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Oxygen diffusion across a sea turtle (Chelonia mydas) egg shell
Comp. Biochem. Physiol., 1972, Vol. 43A, pp. 905 to 909. Pergamon Press. Printed in Great Britain
OXYGEN DIFFUSION ACROSS A SEA TURTLE (CHELOiVIA MYDAS) EGG SHELL* RALPH
A. ACKERMAN
and HENRY
D. PRANGE
Department of Zoology, University of Florida, Gainesville, Florida 32601
(Received 18 March 1972) Abstract-l. The egg shell and outer membrane of the turtle egg have a mean diffusion coefficient K of 6.59 x 10-O cm9 STP set-l cm-a mm Hg-‘. 2. This coefficient is approximately twice that of the chick egg shell and outer shell membrane. INTRODUCTION
SEA TURTLES bury their eggs in nest chambers several feet below the surface of the nesting beach. The sand at this level has been shown to be relatively saturated with water (Bustard & Greenham, 1969). The depth and saturation of the sand probably impede the exchange of gases between the nest chamber and the atmosphere. This assumption is supported by our measurements which show low oxygen levels (18 per cent) and high carbon dioxide levels (‘2 per cent) in the chamber several weeks prior to hatching. Little else has been published on the ecology of the nest or the physiology of gas exchange in the turtle embryo. The gas exchange of the avian embryo is better known (Kutchai & Steen, 1971; Wangensteen & Rahn, 1970-71; Wangensteen et al., 1970-71). While the similarities between the eggs of the two groups are clear, the supposition can be made that the turtle embryo faces a rather different environment and should show different physiological responses. The metabolic rate, blood chemistry or the diffusion of gases through the egg shell and associated membranes could show adaptations to that environment. We report here on the diffusion of oxygen across the egg shell and outer membrane of the embryonic green sea turtle (Chelmia mydm). MATERIALS
AND METHODS
Nineteen green turtle eggs were collected on 29 August, 1971 at The Green Turtle Station, Tortuguero, Costa Rica from a single nest shortly after they were laid. The eggs were packed in sand and hand carried to our laboratory. Through the incubation period the eggs were kept moist and maintained at room temperature (23-25°C). Eighteen of the nineteen eggs hatched 94 days after they were laid. The data used were derived from eight experiments on sections from five eggs. The sections were removed from eggs which had incubated for at least 90 days. Data taken from + This work was supported by a N.I.H. Biomedical Sciences Grant to the Division of Sponsored Research of the University of Florida. 905
906
RALPH
A. ACKERMAN ANDHENRY D. PRANCE
eggs which had hatched indicated very low permeabilities and were excluded from the results. Diffusion was measured with an apparatus similar in design to that used by Kutchai & Steen (1971), see Fig. 1. A section of shell (at least 15 mm dia.) was cut from the top of an egg and the inner shell membrane and other material stripped away. The preparation was
._
shell
Upper
Gas
Lower
u-
Oxygen
chamber
port
chamber
electrode
FIG. 1. Apparatus used for the measurement of oxygen diffusion through the turtle egg shell. then tightly clamped between the two chambers of the apparatus. The upper chamber was flushed continuously with water-saturated air. The lower chamber was flushed with watersaturated nitrogen and then sealed. The partial pressure of oxygen in the lower chamber was detected with a Pb-Ag oxygen electrode (1 mil Teflon membrane) which gave a current output linear with partial pressure. Calculations of permeability were based on the derivation developed by Wangensteen et al. (1970-71) for an ideal two compartment system where the enclosed compartment is well mixed and the principal resistance to gas transfer is the shell preparation. Under these conditions, the equation,
$) =K'A(C, - C,) describes the amount of change of a specific gas where S, are the moles of gas in the enclosed chamber, C, is the concentration of gas in the enclosed chamber (moles cm-3), C,, is the concentration of gas in the open chamber (moles cm-3), A is the area of the shell preparation (cm8), K’ is the permeability of the shell preparation (cm set-l) and t is the time in sec. Conversion of gas concentration to partial pressure and integration yield ln( 1 -P,JP,,) = (-WA/ V)t where P,, the partial pressure of the open chamber, is constant, and P,,the partial pressure of the closed chamber, is 0 at t = 0. V is the volume of the closed chamber. The permeability, K’ (cm se+), can be converted to units more useful for measurement of the volume
TURTLEEGG: OXYGENDIFFUSION
907
of gas transported under specified conditions and corrected to STP so that K’(273/760)
T-l = K (cm3 set-’
cm-* mm Hg-l)
STP,
where T is the absolute temperature (“K) If equation (2) is solved for K’, substitution of K then yields K = (0*359)1n(l -PJP,,)V/tAT.
(3)
The permeability is then calculated from the experimental data.
