Semen must be evaluated as soon as possible
after collection, because changes in temperature, exposure to light, and
exposure to any type of chemicals, lubricants etc. can change sperm
motility and adversely affect fertility. Motility
Motility should be examined as
soon as possible, as motility is the most influenced parameter in the
semen analysis. Use a wooden stick to handle semen, because a wooden
stick is thermo-neutral and will not cold-shock the sperm cells.
Gross motility is examined first.
Mix the semen
sample with a wooden stick, as motile sperm cells will try to swim
upward and dead cells will settle to the bottom.
For gross motility use
2 wooden sticks to place a drop of semen on a warm slide.
Do not use a cover slip and examine the cells under a 10X objective.
The motility is
judged by the swirling motion of the sample.
The swirling pattern will
definitely indicate that there are cells alive.
The swirling looks like
currents and eddies (like a fast motion weather map).
individual cells cannot be made as live cells will carry the dead cells.
The gross motility will tell you though that the cells are alive, and if
the cells are dead when you examine them for individual motility there
has been a handling problem.
- Gross - swirl
pattern in bull, ram and buck.
motility is examined next.
- Individual motility checks for the
progressive movement of the sperm cells.
- Make the sample by placing a
drop of diluent (saline or Na citrate) on a warm slide.
- Place a small
amount of semen into the saline.
You want about 10 cells/high power
field in order to accurately estimate the number of cells that are
progressively moving across the field.
Then place a warm cover slip on
Examine the sample under high dry (40X) power.
examine the sample quickly as the motility changes very rapidly with
heat, light, and cold.
The motility is a very subjective measurement and is
affected by many things, such as diluent (it may be old and hypertonic
etc.), cold, the glassware, urine, soap, prostatic fluid, seminal pH,
and ion composition.
Computerized sperm motility computers (CASA) are
available for $30-50000
- Objective, but they probably do not tell us
that much more than by eye.
Morphology is usually examined with an eosin-nigrosin
(Society for Theriogenology) stain (background stain) to highlight the
The final slide should have
dark and light areas that allow you to view different colored backgrounds as needed when examining the slide.
The slide is made by painting a drop or line of stain on a warm
slide, then using a wooden stick to place a small amount of semen
into the stain; the semen and stain are mixed using another slide, then
slowly push the second slide through the stain and across the first
slide while pressing firmly down .
The goal is to get a dark background, as the stain is a back
round stain and is not intended to stain the cells.
In fact, some cells
will stain red, but this makes no difference in our evaluation.
the cells to be close, but not overlapping.
Examine the cells under 1000X (oil)
to fully assess the morphology.
Count 100 cells and, in a practice situation, you can
just differentiate normal from abnormal cells.
Using eosin-nigrosin all
the cells will appear flat, as if looking at your hand. In order to do a
full spermiogram, differentiate the abnormalities by type.
contrast microscope can be used, but is rare in a practice situation.
phase contrast mount is made by placing a small amount of semen in
formal-buffered saline to kill and preserve the cells.
Then place a drop
of the the formal-buffered saline sample onto a slide and place a cover slip
over the drop.
You need a phase contrast microscope to
examine the specimen. In using the phase-contrast microscope, match the
phase ring with the power that you are using.
The phase contrast acts
like a 'stain', however the cells will float by instead of lying flat.
The cells now look like your hand from the top and side views.
are classified as primary and secondary.
Primary abnormalities are
thought to arise in the testes, whereas secondary abnormalities arise in
the epididymis or ejaculate.
Secondary abnormalities may be just as
Primaries abnormalities decrease through transit and
secondary abnormalities increase through transit.
and minor may be a better clasification.
Major problems cause Early Embryonic Death or prevent fertilization.
example, an acrosome problem prevents zona entry, and a nuclear problem
leads to nonfertilization or EED.
problems such as tail abnormalities etc. stop sperm movement, so the
sperm cell cannot get to egg.
Some abnormalities are compensable and
some are non-compensable.
Problems with sperm cell examinaiton.
The light microscope cannot always detect abnormalities.
sperm cells indicate cellular damage, but the relationship to fertility
is only circumstantial.
Normal Sperm Cell above
Abnormalities of the sperm cells include:
Decapitated sperm (not pictured)- the basal plate is defective;
tails move (wrap around drops); 100% cells involved; occurs in
Loose heads (not pictured)- these may be increased on the first
ejaculate (rusty load) because the tail attachment is frail.
Knobbed or flat
acrosome (not pictured)- the acrosome folds over itself of the apex of the acrosome is
knobbed or flattened. When 20% of the cells have acrosome problems the
result may be infertility in the bull. This condition may be hereditary
in Charolais, Hereford, and Holsteins.
- Wrinkled acrosome - this may reflect a nuclear
problem which prevents zona attachment by the sperm cell. It is a rare
- Pyriform and tapered heads - the
nuclear material is poorly distributed. The defect may be subtle.
