Post-ruminal protein supply and N retention of weaner sheep fed on a basal diet of lucerne hay (Medicago sativa) with increasing levels of quebracho tannins

The ability of low to moderate levels (

Biotoxicity assays of quantum dots in in vitro cultures of Medicago sativa and Medicago truncatula

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

The impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension culture

Abstract Background: Nanotechnology has the potential to provide agriculture with new tools that may be used in the rapid detection and molecular treatment of diseases and enhancement of plant ability to absorb nutrients, among others. Data on nanoparticle toxicity in plants is largely heterogeneous with a diversity of physicochemical parameters reported, which difficult generalizations. Here a cell biology approach was used to evaluate the impact of Quantum Dots (QDs) nanocrystals on plant cells, including their effect on cell growth, cell viability, oxidative stress and ROS accumulation, besides their cytomobility. Results: A plant cell suspension culture of Medicago sativa was settled for the assessment of the impact of the addition of mercaptopropanoic acid coated CdSe/ZnS QDs. Cell growth was significantly reduced when 100 mM of mercaptopropanoic acid -QDs was added during the exponential growth phase, with less than 50% of the cells viable 72 hours after mercaptopropanoic acid -QDs addition. They were up taken by Medicago sativa cells and accumulated in the cytoplasm and nucleus as revealed by optical thin confocal imaging. As part of the cellular response to internalization, Medicago sativa cells were found to increase the production of Reactive Oxygen Species (ROS) in a dose and time dependent manner. Using the fluorescent dye H2DCFDA it was observable that mercaptopropanoic acid-QDs concentrations between 5-180 nM led to a progressive and linear increase of ROS accumulation. Conclusions: Our results showed that the extent of mercaptopropanoic acid coated CdSe/ZnS QDs cytotoxicity in plant cells is dependent upon a number of factors including QDs properties, dose and the environmental conditions of administration and that, for Medicago sativa cells, a safe range of 1-5 nM should not be exceeded for biological applications.

Efeitos do enxofre e do boro e da inoculação em alfafa (Medicago sativa L.)

This paper deals with the results of a pot and plot experiment which was carried out to determine the influence of sulphur and boron and the effect seed inoculation with Rhizobium meliloti in the yield of alfafa. Sulphur was applied as flower of sulphur at the rates of 1,000 and 2,000 kg por hectare; boron was employed in the proportion of 15 kg of borax per hectare; both sulphur and boron were distributed broadcast before planting; the experimental design chosen for the field trial was a latin square of 6 x 6 with the following treatments: Number Treatment 1 Control 2 One dosis S + inoculation 3 Two dosis S +inoculation 4 One dosis S + B + inoculation 5 B + inoculation 6 inoculation The crop supplied four cuttings in an eleven months period. The pot experiment nearly confirmed the plot one. The following conclusions can be drawn: 1. The classification of treatments in a decrescent order was: l.o - two doses S + inoculation; 2.o - one dosis S +inoculation, S + B + inoculation, and B + inoculation (these treatmente were not statistically different); 3.o - control; 4.o - inoculation; 2 The vield due to the treatment two dosis S + inoculation was 22 per cent higher than the control one, a fact that suggests that the S supply in the soil studied ("terra roxa misturada") is not sufficient for the total requirements of alfafa; 3. From an economical point of view the best treatment was: one dosis B + inoculation which permits a net gain of Cr$ 12.527,30 per hectare per year; 4. Based on the mentioned results we recommend in soils of same type the following fertilization for alfafa. 5 tons limestone/hectare 300 kg serranafosfato and 600 kg hiperfosfato/ha 300 kg muriate of potash/ha 15 kg borax/ha and a medium organic manuring if the soil is very poor in organic matter.

On the relationship between C and N fixation in nodulated alfalfa (Medicago sativa)

Legumes such as alfalfa (Medicago sativa L.) are vital N2-fixing crops accounting for a global N2 fixation of ~35 MtNyear-1. Although enzymatic and molecular mechanisms of nodule N2 fixation are now well documented, some uncertainty remains as to whether N2 fixation is strictly coupled with photosynthetic carbon fixation. That is, the metabolic origin and redistribution of carbon skeletons used to incorporate nitrogen are still relatively undefined. Here, we conducted isotopic labelling with both 15N2 and 13C-depleted CO2 on alfalfa plants grown under controlled conditions and took advantage of isotope ratio mass spectrometry to investigate the relationship between carbon and nitrogen turn-over in respired CO2, total organic matter and amino acids. Our results indicate that CO2 evolved by respiration had an isotopic composition similar to that in organic matter regardless of the organ considered, suggesting that the turn-over of respiratory pools strictly followed photosynthetic input. However, carbon turn-over was nearly three times greater than N turn-over in total organic matter, suggesting that new organic material synthesised was less N-rich than pre-existing organic material (due to progressive nitrogen elemental dilution) or that N remobilisation occurred to sustain growth. This pattern was not consistent with the total commitment into free amino acids where the input of new C and N appeared to be stoichiometric. The labelling pattern in Asn was complex, with contrasted C and N commitments in different organs, suggesting that neosynthesis and redistribution of new Asn molecules required metabolic remobilisation. We conclude that the production of new organic material during alfalfa growth depends on both C and N remobilisation in different organs. At the plant level, this remobilisation is complicated by allocation and metabolism in the different organs. Additional keywords: carbon exchange, carbon isotopes, nitrogen fixation, nitrogen 15 isotope

