The mammalian gut is colonized by a variety of microorganisms, leading to the concept that the host and its inhabiting microbiota constitute a “superorganism,” also called holobiont (1
). The symbiotic microbiota is essential for the nutrition and health of the host (2
The microbial ecosystem evolves concomitantly with the host chronological development (3
). Microbiota development is first under the influence of the maternal milk, a substrate containing various microbiota-shaping compounds (4
). The introduction of solid food later in life represents a new step in the dynamic construction of the gut microbiota with the ingestion of components resistant to host digestion, such as plant cell walls and specific starches (6
). This dietary shift modifies the substrates present in the luminal milieu, leading to dramatic changes in the bacterial population in terms of both diversity and composition (7
). As a result of this new gut environment, the microbiota deploys metabolic adaptations with increased capacities to degrade complex plant carbohydrates (3
). Later in life, bacterial communities continue their adaptation to the host diet (10
). For these reasons, the introduction of solid food and diet composition are considered to be major drivers of the microbial succession in the digestive tract (7
Recent studies have shown that precocious supply of solid food before weaning can modulate the establishment of intestinal bacterial communities and enzymatic activities in several mammalian species (12–16
). As a result, the host–microbiota dialogue at the mucosal interface may be affected, such as in lambs, for which inflammation system (12
) and digestive tract development (13
) modulations were observed following early solid food supplementation. Although these studies demonstrated the influence of the timing of solid food introduction on the host-microbiota codevelopment, the understanding of how early-life solid food ingestion affects the gut microbiota trajectory and the long-term host response remains insufficient.
Among the substances that influence the gut microbiome of mammals, dietary fibers are known to affect the microbiota in late childhood or adulthood, as they are important substrates for bacterial fermentations (17
). The term “dietary fiber” encompasses a variety of complex carbohydrates with different physicochemical properties such as resistant starch, nonstarch polysaccharides from plant walls, or nondigestible oligosaccharides (19
). The time required by the developing microbial ecosystems to adapt to specific fibrous carbohydrate substrates remains largely unexplored.
Our approach was to provide a dynamic follow-up of the holobiont, evaluated with various measurements, to provide a comprehensive understanding of the impact of different feeding patterns on mammals. In these species, modeling the effects of early‐life exposure to solid food is particularly challenging, as it is difficult to capture juvenile feeding patterns (food and milk) without disrupting mother–offspring interactions. To bypass this issue, we propose herein to use a neonatal rabbit model, characterized by a short contact time with the nursing doe for suckling (less than 5 min every 24h) (20
). This experimental design allowed us to track the early ingestion of solid food together with a control of milk ingestion. Moreover, 1-week-old rabbits are capable of consuming solid food simultaneously with large amounts of milk (21
), making the newborn rabbit a good model to study the timing of solid food introduction in early life. Finally, rabbits are hindgut fermenters that rely heavily on their gut microbiota for digestion and health, allowing for a relevant study of symbiosis in mammals. As commonly observed in mammals, the rabbit cecal microbiota is dominated by Bacteroidetes
) and followed by others including Proteobacteria
are abundant families of the cecal microbiota, with a distinctive feature being the poor colonization of the gut by Lactobacilli
The present study aimed to further investigate the mechanisms by which precocious solid food ingestion affects microbiota establishment, with attention given to the dietary polysaccharides. We investigated how the introduction of solid food affects intestinal maturation by providing solid nutrients to suckling rabbits as of 3 or 15 days of age. We examined the impact of two types of plant polysaccharides (rapidly fermentable fibers [RFF] and starch) consumed during suckling and thereafter, on the gut microbiota. The effects of early solid feeding and plant polysaccharides ingestion on bacterial communities were assessed at five developmental stages in two intestine sections with distinct physiological functions: the cecum, which contributes greatly to host nutrition (25
), and the appendix vermiformis
, a specialized lymphoid organ at the apex of the cecum (26
). Extensive investigation of the cecal ecosystem was then performed with the assessment of predictive functional profiling and quantitative metabolic signatures. Gene expression in this tissue was analyzed as a proxy of gut health.
