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Cafeteria diet during the gestation period programs developmental and behavioral courses in the offspring
Accepted Manuscript Title: Cafeteria diet during the gestation period programs developmental and behavioral courses in the offspring Authors: Ana Cl´audia Alves Freire Ribeiro, Tatiane Helena Batista, Vanessa Barbosa Veronesi, Alexandre Giusti-Paiva, Fabiana Cardoso Vilela. PII: DOI: Reference:
S0736-5748(18)30009-1 https://doi.org/10.1016/j.ijdevneu.2018.05.001 DN 2258
To appear in:
Int. J. Devl Neuroscience
Received date: Revised date: Accepted date:
8-1-2018 26-4-2018 1-5-2018
Please cite this article as: Ribeiro ACAF, Batista TH, Veronesi VB, Giusti-Paiva A, Vilela. FC, Cafeteria diet during the gestation period programs developmental and behavioral courses in the offspring, International Journal of Developmental Neuroscience (2010), https://doi.org/10.1016/j.ijdevneu.2018.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cafeteria diet during the gestation period programs developmental and behavioral courses in the offspring
Ana Cláudia Alves Freire Ribeiro, Tatiane Helena Batista, Vanessa Barbosa Veronesi,
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Alexandre Giusti-Paiva, Fabiana Cardoso Vilela.*
Departamento de Ciências Fisiológicas, Instituto de Ciências Biomédicas, Universidade
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Federal de Alfenas (Unifal-MG), Alfenas, Minas Gerais, Brazil;
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Programa de Pós-Graduação em Biociências Aplicadas à Saúde, Brazil.
*
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Corresponding author: Fabiana Cardoso Vilela, PhD , Instituto de Ciências Biomédicas
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, Universidade Federal de Alfenas, UNIFAL , Avenida Jovino Fernandes Sales nº 2600 ,
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37130-000, MG, Brazil , Phone: +55 (35) 37011890 , E-mail: [email protected]
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Highlights
Cafeteria diet during gestation altered maternal behavior.
Cafeteria diet altered the onset of physical and neurodevelopmental landmarks.
Maternal cafeteria diet had an impact on emotional behavior in adolescent
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offspring.
Maternal cafeteria diet had an impact on play behavior in adolescent offspring.
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Abstract
The objective of this study was to investigate the effect of exposure to maternal consumption of a hyperenergetic, highly palatable diet, known as the cafeteria diet, during the gestation period on the development and behavior of offspring. For this, we used
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pregnant female mice that were fed a normal or a cafeteria diet during the gestation period. The evaluation of maternal behavior in lactating dams was performed from the
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second to the eighth day postpartum (PND 2 - 8). Weight gain, feed intake, and energy intake were recorded during the gestation period. In the offspring, reflex parameters and physical development were evaluated during the lactation period and when they reached
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adolescence. Behavioral performance was evaluated in light-dark, open-field, and play
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behavior tests. In addition, biochemical parameters of the dams and the adolescent
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offspring were evaluated. The cafeteria diet during gestation altered maternal behavior
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and the onset of physical and neurodevelopmental landmarks and had an impact on emotional and play behavior in adolescent offspring. In conclusion, our results
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demonstrate that exposure to maternal consumption of a cafeteria diet during the gestation
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period can program developmental and behavioral courses in the offspring.
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Keywords: cafeteria diet; offspring; developmental; behavior.
1. Introduction
Maternal nutrition plays a critical role in the offspring's physical growth and behavior (Laus et al., 2011; Black et al., 2013). An adequate supply of nutrients is critical
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for maintenance of growth, as well as, for the development of functions in organic systems (Morgane et al., 1978; Umeta et al., 2003). Thus, an inadequate diet during the gestation period can negatively influence the development of the brain, resulting in changes in its structure and, hence, its function (Morgane et al., 1978; Morgane et al., 1993).
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As a consequence, malnutrition is a very important, nongenetic factor affecting the developmental processes (Morgane et al., 1978; Morgane et al., 1993). Some evidence
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suggests that the in utero environment independently and critically impacts offspring
development and their susceptibility to obesity and disease in later life (Cooper et al.,1996; Chen et al., 2008). However, little is currently known about the behavioral
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effects of feeding a hyperenergetic diet during the gestation period when offspring
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behavior is tested during adolescence.
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The term malnutrition implies that one or more essential nutrients is missing or
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present but in inadequate proportions (Morgane et al., 2002). Among various
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experimental diets of malnutrition, the cafeteria diet, which is considered a hyperenergetic diet, has been well established as an obesogenic diet in rodents and as a
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model of a Western-style diet. In short, a cafeteria diet consists of various hyperenergetic and highly palatable human food items (Rothwell and Stock, 1979; Speight, 2017).
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Exposure to maternal consumption of a cafeteria diet during lactation leads to
reduced anxiety and changes in memory in the offspring when tested at adult age (Wright
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et al., 2011a, 2011b). In another study, a cafeteria diet during the lactation period changed open-field behavior in the developing offspring (Speight et al., 2017). Although these results suggest the nutritional programming of behavior, less is known about the behavioral consequences of prenatal exposure.
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Since maternal nutrition plays a critical role in the offspring's physical growth and behavior, we hypothesize that an inadequate diet during the gestation period, such as consumption of a cafeteria diet, can negatively influence developmental and behavioral courses in the offspring. Therefore, knowing that maternal malnutrition may affect the maturation of the brain and the development of cognitive function, resulting in behavioral
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abnormalities in offspring, the objective of this study was to investigate the effect of exposure to maternal consumption of a cafeteria diet during the gestation period on the
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development and behavior of the offspring. In addition, the maternal behavior of the dams
as well as biochemical parameters of the dams and the adolescent offspring were
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evaluated.
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2. Materials and methods
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2.1 Animals
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Adult Swiss nulliparous female and male mice aged approximately 9 weeks were
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obtained from the Central Animal Facility of the Federal University of Alfenas and were housed in a temperature-controlled room (22ºC) on a 12:12 h light-dark cycle (lights on
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at 7:00 h) with ad libitum access to water and standard laboratory mouse chow. The female mice were time-mated by placing them with sexually experienced male mice (two
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females for one male). The next morning, the presence of vaginal plug was considered as gestation day zero (GD0). The pregnant mice were housed individually and were divided
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into two experimental groups: normal diet (standard laboratory chow) or cafeteria diet. All experimental procedures followed the Ethical Principles in Animal Research adopted by the Ethics Committee on the Use of Animals of the Federal University of Alfenas (protocol 620/2015). The experiments were performed in accordance with good laboratory practice protocols and quality assurance methods.
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2.2 Experimental design During the gestation period (GD 0 - 21), twenty pregnant mice were fed with a normal (n = 10) or cafeteria diet (n = 10). On parturition day (PND 0), the cafeteria diet was replaced with a normal diet. At PND 1, all litters were culled to eight pups (four males and four females). These dams were used for evaluation of maternal behavior (PND
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2 - 8) and locomotor activity (PND 6) in the open field. In addition, weight gain, feed intake, and energy intake were recorded during the gestation period. Their offspring (40
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female and 40 male mice from each group) were used for the experimental procedures
during adolescence (PND 30 - 32). To avoid the litter effect, one couple (1 male and 1 female mouse) from each litter received an ink mark on one hind limb for observation of
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reflex parameters and physical development until PND 21 (n = 10 per group) (Veronesi
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et al., 2017). The other three couples of each litter that were not marked were used to
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evaluate the behavioral performance in the light dark, open field, or play behavior tests
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(n = 10 per group) during adolescence since each animal was used only once for a given
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behavioral evaluation. After these tests, two animals from each test were used for blood collection for biochemical analyzes (n = 8 per group) and removal of perigonadal and
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retroperitoneal fats (n = 8 per group). Other groups of dams (n = 8 per group) were used
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only to collect blood and remove perigonadal and retroperitoneal fats at PND 1.
2.3 Gestational feeding
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The normal diet consisted of commercial chow (Nuvilab® CR1) in the form of
pellets. The cafeteria diet consisted of the following items: Nestlé Classic® milk chocolate, Bauducco® strawberry wafer, Tropeiro® bacon, mozzarella cheese, original Ruffles® potato and commercial chow (Nuvilab® CR1). Foods were offered in the form in which they were purchased, in natura, on the same proportion. The choice of food to
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compose the cafeteria diet was based on previous studies and adapted to availability in the local market (Akyol et al., 2009; Bouanane et al., 2010). The values used to calculate the energy (kJ) were as follows: (total protein in grams) × 4 + (total carbohydrates in grams) × 4 + (total lipids in grams) × 9 = total calories ingested x 4.186 kJ. The composition of the macronutrients and the energy of the foods offered to the animals are
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described in table 1.
