periods of time. Eggs from precocial species possess several common characteristics relative to their func- tional properties. Ostrich eggs breathe totally by diffu-.
PHYSIOLOGY AND REPRODUCTION Eggshell Conductance and Other Functional Qualities of Ostrich Eggs1 V. L. CHRISTENSEN, G. S. DAVIS,2 and L. A. LUCORE Department of Poultry Science, North Carolina State University, Box 7608, Raleigh, North Carolina 27695-7608 ABSTRACT Eggshell conductance was measured and eggshell conductance constants were calculated for eggs from ostriches. In addition, egg water, yolk, albumen, shell, and total solids were measured in an effort to determine the maternal investment of ostrich eggs. The results of the study suggest that the optimal incubator humidity for ostrich eggs is less than 25% to allow a 15% loss of initial egg mass during the 45-d incubation period. This low humidity does not preclude increasing
humidity during the actual hatching process. In addition, incubation temperatures need to be adjusted to allow a longer development time for the embryo to attain an adequate level of maturity to survive the plateau stage in oxygen consumption or to prevent the use of limited energy of the yolk to survive the anoxia of tucking and internal pipping. The optimal incubation temperature of ostrich eggs appears to be between 36.1 and 36.9 C.
(Key words: eggshell, conductance, ostrich)
INTRODUCTION The ostrich belongs to the biological category of birds termed precocial, in which the offspring hatch in a relatively mature state. The maturity of the hatchling demands little additional maternal care posthatching, in contrast to altricial avian species, such as the canary, which feed and care for their young for extended periods of time. Eggs from precocial species possess several common characteristics relative to their functional properties. Ostrich eggs breathe totally by diffusion through pores in the shell (Wangensteen and Rahn, 1970/1971). In ratites, pores are branched to accomplish this function and provide additional ventilation for the significantly larger egg mass (Board, 1980). Because of diffusive respiration, the ostrich embryo is unable to increase its respiration rate to match its metabolic needs as adult birds do (Rahn et al, 1974). Functional conductance properties of the eggshell for respiratory gases must, therefore, be established by the hen at the time of egg formation to meet the subsequent conflicting requirements for oxygen uptake and carbon dioxide and water vapor ventilation for a growing tissue mass (Rahn, 1981). Furthermore, at a time precisely located and coordinated with embryo maturity, the diffusion properties of the eggshell must be exceeded to provide an adequate concentration of carbon dioxide (6%) within
Received for publication December 5, 1995. Accepted for publication July 1, 1996. lr The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the product named nor criticism of similar ones not mentioned. 2 To whom correspondence should be addressed.
the airspace to facilitate the onset of pulmonary respiration (Windle et al, 1938; Windle and Barcroft, 1938; Visschedijk, 1968 a,b,c) or the timing of hatching and pipping (Rahn et al., 1974), and to allow sufficient water to be lost to prevent drowning (15% of the initial egg mass) (Rahn et al., 1979) and create a metabolic hypoxia (14%) within the airspace (Rahn and Paganelli, 1991). Many ostrich producers, especially those who are novices of incubation and hatching, have experienced poor hatchability and livability of embryos and chicks. Because the eggshell conductance and functional qualities of ostrich eggs are not well defined, elucidation of these parameters can result in recommendations for optimal incubation temperature and humidity. Therefore, the proposed objectives of the current study were: 1) to determine the eggshell conductance and eggshell conductance constants of eggs from ostriches; 2) to determine the maternal investment in ostrich eggs by measuring water, yolk, albumen, shell, and total solids in ostrich eggs; and 3) to examine various eggshell components by scanning electron microscopy.
MATERIALS AND METHODS Seven eggs from laying ostrich hens were obtained over a 2-yr period. The eggs were obtained as closely as possible to the time of oviposition. Immediately after receiving each egg, the physical dimensions of the egg and eggshell and functional characteristics were measured as described below. All gravimetric measurements were made to six significant figures. The means and SEM for the physical and functional characteristics were determined by the PROC MEANS procedure of SAS®
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1996 Poultry Science 75:1404-1410
EGGSHELL CONDUCTANCE OF OSTRICH EGGS
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(SAS Institute, 1989). Data for the distribution of shell pores on different regions of the ostrich eggs were analyzed by the PROC ANOV procedure of SAS® (SAS Institute, 1989).
counting to hydrolyze and removed the eggshell membranes. An additional eggshell thickness measurement with eggshell membranes was then made prior to pore counting.
Shell Conductance
Shell Volume
Eggs were placed in a large desiccator, vented to room air, that contained silica gel and were maintained at 25 C for 6 d and were weighed once per day. Following each weighing the silica gel was stirred with a pencil. Vapor pressure from the silica gel was measured using a prepared chart noting absorption of water vapor by the absorbent over a 14-d period. The silica gel was rehydrated at approximately 7-d intervals or when the vapor pressure of the absorbent became critical. The water vapor pressure inside the egg is saturated, and in the desiccator it is essentially zero. Thus, by dividing the daily mass loss by the saturation vapor pressure of 23.86 m m / Hg (25 C) the water vapor conductance expressed in milligrams per day per millimeter of Hg was obtained. Barometric pressure was recorded daily. This value was corrected to a barometric pressure of one atmosphere (Ar et ah, 1974). The daily values obtained for each egg were then averaged.
