Comparative Analysis ofMaternal Care in theHigh-Yawning (HY) andLow-Yawning (LY) SublinesFrom Sprague–Dawley Rats

Araceli Ugarte1

Jose R. Eguibar1,2

Ma. del Carmen Cortes1

Bertha A. Leon-Chavez3

Angel I. Melo4

1Instituto de FisiologıaBenemerita Universidad Autonoma

de Puebla, Puebla, MexicoE-mail: jeguibar@siu.buap.mx

2Secretarıa GeneralBenemerita Universidad Autonoma

de Puebla, Puebla, Mexico

3Facultad de Ciencias QuımicasBenemerita Universidad Autonoma

de Puebla, Puebla, Mexico

4Centro de Investigacion enReproduccion Animal

CINVESTAV-Universidad Autonomade Tlaxcala, Tlaxcala, Mexico

ABSTRACT: High- and low-yawning rats (HY and LY) were selectively bred as afunction of their spontaneous yawning frequency with the LY subline about 2 yawns/hr and the HY 20 yawns/hr. The HY rats have more grooming bouts and travellonger distances in an open field. HY dams spent less time in the nest, retrieved theirpups faster, and show a longer latency to licking and mouthing the pups than the LYor outbred Sprague–Dawley (SD) animals. The percentage of HY dams that hadatypical retrieving was higher, with a lower nest quality, and produced offspringwhose weights were lower than those from the LY subline. We also showed that thepregnant HY dams have fewer pups and the percentage that had lost at least threepups during lactation was higher than the SD and LY dams. In conclusion, HY damsare motivated to take care of their pups, but the ‘‘fine tuning’’ of maternal care isdisturbed. � 2010 Wiley Periodicals, Inc. Dev Psychobiol

Keywords: yawning; grooming; anxiety; retrieving; nest building; inbred rats;lactation; animal model; dopamine; stress

INTRODUCTION

Social interactions among members of a community are

important for their organization and survival. These

interactions need the display of affiliative behaviors such

as maternal care, sexual behavior, and grooming,

compared to the antagonistic ones such as aggression,

isolation, and submissive behaviors. In altricial species,

maternal behavior is the most important and most studied

behavior not only because it allows the immature

offspring to become independent over time, but it is also

a good model for the offspring’s physiological, neuro-

endocrine, and cognitive display as adults (Beach &

Jaynes, 1954; Hofer, 1994). Maternal behavior is the

expression of a series of motor and somatosensory events

by the mother at the end of pregnancy, parturition, and

during the postnatal preweaning period (Rosenblatt, 1967;

Rosenblatt & Lehrman, 1963). In rodents, once the pups

are born, the mother retrieves them to the nest, licks their

bodies and the anogenital region followed by a nursing

posture over them in a highly stereotyped and defined

organization (Gonzalez-Mariscal & Poindron, 2002;

Rosenblatt & Lehrman, 1963). Thus, the offspring receive

warmth, nutrients, protection, and sensory and social

stimulation in the form of social contact with the mother

and their siblings (Beach & Jaynes, 1954; Hofer, 1994;

Levine, Haltmeyer, Karas, & Denenberg, 1967). In rats

and mice, differences in the timing and components of

maternal behavior have been described over the course of

the nurturing period, variations that mediate behavioral

transmission of traits and thus transgenerational or

nongenomic transmission to the offspring (Champagne,

Developmental Psychobiology

Received 10 June 2010; Accepted 16 August 2010Correspondence to: J. R. EguibarContract grant sponsor: VIEP-BUAP G/SAL/2010Contract grant sponsor: CONACyTContract grant number: 106694Contract grant sponsor: PROMEP-BUAPContract grant number: 664Published online in Wiley Online Library

(wileyonlinelibrary.com). DOI 10.1002/dev.20497

� 2010 Wiley Periodicals, Inc.

Francis, Mar, & Meaney, 2003; Fleming et al., 2002).

There are variations in mother–infant interactions within

a same strain, that is, natural variations (Champagne,

Curley, Keverne, & Bateson, 2007; Champagne et al.,

2003; Meaney, 2001). For example, by choosing the

extremes of the populations of the Long-Evans rats and

comparing the frequency that the mothers lick their pup’s

body and genitals, and their nursing posture, Meaney and

his group had found that there are mothers that show high

levels of licking and arched-back nursing (HG-ABN) and

other mothers that show low levels of these behaviors

(LG-ABN; Champagne et al., 2003). Interestingly,

lactating rats from HG-ABN mothers show a low

reactivity of the hypothalamus–pituitary–adrenal (HPA)

axis after exposure to stressful environment, with a small

fear response, a good level of spatial learning, and mainly

spent much time licking and nursing their pups compared

to the LG-ABN dams. These differences are not caused by

the genetic background because cross-fostering studies

have shown that the offspring phenotypes depend on the

mother that reared it (Champagne et al., 2003). Exper-

imentally, it is possible to regulate how the mothers take

care of their offspring. If lactating rats are exposed to a

handling paradigm (pups are removed from the nest for

15min), the dams spend more time licking the body and

genital area of the pup’s when they are returned to the nest

(Meaney et al., 1985; Pryce, Bettschen, & Feldon, 2001).

