Propionibacterium acidipropionici P169 and glucogenic precursors improve rumen fermentation of low-quality forage in beef cattle.

Cattle grazing dormant western rangelands may have a high ruminal acetate to propionate ratio (A:P) and may have low tissue clearance of acetate. Increasing propionate production could shift this ratio and improve animal performance. In Exp. 1, the effect of Propionibacterium acidipropionici P169 (PA) on forage digestibility and VFA production was evaluated in vitro using 2 substrates: 100% dormant warm-season grass extrusa and 50% sorghum-Sudan hay with 50% ground corn (DM basis). The objective of Exp. 2 was to evaluate the effect of PA or calcium propionate supplementation on digestibility, ruminal fermentation, acetate clearance, and BW change. Twelve 2-yr-old, pregnant Brangus heifers (BW = 416 ± 85 kg) were assigned to 1 of 3 treatments. All cattle were fed a basal ration of Old World Bluestem hay (Bothriochloa ischaemum; 5.8% CP and 76.5% NDF, DM basis) at 1.5% BW from d -10 to d 49. Treatments included a protein supplement (CON; 36% CP and 35% RUP, DM basis; 454 g/animal fed twice daily), CON plus 6 × 10(10) cfu PA/animal (BACT), and CON plus 80 g calcium propionate (PROP). After initiation of treatments (d 0), rumen fluid was collected via oral lavage every 3 d and analyzed for VFA, pH, and ammonia. Glucogenic potential of treatments was evaluated with an acetate tolerance test on d 49. In Exp. 1, PA addition increased (IVDMD; P < 0.001) and total VFA (P < 0.001) of 100% dormant warm-season grass extrusa but not 50% sorghum-Sudan hay with 50% ground corn (P ≥ 0.28). The addition of P169 decreased (P < 0.001) acetate, increased propionate (P < 0.001), and decreased A:P ratio (P < 0.001) for both substrates. In Exp. 2, total tract OM and NDF digestibility and ruminal pH, total VFA, and acetate did not differ (P ≥ 0.13) among treatments. Propionate concentration was least (P = 0.001) for CON, intermediate for P169, and greatest for PROP. Conversely, A:P ratio was greatest (P < 0.004) for CON, intermediate for P169, and least for PROP. Acetate clearance did not differ (P = 0.69) among treatments. Propionibacterium acidipropionici P169 increased IVDMD and total VFA of low-quality forage. Supplementation with PA and calcium propionate salts increased propionate and decreased A:P in the rumen. Supplementation of PA represents a potential way to increase ruminal propionate concentration when dormant forages are fed.

Ruminant Nutrition Symposium: The utility of lipid extracted algae as a protein source in forage or starch-based ruminant diets.

Two experiments were conducted to determine the influence of lipid extracted algae (LEA) on OM digestibility, N flow, and rumen fermentation. Six samples of LEA were evaluated representing 2 genus of microalgae (Nannochloropsis spp. [n = 3] or Chlorella spp. [n = 3]). Four dual-flow continuous flow fermenters (2,700 mL) were used in a Latin square design to evaluate LEA in forage or concentrate diets compared with soybean meal. Temperature (39 °C), pH, solid (5%/h) and liquid (10%/h) dilution rates, and feed schedule were maintained constant for all experiments. Each experimental period consisted of 6-d adaptation and 4-d sampling periods. There were 7 treatments consisting of 6 different samples of LEA and a soybean meal control (SOY). Diets for Exp.1 were formulated to be 13.0% CP (DM basis) using either soybean meal or LEA and met or exceeded the requirements of a nonpregnant and nonlactating beef cow (450 kg). The forage portion consisted of sorghum-sudan hay (6.4% CP and 46.2% TDN, DM basis) and alfalfa (26.1% CP and 82.3% TDN, DM basis). Concentrate diets used in Exp. 2 met or exceeded the nutrient requirements of a (400 kg) growing steer and contained 85% fine ground corn and included 7% (DM basis) soybean meal or LEA. Data were analyzed as mixed model considering the effect of each LEA compared with soybean meal. Orthogonal contrasts were used to determine the overall effect of LEA genus vs. SOY. True OM digestibility were not influenced by LEA addition to forage diets (P ≥ 0.08) but increased with Chlorella LEA addition to concentrate diets (P < 0.01) but not Nannochloropsis LEA. Degradation of N was greater for SOY with forage diets and LEA for concentrate diets (P < 0.0001). Total VFA production was greatest for SOY in forage diets and increased when LEA was added to concentrate diets (P < 0.0001). Microbial efficiency did not differ between SOY and LEA in forage diets (P ≤ 0.08). In concentrate diets Nannochloropsis decreased microbial efficiency (P < 0.01). Microbial efficiency results for Chlorella were more variable for Nannochloropsis with 1 Chlorella spp. increasing microbial efficiency by 36% over SOY (P < 0.05) and the other Chlorella spp. decreasing microbial efficiency by approximately 42% compared with SOY (P < 0.01). Overall, the results from both experiments are promising for LEA as a protein feedstuff in ruminant diets. Further research is necessary to fully understand the interactions and consequences of upstream processes and what role algal strain plays in LEA quality.

