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The Changes in Egg Shell Strength During Incubation
The Changes in Egg Shell Strength During Incubation J. VANDERSTOEP AND J. F. RICHARDS 1
Department of Poultry Science, University of British Columbia, Vancouver 8, B.C., Canada (Received for publication September 20, 1969)
E
Since about 95% of the egg shell consists of inorganic matter and 98% of this is in the form of calcium salts (Romanoff and Romanoff, 1949) it would appear 1
Present address: Department of Food Science, University of British Columbia, Vancouver 8, British Columbia.
logical that the removal of calcium by the embryo during incubation would influence thestrength of the shell during that period. The present paper reports the effect of incubation on shell strength as measured by various means and the effect of the shell membranes on shell strength. MATERIALS AND METHODS Experiment 1. A random sample of fresh fertile and infertile eggs laid by U.B.C. Single Comb White Leghorns (S.C.W.L.) was selected. Each of the two groups of eggs was divided into four lots and stored at the following temperatures: a) room temperature (20.1°C, 70°F.) for 3 days, b) room temperature for 12 days, c) refrigerated temperature (9.5°C., 50°F.) for 3 days and d) refrigerated temperature for 12 days. Prior to and after storage, the eggs were weighed and the deformation under a unit load of 500 grams measured (Schoorl and Boersma, 1962). This method is subsequently referred to as partial load deformation (P.L. deformation). The eggs were then incubated. During incubation, deformation was measured on day 5, 10, 12, 14, 16, 17, 18, 19 and 20. The chicks that hatched were identified and body weights recorded over the period from hatching to four weeks of age, at which time they were killed and their sex determined. Experiment 2. In order to overcome the variation in deformation among the eggs in Experiment 1, the second experiment was carried out using eggs from known sources. Six S.C.W.L. pullets near the end of their first year of lay were kept in single
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GG shell strength of infertile eggs has been studied by a number of workers including Tyler (1961), Hunt and Voisey (1966) and Richards and Staley (1967), but relatively little work has been carried out on the strength of shells of fertile incubated eggs. Romanoff (1929), investigating the effect of humidity on the growth and calcium metabolism of the chick embryo, concluded that calcium was probably passing to the embryo from the shell through the shell membranes. Romanoff and Taylor (1949) as cited by Driggers et al. (1951) found that the newly hatched chick, exclusive of the yolk sac, contained about 150 mg. more calcium than the entire contents of the unincubated egg. Driggers et al. (1951) using radioactive calcium, found that the only possible mechanism for the Ca45 to have reached the yolk sac, which was drawn into the chick prior to hatching, was by diffusion from the shell and to a much lesser extent from the egg white. Tyler and Simkiss (1959) felt that the conclusion made by Plimmer and Lowndes (1924) concerning the passage of shell calcium to the embryo through the membrane is probably correct. Johnson and Comar (1955) labelled the albumen calcium and found that shell calcium was starting to be mobilized at about the tenth to eleventh day of incubation.
SHELL STRENGTH AND INCUBATION
The membranes were digested from the shell using boiling 2.5% (w/v) NaOH for three minutes. After rinsing with tap and distilled water, the shell was oven dried. The true shell was then weighed. The shell thickness was measured at a premarked area of the shell equator, using a Starrett anvil-jawed dial micrometer. An additional sample of ten eggs was collected from each hen, weighed, P.L. and Bellows deformation measured, breaking strength determined and amount and thickness of true shell measured. This sample of ten eggs was employed to obtain initial base values of each of the measurements for each hen. Experiment 3. A random sample of 58 eggs was obtained and a P.L. deformation measurement made for each intact egg. A
small hole was drilled in each pole of the egg and the contents blown out using a stream of air. The empty shells were washed with water to remove any remaining albumen adhering to the shell membrane. While the shell was still wet, the deformation was again measured. The shells were then dried to constant weight in an air oven at 125°C. and the deformation measured again. The hole in the small pole of the egg was then sealed by cementing a small piece of shell over the hole, using airplane glue. When the patch was dry, a hot solution of 5% NaOH was run into the shell filling it to overflowing. The hot NaOH solution was kept near boiling during the filling. The NaOH filled shells were kept in an air oven at 120 to 125°C. for 2 to 3 minutes to allow equilibration of NaOH temperature and a further 10 minutes to allow digestion of the membranes. It was found that 10 minutes under these conditions were required for clean removal of the membrane. At the end of the digestion period, the hole covered by the shell patch was redrilled to allow drainage of the NaOH solution. The shells were again washed to clean the inside of the shell and the deformation measured again. The shells were again dried in an air oven at 125°C. and the deformation of the dried membrane-less shells determined. RESULTS AND DISCUSSION
In Experiment 1, P.L. deformation of a random sample of 21 eggs was measured before and after storage. Statistical analysis by paired comparison (Steel and Torrie, 1960) showed no significant difference between the deformation values before or after storage ( P > 5 % ) . Cumulative partial-load deformation change with time of incubation was calculated for each egg by determining the difference in deformation between successive
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cages and were fed a standard laying mash. Two W.L. males were used in the mating and the pullets artificially inseminated twice weekly. The eggs were collected, identified and refrigerated until used. At the appropriate time, three eggs per hen per incubation period were selected and weighed and a partial load deformation value determined. During the incubation period, deformation measurements were made at day 1, 5, 10, 12, 14, 16, 18 and 20. The first three eggs per hen were removed at day 10, the next three at day 12 etc. After removal of the eggs from the incubator the eggs were cracked using the Bellows Valvair Hydrocheck Compression Unit, to measure crushing strength and total deformation (Richards and Staley, 1967). From this data, deformation per 500 gram load was calculated (Bellows deformation) . The contents were removed from the shell, fertility determined, and a subjective assessment made of the looseness of the attachment of shell membranes to the shell.
