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\begin{document}

\title{Maternal exercise during pregnancy modulates genetic and biochemical
damage caused by high consumption of fructose in blood and liver of
offspring}



\author[1]{Marina Magenis}%
\author[1]{Adriani Damiani}%
\author[1]{Isadora  Monteiro}%
\author[1]{Ligia  Dagostin}%
\author[1]{Nicollas  da Silva}%
\author[1]{Rahisa  Scussel}%
\author[1]{Seigo Nagashima}%
\author[1]{Sabine Langie}%
\author[1]{Ricardo Pinho}%
\author[1]{Vanessa de Andrade}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Pregnancy is a period that is characterized by several metabolic and
physiological changes and requires special attention, especially with
regard to the relationship between feeding and fetal development.
Therefore, the objective of this study is to evaluate whether the
practice of voluntary physical exercise in combination with chronic
consumption of fructose from the beginning of life and/or until the
gestational period causes biochemical and genotoxic changes in pregnant
females and in their offspring. 70 Swiss female mice received fructose
in the hydration bottle and/or practiced voluntary physical exercise
(VPE) for 8 weeks (pre-pregnancy/pregnancy). After the lactation period,
the offspring groups were separated by sex. It was observed that the
consumption of fructose affected the food consumption, serum
concentration of fructose and glycemic profile in the mothers and that
the VPE decreases these parameters. In addition, fructose was genotoxic
in the mothers' peripheral tissues and VPE had a preventive effect on
these parameters. The offspring showed changes in food consumption,
serum fructose concentration and body weight, in addition to an increase
in the adiposity index in male offspring in the FRU group and a decrease
in the FRU+VPE group. Fructose lead to hepatic steatosis in the
offspring and VPE was able to decrease the area of steatosis. In
addition, fructose led to genotoxicity in the offspring and VPE was able
to modulate this effect, reducing damages. In conclusion, we observed
that all interventions with voluntary physical exercise had nutritional,
genetic and biochemical benefits of the mother and her offspring.%
\end{abstract}%



\sloppy


\textbf{Maternal exercise during pregnancy modulates genetic and
biochemical damage caused by high consumption of fructose in blood and
liver of offspring}

Marina Lummertz Magenis\textsuperscript{a}, Adriani Paganini
Damiani\textsuperscript{a}, Isadora de Oliveira
Monteiro\textsuperscript{a}, Ligia Salvan Dagostin\textsuperscript{a},
Nicollas dos Santos da Silva\textsuperscript{a}, Rahisa
Scussel\textsuperscript{b}, Seigo Nagashima\textsuperscript{c}, Sabine
A. S. Langie\textsuperscript{d}, Ricardo Aurino
Pinho\textsuperscript{e}and Vanessa Moraes de Andrade\textsuperscript{a}

\textsuperscript{a} Laboratory of Translational Biomedicine, Graduate
Program of Health Sciences, University of Southern Santa Catarina --
UNESC, Crici\selectlanguage{ngerman}úma, SC, Brazil.

\textsuperscript{b} Laboratory of Experimental Pathophysiology, Graduate
Program of Health Sciences, University of Southern Santa Catarina --
UNESC, Criciúma, SC, Brazil.

\textsuperscript{c} Laboratory of Experimental Pathology, Pontifical
Catholic University of Paraná -- PUCPR, Paraná, SC, Brazil.

\textsuperscript{d} Department of Pharmacology \& Toxicology, School for
Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht
University, The Netherlands.

\textsuperscript{e} Laboratory of Biochemistry Exercise Health,
Pontifical Catholic University of Paraná -- PUCPR, Paraná, SC, Brazil.

Corresponding author: Vanessa Moraes de Andrade, Laboratory of
Translational Biomedicine, Graduate Program of Health Sciences,
Department of Health Sciences, University of Southern Santa Catarina,
UNESC, 1105, Universitária Rd, 88806000, Criciúma, SC, Brazil.

Phone: +55 (48) 34 31 2757;

E-mail: vmoraesdeandrade@yahoo.com.br

\textbf{ABSTRACT}

Pregnancy is a period that is characterized by several metabolic and
physiological changes and requires special attention, especially with
regard to the relationship between feeding and fetal development.
Therefore, the objective of this study is to evaluate whether the
practice of voluntary physical exercise in combination with chronic
consumption of fructose from the beginning of life and/or until the
gestational period causes biochemical and genotoxic changes in pregnant
females and in their offspring.~ 70 Swiss female mice received
fructose~in the hydration bottle and/or practiced voluntary physical
exercise (VPE) for 8 weeks (pre-pregnancy/pregnancy). After the
lactation period, the offspring~groups were separated by sex. It was
observed that the consumption of fructose affected the food consumption,
serum concentration of fructose and glycemic profile in the mothers and
that the VPE decreases these parameters. In addition, fructose was
genotoxic~in the mothers' peripheral tissues and VPE had a preventive
effect on these parameters. The offspring showed changes in food
consumption, serum fructose concentration and body weight, in addition
to an increase in the adiposity index in male offspring in the FRU group
and a decrease in the FRU+VPE group. Fructose lead to hepatic steatosis
in the offspring and VPE was able to decrease the area of steatosis. In
addition, fructose led to genotoxicity in the offspring and VPE was able
to modulate this effect, reducing damages. In conclusion, we observed
that all interventions with voluntary physical exercise had nutritional,
genetic and biochemical benefits of the mother and her offspring.

\textbf{Keywords:} Fetal programming; Fructose; Physical exercise;
Genotoxicity; Mutagenicity



\textbf{Introduction}

It is believed that an inadequate intrauterine environment can affect
fetal growth and development, leading to aberrant, permanent programming
of the metabolic and endocrine function (Deodati et al., 2020). Fetal
programming is an adaptive response of the organism to the environment.
This ability is called ``developmental plasticity'' which allows the
development of a spectrum of phenotypes from a single genotype (Lucas et
al., 1991; Gluckman and Hanson, 2004). It allows the organism to respond
to the surrounding environment, especially during early development,
when cells are differentiating and tissues are developing. This
plasticity is based on modifications of cellular pathways leading
altered gene expression and the induction of specific phenotypes, but
without modification of DNA sequence (Hanson and Gluckman, 2008; Barouki
et al., 2012). Thus, early metabolic events during the pre-natal period
may be associated with susceptibility to chronic diseases later in the
adult life (Gluckman et al., 2005).

Maternal obesity and gestational diabetes, for example, have been shown
to provoke long-term detrimental changes in the pathways that regulate
energy balance in offspring. Notably, the increase in obesity and
diabetes, as well as metabolic syndrome and fatty liver disease
coincided with the increase in fructose in recent years (Muriel et al.,
2021). In recent decades the daily intake of free fructose or as high
fructose corn syrup has increased markedly. These forms of fructose are
used in the food industry for their sweetness, palatability, solubility,
low cost and high production efficiency compared to sugar (Cioffi et
al., 2017). Ancestral diets showed that the average fructose intake was
2 kg per year, while the current global average consumption is 25 kg per
year (Kmietowicz, 2012). More recently, fructose consumption has been
estimated at 117 g/day in women and 162 g/day in men in world, and that,
to consume this excess fructose via industrialized foods it is necessary
to consume a 350 mL can of soda a day, while when eating fruits one
would have to consume 1.3 kg of banana, 4.3 kg of strawberry, 800 g of
apple and 900 mL of orange juice a day. In the latest data from Vigitel
Brazil for 2018, it was observed that, in the set of 27 cities
evaluated, the frequency of consumption of five or more groups of
ultra-processed foods (rich in fructose and glucose) was 18.2\%, being
higher among men (21.8\%) than among women (15.1\%) (Ministry of Health,
2019).

Research with animals, showed that after 8 weeks of high fructose
consumption, rats developed features of the metolinic syndrome along
with mitochondrial structural changes (Mamikutty et al., 2014). In this
context, Magenis et al. (2020) evaluated the effects of fructose
treatment during pregnancy and lactation in mice, and observed an
increased in DNA damage, hyperglycemia and hyperlipidemia in females
that received doses of 10 and 20\%/L fructose/day. Corroborating with
this, Magenis et al. (2022) observed that the offspring (males and
females) of these females, in both dosage groups, demonstrated
genotoxicity (assessed by the comet assay) and oxidative stress
(assessed by nitrite concentration, sulfhydryl content, and superoxide
dismutase activity) in peripheral tissues. In addition, they showed
nutritional and metabolic changes due to increased food consumption,
hyperglycemia, hyperlipidemia and metabolic syndrome. Therefore, it was
suggested that high fructose consumption by pregnant females was harmful
to their offspring. However, it is believed that early dietary changes,
both in mothers and children, significantly contribute to alleviate or
assist in the treatment of these pathologies (Frias and Grove, 2012).

In this context, seeking alternatives to protect the damage caused to
fetal programming are necessary in order to search new treatments.
Although, the practice of physical exercise increased among Brazilians
according to Vigitel data (2019), the proportion of women performing
some type of physical activity dropped from 15.7\% in 2013 to 14.2\% in
2018 (Ministry of Health, 2019). More worrying is the sedentary
lifestyle during pregnancy, since only 4.7\% of pregnant Brazilian women
are active throughout this period and 12.9\% of women reported some
physical activity during pregnancy as recommended by the American
College of Obstetricians and Gynecologists (ACOG) (Domingues and Barros,
2007). In addition, it is observed that 50\% of women at reproductive
age and 20--25\% of pregnant women in Europe are overweight or obese at
the first antenatal visit (Stevens et al., 2012).

Guidance for physical exercise during pregnancy has been encouraged and
recommended by the ACOG since the 1990s, but only in 2002 was this
practice recognized as safe and indicated for healthy pregnant women.
Through a systematic review and meta-analysis, Davenport et al. (2018)
observed that pregnant women who exercised have a 39\% reduced chance of
having a baby weighing more than four kilos compared to those who did
not exercise. It was also shown that the practice of exercise during
pregnancy is not related to intrauterine growth restriction, premature
birth and low birth weight, thus being safe and beneficial for the
fetus. The ACOG advises pregnant women, even if they are sedentary, to
perform at least 30 minutes of physical exercise on as many days of the
week as possible (ACOG, 2002). In São Paulo, it was observed in the
pregnant population that only 13\% developed some type of physical
activity, and only half of them performed exercises with frequency
guided by the ACOG (Surita et al., 2014).

So far research in animals observed that maternal exercise before and
during pregnancy does not seem to affect the body composition of
offspring in healthy mice (Kelly et al., 2015). In an animal model of
maternal obesity induced by a high-fat diet and treated with treadmill
exercise, it was observed that the intervention had beneficial effects
in relation to insulin resistance, excess placental lipid deposition and
its hypoxia in offspring (Fernandez-Twinn et al. al., 2017). Moreover,
Brito et al. (2016) observed that maternal physical exercise decreases
DNA damage and oxidative stress caused by doxorubicin in offspring
cardiomyocytes. In addition, Netto et al. (2018) observed that the
practice of swimming in pregnant females with streptozotocin-induced
diabetes by contributed to the reduction of DNA damage and lipid
peroxidation in the mother and in her offspring.

To understand the effect of lifestyle and levels of DNA damage on
pregnancy, further studies are needed, with sample collection from
preconception, pregnancy and its offspring. Thus, the objective of the
study is to evaluate whether the practice of voluntary physical exercise
associated with chronic consumption of fructose from the pre-pregnancy
period and/or until the pregnancy period causes biochemical and
genotoxic alterations in breeding females, as well as genotoxic and
biochemical alterations in their offspring.

\textbf{2. Materials and Methods}

2.1 Animals, ethical principles and experimental design

The animals were obtained from the Animal Center of the University of
Southern Santa Catarina (Unesc, Brazil) and housed in polypropylene
boxes and maintained under a 12 h light/dark cycle with access to plain
water or water containing fructose (20\%/L) in a temperature-controlled
(23 ± 1°C) room. This study was approved by the Ethics Committee of the
University of Southern Santa Catarina (approval No. 023/2019-1) and was
performed in accordance with the ethical principles of the Brazilian
College of Animal Experimentation (COBEA). The experiments followed the
guidelines of ARRIVE (Animal Research: Reporting of In Vivo
Experiments). Moreover, every effort was made to minimize animal
suffering and to reduce the number of animals used in the experiments.
The standard feed of the animals was supplied from the Puro Lab 22PB
laboratory, which corresponds to 2.93kcal/g, 20\%, 10\% and 70\% of the
calories supplied with protein, fat and carbohydrates, respectively

Seventy Swiss female mice with 21 days of life were used, which received
fructose (20\%/L) in the hydration bottle and/or practiced voluntary
physical exercise (VPE) for 8 weeks of treatment (pre-pregnancy).
Twenty-one male 60-day-old Swiss mice were used for copulation, 1 male
placed every 5 females (the males were kept with the females for only 7
days, the period intended for copulation) in order to obtain offspring
(males and females).

