1. Arrange dry goods, aerated water containers, and other supplies.
2. Measure or calculate the gallons of pond water in the anesthesia tub for proper initial dosage of anesthesia.
3. If desired, flush needles and syringes with heparin and recap.
4. Print Index cards with koi identification number using a broad stroke waterproof pen (broad tipped Sharpie).
5. Print identification number on sample containers (vacutainers) using waterproof pen ( fine tipped Sharpie).
6. Place fish in aerated anesthesia water with appropriately dosed amount of anesthesia. 7. Observe fish carefully watching gill retractions. Gill retractions should slow after an initial period of hyperactivity. If necessary, add additional anesthetic agent to water.
8. When adequate anesthesia has been reached (the fish has relaxed, often rolling on its side accompanied by slow gill retractions), carefully transport the fish to the aerated tub of pond water for venipuncture.
9. Roll the koi such that the ventral side (belly) is up. Immobilize the tail area. Different techniques are utilized for different sized koi.
10. Insert the needle in the midline posterior to the anal fin, directing the needle toward the central vertebral body. Personal preferences vary but my preference is to hold the syringe between my thumb and index finger when inserting the needle. The lateral line is usually at or a little deeper than the vertebral body when approaching from the ventral surface. This can be a useful guide if you have missed the vertebral body and gone too deep. Only after it “feels right”, that is the needle is felt to be in the midline and one has penetrated to the vertebral body, is the hand reposition and the plunger withdrawn to create suction for blood flow. If blood flow is not observed, suction is released and the syringe is partially withdrawn and repositioned. If one maintains suction while repositioning the needle, it is very likely the needle will become obstructed with tissue and blood can never be withdrawn. When in doubt, toss the needle and syringe and use a fresh one.
11. After the blood is obtained, swab the venipuncture site with disinfectant.
12. Slowly transfer the blood to the vacutainer or microtainer, taking precautions to minimize splashing or rapid movement of blood through the needle. Hemolysis of the fragile red cells is to be avoided. Protect the specimen from heat (ice bath, etc.).
13. Verify that the tube specimen number and the index card number agree.
14. Place the fish and the index card (with the confirmed specimen number) in the measuring container.
15. Measure the fish as it is recovering from anesthesia.
16. Photograph the fish and index card (with the specimen number up).
17. Record fish description, length, and other pertinent information.
18. Allow the fish to recover from anesthesia in aerated pond water before returning to the pond.
19. Centrifuge specimens for 10 – 15 minutes, then refrigerate or freeze as applicable.
20. Clean and disinfect all equipment as appropriate.
21. Complete paperwork for KHV serology, carefully pack specimens (with icepack) and send by overnight service to laboratory. Avoid timing shipment arrival on weekends or holidays.
22. Await results. All specimens received by the laboratory are usually placed in tissue culture at the same time each week. It takes a full week for the cells to grow, be fixed, stained and counted. Results will not be discussed by phone. They will be sent to a dedicated fax or by mail as indicated on your paperwork. If you need a “veterinarian of record” and do not have one, you may wish to check one of the web based listings for aquatic veterinarians in you area or contact Dr. Ritchie at the University of Georgia in Athens, Georgia, for suggestions.