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In two in vitro experiments with the RUSITEC-apparatus, Brachiaria dictyoneura was tested alone and with legumes at dietary proportions of 1/3, 2/3, or 3/3 of Arachis pintoi (Expt 1) and 1/3 of Arachis pintoi, Cratylia argentea, or Calliandra calothyrsus (Expt 2). In Expt 2, all diets were evaluated with and without 80 mg/g diet of Sapindus saponaria fruits. In Expt 1, the stepwise replacement of the grass by A. pintoi curvi-linearly increased rumen fluid concentrations of ammonia, volatile fatty acids, bacteria, and protozoa. Methane release rates were 1.7, 7.3, 8.8, and 9.0�mmol/day. With increasing legume proportion, more organic matter and protein were degraded, the latter being only partially recovered as ammonia. In Expt 2, 1/3 of A. pintoi basically had the same effects as in Expt 1. Cratylia argentea was less effective in modifying the fermentation pattern. In association with a higher nutrient degradation and rumen ammonia concentration, C. argentea and A. pintoi increased methane release to about 3- and 4-fold levels. Calliandra calothyrsus reduced nutrient degradation and methane release per gram of organic matter degraded. Tannins, predominant in C. calothyrsus, might have affected methanogenesis. Sapindus saponaria reduced methanogenesis by 11% on average in grass-alone and legume-supplemented diets.

Six adult African-type hair sheep (BW = 40.3 +/- 6.3 kg) fitted with ruminal and duodenal cannulas were subjected to four treatments. Sheep were offered basal diets at a rate of 80 g of DM/kg of metabolic BW (equivalent to ad libitum access) consisting either of a low-quality grass hay (Brachiaria dictyoneura, 3.7% CP, DM basis) alone or in combination with a forage legume (Cratylia argentea, 18.6% CP, DM basis) in a 3:1 ratio (DM basis). In addition, 0 or 8 g of DM of Sapindus saponaria fruits (12.0% crude saponins, DM basis) per kilogram of metabolic BW was administered intraruminally. Supplementation of C. argentea increased intakes of OM (+21%; P < 0.01) and CP (+130%; P < 0.001), as well as ruminal fluid ammonia N concentrations (from 2.40 to 8.43 mg/dL; P < 0.001). Apparent OM and N digestibilities were not affected by legume addition, but ADF digestibility decreased by 10% (P < 0.01). Total ruminal VFA concentration was unchanged, but acetate:propionate was lower (P < 0.01) and isobutyrate proportion was greater (P < 0.001) with the legume addition. Legume supplementation increased duodenal flows of total N (+56%; P < 0.001), nonammonia N (+52%; P < 0.001), ruminal escape N (+80%; P < 0.001), and microbial N (+28%; P < 0.05). Microbial efficiency was not affected by legume addition. Supplementation of S. saponaria increased (P < 0.05) dietary OM intake by 14%, but had no effect on CP intake and ruminal fluid ammonia concentration or on OM and N digestion. Digestibility of ADF was decreased (P < 0.01) by 10% with S. saponaria as was acetate:propionate (P < 0.001) and the isobutyrate proportion (P < 0.001). Ruminal protozoa counts increased (P < 0.01) by 67% with S. saponaria. Duodenal N flows were not significantly affected by S. saponaria supplementation, except for microbial N flow (+34%; P < 0.01). Microbial efficiency was greater (P < 0.05) by 63% with the addition of S. saponaria. Few interactions between legume and S. saponaria supplementation were observed. The NDF digestibility was decreased with S. saponaria in the grass-alone diet, but not in the legume-supplemented diet (interaction; P < 0.05). Interactions were absent in ruminal fermentation measures and duodenal N flow, indicating that effects were additive. Results suggest that, even when not decreasing ruminal protozoa count, supplementation of S. saponaria fruits is a beneficial way to improve ruminal VFA profile, microbial efficiency, and duodenal flow of microbial protein in sheep fed tropical grass-alone or grass-legume diets.

<p>En el presente trabajo se estandarizó una técnica de criopreservación en nitrógeno líquido, usando como crioprotector dimetil sulfóxido al 5%, para la preservación de aislados nativos bacterianos de <em>Fibrobacter succinogenes</em>. El respectivo protocolo de criopreservación se evaluó determinando la viabilidad bacteriana por el transcurso de un año. La escala de MacFarland, ampliamente conocida en la estimación de poblaciones bacterianas aerobias, fue validada como técnica de estimación de bacterias anaerobias, usando la técnica de conteo bacteriano por <em>roll-tube </em>(R2 = 0.95). Se utilizaron dos aislados nativos, C7 y C9, de <em>Fibrobacter succinogenes</em>, obtenidos del rumen de bovinos en pastoreo de <em>Brachiaria decumbens</em>, en los Llanos Orientales de Colombia. Quince días después de la exposición de los cultivos al proceso de criopreservación, se apreció una reducción en el número de bacterias viables con relación a la población inicial de cada uno de los aislados (C7 = 9.25 x 108 UFCml-1 vs. 6.15 x 108 UFCml-1 y C9 = 12.51 x 108 UFCml-1 vs. 9.26 x 108 UFCml-1). Sin embargo, no se presentaron diferencias en la población de bacterias viables en los muestreos posteriores (C7 = 6.13 – 6.16 x 108; C9 = 9.26 – 9.35 x 108 UFCml-1). Esta técnica permite mantener la viabilidad bacteriana y puede considerarse como un procedimiento eficiente y de fácil aplicación para la preservación de bacterias ruminales. Además, constituye una herramienta fundamental para el establecimiento de bancos de germoplasma de microorganismos anaerobios ruminales en Colombia. El uso conjunto de las técnicas de criopreservación y la escala de MacFarland, posee ventajas sobre los métodos de preservación y estimación de poblaciones por cultivos activos, por su confiabilidad, eficiencia y bajo costo.</p><p> </p><p><strong>Cryopreservation of native isolates </strong><strong>of ruminal bacteria <em>Fibrobacter </em></strong><strong><em>succinogenes</em></strong><strong>.</strong></p><p>In this study, the technique of cryopreservation in liquid nitrogen to preserve native isolates of the fibrolytic bacteria <em>Fibrobacter succinogenes </em>was standardized. The technique efficiency was evaluated by establishing the bacterium viability during one year. The Mac-Farland scale, widely used to estimate aerobic bacterial populations, was validated by the roll tube technique (R2=0.95). Two native <em>F. succinogenes </em>strains, C7 and C9, were used. These strains were obtained from cattle fed on <em>Brachiaria decumbens </em>in the eastern lowlands of Colombia. Fifteen days after the initiation of the cryopreservation process, a reduction in the number of viable bacteria (CFU ml-1) was registered (C7 from 9.25 x 108 to 6.15 x 108 and C9 from 12.51 x 108 to. 9.26 x 108). However, the bacterial populations did not present differences in subsequent samplings (C7=6.13 – 6.16X108, C9=9.26 – 9.35X108 CFU ml-1, range over a year). Results indicated that the standardized cryopreservation technique is an effective and easy procedure to preserve ruminal bacteria, which would be a very useful tool to create a germplasm bank of Colombian ruminal anaerobic microorganisms. The cryopreservation technique, and the MacFarland scale have an advantage over other methods of preserving bacteria and methods of estimating populations of bacteria by active cultures. The procedures used in this research are reliable and economical techniques using minimal storage spaces and laboratory resources.</p>

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