This research was supported by the National Animal Nutrition Program and Virginia Tech. The technical assistance of Ms. Tara Pilonero, Dr. Julie Settlage, and Dr. Izabelle Teixeira are gratefully acknowledged.

All methods described were approved by the Virginia Tech Institutional Animal Care and Use Committee (IACUC).
1. Personnel
<ol>
	<li>Have at least two assistants with surgical and cow-handling experience.</li>
	<li>Train all assistants at least three times, if possible using cadaveric material, prior to performing procedures associated with this protocol on live animals. 
	NOTE: Training should be conducted by an instructor that has been previously trained and performed the technique.</li>
	<li>Have a large animal veterinarian on-hand for drug administration and in the event that emergency treatment becomes necessary during the procedure. Emergencies that may occur during this procedure include but are not limited to: hemorrhage, α-2 agonist overdose and pulmonary edema, regurgitation and aspiration of food material, pain, and patient resistance requiring further sedation.</li>
</ol>
2. Preparation of the surgical instruments, supplies, and facility
<ol>
	<li>Inventory and purchase all equipment and supplies (see Table of Materials).</li>
	<li>Clean and autoclave surgical drapes, biopsy instruments, scalpel holders, surgical towels, and forceps.</li>
	<li>Have a properly sized squeeze chute and head gate to restrain the cows.</li>
	<li>Establish a work space with a table near the squeeze chute.</li>
	<li>Ensure that the work area is clean and has limited cow through-traffic.</li>
	<li>Organize equipment and supplies in the work space for easy access.</li>
	<li>Have proper lighting inside of the work space.</li>
</ol>
3. Preparation of the animals
<ol>
	<li>One day before the scheduled biopsy, wash and scrub the animal, particularly the udder to remove manure and soiled material.</li>
	<li>Follow procedures for safe and humane restraint of cattle.</li>
	<li>In advance of the biopsy and again at the time of the biopsy, assess the health, physical condition and behavior of the animal. 
	NOTE: A minimum examination includes temperature, pulse, and respiratory rates as well as examination for dermatitis over the proposed surgical site or other areas of bacterial infection. Collect milk samples from each quarter and check for mastitis.</li>
	<li>Use only healthy cows.</li>
	<li>Completely milk the cow.</li>
	<li>Move the animal into the squeeze chute, ideally within 2 h of milking to minimize milk presence in the glands.</li>
	<li>Restrain the animal with a head gate.</li>
</ol>
4. Analgesia and sedation
<ol>
	<li>Place a rope halter on the head of the cow to prevent backward and forward movement.</li>
	<li>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.</li>
	<li>Clean the area of injection with a 70% isopropyl alcohol swab and administer flunixin meglumine (1.1 to 2.2 mg/kg of body weight) intravenously via the jugular vein 15-20 min prior to biopsy. 
	NOTE:In some protocols, nonsteroidal anti-inflammatory drugs are administered after the biopsy if anti-inflammatory drugs will not affect research results.
	<ol>
		<li>Locate the jugular vein.</li>
		<li>Raise the jugular vein by application of pressure at the base of the jugular groove.</li>
		<li>Check to make sure that there are no bubbles in the syringe.</li>
		<li>Insert the needle into the raised jugular vein, and draw 0.5 mL 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.</li>
		<li>Gently remove the needle.</li>
		<li>Apply gauze with gentle pressure to the injection site to prevent bleeding.</li>
	</ol>
	</li>
	<li>Administer xylazine hydrochloride intravenously (0.01 to 0.05 mg/kg of body weight) in the coccygeal vessel approximately 5-10 min prior to biopsy to allow sufficient time for establishment of sedation. 
	CAUTION: Check the animal for signs of pulmonary edema. Clinical signs of pulmonary edema include respiratory distress, severe dyspnea, breathing difficulties, cough, frothy sputum, and blue tongue. If signs of pulmonary edema are observed, it is recommended to use tolazine (2 to 4 mg/kg of body weight) to reverse the xylazine effects.
	<ol>
		<li>Raise the tail and clean the area of injection with a 70% isopropyl alcohol swab.</li>
		<li>Check to make sure that there are no bubbles in the syringe.</li>
		<li>Insert the needle into the tail vessel, draw 0.2 mL of blood into the syringe and mix with the contents to ensure the needle is resident in the vessel. If the needle is resident in the vessel, inject the syringe contents.</li>
