Acclimation to hypothermic incubation in developing chicken embryos (Gallus domesticus) : I. Developmental effects and chronic and acute metabolic adjustments
Department of Biological Sciences, University of North Texas, Denton, TX 76203, USA
* Author for correspondence (e-mail: burggren{at}unt.edu)
Accepted 22 January 2004
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Summary |
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Key words: chicken embryo, Gallus domesticus, thermoregulation, hypothermia, incubation, development, heterokairy
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Introduction |
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Less obvious than the simple lengthening of the embryonic period by chronic
hypothermia, but potentially of considerable physiological importance, is the
impact on both the absolute and relative timing of the onset of different
physiological processes and their regulation. Indeed, temperature changes
during development often have complex affects that go beyond simply
accelerating or decelerating the development of the embryo as a unit
(Bull, 1980;
Temple et al., 2001
;
Gutzke and Crews, 1988
;
Johnston et al., 1996
;
Spicer and Burggren, 2003
).
Susceptible to change in the chronically hypothermic embryo, then, are the
absolute as well as the relative timings of onset of different physiological
process and the systems that regulate them. This phenomenon has recently been
termed `heterokairy' to differentiate it from the rather broadly and loosely
used `heterochrony' (see Spicer and
Burggren, 2003
).
The shift from poikilotherm to homeotherm in bird embryos is an important physiological transition, beginning within the last 20% of incubation. This thermoregulatory transition requires heat-producing metabolism and supporting oxygen transport to mature as essential steps in the precise regulation of body temperature. Such developmental events may be particularly susceptible to temperature-induced qualitative adjustments. In this and the following study on the late stages of incubation of the chicken embryo (Gallus domesticus), we examine both acute and chronic temperature influences on metabolism, changes in blood-oxygen transport supporting metabolism, and thermoregulatory responses of chicken embryos. Our goal is to determine if chronic hypothermia (35°C) alters standard developmental patterns, providing additional evidence for heterokairy. As a self-contained embryo that is very well characterized anatomically and reasonably well understood physiologically, the chicken embryo is an excellent model to examine how the changes in the thermal environment can quantitatively and qualitatively influence the developmental timeline.
In this first study, we test the hypothesis that chronic incubation in hypothermia (35°C) not only lengthens the embryonic period but also alters the relative timing of hatching events, the normal pattern of changes in metabolic activity, and the ability of the embryo to respond physiologically to acute decreases in Ta.
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Materials and methods |
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Populations of eggs were incubated at 38.0°C (control), or 35.0°C
(hypothermic), all at a relative humidity of 60%. To determine the gross
effects of incubation temperature on development, nine or more embryos
incubated at each temperature were staged for developmental maturity on days
1314, 1516, 1718 and 1920
(Hamburger and Hamilton, 1951).
Hypothermic embryos have a slower rate of development than embryos incubated
in control conditions, so staging was completed through hatching to determine
the length of incubation required for the 35°C embryos to reach
developmental stages equivalent to the 38°C embryos.
All subsequent metabolic experiments were conducted on embryos at the following stages: stage 3940, reached on days 1314 for 38°C and days 1516 for 35°C; stage 4142, reached on days 1516 for 38°C and days 1718 for 35°C; stage 4344, reached on days 1718 for 38°C and days 1920 for 35°C.
Rates of survival and timing of hatching
Fertilized eggs (N=40 for 38°C and N=32 for 35°C)
were incubated as described above. Eggs were candled every 2 days (D) from D4
to D18 of incubation, to determine survival. From D19 to D25 of incubation
eggs were candled daily to determine survival at the pre-pip stage, internally
pipped stage, externally pipped stage and hatching success. Survival rates
were calculated as number of eggs alive compared to total number of eggs
incubated at that temperature. Counts of eggs on each day were converted to
relative frequencies and plotted for each day of incubation. Egg counts also
allowed the calculation of percentage survival and the timing of pipping and
hatching events.
