ARTIFICIAL INSEMINATION

Introduction of male reproductive cells into the female reproductive tract by artificial
means is called artificial insemination.
In case of humans
AIH-when husband’s semen is used
AID- when donor’s semen is used.

Lazzaro Splallanzi (1977) –used AI successfully to inseminate a Spanish bitch.

Elias I. Ivanoff – a Russain Researcher- used AI in birds, horses, cattle and sheep.

Mass breeding of cows through AI was first accomplished in Russia.

AI in India: -
First attempted in 1939 by Dr. Sampath Kumar at Palace of Dairy Farm Mysore.

Comprehensive studies on problems on AI with special reference to Indian conditions

began in 1942 at IVRI under a scheme sponsored by ICAR.

The first buffalo calf through AI was born in 1943 at Allahabad Agricultural Institute
in U.P India.
COMPONENTS OF SEMEN

The two components of semen are

a) Spermatozoa

Whole semen as ejaculated appears Viscous, Creamy, Slightly Yellowish or Greyish

Fluid and consists of spermatozoa or sperm suspended in the fluid medium called
Seminal Plasma.

In majority of species including man, mature spermatozoa have filliform structure

owing to the presence of flagellate appendages although non-flagellar forms of sperm
cells are not uncommon in certain lower animals e.g., crustacea nematodes.
Filliform Structure determines the remarkable permeability of the sperm cells which is
best illustrated by the Leakage Phenomenon( The remarkable case with which the even
larger molecules such as cytochrome or hyaluronidase can detach themselves from the
sperm structure and pass with extracelluar environment).

In a typical falgellar spermatozoa there are three regions

1. Sperm Head
2. Middle Piece
3. Tail

Monster Cells (Spermatozoa) :- Containing several nuclei and several tails in a mass of
cytoplasm.
In humans 20% of sperm are of abnormal forms.
Bull and Stallion = percentage is higher. Ram= Percentage is low.
Shape of head:-

Ovoid =Bull, Ram, Boar and Rabbit.

Elongated Cylindrical= Fowl.
Hook Shaped= Mouse and Rat.

Bull Spermatozoa= 68 µ± 3µ in Length

Tail = 50µ
Head=8-10µ
Neck=1µ
Mid Piece=8-10µ
Main Part of head is occupied by nucleus, filled by closely packed Chromatin which
consists largely of Deoxyribonucleoprotein.
Anterior part of nucleus is covered by cap like structure k/a Acrosome.
Another Cap k/a Galea Capitis is a loose protoplasmic structure which envelops the
apical part of sperm head and can break away spontaneously to form Spermatic Veil or
Floatin Cap.
Some consider Acrosome proper and galea capitis as distinct structural entities while
some consider them to be identical.

Neck/Neck Piece: - (most vulnerable and fragile part of spermatozoa).

It is a narrow region which connects sperm head with middle piece.
In the neck, close to the base of sperm nucleus, centrosome is situated which marks the
beginning of Axial Filament (central core of both middle piece and tail).
It consists of 20 filaments which run uninterruptedly through the whole length of middle
piece and tail.
Fibrils are attached in two rings of 9 fibrils each.
The nine members of inner ring are surrounding the other two central fibers (9+9+2
arrangement for Bull spermatozoa.)
These fibrils are responsible for Whip-Like Lashing of Tail.

In the Middle piece (Mid Piece) axial filament is surrounded by Broad Helix also
Known as Spiral Body or Mitochondrial Sheath.

Junction between mid-piece and tail is marked by the presence of Ring Centriole.

Terminal tail piece (3µ) is a solid bundle of fibers.

b) Seminal Plasma
Composite mixture of fluids sectred by Epididymis, Vas Deferens, Ampullae, Prostrate,
Seminal Vesicles, and Cowper’s Glands and other glands located in the urethral canal.

