Artificial breeding methods

Introduction

• Artificial breeding is also called Selective breeding/artificial selection it is the process by which humans breed other animals for particular traits.

• Typically, strains that are selectively bred are domesticated and the breeding is normally done by a professional breeder.

• Bred animals are known as breeds. The offspring of two pure-breed animals but of different breeds is called a crossbreed. (Jersey , Angus, Holstein, Brahman)

History• Recorded as early as the 14th century-Arabian horse breeder, who

transferred the contents of an ejaculate by means of a sponge.

• 1677-Van Leeuwenhoek(Dutch scientist), observed sperm cells in an ejaculate.

• 1780 – Spallanzani (Italian physiologist) did the 1st scientific artificial breeding with dogs and proved that the sperm fraction of the ejaculate is the component which causes fertilization.

• 1914- Ivanov and his co-workers( Russia)- developed the artificial vagina (AV) to facilitate semen collection from bulls.

• 1937- Sorensen (Danish veterinarian) developed the recto-vaginal technique of insemination (used worldwide today).

• 1949- Polge developed techniques for deep freezing of sperm for long term storage

• 1960s-Cassou (France) brought semen processing technology by designing the straw system of packaging deep frozen semen, culminating in the development of the mini-straw in 1969.

Methods of Artificial Breeding

1. Super ovulation and embryo transplantation.

2. Invitro Fertilization

3. Artificial Insemination

Artificial Insemination• Artificial Insemination(AI) is the 2nd most common practice of breeding

livestock-it's the only alternative to breeding livestock next to natural breeding using a male over females.

• Semen from a high quality pedi greed bull is selected and the cows are artificially inseminated.

• There are several mechanical devices to introduce the sperm in the vaginal tracts of the cows.

• Semen collected from bulls is usually kept frozen at temperatures upto -196°C in liquid nitrogen.

• At the appropriate time it is brought to normal temperature and used for insemination.

Semen – collection, processing & storage

• Collection- Dummy cows, electro-ejaculation (passage of a fluctuating current between electrodes on a probe which is placed in the bull’s rectum)

• Processing- About ten million normal, actively moving sperm are required to produce conception using artificial insemination. They must be placed at the correct site in the reproductive tract of a cow which is at the correct stage of the breeding cycle.

1. EVALUATION- A drop of raw sperm is observed under Low magnification to obtain an estimate of sperm concentration and activity, another drop is stained to determine the % of the spermatozoa which were alive and of normal conformation at the time of staining, another test is used to ascertain the density of the raw semen – the number of sperm per ml. A spectrophotometer measures the amount of light passing through the sample of semen diluted at a particular rate.

2. Dilution (Extension): An average ejaculate may contain 5000 million sperm in 5 ml of raw semen. Super-fertile mature bulls may yield up to 12 000 million sperm per ejaculate.

• By natural method this could only produce one calf. • Diluting the ejaculate it can be extended to give 200 or more individual

0.25 ml doses each containing 25 million live sperm. • Only 10 million sperm are required for conception, more than double

this number are placed in the straw to allow for losses during freezing. Glycerol is the cryoprotectant added to protect the sperm during the freezing process.

• Diluents may be based on skim milk, egg yolk citrate or specially prepared chemical diluents such as TRIS with egg yolk.

• Antibiotics are added as a precautionary measure and most diluents have added fructose (fruit sugar) to supply energy to the sperm.

3. PACKING:• More than 200 AB centers around the world use the Cassou or French

straw system for packing semen.• Glass ampoules were used originally but in most countries, these have

been replaced by straws.• The medium straw (0.5 ml volume) was introduced in 1969 and the

‘mini-straw’ (0.25 ml volume) in 1972.• Cassou straws are 133 mm long poly-vinyl chloride tubes. Medium straws

have a 3 mm diameter and mini-straws 2 mm. They are plugged at one end (the double plug end) with a sealing powder which is retained between two cotton plugs.

• By applying a vacuum to this end of the straw, semen can be drawn up the tube and into contact with the sealing powder. As soon as the powder becomes wet it turns into a gel to provide a very effective seal.

• Automatic straw filling and sealing machines use an ultrasonic pulse to seal the other end (the laboratory end).

4. COOLING AND FREEZING:• At body temperature sperm swim about very rapidly, exhaust their

energy reserves in a relatively short time, and die.• If the temperature is reduced, the rate of reaction is also reduced.• Cooling semen to near freezing point slows the sperm down, by

slowing their internal reactions, and extends their life for several days if they are protected by suitable chemicals.

• By further cooling, activity can be effectively stopped to give the sperm an almost indefinite life. Some of the sperm are killed in the freezing process but with correct processing, many survive and will revive on thawing.

5. QUALITY CONTROL: • 24hrs after freezing, a portion of each batch of semen is thawed

and examined under a microscope.• If the batch meets prescribed minimum standards for survival and

motility, it is packed into goblets and transferred to storage units. Periodically, straws from each batch are checked for signs of deterioration in storage.

6. STORAGE:• For ease of handling and to minimize the risk of damage to the

sperm through exposure, straws are always packed in plastic goblets. Semen should only be transferred to and from liquid nitrogen containers in goblets.

• Goblets are usually marked with the bull’s common name and the batch number.

Sperm Sexing• Offspring of choice• Females: Continuous milk production• Males: Meat production• Breeding stocks X-sperm contains 4% more DNA than Y-sperm.