The effect of temperature on length of incubation to internal pipping,
external pipping and hatching was expressed by the temperature quotient
(Q10) calculated using the van't Hoff equation:
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Static O2 measurements at 35°C and 38°C
Six eggs from each incubation temperature, at each stage, were implanted
with thermocouples. Thermocouple wires were inserted immediately beneath the
shell through a 0.5 mm hole, and held in place with a 1 cm2 piece
of tape. Preliminary experiments revealed no detectable thermal gradient from
inside the embryo's body to the shell exterior in incubating eggs.
Consequently, `surface temperature' measured immediately under the shell is
assumed to be embryo temperature. The eggs were placed in an air-tight,
water-tight respirometer (240 ml) through which air was pumped continuously at
7075 ml min1. Water and carbon dioxide were removed
from the outflow air by passing it through DrieriteTM and soda
lime, respectively. Analysis of O2 content of the air and
calculation of oxygen consumption was carried out using an oxygen analyzer
(model FC-1B, Sable Systems Inc., Henderson, NV, USA). The ventilated chamber
was partially submerged in a programmable water bath (ISOTEMP 1028P, Fisher
Scientific, Hampton, NH, USA) and allowed to equilibrate to incubation
temperature for a minimum of 30 min before measurements were started.
Measurements of oxygen consumption were recorded simultaneously with egg
temperature and ambient temperature (Chart software and Powerlab data
acquisition system, ADInstruments, Colorado Springs, CO, USA). All values of
O2 (µl
O2 g1 min1) were expressed on
an embryo mass-specific rather than egg mass-specific basis, unless otherwise
indicated.
Basal O2
measurements made at constant Ta were designated as
`static'
O2
measurements (in contrast to measurements during gradual cooling or warming,
described below). Static measurements were always recorded first at the
chronic incubation temperature of that particular egg. After this initial
measurement (for approximately 30 min), the water bath temperature was changed
at a rate of 3°C h1 to expose the egg to the other
treatment incubation temperature (e.g. 35°C if the incubation temperature
was 38°C) for a minimum of 2 h.
O2 was then
determined for the same egg at the other treatment temperature as described
above.
O2 measurements during gradual cooling
O2 was also
measured during gradual cooling, because the Ta at which
basal
O2 (and
the accompanying heat production) can no longer be maintained during cooling
the Tcrit is an indication of the maturity
of thermoregulatory ability. Hence, eggs (N
6 for each incubation
temperature) with implanted thermocouple wires were placed in metabolic
chambers. Chamber and egg surface temperature were simultaneously recorded as
described above. Static basal
O2 measurements were
determined at the egg's chronic incubation temperature before the start of the
gradual cooling protocol. For those eggs incubated at 38°C, measurement of
O2 at 38°C was
followed by continuous
O2
measurements during gradual cooling (3°C h1) of the
water bath and metabolic chamber to a final egg temperature of 30°C. For
those eggs incubated in hypothermia (35°C), a static
O2 measurement was
determined at 35°C and the temperature of the water bath was then
increased to 38°C. Eggs were allowed a minimum of 2 h at 38°C before
entering the same gradual cooling protocol described for the control eggs
incubated at 38°C. Preliminary measurements indicated that the temperature
in the deep interior of the egg was always identical to surface temperatures
at the cooling rates used in these experiments.
Stage and mass determination
Upon completion of O2
measurements, embryos were killed by placing eggs in a container containing
halothane vapor. Each embryo was then removed from the egg, blotted dry with
towels and its wet mass determined using a microbalance (Denver Instrument
Company, Denver, CO, USA). The ventricle was dissected from the embryo,
blotted dry and weighed. The embryo was staged to confirm that it was at the
expected developmental stage, weighed, and then dried in a 40°C oven for 2
days for subsequent determination of dry mass.
Statistical analysis of O2 and mass data
All data were tested for normality of distributions (ShapiroWilks
normality test) and equality of variances. SAS (Version 8.0) was used to
conduct all statistical analyses. All statistical decisions were made with a
0.05 level of significance and all values are presented as means ±
S.E.M.
Significance of differences between static oxygen consumption measurements at 35°C and 38°C for embryos incubated at the same temperature was determined using a paired t-test. The same oxygen consumption data were tested for significance of differences between incubation temperatures at a particular measurement temperature using independent t-tests.