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2.4 Maternal studies
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2.4.1 Weight gain, feed and energy intake by the dams during gestation
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Weight gain, feed, and energy intake were recorded during the gestation period (n =
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10 per group). Energy intake was based on the amount of each food ingested.
2.4.2 Maternal behavior
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The maternal behavior of lactating mice (n = 10 per group) was scored daily during the first week of lactation (PND 2 - 8). Observations were conducted during three
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72-min periods in the light phase (08:00, 12:00, and 16:00) and one 72-min period in the dark phase (20:00). During each session, maternal behavior was scored every 3 min (25
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observations per four periods per day for a total of 100 observations per dam per day). Six maternal behaviors and three non-maternal behaviors were recorded as follows: (1) licking the pups (either the body surface or the anogenital region), (2) nursing the pups in an arched-back posture, (3) “blanket” posture in which the mother lays over the pups, (4) passive posture in which the mother lies either on her back or side while the pups nurse,
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(5) self-grooming in which dams lick the breasts, (6) nest building, (7) feeding, (8) exploring the cage housing, (8) not exploring (Costa et al., 2013; Carvalho et al., 2016). Behavioral data are reported as percentages of the total number of behavioral observations (number of target behavior observations divided by the total number of all behavioral
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observations × 100).
2.4.3 Locomotor activity evaluation in the open field test
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Locomotor activity was performed at PND 6 (n = 10 per group) for 5 min in an open field box (10 - 12 lux), consisting of an acrylic painted acrylic base 100 x 80 cm and 50 (height) x 60 cm (diameter). Four squares were defined as the center and the eight
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squares along the walls were considered the periphery. Each mouse was gently placed in
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the exact center of the box. Activity was scored as a line crossing when a mouse removed
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all four paws from one square and entered another. Line crossings among the central four
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(Veronesi et al., 2017).
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squares or among the peripheral eight squares of the open field were counted separately
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2.4.4 Tissue collection and biochemical analysis Another group of animals was used for these procedures at PND 1. Normal diet
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and cafeteria diet dams (n = 8 per group) were euthanized by decapitation, and a blood sample was collected in a heparinized tube. The plasma was immediately isolated by
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centrifugation, and duplicate aliquots were stored at -80 °C prior to the biochemical analyses. After the mice were sacrificed, perigonadal and retroperitoneal adipose tissue samples were harvested and weighed. Commercial kits from In Vitro Diagnostica Ltd. (Itabira-MG, Brazil) were used to measure plasma total cholesterol, triglycerides, and glucose by the absorbance method (Orlandi et al., 2015).
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2.5 Offspring studies 2.5.1 Physical and neurobehavioral development The following physical parameters were assessed in one male and one female pup from each litter (n = 10 per group) as follows: pinna unfolding (beginning on PND 2),
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separation of front and rear digits, hair growth, superior and inferior incisor eruption
(beginning on PND 5), and eye opening (beginning on PND 10). The following reflexes
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were also assessed in one male and one female pup from each litter as follows: surface righting (beginning on PND 2), negative geotaxis (turning at least 135º within 30 s of face-down placement on a 45º incline, beginning on PND 4), palmar grasp reflex
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(grasping of a paper clip with forepaws if stroked, beginning on PND 2), rooting (turning
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the head toward the side of the face being stroked with the tip of a cotton swab beginning
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on PND 2), auditory startle (beginning on PND 10), and stimulation of fibrils (beginning
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on PND 12). Pups were briefly removed from their dams between 14:00 - 15:00 for daily
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observation and then immediately returned to their home cage. The mean number of days required for the appearance of each of the above-mentioned parameters was calculated.
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On PND 21, the offspring were weaned, marked with a nontoxic ink, distributed in accordance with their sex, and housed in groups of four per cage (Carvalho et al., 2016;
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Veronesi et al., 2017).
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2.5.2 Light-dark test The apparatus consisted of a Plexiglas rectangular box (48 cm long × 24 cm wide
× 24 cm high) divided into a dark region (24 cm long) and a light region (24 cm long). The light and dark regions were separated by an opening (8.0 × 8.0 cm) that allowed the animals to move between the two compartments. The dark region was made of black
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Plexiglas and covered with a black lid. The light portion was made of white Plexiglas, and a 60 W light was positioned directly over it. Each mouse at PND 30 - 32 (n = 10 per group) was placed in the light compartment and allowed to move freely between the two compartments. The box was carefully cleaned with a 5% ethanol solution after every test. The behavior was video-recorded for a total of 5 min, and the videotapes were scored for
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compartments, and the time of permanence in light (Ribeiro et al., 2016).
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latency to the first transition, number of transitions between the light and dark
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2.5.3 Open field test
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The open field test for the offspring at PND 30 - 32 (n = 10 per group) was
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5% ethanol solution after every test.
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performed as previously described, for the dams. The arena was carefully cleaned with a
2.5.4 Adolescent play behavior
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Adolescent play at PND 30 - 32 was conducted in an arena covered with a 0.5 cm layer of clean bedding. Subjects (n = 10 per group) were acclimatized to the box for two
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consecutive days and on the third day (test day), they were individually housed in standard mouse cages for 3.5 hrs prior to the play session. Two mice of the same group, age, and
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sex, but from different litters, were then placed in the arena and their interactions were recorded for 10 min, the period during which the majority of social interaction occurs. Behaviors were subsequently scored, and the behaviors of interest included time of following (one mouse walks straight behind its partner, keeping pace with the one ahead), time of sniffing (sniffing anywhere on the body), frequency of pushing (pushing
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head/snout underneath the partner's body or squeezing itself between the wall/floor and the partner), and frequency of crawling (crawling over or under the partner's body) (Yang et al., 2009; Manduca et al., 2014).
2.5.5 Body weight, tissue collection, and biochemical analysis of offspring
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The tissue collection and biochemical analysis for the offspring (n = 8 per group)
was performed as previously described, for the dams (Orlandi et al., 2015). In addition,
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at PND 30 - 32, before the animals were decapitated, they were individually weighed.
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2.6 Data analysis
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Data were analyzed using the GraphPad program version 6.0 and are expressed as
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the mean ± standard error of the mean (S.E.M). Statistical comparisons were made using
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two-way repeated measures ANOVA followed by the Bonferroni test (data of weight
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gain, feed and energy intake). The other data were analyzed by Student’s t-test. P-values
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of less than 0.05 (p < 0.05) were considered as statistically significant.
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3. Results 3.1 Weight gain, feed and energy intake by the dams during gestation In Figure 1, it can be observed that pregnant female mice fed a cafeteria diet had a greater weight gain from GD 6 (GD 6: p < 0.05; GD 9: p < 0.01; GD 12 - 18: p < 0.001) compared to the normal diet group (diet factor: F1,126 = 103.2, p < 0.001; day factor:
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F6,126 = 328.5, p < 0.001; interaction: F6,126 = 4.14, p < 0.001). According to the results presented in table 2, dams of both the normal diet group and the cafeteria diet group had
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similar food intake during the gestation period (GD 1 - 18: p > 0.05; diet factor: F1,126
= 0.06, p > 0.05; day factor: F6,126 = 24.08, p < 0.001; interaction: F6,126 = 2.12, p > 0.05). However dams from the cafeteria diet group consumed more energy (kJ) during
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the gestation period (GD 1 - 18: p < 0.001) compared to the normal diet group (diet factor:
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F1,126 = 204.7, p < 0.001; day factor: F6,126 = 8.18, p < 0.001; interaction: F6,126 =
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3.2 Maternal behavior
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3.08, p < 0.01).
The analysis of the maternal behavior performed in PND 2 - PND 8, in figure 2,
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showed that there was an increase in the percentage of maternal parameters, such as licking the pups (p < 0.05), arched nursing (p < 0.01), and nest building (p < 0.05), and a
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reduction of non-maternal parameters such as feeding (p < 0.05) and not exploring (p <
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0.01) in the dams of the cafeteria diet group compared to the normal diet group.
3.3 Locomotor activity evaluation of the dams in the open field test The locomotor activity evaluated in the first week of lactation was not affected by the ingestion of the cafeteria diet compared to dams of the normal diet group, as there
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was no significant difference in the total number of crosses in the open field (normal diet group: 116.8 ± 7.36; cafeteria diet group: 102.3 ± 7.66).
