The Antioxidant Activity of a Commercial and a Fractionated Phycocyanin on Human Skin Cells In Vitro.

The protective effects for cells against chemical and UVA stress of a commercial phycocyanin (PC) for food use and a PC extracted from Arthrospira platensis (Spirulina) in phosphate buffer were assessed. The purity of the commercial PC, spectrophotometrically estimated as A620/A280 and confirmed by HPLC, was higher than that of the fractionated PC (2.0 vs. 1.5) but was twofold less concentrated. The oxygen radical antioxidant capacities (ORACs) of the commercial and fractionated PCs were 12,141 ± 1928 and 32,680 ± 3295 TE/100 g, respectively. The degradation of PCs upon exposure to UVA was spectrophotometrically estimated, and cytotoxicity was evaluated with the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) test on human fibroblasts and keratinocytes. A lower level of reactive oxygen species (ROS) was recorded in the two cell lines incubated with the commercial PC after menadione treatment (p < 0.01) and UVA exposure (p < 0.001) on fibroblasts after 5 min and keratinocytes up to 25 min, compared with controls. Differently, the fractionated PC was not protective and showed significant (p < 0.01) paradoxical prooxidant effects. Overall, the PC for food consumption demonstrated a high safety threshold and antioxidant ability to cells that, along with its coloring power, make it an excellent candidate for cosmetic formulations.

Comparison between Single- and Group-housed Pregnant Sows for Direct and Indirect Physiological, Reproductive, Welfare Indicators and Gene Expression Profiling.

Single- and group-housed pregnant sows were assessed during 8 weeks (4th and 8th to 14th) with regard to physiological, reproductive, welfare indicators and gene expression profiling. Compared to single-housed sows, group-housed sows had decreased non-return to estrus at 56 days after artificial insemination (AI) (83% vs 92%) and farrowing rate (78% vs 88%), respectively. Furthermore, group-housed sows showed a higher degree (p < 0.01) of advantageous physiological indicators, such as albumin (odds ratio [OR] = 4.4), alkaline phosphatase (OR = 1.5), bactericidal (OR = 3.2) and complement (OR = 24.3), and disadvantageous for alanine amino transferase (OR = 0.5), bilirubin (OR = 0.4), lysozyme (OR = 0.3) and C-reactive protein compared to single-housed. Eighty-seven genes related to immune response were underexpressed (log fold change ≤ 1.5; p < 0.05) during the 8th to 14th weeks in the group compared to single-housed sows, which in turn showed an immunomodulatory reduction on the expression of 43 genes during the 11th to 14th compared to the 4th week. Overall, the results were interpreted as indicative of greater comfort state of the group compared to single-housed sows.

Can contamination by major systemic insecticides affect the voracity of the harlequin ladybird?

Systemic neurotoxic insecticides are widely used to control aphid pests worldwide and their potential non-target effects on aphid predators are often unknown. Behavioral responses linked to biological control services are crucial when assessing the compatibility of chemicals with biocontrol organisms. This is particularly relevant for insecticides at low and sublethal concentrations. We studied the acute toxicity and the sublethal effect on the voracity of the generalist predator Harmonia axyridis (Coleoptera: Coccinellidae) caused by the exposure to three systemic insecticides routinely used against aphids. The tested insecticide concentrations were the Lethal Concentration 50% (LC50), 20% (LC20) and 1% (LC1) estimated for the target pest Aphis gossypii (Hemiptera: Aphididae) in a companion study. The survival and the voracity differed among the tested chemicals and concentrations, but only thiamethoxam at LC50 caused a significant predator mortality, and individuals that survived showed a reduced predation rate. The predators showed a density independent functional response after the exposure to most of the insecticide-concentration combinations, while an inverse density dependence of the prey consumption rate was observed for coccinellids exposed to sulfoxaflor and thiamethoxam at their lowest tested concentration. The estimated parameters, i.e., the attack rate and the prey handling time, were affected at higher concentrations by both imidacloprid and sulfoxaflor. These findings stress the importance of carefully evaluating side effects of insecticides at very low concentrations on beneficial arthropods in the risk assessment schemes for sustainable pest control programmes.

In Vitro Assessment of the Antioxidant Properties of Aqueous Byproduct Extracts of Vitis vinifera.

