Quantitative Assessment of Epithelial Proliferation in Rat Mammary Gland Using Artificial Intelligence Independent of Choice of Proliferation Marker.

Epithelial proliferation in the rat mammary gland is recommended in regulatory guidelines as an endpoint for assessment of the in vivo carcinogenic potential of insulin analogues. Epithelial proliferation is traditionally assessed by immunohistochemical staining of a proliferation marker, for example, 5-bromo-2'-deoxyuridine (BrdU) or Ki67, followed by labor-intensive manual counting of positive and negative cells. The aim of this study was to develop and validate an approach for image analysis based on artificial intelligence, which can be used for quantification of proliferation in rat mammary gland, independent of the choice of proliferation marker. Furthermore, the aim was to compare the markers BrdU, Ki67, and phosphorylated histone H3 (PHH3). A sequence of image analysis applications were developed, which allowed for quantification of proliferative activity in the mammary gland epithelium. These endpoints agreed well with manually counted labeling indices, with correlation coefficients in the range ≈0.92-0.93. In addition, all three proliferation markers were significantly correlated and could detect the variation in epithelial proliferation during the estrous cycle. In conclusion, image analysis can be used to quantify epithelial proliferation in the rat mammary gland and thereby replace time-consuming manual counting. Furthermore, BrdU, Ki67, and PHH3 can be used interchangeably to assess proliferation.

An in vitro method for assessment of amino acid bidirectional transport and intracellular metabolic fluxes in mammary epithelial cells.

Understanding uptake of AA by mammary tissue as supply varies is critical for predicting milk component production. Our objective was to develop an in vitro method to quantify cellular uptake, efflux, and intracellular metabolism of individual AA that could be implemented for evaluating these factors when AA supply and profile are varied. Bovine primary mammary epithelial cells were grown to confluency and exposed to medium with an AA profile and concentration similar to lactating dairy cow plasma for 24 h. Cells were then preloaded in medium enriched with 15N-labeled AA for 24 h followed by removal of the 15N-labeled medium and incubation with medium enriched with 13C-labeled AA for 0, 15, 60, 300, 900, 1,800, and 3,600 s. Extracellular free AA and intracellular free and protein-bound AA were analyzed for concentrations and isotopic enrichment by gas chromatography-mass spectrometry. A dynamic, 12-pool model was constructed representing extracellular and intracellular free and protein-bound pools of an AA, and their respective 15N and 13C isotopes. Markov chain Monte Carlo simulation (n = 5,000) was conducted to evaluate prediction errors by deriving standard errors and posterior distributions for rate constants, fluxes, and pools. Cellular Ala influx and efflux were higher than Leu, reflecting Ala role in driving system L transport and the high capacity of sodium-dependent transport. The Ala and Leu turnover rates were 181 and 95, 580 and 857, and 74 and 157% per hour for extracellular, intracellular, and fast protein-bound pools, respectively. The intracellular and extracellular Ala to Leu ratios were quite different, meaning the blood AA profile is not the AA profile provided for protein translation. The high level of exchange and rapid turnover of pools provide a mechanism for matching the AA supplies to the precision necessary for translation. This also understates the importance of using experimental medium similar to what is observed in vivo given that some AA depend on other AA for influx (exchange driven). The average root mean squared prediction error across the isotope enrichments, pools, and concentrations was 9.7 and 14.1% for Ala and Leu, respectively, and collinearity among parameters was low, indicating adequate fit and identifiability. The described model provides insight on individual AA transport kinetics and a method for future evaluation of AA transport and intracellular metabolism when subjected to varying AA supplies.

Short communication: Technologies and milking practices that reduce hours of work and increase flexibility through milking efficiency in pasture-based dairy farm systems.

