In vitro protein breakdown by enzyme extracts of rumen origin: comparison with methods in situ and proteases of Streptomyces griseus

A. Velasquez, and G. Pichard. 2010. In vitro protein breakdown by enzyme extracts of rumen origin: comparison with methods in situ and proteases of Streptomyces griseus. Cien. Inv. Agr. 37(3): 57-70. Proteolytic activity of enzymatic extracts generated from rumen microorganisms cultivated in vitro was evaluated. The incubation of rumen fluid used different substrates to generate a higher enzyme concentration and promote a broad spectrum of hydrolytic activity. The composition of the substrates used in the cultivation of the fluid was enriched in protein, starch or cell wall. Enzyme preparations were evaluated by incubating in 30 mL of buffer 50 mM Tris-HCl (pH 6.5) at 39 degrees C during 48 hours, 100 mg of crude protein from feeds soybean meal, canola meal, sunflower meal, gluten feed, dehydrated alfalfa meal, berseem clover, oat forage and perennial ryegrass. Enzyme extracts from cultivated rumen fluid showed an average protein breakdown of 75.5%, in eight feed samples tested. This value was very close to that measured with the technique of proteases from Streptomyces griseus (74.6% CP), but significantly lower (P <= 0.05) than the one obtained by the in situ methodology (84.8% CP). The technique with extracted rumen enzymes showed higher level of proteolysis in the early hours of incubation (6 H) compared to the other techniques. These results suggest that the enzyme preparations of ruminal origin have the ability to predict degradability of feed proteins in the rumen, particularly in the first phase when most of proteins are hydrolyzed and become available for microbial utilization

Effects of rumen fluid pre-incubation on in vitro proteolytic activity of enzymatic extracts from rumen microorganisms

Enzymatic extracts were prepared from rumen fluid (RF) either directly after collection or after incubation in enriched media and their proteolytic activities were measured and compared The objective of RF in vitro cultivation was to augment enzymatic activity by using diverse incubation media containing different feed substrates to selectively induce microorganisms with essentially unrestricted sources of energy and N The effects on protein degradation in bovine serum albumin (BSA) and soybean meal rapeseed meal dehydrated alfalfa meal and perennial ryegrass were determined Enzymatic extracts were characterized by means of protease zymograms Two adult Holstein Friesian rumen fistulated cows were used as RF donors Standard anaerobic incubation techniques in buffer-mineral media were used and protein degradation was measured as residual true protein after enzymatic incubations Results reveal an effect of RF pre-incubation on protein degradation (P<0 0001) measured in BSA and in feed substrates and also on the hydrolysis rate (P<0 001) measured with native substrates only The enzymatic extracts showed a high potential for proteolytic degradation reaching 785 mg/g crude protein (CP) degraded in BSA after 12h of incubation and 830 mg/g CP degraded in soybean meal with 48h of incubation compared to only 654 mg/g and 414 mg/g in controls The zymograms of these extracts revealed six zones of proteolytic activity with the highest concentration of peptidases in the zone of high molecular weight (117 and 130 kDa) In vitro pre-incubation of rumen fluid in culture media enriched with energy and N sources allowed collection of enzymatic extracts with higher proteolytic activity than extracts obtained directly from fresh rumen fluid (C) 2010 Elsevier B V All rights reserve

Effect of substrate on the in vitro protein digestibility of extracts generated by Saccharomyces cerevisiae

