Physiology & Behavior 141 (2015) 172–179

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Physiology & Behavior

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Severe maternal undernutrition and post-weaning behavior of rabbits☆

Panagiotis E. Simitzis a, George K. Symeon c,⁎, Antonios P. Kominakis a, Iosif A. Bizelis a, Stella E. Chadio b,Zafeiris Abas c, Stelios G. Deligeorgis a

a Department of Animal Breeding and Husbandry, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Greeceb Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Greecec Department of Agricultural Development, Democritus University of Thrace, Greece

H I G H L I G H T S

• Effect of severe prenatal undernutrition (50%) on rabbits' behavior was examined.• Prenatally undernourished (PU) rabbits were less active compared to the others.• The effects were more obvious in PU rabbits, during the final third of pregnancy.

☆ This paper is dedicated to the loving memory of Prof.sor of this project, whohas so unexpectedly passed away dpaper.⁎ Corresponding author at: 75 Iera Odos, 118 55 Athen

E-mail address: symeon@aua.gr (G.K. Symeon).

http://dx.doi.org/10.1016/j.physbeh.2015.01.0250031-9384/© 2015 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 January 2014Received in revised form 19 January 2015Accepted 21 January 2015Available online 22 January 2015

Keywords:Severe maternal undernutritionRabbitsBehavioral patternOpen-field test

The objective of the present experimentwas to investigate the implications of severematernal undernutrition onthe post-weaning behavior of rabbits. Thirty two does were randomly assigned to four groups: the control group(C) that was fed 100% of the recommended energymaintenance requirements throughout pregnancy and lacta-tion and the U1, U2 and U3 groups that were fed 50% of the recommended energy maintenance requirementsbetween the 6th and the 19th day of pregnancy, between the 20th and the 27th day of pregnancy and betweenthe 3rd and the 10th day of lactation, respectively. At the age of 50 and 65 days, behavior of rabbits in cages wasrecorded and rabbits were further subjected to an open-field test; a paradigm used as an indicator of fear andemotional distress.Significant differences were found between the U2 and the other experimental groups for the duration of eatingand drinking, and duration of locomotory and investigatory behaviors (P b 0.05). At the age of 65 days, durationof comfort behaviors was also lower in the U2 compared to the other groups (P b 0.05). On the other hand, du-ration of resting appeared to be the highest in the group of rabbits born from undernourished does between the20th and the 27th day of pregnancy (P b 0.05). Duration of restingwas significantly increased, although frequen-cy and duration of eating and drinking were significantly decreased at the age of 65 days (P b 0.05). Male rabbitshad greater values for locomotory and investigatory behaviors, and duration of eating and drinking compared tothe females (P b 0.01). A decreased activity of rabbits during the light periodwas observed (as expected), where-as an increase in duration of locomotory and investigatory behaviors was observed during the first four and thelast 4 h of the light and dark periods, respectively (P b 0.05).Rabbits born from the U1 and U2 group of does displayed increased rates of latency to leave the start positioncompared to the other groups during the implementation of the open-field test (P b 0.05). No other significantdifferences in the exhibition of the examined behaviors were found between the treatment groups, with the ex-ception of ‘moving forelegs’ activity that had the lowest value in the U3 group of rabbits.It is concluded that severematernal undernutrition (50% of the recommendedmaintenance requirements) espe-cially between the 20th and the 27th day of pregnancy has significant effects on the exhibition of rabbits' post-weaning behavior, since the offspring from the U2 undernourished does were significantly less active comparedto the other experimental groups.

© 2015 Elsevier Inc. All rights reserved.

Zafeiris Abas, scientific supervi-uring thewriting process of this

s, Greece.

