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The transition from pregnancy to lactation (i.e. the transition period) is a time of significant metabolic challenge, with a several-fold increase in a cow's requirement for energy, protein and minerals within days of calving. A successful transition involves the initiation and coordination of changes in multiple tissues that facilitate the provision of these nutrients to the cow and, more specifically, to the mammary gland, often at the considerable expense of other tissues. Failure to coordinate the necessary changes effectively results in transition period maladaptation, which can broadly be grouped into three categories: (1) negative energy balance and metabolic diseases associated with energy metabolism; (2) immune dysfunction and inflammation; and (3) metabolic diseases associated with mineral deficiency. Because reinitiation of ovarian activity, follicle recruitment, ovulation, fertilisation and, potentially, even maternal recognition of pregnancy and implantation occur against the backdrop of this metabolic and immunological disturbance in early lactation, the role of nutrition in ensuring a smooth transition between the pregnant and lactating state is important. In this paper we integrate recent research findings with previous knowledge of the interaction between transition cow metabolism and nutrition and reproductive outcomes, and offer new insights into key elements of successful cow management to avoid transition 'maladaptation' and improve pregnancy rates.

In Table 3 (page 6478), the means and standard errors of the difference (SED) for 9 of the parameters were tabulated as log10 values, rather than in the original units. All P-values and Tukey comparisons are correct. The corrected table is shown below, with corrected parameters and values in bold. The authors regret the errors.

The common practice of increasing dietary energy density during the close-up dry period (last ∼3 wk prepartum) has been recently associated with a higher incidence of metabolic disorders after calving. Despite these reports, over-feeding of metabolizable energy (ME) during the far-off, nonlactating period is a common management policy aimed at achieving optimum calving body condition score (BCS) in pasture-based systems, as cows are generally thinner than total mixed ration cows at the end of lactation. Our hypothesis was that both far-off and close-up overfeeding influence the peripartum adipose tissue changes associated with energy balance and inflammatory state. Sixty mid-lactation, grazing dairy cows of mixed age and breed were randomly allocated to 1 of 2 groups that were managed through late lactation to achieve a low and high BCS (approximately 4.25 and 5.0 on a 10-point scale) at dry-off. The low BCS cows were then overfed ME to ensure that they achieved the same BCS as the higher BCS group by calving. Within each rate of BCS gain treatment, cows were offered 65, 90, or 120% of their pre-calving ME requirements for 3 wk pre-calving in a 2 × 3 factorial arrangement of treatments (i.e., 10 cows/treatment). Subcutaneous adipose tissue was collected via biopsy at −1, 1, and 4 wk relative to parturition. Quantitative PCR was used to measure mRNA and microRNA expression of targets related to adipogenesis and inflammation. Cows overfed in the far-off period had increased expression of miR-143 and miR-378 prepartum (−1 wk) indicating greater adipogenesis, consistent with their rapid gain in BCS following dry-off. Furthermore, the lower postpartum expression of IL6, TNF, TLR4, TLR9, and miR-145, and a higher abundance of miR-99a indicated lower body fat mobilization in early lactation in the same group. In the close-up period, feeding either 65 or 120% of ME requirements caused changes in FASN, IL1B, IL6R, TLR9, and the microRNA miR-143, miR-155, and miR-378. Thei

Preterm delivery is a major obstetric health problem contributing to poor neonatal outcome including low birth weight, respiratory distress syndrome, gastrointestinal, immunologic, central nervous system, hearing, and vision problems. Worldwide, approximately 15 million babies are born prematurely each year. The critical question which remains is how to identify women destined to deliver preterm from those who will achieve a term delivery. Prostaglandins, in all mammals, are important in the parturient process. Increased intrauterine prostaglandin production is associated with labor and in fact prostaglandin E2 (PGE2) or analogs are widely used clinically for cervical ripening and labor induction. Measurements of circulating eicosanoids have been problematic because of the rapid and major clearance by the lungs and then kidneys resulting in very low concentrations in plasma. Moreover, since eicosanoids are produced by all mammalian tissues, the sources of the measured eicosanoids are unknown. Our understanding of how cells communicate has undergone a paradigm shift with the recognition of the role of exosomes in intercellular signaling. Recent publications have identified enzymes and products of arachidonic acid metabolism (eicosanoids) within exosomes. This review will explore the potential roles of exosomes in eicosanoid functions that are critical in preterm labor and delivery.

