Survival Anesthetic and Injection Procedures for
Neonatal Opossums
ZHIQIANG WANG, PHD * AND JOHN L. VANDEBERG, PHD
The laboratory opossum, Monodelphis domestica, differs from most other marsupial species in that females lack a pouch, and
neonates are exposed. This characteristic has fostered the use of this species for research on normal developmental processes
early in life and on experimental perturbations of those processes. However, in vivo experiments on neonates younger than 5 days
old have been hampered by lack of a protocol that is efficient in anesthetizing the mother without causing loss of the litter.
Therefore, the enormous potential for exploring this prototype laboratory marsupial in research on biological features of the
neonatal stages is largely undeveloped. We report here that halothane inhalation through a 50-ml conical tube placed over the
mother’s head succeeded in rapidly inducing anesthesia of the mother while sparing the babies from contact with the anesthetic.
Babies 0 to 2 days old survived injections of saline while their mothers were anesthetized by this procedure. This anesthetic procedure enables broad use of neonatal opossums as models for biomedical research on early developmental processes of mammals.
Monodelphis domestica is a South American marsupial (family:
Didelphidae) that has been fully adapted to laboratory conditions.
It is small (adult female opossums weigh 60 to 100 g and males
90 to 150 g), prolific (mean litter size of 8, as many as 3 litters
annually) and housed and bred in small cages under conditions
that compare favorably with those used for laboratory rodents
(1). Since its introduction as a laboratory animal in 1978, the
opossum model has become widely used for a variety of biomedical research objectives, particularly as models for mammalian
developmental processes.
Born after a brief gestation (13.5 days), newborn opossums
are essentially extrauterine fetuses, equivalent to 12.5-day-old
mouse or 6-week-old human embryos. Although a few organs,
e.g., lungs, are precociously developed, most are not. Development proceeds rapidly in the days after birth (1, 2). By 5 days of
age, the neonates have tripled their birth weight from 100 mg to
300 mg (unpublished data).
As the prototype laboratory marsupial, Monodelphis is used
predominantly for research on normal developmental processes
early in life (i.e., those that occur after birth in marsupials and
before birth in eutherian mammals) and on experimental perturbations of those processes. Examples include: a) research on
function of the embryonic nervous system in long-term cell culture (3); b) healing of the neonatal spinal cord after complete
crushing or transection (4, 5); c) effects of estrogen on testicular development (6); d) ontogeny of skin healing and
development of scarring (7); e) development of neuropeptides,
steroid receptors, and the visual system (8); f) UV-induced melanoma (9); and g) allogeneic and xenogeneic models for
melanoma research (10-12).
Some of these applications depend not only on the early developmental stage of Monodelphis at birth but also on the fact
that females have no pouch, so the neonates are totally exposed
on the abdomen of the mother and can be manipulated easily.
However, most in vivo research on development has used opossums that were at least 5 days old. Although experimental
manipulations of neonates older than 5 days are commonly successful after anesthetizing the mother, younger neonates tend
to release the nipples during anesthesia or when the mother
recovers from anesthesia and behaves in an agitated manner
Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, Texas
78227-5301
*
Corresponding author: Zhiqiang Wang, PhD, Department of Genetics, Southwest Foundation for Biomedical Research, P.O. Box 760549, San Antonio, Texas 78245-0549
Volume 42, No. 5 / September 2003
(unpublished observations). This impediment to experimental
research with 0- to 5-day-old Monodelphis has limited the use of
this species as a model for research on early mammalian development. For example, according to Robinson and Dooley (10),
injection of allogeneic melanoma cells resuspended in 25 µl saline buffer into neonates of 1 to 3 days old resulted in loss of all
pups. That report suggested that the loss was due to the extreme
developmental immaturity of the neonates as well as to the procedures that were used.
We hypothesized that the release of the nipple was due to the
neonate’s becoming anesthetized by inhalation or via respiration through the skin rather than by consumption of milk.
Therefore, we developed a protocol for short-term anesthesia of
Monodelphis mothers by inhalation without simultaneous anesthesia of the neonates. This protocol apparently spares the
neonates from being directly affected by the anesthetic. We describe our anesthetic method and report the results of injection
of five litters of neonates.
