Hippocampal neurogenesis and Arc expression are enhanced in high-fat fed prepubertal female pigs by a diet including omega-3 fatty acids and Bifidobacterium breve CECT8242

Animals and treatments

A total of 43 female piglets from a Duroc pig line (Sus scrofa domesticus) were used in the present study. Animals were born in 11 different litters (i.e., 11 groups of 3–4 littermates). Same sex littermates, from same father and mother, were randomly distributed into 4 experimental groups, using a matched pairs experimental design. After weaning, piglets were transferred to the IRTA pig experimental station, and subjected to the same management procedures as described in Ballester et al. [31] and Jove et al. [32]. Briefly, at 9 weeks of age, animals were located in environmentally monitored facilities, randomly distributed into 4 pens (10–11 animals per pen from different litters) and fed ad libitum for 10 weeks with 4 different dietary treatments giving rise to four different experimental groups: (T1) a conventional (and balanced) growth diet according to Nutrition Resource Centre (NRC) recommendations used as a control diet; (T2) a western-type diet formulated with a high fat and high saccharose content and protein of animal origin (caseinate); (T3) a western-type diet in which 50% of the protein was substituted for protein of vegetal origin (rice hydrolysate) and including 5 × 1010 cfu/day B. breve probiotic CECT8242; and T4) a diet similar to T3, supplemented with omega-3 fatty acid (1 g stearidonic acid and 2 g docosahexaenoic acid per 100 g fat) (Fig. 1). Components and nutritional details about the feed provided to piglets have been previously reported in [33] and detailed in supplementary tables S1 and S2, respectively.

Fig. 1figure 1

Schematic representation of the experimental design. A total of 43 female piglets turning 9 weeks old were randomly distributed into 4 experimental groups, each consisting in a different dietary treatment. T1: conventional diet (N = 11), T2: Western-type diet (high-fat and high-saccharose diet, protein of animal origin) (N = 10), T3: western-type diet with protein of vegetal origin and B. breve probiotic (N = 11), T4: western-type diet with protein of vegetal origin, B. breve probiotic and omega-3 fatty acids (N = 11). After 10 weeks of feeding, blood samples were collected, pigs were slaughtered and brain samples were obtained, as thoroughly explained in the “Materials and methods” section

Each pen had a partly slatted floor (60% solid concrete and 40% slatted), with some sawdust provided on the concrete floor on a regular basis and a natural light cycle, with a minimum of intensity of 40 lx (EU legislation on pig welfare) and 8 h light. The room temperature was maintained at 22 ± 5 °C.

The experiment lasted until pigs reached 19 weeks of age, when animals were slaughtered after an overnight fasting, at IRTA experimental slaughterhouse in totally controlled conditions and in compliance with all welfare regulations. Pigs were weighed individually at the beginning of the experiment and every two weeks during the whole experiment, as well as on the day before slaughter. Daily food intake (FI; kg/day) was controlled individually by means of automatic electronic feeding system (HOKOFARM, IVO-G®, Marknesse, The Netherlands).

Sample collection

Blood samples for biochemical analyses were taken from overnight-fasted animals immediately before slaughter. After serum separation by centrifugation, lipids and other conventional biochemical variables, including triglycerides (TG) (Spinreact, 1001310), total cholesterol (TC) (Spinreact 1001091), LDL cholesterol (Spinreact 41023), and HDL cholesterol (Spinreact 1001096), were measured using commercial kits from Spinreact (Girona, Spain).

Animals were stunned by exposure to 90% CO2 at atmospheric air for 3 min and exsanguinated afterwards. The brain was removed from the skull and dissected to obtain the dorsal part of the left hippocampi, which corresponds to the anterior hippocampus in rodents. The hippocampi were fixed in 4% paraformaldehyde in phosphate buffer saline (PBS), pH 7.4, solution for 4 h and then placed in 15% sucrose in PBS for 3 days at 4 °C followed by 30% sucrose in PBS at 4 °C until they sank. Frozen tissue was cut in a cryostat (Cryocut 1800, with 2020 JUNG microtome) at – 25 °C, to obtain 30 µm sections. Serial sections were collected in six sets, each containing 12 slices, where each slice is 180 µm apart from the next, and stored at – 80 °C until immunohistochemistry staining.

