In as a replacement for Aceto-orcein stain.

Inpresent study aniline blue staining showed large number of sperms inexperimental group were in dark blue color which indicates poor chromatinintegrity of sperm cell fig. 1(b) 1(c) 1(d), whereas the control group sperms appears to be of light blue color showing good integrity ofchromatin as shown in fig 1(a). A positive correlation between chromosomalabnormalities at the time of meiosis that cause disturbance during thetransition of nucleoprotein and percentage of sperm nuclei that stained withaniline blue. The acidic aniline blue stains lysine-rich nucleoprotein ofimmature sperm. During spermatogenesis lysinerich histones are replaced by intermediate nucleoproteins which then arereplaced by arginine and cysteine-rich protamines. Then, abnormal chromosomesegregation at the time of meiosis allows the persistence of lysine-richnucleoproteins in spermatozoa.

Toluidine blue is a basic thiazine metachromatic dye with highaffinity for acidic tissue components.It stains nucleic acids blue and polysaccharides purple and alsoincreases the sharpness of histology slide images. It is especially useful forstaining chromosomes in plant or animal tissues, as a replacement forAceto-orcein stain. TB dye staining method is a simple method and allowsimultaneous evaluation for morphology and condensation of spermatozoa. TB isan ideal dye for routine use in this area.Treatmentof sperm from epididymis and testes using toluidine staining showed sperm cellhead with deep violet color in experimental group which indicates diminishedintegrity fig 2(c) 2(d), while light blue cell head in control group whichshows good chromatin integrity fig 2(a) 2(b).TB is a classic nuclear (cationic) dye which is used for externalmetachromatic and orthochromatic staining of chromatin, which overall isnegatively charged 46. Thus, orthochromatic (light blue) staining is theresult of monomeric dye forms characteristic for either low dye concentrationsor low accessibility of sites on the chromatin.

Metachromatic (purple)staining, on the other hand, is the result of polymeric dye formscharacteristic of cooperative binding to the DNA phosphate residues. The firstsituation corresponds to highly packaged chromatin of mature sperm cells withtheir very low stain ability by external dyes 47. The second situation ariseswhen chromatin proteins are more loosely electrostatically bound to the DNA andcan dissociated from it easily. In turn, DNA–protein interactions depend on theintegrity and superhelicity of the DNA. In agreement with this, nicked DNA wasshown to be more weakly electrostatically bound to chromatin proteins insomatic cells, therefore favouring binding and polymerization of externalthiazine dyes.Theaniline blue staining showed the toxicity in terms of maturation orimmaturation of sperms.

The sperms for this staining were taken from epididymisby squeezing. Ill effects were seen as many sperms taken dark blue stain inexperimental group which is a sign of immature sperm (figure3(b) 3(c)), whereassperm in control group stained reddish pink showing healthy mature sperm figure3(a).Morel et al. (1998) examined morphological disorders in thespermatozoa to show their connection with the nuclear maturity, sperms stainedwith Aniline Blue. Spermatozoa morphological defects differenceswere seen between individuals and they found a significant relationship betweenfrequent of defects and degree of nuclear maturates.

Our study is compatiblewith the knowledge of the literature. Aniline Blue dye provides the ideal imageto measure the rate of acrosome head. Condensation and morphology can beassessed together.Acridineorange is an organic compound. It is used as a nucleic acid-selectivefluorescent cationic dye useful for cell cycle determination. Beingcell-permeable, it interacts with DNA and RNA by intercalation or electrostaticattractions respectively.Furtherreproductive toxicity of silver nanoparticles was confirmed using acridineorange staining.

DNA integrity was checked using this staining. This stainingshowed orange and yellow color in sperms of experimental group which indicatess DNA.fig 4(c) 4(d)whereas the control group sperm took green color which is asign of ds DNA and a healthy sperm4(a) 4(b).SpermDNA damage may occur by at least three putative mechanisms: (i) defectivechromatin condensation during spermiogenesis; (ii) initiation of apoptosis duringspermatogenesis or transport of sperm through male or female genital tract;(iii) by oxidative stress mainly resulting from reactive oxygen species (ROS)produced internally or externally. Endogenous nicks in DNA may be present atspecific stages of spermatogenesis in rodents, during the replacement ofhistones by protamines (McPherson and Longo, 1992).

It is postulated that anendogenous nuclease, topoisomerase II, creates and ligates nicks to providerelief of torsional stress and to aid chromatin rearrangement duringprotamination (McPherson and Longo, 1993). Therefore, the existence ofendogenous nicks in sperm may indicate incomplete maturation.TheAO assay measures the susceptibility of sperm nuclear DNA to acid-induceddenaturation in situ by quantifying the metachromatic shift of AO fluorescencefrom green (native DNA) to Yellow (denatured DNA) and red (denatured DNA). Thefluorochrome AO intercalates into double-stranded DNA as a monomer and binds tosingle-stranded DNA as an aggregate. The monomeric AO bound to native DNAfluoresces green, whereas the aggregated AO on denatured DNA fluorescesYellow/red.Thereactivity of trypan blue is based on the fact that the chromopore isnegatively charged and does not interact with the cell unless the membrane isdamaged. Therefore, all the cells which exclude the dye are viable.

Trypan bluestaining showed the toxicity by giving dark blue color to the experimentalgroup which shows membrane damaged sperm fig{5(b),5(c)} whereas control groupremains colorless showing healthy sperm fig5(a). Condensation and headmorphology of spermatozoa were well-selectable. The middle piece and tail couldbe seen.

The in vitromicronucleus assay is a mutagenic test system for the detection of chemicalswhich induce the formation of small membrane bound DNA fragments i.e.micronuclei in the cytoplasm of interphase cells.

These micronuclei mayoriginate from acentric fragments (chromosome fragments lacking a centromere)or whole chromosomes which are unable to migrate with the rest of thechromosomes during the anaphase of cell division. The purpose of themicronucleus assay is to detect those agents which modify chromosome structureand segregation is such a way as to lead to induction of micronuclei ininterphase cells. As shown in thefigure 7 (a) (b), the number of micronucleus has been observed in the treatmentgroup which signifies the genotoxic effect of silver nanoparticles. A positiveresult from the in vitro micronucleus test indicates that the testsubstance induces chromosome damage or damage to the cell division apparatus.



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