Prostatic Secretion
Colorless and slightly acidic (pH=almost 6.5)
Contains Proteolytic Enzymes (In humans= Fibrinolysin present)
Contains citric acid, acid phosphate. It is main source of Ca in man semen.
Prostatic secretion is also high in content of Zinc.
Seminal Vesicle Secretion
In most of species, the seminal vesicles alone contribute more fluid than rest of accessory
sex glands together.
Compared with prostatic secretion it is Less Acidic and is sometimes distinctly
Alkaline.
Has higher Dry Weight, contains more potassium bicarbonate, acid soluble phosphate
and protein.
Dog and Cat= S.V are absent.
Normally secretion is slightly yellowish but in man and bull it can be deep pigmented
because of the presence of Flavins.
The Reducing Power of Seminal Vesicle Secretion is one of its Most Characteristic
Chemical Properties.
Seminal Vesicles are main source of Fructose in higher animals.

COLLECTION OF SEMEN
Collection from Bull
Factors affecting semen output
a. Age: - Max O/P at 5-8 years of age.
b. Season; -Winters best for total sperm production.
c. Frequency of Ejaculation: - with increased frequency- more total sperms obtained
but with fewer sperms per collection.
d. Pre-ejaculation sexual preparation: -
e. Correct Techniques of Semen Collection
f. Oxytocin: influence sperm o/p.
PRL, Testosterone and Growth Hormone increase immediately after ejaculation.
Scrotal Circumference (SC) or Testicular Size is positively correlated with
Fertility(r=0.58)
Correlation of S.C with Testicular Weight (r=0.95)
It means SC is reliable predictor of the amount of sperm producing tissue within the
testes.
Larger the testis, greater the sperm producing potential.

Methods of obtaining semen from Bull

1. Artificial Vagina
2. Electro Ejaculation.
Selection of Breeding Bulls:
Those bulls whose pedigree are known and are born of a high yielding dam are selected.

Preparation of Young Bull

6-12 Months = Procured
9 Months= Fixation of Nose ring (made of copper)
12 Months = Screened for Vibriosis, Trichomoniasis, Brucellosis, Jhone’s Disease and
T.B.
The bull calves so selected are finally screened Karyologically to detect any
Chromosomal abnormality. It improves fertility by 15%.( Available at TANUVAS)
18 Months= Trained for semen collection.
24 Months= Semen Collected and evaluated before final selection.
Once selected, bulls should be on Balanced Ration comprising sufficient greens.
Maintenance Ration is enough as semen production doesn’t require any extra ration.
Time for Collection
Early morning hours before feeding when bulls are alert and fresh.
Advantage is that it can be used on same day and may raise fertility rate to a certain
extent.

Collection Yard:
Dummy should never be taller than bull.
Angle b/w back of teaser and that of bull should not exceed 45º during ejaculation.

Cleanliness of Bull:
Wet underline is more conducive to the transmission of Microbes than a dry one.
Long hairs at sheath of Penis should be cut to 4-7 c.m.

Selection of Teaser Cow:

Should be of same breed, size and colour of bull.
Physically strong and docile.
Gone through 2-3 Calving’s.
Should not be in estrus or Pregnant.

Pre-Ejaculation Sexual Preparation:

Sexual preparation of bulls may be defined as prolonging the period of stimulation
beyond that adequate for mounting and ejaculation.
Motile sperm output per ejaculation can be increased as much as two fold by giving 2-3
flase mounts( by 10 minutes) before ejaculation.
Stimuli other than false mounting can also be used to increase the sperm output. It
includes
i) Moving the stimulus animal
ii) Exchanging stimulus animal
iii) Changing locations of preparation
iv) Changing the personnel handling the bulls
v) And combination of these.
Undefined stimuli from teaser animal near a bull cause to ejaculate more sperms.
Undefined stimuli are not visual because blinded bulls also respond to sexual preparation
with increased sperm output.
Part of the stimuli must involve olfactory (sense to smell ) mechanism since bulls mount
estrus cows twice as fast as non-estrus cows and yield larger volumes of semen and great
sperm numbers.
Tying of aprons to the bulls(size=65x 62cm)
Apron cloth should be of coated rexin on one side facing penis of the bull.
It should be tied behind the elbow in such a way it doesn’t touch the ground.
Advantages
i) Prevents cross infection from bull to bull
ii) Prevents loss of semen ejaculates
iii) Prevents direct contact with skin of teaser thereby more hygienic ejaculates
obtained.
ARTIFICIAL VAGINA FOR BULL
First AV designed in 1930 by Russian scientists.
Danish Model-most commonly used in USA, India and other countries.( refer to p294
for structure of AV)
Preparation of A.V:-
i) AV for particular bull should be chosen depending upon the length of its
penis.
ii) Water is toxic to spermatozoa.
iii) Water mixed with Methylene Blue Solution-used for testing leakage of
water droplets in AV
iv) Before semen collection warm water between 45 to 50 ºC is filled through the
filling aperture into the space b/w outer cylinder and inner latex to provide an
inner temperature 42 ºC to 46 ºC.( an inside temperature above 47 ºC kills
spermatozoa).
v) Soft paraffin is smeared over inner surface of latex.
vi) Pressure inside AV=45 to 55 mmHg( young bulls require higher pressure)
vii) When pressure is right inner lining of Av should give Star Like appearance.

Semen Collection
Hold AV at 45 º
Collection twice a week (one ejaculate at each time) ; Collection once a week (2
ejaculates/collection)

Electro-ejaculation in bulls:-
First adopted by Battelli for collection of semen from guinea pigs.
In Bulls – by French investigators.
Semen collected by this method are more in volume and less in concentration but there is
no difference in total sperm concentration or fertility.
Alternating current- increasing in voltage gradually from 0 to 5 volts and returning again
to 0 within every 5 to 10 seconds is initially passed. (Rectum is first washed with 6%
NaCl Solution and then probe is inserted upto about 12 inches and held in position of
rectal floor.)
The subsequent stimulations made progressively higher so that at about fifth stimulus a
maximum of 10-15 volts is reached.
Erection and ejaculation occur at 10 to 15 volts when 0.5 to 1 ampere current is
flowed. The source of electric current is AC/220-250 volts/ single phase/ 50 cycles.
Advantages
i) Semen can be collected from males that are too young or old or unable to
mount due to weak or injured legs.
ii) No female or dummy required for collection.
iii) Less chance of contamination.
Disadvantages:-
i) Highly technical and need considerable skill and practice
ii) Semen gets contaminated with urine
iii) Some males resist too much to this method and refuse collection
iv) Sciatic nerves are temporarily affected during the operation but is relatively
minor if some electrodes are kept over ampullar region.
Collection of semen from Buffalo Bull:-
AV Method
AV length =20 to 30 cm. -other aspects same as followed in cow bull.
Buffalo bulls are most susceptible to temperature variations.

Collection of semen from sheep and Goat:-

AV and Electric stimulation methods are the only methods employed for collection of
semen from rams and bucks. AV method is preferred.
Electric stimulation is useful for testing large no. of males for fertility, including teaser
rams or bucks prepared by surgical intervention.
Collection by AV :-( Angle 45 º)
AV used for rams and bucks is similar to that used for bulls.
The temperature of AV just before semen collection should be 42-45 ºC.
In order to prevent cold shock semen collected glasses should be warmed to 30-37 ºC.
The frequency at which semen may be collected depends on the age, condition,
temperament of the animal. Rams may mount and ejaculate 20-25 times or more a day.
At such frequency the volume and concentration of semen (and consequently the no. of
spermatozoa per ejaculate) decreases with successive ejaculates. However, a regime of 3-
5 collections daily for 4-5 day period separated by 2-3 day rest periods should not cause a
marked reduction in semen quality or quantity.
For goat bucks 2-3 collections daily on alternate days can be regarded as a normal
regime. Intervals of 0.5 to 5 hr between successive collections are advisable to obtain
ejaculates of good volume and concentration.
Collection by Electric Stimulation:-
There are different types of electrical stimulators. Those in current use have a bipolar
rectal electrode like the one of Ruakura Ram Probe. This is a self-contained stimulator
operated by batteries giving a 10 por 15 colt output. When the rectum of the male is dry
the 15 volt output is recommended.
The rectal probe is pressed towards the floor of the pelvis by an assistant and short
stimuli (3-8 seconds) are applied at 15-20 second intervals. After several stimuli
accessory gland secretions will flow, followed by semen. When a large amount of clear
fluid is obtained initially this should be discarded to avoid diluting the semen. There is
considerable variation between males in the amount of stimulation required to produce a
satisfactory ejaculate. However, apart from the discomfort and muscular contractions
occurring during treatment there are no permanent ill effects.