Concerted changes in N and C primary metabolism in alfalfa (Medicago sativa) under water restriction

Although the mechanisms of nodule N2 fixation in legumes are now well documented, some uncertainty remains on the metabolic consequences of water deficit. In most cases, little consideration is given to other organs and, therefore, the coordinated changes in metabolism in leaves, roots, and nodules are not well known. Here, the effect of water restriction on exclusively N2-fixing alfalfa (Medicago sativa L.) plants was investigated, and proteomic, metabolomic, and physiological analyses were carried out. It is shown that the inhibition of nitrogenase activity caused by water restriction was accompanied by concerted alterations in metabolic pathways in nodules, leaves, and roots. The data suggest that nodule metabolism and metabolic exchange between plant organs nearly reached homeostasis in asparagine synthesis and partitioning, as well as the N demand from leaves. Typically, there was (i) a stimulation of the anaplerotic pathway to sustain the provision of C skeletons for amino acid (e.g. glutamate and proline) synthesis; (ii) re-allocation of glycolytic products to alanine and serine/glycine; and (iii) subtle changes in redox metabolites suggesting the implication of a slight oxidative stress. Furthermore, water restriction caused little change in both photosynthetic efficiency and respiratory cost of N2 fixation by nodules. In other words, the results suggest that under water stress, nodule metabolism follows a compromise between physiological imperatives (N demand, oxidative stress) and the lower input to sustain catabolism.

Uso de técnicas isotópicas de 15 N aplicadas al estudio de la fijación biológica de nitrógeno en el cultivo de alfalfa (Medicago sativa L.) en La Comarca Lagunera, México

Tesis (Maestría en Ciencias con Especialidad en Microbiología) UANL

Ruminal dry matter and nitrogen degradation in relation to condensed tannin and protein molecular structures in sainfoin (Onobrychis viciifolia) and lucerne (Medicago sativa).

Sainfoin is a temperate legume that contains condensed tannins (CT), i.e. polyphenols that are able to bind proteins and thus reduce protein degradation in the rumen. A reduction in protein degradation in the rumen can lead to a subsequent increase in amino acid flow to the small intestine. The effects of CT in the rumen and the intestine differ according to the amount and structure of CT and the nature of the protein molecular structure. The objective of the present study was to investigate the degradability in the rumen of three CT-containing sainfoin varieties and CT-free lucerne in relation to CT content and structure (mean degree of polymerization, proportion of prodelphinidins and cis-flavanol units) and protein structure (amide I and II bands, ratio of amide I-to-amide II, α-helix, β-sheet, ratio of α-helix-to-β-sheet). Protein molecular structures were identified using Fourier transform/infrared-attenuated total reflectance (FT/IR-ATR) spectroscopy. The in situ degradability of three sainfoin varieties (Ambra, Esparcette and Villahoz) was studied in 2008, during the first growth cycle at two harvest dates (P1 and P2, i.e. 5 May and 2 June, respectively) and at one date (P3) during the second growth cycle (2 June) and these were compared with a tannin-free legume, lucerne (Aubigny). Loss of dry matter (DMDeg) and nitrogen (NDeg) in polyester bags suspended in the rumen was measured using rumen-fistulated cows. The NDeg of lucerne compared with sainfoin was 0·80 v. 0·77 at P1, 0·78 v. 0·65 at P2 and 0·79 v. 0·70 at P3, respectively. NDeg was related to the rapidly disappearing fraction (‘a’) fraction (r=0·76), the rate of degradation (‘c’) (r=0·92), to the content (r=−0·81) and structure of CT. However, the relationship between NDeg and the slowly disappearing fraction (‘b’) was weak. There was a significant effect of date and species×date, for NDeg and ‘a’ fraction. The secondary protein structure varied with harvest date (species×date) and was correlated with the fraction ‘b’. Both tannin and protein structures influenced the NDeg degradation. CT content and structure were correlated to the ‘a’ fraction and to the ‘c’. Features of the protein molecular secondary structure were correlated to the ‘b’ fraction.

