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International Journal of Developmental Neuroscience
journal homepage: www.elsevier.com/locate/ijdevneu
Maternal intake of cashew nuts accelerates reflex maturation and facilitates
memory in the offspring
Marília Ferreira Frazão Tavares de Melo
a
, Diego Elias Pereira
a,⁎
, Morgana Moura Sousa
a
,
Dilian Maise Ferreira Medeiros
a
, Leanderson Tulio Marques Lemos
b
, Marta Suely Madruga
b
,
Nayane Medeiros Santos
a
, Maria Elieidy Gomes de Oliveira
a
, Camila Carolina de Menezes
a
,
Juliana Késsia Barbosa Soares
a
a
Laboratory of Experimental Nutrition, Federal University of Campina Grande, Paraíba, Brazil
b
Department of Food Engineering, Federal University of Paraíba, Paraíba, Brazil
ARTICLE INFO
Keywords:
Fatty acids
Brain development
Lactation
ABSTRACT
Essential fatty acids, being indispensable during the stages of pregnancy, lactation and infancy influence the
transmission of nerve impulses and brain function, and cashew nuts are a good source of these fatty acids. The
objective of this study was to evaluate the effects of cashew nut consumption on reflex development, memory
and profile of fatty acids of rat offspring treated during pregnancy and lactation. The animals were divided into
three groups: Control (CONT), treated with 7% lipid derived from soybean oil; Normolipidic (NL) treated with
7% lipids derived from cashew nuts; and Hyperlipidic (HL) treated with 20% lipids derived from cashew nuts.
Reflex ontogeny, Open-field habituation test and the Object Recognition Test (ORT) were assessed. The profile of
fatty acids in the brain was carried out when the animals were zero, 21 and 60 days old. Accelerated reflex
maturation was observed in animals treated with cashew nuts (p < 0.05). NL presented better memory in the
Open-field habituation test; the NL and HL showed improvement of short-term memory in the ORT, but long
term damage in HL (p < 0.05). The results of the lipid profile of the brain at the end of the experiment showed
an increase in levels of saturated fatty acids and less Docosahexaenoic acid (DHA) in animals of the HL. The data
showed that maternal consumption of cashew nuts can accelerate reflex maturation and facilitate memory in
offspring when offered in adequate quantities.
1. Introduction
Nutrition plays an important role in the body, being responsible for
the maintenance of several essential body functions for life, such as
growth, reproduction and defense (Pang et al., 2012). Nutrients exert a
number of influences on development during pregnancy, lactation and
infancy. In these stages, the body needs proper nutrition so that it can
have satisfactory functioning and development (Allen, 2005).
During this period which is considered critical to development, the
nervous system requires essential nutrients as this is a stage when the
formation of nerve cells, myelination and organization of synapses oc-
curs (Guesry, 1998; Morgane et al., 1993). Thus, the brain becomes
more susceptible to nutritional deficiencies and environmental factors
(Georgieff, 2007).
Nutrients necessaries to the development and maintenance of
neuronal functions are the fatty acids (FA), especially, the poly-
unsaturated fatty acids (PUFA), that are characterized by containing
two or more unsaturations. α-linolenic acid (C18:3 n-3, ALA) and li-
noleic acid (C18:2 n-6, LA) are PUFAs considered essential fatty acids
(EFA). It cannot be synthesized by mammals, and must be obtained
through the diet. N-3 and n-6 are precursor of docosahexaenoic acid
(DHA) and arachidonic acid (ARA) respectively, both are long chain
PUFA (C20-22; LCPUFAs) formed through sequential desaturation,
elongation and partial degradation step (Guillou et al., 2010; Fujii et al.,
2013; Valenzuela et al., 2015; Hatanaka et al., 2016). They are involved
in neurogenesis, synaptic function and psychiatric disorders (Ledesma
et al., 2012;Bazinet and Layé, 2014;Innis, 2008). In this sense, the
quality of lipids in the diet in early life is crucial for neurological de-
velopment, visual development and long-term health (Lauritzen et al.,
2001; Innis, 2008).
http://dx.doi.org/10.1016/j.ijdevneu.2017.06.006
Received 22 June 2017; Accepted 24 June 2017
⁎
Corresponding author.
E-mail addresses: mariliafrazao@hotmail.com (M.F.F.T. de Melo), diegoelias.ufcg@gmail.com (D.E. Pereira), morganamouras@gmail.com (M.M. Sousa),
dilianmaysi@hotmail.com (D.M.F. Medeiros), leanderson_tulio@hotmail.com (L.T.M. Lemos), msmadruga@uol.com.br (M.S. Madruga), nayane.medeiros@hotmail.com (N.M. Santos),
elieidynutri@yahoo.com.br (M.E.G. de Oliveira), camila-carolina@hotmail.com (C.C. de Menezes), julianakessia2@gmail.com (J.K.B. Soares).
International Journal of Developmental Neuroscience 61 (2017) 58–67
Available online 27 June 2017
0736-5748/ © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.
MARK
The main fatty acid involved in membrane phospholipids neuronal
function and visual cells is DHA, particularly during the perinatal and
infant life (Gaete et al., 2002;Valenzuela et al., 2012;Mcnamara et al.,
2010;Bazinet and Layé, 2014). In addition, DHA increases synaptic
plasticity in the hippocampus and improves cognition (Cutuli et al.,
2014; Joffre et al., 2014; Hashimoto et al., 2015).
Several studies have shown that maternal intake of LC-PUFA sources
can improve maturation reflex (Santillán et al., 2010), memory (Ferraz
et al., 2011) and behavior (Soares et al., 2013) in the offspring of an-
imals in several experimental models.