- Giant or small heads - This a a nuclear problem. If
the head is twice normal size the cell is a giant cell.
- Nuclear vacuoles - These distort the shape of the
- Diadem defect - With this you see invaginations in
the nucleus, mostly by the post nuclear cap. The pit lacks DNA. The
condition may be associated with stress in bulls and may come and go as
- Dense proximal droplets - This arises in the
epididymis and indicates maturation problem.
- Stump tail defect (not pictured) - this is an axonemal problem. It
looks like a cytoplasmic drop and has a poor prognosis. The incidence
increases with age. Midpiece defects - You see
lumps on the midpiece that can be confused with proximal drops. Coiled
mainpiece - The mainpiece is coiled within the plasma membrane.
- Dag defect - This is a sterilizing defect that occurs
in the epididymis so is it is actually a secondary abnormality, but it
is a major defect. The condition is inherited and the axoneme is
disrupted (fibrils and helix). You see split, shattered, or fractured
midpiece. The tail may coil and the motility is is low.
- Coiled midpiece (not pictured). This is an epididymal defect, but is
a major defect. The midpiece problems (not Dag).
- Abaxial midpiece - the implantation fossa is
defective. You may see abaxial or double midbpieces; There is generally
a low incidence and fertility is not affected; (normal in stallion).
mainpiece - The mainpiece is coiled within the plasma membrane.
- Teratospermia - the entire cell is degenerative.
tails - the bend in the tail may include a droplet which may be in the
(distal )droplet - some consider this a minor
defect, but in fact it may be a major defect. These cells do not freeze
well because the water in droplet crystalizes and ruptures the cell
Temporal progression of sperm abnormalities after 4
days of scrotal insulation.
|Days after scrotal insulation
||Sperm abnormalities in the ejaculate
||Days return to normal
||Distal midpiece refles, Proximal Droplets
||Miochondrial Sheaths, Detached heads
||coiled principal piece
bulls and rams, the sperm count is estimated by measuring the scrotal
circumference. The scrotal circumference measurement is used because
electroejaculation does not give a physiologic sample that can be
- To count the concentration of semen sample a
hemacytometer can be used.
- The hemacytometer is loaded with a 1:100
dilution of semen (see below how to make a 1:100 dilution,
- a hemacytometer coverslip is placed over the
- the chambers are filled
- the sample
allowed to settle,
- all the sperm heads in the middle big square (the
square with 25 smaller squares (....and another 16 squares within each
of these 25) within the triple lines) are counted.
- The number of sperm
heads counted in a single chamber is multiplied by 106 to give the
concentration of cells/cc.
- Both chambers of the hemacytometer should be
counted and the numbers should not differ by more that 10%.
- The coverslips are expensive so do not
break them or dispose of them!
confusing...look at the diagram.
hemacytometer grid has 9 large squares. The central square has triple lines
around it. Inside the triple lines are 25 smaller squares (also bounded by
triple lines). Within each of the 25 squares are 16 squares (only one of the
25 cells is illustrated having the 16 smaller squares. This is illustrated
below, however the triple lines are only shown on the outside edges of the
middle big square.
is an actual picture of the grid of the 25 squares in the middle big square.
They are all bounded by triple lines. Count every sperm head bounded by
is a picture of one of the 25 squares with the 16 squares inside.
For example, if each of the dots represented a
sperm head, the count on the grid below would be 13 x 106
The 1:100 dilution can be made using a Unopette
diluter or a hand dilution.
The Unopette is easier, but sometimes the
cells will clump, so an accurate count cannot be made.
If using the
Unopette, first puncture the bottle with the sharp point, then draw up
the semen via capillary action into the capillary tube.
The bottle is
then squeezed and the capillary tube inserted into the bottle and the
semen sucked into the bottle.
You can turn the capillary tube over and
use it as an applicator to load the hemacytometer chambers.
- We no longer use the Unopette at LSU
A hand dilution is made by:
diluting 1 part semen with 9 parts
formal-buffered saline to make a 1:10 dilution,
then taking 1 part of
the 1:10 dilution and adding 9 parts of formal-buffered saline to make a
This is a little more time consuming, but the cells tend
not to clump, so you get a more accurate sperm count.
A spectrophotometer can be used to count stallion
semen (and possibly dog semen if the correct software is used).
Basically, the machine measures the amount of light that passed through
a sample and calculates the concentration of cells that the density
Using our machine:
Total sperm numbers.
the concentration X the volume to give the total number of cells in the
- Take that number and multiply by the percent progressively
motile cells to get the total number of progressively motile cells.
that number and multiply by the percent normal cells to get the total
number of normal, motile cells.
- For example: 100
cc X 50million cells/cc = 5 billion cells 5
billion cells X 50% motility = 2.5 billion motile cells 2.5
billion motile cells X 50% normal = 1.25 billion normal, motile cells.
- Total sperm numbers are used in stallions and dogs, but not in bulls,
rams or bucks.