Mammary duct luminal epithelium controls adipocyte thermogenic programme.

Sympathetic activation during cold exposure increases adipocyte thermogenesis via the expression of mitochondrial protein uncoupling protein 1 (UCP1)1. The propensity of adipocytes to express UCP1 is under a critical influence of the adipose microenvironment and varies between sexes and among various fat depots2-7. Here we report that mammary gland ductal epithelial cells in the adipose niche regulate cold-induced adipocyte UCP1 expression in female mouse subcutaneous white adipose tissue (scWAT). Single-cell RNA sequencing shows that glandular luminal epithelium subtypes express transcripts that encode secretory factors controlling adipocyte UCP1 expression under cold conditions. We term these luminal epithelium secretory factors 'mammokines'. Using 3D visualization of whole-tissue immunofluorescence, we reveal sympathetic nerve-ductal contact points. We show that mammary ducts activated by sympathetic nerves limit adipocyte UCP1 expression via the mammokine lipocalin 2. In vivo and ex vivo ablation of mammary duct epithelium enhance the cold-induced adipocyte thermogenic gene programme in scWAT. Since the mammary duct network extends throughout most of the scWAT in female mice, females show markedly less scWAT UCP1 expression, fat oxidation, energy expenditure and subcutaneous fat mass loss compared with male mice, implicating sex-specific roles of mammokines in adipose thermogenesis. These results reveal a role of sympathetic nerve-activated glandular epithelium in adipocyte UCP1 expression and suggest that mammary duct luminal epithelium has an important role in controlling glandular adiposity.

Paternal RLIM/Rnf12 is a survival factor for milk-producing alveolar cells.

In female mouse embryos, somatic cells undergo a random form of X chromosome inactivation (XCI), whereas extraembryonic trophoblast cells in the placenta undergo imprinted XCI, silencing exclusively the paternal X chromosome. Initiation of imprinted XCI requires a functional maternal allele of the X-linked gene Rnf12, which encodes the ubiquitin ligase Rnf12/RLIM. We find that knockout (KO) of Rnf12 in female mammary glands inhibits alveolar differentiation and milk production upon pregnancy, with alveolar cells that lack RLIM undergoing apoptosis as they begin to differentiate. Genetic analyses demonstrate that these functions are mediated primarily by the paternal Rnf12 allele due to nonrandom maternal XCI in mammary epithelial cells. These results identify paternal Rnf12/RLIM as a critical survival factor for milk-producing alveolar cells and, together with population models, reveal implications of transgenerational epigenetic inheritance.

Going Full TeRM: The Seminal Role of Tissue-Resident Macrophages in Organ Remodeling during Pregnancy and Lactation.

During pregnancy and lactation, the uterus and mammary glands undergo remarkable structural changes to perform their critical reproductive functions before reverting to their original dormant state upon childbirth and weaning, respectively. Underlying this incredible plasticity are complex remodeling processes that rely on coordinated decisions at both the cellular and tissue-subunit levels. With their exceptional versatility, tissue-resident macrophages play a variety of supporting roles in these organs during each stage of development, ranging from maintaining immune homeostasis to facilitating tissue remodeling, although much remains to be discovered about the identity and regulation of individual macrophage subsets. In this study, we review the increasingly appreciated contributions of these immune cells to the reproductive process and speculate on future lines of inquiry. Deepening our understanding of their interactions with the parenchymal or stromal populations in their respective niches may reveal new strategies to ameliorate complications in pregnancy and breastfeeding, thereby improving maternal health and well-being.

Alveolar progenitor differentiation and lactation depends on paracrine inhibition of notch via ROBO1/CTNNB1/JAG1.

In the mammary gland, how alveolar progenitor cells are recruited to fuel tissue growth with each estrus cycle and pregnancy remains poorly understood. Here, we identify a regulatory pathway that controls alveolar progenitor differentiation and lactation by governing Notch activation in mouse. Loss of Robo1 in the mammary gland epithelium activates Notch signaling, which expands the alveolar progenitor cell population at the expense of alveolar differentiation, resulting in compromised lactation. ROBO1 is expressed in both luminal and basal cells, but loss of Robo1 in basal cells results in the luminal differentiation defect. In the basal compartment, ROBO1 inhibits the expression of Notch ligand Jag1 by regulating ß-catenin (CTNNB1), which binds the Jag1 promoter. Together, our studies reveal how ROBO1/CTTNB1/JAG1 signaling in the basal compartment exerts paracrine control of Notch signaling in the luminal compartment to regulate alveolar differentiation during pregnancy.

