Multi-omics in vitro study of the salivary modulation of the goat rumen microbiome.

Ruminants are able to produce large quantities of saliva which enter into the rumen and salivary components exert different physiological functions. Although previous research has indicated that salivary immunoglobulins can partially modulate the rumen microbial activity, the role of the salivary components other than ions on the rumen microbial ecosystem has not been thoroughly investigated in ruminants. To investigate this modulatory activity, a total of 16 semi-continuous in vitro cultures with oats hay and concentrate were used to incubate rumen fluid from four donor goats with autoclaved saliva (AUT) as negative control, saliva from the same rumen fluid donor (OWN) as positive control, and either goat (GOAT) or sheep (SHEEP) saliva as experimental interventions. Fermentation was monitored throughout 7 days of incubation and the microbiome and metabolome were analysed at the end of this incubation by Next-Generation sequencing and liquid chromatography coupled with mass spectrometry, respectively. Characterisation of the proteome and metabolome of the different salivas used for the incubation showed a high inter-animal variability in terms of metabolites and proteins, including immunoglobulins. Incubation with AUT saliva promoted lower fermentative activity in terms of gas production (-9.4%) and highly divergent prokaryotic community in comparison with other treatments (OWN, GOAT and SHEEP) suggesting a modulatory effect derived from the presence of bioactive salivary components. Microbial alpha-diversity at amplicon sequence variant (ASV) level was unaffected by treatment. However, some differences were found in the microbial communities across treatments, which were mostly caused by a greater abundance of Proteobacteria and Rikenellacea in the AUT treatment and lower of Prevotellaceae. These bacteria, which are key in the rumen metabolism, had greater abundances in GOAT and SHEEP treatments. Incubation with GOAT saliva led to a lower protozoal concentration and propionate molar proportion indicating a capacity to modulate the rumen microbial ecosystem. The metabolomics analysis showed that the AUT samples were clustered apart from the rest indicating different metabolic pathways were promoted in this treatment. These results suggest that specific salivary components contribute to host-associated role in selecting the rumen commensal microbiota and its activity. These findings could open the possibility of developing new strategies to modulate the saliva composition as a way to manipulate the rumen function and activity.

Enhancing rumen microbial diversity and its impact on energy and protein metabolism in forage-fed goats.

Introduction: This study explores if promoting a complex rumen microbiota represents an advantage or a handicap in the current dairy production systems in which ruminants are artificially reared in absence of contact with adult animals and fed preserved monophyte forage. Methods: In order to promote a different rumen microbial diversity, a total of 36 newborn goat kids were artificially reared, divided in 4 groups and daily inoculated during 10 weeks with autoclaved rumen fluid (AUT), fresh rumen fluid from adult goats adapted to forage (RFF) or concentrate (RFC) diets, or absence of inoculation (CTL). At 6 months of age all animals were shifted to an oats hay diet to determine their ability to digest a low quality forage. Results and discussion: Early life inoculation with fresh rumen fluid promoted an increase in the rumen overall microbial diversity which was detected later in life. As a result, at 6 months of age RFF and RFC animals had higher bacterial (+50 OTUs) and methanogens diversity (+4 OTUs) and the presence of a complex rumen protozoal community (+32 OTUs), whereas CTL animals remained protozoa-free. This superior rumen diversity and presence of rumen protozoa had beneficial effects on the energy metabolism allowing a faster adaptation to the forage diet, a higher forage digestion (+21% NDF digestibility) and an energetically favourable shift of the rumen fermentation pattern from acetate to butyrate (+92%) and propionate (+19%) production. These effects were associated with the presence of certain rumen bacterial taxa and a diverse protozoal community. On the contrary, the presence of rumen protozoa (mostly Entodinium) had a negative impact on the N metabolism leading to a higher bacterial protein breakdown in the rumen and lower microbial protein flow to the host based on purine derivatives urinary excretion (-17% to -54%). The inoculation with autoclaved rumen fluid, as source of fermentation products but not viable microbes, had smaller effects than using fresh inoculum. These findings suggest that enhancing rumen microbial diversity represents a desirable attribute when ruminants are fed forages in which the N supply does not represent a limiting factor for the rumen microbiota.

Presence of Adult Companion Goats Favors the Rumen Microbial and Functional Development in Artificially Reared Kids.

