
==== Front
Vet Med Sci
Vet Med Sci
10.1002/(ISSN)2053-1095
VMS3
Veterinary Medicine and Science
2053-1095
John Wiley and Sons Inc. Hoboken

10.1002/vms3.70027
VMS370027
Original Article
RUMINANTS
Original Article
Ultrasound‐assisted minimally invasive technique for tissue collection from bovine mammary glands
VANG et al.
Vang Alysia L. https://orcid.org/0000-0003-4790-8924
1
Frizzarini Waneska S. https://orcid.org/0000-0002-7901-4004
1
Webster Haylee H. https://orcid.org/0000-0003-0945-2727
1
Cunha Thiago O. https://orcid.org/0000-0003-4356-8651
1
Nelson Kathryn M. 1
Dorea Joao R. R. https://orcid.org/0000-0001-9849-7358
1
Hernandez Laura L. https://orcid.org/0000-0001-7591-5203
1 llhernandez@wisc.edu

1 Department of Animal and Dairy Sciences University of Wisconsin—Madison Madison Wisconsin USA
* Correspondence
Laura L. Hernandez, Department of Animal and Dairy Sciences, University of Wisconsin—Madison, Madison, Wisconsin 53706‐1314, USA.
Email: llhernandez@wisc.edu

18 9 2024
11 2024
10 6 10.1002/vms3.v10.6 e7002724 4 2024
01 4 2023
10 6 2024
© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

Abstract

Background and Aims

The objective of this case report was to describe an ultrasound‐guided, minimally invasive method for longitudinal mammary gland tissue collection from the bovine species.

Materials and Methods

Biopsies were performed on 14 8‐week‐old calves and 113 10‐week‐old calves. A subset of 36 animals had repeated mammary gland biopsies through the first lactation. Mammary gland biopsies were performed using a disposable biopsy punch. The technique was also performed on multiparous cows on other independent research trials.

Results

One‐hundred and thirteen animals healed from the 10‐week biopsies with no complications. Of the 36 animals that received repeated biopsies, one developed mastitis due to premature suture removal and one had recurring mastitis in all quarters. Thirty‐three animals underwent all biopsies during gestation. Thirty of the original 36 are currently in lactation and still undergoing repeated biopsies. The method has also been successfully replicated on multiparous cows in separate studies.

Discussion and Conclusion

The described technique is a safe, reliable method for cattle mammary gland biopsies beginning at eight weeks of age and can be utilized to obtain repeated tissue collection from individual animals. The technique is also straightforward to perform and utilizes simple tools while providing acceptable amounts of tissue for most applications, with low risk for infection and long‐term tissue damage.

Mammary biopsies were performed on animals from 10 weeks of age through the second lactation with disposable biopsy punches. Results demonstrate that the ultrasound‐guided biopsy procedure using disposable biopsy punches is a relatively safe and effective method for repeated mammary tissue collection and allows for collection from young animals. Modification of the method has also allowed for the collection of mammary tissue from older heifers and lactating and dry cows with few complications.

biopsy
bovine
longitudinal
mammary gland
National Institute of Food and Agriculture of the United States Department of Agriculture2020‐67015‐30831 WIS04094 National Institute of Health TrainingR25GM083252 T32GM135066 source-schema-version-number2.0
cover-dateNovember 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:19.09.2024
Vang, A. L. , Frizzarini, W. S. , Webster, H. H. , Cunha, T. , Nelson, K. M. , Dorea, J. R. R. , & Hernandez, L. L. (2024). Ultrasound‐assisted minimally invasive technique for tissue collection from bovine mammary glands. Veterinary Medicine and Science, 10 , e70027. 10.1002/vms3.70027
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pmc1 INTRODUCTION

Tissue collection from research subjects is essential as it allows researchers to investigate gene and protein expression, molecular signalling pathways, metabolic processes, as tissue microstructure. Mammary gland tissue collection in calves and heifers has historically required euthanasia (Brown et al., 2005; Daniels et al., 2009; Geiger et al., 2016; Meyer et al., 2006; Soberon & Van Amburgh, 2017). Although this method has been invaluable in studying development in the bovine species, it limits the ability to collect longitudinal repeated measures and results in either single timepoint measurements or milk production for individual animals (Weng et al., 2017). The minimally invasive technique described has allowed for repeated tissue collections from individual animals from 10 weeks of age through the second lactation with minimal complications.

