Artificial insemination

Definition

• Artificial insemination is when sperm is placed into a female's vagina (intravaginal), or cervix (intracervical) or uterus (intrauterine) using artificial means rather than by natural copulation.

History of Artificial Insemination

• Leeuwenhook - 1677
• Used microscope to see sperm
• Spallenzani - 1780
• Sperm could fertilize
• Claim he produced puppies with AI
• Ivanov (Russia) - 1900
• Developed methods as we know today
• In 1922, he successfully inseminate
• Most work was with horses but did some cattle and pig work
• Artificial vagina is created in 1914 by Amanta
• Denmark - 1933
• First dairy cooperative

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• First US dairy Cooperative - 1937
• First US dairy cooperative in New Jersey
• Dairy cooperatives increase in numbers - 1940’s and 1950’s
• Dairy cooperatives merge and form large companies that dominate cattle AI industry - 1960’s to present
• All turkey’s bred AI - 1960’s to present
• Expansion of swine AI - 1990’s
• Expansion of horse AI - 1990’s
• How about in Ethiopia?
• Around 1967
• Around 120,000 animals are produced (2003)

Purpose of Artificial Insemination?

• Genetic improvement of livestock
• Disease control mechanism
• Possible to increase fertility
• Decrease breeding expense

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Advantages of AI

• Genetic Improvement
• Wide spread use of genetically superior sires
• 1 bull can breed 500,000 cows in a lifetime
• After death, semen can be used
• Oldest frozen semen 40 - 45 years old
• Rapid proof of sire
• Facilitate progeny testing and examines offspring for desired traits
• With natural mating would only have 100’s of offspring
• Availability of sires
• Sires anywhere in world (national level improvement is possible)

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• Reduce danger of bull (male) & and allow to employ low performing bulls (lamed but high performance)
• Reduce the risk of transfer of venereal disease
• Permit cross breeding to change the genetic character
• Reduce the importance of buying many sires
• Improved management
• Availability and possibility of keeping records
• Economics
• Cost of very good sire reduced because extend semen
• Cost to maintain sire’s reduced as don’t need as many to breed all the females
• Possibility of gender control by separating male and female containing spermatozoa
• Provide useful research materials for researchers

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Limitations

• Estrus detection must be good
• There is a need of trained inseminator
• Problems related with availability of required facilities of bull and facilities for collection, dilution, preservation, storage and transportation of semen
• Problems related with long time preservation
• Bull semen the best, other species not as good
• decrease in genetic variability
• Possible dissemination of hereditary defects or uncontrolled (or unknown) diseases
• an increase in the inbreeding coefficient affecting the maternal traits which are particularly sensitive

Processes of artificial insemination

• Collection of semen: see breeding soundness examination
• Evaluation: see breeding soundness examination
• Extension or dilution
• Cryopreservation
• Insemination

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DILUTION/ EXTENSION OF SEMEN

• The ejaculates of most domestic animals contain more sperm than are needed for achieving a pregnancy. Hence, by diluting the semen, it can potentially be used for several inseminations
• Purposes of diluents:
• To preserve the fertilizing capabilities of sperm
• To increase the volume of water

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• The major properties of a semen diluents are:
• Addition of volume: Insemination doses must be prepared in a volume, which is a compromise between ease of handling and an appropriate volume for the site of insemination.
• Buffers: Spermatozoa have a narrow range of tolerance to changes in pH, so provision of buffering capacity is necessary.
• Maintenance of osmotic pressure: Seminal plasma has an osmotic pressure of 285 mOsm
• Energy substrate: Most diluents make some provision of energy substrates for sperm.
• Antimicrobial activity: Antibiotics are added to most semen diluents as a prophylactic measure against the transmission of pathogenic bacteria and to reduce the load of non-pathogenic organisms that contaminate the semen

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Characteristics of good diluents(extender)

• Easily available
• Cheap
• pH equal to semen
• Osmolarity near to semen
• Free from microbial contamination
• Facilitate semen examination through microscope
• Contain balanced nutrients and minerals
• Provide maximum dilution rate

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Importance of diluents/ extenders

• Dilution
• double-distilled water or UHT milk
• protection against cold shock
• Egg yolk (usually around 10% by volume) is commonly used to minimize this. Milk, or a combination of egg yolk and milk, may also be used.
• Cryoprotection
• glycerol
• energy source
• Fructose
• Buffering
• Citrate, phosphate and Tris buffers
• maintenance of osmotic pressure
• sugar
• inhibition of bacteria
• Antibiotics

Semen Extenders

Components Function

1. Egg Yolk Provides a source of nutrients

Buttermilk

Glucose

2. Na Citrate Stabilizes pH

Na Phosphate

3. Glycerol Protects against temperature shock

during cooling and warming

4. Antibiotics Controls metabolic and bacterial activity

(Penicillin, Streptomycin, etc.)

