Semen diluting (semen extension)
Semen collected from farm animals and used for AI is either extended
and used “fresh” within several days of collection or extended and
frozen for use at a later date. The primary purpose of extending
semen is to maximize the number of females that can be inseminated
with a single collection. Furthermore, extending semen also provides
nutrients for the sperm and creates a physiologically safe
environment for sperm to survive so fertility is maximized.
For horses and pigs, the majority of inseminations are conducted
using fresh extended semen. In contrast, in the bull and ram it is not
practical to use fresh semen so the semen is extended, packaged into
small straws, frozen, and stored in liquid nitrogen for future use.
Depending on the species and volume of semen collected during an
ejaculate, the number of inseminations attained after extension
varies from 1 - 4 in the stallion, 5 - 15 in the boar, and 100 - 500 in
the bull.
Functions of extenders.
1. Provides nutrients as a source of energy.
2. Protects against the harmful effects of cooling and freezing,
which take place in liquid nitrogen (-196°C).
3. Provides buffers to prevent harmful shifts in pH.
4. Maintenance of proper osmotic pressure.
5. Increase semen volume so it can be used for multiple
inseminations.
6. Extenders must provide an isotonic environment; hypertonic
solutions dehydrate sperm while hypotonic solutions cause sperm
to swell and rupture.
Components of extenders.
 Buffers - Function to control pH 6.7 to 7.0. Sodium citrate, egg
yolk, tris buffers are commonly used.
 Lipids - Provides protection of sperm membranes from
temperature changes. Skim milk and egg yolks are good
sources of lipids.
 Nutrients - Provide energy for sperm. Fructose and glucose are
typically used.
 Antibiotics - Prevent bacterial growth.
 Glycerol - Is a cryoprotective agent for freezing semen. It
protects against the lethal effects of freezing to prevent
crystallization of water within the sperm cells, which
eventually allows sperm cells to be frozen rapidly. Formation
of ice crystals results in puncture of cell membranes resulting
in the decrease in membrane integrity.
Semen freezing
Sperm can be frozen in all the species. The use of frozen sperm is
limited to conservatory purposes and preservation of genetic
resources. At the beginning, mainly fresh or cooled (5°C) semen was
used in AI. After Polge and co-workers discovered the protective
effect of glycerol during freezing, the technique evolved to the use of
the frozen semen. This technique is used in cattle and sheep. Before
freezing, a mixture of semen and extenders are kept for almost six
hours at 5°C. This enables sperm cells to spread uniformly into the
extenders. Then semen is packed into small “plastic” containers
(straws or pellets). Straws contain 0,25 to 0,5 ml of semen extended
for bulls and 0,5 ml for ram. Straws are first cooled to 5°C. Then, they
are brought down at a controlled rate to -110°C before storage in
liquid nitrogen at –196°C.
Frozen semen is transported and stored in liquid nitrogen tanks
designed specifically for this purpose. As long as semen remains
submerged in liquid nitrogen, the condition of the sperm and its
fertility remains unchanged. Problems can arise when straws are
exposed to elevated temperatures before they are actually needed for
A.I. This may occur if a tank is allowed to run out of liquid nitrogen.
the damaging effects of exposing straws to elevated temperatures are
cumulative and care should be taken to avoid any unnecessary
removal of straws from liquid nitrogen.
Before semen is used, the samples (straws) must be thawed rapidly
by placing them in a warm water bath (30-37°C) for e.g. two minutes.
Straws thawed in warm water should be dried thoroughly to avoid
the possibility of water contacting the semen when the straw is
opened, then they must be used rapidly, because spermatozoa cannot
survive for a long time after thawing. Each straws are marked with
the indication of the animal indentification number and the date of
processing.