		<li>Gently remove the needle.</li>
		<li>Apply gauze with gentle pressure to the injection site.</li>
	</ol>
	</li>
</ol>
5. Preparation of the biopsy site
<ol>
	<li>Have an assistant tie up the tail for the procedure.</li>
	<li>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, Figure 1), and remove any soiled material or manure from the selected biopsy site.</li>
</ol>
<img alt="Udder health issue; inflamed cow udder marked for mastitis check." class="xfigimg" src="/files/ftp_upload/58602/58602fig1v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Image of the bovine udder illustrating the biopsy site. <a href="https://www.jove.com/files/ftp_upload/58602/58602fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Observe and palpate the skin with special attention to identify any large subcutaneous blood vessels in order to avoid these vessels during the biopsy.</li>
	<li>Clip the hair from a 15 cm x 15 cm area around the biopsy site.</li>
	<li>Prepare the biopsy area with povidone-iodine (0.75% available iodine) or chlorhexidine gluconate scrub. Alternate with 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 5 min.</li>
	<li>Use a butterfly infusion set with an 18 G needle to deposit 6 mL of 2% lidocaine hydrochloride subcutaneously at the incision site to create a line-block. Do not penetrate into the deeper tissues. 
	NOTE: Dosage of lidocaine varies between 3 and 8 mL.</li>
	<li>Allow the local anesthetic to diffuse for 3 to 5 min. Perform another repetition of scrub solution and alcohol prior to incision. While waiting, prepare the biopsy instruments.</li>
</ol>
6. Biopsy procedure
<ol>
	<li>Use aseptic techniques when handling the biopsy tools and for the incision.</li>
	<li>Wash hands to remove all visible contamination and apply sterile surgical gloves.</li>
	<li>Arrange the surgical instruments in a sterile area in order of use. Have a number 10 scalpel, sterile gauze, an assembled biopsy instrument, and a sterile towel for hemostasis. 
	NOTE: Follow Procedure 1 to perform a Core biopsy or Procedure 2 to perform a Needle biopsy.</li>
</ol>
<img alt="Assembly of a mechanical apparatus; step-by-step diagram; precision fitting." class="xfigimg" src="/files/ftp_upload/58602/58602fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Schematic representation of assembly and setup of the core biopsy tool described by V. C. Farr, et al.9 Scale bar is 1 cm. <a href="https://www.jove.com/files/ftp_upload/58602/58602fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Core Biopsy Instrument (Farr et al.9)
	<ol>
		<li>Assemble the core biopsy instrument using sterile gloves (Figure 2).
		<ol>
			<li>Place the 7 sterile pieces on a sterile drape (Figure 2A).</li>
			<li>Insert the blade (piece 1) into the docking system on piece 2 (Figure 2B ). 
			CAUTION: Do not place fingers directly in the blade line .</li>
			<li>Insert piece 3 on top of piece 2 (Figure 2C) ensuring that the docking station will align. 
			NOTE: Observe a docking system in the interior wall of piece 4 (Figure 2D).</li>
			<li>Engage the final edge of the blade (piece 1) into the docking system of piece 4 (Figure 2D).</li>
			<li>Push forward piece 4 and observe if piece 4 is next to piece 3 (Figure 2E).</li>
			<li>Insert piece 5 on the device (blade + piece 2) (Figure 2F).</li>
			<li>Ensure that the docking station is aligned (Figure 2G).</li>
			<li>Insert piece 6 into the top of piece 2 (opposite side of the blade) (Figure 2H)</li>
			<li>Ensure that the docking station is aligned (Figure 2H).</li>
			<li>Insert the locking screw (piece 7) into the docking station (Figure 2I).</li>
			<li>Push forward piece 3 (black) to cover the locking screw. 
			CAUTION: Do not place fingers on the blade exit.</li>
			<li>Activate the tool pushing forward piece 4 and observe the blade outside of the tool.</li>
			<li>Pull piece 4 back to retract the blade into the tool (ready to use). 
			NOTE: The blade should be activated only when the tool has penetrated the desired distance inside the tissue to be biopsied.</li>
		</ol>
		</li>
		<li>Ensure that the cow is sufficiently sedated and the biopsy site is sufficiently anesthetized. Pinch the skin to ensure no reaction.</li>
		<li>Make a 2 to 3 cm vertical incision through the skin and subcutaneous tissues from proximal to distal using a number 10 scalpel.</li>
		<li>Attach the biopsy instrument to a cordless drill using sterile technique.</li>
