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In vitro embryo production has grown in recent decades due to its great potential for cattle production. However, the quality of in vitro-produced embryos is lower compared with those produced in vivo. The postfertilization culture environment has a major influence on bovine embryo quality. We hypothesize that the inclusion of the inclusion of alpha-lipoic acid (ALA) in the in vitro culture (IVC) medium during the first 24 h would have positive effects on embryo development in vitro and cryotolerance. The aims of this study were to evaluate the antioxidant effect of ALA in IVC medium for 24 h on bovine zygotes (21 h post in vitro fertilization, IVF), day 2 cleaved embryos (46 h post-IVF), and to assess embryo quality, developmental competence, and cryotolerance after vitrification. In all experiments, IVC medium was the Control, and 2.5 μM ALA was the treatment implemented. Viability and reactive oxygen species (ROS) levels in zygotes and day 2 embryos did not differ from the Control (P 0.05). Supplementation with ALA increased total blastocyst and hatching rates (P 0.05). It also improved embryo quality, evidenced by the increased blastocyst total cell number and the percentage of excellent-quality embryos observed (P 0.05). In embryos cultured with ALA and then vitrified, ALA reduced intracellular ROS levels in warmed blastocysts (P 0.05). In conclusion, ALA supplementation to IVC medium during 24 h is a new advantage in improving embryo quality for assisted bovine reproduction.

Ghrelin is a gut hormone related to energy balance and reproductive functions. The aim of this study was to evaluate the effect of ghrelin antagonist D-Lys3-GHRP-6 (GA) as a potential agent that prevents ghrelin effects during bovine oocyte maturation on progesterone production, cumulus cell (CC) viability, CC DNA damage and embryo development and hatching rates. Ghrelin's potential to induce oxidative stress in cumulus-oocyte complexes (COC) was also evaluated. COCs were cultured for 24 hr in medium without supplementation (C) or supplemented with 60 pM ghrelin (Ghrelin60), Ghrelin60 + 20 pM GA (GA20), Ghrelin60 + 60 pM GA (GA60) or Ghrelin60 + 100 pM GA (GA100) for experiment I. For experiment II, C and Ghrelin60 treatments were used. Differences between C and Ghrelin60 and the linear or quadratic association between GAs on Ghrelin60 were evaluated. Results demonstrated that Ghrelin60 increased progesterone concentration, reduced CC viability, induced CC DNA damage and decreased blastocyst and hatching rate compared with C (p

The aim of this study was to evaluate the association between plasma copper (Cu) concentration and ovarian function during a fixed-time artificial insemination (FTAI) protocol and the effect of parenteral Cu administration (100 mg) at the start of such protocol (day 0) on area of preovulatory follicle (APF); area of corpus luteum (ACL), plasma estradiol (E2), and progesterone (P4) concentrations; CL blood flow (CLBF); and pregnancy rate in beef heifers and cows. In cows, plasma Cu concentration on days 0 and 7 correlated positively with APF. Copper administration increased plasma Cu concentration and decreased APF and plasma E2 concentration (day 9), without modifying ACL, plasma P4 concentration, and CLBF (day 16) in cows. Pregnancy rate was higher in Cu-supplemented cattle on day 41 after FTAI as compared with controls (58.76 and 45.28%, respectively). In conclusion, Cu administration at the beginning of the FTAI protocol increased pregnancy rate in beef heifers and cows, modifying APF and plasma E2 concentration in the latter., Instituto de Genética Veterinaria

The aim of this study was to evaluate the genotoxic and cytotoxic effects of amitraz (AMZ) on the primary culture of bovine cumulus cells (CC) and oocyte nuclear maturation. Cytotoxicity was evaluated by assessing mitochondrial activity with the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Genotoxicity was estimated using the alkaline single cell gel electrophoresis (SCGE) assay. Apoptosis was detected with the Annexin V-affinity assay. The in vitro maturation test was performed in bovine oocytes. To understand AMZ action, glutathione content, superoxide dismutase enzyme activity, and lipid peroxidation were evaluated in CC. Results showed that AMZ lethal concentration (LC 5024h) for bovine CC was 32.55 μg/mL (MTT assay). A 25 μg/mL induced late apoptosis and necrotic cells (p < 0.05); however, DNA damage was decreased at the same concentration (SCGE assay; p < 0.05). A decrease in metaphase II was observed at 25 μg/mL, and degenerate oocytes were observed at 15 and 25 μg/mL (p < 0.05). None of the oxidative stress parameters evaluated showed significant differences. This study contributes to a better understanding of AMZ in this model, suggesting its potential cytotoxicity and impact on bovine reproduction., Instituto de Genética Veterinaria

The effect of doramectin (DOR) was tested on two experimental somatic bovine cells in vitro: peripheral lymphocytes (PL) and cumulus cells (CC). The cytotoxicity and genotoxicity of DOR were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, single cell gel electrophoresis assay (SCGE) and cytokinesis-block micronucleus cytome (CBMN Cyt) assay. Both cells were treated with three concentrations of DOR (20, 40, 60 ng mL–1) for 24 h. The results obtained from PL demonstrated that DOR was able to induce cytotoxic effect and DNA damage with all concentrations tested. Additionally, DOR increased micronuclei (MNi) frequency and nuclear buds (NBuds) with 20, 40, 60 ng mL–1, and nucleoplasmic bridges (NPBs) only with 40 ng mL–1. On the other hand, the three concentrations of DOR were not able to induce cytotoxic effect and DNA damage using SCGE in the bovine CC. Nevertheless, the two higher concentrations of DOR (20, 40 µg mL–1) significantly increased the frequency of micronucleus formation in bovine CC. These results represent the first experimental evidence of genotoxic and cytotoxic effects exerted by DOR on bovine PL and CC. Fil: Anchordoquy, Juan Mateo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Anchordoquy, Juan Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Gambaro, Rocío Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Padula, Gisel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo; Argentina Fil: Seoane, Analia Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Furnus, Cecilia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina

In many species, alpha-lipoic acid (ALA) is essential for embryo development. There, therefore, was investigation of effects of ALA supplementation to culture media for in vitro development of cattle embryos. In Experiment I, there were assessments of embryo production and oxidative status of cattle embryos derived by in vitro maturation and fertilization (IVM/IVF)that were cultured until the blastocyst stage of development using different ALA concentrations (5, 25 and 100 μM), fetal bovine serum (FBS) and amino acids (aa) as well as 20 % oxygen (O2) in the culture atmosphere. In Experiment II, embryos were cultured without FBS, at different ALA concentrations (2.5, 5 and 7.5 μM) and in the presence or absence of aa when there was a 7 % O2 atmosphere. Embryo development rates and blastocyst quality were evaluated. With 20 % O2 concentration, treatment with 100 μM ALA resulted in lesser hatching rates and development to the blastocyst stage (P

The eicosapentaenoic acid (EPA) is an n-3 polyunsaturated fatty acid (PUFA) present in the lipid composition of bovine oocytes. Little is known about the importance of EPA in bovine oocyte maturation and embryo development in vitro. Although previous work suggest that n-3 PUFAs may inhibit oocyte maturation, the available data are inconsistent. In this study, we evaluated the effect of EPA (1, 10, 100 nM) during in vitro maturation (IVM) of bovine oocytes, alone and in combination with vitamin E (VE) or cysteamine (CYS). EPA treatment in IVM decreased oocyte lipid content and affected lipid droplets pattern (P < 0.05). EPA 100 nM reduced oocytes maturation rate (P < 0.05), without affecting cumulus expansion. At the concentrations tested, EPA did not modify embryo development. However, the addition of antioxidants during IVM reduced the levels of reactive oxygen species in the culture system by increasing intracellular glutathione content (P < 0.05). Besides, the combination of EPA with VE or CYS reduced the percentages of MI oocytes after 24 h of IVM (P < 0.05). EPA reduced oocyte lipid content without any detrimental for embryo development. Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Campagna, Anabella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Luchetti, Carolina Griselda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Histología y Embriologías; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina Fil: Carranza Martin, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Pascua, Ana Malen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Anchordoquy, Juan Mateo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Histología y Embriologías; Argentina Fil: Anchordoquy, Juan Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Lombardo, Daniel Marcelo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Histología y Embriologías; Argentina Fil: Seoane, Analia Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Furnus, Cecilia Cristina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina

The in vitro effect of enrofloxacin (EFZ) was tested on two experimental somatic bovine cells in vitro: peripheral lymphocytes (PLs) and cumulus cells (CCs). The cytotoxicity and genotoxicity of this veterinary antibiotic were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, single-cell gel electrophoresis (SCGE) assay, and cytokinesis-block micronucleus cytome (CBMN cyt) assay. Cells were treated during 24 h, and three concentrations were tested (50 μg/mL, 100 μg/mL, 150 μg/mL). When EFZ was tested in PLs, the results demonstrated that the antibiotic was able to induce cell death and DNA damage with all concentrations. In addition, 50 μg/mL and 100 μg/mL EFZ increased frequencies of micronuclei (MNi). On the other hand, the highest EFZ concentration occasioned cellular cytotoxicity in CCs as evidenced by mitochondrial activity alterations. Nevertheless, EFZ was not able to induce DNA damage and MNi in CCs. These results represent the first experimental evidence of genotoxic and cytotoxic effects exerted by EFZ in bovine PLs and CCs., Instituto de Genética Veterinaria

Glutathione (GSH) is an antioxidant synthesized from three constitutive amino acids (CAA): cysteine (Cys), glycine (Gly) and glutamate (Glu). Glutathione plays an important role in oocyte maturation, fertilization andearly embryo development. This study aimed to investigate the effect of Cys (0.6 mM), Gly (0.6 mM) and Glu (0.9 mM) supplementation during in vitro fertilization (IVF) of cattle oocytes. In a Pilot Experiment, de novo synthesis of GSH in bovine zygote was evaluated using a modified TALP medium prepared without MEM-essential and MEM-non-essential amino acids (mTALP): mTALP+CAA (constitutive amino acids); mTALP+CAA+5 mMBSO (buthionine sulfoximide); mTALP+Cys+Gly; mTALP+Cys+Glu and mTALP+Gly+Glu. This evidence led us to investigate the impact of CAA supplementation to TALP medium (with essential and non-essential amino acids) on zygote viability, lipid peroxidation, total intracellular GSH content (include reduced and oxidized form; GSH-GSSG), pronuclear formation in zygotes and subsequent embryo development. IVF media contained a) TALP; b) ALP+Cys+Gly+Glu (TALP+CAA); c) TALP+Cys+Gly; d) TALP+Cys+Glu; e) TALP+Gly+Glu, were used. Total GSH-GSSG concentration was increased in TALP, TALP+CAA, and TALP+Cys+Gly. The viability of zygote was similar among treatments. Lipid peroxidation was increased in zygote fertilized with TALP+Cys+Gly; TALP+Cys+Glu;TALP+Gly+Glu and TALP+CAA. The percentage of penetrated oocytes decreased in TALP+CAA and TALP+Cys+Gly. The cleavage rate was lower in TALP+CAA and TALP+Gly+Glu. The percentage of embryos developing to the blastocyst stage was lower in TALP+Cys+Glu and TALP+CAA. In conclusion, we have demonstrated the synthesis of GSH during IVF. However, Cys, Gly and Glu supplementation to TALPmedium had negative effects on embryonic development. Fil: Anchordoquy, Juan Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Ciencias Básicas. Cátedra de Fisiología; Argentina Fil: Lizarraga, Raúl Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Anchordoquy, Juan Mateo. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Ciencias Básicas. Cátedra de Fisiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Rosa, Diana Esther. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Ciencias Básicas. Cátedra de Fisiología; Argentina Fil: Fabra, Mariana Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Peral Garcia, Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Furnus, Cecilia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina

Zinc (Zn) is a vital trace element for the body and its bioavailability influences numerous reproductive events. However, the mechanisms that regulate Zn homeostasis in the cumulus-oocyte complex (COC) are yet to be elucidated. The aim of this study was to investigate the role of estradiol 17-beta (E2), FSH and LH in Zn homeostasis regulation in bovine COC matured in vitro and Zn transporters gene expression. For this purpose, intracellular Zn levels in oocytes and cumulus cells (CC) were assessed using a Zn-specific fluorescent indicator. In addition, gene expression and sequencing of six Zn transporters (Slc39a6, Slc39a8, Slc39a14, Slc30a3, Slc30a7 and Slc30a9) were assessed. Our results demonstrated that the simultaneous presence of E2, FSH, and LH during oocyte maturation altered intracellular zinc levels and transporters expression in both oocytes and CC. Transporter’s gene expression was different in oocytes and CC, possibly due to cell-specific changes in Zn levels during maturation. The interaction effects of Zn with hormonal treatments influenced the results. This study emphasizes that Slc39a6 is highly sensitive to hormone induction. Overall, the hormonal modulation of Zn homeostasis in the COC was evidenced. Also, a preponderant role of FSH as a modulator of Zn intracellular levels and transporter gene expression is suggested.

