Genotoxic Effects Induced by Cd(+2), Cr(+6), Cu(+2) in the Gill and Liver of Odontesthes bonariensis (Piscies, Atherinopsidae).

Genotoxic effects of Cd(+2), Cr(+6), and Cu(+2) on the gill and liver of the Argentinean Silverside (Odontesthes bonariensis) were studied using the comet assay and in relation with the metal tissue accumulation. Fish were exposed to three waterborne concentrations of each metal for 2 and 16 days. Genotoxicity was assessed by the single cell gel electrophoresis (comet assay). After 2 days, significant increase of the genetic damage index (GDI) was only observed in the gill of fish exposed to Cr(+6) and Cu(+2), and the LOECs were 2160 nM and 921.1 nM, respectively. The gill LOEC for Cd(+2) by 16 days was 9.4 nM. In the liver, LOECs were obtained only for Cd(+2) and Cr(+6) and were 9.4 and 2160 nM, respectively. The three metals were able to induce genotoxic effects at environmentally relevant concentrations and the gill was the most sensitive organ.

The importance of having zinc during in vitro maturation of cattle cumulus-oocyte complex: role of cumulus cells.

The aim of this study was to investigate the influence of zinc (Zn) on the health of cumulus-oocyte complex (COC) during in vitro maturation (IVM). Experiments were designed to evaluate the effect of Zn added to IVM medium on: DNA integrity, apoptosis, cumulus expansion and superoxide dismutase (SOD) activity of cumulus cells (CC). Also, role of CC on Zn transport during IVM was evaluated on oocyte developmental capacity. DNA damage and early apoptosis were higher in CC matured with 0 µg/ml Zn compared with 0.7, 1.1 and 1.5 µg/ml Zn (p < 0.05). Cumulus expansion did not show differences in COC matured with or without Zn supplementation (p > 0.05). Superoxide dismutase activity was higher in COC matured with 1.5 µg/ml Zn than with 0 µg/ml Zn (p < 0.05). Cleavage and blastocyst rates were recorded after IVM in three maturation systems: intact COCs, denuded oocytes with cumulus cells monolayer (DO + CC) and denuded oocytes (DO). Cleavage rates were similar when COC, DO + CC or DO were matured with 1.5 µg/ml Zn compared with control group (p > 0.05). Blastocyst rates were significantly higher in COC than in DO + CC and DO with the addition of 1.5 µg/ml Zn during IVM (p < 0.01). Blastocyst quality was enhanced in COC and DO + CC compared with DO when Zn was added to IVM medium (p < 0.001). The results of this study indicate that Zn supplementation to IVM medium (i) decreased DNA damage and apoptosis in CC; (ii) increased SOD activity in CC; (iii) did not modify cumulus expansion and cleavage rates after in vitro fertilization; (iv) improved subsequent embryo development up to blastocyst stage; and (v) enhanced blastocyst quality when CC were present either in intact COC or in coculture during IVM.

Effect of different manganese concentrations during in vitro maturation of bovine oocytes on DNA integrity of cumulus cells and subsequent embryo development.

Manganese (Mn) is a trace element present in forages and cereals, and its concentration depends on soil status. Manganese deficiency in cattle, goats and ewes not only impairs oestrous cycle but reduces calf birth weight. The achievement of the first oestrus is delayed, and more attempts are necessary to obtain a successful conception. This study was conducted to investigate the effect of the availability of supplemental Mn during IVM on DNA damage of cumulus cells and total glutathione (GSH) content in oocytes and cumulus cells. The effect of supplementary Mn during IVM on subsequent embryo development was also studied. The results reported here indicate (i) DNA damage in cumulus cells decreased with 0, 2, 5 and 6 ng/ml Mn supplementation during IVM (p < 0.05). (ii) Intracellular GSH-GSSG content increased (p < 0.01) with different Mn concentrations in oocytes and cumulus cells. Also, cumulus cell number per cumulus oocyte-complexes (COC) did not differ either before or after IVM. (iii) Addition of Mn to maturation medium resulted in similar cleavage rates (p > 0.05) at 0, 2, 5 and 6 ng/ml Mn. However, subsequent embryo development to blastocyst stage was significantly higher (p < 0.01) in oocytes matured with 5 and 6 ng/ml Mn. (iv) There was also an increase (p < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 5 and 6 ng/ml respect to zero Mn (IVM alone) and 2 ng/ml Mn. This study provides evidence that optimal embryo development to the blastocyst stage was partially dependent on the presence of Mn during IVM. Moreover, the availability of Mn during oocyte maturation ensures 'normal' intracellular GSH content in COCs and protects DNA integrity of cumulus cells.

