Teat morphology across five buffalo breeds: a multi-country collaborative study.

The buffalo (Bubalus bubalis) is a species of worldwide importance, raised to produce milk, meat, and hides, and often used as a working animal in rural contexts with low access to hi-tech solutions. In the present study, 100 lactating buffaloes (50 primiparous and 50 pluriparous) of five popular breeds were recruited to characterize and compare teat morphology. In particular, the focus was put on the Nili Ravi, Mediterranean, Egyptian, Bulgarian Murrah, and Azeri buffaloes raised in Pakistan, Italy, Egypt, Bulgaria, and Iran, respectively. In all countries, a longitudinal cross-section ultrasound was obtained before the milking to measure teat parameters at individual level: overall, teat canal length (TCL) averaged 24.13 mm, teat diameter (TD) 30.46 mm, cisternal diameter (CD) 17.80 mm, and teat wall (TW) 7.12 mm. The most variable trait across breeds was TCL which was positively correlated with CD and TD and negatively with TW, regardless of the teat position (front/rear or left/right). A strong negative correlation was found between TW and CD (- 0.43). The analysis of variance revealed that the fixed effect of breed significantly affected all the traits except TD. In fact, Bulgarian Murrah, Azeri, and Egyptian buffaloes presented the greatest estimate of TCL, whereas NR the smallest (14.70 mm). The TW was maximum in Nili Ravi, Egyptian, and Mediterranean buffaloes, with estimates equal to 8.19, 7.59, and 8.74 mm, respectively. Nili Ravi also showed the greatest TL (82.39 mm). In terms of CD, the lowest least square mean was that of Mediterranean buffaloes (12.14 mm). Primiparous and pluriparous buffaloes differed in terms of TD, TW, and TL, with older animals presenting the highest least square mean. In terms of position, instead, significant differences were observed for TD, CD, and TL when comparing front and rear teats, as left and right teats did not differ. Teat anatomy includes a set of heritable morphological features and is therefore breed-dependent. Differences presented in this study could be attributed to the divergent breeding objective and selective pressure across the five breeds; e.g., in some cases such as Mediterranean buffalo, selection for decades was oriented to improve milk production and milkability and achieve optimal conformation for mechanical milking. A better understanding of the mammary gland anatomical descriptors can be informative of the history of a breed and could provide useful insights to guide possible selection.

Digital infrared thermal imaging of udder skin surface temperature: a novel non-invasive technology to monitor calving process in Murrah buffalo (Bubalus bubalis).

Quantifiable decline in the maternal body temperature during the pre-calving offers the possibilities for predicting the calving that can improve the calving management. As infrared thermography (IRT) is a simple non-contact tool for precise measurement of surface temperature, we investigated the use of IRT to establish thermal signatures around calving in the Murrah buffalo. The IRT of eye, right lateral, left lateral and rear side of udder skin surface temperature (USST) were recorded at 6 h interval from 96 h before the expected date of calving, at the time of calving and 24 h post-calving in Murrah buffaloes (n = 28). In parallel, blood samples were collected for progesterone (P4) assay. The results revealed that the IRT of the eye, right and left lateral and rear side of USST showed a significant decrease in the temperature from 48 h pre-calving till the onset of calving with a ΔT (°C) of 0.56, 0.91, 0.70, and 0.90, respectively. Mean USST significantly declined from 48 h pre-calving with a ΔT of 0.85 °C. The residual temperature of both eye and various ROI of the udder also followed a similar and significant declining trend from 48 to 0 h of calving indicating that circadian influence on the USST was minimum. Plasma P4 concentration significantly decreased from 72 h pre-calving till calving. It is concluded that a marked reduction in the IRT of the USST at 6-12 h pre-calving would be useful in predicting the onset of calving in the Murrah buffalo.

Longitudinal analysis of bovine mammary gland development.