An oleaginous source of LC-PUFA is cashew nuts (Anacardium oc-
cidentale L.) (Akinhanmi, Atasie and Akintokun, 2008). Cashew nuts are
the true fruit of the cashew tree, which are native to North and
Northeast Brazil (Johnson, 1973). It has important nutritional char-
acteristics for the proper functioning of the body since it contains
proteins, lipids, carbohydrates, phosphorus, iron, zinc, magnesium,
polyphenols, tocopherols, phytosterols, squalene, arginine and fiber in
its composition. Cashew nuts have significant levels of saturated (pal-
mitic, stearic, arachidic, behenic and lignoceric), monounsaturated
(oleic and gadoleic) and polyunsaturated FA (α-linoleic and α-lino-
lenic). When roasted, the cashew nut has an elevation of about 50% in
the total lipid content as compared to raw nuts, being 46.28%; n-9 oleic
acid is found in higher amounts (about 68.2–80.4%), with n-6 linoleic
acid at about 20%, and n-3 linolenic acid in less quantity (Akinhanmi
et al., 2008; Venkatachalam and Sathe, 2006; Nagaraja, 1987).
Given the large number of studies that show cognitive improvement
of offspring by maternal consumption of rich LC-PUFAs sources and the
lack of results for the effects of cashew nuts on the CNS, the main ob-
jective of this study was to investigate the effects of cashew nuts in
different concentrations in the diet offered to mothers during pregnancy
and lactation on their offspring’sreflex development and memory, as
well as to assess the profile of fatty acids at different life stages of these
animals.
2. Materials and methods
2.1. Animals and diet
In this study, primiparous Wistar rats at 90 days of age and ap-
proximately 230 g were used, originating from the breeding vivarium of
the Federal University of Pernambuco and kept at the Experimental
Nutrition Laboratory of the Federal University of Campina Grande –
Cuité Campus –Paraiba in a temperature controlled environment of
22 ± 1 °C under 12/12 h light/dark cycle (beginning of the light cycle
at 6:00), humidity ± 65%, with free access to water and feed. The
females were mated and kept in the ratio of two females per male to
obtain lactating rats. After confirmation of pregnancy by vaginal swab,
animals were allocated into individual polypropylene maternity cages
(60 cm long, 50 cm wide and 22 cm high) and received the experi-
mental diet from the first day of gestation until the end of lactation.
After birth, the offspring were standardized to 6 pups, preferably males.
Female pups were used to complete the litter when there was not en-
ough male pups. Only male pups were used to analyse data.
Three groups were formed: Control group (CONT) –Offspring
whose mothers received control diet based on soy oil (7% lipids);
Normolipidic group (NL) –Offspring whose mothers received normo-
lipidic diet containing 7% lipids from cashew nuts; Hyperlipidic group
(HL) –Offspring whose mothers received a high fat diet containing 20%
lipids from cashew nuts. Dams were weighed in the end of lactation and
neonates were weighed weekly, from the first day of life until weaning.
Cashew nuts used for manufacture of experimental diets belonged to
the Anacardium occidentale L species from the city of Caicó/RN, Brazil
(Table 1).
The experimental diets followed the recommendations of the
American Institute of Nutrition AIN-93G (Reeves et al., 1993). The diet
was manufactured by the company Rhoster (São Paulo/Brazil), being
stored under refrigeration. Table 2 discribes diet composition.
After weaning, the offspring started receiving maintenance feed
(Presence
®
) until the end of the experiment.
The experimental protocol followed the ethical recommendations of
the National Institute of Health Bethesda (Bethesda, USA) regarding
animal care, and was approved by the Ethics Committee for Animal Use
of the UFCG under certification number 108–2013 (Fig. 1).
2.2. Breast milk collection
Breast milk was collected according to the adapted methodology
described by Keen et al. (1981). Mothers were separated from their
respective offspring on the 21 st day of lactation one hour before the
procedure. The animals were anesthetized intramuscularly with xyla-
zine hydrochloride (20 mg/kg) and ketamine hydrochloride (50 mg/
kg) and received intraperitoneal injection with 3 IU of oxytocin. Nipple
massage to stimulate milk ejection was made. The milking was done
manually and the milk was housed in eppendorf and frozen for further
analysis.
2.3. Reflex development
Reflex responses were checked daily between 12 pm and 2 pm, from
the 1st to the 21st day after birth. The response was considered con-
solidated when the expected reflex reaction was repeated for three
consecutive days, considering the consolidation day as the 1st day of
appearance. Studied reflexes followed the experimental model estab-
lished by Smart and Dobbing (1971),Table 3. The maximum observa-
tion time considered was 10 s, timed by a KK-2808 Kenko hand digital
timer.
2.4. Behavioral tests
2.4.1. Open-field habituation test
The animals (35 days) were tested in an open field device consisting
of a circular arena (1 m in diameter) closed by white walls (50 cm
high), divided into 17 fields. Each animal was individually placed in the
Table 1
Fatty acids composition of cashew nut.
Fatty acids Cashew nut (%) Standard deviation
Myristic acid C14:0 0,49 0,01
Myristoleic acid C14:1 0,04 0,01
Palmitic acid C16:0 8,86 0,05
Cis-7 hexadecenoic acid C16:1n9c 0,07 0,00
Palmitoleic acid C16:1n7c 0,40 0,01
Heptadecanoic acid C17:0 0,11 0,01
Cis-10-heptadecenoic acid C17:1n7c 0,02 0,00
Stearic acid C18:0 7,02 0,07
Oleic acid C18:1n9c 59,19 0,04
Linoleic acid C18:2n6c 23,28 0,16
Table 2
Diet Composition.