Collection of semen from Boar:-

Pressure is especially important for collecting semen from the boar. The boar ejaculates
when the curled tip of the penis is firmly engaged in the sow’s cervix or in AV specially
made to suit male boar or the operator’s gloved hand and pressure is exerted on the coiled
distal end of the penis throughout the ejaculation.
The ejaculate from the Boar consists of the following four fractions:-
i) Pre-sperm fraction:-
It is thin like water and has no colour. The quantity is about 10ml and is
sperm free.
Helps in rinsing the urethral tract which usually has high bacterial count. This
fraction is always preferred to discard.
ii) Sperm-rich fraction:-
Volume is about=40 to 80ml.
It is dense and milky and contains about 80% Spermatozoa.

iii) Low-sperm fraction:-

150 to 200ml thin fluid with low sperm concentration.
This fluid originates from the secretion of seminal vesicles and prostate gland
which is known as seminal plasma.
iv) Post-sperm fraction:-
It is gelatinous and is secreted from Bulbo-Urethral Glands.
It tends to seal the cervix of the sow during mating, preventing loss of
sperm.

Semen collection by AV in Boar:-

Mating is prolonged in boar varying from 3-25 minutes and waves of high and low
sperm concentrations exist in flow. The tip of the penis locks into the entrance of uterus,
cervix and ejaculation occurs directly into the uterus.
Norwegian type of AV is more common, the length of which is about 18cm while the
diameter is that of Bull.
When the boar mounts the dummy, the collector guides the penis into the AV through the
Y-shaped hole in foam rubber piece. Ejaculation is produced by digital pressure on
the spiral portion of glans penis.
Some 200 cc of semen is collected but of this about 50 cc consist of gelatinous sperm,
and this is strained off before insemination or storage.

Collection of semen from Stallion:-

Methods in current use employ either AV or latex condoms.
The Cambridge or Russian model and the Missouri or American model are
considered suitable.
It is essential practice that the mare should be in full oestrus.
The Missouri and Mississippi models are most popular in USA. Mississippi model is
made of one piece of rubber tubing 91.5 to 122 cm long and 18 to 20 cm in flat diameter.
Water temperature is between 41-45ºC inside the AV.
Prior to ejaculation, the penis should be washed with warm soap water and rinsed with
clean water to remove smegma and other debris on the surface of penis.
Unlike with bovine or ovine spermatozoa, the poor viability of equine spermatozoa
is partly due to poor initial sugar content of semen and its early exhaustion during
metabolism.
The first fraction is of watery consistence and is devoid of spermatozoa. In the next
phase the ejaculate comes from the testes and possess high concentration of
spermatozoa and thereby milky white in appearance. During the third and final
phase a glairy, viscous material resembling white of an egg is expelled. This is the
secretion from the seminal vesicles and cowper’s glands.