Many studies on bovine mammary glands focus on one stage of development. Often missing in those studies are repeated measures of development from the same animals. As milk production is directly affected by amount of parenchymal tissue within the udder, understanding mammary gland growth along with visualization of its structures during development is essential. Therefore, analysis of ultrasound and histology data from the same animals would result in better understanding of mammary development over time. Thus, this research aimed to describe mammary gland development using non-invasive and invasive tools to delineate growth rate of glandular tissue responsible for potential future milk production. Mammary gland ultrasound images, biopsy samples, and blood samples were collected from 36 heifer dairy calves beginning at 10 weeks of age, and evaluated at 26, 39, and 52 weeks. Parenchyma was quantified at 10 weeks of age using ultrasound imaging and histological evaluation, and average echogenicity was utilized to quantify parenchyma at later stages of development. A significant negative correlation was detected between average echogenicity of parenchyma at 10 weeks and total adipose as a percent of histological whole tissue at 52 weeks. Additionally, a negative correlation between average daily gain at 10 and 26 weeks and maximum echogenicity at 52 weeks was present. These results suggest average daily gain and mammary gland development prior to 39 weeks of age is associated with development of the mammary gland after 39 weeks. These findings could be predictors of future milk production, however this must be further explored.

Relationship between blood calcium level and post-milking teat canal closure in dairy cows.

The teat canal-one of the primary defense mechanisms of the udder-ensures the milk flow during milking in bovines and prevents pathogens from entering the udder by forming a barrier through the elastic muscle and keratin layers tightly closing the surrounding area. The current study investigated the effects of blood calcium status on teat closure in cows after milking. The study covered 200 healthy teats, of which 100 were from normocalcemic (NC) cows and 100 were from subclinical hypocalcemic (SCH) cows. Teat canal length (TCL) and width (TCW) were measured with ultrasonography at 0-min pre-milking and 15- and 30-min post-milking. Cylindrically shaped teat canal volume (TCV) was calculated by deriving from TCL and TCW. Time-dependent changes in teat canal closure and their relationships with blood calcium levels were analyzed. The results showed that the calcium level did not affect TCL, TCW, and TCV (P > 0.05) during the 15-min post-milking period. However, TCL (P < 0.001), TCW (P < 0.05), and TCV (P < 0.001) were lower in NC cows than in SCH cows at 30-min post-milking. At 15-min post-milking, no correlation existed between the teat canal closure (ΔTCL, ΔTCW, and ΔTCV) and the blood calcium level, while significant correlations were available between the teat canal closure and the blood calcium level {ΔTCL (r: - 0.288, P < 0.001), ΔTCW (r: - 0.260, P < 0.001), ΔTCV (r: - 0.150, P < 0.05)} at 30-min post-milking. The current study concluded that the blood calcium status significantly impacts the teat canal closure in bovines, and calcium status should be meticulously monitored with the mastitis control program to apply necessary strategic steps.

Ultrasound characterization of mammary gland development in heifer calves fed at two different levels until weaning.

