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2008

The Effects of Incubation Temperature On Hatching Success,Embryonic Use of Energy and Hatchling Morphology in
the Stripe-tailed Ratsnake Elaphe taeniura
WEI GUO DU1,* AND XIANG JI1,2
1

Hangzhou Key Laboratory for Animal Sciences and Technology, Hangzhou Normal
University, 310036, Hangzhou, Zhejiang, P. R. China
2
Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences,
Nanjing Normal University, Nanjing 210097, Jiangsu, P. R. China*Corresponding author E-mail: dwghz@126.com
Abstract.- We incubated eggs of Elaphe taeniura at 22, 24, 27, 30 and 32°C to examine the effects of incubation temperature on hatching success, embryonic use of energy and hatchling morphology. Incubation temperature affected
incubation length and most hatchling traits examined in this study. Incubation length increased nonlinearly as temperature decreased, with the mean incubation length being 101.7 d at 22°C, 86.0 d at 24°C, 66.3 d at 27°C, 53.9 d at
30°C, and 50.5 d at 32°C. Hatching successes were lower at the two extreme temperatures (41.2% at 32°C, and50.0% at 22°C) than at the other three moderate temperatures (78.1–79.3%). Hatchlings from the extreme high incubation temperature (32°C) were smaller in body size and wet body mass. High incubation temperatures resulted in
production of less developed hatchlings that characteristically had less developed carcasses but contained more unutilized yolks. The proportion of energy transferred from the egg contents to the hatchling was 71.1% at 22°C, 80.2%
at 24°C, 81.5% at 27°C, 82.6% at 30°C and 83.9% at 32°C. Taking the lowest hatching success at 32°C and the substantially prolonged incubation lengths at 22°C into account, we conclude that these temperatures are not suitable forembryonic development in E. taeniura. Our data confirm the prediction that there are some thresholds over whichincubation temperatures can affect hatching success, embryonic use of energy and hatchling morphology.
Keywords.- Reptilia, Colubridae, Elaphe taeniura, egg, incubation, temperature, hatchling phenotype.

Introduction
As in other vertebrate and invertebrate taxa, temperature
may profoundly influence embryonic development in
reptiles. Compared to avian embryos, reptilian embryos
can develop under a relatively wide range of temperatures (Birchard, 2004, Booth, 2004). Low temperatures
slow embryogenesis but usually have little lethal effecton embryos; high temperatures result in faster embryonic development (and thus, shortened incubation or gestation length) but often increase embryonic abnormality or
mortality (e.g. Andrews and Rose, 1994; Andrews et al.,
1997; Deeming and Ferguson, 1991; Sexton andMarion, 1974; Shine and Harlow, 1996). Apart from theeffects on the rate of embryonic development andembryonic abnormality or mortality, thermal environments experienced by developing embryos also affect anumber of phenotypic attributes of the hatchling, including morphology (Du and Ji, 2002; Ji and Du, 2001a, b;
Overall, 1994), energy reserves (Du and Ji, 2001),
behavior (Burger, 1991; 1998), post-hatching growth(Braña and Ji, 2000; Du and Ji, 2003; Rhen and Lang,
1995), and gender in species with temperature-dependent sex determination (Janzen and Paukstis, 1991). It© 2008 by Asiatic Herpetological Researchhas been repeatedly reported for oviparous reptiles thateggs incubated at optimal temperatures not only exhibit
high hatching success but also produce good-quality
hatchlings.
The range of optimal temperatures for reptilian
embryos is often narrow and varies not only among but
also within species. For example, the optimal incubation
temperatures fall within the range from 24°C to 26°C in
Xenochrophis piscator (checkered keelback; Ji et al.,2001) and Deinagkistrodon acutus (five-paced pit-viper;
Lin et al., 2005) but, in Elaphe carinata (king ratsnake;Ji and Du, 2001b), Naja atra (Chinese cobra; Ji and Du,
2001a), Rhabdophis tigrinus lateralis (red-necked keelback; Chen and Ji, 2002), Dinodon rufozonatum (redbanded wolf snake; Ji et al., 1999b; Zhang and Ji, 2002),
Ptyas korros (gray ratsnake; Du and Ji, 2002) and P.
mucosus (mucous ratsnake; Lin and Ji, 2004), generally
within the range from 26°C to 30°C. Pelodiscus sinensis(Chinese soft-shelled turtle), however, has a wider range
of optimal incubation temperatures, because temperatures exert no important effects on hatching success andhatchling phenotypes within the range from 24°C to
34°C (Du and Ji, 2003; Ji et al., 2003). In Eumeces chinensis (Chinese skink), eggs from a lower latitudinal
population have a narrower range of optimal incubation

