The mare must be monitored throughout her heat cycle for follicle growth. When ovulation is immanent (38- 44mm), mare should be checked every 6-12 hours to detect ovulation. When ovulation has been confirmed, the semen straws should be thawed, and

inseminated. (Donkey and Zebra mares ovulate between 35mm-40mm.)

DO NOT breed prior to ovulation.

Confirm the cane ID and the straw color on your Frozen Semen Work Sheet. Also check the number of straws per dose. Prior to retrieving the straws from the liquid nitrogen tank, you will need to prepare two (2) water baths. The 1^st bath should be 45° C and the 2^nd bath should be 37° C. Thaw one (1) straw at a time. The straw should be removed from the liquid nitrogen tank and immediately put into the 1^st water bath for 10 seconds. Hold the straw under the water (straw will try to float). Immediately place the straw in the 2^nd water bath for 30 seconds minimum. NEVER let any straws be exposed to outside air temperatures (raised above the frost line in the liquid nitrogen tank) for over 4 seconds unless it is being thawed. Four (4) seconds should give you ample time to retrieve the straw to be thawed. After thawing, shake the air bubble to the sealed end. DO NOT cut the wick end of the straw.

IMMEDIATELY after thawing the straws, artificially inseminate (AI) the mare. Procedure # 2 may be more practical.

Deep uterine insemination: Use ½ ml straw AI pipette. (Flexible models are available thru Minitube of America - 800-646-4882., stylet #17209/1075, pipette #17209/1275) It is important the tip of the pipette is in the uterine horn toward the ruptured follicle.

Use 8-10 ml Next Generation Basic Formula Extender, or equivalent (no antibiotics). Empty all thawed straws into the extender. Mix by inversion. DO NOT SHAKE. Use a standard equine AI pipette and a ALL PLASTIC syringe. Flush the AI tube with 4 ml of plain extender to wash all semen through the pipette.

Do not use abnormal mares.

AI all semen into the uterine horn toward the ruptured follicle. (flexible pipette)

It may be difficult to check motility using AI procedure 1. (Apply enough semen on slide for cover slip to float across entire sample.)

Do not use soap or iodine to clean mare. Use water only.

Monitor the liquid nitrogen level in your tank weekly. In the unlikely event your tank loses all of it’s liquid nitrogen, discard every straw in the tank. Keep your tank in plain view. Ice or frost should not form on outside of tank. If you suspect a tank failure, move semen to another tank immediately. A thawed straw cannot be refrozen.

Keep your liquid nitrogen tank, work sheets and these instructions in a safe place.

Do not allow liquid nitrogen tanks to come in direct contact with concrete, asphalt or another tank.

These instructions are for information purposes only, and should not be used in some States.

Tennessee code annotated 63-12-103 / 9.a. (1) Kentucky revised statutes 321.181 5.a.

If you have any questions contact Allendale Farms LLC at 706-476-5415.

Allendale Farms LLC operates in accordance with HB 347, Title 43 of the Official Code of Georgia Annotated. Ref: 43-50-44 Collection, evaluation and storage of semen. Revised 4/17/03

Revised June 15, 2005