The rate of oxygen consumption during gradual cooling at each stage and each incubation temperature were tested with repeated-measures ANOVA (block design) to determine Tcrit, the temperature at which a significant decrease in rate of oxygen consumption from control values occurred during the cooling protocol. Comparisons of oxygen consumption rates during gradual cooling between stages and incubation temperatures were performed using a one-way ANOVA. StudentNewmanKeuls (SNK) multiple range tests were performed following each ANOVA for post-hoc detection of specific differences between means.
Differences in embryo wet mass, dry mass, and heart mass were determined using ANOVA and SNK multiple range test.
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Results |
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Hypothermic embryos required an incubation time of 23.7 days, an average of 3.1 days longer than 38°C embryos (Table 1). Internal pipping in 35°C embryos occurred an average of 2.9 days later than in normothermal conditions, where it occurred at 22.2 days. Similarly, external pipping occurred at 22.8 days, 3.0 days later than in warmer embryos. The effect of hypothermic incubation on the relative timing of each hatching event was determined by converting the length of each event interval to a percentage of total incubation length, and then determining Q10 (Table 1). Incubation in hypothermic conditions had no effect on the relative timing of each hatching event, as indicated by Q10 values of 1.00 for each event interval. Thus, internal pipping occurred at 93% of development and external pipping at 96% of development, regardless of incubation temperature.
Growth and developmental progress
Embryos incubated at 35°C reached HH 3940 on D15D16 of
incubation (Fig. 2), and by
D17D18 they were at HH 4142. After 1920 days 35°C
embryos were at HH 4344. Developmental progress of hypothermic embryos
in late stages of incubation generally lagged behind 38°C embryos by
23 days.
Growth of embryos at each incubation temperature was determined by measuring both wet and dry embryo mass (Fig. 3). At the earliest stage (3940) there was no significant difference (P>0.05) in wet mass or dry mass between embryos incubated at 35°C (7.60±1.03 g and 0.79±0.13 g, respectively) and 38°C (5.54±0.29 g and 0.51±0.03 g, respectively). All embryos experienced a significant increase in both wet and dry mass from stage 3940 to stage 4142 (15.71±0.81 g and 2.38±0.17 g for 35°C embryos and 13.75±0.40 g and 2.20±0.11 g for 38°C embryos, F=62.29, P<0.01), but there was still no differences attributable to incubation temperature. By stage 4344 the 35°C embryos had a significantly higher wet mass (22.26±0.98 g) than 38°C embryos (19.42±0.51 g) (F=62.29, P<0.01), but there was no significant difference in dry embryo mass (4.16±0.25 g and 4.62±0.36 g, respectively).
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No apparent differences in embryo body mass were detected between incubation temperatures at stage 4142; nonetheless, the wet ventricle mass of 35°C embryos (0.22±0.04 g) was significantly larger than that of the 38°C embryos at stage 4142 (0.10±0.01 g) (F=5.72, P=0.0016), and the ratio of ventricle mass to embryo mass was significantly larger in 35°C embryos at all stages examined (F=30.66, P<0.0001) (Fig. 4). Between stages 4142 and 4344 there was no significant change in ventricle mass in embryos at either incubation temperature. Because embryonic wet mass significantly increased as development progressed, the ratio of ventricle mass to embryo mass decreased significantly in all embryos (F=30.66, P<0.0001).
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Static O2 at 35°C and 38°C
There was a general decrease in mass-specific
O2 with increasing stage
(Fig. 5), with stage
4344 embryos from both incubation temperatures having a significantly
lower
O2 than at stage
3940. Mean mass-specific
O2 for 35°C embryos
ranged from 25±1.7 µl g1 min1 at
stage 3940 to 13±0.35 µl g1
min1 at stage 4344
(Table 2). The mass-specific
O2 of 38°C embryos
ranged from 33±0.82 µl g1 min1
at stage 3940 to 27±2.2 µl g1
min1 at stage 4344
(Table 2). Mass-specific
O2 of 35°C and
38°C embryos at the same developmental stage were not significantly
different.