		<li>Place the drill against the biopsy tool and check if the tool is firmly attached to the drill.</li>
		<li>Ensure adequate restraint by having an individual elevate the tail during all procedure.</li>
		<li>Turn on the drill using clockwise rotation and a low speed. 
		NOTE: The drill is not sterile, and the operator does not remain sterile when using the drill.</li>
		<li>Advance the entire biopsy tool (around 7.5 cm) into the udder through the incision while the drill is rotating the toll.</li>
		<li>Turn off the drill and manually extend piece 4 of the tool.</li>
		<li>Turn on the drill using clockwise rotation and low speed.</li>
		<li>Remove the instrument containing the tissue core from the udder.</li>
		<li>Apply strong pressure immediately to the wound using a sterile towel for at least 20 min. 
		NOTE: Have an assistant perform this using their fist to ensure adequate pressure is applied.</li>
		<li>Remove the tissue from biopsy tool using tweezers.</li>
		<li>Keep the sample in 1x phosphate buffered saline and evaluate the amount of tissue.</li>
		<li>Take the cow's vital signs every 10 min after the biopsy for at least 30 min.</li>
		<li>Check for bleeding after 20 min of pressure on the biopsy site. If there are drips of blood continue to apply pressure for an additional 5-10 min.</li>
		<li>Close the incisions using stainless steel staples at 5 mm intervals after all bleeding has stopped. Use between 5 and 8 staples.</li>
		<li>Apply an aerosol bandage to the biopsy area.</li>
		<li>Observe the animal for 50 min after the procedure.</li>
	</ol>
	</li>
	<li>Needle Biopsy Instrument
	<ol>
		<li>Follow the manufacturer's instructions for the device.</li>
		<li>Remove the biopsy needle from the package using sterile techniques.</li>
		<li>Discard the needle if any damage is observed.</li>
		<li>Attach the needle to the device.</li>
		<li>Close the cover and cock the device.</li>
		<li>Ensure that the cow is sufficiently sedated and the biopsy site is sufficiently anesthetized. Pinch the skin to ensure no reaction.</li>
		<li>Make a 1 to 2 cm vertical incision through the skin and subcutaneous tissues from proximal to distal using a number 10 scalpel. 
		NOTE: The incision for the biopsy needle instrument can be smaller (1-2 cm) than that for the core biopsy instrument.</li>
		<li>Insert the biopsy needle into the incision site (around 10 to 13 cm from skin).</li>
		<li>Activate the biopsy needle device to collect the tissue.</li>
		<li>Remove the needle from the udder.</li>
		<li>Apply immediate, strong pressure to the wound using a sterile towel for at least 20 min.</li>
		<li>Remove the tissue from the biopsy needle using tweezers.</li>
		<li>Keep the sample in 1x phosphate buffered saline and evaluate the amount of tissue.</li>
		<li>Take vital signs every 10 min after the biopsy for at least 30 min.</li>
		<li>Check for bleeding after 20 min of pressure on the biopsy site.</li>
		<li>Close the incisions using stainless steel staples after all bleeding has stopped.</li>
		<li>Apply an aerosol bandage to the biopsy area.</li>
		<li>Observe the animal for 50 min after the procedure.</li>
	</ol>
	</li>
</ol>
7. Post-biopsy animal care
<ol>
	<li>Document all drugs administered to animals. 
	NOTE: Milk was discarded during a period of 36 h after the procedure; meat withdrawal was 4 d. Withdrawal periods for milk and meat may vary depending on the country or jurisdiction and the drugs used. Please check local rules and regulations.</li>
	<li>Check for the presence of blood in the milk for 7 to 10 d after the biopsy.</li>
	<li>Hand strip blood clots from the biopsied quarter at subsequent milkings and ensure that complete milk removal occurs. 
	NOTE: Presence of blood clots from the biopsied quarter for 1-3 milkings following the procedure is expected. Blood in the milk may be observed for 1 to 6 d after the biopsy.</li>
	<li>Observe milk yield, and if possible, the individual daily feed intake until surgical staples have been removed.</li>
	<li>Monitor the animal twice a day for body temperature, respiration and heart rates, and demeanor until surgical staples have been removed.</li>
	<li>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. 
	NOTE: Keep the biopsy site clean and reapply the aerosol bandage every one to three days as needed.</li>
	<li>Remove the staples from the incision site 10 to 14 d after the biopsy, depending on the healing rate.</li>
	<li>Consult a veterinarian if any signs of local or systemic infection are observed.</li>
</ol>