Contrary to the handling paradigm, mothers who had

been isolated from their mothers during infancy spent

less time taking care of her pups (Gonzalez, Lovic,

Ward, Wainwright, & Fleming, 2001; Melo, Hernandez-

Curiel, & Hoffman, 2009; Melo et al., 2006). Different

groups of mice showed remarkable variations in the

expression of maternal behavior (Anisman, Zaharia,

Meaney, & Merali, 1998; Broida & Svare, 1982; Brown,

Mathieson, Stapleton, & Neumann, 1999; Champagne

et al., 2007; Ohta, Shirota, Tohei, & Taya, 2002; Shoji &

Kato, 2006). Thus, lactating female mice of C57BL/6,

CBA/H, C3H/Ico, and CBA/J strains retrieved pups

faster than BALB/c, NBZ, DBA/2, XLII, A/J, and AKR

strains (Carlier, Roubertoux, & Cohen-Salmon, 1982).

Furthermore, DBA/2J females built better nests and spent

more time crouching over and nursing pups (Brown et al.,

1999) than C57BL/6J dams.

A comparison between inbred and outbred mice has

shown that the 129Sv strain had shorter latencies in nest

building, built the nest less frequently, and spent less time

engaged in licking the pups than outbred dams (Broida &

Svare, 1983; Champagne et al., 2007; Meaney, 2001). In

rats, psychogenetic selection has resulted in at least four

different strains that, besides their own phenotype, have

variations in postpartum maternal behaviors; (1) The

Flinders Sensitive Line (FSL), considered a genetic

animal model of depression, spent less time licking the

pups and nursing them (Lavi-Avnon, Yadid, Overstreet, &

Weller, 2005), a shorter latency to first pup retrieval, and

more self-directed behavior than controls (Braw et al.,

2009); (2) spontaneously hypertensive rats (SHR) more

often had an arched and blanket-nursing posture and a

lesser passive-nursing posture, spent less time licking

their pups, and retrieved them more quickly than the

Wistar strain (Myers, Brunelli, Squire, Shindeldecker, &

Hofer, 1989); (3) Roman high (RHA-Verh)- and low

(RLA-Verh)-avoidance sublines of rats were selected and

bred for their rapid response compared to poor acquisition

in a two-way active-avoidance response (Steimer, Escor-

ihuela, Fernandez-Teruel, & Driscoll, 1998). Female rats

of RHA-Verh mothers had a high active avoidance, spent

less time with their young, are more active, and also

assumed the side-nursing position less often than the

RLA-Verhmothers (Driscoll, Fumm,&Battig, 1979); and

(4) Hatano high- (HAA) and low- (LAA)-avoidance

selective-breeding lines from the Sprague–Dawley strain

(Ohta,Matsumoto,Nagao,&Mizutani, 1998) show a high

variation in the expression of maternal behavior, with the

low avoidance (LAA) females having longer latencies for

retrieving the pups, spent less time with them, showed a

decreased amount of milk ejection, a lesser increase in

blood prolactin, and a greater increase of adrenocortico-

trophic hormone (ACTH) than the HAA mothers (Ohta

et al., 2002).

Although the behavioral differences among inbred

groups of mice or rats are attributed to genetic variations,

it has been reported that the genetic–environment

interactions early in life, mainly mediated by maternal

care and their siblings, are the main cause of those

variations (Francis, Szegda, Campbell, Martin, & Insel,

2003; Myers et al., 1989; Ohta et al., 1998; Shoji & Kato,

2009; Steimer & Driscoll, 2005). These data show that,

besides the phenotype used, during inbreeding the process

can generate other changes of the behavioral display that

could be caused by maternal care.

Yawning is a phylogenetically old behavior and

stereotypically shown by reptiles, fish, birds, and

mammals (Walusinski & Deputte, 2004). It consists of a

wide opening of the mouth with a long inspiration,

followed by a short expiration. Yawning can bemodulated

by several peptides such as adrenocorticotropin hormone,

alpha-melanocyte stimulating hormone, and oxytocin,

and also by several neurotransmitters as GABAergic,

dopaminergic, and muscarinic cholinergic systems in

several strains of rats, as well as HYand LY sublines (for

review, see Collins & Eguibar, 2010; Doger, Urba-

Holmgren, Eguibar, &Holmgren, 1989; Eguibar, Barajas,

&Moyaho, 2004; Eguibar, Romero-Carbente,&Moyaho,

2003; Urba-Holmgren, Santos, Holmgren, & Eguibar,

1993). The HY males yawned more and also had more

grooming bouts after exposure to a novel environment

Developmental Psychobiology2 Ugarte et al.

