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Clostridioides difficile is a clinically and agriculturally important organism with diverse metabolic capabilities. Commercially available media types to cultivate C. difficile typically include multiple growth substrates and often selective agents. Under these conditions, it is difficult to determine what the bacteria utilized and which products are derived from which substrates. These experiments compared a commercial broth (Reinforced Clostridium Medium/RCM) to simpler, defined, carbonate-based media types influenced by Robert Hungate. Peptides (tryptone peptone), amino acids (casamino acids), and/or glucose were added to evaluate the growth of C. difficile strains 9689, BAA-1870, and 43597, and the metabolism of the type strain 9689. C. difficile grew to the greatest optical density in the rich RCM broth but produced less ammonia than the tryptone-containing media types. C. difficile utilized all glucose in RCM and T+G media in addition to performing amino acid fermentations, though the volatile fatty acids produced were not necessarily consistent across media type. When cultured in CAA-containing media, 9689 performed very little metabolism and did not grow regardless of supplementation with glucose. These data demonstrated that C. difficile could metabolize substrates and grow in defined, anaerobic, and carbonate-buffered media. Hungate-style media appear to be an acceptable choice for reliable culturing of C. difficile. [ABSTRACT FROM AUTHOR]

Sheep are often utilized as a model ruminant, despite a lack of functional comparisons of rumen bacterial communities and responses during dietary transitions between sheep and cattle. Therefore, an ex vivo study was conducted to evaluate species differences. Rumen fluid was obtained from hay-fed sheep and cattle (n = 3 species−1). Mixed bacterial cell suspensions in buffered media containing 3% w/v ground hay, corn, or combinations (2:1, 1:2) of substrates were incubated (24 h; 39 °C). Suspension pH, lactate, volatile fatty acids (VFA), and digestibility were assessed, functional guilds enumerated, and amylolytic bacteria isolated. Lactate was fully utilized in all hay incubations, and pH did not differ between species (p > 0.75). In contrast, digestibility, lactate accumulation, and pH decline were greater in bovine suspensions fermenting corn (p < 0.01). Streptococcus bovis was the predominant bacteria regardless of species, but total amylolytic bacteria were 10-fold greater in bovine suspensions (p < 0.01). Lactate-utilizing bacteria were 1000-fold greater in bovine than ovine suspensions (p < 0.01). However, total VFA did not differ between species (p > 0.28). Overall, these results demonstrate differential feed utilization capacities in rumen microbial communities of sheep and cattle as well as potential differences in rumen acidosis susceptibility. [ABSTRACT FROM AUTHOR]

Antibiotic resistance has progressively increased over time, thus necessitating the discovery of sustainable, novel antimicrobials. Ammonia producing bacteria (APB) are predominant organisms occupying many niches in the rumens of cattle and soil. The objective of this study was to discover target phytophenolic compounds (PPCs) that can act as replacements for existing antimicrobials, as well as at what concentration ranges may be optimal for inhibition of APB growth. In recognition of this discovery, producers may have alternative antimicrobial compounds for use in managing APB and common silage spoilage organisms. To shed light on this matter, inhibitory concentrations (IC) and ammonia assays were implemented. ICs analyzed the inhibitory action of six structurally similar PPCs: thymol, eugenol, carvacrol, hydro-cinnamic acid (H-CA), trans-cinnamic acid (T-CA), and phloroglucinol. Five APB (SR, MD1, F, BG1, and B14) were exposed to five concentrations of phytochemicals via a 10% v/v serial dilution: 10, 1, 0.1, 0.01, 0.001 mM. Following serial dilution, the bacteria were incubated ~24h at 39°C in a water bath, and visually inspected for growth. Ammonia assays utilized the least, fully inhibitory concentration of each phytochemical identified via the inhibitory concentration experiments. Ammonia concentrations were determined using the phenolic acid/hypochlorite method. All protocols utilized anaerobic sterile technique. All experiments were repeated in triplicate and significant results (P < 0.05) were determined using a one-way ANOVA with Tukey’s post hoc test utilizing OriginPro statistical software. Inhibition results revealed that eugenol, thymol, and carvacrol were successful in inhibiting bacterial growth at a concentration range of 10 mM to 1 mM. However, phloroglucinol was not fully inhibitory at any concentration tested against any bacteria. Eugenol, thymol, carvacrol were fully inhibitory at either 10 mM or 1 mM for all organisms tested except BG1, where it was only inhibitory at 10 mM. Ammonia analyses determined that eugenol and carvacrol greatly reduced ammonia production for all species tested except for B14, a generalist, which had no measurable changes in ammonia for any of the treatments including control. Thymol reduced ammonia production for MD1, BG1, and SR. Additionally, H-CA and T-CA decreased ammonia production of SR compared with control but not as much as eugenol or carvacrol. Results suggested that the growth of APB was generally inhibited by the greatest concentrations of PPCs (10 mM to 1 mM) tested here. Additionally, phloroglucinol did not inhibit bacterial growth therefore it was not included in the ammonia analyses. Eugenol and carvacrol significantly reduced ammonia production in all organisms, therefore indicating possible antimicrobial candidates to be further investigated. In comparison with carvacrol and eugenol, T-CA and H-CA made a modest to negligible reduction in ammonia. Phyto-phenolic compounds that were successful in inhibiting bacterial growth and reducing ammonia production demonstrate several promising target antimicrobials. [ABSTRACT FROM AUTHOR]

