Bovine Mammary Gland Biopsy Techniques

A biopsy, as I think most of you know, is the removal of a bit of tissue from the organ of interest and in this case, it's the mammary gland. The biopsy sample is very useful if you're gonna do things like gene expression analysis, if you're going to do things like to take some of the tissue and do a histological analysis, to look at the structure at the microscopic level of the mammary gland, also very useful to determine things like signaling pathways related to hormone action, for example, so one of the cautions that you need to be aware of when you do any sort of mammary tissue sampling, is have you taken a sample that's truly representative? Because, you may capture a lobule, which would be like a cluster of grapes, or you may capture some individual alveoli that really don't represent the normal, so, the hope that you have when you do your mammary biopsy is that you are collecting parenchymal tissue, which is just a fancy word to say the functional tissue responsible for the production of milk, a sample that's representative.

Most of the sensory nerve endings in the mammary gland are associated with the skin, and so it's important to realize and to appreciate that, once you have done the proper preparation of the area where you're gonna make the initial incision, to anesthetize the skin, then there's very little in the way of any sensory nerve endings associated with the parenchymal tissue itself, and so the discomfort that the animal would feel is mostly associated with the skin. So, there's a variety of things that can be done with this tissue, and it actually allows one to gather more information about how that animal works, as compared to what might be obtained from simply observing what was fed to the animal and the responses in milk protein output or milk component output. All methods described were approved by the Virginia Tech Institutional Animal Care and Use Committee.

One day before the scheduled biopsy, wash and scrub the animal, particularly, the udder, to remove manure and soiled material. Assess the health, physical condition, and behavior of the animal. Use only healthy cows.

Completely milk the cow. Move the animal into the squeeze chute, ideally within two hours of milking to minimize milk presence in the glands. Restrain the animal with a head gate.

Place a rope halter on the head of the cow to prevent backward and forward movement. Pull the animal's head to one side and tie the rope to the squeeze chute using a quick release knot to hold the head in place. Clean the area of injection with a 70%isopropyl alcohol swab and administer flunixin meglumine 1.1 to 2.2 milligrams per kilogram of body weight intravenously, via the jugular vein, 15 to 20 minutes prior to the biopsy.

Locate the jugular vein. Raise the jugular vein by application of pressure at the base of the jugular groove. Check to make sure that there are no bubbles in the syringe.

Insert the needle into the raised jugular vein and draw 0.5 milliliters of blood into the syringe twice and mix with the contents. If no blood shows in the syringe, relocate the needle. If the needle is resident in the vein, inject the contents.

Gently remove the needle, apply gauze with gentle pressure to the injection site to prevent bleeding. Administer xylazine hydrochloride intravenously, 0.01 to 0.05 milligrams per kilogram of body weight in the coccygeal vessel approximately five to 10 minutes prior to the biopsy to allow sufficient time for establishment of sedation. Raise the tail and clean the area of injection with a 70%isopropyl alcohol swab.

Check to make sure that there are no bubbles in the syringe. Insert the needle into the tail vessel, draw 0.2 milliliters of blood into the syringe, and mix with the contents to ensure that the needle is resident in the vessel. If needle is resident, inject the syringe contents.

Gently remove the needle. Apply gauze with gentle pressure to the injection site. It's really important to have an alpha two reversal on hand, like tolazoline, this is because dairy cows are sensitive to the effects of xylazine, and an overdose of xylazine can cause pulmonary edema, which could potentially be fatal for the dairy cow, so watch your cow for signs of over-sedation and if these occur, then the appropriate amount of tolazoline may be given.

We want our cows to be standing during a procedure. Too much xylazine may cause them to sit down. Have an assistant tie up the tail for the procedure, select the biopsy site on the udder, typically in an upper area to minimize collection of connective tissue and to avoid penetration into the gland cistern.

Observe and palpate the skin with special attention to identify any large subcutaneous blood vessels in order to avoid these vessels during the biopsy. Clip the hair from a 15 by 15 centimeter square area around the biopsy site, prepare the biopsy area with a povidone-iodine, 0.75 percent available iodine, or chlorhexidine gluconate scrub. Alternate the 70%isopropyl alcohol at least three times to remove all visible and invisible debris.

Apply the aseptic scrub solution and isopropyl alcohol in a circular motion, using inside-out approach. Ensure that the antiseptic scrub solution remains in contact with the skin for at least five minutes. Use a butterfly infusion set with an 18 G needle to deposit six milliliters of two percent lidocaine hydrochloride subcutaneously at the incision site to create a line block.

Do not penetrate into the deeper tissues. Allow the local anesthetic to diffuse for three to five minutes, perform another repetition of scrub solution and alcohol prior to incision. While waiting, prepare the biopsy instruments.

What you want to do in terms of providing analgesia is desensitize the skin. That's because we have more neural innervation to the skin and subcutaneous tissues, and in the parenchyma, we're really only talking about stretch receptors, and these do not feel surgical pain the same as the skin and the subcutaneous tissues do. In fact, if you end up putting local anesthetic through into the parenchyma, you may increase your risk of bleeding during the procedure.

Use aseptic techniques when handling the biopsy tools and for the incision. Wash hands to remove all visible contamination and apply sterile surgical gloves. Arrange the surgical instruments in a sterile area in order of use.

Have a number 10 scalpel, sterile gauze, and assemble the biopsy instrument and a sterile towel for homeostasis. Procedure one, core biopsy instrument. Assemble the core biopsy instrument using surgical gloves.

