Resumen:
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[EN] he general aim of the present study was to apply a method of fluorescent multiple
analysis developed by the
group TECNOGAM in the study
of the motility and
morphometry
of the spermatic subpopulations ...[+]
[EN] he general aim of the present study was to apply a method of fluorescent multiple
analysis developed by the
group TECNOGAM in the study
of the motility and
morphometry
of the spermatic subpopulations and
improving hypo
-
osmotic test
.
Three studies were conducted
using
frozen semen of Friesian bulls as
animal
model
. In three
studies the image
analysis was
conducte
d with the
ImageJ
free
software,
and the statistical analysis of the
data
using the analysis of variance
(ANOVA), followed by Tukey's test.
In
the s
tudy 1
, the percentage motile
sperm
atozoa in the fluorescent
subpopulations was determined
. The
fluorochromes
allowed a rapi
d determination of the plasma membrane and acrosome integ
rity
of
sperm
. The
subpopulation with intact
plasma membrane
and acrosome
displayed a
hi
gher percentage of mot
ile sperm
with the damaged
membrane were motile. In the
stud
y 2, the morphometric parameters of the head, nucleus and acrosome of the
sperm subpopulations and of 3 treatments: liquid samples immobilized with
formaldehyde (1) and with shock for heat 65°C
-
Trumorph® (2), dried, fixed smears
dyed with IP/PSA (3) were
compared.
The area of the head, nucleus
and acrosome
were lower (P <0,05) in
smears
than it wet mounts
treatment
s.
However, no
significant differences
in the sperm morphometry of the two treatments in the wet
mounts were found.
The subpopulation with
acrosome
normal,
damaged membra
ne
presented significantly higher
measures (P <0,05)
compared
with
the subpopulation
with acrosome
normal, membrane intact
, for both treatments in
wet mounts.
The
percentage of t
he head occupied by the acrosome
in the smears
wereas higher that
those of the treatments in wet mounts.
Finally, in the study 3, the effect of
hypos
-
osmotic test
(HOST), two types of solu
tions based on citrate and TRIS
, a
nd the test
water (
WT) on t
he distribution of the sperm
subpo
pulations. The final
osmolarity
was
adjusted to 150 mOsm/kg in all the cases.
The pH was adjusted to 7.0 based
only on the TRIS solution
. The subpopulations were determined at
5 and 40 min
from incubation to 37°C.
The response to HOST, the state and acrosome
membrane integrit
y were used to establish eight subpopulations
.
For both periods of
incubation, the subpo
pulation
HOST
negative, acrosome
damaged, damaged
membrane was significantly higher
in the WT that in both
HOST
solutions
evaluated
(P <0.05). At
40 min, the subpop
ulat
ion,
HOST
positive, acrosome
i
ntact, intact
membrane was lower
in the WT t
hat in the HOST when citrate
-
based solution was
used
. The new met
hod of multiple fluorescent staining
allowed, for the first time, to
study in the same sperm
atozoa the
integrity of the plasma membrane and
acrosome,
functionality and motility,
obtaining
diffe
rent
the sperm
subpopulations.
The ability of these new methods of analysis to predict sperm fertility will be
addressed in future studies.
[-]
[ES] Se trabajará sobre el desarrollo de nuevos métodos de evaluación de la calidad seminal en rumiantes, que estarán basados en una tinción múltiple fluorescente del espermatozoide, lo que permitirá evaluar simultáneamente ...[+]
[ES] Se trabajará sobre el desarrollo de nuevos métodos de evaluación de la calidad seminal en rumiantes, que estarán basados en una tinción múltiple fluorescente del espermatozoide, lo que permitirá evaluar simultáneamente diferentes parámetros con relevancia funcional y, potencialmente, mejorar la capacidad predictiva de la fertilidad del semen
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