The udder in the bovine species is composed of four separate glands containing alveoli, ducts and connective tissue, which make up the parenchyma. Alveoli are spherical structures responsible for synthesizing and secreting milk and are lined internally with milk‐producing mammary epithelial cells and externally with myoepithelial cells. Groups of alveoli form lobules, which then combine to form lobes. The mammary gland begins developing in utero and continues developing throughout the different stages of the animal's life. Various factors can affect not only development but also function of the mammary gland, including genetics, nutrition, management, disease and infection and environment (Akers, 2017; Cheng & Han, 2020; Cole et al., 2023; Van Amburgh et al., 2019). Although a biopsy sample is not representative of the entire mammary gland, biopsies can give researchers insight into the activities and structural elements of the complex tissue. Currently, there are no repeated measured, longitudinal documented histological images of mammary gland development in the bovine beginning at the calf stage through an entire lactation.

Various methods, such as surgical excision, biopsy needles and core biopsy instruments, have been utilized to perform biopsies in cattle (Daley et al., 2018; de Lima et al., 2016; Farr et al., 1996; Khatun et al., 1992; Knight et al., 1992; Lima et al., 2016; Sheehy et al., 2004; VanKlompenberg et al., 2012; Weng et al., 2017). Notably, these methods have been largely reported in lactating or dry cows, although tissue collection in heifers has involved euthanasia (Brown et al., 2005; Daniels et al., 2009; Geiger et al., 2016; Meyer et al., 2006; Soberon & Van Amburgh, 2017). Use of ultrasound imaging has not only allowed for the collection of mammary tissues from calves and heifers, but it has also aided in identification of large internal blood vessels that cannot be seen on the udder's exterior, thus decreasing the risk of heavy bleeding. As demonstrated previously, ultrasound imaging is effective in examination of internal structures and overall udder health (Flöck & Winter, 2006; Franz et al., 2009; Themistokleous et al., 2023). As the procedures necessary to perform heifer biopsies have yet to be described in literature with sufficient detail for replication, the objective of this work is to describe the use of a minimally invasive biopsy technique in calves, heifers, and cows to allow for replication of mammary gland biopsies in calves with only slight modifications throughout the different life stages. This is critical for evaluation of biological processes related to mammary gland function across the animal's lifespan as it allows for experiments to be designed with repeated measures and longitudinal timeframes in mind.

2 MATERIALS AND METHODS

2.1 10‐week biopsy

2.1.1 Biopsy preparation

Table 1 displays the number of biopsies each animal underwent in the repeated biopsy group. Mammary gland biopsies were performed on 14 8‐week‐old calves and 113 10‐week‐old calves using a novel technique utilizing a disposable biopsy punch. Biopsies were then repeatedly performed on 36 animals through the first lactation. All 36 animals were biopsied at 6, 9 and 12 months of age. Three animals were sold for reproductive issues, and so 33 animals were biopsied during gestation at 5, 7 and 8 months. Thirty‐two animals were biopsied at 1 day in milk (1DIM). One animal was not biopsied at 1DIM due to recurring mastitis in all quarters during gestation. Twenty‐nine animals were biopsied at 45DIM as one animal died unexpectedly due to unknown causes 3DIM after delivering twins and one animal developed severe mastitis after chewing its teats and had to be euthanized. The same 29 animals were biopsied through the first lactation at 90, 150 and 270DIM unless they were ill. Sixteen of those animals are currently undergoing biopsies in their dry period and second lactation.

TABLE 1 Repeated Biopsies. The table displays the biopsies each animal has undergone as well as the reason why the animal no longer underwent biopsies.