1. Introduction

Maternal nutrition can program long-term effects on the offspringwithout necessarily affecting size at birth, since a stimulus or insultat a critical, sensitive period of early life can have permanent

173P.E. Simitzis et al. / Physiology & Behavior 141 (2015) 172–179

consequences on structure, physiology, metabolism and behavior (fetalprogramming) [1]. This discovery of a link between the in utero experi-ences and the later metabolic and behavioral abnormalities or disordersis one of the most important advances in epidemiology research ofrecent years [2].

Maternal stress during gestation can influence the physiological de-velopment of the offspring, although these effects vary considerably, de-pending on the nature, intensity and duration of the stressor as well ason the stage of gestation at which the stressor is effective [3]. There arecritical periods in development when the organism is particularly vul-nerable to these malnutrition effects. The type and magnitude of theseconsequences depend on the stage of brain development at the timewhen themalnutrition is imposed and its duration. Scientific data clear-ly demonstrate that the earlier the dietary insult, the more severe andmore permanent is its effect [4].

Prenatal stress has also programming effects on the hypothalamic–pituitary–adrenocortical (HPA) axis, brain neurotransmitter systemsand cognitive abilities of the offspring, leading to an increase in the ex-hibition of anxiety and emotionality, especially during stressful situa-tions [5–7]. In general, stressors, such as malnutrition, appear toimpose a limitation on the complexity of neuronal circuits and on thefunctional capabilities of the brain. As a result, this brain dysfunctioncan ultimately influence attentional processes and interactions of theorganism with the environment, i.e. a decreased responsiveness toenvironmental stimuli and functional isolation from the environment,leading to various types of learning disabilities [4]. Generally, the under-nourished animal has difficulty coping with novel situations thatrequire adaptive responses and accommodations to the new environ-mental demands, as a result of the inadequate hippocampal formation,which is a prominent archicortical structure of the limbic system thatcomprises a large number of neural components involved in the regula-tion of autonomic and somatic behaviors [8,9].

Rabbit can be classified as a perinatal brain developer like the humaninfant, meaning that the spurt in brain growth and development occursclose to the time of birth [10] and it was therefore chosen as an animalmodel in the present experiment. According to previous studies, alter-ations in the fetal environment, such as severematernal undernutrition,can have crucial long-term consequences on offspring development,physiology and behavior in rat [4]. As a result,we carried out thepresentstudy to determine possible effects of severe prenatal undernutrition(50% of the recommended energy maintenance requirements) duringdifferent time periods in does on the exhibition of their offspring behav-ior at the age of 50 and 65 days.

2. Materials and methods

2.1. Animals

Thirty-two does (Oryctolagus cuniculus) of a hybrid commercialbreed (Hyla Nouvelle Generation), of similar age (7.5 months old) andbody weight (4338 ± 134 g) were purchased from a breeding farm.Upon arrival at the experimental facilities, doeswere kept indoors in in-dividual cages (0.40 × 1.00× 0.39m) and seven days later doeswere ar-tificially inseminated (sperm fromHylamaxbucks). Seven days after theartificial insemination, does were randomly allocated to four groups:the control group (C) (n = 8) that was fed 100% of the recommendedenergy maintenance requirements throughout pregnancy and lactationand the three nutrient restricted groups U1 (n= 8), U2 (n= 8) and U3(n= 8) offered 50% of the recommended energy maintenance require-ments between the 6th and the 19th day of pregnancy, between the20th and the 27th day of pregnancy and between the 3rd and the10th day of lactation, respectively. According to the experimental de-sign, 100% of the recommended energy maintenance requirements ofthe feed were also provided to the undernourished groups before andafter the examined time periods, until weaning (during lactation thedoes were also offered the appropriate amount of feed to cover the

needs for milk production). Maintenance and milk production re-quirements were estimated according to Xiccato and Trocino [11].The diet consisted of a commercial concentrate mixture (in pellets)containing barley (25%), wheat bran (14%), soybean meal (9%), sun-flower meal (10%), alfalfa meal (20%), sugar beet (15%), soybean oil(1%), molasses (3%), limestone (1%), monocalcium phosphate (1%),salt (0.3%), lysine (0.1%), methionine (0.1%), threonine (0.05%), choline(0.15%) and a vitamin & mineral premix (0.2%). Does had free access towater.

