Overview of gastrointestinal microbiota

In recent years, several studies have shown how the gastrointestinal microbiota is closely related to host health and performance, either through metabolic function or protection against pathogens (Maranduba et al., Reference Maranduba, De Castro, De Souza, Rossato, da Guia, Valente, Rettore, Maranduba, De Souza, Do Carmo, Macedo and Silva2015; Pascale et al., Reference Pascale, Marchesi, Marelli, Coppola, Luzi, Govoni, Giustina and Gazzaruso2018). The gastrointestinal microbiota is genetically complex, represented by various species of bacteria, protozoa, fungi, methanogenic archaea, and bacteriophages (Chaucheyras-Durand and Ossa, Reference Chaucheyras-Durand and Ossa2014; Dill-McFarland et al., Reference Dill-McFarland, Breaker and Suen2017; Dias et al., Reference Dias, Marcondes, de Souza, da Mata e Silva, Noronha, Resende, Machado, Mantovani, Dill-McFarland and Suen2018), with many functional and phylogenetic differences. The ruminal microbial ecosystem is more diverse and responsible for several functional niches, including proteolytic, fibrolytic, and lipolytic functions (Clemmons et al., Reference Clemmons, Voy and Myer2019). The counts are in the order of 10¹⁰ CFU ml⁻¹ of ruminal fluid for bacteria, 10⁸ for protozoa, 10⁷ for archaea, and 10³ for fungal spores (Deusch et al., Reference Deusch, Tilocca, Camarinha-Silva and Seifert2015).

Among all microorganisms, bacteria are the most studied for their functional and phylogenetic diversity, reproducibility, and accessibility to genetic material (Clemmons et al., Reference Clemmons, Voy and Myer2019). The bacteria are responsible for approximately half of the microbial genetic material in the rumen (Nagaraja, Reference Nagaraja2016), and may represent even more in other GIT compartments.

In general, the most abundant bacterial phyla in the GIT are Firmicutes (41.22%), Bacteroidota (33.51%), and Proteobacteria (12.15%), but these proportions are variable depending on the age and the GIT compartment (de Oliveira et al., Reference de Oliveira, Jewell, Freitas, Benjamin, Tótola, Borges, Moraes and Suen2013; Myer et al., Reference Myer, Smith, Wells, Kuehn and Freetly2015). In beef steers, Firmicutes were more abundant in the small and large intestines, while Bacteroidota were more abundant in the reticulum-rumen, omasum, and abomasum (de Oliveira et al., Reference de Oliveira, Jewell, Freitas, Benjamin, Tótola, Borges, Moraes and Suen2013). Coincidentally, rumen, omasum, and abomasum had the highest bacterial diversity, and the jejunum was the lowest in bacterial diversity and richness.

In dairy calves, some results are similar. Dias et al. (Reference Dias, Marcondes, de Souza, da Mata e Silva, Noronha, Resende, Machado, Mantovani, Dill-McFarland and Suen2018) also observed higher diversity in the rumen, cecum, and colon compared to the jejunum, and also had fewer taxa shared with other groups, and a higher abundance of the Proteobacteria phylum. As calves aged, the bacterial community became more homogeneous. In newborn calves, Yeoman et al. (Reference Yeoman, Ishaq, Bichi, Olivo, Lowe and Aldridge2018) observed higher abundance of Proteobacteria (41%), Firmicutes (29%), and Bacteroidota (23%), with significant differences between the lumen and mucosal-attached microorganisms. This higher abundance of Proteobacteria was associated to colostrum intake and had a bacterial composition of 42% Proteobacteria, 22% Firmicutes, and 21% Bacteroidota, similar values to gut microbiota. This age-related difference in the composition may be mainly associated to dietary changes occurring throughout the pre-weaning period.

The differences in composition affect the diversity indices (de Oliveira et al., Reference de Oliveira, Jewell, Freitas, Benjamin, Tótola, Borges, Moraes and Suen2013; Dias et al., Reference Dias, Marcondes, de Souza, da Mata e Silva, Noronha, Resende, Machado, Mantovani, Dill-McFarland and Suen2018). Understanding the concept of these indices, as well as the distinction between indigenous (autochthonous) and transient (allochthonous) microorganisms in the GIT microbiota is fundamental to the ecological understanding of colonization, succession, and interaction mechanisms between intestinal microorganisms and hosts (Mackie et al., Reference Mackie, Sghir and Gaskins1999). This distinction is difficult, especially in neonates, as their microbiota is not yet well established, with many microorganisms being transient. Some of these concepts, as well as their definitions, are shown in Table 1.

Table 1.

Concepts, definitions and techniques used in microbial ecology

Adapted from Yeoman and White (Reference Yeoman and White2014); Malmuthuge and Guan (Reference Malmuthuge and Guan2016); Thomas et al. (Reference Thomas, Webb, Ghimire, Blair, Olson, Fenske, Fonder, Christopher-Hennings, Brake and Scaria2017) and Allaband et al. (Reference Allaband, McDonald, Vázquez-Baeza, Minich, Tripathi, Brenner, Loomba, Smarr, Sandborn, Schnabl, Dorrestein, Zarrinpar and Knight2019).

Study tools for assessment of the gut microbiota

Many ruminant studies have investigated the ruminal microbiota since the 1950s, and these studies focused more on the effects of nutritional factors on the diet than on the host relationship. In addition, studies have used culture-based methods for a long time (Hungate, Reference Hungate1966). However, any method that favors the growth of microorganisms over others is biased (Allaband et al., Reference Allaband, McDonald, Vázquez-Baeza, Minich, Tripathi, Brenner, Loomba, Smarr, Sandborn, Schnabl, Dorrestein, Zarrinpar and Knight2019)

Genome sequencing by whole-genome shotgun and later by next-generation sequencing techniques allowed a great evolution and deepening of metagenomics (Koonin et al., Reference Koonin E, Makarova and Wolf2021), as well as in studies and definitions of other ‘omics’ (Table 1). Such techniques have allowed unculturable, and previously unknown, microorganisms to be discovered, and have their function and metabolites analyzed. Some estimates indicate that less than 0.1% of the prokaryotes represented in most environments can grow in culture (Fraser-Liggett, Reference Fraser-Liggett2005; Koonin et al., Reference Koonin E, Makarova and Wolf2021).

Bioinformatics is essential to support the microbiome analysis, applying methodologies from computer science, advanced mathematics, and statistics to the study of biological phenomena (Carriço et al., Reference Carriço, Rossi, Moran-Gilad, Van Domselaar and Ramirez2018; Allaband et al., Reference Allaband, McDonald, Vázquez-Baeza, Minich, Tripathi, Brenner, Loomba, Smarr, Sandborn, Schnabl, Dorrestein, Zarrinpar and Knight2019). Furthermore, the use of popular phylogenetic marker genes such as 16S ribosomal RNA (rRNA; bacteria and archaea), 18S rRNA (protozoa), and the internal transcribed spacer region (ITS; fungi) have aided in the investigation of microbial communities (Smith et al., Reference Smith, Waters, Gómez Expósito, Smidt, Carberry and McCabe2020).

