Journal of Zoo and Wildlife Medicine 49(4): 870–874, 2018

Copyright 2018 by American Association of Zoo Veterinarians

EVALUATION OF ACUPUNCTURE POINTS GOVERNING VESSELS

1 AND 26 ON ANESTHETIC RECOVERY OF EASTERN BOX
TURTLES (TERRAPENE CAROLINA CAROLINA)

Anthony J. Cerreta, B.S., Meghan E. Walker, D.V.M., and Tara M. Harrison, D.V.M., M.P.V.M., Dipl.
A.C.Z.M., Dipl. A.C.V.P.M., Dipl. E.C.Z.M. (ZHM), C.V.A.

Abstract: Intramuscular administration of anesthetic agents in chelonians may result in a prolonged (!1 hr)
return of spontaneous movement and breathing, which increases the probability for peri- and postoperative
complications. The acupuncture point governing vessel (GV)-26 has been demonstrated to reduce anesthetic
recovery times from inhalant anesthesia in other species. In this study, 30 eastern box turtles (EBT; Terrapene
carolina carolina), presented to the Turtle Rescue Team at North Carolina State University’s College of Veterinary
Medicine for treatment of aural abscess, were divided into four groups: control (no treatment); GV-26
acupuncture; GV-1 and GV-26 acupuncture; or GV-1 and GV-26 electroacupuncture. Turtles receiving either
GV-1 and GV-26 acupuncture or GV-1 and GV-26 electroacupuncture had a significantly reduced time to return
of voluntary movement (P ¼ 0.012 and P ¼ 0.006, respectively), a significantly reduced time to response of limb
extension (P ¼ 0.03 and P , 0.001, respectively), and a significantly reduced time to anesthetic recovery (P , 0.05
and P , 0.01, respectively). Therefore, the use of either GV-1 and GV-26 acupuncture or GV-1 and GV-26
electroacupuncture produces significant reductions in anesthetic recovery time in EBTs that have received
injectable anesthetics.
Key words: Acupuncture, anesthesia, eastern box turtle, GV-1, GV-26, Terrapene carolina carolina.

INTRODUCTION carolina).4,10,11 When compared with other turtle

Injectable anesthetic agents are commonly used
for the induction of anesthesia in reptiles. In
chelonians, the slow metabolism of injectable
anesthetics provides adequate anesthesia for mi-
nor surgical procedures; however, it also leads to
extended recovery periods and increases the
chance of morbidity.1,6,12 Furthermore, recoveries
of greater than 1 hr to limb movement and normal
spontaneous breathing after intramuscularly ad-
ministered anesthetic protocols with ketamine
precludes their use in debilitated patients.2 There-
fore, establishing a method for reducing recovery
time of injectable anesthetics with an adjunctive
technique, such as acupuncture, may help to
provide additional benefit and increase the overall
quality of an established anesthetic protocol.
At the Turtle Rescue Team (TRT) based at the
North Carolina State University College of Vet-
erinary Medicine and at other wildlife rehabilita-
tion clinics throughout the United States,
traumatic injury is the most common presentation
for eastern box turtles (EBTs; Terrapene carolina
From the Department of Clinical Sciences, North
Carolina State University, College of Veterinary Medicine,
Raleigh, North Carolina 27607, USA (Cerreta, Walker,
Harrison). Correspondence should be directed to Dr.
Harrison (tara_harrison@ncsu.edu).
species, EBTs have also been more commonly
found to present for nontraumatic conditions,
including aural abscesses and upper respiratory
infections.10 To provide a surgical plane of anes-
thesia that enables debridement of the tympanic
cavity in EBTs with aural abscesses, the TRT
administers 1.5 mg/kg morphine, 7.0 mg/kg
ketamine, and 100 lg/kg dexmedetomidine by
im injection into the tricep muscle complex. This
injectable anesthetic protocol has resulted in
cases requiring positive pressure ventilation for
greater than 1 hr and instances of mortality.
Lower doses of this protocol did not consistently
provide a depth of anesthesia for surgical de-
bridement of the tympanic cavity.
Governing vessel 1 (GV-1) is the starting
acupuncture point of the GV channel that is
expected to enhance the actions of GV-26. GV-1
is an acupuncture point located on the midline,
midway between tip of the coccyx and anus that is
also purported to alleviate pain along the center-
line and back.8,14 Traditionally, GV-26 is used for
emergency and resuscitation. GV-26 is an acu-
puncture point located on midline below the nares
that is used to aid the in the treatment of shock,
cardiopulmonary arrest, and apnea.3,7,13 Further-
more, administration of GV-26 in common snap-
ping turtles (Chelydra serpentina) reduced overall
recovery times following inhalant isoflurane an-