Rabbit kits were born after 30.7 ± 0.3 days of gestation. At thesecond day post partum, kits were cross-fostered within treatmentgroups in order to ensure equal litter sizes of ten pups per doe.After weaning (35th day of age), sixty-four (32 animals per sex) ofa total of 317 rabbits were randomly selected and kept indoors in in-dividual cages (0.41 × 0.33 × 0.29 m) with wire mesh floors, underthe following environmental conditions (temperature: 22.5 ±3.5 °C; relative humidity: 50 ± 20%; lightning: 0:00–12:00 h/12:01–24:00 h light/dark cycle). Each cage was equipped with ametal feeder and an automatic nipple drinker. Feed was providedad libitum and rabbits had free access to water. The diet consistedof a commercial concentrate mixture (in pellets) containing barley(17%), wheat bran (23%), soybean meal (7%), sunflower meal (11%),alfalfa meal (20%), sugar beet (15%), soybean oil (1%), molasses (3%),limestone (1%), monocalcium phosphate (1%), salt (0.4%), lysine (0.1%),methionine (0.1%), threonine (0.05%), choline (0.15%) and a vitamin &mineral premix (0.2%).

The methods used in the present experiment were approved bythe Research Ethics Committee of the Department of Animal Scienceand Aquaculture of the Agricultural University of Athens under theguidelines of “Council Directive 86/609/EEC regarding the protectionof animals used for experimental and other scientific purposes”.

2.2. Behavioral recordings of offspring in their cages

At the age of 50 and 65 days, behavior elements of thirty-two rabbits(four males and four females from each treatment group) were record-ed on three consecutive days. The behaviors observed could be catego-rized in the five following groups: maintenance (eating and drinking),resting, comfort and locomotory–investigatory. The definitions ofthese behavioral patterns are [12,13]:

• Frequency of eating: Oral seizing of feed and chewing/munching of itor having the muzzle over the feeder for over 10 s

• Duration of eating (min): Cumulative duration of acts of eating in afeeder

• Frequency of drinking: Drinking water from nipple drinkers• Duration of drinking (min): Cumulative duration of acts of drinking ina nipple drinker

• Resting: Sleeping, lying at any position (unsleeping, stretched) andsitting

• Comfort behaviors: Any behavior form connected with the own bodyof the animal (washing, licking, starching)

• Locomotory and investigatory behaviors: Any voluntary change of po-sition (treading, running, hopping, prancing) and behavior forms con-nected to the cage or to its equipments (rubbing, licking, grawing,smelling, marking with the chin).

The behavior of the rabbits was recorded using four video cam-eras with infrared lighting (TX-1430OA, Turbo-X). Each camerawas placed in a fixed position in order to record behavior patternsin 8 cages of rabbits. The recorded data were stored in a digital videorecorder equipped with a hard disk (TX168, Telexper Inc., USA). Oneperson decoded all the recordings and was blind to the group for allthe behavioral measurements.

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Fig. 1. Effect of gestational maternal undernutrition on live weight (g) of rabbit offspringsfrom 0 to 70 days of age (C: control, U1, U2 and U3: maternal undernutrition (50%) be-tween the 6th and the 19th day of pregnancy, between the 20th and the 27th day of preg-nancy and between the 3rd and the 10th day of lactation, respectively). * s.e. lines arepresent but not visible due to scale range; U3 group was significantly lighter comparedto the other three groups (C, U1, U2) at the age of 7, 14, 21 and 28 days old (P b 0.05).

174 P.E. Simitzis et al. / Physiology & Behavior 141 (2015) 172–179

2.3. Open-field test

At the age of 50 and 65 days old, rabbits not included in the behav-ioral recordings in home cages (four males and four females from eachtreatment group) were tested in an open field once a day on 2 consecu-tive days between 08:00 am and noon (under natural light). Open-fieldtest is generally used as a paradigm to measure emotionality and as anindicator of fear [14,15].

