Cryopreservation of rooster sperm

CRYOPRESERVATION OF
ROOSTER SPERMATOZOA
Presented By:
K.PRANAY KUMAR
RVM/15-39
M.V.Sc Scholar
Veterinary Physiology
K.PRANAY KUMAR 1ROOSTER SPERM CRYOPRESERVSTION

Preamble
Definition
Prospects of cryopreservation
Principles of cryopreservation and cryoinjury
Rooster sperm cryopreservation
Future needs
2/18/2017 2ROOSTER SPERM CRYOPRESERVSTION

Introduction
Cryopreservation is a process that maintains
cellular life at sub-zero temperatures for an
extended period of time by arresting all the
metabolic activities.

WHY....
• Ex-situ management of genetic diversity in
birds.
• Breed reconstruction, creation of synthetic
breeds.
• Cryobanking of spermatozoa.
 Conventional methods of preservation are prone
to possible catastrophic losses like epidemic
diseases, climate & natural disasters.

• Liquid Nitrogen (LN2) is the most widely used
cryopreservation material.
Why LN2 :
 Chemically inert
 Non inflammable
 Non toxic
 Easily available
 Relatively low cost

Cryopreservation protocol has a number of
potentially damaging stresses:
• Change in temperature.
• Osmotic and toxic stress.
• Formation and dissolution of ice.

Cryobiology principles:
 Cells must be frozen in such a way that little or none
of their water freezes intracellularly.
 Cells must be thawed in such a way that
recrystallization is avoided.
 CPA concentration must not exceed osmotic
tolerance limits of cells.

• 0.5 µm width and 100 µm length and approximate
volume of 100 µm³ (Etches, 1996).
• Different regions: head, mid piece, principle piece &
end piece.
• The rooster spermatozoon’s nucleus is thin, long and
cylindrical, giving the head of the cell a bullet-like
shape (Etches, 1996).
• Homogametic haploid cells with highly condensed
chromatin.

A comparison...
Acrosomal cap
nucleus
Mid piece
principal piece
End piece
annulus
Neck

Scanning electron micrograph of
chicken sperm cells. The
constriction at the anterior end of
the sperm cell (A) marks posterior
boundary of the acrosome. The
nucleus extends posteriorly from
(A) to the neck region (N), which
marks the anterior end of the
midpiece. The midpiece, site of
the anterior portion of the
axoneme and the highly modified
mitochondria, extends back to the
raised annulus (U). The tail of the
sperm extends from the annulus
to the cell’s termination. At the
nucleus, chicken sperm are about
0.5 μm in diameter, with the
overall length of the cell
approximately 90 μm. Bar = 2 μm.
(Bahr, J.M., Bakst, M.R., 1987.)

STEPS IN CRYOPRESERVATION
Collection of semen
Cooling of spermatozoa to 5 ̊C
Addition of diluent and CPA
Freezing of sperm
Storing in LN2

• Abdominal massage method (Burrows and
Quinn, 1937).

• Freshly collected sperm are immediately diluted with diluent Which
keeps sperm alive for long periods of time. (Burrows and Quinn,
1937; Lake, et al., 1978; Sasaki, et al., 2010).
• The sperms are cooled from body temperature (41.5°C) to 5°C over
ice (Mazur, 1984).
• Rooster sperms do not undergo cold shock (Steponkus, et al., 1983;
Parks & Lynch, 1992).

Cold shock
• Cold shock is defined as the insult a sperm
undergoes when cooled too quickly prior to
freezing due to the phase changes in lipids
and altered functional state of membranes
(Pickett & Komarek, 1967).
• Integral membrane proteins are clustered by
lipid phase separation and this may alter the
function.

• Cryoprotective agent (CPA) is used to protect
biological tissues from freezing damage.
• They act by lowering the freezing temperature
and increase the viscosity there by preventing
cryoinjury to cells.