Ultrasound technologies allow for a non-invasive assessment of mammary gland (MG) development, the differentiation between the tissue types of the MG, and the evaluation of changes in its composition. This study aimed to work out a detailed description of the different stages of MG development that are visually discernible by ultrasonography for providing a template to classify the different structures. With this basis, the qualitative categorization of the developmental stage, as well as further quantitative assessments via pixel densities in the structures of interest, should be facilitated. Ultrasonic images were acquired from all four quarters of 37 German Holstein heifer calves fed either at a high feeding level of milk replacer (MR; 14% solids) at 10 L/day (1.4 kg MR/day; HI, n = 18) or at a restrictive low level of 5.7 L/day (0.8 kg MR/day; RES, n = 19) until linear weaning from week 13 to 14 of life. Ultrasound MG scans were performed first in week 3 of life, fortnightly from week 8-16, and in week 20 of life, in standing position, of each quarter, using a B-mode ultrasound device equipped with a linear probe (18 MHz). The developmental stages of the mammary gland parenchyma (PAR), visible in ultrasound images, obtained over 20 weeks of life, were categorized, described, and drawn by hand. On this basis, a template for classifying the visible categories of mammary PAR development and its surrounding tissue (SURR), and for measuring their pixel brightness was created thus providing an ultrasonographic atlas of the developing bovine MG, describing 11 categories. The ultrasound images were further classified by PAR structure, and pixel brightness was measured in PAR and SURR by using ImageJ Fiji. The difference in pixel brightness between PAR and SURR, the delta (Δ) pixel value was calculated. With increasing age, higher atlas categories of PAR developmental stages were shown. Pixel values, i.e. the brightness of PAR, its SURR, and Δ pixel value changed with age but were neither affected by the feeding group nor by a group × time interaction. With progressing PAR development, its pixel brightness increased from week 10 to 20 of life, i.e., the PAR became more hyperechoic since it spread and grew into its SURR. The atlas can serve as a template for the categorization and qualitative assessment of MG structures and for the quantitative assessment of PAR's development by measuring pixel brightness. With our study, we could show the structural development in PAR as well as in SURR in MG simultaneously in early life and confirm the spreading of PAR into its SURR by ultrasound scanning.

Mouse Mammary Gland Whole Mount Density Assessment across Different Morphologies Using a Bifurcated Program for Image Processing.

Mammographic density is associated with increased breast cancer risk. Conventional visual assessment of murine mouse models does not include quantified total density analysis. A bifurcated method was sufficient to obtain relative density scores on a broad range of two-dimensional whole mount images that contained both normal and abnormal findings. Image processing techniques, including a ridge operator and a gaussian denoising method, were used to isolate background away from mammary epithelium and use mean pixel intensity to represent mammary density on genetically engineered mouse models for breast cancer in mice 4 to 29 months of age. The bifurcated method allowed for application of an optimal image processing approach for the structural elements present in the whole mount images. Gaussian denoising was the optimal approach when more dense lobular growth and tertiary branching dominate and a ridge operator when epithelial growth was more sparse and secondary branching was the more dominant structural feature. The two processing approaches were combined in a single experimental flow program using an initial image density measurement as the decision point between the two approaches. Higher density was associated with lobular growth, tertiary branching, fibrotic stroma, and presence of cancer. The significance of the study is development of a readily accessible program for digital assessment of mammary gland whole mount density across a range of mammary gland morphologies.

Intravital Subcellular Microscopy of the Mammary Gland.

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676 or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs ≥ 0.7 µm. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 µm or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid.

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

Morphology of the mammary gland in romanian buffalo.

The gross morphology of the mammary gland in buffalo is the foundation that is necessary for the process to improve the species for agricultural use. This study determined the mammary gland's anatomical, histological and imaging structure in the Romanian buffalo. The research material was represented by ten udders, five udders from adult buffaloes and five udders from young females slaughtered in the abattoir. The udders were collected immediately after slaughtering the animals, then transported to USAMV Cluj-Napoca, Romania, examined macroscopically, histologically and through imagistic methods. The morphological examination tracked the topography, external conformation, vascularization and innervation of the mammary gland in the buffalo. The histological examination highlighted differences between the ductoacinary structures of the mammary gland parenchyma in adult buffaloes compared with young females. The imaging examination showed the detachment of the middle mammary artery and the arterial anastomoses established between the mammary arteries on the left side of the udder and those on the right side and between the arteries of the quarters on the same side. This research can be theoretical and practical support to other studies in establishing the most accurate diagnosis and therapeutic protocol in various clinical pathologies of the udder in buffalo.

Sonographic and histological udder parenchyma changes after intramammary infection of sheep with Mycoplasma agalactiae.

