546
KRISTAL AND NISHITA
or placentophagia alone, on the subsequent response
toward donor placenta of nonplacentophagic virgins.
In addition, the effect of this experience on the in-
duction rate of pup-directed maternal behavior (ma-
ternal sensitization latency) was assessed.
METHOD
General Design
Each rat, after we determined that she would neither eat pla-
centa nor retrieve pups, was exposed to a rat giving birth and
eating delivered placenta, to a nonpregnant rat eating donor pla-
centa, or to a nonpregnant rat eating chow. During the observa-
tion period, the subject was presented with a donor placenta that
she could see, smell, and eat (access condition), or see and smell,
but not eat (no-access condition). Each stimulus rat, in the center
compartment, was flanked by a pair of observers, one with access
to placenta and the other without. The duration of exposure to
nonpregnant stimulus rats was determined by the duration of de-
livery of the parturient rats. On the day after observing the stim-
ulus rat, each subject was given a placentophagia test (Test 1) and
her response was recorded. Two days later, each rat began a period
of constant exposure to foster pups (concaveation) to determine
the rate of onset of pup-induced maternal behavior (maternal
sensitization latency) (Cosnier & Couturier, 1966; Rosenblatt,
1967). Eight days later, whether or not the subject became mater-
nal, she was given another single placentophagia test (Test 2).
The sets of six subjects exposed to the initial conditions together
(the access rat and the no-access rat in each of the three sets) were
always tested as a group on the same day.
Subjects
One hundred and ten virgin female Long-Evans rats, determined
in pretests to be nonplacentophagic and not spontaneously mater-
nal, served as subjects. All were about 75 days old and weighed
about 225 g at their entry into the experiment. Thirty-eight of these
rats were purchased at 7 weeks of age from a commercial supplier
(Charles River Breeding Laboratories); the remaining 72 were born
and raised in our laboratory and were the daughters of Long-
Evans rats purchased from the same supplier. The rats from the
two sources were distributed evenly across groups. An additional
55 Long-Evans females served as social stimuli for the subjects.
When not in the experimental chamber, each rat was housed in-
dividually in a 45 × 19 × 25 cm hanging wire-mesh cage, which
was fitted with a food hopper containing Charles River Rat/
Mouse/Hamster Formula 3000, and with a water bottle. Food and
water were available ad lib. The entire colony was on a 14:10
day: night cycle, with the day phase beginning at 0600 h (EST).
Procedure
Placentophagia Pretest
When each rat was about 75 days old, we began daily examina-
tion of the cells in the vaginal smear to verify normal ovarian cy-
clicity. After the rat had undergone at least two consecutive nor-
mal estrous cycles, she was tested for her response to donor pla-
centa. Placentophagia tests were conducted during the third quarter
of the light cycle (1200 to 1500 h). Each rat was allowed to habitu-
ate for 2 h to a wire-mesh cage in the testing room. Food was
not available during the 2-h habituation period or during the test.
Water was not available for the last 15 min of the habituation
period or during the test. The test consisted of 15 min of exposure
to a donor placenta presented in an untippable glass dish. If the
rat ate it, she was designated a placentophage and returned to the
colony. (These placentophages served as stimulus rats during the
observation sessions.) If not, she underwent the same procedure
the next day and, if necessary, on the 3rd day. If she did not eat
on the third exposure, she was designated a nonplacentophage.
Previous research has indicated that the response to placenta is
dichotomous in virgins; either they eat it almost immediately or
they appear to avoid it. Furthermore, eaters generally do so on
the first exposure, and once a rat eats in this type of test, she re-
liably eats placenta afterward when it is made available (Kristal,
1980; Kristal & Graber, 1976). On the other hand, a small pro-
portion of nonplacentophages switch after stressful experiences,
so an additional single pretest (fourth placenta exposure), con-
firming nonplacentophagia, was conducted and is described below.
Donor placentas were obtained surgically on Day 22 of preg-
nancy from CO1-killed Long-Evans multiparae. The placentas,
placed three or four to a vial along with a few drops of physio-
logical saline, were then immediately frozen at -20°C. When
needed, the vial was rapidly thawed and the contents warmed to
about 37°C for presentation.
Spontaneous Retrieval Test
A small proportion of virgin rats (extremely small in the Long-
Evans strain) behave maternally toward pups as soon as the pups
are presented. A few of these spontaneous retrievers distributed
unevenly among the groups could bias the results, whereas a larger
number might be expected to be distributed by chance more evenly
and need not affect the results. Since the expected number was
small and was likely to be distributed unevenly, we decided to
identify and remove spontaneous retrievers from the pool of po-
tential subjects.
Approximately I week after the placentophagia pretest, each rat
was placed in a wire-mesh-covered 26.7 x 52.1 × 30.5 cm glass
chamber (10-gal aquarium) containing food, water, and 3 cm of
coarse sawdust and was allowed to habituate to the new surround-
ings for 24 h. The aquaria were all kept in a separate test room.
At about 0900 h, each rat was presented with four 3- to 8-day-old
pups, which had been handled only with disposable plastic gloves.
The presenter scattered the pups in the half of the aquarium that
did not contain the rat’s sleeping area. The rat was then observed
for 15 min; if she retrieved the pups to a central site or even just
carried them around the cage, she was designated a spontaneous
retriever and was removed from the experiment. Only one spon-
taneous retriever was found.
Fourth Placenta Exposure
To avoid the possibility that the spontaneous retrieval test was
stressful enough to cause some nonplacentophages to become
placentophages (Kristal et al., 1981), a single placentophagia test
was conducted about I week after the spontaneous retrieval test.
This test was actually a 4th day of placentophagia pretest, con-
ducted several days, rather than I day, after the third. If a
rat ate placenta on this fourth exposure, she was removed from
the experiment. None of the rats that had come this far in the pre-
testing had to be removed on the basis of the fourth placenta pre-
sentation.
The Effects of Observing Stimulus Rats
Apparatus. Three IO-gal aquaria were modified by inserting in
each two perforated .25-in. Plexiglas panels, so that each aquarium
was divided into three compartments, each measuring 26.7 cm
long × 17.4 cm wide x 30.5 cm high. The panels were perforated
with 65 .5-cm holes. Each of the three compartments was fitted
with a water bottle and drinking tube; the two end compartments
each contained a 4.5 × 5.4 x 3.5 cm Plexiglas feeder with a
removable, perforated Plexiglas lid. The feeder was secured to the
floor of the compartment. The aquaria were covered with lids of
.5-in. mesh hardware cloth.
Each aquarium was housed in a sound-attenuating ventilated
41.9 × 92.1 × 40.0 cm box. Each box was fitted with a 4.5-in.
exhaust fan, was lined with 1.25 cm of acoustic insulation, was
lit by a timer-operated 7.5-W incandescent bulb, and had a one-
way viewing port in the door.