Equine breeding program
Animal Science Department, Iowa State University
BREEDING PROCEDURES
1. Tease mare with stallion and record mares reception to advances of stallion supporting receptivity to stallion
Tease Code:
(1) Resistance – Mare is visibly resistant to stallion
(2) Indifferent – Mare is indifferent to stallion
(3) Interested – Mare is slightly interested in stallion; urinates, winks vulva
(4) Winks vulva, urinates – Mare is interested in stallion; urination, profuse vulva activity
(5) Profuse urination & vulva activity – Mare is greatly interested in stallion; profuse urination, squatting
2. Ultrasound mare to check if she is in transition, starting cycling.
IN-HAND BREEDING OR LIVE COVER
1. Tease mare in stocks. If she shows sufficient signs of estrus, prepare the mare for breeding
2. Preparation of mare for breeding
Wrap top of tail with bandage.
Wash buttocks and perineum.
After positioning in a suitable area for breeding, a twitch may be applied to the mare’s nose if necessary. A front leg may, if necessary, be held or strapped in the flexed position.
3. Lead stallion to mare at a 45-degree angle from the rear on her near side. Do not allow the stallion to charge or become unruly. Allow the stallion to sniff and touch the mare’s flanks and hindquarters, being restrained from any attempts to bite or to mount before he has achieved an erection. When stallion is ready to mount, the attendant pulls the leading rein over the mare’s back.
4. After ejaculation, allow the stallion to dismount. Remove the mare’s twitch and quickly pull her to her own near side.
ARTIFICIAL INSEMINATION
Preparation for breeding: the incubator
1. Check that the incubator is set at 37 degrees C. All equipment to be in contact with the semen should be stored within: Glass slides and cover slips, pipettes, graduated cylinders, collection bottles (labeled for each stallion), filters, sterile lube for A.V., insemination pipettes
2. Prepare extender and place in incubator.
Collecting the Stallion
A. Preparing the artificial vagina
1. Lubricate center of bladder with warm sterile lube by placing a generous amount of lube on a palpation sleeve and inserting the sleeve 2/3 of the way into the bladder, smearing the lube as you go. Leave the sleeve in the bladder to prevent the lube from drying out.
2. Fill the bladder with water. Water temperature should be 118-120 degrees F. Fill to 11-12 lbs. of pressure.
The pressure should be adjusted to suit the individual stallion. As a general rule, you should be able to insert your hand in the AV and make a fist comfortably.
3. Place the collection bottle on the end of the AV.
4. Place the leather jacket around the latex bladder.
B. Collection
1. Tease mare in stocks
2. Stallion a. Obtain erection b. Washing
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Warm water 42 o C (108 o F)
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Clean container
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Safety
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Preputial ring
 “Bean” in fossa glandis and urethral sinus
3. Tease again
4. Check artificial vagina
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Adjust pressure if necessary
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Check temperature and lubrication
5. Collection
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Allow stallion to mount phantom

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If stallion does not mount a phantom, allow stallion to mount a mare in estrus. The mare should be restrained with hobbles and a twitch.
6. Presentation of artificial vagina
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Deflect penis into artificial vagina
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Proper angle
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Resistance
7. Ejaculation
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Flagging
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Slowly lower A.V.
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Remove A.V.
8. Release water from artificial vagina
C. Semen handling & evaluation
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Remove collection bottle
Remove gel filter
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Pour semen into warm graduated cylinder
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Protect sample from sunlight at all times
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Measure and record volume, motility and concentration
D. Prepare mare for insemination
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Assemble all equipment
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Restrain mare in stocks
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Wrap tail
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Wash and rinse (3 times). Always begin washing at vulva and continue outwards (or expanding) circular pattern.
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Dry with paper towel
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Draw semen into syringe/pipette
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Put on sterile sleeve
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Apply sterile lubricant
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Introduce hand/pipette into vagina
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Place index finger into cervix & guide pipette through cervix
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Withdraw finger from cervix and hold cervix closed around pipette
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Deposit semen into the uterus by depressing syringe plunger
Cleaning Protocol
A. Artificial vagina liner washing protocol
1. Drain water from AV.
2. After the stallion has been collected, the AV liner should be placed in a nolvassan solution (3 oz. /gal of warm water) and washed.
3. Rinse the liner three times with cold tap water.
4. Rinse the liner in 70% ethyl alcohol, place paper towels in center of bladder and hang in a cabinet.
B. Glassware cleaning
1. Rinse all glassware in cold tap water prior to being washed.
2. Wash glassware in hot, soapy (Alconex) water.
3. Rinse glassware 3X in warm tap water.
4. Dry glassware.