Our findings highlighted that the bacterial communities of rabbit pups quickly responded to the precocious ingestion of solids with both taxonomic and metabolic changes. Although starter feeding resulted in an acceleration of the gut microbiota toward a steady state and increased acetate and butyrate levels early in life, impacts on microbiota activities and host remained moderate after weaning. Functional analysis revealed bacterial specialization depending on the type of polysaccharides ingested throughout the weaning transition. Taken together, those results underlined key factors to modulate the gut microbiota trajectories (either maturation rate or endpoints).
Postnatal ingestion of solid food is known to be one major influential factor of microbiota implantation in mammals (11
). Recent research suggests that early solids ingestion (i.e., early food diversification) could be beneficial for health (31
), presumably through a reshaping of gut microbial colonization (32
). Our study aimed to analyze further how early-life ingestion of solid foods affects hindgut microbiota implantation. Two dietary approaches were used to broaden our understanding of microbiota engineering strategies: on one hand, a short and early nutritional intervention was performed to investigate the effect of the timing of solid food introduction, and on the other hand, a long-term dietary modulation enabled us to study dietary polysaccharide effects on the holobiont development.
Overall, precocious ingestion of starch-rich solid food accelerated microbiota maturation in both cecum and appendix. Indeed, when rabbits had early access to solid food enriched in starch, we observed more diverse bacterial communities the week preceding the weaning. Faster stabilization of the microbiota with early feeding practice was also evidenced in the STA+ group compared to the STA–. This is in agreement with previous studies (15
) indicating the potential for an earlier introduction of solid feed to reduce the time required for the stabilization of the hindgut bacterial community. Such an enhancement may be advantageous later in life by improving the digestive capacity to cope with dietary shifts (37
), by providing the level of microbiota diversity required to inhibit allergic and autoimmune disorders (32
), and by stimulating normal components of the immune system (39
). But because first gut colonizers play a pivotal role in fermenting the numerous substances present in the milk (40
), a precipitated replacement of these bacterial communities may also be detrimental for the host’s health, as suggested by prospective human studies (15
). When looking at the cecal tissue, decreased expression of the B lymphocytes activator gene TNFSF13B
was observed at the end of gel supplementation, possibly in response to bacterial signaling (42
). However, the downregulation of this gene involved in the adaptive immunity did not modify the cecal IgA content of suckling rabbits. Moreover, it was proposed that a transient reduction of the mucosal IgA content before weaning might be beneficial for the colonization by segmented filamentous bacteria (43
). Decreased plasma IgG content after precocious ingestion of starch-rich solid food also pointed out immune modulations due to early solid food introduction. Whether these changes in the humoral immunity can be beneficial for the host needs to be further elucidated. Histological examination of intestinal epithelium, at the interface of the microbiota and the gut mucosal immunity, would help us to investigate further this question. Still, these findings confirmed the importance of early-life solid food ingestion on the host–bacterial symbiosis.
Early ingestion of starch-rich diet promptly stimulated the growth of species belonging to the Ruminococcaceae
family in the appendix, despite concomitant ingestion of large quantities of milk that might create a niche constraint on the gut microbiome (5
). This compositional change is in line with the pattern observed after later introduction of solid food in young mammals (46
). The cecal luminal metabolome was also modified after solid food introduction, highlighting a quick adaptation of the developing microbiota activity to new dietary substrates and environmental transitions. Notably, the fermentative activities, assessed by cecal acetate and butyrate concentrations, transiently increased after the ingestion of small quantities of solid food. The levels of several amino acids also increased when plant-based food was introduced in the diet at an early stage. Although those amino acids can be of different origins (48
), we hypothesize that poorly digestible plant protein from solid food partly reached the cecum, thus precociously modifying the amino acids content of the hindgut environment, whereas milk proteins are highly digested in the upper gastrointestinal tract (49
). These modulations of the substrates available in the cecal luminal content likely explains some long-term effect of early life solid food ingestion on taxonomic modifications, such as a drop in the Bacteroidaceae
family, a “milk-oriented microbiota” characteristic taxon (50
). After the establishment of the pioneer species, early supply of solid food was thus found to drive the second colonization of gut communities.