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Each group of embryos, regardless of incubation temperature or stage, experienced a significant change in oxygen consumption after 2 h at the opposite incubation temperature (Matched-pairs t-tests with the probability for each group; P<0.018, Fig. 5).
O2 during acute cooling
Some embryos from each stage and each incubation temperature were able to
increase O2 briefly
during the earliest stages of the cooling protocol. However, all embryos
eventually experienced a significant decline in
O2 at some point during
the gradual 8°C drop in Ta
(Fig. 6). The youngest embryos
of each incubation temperature had significantly higher
O2 values than the older
embryos (Fig. 7). This was
evident for the entire duration of the cooling protocol in the 35°C
embryos (Fig. 7A), and until
Ta fell to 36°C in the 38°C-incubated embryos
(Fig. 7B). There were no
significant differences in
O2 during gradual cooling
between stages 4142 and 4344 for either incubation temperature,
except for the initial static measurement at 35°C for the 35°C
embryos.
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Although there are no significant differences in gradual cooling
O2 between the 35°C
and 38°C embryos compared at each stage, there are important differences
in the temperature (Tcrit) at which they first experienced
a significant decline in
O2 from the baseline rate
at 38°C (Fig. 7).
Fig. 7A shows that the youngest
embryos (stage 3940) experienced a significant decrease in
O2 when
Ta reached 34°C. By stage 4142
(Fig. 7B) the 38°C embryos
had a Tcrit of 34°C, while 35°C embryos had a
higher Tcrit of 36°C. Just prior to hatching at stage
4344 (Fig. 7C), 38°C
embryos can endure a drop in Ta of 8°C
(Tcrit of 30°C) before suffering a significant
decrease in
O2, but the
stage 4344 35°C embryos were not as efficient, showing a
significant decline at a Tcrit of just 36°C
1/4 the temperature decrease at which the 38°C began to show a
O2 decline.
O2 values for each of
these groups at 38°C and at Tcrit are summarized in
Table 3.
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Discussion |
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Importantly, we hypothesized that incubation at 35°C would not only lengthen the developmental timeline, but would also change development qualitatively. Yet our data show that incubation temperature had no effect on the relative timing of internal and external pipping in the chicken embryo, two critical landmarks in avian development. There was a slight difference in the overall rates of survival between embryos incubated at 35°C and 38°C (31% and 48%, respectively) but, overall, temperature had little effect on survival of the chicken embryos throughout incubation. Moreover, small numbers of embryos at each temperature died at approximately the same points in development (Fig. 1A,B). These identical mortality rates during early incubation suggest that the embyros experienced a common failure of an organ or a system at a key point in development.
Incubation temperature and embryonic growth
Embryonic wet mass for 38°C embryos at HH 3940 and HH
4142 closely resembled previously reported values
(Tazawa et al., 1971;
Dzialowski et al., 2002
). The
difference in wet body mass at HH 4344 between incubation temperatures
in the present study is accounted for by the higher total body water content
in the 35°C embryos. Calculations of % total body water from the wet and
dry mass data confirmed that at stage 4344 the 35°C the wet mass of
the embryo consisted of 81% water, compared to only 76% in the 38°C
embryos. Dzialowski et al.
(2002
) reported similar values
of total body water in D18 (HH 44) embryos at 81.8%. Their study also
determined that D12 embryos (HH 38) contained 91.7% body water, a value
comparable to that in our study for HH 3940 embryos incubated at both
35°C (89.6%) and 38°C (90.8%).
Overall, these data reveal that embryo growth is proportional to
developmental stage, irrespective of how long it takes to reach that stage.
This emphasizes the importance of making comparisons at equivalent stages of
development, rather than solely at arbitrary periods of development defined
chronologically. Because the development of embryos and organ systems is
non-linear, absolute time is not a good descriptor when comparing embryos
incubated at different temperatures. The concept of `degree days' (days of
development x the temperature of development) has been used to normalize
the effect of temperature on development and thus address the discrepancies
between absolute time and developmental time (see
Pritchard et al., 1996;
Weltzien et al., 1999
).