than LY rats (Eguibar &Moyaho, 1997). The HY subline

is also more active in an open-field arena (Moyaho,

Eguibar, & Diaz, 1995). In addition, after wetting the HY

showed a disorganized grooming-chain sequence com-

pared to the LY animals with a clear cephalocaudal

organization, similar to that obtained in other strains of

rats (Moyaho et al., 1995). These observations suggest

that the early life experience, such as maternal care and

lactation, could be the cause of the differences among the

sublines. In our experiments, we analyzed maternal care

toward their own offspring of HY and LY dams and

compared them with outbred Sprague–Dawley dams

during the early-to-middle lactation period. In a second

experiment, we compared the number of pups at

parturition and weaning and the fertility index of the

females of all groups.

METHODS

Subjects to Study

The subjects were outbred Sprague–Dawley (SD) HY and

LY females of 90–100 days old obtained and bred in our animal

room facilities with a control temperature (21� 2�C) and

relative humidity (30–45%) with a 12:12 light/dark schedule,

with lights on at 0700. Balanced rodent pellets (Zeigler,

Gardners, PA) and tap water were provided ad libitum.

At the Institute of Physiology of theBenemeritaAutonomous

University of Puebla,Mexico,we selectively inbred two sublines

from Sprague–Dawley rats with a high- and low-spontaneous

yawning frequency (Urba-Holmgren et al., 1990). The high-

yawning rats (HY) had a mean frequency of 20 yawns/hr and

were obtained by an inbreeding process of more than 70

generations. The low-yawning rats (LY) were inbred for more

than 60 generations and have a mean spontaneous yawning

frequency of around 2 yawns/hr (Urba-Holmgren et al., 1990),

with the males yawning more frequently than the females

(Moyaho, Barajas, Ugarte, & Eguibar, 2009).

All procedures described in this study were in accordance

with the Mexican guidelines NOM-062-200-ZOO-1999 for the

care and use of laboratory animals, which are in accordancewith

the NIH Guide for the Care and Use of Laboratory Animals

(HHS 85-23; Clark, 2002), and were approved by the University

Animal Care and Use Committee.

Mating

At 90–100 days old, the nulliparous SD, LY, and HY female rats

were placed in reproduction units with a sexually experienced

male of the same group. Every day all females are visually

inspected and the presence of a vaginal plug was taken as the

beginning of pregnancy. The male was then removed from the

cage.

Experimental Procedure

Experiment 1. On pregnancy days 16–18, each female was

placed in a transparent Plexiglas cage (32 cm� 47 cm� 20 cm)

which allowed us to observe all the behavioral repertoire of the

female. The females were provided with paper towel strips for

building a nest, and then were checked daily for parturition. The

nest quality was rated on a 5-point scale ranging from 0 to 4,

modified from Lisk et al. (Lisk, Pertlow, & Friedman, 1969).

A score of 0 was given when no nest was built by the female.

A score of 1 was given when nesting material was present in a

corner of the cage, but no organized nest was built. A score of

2 was given when some kind of organization of nesting material,

such as semicircular organization, was made in a corner of a

cage, but nowalls ormore complex structureweremade. A score

of 3was givenwhen a complete circular or semicircular nestwith

walls was built and a score of 4 was given to a full nest with tall

walls. Newborn litters found up to 1200 each day were

designated as born on that day (Day 0). Only the females that

mated successfully and had at least 7–8 pups per litter at

parturition were used. The litters were culled to eight pups, with

the gender distribution kept as equal as possible in each litter. All

testing was done between 1000 and 1300 inside the same animal

room tominimize the stress response. On postpartumday 1, pups

were removed from the nest for 2–3min, weighed, and returned

to the opposite corner to where the nest had been built. Maternal

behavior was immediately videotaped for 15min. The same

procedure was done at postpartum days 3, 5, 7, and 9 using the

Observer video Pro software v. 5.0 (Noldus Information

Technology, Amsterdam, the Netherlands). We measured the

time to retrieve the first pup and each of the siblings to the nest,

the time to pup licking, nest-building, nursing, and pup

mouthing. Additionally, we recorded the nest quality and also

the frequency and duration of each maternal behavior, which

were (A) retrieval of pups, (B) licking of pup bodies, (C) licking

of pup anogenital region, (D) crouching (the female adopting a

high or low nursing posture), (E) nest building, (F) being close

to the pups (the time the female spent in close proximity

within 5 cm), (G) nest-height, (H)mouthing and sniffing the pups

(rearrangement of the pups inside the nest by the dam), and

(I) sniffing the pups. In addition, other nonmaternal behaviors,

such as running, walking, jumping, eating, self-grooming, and

rearings, were also recorded.