Furthermore, especially for taxonomic classification of the 16S rRNA gene, reference databases are widely available, such as the Ribosomal Database Project (RDP), SILVA, GreenGenes, RefSeq, and the Genome Taxonomy Database (GTDB; Smith et al., Reference Smith, Waters, Gómez Expósito, Smidt, Carberry and McCabe2020). For classifying methanogens there are specific databases such as the Rumen and Intestinal Methanogen Database (RIM-DB; Seedorf et al., Reference Seedorf, Kittelmann, Henderson and Janssen2014) and the Neocallimastigomycota, and more recently the GlobalFungi for fungi (Větrovský et al., Reference Větrovský, Morais, Kohout, Lepinay, Algora, Awokunle Hollá, Bahnmann, Bílohnědá, Brabcová, D'Alò, Human, Jomura, Kolařík, Kvasničková, Lladó, López-Mondéjar, Martinović, Mašínová, Meszárošová, Michalčíková, Michalová, Mundra, Navrátilová, Odriozola, Piché-Choquette, Štursová, Švec, Tláskal, Urbanová, Vlk, Voříšková, Žifčáková and Baldrian2020). According to Allaband et al. (Reference Allaband, McDonald, Vázquez-Baeza, Minich, Tripathi, Brenner, Loomba, Smarr, Sandborn, Schnabl, Dorrestein, Zarrinpar and Knight2019), metagenomic approaches lose many rare bacteria not well represented in reference databases. This could be a problem, as the reference database can interfere in the interpretation of microbiota analysis (Pollock et al., Reference Pollock, Glendinning, Wisedchanwet and Watson2018). As shown by Sanford et al. (Reference Sanford, Lloyd, Konstantinidis and Löffler2021), SILVA and GTDB are both well-updated databases; however, Greengenes and RDP became outdated since few years ago. In the study by Smith et al. (Reference Smith, Waters, Gómez Expósito, Smidt, Carberry and McCabe2020), considering different databases in conducting 16S rRNA amplicon sequencing taxonomic classification of ruminal microbiota, it was concluded that RefSeq + RDP and SILVA were the most appropriate reference databases due to superior classification at the genus level, even with limitations.

Even with several limitations, such as financial limitations and microbiome analysis, we cannot deny that this did not slow down the publication of studies in the area, both for humans and ruminants. Using the search data cited by Malmuthuge et al. (Reference Malmuthuge, Griebel and Guan2015a), and comparing publications over the interval 2010–2020, we observed an increase of more than six times in the number of studies published for humans and more than 10 times for ruminants, depending on the search term used. Despite this higher publication rate for ruminants, the numbers of studies are well below those for humans. These increased publication numbers suggest that there is still a significant amount of research to be conducted on the microbiota.

Microbial colonization of the GIT

Microbial colonization is influenced by a series of factors associated with the host, the microbiota, and external factors. The external factors are the most diverse, such as maternal microbiota, birth type, environment, colostrum source and feeding time, liquid and solid diets, and antibiotic treatment during early life (Malmuthuge et al., Reference Malmuthuge, Griebel and Guan2015a). Host-related factors have been cited as luminal pH, feeding retention time, and immune defense mechanisms, as well as microbiota-related factors such as adhesion, survival mechanisms under an oxygen gradient, and mechanisms to obtain nutrients from the host (Adlerberth and Wold, Reference Adlerberth and Wold2009). Some authors have suggested that prepartum events may influence newborn GIT colonization (Ley et al., Reference Ley, Backhed, Turnbaugh, Lozupone, Knight and Gordon2005; Dominguez-Bello et al., Reference Dominguez-Bello, Costello, Contreras, Magris, Hidalgo, Fierer and Knight2010; Elolimy et al., Reference Elolimy, Alharthi, Zeineldin, Parys, Helmbrecht and Loor2019).

Until recently, newborn ruminants were commonly believed to have been born with sterile GITs (Taschuk and Griebel, Reference Taschuk and Griebel2012; Malmuthuge et al., Reference Malmuthuge, Griebel and Guan2015a), and that these animals had the first contact with microorganisms during birth because, on fetal development, the placental barrier prevented them from having contact with the fetus. However, meconium samples collected immediately or shortly after birth showed diverse and low-abundant microbiota (Alipour et al., Reference Alipour, Jalanka, Pessa-Morikawa, Kokkonen, Satokari, Hynönen, Iivanainen and Niku2018; Elolimy et al., Reference Elolimy, Alharthi, Zeineldin, Parys, Helmbrecht and Loor2019), suggesting microbial colonization in the intestinal tract during the fetal period. Recently, a pioneering bovine study by Guzman et al. (Reference Guzman, Wood, Egidi, White-Monsant, Semenec, Grommen, Hill-Yardin, De Groef and Franks2020) found a high density of bacterial genetic material in the fetal GIT and amniotic fluid between 5 and 7 months of gestation, indicating a possible pre-natal cow-to-fetus transmission across the placental barrier. Bi et al. (Reference Bi, Tu, Zhang, Wang, Zhang, Suen, Shao, Li and Diao2021) also observed a low diversity microbiome in the intestine of goat fetus, composed of bacteriophages, viruses, and bacteria carrying antibiotic resistance genes. Despite these results, both human and rodent studies are still inconclusive and create considerable discussion, indicating no presence of bacterial genetic material during fetal development (Collado et al., Reference Collado, Rautava, Aakko, Isolauri and Salminen2016; Hornef and Penders, Reference Hornef and Penders2017; Theis et al., Reference Theis, Romero, Greenberg, Winters, Garcia-Flores, Motomura, Ahmad, Galaz, Arenas-Hernandez and Gomez-Lopez2020).

At birth, colonization rapidly continues by an abundant and diverse microbial community (Jami et al., Reference Jami, Israel, Kotser and Mizrahi2013). The GIT colonization is a dynamic process and has a considerable fluctuation in the animal's early life. The processes related to the microbe establishment are complex, involving microbial succession and interactions between microorganisms and the animal, resulting in dense and stable populations inhabiting specific regions in the GIT. Colonization can continue until weaning because the microbial diversity, richness, structure, and composition of the GIT throughout the pre-weaning period are often constantly changing (Oikonomou et al., Reference Oikonomou, Teixeira, Foditsch, Bicalho, Machado and Bicalho2013; Klein-Jöbstl et al., Reference Klein-Jöbstl, Schornsteiner, Mann, Wagner, Drillich and Schmitz-Esser2014a; Dias et al., Reference Dias, Marcondes, Noronha, Resende, Machado, Mantovani, Dill-McFarland and Suen2017; Virgínio Júnior et al., Reference Virgínio Júnior, Coelho, de Toledo, Montenegro, Coutinho and Bittar2021). After this period, the microbiota become stabilized and resilient to change, being altered only by temporary changes in diet but possibly returning to the previous condition (Weimer, Reference Weimer2015).

From an ecological point of view, gastrointestinal colonization represents the assembly of a new microbial community (Costello et al., Reference Costello, Stagaman, Dethlefsen, Bohannan and Relman2012). Some older studies and reviews describe the colonization as occurring in stages (Rey et al., Reference Rey, Enjalbert, Combes, Cauquil, Bouchez and Monteils2014; Malmuthuge et al., Reference Malmuthuge, Griebel and Guan2015a), however more recent studies show colonization to be much more dynamic from birth to weaning.

The inoculum sources of microorganisms are the most diverse. However, diet and frequency of supply are the most critical factors for establishing and maintaining the stability of microbial populations (Fonty et al., Reference Fonty, Gouet, Jouany and Senaud1987), as inoculation pathways of microorganisms and also serving as substrates for growth. However, it is not clear how microbiota−host interactions occur during the early life of ruminants, and if these interactions have any influence on the performance and health of the animal.

