Validation of a Field Based Chromatin Dispersion Assay to Assess Sperm DNA Fragmentation in the Bottlenose Dolphin (Tursiops truncatus)

Over the last two decades, there have been significant advances in the use of assisted reproductive technology for genetic and reproductive management of captive dolphin populations, including evaluation of sperm DNA quality. This study validated a customized sperm chromatin dispersion test (SCDt) for the bottlenose dolphin (Tursiops truncatus) as a means of assessing sperm DNA damage both in the field and in the laboratory. After performing the SCDt, two different sperm morphotypes were identified: (i) sperm with fragmented DNA showed large haloes of dispersed DNA fragments emerging from a compact sperm nucleoid core and (ii) sperm containing non‐fragmented DNA displayed small compact haloes surrounded by a dense core of non‐dispersed DNA and protein complex. Estimates of sperm DNA fragmentation by means of SCDt were directly comparable to results obtained following a two‐tailed comet assay and showed a significant degree of correlation (r = 0.961; p < 0.001). This investigation also revealed that the SCDt, with minor modifications to the standard protocol, can be successfully conducted in the field using a LED florescence microscopy obtaining a high correlation (r = 0.993; p = 0.01) between the data obtained in the laboratory and in the field.     

In situ micro-spectrophotometric and micro- spectrocolorimetric investigation of vacuolar pigments in flowers of cultivars of carnation (.Dianthus caryophyllus)

Living epidermal cells of flower petals of carnations have been surveyed for their native colour by in situ spectral (350-750 nm wavelengths) and colorimetric investigations (CIE- Lab, L* C* h system) of their vacuolar pigment content with a micro-spectrophotometer. Vacuolar solutions in cultivars with yellow based colours generally display high Absorbances (close to, or over A = 2 under a 25 |xm optical depth) in the near-UV area only: at 360-375 nm for ivory or pale yellow colours (flavonols only) and additionally at 380- 395 nm (chalcone glycosides) for yellow ones. Carnation cultivars with cyanic colours are accumulating anthocyanins, namely monosides and diosides of pelargonidin and cyani- din, along with flavonol glycosides acting as co-pigments. Besides their main À.max in the 520-550 nm area, frequently appearing at lower wavelengths than in those of diosides, spectra of solutions of monosides are characterized by an additional peak or shoulder around 450 nm. Cyanidin derivates often exhibit higher X.max wavelengths than those recorded in spectra of corresponding pelargonidin derivates. In the spectra of solutions of most classes of these anthocyanins, the visible Xmax position varies considerably, according to the relative concentrations of pigment (measured as the Absorbance at the visible Àmax) to co-pigment (appreciated by the Absorbance at the near-UV Xmax): generally, the higher this ratio, the longer is the visible Xraax wavelength. In situ spectral recording of individual vacuolar sap also reveals that different cells in the same petal epidermis are accumulating, in some cultivars, distinct pigment mixtures (diosides vs. monosides for instance) originating different colours. Colours of the vacuolar solutions have been colorimetrically described in the CIELab system in terms of hue (hab), saturation (Chroma, C*) and intensity (Lightness, L*). Generally, as the Absorbance at X.max, i.e. the pigment concentration, increases, Lightness decreases accordingly. Chroma mainly depends on the general shape of the spectra in the visible area. The more complex relationships of hue with spectral characteristics and the nature of the pigment mixture are discussed. Finally, the comparison of L* C* h colorimetric data calculated for two CIE illuminants (D65 and A) shows how the same pigment mixture (and consequently the further colour of intacts petals) appears to be coloured differently according to the lighting conditions of the visual observation.