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Asiatic Herpetological Research, Vol. 11

temperatures than do those from a higher latitudinal
population, primarily because of more stable thermal
environments in the former population (Ji et al., 2002).
Overall, previous studies suggest that optimal temperatures for developing embryos differ among reptiles differing in habitat use and/or distributional range.
The stripe-tailed ratsnake Elaphe taeniura is a large
sized (to 1800 mm SVL [snout-vent length]) oviparous
colubrid snake that ranges from the central and southern
provinces of China to Korea, Burma, Laos, Vietnam andIndia (Zhao, 1993). Wild population of this snake havedeclined dramatically due to habitat loss and over-harvesting over the past two decades (Zhao, 1998).
Fecundity, reproductive output and embryonic mobilization of energy and material during incubation have beenreported for snakes from Zhoushan Islands (Ji et al.,1999a; 2000). Because eggs were never incubated at
multiple temperatures, the range of incubation temperatures optimal for developing embryos of E. taeniura
remains unknown. To fill this gap, we incubated eggs
produced by females from a southern population(Guangxi, China) at five constant temperatures ranging
from 22°C to 32°C. Specifically, our aims are to (1)examine the effects of incubation temperatures on hatching success, embryonic use of energy and hatchling morphology, and (2) determine the range of optimal temperatures for embryos of E. taeniura.

Materials and Methods
We obtained 13 gravid E. taeniura (SVL: 1220–1690
mm; postoviposition body mass: 277.9–755.0 g) in June
1998 from a private hatchery in Guilin (Guangxi, southern China), and brought them to our laboratory inHangzhou, where they were maintained in a 2000 x 800
x 800 (length x width x height) mm wire cage placed ina room inside which air temperatures were never higher
than 30°C. Food (eggs of Coturnix coturnix) and water
were provided ad libitum. The snakes laid eggs between

23 June and 3 July (clutch size: 8.8±0.4, range: 7–11).
We collected the eggs within a few hours after beinglaid. Each egg was measured (to the nearest 0.01 mm)
for length and width with a Mitutoyo digital caliper andweighed (to the nearest 1 mg) on a Mettler balance. One
freshly laid egg from each clutch was dissected to determine the composition of eggs. Egg contents (yolk plus
embryo) were placed in pre-weighed glass dishes, andweighed. Shells were briefly rinsed, dried by blottingwith paper towels and weighed. Egg contents and shells
were weighed again after oven drying to constant massat 65°C. The remaining eggs were incubated systematically at five constant temperatures (22, 24, 27, 30, and32 [± 0.3]°C); such that eggs from single clutches were
distributed almost equally among the five temperature
treatments.
Eggs were individually incubated in covered plastic
jars containing known amounts of vermiculite and distilled water at approximately -12 kPa water potential(vermiculite: water = 1:2). One-third of the egg wasburied lengthwise in the incubating substrate, with thesurface near the embryo exposed to air inside the jar.
Jars were equally assigned to five incubators (Guanzhou
medical instrument, China), with incubation temperatures set at 22, 24, 27, 30, and 32 (± 0.3)°C, respectively.
We moved jars among the shelves in the incubator daily
according to a predetermined schedule to minimize any
effects of thermal gradients inside the incubator. Jars
were weighed on alternate days, and distilled water wasadded evenly into substrates when necessary to compensate for evaporative losses and water absorbed by eggs,
thereby maintaining the substrate water potential constant.
The duration of incubation, measured as the numberof days to pipping, was recorded for each egg.
Hatchlings were collected, measured (for SVL and taillength), and weighed a few hours after hatching, andthen euthanized by freezing to -15°C for determinationof composition and sex. The killed hatchlings were separated into carcass, residual yolk and fat bodies. The