RESULTS
The permeability coefficient for oxygen (K,,) was calculated at the time where the Pot in the enclosed chamber equaled one half of the PO, in the open chamber. The mean Kos for the eight runs was 6.59 x 10~~ ( f 3*08 x 1O-6 S.D.). The data from a typical run are shown in Fig. 2. When these data are transformed to the terms of equation (2) and plotted semi-logarithmically (Fig. 3) it can be seen that they fit very closely the linear relation which, with -K’A/Y as the slope, is predicted by the derivation for an ideal system.
.
.
.* . . . .
.
.
.
.
. . . . .
Ii
Jo
20
Time
,
40
A
min
FIG. 2. Data from a representative experiment showing change in partial pressure of oxygen in the lower chamber as oxygen diffuses through a 177 mm= section of turtle egg shell. At time zero lower chamber filled with water-saturated nitrogen at one atmosphere pressure; upper chamber continuously flushed with water saturated air at one atmosphere pressure. 31
908
RALPH
A. ACKERMAN ANDHENRY D. PRANCE
. ,_
pe PO
2
. \
. \ t.
. . \
O.II 0
20
D
Time
,
30
40
50
mln
FIG. 3. Semilog plot of the data from Fig. 2. The straight line represents rate of diffusion predicted for an ideal system by the equation ln(l-PJP,) = (-KA/V)t where P, is the PO, in the lower chamber, PO is the PO, in the upper chamber, K is the diffusion coefficient, A is the area of the egg shell preparation and Y is the volume of the lower chamber. DISCUSSION
Our data show that the mean diffusion coefficient for the turtle egg shell (6.59 x 10” + 3.08 x 10 --8 S.D.) is about twice the value for the shell of a chick embryo (3.1 x 10~~f 1*2x 10ms S.D.) found by Wangensteen et al. (1970-71). These authors have also shown that the outer shell membrane has little or no effect on K in the chick. We have made this assumption for our turtle preparations. If we assume also that the outer shell membranes of both eggs are of similar thickness and subtract the outer membrane of the turtle from the total preparation thickness, the average thickness for the shell becomes about 0.33 mm. This is nearly identical to the thickness of the chick egg shell (O-30 mm). If we assume that gas transfer is by gas filled pores, as did Wangensteen et al. (1970-71) for the chick egg, the turtle egg shell must have more pores per unit area or substantially larger pores than the chick egg shell. The turtle egg shell is probably thicker during the earlier stages of incubation. Bustard et al. (1969) report that the shell in the sea turtle egg is the primary source of calcium for the embryo as it develops. All of our eggs were taken from the top of the egg mass and were presumably the last eggs laid. Although all were from the same female, one hundred or more are laid at one time and differing amounts of calcium may be put into the shells. This variation, together with the scaling from the external surface which we observed in some eggs shortly before they hatched,
TURTLEEGG: OXYGENDIFFUSION
909
may account for some of the variation in our data. A much larger sample size and more controlled incubation conditions may resolve this problem. Green turtle eggs normally incubate at 30°C and hatch 61 days after they are laid (Carr & Hirth, 1961). The longer incubation time of our eggs was probably due to the lower temperature at which we maintained them. The hatching success (approximately 95 per cent) suggests that the shell had not deteriorated to any important degree during the prolonged incubation. We believe, therefore, that our data should be applicable to eggs near hatching which have incubated under natural conditions. Despite the twofold difference, the permeability of the turtle and chick egg shell is of the same order of magnitude. The similarity between two very different but evolutionarily related animals suggests that the nest conditions may be more similar than supposed or the shell as a diffusion barrier may be rather rigid from an evolutionary point of view. Acknowledgements-We would like to thank Dr. Archie Carr and the Caribbean Conservation Corporation for their assistance in this study. REFERENCES BUSTARDH. R. & GREENHAMP. (1968) Physical and chemical factors affecting hatching in the green sea turtle, Chelonia my&s (L.). Ecology 49,270-276. BUSTAFUI H. R., SIMKI~~K. & JENKINSN. K. (1969) Some analyses of artificially incubated eggs and hatchlings of green and loggerhead sea turtles. J. 2001. Lond. 158, 311-315. CARRA. & HIRTH H. (1961) Social facilitation in green turtle siblings. A&n. Behao. 9, 68-70. KUTCHAIH. & STEENJ . B. (1971) Permeability of the shell and the shell membranes of hen’s eggs during development. Resp. Physiol. 11, 26.5-278. WAGENSTEEN0. D. & RAHN H. (1970-71) Respiratory gas exchange by the avian embryo. Resp. Physiol. 11, 31-45. WANGENSTEEN 0. D., WILSON D. & RAHN H. (1970-71) Diffusion of gases across the shell of the hen’s egg. Resp. Physiol. 11, 16-30. Key Word Index-green coefficient.