ID	10wk	6mo	9mo	12mo	5moG	7moG	8moG	1DIM	45DIM	90DIM	150DIM	270DIM	1WPD	3WPD	5WPD	1DIM	45DIM	90DIM	
1387																			
1388																			
1389																			
1390																			
1391																			
1392					Sold due to reproductive issues.	
1393																	
1394																			
1395									Euthanized due to teat chewing.	
1396										Unrelated health problems.							
1397																			
1398																			
1399																			
1400																			
1401																			
1402																			
1403																			
1404					Sold due to reproductive issues and mastitis complications.	
1405																			
1406																			
1498														
1500													
1501														
1503					Sold due to reproductive issues.	
1505														
1506														
1507													
1508									Died due to unknown cause.							
1509												
1510							Sold due to recurring mastitis.	
1511														
1512														
1513														
1514														
1515														
1516														
Labels: 10wk‐10 weeks of age; 6mo‐6 months of age; 9mo‐9 months of age; 12mo‐12 months of age; 5moG‐5 months gestation; 7moG‐7 months gestation; 8moG‐8 months gestation; 1DIM‐1 day in milk; 45DIM‐45 days in milk; 90DIM‐90 days in milk; 150DIM‐150 days in milk; 270DIM‐270 days in milk; 1WPD‐1 week post‐dry; 3WPD‐3 weeks post‐dry; 5WPD‐5 weeks post‐dry.

Pink: biopsies performed; Gray: biopsies not performed; White: biopsies to be performed

John Wiley & Sons, Ltd.

Prior to biopsies, the animals were weighed to determine drug dosage and udders palpated to determine if the mammary gland was appropriately sized for a biopsy. Health parameters were collected for all animals undergoing biopsies to ensure proper health status prior to procedure (i.e. heart rate, respiratory rate and rectal temperature). Calves were restrained and xylazine hydrochloride (20 mg/mL) was administered via the jugular vein at a dose of 0.10 mg/kg bodyweight. Animals typically lie down within 10 min. A folded towel was placed under the calf's head to prevent regurgitation, and another towel was placed over its eyes. With the calf in right lateral recumbency, the left rear leg was raised and held by an assistant. The entire surface of the udder and inner region of the left leg adjacent to the left rear quarter were clipped of hair. The mammary gland was located by ultrasound (Mindray Z5 Ultrasound, Mindray 65C15EA 6.5 MHz Micro‐Convex Ultrasound Transducer, Mindray North America) and palpation (Figure 1a). The clipped area was cleaned of visible debris using 70% ethanol. The area was then scrubbed three times with alternating povidone–iodine solution and 70% ethanol solution. Lidocaine hydrochloride 2% was administered subcutaneously and the area was scrubbed twice more to further sterilize the biopsy site as well as disperse the lidocaine (see Supporting Information).

FIGURE 1 Photos from 10‐week biopsy of a calf. (a) The animal's ultrasound video of the biopsied quarter with the parenchyma outlined in white. The white bar denotes 1 cm. (b) The surgeon grasping the mammary gland for the incision. (c) A 0.5–1 cm vertical incision is made. (d) The 2 mm punch being placed at the incision site. (e) The suction device used to assist in tissue collection. (f) Tissue that may be collected from a biopsy at 10 weeks of age. The white bar indicates 1 cm.

2.1.2 Biopsy

With sterile gloves, the mammary gland was gripped by hand as shown in Figure 1b. A 0.5–1 cm vertical incision was made in the skin and a 2 mm disposable biopsy punch (Integra Miltex 2 mm biopsy punch) was inserted into the mammary gland by applying pressure and turning the punch tool clockwise 0.5–1 cm into the back end of the mammary gland opposite of the teat connection to avoid damaging the developing cistern and surrounding structures (Figure 1b–d). A suction device consisting of a catheter adaptor, extension line and 20 mL syringe was used to ensure the tissue was extracted with the biopsy tool (Figure 1e). The tissue was removed from the punch with sterile forceps and rinsed with sterile saline. Tissue size was typically 1 mm by 0.5–1 cm (Figure 1f). The tissue was then placed in an Eppendorf tube and frozen in liquid nitrogen or dry ice.

2.1.3 Closure

Blood at the incision site was cleaned with sterile gauze and a few drops of Vetbond tissue adhesive were applied as the wound edges were pinched together lightly. Due to the small size of incision at 10 weeks of age, sutures were not necessary. Aluspray Aerosol Bandaid was applied to the biopsy site and intravenous flunixin meglumine was administered at a dose of 1.1 mg/kg bodyweight via the jugular vein.

2.1.4 Postoperative care

The animal was placed in a recovery area until they stood on their own, typically within 30 min. Once the animal stood on its own, it was returned to its pen. The animal was then supervised periodically to ensure it was eating and drinking normally. Rectal temperatures were collected, and the incision site checked for signs of infection daily for 3 days post‐biopsy.

