Assessment of chromatin maturity in human spermatozoa: useful aniline blue assay for routine diagnosis of male infertility.
A double-blinded comparison of in situ TUNEL and aniline blue versus flow cytometry acridine orange for the determination of sperm DNA fragmentation and nucleus decondensation state index.
Granulosa cells were mostly viable and their nuclei were euochromatic in appearance even in a 1000 [micro]g/ml DSAE concentration and a small population of granulosa cells showed either condensed chromatin after aniline blue
staining or apoptotic signs after Ao/Eb staining (Table 1, Figures 1 and 3).
(19) Aniline blue
stains lysine rich nucleoproteins, which is another sign of deterioration of chromatin condensation and was shown to correlate with AO staining.
Overall, the Alexander's Stain assay gave the highest estimate of viability (82-99%), whereas viability determined by other methods ranged from 34 to 98% (FCR), 57 to 95% (B & K), and 51 to 88% (B & K + aniline blue), primarily depending on the cultivar and glyphosate treatment.
Inclusion of aniline blue in the medium was useful for distinguishing between true pollen tubes (containing callose) and pollen grains which simply burst.
Pollen viability was high, with [greater than]95% of grains staining darkly with aniline blue
cuspidata plants per selection treatment was analyzed in a drop of aniline blue
in lactophenol under a light microscope.
Sperm nuclear instability and staining with aniline blue
: abnormal persistence of histones in spermatozoa in infertile men.
Grade A Grade Grade C B Test r p-value r p-value r p-value Sperm function parameters Acridine 0.05 NS -0.06 NS -0.006 NS Orange % (a) Aniline Blue
Aniline Solution([dagger]) Sucrose blue g mg A 84.6 10 B 84.6 10 C 10 D 10 E 56.0 10 Aniline free Solution([dagger]) Water base Methanol DMSO mL A 100 B 100 C 80 20 D 15 75 10 E 57 43 pH Solution([dagger]) adjustment([double dagger]) A [K.sub.2]HP[0.sub.4]/ [K.sub.3]P[O.sub.4] B NaHC[0.sub.3] C NaHC[O.sub.3] D NaHC[O.sub.3] E NaHC[0.sub.3] ([dagger]) Solution D was prepared by mixing water with DMSO and then adding aniline blue
and aniline free base.
Pistils were cleared in aqueous clearing medium (136 [micro]W aniline blue
and 2.46 M sucrose, pH adjusted to 9.5 with 1 M NaHC[O.sub.3]), mounted between cover slips in sagittal orientation, covered with clearing medium and a cover slip, and analyzed for MMC callose with an Olympus photomicroscope (Olympus America, Inc., Melville, NY) with phase contrast optics and a vertical epifluorescence (UG-1 exciter filter, Y-455 dichroic mirror, L-435 barrier filter) illuminator (Leblanc et al., 1995, Peel et al., 1997).