3.4 Tissue collection and biochemical analysis of dams Table 3 shows that in postpartum dams that were fed a cafeteria diet there was an
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increase in the weight of retroperitoneal and perigonadal fats (p < 0.001) and also an increase in glucose plasma levels (p < 0.001), triglycerides (p < 0.05), and total
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cholesterol (p < 0.05) compared to the dams of the normal diet group.
3.5 Physical and neurobehavioral development of offspring
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A summary of developmental features in male and female pups from the normal
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and cafeteria diet groups is shown in figure 3. Physical developmental landmarks for male
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mice (Fig. 3A) appeared later in the offspring from the cafeteria diet group than in those
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from the normal diet group: hair appearance (p < 0.05), superior incisor eruption (p <
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0.001), and eye opening (p < 0.05). In addition, male offspring from the cafeteria diet group exhibited late reflex development (Fig. 3B), including palmar grasp (p < 0.01) and
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stimulation of fibrils (p < 0.001) compared to male offspring from the normal diet group. Physical developmental landmarks for females (Fig. 3C) appeared later in the offspring
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from the cafeteria diet group than in those from the normal diet group: hair appearance (p < 0.05), superior incisor eruption (p < 0.001), and eye opening (p < 0.001). In addition,
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female offspring from the cafeteria diet group exhibited late reflex development (Fig. 3D) including palmar grasp (p < 0.001), rooting (p < 0.05), and stimulation of fibrils (p < 0.001) compared to female offspring from the normal diet group.
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3.6 Light-dark and open field tests in adolescent offspring Figure 4 shows the behavioral performance of the adolescent male and female offspring in the light-dark and open field tests. With respect to the light-dark test, male offspring from the cafeteria diet group had a decrease in latency for the first transition (p < 0.05, Fig. 4A), an increase in the number of transitions (p < 0.001, Fig. 4B) and an
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increase in permanence time in the dark side (p < 0.05, Fig. 4C) compared to the normal
diet group. In the same test, female offspring from the cafeteria diet group had a decrease
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in latency for the first transition (p < 0.05, Fig. 4E) when compared to the normal diet group. There were no differences in the total number of entries in the open field in male
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3.7 Play behavior test in adolescent offspring
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and female offspring between the experimental groups (p > 0.05, Fig. 4D and H).
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The effect of exposure to maternal consumption of a cafeteria diet during the
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gestation period on adolescent play behavior is shown in Figure 5. Male offspring from
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the cafeteria diet group exhibited reduced frequency of pushing (p < 0.01, Fig. 5B), time following (p < 0.05, Fig. 5C), and time sniffing (p < 0.05, Fig. 5D) compared to the
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normal diet group. Female offspring from the cafeteria diet group exhibited reduced frequency of crawling (p < 0.001, Fig. 5E), pushing (p < 0.001, Fig. 5F), time following
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(p < 0.001, Fig. 5G), and time sniffing (p < 0.01, Fig. 5H) compared to the normal diet
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group.
3.8 Body weight, tissue collection, and biochemical analysis in adolescent offspring Table 4 shows that in PND 30 both the normal and the cafeteria diet male and female offspring had similar weights. Male and female offspring from the cafeteria diet group had an increase in the weight of retroperitoneal and perigonadal fats (p < 0.05), an
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increase in glucose plasma levels (p < 0.05 and p < 0.01, respectively), and total
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cholesterol (p < 0.05) compared to offspring from the normal diet group.
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4. Discussion Exposure to maternal consumption of a cafeteria diet during the gestation period in mice has important implications for long-term behavior in offspring. In the current study, we demonstrated that the cafeteria diet during gestation both altered maternal
impact on emotional and play behavior in adolescent offspring.
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behavior and the onset of physical and neurodevelopmental landmarks and had a negative
Moreover, our study demonstrated that pregnant female mice fed a cafeteria diet
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had weighted more and this must have occurred due to higher energy consumption. Actually, cafeteria feeding, however during the lactation period, led to a significant increase in energy intake in lactating rats that was due to overconsumption of sugar and
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fat (Speight et al., 2017). Another study showed that male mice fed for 15 weeks with
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cafeteria diet had an increase in body weight and energy intake (Zeeni et al., 2015). In
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addition, cafeteria feeding during the pre-pregnancy and pregnancy periods was effective
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in inducing obesity, as demonstrated by increased fat deposit weights and total body fat
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in rats (Akyol et al., 2009). These findings are in line with our results as dams fed the cafeteria diet had higher fat deposits.
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In contrast, the cafeteria diet during gestation did not change the weight of the male and female offspring in adolescence. However, these offspring showed an increase
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in fat deposits, plasma glucose, and total cholesterol levels. Indeed, standard animal models in which dams are fed an obesogenic high fat diet throughout pregnancy and
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lactation demonstrate that exposure of the offspring throughout this period results in increased adiposity in the offspring of mice and rats, respectively (Samuelsson et al., 2008; Nivoit et al., 2009). Mice offspring from dams fed a maternal obesity-inducing, hyperenergetic diet before and during pregnancy have been shown to exhibit disturbed glucose and lipid homeostasis and greater adiposity (Samuelsson et al., 2008).
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Since the cafeteria diet has been well established as an obesogenic diet in rodents (Rothwell and Stock, 1979), increases in plasma glucose, triglycerides, and total cholesterol levels were to be expected. Indeed, dams fed a cafeteria diet during pregnancy showed an increase in these biochemical parameters. A behavioral effect of gestation diet could be due to the direct nutritional effects
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of the diet itself, but could also be mediated by a change in maternal behavior (Speight et
al., 2017). Thus, we also explored maternal behavior following exposure to the diet during
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gestation. The analysis of maternal behavior demonstrated an increase in the time of
licking the pups and of arched position permanence. In another study, dams fed a cafeteria diet during lactation showed an increase in licking time compared to control dams
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(Speight et al., 2017); thus, suggesting that the diet cafeteria during the gestation or
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lactation period may alter the course of maternal behavior.
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In our study some parameters of physical and neurobehavioral development were
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delayed in both male and female offspring. Birth through 21 days is a time of active brain
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growth, extension of neuronal processes, migrating oligodendrocytes and myelination (Graf et al., 2016). Possibly, the deficit in neurobehavioral reflex ontogeny should be
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associated with delayed myelination since previous studies have shown that maternal high fat diet prior to and during pregnancy is associated with decreased myelination in some
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brain regions (Graf et al., 2016). In addition, in another study, it was demonstrated that the behavioral abnormalities in neurological reflex tasks were consistent with delayed
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myelination of subcortical white matter (Wu et al., 2008). It has also been established that impaired development may represent a predictive
factor of behavioral modifications in adulthood (Heyser, 2004; Berk, 2006) and through the evaluation of neonatal reflexes, it is possible to identify the persistence or absence of reflexes and detect developmental delay (Schuch et al., 2016). Physical and
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neurobehavioral development are considered a sign of brain maturation and follow a sequence of the appearance of reflexes and maturation of motor skills (Fox, 1965; Heyser, 2004). Furthermore, maternal malnutrition appears to have both affected offspring development and impacted emotional and play behavior in male and female adolescent
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offspring. This is the first study demonstrating the effect of exposure to maternal
consumption of a cafeteria diet during the gestation period on behavioral performance of
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adolescent offspring in the light-dark test.
