ARTIFICIAL INSERMINATION

1. 3.0 ARTIFICIAL INSEMINATION
Definition:
A process by which sperm are collected from the male,
processed, stored and artificially introduced into the
female reproductive tract for the purpose of conception.
1) Allows for widespread use of genetically superior
sires that are too expensive to farmers to purchase.
2) Provides for faster and increased genetic
improvement in cattle for improved herd performance
and productivity.
3) Accelerated progeny testing of sires to determine
their genetic potential.
ADVANTAGES OF AI

2. 6) Allows for the elimination of dangerous dairy
bulls from the farm.
7) Reduces the number of bulls in the herd.
8) Allows to use larger and heavier bulls on
smaller animals without the danger of injury to
the females.
9) Introduction of new genetic material via
importation of semen from outside the country
(no expensive handling, quarantine, and shipping costs of live animals).
10) Frozen semen can be stored and used long
after the donor or sire is dead / injured sire
that is unable to breed naturally.
11) Allows for more efficient use of estrous
synchronization.

3. Disadvantages of AI
1. Requires well trained operators and special
equipments.
2. Requires more time than natural services.
3. Necessitates the operator to have knowledge of
anatomy and physiology of reproductive system of
animals.
4. Lower fertility (May occur in case of Improper cleaning of the
instruments and insanitary conditions).
5. Difficult preservation and transportation of semen
(Under severe climatic conditions like those prevailing in most
parts of tropics).

4. A) COLLECTION OF SEMEN
The semen collection area should be as close as
possible to the semen evaluation laboratory (not more
than 30 m).
Common methods for collecting semen are:
1. Massaging vesicular gland and ampullae.
2. Electro-ejaculation.
3. Artificial vagina (AV).

5. 1: Massage vesicular gland and ampullae Method
• This method is the simplest technique of semen collection.
• The operator (wearing long sleeve gloves) gently inserts
his lubricated hand/ fingers of forearm into the male’s
rectum, empty the feces, gently massage the seminal
vesicles/ampullae by backward and downward strokes
toward the urethra and a cloudy fluid containing
spermatozoa will be expelled.
• This method is seldom used because of poor response
(some bulls), and dirty semen collections (contamination
from the prepucial hairs).
• Commonly used to collect semen from cock, turkey and
dog.

6. 2. Electro-ejaculation method.
• Ejaculation of semen is brought about by inserting a probe or
electrode in male’s rectum stimulating nerves of the
reproductive system by gradually increasing voltage in rhythmic
fashion for a short period.
• Many types of electro-ejaculators in the market which are either
operated only on battery or battery cum-electric transistorized
circuits (battery connected to electricity).
Preparation/precaution
 The bull should be restrained in a crush.
 The ground surface of the crush should provide good footing.

7. The electro-ejaculator
1) Probe 2) betray or power supply 3) monitor
1
3
2

8. Advantages
1. Semen can be collected from males that are too young or old
or unable to mount due to weakened or injured legs.
2. No female or dummy is required for collecting the semen.
3. Less chance of bacterial contamination.
Disadvantages
a) Highly technical and needs considerable skill and practice.
b) Semen generally gets contaminated with urine.
c) Some males resist to this method and refuse collection.
d) Sciatic nerves are temporarily affected during the operation.

9. 3: Artificial vagina method.
 The artificial vagina (AV) method is most widely used today for
the collection of bull semen.
 There are different kinds of artificial vagina for different classes
of animals.
 The AV for cattle and buffaloes consists of:
a) An outer heavy rubber cylinder.
b) Inner sleeves of rubber.
c) Semen receiving cone.
d) Semen collecting vial made of glass or plastic (usually is
graduated).

10. 1
1
2
3
4
Parts of Artificial vagina: (1) Outer rubber cylinder,
(2) Inner sleeves, (3) Semen receiving cone,
(4) Semen collecting vial

11. Preparation of AV for semen collection
 The AV should be cleaned completely after each collection.
 Prior to collection the inner sleeve is put into the outer cylinder and
both the ends of inner sleeves are reflected over the cylinder forming
a watertight space between them.
 Then water at 40 –50oC is filled in the created space between the
outer cylinder and inner sleeve.
 The large end of semen receiving cone is attached over one of the
ends of this water jacketed and tightly secured with the help of rubber
bands.
 Then the semen collecting vial is attached at the smaller end of
receiving cone and is covered with a warmed insulating jacket.
 The inner sleeve is then lubricated with sterilized jelly using glass rod.
 Modem AV types have air screw along with the water screw for
blowing the air between the two layers to create the desired pressure.