Newborn dairy ruminants are usually separated from their dams after birth and fed on milk replacer. This lack of contact with adult animals may hinder the rumen microbiological and physiological development. This study evaluates the effects of rearing newborn goat kids in contact with adult companions on the rumen development. Thirty-two newborn goat kids were randomly allocated to two experimental groups which were reared either in the absence (CTL) or in the presence of non-lactating adult goats (CMP) and weaned at 7 weeks of age. Blood and rumen samples were taken at 5, 7, and 9 weeks of age to evaluate blood metabolites and rumen microbial fermentation. Next-generation sequencing was carried out on rumen samples collected at 7 weeks of age. Results showed that CTL kids lacked rumen protozoa, whereas CMP kids had an abundant and complex protozoal community as well as higher methanogen abundance which positively correlated with the body weight and blood ß-hydroxybutyrate as indicators of the physiological development. CMP kids also had a more diverse bacterial community (+132 ASVs) and a different structure of the bacterial and methanogen communities than CTL kids. The core rumen bacterial community in CMP animals had 53 more ASVs than that of CTL animals. Furthermore, the number of ASVs shared with the adult companions was over 4-fold higher in CMP kids than in CTL kids. Greater levels of early rumen colonizers Proteobacteria and Spirochaetes were found in CTL kids, while CMP kids had higher levels of Bacteroidetes and other less abundant taxa (Veillonellaceae, Cyanobacteria, and Selenomonas). These findings suggest that the presence of adult companions facilitated the rumen microbial development prior to weaning. This accelerated microbial development had no effect on the animal growth, but CMP animals presented higher rumen pH and butyrate (+45%) and ammonia concentrations than CTL kids, suggesting higher fibrolytic and proteolytic activities. CMP kids also had higher blood ß-hydroxybutyrate (+79%) and lower blood glucose concentrations (-23%) at weaning, indicating an earlier metabolic development which could favor the transition from pre-ruminant to ruminant after the weaning process. Further research is needed to determine the effects of this intervention in more challenging farm conditions.

Inoculation with rumen fluid in early life accelerates the rumen microbial development and favours the weaning process in goats.

BACKGROUND: Newborn ruminants possess an underdeveloped rumen which is colonized by microorganisms acquired from adult animals and the surrounding environment. This microbial transfer can be limited in dairy systems in which newborns are separated from their dams at birth. This study explores whether the direct inoculation of fresh or autoclaved rumen fluid from adult goats to newborn kids has a beneficial effect on rumen microbial development and function. RESULTS: Repetitive inoculation of young kids with fresh rumen fluid from adult goats adapted to forage (RFF) or concentrate diets (RFC) accelerated microbial colonization of the rumen during the pre-weaning period leading to high protozoal numbers, a greater diversity of bacterial (+ 234 OTUs), methanogens (+ 6 OTUs) and protozoal communities (+ 25 OTUs) than observed in control kids (CTL) without inoculation. This inoculation also increased the size of the core bacterial and methanogens community and the abundance of key rumen bacteria (Ruminococcaceae, Fibrobacteres, Veillonellaceae, Rikenellaceae, Tenericutes), methanogens (Methanobrevibacter ruminantium, Methanomicrobium mobile and Group 9), anaerobic fungi (Piromyces and Orpinomyces) and protozoal taxa (Enoploplastron, Diplodinium, Polyplastron, Ophryoscolex, Isotricha and Dasytricha) before weaning whereas CTL kids remained protozoa-free through the study. Most of these taxa were positively correlated with indicators of the rumen microbiological and physiological development (higher forage and concentrate intakes and animal growth during the post-weaning period) favoring the weaning process in RFF and RFC kids in comparison to CTL kids. Some of these microbiological differences tended to decrease during the post-weaning period, although RFF and RFC kids retained a more complex and matured rumen microbial ecosystem than CTL kids. Inoculation with autoclaved rumen fluid promoted lower development of the bacterial and protozoal communities during the pre-weaning period than using fresh inocula, but it favored a more rapid microbial development during the post-weaning than observed for CTL kids. CONCLUSIONS: This study demonstrated that inoculation of young ruminants with fresh rumen fluid from adult animals accelerated the rumen microbial colonization which was associated with an earlier rumen functional development. This strategy facilitated a smoother transition from milk to solid feed favoring the animal performance during post-weaning and minimizing stress.