Sub-optimal cattle embryo development to the blastocyst stage still is a problem when conducting in vitro production (IVP) procedures. Supplementation of in vitro maturation (IVM) medium with omega 3-polyunsaturated eicosapentaenoic acid (EPA) is an approach that might have positive effects on lipid metabolism of cattle oocytes, potentially improving subsequent embryo development. The aim of this study was to evaluate effects of EPA addition to serum-free IVM medium on pronuclear formation after in vitro fertilization, cleavage, and blastocyst rates. Effects of EPA on lipid accumulation and intracellular reactive oxygen species (ROS) generation with IVP of cattle embryos was also investigated. In all experiments, cumulus-oocyte complexes were matured in IVM medium supplemented with 0 nM, 1 nM, or 1 μM EPA for 24 h. Pronuclear formation, cleavage, and blastocyst rates were similar for embryos when there was supplementation of EPA at all concentrations to those of the control group (P0.05). The inclusion of 1 nM EPA in medium resulted in a greater lipid content and less intracellular ROS in day 8-embryos compared with those of the Control group (P0.05). There were no differences, however, when there was inclusion of 1 μM EPA compared to embryos of the Control group at the day 8 developmental stage (P0.05). In conclusion, supplementation with IVM medium with the 1 nM EPA concentration resulted in a lesser blastocyst lipid and intracellular ROS concentration, without modifying embryo development, therefore, EPA could be a desirable supplement to improve embryo quality in cattle.

In human tumor cells, experimental and clinical evidence indicates that some factors involved in signal transductionand cell growth can also modulate the response to chemotherapeutic treatment. The aim of the presentstudy was to investigate the role of folic acid (FA) as a modulator of carboplatin (CBDCA) activity.Genotoxicity and cytotoxicity induced by CBDCA alone and in combination with FA were assessed in culturedHeLa cells. We used comet assay, mitotic index analysis, MTT and NR assays, cytokinesis-block micronucleuscytome assay and annexin V-IP as different cytotoxicity and genotoxicity approaches for human cervical carcinomacell line studies.The results showed that addition of 900 nMFA together with 40.4mMCBDCA enhanced the activity of the platinumcompound,increasing its effect on cell death by nearly 20%, as evidenced by theMTT and NR assays. Moreover,not only higher levels of DNA and chromosomal damage were reached but also the number of necrotic andapoptotic cells were significantly increased when cell cultures were treated with the combined procedure.This situation opens the possibility to explore the use of FA in platinum-based chemotherapy protocols to reducethe platinum doses for patient treatment and decrease the chance of developing the known side effects withoutlosing biological activity. Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Ponzinibbio, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Padula, Gisel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: de Luca, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Golijow, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Seoane, Analia Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina

Cytotoxic and genotoxic effects of flurochloridone (FLC) and its formulations Twin Pack Gold® and Rainbow® were evaluated in CHO-K1 cells. Using the alkaline single-cell gel electrophoresis (SCGE) assay, we observed that FLC (15 μg/ml), Twin Pack Gold® or Rainbow® induced primary DNA damage, increasing the frequency of damaged nucleoids. Vitamin E pretreatment did not modify the effect. Decreased cell viability was observed only in Twin Pack Gold®-treated cultures and was significantly ameliorated by vitamin E. Post-treatment of herbicide-damaged CHO-K1 cells with the enzymes Endo III or Fpg did not increase FLC-, Twin Pack Gold®-, or Rainbow®-induced DNA damage. These results demonstrate that neither FLC nor FLC-based formulations induce DNA damage through hydroxyl radical or lipid alkoxyl radical production, and that the induced DNA lesions were not related to oxidative damage at the purine/pyrimidine level. Our observations strongly suggest that the cytotoxic effects observed after Twin Pack Gold® exposure are due to the excipients contained within the technical formulation rather than FLC itself. Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina

Zinc (Zn) is required for normal reproductive performance in cattle. The aim of this study was to evaluate the effect of subcutaneous injection of 400 mg Zn at the beginning of fixed-time artificial insemination (FTAI) on preovulatory follicle and corpus luteum (CL) size, plasma estradiol (E2) and progesterone (P4) concentrations, and pregnancy rates in beef cows. Copper (Cu) concentration and alkaline phosphatase (ALP) activity in plasma were also evaluated. Zinc supplementation at the beginning of the FTAI protocol (day 0) increased the area of preovulatory follicle (APF, day 9; P = 0.042) and plasma P4 concentration (day 16; P = 0.01), whereas plasma E2 concentration (day 9) and area of CL (ACL; day 16) were not modified by Zn supplementation in cows with adequate plasma Zn concentration. Zinc supplementation in Zn-deficient cows increased ACL with respect to controls (P = 0.048) but did not modify plasma E2 concentration. Pregnancy rate on day 41 after FTAI was higher in cows supplemented with Zn compared with controls (80.95% and 51.61%, respectively; P = 0.042). Plasma Zn and Cu concentrations on days 7, 9, and 16 were not affected by Zn supplementation. In conclusion, the results obtained in the present study determined that parenteral Zn supplementation at the beginning of the FTAI protocol increased preovulatory follicle size, plasma P4 concentration, and pregnancy rates in beef cows., Facultad de Ciencias Veterinarias, Instituto de Genética Veterinaria