Females were initially divided into 4 treatment groups (G) starting
pre-pregnancy: G1- Water: females that received water and did not
practice voluntary physical exercise (n=10); G2 -- Water + VPE: females
that received water and practiced voluntary physical exercise (n=20); G3
--FRU: females that received 20\% fructose/L (n=10); G4 --FRU + VPE:
females that received 20\% fructose/L and practiced voluntary physical
exercise (n=30) (Figure 1). Blood samples from the mothers were
collected pre-pregnancy (week 8), during pregnancy (week 12) and
post-pregnancy (week 15) via tail venipuncture in heparin tubes.

Subsequently they were subdivided into 7 experimental groups for the
period of copulation, pregnancy and lactation: G1 - Water: females that
received water and did not practice voluntary physical exercise until
the end of pregnancy (n=10); G2a -- Water + VPE: females that received
water and practiced voluntary physical exercise until pregnancy (n=10);
G2b -- Water + VPE/Water: females that received water and practiced
voluntary physical exercise until copulation and then stopped the VPE
and continued only with water until the pregnancy period (n=10); G3 --
FRU: females that received 20\% fructose/L (n=10); G4a -- FRU + VPW:
females that received 20\% fructose/L and practiced voluntary physical
exercise until pregnancy (n=10); G4b --FRU + VPE/Water + VPE: females
that received 20\% fructose/L and practiced voluntary physical exercise
until copulation, after this period, they stopped fructose consumption
and continued receiving only water and practicing voluntary physical
exercise during pregnancy (n=10); G4c --FRU + VPE/Water: females that
received 20\% fructose/L and practiced voluntary physical exercise until
copulation, after stopped fructose consumption and voluntary physical
exercise and received only water during pregnancy (n=10) (Figure 1).

After the lactation period, the offspring (males and females, separated
by sex) were separated into the same 7 offspring groups (OG) in
correspondence to the treatments performed on the mother females. The
mothers were euthanized and tissues were collected for further analysis.
The offspring receive standard show and water until 60 days of age, at
which point they were sacrificed and tissues were collected for
genotoxic and biochemical tests (Figure 1).

2.2 Treatments

\emph{2.2.1 Fructose}

Mothers received drinking water or fructose at a concentration of 20\%/L
ad libitum in hydration bottles during pre-pregnancy (8 weeks of
treatment), copulation (7 days), pregnancy (21 days) and lactation (21
days) periods. These dosages and treatment time were chosen according to
data previously described in the literature (Mamikutty et al., 2014;
Magenis et al., 2020). The route of administration and the period of
treatment were chosen in order to maintain the relevance of the
applicability of human studies. Fructose (Sigma Aldrich) was dissolved
in drinking water and prepared three times a week at a concentration of
20\%/L (Magenis et al., 2020).

\emph{2.2.2 Voluntary Physical Exercise (VPE)}

Voluntary physical exercise was performed using a voluntary running
wheel placed inside the cages during the treatment time (varying
according to each group), thus allowing free access by the animals. The
exercise mileage of the animals was controlled through an infrared
equipment placed next to the wheel and was divided by the animals
present in the cage (5 animals) (Muller et al., 2011). This analysis was
performed in the pre-pregnancy period (8th week) and during pregnancy
(12th week). The females during the pre-pregnancy period ran an average
of 3143 meters/day and in the gestation period 5649m/day, and this
amount traveled was an estimate per animal.

2.3 Body composition and food consumption of animals

The mothers were weighed throughout the experiment according to their
periods (pre-pregnancy at week 8, at the end of pregnancy at week 12 and
post-pregnancy at week 15) and the offspring (males and females) at ages
(7, 14, 21, 30 and 60 days of age). Food consumption (food and fructose
solution) was calculated weekly by weighing the total amount of food (g)
and liquids (mL) given to the animals and subtracting the food (g) and
liquids (mL) from the remainder in the cage and hydration bottle
throughout the experiment (Diniz et al., 2004; Diniz et al., 2005). From
this, the daily calories ingested on the day were calculated, since the
chow provides 3.36 kcal/g and the fructose solution 4 kcal/mL (Kending
et al., 2014).

To assess the adiposity index of the animals, epididymal, mesenteric,
perirenal and retroperitoneal adipose tissue were extracted to calculate
the adiposity index (gram of fat/gram body weight x 100). This
evaluation was performed after euthanasia of the offspring (males and
females) at 60 days of age.

2.4 Biochemical analyses

\emph{2.4.1 High Performance Liquid Chromatography (HPLC)}

The quantification of serum fructose levels was determined by High
Performance Liquid Chromatography (HPLC) (Rahman et al., 2008) and was
carried out in serum samples from pre-pregnancy (week 8) and pregnant
mothers (week 12) and their offspring (males and females) at 7 and 60
days of life. Serum samples were obtained by centrifugation. The
chromatographic system was a Shimadzu Prominence HPLC system, equipped
with an LC-20AT pump coupled to a SIL-20AHT autosampler, a CTO-20A
thermostat and a SPD-20A UV-Vis detector, in addition to the LC Solution
software (Shimadzu, Kyoto, Japan). Chromatographic separations were
performed on an Ascentis® C18 chromatographic column, reference code
581305 (250 mm × 2.1 mm, 5 \selectlanguage{greek}μ\selectlanguage{english}m; Supelco\selectlanguage{ngerman}®, Bellefonte, PA, USA) and the
column temperature was 30°C. The injection volume of the samples was 20
\selectlanguage{greek}μ\selectlanguage{english}L. The mobile phase was eluted in isocratic profile and consisted of
HPLC grade acetonitrile and ultra-pure water (Milli-Q) (55:45, v/v). The
mobile phase flow rate was 0.4 mL/min and detection was performed at a
wavelength of 195 nm. The identification of fructose in the samples was
by comparing the retention time in the standard solution. The results
were quantified by means of a calibration curve (r\selectlanguage{ngerman}² \selectlanguage{english}[?]0.99) prepared with
1.25, 2.5, 5, 10 and 20 mg/mL of fructose standards and the respective
peak areas.

\emph{2.4.2 Fasting glucose and insulin tolerance test (ITT)}

To assess fasting blood glucose, blood was collected through a small
incision at the tip of the tail of the animals, after fasting period of
6 hours (Ayala et al., 2010), and blood glucose concentration was
measured using a glucometer for determination of fasting blood glucose
(mother females and their offspring).

The ITTs were only performed for the mothers pre- and post-pregnancy
(weeks 8 and 12), since this test cannot be performed during pregnancy
due to insulin administration. In the offspring (males and females),
this test was performed when they completed 60 days of life. After that,
all animals received a dose of 2U/kg of intraperitoneal insulin and
subsequent blood glucose measurements at times 5, 10, 15, 20, 25 and
30min post-insulin injection were performed (adapted from Wang and Liao,
2012). The animals that presented glucose values below 30mg/dL during
the test were kept warm and received intraperitonially glucose. For
demonstration and data analysis, they were presented as absolute glucose
data (mg/dL), as area under the ITT curve calculated by dividing glucose
data (mg/dL) by the time post-insulin injection (0 to 30min).

2.5 Assessing genome damage

\emph{2.5.1. Comet assay to assess DNA strand breaks}

The comet assay was performed under alkaline conditions as described by
Collins et al. (2023). Blood was collected using a tip and a
micropipette and placed in heparinized and refrigerated microtubes,
while liver samples were dissected and immersed in refrigerated
phosphate buffer (PBS). Liver samples of about 3x3mm were individually
homogenized with the aid of a syringe, through the back and forth
movement, in order to obtain a cell suspension.

Blood cells (5 \selectlanguage{greek}μ\selectlanguage{english}L whole blood aliquots) and dissociated liver cell
suspensions (10 \selectlanguage{greek}μ\selectlanguage{english}L aliquots) were embedded in low melting point agarose
(0.75\%, w/v, 115 \selectlanguage{greek}μ\selectlanguage{english}L or 110 \selectlanguage{greek}μ\selectlanguage{english}L, respectively). The mixture was added to
a microscope slide pre-coated with 1.5\% normal melting point agarose,
covered with a coverslip and then placed in the refrigerator for
approximately 5 minutes at 4\selectlanguage{ngerman}°C for solidification. Soon after, the
coverslips were carefully removed and the slides immersed in lysis
buffer (2.5M NaCl, 100mM EDTA and 10mM Tris, pH 10.0-10.5, with the
addition of 1\% Triton X - 100 and 10\% DMSO) at 4ºC for a minimum of 12
hours and a maximum of 48 hours.

The slides were incubated in alkaline solution (300mM NaOH and 1mM EDTA,
pH\textgreater{}13) for 20 minutes for DNA unfolding, followed by
electrophoresis at \textasciitilde{} 1V/cm for approximately 20 minutes.
All these steps were performed under weak yellow indirect light.
Afterwards, the slides were neutralized with 0.4M Tris (pH 7.5) and, at
the end, the DNA was stained by Syber Gold (1:10.000) (Invitrogen, USA)
for further analysis.

Evaluation of 100 cells per individual and per tissue (50 cells in each
duplicated slide) was performed. Resulting comet shapes were visually
evaluated, being classified into five classes, according to the shape
and size of the comet tail, with the classification for absence of tail
considered 0, up to 4 when almost all DNA has moved to the tail, leaving
a tiny comet head (Collins et al., 1997). In this way, we have a Damage
Index for each animal ranging from zero (100 X 0 = 0; 100 cells observed
completely without damage) to 400 (100 X 4 = 400; 100 cells observed
with maximum damage).

International guidelines and recommendations for the comet assay
consider the visual scoring of 100 comets to be a well-validated
assessment method. It has a high correlation with computer image
analysis (Collins et al., 1997). Negative and positive controls in in
whole peripheral blood cells from healthy individuals were used for each
assay to ensure the reliability of the procedure. Damage index of
negative control = 13.00 ± 4.6 and Damage index of positive control:
105.30 ± 22.7). All slides were coded for blind analysis. The aspects of
the comet assay procedures were reported following the guidelines of
MIRCA (Minimum Information for Reporting on the Comet Assay) (Moller et
al., 2020).

\emph{2.5.2 The enzyme modified comet assay to detect DNA oxidation}

An enzyme-modified comet assay was performed as described by Collins et
al. (2023), only with peripheral blood of the mothers. For the
measurement of oxidized purines, there is an additional step included
after the lysis: the slides were washed three times with ice-cold buffer
(0.1 M KCl, 0.5 mm Na2 EDTA, 40 mm HEPES, and 0.2 mg/ml bovine serum
albumin; pH 8.0) and incubated for 40 min at 37°C with
formamidopyrimidine DNA glycosylase (FPG) (Uniscience®, 1:1000) or
buffer. Further steps were performed as mentioned for the comet assay
under alkaline conditions (Collins et al., 2023). Net FPG sensitive
sites were calculate as the difference between the samples incubated
with FPG and the samples incubated with buffer; Damage Index of
sample\textsubscript{x}/FPG -- Damage Index of
sample\textsubscript{x}/buffer.

\emph{2.5.3 Micronucleus (MN) test to assess chromosome damage}

This test was performed according to the guidelines of the GeneTox
program of the US Environmental Protection Agency (Mavournin et al.,
1990; Krishna e Hayashi, 2000) and following the new OECD test guideline
(TG) 474 for the in vivo mammalian erythrocyte micronucleus (MN) test
(29 July 2016).

Bone marrow was extracted from animal femurs, and a smear was deposited
directly onto a microscope slide using a drop of fetal calf serum. The
slides were subjected to Giemsa staining (5\%) then air-dried and coded
for blinded analysis. To avoid false negative results and as a toxicity
measure, the ratio of polychromatic to normochromatic erythrocytes
(PCE/NCE) was determined for 500 erythrocytes per animal. The presence
of the MN was determined in 4000 erythrocytes per animal (2000 from each
of the two slides) by bright field microscopy (1000× magnification with
immersion oil). For individual animals, mean values of MnPCE were used
as the experimental units, and variation was based on differences among
animals within the same group.