A study indicated an anxiogenic effect of an obesogenic maternal diet observed in the elevated plus maze test in adult rats. However, the authors used two different high fat
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diets (high-saturated-fat and high-trans-fat diet) for 4 weeks prior to mating, and remained
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on the diet throughout pregnancy and lactation (Bilbo and Tsang, 2010). In contrast, it
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has been shown that the pre-gestational, gestational, and lactational maternal cafeteria
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diet programs behavioral courses in the offspring of rats; with the lactational cafeteria
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diet reducing anxiety in the male offspring on the elevated plus maze test (Wright et al., 2011). In addition, adolescent offspring exposed to a cafeteria diet during lactation did
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not present altered behavioral performance in the elevated plus maze (Speight et al., 2017). In view of this, it may be observed that there are controversial data regarding the
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programming by the maternal diet; however, it must be considered that the periods and types of diets differ among the several studies. The data of the present study demonstrate
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that maternal gestation cafeteria diet programs behavior in the adolescent offspring by increasing anxiety-like behavior assessed on the light-dark test, because there was a reduction in latency for the first transition in males and females and an increase in the permanence time on the dark side of the box in males. Furthermore, our results showed that there were no changes in the locomotor activity of these animals. Conversely,
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adolescent offspring of rats exposed to a cafeteria diet, but during lactation, demonstrated an increase in locomotor activity in the open field (Speight et al., 2017). Another interesting finding of our study was a reduction of play behavior in adolescent offspring whose mothers were fed a cafeteria diet during gestation. To our knowledge, this is the first report concerning exposure to maternal consumption of a cafeteria diet
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during the gestation period programming play behavior. Play behavior is a prevalent
behavior during a short period of time in rodents, declining with the beginning of sexual
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maturity, and may serve to equip the animals with some basic skills and strategies
essential for a variety of behaviors that are expressed in adulthood (Camargo and Almeida, 2005). In addition, play behavior is one of the earliest forms of non-mother
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directed social behavior in rodents, and is considered to be a vigorous form of social
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interaction in young mammals (Vanderschuren et al., 1997; Spear, 2000). Most young
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mammals spend a substantial part of maturation engaged in play with peers. This ability
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to participate in social play is a principal indicator of healthy development (Trezza et al.,
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2010). Social behaviors are relevant to normal cognitive and social development and have been utilized as behavioral biomarkers for altered development in both rodent models and
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humans (Blake and McCoy, 2015). Our results suggest that changes in the expression of social play in the offspring is possibly due to the result of developmental retardation and
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neurological changes produced by the gestational cafeteria diet. Moreover, the evaluation of social interactions, which may lead to increases in pleasure and impact future decision-
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making, seems to involve reward-related processing (Izuma et al., 2008). Specific types of social behavior can be rewarding and an association between social approach and reward provides a conceptually powerful mechanism by which approach behaviors can be initiated and maintained (Panksepp and Lahvis, 2007). Thus, the prenatal cafeteria diet could have changed the reward value of peers in the play interaction test.
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Given the data obtained from the offspring, it can be observed that the increase in maternal behavior did not influence the offspring behavior, since the offspring had their behavior impaired even with the increase in maternal behavior. In conclusion, our results demonstrate that exposure to maternal consumption of a cafeteria diet during the gestation period can program developmental and behavioral
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courses in the offspring. Considering that psychiatric diseases are increasingly seen as
developmental disorders, maternal malnutrition could be a contributing factor for
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psychiatric disorders present in the offspring.
Disclosure statement
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Contributors: All authors contributed equally.
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Funding
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This work was supported by Fundação de Amparo a Pesquisa de Minas Gerais
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(FAPEMIG #01483/2013, AG-P) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, #456078/2014-2; FCV). The FAPEMIG and CNPq had no further
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role in the design of the study; the collection, analysis, and interpretation of the data; the
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writing of the report; or the decision to submit the paper for publication.
Conflict of interest
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No potential conflict of interest was reported by the authors. The authors report no biomedical financial interests or potential conflicts of interest. Acknowledgements We are grateful for the excellent technical support of José dos Reis Pereira.
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35. Wright T., Langley-Evans S.C., Voigt J.P., 2011. The impact of maternal cafeteria diet on anxiety-related behaviour and exploration in the offspring. Physiol. Behav.
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38. Zeeni N., Dagher-Hamalian C., Dimassi H., Faour W.H., 2015. Cafeteria diet-fed mice is a pertinent model of obesity-induced organ damage: a potential role of inflammation. Inflamm. Res. 64, 501-512.
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Figure Legends
Figure 1: Effect of the normal or cafeteria diet during the gestation period on weight gain (grams) of the dams during gestation. Data represent the mean (± S.E.M.) of 10 animals per group. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the normal diet group.
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Data analyzed by two-way repeated measures ANOVA followed by the Bonferroni test.
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Figure 2: Effect of the normal or cafeteria diet during the gestation period on percentage
of maternal behavior (maternal parameters and non-maternal parameters) of lactating mice in postnatal day (PND) 2-PND 8. Each column represents the mean (± S.E.M.) of
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10 animals per group. *p < 0.05 and **p < 0.01 compared to the normal diet group. Data
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analyzed by Student’s t-test.
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Figure 3: Effect of the normal or cafeteria diet during the gestation period on physical and
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reflex developmental parameters in pups: (A and B) male offspring and (C and D) female offspring. Each column represents the mean (± S.E.M.) of 10 animals per group. *p <
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0.05 and ***p < 0.001 compared to offspring of the normal diet group. Data analyzed by Student’s t-test.
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Figure 4: Effect of the normal or cafeteria diet during the gestation period on behavioral performance in the light-dark and open field tests of adolescent male and female offspring: (A and E) latency in seconds to first transition, (B and F) number of transitions,
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(C and G) time in seconds of permanence in the dark, and (D and H) total number of entries in open field test. Each column represents the mean (± S.E.M.) of 10 animals per group. *p < 0.05 and ***p < 0.001 compared to offspring of the normal diet group. Data analyzed by Student’s t-test.
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Figure 5: Effect of the normal or cafeteria diet during the gestation period on play behavior test of adolescence male and female offspring: (A and E) frequency of crawling, (B and F) frequency of pushing, (C and G) time in seconds of following, and (D and H) time in seconds of sniffing. Each column represents the mean (± S.E.M.) of 10 animals per group. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to offspring of the normal
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A
N
U
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diet group. Data analyzed by Student’s t-test.
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Table 1: Energy and macronutrient content of the cafeteria diet per 100 grams Protein
Carbohydrats
Lipids
(kJ)
(grams)
(grams)
(grams)
2.260,44
4,8
60
31,2
865,25
20
3,3
20
Potato chips
2.354,63
6,25
45,8
38,8
Bacon
1.611, 61
16,5
0
Wafer biscuit
2.218,58
4,7
63,3
Chow diet
1.391,85
20
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53
4,5
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Cheese
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Chocolate
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Energy
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Table 2: Feed and energy intake of dams during gestation Feed intake
Feed intake
Energy intake
Energy intake
days
(grams)
(grams)
(kJ)
(kJ)
Normal diet
Cafeteria diet
Normal diet
Cafeteria diet
6.59±0.22
80.45±2.76
135.26±5.53***
8.47±0.32
7.88±0.14
105.27±4.76
143.87±7.08***
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Gestation
7.04±0.35
6°
7.46±0.32
7.03±0.22
89.69±3.67
128.89±5.73***
9°
7.98±0.22
6.93±0.16
98.67±3.17
135.60±5.93***
12°
8.28±0.25
7.80±0.25
101.08±2.99
151.60±6.01***
15°
8.97±0.52
8.86±0.22
109.47±7.49
161.99±5.99***
18º
10.14±0.31
8.34±0.50
124.74±4.28
140.56±6.96***
1°
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3°
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Values are the mean ± SEM (Student’s t-test) of 10 animals per group. *** p < 0.001 compared to the normal diet group. Data analyzed by two-way repeated measures ANOVA followed by the Bonferroni test.
Table 3: Adipose tissue and biochemical determination in dams of the normal or cafeteria
Normal diet
Cafeteria diet
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Adipose tissue (g/10g b.w.) Retroperitoneal fat
0.08±0.01
0.26±0.01***
Perigonadal fat
0.15±0.01
0.43±0.03***
Triglycerides (mg/dl)
159.20±6.10
Total cholesterol (mg/dl)
72.72±1.63
173.00±6.55*** 183.70±6.49* 80.72±3.18*
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125.50±6.28
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Biochemical determinations Glucose (mg/dl)
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diet groups at PND1
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Values are the mean ± SEM (Student’s t-test) of 8 animals per group. * p < 0.05 and ***
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p < 0.001 compared to the normal diet group. Data analyzed by Student’s t-test.
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Table 4: Body weight, adipose tissue, and biochemical determinations of male and female offspring of the normal or cafeteria diet dams at PND 30 - 32 Offspring cafeteria diet
Male
Female
Male
Female
30.76±0.34
27.47±0.42
31.07±6.36
27.80±0.76
Retroperitoneal fat
0.03±0.00
0.04±0.00
0.04±0.00*
0.07±0.00*
Perigonadal fat
0.07±0.00
0.08±0.00
0.08±0.00*
1.00±0.00*
Glucose (mg/dl)
110.90±4.68
117.40±3.75
133.30±7.29*
146.90±7.52**
Triglycerides (mg/dl)
247.40±15.19
252.80±11.65
237.40±16.66
217.80±14.04
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Offspring normal diet
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Body weight
Adipose tissue (g/10g b.w.)