Effects of copper sulphate concentrations during in vitro maturation of bovine oocytes.

THE OBJECTIVES WERE TO EVALUATE: 1) copper (Cu) concentrations in plasma and follicular fluid (FF) from cattle ovaries; 2) the effects of supplemental Cu during in vitro maturation (IVM) on DNA damage of cumulus cells and glutathione (GSH) content in oocytes and cumulus cells; and 3) supplementary Cu during IVM on subsequent embryo development. Copper concentrations in heifer plasma (116 ± 27.1 µg/dL Cu) were similar (P > 0.05) to concentrations in FF from large (90 ± 20.4 µg/dL Cu) and small (82 ± 22.1 µg/dL Cu) ovarian follicles in these heifers. The DNA damage in cumulus cells decreased with supplemental Cu concentrations of 4 and 6 µg/mL (P < 0.01) in the IVM medium (mean ± SEM index of DNA damage was: 200.0 ± 27.6, 127.6 ± 6.0, 46.4 ± 4.8, and 51.1 ± 6.0 for supplementation with 0, 2, 4, and 6 µg/mL Cu respectively). Total GSH concentrations increased following supplementation with 4 µg/mL Cu (4.7 ± 0.4 pmol in oocytes and 0.4 ± 0.04 nmol/10(6) cumulus cells) and 6 µg/mL Cu (5.0 ± 0.5 pmol in oocytes and 0.5 ± 0.05 nmol/10(6) cumulus cells, P < 0.01) compared with the other classes. Cleavage rates were similar (P ≥ 0.05) when Cu was added to the IVM medium at any concentration (65.1 ± 2.0, 66.6 ± 1.6, 72.0 ± 2.1, and 70.7 ± 2.1 for Cu concentrations of 0, 2, 4, and 6 µg/mL). Percentages of matured oocytes that developed to the blastocyst stage were 18.7 ± 0.6, 26.4 ± 0.03, and 29.0 ± 1.7% for 0, 2, and 4 µg/mL Cu, and was highest (33.2 ± 1.6 %) in oocytes matured with 6 µg/mL Cu (P > 0.01). There was an increase (P > 0.05) in mean cell number per blastocyst obtained from oocytes matured with 4 and 6 µg/mL Cu relative to 0 Cu (IVM alone) and 2 µg/mL Cu. In conclusion, Cu concentrations in the FF and plasma of heifers were similar. Adding copper during oocyte maturation significantly increased both intracellular GSH content and DNA integrity of cumulus cells. Since embryo development was responsive to copper supplementation, we inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Cu concentrations during IVM.

Effect of increasing zinc sulphate concentration during in vitro maturation of bovine oocytes.

The objective was to investigate the effects of supplementary zinc (Zn) during in vitro maturation (IVM) of bovine oocytes. The DNA damage in cumulus cells was low with supplemental Zn concentrations of 1.1 and 1.5 µg/mL in the IVM medium (mean ± SEM index of DNA damage was 67.52 ± 9.32, 68.52 ± 13.34, 33.80 ± 4.89, and 34.65 ± 7.92 for supplementation with 0, 0.7, 1.1, and 1.5 µg/mL Zn, respectively; P < 0.01). Total glutathione concentrations did not differ following Zn supplementation of 1.1 and 1.5 µg/mL (3.7 ± 0.4 vs. 4.0 ± 0.5 pmol, respectively, in oocytes; and in cumulus cells, 0.5 ± 0.04 nmol/10(6) cells, combined for both treatments), but were greater (P < 0.01) than supplementation with 0.7 µg/mL (1.8 ± 0.5 pmol in oocytes and 0.2 ± 0.02 nmol/10(6) cumulus cells). Cleavage rate increased (P < 0.05) when Zn was added to the IVM medium at any concentration (67.16 ± 1.17, 73.15 ± 1.15, 74.05 ± 1.23, and 72.76 ± 0.74 for 0, 0.7, 1.1, and 1.5 µg/mL Zn). For these concentrations, subsequent embryo development to the blastocyst stage was 17.83 ± 2.15, 21.95 ± 0.95, 27.65 ± 1.61, and 30.33 ± 2.78%, highest (P < 0.01) in oocytes matured with 1.5 µg/mL Zn. There was an increase (P < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 1.1 and 1.5 µg/mL Zn relative to 0 Zn (IVM alone) and 0.7 µg/mL Zn. In conclusion, Zn during oocytes maturation significantly affected intracellular GSH content and DNA integrity of cumulus cells, and improved preimplantational embryo development. We inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Zn concentrations.

Association between copper deficiency and DNA damage in cattle.

