In a simple method and allow simultaneous evaluation

present study aniline blue staining showed large number of sperms in
experimental group were in dark blue color which indicates poor chromatin
integrity 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 of
chromatin as shown in fig 1(a). A positive correlation between chromosomal
abnormalities at the time of meiosis that cause disturbance during the
transition of nucleoprotein and percentage of sperm nuclei that stained with
aniline blue. The acidic aniline blue stains lysine-rich nucleoprotein of
immature sperm. During spermatogenesis lysine
rich histones are replaced by intermediate nucleoproteins which then are
replaced by arginine and cysteine-rich protamines. Then, abnormal chromosome
segregation at the time of meiosis allows the persistence of lysine-rich
nucleoproteins in spermatozoa.

Toluidine blue is a basic thiazine metachromatic dye with high
affinity for acidic tissue components.

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It stains nucleic acids blue and polysaccharides purple and also
increases the sharpness of histology slide images. It is especially useful for
staining chromosomes in plant or animal tissues, as a replacement for
Aceto-orcein stain. TB dye staining method is a simple method and allow
simultaneous evaluation for morphology and condensation of spermatozoa. TB is
an ideal dye for routine use in this area.

of sperm from epididymis and testes using toluidine staining showed sperm cell
head with deep violet color in experimental group which indicates diminished
integrity fig 2(c) 2(d), while light blue cell head in control group which
shows good chromatin integrity fig 2(a) 2(b).

TB is a classic nuclear (cationic) dye which is used for external
metachromatic and orthochromatic staining of chromatin, which overall is
negatively charged 46. Thus, orthochromatic (light blue) staining is the
result of monomeric dye forms characteristic for either low dye concentrations
or low accessibility of sites on the chromatin. Metachromatic (purple)
staining, on the other hand, is the result of polymeric dye forms
characteristic of cooperative binding to the DNA phosphate residues. The first
situation corresponds to highly packaged chromatin of mature sperm cells with
their very low stain ability by external dyes 47. The second situation arises
when chromatin proteins are more loosely electrostatically bound to the DNA and
can dissociated from it easily. In turn, DNA–protein interactions depend on the
integrity and superhelicity of the DNA. In agreement with this, nicked DNA was
shown to be more weakly electrostatically bound to chromatin proteins in
somatic cells, therefore favouring binding and polymerization of external
thiazine dyes.

aniline blue staining showed the toxicity in terms of maturation or
immaturation of sperms. The sperms for this staining were taken from epididymis
by squeezing. Ill effects were seen as many sperms taken dark blue stain in
experimental group which is a sign of immature sperm (figure3(b) 3(c)), whereas
sperm in control group stained reddish pink showing healthy mature sperm figure

Morel et al. (1998) examined morphological disorders in the
spermatozoa to show their connection with the nuclear maturity, sperms stained
with Aniline Blue. Spermatozoa morphological defects differences
were seen between individuals and they found a significant relationship between
frequent of defects and degree of nuclear maturates. Our study is compatible
with the knowledge of the literature. Aniline Blue dye provides the ideal image
to measure the rate of acrosome head. Condensation and morphology can be
assessed together.

orange is an organic compound. It is used as a nucleic acid-selective
fluorescent cationic dye useful for cell cycle determination. Being
cell-permeable, it interacts with DNA and RNA by intercalation or electrostatic
attractions respectively.

reproductive toxicity of silver nanoparticles was confirmed using acridine
orange staining. DNA integrity was checked using this staining. This staining
showed orange and yellow color in sperms of experimental group which indicate
ss DNA.fig 4(c) 4(d)whereas the control group sperm took green color which is a
sign of ds DNA and a healthy sperm4(a) 4(b).

DNA damage may occur by at least three putative mechanisms: (i) defective
chromatin condensation during spermiogenesis; (ii) initiation of apoptosis during
spermatogenesis 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 at
specific stages of spermatogenesis in rodents, during the replacement of
histones by protamines (McPherson and Longo, 1992). It is postulated that an
endogenous nuclease, topoisomerase II, creates and ligates nicks to provide
relief of torsional stress and to aid chromatin rearrangement during
protamination (McPherson and Longo, 1993). Therefore, the existence of
endogenous nicks in sperm may indicate incomplete maturation.

AO assay measures the susceptibility of sperm nuclear DNA to acid-induced
denaturation in situ by quantifying the metachromatic shift of AO fluorescence
from green (native DNA) to Yellow (denatured DNA) and red (denatured DNA). The
fluorochrome AO intercalates into double-stranded DNA as a monomer and binds to
single-stranded DNA as an aggregate. The monomeric AO bound to native DNA
fluoresces green, whereas the aggregated AO on denatured DNA fluoresces

reactivity of trypan blue is based on the fact that the chromopore is
negatively charged and does not interact with the cell unless the membrane is
damaged. Therefore, all the cells which exclude the dye are viable. Trypan blue
staining showed the toxicity by giving dark blue color to the experimental
group which shows membrane damaged sperm fig{5(b),5(c)} whereas control group
remains colorless showing healthy sperm fig5(a). Condensation and head
morphology of spermatozoa were well-selectable. The middle piece and tail could
be seen.

The in vitro
micronucleus assay is a mutagenic test system for the detection of chemicals
which induce the formation of small membrane bound DNA fragments i.e.
micronuclei in the cytoplasm of interphase cells. These micronuclei may
originate from acentric fragments (chromosome fragments lacking a centromere)
or whole chromosomes which are unable to migrate with the rest of the
chromosomes during the anaphase of cell division. The purpose of the
micronucleus assay is to detect those agents which modify chromosome structure
and segregation is such a way as to lead to induction of micronuclei in
interphase cells.

As shown in the
figure 7 (a) (b), the number of micronucleus has been observed in the treatment
group which signifies the genotoxic effect of silver nanoparticles. A positive
result from the in vitro micronucleus test indicates that the test
substance induces chromosome damage or damage to the cell division apparatus.



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