Experiment 2. To record the number of females that success-

fully mated (fertility), the size of the litter at birth, and the

mortality at weaning, we used another cohort of females from

each group. As for the first cohort, each female was placed in an

individual Plexiglas cage at the end of pregnancy and checked

daily for parturition. All the females that mated successfully

were recorded and used independently of the number of pups per

litter at birth. On the day of parturition (Day 0), the number of

pups was recorded and returned to the nest andwas not disturbed

until weaning (about P22). The mothers were treated for

cleaning, food, and water as for every rat in the vivarium. At

weaning, the number of pups was again determined, and the

number of dams that lost at least three pups.

Statistical Analysis

Because the data did not always show homogeneity of

variance, the latencies to retrieve the first, the second, the third,

and the last pup of the litter to the nest and the average latency to

Developmental Psychobiology Maternal Care in High- and Low-Yawning Rats 3

began each behavior during the first 3 days of testing (postpartum

days 1 and 3 (1þ 3)) and during the last 2 days of testing

(postpartum days 7 and 9 (7þ 9)) were compared using

nonparametric statistics, the Kruskall–Wallis test, an ANOVA,

and a w2 test for three-group analyses, and the Mann–Whitney

U-test, and a w2 test for two-group analyses. The sameprotocol of

analysis was used to compare the frequency and duration of the

different components of maternal behavior (nursing, body and

genital licking, and nest-building), the nonmaternal behaviors,

and the number of pups at birth and at weaning. The w2 test was

used to compare the proportion of females that displayed

atypical retrievings, re-retrievings, nest building of high and

low quality, and those that lost at least three pups at weaning.

P-values of<.05 were accepted as statistically significant. Data

were analyzed using SPSS software (Version 11.0 for Windows

Vista).

RESULTS

Experiment 1

Latency for Retrieving Pups and the Total Time for

Retrieving All the Litter. Figure 1 showed that the

latency to retrieve to the nest the first pup (P<.02), second

(P<.04), third (P<.03), and the last pup (P<.01) were

different among the groups. The post hoc comparisons

showed that the HY dams retrieved the first, second, third,

and the last pup more quickly than the SD dams (P<.05,

P<.008, P<.006, and P<.005, respectively). Further-

more, to analyze with detail the latency to retrieve the first

pup, we compared them during the first 3 days of lactation

(1þ 3) compared tomiddle lactation period (7þ 9). There

were marginal group differences during the last 2 days of

testing (P¼.07; Fig. 2A), but not in the first 2 days of

testing. The post hoc comparisons showed that the HY

dams retrieved the first pups more rapidly than the SD

dams (P<.05) and the LY dams (P<.05).

Latency to Begin Maternal Behavior. To compare the

differences among HY, LY, and SD dams at the beginning

of each of the maternal characteristics in the early (1þ 3)

compared to middle lactation (7þ 9), we averaged the

latency of each behavior from the first days of lactation

compared to middle lactation. There were no significant

differences in the latency to begin nursing, body and

genital licking, nest building, or mouthing in the first

2 days among groups. However, there were group differ-

ences in the last 2 days in the latency of body licking

(P<.01, Fig. 2C), genital licking (P<.05, Fig. 2D), and

mouthing the pups (P<.003, Fig. 2F). Figure 2C and D

shows the latency to lick the pup’s body and genitals were

longer in the HY dams than in the LY dams (P<.02 and

<.03, respectively) and the SD dams (P<.03 and <.03,

for both comparisons). In contrast, the HYmothers began

mouthing the pups faster than the LY and SD mothers

(P<.04 and <.001; Fig. 2E).

Maternal Behavior. There were group differences in the

total time to retrieve (P<.03; Fig. 3A), with the HY

quicker than the SD dams (P<.007). This is also truewith

the time that the mothers spent inside the nest, which is

greater in theHY subline (P<.03; Fig. 3E). Therewere no

significant differences in the total time of nursing, body

and genital licking, or nest building among groups.

Subsequent analyses showed that the HY mothers spent

less time retrieving all pups and inside the nest compared

to the SD dams (P<.03 and <.03, respectively). The LY

dams spent less time inside the nest than the SD dams

(P<.02, Fig. 3E). Furthermore, as shown in Table 1, only

10% of the HY mothers built a maternal nest of high

quality, that is, a compact nest at least 5-cm high; score:

(3–4), which was significantly less compared to that of

90% of the SD and 50% of the LY dams (P<.0001 and

<.05 in the other two comparisons). In addition, the

percentage of LY dams that built high-quality nests was

significantly lower than the SD dams (P<.05; see also

Table 1).

Atypical Maternal Behavior

Reretrievings. Under our conditions and without any

selection of dams, some rats from all groups made the

reretrieving behavior that is characterized by taking the

pups outside the nest (once theywere retrieved) and after a

variable time the dams returned them to the nest. Because

almost all females showed at least one reretrieving during

the period of observation, we computed as reretrieving if

the dam showed this behavior three ormore times during 3

or more days. Eighty percent of the HY dams reretrieve

pups, which was higher than the SD dams of just 40%

(P<.05; Fig. 4A) and 50% in the LY subline (P<.05;

Fig. 4A).

