Use of lignin extracted from different plant sources as standards in the spectrophotometric acetyl bromide lignin method

A nongravimetric acetyl bromide lignin (ABL) method was evaluated to quantify lignin concentration in a variety of plant materials. The traditional approach to lignin quantification required extraction of lignin with acidic dioxane and its isolation from each plant sample to construct a standard curve via spectrophotometric analysis. Lignin concentration was then measured in pre-extracted plant cell walls. However, this presented a methodological complexity because extraction and isolation procedures are lengthy and tedious, particularly if there are many samples involved. This work was targeted to simplify lignin quantification. Our hypothesis was that any lignin, regardless of its botanical origin, could be used to construct a standard curve for the purpose of determining lignin concentration in a variety of plants. To test our hypothesis, lignins were isolated from a range of diverse plants and, along with three commercial lignins, standard curves were built and compared among them. Slopes and intercepts derived from these standard curves were close enough to allow utilization of a mean extinction coefficient in the regression equation to estimate : lignin concentration in any plant, independent of its botanical origin. Lignin quantification by use of a common regression equation obviates the steps of lignin extraction, isolation, and standard curve construction, which substantially expedites the ABL method. Acetyl bromide lignin method is a fast, convenient analytical procedure that may routinely be used to quantify lignin.

Anatomia quantitativa da folha e do colmo de Brachiaria brizantha (Hochst. ex A. Rich.) Stapf e B. humidicola (Rendle) Schweick

As características da digestão das forragens podem ser mais bem compreendidas quando complementadas com estudos de anatomia dos tecidos de plantas forrageiras. Com o objetivo de agregar novas informações ao tema, determinou-se a área dos diversos tecidos presentes na folha e no colmo de Brachiaria brizantha e B. humidicola, em três diferentes níveis de inserção no perfilho. O experimento foi conduzido em casa de vegetação, com plantas mantidas em vasos plásticos contendo areia esterilizada e recebendo solução nutritiva de macro e micronutrientes. Após 70 dias do corte de uniformização das parcelas (vasos), coletou-se o segmento do colmo e da folha (limbo e bainha) situado na porção mediana de cada um dos três diferentes níveis de inserção do perfilho estudado. As amostras foram emblocadas em GMA, seccionadas em micrótomo e o laminário fotomicrografado. As fotografias foram digitalizadas e as áreas de tecidos, mensuradas com software Image Tool. Observou-se que o limbo das folhas basilares apresentou menor área de tecido vascular lignificado. Maior área de tecido parenquimático foi observada na bainha e no colmo da B. brizantha. em relação à B. humidicola, a B. brizantha apresentou maior área de tecido vascular lignificado no limbo, bainha e colmo. Maior distância entre os feixes vasculares foi observada no limbo e na bainha, sendo o número de feixes maior no limbo e no colmo.

The inclusion of forage mixtures in the diet of growing dairy heifers: impacts on digestion, energy utilisation, and methane emissions