Artificial rearing remains an integral practice in sheep production largely due to the unique prolificacy of ewes as opposed to other species. Research has demonstrated slower growth and reduced performance of lambs reared on milk replacer when compared with conventionally reared lambs, but little is known about the functional development of the rumen microbial community. The objective of this study was to evaluate differences in functional rumen microbiological characteristics between conventionally (CR) and artificially (AR) reared lambs. We hypothesized that AR lambs would have slower rumen microbial community development than CR lambs, with consequent impacts on circulating metabolites. Polypay ewes (n = 9) bearing triplets were selected for the study. One lamb from each set was removed from the ewe 24 h after birth and placed on milk replacer. AR lambs were weaned on d 45 and CR lambs were weaned on d 60. A standard concentrate creep diet and alfalfa hay was made available ad libitum to both CR and AR lambs throughout the entire pre-weaning period. Rumen samples were collected via oral intubation and blood samples via jugular venipuncture on d 2 (blood only), 15, 45, and 60. Rumen bacterial functional guilds were enumerated via serial dilution and serum metabolites quantified via LC-MS. Data were analyzed using PROC MIXED in SAS (v. 9.4, SAS Inst. Inc., Cary, NC). No differences between treatments were found for average daily gain (gּ·d-1) over the pre-weaning period (P = 0.12). Cellulolytic bacteria in the rumen increased during the pre-weaning period (P < 0.01), with proliferation noted for both treatments from d 15 to d 45 (P < 0.05). However, the magnitude of this proliferation differed between pre-weaning management, with a 1,000-fold increase for CR lambs, but only a 10-fold increase for AR lambs from d 15 to d 45 (P < 0.05). This resulted in a 100-fold difference between treatments on d 45 which persisted to d 60 (P < 0.05). Circulating β-hydroxybutyrate concentrations also increased over the pre-weaning period (P < 0.01), and were consistently less for AR (134.17 ± 15.04 mmolּ·L-1) than CR lambs (209.21 ± 15.04 mmolּ·L-1; P = 0.01). Conversely, circulating glucose decreased in both AR and CR lambs from d 2 to 60 (P = 0.06), with CR lambs having decreased concentrations detected on d 2 and 45 (P < 0.05). These results indicate potential delays in rumen development of AR lambs, supported by the differences observed in rumen cellulolytics and circulating metabolites when compared with CR lambs. These results could suggest implications for impaired dietary utilization and subsequent growth of AR lambs post-weaning, especially those managed on high-forage diets. [ABSTRACT FROM AUTHOR]