A recent study showed what are the main sources of microorganisms in the gut of lambs, comparing to animals that suckled directly to their dams to those that were bottle-fed (Bi et al., Reference Bi, Cox, Zhang, Suen, Zhang, Tu and Diao2019). The study showed that in lambs suckling their dam, the initial intestinal microbiota were more similar to that present in the teats (43%) and the environment (28%). In comparison, bottle-fed lambs were more similar to microorganisms in the vagina (46%), in the ambient air (31%), and the floor of the pen (12%).

The first contact extra-uterine with microorganisms occurs as the animal passes through the vaginal canal. Klein-Jöbstl et al. (Reference Klein-Jöbstl, Quijada, Dzieciol, Feldbacher, Wagner, Drillich, Schmitz-Esser and Mann2019) have observed some variation in the fecal microbiota of neonatal calves at 48 h, but closer to the vaginal microbiota of the dam than to the microbiota of the dam's mouth and feces, or even colostrum. Although the authors did not assess environmental influence, an important factor in post-natal colonization. Yeoman et al. (Reference Yeoman, Ishaq, Bichi, Olivo, Lowe and Aldridge2018) also observed greater proximity of the vaginal microbiota to the ruminal microbiota 72 h after birth, including fibrolytic ruminal bacteria, as well as methanogenic archaea, potentially indicating a role for the vagina in populating the developing reticulum-rumen with microbes important to the nutrition of the adult animal. Less than 20 min after birth, archaea (Methanobrevibacter spp., M. mobile, and M. votae), fibrolytic bacteria (F. succinogenes, Ruminococcus flavefaciens, and P. ruminicola), and Geobacter spp. (phylum Proteobacteria) were detected in GITs and calf feces (Guzman et al., Reference Guzman, Bereza-Malcolm, De Groef and Franks2015). Barden et al. (Reference Barden, Richards-Rios, Ganda, Lenzi, Eccles, Neary, Oultram and Oikonomou2020) compared the fecal and oral microbiota of beef and dairy cows and calves, and up to 4 weeks, the calves' fecal microbiota were closer to the cows' oral microbiota. Overall, no marked differences were observed in the calves' microbiota, indicating that vertical transmission through feces may be low.

While studies do not indicate colostrum as a major source for gut microbiota colonization, it is important to remember that colostrum represents an important substrate for the growth of commensal bacteria, accelerating bacterial colonization in the calf's small intestine. Calves that received fresh colostrum reached a total bacterial density of 10¹⁰ copies of the 16S rRNA  gene g⁻¹ at 12 h of life, while calves that did not receive colostrum reached only 10⁸ copies of the 16S rRNA gene g⁻¹, and have abundant E. coli both in the lumen and mucosal (Malmuthuge et al., Reference Malmuthuge, Chen, Liang, Goonewardene and Guan2015b), and this can have serious consequences in the calves' performance. Besides, colostrum feeding may result in a higher prevalence of Bifidobacterium in the small intestine during the first 12 h of life (Malmuthuge et al., Reference Malmuthuge, Chen, Liang, Goonewardene and Guan2015b). However, Fischer et al. (Reference Fischer, Song, He, Haines, Guan and Steele2018) observed a higher abundance of this genus with colostrum supply soon after birth. This increased prevalence may explain the reduced incidence of enteric infections (Godden et al., Reference Godden, Smolenski, Donahue, Oakes, Bey, Wells, Sreevatsan, Stabel and Fetrow2012), indicating that colostrum plays a crucial role in establishing a healthy GIT.

According to age and feeding rate, among other variables, 5–20% of the dry matter (DM) intake of whole milk is not digested (Guilloteau et al., Reference Guilloteau, Toullec, Grongnet, Patureau-Mirand, Prugnaud and Sauvant1986; Hill et al., Reference Hill, Bateman, Aldrich and Schlotterbeck2010). In the case of milk replacers, these quantities may be even higher. Although this is presumably a higher flow of fermentable nutrients in the posterior intestine of the pre-ruminant calf, limited information is available about how early microbial activity can affect gut development and health (Oikonomou et al., Reference Oikonomou, Teixeira, Foditsch, Bicalho, Machado and Bicalho2013) compared to ruminal development. Therefore, during the calf pre-ruminant phase, the increased supply of fermentable substrate may result in bacterial colonization and establishment of fermentation in the posterior intestine, which is equally or more relevant than in the anterior intestine for the health and survival of the newborn (Castro et al., Reference Castro, Gomez, White, Loften, Drackley and Mangian2016).

Bacterial colonization has been the focus of different studies in recent years. Facultative anaerobes, such as Streptococcus and Enterococcus, are known as early colonizers and convert the GIT into a completely anaerobic environment (Adlerberth and Wold, Reference Adlerberth and Wold2009; Jami et al., Reference Jami, Israel, Kotser and Mizrahi2013). These changes in the gastrointestinal microbiota are dynamic in the first 12 weeks of life, increasing diversity, richness, and evenness over the pre-weaning period (Li et al., Reference Li, Connor, Li, Baldwin VI and Sparks2012; Rey et al., Reference Rey, Enjalbert, Combes, Cauquil, Bouchez and Monteils2014), including the appearance of new species such as R. flavefaciens and Fibrobacter species and reducing the abundance of Bifidobacterium, Enterobacteriaceae, Streptococcus, and Lactobacillus species (Uyeno et al., Reference Uyeno, Sekiguchi and Kamagata2010; Li et al., Reference Li, Connor, Li, Baldwin VI and Sparks2012).

Archaea are difficult to detect in calf feces until week 4 of age, and fungi appear 1 week after birth but below detection until weaning (Dill-McFarland et al., Reference Dill-McFarland, Breaker and Suen2017). In a microscopic evaluation, Virgínio Júnior et al. (Reference Virgínio Júnior, de Azevedo, Ornelas, de Oliveira, Geraseev and Duarte2016) also observed no fungi attached to the fiber in calves at 60 days of age. Dias et al. (Reference Dias, Marcondes, Noronha, Resende, Machado, Mantovani, Dill-McFarland and Suen2017) have identified fungi and archaea in calves at 7 days of age, even before the starter concentrate supply. The absence of starter concentrate does not seem to be a limiting factor to the archaea. Zhou et al. (Reference Zhou, Chen, Griebel and Guan2014) have observed an abundance of Methanobrevibacter in the dairy calf GIT, and the higher diversity in the rumen. Malmuthuge et al. (Reference Malmuthuge, Liang and Guan2019) have found less than 0.1% of fungi in the ruminal environment of newborn calves. Archaea were not detected.