Developmental Psychobiology

FIGURE 1 Latency for retrieving the first, second, third, and

last pup of the litter by each group of dams. In all tested days, the

HY dams (dark bars) were significantly faster than the LY (gray

bars) and Sprague–Dawley (open bars) rats. �P<.05 and��P<.01. Data are the mean� SE of 10 rats in each group.

4 Ugarte et al.

Atypical retrieving behavior. Mothers were considered

to show atypical retrieving when they retrieved at

least three pups by holding them by their leg, mouth, or

belly during testing, and during 3 or more days. The 80%

of the HY dams that had atypical retrieving was

significantly higher than the 30% of the SD and LY

mothers (P<.0001 and <.02; Fig. 4B). A statistical

tendency in the percentage of the HY dams that showed

atypical retrieval was higher than for the LY mothers

(P¼.06).

Nonmaternal Behaviors. Most of the mothers showed

nonmaternal behaviors, such as exploring, digging the

cage, hanging on the top of the cage, and self-grooming.

We did find group differences for the exploration time of

the maternal cage (P<.007; Fig. 5A), with a post hoc

comparison showing that the SD dams spent less time

exploring their cage than the HY dams (P<.002) and LY

dams (P<.03). In addition, the SDmothers spent less time

engaging in self-grooming than the LYmothers (P<.05),

but not the HY dams (Fig. 5B).

Developmental Psychobiology

FIGURE 2 Latencies to begin each of thematernal components in the early compared to themiddle

lactation days. (A) Latency for retrieving of all pups to the nest. (B) Latency to nursing the pups.

(C) Latency to body licking. (D) Latency to genital licking. (E) Latency to start nest building.

(F) Latency to mouthing the pups. Note that retrieving and mouthing the pups is statistically lower in

the HY dams (upper triangles) compared to the Sprague–Dawley (filled circle) and LY (filled lower

triangles) dams (�P<.05). The HY dams lick the body and genitals of the pups similar to the other two

groups of rats (�P<.05).

Maternal Care in High- and Low-Yawning Rats 5

Experiment 2

Fertility. Although not significant, only 68% of the HY

females became pregnant and these data were lower than

in the SD and LY females, who had 82% pregnancy.

Weight of pups. As shown in Table 2, there were group

differences for individual body weight on postnatal day

(PND) 1, 5, 9, 14, and 18 (P<.001 in each age), with post

hoc comparisons showing that the body weights of the

offspring in PNDs 1, 5, 9, 14, and 18 from the HY dams

were lower than the SD dams (P<.0001 in each age).

Similarly, body weights of the offspring of PNDs 1, 5, 9,

14 (P<.0001), and 18 (P<.02) from the HY dams were

lower than those from the LY dams. The body weight of

Developmental Psychobiology

FIGURE 3 Time spent by the dams displaying each maternal component. (A) Retrieving,

(B) nursing, (C) body licking, (D) genital licking, (E) inside nest, and (F) nest building. The time

spent by the dams in inside nest was statistically lower in the HY and LY sublines compared to the

Sprague–Dawley dams (�P<.05). Data are the mean� SE of 10 rats in each group.

Table 1. Percentage of FemalesThatBuilt Nests ofLowand

High Quality

Nest Quality

Score SD LY HY P-Value

Low (1–2) 10 50 90 <.0001 vs. HY

<.05 vs. LY

<.05 vs. HY

High (3–4) 90 50 10 <.0001 vs. HY

<.05 vs. LY

<.05 vs. HY

SD, Sprague–Dawley; HY, high-yawning; LY, low-yawning.

Note. All comparisons are done using the w2 test, followed by a Tukey

test.

6 Ugarte et al.

the offspring of PNDs 14 and 18 from the LY dams was

lower than the offspring from the SD dams (P<.009

and <.002). When we compared the difference in the

percentage of the pup’s body weight from the HYand SD

mothers, we also showed that the HY offspring had 16%

(PND1), 19% (PND5), 21% (PND9), 17% (PND14), and

26% (PND18) lowerweights than those obtained from the

Sprague–Dawley rats. To determine whether the low

weight of the HY pups at weaning remained until

adulthood, the body weight of females from the LY and

HY sublines of 75 and 90 days old were recorded. The

body weight of the HY female rats was lower than the LY

animals at both ages (P<.05, data not shown).

Number of Pups Per Litter at Birth and Weaning.

There were group differences among the groups of the

number of pups per litter at birth (P<.03) and number of

pups weaned (P<.0001).Whenwe compared the number

of pups per litter for the HY mothers it was significantly

lower than those of theLYdams (P<.01) and compared to

the SD dams (P¼.06; see Fig. 6). Furthermore, the

number of pups at weaning time from theHYmothers was

significantly lower than from the SD and LY mothers

(P<.002 and<.0001). In addition, the percentage of HY

mothers that lost at least three pups during lactation was

higher than that of the SD and LY mothers (P<.03 and

<.006).