Effects of nordihydroguaiaretic acid on in vitro fermentation profiles of rumen bacteria.

Nordihydroguaiaretic acid (NDGA) is a secondary plant metabolite with antimicrobial properties, and therefore may have potential as a rumen modifier. Two in vitro experiments were conducted to determine the usefulness of NDGA as a rumen modifier. Exp. 1 evaluated the effect of adding 0, 5, 10, 50, and 100 mg/mL NDGA on growth of pure and mixed cultures of rumen bacteria. Growth of all cultures except Butyrivibrio fibrisolvens H17c was inhibited at 50 mg/mL NDGA (P < 0.05). Cultures from whole rumen fluid and B. fibrisolvens H17c were inhibited with 100 mg/mL NDGA (P < 0.05). Exp. 2 evaluated additions of NDGA on IVDMD (48 h) and VFA production. Three dietary substrates simulating different resources available for livestock production and 5 concentrations of NDGA were compared with monensin (47.5 µg/mL, MON, Elanco Animal Health, Indianapolis, IN). Substrates included (DM basis) 100% meadow hay (H100), 50% alfalfa with 50% ground corn (H50), and 90% ground corn with 10% alfalfa (H10). Treatments were 0 (Control; CON), 20, 40, 60, 80 µg/mL NDGA and MON. Treatment means were compared using 2 single degree of freedom contrasts (0 µg/mL NDGA vs. MON and NDGA vs. MON) and orthogonal polynomial contrasts within NDGA concentrations. Monensin fermented with H100 had the least (P < 0.01) IVDMD. A linear increase in IVDMD was observed for H50 (P < 0.01) but not H10 or 100 (P > 0.40). Acetate was quadratic for all substrates tested with NDGA (P < 0.01) and adding NDGA vs. MON resulted in 9% greater values (P < 0.01). Propionate increased by addition of MON compared with CON, which was opposite for acetate. Propionate showed the greatest increase with addition of MON and was dependent on diet vs. CON (H100 vs.H50 vs. H10; 22.5%, 44.4%, and 30.2%, respectively). When H100 was used, total VFA declined linearly by 61% with increasing NDGA (P < 0.01), whereas H50 and H10 were quadratic (P < 0.01) with the greatest total VFA resulting from 40 and 80 mg/mL NDGA for H50 and H10, respectively. Addition of NDGA tended to decrease total VFA (P =0.06) for H100 and H10 by 18.5% and 9.0%, respectively; however, H50 did not differ (P = 0.82) compared with MON. Butyrate increased linearly with increasing NDGA for H10 (P < 0.03) and quadratic for H50 and H100 (P < 0.01). The lowest overall acetate:propionate ratio was obtained with addition of MON to H10 (1.35) and the greatest ratio resulted from adding 60 µg/mL NDGA to H100 (3.63). Rumen fermentation was responsive to NDGA, and the response is dependent on diet.

Technical note: Bacterial diversity and fermentation end products in rumen fluid samples collected via oral lavage or rumen cannula.

