Cellular proliferation and apoptosis in Staphylococcus aureus-infected heifer mammary glands experiencing rapid mammary gland growth.

Intramammary infections in nonlactating mammary glands are common and can occur during periods of rapid mammary epithelial cell (MEC) accumulation, which may ultimately reduce total MEC numbers. Reduced MEC numbers, resulting from impaired MEC proliferation and increased cellular apoptosis, are expected to reduce future milk yields. The objective of this study was to measure the degree of cellular proliferation and apoptosis in the epithelial and stromal compartment of uninfected and Staphylococcus aureus-infected mammary glands hormonally induced to grow rapidly. Nonpregnant heifers (n = 8) between 11 and 14 mo of age were administered supraphysiological injections of estradiol and progesterone for 14 d. One mammary gland of each heifer was randomly selected and infused with Staph. aureus (CHALL) while another mammary gland was designated as an uninfected control on d 8 of injections. Mammary tissues were collected on the last day of hormonal injections from center and edge parenchymal regions and subject to proliferation assessment via Ki-67 staining and apoptotic assessment via terminal deoxynucleotidyl transferase dUTP nick-end labeling. Differences in cellular proliferation between CHALL and uninfected control quarters were not apparent, but proliferation of MEC was marginally greater in edge parenchyma than in center parenchyma. Coincidently, CHALL quarters experienced a greater percentage of apoptotic MEC and lower percentage of stromal cells undergoing apoptosis than uninfected control quarters. This study also provides the first insight into the mechanisms that allow the mammary fat pad to be replaced by expanding mammary epithelium as edge parenchyma contained a greater percentage of apoptotic stromal cells than center parenchyma. When taken together, these data suggest that Staph. aureus intramammary infection impairs mammary epithelial growth through reductions in MEC number and by preventing its expansion into the mammary fat pad. These factors during periods of rapid mammary growth are expected to impair first lactation milk yield.

Histological tissue structure alterations resulting from Staphylococcus aureus intramammary infection in heifer mammary glands hormonally induced to rapidly grow and develop.

Intramammary infections (IMI) are common in nonlactating dairy cattle and are expected to impair mammary growth and development and reduce future milk production. The objective of this study was to histologically evaluate how IMI alter tissue structure in growing and developing heifer mammary glands. A total of 18 nonpregnant, nonlactating heifers between 11 and 14 mo of age were used in the present study. Heifers received daily supraphysiological injections of estradiol and progesterone for 14 d to stimulate rapid mammary growth and development. One-quarter of each heifer was subsequently infused with Staphylococcus aureus (CHALL) while a second quarter served as an uninfected control (UNINF). Heifers were randomly selected and euthanized either the last day of hormonal injections to observe IMI effects on mammary gland growth (GRO), or 13 d post-injections, to observe IMI effects on mammary development (DEV). Mammary tissues were collected from the center and edge parenchymal regions of each mammary gland for morphometric tissue area evaluation. For GRO tissues, CHALL quarters had less epithelial tissue area and marginally more intralobular stroma tissue area than UNINF quarters. Tissue areas occupied by luminal space, extralobular stroma, adipose, and lobular tissue were similar. For DEV tissues, area occupied by epithelium, luminal space, intralobular stroma, and extralobular stroma did not differ between quarter treatments, but UNINF quarters had more adipose tissue area and marginally less lobular area than CHALL quarters. Results indicate that IMI in growing and developing mammary glands reduces mammary epithelial growth and alters mammary gland development by impairing epithelial branching into the mammary fat pad. Taken together, these tissue changes before calving may have adverse effects on milk production. Therefore, an important focus should be placed on improving udder health in replacement heifers through management strategies that mitigate the deleterious effects of IMI and promote the positive development of the mammary gland.

Use of commercial somatic cell counters to quantify somatic cells in non-lactating bovine mammary gland secretions.