Air exposed silages can harbor spoilage and pathogenic microbes which can reduce the palatability, nutritive quality and microbial safety of these feedstuffs. Accordingly, effective, environmentally friendly interventions are sought to rescue the biological and economical value of spoiled silages. The objectives of this study were to test the effects of treating air exposed corn silage with LipoVital GL-45, a Berg + Schmidt GmbH & Co. KG (Hamburg, Germany) product containing 45% monoglyceride of lauric acid (mixed within a 50% di/triglyceride base) alone or mixed with either Chinook or Galena hops or with an experimental extract containing hops β-acids. All treatments were administered as a warm water spray to deliver 1.6% product to the silage on a dry matter basis. Silage samples collected after 24 h ambient incubation were serially diluted in 0.4 M sodium phosphate buffer (pH 6.4) and plated to 3M Petrifilm for enumeration of yeast and molds and total aerobes as well as to Rogosa SL for enumeration of lactic acid bacteria and streptococci. Fluid samples from sampled silage were also inoculated in 10-fold increments to triplicate sets of tubes containing anaerobically prepared Reinforced Clostridial agar supplemented cellobiose and xylose (0.05% wt/vol each) for most probable number enumeration of total anaerobes. Populations of presumptive spore-forming aerobes and anaerobes were measured as described above except inocula were heated to 80°C for 10 min to kill vegetative cells. An analysis of variance (Statistix Analytical Software, Tallahassee, FL, USA) revealed no treatment effects on yeasts and mold populations (P > 0.05) after 24 h air exposure of the silage, with counts averaging (± SD) 5.0 ± 0.7 log10 CFU/g. Silage treated with combinations of LipoVital GL-45 with Galena hops or the β-acid extract had 0.8 to 0.9 log10 units fewer (P < 0.05) lactic acid bacteria after 24 h air exposure than untreated controls (7.4 ± 0.1 log10 CFU/g). Similarly, silage treated with the LipoVital GL-45/Galena hops combination had 0.6 to 0.7 log10 units fewer (P < 0.05) heat-sensitive and presumptive spore-forming aerobes after 24 h air exposure when compared with controls (5.5 ± 0.2 and 5.2 ± 0.2 log10 CFU/g, respectively). Conversely, most probable numbers of nonheated anaerobe populations were lower (P < 0.05) in silage treated with LipoVital GL-45 combined with the β-acid extract when compared with controls (8.6 ± 0.x and 9.9 ± 0.7 log10 cells/g) with all other treatments being similar to controls. Populations of presumptive spore-forming anaerobes in the air exposed silage were unaffected by treatments, averaging 8.3 ± 0.7 log10 cells/g. Results warrant further research to investigate whether optimized dosages and formulations of LipoVital GL-45, Galena hops and β-acid extracts may ultimately yield treatments to prevent spoilage of air-exposed silages [ABSTRACT FROM AUTHOR]

Pre-weaning strategies, such as artificially raising on milk replacer, can impact lamb quality. Limited studies have examined the influence of artificial raising on color attributes of lamb. The aim of this study was to evaluate how artificially raising on milk replacer affects the color and oxidative stability of longissimus lumborum (LL) muscle from ram lambs finished on a high-concentrate diet. For a 60-day pre-weaning period, Polypay ram lambs were reared either conventionally with ewes (CR; n = 5) or artificially on milk replacer (MR; n = 5). After 60 days, the lambs were finished on a high concentrate diet (85:15 concentrate: forage) containing 78% cracked corn and 22% pre-mixed protein pellet as the concentrate source as well as 15% orchard grass pellet as the forage source. The lambs were fed ad libitum high-concentrate diet until the target body weight of 59 kg. The lambs were harvested, and the LL muscles from both sides of the carcasses (24 h post-mortem) were fabricated into 2.5-cm thick chops. Carcass characteristics were recorded during harvesting and fabricating. The chops were placed on polystyrene trays, overwrapped with oxygen-permeable polyvinyl chloride film, and stored at 2°C in the darkness for 0, 3, and 6 d. Instrumental color, color stability (R630/580), pH, lipid oxidation, and myoglobin reducing activity of the chops were evaluated on the storage days. A split-plot design was employed to analyze the data. Pre-weaning management was the main plot, while the storage time was considered as the sub-plot. The LL muscles from each lamb carcass were considered as the experimental unit, whereas the sub-plot experimental units were the chops allocated for 0, 3, or 6 d storage. The data were analyzed using the PROC GLIMMIX. The least square means were separated utilizing the PDIFF option and adjusted with the Tukey-Kramer method. The differences among means were detected at a 5% level. There was no effect (P > 0.05) of pre-weaning management on carcass characteristics, surface redness (a* value), lightness (L* value), yellowness (b* value), color stability (R630/580), pH, or metmyoglobin reducing activity of LL chops. Color stability (R630/580) and metmyoglobin reducing activity declined (P < 0.05) during storage in both CR and MR chops. In contrast, lipid oxidation, lightness (L* value), yellowness (b* value), and hue value of the chops increased (P < 0.05) in both treatments during storage. Artificial raising on milk replacer for 60 d pre-weaning period did not influence color and oxidative stability of lambs finished on a high-concentrate diet. Carcass characteristics, color stability, and lipid oxidation were comparable in lambs raised through conventional method and those raised artificially on milk-replacer. These findings indicate that milk-replacer could be employed as a practical approach for artificial rearing in lamb production. [ABSTRACT FROM AUTHOR]

