Incubation temperatures and gender

[gtm]

Thanks Dan - very interesting.

[egyptiandan64]

Here's the study in Hermanns Ed
INCUBATION PERIOD AND SEX RATIO OF
Testudo hermanni boettgeri
Bert T. Eendebak
TORTOISE STUDY CENTRE OOSTERBEEK
ABSTRACT
In many tortoise and turtle species the incubation temperature of the egg determines the sex of the
hatchling. For Hermann's tortoises (T. hermanni) however, such data are fragmentary or
unpublished. In this paper, duration of incubation of hatchlings from 312 eggs laid by a group of 10
female T. hermanni boettgeri are described. This incubation period did not vary linearly with
incubation temperature but rather was independent of temperature in the range 29°C to 34°C, being
~57 days throughout. Incubation period changed abruptly to ~83 days for temperatures of 26°C with
the transition range of temperatures lying between 26°C and 28°C. No significant correlation
between incubation period and other environmental parameters could be detected. Sex ratios were
also measured as a function of incubation temperature, with a sex ratio of 50% at a threshold
temperature of 31.5°C and an abrupt change from virtually all males to all females over a narrow
temperature range.
INTRODUCTION
The influence of temperature on the incubation period and sex of chelonians, especially aquatic
species, has been studied by different authors. Extensive studies on Emys orbicularis have been
published by Pieau (1971) and on various Emydinae species by Bull et al. (1982a). A recent review
of temperature-dependent sex determination (TSD) in squamate reptiles is given by Spotila et al.
(1994) and of TSD in turtles by Ewert et al. (1994). However little detailed work has been published
on measurements of the effect of the incubation temperature on the incubation period and sex ratio
of tortoises. In this paper the author reports on a study of the incubation of a total of 741 eggs of
T. hermanni boettgeri from which 312 tortoises hatched successfully. These eggs were laid during
the period 1982-1992 by a group of 10 female T. hermanni boettgeri kept in an outdoor terrarium.
The study was planned to investigate the effects of environmental parameters on the incubation
period and sex ratio of T. hermanni boettgeri. Apart from the temperature, many other parameters
were studied, e.g. characteristics of the parental female, nest location, time of nesting, order of laying
within a clutch, egg weight and humidity during the incubation period.
MATERIALS AND METHODS
Nesting by the colony of captive T. hermanni boettgeri took place in the months of May, June and
July, usually between 10 am and 12 noon. All eggs were removed from the nests, marked with a
pencil, weighed, inspected, and placed in incubators within one to three hours after laying. Three
different types of incubators were used. One incubator used bi-metal control, whereas the other two
used NTC-sensed temperature elements and a temperature sensing bridge or TRIAC triggering
circuit as control units. All these incubators usually regulated the intended temperature to within
about 0.2°C. To achieve this result the bi-metal controller had to be adjusted almost daily.
Within an incubator the eggs were placed on a foam rubber subsoil and positioned in a Latin square
design. Horizontal and vertical thermal gradients within the incubator were minimised by careful
distribution of the heating cables. Variation from the intended temperature was limited to ~0.2°C, as
monitored by movable thermocouple probes. In view of the control of temperature gradients the
approach of the Latin square design as intended by Bull et al. (1982b) is not necessary. To reduce
the influence of unknown genetic or environmental parameters the eggs of one clutch were equally
distributed over the three incubators.
The "incubation period" as used in this study is defined as the time interval between ovi-position and
emergence of the full-grown hatchling. The length of time from pipping of the eggshell to the
emergence of the hatchling is about one day. Therefore the influence on the determination of the
incubation period in small. Immediately after laying, the eggs were inspected for the possible
development of blood-vessels which are normally visible after 6-8 days of incubation. No sign of
blood-vessels were detected, so a hypothetical developmental process within the female is unlikely
and will practically not influence the results. In exceptional cases (~2%) the hatchling did not hatch
after the expected time. In these cases the egg was opened by hand two weeks after the end of the
predetermined incubation period. These exceptional cases were not included in the used data.
The hatchlings were marked individually by making notches in the marginal scutes according to the
system used by the Charles Darwin Station on Santa Cruz Island (Thonton, 1991). Most of these
hatchlings were distributed among members of the Dutch Tortoise Association. Only a relatively
small number (~25%, random selected) were kept until sex determination could take place. These
juveniles were kept in the same outdoor terrarium as the colony of full-grown T. hermanni boettgeri.
The sex of the juveniles was determined by external characteristics such as the shape of the tail,
carapace, plastron or anal scutes (Stubbs et al., 1981). In most cases this determination can take
place with a carapace length of ~10 cm at an age of 3-4 years. Juveniles were defined as male or
female not before at least two different sex characteristics, in general the shape of the tail and anal
scutes, were deciding.
RESULTS
Incubation period
In this study incubation periods of T. hermanni boettgeri were measured at constant incubation
temperatures. From the 741 eggs incubated at a constant temperature, 515 eggs were visibly