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The authors have nothing to disclose.

Core and needle biopsy methods were described in this protocol9,16. A detailed evaluation of the animal health and incidence of mastitis14 before the biopsy is required for both procedures. For research purposes, performing the technique in animals with obvious signs of inflammation or infectious diseases should be avoided. This will reduce the risk of complications during and after a biopsy. All biopsy instruments and devices should be clean, disinfected, and sterilized to avoid contamination of the biopsy site. Before the procedure, it is necessary to minimize surgical site infections (SSI). In general, SSI is associated with animal morbidity, lost performance, and higher production costs in dairy cows. Studies previously reported methods to prevent SSI due to biopsy including clipping the hair around the incision site, washing the biopsy area to remove contamination9,14, and using antiseptic agents (70% alcohol, iodine surgical scrub9, 2% chlorhexidine acetate solution14, 10% povidone-iodine8) for skin preparation. In some studies, administration of antibiotics was adopted during7 or immediately after the biopsy9; however, antibiotic prophylaxis was not used in the present work, and no infections of the biopsy site were observed. To prevent contamination of the biopsy wound, the tail should be secured to prevent contact with the biopsy site until the aerosol bandage has been applied. In this protocol, staples are removed about 10 to 14 d after the biopsy.
Prior to a core biopsy, it is important to properly set up the tool and attach it to the drill; choose a slow rotation speed, select the forward rotation, and do not use the reverse mode of the drill. During the procedure, it is important to use digital pressure on both sides of the incision to keep the skin edges apart and have a long enough incision to allow tool entry without contacting the skin or connective tissue. If this procedure is not done, a drag on the incision edges during rotation of the core biopsy instrument may occur and cause additional skin tissue trauma which will increase the risk of infection and may delay wound healing. The present biopsy procedures described in this protocol were performed by a board-certified large animal surgeon. The procedures were performed successfully (Figure 5). Both techniques are relatively easy and fast to perform as compared to the surgical excision procedure.
Bleeding from the biopsy site are common after MG biopsy in dairy cows8,9,10,14. In the present protocol, bleeding observed was minimal (Figure 4), which may be due to application of adequate (strong) pressure on the wound immediately after the procedure. Strong pressure for at least 20 min is required, and it may be required for more than 30 min in some cases. If moderate to severe bleeding is observed after application of pressure to the biopsy site, it is recommended to continue pressure, and immediately contact a veterinarian.
As hemostasis is an important factor for reducing cow morbidity, one study packed hemostatic pads into the biopsy site to control bleeding9. However, use of hemostatic pads in such a manner has a high potential risk for microbiological contamination in the dairy farm environment. Another study14 applied manual pressure to the biopsy site between repeated biopsies and after biopsy and skin closure, and applied ice to the site for at least 2 h following the biopsy. In the present protocol, strong pressure applied to the biopsy site for 20 to 25 min was adequate to control bleeding.
A successful biopsy technique should result in minimum blood in the milk which persists for a short period of time after the procedure. To avoid an interruption of milk secretion during milk letdown and mastitis infections, intramammary blood clots should be removed by hand stripping from the biopsied quarter10,14. In the present protocol, blood was observed in the milk up to 48 h after the biopsy. However, in a study that used a larger number of animals, blood in the milk was observed in the majority of cows for less than 6 days. Few animals showed blood in the milk after 6 days8. For this reason, daily observation of milk appearance is necessary for 6 d after the procedure. Animals did not exhibit any signs of mastitis infection when a large tissue sample was obtained14. However, previous research which performed repeated biopsies on the same animal using a needle found that mastitis infection incidence was approximately 12% following the procedure8. In the present protocol, neither animal had visual signs of clinical mastitis infection after the procedure. There is also a slight chance that the incision site may become infected after the biopsy.
Tolazoline hydrochloride, a drug that reverses the effects of sedation, should be available in the event of an overdose from xylazine. An excessive dose of xylazine may cause pulmonary edema. Clinical signs of pulmonary edema include respiratory distress, severe dyspnea, breathing difficulties, cough, and frothy sputum. If signs of pulmonary edema are observed, it is recommended to use tolazine (2 to 4 mg/kg of body weight) to reverse the xylazine effects.
The present protocol describes the technique to perform both a needle biopsy and core biopsy. In general, the advantage of a core biopsy as compared to needle biopsy is the larger tissue sample (0.75 to 1 g)9 with minimum negative effect on udder health. The needle biopsy is a less invasive method than the core biopsy instrument. However, multiple biopsy attempts to obtain a larger amount of tissue using a needle biopsy procedure may increase the risk of major bleeding after the procedure, as well as blood clots in the milk. Both techniques caused minimal cow morbidity and were easily achieved with minimal training of surgical personnel. A short-term change in daily milk yield (8 to 10 % decrease) and its composition9 and feed intake reduction14 is expected after the core and needle biopsies, respectively. Limitations of the needle biopsy procedure are the small volume of tissue removed with the needle. The instrument needs to be activated only when the needle is within the tissue to be biopsied and multiple attempts often are necessary to obtain adequate amounts of tissue, which increases the risk of blood in the milk and mastitis after the procedure. Limitations of the core biopsy include a higher risk of bleeding after the procedure if a large amount of tissue (&#62;200 mg) is obtained. In addition, the biopsy tool is more difficult to assemble requiring adequate training before use.

