SEMEN DILUTION AND ITS PRESERVATION

Dr. Abhijeet Fernandes

Assistant Professor
VGO
 SEMEN DILUTION/ EXTENSION OF SEMEN

• The ejaculates of most domestic animals contain more spermatozoa than are
needed for achieving a pregnancy.

• By diluting the semen, it can potentially be used for several inseminations.

• Dilutor/ extender: It is a nutrient media used for the dilution of semen to increase
the life of semen and volume of the semen.

• Semen dilutor is a liquid diluent which is added to semen to preserve its fertilizing
ability.
 Objectives of dilution/ extending of semen
• To increase the number of service of the females from one ejaculate of the male-
addition of volume

• To maintain the viability of the sperms during storage period

• To protect the sperms against possible damage by toxic end products of sperm
metabolism.

 Since spermatozoa do not survive in neat semen for a long period of time even at low
temp., it is necessary to improve the media surrounding sperm by furnishing a supply
of energy and providing protection against by-product of metabolism & temp.
changes.
 BASIC PRINCIPLES/ PROPERTIES OF SEMEN EXTENDER
• Maintains proper osmotic pressure of approximately 285 mili osmoles (which is
equivalent to blood, semen and milk)
• Provision for buffering substances to maintain a nearly neutral pH—more important
for chilled storage of semen (TRIS, HEPES)
• Provision of energy source
• Provision for protection against cold shock/ low temp.
• Antibiotics to cover broad range of bacteria
• Must have balanced proportion of mineral elements essential for life of sperm cell
• Components should be easily available & economical
• Should not hinder during the microscopic examination of sperm
• Must preserve the integrity of sperm
• Must extend the life of spermatozoa
Hence, precise weighing of chemicals is absolutely necessary to ensure correct
composition. The pH is a good indicator and it should be assessed frequently to
ascertain the quality of semen.
The sperm concentration is determined and the dilution rate is calculated.
Dilution should be done within 10 minutes after collection. Delaying the dilution
adversely affects the semen quality.
Both the semen and diluent should be at the same temperature (30°c) at the time of
dilution.
Semen is diluted with equal quantity of diluent immediately after collection, poured
slowly and gently along the sides of the collection tube and then the whole content is
emptied into the pre-measured remaining diluent with caution
Storage of spermatozoa outside the animal body therefore involves
Delaying the senescence.
Slowing down the metabolic processes
Supply of nutritive materials
Provision of substances which prevents autointoxication of metabolic products
 Basic component of extender
• Buffer- maintain pH & osmotic pressure (2.9 % sodium citrate, Tris)
• Nutrient- Fructose, glucose
• Antibiotics- to cover broad range of [Link] Penicillin & streptomycin, GTLS
(Penicillin-G Na - 1000 IU, Streptomycin - 1 mg, Sulphanilamide - 3 mg/ ml)
• Cold shock preventer-
• Lecithin in egg yolk
• Phospholipid in milk
• Glycerol in deep freezing

Precaution:
• Dilutors should be aseptically with analytical grade chemicals
• Pure substances & clean, sterilized equipments should be used.
• Handling of semen should be done carefully to avoid cold shock, overheating and
contamination with urine, dust & water, exposure to direct sunlight and excessive
agitation.
 Semen dilutors

Preservation at room Preservation at Preservation at ultra

temp. (18-30o c) refrigerator temp. (4-6oc) temp. (-79 to -196o c)

1. Coconut milk extender (CME) 1. Egg yolk phosphate (EYP) 1. Glycerolated egg
2. Caprogen 2. Egg yolk citrate (EYC) yolk citrate
3. Illini variable temp. (IVT) 3. Egg yolk glucose bicarbonate 2. Glycerolated egg
4. Cornell university extender (EYGB) yolk TRIS
(CUE) 4. D2/ D3 dilutor
5. Carbonate phosphate method 5. Tris buffered egg yolk
6. Millovanov dilutor 6. Milk dilutor
7. Citric acid whey
 Preservation at room temperature

Principle: the dilutor in which spermatozoa are suspended provide the condition
which inhibits those pathways that are detrimental to their survival at higher temp.
• Prevention of sperm metabolism either by CO2 gassing or producing CO2 by
chemical reaction (reversible inhibitor)
• Motility & fertility of spermatozoa can be prolonged by proper control of CO2
• IVT is saturated with CO2 by bubbling gas through it for 10 min. or until pH
reaches 6.3
• In CUE, citric acid reacts with bicarbonate which releases sufficient CO2 to
preserve sperm.
 Always add diluent to semen not vice a versa.
 Preservation at refrigerator temp.

