Influence of maternal and postnatal diets on body weight

Table 1 shows anthropometric data of rats used in this study which was a subset of animals from our previous study [17]. The Caf diet increased maternal consumption of sugar, saturated fat and protein, relative to chow diet [17]. Prior to mating, mothers fed the Caf diet had significantly elevated adiposity but no significant change in fasting blood glucose. When euthanized after lactation, RP fat mass was still elevated in Caf versus control dams, with no difference in body weight or girth [17].

Table 1 Anthropometric data of animals used in this study

Mothers body weight from diet day 30 to the end of lactation was analysed by two-way repeated measures ANOVA. Caf diet consumption significantly increased dams body weight across time (main effect of maternal diet, F = 19.87, p < 0.001); and main effect of time (F = 283.5, p < 0.0001) with significant interaction between maternal diet and time (F = 38.46, p < 0.0001) (Table 1). Retroperitoneal (RP) fat in Caf mothers was significantly heavier than Chow mothers (t = 2.20, p < 0.05). For weanlings, maternal diet and sex did not affect body weight. Interestingly, RP fat mass differed according to maternal diet (main effect of maternal diet, F = 21.8, p < 0.01) and sex (main effect of sex, F = 11.4, p < 0.01), however there was no significant maternal diet × sex interaction. At 7 weeks, body weight differed according to postnatal diet (main effect of postnatal diet, F = 70.0, p < 0.01) and sex (main effect of sex, F = 589.6, p < 0.01). Maternal diet did not affect body weight nor was there any maternal diet, postnatal diet and sex interactions. At 14 weeks, significant sex × maternal diet interaction on RP fat (F = 6.17, p < 0.05) and body weight (F = 4.3, p < 0.05) indicated that maternal diet differentially affected males and females. Also, there was a significant sex × postnatal diet interaction (F = 31.66, p < 0.01) for RP fat mass. However, there was no significant three-way (maternal diet × postnatal diet × sex) or two-way (maternal diet × postnatal diet) interaction on body weight and RP fat.

For additional phenotypic data of mothers and offspring, overall macronutrient intake and 24 h food consumption in the dams, see the related study [17] and our previous work [28]. Gut microbial diversity of each group was assessed by 16S rRNA genes from fecal samples collected at times shown in Fig. 1.

Lasting effects of maternal obesogenic diet consumption on gut microbiota α-diversity in mothers and offspring

We first examined α-diversity measures across groups and time. Caf diet consumption significantly depleted species richness, evenness and the Shannon diversity index in Caf mothers (t = 3.05, p < 0.01, Fig. 2A; t = 3.84, p < 0.01, Fig. 2B; and t = 3.42, p < 0.01, Fig. 2C respectively).

Fig. 2
figure 2

α-diversity across groups and time. A Species Richness; B Evenness; and C Shannon Index. **p < 0.01 independent t test; *p < 0.05 main effect of maternal diet. #p < 0.05; ##p < 0.01; ###p < 0.001 by Post hoc comparisons. Data are displayed as mean ± SEM. Chow: chow diet; Caf: Cafeteria diet; ChowChow: maternal chow diet and postnatal chow diet; ChowCaf: maternal chow diet and postnatal Caf diet; CafChow: maternal Caf diet and postnatal chow diet; CafCaf: maternal Caf diet and postnatal Caf diet

At weaning, two-way ANOVA (maternal diet × sex) indicated that maternal Caf diet consumption significantly reduced species richness (F (1,32) = 7.475, p = 0.01), evenness (F (1,32) = 7.347, p = 0.011) and the Shannon diversity index (F (1,32) = 8.290, p = 0.007) regardless of offspring sex, with no significant interactions between maternal diet and sex (Fig. 2A–C). There was no significant cage effect on weaner’s species richness, evenness and Shannon index.

Adult offspring microbiota composition at 7- and 14-week timepoints was analyzed by 2 (maternal diet) × 2 (postnatal diet) × 2 (time) × 2 (sex) factorial ANOVA. A significant three-way interaction between maternal diet, postnatal diet and time were found for species richness (F (1,161) = 7.550, p = 0.007), evenness (F (1,161) = 14.217, p < 0.001) and Shannon index (F (1,161) = 12.547, p = 0.001), while offspring sex did not interact with these variables (Additional file 1: Fig. S1).

