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).