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The genotoxic and nephrotoxic mycotoxin Ochratoxin A (OTA) has also been reported to have adverse effects on oocyte maturation and embryo development. Previous studies on the effects of OTA on female fertility have used micromolar concentrations, but no information is available to date on effects in a more relevant nanomolar range. This study used a juvenile sheep model to evaluate the effects of oocyte exposure to low levels of OTA on maturation, fertilization, and embryo development. Further, it was investigated whether different mechanisms of action of OTA could be responsible for varying toxic effects at different levels of exposure. Cumulus-oocyte-complexes (COCs) were exposed to 10 μmol/L–0.1 nmol/L OTA during in vitro maturation and evaluated for cumulus viability, oocyte maturation, and bioenergetic/oxidative status. COCs were subjected to in vitro fertilization, embryo culture, and embryo quality assessment via morphology, viability, bioenergetic/oxidative status, and time-lapse monitoring. At micromolar concentrations, OTA induced cytotoxic effects, by reducing cumulus expansion and oocyte maturation. OTA altered temporospatial dynamics of zygote pronuclear formation and embryo morphokinetics. Blastocysts, even morphologically normal, were found to undergo collapse events, which were probably related to boosted blastocyst mitochondrial activity. At nanomolar concentrations, OTA did not affect COC morpho-functional parameters, but impaired oocyte ability to prevent polyspermy and increased blastocyst apoptosis. In conclusion, in the female germ cell, cytotoxic nonspecific effects characterize OTA-induced toxicity at high exposure levels, whereas fine tuning-mode effects, not associated with altered cell viability and integrity, characterize OTA toxic action at low levels. Electronic supplementary material The online version of this article (10.1007/s12550-020-00410-y) contains supplementary material, which is available to authorized users.

Beauvericin (BEA) is a member of the enniatin family of mycotoxins which has received increasing interest because of frequent occurrence in food and feed. By its ionophoric properties, BEA is able to alter membrane ion permeability uncoupling oxidative phosphorylation. It was also shown to alter oocyte mitochondrial function. In this study, the effects of BEA at 0.5, 1, ,3 and 5 μmol/L on expression of genes coding for key proteins of the mitochondrial chain in ovine oocytes and cumulus cells were evaluated at different time points of in vitro maturation (IVM), germinal vesicle (GV; t = 0), metaphase I (MI; t = 7 h), and metaphase II (MII; t = 24 h). The expression of nuclear (TFAM, NDUFA12, UQCRH, COX4, ATP5O) and mitochondrial (ND1, COX1, COX2, ATP6, ATP8) genes coding for proteins of Complexes I, III, IV, and V was analyzed by qRT-PCR. After BEA exposure, perturbed expression of all genes was observed in cumulus cells and in oocytes at the MI stage (7 h IVM). Expression of ND1, UQCRH, COX4 and ATP5O was downregulated in cumulus cells and upregulated in oocytes starting from 0.5 μmol/L BEA. Expression of TFAM, NDUFA12, COX1, COX2, ATP6, and ATP8 was upregulated starting from 1 μmol/L in cumulus cells and from 3 μmol/L in oocytes. Cumulus cells and oocytes displayed different gene expression patterns upon BEA exposure. The downregulation in cumulus cells of four genes coding for proteins of mitochondrial complexes could represent a major toxic event induced by BEA on the cumulus-oocyte complex which may result in mitochondrial functional alteration.

An assessment of the natural ochratoxin A (OTA) exposure of seven Martina Franca jennies was carried out by analyzing blood and milk samples collected close to and after delivery. A total of 41 and 34 blood samples were collected from jennies and foals, respectively, and analyzed by ELISA. A total of 33 milk samples were collected from jennies and analyzed by the HPLC/FLD method based on IAC clean-up. Furthermore, 53 feed samples were collected from January to September and analyzed by a reference method (AOAC Official Method No. 2000.03) for OTA content. Feed samples showed OTA levels up to 2.7 ng/g with an incidence of 32%, while the OTA incidence rate in jennies&rsquo, blood samples was 73%, with a median value of 97 ng/L and concentrations ranging from &lt, LOD to 6000 ng/L. A seasonal effect on OTA levels in positive blood samples was observed, with increases in the 53% of the positive ones from April to June. Concerning foals, the incidence rate of blood samples was 50%, with a median value of 52 ng/L, and concentrations ranged from &lt, LOD to 4034 ng/L. The incidence of milk samples was 36%, with levels ranging from &lt, LOD to 82 ng/L. In conclusion, the results showed a natural exposure of jennies and foals to OTA, and its presence in jenny milk could pose a risk for human newborns, considering its well-known nutritional and health properties.

Seventy-one (71) food samples were analyzed for the mycotoxin fumonisin by a monoclonal antibody based competitive enzyme-linked immunosorbent assay (ELISA). Fumonisins were detected primarily in corn-based products with 7/12, 2/2 and 1/3 and 1/7 yellow cornmeal, blue cornmeal, corn muffin mix, and mixed grain cereal samples yielding positive results, respectively. When the positive samples and randomly selected negative samples were assessed by other methods, correlations (r values) between ELISA and gas chromatography-mass spectrometry (GC-MS), ELISA and high-pressure liquid chromatography (HPLC) and GC-MS and HPLC were 0.478 (p0.05), 0.512 (p0.05), and 0.946 (p0.01), respectively. The results suggested that although the immunoassay could be used for screening of fumonisin in food samples, higher estimates were attained by ELISA than by the other two methods particularly in the more contaminated samples. These observations may result from differences in sample preparation among the methods or because of the presence of structurally related compounds in extracts that are detectable by ELISA but not the other two methods.

Beauvericin (BEA) is a mycotoxin produced by Beauveria bassiana and Fusarium species recently reported as toxic on porcine oocyte maturation and embryo development. The aim of this study was to assess, in the juvenile sheep, whether its effects are due to alterations of oocyte and/or embryo bioenergetic/oxidative status. Cumulus-oocyte-complexes (COCs) were exposed to BEA during in vitro maturation (IVM), evaluated for cumulus cell (CC) apoptosis, oocyte maturation and bioenergetic/oxidative status or subjected to in vitro fertilization (IVF) and embryo culture (IVEC). Oocyte nuclear maturation and embryo development were assessed after Hoechst staining and CC apoptosis was analysed by terminal deoxynucleotidyl transferase-mediated dUTP nick-End labeling assay and chromatin morphology after Hoechst staining by epifluorescence microscopy. Oocyte and blastocyst bioenergetic/oxidative status were assessed by confocal microscopy after mitochondria and reactive oxygen species labelling with specific probes. BEA showed various toxic effects, that is, short-term effects on somatic and germinal compartment of the COC (CCs and the oocyte) and long-term carry-over effects on developing embryos. In detail, at 5 µM, it significantly reduced oocyte maturation and immature oocytes showed increased late-stage (Type C) CC apoptosis and DNA fragmentation while matured oocytes showed unaffected CC viability but abnormal mitochondrial distribution patterns. At lower tested concentrations (3-0.5 µM), BEA did not affect oocyte maturation, but matured oocytes showed reduced mitochondrial activity. At low concentrations, BEA impaired embryo developmental capacity and blastocyst quality after IVF and IVEC. In conclusion, in the juvenile sheep, COC exposure to BEA induces CC apoptosis and oocyte mitochondrial dysfunction with negative impact on embryo development.

Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer with endocrine-disrupting properties. In this study, we used an equine model to investigate DEHP concentrations in ovarian follicular fluid (FF), and to determine the effects of exposure of oocytes to potentially toxic concentrations of DEHP during in vitro maturation (IVM) on embryo development and quality. Embryo development was evaluated using time-lapse monitoring (TLM), a photomicroscopic tool that reveals abnormalities in cleavage kinetics unobservable by conventional morphology assessment. Blastocyst bioenergetic/oxidative status was assessed by confocal analysis. The possibility that verbascoside (VB), a bioactive polyphenol with antioxidant activity, could counteract DEHP-induced oocyte oxidative damage, was investigated. DEHP was detected in FF and in IVM media at concentrations up to 60 nM. Culture of oocytes in the presence of 500 nM DEHP delayed second polar body extrusion, reduced duration of the second cell cycle, and increased the percentage of embryos showing abrupt multiple cleavage, compared with controls. Mitochondrial activity and intracellular levels of reactive oxygen species were reduced in blastocysts from DEHP-exposed oocytes. VB addition during IVM limited DEHP-induced blastocyst damage. In conclusion, DEHP is detectable in equine FF and culture medium, and oocyte exposure to increased concentrations of DEHP during IVM affects preimplantation embryo development. Moreover, TLM, reported for the first time in the horse in this study, is an efficient tool for identifying altered morphokinetic parameters and cleavage abnormalities associated with exposure to toxic compounds.

In this study, the naturally debittered table olives cv Bella di Cerignola were studied in order to (i) characterize their phenolic composition; (ii) evaluate the polyphenols bioaccessibility; (iii) assess their absorption and transport, across Caco2/TC7. LC-MS/MS analysis has confirmed the presence of hydroxytyrosol acetate, caffeoyl-6?-secologanoside, and comselogoside. In vitro bioaccessibility ranged from 7% of luteolin to 100% of tyrosol, highlighting the flavonoids sensitivity to the digestive conditions. The Caco2/TC7 polyphenols accumulation was rapid (60 min) with an efficiency of 0.89%; the overall bioavailability was 1.86% (120 min), with hydroxytyrosol and tyrosol the highest bioavailables, followed by verbascoside and luteolin. In the cells and basolateral side, caffeic and coumaric acids metabolites, probably derived from esterase activities, were detected. In conclusion, the naturally debittered table olives cv Bella di Cerignola can be considered as a source of bioaccessible, absorbable, and bioavailable polyphenols that, for their potential health promoting effect, permit inclusion of table olives as a functional food suitable for a balanced diet.

Although the cross-kingdom transfer of vegetable miRNAs (miRNAs) in mammalian species, including humans, is still controversial, recent studies have rejected this theory. Based on these recent studies, we hypothesized that artichoke-derived miRNAs (cca-miRNAs) are not adsorbed into human intestinal cells after cooking and in vitro digestion. In order to test this hypothesis, we evaluated miRNA (cca-miRNAs) in the edible part of globe artichokes (head portion), after cooking and digestion by an in vitro digestion system. The cca-miRNA levels were analyzed by real-time PCR (RT-qPCR), and those that withstood cooking and digestion conditions were further analyzed for their bioavailability using an in vitro system (Caco-2/TC7 cell clone). We detected 20 cca-miRNAs after cooking, 5 of which were statistically down-regulated in comparison with uncooked samples. Only 4 cca-miRNAs were found after in vitro digestion. By using scanning electron microscopy (SEM), we also evaluated the extracellular vesicles (EVs) in homogenized artichoke as possible miRNA transporters. However, approximately 81% were degraded after cooking, while the remaining EVs had changed shape from round to elliptical. Finally, we detected no cell-free cca-miRNAs, miRNAs bound to protein complex, and no cca-miRNAs encapsulated in EVs inside Caco-2 cells or in basolateral medium after bioavailability experiments. In conclusion, the data from the present study agrees with recent findings that the human small intestine does not uptake dietary miRNAs from raw or cooked artichoke heads. (C) 2018 Elsevier Inc. All rights reserved.

This investigation shows the influence of the fermentation process started with the probiotic Lactobacillus paracasei LMG P-22043 (PF) on the fate of health promoting polyphenols in artichoke in comparison with not started fermented artichokes (NSF). The fermentation process did not influence significantly the polyphenol compositions. However, low variations mainly related to the isomerization process of dicaffeoylquinic acids, probably due to the different pH values of the two samples, were observed. Further, the total polyphenol bioaccessibility of PF artichoke was lower (34%) than of NSF artichoke (64%) in small intestine. Moreover, both polyphenol isomerization and bioaccessibility influenced the antioxidant activity that was significantly lower for PF artichoke than for NSF ones. These effects were probably related to the bioprotective process exerted by the probiotic strain on the food matrix, with the consequent limited release of polyphenols in the digestion liquids, making them available for further metabolism and absorption in the colon.

In this study, the quali-quantitative composition of hydrophilic (phenolic acids) and lipophilic (isoprenoids) extracts from whole-meal flour of five elite Italian durum wheat cultivars was determined. Significant differences in the content of bioactive compounds were observed among the wheat extracts, in particular concerning the content of bound phenolic acids, lutein and β-tocotrienols. The cultivars Duilio and Svevo showed the highest amount of phenolic acids and isoprenoids, respectively. Extracts were evaluated for their anti-inflammatory activity on HT-29 human colon cells by measuring the levels of interleukin 8 (IL-8) and transforming growth factor β1 (TGF-β1). Durum wheat extracts significantly inhibited the secretion of the pro-inflammatory IL-8 mediator at 66 µg/mL of phenolic acids and at 0.2 µg/mL of isoprenoids. Conversely, the secretion of the anti-inflammatory mediator TGF-β1 was not modified by neither hydrophilic nor lipophilic extracts. These results provide further insight into the potential of durum wheat on human health suggesting the significance of varieties with elevated contents of bioactive components.

Ingestion of food is considered a major route of exposure to many contaminants including mycotoxins. The amount of mycotoxin resisting to the digestion process and potentially absorbable by the systemic circulation is only a smaller part of that ingested. In vitro digestion models turn useful for evaluating mycotoxins bioaccessibility during the intestinal transit and can be intended as a valuable tool for the assessment of mycotoxin bioavailability in food. In this paper we describe a study aimed at investigating toxicity of in vitro gastro-duodenal digests of mycotoxin contaminated bread collected along the digestion time-course. Toxicity tests were carried out on a sensitive RPMI lymphoid B cell line chosen as the most suitable lineage to assess toxicity retained by gastro-duodenal digests. In parallel, a chemical quantification of T-2 and HT-2 toxins contaminating the bread digests was accomplished during the gastric and duodenal transit. The digestive fluids undergoing chemical and toxicological analysis were collected at the beginning and end of gastric phase, and after completion of the duodenal phase. Results proved that a correlation between HT-2 content and toxicity did exist although a more persistent toxic activity was displayed in the later stage of the duodenal phase. This persistent toxicity might be explained by the co-occurrence of unknown HT-2-related conjugates or metabolites formed during digestion.