Table 1. The effect of temperature on incubation duration, hatching success, and sex ratio in Elaphe taeniura. Data on
incubation duration are expressed as mean±1 SE.
Duration of incubation (d)Hatching success (%)

Sex ratio(♀♀/♂♂)

22

101.7±1.3

50.0 (6/12)

2/4

24

86.0±0.6

78.1 (25/32)

12/13

27

66.3±0.7

79.2 (19/24)

13/6

30

53.9±0.4

79.3 (23/29)

11/12

32

50.5±0.5

41.2 (7/17)

2/5

Temperature (°C)

18

a

ab
17

a

a

3.9

ab

19

25
16

23

7

6

15

b

14

a

3.3
3.0

6

2.7

a

a

25

19

ab

2.4

23

b

2.1
7

1.8
1.5

5.0
4.8
4.6
4.4

2.4

4.2
4.0

Snout-vent length (mm)

a

3.6

b

Yolk sac Dry mass (g)Dry body mass (g)

5.2

420

c

ab

400

a

a
ab

380
360

b

a

2.0

ab

1.6
1.2

b

ab

b

.8
.4
0.0

b

340
320
300

105

d

100

a

a

95
90

Fat body dry mass (g)1.0Tail length (mm)2008

Asiatic Herpetological Research, Vol. 11

Carcass dry mass (g)Wet body mass (g)

26

a

b

b

85
80
75
70
20

22

24

26

28

30

32

34

Temperature (oC)

Figure 1. Adjusted means (±1 standard error) for bodymass, SVL and tail length of hatchlings from different
incubation temperatures, with initial egg mass being set
at 23.5 g. Adjusted means with different letters differ significantly. Numbers under the error bars in the upper plot
are sample sizes, and are applicable to the other twoplots.

three components of the hatchling were dried in an oven
(65°C) to constant mass, weighed and preserved frozen
for later analyses. We determined the sex of hatchlings
by pressing on both sides of the ventral tail base withforceps to record the presence or absence of hemipenes;
hatchlings with everted hemipenes were recorded as
males.
We extracted non-polar lipids from dried samples of
egg contents and hatchlings in a Soxhlet apparatus for aminimum of 5.5 h using absolute ether as solvent. The
amount of lipids in each sample was determined by sub-

c

.9
.8
.7
.6
.5
20

22

24

26

28

30

32

34

0

Temperature ( C)

Figure 2. Carcass, yolk sac and fat bodies of hatchling
Elaphe taeniura incubated at different temperatures.
Graphs show adjusted mean (± standard error), with initial egg mass as the covariate. Adjusted means with different letters above the error bars differ significantly.
Numbers under the error bars in the upper graph aresample sizes, and apply to all graphs within this figure.

tracting the lipid-free dry mass from the total sample dry
mass. We determined energy density of each dried sample using a GR-3500 adiabatic bomb calorimeter(Changsha Instruments, China).
All data were tested for normality using
Kolmogorov-Smirnov test and for homogeneity of variance using Bartlett’s test. Parametric analyses were used
to analyze data when the assumptions for these analyses
were met; otherwise, nonparametric analyses were used.
Values are presented as mean ± 1 standard error, and thesignificance level is set at α = 0.05.