fertilized, from which 312 tortoises hatched successfully. Table 1 shows the results of incubation
temperatures of 25°C up to 34°C. Other environmental parameters such as parental female, nest
location, time of nesting, order of laying within a clutch, egg weight and humidity during the
incubation period were studied, but no significant correlation of these parameters with the incubation
period could be found. The numbers however were too small to justify definite conclusions. The eggs
were weighed with an accuracy of ± 0.5 g. Loss of weight of eggs during incubation is on the
average 30-40%. In 4 out of 120 clutches exceptional loss of weight (more than 60%) resulted in
either dead embryos or rather short incubation periods. These exceptions are not included in the
numbers given in Table 1. The results, also presented in Figure 1, show a relatively abrupt change
from a low-temperature incubation period of ~83 days to a high-temperature period of ~57 days. The
transition period is defined as the average of those two periods, and the corresponding temperature
is called the transition temperature. A possible curve that correlates the measure points was found to
be the following mathematical approximation;
y = 70 - 26arctan(3(x-27.5))p-1 [1]
where: y = incubation period [days]
x = incubation temperature [°C]
27.5 = transition temperature [°C]
70 = transition period [days]
Mortality rates for incubation temperatures of 26°C up to 32°C were in the range 20% to 30%. For
incubation temperatures of 25°C, 33°C and 34°C the mortality rate was about 50%, whereas at 24°C
and 35°C (~20 eggs) the mortality rate was almost 100%. Hatchlings incubated at a constant
temperature of 24°C and 25°C are generally small, weak and not able tot hatch successfully, so the
definition of the incubation period at these temperatures is questionable. For that reason the exact
shape of the curve for incubation temperatures below 27°C in Figure 1 cannot be determined.
Sex ratio
During the study it was found that in some cases T. hermanni boettgeri incubated at temperatures of
30°C - 33°C showed their secondary sex characteristics only after a long time. In exceptional cases it
took 8-10 years, which is long compared with the normal situation. This effect can lead to a maleoriented
shift in the measured sex ratio if not all the hatchlings of a specific year (or even better a
specific clutch) are sexed definitively. For that reason only the number of males and females of
clutches from which the sex of all hatchlings was determined were taken into account. A random
selected group of ~25% of the hatchlings (72 out of 312) were kept until sex determination could take
place. Table 2 and Figure 2 give some results of the measured sex ratios (defined as the percentage
male hatchlings) of 72 T. hermanni boettgeri. Figure 2 shows a sharp transition between 30°C and
33°C, with a sex ratio of 50% at a temperature of 31.5°C. The temperature producing a sex ratio of
50% is defined as the threshold temperature.
DISCUSSION
Detailed information on the influence of incubation temperatures on the incubation period and sex
ratio has been reported for Emys orbicularis by Pieau (1971), Pieau and Dorizzi (1981) and for
various Emydinae species by Bull et al. (1982a). Since then temperature dependent sex
determination (TSD) is described in about eight families of turtles. A recent review of TSD is given by
Ewert et al. (1994). For T. hermanni only sparse measurements are reported. Kirsche (1967) found
an average incubation period of 63 days for an incubation temperature of 28°C - 30°C, which is in
accordance with the results of this study. Much longer periods of more than 100 days are reported
for wild populations by Swingland & Stubbs (1985). In the authors outdoor terrarium, some
T. hermanni boettgeri hatched after 110 to 120 days, thanks to a relatively warm and dry Dutch
summer.
In this study incubation temperatures of 24°C to 34°C were used. Constant temperatures below 26°C
and above 32°C lead to increasing mortality rates.
It appears that damaging incubation temperatures are similar to those that are avoided in wild
populations by careful nesting site selection (Meek and Avery (1988), Willemsen (1991)).
The incubation period was almost constant in the range 29°C to 34°C, being ~57 days throughout.
This result indicates that embryonic developmental rates are almost constant in that range. A
mortality rate of almost 100% at an incubation temperature of 24°C indicates that the minimum
temperature for embryonic development for T. hermanni boettgeri is about 23°C to 24°C. Using
these results to test the model suggested by Georges (1989) to predict hatchling sex ratios when
nest temperatures fluctuate will be difficult. In nature nest temperatures will regularly drop below this
minimum temperature of embryonic development.
Sex determination of hatchlings that were incubated at temperatures between 30°C and 33°C was in
some exceptional cases possible only after a long time. This phenomenon can be explained by the
study of Pieau (1975), who has reported on the formation of intersexes of Emys orbicularis,
incubated at a threshold temperature of 28°C to 29°C. Observations of the author however indicate
that "external" sex characters will appear with time in almost all cases. Zaborski et al. (1982)
suggested the possibility that some individuals which were intersexes as hatchling become
phenotypic females afterwards. Whether sex ratio persist until appearance of sexual dimorphism
cannot be determined because sex determination was based only upon external sex characteristics.
Comparison with the results for the emydines shows a similar change from virtually all males to all