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J. VANDEESTOEP AND J. F. RICHARDS
TABLE 1.—Average cumulative partial load deformation changes of fertile and infertile eggs during incubation after pre-incubation storage at room and refrigerated temperatures for 3 and 12 days Incubation time (days)
Treatment 3 days— room
3 days— refrig.
12 days— room
12 days— refng.
Fertile eggs microns
s
0.32 1.44 2.24 2.74 6.06 8.42 10.85 12.75 («=21)
0.52 1.03 1.29 2.06 4.73 6.60 9.06 9.68 (« = 21)
5 10 12 14 16 17 18 19
0.33 0.25 1.47 1.52 1.74 1.71 1.66 1.88 («=9)
Infertile eggs 0.85 0.35 1.45 0.35 1.45 0.35 1.00 0.70 1.60 0.98 2.57 0.88 2.64 0.88 2.29 0.90 (m = 10) (» = 10)
1.00 1.28 2.50 3.19 5.63 7.25 10.37 11.81 («=8)
0.14 0.54 1.37 1.81 4.32 7.21 9.29 11.49 (n=22) -0.75 -0.08 0.40 1.38 1.73 1.38 1.55 1.53 (» = 10)
times of measurement and totaling these differences. Then average cumulative deformation was calculated for each treatment and time of incubation (Table 1). Although storage had no effect on the initial deformation value, the P.L. cumulative deformation values during incubation were almost invariably lower for refrigerated fertile eggs than for those stored at room temperature. The cumulative deformation of infertiles remained fairly constant during the incubation period. Average cumulative P.L. deformation for all treatments combined increased slowly up to day 10, more rapidly from day 10 to 14 at a greatly increased rate from day 14 to 18 and at a slightly but consistently reduced rate from day 18 to 20 (Fig. 1). This relationship is identical to that found by Richards and Robinson (1967). The pattern of deformation change may reflect the chemical and morphological changes that are taking place in the developing embryo. Kroon (1940) noted
For individual hen data, cumulative P.L. deformation change with time showed the typical increase in rate of change. The average cumulative P.L. deformation change during incubation was similar to the relationship found in Experiment 1 (curve A, Fig. 3). A similar relationship
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10 12 14 16 17 18 19
a considerable increase in wet weights and ash contents of embryos on the 1213th day and that at day 17 the wet weight started to increase less rapidly but that ash continued to increase rapidly. Johnson and Comar (1955) reported that their results indicated that shell calcium was starting to be mobilized at about 1011 days. In Experiment 2, three eggs per hen per incubation period were initially used, but due to infertility and early embryonic death, some of the measurements were invalid and were discarded. The actual P.L. deformation values of the remaining eggs indicated a general trend of increasing deformation as incubation time increased. Using these values to compute an overall average deformation value for all hens gave curve A of Figure 2. This curve is similar to Figure 1 with the exception that in the present case there is a decrease starting at day 18. It is felt that this is due to the fact that data from different eggs were used to calculate the average value at each incubation time. To support this contention an average deformation value based on only those eggs incubated to hatching was calculated and plotted (curve B of Figure 2). The deformation value at each incubation time in this instance is an average of the measurements at that time on the same eggs. This curve shows the typical increase in deformation starting at day 10 with a sharp increase in rate of change at day 14 and a decline in rate of increase between day 18 and 20.
279
SHELL STRENGTH AND INCUBATION
o
o FERTILES
o
o INFERTILES
10 INCUBATION
12
14
16
18
20
PERIOD (DAYS)
FIG. 1. Average cumulative partial load deformation change of egg shells during incubation. Exp. 1
was obtained using only those eggs incubated to hatching in the calculation of averages (curve B, Fig. 3). Using the Bellows compression unit, crushing strength and total deformation were determined on the eggs as they were removed from the incubator. From this data deformation per 500 gram load was computed. Average deformation increased inconsistently during incubation, the inconsistency probably being due to the inclusion of different eggs in the calculation of the averages (Table 2). Generally the Bellows deformation value for any particular egg as computed from crushing strength and total defor-
mation data was higher than the P.L. deformation value. In an attempt to calculate a cumulative deformation change (Bellows method), the deformation at each incubation time was subtracted from the average deformation of the base pool eggs. These values also increased inconsistently during incubation (Table 2). A plot of the average P.L. deformation, crushing strength, percent shell and shell thickness (Y) vs. time (X) is presented in Fig. 4. Analyses of the relationships among these variables were performed on data up to and including the eighteenth day of incubation. Data for the twentieth day
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8
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J. VANDERSTOEP AND J. F. RICHARDS
10
12
14
16
18
20
FIG. 2. Partial load deformation change of egg shells during incubation for Exp. 2. Curve A represents the change in P.L. deformation as an average for all hens. Curve B is an average for those eggs incubated to hatching.
were excluded because of the inconsistency introduced by egg to egg variation. Analysis of P.L. deformation vs. crushing strength indicated a highly significant correlation (r=—.853). Similar analyses of percent shell and shell thickness vs. crushing strength indicated highly significant correlation coefficients (r) of +0.773 and +0.778 respectively. Analysis of average values for P.L. deformation, percent shell and shell thickness vs. crushing strength indicated highly significant correlation coefficients (r) of -0.968, +0.986 and +0.985 respectively.