870
 CERRETA ET AL—BOX TURTLE ACUPUNCTURE ANESTHETIC RECOVERY
Figure 1. Illustrates the site of governing vessel 1
acupuncture needle insertion in an eastern box turtle at
the center of the ‘‘X.’’
esthesia.5 The usefulness of the combination of
GV-1 and GV-26 acupuncture points in reducing
anesthetic recovery times has not been conducted
previously. The aim of this study was to evaluate
for decreasing recovery time following intramus-
cular injectable anesthesia in EBTs by comparing
the efficacy of the GV-26 acupuncture point to the
combination of GV-26 and GV-1 acupuncture
points and to further evaluate for the use of
acupuncture versus electroacupuncture stimula-
tion. This study specifically tests hypotheses in
comparison to controls on how the use of GV-1
and GV-26 acupuncture would decrease recovery
time and that the use of electroacupuncture would
further potentiate this effect.
MATERIALS AND METHODS
Thirty EBTs were used in this study. Procedures
performed were in accordance with a North
Carolina State University, Institutional Animal
Care and Use Committee–approved protocol.
Turtles were divided into four groups (control,
needle-only acupuncture at GV-26, needle-only
acupuncture at GV-1 and GV-26, and needle
electroacupuncture at GV-1 and GV-26), with
consecutive additions across this series of four
groups on the basis of the day of presentation to
the TRT. All turtles in this study were brought in
by members of the public, and a complete
physical examination was performed on each
animal. A lateral swelling of the tympanum that
was pale in color, suspect of an aural abscess, was
identified. The set of turtles (n ¼ 30) consisted of
19 males and 11 females weighing 275.9 6 126.3 g
(mean 6 SD) and were housed individually in 50 3
30 315-cm containers with adequate access to
871
water. They were fed and cleaned at least every
other day. A controlled temperature (22.9–23.88C)
was maintained in the housing area, and the
minimum and maximum temperatures were re-
corded daily.
Turtles were anesthetized by using a previously
established anesthetic protocol that was modified
to provide a surgical plane of anesthesia: 1.5 mg/
kg morphine (15 mg/ml, West-Ward Pharmaceu-
ticals, Eatontown, NJ 07724, USA); 7.0 mg/kg
ketamine (Ketalar, 100 mg/ml, Pfizer, New York,
NY 10017, USA); and 100 lg/kg dexmedetomi-
dine (Dexdomitor, 0.5 mg/ml, Zoetis, Florham
Park, NJ 07054, USA) by im injection into the
tricep muscle complex.9 Within 24 hr of presen-
tation at the TRT, surgical removal of aural
abscesses were performed via a ventral border
incision from the 9 to 3 o’clock position along the
entire thickness of the tympanum. A horizontal
incision across the center of the tympanum was
then performed to allow for removal of inflam-
matory debris. The dexmedetomidine was re-
versed with 0.5 mg/kg atipamezole (Antisedan, 5
mg/ml, im, Pfizer) at the conclusion of the
procedure. Heart rates were measured with vas-
cular Dopplers, and limb extensions were as-
sessed as a binary outcome every 5 min.
Voluntary movement, characterized by movement
of the head, neck, or limbs irrespective of
stimulation, was recorded at the time of anesthet-
ic recovery by two observers using a standardized
protocol. Data were collected until the time of
recovery (Tidal Wave Sp Novametrix Capno-
graph, DRE Medical Equipment, Louisville, KY
40223, USA; millimeters of mercury).
After reversal with atipamazole, one of four
actions were performed based on the date of
intake for each animal. Turtles received either
GV-26 acupuncture (4 turtles), GV-1 and GV-26
acupuncture (6 turtles), or GV-1 and GV-26
electroacupuncture (10 turtles; ES-130, ITO,
Tokyo, Japan). Acupuncture was performed by
two individuals after being trained by a certified
veterinary acupuncturist. Control animals re-
ceived no additional therapies after reversal with
atipamazole (10 turtles). GV-1 and GV-26 needle
stimulation was performed by using a 36-ga
stainless-steel disposable acupuncture needle
(Jing Tang sterile acupuncture needle, size #36,
JiaJian, Medical Instrument Co., Ltd., Wuxi,
China) placed in the depression between the anus
and base of the tail (GV-1) and midline at the
ventral margin of the nares (GV-26; Figs. 1, 2).
Electrical stimulation for electroacupuncture was