females over a narrow temperature range. The threshold temperature for T.hermanni however was
found in this study to be 31.5°C instead of 28°C to 30°C for Emydinae studied by Bull et al. (1982a).
As suggested by Bull and Vogt (1979), different mortality rates of the embryos are not likely to occur
because mortality rates were independent of the incubation temperatures within the region of 26°C to
33°C.
Based on the effect of temperature on sex determination one could expect that relatively cold regions
or periods may lead to temporarily lower threshold temperatures or favored male differentiation. This
theory is not supported by field investigations as performed for Emydinae by Bull et al. (1982b). This
lack of evidence could be explained by compensation with other environmental effects. However in
this study the incubation temperature was the only environmental parameter that had a detectable
influence on the incubation period and sex ratio. Other parameters, such as genetically determined
differences, nest location, time of nesting, order within a clutch, egg weight, and humidity during the
incubation were investigated, but no correlations with the incubation period or sex ratio were found.
Further investigations are needed to confirm these preliminary conclusions.
The most conspicuous difference with incubation in nature are the dayly and seasonal variations in
the incubation temperatures versus the constant incubation temperatures used in this study. In
nature during daylight the soil temperature at the egg location can be up to 18°C higher than at night
(Pieau, 1982) and this effect may easily overrule the influence of a small change in a constant
average temperature. The results of this study show that fluctuations of the environmental
temperature above 28°C does not influence the development rate, but can have a large effect on sex
ratios. To what extent the number of hours of incubation or the embryonic development above the
threshold temperature are decisive for sex determination is still an open question.
For that reason this study is being continued by an investigation on the influence of variations of the
incubation temperature on the sex ratio of T. hermanni boettgeri.
REFERENCES
- Bull, J.J. & Vogt, R.C. (1979) Temperature dependent sex determination in turtles. Science
206, 1186-1188.
- Bull, J.J. (1980) Sex determination in reptiles. Quart.Rev.Biol. 55, 3-21.
- Bull, J.J., McCoy, C.J. & Vogt, R.C. (1982a) Sex determining temperatures in turtles: A
geographic comparison. Evolution 36(2), 326-332.
- Bull, J.J., Vogt, R.C. & Bulmer, M.G. (1982b) Heritability of sex ratio in turtles with
environmental sex determination. Evolution 36(2), 333-341.
- Hailey, A. (1990) Adult survival and recruitment and the explanation of an uneven sex ratio in a
tortoise population. Can.J.Zool. 68, 547-555.
- Ewert, M.A., Jackson, D.R. & Nelson, C.E. (1994) Patterns of temperature-dependent sex
determination in turtles. J.Exp.Zool. 270, 3-15.
- Georges, A. (1989) Female turtles from hot nests: is it duration of incubation or proportion of
development at high temperatures that matters? Oecologia 81, 323-328.
- Kirsche, W. (1967) Zur Haltung, Zucht, und Ethologie der Griechische Landschildkröte.
Salamandra 3, 36-66.
- Kirsche, W. (1979) The housing and regulator breeding of mediterranean tortoises. Int. Zoo
Ybk 19, 42-49.
- Meek, R., Avery, R.A. (1988) Thermoregulation in chelonians. Herpetological Journal Vol 1,
253-259.
- Pieau, C. (1971) Sur la proportion sexuelle chez les embryons de deux cheloniens issus
d'oeufs incubes artificiellement. C.r.hebd.Séanc.Acad.Sci. Paris (D) 272, 3071-3074.
- Pieau, C. (1975) Donnees recentes sur la differentiation en fonction de la temperature chez les
embryons d'Emys orbicularis. Bull.Soc.Zool.France 101, suppl.4, 46-53.
- Pieau, C. & Dorizzi, M. (1981) Determination of Temperature sensitive Stages for Sexual
Differentation of the Gonads in Embryos of the Turtle Emys Orbicularis. Journal of Morphology
170, 373-382.
- Pieau, C. (1982) Modalities of the Action of Sexual Differentiation in Field Developing Embryos
of the European Pond Turtle Emys orbicularis. The Journal of Experimental Zoology 220, 353-
360.
- Spotila, J.R., Spotila, L.D. & Kaufer, N.F. (1994) Molecular mechanism of TSD in reptiles: A
search for the magic bullet. J.Exp.Zool. 270, 117-227.
- Stubbs, D., Hailey, A., Tyler, W. & Pullford, E. (1981) Expedition to Greece 1980. University of
London Natural Society.
- Swingland, I.R. & Stubbs, D. (1985) The ecology of a Mediterranean tortoise (Testudo
hermanni): Reproduction. J. Zool. Lond. (A) 205, 595-610.
- Thonton, I (1971) Darwin's islands. The natural history press, New York.
- Willemsen, R.W. (1991) Differences in thermoregulation between T. hermanni and
T. marginata and their ecological significance. Herpetological Journal Vol 1, 559-567.
- Zaborski, P., Dorizzi, M. & Pieau, C. (1982) H-Y Antigen Expression in temperature sexreversed
turtles (Emys orbicularis). Differentiation 22, 73-78.
TABLES AND FIGURES
Table 1 : Incubation period of Testudo hermanni boettgeri
Incubation
Temperature
[°C]
Number of eggs Mean
Incubation
Period [days]
Standard
deviation
25 2 82.0 0.0
26 7 83.0 3.6
27 3 72.0 2.7
28 20 65.7 3.2
29 10 56.1 0.9
30 31 57.8 1.3
31 48 57.7 2.9
32 64 55.6 3.7
33 101 56.1 3.3
34 26 56.4 3.2
Table 2 : Sex ratios of Testudo hermanni boettgeri
Incubation
Temperature
[°C]
Number of males Number of
females
Sex ratio
%
25 2 0 100
26 4 0 100
28 5 0 100
30 11 0 100
31 11 3 79
32 6 17 26
33 0 11 0
34 0 4 0
Fig. 1 shows the incubation period of Testudo hermanni boettgeri as a function of the incubator
temperature. Data are shown as mean and S.D
Fig. 2 shows the sex ratio of Testudo hermanni boettgeri as a function of the incubator temperature.
Data are shown as mean sex ratios.