12. a) Clean and disinfect the mounting floor.
b) Clean the hindquarters of the teaser/live teaser or dummy.
c) Hold the preputial area of mounting animal with your left hand
while your right hand holding the AV (The hand of the person
collecting the semen should not come into contact with the
animal’s penis. Disposable gloves should be worn by the
collector and changed for each collection).
d) Direct the protruded animal’s penis into the AV and hold it until
complete trust is attained.
e) After semen collection, the tube should be left attached to the
cone and within its sleeve until it has been removed from the
collection room to the laboratory for evaluation.
Semen collection procedure using AV:

13. • Collection of semen with the artificial vagina.
• The left hand touches only the preputial skin, not the penis itself.
f) The semen should be transferred to a water bath maintained
at 33-36oC immediately after collection.

14. Advantages of artificial vagina method
1. The ejaculate is collected in uncontaminated, natural state.
2. It is free from the extraneous secretions.
3. It is disease free; sterile conditions of the apparatus ensure
disease control.
4. The viability of the sperm is better.
5. No female is needed if dummy is used successful.
Disadvantages
1. It requires equipment and a teaser animal or dummy.
2. There is increased danger to the collector.
3. It can be very time consuming if the bull is uncooperative or
hesitant

15. B) EVALUATION OF SEMEN
Parameter for evaluation of semen
1. Visual evaluation for volume, color, consistency/density,
smell/odor and for presence of foreign material should
be made and recorded.
2. Microscopic evaluation for motility: mass activity and
individual motility is done using a phase contrast
microscope.
3. Determination of concentration; sing either Hemocytometer
or a calibrated photometer.
4. Evaluation of live /dead counts: Buffered Nigrosin eosin solution is
mixed with a drop of semen and smeared on a glass.
5. Morphological studies for head and tail abnormalities, and
acrosomal abnormalities.

16. Guide values for normal semen characteristics in the bulls:
a) Motility (moving actively forward): > 50%
b) Concentration: > 500 million /ml
c) Live sperm: > 50%
d) Abnormal sperm heads: < 20%
e) Proximal droplets: < 4 %; Distal droplets: < 4%
f) Tailless: < 15%; singly bent tails: < 8%; Double bent
tails: < 4%; Coiled tails: < 3%
g) Cells other than spermatozoa: none, or very few
leucocytes or epithelial cells.

17. C) SEMEN DILUTION / EXTENSION
• The main reason for extending (diluting) semen is to
increase the number of females serviced from one
ejaculation.
• A normal ejaculate from a dairy bull contains 5 - 10
billion sperm which can be used to inseminate 300 to
1000 cows if fully extended (15 to 20 million
spermatozoa per straw for deep frozen semen).
• Dilution rate depends on quality of the ejaculate,
number of sperm cells, percent alive and mobility.

18. Characteristics of semen extender:
1. Provide nutrient and increase the volume without
reducing sperm cell viability.
2. Provide buffer effects to semen (pH 6.7 – 7.0).
3. Have a proper osmotic pressure and balance of
ingredients.
4. Inhibit bacterial growth:- inclusion of antibiotics with a
bactericidal activity (e.g. Gentamicin, Tylosin, Lincomycin-
spectinomycin, Penicillin, Streptomycin etc in different
combinations).
5. Protect against the harmful effects of cooling/ freezing
(cold-shock). A common cryoprotectant is glycerol.

