Morphology and morphometry of feline bone marrow-derived mesenchymal stem cells in culture

Mesenchymal stem cells (MSC) are increasingly being proposed as a therapeutic option for treatment of a variety of different diseases in human and veterinary medicine. Stem cells have been isolated from feline bone marrow, however, very few data exist about the morphology of these cells and no data were found about the morphometry of feline bone marrow-derived MSCs (BM-MSCs). The objectives of this study were the isolation, growth evaluation, differentiation potential and characterization of feline BM-MSCs by their morphological and morphometric characteristics. in vitro differentiation assays were conducted to confirm the multipotency of feline MSC, as assessed by their ability to differentiate into three cell lineages (osteoblasts, chondrocytes, and adipocytes). To evaluate morphological and morphometric characteristics the cells are maintained in culture. Cells were observed with light microscope, with association of dyes, and they were measured at 24, 48, 72 and 120h of culture (P1 and P3). The non-parametric ANOVA test for independent samples was performed and the means were compared by Tukey's test. On average, the number of mononuclear cells obtained was 12.29 (±6.05x10(6)) cells/mL of bone marrow. Morphologically, BM-MSCs were long and fusiforms, and squamous with abundant cytoplasm. In the morphometric study of the cells, it was observed a significant increase in average length of cells during the first passage. The cell lengths were 106.97±38.16µm and 177.91±71.61µm, respectively, at first and third passages (24 h). The cell widths were 30.79±16.75 µm and 40.18±20.46µm, respectively, at first and third passages (24 h).The nucleus length of the feline BM-MSCs at P1 increased from 16.28µm (24h) to 21.29µm (120h). However, at P3, the nucleus length was 26.35µm (24h) and 25.22µm (120h). This information could be important for future application and use of feline BM-MSCs.

Reproductive efficiency of asymptomatic Theileria equi carriers mares submitted to an embryo transfer program

This study aimed to assess and evaluate the effects of Theileria equi infection on embryonic recovery, gestation and early embryonic loss. Thirteen Mangalarga Marchador Theileria equi positive donors (diagnosed through nested-PCR) and 40 embryos receptors were used. Donors were submitted to two embryo collections in two consecutive estrous cycles (GId); after, the same mares were treated with imidocarb dipropionate (1.2mg/kg IM.) in order to collect more embryos in two more estrous cycles (GIId). Receptors were divided into two groups (control and with treated) with 20 animals each, where one group was the control (GIr) and the other one (GIIr) treated with 1.2mg/kg IM of imidocarb dipropionate assessing the gestation rate at 15, 30, 45 and 60 days. After 52 embryo collections, the embryonic recovery rates were 53.84% (14/26) and 65.38% (17/26) (p> 0.05) for GId and GIId, respectively. The gestation rate was 70% (14/20) (p>0.05) at 15, 30, 45 and 60 days in group GIr and for GIIr was 85% (17/20) (p>0.05) at 15 days, 80% (16/20) (p>0.05) at 30, 45 and 60 days. The treatment with imidocarb dipropionate did not cause significant improvement in the reproductive efficiency at an ET program.

Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos

Abstract In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation.

Density and sowing season of two Brachiaria species on the soybean culture

The aim of this study was to evaluate the growth and yield of soybean cultivar M-8766 in consortium with Brachiaria brizantha. BRS Piata and Brachiaria ruziziensis at different densities and sowing dates. The experimental design was randomized blocks with treatments arranged in a factorial 2 x 2 x 3 with four replications. Used as factors grass species (Brachiaria brizantha Piata and Brachiaria ruziziensis BRS) intercropped with soybean M-8766, sowing dates (12 and 24 days after soybean emergence) and three seeding rates (0, 5, 10 kg ha-1 of seed). At 71 days after soybean emergence were evaluated plant height, stem diameter, dry mass of leaves, stems and shoots, and 4 months after sowing determined the weight of 100 grains and soybean yield. The results showed that when seeded at a density of 10 kg ha-1 at 12 and 24 DAE soy, Brachiaria brizantha. BRS Piata caused reduction in yield in the order of 6.71% and 3.03% respectively, while the Brachiaria ruziziensis was one that caused a greater reduction in productivity in the order of 13.42 and 16.23%, respectively, of these values expression when considering the price of soybean sack. B. ruziziensis expressed less competitive with soybean. However, the large biomass production of this grass provides deployment system till the next harvest.