To attract and retain quality employees, dairy farms must be competitive with other workplaces offering more conventional hours of work. Milking requires significant labor input and influences the start and end times of the working day, affecting flexibility to suit employee needs or availability. The use of labor-saving technology and milking management strategies could help with this challenge. Previous studies have used scenario modeling in attempt to quantify the value of in-parlor technologies, however, they have relied on assumptions about the effect of the technologies on labor in the dairy. Similarly, the effect of management strategies on work patterns, such as flexible milking intervals (changing the timing of milking), has not been evaluated. The aims of this study were to (1) quantify the milking labor requirements in a range of pasture-based dairy farm systems and (2) identify practices or technologies that facilitate efficient milking. A telephone survey of 500 dairy farmers in New Zealand was conducted during April and May 2018, with questions asked about milking practices and technology use. Predictive analysis showed that at peak lactation, milking required between 17 and 24 h/wk per worker for farms milking twice a day, representing 43 to 58% of a conventional 40-h work week, depending on parlor type (herringbone or rotary), the number of clusters, and herd size. Using milking intervals of 8 and 16 h (intervals between milkings), compared with the more usual 10 and 14 h, largely avoided starting milking before 0500 h. Eight percent of herds were milked once a day, which required between 7 and 14 h/wk per worker (18-35% of a 40-h week). ANOVA showed that for metrics that related to people (labor efficiency and work routine), using automatic teat spraying had a positive effect on efficiency. Having both automatic cluster removers and drafting were associated with longer milking times in terms of throughput and row/rotation time compared with using drafting only. The results highlight considerable opportunity to reduce the number of hours those milking (employers and employees) spend in the parlor and increase staff time flexibility through milking (e.g., intervals between milkings) and parlor management (e.g., row/rotation time) and use of specific technologies. This study provides useful data for those wishing to analyze the likely value of an in-parlor automation technology or management practice for an individual situation.

Role of Liver X Receptor in Mastitis Therapy and Regulation of Milk Fat Synthesis.

Mastitis is important disease that causes huge economic losses in the dairy industry. In recent years, antibiotic therapy has become the primary treatment for mastitis, however, due to drug residue in milk and food safety factors, we lack safe and effective drugs for treating mastitis. Therefore, new targets and drugs are urgently needed to control mastitis. LXRα, one of the main members of the nuclear receptor superfamily, is reported to play important roles in metabolism, infection and immunity. Activation of LXRα could inhibit LPS-induced mastitis. Furthermore, LXRα is reported to enhance milk fat production, thus, LXRα may serve as a new target for mastitis therapy and regulation of milk fat synthesis. This review summarizes the effects of LXRα in regulating milk fat synthesis and treatment of mastitis and highlights the potential agonists involved in both issues.

Zymography in Multiwells for Quality Assessment of Proteinases.

Zymography is a well-standardized protocol for the qualitative assessment and analysis of proteinases under specified conditions. However, analysis of a large number of samples simultaneously becomes a challenge when the zymography is carried out by the usual protocol of electrophoresis. This can be overcome by assaying the matrix-degrading proteinases in substrate-impregnated gels in multiwells. Enzymes are copolymerized with 300 mL of 10% acrylamide impregnated with gelatin substrate and incubated for 16 h. The gels are then stained with Coomassie blue, destained with water, and visualized with the naked eye. The intensity; if needed can be measured with a densitometer or gel documentation system. This method has been tested for bacterial collagenases as well as some matrix-degrading metalloproteinases that were purified from rat mammary gland. It can also be used to characterize the enzymes with respect to the type and concentration of the cations required for activity and the role of other regulatory molecules that may affect the enzyme activity. The added advantage of this method is that the electrophoresis set up and electricity is not needed for the procedure.

Functional assessment of CTCF sites at cytokine-sensing mammary enhancers using CRISPR/Cas9 gene editing in mice.

The zinc finger protein CTCF has been invoked in establishing boundaries between genes, thereby controlling spatial and temporal enhancer activities. However, there is limited genetic evidence to support the concept that these boundaries restrict the search space of enhancers. We have addressed this question in the casein locus containing five mammary and two non-mammary genes under the control of at least seven putative enhancers. We have identified two CTCF binding sites flanking the locus and two associated with a super-enhancer. Individual deletion of these sites from the mouse genome did not alter expression of any of the genes. However, deletion of the border CTCF site separating the Csn1s1 mammary enhancer from neighboring genes resulted in the activation of Sult1d1 at a distance of more than 95 kb but not the more proximal and silent Sult1e1 gene. Loss of this CTCF site led to de novo interactions between the Sult1d1 promoter and several enhancers in the casein locus. Our study demonstrates that only one out of the four CTCF sites in the casein locus had a measurable in vivo activity. Studies on additional loci are needed to determine the biological role of CTCF sites associated with enhancers.

A decision analysis model for KEGG pathway analysis.