• Diluents/ extenders for refrigerated semen
1. Eggyolk-phosphate diluter
2. Eggyolk citrate diluter
3. Skimmed milk-eggyolk diluter
4. Coconut-milk extender
• Diluents/ extenders for frozen semen
• Tris diluter
• Eggyolk diluter
• Milk diluter
• Eggyolk citrate diluter

Procedure for dilution

• Preserve the semen in buffered diluents at the same temperature, pH, osmolarity
• Dilution shall be made by taking precautions to prevent damages of spermatozoa from agitation, light, radiation, temperature
• frozen or liquid semen can be stored at 5^oC in refrigerator

• Unfrozen semen
• Collect the semen
• Transfer the semen sample tube in water bath (30-34^oC)
• Examine the semen for motility, number, morphology
• Initially dilute 1:5 ratio in a flask
• Transfer the flask in a beaker with warm water
• Transfer the beaker with flask containing semen to the refrigerator at 5^oC
• Keep it there for 1 - 1:30hr to reach the flask temperature to 5^oC
• Dilute the semen finally to 1:10 ratio
• Transfer it in semen ampule, seal it, label it
• Keep it there until use

• Frozen semen (cryo-preservation)
• Collect the semen
• Transfer the semen sample tube in water bath (32-37^oC)
• Examine the semen for motility, number, morphology
• Initially dilute to 1:5 ratio in a flask
• Transfer the flask in a beaker with warm water
• Transfer the beaker with flask containing semen to the refrigerator at 5^oC
• Keep it there for 1 to 1:30hr to reach the flask temperature to 5^oC
• Dilute the semen by using 50% the total diluent
• Keep the semen at 5^oC for 4-5hr
• Mix the remaining 50% diluents and with 15% glycerol, and add it to the initially 50% diluted semen
• Transfer it in semen in to either glass slide, polyvinyl chloride straw or form pellete: then seal it, label it
• Preservation

Preservation methods

• Dry ice and alcohol
• Vacuum jar
• Dry ice storage chest
• Mechanical refrigerator
• Liquid air and CO2
• Liquid nitrogen (-179oC)

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Insemination

Equipment Necessary to Artificial Insemination

• Liquid nitrogen refrigerator,
• Pipettes,
• Syringes or poly bulbs
• The sheaths go with the french gun
• A thaw box
• A thermos (there are special thermos' to thaw straws in warm water)
• Plastic gloves
• A pair of scissors or toe nail clippers and scribe
• Paper towels
• Lubricant
• Disinfectant
• A kit to put them all in
• SELF CONFIDENCE

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AI Method

1. Speculum

• Insert plastic speculum to reproductive tract as far as cervix
• Deposit semen in uterus via small gun with syringe on end
• It is generally used on ewes.
• Used in sows, but not effective as recto-vaginal method

2. Rectovaginal

• Insert hand to rectum & locates cervix via wall of rectum
• Insert inseminating gun & guide to pass the cervix.
• Semen is deposited in the uterus.
• Rectovaginal method is generally used with cows.

3. Artificial Penis

• Artificial penis is inserted into the reproductive tract.
• Deposit semen in uterus once the penis passes the cervix.
• The artificial penis is used with sows.

AI in different species of animals

A. Cattle

• Collection: AV and electro-ejaculation
• Extension: either egg yolk or skimmed milk
• Preservation: most semen is cryopreserved, although some is used after simple extension and chilling to 4°C.
• Dose: Ability to perform an intrauterine insemination in cattle means that a relatively low dose of sperm is required to achieve acceptable pregnancy rates.
• Frozen method, typically, 20–30 million sperm that are required in each insemination dose, 6–7 million survive freezing

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• Insemination
• Time of insemination:
• Cows ovulate at 12 hours after the end of the estrus period.
• Ideal time for insemination is 6–24 hour prior to ovulation
• Optimum insemination times achieved in practice are on the same day (morning or afternoon) where oestrus is first observed in the morning, or on the morning of the next day, where oestrus is first observed in the afternoon
• Method of insemination: recto-vaginal method
• Site of deposition:
• inseminate just into the short uterine body.
• Insemination into the cervix produces a lower fertilization rate