Aqueous extracts were obtained at low temperature with the Naviglio technology from grapevine stalks (Merlot), marc (Merlot and Cabernet Sauvignon) and leaves (Merlot) as typical byproducts of winemaking industry, and their properties were evaluated cytofluorometrically on human dermal fibroblasts. Leaf extracts had the greatest total phenolic ((47.6±3.5) mg/g) and proanthocyanidin ((24.2±0.1) mg/g) contents compared to the others. The preliminary colorimetric MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay individuated two consecutive non-toxic volume fractions of each extract (from 0.8 to 12.8%) that were adopted for three cytofluorometric tests. The first cell membrane test did not evidence any harmful effects against plasma membranes at the two non-toxic volume fractions. The second mitochondrial membrane test showed a decreased (p<0.01) percentage of cells ((15.7±8.3) vs (32.5±1.3) %) with active polarized mitochondrial membranes at the higher non-cytotoxic volume fractions of extracts from Cabernet Sauvignon marc in response to 4.5 mM H2O2, and from Merlot stalks (p<0.05) at 1.5 mM H2O2 ((49.3±6.1) vs (64.6±2.4) %) and without H2O2 ((89.7±2.4) vs (96.9±1.8) %), compared to the controls submitted to the same H2O2 concentration. Conversely, mitochondrial activity of leaf extracts significantly (p<0.05) increased ((96.3±1.8) and (96.4±1.4) %) after treatment with 0.5 mM H2O2 at both non-cytotoxic volume fractions compared to control ((88.2±1.1) %). Finally, as evidenced by the third oxidative status test, stalk extracts did not evidence relevant effects on the cellular oxidative state, while the extracts of marc and leaves demonstrated significantly medium (p<0.05) to highly (p<0.001) positive effects following exposure to H2O2 ranging from 0.5 to 4.5 mM, compared to controls.

Cryopreservation of stallion semen: laboratory assessment of sperm injuries after cushioned centrifugation and freezing with conventional and alternative directional freezing methods.

Fresh 36 ejaculates of 13 stallions were split into two volumes, centrifuged with and without cushion and frozen with Conventional and two prototype, Drum and Directional, methods using 0.5 ml straws for the Conventional and Drum, and 2 ml flat straws for both the Drum and Directional. Cushioned centrifugation increased total motility (61.2 ± 18.6% vs. 57.5 ± 18.6%; P < 0.001) and mean velocity (84.3 ± 15.6% vs. 83.2 ± 13.8%; P < 0.05) when compared to not cushioned centrifugation, estimated after cooling the sperm at 4°C for 90 min before freezing. Cushioned centrifugation also increased (P < 0.001) spermatozoa with polarized mitochondrial membranes (46.8 ± 11.4% vs. 43.4 ± 10.6%) and intact plasmatic/acrosomal membranes (41.0 ± 11.2% vs. 38.5 ± 11.3%) of frozen/thawed sperm, with respect to not cushioned centrifugation. However, no effects of the centrifugation were evidenced for classical kinetic parameters. Flat straws had negative effect for almost all the parameters analyzed at thawing (T,) and after 3 hours' incubation at 37°C (T1), while the Drum method with Paillettes did not show appreciable affects. The variability among stallions was relevant (5% to 69% variance for kinetics and membrane status), while the variability among ejaculates was minor (9% to 28%). Factorial analysis identified three relevant, factors with different informational content: Factor 1 represented by membranes status, Factor 2 by kinetics estimated at T0, and Factor 3 by kinetics estimated at T1. Cushioned centrifugation had some beneficial effects for the membrane status of the frozen/thawed sperm, while the use of flat straws needs to be improved.

Experimental evidence of a buoyant mass difference between bovine spermatozoa bearing X- and Y-chromosomes using a micromechanical resonator.

Flow cytometry is to date the only commercially viable technique for sex preselection of mammalian spermatozoa, measuring the different DNA content in X- and Y-chromosome bearing spermatozoa. Here we present experimental evidence of a measurable difference between bovine spermatozoa bearing X- and Y-chromosomes based on their buoyant mass. Single cells of two populations of flow-cytometrically sorted spermatozoa were analyzed by means of a micromechanical resonator, consisting of a suspended doubly-clamped microcapillary. Spermatozoa buoyant mass is related to the transitory variation in vibration phase lag, caused by the passage through the sensitive area of a single sperm cell suspended in a fluid. Data analysis shows two well-separated distributions and provides evidence of the sensor capabilities to detect the buoyant mass of single cells with such accuracy to distinguish X- and Y-chromosome bearing spermatozoa. These preliminary results suggest the possibility to develop an intriguing technique alternative to flow cytometry in the field of sperm sorting.

