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ABSTRACT
Intramammary infections are common in nonlactating 
dairy cattle and have been shown to disrupt mammary 
tissue architecture in nonpregnant heifers. However, 
their effect on mammary development during pregnancy 
remains unclear. This study assessed the effects of IMI on 
mammary gland development in pregnant dairy heifers 
during late gestation. The study used 21 pregnant Hol-
stein heifers, divided across 3 gestational stages (~5.75, 
6.75, and 7.75 mo of gestation; corresponding to 180 ± 
2, 208 ± 2, and 238 ± 2 d pregnant, respectively). Us-
ing a contralateral quarter-pair design, a single culture-
negative quarter of each heifer was infused with saline 
(SAL), and the contralateral quarter was challenged with 
5,000 cfu of Staphylococcus aureus (CHALL). Mammary 
secretion samples were collected at various time points 
until tissue harvest at 21 d postchallenge, when animals 
were 6.5, 7.5, and 8.5 mo pregnant. Mammary tissue 
samples from the center and edge parenchymal regions 
were collected and evaluated for immune cell infiltration 
and tissue morphometry. Secretions from CHALL quar-
ters had greater SCC and a greater proportion of neu-
trophils compared with SAL quarters. Mammary tissues 
from CHALL quarters exhibited increased immune cell 
infiltration in both the luminal and intralobular stromal 
regions and lower secretion score compared with SAL, 
regardless of gestational stage. Additionally, tissues 
from animals at later gestational stages showed reduced 
adipose tissue area and larger lobular areas, regardless of 
quarter treatment. At 8.5 mo of pregnancy, luminal areas 
in the edge regions of CHALL quarters were nearly 50% 
smaller than in SAL quarters, suggesting an increased risk 
to restricting milk accumulation and secretion capacity 
in the mammary gland. Additionally, in 7.5-mo pregnant 
heifers, CHALL quarters showed decreased epithelial ar-
eas and increased intralobular stromal areas in the central 
region. Lobular, adipose, and extralobular stromal areas 
did not differ markedly between CHALL and SAL quar-
ters. Overall, the results of this study indicate that IMI 
induces tissue damage in mammary glands of pregnant 
heifers, with a greater effect during late gestation, and 
that the IMI-induced changes in tissue architecture were 
not consistent across all tissue mammary gland regions 
or gestational ages.
Key words: mastitis, pregnancy, mammogenesis, 
Staphylococcus aureus
INTRODUCTION
Mastitis, defined as an inflammatory condition of 
the mammary gland, is almost exclusively resultant of 
an IMI by a microorganism in cattle. Mastitis is one of 
the most economically important diseases in the US and 
global dairy industries, and most financial losses result 
from reduced milk production of affected mammary 
glands (Hogeveen et al., 2019; Rasmussen et al., 2024). 
Although mastitis is often assumed to affect lactating 
cows, many studies have documented IMI in nulliparous 
and primigravid heifers (Minett et al., 1933; Oliver and 
Mitchell, 1983; Trinidad et al., 1990b). Such IMI often 
go unnoticed because they are predominantly subclini-
cal and heifer udders are not inspected multiple times 
per day as with lactating cows during routine milking. 
Quarter IMI prevalen
ce in gravid dairy heifers has been reported to range 
from 26% (Larsen et al., 2021) to 75% (Trinidad et al., 
1990b), with a weighted average of ~43% (Enger, 2018). 
Heifers with IMI during the first pregnancy can be less 
productive than healthy herdmates (Oliver et al., 2003) 
and have a greater risk of culling due to higher SCC (De 
Vliegher et al., 2005), highlighting the potential impact 
of gestational IMI on early lactation performance and 
herd longevity. Despite these long-term implications, the 
consequences of IMI on mammary growth and develop-
ment during pregnancy remain unclear and unexplored.
Complete development of a functioning mammary 
gland is central to lactational success and performance 
Impact of intramammary infections on mammary gland 
development in pregnant dairy heifers during late gestation
M. X. S. Oliveira,1 C. S. Gammariello,1 P. H. Baker,1 K. M. Enger,1 S. K. Jacobi,1 and B. D. Enger1* 
1Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691
2Department of Animal Sciences, The Ohio State University, Columbus, OH 43210
J. Dairy Sci. TBC
https://doi.org/10.3168/jds.2025-26734
© TBC, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. 
