LH surge in Nelore cows (Bos indicus), after induced estrus or after ovarian superestimulation

Considering that there is limited information about the preovulatory LH surge in Zebu cattle (Bos indicus), the purpose of the present work was to assess the LH surge in Nelore cows during the estrous cycle and after ovarian superestimulation of ovarian follicular development with FSH. This information is particularly important to improve superovulatory protocols associated with fixed-time artificial insemination. Nelore cows (n=12) had their estrus synchronized with an intravaginal device containing progesterone (CIDR-B) associated with estradiol benzoate administration (EB, 2.5 mg, i.m., Day 0). Eight days later all animals were treated with PGF2alpha (Day 8) in the morning (8:00 h) and at night, when CIDR devices were removed (20:00 h). Starting 38h after the first PGF2alpha injection, blood sampling and ovarian ultrasonography took place every 4h, during 37 consecutive hours. Frequent handling may have resulted in a stress-induced suppression of LH secretion resulting in only 3 of 12 cows having ovulations at 46.7+/-4.9 and 72.3+/-3.8 h, respectively, after removal of CIDR-B. Thirty days later, the same animals received the described hormonal treatment associated with FSH (Folltropin), total dose=200 mg) administered twice a day, during 4 consecutive days, starting on Day 5. Thirty-six hours after the first injection of PGF2alpha, to minimize stress, only seven blood samples were collected at 4h interval each, and ultrasonography was performed every 12 h until ovulation. In 11 of 12 cows (92%) the LH surge and ovulation were observed 34.6+/-1.6 and 59.5+/-1.9 h, respectively, after removal of progesterone source. The maximum values for LH in those animals were 19.0+/-2.6 ng/ml (mean+/-S.E.M.). It is concluded that, in Nelore cows submitted to a ovarian superstimulation protocol, the LH surge occurs approximately 35 h after removal of intravaginal device containing progesterone, and approximately 12h before the LH surge observed after an induced estrus without ovarian superstimulation.     

Effect of recombinant bovine somatotropin (bST) on follicular population and on in vitro buffalo embryo production

The objective of this study was to evaluate the effect of bovine somatotropin (bST) on ovarian follicular population in buffalo heifers and its influence on oocyte quality, recovery rates and in vitro embryo production. We tested the hypothesis that bST treatment in buffalo females submitted to an ovum pick-up (OPU) program would improve the number of follicles recruited, oocyte quality and in vitro embryo production. A total of 10 heifers were assigned into two treatment groups: group bST (n=5; receiving 500 mg of bST in regular intervals) and control group (n=5; without additional treatment). Both groups were subjected to OPU sessions twice a week (every 3 or 4 days), for a total of 10 sessions per female, although due to procedural problems, only the first five OPU sessions produced embryos. The number of follicles and the diameters were recorded at all OPU sessions. The harvested oocytes were counted and classified according to their quality as either A, B, C, D or E, with A and B considered good quality. Cleavage and blastocyst production rates were evaluated 2 and 7 days after in vitro fertilization, respectively. The bST treatment increased the total number of antral follicles (>3mm in diameter; 12.2 compared with 8.7; p<0.05) and of small antral follicles (<5mm; 9.1 compared with 6.5; p<0.05) per OPU session. The bST also tended to increase the number of oocytes recovered per session (5.2 compared with 4.1; p=0.07), and enhanced the percentage of good quality oocytes (48.8% compared with 40.6%; p=0.07). bST showed no effect on cleavage and blastocyst production rates (p>0.05). The significant effects of performing repeated OPU sessions were decreasing the follicular population (p<0.001) as well as the number of follicles aspirated (p<0.001), and oocytes recovered (p<0.02). In conclusion, bST treatment improves the follicular population, demonstrating its possible application in buffalo donors submitted to OPU programs.     