applied to the needles at a continuous 1.4-Hz
872 JOURNAL OF ZOO AND WILDLIFE MEDICINE
Figure 2. Illustrates the site of governing vessel 26
acupuncture needle insertion in an eastern box turtle at
the center of the ‘‘X.’’
frequency, using a paired wire lead until the time
of recovery.
Recovery from anesthesia was defined at the
time of voluntary motor function. Voluntary
motor function was defined as a purposeful limb
extension with either the forelimbs or hindlimbs.
The times to return of reflexes were recorded from
the time of atipamazole administration and de-
fined as limb withdraw in response to a manual
toe pinch at an extremity. The treatment groups
were evaluated for differences in the time to
voluntary motor function and return of reflexes
with independent sample Kruskal-Wallis tests
(SPSS, Version 24, IBM Corporation, Armonk,
NY 10504, USA). A two-way analysis of variance
(repeated measures) followed by Tamhane T2
post hoc test was performed to evaluate for
differences in heart rate from 0 to 30 min on the
basis of the factor of acupuncture treatment and
control groups and the factor of time of repeated
measurement (SPSS, Version 24). Independent
sample t-tests compared recovery time (time at
which heart rate was last measured) for the
treatment groups to the control group. Statistical
significance was considered if P , 0.05.
RESULTS
The average mean total surgery time was 13.4 þ
8 min, with no significant difference between the
groups (P ¼ 0.287). EBTs receiving either GV-26,
GV-1 and GV-26 acupuncture, or GV-1 and GV-
26 electroacupuncture had significantly faster
times to return of voluntary movement than
turtles receiving the control treatment (P ¼
0.022), which corresponds to a more rapid
recovery from anesthesia. Turtles receiving GV-
1 and GV-26 acupuncture or GV-1 and GV-26
electroacupuncture had a significantly reduced
time to return of voluntary movement than
control animals (P ¼ 0.012 and P ¼ 0.006,
respectively). GV-1 and GV-26 electroacupunc-
ture resulted in a further decrease in the time to
voluntary movement; however, the difference was
not statistically significant when compared with
the methods of treatment other than the control
(P ¼ 0.777). EBTs in the group receiving GV-26
acupuncture alone did not have a significantly
reduced time to return of voluntary movement (P
¼ 0.228).
Turtles receiving either GV-26, GV-1 and GV-
26 acupuncture, or GV-1 and GV-26 electro-
acupuncture had significantly reduced time to
return of response to limb extension versus the
control animals (P , 0.001). EBTs in the groups
receiving either GV-1 and GV-26 acupuncture or
GV-1 and GV-26 electroacupuncture had a sig-
nificantly reduced time to response of limb
extension (P ¼ 0.03 and P , 0.001, respectively).
GV-26 acupuncture alone did not have a signifi-
cantly reduced time to response of limb extension
(P ¼ 0.39). Results and P-values are displayed in
Table 1.
Turtles receiving GV-26 acupuncture had a
significantly greater heart rate on average than
the control group (P , 0.05) for the analyzed
periods of 20, 25, and 30 min of treatment, based
on 21 turtles (nine control, two GV-26, four GV-1
and GV-26 acupuncture, and six GV-1 and GV-26
electroacupuncture) that remained in an anesthet-
ic state during which the heart rate was measured
(Fig. 3). Recovery times from anesthesia signifi-
cantly differed for the GV-26 and GV-1 group
(34.2 6 9.7 SD, P , 0.05) and the GV-1 and GV-
26 electroacupuncture group (29.5 6 12.1 SD, P ,
0.01) compared with the control (54.5 6 17.9 SD).
There was not a significant difference in recovery
time for the GV-26 group (43.8 6 30.7 SD, P ¼
0.42). No complications were noted during the
duration of the study. All 30 turtles were released
into the wild in the same vicinity in which they
were found or have been placed in rehabilitation
facilities until spring when they could be safely
released.
DISCUSSION
In reptiles, intramuscular administration of
anesthetic agents may result in a prolonged time
(!1 hr) to the return of spontaneous movement
and breathing. Inconsistent uptake of drugs from
the muscle may impair drug metabolism or
elimination or both, leading to protracted clinical
 CERRETA ET AL—BOX TURTLE ACUPUNCTURE ANESTHETIC RECOVERY 873

