Course : FAPH 413 (II) Farm animal reproduction and obstetrics II (Male
reproduction and artificial insemination) Sem. 07, 30 h.
Module II: Semen evaluation, Processing and Artificial Insemination
techniques
Year of study: 04
Lecture: 02
Artificial Insemination in cattle
Dr. Basil Alexander, PhD.
Department of Farm Animal Production,
Faculty of Veterinary Medicine and Animal Science,
University of Peradeniya, Peradeniya, Sri Lanka
Email : pabda@pdn.ac.lk; phone: 081-239 5927
Artificial Insemination technique (AI) in cattle
Artificial insemination (AI) was first tried in 1800s, but its widespread use started
by 1950s after the invention of successful semen freezing techniques. In Sri
Lanka the first AI was carried out by Mahamooth in 1938 and its field application
commenced by 1951 (Mahamooth, 1955).
Advantages and disadvantages of AI
Advantages
1. It is a relatively inexpensive procedure
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2. Increases the production: AI on dairy cattle increases their production,
usually 10-16 kg more fat and protein can be obtained from these animals
compared to naturally bred dairy population.
3. Disease control: the practice of AI and the use of licensed semen
eliminate the venereal diseased from a herd. Usually these diseases are
spread by the bull in a naturally bred environment.
4. Bull availability: Farmer has a wide choice of selecting a bull within each
breed and helps in flexibility in breeding programs
5. Safety: dangerous bulls can be eliminated from the farm.
6. Better record keeping: AI practice uses better recording system and it will
help good management of the farm.
Disadvantages:
1. Careless bull selection, semen processing, storage and careless AI
practice may result in damage to the health of cows or to the herd.
2. Deep uterine insemination of a pregnant cow results in abortion
3. Some inconvenience in handling and restraining cows for AI
Semen collection and processing for AI
Usually semen is collected from bulls when they are around 12 months old. Bulls
can be used in a weekly roster to collect semen or according to the demand of
the semen of the particular bull. Most AI centers adopt a similar routine of semen
collection from bulls.
Semen collection
In AI centers, semen is collected from bulls using an artificial vagina providing a
standered environment. Live females, steers or other bulls are used as teaser
animals. Two ejaculates are collected from each bull on a collection day and
average of 4 ml of semen may be collected for one ejaculate. Each ejaculate
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contains anything between 500 and 2,000 millions of sperms per milliliter. A
small sample is taken from the ejaculated sample and diluted for analysis. The
sample to be processed should have the following criteria:
a) Motility > 50%
b) Concentration > 500 million/ml
c) Live sperm > 50%
d) Abnormal sperm heads < 20% (good bulls have 8-12% abnormal
sperm heads)
e) Proximal droplets < 4%; distal droplets < 4%
f) Tailless < 15%; singly bent tails < 8%, double bent tails < 4%;
coiled tails < 3%
g) Cell other than spermatozoa: none or very few leucocytes or
epithelial cells.
If the semen sample does not have these criteria, it will be discarded. A good
bull without any reproductive problem gives semen with progressive motility
>70% and head abnormalities below 10%.
Diluted sample is analyzed using a computerized semen analyzer. The
machine calculates the total sperm count and sperm concentration. Using
these initial concentrations, the sperm dose per semen straw will be
calculated considering the amount of semen extender to be added.
Semen processing
Collected semen is processed either deep frozen semen or liquid semen (fresh
semen/chilled semen).
1. Deep frozen semen
This is the most common technique used by most of the semen
processing centers. The technique involves the processing of semen
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using different extenders and freezing at –1960C which maintains the
semen fertility indefinitely. Semen from superior bulls can be collected and
stored for longer time. Successful AIs have been carried out using semen
stored for over 20 years, and many calves have been born. The semen
diluent is made to protect the sperm cell from damage that occurs during
freezing and thawing. It maintains pH level, osmotic pressure and supply
energy, extends the volume and inhibits bacterial growth. One of the
disadvantages of deep frozen semen is that 30-40% of sperm are killed in
the process of freezing and thawing.
During the processing step, the semen sample is diluted in five
steps using recommended diluents. For deep frozen semen some
common diluents are :
h) egg yolk – citrate –glycerol extenders
i) skimmed milk – egg yolk extenders
j) Tris-buffer based diluents (synthetic diluents)
TRILADYL (Minitube), Bioxcell (IMV technologies), and ANDROMED
(Minitube) are commercially available preparations that can be used as
synthetic diluents. TRILADYL has 1 x Triladyl concentrate + 1 x egg
yolk + 3 x Dist. Water. TRILADYL contains lactose as non-permeating
and glycerol as a permeating cryoprotectant. These two agents bind
and compete with water and reduce the freezing point of the liquid.