Fermentation acids inhibited the growth and ammonia production of the amino-acid-fermenting bacterium Clostridium sporogenes MD1, but only when the pH was acidic. Such inhibition was traditionally explained by the ability of fermentation acids to act as uncouplers and decrease protonmotive force ([DELTA]p), but C. sporogenes MD1 grows even if the [DELTA]p is very low. Cell suspensions incubated with additional sodium chloride produced ammonia as rapidly at pH 5.0 as at pH 7.0, but cells incubated with additional sodium lactate were sensitive to even small decreases in extracellular pH. Similar results were obtained if the sodium lactate was replaced by sodium acetate or propionate. When extracellular pH declined, [DELTA]pH increased even if sodium lactate was present. The cells accumulated intracellular lactate anion when the pH was acidic, and intracellular glutamate declined. Because amino acid deamination is linked to a transamination reaction involving glutamate dehydrogenase, the decrease in ammonia production could be explained by the decrease in intracellular glutamate. This latter hypothesis was consistent with the observation that extracellular glutamate addition restored amino acid deamination even though glutamate alone did not allow for the generation of ammonia.

Artificially rearing lambs on milk replacer impacts development of the ruminal microbiome. The objective was to evaluate the extent to which pre-weaning management influences the ruminal microbiome of lambs during finishing. A finishing study was conducted using 32 Polypay conventionally-raised (CR; n = 10 rams, 6 ewes) or artificially raised (AR; n = 10 rams, 6 ewes) lambs weaned at 56 d of age. Following weaning, lambs were adapted to a HC (85:15 concentrate:forage) diet for 35 d. Lambs were then blocked by sex and body weight and randomly assigned to either continue receiving HC or abruptly transition to a HF (50:50 concentrate:forage) diet. Individually-housed lambs were fed ad libitum through a target slaughter weight (rams: 59.0; ewes: 54.4 kg). Rumen fluid samples were collected via oral intubation on d 0, 14, 28, and when lambs reached slaughter weight. Analysis of 16S rRNA gene sequences was conducted in QIIME 2 (v.2023.2). Taxonomy was assigned with SILVA (SSU 138). Diversity was analyzed by PERMANOVA. Random forest classification was applied to determine if pre-weaning management and diet could be predicted using microbial composition. Alpha diversity did not differ in CR vs. AR lambs at the initial sampling (P > 0.22), but species richness was greater in HR lambs regardless of pre-weaning management from d 14 through slaughter (P < 0.01). Beta diversity did differ between CR and AR at the initial sampling (P < 0.02). The influence of pre-weaning management was still present in weighted UniFrac distances by d 14 (P = 0.09), but effects of pre-weaning management were no longer apparent from d 28 through slaughter (P > 0.28). Similarly random forest modeling was able to predict pre-weaning management in initial samples (accuracy: 0.75 ± 0.15). Accuracies decreased over the study period (d 14: 0.58 ± 0.07; d 28: 0.50 ± 0.15; slaughter: 0.45 ± 0.07), whereas model performance improved for prediction of diet (d 14: 0.83 ± 0.15; d 28: 0.87 ± 0.12; slaughter: 0.97 ± 0.07). The most important feature in predicting pre-weaning management at initial sampling belonged to Ruminococcus, and 9 ASV assigned to Prevotella were among the most important features in the model. Five features were present in the top 20 most important features for both the initial and finishing models, and included ASV assigned to Streptococcus, Lactobacillus, and Bifidobacterium. These results illustrated continued impacts of early-life management on ruminal microbiome composition at the beginning of the finishing period, even though CR and AR lambs had been adapted to the same diet for more than 1 mo. While pre-weaning effects were muted over the course of finishing as diet became the predominant factor, the influence of early-life management was still detectable by the end of the finishing period. [ABSTRACT FROM AUTHOR]