Ingredients (g/100 g) Control diet Normolipidic diet Hiperlipidic diet
Cashew nut –16.64 42.43
Soybean oil 7.00 ––
Casein 20.00 16.87 11.06
Sucralose 10.00 10.00 10.00
Cornstarch 52.95 48.23 31.11
Fiber 5.00 4.38 0.35
Minerais mix 3.50 3.50 3.50
Coline 0.25 0.25 0.25
L-cystine 0.30 0.30 0.30
Vitamins mix 1.00 1.00 1.00
M.F.F.T. de Melo et al. International Journal of Developmental Neuroscience 61 (2017) 58–67
59
center of the device for 10 min for ambulation observation. The pro-
cedure was repeated after seven days. The device was sanitized with a
solution of 10% alcohol after each behavior session. The sessions were
recorded with a video camera installed on the ceiling (Leussis and
Bolivar, 2006; Gamberini et al., 2015).
2.4.2. Object recognition test
The object recognition test was performed 3 days after the open
field habituation test, using the same open field arena.
This test consists of 4 steps: habituation to the experimental arena,
training session, test session and retest session. During habituation, the
animals freely explored the arena for 5 min in the absence of any spe-
cific stimulus. In the training session, animals were placed in the arena
containing two different objects (A1 and A2) for free exploration for
10 min, for the animal to recognize and identify the A1 object as fa-
miliar. After 180 min, a testing session took place to evaluate short-
term memory, in which the animals were placed in the arena containing
A1 (familiar object) and A3 (novel object) to explore freely for 5 min.
After seven days, the animals were subjected to retesting in order to
assess long-term memory. The animals were placed in the arena to
freely explore A1 (familiar object) and A4 (novel object) objects.
Exploration was defined as sniffing or touching the object with their
nose and/or front legs (Rachetti et al., 2013)(Fig. 2).
The objects and equipment were cleaned with 10% alcohol after
each animal exposure to the arena. The objects used were suitable for
pets. The sessions were filmed with a video camera installed on the
ceiling. After behavioral analysis, animals were anesthetized and the
brains were carefully removed. Results were analyzed as the total time
spent exploring the objects and the novel object/total familiar + novel
object ratio (Gustavsson et al., 2010).
2.5. Determination of fatty acid profile in the brain
2.5.1. Brain collection of the offspring
To extract the brain in the day of birth, the neonates were decapi-
tated and the others animals were intramuscularly anesthetized with
xylazine chloridrate (20 mg/kg) and ketamine chloridrate (50 mg/kg).
Soon after, the brains were removed and placed in sterilized collecting
jars and then stored in a freezer at −20 °C until analysis.
Three collections were carried out: on the day of birth, on the day of
weaning (21 days) and when the animals reached 60 days old.
2.5.2. Lipidic extraction
2 g of each sample were weighed in a 50 ml beaker (wet sample)
and added to 30 ml of chloroform:methanol mixture (2:1). After this
addition, the content was transferred to a deep glass container with the
side covered with aluminum foil and stirred for 2 min with the help of
grinder. The triturate was filtered through qualitative filter paper into a
100 ml graduated cylinder with a polished mouth. Next, the vessel
walls were washed with an additional 10 ml of chloroform:methanol
which was also filtered with the previous volume. The volume of the
filtered extract of the graduated cylinder was recorded with the grad-
uated cylinder closed. 20% of the final volume of the filtered extract
was added to 1.5% sodium sulfate. Then, the mixture was stirred with
the graduated cylinder closed and given time for the phases to separate.
It was observed that the upper phase was approximately 40% and the
bottom 60% of the total volume. The volume of the lower phase was
recorded and then the upper phase was discarded by suction with a
graduated pipette. For lipid quantification, an extract aliquot of 5 ml
(lower phase) was separated with a volumetric pipette and transferred
to a previously weighed beaker. This beaker was placed in an oven at
105 °C so the solvent mixture could evaporate, being careful that the fat
would not be degraded by heat. After cooling in a desiccator, the beaker
was weighed and the fat residue weight was obtained from the differ-
ence (Folch et al., 1957).
2.6. Fatty acids methylation
In the sample treatment, methylation of fatty acids present in the
lipid extract was carried out following the methodology described by
Hartman and Lago (1973). An aliquot of the lipid extract was taken,
calculated for each sample according to the fat content found in the
lipid measurement, and performed according to the Folch, Less and
Stanley method (1957), adding 1 ml of internal standard (C19:0) and a
saponification (KOH) solution. This solution was subsequently brought
to heating under reflux for 4 min. Esterification solution was added
Fig. 1. Outlines the experimental protocol specifying the days that the
procedures were performed.
Experimental protocol. Time sequence (days) of experiments conducted
with the offspring of Wistar rats treated during pregnancy and lactation
with control diet (CONT), normolipidic diet with cashew nuts (NL) or
hyperlipidic diet with cashew nuts (HL). (*) Days of brain collection for
the lipidic profile analysis.
Table 3
Description of the reflex test.
Reflex Stimulus Response
Palmar grasp (PG) Light percussion on the palm of the right foreleg Quick bending of ankles.
Righting (RR) The rat is placed in supine position on a surface. Return to the prone position with all paws in 10s.
Cliff-avoidance (CA) The rat is placed on a flat and high surface (table), with legs towards
the extremity.
Moves to one side and walks in the opposite direction to the edge
Vibrissa-placing (VP) The animal is suspended by the tail and its vibrissae lightly touch the
edge of a flat surface.
Both front legs are placed on the table, performing march movements.
Negative-geotaxis (GN) The rat is placed at the center of an inclined ramp with head facing
downwards
Body spin at an angle of 180°, positioning head upwards.
Auditory-startle response (AS) Intense and sudden sound stimulus Retraction of anterior and posterior legs, with rapid and involuntary
body immobilization
Free-fall righting (FFR) Held by the four legs, at a height of 30 cm, it is released in free fall on
a synthetic foam bed.
Position recovery during freefall on the surface supported on four paws.