2.6 Histological analysis

The liver of the offspring (males and females) were sectioned,
immediately immersed in 4\% paraformaldehyde (PFA) fixative solution and
buffered for 48 hours for subsequent histological processing. The
material was embedded in paraffin and cut with a microtome, obtaining 4
\selectlanguage{greek}µ\selectlanguage{english}m thick sections. Slides were stained with hematoxylin \& eosin (H\&E)
staining for image acquisition and histopathological analysis of liver
histoarchitecture.

To analyze protein expression in an automated way, the technique of
color morphometry was used, which was adapted to obtain the intensity
and destruction of nuclear expression of the protein. Therefore, the
H\&E stained slides were submitted to the Axio
Scan.Z1\selectlanguage{ngerman}\textsuperscript{®}(ZEISS, Jena, Germany) to capture images of
all sample areas. After pre-processing these images with Adobe Photoshop
CS6 v 13.0 \textsuperscript{®} (Adobe, San Jose, CA), in order to
improve quality and separate images, they were analyzed with the Image
Pro Plus v.4.5.0.29 (Media Cybernetics, Rockville, MD) software using
the color morphometry tool. Initially, the software analyzed the total
distribution of the white steatosis area in the tissue. With the help of
the software, the steatosis area was marked in red. This method of
marking a particular structure based on its color was called ``mask''.

The software automatically provided the marked areas, quantified in
square micrometers, through the following formula: (value of the white
steatosis area/value of the image field area). Subsequently, the results
were transported to an excel spreadsheet and quantified.

2.7 Statistical analysis

Data are expressed as mean and standard deviation of the mean (mean ±
SD). For statistical analysis, the normality of the variables was
analyzed using the Bartlett test. For non-parametric variables,
Kruskal-Wallis was used followed by Dunn's post hoc. For parametric
variables, one-way variance (ANOVA) by Tukey's post hoc was used, and
for specific comparisons between groups, the student t-test was used.

For the evaluation of the mothers, food consumption and body weight,
Kruskal-Wallis followed by Dunn's post hoc were used. Fasting blood
glucose, insulin tolerance test, and the comet assay were analyzed by
one-way ANOVA with Tukey's post hoc. Differences in the fructose serum
levels and micronucleus test were tested with the student t-test.

For the evaluation of the offspring (males and females), food
consumption, fructose serum levels, fasting glucose, insulin tolerance
test, adiposity index and comet assay, ANOVA with Tukey's post hoc was
used. Micronucleus test were analyzed with the student t-test. Body
weight and histological parameters were tested by Kruskal-Wallis
followed by Dunn's post hoc. Differences in micronuclei was tested by
student t-test.

Differences between groups were considered significant when P values
were less than 0.05 (p\textless{}0.05), using the GraphPad Prism 5.0
program.

\textbf{3 Results}

\textbf{3.1 Characteristics of the mothers}

\emph{3.1.1 Food consumption, body weight and fructose uptake}

During the treatment period, the amounts of liquids, animal feed and
daily calories ingested by the mothers were measured, in addition to the
monitoring of body weight.

Table 1 shows the average of these parameters by group during the three
periods.

In the pre-pregnancy period, an increase in liquid consumption was
observed in the water + VPE group and in the FRU group in relation to
the water group (p\textless{}0.05). Concerning food consumption, only
the water + VPE group demonstrated an increase in relation to the water
group (p\textless{}0.05). However, daily caloric consumption was
increased in the FRU group compared to the water group (Table 1 and
Figure 2). Regarding body weight, no statistically significant
difference was observed among the groups (Table 1).

During pregnancy, liquid consumption increased in the FRU group compared
to the water group, and the groups FRU + VPE/Water + VPE and
FRU+VPE/Water consumed less water compared to the FRU group
(p\textless{}0.05). Food consumption was only decreased in the FRU +
VPE/Water group in comparison to the FRU + VPE/Water + VPE group
(p\textless{}0.05). Regarding caloric consumption, an increase was
observed in the FRU group compared to the water group (p\textless{}0.05)
(Table 1 and Figure 2). Regarding body weight, there was no
statistically significant difference among the groups (Table 1).

In the post-pregnancy period, there was no statistically significant
difference between the groups in the consumption of liquids, feed and
calories. Apart from the caloric consumption of the FRU group, which was
increased compared to the water group (p\textless{}0.05) (Table 1 and
Figure 2). In terms of body weight, it was observed that the groups FRU
+ VPE/Water + VPE and FRU+VPE/Water had a decreased weight compared to
the FRU group (p\textless{}0.05) (Table 1).

The average consumption of daily calories consumed via the liquids and
feed in the pre-pregnancy, pregnancy and post-pregnancy period, is
presented in figure 2.

In addition to the consumed liquid and food containing fructose (Table
1), the concentration of fructose in the serum of the mothers was
quantified in the pre-pregnancy and pregnancy period through HPLC. In
the pre-pregnancy period, it was observed that the FRU group had higher
serum fructose concentrations compared to the water group
(p\textless{}0.05) and the FRU+VPE group showed decreased levels
compared to the FRU group (p\textless{}0.05) (figure 3). The same
happened in the pregnancy period, where the FRU group showed increased
the serum concentrations of fructose in comparison to the water group,
while the interventions FRU+VPE and FRU+VPE/Water + VPE reduced this
concentration compared to the FRU group (p\textless{}0, 05) (figure 3).
These evaluations were not performed in the post-pregnancy period due to
sampling problems.

\emph{3.1.2 Effect on blood glucose and insulin sensitivity}

During the treatment period, the glycemic profile was evaluated through
fasting glucose (Table 2) and the insulin tolerance test (Figure 4).

Fasting blood glucose levels pre-pregnancy were increased in the FRU
group compared to the water group, whereas the FRU+VPE group showed a
decrease in glucose levels compared to the FRU group (p\textless{}0
.05). During pregnancy, blood glucose increased in the FRU group in
comparison to the water group, while FRU+VPE/Water + VPE and
FRU+VPE/Water groups showed decreased glucose levels compared to the FRU
group (p\textless{}0.05). Regarding the post-pregnancy period, the water
+ VPE group showed higher glucose levels in comparison to the water
group, whereas the interventions FRU+VPE/Water+VPE and FRU+VPE/Water
increased these levels in relation to the FRU+VPE group
(p\textless{}0.05) (Table 2).

In the pre-pregnancy period, an increase in AUC in the FRU group was
observed compared to the water group and a decrease in the FRU+VPE group
compared to the FRU group (p\textless{}0.05), indicating that at the
time of mating the females were resistant the insulin. However, in the
post-pregnancy period, there was no statistically significant difference
between the groups, since the glycemic curve of the animals was similar
(Figure 4).

\emph{3.1.3 Effect on DNA strand breaks and DNA oxidation}

In the pre-pregnancy period, in both strand breaks and oxidized purine
levels increased in the FRU group compared to the water group (Figure
5A; p\textless{}0.05). A decrease in oxidized DNA damage was observed in
the FRU+VPE treated group compared to the FRU group (p\textless{}0.05).

The same happened in the pregnancy period, an increase in strand breaks
and oxidized DNA damage was observed in the FRU group compared to the
water group (Figure 5B; p\textless{}0.05). Regarding the interventions,
it was observed that there was a decrease in strand breaks and oxidized
DNA in the FRU+VPE, FRU+VPE/Water + VPE and FRU+VPE/Water group in
comparison to the FRU group (p \textless{}0.05).

In the post-pregnancy period, we observed an increase in strand breaks
and oxidized DNA damage in the FRU group compared to the water group
(Figure 5C; p\textless{}0.05). In addition, a decrease in strand breaks
was observed in the FRU+VPE, FRU+VPE/Water + VPE and FRU+VPE/Water
groups compared to the FRU group (p\textless{}0.05).

In the liver (post-pregnancy period), an increase in DNA damage
(detected as strand breaks) was observed in the FRU group in comparison
to the water group and a reduction in DNA damage was observed in the
FRU+VPE/Water group compared to the FRU group (Figure 6 ;
p\textless{}0.05).

\par\null

\emph{3.1.4 Levels of chromosome damage}

To evaluate whether the consumption of fructose and the practice of
voluntary physical exercise causes additional genotoxic effects in the
mothers in the post-pregnancy period, the micronucleus test was
performed on bone marrow cells (Table 3).

Significantly higher levels of MnPCE were observed in mothers treated
with FRU in comparison to the water group (p\textless{}0.05). In
addition, a decrease in the number of MnPCE was observed in the FRU+VPE,
FRU+VPE/Water + VPE and FRU+VPE/Water group compared to the FRU group
(p\textless{}0.05). Regarding the PCE/NCE ratio, no statistically
significant differences were observed between the groups, demonstrating
that the production of erythrocytes was occurring normally in the bone
marrow, with no evidences of cytotoxicity.

\textbf{3.2 The effect of maternal fructose consumption and exercise on
the offspring}

\emph{3.2.1 Fructose uptake by the offspring and food consumption, body
weight}

Knowing that there was a consumption of fructose by the matrices females
(Table 1), we sought to quantify its concentration in the serum of the
offspring at 7 days of life (lactation period) and at 60 days (adult
life) through HPLC (Figure 7).

Regarding the offspring with 7 days of life, we observed in both (males
and females) that there was an increase in fructose concentration in the
FRU group in relation to the water group and a decrease in fructose
concentration in the FRU+VPE, FRU+VPE /Water+VPE and FRU+VPE/Water
groups in relation to the FRU group (p\textless{}0.05). In the
60-day-old offspring (males and females), we did not find statistically
significant differences. However, we observed that there is an increase
in the concentration of serum fructose in the offspring during the
period of exposure via mother.

During the life follow-up period, the amount of animal food and daily
calories ingested by the offspring were measured (Table 4). In addition,
body weight was monitored during 7, 14, 21, 30 and 60 days of life,
these results are shown in table 5.

Regarding food consumption, it is observed that both offspring (males
and females) had higher consumption of chow in the water + VPE group
compared to the water group (p\textless{}0.05). Furthermore, it was
observed that the FRU+VPE/Water+VPE group had a decrease in consumption
compared to the FRU+VPE group (p\textless{}0.05). Regarding daily
calories, statistical differences were observed only in the male
offspring, where the FRU+VPE/Water + VPE group showed a reduction in
calories compared to the FRU+VPE group (p\textless{}0.05).

In addition to the consumption of liquids, the weight gain was also
analyzed in the animals throughout their lives (Table 5).

In the female offspring, we observed that at 7 and 14 days of age the
FRU group had an increase in body weight in relation to the water group.
However, at 7 days a decrease in weight in the FRU+VPE group compared to
the FRU group (p\textless{}0.05) was observed. At 21 days of age, was
observed only an increase in weight in the FRU+VPE/Water group in
relation to the FRU+VPE group (p\textless{}0.05). At 30 days of age,
there was a decrease in body weight in the FRU+VPE group in relation to
the FRU group (p\textless{}0.05). Finally, at 60 days of life, an
increase in body weight in the FRU group in relation to the water group
(p\textless{}0.05) was also observed, whereas the FRU + VPE and
FRU+VPE/Water+VPE groups showed weight reduction compared to the FRU
group (p\textless{}0.05). In addition, an increase in body weight was
observed in the FRU+VPE/Water group in relation to the FRU+VPE group
(p\textless{}0.05). Thus, we observed in the female offspring that the
offspring from the FRU group has an increase in body weight, especially
in the first days of life and in adulthood, since no differences were
observed throughout life. Furthermore, we observed that interventions
can decrease this body weight.

In the male offspring, at 7 days of age, we observed an increase in body
weight in the FRU group in relation to the water group
(p\textless{}0.05) and a decrease in body weight in the FRU+VPE and
FRU+VPE/Water in relation to the FRU group (p\textless{}0.05).
Furthermore, an increase in body weight was found in the
FRU+VPE/Water+VPE group in relation to the FRU+VPE group
(p\textless{}0.05). At 14 and 21 days of age, an increase in body weight
in the FRU group in relation to the water group (p\textless{}0.05) was
observed. At 30 and 60 days of life, no statistically significant
differences were observed. Thus, we observed in male offspring that the
weight is changed until 21 days of age and then it is brought to
eutrophy in relation to the other groups.

\emph{3.2.2 The effect on the offspring's body adiposity}

To assess whether the FRU increases the adipose tissue of the animals
and whether the VPE could reverse this situation, the body adiposity
index was calculated (figure 8) by weighing the epididymal, mesenteric,
retroperitoneal and perirenal fats.

