Comparison of the TBARS Assay and BODIPY C11 Probes for Assessing Lipid Peroxidation in Red Deer Spermatozoa

Several methods are used to measure lipid peroxidation (LPO) in spermatozoa. The objective of this study was comparing the thiobarbituric acid reactive species (TBARS) method and the BODIPY 581/591 C₁₁ (B581) and BODIPY 665/676 C₁₁ (B665) fluorescent probes to measure induced peroxidative damage in thawed epididymal spermatozoa from Iberian red deer. Samples from three males were thawed, pooled, diluted in PBS, incubated at room temperature and assessed at 0, 3, 6 and 24 h under different experimental conditions: Control, hydrogen peroxide (H₂O₂) 0.1 mm or 1 mm , or tert‐butyl hydroperoxide (TBH) 0.1 mm or 1 mm . LPO was assessed by the TBARS assay [malondialdehyde (MDA) detection] and by the fluorescence probes B581 and B665 (microplate fluorimeter and flow cytometry). Increasing MDA levels were only detectable at 1 mm of TBH or H₂O₂. Both fluorescence probes, measured with fluorometer, detected significant increases of LPO with time in all treatments, except Control. Flow cytometry allowed for higher sensitivity, with both probes showing a significant linear relationship of increasing LPO with time for all oxidizing treatments (p < 0.001). All methods showed a good agreement, except TBARS, and flow cytometry showed the highest repeatability. Our results show that both B581 and B665 might be used for LPO analysis in Iberian red deer epididymal spermatozoa, together with fluorometry or flow cytometry. Yet, the TBARS method offered comparatively limited sensitivity, and further research must determine the source of that limitation.     

Inhibition of resistance plasmid transfer in Escherichia coli by ionophores, chlortetracycline, bacitracin, and ionophore/antimicrobial combinations

Medicinal feed additives bacitracin, chlortetracycline (CTC), laidlomycin, lasalocid, and salinomycin inhibited the transfer of multiresistance-conferring plasmid pBR325 (Tet(r) Amp(r) Cp(r), 6.0 kb) into selected gram-negative strains with the use of an in vitro model. High concentrations of ampicillin-sensitive competence-pretreated Escherichia coli HB 101 cells were exposed to 10% (v/v) of 1:10 dimethyl sulfoxide/agent : water containing test mixtures for 0.5 hr prior to plasmid addition and transforming conditions. Transformation was inhibited for all antimicrobials and showed a positive association wich higher concentration. Additional testing of ionophore compounds separately and in combination with bacitracin, chlortetracycline, lincomycin, roxarsone, tylosin, and virginiamycin at representative feed concentrations demonstrated 80.6% to >99.9% inhibition (P < 0.001) of resistance transfer. Bacitracin alone inhibited transformation within the range of 50-500 ppm. No increase in resistance transfer was observed when poultry-derived and reference gram-negative isolates having low or no transformation efficiency were additionally tested. The results suggest that these compounds, at relevant concentrations used in animal feed, may interfere with cell envelope-associated DNA uptake channels or other transformation competence mechanisms. Through these mechanisms, ionophores and cell membrane-interactive feed agents such as CTC and bacitracin may act to inhibit resistance transfer mechanisms within poultry and livestock.     

Effects of Cryopreservation on Bull Spermatozoa Distribution in Morphometrically Distinct Subpopulations

Assisted sperm morphometry analysis (ASMA) was used in this study to determine the effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations. Ejaculates were collected from five bulls and were divided. One portion was diluted at 30 degrees C in a skim milk-egg yolk medium, containing glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 200 sperm heads were analysed from each sample by means of the Sperm-Class Analyser (SCA), and the mean measurements recorded. Our results showed that applying the ASMA technology and multivariate cluster analyses, it was possible to determine that three separate subpopulations of spermatozoa with different morphometric characteristics coexist in bull ejaculates (large, average and small spermatozoa). The mean values of each sperm head dimension among the three subpopulations of spermatozoa were significantly different (p < 0.001). Besides, there were significant (p < 0.001) differences in the distribution of these three sperm subpopulations between fresh and thawed samples. Thus, the percentage of representation of the subpopulation that includes those spermatozoa whose dimensions are the biggest, decreased from 52.06% in extended fresh samples to 15.51% in the thawed ones. Contrarily, the percent of representation of the subpopulation containing the smallest spermatozoa, increased from 8.70% in extended fresh samples to 34.04% in the thawed ones. In conclusion, the present study confirms the heterogeneity of sperm head dimensions in bull semen, heterogeneity that vary through the cryopreservation procedure.     

Development of rabbit visual cortex: late appearance of a class of receptive fields

In young rabbits before the age at which the eyes open, only three of the seven receptive field types described in the adult visual cortex are detectable. The remaining four receptive field types-which share the property of having radially asymmetric fields-appear later, coincident with a decline in the percentage of cells that are visually responsive but not classifiable as to receptive field type.     

Photopolymerization and Mass-Independent Sulfur Isotope Fractionations in Carbon Disulfide

Irradiation of gaseous carbon disulfide [CS2(g)] at 313 nanometers produces a dark brown aerosol of (CS2)x. Its thermal decomposition products include disulfur (S2), carbon monosulfide (CS), and (CS)x. The photopolymerization process is accompanied by a large mass-independent isotopic fractionation of sulfur (a 5 to 10 per mil sulfur-33 excess and a 61 to 84 per mil sulfur-36 deficit). Excess sulfur-33 has been observed in several classes of meteorites. Photochemical production of (CS2)x may be important in the origin and evolution of cosmochemical environments such as the presolar nebula, meteorites, asteroids, and planetary atmospheres.     

DNA Status on Thawed Semen from Fighting Bull: A Comparison Between the SCD and the SCSA Tests

The assessment of sperm chromatin status is compulsory in a complete spermiogram. Here we applied the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test to assess the chromatin status of three fighting bulls. Cryopreserved semen (two straws/bull) were analysed by duplicate after thawing and after 6 h at 37°C with and without oxidative stress (1 m m FE²⁺). Results (SCD: percentage of spermatozoa with halo; SCSA: SD-DFI, %DFI and HDS) were analysed for differences between bulls and treatments, sensitivity and specificity (receiver operating characteristic curves) and repeatability (repeatability coefficients as 2SD of duplicate differences).%DFI for the three bulls was below 2% at 0 h, indicating no risk for fertility according to previous reports. It increased slightly for two of the bulls after FE²⁺ treatment (%DFI < 5%) and more pronouncedly for the other bull (C, %DFI∼10%), which merits further investigation. SCD rendered higher percentage of halos for bull C, but could not discriminate between samples with and without oxidizing treatment (AUC: 0.52). SCSA (%DFI) showed a high discriminating ability between treatments (AUC: 0.96). The repeatability coefficient was also higher for SCD (5.9) than for %DFI (1.8), indicating lower repeatability for SCD. Overall, %DFI might be the most useful parameter for assessing sperm chromatin on fighting bull. SCD might yield different information than SCSA, hence further research is warranted.