turtle; Cheloniu mydas; oxygen diffusion; turtle egg; diffusion
OXYGEN DIFFUSION ACROSS A SEA TURTLE (CHELOiVIA MYDAS) EGG SHELL* RALPH
A. ACKERMAN
and HENRY
D. PRANGE
Department of Zoology, University of Florida, Gainesville, Florida 32601
(Received 18 March 1972) Abstract-l. The egg shell and outer membrane of the turtle egg have a mean diffusion coefficient K of 6.59 x 10-O cm9 STP set-l cm-a mm Hg-‘. 2. This coefficient is approximately twice that of the chick egg shell and outer shell membrane. INTRODUCTION
SEA TURTLES bury their eggs in nest chambers several feet below the surface of the nesting beach. The sand at this level has been shown to be relatively saturated with water (Bustard & Greenham, 1969). The depth and saturation of the sand probably impede the exchange of gases between the nest chamber and the atmosphere. This assumption is supported by our measurements which show low oxygen levels (18 per cent) and high carbon dioxide levels (‘2 per cent) in the chamber several weeks prior to hatching. Little else has been published on the ecology of the nest or the physiology of gas exchange in the turtle embryo. The gas exchange of the avian embryo is better known (Kutchai & Steen, 1971; Wangensteen & Rahn, 1970-71; Wangensteen et al., 1970-71). While the similarities between the eggs of the two groups are clear, the supposition can be made that the turtle embryo faces a rather different environment and should show different physiological responses. The metabolic rate, blood chemistry or the diffusion of gases through the egg shell and associated membranes could show adaptations to that environment. We report here on the diffusion of oxygen across the egg shell and outer membrane of the embryonic green sea turtle (Chelmia mydm). MATERIALS
AND METHODS
Nineteen green turtle eggs were collected on 29 August, 1971 at The Green Turtle Station, Tortuguero, Costa Rica from a single nest shortly after they were laid. The eggs were packed in sand and hand carried to our laboratory. Through the incubation period the eggs were kept moist and maintained at room temperature (23-25°C). Eighteen of the nineteen eggs hatched 94 days after they were laid. The data used were derived from eight experiments on sections from five eggs. The sections were removed from eggs which had incubated for at least 90 days. Data taken from + This work was supported by a N.I.H. Biomedical Sciences Grant to the Division of Sponsored Research of the University of Florida. 905
906
RALPH
A. ACKERMAN ANDHENRY D. PRANCE
eggs which had hatched indicated very low permeabilities and were excluded from the results. Diffusion was measured with an apparatus similar in design to that used by Kutchai & Steen (1971), see Fig. 1. A section of shell (at least 15 mm dia.) was cut from the top of an egg and the inner shell membrane and other material stripped away. The preparation was
._
shell
Upper
Gas
Lower
u-
Oxygen
chamber
port
chamber
electrode
FIG. 1. Apparatus used for the measurement of oxygen diffusion through the turtle egg shell. then tightly clamped between the two chambers of the apparatus. The upper chamber was flushed continuously with water-saturated air. The lower chamber was flushed with watersaturated nitrogen and then sealed. The partial pressure of oxygen in the lower chamber was detected with a Pb-Ag oxygen electrode (1 mil Teflon membrane) which gave a current output linear with partial pressure. Calculations of permeability were based on the derivation developed by Wangensteen et al. (1970-71) for an ideal two compartment system where the enclosed compartment is well mixed and the principal resistance to gas transfer is the shell preparation. Under these conditions, the equation,
$) =K'A(C, - C,) describes the amount of change of a specific gas where S, are the moles of gas in the enclosed chamber, C, is the concentration of gas in the enclosed chamber (moles cm-3), C,, is the concentration of gas in the open chamber (moles cm-3), A is the area of the shell preparation (cm8), K’ is the permeability of the shell preparation (cm set-l) and t is the time in sec. Conversion of gas concentration to partial pressure and integration yield ln( 1 -P,JP,,) = (-WA/ V)t where P,, the partial pressure of the open chamber, is constant, and P,,the partial pressure of the closed chamber, is 0 at t = 0. V is the volume of the closed chamber. The permeability, K’ (cm se+), can be converted to units more useful for measurement of the volume
TURTLEEGG: OXYGENDIFFUSION
907
of gas transported under specified conditions and corrected to STP so that K’(273/760)
T-l = K (cm3 set-’
cm-* mm Hg-l)
STP,
where T is the absolute temperature (“K) If equation (2) is solved for K’, substitution of K then yields K = (0*359)1n(l -PJP,,)V/tAT.
(3)
The permeability is then calculated from the experimental data.