EXAMINATION AND EVALUATION OF SEMEN

Semen:- Semen is a suspension of spermatozoa in seminal fluid. It is opaque, white to
light cream colored fluid. The spermatozoa are generated in the testes and are stored in
the epididymis whereas the seminal plasma is contributed by the secretory fluids
produced in the accessory organs like epididymis, prostate gland, seminal vesicles and
Cowper’s gland.
A) Macroscopic and Physical Tests:-
i) Volume (cc)
Bull=5-8
Stallion=100
Boar=200
Cock= 0.6
ii) Colour:-
Yellow= Pus and Urine
Pinkish or reddish= Admixture of fresh blood
Deep Red and Brownish= Degenerative blood tissue
Greenish= Purulent Degeneration
iii) Consistency and cloudiness:- gives an indication of colour and consistency.
Thick creamy= Excellent
Thin creamy= very good
Thick milky= good
Think milky= fair
Watery= extremely poor.
iv) Osmotic Pressure:-
Determinations of the osmotic pressure in terms of freezing point depression
(in Centigrade)
Bull= 0.54-0.73
Ram=0.55 to 0.70
Stallion= 0.58-0.62

Specific gravity of sperm cells is due to highly condensed nuclear and

protoplasmic protein whereas the specific gravity of seminal plasma is
because of actual osmotic pressure exerted by electrolytes and is thus
related to depression in freezing point.
 v) Specific gravity:-
The average specific gravity of whole semen in
a) Man=1.028
b) Dog=1.011
c) Bull=1.035
Fluctuations arise due to variable ration between sperm and seminal
plasma.
Seminal plasma in bull is so much lighter than the spermatozoa (1.240-
1.334), therefore specific gravity of semen is found to be directly
proportional to sperm concentration.
In case of bull, if specific gravity is low it means sperm concentration is
low and of poor quality whereas high values indicate good density and
good quality.
vi) Electro-conductivity:-
Spermatozoa has a small and directly opposite charges at head and tail. Its
magnitude depends largely on the concentration of various positively and
negatively charged ions in the surrounding medium.
Values of electro-conductivity at 25ºC expressed in reciprocal ohms x 10 -4=
a) Bull=89.5-116.3
b) Ram=48.5-80.5
c) Stallion=111.3-129.5

B) MICROSCOPIC TESTS:-
i) Counting of sperms(by Heamacytometer):- page 303

ii) Motility of spermatozoa:-

0=No motility
+ = less than 20 % of the sperms showing progressive motion
++ = 20-40% showing progressive movement but no wave.
+++ = 44-60% showing progressive movement with slow wave
++++ =60-80% showing progressive movement with wave more intense.
+++++ =80-100% showing progressive movement with rapid waves
As a rule +++ or more are recommended for A.I purposes.
The movement of sperms may be
a) Progressive or rapid
b) Rotatory
c) Oscillatory.
iii) Live and dead count:-
One drop of semen in mixed with 2 drops of 50% Eosin solution in distilled
water, one drop of 10% Nigrosin added and mixed.(semen and stain should be
at same temp.)
Live spermatozoa= appear unstained
Dead spermatozoa= stained pink against brownish purple background
 iv) Morphological abnormalities:-
The sperms may be abnormal w.r.t their
a) Head-Micro-head; Mega-head; altered shape including pear-shaped head;
double head; detached galea capitis; abnormal staining reactions.
b) Neck-Rupture or absence of attachment to head; fixation misplaced to one
side; presence of protoplasmic droplet.
c) Middle piece-Enlarged, narrowed, adherent protoplasmic droplet coiled at
anterior end.
d) Tail-coiled at anterior end, split, broken at junction with mid-piece,
looping.
In normal bull semen %age of abnormal sperms should not exceed 15-20%.
The presence of 2-3% of spermatozoa with proximal protoplasmic droplets is considered
abnormal.
The no. of abnormal sperms are influenced by various reasons:-
a) Season of the year-high in the rains during May and June, with young bulls it is
higher in summer but in old one it is higher in winter.
b) Previous coitus or collection-when the sexual rest between the ejaculations is long
there will be an abnormal number.
c) Methods of collection-minimum with AV and maximum with Sponge method.

