EREC-AH-001v1 October 2017 Page 1 of 6

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1 EREC-AH-001v1 October 2017 Page 1 of 6 Standard Operating Procedure Edisto Research and Education Center Clemson University Section I: Title SOP ID: EREC-AH-001v1 October 2017 Title: Synchronization, Breeding, and Artificial Insemination Author(s): John Parrish, DVM, University Veterinarian; Scott Sell, EREC Cattle Farm Manager; Gillian Tuttle, EREC Cattle Farm Technician Approval Signatures: University and Attending Veterinarian / Date Facility Manager / Date Disclaimer: This SOP has been prepared and approved for sole use by the Clemson University Edisto Research and Education Center Cattle Farm and may not be applicable to other facilities. Changes to this SOP require approval by the University Veterinarian. Reviewed by Signature Date

2 EREC-AH-001v1 October 2017 Page 2 of 6 Section II: Procedure The Principal Investigator and Cattle Technicians will be responsible for coordinating the artificial insemination program and the assignment of bulls to groups of cows for natural service/clean up. Synchronization Protocols 7-Day Protocol Cows 1. Insert CIDRS according to package instructions and inject 2cc of GnRH 2. In 7 days, remove the CIDRS and inject 5cc of PG to 66 hours after CIDR removal and PG injection, perform Timed Artificial Insemination (TAI) and inject 2cc of GnRH Heifers 1. Insert CIDRS according to package instructions and inject 2 cc of GnRH 2. In 7 days, remove the CIDRS and inject 5cc of PG to 56 hours after removal of CIDRS and PG injection, perform TAI and inject 2cc of GnRH 5-Day Protocol Cows 1. Insert CIDRS according to package instructions and inject 2cc of GnRH 2. In 5 days, remove CIDRS and inject 5cc of PG. 3. In 8 hours, inject 5cc of PG intramuscularly and remove CIDRS hours after the first PG injection, perform TAI and inject 2cc of GnRH Heifer 1. Insert CIDRS according to package instructions and inject 2cc of GnRH 2. In 5 days, remove CIDRS and inject 5cc of PG. 3. In 8 hours, inject 5cc of PG intramuscularly and remove CIDRS hours after the first PG injection, perform TAI and inject 2cc of GnRH

3 EREC-AH-001v1 October 2017 Page 3 of 6 Heat Checking 1. Only trained personnel on an active animal use protocols will be allowed to heat check. 2. Heat checkers should carry pen and paper to record cows that show signs of heat. 3. Heat checking will be done at least twice per day; first thing in the morning and in the late afternoon. 4. Observe the herd for cows that stand for another cow to mount and ride (standing estrus). Record the ID numbers of cows in standing estrus. 5. Observe cows for secondary signs of estrus. a. Estrotect patches appear rubbed off b. Roughened fur on the tail head c. Vaginal mucus d. Muddy sides e. Mounting behaviors 6. Record the ID numbers of cow exhibiting secondary signs of estrus. Artificial Insemination (A.I) Cows should be assigned to a bull prior to time of breeding. 1. Artificial insemination should be done after heat checking is completed 2. Cows should be bred 12 hours after standing estrus is observed. If a cow is still exhibiting standing estrus 24 hours after breeding, re-breed. 3. Maintain records of all breedings in CattleMax. 4. Artificial insemination equipment and supplies. a. Thaw Bath. i. Plug the thaw bath in prior to heat checking to allow it to warm to temperature. b. Breeding kit. i. Keep the breeding kit clean and stocked with supplies. Procedure for thawing semen: 1. Fill the thaw unit with water and plug it in. 2. Locate the thaw unit next to the nitrogen refrigerator to reduce the time a straw may be exposed to air. In 15 seconds semen reaches the critical zone, or the temperature at which sperm damage could occur if the semen is recooled. 3. Uncap the refrigerator and quickly locate the canister that holds the straw rack needed. Lift the handle out of the index ring in the refrigerator neck, raising the canister only as high as necessary to safely pick up the desired straw rack. You should be able to locate and retrieve the appropriate straw by keeping the canister at least 1 1/4 inches from the top. If the correct rack cannot be located within 10 seconds, lower the canister into the refrigerator for seconds and then bring it back up again.