• Refrigerator temp. lowers the metabolic rate of spermatozoa & in this way it
extends the fertile life of spermatozoa.
• Semen preserved at 5c can be utilised for 2-4 days.
• In EYC, citrate acts as chelating agent that binds calcium & other heavy metals,
also disperses fat globules in the yolk to improve visualization under microscope.
• Lactenin is normally present in milk which is toxic to the spermatozoa. So to use
milk as dilutent, it should be heated at 92 °C for 10 min to inactivate the toxic
compound.
• Egg yolk is added at the rate of 20% to the above buffer solution (20 mL egg yolk
+ 80 mL of buffer)
 EXTENDERS FOR FREEZING OF SEMEN

Successful freezing of semen will depend upon prevention of damage caused by

• Internal ice crystal formation that affects the structure of the spermatozoa.
• The increase in solute concentration as water is withdrawn from the suspension
medium.
• Tris is commonly called as universal diluents which prolongs the life of sperm at
room temperature, penetrate sperm and acts as intra cellular buffer, less toxic at
the critical low freezing temperature.
• Addition of fructose provides energy, prevent agglutination, maintain required
osmotic tension and electronic balance and gives better cryoprotection during deep
freezing.
• Glycerol is the most widely and commonly used cryoprotective agent for bull
spermatozoa
• It was the spectacular discovery of Polge in 1949 that showed that the death of
spermatozoa could be avoided if the cells were suspended in a medium containing
glycerol.
• The possible modes of action of glycerol are:
• Modifies the size and shape of ice crystals formed.
• Binds water and decrease freezing point of solution and less ice is formed.
• Acts through salt buffering mechanism.
• Reduces solute concentration.
• Prevents denaturation of proteins and rupture of plasma membrane.
 Advantages of Frozen semen
• It permits maximum utilization of semen collected from a sire.
• Long term preservation of semen for any time period is possible.
• Semen can be used even after death of the bull.
• Transport of semen is easy even to international level.
• Transport cost is frozen semen is less when compared to liquid semen.
• Selective breeding is possible as per the wish of the owner.
• It eliminates the maintenance of breeding bull in a herd thereby economic benefit to
the owner is ensured.
• Number of bulls required for breeding purpose can be minimized.
• Aids in progeny testing of bulls.
• Round the clock facility is possible even at villages.
• Door step insemination even at remote places is possible.
 Protective mechanism of Egg yolk
• When semen is brought from 30° to 5°c, there is considerable damage to the
spermatozoa, characterized by greater contraction of lipoprotein sheath of
spermatozoa than cell contents & leading to leakage of intracellular content.
• All these changes occur due to phase transition of plasma membrane from fluid to gel
by lowering of temp.
• This phase changes causes irregular clustering of membrane proteins. This whole
process is called as cold shock.
• Egg yolk offers protection due to presence of LDL (lecithin) which forms a layer
around the damages plasma membrane & replace the phospholipids.
• LDL & cholesterol incorporate into membrane & reduce the gel forming tendency.
• For effective action of yolk, temp. should be reduced from 30 ° C to 5 ° C @ 1 ° C
/min.
• When spermatozoa is cooled to 5 ° C they are subjected to cold shock, which causes
leakage of intracellular enzymes and other materials present in the spermatozoa.
• This damage can be prevented by providing addition of lecithin, protein, lipoprotein
and similar compounds present in the egg yolk.
• In addition the glucose, proteins and vitamins present in the egg are utilized by the
spermatozoa and also protect the enzymes and antiagglutinic factors present seminal
plasma.