Danny

[EJ]

Where was that paper published?

[egyptiandan64]

I have no clue Ed The PDF file I saved doesn't say and I have no clue where I got it. You could google the article, that might help.

Danny

[mlepage15]

I always thought that it was like in humans, that the parents detrmined the sex. Never in a thousand years would I have guessed the incubation temperatures had anything to do with it

[EJ]

I beleive that is a mean temperature.

'Sex ratios were
also measured as a function of incubation temperature, with a sex ratio of 50% at a threshold
temperature of 31.5°C and an abrupt change from virtually all males to all females over a narrow
temperature range.'

Which was my point. There is a range even though it is going to be a narrow range.

It also should be noted that this range might shift dependent on the specific individuals.

As a case in point I have had specific leopards that laid eggs that hatched in the range of 90 to 120 days... one female about 8 clutches. I had another leopard whos eggs hatched in the range of 150 to 180 days with as many clutches... I hope you can see the paralell.

Ed

[Ajay]

It's been translated into French and German. It's now stored on one website: www.schildpadden.akkido.com
Schildkroten means tortoise in German so guessing Swiss maybe? It's interesting anyway. Any more gems like that hidden away Danny?

Yes EJ, there is a range and Danny is correct that 31.5 is the threshold. So 5 QI points to you both and I hope we answered the OP's question

What's are the main differences between incubating the eggs of different species? Just out of interest and to expand on the original topic (and hijack the thread lol)

[EJ]

Not at all... So now when you hear or read TSD in tortoises you'll know it is Temperature Sex Determined not not some form of disease.

Tortoises as well as many reptiles (not all) do not have XY chromosomes. It is the temperature that determins the sex. It's pretty interesting.

Do a google search for Temperature Sex Determination and Gonadal Sex Determination... the net's a wonderful thing.

Originally Posted by mlepage15

I always thought that it was like in humans, that the parents detrmined the sex. Never in a thousand years would I have guessed the incubation temperatures had anything to do with it

[EJ]

Different species have different 'cross over' temperatures.

My point was that diffierent individuals might have different crossover temperatures.

(definately Dutch)(interesting site though)
(got it Danny, CCB)

Originally Posted by Ajay

It's been translated into French and German. It's now stored on one website: www.schildpadden.akkido.com
Schildkroten means tortoise in German so guessing Swiss maybe? It's interesting anyway. Any more gems like that hidden away Danny?

Yes EJ, there is a range and Danny is correct that 31.5 is the threshold. So 5 QI points to you both and I hope we answered the OP's question

What's are the main differences between incubating the eggs of different species? Just out of interest and to expand on the original topic (and hijack the thread lol)

[Ajay]

Originally Posted by EJ

Different species have different 'cross over' temperatures.

My point was that diffierent individuals might have different crossover temperatures.

Exactly, thus the range, I knew what you meant

mlepage: Fascinating isn't it? if a little complicated