Extensive metabolic, physiological, and immunological changes are associated with calving and the onset of lactation. As a result, cows transitioning between pregnancy and lactation are at a greater risk of metabolic and infectious diseases. The ability of neutrophils to mount an effective immune response to an infection is critical for its resolution, and increasing evidence indicates that precalving nutrition affects postpartum neutrophil function. The objectives of the current study were to investigate the effect of 2 precalving body condition scores (BCS; 4 vs. 5 on a 10-point scale) and 2 levels of feeding (75 vs. 125% of estimated maintenance requirements) on gene expression in circulating neutrophils. We isolated RNA from the neutrophils of cows (n = 45) at 5 time points over the transition period: precalving (−1 wk), day of calving (d 0), and postcalving at wk 1, 2, and 4. Quantitative reverse transcriptase PCR with custom-designed primer pairs and Roche Universal Probe Library (Roche, Basel, Switzerland) chemistry, combined with microfluidics integrated fluidic circuit chips (96.96 dynamic array), were used to quantify the expression of 78 genes involved in neutrophil function and 18 endogenous control genes. Statistical significance between time points was determined using repeated measures ANOVA with Tukey-Kramer multiple-testing correction to determine treatment effects among weeks. Precalving BCS altered the inflammatory state of neutrophils, with significant increases in overall gene expression of antimicrobial peptides (BNBD4 and DEFB10) and the anti-inflammatory cytokine IL10, and significantly decreased expression of proinflammatory cytokine IL23A in thinner cows (BCS 4) compared with cows calving at BCS 5. Feeding level had a time-dependent effect on gene expression; for example, increased expression of genes involved in leukotriene synthesis (PLA2G4A and ALOX5AP) occurred only at 1 wk postcalving in cows overfed (125% of requirements) precalvin

Hepatic gluconeogenesis is essential for maintenance of whole body glucose homeostasis and glucose supply for mammary lactose synthesis in the dairy cow. Upregulation of the gluconeogenic enzyme pyruvate carboxylase (PC) during the transition period is vital in the adaptation to the greater glucose demands associated with peripartum lactogenesis. The objective of this study was to determine if PC transcription in hepatocytes is regulated by DNA methylation and if treatment with a nonsteroidal anti-inflammatory drug (NSAID) alters methylation of an upstream DNA sequence defined as promoter 1. Dairy cows were left untreated (n = 20), or treated with a NSAID during the first 5 d postcalving (n = 20). Liver was biopsied at d 7 precalving and d 7, 14, and 28 postcalving. Total PC and transcript specific gene expression was quantified using quantitative PCR and DNA methylation of promoter 1 was quantified using bisulfite Sanger sequencing. Expression of PC changed over the transition period, with increased expression postcalving occurring concurrently with increased circulating concentration of nonesterified fatty acids. The DNA methylation percentage was variable at all sites quantified and ranged from 21 to 54% across the 15 CpG dinucleotides within promoter 1. The DNA methylation at wk 1 postcalving, however, was not correlated with gene expression of promoter 1-regulated transcripts and we did not detect an effect of NSAID treatment on DNA methylation or PC gene expression. Our results do not support a role for DNA methylation in regulating promoter 1-driven gene expression of PC at wk 1 postcalving. Further research is required to determine the mechanisms regulating increased PC expression over the transition period.

An increase in intrauterine prostaglandin production is critical for the onset and progression of labor in women and indeed all mammalian species studied. Endocannabinoids can act as substrates for enzymes of the prostaglandin biosynthetic pathways and can be utilized to generate other related compounds such as prostamides. The end products are indistinguishable by radioimmunoassay. We have separated such compounds by mass spectrometry. We now show that inflammatory stimuli such as LPS and proinflammatory cytokines act differentially on these pathways in human choriodecidua and preferentially create drive through to prostaglandin end products. These findings create doubt about the interpretation of data on prostaglandin biosynthesis in intrauterine tissues from pregnant women especially in the presence of an infection. The possibility is raised that separation of these products might reduce variability in results and lead to potential uses for their measurement in the diagnosis of preterm labor.