• The value of the yolk of the hen’s egg as a diluting medium in semen preservation
was found by Philips during 1939.
• The active principle in yolk responsible for this is now thought to be lecithin or a
similar phospholipid occurring either free or in combination with protein.
• The hydrogen peroxide formed, which is toxic to the sperm, may be destroyed by
additions of the enzyme catalase to the yolk diluents, under anaerobic conditions the
problem does not occur.
 CRYOPROTECTANTS
• Cryoprotective agents can be divided into permeating and nonpermeating agents. Permeating
agents such as glycerol can move across cellular membranes and modulate the rate and extent
of cellular dehydration during freezing-induced membrane phase transitions.
• Non-permeating protectants including sugars and proteins are used as bulking agents and to
increase the glass transition temperature of the freezing extender.
• Both, permeating and non-permeating protectants form a protective glassy state during
freezing preserving biomolecular and cellular structures.
1. Permeating CPAs (penetrate the plasma membrane)
• Such as dimethylacetaldehyde; dimethyl sulfoxide, glycerol, ethylene glycol.
• They stabilize cell plasma membrane proteins and reduce concentrations of electrolytes.
2. Nonpermeating CPAs (unable to penetrate the plasma membrane)
• Such as albumins, dextrans, egg yolk citrate, hydroxyethyl, polyethylene glycols, polyvinyl
pyrollidone, fructose and sucrose.
• They minimize intracellular crystallization by increasing viscosity of the sample
 Principle of cryopreservation
• Cryopreservation includes various steps such as dilution, cooling, freezing & thawing.
• The phenomenon which causes damage to sperm cells during this are mainly cold
shock, solution effect & intracellular ice formation.
• Freezing rate directly related to water permeability of the sperm membrane.
• Extracellular ice formed during freezing leads to increased osmotic pressure in the
unfrozen medium, which in turn pull more water out of the cell..
• The concentration of salt increase both intra & extracellularly as water freezes.
• Rapid freezing causes structural damage by intracellular ice formation & too slow
freezing rate causes salt damage to proteins (solution effect).
• The survivability of the spermatozoa depends upon the optimum cooling rate, which
results in less intracellular dehydration, less intracellular solute concentration & less
shrinkage of the cell.
• It is also essential to add a cryoprotectant in the extender because they modify ice
crystal formation during freezing & thawing.
 Physiology of cryopreservation
• There are two types of fluid involved in semen freezing (one inside the sperm cell & other
found surrounding the sperm which is a mixture of seminal plasma & extender).
• When temp. of semen falls below the freezing point, ice crystal starts to form firstly
extracellularly as it is more near to LN2 vapour.
• As ice crystal starts to form, conc. of solute in extracellular fluid will increase. Ice crystals do
not extend I/C at this stage, as they are excluded by CM.
• Now, the condition becomes hypertonic outside sperm cell, therefore I/C comes outside by
osmosis to maintain the equilibrium resulting in variable degree of dehydration followed by
formation of I/C ice crystals.
• During freezing, sperm cells are damaged by high concentration of solutes (solution effect)
inside the sperm cells & formation of ice crystals especially I/C.
• During fast freezing, sperms are damaged mainly due to I/C ice crystals as there is very little
time for water to move from inside the sperm outside through semipermeable membrane to
create equilibrium.
• During slow freezing, sperm are mainly damaged by solution effect because sperm cells get
enough time for water movement from inside to outside resulting in high conc. of solute
inside sperm cell but small ice crystal formed I/C.
 Cryopreservation