The association between teat shape and bimodal milk ejection in Holstein dairy cows.

Our objectives were to investigate the association of teat shape with (1) bimodality, (2) incremental milk flow rates (average flow rates during the first 15 s, 15-30 s, 30-60 s, and 60-120 s of milking), and (3) peak milk flow rate in Holstein dairy cows milked 3 times/d. In this prospective cohort study, we analyzed 220,928 milking observations that were collected from 2,520 cows from a single dairy herd over a period of 31 d. Teat shape was visually assessed and classified into 1 of 4 categories as follows: (1) triangular barrel and pointed teat end (TP), (2) square barrel and round teat end (SR), (3) square barrel, round teat end, and flat in the area of the teat orifice (SRF), and (4) square barrel and flat teat end (SF). Individual cow-level milk flow rates were obtained using electronic on-farm milk meters. We considered bimodality to be present if any of the incremental milk flow rates (flow rates during 15-30 s, 30-60 s, or 60-120 s) were lower than any of the previous rates (flow rates during the first 15 s, 15-30 s, or 30-60 s). The frequency distribution of cows with different teat shapes was as follows: TP, 96 (3.8%); SR, 1,751 (69.5%); SRF, 617 (24.5%); and SF, 56 (2.2%). A generalized linear mixed model revealed differences in the odds of bimodality among cows with different teat shapes. Compared with cows in category SR, the odds (95% CI) of bimodality of cows in other categories were as follows: TP, 0.68 (0.48-0.97); SF, 1.96 (1.21-3.19); and SRF, 1.46 (1.23-1.72). General linear mixed models indicated an association between teat shape and all incremental milk flow rates, with higher milk flow rates being present for cows with teat shapes in the SRF and SF categories. The general linear mixed model for the dependent variable peak milk flow rate indicated an association between teat shape and peak milk flow rate. Least squares means and 95% CI were 5.1 (4.9-5.3) kg/min for TP, 5.8 (5.5-6.1) kg/min for SF, 5.6 (5.5-5.7) for SRF, and 5.3 (5.3-5.4) for SR, respectively. We conclude that in this study cohort, bimodality is more likely to occur in cows with flat teat ends compared with those with a round teat ends. We attribute this relationship to the difference in milking speed across cows with different teat shapes. Teat shape may serve as a useful phenotype to identify cows that are more likely to exhibit bimodality. Because our study population was unique, future studies considering different circumstances such as breed, milking system, and milking routine are needed before results can be extrapolated.

Advancing mastitis assessment in dairy bovines via short milking tube thermography: A seasonal perspective.

In India, where dairy production leads globally, infrared thermography (IRT) and short milking tube thermography specifically are vital for managing mastitis. Therefore, the present study focuses on thermal imaging of the udder and short milking tube (SMT) of the milking machine during the peak milking process of Sahiwal cows and Murrah buffaloes during winter, summer, rainy and autumn seasons to identify sub-clinical (SCM) and clinical mastitis (CM) cases using the Darvi DTL007 camera. The udder health was assessed using the California Mastitis Test, Somatic Cell Count (SCC) and IRT throughout the year. Log10SCC and thermogram analysis revealed a difference (p < 0.01) between healthy, SCM, and CM cases during different seasons in both breeds. Further results showed an increase (p < 0.01) in SMT thermograms of SCM and CM cases compared to healthy quarters in Sahiwal cows during winter, summer, rainy, and autumn were 4.26 and 7.51, 2.37 and 4.47, 2.20 and 3.64, 2.90 and 4.94 ºC, respectively and for Murrah buffaloes were 3.56 and 5.55, 2.70 and 3.81, 1.72 and 3.10, 3.14 and 4.42ºC, respectively. The highest degree of increase in milking udder skin surface temperature and SMT of SCM and CM cases compared to healthy quarters was observed during the winter and the least during the rainy season. Thus, regardless of the seasons examined in this study, SMT thermograms effectively assessed SCM and CM.

Proliferation and apoptosis dynamics of the normal canine mammary gland during the oestrous cycle evaluated by Ki-67 and Caspase-3 indexes.

