Isoflavone Containing Legumes Mitigate Ergot Alkaloid-Induced Vasoconstriction in Goats (Capra hircus).

Ergot alkaloids produced by a fungal endophyte that infects tall fescue (Lolium arundinaceum; (E+ TF) can induce constriction of the vasculature in ruminants, resulting in "fescue toxicosis". Legumes contain isoflavones that have been demonstrated to prevent and reverse E+ TF vasoconstriction. Several legumes are conventionally utilized in ruminant production, but can vary in both isoflavone concentration and composition. A feeding study was conducted to determine if isoflavone supplementation via red clover (Trifolium pratense), white clover (Trifolium repens), or soybean (Glycine max) meal can alleviate vasoconstriction when wether goats were challenged with E+ TF seed. The basal diet was chopped grass hay ad libitum. Carotid luminal areas were obtained pre- and post-ruminal infusions of E+ TF seed (15 µg kg BW−1 ergovaline + ergovalanine ± red clover, white clover, or soybean meal at 2.61 mg kg BW−1). When goats were challenged with E+ TF seed, the mean carotid luminal areas decreased by 56.1% (p < 0.01). All treatments were able to partially mitigate vasoconstriction, with red clover being the most effective (+39.8%), and white clover and soybean meal eliciting an intermediate response (+30%, p < 0.01). Results indicate that legumes can relax vasoconstriction in goats consuming ergot alkaloids, despite differences in isoflavone profile and concentrations.

Effect of biochanin A on the rumen microbial community of Holstein steers consuming a high fiber diet and subjected to a subacute acidosis challenge.

Subacute rumen acidosis (SARA) occurs when highly fermentable carbohydrates are introduced into the diet, decreasing pH and disturbing the microbial ecology of the rumen. Rumen amylolytic bacteria rapidly catabolize starch, fermentation acids accumulate in the rumen and reduce environmental pH. Historically, antibiotics (e.g., monensin, MON) have been used in the prevention and treatment of SARA. Biochanin A (BCA), an isoflavone produced by red clover (Trifolium pratense), mitigates changes associated with starch fermentation ex vivo. The objective of the study was to determine the effect of BCA on amylolytic bacteria and rumen pH during a SARA challenge. Twelve rumen fistulated steers were assigned to 1 of 4 treatments: HF CON (high fiber control), SARA CON, MON (200 mg d-1), or BCA (6 g d-1). The basal diet consisted of corn silage and dried distiller's grains ad libitum. The study consisted of a 2-wk adaptation, a 1-wk HF period, and an 8-d SARA challenge (d 1-4: 40% corn; d 5-8: 70% cracked corn). Samples for pH and enumeration were taken on the last day of each period (4 h). Amylolytic, cellulolytic, and amino acid/peptide-fermenting bacteria (APB) were enumerated. Enumeration data were normalized by log transformation and data were analyzed by repeated measures ANOVA using the MIXED procedure of SAS. The SARA challenge increased total amylolytics and APB, but decreased pH, cellulolytics, and in situ DMD of hay (P < 0.05). BCA treatment counteracted the pH, microbiological, and fermentative changes associated with SARA challenge (P < 0.05). Similar results were also observed with MON (P < 0.05). These results indicate that BCA may be an effective alternative to antibiotics for mitigating SARA in cattle production systems.

Evaluation of oral citrulline administration as a mitigation strategy for fescue toxicosis in sheep.

Gestating ewes consuming ergot alkaloids, from endophyte-infected (E+) tall fescue seed, suffer from intrauterine growth restriction and produce smaller lambs. Arginine (Arg) supplementation has been shown to increase birth weight and oral citrulline (Cit) administration is reported to increase arginine concentrations. Two experiments were conducted to: 1) evaluate if oral supplementation with Cit or water, to ewes consuming E+ fescue seed, increases lamb birth weight and 2) determine the effectiveness of Cit and citrulline:malate as an oral drench and elevating circulating levels of Cit to determine levels and dose frequency. In experiment 1, gestating Suffolk ewes (n = 10) were assigned to one of two treatments [oral drench of citrulline-malate 2:1 (CITM; 81 mg/kg/d of citrulline) or water (TOX)] to start on d 86 of gestation and continued until parturition. Ewes on CITM treatment had decreased (P < 0.05) plasma Arg and Cit concentrations during gestation. At birth, lambs from CITM ewes had reduced (P < 0.05) crude fat and total fat but did not differ (P > 0.05) in birth weight from lambs born to TOX ewes. In experiment 2, nonpregnant Suffolk ewes (n = 3) were assigned to either oral citrulline (CIT; 81 mg/kg/d), citrulline-malate 2:1 (CITM; 81 mg/kg/d of citrulline), or water (CON) drench in a Latin Square design for a treatment period of 4 d with a washout period of 3 d. On d 4, blood samples were collected at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 18 h post drench. Oral drenching of CIT and CITM increased (P < 0.0001) Cit concentrations within 2 h and levels remained elevated for 6 h. Apparent half-life of elimination for CIT and CITM were 8.484 and 10.392 h, respectively. Our results show that lamb birth weight was not altered with a single oral drench of citrulline-malate; however, lamb body composition was altered. The level and frequency of citrulline dosing may need to be greater in order to observe consistent elevation of Cit/Arg concentrations to determine its effectiveness in mitigating fescue toxicosis.