M.F.F.T. de Melo et al. International Journal of Developmental Neuroscience 61 (2017) 58–67
60
immediately after, returning the solution to heating under reflux for 3
more minutes. Next, the sample was allowed to cool before subsequent
washings with ether, hexane and distilled water, finally obtaining an
extract (with the methyl esters and solvents), which was conditioned
into a properly identified amber glass until complete drying of the
solvents. After drying, a suspension in 1 ml of hexane was made and
packaged into a vial for further chromatographic analysis. The aliquots
of saponification and esterification solutions were determined ac-
cording to the methodology described by Hartman and Lago (1973).
2.6.1. Gas chromatography analysis
A gas chromatograph (VARIAN 430-GC, California, EUA), coupled
to a capillary column of fused silica (CP WAX 52 CB, VARIAN,
California, EUA) with dimensions of 60 m × 0.25 mm and 0.25 mm
film thickness was used with helium as carrier gas (Flow rate of 1 ml/
min). The initial oven temperature was 100 °C programmed to reach
240 °C, increasing 2.5 °C per minute for 30 min, totaling 86 min. The
injector temperature was maintained at 250 °C and the detector at
260 °C. 1.0 μl aliquots of esterified extract were injected in a Split/
Splitless injector. The chromatograms were recorded using Galaxie
Chromatography Data System software.
Fatty acids were identified by comparing retention times of the
methyl esters of the samples with Supelco Mix C4-24/C19 standards.
The fatty acids results were quantified by normalizing the areas of the
methyl esters and are expressed in percentage by area.
2.7. Statistical analyses
Reflex maturation and memory were analyzed with one-way Anova
followed by Kruskal–Wallis test. For the rest of the data, One-Way
ANOVA followed by the Tukey test was used. Differences were con-
sidered significant when p < 0.05. The Sigma Start software was used
for data analysis.
3. Results
3.1. Body weight
In the end of the lactation, the body weight of HL (161,25 ± 16,77)
was increased compared with CONT (242,94 ± 22,45) and NL
(266,00 ± 21,81) (p < 0.05).
Analyzing offspring body weight, data showed that HL had de-
creased on days 1, 7 and 14 compared to CONT and in day 21 versus
CONT and NL (p < 0.05)(Fig. 3).
3.2. Reflex maturation
Regarding ontogeny of reflex responses, acceleration of reflexes was
found in PG, VP, CA, GN, AS, FFR of the NL in relation to CONT
(p < 0.05). In comparing HL to NL, we found a delay in the dis-
appearance of PG and RR and AS (p < 0.05). Our results also showed
Fig. 2. The figure (A) represents the habituation
session on the open field. The animals were placed in
the central area to explore the environment. The
number of crossings between the open field areas
were recorded. Each session lasted 3 min. In the
figure (B) was demonstrated how the object re-
cognition task was performed. The animals were
exposed to the arena on the open field containing 2
objects (A1- familiar and A2- unfamiliar). The time
of exploration was recorded. It was made during
10 min. Figure (C) demonstrates the short-term ob-
ject recognition test. Three hours after the first
training session the animal was inserted into the
open field. The family object A1 was maintained and
an unfamiliar (A3) object was changed. The time
needed was 5 min. Figure (D) –demonstrated how
the task of object recognition for long-term memory
was performed. Seven days after the short term
memory test the animals were inserted into the open
field. The familiar object A1 was kept and a new
object was inserted (A4- unfamiliar object). The time
required for the test was 5 min.
Fig. 3. Body weight of pups rats treated during pregnancy and lactation. Data were ex-
pressed as mean ± SEM. * = versus control group (CONT); # = versus normolipid
group (NL); Hyperlipidic group with cashew nuts (HL).
M.F.F.T. de Melo et al. International Journal of Developmental Neuroscience 61 (2017) 58–67
61
acceleration in the day of PG disappearance and emergence of CA in
neonates of the HL compared to CONT (p < 0.05) (Table 4). HL pre-
sented delay compared to NL in PG, RR and AS reflexes (p < 0.05).
3.3. Open-field habituation test
Ambulation parameter analysis in the Open-field habituation test
showed a statistically significant difference for the NL group between
the 1st and 2nd exposures (101.80 ± 8.78 and 66.30 ± 7.43) (Fig. 4).
3.4. Object recognition test
The exploration ratio was higher in NL and HL compared with
CONT during the short term (p < 0.05). For the longer term, ex-
ploration time was decreased in HL compared to both CONT and NL
(Fig. 5).
3.5. Fatty acids composition in breast milk
Analyzing the composition of breast milk was observed that the
total of myristic acid were decreased in both groups treated with
cashew nuts (p < 0.05). By the other hand, stearic acid was higher in
HL compared to CONT. The total of saturated fatty acids in HL com-
pared to CONT was 68%.
The oleic and eicosenoic acid (MUFA) were increased in NL versus
Control group. HL showed decreased levels of palmitoleic compared
with CONT and oleic acid compared to all groups (p < 0.05). The total
of monounsaturated fatty acids in NL was 39% higher and HL was
41,12% lower compared to CONT.
The total of PUFA in breast milk was higher in HL (56%) compared
to Control group. Dihomo gamma linoleic, eicosatrienoic, arachidonic
acids and DHA were increased compared to CONT (p < 0.05) Table 5.
3.6. Fatty acids composition of offspring brain on the day of birth
We analyzed offpring brain fatty acids composition in the first day
of life. Data showed that NL had increased pentadecanoic and hex-
acoisanoic acid compared to CONT (p < 0.05). HL presented decrease
in myristic, palmitic and stearic acid compared to CONT (p < 0.05).
The total of MUFA was 25% higher in HL compared with CONT, and
34% compared to NL. Oleic acid (C18:1) was increased in HL compared
to all groups (p < 0.05).
Level of linoleic acid was higher in HL compared to NL (p < 0.05).
DHA was lower in both groups treated with cashew nuts compared to
CONT (p < 0.05). NL and HL showed that total of PUFA in the brain of
the newborn in the day of life was 8% and 16% lower respectively
compared to CONT (Table 5).