Extensive metabolic and physiologic changes occur during the peripartum, concurrent with a high incidence of infectious disease. Immune dysfunction is a likely contributor to the increased risk of disease at this time. Studies using high-yielding, total mixed ration-fed cows have indicated that neutrophil function is perturbed over the transition period; however, this reported dysfunction has yet to be investigated in moderate-yielding, grazing dairy cows. Therefore, we investigated changes in the expression of genes involved in neutrophil function. Blood was collected from cows at 5 time points over the transition period: precalving (−1 wk; n = 46), day of calving (d 0; n = 46), and postcalving at wk 1 (n = 46), wk 2 (n = 45), and wk 4 (n = 43). Neutrophils were isolated by differential centrifugation and gene expression was investigated. Quantitative reverse transcriptase PCR with custom-designed primer pairs and Roche Universal Probe Library (Roche, Basel, Switzerland) chemistry, combined with microfluidics integrated fluidic circuit chips (96.96 Dynamic Array, San Francisco, CA) were used to investigate the expression of 78 genes involved in neutrophil function and 18 endogenous control genes. Statistical significance between time points was determined using a repeated measures ANOVA. Genes that were differentially expressed over the transition period included those involved in neutrophil adhesion (SELL, ITGB2, and ITGBX), mediation of the immune response (TLR4, HLA-DRA, and CXCR2), maturation, cell cycle progression, apoptosis (MCL1, BCL2, FASLG, and RIPK1), and control of gene expression (PPARG, PPARD, and STAT3). We noted reduced gene expression of proinflammatory cytokines (IFNG, TNF, IL12, and CCL2) on the day of calving, whereas anti-inflammatory cytokine gene expression (IL10) was upregulated. Increased gene expression of antimicrobial peptides (BNBD4, DEFB10, and DEFB1) occurred on the day of calving. Collectively, transcription profiles are indicativ

Cell-to-cell communication in physiological and pathological conditions may be influenced by neighboring cells, distant tissues, or local environmental factors. Exosomes are specific subsets of extracellular vesicles that internalize and deliver their content to near and distant sites. Exosomes may play a role in the maternal-embryo crosstalk vital for the recognition and maintenance of a pregnancy; however, their role in dairy cow reproduction has not been established. This study aimed to characterize the exosome profile in the plasma of 2 strains of dairy cow with divergent fertility phenotypes. Plasma was obtained and characterized on the basis of genetic ancestry as fertile (FERT; <23% North American genetics, New Zealand Holstein-Friesian strain, n = 8) or subfertile (SUBFERT; >92% North American genetics, North American Holstein-Friesian strain, n = 8). Exosomes were isolated by differential and buoyant density centrifugation and characterized by size distribution (nanoparticle tracking analysis, NanoSight NS500, NanoSight Ltd., Amesbury, UK), the presence of CD63 (Western blot), and their morphology (electron microscopy). The total number of exosomes was determined by quantifying the immunoreactive CD63 (ExoELISA kit, System Biosciences), and the protein content established by mass spectrometry. Enriched exosome fractions were identified as cup-shape vesicles with diameters around 100 nm and positive for the CD63 marker. The concentration of exosomes was 50% greater in FERT cows. Mass spectrometry identified 104 and 117 proteins in FERT and SUBFERT cows, of which 23 and 36 were unique, respectively. Gene ontology analysis revealed enrichment for proteins involved in immunomodulatory processes and cell-to-cell communication. Although the role of exosomes in dairy cow reproduction remains to be elucidated, their quantification and content in models with divergent fertility phenotypes could provide novel information to support both physiological and genetic a

Background: A transcriptomic approach was used to evaluate potential interactions between prepartum body condition score (BCS) and feeding management in the weeks before calving on hepatic metabolism during the periparturient period. Methods: Thirty-two mid-lactation grazing dairy cows of mixed age and breed were randomly allocated to one of four treatment groups in a 2×2 factorial arrangement: two prepartum BCS categories [4.0 (thin, BCS4) and 5.0 (optimal, BCS5); based on a 10-point scale], and two levels of energy intake during the 3weeks preceding calving (75 and 125% of estimated requirements). Liver samples were obtained at 7, 7, and 28 d relative to parturition and subsequent RNA was hybridized to the Agilent 44K Bovine (V2) Microarray chip. The Dynamic Impact Approach was used for pathway analysis, and Ingenuity Pathway Analysis was used for gene network analysis. Results: The greater number of differentially expressed genes in BCS4 cows in response to prepartum feed allowance (1071 vs 310, over the entire transition period) indicates that these animals were more responsive to prepartum nutrition management than optimally-conditioned cows. However, independent of prepartum BCS, pathway analysis revealed that prepartal feeding level had a marked effect on carbohydrate, amino acid, lipid, and glycan metabolism. Altered carbohydrate and amino acid metabolism suggest a greater and more prolonged negative energy balance postpartum in BCS5 cows overfed prepartum. This is supported by opposite effects of prepartum feeding in BCS4 compared with BCS5 cows in pathways encompassing amino acid, vitamin, and co-factor metabolism. The prepartum feed restriction ameliorates the metabolic adaptation to the onset of lactation in BCS5 cows, while detrimentally affecting BCS4 cows, which seem to better adapt when overfed. Alterations in the glycosaminoglycans synthesis pathway support this idea, indicating better hepatic health status in feed-restricted BCS5 and overfed BCS

