Rat Sperm Cryopreservation and Thawing
Protocols using CARD FERTIUP® Rat Sperm
Cryopreservation Agent
１． Sperm cryopreservation protocol

Rat Sperm
Cryopreservation
Agent (CPA)

A. Place 3mL of sperm cryopreservation agent (CPA) at room temperature
into a 35mm culture dish. Transfer two cauda epididymides taken fromone male rat into the agent.
※When using one cauda epididymis, put 1.5mL CPA at room
temperature into a 35mm culture dish.

Cauda epididymides

B. Cut the cauda epididymides in two using scissors and a pair of forceps
as shown in ①. Next, make further cuts in each piece of caudal tissue asshown in ② and ③.
C. Place the culture dish containing the dissected cauda epididymides on atin tray set on top of crushed ice. Leave the dish to cool and incubate for10 minutes.

CulturedishD. Connect the end of a straw to a straw connector attached to a 1mL
syringe.

Crushed ice
Cool and incubate
for 10 minutes

E. Load the sperm suspension into the straw following the method below:a）Aspirate approx. 1cm of mHTF.
b）Aspirate approx. 1cm of air, then approx. 150μL of the spermsuspension.
c）Pull back the 1mL syringe plunger until the mHTF reaches the cottonplug.
d）Seal the ends of the straw with an impulse sealer.
F. Place straws containing the sperm suspension on the tin tray set on topof crushed ice. Leave the suspension to incubate for 30 minutes.
G. Line up straws containing sperm suspension on top of a plate which
has been pre-cooled on a bed of ice. Place the plate on top of a Styrofoam
float which is floating on liquid nitrogen. Leave the plate there for 10minutes, then immerse the straws into the liquid nitrogen and transfer
them to a triangular cassette for storage.

Dissect the cauda
epididymides inside the CPA

Tin trayStyrofoam container

Connector

1ml syringe

StrawMarker

Cotton plug

1cm mHTFa）b）
c, d）

1cm Air
150μL CPASeal

Tin trayH. Store the triangular cassette containing the straws in a liquid nitrogen
tank.

Straw

Crushed ice

Cool for 30 minutes

Appendix: Sperm suspension straw cooling diagram
Styrofoam float

FPlateGPlate

4 cm
Liquid nitrogen

Liquidnitrogentank

Tin tray
Overhead view↔

Styrofoam float

Bird's eye view
Storage in a liquid nitrogen tank

２．Frozen rat sperm thawing protocol

Remove a frozen straw from theliquid nitrogen tankStyrofoam float

I. Remove a frozen straw from the liquid nitrogen storage tank.
Place the straw in a Styrofoam float (in a water bathmaintained at 37℃) and leave it for 15 minutes. During thistime, dispense 1.0mL of mHTF into a 1.5mL centrifuge tube
and equilibrate it at 37℃.

50mL
centrifuge tube

J. Remove the straw from the float. Use fine tissues to wipe any
water from the straw. Use a paper towel to wipe any waterfrom the straw. Next, cut the end of the straw closest to thesperm suspension and transfer the suspension into thebottom of the 1.0mL mHTF within the centrifuge tube. Lay
the tube on its side and place it in an incubator (37℃) for 30minutes.

37℃ water bath,50mL centrifuge tube

Transfer the sperm suspension into
the bottom of the 1.0mL mHTF
within the 1.5mL centrifuge tube

Lay the tube on its side and place it inan incubator (37℃) for 30 minutes

Centrifuge at 300g
for 60 seconds

K. After 30 minutes, slowly invert the centrifuge tube 2-3 times
to mix the contents, then centrifuge it at 300g for 60 seconds.

L. Take a pipette with a wide opening and gently insert it into
the bottom of the centrifuge tube. Aspirate 50μL mHTF
along with the sperm pellets at the bottom of the tube, thentransfer them to a 200μL drop of CARD Rat In Vitro
Fertilization Medium in a culture dish.

Place the straw in thefloat for 15 minutes

Liquidnitrogentank

Aspirate 50μL mHTF along with sperm pellets from thebottom of the tube using a pipette with a wide opening

Paraffin liquid

Suspend the sperm pellets in 200μL
IVF Medium

Sperm pellets

Leave for 30 minutes at 37℃

M. After leaving the dish in an incubator for 30 minutes,
remove dead sperm and 125μL of medium from the mHTF
drop using a pipette with a fine opening. Two hours later,
introduce obtained oocytes into the drop for insemination.

Remove dead spermand 125μL medium
Paraffin liquid

Dead sperm

Live sperm

Two hours later, introduce obtained oocytes
into the drop for insemination

Thesis title： Establishment of sperm cryopreservation and in vitro fertilisation protocols for rats
Authors： Naomi Nakagata, Nobuyuki Mikoda, Satohiro Nakao, Ena Nakatsukasa& Toru Takeo
Journal ： Scientific Reports
doi ： https://doi.org/10.1038/s41598-019-57090-7
URL ： https://www.nature.com/articles/s41598-019-57090-7
Manufacturer ; KYUDO Co. Ltd.
TEL : +81-942-82-6519 FX : +81-942-85-3175
E-mail: fertiup@kyudo.co.jp URL : http://www.kyudo.co.jp/※FERTIUP is our registered trademark.