It would appear from the data so far that P.L. deformation, percent shell and shell thickness are accurate indicators of shell strength. Regression of each of the four variables (Y) against time (X) gave slopes (b) and correlation coefficients (r) as reported in Table 3. Using all observations in the analysis resulted in highly significant ( P < .01)' correlation coefficients (r) for P.L. deformation and crushing strength, a significant correlation (P<.05) for shell thickness and a non-significant correlation ( P > .05) for percent shell. However, when average values were used deformation,
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8
INCUBATION PERIOD (DAYS)
281
SHELL STRENGTH AND INCUBATION
12
CURVE
A
10 CURVE B
z o < O
6
IU
Q
^ 4
i 2 •
6
8 INCUBATION
10
12
PERIOD
(DAYS)
14
16
18
20
FIG. 3. Average cumulative partial load deformation change of egg shells during incubation. Exp. 2. Curve A represents the change in P.L. deformation as an average for all hens. Curve B is an average for those eggs incubated to hatching. crushing strength and percent shell were significantly (P < 0.05) correlated to incubation time and the correlation of shell thickness with incubation time was highly significant ( P < 0 . 0 1 ) . I t is apparent from Fig. 4 t h a t the relationships of t h e variables ( F ) to time (X) are not linear even when d a t a for t h e twentieth day are excluded. Correlation coefficients increased and in most cases t h e confidence level increased when t h e quadratic equation F = a - r - J X + c ( X 2 ) was fitted to the d a t a (Table 4). T h e regression and correlation analyses, combined with Fig. 4 indicate t h a t defor-
mation, crushing strength, percent shell and shell thckness measurements are capable of detecting a change in shell strength over time during incubation. TABLE 2.—Average Bellows deformation and average cumulative Bellows deformation change of egg shells during incubation Incubation time
Deformation (n.)
Cumulative deformation change (/».)
10 12 14 16 18 20
35.49 41.28 39.10 42.80 46.27 42.52
5.20 9.75 8.92 11.75 14.95 11.60
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s
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J. VANDERSTOEP AND J. F. RICHARDS
16
18
20
0.010
INCUBATION PERIOD (DAYS)
FIG. 4. Change in partial load deformation, crushing strength, % shell and shell thickness of egg shells during incubation. Exp. 2. Values of repeatability of measurement or precision were established as 0.25/*., 10.0 gms., 0.10 percent, and 0.0002 inches for deformation, crushing strength, percent shell and shell thickness respectively (Richards and Vanderstoep, 1968). A measure of sensitivity was then denned as the change per day as a percent of precision. The regression coefficients (b) from the analysis of averages (Table 3) were used as a measure of the amount of change per day. This procedure resulted in sensitivities of 490, 250, 52 and 52% for deformation, crushing strength, percent shell and shell thickness respectively. This indi-
cates that the sensitivity of percent shell and shell thickness is much lower than that of deformation and crushing strength. Thus crushing strength and particularly deformation are superior measures of the change in shell strength over time during incubation. It might be possible to explain the insensitivity of shell thickness by the work of Tyler and Simkiss (1959), who showed that during incubation, the outer shell membrane comes away from the shell. Rather than coming away cleanly, the membrane carries with it ends of the mammillary knobs including the mam-
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14
283
SHELL STRENGTH AND INCUBATION
TABLE 4.—Multiple regression coefficients and correla-
tion coefficients for each shell parameter (F) vs. time of incubation (X) All observations (» = 74) b
c
r
5.44 + 237.49 + 0.21 +0.13X10"*
+ 0.24 — 12.80 -0.92X10-2 -0.84X10-S
+0.44** -0.574" -0.203n.s. -0.244*
Variable (Y) Deformation Crushing Strength Percent Shell Shell Thickness
Average (»=5)
Deformation Crushing Strength Percent Shell Shell Thickness
r
b
c
5.66 + 246.80 + 0.21 +0.45X10-<
+ 0.25 13.12 -0.93X10-2 -0.54X10-'
II
Variable (F)
** (P<.01) * (P<-05) n.s. =non-significant.
sis of a two-tailed paired comparison /-test (Table 5). The mean P.L. deformation value for the intact eggs was not significantly different from the mean value for the same eggs with the membrane removed but with shells still wet. The mean values for the eggs with contents removed and the shells dried and the eggs with the membrane removed and the shells dried were not significantly different. There was, however, a highly significant difference TABLE 5.—Mean partial load deformation values and
TABLE 3.—Simple linear regression coefficients and
correlation coefficients for each shell parameter (F) vs. time of incubation {X) All observations (» = 74)
results of paired comparison t-tests between pairs of shell treatments Treatment comparison
drfomation
'"value
-ft-
Variable (F) Deformation Crushing Strength Percent Shell Shell Thickness
b
r
+ 1.2582 +0.405** -121.343 -0.558** 0.0518 -0.187n.s. 0.00109 -0.240* Average (« = 5)
Variable (F)
b
r
Deformation Crushing Strength Percent Shell Shell Thickness
+ 1.2295 -120.50 0.052 0.00105
+0.900* -0.956* -0.918* -0.988**
** (P<.01) * (P<.05) n.s. = non-significant.
Intact vs. membrane removed and wet
30.00 29.83
1.07 n.s.
Intact vs. membrane removed and dry
30.00 29.50
3.57"
Contents removed and wet vs. contents removed and dry
30.36 29.38
6.06**
Contents removed and wet vs. membrane removed and dry
30.36 29.50
5.02**
Contents removed and dry vs. membrane removed and wet
29.38 29.83
2.59*
Contents removed and dry vs. membrane removed and dry
29.38 29.50
-0.77 n.s.
Membrane removed and wet vs. membrane removed and dry
29.83 29.50
2.27*
* (P<0.05) " (P<0.01) n.s. =non-significant.