The in vitro effect of enrofloxacin (EFZ) was tested on two experimental somatic bovine cells in vitro: peripheral lymphocytes (PLs) and cumulus cells (CCs). The cytotoxicity and genotoxicity of this veterinary antibiotic were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, single-cell gel electrophoresis (SCGE) assay, and cytokinesis-block micronucleus cytome (CBMN cyt) assay. Cells were treated during 24 h, and three concentrations were tested (50 μg/mL, 100 μg/mL, 150 μg/mL). When EFZ was tested in PLs, the results demonstrated that the antibiotic was able to induce cell death and DNA damage with all concentrations. In addition, 50 μg/mL and 100 μg/mL EFZ increased frequencies of micronuclei (MNi). On the other hand, the highest EFZ concentration occasioned cellular cytotoxicity in CCs as evidenced by mitochondrial activity alterations. Nevertheless, EFZ was not able to induce DNA damage and MNi in CCs. These results represent the first experimental evidence of genotoxic and cytotoxic effects exerted by EFZ in bovine PLs and CCs.

The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus–oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes., Instituto de Genética Veterinaria

We evaluated the role of oxidative stress in the genotoxic damage induced by imazethapyr (IMZT) and its formulation Pivot® in mammalian CHO-K1 cell line. Using the alkaline comet assay, we observed that a concentration of 0.1 μg/mL of IMZT or Pivot® was able to induce DNA damage by increasing the frequency of damaged nucleoids. To test whether the DNA lesions were caused by oxidative stress, the DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), which convert base damage to strand breaks, were used. Our results demonstrate that after treatment of CHO-K1 cells with the pure active ingredient as well as the commercial formulation Pivot®, an increase in DNA strand breaks was observed after incubation of both Endo III and Fpg enzymes, indicating that both compounds induce DNA damage involving both pyrimidine and purine-based oxidations, at least in CHO-K1 cells. Our findings confirm the genotoxic potential of IMZT and suggest that this herbicide formulation must be employed with great caution, especially not only for exposed occupational workers but also for other living species., Facultad de Ciencias Naturales y Museo

Adequate dietary intake of copper (Cu) is required for normal reproductive performance in cattle. The objective of this study was to investigate the pregnancy rates from cattle with deficient, marginal and adequate Cu plasma concentration at the beginning of artificial insemination protocol. Moreover, we determined Cu concentrations present in bovine oviductal fluid (OF), and the effects of Cu on fertilizing ability of bovine spermatozoa. Also, the presence of Cu transporter, SLC31A1 (also known as CTR1), in spermatozoa and in vitro matured oocyte were investigated. We found no differences in pregnancy rates among animals with adequate, marginal, and deficient Cu concentrations measured in plasma at the beginning of fixed-time artificial insemination (FTAI) protocol. Copper concentrations in OF were 38.3 ± 2.17 μg/dL (mean ± SEM) regardless of cupremia levels. The addition of 40 μg/dL Cu to IVF medium enhanced total and progressive motility, sperm viability, functional sperm membrane integrity (HOST), sperm–zona binding, and pronuclear formation. On the other hand, the presence of Cu in IVF medium did not modify acrosome integrity and cleavage rates after IVF, but impaired blastocyst rates. Cu transporter SLC31A1 was detected in bovine spermatozoa in the apical segment of acrosome, and in the oocyte matured in vitro. In conclusion, the results obtained in the present study determined that cupremia levels at the beginning of FTAI protocol did not influence the pregnancy rates at 60 d after insemination. The presence of CTR1 in bovine mature oocyte and spermatozoa, as well as the beneficial effect of Cu on sperm quality would suggest an important role of this mineral during the fertilization process. Fil: Anchordoquy, Juan Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria ; Argentina Fil: Anchordoquy, Juan Mateo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria ; Argentina Fil: Pascua, Ana Malen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria ; Argentina Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria ; Argentina Fil: Peral Garcia, Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria ; Argentina Fil: Furnus, Cecilia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria ; Argentina

The in vitro effects of S-metolachlor and its formulation Twin Pack Gold® (96% a.i.) were evaluated in human hepatoma (HepG2) cells. Cytokinesis-blocked micronucleus cytome (CBMN-cyt) and MTT assays as well as Neutral Red uptake were employed for genotoxicity and cytotoxicity evaluation. Activities were tested within the concentration range of 0.25–15 μg/ml S-metolachlor for 24 h of exposure. Both compounds rendered a minor reduction in the NDI although not reaching statistical significance. Results demonstrated that the S-metolachlor was not able to induce MNs. On the other hand, 0.5–6 μg/ml Twin Pack Gold® increased the frequency of MNs. When cytotoxicity was estimated, S-metolachlor was not able to induce either a reduction of lysosomal or mitochondrial activity. Contrarily, whereas 1–15 μg/ml Twin Pack Gold® induced a significant reduction of mitochondrial activity, all tested concentrations of the formulated product induced a significant decrease of lysosomal performance as a function of the concentration of the S-metolachlor-based formulation titrated into cultures. Genotoxicity and cytotoxicity differences obtained with pure S-metolachlor and the commercial S-metolachlor-based formulation indicate that the latter may contain additional unsafe xenobiotics and support the concept of the importance of evaluating not only the active principle but also the commercial formulation when estimating the real hazard from agrochemicals. Fil: Nikoloff, Noelia. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Escobar, Luciana Ines. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Soloneski, Sonia Maria Elsa. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Larramendy, Marcelo Luis. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The aim of this study was to investigate the cytotoxic and genotoxic effects of high copper (Cu) concentrations on bovine cumulus cells (CCs) cultured in vitro.We evaluated the effect of 0, 120, 240, and 360 μg/dL Cu added to in vitro maturation (IVM) medium on CC viability assessed by the trypan blue (TB)–fluorescein diacetate (FDA) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, apoptosis, and DNA damage. Differences in cell viability assessed by TB–FDAwere not significant among CC treated with 0, 120, 240, and 360 μg/dL Cu. However, mitochondrial activity assessed by MTT was lower in CC cultured with 120, 240, and 360 μg/dL Cu as compared with the control (p < 0.01). Percentages of apoptotic cells were higher when CCs were treated with 120, 240, and 360 μg/dL Cu (p < 0.05) due to higher frequencies of late apoptotic cells (p < 0.05). The frequency of live cells diminished in a dose-dependent manner when Cu was added to the culture medium. Whereas genetic damage index (GDI) increased significantly in CC cultured in the presence of 240 and 360 μg/dL Cu (p ˂ 0.05), DNA damage increased at all Cu concentrations tested (p ˂ 0.05). These results indicate that Cu induces cytotoxic and genotoxic effects in bovine CC., Facultad de Ciencias Veterinarias, Instituto de Genética Veterinaria, Facultad de Ciencias Médicas