RESULTS
The permeability coefficient for oxygen (K,,) was calculated at the time where the Pot in the enclosed chamber equaled one half of the PO, in the open chamber. The mean Kos for the eight runs was 6.59 x 10~~ ( f 3*08 x 1O-6 S.D.). The data from a typical run are shown in Fig. 2. When these data are transformed to the terms of equation (2) and plotted semi-logarithmically (Fig. 3) it can be seen that they fit very closely the linear relation which, with -K’A/Y as the slope, is predicted by the derivation for an ideal system.
.
.
.* . . . .
.
.
.
.
. . . . .
Ii
Jo
20
Time
,
40
A
min
FIG. 2. Data from a representative experiment showing change in partial pressure of oxygen in the lower chamber as oxygen diffuses through a 177 mm= section of turtle egg shell. At time zero lower chamber filled with water-saturated nitrogen at one atmosphere pressure; upper chamber continuously flushed with water saturated air at one atmosphere pressure. 31
908
RALPH
A. ACKERMAN ANDHENRY D. PRANCE
. ,_
pe PO
2
. \
. \ t.
. . \
O.II 0
20
D
Time
,
30
40
50
mln
FIG. 3. Semilog plot of the data from Fig. 2. The straight line represents rate of diffusion predicted for an ideal system by the equation ln(l-PJP,) = (-KA/V)t where P, is the PO, in the lower chamber, PO is the PO, in the upper chamber, K is the diffusion coefficient, A is the area of the egg shell preparation and Y is the volume of the lower chamber. DISCUSSION
Our data show that the mean diffusion coefficient for the turtle egg shell (6.59 x 10” + 3.08 x 10 --8 S.D.) is about twice the value for the shell of a chick embryo (3.1 x 10~~f 1*2x 10ms S.D.) found by Wangensteen et al. (1970-71). These authors have also shown that the outer shell membrane has little or no effect on K in the chick. We have made this assumption for our turtle preparations. If we assume also that the outer shell membranes of both eggs are of similar thickness and subtract the outer membrane of the turtle from the total preparation thickness, the average thickness for the shell becomes about 0.33 mm. This is nearly identical to the thickness of the chick egg shell (O-30 mm). If we assume that gas transfer is by gas filled pores, as did Wangensteen et al. (1970-71) for the chick egg, the turtle egg shell must have more pores per unit area or substantially larger pores than the chick egg shell. The turtle egg shell is probably thicker during the earlier stages of incubation. Bustard et al. (1969) report that the shell in the sea turtle egg is the primary source of calcium for the embryo as it develops. All of our eggs were taken from the top of the egg mass and were presumably the last eggs laid. Although all were from the same female, one hundred or more are laid at one time and differing amounts of calcium may be put into the shells. This variation, together with the scaling from the external surface which we observed in some eggs shortly before they hatched,
TURTLEEGG: OXYGENDIFFUSION
909
may account for some of the variation in our data. A much larger sample size and more controlled incubation conditions may resolve this problem. Green turtle eggs normally incubate at 30°C and hatch 61 days after they are laid (Carr & Hirth, 1961). The longer incubation time of our eggs was probably due to the lower temperature at which we maintained them. The hatching success (approximately 95 per cent) suggests that the shell had not deteriorated to any important degree during the prolonged incubation. We believe, therefore, that our data should be applicable to eggs near hatching which have incubated under natural conditions. Despite the twofold difference, the permeability of the turtle and chick egg shell is of the same order of magnitude. The similarity between two very different but evolutionarily related animals suggests that the nest conditions may be more similar than supposed or the shell as a diffusion barrier may be rather rigid from an evolutionary point of view. Acknowledgements-We would like to thank Dr. Archie Carr and the Caribbean Conservation Corporation for their assistance in this study. REFERENCES BUSTARDH. R. & GREENHAMP. (1968) Physical and chemical factors affecting hatching in the green sea turtle, Chelonia my&s (L.). Ecology 49,270-276. BUSTAFUI H. R., SIMKI~~K. & JENKINSN. K. (1969) Some analyses of artificially incubated eggs and hatchlings of green and loggerhead sea turtles. J. 2001. Lond. 158, 311-315. CARRA. & HIRTH H. (1961) Social facilitation in green turtle siblings. A&n. Behao. 9, 68-70. KUTCHAIH. & STEENJ . B. (1971) Permeability of the shell and the shell membranes of hen’s eggs during development. Resp. Physiol. 11, 26.5-278. WAGENSTEEN0. D. & RAHN H. (1970-71) Respiratory gas exchange by the avian embryo. Resp. Physiol. 11, 31-45. WANGENSTEEN 0. D., WILSON D. & RAHN H. (1970-71) Diffusion of gases across the shell of the hen’s egg. Resp. Physiol. 11, 16-30. Key Word Index-green coefficient.
turtle; Cheloniu mydas; oxygen diffusion; turtle egg; diffusion