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miliary cores. This could reduce shell strength considerably, but it would be by localized removal of the shell material rather than uniform removal. The fact that the measuring surface of the dial micrometer is large in comparison to the mammillary core areas, could account for the relatively minor change in shell thickness over time. Finally, a rather general and superficial study was made of the loosening of the shell membranes during incubation. As a general observation, it can be said that a very slight amount of loosening had started by the tenth day, and that by the sixteenth day the membranes were quite loose and extremely loose by the eighteenth day. This general observation agrees with the work carried out by Tyler and Simkiss (1959). Experiment 3 was carried out to determine if the change in P.L. deformation which occurs during incubation can be accounted for solely by changes in the shell itself, as suggested by the work of Johnson and Comar (1955) or whether membrane loosening influenced the readings. The analysis of the data was on the ba-
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J. VANDERSTOEP AND J. F. RICHARDS
SUMMARY
It has been found that during incubation, shell strength, as measured by deformation under a unit load, decreased with time. The rate of change in deformation increased most rapidly during the period from the 14th to the 18th day and decreased in rate between the 18th and 20th day. Although deformation, shell weight as a percentage of initial egg weight and
shell thickness at any particular time during incubation were found to be well correlated to crushing strength, deformation was more sensitive than percent shell and shell thickness to changes in shell strength during incubation. By measuring the deformation of the eggs intact, with contents removed and with shell membranes removed, it was found that shell membranes loosened during the period of greatest shell strength change but did not affect the shell strength. ACKNOWLEDGEMENTS
The financial assistance of the National Research Council of Canada in this study is appreciated. REFERENCES Driggers, J. C , R. L. Shirley, G. K. Davis and N. R. Mehrhof, 1951. The transference of radioactive calcium and phosphorus from hen to chick. Poultry Sci. 30: 199-204. Hunt, J. R., and P. W. Voisey, 1966. Physical properties of egg shells. 1. Relationship of resistance to compression and force at failure of egg shells. Poultry Sci. 45: 1398-1404. Johnson, P. M., and C. L. Comar, 1955. Distribution and contribution of calcium from the albumen yolk and shell to the developing chick embryo. Am. J. Phys. 183: 365-370. Kroon, D. B., 1940. Distribution of Ca and P during the growth of the chicken embryo. Acta Brevia Neerlandia, X: 128-129. Lee, J., J. F. Richards and L. M. Staley, 1968. Influence of several factors on egg shell strength and its prediction. Can. J. An. Sci. 48: 163-169. Plimmer, R. H. A., and J. Lowndes, 1924. The changes in the lime content of the hen's egg during development. Biochem. J. 18: 1163. Richards, J. F., and M. L. Robinson, 1967. Unpublished data. Richards, J. F., and L. M. Staley, 1967. The relationship between crushing strength, deformation and other physical measurements of the hen's egg. Poultry Sci. 46: 430-437. Richards, J. F., and J. Vanderstoep, 1968. Unpublished data. Romanoff, A. L., 1929. Effect of humidity on the growth, calcium metabolism and mortality of the chick embryo. J. Exp. Zool. 5: 343-348.
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between the mean values for the intact eggs and the eggs with the membrane removed and the shells dried. There was a significant difference between the values for the eggs with contents removed and shells dried and the eggs with the shell membranes removed but the shell still wet. Highly significant differences existed between the values for the eggs with contents removed and shells wet and those with the shell membranes removed and the shells dried. I t appears from the above data that shell membranes do not influence shell strength but rather that the differences obtained are due to the wetness of the shell. This is substantiated by the fact that highly significant differences were found between the eggs with contents removed and the shells wet and those with the shells dried. Further there were significant differences between values for the eggs with shell membranes removed and shells wet and those with the shells dried. These facts substantiate the previously reported finding that mositure weakens the shell (Tyler and Geake, 1964; and Leeetal., 1968). The results of the study of the effect of membranes on shell strength are similar to those reported by Tyler and Geake (1964) even though methods of membrane removal and strength evaluation were different.
SHELL STRENGTH AND INCUBATION Romanoff, A. L., and A. J. Romanoff, 1949. The Avian Egg. Chapman and Hall Ltd. London. Romanoff, A. L., and L. W. Taylor, 1949. Fertility and Hatchability of Chicken and Turkey Eggs. J. Wiley and Sons Inc., N. Y., p. 432. Schoorl, P., and H. Y. Boersma, 1962. Research on the quality of the egg shell. Proc. 12th World's Poultry Cong.: 432-435. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Company.ync, New York.
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Tyler, C , 1961. Shell strength: Its measurement and relationship to other factors. Brit. Poultry Sci. 2: 3-19. Tyler, C , and F. H. Geake, 1964. The testing of methods for cracking egg shells, based on paired readings from individual eggs and the measurement of some effects of various treatments. Brit. Poultry Sci. 5: 19-28. Tyler, C , and K. Simkiss, 1959. Studies on egg shells. XII. Some changes in the shell during incubation. J. Sc. Food Agric. 11: 611-615.
D. E. TURK AND J. F. STEPHENS 3 Poultry Science Department Clemson University, Clemson, S. C. 29631 (Received for publication September 22, 1969)
W
E HAVE previously described the effects of several types of chicken coccidiosis upon the absorption of orally administered zinc-65, iodine-131-labeled oleic acid, proteins, and amino acids into the bloodstreams of growing birds (Turk and Stephens, 1966; 1967 a,b; 1969 a,b). In all of these studies, acute infections of single coccidial species were allowed to completetheircycles. In the field, however, coccidial infections are not usually allowed to run their courses, but are treated in order to minimize the adverse effects of the disease. The ability of the coccidial treatments to ameliorate the severe effects that coccidiosis has upon nutrient absorption is unknown, therefore a series of trials 1
This study was supported in part by Public Health Service Research grant #AM-09189 from the National Institute of Arthritis and Metabolic Diseases. 2 Published with the approval of the Director of the S. C. Agricultural Experiment Station as technical contribution number 822. 3 Present address: Poultry Science Department, The Ohio State University, Columbus, Ohio 43210.
was undertaken to determine the effect of an interrupted coccidial infection upon the ability of young birds to absorb zinc. Data pertaining to growth rate and intestinal damage, as well as Zn absorption, were collected to provide information on the severity of the induced infection, the effects of the treatments on the severity of the infection, and the relationship of these variables to the rate of absorption of orally administered Zn-65. Sulfaquinoxaline was used to interrupt the induced E. necatrix infection since this is an effective coccidiostat for treatment of E. necatrix infections (Grumbles et al., 1948). PROCEDURE
Day-old commercial broiler male chicks were raised to four weeks of age in electrically heated batteries with ad libitum access to a non-medicated corn-soy chick starting ration and to tap water. At four weeks of age, the birds were weighed and assigned at random to one of the experimental groups. One group in each trial was re-
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Eimeria Necatrix and Zinc Absorption in the Chick: Effect of Sulfaquinoxaline Treatment of the Infection12
Department of Poultry Science, University of British Columbia, Vancouver 8, B.C., Canada (Received for publication September 20, 1969)
E
Since about 95% of the egg shell consists of inorganic matter and 98% of this is in the form of calcium salts (Romanoff and Romanoff, 1949) it would appear 1
Present address: Department of Food Science, University of British Columbia, Vancouver 8, British Columbia.