Isoflavone supplementation, via red clover hay, alters the rumen microbial community and promotes weight gain of steers grazing mixed grass pastures.

Biochanin A, an isoflavone present in the pasture legume red clover (Trifloium pratense L.), alters fermentation in the rumen of cattle and other ruminants. Biochanin A inhibits hyper-ammonia-producing bacteria and promotes cellulolytic bacteria and fiber catalysis in vitro and ex vivo. Consequently, biochanin A supplementation improves weight gain in grazing steers. Red clover contains biologically active isoflavones that may act synergistically. Therefore, the objective was to evaluate the effect of two levels of red clover hay on growth performance and the microbial community in growing steers grazing mixed grass pastures. A grazing experiment was conducted over 2 early growing seasons (2016 and 2017) with 36 cross-bred steers and twelve rumen-fistulated, growing Holstein steers for evaluation of average daily gain and rumen microbiota, respectively. Steers were blocked by body weight and assigned to pastures with one of four treatments: 1) pasture only, 2) pasture + dry distillers' grains (DDG), 3) pasture + DDG + low level of red clover hay (~15% red clover diet), or 4) pasture + DDG + high level of red clover hay (~30% red clover diet). DDG were added to treatments to meet protein requirements and to balance total protein supplementation between treatments. All supplementation strategies (DDG ± red clover hay) increased average daily gains in comparison to pasture-only controls (P < 0.05), with a low level of red clover supplementation being the most effective (+0.17 kg d-1 > DDG only controls; P < 0.05). Similarly, hyper-ammonia-producing bacteria inhibition (10-100-fold; P < 0.05), fiber catalysis (+10-25%; P < 0.05) and short chain fatty acid concentrations were greatest with the low red clover supplement (+~25%; P < 0.05). These results provide evidence that lower levels or red clover supplementation may be optimal for maximizing overall microbial community function and animal performance in grazing steers.

Effect of Starch Source in Pelleted Concentrates on Fecal Bacteria in Prepartum and Postpartum Mares.

Dietary starch source has been shown to affect fecal bacterial communities of horses fed minimally processed cereal grains. However, processing may increase foregut starch digestibility, reducing effects of starch source on fecal bacterial communities. This study aimed to determine the effect of starch source in pelleted concentrates on fecal Lactobacillus spp., amylolytic bacteria, and cellulolytic bacteria in broodmares mares, during the prepartum and postpartum period. Thoroughbred mares (n = 18) were paired by last breeding date then randomly assigned to either an oat-based or a corn and wheat middlings-based pelleted concentrate fed with forage. Mares were fed their assigned concentrates beginning on 310 days of gestation, and fecal samples were collected at 324 days of gestation, before parturition, 1 day, 14 days, and 28 days postpartum. Fecal samples were enumerated by serial dilution and inoculation into selective, enriched media for Lactobacillus spp., amylolytic bacteria, and cellulolytic bacteria. Data were log transformed then analyzed using a mixed model ANOVA with repeated measures (SAS 9.3) to test the main effects of treatment, time of sample, and treatment by time interaction. Starch source did not affect enumerated bacterial communities (P > .05); thus, pelleting concentrates may alter some of the effects of starch sources on the hindgut microbiota. Sample date did not affect amylolytic bacteria (P > .05); however, lactobacilli and cellulolytic bacteria decreased 1 day postpartum (P < .05). Although we did not observe an effect of starch source on fecal bacteria in mares, parturition did appear to alter the hindgut microbiota.

Effect of maternal diet on select fecal bacteria of foals.