Synthesis, biological, and theoretical evaluations of new 1,2,3-triazoles against the hemolytic profile of the Lachesis muta snake venom

The current treatment used against envenomation by Lachesis muta venom still presents several side effects. This paper describes the synthesis, pharmacological and theoretical evaluations of new 1-arylsulfonylamino-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid ethyl esters (8a–f) tested against the hemolytic profile of the L. muta snake venom. Their structures were elucidated by one- and two-dimensional NMR techniques (¹H, APT, HETCOR ¹J_CH and ⁿJ_CH, n = 2, 3) and high-resolution electrospray ionization mass spectrometry. The series of triazole derivatives significantly neutralized the hemolysis induced by L. muta crude venom presenting a dose-dependent inhibitory profile (IC₅₀ = 30−83 μM) with 1-(4′-chlorophenylsulfonylamino)-5-methyl-1H-[1,2,3]-triazole-4-carboxylic acid ethyl ester (8e) being the most potent compound. The theoretical evaluation revealed the correlation of the antiophidian profile with the coefficient distribution and density map of the Highest Occupied Molecular Orbitals (HOMO) of these molecules. The elucidation of this new series may help on designing new and more efficient antiophidian molecules.     

Cell wall adaptations of planktonic and biofilm <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> cells to growth on C5 to C16 <Emphasis Type="Italic">n</Emphasis>-alkane hydrocarbons

Rhodococcus erythropolis was found to utilize C5 to C16 n-alkane hydrocarbons as sole source of carbon and energy when growing as planktonic or biofilm cells attached to polystyrene surfaces. Growth rates on even numbered were two- to threefold increased relatively to odd-numbered n-alkanes and depended on the chain length of the n-alkanes. C10-, C12-, C14-, and C16-n-alkanes gave rise to two- to fourfold increased maximal growth rates relative to C5- to C9-hydrocarbons. In reaction to the extremely poor water solubility of the n-alkanes, both planktonic and biofilm cells exhibited distinct adaptive changes. These included the production of surface active compounds and substrate-specific modifications of the physicochemical cell surface properties as expressed by the microbial adhesion to hydrocarbon- and contact angle-based hydrophobicity, as well as the zeta potential of the cells. By contrast, n-alkane-specific alterations of the cellular membrane composition were less distinct. The specificity of the observed autecological changes suggest that R. erythropolis cells may be used in the development and application of sound biocatalytic processes using n-alkanes as substrates or substrate reservoirs or for target-specific bioremediation of oils and combustibles, respectively.     

Mycobacterium tuberculosis expresses methionine sulphoxide reductases A and B that protect from killing by nitrite and hypochlorite

Methionine sulphoxide reductases (Msr) reduce methionine sulphoxide to methionine and protect bacteria against reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Many organisms express both MsrA, active against methionine-( S )-sulphoxide, and MsrB, active against methionine-( R )-sulphoxide. Mycobacterium tuberculosis (Mtb) expresses MsrA, which protects Δ msrA-Escherichia coli from ROI and RNI. However, the function of MsrA in Mtb has not been defined, and it is unknown whether Mtb expresses MsrB. We identified MsrB as the protein encoded by Rv2674 in Mtb and confirmed the distinct stereospecificities of recombinant Mtb MsrA and MsrB. We generated strains of Mtb deficient in MsrA, MsrB or both and complemented the mutants. Lysates of singly deficient strains displayed half as much Msr activity as wild type against N -acetyl methionine sulphoxide. However, in contrast to other bacteria, single mutants were no more vulnerable than wild type to killing by ROI/RNI. Only Mtb lacking both MsrA and MsrB was more readily killed by nitrite or hypochlorite. Thus, MsrA and MsrB contribute to the enzymatic defences of Mtb against ROI and RNI.     

Ovicidal effect of nematophagous fungi on Taenia taeniaeformis eggs

This work evaluated the ovicidal effect of the nematophagous fungi Monacrosporium sinense (SF53), Monacrosporium thaumasium (NF34) and Pochonia chlamydosporia (VC1) on Taenia taeniaeformis eggs in laboratory conditions. T. taeniaeformis eggs were plated on 2% water-agar with the grown isolates and control without fungus and examined at seven and fourteen days post-inoculation. At the end of the experiment, P. chlamydosporia showed ovicidal activity (P < 0.01) on T. taeniaeformis eggs unlike the other two species, mainly for internal egg colonization with percentage results of 32.2–54.0% at 7th and 14th day, respectively. The other fungi only showed lytic effect without morphological damage to eggshell. Results demonstrated that P. chlamydosporia was in vitro effective against Taenia taeniaeformis eggs unlike the other fungi. In this way, the use of P. chlamydosporia is suggested as a potential biological control agent for eggs of this cestode.     