Egg yolk has lipoproteins and phospholipids binds to cell membranes
and provide protection against the cold and osmotic shock through the
critical temperature range. It also contains TRIS and citric acid as pH
buffers and 4 diffrent antibiotics (Tylosin, spectinomycin, gentamycin
and lincomycin).
After semen is frozen, post-freeze examination is carried out to
evaluate whether the semen can withstand the freezing and thawing
conditions. If the spermatozoa have >40% progressive forward motility,
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it is assumed that the semen is in good quality for AI. In most of the AI
centers, one semen straw contains 15 - 20 million spermatozoa as one
cow dose.
A standard daily routine of a semen-processing center should be as
follows:
1. Diluent preparation
2. Raw semen collection and evaluation
3. Extender A is added to semen 1:1 and allowed to cool to room
temperature (approximately 20 minutes)
4. Complete dilution with extender A at room temperature and
placed in 40C for 4 hours.
5. Extender B is held at 40C and added in two steps, 30% and
70% at the same temperature.
6. Fill the straws and label them
7. Place the straws on freezing racks in liquid nitrogen vapor to, --1400C for about 10 minutes. (Straws should be 5 cm above the
liquid nitrogen surface at – 1960C).
8. Place racks in liquid nitrogen at – 1960C.
9. Collect straws with gloved hand and store in goblets in liquid
nitrogen
10. Wash all the equipment for next day.
In this procedure, usually the extender is added in two fractions.
Fraction A contains no glycerol and fraction B contains 14%
glycerol. The final concentration of glycerol is 7%. The 4-hour
period cooling allows the antibiotics to work before their action is
inhibited by glycerol coming from the fraction B.
Process semen is packaged into 0.25 ml (Mini straws) or into 0.5 ml
(medium straws) and contains atleast 20 million spermatozoa per
each cow dose.
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2. Chilled semen.
Chilled semen is processed in two steps using an egg-yolk based diluents.
Processed semen is packaged in to 1.0 ml vials or ampules containing 1520 million spermatozoa and some times this dose can be reduced to 5
million. Processed chilled semen is stored at 40C in refrigerator and
transported in insulated containers at the same temperature. Usually the
fertility rate is about 2% high when chilled semen is used compared to
frozen semen. One disadvantage is that chilled semen can be used only 3
to 4 days from initial processing. Different distribution system is required
and stockpiles of semen would not build up.
When to inseminate a cow
Heat detection
Accurate heat detection is necessary for efficient reproductive management
and to maintain a 12-month calving interval. Under herd conditions, farmers
should observe their animals for heat signs at least 2-3 times a day other than
the milking and feeding times. Two hours after milking, more animals can be
seen in heat than in the shed or laneways. One or more signs can be seen in
a cow approaching heat.
1. Pre-heat signs: restlessness, separates from herd, ear movements,
attempts to mount others, clear mucus, reduced milk production and
bellowing.
2. Standing heat: stands still when mounted; other signs are clear and
copious mucus, vulva enlarged and swollen, rests head on back of
other animals, tail head roughened.
3. Post-heat (2-3 days after start of heat) : moves away when mounted,
tired and lying when others are mounting. Clear or bloody mucus on
tail or legs can be seen. Economic loss is high if heat signs are not
properly identified and due to missed heat.
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When to inseminate
Ideal time to inseminate a cow is 12 hours after onset of heat or first seen
in heat. If the cow is first seen in heat in the morning, she should be inseminated
in the afternoon of the same day. If she is first seen in heat in the afternoon or
evening, she should be inseminated in the next morning. It has also been shown
recently that if an AI technician visits a farm, and If an animal is seen in heat, she
should be inseminated at the first visit. Theoretically, the best time to inseminate
a cow lies between 6 and 18 hours after detection of heat. The average heat
duration is 18 hours and ovulation usually occurs 12 hours after the end of heat.
The egg must be fertilized within 10 h of ovulation and delay results in death of
the ovum. Sperm should spend some time in the female reproductive tract to
complete capacitation. Capacitated sperm is able to fertilize the egg.
Education of the farmer for heat detection methods, adequate feeding,
observation of cows for heat signs, identification of cows truly in heat and record
the timing of heat observation and inform the AI technician about the time of first
observation are also important in efficient cattle farming.