This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-25. Nonstandard abbreviations are available in the Notes.
Received April 9, 2025.
Accepted August 16, 2025.
*Corresponding author: enger.5@osu.edu
https://orcid.org/0000-0003-1368-8716
https://orcid.org/0009-0005-7188-7408
https://orcid.org/0000-0003-4943-9279
https://orcid.org/0000-0002-8295-4128
https://orcid.org/0000-0002-2374-2280
https://orcid.org/0000-0001-7760-3107
https://adsa.org/jds-abbreviations-25
mailto:enger.5@osu.edu
Journal of Dairy Science Vol. TBC No. TBC, TBC
(Capuco and Akers, 2009). Accretion of mammary epi-
thelial cells is exponential during first pregnancy, with 
the greatest accretion of cells occurring during the last 
trimester of pregnancy (Swanson and Poffenbarger, 
1979). Substantial development of the epithelial tissue 
occurs, with epithelium branching and infiltrating the 
fat pad. Notably, this crucial phase of mammary growth 
and development coincides with the greatest prevalence 
of IMI in gravid heifers (Trinidad et al., 1990b). For 
instance, Fox et al. (1995) reported an increase in the 
percentage of infected quarters from 29% during the first 
trimester of gestation to 33% in the second trimester and 
45% in the third trimester. Impediments to the growth 
and development of the mammary gland during first 
pregnancy are anticipated to negatively affect mammary 
tissue maturation and subsequent milk production.
Few have investigated the effects of IMI on the mam-
mary glands of heifers. Boddie et al. (1987) observed 
leukocyte infiltration and histological alterations in nul-
liparous heifer mammary glands infected with Staphy-
lococcus aureus and coagulase-negative staphylococci. 
Trinidad et al. (1990a) subsequently reported that IMI in 
nulligravid heifer mammary glands resulted in increased 
infiltration of lymphocytes and neutrophils in the stromal 
and luminal regions of infected mammary glands; the in-
fected mammary glands also had a greater stromal area 
and less luminal space and epithelial tissue areas. A recent 
study by Baker et al. (2023b) reported that Staphylococ-
cus aureus–infected quarters in nulliparous, nonpregnant 
heifers, whose mammary glands were stimulated to grow 
via supraphysiological injections of estrogen and pro-
gesterone, exhibited increased immune cell infiltration, 
reduced epithelial area, and more intralobular stromal 
areas than uninfected quarters. The same Staphylococ-
cus aureus–infected quarters had a greater percentage of 
apoptotic mammary epithelial cells and a decreased in 
the percentage of apoptotic stromal cells than uninfected 
quarters (Baker et al., 2023a), suggesting stromal tissues 
were failing to regress and lumen spaces were failing to 
increase. Although previous works indicate how IMI af-
fect mammary glands in native and hormonally treated 
nulliparous heifers, how IMI affects maturing mammary 
tissue of primigravid heifers is unknown and unexplored. 
Thus, further research is needed to elucidate the effects 
of IMI on mammary development during the critical pe-
riod of pregnancy in heifers.
The objectives of this study were to (1) evaluate how 
Staphylococcus aureus IMI affects mammary secretion 
SCC and differential cell counts, (2) assess immune cell 
infiltration in the developing mammary gland tissues ofpregnant dairy heifers at different stages of late gesta-
tion, and (3) determine whether IMI induces architec-
tural changes in mammary tissue as measured by histo-
morphometry. We hypothesized that IMI would impair 
mammary development by restricting epithelial tissue 
expansion and delaying stromal tissue regression.
MATERIALS AND METHODS
Animal Selection and Study Design
A total of 21 pregnant Holstein heifers were selected 
from The Ohio State University Krauss Dairy Farm. 
Seven heifers from 3 distinct gestational stages (~5.75, 
6.75, and 7.75 mo of gestation; corresponding to 180 
± 2, 208 ± 2, and 238 ± 2 d gravid, respectively) were 
used. Animals joined the study in 3 different cohorts 
containing 9, 6, and 6 animals, respectively, from 
May 2022 to May 2023; equal representation of each 
gestational state was present for each individual co-
hort. Heifers were eligible for study inclusion if they 
were free of IMI in at least 2 contralateral mammary 
glands and were clinically healthy. Heifer quarters 
were aseptically sampled thrice, with 2 d between 
each sampling immediately before the start of the trial. 