Robustness of sludge enriched with short SBR cycles for biological nutrient removal

In this study, it is proposed that short sequencing batch reactor (SBR) cycles select and maintain a robust and active biomass, able to cope with typical disturbances occurring in wastewater treatment plants. In order to test this hypothesis, an SBR system was subjected to COD, N and P shock loads. It was shown that the sludge enriched in the SBR operated with short cycles was able to rapidly recover from the tested disturbances. COD and N removal recovered within 1–2 days for shock loads of 10 times the standard concentration. The P removal took up to 2–3 sludge ages to fully recover from the COD spike, but the enhanced biological phosphorus removal (EBPR) performance was still able to be totally re-established after each of the tests, even in theoretically adverse conditions for the growth of polyphosphate accumulating organisms.     

Genetic Diversity and Variation Among Botanical Varieties of Old Portuguese Wheat Cultivars Revealed by ISSR Assays

Inter-simple sequence repeats (ISSRs) were used for genetic diversity analyses of an Old Portuguese wheat collection. Eighteen primers produced 96.3 and 98.5% of ISSR polymorphism in bread and durum wheat cultivars, respectively. The unweighted pair group method with arithmetical averages (UPGMA) phenogram clearly split all cultivars based on their species/ploidy, reflecting a defined genetic structure. ISSRs revealed high genetic diversity at interspecific, intraspecific, and intercultivar levels. Thirty-three exclusive ISSR markers were found. Cultivars were clustered according to their botanical varieties and, in a few cases, with their homonym(s). No statistically significant differences were found between genetic diversity parameters of durum and bread wheat, probably due to high intraspecific diversity. Similar analyses were performed among botanical varieties, and their relationships were defined. Cladograms resembled UPGMA clustering. This highly genetically diverse Old wheat collection will be conserved and maintained, and it could be further used in breeding programs to widen the narrow genetic basis of modern wheat varieties and to avoid the loss of rare and unique alleles.     

On the molecular mass of the extracellular hemoglobin of Glossoscolex paulistus: Analytical ultracentrifugation reexamination

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted by subunits containing heme groups with molecular masses (M) in the range of 15 to 19 kDa, monomers of 16 kDa (d), and trimers of 51 to 52 kDa (abc) linked by nonheme structures named linkers of 24 to 32 kDa (L). HbGp is homologous to Lumbricus terrestris hemoglobin (HbLt). Several reports propose M of HbLt in the range of 3.6 to 4.4 MDa. Based on subunits M determined by mass spectrometry and assuming HbGp stoichiometry of 12(abcd)₃L₃ (Vinogradov model) plus 144 heme groups, a value of M for HbGp oligomer of 3560 kDa can be predicted. This value is nearly 500 kDa higher than the unique HbGp M value reported in the literature. In the current work, sedimentation velocity analytical ultracentrifugation (AUC) experiments were performed to obtain M for HbGp in oxy and cyano-met forms. s⁰_20,w values of 58.1 ± 0.2 S and 59.6 ± 0.2 S, respectively, for the two oxidation forms were obtained. The ratio between sedimentation and diffusion coefficients supplied values for M of approximately 3600 ± 100 and 3700 ± 100 kDa for oxy and cyano-met HbGp forms, respectively. An independent determination of the partial specific volume, V_bar, for HbGp was performed based on density measurements, providing a value of 0.764 ± 0.008, in excellent agreement with the estimates from SEDFIT software. Our results show total consistency between M obtained by AUC and recent partial characterization by mass spectrometry. Therefore, HbGp possesses M very close to that of HbLt, suggesting an oligomeric assembly in agreement with the Vinogradov model.