The document discusses cryopreservation of rooster spermatozoa. It outlines the steps involved, including collection of semen, cooling to 5°C, addition of cryoprotectants like glycerol, freezing typically using liquid nitrogen, and thawing. Cryopreservation aims to arrest cellular metabolism and prevent damage during freezing and thawing. While research continues to improve methods, current cryopreservation of rooster sperm has variable success and is not widely used commercially. Future work could optimize techniques using different cryoprotectants and freezing protocols.

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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