3.7. Fatty acids composition in offspring brain after weaning (21 days of
life)
Evaluating the levels of saturated fatty acids in the brain of the rats
at 21 days of life, we observed that levels of palmitic, stearic and hex-
acoisanoic acids of NL and HL were decreased compared to CONT, but
arachidic acid was higher (p < 0.05). However, the total of saturated
fatty acids between the all groups were similar, as shown in Table 5.
NL and HL showed about 20% more total of MUFA than CONT. The
main fatty acid increased was oleic acid (p < 0.05).
Total of PUFA were decreased in NL (8%) and HL (16%) compared
to CONT. DHA was lower in both groups treated with cashew nuts
(p < 0.05) Table 5.
3.8. Fatty acids composition of brain in adulthood
The saturated fatty acids contained in the brain of adults rats which
mothers were treated with cashew nuts were increased in myristic,
palmitic and hexacoisanoic acids and decreased in steric and arachidic
acids (p < 0.05). The total of saturated fatty acids in HL was about
14% higher than CONT and NL.
The total of MUFA in the brain of HL was higher than NL and CONT
about 21%. Palmitoleic acid was increased in HL compared to the
others groups and oleic and eicosenoic acids were decreased
(p < 0.05).
The fatty acids eicosadienoic and eicosatrienoic were higher than
NL and CONT. DHA was decreased in HL compared to the others groups
(p < 0.05).
4. Discussion
In the current study, the results indicate that the consumption of a
normolipidic diet with cashew nuts as the main source of LC-PUFA
during pregnancy and lactation was able to influence the development
of certain reflex responses in newborns and improve performance on
memory tasks in adolescent rats. However, the hyperlipidic diet with
cashew nuts did not promote such beneficial long-term effects, de-
monstrating that both the quantity and quality of lipids exert influence
on animal development.
Data showed significant advancing effects in reflex maturation in
both groups treated with cashew nuts. During early life, the nervous
system has a growth spurt and lipids are directly involved in its proper
functioning (Morgane et al., 1993;; Salvati, 1996). Offspring received
LC-PUFA primarily through the placenta and then through breast milk
during lactation. The deficiency of fatty acids such as DHA at this stage
Table 4
Reflex ontogenesis of neonates rats whose mothers received a ration with 7% from soy-
bean oil (n = 19), 7% from cashew nuts (n = 14) and 20% from cashew nuts (n = 11)
during pregnancy and lactation. Data expressed as median values (minimum and max-
imum). Considering: ª = Day of response disappearance and
b
= Day of response ap-
pearance. Groups: CONT (Control group), NL (Normolipidic group), HL (Hyperlipidic
group). The Mann-Whitney Rank Sum Test with a significance level of P < 0.001 was
used for statistical analysis. * versus control group;
#
versus normolipidic group.
Reflexes Groups
CONT NL HL
Palmar Grasp
a
8(6–10) 4 (2–5)* 6 (4–7)*
#
Righting Reflex
b
3(1–10) 3 (1–5) 3 (2–8)
#
Vibrissa Placing
b
11 (8–12) 8,5 (5–10)* 10 (9–11)
CliffAvoidance
b
10 (6–13) 6 (4–11)* 7 (6–11) *
Negative Geotaxis
b
15 (10–19) 14 (11–21) 13 (11–17)
Auditory Startle
b
12 (11–15) 11 (10–12)* 12 (12–14)
#
Free-Fall Righting
b
16 (13–19) 14 (13–16) 14 (12–17)
Fig. 4. Influence of maternal diet containing cashew nuts on total ambulation in the
Open-field habituation test of rat offspring. Values are expressed as mean ± SD. CONT
Group: treated with 7% soybean oil; NL Group: treated with 7% of cashew nuts as lipidic
source; HL Group: treated with 20% of cashew nuts as lipidic source. One way ANOVA
followed by Kruskal Wallis test. *p < 0.05 versus 1st exposure to the open field.
M.F.F.T. de Melo et al. International Journal of Developmental Neuroscience 61 (2017) 58–67
62
may induce delayed visual development, cognitive impairment and
damage the cerebellum structure (Haag, 2003).
Reflex maturation is related to adequate visual and motor devel-
opment (Allam and Albo-Eleneen, 2012) which depends on neurogen-
esis, myelination, synapses and neurotransmitter action (Schiefermeier
and Yavin, 2002;; Morgane at al., 1993). In the present study, DHA
(C22: 6) was present in the brain tissue of all animals, both at the end of
pregnancy and during lactation. DHA usually is deposited in the cere-
bral membranes and is related to the functioning of the vision and
cellular signaling. Its accumulation in the membranes occurs mainly in
early life (Calder, 2016). Although found less in animals treated with
cashew nuts compared to the control group, these levels along with
other fatty acids were able to induce an acceleration in the reflex ma-
turation of offspring. This acceleration was more pronounced in NL
than in HL. In addition, essential FA in the diet, including myristic,
palmitoleic, stearic, and oleic acids are involved in myelin synthesis in
the brains of neonatal rats and the mediators responsible for this in-
creased myelin synthesis are also associated with accelerated reflex
maturation (Salvati et al., 1996; Soares et al., 2014). SFA levels in the
brain of the offspring at the end of lactation was similar between the
investigated groups; however, in the weaning, the levels of MUFA in the
animals treated with the cashews were about 20% higher than those
from the control group. These findings suggest that MUFA levels can
also directly interfere in reflex development. A study assessed the effect
of maternal consumption of olive oil (a source of MUFA) and found
reduced oxidative damage in the brain tissue of the offspring and im-
proved neurotrophic gene factor expression in rats treated during
pregnancy, lactation and after weaning (Pase et al., 2015). This re-
duction in oxidative damage protects brain tissues and helps maintain
mental health (Haag, 2003).