A biopsy is a procedure to harvest a core section of tissue from subjects for medical or research purposes. It is a minimally invasive and widely used technique to collect tissue as an alternative to euthanasia1 to allow analysis of tissue responses to treatment or other factors of interest. Samples of bovine mammary gland (MG) tissues are essential for dairy research to study gene and protein expression, histology, the organization of cellular organelles, signaling pathways, and metabolic processes in response to changes in management or the environment. Additionally, MG tissue is needed to diagnose and research certain infectious diseases, such as bovine mastitis caused by bacterial infections2.
The udder of cattle contains four separate glands and each gland contains an independent milk secretory system collectively called parenchyma. The alveoli, ducts, and connective tissue are present in the mammary parenchyma. The alveoli are microscopic spherical hollow structures that are composed of epithelial cells in the internal surface (luminal) and specialized myoepithelial cells on the external surface (basal). The alveoli are responsible for synthesis and secretion of milk. The fibrous connective tissue present in the parenchyma can separate a group of alveoli from other groups of alveoli, and each group is called a lobule3. Several factors can affect the development and function of the MG including animal physiology, nutrition, management, genetics, and the environment4. Mastitis also is a critical factor that negatively affects MG function and milk quality5. The toxins released by mastitic pathogens can cause damage to the alveoli and induce necrosis resulting in biochemical and histological changes in the MG tissue6. Thus, the histology and metabolic processes of each alveoli or lobule in the MG may be significantly different from others. As such, biopsies that are representative samples of the entire mammary gland are desirable. Small biopsies may only capture a single lobule or individual alveolus, limiting the resulting scientific or diagnostic information. For research purposes, it is necessary to be aware that a mammary biopsy provides a &#39;snapshot&#39; of tissue characteristics but a biopsy cannot adequately characterize total mammary function in the absence of estimates of total mammary gland mass.
Several biopsy tools have been developed over the last 30 years for human use. Currently, adaptations of those instruments are available for use with animals. For dairy cattle, samples of MG tissue have been obtained using different techniques including surgical excision (blunt dissection)7, biopsy needles1,8, and core biopsy instruments9,10. Thus, MG biopsy techniques in lactating dairy cows have transitioned from procedures using recumbent sedation with surgical dissection using electrocautery hemostasis in 19927 to collection of core biopsies under standing sedation9,10,11,12. Surgical biopsy is an invasive method, which can be expensive and have a higher incidence of complications such as hematoma, wound problems, and tumor spread13. Currently, core biopsy and needle biopsy (also known as a tru-cut biopsy) have been widely adopted as alternatives to surgical biopsy. The advantages of core and needle biopsies compared to surgical biopsy include: the procedure is minimally invasive; major complications are rare; general anesthesia is not required; the procedure is relativley rapid; the recovery time is short; there is minimal negative effects on udder health, and only short-term effects on milk yield and composition8,9,10; and the cost is less than surgical biopsy13.