INTRODUCTION: The sonographic findings of the udder parenchyma and udder lymph nodes in 30 lactating sheep after experimental infection with Mycoplasma agalactiae are described. The objective of the study was to describe infection related changes in the udder parenchyma and udder lymph nodes using physical, sonographic, and histological examination and to detect associations between sonographic and histological changes of the tissues. Animals were intramammarily infected with different mutant cocktails and the wild type PG2. One group served as a negative control. A 15 MHz linear transducer (Esaote MyLab 30 CV, Esaote, Florence, Italy) was used for sonographic examinations. Compared with the uninfected control group with homogeneously granular parenchyma, the udder lymph nodes were larger and the udder parenchyma was more inhomogeneous and partially hyperechoic. The corresponding histological findings in infected mammary glands comprised proliferation of interstitial connective tissue, non-purulent interstitial mastitis, and purulent galactophoritis. The infected udder lymph nodes showed reactive hyperplasia. The findings obtained in this study may improve the diagnosis of Mycoplasma mastitis in sheep.

INTRODUCTION: Les constatations échographiques sur le parenchyme de la mamelle et des ganglions lymphatiques de la mamelle chez 30 brebis en lactation après une infection expérimentale avec Mycoplasma agalactiae sont décrits. L'objectif de l'étude était de décrire les modifications liées à l'infection dans le parenchyme mammaire et les ganglions lymphatiques de la mamelle à l'aide d'un examen physique, échographique et histologique et de détecter les associations entre les altérations échographiques et histologiques des tissus. Les animaux ont été infectés par voie intramammaire avec différents cocktails de mutants et le type sauvage PG2. Un groupe a servi de contrôle négatif. Une sonde linéaire de 15 MHz (Esaote MyLab 30 CV, Esaote, Florence, Italie) a été utilisé pour les examens échographiques. Comparativement au groupe témoin non infecté avec un parenchyme granulaire homogène, les ganglions lymphatiques de la mamelle étaient plus gros et le parenchyme de la mamelle était plus inhomogène et partiellement hyperéchogène. Les résultats histologiques correspondants dans les glandes mammaires infectées comprenaient une prolifération de tissu conjonctif interstitiel, une mammite interstitielle non purulente et une galactophorite purulente. Les ganglions lymphatiques de la mamelle infectée présentaient une hyperplasie réactive. Les résultats obtenus dans cette étude peuvent améliorer le diagnostic de la mammite à Mycoplasma chez le mouton.

Quantitative measurement of udder oedema in dairy cows using ultrasound to monitor the effectiveness of diuretic treatment with furosemide.

INTRODUCTION: The aim of this study was to record the course of peripartal udder oedema with ultrasonography in dairy cows and to investigate the therapeutic effects of diuretic furosemide. For this purpose, a device with a force sensor for the ultrasound probe was developed, which ensured the generation of data under similar pressure conditions and thereby repeatable and comparable results. In a pretrial with ten cows, ultrasonographic measurements were performed daily at four locations per udder quarter beginning 14 days ante partum until 14 d post partum. The main study included 50 dairy cows. The experimental group (n=25) received 500 mg furosemide intramuscularly on the day of calving, as well as on the first and second day post partum. The control group (n=25) was treated with 10 mL 0,9 % sodium chloride solution (NaCl) at the same timepoints. The experimental period was extended from 21 days ante partum until 21 days post partum and included 15 ultrasonographic measurements at three-day intervals. The measurements were performed at the teat base, which turned out to be the most suitable location in the pretrial. Quarter milk samples were collected on the day of calving, 7 and 14 days post partum. The average thickness of the udder oedema between the group treated with furosemide and the control group did not differ. In conclusion, a method for the ultrasonographic measurement of udder oedema under comparable applied forces was established. The teat base turned out to be a suitable location to monitor the characteristic temporal course of udder oedema. In the present study, treatment with furosemide did not have a measurable, positive effect on the severity of the udder oedema post partum in the study animals. Finally, further studies with a higher sample size are necessary to confirm this relationship.