The maternal diet with cashew nuts provided a variety of fatty acids
that allowed for excellent formation of the nervous system in the off-
spring as shown by the results. Accelerated reflex maturation was ob-
served in the animals treated during lactation with goat milk, which is
also a source of different fatty acids (Soares et al., 2014). An opposing
result was observed in a study using a maternal diet containing Buriti
oil, showing retardation in reflex maturation (Medeiros et al., 2015).
Another work using soy oil and sunflower oil found even more re-
tardation and acceleration in investigated reflex maturation (Santillán
et al., 2010). An inadequate amount of maternal FA in the diet, or in
excess or deficiency may result in changes in the proportion of satu-
rated and unsaturated FA in the body, leading to alterations in brain
development (Soares et al., 2009). Supplementation with arachidonic
acid in newborn mice induced the increase of this fatty acid in the brain
of these animals and improved their motor activity (Hatanaka et al.,
2016). However, arachidonic acid present in breast milk was not de-
posited in the brain tissue either during pregnancy or after birth.
Therefore, it is important that both the quantity and the quality of FA
are balanced in the diet.
In addition to investigating the neurological development of new-
borns, the present study also evaluated the influence of cashew nuts on
memory tasks, as adequate amounts of LC-PUFA provide integrity of
cell membranes and form myelin on neurons and glia cells, being fun-
damental to ensure normal brain function in individuals for learning
and cognition (Yehuda et al., 2006; Soares et al., 2009). Memory ac-
quisition is a process involving molecular and cellular events activated
immediately after the training session and after a few hours to a few
days of the task, occurring in specific brain regions such as the hippo-
campus (Bekinschtein et al., 2010; Medina et al., 2008; Bach et al.,
2014). Neurogenesis especially occurs in the dentate gyrus of the hip-
pocampus, where recently formed neurons are recruited to pre-existing
neural circuits, being involved in the hippocampus-dependent learning
processes (Drapeau et al., 2003; Fernandes et al., 2011). Thus, a defi-
ciency in FA composition in the maternal diet is associated with cog-
nitive impairment and mental illnesses such as Alzheimer's disease,
attention-deficit hyperactivity disorder (ADHD), depression and schi-
zophrenia (Haag, 2003; Ozias et al., 2007; Mucci et al., 2015).
Molecular mechanisms through which LC-PUFA affect the memory
of animals has not yet been fully clarified, but it is believed that the
glutamatergic system is involved in hippocampus-related improvement
of cognitive function caused by DHA (Cao et al., 2009) through the
activation of N-methyl-D-aspartate glutamate receptors (NMDA) which
are involved in the induction of long- term potentiation (LTP) and brain
derived neurotrophic factor (BDNF) synthesis (Collingridge et al., 2013;
Bekinschtein et al., 2007; Bach et al., 2014). Other evidence also sug-
gests that the cholinergic system is involved in this LTP modulation
process and synaptic plasticity, as the AA and DHA increase the release
of acetylcholine and improve learning ability in experimental animals
(Das, 2003). AA was not found in rat brain from birth until 60 days of
life in the presente work. However, eicosatrienoic acid (C20:3n3) was
found. Some fatty acids as oleic acid (presented in cashew nut diet),
may be converted by the action of the enzyme Δ6 dessaturase in eico-
satrienoic acid (Smit et al., 2004; Rivers and Frankel, 1981). These finds
could explain the presence of this fatty acid in the breast milk brain of
these animals.
Two methods that assess cognitive performance of animals (ado-
lescent rats) were used in this study due to the importance of the brain
in animals’cognition and behavior, being the Open-field habituation
test (OFH) and the Object Recognition Test (ORT).
The Open-field habituation test measures non-associative learning
ability through long-term animal habituation. This is represented by
decreased locomotor activity resulting from repeated exposure of the
animal to the same environment, which is seen as a memory facilitation
index (Rachetti et al., 2013). Our results showed that maternal con-
sumption of cashew nuts induced habituation in the open field in the
normolipidic group, similar to that found in the study by Rachetti et al.
(2013), in which the animals received diets with fish oil as a source of
LC-PUFA. However, this effect was not observed in the HL, corrobor-
ating the results obtained by Venna et al. (2009), where animals treated
with high LC-PUFA concentration diet showed no significant difference
in locomotor activity. These findings can be explained by the profile of
fatty acids in the brains of the analyzed offspring. It can be observed
that there was a similarity in the PUFA levels at the end of experiment
period when these tests were performed. However, the amounts of DHA
and MUFA were similar between CONT and NL in the end of the ex-
periment, but lower in HL. These data demonstrate that both DHA and
MUFA levels directly interfere in the development of animal memory as
mentioned in literature (Pase et al., 2015), and that pregnant and
Fig. 5. Influence of cashew nuts on short and long term memory in the
exploration time/rate of the Object Recognition Test in rats. Values are
expressed as mean ± SD. CON = control group (n = 17);
NL = normolipidic group treated with 7% of cashew nuts (n = 15);
HL = hyperlipidic group treated with 20% of cashew nuts (n = 9). One-
way ANOVA followed by Kruskal Wallis test. * = versus control group; **
versus all groups.
M.F.F.T. de Melo et al. International Journal of Developmental Neuroscience 61 (2017) 58–67
63
Table 5
Fatty acids composition of breast milk and cerebral tissue of offspring treated during gestation and lactation with 7% of soybean oil (CONT), 7% of cashew nuts (NL) and 20% of cashew nuts (HL).