logical that the removal of calcium by the embryo during incubation would influence thestrength of the shell during that period. The present paper reports the effect of incubation on shell strength as measured by various means and the effect of the shell membranes on shell strength. MATERIALS AND METHODS Experiment 1. A random sample of fresh fertile and infertile eggs laid by U.B.C. Single Comb White Leghorns (S.C.W.L.) was selected. Each of the two groups of eggs was divided into four lots and stored at the following temperatures: a) room temperature (20.1°C, 70°F.) for 3 days, b) room temperature for 12 days, c) refrigerated temperature (9.5°C., 50°F.) for 3 days and d) refrigerated temperature for 12 days. Prior to and after storage, the eggs were weighed and the deformation under a unit load of 500 grams measured (Schoorl and Boersma, 1962). This method is subsequently referred to as partial load deformation (P.L. deformation). The eggs were then incubated. During incubation, deformation was measured on day 5, 10, 12, 14, 16, 17, 18, 19 and 20. The chicks that hatched were identified and body weights recorded over the period from hatching to four weeks of age, at which time they were killed and their sex determined. Experiment 2. In order to overcome the variation in deformation among the eggs in Experiment 1, the second experiment was carried out using eggs from known sources. Six S.C.W.L. pullets near the end of their first year of lay were kept in single
276
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GG shell strength of infertile eggs has been studied by a number of workers including Tyler (1961), Hunt and Voisey (1966) and Richards and Staley (1967), but relatively little work has been carried out on the strength of shells of fertile incubated eggs. Romanoff (1929), investigating the effect of humidity on the growth and calcium metabolism of the chick embryo, concluded that calcium was probably passing to the embryo from the shell through the shell membranes. Romanoff and Taylor (1949) as cited by Driggers et al. (1951) found that the newly hatched chick, exclusive of the yolk sac, contained about 150 mg. more calcium than the entire contents of the unincubated egg. Driggers et al. (1951) using radioactive calcium, found that the only possible mechanism for the Ca45 to have reached the yolk sac, which was drawn into the chick prior to hatching, was by diffusion from the shell and to a much lesser extent from the egg white. Tyler and Simkiss (1959) felt that the conclusion made by Plimmer and Lowndes (1924) concerning the passage of shell calcium to the embryo through the membrane is probably correct. Johnson and Comar (1955) labelled the albumen calcium and found that shell calcium was starting to be mobilized at about the tenth to eleventh day of incubation.
SHELL STRENGTH AND INCUBATION
The membranes were digested from the shell using boiling 2.5% (w/v) NaOH for three minutes. After rinsing with tap and distilled water, the shell was oven dried. The true shell was then weighed. The shell thickness was measured at a premarked area of the shell equator, using a Starrett anvil-jawed dial micrometer. An additional sample of ten eggs was collected from each hen, weighed, P.L. and Bellows deformation measured, breaking strength determined and amount and thickness of true shell measured. This sample of ten eggs was employed to obtain initial base values of each of the measurements for each hen. Experiment 3. A random sample of 58 eggs was obtained and a P.L. deformation measurement made for each intact egg. A
small hole was drilled in each pole of the egg and the contents blown out using a stream of air. The empty shells were washed with water to remove any remaining albumen adhering to the shell membrane. While the shell was still wet, the deformation was again measured. The shells were then dried to constant weight in an air oven at 125°C. and the deformation measured again. The hole in the small pole of the egg was then sealed by cementing a small piece of shell over the hole, using airplane glue. When the patch was dry, a hot solution of 5% NaOH was run into the shell filling it to overflowing. The hot NaOH solution was kept near boiling during the filling. The NaOH filled shells were kept in an air oven at 120 to 125°C. for 2 to 3 minutes to allow equilibration of NaOH temperature and a further 10 minutes to allow digestion of the membranes. It was found that 10 minutes under these conditions were required for clean removal of the membrane. At the end of the digestion period, the hole covered by the shell patch was redrilled to allow drainage of the NaOH solution. The shells were again washed to clean the inside of the shell and the deformation measured again. The shells were again dried in an air oven at 125°C. and the deformation of the dried membrane-less shells determined. RESULTS AND DISCUSSION
In Experiment 1, P.L. deformation of a random sample of 21 eggs was measured before and after storage. Statistical analysis by paired comparison (Steel and Torrie, 1960) showed no significant difference between the deformation values before or after storage ( P > 5 % ) . Cumulative partial-load deformation change with time of incubation was calculated for each egg by determining the difference in deformation between successive
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cages and were fed a standard laying mash. Two W.L. males were used in the mating and the pullets artificially inseminated twice weekly. The eggs were collected, identified and refrigerated until used. At the appropriate time, three eggs per hen per incubation period were selected and weighed and a partial load deformation value determined. During the incubation period, deformation measurements were made at day 1, 5, 10, 12, 14, 16, 18 and 20. The first three eggs per hen were removed at day 10, the next three at day 12 etc. After removal of the eggs from the incubator the eggs were cracked using the Bellows Valvair Hydrocheck Compression Unit, to measure crushing strength and total deformation (Richards and Staley, 1967). From this data, deformation per 500 gram load was calculated (Bellows deformation) . The contents were removed from the shell, fertility determined, and a subjective assessment made of the looseness of the attachment of shell membranes to the shell.
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J. VANDEESTOEP AND J. F. RICHARDS
TABLE 1.—Average cumulative partial load deformation changes of fertile and infertile eggs during incubation after pre-incubation storage at room and refrigerated temperatures for 3 and 12 days Incubation time (days)
Treatment 3 days— room
3 days— refrig.