This was calculated from the relationship Volume = A x L; where A = surface area of the egg (square centimeters); and L = thickness of the shell (centimeters) (Rahn et ah, 1981).
A specially designed egg holder for clamping eggs replaced the scale pan of a Mettler balance (Rahn et al., 1981). With this device, eggs were weighed dry and weighed again while submerged in a beaker of distilled water (making appropriate tare corrections for egg holder in air and water). The volume was obtained by Archimedes' principle using the difference in mass divided by the density of water at the water temperature.
Initial Egg Mass Eggs were candled to visualize the air cell. The eggshell and air cell were punctured with a hypodermic needle and injected with distilled water, which displaced the air volume. Initial egg mass was the mass of the egg immediately following injection.
Shell Weights Eggs were emptied, internally washed with water to remove all albumen, and dried for several days before weighing. Shell weights include the shell membrane.
Shell Thickness Shells were broken to obtain representative areas except at the pointed end. With ball pointed calipers, 12 areas on each egg were measured to four significant figures and averaged. Shell thickness also includes membrane thickness. The shells were subsequently boiled for 10 min in a 5% NaOH solution preparatory to pore
Representative sections of the shells were boiled in 5% NaOH for 5 to 10 min to remove cuticle and membranes. After washing and drying, these pieces were etched for 10 to 15 s in concentrated nitric acid and again washed and dried. The shell sections were painted on the inside with a concentrated aqueous solution of methylene blue. The solution taken up by the pores then could be seen easily with the aid of a dissecting microscope. The pores of 1 cm 2 were counted. Twenty areas for each egg were used to establish a mean value, for each of three areas, (i.e., the top, equator, and bottom of each egg (a total of 60 areas per egg), which was multiplied by the total surface area of the egg to estimate the total number of pores per egg.
Surface Area of Egg The surface area of the egg was estimated from the allometric relationship in which area (square centimeters) = 4.835 W 0 - 662 and W = initial egg mass (Paganelli et al, 1974).
Total Pore Area Total pore area was calculated from the relationship Ap = 0.447 G x L; where Ap = total effective pore area (square millimeters); G = water vapor conductance, milligrams per day per millimeter of Hg; L = length of the pore or shell thickness (millimeters); and 0.447 is a constant at 25 C based on the diffusion coefficient of water vapor, the gas constant R, and the absolute temperature T (Rahn et al., 1974).
Pore Radius Pore radius was calculated from the relationship R = x IT; where R = effective pore radius, assuming that the pores have a constant diameter (micrometers); Ap = total effective pore area (square millimeters); N = number of pores in egg, ir = 3.14; and 1000 = constant to adjust the dimensions to micrometers.
VAp/N
Egg Components Eggs were separated carefully into yolk, albumen, and shell, and placed into tared vessels. All components were
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Volume Determination
Pore Counts
CHRISTENSEN ET AL.
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weighed wet then dried at 65 C for several days until a constant weight was achieved. The terminal values were used as the dried value for each component.
Electron
Microscopy
Ostrich eggshell surfaces and membranes of two eggs were examined by scanning electron microscopy. An ostrich egg was broken into 0.5 cm or smaller pieces by fracturing the shell with a hammer. Shell samples were randomly collected and fixed in 3% glutaraldehyde for 24 h. All membrane samples were lightly rinsed with distilled water and fixed in 3% glutaraldehyde for 2 h. The specimens were critical point dried for 15 min. The specimens were sputter coated with approximately 18 pM of gold palladium alloy and examined on a Phillips 505T scanning electron microscope at 7 KV.
Electron micrographs of ostrich eggshell and egg membranes are shown in Figures 1 to 5. Similar chicken
FIGURE 1. Cross section of the ostrich egg showing the branched pore system (a), palisade (b), and cone (c) layers of the shell. Artifacts (x) are cuticle fused by gold palladium sputter coating.
FIGURE 2. Surface of an ostrich egg revealing cracked cuticle.