Too rapid Too slow Optimum

Less dehydration More dehydration

Less dehydration
of sperm cells of sperm cells

More no. & large Less I/C ice crystal Less I/C ice crystal
sized I/C ice crystal formation but higher formation
formation I/C salt/solute conc.

Mechanical damage to Salt damage the Minimum damage to

internal membrane as membranous internal membrane
well as CM proteins as well as CM

Less survival of Less survival of Better survival of

spermatozoa spermatozoa spermatozoa
 Factors affecting quality and quantity of semen
1. Sexual maturitity
• The process of spermatogenesis is initiated just before puberty, the spermatogenesis
is at it’s peak during sexual maturity.
2. Senile changes
• The age of the male livestock affects the semen production such as spermatozoal
concentration and seminal volume.
3. Season
• The seasonal variation is observed on the process of spermatogenesis as well as
plasmogenesis . in male live stock via., buffalo –bull, stallion ,ram.
• The quantity of semen ejaculate is greatly declined due to adverse summer season in
buffalo bull which is probably due to less activity of accessory gland (seminal
vesicle).
4. Climate
• The environmental temperature, relative humidity and wind flow rate have a great
effect on the process of spermatogenesis.
• The extreme hot or extreme cold has adverse effect on spermatogenesis and semen
quality.
• The day length also affects the spermatogenesis among ram in cold countries
5. Nutrition
• The under nourished male livestock especially in terms of protein, A, D and E
vitamins and certain trace elements (iron ,cobalt, copper, phosphorus)have
adversely affect the semen quality.
6. Endocrine
• The testicle and pituitary gland play an important role in spermatogenesis through
production of testosterone and ABP from male gonad and LH/ICSH and FSH from
hypophysis.
7. Hormone
• The androgen is the principle hormone for initial impetus for spermatogenesis.
• The androgen is secreted by leydig cells under the control of LH/FSH . the FSH also
helps in the spermatogenesis through influencing the production of ABP (Androgen
Binding Protein) by sertoli cell . Hence the level of above hormone is vital.
8. Pshycological factors
• The pshycho –neuric and pshyco-somatic factors also affect semen production.
9. Sexual stimulant
• If the male livestock is placed near it’s female sexual partner then it will stimulates
the process of semen production through pheromone and various extraneous
stimulies.
10. Semen collection technology
• The quantity and quality of semen can be improved through proper semen collection
techniques via., sexual preparation and sexual stimulation prior to ejaculation.
• The proper temperature, lubrication and pressure of artificial vagina, site of semen
collection , dummy and attendant affects the semen production.
• The frequency of semen collection is an important factor for semen quality.
• The method of semen collection techniques (AV, electroejaculation, massage
method) also affect the semen production .
11. Management factors
• The care and management of male livestock directly affects the quantity and
quality of semen ejaculate .The exercise of animal also affects it.
12. Health
• The general and reproductive health conditions of male livestock affects the semen
production.
13. Genetics
• The species , subspecies and breed affect the quantity and quality of semen
ejaculate .
14. Individual variation
• The production of semen chiefly depends on the individual male livestock
 Bull semen freezing protocol
1. Preparation of extender
• Prepare the extender on the day of collection or on the previous day in the
evening.
• Keep the extender in water bath at 32-35 c before start of collection.
2. Semen collection
• Collected in the early hours of morning before feeding.
• Two ejaculates form each bull should be collected on the day of collection &
interval between the two collections should be 10-12 min.
3. Semen evaluation
• After receiving neat semen, it should be kept in a water bath at 32-35 c under
laminar unit, after recording the volume.
• Evaluate semen for volume, colour, consistency, mass motility, individual motility
& conc.
• place a drop of semen on glass slide & observe under microscope for mass motility.
Ejaculates having ≥ +3 motility are selected for further processing.
• Sperm conc. & volume of extender can be determined with the help of auto diluter &
digital photometer.
4. Initial dilution
• Till the final dilution rate is decided, semen in diluted with an equal quantity of diluent &
kept in same water bath.
• The progressive motility of extended semen in assessed with the help of phase contrast
microscope & the ejaculate showing > 70% motility is taken for further processing.
5. Final dilution
• The dilutor & ejaculate should be kept in water bath at same temp. (32 c)
• After calculating the final vol. of dilutor, the remainder of diluent is added to pre diluted
semen so that each straw should contain 20 million progressive motile sperm.
• Then the diluted semen is placed in a cold cabinet where it reaches 4 c within 1-1.5 hrs.
this slow cooling prevents sperm from cold shock & egg yolk plays important role to
prevent cold shock.
6. Equilibration of semen
• The pre-freeze storage of diluted semen is k/a “equilibration” of diluted semen.
• It refers to the time from adding the glycerol to extender until freezing. To extend the fertile
life of sperm, temp. is reduced to decrease the metabolic rate.
• After initial cooling period from 32 to 4c, the extended semen should be allowed equilibrate
at 4c for at least 4 hrs.
• During this, glycerol penetrate the sperm followed by membrane stabilisation upon exposure
to low temp.
• During this, straws are filled with extended semen, sealed & placed on the racks for freezing
in cold cabinet.
7. Printing of straws
• Bull no., breed, name of semen station & date of collection are printed on the straw before
filling followed by UV sterilization
8. pre-freeze evaluation
• At the end of equilibration, semen samples are evaluated for pre-freeze motility.
• Ejaculate showing ≥ 70% pre-freeze motility are selected for freezing.
9. Filling & sealing of straws
• Can be filled manually & automatically with machines.
• Semen is sucked into the straws & on touching the polyvinyl alcohol powder plug at one
end & impervious seal is made.
10. Racking
• After filling, straws are spread over freezing rack inside the cold cabinet at the same
temp.
11. Freezing
• Vapour freezing is carried out in a wide mouthed container especially designed for
freezing of straws.
• Equilibrated straws are placed 4-5 cm above the level of LN2 to expose them to vapours
of LN2.
• The lid is placed over the container for settling the vapour. The temp. at this height is -
180c, when the temp. of semen reaches -130c to -150c by about 10-15 min., straws are
are immediately transferred to pre-cooled goblet.
• Then these goblets are now carefully transferred into LN2 container for further storage.
 Handling of Semen
• Storage of straws may be so arranged that the doses can be quickly sorted batchwise,
bullwise and for the dispatch to various AI Centres or farms for subsequent
utilization.
• If the doses are exposed to environmental temperature permitting them to reach -80
OC to -100 OC, irreversible injury to the sperm cell can occur.