BACKGROUND: The knowledge base-driven pathway analysis is becoming the first choice for many investigators, in that it not only can reduce the complexity of functional analysis by grouping thousands of genes into just several hundred pathways, but also can increase the explanatory power for the experiment by identifying active pathways in different conditions. However, current approaches are designed to analyze a biological system assuming that each pathway is independent of the other pathways. RESULTS: A decision analysis model is developed in this article that accounts for dependence among pathways in time-course experiments and multiple treatments experiments. This model introduces a decision coefficient-a designed index, to identify the most relevant pathways in a given experiment by taking into account not only the direct determination factor of each Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway itself, but also the indirect determination factors from its related pathways. Meanwhile, the direct and indirect determination factors of each pathway are employed to demonstrate the regulation mechanisms among KEGG pathways, and the sign of decision coefficient can be used to preliminarily estimate the impact direction of each KEGG pathway. The simulation study of decision analysis demonstrated the application of decision analysis model for KEGG pathway analysis. CONCLUSIONS: A microarray dataset from bovine mammary tissue over entire lactation cycle was used to further illustrate our strategy. The results showed that the decision analysis model can provide the promising and more biologically meaningful results. Therefore, the decision analysis model is an initial attempt of optimizing pathway analysis methodology.

Isolation of RNA from milk somatic cells as an alternative to biopsies of mammary tissue for nutrigenomic studies in dairy ewes.

Nutrigenomic studies of mammary lipogenesis in ruminants often rely on the use of mammary tissue (MT) collected either by biopsy or at slaughter. However, isolating RNA from milk would be a useful and cost-effective technique that may avoid distress to the animal and facilitate the collection of samples in time series experiments. This assay was therefore conducted to test the hypothesis that RNA extracted from milk somatic cells (MSC) in dairy sheep would be a feasible alternative to the performance of MT biopsies for nutrigenomic analyses. To meet this objective, 8 lactating Assaf ewes were divided in 2 groups and offered a total mixed ration without supplementation (control) or supplemented with 2.4% dry matter of fish oil, which was known not only to elicit milk fat depression but also to downregulate the expression of some candidate genes involved in mammary lipogenesis. Total RNA was extracted from MSC and biopsied MT to examine whether the potential changes in the abundance of transcripts was similarly detected with both RNA sources. Milk fatty acid profile was also analyzed by gas chromatography, and variations in mRNA abundance were determined by reverse transcription quantitative PCR. Values of RNA integrity number were always ≥7.7. The expected and designed decrease of milk fat concentration with fish oil (-29%), was associated with a lower transcript abundance of genes coding for enzymes involved in fatty acid activation (ACSS1), de novo synthesis (ACACA and FASN), uptake from plasma lipids (LPL), and esterification of fatty acids to glycerol (LPIN1), as well as of a transcription factor that may regulate their expression (INSIG1). Stable mRNA levels were showed in other candidate genes, such as FABP3, GPAT4, or SCD. Changes due to the dietary treatment were similarly detected with both RNA sources (MSC and MT biopsies), which supports the initial hypothesis and would validate the use of milk as an alternative RNA source for nutrigenomic analyses in dairy sheep.

Assessment of the proliferative capacity of the flavanones 8-prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and naringenin in MCF-7 cells and the rat mammary gland.

8-Prenylnaringenin (8-PN) and naringenin (Nar) are phytoestrogens found in food items and nutritional supplements, while 6-(1.1-dimethylallyl)naringenin (6-DMAN) is a component of an African plant. Besides their assumed beneficial effects they may promote mammary and endometrial cancer. We therefore assessed their proliferative and estrogenic potential on the mammary gland in vitro and in vivo. In competitive estrogen receptor (ER) ligand binding assays 8-PN displayed a high relative binding affinity for both ERs with a preference for ERα and had the strongest mitotic effect on MCF-7 cells among the test substances. In a three day exposure in young adult ovariectomized female rats 15 mg/kg 8-PN had the highest capacity to increase the number of terminal end buds (TEB) in the mammary gland and stimulated expression of proliferation markers in epithelial ductal cells, followed by 6-DMAN and Nar, but overall their capacity to stimulate proliferation was weak in comparison to 17ß-Estradiol (E2).

Characterisation and expression profile of the bovine cathelicidin gene repertoire in mammary tissue.