Measurement of the somatic cell count (SCC) in milk is commonly used to detect mastitis in lactating dairy cows. Many techniques and tools have been developed and adapted to quantify milk SCC, but few tools have been evaluated in their ability to enumerate somatic cells in non-lactating bovine mammary secretions. This limits the tools available for detecting mastitis in non-lactating animals. The objective of these studies was to evaluate methods of somatic cell quantification, originally developed for milk, in their ability to quantify the SCC in non-lactating bovine mammary secretions when compared to the gold standard microscopic quantification method. Two experiments were conducted. In a first experiment, 222 mammary secretions were collected and diluted 1:10 with PBS. Cells in these suspensions were quantified microscopically and with a DeLaval Cell Counter. Microscopic SCC (MSCC) ranged from 1.9 × 106 to 259.5 × 106 cells/mL while DeLaval Cell Counter SCC (DSCC) ranged from 1.8 × 106 to 27.0 × 106 cells/mL; a measurement of agreement between the 2 measures, based on the Lin's Concordance Correlation Coefficient (CCC) suggested moderate agreement between measures (CCC = 0.60). In a second experiment 72 mammary secretions were collected and diluted 1:50 in PBS. Somatic cells in these suspensions were quantified microscopically, with a DeLaval Cell Counter, and by a DHIA laboratory using a Fossomatic™ FC. MSCC ranged from 1.6 to 47.5 × 106 cells/mL, DSCC ranged from 1.0 to 35.7 × 106 cells/mL, and Fossomatic SCC (FMSCC) ranged from 1.6 to 46.7 × 106 cells/mL. CCCs of 0.81 and 0.88 resulted when DSCC and FMSCC were paired with the MSCC, respectively. The results of this work indicate that a significantly greater concentration of somatic cells exist in non-lactating mammary secretions and dilution of these mammary secretions influences accuracy of SCC estimates. Future studies seeking to quantify somatic cells in mammary secretions from non-lactating cows should identify the most appropriate dilution factors specific to each method of measure, given that these two factors will influence the accuracy of SCC estimates. Development of a standardized approach for quantifying somatic cells in non-lactating dairy animals such as heifers and cows, via a rapid automated counter, can allow for the detection of mastitis in non-lactating dairy animals.

Effects of Staphylococcus aureus intramammary infection on the expression of estrogen receptor α and progesterone receptor in mammary glands of nonlactating cows administered estradiol and progesterone to stimulate mammary growth.

Intramammary infections (IMI) are prevalent in nonlactating dairy cattle and are known to alter mammary structure and negatively affect the amount of mammary epithelium in the gland. Mechanisms responsible for the observed changes in mammary growth during an IMI are poorly understood, yet the importance of the key mammogenic hormones driving mammary growth is well recognized. This study's objective was to characterize the expression of estrogen receptor α (ESR1) and progesterone receptor (PGR) in mammary glands stimulated to grow and develop in the presence or absence of an IMI as well as preliminarily characterize myoepithelial cell response to IMI. Mammary growth was stimulated in 18 nonpregnant, nonlactating dairy cows using subcutaneous estradiol and progesterone injections, and 2 culture-negative quarters of each cow were subsequently infused with either saline (n = 18) or Staphylococcus aureus (n = 18). Mammary parenchyma tissues were collected 5 d (n = 9) or 10 d (n = 9) postchallenge and examined using immunofluorescence microscopy to quantify positive nuclei and characterize staining features. There tended to be a greater number of ESR1-positive nuclei observed across 8 random mammary parenchyma fields of view in saline quarters than in Staph. aureus quarters (201 vs. 163 ± 44 nuclei). Saline quarters also contained a greater number of PGR-positive nuclei (520 vs. 440 ± 45 nuclei) and myoepithelial cells (971 vs. 863 ± 48 nuclei) than Staph. aureus-challenged quarters. However, when ESR1, PGR, and myoepithelial nuclei counts were adjusted for Staph. aureus quarters containing less epithelium, differences between quarter treatments abated. The examined ESR1 and PGR staining characteristics were similar between saline and Staph. aureus quarters but were differentially affected by day of tissue collection. Additionally, nuclear staining area of myoepithelial cells was greater in Staph. aureus quarters than in saline quarters. These results indicate that IMI had little effect on the number or staining characteristics of ESR1- or PGR-positive nuclei relative to epithelial area, but myoepithelial cells appear to be affected by IMI and the associated inflammation in nonlactating mammary glands that were stimulated to grow rapidly using mammogenic hormones. Accordingly, reductions in mammary epithelium in affected glands are not suspected to be resultant of alterations in the number or staining characteristics of ESR1- or PGR-positive mammary epithelial cells.

Apoptosis and proliferation in Staphylococcus aureus-challenged, nonlactating mammary glands stimulated to grow rapidly and develop with estradiol and progesterone.

Bovine mastitis is a common and costly disease in the dairy industry and is known to negatively affect the amount of epithelium in nonlactating mammary glands. Despite this recognition, an understanding of the mechanisms contributing to reductions in epithelium is lacking. The objective of this study was to evaluate cellular apoptosis and proliferation in uninfected and Staphylococcus aureus-infected mammary glands that were stimulated to rapidly grow and develop. Estradiol and progesterone injections were administered to 18 nonlactating dairy cows to induce mammary growth, and 2 quarters from each animal were infused with saline or Staph. aureus. Mammary tissues were collected at 5 (n = 9) and 10 d (n = 9) postinfusion and examined using quantitative bright field and florescent immunohistochemistry. Staphylococcus aureus mammary glands tended to have a greater number of mammary epithelial cells undergoing apoptosis than saline quarters. In the stromal compartment, challenged quarters contained a lower proportion of cells undergoing apoptosis than saline quarters overall; however, cell types undergoing apoptosis were differentially affected. Staphylococcus aureus quarters contained a lesser percentage of apoptotic fibroblasts while also containing more nonapoptotic immune cells than saline quarters in the intralobular stroma compartment. A similar number of proliferating epithelial cells were present in Staph. aureus and saline mammary tissues, but more proliferating cells were present in the intralobular stroma compartment of Staph. aureus-infused quarters than those infused with saline. When these cellular responses are considered together, it indicates that changes in cellular apoptosis and proliferation contribute to changes in the gland structure by potentiating the expansion of the intralobular stromal compartment, via cellular accumulation, and limiting the amount of epithelium due to increases in cellular apoptosis in affected glands. Reductions in mammary epithelium are expected to reduce future milk yields and productive herd life.

