Localized mammary gland changes in milk composition and venous blood metabolite concentrations result from sterile subclinical mastitis

Subclinical mastitis reduces milk yield and elicits undesirable changes in milk composition, but the mechanisms resulting in reduced milk production in affected mammary glands are incompletely understood. This study investigated the effects of sterile inflammation on mammary gland metabolism by assessing changes in milk and venous blood composition. Mid-lactation primiparous Holstein cows (n = 4) had udder halves randomly allocated to treatments; quarters of 1 udder half were infused with 2 billion cfu of formalin fixed Staphylococcus aureus ( FX-STAPH ) and quarters of the opposite udder half infused with saline ( SAL ). Blood samples were collected from the right and left subcutaneous abdominal veins in 2.6 h intervals until 40 h post challenge and analyzed for blood gas and metabolite concentrations. Milk from FX-STAPH udder halves had significantly increased SCS by first milking at 8 h post-challenge. By 16 h post-challenge, FX-STAPH udder halves had increased concentrations of protein and lactate and lower lactose concentrations than SAL udder halves. Milk fat concentrations, milk yields, energy corrected milk yields, and the ferric reducing antioxidant power of milk were not significantly different between SAL and FX-STAPH udder halves. Venous blood of FX-STAPH halves had marginally greater concentrations of saturated O 2 , partial pressures of O 2 , and glucose concentrations than SAL halves. Conversely, total and partial pressures of CO 2 did not differ between udder half treatments suggesting a shift in local metabolite utilization in FX-STAPH udder halves. These results indicate that changes in milk composition resulting from mastitis are accompanied by changes in some key blood metabolite concentrations. The shift in venous blood metabolite concentrations, along with the marked increase in milk lactate, suggests that local mammary tissue and/or recruited and immune cells alters metabolite usage in mammary tissues. Future studies are needed to quantify the uptake of key milk precursors during mastitis.


INTRODUCTION
Mastitis is inflammation of the mammary gland and remains a common disease in the dairy industry.Subclinical mastitis is responsible for significant monetary losses with the greatest monetary losses arising from decreased milk production and reduced quality of milk from affected mammary glands (Janzen, 1970;Blosser, 1979;Huijps et al., 2008).
Milk composition is significantly altered during mastitis.Concentrations of lactose, milk-derived proteins (i.e., caseins, α-lactalbumin, β-lactoglobulin), and typically fat are reduced (Schultz, 1977;Antanaitis et al., 2021), while blood-borne electrolytes (e.g., sodium, chloride) and serum proteins (e.g., bovine serum albumin, immunoglobulins) increase in milk from affected quarters (Bansal et al., 2005;Eckersall et al., 2006).The changes in milk composition that occur during mastitis transpire during periods of marked neutrophil diapedesis from the blood into the mammary gland lumen (Akers and Nickerson, 2011).During periods of acute neutrophil infiltration, blood constituents may leak into milk resulting in the presence of blood-derived products in milk (Wellnitz et al., 2011;Rainard et al., 2022).While the influx of immune cells, primarily neutrophils, during the early the inflammatory response is associated with the changes in milk composition, the specific mechanisms that alter milk composition and are responsible for decreased milk synthesis are not fully settled.For instance, our group elicited marked leukocyte recruitment into the mammary gland after intramammary oyster glycogen challenge (producing SCC >3,000,000) and only minor changes in milk composition resulted; changes in milk yield were absent during the 3-d study (Enger et al., 2023).
Maximal milk synthesis at a given secretory capacity for an individual animal is contingent upon the unrestricted delivery of substrates and milk precursors to the mammary gland.Glucose, amino acids, fatty acids, vitamins, minerals and other substrates diffuse from the blood into the interstitial space of the mammary gland and are taken up by the mammary epithelial cells for milk synthesis (Akers, 2002).Glucose uptake is especially important.The majority of glucose that is taken up by the mammary gland is used for lactose synthesis (Zhao, 2014).Because lactose is the chief osmoregulator of milk secretion (Stacey et al., 1995), the amount of lactose synthesized by the mammary gland directly affects total milk volume.Accordingly, if lactose synthesis is increased or decreased, total milk volume and yield respond in kind.Because of this relationship, the rate of lactose synthesis and secretion fundamentally underpins total milk yield.
Mammary gland blood flow increases during mastitis (Dhondt et al., 1977), which may indicate that the mammary gland has an increased substrate and energy demand during the diseased state.The initial innate immune response during mastitis is largely formed by neutrophil recruitment and activation.Neutrophils are highly glycolytic (Sadiku et al., 2021) and use glucose for energy to internalize and inactivate pathogens with reactive oxygen species and anti-bacterial peptides (Paape, 2003).We recently proposed that the increased energy demands of neutrophils during mastitis may increase the competition for glucose in the mammary gland (Enger, 2019).Glucose and other nutrients could therefore be potentially sequestered by the immune response, reducing substrates available for milk component synthesis.Specifically, reduced availability of glucose for incorporation into lactose may be a controlling factor in the reduction of milk yield and milk lactose concentration during mastitis.
Milk compositional responses during mastitis are relatively consistent for lactose but are less so when it comes to protein and fat.Milk fat concentrations have been reported to minorly decrease and then markedly increase in response to an endotoxin challenge (Shuster et al., 1991).Conversely, cross sectional studies report that milk fat concentrations are reduced during mastitis (Philpot, 1967;Randolph and Erwin, 1974), but rapid reductions in milk yield during mastitis can increase milk fat concentrations (Schultz, 1997).Similarly, assessing changes in total milk protein content during mastitis is complex.Generally, whey protein content is increased while casein content is reduced (Schultz, 1977).Nonetheless, the competing changes in whey and casein protein fractions can result in mastitic milk having a similar total protein content relative to normal milk (Haenlein et al., 1972), or as more typical, be greater (Akers and Thompson, 1987;Enger et al., 2023).Because of the complex and situationally dependent responses of milk fat and protein during mastitis, we focused our first investigation on the potential competition for milk substrates on glucose, and additionally assessed blood biochemical measures that are related to tissue metabolism.The hypothesis of this study was that mastitis-induced alterations in milk synthesis and secretion are accompanied by local changes in mammary gland metabolism and blood-tissue substrate exchange.The objective was to quantify blood glucose concentrations and assess the chemistry of venous blood exiting healthy and sub-clinically inflamed mammary glands to determine if localized mammary gland inflammation alters blood-tissue substrate exchange and is related to changes in milk composition.