Bacterial screening followed the National Mastitis 
Council’s guidelines (Adkins et al., 2017), in which 
a 10-μL aliquot of fresh secretion was streaked onto 
blood agar (Columbia Blood Agar, Hardy Diagnostics, 
Santa Maria, CA), incubated at 37°C, and examined at 
24 and 48 h. Samples were classified as culture posi-
tive if 3 or more colonies of similar morphology were 
observed. Selected heifers were moved to the Ohio 
Agricultural Research and Development Beef Research 
Center (Wooster, OH) 3 d before the start of the experi-
ment. To be eligible for inclusion in the study, heifers 
were required to have both rear or both fore mammary 
quarters be culture-negative. If both fore and rear pairs 
were eligible, the random number generator function 
in Microsoft Excel was used to determine whether the 
fore or rear pair would be used. Within the selected 
pair, one quarter was randomly assigned to receive a 
Staphylococcus aureus challenge (CHALL), and the 
contralateral quarter received a sterile phosphate-buff-
ered saline infusion (SAL), using the same randomiza-
tion method. This approach, which involved treating 
one quarter per udder half, differs from the traditional 
split-udder design where 2 ipsilateral quarters receive 
the same treatment. Nonetheless, assigning CHALL 
and SAL treatments to contralateral quarters within 
the same animal enabled direct within-animal compari-
sons, minimizing interanimal variability. Mammary 
secretion samples were collected on d 0, 1, 2, 8, 14, 
and 20 relative to challenge day and were evaluated for 
bacteriology, total SCC, and somatic cell differential 
counts. Heifers were transported to The Ohio State 
University’s Meat Laboratory (Columbus, OH) on the 
afternoon of d 20 and were stunned by captive bolt and 
Oliveira et al.: INTRAMAMMARY INFECTION PREGNANT HEIFERS
Journal of Dairy Science Vol. TBC No. TBC, TBC
euthanized via exsanguination for tissue collection the 
following morning (d 21). Whole udders were immedi-
ately removed, and mammary parenchyma tissues were 
sampled from CHALL and SAL at 2 regions: (1) the 
edge parenchyma, proximal to the abdominal wall and 
bordering the fat pad, and (2) the center parenchyma, 
located slightly above the gland cistern and proximal 
to the teat end. At the time of tissue collection, animals 
in their respective gestational groups were 6.5, 7.5, and 
8.5 mo pregnant.
Intramammary Challenge
Staphylococcus aureus was selected as the challenge 
organism for the present study due to its importance as a 
common and persistent cause of subclinical and chronic 
IMI, as well as its predictability to establish an IMI that 
would last the study duration. It is frequently isolated 
from infected quarters in heifers (Trinidad et al., 1990b; 
Fox et al., 1995; Fox, 2009) and is known to induce ar-
chitectural changes in mammary tissue (Nickerson and 
Heald, 1981; Trinidad et al., 1990a; Baker et al., 2023b). 
The Staph. aureus novel strain isolated by Smith et al. 
(1998) was used as the challenge organism in this study, 
as previously detailed by Enger et al. (2018). Briefly, 
trypticase soy broth was inoculated with a single Staph. 
aureus colony and incubated at 37°C in an orbital shak-
ing incubator for 6 h. The bacteria were washed thrice 
and adjusted to a concentration of 5 × 108 cfu/mL. The 
culture was then serially diluted to a target concentra-
tion of 5,000 cfu/mL. Tuberculin syringes were loaded 
with 1-mL of the target dilution suspension, placed on 
ice, and transported to the animal unit for intramam-
mary infusion using a sterile plastic cannula (Becton, 
Dickson and Company, Franklin Lakes, NJ). The dos-
age of Staph. aureus infused into each mammary gland 
was 8,200, 6,600, and 5,600 cfu for cohorts 1, 2, and 3, 
respectively. Contralateral SAL quarters were infused 
with 1 mL of PBS. Before intramammary infusion, teats 
were first immersed in an iodine solution (Teat-Kote 10/
III, GEA Farm Technologies Inc., Naperville, IL) and 
wiped clean, then sprayed with a chlorhexidine solution 
(Fight Bac, Deep Valley Farm, Brooklyn, CT) before 
being wiped clean, and then teat ends were scrubbed 
with an 70% ethanol-soaked cotton square. All mam-
mary infusions were conducted via the partial insertion 
technique (Boddie and Nickerson, 1986).
