Tools to understand the ruminal microbiome

Colonization and establishment of the rumen microbiota – opportunities to influence productivity and methane emissions


The gastrointestinal (GIT) microbiota of ruminants cannot be dissociated from the host animal. Gut symbiotic microbes have a critical role in the interaction of the host animal with the surrounding environment, providing fundamental nutritional, immunological and protection services. As for other essential body 'organs', the GIT microbiota undergoes a series of development stages from early stages of formation until maturity. Differently from the embryogenesis process; however, the development of the GIT microbiota starts in earnest at birth and it is characterized by a succession of dynamic communities in the early stages of life (Savage, 1977; Jami et al., 2013; Rey et al., 2014). This process of acquisition of various microbial populations and their evolution within the ecosystem is essential for the correct functioning and interaction of the microbiota with the host (Costello et al., 2012).

Determinism is a strong driver dictating the microbial community structure of the GIT of animals as there is a strong selection by the diet, anatomy and © Burleigh Dodds Science Publishing Limited. 2020. All rights reserved.

gut physico-chemical conditions (Ley et al., 2008). Yet, stochastic and historical events also influence the assemblage of the GIT microbiota that may have lasting effects in ruminants (Yanez-Ruiz et al., 2010; Morgavi et al., 2015; Moral's and Mizrahi, 2019). In this chapter, we review current information in the establishment of the microbiota in the rumen and posterior intestinal tract in young ruminants and its modulation for promoting health and favouring desirable phenotypes.

Establishment of the rumen microbiota

As the composition of the rumen microbiota directly influences the digestive and metabolic performance of the host animal, many studies have explored the microbial colonization of the rumen from birth to adulthood. These include early work using cultural methods (Fonty et al., 1983, 1988) to more recent studies using high-throughput sequencing methods in calves, lambs and goat kids(Jami et al., 2013; Rey et al., 2014; Guzman et al., 2015; Wang et al., 2017b; Abecia et al„ 2018; Dias et al., 2018). The developing rumen in the newborn ruminant may provide a unique opportunity to manipulate the symbiotic microbiota fora long-lasting impact in the adult ruminant (Yanez-Ruiz et al., 2015).

Colonization: from birth (pre-ruminant) to a fully functional rumen

Recent reviews describe the microbial community successions that occur in the rumen from birth to weaning and after, when animals feed exclusively on solid feeds (Malmuthuge et al., 2015; Yanez-Ruiz et al., 2015; Meale et al., 2017a). Functional populations, as well as taxa present in adult rumens, appear very early after birth, in a progressive way and in a defined sequence. Several studies monitored the establishment of the rumen bacterial community in calves from birth to weaning using high-throughput sequencing and qPCR approaches (Jami et al., 2013; Rey et al., 2014; Guzman et al„ 2015). They show that rapid changes occur in the composition of the rumen bacterial community during the first days of life. Proteobacteria and Streptococcus- related sequences are proportionally abundant in 1-3-day old calves and are rapidly replaced by strictly anaerobic bacterial taxa (Jami et al., 2013). Proteobacteria are then gradually replaced by Bacteroidetes as the animal grows, Firmicutes being present from early age to adulthood (Table 1). These results are in accordance with early studies using culture techniques reporting that aerobic and facultative anaerobic bacteria establish first (Fonty et al., 1987). Notwithstanding, strict anaerobes that are important for function in the mature rumen, such as cellulolytic bacteria and methanogenic archaea are already present in the rumen at 1 or 2 days after birth (Fonty et al., 1987; Gagen et al., 2012; Jami et al., 2013; Guzman et al., 2015). Methanogenic archaea


3 days

7 days

14 days

28 days

42 days

6 months

2 years


























































Data collected from Li et al. (2012), Jami et al. (2013), Rey et al. (2014), Yanez-Ruzi et al. (2015), Abecia et al. (2018).