The open field measured 2 × 2 m, was surrounded by 80-cm highpolished plywood walls and was situated in a corner of the barn inwhich the rabbits were housed. Each test lasted 5 min and after itsend, the box, floor and walls were wiped with a cloth moistened withan odorless detergent to remove odor traces. For testing, the rabbitswere taken out of their pens, placed in a wooden box and carried tothe open field. The box was placed in the center of the test area,where the rabbits were released without human contact. To minimizefurther human contact, experimenter stayed away from the field duringtesting and behavior was continuously video-recorded.

The following behavioral elements were recorded during the open-field test [16]:

• Latency: Time to first leaving of the central starting area of 30 × 30 cm• Sitting still: No movement of head, body or legs• Standing stretched: The forelegs are forward, the hindlegs stay inplace and the heels are visible behind the body

• Moving forelegs: The forelegs move across the floor whereas thehindlegs stay in place

• Walking: Forelegs as well as hindlegs move alternately• Exploration: Head movements indicating investigation of the envi-ronment. Exploration is an independent category, but it can occur to-gether with standing stretched, moving forelegs or walking

• Rearing: Forefeet are raised from the floor, the rabbit stands on itsheels.

• Digging: Scratching with the forelegs on the floor or wall

The behavior of the rabbits during the open-field test was recordedusing a Panasonic PV120 video camera fitted with a 3.6 mm lens. Thecamera was placed in a fixed position in order to continuously recordbehavior patterns in the open field. The recorded data were also storedin the digital video recorder and the observerwas also blind to group forall the behavioral measurements as previously described.

2.4. Statistical analysis

Significant differences from normal distribution for variables wereassessed using the normal quartile plots alongwith values for skewnessand kurtosis. The majority of the studied variables reasonably followedANOVA assumptions i.e. normal distribution (ND) and homogeneity ofvariances within the class effects. For these parameters, standardmulti-factor analysis of variance was employed treating treatment, sex andage of testing and as fixed effects (time period of the day was also in-cluded in the case of the cage's behavioral recordings). However, therewere certain variables that clearly deviated from ND and generalizedmixed models were therefore employed. More specifically, “durationof eating”, “duration of drinking”, ‘locomotory and investigatory behav-iors’, ‘moving forelegs’, ‘rearing’, ‘standing stretched’ and ‘digging’wereanalyzed via zero-inflated negative binomial models. Frequencies ofeating and drinking measured during the cages' behavioral recordingswere analyzed by the application of logistic regression models. Here, acumulative logitmodel and the Fisher's ScoringOptimization Techniquefor obtaining maximum likelihood parameter estimates were applied.In this case, values are presented in contrast with zero, used as a refer-ence value. A negative value corresponds to a lower and a positive to agreater value compared to the reference. All models for the cages' be-havioral recordings included the effects of treatment, the sex, the hour

of the day and the age of testing as fixed effects while behavioral pat-terns during the open-field test were analyzed treating the treatment,the sex and the age of testing as the only fixed effects. Interaction be-tween treatment and age of testing was examined and was includedin our models only when it was significant in the cases of ‘comfort be-haviors’ and ‘locomotory and investigatory behaviors’measured duringthe cages' behavioral recordings. The probability level formean compar-isons was set at 0.05. All the above analyses were carried out using theappropriate procedures in the statistical package of Sas/Stat [17].