Materials and Methods
Animals. M. domestica is broadly distributed in several South
American countries, including Brazil, Bolivia, and Paraguay. The
animals used in this study were produced in the colony maintained by the Southwest Foundation for Biomedical Research
(SFBR; San Antonio, Texas). The animals are housed at 23.5 to
26.5°C in 43 cm × 22 cm × 13 cm polycarbonate cages (mouse
“shoebox” cages) with wood shavings for bedding. Each nursing
mother was provided a nest box and shredded paper toweling
for nesting material. Feed (Reproduction Diet, National Complete Fox Food Pellets, Milk Specialties Co., New Holstein, Wis.)
and acidified water are provided ad libitum (1). All procedures
described in this article were preapproved by the SFBR Institutional Animal Care and Use Committee.
Anesthesia by inhalation. The procedures were conducted in
a fume hood. Approximately 2 ml of halothane was added to
cotton balls in a 2-liter beaker. The cotton balls then were covered with a dry paper towel to prevent wetting the animals with
anesthetic. A glass cover was placed over the beaker for 3 to 5 min
to allow equilibration of the anesthetic within the beaker. The
mother with a neonatal litter then was gently suspended upside
down by holding its tail, so that its body was 1/3- to 1/2-way into
the beaker, while keeping the babies outside the beaker. In about
30 sec, the animal was lightly anesthetized and put on the surface
of the fume hood.
CONTEMPORARY TOPICS © 2003 by the American Association for Laboratory Animal Science
41
Table 1. Results of neonatal injections
Litter
1
2
3
4
5
Figure 1. Female opossum being anesthetized with halothane by using a
conical tube as a nose cone.
Age at
injection
(days)
0
1
2
2
2
Injection
volume
(µl)
5–10
15–20
10–15
15–20
25–30
Litter size
pre-injection
8
9
10
11
11
Pups remaining at
no. days after injection
1
2
4
7
8
9
10
11
7
8
9
10
11
4
8
9
10
11
4
8
9
10
11
4
the animal in the beaker containing halothane and covered with
glass plate. The mother was removed from the beaker immediately after muscle relaxation was achieved.
Results
Figure 2. One-day-old pups attached to their mother’s teats. Handles of
surgical scissors or clamps were placed over the limbs of the mother to
maximize exposure of the neonates.
A 50-ml conical tube, pre-equilibrated with 1 ml halothane on
a cotton ball, was placed over the head of the animal (Fig. 1).
Because of the aeroirritant nature of halothane, the animals tried
to avoid inhalation after they had squeezed their heads into the
tube. About three to five attempts generally were required for
each animal to reach the desired state of anesthesia. The animal
was closely monitored for breathing; when the animal appeared
to be relaxed, it was removed from the conical tube and laid on
its back. The time required to reach this state of anesthesia was
about 1 to 2 min.
Injection of neonates. Opossum mothers lack a pouch, so when
a mother is laid on her dorsal surface, the neonates are fully
exposed, each attached to a nipple. In order to enable one person to perform the injections, handles of surgical scissors or
clamps were placed over the limbs of the animal to ensure maximum exposure of the neonates (Fig. 2). A 29-gauge needle
attached to a 0.3-ml insulin syringe was used for the injections.
Phosphate buffered saline (PBS) of varying volumes was injected
subcutaneously into the neonates of five litters (Table 1). The
size of the litters ranged from 8 to 11 pups. To avoid unnecessary disturbance of the mother, a pair of fine forceps was used to
hold each baby for the injection. After completion of the procedure and after the mother was fully awake and ambulatory, the
mother was returned to its original cage and maintained under
the conditions prior to the procedures. The litters were observed
for survival on days 1, 2, 4, and 7 after the injections.