Differential gene expression in cumulus oocyte complexes collected by ovum pick up from repeat breeder and normally fertile Holstein Friesian heifers.

The aim of this study was to establish whether perturbed gene expression during cumulus oocyte development causes repeat breeding in cattle. In this study, a repeat breeder was defined as a normal estrous cycling animal that did not become pregnant after three inseminations despite the absence of clinically detectable reproductive disorders. Transcripts of genes extracted from cumulus oocyte complexes (COC) that were collected from three repeat breeder and three normally fertile Holstein Friesian heifers were compared. Up to 40 COC were collected from each heifer by means of repeated sessions of ovum pick up in the absence of hormonal stimulation; immediately plunged into liquid nitrogen; and stored at -80°C until analysis. For each heifer, RNA was extracted from the pooled COC and hybridized on GeneChip(®) Bovine Gene Array (Affymetrix). Analysis of gene expression profiles of repeat breeder and control COC showed that 178 genes were differentially expressed (log2 fold change>1.5). Of these genes, 43 (24%) were up-regulated and 135 (76%) were down-regulated in repeat breeder relative to control heifers. This altered pattern of expression occurred in genes involved in several cellular biological processes and cellular components such as metabolism, angiogenesis, substrate/ion transport, regulation/signaling, cell adhesion and cytoskeleton. From these, 13 genes potentially involved in cumulus oocyte growth were subjected to validation by qRT-PCR and nine genes (annexin A1, ANXA1; lactoferrin, LTF; interferon stimulated exonuclease 20kDa, ISG20/HEM45; oxidized low density lipoprotein receptor 1, OLR1; fatty acid desaturase 2, FADS2; glutathione S-transferase A2 and A4, GSTA2 and GSTA4; glutathione peroxidase 1, GPX1; endothelin receptor type A, EDNRA) were confirmed to be differentially expressed. This study identified potential marker genes for fertility in dairy cattle.

Clear coherent imaging in turbid microfluidics by multiple holographic acquisitions.

Recently it has been demonstrated that digital holography is a powerful means allowing imaging of both amplitude and phase objects in turbid flowing media. However, in quasi-static turbid microfluidics, multiple scattering contributions through the colloids superimpose coherently to the recording device, resulting in speckle noise and hindering a clear vision of the objects. In this Letter we exploit the Brownian motion of the colloidal particles to get multiple uncorrelated holograms, and we combine them to reduce the speckle contrast. In this way we get a multi-look gain without losing image resolution.

Microscopy imaging and quantitative phase contrast mapping in turbid microfluidic channels by digital holography.

We show that sharp imaging and quantitative phase-contrast microcopy is possible in microfluidics in flowing turbid media by digital holography. In fact, in flowing liquids with suspended colloidal particles, clear vision is hindered and cannot be recovered by any other microscopic imaging technique. On the contrary, using digital holography, clear imaging is possible thanks to the Doppler frequency shift experienced by the photons scattered by the flowing colloidal particles, which do not contribute to the interference process, i.e. the recorded hologram. The method is illustrated and imaging results are demonstrated for pure phase objects, i.e. biological cells in microfluidic channels.

The usefulness of combining traditional sperm assessments with in vitro heterospermic insemination to identify bulls of low fertility as estimated in vivo.

To date, no single in vitro assessment can estimate bull fertility. This research was aimed at evaluating the ability of a series of laboratory assessments to assign 50 Holstein Friesian bulls grouped as low (ER-NRR<-1.5), medium (-0.5

Dynamic DIC by digital holography microscopy for enhancing phase-contrast visualization.