2.1.5 Follow‐up

The biopsy site was assessed again 7 days post‐biopsy and imaged by ultrasound 2 weeks post‐biopsy to check for abnormalities. Incision sites were fully healed by the end of 2 weeks.

2.2 Six months of age through lactation

2.2.1 Biopsy preparation

Prior to biopsies, the animals were weighed to determine drug dosage. Health parameters were collected for all animals undergoing biopsies to ensure all animals were healthy as performed in calves. Animals were restrained using a chute and xylazine hydrochloride (20 mg/mL) was administered intravenously via the coccygeal vessels at a dose of 6–10 mg pre‐gestation, starting with lower doses for animals which were smaller in size or more sensitive to xylazine. Atipamezole was administered intramuscularly to animals, if necessary, at a dose of 1 mg/kg bodyweight. During gestation and through the first lactation, fixed amounts of 10 mg xylazine and 10 mg butorphanol were administered intravenously. Butorphanol was added to the protocol at 6 months of age as an additional analgesic per veterinarian recommendation. Beyond the first gestation, xylazine and butorphanol were both administered at a dose of 0.02 mg/kg intravenously.

The animals stood for the procedure after 10 weeks of age and biopsies were performed in cattle chutes. The udder and the inner regions of the leg adjacent to the udder were clipped of hair (Figure 2). Ultrasound was then used to identify the biopsy site, avoiding large blood vessels and the cistern (Figure 2a,b). The skin was cleaned of visible debris with 70% ethanol before scrubbing three times with alternating povidone–iodine solution and 70% ethanol solution (Figure 2c). Lidocaine hydrochloride 2% was administered subcutaneously, 2 mL before gestation, 2–3 mL during gestation depending on the udder size and 5 mL during lactation. The surgical site was then scrubbed twice more to further sterilize the biopsy site and disperse the lidocaine.

FIGURE 2 Images from a cow at 45DIM. (a and b) Ultrasound imaging of the left rear quarter to identify the biopsy location, avoiding major blood vessels and the cisternal space. The white bar denotes 1 cm. (c) The biopsy site is marked by permanent marker, scrubbed and lidocaine is administered subcutaneously. (d) A 1–1.5 cm vertical incision is made. (e) A 6 mm punch is used to collect the tissue. (f) The tissue collected from the animal was rinsed with sterile saline. The white bar denotes 1 cm in length. (g) The incision site is closed with three sutures total about 4 mm apart. (h) The surgical site is sprayed with Aluspray and fly spray. (i) The same animal's udder 45 days post‐biopsy in which the surgical site is fully healed.

2.2.2 Biopsy

A 1–1.5 cm vertical incision site was made and a 6 mm disposable biopsy punch (Integra Miltex 6 mm biopsy punch) was inserted by applying pressure and turning the punch tool clockwise 3–4 cm into the mammary gland ending with a scooping motion at a 45‐degree angle to cut the end of the tissue piece (Figure 2d,e). The 6 mm disposable biopsy punch was used after 10 weeks of age, although from 6 to 12 months of age the punch size can range between 4 and 6 mm and during gestation and lactation the biopsy punch size can range between 6 and 8 mm. The punch was then removed from the animal and the tissue was removed from the punch using sterile tissue forceps. If necessary, sterile tissue forceps were used to carefully extract the tissue from the udder if it was not removed with the punch. A sterile drape‐covered icepack was used to hold pressure for 10 min to achieve haemostasis and the incision was sutured. If haemostasis was not achieved after 10 min, pressure was held again for 10 min before suturing. Of 511 biopsies, 13 required an additional 10 min of ice pressure. If after the total 20 min of pressure haemostasis is not achieved, the incision should be sutured, and pressure held once more until haemostasis is achieved. This was not necessary in the biopsies reported in this text. Tissue size was typically 4–5 mm by 2–4 cm (Figure 2f). The tissue was rinsed with sterile saline and placed in an Eppendorf and frozen in liquid nitrogen or dry ice. If tissue was going to be used for histology, it was placed in 10% buffered formalin for 24 h at room temperature.

