5. Glassware, containers, and/or tubes for diluting and handling
of sperm. Ordinary polypropylene conical tubes for 50 mL can
be used.
6. Diluents for diluting and storage of semen. Details on preparing the following diluents are given in Subheading 3: INRA-82
for primary dilution and INRA-82 freezing extender for equine
semen, TRIS-egg yolk (TEY-1 and TEY-2) extenders for
bovine semen, and Beltsville Thawing Solution (BTS) and
lactose-egg yolk (LEY cooling and freezing) extenders for
porcine semen (see Note 2).
7. For use with egg yolk: Equex paste (from Minitube).
8. If preparing clarified egg yolk, a temperature-controlled centrifuge is needed that can be used at 10,000 Â g and 4
C, with
accompanying tubes.
9. If performing centrifugation processing of sperm, a centrifuge
is needed that allows for centrifugation at 600 Â g for stallion
and 2400 Â g for boar sperm. For boar sperm, a temperaturecontrolled device is preferred, which allows for centrifugation
at 17
C.
10. If performing high-speed cushioned centrifugation, cushionfluid (e.g., OptiPrep/iodixanol) is needed.
11. Device for removing the supernatant after centrifugation (vacuum pump or syringe).
2.2 Materials
and Equipment
for Cooling,
Cryopreservation,
and Thawing of Sperm
Samples
1. Cryoprotective gloves, goggles, and tweezers for save handling
of liquid nitrogen and straws.
2. Thermocouple (e.g., from Fluke) for measuring cooling rates.
3. Straws made of polypropylene or polyvinyl chloride (e.g., from
IMV Technologies or Minitube). Typically, straws that fit
0.25 mL are used for bull sperm and straws for 0.5 mL for
stallion and boar sperm.
4. Device for labeling of straws or permanent marker.
5. Device(s) for (semi-)automated filling and sealing of straws.
Alternatively, for filling straws manually, a 1 mL syringe can be
used with a small piece of tubing attached on the opening to fit
in a straw. Sealing can also be done using heat, sealing powder,
or glass balls.
6. Cooling cabinet or refrigerator/cold room for handling of
materials at 5
C.
7. Controlled rate freezer (e.g., model “Icecube” from Minitube
or the “MiniDigitcool” from IMV Technologies), with racks
for straws. Alternatively, a Styrofoam box with liquid nitrogen
can be used with racks placed at a fixed position above the
liquid nitrogen level (see Fig. 1). Also, a “floating freeze rack”
can be used that has Styrofoam floaters for placing directly on
Cryopreservation of Sperm from Domestic Livestock
367
of sperm. Ordinary polypropylene conical tubes for 50 mL can
be used.
6. Diluents for diluting and storage of semen. Details on preparing the following diluents are given in Subheading 3: INRA-82
for primary dilution and INRA-82 freezing extender for equine
semen, TRIS-egg yolk (TEY-1 and TEY-2) extenders for
bovine semen, and Beltsville Thawing Solution (BTS) and
lactose-egg yolk (LEY cooling and freezing) extenders for
porcine semen (see Note 2).
7. For use with egg yolk: Equex paste (from Minitube).
8. If preparing clarified egg yolk, a temperature-controlled centrifuge is needed that can be used at 10,000 Â g and 4
C, with
accompanying tubes.
9. If performing centrifugation processing of sperm, a centrifuge
is needed that allows for centrifugation at 600 Â g for stallion
and 2400 Â g for boar sperm. For boar sperm, a temperaturecontrolled device is preferred, which allows for centrifugation
at 17
C.
10. If performing high-speed cushioned centrifugation, cushionfluid (e.g., OptiPrep/iodixanol) is needed.
11. Device for removing the supernatant after centrifugation (vacuum pump or syringe).
2.2 Materials
and Equipment
for Cooling,
Cryopreservation,
and Thawing of Sperm
Samples
1. Cryoprotective gloves, goggles, and tweezers for save handling
of liquid nitrogen and straws.
2. Thermocouple (e.g., from Fluke) for measuring cooling rates.
3. Straws made of polypropylene or polyvinyl chloride (e.g., from
IMV Technologies or Minitube). Typically, straws that fit
0.25 mL are used for bull sperm and straws for 0.5 mL for
stallion and boar sperm.
4. Device for labeling of straws or permanent marker.
5. Device(s) for (semi-)automated filling and sealing of straws.
Alternatively, for filling straws manually, a 1 mL syringe can be
used with a small piece of tubing attached on the opening to fit
in a straw. Sealing can also be done using heat, sealing powder,
or glass balls.
6. Cooling cabinet or refrigerator/cold room for handling of
materials at 5
C.
7. Controlled rate freezer (e.g., model “Icecube” from Minitube
or the “MiniDigitcool” from IMV Technologies), with racks
for straws. Alternatively, a Styrofoam box with liquid nitrogen
can be used with racks placed at a fixed position above the
liquid nitrogen level (see Fig. 1). Also, a “floating freeze rack”
can be used that has Styrofoam floaters for placing directly on
Cryopreservation of Sperm from Domestic Livestock
367