can be enumerated in the immature rumen of lambs at 2-4 days, well before the consumption of solid feeds, and after two weeks, their concentration is equivalent to that found in adult animals (Fonty et al., 1987; Morvan et al., 1994). Although not detected by culture, a low-abundant but diverse population of methanogens (predominantly Methanobrevibacter spp.) was identified using molecular methods in lambs placed into sterile isolators 17 h after birth (Gagen et al., 2012). A recent study in goat kids also indicated that active methanogens colonized the rumen at one day of life, Methanobrevibacter, Methanosphaera (both Methanobacteriales order) and Candidatus Methanomethylophilus (Methanomassiliicoccales order) being the top three genera (Wang et al., 2017b). There are four major methanogenic orders usually found in the rumen: Methanobacteriales, Methanomicrobiales, Methanosarcinales and Methanomassiliicoccales (Janssen and Kirs, 2008). All these are abundantly present in calves from day 1 to 2 weeks of age, whereas only Methanobacteriales and Methanomassiliicoccales could be qPCR-detected in the mature rumen (Friedman et al., 2017). Based on substrate utilization for methanogenesis, the authors suggest that the early methanogenic community may be characterized by a high activity of methylotrophic methanogenesis, likely performed by members of the order Methanosarcinales. Eukaryotic microorganisms also establish sequentially. Anaerobic fungi can be enumerated in the rumen of lambs by 8-10 days after birth (Fonty et al., 1987). Anaerobic fungi, which are cellulolytic, are thus present in the rumen long before the animal ingests solid feeds regularly. Ciliates are detected from 2 to 3 weeks of age, with Entodinium establishing first (15-20 days), then Polyplastron, Eudiplodinium and Epidinium (20-25 days) and finally Isotricha (50 days) (Fonty et al., 1988). In contrast to bacteria and archaea, protozoa do not establish when newborns are isolated from their dams shortly after birth (Fonty et al., 1988; Chaucheyras-Durand et al., 2019). In addition, ciliate protozoa require the presence of a complex microbiota to establish (Fonty et al., 1983, 1988). Figure 1 shows the main colonization events by groups of microbes in lamb's rumen throughout the suckling period and up to the end of weaning.

Colonization of the rumen wall by epimural bacteria is also age-related, with sequential diversification of bacterial morphotypes (Rieu et al., 1990). The phylum Proteobacteria is dominant on the rumen epithelium with an important contribution of the genus Escherichia (Jiao et al„ 2015; Wang et al., 2017a). As for the lumen, the abundance of Proteobacteria associated with rumen epithelium decreases, and that of Firmicutes and Bacteroidetes increases with age (Jiao et al., 2015).

Large differences between digesta and epimural bacterial communities have been observed in the rumen of pre-weaned calves, with higher abundances of Prevotella and lower abundances of Bacteroidetes in digesta compared with epimural bacteria (Malmuthuge et al., 2014).

Microbial colonization of the lamb rumen

Figure 1 Microbial colonization of the lamb rumen. Colonization of the rumen by microbial groups detected by cultural and molecular methods. Lambs were kept with their dams. Arrows indicate the start of the colonization period.

Although the focus of this chapter is on the rumen compartment, we will mention some aspects of the microbiota of other GIT sections when relevant and/or available. While the rumen microbial community evolution with age has been well studied, information on the other pre-gastric compartments (reticulum and omasum) is scarce. Recent studies analysed fluid samples from these organs in goat kids from 3 to 56 days after birth (Lei et al., 2018) or in calves from birth to 21 days (Yeoman et al., 2018). As for the rumen, Proteobacteria gradually decrease with age while the relative abundance of Bacteroidetes increases.

For the post-gastric compartments, a surprisingly diverse microbiota is also described in the first hours post-delivery (Alipour et al., 2018). Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes dominate the newborn's rectal microbiota but composition rapidly changes in the early postnatal life. Pioneer studies reported E. coli and Streptococcus to be the firsts to colonize all GIT regions in calves and lambs few hours after birth (Smith, 1965) and culture- independent studies confirmed their high abundance in rectal microbiota one day after birth (Alipour et al., 2018). As for the rumen (Jami et al., 2013), it is assumed that these facultative anaerobes scavenge oxygen and render the environment suitable for strictly anaerobic gut microbes. Lactobacilli take advantage of these conditions and colonize all intestinal sections of one-day- old ruminants (Smith, 1965). Proteobacteria, Firmicutes and Bacteroidetes are the prevalent phyla in all anatomical locations within the first 3 weeks of life (Malmuthuge et al., 2014; Alipour et al., 2018; Yeoman et al., 2018). Firmicutes dominate distal parts of the GIT in young ruminants (colon and faeces), whereas Bacteroidetes abundance is higher in the reticulum, rumen, omasum and abomasum (Malmuthuge et al., 2014; Yeoman et al., 2018). It should be noted that while Firmicutes and Bacteroidetes are more abundant in luminal contents, Proteobacteria dominate mucosal samples (Yeoman et al., 2018). This niche specialization for mucosa-associated populations is certainly driven by environmental conditions (presence of trace oxygen) and available substrates such as mucins. Interestingly, richness and а-diversity in luminal samples increase with age in most anatomical sections, whereas no such trend is observed for mucosa-associated populations. Nevertheless, mucosa-associated bacterial communities in the small and large intestine are more diverse than digesta- associated communities (Malmuthuge et al., 2014). Though the post-gastric intestinal tract has not received much attention this far, there is evidence that its microbiota plays a crucial role in older animals' health and performance. For instance, lactic acid bacteria increase IgA production by stimulating host's adaptive immune system, boosting calves' active immunity by the time when passive immunity from colostrum decline (Corthesy et al., 2007).

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