2.2.3 Closure

Due to the increased size of the incisions, Vetbond tissue adhesive was not suitable for closure beyond 10 weeks of age. The skin incision was closed with #2‐0 nylon monofilament suture with an FS (for skin) reverse cutting needle in an interrupted pattern spaced at about 4 mm before lactation (Figure 2g). During lactation, the skin incision was closed with #2‐0 poliglecaprone 25 FS‐1 reverse cutting‐edge needle in an interrupted pattern spaced at 4 mm (Figure 2g). The udder was then cleaned of blood and the incision site was sprayed with Aluspray Aerosol Bandaid (Figure 2h). Fly spray was also applied during warmer weather. Intravenous flunixin meglumine was administered at a dose of 1.1 mg/kg bodyweight via the jugular vein in addition to butorphanol. During the transition period, flunixin meglumine was not administered as it has been shown to lead to decreased milk production and increased odds of having retained placenta as well as increased risk of metritis (Newby et al., 2017).

2.2.4 Postoperative care

Animals were fed after being released from the chute to encourage standing for at least 15 min to allow for further coagulation at the biopsy site. The incision site and body temperature were checked daily for 3 days post‐biopsy for signs of swelling or infection. The animals were also observed to ensure normal rumination. The sutures were removed 7–10 days after the biopsy. The skin incisions fully healed within 2–3 weeks.

2.2.5 Follow‐up

The biopsy site was palpated 4 weeks post‐biopsy and imaged by ultrasound during the next biopsy to check for any abnormalities (Figures 2i and 3).

FIGURE 3 Images from a 5‐week dry cow. (a) An external image displaying incision sites 2 weeks (black arrow) and 4 weeks post‐biopsy (white arrow). (b) An ultrasound image of the incision site 2 weeks after a biopsy procedure in which there is an indication of the procedure internally superficial to the gland and indicated by the white arrow. (c) An ultrasound image of the incision site 4 weeks after a biopsy in which there is no longer an indication of the biopsy procedure having been performed internally. The scale bar indicates 1 cm.

2.3 Statistical analysis

Linear mixed models with repeated measures (R Version 4.0.4.) were utilized to analyse milk production in the days surrounding the biopsy procedures, including the fixed effect of day (in relation to the procedure) and the random effect of animal (Figure 4).

FIGURE 4 Milk production (kg) from 5 days before biopsy through 5 days after the biopsies at 45, 90, 150 and 270 days in milk (DIM) in animals that were repeatedly biopsied. Milk production was decreased on Days 1, 2 and 3 (p < 0.05) compared to Days −5, −4, −3, −2, −1 and 0, although production was restored by Days 4 and 5 post‐biopsy (p > 0.05).

3 RESULTS

3.1 Intraoperative complications

Two animals collapsed prior to the biopsy procedures pre‐gestation due to sensitivity to the xylazine hydrochloride administered. Atipamezole was administered intramuscularly at a dose of 0.02 mg/kg bodyweight, and the animals recovered shortly after. These animals were administered reduced doses of xylazine at subsequent biopsies.

The disposable biopsy punch tool broke during two biopsies. However, the pieces were retrieved, and the biopsy continued normally with no further complications.

No blood transfusions were required for any animals at any point as bleeding was minimal. One case out of 511 biopsies resulted in heavier bleeding, although not life‐threatening. With ice pressure, haemostasis was achieved.

3.2 Postoperative complications

Of the 511 biopsies performed, there were 17 instances of incisional infections due to exposure to manure. With the application of triple antibiotic and Aluspray Aerosol Bandaid over the course of several days the infections cleared up and the incision site healed. One animal developed mastitis due to its incision site reopening shortly after the 6‐month biopsy. Treatment with Spectramast Dry was attempted, although the condition of the quarter did not improve; therefore, the animal was sold due to breeding difficulties. After this incident, sutures were used in place of Vetbond tissue adhesive for biopsies after 10 weeks of age to reduce the risk of wound splitting. Another animal had chronic mastitis in all quarters during gestation and early lactation in biopsied and non‐biopsied quarters; therefore, biopsies were discontinued, and it was also sold. Six biopsies resulted in clinical mastitis that could be treated.