Intensive farming focusing on monoculture grass species to maximise forage production has led to a reduction in the extent and diversity of species-rich grasslands. However, plant communities with higher species number (richness) are a potential strategy for more sustainable production and mitigation of greenhouse gas (GHG) emissions. Research has indicated the need to understand opportunities that forage mixtures can offer sustainable ruminant production systems. The objective of the two experiments reported here were to evaluate multiple species forage mixtures in comparison to ryegrass-dominant pasture, when conserved or grazed, on digestion, energy utilisation, N excretion, and methane emissions by growing 10–15 month old heifers. Experiment 1 was a 4 × 4 Latin square design with five week periods. Four forage treatments of: (1) ryegrass (control); permanent pasture with perennial ryegrass (Lolium perenne); (2) clover; a ryegrass:red clover (Trifolium pratense) mixture; (3) trefoil; a ryegrass:birdsfoot trefoil (Lotus corniculatus) mixture; and (4) flowers; a ryegrass:wild flower mixture of predominately sorrel (Rumex acetosa), ox-eye daisy (Leucanthemum vulgare), yarrow (Achillea millefolium), knapweed (Centaurea nigra) and ribwort plantain (Plantago lanceolata), were fed as haylages to four dairy heifers. Measurements included digestibility, N excretion, and energy utilisation (including methane emissions measured in respiration chambers). Experiment 2 used 12 different dairy heifers grazing three of the same forage treatments used to make haylage in experiment 1 (ryegrass, clover and flowers) and methane emissions were estimated using the sulphur hexafluoride (SF6) tracer technique. Distribution of ryegrass to other species (dry matter (DM) basis) was approximately 70:30 (clover), 80:20 (trefoil), and 40:60 (flowers) for experiment 1. During the first and second grazing rotations (respectively) in experiment 2, perennial ryegrass accounted for 95 and 98% of DM in ryegrass, and 84 and 52% of DM in clover, with red clover accounting for almost all of the remainder. In the flowers mixture, perennial ryegrass was 52% of the DM in the first grazing rotation and only 30% in the second, with a variety of other flower species occupying the remainder. Across both experiments, compared to the forage mixtures (clover, trefoil and flowers), ryegrass had a higher crude protein (CP) content (P < 0.001, 187 vs. 115 g kg −1 DM) and DM intake (P < 0.05, 9.0 vs. 8.1 kg day −1). Heifers in experiment 1 fed ryegrass, compared to the forage mixtures, had greater total tract digestibility (g kg −1) of DM (DMD; P < 0.008, 713 vs. 641) and CP (CPD, P < 0.001, 699 vs. 475), and used more intake energy (%) for body tissue deposition (P < 0.05, 2.6 vs. −4.9). For both experiments, heifers fed flowers differed the most compared to the ryegrass control for a number of measurements. Compared to ryegrass, flowers had 40% lower CP content (P < 0.001, 113 vs. 187 g kg −1), 18% lower DMD (P < 0.01, 585 vs. 713 g kg −1), 42% lower CPD (P < 0.001, 407 vs. 699 g kg −1), and 10% lower methane yield (P < 0.05, 22.6 vs. 25.1 g kg −1 DM intake). This study has shown inclusion of flowers in forage mixtures resulted in a lower CP concentration, digestibility and intake. These differences were due in part to sward management and maturity at harvest. Further research is needed to determine how best to exploit the potential environmental benefits of forage mixtures in sustainable ruminant production systems.

Digestion, rumen fermentation and circulating concentrations of insulin, growth hormone and IGF-1 in steers fed diets based on different proportions of maize silage and grass silage

Replacing grass silage with maize silage results in a fundamental change in the ratio of structural to non-structural carbohydrates with commensurate changes in rumen fermentation patterns and nutrient utilisation. This study investigated the effects of feeding four forage mixtures, namely grass silage (G); 67 g/100 g grass silage133 g/100 g maize silage (GGM); 67 g/100 g maize silage133/100 g grass silage (MMG); maize silage (M) to four ruminally and duodenally canulated Holstein Friesian steers. All diets were formulated to be isonitrogenous (22.4 g N/kg DM) using a concentrate mixture. Dietary dry matter (DM) and organic matter (OM) digestibility increased with ascending maize silage inclusion (P,0.1) whereas starch and neutral detergent fibre digestibility declined (P,0.05). Ratio of non-glucogenic to glucogenic precursors in the rumen fluid increased with maize silage inclusion (P,0.01) with a commensurate reduction in rumen pH (P,0.05). Mean circulating concentrations of insulin were greatest and similar in diets MMG and GGM, lower in diet M and lowest in diet G (P,0.01). There were no effects of diet on the mean circulating concentration of growth hormone (GH), or the frequency, amplitude and duration of GH pulses, or the mean circulating concentrations of IGF-1. Increasing levels of DM, OM and starch intakes with the substitution of grass silage with maize silage affected overall digestion, nutrient partitioning and subsequent circulating concentrations of insulin.