C) Chemical Tests:-
a) Fructolysis:- the main anaerobic source of energy in bull and ram semen in
fructose.(discovered by Mann).
Normally fructose is metabolized by active cells anaerobically when incubated at
37ºC. Fructose is not utilized by either azoospermic(devoid of sperm) or
necrospermic (immotile) semen. The rate of fructolysis is thus used to distinguish
normal from subnormal activity.
The anaerobic incubation of freshly ejaculated semen is accompanied by a
gradual decline in content of fructose with simultaneous accumulation of Lactic
Acid.
Index of fructolysis: - photometric method for measurement of sperm fructolysis.
Defined as amount of fructose (in mg) utilized by 10-9 spermatozoa in one hour at
37 ºC.
In Bull the index of fructolysis is about 1-2, and is correlated with both the
concentration and motility of spermatozoa.
b) Respiratory Coefficient:-
Walton first suggested measurement of oxygen uptake in bull semen as a method
for the appraisal of semen quality.
In the presence of oxygen, semen shows a considerable respiratory activity which
is correlated both with concentration and motility of spermatozoa.
Sperm respiration is expressed in terms of ZO2, a coefficient introduced by
Redenz to denote µlO2 taken up by 108 sperm cells during one hour at 37 ºC.
ZO2 values reported by Lardy and Philips are for Bull semen-21; Ram-22; Rabbit-
11; and cock-7
 c) Methylene Blue reduction test:-
This test is made by mixing 0.2cc of semen with 0.5cc of egg yolk citrate dilutor
and then adding to this 0.1cc of methylene blue solution( 50mg in 100cc of
sodium citrate dilutor). The mixture is then placed in a water bath at 38 ºC. A
control blue with methylene blue is also kept for comparison.
A good quality semen should reduce the colour in 3.5 to 6 minutes, while the poor
quality semen may take more time to reduce.
d) Hydrogen ion concentration.
The reaction of freshly ejaculated semen may become alkaline at first, unless
precautions are taken to prevent loss of CO2.
Specimens containing fructose and high concentration of spermatozoa there is a
rapid decrease of pH owing to fructolysis and accumulation of lactic acid.
Excessive initial alkalinity of semen in some species, notably in bulls and rams,
often accompanies low fertility ,the alkaline reaction being associated with
absence or low concentration of sperm and with a correspondingly higher
proportion of seminal plasma.
D) BACTERIOLOGICAL TESTS:-
Diptheroids are predominant organism found in semen followed by Staphylococci.
Pseudomonas araginosa and coliform organisms were present occasionally.
E. Coli –agglutination of bull spermatozoa.
Bacterial count from artificial vagina ranged from 100 to 960,000 organisms per ml.
Generally plate count is first done and afterwards where necessary the differential
counts are made only to know the type of organism in the sample.