Varying dietary protein and fat elicits differential transcriptomic expression within stress response pathways in preweaned Holstein heifers.

Increases in milk replacer dietary energy subsequently increase growth and weight in preweaned dairy heifers. However, the underlying effects of dietary component increases on key functional pathways have yet to be fully investigated. Elucidating these relationships may provide insights into the mechanisms through which protein and fat are partitioned for tissue growth and metabolism. We hypothesized that genes within key growth and metabolic pathways would be differentially expressed between calves fed a protein- and fat-restricted diet and calves fed a protein- and fat-enhanced diet. The objectives of this study were to (1) identify genes differentially expressed between dietary restricted calves and enhanced calves and (2) determine the key regulatory pathways influenced by these genes. Preweaned Holstein heifers (n = 12; 6 ± 0.02 d of age) were randomly assigned to 1 of 2 milk replacer diets: enhanced (28.9% crude protein, 26.2% fat; n = 6) or restricted (20.9% crude protein, 19.8% fat; n = 6). Growth measures included average daily gain and gain-to-feed ratio. After 56 d, calves were killed for tissue collection. Samples from longissimus dorsi, adipose, and liver tissues were collected and RNA was isolated for RNA sequencing analysis. The MIXED procedure of SAS (SAS Institute Inc., Cary, NC) was used to evaluate relationships of growth with dietary energy. Fixed effects included date of collection and time (day). Random effects included sire and birth weight. The RNA sequencing analysis was performed using CLC Genomics Workbench (Qiagen, Germantown, MD), and the Robinson and Smith exact test was used to identify differentially expressed genes between diets. The Protein Analysis Through Evolutionary Relationships (PANTHER) database was then used to identify functional categories of differentially expressed genes. Enhanced calves had increased growth rates and feed efficiency compared with restricted calves (average daily gain = 0.76 and 0.22, respectively; gain-to-feed ratio = 0.10 and 0.06, respectively). There were 238 differentially expressed genes in adipose, 227 in longissimus dorsi, and 40 in liver. We identified 10 genes concordant among tissues. As expected, functional analyses suggested that the majority of genes were associated with metabolic or cellular processes, predominantly cell communication and cell cycle. Overall, it appears that varying levels of dietary protein and fat influence calf growth and development through metabolic processes, including oxidative phosphorylation and glyceroneogenesis. However, protein- and fat-restricted calves appeared to experience metabolic stress at a cellular level, as evidenced by an upregulation in stress response pathways, including genes in the p53 pathway. Calves could be fed at a higher level of protein and fat to decrease the prevalence of metabolic stress at the cellular level, but evidence indicating the presence of inflammatory stress and adipose fibrosis in enhanced calves prompts further investigation of the effects of milk replacer component levels.

Higher plane of nutrition pre-weaning enhances Holstein calf mammary gland development through alterations in the parenchyma and fat pad transcriptome.

BACKGROUND: To reduce costs of rearing replacement heifers, researchers have focused on decreasing age at breeding and first calving. To increase returns upon initiation of lactation the focus has been on increasing mammary development prior to onset of first lactation. Enhanced plane of nutrition pre-weaning may benefit the entire replacement heifer operation by promoting mammary gland development and greater future production. METHODS: Twelve Holstein heifer calves (< 1 week old) were reared on 1 of 2 dietary treatments (n = 6/group) for 8 weeks: a control group fed a restricted milk replacer at 0.45 kg/d (R, 20% crude protein, 20% fat), or an accelerated group fed an enhanced milk replacer at 1.13 kg/d (EH, 28% crude protein, 25% fat). At weaning (8 weeks), calves were euthanized and sub-samples of mammary parenchyma (PAR) and mammary fat pad (MFP) were harvested upon removal from the body. Total RNA from both tissues was extracted and sequenced using the Illumina HiSeq2500 platform. The Dynamic Impact Approach (DIA) and Ingenuity Pathway Analysis (IPA) were used for pathway analysis and functions, gene networks, and cross-talk analyses of the two tissues. RESULTS: When comparing EH vs R 1561 genes (895 upregulated, 666 downregulated) and 970 genes (506 upregulated, 464 downregulated) were differentially expressed in PAR and MFP, respectively. DIA and IPA results highlight a greater proliferation and differentiation activity in both PAR and MFP, supported by an increased metabolic activity. When calves were fed EH, the PAR displayed transcriptional signs of greater overall organ development, with higher ductal growth and branching, together with a supportive blood vessel and nerve network. These activities were mediated by intracellular cascades, such as AKT, SHH, MAPK, and Wnt, probably activated by hormones, growth factors, and endogenous molecules. The analysis also revealed strong communication between MFP and PAR. CONCLUSION: The transcriptomics and bioinformatics approach highlighted key mechanisms that mediate the mammary gland response to a higher plane of nutrition in the pre-weaning period.