The in vitro crude protein (CP) digestibility (D-ivcp), true protein digestibility (D-tp) and degradation rate (kd(cp)) were measured in different protein extracts of Saccharomyces cerevisiae (Sc) yeast. These extracts were generated through a biotechnological method (solid state fermentation). Wheat straw (TWS), barley straw (TBS), chili stubble (TCS), oats hull (TOH) and starch-glucose powder (TSP; control) were used as substrates for fermentation by Sc, and their effect on the aforementioned kinetic parameters in the generated cellular biomass was evaluated. In 12 mL cultivation medium, 2 mL Sc solution inoculum (6.7x10(6) spores mL(-1)) was added together with 2 g vegetable substrate (dry) and additional nitrogen source, and the mixture was incubated for 7 days at a constant temperature (26 degrees C at pH 5) under aerobic conditions. The average CP content of the yeast biomass (DM) was of 45%. The D-ivcp and D-tp were analyzed (two phases: pepsin/trypsin-pancreatin) after the extraction of the yeast biomass, and an effect of the fermented substrate on these values (P <= 0.001 and P <= 0.01, respectively) was observed. The greatest digestibility (D-ivcp) was obtained with TSP (78.9% CP) and the lowest with TCS (67.1% CP). In addition, differences in the kd(cp) between the treatments were also observed (P <= 0.01). The high contents of CP, D-ivcp (71.8% on average) and kd(cp) (16.4% h(-1) on average) suggest that Sc yeast biomass has the potential to become an alternative for the production of animal protein supplements

Lupin and pea extrusion decreases the ruminal degradability and improves the true ileal digestibility of crude protein

The aim of this work was to evaluate the effect of the extrusion of dehulled lupins (Lupinus albus L.) and peas (Pisum sativum L.) on the ruminal degradability and intestinal digestibility of their protein contents. Ruminal degradability was evaluated in situ in the rumens of two fistulated cows. The true ileal crude protein (CP) digestibility was evaluated via a bioassay with Sprague Dawley laboratory rats as the animal model. Extrusion caused soluble fraction decreases in both feeds (P0.05). Extrusion decreased the effective degradability (ED) of lupins by 12 % (P0.05). The extrusion process had a greater impact on the reduction of the effective degradability in lupins than it did in peas. Nevertheless, extrusion increased the true ileal CP digestibility of the ruminal postfermentation residues in both feeds

Empirical attributes and limitations of methodologies for predicting the degradability of ruminal protein

The object of the present review is to analyze the methodologies that are commonly used to estimate protein degradability in the rumen, focusing on their attributes and limitations to offer suggestions for improving their use. This information is essential for selecting food types when formulating feed diets. A reliable prediction of the digestibility of ruminal proteins is basic information necessary for optimizing the use of nitrogenous sources because digestibility can translate, on the one hand, into higher yields of milk, milk protein, meat or wool and, on the other hand, into lower excretion of nitrogenous compounds into the environment; it also has an impact on animal health and welfare. Traditionally, the digestibility of feed proteins in the rumen has been predicted by in vivo, in situ and in vitro methods, but other techniques based on infrared spectroscopy have been developed, notably the NIRS and FTIR methods. All of these techniques present limitations, such as a disturbance factor or a source of error, that may result in inaccurate predictions. The in situ and in vitro methods, which use enzyme extracts of ruminal origin, and FTIR probably have the greatest advantages, but they need to be perfected through further research

Improvement in nutritional quality of fibrous food via in vitro digestion by Aspergillus niger

In a first experiment, the effect of in vitro incubation with Aspergillus niger (An) on the chemical composition of different fibrous substrates was studied. In a second experiment, the effect of incubation time (0, 72 and 144 h) on in vitro digestion with An of dry matter (IVDMD), neutral detergent fiber (DVNDF) and acid detergent fiber (DVADF) was evaluated for different substrates. Wheat straw, WS; barley straw, BS; oat hulls, OH; dehydrated alfalfa, DA; and dehydrated ryegrass, DB substrates were evaluated. In both experiments, incubations were performed in 250 mL Erlenmeyer flasks; 2 g of substrate was added to a culture medium (pH = 6), and incubated at 28 degrees C with constant ventilation. The An dose consisted of 3 mL of a solution of 5.3 x 10(6) spores mL(-1) per flask. Incubation time for the first experiment was 144 h. After incubation, the contents of the flasks were homogenized in a blender for one minute. Subsequently, bromatological analysis was conducted without separating the An biomass. No interaction effects among any studied variables were observed. An incubation effected cell wall (NDF) and lignocellulose (ADF) content. An increase in true protein (TP) and soluble nitrogen (SN) was observed for all substrates tested, but responses differed depending on the type of substrate. The largest increases in TP were observed in DA and DB substrates (0.55 and 0.63% DM, respectively). IVDMD, DVNDF and DVADF were affected by the type of substrate and incubation time. The highest value of IVDMD144 was observed in the DAAn treatment (49.47% DM), followed by the DBAn treatment, with a value of 45.51% DM. OHAn showed the lowest value of IVDMD144 (29.38% DM). The results suggest that An possesses fibrolytic and metabolic potential for improving the nutritional value of fibrous foods through of the digestion of structural carbohydrates and the liberation of nitrogenous fractions embedded in cell walls, producing a change in the chemical composition and the potential digestibility of treated foods