Dilution of semen:-
Always add diluent to semen.
Helps to increase volume so a larger no. of females can be mated.
The agents that comprise good extending media have following functions:
1. Provide nutrient as a source of energy.
2. Provide lipoproteins and/or lecithin to protect sperm against low-temp. shock
3. Provide a buffer to prevent harmful shifts in pH as lactic acid is formed.
4. Maintain the proper osmotic pressure and electrolyte balance.
5. Inhibit bacterial growth.
6. Provide a source of reducing substances to protect the sulfhydryl containing cellular
enzymes.
7. Provide a buffer for a proper balance of mineral elements essential to the life of sperm
cells.
8. Increase the volume of pure semen so that it can be used for multiple inseminations.
Functions of common extender ingredients:-
1. Glucose: Provides Energy.
2. Egg yolk and Milk:- protect against cold shock of the sperm cells as they are cooled
from body temperature to 5ºC and by providing lecithin, proteins, lipoproteins and
similar compounds found in egg yolk or milk. These substances also contain nutrients
utilized by sperm.
 3. Buffers:- used for maintain neutral pH and osmotic pressure of approximately 300 milli-
osmoles, which is equivalent to that of semen, blood plasma and milk. This has resulted
in number of diluents for various species.
4. Antibiotics:- Penicillin, Streptomycin are polymycin B used.
Procaine Penicillin is toxic to spermatozoa.
Addition of glycerol reduces the efficacy of antibiotics.
Antibiotic will not completely eliminates C. pyogenes, Brucella, T. foetus,
Mycobacterium, Rickettsiae and Fungi, hence addition of antibiotics is not an extra
precaution and cannot be taken as an effective tool to permit use of semen from infected
bulls or unhygienic semen processings.
5. Glycerol:- Most widely used cryoprotective agent for bull spermatozoa particularly
for frozen semen. The possible modes of action proposed are :-
a) Modifies size and shape of ice crystals formed.
b) Binds water and decrease freezing point of solution and less ice is formed.
c) Prevents denaturation of proteins and rupture of plasma membrane.
d) Reduces the soluble concentrations.
6. Fructose:- provides glycosable substrate for the sperm, prevents sperm agglutination,
maintains required osmotic tension and electrolyte balance and gives added
cryoprotection during deep freezing.
Composition of Extenders:-
While preparing diluents either for liquid/chilled semen or for frozen , the following basic
principles should be borne in mind:
1. Accurate weighting of various recommended ingredients of the dilutor to provide exact
osmotic tension, pH and electrolyte levels.
2. Exclusion of toxic materials and simultaneous inclusion of protective components.
3. Rigid control on pathogenic contamination.
4. Decide dilution rate so as to provide optimum number of viable spermatozoa per dose at
the time of insemination
5. Maintain absolute sanitation of the room, technician, all metal and glass equipment and
use quality reagents.

Dilutors for Bull

Whatever may be diluents, always add diluents to semen, never the reverse, as this may cause
shock to the spermatozoa and reduce their motility.
A. Milk or skim milk based
B. Egg-yolk based
C. Coconut milk based
D. Other commercial diluents.
A.Milk Based Dilutors
The foremost objection to whole milk as dilutor is that the fat globules make microscopic
examination difficult.
Use of skim milk or even homogenized milk or milk of goats ,which is naturally homogenized
can partially solve the problem.
Fresh milk has got lactenin, an antibacterial substance present in milk albumin. It also exerts
effect on spermatozoa. Therefore fresh milk if heated to about 92-95 ºC for 10 minutes ,its sperm
killing ability reduces to a great extent.
B.Egg-yolk Dilutors:-
Egg-yolk citrate is one of the commonly used semen extender. Only the yolk portion is used
because egg white contains a substance(lysozyme) toxic to spermatozoa.
The eggs used to provide the yolk should not be more than 4 to 5 days old.
The egg-yolk-tris-fructose-citrate diluent should be freshly prepared on each day of before use.
Tris Buffer Diluent:- It is used as common extender for frozen semen.
C.Cocconut Milk Based Dilutors:-
D.Other Commercial Diluents:-
Recently at NDRI Karnal Haryana, a diluent named as ‘Citrate Acid Whey’ (CAW) at pH 6.8
found to be best diluent for preservation of buffalo semen.

Dilutors for Sheep and Goat: -

The diluents commonly used for dilution or ram semen (i) tris, or (ii) citrate as buffers, glucose
and fructose as the energy source and egg yolk to protect the sperm cell membrane against cold
shock.
These diluents are also used for goat semen, but with a reduced egg yolk content to avoid a
reaction which occurs due to the egg yolk coagulating enzyme present in the seminal plasma of
goat bucks and it is higher when semen is collected by electro-ejaculation. This problem can be
overcome by one of the following methods: - i) using a low concentration of egg yolk in diluent,
ii)using a medium containing no egg yolk (for example milk) or iii) removal of seminal plasma,
and thereby the enzyme, be centrifugation.
Two most common recommended diluents are;-
Egg-yolk-tris-fructose diluent and ii) Egg-yolk-glucose-citrate diluent.