The colonization of ciliated protozoa depends on contact with other adult ruminants and can be detected in the first weeks of calf life (Dehority, Reference Dehority1998). If contact with adult ruminants occurs, protozoa populations develop between 15 and 20 days of age (Fonty et al., Reference Fonty, Senaud, Jouany and Gouet1988). Malmuthuge et al. (Reference Malmuthuge, Liang and Guan2019) also did not detect protozoa in the rumen of newborn calves. Virgínio Júnior et al. (Reference Virgínio Júnior, de Azevedo, Ornelas, de Oliveira, Geraseev and Duarte2016) have found no protozoa in dairy calves weaned at 60 days and attributed it to out of contact to other ruminants. Minato et al. (Reference Minato, Otsuka, Shirasaka, Itabashi and Mitsumori1992) found some genera such as Entodinium in calves at 8–10 weeks. Relatively limited information is available on the colonization of protozoa in the rumen of calves, and even a relatively recent review (Newbold et al., Reference Newbold, de la Fuente, Belanche, Ramos-Morales and McEwan2015), has no mention of colonization in dairy and beef calves. In the only study in beef calves at 6–8 months of age, Duarte et al. (Reference Duarte, Abrão, Oliveira Ribeiro, Vieira, Nigri, Silva, Virgínio Júnior, Barreto and Geraseev2018) found a diversity of protozoal genera in the rumen of Nelore calves, but these calves are too old for comparisons to calves up to 60 days. Protozoa colonization may be delayed compared to bacteria because protozoa are sensitive to the lower ruminal pH characteristics of pre-weaning calves possibly to milk escape and subsequent lactic fermentation, as mentioned by Dias et al. (Reference Dias, Marcondes, Noronha, Resende, Machado, Mantovani, Dill-McFarland and Suen2017). The rapid establishment of protozoa requires a well-structured bacterial population. Bacteria promote physico−chemical characteristics in the ruminal environment (Fonty et al., Reference Fonty, Senaud, Jouany and Gouet1988), and are a nitrogen source for growth of protozoa (Williams and Coleman, Reference Williams and Coleman1997).

The gastrointestinal microbiota are continually changing as calves increase the intake of a solid diet and are weaned, with the eventual establishment of a mature microbial community (Meale et al., Reference Meale, Li, Azevedo, Derakhshani, Plaizier, Khafipour and Steele2016; Dill-McFarland et al., Reference Dill-McFarland, Breaker and Suen2017). The development of diverse microbiota in young ruminants sets the ecosystem and initiates fermentative digestion in these animals (Fonty et al., Reference Fonty, Jouany, Chavarot, Bonnemoy and Gouet1991). The flaw in establishing healthy microbiota can result in bowel disorders and immune function impairment (Czarnecki-Maulden, Reference Czarnecki-Maulden2008).

Importance of microbiota to the physiology of young ruminants

Ruminants have established symbiotic relationships with different microorganisms, and which degrade and metabolize different nutrients such as fibrous and non-fibrous carbohydrates and proteins. This symbiosis occurs because mainly the ruminal environment is suitable to allow microorganisms to establish and develop, and to provide ammonia, amino acids, vitamins, and SCFA to the animal through the fermentation of diet components (Hungate, Reference Hungate1966; Rey et al., Reference Rey, Enjalbert, Combes, Cauquil, Bouchez and Monteils2014).

Ruminal microorganisms produce glycogenic, lipogenic, and aminogenic precursors that contribute to regulating energy and protein metabolism in cattle (Hungate, Reference Hungate1975), and provide the host approximately 70 and 60% of its daily energy and protein needs, respectively, through fermentation (Flint and Bayer, Reference Flint and Bayer2008; Yeoman and White, Reference Yeoman and White2014). Therefore, studies on microbiota in ruminants have always focused on ruminal microbiota to understand the effects on meat and milk production (Malmuthuge et al., Reference Malmuthuge, Griebel and Guan2015a).

However, in the last few years, the topic of microbiota has gained importance in studies with ruminants, with the main focus on the effect of different diets. The gut microbiota also provide crucial physiological characteristics that the host organism would not be able to develop by itself (Gill et al., Reference Gill, Pop, DeBoy, Eckburg, Turnbaugh, Samuel, Gordon, Relman, Fraser-Liggett and Nelson2006; Neish, Reference Neish2009).

Although numerous studies in humans and rats report the importance of early intestinal microbiota in the host's future health, few studies try to understand the role of intestinal colonization in the development of GIT and the health of neonatal ruminants. Moreover, ruminal and intestinal development and the establishment of microbiota have always been studied as separate aspects of ruminant biology. Only a few reports aimed to understand possible interactions between these two compartments (Malmuthuge et al., Reference Malmuthuge, Griebel and Guan2015a).

The gut microbial colonization at the beginning of life is essential to the development of metabolic functions, maturation of the immune system, and health of calves (Gomez et al., Reference Gomez, Arroyo, Costa, Viel and Weese2017; Kouritzin and Guan, Reference Kouritzin and Guan2017), and may have long-term health effects (Conroy et al., Reference Conroy, Shi and Walker2009). A stable commensal community protects the host from invasive pathogens (Kamada et al., Reference Kamada, Chen, Inohara and Núñez2013), and expands the biosynthetic capacity of the host by improving nutrient uptake (Karasov et al., Reference Karasov, Martínez del Rio and Caviedes-Vidal2011).

The simple provision of starter concentrate during the pre-weaning period increased the abundance of Methanosphaera spp., an archaea efficient in producing enteric methane (Dias et al., Reference Dias, Marcondes, Noronha, Resende, Machado, Mantovani, Dill-McFarland and Suen2017), and has a marked effect, not only by providing bacterial growth substrate, but also by altering the cecal microbiota, increasing the abundance of Allistipes, Parabacteroides, Parassutterella and Butyricimonas. Those microorganisms associated with digestion of fiber and other carbohydrates and butyrate production, and decreased the gene expression related to inflammatory factors, such as IL-12, TNF-α (Sun et al., Reference Sun, Mao, Zhu and Liu2019). In the review by Steele et al. (Reference Steele, Penner, Chaucheyras-Durand and Guan2016), the authors have shown how important butyrate produced by microorganisms is for barrier protection, stimulating the growth of both ruminal and intestinal epithelium.

The resident microbiota represent a barrier to the invasion and proliferation of exogenous and opportunistic pathogenic microorganisms (Hooper et al., Reference Hooper, Bry, Falk and Gordon1998). This effect occurs by the production of antagonist substances (bacteriocins), production of metabolites and toxic products (SCFA and H₂S), maintenance of low oxide reduction potential, and depletion of nutrients necessary for the maintenance of pathogens (Savage, Reference Savage1977; Bezirtzoglou, Reference Bezirtzoglou1997).

Most of the intestinal bacterial species are probiotic species such as Lactobacillus, Bifidobacterium, Enterococcus faecium, and Bacillus species (Ballou, Reference Ballou2011). Dias et al. (Reference Dias, Marcondes, de Souza, da Mata e Silva, Noronha, Resende, Machado, Mantovani, Dill-McFarland and Suen2018) have shown a very peculiar distribution in the GIT. Bifidobacterium had a similar abundance in the rumen, jejunum, and cecum, without differing in age, while Lactobacillus was abundant in the jejunum and Faecalibacterium in the cecum and colon, both decreasing over age. These species play a beneficial role in the production of vitamins, essential amino acids, and other favorable metabolic substrates (Leahy et al., Reference Leahy, Higgins, Fitzgerald and Sinderen2005), in addition to supporting nutrient metabolism (Sommer et al., Reference Sommer, Ståhlman, Ilkayeva, Arnemo, Kindberg, Josefsson, Newgard, Fröbert and Bäckhed2016; Wu et al., Reference Wu, Compher, Chen, Smith, Shah, Bittinger, Chehoud, Albenberg, Nessel, Gilroy, Star, Weljie, Flint, Metz, Bennett, Li, Bushman and Lewis2016), and assisting the host's immune system (Lukens et al., Reference Lukens, Gurung, Vogel, Johnson, Carter, McGoldrick, Bandi, Calabrese, Walle, Lamkanfi and Kanneganti2014). Lactobacillus and Bifidobacterium, as well as Faecalibacterium species, can modulate the immune system and inflammatory response (De Simone et al., Reference De Simone, Ciardi, Grassi, Gardini, Tzantzoglou, Trinchieri, Moretti and Jirillo1992; Oikonomou et al., Reference Oikonomou, Teixeira, Foditsch, Bicalho, Machado and Bicalho2013). These bacteria can prevent the binding of pathogens to intestinal epithelial cells (Duffy et al., Reference Duffy, Zielezny, Riepenhoff-Talty, Dryja, Sayahtaheri-Altaie, Griffiths, Ruffin, Barrett and Ogra1994), and stimulate the production of IgA (Fukushima et al., Reference Fukushima, Kawata, Hara, Terada and Mitsuoka1998).