A study was conducted to determine if sampling rumen contents via a ruminal cannula or oral lavage tube would yield similar denaturing gradient gel electrophoresis profiles of the bacterial community. Two species of ruminally cannulated animals were used for this study (cattle, n = 2; sheep, n = 3). All animals were allowed ad libitum access to feed. Cattle were fed baled unprocessed sorghum-sudan hay (12% CP, 68% NDF; DM basis), whereas sheep were maintained on chopped alfalfa (18% CP, 40% NDF; DM basis). Ruminal fluid was collected (approximately 20 mL) once per week for 3 wk from each animal using a poly tube equipped with a suction strainer with a hand-held suction pump through the rumen cannula or oral cavity. The denaturing gradient gel electrophoresis analysis demonstrates that yield of bacterial diversity was not different between the 2 sampling methods (P = 0.73). When samples were grouped according to band pattern similarity, groups were most stable according to individual animal and species rather than sampling method. Total VFA and molar proportions of individual VFA did not differ by sampling method (P > 0.40). Additionally, rumen ammonia concentrations were similar for both sampling methods (19.3 vs. 19.1 mM +/- 8.0 for cannula vs. lavage, respectively; P = 0.98). These data indicate that rumen samples collected via oral lavage or rumen cannula yield similar results. This knowledge will allow sample collection from a greater population of animals and an ability to maintain the value of research livestock that can be lost due to the surgical implantation of a ruminal cannula.

Physiological and digestive effects of Neotyphodium coenophialum-infected tall fescue fed to lambs.

The digestive responses and degradation of ergovaline and production of lysergic acid in the rumen of sheep offered Neotyphodium coenophialum-infected tall fescue straw at 2 ergovaline levels were investigated. Six crossbred wethers (56 +/- 3.0 kg of BW) were used in a randomized crossover design involving 2 treatments, for a total of 6 observations per treatment. The experiment consisted of two 28-d feeding periods with a 14-d washout period between them. The treatments were 1) tall fescue straw containing <0.010 mg of ergovaline/kg (E-), and 2) tall fescue straw containing 0.610 mg of ergovaline/kg (E+). Feed, orts, and feces were measured and analyzed for DM, ADF, and CP, and used to determine digestibilities. Feed and water intake were monitored throughout the feeding periods. Body weight and serum prolactin levels were measured at the beginning and end of each feeding period. Ruminal fluid was sampled 3 times (d 0, 3, and 28) during each 28-d feeding period for determination of ergovaline, lysergic acid, ammonia, and pH. Samples were collected before feeding (0 h) and at 6 and 12 h after feeding. Total fecal and urine collection commenced on d 21 and continued until d 25 of each feeding period. Ruminal ammonia, ruminal pH, and rectal temperature were not influenced by ergovaline concentration (P > 0.10). Digestion of DM, ADF, and CP was not different between treatments (P > 0.10). Daily water intake was less for the E+ diet (2.95 vs. 2.77 L/d; P < 0.05) as was serum prolactin (22.9 vs. 6.4 ng/mL; P < 0.05). Ergovaline concentration in ruminal fluid increased over sampling days at each sampling time (P < 0.05). Lysergic acid concentration in ruminal fluid increased over time from d 0 to 3 (P < 0.05) but was not different between d 3 and 28 (P > 0.10). In the E+ treatment, ergovaline was not detectable in the urine, whereas the concentration in the feces was 0.480 mg/kg. Lysergic acid was detected in the diet of the E+ treatment at 0.041 g/kg, lysergic acid in the urine was 0.067 mg/kg and in the feces was 0.102 mg/kg. The apparent digestibility of the alkaloids was 64.2% for ergovaline and -12.5% for lysergic acid. Approximately 35% of dietary ergovaline and 248% of dietary lysergic acid were recovered in the feces and urine. The appearance of lysergic acid in the feces, urine, and ruminal fluid is likely due to microbial degradation of ergovaline in the rumen and further breakdown in the lower digestive tract.

Effects of initial and extended exposure to an endophyte-infected tall fescue seed diet on faecal and urinary excretion of ergovaline and lysergic acid in mature geldings.