Dilutor for stallion:-

It is very much in practice to use undiluted stallion semen for insemination of mares when the
number of mares to a stallion is very limited. When large numbers of mares are assigned to one
stallion, the semen has to be diluted and extended for use either for a single or repeated
inseminations.
On account of sensitivity, the dilutor should be added to semen at body temperature by slow
rotating motions as soon as collection is obtained. Before this operation viscous fraction is
separated from sperm bearing fraction.
Diluted semen is held in water bath at 20-27 ºC and is used for inseminations within a few hours
of extension. When egg yolk dilutor is used the cooling process can be very rapid.
The egg-yolk is freed of albumin membrane and chalazae. The dilution rate varied from 1:3 to
1:5. Fairly good results are obtained even after 48 hours storage and long transport.

Insemination Techniques:-

Isemination in cow:- 1. Speculum method 2. Recto-vaginal Method

In speculum method semen is deposited in cervix.
Recto-vaginal method:- is widely used. By this technique, it is possible to carry out intra-uterine
an insemination which is not possible by the speculum method.
A cow usually remains in heat for 12-24hrs and should be inseminated between 4-6 hours before
the end of estrus. Cows first seen in estrus in the morning may be inseminated in the afternoon of
the day; those first observed in estrus in the afternoon should be inseminated in the next
morning.
The semen is deposited either deep in the cervix or at beginning of the body of uterus. Too deep
deposition in the uterine horns or deposition inside the beginning of cervix or in vagina should be
avoided for better performances.
Insemination in Buffalo:-
The estrus cycle is about 21 days and ‘standing’ estrus is usually less than 24 hours. Estrus
commences towards late evening with peak sexual activity during the night and early morning
hours. Matings are noted less frequently during day light. Ovulation is spontaneous and usually
occurs 15-18 hours after the end of estrus. Clear signs of estrus are not as pronounced in
cattle.
Heterosexual behavior or standing to be mounted by a male is the most reliable sign of
estrus in the buffalo because homosexual behavior or standing to be mounted by other females
is observed only occasionally.
Most insemination are performed between 12 and 14 hours from the onset of estrus (the duration
of estrus is 32 hours on an average)

Insemination in sheep:- sheep are seasonally polyestrus, having three main mating seasons
viz. March to April or summer; June to July or autumn ; and September to October or
post monsoon. Two commonly used methods are i) inseminating pit ii) holding over a rail.
The use of inseminating crate is now obsolete due to many obvious reasons.
The volume of insemination is very important, for example, insemination of more than 0.2ml
of semen into the cervix of the ewe or doe is of no advantage as the semen would flow back into
the vagina. Recommended inseminate volumes for sheep and goat are the following: -
Vaginal Insemination0.30-0.50ml
Cervical Insemination0.05-0.20ml
Intrauterine Insemination 0.05-0.10ml.
These volumes should contain at least the recommended minimum no. of motile spermatozoa.
For sheep and goat many more spermatozoa are needed than for insemination of cow.
As a general rule, less spermatozoa are required for uterine than for cervical insemination
and less for cervical than vaginal inseminations. The minimum safe limit for the no. of
motile spermatozoa per inseminate is given below: -
Type of semen
Fresh Liquid-stored Frozen-stored
Vaginal Insemination 300 Not effective Not effective
Cervical Insemination 100 150 180
Intrauterine (goat only) 60 60 65

Insemination in pigs
Young females exhibit estrus for the first time at about 6 months, but because of small litters
resulting from matings during the first few heats, they are not generally served until they are
about 8 months old.
Insemination in horses
In india, early spring is considered the best breeding season for horses. There is less mortality in
spring foals as compared to winter foals due to warm weather.
It has been estimated for optimum fertility between 1 and 2 billion living sperms should enter the
uterus and volume of the semen should be 10-50 ml according to the size of mare.
Without dilution 4 mares can be inseminated from the same ejaculate.