Adaptive value of ryegrass biotypes with low-level resistance and susceptible to the herbicide fluazifop and competitive ability with the wheat culture

Ryegrass is the main weed in wheat crop, causing yield loss due to competition by environmental resources. The objectives of this study were to estimate the fitness cost of ryegrass biotypes with low-level resistance and susceptible to fluazifop and to investigate the relative competitive ability of these biotypes between themselves and against the crop. Thus, fitness cost and competitive ability experiments were conducted under greenhouse conditions. For the fitness cost experiments, the low-level resistant ryegrass biotypes and those susceptible to fluazifop were used. For competitive ability, the treatments were arranged in a replacement series, with five proportions of the wheat cultivar FUNDACEP Horizonte and the low-level resistant and susceptible ryegrass biotypes 100:0, 75:25, 50:50, 25:75 and 0:100. Competitive analysis was carried out through diagrams applied to the replacement experiments and use of relative competitiveness indices. Variables evaluated were plant height, in the fitness cost experiment, and leaf area and shoot dry biomass in both experiments. The ryegrass biotypes show overall similar fitness cost and competitive ability. The wheat cultivar FUNDACEP Horizonte is superior in competitive ability to the ryegrass biotype with low-level resistance and equivalent to the susceptible biotype.

Infestation maps and spatial stability of main weed species in maize culture

A study on the spatial distribution of the major weeds in maize was carried out in 2007 and 2008 in a field located in Golegã (Ribatejo region, Portugal). The geo-referenced sampling focused on 150 points of a 10 x 10 m mesh covering an area of 1.5 ha, before herbicide application and before harvest. In the first year, 40 species (21 botanical families) were identified at seedling stage and only 22 during the last observation. The difference in species richness can be attributed to maize monoculture favouring reduction in species number. Three of the most representative species were selected for the spatial distribution analysis: Solanum nigrum, Chenopodium album and Echinochloa crus-galli. The three species showed an aggregated spatial pattern and spatial stability over both years, although the herbicide effect is evident in the distribution of some of them in the space. These results could be taken into account when planning site-specific treatments in maize.

In vitro culture of Phyllanthus stipulatus (Euphorbiaceae)

An efficient micropropagation protocol was developed for the medicinal plant Phyllanthus stipulatus (Euphorbiaceae) using nodal segments for axillary shoot proliferation. Maximum multiplication rates (8-9 shoots per explant) was achieved on MS media supplemented with either 2.5-5.0 muM IBA. The best basal media for axillary shoot proliferation when 0.62 muM BA was supplemented were MS, MS/2 and AR (4-5 shoots per explant). Rooting was achieved with 100% of the microshoots on MS medium without growth regulators. Regenerated plants were successfully acclimatized and about 88% of plantlets survived under ex vitro conditions. Flowering was observed in 81% of the ex vitro grown plantlets after 12 weeks of acclimatization. High frequency callus initiation and growth was achieved when nodal segment explants were inoculated either in the vertical position, in the light on MS medium supplemented with 5.0 muM NAA or horizontally oriented, in the dark on MS supplemented with 5.0 muM NAA or 1.25-5.0 muM BA or 2iP. Root cultures were successfully established on MS medium containing 1.1 muM NAA. The optimized micropropagation, callus and root culture protocols offer the possibility to use cell/organ culture techniques for vegetative propagation and secondary metabolism studies.

Histological changes in banana explants, cv. Nanicão (Musa spp., Group AAA), submitted to different auxins for induction of somatic embryogenesis

The present work analyzes the behavior of banana explants, cv. Nanicão (Musa spp. Group AAA) regarding somatic embryogenesis induction treatments with several auxins. Longitudinal segments of shoot meristematic apices of micropropagated banana plantlets cultivated and rooted in vitro were introduced in culture medium containing dicamba, picloram, 2,4-D or NAA in different concentrations. Explant samples were collected at 0, 7 and 10 days and prepared for light microscopy. Histological sections were used for comparison of the histological changes occurring after induction treatment with different auxins. Embryogenic response was observed only in treatments with picloram or dicamba, with distinct embryogenic regions observed at 14 and 21 days in culture, respectively. Histological sections of embryogenic regions of the explant at 26 days in culture revealed the formation of meristematic regions, structures with multiple root meristems, and somatic embryos at early globular stages. Embryo-like structures morphologically similar to Musa balbisiana zygotic embryos were sectioned and showed a lack of apical meristems and absence of procambial differentiation. These results indicate the induction of non-functional somatic embryos and the need for more studies on developmental aspects and maturation treatments for optimization of the process.