INTRODUCTION: Le but de la présente étude était d'enregistrer l'évolution de l'œdème de la mamelle périnatal par échographie chez les vaches laitières et d'étudier les effets thérapeutiques du diurétique furosémide. À cette fin, un appareil avec un capteur de force pour la sonde à ultrasons a été développé, ce qui a assuré la génération de données dans des conditions de pression similaires et ainsi des résultats reproductibles et comparables. Dans un essai préliminaire avec dix vaches, des mesures échographiques ont été effectuées quotidiennement à quatre endroits par quartier à partir de 14 jours ante partum jusqu'à 14 jours post partum. L'étude principale portait sur 50 vaches laitières. Le groupe expérimental (n=25) a reçu 500 mg de furosémide par voie intramusculaire le jour du vêlage ainsi que les premier et deuxième jours post-partum. Le groupe témoin (n = 25) a été traité avec 10 ml de solution de chlorure de sodium à 0,9 % (NaCl) aux mêmes moments. La période expérimentale a été étendue de 21 jours ante partum jusqu'à 21 jours post partum et comprenait 15 mesures échographiques à trois jours d'intervalle. Les mesures ont été effectuées à la base du trayon, qui s'est avérée être l'emplacement le plus approprié dans la phase préliminaire. Des échantillons de lait de chaque quartier ont été prélevés le jour du vêlage, 7 et 14 jours post-partum. L'épaisseur moyenne de l'œdème de la mamelle entre le groupe traité au furosémide et le groupe témoin ne différait pas. En conclusion, une méthode de mesure échographique de l'œdème de la mamelle sous des forces appliquées comparables a été établie. La base du trayon s'est avérée être un endroit approprié pour surveiller l'évolution temporelle caractéristique de l'œdème de la mamelle. Dans la présente étude, le traitement au furosémide n'a pas eu d'effet positif mesurable sur la gravité de l'œdème de la mamelle post-partum chez les animaux de l'étude. Enfin, d'autres études avec une taille d'échantillon plus élevée sont nécessaires pour confirmer cette relation.

Multiscale imaging of basal cell dynamics in the functionally mature mammary gland.

The mammary epithelium is indispensable for the continued survival of more than 5,000 mammalian species. For some, the volume of milk ejected in a single day exceeds their entire blood volume. Here, we unveil the spatiotemporal properties of physiological signals that orchestrate the ejection of milk from alveolar units and its passage along the mammary ductal network. Using quantitative, multidimensional imaging of mammary cell ensembles from GCaMP6 transgenic mice, we reveal how stimulus evoked Ca2+ oscillations couple to contractions in basal epithelial cells. Moreover, we show that Ca2+-dependent contractions generate the requisite force to physically deform the innermost layer of luminal cells, compelling them to discharge the fluid that they produced and housed. Through the collective action of thousands of these biological positive-displacement pumps, each linked to a contractile ductal network, milk begins its passage toward the dependent neonate, seconds after the command.

Magnetic resonance angiography reveals increased arterial blood supply and tumorigenesis following high fat feeding in a mouse model of triple-negative breast cancer.