A rotary and reciprocating scintillator based fast-ion loss detector for the MAST-U tokamak.

The design and unique feature of the first fast-ion loss detector (FILD) for the Mega Amp Spherical Tokamak - Upgrade (MAST-U) is presented here. The MAST-U FILD head is mounted on an axially and angularly actuated mechanism that makes it possible to independently adapt the orientation [0°, 90°] and radial position [1.40 m, 1.60 m] of the FILD head, i.e., its collimator, thus maximizing the detector velocity-space coverage in a broad range of plasma scenarios with different q95. The 3D geometry of the detector has been optimized to detect fast-ion losses from the neutral beam injectors. Orbit simulations are used to calculate the strike map and predict the expected signals. The results show a velocity-space range of [4 cm, 13 cm] in gyroradius and [30°, 85°] in pitch angle, covering the entire neutral beam ion energy range. The optical system will provide direct sight of the scintillator and simultaneous detection with two cameras, giving high spatial and temporal resolution. The MAST-U FILD will shed light on the dominant fast-ion transport mechanisms in one of the world's two largest spherical tokamaks through absolute measurements of fast-ion losses.

Excess deposition of collagen in mammary glands of tamoxifen-treated Holstein heifers is associated with impaired mammary growth.

It is established that the ovary and estrogen are essential to bovine mammary development with the onset of puberty. Recent studies have shown that ovariectomy in the very early prepubertal period, well before onset of puberty, also dramatically impairs mammary growth. Similarly, prepubertal heifers treated with the antiestrogen tamoxifen (TAM) also exhibit markedly impaired mammary growth in correspondence with reduced estrogen receptor α (ESR1) expression. Our objective was to evaluate the effect of TAM on the mammary stroma and specifically to determine if the reported decrease in mammary development was related to changes in TAM-induced alterations in the stroma surrounding the mammary parenchyma. Briefly, 16 Holstein heifers calves were randomly assigned to one of 2 treatment groups: TAM-injected or control. Calves were administered TAM (0.3 mg kg1 d1) or placebo from 28 to 120 d of age. At day 120, calves were euthanized and udders removed. Mammary tissue from near the boundary between the parenchyma and surrounding mammary fat pad was collected for histology and morphometric analysis, expression of selected extracellular matrix-related genes, and quantitation of stromal collagen deposition by study of Sirius Red-stained tissue sections imaged with polarized light. Compared with tissue from control heifers, TAM heifers frequently exhibited areas with abundant fibroblasts and mesenchymal cells especially within the intralobular stroma, as well as less complex ductal structures. Among the array of extracellular matrix-related genes tested, only a small difference (P < 0.05) in expression of laminin was found between treatments. The relative tissue area occupied by stromal tissue was not impacted by treatment. However, the deposition of collagen within the stromal tissue was more than doubled (P < 0.0001) in TAM-treated heifers. These data suggest that blocking ESR1 expression with TAM allows for excessive collagen deposition in the stroma surrounding the developing epithelial structures and that this interferes with both the degree of overall mammary parenchymal development, as well as the pattern of normal ductal morphogenesis.

Technical note: p40 antibody as a replacement for p63 antibody in bovine mammary immunohistochemistry.

Tumor protein 63 (p63) is a nuclear antigen found in basal epithelial cells. To date, 10 isoforms of p63 have been identified, falling into 2 major groups identified by presence or absence of an N-terminal transactivation domain (TAp63 and ΔNp63, respectively). Literature suggests that ΔNp63 is the predominant form expressed in basal epithelial cells and myoepithelial cells (MYEC). The mouse anti-p63 antibody, clone 4B1E12, has been used as a specific nuclear marker for bovine MYEC. Unfortunately, this antibody is no longer commercially available. A new mouse monoclonal antibody, clone BC28, specific to ΔNp63 (designated p40) has been developed. We hypothesized that the p40 antibody would be an appropriate substitution as a MYEC and epithelial basal cell marker. An array of archived formalin-fixed, paraffin-embedded bovine tissues were subjected to immunohistochemical staining for either p40 or p63, with a subset being dual stained for direct comparison. Positive staining for p40 and p63 was observed in serial sections of mammary, skin, rumen, salivary gland, ureter, and bladder. As predicted, negative staining for p40 and p63 was observed in testis and intestine. Dual staining for p40 and p63 in calf mammary (n = 4), lactating mammary (n = 4), rumen (n = 4), and skin (n = 4) showed nearly 100% agreement. Thus, we established that the mouse monoclonal antibody, clone BC28, is a suitable replacement for anti-p63, clone 4B1E12, as a marker of MYEC and basal epithelial cells in bovine tissues.