White table grape cv. Italia is a typical component of the Mediterranean diet and a source of phenolic compounds, particularly abundant in the skin portion. The aim of this study was to characterize the phenolic profile of the table grape skin and to assess its stability after the in vitro digestion process. The main phenolic compounds identified by the HPLC-DAD analysis were: procyanidin B1, caftaric acid, catechin, coutaric acid, quercetin 3-glucuronide and quercetin 3-glucoside. All compounds showed a good stability after in vitro digestion (from 43 to 80%). Moreover, the influence of grape skin polyphenols on the modulation of ROS and GSH levels was evaluated in basal and in stressed conditions on human intestinal cells (HT-29). In basal conditions, a higher polyphenol concentrations exerted pro-oxidant effect corresponding to high ROS level and low GSH content. This effect was probably due to the polyphenolic oxidation in cell culture condition with consequent production of hydrogen peroxide. Otherwise, in stressed conditions, grape skin polyphenols exerted antioxidant effects up to 1.3 × 10−6 μg/g and restored the stress-related GSH reduction. The in vitro digestion process attenuated the biological effect of grape skin polyphenols on intestinal cell line (HT-29). In conclusion, grape skin polyphenols showed different behavior in relation to their concentrations and to the intracellular ROS levels.

The effects of fermentation by autochthonous microbial starters on phenolics composition of Apulian table olives, Bella di Cerignola (BDC), Termite di Bitetto (TDB) and Cellina di Nardo (CEL) were studied, highlighting also the cultivars influence. In BDC with starter, polyphenols amount doubled compared with commercial sample, while in TDB and CEL, phenolics remain almost unchanged. The main phenolics were hydroxytyrosol, tyrosol, verbascoside and luteolin, followed by hydroxytyrosol-acetate detected in BDC and cyanidine-3-glucoside and quercetin in CEL. Scavenger capacity in both DPPH and CAA assays, assessed the highest antioxidant effect for CEL with starters (21.7 mg Trolox eq/g FW; 8.5 μmol hydroxytyrosol eq/100 g FW). The polyphenols were highly in vitro bioaccessible (>60%), although modifications in their profile, probably for combined effect of environment and microorganisms, were noted. Finally, fermented table olives are excellent source of health promoting compounds, since hydroxytyrosol and tyrosol are almost 8 times more than in olive oil.

The raw materials used in the production of bakery products are the main source of sporeforming bacteria associated with the alteration of "bread rope" which occurs predominantly in humid hot weather and which results in significant economic losses in the bakery product sector (Valerio et al., 2012 , Int. J. Food Microbiol, 156, 278-285). The spores, surviving the cooking process, germinate and the vegetative cells start a degrading process of starch and bread proteins leading to the formation of slime. In the present study a screening test was carried out on 176 isolates from raw materials to evaluate which species could cause alteration in bread. Bacillus amyloliquefaciens, together with B. subtilis and B. pumilus, were the main species capable of causing the spoilage. Moreover, among the identified species, strains belonging to the B. cereus group account for 17%, of which 38% were found to be able to cause bread rope. The B. cereus group strains belong to the phylogenetic groups III and IV associated with high risk of intoxication, particularly those of group III, for which a high thermal resistance of the spores has been demonstrated during a test that simulates the thermal profile of the baking process (De Bellis et al., 2015, Int. J. Food Microbiol., 197, 30-39). To estimate the risk of bread spoilage during shelf-life, Symp Previus tool was used: cardinal parameters and growth / no growth boundaries of three strains were determined for three B. amyloliquefaciens strains isolated from bread with clear alteration symptoms, wheat and semolina. In addition, challenge tests were conducted by inoculating the spores of one strain in the mixing phase: various bread formulations were prepared to evaluate their effect on bacterial growth and the experimental results were compared with silica simulations. Finally, to estimate the probability of product contamination during shelf-life, two storage temperatures and a threshold of 5 log cfu / g were considered, a value which could cause alteration and / or represent a risk to consumer health. The characterization of the growth behavior of the three strains showed a difference in the contamination probability and subsequent alteration of the product, demonstrating the need to consider biological variability in predictive microbiological studies in order to obtain a realistic estimate of the risk of contamination. In conclusion, this study demonstrated the applicability of predictive microbiological instruments, known for assessing the risk of food contamination from pathogenic microorganisms, to the study of the behavior of alterative microorganisms to control the quality of food (Valerio et al. 2015 Food Microbiol., 45, 2-9).

Cadmium is a highly toxic heavy metal with negative effects on oocyte fertilization. The aim of this study was to analyse whether cadmium-induced impairment of fertilization is caused by mitochondria dysfunction and oxidative stress in the cumulus-oocyte complex (COC). Preliminarily, 19 trace element levels were measured in ovaries from juvenile and adult ewes and age-related cadmium ovarian bioaccumulation at nanomolar concentrations was found. COCs from juvenile and adult ewes, exposed during in vitro maturation to 1 nM or 100 nM CdCl2, and subjected to in vitro fertilization showed significantly lower fertilization rates in exposed COCs compared with controls. In vitro matured exposed and control COCs underwent confocal microscopy analysis of mitochondria activity and reactive oxygen species (ROS) levels and lipid peroxidation (LPO) assay at cumulus cell and oocyte level. In both age groups, cadmium at nanomolar concentrations induced cumulus-oocyte mitochondria over-activity and oxidative damage which were related to impaired oocyte fertilization. (C) 2017 Elsevier Inc. All rights reserved.