Mammary Secretion Collection and Processing
Mammary secretion samples were aseptically col-
lected from heifers following the procedures of Enger et 
al. (2018) and expressed into sterile 5-mL round-bottom 
polystyrene tubes. Samples were immediately placed on 
ice for transport to the laboratory for bacterial culture 
and somatic cell quantification and differentiation.
First, mammary secretions were streaked for isola-
tion using a 10-µL aliquot on blood agar following 
standard procedures (Adkins et al., 2017) and incubated 
at 37°C. Smears were examined at 24 and 48 h, and 
resultant microbial growth was subject to biochemical 
examinations; Staphylococcus spp. were classified as 
Staph. aureus based on a coagulase test. Second, so-
matic cells were microscopically enumerated using the 
methodology described by Enger et al. (2020). Secre-
tion samples were diluted 1:10 with PBS containing 
2.2% BSA, vortexed for a few seconds, spread onto 
milk somatic cell counting slides (Bellco Glass Inc., 
Vineland, NJ), dried, and stained with Newman's Modi-
fied Stain Solution (Sigma-Aldrich Corporation, Saint 
Louis, MO). A 5-mm square reticle (Microscope World, 
Carlsbad, CA) was used to count stained cells under oil 
immersion, producing a strip width of 0.050 mm for the 
microscope-reticle combination. The SCC of the undi-
luted secretion sample was calculated using enumerated 
smears, and these SCC were averaged to create a single 
SCC estimate for each secretion sample.
The methods for differentiating secretion somatic 
cells were previously described (Enger et al., 2018). In 
short, duplicate smears were created using 10 to 20 μL of 
fresh mammary secretion and 50 μL of PBS with 2.2% 
BSA. A Shandon CytoSpin 2 (Thermo Fisher Scientific, 
Waltham, MA) was used to prepare the smears at 110 × 
g for 10 min. Slides were dried, stained with Wright–
Giemsa stain (Electron Microscopy Sciences, Hatfield, 
PA), and then immersed into stain-primed phosphate 
buffer (6.8 pH; Electron Microscopy Sciences). Slides 
were rinsed, dried, and coverslipped. Somatic cells were 
differentiated as (1) neutrophils, (2) macrophages, which 
could not be differentiated from epithelial cells, and (3) 
lymphocytes. A total of 100 cells were differentiated for 
each duplicate smear, resulting in 200 cells being differ-
entiated and used to calculate percentages for each cell 
type. Secretion SCC and differential cell counts were 
assessed to confirm that an immune response was elic-
ited in response to Staph. aureus intramammary infusion 
while remaining absent inSAL quarters. This analysis 
served as a secondary validation, alongside bacteriology 
results, to ensure the success of quarter treatments.
Mammary Tissue Sampling and Processing
Tissue samples were collected as in Baker et al. 
(2023b). Sampled tissues were immediately placed in 
10% formalin, fixed for 48 h, and subsequently trans-
ferred and stored in 70% ethanol. Fixed tissue tissues 
were processed and embedded in paraffin by the Ohio 
State Veterinary School Histopathology Laboratory.
Oliveira et al.: INTRAMAMMARY INFECTION PREGNANT HEIFERS
Journal of Dairy Science Vol. TBC No. TBC, TBC
Mammary Tissue Histologic Analysis
Paraffin-embedded tissues were sectioned and stained 
with hematoxylin and eosin following the procedures 
of Tucker et al. (2016). Stained sections were visual-
ized and imaged as before (Baker et al., 2023b). Our 
tissue evaluation followed a multistep approach, first 
using a course evaluation of tissue structure areas (i.e., 
quantifying lobular, adipose, and extralobular stroma) 
and then a secondary, intralobular specific, tissue area 
assessment (i.e., quantifying intralobular epithelial, in-
tralobular stroma, and intralobular luminal). One tissue 
section was examined for each unique heifer, mammary 
gland, and parenchymal region combination. Images 
were acquired for 3 randomly selected fields of view at 
40× magnification and for 8 fields at 100× magnifica-
tion. The summed total area imaged for the three 40× 
magnification and eight 100× magnification images was 
13.0 mm2 and 5.7 mm2, respectively. Mammary tissue 
structures were manually traced using CellSens imag-
ing software (Olympus Corp.) to yield the area of each 
traced object. For 40× images, adipose and all epithelial 
structures and internalized luminal spaces (lobules and 
ducts) were traced and measured (Figure 1A); extralob-
ular stromal tissue area was calculated by difference 
and identified as tissue not labeled as either adipose 
or epithelia/luminal areas. For 100× images, lobules, 
intralobular epithelium, and intralobular lumens were 
traced and measured. Intralobular stromal tissue area 
was determined by subtracting the epithelial area from 
the total lobular area (Figure 1B). Tissue areas values 
are expressed as the area occupied (mm2) per 1 mm2 of 
evaluated mammary tissue.