3. Results

3.1. Rabbit pups' weights at birth and weaning

Birth weight of kits was not significantly influenced by the maternalundernutrition. As it was demonstrated, the birth weights (g) for theoffspring born from the control and the undernourished mothers (LSmeans ± s.e.m.) were: 57.1 ± 3.3 vs 55.7 ± 3.6 vs 48.1 ± 3.0 vs58.9 ± 3.3, for the C, U1, U2 and U3 groups, respectively. Offspringsfrom the U3 group were lighter than the other three experimentalgroups from day 7 to day 28 post partum while no significant weight(g) differences were observed at weaning (35th day) and at the agesof testing (50th and 65th days); 786 ± 33 vs 727 ± 42 vs 740 ± 30 vs650 ± 36, 1537 ± 46 vs 1539 ± 57, 1562 ± 47 vs 1410 ± 52 and2178 ± 50 vs 2197 ± 63 vs 2222 ± 51 vs 2070 ± 67 for the C, U1, U2and U3 groups, respectively (Fig. 1).

3.2. Behavioral recordings of offspring in their cages

Control rabbits appeared to display a greater value for the frequencyof eating than the rabbits born to undernourished does (P b 0.05)(Fig. 2). As it is also illustrated in Fig. 2, frequency of drinking appearedto be higher in the C and U1 compared to the U2 and U3 groups of rab-bits, with the rabbits born from undernourished does between the 20thand the 27th day of pregnancy having the lowest value (P b 0.05).

A significant reduction in the frequency and duration of eating anddrinking and an increase in duration of resting were observed with in-creasing age (P b 0.05) (Fig. 3). Duration of eating and drinking and lo-comotory and investigatory behaviors had the lowest and resting hadthe highest value in the U2 group of rabbits (P b 0.05). In addition, al-though duration of comfort behaviors did not differ among the treat-ment groups at the age of 50 days, the U2 group displayed the lowestvalue at the age of 65 days (P b 0.05) (Fig. 4). At the same time, malerabbits had greater values for the duration of eating, drinking and loco-motory and investigatory behaviors but lower values for the frequencyof eating compared to the females (P b 0.05) (Fig. 5).

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Fig. 2. Effect of treatment on behavioral patterns of rabbits in cages (per hour) after continuous 24-h recording on three consecutive days (least squares means± s.e.) (C: control, U1, U2andU3:maternal undernutrition (50%) between the6th and the19th day of pregnancy, between the 20th and the27h day of pregnancy andbetween the3rd and the10thday of lactation,respectively). a,b,c Means within treatment with different superscripts are significantly different (P b 0.05). For frequencies of eating and drinking a reference value (estimate— 95% con-fidence limits) is alternatively given (logistic regressionmodel). Values are presented in contrastwith the zero (0), used as a reference value. A negative value corresponds to a lower and apositive to a greater value compared to the reference.

175P.E. Simitzis et al. / Physiology & Behavior 141 (2015) 172–179

Significant differences were also observed during the time periodswithin the day (Fig. 6). In detail, the activity of rabbits was generally in-creased during the dark period (1–12 h), although value for the dura-tion of locomotory and investigatory behavior was the highest duringthe first hour of the light period (P b 0.05). Frequency of eating anddrinking had lower values during the light (1–10 h) compared to thedark period (P b 0.05). At the same time, duration of eating and drinkingappeared to decrease especially between the 5th and 10th hour (lightperiod) (P b 0.05). Comfort behaviors had their lowest values duringthe last hours of dark period (21–24 h) and the second hour of thelight period (P b 0.05). On the other hand, rates of resting behaviorwere increased during the light period and especially between thesixth and the tenth hour of light period (P b 0.05) (Fig. 6).

3.3. Open-field test

Prenatally undernourished rabbits displayed increased rates for thelatency compared to the other two groups (P b 0.05). Values for sittingstill, standing stretched, walking, exploration, rearing and diggingwere not significantly different among the experimental groups,

although values for walking tended to be lower in the U1 and U2 com-pared to the C and U3 group of rabbits (Fig. 7). Finally, the exhibition ofthe ‘moving forelegs’ activity appeared to be lower in the U3 comparedto the other groups (P b 0.05).