Breast cancer is the second most commonly diagnosed malignancy among women globally. Past MRI studies have linked a high animal fat diet (HAFD) to increased mammary cancer risk in the SV40Tag mouse model of triple-negative breast cancer. Here, serial MRI examines tumor progression and measures the arterial blood volume feeding mammary glands in low fat diet (LFD) or HAFD fed mice. Virgin female C3(1)SV40Tag mice (n = 8), weaned at 3 weeks old, were assigned to an LFD (n = 4, 3.7 kcal/g, 17.2% kcal from vegetable oil) or an HAFD (n = 4, 5.3 kcal/g, 60% kcal from lard) group. From ages 8 to 12 weeks, weekly fast spin echo MR images and time-of-flight (TOF) MR angiography of inguinal mammary glands were acquired at 9.4 T. Following in vivo MRI, mice were sacrificed. Inguinal mammary glands were excised and fixed for ex vivo MRI and histology. Tumor, blood, and mammary gland volumes for each time point were measured from manually traced regions of interest; tumors were classified as invasive by histopathology-blinded observers. Our analysis confirmed a strong correlation between total tumor volume and blood volume in the mammary gland. Tumor growth rates from weeks 8-12 were twice as high in HAFD-fed mice (0.42 ± 0.14/week) as in LFD-fed mice (0.21 ± 0.03/week), p < 0.004. Mammary gland blood volume growth rate was 2.2 times higher in HAFD mice (0.29 ± 0.11/week) compared with LFD mice (0.13 ± 0.06/week), p < 0.02. The mammary gland growth rate of HAFD-fed mice (0.071 ± 0.011/week) was 2.7 times larger than that of LFD-fed mice (0.026 ± 0.009/week), p < 0.01. This is the first non-invasive, in vivo MRI study to demonstrate a strong correlation between an HAFD and increased cancer burden and blood volume in mammary cancer without using contrast agents, strengthening the evidence supporting the adverse effects of an HAFD on mammary cancer. These results support the potential future use of TOF angiography to evaluate vasculature of suspicious lesions.

Different vacuum levels, vacuum reduction during low milk flow, and different cluster detachment levels affect milking performance and teat condition in dairy cows.

Traditionally, machine milking is performed at a constant vacuum supply. The system vacuum has to be set high enough to allow a sufficiently high vacuum at the teat end, despite the inevitable vacuum drop caused by milk flow. This leads to an increased vacuum load on the teat, especially when milk flow ceases at the end of milking. We tested the hypothesis that a milk flow-controlled adaptation of vacuum settings during milking allows even higher vacuum levels than are usually recommended during the period of high milk flow if the vacuum is reduced during low milk flow. Combined with a high cluster detachment flow rate level, increased milking performance is expected without an increased effect on teat tissue. Ten Holstein dairy cows were milked with a bucket milker with the claw vacuum adjusted in the absence of milk flow at a regular (43 kPa) and high (48 kPa) claw vacuum, with and without vacuum reduction during low milk flow (<2 kg/min), and combined with different cluster detachment levels (0.2, 0.6, and 1 kg/min). Each treatment was applied in each cow during 4 subsequent milkings in a randomized crossover design. Both claw vacuum and milk flow were continuously recorded throughout milking. Teat tissue thickness was measured using a cutimeter and teat wall diameter was measured by B-mode ultrasonography at 5 min after the end of milking. Milk yield was not affected by either vacuum settings or detachment levels. Machine-on time in treatments with vacuum reduction was shorter at high than at low vacuum and decreased with increasing detachment levels. Average milk flow was higher at high than at low vacuum and reached highest values in milkings without vacuum reduction at both vacuum levels. The average milk flow was higher at a cluster detachment of 1 kg/min than at 0.2 kg/min. However, both teat tissue thickness and (as a tendency) teat wall diameter at 5 min after cluster detachment were higher in milkings at high vacuum without vacuum reduction compared with all other treatments. In conclusion, high claw vacuum up to 48 kPa increases milking performance because of higher milk flow and reduced machine-on time. Negative effects of high vacuum on teat tissue are prevented by reducing vacuum during low milk flow (<2 kg/min) at the start and end of milking. Additionally, using a high cluster detachment level reduces machine-on time without a loss of harvested milk.

Blood perfusion of teat tissue in dairy cows: Changes associated with pre-milking stimulation and machine milking.