One core biopsy procedure described in 1996 used a sterile, stainless steel cannula with a removable retractable blade to remove a representative amount of tissue from the bovine MG without general anesthesia9,10,11,12. During the procedure, the instrument was attached to a cordless drill to create a low speed, rotational motion which cleanly cut a tissue core as the tool was advanced into the tissue. The benefit was a larger tissue sample (70 mm x 4 mm in diameter, about 0.75 to 1 g) 9. A recent study10 showed that the biopsy procedure described by V. C. Farret al.9 can be used to perform repeated MG tissue collections without negative impact on performance and udder health of lactating dairy cows. Most recently, a study14 was carried out in dairy cows to evaluate repeated biopsies of the MG using a larger trocar (31 cm long, outer diameter of 9.5 mm, inner diameter of 8 mm) with vacuum applied to an internal stainless steel cannula to collect the biopsy. This method used sedation (xylazine) and local anesthesia (2% lidocaine hydrochloride).
The needle biopsy is another technique to collect mammary tissue. Several studies have adopted this technique. One study1 used sedation (detomidine)and local anesthetic (1% lidocaine) in the procedure. After the biopsy, the cows received a prophylactic antibiotic treatment. The mammary gland was manually massaged before and after the milking. Blood in the milk was observed for up to 84 h after the biopsy. The amount and composition of the milk were affected for a short period of time. Recently, a study used a biopsy needle to perform repeated MG biopsies in dairy cows8. Sedation (1% acepromazine, intramuscular) and local anesthesia (2% lidocaine hydrochloride, subcutaneous) were administered to the animals. The animals did not receive intramammary drugs or antibiotics before or after the procedure, and there were no signs of infection at the biopsy site during the post-surgical period. In this study, repeated bovine MG biopsies using a needle had a minor negative impact on milk production and udder health of dairy cows. In general, the needle biopsy seems to be a less invasive method than the core biopsy instrument. However, as noted previously, it is essential for the biopsy technique to harvest a representative sample of the MG tissue. The limitation of a needle biopsy is that a small amount of bovine MG tissue is obtained (about 20 to 25 mg)8,15.
Almost all of the studies used a combination of &#945;-2 agonist sedation and local anesthetic8,9,10, whether the biopsy was via a needle or a larger core. In the bovine MG, most nerve endings are associated with the skin. Innervation to the parenchymal tissue is largely via stretch receptors with sparse Type A nerve fibers to detect sharp surgical pain. As a result, physiological mechanisms of pain due to surgical manipulation of the MG is via skin and subcutaneous tissues3 and not deep tissues such as parenchymal tissue. Therefore, for biopsy procedures, it is only necessary to locally anesthetize the skin and subcutaneous tissues, as infiltration of local anesthetic into the deeper tissues does not significantly reduce surgical pain. After an appropriate preparation of the biopsy area, animal discomfort is, primarily, associated with restraint.
Collection of larger core samples may increase hematoma formation and increase the risk of infection within the MG parenchyma. Therefore, peri-operative protocols often include administration of parenteral antibiotics7, although that is not universals8. Achieving hemostasis is also an important factor for reducing cow morbidity. In the aforementioned study using a large core biopsy instrument14, manual pressure was applied to the biopsy site and a cow bra was used to apply ice to the wounds for at least 2 h following the procedure. Despite the large amount of tissue harvested, only slight reductions in feed intake and milk yield were observed, and the procedure was repeated every three weeks without negative effects on cow health.
Researchers performing MG biopsy in dairy cows need to consider the diagnostic or analytical quality of the resulting biopsy, ease of technique, and cow morbidity. Accurate and pre-planned surgical techniques are imperative to achieve these goals. To date, MG biopsy studies have focused on describing biopsy outcomes, as opposed to describing the biopsy technique itself, and descriptions for lactating dairy cows lack sufficient detail to allow replication. Thus, the objective of this work was to describe both needle biopsy and larger core biopsy techniques in sufficient detail to allow safe and humane replication of MG biopsy of cattle.