Rabbits tended to spend more time sitting still at the age of 65 thanof 50 days, and time spent moving forelegs, standing stretched and la-tency were significantly decreased by the test age (P b 0.01). Valuesfor walking, exploration, rearing and digging were not influenced bythe age (Fig. 7).

4. Discussion

4.1. Rabbit pups' weights at birth and weaning

Birthweight of pupswasnot significantly reduced after thematernalundernutrition during gestation, although pups born from the U2 groupof does tended to have the lowest values. Moreover, although this ten-dency also appeared at weaning and at the testing ages (50 and65 days old), no significant effect of maternal undernutrition duringgestation on the body weight of pups was found. On the contrary, the

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Fig. 3.Effect of age (50 or 65 days) on behavioral patterns of rabbits in cages (per hour) after continuous24-h recording on three consecutive days (least squaresmeans± s.e.). a,b,d,e Meanswithin age with different superscripts are significantly different (P b 0.05).

176 P.E. Simitzis et al. / Physiology & Behavior 141 (2015) 172–179

maternal undernutrition during lactation resulted in a significant de-crease of the U3 offsprings' weight from the 7th till the 28th day ofage, a difference that was diminished though inweaning and thereafter.In general, maternal undernutrition (irrespective of the imposed feedrestriction level) negatively affects the weight of kits at birth. Fetalweights of kits were reduced when maternal feed intake was severelyrestricted (15 g instead of 150 g per day) between the 6th and the18th dayof pregnancy [18]. At the same time, feed restriction (60%) dur-ing the second half of does' pregnancy appears to significantly decreasethe litter weight at birth [19]. Fetal body weights were also reduced atdoes' feed levels of ≤75 g/day between the 7th and 19th day of preg-nancy [20]. In a previous experiment conducted by our experimentalteam, pups born from does offered 75% of the recommended energymaintenance requirements between the 6th and 26th day of pregnancywere lighter compared to pups born from ad libitum fed does [21]. Thelack of significance in the present experiment is probably due to differ-ences between ad libitum and 100% of the recommended energy main-tenance requirement feeding regime. The present results are in linewith those reported in rats, in which offspring from undernourishedmothers (30% of ad libitum intake, throughout pregnancy) were signif-icantly lighter at birth than pups born to ad libitum fed dams [2]. The

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same applies also for the maternal undernutrition during lactation,where in rats any significant differences in body weight were dimin-ished after weaning [22].

4.2. Behavioral recordings of offspring in their cages

The frequency of eating was higher in the control compared to theundernourished groups of rabbits (Fig. 2). As it is also illustrated inFig. 2, frequency of drinking, duration of eating and drinking, and loco-motory and investigatory behaviors appeared to have the lowest andresting the highest value in the group of rabbits born from undernour-ished does between the 20th and the 27th day of pregnancy (P b

0.05). In addition, the U2 group displayed the lowest value for the dura-tion of comfort behaviors at the age of 65 days (P b 0.05) (Fig. 4).

Prenatal stressors, such as severematernal undernutrition, can causealterations of the hypothalamic–pituitary–adrenocortical (HPA) axisand brain neurotransmitter systems in the offspring [23]. These effectsare long-lasting and lead to permanent deficits in learning and behavior[24,25]. The significant differences in the exhibition of rabbit behaviorelements between theU2 and the other experimental groups could pos-sibly be attributed to the severe undernutrition (50% of the ad libitum

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Fig. 5. Effect of sex on behavioral patterns of rabbits in cages (per hour) after continuous 24-h recording on three consecutive days (least squaresmeans± s.e.). a,b Means within sexwithdifferent superscripts are significantly different (P b 0.05). For frequencies of eating and drinking a reference value (estimate — 95% confidence limits) is alternatively given (logistic re-gression model). Values are presented in contrast with the zero (0), used as a reference value. A negative value corresponds to a lower and a positive to a greater value compared to thereference.