Semen Handling & Evaluation
Semen must be evaluated as soon as possible after collection, because changes in temperature, exposure to light, and exposure to any type of chemicals, lubricants etc. can change sperm motility and adversely affect fertility.
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Remove collection bottle
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Remove gel filter
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Pour semen into warm cylinder
Parameter Normal Values
Ejaculate volume
Sperm concentration
Ejaculate volume
Total sperm per ejaculate
Progressive motility
70 ml (range 30-250 ml)
120 million/ml (range 30-600 million/ml)
70 ml (range 30-250 ml)
Typically 7-10 billion
Morphology
Gross appearance of the Ejaculate
Volume
Greater than 60%
Greater than 60% normal
Measure total gel-free volume.
Motility - Motility should be examined as soon as possible, as motility is the most influenced parameter in the semen analysis.
Gross motility
Mix the semen sample with a wooden stick, as motile sperm cells will try to swim upward and dead cells will settle to the bottom.
Place a drop of semen on a warm slide.
Do not use a cover slip and examine the cells under a 10X objective.
The motility is judged by the swirling motion of the sample.
Individual motility
Individual motility checks for the progressive movement of the sperm cells (PMS)
Dilute semen (1:20 = 0.25 ml sperm cells + 4.75 ml extender).
Place a drop of semen on a warm slide. Then place a warm cover slip on the drop.
Examine the cells under a 10X objective.
You must examine the sample quickly as the motility changes very rapidly with heat, light, and cold.
Motility is a very subjective measurement and is affected by many things, such as temperature of extender, cold, the glassware, urine, soap, prostate fluid, seminal pH, and ion composition.
Concentration – Measuring the number of spermatozoa
HRI Counterpoint
Zero-formalin solution, Add 100 ul semen, Mix, Read sample
Million/ml
Hemacytometer
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The hemacytometer is loaded with a 1:100 dilution of semen to make a 1:100 dilution,
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A hemacytometer cover slip is placed over the chambers.
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The chambers are filled
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The sample is allowed to settle,
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All the sperm heads in the middle big square (the square with 25 smaller squares (....and another 16 squares within each of these 25) within the triple lines) are counted.
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The number of sperm heads counted in a single chamber is multiplied by 10 6 to give the concentration of cells/cc.
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Both chambers of the hemacytometer should be counted and the numbers should not differ by more that 10%.
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The cover slips are expensive so do not break them or dispose of them!
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The 1:100 dilutions can be made using a Unopette diluter or a hand dilution.
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The Unopette is easier, but sometimes the cells will clump, so an accurate count cannot be made.
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If using the Unopette, first puncture the bottle with the sharp point, then draw up the semen via capillary action into the capillary tube.
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The bottle is then squeezed and the capillary tube inserted into the bottle and the semen sucked into the bottle.
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You can turn the capillary tube over and use it as an applicator to load the hemacytometer chambers.

Morphology
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Morphology is usually examined with an eosin-nigrosin stain (background stain) to highlight the cells.
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Paint a drop or line of stain on a warm slide, then using a wooden stick place a small amount of semen into the stain; the semen and stain are mixed using another slide, and then slowly push the second slide through the stain and across the first slide while pressing firmly down.
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The goal is to get a dark background, as the stain is a background stain and is not intended to stain the cells. In fact, some cells will stain red, but this makes no difference in our evaluation.
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You want the cells to be close, but not overlapping.
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The final slide should have dark and light areas that allow you to view different colored backgrounds as needed when examining the slide.
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Examine the cells under 1000X (oil) to fully assess the morphology.
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Count 100 cells and, in a practice situation, you can just differentiate normal from abnormal cells.
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Using eosin-nigrosin all the cells will appear flat, as if looking at your hand.
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A phase contrast mount is made by placing a small amount of semen in formal-buffered saline to kill and preserve the cells.
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Then place a drop of the formal-buffered saline sample onto a slide and place a cover slip over the drop.
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The phase contrast acts like a 'stain', however the cells will float by instead of lying flat.
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The cells now look like your hand from the top and side views.
Semen Extenders
Composition
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Semen extenders provide substances for the metabolic activity of the spermatozoa, buffer against changes in acidity and protect against cold shock. Glucose is the primary nutritive component of most extenders.
Depending on the particular recipe, egg yolk or milk normally provides the protective effect against cold shock.
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Most commercially available extenders rely on non-fat dry skim milk as a base.
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Antibiotics are usually added to the extender to inhibit growth of bacteria in the semen during storage.
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The antibiotic polymyxin B is not suitable for storage of semen; therefore it is not used in extenders for transported semen.
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From practical standpoint antibiotics such as ticarcillin, gentamicin or amikacin all give satisfactory results and are commonly used.
Osmolarity and acidity
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After preparing an extender, both pH and osmolarity should be checked before use. If instruments are not available to check pH and osmolarity, the extender should be tested to verify that sperm viability is maintained before the extender is used for shipping.
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Some antibiotics may significantly alter the pH of the extender and sodium bicarbonate must be added to restore it to a suitable range.
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Properly stored, the semen extender will be preserved for 3 to 6 months.
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Commercially available extenders are very easy to use and are formulated to provide the correct pH and osmolarity. They usually consist of a packet of dry ingredients and a small vial of diluent which are mixed together at the time of use. Most are available in convenient 100 to 125 ml sizes so that extender is made as needed rather than pre-made and frozen.