Myoepithelial cell differentiation markers in prepubertal bovine mammary gland: effect of ovariectomy.

We reported previously that ovariectomy alters prepubertal development of mammary myoepithelial cells (MC) by mechanisms that are not well understood. Therefore, in the present study, we analyzed expression of 2 myoepithelial differentiation markers, α-smooth muscle actin (SMA) and the common acute lymphoblastic leukemia antigen (CD10), in mammary parenchymal tissue from intact (INT) and ovariectomized (OVX) heifers. On d 40, Holstein heifers underwent either an ovariectomy (OVX; n=16) or a sham (INT; n=21) operation. At 55, 70, 85, 100, 130, and 160 d of age, tissues were collected, and multispectral imaging was used to quantify immunofluorescent staining for myoepithelial cell (MC) markers. Fluorescent intensity (FI) of the markers was normalized against a control sample. In the basal epithelial layer, CD10 FI was less and SMA FI was greater in OVX than INT. The ratio of SMA to CD10 FI, as a proxy indicator for MC differentiation, was greater in tissue from OVX compared with INT heifers after 55 d of age. The staining for SMA was frequently more intense along the basal aspect of cells, whereas CD10 expression was localized on the apical surface of the MC. In mammary tissue from both INT and OVX heifers, we observed basal cells that were negative for both CD10 and SMA, some of which appeared to span the distance from basement membrane to the ductal lumen. Interestingly, we also observed CD10+ cells adjacent to the ductal lumen, a situation that was more prevalent in OVX than in INT heifers. Also, ovariectomy affects MC expression of both SMA and CD10, as well as the pattern of MC development. Myoepithelial cells are known to limit parenchymal growth in other species. Involvement of MC as regulators of prepubertal bovine mammary development is worthy of further investigation.

Effects of stress on endocrine and metabolic processes and redirection: cross talk between subcellular compartments.

Recent advances in genome analysis and biochemical pathway mapping have advanced our understanding of how biological systems have evolved over time. Protein and DNA marker comparisons suggest that several of these systems are both ancient in origin but highly conserved into today's evolved species. However, remnants of some of the more ancient functions of these chemical systems can run in conflict with the functions that those same pathways serve in complex organisms and tissue systems today. Relevant to the present topic, nitric oxide (NO) and superoxide anion (O(2)(•-)), ancient cellular molecules in evolutionary terms, are recognized today as both necessary for the well-being and stable health of cells but also injurious to cells as elaborated in conjunction with the cellular stress response. Why the dichotomy? This question underlies one of the basic issues challenging researchers as well as practitioners in their approach to disease management. The fundamental proinflammatory response of the innate immune system of the host is needed for pathogen control but can be injurious to tissues from "collateral damage" from NO- and O(2)(•-)-derived reactive molecules capable of affecting protein function via post-translational chemical modification. This review highlights newer aspects of the biochemistry of the NO- and O(2)(•-)-mediated innate proinflammatory response and further show how protein and tissue damage via overproduction of reactive nitrogen and oxygen intermediary molecules such as peroxynitrite (ONOO(-)) might be targeted to specific epitopes of proteins. Changes in the regulation of metabolism in response to proinflammatory disease states are discussed for GH signal transduction and tissue specificity.

Bovine mammary stem cells: cell biology meets production agriculture.

Mammary stem cells (MaSC) provide for net growth, renewal and turnover of mammary epithelial cells, and are therefore potential targets for strategies to increase production efficiency. Appropriate regulation of MaSC can potentially benefit milk yield, persistency, dry period management and tissue repair. Accordingly, we and others have attempted to characterize and alter the function of bovine MaSC. In this review, we provide an overview of current knowledge of MaSC gained from studies using mouse and human model systems and present research on bovine MaSC within that context. Recent data indicate that MaSC retain labeled DNA for extended periods because of their selective segregation of template DNA strands during mitosis. Relying on this long-term retention of bromodeoxyuridine-labeled DNA, we identified putative bovine MaSC. These label-retaining epithelial cells (LREC) are in low abundance within mammary epithelium (<1%). They are predominantly estrogen receptor (ER)-negative and localized in a basal or suprabasal layer of the epithelium throughout the gland. Thus, the response of MaSC to estrogen, the major mitogen in mammary gland, is likely mediated by paracrine factors released by cells that are ER-positive. This is consistent with considerable evidence for cross-talk within and between epithelial cells and surrounding stromal cells. Excision of classes of cells by laser microdissection and subsequent microarray analysis will hopefully provide markers for MaSC and insights into their regulation. Preliminary analyses of gene expression in laser-microdissected LREC and non-LREC are consistent with the concept that LREC represent populations of stem cells and progenitor cells that differ with regard to their properties and location within the epithelial layer. We have attempted to modulate the MaSC number by infusing a solution of xanthosine through the teat canal and into the ductal network of the mammary glands of prepubertal heifers. This treatment increased the number of putative stem cells, as evidenced by an increase in the percentage of LREC and increased telomerase activity within the tissue. The exciting possibility that stem cell expansion can influence milk production is currently under investigation.