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Table 2. Lipids and energy in egg contents and shell dry mass of hatched eggs and freshly laid eggs in Elaphe taeniura.
Data are expressed as adjusted mean ±1 SE, with initial egg mass as the covariate.
Egg contents(n = 11)

Hatchlings
22°C (n = 6)

24°C (n = 25)

27°C (n = 19)

30°C (n = 23)

32°C (n = 7)

Lipid mass (g)1.956±0.079

1.082±0.047

1.269±0.039

1.316±0.048

1.362±0.046

1.387±0.101

Energy (kJ)

143.9±4.3

102.3±3.9115.4±2.5117.3±3.1118.9±2.6120.8±4.9

Shell dry mass (g)1.555±0.031

1.401±0.075

1.411±0.011

1.408±0.029

1.380±0.029

1.397±0.030

Results
The mean values for incubation length varied considerably among the five temperature treatments (ANOVA;
F4,75 = 687.2, p < 0.0001). Incubation length decreased
as incubation temperature increased, but not in a linear
pattern. The mean incubation length was shortened by
3.5 d from 30°C to 32°C, 12.4 d from 27°C to 30°C,
19.7 d from 24°C to 27°C, and 15.7 d from 22°C to 24°C
(Table 1). Incubation temperature affected hatching success (G-test, G = 11.51, df = 4, p < 0.05), but not thesexual phenotype of hatchlings (G = 4.73, df = 4,
p > 0.25). Hatching successes were apparently lower at
the two extreme temperatures (22°C and 32°C) than at
the other three moderate temperatures (24, 27, and30°C) (Table 1).
There were no between-sex differences in all examined hatchling variables (all p > 0.05), so we pooled datafor both sexes. All examined hatchling variables, except
yolk sac, were positively correlated with initial eggmass). ANCOVAs with initial egg mass as the covariate
showed that incubation temperatures significantly
affected wet body mass (F4,74 = 3.67, p < 0.01; Fig. 1a),
SVL (F4,74 = 4.21, p < 0.01; Fig. 1c) and tail length (F4,74= 4.31, p < 0.01; Fig. 1d) of hatchlings, but not hatchling
dry body mass (F4,74 = 0.38, p > 0.05; Fig. 1b). Meanvalues for wet body mass, SVL and tail length were allsmaller in hatchlings incubated at 32°C than in hatchlings incubated at the other four temperatures (Fig. 1a, c,d). Hatchlings from different incubation temperaturesdiffered in carcass mass (ANCOVA with the initial egg
mass as the covariate: F4,74 = 6.36, p < 0.001; Fig. 2a)and yolk sac dry mass (ANOVA: F4,75 = 4.79, p < 0.01;
Fig. 2b), but not fatbody dry mass (F4,74 = 0.37,
p > 0.05; Fig. 2c). Hatchlings from 32°C had lighter carcass dry mass but larger residual yolk sac, whereas
hatchlings from 22°C had heavier carcass but smaller
residual yolk sac (Fig. 2a, b).
Energy contents (F4,74 = 2.78, p < 0.05) and nonpolar lipids (F4,74 = 2.80, p < 0.05) differed among
hatchlings from different incubation temperatures, withhatchlings incubated at 22°C containing lower quantities
of energy and non-polar lipid than did those incubated atother four temperatures (Table 2). Conversion efficiencyof energy during incubation at 22°C (71.1%) was thus
lower than those at 32, 30, 27, and 24°C (83.9%,
82.6%, 81.5%, and 80.2%). Similarly, conversion efficiency of lipid at 22°C (55.3%) was lower than those at
32, 30, 27, and 24°C (70.9%, 69. %, 67.3% and 64.9 %).
Additionally, shells of hatched eggs were significantly
lighter than shells of freshly laid eggs (F5,84 = 3.19, p <0.05; Table 2).

