RESUMEN
Aqueous extracts were obtained at low temperature with the Naviglio technology from grapevine stalks (Merlot), marc (Merlot and Cabernet Sauvignon) and leaves (Merlot) as typical byproducts of winemaking industry, and their properties were evaluated cytofluorometrically on human dermal fibroblasts. Leaf extracts had the greatest total phenolic ((47.6±3.5) mg/g) and proanthocyanidin ((24.2±0.1) mg/g) contents compared to the others. The preliminary colorimetric MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay individuated two consecutive non-toxic volume fractions of each extract (from 0.8 to 12.8%) that were adopted for three cytofluorometric tests. The first cell membrane test did not evidence any harmful effects against plasma membranes at the two non-toxic volume fractions. The second mitochondrial membrane test showed a decreased (p<0.01) percentage of cells ((15.7±8.3) vs (32.5±1.3) %) with active polarized mitochondrial membranes at the higher non-cytotoxic volume fractions of extracts from Cabernet Sauvignon marc in response to 4.5 mM H2O2, and from Merlot stalks (p<0.05) at 1.5 mM H2O2 ((49.3±6.1) vs (64.6±2.4) %) and without H2O2 ((89.7±2.4) vs (96.9±1.8) %), compared to the controls submitted to the same H2O2 concentration. Conversely, mitochondrial activity of leaf extracts significantly (p<0.05) increased ((96.3±1.8) and (96.4±1.4) %) after treatment with 0.5 mM H2O2 at both non-cytotoxic volume fractions compared to control ((88.2±1.1) %). Finally, as evidenced by the third oxidative status test, stalk extracts did not evidence relevant effects on the cellular oxidative state, while the extracts of marc and leaves demonstrated significantly medium (p<0.05) to highly (p<0.001) positive effects following exposure to H2O2 ranging from 0.5 to 4.5 mM, compared to controls.
RESUMEN
Fresh 36 ejaculates of 13 stallions were split into two volumes, centrifuged with and without cushion and frozen with Conventional and two prototype, Drum and Directional, methods using 0.5 ml straws for the Conventional and Drum, and 2 ml flat straws for both the Drum and Directional. Cushioned centrifugation increased total motility (61.2 ± 18.6% vs. 57.5 ± 18.6%; P < 0.001) and mean velocity (84.3 ± 15.6% vs. 83.2 ± 13.8%; P < 0.05) when compared to not cushioned centrifugation, estimated after cooling the sperm at 4°C for 90 min before freezing. Cushioned centrifugation also increased (P < 0.001) spermatozoa with polarized mitochondrial membranes (46.8 ± 11.4% vs. 43.4 ± 10.6%) and intact plasmatic/acrosomal membranes (41.0 ± 11.2% vs. 38.5 ± 11.3%) of frozen/thawed sperm, with respect to not cushioned centrifugation. However, no effects of the centrifugation were evidenced for classical kinetic parameters. Flat straws had negative effect for almost all the parameters analyzed at thawing (T,) and after 3 hours' incubation at 37°C (T1), while the Drum method with Paillettes did not show appreciable affects. The variability among stallions was relevant (5% to 69% variance for kinetics and membrane status), while the variability among ejaculates was minor (9% to 28%). Factorial analysis identified three relevant, factors with different informational content: Factor 1 represented by membranes status, Factor 2 by kinetics estimated at T0, and Factor 3 by kinetics estimated at T1. Cushioned centrifugation had some beneficial effects for the membrane status of the frozen/thawed sperm, while the use of flat straws needs to be improved.