Triennial Lactation Symposium: Bovine mammary epithelial cell lineages and parenchymal development.

Mammary development proceeds from an aggregation of cells in the ventral ectoderm to the establishment of an elaborate tree of alveoli, ducts, and cisternae. However, despite abundant data on endocrine regulation of ruminant mammary growth, we know comparatively little about cell lineages, expression of differentiation markers, and plasticity in mammary cell phenotype. Histologic analyses have revealed cell populations with distinct histochemical profiles, but functional assessment of cell populations during development has been limited to analysis of proliferation and frequency estimations of morphotypes. The lack of transplantation models, limited availability of validated antibodies with reactivity to bovine antigens, and similar technical challenges have generally hindered the pace of discovery, but the application of new technologies such as laser microdissection, transcriptional profiling, and multispectral image analysis are yielding important clues into bovine mammary cell ontogeny and developmental regulation. Our analyses have shown that prepubertal ovariectomy affects epithelial architecture, increases the proportion of cells expressing the estrogen receptor, and increases myoepithelial cell development, all concomitant with a dramatic reduction in the mass of parenchymal tissue. Our observations point to a dual role for ovarian secretions in the control of not only the rate of epithelial development, but also the nature of the parenchymal development. The balance of stimulus and inhibition pathways cooperatively regulates mammary growth. The increased reliance on objective staining analyses and quantitative approaches will ensure broader repeatability, application, and extension of the findings regarding the impact of the ovary and other regulatory entities and factors. Advances in understanding the ontogeny of mammary epithelial cells, coupled with established and increasing knowledge of endocrine factors affecting mammary development, may yield intervention strategies to improve dairy profitability.

Neither bovine somatotropin nor growth hormone-releasing factor alters expression of thyroid hormone receptors in liver and mammary tissues.

Physiological effects of thyroid hormones are mediated primarily by binding of triiodothyronine to specific nuclear receptors. Organ-specific changes in production of triiodothyronine from its prohormone, thyroxine, have been hypothesized to target the action of thyroid hormones on the mammary gland and play a role in mediating or augmenting a galactopoietic response to bovine somatotropin (bST). Additionally, tissue responsiveness to thyroid hormones may be altered by changes in the number or affinity of nuclear receptors for thyroid hormones. In the present study, effects of bST and bovine growth hormone-releasing factor (bGRF) on thyroid hormone receptors in liver and mammary gland were studied. Lactating Holstein cows received continuous infusions of bST or bGRF for 63 d or served as uninfused controls. Nuclei were isolated from harvested mammary and liver tissues and incubated with [(125)I]-triiodothyronine. Treatments did not alter the capacity or affinity of specific binding sites for triiodothyronine in liver or mammary nuclei. Evaluation of transcript abundance for thyroid hormone receptors showed that isoforms of thyroid hormone receptor or retinoid receptor (which may influence thyroid receptor action) expressed in the mammary gland were not altered by bST or bGRF treatment. Data do not support the hypothesis that administration of bST or bGRF alters sensitivity of mammary tissue by changing expression of thyroid hormone receptors.

Characterization of glucagon-like peptide 2 pathway member expression in bovine gastrointestinal tract.

Glucagon-like peptide 2 (GLP-2), secreted by enteroendocrine cells, has several physiological effects on the intestine of monogastric species, including promotion of growth of intestinal epithelium, reduction of epithelial cell apoptosis, and enhancement of intestinal blood flow, nutrient absorption, and epithelial barrier function. The regulatory functions of GLP-2 in the ruminant gastrointestinal tract (GIT) have not been well studied. The objectives of this investigation were to characterize the mRNA expression of 4 members of the GLP-2 pathway throughout the bovine GIT, including (1) proglucagon (GCG), the parent peptide from which GLP-2 is derived through cleavage by prohormone convertase; (2) prohormone convertase (PCSK1); (3) GLP-2 receptor (GLP2R); and (4) dipeptidyl peptidase IV (DPP4), the enzyme that inactivates GLP-2. Gene expression was evaluated in rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, and rectum collected at slaughter from prepubertal heifers, mature cows in early, mid, and late lactation, and nonlactating cows (n=3 per stage) by a gene expression profiling assay. In addition, mRNA expression of 14 genes involved in nutrient transport, enzyme activity, blood flow, apoptosis, and proliferation were evaluated in the 9 GIT tissues for their association with GCG and GLP2R mRNA expression. Immunohistochemistry was used to localize GLP2R protein in tissues of the lower GIT. Results indicated that mRNA expression of GCG, PCSK1, GLP2R, and DPP4 varies across the 9 GIT tissues, with greatest expression in small and large intestines, and generally nondetectable levels in forestomachs. Expression of DPP4 and GLP2R mRNA varied by developmental stage or lactational state in intestinal tissues. Expression of GCG or GLP2R mRNA was correlated with molecular markers of proliferation, apoptosis, blood flow, enzyme activity, and urea transport, depending on the tissue examined, which suggests a potential for involvement of GLP-2 in these physiological processes in the ruminant GIT. The GLP2R protein was expressed in intestinal crypts of the bovine GIT, which is consistent with the distribution in monogastric species. Our findings support a functional role of the GLP-2 pathway in bovine GIT and the potential for use of GLP-2 as a therapy to improve intestinal function and nutrient absorption in ruminants.