However, as Spicer and Burggren
(2003
) and Burggren and
Crossley (2002
) emphasize,
temperature effects on development are complex, selective and not easily
unraveled.
In contrast to whole embryo mass, ventricular mass showed evidence of
complex temperature effects. The ventricle mass of stage 4344 embryos
incubated at 38°C (0.10g±0.01) closely resembled the heart mass
data for D18 chicken embryos (0.12g±0.01) obtained by Dzialowski et al.
(2002). Our study revealed that
both ventricle mass and the size of the ventricle relative to embryo mass were
significantly higher in the 35°C embryos
(Fig. 4) at stage 4142.
Previous studies examining the effects of hypoxic stress on the development of
the chicken embryo saw increases in heart mass of D12 embryos that were
exposed to hypoxia between days 6 and 12 of incubation
(Dzialowski et al., 2002
). That
study, however, was unable to show a similar response in D18 embryos or in
hatchlings. Future experiments to examine the differential effects of
incubation temperature on heart mass will help to unravel why hypothermic
incubation leads to cardiac hypertrophy.
Temperature and O2
O2 measurements
obtained in the present study agree with previous measurements for D12, 14, 16
and 18 chicken embryos incubated at 38°C
(Table 2). However, this is the
first study to complete measurements of
O2 in embryos chronically
incubated at 35°C. The mass-specific
O2 of stage 4344
embryos incubated at 35°C appears low in comparison with measurements from
38°C embryos at the same stage in this and previous studies. The
significantly larger embryo wet mass of the 35°C embryos contributes to
this difference, but this group also demonstrated a large amount of variation
in
O2. Observations made
during incubation indicated that the chorioallantoic membrane (CAM) in the
35°C embryos failed to line the entire inner surface of the shell.
Although CAM surface area data was not collected in these experiments, a
smaller surface area for gas exchange might have an impact on the embryos with
the largest metabolic demand, i.e. HH 4344 embryos. The importance of
CAM surface area is debated. Okuda and Tazawa
(1988
) covered up to 50% of the
chicken egg with epoxy, effectively reducing the surface area of the CAM able
to exchange gases with the environment. They found that the reduction in gas
conductance reduced
O2.
In contrast, Wagner-Amos and Seymour
(2002
) reported that metabolic
activity was not significantly correlated with reductions in gas conductance,
accomplished by applying wax to the shell.
Incubation temperature did not profoundly affect basal
O2 of chicken embryos in
the final stages of development. The very similar
O2 exhibited by 35°C-
and 38°C-incubated embryos at HH 4344 and the general trend of a
decrease in mass-specific oxygen consumption with development may both be
explained by the internal O2 levels late in development. In such
late stages,
O2 of the
embryo is constrained by O2 diffusion rates across the shell.
Reduced embryonic mass-specific
O2 results because the
embryo continues to grow at the expense of establishing hypoxia within the egg
(Romijn and Lokhorst, 1951
;
Wagensteen et al., 1970
;
Rahn et al., 1974
;
Ar et al., 1980
;
Tazawa, 1980
). If late stage
chicken embryos are provided with increased O2 (hyperoxic
environment), they increase their metabolic activity
(Tazawa et al., 1992
). An
examination of the early stages of development would probably reveal lower
O2 in embryos incubated
at 35°C, supporting the slower rate of growth and development and the
increased length of the developmental timeline.
Temperature and thermoregulatory activity
A homeotherm must be able to regulate precisely endogenous heat production
as well as heat loss in order to maintain a stable body temperature in the
face of fluctuating ambient temperatures. Chicken embryos are certainly not
homeotherms, but at some point in very late development the embryo makes the
transition from poikilothermy to homeothery, using elevated
O2 to produce heat used
to maintain body temperature. Tazawa et al.