Methods of sexing sperm1. Gradient swim down procedure:• An albumin gradient is used to separate X- and Y-sperm• Y-sperm is smaller in size and has greater downward swimming

velocity• Sperm (22% v) at the bottom of the• column are rich in Y-sperm.

2. Surface antigenic differences:• H-Y antigen as the marker• Sex-specific proteins (SSPs)• Non-SSPs are removed immunologically• Ab against SSPs are used to identify a specific sperm

3. Free-flow electrophoresis:• Presence of electric charge on surface of sperm• X-sperm differs from Y-sperm based on surface charge of the

sperm• An electric field is used to separate the sperm in two major classes.

4. Volumetric differences:• X-sperm is larger than Y-sperm• Difference in sperm head volume• Interference microscopy and image analysis• Flow cytometer is used to separate the sperm.

5. Centrifugal countercurrent distribution:• Aqueous two-phase system• Chromatography procedure aided with Centrifugation• Stationary: Lower phase

Flow cytometer

Stains Used• Hoechst 33342-binds to the minor groove of the DNA helix within

the cell and allows them to show up in the flow cytometer and their measurements taken.

• The identification of dead or living sperm is also done prior to sorting. The dead sperm are identified by their uptake of a type of food coloring, which is a membrane impermeant dye.

• Quinacrine stain

How to Artificially Inseminate Cows and Heifers

1. The Females are watched, then are taken In- for signs of estrus, Females go into heat once every ~21 days, and the heat periods themselves last for 24 hours.

• physiological, behavioral and physical signs of heat.

• Most heat periods start or end around dusk or dawn

2.

12 hrs after estrus is when females should be AI'd. This time period is when the female ovulates, sending an ovum out to the fallopian tube to await fertilization from sperm from a bull.

3. Calmly, with proper handling practices the heifers/cows are put in to the working alley to the squeeze chute (or alleyway with just

a head gate).

• Preparations Prior to Insemination.

 

• Prepare a water bath-34-35°C .

• Identify which canister contains the semen you need. Remove the canister from its storage location to the center of the tank. Grasp the desired cane immediately.

• Shake the straw to remove excess liquid nitrogen

(nitrogen quickly goes into a gaseous state when

Exposed to air and warmer temperatures).

Immediately place the straw in prepared

thermos of water for40-45 seconds.

Put the can back in the canister by raising the

canister up and over the cane and return the

canister to its storage position.

Keep AI gun ready by having it pre-assembled.

The AI gun should not be extremely hot nor cold

to the touch.

• Remove the straw from the thermos and wipe it dry with a paper towel. It should be completely dry before you do anything else with it. Flick your wrist slightly, while holding the crimped end, to adjust the air bubble in the straw. The flick should move the bubble to the end you are holding

• Put the straw in the rod. Clip 1/2" (or 1 cm) from the crimped end of the straw. Use sharp scissors or specially designed straw-cutter scissors should be used, and cut in the area that the air bubble is located.

Artificially Inseminating the Female Bovine

• The tail is moved and the cow is cleaned to remove any excess manure and debris from the vulva.

• The gun is unwrapped and then inserted at a 30 degree angle into the cow's vulva.

• The left hand is inserted into the rectum to check for the location of the end of the AI gun.

• The cervix is grasped with the hand in the rectum of the cow and is held steadily while the rod is thread into the cervix of the cow.

• When the rod is all the way through the cervix, the location is checked with index finger. The rod should be only ½ to ¼ of an inch into the uterus.

• Slowly the plunger is depressed at the end where the right hand is so that ½ of the straw’s contents is deposited. Recheck and deposit the remaining ½ .

Advantages Many females may be inseminated from semen from one ejaculate. 

Advantage of using a sire that is owned (and not to be sold) by someone

else that is proven to be very productive in producing highly viable

young for your herd 

Choice of using a sire of your choice according to what kind of herd/flock

you have or the individual "needs" of females to improve your herd.

Time of parturition (birth) can be synchronized in a herd to when forage

and feedstuffs are most available and the weather is good for babies to

survive.

Advantages Availability of proven sires for better calf/lamb Limits risks of females getting STDs from used sire Ability to use sexed semen to increase chance of getting offspring that is

either male or female depending on type of stock.  Limits the need to house and feed a male, since all you need is frozen

semen.  Synchronization using hormones enables a herd of females to be AI'd all

on one or two days. Livestock of good genetic stock (high milk producers, good meat

producers, fast and efficient growth, low disease potential) that otherwise could not breed due to behavioral or anatomical/physiological factors can have their genetic material reproduced and pass on their good genes.

Disadvantages

Need facilities to perform AI on livestock 

Need experience plus training for a producer to perform AI on

livestock 

If producer has no experience, need to hire an AI technician. Some

technicians are not available for certain areas. 

If producer wants to AI own herd, start-up costs are high because of

the materials and tools needed for AI are costly. 

Disadvantages The insemination may not produce live offspring .

The hormonal manipulation will cause adverse side effects in the

breeding female and shorten her potential breeding life.

Labour intensive due to need for watching herd at least twice daily

for signs of estrus activity and for the AI'ing itself. 

Risk of infection in vagina or uterus if person is forceful or doesn't

really know what they're doing. 

Conception rates of females are lowered if they are handled roughly

prior to being AI'd due to stress.

ThanK You !