• This injury is thought to be due to recrystallization of frozen semen. Though the

straw offers us many advantages primarily due to its geometry, yet the thermal errors
in handling the straws may also lead to rapid rise in semen temperature due to the
same high surface to volume ratio.
• It is therefore, important to have freezing and thawing procedures closely in tune
with the thermal requirements for optimum semen handling in storage.
• Handling semen doses in nitrogen vapour is safe only if the vapour is sufficiently
cold and the exposure is of short duration.
• Use of exhaust fans to remove vapour to improve the vision into storage tanks will
greatly increase the temperature of exposed doses. Bubbling nitrogen or cold nitrogen
through the tank will accomplish the same task safely.
 Thawing
• The rate of thawing used on the farm has important bearing on the viability of
spermatozoa.
• The optimum thawing rate should be assessed with respect to the entire semen
processing system.
• To avoid the recrystallization zone, thawing should be rapid. Thawing rate depends
on thermal history of frozen semen, type of packaging, duration, ambient
temperature.
• The commonly used method of thawing frozen dose is to plunge it into water. Time
allowed for this depends upon the temperature of water and the packaging system.
• Under field conditions thawing is done at a temperature of 35-37C water (15 sec.).
• Though experimentally higher thaw rates have shown improved viability, yet very
fast rates can not be recommended in the field at present because slight errors in
terms of prolonged exposure to high temperature can cause overheating of sperm and
hence loss of viability.
 Handling of semen in LN2
• In the upper half of the neck tube of LN2 container, high temp. exist.
• Time spent in removing semen from the LN2 tank must be kept to a min. to reduce
semen damage.
• If the entire canister of semen is withdrawn above the frost line (3-4 inches from the
top), all the straws of semen will be damaged.
• Thermal injury to sperm is permanent & can’t be corrected by returning semen to liquid
nitrogen.
• Identify the canister contains the desired semen with the help of attached tags to the
canisters.
• Remove the canister from its storage position to the middle of tank.
• Raise the canister just high enough in the neck region to grasp the desired straw.
• Grasp the straw & immediately lower the canister to the tank floor.
• Use tweezers or long pre-cooled forceps to remove the straw within 10 sec. from the
time of canister is raised into position, then immerse it in 37c water.
• If it takes > 15 sec to take out the desired no. of straws, the canister should be
lowered back into the tank to cool completely & again after sometimes take out
container.
• After the straw is taken out of the container, it should be given little jerk to remove
any liquid nitrogen inside.
• Straw should be taken out from the water and should be wiped clean with a neat cloth
or cotton after ascertaining the bull number printed on the straw.
• With mild jerks bring air bubble in the straw towards the lab seal in French straw
holding by the lab plug.
• Bring the insemination gun to the proper temperature by rubbing it with a piece of
cloth or paper. This is important in the winter months.
• Load the gun by keeping the factory plug downwards
• Universal gun can be used for all types of straws with the help of universal sheath,
the gun can also he used for fluid semen insemination with a special type of sheath
• Hold the straw vertically and cut just above the semen level with clean scissors. The
cut should be at right angle and sharp. Straw should not get pressed at the cut end.
• Cover the gun with suitable sheath and secure it with the securing ring at the base.
Sheath must have a cut at the lower end for proper fixing. Ensure that the tip of the
straw has got tightly fixed in the mouth of the sheath and there is no gap.

• Frozen semen generally packaged in plastic tubes of various capacities and sizes i.e.,
Medium French straw, Mini French straw and Mini tub.

• Avoid splashing, overflowing, violent boiling and direct contact with LN

• Use gloves while handling liquid nitrogen, frozen semen or racks etc.

• If it falls on body, it causes cold burns and frost bite. If so, flood with cold water and
apply cold compressor bandage
 Packing of frozen semen

Ampoule Semen straw Pellet

French straw German straw US straw

Large Medium Mini

Type of straw Volume (ml) Length (mm) Diameter (mm) Surface area (mm2) Made up of

French large 1.2 133 4.2 - PVC

French medium 0.5 133 2.8 1152 PVC
French mini 0.25 133 2.2 823 PVC
German 0.4 90 2.9 555 Plastic
US 0.25-0.5 57-127 2.9 - Polypropylene
• (7079) GADVASU. Dept of Vet Gynaecology & Obstetrics. Tutorial on
Vet Andrology & Artificial Insemination. - YouTube