Influence of Caffeine and Chondroitin Sulfate on Swine Sperm Capacitation and In Vitro Embryo Production

Background: The establishment of an in vitro production (IVP) of embryo in swine allows the generation of embryos with the same quality as in vivo produced embryos with less costs and time. In order to achieve successful fertilization under normal circumstances in vivo, mammalian spermatozoa must first undergo capacitation and then acrosome reaction. The purpose of this study was compared the efficacious of IP/CFDA fluorescence and Coomassie Blue G (CB) staining to detect capacitated sperm cells in refrigerated and fresh semen. Morever, it was investigated the efficacious of caffeine and chondroitin sulphate to promote in vitro sperm capacitation and in vitro embryo produced (IVP) of swine embryos. Materials, Methods & Results: A sperm-rich fraction from ejaculate was obtained using the gloved-hand method and the gel-free fraction was separated using sterile gauze. The semen was diluted in BTS at a final concentration of 1.5 x 10(8) cells/mL. The sperm suspension was incubated for 2 h at 25 degrees C, refrigerated and maintained for 1 h at 15-18 degrees C (refrigerated group) or used immediately (fresh group). Sperm capacitation was assessed by IP/CFDA fluorescence and CB staining for both fresh and refrigerated semen. For PI/CFDA evaluation, a final solution containing 1.7 mM formaldehyde, 7.3 mM PI and 20 mM CFDA in 950 mu L saline was prepared. In the dark, 40 mu L PI/CFDA final solution was added to 10 mu L semen and after 8 min, slides were analyzed on epifluorescence microscopy. For CB evaluation, sperm cells were fixed in 4% paraformaldehyde for 10 min and centrifuged twice at 320 x g in ammonium acetate pH 9 for 8 min. A smear was made and stained with 2.75 mg/mL CB in solution containing 12.5% methanol, 25% glacial acetic acid and 62.5% water, for 2 min. The smear was washed in running water, air dried and sealed with Permount (R), diluted 2:1 in xilol to avoid staining oxidation. Our results showed that refrigeration did not affect sperm capacitation and comparing staining methods, the PI/CFDA combination was more efficient to detect capacitated sperm, when compared to CB staining. In experiment 2, we evaluated the effect of different incubation time (1 - 5 h) with chondroitin sulfate and caffeine on sperm capacitation. For in vitro fertilization, oocytes were obtained from slaughterhouse ovaries. Oocytes with a thick and intact cumulus oophurus layer and cytoplasm with homogenous granules were selected for in vitro maturation for 44 h. According to the results of experiment 2, it was used for in vitro fertilization refrigerated semen was capacitated with 50 mu g/mL chondroitin sulfate for 2 h or capacitated with 5 mu g/mL caffeine for 3 h. Six hours after insemination, cumulus oophorus cells were mechanically removed and oocytes were washed and incubated in microdrops of culture medium. Embryo development after fertilization with sperm capacitated with caffeine or chondroitin sulfate was evaluated on days 3, 5 and 7 of culture. No differences were observed in days 3 or 5 of in vitro culture. However, it was observed an increase on blastocyst rate on Day 7 of culture when caffeine was used as the capacitor agent. Discussion: Molecular basis of sperm capacitation is still poor understood. Sperm capacitation can occur in vitro spontaneously in defined media without addition of biological fluids. We observed that sperm capacitation increased as incubation period enlarged and it was observed using Coomassie blue G and PI/CFDA for fresh semen and for refrigerated semen. It can be concluded that the cooling of semen did not change their pattern of sperm capacitation and this is best assessed by IP/CFDA than by CB. In addition to the use of caffeine in sperm capacitation produces more blastocysts than the chondroitin sulfate after in vitro fertilization.

Cortisol plasmático como indicador de estresse em colopexias laparoscópicas com implante de tela de polipropileno em cães

The realization of conventional surgical procedures, even using the recommended doses of analgesics drugs, generally are associated with a long recovery period, post-surgical trauma, stress and pain in animals. That can vary according with the procedure, extension and time of the surgery. Considering the increased interest in procedures that minimize surgical stress and the recovery period after the surgical procedure in dogs, this present work was developed to evaluate a new technique of laparoscopic colopexy with the alterations of cortisol levels, heart rate and respiratory frequencies in dogs. For this study, nine female dogs were submitted to a colopexy. Blood samples and data of heart rate and respiratory frequency were sampled in intervals of 10min. from the preoperative period until the end of the procedure. After, these parameters were assessed every three hours for 12 hours. Cortisol levels were measured using an ELISA Kit for cortisol. Results were analyzed by ANOVA and comparisons were made using Tukey's test. Significance levels was set at p <= 0.05. The increase of the heart rate, respiratory frequency and cortisol levels were mainly observed in the period after shaving and after the end of the anesthesia. Values were kept stable during all the surgery, and coming to the basal conditions after nine hours in the post-operative period. The laparoscopic colopexy is a stress-associated surgery, but it produces lower increasing of cortisol levels returning to normal in 9 hours after the surgery.