MATERIALS AND METHODS
The animal use and animal procedures were reviewed and approved by The Ohio State University Institutional Animal Care and Use Committee (Protocol 2022A00000020).

Animal selection and study design
Four mid-lactation primiparous Holstein cows (mean DIM = 114, SD = 28.5)were used in a study that occurred from December 5th-15th, 2022.The cows were adapted to tie stalls for at least 7 d before study commencement and had no previous history of clinical mastitis and a SCC <25,000 cells/mL at time of enrollment.Due to logistical considerations, only 2 cows were enrolled at a time resulting in 2 animal cohorts, each containing 2 cows.No sample size calculation was performed.The experimental design and sampling regimen were identical for both cohorts and is illustrated in Figure 1.Three d before the intramammary infusions and commencement of intensive sampling (d −3), cows were moved to new tie stalls that utilized a milk in place design; cows were fed twice daily and milked with a Surge RX quarter milker to separate the milk from the right and left udder halves.Upon moving to the new tie stalls, milking frequency was increased from 2x/d to 3x/d and contralateral udder halves were assessed for equal milk production.Udder half milk yields at individual milkings did not differ by more than 0.5 kg within each cow before intramammary challenge on d 0. On d −1, 4 catheters were placed in each cow: 1 in the left subcutaneous abdominal vein, 1 in the right subcutaneous abdominal vein, 1 in the jugular vein, and 1 in the aural artery.The jugular and aural catheters were placed for data collection measures unrelated to the present study.On d 0, immediately after 1st milking, each cow received intramammary infusions of 2 billion cfu of formalin-fixed Staphylococcus aureus (FX-STAPH) into both quarters of a randomly selected udder half; saline (SAL) was infused into the quarters of the contralateral udder half.Blood sampling began 1 h 20 min after intramammary infusions.Sampling increments were designed to capture 3 time points between each milking, yielding equal sampling increments of 2 h 40 min.Accordingly, 15 blood collection time points resulted by the termination of the study, which was 40 h post intramammary infusions (Figure 1).After the final milking, mammary tissues were collected via biopsy to qualitatively assess mammary tissue histopathology.Biopsies were performed under anesthesia using a cocktail of 40 mg of ketamine (Dechra Veterinary Products, Overland Park, KS), 20 mg of xylazine (Akorn Inc., Lake Forest, IL), and 10 mg of butorphanol tartrate (Zoetis Manufacturing and Research, Girona, Spain) that was administered intramuscularly.The biopsy site was anesthetized using 2 mL of 2% lidocaine (MWI Animal Health, Boise, ID), which was administered just under the skin.Biopsy procedures were similar to those detailed by Farr et al. (1996) and demonstrated by Daley et al. (2018), and used a biopsy tool like the one developed by Farr et al. (1996).Mammary tissues were collected from each rear quarter of the 2 cows in the last cohort.Samples were collected and fixed in formalin for 48 h before being embedded in paraffin, sectioned, and stained using hematoxylin and eosin.Biopsies were attempted with the 2 cows in the first cohort, but technical issues with the biopsy tool thwarted successful tissue collection.