Table olives are a typical food of the Mediterranean diet and an important source of phenolic compoundswith high biological potential for human health. Their concentrations (?1-2% of FW) confer to table olives antioxidant, anti-inflammatory, and antitumoral properties. The polyphenols content and composition in table olives can be affected by several factors, such as cultivars, climate, fruits ripeness, and, mainly, the processing methods. Among the de-bittering processes, the Greek method represents a spontaneous fermentation procedure that is driven by a mixed population of microorganisms, mainly consisting of yeasts and lactic acid bacteria (LAB). In this work, the effects of fermentation by autochthonous yeast and LAB starters on phenolics composition of Apulian table olives, Bella di Cerignola (BDC), Termite di Bitetto (TDB) and Cellina di Nardò (CEL) were studied in comparison with the commercial products. The samples were characterized by HPLC-DAD for their polyphenols composition; 18 compounds were identified and the cultivar related effect was highlighted. The main identified phenolics were hydroxytyrosol, tyrosol, verbascoside and luteolin, followed by hydroxytyrosol-acetate detected in BDC and cyanidine-3-glucoside and quercetin in CEL. Further, the fermentation using selected LAB and yeasts influenced differently the composition and amount of polyphenols of the three cultivars, in particular the BDC amount was doubled compared with the commercial sample. Instead, for TDB and CEL, the treatment did not influence the polyphenols composition. It is noteworthy that the use autochthonous microbial starter to drive table olives fermentation process allows to maintain stable or increases polyphenols concentration in fermented table olives, significantly reducing the time necessary for de-bittering and improving organoleptic and sensory characteristics of the final product. Scavenger capacity in both DPPH and CAA assays, assessed the highest antioxidant effect for CEL with starters (21.7 mg Trolox eq/g FW; 8.5 ?mol hydroxytyrosol eq/100g FW). Moreover, the polyphenols were highly in vitro bioaccessible (>60%), although modifications in their profile, probably for combined effect of environment and microorganisms, were noted. Finally, fermented table olives are excellent source of health promoting compounds. Indeed, hydroxytyrosol and tyrosol are almost 8 times more than in olive oil for which a nutritional EU claim (No 433/2012 of 23 May 2012) that attributes the protective effect from oxidative stress by polyphenols on blood lipids, has been established.

Artichoke (Cynara cardunculus (L.) subsp. scolymus Hayek) is an important component of the Mediterranean diet, and a good source of health-promoting compounds, including polyphenols. The main phenolic compounds of artichoke are 5-O-caffeoylquinic acid (chlorogenic acid), and 3,5-O-, 1,5-O-dicaffeoylquinic acids and its beneficial effects are linked to their antioxidant activity that can be affected by their stability under gastro-intestinal conditions, during digestion. This study aimed to assess the antioxidant activity induced by artichoke head extract, and its main individual polyphenols, on a stimulated HT-29 cell line, using a DCFH-DA probe. In addition, influences of in vitro digestion on artichoke head and its individual polyphenols towards antioxidant activity, were tested. Artichoke extract showed higher antioxidant activity than the individual polyphenols, even after in vitro gastro-intestinal digestion. In addition, in vitro digestion did not modify the antioxidant activity of artichoke polyphenols, except for 1,5-O-dicaffeoylquinic acid, which proved to be the least active.

The intestinal absorption of verbascoside (VB), a phenylpropanoid glycoside, extracted and purified from olive mill waste water (OMWW), was investigated using viable and healthy human colonic tissues, mounted in an Ussing chamber. Additionally, VB absorption and transport through the intestinal mucosa were quantified using permeability coefficients. VB absorption was time-dependent and varied in relation to the specific colonic segments considered. In particular, major uptake/absorption (0.50 μg/cm 2 ) occurred between 5 and 15 min in the proximal tract of the colon, followed by descending colon (0.38 μg/cm 2 ) between 30 and 60 min, and sigmoid–rectum colon (0.34 μg/cm 2 ) at 60 min. Overall, VB was absorbed rapidly, with an average uptake of 0.29 μg VB per cm 2 , corresponding to a total accumulation efficiency of ~ 0.12%. Moreover, the presence of the VB in the basolateral side supported the hypothesis of its bioavailability in the extent of 0.1%. In addition, the permeability coefficient calculation has contributed in a deeper understanding of VB absorption and transport across the human intestinal barrier and could be utilized for other polyphenols present in food and in dietary supplements.

The effects of verbascoside (VB), added at nanomolar concentrations during in vitro maturation (IVM) of juvenile sheep oocytes, on in vitro embryo development and its mechanisms of action at the oocyte level were analyzed. Developmental rates, after IVM in the presence/absence of VB (1nM for 24h; 1nM for 2h; 10nM for 2h), were evaluated. The bioenergetic/oxidative status of oocytes matured after IVM in the presence/absence of 1nM VB for 24h was assessed by confocal analysis of mitochondria and reactive oxygen species (ROS), lipid peroxidation (LPO) assay, and quantitative PCR of bioenergy/redox-related genes. The addition of 1nM VB during 24h IVM significantly increased blastocyst formation and quality. Verbascoside reduced oocyte ROS and LPO and increased mitochondria/ROS colocalization while keeping mitochondria activity and gene expression unchanged. In conclusion, supplementation with nanomolar concentrations of VB during IVM, in the juvenile sheep model, promotes embryo development by protecting the oocyte against oxidative stress.

Flow cytometry is a useful tool that provides an accurate, objective and rapid evaluation of semen quality. The use of this technique could significantly improve the quality of buffalo semen samples used in artificial insemination. This study was carried out to evaluate, by flow cytometry, frozen-thawed buffalo spermatozoa quality parameters such as sperm viability by SYBR-14/propidium iodide staining; mitochondrial function by JC-1 potentiometric probe; sperm chromatin stability (SCSA) by acridine orange; and acrosome reaction (AR) by FITC-PNA staining. Semen samples from five Italian Mediterranean buffalo bulls were used. Sperm viability was not different between bulls and ranged from 33.4% to 43.6%. A consistent rate (55.1 ± 10.8%) of sperm cells showed high mitochondrial membrane potential (Δψ(high)), with no significant differences between subjects. Sperm chromatin structure assay differed significantly between the five buffalo bulls; moreover, data showed high stability within each buffalo. DNA fragmentation indexes (DFI), such as %-DFI, -DFI, SD-DFI, were 11.2 ± 8.6, 153.3 ± 24.6 and 81.6 ± 21.2, respectively. Regarding AR, the percentage of acrosome-reacted live (ARL) and acrosome-reacted dead (ARD) spermatozoa was 0.3 ± 0.2 and 15.3 ± 5.5, respectively. This functional parameter differed significantly between buffalo bulls and showed high stability. Following to Ca(2+) ionophore A23187 for 3 h, AR significantly differed between subjects and was characterized by an increase in both ARL (10.8%) and ARD population (22.0%). This study indicates that flow cytometry could be a useful tool for a quick multiparametric evaluation of sperm quality in buffalo. In particular, SCSA and AR resulted in sperm functional parameters sensitive enough for the diagnosis of frozen-thawed semen fertilizing potential.

Olive oil processing industries generate substantial quantities of phenolic-rich byproducts, which could be valuable natural sources of antioxidants. This work is focused on the recovery and structural characterization of antioxidant compounds from olive mill wastewater (OMWW), a polluting byproduct of the olive oil production process. Phenolics were extracted from the waste material using a membrane technology coupled to low-pressure gel filtration chromatography on a Sephadex LH-20. The LH-20 fraction was, in turn, characterized for its phenolic composition by HPLC-DAD-MS/MS analyses. Verbascoside, isoverbascoside, β-hydroxyverbascoside, β-hydroxyisoverbascoside, and various oxidized phenolics were identified. Uptake of verbascoside, purified from the LH-20 fraction, by HT-29 cells, an established model system for studying drug transport properties, was also assayed. Finally, the antioxidant activities of the LH-20 fraction and verbascoside were characterized by two different techniques. Individual verbascoside was more active as a scavenger of reactive oxygen species and as a chemopreventive agent protecting low-density lipoproteins from oxidative damage than the LH-20 fraction.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm motility by CASA system. Fumonisin B1 did not affect viability of fresh spermatozoa after 2 h exposure up to 25 μM. Damage on sperm chromatin structure was observed only in one frozen sample after exposure up to 2.5 × 10 −5 μM FB1 without associated increase of ROS. Increase of ROS, at FB1 levels up to 2.5 × 10 −4 μM, was found on another frozen-thawed sperm sample, may be as a consequence of seminal plasma removal. At 7.5 and 15 μM, FB1 induced reduction of total and progressive motility.