Table 1. Anesthetic recovery parameters for eastern box turtles receiving no acupuncture, governng vessel
(GV)-1 and GV-26 acupuncture, or GV-1 and GV-26 electroacupuncture, following reversal with atipamazole.a

GV-26 GV-1 and GV-26 GV-1 and GV-26

Recovery parameter Control acupuncture acupuncture electroacupuncture

Time to voluntary 41.3 6 19.45 28.5 6 23.27 16.83 6 5.88 16.8 6 8.18
movement (min) — P ¼ 0.228 P ¼ 0.012 P ¼ 0.006
Time to response of 32 6 17.96 23.75 6 14.68 12.33 6 6.28 7.6 6 3.31
limb extension (min) — P ¼ 0.394 P ¼ 0.030 P ¼ 0.000
a
Data are expressed as mean 6 SD in minutes.

effects. The extended monitoring period can
increase the probability for post-procedural mor-
bidity. Intravenous administration of anesthetic
agents may be preferable to increase the predict-
ability of drug action; however administering
drugs intravenously in hypovolemic chelonians
can be challenging and may require sedation to
access the jugular vein for catheter placement.
GV-1 and GV-26 acupuncture and GV-1 and
GV-26 electroacupuncture were associated with
significantly reduced time to return of voluntary
movement and significantly reduced time to
response of limb extension, therefore resulting in
a significantly reduced time to anesthetic recovery
following intramuscular injectable anesthesia.
Needle location is most likely responsible for
the reported therapeutic benefit rather than
electrical stimulation alone. Furthermore, be-
cause some practitioners may not have access to
an electrical stimulation unit, these results pro-
vide evidence that acupuncture alone will hasten
recovery from an a-2 agonist and ketamine
intramuscular injectable anesthetic protocols.
The mechanism of action responsible for faster
recovery from anesthesia with GV-26 acupunc-
ture has been postulated to be attributed to a
neurorespiratory or adrenergic mechanism.5 Elec-
troacupuncture is said to further stimulate the
response of acupuncture. However, it was the set
of turtles receiving GV-26 acupuncture that had a
significantly greater heart rate on average than the
control group for an intermediate period leading
to recovery (20 to 30 min). Further studies are
required to better account for the effect of
acupuncture in this species. This study design
was based on large effect differences, and a
greater sample size would aid in greater detection
of differences having a lesser effect, in addition to
further verification of these differences observed.
After administration of injectable anesthetics,
turtles relaxed from cranial to caudal sides. Motor
function returned in the opposite direction during

Figure 3. Estimated marginal mean heart rates over time, postreversal with atipamazole, in eastern box turtles
receiving no acupuncture, governing vessel (GV)-1 and GV-26 acupuncture, or GV-1 and GV-26 electro-
acupuncture.
874 JOURNAL OF ZOO AND WILDLIFE MEDICINE
recovery, as previously described.2 Response to
limb extension was observed in all animals prior
to the return to voluntary movement.
EBTs undergoing surgery were not intubated
for the procedure, which prevented the sampling
of expired gases. Additional studies that include
blood gas analysis may provide further insight
into the physiologic effects that the treatment
groups had on the body.
CONCLUSION
Prolonged recovery from anesthesia in reptiles
increases the probability for post-procedural
morbidity. GV-1 and GV-26 acupuncture or GV-
1 and GV-26 electroacupuncture can be used as
an adjunctive method to reduce anesthetic recov-
ery time in EBTs that have received injectable
anesthetics. Additionally, more investigation
needs to be performed on other injectable anes-
thetic protocols.
Acknowledgments: The authors thank the
members of the North Carolina State University
College of Veterinary Medicine’s Turtle Rescue
Team for case management and rehabilitation of
injured turtles. The authors would like to thank
Michelle Kordupel for her involvement in this
project. The authors also thank Drs. Huisheng
Xie and Scott Harrison for consultation on this
project.
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Accepted for publication 25 July 2018