According to the results obtained in the figure 8, it was possible to
verify that in the female offspring there was no statistically
significant difference between the groups. However, in males, an
increase in body adiposity index was observed in the FRU in relation to
the water group (p\textless{}0.05) and a decrease in the FRU+VPE in
relation to the FRU group (p\textless{}0.05).

\emph{3.2.3 Fasting blood glucose and insulin sensitivity of the
offspring}

The glycemic profile was evaluated in the offspring (females and males)
through fasting glucose (Table 6) and the insulin tolerance test (Figure
9).

A decrease in fasting glucose was observed in offspring females in the
FRU+VPE/Water+VPE group compared to the FRU group and FRU+VPE
(p\textless{}0.05). In males, no significant differences were observed.

In the oral insulin tolerance test (figure 9), no significant
differences were found in females, showing that the offspring were not
insulin resistant. In offspring males, we observed a decrease in the
area under the curve in the FRU+VPE/Water+VPE group compared to the
fructose group and the FRU+VPE group (p\textless{}0.05). However, the
fructose group did not demonstrated insulin resistance.

\emph{3.2.4 Levels of hepatic steatosis in the offspring}

To evaluate hepatic steatosis, histology of the liver of the offspring
(female and male) was performed and the quantification was through the
percentage of the steatosis area as observed in the graph and in the
morphology of the liver cells, evidenced by the black arrows (figure
10).

In female offspring, was observed an increase in the area of steatosis
in the FRU group in relation to the water group (p\textless{}0.05). The
treated groups, FRU+VPE, FRU+VPE/Water+VPE and FRU+VPE/Water reduced the
area of steatosis in relation to the FRU group (p\textless{}0.05). In
addition, it was observed that the FRU+VPE/Water+VPE group decreased in
relation to the FRU+VPE group, already and the FRU+VPE/Water group
increased in relation to the FRU+VPE/Water+VPE (p\textless{}0.05).

In male offspring, there was a decrease in the area of steatosis in the
FRU group compared to the control group (p\textless{}0.05). The treated
groups, FRU+VPE, FRU+VPE/Water+VPE and FRU+VPE/Water reduced the area of
steatosis in relation to the FRU group (p\textless{}0.05). In the
FRU+VPE/Water group there was also a reduction in the area of steatosis
in relation to the FRU+VPE and FRU+VPE/Water+VPE group
(p\textless{}0.05).

Thus, our results demonstrate that the VPE made by the mothers exposed
to fructose before and during pregnancy was able to decrease the area of
hepatic steatosis in the offspring (females and males).

\emph{3.2.5 Levels of DNA damage in the offspring}

The alkaline comet assay was performed on peripheral tissues (blood and
liver) in the offspring (females and males) using the damage index
parameter (0-400) (figure 11).

In the evaluation of the female offspring, in the blood (figure 11), our
results demonstrated an increase in DNA damage index in the FRU group in
relation to the water group (p\textless{}0.05). In addition, was
observed a decrease in DNA damage in the FRU+ VPE, FRU+VPE/Water+VPE and
FRU+VPE/Water, in relation to the FRU group (p\textless{}0.05). No
significant differences were found in the liver.

In the male offspring was found an increase in DNA damage in the
water+VPE and FRU group in relation to the water group
(p\textless{}0.05) in the blood. A decrease in DNA damage in the
FRU+VPE, FRU+VPE/Water+VPE and FRU+VPE/Water groups compared to the FRU
group (p\textless{}0.05) was observed. Regarding the liver, an increase
in DNA damage was observed in the FRU group when compared to the water
group and a decrease in the damage index in FRU+VPE, FRU+VPE/Water+VPE
and FRU+VPE/Water in relation to the FRU group (p\textless{}0.05).

\emph{3.2.6 Levels of chromosome damage in the offspring}

Table 7 presents the results of the micronucleus test in the female and
male offspring, evaluating whether the consumption of fructose and the
practice of voluntary physical exercise in the mothers causes mutagenic
effects in the bone marrow cells of the offspring.

Significantly higher levels of MnPCE were observed in the female and
male offspring from mothers treated with FRU in comparison to the water
group (p\textless{}0.05). Regarding the PCE/NCE ratio, no statistically
significant differences were observed between the groups, demonstrating
that the production of erythrocytes was occurring normally in the bone
marrow, with no evidence of cytotoxicity.

\textbf{4. Discussion}

Fetal programming during pregnancy can generate a healthy or poor
environment for the offspring (Deodati et al., 2020). Maternal obesity
and gestational diabetes, for example, have been shown to provoke
long-term changes in the mechanisms that regulate energy balance in
offspring. The increase in childhood obesity and diabetes in children is
often attributed to an increase in calorie-dense diets, reduced levels
of physical activity and exercise frequency by the mother. However, it
is believed that early dietary changes, both in mothers and children,
significantly contribute to alleviate or assist in the treatment of
these pathologies (Frias and Grove, 2012). In this context, the current
study evaluated the effects of maternal chronic consumption of fructose
(20\%) during pregnancy and early-life on the genome and metabolism of
the mothers and their offspring, and assessed the modulating effect of
interventions with voluntary physical exercise.

4.1 Maternal fructose consumption increases their own and their
offsprings' caloric intake, while exercise reduces it

In our study, the fructose serum concentration of the mothers in the
groups treated with fructose was higher compared to those consuming
water, confirming proper uptake of the fructose by the body, and a
decrease was observed in the groups doing voluntary physical exercise.
The higher exposure to fructose before and during pregnancy increased
the liquid consumption of the mothers, and that intervention with
voluntary physical exercise was able to reduce this 20\%/L fructose
liquid consumption, especially in the groups FRU+VPE/Water+VPE and
FRU+VPE/Water. Food consumption seemed to be more associated with doing
exercise, as only slight non-significant increases were observed for the
fructose only group (G3) compared to the water only group (G1). However,
caloric intake did increase significantly in the fructose consuming
groups during all the study periods.

Corroborating with our results, Magenis et al. (2020) observed that
fructose consumption at 10 and 20\%/L in mice can lead to an increase in
food and caloric intake in female mice during pregnancy, as well as,
increase in food consumption in offspring of mothers that were exposed
to fructose. Clayton et al. (2015) suggest that maternal fructose (10\%)
consumption may alter maternal responsiveness to leptin and lead to
impaired signaling in the fetus, thus altering food consumption. The
imbalance of this hormone can affect appetite regulation and lead to
insufficient satiety, increasing food intake (Kao et al., 2021). It is
further suggested that fructose consumption may be a significant factor
in the obesity epidemic since human studies using fructose
concentrations ranging from 7.5\% to 25\% of caloric intake have
demonstrated that its long-term consumption may be associated with the
development of hyperlipidemia and insulin resistance (Vos et al., 2008;
Stanhope et al., 2015).

Physical exercise can bring benefits in food control as observed in this
study. Platt et al. (2015) observed that the physical exercise of the
voluntary wheel did not change the caloric consumption of pregnant
females, as in our study. Hsu et al. (2021) evaluated the effects of
aquatic exercise in mice fed with a high-fructose diet and observed that
fructose increases food consumption and that physical exercise decreases
this consumption when associated with fructose.

In the offspring (male and female), an increase in serum fructose
concentration was observed during the lactation, showing that maternal
fructose exposure passes via the placental barrier and that exposure
continues via breast milk to offspring. However, upon reaching adulthood
and without maternal exposure, levels returned to normal. These results
prove that fructose was taken up by the mothers, and that the offspring
were exposed to fructose via the mothers during pregnancy and lactation.
When fructose was consumed in combination with voluntary physical
exercise, these levels decreased. As suggested by Egli et al. (2016)
exercise performed immediately after fructose ingestion increases
fructose oxidation and suppresses fructose and glycogen storage.

Indeed, we observed that food consumption, and caloric intake
(especially for the males), was lower in the FRU+VPE/Water+VPE
intervention compared to the FRU group, showing that stopping fructose
and continuing to practice physical exercise during pregnancy has better
regulation of food consumption in the offspring. Interestingly, the
uterine environment may influence taste preferences and healthy eating
in offspring. The flavors of the maternal diet are found in the amniotic
fluid and consequently ingested by the fetus, thus flavors experienced
in the intrauterine period determine a preference that persists into
childhood or even into adulthood, along with possible complications such
as type 2 diabetes mellitus and obesity (Trout and Effinger, 2012). Koo
et al. (2021) have observed the negative effects of maternal fructose
intake during pregnancy and lactation on their offspring, leading to the
appearance of metabolic syndrome, suggesting that nutritional status and
food intake during pregnancy and lactation may affect fetal programming
(Koo et al., 2021).

4.2 The effects on body composition in the mothers and their offspring

As changes were found in food consumption, as well as in levels of
exposure with physical exercise, we sought to understand body weight and
body composition in mothers and their offspring. Regardless the increase
in maternal caloric intake, no significant increases in maternal weight
was observed. In the post-pregnancy period a decrease was observed,
which may be due to the postpartum period. Indeed in our previous study
by Magenis et al. (2020), this weight drop was observed in the lactation
period, as expected due to the physiological demand postpartum with the
birth of offspring. However, the weight of the fructose group remained
high post-pregnancy. Interestingly, the exercise interventions (G4a, G4b
and G4c) reduced maternal weight (especially the ``FRU+VPE/Water+VPE''
and ``FRU+VPE/Water'' groups compared to the FRU group) and brought it
down to similar weights as in the water treatment groups.

The weight of the offspring was followed up from 7 days after birth
until adulthood at 60 days. In female offspring, the maternal fructose
exposure lead to increased body weight in early life (significant at 7
and 14 days of age) and in adulthood (60 days of age). While maternal
exercise was able to decrease and modulate the offsprings' body weight,
especially in adulthood. Although, the adiposity index of the female
offspring showed an increase for the fructose group, this difference was
not statistically significant compared to the water group. In the male
offspring an increase in body weight was mainly observed early in life
(7-14 days of age) and the maternal exercise only showed a significant
reduction in body weight at 7 days of age. Regardless, maternal fructose
consumption led to an increase in the male offspring's adiposity index
(OG3 = FRU group) and maternal exercise significantly decreased
adiposity in the FRU+VPE group (OG4a) at 60 days of life.

It is known that fructose can increase body weight in offspring, as
observed in our previous study by Magenis et al. (2022), where the
offspring were followed up to 30 days of age and an increase in body
weight was observed. Furthermore, it is suggested, as observed in our
current study, that physical exercise can reduce intrahepatic lipids and
abdominal fat with or without weight loss (Thyfault and Rector, 2020).
It is believed that physical exercise is able to increase adipogenesis
in brown adipose tissue, favoring mitochondrial biogenesis with an
increase in PGC-1alpha and UCP1 in male and female offspring of female
mice that performed exercise during pregnancy (Son et al. 2020).
Overall, our current results indicate that the maternal fructose
consumption can change the weight and adiposity of the offspring
throughout life, and that maternal voluntary physical exercise is able
to reduce these adverse effects.

4.3 The effect on disease risk markers

The alarming increase in metabolic problems, such as non-alcoholic fatty
liver disease (NASH), metabolic syndrome and type 2 diabetes mellitus
are worrying and studying the effects of life-style interventions, such
as physical exercise, is of great importance for their prevention and
treatment (Muriel et al., 2021). In view of this, the glycemic profile
of mothers and offspring and liver steatosis in the offspring were
evaluated in the current study.

\emph{4.3.1 The effects on insulin resistance in mothers and their
offspring}

Factors associated with insulin resistance include obesity, particularly
abdominal obesity, increased waist circumference, familial history of
type 2 diabetes, sedentary lifestyle, hypertension, and fatty liver.
Furthermore, is insulin resistance also characterized by
hypertriglyceridemia, lower concentrations of high-density lipoprotein
(HDL) cholesterol in blood, and increased inflammation. As a result,
glucose metabolism deteriorates, resulting in hyperglycemia, impaired
glucose tolerance or overt type 2 diabetes mellitus. Weight loss and
regular moderate intensity physical activity/exercise are significant
factors for insulin resistance prevention and/or treatment
(Papakonstantinou et al., 2022). In our work, we used voluntary physical
exercise as an intervention and it was observed to improve the glycemic
profile in the mothers and their offspring.

The glycemic profile in mothers was altered with FRU and the VPE had the
potential to modulate this effect, in different periods. Regarding
fasting glycemia and insulin tolerance test, in pre-pregnancy mothers we
observed an increase in fasting glycemia and insulin resistance in the
FRU group, while in combination with modulation the exercise
intervention (FRU+VPE group) there was a decrease in fasting glycemia
and modulation of insulin resistance. During pregnancy, we observed a
similar change with a decrease in fasting glucose in response to the
physical activity interventions (FRU+VPE/Water+VPE and FRU+VPE/Water
groups). In contrast, in the post-pregnancy period, an increase in
fasting glucose was observed in the Water+VPE group, however we did not
observe insulin resistance in any group during this period.