BACKGROUND: Cathelicidins comprise a major group of host-defence peptides. Conserved across a wide range of species, they have several functions related to host defence. Only one cathelicidin has been found in humans but several cathelicidin genes occur in the bovine genome. We propose that these molecules may have a protective role against mastitis. The aim of this study was to characterise the cathelicidin gene-cluster in the bovine genome and to identify sites of expression in the bovine mammary gland. RESULTS: Bioinformatic analysis of the bovine genome (BosTau7) revealed seven protein-coding cathelicidin genes, CATHL1-7, including two identical copies of CATHL4, as well as three additional putative cathelicidin genes, all clustered on the long arm of chromosome 22. Six of the seven protein-coding genes were expressed in leukocytes extracted from milk of high somatic cell count (SCC) cows. CATHL5 was expressed across several sites in the mammary gland, but did not increase in response to Staphylococcus aureus infection. CONCLUSIONS: Here, we characterise the bovine cathelicidin gene cluster and reconcile inconsistencies in the datasets of previous studies. Constitutive cathelicidin expression in the mammary gland suggests a possible role for these host defence peptides its protection.

Assessment of ORAI1-mediated basal calcium influx in mammary epithelial cells.

BACKGROUND: The entry of calcium ions into mammary gland epithelial cells is one of the least well-understood processes in the transport of calcium into milk during lactation. The store-operated calcium entry channel ORAI1, has been suggested as a potential mechanism for the entry of Ca(2+) into mammary gland epithelial cells from the maternal blood supply during lactation. The down regulation of the canonical ORAI1 activator STIM1 during lactation suggests that other known ORAI activators such as STIM2 and SPCA2 may be important during lactation. RESULTS: Differentiation of HC11 mammary gland epithelial cells was associated with enhanced basal Ca(2+) influx. Silencing of Orai1 abolished this enhancement of Ca(2+) influx. Stim2 had a modest effect on Ca(2+) influx in this in vitro model of lactation, whereas Stim1 and Spca2 silencing had no effect. Despite pronounced increases in Spca2 mRNA during lactation there was no change in the generation of the alternative splice product generated by Mist1, which increases during lactation. CONCLUSIONS: These studies support the hypothesis that lactation is associated with a remodelling of Ca(2+) influx and this is associated with enhancement of basal Ca(2+) influx. This enhanced Ca(2+) influx appears to occur through the calcium channel Orai1.

Assessment of ABCG2-mediated transport of xenobiotics across the blood-milk barrier of dairy animals using a new MDCKII in vitro model.

The ATP-binding cassette (ABC) efflux transporter ABCG2 represents the main route for active secretion of drugs and toxins across the blood-milk barrier, thereby producing a potential health risk for dairy consumers through formation of relevant residues in milk. However, no suitable in vitro model is as yet available to systematically investigate ABCG2-mediated transport of xenobiotics into milk of dairy animals. We recently cloned ABCG2 from the lactating mammary gland of dairy cows (bABCG2) and goats (cABCG2). Thus, the objective of this study was to generate a suitable blood-milk barrier in vitro model using polarized MDCKII monolayers stably expressing mammary bABCG2 or cABCG2. ABCG2 protein was localized by confocal microscopy to the apical and lateral plasma membrane of polarized MDCKII cells. Intact barrier function of MDCKII-bABCG2 and MDCKII-cABCG2 monolayers was confirmed by determination of cell permeability of transcellular marker propranolol and paracellular marker atenolol which was ≤1 %. In flux assays, ABCG2 substrate 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) showed preferential basolateral to apical (B > A) transport in ABCG2-MDCKII cells. This apically directed PhIP transport was significantly inhibited by ABCG2 inhibitor fumitremorgin C (FTC) or the flavonoid equol. PhIP B > A transport in MDCKII-bABCG2 monolayers was additionally decreased by ABCG2 inhibitor Ko143. The fluoroquinolone antibiotic enrofloxacin was identified as a substrate of ruminant mammary ABCG2. The analgesic drug sodium salicylate was shown to be substrate of bABCG2 but not of cABCG2. Thus, the generated mammary ABCG2-expressing MDCKII cells represent a valuable tool to study active secretion of drugs and toxins into milk.

Effect of fur removal on the thermal conductance and energy budget in lactating Swiss mice.