(1988
) slowly cooled
late-stage embryos from 38°C to 30°C over a period of 8 h, minimizing
the imbalance between heat loss and heat production. They noted that embryos
as young as stage 43 were able to maintain a maximal oxygen consumption until
the ambient temperature reached 34°C, and proposed that the embryo was an
apparent poikilotherm, unable to maintain body temperature, only because of
the thermal constraints of the egg environment, not because they lacked the
mechanisms required for regulating metabolic activity. In support of the
contentions of Tazawa et al.
(1988
), we propose two lines
of evidence suggesting the onset of thermoregulatory ability in late-stage
embryos: (1) the ability of some individual embryos actually to increase
briefly their
O2 by
510% upon a 1°C drop in Ta and (2) the ability
of the embryo generally to maintain
O2 during an 8°C drop
in Ta.
Incubation temperature appears to modify the developmental onset of the
chicken embryo's ability to trigger endogenous heat production as part of
developing thermoregulatory mechanisms. Specifically, hypothermic incubation
delays the onset of the embryo's ability to maintain stable
O2 in the face of acutely
declining temperature. The youngest embryos examined from both incubation
temperatures significantly reduced
O2 at a
Tcrit of 34°C (Fig.
7A). By stages 4142 and 4344, the 35°C embryos
experienced significant decreases in metabolic activity earlier, but only at
36°C. The ability of the 38°C embryos to maintain
O2 improved with
continued development, and by stage 4344 they experienced a significant
decline in oxygen consumption only after a full 8°C decline in egg surface
temperature, thus surpassing the performance reported for chicken embryos by
Tazawa et al. (1988
). While
some of the statistically significant differences individually may be of
limited biological significance, collectively these data show that over all
incubation conditions, hypothermic incubation temperatures produce embryos
that are less efficient in responding physiologically to acute
Ta decreases.
Important differences exist between measurements of static
O2 made after longer
periods of exposure (2 h) to altered ambient temperature compared with the
`gradual cooling' experiments. First, after longer exposure, the temperature
effect on
O2 was the same
for all stages and both incubation temperatures, with Q10 values
ranging from 1.49±0.08 to 1.57±0.17. Although there is something
inherently different about the 38°C embryos that allows them to better
resist gradual decreases in ambient temperature, these data suggest this
effort is short-lived, lasting for only a few hours. The constraints imposed
by the egg environment include a complete lack of insulation
(Whittow and Tazawa, 1991
),
high thermal conductance (Tazawa et al.,
1988
) and limited diffusion of respiratory gases
(Wagensteen et al., 1970
;
Rahn et al., 1974
). Each of
these factors is likely to contribute to an eventual overwhelming demand on
metabolic heat production, such that after 2 h of exposure there is no
difference between embryos incubated at 35°C and 38°C. Secondly, these
Q10 values are also lower than those that describe the effect of
temperature on
O2 during
gradual cooling over the same temperature range. With a Q10 as high
as 1.88, temperature has a greater effect on
O2 during the initial
stages of cooling. The lower Q10 after a longer period of exposure
implies that the exponential decrease in oxygen consumption initially
overshoots the appropriate
O2 but compensates within
the 2 h period of this experiment (Tazawa
et al., 1989
).
Incubation temperature and heterokairy
Chicken embryos incubated under hypothermic conditions at 35°C appear
to follow the same relative developmental timeline as embryos incubated at
38°C. Embryo masses were the same,
O2 values were similar,
and internal and external pipping occurred at the same relative points along
the developmental timeline. These responses would suggest that temperature, in
fact, does not induce heterokairy, at least as it relates to changes in the
relative timing of the onset of key physiological processes and their control
that specifically affect growth or the maturation of the respiratory system
(judging from the timing of pipping events). Yet, the different responses of
35°C and 38°C embryos to gradual cooling reveal significant effects of
chronic hypothermic incubation on the maturity of physiological components
required for endogenous heat production for thermoregulation. These
ontogenetic differences support the concept of heterokairy, but additional
experiments will be required to determine which regulatory components are
responsible for the differences between 35°C and 38°C embryos.
Temperature has been shown to have complex and selective effects on physiological and metabolic development in chicken embryos. In the companion paper we show how blood O2 transport properties are similarly affected in complex ways by incubation temperature.
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Acknowledgments |
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References |
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