Dietary fiber fractions represent major modulators of digestive physiology due to their influence on the nutrient rate of passage, mucosal functionality, and gut microbiota (51
). The particularity of this study was to modulate starch and digestible fiber fractions only while maintaining a balanced diet. Our results showed that the type of dietary complex plant carbohydrates shaped both composition and functions of the hindgut microbiota, with major effects observed when the ingestion of solid food became predominant over milk. Not surprisingly, the type of polysaccharides ingested modulated the distribution of species from Firmicutes
phyla, which encompass plant-degrading bacteria (52
). At the family level, we found that the distinct carbohydrates contents of STA and RFF diets drove a differential establishment of Lachnospiraceae
, presumably because they encompass two plant degrader groups characterized by different carbohydrate-active enzymes within their genomes (53
). When rabbits ingested high amounts of starch throughout their lives, the cecal communities reached a mature state faster (27
). We hypothesized that resistant starch (54
), which has the ability to be quickly fermented and to modulate the gut microbiota (10
), may have contributed to this stabilization. Indeed, glucose is readily absorbed in the rabbit small intestine (55
), and the higher levels of free glucose observed in the cecum of rabbits that received the enriched-starch diet could be related to the bacterial degradation of starch resistant to host digestion. Diet enrichment with RFF was associated with decreased diversity in the cecum and appendix, with a lower InvSimpson index outlining the dominance of some abundant species in those ecosystems. This can be attributed to the specific dietary content of RFF diet but also can be due to an improved fiber fraction digestibility as reported when increasing digestible fiber to starch ratio (56
). Interestingly, higher levels of cecal methanol were found when rabbits received higher quantities of RFF. This must be explained by high dietary concentrations of pectins, a substrate that can be fermented into methanol by specific intestinal bacteria such as members of the Bacteroides
). Consistently, the RFF diet was associated with increased cecal acetate content before weaning, in line with the pectin in vitro
fermentation process assessed in human stools (58
). PICRUSt predictions were sketchy and should be treated with caution. However, these predictions seem in line with our hypothesis since increased expression of glycans-degradation pathways was observed in the cecal microbiome of the rabbits that received the RFF diet. In terms of host response, lifelong ingestion of a diet rich in RFF was associated with increased gene expression of ALPI
, considered as a marker of epithelial differentiation and a regulator of intestinal inflammation (59
). Moreover, animals that ingested more RFF had heavier cecum, which may highlight faster development of this organ in response to fermentable nonstarch polysaccharides.
Interestingly, we observed different distribution drivers of microbial populations in the two contiguous digestive segments investigated, even though these sections do not differentiate from each other during the neonatal phase (60
). Cecal communities were mostly affected by the type of polysaccharides substrates provided, with pronounced effects on alpha diversity and OTU composition once solid food ingestion overtook milk consumption. In contrast, the composition of the appendix microbiota was substantially influenced by food ingestion stimulation in early life. These differential effects can be linked to the dualism of functions between the cecum and appendix, respectively related to nutrition and immunity (26
). Differences in peristaltic movements may also explain these distinct microbiota (61
): the narrow lumen of the appendix limits the nutrient flow, which could induce an inertia of the microbiota. The appendix has long been considered as a degenerating organ, but it could represent a reservoir for beneficial bacteria that can reinoculate the bowel (62
). Moreover, the microbial communities of the gut-associated lymphoid tissue are essential for the immune system development (63
). We thus believe that the effects of early-life stimuli on the colonization of lymphoid organs deserve increased attention.
We demonstrated that very early ingestion of solid food in infant rabbits, although in small quantities, induced changes in gut microbiota colonization and activity, with an acceleration of the ecological species succession and increased production of short-chain fatty acids. Postnatal solid ingestion showed more impact on the appendix microbiota pattern, whereas the type of dietary plant polysaccharides mainly modified microbiota composition and functions in the cecum. In sum, we showed that the gut microbiota development trajectory was partly modified by the type of plant polysaccharides ingested, whereas its maturation speed was more dependent on the timing of solid food introduction. However, no strong impact on the endpoint studied was achieved, suggesting a dilution of the effects observed at maturity. Those results evidence the possibility of shaping the developing microbiota with nutrition leverage. An important matter to resolve in future research will be to understand the implication of early modifications of the microbiota on the young mammal immune response in a challenging environment.