Bovine Mammary Gland Biopsy Techniques.

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

A 100-Year Review: Mammary development and lactation.

What is old is new again-and with respect to the study of the mammary development and function in dairy animals, the expression resonates. Many of the mammary and milk production questions raised in the early years of the Journal of Dairy Science apply today. To be sure, scientists have filled in many details regarding, for example, identification of hormones and growth factors important in the control of mammary growth, the onset of copious milk production at calving, and maintenance of lactation. Early years focused on identification and subsequent availability of classic mammogenic, lactogenic, and galactopoietic hormones (e.g., steroids, prolactin, and growth hormone). The advent of sensitive assays to measure concentrations of these hormones and, subsequently, myriad growth factors in blood, milk, and tissues, allowed creation of multiple hypotheses to explain mammary cell proliferation and regulation of function. It is also apparent that we understand many of the fundamentals of milk removal, milking frequency, milking management, and milk ejection for successful lactation. However, some questions remain. Are the principles that were identified when cows produced markedly less milk still valid for the high-producing cows of today and the future? What mechanism(s) explain the positive effects of early increased milking frequency on subsequent milk production? Can the persistency of lactation be improved (secretory cell number vs. secretory cell function) or does early management "program" future mammary development or productivity (epigenetics, immune responsiveness, other)? The explosion of tools and techniques (Southern and Northern blots, PCR, and the "-omics" revolution) has driven an almost overwhelming evaluation of cellular and molecular functions in the mammary gland and other tissues. One key may be the discovery of a "Rosetta stone" that will allow understanding of this mass of detailed information on gene expression, cell signaling, and so on. Many scientists can now better appreciate the difficulty of the dairy farmer seeking to process DHIA or Dairy Comp 305 data, milking data, weights, feeding reports, pedometer readings, or genomic evaluations to manage their operations.

Feeding an enhanced diet to Holstein heifers during the preweaning period alters steroid receptor expression and increases cellular proliferation.

Preweaning diet and estradiol treatment alters mammary development. Our objectives were to study the effects of diet and estradiol on proliferation of mammary epithelial cells and expression of estrogen receptor α (ESR1) and progesterone receptors (PGR) in these cells. Thirty-six Holstein heifer calves were raised on (1) a control milk replacer fed at 0.44 kg of powder/head per day, dry matter (DM) basis (restricted, R; 20.9% crude protein, 19.8% fat, DM basis), or (2) an enhanced milk replacer fed at 1.08 kg of powder/head per day, DM basis (Enhanced, EH; 28.9% crude protein, 26.2% fat, DM basis). Milk replacer was fed for 8 wk. At weaning, a subset (n = 6/diet) of calves were euthanized and had tissue harvested. Remaining calves received estradiol implants (E2) or placebo and were euthanized at wk 10 to harvest tissue. Treatments were (1) R, (2) R + E2 (R-E2), (3) EH, and (4) EH + E2 (EH-E2). One day before euthanasia calves were given bromo-2'-deoxyuridine (BrdU; 5 mg/kg of body weight). At euthanization, mammary parenchyma was removed and fixed. Tissue sections from zone 1 (cisternal), 2 (medial), and 3 (distal) within the mammary gland were stained with hematoxylin and eosin and antibodies to measure expression of ESR1, PGR, and incorporation of BrdU. At wk 8, R-fed calves had more PGR-expressing cells in distal parenchyma; however, PGR expression intensity was greater in EH-fed calves. The proportion of cells expressing ESR1 was not affected by diet, but expression intensity (receptors per positive cell) was greater in EH-fed calves across all zones (62-81%). Overall, the percent BrdU-positive epithelial cells was 2 and 0.5 fold greater for EH-fed calves in zone 2 and 3. The proportion of labeled cells was greater in terminal ductal units than in subtending ducts, and treatment effects were more evident in terminal ductal units. At wk 10, calves treated with estradiol had 3.9-fold greater PGR expression intensity. The intensity and percent of cells expressing ESR1 was lowest in estradiol-treated calves. Overall, estradiol-treated calves had the greatest number of proliferating epithelial cells. Moreover, in zone 3, EH-E2 calves had a higher percentage of proliferating cells than in all other treatments. Results indicate both diet and estradiol administration alter proliferation rates of the mammary epithelium and that changes in expression of ESR1 and PGR are involved in enhanced mammary development. The data support our hypothesis that enhanced preweaning feeding increases the mammary tissue responsiveness to mammogenic stimulation.

The influence of postnatal nutrition on reproductive tract and endometrial gland development in dairy calves.