<table><tbody><tr><td>70% Isopropyl alcohol&amp;nbsp;</td><td>Walmart&amp;nbsp;</td><td>565106257</td><td>1 L</td></tr><tr><td>Aerosol spray to kill and repel flies</td><td>MWI</td><td>14339</td><td>1 bottle (if necessary)</td></tr><tr><td>Biopsy needles, 12 G &amp;times; 16 cm</td><td>C. R. Bard</td><td>MN1216</td><td>2</td></tr><tr><td>Butterfly infusion set (18 G needle)</td><td>Fisher</td><td>22-258087</td><td>1</td></tr><tr><td>Cell culture dishes</td><td>Fisher</td><td>08-772E</td><td>4</td></tr><tr><td>Cordless drill (low speed)</td><td>Hitachi&amp;nbsp;</td><td>&amp;nbsp;DS10DFL&amp;nbsp;</td><td>1</td></tr><tr><td>Core biopsy instrument, Farr et al. (1996)</td><td></td><td></td><td>2, custom metal fabrication. To request the tool contact the authors.</td></tr><tr><td>Cows</td><td></td><td></td><td>cattle healthy</td></tr><tr><td>Flunixin meglumine</td><td>MWI/VetOne</td><td>501018</td><td>1.1 to 2.2 mg/kg of body weight</td></tr><tr><td>Folding table&amp;nbsp;&amp;nbsp;</td><td>Amazon</td><td></td><td>1</td></tr><tr><td>Forceps</td><td>Fisher</td><td>09-753-50</td><td>1</td></tr><tr><td>Gloves non-sterile&amp;nbsp;</td><td>Fisher</td><td>17-200-845</td><td>9, size dependent</td></tr><tr><td>Hard brush&amp;nbsp;</td><td>Sullivan Supply</td><td>18270</td><td>1, to wash the cows</td></tr><tr><td>Head gate&amp;nbsp;</td><td></td><td></td><td>1, customized for dairy cows</td></tr><tr><td>Lidocaine hydrochloride 2% injectable&amp;nbsp;</td><td>MWI/VetOne</td><td>510212</td><td>6 mL was used,&amp;nbsp; from 3 to 8 mL</td></tr><tr><td>Livestock body wash</td><td>eZall Technologies</td><td>39384</td><td>1, to wash the cows</td></tr><tr><td>Needle 18 G&amp;nbsp; (1 to 1/5&amp;ldquo;)</td><td>Fisher</td><td>14-821-15A</td><td>6</td></tr><tr><td>Needle 20 G (1 to 1/5&amp;ldquo;)</td><td>Fisher</td><td>14-815-526</td><td>6</td></tr><tr><td>Phosphate buffered saline (0.9%)</td><td>Fisher</td><td>20-012-043</td><td>1 L</td></tr><tr><td>Povidone Iodine ointment</td><td>Jeffers</td><td>#VED1A</td><td>500 g (if necessary)</td></tr><tr><td>Povidone iodine scrub solution (0.75% iodine)</td><td>MWI/VetOne</td><td>510094</td><td>1 L</td></tr><tr><td>Reusable biopsy instrument&amp;nbsp;</td><td>C. R. Bard</td><td>MG1522</td><td>1</td></tr><tr><td>Rope halter</td><td>Nasco farm and ranch</td><td>C10852</td><td>2, cow size</td></tr><tr><td>Scalpel blades (#10)</td><td>MWI</td><td>033870</td><td>2</td></tr><tr><td>Scalpel holders (#3)</td><td>MWI</td><td>602008</td><td>1</td></tr><tr><td>Self seal autoclave pouch 10 x 5.5 in.</td><td>Fisher</td><td>19-130-0038</td><td>1 case of 800</td></tr><tr><td>Sterile cotton gauze sponges</td><td>Fisher</td><td>22-037-986</td><td>4</td></tr><tr><td>Sterile gauze pads</td><td>Fisher</td><td>19-090-735</td><td>4</td></tr><tr><td>Sterile surgical gloves&amp;nbsp;</td><td>Surgical gloves</td><td>19-166-679</td><td>3,&amp;nbsp; size dependent</td></tr><tr><td>Sterile surgical towels</td><td>Fisher</td><td>50-118-0339</td><td>6</td></tr><tr><td>Stethoscope</td><td>Littmann Master Classic II</td><td>1392</td><td>1</td></tr><tr><td>Surgical clipper</td><td>Oster A5</td><td>078005-010-003</td><td>1, preparation of the animal for biopsy</td></tr><tr><td>Surgical clipper blades (#40 )</td><td>Oster&amp;nbsp;</td><td>78919-016</td><td>1, preparation of the animal for biopsy</td></tr><tr><td>Surgical staple remover</td><td>MWI</td><td>541</td><td>1</td></tr><tr><td>Surgical stapler pre-loaded</td><td>MWI</td><td>17713</td><td>5</td></tr><tr><td>Syringes of 10 mL</td><td>Fisher</td><td>14-823-224</td><td>3</td></tr><tr><td>Syringes of 2 mL</td><td>Fisher</td><td>22-028854</td><td>3</td></tr><tr><td>Syringes of 20 mL</td><td>Fisher</td><td>22-034507</td><td>3</td></tr><tr><td>Syringes of 5 mL</td><td>Fisher</td><td>22-028855</td><td>3</td></tr><tr><td>Thermometer</td><td>Agri-Pro Enterprises Inc</td><td>72000</td><td>1</td></tr><tr><td>Tolazine hydrochloride</td><td>Akorn Animal Health</td><td>61311-486-10</td><td>2 to 4 mg/kg of body weight (if necessary)</td></tr><tr><td>Tweezer&amp;nbsp;</td><td>Fisher</td><td>14-955-025</td><td>1, for handle the tissue sample</td></tr><tr><td>Water hose</td><td>Miracle-Gro, Walmart</td><td>554990501</td><td>1, to wash the cows</td></tr><tr><td>Water-resistant aerosol bandage (aluminum powder 40 mg)</td><td>MWI</td><td>010728</td><td>1 bottle&amp;nbsp;</td></tr><tr><td>Xylazine hydrochloride</td><td>MWI/VetOne</td><td>510650</td><td>0.01 to 0.05 mg/kg of body weight</td></tr></tbody></table>