Effect of the type of substrate on the chemical composition and productivity of a protein concentrate of yeast origin

A. Velasquez, R. Arias, and M. Toneatti 2012. Effect of the type of substrate on the chemical composition and productivity of a protein concentrate of yeast origin. Cien. Inv. Agr. 39(3): 425-434. Extracts of Saccharomyces cerevisiae (Sc) yeast were generated using a biotechnological solid-state fermentation method. The organic material used for fermentation consisted of a variety of fibrous substrates of agricultural origin, and their effect on the chemical composition and productivity of cellular biomass generated was evaluated. The substrates studied included the following: wheat straw, WS; barley straw, BS; chili stubble, CS; oat hull, OH and starch powder, SP (control). For incubation with Sc, 2 g of (dry) vegetable substrate were added to 12 mL of cultivation medium. The fermentation time was seven days under aerobic conditions (pH 5, 26 C). After extraction of the yeast biomass, its chemical composition and productivity were analyzed. The results showed an effect (P <= 0.01) of the type of vegetable substrate fermented on the crude protein (CP) and true protein (TP) contents of the yeast biomass. The highest concentration of CP was obtained by fermentation of the control TSP (treatment with the starch powder substrate) (506.3 g kg(-1) DM), followed by the treatment TOH (474.5 g kg(-1) DM). The productivity of the yeast biomass was affected by the type of substrate fermented (P <= 0.001). The highest value was obtained with TSP (327.10 mg g(-1)), followed by TOH (treatment with the oat hull substrate) (207.08 mg g-1). The lowest efficiency was measured in TCS (156.30 mg g(-1)). On the basis of these results, it may be inferred that an extract of high CP can be obtained from yeast (Sc) by the fermentation of fibrous substrates

Simulation of the nitrogen use efficiency in pasture-finished steers in southern Chile

The high content of crude protein (CP) of pastures in southern Chile creates a nutritional imbalance that results in large amount of nitrogen (N) being excreted into the environment. Two regression equations and a dynamic model, available from the literature, were utilized to simulate the N use efficiency (NUE) in pasture-finished steers. A medium frame animal (Angus breed) with an initial and final body weight (BW) of 350 and 500 kg, respectively, was used to estimate nutrient requirements and excretions. Average DMI was fixed at 2.2% of animal BW with 150 days on feed (October to February). Energy and protein requirements, as well as the retained protein, were estimated by using Beef NRC software (tabular system level 1), while the nutritional characteristics of the pasture were obtained from studies based in Chile. The quality of the pastures was good enough to allow daily weight gain (GDP) of 1.0 kg/d. However, metabolizable protein balance was positive across the whole finishing period (average of 149 g/d). Total production of microbial protein increases with time because of the greater DMI, even when the PC content in forage decreases with time. This also explains the lower contribution of DIP during the last months of the finishing period. The estimated N intake was 123 g/d, whereas N excretion was 106 g/d. In summary, the combination of high levels of CP of Chilean southern pastures and the low NUE of the finishing cattle (13.4 - 16.3%), represent a strong challenge in terms of contamination and production

Effect of High Potency Growth Implants on Average Daily Gain of Grass-Fattened Steers