Place the seven sterile pieces on a sterile drape. Insert the blade into the docking system on piece two. Insert piece three on top of piece two, ensuring that the docking station will align.

Engage the final edge of the blade into the docking system of piece four. Push forward piece four and observe if piece four is next to piece three. Insert piece five on the device.

Ensure that the docking station is aligned. Insert piece six into the top of piece two. Ensure that the docking station is aligned.

Insert the locking screw into the docking station. Push forward piece three to cover the locking screw. Activate the tool, pushing forward piece four and observe the blade outside of the tool.

Pull piece four back to retract the blade into the tool. Ensure that the cow is sufficiently sedated and the biopsy site is sufficiently anesthetized, pinch the skin to ensure no reaction. Make a two to three centimeter vertical incision through the skin and subcutaneous tissues from proximal to distal using a number 10 scalpel.

Attach the biopsy instrument to a cordless drill using sterile techniques. Place the drill against the biopsy tool and check if the tool is firmly attached to the drill. Ensure adequate restraint by having an individual elevate the tail during all procedures.

Turn on the drill using clockwise rotation and a low speed. Advance the entire biopsy tool around 7.5 centimeters into the udder through the incision while the tool is rotating. Turn off the drill and manually extend piece four of the tool, turn on the drill using clockwise rotation and a low speed, remove the instrument containing the tissue core from the udder.

Apply strong pressure immediately to the wound using a sterile towel for at least 20 minutes. Insert the cannula into the tissue. Push the piece forward to activate the tool.

Rotate in a slow clockwise rotation. Tissue sample is now inside of the cannula. Remove the tissue from the biopsy tool using tweezers.

Keep the sample in a one X phosphate buffered saline and evaluate the amount of tissue. Take the cow's vital signs every 10 minutes after the biopsy for at least 30 minutes. Check for bleeding after 20 minutes of pressure on the biopsy site.

If there are drips of blood, continue to apply pressure for an additional five to 10 minutes. Close the incisions using stainless steel staples at five millimeter intervals after all bleeding has stopped. Apply an aerosol bandage to the biopsy area.

Observe the animal for 50 minutes after the procedure. Procedure two, needle biopsy instrument. Follow the manufacturer's instructions for the device.

Remove the biopsy needle from the package using sterile techniques. Discard the needle if any damage is observed. Attach the needle to the device.

Close the cover and cock the device. Ensure that the cow is sufficiently sedated and the biopsy site is sufficiently anesthetized, pinch the skin to ensure no reaction. Make a one to two centimeter vertical incision through the skin and subcutaneous tissues from proximal to distal using a number 10 scalpel.

Insert the biopsy needle into the incision site, around 10 to 13 centimeters from the skin. Activate the biopsy needle device to collect the tissue. Remove the needle from the udder.

Apply immediate strong pressure to the wound using a sterile towel for at least 20 minutes. After removing the tissue from the needle, follow the procedure described previously to close the incision. The entire procedure takes about 45 minutes to finish, per animal.

Blood clots need to be stripped out from the quarter biopsied. Also, it's necessary to observe the milk yield and the food intake, if possible. Always ensure the health and the wellbeing of the animal before, during, and after the procedure.

Document all drugs administered to the animals. Check for the presence of blood in the milk for seven to 10 days after the biopsy. Hand-strip blood clots from the biopsied quarter at subsequent milkings and ensure that complete milk removal occurs.

Observe milk yield, and if possible, the individual daily feed intake until surgical staples have been removed. Monitor the animal twice a day for body temperature, respiration and heart rates, and demeanor, until surgical staples have been removed. Check the biopsy site twice daily for swelling, tenderness, and any signs of drainage until the surgical staples have been removed.

If these are observed, consult a veterinarian. Remove the staples from the incision site 10 to 14 days after the biopsy, depending on the healing rate. Consult a veterinarian if any signs of local or systemic infection are observed.

So, as soon as the biopsy is taken, you've interrupted the blood and therefore the nutrient and oxygen supply to that tissue, and so if you aren't very quick at removing that tissue core, and somehow stopping the tissue activity, the concentrations that you might measure at a later point in time in the laboratory may not be truly reflective of what was going on in that animal at the time of the biopsy. Typically, we would rapidly remove the tissue, and flash freeze it in liquid nitrogen to try to preserve that activity. The other thing that one can do with that tissue, and this is actually something that we did do with the tissue that we collected, was one can isolate and grow the cells that are present in that tissue in a culture system.

In our case, we're interested in the epithelial cells that are within that tissue, because those are the cells that actually synthesize and secrete the milk components, and so, those cells from those tissue pieces were placed in culture, and actually, they're still growing today. The animals were observed twice a day for 10 days after the procedure. No complications occurred during the procedure or in the post operative period, with vital parameters of the cows remaining within normal limits.

The linear models show that 10 days before the biopsy, the animals produced 23.35 kilograms of milk per day. The animals did not show significant changes in the milk yield from 10 days before to 10 days after the biopsy. The bovine mammary gland biopsy is a minimally invasive procedure, allowing the animal to have a short recovery time.

Therefore, this biopsy procedure can be used in many nutrition trials and other types of trials where we might use repeated measures on the animals, so for example, if we have four periods with four different nutritional treatments, we could easily biopsy these cows four different times at the end of each period, allowing perhaps a week of rest time in between each period, to ensure that the animals return back to their normal production.