Understanding the normal physiology of the canine mammary gland (CMG) is crucial, as it provides a foundational reference for understanding canine mammary neoplasms. The relation between the Proliferation Index (PI) indicated by Ki-67 expression, along with the Apoptotic Index (AI) determined through Caspase-3 expression during the oestrous cycle, is inadequately documented in existing literature. This study seeks to offer insights into the interplay between PI and AI in the CMG across oestrous cycle phases. An extensive investigation was conducted on a diverse case series of bitches (n = 18). Oestrous cycle stages were determined through vaginal cytology, histological examination of the reproductive tract and serum progesterone and oestradiol concentrations. The entire mammary chain was histologically examined, and proliferation and apoptosis were assessed via double immunohistochemistry employing anti-Ki-67 and Caspase-3 antibodies. PI and AI were evaluated through a systematic random sampling approach, counting a minimum of 200 cells for each cell type. There was a significantly higher PI during early dioestrus in all mammary gland components, with a greater proportion of positive cells observed in epithelial cells compared to stromal cells. The highest PI was detected in epithelial cells within the end buds. Significant differences were found in Ki-67 labelling across the cranial mammary glands. A positive and strong correlation was noted between progesterone concentration and PI in epithelial cells. The AI remained consistently low throughout the oestrous cycle, with few differences observed across histological components. Caspase-3 labelling displayed the highest positivity in caudal mammary pairs. A negative and moderate correlation was identified between progesterone concentration and AI in interlobular mesenchymal cells. This study highlights the influence of endocrine regulation on cell proliferation indices in mammary tissue, emphasizing the need to consider these hormonal variations in toxicopathological studies involving canine mammary gland.

Laminin-binding integrins are essential for the maintenance of functional mammary secretory epithelium in lactation.

Integrin dimers α3/ß1, α6/ß1 and α6/ß4 are the mammary epithelial cell receptors for laminins, which are major components of the mammary basement membrane. The roles of specific basement membrane components and their integrin receptors in the regulation of functional gland development have not been analyzed in detail. To investigate the functions of laminin-binding integrins, we obtained mutant mice with mammary luminal cell-specific deficiencies of the α3 and α6 integrin chains generated using the Cre-Lox approach. During pregnancy, mutant mice displayed decreased luminal progenitor activity and retarded lobulo-alveolar development. Mammary glands appeared functional at the onset of lactation in mutant mice; however, myoepithelial cell morphology was markedly altered, suggesting cellular compensation mechanisms involving cytoskeleton reorganization. Notably, lactation was not sustained in mutant females, and the glands underwent precocious involution. Inactivation of the p53 gene rescued the growth defects but did not restore lactogenesis in mutant mice. These results suggest that the p53 pathway is involved in the control of mammary cell proliferation and survival downstream of laminin-binding integrins, and underline an essential role of cell interactions with laminin for lactogenic differentiation.

Integrated morphodynamic signalling of the mammary gland.

The mammary gland undergoes a spectacular series of changes as it develops, and maintains a remarkable capacity to remodel and regenerate for several decades. Mammary morphogenesis has been investigated for over 100 years, motivated by the dairy industry and cancer biologists. Over the past decade, the gland has emerged as a major model system in its own right for understanding the cell biology of tissue morphogenesis. Multiple signalling pathways from several cell types are orchestrated together with mechanical cues and cell rearrangements to establish the pattern of the mammary gland. The integrated mechanical and molecular pathways that control mammary morphogenesis have implications for the developmental regulation of other epithelial organs.

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.

A randomized controlled trial to study the effects of an automated premilking stimulation system on milking performance, teat tissue condition, and udder health in Holstein dairy cows.