Introduction Gestational diabetes mellitus (GDM) is characterised by maternal glucose intolerance and insulin resistance during pregnancy. Myostatin, initially identified as a negative regulator of muscle development may also function in the regulation of placental development and glucose uptake. Myostatin expression in placentae of GDM complicated pregnancies is unknown. However, higher myostatin levels occur in placentae of pregnancies complicated with preeclampsia. We hypothesise that myostatin will be differentially expressed in GDM complicated pregnancies.Methods Myostatin concentrations (ELISA) were evaluated in plasma of presymptomatic women who later developed GDM and compared to plasma of normal glucose tolerant (NGT) women. Furthermore, myostatin protein expression (Western blot) was studied in placentae of pregnant women with GDM (treated with diet or insulin) compared to placentae of NGT women.Results No significant difference in myostatin concentration was seen in plasma of pre-symptomatic GDM women compared to NGT women. In placenta significant differences in myostatin protein expressions (higher precursor; p < 0.05and lower dimer: p < 0.005) were observed in GDM complicated compared to NGT pregnancies. Furthermore, placentae of GDM women treated with insulin compared to diet have higher dimer (p < 0.005) and lower precursor (p < 0.05). Compared to lean women, placentae of obese NGT women were lower in myostatin dimer expression (p < 0.05).Discussion Myostatin expression in placental tissue is altered under stress conditions (e.g. obesity and abnormal glucose metabolism) found in pregnancies complicated with GDM. We hypothesise that myostatin is active in these placentae and could affect glucose homoeostasis and/or cytokine production thereby altering the function of the placenta.

Previous research results have indicated an increase in pregnancy rate in pasture-grazed cows treated with a nonsteroidal antiinflammatory drug (NSAID) 3 to 4. wk postcalving, when a high proportion of nucleated cells from within the uterus were polymorphonucleated; however, no effect on milk production was detected. It was hypothesized that this lack of effect on milk production was because the administration of the NSAID was too late after calving. The aims of the current study were to evaluate the timing of administering a propionic acid-derived NSAID (i.e., carprofen) on milk production, metabolic status, uterine health, and reproductive performance. Six-hundred and thirty-nine cows (134 primiparous and 505 multiparous) calving between July 4 and September 5, 2012, in 2 herds (herd 1: n. = 228; herd 2: n. = 411) were enrolled. Using a randomized block design, cows were allocated to 1 of 3 treatment groups as they calved: (1) no treatment (control; n. = 221), (2) NSAID administered on d 1, 3, and 5 postcalving (early; n. = 214), and (3) NSAID administered on d 19, 21, and 23 postcalving (late; n. = 204). Milk production and composition, and body condition were determined weekly. Blood was sampled at 4 time points (1 precalving and 3 postcalving) to determine the effects of treatment on indicators of metabolic health and energy status. Uterine health was determined by measuring the proportion of nucleated cells that were polymorphonucleated following cytobrush sampling of the uterus between d 13 to 24 and d 30 to 49 postcalving. Irrespective of timing of application, NSAID did not affect milk production, body weight, or body condition during early lactation. Treatment with an NSAID 19 to 23. d postcalving increased the proportion of cows submitted for breeding during the first 3. wk of the seasonal breeding program (control: 85%, early: 83%, and late: 92%), but did not affect conception or pregnancy rates. No detectable effect of treatment on uterine health or

The objective of this study was to determine if free AA concentrations in uterine luminal fluid (ULF) and plasma differed between dairy cow strains that differ phenotypically for fertility and to evaluate the effect of the presence of a conceptus on ULF AA concentrations. Uterine luminal fluid was obtained postmortem from cows characterized on the basis of genetic ancestry as fertile (n. = 11) or subfertile (n. = 11) strains. At slaughter, cows were at a similar stage of lactation (fertile, 85. ±. 1. d and subfertile, 87. ±. 1. d postpartum, respectively). Cows were slaughtered on either d 17 of the estrous cycle [nonpregnant (n. = 10): fertile n. = 5; subfertile n. = 5] or d 17 of pregnancy [10. d after embryo transfer, which was undertaken 7. d after estrus (n. = 12, pregnant): fertile n. = 6, subfertile n. = 6]. Uterine luminal fluid was collected from each uterine horn of the pregnant (gravid and nongravid horns) and nonpregnant (horn ipsilateral and contralateral to the corpus luteum) cows. Plasma harvested on the day of slaughter and ULF samples were analyzed for AA determination using HPLC. The main effects of genetic strain, reproductive status, and their interactions on ULF and plasma AA content were tested. Additionally, the effect of uterine horn on ULF AA was tested for the pregnant and nonpregnant cows. Reproductive status had the greatest effect on AA concentrations in ULF. The concentrations of Leu, Met, Phe, Val, 1-methyhistidine, Asp, essential, ketogenic, and branched-chain AA, and those AA classified as both glucogenic and ketogenic were greater in the ULF collected from pregnant cows, with taurine being lower. Additionally, we observed effects of uterine horn and genetic strain × uterine horn interaction for ULF AA concentrations. Concentrations of the essential AA plus Met and Phe were greater in the ULF from the gravid horn, irrespective of strain. The ULF from the gravid horn of fertile cows contained the greatest concentrations of nonessen