The purpose of this study was to assess the natural exposure of male horses (Equus caballus) to the mycotoxin zearalenone (ZEA) by using the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on sperm chromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatin structure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n = 27) horses were tested on frozen-thawed spermatozoa to evaluate the toxic effect of mycotoxin on their chromatin structure by flow cytometry. Different parameters of the DNA fragmentation index (DFI), such as the mean ( X ¯ - DFI ), the percentage (%-DFI), and the standard deviation (SD-DFI), were analyzed. Urine samples showed a mean level of 32.3 ng/mL ZEA with significantly higher concentrations in northeastern European samples than in Italian samples, probably in relation to climatic and feeding differences. The toxic effects of ZEA-containing urine samples on SCSA parameters were found at low ZEA concentrations and were mainly observed in Italian samples. By using mycotoxin standards, ZEA, α-zearalenol, and β-zearalenol proved to be more toxic compounds for sperm chromatin stability than other tested derivatives. A nongenomic mechanism of action can be hypothesized.

Farm animals are exposed to zearalenone through the feed because of the widespread occurrence of this mycotoxin in cereals and clinical reproductive disorders due to mycotoxin effects are often reported in farm animal species. This review describes the metabolism, the mechanistic aspects, the clinical reproductive symptoms and the in vitro effects on functional parameters of oocytes and sperm cells induced by zearalenone and its derivatives in farm animals. The studies on in vitro effects allow to understand the action mechanisms of mycotoxins and, sometime, to explain the in vivo symptoms. The impairment of semen quality and female reproductive function induced by zearalenone could be a factor responsible for the reproductive failure in farm animals.

Raw materials used in bread-making process may be a rich source of spore-forming bacteria whose presence after cooking may represent a spoilage concern for bakery industries and a risk to consumer health. The aim of this study was to investigate the toxigenic potential of 54 spore-forming bacterial strains isolated from bread ingredients and bread, mainly of the Bacillus genus, and their resistance to a thermal treatment reproducing the bread cooking process to ascertain if they could represent a health concern for consumers. The potential toxigenicity of the strains was evaluated by screening the cytotoxic activity on HT-29 cells using bacterial culture filtrates after growing bacterial cells in BHI and in the bread-based medium BEB. The results showed a high cytotoxic activity of B. cereus strains, although it was lower in BEB medium. PCR analyses detected the presence of genes involved in the production of NHE, HBL or CytK toxins in B. cereus strains, while none of the tested strains contained the gene for cereulide production. Production of NHE and HBL toxins was also confirmed by specific immunoassays only for B. cereus strains. Cytotoxic activity of 13 strains belonging to B. amyloliquefaciens (7 strains), Paenibacillus spp. (3) B. mojavensis (1), B. simplex (1) and B. pumilus (1) was also detected. Interestingly, B. cereus strains assigned to phylogenetic group IV exhibited a thermal resistance markedly lower than B. cereus group III; furthermore, B. amyloliquefaciens strains almost completely survived the heat treatment, but showed a low cytotoxic activity. It is also relevant that single strains belonging to B. mojavensis and B. simplex showed a cytotoxic activity higher after growth in BEB than in BHI and a spore resistance enough to survive the bread cooking process. In conclusion, our study indicates that spore-forming bacteria could represent a risk to consumer health related to strains able to produce toxic substances and to survive bread cooking conditions.

Fusarium is a genus frequently contaminating different agricultural products and is also a renown producer of Trichothecenes1. This group of mycotoxins can be further subdivided into subgroups with T-2/HT-2 toxins and deoxynivalenol being the main representatives of the type A and B respectively. In general Trichothecenes are responsible for a wide variety of toxic effects in humans and animals. Within the type class A, in particular, T-2 and HT-2 toxins represent the most acutely toxic compounds2 and have been lately reviewed by EFSA3. In human health risk assessment, ingestion of contaminated food is considered the major route of human exposure to these compounds although only a smaller amount will be further available for absorption during its transit along the gut4. In vitro digestion models have been used to evaluate mycotoxins bioaccessibility during the intestinal transit. As a result, the bioaccessibility can be calculated representing a measure for the assessment of mycotoxin bioavailability in food. Some investigations have been already carried out in the past by our group in order to assess T-2 and HT-2 bioaccessibility in bread chosen as reference food model5 In this note we describe a study aimed to investigate toxicity of gastro-duodenal digests of mycotoxins contaminated bread samples collected along the digestion time-course, assayed on RPMI lymphoid B cell line, as a sensitive cellular model. In parallel, a chemical quantification of T-2 and HT-2 toxins contaminating the bread digests was accomplished along the gastric and duodenal phase. The digestive fluids undergoing chemical and toxicological analysis, were collected at the beginning and end of the gastric phase, and after completion of the duodenal phase Results proved that a correlation between HT-2 content and toxicity did exist in digestive fluids although a more persistent toxic activity was noticed in the later stage of the duodenal phase.

Artichoke is a rich source of health promoting compounds such as polyphenols, important for their pharmaceutical and nutritional properties. In this study, the potential for bioavailability of the artichoke polyphenols was estimated by using both in vitro digestion and Caco-2 human intestinal cell models. In vitro digestive recoveries (bio-accessibility) were found to be 55.8% for total artichoke phenolics and in particular, 70.0% for chlorogenic acid, 41.3% for 3,5-O-dicaffeoylquinic acid, and 50.3% for 1,5-O-dicaffeoylquinic acid, highlighting potential sensitivity of these compounds to gastric and small intestinal digestive conditions. Uptake of artichoke polyphenols was rapid with peak accumulation occurring after 30 min with an efficiency of 0.16%, according to the poor uptake of dietary polyphenols. Some compounds, such as coumaric acid, caffeic acid and caffeic acid derivatives, were also detected in the basolateral side assuming an extra and intracellular esterases activities on chlorogenic acid. Only apigenin-7-O-glucoside was absorbed and transported through the Caco-2 monolayer demonstrating its bioavailability in the extent of 1.15% at 60 min. In addition, permeability coefficient (Papp=2.29 x 10-5 cm/sec), involving apical to basolateral transport of apigenin 7-O-glucoside, was calculated to facilitate estimation of absorption and transport through Caco-2 monolayer. Finally, the mono and dicaffeoylquinic acids present in artichoke heads, exert an antioxidant activity on human low density lipoprotein system correlated to their chemical structure. In conclusion, the utilized in vitro models, although not fully responding to the morphological and physiological features of human in vivo conditions, could be a useful tool for investigating mechanistic effects of polyphenols released from food matrix.