An integrated view on how the management of the dry period length of lactating cows could affect mammary biology and defence.

The dry period is necessary to facilitate cell turnover in the bovine mammary gland and to optimize milk production in the next lactation. An 8-week dry period has long been the golden standard of management for dairy cows. Genetic improvements and new management technologies have led to higher milk production and a need for re-evaluation of the dry period length. Over the last decade, dry period length has been proposed to be shortened or eliminated mainly from an economic point of view. However, the influence of modified dry period length on the immune defence of the bovine mammary gland and the occurrence of new intramammary infections has not yet been appreciated. The objective of this review is to discuss the bovine mammary gland biology, defence and systemic health when the dry period length is modified. Shortening or eliminating the dry period may minimize or remove the impact of milk accumulation at dry off, thereby lessening the immunodeficiency of the dam that is characteristic of this period. Composition of mammary secretions may change and the extent of tissue remodelling may be reduced when the dry period is reduced or eliminated. Additionally, impact of the dry period length on energy and nutritional status, and on hormonal and local regulatory factors, lead us to hypothesize that changing the dry period length might also affect the response to intramammary infection. It is concluded that there is a need to integrate mammary gland biology and defence mechanisms in studies dealing with modified dry period lengths.

Technical note: A rapid method for 5-bromo-2'-deoxyuridine (BrdU) immunostaining in bovine mammary cryosections that retains RNA quality.

A rapid method of 5-bromo-2'-deoxyuridine (BrdU) immunostaining was developed in cryosections of bovine mammary tissue while preserving RNA quality of the stained section. A thymidine analog that is incorporated into DNA of proliferating cells, BrdU serves as a proliferation marker. Immunostaining of BrdU-labeled cells within a histological section requires heat, enzymatic or chemical-mediated antigen retrieval to open double-stranded DNA, and exposure to the BrdU antigen. Although these established treatments permit staining, they preclude use of cells within the tissue section for further gene expression experiments. Additionally, long antibody incubations and washing steps lead to extensive RNA degradation and elution. A protocol was developed for immunolocalization of BrdU-labeled cells in cryosections of bovine mammary tissue, which does not require harsh DNA denaturation and preserved RNA integrity and quantity. This protocol used an initial acetone:polyethylene glycol 300 [9:1 (vol/vol)] fixation (2 min) followed by staining with methyl green (0.5% aqueous; 2 min) to stabilize macromolecules, antigen retrieval with deionized formamide (70% in nuclease-free phosphate buffered saline; 4 min incubation), antibody incubation in the presence of RNase inhibitors (5 min), and minimal washing to facilitate recovery of RNA from cells from the stained sections. Applicability of this protocol to other nuclear antigens was evaluated by testing its suitability for staining estrogen receptor alpha and Ki-67 antigen. In both cases, use of the protocol provided good immunostaining and tissue morphology. The RNA quality of estrogen receptor alpha- and Ki-67-stained sections was not evaluated. Quality of the isolated RNA from BrdU-stained sections was evaluated by micro-fluidic electrophoresis and its utility was confirmed using quantitative reverse transcription-PCR. Staining intensity obtained with this labeling protocol was similar to that obtained using conventional immunohistochemistry protocols. When coupled with laser microdissection and RNA or cDNA amplification, this immunostaining protocol provided a means for future transcriptome analysis of BrdU-labeled cells within a complex tissue.

Effects of increased milking frequency on metabolism and mammary cell proliferation in Holstein dairy cows.