Immune Cell Infiltration and Luminal 
Secretion Scoring
The acquired 40× images were used for a secondary 
assessment to quantify (1) the extent of immune cell in-
filtration in both intralobular stromal and luminal tissue 
and (2) the composition of luminal secretions. Scoring 
followed the procedures detailed by Baker et al. (2023b). 
Briefly, a scorer blinded to treatments graded luminal 
and intralobular stromal infiltration on a scale ranging 
from 1 to 3. A score of 1 indicated the absence of immune 
cells, 2 denoted their presence in half of the available 
area, and 3 signified their presence in over two-thirds of 
the respective areas. Luminal secretion score evaluations 
were used to assess the composition of lumen contents 
as a means of estimating secretory epithelial activity. 
Luminal secretion scores ranged from 1 to 4. A score of 
1 indicated lumens devoid of fat globules, 2 indicated 
less than half of luminal spaces contained fat globules, 3 
indicated over half of luminal spaces contained fat glob-
ules, and a score of 4 was assigned to distended lumens 
containing protein granules and fat globules.
Statistical Analysis
Power Analysis. A power analysis was conducted us-
ing PROC POWER in SAS 9.4 (SAS Institute Inc., Cary, 
NC), based on previously mammary gland morphology 
data (Enger et al., 2018). Power calculations were made 
after considering means and SD reported by Howe et al. 
(1975), Nickerson and Heald (1981), and Trinidad et al. 
(1990a). Mean luminal areas of SAL and CHAL quarters 
were predicted to be 20% for SAL quarters and 13% for 
Oliveira et al.: INTRAMAMMARY INFECTION PREGNANT HEIFERS
Figure 1. Representative mammary tissue sections from pregnant heifers stained with hematoxylin and eosin. A: Lobules (yellow) and adipose 
tissue (black) are outlined, with extralobular stromal areas calculated as the total area minus lobular and adipose areas. Scale bar = 200 μm. B: 
Lobular (yellow), epithelial (blue), and luminal (red) areas are delineated to estimate tissue components, with intralobular stromal areas calculated 
as lobular area minus epithelial and luminal areas. Scale bar = 100 μm.
Journal of Dairy Science Vol. TBC No. TBC, TBC
CHALL quarters. Assuming a SD of 6.0, a power of 0.80, 
and an α level of 0.05, a minimum of 16 animals was 
required to detect such a difference. The within-animal 
design of comparing treatments within the same heifer 
would reduce unexplained variation. We have used simi-
lar approaches in our previous studies investigating IMI 
in dry cows (Enger et al., 2018) and nulliparous heifers 
(Baker et al., 2023b).
Somatic Cell Counts and Differentials. Secretion 
SCC and differentials were analyzed using the GLIM-
MIX procedure of SAS (Version 9.4, SAS Institute, Cary, 
NC). Separate models were constructed for each of the 
following outcome variables: (1) log10-transformed SCC, 
(2) neutrophil percentage, (3) macrophage percentage, 
and (4) lymphocyte percentage. For each model, the ex-
planatory variables included gestational stage, trial day, 
quarter treatment, and the interaction between quarter 
treatment and trial day. All models were specified as lin-
ear mixed models assuming a normal distribution with an 
identity link function. Fixed effects were chosen a priori 
based on experimental design and biological relevance. 
Random effects included cohort and heifer nested within 
cohort. Trial day was modeled as a repeated measure, and 
covariance structures (i.e., variance components, com-
pound symmetry, and unstructured) were selected based 
on the lowest Akaike information criterion values and 
residual distribution. Residuals were visually inspected 
for each model, and outliers were removed if the residual 
exceeded 3.5 SD from the mean. This threshold was se-
lected to prevent undue influence from extreme values 
while preserving biologically meaningful variation and 
resulted in the removal of 4 observations for the log10 
SCC model, 5 observations for the neutrophil percent-
age model, 3 observations for the macrophage model, 
and 5 observations for the lymphocyte model. The Ken-
ward–Roger denominator degrees of freedom correction 
was applied to all models (Kenward and Roger, 1997). 