AI in heifers
Insemination may be sometime more difficult as the cervix is smaller in heifers
than cows. The semen to be used should help easy calving in heifers and in dairy
heifers usually jersey semen is used in first AI resulting in smaller calves and
easy birth. Heat detection in heifers is somewhat difficult and estrus
synchronization methods may have some benefit.
Factors affecting conception rates
Many factors affect the conception rate of a herd. Conception rate is the
percentage of pregnant animals resulted from AI. Semen handling and
insemination techniques directly affect the conception rates. Nutrition also plays
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a role in conception rates. The following reasons can be listed as factors
affecting conception rates in artificially bred population:
1.
Semen handling: handling of deep frozen semen is very
important in an AI program. Frozen semen should be thawed in
warm water (320c to 370C) for 30 seconds to ensure minimal
damage to the spermatozoa and ensure the semen reaches the
temperature of the warm water. Exposing the semen to sunlight,
dust, water, chemicals, sudden temperature changes or rough
handling may affect the semen quality resulting low conception
rates.
2.
Insemination technique: If the gun is withdrawn rapidly results in
semen flow back into the vagina. Passing the gun too far in the
uterus or forward or excessive movements of the AI gun inside
the uterus may damage the uterus lining. Poor hygiene resulting
in contamination of the gun and risk of infection of the uterus.
3.
Semen placement: semen should be placed correctly in the
reproductive tract. Usually one third of the semen dose is
deposited in the body of the uterus and the rest is deposited 1-2
cm back from the first point and in the cervix.
4.
Discharge and infections of the uterus: cows showing any
abnormal discharge or infection in the uterus are unlikely to
conceive.
5.
Errors in heat detection: inseminating too early or too late,
insemination of cows not in heat, inseminating the wrong cow will
decrease conception rates.
6.
Trace elements and minerals: In some areas trace elements and
minerals can affect conception rates.
Equipment required for artificial insemination
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1. Small nitrogen field tank: aluminum vacuum-insulated vessel is needed
to hold semen and liquid nitrogen
2. Canister: a removable cylinder with a mesh or solid bottom to hold
semen straws in the tank. Its long hooked handle helps find semen
straws inside the tank and allows access from the mouth of the tank.
3. Mini-goblet: it is a plastic cylinder with a sealed bottom, fits into the
canister. It can hold up to twenty-five straws emerged in liquid nitrogen.
4. Straws: each straw has enough spermatozoa to inseminate a cow. The
mini- straw is 0.25 ml and usually contains 20 million spermatozoa and
40% will be live after thawing. In medium straw (0.5 ml) the same
number of spermatozoa is available. Medium straws should be
handled in the same way as mini-straws and a separate AI gun is used
for medium straw insemination.
5. Vacuum flask for hot water and a thawing flask and thermometer
6. Gloves, tweezers, insemination guns, plastic sheaths, scissors, paper
towels, soap and record books. Inseminator requires a protective
clothing and a watch to monitor thawing time
In artificial insemination hygiene plays a major role and it prevents
spread of certain diseases among cattle. In any case contaminants
should not be introduced into the uterus of the cow, as it is a good
source of infection. Inseminators careless with hygiene can spread
diseased to cows as well as from cow to inseminator. Begin with clean
equipment and keep them clean result in good conception rates and
good AI practice.
Insemination technique
Thawing of semen:
1. Check the water temperature in the thawing flask and adjust it to
320 C - 380C. Most often, AI technicians use 350C temperature
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in the warm water and the semen straw is thawed for 20-30
seconds. Thawing semen at 400C or above will ‘cook’ the
semen. Ensure the water container is deep enough to maintain
a water level that only cover 4/5th of the straw. If the water is too
deep, and the end of the straw is broken water may enter into
the straw and kill the semen.
2. Remove the neck plug from the tank
3. Select the canister containing the type of semen required. If the
location of the semen is known previously, it helps find the
semen straw quickly avoiding unnecessary handling of other
straws. It is better to keep a semen location note or a diary with
the tank.
4. Lift the canister to the top of the frost line in the neck of the tank.
Usually the frost line is located about 50mm below the top of the
neck. Never bring the canister up to the top of the neck.
5. Select the particular straw from the mini-goblets using the
tweezers. Identify the straw clearly. Always use tweezers to hold
semen straw. It should be noted that the goblet should be full of
liquid nitrogen and the canister may be held in the neck of the
tank for up to 45 seconds before being returned to the bottom of
the canister.
6. The straw is held from its laboratory end and give a flick as this
will dislodge any liquid nitrogen that has accumulated in the
manufacture’s end of straw.