The limits to sustained energy intake (SusEI) are important because they determine the ceiling restricting all the competing physiological processes. A recent hypothesis is that SusEI is constrained by the capacity to dissipate heat. However some previous data for Swiss mice are inconsistent with this hypothesis. To examine the role of limits to heat dissipation on SusEI, the body temperature, thermal conductance and lactation performance were measured in dorsally shaved Swiss mice. Shaving reduces external insulation and the heat dissipation limitation hypothesis predicts such animals should be capable of eating more food and raising heavier litters. Shaved mice had a significantly higher thermal conductance and a faster reduction in body temperature following noradrenaline injection. At peak lactation, shaved mice spent more time in feeding behaviour, and increased food intake above that observed in non-shaved controls, indicating that limits on SusEI might be imposed by the capacity to dissipate heat. However, shaved females did not spend more time suckling their pups, and did not raise heavier litters, which is inconsistent with the expectations of the heat dissipation limitation hypothesis. The strong correlations between resting, feeding and suckling behaviour at peak lactation suggested that there might be a trade-off in the time distribution between the behavioural patterns. These data suggest that limits on performance may be set at different levels in different strains or species. In MF1 mice studied previously the limit on milk production imposed by maximal mammary secretion capability may lie above that for heat dissipation, hence when the latter was increased the mice produced more milk and raised heavier litters. In Swiss mice the opposite might be the case. Hence when the heat dissipation capacity was increased this did not translate into heavier litters, i.e. supporting the peripheral limitation hypothesis. Further work in a range of additional species or strains will be necessary to establish whether the more normal condition is for SusEI in animals during late lactation to be set by combined peripheral demands or by the heat dissipation capacity.

High rates of mammary tissue protein turnover in lactating goats are energetically costly.

The high energetic demands and metabolism of amino acids (AA) within the lactating mammary gland have been ascribed to the requirements for milk component synthesis and tissue maintenance. Our objective in this work was to assess rates of protein synthesis from several AA so that the energetic costs of tissue maintenance could be better reflected. Lactating goats (n = 4) were given staggered infusions of 5 labeled forms of phenylalanine (Phe) initiated at 30, 12, 9, 6, and 3 h before goats were killed. [5-(13)CH(3)] Methionine (Met), [1-(13)C] leucine, and [1-(13)C] valine were also infused for 30 h, during which time, the glands were milked hourly and arteriovenous flux measurements were performed the last 6 h. A dynamic, compartmental model capable of simulating fluxes of AA through extracellular and intracellular free, slow and fast turnover tissue-bound, and milk protein pools was developed and fitted to the observed data. The udder removed 81% of the Phe present in plasma using 31% for milk protein synthesis and releasing 66% back into plasma. Transamination accounted for 40% of Phe flux in the mammary and transmethylation accounted for a portion of mammary Met flux. Mammary tissue protein synthesis was >300% the value of milk protein synthesis with fractional protein synthesis rates >130%/d. Assuming 4 mol of ATP/mol of peptide bond formed, we estimate that approximately 50% of ATP generated by the lactating mammary glands is used for synthesis of tissue (nonmilk) protein.

Assessment of antimicrobial transfer from treated to untreated mammary gland quarters by use of high-pressure liquid chromatography for detection of cloxacillin in milk samples from nonlactating dairy cows.

OBJECTIVE: To determine whether half-udder intramammary infusion of cloxacillin results in transfer of cloxacillin from treated to untreated mammary gland quarters within nonlactating cows, and, if so, at what concentrations, and to determine whether selection of ipsilateral versus diagonal-contralateral quarters for treatment affects cloxacillin transfer among quarters. ANIMALS: 20 Holstein-Friesian cows from a dairy herd. PROCEDURES: A within-cow half-udder comparison trial was used in which 2 of 4 mammary gland quarters (ipsilaterally or diagonally) received an intramammary infusion of cloxacillin on day 1 of the nonlactating period. Three days later, milk samples were taken from all untreated quarters and high-pressure liquid chromatography was used to detect and quantify milk cloxacillin concentrations. RESULTS: Cloxacillin was detected in 25% of all untreated mammary gland quarters. Mean cloxacillin concentration in untreated quarters was below minimum inhibitory concentrations for targeted mastitis pathogens. No significant difference in cloxacillin concentrations was found in the ipsilateral or diagonal treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE: Within-cow half-udder comparison trials are valid for antimicrobial trials in nonlactating cows, although transfer of antimicrobials does occur in trace concentrations. Ipsilateral or diagonal-contralateral treatment designs perform similarly. This type of design is economical for researchers, although care must be taken to account for within-cow clustering of mammary gland quarter data.