To clarify the source of these interactions, we compared the effects of Caf diet on offspring α-diversity measures over time using post hoc comparisons applying the Bonferroni correction. Species richness was significantly reduced in the CafCaf group at 7 weeks compared with ChowChow group at 7 and 14 weeks (p < 0.01 and p < 0.001 respectively) and CafCaf group at 14 weeks (p < 0.01). By 14 weeks, intriguingly, species richness in CafCaf group was no longer different from other groups (Fig. 2A). Similar effects were seen for evenness and Shannon index measures, which were both significantly reduced in the CafCaf group at 7 weeks compared to ChowChow group at 7 and 14 weeks and CafCaf group at 14 weeks. At 14 weeks, evenness and Shannon diversity did not differ between groups (Fig. 2B–C). Finally, we entered cage as a covariate in the three-way analyses. For 7 and 14 week measures, there was some evidence that species richness differed between cages (p = 0.03) but critically, this factor did not interact with maternal and offspring diet factors (all ps > 0.05), and thus did not appear to determine the results of interest.

Next, we examined the effect of diet switch (maternal diet ≠ postnatal diet) on offspring α diversity measures over time. Offspring from Caf dams switched to chow at weaning (CafChow group) did not differ in α diversity measures (species richness, evenness and Shannon index) relative to the ChowChow group at 7 and 14 weeks (Fig. 2A–C). Likewise, offspring from chow dams switched to Caf (ChowCaf group) did not differ in α-diversity measures from ChowChow group at 7 and 14 weeks. Evenness and Shannon index in the ChowCaf group at 14 weeks was significantly increased compared with the CafCaf group at 7 weeks (Fig. 2B–C).

β-diversity of gut microbiota shifted over time in a host age-dependent manner

We first examined the impact of chow and Caf diet on gut microbial communities of mothers, and offspring at weaning, 7 and 14 weeks; all groups clustered differently regardless of diet type at the OTU level, as indicated by non-metric multidimensional scaling (NMDS) plots (Fig. 3A and B; and Additional file 3: Fig. S3A–D) and PCO (Additional file 2: Fig. S2). PERMANOVA analyses (999 permutations) confirmed significant differences in β-diversity between Chow and Caf mother (F(1,18) = 6.7575, p = 0.001) and between Chow weaner and Caf weaner (F(1,33) = 6.4525, p = 0.001) (Table 2). There was no significant interaction between maternal diet and sex on β-diversity of weanlings.

Fig. 3
figure 3

β-diversity. Non-metric multidimensional scaling (NMDS) plots following square root transformation and Bray–Curtis resemblance of relative abundance data at the OTU level. A NMDS for Weaner (Chow or Caf), 7wks (ChowChow or CafCaf) and 14wks (ChowChow or CafCaf); Father (Chow only), Mother (Chow or Caf) are also shown. B NMDS for Weaner (Chow or Caf), 7wks (ChowCaf or CafChow) and 14 wks (ChowCaf or CafChow); Father (Chow only), Mother (Chow or Caf) are also shown. Chow: chow diet; Caf: Cafeteria diet; ChowChow: maternal chow diet and postnatal chow diet; ChowCaf: maternal chow diet and postnatal Caf diet; CafChow: maternal Caf diet and postnatal chow diet; CafCaf: maternal Caf diet and postnatal Caf diet

Table 2 Summary of PERMANOVA analyses

Results from PERMANOVA (999 permutations) analyses are summarised in Table 2. For β-diversity of offspring at 7 and 14 weeks, we performed PERMANOVA using a 2 (maternal diet) × 2 (postnatal diet) × 2 (time) × 2 (sex) design. The analysis indicated a significant 3-way interaction between maternal diet, postnatal diet and time (F(1,154) = 2.0437, p = 0.005) (Table 2). PERMDISP (variation of Bray–Curtis similarities) confirmed no systematic differences in sample dispersion (Mother: F(1,18) = 0.0267, p = 0.889; Weaner: F(1,35) = 2.213, p = 0.169; 7 and 14 weeks: F(7, 162) = 1.444, p = 0.262). Pairwise comparisons between offspring groups at 7 and 14 weeks confirmed that microbiota composition significantly differed between all groups (largest p < 0.033).