In the female offspring, we did not observe changes in fasting blood
glucose in the fructose group, but a possible modulation in the
FRU+VPE/Water+VPE group, due to the decrease in blood glucose. However,
in males no differences were found, only a decrease in the area under
the curve in the insulin tolerance test. With these results, we observed
that fructose exposure can cause a glycemic imbalance in the mothers and
that physical exercise may have the potential to modulate, but not
enough to be a modulator of insulin resistance in offspring.

Similar to our result, Mamikutty et al. (2014) observed that fructose
consumption at doses of 20\% and 25\% for a period of 8 weeks was able
to alter food consumption and lead to metabolic disorders such as
obesity, dyslipidemia, hypertension and hyperglycemia. This change in
the glycemic profile and insulin resistance is due to the fact that,
unlike glucose, fructose does not stimulate insulin secretion from
pancreatic \selectlanguage{greek}β \selectlanguage{english}cells. Furthermore, reduced insulin sensitivity in the
state of hypertriglyceridemia can lead to the formation of hyperglycemia
(Bray et al., 2004). Furthermore, excess fructose can increase the
production of its triosis phosphate metabolites and enter the Krebs
cycle for intrahepatic oxidation or synthesis of glucose, lactate and/or
lipids (Horst and Serlie, 2017). The change in the gestational period,
on the other hand, corroborates the results found in the study by
Magenis et al. 2020 and 2022, where the consumption of fructose during
the gestational period in mice was able to increase the fasting glucose
of the females and offspring. However, we did not observe insulin
resistance in the post-pregnancy period and in the offspring, and the
same was found in previous studies (Magenis et al., 2020; Magenis et
al., 2022).

Pregnancy is characterized by myriad metabolic adaptations that affect
glucose levels and may also alter the effects of exercise on glycemic
control. Glucose is the primary source of fetal energy and is required
for optimal fetal growth and development. Accordingly, hormonal changes
during pregnancy and in the placenta drive preferential feto-placental
glucose delivery, since maternal glucose production increases and
maternal insulin sensitivity is reduced as pregnancy progresses,
particularly at the level of skeletal muscle, in order to shift glucose
to the developing fetus (Motola and Artal, 2016; Barbour et al. 2007).
We believe that the mechanisms of fructose metabolic disorders are more
associated with an increase in free fatty acid, so there is an increase
in lipotoxicity and, consequently, oxidative stress and inflammation,
often not causing type 2 diabetes mellitus initially, only a glycemic
imbalance (Lipke et al. al., 2022).

Based on this assumption, physical exercise is necessary as an adjuvant
for the prevention and treatment of obesity and associated
comorbidities. To date, studies show that resistance and aerobic
exercises are recommended as effective treatments for people with
obesity and type 2 diabetes mellitus (Yang et al., 2014; Xiao and Fu,
2015; Villareal et al., 2017; Garc\selectlanguage{ngerman}ía-Hermoso et al., 2018). Furthermore,
the ACOG recommends the practice of physical exercise during the pre-
and gestational period in order to prevent gestational diabetes (ACOG,
2018). Among healthy pregnant women, regular exercise during pregnancy
reduces insulin resistance and upregulates skeletal muscle glucose
transporter GLUT4, (Davenport et al., 2018). Although our glycemic
results are not conclusive regarding insulin resistance, we suggest that
physical exercise has the potential to control the mother's fasting
glucose and can bring benefits to her offspring, as described in the
literature.

\emph{4.3.2 The risk of fatty liver disease in the offspring}

It has been previously shown that excessive fructose consumption leads
to dysfunctions in various tissues and organs, including liver, adipose
tissue, pancreatic, skeletal muscle, kidney, heart, brain, and intestine
(Muriel et al.,2021). The primary metabolites of fructose metabolism are
produced in the liver and secreted into the circulation, directly
affecting tissue and organ functions (Zhang et al., 2019). In humans,
70\% of fructose is metabolized in the liver, so a high fructose diet
induces hepatic - fatty acid synthesis and triglyceride accumulation
(Muriel et al., 2021). Since uncontrolled lipid metabolism is associate
with the development of non-alcoholic fatty liver disease (NASH) hepatic
steatosis was studied in the offspring. It is a term widely used to
describe excessive fatty infiltration in the liver in the absence of
alcohol, autoimmune disorders, or viral hepatitis; it is attributed to
obesity, high sugar and fat consumption, and sedentarism (Horst and
Serlie, 2017). The current study observed that maternal fructose
consumption during pregnancy leads to an increased area of steatosis in
the livers of male and female offspring, compared to the water groups.

Furthermore, it is suggested that physical exercise is capable of
decreasing intrahepatic lipids and the formation of lipid droplets,
decreasing free fatty acids and the effects of lipotoxicity, since
increased levels of free fatty acids can be toxic to the body cells, and
their sequestration by lipid droplets provides a buffering capacity that
prevents lipotoxicity (Olzmann \& Carvalho, 2019). Indeed in our current
study, the maternal physical exercise interventions were able to reduce
hepatic steatosis, demonstrating that the practice of voluntary physical
exercise by the mothers has a hepatic protective effect in the
offspring.

4.5 Fructose consumption enhances genome damage and VPE can prevent this

The metabolization of fructose results in liver glycogen and de novo
lipogenesis of free fatty acids (FFA) that can cause lipotoxicity (Zhang
et al., 2019), which is defined as the deleterious effect caused by high
concentrations of lipids and lipid derivatives that manifest as a set of
metabolic disorders in non-adipose tissue cells. The mechanisms involved
in lipotoxicity (also known as lipotoxic effects) include oxidative
stress, endoplasmic reticulum (ER) stress, and induction of inflammation
(Lipke et al., 2022). Since lipid peroxidation and oxidative stress can
cause damage to the genome, the genotoxic and mutagenic effects of the
fructose consumption was evaluated in the blood and liver of the mothers
and their offspring through the comet assay and micronucleus test.

In our study, the exposure to fructose led to increased DNA damage in
mothers and offspring and the intervention with physical exercise was
able to modulate and reduce the detected genome damage. In the mothers
increased DNA oxidation and DNA strand-breaks (considered as markers of
short-term exposures) were observed in both the blood and liver in
response to fructose consumption (during all the (pre/post)pregnancy
periods), as well as an increased number of MN (marker of long-term
exposures). Maternal fructose consumption led to increased DNA damage
(markers of short-term damage) in the blood of the offspring, while DNA
damage levels in the liver were only significantly increased in male
offspring due to fructose. The number of MN (marker of long-term
exposures) was increased in both female and male offspring from mothers
who consumed fructose. The VPE intervention led to the reduced
genotoxicity of fructose in mothers and offspring. Overall these results
indicate that the consumption of high concentrations of fructose in a
fetal programming model was genotoxic in the mother and their offspring
and that physical exercise was capable to protect genomic stability. The
genotoxic results found in liver in the offspring corroborate the
results found in histology, where we observed an increase in the area of
steatosis in the FRU group and a decrease in FRU+VPE.

Similar to these results, our previous studies also showed that fructose
leads to genomic instability in the mothers and their offspring (Magenis
et al., 2020; Magenis et al., 2022). The fructose can lead to decreased
expression of hepatic PGC-1alpha, a regulator of mitochondrial
biogenesis in fructose-fed rats, due to increased ROS and DNA damage
(Cioffi et al., 2017).

Diet rich in fructose promotes ROS imbalance through the simultaneous
increase in ROS production and the negative regulation of antioxidant
defense mechanisms, having as a result, widespread damage to
macromolecules such as lipids, proteins and DNA (Cioffi et al., 2017).
Furthermore, we suggest that maternal voluntary exercise has the ability
to positively modulate the antioxidant defense system, thus decreasing
the action of ROS and other reactive species that lead to DNA damage
(Saiyin et al., 2019). Tryfidou et al. (2020) also showed that physical
exercise can decrease inflammation, ROS and increase mitochondrial
biogenesis, thus improving genomic instability. Corroborating, Kasper et
al. (2021) showed that maternal physical exercise mediates hepatic
metabolic programming via activation of the AMPK-PGC1\selectlanguage{greek}α \selectlanguage{english}axis in the
offspring of obese mothers, which lead to reduced DNA damage in liver.

Conclusion

In this study, we sought to evaluate the different combinations and
durations of interventions with voluntary physical exercise during the
gestational period. Although, we did not find similar combinations of
interventions in the literature as in our metabolic programming model,
it has been reported that combining a change in eating habits and
lifestyle make the benefits more effective in healthy adults
(Martinez-Avila et al., 2020; Lelis et al., 2020).

We showed that the practice of physical exercise in the control group
did not lead to maternal and offspring changes, ie, it does not cause
harm. Overall, we observed that maternal voluntary physical exercise has
a protective effect on genomic stability in mothers and their offspring,
reversing the adverse effects observed in blood and liver in response to
the chronic high consumption of maternal fructose. In addition, we
provide evidence for metabolic programming caused by the various
maternal treatments that have a lasting effect in the offspring until
adulthood. However, the intervention with VPE was not able to reach the
point of returning to the similar levels as the controls. It is possible
that the effects on pregnant mice, as well as the transplacental effect
of the direct passage of fructose, confer indirect and direct metabolic
risk to the offspring later in life. However, more studies are needed in
order to understand its long-term effects on the adult life of the
offspring and also to study other metabolic pathways.

To conclude, our data indicate that, regardless the period during which
the exercise was performed (pre-, post- or during pregnancy), the
practice of exercise during pregnancy becomes essential to obtain the
observed metabolic and genomic benefits for the mother and offspring,
and that when combined with changing to water, the benefits are even
better.

\textbf{Acknowledgments:} The authors would also like to thank the
Graduate Program in Health Sciences (PPGCS), Coordination for the
Improvement of Higher Education Personnel (CAPES); National Council of
Technological and Scientific Development (CNPq -- 304203/2018-1); Santa
Catarina Research and Innovation Support Foundation (FAPESC); and the
University of Southern Santa Catarina (UNESC).

\textbf{Conflicts of Interest:} The authors declare no conflict of
interest.

\textbf{Funding:} This work was not funded.

\textbf{Data Accessibility Statement:} The animals body composition and
food consumption, genome damage assessment, fasting glycemia and insulin
tolerance test (ITT) are in the Microsoft Excel and Prism - GraphPad
database.

Histological analyzes were performed using the Axio Scan.Z1\selectlanguage{ngerman}® (ZEISS,
Jena, Germany) to capture images of all sample areas. After
pre-processing these images with Adobe Photoshop CS6 v 13.0 ® (Adobe,
San Jose, CA) in order to improve quality and separate the images, they
were analyzed with Image Pro Plus v.4.5.0.29.

\textbf{Benefit-Sharing Statement:} A research collaboration was
developed with scientists from the countries providing genetic samples,
all collaborators are included as co-authors, the results of research
have been shared with the provider communities and the broader
scientific community (see above), and the research addresses a priority
concern, in this case the conservation of organisms being studied. More
broadly, our group is committed to international scientific
partnerships, as well as institutional capacity building.

\textbf{Author Contributions:}

\emph{Designed research:} Marina Lummertz Magenis and Vanessa Moraes de
Andrade

\emph{Performed research:} Marina Lummertz Magenis, Adriani Paganini
Damiani, Isadora de Oliveira Monteiro, Ligia Salvan Dagostin and
Nicollas dos Santos da Silva\textsuperscript{.}

\emph{Contributed new reagents or analytical tools} : Rahisa Scussel,
Seigo Nagashima and Ricardo Aurino Pinho.

\emph{Analyzed data:} Marina Lummertz Magenis, Sabine A. S. Langie and
Vanessa Moraes de Andrade.

\emph{Wrote the paper:} Marina Lummertz Magenis, Sabine A. S. Langie and
Vanessa Moraes de Andrade.

