Effects of Growth Hormone on In Situ Culture of Bovine Preantral Follicles are Dose Dependent

The objective of this study was to evaluate different concentrations of growth hormone (GH) on the development of bovine preantral follicles cultured included in the ovarian tissue (in situ) on the rates of morphologically normal, viable, primordial and developing follicles, as well as the oocyte and follicle diameter and ultrastructural analysis. Ovarian fragments collected from cows with no cross‐breeds defined were cultured in situ for 1 and 7 days in minimal essential medium (α‐MEM+) supplemented with different concentrations of recombinant human GH (0, 10, 25, 50 ng/ml). The ovarian fragments non‐cultured (control) and cultured were processed for classic histology, mechanical isolation and electron transmission microscopy (MET). The parameters underwent anova (Tukey′s and Dunnett′s tests) and chi‐square test (χ²). After 7 days of culture, the treatment with 50 ng/ml GH showed no differences with fresh control (p > 0.05) and had greater effectiveness than in the 0, 10 and 25 ng/ml GH concentrations of the morphologically normal follicles. Regarding the primordial follicles, a reduction was observed in the 50 ng/ml GH concentration concomitant with the significant increase in developing follicles, differing from both the fresh control and the other GH concentrations tested. In addition, 50 ng/ml GH showed a larger follicle and oocyte diameter when compared to the other treatments cultured. Similar structures were ultrastructurally observed in the control group, 50 ng/ml GH. Follicles cultured in 10 ng/ml GH showed nuclear invagination, vacuoles and lesioned basal membrane. Hence, it is concluded that 50 ng/ml GH is the most effective concentration for the development of preantral follicles cultured in situ.     

Automated strong cation exchange extraction of fatty acid esters of 3-(N-phenylamino)-1,2-propanediol from oil samples for routine quantification by HPLC-APCI/MS/MS.

Fatty acid esters of 3-(N-phenylamino)-1,2-propanediol are currently considered the best chemical markers of toxic oils related to the Spanish toxic oil syndrome. Recent research in this area has undertaken the exhaustive and quantitative characterization of these compounds in oils collected during the epidemic outbreak. Current methods developed in this laboratory are based on solid phase extraction (SPE) using SCX cartridges followed by HPLC-APCI/MS/MS quantification. To circumvent the long and tedious extraction procedure, the SPE protocol was adapted for automatic extraction and the problems derived from the use of the immiscible solvents required for the SCX extraction were solved. Linearity of the analytical method was found in the same range as for the manual method. Extraction recoveries were 87 and 75% for 2-hydroxy-3-(N-phenylamino)propyl linoleate and 2-(linoleyloxy)-3-(N-phenylamino)propyl linoleate, respectively, and the corresponding coefficients of variation were approximately 1%, greatly improving reproducibility over manual procedures.     

Habitat selection and conservation of an endemic spanish lizard Algyroides marchi (reptilia, lacertidae)

We study the habitat selection of Algyroides marchi, a small lizard endemic to the southeastern mountains of the Iberian Peninsula whose populations are concentrated in a few small localities. Three spatial scales of study were used to analyse the different factors affecting the species distribution patterns: regional scale, local scale and within-habitat use of different substrates. The results obtained at these different spatial scales showed a high consistency, suggesting that the same ecological pressures probably acted at different scales. Localities occupied by A. marchi were characterized by high altitude, high geomorphological complexity, northern aspect, high cover of large rocks, and presence of water. Density of A. marchi in a favourable locality was 213 adult-subadult lizards/ha. Abundance and use of space of A. marchi (at local and individual levels) were directly associated with extent of water (streams and small pools) and the presence of large rocks, and inversely with hours of direct solar radiation and cover of small stones and vegetation. Considering the habitat selection of A. marchi and the management practices within its geographical range, potential risks for the conservation of the species are identified.     

Effects of IGF‐1 on In Vitro Culture of Bovine Preantral Follicles are Dose‐Dependent

This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 (IGF‐1) in the development, survival and ultrastructure of the bovine preantral follicles cultured in situ. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7 days in 1 ml of α‐MEM⁺, supplemented with different concentrations of human recombinant IGF‐1 (0, 30, 70 and 100 ng/ml), in an incubator at 37°C and 5% of CO₂ in 24‐well plates with total replacement of the medium every 2 days. Non‐cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7 days were processed for classic histology, mechanical isolation and electron transmission microscopy (ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF‐1 30 ng/ml treatment was similar to the fresh control (p > 0.05) both on the day 1 and on the day 7 of in vitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p > 0.05). After 7 days of culture, the IGF‐1 30 ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p < 0.05). Also, after 7 days of culture, IGF‐1 30 ng/ml presented a higher follicular diameter when compared to the control and other concentrations of IGF‐1 tested. Ultrastructurally, the non‐cultured control and IGF‐1 30 ng/ml, after 7 days of culture, showed conserved oocytes, nuclei and organelles. Hence, it is concluded that IGF‐1 30 ng/ml was the most efficient concentration for the development of bovine preantral follicles cultured in vitro.     