Catheter placement and maintenance
Catheters were placed by a veterinarian using the same anesthesia cocktail described earlier and a local anesthetic (i.e., a 0.5 -1.0 mL bleb of 2% lidocaine was administered at each catheter site before placement).Catheters were maintained throughout the trial by flushing ~10 mL of heparinized saline (20 IU/mL) at least every 8 h, and after every blood collection.Venous catheters were 16 gauge and 130 mm in length (cat # J0458B, Jorgensen Laboratories Inc., Loveland, Co), while the arterial catheter was 18 gauge and 51 mm in length (cat # SR-OX1851CA, Terumo Medical Products, Somerset, NJ).If a catheter lost patency, it was replaced as needed.Notably, sedation was not used during replacement of catheters.

Intramammary challenge
We conducted a pilot study to identify a suitable sterile inflammatory agent and dosage.Formalin-fixed Staph.aureus was chosen because we observed that it elicited changes in milk composition and caused localized clinical mastitis with the absence of a febrile response (Gammariello and Enger, unpublished data).The Staph.aureus Novel strain (Smith et al., 1998) was prepared as we have done previously (Enger et al., 2018) and the culture suspension was fixed with 0.5% formalin for 24 h in a shaking incubator at 25°C.Immediately before fixation, an aliquot of grown culture was collected and used to enumerate the concentration and total number of Staph.aureus that was fixed.The formalin-fixed Staph.aureus suspension was confirmed to be non-viable via aerobic culture.The fixed Staph.aureus were washed thrice with sterile PBS (Hyclone Laboratories, Logan, UT) before 0.75-1.5 mL (volume dependent upon the earlier cfu enumeration procedure) was added to 18.5 −19.25 mL aliquots of PBS for an approximate total volume of 20 mL per intramammary infusion.
Sterile PBS (Hyclone Laboratories) and formalinfixed Staph.aureus intramammary infusions were administered immediately following 1st milking on d 0. Infusion procedures followed those detailed elsewhere (Enger et al., 2018) and utilized the partial insertion method where the tip of the cannula was inserted approximately 3 mm into the teat canal.Infusate volumes were 20 mL for all quarters.Mammary glands were briefly massaged to disperse the infusate after infusion.For 2 d post-infusion, clinical signs of mastitis were assessed by taking rectal temperatures, checking for redness/swelling of the udder, and checking for the presence of milk clots at every milking.

Milk collection and processing
Fresh whole milk samples were collected from the left and right udder halves and submitted to a local DHIA laboratory to measure milk SCC and composition using a Bentley Fourier Transform Spectrometer and Flow Cytometer (Bentley Instruments, Chaska, MN).Samples collected for DHIA analysis were placed in the DHIA provided tubes, which contained a preservative pellet (44% Bronopol + 2% Natamycin) and were stored at 5°C until analysis; samples were picked up and analyzed within 3 d of collection.Additional whole milk samples were collected and centrifuged for 20 min at 2,000 x g at 4°C to separate skim, fat, and cell pellet fractions.To achieve removal of only the skim fraction, a needle was used to pierce the polypropylene tube just above the cell pellet (Kitchen et al., 1978) and the skim was subsequently aliquoted into 1.5 mL microcentrifuge tubes.Milk samples used to isolate the skim fraction were processed immediately after milking and stored at −20°C until subsequent analysis.