Anesthesia by conventional procedures. When her pups were
1 day old, as a control, we anesthetized a mother carrying a litter
of 11 by using a conventional anesthetic method that is typically
used for brief procedures with adult Monodelphis, i.e., placing
42
CONTEMPORARY TOPICS © 2003 by the American Association for Laboratory Animal Science
When the mother that was subjected to conventional anesthesia was placed on her dorsal surface, we observed that 8 of the 11
neonates were detached from the mother. Upon injection of
10 µl of PBS buffer, the three remaining babies also became
detached. Efforts to reattach the neonates failed, and the entire
litter was lost.
The neonates of all of the mothers anesthetized using the newly
described method remained attached to nipples throughout the
injection procedure. Generally, the animals were maintained
under the anesthetized relaxed state for about 2 to 5 min, which
was enough time to conduct the injections. The conical tube
was replaced loosely over the animal’s head for extended anesthesia if more time was required to complete the procedure.
We injected 49 neonates belonging to five litters with PBS. We
injected one litter on the day of birth, one when the pups were 1
day old, and three when they were 2 days old (Table 1). Litter 1
consisted of eight 0-day-old babies, which were naturally distributed apart from each other over the mammary region of the
mother. The small size [weight, 100 mg (mean value [1]); length,
0.75 cm (mean value, unpublished data)] and the delicate membrane-like skin of the 0-day-old babies warranted extremely
cautious handling. The volume of PBS injected varied from 5 to
10 µl. All injections were made subcutaneously by horizontally
inserting the 29-gauge needle through the skin without entering the underlying muscular layer. The skin, although
underdeveloped, could still retain the injected saline. As shown
in Table 1, no mortality occurred in litter 1 as a consequence of
the injections.
Litter 2 comprised nine 1-day-old babies. Like the 0-day-old
pups, the 1-day-old babies were still immature, but slightly larger.
Their skin is slightly more developed, and slightly larger volumes
of fluid could be injected without appreciable loss through the
injection site. Because they are larger, the 1-day-old babies are
positioned more closely to each other (Fig. 2). For the pups in
this litter, we injected 15 to 20 µl PBS, and all nine babies appeared to be normal 1 week after injection.
Litters 3 through 5 were 2 days old and more mature than
litters 1 and 2. These three litters were injected with 10 to 15 µl,
15 to 20 µl, and 25 to 30 µl PBS, respectively. Although litters 3
and 4 were intact 1 week after injection, 4 of the 11 babies of
litter 5 were lost 1 day after injection, and another three were
lost the following day (Table 1). We do not know the cause of
death of the babies because it is not uncommon for mother opossums to cannibalize young under normal undisturbed situations.
However, this result suggests that 15 to 20 µl is a safe volume for
subcutaneous injection of neonates older than 1 day and that at
least some 2-day-old opossums can tolerate 25 to 30 µl.
Litters that survived 1 week after injection were returned to
the colony for other research purposes. They were not monitored further because loss of pups beyond this age was not likely
to be related to the injections.
Volume 42, No. 5 / September 2003
Discussion
After we conducted the experiments reported here, we became
aware of a similar method that was developed for anesthetizing
rats (13). However, that method requires restraining the rat by
holding its back. Obviously, the restraining effort and the forceful
struggling of the opossum mother to avoid inhaling halothane
would stress both the mother and the neonates. Therefore, our
protocol, which involves lowering the mother upside down into
the halothane-containing beaker for < 30 sec to lightly anesthetize
the animal before applying the nose cone, has proven to be quick
and effective for Monodelphis mothers with neonates for brief procedures. However, investigators may find it advantageous to use a
calibrated halothane precision vaporizer if they have access to one,
particularly for longer-term sedation.
Other methods have been developed to anesthetize
Monodelphis with litters, e.g., combined use of a mixture of drugs
including pentobarbital, atropine and ketamine, etc. (14). However, injectable anesthesia is only required for prolonged
procedures, and because inhalation anesthesia of the mother is
required initially to partially anesthetize the animal before injections are performed, that complex method takes longer for
both anesthetic induction and recovery. In addition, preheated
heating pads are required during recovery to prevent hypothermia. Poor temperature regulation of the mother and prolonged
recovery may be detrimental to the survival of the neonates.