Ovule ontogeny of Tabebuia pulcherrima Sandwith (Bignoniaceae): embryo sac development

The chalazal megaspore develops in a Polygonum-type embryo sac. The amyloplast-rich endothelium is partially degraded during the expansion of the micropylar portion of the megagametophyte. Organization of the mature embryo sac is determined by the patterns of vacuolation, nuclear migration, spindle orientation and cellularization. The egg cell is slightly chalazal in relation to the synergids, and its micropylar end does not touch the micropylar channel. At the chalazal pole of the egg apparatus, the common walls between the synergids, the egg and central cells, despite their tenuity, are present in the mature megagametophyte. The polar nuclei do not fuse before fertilization and the antipodals are persistent until the first stages of endosperm formation. The taxonomic significance of some embryological characters for the Bignoniaceae is discussed.

Effect of abscisic acid on the mobilisation of galactomannan and embryo development of Sesbania virgata (Cav.) Pers. (Leguminosae - Faboideae)

Galactomannans (GM) are storage cell wall polysaccharides present in endospermic seeds of legumes. They are thought to be storage polymers, since it has been observed for a few species (among them Sesbania virgata) that they are completely broken down after germination and their products are transferred to the growing embryo. We examined the effect of 10-4 M abscisic acid (ABA) on the degradation of galactomannan in isolated endosperms and intact seeds of S. virgata. We found that after seed germination the initial embryo growth was retarded. Ultrastructural analysis showed that the embryo is completely surrounded by an endosperm which displays very thick galactomannan-containing cell walls. Although an inhibitory effect has been observed on the increase of fresh mass of the embryo, the effect of ABA on the dry mass was weaker and transitory (from 48 to 96 h). Endosperm dry mass and galactomannan degradation were significantly inhibited and the activity of alpha-galactosidase was strongly affected. The addition of ABA before and/or after the start of mobilisation in intact seeds or isolated endosperms, showed that whereas addition before mobilisation did not affect dry mass decrease in intact seeds, it was strongly affected in isolated endosperms. On the other hand, whereas it affected embryo fresh mass increase in intact seeds, but not in isolated embryos, no significant effect was observed on dry mass. These results suggest that ABA affects galactomannan degradation and by doing so, prevents water absorption by the embryo, rather than affect its dry mass. As ABA has been detected in the endosperm of seeds of S. virgata, it is proposed that it probably acts as a modulator of galactomannan mobilisation and consequently synchronises it with early growth of the embryo.

Effects of iron-ore particles on propagule release, growth and photosynthetic performance of Sargassum vulgare C. Agardh (Phaeophyta, Fucales)

The effect of iron-ore particles on the propagule release and growth of Sargassum vulgare C. Agardh was tested under treatments with different concentrations of iron-ore particles: 0.1, 1.0, 10.0 g.L-1 and a solution of 10.0 g.L-1 of filtered iron-ore. Filtered seawater was used as control. Photosynthesis vs. irradiance (P-I) curves were calculated for S. vulgare in the presence of iron-ore and in seawater. There was no significant difference in the number of propagules released by the receptacles or in the percentage of zygote formation among the treatments. The released propagules acted like aggregation centers for the particles, those more heavily coated with iron (10.0 g.L-1) exhibiting the highest sinking velocity (32.6 ± 9.8 mm.s-1). No difference in the percentage of embryo survival was detected during the first week in culture. After four weeks the embryos grew in all treatments. Maximum frond development (5.3 ± 0.8 mm) was observed in treatment of seawater enriched with Provasoli's medium (PES) while initial filoids did not develop in three treatments without PES and with iron-ore (0.1 g.L-1, 1.0 g.L-1 and 10.0 g.L-1). The values for Pmax, alpha and respiration showed no significant differences between the P-I curves. The calculated value for I K was 106.26 µmol.m-2.s-1 to the control curve and 981.49 µmol.m-2.s-1 to the iron-ore curve. The results indicate that the iron-ore particles in high concentration reduce the growth of S. vulgare as they recovered the embryos, juveniles and young plants. In contrast, the presence of the particles did not affect the release of gametes, percentage of zygote formation or the percentage of embryo survival.