19. Diluents types and extension ratio depends on the type
of semen produced: Deep Frozen Semen (DFS), Chilled
Semen (CS) or Room Temperature Semen (RTS).
1. For DFS the recommended diluents are:
a) Egg yolk - glycerol extenders
b) Skimmed milk – glycerol extenders
c) Tris-buffer based diluents (synthetic)
2. For CS and RTS the recommended diluents are:
a) Coconut milk - glycerol extender
b) Egg yolk - citrate - glycerol extenders
c) Caprogen extender (common in New Zealand; not yet
tested under African conditions)

20. Three types of packing
1. Glass ampules; This hold 1.0ml of extended semen. The
ampules are sealed in a gas flame and placed in a bath of 95%
ethyl alcohol and controlled amount of CO2. The semen is kept
at -790C for long time by daily addition of ice.
2. Straws: two types (Mini straw- 0.25 ml & Medium straw- 0.50 ml).
The major advantage is that the sperm cells can be frozen
more uniformly, less storage space is required and few sperms
are left in the straw upon insemination.
3. Pellets. Composed of lactose (11%), distilled water (18.5%),
egg yolk (20%), glycerol (5%) and antibiotics.
≈ 0.1 - 0.2 ml of extended semen containing 12 to 30 million
spermatozoa is put directly in dipressions made in a block of
dry ice and then transferred to storage tubes and put directly
into liquid nitrogen.
D: SEMEN PACKINGS

21. E: SEMEN STORAGE AND RETRIVAL
There are three methods of storing semen:
1. Fresh liquid semen can be successfully stored for
1 to 4 days at 40 Celsius (C) or 400 Fahrenheit (F).
2. Dry ice and alcohol (- 700C or -100 degrees F)
3. Liquid nitrogen (-320 degrees F/ -1960C).

22. A cross-section diagram of liquid nitrogen tank used to store semen.

23. The semen storage tank is a large vacuum-sealed metal bottle
with an extremely efficient insulation system. Although semen
storage tanks are well constructed, they still are susceptible to
damage from mishandling.
1. Semen tanks should be kept in clean, dry, and well-ventilated.
2. Avoid excessive movement of the tank. Any abnormal
movement of the neck tube may cause an excessive swinging
motion leading to crack of the tube.
3. Protect the tank from corrosion by keeping it elevated above
concrete or wet floors (Use boards or pallets).
4. Pick a location that is safe from children and vandals (but do
not hide the tank).
5. Finally, always be watchful for a lid that is left off and for frost
or sweat on the tank. Frost indicates that the vacuum
insulation has been lost, and liquid nitrogen has been or is
evaporating rapidly.
Liquid Nitrogen Tank Management

24. • Remove the canister from its storage position to the
middle of the tank.
• Raise the canister up to the neck region of tank, bend
it up to a 45º angle, and then pull it up until you see
the desired goblet of semen.
• Pull the desired goblet, grasp the cane and raise it until
the straws are seen.
• Hold the cane and canister with one hand, with your
forceps or tweezers remove the desired straw.
• If it takes more than 10 seconds to retrieve the straw,
lower the canister back into the tank and allow to re-
cool for 20 to 30 seconds.
Retrieving and Thawing Semen

25. • Shake the straw gently to remove excess liquid
nitrogen and promptly place in 95º F (35 - 37º C) water
to thaw for 10- 60 seconds (at least 45 seconds).
• Lower the cane of semen back into the canister and
return the canister to its storage position immediately
after straw removal.
• To remove straws from bottom of goblet, hold cane
and canister in one hand while the other hand picking
the straws.
• The canister containing frozen straws should not be
brought beyond the neck of the container.

26. Frost
line

27. AI Equipment
ARTIFICIAL INSEMINATION PROCEDURE IN
COWS
1. Forceps/tweezers:- for removing straws from the tank.
2. Thermometer:- for measuring temperature of water used for
thawing straws.
3. Sharp stainless steel scissors:- for cutting straws.
4. Paper towels:- for cleaning vulva, and wiping of scissors and
straws.
5. Insemination gun:- to expel the semen in female reproductive
tract
 Has three parts: hollow catheter, plunger and O-ring.
 Three types: 0.5ml straw guns, 0.25ml straw guns, and
universal straw guns(0.25 ml and 0.5ml straws)

28. 6. Disposable plastic sheaths – for securing straws to gun using O-ring
• Two types of sheath:
a) Split sheathes are designed for use with O-ring guns.
b) Non-split sheaths for use with spiral or Kombicolor guns.
7. Disposable plastic insemination gloves:- for protection of
both inseminator and animal.
Split sheaths Non- split sheath
Plastic inserts