The potential of measuring serum amyloid A in individual ewe milk and in farm bulk milk for monitoring udder health on sheep dairy farms.

The aim of the study was to determine the diagnostic value of measuring serum amyloid A (SAA) concentrations in milk of individual ewes and in farm bulk milk for monitoring udder health. Udder health was calculated by examining a randomly selected group of seven flocks at each farm visit by means of California mastitis test and bacteriological examination of 5749 milk samples. SAA was determined additionally in 267 randomly selected milk samples from six flocks. Thirty-one bulk milk samples from these farms were tested for SCC and SAA levels. Subclinical infections were detected in 29.5% of samples whereas no clinical infections were observed. Intramammary infected udder halves showed significantly elevated SAA concentrations (121.3+/-25.3 microg/ml) in milk compared to the levels of healthy udder halves (8.0+/-1.9 microg/ml; p<0.001). SAA was significantly elevated in sheep with elevated CMT scores and positive bacteriological results. Bulk milk SAA levels ranged from 18.6+/-6.7 to 37.4+/-14.1 microg/ml and showed a positive correlation with bSCC (r=0.38, p=0.018) but not with percent infected glands (r=0.022, p=0.453). This study demonstrated that SAA levels in milk can be used to detect subclinical mastitis in individual ewes whereas further investigations are needed to determine the value of measuring SAA in bulk milk for monitoring flock udder health.

Immunolocalization of the smooth muscle-specific protein calponin in complex and mixed tumors of the mammary gland of the dog: assessment of the morphogenetic role of the myoepithelium.

The immunohistochemical expression of the smooth muscle-specific protein calponin was studied to assess the contribution of myoepithelial cells to the histogenesis of spindle cells of complex and mixed tumors of the mammary gland of the dog and the origin of cartilage and bone in mixed tumors. Formalin-fixed tissues from 55 benign and malignant tumors (49 also containing surrounding normal mammary gland) were evaluated. Periacinar and periductal myoepithelial cells of all the 49 normal mammary glands were diffusely stained by the anti-human calponin monoclonal antibody. Calponin was found in 53 (98%) of the tumors studied, reacting with the myoepithelium-like cells of 86% of benign tumors and their remnants in 85% of malignant tumors. Five different types of calponin-immunoreactive myoepithelial cells were identified: hypertrophic myoepithelial cells. fusiform cells, stellate myoepithelial cells, rounded (myoepithelial) cells, and chondroblasts. Differences in staining intensity and staining pattern among these five types of cells suggested a transition of myoepithelial cells to chondroblasts. Stromal myofibroblasts also showed calponin immunoreactivity, but they did not react with a cytokeratin 14 monoclonal antibody, which recognizes myoepithelial cells in mammary gland. Calponin appears to be a very sensitive marker of normal and neoplastic myoepithelium in the canine mammary gland, and its identification in different cell types of complex and mixed tumors of the mammary gland of the dog suggests a major histogenetic role for myoepithelial cells.

Impact of mastitis control measures on milk production and mastitis indicators in smallholder dairy farms in Kiambu district, Kenya.

Bovine mastitis and mastitis control were investigated on smallholder farms in central Kenya. After an initial observational study, a clinical trial to assess the impact of three different mastitis control strategies--(1) improved udder hygiene, (2) treatment of subclinical cases, and (3) a combination of these--was conducted on 100 randomly selected farms with 332 lactating cows. Before the implementation of control measures, the milk yield was low (mean 6.5 kg/day; median 6 kg/day) and somatic cell counts (SCC) were high, with 80% and 43% of cows having milk with SCC greater than 250 x 10(3) cells/ml and 600 x 10(3) cells/ml, respectively. Infectious pathogens were also commonly isolated, with 63% of cows being positive for pathogenic bacteria. Neither intervention strategy alone had any effect on mastitis indicators or milk yield. In combination, the measures had some impact, lowering the prevalence of contagious pathogens by 18%, but this was not reflected in a significantly increased milk yield, lowered SCC or reduced incidence of clinical mastitis.