Results of previous studies have shown that increased milking frequency (IMF) during early lactation results in increased milk yield not only during the period of IMF but also after cows have returned to a decreased milking frequency. The cellular mechanisms underpinning this increased milk yield and the overall effects of IMF on metabolism have not been well characterized. The objective of this study was to determine the effects of IMF on metabolism and mammary epithelial cell proliferation in dairy cows. Thirty primiparous and 30 multiparous Holstein cows were assigned randomly at calving to 1 of 2 treatments. The control group was milked twice daily (2x) for 119 d, whereas the IMF group was milked 4 times daily (4x) from d 2 postcalving until d 21 and then 2x from d 22 until d 119. Overall milk yield did not differ between treatments throughout the 119 d monitored; however, the interaction of treatment by week was significant in that IMF cows yielded 4.8kg/d more milk than control cows during wk 2 and 3 and had similar levels of milk yield during the remainder of the study period. Reanalysis of data excluding data from cows subjected to mammary biopsy suggested that the mammary biopsy procedure contributed to the lack of overall responses of milk yield, but that responses overall to IMF were greater in primiparous cows compared with multiparous cows. Plasma nonesterified fatty acid concentrations were elevated in multiparous cows subjected to IMF during the period of IMF, but were not influenced by treatment in primiparous cows. Plasma beta-hydroxybutyrate concentrations were not affected by treatment. Mammary tissue was collected by biopsy in a subset of cows (n=8 cows per parity and treatment) at calving and at d 21 and 75 postpartum and used for immunohistochemical localization of the cell proliferation antigen, Ki67. Effects of treatment on mammary epithelial cell proliferation were not significant, suggesting that other mechanisms must be responsible for carryover effects of IMF on lactational performance.

Dietary genistein stimulates mammary hyperplasia in gilts.

The possible role of the phytoestrogen genistein on prepubertal development of mammary glands, hormonal status and bone resorption was investigated in gilts. Forty-five gilts were fed a control diet containing soya (CTLS, n = 15), a control diet without soya (CTL0, n = 15) or the CTLS diet supplemented with 2.3 g of genistein daily (GEN, n = 15) from 90 days of age until slaughter (day 183 ± 1). Both basal diets were isonitrogenous and isocaloric. Jugular blood samples were obtained on days 89 and 176 to determine concentrations of isoflavone metabolites (on day 176 only), prolactin, estradiol, progesterone, insulin-like growth factor 1 (IGF1), and N-telopeptide of type I collagen (NTx; on day 176 only). At slaughter, mammary glands were excised, parenchymal and extraparenchymal tissues were dissected, and composition of parenchymal tissue (protein, fat, dry matter (DM), DNA) was determined. Histochemical analyses of mammary parenchyma were performed. Dietary genistein increased parenchymal protein (P < 0.05) while decreasing DM (P < 0.05) and tending to lower fat content compared with the CTLS, but not the CTL0, diet. There was more parenchymal DNA (1.26 v. 0.92 mg/g, P < 0.05) in GEN than CTLS gilts, likely reflecting an increase in the quantity of mammary epithelial cells. Circulating concentrations of genistein were increased in GEN gilts (P < 0.001) but concentrations of hormones or NTx (indicator of bone collagen resorption) were not affected by GEN (P > 0.1). Percentage of estradiol receptor alpha (ERα)-positive epithelial cells was lower (P < 0.05) in GEN than CTLS gilts, whereas 5-bromo-2'-deoxyuridine labeling index was unaltered (P > 0.1). Transcript levels for ERα, ERß, IGF1, epidermal growth factor (EGF), epidermal growth factor receptor and transforming growth factor alpha were not altered by treatments. Supplementation of the diet with genistein during the growing phase in gilts, therefore, led to hyperplasia of mammary parenchymal tissue after puberty; yet, even though circulating genistein was increased, this was not accompanied by changes in mammary expression of selected genes or circulating hormone levels.

Effects of milk replacer formulation on measures of mammary growth and composition in Holstein heifers.

Overfeeding prepubertal heifers may impair mammary parenchymal growth and reduce milk production, but evidence suggests that increased intake of a high-protein milk replacer before weaning may be beneficial. This study was designed to evaluate effects of milk replacer (MR) composition on mass and composition of mammary parenchyma and fat pad, growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis gene expression, and putative mammary epithelial stem cells. Specifically, we hypothesized that positive effects of faster rates of gain during the preweaning period alter the development, persistence, or activity of populations of putative mammary epithelial stem cells, possibly through involvement of GH/IGF-I axis molecules. Twenty-four newborn heifers were fed 1 of 4 MR diets (n = 6/diet): control [20% crude protein (CP), 21% fat MR fed at 441 g of dry matter (DM)/d], high protein, low fat (28% CP, 20% fat MR fed at 951 g of DM/d), high protein, high fat (27% CP, 28% fat MR fed at 951 g of DM/d), and high protein, high fat+ (27% CP, 28% fat MR fed at 1,431 g of DM/d). Water and starter (20% CP, 1.43% fat) were offered ad libitum. Animals were killed on d 65 and mammary tissue was subjected to biochemical, molecular, and histological examination. No differences in mammary parenchymal mass or composition, with or without adjusting for empty body weight, were detected. Mass was increased and composition of the mammary fat pad was altered by nutrient intake. No diet differences in putative mammary epithelial stem cell abundance or abundance of transcripts for genes of the GH/IGF-I axis were detected. In this study, growth of the mammary epithelium, size of the mammary epithelial stem cell population, and components of the GH/IGF-I axis did not depend on diet. However, an underlying positive correlation between telomerase, a marker of mammary stem cells, and growth of the mammary parenchyma was detected. Implications of diet-induced effects on mammary fat pad and possible effects on subsequent development and function remain to be determined.