Melhoramento genético de leguminosas de clima temperado - alfafa (Medicago sativa L) e cornichão (Lotus corniculatus L) para aptidão ao pastejo

O presente trabalho foi realizado com o objetivo de avaliar e selecionar genótipos de alfafa crioula e de cornichão cultivar São Gabriel para aptidão ao pastejo. Neste trabalho, foi proposto o termo aptidão ao pastejo em substituição à resistência ao pastejo. O procedimento padrão utilizado no processo de seleção e de caracterização dos genótipos constou de experimentos nos quais as plantas eram submetidas a uma pressão de pastejo elevada e contínua (3-5 cm de altura do resíduo). Houve diferença (P<0,05) na sobrevivência das diferentes populações de alfafa crioula, evidenciando variabilidade para a aptidão ao pastejo, o que não se evidenciou de forma consistente para o cornichão, que, no entanto, apresentou uma maior porcentagem de sobrevivência em comparação com a alfafa. A avaliação das características morfofisiológicas em alfafa não evidenciou diferenças (P>0,05) na contração das coroas, área da coroa e da raiz, área foliar residual e específica, número de hastes por planta, tipo de haste predominante (basilar ou axilar) e reservas orgânicas. Foram evidenciadas diferenças (P<0,05) no comprimento do entrenó e no índice de gemas específicas, proposto neste trabalho como descritor funcional da aptidão ao pastejo. Ensaios em casa-de-vegetação identificaram um marcador morfológico (altura do primeiro nó) nas plântulas de alfafa e de cornichão, capaz de separar precocemente os genótipos contrastantes quanto à aptidão ao pastejo. Foi comparada a efetividade das estirpes de rizóbio recomendadas para o cornichão com a de novas estirpes isoladas, demonstrando a possibilidade de obtenção de estirpes mais eficientes.

Modelo morfogênico da produção potencial de flores em alfafa (Medicago sativa L.)

Assegurando-se condições não limitantes de radiação incidente, água e nutrientes, acompanhou-se, em casa-de-vegetação, o desenvolvimento de plantas isoladas da cultivar Europe de alfafa, desde a emergência até o estádio primeiros legumes maduros. Medidas semanais permitiram quantificar o ritmo de formação de fitômeros da haste principal e suas ramificações primárias, assim como o surgimento de inflorescências e seu número de flores. Paralelamente, plantas mantidas nas mesmas condições foram amostradas para determinar a evolução e repartição da biomassa formada. Independentemente do tipo de haste, a indução floral provoca uma diminuição na velocidade de emissão de fitômeros concomitante com uma redução progressiva no tamanho da folha e no número de flores por inflorescência. As diferentes hastes (haste principal – HP e as ramificações – R1 a Rn) apresentam comportamento distinto quanto a todos os parâmetros estudados. Com base no filocrono, identificou-se um grupo de maior velocidade formado pela HP, e R1 a R4, enquanto as demais são mais lentas. A velocidade de desenvolvimento foi a variável mais pertinente para explicar a maior biomassa e o maior número de flores produzidas pelas hastes do primeiro grupo. Um modelo de produção de flores por planta baseado na temperatura média diária é proposto, integrando sub-modelos de desenvolvimento de cada grupo de hastes. Validado com dados independentes da mesma cultivar e da cultivar Cinna, o modelo mostrou-se adequado como referencial da produção potencial de flores por planta.

Efeito do emurchecimento e da adição de fubá na degradabilidade in situ da silagem de alfafa (Medicago sativa L.)

O objetivo deste ensaio foi avaliar o efeito do emurchecimento e da adição de fubá durante o processo de ensilagem da alfafa sobre a degradabilidade in situ da matéria seca (MS), da fibra em detergente neutro (FDN) e da proteína bruta (PB). Foram utilizados três bovinos adultos mestiços fistulados no rúmen, nos quais foram incubados seis tipos de silagens (1-alfafa fresca; 2-alfafa fresca +5% de fubá; 3-alfafa fresca +10% de fubá; 4-alfafa emurchecida; 5-alfafa emurchecida + 5% de fubá ; 6-alfafa emurchecida + 10% de fubá) durante três tempos de incubação (6, 24, 96 horas). O delineamento experimental foi em parcelas subdivididas, com tratamentos em parcelas, tempos em subparcelas e animais como blocos. A adição de fubá à silagem aumentou (P<0,05) a degradação da MS e FDN com o tempo de incubação. O emurchecimento proporcionou diminuição (P<0,05) na degradabilidade dos nutrientes, entretanto esse efeito foi ligeiramente contornado pela adição de 10% de fubá. Os tratamentos com a adição de fubá e o emurchecimento proporcionaram maior taxa de degradabilidade da MS de fração b, entretanto o emurchecimento diminuiu o potencial de degradação a, enquanto que a adição de níveis de fubá não alterou a degradabilidade da MS dessa fração. A degradação da fração b da FDN aumentou com a adição de fubá e diminuiu com o emurchecimento, entretanto houve aumento dessa fração quando se adicionou fubá à forragem emurchecida. A degradação da fração a da PB aumentou com a adição de fubá e diminuiu com o emurchecimento; para a fração b ocorreu o inverso. Concluiu-se que a prática do emurchecimento, por reduzir a fração solúvel da forragem, diminui a degradabilidade dos nutrientes da silagem de alfafa, que no entanto pode ser melhorada com a adição de fubá.

