Characterization of bovine ruminal and equine cecal microbial populations enriched for enhanced nitro-toxin metabolizing activity.

The nitrotoxins 3-nitro-1-propionic acid (NPA) and 3-nitro-1-propanol (NPOH) are produced by a wide variety of leguminous plants, including over 150 different species and varieties of Astragalus potentially grazed by livestock. These toxins are known to be detoxified by at least one ruminal bacterium but detoxification by bacteria from other gut habitats is not known. In the present study, mixed populations of bovine ruminal and equine cecal microbes were enriched for NPA-metabolizing bacteria via consecutive 24-72 h culture in a basal minimal rumen fluid-based medium supplemented with 4.2 mM NPA and H2 as the energy source. Rates of NPA metabolism by the respective populations increased from 58.4 ± 4.8 and 8.6 ± 11.6 nmol NPA/mL per h during initial culture to 88.9 ± 30.6 and 50.2 ± 30.9 nmol NPA/mL per h following enrichment. Results from 3-tube most probable number tests indicated that numbers of NPA-degrading microbes increased 2.1 and 1.8 log10 units during enrichment from numbers measured pre-enrichment (3.9 × 10³ and 4.3 × 10¹ cells/mL for ruminal and equine cecal populations, respectively). Hydrogen, formate, and to a lesser extent, DL-lactic acid, served as electron donors to the enriched populations and CO2 or formate were needed to maintain high rates of NPA-metabolism. The NPA-enriched populations were able to metabolize nitrate which, being a preferred electron acceptor, was antagonistic to NPA metabolism. Supplemental NPA was inhibitory to methanogenesis. Fermentation balance estimates indicated that only 47.6% of carbon available in potential substrates was recovered in headspace CO2, volatile fatty acids or unmetabolized NPA after 72 h incubation of NPA-enriched populations that had metabolized 98% of 8.4 mM added NPA. Overall, these results reveal low level carriage of NPA-metabolizing, CO2 or formate-requiring bacterial populations in the equine cecum yet support the concept that Denitrobacterium detoxificans-like organisms may well be the functional agents of NPA and NPOH detoxification in the populations studied here.

Effects of crude protein content on intake and digestion of coastal bermudagrass hay by horses.

This study was conducted to determine the effects of forage crude protein (CP) level on intake and digestion of Coastal bermudagrass hay by horses. Four cecally fistulated geldings were used in a 4 × 4 Latin square design with four treatments and four periods. Horses were fed one of four Coastal bermudagrass hays consisting of 7%, 10%, 13%, or 16% CP during each of the four 15-d periods. Intake and apparent digestibility were determined for each horse at the end of each period by total fecal collection. In addition, cecal fluid and blood samples were collected on the last day of each period for the determination of cecal ammonia, cecal pH, plasma urea nitrogen (PUN), and plasma glucose concentrations. Data were analyzed using PROC MIXED of SAS. CP concentration of Coastal bermudagrass hay influenced equine intake and digestion. Increasing CP concentration linearly increased digestible organic matter intake (DOMI) from 3.79 to 5.98 kg/d for 7% and 16% CP hay, respectively (P = 0.04). Furthermore, as the forage CP level increased, CP intake increased linearly (P < 0.01). The forage CP level had no effect on forage dry matter intake. Quadratic effects (P ≤ 0.05) were observed for forage OM, neutral detergent fiber, acid detergent fiber, and digestible energy. Overall digestibility was lowest for the 7% CP hay and highest for the 10% CP hay. Cecal pH remained above 6.62 irrespective of treatment and time, indicating that cecal pH was suitable for microbial growth. As the forage CP level increased, cecal ammonia concentration increased linearly from 0.03 mM for the 7% to 1.74 mM for the 16% CP hay (P < 0.01). Concentration of plasma glucose also linearly increased (P = 0.04) from 68.77 to 73.68 mg/dL as CP concentration increased from 7% to 16% CP. PUN exhibited a quadratic effect as concentration increased (P < 0.01) from 4.34 to 5.61 mM for the 7% and 16% CP hays, respectively. Overall, the 10% CP hay had the highest digestibility due to its higher OM digestion. As forage OMI increased, digestible OM increased until physiological capacity for digestion is exceeded. At that point, digestion will decline with the decrease in OMI, explaining the lower digestion for other forages fed.