The GIT development and homeostasis depend on the dynamics between gut microorganisms and the immune system (Sansonetti and Medzhitov, Reference Sansonetti and Medzhitov2009). The immune system ‘learns’ from the microbiota to recognize beneficial and pathogenic microorganisms, and thus quickly sends a response against harmful pathogens (Candela et al., Reference Candela, Maccaferri, Turroni, Carnevali and Brigidi2010).

On the other hand, other microorganisms are defined as causing pathology and virulence to the host, but all can cause endotoxemia under certain circumstances (Mackie et al., Reference Mackie, Sghir and Gaskins1999; Brooks et al., Reference Brooks, Carroll, Butel and Morse2007). As a consequence, beneficial microorganisms, such as species Bifidobacterium species limit the pathogenic agents' growth, e.g. E. coli, Clostridium perfringens, Campylobacter, Listeria, Shigella, and Vibrio cholerae (Gibson and Wang, Reference Gibson and Wang1994).

The gut microbiota's protective potential is reduced in stressful events, such as changes in diet, weaning, transportation, environmental changes, and contact with multiple infectious agents, causing dysbiosis and, consequently, the high incidence of bowel diseases in calves (Fleige et al., Reference Fleige, Preißinger, Meyer and Pfaffl2007). In addition to affecting the ruminal and fecal microbiota, weaning decreased bacteria, such as Bifidobacterium, Lactobacillus, Faecalibacterium, and Blautia, and reduced permeability in the rumen, duodenum, and jejunum (Wood et al., Reference Wood, Palmer, Steele, Metcalf and Penner2015; Meale et al., Reference Meale, Chaucheyras-Durand, Berends, Guan and Steele2017). Changes in intestinal permeability may be the cause of diarrhea incidence in dairy calves (Araujo et al., Reference Araujo, Yunta, Terré, Mereu, Ipharraguerre and Bach2015).

Microbiota and ruminal development in dairy calves

The ruminal microbiome regulates the fermentation processes and, consequently, the diversity and functional capacity to support the digestive efficiency of ruminants (Abecia et al., Reference Abecia, Ramos-Morales, Martínez-Fernandez, Arco, Martín-García, Newbold and Yáñez-Ruiz2014). However, the rumen-reticulum of neonatal ruminants is small and non-functional, and will only develop during the pre-weaning period as the animal starts feeding on starter concentrate of adequate quantity and quality (Davis and Drackley, Reference Davis and Drackley1998; Baldwin et al., Reference Baldwin, McLeod, Klotz and Heitmann2004).

Stater concentrate composition for dairy calves should be between 20 and 22% CP, 80% TDN, 15–25% NDF, and 6–20% ADF (Davis and Drackley, Reference Davis and Drackley1998). Minimum levels of NDF and ADF ensure healthy ruminal epithelium, and maximum levels ensure the inclusion of highly digestible ingredients. The inclusion of higher levels of NDF does not depress the intake, animal performance, or ruminal development using a high-quality fiber source, such as soybean hulls (Poczynek et al., Reference Poczynek, de Toledo, da Silva, Silva, Oliveira, Coelho, Virgínio Júnior, Polizel, Costa and Bittar2020).

The solid diets intake increases microbial populations' development, resulting in higher production of SCFA (Baldwin et al., Reference Baldwin, McLeod, Klotz and Heitmann2004). Depending on the type and quality of the diet fed to the calves, the GIT's morphophysiology may be altered (Li et al., Reference Li, Sparks, Connor and Li2011; Toledo et al., Reference Toledo, da Silva, Poczynek, Coelho, Silva, Polizel, Reis, Virgínio Júnior, Millen and Bittar2020). In addition, the provision of drinking water is essential to maintain ruminal activity. Wickramasinghe et al. (Reference Wickramasinghe, Kramer and Appuhamy2019) have reported the highest digestibility of NDF, ADF, and were more efficient than calves receiving water at day 17 or later.

Bacteria are the most significant ruminal microorganisms, mainly producing SCFA and microbial proteins, as well as some other metabolites such as biohydrogenated lipids (Clemmons et al., Reference Clemmons, Voy and Myer2019). Bacteria are key to mature ruminal function and can be detected one day after birth, before the rumen is mature, or even before the solid diet is consumed (Jami et al., Reference Jami, Israel, Kotser and Mizrahi2013; Dias et al., Reference Dias, Marcondes, Noronha, Resende, Machado, Mantovani, Dill-McFarland and Suen2017). The bacterial population increases with animal age and intake of solid diet (Dill-McFarland et al., Reference Dill-McFarland, Breaker and Suen2017; Dias et al., Reference Dias, Marcondes, Noronha, Resende, Machado, Mantovani, Dill-McFarland and Suen2017). Thus, as soon as calves consume solid feed, the faster the microbial composition and gastrointestinal function become similar to those seen in adult animals (Pitta et al., Reference Pitta, Pinchak, Dowd, Osterstock, Gontcharova, Youn, Dorton, Yoon, Min, Fulford, Wickersham and Malinowski2010; Li et al., Reference Li, Connor, Li, Baldwin VI and Sparks2012; Dill-McFarland et al., Reference Dill-McFarland, Breaker and Suen2017).

The increase in ruminal development is associated to starter intake resulting in the production of SCFA, especially butyric and propionic acids (Baldwin et al., Reference Baldwin, McLeod, Klotz and Heitmann2004). Acetate, which is not related to the changes observed in ruminal development, has a low molar ratio for the first 2 months and increases until 9 months of age as forage intake increases (Davis and Drackley, Reference Davis and Drackley1998; Suarez-Mena et al., Reference Suarez-Mena, Heinrichs, Jones, Hill and Quigley2016), and is not related to the changes observed in ruminal development.

The concentration and absorption of butyrate and propionate in the rumen provide chemical stimulation necessary for ruminal epithelium proliferation (Khan et al., Reference Khan, Lee, Lee, Kim, Kim, Park, Baek, Ha and Choi2008, Reference Khan, Bach, Weary and von Keyserlingk2016; Suarez-Mena et al., Reference Suarez-Mena, Heinrichs, Jones, Hill and Quigley2016). Some studies suggest that ruminal papillae proliferation is associated with the direct effect of butyrate and propionate on gene expression (Gálfi et al., Reference Gálfi, Neogrády and Sakata1991; Steele et al., Reference Steele, Penner, Chaucheyras-Durand and Guan2016).