DISCUSSION

In this study we compared the maternal and nonmaternal

behaviors of primiparous female rats of the HY and LY

inbred sublines compared to outbred Sprague–Dawley

rats during early-to-middle lactation. Our results showed

that HY mothers express different patterns of maternal

and nonmaternal behaviors compared to the Sprague–

Dawley and LY dams. In addition, the number of pups per

Developmental Psychobiology

FIGURE 4 Percentage of dams that did reretrieving and had

atypical retrievings. (A) All groups of rats show reretrieving, but

80% of the HY dams do and only 50% of the LYand 40% of the

Sprague–Dawley dams do this (�P<.05). (B) The atypical

retrieving is 80% greater in the HY dams than the Sprague–

Dawley dams that do not display this behavior (�P<.05) and the

LYdamswith just 30% (P¼.06).&Significantly different among

LYand Sprague–Dawley rats.

FIGURE 5 Time spent by the dams in nonmaternal behaviors.

(A) The total amount of exploration is greater in the HYand LY

dams compared to the Sprague–Dawley rats (�P<.05). (B)

The time spent in self-grooming is longer in the HY and LY

sublines compared to the Sprague–Dawley dams (�P<.05).&Significantly different among LYand Sprague–Dawley rats.

Maternal Care in High- and Low-Yawning Rats 7

litter at birth and weaning from the HYmothers was lower

compared to the other groups of rats.

The results showed that the time engaged in nursing

and licking the pups did not significantly differ among the

groups, but the HYand LY mothers spent less time inside

the nest than the Sprague–Dawley dams. In contrast, the

HY dams retrieve all pups to the nest more quickly than

the Sprague–Dawleymothers. These results are similar to

that obtained in mice from the C57BL/6, CBA/H, C3H/

Ico, andCBA/J strains, which retrieved pupsmore quickly

than the BALB/c, NBZ, DBA/2, XLII, A/J, and AKR

strains (Carlier et al., 1982). The HY dams not only

retrieve the pups more quickly, but most of them made

atypical retrievings and also displayed reretrieving and

built the nest more rapidly, but with lower quality. These

results match with those found in the 129Sv inbred strain

that had shorter latencies in nest building, built the nest

less frequently, and spent less time in licking the pups

(Champagne et al., 2007). The above data suggest that

mothers are maternally motivated but the ‘‘fine tuning’’ of

the expression of all maternal characteristics are dis-

turbed, similar to that already reported in the organization

of grooming bouts in HY rats (Eguibar & Moyaho, 1997;

Moyaho et al., 1995). Interestingly, when we compared

the latency of each maternal component in the early

compared to themiddle lactation, we showed thatmothers

from all groups showed similar latencies during the early

lactation. During the middle lactation the latency to

retrieve the pups, mouthing them, and building the nest by

theHYdamswas shorter, but the latencies to begin licking

the body and genital areas of the pups were longer than

that of the LYand Sprague–Dawley dams.

The HY, the LY, and the Sprague–Dawley dams

showed normal maternal motivation because once they

get cues from the pups, they established contact with them

(appetitive component), then they walk around the

maternal cage with the pups in their mouth and put them

in a different place and later on reretrieve them to the nest.

This shows that they have enough motivation to engage in

a behavioral interaction with a specific goal object, and

they retrieve them to the nest (consummatory compo-

nent), but their ability to show a specific behavior, that is,

properly retrieve the pups is not adequate after putting

them outside the nest, reretrieve them and also with

atypical retrieving suggesting a disorganized pattern in

the global organization of maternal care in HY rats

(Everitt, 1990; Numan, Fleming,&Levy, 2006; Numan&

Insel, 2003; Timberlake & Silva, 1995). The HY males

also have a disorganized sequence of their grooming bouts

because they showed caudocephalic or lateralcaudal

sequences, instead of the cephalocaudal sequences shown

by the LY rats and other rodent species (Berridge, 1990;

Developmental Psychobiology

Table 2. Pup Body Weight on Different Lactation Days

LactationDayGroup

SD LY HY P-Value

1 7.1� .5 7.3� .3 6.0� .1 <.005 vs. HY

<.000 vs. HY

5 12.9� .6 12.4� .4 10.4� .2 <.001 vs. HY

<.002 vs. HY

9 21.1� .5 20.6� .8 16.5� .5 <.001 vs. HY

<.001 vs. HY

14 26.7� .5 23.9� .7 21.8� .6 <.01 vs. LY

<.025 vs. HY

18 35.9� .8 30.2� 1.1 26.4� .9 <.0001 vs. HY

<.02 vs. HY

SD, Sprague–Dawley; HY, high-yawning; LY, low-yawning.