In vitro somatic embryogenesis and adventitious root initiation have a common origin in eggplant (Solanum melongena L.)

Somatic embryogenesis was induced from cotyledon explants of eggplant cultured on MS medium supplemented with 54 µM NAA. Anatomical analysis of somatic embryo initiation and development was performed during the first four weeks. Proembryo formation was observed after the second day of culture, directly from perivascular cells or via pro-embryogenic masses derived from indeterminate meristematic masses (IMMs) originated in the vascular tissue. Those IMMs also gave rise to root primordia after 10 days of culture. The origin of embryos is discussed as well as the similarities between somatic embryogenesis and adventitious root formation.

Anomalous somatic embryos in Acca sellowiana (O. Berg) Burret (Myrtaceae)

Somatic embryogenesis represents a valuable tool for the studies on the basic aspects of plant embryo development. Today this process is used as a potencial technique for large-scale plant micropropagation although, so far, it has been applied to only a small number of species. However, when somatic embryos are malformed they are considered economically useless. In Acca sellowiana (O. Berg) Burret, an important fruit-producing crop, large amounts of anomalous somatic embryos (76.3%) were found just after 40 days of culture of explants in a 2,4-D containing medium. Among the anomalous forms found in the cotiledonary stage, 12.2% consisted of fused embryos, 40.4% displayed fused cotyledons, 13.0% presented supernumerary cotyledons, and 10.7% showed absence or poorly developed cotyledons, including those without the shoot apical meristem. Histological analyses indicated that the altered embryos were formed either directly from cotyledons, hypocotyl and radicle of the zygotic embryos used as explants, or indirectly from calli formed from these tissue parts. It is suggested that the formation of anomalous somatic embryos, as well as a low frequency of conversion into emblings reflect physiological and/or genetic disturbances triggered by the presence of 2,4-D in the medium. In vitro experimental alternative approaches are discussed in order to lessen the occurrence of malformed somatic embryos.

In vitro culture at low temperature and ex vitro acclimatization of Vriesea inflata an ornamental bromeliad

(In vitro culture at low temperature and ex vitro acclimatization of Vriesea inflata an ornamental bromeliad). In vitro culture by seeds is a technique for preservation of threatened species because it may provide a large number of plants with genetic diversity. The bromeliad Vriesea inflata (Wawra) Wawra, an ornamental bromeliad, is extensively and illegally collected from the nature and must be preserved. It is possible to form plant threatened collections in vitro by reducing the temperature of culture, while occupying little space, with the consequent reduction of maintenance costs. This work evaluated the influence of temperature on in vitro growth and morphology of plants of V. inflata, with the aim of establishing a slow growth-rate and analyzing the ex vitro acclimatization. Seeds were germinated in vitro in Murashige and Skoog (MS) medium, with macronutrients reduced to 50% (MS/2). After three months the plants were transferred to flasks of new same medium and kept in two germination chambers with the temperature adjusted to 15 °C and to 28 °C. After 24 months the plants were subject to biometric, photosynthetic pigments content and anatomical analyses. Results showed that plants maintained at 15 °C were smaller than those at 28 °C. Nevertheless, there were no alterations in pigments content, anatomy. In both treatments there was a survival rate of 100%. This work showed that plants of this species can be kept in vitro at 15 °C with the aim of forming a slow-growth collection, thereby seeking its preservation, and can be transferred to growth at ex vitro condition to achieved 100% survival rate.

Intra and interspecific variability of in vitro culture response in Lycopersicon (tomatoes)

Intra and interspecific variability was measured in the genus Lycopersicon for the traits: productivity rate (PR, total number of regenerated shoots/total number of cultures), regeneration percentage (%R, number of cultures regenerating shoots or primordia/total number of cultures) and callus percentage (%C, number of cultures only producing callus/total number of cultures). Leaf explants from various genotypes of L. esculentum, L. esculentum var. cerasiforme, L. pimpinellifolium and L. peruvianum were placed on Murashige and Skoog (Physiol. Plant. 15: 473-493, 1962) medium + 0.175 mg/l IAA + 2.25 mg/l BA. Significant differences among species and among genotypes within the same species were found, while genotypes from different species showed similar responses.