Besides anatomical changes, changes in the site of digestion and absorption occur, which shift from the intestine to the rumen. The calf's energy metabolism is altered, beginning to use more SCFA and less glucose. The development of other digestive compartments also occurs with changes in intestinal enzyme activity, reduction in lactase activity, and increase in maltase as well as in the development of the salivary system and rumination behavior (Baldwin et al., Reference Baldwin, McLeod, Klotz and Heitmann2004; Khan et al., Reference Khan, Bach, Weary and von Keyserlingk2016).

Feeding a high-starch starter, despite the fermentative benefit for ruminal development, can cause metabolic disorders such as acidosis (Daneshvar et al., Reference Daneshvar, Khorvash, Ghasemi, Mahdavi, Moshiri, Mirzaei, Pezeshki and Ghaffari2015). The high production and low absorption/clearance of SCFA reduce the ruminal pH, resulting in reduced fiber digestibility by reducing fibrolytic bacteria (Russell and Wilson, Reference Russell and Wilson1996). This disorder, affecting developing calves, can change the establishment of the microbiota. However, the use of ingredients rich in non-starch polysaccharides (NSP), such as soybean hulls, which do not increase lactic acid concentration and, consequently, do not decrease pH, would be an alternative for inclusion in diets.

Forage or rougher feed promotes the development of ruminal muscle, maintains the integrity of the ruminal epithelium (Baldwin et al., Reference Baldwin, McLeod, Klotz and Heitmann2004), and keeps the ruminal pH higher due to the larger particle size and fermentation profile (Terré et al., Reference Terré, Pedrals, Dalmau and Bach2013). Maintaining a higher ruminal pH benefits microbial populations typically associated with forage, which in turn alter the production of butyrate and propionate to acetate.

Many recent studies have demonstrated that access to chopped hay promotes starter concentrate intake in calves, contrary to traditional recommendations (Jiao et al., Reference Jiao, Lu, Forster, Zhou, Wang, Kang and Tan2016; Toledo et al., Reference Toledo, da Silva, Poczynek, Coelho, Silva, Polizel, Reis, Virgínio Júnior, Millen and Bittar2020). Castells et al. (Reference Castells, Bach, Aris and Terré2013) have conducted a meta-analysis and concluded that no difference in the intestinal filling was found between calves not consuming forage and those consuming forage up to 5% of total solid feed intake. However, the effect of forage feeding on the long-term microbial ruminal ecosystem has not yet been determined.

Despite all this discussion, relatively limited information about the relationship of the early microbiota to ruminal development is still available. Recently, Pan et al. (Reference Pan, Li, Li, Zhao, Wang, Chen and Jiang2021) have analyzed ventral sac rumen wall gene expression and the ruminal microbiome of goats until 56 d (weaning). The animals were fed only whole milk until 24 d, receiving the solid diet at 25 d. The analysis showed that the differentially expressed genes of the ruminal transcriptome related to two distinct phases, immune phase (1–14 d) and metabolic phase (21–56 d). In addition, they observed changes in the ruminal microbiome (at 42 d), with increased abundance of Selenomonas spp., Prevotella spp. and Ruminococcus, and increased expression of bacterial genes related to biosynthesis and glycolysis/glyconeogenesis activities. Based on this, the authors understood that ruminal development in the first phase is more likely a programmed process than continuously stimulated by the solid diet and the microbiome, but can be activated by microbial colonization at birth. The intense functional correlations between rumen genes and the microbiome are related to ruminal pH homeostasis, nitrogen metabolism, and immune response during early ruminal development. This study brings a new insight into the interactions between host and microbiota, however, there are some questions about the possible effect of the microbiota in activating the immune phase, and the expression of these genes to the animal has earlier access to a solid diet. Or if there is any effect of the liquid diet in this period.

Gut microbiota and health of dairy calves

Commensal bacteria in the intestine protect against the invasion of pathogenic microorganisms (Kamada et al., Reference Kamada, Chen, Inohara and Núñez2013; Kouritzin and Guan, Reference Kouritzin and Guan2017), stimulate and maintain the integrity of the gut mucosal barrier, and produce a variety of beneficial metabolic substrates (Leahy et al., Reference Leahy, Higgins, Fitzgerald and Sinderen2005; O'Connell Motherway et al., Reference O'Connell Motherway, Zomer, Leahy, Reunanen, Bottacini, Claesson, O'Brien, Flynn, Casey, Moreno Munoz, Kearney, Houston, O'Mahony, Higgins, Shanahan, Palva, de Vos, Fitzgerald, Ventura, O'Toole and van Sinderen2011). Change in microbial composition leading to shifts in the proportion of commensal and pathogenic bacteria is called dysbiosis and is often related to gastrointestinal disorders, metabolic and immunological alterations, and generalized effects on the organism (Maynard et al., Reference Maynard, Elson, Hatton and Weaver2012; Zeineldin et al., Reference Zeineldin, Aldridge and Lowe2018).

Nutrition is critical in maintaining and modifying the gut microbial composition. Besides the previously comments about supplying colostrum (Malmuthuge et al., Reference Malmuthuge, Chen, Liang, Goonewardene and Guan2015b; Fischer et al., Reference Fischer, Song, He, Haines, Guan and Steele2018), the liquid diet plays a very important role during the pre-weaning period. The feeding of a liquid diet to the newborn has considerable implications on the development of gut microbiota. Several of its constituents, such as oligosaccharides, select for highly adapted intestinal microbiota dominated by Bifidobacterium species (Pacheco et al., Reference Pacheco, Barile, Underwood and Mills2015). However, the type and availability of nutrients in the milk replacers are different from those observed in whole milk, and can significantly impact the colonization of the animal's intestine.

Whole milk is the best feed for pre-weaning calves, both nutritionally and to stimulate the microbiota (Zou et al., Reference Zou, Wang, Deng, Cao, Li and Wang2017; Virgínio Júnior et al., Reference Virgínio Júnior, Coelho, de Toledo, Montenegro, Coutinho and Bittar2021); however, many dairy herds still insist on feeding waste milk to calves. Deng et al. (Reference Deng, Wang, Zou, Azarfar, Wei, Ji, Zhang, Wu, Wang, Dong, Xu, Shao, Xiao, Yang, Cao and Li2017), Zou et al. (Reference Zou, Wang, Deng, Cao, Li and Wang2017), and Pereira et al. (Reference Pereira, Carroll, Lima, Foditsch, Siler, Bicalho and Warnick2018) have already demonstrated that in addition to altering the intestinal microbiota, animals fed waste milk had a higher abundance of pathogenic microorganisms such as Odoribacter and Fusobacterium, and antibiotic residues in the feces. Pasteurization and acidification can be an alternative in reducing the bacterial load (Deng et al., Reference Deng, Wang, Zou, Azarfar, Wei, Ji, Zhang, Wu, Wang, Dong, Xu, Shao, Xiao, Yang, Cao and Li2017; Zou et al., Reference Zou, Wang, Deng, Cao, Li and Wang2017), but there are no ways to reduce antibiotic residues after milking.

Also, bacterial genera Bifidobacterium, Faecalibacterium, and Megamonas were associated with healthy calves and more weight gain (Oikonomou et al., Reference Oikonomou, Teixeira, Foditsch, Bicalho, Machado and Bicalho2013; Gomez et al., Reference Gomez, Arroyo, Costa, Viel and Weese2017; Virgínio Júnior et al., Reference Virgínio Júnior, Coelho, de Toledo, Montenegro, Coutinho and Bittar2021), whereas unhealthy calves were associated to Escherichia, Shigella and Fusobacterium genera (Gomez et al., Reference Gomez, Arroyo, Costa, Viel and Weese2017; Virgínio Júnior et al., Reference Virgínio Júnior, Coelho, de Toledo, Montenegro, Coutinho and Bittar2021) and had lower microbial diversity on the gut (Oikonomou et al., Reference Oikonomou, Teixeira, Foditsch, Bicalho, Machado and Bicalho2013; Ma et al., Reference Ma, Villot, Renaud, Skidmore, Chevaux, Steele and Guan2020).