The aim of the present study was to investigate the effects of eicosapentaenoic acid (EPA) supplementation during in vitro maturation (IVM) of bovine oocytes. The concentrations tested in all experiments were 1 nM, 1 μM, and 1 mM EPA. The effect of EPA was evaluated on cumulus-oocyte complexes (COC) by oocyte maturation (cumulus expansion area and oocyte nuclear maturation), genotoxicity [single cell gel electrophoresis (SCGE)], and cytotoxicity (apoptosis, viability, and MTT assays) end points. The maturation parameters were affected by exposure of COC to different EPA concentrations in the IVM medium. Cumulus expansion area increased in the presence of 1 nM EPA (P 0.05) whereas addition of 1 nM EPA (P 0.05) decreased cumulus expansion after 24 h of IVM. Moreover, the maturation rate significantly decreased when 1 mM of EPA was assayed (P 0.001). EPA at 1 nM induced genotoxic and cytotoxic effects on bovine cumulus cells (CC) and primary DNA lesions (P 0.001). A significant increase in the frequency of apoptotic (P 0.01) and necrotic (P 0.001) cells was observed after 24 h of treatment with 1 nM, 1 μM, and 1 mM EPA. Mitochondrial activity was altered with 1 mM EPA (P 0.001). We inferred that optimal oocyte quality was partially dependent on the presence of adequate EPA concentrations; EPA could be beneficial to improve oocyte quality in the maturation process, because low concentration tested (1 nM EPA) improved cumulus expansion.

The in-vitro effects of flurochloridone and its formulations Twin Pack Gold® (25% a.i.) and Rainbow® (25% a.i.) were evaluated in Chinese Hamster Ovary K1 (CHO-K1) cells. The cytokinesis-block micronucleus cytome (CBMN-cyt) and single-cell gel electrophoresis (SCGE) assays were used. The activities were tested within the range of final concentrations of 0.25-15 μg flurochloridone/mL. The results demonstrated that both the flurochloridone and Rainbow® were not able to induce micronuclei (MN). On the other hand, Twin Pack Gold® only increased the frequency of MN at 5 μg/mL. Furthermore, 10 and 15 μg/mL of both formulations resulted in a cellular cytotoxicity demonstrated by alterations in the nuclear division index and cellular death. SCGE assay appeared to be a more sensitive bioassay for detecting primary DNA strand breaks at lower concentrations of flurochloridone than MN did. A marked increase in the genetic damage index was observed when 5 and 15 μg/mL of both flurochloridone and Rainbow® but only when 15 μg/mL of Twin Pack Gold® were used. This is the first report demonstrating that flurochloridone and its two commercial formulations are able to induce single-strand DNA breaks in vitro on mammalian cells.

The in vitro effects of flurochloridone (FLC) and its formulations Twin Gold Pack® (25% a.i.) and Rainbow® (25% a.i.) were evaluated on Chinese hamster ovary (CHO-K1) cells by genotoxicity [sister chromatid exchange (SCE)] and cytotoxicity [cell-cycle progression, proliferative rate index (PRI), mitotic index (MI), MTT, and neutral red] end points. Cells were treated for 24. h within the 0.25-15 μg/ml concentration range. FLC and Twin Pack Gold® induced a significant and equivalent increase in SCEs regardless of the concentration. Rainbow®-induced SCEs at concentrations higher than 2.5 μg/ml; however, the increases were always lower than those induced by FLC and Twin Pack Gold®. For all compounds, the PRI decreased as a function of the concentration titrated into cultures. Whereas only the highest FLC and Twin Pack Gold® concentrations induced a significant reduction of the MI, all tested Rainbow® concentrations induced MI inhibition. Overall, the results demonstrated that although all compounds were not able to reduce the lysosomal activity, the mitochondrial activity was diminished when the highest concentrations were employed. These observations represent the first study analyzing the genotoxic and cytotoxic effects exerted by FLC and two formulated products on mammalian cells in vitro, at least on CHO-K1 cells. Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina

The purpose of this study was to further investigate the cytotoxic and genotoxic effects of dicamba and Banvel® employing the cytokinesis-block micronucleus cytome (CBMN-cyt) assay estimated by the analysis of the nuclear division index (NDI), the frequency of micronucleus (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs). Besides, for mechanism of MN induction CREST anti-kinetochore antibody analysis was performed. The activities of both compounds were tested within the range of 50–500 μg/ml on Chinese hamster ovary (CHO-K1) cells. Overall, dicamba and Banvel® produced a NDI dose-dependent decrease but the response was statistically significant only in cultures treated with Banvel® at a 100–500 μg/ml concentration range. A dose-dependent induction of MN was observed after dicamba- and Banvel®-treatments within the 50–400 μg/ml and 50–500 μg/ml concentration-ranges, respectively. Induction of NPBs and NBUDs was significantly enhanced by both test compounds. The NPBs/MN ratio values found for dicamba and Banvel® were 0.04–0.11 and 0.05–0.18, respectively. Results clearly demonstrated that dicamba and Banvel® exerted both cyto- and genotoxic damage on CHO-K1 cells. Furthermore, the CBMN-cyt assay employed confirmed our previous investigations concerning the cellular and DNA damaging capabilities of dicamba and highlights that both clastogenic and aneugenic mechanisms are implicated in the MN induction. Fil: González, N.V.. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Larramendy, Marcelo Luis. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Acute lethal and sublethal toxicity of the imidazolinone imazethapyr (IMZT)-based commercial formulation herbicide Pivot H® (10.59% IMZT) was evaluated on Hypsiboas pulchellus tadpoles. Whereas mortality was used as the end point for lethality, frequency of micronuclei (MNs) and other nuclear abnormalities as well as DNA single-strand breaks evaluated by the single cell gel electrophoresis assay were employed to test genotoxicity. Behavioral, growth, developmental, and morphological abnormalities were also employed as sublethal end points. Mortality studies revealed equivalent LC50 (96h) values of 1.49mg/L (confidence limit, 1.09-1.63) and 1.55mg/L (confidence limit, 1.51-1.60) IMZT for Gosner stage (GS) 25 and GS36, respectively. Behavioral changes, i.e., irregular swimming and immobility, as well as a decreased frequency of keratodonts were observed. The herbicide increased the frequency of MNs in circulating erythrocytes of tadpoles exposed for 48h to the highest concentration assayed (1.17mg/L). However, regardless of the concentration of the herbicide assayed, an enhanced frequency of MNs was observed in tadpoles exposed for 96h. The herbicide was able to induce other nuclear abnormalities, i.e., blebbed and notched nuclei, only when tadpoles were exposed for 96h. In addition, we observed that exposure to IMZT within the 0.39-1.17mg/L range increased the genetic damage index in treatments lasting for both 48 and 96h. This study represents the first evidence of acute lethal and sublethal effects exerted by IMZT on amphibians. Finally, our findings highlight the properties of this herbicide that jeopardize nontarget living species exposed to IMZT. Fil: Pérez Iglesias, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Natale, Guillermo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina

Acute toxicity and genotoxicity of imidacloprid (IMI) was evaluated on Hypsiboas pulchellus (Anura: Hylidae) tadpoles exposed under laboratory conditions. A lethal effect was used as the end point for lethality, whereas the frequency of micronuclei (MNs) and DNA single-strand breaks evaluated by the single cell gel electrophoresis assay were employed as end points for genotoxicity. Experiments were performed on tadpoles at stage 36 (range, 35–37) according to the classification proposed by Gosner. Mortality studies revealed an LC50 (96 h) value of 84.91 mg/L IMI (95% confidence limits, 77.20–93.04). While increased frequency of MNs was observed when 15 and 30 mg/L were assayed for 48 h, only 15 mg/L increased the frequency of MNs in tadpoles exposed for 96 h. Furthermore, other nuclear abnormalities, i.e., binucleated cells and blebbed and notched nuclei, were induced in tadpoles exposed for both 48 h when treated with 15 mg/L and 96 h when treated with 15 and 30 mg/L. An increase in the genetic damage index was observed in tadpoles treated with 30 mg/L for 48 and 96 h. This study represents the first evidence of acute lethal and sublethal effects exerted by IMI on tadpoles of an amphibian species native to Argentina. Finally, our findings highlight the hazardous properties of this insecticide for nontarget living species exposed to this agrochemical. Fil: Ruiz de Arcaute, Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Pérez Iglesias, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Natale, Guillermo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigaciones del Medio Ambiente. La Plata; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina

Agrochemicals represent one of the most important sources of environmental pollution. Although attempts to reduce agrochemical use through organic agricultural practices and the use of other technologies to control pests continue, the problem is still unsolved. Recent technological advances in molecular biology and analytical science have allowed the development of rapid, robust, and sensitive diagnostic tests (biomarkers) that can be used to monitor exposure to, and the effects of pollution. One of the major goals of our research laboratory is to evaluate comparatively the genotoxic and cytotoxic effects exerted by several pure agrochemicals and their technical formulations commonly used in Argentina on vertebrate cells in vitro and in vivo employing several end-points for geno and cytotoxicity. Among them are listed the herbicides dicamba and flurochloridone, the fungicide zineb, the insecticides pirimicarb and imidacloprid. Overall, the results clearly demonstrated that the damage induced by the commercial formulations is in general greater than that produced by the pure pesticides, suggesting the presence of deleterious components in the excipients with either a putative intrinsic toxic effect Larramendy et al. 4 or with the capacity of exacerbating 52 the toxicity of the pure agrochemicals, or both. Accordingly, the results highlight that: 1) A complete knowledge of the toxic effect/s of the active ingredient is not enough in biomonitoring studies; 2) Pesticide/s toxic effect/s should be evaluated assaying to the commercial formulation available in market; 3) The deleterious effect/s of the excipient/s present within the commercial formulation should not be either discarded nor underestimated, and 4) A single bioassay is not enough to characterize the toxicity of a agrochemical under study., Facultad de Ciencias Naturales y Museo

