Dietary energy sources and levels shift the multi-kingdom microbiota and functions in the rumen of lactating dairy cows

Background: Dietary energy source and level in lactation diets can profoundly affect milk yield and composition.Such dietary effects on lactation performance are underpinned by alteration of the rumen microbiota, of which bacteria, archaea, fungi, and protozoa may vary differently. However, few studies have examined all the four groups of rumen microbes. This study investigated the effect of both the level and source of dietary energy on rumen bacteria, archaea, fungi, and protozoa in the rumen of lactating dairy cows. A 2 × 2 factorial design resulted in four dietary treatments: low and high dietary energy levels(LE: 1.52–1.53;and HE: 1.71–1.72 Mcal/kg dry matter) and two dietary energy sources(GC: finely ground corn;and SFC: steam-flaked corn). We used a replicated 4 × 4 Latin square design using eight primiparous Chinese Holstein cows with each period lasting for 21 d. The rumen microbiota was analyzed using metataxonomics based on kingdom-specific phylogenetic markers [16 S r RNA gene for bacteria and archaea, 18 S r RNA gene for protozoa, and internally transcribed spacer 1(ITS1) for fungi] followed with subsequent functional prediction using PICRUSt2.Results: The GC resulted in a higher prokaryotic(bacterial and archaeal) species richness and Faith's phylogenetic diversity than SFC. For the eukaryotic(fungi and protozoa) microbiota, the LE diets led to significantly higher values of the above measurements than the HE diets. Among the major classified taxa, 23 genera across all the kingdoms differed in relative abundance between the two dietary energy levels, while only six genera(none being protozoal)were differentially abundant between the two energy sources. Based on prokaryotic amplicon sequence variants(ASVs) from all the samples, overall functional profiles predicted using PICRUSt2 differed significantly between LE and HE but not between the two energy sources. Fish Taco analysis identified Ruminococcus and Coprococcus as the taxa potentially contributing to the enriched KEGG pathways for biosynthesis of amino acids and to the metabolisms of pyruvate, glycerophospholipid, and nicotinate and nicotinamide in the rumen of HE-fed cows. The co-occurrence networks were also affected by the dietary treatments, especially the LE and GC diets, resulting in distinct co-occurrence networks. Several microbial genera appeared to be strongly correlated with one or more lactation traits.Conclusions: Dietary energy level affected the overall rumen multi-kingdom microbiota while little difference was noted between ground corn and steam-flaked corn. Some genera were also affected differently by the four dietary treatments, including genera that had been shown to be correlated with lactation performance or feed efficiency.The co-occurrence patterns among the genera exclusively found for each dietary treatment may suggest possible metabolic interactions specifically affected by the dietary treatment. Some of the major taxa were positively correlated to milk properties and may potentially serve as biomarkers of one or more lactation traits.

Rumen methanogens and mitigation of methane emission by anti-methanogenic compounds and substances

Methanogenic archaea reside primarily in the rumen and the lower segments of the intestines of ruminants, where they utilize the reducing equivalents derived from rumen fermentation to reduce carbon dioxide, formic acid, or methylamines to methane（CH_4）. Research on methanogens in the rumen has attracted great interest in the last decade because CH_4 emission from ruminants contributes to global greenhouse gas emission and represents a loss of feed energy. Some DNA-based phylogenetic studies have depicted a diverse and dynamic community of methanogens in the rumen. In the past decade, researchers have focused on elucidating the underpinning that determines and affects the diversity, composition, structure, and dynamics of methanogen community of the rumen. Concurrently, many researchers have attempted to develop and evaluate interventions to mitigate enteric CH_4 emission. Although much work has been done using plant secondary metabolites, other approaches such as using nitrate and 3-nitrooxy propanol have also yielded promising results. Most of these antimethanogenic compounds or substances often show inconsistent results among studies and also lead to adverse effects on feed intake and digestion and other aspects of rumen fermentation when fed at doses high enough to achieve effective mitigation. This review provides a brief overview of the rumen methanogens and then an appraisal of most of the antimethanogenic compounds and substances that have been evaluated both in vitro and in vivo. Knowledge gaps and future research needs are also discussed with a focus on methanogens and methane mitigation.