According to Klein-Jöbstl et al. (Reference Klein-Jöbstl, Iwersen and Drillich2014b), diarrhea is a complex multifactorial disease with numerous infectious and noninfectious factors, and diarrhea is cited in several studies worldwide as the leading cause of morbidity and mortality in calves during the pre-weaning period (Lanz Uhde et al., Reference Lanz Uhde, Kaufmann, Sager, Albini, Zanoni, Schelling and Meylan2008; Bartels et al., Reference Bartels, Holzhauer, Jorritsma, Swart and Lam2010; dos Santos and Bittar, Reference dos Santos and Bittar2015; Mohammed et al., Reference Mohammed, Marouf, Erfana, El-Jakee, Hessain, Dawoud, Kabli and Moussa2019; Fruscalso et al., Reference Fruscalso, Olmos and Hötzel2020). Among the pathogens commonly associated with diarrhea are E. coli and Salmonella spp. (Izzo et al., Reference Izzo, Kirkland, Mohler, Perkins, Gunn and House2011), such as bovine rotavirus group A (BRV-A), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV), Clostridium perfringens type C, and Cryptosporidium parvum (Saif and Smith, Reference Saif and Smith1985; Singh et al., Reference Singh, Sharma, Kumar, Banga, Aulakh, Gill and Sharma2006; Gulliksen et al., Reference Gulliksen, Jor, Lie, Hamnes, Løken, Åkerstedt and Østerås2009; Cho et al., Reference Cho, Han, Wang, Cooper, Schwartz, Engelken and Yoon2013; Cruvinel et al., Reference Cruvinel, Ayres, Zapa, Nicaretta, Couto, Heller, Bastos, Cruz, Soares, Teixeira, de Oliveira, Fritzen, Alfieri, Freire and Lopes2020). Farm management can also be responsible for the frequency and spreading of these pathogens, such as poor hygiene in milking equipment, buckets, feeding bottles, shelters, or even poor water quality, or related to the number of animals on the farm and the presence of other animal species (Klein-Jöbstl et al., Reference Klein-Jöbstl, Iwersen and Drillich2014b; Mohammed et al., Reference Mohammed, Marouf, Erfana, El-Jakee, Hessain, Dawoud, Kabli and Moussa2019).

To decrease the use of antimicrobials in the farms, alternative treatments have grown in recent years. The use of natural products, such as probiotics, prebiotics, herbal extracts, enzyme blends, among others, has been commonly used in studies to treat diarrhea. Signorini et al. (Reference Signorini, Soto, Zbrun, Sequeira, Rosmini and Frizzo2012) reviewed the results of different studies using lactic acid bacteria (LAB) in calf feed, and only in studies using whole milk the LAB were effective in reducing the diarrheal incidence and increasing the ratio of LAB to coliforms. Probably the lack of effect on milk replacer feeding may be related to the different compositions among commercial products, and would make it difficult to provide a more specific response to the effects of LAB.

Cangiano et al. (Reference Cangiano, Yohe, Steele and Renaud2020) reviewed the strategic use of prebiotics in calf nutrition such as fructooligosaccharides, galactosaccharides, mannanooligosaccharides (MOS), and have showed that prebiotics have been little used in ruminant nutrition, compared to probiotics. Most of the prebiotic's studies have been on calves, primarily MOS, and have been effective in improving performance and gut health. However, some studies with beta-glucans have also shown promise (Ma et al., Reference Ma, Tu, Zhang, Guo, Deng, Zhou, Yun and Diao2015; Kido et al., Reference Kido, Tejima, Haramiishi, Uyeno, Ide, Kurosu and Kushibiki2019). However, there is still a lack of studies to show further results or to indicate the best time to administer this prebiotics.

Other studies using compounds containing different microorganisms and enzyme blends, (Renaud et al., Reference Renaud, Kelton, Weese, Noble and Duffield2019), red propolis (Slanzon et al., Reference Slanzon, de Toledo, da Silva, Coelho, da Silva, Cezar and Bittar2019), and lysolecithin (Reis et al., Reference Reis, Toledo, da Silva, Poczynek, Fioruci, Cantor, Greco and Bittar2021) have shown potential effect in reducing diarrhea incidence in dairy calves. However, the effect on microbiota has not been reported. Fecal microbiota transplantation has also been shown to be a technique with beneficial effects, such as reduced fecal amino acid concentration, improved weight gain, and growth (Kim et al., Reference Kim, Whon, Sung, Jeong, Jung, Shin, Hyun, Kim, Lee, Lee and Bae2021). Ruminal transfaunation is also a similar technique, using ruminal contents from healthy cows to cows with ruminal disorders, and in addition to repopulating the rumen, it also provides various nutrients and energy to the microbiome (DePeters and George, Reference DePeters and George2014). However, the study by Yu et al. (Reference Yu, Shi, Yang, Gao, Chen, Cao, Yu and Wang2020) was the most promising, using freeze-dried ruminal fluid, they have observed in goats a higher digestibility of DM, crude protein during pre-weaning, and after weaning, improved starch digestibility. Bu et al. (Reference Bu, Zhang, Ma, Park, Wang, Wang, Xu and Yu2020) have observed a reduction in the overall incidence of diarrhea (91 vs 69 d) in animals receiving fresh ruminal fluid compared to those receiving autoclaved ruminal fluid.

Despite there is an increasing number of microbiota studies, there is still limited knowledge about the gut microbiota in dairy calves, and the mechanisms of influence of these microbiota on development and gut health are still unclear and undefined.

Diet as a factor in the manipulation of the gastrointestinal microbiota

The composition of the GIT microbial community is influenced by both the diet and the animal's age. Age is a factor in increasing diversity and uniformity (Jami et al., Reference Jami, Israel, Kotser and Mizrahi2013). On the other hand, diet plays a significant role in defining the structure of the ruminant microbiota and the general health of the GIT (Kreikemeier et al., Reference Kreikemeier, Harmon, Peters, Gross, Armendariz and Krehbiel1990). Diet is an influential contributing factor in establishing GIT microbiota and can serve as a tool to change the microbiota in early life (Dill-McFarland et al., Reference Dill-McFarland, Weimer, Breaker and Suen2019). The manipulation of GIT microbiota in the pre-weaning period may result in the effective establishment of the desired microbiota that remains stable until maturity (Malmuthuge and Guan, Reference Malmuthuge and Guan2016). According to Dias et al. (Reference Dias, Marcondes, Noronha, Resende, Machado, Mantovani, Dill-McFarland and Suen2017), manipulation of the ruminal microbiota at the pre-weaning period may be possible with dietary interventions, besides being useful to design strategies to promote colonization of specific microorganisms associated to better development of the calf. This allows for the selection of beneficial microorganisms, supporting their increased growth, and reducing pathogenic microorganisms that cause various diseases in calves, such as diarrhea.