Los estudios llevados a cabo en el presente trabajo de Tesis Doctoral, tuvieron como objetivo general, evaluar la capacidad deletérea del herbicida flurocloridona y de dos de sus formulaciones comerciales Twin Pack Gold® y Rainbow® empleadas en el agro de nuestro país conteniendo un 25% del principio activo. Estos formulados se emplean en el tratamiento de cultivos agrícolas como algodón, apio, arveja, avena, cebada, centeno, lenteja, perejil, zanahoria, caña de azúcar, girasol, papa y trigo, entre otros. Para evaluar la toxicidad del mencionado herbicida y de sus formulaciones comerciales, se emplearon como modelo de estudio in vitro dos líneas celulares de mamífero (CHO-K1 y HepG2) y como modelo in vivo larvas del anfibio anuro Rhinella arenarum. Los efectos genotóxicos de dichos herbicidas, fueron investigados mediante el estudio de biomarcadores de efecto, analizando la frecuencia de intercambios de cromátidas hermanas (ICHs) y los ensayos cometa (EC) y de micronúcleos (MN). En la línea celular CHO-K1 todos los compuestos ensayados fueron capaces de inducir ICHs con concentraciones de 0,25-15 μg/ml. La capacidad de los compuestos para inducir ICHs fue: Twin Pack Gold® > FLC > Rainbow®. El formulado comercial Twin Pack Gold® fue el único compuesto capaz de incrementar la frecuencia de MN con la concentración de 5 μg/ml. Además, las mayores concentraciones ensayadas de ambos formulados (10-15 μg/ml) resultaron ser citotóxicas tal cual fue evidenciado a través de cambios morfológicos, pérdidas de la integridad de la membrana plasmática y alteraciones nucleares que no permitieron monitorear la frecuencia de MN. Todos los compuestos fueron capaces de inducir rupturas de cadena simple y/o sitios álcali sensibles en el ADN, evidenciado mediante la variante alcalina del EC con todas las concentraciones ensayadas. En células HepG2, al igual que en las células CHO-K1, sólo el formulado Twin Pack Gold® fue capaz de incrementar la frecuencia de MN a la concentración de 5 μg/ml, no pudiéndose monitorear la frecuencia de MN con las concentraciones de 10-15 μg/ml de ambos formulados comerciales debido a la aparición de alteraciones celulares como ocurrió en el sistema celular CHO-K1. El análisis de los compuestos estudiados evidenció que fueron capaces de inducir rupturas de cadena simple y/o sitios álcali sensibles en el ADN con todas las concentraciones ensayadas lo cual fue evidenciado mediante el análisis de los nucleoides obtenidos en el EC. Los efectos citotóxicos fueron evaluados mediante el análisis del índice de proliferación celular (IPC), el índice mitótico (IM), el índice de división nuclear (IDN), los ensayos colorimétricos de captación de Rojo Neutro y MTT (RN y MTT, respectivamente) y el estudio de muerte celular programada o apoptosis. En la línea celular CHO-K1 los resultados revelaron que el IPC decrece en función de la concentración empleada de todos los compuestos ensayados y para una concentración dada, la capacidad de ambas formulaciones comerciales de inducir un retraso en la progresión del ciclo celular siempre fue mayor que la inducción producida por el principio activo FLC. Asimismo, se observó actividad mitodepresiva ejercida tanto por el principio activo como por los formulados comerciales. Asimismo, se observó una marcada reducción de la actividad mitocondrial con las mayores concentraciones ensayadas de todos los compuestos. Cuando se realizó el ensayo de RN únicamente la formulación comercial Rainbow® ejerció efecto citotóxico a nivel lisosomal. El estudio de la viabilidad celular evidenció que los formulados comerciales resultaron ser más citotóxicos que el principio activo FLC. En células HepG2 se observó que solamente los formulados comerciales fueron capaces de inducir una alteración de la actividad mitocondrial y lisosomal. Además, se observó inducción de apoptosis cuando las células fueron expuestas durante 24 h a todos los compuestos. En sistemas in vivo, de larvas de R. arenarum, se determinó una CL5096 h con valores de 2,96 mg/L para Twin Pack Gold® y de 2,85 mg/L para Rainbow®. Sólo el formulado Rainbow® fue capaz de incrementar a las 48 h de exposición la frecuencia de MN en eritrocitos circulantes con la concentración de 0,71 mg/L. Por el contrario, ambos formulados fueron capaces de inducir efecto genotóxico con todas las concentraciones empleadas tanto a las 48 como a las 96 h de exposición evaluadas mediante el EC. Teniendo en cuenta los resultados obtenidos, se propuso que la respuesta diferencial encontrada en cada modelo de estudio frente a los compuestos podría deberse a la presencia de xenobióticos de naturaleza desconocida presentes en el excipiente de las formulaciones comerciales, así como a variaciones en la sensibilidad diferencial de los modelos en estudio. Los resultados de los estudios de mortalidad, genotoxicidad y citotoxicidad de FLC, Twin Pack Gold® y Rainbow® obtenidos en el desarrollo de la presente Tesis Doctoral, representan una evidencia concreta de que el herbicida FLC debe ser tenido en cuenta como un agente inductor de daño genotóxico y citotóxico, y debería ser clasificado, según los valores de toxicidad aguda CL5096 h obtenidos mediante la exposición de larvas de R. arenarum a Twin Pack Gold® y Rainbow®, como compuesto tóxico (categoría II), siguiendo la clasificación propuesta por Instituciones reguladoras tales como OECD y UN. Asimismo, se sugiere la necesidad de una correcta evaluación toxicológica de FLC y de las formulaciones comerciales existentes del herbicida antes que continúen siendo aplicadas masivamente en nuestro país y en el mundo, representando un factor de riesgo para los organismos expuestos, incluyendo al ser humano., Directores de la tesis: Sonia Soloneski y Marcelo L. Larramendy, Facultad de Ciencias Naturales y Museo

Acute toxicity and genotoxicity of the flurochloridone (FLC)-containing commercial formulation herbicides Twin Pack Gold® (25 percent a.i.) and Rainbow® (25 percent a.i.) were evaluated on Rhinella arenarum (Anura: Bufonidae) tadpoles exposed under laboratory conditions. Lethal effect was evaluated as end point for lethality, whereas frequency of micronuclei (MN) and single cell gel electrophoresis (SCGE) were employed as end points for genotoxicity. Lethality studies revealed equivalent LC-5096 h values of 2.96 and 2.85 mg/L for Twin Pack Gold® and Rainbow®, respectively. Twin Pack Gold® did not induce DNA damage at the chromosomal level, whereas Rainbow® increased the frequency of MN only when the lowest concentration (0.71 mg/L) was used. However, all concentrations of Twin Pack Gold® and Rainbow® increased the frequencies of primary DNA lesions estimated by alkaline SCGE. This study represents the first evidence of the acute toxic and genotoxic effects exerted by two FLC-based commercial formulations, Twin Pack Gold® and Rainbow®, on tadpoles of an amphibian species native to Argentina under laboratory conditions. Finally, our findings highlight the importance of minimizing the impacts on nontarget living species exposed to agrochemicals. Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Natale, Guillermo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentina Fil: Marino, Damian Jose Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina

The neonicotinoid insecticide imidacloprid (IMI) affects the insect central nervous system and is successfully applied to control pests for a variety of agricultural crops. In the current study, acute toxicity and genotoxicity of the IMI-containing commercial formulation insecticide Glacoxan Imida (35 percent IMI) was evaluated on Hypsiboas pulchellus (Anura: Hylidae) tadpoles exposed under laboratory conditions. A lethal effect was evaluated as the end point for lethality, whereas micronucleus (MN) frequency and DNA single-strand breaks evaluated by the single cell gel electrophoresis (SCGE) assay were employed as end points for genotoxicity. Sublethal end points were assayed within the 12.5–37.5 mg/L IMI concentration range. Experiments were performed on tadpoles at stage 36 (range, 35–37) according to the classification proposed by Gosner. Lethality studies revealed an LC50 96 h value of 52.622 mg/L IMI. Increased frequency of MNs was only observed when 25.0 mg/L was assayed for 96 h, whereas no other nuclear abnormalities were induced. Increase of the genetic damage index was observed at 48 h of treatment within the 12.5–37.5 mg/L concentration range, whereas an increased frequency of DNA damage was observed only in tadpoles treated with 37.5 mg/L IMI for 96 h. This study represents the first evidence of the acute lethal and genotoxic effects exerted by IMI on tadpoles of an amphibian species native to Argentina under laboratory conditions. Fil: Pérez Iglesias, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Ruiz de Arcaute, Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Nikoloff, Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Dury, Lucía. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Soloneski, Sonia Maria Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina Fil: Natale, Guillermo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentina Fil: Larramendy, Marcelo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina

In vitro effects of flurochloridone (FLC) and its formulations Twin Pack Gold® [25% active ingredient (a.i.)] and Rainbow® (25% a.i.) were evaluated in HepG2 cells. Whereas cytokinesisblocked micronucleus cytome (CBMN-cyt) and single-cell gel electrophoresis (SCGE) assays were employed for genotoxicity, MTT, neutral red, and apoptosis detections were used for cytotoxicity evaluation. Activities were tested within the concentration range of 0.25-15 µg/ml FLC. Results demonstrated that neither FLC nor Rainbow® was able to induce MNs. On the other hand, 5 µg/ml Twin Pack G old® only increased MN frequency. Furthermore, 10 and 15 µg/ml of both formulations resulted in cellular cytotoxicity demonstrated by alterations in the nuclear division index and cellular death. A marked increase in the genetic damage index was observed after treatment with all compounds. SCGE assay appeared to be more sensitive bioassay for detecting primary DNA strand breaks at lower concentrations of FLC than did MN. Our results reveal that FLC and its two formulations trigger apoptosis on HepG2 cells. The results represent the first experimental evidence of the in vitro apoptogenic role exerted on mammalian cells by FLC and the FLC-based formulations Rainbow® and Twin Pack Gold®, at least on HepG2 cells. Fil: Nikoloff, Noelia. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Larramendy, Marcelo Luis. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Soloneski, Sonia Maria Elsa. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Citología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The in-vitro effects of flurochloridone and its formulations Twin Pack Gold® (25% a.i.) and Rainbow® (25% a.i.) were evaluated in Chinese Hamster Ovary K1 (CHO-K1) cells. The cytokinesis-block micronucleus cytome (CBMN-cyt) and single-cell gel electrophoresis (SCGE) assays were used. The activities were tested within the range of final concentrations of 0.25-15 μg flurochloridone/mL. The results demonstrated that both the flurochloridone and Rainbow® were not able to induce micronuclei (MN). On the other hand, Twin Pack Gold® only increased the frequency of MN at 5 μg/mL. Furthermore, 10 and 15 μg/mL of both formulations resulted in a cellular cytotoxicity demonstrated by alterations in the nuclear division index and cellular death. SCGE assay appeared to be a more sensitive bioassay for detecting primary DNA strand breaks at lower concentrations of flurochloridone than MN did. A marked increase in the genetic damage index was observed when 5 and 15 μg/mL of both flurochloridone and Rainbow® but only when 15 μg/mL of Twin Pack Gold® were used. This is the first report demonstrating that flurochloridone and its two commercial formulations are able to induce single-strand DNA breaks in vitro on mammalian cells.

In Argentina, there are numerous native species which are an important source of natural products and which are traditionally used in medicinal applications. Some of these species are going through an intense extraction process in their natural habitat which may affect their genetic diversity. The aim of this study was to establish vegetative propagation systems for three native forestal species of medicinal interest. This will allow the rapid obtainment of plants to preserve the germplasm. This study included the following species which are widely used in folk medicine and its applications: Erythrina crista-galli or "seibo" (astringent, used for its cicatrizant properties and for bronchiolitic problems); Acacia caven or "espinillo" (antirheumatic, digestive, diuretic and with cicatrizant properties) and Salix humboldtiana or "sauce criollo" (antipyretic, sedative, antispasmodic, astringent). The methodology included the micropropagation of seibo, macro and micropropagation of Salix humboldtiana and the somatic embryogenesis of Acacia caven. The protocol for seibo regeneration was adjusted from nodal sections of seedlings which were obtained from seeds germinated in vitro. The macropropagation through rooted cuttings of "sauce criollo" was achieved and complete plants of this same species were obtained through both direct and indirect organogenesis using in vitro cultures. The somatic embryogenesis for Acacia caven was optimized and this led to obtain a high percentage of embryos in different stages of development. We are able to support the conservation of native forest resources of medicinal use by means of vegetative propagation techniques.

In Argentina, there are numerous native species which are an important source of natural products and which are traditionally used in medicinal applications. Some of these species are going through an intense extraction process in their natural habitat which may affect their genetic diversity. The aim of this study was to establish vegetative propagation systems for three native forestal species of medicinal interest. This will allow the rapid obtainment of plants to preserve the germplasm. This study included the following species which are widely used in folk medicine and its applications: Erythrina crista-galli or "seibo" (astringent, used for its cicatrizant properties and for bronchiolitic problems); Acacia caven or "espinillo" (antirheumatic, digestive, diuretic and with cicatrizant properties) and Salix humboldtiana or "sauce criollo" (antipyretic, sedative, antispasmodic, astringent). The methodology included the micropropagation of seibo, macro and micropropagation of Salix humboldtiana and the somatic embryogenesis of Acacia caven. The protocol for seibo regeneration was adjusted from nodal sections of seedlings which were obtained from seeds germinated in vitro. The macropropagation through rooted cuttings of "sauce criollo" was achieved and complete plants of this same species were obtained through both direct and indirect organogenesis using in vitro cultures. The somatic embryogenesis for Acacia caven was optimized and this led to obtain a high percentage of embryos in different stages of development. We are able to support the conservation of native forest resources of medicinal use by means of vegetative propagation techniques., Centro Experimental de Propagación Vegetativa