Mechanical forces during machine milking of dairy cows evoke circulatory impairment of the teat tissue that may affect the teats' defense mechanisms against mastitis pathogens. Ample research describes dimensional changes of different teat traits after machine milking, whereas reports that describe changes in blood circulation of dairy cows' teats are limited. Therefore, the objectives of this study were to (1) describe changes in teat blood circulation that occur after pre-milking teat stimulation and machine milking and (2) study the effect of 2 different milking liners on machine milking-induced changes in teat blood flow. In a randomized trial, Holstein dairy cows were stratified by parity, stage of lactation, and average daily milk yield during the previous week, and allocated to 1 of 2 treatment groups. Treatment consisted of 1 milking observation with either a round or multisided concave milking liner. Teat scans were taken of the left front and the right hind teats using power Doppler ultrasonography. Imaging occurred before pre-milking udder preparation (T1), after completion of pre-milking udder preparation but before milking-unit attachment (T2), and immediately after unit detachment (T3). Perfusion intensity measurements from teat scans were performed with a commercially available software program. Data from 109 cows were analyzed. A general linear mixed model showed differences in perfusion intensity between time points. Least squares means (95% confidence intervals) for T1, T2, and T3, respectively, were 0.035% (0.026-0.047), 0.124% (0.093-0.164), and 0.095% (0.073-0.124). Conversely, no statistically significant differences between treatment groups were observed. We conclude that teat blood circulation is subjected to several influences, including inherent circulatory regulation mechanisms, as well as extrinsic factors such as machine milking. Future research is warranted to decipher the magnitude of their influence and to further our understanding of how these changes relate to the susceptibility to intramammary infection and milking performance.

3-D-Printed Registration Phantom for Combined Ultrasound and Optical Imaging of Biological Tissues.

Efforts to develop quantitative ultrasound biomarkers would benefit from comparisons between ultrasound data and higher-resolution images of the tissue microstructure, such as from optical microscopy. However, only a few studies have used these methods for multiscale imaging because it is difficult to register low-resolution (>100 µm) ultrasound images to high-resolution microscopy images. To address this need, we have designed a 3-D-printed registration phantom that is made of a hard fluorescent resin, fits into a glass-bottom dish and can be used to calculate a coordinate system transform between ultrasound and optical microscopy. We report the phantom design, a registration protocol and an example registration using 18.5-MHz ultrasound and second harmonic generation microscopy. We evaluate the registration precision, achieving standard deviations smaller than the ultrasound resolution across all axes, and illustrate on a mouse mammary gland that this method yields results superior to those of manual landmark registration.

Tantalum oxide nanoparticles as versatile contrast agents for X-ray computed tomography.

Here, we describe the synthesis, characterization and in vitro and in vivo performance of a series of tantalum oxide (TaOx) based nanoparticles (NPs) for computed tomography (CT). Five distinct versions of 9-12 nm diameter silane coated TaOx nanocrystals (NCs) were fabricated by a sol-gel method with varying degrees of hydrophilicity and with or without fluorescence, with the highest reported Ta content to date (78%). Highly hydrophilic NCs were left bare and were evaluated in vivo in mice for micro-CT of full body vasculature, where following intravenous injection, TaOx NCs demonstrate high vascular CT contrast, circulation in blood for ∼3 h, and eventual accumulation in RES organs; and following injection locally in the mammary gland, where the full ductal tree structure can be clearly delineated. Partially hydrophilic NCs were encapsulated within mesoporous silica nanoparticles (MSNPs; TaOx@MSNPs) and hydrophobic NCs were encapsulated within poly(lactic-co-glycolic acid) (PLGA; TaOx@PLGA) NPs, serving as potential CT-imagable drug delivery vehicles. Bolus intramuscular injections of TaOx@PLGA NPs and TaOx@MSNPs to mimic the accumulation of NPs at a tumor site produce high signal enhancement in mice. In vitro studies on bare NCs and formulated NPs demonstrate high cytocompatibility and low dissolution of TaOx. This work solidifies that TaOx-based NPs are versatile contrast agents for CT.

The utility of the "Glowing Head" mouse for breast cancer metastasis research.

