Project
protocol
-
Contents
Workflow
and
sampling
Step |
Procedure |
Equipment |
Data
collected* |
1 |
Mice
euthanized
and
weighed
(see
Odet1) |
- |
- |
2 |
Reproductive
organs
harvested
and
weighed
(see
Odet1) |
- |
- |
3 |
Testis
fixed,
prepared
for
histology,
and
imaged
(see
Odet1) |
- |
- |
4 |
Sperm
harvested
from
right
cauda
epididymis
and
counted
(see
Odet1) |
- |
- |
5 |
Sperm
harvested
from
left
cauda
epididymis
for
in
vitro
capcitation
study
on
sperm
motility
|
Microscope
(Computer
Assisted
Sperm
Analysis
(CASA)) |
Motility
analysis |
6 |
Sperm
from
left
cauda
epididymis
prepared
to
assess
morphology
(see
Odet1) |
- |
- |
7 |
Sperm
from
left
cauda
epididymis
prepared
to
measure
lactate
production
(see
Odet1) |
- |
- |
*Supplementary
data
are
available
for
this
project.
See Odet2 downloads.
Equipment
and
supplies
- Hamilton
Thorne
CEROS
imaging
system
with
version
12.3
IVOS
software
(Goodson
et
al.
2011)
for
computer-assisted
sperm
analysis
(CASA)
- CASAnova
(a
support
vector
machines
program
based
on
CASA
parameters
of
CD1
sperm)
- Leja
chambers
(100
µm-depth)
(Leja,
Netherlands)
- 5% CO2 tissue culture incubator
Reagents
and
solutions
- Human
tubal
fluid
medium (HTF):
101.6
mM
NaCl,
4.7
mM
KCl,
0.37
mM
KH2PO4,
0.2
mM
MgSO4·7H2O,
2
mM
CaCl2,
25
mM
NaHCO3,
2.78
mM
glucose,
0.33
mM
pyruvate,
21.4
mM
lactate,
5
mg/mL
of
bovine
serum
albumin,
100
U/mL
of
penicillin
G,
and
0.1
mg/mL
of
streptomycin
(Goodson
et
al.
2011).
- Bovine
serum
albumin
(BSA)
Procedure:
Sperm
motility
- Mice
are
euthanized
by
CO2
asphyxiation
followed
by
cervical
dislocation.
- Reproductive
organs
are
harvested
and
weighed
(Odet1).
- The
left
cauda
epididymis
is
clipped
with
iris
scissors
and
incubated in
human
tubal
fluid
medium (HTF)
at
37°C
(5%
CO2)
for
10
min.
- Sperm
suspensions
are
diluted
with
HTF
and
incubated
for
2
h
at
37°C
(5%
CO2).
- Motility
is
assessed
at
30
min
intervals
throughout
the
in
vitro
capacitation
period
by
computer-assisted
sperm
analysis
(CASA)
using
Leja
chambers.
- Sperm
tracks
(90
frames,
1.5
s)
and
kinetic
parameters
for
individual
sperm
are
captured
at
60
Hz
using
motility
analysis
parameters
(mouse
2)
recommended
by
Hamilton
Thorne
Biosciences,
except
that
slow
sperm
are
considered
motile.
- Tracks
in
10
fields
are
typically
recorded
for
each
mouse;
motility
measurements
are
captured.
- CASAnova
is
used
to
classify
individual
sperm
into
one
of
five
motility
groups. Classification is based on Support Vector Machines equations incorporating independent CASA parameters for each sperm track.
Data
collected
by
investigator
CASA parameters for individual sperm tracks and means for each sample:
- average
path
velocity
(VAP)
- straight
line
velocity
(VSL)
- curvilinear
velocity
(VCL)
- amplitude
of
lateral
head
displacement
(ALH)
- beat
cross
frequency
(BCF)
- straightness
(STR) = VSL/VAP
- linearity
(LIN) = VSL/VCL
Sperm
motility
groups
classification:
- percentage
of
sperm
defined
as
progressive
- percentage
of
sperm
defined
as
intermediate
- percentage
of
sperm
defined
as
hyperactivated
- percentage
of
sperm
defined
as
slow
- percentage
of
sperm
defined
as
weakly
motile
- percentage
of
sperm
considered
vigorous
=
progressive
+
intermediate
+
hyperactivated
Goodson
SG,
Zhang
Z,
Tsuruta
JK,
Wang
W,
O'Brien
DA.
Classification
of
mouse
sperm
motility
patterns
using
an
automated
multiclass
support
vector
machines
model.
Biol
Reprod.
2011
84(6):
1207-15.
|