Assessment of the cavitation erosion resistance in high nitrogen austenitic stainless steels: study of the wear mechanisms

Specimens of a UNS S31803 steel were submitted to high temperature gas nitriding and then to vibratory pitting wear tests. Nitrided samples displayed fully austenitic microstructures and 0.9 wt. % nitrogen contents. Prior to pitting tests, sample texture was characterized by electron backscattering diffraction, EBSD. Later on, the samples were tested in a vibratory pit testing equipment using distilled water Pitting tests were periodically interrupted to evaluate mass loss and to characterize the surface wear by SEM observations. At earlier pit erosion, stages intense and highly heterogeneous plastic deformation inside individual grains was observed. Later on, after the incubation period, mass loss by debris detachment was observed. Initial debris micro fracturing was addressed to low cycle fatigue. Damage started at both sites, inside the grains and grain boundaries. The twin boundaries were the most prone to mass-loss incubation. Grains with (101) planes oriented near parallel to the sample surface displayed higher wear resistance than grains with other textures. This was attributed to lower resolved stresses for plastic deformation inside the grains with (101)

Microbial activity and community composition in saline and non-saline soils exposed to multiple drying and rewetting events

The effects of drying and rewetting (DRW) have been studied extensively in non-saline soils, but little is known about the impact of DRW in saline soils. An incubation experiment was conducted to determine the impact of 1-3 drying and re-wetting events on soil microbial activity and community composition at different levels of electrical conductivity in the saturated soil extract (ECe) (ECe 0.7, 9.3, 17.6 dS m(-1)). A non-saline sandy loam was amended with NaCl to achieve the three EC levels 21 days prior to the first DRW; wheat straw was added 7 days prior to the first DRW. Each DRW event consisted of 1 week drying and 1 week moist (50% of water holding capacity, WHC). After the last DRW, the soils were maintained moist until the end of the incubation period (63 days after addition of the wheat straw). A control was kept moist (50% of WHC) throughout the incubation period. Respiration rates on the day after rewetting were similar after the first and the second DRW, but significantly lower after the third DRW. After the first and second DRW, respiration rates were lower at EC17.6 compared to the lower EC levels, whereas salinity had little effect on respiration rates after the third DRW or at the end of the experiment when respiration rates were low. Compared to the continuously moist treatment, respiration rates were about 50% higher on day 15 (d15) and d29. On d44, respiration rates were about 50% higher at EC9.7 than at the other two EC levels. Cumulative respiration was increased by DRW only in the treatment with one DRW and only at the two lower EC levels. Salinity affected microbial biomass and community composition in the moist soils but not in the DRW treatments. At all EC levels and all sampling dates, the community composition in the continuously moist treatment differed from that in the DRW treatments, but there were no differences among the DRW treatments. Microbes in moderately saline soils may be able to utilise substrates released after multiple DRW events better than microbes in non-saline soil. However, at high EC (EC17.6), the low osmotic potential reduced microbial activity to such an extent that the microbes were not able to utilise substrate released after rewetting of dry soil.

In Vitro Simultaneous Transfer of Lipids to HDL in Coronary Artery Disease and in Statin Treatment

The exchange of lipids with cells and other lipoproteins is a crucial process in HDL metabolism and for HDL antiatherogenic function. Here, we tested a practical method to quantify the simultaneous transfer to HDL of phospholipids, free-cholesterol, esterified cholesterol and triacylglycerols and to verify the lipid transfer in patients with coronary artery disease (CAD) or undergoing statin treatment. Twenty-eight control subjects without CAD, 27 with CAD and 25 CAD patients under simvastatin treatment were studied. Plasma samples were incubated with a donor nanoemulsion prepared by ultrasonication of the constituent lipids and labeled with radioactive lipids; % lipids transferred to HDL were quantified in the HDL-containing supernatant after chemical precipitation of non-HDL fractions and the nanoemulsion. The assay was precise and reproducible. Increase of temperature (4-37 A degrees C), of incubation period (5 min to 2 h), of HDL-cholesterol concentration (33-244 mg/dL) and of mass of nanoemulsion lipids (0.075-0.3 mg/mu L) resulted in increased lipid transfer from the nanoemulsion to HDL. In contrast, increasing pH (6.5-8.5) and albumin concentration (3.5-7.0 g/dL) did not affect lipid transfer. There was no difference between CAD and control non-CAD with regard to the lipid transfer, but statin treatment reduced the transfer to HDL of all four lipids. The test herein described is a valid and practical tool for exploring an important aspect of HDL metabolism.