Digestion, rumen fermentation and circulating concentrations of insulin, growth hormone and IGF-1 in steers given maize silages harvested at three stages of maturity

Advancing maturity of forage maize is associated with increases in the proportion of dry matter (DM) and starch and decreases in the proportions of structural carbohydrates in the ensiled crop. Three maize silages (286 (low, L), 329 (medium, M) and 379 (high, H) g DM per kg fresh weight) plus a concentrate formulated to give isonitrogenous intakes were offered to Holstein-Friesian steers fitted with a cannula in the dorsal sac of the rumen and a 'T' piece cannula in the proximal duodenum in an experiment with a cross-over design that allowed four collection periods. Nutrient flow to the duodenum was estimated using chromium-EDTA. Steers consumed approximately 0(.)6 kg DM per day less of diet L compared with the other two diets (P=0(.)026), resulting in less DM being digested (P=0(.)005) but digestibility did not differ between diets. Similar results were obtained for organic matter. There were no differences between diets in the intake or digestibility of neutral-detergent fibre. Intake, duodenal flow and faecal output of starch were greater for steers offered diets M and H compared with those given diet L (P < 0(.)05). In all diets rumen digestion contributed to over 90% of total digestion of starch, although rumen digestibility declined significantly with advancing maize maturity (P=0(.)002). Molar proportions of acetic acid were higher in diet H (P < 0(.)05) whilst proportions of propionic acid and n-butyric acid were higher in diets M and L. There were no significant differences between diets in mean rumen pH or ammonia concentrations. Mean circulating concentrations of insulin were higher (P=0(.)009) in cattle given diets L and M compared with diet H. There were no differences between diets in the mean circulating concentration of growth hormone, or the frequency, amplitude and duration of growth hormone pulses, or the mean circulating concentrations of IGF-1. Changes in forage composition that accompany advancing maize maturity affect overall silage digestion and circulating concentrations of insulin.

The effect of condensed tannins in fresh sainfoin (Onobrychis viciifolia) on in vivo and in situ digestion in sheep

This study focused on effects of structure, content and biological activity of condensed tannins (CT) in leaves, stems and whole plant of sainfoin (Onobrychis viciifolia) on its in vivo and in situ digestive characteristics in sheep. Sainfoin was studied as fresh forage during the first vegetation cycle at two phenological stages (i.e., end of flowering and green seeds) and during the second vegetation cycle (i.e., start of flowering). The feeding experiment used 12 sheep; with six dosed, through the rumen cannula, with polyethylene glycol (PEG) to neutralise CT effects. Organic matter digestibility (OMD), total tract N disappearance and N balance were measured in sheep fed the whole plant. The residues of dry matter (DM) and N from nylon bags suspended in the rumen were determined on leaves and stems. Intestinal digestibility was measured using other, intestinally fistulated sheep. PEG addition and vegetation cycle increased total tract N digestibility (P<0.001) but PEG affected OMD only at the end of flowering. PEG inactivated the CT and increased urinary N excretion (P<0.05) but this was offset by lower faecal N excretion (P<0.001). Feeding sainfoin can be used to alter the form of excreted N (i.e., urine vs faeces) and thus potentially reduce environmental N pollution without affecting body N retention. Kinetic studies of total N, ammonia N (NH3-N) and volatile fatty acids (VFA) in rumen fluid were made before and 1.5, 3 and 6 h after feeding. Sainfoin CT decreased rumen fluid soluble N (P<0.05) and NH3-N (P<0.01). Ruminal N disappearance (DisN) of leaves or stems was lower in the presence of active CT compared to PEG-inactivated CT (P<0.001) for both vegetation cycles. PEG also increased intestinal digestibility (P<0.05) of leaves and stems. Leaves had lower ruminal DisN, but higher N disappearing from intestine than stems. The biological activity and content of CT in the whole plant decreased as phenological stage increased. Prodelphinidin:procyanidin (PD:PC) ratios of leaves varied with vegetation cycle and phenological stage. The molecular size of CT in the whole plant, as indicated by their mean degree of polymerisation (mDP), was lowest at the start of flowering and coincided with the higher biological activity and content of CT.