Because of the small size of neonates and the underdeveloped
state of their skin, injected volumes should be minimal. However,
we caution that the volumes stated in this article denote the injected volume and not necessarily the actual volume retained
subcutaneously by the neonates. A fraction of the injected volume
may be lost by postinjection leakage at the injection site, spontaneously or as a consequence of licking by the mother. Therefore,
we suggest that investigators adjust the volume to be slightly greater
than the volume expected to be retained subcutaneously.
In summery, the high survival rate of neonatal pups injected
after we used this maternal anesthetic regimen enhances the
value of M. domestica as a model of early mammalian development and reduces the number of animals needed.
Acknowledgment
We thank the Robert J. Kleberg, Jr., and Helen C. Kleberg Foundation for supporting this research.
Volume 42, No. 5 / September 2003
References
1. VandeBerg, J. L. 1999. The laboratory opossum (Monodelphis
domestica), p. 193-209. In T. Poole and P. English (ed.), UFAW handbook on the management of laboratory animal, 7th ed., vol. 1:
terrestrial vertebrates. Blackwell Science Ltd., Oxford, U.K.
2. Mate, K. E., E. S. Robinson, J. L. Vandeberg, et al. 1994. Timetable
of in vivo embryonic development in the gray short-tailed opossum
(Monodelphis domestica). Mol. Reprod. Dev. 39:365-374.
3. Stewart, R. R., D. J. Zou, J. M. Treherne, et al. 1991. The intact
central nervous system of the newborn opossum in long-term culture: fine structure and GABA-mediated inhibition of electrical
activity. J. Exp. Biol. 161:25-41.
4. Saunders, N. R., A. Deal, G. W. Knott, et al. 1995. Repair and recovery following spinal cord injury in a neonatal marsupial
(Monodelphis domestica). Clin. Exp. Pharmacol. Physiol. 8:518-526.
5. Saunders, N. R., P. Kitchener, G. W. Knott, et al. 1998. Development of walking, swimming and neuronal connections after
complete spinal cord transection in the neonatal opossum,
Monodelphis domestica. J. Neurosci. 18:339-355.
6. Fadem, B. H. and J. V. Tesoriero. 1986. Inhibition of testicular
development and feminization of the male genitalia by neonatal
estrogen treatment in a marsupial. Biol. Reprod. 34:771-776.
7. Armstrong, J. R. and M. W. Ferguson. 1995. Ontogeny of the skin
and the transition from scar-free to scarring phenotype during
wound healing in the pouch young of a marsupial, Monodelphis
domestica. Dev. Biol. 169:242-260.
8. Kuehl-Kovarik, C., D. S. Sakaguchi, J. Iqbal, et al. 1995. The gray
short-tailed opossum: a novel model for mammalian development.
Lab. Anim. 24:24-29.
9. Robinson, E. S., G. B. Hubbard, G. Colon, et al. 1998. Low dose
UVB exposure of suckling young can lead to widespread melanoma
in the opossum model. Int. J. Exp. Path. 79:235-244.
10. Robinson, E. S. and T. P. Dooley. 1995. A new allogeneic model
for metastatic melanoma. Eur. J. Cancer. 31A:2302-2308.
11. Fadem, B. H. and H. Z. Hill. 1985. The gray opossum (Monodelphis
domestica): a marsupial model for xenogeneic neoplasms. Cancer
Lett. 27:233-238.
12. Fadem, B. H., H. Z. Hill, C. A. Huselton, et al. 1988. Transplantation, growth, and regression of mouse melanoma xenografts in
neonatal marsupials. Cancer Invest. 6:403-408.
13. Martinic, G. and J. Taylor. 1993. Dorsal metatarsal, penile, and sublingual vein injections of anesthetized rats using a simplified
inhalation anesthetic. Lab. Anim. 22:38-44.
14. Robinson, E. S. and J. L. VandeBerg. 1994. Blood collection and
surgical procedures for the laboratory opossum (Monodelphis
domestica). Lab. Anim. Sci. 44:63-68.
CONTEMPORARY TOPICS © 2003 by the American Association for Laboratory Animal Science
43