Effects of body weight and nutrition on histological mammary development in Holstein heifers.

Our objective was to determine the effects of rate of gain and body weight (BW) on development of the mammary parenchyma. Mammary tissue samples were collected from heifers (n = 72) reared on 1 of 2 dietary treatments (restricted, 650 g/d of daily gain; or elevated, 950 g/d of daily gain) and slaughtered at 100, 150, 200, 250, 300, or 350 kg of BW. Mammary samples were excised, preserved, prepared for histology, and stained with hematoxylin and eosin. Digital images of tissue sections were captured for analysis. Tissue areas occupied by the interlobular and intralobular stroma, epithelium, and lumen were measured (mum(2)). The numbers of epithelial and luminal structures per image were tabulated to measure the complexity of ductal development. Mean percentages of mammary parenchyma occupied by the interlobular stroma, epithelium, lumen, and intralobular stroma were 29, 20, 7, and 43%, respectively. Percentage of area occupied by the intralobular stroma was affected by BW and was lower for 100-kg heifers compared with heifers 200 kg and heavier (33 +/- 4 vs. 46 +/- 4), but the percentage of area occupied by other tissue elements did not differ by BW or treatment, nor was there an interaction. However, the numbers of both epithelial (8.3 +/- 4 vs. 47 +/- 4) and luminal-containing (6 +/- 4 vs. 38 +/- 4) structures per image increased markedly between 100 and 350 kg of BW, irrespective of diet. For heifers slaughtered between 100 and 350 kg of BW, alterations in the rate of gain between 650 and 950 g/d, accomplished by feeding varying amounts of the same diet, had no significant effect on tissue characteristics or the pattern of mammary parenchymal development. These data emphasize the importance of BW and age in determining developmental characteristics of the heifer mammary parenchyma and suggest that the rate of gain per se has a minimal impact on histological development, and thus do not support the hypothesis that rate of gain has a direct negative impact on ductal development.

Evaluation of antioxidant and proinflammatory gene expression in bovine mammary tissue during the periparturient period.

The incidence and severity of mastitis can be high during the period of transition from pregnancy to lactation when dairy cattle are susceptible to oxidative stress. Oxidative stress may contribute to the pathogenesis of mastitis by modifying the expression of proinflammatory genes. The overall goal of this study was to determine the relationship between critical antioxidant defense mechanisms and proinflammatory markers in normal bovine mammary tissue during the periparturient period. Mammary tissue samples were obtained from 12 cows at 35, 20, and 7 d before expected calving and during early lactation (EL, 15 to 28 d in milk). Enzyme activities for cytosolic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase were relatively low during the dry period, but increased during EL, whereas activity of thioredoxin reductase 1 did not change significantly as a function of time. In contrast, gene expression for these antioxidant selenoproteins and for heme oxygenase-1 gradually decreased as parturition approached and then increased during EL. The expression of intercellular vascular adhesion molecule-1 and vascular cell adhesion molecule-1 followed a similar trend where mRNA abundance gradually declined as parturition approached with a slight rebound in EL. Gene expression of the pro-oxidant, 15-lipoxygenase 1, which is known to increase during times of oxidative stress, also increased dramatically in mammary tissue from EL cows. Expression of the proinflammatory cytokines, IL-1beta, IL-6, and IL-8 did not change significantly during the periparturient period. Strong positive correlations were found between several antioxidant enzymes (cytosolic glutathione peroxidase, thioredoxin reductase 1, and heme oxygenase-1) and vascular adhesion molecules (intercellular vascular adhesion molecule-1, vascular cell adhesion molecule-1) suggesting a protective response of these antioxidants to an enhanced proinflammatory state. Ability to control oxidative stress through manipulation of key antioxidant enzymes in the future may modify the proinflammatory state of periparturient cows and reduce incidence and severity of some diseases such as mastitis.

Effects of milk replacer composition on growth, body composition, and nutrient excretion in preweaned Holstein heifers.