Blood collection and processing
Blood samples were simultaneously collected from the subcutaneous abdominal veins at each blood collection time point.To collect blood samples, catheter lines were first flushed by drawing approximately 10 mL of fresh blood through the catheter and extension set and discarded.Immediately following flushing, blood samples were collected in 6 mL lithium heparin tubes and 10 mL EDTA tubes and gently inverted at least 5x following collection.After blood samples were collected, catheters were flushed with ~10 mL of heparinized saline.
Blood from the lithium heparin tubes was immediately analyzed using a hand-held blood chemistry analyzer (i-STAT Alinity V, Zoetis, Parsipanny, NJ).Whole blood was drawn from the lithium heparin tube into a syringe and needle and 2-3 drops of blood were loaded into CG4+ Cartridges and CHEM8+ Cartridges.Blood was handled to maintain anaerobic conditions and protected from air because blood gas measures were being assessed.
Blood in the EDTA tubes was immediately placed in a refrigerator after collection.The collected EDTA samples were processed within 15 min after being placed in the refrigerator and were centrifuged at 1,600 x g for 15 min at 4°C.Plasma was aliquoted into 1.5 mL tubes and stored at −20°C until further analysis.

Lactate analysis
Notably, blood lactate levels were initially measured using the i-STAT Alinity blood analyzer, but most samples fell below the limit of detection (0.3 mmol/L), so stored plasma samples were used to quantify blood lactate concentrations instead.A fluorometric assay was adapted from Shangraw et al. (2020) who used and adapted procedures from Shapiro and Silanikove (2011) to measure L-lactate in blood plasma and skim milk samples.Plasma samples were diluted 50:50 with water and milk samples were left undiluted.Solution A consisted of β-NAD+ (Sigma, cat.# NO632) and L-Lactic Dehydrogenase from bovine heart (Sigma, cat.# L3916) in a phosphate buffer solution.Solution A was combined with the samples and standards in a black 96-well plate and incubated at 37°C for 30 min.Solution B consisted of resazurin and diaphorase enzyme from Clostridium kluyveri (Sigma, cat.# D2197) in a buffer solution and was added to the plate and incubated at room temperature for 30 min.Fluorescence was measured immediately afterward on a fluorometric plate reader at Ex/Em = 530/590.

Ferric antioxidant reducing power (FRAP)
Plasma antioxidant capacity was measured via the FRAP assay using a protocol adapted from Shangraw et al. (2020).Plasma samples were diluted 50:50 with water and then combined with freshly prepared FRAP solution and incubated at 37°C in a clear 96-well plate.The FRAP reaction occurs within minutes so absorbance values were measured at 15 m at 593 nm.
Skim milk samples were analyzed via the FRAP assay using a protocol from Reichler (2023).In brief, 300 μL of each sample was combined with 4.5 mL of freshly prepared FRAP solution in a glass tube and incubated in a water bath at 37°C for exactly 4 m.Following incubation, the supernatant was filtered through a syringe filter and absorbance was immediately measured in cuvettes at 593 nm.

Statistical Analysis
All response variables (SCC, milk composition, milk yield, blood metabolites, lactate and FRAP assay results) were analyzed using PROC GLIMMIX (SAS 9.4, Cary, NC) in separate models.Total yield of milk components were calculated using concentrations of milk components and udder half milk weights.The density of milk was assumed to be ρ = 1.03 g/cm 3 .Milk SCC was transformed and analyzed as milk SCS to satisfy the assumption of equal variance.Total somatic cell count was also transformed using the natural logarithm to satisfy the assumption of equal variance.Fixed effects in the models were udder half treatment (n = 2), time point (n = 6 for milk measures and n = 15 for blood measures), and the interaction of treatment by time; cohort and cow nested within cohort were designated as random effects.Measures were repeated on udder half treatments interacting with cow over time.The denominator degrees of freedom adjustment developed by Kenward and Roger (1997) was used in all models.Treatment least squares means estimated by the model were contrasted within time points using the slice procedure.The following covariance structures were tested for each response variable; compound symmetry, heterogenous compound symmetry, first order autoregressive, heterogenous first order autoregressive, and toeplitz.A covariance structure was selected per response variable according to the lowest Akaike Information Criterion values.
For clarity, brevity, and most importantly, to prevent inappropriate extrapolations, time point main effects are not reported.This is because the present study was not designed to assess how cows alone respond to Staph.aureus challenge over time.Here, management and experimental factors are confounded with time, and it is not possible to establish if changes over time are due to the effect of time since Staph.aureus challenge, or due to extraneous management and experimental factors (e.g., compelling cows to stand for blood collection, sleep deprivation, changes in feeding behavior, etc).Including cows that would have only been infused with saline and subjecting them to the same experimental procedures would have allowed such a comparison.Conversely, the interactive effect of treatment by time was a focus of this study and is detailed in the following results.Differences were considered significantly different when P ≤ 0.05 and marginally different when P ≤ 0.15.