Uterine gland development occurs after birth in cattle and other mammals. The timeline of gland development has been described in various species, but little is known about how postnatal diet influences uterine gland development. This is especially concerning in dairy heifers, where a variety of milk replacer and whole milk nutrition options exist. Little work also exists in cattle to describe how early exposure to steroids influences reproductive tract and uterine gland development. The objective of this work was to determine the effects of early postnatal plane of nutrition and estrogen supplementation on uterine gland development in calves. In both studies, Holstein heifer calves were assigned to restricted milk replacer (R-MR) or enhanced milk replacer (EH-MR) diets. In study 1, calves (R-MR, n = 6; EH-MR, n = 5) were euthanized at 8 wk. In study 2, calves were weaned at 8 wk and administered estradiol (R-MR, n = 6; EH-MR, n = 6) or placebo (R-MR, n = 6; EH-MR, n = 5) for an additional 14 d before euthanasia. Average daily gain and final body weight was greater in both studies in heifers fed the enhanced diet. At 8 wk, EH-MR calves had a greater number of glands and a smaller average gland size, but total gland area was not different from the R-MR group. At 10 wk, uterine gland number and size were not affected by diet or estrogen. Expression profiles of several paracrine mediators of gland development were examined. Increases in transcript abundance for IGF1 and IGFBP3 and a decrease in abundance of WNT7A were detected in calves fed the enhanced diet at 8 wk of age. Plane of nutrition did not affect transcript profiles at 10 wk of age, but estradiol supplementation decreased MET and WNT7A transcript abundance. To conclude, heifer calves on a restricted diet exhibited a uterine morphology and transcript profile suggestive of delayed uterine gland development. These changes appear to be corrected by wk 10 of life. Also, this work provides evidence supporting the contention that early estradiol exposure has detrimental effects on uterine gene expression.

TRIENNIAL LACTATION SYMPOSIUM/BOLFA: Plasticity of mammary development in the prepubertal bovine mammary gland.

Although peripubertal mammary development represents only a small fraction of the total mass of mammary parenchyma present in the udder at the end of gestation and into lactation, there is increasing evidence that the tissue foundations created in early life can affect future mammary development and function. Studies on expression of estrogen and progesterone receptors seem to confirm the relevance of these steroids in prepubertal mammary development, but connections with other growth factors, hormones, and local tissue factors remain elusive. Enhanced preweaning feeding in the bovine appears to enhance the capacity of mammary tissue to response to mammogenic stimulation. This suggests the possibility that improved early nutrition might allow for creation of stem or progenitor cell populations to better support the massive ductal growth and lobulo-alveolar development during gestation. Increasing evidence that immune cells are involved in mammary development suggests there are unexpected and poorly understood connections between the immune system and mammary development. This is nearly unexplored in ruminants. Development of new tools to identify, isolate, and characterize cell populations within the developing bovine mammary gland offer the possibility of identifying and perhaps altering populations of mammary stem cells or selected progenitor cells to modulate mammary development and, possibly, mammary function.

Impaired mammary development in tamoxifen-treated prepubertal heifers is associated with altered development and morphology of myoepithelial cells.

Prepubertal mammary development involves elongation and branching of ducts and stromal tissue remodeling. This process is closely linked with ovarian and pituitary hormones, growth factors, and local regulators. Accumulating evidence suggests that the myoepithelial cells also play a role in ductal development in addition to their well-recognized importance in the milk ejection reflex. Following reports that myoepithelial cells changed in correspondence with decreased mammary growth after ovariectomy of prepubertal heifers, we evaluated myoepithelial cells in mammary tissue collected from prepubertal heifers treated with the antiestrogen tamoxifen. Briefly, heifers were given placebo (n=7) or tamoxifen (n=8; 0.3mg/kg per day) beginning on d 28 of life until the animals were euthanized on d 120. Tissues were collected from each of 3 zones (near the gland cistern, midway between the gland cistern and mammary fat pad, and at the interface of the parenchyma and mammary fat pad). Samples were processed to measure expression of transformation-related protein 63 (p63), smooth muscle actin, and common acute lymphoblastic leukemia antigen. We found that smooth muscle actin and common acute lymphoblastic leukemia antigen were expressed in the cytoplasm and p63 in the nuclei of myoepithelial cells. In concert with a 50% impairment in mammary growth after tamoxifen, we found that the number of myoepithelial cells around developing mammary ducts was reduced. But the average intensity of p63 expression per nucleus was not affected. We used the very distinct and exclusive staining of p63 in myoepithelial cell nuclei to capture hundreds of nuclear images for subsequent analysis using CellProfiler software. From this image analysis, we found that the area of myoepithelial cell nuclei and perimeter distances were reduced by tamoxifen. When nuclei were classified based on nuclear shape (eccentricity), we found differences in area, perimeter, and patterns of p63 expression based on Zernike number evaluations as well as treatment differences within each shape classification. These data provide support to the concept that myoepithelial cells are also the involved in mammary development in the prepubertal bovine mammary gland and that use of multispectral imaging combined with image analysis software can provide quantitative data to better understand the complex cellular interactions that ultimately regulate mammary morphogenesis in the bovine.