12 days— room
12 days— refng.
Fertile eggs microns
s
0.32 1.44 2.24 2.74 6.06 8.42 10.85 12.75 («=21)
0.52 1.03 1.29 2.06 4.73 6.60 9.06 9.68 (« = 21)
5 10 12 14 16 17 18 19
0.33 0.25 1.47 1.52 1.74 1.71 1.66 1.88 («=9)
Infertile eggs 0.85 0.35 1.45 0.35 1.45 0.35 1.00 0.70 1.60 0.98 2.57 0.88 2.64 0.88 2.29 0.90 (m = 10) (» = 10)
1.00 1.28 2.50 3.19 5.63 7.25 10.37 11.81 («=8)
0.14 0.54 1.37 1.81 4.32 7.21 9.29 11.49 (n=22) -0.75 -0.08 0.40 1.38 1.73 1.38 1.55 1.53 (» = 10)
times of measurement and totaling these differences. Then average cumulative deformation was calculated for each treatment and time of incubation (Table 1). Although storage had no effect on the initial deformation value, the P.L. cumulative deformation values during incubation were almost invariably lower for refrigerated fertile eggs than for those stored at room temperature. The cumulative deformation of infertiles remained fairly constant during the incubation period. Average cumulative P.L. deformation for all treatments combined increased slowly up to day 10, more rapidly from day 10 to 14 at a greatly increased rate from day 14 to 18 and at a slightly but consistently reduced rate from day 18 to 20 (Fig. 1). This relationship is identical to that found by Richards and Robinson (1967). The pattern of deformation change may reflect the chemical and morphological changes that are taking place in the developing embryo. Kroon (1940) noted
For individual hen data, cumulative P.L. deformation change with time showed the typical increase in rate of change. The average cumulative P.L. deformation change during incubation was similar to the relationship found in Experiment 1 (curve A, Fig. 3). A similar relationship
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10 12 14 16 17 18 19
a considerable increase in wet weights and ash contents of embryos on the 1213th day and that at day 17 the wet weight started to increase less rapidly but that ash continued to increase rapidly. Johnson and Comar (1955) reported that their results indicated that shell calcium was starting to be mobilized at about 1011 days. In Experiment 2, three eggs per hen per incubation period were initially used, but due to infertility and early embryonic death, some of the measurements were invalid and were discarded. The actual P.L. deformation values of the remaining eggs indicated a general trend of increasing deformation as incubation time increased. Using these values to compute an overall average deformation value for all hens gave curve A of Figure 2. This curve is similar to Figure 1 with the exception that in the present case there is a decrease starting at day 18. It is felt that this is due to the fact that data from different eggs were used to calculate the average value at each incubation time. To support this contention an average deformation value based on only those eggs incubated to hatching was calculated and plotted (curve B of Figure 2). The deformation value at each incubation time in this instance is an average of the measurements at that time on the same eggs. This curve shows the typical increase in deformation starting at day 10 with a sharp increase in rate of change at day 14 and a decline in rate of increase between day 18 and 20.
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SHELL STRENGTH AND INCUBATION
o
o FERTILES
o
o INFERTILES
10 INCUBATION
12
14
16
18
20
PERIOD (DAYS)
FIG. 1. Average cumulative partial load deformation change of egg shells during incubation. Exp. 1
was obtained using only those eggs incubated to hatching in the calculation of averages (curve B, Fig. 3). Using the Bellows compression unit, crushing strength and total deformation were determined on the eggs as they were removed from the incubator. From this data deformation per 500 gram load was computed. Average deformation increased inconsistently during incubation, the inconsistency probably being due to the inclusion of different eggs in the calculation of the averages (Table 2). Generally the Bellows deformation value for any particular egg as computed from crushing strength and total defor-
mation data was higher than the P.L. deformation value. In an attempt to calculate a cumulative deformation change (Bellows method), the deformation at each incubation time was subtracted from the average deformation of the base pool eggs. These values also increased inconsistently during incubation (Table 2). A plot of the average P.L. deformation, crushing strength, percent shell and shell thickness (Y) vs. time (X) is presented in Fig. 4. Analyses of the relationships among these variables were performed on data up to and including the eighteenth day of incubation. Data for the twentieth day
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8
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J. VANDERSTOEP AND J. F. RICHARDS
10
12
14
16
18
20
FIG. 2. Partial load deformation change of egg shells during incubation for Exp. 2. Curve A represents the change in P.L. deformation as an average for all hens. Curve B is an average for those eggs incubated to hatching.
were excluded because of the inconsistency introduced by egg to egg variation. Analysis of P.L. deformation vs. crushing strength indicated a highly significant correlation (r=—.853). Similar analyses of percent shell and shell thickness vs. crushing strength indicated highly significant correlation coefficients (r) of +0.773 and +0.778 respectively. Analysis of average values for P.L. deformation, percent shell and shell thickness vs. crushing strength indicated highly significant correlation coefficients (r) of -0.968, +0.986 and +0.985 respectively.