Avaliação do Potencial para Ensilagem de Cultivares de Alfafa (Medicago sativa L.)

The objective of this trial was to study the ensilage potential of alfalfa (Medicago sativa L,.). The material used in the evaluation was harvested from an experiment in a complete randomized blocks design with three replicates, with seventeen alfalfa cultivars, Criouil, Florida 77, P555, P581, Moapa, Gilboa, 34 Linea, C/W 8754, C/W 4468, C/W 86, C/W 8746, Hunter Field, Trifecta, Sequel, CUF 101, Aurora e Siriver, during three years. The harvest used for the determinations was collected at 30(th) day of plants development and the material was submitted to two treatments: no wilting - Fresh alfalfa frozen immediately after harvest; wilting fresh alfalfa exposed to sun by three hours to get 35% DM. The dry matter content (DM), water soluble carbohydrates (WSC) and buffer capacity (BC) were evaluated in rhp forages Wilting increased the dry matter content (39,98%), however there were not differences among cultivars. The same occurred for the WSC content, with interaction among cultivars and treatments. The alfalfa cultivars, when submitted to effective wilting practice, presented appropriated characteristics of dry matter and water-soluble carbohydrates for ensilage. Wilting had no effect on alfalfa buffer capacity.

Produção e Distribuição de Matéria Seca e Composição Bromatológica de Cultivares de Alfafa (Medicago sativa L.)

The objective of this research was to evaluate dry matter production (DMP), seasonal distribution (SD) and chemical composition of crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) of seventeen alfalfa cultivates: Creole, Florida 77, P555, P581, Moapa, Gilboa, 34 Linea, C/W 8754, C/W 4468, C/W 86, C/W 8746, Hunter Field, Trifecta, Sequel, CUF 101, Aurora e Siriver. A complete randomized blocks experimental design, with three replicates in a split plot on time scheme, was used. For DMP and SD, the cultivates were in the main plot and cuts in the subplots. For chemical composition, cultivates were also the main plots, and seasons of the year, summer and winter, the subplots. There were no differences among cultivates for the annual average dry matter production (12,08 t/[ha.year]) and SD (35% in the winter). There were differences in the chemical composition, among cultivates for CP and significant interaction among cultivates and the seasons of year for NDF and ADF. All cultivates showed similar potential production and seasonal distribution of dry matter. The alfalfa quality in the winter was better than in the summer.

Influência do método de fenação e tempo de armazenamento sobre a composição bromatológica e ocorrência de fungos no feno de alfafa (Medicago sativa, L. cv. Flórida 77)

O experimento objetivou estudar a influência de 24 tratamentos resultantes do arranjo fatorial 6 x 4, referente a seis métodos de fenação e quatro tempos de armazenamento em delineamento inteiramente casualizado, com cinco repetições para análise bromatológica e quatro para determinação de fungos. Os métodos de fenação consistiram em: T1 - (sombra, E) secagem à sombra, a forrageira foi levada para o galpão imediatamente após o corte e mantida espalhada (E); T2 e T3 - o material permaneceu ao sol até perder 50% do peso e posteriormente foi seco à sombra, sendo o T2 espalhado (sol 50%, E) e o T3 amontoado - A (sol 50%, A); T4 e T5, consistiram do recolhimento da forragem para completar sua secagem à sombra, após viragem e perda de 60% de peso sob exposição ao sol, T4 espalhada (sol 60%, E), T5 amontoada (sol 60%, A) e o T6 perda de 80% do peso sob exposição ao sol e posteriormente amontoado à sombra (sol 80%, A). Os quatro tempos de armazenamento foram: 0, 15, 30 e 60 dias. Verificou-se que os fenos nos quais o material ficou mais tempo exposto ao sol apresentaram pior qualidade em termos de PB, FDN e FDA. A ocorrência de fungos foi maior no feno seco à sombra (T1) e naqueles em que a forragem foi amontoada, devido à ausência de aeração para retirada de umidade. O método mais adequado para a conservação da alfafa consistiu na exposição da forragem ao sol até a perda de 50% do peso da forragem original, com posterior secagem do material espalhado à sombra.