Biochemical determinations
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Total cholesterol (mg/dl)
84.44±2.93
77.32±3.24
97.44±3.68*
87.04±2.29*
Values are the mean ± SEM (Student’s t-test) of 8 animals per group. * p < 0.05 and **
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A
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p < 0.01 compared to the normal diet group. Data analyzed by Student’s t-test.
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S0736-5748(18)30009-1 https://doi.org/10.1016/j.ijdevneu.2018.05.001 DN 2258
To appear in:
Int. J. Devl Neuroscience
Received date: Revised date: Accepted date:
8-1-2018 26-4-2018 1-5-2018
Please cite this article as: Ribeiro ACAF, Batista TH, Veronesi VB, Giusti-Paiva A, Vilela. FC, Cafeteria diet during the gestation period programs developmental and behavioral courses in the offspring, International Journal of Developmental Neuroscience (2010), https://doi.org/10.1016/j.ijdevneu.2018.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cafeteria diet during the gestation period programs developmental and behavioral courses in the offspring
Ana Cláudia Alves Freire Ribeiro, Tatiane Helena Batista, Vanessa Barbosa Veronesi,
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Alexandre Giusti-Paiva, Fabiana Cardoso Vilela.*
Departamento de Ciências Fisiológicas, Instituto de Ciências Biomédicas, Universidade
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Federal de Alfenas (Unifal-MG), Alfenas, Minas Gerais, Brazil;
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Programa de Pós-Graduação em Biociências Aplicadas à Saúde, Brazil.
*
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Corresponding author: Fabiana Cardoso Vilela, PhD , Instituto de Ciências Biomédicas
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, Universidade Federal de Alfenas, UNIFAL , Avenida Jovino Fernandes Sales nº 2600 ,
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37130-000, MG, Brazil , Phone: +55 (35) 37011890 , E-mail: [email protected]
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Highlights
Cafeteria diet during gestation altered maternal behavior.
Cafeteria diet altered the onset of physical and neurodevelopmental landmarks.
Maternal cafeteria diet had an impact on emotional behavior in adolescent
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offspring.
Maternal cafeteria diet had an impact on play behavior in adolescent offspring.
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Abstract
The objective of this study was to investigate the effect of exposure to maternal consumption of a hyperenergetic, highly palatable diet, known as the cafeteria diet, during the gestation period on the development and behavior of offspring. For this, we used
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pregnant female mice that were fed a normal or a cafeteria diet during the gestation period. The evaluation of maternal behavior in lactating dams was performed from the
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second to the eighth day postpartum (PND 2 - 8). Weight gain, feed intake, and energy intake were recorded during the gestation period. In the offspring, reflex parameters and physical development were evaluated during the lactation period and when they reached
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adolescence. Behavioral performance was evaluated in light-dark, open-field, and play
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behavior tests. In addition, biochemical parameters of the dams and the adolescent
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offspring were evaluated. The cafeteria diet during gestation altered maternal behavior
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and the onset of physical and neurodevelopmental landmarks and had an impact on emotional and play behavior in adolescent offspring. In conclusion, our results
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demonstrate that exposure to maternal consumption of a cafeteria diet during the gestation
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period can program developmental and behavioral courses in the offspring.
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Keywords: cafeteria diet; offspring; developmental; behavior.
1. Introduction
Maternal nutrition plays a critical role in the offspring's physical growth and behavior (Laus et al., 2011; Black et al., 2013). An adequate supply of nutrients is critical
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for maintenance of growth, as well as, for the development of functions in organic systems (Morgane et al., 1978; Umeta et al., 2003). Thus, an inadequate diet during the gestation period can negatively influence the development of the brain, resulting in changes in its structure and, hence, its function (Morgane et al., 1978; Morgane et al., 1993).
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As a consequence, malnutrition is a very important, nongenetic factor affecting the developmental processes (Morgane et al., 1978; Morgane et al., 1993). Some evidence
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suggests that the in utero environment independently and critically impacts offspring
development and their susceptibility to obesity and disease in later life (Cooper et al.,1996; Chen et al., 2008). However, little is currently known about the behavioral
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effects of feeding a hyperenergetic diet during the gestation period when offspring
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behavior is tested during adolescence.
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The term malnutrition implies that one or more essential nutrients is missing or
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present but in inadequate proportions (Morgane et al., 2002). Among various
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experimental diets of malnutrition, the cafeteria diet, which is considered a hyperenergetic diet, has been well established as an obesogenic diet in rodents and as a
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model of a Western-style diet. In short, a cafeteria diet consists of various hyperenergetic and highly palatable human food items (Rothwell and Stock, 1979; Speight, 2017).
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Exposure to maternal consumption of a cafeteria diet during lactation leads to
reduced anxiety and changes in memory in the offspring when tested at adult age (Wright
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et al., 2011a, 2011b). In another study, a cafeteria diet during the lactation period changed open-field behavior in the developing offspring (Speight et al., 2017). Although these results suggest the nutritional programming of behavior, less is known about the behavioral consequences of prenatal exposure.
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Since maternal nutrition plays a critical role in the offspring's physical growth and behavior, we hypothesize that an inadequate diet during the gestation period, such as consumption of a cafeteria diet, can negatively influence developmental and behavioral courses in the offspring. Therefore, knowing that maternal malnutrition may affect the maturation of the brain and the development of cognitive function, resulting in behavioral
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abnormalities in offspring, the objective of this study was to investigate the effect of exposure to maternal consumption of a cafeteria diet during the gestation period on the
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development and behavior of the offspring. In addition, the maternal behavior of the dams
as well as biochemical parameters of the dams and the adolescent offspring were
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evaluated.
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2. Materials and methods
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2.1 Animals
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Adult Swiss nulliparous female and male mice aged approximately 9 weeks were
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obtained from the Central Animal Facility of the Federal University of Alfenas and were housed in a temperature-controlled room (22ºC) on a 12:12 h light-dark cycle (lights on
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at 7:00 h) with ad libitum access to water and standard laboratory mouse chow. The female mice were time-mated by placing them with sexually experienced male mice (two
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females for one male). The next morning, the presence of vaginal plug was considered as gestation day zero (GD0). The pregnant mice were housed individually and were divided
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into two experimental groups: normal diet (standard laboratory chow) or cafeteria diet. All experimental procedures followed the Ethical Principles in Animal Research adopted by the Ethics Committee on the Use of Animals of the Federal University of Alfenas (protocol 620/2015). The experiments were performed in accordance with good laboratory practice protocols and quality assurance methods.
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2.2 Experimental design During the gestation period (GD 0 - 21), twenty pregnant mice were fed with a normal (n = 10) or cafeteria diet (n = 10). On parturition day (PND 0), the cafeteria diet was replaced with a normal diet. At PND 1, all litters were culled to eight pups (four males and four females). These dams were used for evaluation of maternal behavior (PND
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2 - 8) and locomotor activity (PND 6) in the open field. In addition, weight gain, feed intake, and energy intake were recorded during the gestation period. Their offspring (40
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female and 40 male mice from each group) were used for the experimental procedures
during adolescence (PND 30 - 32). To avoid the litter effect, one couple (1 male and 1 female mouse) from each litter received an ink mark on one hind limb for observation of
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reflex parameters and physical development until PND 21 (n = 10 per group) (Veronesi
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et al., 2017). The other three couples of each litter that were not marked were used to
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evaluate the behavioral performance in the light dark, open field, or play behavior tests
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(n = 10 per group) during adolescence since each animal was used only once for a given
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behavioral evaluation. After these tests, two animals from each test were used for blood collection for biochemical analyzes (n = 8 per group) and removal of perigonadal and
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retroperitoneal fats (n = 8 per group). Other groups of dams (n = 8 per group) were used
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only to collect blood and remove perigonadal and retroperitoneal fats at PND 1.
2.3 Gestational feeding
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The normal diet consisted of commercial chow (Nuvilab® CR1) in the form of
pellets. The cafeteria diet consisted of the following items: Nestlé Classic® milk chocolate, Bauducco® strawberry wafer, Tropeiro® bacon, mozzarella cheese, original Ruffles® potato and commercial chow (Nuvilab® CR1). Foods were offered in the form in which they were purchased, in natura, on the same proportion. The choice of food to
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compose the cafeteria diet was based on previous studies and adapted to availability in the local market (Akyol et al., 2009; Bouanane et al., 2010). The values used to calculate the energy (kJ) were as follows: (total protein in grams) × 4 + (total carbohydrates in grams) × 4 + (total lipids in grams) × 9 = total calories ingested x 4.186 kJ. The composition of the macronutrients and the energy of the foods offered to the animals are
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described in table 1.