bovine mammary gland biopsy techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

In the present protocol, the core biopsy technique produced tissue sample of 200 to 600 mg while the needle biopsy produced samples of 10 to 30 mg per biopsy. The animals were observed twice a day for 10 d 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 (average respiratory rate = 31.4 ± 7.04 (SD) breaths per min, average heart rate = 75.9 ± 8.9 beats per min, and average rectal temperature = 38.2 ± 0.68 °C, Figure 3).
Following manual pressure on the biopsy area to achieve hemostasis (about 25 min), the open wound showed minimal bleeding (Figure 4). The cows were fed the same diet ad libitum, and the animals were kept in the same housing facility during the post-operative period. The animals did not show signs of infection at the biopsy site; there was no pain, bleeding, swelling, drainage, or elevated temperature (Figure 5). The wound healed within 8 to 10 d of the biopsy (Figure 5C).
One animal developed minor skin irritation due to wound rubbing on day 8 after the procedure. This cow was treated by cleaning the biopsy site using povidone-iodine solution and 70% isopropyl alcohol. The stainless steel staples were removed to allow drainage and povidone iodine ointment (1%) was applied twice a day on the biopsy site for 2 to 3 d. The skin irritation disappeared 2 d after applying povidone-iodine ointment and the wound healed with no further complications.
Blood clots were removed by hand stripping of milk from the biopsied quarter prior to machine milking. In the present protocol, the presence of blood clots in the milk was observed for up to three milkings (about 36 h) after the biopsy. Blood contamination of milk was observed for up to 48 h after the biopsy. There were no visual differences observed between core and needle biopsies relative to the amount of blood in the milk. The animals did not show a significant decrease in milk yield after the procedure (Figure 6). Average milk composition was 4.37% fat, 3.34% protein, and 4.64% lactose. The average milk somatic cell count (SCC) was less than 200,000 cells per mL.
The MG tissue obtained can be used for different research purposes such as primary cell cultures (Figure 7).
<img alt="Breathing, heart rate, temperature data post-biopsy; comparative graph for core vs. needle tools." class="xfigimg" src="/files/ftp_upload/58602/58602fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Evaluation of animal health indicators after the bovine mammary gland biopsy. <a href="https://www.jove.com/files/ftp_upload/58602/58602fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cow hindquarters with udder swelling, indicating possible mastitis; used in veterinary diagnostics." class="xfigimg" src="/files/ftp_upload/58602/58602fig4v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Biopsy site appearance after manual pressure was applied to the biopsy area to achieve hemostasis. <a href="https://www.jove.com/files/ftp_upload/58602/58602fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Needle vs. core biopsy wound healing over 10 days; comparative photographic study of skin recovery." class="xfigimg" src="/files/ftp_upload/58602/58602fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5. Biopsy site photographs. (A) Biopsy site 3 d after the procedure. (B) Biopsy site 6 d after the procedure. (C) Biopsy site 10 d after the procedure illustrating that the wound was adequately healed. <a href="https://www.jove.com/files/ftp_upload/58602/58602fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Milk yield chart with linear regression; biopsy effects by tool and day; statistical graph." class="xfigimg" src="/files/ftp_upload/58602/58602fig6.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6. Daily milk production before and after the bovine mammary gland biopsy (n = 3 cows). A linear model estimated that 10 days before the biopsy the animals produced 23.35 kg of milk per day. The milk yield did not change significantly (P &gt; 0.05) from 10 days before to 10 days after the biopsy although a slight increase in milk yield was observed (increase of 0.0005 kg/d of milk for each day from 10 days before the biopsy). Three animals were used in this protocol; one animal was biopsied twice (Animal 1: core biopsy on the left side of the udder and needle biopsy on the right side of the udder) and two animals were biopsied once (Animal 2 and 3: only on the right side of the udder using the core or needle procedure). <a href="https://www.jove.com/files/ftp_upload/58602/58602fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell culture microscopy image showing compact growth pattern and cellular confluence at 100 μm scale." class="xfigimg" src="/files/ftp_upload/58602/58602fig7.jpg" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
Figure 7. Representative images of primary bovine mammary epithelial cells culture. Scale bar is 100 µm. <a href="https://www.jove.com/files/ftp_upload/58602/58602fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Bovine Mammary Gland Biopsy Techniques at JoVE.com

Bovine Mammary Gland Biopsy Techniques

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, ...

Research

JoVE Journal

Biology

14.1K Views.  University of Kentucky. Een biopsie, zoals ik denk dat de meesten van jullie weten, is het verwijderen van een beetje weefsel uit het orgaan van belang en in dit geval is het de borstklier. De biopsie monster is zeer nuttig als je gaat om dingen te doen zoals genexpressie analyse, als je gaat om dingen te doen zoals om een deel van het weefsel te nemen en een histologische analyse te doen, om te kijken naar de structuur op het microscopisch niveau van de borstklier, ook zeer nuttig om dingen te bepalen zoals signale...

Video: Bovine Mammary Gland Biopsy Techniques

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Cancer Research

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

Medicine

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

Biochemistry

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

Developmental Biology

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals.
The aim was to establish in vivo sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions.
Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

This article describes bronchoscopic techniques in the bovine lung under experimental conditions, i.e. bronchoscopically guided inoculation, bronchoalveolar lavage, bronchial brushing, and transbronchial lung biopsy.

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as ...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Bovine Mammary Gland Biopsy Techniques

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