Discussion
As in numerous other reptiles, thermal environments
experienced by developing embryos affect hatching success, incubation length, embryonic expenditure of energy, and linear dimensions (SVL and tail length) andbody composition of hatchlings in Elaphe taeniura. Our
results provide support for the previous conclusion that
reptilian embryos developing at relatively low or moderate temperatures produce well developed and thus, larger hatchlings (e.g. Deeming and Ferguson, 1991; Du andJi, 2002; Ji and Du, 2001a, b). The larger hatchling size
has an association with the greater carcass dry mass
(Chen and Ji, 2002; Du and Ji, 2002; Ji et al., 1997; Ji
and Sun, 2000). Data from the current study proved that
this conclusion is also true in E. taeniura (Fig. 1; Fig. 2).
The finding that more yolks remain unutilized at hatching when eggs are incubated at high temperatures has
been reported for nearly all reptiles studied to-date (e.g.,
Beuchat, 1988; Du and Ji, 2002; Ji and Du, 2001a, b; Linet al., 2005; Phillips et al., 1990; Phillips and Packard,
1994). In E. carinata (Ji et al., 1997), E. taeniura (Ji etal., 1999a), D. rufozonatum (Ji et al., 1999b) and P. korros (Ji and Sun, 2000), hatchlings exhibit a substantial
increase in size (SVL) during the first post-hatchingdays due to the transfer of resources in the residual yolk
into carcass.
The proportion of lipids transferred from the egg
contents into the hatchling was noticeably lower thanthose of energy. Given that mass-specific energy density
is much higher in lipids than in proteins and carbohydrates, this result provides evidence that lipids are themain source of energy for embryonic development.

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Hatchling size and mass were primarily determined by
the embryonic expenditure of energy during incubation
after removing the influence of variation in initial egg
mass (Du and Ji, 2001, 2003; Ji and Du, 2001a, b).
Given that the total energy invested in an egg is fixed,
any increase in embryonic expenditure of energy during
incubation may inevitably result in production of small
sized or lighter hatchlings. Incubating eggs at low temperatures or high temperatures may increase energy
expenditure for embryonic development due to theincreased incubation length and/or embryonic metabolism (Booth, 1998; Booth and Astill, 2001; Packard andPackard, 1988). Consequently, eggs incubated at moderate temperatures usually produce larger and heavier
hatchlings than did those at low or high temperatures.
A prolonged exposure of eggs of E. taeniura to temperatures lower than 24°C or higher than 30°C may havea detrimental effect on embryonic development, as indicated by the fact that hatching success decreases dramatically at temperatures outside this temperature range(Table 1). The mean incubation length 30°C is 53.9
days, approximately 3.4 days longer than that at 32°C,
so the ecological disadvantage of the increased incubation length (and thus, decreased growth period prior tothe onset of the first winter) due to a decrease in incubation temperature from 32°C to 30°C is less pronounced.
Considering that less developed hatchlings are produced
at 32°C and that hatching success is low at this temperature, we conclude that the temperature of 32°C is outside the range of optimal temperatures for incubating
eggs of E. taeniura. Eggs incubated at 24°C exhibit highhatching success and produce well developed hatchlings. However, the majority of hatchlings from eggs
incubated at 24°C appear between late September andmid-October, so the growth prior to the onset (lateNovember) of the first winter for these hatchlings isabout 1.5–2 months. As incubation length increasingly
increases as temperature decreases in E. taeniura (Table1), we expect that the disadvantage of incubating eggs ofE. taeniura at temperatures lower than 24°C can be very
pronounced due to the increasingly prolonged incubation length. For eggs of reptiles incubated under natural
conditions, the prolonged incubation length at low temperatures also increases exposure of eggs to the effects
of adverse biotic (increased microbial contamination)
and abiotic factors(extreme thermal and hydric conditions) in the incubation environment of the eggs, which
potentially reduces hatching success. Thus, the temperature of 24°C is suitable but not optimal for incubating
eggs of E. taeniura. Taking the energy expenditure during incubation, the rate of embryonic development andhatchling phenotypes into account, we consider thattemperatures around 27°C are optimal for incubating
eggs of E. taeniura.

2008

Acknowledgments
This work was supported by a grant from Zhejiang
Provincial Natural Science Foundation. Many thanks
were given to X.-Z. Hu, W.-Q. Xu, and J.-Q. Du for theirassistant in the laboratory.

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Submitted: 17 November 2006
Accepted: 22 September 20072008