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Ricardo de la Vega 1, Irini S Parastatidou 2, Roberto Ru\selectlanguage{ngerman}íz-Barquín 3,
Attila Szabo 4 5. Exercise Addiction in Athletes and Leisure Exercisers:
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Saiyin T, Engineer A, Greco ER, Kim MY, Lu X, Jones DL, Feng Q. Maternal
voluntary exercise mitigates oxidative stress and incidence of
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Soohyeon Koo,1,2,3,* Mina Kim,1,2,* Hyun Min Cho,1,2 and Inkyeom Kim.
Maternal high-fructose intake during pregnancy and lactation induces
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McIntire RK, Gutierrez HR, Cowan M, Paciorek CJ, Farzadfar F, Riley L,
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Surita FG, Nascimento SL, Silva JLP. Exercício físico e gestação. Rev
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TA, Fluckey JD, Carson JA, Dridi S, Washington TA, Greene NP. PGC-1\selectlanguage{greek}α\selectlanguage{english}4
gene expression is suppressed by the IL-6---MEK---ERK 1/2 MAPK
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Villareal DT, Aguirre L, Gurney AB, Waters DL, Sinacore DR, Colombo E,
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Wang CY, Liao JK. A mouse model of diet-induced obesity and insulin
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Xiao T, Fu Y-F. Resistance training vs. aerobic training and role of
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Yang Z, Scott CA, Mao C, Tang J, Farmer AJ. Resistance exercise versus
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Yi-Ju Hsu 1, Mon-Chien Lee 1, Chi-Chang Huang 1, Chun-Sheng Ho. The
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\textbf{Tables}

\textbf{Table 1.} Food consumption and body weight of the mothers that
received water or fructose and/or practiced voluntary physical exercise
pre-pregnancy, during pregnancy and post-pregnancy.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
& \textbf{Treatment} & \textbf{Liquid (mL/day)} & \textbf{Food (g/day)}
& \textbf{Calories (kcal/day)} & \textbf{Body weight (g)}\tabularnewline
\midrule
\endhead
\textbf{Pre-pregnancy} & Week 0-8 & & & &\tabularnewline
G1 & Water & 5.8 \selectlanguage{ngerman}± 1.09 & 4.5 \selectlanguage{ngerman}± 0.72 & 16.7 \selectlanguage{ngerman}± 2.55 & 30.8 \selectlanguage{ngerman}±
3.23\tabularnewline
G2 & Water + VPE & 7.2 \selectlanguage{ngerman}± 1.26 \textsuperscript{a} & 5.6 \selectlanguage{ngerman}±
0.40\textsuperscript{a} & 19.3 \selectlanguage{ngerman}± 2.52 & 30.7 \selectlanguage{ngerman}± 1.67\tabularnewline
G3 & FRU & 9.0 \selectlanguage{ngerman}± 3.88\textsuperscript{a} & 4.9 \selectlanguage{ngerman}± 0.42 & 39.6 \selectlanguage{ngerman}±
21.7\textsuperscript{a} & 39.3 \selectlanguage{ngerman}± 8.33\tabularnewline
G4 & FRU + VPE & 7.3 \selectlanguage{ngerman}± 1.89 & 5.0 \selectlanguage{ngerman}± 0.49 & 36.8 \selectlanguage{ngerman}± 15.37 & 30.8 \selectlanguage{ngerman}±
3.02\tabularnewline
\textbf{Pregnancy} & Week 9-12 & & & &\tabularnewline
G1 & Water & 10.9 \selectlanguage{ngerman}± 2.22 & 7.4 \selectlanguage{ngerman}± 2.13 & 28.5 \selectlanguage{ngerman}± 7.77 & 40.8 \selectlanguage{ngerman}±
1.78\tabularnewline
G2a & Water + VPE & 9.28 \selectlanguage{ngerman}± 2.37 & 7.5 \selectlanguage{ngerman}± 0.72 & 25.9 \selectlanguage{ngerman}± 3.05 & 40.6 \selectlanguage{ngerman}±
1.95\tabularnewline
G2b & Water + VPE/Water & 10.7 \selectlanguage{ngerman}± 3.14 & 7.3 \selectlanguage{ngerman}± 1.04 & 26.7 \selectlanguage{ngerman}± 4.78 & 40.8
\selectlanguage{ngerman}± 3.41\tabularnewline
G3 & FRU & 14.6 \selectlanguage{ngerman}± 5.8\textsuperscript{a} & 7.8 \selectlanguage{ngerman}± 0.70 & 57.9 \selectlanguage{ngerman}±
12.4\textsuperscript{a} & 45.4 \selectlanguage{ngerman}± 4.40\tabularnewline
G4a & FRU + VPE & 13.0 \selectlanguage{ngerman}± 5.17 & 7.0 \selectlanguage{ngerman}± 0.72 & 59.7 \selectlanguage{ngerman}± 15.4 & 40.6 \selectlanguage{ngerman}±
5.49\tabularnewline
G4b & FRU + VPE/ Water + VPE & 10.0 \selectlanguage{ngerman}± 2.86\textsuperscript{b} & 9.0 \selectlanguage{ngerman}±
1.11 & 54.4 \selectlanguage{ngerman}± 11.4 & 41.5 \selectlanguage{ngerman}± 4.08\tabularnewline
G4c & FRU + VPE/Water & 9.9 \selectlanguage{ngerman}± 2.96\textsuperscript{b} & 6.6 \selectlanguage{ngerman}±
0.25\textsuperscript{c} & 47.7 \selectlanguage{ngerman}± 10.0 & 41.3 \selectlanguage{ngerman}± 4.29\tabularnewline
\textbf{Post-pregnancy} & Week 12-15 & & & &\tabularnewline
G1 & Water & 16.4 \selectlanguage{ngerman}± 4.06 & 9.6 \selectlanguage{ngerman}± 2.80 & 30.1 \selectlanguage{ngerman}± 8.14 & 35.3 \selectlanguage{ngerman}±
1.15\tabularnewline
G2a & Water + VPE & 14.3 \selectlanguage{ngerman}± 4.11 & 15.8 \selectlanguage{ngerman}± 2.42 & 55.2 \selectlanguage{ngerman}± 8.74 & 34.4 \selectlanguage{ngerman}±
0.89\tabularnewline
G2b & Water + VPE/Water & 14.0 \selectlanguage{ngerman}± 3.71 & 12.0 \selectlanguage{ngerman}± 3.47 & 31.7 \selectlanguage{ngerman}± 12.0 & 37.8
\selectlanguage{ngerman}± 2.77\tabularnewline
G3 & FRU & 18.5 \selectlanguage{ngerman}± 5.19 & 13.8 \selectlanguage{ngerman}± 2.06 & 97.0 \selectlanguage{ngerman}± 6.46\textsuperscript{a} &
48.2 \selectlanguage{ngerman}± 8.34\tabularnewline
G4a & FRU + VPE & 15.5 \selectlanguage{ngerman}± 5.50 & 13.1 \selectlanguage{ngerman}± 3.77 & 75.2 \selectlanguage{ngerman}± 28.5 & 35.5 \selectlanguage{ngerman}±
4.79\textsuperscript{b}\tabularnewline
G4b & FRU + VPE/ Water + VPE & 14.4 \selectlanguage{ngerman}± 4.25 & 9.7 \selectlanguage{ngerman}± 3.11 & 86.3 \selectlanguage{ngerman}± 16.08 &
33.6 \selectlanguage{ngerman}± 3.78\textsuperscript{b}\tabularnewline
G4c & FRU + VPE/Water & 17.2 \selectlanguage{ngerman}± 6.94 & 11.8 \selectlanguage{ngerman}± 3.67 & 86.11 \selectlanguage{ngerman}± 18.7 & 32.4
\selectlanguage{ngerman}± 1.81\textsuperscript{b}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Data are expressed as mean ± standard deviation of the mean (n=10
animals per group). FRU- Fructose; VPE- voluntary physical
exercise.\textsuperscript{a} Significant difference in relation to the
water group (p \textless{} 0.05, Kruskal-Wallis, \emph{post}
\emph{hoc}Dunn's);\textsuperscript{b}Significant difference in relation
to the fructose group (p \textless{} 0.05, Kruskal-Wallis,
\emph{posthoc} Dunn's); \textsuperscript{c}Significant difference in
relation to the group FRU + VPE/ Water + VPE (p \textless{} 0.05,
Kruskal-Wallis, \emph{post} \emph{hoc} Dunn's);

\textbf{Table 2.} Fasting blood glucose (mg/dL) of mothers during the
various stages, while or after having received water or fructose and/or
practiced voluntary physical exercise during pre-pregnancy and/or
pregnancy.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& \textbf{Treatment} & \textbf{Glucose (mg/dL)}\tabularnewline
\midrule
\endhead
\textbf{Pre-pregnancy} & Week 0-8 &\tabularnewline
G1 & Water & 109.0 \selectlanguage{ngerman}± 5.91\tabularnewline
G2 & Water + VPE & 115.4 \selectlanguage{ngerman}± 15.7\tabularnewline
G3 & FRU & 142.5 \selectlanguage{ngerman}± 18.2\textsuperscript{a}\tabularnewline
G4 & FRU + VPE & 116.7 \selectlanguage{ngerman}± 13.7\textsuperscript{b}\tabularnewline
\textbf{Pregnancy} & Week 9-12 &\tabularnewline
G1 & Water & 104.3 \selectlanguage{ngerman}± 9.29\tabularnewline
G2a & Water + VPE & 115.3 \selectlanguage{ngerman}± 7.89\tabularnewline
G2b & Water + VPE/Water & 106.8 \selectlanguage{ngerman}± 14.2\tabularnewline
G3 & FRU & 131.0 \selectlanguage{ngerman}± 5.24\textsuperscript{a}\tabularnewline
G4a & FRU + VPE & 110.6 \selectlanguage{ngerman}± 5.59\tabularnewline
G4b & FRU + VPE/ Water + VPE & 101.4 \selectlanguage{ngerman}±
15.4\textsuperscript{b}\tabularnewline
G4c & FRU + VPE/Water & 92.83 \selectlanguage{ngerman}± 12.1\textsuperscript{b}\tabularnewline
\textbf{Post-pregnancy} & Week 12-15 &\tabularnewline
G1 & Water & 119.6 \selectlanguage{ngerman}± 1.47\tabularnewline
G2a & Water + VPE & 151.8 \selectlanguage{ngerman}± 7.29\textsuperscript{a}\tabularnewline
G2b & Water + VPE/Water & 139.6 \selectlanguage{ngerman}± 10.0\tabularnewline
G3 & FRU & 130.0 \selectlanguage{ngerman}± 9.59\tabularnewline
G4a & FRU + VPE & 107.2 \selectlanguage{ngerman}± 24.6\tabularnewline
G4b & FRU + VPE/ Water + VPE & 141.0 \selectlanguage{ngerman}±
13.9\textsuperscript{c}\tabularnewline
G4c & FRU + VPE/Water & 140.3 \selectlanguage{ngerman}± 15.6\textsuperscript{c}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Data are expressed as mean ± standard deviation of mean fasting blood
glucose (n=6 animals per group). FRU- Fructose; VPE- voluntary physical
exercise. \textsuperscript{a}Significant difference in relation to the
water group (p \textless{} 0.05, ANOVA, \emph{post hoc}
Tukey's);\textsuperscript{b}Significant difference in relation to the
fructose group (p \textless{} 0.05, ANOVA, \emph{post hoc}
Tukey's);\textsuperscript{c}Significant difference in relation to the
FRU + VPE group (p \textless{} 0.05, ANOVA, \emph{post hoc} Tukey's).

Table 3. Number of micronucleated polychromatic erythrocytes (MnPCE)
observed in bone marrow samples from mothers post-pregnancy, after they
had received water or fructose and/or practiced voluntary physical
exercise pre-pregnancy and/or during pregnancy.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\textbf{Treatment} & \textbf{MnPCE} & \textbf{PCE/NCE}\tabularnewline
\midrule
\endhead
Water & 0.33 \selectlanguage{ngerman}± 0.51 & 0.53 \selectlanguage{ngerman}± 0.02\tabularnewline
Water + VPE & 0.75 \selectlanguage{ngerman}± 0.95 & 0.53 \selectlanguage{ngerman}± 0.03\tabularnewline
Water + VPE/Water & 0.83 \selectlanguage{ngerman}± 0.75 & 0.54 \selectlanguage{ngerman}± 0.01\tabularnewline
FRU & 6.0 \selectlanguage{ngerman}± 1.15\textsuperscript{a} & 0.56 \selectlanguage{ngerman}± 0.02\tabularnewline
FRU + VPE & 3.25 \selectlanguage{ngerman}± 1.70\textsuperscript{b} & 0.52 \selectlanguage{ngerman}± 0.03\tabularnewline
FRU + VPE/ Water + VPE & 3.60 \selectlanguage{ngerman}± 1.67\textsuperscript{b} & 0.53 \selectlanguage{ngerman}±
0.02\tabularnewline
FRU + VPE/Water & 3.40 \selectlanguage{ngerman}± 1.51\textsuperscript{b} & 0.53 \selectlanguage{ngerman}±
0.02\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Data are expressed as mean of 4000 cells analyzed per sample ± standard
deviation of the mean (n=8 animals per
group).\textsuperscript{a}Significant difference in relation to the
water group (p\textless{} 0.05, Test \emph{t}
-student);\textsuperscript{b}Significant difference in relation to the
fructose group (p \textless{} 0.05, Test \emph{t} -student). MnPCE:
micronucleated polychromatic erythrocytes; PCE/NCE: the ratio of
polychromatic to normochromatic erythrocytes; FRU- Fructose; VPE-
voluntary physical exercise.