Adult horses depend on the microbial community in the hindgut to digest fiber and produce short-chain fatty acids that are use for energy. Colonization of the foal gastrointestinal tract is essential to develop this symbiosis. However, factors affecting colonization are not well understood. The objectives of this study were to evaluate the age-related changes and effects of maternal diet on select fecal bacterial groups in foals from 1 to 28 d of age. Thoroughbred foals (n = 18) were from dams fed forage and one of two concentrates: an oat-based (OB) or corn and wheat middlings-based (CWB) pelleted concentrate. The mares had access to assigned concentrates, along with a mixed hay and cool-season grass pasture, 28 d before and 28 d after parturition. Fecal samples were collected from foals at 1 d (14 to 36 h), 4, 14, and 28 d after birth. Fecal samples were serially diluted with phosphate-buffered saline before inoculation of enriched, selective media to enumerate Lactobacillus spp., amylolytic bacteria, and cellulolytic bacteria. Enumeration data were log-transformed then analyzed with mixed model analysis of variance with repeated measures (SAS 9.3) to test the main effects of maternal diet (OB or CWB), time of sample, and interaction between maternal diet and time. Cellulolytic bacteria first appeared in foal feces between 4 and 14 d of age and increased with age (P < 0.05). Amylolytic bacteria and lactobacilli were abundant at 1 d and then increased with age (P < 0.05). There was an interaction between maternal diet and time for Lactobacillus spp. with OB foals having more lactobacilli than CWB foals at 1 and 4 d (P < 0.05); however, there were no differences observed at 14 d (P > 0.05). Maternal diet did not influence amylolytic or cellulolytic bacteria (P > 0.05). These results indicate that colonization of the hindgut is a sequential process beginning early in the foal's life and that maternal diet may influence some bacteria in the gastrointestinal tract of foals.

Biochanin A improves fibre fermentation by cellulolytic bacteria.

AIMS: The objective was to determine the effect of the isoflavone biochanin A (BCA) on rumen cellulolytic bacteria and consequent fermentative activity. METHODS AND RESULTS: When bovine microbial rumen cell suspensions (n = 3) were incubated (24 h, 39°C) with ground hay, cellulolytic bacteria proliferated, short-chain fatty acids were produced and pH declined. BCA (30 µg ml-1 ) had no effect on the number of cellulolytic bacteria or pH, but increased acetate, propionate and total SCFA production. Addition of BCA improved total digestibility when cell suspensions (n = 3) were incubated (48 h, 39°C) with ground hay, Avicel, or filter paper. Fibrobacter succinogenes S85, Ruminococcus flavefaciens 8 and Ruminococcus albus 8 were directly inhibited by BCA. Synergistic antimicrobial activity was observed with BCA and heat killed cultures of cellulolytic bacteria, but the effects were species dependent. CONCLUSIONS: These results indicate that BCA improves fibre degradation by influencing cellulolytic bacteria competition and guild composition. SIGNIFICANCE AND IMPACT OF THE STUDY: BCA could serve as a feed additive to improve cellulosis when cattle are consuming high-fibre diets. Future research is needed to evaluate the effect of BCA on fibre degradation and utilization in vivo.

Exogenous lactobacilli mitigate microbial changes associated with grain fermentation (corn, oats, and wheat) by equine fecal microflora ex vivo.

Cereal grains are often included in equine diets. When starch intake exceeds foregut digestion starch will reach the hindgut, impacting microbial ecology. Probiotics (e.g., lactobacilli) are reported to mitigate GI dysbioses in other species. This study was conducted to determine the effect of exogenous lactobacilli on pH and the growth of amylolytic and lactate-utilizing bacteria. Feces were collected from 3 mature geldings fed grass hay with access to pasture. Fecal microbes were harvested by differential centrifugation, washed, and re-suspended in anaerobic media containing ground corn, wheat, or oats at 1.6% (w/v) starch and one of five treatments: Control (substrate only), L. acidophilus, L. buchneri, L. reuteri, or an equal mixture of all three (107 cells/mL, final concentration). After 24 h of incubation (37°C, 160 rpm), samples were collected for pH and enumerations of total amylolytics, Group D Gram-positive cocci (GPC; Enterococci, Streptococci), lactobacilli, and lactate-utilizing bacteria. Enumeration data were log transformed prior to ANOVA (SAS, v. 9.3). Lactobacilli inhibited pH decline in corn and wheat fermentations (P < 0.0001). Specifically, addition of either L. reuteri or L. acidophilus was most effective at mitigating pH decline with both corn and wheat fermentation, in which the greatest acidification occurred (P < 0.05). Exogenous lactobacilli decreased amylolytics, while increasing lactate-utilizers in corn and wheat fermentations (P < 0.0001). In oat fermentations, L. acidophilus and L. reuteri inhibited pH decline and increased lactate-utilizers while decreasing amylolytics (P < 0.0001). For all substrates, L. reuteri additions (regardless of viability) had the lowest number of GPC and the highest number of lactobacilli and lactate-utilizers (P < 0.05). There were no additive effects when lactobacilli were mixed. Exogenous lactobacilli decreased the initial (first 8 h) rate of starch catalysis when wheat was the substrate, but did not decrease total (24 h) starch utilization in any case. These results indicate that exogenous lactobacilli can impact the microbial community and pH of cereal grain fermentations by equine fecal microflora ex vivo. Additionally, dead (autoclaved) exogenous lactobacilli had similar effects as live lactobacilli on fermentation. This latter result indicates that the mechanism by which lactobacilli impact other amylolytic bacteria is not simple resource competition.