Comparative reproductive biology of two southwestern Atlantic populations of the hermit crab Pagurus exilis (Crustacea: Anomura: Paguridae)

Pagurus exilis (Benedict, 1892) is an endemic South Atlantic hermit crab with a distribution ranging from Rio de Janeiro State, Brazil, to Buenos Aires Province, Argentina. The present study analyzed the reproduction of two populations at the extremes of this geographical distribution, and compared their reproductive period, fecundity, and changes in egg size and egg volume during the incubation period in order to assess the variability over this latitudinal range. Hermit crabs were collected monthly over a 2-year period. In total, 108 females were analyzed for Brazil (44 non-ovigerous and 64 ovigerous), and 141 for Argentina (87 non-ovigerous and 54 ovigerous). Reproduction in Brazil occurs year-round, with peaks in the fall and winter seasons; in Argentina reproduction occurs only in spring and summer. The Brazilian ovigerous females were significantly larger than the Argentina ones (Brazil: SL = 5.33 +/- 1.45 mm; Argentina: SL = 4.15 +/- 0.52 mm; P < 0.001). The fecundity was 1447 +/- 831 eggs (317 to 2885) in Brazil and 987 +/- 711 eggs (114 to 2665) in Argentina, with a trend towards higher fecundity in Brazil. Eggs in the Argentina population were larger than those in Brazil for all the three stages investigated, and no changes in egg volume were found during egg development for both populations. The reproductive traits of the two populations showed some important differences, which may reflect adaptations to local environmental conditions, demonstrating a high plasticity of reproductive features of the species in Brazilian and Argentine waters. The strategy adopted by the Argentina population involves a lower production of larger eggs compared to the population in Brazil; this lower production is associated with reproduction in cold-water regions.

Reproductive strategy of the snapping shrimp Alpheus armillatus H. Milne-Edwards, 1837 in the South Atlantic: fecundity, egg features, and reproductive output

The family Alpheidae, composed by shrimps of relatively small size, popularly known as snapping shrimps, is the one of the most diverse decapod groups. These shrimps are found worldwide and Occur in tropical and subtropical waters, from the intertidal zone to great depths. We investigated reproductive aspects of Alpheus armillatus, in order to gather information on egg production, aiming to enhance knowledge of its reproductive strategies in a population in an intertidal area of the South Atlantic. Ovigerous females were collected under rocks, in May and July 2006 (dry season) and in November 2006 and March 2007 (rainy season). Egg production and reproductive output were analyzed and compared seasonally and during the period of embryonic development. Females measured on average 11.28 mm CL with a mean of 763 eggs and 0.11 mm(3) egg volume. The egg volume of this population was smaller than previous estimates for other species of snapping shrimps, but the mean egg number was higher. The volume of eggs doubled during the incubation period, but despite this increase, no significant loss of eggs was observed. Alpheus armillatus invests oil average about 12% of body weight in reproduction. The proportional investment in egg production IS Significantly higher in the rainy season when compared with the dry season (17.9% vs 4.8%), correlated with higher temperatures and increased food availability at this time. Our results corroborated the hypothesis of a pattern of egg production influenced by environmental conditions and intraspecific variability among the family Alpheidae, as a function of the biogeographic region.

Acoustic hypersensitivity in adult rats after neonatal ventral hippocampus lesions