Twenty-four newborn Holstein heifer calves were fed 1 of 4 milk replacers (MR): control (20% CP, 21% fat; MR fed at 441 g/d); high protein/low fat (HPLF; 28% CP, 20% fat; MR fed at 951 g/d); high protein/high fat (HPHF; 27% CP, 28% fat; MR fed at 951 g/d); and HPHF MR fed at a higher rate (HPHF+; 27% CP, 28% fat; MR fed at 1,431 g/d). Dry calf starter (20% CP, 1.43% fat) composed of ground corn (44.4%), 48% CP soybean meal (44.4%), cottonseed hulls (11.2%), and molasses (1.0%) was offered free choice. Heifers were obtained from a commercial dairy, blocked by groups of 8 in the order acquired, and randomly assigned to treatments within group. Upon arrival at the research farm, heifers were fed the control for 2 feedings. Treatments were imposed when heifers were 4 +/- 1 d of age. Heifers were on study for 61 +/- 1 d. Body weight and body size measures were taken weekly. Four-day total collection of feed refusals, feces, and urine was initiated at 57 +/- 1 d of age. Heifers were slaughtered at the end of the collection period to evaluate body composition. Preplanned contrasts were used to compare control to all, HPLF to HPHF, and HPHF to HPHF+. Heifers fed the control diet consumed more starter than those fed other treatment diets, but their total dry matter intake and apparent dry matter digestibility were lowest. Fecal output was highest in heifers fed the control diet, whereas urine output and urine N excretion were lowest. Nitrogen intake and urine N excretion were greater for heifers fed HPHF+ compared with HPHF but were not affected by MR fat content (HPLF vs. HPHF). Retention (g/d) of N and P was greater in heifers fed all nutrient-dense diets compared with those fed the control diet, but was not improved by increasing fat in the milk replacer (HPLF vs. HPHF) or by increasing the amount fed. Addition of fat to the milk replacer (HPLF vs. HPHF) increased empty body weight fat content without improving average daily gain or frame measures. Increasing the volume fed (HPHF vs. HPHF+) increased growth rate and empty body weight, but HPHF+ heifers were neither taller nor longer and their carcasses contained more fat. Clear improvements in growth and nutrient retention were observed with more nutrient-dense diets, but most of the improvements were seen with the increased protein intake relative to the control MR; adding fat to the high protein MR did not further improve lean tissue gain.

Regulation of mammary gland sensitivity to thyroid hormones during the transition from pregnancy to lactation.

Thyroid hormones are galactopoietic and help to establish the mammary gland's metabolic priority during lactation. Expression patterns for genes that can alter tissue sensitivity to thyroid hormones and thyroid hormone activity were evaluated in the mammary gland and liver of cows at 53, 35, 20, and 7 days before expected parturition, and 14 and 90 days into the subsequent lactation. Transcript abundance for the three isoforms of iodothyronine deiodinase, type I (DIO1), type II (DIO2) and type III (DIO3), thyroid hormone receptors alpha1 (TRalpha1), alpha2 (TRalpha2) and beta1 (TRbeta1), and retinoic acid receptors alpha (RXRalpha) and gamma (RXRgamma), which act as coregulators of thyroid hormone receptor action, were evaluated by quantitative RT-PCR. The DIO3 is a 5-deiodinase that produces inactive iodothyronine metabolites, whereas DIO1 and DIO2 generate the active thyroid hormone, triiodothyronine, from the relatively inactive precursor, thyroxine. Low copy numbers of DIO3 transcripts were present in mammary gland and liver. DIO2 was the predominant isoform expressed in mammary gland and DIO1 was the predominant isoform expressed in liver. Quantity of DIO1 mRNA in liver tissues did not differ with physiological state, but tended to be lowest during lactation. Quantity of DIO2 mRNA in mammary gland increased during lactation (P < 0.05), with copy numbers at 90 days of lactation 6-fold greater than at 35 and 20 days prepartum. When ratios of DIO2/DIO3 mRNA were evaluated, the increase was more pronounced (>100-fold). Quantity of TRbeta1 mRNA in mammary gland increased with onset of lactation, whereas TRalpha1 and TRalpha2 transcripts did not vary with physiological state. Conversely, quantity of RXRalpha mRNA decreased during late gestation to low levels during early lactation. Data suggest that increased expression of mammary TRbeta1 and DIO2, and decreased RXRalpha, provide a mechanism to increase thyroid hormone activity within the mammary gland during lactation.

Ontogenic and nutritional regulation of steroid receptor and IGF-I transcript abundance in the prepubertal heifer mammary gland.