The mature rumen is well known to be resilient to dietary changes. Any changes in the microbiota composition may be temporary, only occurring in the period of supplementation (Weimer, Reference Weimer2015). This fact suggests that microbiota manipulations can be challenging as they it is resilient (Abecia et al., Reference Abecia, Martínez-Fernandez, Waddams, Martín-García, Pinloche, Creevey, Denman, Newbold and Yáñez-Ruiz2018). In contrast, neonatal microbiota are more sensitive to external pressures, such as antibiotics \ and changes in the composition of the liquid or solid diet (Mulder et al., Reference Mulder, Schmidt, Lewis, Delday, Stokes, Bailey, Aminov, Gill, Pluske, Mayer and Kelly2011; Malmuthuge et al., Reference Malmuthuge, Griebel and Guan2015a). Thus, it is necessary to evaluate the GIT microbiota in the same individual during its entire development from calf to cow, to determine the best moment to the microbiota manipulation would be more efficient in improving future milk production.

External factors in early life cause changes in the composition of the gastrointestinal microbiota and this effect may persist into adulthood, in addition to causing an effect on the animal phenotype (Russell et al., Reference Russell, Gold, Hartmann, Willing, Thorson, Wlodarska, Gill, Blanchet, Mohn, McNagny and Finlay2012; Abecia et al., Reference Abecia, Martín-García, Martínez, Newbold and Yáñez-Ruiz2013). Thus, manipulating the gastrointestinal microbial community in neonates can allow a desirable microbial composition to establish itself in adult animals. This manipulation can in the future obtain animals producing less methane, having greater efficiency in milk production, or weight gain rates.

Recent, studies have shown how effective different diets can be on the microbiota. Malmuthuge et al. (Reference Malmuthuge, Chen, Liang, Goonewardene and Guan2015b) and Fischer et al. (Reference Fischer, Song, He, Haines, Guan and Steele2018) have already shown how crucial is colostrum feeding or the timing of feeding influences the gut microbiome in early life. However, the effect of early management on the microbiome is still understudied. Colostrum powder, frozen colostrum, and colostrum enrichment are all routinely used on farms, but the impact on the microbiota is not well understood.

The liquid diet also has a significant impact on the intestinal microbiota. Whole milk should be the first choice to feed calves, and its composition has proven ideal to limit the growth of pathogenic microorganisms (Pacheco et al., Reference Pacheco, Barile, Underwood and Mills2015). Acidification or pasteurization of whole or waste milk have proven to be good alternatives, with good results in performance, besides stimulating the growth of commensal and probiotic bacteria, and decreasing the incidence of diarrhea (Deng et al., Reference Deng, Wang, Zou, Azarfar, Wei, Ji, Zhang, Wu, Wang, Dong, Xu, Shao, Xiao, Yang, Cao and Li2017; Coelho et al., Reference Coelho, Silva, Silva, da Silva, Cezar, Slanzon, Miqueo, de Toledo, Bittar and De Toledo2020; Virgínio Júnior et al., Reference Virgínio Júnior, Coelho, de Toledo, Montenegro, Coutinho and Bittar2021).

Milk replacers are the main liquid diet in many dairy herds, however, with a wide range of ingredients, such as dairy products and vegetable sources (Bittar et al., Reference Bittar, da Silva and Chester-Jones2018), that can affect the gastrointestinal microbiota. Virgínio Júnior et al. (Reference Virgínio Júnior, Coelho, de Toledo, Montenegro, Coutinho and Bittar2021) noted that the milk replacer composition used in one study may have negatively affected the initial microbiota, predisposing these animals to be more susceptible to diarrhea. Badman et al. (Reference Badman, Daly, Kelly, Moran, Cameron, Watson, Newbold and Shirazi-Beechey2019) also observed that the different composition impacts the initial microbiota up to 7 d.

The addition of prebiotics, probiotics, and additives to liquid diets is a beneficial alternative in modulating the rumenal and intestinal microbiomes, decreasing diarrhea incidence, modulating the immune response, promoting higher performance and gut health. O'Hara et al. (Reference O'Hara, Kelly, McCabe, Kenny, Guan and Waters2018) adding sodium-butyrate to whole milk, observed higher SCFA concentration in the colon, as well as a higher abundance of Phascolarctobacterium, considered a probiotic bacteria. Other studies, even if not evaluating the microbiome, have high expectations in modulating the gut microbiota (Slanzon et al., Reference Slanzon, de Toledo, da Silva, Coelho, da Silva, Cezar and Bittar2019; Reis et al., Reference Reis, Toledo, da Silva, Poczynek, Fioruci, Cantor, Greco and Bittar2021).

The concentrate supply also modulated the ruminal microbiota (Dias et al., Reference Dias, Marcondes, Noronha, Resende, Machado, Mantovani, Dill-McFarland and Suen2017). Changes in the solid diet, such as the forage/concentrate ratio, can cause changes in the ruminal bacterial community at the beginning of life and persist for up to 4 months (Yáñez-Ruiz et al., Reference Yáñez-Ruiz, Macías, Pinloche and Newbold2010), as diets probably have higher NDF contents or even the supplementation of hay or whole-grain corn.

A recent study by Dill-McFarland et al. (Reference Dill-McFarland, Weimer, Breaker and Suen2019) evaluated the fecal microbiota at 2, 4, and 8 weeks, and at 1 and 2 years, and the rumen at week 8 (from males that were slaughtered) and cannulated females at 1 and 2 years. A calf starter, corn silage, and a diet containing 25/75 concentrate and corn silage were fed to calves in the pre-weaning period. This study has shown off early intervention does not have as guaranteed long-term effects on the microbiota or milk production. However, some authors have reported that there is great inter-individual variability even in groups receiving the same diet (Klein-Jöbstl et al., Reference Klein-Jöbstl, Schornsteiner, Mann, Wagner, Drillich and Schmitz-Esser2014a; Zhou et al., Reference Zhou, Chen, Griebel and Guan2014; Virgínio Júnior et al., Reference Virgínio Júnior, Coelho, de Toledo, Montenegro, Coutinho and Bittar2021), so the differences in the ruminal microbiome may not be so accurate. Another question may be regarding the Greengenes database, as shown in the second topic, which has not been updated for a long time. So far, the Dill-Mcfarland study is the only study that has been up to 2 years old, but its results deserve to be discussed with more care and attention.

Another study that found similar results, Virgínio Júnior et al. (Reference Virgínio Júnior, Coelho, de Toledo, Montenegro, Coutinho and Bittar2021) evaluating the fecal microbiota of calves receiving different liquid diets (milk replacer, whole milke, and acidified whole milk), observed at weaning (week 8), that the bacterial community structure has similarities among treatments, and after the animals were housed in a pasture at week 10, this similarity was more evident, with few differences in composition after weaning. Thus, indicating that perhaps the environment is a factor to be considered as a modulator or leveler of the gastrointestinal microbiome, as well as the starter intake.

However, we cannot deny the fact that dietary modulation associated with management practices may affect the GIT microbial composition in neonatal calves. Thus, it can improve health status, prevent colonization by pathogenic microorganisms, and reduce inflammatory reactions. It is not yet clear if the effects of modulation are can persist in the microbiota of mature animals. Perhaps future studies on dietary modulation in the GIT microbiota should consider the effect of the environment. In addition, should also make use of integrating different molecular techniques, such as metabolomics, transcriptomics, and proteomics, which, also correlating microbiome data with performance, intake, metabolism, health, and behavior data. Only then, will we be able to make more strategic decisions regarding gut microbiota modulation.