Constituents of cryoprotective medium
• Permeating cryoprotective agent: Glycerol, ethylene glycol,
dimethyl sulfoxide, propylene glycol and methylformamide.
• Nonpermeating cryoprotective agents: Long chain polymers or
sugars (methylcellulose, sucrose, raffinose, trehalose).
• Plasma membrane protecting agents: Egg yolk, milk, soy
lecithin and albumin.
• Chelating agents: EDTA and citrate chelate.
• pH Buffers: Glycine, Sodium citrate, TRIS.
• Free radical scavenger (anti-lipid peroxidation agents){Improve
post-thaw motility and acrosomal integrity} : Butylated
hydroxytoluene, glutathione and dithiothreitol
• Antibiotics: Gentamicin.

CRYOPROTECTANTS
CRYOPROTECTANTS
(CPA)
PERMEATING
(P-CPA)
EXERT THEIR
PROTECTIVE
COLLIGATIVE ACTION
NONPERMEATING
(NP-CPA)
EFFECT THROUGH
OSMOTIC
DEHYDRATION

• Penetrating CPA are small, non-ionic molecules that
lipophilic and highly miscible with water, giving them
the ability to easily penetrate the cell membrane.
Ex: Glycerol, DMSO, PG, EG, MF
• Nonpenetrating CPA are high molecular weight
substances which increase the viscosity and lower the
freezing point of extra cellular fluid and promote
cellular dehydration.
Ex: Sugars (sucrose, trehalose)
– They augment the effectiveness of permeating CPA
or permit use of lower concentration of CPA.

CRYOPROTECTANT REFERENCE
Glycerol (Gly) Lake et al., 1981; Wishart, 1995;
Watanabe and Terada, 1980; Seigneurin
and Blesbois (1995);Terada et al., 1989;
Ansah and Buckland, 1983; Bacon et al.,
1986; Buss, 1993
Ethyleneglycol (EG) Kurbatov et al., 1980
Dimethylacetamide (DMA) Kurbatov et al., 1984; Tselutin et al., 1999
Hubner and Schramm, 1988
Dimethylsulphoxide (DMSO) Bakst and Sexton, 1979
Dimethyl formamide (DMF) Schramm (1991)
N-methyl acetamide (N-MA) Hanzawa et al. (2006); Sasaki et al (2010)

Efficacy of different cryoprotectants towards chicken spermatozoa
REFERENCE PARAMETER EFFICACY RANKING
Maeda et al. 1984 morphology after freezing Gly>EG>DMSO
Sakhatsky et al., 1988 integrity after freezing Gly>EG=DMSO
Lake and Ravie, 1984 fertility after freezing DMA>EG =DMSO
Kurbatov et al., 1986 fertility after freezing EG>DMSO
Hubner and Schramm,
1988
fertility after freezing DMA>EG
Tajima et al., 1990 fertility after freezing Gly>DMSO>DMA
Haije, 1990 fertility after freezing Gly>DMSO

• Cooling between -6°C to -15°C is the most critical part of
freezing, as this is when water in the diluent is transitioning to
the crystalline state (Mazur & Cole, 1989; Hammerstedt,
1995).
• Unfrozen channels remain between the ice crystals which
contain high salt concentrations.
• Spermatozoa that are able to tolerate the high salt
environment survive in these unfrozen channels.

• Cryoprotectants lower the freezing point of the
diluent, increase the volume of the unfrozen fraction,
decrease high salt concentrations in this fraction and
alter the formation of ice crystals (Amann & Pickett,
1987).
• Once the spermatozoa reach -50°C, the unfrozen
fraction and the sperm vitrify and become inert.
Therefore, sperm can be plunged into liquid nitrogen
after reaching -50°C, and can be held in storage at
-196°C indefinitely (Graham, 1996).

Freezing: slow freezing
• Cells are frozen with gradual decrease in
temperature 1-10 ̊ C/min to -35 ̊C.
• Permits flow of water from the cells to the
outside, thereby promoting extracellular
crystallization avoiding lethal intracellular ice
formation.

Freezing: rapid freezing
• Currently fast cooling rates are preferred
20-100 ̊C/min (Varadi. et al.,2013; Long. et
al.,2014).
• Optimal rapid freezing avoids excessive
dehydration and shrinkage of cells.