The objectives were to examine the effect of an automated premilking stimulation (APS) by means of a high pulsation frequency (300 cycles/min) without a reduction of the vacuum in the pulsation chamber or claw piece on (1) milking performance, (2) teat tissue condition, and (3) udder health in dairy cows. In a randomized controlled field study, Holstein cows (n = 427) from 1 commercial dairy farm with a milking schedule of 3 times per day were assigned to treatment and control groups over a 90-d period. Treatments consisted of a maximum of 80 s (APS80) or 99 s (APS99) of mechanical stimulation at a pulsation rate of 300 pulses per minute and a ratio of 25:75 (no reduction of the pulsation chamber or milking vacuum). Cows in the control group (CON) received traditional premilking stimulation by means of manual forestripping for 8 s. Milking characteristics were documented with on-farm milk meters. Short- and long-term changes in teat tissue condition induced by machine milking were assessed visually on a weekly basis. Composite milk samples were analyzed once per month to determine somatic cell count. Generalized linear mixed models were used to study the effect of the treatment on the outcome variables. We observed no meaningful differences in milk yield or milking unit-on time. Least squares means and their 95% confidence intervals (95% CI) for cows in the APS80, APS99, and CON groups were 13.5 (13.1-14.0), 13.2 (12.8-13.7), and 13.2 (12.8-13.7) kg for milk yield and 222 (213-231), 219 (210-228), and 223 (214-232) s for milking unit-on time, respectively. The effect of treatment on bimodality was modified by milk yield such that the odds of bimodality increased in the treatment groups with increasing milk yield. Compared with cows in the CON group, the odds ratios (95% CI) of bimodality were 1.08 (0.62-1.89) in the APS80 group and 0.89 (0.55-1.42) in the APS99 group at a milk yield of 11 kg and 2.0 (1.24-3.22) in the APS80 group and 2.08 (1.29-3.35) in the APS99 group at a milk yield of 16 kg. We observed differences in short- and long-term changes in teat tissue condition between the treatment and control groups. Compared with cows in the CON group, the odds (95% CI) of short-term changes were 1.87 (1.35-2.58) for the APS80 group and 1.49 (1.08-2.07) for the APS99 group, and the odds of long-term changes were 1.52 (1.24-1.85) for cows in the APS80 group and 1.59 (1.31-1.94) for cows in the APS99 group. The least squares means (95% CI) for somatic cell counts (log10-transformed) were 4.74 (4.68-4.81) for the APS80 group, 4.77 (4.71-4.83) for the APS99 group, and 4.79 (4.73-4.86) for the CON group. We conclude that the APS system tested here had no negative effects on milk yield or milking unit-on time. However, differences in bimodality and teat tissue condition suggest that the APS system did not provide sufficient stimulation to facilitate a gentle milk harvest and adversely affects teat tissue condition.

Effect of mechanical premilking stimulation on milking duration in late lactation.

This study documents the effect of mechanical prestimulation on the milking duration of pasture-based cows in late lactation to better harness increased capacity of automation in the milk harvesting process. Premilking stimulation, provided via manual or mechanical means, has been shown to promote the milk letdown reflex and assist in achieving quick, comfortable, and complete milk removal from the udder. The literature is lacking knowledge on the effect of mechanical premilking stimulation on milking duration, especially in late lactation and in pasture-based systems, and many pasture-based farms do not practice a full premilking routine because of a lack of labor availability. The current study addresses this gap in knowledge. In this study, we tested 2 treatments: (1) the No Stim treatment used normal farm milking settings with no premilking preparation and (2) the Stim treatment used 60 s of mechanical premilking stimulation, with a rate of 120 cycles per minute and a pulsator ratio of 30:70 on cluster attachment. Once the 60 s of stimulation had elapsed, normal milking settings resumed for the remainder of the milking. Sixty cows were enrolled in the study, which ran for 20 d. The effect of treatment on a.m. milking duration was significant, a.m. milking duration for Stim was 12 s shorter than that of No Stim. The effect of treatment on p.m. milk duration was not significant. Treatment had no effect on a.m./p.m. milk yields, average milk flowrates or peak milk flowrates. Significant differences emerged between treatments on a.m. and p.m. dead time (time from cluster attachment to reach a milk flowrate of 0.2 kg/min). The a.m. and p.m. dead times were 6 s shorter for Stim compared with No Stim. The time taken to achieve peak milk flowrate (time to peak) at morning milking was 7 s shorter for Stim compared with No Stim, and treatment yielded no significant effects on time to peak at p.m. milkings. Treatment also had no significant effect on log10 somatic cell count. Although the percentage of congested teat-ends and teat-barrels was numerically lower for Stim versus No Stim, no statistical differences were detected across these measures. Based on the results of the study, we found merit in applying 60 s of mechanical pre-stimulation at a.m. milking from a milking duration perspective. However, the strategy was not as successful for the p.m. milking. Analysis of the milk flowrate profiles recorded during the study suggest potential utility in employing different machine settings for various milkings based on anticipated yield and level of udder fill.