Evaluation of dietary trace mineral supplementation in young horses challenged with intra-articular lipopolysaccharide.

Sixteen weanling Quarter Horses (255 ± 22 kg) were utilized in a 56-d trial to evaluate the effects of trace mineral (TM) source on intra-articular inflammation following a single acute inflammatory insult. Horses were stratified by age, sex, and BW and then randomly assigned to dietary treatment: concentrate formulated with Zn, Mn, Cu, and Co as inorganic sources (CON; n = 8) or complexed TMs (CTM; n = 8). Added TM were formulated at iso-levels across treatments and intakes met or exceeded NRC requirements. Horses were offered 1.75% BW (as-fed) of treatment concentrate and 0.75% BW (as-fed) coastal Bermudagrass hay. Growth measurements were collected on days 0, 28, and 56, and plasma was collected biweekly for determination of Mn, Cu, Zn, and Co concentrations. On day 42, carpal joints were randomly assigned to receive injections of 0.5 ng lipopolysaccharide (LPS) or sterile lactated Ringer's solution (LRS; contralateral control). Synovial fluid was collected at preinjection hours (PIH) 0, and 6, 12, 24, 168, and 336 h post-injection and analyzed for TM concentration, prostaglandin E2 (PGE2), carboxypeptide of type II collagen (CPII), collagenase cleavage neopeptide (C2C), and aggrecan chondroitin sulfate 846 epitope (CS846). Data were analyzed using the MIXED procedure of SAS. Results showed a TM source × LPS × h effect for synovial fluid Co, Cu, and Se (P < 0.05); concentrations of TM peaked at hour 6 and decreased to preinjection values by hour 168 in both CON and CTM-LPS knees. A delayed peak was observed at hour 12 for CTM-LRS. Peak synovial fluid Cu and Se concentrations were higher in LPS knees, and Co was highest in CTM-LPS. A TM source × h interaction was observed for Zn (P < 0.05); concentrations peaked at hour 6 in CON vs. hour 12 for CTM. An LPS × h interaction was observed for Mn (P < 0.01); synovial concentration peaked at hour 6 in LPS knees compared with hour 24 in LRS. Synovial PGE2, C2C, CPII, and CS846 concentrations were greater with LPS (P ≤ 0.01), and C2C was greater (P < 0.01) in CTM compared with CON. Concentrations of CPII and PGE2 were unaffected by diet. A TM source × h × LPS interaction was observed for CS846 (P = 0.02). Concentrations of CS846 in CTM peaked at 12 h, whereas CON peaked at a lower concentration at 24 h (P < 0.05). Data indicate sufficient intake of a complexed TM source may support cartilage metabolism through increased aggrecan synthesis and type II collagen breakdown following an intra-articular LPS challenge in growing horses.

Influence of housing type on the cecal environment of horses.