Cattle hypocuprosis is the second most widespread mineral deficiency affecting grazing cattle. The consequences of hypocuprosis include a failure of copper metalloenzymes, many of which form part of the antioxidant defence system. This work focuses on the association between copper (Cu) plasma concentration and DNA damage in Aberdeen Angus cattle. Two-hundred and ninety-nine heparinized blood samples from 2-year-old Aberdeen Angus cows were obtained from different farms located in the Salado River basin, Argentina. Plasma copper level analysis was carried out in whole samples, while cytogenetic analysis and single cell gel electrophoresis assay (comet assay) were carried out in 82 and 217 samples, respectively. Cytogenetic analysis showed a significant increase in the frequency of abnormal metaphases in moderate/severe hypocupremic groups (groups B and C) in relation to the normocupremic group (group A) (4.5 and 1.5 abnormal metaphases/100 cells, respectively, P < 0.01). The Spearman correlation test showed a negative association between cupremic values and the yield of chromosomal aberrations (r = -0.708, P < 0.0001). In the comet assay greater migration was observed in cells from the hypocupremic group, from a median of 54 in the severe hypocupremic group to 31 in the normocupremic group (P < 0.01). Accordingly, the Spearman correlation test showed a significant positive relationship between copper levels and cells without DNA migration and a significant negative relationship between copper levels and cells with a tiny tail (P < 0.0001 in both cases). The results obtained show that hypocupremia in cattle is associated with an increase in the frequency of chromosomal aberrations as well as in DNA migration as assessed by the comet assay. Whereas the comet assay could differentiate copper plasma level groups, chromosomal aberrations only detected differences between normal and hypocupremic animals. The increase of DNA damage found in hypocupremic animals could be explained by higher oxidative stress suffered by these animals.

Incidence of 1/29 translocation in Bolivian Creole and Brahman Yacumeño cattle.

In Bolivia, four different Creole cattle breeds can be found, as well as other European and Zebu breeds adapted to local environments. The relationship between the occurrence of the 1/29 translocation and subfertility is well known, and analysis of Y chromosome morphology is useful to determine a possible introgression with Bos indicus. The incidence of the 1/29 translocation was analyzed in four Bolivian Creole cattle breeds and the Brahman Yacumeño population, as well as on four farms with phenotypical Creole-type cattle. In 259 (164 dams and 95 sires) Bolivian Creole cattle, 10.42% of the individuals demonstrated the 1/29 translocation, with a variation from 0 to 28.20% between the breeds. In contrast, 43 (19 dams and 24 sires) Yacumeño Brahman and the Creole-type cattle did not show the centric fusion. The highly significant differences between Creole cattle breeds in relation to the incidence of 1/29 translocation could be a consequence of factors such as founder group, genetic drift, and selection. The low frequency observed in the Saavedreñio Creole dairy cattle might be explained by its breeding under a more intensive system, and selection according to milk yield and fertility traits. Finally, no relation between acrocentric Y chromosomes and 1/29 translocation was observed.

DNA damage induced by copper deficiency in cattle assessed by the Comet assay.

Cattle hypocuprosis is a well-known endemic disease in several parts of the world. In a previous paper, the clastogenic effect of copper deficiency in cattle has been described although the occurrence of DNA damage was not directly tested. For this reason, the relation between DNA damage assessed by the Comet assay and Cu plasma concentration was studied in Aberdeen Angus cattle. Blood samples were obtained in heparinized Vacutainer tubes from 28 female Aberdeen Angus cows during pregnancy or immediately after to give birth. Each sample was divided into two aliquots for Comet assay and Cu plasma determination, respectively. From the 28 cattle sampled, 17 were normocupremic and 11 were hypocupremic. Results obtained showed that whereas the average plasma Cu level in normocupremic cattle was 67.6 microg/dl, in hypocupremic cattle it was 32.1 microg/dl. The increase of DNA damage was mostly evidenced by the decrease of comet degree 1 cells and an increase of comet degree 2 cells. Correlation analysis comparing plasma Cu levels and degree 1 cells showed a correlation coefficient 0.72 (P<0.01). The comparison between plasma Cu levels and comet degree 2 cells was -0.65 (P<0.01). The comparison between plasma Cu levels and the comet length-head diameter medians determined in 23 out of 28 animals showed a correlation coefficient of -0.54 (P<0.01). The induction of DNA damage was clearly supported by the fact that the decrease of plasma Cu levels was correlated with the increase of comet length-head diameter. These findings could be considered as a contribution to the hypothesis that DNA and chromosome damage are a consequence of the higher oxidative stress suffered by hypocupremic animals.