Effect of Dietary Starch Source and Concentration on Equine Fecal Microbiota.

Starch from corn is less susceptible to equine small intestinal digestion than starch from oats, and starch that reaches the hindgut can be utilized by the microbiota. The objective of the current study was to examine the effects of starch source on equine fecal microbiota. Thirty horses were assigned to treatments: control (hay only), HC (high corn), HO (high oats), LC (low corn), LO (low oats), and LW (low pelleted wheat middlings). Horses received an all-forage diet (2 wk; d -14 to d -1) before the treatment diets (2 wk; d 1 to 14). Starch was introduced gradually so that horses received 50% of the assigned starch amount (high = 2 g starch/kg BW; low = 1 g starch/kg BW) by d 4 and 100% by d 11. Fecal samples were obtained at the end of the forage-only period (S0; d -2), and on d 6 (S1) and d 13 (S2) of the treatment period. Cellulolytics, lactobacilli, Group D Gram-positive cocci (GPC), lactate-utilizers and amylolytics were enumerated. Enumeration data were log transformed and analyzed by repeated measures ANOVA. There were sample day × treatment interactions (P < 0.0001) for all bacteria enumerated. Enumerations from control horses did not change during the sampling period (P > 0.05). All treatments except LO resulted in increased amylolytics and decreased cellulolytics, but the changes were larger in horses fed corn and wheat middlings (P < 0.05). Feeding oats resulted in increased lactobacilli and decreased GPC (P < 0.05), while corn had the opposite effects. LW had increased lactobacilli and GPC (P < 0.05). The predominant amylolytic isolates from HC, LC and LW on S2 were identified by 16S RNA gene sequencing as Enterococcus faecalis, but other species were found in oat fed horses. These results demonstrate that starch source can have a differential effect on the equine fecal microbiota.

Diarrhea-associated pathogens, lactobacilli and cellulolytic bacteria in equine feces: responses to antibiotic challenge.

Antibiotics are important to equine medicine, but antibiotic-associated diarrhea (AAD) can lead to poor performance and even mortality. AAD is attributed to disruption of the hindgut microbiota, which permits proliferation of pathogenic microbes. The goal of this study was to evaluate the effects of common antibiotics on cellulolytic bacteria, lactobacilli, and AAD-associated pathogens in the feces of healthy horses. Fifteen horses were assigned to three treatment groups (blocked by age and sex): control (no antibiotics), trimethoprim-sulfadiazine (PO), or ceftiofur (IM). Fecal samples (n=8 per horse) were taken during dietary adaptation (3 weeks), antibiotic challenge (1 week), and withdrawal (1 week). Bacteria were enumerated by serial dilution and viable count. Cellulolytic bacteria decreased by >99% during administration of either antibiotic (P<0.0001) and were still less than controls at the end of the withdrawal period (P<0.0001). Fecal samples from horses challenged with ceftiofur had 75% fewer lactobacilli than those from control horses at the end of the antibiotic challenge period (P<0.05). Antibiotic challenged horses also shed more salmonella than control horses (P<0.05). Antibiotics had no effect on the number of Clostridium perfringens isolates. There was no detectable Clostridium difficile during adaptation or in any control horse. C. difficile increased (P<0.0001) to approximately 10(4)cfu/g when horses were challenged with antibiotics, and were still detectable 1 week after withdrawal. These results indicate that antibiotics can disrupt the normal gastrointestinal microbiota and allow proliferation of Salmonella spp. and C. difficile.