The interplay of continuous milk ejection and milking system with and without prestimulation at different vacuum settings.

Efficient machine milking requires an optimal interaction of alveolar milk ejection in the udder and milk removal by the milking machine. The aim of the present study was to test whether the equilibrium between continuous milk ejection and milk removal can also be maintained at very fast milking through a particularly high vacuum. Eight Holstein dairy cows were milked at 42, 52, or 60 kPa, with (PS) or without (nPS) prestimulation. Each of the 6 treatments was conducted at 2 afternoon milkings in each animal. The prestimulation lasted 40 s and consisted of forestripping and teat cleaning. The cluster attachment followed after a 20-s latency period. Throughout each milking, B-mode ultrasound videos of the gland cistern of 1 front quarter as well as milk flow and claw vacuum curves were recorded. Total milk yield was neither affected by nPS or PS nor by the vacuum level. Milk removed within the first minute and the first 2 min of milking and average milk flow were higher, and the duration of incline and time until peak milk flow were shorter at PS than at nPS milkings at all vacuum levels. Machine-on time was shorter at PS than at nPS milkings, although only at 42 and 52 kPa vacuum, obviously caused by the high percentage of bimodalities occurring in nPS milkings (17% bimodalities in PS vs. 92% bimodalities in nPS milkings). The frequency of bimodalities was higher at high than at low vacuum both in PS and nPS milkings. Peak flow rate and average milk flow were both higher at higher vacuum levels. The duration of milk flow plateau was shorter at 60 kPa than at 42 kPa milkings. At the highest vacuum (60 kPa), the shorter plateau phase indicated a declining milk ejection rate toward the end of the plateau phase, and milk ejection could no longer keep up with the fast milk removal; hence, a higher milking efficiency at a higher vacuum level could only be achieved as long as the gland cistern remained sufficiently filled by the continuous milk ejection. The ultrasound imaging confirmed this finding as the duration of cisternal area plateau in the recorded front quarter was shorter at high than at low vacuum. Thus, the highest vacuum of 60 kPa did not cause a shorter machine-on time than 52 kPa. In conclusion, milking at a very high vacuum can increase milking efficiency compared with a low vacuum. However, a vacuum reduction at the start and toward the end of milking is required to prevent overmilking if milking is performed at a very high vacuum.

Graduate Student Literature Review: The challenge of drying-off high-yielding dairy cows.

The cessation of lactation (i.e., dry-off) in dairy cattle is an area of research that has received much focus in recent years. The dry period is necessary to optimize tissue remodeling of the mammary gland, but represents a stressful event, incorporating several changes in daily routine, diet, and metabolism. Moreover, the high milk yields achieved by modern cows in late gestation exacerbate the need for relevant manipulations in the days around dry-off, as excessive accumulation of milk might jeopardize the success of the dry period, with potential negative effects on future lactation. Production levels over 15 kg/d are an additional risk factor for udder health, delay mammary involution, and worsen metabolic stress and inflammatory responses. Furthermore, the pressure to reduce antibiotic usage in farm animals has resulted in increased attention on the dry period, given that historically most dairy cattle were provided prophylactic intramammary antibiotic treatment at dry-off as a means to reduce the risk of intramammary infections in the subsequent lactation. Several strategies have been proposed over the years to cope with these challenges, aiming to gradually reduce milk yield before dry-off, promoting at the same time the start of mammary involution. Among them, the most common are based on feed or nutrient restriction, a decrease in milking frequency, or administration of prolactin inhibitors. These practices have different capacities to reduce milk yield through different mechanisms and entail several implications for udder health, animal welfare, behavior, endocrine status, metabolism, and inflammatory conditions. The present review aims to provide a comprehensive overview of the dry-off phase in high-yielding cows and of the impact of high milk production at dry-off, and to describe possible strategies that might be implemented by farmers and veterinarians to optimize this critical phase in an integrated way.

Taurine alleviates heat stress-induced mammary inflammation and impairment of mammary epithelial integrity via the ERK1/2-MLCK signaling pathway.