Simple Summary Improving efficiency in beef cattle production requires the adoption of technologies that are low cost but high in return. High potency growth-promoting implants (HGPs) are widely used under feedlot conditions but there are few reports of their use under grazing conditions. We conducted a trial to assess whether the use of high potency HGPs have advantages for grass-finished cattle. Our results were similar to those reported for feedlot cattle, showing that the quality of the pastures was good enough to take advantage of the HGP technology, generating a marginal income that justifies the use of technology under grazing conditions. High potency growth promoter implants (HGPs) are widely used under feedlot conditions but there are few reports under grazing conditions. The study's goal was to assess the effect of HGPs on the average daily gain of steers fattened in pastures. A total of 57 crossbreed steers (Hereford x Angus)-initial body weight = 356.65 kg +/- 5.04 (SEM)-were randomly allocated to one of three groups: Control without HGP (n = 19), Synovex group (n = 17), and Revalor group (n = 21). The fattening period was 67 days using paddocks of Lolium perenne L. and Trifolium repens L. Body weight was recorded three times in the period. The data were analyzed using an analysis of covariance with a level of significance of 5%. The average daily gain (ADG) (1.55 +/- 0.07 and 1.48 +/- 0.09 kg/d) and the total weight gain (103.4 +/- 4.9 and 99.2 +/- 5.8 kg) were similar for Revalor and Synovex, respectively (p > 0.05). Moreover, HGP groups showed higher ADG and total weight gain (p < 0.01) than the control group (ADG = 0.93 +/- 0.08 kg/d and a total weight gain of 62.2 +/- 5.2 kg). Final body weights were 527.8 +/- 8.5 kg and 512.2 +/- 9.9 kg for Revalor and Synovex, respectively; and 479.9 +/- 10.1 kg for Control. In conclusion, grazing-finished steers showed better performance when high potency HGPs were used, improving the ADG and final live body weight, generating a marginal income that justifies the use of the technology under grazing condition

Dietary preference of European wild boar (Sus scrofa L.) grazing grass and legume at two contrasting plant heights: A pilot study

There were two objectives: checking a methodology to test diet preferences in a natural environment and secondly to assess diet preferences of European wild boar in terms of species and plant characteristics when offered in a situation with minimal physical constraints. Five hundred pots (100 mm upper diameter, 450 cc volume) were completely filled with soil. Half of the pots were sown with red clover (Trifolium pretense) and half with hybrid ryegrass (Lolium hybridum Hausskn). All pots were irrigated, periodically cut, and maintained outdoors. Then 220 leaves of ryegrass, 201 leaflets of clover and 188 petioles of clover were used to establish a relationship between plant structure dimensions and their dry matter mass. The preference study was conducted in a fenced area (18 m length and 11,1 m width) with a pasture cut to a height of 3 cm (lowest height possible to cover the soil). Four transects were marked separated 2 m from each other, and 16 holes within each transect were made 1 m apart, in which the pots were buried and anchored. Treatments consisted of the factorial combination of two plant species (red clover and hybrid ryegrass) and two plant heights (12 and 18 cm, tall and short, respectively), randomized within each transect (block). During three days, four nose-ringed wild boars grazed the experimental area for one hour and the treatments which they were grazing were recorded every two minutes. Plants structures were measured pre -and post-grazing from marked plants (green thread tied at the base) in each pot to detect consumption and to estimate the amount consumed. Strong correlations were found between plant structures and their dry mass (R-2 between 0,83 and 0,89). The methodology was able to show that more dry matter was apparently consumed from clover than ryegrass (P < 0.001) and clover had a greater probability of being grazed than ryegrass (P < 0.05). Also, the tall plants were consumed more than the short plants (P < 0.001) with individual tall plants having a higher probability of being grazed (P < 0.05). It is concluded that the novel method proposed was able to detect the diet preferences of wild boar during grazing. Additionally, in this study European wild boar discriminated between plant species and between plant heights, preferring legume over ryegrass and taller over shorter plants. However, more studies with a greater number of animals are necessary to validate these results