Rats with a bilateral neonatal ventral hippocampus lesion (NVHL) are used as models of neurobiological aspects of schizophrenia. In view of their decreased number of GABAergic interneurons, we hypothesized that they would show increased reactivity to acoustic stimuli. We systematically characterized the acoustic reactivity of NVHL rats and sham operated controls. They were behaviourally observed during a loud white noise. A first cohort of 7 months` old rats was studied. Then the observations were reproduced in a second cohort of the same age after characterizing the reactivity of the same rats to dopaminergic drugs. A third cohort of rats was studied at 2, 3, 4, 5 and 6 months. In subsets of lesioned and control rats, inferior colliculus auditory evoked potentials were recorded. A significant proportion of rats (50-62%) showed aberrant audiogenic responses with explosive wild running resembling the initial phase of audiogenic seizures. This was not correlated with their well-known enhanced reactivity to dopaminergic drugs. The proportion of rats showing this strong reaction increased with rats` age. After the cessation of the noise, NVHL rats showed a long freezing period that did neither depend on the size of the lesion nor on the rats` age. The initial negative deflection of the auditory evoked potential was enhanced in the inferior colliculus of only NVHL rats that displayed wild running. Complementary anatomical investigations using X-ray scans in the living animal, and alizarin red staining of brain slices, revealed a thin layer of calcium deposit close to the medial geniculate nuclei in post-NVHL rats, raising the possibility that this may contribute to the hyper-reactivity to sounds seen in these animals. The findings of this study provide complementary information with potential relevance for the hyper-reactivity noted in patients with schizophrenia, and therefore a tool to investigate the underlying biology of this endophenotype. (C) 2009 Elsevier B.V. All rights reserved.

Childhood radiation-associated atypical meningioma with novel complex rearrangements involving chromosomes 1 and 12

Meningiomas are recognized as the most common late complication following radiotherapy. However, cytogenetic studies in childhood atypical radiation-induced meningioma are sporadic, mainly because this condition generally occurs after a long latent period. In the present study we show the results of conventional and molecular cytogenetics in a 14-year-old boy with a secondary atypical meningioma. Apart from numerical changes, we found complex aberrations with the participation of chromosomes 1, 6 and 12. The invariable presence of loss of 1p was demonstrated by fluorescent in situ hybridization (FISH) analysis with probes directed to telomeric regions and by comparative genome hybridization (CGH). Previous cytogenetic studies on adult spontaneous and radiation-associated meningiomas showed loss of chromosome 22 as the most frequent change, followed by loss of the short arm of chromosome 1. To the best of our knowledge this is the first report of highly complex chromosome aberrations in the pediatric setting of meningioma.

Spontaneous Interblink Time Distributions in Patients with Graves` Orbitopathy and Normal Subjects

PURPOSE. To determine the shape of spontaneous interblink time interval distributions obtained in a long observation period in normal subjects and patients with Graves` orbitopathy. METHODS. The magnetic search coil technique was used to register the spontaneous blinking activity during 1 hour of video observation of two groups of 10 subjects each (normal controls aged 27-61 years, mean +/- SD = 46.0 +/- 13.6; patients with Graves` orbitopathy aged 33-61 years, mean +/- SD +/- 46.7 +/- 8.9). The spontaneous blink rate of each subject was calculated for the entire period of observation and for 56 five-minute bins. Histograms of the interblink time interval were plotted for each measurement of blink rate. RESULTS. Neither the overall mean blink rate (controls, 19.8 +/- 4.9; Graves`, 17.6 +/- 5.4) nor the interblink time (controls, 5.2 +/- 3.1, Graves`, 7.9 +/- 3.5) differed between the two groups. There was a large variation of both measurements when the 5-minute bins were considered. The interblink time distribution of all subjects was highly positively skewed when the 1-hour period was measured. A significant number of the 5-minute bin distributions deviated from the overall pattern and became symmetric. CONCLUSIONS. The normal blinking process is characterized by highly positively skewed interblink time distributions. This result means that most blinks have a short time interval, and occasionally a small number of blinks have long time intervals. The different patterns of distribution described in the early literature probably represent artifacts because of the small samples analyzed. (Invest Ophthalmol Vis Sci. 2011;52:3419-3424) DOI:10.1167/iovs.10-7060

Biological aspects of Amblyomma brasiliense (Acari : Ixodidae) under laboratory conditions

Although Amblyomma brasiliense Arag (a) over tildeo 1908 has been reported as one of the most aggressive ticks to humans in Brazil, information about the biology of this tick species is virtually inexistent. This work reports data on the life cycle of A. brasiliense fed on rabbits and pigs and maintained in an incubator at 20 degrees C, 90% RH and 12 h of light for off-host development. Tick yield of adult females fed on pigs and rabbits was 81.2% and 58.3%, respectively. Females fed on pigs had mean engorgement weight of 862.3 mg and egg mass of 208 mg, while females fed on rabbits had mean engorgement weight of 606.1 mg and egg mass of 160 mg; these values did not differ statistically between host species. Feeding period of female ticks fed on pigs (10 days) was significantly shorter than that on rabbits (17 days). Mean preoviposition period was slightly longer (35.9 days) for ticks fed on pigs than on rabbits (30 days). The minimum incubation period of eggs of ticks from both host species was similar and over 100 days. Egg production efficiency was low for females fed on both hosts (less than 30% and 20% for ticks from pigs and rabbits, respectively). More than 55% of larvae and 79% of nymphs fed on rabbits, set free inside the feeding chambers, engorged successfully. These ticks attained an engorgement weight of 1.3 and 18.2 mg, respectively, and fed for approximately 5 days. The minimum pre-molt period was 30 days for engorged larvae and over 44 days for nymphs. Molting success was low, less than 50% in the case of larvae and less than 20% for nymphs. Further studies are required to better determine the off-host requirements of this tick species.