Statistical significance was declared when P ≤ 0.05, and 
marginal significance when P ≤ 0.10.
Tissue Areas. Tissue areas were analyzed using PROC 
GLIMMIX; separate statistical models were constructed 
for the following outcomes: lobular area, adipose tissue 
area, extralobular stroma area, intralobular stroma area, 
intralobular epithelium arear, and intralobular luminal 
space. All models accounted for a 3-way factorial treat-
ment design, incorporating the fixed, explanatory effects 
of gestational stage, quarter treatment, mammary region, 
and all possible interactions. The treatment structure was 
embedded within a split-split-plot design, where heifer 
served as the whole plot, mammary glands as the first 
split, and mammary regions as the second split. Random 
effects included cohort and heifer within cohort. To ac-
count for variability in adipose tissue across gestational 
ages, residual heterogeneity by age was specified to meet 
model assumptions. Statistical significance was declared 
when P ≤ 0.05, and marginal significance when P ≤ 0.10.
Tissue Scores. The modeling approach for immune 
cell infiltration and luminal secretion scoring followed 
used the exact approach and structure described for tis-
sue areas. Fixed effects included gestational stage, quar-
ter treatment, mammary region, and their interactions. 
Statistical significance was set at P ≤ 0.05, and marginal 
significance at P ≤ 0.10.
RESULTS
Baseline MeasuresMean body weights at the time of enrolment were 
600 ± 24 kg for the 6.5-mo gravid heifer groups, 623 
± 24 kg for the 7.5-mo gravid heifer group, and 651 ± 
25 kg for the 8.5-mo gravid heifers. All animals were 
clinically healthy and did not display any observable 
signs of disease.
Success of Intramammary Challenge
Staphylococcus aureus infusion established IMI in all 
challenged quarters that lasted the study duration, and 
all SAL quarters remained bacteriologically negative 
throughout. Clinical signs of mastitis, such as clots and 
flakes in the mammary secretion, were observed in ~20% 
of all CHALL glands; 1 heifer developed a mild fever 1 
d after intramammary infusions (zenith of 40.2°C) but 
remained afebrile thereafter. One heifer in the 8.5-mo 
pregnant group calved before tissue collection. Data from 
this animal were not included in the statistical analysis, 
yielding a final total of 20 CHALL and 20 SAL quarters.
Total and Differential Secretion Somatic Cell Counts
Mean log10 SCC was influenced by quarter treatment 
with trail day interaction (PINFECTION PREGNANT HEIFERS
Figure 3. Mean immune cell infiltration scores and luminal secretion 
scores for uninfected (SAL, solid blue bar) and Staphylococcus aureus–
infected (CHALL, textured red bar) are presented in Panel A. Error 
bars represent the SEM, and asterisks indicate significant differences 
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one receptors (PGR). Domest. Anim. Endocrinol. 54:95–105. https: / 
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ORCIDS
M. X. S. Oliveira, https: / / orcid .org/ 0000 -0003 -1368 -8716
C. S. Gammariello, https: / / orcid .org/ 0009 -0005 -7188 -7408
P. H. Baker, https: / / orcid .org/ 0000 -0003 -4943 -9279
K. M. Enger, https: / / orcid .org/ 0000 -0002 -8295 -4128
S. K. Jacobi, https: / / orcid .org/ 0000 -0002 -2374 -2280
B. D. Enger https: / / orcid .org/ 0000 -0001 -7760 -3107
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https://doi.org/10.1146/annurev-resource-100518-093954
https://doi.org/10.3168/jds.S0022-0302(75)84649-2
https://doi.org/10.3168/jds.S0022-0302(75)84649-2
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https://doi.org/10.3168/jds.S0022-0302(84)81510-6
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https://doi.org/10.1038/sj.cdd.4400878
https://doi.org/10.1038/sj.cdd.4400878
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https://doi.org/10.1111/j.1439-0531.2012.02102.x
https://doi.org/10.3168/jds.S0022-0302(03)73702-3
https://doi.org/10.3168/jds.S0022-0302(83)81916-X
https://doi.org/10.3168/jds.S0022-0302(83)81916-X
https://doi.org/10.3168/jds.2018-15668
https://doi.org/10.1023/A:1020343717817
https://doi.org/10.3168/jds.2011-4289
https://doi.org/10.3168/jds.2011-4289
https://doi.org/10.3168/jds.2023-24626
https://doi.org/10.3168/jds.2021-20776
https://doi.org/10.2460/javma.1998.212.04.553
https://doi.org/10.2460/javma.1998.212.04.553
https://doi.org/10.3168/jds.S0022-0302(79)83313-5
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https://doi.org/10.3168/jds.S0022-0302(90)78652-3
https://doi.org/10.1016/j.domaniend.2015.10.002
https://doi.org/10.1016/j.domaniend.2015.10.002
https://orcid.org/0000-0003-1368-8716
https://orcid.org/0009-0005-7188-7408
https://orcid.org/0000-0003-4943-9279
https://orcid.org/0000-0002-8295-4128
https://orcid.org/0000-0002-2374-2280
https://orcid.org/0000-0001-7760-3107
Journal of Dairy Science Vol. TBC No. TBC, TBC
A
PP
EN
D
IX
Oliveira et al.: INTRAMAMMARY INFECTION PREGNANT HEIFERS
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7:109–121. 