Response to challenge
No cow developed a febrile response in response to intramammary infusion (mean body temperature = 38.4°C,SD = 0.64°C, range 37.8 -39.0°C), and no quarters exhibited signs of redness or swelling.Aside from a single FX-STAPH quarter that produced 1 large milk flake at 32 h post-challenge, no other quarters exhibited signs of clinical mastitis throughout the study.
Milk SCS are presented in Figure 2A.Milk SCS was significantly affected by udder half treatment (P < 0.01) and the interaction of udder half treatment with time (P < 0.01).Milk SCS of FX-STAPH and SAL udder halves were comparable at challenge (P = 0.68), but SCS of FX-STAPH udder halves were greater than SAL udder halves by 8 h post-challenge (P < 0.01) and remained greater than SAL udder halves for all subsequent milkings (P < 0.01).Total somatic cells are presented in Figure 3B.Total somatic cells were significantly affected by udder half treatment (P < 0.01) and the interaction of udder half treatment with time (P < 0.01).Total somatic cells of FX-STAPH udder halves were comparable to SAL udder halves at challenge (P = 0.92), marginally increased by 8 h post challenge (P = 0.09) and greater for the duration of the study (P < 0.01).

Milk composition and yield
Milk component concentrations and milk yields are presented in Figure 2B-F.Milk lactose content was similar between udder half treatments at infusion (P = 0.87), but were significantly lower in FX-STAPH udder halves by 16 h post-challenge (P = 0.01) and remained lower than SAL udder halves for the remainder of the study (P ≤ 0.03).Protein content was also similar between udder half treatments at challenge (P = 0.72), but FX-STAPH udder half protein content was marginally greater than SAL udder halves by 16 h post-challenge (P = 0.09) and remained greater than SAL udder halves for all subsequent milkings (P ≤ 0.03).Milk fat content, milk yields, and energy corrected milk yields, were not significantly affected by udder half treatment (P ≥ 0.30), nor the effect of udder half treatment interacting with time (P ≥ 0.81).
Milk component yields are presented in Figure 3C-E.Total yields of fat and protein did not significantly differ between udder half treatments (P ≥ 0.35) and were not affected by the interaction of udder half treatment with time (P ≥ 0.95).On average, lactose yields per milking were marginally lower in FX-STAPH udder halves than SAL udder halves (0.251 vs. 0.263 kg ± Gammariello et al.: SUBCLINICAL MASTITIS EFFECTS ON MILK AND BLOOD 0.0114 kg; P = 0.15) and were marginally lower in FX-STAPH udder halves than SAL udder halves at 24 h (P = 0.09) and 32 h (P = 0.14) post-infusion.
Skim milk lactate concentrations were affected by udder half treatment (P < 0.01) and the interaction between udder half treatment and time (P = 0.03; Figure 4A).Lactate concentrations were similar between udder half treatments at challenge (P = 1.00), but were greater in FX-STAPH udder halves than SAL udder halves by 16 h post-challenge (P < 0.01) and remained greater than SAL udder halves throughout the study's duration (P ≤ 0.04).Milk lactate yields (Figure 4B) followed the same pattern and were similar between udder half treatments at challenge (P = 0.93), but were greater in FX-STAPH udder halves than SAL udder halves by 16 h post-challenge (P < 0.01) and remained greater for all subsequent milkings (P ≤ 0.06).
Skim milk FRAP values (Figure 5A) did not differ between udder half treatments (P = 0.81) and were not affected by the interaction of udder half treatment by time (P = 0.96).
Venous blood plasma FRAP values (Figure 5B) did not differ between udder half treatments (P = 0.22) and were not affected by time interacting with udder half treatment (P = 1.00).

