightly lower than 95%. These results suggest that, while the composition of the protein pattern of different quality whole whites appears the same, there is a difference between quality levels in the distribution of ovomucin in the layers of white. No differences were noticed in the concentrations of ovomucin in the outer thin white layers calculated on either basis or in the inner thin white layers as percent wet sample. Hill et al. (1949) pooled the two thin white layers for analysis, since preliminary estimations using the ovomucin method of Conrad and Philipps (1938) failed to yield a measurable precipitate from the inner thin white. However, a definite precipitate was obtained with the method employed for this study. While definite differences in the major proteins determined by the paper electrophoretic analysis (Table 4) were found between egg white layers within quality levels, none were found between quality levels. Results were calculated as percent of total protein in the sample as measured by total dye absorbed by all zones on the strip. This would not discredit the ovomucin findings since it is doubtful if ovomucin is represented by the non-mobile fraction in the electrophoretic determination, or if it is even present in the sample applied to the strips because of its relative insolubility. These results would suggest, then, that the soluble proteins determined by this method are distributed similarly within the whole white of high and low quality fresh eggs. It has long been recognized that the ph of the egg white is related to the appearance of eggs which have been stored. Excised portions of middle thick white were subjected to ph determinations in this study since it was believed that this is the layer of white having the major influence on the Haugh unit score and, thus, would reflect any difference in this factor. The results presented in Table 5 indicated no difference in this respect in fresh eggs. The results presented in this study and the physical measurements reported in Part 1 suggest a possible mechanism for the differentiation of quality during the formation of eggs in the hen's oviduct. It would appear that viscous unstratified secretion of the magnum is similar with reference to the composition of the total protein pattern irrespective of the quality of the fully formed egg. Each protein constituent is proportionately present in relation to the TABLE 5. ph of the middle thick layer of white from high and low quality fresh eggs Low High Observed t * * Denotes significance at 5% level or better; expected t at 0.05 probability for 10 d. f. is

7 ALBUMEN COMPOSITION AND EGG QUALITY 1543 High Quality Samples Low Quality Sample Layer of White tic. 1. Distribution of total white, total protein, and total ovomucin among layers of egg white. amount of total protein secreted. As the magnum secretion is stratified into the layers of white, there is less differentiation of this secretion in the higher quality eggs, based on obesrvations reported in Part 1 of this series (Figure 1). During this stratification, a disproportionate distribution of the individual soluble proteins occurs in all eggs regardless of quality. Ovomucin is distributed similarly to the other proteins, although not in direct relation to the total protein, possibly due to its peculiar properties and the action to which it may be subjected. This hypothesis is supported by the additional observation (Figure 1) that a larger proportion of the total protein remained in the middle thick white of higher quality eggs (62.51% vs %) and a smaller proportion was transferred to the inner thin white (19.93% vs %) and the outer thin white (17.56% vs %). The middle thick white of higher quality eggs contained a greater proportion of the total ovomucin determined (76.68% vs %), while the inner and outer thin white layers contained a lesser proportion (16.37% vs % and 6.95% vs. 8.09%, respectively). If it is accepted that ovomucin is chiefly responsible for the upstanding appearance of egg white, its possible interactions with other proteins notwithstanding, it would follow that the relative amount of this protein in the middle thick white layer of a laid egg is important in determining appearance or Haugh unit score. The cause or process which governs the relative amount of ovomucin, as well as other proteins, may be the extent of differentiation of the initial magnum secretion as suggested above. The degree of differentiation into layers of white may be determined by the extent of two principal processes occurring in the oviduct: the physical action of rotation during which the chalazae and inner thin white are formed, and the dissolution of protein from the middle thick white by the uterine secretion. It would appear that both of these processes have occurred to a greater extent in the formation of the lower quality eggs examined in this study. As has been mentioned before, inner thin white differentiation appeared to be less in higher quality whites, as judged by the relative amounts of the different layers of white in the laid egg. Although the concentration of protein in the outer thin white layers was the same, regardless of quality, the protein in solution in this layer of low quality whites necessarily was a larger proportion of the total protein because of the relatively larger quantity of outer thin white. The amount of water in these secretions, as it influences the concentration of the magnum secretion and the amount of the uterine secretion, may be the principal factor governing the extent of layer differentiation. A more dilute magnum secretion, if such exists in the case of hens laying lower quality eggs, may result in de-