Differential effects of vegetation restoration in Mediterranean abandoned cropland by secondary succession and pine plantations on bird assemblages

Two contrasting trajectories for vegetation restoration in agricultural landscapes are secondary succession following cropland abandonment that can regenerate woodlands (passive restoration) and conversion of cropland to tree plantations (active restoration), which have mostly focused on pine species in the Mediterranean Basin. We compared the effects of these two contrasting trajectories of vegetation restoration on bird assemblages in central Spain. Vegetation structure differed in the two restoration trajectories, pine plantations attaining higher tree cover and height (31% and 4.1 m, respectively) but lower strata complexity than secondary shrubland and holm oak woodland (which attained 10% and 1.4 m of tree cover and height, respectively). Bird species richness differed in stands under active or passive restoration trajectories, the former collecting a higher total number of species (4.2 species per 0.78 ha plot) than the latter (3.5 species per plot). The number of forest species increased with vegetation maturity in both restoration trajectories, but especially in stands under active restoration. The occurrence of woodland generalist species increased and of species inhabiting open habitats decreased in actively restored stands, being some of these latter species of high conservation priority in the European context but relatively common at the regional level. Bird species inhabiting pine plantations had broader habitat breadth at the regional level than those inhabiting secondary shrublands and woodlands. Maximum regional density did not differ between both restoration trajectories, but it increased with development of the herbaceous layer only at the secondary succession trajectory. The relative importance of species of European biogeographic origin was higher in mature pine plantations (58.9% of total bird abundance) than in mature holm oak woodlands (34.4%), whereas that of Mediterranean species was considerably higher in the latter (40.1%) than in the former (20%). Bird assemblages of relatively small patches of pine plantations are unable to reflect the regional avifauna, in contrast with the relationships between local and regional assemblage characteristics that can be found in isolated natural forests. We conclude that programs of vegetation restoration should base upon a range of approaches that include passive restoration, active restoration with a variety of tree and shrub species, and mixed models to conciliate agricultural production, vegetation restoration and conservation of target species.     

Influence of corpus luteum and ovarian volume on the number and quality of bovine oocytes

In order to evaluate whether ovarian volume, presence and diameter of the corpus luteum (CL) have effects on the number and quality of bovine recovered oocytes, 110 ovaries were obtained from the slaughterhouse. Cumulus oocytes complex were aspirated and evaluated under stereomicroscope. Oocytes were counted and classified according to their quality (Grades I, II, III and IV). Ovarian volume was weakly correlated to the number of good quality oocytes (P < 0.05). Ovaries with CL showed greater numbers of good quality oocytes than ovaries without CL (P < 0.05). Further, presence of CL and its diameter positively influenced the probability of recovering good quality oocytes (P < 0.05). In conclusion, ovarian volume is not a good parameter itself to predict important ovarian characteristics; moreover, analysis of CL, its presence and diameter, may be a good tool to improve efficiency on in vitro embryo production programs.     

Assessment of the bacterial community diversity associated with the queen conch Strombus gigas (Linnaeus, 1758) from the Caribbean coast of Colombia using denaturing gradient gel electrophoresis and culturing

The biotic diversity of Strombus gigas has not been thoroughly studied despite the status of the queen conch as an important ecological resource. The bacteria associated with the conch influence their host in several ways, including through the metabolism of nutrients, protection against invasive bacteria and the regulation of the physical conditions. In this study, conventional microbiological methods and molecular tools such as denaturing gradient gel electrophoresis (DGGE) were used to assess the composition of the bacterial communities associated with the queen conch (S. gigas) in wild and captive habitats in Colombia. A genetic analysis of the bacterial communities revealed a high level of diversity based on the large number of bands detected using DGGE. In addition, differences in bacterial community structure were found between the conchs in captivity and the wild populations. The dominant phylogenetic affiliations of the bacteria, as determined using 16S rRNA gene sequencing, were grouped into four classes, namely, Betaproteobacteria (16%), Gammaproteobacteria (70%), Firmicutes (7%) and Actinobacteria (2%). These groups are related to host defence processes and the decomposition of organic matter. The 16S rDNA sequence analysis of the cultured bacteria and the resulting DGGE profiles are useful tools for characterizing the diversity of the bacteria associated with the analyzed conchs.