Some animals had bloody milk post‐biopsy, although the presence of blood in the milk cleared up within 36–48 h post‐biopsy. Typically, if an animal had bloody milk at one biopsy, it tended to have bloody milk in other biopsies. Milk production was decreased 3 days post‐biopsy (p < 0.05), although milk production was restored by Days 4 and 5 (p > 0.05; Figure 4). Milk was withheld for a minimum of 3 days after the procedure due to medication administration during the biopsies. Two animals had blind quarters at parturition in the quarter that was biopsied at 10 weeks of age, although it is unclear as to the cause as one animal has scar tissue blocking the milk from exiting the cistern and the other animal did not undergo ultrasound imaging at calving to determine the cause. It is possible that the biopsy at 10 weeks of age resulted in defects in these quarters, although blind quarters can occur in heifers due to suckling by other heifers (Swett et al., 1939).

3.3 Outcomes

Of 511 biopsies performed, one biopsy resulted in serious complications as discussed previously. Animals which underwent biopsies at 10 weeks of age, in addition to those who have undergone repeated biopsies across their lifespan, have lactated normally. The udders of the animals that underwent repeated biopsies have superficial scarring, although in most animals the scarring consists of very faint lines where the incisions were made. The animal that chewed its teat had its udder dissected upon necropsy, and no visible scarring from biopsies was detected internally.

4 DISCUSSION

The results of this study demonstrated that the ultrasound‐guided biopsy procedure using disposable biopsy punches was a relatively safe and effective method for mammary gland tissue collection. The use of ultrasound minimizes the risk of performing the biopsy near the cistern and large blood vessels, minimizing cisternal damage as well as bleeding. Utilization of the disposable biopsy punch allows for the collection of large amounts of tissue with simple tools that do not need to be assembled, nor cause extensive tissue damage, and require small incision sites ranging from 0.5 to 1.5 cm in length depending on the punch size.

Although the goal of the study was to develop a biopsy method appropriate for young animals to reduce the need to euthanize animals for tissue collection and to allow for repeated measures analysis of longitudinal studies, the method was modified and used for mature animals to develop a single method suitable through the animal's lifetime. The described biopsy method can be used for both clinical diagnoses as well as for research purposes in animals as young as 8 weeks of age. In addition, as this method was successfully used to obtain tissue from calves, it can likely be used in other species with smaller udders, such as sheep and goats.

A limitation of this method of tissue collection was the size of the tissue collected. As we are collecting a tissue biopsy, the tissue may not be representative of the entire gland. The advantage of the biopsy is that the tissue is still invaluable as it provides insight into mammary gland development and cellular activities that are critical for mammary gland function due to treatment manipulations. Typically, only one biopsy punch was necessary for the amount of tissue needed for the experiment, although it is possible to increase the number of punches if more tissue is required.

AUTHOR CONTRIBUTIONS

Alysia L. Vang: Investigation; conceptualization; writing—original draft; methodology; data curation; formal analysis. Waneska S. Frizzarini: Writing—review & editing; investigation; methodology; data curation. Haylee H. Webster: Investigation; writing—review & editing; data curation. Thiago O. Cunha: Investigation; writing—review & editing; data curation. Kathryn M. Nelson: Conceptualization; supervision. Joao R.R. Dorea: Resources; project administration; formal analysis; writing—review & editing; supervision; funding acquisition. Laura L. Hernandez: Writing—review & editing; resources; supervision; project administration; conceptualization; investigation; funding acquisition; methodology.

ETHICS STATEMENT

All procedures were approved by the Animal Use and Care Committee of the University of Wisconsin—Madison (A006270‐R01). The same individual performed all biopsies.

Supporting information

Supporting Information

ACKNOWLEDGEMENTS

Thank you to Dr. Michael Maroney for assisting in early biopsies. Thank you to Dr. Holly Hovanec and RARC staff for caring for the animals. Thank you to the staff at the Blaine Dairy Cattle Center as well as the Marshfield Agricultural Research Station for their support for research and care for the animals. Thank you to Negreiro A and Menezes GL for occasionally assisting in biopsies. A very special thank you to the exceptional heifers on trial, as their contribution is invaluable. Research reported in this publication was supported by the National Institute of Food and Agriculture of the United States Department of Agriculture under award numbers 2020‐67015‐30831 and WIS04094. Alysia L. Vang was supported by National Institute of Health Training grants R25GM083252 and T32GM135066.

DATA AVAILABILITY STATEMENT

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable.
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