https: / / doi .org/ 10 .1023/ A: 1020343717817 .
Quesnell, R. R., S. Klaessig, J. L. Watts, and Y. H. Schukken. 2012. 
Bovine intramammary Escherichia coli challenge infections in late 
gestation demonstrate a dominant antiinflammatory immunological 
response. J. Dairy Sci. 95:117–126. https: / / doi .org/ 10 .3168/ jds .2011 
-4289.
Rasmussen, P., H. W. Barkema, P. P. Osei, J. Taylor, A. P. Shaw, B. 
Conrady, G. Chaters, V. Muñoz, D. C. Hall, O. O. Apenteng, J. 
Rushton, and P. R. Torgerson. 2024. Global losses due to dairy cattle 
diseases: A comorbidity-adjusted economic analysis. J. Dairy Sci. 
107:6945–6970. https: / / doi .org/ 10 .3168/ jds .2023 -24626.
Shangraw, E. M., and T. B. McFadden. 2022. Graduate Student Lit-
erature Review: Systemic mediators of inflammation during mastitis 
and the search for mechanisms underlying impaired lactation. J. 
Dairy Sci. 105:2718–2727. https: / / doi .org/ 10 .3168/ jds .2021 -20776 .
Smith, T. H., L. K. Fox, and J. R. Middleton. 1998. Outbreak of mastitis 
caused by one strain of Staphylococcus aureus in a closed dairy herd. 
J. Am. Vet. Med. Assoc. 212:553–556. https: / / doi .org/ 10 .2460/ javma 
.1998 .212 .04 .553.
Swanson, E. W., and J. I. Poffenbarger. 1979. Mammary gland devel-
opment of dairy heifers during their first gestation. J. Dairy Sci. 
62:702–714. https: / / doi .org/ 10 .3168/ jds .S0022 -0302(79)83313 -5.
Trinidad, P., S. C. Nickerson, and R. W. Adkinson. 1990a. Histopathol-
ogy of staphylococcal mastitis in unbred dairy heifers. J. Dairy Sci. 
73:639–647. https: / / doi .org/ 10 .3168/ jds .S0022 -0302(90)78715 -2.
Trinidad, P., S. C. Nickerson, and T. K. Alley. 1990b. Prevalence of 
intramammary infection and teat canal colonization in unbred and 
primigravid dairy heifers. J. Dairy Sci. 73:107–114. https: / / doi .org/ 
10 .3168/ jds .S0022 -0302(90)78652 -3.
Tucker, H. L. M., C. L. M. Parsons, S. Ellis, M. L. Rhoads, and R. M. 
Akers. 2016. Tamoxifen impairs prepubertal mammary development 
and alters expression of estrogen receptor α (ESR1) and progester-
one receptors (PGR). Domest. Anim. Endocrinol. 54:95–105. https: / 
/ doi .org/ 10 .1016/ j .domaniend .2015 .10 .002.