Effects of 3-nitrooxypropanol on methane emission,digestion, and energy and nitrogen balance of lactating dairy cows

The objective was to measure effects of 3-nitrooxypropanol (3NP) on methane production of lactating dairy cows and any associated changes in digestion and energy and nitrogen metabolism. Six Holstein-Friesian dairy cows in mid-lactation were fed twice daily a total mixed ration with maize silage as the primary forage source. Cows received 1 of 3 treatments using an experimental design based on two 3 × 3 Latin squares with 5-wk periods. Treatments were a control placebo or 500 or 2,500 mg/d of 3NP delivered directly into the rumen, via the rumen fistula, in equal doses before each feeding. Measurements of methane production and energy and nitrogen balance were obtained during wk 5 of each period using respiration calorimeters and digestion trials. Measurements of rumen pH (48 h) and postprandial volatile fatty acid and ammonia concentrations were made at the end of wk 4. Daily methane production was reduced by 3NP, but the effects were not dose dependent (reductions of 6.6 and 9.8% for 500 and 2,500 mg/d, respectively). Dosing 3NP had a transitory inhibitory effect on methane production, which may have been due to the product leaving the rumen in liquid outflow or through absorption or metabolism. Changes in rumen concentrations of volatile fatty acids indicated that the pattern of rumen fermentation was affected by both doses of the product, with a decrease in acetate:propionate ratio observed, but that acetate production was inhibited by the higher dose. Dry matter, organic matter, acid detergent fiber, N, and energy digestibility were reduced at the higher dose of the product. The decrease in digestible energy supply was not completely countered by the decrease in methane excretion such that metabolizable energy supply, metabolizable energy concentration of the diet, and net energy balance (milk plus tissue energy) were reduced by the highest dose of 3NP. Similarly, the decrease in nitrogen digestibility at the higher dose of the product was associated with a decrease in body nitrogen balance that was not observed for the lower dose. Milk yield and milk fat concentration and fatty acid composition were not affected but milk protein concentration was greater for the higher dose of 3NP. Twice-daily rumen dosing of 3NP reduced methane production by lactating dairy cows, but the dose of 2,500 mg/d reduced rumen acetate concentration, diet digestibility, and energy supply. Further research is warranted to determine the optimal dose and delivery method of the product. Key words: 3-nitrooxypropanol, methane, digestion, rumen, dairy cow

Digestion of feed fractions and intake of heifers fed hydrolyzed sugarcane stored for different periods

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Comparative in vitro evaluation of forage legumes (prosopis, acacia, atriplex, and leucaena) on ruminal fermentation and methanogenesis

Two experiments in vitro were conducted to evaluate four Egyptian forage legume browses, i.e., leaves of prosopis (Prosopis juliflora), acacia (Acacia saligna), atriplex (A triplex halimus), and leucaena (Leucaena leucocephala), in comparison with Tifton (Cynodon sp.) grass hay for their gas production, methanogenic potential, and ruminal fermentation using a semi-automatic system for gas production (first experiment) and for ruminal and post ruminal protein degradability (second experiment). Acacia and leucaena showed pronounced methane inhibition compared with Tifton, while prosopis and leucaena decreased the acetate:propionate ratio (P<0.01). Acacia and leucaena presented a lower (P<0.01) ruminal NH3-N concentration associated with the decreasing (P<0.01) ruminal protein degradability. Leucaena, however, showed higher (P<0.01) intestinal protein digestibility than acacia. This study suggests that the potential methanogenic properties of leguminous browses may be related not only to tannin content, but also to other factors.

