Ad libitum consumption of milk supplemented with omega 3, 6, and 9 oils from infancy to middle age alters behavioral and oxidative outcomes in male mice

Abstract

We tested the hypothesis that administration of omega (ω)-9, ω-3, and ω-6 to mice can prevent oxidative alterations responsible for behavioral and cognitive alterations related with aging. Twenty-eight-day-old mice received skim milk (SM group), SM enriched with omega oil mixture (EM group), or water (control group) for 10 and 14 months, equivalent to middle age. Mice were evaluated for behavioral alterations related to depression and memory and oxidative status [brain levels of thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), and myeloperoxidase (MPO)]. The 10-month EM group increased immobility time during the forced swimming test compared with control, indicating increased stress response. The 14-month SM- and EM-treated groups increased sucrose consumption compared with control, showing an expanded motivational state. The 14-month SM group decreased the number of rearings compared with the 14-month control and EM groups. The number of entries and time spent in the central square of the open field was higher in the 10-month EM group than in the control, revealing an anxiolytic-like behavior. TBARS decreased in the hippocampus and striatum of the 10-month EM group compared with the control. A similar decrease was observed in the striatum of the 10-month SM group. GSH levels were higher in all 14-month treated groups compared with 10-month groups. MPO activity was higher in the 14-month EM group compared with the 14-month control and SM groups, revealing a possible pro-inflammatory status. In conclusion, omega oils induced conflicting alterations in middle-aged mice, contributing to enhanced behavior and anxiolytic and expanded motivational state, but also to increased stress response and pro-inflammatory alterations.

Figure 1. Timeline of the experimental design of the study.

Figure 2. Alternations in the Y maze (A), immobility time in forced swimming (B), and sucrose preference (C). Data are reported as means±SE of 6-12 animals/group. Data were analyzed by two-way ANOVA considering the factors “time of administration” (10 and 14 months) and “milk supplementation” (water, skim milk, and enriched milk). **P<0.01, ****P<0.0001.

Figure 3. Representation of behavioral parameters obtained in the open field test. Number of crossings (A) and number of rearings (B) during 5 min, rearing time (C), and number of entries (D) and time spent (E) in the central square of the field. Each bar represents means±SE of 6-12 animals/group. Data were analyzed by two-way ANOVA considering the factors “time of administration” (10 and 14 months) and “milk supplementation” (water, skim milk, and enriched milk). *P<0.05, **P<0.01.

Figure 4. Lipid peroxidation in the hippocampus (A) and striatum (B). Each bar represents means±SE of 6-12 animals/group. Data were analyzed by two-way ANOVA considering as factors “time of administration” (10 and 14 months) and “milk supplementation” (water, skim milk, and enriched milk). *P<0.05, ***P<0.001, ****P<0.0001.

Figure 5. GSH levels in the hippocampus (A) and striatum (B). Each bar represents means±SE of 6-12 animals/group. Data were analyzed by two-way ANOVA considering as factors “time of administration” (10 and 14 months) and “milk supplementation” (water, skim milk, and enriched milk). *P<0.05 and ***P<0.001.

Figure 6. Myeloperoxidase activity in the hippocampus (A) and striatum (B). Each bar represents means±SE of 6-12 animals/group. Data were analyzed by two-way ANOVA considering as factors “time of administration” (10 and 14 months) and “milk supplementation” (water, skim milk, and enriched milk). *P<0.05, **P<0.01, and ***P<0.001.

Figure 7. Weight of mice during treatment protocol (A) and average liquid intake per mouse (B). Each bar represents means±SE of 6-12 animals/group. Data were analyzed by two-way ANOVA. *P<0.05, ***P<0.001 compared with the control group in each day of treatment.