29. Types of AI guns
O-ring Spiral Kombicolor
stainless steel scissors
Tweezers

30. • Use tweezers or forceps to remove the desired straw from the
goblet and place it in the thaw water.
• Shake the straw gently to remove excess liquid nitrogen and
promptly place in 95º F (35- 37º C) water for 10- 60 second (at
least 45 seconds).
• Thawed straws may remain viable in thawing water only for 10
to 15 minutes.
• By using fingers remove the straw from the thawing flask and
wipe/dry it with a paper towel.
• Check the identification of the straw: Name, Number and
Breed of the bull printed on the straw. If the wrong straw has
been selected it must be discarded “Never replace a thawed
straw in liquid nitrogen”
Thawing of straw

31. • Choose a sheltered area for loading the guns and
inseminating cattle (help curb/control undesirable semen
temperature fluctuations).
• Wash hands prior to loading guns.
• Pre-warm the Insemination gun by stroking it vigorously with
your hand five or six times.
• Pull back the plunger to about 120 – 180 mm to allow for
insertion of the straw.
• Remove the straw (holding by the end) from the thaw water
and wipe it completely dry with a paper towel.
• Check to see that the straw contains semen and that a small
bubble is at the crimped end/ laboratory end (If the bubble is not
at the crimped end, gently tap the straw until the bubble moves to its
correct position).
Loading the Insemination Gun

32. • Place the manufacture’s/industry’s end (cotton plug end) of the
straw into the gun as far as it will go. It will stop at exactly the
right depth/at in-built stop.
• Wipe the scissors with a paper towel and cut the straw end of
the straw at a right angle ¼ mm below the lab seal.
• Select a plastic sheath and place it over the barrel of the gun.
• Push the sheath through the beveled centre hole of the
unlocking O-ring and firmly attach the sheath and gun together
with the O - ring.
• Inspect the straw end of the gun to ensure a proper seal
between the plastic insert and straw.
• Put the loaded AI gun in your mouth or wrap it in a paper
towel/plastic sheath cover to provide both thermal and hygienic
protection.

33. Cut straws ¼ mm below lab seal at 90º angle. Slide sheath over AI gun
Lock sheath and gun together with the o-ring Check for proper seal between straw and sheath.

34. Insemination procedure
 The recto-vaginal technique is the most commonly used
method to artificially inseminate cattle.
 Regardless of whether you are left or right handed, it is
recommended that you use your left hand in the rectum to
manipulate the reproductive tract and the right hand to
manipulate the insemination gun.
 AI steps:
1. Restrain the animal to be inseminated.
2. Wear/put on an obstetrical glove on your left hand and
lubricated it with soft obstetrical gel.
3. Raise the tail of the cow with your right hand and Place the

35. 4. Cup together the fingers of your left hand in a pointed fashion
and insert it in the rectum up to the wrist and gently massage
the rectum.
5. Gently wipe the vulva with a paper towel to remove excess
manure and debris.
6. Grasp/hold the cervix with finger tips of your left hand
(Cervix is the hard cylindrical part of female reproductive tract)
7. With your left hand press down directly on top of the vulva.
(This will spread the vulva lips allowing clear access to insert the gun
tip several inches into the vagina before contacting the vaginal walls).
8. Insert the gun between the lips of vulva into the vagina at 45 º
upward angles to avoid entering the urethral opening and
bladder. (Raise the rear end of the gun to a somewhat level position
and gently push the gun through the vagina until you reach the
surface of cervix which has a distinct gristly sensation on the gun

36. 9. When the gun reaches the anterior part of cervix, locate the
external opening of the cervix with your thumb and then push
the vagina and cervix forward and away from you to straighten
the vaginal folds (Use your palm and two fingers; third and fourth
fingers of your palpating hand to guide the gun tip to the cervical
opening).
10.With gentle forward pressure on the gun, locate the opening of
cervix and the gun will freely slide forward until it contacts the
second cervical ring, and by using the flexibility of your wrist,
twist and bend the cervix until you feel the second, and all
rings have been passed by the gun tip (when all rings of the
cervix have been cleared, the gun should slide forward freely with
little resistance. Since the uterine wall is very thin, you will once again
be able to clearly feel the insemination gun).