Short communication: Initial evidence supporting existence of potential rumen epidermal stem and progenitor cells.

The bovine rumen epidermis is a keratinized multilayered tissue that experiences persistent cell turnover. Because of this constant cell turnover, epidermal stem cells and their slightly more differentiated daughter cells, epidermal progenitor cells, must exist in the stratum basale of rumen epidermis. To date, these 2 epidermal cell populations and any unique cellular markers they may possess remain completely uncharacterized in the bovine rumen. An important first step in this new research area is the demonstration of the relative abundance and existence of markers for these cells in rumen tissue. A related second step is to document rumen epidermal proliferative responses to an extrinsic signal such as nutrient concentration within the rumen. The objectives of this experiment were to evaluate the extrinsic effect of diet on (1) gene expression of 6 potential rumen epidermal stem or progenitor cell markers and (2) rumen epidermal cell proliferation within the stratum basale. Twelve preweaned Holstein heifers were fed either a restricted diet (R) or an enhanced diet (EH). Animals on R received a milk replacer (MR) diet fed at 0.44kg of powder dry matter (DM)/d (20.9% crude protein, 29.8% fat, DM basis) and EH received MR at 1.08kg of powder dry matter/d (28.9% crude protein, 26.2% fat, DM basis). All calves had access to a 20% crude protein starter and were weaned during wk 7 of the experiment. Lifetime DM intake was 0.73kg of DM/calf per day for R (5.88 Mcal of net energy/calf per day) and 1.26kg of DM/calf per day for EH (10.68 Mcal of net energy/calf per day). Twenty-four hours before slaughter heifers received an intravenous dose of 5-bromo-2'-deoxyuridine to label proliferating cells. Heifers were slaughtered at 8 wk of age, and rumen samples from the ventral sac region were obtained and stored in RNA preservative and processed for routine histology. Quantitative real-time reverse transcriptase PCR was used to analyze relative abundance of genes. Candidate genes for markers of epidermal stem and progenitor cells were ß1-integrin (ITGB1), tumor protein p63 (TP63), keratin-14 (KRT14), Notch-1 (NOTCH1), Leu-rich repeat-containing G protein-coupled receptor 5-expressing (LGR5), and musashi-1 (MSI1). All genes were detected in the rumen tissue; ITGB1 was increased in EH compared with R. 5-Bromo-2'-deoxyuridine immunohistochemistry revealed that both R and EH rumen tissue had proliferating cells within the stratum basale of the rumen epidermis at the time of analysis. The EH diet resulted in an additive effect on cell proliferation. The percentage of cells in the stratum basale synthesizing DNA in preparation for mitosis nearly doubled (23.8±2.4% for EH vs. 14.7±2.0% for R) compared with calves fed R. This work represents the first attempt at characterizing rumen epidermal stem and progenitor cells. We demonstrated the relative abundance and existence of potential markers in rumen tissue and showed a rumen epidermal proliferative response to the extrinsic stimulus of nutrient concentration in the form of diet.

Feeding a higher plane of nutrition and providing exogenous estrogen increases mammary gland development in Holstein heifer calves.

Feeding heifers a higher plane of nutrition postweaning but before puberty can negatively affect mammary gland development and future milk yield. However, enhanced nutrition preweaning may promote development and future production. Our objectives were to determine the effects of enhanced feeding preweaning and exogenous estrogen immediately postweaning on mammary gland development and the composition of the mammary parenchyma (PAR) and mammary fat pad (MFP). Thirty-six Holstein heifer calves (<1 wk old) were reared on 1 of 2 dietary treatments for 8 wk: (1) a restricted milk replacer fed at 0.45 kg/d (R; 20% crude protein, 20% fat), or (2) an enhanced milk replacer fed at 1.13 kg/d (EH; 28% crude protein, 25% fat). Upon weaning, calves from each diet (n=6) were given either a placebo or estrogen implant for 2 wk, creating 4 treatments: R, R + estrogen (R-E2), EH, and EH + estrogen (EH-E2). Calves were housed individually with ad libitum access to water. Starter feeding began at wk 5 and was balanced between treatments. Udders were evaluated by palpation and physical measurements weekly. Subsets of calves were killed at weaning (n=6 per diet) and at the conclusion of the trial (n=6 per treatment). Udders were removed, dissected, and weighed. At wk 8, EH calves had longer front and rear teats. Providing estrogen to EH calves increased the length of rear teats during wk 9 and 10. Enhanced-fed calves had 5.2-fold more trimmed mammary gland mass than R calves. Providing estrogen to EH calves further increased mammary gland weight. Masses of PAR and MFP were markedly greater for EH calves than for R calves (e.g., 7.3-fold greater PAR tissue). Estrogen increased the mass of both PAR and MFP in EH calves. Feeding a higher plane of nutrition increased total protein, DNA, and fat in the MFP and total protein and DNA in the PAR. Dual-energy x-ray absorptiometry estimates of mammary fat mass were highly correlated with biochemical analyses of fat content. From histological study, we observed that the degree of expansion of epithelium into the adjacent stromal tissue and the complexity of ductal development were minimal in R, increased in EH, and increased by estrogen in both dietary treatments. Results provide compelling evidence that preweaning nutrition and estrogen administration immediately postweaning markedly increase mammary gland development in dairy calves. Cellular and molecular mechanisms responsible for these differences are currently under study.