Step-wise freezing
• A programmable freezing machine was used
(Blanco et al.,2012; Santiago-Moreno, 2011;
Blesbois,2007).
• Frozen at 7 ̊C/min to -35 ̊C, then -60 ̊C/min to
-140 ̊C in a programmable freezer.

VITRIFICATION
• The term “vitrification” refers to any process
that results in “glass formation”
(transformation of a liquid into a solid without
crystallization).
• Water solidifies into an amorphous ‘glassy’
state.
• If vitrification does not occur, the aqueous
solution is too concentrated to permit ice
crystal nucleation.

VITRIFICATION

SYSTEM PRINCIPLE REFERENCE
Pellet formation 1 0.2 ml volumes of semen
dropped onto a depression
in solid CO2 then
transferred into liquid
nitrogen.
Watanabe and Terada,
1980; , Terada et al., 1989.
Pellet formation 2 0.2 ml semen dropped
onto a fluoroplastic plate
held in liquid nitrogen
vapour at -70 ̊ C then
transferred into liquid
nitrogen.
Kurba tov et al., 1984;
Tselutin et al., 1995.
Pellet formation 0.2 ml volumes of
equilibrated semen are
dropped directly into liquid
nitrogen.
Yerashevich, 1990; Tselutin
et al., 1995.

Rotation method 1 ml volumes within a 10
ml capacity glass vial are
rotated in liquid nitrogen
vapour at -70 ̊ C before
plunging into liquid
nitrogen.
Kurbatov et al., 1984.
Programmable freezer Samples within glass
ampoules, plastic cryovials,
straws are cooled from
5 to -35 ̊C at between
1 and 10 ̊C/min, before
plunging into liquid
nitrogen.
Lake and Ravie, 1984;
Wishart, 1995; Seigneurin
and Blesbois, 1995.
0.5 ml straws Straws were frozen for
30 min by exposure to LN2
vapours 4-4.5 cm above
the surface of LN2.
Sasaki et al, 2010; Hong Jo
Lee, 2012.

• The process of changing the state of a
substance from frozen solid state to liquid
state by gradual warming.
• As the sperms are thawed, their membranes
will leave their vitrification state, as well as
undergo the membrane phase transition to a
more fluid state.
• Here, membrane damage from previous steps
will manifest when the plasma membrane
reverts back to its normal state (Hammerstedt,
et al., 1990).

• Many freezing methods with different CPAs like glycerol,
DMA, DMSO & MA and different types of sperm packaging
techniques (with slow & rapid freezing procedures) (Blanco. et
al.,2102; Blesbois. et al.,2007; Sasaki. et al., 2010; Tselutin. et
al.,1999 ).
• Rooster spermatozoa cryopreservation, although successful, is
associated with extremely variable fertility (Lake,1986;
Hammerstedt & Graham, 1992; buss, 1993; Long, et al., 210).
• Studies on influence of freezing on avian sperm physiology.

• Recent advances in cryopreservation technology for
poultry semen have resulted in the emergence of
cryobanking, which is now being developed in an
increasing number of countries (see Blackburn,
2006;Woelders et al., 2006; Blesbois et al.,2007,
2010).
• Many cryopreservation diluents have been
developed, and each requires its own specialised
protocol, utilizing different freezing rates, sperm
concentration(Lake & Stewart, 1978; Caudhuri &
Lake, 1988; Tajima, et al., 1989; Sasaki, et al., 2010).

References:
• A Review Article of Artificial Insemination in Poultry
2016
• Current status in avian semen preservation.
Blesbois2007
• Possibilities for preserving genetic resources.
Benesova2016
• Storage of poultry semen. Donoghue2000
• FAO cryoconservation of animal genetic resources.
• Animal Genetic Resources Conservation poultry2006
• Avian semen cryopreservation. Long 2006
• Avian Genetic Stock Preservation. Fulton2006

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Cryopreservation of rooster sperm

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