Histopathological evaluation
Images of tissues collected from SAL and FX-STAPH mammary glands are presented in Figure 6.Tissues from SAL mammary glands exhibited open alveolar lumens and contained secretory epithelial cells that exhibited a pronounced abundance of secretory components near the apical membrane, giving the apical edge a semi-translucent and lacy appearance that is characteristic of highly active secretory epithelial cells.Immune cells were only observed intermittently in stromal and parenchymal tissue compartments of SAL mammary glands.In comparison, some epithelial lumens of FX-STAPH glands were markedly infiltrated by leukocytes, while some epithelial lumens were not and appeared entirely unaffected.Leukocyte infiltration was also apparent in the intralobular stromal areas of FX-STAPH mammary tissues.Epithelial structures containing large numbers of leukocytes had epithelial cells that appeared to be degraded and lacked the normal lacy and translucent apical edge that is apparent in highly secretory epithelium.Instead, some epithelial cells had either large vacuoles or large lipid droplets that had not been secreted and were accumulating within the cytoplasm.

DISCUSSION
The objective of this experiment was to quantify the concentration of glucose and assess the chemistry of venous blood exiting healthy and inflamed mammary glands to evaluate if mammary gland inflammation alters blood-tissue substrate exchange and is associated with changes in the composition of milk that occur during mastitis.We chose to use a split udder model because the blood entering each udder half within a cow is identical in composition, which allows us to attribute disparities in venous blood composition to changes in blood-tissue substrate exchange resulting from treatment.An important limitation in this experiment is that neither the directionality of substrate exchange (i.e., blood to tissue or tissue to blood) nor the magnitude of uptake (i.e., mass uptake) can be determined.
The marked increase of SCS in FX-STAPH udder halves in response to intramammary infusion compared with the stable SCS of SAL udder halves indicates an immune response was generated in FX-STAPH udder halves and apparently absent in SAL udder halves.Total somatic cells of FX-STAPH udder halves also followed a similar pattern.Overall, the lack of a febrile response suggests that a notable systemic inflammatory response was absent, as 7 out of 8 FX-STAPH infused quarters only exhibited subclinical mastitis except for a single quarter producing a single milk flake at 1 milking.Based on these outcomes, we conclude that the single infusion of FX-STAPH into ipsilateral quarters generated an inflammatory state that was primarily isolated to their respective udder half.
The reduced lactose content, marginal reduction in lactose yield, and increased milk protein content of FX-STAPH udder halves relative to SAL udder halves was expected.We and others have reported similar reductions in lactose content (Fox et al., 1986;Akers and Thompson, 1987;Enger et al., 2023) and increases in milk protein content (Akers andThompson, 1987, Enger et al., 2023) after intramammary infusion of agents intended to elicit sterile immune cell recruitment and mastitis.Milk compositional changes resulting from mastitis partly arise from blood components leaking into milk, and from an increased synthesis of locally mammary produced proteins.For instance, BSA, immunoglobulins (Schultz, 1977;Bannerman et al., 2003), and LPS-binding protein (Bannerman et al., 2003) increase in milk, presumably via leakage from the blood when epithelial barrier integrity is disrupted.Additionally, BSA (Shamay et al., 2005) and other whey proteins like lactoferrin (Molenaar et al., 1996) can be locally synthesized by mammary tissues and the increased abundance of these proteins in mastitic milk (Harmon et al., 1976) is also resultant of local mammary gland synthesis.We suspect the increased milk protein content observed for FX-STAPH udder halves resulted via leakage from blood, and increased local synthesis of whey proteins.Such increases in whey protein content may be occurring when casein synthesis is not markedly impacted, which may be occurring here  given the 1 time infusion of a sterile irritant that would limit duration of the inflammatory response.
Lactose is a key osmoregulator of milk secretion, and a primary determinant of milk volume (Holt, 1983;Stacey et al., 1995).The milk abundance of α-lactalbumin, a key subunit of lactose synthase, is reduced during mastitis (Harmon, 1955;Bortree et al., 1962) and a reduction in α-lactalbumin expression during mastitis aligns with the reduced lactose content and marginal reduction in lactose yield observed here.Notably, however, milk ion concentrations increase during mastitis due to disruptions in mammary epithelium integrity (Kitchen, 1981;Gaucheron, 2005), which draws water into the alveolar lumen.The result is that osmolarity of milk from mastitic mammary glands is similar to that of healthy mammary glands (Kitchen, 1981).Our result that milk volume was maintained while lactose yield was marginally reduced for FX-STAPH udder halves suggests that local lactose synthesis may have been suppressed.Conversely though, leakage of lactose into blood when the epithelial integrity is compromised during mastitis is expected (Wellnitz and Bruckmaier, 2021) and may be the reason lactose yield was reduced for FX-STAPH udder halves.Milk yield was not significantly lower in FX-STAPH than SAL udder halves.It is plausible that an immune response of greater magnitude and/or duration would have induced a milk yield response.Still, the resilience and flexibility of mammary gland productivity is highlighted in these results because marked immune cell recruitment and infiltration only changed milk composition, even considering the histopathological alterations observed at 40 h post-challenge.Reasons for the results here cannot be definitively concluded but it is plausible that the single infusion of killed Staph.aureus resulted in only minor activation of the immune system before being flushed out by milking.Future works are needed to develop a sterile mastitis model that results in notable reduced milk production of only challenged mammary glands because the metabolic mechanisms are likely to differ between these resilient and compromised states.