Immunohistochemistry

Immunohistochemistry labeling protocols were performed by free-floating method. For DCX detection, sections were post-fixed in 2% formaldehyde in PBS for 20 min. Endogenous peroxidase was inactivated by incubation in 0.3% H2O2 in PBS and the blocking and permeabilization steps consisted of incubation in 0.1% BSA, 0.05% Tween 20 in tris-buffered saline pH 7.6 (TBS) for 30 min plus 30 min in donkey serum 1:200 in Tween 20 in TBS (TBS-T) at room temperature (RT). Sections were incubated in primary antibody anti-DCX (1:600, sc 8066 Santa Cruz) in 0.1% BSA TBS-T, for 1 h at RT and ON at 4 °C. The secondary antibody donkey anti-goat biotin (1:500, Jackson Immunosearch) was applied for 1 h at RT followed by incubation with SA-HRP (1:1800, Perkin Elmer) for 2 h at RT and visualized with diaminobenzidine (DAB) using a DAB substrate kit (Vector, Burlingame, USA). For Arc detection, sections were post-fixed using 3% formaldehyde in PBS for 20 min. After incubation in 0.3% H2O2 in PBS, tissues were permeabilizated with PBS-T, PBS and PBS-T, and transferred to the blocking solution (0.1% BSA, PBS-T) for 30 min at RT. Sections were then incubated with mouse anti-Arc antibody (1:60 sc 166,461 Santa Cruz, EUA) for 4 h at RT and 72 h at 4 °C. Subsequently, the secondary antibody anti-mouse IgG biotin (1:100, BA-2001, Vector, EUA) was applied for 1 h at RT followed by incubation with SA-HRP (1:3600, Pierce 21124) for 2 h at RT and DAB detection. Finally, sections were mounted onto slides, desiccated at RT 24 h, dehydrated and coverslipped with Pertex mounting medium (Sigma, Aldrich).

Image acquisition and analysis

Images were obtained using a digital camera (OlympusXC-50) coupled to OlympusVanox-T microscope. Images were analyzed with the Image J v1.50i® (Wayne Rasband, National Institutes of Health, EUA) free software.

For DCX analysis, images were obtained using a 20X objective. DCX labeling was measured as number of immunopositive cells/mm using regions of interest (ROIs) along different DG layers. DCX-positive cells were recorded in the crest, the suprapyramidal blade (SP) and the infrapyramidal blade (IP) of the DG. Appropriate gray threshold and particle sizes for DCX cell countings were set for each area and maintained for all subjects. Particle sizes were set to discriminate isolated cells from several overlapping cells, which were manually identified. For Arc analysis, images were obtained using a 4X objective and Arc-positive labeling was recorded in different regions of interest (ROIs) of each hippocampal section: the suprapyramidal blade (SP), the infrapyramidal blade (IP) of the DG, and the hilus, CA1, CA2 and CA3 hippocampal regions. Arc grayscale intensity levels were measured using circular ROIs as area labeled per mm2. To remove background noise, each image was digitally smoothed and subtracted from the original. The number of cell countings from DCX or Arc labeling intensity in every region was averaged from three sections from each subject.

Data analysis

The statistical computer package IBM SPSS Statistics 25.0 was used to process the data. Physiological, biochemical and immunohistological variables were analyzed by univariate one-way ANOVA or Kruskal Wallis test for nonparametric data, except for DCX and Arc immunostaining of DG subregions that were conducted by using GLM repeated measures model. After Levene’s test, post hoc tests were performed for detailed multiple comparisons, using Bonferroni or Tukey HSD if equal variances were assumed (respectively, for univariant or GLM repeat measures) or Games–Howell if they were not assumed. The Spearman’s correlation coefficient rho was used to analyze correlated patterns of DCX and Arc measurements with body weight, TG, TC, LDL, HDL, TC/HDL, along with linear regression line modeling of the main correlations. Significance was considered when p value < 0.05.

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