Note. Data are the mean� SE. The data is in grams.

FIGURE 6 Fertility index and numbers of pups per litter at

birth in the three groups of rats. The Sprague–Dawley (open

bars) and LY (gray bars) rats have more pups at parturition and

after the weaning period than the HY rats (filled bars, �P<.05).

Data from the LYand HY rats are the mean� SE of 15 rats and

for the Sprague–Dawley rats are the mean� SE of 10 rats.

8 Ugarte et al.

Moyaho et al., 1995). These alterations could be caused, at

least in part, to a greater number of D1 dopaminergic

receptors in the ventral striatum in theHYcompared to the

LY animals (Diaz-Romero, Arias-Montano, Eguibar, &

Flores, 2005). Matell, Berridge, and Wayne-Aldridge

(2006) showed that the grooming syntactic chains can be

altered after a lesion of the striatum or changing the

dopaminergic transmission in the nigrostriatal pathway. It

is well known that the basal ganglia play a crucial role in

the organization, timing, and coordination of motor

sequences including grooming (Cromwell & Berridge,

1996). Furthermore, systemic administration of SCH-

23390, a specific dopaminergic D1 antagonist, produced a

disruption ofmaternal care causing themother to leave the

pups outside the nest, so reretrieving them (Byrnes,

Rigero, & Bridges, 2002). This also happens with intra-

accumbens injection of cis-flupenthixol, which inhibits

maternal retrieving and licking the pups but enhances

nursing behavior in lactating Long-Evans rats (Keer &

Stern, 1999). The maternal-care deficits caused by

haloperidol can be restored by demanding pups (12-hr

deprived), showing that pups can reverse the effects

produced by the dopaminergic antagonist and by

bromocriptine, a dopaminergic agonist that produced an

opposite effect (Pereira & Ferreira, 2006). Because the

HY rats showed an increase of D1 receptors in the ventral

striatum (Diaz-Romero et al., 2005) and a decrease in the

dopamine levels in the nucleus accumbens (unpublished

data), we suggest that dopamine changes could be

responsible for the alterations in maternal care in the

HY dams.

Recently, it has been reported that the mother not only

gives somatosensorial stimulation but also gives growth

factors such as prolactin and growth hormone through the

milk that could act in concert to aid growth, weight gain,

and glucose homeostasis in the perinatal period (Fleenor

et al., 2005). Prolactin plays a fundamental role not only to

supportmilk production but also in the developmental and

maturation of the pups (Melo et al., 2009). The above data

suggest that because that HYoffspring never gain normal

weight during lactation it could be that these dams

produce milk of lower quantity or quality. It is also

possible that humoral factors such as growth, oxytocin,

and thyroid hormones could be responsible for the lower

rate of body-weight gain in the HY offspring (Bautista,

Boeck, Larrea, Nathanielsz, & Zambrano, 2008; Glinoer,

1997; Hapon, Simoncini, Via, & Jahn, 2003; Valdez,

Penissi, Deis, & Jahn, 2007).

A relationship between high emotionality and a deficit

in the expression ofmaternal licking and grooming aswell

as arched-back nursing posture has been demonstrated

(Francis, Diorio, Liu, & Meaney, 1999; Gonzalez, Lovic,

Ward, Wainwright, & Fleming, 2001; for review, see

Numan & Insel, 2003). Female rats that are isolated early

in life had a high stress response and show a deficit in the

expression of maternal behavior, and these characteristics

are transmitted to the next generation (Gonzalez et al.,

2001).

Because the HY offspring have lower weights during

lactation that persists until adulthood, it is possible that

behavioral disturbances found in these rats could be

caused by differences in the maternal care, including

grooming and the arched-back posture. There are some

reports that partially support this hypothesis, that is,

mothers that were undernourished during early life had an

abnormal maternal care, as we found in the HY mothers.

Thus, these dams show a decrease in nest quality and

nursing time, an increase in the latency to retrieve pups,

and atypical retrievings that can even produce sonic

distress in the pups (Regalado, Torrero, & Salas, 1999;

Salas, Torrero, Regalado, & Perez, 2002; Salas, Torrero,

& Pulido, 1984; Smart, 1976). The HYmothers also had a

lower nest-building rating and showed atypical retriev-

ings. Rosenblatt and Lehrman (1963) reported that when a

female cannot maintain a stable nest, she retrieves the

pups too many times and deposits them anywhere in the

cage, similar to that made byHYand LY dams, suggesting

that the sublines had a disorganized pattern of maternal

care.