• Standard technique of insemination
• Remove the straw from the container
• Thaw by using hot water at 37^oC for 5min
• Insert the straw in to the catheter
• Grasp the cervix through rectum with the left hand.
• A catheter, into tip of which a paillette of semen has been inserted is then passed into the vagina and manipulated into and through the cervix by the right hand.
• Catheter is initially inserted pointing upwards at an angle of about 30° to avoid entering urethral meatus or fossa, and then moved horizontally until it engages in external os of the cervix

B. Sheep

• Sheep is less amenable to AI than the cow, since oestrus cannot readily be detected in the absence of rams
• Insemination is less straightforward and ovine semen is less easy to freeze than bovine semen.
• Most important limitation in the method of insemination, since it is difficult to achieve an intrauterine insemination because the cervical canal of the ewe is so tortuous
• Collection: AV, Electro-ejaculation, siphoning or spooning
• Preservation: Cryo-preservation is not common because;
1. The sperm couldn’t withstand the chilling temperature
2. The cervix is better suited for fresh semen than the frozen once.

• Mostly fresh semen is used for insemination either diluted or as a raw
• Diluents that are in routine use for unfrozen semen include buffers that contain glucose, egg yolk–citrate or egg yolk–phosphate solutions, or heat-treated cows’ milk
• After dilution, the semen is cooled and stored at either +15°C or +4°C until used.
• The semen has to be used within 8 hours of collection, as fertility declines substantially after this time

• Insemination
• Methods: intravaginal, intracervical, transcervical, intrauterine, laparoscopic intrauterine
• Vaginal insemination deposits semen into the cranial part of the vagina, without attempting to locate the cervix.
• The requirements of this method, in terms of both technical proficiency and handling facilities for the sheep, are minimal
• this method requires large numbers of spermatozoa per insemination and is not really amenable for use with stored semen
• The ideal timing of insemination is before ovulation, i.e. 12–18 hours after the onset of oestrus

• Intracervical insemination is best achieved with the hindquarters of the ewe elevated.
• After cleaning of the perineum, the vagina is opened with a duck-billed speculum and the cervix located
• Insemination catheter is then inserted as far as into the cervix. Penetration of cervix is typically 0–2 cm.
• Conception rate is highly correlated with depth of penetration &, hence inseminator technical proficiency
• Ideal time for insemination is 15–17 hours after the onset of detected oestrus
• Direct intrauterine, laparoscopic insemination was developed to overcome many of the difficulties of intravaginal and intracervical insemination. In this method, ewes are restrained in a cradle and laparoscopy is performed close to the udder, whereupon the uterus is located and semen injected into the uterine lumen

3. Goats

• Similar with sheep.
• It is much easier to achieve an intrauterine insemination via the cervix of the goat than in the ewe.
• Moreover, conception rates to frozen semen tend to be much higher in the goat than in the sheep
• Direct insemination, caprine semen can be extended in a skimmed milk–glucose diluent or, after removal of seminal plasma, in an egg yolk–citrate or egg yolk–tris–fructose diluent
• Cryopreservation of goat semen differs from that of the ram in one important aspect: that is seminal plasma must be removed before dilution in media that contain egg yolk.

• As a diluent/extender, either been based upon skimmed milk, in which the sperm survive adequately, or the seminal plasma has been removed before using egg yolk-based diluents.
• It is because of the reason that phospholipase that is secreted by the bulbourethral gland coagulates egg yolk media and hydrolyses lecithin to fatty acids and spermicidal lysolecithins
• Where yolk-based diluents are to be used, washing the spermatozoa is achieved by dilution in Krebs–Ringer phosphate solution and centrifugation.

• Swine
• AV
• Dummy sow method
• Boar semen has three fractions
• Pre-sperm – low sperm concentration, translucent
• Sperm rich – whitish/milky
• Post-sperm - translucent
• Dummy-sow method
• Since there is a presence of cork screw penis and ridge cervix, pressure is more important that heat for ejaculation
• Allow the boar to mount the sow
• Once the penis is protruded, the semen collector hold and grip the head of the penis in his hand.
• This gives the boar a feeling that the penis is in the cervix
• Collect only sperm rich component

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• Dogs
• AV
• Gloved hand method
• Syringe
• Syringe
• Allow the male to mount the bitch
• Hold erected penis at the bulb of the glans with mild pressure
• Collect the semen using syringe after removing the piston

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