Eight previously cecally cannulated Quarter Horse geldings were utilized in a crossover design with two 28-d periods with a 21-d washout period between to evaluate the influence of housing on the cecal environment and dry matter intake (DMI). Horses were adapted to diet and housing from day 1 to 19, DMI was determined from day 20 to 24, and cecal fluid was collected on day 28. Horses were paired by age and body weight (BW) and randomly assigned to treatment. Treatments consisted of housing horses individually in stalls or group housed in a pen. Regardless of treatment, all horses were individually fed a pelleted concentrate at 1% BW (as fed) offered twice daily 12 h apart. All horses had ad libitum access to coastal bermudagrass hay (Cynodon dactylon). Hay was offered to stalled horses initially at 2% BW (as fed) and then adjusted based on 120% of a previous 3-d average of voluntary intake. A dual marker system was used to estimate forage consumption in all horses, using titanium dioxide (TiO2) as the external marker and acid detergent insoluble ash (ADIA) as the internal marker. TiO2 was offered at 10 g/d for 10 d with fecal samples collected on the final 4 d at 12-h intervals advancing by 3 h each day to account for diurnal variation. Cecal samples were collected on day 28, 4 h after the morning meal and immediately analyzed for pH, total anaerobic and lactic acid bacteria populations, methane and ammonia concentrations, as well as volatile fatty acid (VFA) concentrations. Data were analyzed using the PROC GLM procedure of SAS with the model containing effects for horse, period, and treatment. Cecal pH was affected by housing (P = 0.02) with group-housed horses having lower cecal pH values compared with stalled horses (6.52 vs. 6.69, respectively). There was no influence of housing on populations of total anaerobic or lactic acid bacteria. Furthermore, housing did not influence cecal concentrations of VFA or methane and ammonia concentrations. Estimates of voluntary forage DMI were greater for group-housed horses (P = 0.04) than stalled (8.47 and 5.17 ± 0.89 kg DM/d, respectively). In conclusion, confinement housing did not, with the exception of pH, alter cecal environment of a horse when similar diets were offered but did affect forage consumption.

Evaluation of conjugated linoleic acid supplementation on markers of joint inflammation and cartilage metabolism in young horses challenged with lipopolysaccharide.

Seventeen yearling Quarter Horses were used in a randomized complete block design for a 56-d trial to determine ability of dietary CLA to mitigate joint inflammation and alter cartilage turnover following an inflammatory insult. Horses were blocked by age, sex, and BW, and randomly assigned to dietary treatments consisting of commercial concentrate offered at 1% BW (as-fed) supplemented with either 1% soybean oil (CON; n = 6), 0.5% soybean oil and 0.5% CLA (LOW; n = 5; 55% purity; Lutalin, BASF Corp., Florham Park, NJ), or 1% CLA (HIGH; n = 6) top-dressed daily. Horses were fed individually every 12 h and offered 1% BW (as-fed) coastal bermudagrass (Cynodon dactylon) hay daily. This study was performed in 2 phases: phase I (d 0 to d 41) determined incorporation of CLA into plasma and synovial fluid; phase II (d 42 to d 56) evaluated potential of CLA to mitigate intra-articular inflammation and alter cartilage metabolism. Blood and synovial fluid were collected at 7- and 14-d intervals, respectively, to determine fatty acid concentrations. On d 42, carpal joints within each horse were randomly assigned to receive intra-articular injections of 0.5 ng lipopolysaccharide (LPS) derived from Escherichia coli 055:B5 or sterile lactated Ringer's solution. Synovial fluid samples were obtained at preinjection h 0 and 6, 12, 24, 168, and 336 h postinjection, and analyzed for prostaglandin E2 (PGE2), carboxypeptide of type II collagen (CPII), and collagenase cleavage neopeptide (C2C). Data were analyzed using PROC MIXED procedure of SAS. Horses receiving the CON diet had undetectable levels of CLA for the duration of the study. A quadratic dose response was observed in concentrations of CLA in plasma and synovial fluid (P < 0.01). A negative quadratic dose response was observed for plasma arachidonic acid (20:4) with a reduction in concentration to d 14 in HIGH horses (P = 0.04). Synovial fluid 20:4 tended to decrease in horses receiving the HIGH diet (P = 0.06). Post LPS injection, synovial PGE2 was not affected by dietary treatment (P = 0.15). Synovial C2C was lower in HIGH horses (P = 0.05), and synovial CPII tended to be greater in LOW horses than HIGH and CON horses (P = 0.10). In conclusion, dietary CLA incorporated into plasma and synovial fluid prior to LPS challenge. Dietary CLA did not influence inflammation; however, there was a reduction in cartilage degradation and an increase in cartilage regeneration.