Heat stress leads to milk production losses and mammary gland inflammation, which may be associated with mammary epithelium damage. Taurine is one of the most abundant free amino acids in mammals which has anti-inflammatory properties. This study aimed to explore the effect of taurine pretreatment on heat stress-induced mammary epithelial integrity disruption and inflammatory damage. In our first experiment on dairy cows our results showed that compared with animals under autumn thermoneutral condition (THI = 62.99 ± 0.71), summer heat stress (THI = 78.01 ± 0.39) significantly reduced milk yield and disrupted mammary epithelial integrity as revealed by increased concentrations of serotonin and lactose in plasma, and increased levels of SA and Na+/K+ in milk. In our second study, 36 lactating mice were randomly divided into three groups (n = 12) for a 9d experiment using a climate chamber to establish a heat stress model. Our findings suggest taurine pretreatment could attenuate heat stress-induced mammary histopathological impairment, inflammation response, and enhance mammary epithelium integrity, which was mainly achieved by promoting the secretion of ZO-1, Occludin, and Claudin-3 through inhibiting activation of the ERK1/2-MLCK signaling pathway in the mammary gland. Overall, our findings indicated that heat stress induced mammary epithelium dysfunction in dairy cows, and emphasized the protective effect of taurine on mammary health under heat stress conditions using a mouse model, which may be achieved by alleviating the mammary epithelium integrity damage and inflammation response.

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.

Symposium review: Environmental effects on mammary immunity and health.

Environmental effects on pathogen abundance and access are precursors to mastitis. Indeed, high heat and humidity, and unsanitary housing and equipment, are associated with greater pathogen load and exposure. Although less is known about effects of environment on a cow's ability to resist infection, several indicators suggest that it can affect pathogen responses. Mastitis incidence and bulk tank somatic cell count vary with season, typically peaking in summer. Recent controlled studies have revealed that heat stress exposure results in changes in the microbiome of the cow and her environment, which may relate to negative effects on milk quality and cow health. Alternatively, specific pathogen loads may vary based on housing dynamics rather than associations with physical environment. Indeed, housing-related stressors, such as overcrowding and social group challenge, influence secretion of glucocorticoids, thus affecting pathogen resistance in the cow. Two key seasonal variables are photoperiod and temperature, specifically the heat stress consequent to elevated temperature and humidity. Shifts in light duration regulate immune function in other species, but apparently have limited effect on udder health of lactating cows. In contrast, in dry cows, short days increase peripheral blood mononuclear cell number and are associated with lower somatic cell count in the next lactation, compared with long days. With heat stress, elevated body temperature directly affects expression of immune-related genes in mammary tissue. Responses depend on duration of exposure and feature acute upregulation of immune-signaling pathways, followed by enrichment of other immune-related pathways after prolonged exposure. Most responses are transient and recover within 1 wk. Functionally, heat stress impairs some aspects of acquired immunity in dry cows, including antigen responses and lymphocyte proliferation, but apparently not innate immune function. However, heat stress in late gestation reduces neutrophil phagocytosis and killing in vitro, and neutrophils in circulation are reduced in vivo as are responses to pathogen challenge in the subsequent lactation. A holistic understanding of the complex interplay of environment, pathogens, and host is needed to inform advances in this area.

Risk factors for delayed milk ejection in Holstein dairy cows milked 3 times per day.