The expression of cellular reporters to label cancer cells, such as green fluorescent protein (GFP) and luciferase, can stimulate immune responses and effect tumor growth. Recently, a mouse model that expresses GFP and luciferase in the anterior pituitary gland was generated to tolerize mice to these proteins; the "Glowing Head" mouse. Mice were obtained from a commercial vendor, bred, and then used for tumor growth and metastasis studies. The transgene expression of luciferase was assessed within tumor-naïve mice as well as mice with mammary tumors or metastases. Tumor-free mice with white fur, compared to black fur, allowed for stronger luciferase transgene expression to be observed in the pituitary, sternum, and femur. Growth of four different luciferase-expressing mouse cancer cell lines readily occurred in the mammary gland. Though sternum expression of the luciferase transgene occurred in cancer-free mice, growth or death of luciferase positive cancer cells in the lung could be observed. Liver metastases seeded by portal vein injections of luciferase positive cancer cell lines were completely distinct from luciferase transgene expression. Though lung and brain metastasis studies have limitations, the Glowing Head mouse can be useful to inhibit immune system rejection of luciferase or GFP expressing cancer cells. This mouse model is most beneficial for studies of mammary tumors and liver metastases.

Deep imaging reveals new insights into mammary gland architecture and breast cancer susceptibility.

The abundance of adipose tissue in the mammary gland obscures vision of the 3-dimensional architecture. Hitchcock et al. employed a new technique of deep tissue imaging that has enabled visualisation of dynamic interactions between mammary gland epithelial and immune cells with unprecedented 3-dimensional clarity. Deep imaging will help further our understanding of the complex biological interactions that underpin both normal mammary gland development and the susceptibility of this tissue to cancer. This knowledge will assist the development of much-needed prevention strategies to reduce the incidence of breast cancer. Comment on: https://doi.org/10.1111/febs.15126.

Short communication: Teat wall diameter and teat tissue thickness in dairy cows are affected by intramammary pressure and by the mechanical forces of machine milking.

The objective of this study was to validate and apply 2 different methods to record changes in teat tissue related to machine milking. Teat wall diameter was measured via B-mode ultrasound cross sectioning with a 7.5-MHz linear probe. Teat tissue thickness was measured using a cutimeter (spring-loaded caliper, spring constant 6.5 N/cm, 0.5 N at closed jaws). Both methods were applied at the teat barrel, 2 cm above the teat tip. In experiment 1, 24 teats from freshly slaughtered cows were used to perform ultrasound imaging (12 teats) or cutimeter measurements (12 teats) while the teat cisterns were filled with water to increase the intracisternal pressure from 0 to 30 kPa in steps of 1 kPa. Teat tissue thickness did not change at an intracisternal pressure from 0 to 10 kPa but increased with intracisternal pressure at levels >10 kPa. In contrast, teat wall diameter decreased with intracisternal pressure between 0 and 7 kPa but did not significantly change at a pressure ≥7 kPa up to 30 kPa. Significant Pearson correlation coefficients between intracisternal pressure and teat wall diameter were observed from 0 to 7 kPa (r = -0.38), and between intracisternal pressure and teat tissue thickness from 10 to 30 kPa (r = 0.45). In experiment 2, ultrasound and cutimeter measurements were performed in 12 lactating Holstein cows. Measurements before and during milking, immediately after cluster removal, with normal milking or with a 5-min overmilking, were performed and continued at 5-min intervals for 60 min and at 10-min intervals until 120 min. Additionally, with the 5-min overmilking treatment, measurements were continued at 60-min intervals up to 10 h after milking. Teat wall diameter decreased in response to milk ejection, followed by a continuous increase during the course of milking, with highest values after 5 min overmilking. Teat tissue thickness did not change during milking but was significantly increased after overmilking. Teat wall diameter and teat tissue thickness recovered to premilking levels within 35 min after normal milking and within 60 min after overmilking. Until 10 h after overmilking, the teat wall diameter decreased steadily, whereas teat tissue thickness was unfluctuating. In the physiologically relevant range of intramammary pressure, ultrasound measurements of the teat wall were affected by both intramammary pressure and mechanical forces, whereas cutimeter measurements were not affected by the intramammary pressure.