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2.4 Maternal studies
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2.4.1 Weight gain, feed and energy intake by the dams during gestation
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Weight gain, feed, and energy intake were recorded during the gestation period (n =
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10 per group). Energy intake was based on the amount of each food ingested.
2.4.2 Maternal behavior
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The maternal behavior of lactating mice (n = 10 per group) was scored daily during the first week of lactation (PND 2 - 8). Observations were conducted during three
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72-min periods in the light phase (08:00, 12:00, and 16:00) and one 72-min period in the dark phase (20:00). During each session, maternal behavior was scored every 3 min (25
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observations per four periods per day for a total of 100 observations per dam per day). Six maternal behaviors and three non-maternal behaviors were recorded as follows: (1) licking the pups (either the body surface or the anogenital region), (2) nursing the pups in an arched-back posture, (3) “blanket” posture in which the mother lays over the pups, (4) passive posture in which the mother lies either on her back or side while the pups nurse,
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(5) self-grooming in which dams lick the breasts, (6) nest building, (7) feeding, (8) exploring the cage housing, (8) not exploring (Costa et al., 2013; Carvalho et al., 2016). Behavioral data are reported as percentages of the total number of behavioral observations (number of target behavior observations divided by the total number of all behavioral
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observations × 100).
2.4.3 Locomotor activity evaluation in the open field test
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Locomotor activity was performed at PND 6 (n = 10 per group) for 5 min in an open field box (10 - 12 lux), consisting of an acrylic painted acrylic base 100 x 80 cm and 50 (height) x 60 cm (diameter). Four squares were defined as the center and the eight
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squares along the walls were considered the periphery. Each mouse was gently placed in
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the exact center of the box. Activity was scored as a line crossing when a mouse removed
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all four paws from one square and entered another. Line crossings among the central four
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(Veronesi et al., 2017).
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squares or among the peripheral eight squares of the open field were counted separately
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2.4.4 Tissue collection and biochemical analysis Another group of animals was used for these procedures at PND 1. Normal diet
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and cafeteria diet dams (n = 8 per group) were euthanized by decapitation, and a blood sample was collected in a heparinized tube. The plasma was immediately isolated by
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centrifugation, and duplicate aliquots were stored at -80 °C prior to the biochemical analyses. After the mice were sacrificed, perigonadal and retroperitoneal adipose tissue samples were harvested and weighed. Commercial kits from In Vitro Diagnostica Ltd. (Itabira-MG, Brazil) were used to measure plasma total cholesterol, triglycerides, and glucose by the absorbance method (Orlandi et al., 2015).
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2.5 Offspring studies 2.5.1 Physical and neurobehavioral development The following physical parameters were assessed in one male and one female pup from each litter (n = 10 per group) as follows: pinna unfolding (beginning on PND 2),
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separation of front and rear digits, hair growth, superior and inferior incisor eruption
(beginning on PND 5), and eye opening (beginning on PND 10). The following reflexes
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were also assessed in one male and one female pup from each litter as follows: surface righting (beginning on PND 2), negative geotaxis (turning at least 135º within 30 s of face-down placement on a 45º incline, beginning on PND 4), palmar grasp reflex
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(grasping of a paper clip with forepaws if stroked, beginning on PND 2), rooting (turning
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the head toward the side of the face being stroked with the tip of a cotton swab beginning
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on PND 2), auditory startle (beginning on PND 10), and stimulation of fibrils (beginning
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on PND 12). Pups were briefly removed from their dams between 14:00 - 15:00 for daily
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observation and then immediately returned to their home cage. The mean number of days required for the appearance of each of the above-mentioned parameters was calculated.
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On PND 21, the offspring were weaned, marked with a nontoxic ink, distributed in accordance with their sex, and housed in groups of four per cage (Carvalho et al., 2016;
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Veronesi et al., 2017).
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2.5.2 Light-dark test The apparatus consisted of a Plexiglas rectangular box (48 cm long × 24 cm wide
× 24 cm high) divided into a dark region (24 cm long) and a light region (24 cm long). The light and dark regions were separated by an opening (8.0 × 8.0 cm) that allowed the animals to move between the two compartments. The dark region was made of black
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Plexiglas and covered with a black lid. The light portion was made of white Plexiglas, and a 60 W light was positioned directly over it. Each mouse at PND 30 - 32 (n = 10 per group) was placed in the light compartment and allowed to move freely between the two compartments. The box was carefully cleaned with a 5% ethanol solution after every test. The behavior was video-recorded for a total of 5 min, and the videotapes were scored for
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compartments, and the time of permanence in light (Ribeiro et al., 2016).
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latency to the first transition, number of transitions between the light and dark
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2.5.3 Open field test
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The open field test for the offspring at PND 30 - 32 (n = 10 per group) was
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5% ethanol solution after every test.
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performed as previously described, for the dams. The arena was carefully cleaned with a
2.5.4 Adolescent play behavior
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Adolescent play at PND 30 - 32 was conducted in an arena covered with a 0.5 cm layer of clean bedding. Subjects (n = 10 per group) were acclimatized to the box for two
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consecutive days and on the third day (test day), they were individually housed in standard mouse cages for 3.5 hrs prior to the play session. Two mice of the same group, age, and
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sex, but from different litters, were then placed in the arena and their interactions were recorded for 10 min, the period during which the majority of social interaction occurs. Behaviors were subsequently scored, and the behaviors of interest included time of following (one mouse walks straight behind its partner, keeping pace with the one ahead), time of sniffing (sniffing anywhere on the body), frequency of pushing (pushing
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head/snout underneath the partner's body or squeezing itself between the wall/floor and the partner), and frequency of crawling (crawling over or under the partner's body) (Yang et al., 2009; Manduca et al., 2014).
2.5.5 Body weight, tissue collection, and biochemical analysis of offspring
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The tissue collection and biochemical analysis for the offspring (n = 8 per group)
was performed as previously described, for the dams (Orlandi et al., 2015). In addition,
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at PND 30 - 32, before the animals were decapitated, they were individually weighed.
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2.6 Data analysis
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Data were analyzed using the GraphPad program version 6.0 and are expressed as
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the mean ± standard error of the mean (S.E.M). Statistical comparisons were made using
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two-way repeated measures ANOVA followed by the Bonferroni test (data of weight
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gain, feed and energy intake). The other data were analyzed by Student’s t-test. P-values
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of less than 0.05 (p < 0.05) were considered as statistically significant.
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3. Results 3.1 Weight gain, feed and energy intake by the dams during gestation In Figure 1, it can be observed that pregnant female mice fed a cafeteria diet had a greater weight gain from GD 6 (GD 6: p < 0.05; GD 9: p < 0.01; GD 12 - 18: p < 0.001) compared to the normal diet group (diet factor: F1,126 = 103.2, p < 0.001; day factor:
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F6,126 = 328.5, p < 0.001; interaction: F6,126 = 4.14, p < 0.001). According to the results presented in table 2, dams of both the normal diet group and the cafeteria diet group had
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similar food intake during the gestation period (GD 1 - 18: p > 0.05; diet factor: F1,126
= 0.06, p > 0.05; day factor: F6,126 = 24.08, p < 0.001; interaction: F6,126 = 2.12, p > 0.05). However dams from the cafeteria diet group consumed more energy (kJ) during
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the gestation period (GD 1 - 18: p < 0.001) compared to the normal diet group (diet factor:
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F1,126 = 204.7, p < 0.001; day factor: F6,126 = 8.18, p < 0.001; interaction: F6,126 =
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3.2 Maternal behavior
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3.08, p < 0.01).
The analysis of the maternal behavior performed in PND 2 - PND 8, in figure 2,
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showed that there was an increase in the percentage of maternal parameters, such as licking the pups (p < 0.05), arched nursing (p < 0.01), and nest building (p < 0.05), and a
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reduction of non-maternal parameters such as feeding (p < 0.05) and not exploring (p <
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0.01) in the dams of the cafeteria diet group compared to the normal diet group.
3.3 Locomotor activity evaluation of the dams in the open field test The locomotor activity evaluated in the first week of lactation was not affected by the ingestion of the cafeteria diet compared to dams of the normal diet group, as there
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was no significant difference in the total number of crosses in the open field (normal diet group: 116.8 ± 7.36; cafeteria diet group: 102.3 ± 7.66).
