“Cryopreservation of Rooster Spermatozoa’’

1. COLLEGE OF VETERINARY AND ANIMAL SCIENCEs
S.V.B.P.UNIVERSITYOFAG.& TECH.,MEERUT.
Course Advisor
Dr. Rakesh Kr. Singh
Presented By.
Surya Kant (v -3098/14)
SEMINAR
ON
“Cryopreservation of Rooster Spermatozoa’’
Tracking Programme
Cryobiology of Gametes
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2. Introduction
o Cryo is Greek word (krayos – frost)
o It literally means preservation in “frozen state”
o The principle – to bring cells or tissue to a zero
metabolism and non dividing state by reducing the
temperature in the presence of cryoprotectant
o Cryopreservation is a process that maintains cellular life at
sub-zero temperature for an extended period of time by
arresting all the metabolic activities
o It can be done :
 Over solid carbon dioxide (at -79 degree)
 Low temperature deep freezer (at -80 degree)
 In vapor phase nitrogen (at -150 degree)
 In liquid nitrogen (at -196 degree)
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3. 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
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4. Cont….
 Liquid nitrogen (LN2) is the most widely used
cryopreservation material
Why LN2 :
 Chemically inert
 Non inflammable
 Non corrosive
 Non toxic
 Easily available
 Relatively low cost
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5. CRYOPRESERVATION AND CRYOINJURY
 Cryopreservation protocol has a number of
potentially damaging stresses :
 Change in temperature
 Osmotic and toxic stress
 Formation and dissolution of ice
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7. 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
 This can be achieved by controlling intracellular
and extracellular movement of solutes and water
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8. Rooster Spermatozoa
 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
 Homogametic haploid cells with highly condensed
chromatin
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r

10. STEPS IN CRYOPRESERVATION
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11. Collection of Semen
• Abdominal massage method (Burrows and Quinn, 1937)
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12. Cooling of spermatozoa to 5˚c
 Freshly collected sperm are immediately diluted
with diluent which keeps sperm alive for long
periods of time (Burrow and Quinn,1937; Sasaki et al.,2010)
 The sperms are cooled from body temperature
(41.5˚c) to 5˚c over ice
 Rooster sperms do not
undergo cold shock
(Parks & Lynch, 1992)
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 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 lipid and
altered functional state of membranes
 Integral membrane proteins are clustered by
lipid phase separation and this may alter the
function

14. Adding of Cryoprotectant
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 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

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Constituents of cryoprotective medium
• Permeating cryoprotective agent : Glycerol, ethylene
glycol, dimethyl sulfoxide, propylene glycol and
methylformamide
• Non-permeating 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

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CRYOPROTECTANTS

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• 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, Ethyleneglycol
• Non-penetrating 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
 Glycerol(Gly), Ethyleneglycol(EG),
Dimethylacetamide(DMA), Dimethylsulphoxide(DMSO),
Dimethylformamide(DMF), N-methyl acetamide(N-MA)
 CPA used in rooster semen cryopreservation :

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19. Freezing of sperm
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 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)
 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

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 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)

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Freezing
 Slow freezing :
o Cells are frozen with gradual decrease in temerature
1-10˚C/min to -35˚C
o Permits flow of water from the cells to the outside, Thereby
promoting extracellular crystallization avoiding lethal
intracellular ice formation
 Rapid freezing :
o Currently fast cooling rates are preferred 20-100˚C/min
(Varadi. et al.,2103; Long et al.,2014)
o Optical rapid freezing avoids excessive dehydration and
shrinkage of cells
 Step-wise freezing :
o A programmable freezing machine was used (Blanco et al.,2012)
o Frozen at 7˚C/min to -35˚C , then -60˚C/min to -140˚C in a
programmable freezer

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Vitrification
o The term “vitrification” refers to any process that results
in “glass formation” (transformation of a liquid into a
solid/glass without crystallization below the threshold
temperature)According to this definition, cells that are
properly slow frozen become “vitrified”
o In a wider sense, the embedding of material in a glassy
matrix is also called ‘vitrification’
o Water solidifies into an amorphous
‘glassy’ state
o If vitrification does not occur, the
aqueous solution is too concentrated
to permit ice crystal nucleation
(Rapid freezing)

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Thawing
• The process of changing the state of a substance
from frozen solid state to liquid state by gradual
warming (water bath @ 37˚c)
• 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 it’s normal state
Thawing of frozen sperm

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Current Status of Cryopreservation
o Currently, no artificial insemination using cryopreserved
rooster spermatozoa is being used in the commercial poultry
industry in the United States (Donoghue & Wishart, 2000)
o Recent studies have investigated low molecular weight
cryoprotectants that would not need to be removed from the
rooster spermatozoa prior to insemination (Van Voorst &
Leenstra, 1995; Tselutin, 1999; Blesbois, et al., 2007; Sasaki, et al., 2010)
o This new method of freezing rooster spermatozoa could be
easily incorporated into the industry, as straws would merely
need to be thawed and the sperm inseminated

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o Cryobanking valuable poultry genetics across the world has
become a pertinent issue and there is a need for establishing
genetic reservoirs of cryopreserved livestock gametes,
embryos and other tissues (Boettcher, et al., 2005; Blackburn, 2006;
Woelders, et al., 2006; Danchin-Burge, et al., 2011)
o Other technologies, including poultry gonadal tissue
cryopreservation, are emerging as other methods to preserve
valuable genetics from both male and female chickens (Song &
Silversides, 2008)
o This technology is very labor-intensive and expensive,
leaving improvement for adaption outside the research
laboratory, but has high potential for use in cryobanking

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Future needs
• Many freezing methods with different CPAs like
glycerol, DMA, DMSO and different types of sperm
packaging techniques (with slow and rapid freezing
procedures) (Blanco et al.,2012)
• Rooster spermatozoa cryopreservation, although
successful, is associated with extremely variable
fertility
• Studies on influence of freezing on avain sperm
physiology

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• 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 (Blesbois et al.,2007,
2010)
• Many cryopreservation diluents have been
developed, and each requires it’s own specialised
protocol, utilizing different freezing rates, sperm
concentration (Sasaki et al.,2010)

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