Published: December 23rd, 2018
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, 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.
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 'snapshot' 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 α-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.
All methods described were approved by the Virginia Tech Institutional Animal Care and Use Committee (IACUC).
2. Preparation of the surgical instruments, supplies, and facility
3. Preparation of the animals
4. Analgesia and sedation
5. Preparation of the biopsy site
Figure 1. Image of the bovine udder illustrating the biopsy site. Please click here to view a larger version of this figure.
6. Biopsy procedure
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. Please click here to view a larger version of this figure.
7. Post-biopsy animal care
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).
Figure 3. Evaluation of animal health indicators after the bovine mammary gland biopsy. Please click here to view a larger version of this figure.
Figure 4. Biopsy site appearance after manual pressure was applied to the biopsy area to achieve hemostasis. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 > 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). Please click here to view a larger version of this figure.
Figure 7. Representative images of primary bovine mammary epithelial cells culture. Scale bar is 100 µm. Please click here to view a larger version of this figure.
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 (>200 mg) is obtained. In addition, the biopsy tool is more difficult to assemble requiring adequate training before use.
The authors have nothing to disclose.
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.
|70% Isopropyl alcohol
|Aerosol spray to kill and repel flies
|1 bottle (if necessary)
|Biopsy needles, 12 G × 16 cm
|C. R. Bard
|Butterfly infusion set (18 G needle)
|Cell culture dishes
|Cordless drill (low speed)
|Core biopsy instrument, Farr et al. (1996)
|2, custom metal fabrication. To request the tool contact the authors.
|1.1 to 2.2 mg/kg of body weight
|9, size dependent
|1, to wash the cows
|1, customized for dairy cows
|Lidocaine hydrochloride 2% injectable
|6 mL was used, from 3 to 8 mL
|Livestock body wash
|1, to wash the cows
|Needle 18 G (1 to 1/5“)
|Needle 20 G (1 to 1/5“)
|Phosphate buffered saline (0.9%)
|Povidone Iodine ointment
|500 g (if necessary)
|Povidone iodine scrub solution (0.75% iodine)
|Reusable biopsy instrument
|C. R. Bard
|Nasco farm and ranch
|2, cow size
|Scalpel blades (#10)
|Scalpel holders (#3)
|Self seal autoclave pouch 10 x 5.5 in.
|1 case of 800
|Sterile cotton gauze sponges
|Sterile gauze pads
|Sterile surgical gloves
|3, size dependent
|Sterile surgical towels
|Littmann Master Classic II
|1, preparation of the animal for biopsy
|Surgical clipper blades (#40 )
|1, preparation of the animal for biopsy
|Surgical staple remover
|Surgical stapler pre-loaded
|Syringes of 10 mL
|Syringes of 2 mL
|Syringes of 20 mL
|Syringes of 5 mL
|Agri-Pro Enterprises Inc
|Akorn Animal Health
|2 to 4 mg/kg of body weight (if necessary)
|1, for handle the tissue sample
|1, to wash the cows
|Water-resistant aerosol bandage (aluminum powder 40 mg)
|0.01 to 0.05 mg/kg of body weight
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