Evaluation of chromatin integrity in human sperm using acridine orange staining with different fixatives and after cryopreservation.

Staining of cells with acridine orange (AO) has been widely accepted as a predictor of DNA damage in many cell types. Because of variability of protocols used in previous studies, the AO staining technique has not been widely accepted as a screening test to predict DNA damage in human sperm. In order to further validate the use of AO staining, sperm were evaluated using numerous variations in the staining protocol. This study also elucidated the effects of cryopreservation on sperm DNA. Sperm fixation in Carnoy's solution showed significantly (P < 0.05) more DNA damage (29.9 +/- 4.5%) than 2% glutaraldehyde (14.4 +/- 2.1%), 4% paraformaldehyde (5.5 +/- 1.7%), no fixation (15.8 +/- 4.3%) but did not differ from Diff Quik solution (19.2 +/- 5.8%). No difference was observed for sperm DNA damage assessment using a 0.2 m (15.5 +/- 3.2%) or 0.3 m (14.9 +/- 3.3%) concentration of Na(2)HPO(4).7H(2)O in the AO staining solution. Frozen-thawed semen samples showed increased damage to sperm DNA under both Carnoy's (fresh: 10.9 +/- 1.3%; frozen: 30.8 +/- 2.9%; P < 0.05) and Diff Quik fixation (fresh: 6.2 +/- 0.8; frozen: 17.1 +/- 2.5%P < 0.05). Present data also showed that spermatozoa from some individuals are more prone to DNA damage after freezing and thawing procedures than others. In conclusion, Carnoy's fixative provides a better predictive value for DNA damage to sperm using AO staining. Additionally, cryopreservation increased damage to the sperm DNA.

Repellence of the tick Rhipicephalus appendiculatus by the grass Melinis minutiflora.

In a study to develop anti-tick pastures, the climbing behaviour of Rhipicephalus appendiculatus on Melinis minutiflora (molasses grass) was investigated. Experiments were done with cut green stems of grass, grass dried in the shade, grass dried in sunshine, grass washed in solvent and grass growing in a study plot. In all cases a common pasture grass, Pennisetum clandestinum (Kikuyu grass), was used as control. All instars of the tick avoided climbing on the green M. minutiflora whereas most larvae, nymphs and adults climbed on the control green P. clandestinum grass. More ticks climbed on the stems dried in the sunshine than on air-dried grass. Acetone was found to be the best of 5 solvents used to extract the tick-repellent substance. Possibilities of using M. minutiflora as part of an integrated tick control package are discussed.

Improvements in the thin-layer chromatography of natural products. I. Thin-layer chromatography of the aflatoxins.

During the period which has elapsed since the aflatoxins were first isolated, one of the main problems has been the separation of the individual aflatoxins in pure form from aflatoxin-containing extracts. This separation has been best effected by thin-layer chromatography, and in this paper we describe how some of the difficulties may be overcome by using an appropriate combination of solvent system and silica gel preparation. For the examination of aflatoxin-containing extracts from the mycelia of Aspergillus flavus moulds, an initial freeze-drying step has been found to improve appreciably the quality of the chromatograms obtained.