3.4 Tissue collection and biochemical analysis of dams Table 3 shows that in postpartum dams that were fed a cafeteria diet there was an
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increase in the weight of retroperitoneal and perigonadal fats (p < 0.001) and also an increase in glucose plasma levels (p < 0.001), triglycerides (p < 0.05), and total
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cholesterol (p < 0.05) compared to the dams of the normal diet group.
3.5 Physical and neurobehavioral development of offspring
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A summary of developmental features in male and female pups from the normal
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and cafeteria diet groups is shown in figure 3. Physical developmental landmarks for male
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mice (Fig. 3A) appeared later in the offspring from the cafeteria diet group than in those
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from the normal diet group: hair appearance (p < 0.05), superior incisor eruption (p <
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0.001), and eye opening (p < 0.05). In addition, male offspring from the cafeteria diet group exhibited late reflex development (Fig. 3B), including palmar grasp (p < 0.01) and
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stimulation of fibrils (p < 0.001) compared to male offspring from the normal diet group. Physical developmental landmarks for females (Fig. 3C) appeared later in the offspring
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from the cafeteria diet group than in those from the normal diet group: hair appearance (p < 0.05), superior incisor eruption (p < 0.001), and eye opening (p < 0.001). In addition,
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female offspring from the cafeteria diet group exhibited late reflex development (Fig. 3D) including palmar grasp (p < 0.001), rooting (p < 0.05), and stimulation of fibrils (p < 0.001) compared to female offspring from the normal diet group.
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3.6 Light-dark and open field tests in adolescent offspring Figure 4 shows the behavioral performance of the adolescent male and female offspring in the light-dark and open field tests. With respect to the light-dark test, male offspring from the cafeteria diet group had a decrease in latency for the first transition (p < 0.05, Fig. 4A), an increase in the number of transitions (p < 0.001, Fig. 4B) and an
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increase in permanence time in the dark side (p < 0.05, Fig. 4C) compared to the normal
diet group. In the same test, female offspring from the cafeteria diet group had a decrease
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in latency for the first transition (p < 0.05, Fig. 4E) when compared to the normal diet group. There were no differences in the total number of entries in the open field in male
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3.7 Play behavior test in adolescent offspring
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and female offspring between the experimental groups (p > 0.05, Fig. 4D and H).
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The effect of exposure to maternal consumption of a cafeteria diet during the
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gestation period on adolescent play behavior is shown in Figure 5. Male offspring from
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the cafeteria diet group exhibited reduced frequency of pushing (p < 0.01, Fig. 5B), time following (p < 0.05, Fig. 5C), and time sniffing (p < 0.05, Fig. 5D) compared to the
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normal diet group. Female offspring from the cafeteria diet group exhibited reduced frequency of crawling (p < 0.001, Fig. 5E), pushing (p < 0.001, Fig. 5F), time following
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(p < 0.001, Fig. 5G), and time sniffing (p < 0.01, Fig. 5H) compared to the normal diet
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group.
3.8 Body weight, tissue collection, and biochemical analysis in adolescent offspring Table 4 shows that in PND 30 both the normal and the cafeteria diet male and female offspring had similar weights. Male and female offspring from the cafeteria diet group had an increase in the weight of retroperitoneal and perigonadal fats (p < 0.05), an
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increase in glucose plasma levels (p < 0.05 and p < 0.01, respectively), and total
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cholesterol (p < 0.05) compared to offspring from the normal diet group.
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4. Discussion Exposure to maternal consumption of a cafeteria diet during the gestation period in mice has important implications for long-term behavior in offspring. In the current study, we demonstrated that the cafeteria diet during gestation both altered maternal
impact on emotional and play behavior in adolescent offspring.
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behavior and the onset of physical and neurodevelopmental landmarks and had a negative
Moreover, our study demonstrated that pregnant female mice fed a cafeteria diet
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had weighted more and this must have occurred due to higher energy consumption. Actually, cafeteria feeding, however during the lactation period, led to a significant increase in energy intake in lactating rats that was due to overconsumption of sugar and
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fat (Speight et al., 2017). Another study showed that male mice fed for 15 weeks with
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cafeteria diet had an increase in body weight and energy intake (Zeeni et al., 2015). In
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addition, cafeteria feeding during the pre-pregnancy and pregnancy periods was effective
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in inducing obesity, as demonstrated by increased fat deposit weights and total body fat
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in rats (Akyol et al., 2009). These findings are in line with our results as dams fed the cafeteria diet had higher fat deposits.
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In contrast, the cafeteria diet during gestation did not change the weight of the male and female offspring in adolescence. However, these offspring showed an increase
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in fat deposits, plasma glucose, and total cholesterol levels. Indeed, standard animal models in which dams are fed an obesogenic high fat diet throughout pregnancy and
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lactation demonstrate that exposure of the offspring throughout this period results in increased adiposity in the offspring of mice and rats, respectively (Samuelsson et al., 2008; Nivoit et al., 2009). Mice offspring from dams fed a maternal obesity-inducing, hyperenergetic diet before and during pregnancy have been shown to exhibit disturbed glucose and lipid homeostasis and greater adiposity (Samuelsson et al., 2008).
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Since the cafeteria diet has been well established as an obesogenic diet in rodents (Rothwell and Stock, 1979), increases in plasma glucose, triglycerides, and total cholesterol levels were to be expected. Indeed, dams fed a cafeteria diet during pregnancy showed an increase in these biochemical parameters. A behavioral effect of gestation diet could be due to the direct nutritional effects
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of the diet itself, but could also be mediated by a change in maternal behavior (Speight et
al., 2017). Thus, we also explored maternal behavior following exposure to the diet during
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gestation. The analysis of maternal behavior demonstrated an increase in the time of
licking the pups and of arched position permanence. In another study, dams fed a cafeteria diet during lactation showed an increase in licking time compared to control dams
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(Speight et al., 2017); thus, suggesting that the diet cafeteria during the gestation or
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lactation period may alter the course of maternal behavior.
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In our study some parameters of physical and neurobehavioral development were
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delayed in both male and female offspring. Birth through 21 days is a time of active brain
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growth, extension of neuronal processes, migrating oligodendrocytes and myelination (Graf et al., 2016). Possibly, the deficit in neurobehavioral reflex ontogeny should be
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associated with delayed myelination since previous studies have shown that maternal high fat diet prior to and during pregnancy is associated with decreased myelination in some
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brain regions (Graf et al., 2016). In addition, in another study, it was demonstrated that the behavioral abnormalities in neurological reflex tasks were consistent with delayed
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myelination of subcortical white matter (Wu et al., 2008). It has also been established that impaired development may represent a predictive
factor of behavioral modifications in adulthood (Heyser, 2004; Berk, 2006) and through the evaluation of neonatal reflexes, it is possible to identify the persistence or absence of reflexes and detect developmental delay (Schuch et al., 2016). Physical and
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neurobehavioral development are considered a sign of brain maturation and follow a sequence of the appearance of reflexes and maturation of motor skills (Fox, 1965; Heyser, 2004). Furthermore, maternal malnutrition appears to have both affected offspring development and impacted emotional and play behavior in male and female adolescent
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offspring. This is the first study demonstrating the effect of exposure to maternal
consumption of a cafeteria diet during the gestation period on behavioral performance of
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adolescent offspring in the light-dark test.