7. Place the straw in the thawing water for 30 seconds. Thawing
long time does not cause harm to the semen, but the straw
should be used within 20 min of removing from the tank. Once
thawed the semen should not be refrozen.
Loading the gun
1. Tear off approximately 20 cm of paper.
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2. Remove the straw from the thawing flask and dry it with a paper
towel. Hold the straw from laboratory end and draw It through
the paper once. Grasp the straw from the manufacture’s end
and wipe the straw. Hold the straw from manufacturer’ s end
after drying is completed.
3. Check the name of the bull recorded on the straw.
4. Remove the insemination gun from the kit.
5. Pull back the plunger for 120 to 180 mm (approximately 6
inches).
6. Holding the straw by the end, insert the manufacture’s end into
the AI gun until it stops inside the gun. There is an inbuilt stop
inside the AI gun to prevent the straw going too far.
7. Hold the loaded gun vertically at eye-level, cut the laboratory
end of the straw by scissors. Keep the cut at horizontal at all
times and 10 mm above the end of the gun.
8.
Clean the scissors and keep in the AI kit box.
9.
Select a sheath from its protective package and place it over
the barrel of the AI gun. Handle only the split end of the sheath
keeping the cow end clean.
10. Push the sheath through the plastic ring and fix it to the conical
side of the AI gun. The sheath prevents disease contamination
and holds the straw in place during semen deposition. The
sheath and the gun should be secured otherwise semen will
leak into the gun.
11. While holding the gun vertically, press the plunger of the gun
until the semen drop is just visible at the end of the gun. This
reduces the unnecessary stretching of fingers needed to handle
the gun.
12. Keep the AI gun in your mouth or keep it on the AI box,
ensuring that the cow end should not contact anything.
13. Place the paper in your back pocket.
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Inseminating the cow
1. Put the glove on your hand and wet it with water or glove lubricant.
2.
Carry the gun in your mouth ensuring the cow end does not touch any
surface or wall etc..
3.
Make the cow aware of your presence.
4.
Retrieve the paper from your pocket.
5.
Hold the tail to a side and smear lubricant across the anus of the cow.
Form a cone with the gloved fingers and insert your hand into the rectum.
Pause at this time allows the relaxation of the anus by gentle revolving
your fingers. The wide part of your hand then be inserted further without
dragging the rough dry skin surrounding the anus. Avoid rough sudden
entry.
6.
Thoroughly clean the vulva at this time using the paper towel to remove
all the dirt.
7.
Exert pressure downwards while the hand in the rectum, which allows the
lips of the vulva to wide open and presenting a clean area for the insertion
of the gun.
8.
Insert the gun cleanly between the lips of the vulva into the vagina. Take
care to avoid it going to the bladder.
9.
Gently push the gun forward in the vagina until you reach the surface of
the cervix, which has ‘grating’ feel.
10. Hold the cervix in your fingertips and maintain a forward pressure on the
gun and manipulate the cervix so that in a manner that the gun passes
through the cervix. If you are unable to hold the cervix try to pin it against
the pelvis.
11. While passing the gun forward in the cervix, locate the anterior end of the
cervix using your index finger. Gently push the gun until it reaches your
finger and do not go beyond this limit. Avoid deep penetration of the
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uterus as the gun may cause damage and possibly infection can occur in
the uterus. In the case of pregnant cow, this can cause abortion. (Usually
5% of the cows show heat although they are pregnant).
12. Gently express the semen at the body of uterus. Drop one third of the
volume in the body of uterus and the rest is deposited in the anterior 1cm
of the cervix. Take care not to withdraw the gun too back.
13. Do not spit the semen inside the uterus but gently express it inside the
uterus.
14. Pause before withdrawing the gun helps the semen to get away and
slowly withdraw the gun from the cervix. Rapid withdrawal can suck the
semen into the vagina.
15. Remove the gun slowly from the vagina.
16. Slowly withdraw the arm from the rectum of the cow.
17. Loosen the locking ring on the gun and remove the sheath.
18. Dispose the straw, sheath, glove and paper in the waste bin.
19. All the equipment should be cleaned before being returned them to the
AI box.
References:
1. Hafez ESE (Ed.) (1987), Reproduction in farm animals, 5th edition, Lea
and Febiger, USA.
2. Ian Lewis et al (Ed.) (1996). Cattle breeding technologies. Genetics
Australia, Bacchus Marsh, Australia.
3. Guidelines and recommendations for improving artificial breeding of cattle
in Africa (2003). IAEA, Working document.
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