ORCIDS
M. X. S. Oliveira, https: / / orcid .org/ 0000 -0003 -1368 -8716
C. S. Gammariello, https: / / orcid .org/ 0009 -0005 -7188 -7408
P. H. Baker, https: / / orcid .org/ 0000 -0003 -4943 -9279
K. M. Enger, https: / / orcid .org/ 0000 -0002 -8295 -4128
S. K. Jacobi, https: / / orcid .org/ 0000 -0002 -2374 -2280
B. D. Enger https: / / orcid .org/ 0000 -0001 -7760 -3107
Oliveira et al.: INTRAMAMMARY INFECTION PREGNANT HEIFERS
https://doi.org/10.3168/jds.S0022-0302(95)76786-8
https://doi.org/10.3168/jds.S0022-0302(94)77153-8
https://doi.org/10.3168/jds.S0022-0302(94)77153-8
https://doi.org/10.1146/annurev-resource-100518-093954
https://doi.org/10.1146/annurev-resource-100518-093954
https://doi.org/10.3168/jds.S0022-0302(75)84649-2
https://doi.org/10.3168/jds.S0022-0302(75)84649-2
https://doi.org/10.2460/ajvr.1981.42.05.743
https://doi.org/10.3168/jds.S0022-0302(84)81510-6
https://doi.org/10.3168/jds.S0022-0302(84)81510-6
https://doi.org/10.2307/2533558
https://doi.org/10.3168/jds.2021-20819
https://doi.org/10.3168/jds.2021-20819
https://doi.org/10.1038/sj.cdd.4400878
https://doi.org/10.1038/sj.cdd.4400878
https://doi.org/10.1016/S0368-1742(33)80018-X
https://doi.org/10.1016/S0368-1742(33)80018-X
https://doi.org/10.2460/ajvr.1981.42.08.1351
https://doi.org/10.2460/ajvr.1981.42.08.1351
https://doi.org/10.1111/j.1439-0531.2012.02102.x
https://doi.org/10.1111/j.1439-0531.2012.02102.x
https://doi.org/10.3168/jds.S0022-0302(03)73702-3
https://doi.org/10.3168/jds.S0022-0302(83)81916-X
https://doi.org/10.3168/jds.S0022-0302(83)81916-X
https://doi.org/10.3168/jds.2018-15668
https://doi.org/10.1023/A:1020343717817
https://doi.org/10.3168/jds.2011-4289
https://doi.org/10.3168/jds.2011-4289
https://doi.org/10.3168/jds.2023-24626
https://doi.org/10.3168/jds.2021-20776
https://doi.org/10.2460/javma.1998.212.04.553
https://doi.org/10.2460/javma.1998.212.04.553
https://doi.org/10.3168/jds.S0022-0302(79)83313-5
https://doi.org/10.3168/jds.S0022-0302(90)78715-2
https://doi.org/10.3168/jds.S0022-0302(90)78652-3
https://doi.org/10.3168/jds.S0022-0302(90)78652-3
https://doi.org/10.1016/j.domaniend.2015.10.002
https://doi.org/10.1016/j.domaniend.2015.10.002
https://orcid.org/0000-0003-1368-8716
https://orcid.org/0009-0005-7188-7408
https://orcid.org/0000-0003-4943-9279
https://orcid.org/0000-0002-8295-4128
https://orcid.org/0000-0002-2374-2280
https://orcid.org/0000-0001-7760-3107
Journal of Dairy Science Vol. TBC No. TBC, TBC
A
PP
EN
D
IX
Oliveira et al.: INTRAMAMMARY INFECTION PREGNANT HEIFERS
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	Impact of intramammary infections on mammary gland development in pregnant dairy heifers during late gestation
	INTRODUCTION
	MATERIALS AND METHODS
	Animal Selection and Study Design
	Intramammary Challenge
	Mammary Secretion Collection and Processing
	Mammary Tissue Sampling and Processing
	Mammary Tissue Histologic Analysis
	Immune Cell Infiltration and Luminal Secretion Scoring
	Statistical Analysis
	RESULTS
	Baseline Measures
	Success of Intramammary Challenge
	Total and Differential Secretion Somatic Cell Counts
	Immune Cell Infiltration and Luminal Secretion Scores
	Coarse Evaluation of Mammary Tissues
	Intralobular Specific Evaluation
	DISCUSSION
	Total and Differential Secretion Somatic Cell Counts
	Immune Cell Infiltration and Luminal Secretion Scores
	Coarse Evaluation of Mammary Tissues
	Intralobular Specific Evaluation
	Limitations and External Validity
	CONCLUSIONS
	NOTES
	REFERENCES