37. 9. Retract your gloved hand until it lies on top of the
cervix. With your index finger, locate the far
end/anterior end of the cervix. Pull back the gun until
you feel that the tip is directly underneath your finger
near the internal opening of the cervix.
10. Raise your finger and slowly push the plunger of the
gun so that drops of semen fall directly into the uterine
body. Uterine contractions will then transport
spermatozoa forward to the horns and oviducts with a
good distribution of both sides.

38. Insertion of the breeding gun into the vagina Grasp the cervix and push it forward to
straighten vaginal folds
Grasp the external opening to the cervix with
the thumb and guide the gun tip into the cervix
Use your index finger to check gun
placement then push the plunger slowly to
deposit semen into the uterine body.

39. With proper A.I. technique and gun
placement, semen will be deposited in
the uterine body and contractions will
transport spermatozoa forward to the
horns and oviducts.
In this case semen is deposited halfway
through the cervix, sperms will
encounter difficult in passing the
cervical folds.
If the gun is more than 1
inch through the cervix, all
the semen will be deposited
into only one horn.

40.  Maintaining a twelve month calving interval depends on good
heat detection and correct timing of insemination.
 The first step in successful heat detection program is
adequate identification of all animals.
 The best times to watch for heat are first thing in the morning,
before milking and feeding, early afternoon and late in the
evening after the animals are milked and have finished eating.
 The signs of heat to look for is cow to stand still while mounted
by others (The primary sign of heat)
HEAT DETECTION AND TIMING OF INSEMINATION FOR
MAXIMUM CONCEPTION

41. 1. Duration of standing estrus is approximately 8 - 16 hrs.
2. Ovulation occurs 24 - 30 hrs after the onset of estrus or 10- 12
hours after the end of estrus.
3. Life span of the sperm in the females’ reproductive system is
16 - 24 hrs. This also include the sperm capacitation period
lasting for 6 hours in cattle.
4. The fertile life of the ovulated egg is 10 – 20 hrs but the most
fertile life is 8-10 hrs for egg to develop into normal embryo
(The likelihood of embryonic death increases as the time beyond this
interval increases).
5. Viable sperm should be at the site of fertilization awaiting the
arrival of the freshly ovulated egg. Breeding either too early or
too late allows an aged sperm or an aged egg to interact at the
site of fertilization and will result in poor conception.
Important physiologic factors to consider before IA

43. FACTORS AFFECTING CONCEPTION RATE AFTER AI IN COWS
1. Improper technique associated with AI:
a) Timing of Insemination: for maximum conception rate, cow
should be bred at a time that will ensure that healthy, living
sperm are present at the site of fertilization when the
unfertilized egg arrives.
b) Site of semen deposition: semen should be deposited into
the body of the uterus.
2. Role of inseminator:
a) Poor storage, handling, thawing of semen as well as post
thawing handling of straws prior to insemination.
b) Improper cutting of straw resulting into back flow of semen
between the sheath and the straw rather than going inside
the uterus.

44. 3. Inaccurate estrus detection:
• Proper timing of insemination results in a large number of
healthy sperm being available to fertilize the egg soon after
its release.
• Too early insemination results into many sperm cells dying
/aging before the cow ovulates.
• Too late insemination results into the egg will have aged /
deteriorated and died before sperm reaches the site of
fertilization (Ampullary-isthmic junction).
4. Cow’s factors:
• Age of female animal: low conception rate in older cows.
• Level of milk production: low conception rate in high milk
producers.
• Postpartum problems such as retained placenta,
reproductive diseases and metabolic diseases (milk fever).

45. 5. Bull fertility:
• Fertility of bulls vary greatly (e.g. conditions of storage,
transport and thawing).
• One bull’s semen may last for short period in female
reproductive system while other bull’s semen last long (one
successfully fertilize if used 10 hrs after the onset of estrus whereas
another bull may fertilize the ovum only if used closer to 16 hrs after
the beginning of estrus).
6. Heat stress:
 Conception rate decline when both ambient temperatures
exceed critical range of 27 – 310C and cattle rectal
temperate rises above 390C (sign of heat stress).
7. Nutritional problems:
•Reduced energy intake
•Protein deficiency
•Mineral deficiency (Phosphorus, Selenium etc).