Le micotossine, frequenti contaminanti degli alimenti, rappresentano un serio problema per la salute umana e animale. Lo studio sulla tossicità indotta da naturali livelli di esposizione alle micotossine è necessario per una valutazione del rischio di tali sostanze. Recentemente è stato individuato per alcune micotossine, quali le fumonisine (FBs) e l'ocratossina A (OTA), l'induzione di stress ossidativo come principale e precoce meccanismo di tossicità in studi in vitro. Si è voluto verificare se questo meccanismo di tossicità fosse presente in altri organi target e fosse determinato da naturali livelli di esposizione. Nel caso delle FBs lo studio è stato condotto sull'intestino, principale target di esposizione. Si è utilizzato un modello che ha riprodotto fisiologicamente l'esposizione intestinale alle micotossine con gli alimenti; in particolare sono stati sottoposti a un processo di digestione di vitro (1) campioni di mais naturalmente o artificialmente contaminati da FBs e i campioni di chimo ottenuti sono stati utilizzati per una breve esposizione di porzioni di intestino (umano e di ratto) tramite la camera di Ussing. E' stata registrata una riduzione del parametro elettrico della corrente di cortocircuito in porzioni di intestino esposti a chimo senza FBs (conseguente all'osmolalità del chimo). Questa variazione del trasporto ionico non si è ritrovata in porzioni di intestino esposti a chimo contenente FBs (probabilmente per interferenza del trasporto ionico del Na+) con conseguente sbilancio idrico-salino. Sui campioni di intestino esposti ai diversi campioni di chimo, si è anche osservato un aumento dei livelli di malondialdeide (MDA), marker di tossicità dello stress ossidativo fortemente influenzato dall'effetto della digestione, dai livelli di FBs e dall'origine dei campioni intestinali (ratto e uomo). Questi risultati testimoniamo come lo stress ossidativo rappresenti anche in studi ex vivo su porzioni di intestino il principale meccanismo di tossicità (2, 3). Nel caso dell'OTA, l'induzione dello stress ossidativo, evidenziato in diversi modelli cellulari, è stato anche ritrovato in cellule mesenchimali staminali ottenute da cordone ombelicale prelevato da cagne, un utile modello in vitro per valutare la tossicità riproduttiva e dello sviluppo fetale. Dopo esposizione a livelli di OTA sovrapponibili a quelli serici riportati in bibliografia (da0.25 pM a 25 nM), si è osservato un effetto inibitorio dei parametri cinetici relativi alla proliferazione cellulare e al doubling time a tutte le concentrazioni di OTA testate. L'effetto tossico indotto dall'OTA sulla proliferazione era secondario all' induzione di un processo apoptotico, osservato tramite analisi confocale della condensazione e frammentazione della cromatina. Il danno cromatinico era conseguente a stress ossidativo, come evidenziato dall'aumento di fluorescenza generato dal danno ossidativo del DNA indotto dall'OTA e valutato con il kit 8-hydroxy-2'-deoxyguanosine (Biotrin) (4Rutigliano et al, 2015). Il riscontro di questo effetto tossico indotto da bassi livelli di OTA, simili a quelli serici, potrebbe essere correlato con l'elevato effetto embriotossico e teratogeno dell'OTA riportato in bibliografia. Parte del lavoro è stato finanziato dal progetto MycoRed EC KBBE -2007-222690-2. 1) Versantvoort, C.H., Oomen, A., Van de Kamp, E., Rompelberg, C.J.M., Sips, A.J.A.M., 2005. Applicability of an in vitro digestion model in assessing the bioaccessibility of mycotoxins from food. Food Chem. Toxicol. 43, 31-40. ; 2) Minervini F., Debellis L, Garbetta A., De Girolamo A., Schena R., Portincasa P., Visconti A., 2014. Influence on functional parameters of intestinal tract induced by short-term exposure to fumonisins contaminated corn chyme samples. Food ChemToxicol 66,166-172. 3) Garbetta A., Debellis L., De Girolamo A,. Schena R ,. Visconti A., Minervini F.,2015. Dose-dependent lipid peroxidation induction on ex vivo intestine tracts exposed to chyme samples from fumonisins contaminated corn samples. Toxicol in Vitro 29, 1140-1145.4) Rutigliano L., Valentini L., Martino N. A., Pizzi F., Zanghì A., Dell'Aquila M. E., Minervini F. 2015. Ochratoxin A at low concentrations inhibits in vitro growth of canine umbilical cord matrix mesenchymal stem cells through oxidative chromatin and DNA damage. Reprod. Toxicol. 57, 121-129.

Ochratoxin A (OTA) exposure during pregnancy in laboratory animals induces delayed/abnormal embryo development. Foetal adnexa-derived mesenchymal stem cells (MSCs) could help evaluate the developmental risk of exposure to chemicals in advanced gestational age. We tested the effects of OTA at concentrations ranging from 2.5×10(-4) to 25nM on growth parameters of canine umbilical cord matrix (UCM)-derived MSCs. The hypothesis that oxidative chromatin and DNA damage could underlie OTA-mediated cell toxicity was also investigated. After in vitro exposure, OTA significantly decreased cell density and increased doubling time in a passage- and concentration-dependent manner and no exposed cells survived beyond passage 5. Significantly higher rates of cells showed condensed and fragmented chromatin and oxidized DNA, as assessed by OxyDNA assay. These findings showed that in vitro exposure to OTA, at picomolar levels, perturbs UCM-MSC growth parameters through oxidative chromatin and DNA damage, suggesting possible consequences on canine foetal development.

Ochratoxin A (OTA) exposure during pregnancy in laboratory animals induces delayed/abnormal embryodevelopment. Foetal adnexa-derived mesenchymal stem cells (MSCs) could help evaluate the devel-opmental risk of exposure to chemicals in advanced gestational age. We tested the effects of OTA atconcentrations ranging from 2.5 × 10-4to 25 nM on growth parameters of canine umbilical cord matrix(UCM)-derived MSCs. The hypothesis that oxidative chromatin and DNA damage could underlie OTA-mediated cell toxicity was also investigated. After in vitro exposure, OTA significantly decreased celldensity and increased doubling time in a passage- and concentration-dependent manner and no exposedcells survived beyond passage 5. Significantly higher rates of cells showed condensed and fragmentedchromatin and oxidized DNA, as assessed by OxyDNA assay. These findings showed that in vitro exposureto OTA, at picomolar levels, perturbs UCM-MSC growth parameters through oxidative chromatin andDNA damage, suggesting possible consequences on canine foetal development.

