LN2

2.  To understand about cryogenic container and its handling.
 To know the precautions to be taken during transportation of
cryogenic containers.
 To know the safety measures to be followed while handling
cryogenic agents.
 To know how to measure liquid nitrogen in a cryogenic
container.
 To know the different modes of transport of frozen semen to
the field level.

3. 1. Liquid nitrogen container with frozen semen.
2. Dip stick
3. A.I gun (medium/mini)
4. Plastic sheaths
5. Semen straw holding forceps
6. Thawing device and Thermometer
7. Clean absorbent cotton
8. Clean and sharp scissors
9. A clean and hygienic tray for carrying loaded
A.I gun
10. Soap and clean towel

4. Cryogenics : Cryogenics is a branch of physics
that deals with production and effects of very
low temperature.
Cryopreservation/Cryoconservation: Is a
process where cells, whole tissue, or any
other substance are preserved by cooling to
sub-zero temperatures.
e.g. spermatozoa, oocytes, embryo, tissues
etc

5. Semen cryopreservation: Is a procedure to
preserve sperm cells at sub-zero
temperatures which can be used
successfully and indefinitely .

6. Cryogen: Is a substance with a very low boiling
point, used to lower the temperature of the
tissues to be frozen.
Different types of cryogens are:
1. Liquid nitrogen
2. Carbon dioxide
3. Liquid helium
4. Nitrous oxide
5. Liquid oxygen
6. Liquid argon

7.  It is nitrogen in liquid state
at an extremely low
temperature.
 Colourless,odourless clear
liquid.
 Boiling point : -196°C

8. Features of LN2 containers
 Delicate by construction
 Double layered vessel
 Outer chamber- aluminium
 Inner chamber- is suspended
by the neck tube and the wall is
coated with high quality
insulating material.
 Neck tube: non-metallic,
delicate and bad conductor of
heat.

10. Frostline

11.  Vacuum- is created in
between the inner and the
outer chambers
 Container sizes:
3, 20, 35, 60 ltr

12. 1. Should be kept in a well ventilated and cold place.
2. Avoid direct contact with hard floor, use rubber/jute
mats.
3. Avoid moisture on floor.
4. Avoid injuries, drilling and scrapping.
5. Vacuum knob should not be disturbed
unnecessarily

13. 6. Never roll/swing the LN2 container.
7. Use trolley during transportation.
8. Do not put LN2 containers one over the
other.
9. Never change lid from other containers.
10. Always keep the lid loaded over the
container.

14. 11. Do not interchange canisters from other
LN2 cans.
12. Fill the LN2 slowly into the cans. Semen
containers should be periodically topped with
liquid nitrogen (min 15cm).
13. Make regular checks of the LN2 containers.

15. Dip sticks are used for
measuring the level of liquid
nitrogen in the LN2container.

16. Dip the dip stick in the cryocan to reach the
bottom
should remain in the tank for about 10 sec
Remove and wave in air
Frost indicates the level of LN2
.

17. 1. Unnecessary and frequent measuring of the
LN2 should be avoided.
2. Dip stick should not be kept loose but hanged
through the wall.

18. A.I gun should match
the straw of frozen
semen.
French mini , German
Contains cylinder of
the gun, piston wire
and lock.

19. 1. Avoid bending of A.I gun and piston wire.
2. A.I gun should always be kept clean and
hygienic.
3. A.I gun must be kept in plastic
container/carrier.

20. Plastic sheaths should match the A.I gun
and the semen straw.
The sheaths should always remain in the
polythene pocket.
The sleeve on each sheath should be
removed just before introducing it into
the vulva/vagina
**Double sheath breeding provides both hygienic
and thermal protection

21. Straw holding forceps is used
to take out the desired
number of straws from LN2.

23. Proper semen handling is essential to maintain
optimum reproductive performance of AI breeding
program
AI with frozen semen is done by using PVC straws.
 A. Glass ampoules were used until about 1970
 B. Plastic straws:
 a. Size: 0.5 ml with 113 mm long and 2.8 mm in
diameter (In Europe 0.25 or 0.3 ml straws are used)
b. Straw preparation: One end of straw contains 3-
parts plug: Cotton- Polyvinyl alcohol powder-
Cotton

24. Keep all the necessary
equipment ready
Raise the canister of
desired breed to the
neck of the container.
Take out the required
no. of straws as early
as possible using the
forceps
Frostline

25. Thaw the semen straw in
thawing unit
Temp: 35 to 37°C
Duration: 30 to 60 sec
Remove the straw and wipe
out water content using
absorbent material.
Hold the straw vertically with
the cotton plug downwards
Slowly tap at the air
space/region

26.  Cut the straw at right
angle to remove the
AI centre seal
 Withdraw the piston
wire of the AI gun and
place the straw
 Fix the plastic sheath
over the AI gun
 Set the lock securely
over the plastic sheath

28. Proper seal between straw and
sheath is essentially important.

29. Proper site of
deposition of frozen
thawed semen