Digestion of feed fractions and intake of heifers fed hydrolyzed sugarcane stored for different periods

The objective of this study was to evaluate, in Nellore heifers, intake and digestibility of hydrolyzed sugarcane stored for different periods. The experimental design used was a 4 × 4 Latin square, four diets, four Nellore heifers with ruminal cannulas (initial body weight 285.4±23.08 kg and average initial age 14 months) and four periods of 21 days. The diets were composed by fresh sugarcane (time zero) or hydrolyzed sugarcane with addition of 0.5% of hydrated lime, stored for 24, 48 or 72 hours, as the unique forage. Intake and digestibility of feed fractions, nitrogen balance, microbial synthesis efficiency, total number of ruminal protozoans and ammoniacal nitrogen did not significantly change by storing sugarcane with addition of 0.5% of hydrated lime. Sugarcane pH varied quadratically for storage time, with maximum pH of 7.02 after 24 hours from lime addition. Ruminal liquid pH values were higher for heifers fed fresh sugarcane, in comparison with those fed hydrolyzed sugarcane. Sugarcane treated with 0.5% of hydrated lime stored for up to 72 hours does not change ruminal digestion to alter the amount of feed consumed by pubescent Nellore heifers. Thus, lime is a viable technology, once it allows long-duration storage and bee control on treated forage, which contributes to animal feeding logistics.

Afferent Connections to the Rostrolateral Part of the Periaqueductal Gray: A Critical Region Influencing the Motivation Drive to Hunt and Forage

Previous studies have shown that a particular site in the periaqueductal gray (PAG), the rostrolateral PAG, influences the motivation drive to forage or hunt. To have a deeper understanding on the putative paths involved in the decision-making process between foraging, hunting, and other behavioral responses, in the present investigation, we carried out a systematic analysis of the neural inputs to the rostrolateral PAG (rlPAG), using Fluorogold as a retrograde tracer. According to the present findings, the rlPAG appears to be importantly driven by medial prefrontal cortical areas involved in controlling attention-related and decision-making processes. Moreover, the rlPAG also receives a wealth of information from different amygdalar, hypothalamic, and brainstem sites related to feeding, drinking, or hunting behavioral responses. Therefore, this unique combination of afferent connections puts the rlPAG in a privileged position to influence the motivation drive to choose whether hunting and foraging would be the most appropriate adaptive responses. Copyright (C) 2009 Sandra Regina Mota-Ortiz et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Ants Can Learn to Forage on One-Way Trails

The trails formed by many ant species between nest and food source are two-way roads on which outgoing and returning workers meet and touch each other all along. The way to get back home, after grasping a food load, is to take the same route on which they have arrived from the nest. In many species such trails are chemically marked by pheromones providing orientation cues for the ants to find their way. Other species rely on their vision and use landmarks as cues. We have developed a method to stop foraging ants from shuttling on two-way trails. The only way to forage is to take two separate roads, as they cannot go back on their steps after arriving at the food or at the nest. The condition qualifies as a problem because all their orientation cues-chemical, visual or any other - are disrupted, as all of them cannot but lead the ants back to the route on which they arrived. We have found that workers of the leaf-cutting ant Atta sexdens rubropilosa can solve the problem. They could not only find the alternative way, but also used the unidirectional traffic system to forage effectively. We suggest that their ability is an evolutionary consequence of the need to deal with environmental irregularities that cannot be negotiated by means of excessively stereotyped behavior, and that it is but an example of a widespread phenomenon. We also suggest that our method can be adapted to other species, invertebrate and vertebrate, in the study of orientation, memory, perception, learning and communication.