\textbf{Table 4.} Food consumption of offspring (females and males) born
to mothers that received water or fructose (FRU) and/or practiced
voluntary physical exercise (VPE) during pre-pregnancy and/or pregnancy.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& \textbf{Treatment} & \textbf{Food (g/day)} & \textbf{Calorie
(kcal/day)}\tabularnewline
\midrule
\endhead
\textbf{Female} & & &\tabularnewline
OG1 & Water & 3.6 \selectlanguage{ngerman}± 0.92 & 14.6 \selectlanguage{ngerman}± 2.55\tabularnewline
OG2a & Water + VPE & 5.8 \selectlanguage{ngerman}± 1.90\textsuperscript{a} & 17.6 \selectlanguage{ngerman}±
4.64\tabularnewline
OG2b & Water + VPE/Water & 4.1 \selectlanguage{ngerman}± 0.95 & 13.2 \selectlanguage{ngerman}± 2.3\tabularnewline
OG3 & FRU & 5.1 \selectlanguage{ngerman}± 1.29 & 18.8 \selectlanguage{ngerman}± 8.46\tabularnewline
OG4a & FRU + VPE & 5.4 \selectlanguage{ngerman}± 1.71 & 19.3 \selectlanguage{ngerman}± 6.62\tabularnewline
OG4b & FRU + VPE/ Water + VPE & 3.7 \selectlanguage{ngerman}± 1.09\textsuperscript{b} & 12.2 \selectlanguage{ngerman}±
3.0\tabularnewline
OG4c & FRU + VPE/Water & 4.4 \selectlanguage{ngerman}± 1.42 & 16.9 \selectlanguage{ngerman}± 5.27\tabularnewline
\textbf{Male} & & &\tabularnewline
OG1 & Water & 3.6 \selectlanguage{ngerman}± 0.92 & 12.4 \selectlanguage{ngerman}± 3.63\tabularnewline
OG2a & Water + VPE & 5.8 \selectlanguage{ngerman}± 2.42\textsuperscript{a} & 17.7 \selectlanguage{ngerman}±
3.87\tabularnewline
OG2b & Water + VPE/Water & 4.1 \selectlanguage{ngerman}± 0.95 & 16.5 \selectlanguage{ngerman}± 5.06\tabularnewline
OG3 & FRU & 5.1 \selectlanguage{ngerman}± 1.29 & 17.1 \selectlanguage{ngerman}± 4.36\tabularnewline
OG4a & FRU + VPE & 5.4 \selectlanguage{ngerman}± 1.71 & 22.1 \selectlanguage{ngerman}± 7.75\tabularnewline
OG4b & FRU + VPE/ Water + VPE & 3.7 \selectlanguage{ngerman}± 1.09\textsuperscript{b} & 14.9 \selectlanguage{ngerman}±
6.36\textsuperscript{b}\tabularnewline
OG4c & FRU + VPE/Water & 4.4 \selectlanguage{ngerman}± 1.4 & 15.8 \selectlanguage{ngerman}± 4.11\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Data are expressed as mean ± standard deviation of mean (n=10 animals
per group). \textsuperscript{a}Significant difference in relation to the
water group (p \textless{} 0.05, ANOVA , \emph{post hoc}
Tukey's);\textsuperscript{b}Significant difference in relation to the
FRU + VPE group (p \textless{} 0.05, ANOVA, \emph{post hoc} Tukey's).

\textbf{Table 5.} Body weight (g) at different ages of offspring
(females and males) born to mothers that received water or fructose
and/or practiced voluntary physical exercise during pre-pregnancy and/or
pregnancy.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Treatment}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{7} \textbf{days (g)}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{14} \textbf{days (g)}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{21} \textbf{days (g)}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{30} \textbf{days (g)}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{60} \textbf{days (g)}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Female}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
OG1\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Water\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5.2 \selectlanguage{ngerman}± 0.49\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
6.5 \selectlanguage{ngerman}± 0.54\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
19.6 \selectlanguage{ngerman}± 0.52\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
26.6 \selectlanguage{ngerman}±1.91\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
30.1 \selectlanguage{ngerman}± 2.59\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
OG2a\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Water + VPE\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5.0 \selectlanguage{ngerman}± 0.47\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
7.6 \selectlanguage{ngerman}± 0.57\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
17.8 \selectlanguage{ngerman}± 0.72\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
24.0 \selectlanguage{ngerman}± 1.27\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
31.3 \selectlanguage{ngerman}± 2.16\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
OG2b\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Water + VPE/Water\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5.8 \selectlanguage{ngerman}± 0.33\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
7.0 \selectlanguage{ngerman}± 0.73\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
18.8 \selectlanguage{ngerman}± 2.29\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
27.5 \selectlanguage{ngerman}± 1.48\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
33.0 \selectlanguage{ngerman}± 1.62\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
OG3\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
FRU\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
7.8 \selectlanguage{ngerman}± 1.05\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
10.7 \selectlanguage{ngerman}± 1.16\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
24.4 \selectlanguage{ngerman}± 0.90\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
28.2 \selectlanguage{ngerman}± 0.95\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
37.3 \selectlanguage{ngerman}± 2.47\textsuperscript{a}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
OG4a\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
FRU + VPE\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5.7 \selectlanguage{ngerman}± 1.46\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
7.2 \selectlanguage{ngerman}± 1.70\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
18.4 \selectlanguage{ngerman}± 2.28\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
21.97 \selectlanguage{ngerman}± 1.3\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
31.3 \selectlanguage{ngerman}± 2.45\textsuperscript{b}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
OG4b\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
FRU + VPE/ Water + VPE\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
6.6 \selectlanguage{ngerman}± 0.84\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
8.3 \selectlanguage{ngerman}± 1.63\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
19.9 \selectlanguage{ngerman}± 2.12\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
24.6 \selectlanguage{ngerman}± 0.49\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
30.6 \selectlanguage{ngerman}± 1.25\textsuperscript{b}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
OG4c\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
FRU + VPE/Water\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
6.4 \selectlanguage{ngerman}± 0.54\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
8.1 \selectlanguage{ngerman}± 0.98\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
24.5 \selectlanguage{ngerman}± 1.3\textsuperscript{c}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
26.1 \selectlanguage{ngerman}± 0.68\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
34.4 \selectlanguage{ngerman}± 2.47\textsuperscript{c}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Male}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
OG1\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Water\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5.6 \selectlanguage{ngerman}± 0.64\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
6.2 \selectlanguage{ngerman}± 0.66\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
20.6 \selectlanguage{ngerman}± 1.35\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
28.6 \selectlanguage{ngerman}± 4.02\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
32.5 \selectlanguage{ngerman}± 4.11\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
OG2a\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Water + VPE\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5.9 \selectlanguage{ngerman}± 0.91\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
7.5 \selectlanguage{ngerman}± 1.29\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
16.8 \selectlanguage{ngerman}± 1.05\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
27.5 \selectlanguage{ngerman}± 1.79\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
35.9 \selectlanguage{ngerman}± 1.82\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
OG2b\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Water + VPE/Water\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5.4 \selectlanguage{ngerman}± 0.92\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
6.8 \selectlanguage{ngerman}± 1.09\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
21.4 \selectlanguage{ngerman}± 0.89\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
27.5 \selectlanguage{ngerman}± 2.43\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
36.3 \selectlanguage{ngerman}± 2.26\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
OG3\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
FRU\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
7.8 \selectlanguage{ngerman}± 1.17\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
10.2 \selectlanguage{ngerman}± 1.19\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
29.1 \selectlanguage{ngerman}± 2.55\textsuperscript{a}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
29.7 \selectlanguage{ngerman}± 6.0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
34.1 \selectlanguage{ngerman}± 3.01\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
OG4a\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
FRU + VPE\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5.7 \selectlanguage{ngerman}± 1.32\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
9.3 \selectlanguage{ngerman}± 5.77\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
24.2 \selectlanguage{ngerman}± 1.06\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
26.5 \selectlanguage{ngerman}± 2.35\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
36.0 \selectlanguage{ngerman}± 3.35\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
OG4b\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
FRU + VPE/ Water + VPE\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
7.2 \selectlanguage{ngerman}± 1.03\textsuperscript{c}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
10.0 \selectlanguage{ngerman}± 2.44\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
24.3 \selectlanguage{ngerman}± 2.0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
27.6 \selectlanguage{ngerman}± 5.42\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
36.0 \selectlanguage{ngerman}± 1.37\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
OG4c\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
FRU + VPE/Water\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
6.1 \selectlanguage{ngerman}± 0.40\textsuperscript{b}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
7.0 \selectlanguage{ngerman}± 1.09\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
24.7 \selectlanguage{ngerman}± 1.45\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
28.5 \selectlanguage{ngerman}± 2.11\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
37.5 \selectlanguage{ngerman}± 2.73\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Data are expressed as mean ± standard deviation of mean (n=10 animals
per group). FRU- Fructose; VPE- voluntary physical
exercise.\textsuperscript{a}Significant difference in relation to the
water group (p \textless{} 0.05, Kruskal-Wallis, \emph{post} \emph{hoc}
Dunn's);\textsuperscript{b}Significant difference in relation to the FRU
group (p \textless{} 0.05, Kruskal-Wallis, \emph{post} \emph{hoc}
Dunn's);\textsuperscript{c c}Significant difference in relation to the
FRU + VPE group (p \textless{} 0.05, Kruskal-Wallis, \emph{post}
\emph{hoc}Dunn's).

\textbf{Table 6.} Fasting blood glucose (mg/dL) of offspring (females
and males) of mothers that received water or fructose and/or practiced
voluntary physical exercise during pre-pregnancy and/or pregnancy.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& \textbf{Treatment} & \textbf{Glucose (mg/dL)}\tabularnewline
\midrule
\endhead
\textbf{Female} & &\tabularnewline
& Water & 139.8 \selectlanguage{ngerman}± 17.1\tabularnewline
& Water + VPE & 139.6 \selectlanguage{ngerman}± 17.6\tabularnewline
& Water + VPE/Water & 145.5 \selectlanguage{ngerman}± 26.4\tabularnewline
& FRU & 158.8 \selectlanguage{ngerman}± 4.92\tabularnewline
& FRU + VPE & 159.4 \selectlanguage{ngerman}± 12.5\tabularnewline
& FRU + VPE/ Water + VPE & 123.6 \selectlanguage{ngerman}±
9.4\textsuperscript{a.b}\tabularnewline
& FRU + VPE/Water & 149.8 \selectlanguage{ngerman}± 23.6\tabularnewline
\textbf{Male} & &\tabularnewline
& Water & 131.2 \selectlanguage{ngerman}± 12.6\tabularnewline
& Water + VPE & 121.4 \selectlanguage{ngerman}± 12.3\tabularnewline
& Water + VPE/Water & 135.6 \selectlanguage{ngerman}± 11.7\tabularnewline
& FRU & 130.6 \selectlanguage{ngerman}± 15.9\tabularnewline
& FRU + VPE & 133.5 \selectlanguage{ngerman}± 9.0\tabularnewline
& FRU + VPE/ Water + VPE & 131.6 \selectlanguage{ngerman}± 21.2\tabularnewline
& FRU + VPE/Water & 125.0 \selectlanguage{ngerman}± 5.0\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Data are expressed as mean ± standard deviation of mean (n=6 animals per
group). FRU- Fructose; VPE- voluntary physical
exercise.\textsuperscript{a}Significant difference in relation to the
fructose group (p \textless{} 0.05, ANOVA, \emph{post hoc}
Tukey's);\textsuperscript{b}Significant difference in relation to the
FRU + VPE group (p \textless{} 0.05, ANOVA, \emph{post hoc} Tukey's).