It would appear from the data so far that P.L. deformation, percent shell and shell thickness are accurate indicators of shell strength. Regression of each of the four variables (Y) against time (X) gave slopes (b) and correlation coefficients (r) as reported in Table 3. Using all observations in the analysis resulted in highly significant ( P < .01)' correlation coefficients (r) for P.L. deformation and crushing strength, a significant correlation (P<.05) for shell thickness and a non-significant correlation ( P > .05) for percent shell. However, when average values were used deformation,
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8
INCUBATION PERIOD (DAYS)
281
SHELL STRENGTH AND INCUBATION
12
CURVE
A
10 CURVE B
z o < O
6
IU
Q
^ 4
i 2 •
6
8 INCUBATION
10
12
PERIOD
(DAYS)
14
16
18
20
FIG. 3. Average cumulative partial load deformation change of egg shells during incubation. Exp. 2. Curve A represents the change in P.L. deformation as an average for all hens. Curve B is an average for those eggs incubated to hatching. crushing strength and percent shell were significantly (P < 0.05) correlated to incubation time and the correlation of shell thickness with incubation time was highly significant ( P < 0 . 0 1 ) . I t is apparent from Fig. 4 t h a t the relationships of t h e variables ( F ) to time (X) are not linear even when d a t a for t h e twentieth day are excluded. Correlation coefficients increased and in most cases t h e confidence level increased when t h e quadratic equation F = a - r - J X + c ( X 2 ) was fitted to the d a t a (Table 4). T h e regression and correlation analyses, combined with Fig. 4 indicate t h a t defor-
mation, crushing strength, percent shell and shell thckness measurements are capable of detecting a change in shell strength over time during incubation. TABLE 2.—Average Bellows deformation and average cumulative Bellows deformation change of egg shells during incubation Incubation time
Deformation (n.)
Cumulative deformation change (/».)
10 12 14 16 18 20
35.49 41.28 39.10 42.80 46.27 42.52
5.20 9.75 8.92 11.75 14.95 11.60
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s
282
J. VANDERSTOEP AND J. F. RICHARDS
16
18
20
0.010
INCUBATION PERIOD (DAYS)
FIG. 4. Change in partial load deformation, crushing strength, % shell and shell thickness of egg shells during incubation. Exp. 2. Values of repeatability of measurement or precision were established as 0.25/*., 10.0 gms., 0.10 percent, and 0.0002 inches for deformation, crushing strength, percent shell and shell thickness respectively (Richards and Vanderstoep, 1968). A measure of sensitivity was then denned as the change per day as a percent of precision. The regression coefficients (b) from the analysis of averages (Table 3) were used as a measure of the amount of change per day. This procedure resulted in sensitivities of 490, 250, 52 and 52% for deformation, crushing strength, percent shell and shell thickness respectively. This indi-
cates that the sensitivity of percent shell and shell thickness is much lower than that of deformation and crushing strength. Thus crushing strength and particularly deformation are superior measures of the change in shell strength over time during incubation. It might be possible to explain the insensitivity of shell thickness by the work of Tyler and Simkiss (1959), who showed that during incubation, the outer shell membrane comes away from the shell. Rather than coming away cleanly, the membrane carries with it ends of the mammillary knobs including the mam-
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14
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SHELL STRENGTH AND INCUBATION
TABLE 4.—Multiple regression coefficients and correla-
tion coefficients for each shell parameter (F) vs. time of incubation (X) All observations (» = 74) b
c
r
5.44 + 237.49 + 0.21 +0.13X10"*
+ 0.24 — 12.80 -0.92X10-2 -0.84X10-S
+0.44** -0.574" -0.203n.s. -0.244*
Variable (Y) Deformation Crushing Strength Percent Shell Shell Thickness
Average (»=5)
Deformation Crushing Strength Percent Shell Shell Thickness
r
b
c
5.66 + 246.80 + 0.21 +0.45X10-<
+ 0.25 13.12 -0.93X10-2 -0.54X10-'
II
Variable (F)
** (P<.01) * (P<-05) n.s. =non-significant.
sis of a two-tailed paired comparison /-test (Table 5). The mean P.L. deformation value for the intact eggs was not significantly different from the mean value for the same eggs with the membrane removed but with shells still wet. The mean values for the eggs with contents removed and the shells dried and the eggs with the membrane removed and the shells dried were not significantly different. There was, however, a highly significant difference TABLE 5.—Mean partial load deformation values and
TABLE 3.—Simple linear regression coefficients and
correlation coefficients for each shell parameter (F) vs. time of incubation {X) All observations (» = 74)
results of paired comparison t-tests between pairs of shell treatments Treatment comparison
drfomation
'"value
-ft-
Variable (F) Deformation Crushing Strength Percent Shell Shell Thickness
b
r
+ 1.2582 +0.405** -121.343 -0.558** 0.0518 -0.187n.s. 0.00109 -0.240* Average (« = 5)
Variable (F)
b
r
Deformation Crushing Strength Percent Shell Shell Thickness
+ 1.2295 -120.50 0.052 0.00105
+0.900* -0.956* -0.918* -0.988**
** (P<.01) * (P<.05) n.s. = non-significant.
Intact vs. membrane removed and wet
30.00 29.83
1.07 n.s.
Intact vs. membrane removed and dry
30.00 29.50
3.57"
Contents removed and wet vs. contents removed and dry
30.36 29.38
6.06**
Contents removed and wet vs. membrane removed and dry
30.36 29.50
5.02**
Contents removed and dry vs. membrane removed and wet
29.38 29.83
2.59*
Contents removed and dry vs. membrane removed and dry
29.38 29.50
-0.77 n.s.
Membrane removed and wet vs. membrane removed and dry
29.83 29.50
2.27*
* (P<0.05) " (P<0.01) n.s. =non-significant.