It is important to emphasize that body weights of

undernourished pups from 4 to 20 days old were about

20–50% lower than well-nourished rats (Bautista et al.,

2008; Salas et al., 1984, 2002;Wiener, Fitzpatrick, Levin,

Smotherman,&Levine, 1977; Zambrano et al., 2005).We

found that bodyweights in theHYoffspringwere 16–25%

lower during lactation compared to the Sprague–Dawley

and LY animals. Because of the ad libitum disposition of

rodent food pellets the disturbed expression of maternal

behavior in HY dams are not because they are being

underfed during neonatal period, but it is probably

generated by maternal care. These deficits in birth weight

can be corrected by improving postnatal nutrition, as

reported in Wistar Kyoto rats, a good model of anxiety

responses (Romano, Wark, Owens, & Wlodek, 2009). In

our experiments the animals have free access to food

(Zeigler) with 22% protein, but they did not reach weights

similar to Sprague–Dawley pups surely because of their

genetic background (Moyaho et al., 2009). The ‘‘fetal

origin hypothesis’’ proposes that prenatal environmental

exposures, includingmaternal stress, could have sustained

effects across the lifespan (Kinsella & Monk, 2009). A

positive correlation of food ingestion during pregnancy

and low body weight in their offspring has been

demonstrated (Massaro, Levitsky, & Barnes, 1974;

Passos, Ramos, & Moura, 2000), including women with

a poor diet before and during pregnancy who had babies

with a low birth weight (Lechtig et al., 1975), and where

the frequency of infant mortality is four times higher than

Developmental Psychobiology Maternal Care in High- and Low-Yawning Rats 9

normal birth weight babies (Habicht, Yarbrough, Lechtig,

& Klein, 1973).

Comparisons among inbred strains of rodents are

important to determine the effect of environmental factors

over behavioral traits made under laboratory conditions.

Thus, environmental manipulations such as maternal

separation early in life, handling, and enriched environ-

ments clearly affect subsequent juvenile or adult perform-

ances (Fleming et al., 2002). As in many other inbred

strains of rodents, work has focused on the participation of

genetic and epigenetic factors involved in the develop-

ment of specific behaviors (Francis et al., 2003). The HY

offspring had fewer contacts with their mothers, receive

less grooming, and were retrieved carelessly (atypical

retrievings and reretrievings), similar to that reported in

stressed mothers (Salas et al., 2002). The HY mothers

have fewer pups per litter at parturition, most of them lost

pups during lactation, and themeanweight of their pups is

lower from birth to weaning compared to the LY and

Sprague–Dawley offspring. These behavioral differences

could be caused by some metabolic, hormonal, or

emotional issues during pregnancy or lactation (Fleenor

et al., 2005; Glinoer, 1997; Hapon et al., 2003; Ozzane &

Hales, 1999; Shono, Imagima, Zakaria, & Suita, 1999).

Pups, exposed to dexamethasone by its injection into their

mothers during pregnancy, produced an offspring with

lower weight and chronic hyperactivity of the HPA gland

axis. These pups had higher plasma-corticosterone levels

with an upregulation of hepatic gluconeogenesis and

insulin resistance suggesting that glucocorticoids levels

are a key factor formetabolic activity as adults (Buhl et al.,

2007; Burlet et al., 2005).

The release of oxytocin in the paraventricular nucleus

(PVN) of the hypothalamus at parturition probably

facilitates a positive feedback in both parvocellular and

magnocellular neurons to coordinate the high levels of

oxytocin release that are important for the generation of

maternal behavior, infant recognition, and bonding

(Carter & Keverne, 2002). This is also true for yawning

expression because the release of oxytocin in the PVN is a

key factor for the generation of yawning, not only by this

peptide, but also in this part of the brain the dopaminergic,

excitatory amino acids, nitric oxide, GABA, and opioid

receptors converge to increase yawning frequency,

suggesting that neural mechanisms in the hypothalamus

are important regulators for yawning and pair bonding (for

review, see Collins & Eguibar, 2010). Preliminary results

showed that HY rats yawnedmore after the i.c.v. injection

of oxytocin than the LY animals, but with similar

grooming scores (unpublished data), suggesting different

sensitivities in the neural pathways that mediate these

behaviors. In future experiments, we will address oxy-

tocin levels during parturition and lactation in both

sublines.

In conclusion, our results were that the HY dams

showed a different organization of maternal care with a

reduced litter size and lower weights of pups at parturition

and weaning. These changes can be caused by hormonal

or neural mechanisms, which are able to alter somato-

sensory stimulation of the pups and also can produce

hormonal and metabolic changes that ultimately are

responsible for different behavioral characteristics of HY

rats, such as yawning and grooming sequences in the

adults.

NOTES

This work was supported by grants from VIEP-BUAP G/SAL/

2010 and CONACyT 106694 to J.R.E. and also by Dr. Enrique

Aguera-Ibanez, Rector, BUAP. A. Ugarte was supported by

PROMEP-BUAP No. 664. This work is part of the thesis of A.

Ugarte in partial fulfillment of requirements for aMasters degree

at the Universidad Autonoma of Tlaxcala. We thank Dr. Ellis

Glazier for editing the English language.

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