Specific inhibition of Streptococcus bovis by endolysin LyJH307 supplementation shifts the rumen microbiota and metabolic pathways related to carbohydrate metabolism

Background:Endolysins,the bacteriophage-originated peptidoglycan hydrolases,are a promising replacement for antibiotics due to immediate lytic activity and no antibiotic resistance.The objectives of this study were to investigate the lytic activity of endolysin LyJH307 against S.bovis and to explore changes in rumen fermentation and microbiota in an in vitro system.Two treatments were used:1)control,corn grain without LyJH307;and 2)LyJH307,corn grain with LyJH307(4 U/mL).An in vitro fermentation experiment was performed using mixture of rumen fluid collected from two cannulated Holstein steers(450±30 kg)and artificial saliva buffer mixed as 1:3 ratio for 12 h incubation time.In vitro dry matter digestibility,pH,volatile fatty acids,and lactate concentration were estimated at 12 h,and the gas production was measured at 6,9,and 12 h.The rumen bacterial community was analyzed using 16S rRNA amplicon sequencing.Results:LyJH307 supplementation at 6 h incubation markedly decreased the absolute abundance of S.bovis(approximately 70% compared to control,P=0.0289)and increased ruminal pH(P=0.0335)at the 12 h incubation.The acetate proportion(P=0.0362)was significantly increased after LyJH307 addition,whereas propionate(P=0.0379)was decreased.LyJH307 supplementation increased D-lactate(P=0.0340)without any change in L-lactate concentration(P>0.10).There were no significant differences in Shannon’s index,Simpson’s index,Chao1 estimates,and evenness(P>0.10).Based on Bray-Curtis dissimilarity matrices,the LyJH307 affected the overall shift in microbiota(P=0.097).LyJH307 supplementation induced an increase of 11 genera containing Lachnoclostridium,WCHB1-41,unclassified genus Selenomonadaceae,Paraprevotella,vadinBE97,Ruminococcus gauvreauii group,Lactobacillus,Anaerorhabdus furcosa group,Victivallaceae,Desulfuromonadaceae,and Sediminispirochaeta.The predicted functional features represented by the Kyoto Encyclopedia of Genes and Genomes pathways were changed by LyJH307 toward a decrease of carbohydrate metabolism.Conclusions:LyJH307 caused a reduction of S.bovis and an increase of pH with shifts in minor microbiota and its metabolic pathways related to carbohydrate metabolism.This study provides the first insight into the availability of endolysin as a specific modulator for rumen and shows the possibility of endolysin degradation by rumen microbiota.

Heat stress impacts the multi-domain ruminal microbiota and some of the functional features independent of its effect on feed intake in lactating dairy cows

Background:Heat stress(HS)affects the ruminal microbiota and decreases the lactation performance of dairy cows.Because HS decreases feed intake,the results of previous studies were confounded by the effect of HS on feed intake.This study examined the direct effect of HS on the ruminal microbiota using lactating Holstein cows that were pair-fed and housed in environmental chambers in a 2×2 crossover design.The cows were pair-fed the same amount of identical total mixed ration to eliminate the effect of feed or feed intake.The composition and structure of the microbiota of prokaryotes,fungi,and protozoa were analyzed using metataxonomics and compared between two thermal conditions:pair-fed thermoneutrality(PFTN,thermal humidity index:65.5)and HS(87.2 for daytime and 81.8 for nighttime).Results:The HS conditions altered the structure of the prokaryotic microbiota and the protozoal microbiota,but not the fungal microbiota.Heat stress significantly increased the relative abundance of Bacteroidetes(primarily Gram-negative bacteria)while decreasing that of Firmicutes(primarily Gram-positive bacteria)and the Firmicutes-toBacteroidetes ratio.Some genera were exclusively found in the heat-stressed cows and thermal control cows.Some co-occurrence and mutual exclusion between some genera were also found exclusively for each thermal condition.Heat stress did not significantly affect the overall functional features predicted using the 16S rRNA gene sequences and ITS1 sequences,but some enzyme-coding genes altered their relative abundance in response to HS.Conclusions:Overall,HS affected the prokaryotes,fungi,and protozoa of the ruminal microbiota in lactating Holstein cows to a different extent,but the effect on the structure of ruminal microbiota and functional profiles was limited when not confounded by the effect on feed intake.However,some genera and co-occurrence were exclusively found in the rumen of heat-stressed cows.These effects should be attributed to the direct effect of heat stress on the host metabolism,physiology,and behavior.Some of the“heat-stress resistant”microbes may be useful as potential probiotics for cows under heat stress.