Many man-made chemicals (pesticides) and naturally occurring compounds (mycotoxins and phytoestrogens) can enter the food chain and bind to estrogen receptors (ERs). Mycotoxins, including zearalenone (ZEA) and its derivatives, can occur worldwide in cereals and cause several health disorders. In order to characterize the estrogenic activity of zearalenone and its derivatives (alpha-zearalenol (alpha-ZEA), beta-zearalenol (beta-ZEA), alpha-zearalanol (alpha-ZAL) and beta-zearalanol (beta-ZAL)), the proliferation of ER-positive (MCF-7) and ER-negative (MDA-MB-231) human breast cancer cell lines was measured. After exposure at levels ranging from 0.1 pM to 0.1microM, cell proliferation (E-screen assay) was evaluated by MTT test through estrogenic parameters. On the MCF-7 cell line, estrogenic concentration that induced 50% cellular proliferation (EC(50)) of beta-zearalenol was statistically higher (5.2 x 10(-3)microM) than those of other zearalenone-related compounds, in agreement with other authors. All mycotoxins showed similar estrogenic parameters, with the exception of alpha-zearalenol that induced a higher proliferative effect (PE=2.6) and relative proliferative potency (RPP=7). Since MCF-7 contains both ERalpha and ERbeta-positive cells, at the mRNA and protein level, the estrogenic activity induced by mycotoxins may be ER-mediated, particularly through ERalpha that was the predominant ER subtype in these cells. A partial antagonism of mycotoxin-related estrogenic proliferation was seen when tamoxifen was used, confirming a receptor-dependent estrogenic response. MDA-MB-231 cells did not show ERs and after exposure to mycotoxins or 17beta-estradiol marginal PE values related to growth variability of MDA-MB-231 were found. Further studies are needed to understand in human tissues the mechanisms of action of ZEA and its derivatives that may be found as contaminants in the human diet.

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON) and fumonisin B1 (FB1) were studied in the K562 human erythroleukemia cell line using the Trypan Blue, MTT and BrdU uptake analyses of cytotoxicity, cell metabolism and cell proliferation, respectively. Nuclear staining with propidium iodide and DNA analysis by flow cytometry were used to identify apoptosis and cell cycle distribution. By the MTT and BrdU tests, both NIV and DON were significantly more toxic than FB1 by at least one order of magnitude, with ID50s ranging from 0.5 microM for NIV to 70 microM for FB1. The MTT test indicated that NIV was significantly (approximately four times) more toxic than DON. In contrast, the Trypan Blue test did not reveal any effects of mycotoxin exposure suggesting that, at the concentrations tested, NIV, DON and FB1 did not induce cytotoxicity through plasma membrane damage. Cell cycle analysis suggested apoptotic cytotoxicity, revealing 100% cellular debris at the highest concentrations of NIV and DON and approximately 2.9 times more debris than control at the highest FB1 concentration. Morphological evidence of apoptosis was related to the toxicity of the substances, such that the more toxic NIV and DON resulted in more late stage apoptotic events than FB1. This study suggests that human blood cells are sensitive to mycotoxin exposure, that NIV is more toxic than DON which is more toxic than FB1, and that DNA damage and apoptosis rather than plasma membrane damage and necrosis may be responsible for the observed cytotoxicity.

Verbascoside (VB) is a bioactive polyphenol from olive oil mill wastewater with known antioxidant activity. Oxidative stress is an emerging problem in assisted reproductive technology (ART). Juvenile ART is a promising topic because, in farm animals, it reduces the generation gap and, in human reproductive medicine, it helps to overcome premature ovarian failure. The aim of this study was to test the effects of VB on the developmental competence of ovine prepubertal oocytes and the bioenergetic/oxidative stress status of fresh and vitrified oocytes. In fresh oocytes, VB exerted prooxidant short-term effects, that is, catalase activity increase and uncoupled increases of mitochondria and reactive oxygen species (ROS) fluorescence signals, and long-term effects, that is, reduced blastocyst formation rate. In vitrified oocytes, VB increased ROS levels. Prooxidant VB effects in ovine prepubertal oocytes could be related to higher VB accumulation, which was found as almost one thousand times higher than that reported in other cell systems in previous studies. Also, long exposure times of oocytes to VB, throughout the duration ofin vitromaturation culture, may have contributed to significant increase of oocyte oxidation. Further studies are needed to identify lower concentrations and/or shorter exposure times to figure out VB antioxidant effects in juvenile ARTs.

[object Object]The gastrointestinal tract is the main target of exposure to mycotoxin fumonisin B1 (FB1), common natural contaminant in food. Previous studies reported that proliferating cells are more sensitive than confluent cells to the toxic effect of FB1. This study aims to investigate, by dose- and time-dependent experiments on human colon proliferating intestinal cell line (HT-29), the modifications induced by FB1 at concentrations ranging from 0.25 to 69 lM. The choice of highest FB1 concentration considered the low toxicity previously reported on intestinal cell lines, whereas the lowest one corresponded to the lower FBs levels permitted by European Commission Regulation. Different functional parameters were tested such as cell proliferation, oxidative status, immunomodulatory effect and changes in membrane microviscosity. In addition FB1- FITC localization in this cell line was assessed by using confocal laser scanning microscopy. Lipid peroxidation induction was the main and early (12 h) effect induced by FB1 at concentrations ranging from 0.5 to 69 lM, followed by inhibition of cell proliferation (up to 8.6 lM), the immunomodulatory effect (up to 17.2 lM), by assessing IL-8 secretion, and increase in membrane microviscosity (up to 34.5 lM). The toxic effects observed in different functional parameters were not dose-dependent and could be the consequence of the FB1 intracytoplasmatic localization as confirmed by confocal microscopy results. The different timescales and concentrations active of different functional parameters could suggest different cellular targets of FB1.

Fifty-four spore-forming bacterial strains isolated from bread ingredients and bread, mainly belonging to the genus Bacillus (including Bacillus cereus), together with 11 reference strains were investigated to evaluate their cytotoxic potential and heat survival in order to ascertain if they could represent a risk for consumer health. Therefore, we performed a screening test of cytotoxic activity on HT-29 cells using bacterial culture filtrates after growing bacterial cells in Brain Heart Infusion medium and in the bread-based medium Bread Extract Broth (BEB). Moreover, immunoassays and PCR analyses, specifically targeting already known toxins and related genes of B. cereus, as well as a heat spore inactivation assay were carried out. Despite of strain variability, the results clearly demonstrated a high cytotoxic activity of B. cereus strains, even if for most of them it was significantly lower in BEB medium. Cytotoxic activity was also detected in 30% of strains belonging to species different from B. cereus, although, with a few exceptions (e.g. Bacillus simplex N58.2), it was low or very low. PCR analyses detected the presence of genes involved in the production of NHE, HBL or CytK toxins in B. cereus strains, while genes responsible for cereulide production were not detected. Production of NHE and HBL toxins was also confirmed by specific immunoassays only for B. cereus strains even if PCR analyses revealed the presence of related toxin genes also in some strains of other species. Viable spore count was ascertained after a heat treatment simulating the bread cooking process. Results indicated that B. amyloliquefaciens strains almost completely survived the heat treatment showing less than 2 log-cycle reductions similarly to two strains of B. cereus group III and single strains belonging to Bacillus subtilis, Bacillus mojavensis and Paenibacillus spp. Importantly, spores from strains of the B. cereus group IV exhibited a thermal resistance markedly lower than B. cereus group III with high values of log-cycle reductions. In conclusion, our results indicate that spore-forming bacteria contaminating bread ingredients and bread could represent a source of concern for consumer health related to the presence of strains, such as strains of B. cereus group III and single strains of other species, showing the ability to produce toxic substances associated to a thermal resistance enough to survive the bread cooking conditions.