Heteroduplex formation and S1 digestion for mapping alternative splicing sites

The identification of alternatively spliced transcripts has contributed to a better comprehension of developmental mechanisms, tissue-specific physiological processes and human diseases. Polymerase chain reaction amplification of alternatively spliced variants commonly leads to the formation of heteroduplexes as a result of base pairing involving exons common between the two variants. S1 nuclease cleaves single-stranded loops of heteroduplexes and also nicks the opposite DNA strand. In order to establish a strategy for mapping alternative splice-prone sites in the whole transcriptome, we developed a method combining the formation of heteroduplexes between 2 distinct splicing variants and S1 nuclease digestion. For 20 consensuses identified here using this methodology, 5 revealed a conserved splice site after inspection of the cDNA alignment against the human genome (exact splice sites). For 8 other consensuses, conserved splice sites were mapped at 2 to 30 bp from the border, called proximal splice sites; for the other 7 consensuses, conserved splice sites were mapped at 40 to 800 bp, called distal splice sites. These latter cases showed a nonspecific activity of S1 nuclease in digesting double-strand DNA. From the 20 consensuses identified here, 5 were selected for reverse transcription-polymerase chain reaction validation, confirming the splice sites. These data showed the potential of the strategy in mapping splice sites. However, the lack of specificity of the S1 nuclease enzyme is a significant obstacle that impedes the use of this strategy in large-scale studies.

Partial microwave-assisted wet digestion of animal tissue using a baby-bottle sterilizer for analyte determination by inductively coupled plasma optical emission spectrometry

A procedure for partial digestion of bovine tissue is proposed using polytetrafluoroethylene (PTFE) microvessels inside a baby-bottle sterilizer under microwave radiation for multi-element determination by inductively coupled plasma optical emission spectrometry (ICP OES). Samples were directly weighed in laboratory-made polytetrafluoroethylene vessels. Nitric acid and hydrogen peroxide were added to the uncovered vessels, which were positioned inside the baby-bottle sterilizer, containing 500 mL of water. The hydrogen peroxide volume was fixed at 100 mu L The system was placed in a domestic microwave oven and partial digestion was carried out for the determination of Ca, Cu, Fe. Mg, Mn and Zn by inductively coupled plasma optical emission spectrometry. The single-vessel approach was used in the entire procedure, to minimize contamination in trace analysis. Better recoveries and lower residual carbon content (RCC) levels were obtained under the conditions established through a 2(4-1) fractional factorial design: 650 W microwave power, 7 min digestion time, 50 mu L nitric acid and 50 mg sample mass. The digestion efficiency was ascertained according to the residual carbon content determined by inductively coupled plasma optical emission spectrometry. The accuracy of the proposed procedure was checked against two certified reference materials. (C) 2009 Elsevier B.V. All rights reserved.

Adapting the CROPGRO perennial forage model to predict growth of Brachiaria brizantha

Warm-season grasses are economically important for cattle production in tropical regions, and tools to aid in management and research of these forages would be highly beneficial. Crop simulation models synthesize numerous physiological processes and are important research tools for evaluating production of warm-season grasses. This research was conducted to adapt the perennial CROPGRO Forage model to simulate growth of the tropical species palisadegrass [Brachiaria brizantha (A. Rich.) Stapf. cv. Xaraes] and to describe model adaptation for this species. In order to develop the CROPGRO parameters for this species, we began with values and relationships reported in the literature. Some parameters and relationships were calibrated by comparison with observed growth, development, dry matter accumulation and partitioning during a 2-year experiment with Xaraes palisadegrass in Piracicaba, SP, Brazil. Starting with parameters for the bahiagrass (Paspalum notatum Flugge) perennial forage model, dormancy effects had to be minimized, and partitioning to storage tissue/root decreased, and partitioning to leaf and stem increased to provide for more leaf and stem growth and less root. Parameters affecting specific leaf area (SLA) and senescence of plant tissues were improved. After these changes were made to the model, biomass accumulation was better simulated, mean predicted herbage yield per cycle was 3573 kg ha(-1), with a RMSE of 538 kg DM ha(-1) (D-Stat = 0.838, simulated/observed ratio = 1.028). The results of the adaptation suggest that the CROPGRO model is an efficient tool to integrate physiological aspects of palisadegrass and can be used to simulate growth. (C) 2010 Elsevier B.V. All rights reserved.