FATTY
ACIDS
BREAST MILK BRAIN –1 st DAY OF LIFE BRAIN –21 st DAY OF LIFE BRAIN –60th DAY OF LIFE
NO. OF
CARBO-
N ATOM
CONTROL NL HL CONTROL NL HL CONTROL NL HL CONTROL NL HL
SATUR-
AT-
ED
Myristic C14:0 2.95 ± 0.01a 1.92 ± 0.02b 0.37 ± 0.01c 1.82 ± 0.01a 1.75 ± 0.02a 1.66 ± 0.02b 0.54 ± 0.01a 0.45 ± 0.02a 0.36 ± 0.01b 0.23 ± 0.02a 0.14 ± 0.01b 0.36 ± 0.03c
Pentad-
eca-
noic
C15:0 0.16 ± 0.02a 0,20 ± 0.01a 0.00 a 0.11 ± 0.01a 1.93 ± 0.01b 0,00 ac ––– – ––
Palmitic C16:0 24.91 ± 0.03a 22.26 ± 0.01a 24.31 ± 0.01a 32.56 ± 0.03a 32.02 ± 0.02a 29.58 ± 0.01b 28.19 ± 0.02a 25.08 ± 0.01b 24.40 ± 0.02b 22.07 ± 0.02a 21.41 ± 0.03a 24.40 ± 0.0-
3b
Stearic C18:0 4.37 ± 0.02a 4.65 ± 0.01a 19.93 ± 0.02b 17.28 ± 0.03a 16.60 ± 0.02a 15.49 ± 0.01b 22.25 ± 0.02a 20.17 ± 0.02b 19.91 ± 0.01b 22,06 ± 0.03a 21,13 ± 0.03a 19,93 ± 0.0-
3c
Arachid-
ic
C20:0 0.08 ± 0.01a 0.10 ± 0.01a 0.27 ± 0.02a –––0.00a 0.34 ± 0.02b 0.28 ± 0.01ab 0,49 ± 0.01a 0,43 ± 0.01a 0,28 ± 0.01b
Behenic C22:0 2.45 ± 0.03a 1.62 ± 0.01a 0.00a ––––0.12 ± 0.02 ––––
Hexaco-
isa-
noic
C23:0 –– 6.22 ± 0.01 3.50 ± 0.02a 6.45 ± 0.01b 6.29 ± 0.02b 1.70 ± 0.02a 6.06 ± 0.03b 6.26 ± 0.02b 0,00 a 2,52 ± 0.02b 6,25 ± 0.02c
TOTAL 34,90 30,73 51,10 55,27 58,75 53,01 52,68 52,24 51,20 44,85 45,62 51,22
MONO-
UN-
SA-
TU-
RA-
TED
Tetrade-
cen-
oic
C14:1 0.10 ± 0.01a 0.07 ± 0.01a 0,00a –0.11 ± 0.01 ––––0,00 a 2.72 ± 0.02b 2.80 ± 0.03b
Pentad-
ece-
noic
C15:1 –– 2.79 ± 0.03 1.72 ± 0.02a 0,00b 1.87 ± 0.02a 2,16 ± 0.02a 3,03 ± 0.02b 2,80 ± 0.02ab 2.31 ± 0.02a 0,00b 0,00b
Palmito-
leic
C16:1 4.24 ± 0.02a 4.09 ± 0.01a 0.43 ± 0.01b 1.78 ± 0.03a 1.61 ± 0.01ab 1.52 ± 0.01b 0,56 ± 0.01a 0,47 ± 0.01ab 0,43 ± 0.01b 0.33 ± 0.01a 0.34 ± 0.01a 0.43 ± 0.02b
Oleic C18:1n9 33.06 ± 0.03a 47.88 ± 0.02b 18.41 ± 0.01c 15.33 ± 0.01a 15.77 ± 0.02a 19.88 ± 0.01b 16,00 ± 0.03a 19,15 ± 0.03b 18,41 ± 0.03b 23.51 ± 0.04a 22.82 ± 0.02a-
b
18.37 ± 0.0-
2c
Eicosen-
oic
C20:1n9 0.36 ± 0.02a 0.66 ± 0.01b 0.69 ± 0.01b ––0.29 ± 0.03 ––0.68 ± 0.01 2.14 ± 0.02a 2.10 ± 0.02a 0.71 ± 0.01b
TOTAL 37,92 52,89 22,33 18,83 17,49 23,55 18,72 22,65 22,33 28,29 27,97 22,32
POLYU-
NS-
AT-
UR-
AT-
ED
Linoleic C18:2n6 14.42 ± 0.03a 9,75 ± 0.03a 1,37 ± 0.02b 1,20 ± 0.01ab 0,80 ± 0.01a 1,70 ± 0.01b 1,14 ± 0.02ab 1,02 ± 0.01a 1,37 ± 0.02b 0,86 ± 0.01ab 0,79 ± 0.01a 1,36 ± 0.01b
Gamma
Lin-
ole-
nic
C18:3n6 0,14 ± 0.01a 0,09 ± 0.01ab 0,00b –––0,19 ± 0.01 –––––
Alpha C18:3n3 0,76 ± 0.02a 0,10 ± 0.01ab 0,00b – ––––––––
(continued on next page)
M.F.F.T. de Melo et al. International Journal of Developmental Neuroscience 61 (2017) 58–67
64
Table 5 (continued)
FATTY
ACIDS
BREAST MILK BRAIN –1 st DAY OF LIFE BRAIN –21 st DAY OF LIFE BRAIN –60th DAY OF LIFE
NO. OF
CARBO-
N ATOM
CONTROL NL HL CONTROL NL HL CONTROL NL HL CONTROL NL HL
Lin-
ole-
nic
Eicosad-
ie-
noic
C20:2n6 0,72 ± 0.01a 0,33 ± 0.02a 4,49 ± 0.03c 3,45 ± 0.02a 3,44 ± 0.01a 2,85 ± 0.02b 3,63 ± 0.02a 3,86 ± 0.02a 4,51 ± 0.02 3,44 ± 0.02a 3,89 ± 0.02a 4,52 ± 0.02b
Dihomo
Ga-
m-
ma
Lin-
ole-
ic
C20:3n6 0,15 ± 0.02a 0,15 ± 0.01a 0,43 ± 0.01b 12,13 ± 0.03a 13,16 ± 0.03a 0,33 ± 0.01b 12,18 ± 0.03 13,40 ± 0.03 0,44 ± 0.01a 0,39 ± 0.01a 0,44 ± 0.01a
Eicosat-
rie-
noic
C20:3n3 0,61 ± 0.03a 0,77 ± 0.02a 13,41 ± 0.03b ––11,72 ± 0.03 –12,48 ± 0.03 –9,82 ± 0.02a 10,56 ± 0.03a-
b
13,41 ± 0.0-
3b
Arachid-
onic
C20:4 0,38 ± 0.01a 0,12 ± 0.01b 0,00b – ––––––––
Docasa-
He-
xae-
noic
C22:6n3 0,10 ± 0.02a 0,24 ± 0.02a 6,71 ± 0.02b 9,12 ± 0.02a 6,36 ± 0.02b 5,04 ± 0.02b 11,00 ± 0.03a 6,26 ± 0.02b 6,72 ± 0.02b 10,49 ± 0.03a 10,77 ± 0.03a 6,73 ± 0.02b
TOTAL 16,91 11,44 26,42 25,90 23,76 21,64 28,15 23,61 26,01 25,05 26,40 26,46
AGP/
AGS
0,48a 0,37ab 1,93b 0,47 0,40 0,41 0,53 0,45 0,51 0,56 0,58 0,52
N6 15,43a 10,32ab 6,29b 16,78a 17,40a 4,88b 17,14a 4,88b 19,28a 4,74a 5,07ab 6,32b
N3 1,47a 1,11a 20,12b 9,12ab 6,36a 16,76b 11,00ab 18,74a 6,72b 20,31a 21,33a 20,14a
N9 33,42ab 48,54b 19,11a 15,33a 15,77a 20,17b 16,00a 19,15b 19,09b 25,65a 24,92a 19,08b
N6/N3 10,49a 9,29a 0,31b 1,83a 2,73ab 0,29b 1,55a 0,26ab 2,86b 0,23 0,23 0,31
M.F.F.T. de Melo et al. International Journal of Developmental Neuroscience 61 (2017) 58–67
65
lactating animals treated with larger quantities of cashew nuts in the