Delayed milk ejection, manifested most often as bimodal milk flow, occurs when the cisternal milk fraction is removed before the alveolar milk reaches the gland cistern. It is thought to be a consequence of not meeting cows' physiological needs, due to insufficient premilking teat stimulation, inadequate timing of milking unit attachment, or both. It has been associated with decreased milking efficiency, reduced milk yield, and impaired teat and udder health. Traditionally, portable electronic milk meters have been used to assess the presence of delayed milk ejection in dairy cows. By contrast, incremental milk flow rates from on-farm milk meters and their suitability as a measure to assess delayed milk ejection have not been studied by rigorous methods. The objectives were (1) to describe a protocol for identification of cows with chronically delayed milk ejection (CDME) and (2) to investigate risk factors for CDME using incremental milk flow rates obtained from automated on-farm milk meters. In a retrospective case control study, milk flow data from a 4,300-cow dairy with a thrice-daily milking schedule were obtained over a 1-wk period. Incremental milk flow rates (0-15 s, 15-30 s, 30-60 s, and 60-120 s) were used to identify cows with delayed milk ejection. Cases of CDME were defined as presence of delayed milk ejection at all 21 milking observations. Cows that had no delayed milk ejection at any of the same 21 milking observations were included as controls. A total of 171 cases and 393 controls were included in the study based on these criteria. A logistic regression model was used to evaluate associations of the following risk factors with CDME: parity (1, 2, ≥3), stage of lactation (<100, 101-200, >200 DIM), presence of a nonlactating quarter, milk somatic cell count, average daily milk production, and health and management events. Parity and CDME were associated such that compared with cows in their third or greater lactation, the odds (95% confidence intervals, 95% CI) of CDME were 1.27 (0.71-2.25) for cows in their first and 4.77 (2.47-9.22) for animals in their second lactation. The odds of CDME increased with increasing stage of lactation, with an odds ratio of 0.20 (0.11-0.36) for early and 0.28 (0.15-0.52) for mid-lactation animals, respectively, compared with late lactation cows. A 1-kg increase in average daily milk production was associated with decreased odds of CDME [odds ratio (95% CI): 0.89 (0.87-0.92)]. A lameness event during the study period increased the odds of CDME [odds ratio (95% CI): 8.04 (1.20-53.83)], as did a vaccination event 1 wk before the study period [odds ratio (95% CI): 4.07 (0.99-16.71)]. This study confirmed associations between CDME and previously reported risk factors and identified several previously less rigorously investigated health and management events that could be associated with CDME. Incremental milk flow rates from individual cows serve as an automated tool to evaluate milk flow dynamics. This information could be used to improve individual premilking udder preparation to meet the animal's physiological requirements, improve teat and udder health, and enhance parlor efficiency.

Small RNA deep sequencing reveals the expressions of microRNAs in ovine mammary gland development at peak-lactation and during the non-lactating period.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in mammary gland development and lactation in livestock. Little is known about the roles of miRNAs in ovine mammary gland development, hence in this study the expression profiles of miRNAs of the mammary gland tissues of ewes at peak-lactation and during the non-lactating period were investigated using RNA sequencing. A total of 147 mature miRNAs were expressed in the two periods. Compared with peak-lactation, eight miRNAs in the non-lactating ewe mammary gland were significantly up-regulated, whereas fifteen miRNAs were down-regulated. A KEGG analysis revealed that the target genes of the up-regulated miRNAs were significantly enriched in lysosome, Wnt and MAPK signaling pathways, while the target genes of down-regulated miRNAs were significantly enriched in the PI3K-Akt signaling pathway, protein processing in endoplasmic reticulum and axon guidance. These results suggest that further study of the differentially expressed miRNAs could provide a better understanding of the molecular mechanisms of mammary development and lactation in sheep.

An Intravital Microscopy Toolbox to Study Mammary Gland Dynamics from Cellular Level to Organ Scale.

The architecture of the mouse mammary gland is highly dynamic and constantly remodeled during pubertal development and estrous cycle-driven sprouting and regression of alveolar side branches. During each of these developmental stages, turnover is driven by distinct subsets of mammary epithelial cells. Extensive previous research has shed light on the unique morphological and cell biological characteristics of each stage. However, technological shortcomings failed to capture the dynamics and single-cell contributions to mammary remodeling. Here, we developed in vivo imaging strategies to follow the same mammary ducts over time and quantify the dynamics of mammary gland growth and remodeling from single-cell level to organ scale. Using a combination of intravital microscopy and genetic reporter systems we show how proliferative heterogeneity drives ductal morphogenesis during different developmental stages. To visualize pubertal growth at the cellular level, we performed long-term time-lapse imaging of extending terminal end buds through a mammary imaging window. We show that single-cells within the terminal end buds are extremely motile and continuously exchange position whilst the duct is elongating. To visualize short-term remodeling in the adult mammary gland at the single cell level, we performed multi-day intravital imaging in photoconvertible Kikume Green-Red mice and fluorescent ubiquitination-based cell cycle indicator mice. We demonstrate that the contribution of single-cells to estrous-driven remodeling is highly variable between cells in the same micro-environment. To assess the effects of this dynamic proliferative contribution on the long-term stability of tissue architecture, we developed a repeated skin flap method to assess mammary gland morphology by intravital microscopy over extended time spans for up to six months. Interestingly, in contrast to the short-term dynamic remodeling, the long-term morphology of the mammary gland remains remarkably stable. Together, our tool box of imaging strategies allows to identify and map transient and continuing dynamics of single cells to the architecture of the mammary gland.