Mammary Biopsy Protocol

MAMMARY BIOPSY PROTOCOL

By Juan J. Loor, Ph.D., Postdoctoral Research Associate, Department of Animal Sciences, University of Illinois, Urbana

Objective: Obtain a representative sample of glandular tissue to evaluate patterns of gene expression in cows pre- and post-partum.

Procedures:

1. Cows will be mildly sedated with a 0.50 mL injection of Xyla-Ject (Xylazine; 20 mg/mL) administered via the coccygeal artery/vein. Those cows that seem more difficult to work with should receive a larger dose. We have successfully used a dose of 0.80 mL.

Approximately 10-15 minutes after administration, cows will start groaning and salivating which would indicate the sedative has taken effect. Tails will be tied and hind- legs restrained during the procedure, which should not last more than 20-30 minutes. Milk should be discarded during lactation due to presence of blood.

2. Clip a 10 cm2 area of skin at a midpoint on the right rear (or left rear) gland, wash, and sterilize three times with 70% alcohol and iodine surgical scrub.

3. Anesthetize area with a subcutaneous injection of 4 mL Lidocaine (20 mg/mL Lidocaine 2% soln.; Abbott Laboratories, North Chicago, IL). Try to form a “line block” by injecting half the dose of Lidocaine above and below the projected incision site.

4. Make a 1 cm incision with scalpel through the skin and gland capsule avoiding any large subcutaneous blood vessels.

Hemostats and sterile gauze should be available in the event a vessel is punctured. Apply pressure to the area until blood loss ceases and the clamp the wound if necessary. If bleeding persists, the cow should be kept in the chute to monitor prior to returning to the barn.

5. Mammary tissue is obtained using a 12-gauge (2.1 mm x 15.2 cm) Precision Cut biopsy needle (Becton Dickinson Co., Rutherford, NJ). However, due to small sample size (20 to 25 mg) that could be obtained with this instrument multiple penetrations of the mammary gland may be required. We have collected 250-450 mg tissue with 11-15 penetrations. Aim needle at slightly different angles to avoid going into the same site repeatedly.

6. To control bleeding, a 3-  5-cm plug of Surgicel (absorbable hemostat; Johnson & Johnson Medical Inc., Arlington, TX) should be inserted into the wound. 7. The skin incision is then closed with 11 m.m. Michel Wound clips (cat#9534503; http://www.henryschein.com/VETERINARY/default.asp) using an applicator/remover also supplied by the same manufacturer. A coating of Prodine (Phoenix Pharmaceutical, Inc., St. Joseph, MO), or any iodine ointment, should then be applied to the wound.

8. Banamine (1.1 mg/kg body weight), or another analgesic, should be given intramuscularly immediately after biopsy to alleviate post-surgical discomfort. An antibiotic also could be given at this stage if concerned about development of mastitis.

9. All cows need to be monitored post-op. Within 2 h of biopsy, lactating cows should be machine-milked and hand stripped to remove intrammamary blood clots. Cows should be hand-stripped as required at each milking over the next 4 to 7 d until all blood clots are removed.

IMPORTANT: Body temperature, feed intake, and water consumption should be monitored. Surgical staples should be removed once wound has healed. The most important thing when monitoring the udder is to ensure there are no signs of inflammation at the incision site.