In prepubertal cattle, mammary development is characterized by the growth of an epithelial-rich parenchyma (PAR) into the mammary fat pad (MFP). This proliferation and accumulation of mammary epithelial cells require estrogen. Paradoxically, both epithelial cell proliferation and PAR accumulation rate decline with rising plasma estrogen as puberty approaches. The possibility that variation in abundance of estrogen receptors (ERs) in PAR or MFP accounts for a portion of these effects has not been examined in cattle. Additionally, we recently demonstrated that MFP is highly responsive to exogenous estrogen, suggesting that this tissue may play a role in coordinating estrogen's effects on PAR; however, the developing bovine MFP has yet to be studied in detail. To address these hypotheses, Holstein heifers were assigned to planes of nutrition supporting body growth rates of 950 (E) or 650 (R) g/day and harvested every 50 kg from 100 to 350 kg body weight (BW). Post-harvest, their mammary glands were dissected into PAR and MFP compartments. Transcript abundance of genes encoding members of the ER family (ERalpha, ERbeta, and estrogen-related receptor alpha-1 (ERRalpha)) and estrogen-responsive genes (IGF-I and progesterone receptor (PR)) were measured in both mammary compartments by quantitative real-time RT-PCR. Significant expression was detected for all genes in both compartments, with the exception of the ERbeta gene. Transcript abundance of both ERalpha and IGF-I decreased linearly with increasing BW within both compartments. ERRalpha and PR expressions decreased with increasing BW in PAR but not in MFP. Nutrition stimulated ERalpha and ERRalpha expression in the PAR but had no effect on IGF-I or PR in either PAR or MFP. Overall, ERalpha and IGF-I transcript abundance are consistent with the drop in mammary epithelial cell proliferation and PAR accretion observed over development, but do not support a negative effect of nutrition on PAR growth.

Regulation of gene expression in the bovine mammary gland by ovarian steroids.

It is well established that estrogen is required for mammary epithelial cell proliferation and ductal development in the growing animal, and that lobuloalveolar development during gestation is dependent on progesterone. The effects of these steroid hormones on gene expression in the mammary gland are mediated primarily by their respective nuclear hormone receptors, which function as hormone-bound transcription factors. To gain insight into how estrogen and progesterone regulate mammary gland growth and function in cattle, we and others have characterized the expression patterns of their cognate nuclear hormone receptors in the bovine mammary gland throughout development, pregnancy, and lactation. This work has identified a lack of expression of estrogen receptor beta and a greater abundance of progesterone receptor during lactation in the bovine mammary gland, compared with the rodent gland. We speculate that interactions among the estrogen receptor isoforms that regulate progesterone receptor expression may contribute to these species differences. Further, demonstrated expression of substantial quantities of estrogen receptor within the prepubertal bovine mammary fat pad, along with coordinated insulin-like growth factor-I expression, suggests that this tissue may stimulate parenchymal growth via an estrogen-responsive paracrine mechanism. In addition, the recent availability of bovine genomic sequence information and microarray technologies has permitted the study of global gene expression in the mammary gland in response to the steroid environment. We have identified more than 100 estrogen-responsive genes, of which the majority are novel estrogen gene targets. Estrogen-induced changes in gene expression were consistent with increased mammary epithelial cell proliferation, increased extracellular matrix turnover in parenchyma, and increased extracellular matrix deposition in the fat pad. A comparison of estrogen-responsive genes in the mammary glands of humans, mice, and cattle suggests considerable variation among species, as well as potential differences in regulatory elements in common estrogen receptor gene targets. Continuing studies using advanced molecular techniques should assist in elucidating the complex regulation of mammary function at the transcript level.

Effects of dietary supplementation with flax during prepuberty on fatty acid profile, mammogenesis, and bone resorption in gilts.

The possible role of dietary flax on pre-pubertal development of mammary glands and bone resorption was investigated in gilts. Fifty-seven gilts were fed 1 of 4 diets from 88 d of age until slaughter (d 212 +/- 1). Diets were control without flax (n = 14); 10% flaxseed supplementation (n = 13); 6.5% flaxseed meal supplementation (n = 15); and 3.5% flaxseed oil supplementation (n = 15). All diets were isonitrogenous and isocaloric. Jugular blood samples were obtained on d 78 and 210 to establish the fatty acid profile and to determine the concentrations of prolactin, estradiol, and cross-linked N-telopeptides of type I collagen. At slaughter, the mammary glands were excised, parenchymal and extraparenchymal tissues were dissected, and the composition of the parenchymal tissue (protein, fat, DM, and DNA) was determined. Histochemical analyses of the mammary parenchyma were performed, and fatty acid profiles in the extraparenchymal tissue were evaluated. Dietary flax increased (P < or = 0.001) the concentrations of PUFA and decreased those of SFA (P < 0.01) and MUFA (P < or = 0.001) in plasma and extraparenchymal tissues, which was largely due to the inclusion of 10% flaxseed or 3.5% flaxseed oil (P < or = 0.01) but not 6.5% flaxseed meal. Circulating concentrations of prolactin and estradiol were unaltered by treatments (P > 0.1), but concentrations of cross-linked N-telopeptides of type I collagen tended to be greater (P < 0.1) in flax-supplemented gilts. The DM content of parenchymal tissue was the only mammary compositional value affected, showing an increase with flax addition (P < 0.05). No change (P > or = 0.1) in the bromodeoxyuridine labeling index or estrogen receptor localization was observed with treatments. Dietary supplementation with flax as seed, meal, or oil, therefore, brought about the expected changes in the fatty acid profile but had no beneficial effects on mammary development or bone resorption.