Table 7. Number of micronucleated polychromatic erythrocytes (MnPCE)
observed in bone marrow samples from offspring (males and females) of
mothers that received water or fructose and/or practiced voluntary
physical exercise during pre-pregnancy and/or pregnancy\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& \textbf{Treatment} & \textbf{MnPCE} & \textbf{PCE/NCE}\tabularnewline
\midrule
\endhead
\textbf{Female} & & &\tabularnewline
OG1 & Water & 0.16 \selectlanguage{ngerman}± 0.40 & 0.52 \selectlanguage{ngerman}± 0.02\tabularnewline
OG2a & Water + VPE & 0.17 \selectlanguage{ngerman}± 0.41 & 0.54 \selectlanguage{ngerman}± 0.03\tabularnewline
OG2b & Water + VPE/Water & 0.40 \selectlanguage{ngerman}± 0.54 & 0.54 \selectlanguage{ngerman}± 0.02\tabularnewline
OG3 & FRU & 2.5 \selectlanguage{ngerman}± 1.87\textsuperscript{a} & 0.54 \selectlanguage{ngerman}± 0.03\tabularnewline
OG4a & FRU + VPE & 1.83 \selectlanguage{ngerman}± 0.75 & 0.53 \selectlanguage{ngerman}± 0.02\tabularnewline
OG4b & FRU + VPE/ Water + VPE & 1.66 \selectlanguage{ngerman}± 0.81 & 0.51 \selectlanguage{ngerman}± 0.02\tabularnewline
OG4c & FRU + VPE/Water & 1.80 \selectlanguage{ngerman}± 0.83 & 0.54 \selectlanguage{ngerman}± 0.02\tabularnewline
\textbf{Male} & & &\tabularnewline
OG1 & Water & 1.28 \selectlanguage{ngerman}± 0.95 & 0.52 \selectlanguage{ngerman}± 0.02\tabularnewline
OG2a & Water + VPE & 1.16 \selectlanguage{ngerman}± 0.75 & 0.53 \selectlanguage{ngerman}± 0.02\tabularnewline
OG2b & Water + VPE/Water & 1.75 \selectlanguage{ngerman}± 1.25 & 0.53 \selectlanguage{ngerman}± 0.02\tabularnewline
OG3 & FRU & 3.66 \selectlanguage{ngerman}± 1.21\textsuperscript{a} & 0.51 \selectlanguage{ngerman}± 0.01\tabularnewline
OG4a & FRU + VPE & 2.14 \selectlanguage{ngerman}± 2.54 & 0.52 \selectlanguage{ngerman}± 0.02\tabularnewline
OG4b & FRU + VPE/ Water + VPE & 2.16 \selectlanguage{ngerman}± 1.72 & 0.49 \selectlanguage{ngerman}± 0.09\tabularnewline
OG4c & FRU + VPE/Water & 2.14 \selectlanguage{ngerman}± 2.11 & 0.53 \selectlanguage{ngerman}± 0.02\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Data are expressed as mean of 4000 cells analyzed per sample ± standard
deviation of the mean (n=8 animals per
group).\textsuperscript{a}Significant difference in relation to the
water group (p\textless{} 0.05, Test \emph{t} -student). MnPCE:
micronucleated polychromatic erythrocytes; PCE/NCE: the ratio of
polychromatic to normochromatic erythrocytes; FRU- Fructose; VPE-
voluntary physical exercise.

\textbf{Figures}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 1} . Experimental Design. The mothers were divided into
four experimental groups during the pre-pregnancy period, until week 9:
G1- Water: females that received water and did not practice voluntary
physical exercise (n=10); G2 -- Water + VPE: females that received water
and practiced voluntary physical exercise (n=20); G3 --FRU: females that
received 20\% fructose/L (n=10); G4 --FRU + VPE: females that received
20\% fructose/L and practiced voluntary physical exercise (n=30). After
this period, interventions were carried out for which the females were
subdivided into 7 groups: G1 - Water: females continued to receive water
and did not practice voluntary physical exercise until the end of
pregnancy (n=10); G2a -- Water + VPE: females that received water and
practiced voluntary physical exercise until the end of pregnancy (n=10);
G2b -- Water + VPE/Water: females that received water and practiced
voluntary physical exercise until copulation and then stopped the VPE
and continued only with water until the end of the pregnancy period
(n=10); G3 -- FRU: females that continued to receive 20\% fructose/L
(n=10); G4a -- FRU + VPW: females that received 20\% fructose/L and
practiced voluntary physical exercise until the end of pregnancy (n=10);
G4b --FRU + VPE/Water + VPE: females that received 20\% fructose/L and
practiced voluntary physical exercise until copulation, after this
period stopped fructose consumption and continued receiving only water
and practiced voluntary physical exercise during pregnancy (n=10); G4c
-- FRU + VPE/Water: females that received 20\% fructose/L and practiced
voluntary physical exercise until copulation, after which they stopped
fructose consumption and voluntary physical exercise and received only
water during pregnancy (n=10). *During lactation only fructose
supplementation was continued, while VPE was stopped due to limited
space in the cages of the mothers and the pups. After weaning (week 15),
the offspring continued to be monitored according to the seven
experimental groups, referred to as offspring groups (OG). FRU:
fructose; VPE: Voluntary Physical Exercise;

blood collection from mother;

assessment of VPE;

blood collection from offspring.

\textbf{Figure 2.} Caloric intake (kcal) of mothers during the various
stages, while or after receiving water or fructose (FRU) and/or
practiced voluntary physical exercise (VPE) during pre-pregnancy and/or
pregnancy. Data are expressed as mean \selectlanguage{ngerman}± standard deviation of mean
consumption of calories (n=10 animals per
group).\textsuperscript{a}Significant difference in relation to the
water group (p \textless{} 0.05, Kruskal-Wallis, \emph{post hoc}
Dunn's).

\textbf{Figure 3} . Fructose concentration (mg/mL) determined by high
performance liquid chromatography in the serum of mothers that received
water or fructose (FRU) and/or practiced voluntary physical exercise
(VPE) during pre-pregnancy and/or pregnancy. Data are expressed as mean
± standard deviation of mean (n=6 animals per
group).\textsuperscript{a}Significant difference in relation to the
water group (p \textless{} 0.05, Test \emph{t} -student);
\textsuperscript{b}Significant difference in relation to the fructose
group (p \textless{} 0.05, Test \emph{t} -student).

\textbf{Figure 4.} Insulin tolerance test of mothers that received water
or fructose (FRU) and/or practiced voluntary physical exercise (VPE)
during pre-pregnancy and/or pregnancy. Data are expressed as mean ±
standard deviation of mean (n=6 animals per
group).\textsuperscript{a}Significant difference in relation to the
water group (p \textless{} 0.05, ANOVA, \emph{post hoc} Tukey's);
\textsuperscript{b}Significant difference in relation to the fructose
group (p \textless{} 0.05, ANOVA, \emph{post hoc} Tukey's). AUC: Area
under the curve. Based on the graphs on the left, the areas under the
curves were computed and presented in the graphs on the right in the
insulin tolerance test.

\textbf{Figure 5.} DNA damage index in peripheral blood cells of mothers
that received water or fructose (FRU) and/or practiced voluntary
physical exercise (VPE) during pre-pregnancy and/or pregnancy. Data are
expressed as mean ± standard deviation of the mean (n=8 animals per
group). 5A: peripheral blood in pre-pregnancy; 5B: peripheral blood in
pregnancy; 5C: peripheral blood in
post-pregnancy.\textsuperscript{a}Significant difference in relation to
the water group (p \textless{} 0.05, ANOVA, \emph{post hoc}
Tukey's);\textsuperscript{b}Significant difference in relation to the
fructose group (p \textless{} 0.05, ANOVA, \emph{post hoc} Tukey's).
FPG: formamidopyrimidine DNA glycosylase.

\textbf{Figure 6.} DNA damage index (assessed as strand breaks) in liver
cells of mothers that received water or fructose (FRU) and/or practiced
voluntary physical exercise (VPE) during pre-pregnancy and/or pregnancy.
Data are expressed as mean ± standard deviation of the mean (n=8 animals
per group). \textsuperscript{a}Significant difference in relation to the
water group (p \textless{} 0.05, ANOVA, \emph{post hoc}
Tukey's);\textsuperscript{b}Significant difference in relation to the
fructose group (p \textless{} 0.05, ANOVA, \emph{post hoc} Tukey's).

\textbf{Figure 7} . Fructose concentration (mg/mL) determined by high
performance liquid chromatography of the offspring (females and males)
of mothers that received water or fructose and/or practiced voluntary
physical exercise during the pre-pregnancy period /or pregnancy. Data
are expressed as mean ± standard deviation of mean (n=6 animals per
group). FRU- Fructose; VPE- voluntary physical
exercise.\textsuperscript{a}Significant difference in relation to the
water group (p \textless{} 0.05, ANOVA, \emph{post hoc}
Tukey's);\textsuperscript{b}Significant difference in relation to the
FRU group (p \textless{} 0.05, ANOVA, \emph{post hoc} Tukey's).

\textbf{Figure 8.} Body adiposity index of offspring (females and males)
of mothers that received water or fructose and/or practiced voluntary
physical exercise during pre-pregnancy and/or pregnancy. Data are
expressed as mean ± standard deviation of mean (n=6 animals per group).
FRU- Fructose; VPE- voluntary physical
exercise.\textsuperscript{a}Significant difference in relation to the
water group (p \textless{} 0.05, ANOVA, \emph{post hoc} de
Tukey's);\textsuperscript{b}Significant difference in relation to the
FRU group (p \textless{} 0.05, ANOVA, \emph{post hoc} de Tukey's).

\textbf{Figure 9.} Insulin tolerance test of offspring (females and
males) of mothers that received water or fructose and/or practiced
voluntary physical exercise during pre-pregnancy and/or pregnancy. Data
are expressed as mean ± standard deviation of mean (n=6 animals per
group). FRU- Fructose; VPE- voluntary physical
exercise.\textsuperscript{a}Significant difference in relation to the
FRU group (p \textless{} 0.05, ANOVA, \emph{post hoc}
Tukey's);\textsuperscript{b}Significant difference compared to the FRU +
VPE group (p \textless{} 0.05, ANOVA, \emph{post hoc} Tukey's). AUC:
area under the curve.

\textbf{Figure 10.} Histological profile of the hepatic tissue of the
offspring (females and males) of mothers that received water or fructose
and/or practiced voluntary physical exercise during pre-pregnancy and/or
pregnancy. The upper pannel shows the representative images of female
and male offspring. The lower pannel shows the quantified percentage of
steatosis of female and male offspring. Representative images of
histological photomicrographs of cross-sections of the liver of the
animals. A: Water; B: Water + VPE; C: Water + VPE/Water; D: FRU; E: FRU
+ VPE; F: FRU + VPE/Water + VPE; G: FRU + VPE/Water. Data are expressed
as mean ± standard deviation of mean (n=8 animals per group). FRU-
Fructose; VPE- voluntary physical
exercise.\textsuperscript{a}Significant difference in relation to the
water group (p \textless{} 0.05, Kruskal-Wallis, \emph{post hoc}
Dunn's);\textsuperscript{b}Significant difference in relation to the FRU
group (p \textless{} 0.05, Kruskal-Wallis, \emph{post hoc}
Dunn's);\textsuperscript{c}Significant difference in relation to the FRU
+ VPE group (p \textless{} 0.05, Kruskal-Wallis, \emph{post hoc}
Dunn's);\textsuperscript{d}Significant difference compared to the FRU +
VPE/Water + VPE group (p \textless{} 0.05, Kruskal-Wallis, \emph{post
hoc} Dunn's).

\textbf{Figure 11.} DNA damage index in peripheral tissue cells of the
offspring (female and male) of mothers that received water or fructose
(FRU) and/or practiced voluntary physical exercise (VPE) during
pre-pregnancy and/or pregnancy. Data are expressed as mean ± standard
deviation of mean (n=8 animals per group).\textsuperscript{a}Significant
difference in relation to the water group (p \textless{} 0.05, ANOVA,
\emph{post hoc} Tukey's);\textsuperscript{b}Significant difference in
relation to the FRU group (p \textless{} 0.05, ANOVA, \emph{post hoc}
Tukey's).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-5/Figure-5}

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\textbf{Hosted file}

\verb`Tables.docx`  available at \url{https://authorea.com/users/659248/articles/663256-maternal-exercise-during-pregnancy-modulates-genetic-and-biochemical-damage-caused-by-high-consumption-of-fructose-in-blood-and-liver-of-offspring}

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