Prepubertal tamoxifen treatment affects development of heifer reproductive tissues and related signaling pathways.

Prepubertal exposure of the developing ovaries and reproductive tract (RT) to estrogen or xenoestrogens can have acute and long-term consequences that compromise the reproductive performance of cattle. This research examined effects of the selective estrogen receptor modulator tamoxifen (TAM) on gene and protein abundance in prepubertal ovaries and RT, with a particular focus on signaling pathways that affect morphology. Tamoxifen was administered to Holstein heifer calves (n=8) daily (0.3mg/kg subcutaneously) from 28 to 120 d of age, when tissues were collected. Control calves (n=7) received an equal volume of excipient. Weight, gross measurements, and samples of reproductive tissues were collected, and protein and mRNA were extracted from snap-frozen samples of vagina, cervix, uterus, oviduct, ovary, and liver. Neither estradiol nor insulin-like growth factor I (IGFI) concentrations in the serum were affected by TAM treatment. Tamoxifen treatment reduced ovarian weight independently from effects on antral follicle populations, as there was no difference in visible antral follicle numbers on the day of collection. Estrogen receptor α (ESR1) and ß (ESR2) mRNA, ESR1 protein, IGFI, progesterone receptor, total growth hormone receptor, WNT4, WNT5A, and WNT7A mRNA, in addition to mitogen-activated protein kinase (MAPK) and phosphorylated MAPK proteins were affected differently depending on the tissue examined. However, neither IGFI receptor mRNA nor protein abundance were affected by TAM treatment. Results indicate that reproductive development in prepubertal Holstein heifer calves is TAM-sensitive, and that bovine RT and ovarian development are supported, in part, by estrogen receptor-dependent mechanisms during the period studied here. Potential long-term consequences of such developmental disruption remain to be defined.

Growth, intake, and health of Holstein heifer calves fed an enhanced preweaning diet with or without postweaning exogenous estrogen.

Research has shown that changes in nutrition both before and after weaning can affect mammary development. Additionally, estrogen is known to be a potent mammogenic stimulant. Our objectives were to determine effects of altered preweaning feeding and exogenous estradiol postweaning on growth, intake, and health. Thirty-six Holstein heifer calves were reared on (1) a restricted milk replacer (MR) diet fed at 0.44kg powder dry matter (DM)/day [R; 20.9% crude protein (CP), 19.8% fat, DM basis], or (2) an enhanced MR fed at 1.08kg powder DM/d (EH; 28.9% CP, 26.2% fat, DM basis). The MR feeding was reduced 50% during wk 8 to prepare for weaning. Starter was offered after wk 4 but balanced between treatments. Body weight and frame were measured weekly with intakes and health monitored daily. At weaning, a subset of calves were slaughtered (n=6/diet). Enhanced-fed calves had greater carcass, thymus, liver, spleen, and mammary gland (parenchyma and mammary fat pad) weights. The EH calves also had greater average daily gain (ADG) starting during wk 1 (0.36 vs. -0.06kg/d) and lasting through wk 7 (1.00 vs. 0.41kg/d). Remaining calves received estrogen implants or placebo and were slaughtered at the end of wk 10, creating 4 treatments: (1) R, (2) R + estrogen (R-E2), (3) EH, and (4) EH + estrogen (EH-E2). Postweaning ADG was similar between R, EH, and EH-E2 calves, but greater in R-E2 calves than E calves. The EH-E2 calves had the heaviest mammary glands, and R-E2 calves had heavier mammary glands than R calves. The EH calves consumed more MR DM, CP, and fat preweaning. The R-fed calves consumed more starter DM preweaning. Fecal score was greater for EH calves (1.74 vs. 1.50) preweaning, but days medicated did not differ. Fecal scores were lower for R-E2 calves postweaning. Improved preweaning feeding of calves increased body weights and frame measures. Differences in body weights remained postweaning. Enhanced-fed calves showed greater ADG during the preweaning period but not postweaning. Exogenous estrogen may elicit diet-dependent growth responses. Analysis of collected samples will allow determination of cellular and molecular processes responsible for the marked differences in mammary development observed.