Glucose concentrations, partial pressures of oxygen, and saturated oxygen concentrations were the only blood components that differed between FX-STAPH and SAL udder treatments.These results may have occurred from 2 possible mechanisms.During mastitis, cells and tissues in the mammary gland may have increased energy requirements to support an effective immune response and a shift in metabolism toward conserving glucose may result (Gross et al., 2015).The increased glucose concentrations in venous blood of FX-STAPH udder halves may be reflective of such a shift.Additionally, the greater oxygen and glucose concentrations may be resultant of changes in mammary gland blood flow because mammary gland blood flow increases during mastitis (Dhondt et al., 1977).Cant and McBride (1995) modeled and discussed the paradoxical relationship that exists between increased blood flow and reduced metabolite exchange if changes in vascular permeability and capillary dilation are insufficient to maintain the efficiency of metabolite exchange with the surrounding tissues.In other words, if blood velocity is too great, substrate exchange efficiency is reduced.Importantly though, a reduction in exchange efficiency does not automatically translate to a reduction in mass delivery and changes in blood flow could overcome diminished exchange efficacy.The increased blood flow that is expected to occur during mastitis may have reduced exchange efficiency and thereby increased venous concentrations of glucose and O 2 exiting FX-STAPH udder halves.Future works should assess and measure mammary gland blood flow responses during mastitis to better quantify the uptake of milk precursors.
Neutrophil metabolism is best characterized in other species and heavily utilizes glycolysis and the TCA cycle (Jeon et al., 2020) which results in significant lac-tate production.Increased concentrations of lactate in milk of mastitic mammary glands has been repeatedly reported (Davis et al., 2004;Lehmann et al., 2015).The appearance of lactate in milk of FX-STAPH udder halves may have resulted from: 1) leakage of lactate from interstitial fluid and blood (Wellnitz and Bruckmaier, 2021), 2) mammary cells and tissues experiencing altered glucose metabolism (Silanikove et al., 2011), or 3) neutrophils employing anerobic (Sbarra and Karnovsky, 1959), or aerobic (Goetzl and Austen, 1974) glycolysis, both of which can yield lactate in the neutrophil.
Determining the origin of lactate that appears in milk during mastitis is important for assessing localized changes in tissue metabolism given lactate is a byproduct of anaerobic glycolysis.Lactate concentrations are greater in blood than milk, therefore leakage following a concentration gradient is expected when mammary epithelium barrier integrity is compromised.If leakage was the primary mode in which lactate concentrations in milk were increased, glucose from the blood would be expected to follow similar diffusion via disparities in concentration gradients.Glucose is only twice the size of lactate, and blood glucose concentrations are markedly greater than in milk.On the contrary though, milk glucose concentrations are reduced during mastitis, even in mammary glands that are culture negative (Marschke and Kitchen, 1984).The disconnect between these patterns does not support the notion that the primary source of lactate in milk is due to leakage from blood and suggest there is local mammary gland production of lactate during mastitis.Indeed, others have observed greater concentrations of lactate in milk of mastitic mammary glands than in the blood of affected cows (Lehmann et al., 2013).These observations suggest that there is local production of lactate in the mammary gland, most likely from altered glucose metabolism, but additional studies are needed to confirm this hypothesis.
Lactate can be produced from glucose even in the presence of oxygen due to the Warburg effect (Kocianova et al., 2022).The Warburg effect has been observed in cancerous cells (Koppenol et al., 2011), highly proliferative mammalian cells (Hosios et al., 2016), and immune cells (Medzhitov, 2015) where aerobic glycolysis occurs to rapidly generate ATP and maximize carbon utilization.However, to our knowledge, no studies have confirmed the Warburg effect occurs in bovine neutrophils or mammary tissues.The FX-STAPH udder halves were not believed to be hypoxic based on the venous oxygen and carbon dioxide measures observed here, yet milk lactate was significantly increased in inflamed udder halves.The disconnect observed here further suggest altered metabolism in local mammary tissue during mastitis.Perhaps the most striking indication that there were changes in local tissue metabolism here is that venous oxygen concentrations were greater in FX-STAPH halves than SAL, but carbon dioxide concentrations were unaffected.Generally, if local tissue metabolism was largely unchanged, the ratios of these 2 key substrates would not be expected to differ.Importantly though, we cannot assess the potential contribution of reactive oxygen species being neutralized by catalase and thereby increase blood oxygen concentrations.Milk concentrations of catalase are greatly increased during mastitis (Kitchen, 1981).Overall, future studies are needed to identify the source of lactate in milk of inflamed mammary glands.
It is important to note that certain venous blood measures, such as oxygen concentrations, already appeared to be different at 1.3 h post-challenge (data not shown).We initially thought obtaining a venous blood sample only 1.3 h after intramammary infusion would serve as an acceptable baseline sample, but Dhondt et al. (1997) noted that changes in mammary blood flow resulting from LPS challenge are at their zenith by only 2 h post-challenge.It is possible that mammary blood flow was already increased by 1.3 h after intramammary infusion and influenced the observed blood gas measures.