177P.E. Simitzis et al. / Physiology & Behavior 141 (2015) 172–179

intake) imposed in the does between the 20th and 27th day of pregnan-cy (final third; stage of rapid development of fetuses). Frequency ofeating and drinkingwas also decreased in rabbits born fromundernour-ished does offered 75% of the recommended energy maintenance re-quirements between the 6th and 26th day of pregnancy at the age of50 and 65 days old [21]. Rat pups from severely undernourishedmothers (30% of the ad libitum intake) also appear to be significantlyless active compared to the offspring born to ad libitum fedmothers [2].

A significant reduction in the frequency of eating and drinking andan increase in duration of resting were observed with increasing age(Fig. 3), as it was also previously shown in rabbits at the ages of 50and 65 days old [21]. The results of the present study showed that rab-bits generally spent about 15% of their time displaying comfort behav-iors (Fig. 4). These findings are in accordance with previous studies[21,26], where rabbits spend about 16–17% of the time grooming them-selves. Moreover, on average, fattening rabbits between 6 and 10weeksof age spend 60% of their time resting, 10–15% feeding and 25–30%displaying other activities [13,21]. Housing of rabbits in individualcages severely restricts their freedom to display their natural locomoto-ry activities and as a result, rabbits spend most of their time in ‘sternalrecumbency’ such as sitting and lying [27].

At the same time, male rabbits had greater values for the duration ofeating, drinking and locomotory and investigatory behaviors but lower

values for the frequency of eating compared to the females (P b 0.05)(Fig. 5). A possible explanation is that the brain and the respective reg-ulatory pathways develop differently inmales and females under the in-fluence of sex steroid hormones during the perinatal period [28].However, in contrast with the findings of the present study, no signifi-cant differences were found when comparing the whole pattern of be-haviors of males and females in previous studies [12,21].

Significant differences were also observed during the hours withinthe day (Fig. 6). In detail, the activity of rabbits was generally increasedduring the dark period, while rates of resting behavior were increasedduring the light period and especially between the sixth and the tenthhour of light. Gunn and Morton [12] have already demonstrated thatsleeping begins at the start of the light cyclewhen there is an immediateincrease and the main sleeping period forms a 9 h band during the day.Frequency and duration of eating and drinking and duration of comfortbehaviors had greater values during the dark compared to the light pe-riod (Fig. 3), a finding that has been already demonstrated in previousstudies [21] and is related with rabbit's nocturnal nature [12].

The display of investigatory and locomotory behaviors was thegreatest during the first and the last hour of the light and dark periods,respectively, a finding that is in accordance with Abdelfattah et al. [29]who have shown that weaned rabbits move and explore more early inthe morning compared to the other periods of the day. The ancestor of

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40

50

60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Resting (min) by hour

0

2

4

6

8

10

12

14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Comfort behaviors (min) by hour

-1

0

1

2

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Locomotory and Investigatory Behaviors (min) by hour

Fig. 6. Behavioral patterns of rabbits in cages (least squares means± s.e.) as affected by the hourwithin the day. For frequencies of eating and drinking a reference value (estimate— 95%confidence limits) is alternatively given (logistic regression model). Values are presented in contrast with the zero (0), used as a reference value. A negative value corresponds to a lowerand a positive to a greater value compared to the reference.

178 P.E. Simitzis et al. / Physiology & Behavior 141 (2015) 172–179

the domesticated rabbit, the European wild rabbit, is mainly active dur-ing the dusk, night and dawn, because as a prey-animal its chances toescape from predators are higher during these time periods of the day

-12

-10

-8

-6

-4

-2

0

2

4

6

Duration of Latency, Standing stretched, Moving forelegs, Rearing and Digging (s) by treatment

C U1 U2 U3

Laten Standing

stretched

Moving

forelegs

Rearing Digging

bbaa

a a a

b

1

1

2

-4

-3

-2

-1

0

1

2

3

4

Duration of Latency, Standing stretched, Moving forelegs, Rearing and Digging (s) by age

50d 65d

Latency Standing

stretched

Moving

forelegs

Rearing Digging

c dc d

c

d

1

1

2

Fig. 7.Behavioral elements (s) during the open-field test (least squaresmeans± s.e.). a,bMeanswithin age of testing with different superscripts are significantly different (P b 0.01). C: control,nancy, between the 20th and the 27 h day of pregnancy and between the 3rd and the 10th dayand the interaction between treatment and age were found.