Continuous Caf diet consumption altered abundance of OTUs

Figure 4 shows representative taxa on phylum (A) and genus (B) levels in each group. The top 100 OTUs were selected to generate heat maps on phylum (A) and genus level (B), and the heatmaps were normalised by row. At phylum level, Firmicutes was more abundant in both chow and Caf mothers compared with Bacteroidetes; on the other hand, Bacteroides was more abundant in both chow and Caf weanlings compared with Firmicutes. Proteobacteria and Verrucomicrobia were highly abundant in chow and Caf weanlings respectively.

Fig. 4
figure 4

Representative taxa on the phylum and genus levels in each group. Microbial taxa top 100 OTUs were selected to generate heat maps on phylum level (A) and genus level (B). The relative abundance of phylum and genus levels were normalised by row

Next, DESeq2 and LEfSe analyses were used to identify OTUs that were differentially enriched or depleted by Caf diet exposure between groups and across time. To assess the effects of diet exposure, we selected OTUs only if the adjusted p value was < 0.05 from both DESeq2 and LEfSe analyses. Enriched/depleted OTUs meeting these criteria were then assessed and the top 100 OTUs were selected. In mothers, a total of 16 OTUs (10 depleted, 6 enriched) were significantly affected by Caf diet (Fig. 5A). Maternal Caf diet was associated with depletion of several OTUs in Lactobacillus and Alloprevotella genera, while several OTUs in the Blautia and Ruminococcus genera were enriched. At weaning, 8 OTUs were enriched while 31 were depleted in the offspring of Caf dams relative to those from chow dams (Fig. 5B). There were 9 OTUs commonly affected by maternal Caf diet in both mothers and weanlings (see underlined taxa in Fig. 5A and B; E). At 7 weeks, 48 OTUs were significantly affected in CafCaf compared to ChowChow offspring, while at 14 weeks, 9 OTUs differed significantly in abundance in CafCaf compared to ChowChow offspring (Fig. 5C and D). The relative abundance of Ruminococcus_Otu00086 was consistently and significantly decreased in CafCaf offspring at 3, 7 and 14 weeks (Fig. 5F).

Fig. 5
figure 5

OTUs significantly altered between groups. Microbial taxa among the top 100 OTUs identified to significantly differ in abundance between A mothers (Chow vs. Caf); B weanlings (Chow vs Caf), underlined OTUs indicate 9 common taxa between mothers and weanlings; C offspring at 7 weeks (ChowChow vs CafCaf); and D offspring at 14 weeks (ChowChow vs. CafCaf) by DESeq2 (padj < 0.05) and LEfSe (LDA Score > 2.0, p < 0.05). In DESeq2, negative (red) Log2foldchange value denotes decreased abundance and positive (green) Log2foldchange value denotes increased abundance in A Caf mother; B Caf weaner; C 7 week offspring in CafCaf group; and D 14 week offspring in CafCaf group. E Differential OTUs 7 OTUs and 30 OTUs were uniquely altered in Mothers and Weanlings by maternal Caf diet, with 9 common OTUs in Caf Mothers and Caf Weanlings. F Relative abundance of Ruminococcus_Otu00086 in 3-, 7- and 14-week offspring fed Caf diet compared with offspring fed chow diet. Note differences were significant using both LEfSe and DESeq2

We further explored OTUs affected by postnatal diet switch by comparing ChowChow and ChowCaf groups. There were 22 OTUs at 7 weeks and 45 OTUs at 14 weeks that significantly differed in abundance between ChowCaf and ChowChow groups by Log2foldchange and LDA score. Fifteen OTUs were commonly affected at both 7 and 14 weeks, characterised by significantly depleted abundance of Alistipes, Alloprevotella and Prevotella genera and increased abundance of Phascolarctobacterium and Ruminococcaceae genera (Table 3). On the other hand, 32 OTUs at 7 weeks and 33 OTUs at 14 weeks significantly differed in abundance between CafChow and CafCaf groups; 14 OTUs were altered at both timepoints, with decreased abundance of Ruminococcus and Lachnospiraceae genera and increased abundance of Lactobacillus and Collinsella genera in the CafCaf group (Table 4). For the comparison between ChowChow and CafChow groups, no OTUs survived FDR correction.