4 EREC-AH-001v1 October 2017 Page 4 of 6 4. As you lift the canister, place the handle between your first and second fingers with your palm toward the refrigerator. Hold the canister against the tube neck. 5. With the free gloved hand, reach into the neck tube to pick up the desired rack. Raise the rack only far enough to make the uppermost straws available. Grasp the rack between your thumb and forefinger of the hand holding the canister. 6. With the specially designed tweezers lift the straw straight up and out of the rack. Quickly place the straw in thaw water and, at the same time, lower the rack and the canister in its proper slot in the neck ring. 7. Recap the refrigerator. 8. For top fertility, always thaw the straws for at least 30 second but not for more than 15 minutes. Loading the syringe 1. Warm the inseminating syringe by rubbing with a clean paper towel. 2. Place 2-3 paper towels in a pocket for cleaning the cow s vulva before insemination. 3. Remove the straw from the thaw unit or the thermos after a minimum of 30 seconds in thaw water. 4. Dry the straw with a clean paper towel. This will prevent water droplets from contaminating the semen and possibly killing the sperm. 5. Double check the bull identification to be sure you have the correct sire assigned to the cow to be inseminated. Each straw is printed with the bull s registered number, breed code, AI stud number, and a four digit collection number. 6. All the air in the semen column must be at the crimped sealed end. This can be accomplished by slightly flicking your wrist while holding the straw at the crimp-sealed end. 7. Cut off the crimped-sealed straw end with a cutter or the straw scissors. Cut the straw square so the straw will seal within the sheath. 8. There are two styles of stainless steel inseminating syringes available the spiral and the O ring syringes. Each type has sheaths which are uniquely suited to its styling. These sheaths are plastic tubes that hold the semen-filled straw in place and keep the syringe clean while you are inseminating. Some sheaths have plastic adapters about an inch from the open end. They insure proper seating of the straw and also prevent semen from flowing back into the sheath. For sheaths with adapters: a) Insert cut end of the straw into adapter. Hold the sheath at the adapter with the thumb and first finger, push the straw into the adapter until you feel a snug fit. b) Pull back the syringe plunger about 5 inches. c) Place the sheath containing straw over syringe barrel, the straw goes inside the syringe barrel. d) Secure sheath by screwing onto syringe or by twisting O ring onto the end of the sheath. e) Depress plunger slowly to remove air space in outer end of straw.

5 EREC-AH-001v1 October 2017 Page 5 of 6 For sheaths without adapters: a) Pull out the syringe plunger about 5 inches. b) Insert the straw into the open end of syringe barrel, the cotton-plug end goes in first. About 1 inch of the straw should be outside the barrel. c) Pass the open end of the sheath over tip of the straw and slide it down the entire barrel of the syringe. d) Secure the sheath by twisting the O ring over the end of the sheath. e) Depress the plunger slowly to remove air space in outer end of straw. Inseminating the Cow: 1. Load the cow into a squeeze chute with a bar behind her. 2. Protect the syringe from cooler temperatures and direct sunlight. The best place to keep a loaded syringe is in the coveralls, shirt or jacket you are wearing. 3. Put a plastic shoulder-length glove on the arm you will work with in the cow. 4. Use ample AI lubricant or K-Y Jelly on the hand and arm of the glove. 5. Pick up the cow s tail and move it toward the outside of the arm that will enter the cow. Do this with the hand that does not have the glove on. Use a paper towel to avoid direct contact with manure. 6. With the gloved hand, gently rub the anal area to relax the muscles. Then, through the anal opening, insert first one, then several fingers held together. With your fingers forming a cone-shape and by pulling on the cow s tail to provide an equal and opposite force you will be able to push your hand through the anus into the rectum. 7. Once your hand is inserted wrist-deep, open your hand from the coned position, push down and palpate for the cervix. 8. After locating the cervix, determine its width, length and consistency. Then pick it up with your hand. To do this, approach the cervix from the side, sweeping the edge of your hand under it. Then, turn your hand so the fingertips extend well under the cervix. Now you should be able to lift the cervix and, at the same time, grasp it between your thumb and fingers. 9. Palpation and manipulation of the reproductive trace should be done gently to prevent damage to the rectum or the reproductive tract. 10. Wipe the area around the vulva and the inside lips with a clean paper towel. 11. Enter the tip through the vulva at a 45º angle. Pass the syringe forward several inches until it encounters the roof of the vagina then, level it out. 12. If the syringe tip gets caught in a vaginal fold, back off and redirect the syringe. It helps to push the cervix forward with the hand that is in the rectum. 13. When the syringe reaches the cervix manipulate the cervix and the syringe to get lined up to pass the syringe through the cervix. 14. Manipulation of the syringe through the vagina and the cervix should be done gently to prevent damage to the reproductive tract. Blood from damage to the reproductive tract will decrease pregnancy rates. 15. Stop the syringe at the point where the front end of the cervix joins the body of the uterus. This is the area where the gristly cervical tissue abruptly changes into softer,

6 EREC-AH-001v1 October 2017 Page 6 of 6 spongier uterine tissue. By pressing down slightly with your fingertip, you will be able to feel the syringe tip as if passes through the last cervical ring. 16. Semen should be deposited slowly. It s a good habit to inject half the semen, and then recheck to be sure you are in the correct location before depositing the rest of the semen. 17. After all semen is deposited, slowly withdraw the syringe and your arm. 18. Remove glove and plastic sheath and discard in trash. 19. Complete the breeding records immediately after the cow or heifer is inseminated.