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).