AIM: To determine the amount of ergovaline and lysergic acid retained or excreted by geldings fed endophyte-infected seed containing known concentrations of these alkaloids, and the effects of exposure time on clinical expression of toxicosis. METHODS: Mature geldings (n=10) received diets containing either endophyte-free (E-) or endophyte-infected (E+) tall fescue seed during three experimental phases. The first phase (Days -14 to -1) was an adaptation phase, to allow all horses to adapt to a diet containing E- tall fescue seed. The second (Days 0 to 3) was the initial exposure phase to E+ tall fescue seed, used for the delivery of ergovaline and lysergic acid at 0.5 and 0.3 mg/kg of diet, respectively, to test the initial effects of exposure on routes and amounts of elimination of alkaloid. During this phase, half the geldings were exposed to an E+ diet while the rest served as controls by remaining on the E- diet. Once assigned to treatments, geldings remained on the same diet through the third phase (Days 4 to 21), which served as the extended exposure phase. Total outputs of faeces and urine were collected within each phase, to determine retention of ergovaline and lysergic acid and nutrient digestibility. Serum was collected weekly and analysed for activities of enzymes and concentrations of prolactin. Bodyweights (BW) and rectal temperatures were recorded weekly. RESULTS: BW, rectal temperature, enzyme activities and concentrations of prolactin in serum, and nutrient digestibility were not affected by treatment. Total intake of ergovaline by geldings on the E+ diet was 3.5 and 3.6 (SE 0.20) mg/day, and 2.1 and 2.3 (SE 0.11) mg/day were not accounted for in initial and extended phases, respectively. Lysergic acid was excreted in the urine (4.0 and 4.9 (SE 0.97) mg/day) and faeces (2.5 and 2.7 (SE 0.35) mg/day) at greater amounts than that consumed (2.0 and 1.9 (SE 0.09) mg/day) during the initial and extended exposure phases, respectively. Animals exposed to E+ seed for a period of 20 days appeared to excrete more (1.5 vs 1.2 mg/day; SE 0.08; p=0.03) ergovaline in the faeces than those exposed for only 4 days. CONCLUSIONS: Exposure time to the ergot alkaloids had a limited effect on the route of elimination or the amounts of ergovaline or lysergic acid excreted by horses. The primary alkaloid excreted was lysergic acid, and urine was the major route of elimination. These data will aid future research to improve animals' tolerance to toxic endophyte-infected tall fescue.

Detection of lysergic acid in ruminal fluid, urine, and in endophyte-infected tall fescue using high-performance liquid chromatography.

Ergot alkaloids present in endophyte-infected tall fescue induce fescue toxicosis in livestock consuming the plant. The lysergic acid (LA) ring structure is a common moiety among the ergot alkaloids. Little is known about the bioavailability of LA because of limitations in available analytical protocols. Thus, a high-performance liquid chromatography procedure was developed to analyze biological matrices for LA. The biological matrices of interest were tall fescue straw and seed, and ruminant feces, urine, and ruminal fluid. Lysergic acid was added to each matrix at a high (150 ng/ml) or low (30 ng/ml) level. Using the high-level addition, the greatest recovery of LA was obtained from ruminal fluid, feces, and urine (P < 0.05), with an average 85.1% recovered. At the low level, a greater recovery of added LA was observed in the ruminal fluid, urine, and feces (82.1%; P < 0.05) than that in the other 2 matrices (62.6%). The limit of quantitation (LOQ) in ruminal fluid and urine was 5.5 and 18.4 ng/ml, respectively. Seed, straw, and feces had higher LOQ (24.2, 14.5, and 36.0 ng/g, respectively). Limit of detection (LOD) was 1.64, 10.80, 4.35, 5.52, and 7.26 ng/g for ruminal fluid, feces, urine, seed, and straw, respectively. To test the assay in vivo, samples of ruminal fluid and urine were collected from steers consuming a diet containing 400 ng of ergovaline/g and 30 ng of LA/g. All matrices sampled resulted in levels above the LOD and LOQ for the assay, indicating that this assay is sufficiently sensitive for use in assessing the bioavailability of LA.

Molecular analysis of a consortium of ruminal microbes that detoxify pyrrolizidine alkaloids.

Members of a consortium of bacteria, isolated from the rumen of sheep, that degrades pyrrolizidine alkaloids (PAs) found in tansy ragwort (Senecio jacobaea) were characterized. An enrichment of ruminal bacteria was isolated from a sample of ruminal fluid using standard anaerobic techniques. The PA degradative capacity of the enrichment was tested by spiking purified PA extract from tansy ragwort. Length heterogeneity analysis by PCR (LH-PCR) and restriction fragment length polymorphism (RFLP) analysis was used to identify members of the consortium. Phylogenetic analysis of the 16S rDNA gene revealed differing results based on the molecular method used. LH-PCR identified 7 different organisms in 3 groups while RFLP identified 6 organisms with differing banding patterns in 5 groups. After the phylogenetic analyses of both methods were combined, the combined isolates represented 6 groups. The majority of the members of this consortium are <97.0% homologous with known bacteria, indicating this consortium may contain novel organisms able to detoxify PAs found in tansy ragwort. Further understanding of the metabolic pathways used by this consortium to degrade PAs could lead to the use of the consortium as a probiotic therapy for livestock and horses afflicted with tansy ragwort toxicosis.