Effect of continuous milking and bovine somatotropin supplementation on mammary epithelial cell turnover.

Objectives were to determine effects of continuous milking (CM) and bovine somatotropin (bST) administration on 1) mammary epithelial cell (MEC) proliferation, apoptosis, and ultrastructure during late gestation and early lactation, 2) expression of genes associated with proliferation, and apoptosis in mammary epithelial cells, and 3) milk yield and composition. Second-gestation, first dry-period cows were randomly assigned to either continuous bST throughout late gestation and early lactation (+bST; n = 4) or no bST (-bST; n = 4) administration. Within each animal, udder halves were randomly assigned to CM or a 60-d dry period (control) treatment. Daily milk yield and weekly milk composition were measured during the last 60 d of gestation in CM halves and from 1 to 30 d postpartum for both halves. Mammary biopsies were obtained at -20 +/- 7, -8 +/- 3, +1 +/- 0, +7 +/- 0, and +20 +/- 0 d (mean +/- standard error) relative to parturition. Prepartum half-udder milk yield was greater in +bST cows than in -bST cows (9.9 vs. 8.2 kg/d) and postpartum half-udder milk yields were dramatically reduced in CM halves compared with control halves (10.6 vs. 22.2 kg/d), regardless of bST treatment. Proliferation of MEC was reduced in CM halves at -8 d (2.7 vs. 5.4%). Apoptosis of MEC was elevated during early lactation for d +1 and +7 in control halves, but was only increased at d +1 in CM halves. Turnover of MEC was not affected by bST. Ultrastructure data indicated complete involution of the control half and lactation maintenance in CM glands (d -20). By d -8, control tissue contained alveoli in an immature secretory state, but CM tissue contained both lactating and immature alveoli. Postpartum ultrastructure parameters were similar between halves until d 20 when control tissue was composed of a homogeneous population of lactating alveoli, but CM tissue contained lactating, engorged, and resting alveoli. Expression of CCAAT/enhancer binding protein-beta (CEBP-beta), cyclin D1, and bcl(2) were up-regulated during late gestation, but did not differ between control and CM halves. Expression of alpha-lactalbumin was increased in CM halves during late gestation, but was not different in CM and control tissue after parturition. Other genes evaluated (bax, insulin-like growth factor binding protein 5, ATP-binding cassette 1, and p27) were not differentially expressed at any timepoints evaluated. Results indicate that CM reduced subsequent half-udder milk yield in primiparous cows through altered MEC turnover and secretory capacity. Negative effects of CM on the subsequent lactation were not alleviated by bST supplementation.

Developmental and nutritional regulation of the prepubertal heifer mammary gland: I. Parenchyma and fat pad mass and composition.

Prior to puberty, elevated nutrient intake has been shown to negatively affect prepubertal mammary development in the heifer. The objective of this study was to evaluate the effects of increased nutrient intake on mammary development in Holstein heifers at multiple body weights from birth through puberty. Specifically, this study evaluated the effects of nutrient intake and body weight at harvest on 1) total weight and DNA content of the parenchyma (PAR) and mammary fat pad (MFP) and 2) PAR and MFP composition. Starting at 45 kg of body weight, heifers (n = 78) were assigned to either a restricted (R) or elevated (E) level of nutrient intake supporting 650 (R) or 950 (E) g/d of body weight gain. Heifers were harvested at 50-kg increments from 100 to 350 kg of body weight. Mammary fat pad weight and DNA content were greater in E- than in R-heifers. Additionally, E-heifers had a greater fraction of lipids and a smaller fraction of protein in their MFP than did R-heifers. Parenchyma weight and DNA were lower in E- than in R-heifers; however, when analyzed with age as a covariate term, treatment was no longer a significant term in the model. Level of nutrient intake had no effect on the lipid, protein, or hydroxyproline composition of the PAR. Collectively, these data demonstrate that PAR is refractory to the level of nutrient intake whereas MFP is not. Furthermore, the covariate analysis demonstrated that age at harvest, not the level of nutrient intake, was the single greatest determinant of total PAR DNA content.