diet induce damage to the memory of their offspring.
The Object Recognition Test was carried out in order to assess short
and long-term declarative memory of the animals (Ennaceur and
Delacour, 1988; Bruin and Pouzet, 2006; Kamei et al., 2006; Aisa et al.,
2007; Vickers et al., 2009), which consists of a non-rewarded task based
on the natural tendency of rodents to explore a new object more in-
tensely than a familiar object in a familiar area, thus indicating re-
cognition memory (Ennaceur and Delacour, 1988; Nava-Mesa et al.,
2013; Barbosa et al., 2013). This task depends on the proper func-
tioning of the hippocampus (Mumby et al., 2005), the perirhinal cortex
(Winters and Bussey, 2005), and the insular cortex (Bermudez-Rattoni
et al., 2005).
This study showed that the normolipidic and hyperlipidic groups
showed a higher rate of object exploration for short term memory than
animals treated with soy oil. However in the long term, the normo-
limpidic group was similar to the control group and only HL showed a
decrease in such exploration. These results coincide with what Rachetti
et al. (2013) found in their experiment of offering fish oil during
pregnancy, finding that the group receiving fish oil showed better
performance when subjected to the object recognition test. Also,
Fernandes et al. (2011) proved that flaxseed offered to rats during
pregnancy and lactation promoted better cognitive performance when
compared to control group, thus highlighting the importance of these
essential fatty acids in neurological development. However, animals
whose maternal diet contained higher levels of saturated fatty acids
presented damage in the memory and learning ability (Souza et al.,
2012; Yu et al., 2010). At the end of the experiment, brain tissue from
HL animals had 13% less saturated fatty acids compared to the other
groups, which may explain the cognitive impairment displayed.
Moreover, DHA levels (C22:6n3) were about 36% lower in this group
compared to the other groups. The amount of DHA in the brain was
increased in the offspring of animals treated with flaxseed diet, being
correlated with better spatial memory performance (Fernandes at al.,
2011). These fatty acids are essential for brain development and
memory because they modulate synaptic plasticity, improving learning
ability. Furthermore, one study showed that hyperlipidic maternal diets
lead to cognitive impairment in the adulthood of offspring (Noronha
et al., 2017). It is believed that the molecular mechanism of this dis-
order is related to inflammatory responses in the CNS (Valladolid-
Acebes et al., 2013) and with the reduction of BDNF in the hippo-
campus, which is consequently associated to induced cognitive im-
pairment (Alzoubi et al., 2009).
In this study, the profile of brain fatty acids in the offspring of an-
imals treated with cashew nuts in the initial stage of their life was
measured at different moments. This relationship helps us to under-
stand how the lack of different fatty acids can be modified throughout
life and how brain function can be improved or impaired in newborns
(by assessing reflex maturation), as well as in adolescents (by assessing
memory).
5. Conclusion
The maternal diet with cashew nuts offered a variety of fatty acids
(first through the placenta and then through breast milk) that allowed
proper formation of the brain tissue in the group that consumed the
normolipidic diet, inducing accelerated maturation of the nervous
system. This acceleration occurred less intensively in the hyperlipidic
group.
In fact, the normolipidic diet treatment with cashew nuts was able
to prevent memory deficits in an associative task (object recognition in
short time) and also in a non-associative task (open field habituation).
On the other hand, when the supply of cashew nuts was increased in the
hyperlipidic group, an increase of short-term memory was observed;
however, with a decrease in long-term memory.
Based on data from this study, we can infer that maternal
consumption of cashew nuts during pregnancy and lactation is bene-
ficial for the neural development of offspring when offered in adequate
quantities/amounts.
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