cryopreservation of germplasm, sperm and oocytes

1. CRYOPRESERVATION OF
GERMPLASM, SPERM
AND OOCYTES
SUBMITTED BY
ABHISHEK
ROLL NO. 47
M.SC FINAL
SUBMITTED TO;
DR. ANITA YADAV
Department of biotechnology,
Kuk

2. CRYOPRESERVATION
Cryopreservation Drives From The Greek Word cryo meaning
‘cold’.
Thus it refers to the preservation of biological tissue in sub zero
temperature, typically -196 C
At these temperatures, all biological activities of cells and tissues is
effectively stopped or ceased.
Cryogenic storage at very low temperature is presumed to provide
an indefinite, if not near infinite, longevity to cells.
Pre –implantation embryo, oocytes, spermatozoa, ovarian tissue can
be cryopreserved.

3. BENEFITS OF
CRYOPRESERVATION
Effective mean to conserve the germ plasms of endangered
species.
Fertility preservation.
Methods to reduce multiple pregnancies.
Large range of stocks available
Easy disease- free exchange of stocks, nationally and
internationally
Stocks remain viable indefinitely.
Safety from disease, genetic contamination and breeding failure

4. WHAT YOU NEED FOR
CRYOPRESERVATION
Liquid nitrogen (liquid phase of vapor phase)
• Characteristics of liquid nitrogen:
- Chemically inert, Relatively low cost
-Non-Toxic, Non-flammable, Readily available
Cryoprotectant: organic or inorganic additive which will protect the cell
from freezing injuries during cryopreservation.
•Characteristicss of cryoprotectants:
-should easily penetrate into cell
-Non- electrolyte, Easy misible with water
-E.g: Glycerol, DMSO, PVP, PEG etc.
Cryofreezer

5. CRYOPRESERVATION OF
SPERM
•It is commonly known as sperm banking.
•Sperm banking is a procedure to preserve sperm cells for future use.
•The first successful cryopreservation of spermatozoa was initiated over 50 year ago.
•For human sperm, the longest reported successful storage is 22 years.
•It can be used for sperm donation where the recipient wants the treatment in a
different time or place or for men undergoing a vasectomy to still have the option to
have children.
•It is also useful for men suffering from azoosprmia (Lack of motile sperm) and
Gonadial cancer.
•These frozen sperm can be used in association with one of the Assisted
Reproductive Techniques(ART) to induce pregnancy.

6. PROCESS OF SPERM
CRYOPRESERVATION
Obtaining semen sample:
- Semen sample are collected in sterile container.
-The semen so collected is suspended in 10-20% glycerol in egg yolk buffer.
Semen analysis:
-Semen samples are analyzed for volume, viscosity and PH level, and microscopically evaluated to
determine motility, sperm count and morphology.
Freezing:
-Freezing of semen is done either in straw or ampoules.
-Freezing can be achieved in three ways:
1. Slow freezing
2. Rapid freezing

7. 1.Slow freezing: liquid nitrogen is poured into the tank and cooling rate is obtained from 20-
80 degree C at rate of 1.5 degree C/min and then at 6 degree C/min at completion of the
freezing the straws are removed and stored into nitrogen at -196 degree C. Thus take about
40 min.
2.Rapid freezing: Required direct contact between straws and the nitrogen vapors for 8-10
min. and immersion in liquid nitrogen at -196 degree C.
. The sample is initially mixed drop wise manner with equal volume cold cry protectant the
mixture is loaded into the straws and left to incubate at 4 degree C for 10 min.
.The straws are then placed at distance of 15-20 cm above the level of liquid nitrogen (-80
degree C )for 15 min. after the stage, the straws are immersed in liquid nitrogen.
Thawing:
. It is done by putting ampoule containing the sample in a warm water bath (35 to 40 degree
C)
. Frozen tips of the sample in tubes or ampoules are plunged into the warm water with a
vigorous swirling action just to the point of ice disappearance.
. Just a point of thawing quickly transfer the tubes to a water bath maintained at room
temperature and contained the swirling action for 15 sec to cool the warm walls of the tube.

8. CRYOPRESERVATION OF
OOCYTES
• Cryopreservation of oocytes is also
known as Egg freezing is a method
used to save women’s ability to get
pregnant in the future.
• Eggs harvested from your ovaries are
frozen unfertilized and stored for later
use.
• A frozen eggs can be thawed,
combined with sperm in a lab and
implanted in your uterus (in vitro
fertilization)

9. WHY IT’S DONE
• Egg freezing might be an option if you’re not ready to become
pregnant now but want to try to make sure you can get pregnant
later.
• unlike with fertilized egg freezing (embryo cryopreservation), eggs
freezing doesn’t require sperm because the eggs aren’t fertilized
before they are frozen.

10. YOU MIGHT CONSIDER EGG
FREEZING IF;
• you have a condition or circumstance
that can affect your fertility.
like sickle cell anemia, autoimmune
disease
• you are undergoing in vitro
fertilization.
when undergoing in vitro fertilization,
some people prefer egg freezing to
embryo freezing.
• you need treatment for cancer or
another illness that can affect your
ability to get pregnant.
such as radiation or chemotherapy –
can harm your fertility
• you wish to preserve younger
eggs now for future use.
freezing eggs at younger age
might help you get pregnant
when you’re ready.

11. RISKS
• Condition related to the use of fertility drugs:
Rarely use of injectable fertility drugs can cause your ovaries to become swollen and
painful soon after ovulation or egg retrieval.
• Egg retrieval procedure complication:
Rarely, use of an aspirating needle to retrieval eggs causes bleeding, infection or
damage to the bowel bladder or a blood vessel
• Emotional risks:
Egg freezing can provide hope for a future pregnancy, but there’s no guarantee of
success.

12. PROCEDURE
OVARIAN STIMULATION
Synthetic hormones to
stimulate ovaries to
produce multiple eggs.
EGG RETRIEVAL
Egg retrieval is done under
sedation. A common approach
is transvaginal ultrasound
aspiration.
FREEZING
Shortly after your unfertilized eg
are harvested, they’re cooled to
sub zero temp. to preserve them
for future use.

13. RESULTS
• Use frozen eggs, they’ll be
thawed, fertilized with sperm in
lab and implanted in your or a
gestational carrier’s uterus.
• Using a fertilization technique
called intracytoplasmic sperm
injection (ICSI)

14. REFERENCE
• Molecular biotechnology; principle and application of recombinant DNA, Bernard
R.Glick, jack j. Pasternak, and Chery L. Patten
• Pegg DE (January 1, 2007). "Principles of cryopreservation". Methods in Molecular
Biology.
• Mazur P (May 1970). "Cryobiology: the freezing of biological systems". Science.
• Deller RC, Vatish M, Mitchell DA, Gibson MI (February 3, 2014). "Synthetic
polymers enable non-vitreous cellular cryopreservation by reducing ice crystal
growth during thawing". Nature Communication