Sperm cryopreservation protocols vary among animal species
owing to their historical development and inherent particularities
that require specific extenders used for refrigeration and freezing
[4, 5]. Generally, sperm freezing extenders include non-permeating
cryoprotectants (milk and/or egg yolk), a penetrating/permeating
cryoprotectant (typically glycerol), a buffering compound (e.g.,
TRIS or HEPES), sugars (glucose, lactose, raffinose, sucrose, or
trehalose), nutrients and salts (including sodium citrate, citric acid),
as well as antibiotics.
Despite species-specific differences in extender composition,
the main processing steps for cryopreservation of sperm from
domestic species are very similar. After collection, using an artificial
vagina, semen is directly diluted with at least one volume of
pre-warmed (37
C) primary extender. This diluent contains salts,
sugars, proteins, and lipids. After primary dilution and cooling to
room temperature, sperm samples can be subjected to centrifugation to remove part of the seminal plasma and nonviable sperm, as
well as for concentrating the sample. Thereafter, the sperm concentration can be adjusted, and freezing extender is added. In addition
to the components present in the primary diluent, the freezing
extender contains a penetrating protectant like glycerol. Sperm
samples need to be slowly cooled (~0.1
C/min) from room temperature to 5
C, after which they can be packaged in plastic straws
(of 0.25 or 0.5 mL). The latter can be done (semi-)automatically,
after which specimens need to be sealed. Freezing is done by
placing the straws in horizontal position on metal racks. The racks
can be placed either in a controlled rate freezer or at a defined
distance in the vapor phase above liquid nitrogen in a closed polystyrene box. Sperm samples are typically frozen at cooling rates
ranging from 10 to 100
C/min [6–8].
In this chapter, we describe cryopreservation protocols for
bovine, equine, and porcine sperm. The main processing steps
that sperm cryopreservation approaches have in common are
described as well as species-specific differences in processing steps
and extender composition.
2 Materials
2.1 Materials
for Processing of Raw
Semen and Dilution
1. Standard laboratory equipment and supplies.
2. Phase contrast microscope (with heated stage), microscope
glass slides, and cover slips.
3. Hemocytometer for determining sperm concentration (see
Note 1).
4. Water bath set at 37
C, for pre-warming of diluents.
366
Harrie ¨ tte Oldenhof et al.
owing to their historical development and inherent particularities
that require specific extenders used for refrigeration and freezing
[4, 5]. Generally, sperm freezing extenders include non-permeating
cryoprotectants (milk and/or egg yolk), a penetrating/permeating
cryoprotectant (typically glycerol), a buffering compound (e.g.,
TRIS or HEPES), sugars (glucose, lactose, raffinose, sucrose, or
trehalose), nutrients and salts (including sodium citrate, citric acid),
as well as antibiotics.
Despite species-specific differences in extender composition,
the main processing steps for cryopreservation of sperm from
domestic species are very similar. After collection, using an artificial
vagina, semen is directly diluted with at least one volume of
pre-warmed (37
C) primary extender. This diluent contains salts,
sugars, proteins, and lipids. After primary dilution and cooling to
room temperature, sperm samples can be subjected to centrifugation to remove part of the seminal plasma and nonviable sperm, as
well as for concentrating the sample. Thereafter, the sperm concentration can be adjusted, and freezing extender is added. In addition
to the components present in the primary diluent, the freezing
extender contains a penetrating protectant like glycerol. Sperm
samples need to be slowly cooled (~0.1
C/min) from room temperature to 5
C, after which they can be packaged in plastic straws
(of 0.25 or 0.5 mL). The latter can be done (semi-)automatically,
after which specimens need to be sealed. Freezing is done by
placing the straws in horizontal position on metal racks. The racks
can be placed either in a controlled rate freezer or at a defined
distance in the vapor phase above liquid nitrogen in a closed polystyrene box. Sperm samples are typically frozen at cooling rates
ranging from 10 to 100
C/min [6–8].
In this chapter, we describe cryopreservation protocols for
bovine, equine, and porcine sperm. The main processing steps
that sperm cryopreservation approaches have in common are
described as well as species-specific differences in processing steps
and extender composition.
2 Materials
2.1 Materials
for Processing of Raw
Semen and Dilution
1. Standard laboratory equipment and supplies.
2. Phase contrast microscope (with heated stage), microscope
glass slides, and cover slips.
3. Hemocytometer for determining sperm concentration (see
Note 1).
4. Water bath set at 37
C, for pre-warming of diluents.
366
Harrie ¨ tte Oldenhof et al.