[30]. As it has been found, rabbits fed ad libitum spendmore time feed-ing during the night than the day at a percentage of 61 vs 39%, respec-tively [31].

0

50

00

50

00

Duration of Sitting still, Walking and Exploration (s) by treatment

C U1 U2 U3

Sitting still Walking Exploration

0

40

80

20

60

00

Duration of Sitting still, Walking and Exploration (s) by age

50d 65dSitting still Walking Exploratio

n

within treatmentwith different superscripts are significantly different (P b 0.05). c,dMeansU1, U2 and U3: maternal undernutrition (50%) between the 6th and the 19th day of preg-of lactation, respectively. Latency is presented as log values. No significant effect of the sex

179P.E. Simitzis et al. / Physiology & Behavior 141 (2015) 172–179

4.3. Open-field test

Prenatally undernourished rabbits displayed increased rates for thelatency compared to the other two groups (Fig. 7). Latency can beused as a measure of elapsed time between the onset of a stimulusand the initiation of the response and is regarded as a reliable markerof fear response [32]. In open field studies, “emotionality” is expressedas the extent of locomotion or activity and high activity correspondswith low emotionality and vice versa [33]. The factor “fear” could be re-lated with the high emotionality and the time spent standing stretchedby the rabbit [16]. Although, significant differences in fear responsesand emotionality during the open-field test were expected accordingto the existing data [4,34,35], the exhibition of the majority of the ob-served behavioral elements did not significantly differ between the un-dernourished and control rabbits. In detail, values for sitting still,standing stretched, walking, exploration, rearing and digging were notsignificantly different among the experimental groups, although valuesfor walking tended to be lower in the U1 and U2 compared to the C andU3 groups of rabbits (Fig. 7).

Rabbits tended to spend more time sitting still at the age of 65 thanof 50 days, and time spent moving forelegs, standing stretched and la-tency were significantly decreased by the testing age. Values for walk-ing, exploration, rearing and digging were not influenced by the age oftesting (Fig. 7). On the other hand, time spentmoving forelegs, standingstretched and latency were significantly decreased with increasing age,as it was also found in previous studies [16,21]. A possible explanationof the above results is that activity in an open-field test declineswith in-creasing age, as a result of the habituation to the test environment [36].

5. Conclusion

It is concluded that severe maternal undernutrition (50% of the adlibitum intake) especially between the 20th and 27th day of pregnancyhad generally detrimental effect on the exhibition of rabbit post-weaning behavior. Significant differences were found in specific behav-ioral patterns recorded in the cages, such as lower values for the fre-quency and duration of eating and drinking and locomotory andinvestigatory behaviors in the U2 compared to the other experimentalgroups. Duration of comfort behaviors also appeared to be the lowestin the group of rabbits born from undernourished does (between the20th and 27th day of pregnancy) at the age of 65 days compared tothe other groups. Rabbits born from prenatally undernourished doesalso displayed reduced rates of the latency compared to the controlgroup during the open-field test. However, further experimentation iswarranted to fully understand themechanisms that connect severema-ternal undernutrition with the exhibition of rabbit behavior.

Acknowledgments

This research projectwas implementedwithin the framework of theAction «Supporting Postdoctoral Researchers» of the Operational Pro-gram “Education and Lifelong Learning” (Action's Beneficiary: GeneralSecretariat for Research and Technology), and is co-financed by theEuropean Social Fund (ESF) and the Greek State (LS9 (1678)).

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