Role of the left aortic arch and blood flows in embryonic American alligator (Alligator mississippiensis) Page: 394
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394 J Comp Physiol B (2011) 181:391-401
timepoint was selected because embryos were predicted to
be large enough for instrumentation of the great blood
vessels. Eggs were placed in a temperature-controlled
surgical chamber at 30C under normoxic conditions, and a
portion of the eggshell removed under the dissection
microscope. Embryos were then anesthetized (confirmed as
non-responsiveness to mechanical stimulation) with iso-
flurane (Isoflo; Abbott laboratories, North Chicago, IL,
USA). The embryo's ventral surface was exposed, and a
2-cm midline incision made through the scales. Skin was
blunt dissected away from the underlying musculature, and
the pericardium and great vessels exposed by cutting
through the musculature and the sternum. The pericardium
was opened to expose the ventricles and great vessels.
During the surgical preparation and experimentation, the
embryo's head was contained within the amniotic fluid.
Blood flow in the right aorta (QRAo), subclavian artery
(QcCA), common carotid artery (QCCA) and left aorta
(QLAo) were determined using crystal doppler Transonic
flow probes (0.7, 1.0 or 2.0 mm) under sequential norm-
oxic and hypoxic conditions. Gas composition was moni-
tored with an oxygen analyzer (S-3AI) and altered using a
Sechrist Air-Oxygen mixer (3500HL) downstream of
compressed N2 and air. QLAo was determined with a single
flow probe (0.7 or 1.0 mm), and QRAo, QSCA and QCCA
were determined with one or two flow probes, either all
three vessels simultaneously within a 2.0 mm probe or the
RAo and SCA simultaneously in a 1.0 mm probe and the
CCA separately in a 0.7 or 1.0 mm probe. After 10-min of
stable flows, normoxic-incubated embryos were exposed to
acute hypoxia (14.3 1.6% 02; mean SEM) for 10 min,
and blood flows were monitored during hypoxic exposure
and for 5-10 min following a return to normoxia. For hyp-
oxic-incubated embryos, stable blood flows were monitored
for at least 10 min in normoxia, and subsequently embryos
were exposed to hypoxia (14.4 1.4% 02) for the
remainder of the experiment, 10-20 min. Blood flow was
monitored using a Transonic T206 or T106, and blood flow
monitored using a PowerLab data recording system con-
nected to a Macintosh computer running Chart software,
and data recorded at 100 Hz. fH was determined with a
software tachograph. Following completion of each trial, the
embryo was euthanized with an overdose of isoflurane, and
embryonic wet mass and wet heart mass, atria and ventricles,
were determined 0.001 g using the analytical balance.
Occlusion of the left aorta (LAo) and V02
in alligator embryos
At 95% of development, between 67 and 69 days post-
laying, normoxic-incubated eggs were removed from theirincubators and candled to determine orientation of the
embryo (N = 8, 4 sham-operated and 4 experimental,
LAo-occluded). This developmental timepoint was selec-
ted because it represents the point of maximal metabolic
rate prior to lung ventilation. Eggs were placed in a tem-
perature-controlled surgical chamber at 30C under norm-
oxic conditions, and a portion of the eggshell removed
under the dissection microscope. Embryos were then
anesthetized (confirmed as non-responsiveness to
mechanical stimulation) with sodium pentobarbital
(50 mg kg-'; Abbott Laboratories, North Chicago, IL,
USA). The embryo's ventral surface was exposed, and a 2-
cm midline incision made through the scales. Skin was
blunt dissected away from the underlying musculature, and
the pericardium and great vessels were exposed by cutting
through the musculature and the sternum. The pericardium
was not opened. During the surgical preparation and
experimentation, the embryo's head was contained within
the amniotic fluid. For LAo-occluded embryos, the LAo
was isolated downstream of the great vessel truncus and
occluded with 4-0 sterile silk suture (Ethicon, Somerville,
NJ, USA). For the sham group, the surgical procedure was
identical to the LAo-occluded group, but the LAo was not
isolated and no suture wrapped around it. The musculature
and skin were seen shut in succession with 3-0 silk suture
(Ethicon). A portion of a rubber glove was carefully glued
around the open part of the shell, making sure the seal was
air-tight to prevent desiccation. Although it has been
demonstrated that occlusion of the LAo upstream of the
FoP is necessary to completely eliminate the capacity for
systemic blood to re-enter the systemic circulation from the
right ventricle in alligators (Farmer et al. 2008; Eme et al.
2009; Eme et al. 2010), this delicate surgical procedure was
not reliably repeatable in ovo, and its success was not
verifiable (e.g., using H2-electrode technique in Eme et al.
2009). Therefore, we only occluded the LAo well down-
stream of the Foramen, just downstream of the great vessel
truncus.
Immediately following surgery, eggs were returned to
the 30C walk-in, constant temperature room for experi-
mentation. As the embryo recovered from surgery
(~ 2-3 h post operation) and two additional days following
surgery, V02 was determined using methods modified from
Warburton et al. (1995). Eggs were individually sealed for
16-30 min (1-2% fall in oxygen concentration) within an
air-tight, 250 ml mason jar/respirometry chamber that had
been flushed with fresh air at 30C just prior to introduction
and sealing of the egg within it. Hubs from 20-gauge
needles were attached into the lids and connected to a
3-way stopcock to provide access to jar air. 60-ml syringes
were used to sample air within the chamber at the end of
the trial. In order to analyze gas samples from theSpringer
394
J Comp Physiol B (2011) 181:391-401
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Eme, John; Crossley, Dane A., II & Hicks, James W. Role of the left aortic arch and blood flows in embryonic American alligator (Alligator mississippiensis), article, October 30, 2010; New York, NY. (https://digital.library.unt.edu/ark:/67531/metadc958741/m1/4/: accessed April 24, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT College of Arts and Sciences.