A study indicated an anxiogenic effect of an obesogenic maternal diet observed in the elevated plus maze test in adult rats. However, the authors used two different high fat
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diets (high-saturated-fat and high-trans-fat diet) for 4 weeks prior to mating, and remained
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on the diet throughout pregnancy and lactation (Bilbo and Tsang, 2010). In contrast, it
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has been shown that the pre-gestational, gestational, and lactational maternal cafeteria
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diet programs behavioral courses in the offspring of rats; with the lactational cafeteria
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diet reducing anxiety in the male offspring on the elevated plus maze test (Wright et al., 2011). In addition, adolescent offspring exposed to a cafeteria diet during lactation did
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not present altered behavioral performance in the elevated plus maze (Speight et al., 2017). In view of this, it may be observed that there are controversial data regarding the
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programming by the maternal diet; however, it must be considered that the periods and types of diets differ among the several studies. The data of the present study demonstrate
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that maternal gestation cafeteria diet programs behavior in the adolescent offspring by increasing anxiety-like behavior assessed on the light-dark test, because there was a reduction in latency for the first transition in males and females and an increase in the permanence time on the dark side of the box in males. Furthermore, our results showed that there were no changes in the locomotor activity of these animals. Conversely,
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adolescent offspring of rats exposed to a cafeteria diet, but during lactation, demonstrated an increase in locomotor activity in the open field (Speight et al., 2017). Another interesting finding of our study was a reduction of play behavior in adolescent offspring whose mothers were fed a cafeteria diet during gestation. To our knowledge, this is the first report concerning exposure to maternal consumption of a cafeteria diet
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during the gestation period programming play behavior. Play behavior is a prevalent
behavior during a short period of time in rodents, declining with the beginning of sexual
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maturity, and may serve to equip the animals with some basic skills and strategies
essential for a variety of behaviors that are expressed in adulthood (Camargo and Almeida, 2005). In addition, play behavior is one of the earliest forms of non-mother
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directed social behavior in rodents, and is considered to be a vigorous form of social
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interaction in young mammals (Vanderschuren et al., 1997; Spear, 2000). Most young
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mammals spend a substantial part of maturation engaged in play with peers. This ability
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to participate in social play is a principal indicator of healthy development (Trezza et al.,
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2010). Social behaviors are relevant to normal cognitive and social development and have been utilized as behavioral biomarkers for altered development in both rodent models and
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humans (Blake and McCoy, 2015). Our results suggest that changes in the expression of social play in the offspring is possibly due to the result of developmental retardation and
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neurological changes produced by the gestational cafeteria diet. Moreover, the evaluation of social interactions, which may lead to increases in pleasure and impact future decision-
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making, seems to involve reward-related processing (Izuma et al., 2008). Specific types of social behavior can be rewarding and an association between social approach and reward provides a conceptually powerful mechanism by which approach behaviors can be initiated and maintained (Panksepp and Lahvis, 2007). Thus, the prenatal cafeteria diet could have changed the reward value of peers in the play interaction test.
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Given the data obtained from the offspring, it can be observed that the increase in maternal behavior did not influence the offspring behavior, since the offspring had their behavior impaired even with the increase in maternal behavior. In conclusion, our results demonstrate that exposure to maternal consumption of a cafeteria diet during the gestation period can program developmental and behavioral
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courses in the offspring. Considering that psychiatric diseases are increasingly seen as
developmental disorders, maternal malnutrition could be a contributing factor for
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psychiatric disorders present in the offspring.
Disclosure statement
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Contributors: All authors contributed equally.
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Funding
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This work was supported by Fundação de Amparo a Pesquisa de Minas Gerais
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(FAPEMIG #01483/2013, AG-P) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, #456078/2014-2; FCV). The FAPEMIG and CNPq had no further
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role in the design of the study; the collection, analysis, and interpretation of the data; the
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writing of the report; or the decision to submit the paper for publication.
Conflict of interest
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No potential conflict of interest was reported by the authors. The authors report no biomedical financial interests or potential conflicts of interest. Acknowledgements We are grateful for the excellent technical support of José dos Reis Pereira.
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Figure Legends
Figure 1: Effect of the normal or cafeteria diet during the gestation period on weight gain (grams) of the dams during gestation. Data represent the mean (± S.E.M.) of 10 animals per group. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the normal diet group.
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Data analyzed by two-way repeated measures ANOVA followed by the Bonferroni test.
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Figure 2: Effect of the normal or cafeteria diet during the gestation period on percentage
of maternal behavior (maternal parameters and non-maternal parameters) of lactating mice in postnatal day (PND) 2-PND 8. Each column represents the mean (± S.E.M.) of
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10 animals per group. *p < 0.05 and **p < 0.01 compared to the normal diet group. Data
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analyzed by Student’s t-test.
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Figure 3: Effect of the normal or cafeteria diet during the gestation period on physical and
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reflex developmental parameters in pups: (A and B) male offspring and (C and D) female offspring. Each column represents the mean (± S.E.M.) of 10 animals per group. *p <
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0.05 and ***p < 0.001 compared to offspring of the normal diet group. Data analyzed by Student’s t-test.
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Figure 4: Effect of the normal or cafeteria diet during the gestation period on behavioral performance in the light-dark and open field tests of adolescent male and female offspring: (A and E) latency in seconds to first transition, (B and F) number of transitions,
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(C and G) time in seconds of permanence in the dark, and (D and H) total number of entries in open field test. Each column represents the mean (± S.E.M.) of 10 animals per group. *p < 0.05 and ***p < 0.001 compared to offspring of the normal diet group. Data analyzed by Student’s t-test.
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Figure 5: Effect of the normal or cafeteria diet during the gestation period on play behavior test of adolescence male and female offspring: (A and E) frequency of crawling, (B and F) frequency of pushing, (C and G) time in seconds of following, and (D and H) time in seconds of sniffing. Each column represents the mean (± S.E.M.) of 10 animals per group. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to offspring of the normal
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N
U
SC R
IP T
diet group. Data analyzed by Student’s t-test.
27
28
A ED
PT
CC E
IP T
SC R
U
N
A
M
Table 1: Energy and macronutrient content of the cafeteria diet per 100 grams Protein
Carbohydrats
Lipids
(kJ)
(grams)
(grams)
(grams)
2.260,44
4,8
60
31,2
865,25
20
3,3
20
Potato chips
2.354,63
6,25
45,8
38,8
Bacon
1.611, 61
16,5
0
Wafer biscuit
2.218,58
4,7
63,3
Chow diet
1.391,85
20
36 29
53
4,5
A
N
U
Cheese
SC R
Chocolate
IP T
Energy
M
Table 2: Feed and energy intake of dams during gestation Feed intake
Feed intake
Energy intake
Energy intake
days
(grams)
(grams)
(kJ)
(kJ)
Normal diet
Cafeteria diet
Normal diet
Cafeteria diet
6.59±0.22
80.45±2.76
135.26±5.53***
8.47±0.32
7.88±0.14
105.27±4.76
143.87±7.08***
PT
ED
Gestation
7.04±0.35
6°
7.46±0.32
7.03±0.22
89.69±3.67
128.89±5.73***
9°
7.98±0.22
6.93±0.16
98.67±3.17
135.60±5.93***
12°
8.28±0.25
7.80±0.25
101.08±2.99
151.60±6.01***
15°
8.97±0.52
8.86±0.22
109.47±7.49
161.99±5.99***
18º
10.14±0.31
8.34±0.50
124.74±4.28
140.56±6.96***
1°
A
CC E
3°
29
Values are the mean ± SEM (Student’s t-test) of 10 animals per group. *** p < 0.001 compared to the normal diet group. Data analyzed by two-way repeated measures ANOVA followed by the Bonferroni test.
Table 3: Adipose tissue and biochemical determination in dams of the normal or cafeteria
Normal diet
Cafeteria diet
SC R
Adipose tissue (g/10g b.w.) Retroperitoneal fat
0.08±0.01
0.26±0.01***
Perigonadal fat
0.15±0.01
0.43±0.03***
Triglycerides (mg/dl)
159.20±6.10
Total cholesterol (mg/dl)
72.72±1.63
173.00±6.55*** 183.70±6.49* 80.72±3.18*
A
N
125.50±6.28
U
Biochemical determinations Glucose (mg/dl)
IP T
diet groups at PND1
M
Values are the mean ± SEM (Student’s t-test) of 8 animals per group. * p < 0.05 and ***
ED
p < 0.001 compared to the normal diet group. Data analyzed by Student’s t-test.
PT
Table 4: Body weight, adipose tissue, and biochemical determinations of male and female offspring of the normal or cafeteria diet dams at PND 30 - 32 Offspring cafeteria diet
Male
Female
Male
Female
30.76±0.34
27.47±0.42
31.07±6.36
27.80±0.76
Retroperitoneal fat
0.03±0.00
0.04±0.00
0.04±0.00*
0.07±0.00*
Perigonadal fat
0.07±0.00
0.08±0.00
0.08±0.00*
1.00±0.00*
Glucose (mg/dl)
110.90±4.68
117.40±3.75
133.30±7.29*
146.90±7.52**
Triglycerides (mg/dl)
247.40±15.19
252.80±11.65
237.40±16.66
217.80±14.04
CC E
Offspring normal diet
A
Body weight
Adipose tissue (g/10g b.w.)
Biochemical determinations
30
Total cholesterol (mg/dl)
84.44±2.93
77.32±3.24
97.44±3.68*
87.04±2.29*
Values are the mean ± SEM (Student’s t-test) of 8 animals per group. * p < 0.05 and **
A
CC E
PT
ED
M
A
N
U
SC R
IP T
p < 0.01 compared to the normal diet group. Data analyzed by Student’s t-test.
31