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RESULTS AND DISCUSSION
egg structures that have been previously examined by electron microscopy were also apparent in the ostrich eggs (Tullett, 1984). The ostrich egg contains a cuticle, pores, and palisade, cone, and mammillary layers, as well as an outer and inner shell membrane. Comparisons between chicken and ostrich eggs that are mentioned below were made based on previous electron micrographs from our laboratory (Lucore et ah, 1995) and from work presented by Solomon (1991). As shown in Figure 1, the ostrich egg has pores within the shell, but they are branched rather than the straight pore structure observed in the chicken egg. The ostrich egg pore starts with one opening on the surface of the shell that then branches out within the shell. Although many pore branches open into the interior of the shell, some do not. The palisade, cone, and mammillary layers are present (Figure 1). The cuticle appears as a highly fissured surface (Figure 2). No pores are visible from the outer surface of the shell. Figure 3 shows the palisade layer, which reveals vesicular holes and the crystalline structure of the shell. Viewing the cone layer from the interior of the egg (Figure 4), the fusion between the cone and palisade layer is less than
EGGSHELL CONDUCTANCE OF OSTRTCH EGGS
The data collected in the present study are potentially useful to the ostrich breeder in several ways. The measurements infer changes that could be made in the incubation procedures of ostrich eggs that could improve hatchability of ostrich eggs and improve hatchling quality. Our values for eggshell conductance were lower
than previously published values (Ar and Rahn, 1978). One reason may be that the age of hen and genetics within a species have been shown to influence eggshell conductance (Christensen and Nestor, 1994; Christensen et al, 1995). Three measurements of eggs have been shown to be interdependent, i.e., they always change on an interspecies basis in proportion to one another. The interdependence suggests allometry among three egg measurements (Ar and Rahn, 1978). This relationship has been described in mathematical form as a conductance constant (k). The formula is: k = (G x I)/W; where G = eggshell conductance; I = the incubation period of the egg; and W = the initial egg mass (Ar and Rahn, 1978). We measured a k value of 3.28 in the current study (Table 3). This finding suggests that the incubation period (I) of ostrich eggs, based on the egg mass (W) measured in the present study, may be shorter than would be predicted from the conductance relationship. There is confusion among authors about the incubation period for ostrich eggs. Some indicate 45 d (Ar and Rahn, 1978) but others have suggested 42 d (Swart et al., 1987). For purposes of our calculations, we assumed that
FIGURE 3. Palisade layer reveals vesicular holes (v), and crystalline structure of the shell.
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that observed in chicken eggs (Tullett, 1984). This reduction in fusion is probably due to the branched pore system of ostrich eggs, which requires more interior surface areas. Figure 5 shows the filament structures of the outer shell membrane. Similar to chickens (Tullett, 1984), the outer membrane has thicker filaments than the inner membrane. Overall, the structures of the ostrich egg are similar to those of the chicken egg (Solomon, 1991). However, each structure of the ostrich egg differs slightly from its smaller counterpart. The physical dimensions of the ostrich eggs are given in Table 1. The eggs are considerably larger than other poultry eggs measured previously for eggshell conductance determination. The values for dimensions agree with those published by Ar et al. (1974). The physical dimensions of the ostrich eggshell are given in Table 2. The eggshell represents about 19% of the total egg mass, which is higher than that of most poultry species (Ar et al, 1974). The functional characteristics observed in this study for ostrich eggs were less than those reported by Ar et al. (1974) (Table 3). In addition, the eggshell conductance constant of ostrich eggs computed in the current study (3.28) is lower than that of chickens (5.61), turkeys (4.31), and Japanese quail (5.41), or ostrich (5.10) (Ar and Rahn, 1978). The reason for these differences is unknown. Functional pore area, pore number, and pore radii are all less than would be predicted compared to other species. The distribution of pores seen on the ostrich eggs indicate that they belong to the class of birds in which pores are more numerous on the air cell end of the egg than at the equator or at the small or pointed end of the egg (Table 4). Various species display the converse of this pattern or display equal numbers of pores on all areas of the shell (Tullett and Board, 1977). The percentage of each egg component found in the ostrich eggs examined was also different than that observed for other domestic species (Solomon, 1991) (Tables 5 and 6). The ostrich eggs in the current study were about 66% water and 34% solids, which is typical of eggs from domestic species. However, the form in which the solids were found differed from the typical pattern found in other species (Vleck, 1991). The eggs were 27% yolk and 54% albumen based on wet weight. Ostrich eggs appear to have greater percentages of shell and albumen at the expense of yolk compared to other species. Chickens, for example, have about 35% yolk at the beginning of incubation (Christensen et al., 1995). Percentage shell observed (19%) was also greater than observed in most poultry species (11 to 12%) (Christensen and Nestor, 1994; Christensen et al., 1995).
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CHRISTENSEN ET AL. TABLE 1. Physical dimensions of ostrich eggs (x ± SEM, n = 7)
Egg mass
Volume
Density
(g) 1,470.8 + 108
(cm3) 1,325.7 ± 107
3
(g/ori )
1.11 ± 0.03
Surface area
Width
(cm2) 604.1 ± 29
12.7 ± 0.6
Length — (cm) 16.0 ± 0.5
TABLE 2. Physical dimensions of ostrich eggshells (x ± SEM, n == 7)
Weight
Thickness with membrane
Membrane
(g) 242.5 ± 38
Thickness without membrane
Volume
3
(cm) 0.19 ± 0.03
0.14 + 0.04
Density
0.17 + 0.03
(cm3) 105 ± 16
(g/cm ) 0.23 + 0.01
Pore
Conductance constant
Conductance (mg H 2 0 / d / m m Hg) 106.1 ± 44
Area 2
(mm ) 78.9 ± 21
3.28 + 1.4
Length
Number
(cm) 0.19 + 0.03
11,196 + 8.640
Radius
TABLE 4. Distribution of pores per square centimeter on regions of ostrich eggs (x ± SEM, n = 7)
Region of egg Top
Bottom
Middle A
B
20.2 ± 2.0 A_c
17.7 ± l.ic
18.3 ± 1.6
Means with no common superscript differ significantly (P