Background The aim of this study was to investigate the in vitro effects of the Fusarium fungus-derived mycotoxin, zearalenone and its derivatives alpha-zearalenol and beta-zearalenol on motility parameters and the acrosome reaction of stallion sperm. Since the toxic effects of zearalenone and its derivatives are thought to result from their structural similarity to 17beta-estradiol, 17beta-estradiol was used as a positive control for 'estrogen-like' effects. Methods Stallion spermatozoa were exposed in vitro to zearalenone, alpha-zearalenol, beta-zearalenol or 17beta-estradiol at concentrations ranging from 1 pM - 0.1 mM. After 2 hours exposure, motility parameters were evaluated by computer-assisted analysis, and acrosome integrity was examined by flow cytometry after staining with fluoroscein-conjugated peanut agglutinin. Results Mycotoxins affected sperm parameters only at the highest concentration tested (0.1 mM) after 2 hours exposure. In this respect, all of the compounds reduced the average path velocity, but only alpha-zearalenol reduced percentages of motile and progressively motile sperm. Induction of motility patterns consistent with hyperactivation was stimulated according to the following rank of potency: alpha-zearalenol >17beta-estradiol > zearalenone = beta-zearalenol. The hyperactivity-associated changes observed included reductions in straight-line velocity and linearity of movement, and an increase in the amplitude of lateral head displacement, while curvilinear velocity was unchanged. In addition, whereas alpha- and beta- zearalenol increased the percentages of live acrosome-reacted sperm, zearalenone and 17beta-estradiol had no apparent effect on acrosome status. In short, alpha-zearalenol inhibited normal sperm motility, but stimulated hyperactive motility in the remaining motile cells and simultaneously induced the acrosome reaction. Beta-zearalenol induced the acrosome reaction without altering motility. Conversely, zearalenone and 17beta-estradiol did not induce the acrosome reaction but induced hyperactive motility albeit to a different extent. Conclusions Apparently, the mycotoxin zearalenone has 17beta-estradiol-like estrogenic activity that enables it to induce hyperactivated motility of equine sperm cells, whereas the zearalenol derivatives induce premature completion of the acrosome reaction and thereby adversely affect stallion sperm physiology. The alpha form of zearalenol still possessed the estrogenic ability to induce hyperactivated motility, whereas its beta stereo-isomere had lost this property.

Olive oil production generates large amounts of recalcitrant compounds, the olive oil mill wastewater (OMWW), which represent one of the most contaminating effluents among those produced by the agrofood industries. Nowadays, this view has changed to one that recognizes the waste as a low-cost starting material rich in bioactive compounds, particularly biophenols, that can be extracted and applied as natural antioxidants for the food and pharmaceutical industries. The data reported in this paper indicate that the OMWW extracts, besides low molecular weight antioxidant phenolics such as tyrosol and hydroxytyrosol, also contain phenolics with a molecular weight in the range of 600-5000 Da, which exhibit efficient scavenging activities against hydroxyl and peroxyl radicals. This group of phenolics includes, besides verbascoside, isoverbascoside, and an oxidized form of verbascoside, a number of higher molecular weight phenolics arising from oxidative polymerization of hydroxytyrosol and caffeic acid. Overall, these higher molecular weight phenolics prove to be, in some in vitro tests, more efficient scavengers of hydrophilic hydroxyl radicals than hydroxytyrosol, which could be used for industrial applications as natural nontoxic antioxidants.

Zearalenone (ZEA) is a fusariotoxin naturally occurring in crops with known estrogenic activity in swine, the most sensitive known species. The metabolism by swine of ZEA, principally into alpha-zearalenol (alpha-ZOL), is considered as a bio-activation because of its high affinity with estrogenic receptors. Discordant data on male reproductive failures induced by ZEA in vivo are described. In this study, we evaluated the effects to boar spermatozoa when they are exposed in vitro to ZEA and its derivatives (alpha-ZOL, beta-ZOL). We analyzed viability, apoptosis (terminal deoxynucleotidyltransferase dUTP nick end-labelling (TUNEL)), sperm chromatin stability (sperm chromatin structure assay (SCSA)) and motility (using computer-aided sperm analysis (CASA)). Each mycotoxin influenced a specific function of spermatic cells. alpha-Zearalenol and ZEA, at picomolar levels, negatively influenced chromatin structure stability and viability, respectively, whereas beta-ZOL negatively influenced the sperm motility at micromolar levels. This study is the first using these direct measures of sperm integrity to show the potential for an adverse effect of ZEA exposure on boar fertility.

Verbascoside (VB), a bioactive polyphenol from olive mill wastewater with known antioxidant activity, was shown to act as a pro-oxidant molecule, by impairing energy/redox status and embryo developmental competence of prepubertal ovine oocytes when added at micromolar concentrations in a continuative 24-h in vitro maturation (IVM) exposure protocol (1). The aim of the present study was to determine whether a lower (nanomolar) VB concentration and a shorter exposure time (2 v. 24 h) during IVM may improve the maturation rates of prepubertal ovine oocytes and their subsequent embryonic development in vitro. Cumulus-oocyte complexes derived from the ovaries of slaughtered 1-mo-old prepubertal sheep oocytes underwent IVM in TCM 199 with 10% oestrus sheep serum, 0.1 IU mL–1 of FSH/LH, and 100 µM cysteamine, in 5% CO2 in air at 38.5°C for 24 h. Based on our previous results (Dell'Aquila et al. 2014 Biomed. Res. Int. 2014, 878062), VB was added in the IVM medium at 1.03 nM, and 2 incubation times (24 and 2 h) were tested. In the 2-h exposure group, after 2 h of exposure to VB, oocytes were washed and cultured up to 24 h without VB. A group of oocytes were cultured in absence of VB, as controls. Matured oocytes were fertilized with frozen-thawed ram semen in SOF medium for 22 h and zygotes were cultured in vitro for 8 days. Metaphase II (MII) cleavage and blastocyst rates were analysed by Chi-squared test. Embryo quality was evaluated by staining and total cell count of the blastocyst and analysis of variance (ANOVA) was applied. Differences were considered to be significant when P 0.05). However, this treatment allowed to obtain significantly higher rates of cleaved embryos/MII oocytes (156/196, 80% v. 165/226, 73%; respectively; P