Differential image contrast (DIC), through the numerical managing and manipulation of complex wavefronts obtained by digital holography (DH), is investigated. We name the approach Dynamical Differential Holographic Image Contrast (DDHIC). DDHIC dispenses from special optics and/or complex setup configurations with moveable components, as usually occurs in classical DIC, that is not well-suited for investigating objects experiencing dynamic evolution during the measurement. In fact, the technique presented here, is useful for floating samples since it allows, from a single recording, to set a posteriori the best conditions for DIC imaging in conjunction with the numerical focusing feature of DH. By DDHIC, the movies can be easily built-up to offering dynamic representation of phase-contrast along all directions, thus improving the visualization. Furthermore, the dynamic representation is useful for making the proper choice of other key parameters of DIC such as the amount of shear and the bias, with the aim to optimize the visualized phase-contrast imaging as favorite representation for bio-scientists. Investigation is performed on various biological samples.

[Technical variant of mechanical prolapsectomy: transanal prolapsectomy with anoscopy]. / Varianti tecniche della prolassectomia meccanica: la TPA.

Mechanical mucoprolapsectomy for surgical treatment of rectal prolapse and haemorrhoidal pathology is a widespread practice that has been consolidated by the experience of many medical centres. We have been using a variant system (TPA, transanal prolapsectomy with anoscopy) for about two years. The main feature of this technique is the access route. We performed 51 transanal mucoprolapsectomies over a period of 20 months. During the surgical intervention, in rachianaesthesia, patients are placed in a prone position with minimal snapping of the operating cot. The use of a mobile, versatile tool ("the beak") for constructing the purse-string suture makes for very good manageability. Continuous visual and digital control of each operating step permits immediate correction where necessary. This technique is safe and effective and affords the possibility of single-operator intervention, the second operator proving superfluous in all stages of the operation.

[Implementation of transanal endoscopic microsurgery (TEM) in Italy: a multicentre investigation]. / Applicazione della TEM in Italia: indagine multicentrica.

Transanal endoscopic microsurgery (TEM), which was first introduced several years ago, allows the excision of rectal tumours not susceptible to a more traditional endoscopic approach or as an alternative to highly invasive or debilitating procedures. We surveyed the effective implementation of TEM in Italy, the indications adopted by each surgical department and the technical results achieved. We contacted 34 surgical departments and analysed the answers given by 17 centres (those actually using the technique = 50%). Most of these are situated in Northern Italy. A total of 1208 procedures were declared (84% of them performed in 6 centres). The most frequent declared indications were adenomas and T1 and T2 carcinomas (741 cases). The contraindications have to do with the staging, localisation and size of the neoplasms. The mean operative time reported by most of the centres ranged from 60 to 90 minutes. The most frequent complication in 13 departments was haemorrhage. The conclusions reported by some of the surgeons contacted are useful. The implementation of TEM is confined to only a few centres with a large number of treated cases. Overall analysis of the data raised many questions needing to be answered, especially with regard to the proper use of a surgical technique that is difficult but not impossible to implement.

In vitro fertilisation with frozen-thawed bovine sperm sexed by flow cytometry and validated for accuracy by real-time PCR.

The methodologies used for cytometric sorting of fresh spermatozoa never allowed a clear resolution of sexual chromosomes of frozen-thawed semen. To devise a novel method for the production of bovine predefined sexed embryos using frozen-thawed semen, sorting efficiency of different protocols was studied using a new quantitative real-time PCR method to verify the purity of sexed semen. To this aim, after Percoll separation, frozen-thawed samples were stained at different temperatures and concentrations of Hoechst 33342 using a short-incubation time. The concentration of Hoechst 33342 of 500 mug/ml at a temperature of 37 degrees C provided good and stable fluorescence signals. Preventing the sperm clustering by adding 0.6% BSA in the 90% Percoll fraction led to X-bearing sperms purity of 91+/-2%. Thereafter, sorted sperms were used for in vitro fertilisation (IVF). Despite the lower cleavage rates reported in the sorted groups when compared with the control groups (40 vs 48%, P<0.01), blastocyst formation in the sorted and control groups was not different (27 vs 24% of the cleaved respectively). The PCR analysis of 30 blastocysts confirmed 26 embryos to be correctly sexed (87%). Transfer of two embryos per recipient into six synchronised heifers resulted in four pregnancies. Two abortions occurred at day 60, while two pregnancies went to term delivering two female calves. In conclusion, high purity and repeatability of sorting was obtained with frozen-thawed bull semen that was subsequently used for IVF giving rise to viable embryos and offspring. In addition, real-time PCR revealed to be an optimal support for these studies, providing a rapid and reliable estimation of flow cytometric efficiency.