Avaliação causal do "efeito proteína" sobre a atividade microbiana em substratos fibrosos insolúveis

• Autor(a) principal: Carvalho, Isabela Pena Carvalho de
• Data de Publicação: 2009
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/5614
• topic: Carboximetilcelulase; Efeito proteína; Fibra em detergente neutro; Glutamato desidrogenase; Carboxymethilcellulase; Protein effect; Neutral detergent fiber; Glutamate dehydrogenase; CNPQ::CIENCIAS AGRARIAS::ZOOTECNIA::NUTRICAO E ALIMENTACAO ANIMAL

The objective in this trial was to evaluate the effects of supplementation with nitrogenous compounds sources on insoluble fiber utilization of through acid lactic bacteria (LAB) growth, microbial protein production, and activity of the enzymes carboxymethilcellulase (CMC) and glutamate dehydrogenase (GDH). Three experiments were carried out. At the first one, four incubations were performed using the ruminal fluid taken from a Holstein x Zebu steer fitted with ruminal canullae and fed signal grass (Brachiaria decumbens Stapf.) pasture plus 200 g/day of supplemental protein. The fluid was collected and centrifuged. The microbial pellet was re-suspended in a basal media and used as inoculum. Eight treatments were evaluated: Cellulose, Cellulose plus Casein, Cellulose plus Soy Peptone, Cellulose plus Urea:Ammonium Sulfate (9:1), Starch, Starch plus Casein, Starch plus Soy Peptone, and Starch plus Urea:Ammonium Sulfate (9:1). The flasks were kept at 39°C with orbital shaking (70 rpm). Successive incubations were done to select the microorganisms according to the energy and nitrogenous sources. After 24 hours of incubation, on the third and fourth transfers, the pH were evaluated and the resulting fluid was diluted and plated on a selective media for LAB according to spread plate technique. After 48 hours the colonies forming units (CFU/mL) were counted. It wasperformed a test to detect the presence of inhibitory substances on the inhibitory activity by using the LAB obtained in the previous procedure. The growth of LAB was favored (P<0.05) when starch was used as energy source compared to cellulose. The true protein sources (soy peptone and casein) increased the growth of these bacteria (P<0.05) compared to control and urea. The pH was not affected by energy source (P>0.05), but the inclusion of urea resulted in higher pH (P<0.05) compared to other sources of nitrogenous compounds. None inhibitory activity was detect on LAB cultures. In the second experiment, three incubations were performed according to the experimental procedures described above. Four treatments were evaluated: Cellulose, Cellulose plus Casein, Cellulose plus Urea:Ammonium Sulfate (9:1), and Cellulose plus Casaminoacids. After the first transfer, a flask of each treatment was taken at 6, 12 and 24 hours of incubation. The microbial enzymes were extracted and the activities of CMC and GHD were evaluated. A second group of flasks was incubated in order to obtain samples at 6, 12 and 24 hours to quantify the concentration of ammonia nitrogen (AN) and microbial protein. In all incubation times higher AN concentration was observes for urea (P<0.05). At 12 and 24 hours of incubation the sources of true protein (casein and casaminoacids) caused increase in AN concentrations compared to control (P<0.05). In a general, the concentration of microbial protein was stable among treatments. The activity of CMC was higher with the use of urea and casaminoacids (P<0.05) compared to control and casein. Supplementation with casaminoacids implied higher activity of GDH (P<0.05) compared to control at 6 hours of incubation, with intermediate positions for casein and urea. At 12 hours, the activity of GDH was also stimulated by casein (P<0.05). At 24 hours the activity of GDH was not different among treatments (P>0.05). In the third experiment three Holstein x Zebu steers, with average initial body weight of 350 kg, fitted with ruminal canullae and kept in individual stalls, were used. The animals were fed Tifton hay (Cynodon sp.), ad libitum. The treatments were: control, without supplementation; supplementation with non-protein nitrogenous compounds (urea: ammonium sulfate, 9:1); and supplementation with true protein (albumin). The level of supplementation with nitrogenous compounds was 1 g/kg body weight in crude protein. The experiment was carried out according to a 3 x 3 Latin square design, with three experimental periods lasting five days each, being three days for the adaptation of animals to supplementation. In the fourth day of each experimental period, samples of rumen fluid were collected at 6, 12 and 24 hours after supplementation to evaluate pH and ruminal AN concentration. At same time, it was carried out an in situ incubation procedure in order to quantify the activity of the enzymes CMC and GHD. The average of the ruminal pH was 6.55, with no differences among treatments and time of evaluation (P>0.05). In a general way, the activity of GHD was increased between times of evaluation (P<0.05). However, there were no differences among treatments (P>0.05). The control treatment show higher activity of the CMC compared to treatments with supplementation (P<0.05). However, supplementation with urea implied higher activities of the CMC than albumin (P<0.05). The sources of nitrogen compounds (urea and albumin) caused increase in RAN concentrations compared to control (P<0.10). The concentrations of RAN decreased from the first moment of evaluation (P<0.10). From those results, it can be concluded that true protein sources cause deleterious effects on utilization of insoluble fibrous substrates because they stimulate the growth of nonstructural carbohydrates degrading bacteria.