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miliary cores. This could reduce shell strength considerably, but it would be by localized removal of the shell material rather than uniform removal. The fact that the measuring surface of the dial micrometer is large in comparison to the mammillary core areas, could account for the relatively minor change in shell thickness over time. Finally, a rather general and superficial study was made of the loosening of the shell membranes during incubation. As a general observation, it can be said that a very slight amount of loosening had started by the tenth day, and that by the sixteenth day the membranes were quite loose and extremely loose by the eighteenth day. This general observation agrees with the work carried out by Tyler and Simkiss (1959). Experiment 3 was carried out to determine if the change in P.L. deformation which occurs during incubation can be accounted for solely by changes in the shell itself, as suggested by the work of Johnson and Comar (1955) or whether membrane loosening influenced the readings. The analysis of the data was on the ba-
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J. VANDERSTOEP AND J. F. RICHARDS
SUMMARY
It has been found that during incubation, shell strength, as measured by deformation under a unit load, decreased with time. The rate of change in deformation increased most rapidly during the period from the 14th to the 18th day and decreased in rate between the 18th and 20th day. Although deformation, shell weight as a percentage of initial egg weight and
shell thickness at any particular time during incubation were found to be well correlated to crushing strength, deformation was more sensitive than percent shell and shell thickness to changes in shell strength during incubation. By measuring the deformation of the eggs intact, with contents removed and with shell membranes removed, it was found that shell membranes loosened during the period of greatest shell strength change but did not affect the shell strength. ACKNOWLEDGEMENTS
The financial assistance of the National Research Council of Canada in this study is appreciated. REFERENCES Driggers, J. C , R. L. Shirley, G. K. Davis and N. R. Mehrhof, 1951. The transference of radioactive calcium and phosphorus from hen to chick. Poultry Sci. 30: 199-204. Hunt, J. R., and P. W. Voisey, 1966. Physical properties of egg shells. 1. Relationship of resistance to compression and force at failure of egg shells. Poultry Sci. 45: 1398-1404. Johnson, P. M., and C. L. Comar, 1955. Distribution and contribution of calcium from the albumen yolk and shell to the developing chick embryo. Am. J. Phys. 183: 365-370. Kroon, D. B., 1940. Distribution of Ca and P during the growth of the chicken embryo. Acta Brevia Neerlandia, X: 128-129. Lee, J., J. F. Richards and L. M. Staley, 1968. Influence of several factors on egg shell strength and its prediction. Can. J. An. Sci. 48: 163-169. Plimmer, R. H. A., and J. Lowndes, 1924. The changes in the lime content of the hen's egg during development. Biochem. J. 18: 1163. Richards, J. F., and M. L. Robinson, 1967. Unpublished data. Richards, J. F., and L. M. Staley, 1967. The relationship between crushing strength, deformation and other physical measurements of the hen's egg. Poultry Sci. 46: 430-437. Richards, J. F., and J. Vanderstoep, 1968. Unpublished data. Romanoff, A. L., 1929. Effect of humidity on the growth, calcium metabolism and mortality of the chick embryo. J. Exp. Zool. 5: 343-348.
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between the mean values for the intact eggs and the eggs with the membrane removed and the shells dried. There was a significant difference between the values for the eggs with contents removed and shells dried and the eggs with the shell membranes removed but the shell still wet. Highly significant differences existed between the values for the eggs with contents removed and shells wet and those with the shell membranes removed and the shells dried. I t appears from the above data that shell membranes do not influence shell strength but rather that the differences obtained are due to the wetness of the shell. This is substantiated by the fact that highly significant differences were found between the eggs with contents removed and the shells wet and those with the shells dried. Further there were significant differences between values for the eggs with shell membranes removed and shells wet and those with the shells dried. These facts substantiate the previously reported finding that mositure weakens the shell (Tyler and Geake, 1964; and Leeetal., 1968). The results of the study of the effect of membranes on shell strength are similar to those reported by Tyler and Geake (1964) even though methods of membrane removal and strength evaluation were different.
SHELL STRENGTH AND INCUBATION Romanoff, A. L., and A. J. Romanoff, 1949. The Avian Egg. Chapman and Hall Ltd. London. Romanoff, A. L., and L. W. Taylor, 1949. Fertility and Hatchability of Chicken and Turkey Eggs. J. Wiley and Sons Inc., N. Y., p. 432. Schoorl, P., and H. Y. Boersma, 1962. Research on the quality of the egg shell. Proc. 12th World's Poultry Cong.: 432-435. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Company.ync, New York.
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Tyler, C , 1961. Shell strength: Its measurement and relationship to other factors. Brit. Poultry Sci. 2: 3-19. Tyler, C , and F. H. Geake, 1964. The testing of methods for cracking egg shells, based on paired readings from individual eggs and the measurement of some effects of various treatments. Brit. Poultry Sci. 5: 19-28. Tyler, C , and K. Simkiss, 1959. Studies on egg shells. XII. Some changes in the shell during incubation. J. Sc. Food Agric. 11: 611-615.
D. E. TURK AND J. F. STEPHENS 3 Poultry Science Department Clemson University, Clemson, S. C. 29631 (Received for publication September 22, 1969)
W
E HAVE previously described the effects of several types of chicken coccidiosis upon the absorption of orally administered zinc-65, iodine-131-labeled oleic acid, proteins, and amino acids into the bloodstreams of growing birds (Turk and Stephens, 1966; 1967 a,b; 1969 a,b). In all of these studies, acute infections of single coccidial species were allowed to completetheircycles. In the field, however, coccidial infections are not usually allowed to run their courses, but are treated in order to minimize the adverse effects of the disease. The ability of the coccidial treatments to ameliorate the severe effects that coccidiosis has upon nutrient absorption is unknown, therefore a series of trials 1
This study was supported in part by Public Health Service Research grant #AM-09189 from the National Institute of Arthritis and Metabolic Diseases. 2 Published with the approval of the Director of the S. C. Agricultural Experiment Station as technical contribution number 822. 3 Present address: Poultry Science Department, The Ohio State University, Columbus, Ohio 43210.
was undertaken to determine the effect of an interrupted coccidial infection upon the ability of young birds to absorb zinc. Data pertaining to growth rate and intestinal damage, as well as Zn absorption, were collected to provide information on the severity of the induced infection, the effects of the treatments on the severity of the infection, and the relationship of these variables to the rate of absorption of orally administered Zn-65. Sulfaquinoxaline was used to interrupt the induced E. necatrix infection since this is an effective coccidiostat for treatment of E. necatrix infections (Grumbles et al., 1948). PROCEDURE
Day-old commercial broiler male chicks were raised to four weeks of age in electrically heated batteries with ad libitum access to a non-medicated corn-soy chick starting ration and to tap water. At four weeks of age, the birds were weighed and assigned at random to one of the experimental groups. One group in each trial was re-
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Eimeria Necatrix and Zinc Absorption in the Chick: Effect of Sulfaquinoxaline Treatment of the Infection12