8 1544 J. H. SKALA AND M. H. SWANSON creased viscosity and greater rotation of white about the yolk causing more inner thin white formation and proportionately more ovomucin removal. It is possible that the concentration of the magnum secretion is higher in hens laying higher quality eggs, although no direct observations were made in this study. This would be contrary to the findings of Sturkie and Polin (1954); however, their results may be confounded with the effect of the different ages of birds employed. It is also possible that the greater dilution found in lower quality egg whites may result from diffusion of a greater amount of the uterine secretion. The overall apparent dilution of lower quality laid whites may indeed result from properties and action of both secretions. The amount of uterine secretion, low in nitrogenous constituents initially, could both influence the amount of protein removed from the thick white and contribute to the water content of other layers through diffusion. The net result of excess dilution and subsequent layer formation would be an egg with less middle thick white in proportion to total white, a more dilute middle thick white with proportionately less structural ovomucin, and a lower quality egg as judged by Haugh unit evaluation or condition of the white. SUMMARY Certain chemical constituents of the white of eggs varying in initial quality (Haugh unit basis) were determined. Eggs were obtained from a commercial strain of White Leghorn hens as described in Part 1 of this series. The whole whites and the middle thick and inner thin white layers from higher quality eggs contained higher concentrations of total solids and total nitrogen, but this was not true of the outer thin layer. There was no difference in the total ash or sodium concentrations in whole whites between qualities, nor in the ph of the middle thick white. No differences in the composition of the protein complex of different quality whole whites or white layers in terms of major proteins determined by horizontal open strip paper electrophoresis was demonstrated. Higher quality whole whites contained a higher concentration of apparent ovomucin (by precipitation) as percent of total white but not as percent of total protein. Higher quality middle thick whites contained higher concentrations of ovomucin both as percent of total layer and of total protein. No difference was noted in the outer thin whites, but it appeared that the percent ovomucin of total protein was lower in the inner thin whites of higher quality eggs. These results suggested that the extent of egg white layer differentiation and subsequent distribution of ovomucin between the layers may be important to the ultimate initial appearance or quality of the broken-out egg. A hypothetical mechanism for the cause of variation in initial egg quality was suggested. REFERENCES Bailey, L. H., Some observations on methods of ashing cereal products. Cereal Chem. 14: Balls, A. K., and S. R. Hoover, Behavior of ovomucin in the liquefaction of egg white. Ind. Eng. Chem. 32: Bandemer, J. E., and S. L. Evans, Separation of egg white proteins by paper electrophoresis. Agric. Food Chem. 4: Charkey, L. W., E. Dyar and H. S. Wilgus, Jr., The nutrient content of high and low quality fresh eggs. 1. Total solids, total nitrogen, and 1 ( ) tryptophane. Poultry Sci. 26: Conrad, R. M., and R. E. Phillips, The formation of the chalazae and inner thin white

9 ALBUMEN COMPOSITION AND EGG QUALITY 154S in the hen's egg. Poultry Sci. 17: 143r-146. Conrad, R. M., and H. M. Scott, Differences between high and low quality fresh eggs. Poultry Sci. 21: Cotterill, O. J., and A. R. Winter, Egg white lysozyme 1. Relative lysozyme activity in fresh eggs having low and high interior quality. Poultry Sci. 33: Feeney, R. E., E. D. Ducay, R. B. Silva and L. R. MacDonnell, Chemistry of shell egg deterioration: the egg white proteins. Poultry Sci. 31: Hawthorne, J. R., The action of egg white lysozyme on ovomucoid and ovomucin. Biochem. Biophys. Acta, 6: Hill, E. G., G. R. Burton and L. W. Charkey, The nutrient content of high and low quality fresh eggs. III. Mucin in relation to PROGESTERONE is produced by the corpus luteum in mammals and functions synergistically with estrogen to maintain the uterus throughout pregnancy. In sufficient amounts progesterone inhibits estrus and ovulation in mammals. In the domestic fowl progesterone is apparently produced in the reproductive organs and functions in the regulation of egg production. Fraps, Hooker and Forbes (1949) reported that progesterone was found in the blood of laying hens, non-laying hens, and cocks, but not in capons which suggest that 'Florida Agricultural Experiment Stations, Journal Series No Poultry Research Branch, Animal Husbandry Research Division, ARS, USDA, Athens, Ga. ' Department of Animal Science. tryptophan. Poultry Sci. 28: North Central Marketing Regional Project, NCM-7, Outlines for evaluation of poultry products. Chem. Methods, p. 9. Skala, J. H., and M. H. Swanson, Studies of variation in initial quality of chicken eggs. 1. Physical measurements of albumen and yolks. Poultry Sci. 41: Spalding, J. L., Quick ash determination by magnesium acetate alcohol method. Cereal Chem. 7: Sturkie, P. A., and D. Polin, Role of the magnum and uterus in the determination of albumen quality of laid eggs. Poultry Sci. 33: Wilcox, F. H., Jr., Evidence for association between the lysozyme level and the quality of egg white. Poultry Sci. 34: The Effects of Progesterone and Progestational Compounds upon the Production of Laying Pullets 1 R. E. COOK, 2 AND A. C. WARNICK 3 Florida Agricultural Experiment Stations, Gainesville, Florida (Received for publication February 23, 1962) it is produced in the male and female gonads. Previous reports have indicated that progesterone affects the reproductive system of the fowl differently depending upon the levels of treatment and method of administration. Stimulation of testicular growth in immature pigeons was reported by Kar (1949) from injections of small amounts of progesterone. Nalbandov (1956) observed that immature pullets treated with progesterone matured earlier than their controls. Some stimulation of production in Empire White turkeys was observed by van Tienhoven (1958) following periods of broodiness interrupted by progesterone treatment. Cessation of egg production from ad-