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 X 10(6) cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 degrees C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced. (C) 2010 Elsevier Inc. All rights reserved.

Detection of dengue viral RNA in Aedes aegypti (Diptera : Culicidae) exposed to sticky lures using reverse-transcriptase polymerase chain reaction

Active surveillance for dengue (DEN) virus infected mosquitoes can be an effective way to predict the risk of dengue infection in a given area. However, doing so may pose logistical problems if mosquitoes must be kept alive or frozen fresh to detect DEN virus. In an attempt to simplify mosquito processing, we evaluated the usefulness of a sticky lure and a seminested reverse-transcriptase polymerase chain reaction assay (RT-PCR) for detecting DEN virus RNA under laboratory conditions using experimentally infected Aedes aegypti (L.) mosquitoes. In the first experiment, 40 male mosquitoes were inoculated with 0.13 mul of a 10(4) pfu/ml DEN-2 stock solution. After a 7-d incubation period, the mosquitoes were applied to the sticky lure and kept at room temperatures of 23-30 degreesC. Following 7,10,14, and 28 d application, 10 mosquitoes each were removed from the lure pooled and assayed for virus. DEN virus nucleic acid was clearly detectable in all pools up to 28 d after death. A second study evaluated sensitivity and specificity using one, two, and five DEN-infected mosquitoes removed after 7, 10, 14, 21 and 30 d application and tested by RT-PCR. All four DEN serotypes were individually inoculated in mosquitoes and evaluated using the same procedures as experiment 1. The four serotypes were detectable in as few as one mosquito 30 d after application to the lure with no evidence of cross-reactivity. The combination of sticky lures and RT-PCR show promise for mosquito and dengue virus surveillance and warrant further evaluation.

Determination of reactivity rates of silicate particle-size fractions

The efficiency of sources used for soil acidity correction depends on reactivity rate (RR) and neutralization power (NP), indicated by effective calcium carbonate (ECC). Few studies establish relative efficiency of reactivity (RER) for silicate particle-size fractions, therefore, the RER applied for lime are used. This study aimed to evaluate the reactivity of silicate materials affected by particle size throughout incubation periods in comparison to lime, and to calculate the RER for silicate particle-size fractions. Six correction sources were evaluated: three slags from distinct origins, dolomitic and calcitic lime separated into four particle-size fractions (2, 0.84, 0.30 and <0.30-mm sieves), and wollastonite, as an additional treatment. The treatments were applied to three soils with different texture classes. The dose of neutralizing material (calcium and magnesium oxides) was applied at equal quantities, and the only variation was the particle-size material. After a 90-day incubation period, the RER was calculated for each particle-size fraction, as well as the RR and ECC of each source. The neutralization of soil acidity of the same particle-size fraction for different sources showed distinct solubility and a distinct reaction between silicates and lime. The RER for slag were higher than the limits established by Brazilian legislation, indicating that the method used for limes should not be used for the slags studied here.

Vector competence experiments with Rocio virus and three mosquito species from the epidemic zone in Brazil

First-generation progeny of field-collected Psorophora ferox, Aedes scapularis, and Aedes serratus from the Rocio encephalitis epidemic zone in S.Paulo State, Brazil, were tested for vector competency in the laboratory. Psorophora ferox and Ae. scapularis are susceptible to per os infection with Rocio virus and can transmit the virus by bite following a suitable incubation period. Oral ID50S for the two species (10(4.1) and 10(4.3) Vero cell plaque forming units, respectively) did not differ significantly. Infection rates in Ae. serratus never exceeded 36%, and, consequently, an ID50 could not be calculated for this species. It is unlikely that Ae. serratus is an epidemiologically important vector of Rocio virus. The utility of an in vitro feeding technique for demonstrating virus transmission by infected mosquitoes and difficulties encountered in working with uncolonized progeny of field-collected mosquitoes are discussed.