4 Notes
1. For research purposes, a shelf freeze-dryer may serve better
because it would allow more precise controlling of different
parameters such as holding temperature, heating during primary and secondary drying, and avoiding collapse temperature.
2. Pre-scored ampules with a barcode might be helpful for errorfree maintenance of sample inventory.
3. Motile sperm from different species can be separated by a
discontinuous percoll density gradient [45] or similar gradient
systems [26]. Alternatively, an aliquot of liquefied semen can be
transferred to the bottom of a 1.5 mL microcentrifuge tube
containing 1 mL of freeze-drying solution and then be incubated at 37
C for 10 min for dispersion. Next, the upper part
of the freeze-drying solution containing motile sperm can
carefully be aspirated and distributed into freeze-drying glass
ampules [34]. It might be helpful to determine the concentration of the separated motile sperm and adjust it to 0.5 Â 10
5
sperm/mL before its distribution into the glass ampules.
4. It is important to work quickly at this step in order to prevent
evaporative drying of cauda epididymis and sperm. Also, only
sterile solutions, tools, and supplies should be used at this step
and throughout the procedure.
5. Freeze-dried sperm samples can be shipped and stored at ambient temperature for up to few weeks without any significant
adverse effect.
6. Before ICSI, it is important to wash sperm in another drop of
the HEPES-buffered medium containing 12% PVP to minimize the introduction of the calcium chelator (i.e., EDTA or
EGTA) into the oocyte cytoplasm because it may interfere with
normal activation of the microinjected oocyte.
References
1. Walters EM, Benson JD, Woods EJ, Critser JK
(2009) The history of sperm cryopreservation.
In: Pacey AA, Tomlinson MJ (eds) Sperm
banking: theory and practice. Cambridge University Press, Cambridge, UK, pp 1–17
2. Polge C, Smith AU, Parkes AS (1949) Revival
of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666
3. Polge C (1952) Fertilizing capacity of bull
spermatozoa after freezing at 79 degrees
C. Nature 169:626–627
4. Stewart DL (1951) Storage of bull spermatozoa at low temperatures. Vet Rec 63:65–66
5. Bunge RG, Sherman JK (1953) Fertilizing
capacity of frozen human spermatozoa. Nature
172:767–768
6. Sherman JK (1990) Cryopreservation of
human semen. In: Keel BA, Webster BW (eds)
Handbook of the laboratory diagnosis and
treatment of infertility. CRC Press, Boston,
MA, pp 229–260
7. Curry MR (2000) Cryopreservation of semen
from domestic livestock. Rev Reprod 5:46–52
8. Mazur P, Leibo SP, Seidel GE (2008) Cryopreservation of the germplasm of animals used
in biological and medical research: importance,
728
Levent Keskintepe and Ali Eroglu
1. For research purposes, a shelf freeze-dryer may serve better
because it would allow more precise controlling of different
parameters such as holding temperature, heating during primary and secondary drying, and avoiding collapse temperature.
2. Pre-scored ampules with a barcode might be helpful for errorfree maintenance of sample inventory.
3. Motile sperm from different species can be separated by a
discontinuous percoll density gradient [45] or similar gradient
systems [26]. Alternatively, an aliquot of liquefied semen can be
transferred to the bottom of a 1.5 mL microcentrifuge tube
containing 1 mL of freeze-drying solution and then be incubated at 37
C for 10 min for dispersion. Next, the upper part
of the freeze-drying solution containing motile sperm can
carefully be aspirated and distributed into freeze-drying glass
ampules [34]. It might be helpful to determine the concentration of the separated motile sperm and adjust it to 0.5 Â 10
5
sperm/mL before its distribution into the glass ampules.
4. It is important to work quickly at this step in order to prevent
evaporative drying of cauda epididymis and sperm. Also, only
sterile solutions, tools, and supplies should be used at this step
and throughout the procedure.
5. Freeze-dried sperm samples can be shipped and stored at ambient temperature for up to few weeks without any significant
adverse effect.
6. Before ICSI, it is important to wash sperm in another drop of
the HEPES-buffered medium containing 12% PVP to minimize the introduction of the calcium chelator (i.e., EDTA or
EGTA) into the oocyte cytoplasm because it may interfere with
normal activation of the microinjected oocyte.
References
1. Walters EM, Benson JD, Woods EJ, Critser JK
(2009) The history of sperm cryopreservation.
In: Pacey AA, Tomlinson MJ (eds) Sperm
banking: theory and practice. Cambridge University Press, Cambridge, UK, pp 1–17
2. Polge C, Smith AU, Parkes AS (1949) Revival
of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666
3. Polge C (1952) Fertilizing capacity of bull
spermatozoa after freezing at 79 degrees
C. Nature 169:626–627
4. Stewart DL (1951) Storage of bull spermatozoa at low temperatures. Vet Rec 63:65–66
5. Bunge RG, Sherman JK (1953) Fertilizing
capacity of frozen human spermatozoa. Nature
172:767–768
6. Sherman JK (1990) Cryopreservation of
human semen. In: Keel BA, Webster BW (eds)
Handbook of the laboratory diagnosis and
treatment of infertility. CRC Press, Boston,
MA, pp 229–260
7. Curry MR (2000) Cryopreservation of semen
from domestic livestock. Rev Reprod 5:46–52
8. Mazur P, Leibo SP, Seidel GE (2008) Cryopreservation of the germplasm of animals used
in biological and medical research: importance,
728
Levent Keskintepe and Ali Eroglu