Table 3 Common OTUs altered significantly in ChowCaf compared with ChowChow offspring at both 7 and 14 weeks
Table 4 Common OTUs altered significantly in CafCaf compared with CafChow offspring at both 7 and 14 weeks

Contribution of maternal and paternal gut microbial community to composition of offspring gut microbiota

We used SourceTracker [26] to assess any contributions of maternal and paternal gut microbial community to offspring. This revealed differential contributions of species associated with the gut microbiota from Chow mothers, Caf mothers, and fathers, to offspring gut microbiota. Pooled Chow mothers, Caf mothers and fathers were treated as separate sources contributing organisms to offspring gut microbial communities at weaning, 7 and 14 weeks of age (Fig. 6A–C). SourceTracker suggested that the microbiota composition of Caf mothers made a larger contribution to both Chow and Caf weanlings’ gut microbiota than did that of Chow mothers (Additional file 4: Fig. S4). Chow mothers’ contribution to offspring microbiota increased upon switching to postnatal chow diet over time, with offspring showing an increase in chow diet associated species. To a lesser extent, a similar trend was observed for offspring in the CafCaf group. On the other hand, Caf mothers’ contribution to offspring decreased upon switching to postnatal chow diet over time, and this was also observed in the CafCaf group. Paternal contribution to offspring gut microbiota was greatest in weanlings and decreased over time across groups (Additional file 4: Fig. S4).

Fig. 6
figure 6

SourceTracker analyses. Chow mothers, Caf mothers and Chow fathers were pooled separately and computed as sources that can contribute to offspring gut microbial communities. A shows offspring consuming same diet as mother; Chow and Caf weanlings, ChowChow and CafCaf offspring at 7 and 14 weeks. B compares offspring of Chow mothers; Chow weanlings, ChowChow and ChowCaf offspring at 7 and 14 weeks. C compares offspring of Caf mothers; Caf weanlings, CafChow and CafCaf offspring at 7 and 14 weeks. Individual rats are indicated on X axis (number indicates mother id, M or F indicates male or female). D Relative contribution of top 20 OTUs of Chow mother, Caf mother and Chow father gut microbiota to offspring gut microbiota

Figure 6D shows the contributions of top 20 OTUs from Chow and Caf mothers and fathers to offspring gut microbiota. SourceTracker indicated differential transmission according to maternal diet; for Caf mother microbiota, this tended towards Bacteroides_Otu00001 and Bacteroides_Otu00002, while the contribution of Chow mother microbiota tended towards Lachnospiraceae_unclassified_Otu00006, Prevotella_Otu00007, Lactobacillus_Otu00008 and Alloprevotella_Otu00009 (Fig. 5D). There was a greater influence of Caf mother (up to 22%) compared with Chow mother (up to 7%) and father (up to 4%) contribution to offspring (Fig. 6D). FASTA sequences of these OTUs were blasted against whole genome shotgun contigs (wgs) to identify bacterial species in the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The BLAST search showed that Bacteroides_Otu00001 had similarity to Bacteroides vulgatus (Phocaeicola vulgatus) (98.8%), Bacteroides_Otu00002 had similarity to Bacteroides acidifaciens (98.8%), Lactobacillus_Otu00008 had similarity to Lactobacillus murinus (Ligilactobacillus murinus) (98.4%) and Alloprevotella_Otu00009 had similarity to Alloprevotella rava (88.4%), whereas BLAST search for Lachnospraceae_unclassified_Otu00006 and Prevotella_Otu00007 did not result in identifiable species.

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