Here, we provide evidence for a functional role of artemin (ARTN) in progression of endometrial carcinoma (EC). Increased ARTN protein expression was observed in EC compared with normal endometrial tissue, and ARTN protein expression in EC was significantly associated with higher tumor grade and invasiveness. Forced expression of ARTN in EC cells significantly increased total cell number as a result of enhanced cell cycle progression and cell survival. In addition, forced expression of ARTN significantly enhanced anchorage-independent growth and invasiveness of EC cells. Moreover, forced expression of ARTN increased tumor size in xenograft models and produced highly proliferative, poorly differentiated, and invasive tumors. The ARTN-stimulated increases in oncogenicity and invasion were mediated by increased expression and activity of AKT1. Small interfering RNA-mediated depletion or antibody inhibition of ARTN significantly reduced oncogenicity and invasion of EC cells. Thus, inhibition of ARTN may be considered as a potential therapeutic strategy to retard progression of EC. (Endocrinology 151: 909-920, 2010)

It is accepted that both natural and synthetic endocrine disruptors are present in the food we eat, and that they are likely to have a pharmacological impact on the consumer. The magnitude of this impact is a cause of great controversy at present. Much of the debate has focused on the direct impact of endocrine disruptors on the consumer; this chapter speculates on possible impacts on the developing fetus that might lead to effects that manifest themselves much later in life.

Directed by microarray analyses, we report that autocrine human growth hormone (hGH) increased the mRNA and protein expression of DNA methyltransferase 1 (DNMT1), DNMT3A and DNMT3B in mammary carcinoma cells. Autocrine hGH stimulation of DNMT3A and DNMT3B expression was mediated by JAK2 and Src kinases, and treatment of mammary carcinoma cells with the DNMT inhibitor, 5'-aza-2'-deoxycytidine (AZA), abrogated autocrine hGH-stimulated cellular proliferation, apoptosis and anchorage-independent growth. AZA reversed the epitheliomesenchymal transition of mammary carcinoma cells induced by autocrine hGH, to an epithelioid morphology and abrogated cell migration stimulated by autocrine hGH. Autocrine hGH-stimulated hypermethylation of the first exon of the PLAKOGLOBIN gene and AZA abrogated the ability of autocrine hGH to repress plakoglobin gene transcription. Small interfering RNA (siRNA)-mediated depletion of the individual DNMT molecules did not release autocrine hGH repression of PLAKOGLOBIN promoter activity nor did individual DNMT depletion affect autocrine hGH-stimulated migration. However, concomitant siRNA-mediated depletion of both DNMT3A and DNMT3B abrogated hypermethylation of the PLAKOGLOBIN gene stimulated by autocrine hGH and subsequent repression of plakoglobin gene transcription and increased cell migration. Thus, the autocrine hGH-stimulated increases in DNMT3A and DNMT3B expression mediate repression of plakoglobin gene transcription by direct hypermethylation of its promoter and consequent phenotypic conversion of mammary carcinoma cells. Autocrine hGH, therefore, utilizes DNA methylation as a mechanism to exert its oncogenic effects in mammary carcinoma cells.

Trefoil factors (TFFs), in particular TFF1, are classical estrogen-regulated genes and have served as markers of estrogen gene regulation by various environmental estrogens. TFFs are, also regulated by several other factors including growth hormone (hGH), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF) and various oncogenic stimuli. TFFs are secreted proteins present in serum and possess the potential to act as growth factors promoting cell survival, anchorage-independent growth and motility. Recent compelling evidence has emerged from experimental and clinical studies to indicate a pivotal role of TFFs in oncogenic transformation, growth and metastatic extension of common human solid tumours. This review will summarize the current evidence for the involvement of TFFs in human cancer.