CONCLUSIONS
The results of this study indicate that sterile leukocyte recruitment elicits changes in metabolite concentrations in venous blood and milk of subclinically inflamed mammary glands.Milk of inflamed udder halves had decreased concentrations of lactose, increased concentrations and total yield of lactate, and increased protein concentrations relative to uninflamed udder halves, but no significant treatment differences in milk yield were observed.The marked increase of lactate in mastitic milk may be primarily due to anaerobic utilization of glucose by recruited neutrophils for effector functions and/or local changes in mammary tissue metabolism rather than blood lactate leaking into milk.Venous blood of inflamed mammary glands yielded increased concentrations of oxygen and glucose but had no detectable changes in carbon dioxide concentrations, indicating local metabolite consumption and production is altered.Future works should aim to quantify blood substrate uptake in mammary glands during mastitis to further elucidate these mechanisms.

Figure 1 .
Figure 1.Study timeline for the 4 primiparous cows used.Timeline indicates when catheter placement, intramammary infusions of saline and formalin-fixed Staphylococcus aureus, and mammary biopsies occurred.Red lines denote milking time points occurring every 8 h; asterisk denotes blood sampling time point every 2 h 40 min beginning 1 h 20 min after milking and intramammary infusions.

Figure 3 .
Figure 3. Mean milk yiel[INSERT Figure 001]d (A), total somatic cells (B), lactose (C), fat (D), and protein (E) yields of udder halves that received intramammary infusions of either saline (SAL, solid blue line, n = 4) or formalin-fixed Staphylococcus aureus (FX-STAPH, dashed red line, n = 4) at time point 0. Milk yields in A are the same as those in Figure 2E and are re-presented for comparative purposes to highlight the paralleling relationship between milk yields and total yields of lactose, fat, and protein.Error bars denote the SEM * indicates P ≤ 0.05 and † indicates P ≤ 0.15 for SAL vs. FX-STAPH comparisons within time point.

Figure 4 .
Figure 4. Lactate concentrations of skim milk (A), total milk lactate yields (B) and venous plasma lactate concentrations (C) are presented for udder halves that were infused with either saline (SAL, solid blue line, n = 4) or formalin-fixed Staphylococcus aureus (FX-STAPH, dashed red line, n = 4) at time point 0. Error bars denote the SEM * indicates P ≤ 0.05 and † indicates P ≤ 0.15 for SAL vs. FX-STAPH comparisons within time point.

Figure 6 .
Figure 6.Images of mammary tissues collected from mammary glands infused with saline (A) and formalin-fixed Staphylococcus aureus (B) at 40 h post intramammary infusion.Black scale bar = 100 μm.
Gammariello et al.: SUBCLINICAL MASTITIS EFFECTS ON MILK AND BLOOD

Table 1 .
Gammariello et al.: SUBCLINICAL MASTITIS EFFECTS ON MILK AND BLOOD Composition of whole venous blood collected from the subcutaneous abdominal veins of saline (SAL) and formalin-fix Staphylococcus aureus (FX-STAPH) infused udder halves