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, &#945;-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&#39;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="Figure 1" class="xfigimg" src="/files/ftp_upload/58602/58602fig1v2.jpg" /> 
Figure 1. Image of the bovine udder illustrating the biopsy site.&#160;<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="Figure 2" class="xfigimg" src="/files/ftp_upload/58602/58602fig2.jpg" /> 
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.&#160;<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&#39;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&#39;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:&#160;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.

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			<td height="21" style="height:21px;width:451px;">70% Isopropyl alcohol&#160;</td>
			<td style="width:242px;">Walmart&#160;</td>
			<td style="width:123px;">565106257</td>
			<td style="width:306px;">1 L</td>
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			<td>1 bottle (if necessary)</td>
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			<td>1</td>
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			<td>1</td>
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			<td>2, custom metal fabrication. To request the tool contact the authors.</td>
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			<td>1.1 to 2.2 mg/kg of body weight</td>
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			<td height="21" style="height:21px;width:451px;">Needle 18 G&#160; (1 to 1/5&#8220;)</td>
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			<td style="width:242px;">Jeffers</td>
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			<td height="21" style="height:21px;width:451px;">Reusable biopsy instrument&#160;</td>
			<td style="width:242px;">C. R. Bard</td>
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			<td>Fisher</td>
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			<td>1, preparation of the animal for biopsy</td>
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			<td height="21" style="height:21px;width:451px;">Surgical clipper blades (#40 )</td>
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			<td height="21" style="height:21px;width:451px;">Surgical staple remover</td>
			<td style="width:242px;">MWI</td>
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			<td height="21" style="height:21px;width:451px;">Surgical stapler pre-loaded</td>
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			<td>5</td>
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			<td height="21" style="height:21px;width:451px;">Syringes of 10 mL</td>
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			<td height="21" style="height:21px;width:451px;">Syringes of 2 mL</td>
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			<td height="21" style="height:21px;width:451px;">Syringes of 5 mL</td>
			<td>Fisher</td>
			<td>22-028855</td>
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			<td height="21" style="height:21px;width:451px;">Thermometer</td>
			<td style="width:242px;">Agri-Pro Enterprises Inc</td>
			<td style="width:123px;">72000</td>
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			<td height="21" style="height:21px;width:451px;">Tolazine hydrochloride</td>
			<td style="width:242px;">Akorn Animal Health</td>
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			<td>2 to 4 mg/kg of body weight (if necessary)</td>
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			<td>Fisher</td>
			<td style="width:123px;">14-955-025</td>
			<td>1, for handle the tissue sample</td>
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			<td height="21" style="height:21px;width:451px;">Water hose</td>
			<td style="width:242px;">Miracle-Gro, Walmart</td>
			<td style="width:123px;">554990501</td>
			<td>1, to wash the cows</td>
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			<td height="21" style="height:21px;width:451px;">Water-resistant aerosol bandage (aluminum powder 40 mg)</td>
			<td style="width:242px;">MWI</td>
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			<td>1 bottle&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Xylazine hydrochloride</td>
			<td style="width:242px;">MWI/VetOne</td>
			<td style="width:123px;">510650</td>
			<td>0.01 to 0.05 mg/kg of body weight</td>
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</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 &#177; 7.04 (SD) breaths per min, average heart rate = 75.9 &#177; 8.9 beats per min, and average rectal temperature = 38.2 &#177; 0.68 &#176;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="Figure 3" class="xfigimg" src="/files/ftp_upload/58602/58602fig3.jpg" /> 
Figure 3.&#160;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="Figure 4" class="xfigimg" src="/files/ftp_upload/58602/58602fig4v2.jpg" /> 
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="Figure 5" class="xfigimg" src="/files/ftp_upload/58602/58602fig5.jpg" /> 
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="Figure 6" class="xfigimg" src="/files/ftp_upload/58602/58602fig6.jpg" /> 
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 &#62; 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="Figure 7" class="xfigimg" src="/files/ftp_upload/58602/58602fig7.jpg" /> 
Figure 7. Representative images of primary bovine mammary epithelial cells culture. Scale bar is 100 &#181;m. <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, ...

Nanyang Technological University

Research

JoVE Journal

Biology

14.0K Views.  University of Kentucky. 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.

Video: Bovine Mammary Gland Biopsy Techniques

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Dendra2 Photoswitching through the Mammary Imaging Window

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.

Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4  has resulted in important insights into ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

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 ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...