Red clover (RC) contains bioactive isoflavones. Supplementing RC as a functional feed (as little as 7.5% of the total diet) has been shown to alter ruminal fermentation and improve feed efficiency in ram lambs fed high-concentrate finishing diets. The objectives of this study were to determine if reduced RC inclusion will produce similar rumen microbiological effects. It was hypothesized that RC supplementation below 7.5% RC will alter rumen microbial community composition during the finishing period. Sixteen individually housed Polypay ram lambs (age; 134 ± 2.5 d; initial body weight: 31 ± 0.53 kg) were used in a complete block design and fed one of four isocaloric and isonitrogenous diets (85:15 concentrate:roughage ratio; n = 4 rams diet): a control diet in which the roughage component (15.0% wt/wt of total diet) consisted of orchardgrass hay, 2.5%-RC (roughage: 2.5% RC; 12.5% orchardgrass wt/wt), 5.0%-RC (roughage: 5.0% RC; 10% orchardgrass wt/wt), and 7.5%-RC (roughage: 7.5% RC; 7.5% orchard grass wt/wt). Rumen fluid was collected via oral intubation at 0, 2, 4, 6, and 8 h post-feeding at the end of the adaptation period (14 d; all rams on control diet) and at 21-d intervals during the trial. Rumen functional bacterial guilds, including hyper-ammonia-producing bacteria (HAB), amylolytic, lactate-utilizing (LU), and cellulolytic bacteria were enumerated via serial dilution at 4 h post-feeding. Data were analyzed using PROC MIXED with repeated measures in SAS (v.9.4, SAS inst. Inc., Cary, NC). There were no differences between diets on d 0 for any variable (P > 0.05). At d 21, all RC-containing diets exhibited a 10- to 100-fold reduction in ruminal HAB compared with the control (P < 0.05). While suppression of HAB was no longer apparent by d 42 in 2.5%-RC, HAB had decreased 100-fold (109 to 107) in lambs fed the 5.0%-RC and 7.5%-RC diets (P < 0.01). No differences in amylolytic bacteria were detected on d 21 (P > 0.05), but were 10-fold less in all diets containing RC by d 42 in comparison with control (109 to 108; P = 0.08). In contrast, LU increased by d 21 and were 100-fold greater (106 to 108) by d 42 when compared with the control for all levels of RC supplementation (P < 0.05). Cellulolytic bacteria also increased across the study period in all lambs fed RC diets, with 100 fold greater cellulolytics (106 to 108) by d 42 (P < 0.05). Consistent with the hypothesis, supplementing RC at levels as low as 2.5% wt/wt of the total diet decreased protein-wasting HAB and amylolytic bacteria while coincidingly increasing LU and cellulolytic bacteria in the rumen. These results indicate that adding red clover to finishing diets has the potential to increase lamb growth and feed efficiency through a functional feed action. [ABSTRACT FROM AUTHOR]

Ewes transitioning from late gestation to early lactation face a number of challenges including physiological stress, dietary changes, and metabolic adaptations, particularly when bearing and rearing multiple offspring. Furthermore, rumen microbial composition and function has been correlated with milk production and nutrient density in transitioning dairy cows. However, limited research has evaluated the rumen microbial community of ewes during early lactation. The objective of this study was to conduct a functional microbiological survey of the ewe rumen from parturition to weaning. We hypothesized that ewes would experience fermentative disturbances post-parturition and that the rumen microbiota, and consequently, circulating metabolites, would recover over time. Nine multiparous Polypay ewes bearing triplets were fed a lactation diet consisting of alfalfa hay and concentrate mix to meet protein and energy requirements. One lamb of each triplet set was randomly selected and placed on milk replacer 24 h after birth. The two lambs remaining on the ewe were weaned at 56 d of age. Blood and rumen samples from ewes were collected via jugular venipuncture and oral intubation, respectively, at 2, 7, and 15 d of lactation in addition to at the time of weaning. Rumen bacterial functional guilds were enumerated via serial dilution. Ruminal short-chain fatty acids (SCFA) were quantified via HPLC and serum metabolites by LC-MS. All data were analyzed using PROC Mixed with repeated measures in SAS (v. 9.4, SAS Inst. Inc., Cary, NC). Total starch-utilizing bacteria remained steady during early lactation but increased 100-fold by weaning (P = 0.03). In contrast, the starch-utilizing guild sub-population lactobacilli increased 10-fold by d 15 of lactation (P < 0.01) and remained constant through weaning (P > 0.05). No differences were found in the viable number of rumen cellulolytic bacteria (P = 0.37) throughout the study. Ruminal propionate concentrations increased 35% (P = 0.04) from d 2 to weaning and serum propionate (36.8 ± 4.42 µM; P = 0.08) and glucose (59.8 ± 2.97 mg dL-1; P < 0.01) peaked on d 15 of lactation. While ruminal acetate did not fluctuate in early lactation, it increased 58% by weaning (P < 0.01) leading to an 88% increase in serum acetate (P < 0.01). Ruminal butyrate increased 49% by d 15 (P = 0.01) and this was reflected in a 7-fold increase in serum butyrate (P = 0.02). However, serum P-hydroxybutyrate did not differ from parturition through weaning (P = 0.51). Results of this experiment demonstrate that rumen bacterial functional guilds, as well as rumen and serum metabolites, were lowest two days post-parturition and increased over the course of lactation. This may suggest that ruminal fermentation is suppressed post-parturition, which could have implications for early lactation management strategies of triplet-bearing ewes. [ABSTRACT FROM AUTHOR]