Metabolomic evolution of the postpartum dairy cow uterus.

High rates of early pregnancy loss are a critical issue in dairy herds, particularly in seasonal, grazing systems. Components of the uterine luminal fluid (ULF), on which the early embryo depends for sustenance and growth, partly determine early pregnancy losses. Here, changes in ULF from early to mid-postpartum in crossbred dairy cows were explored, linking them with divergent embryo development. For this, the uteri of 87 cows at Day 7 of pregnancy at first and third estrus postpartum were flushed to collect ULF. Eighteen metabolites (chiefly organic acids and sugars) significantly varied in abundance across postpartum, indicating a molecular signature of physiological recovery consistent of the upregulation of pyrimidine metabolism and glycerophospholipid metabolism, and downregulation of pentose phosphate and taurine metabolism pathways. Joint pathway analysis of metabolomics data and a previously generated proteomics data set on the same ULF samples suggests key links between postpartum recovery and subsequent successful embryo development. These include upregulation of VEGFA and downregulation of metabolism, NRF2, T-cell receptor, which appear to improve the ULF's capacity of sustaining normal embryo development, and a putative osmo-protectant role of beta-alanine. These relationships should be further investigated to develop tools to detect and reduce early pregnancy loss in dairy cows.

Protein modifications due to homogenisation and heat treatment of cow milk.

This research paper aimed to locate protein modifications caused by treatment of milk and determine if the modification locations were consistent. The majority of milk for consumption is homogenised using pressure and heat, and this causes changes in the location of proteins in the milk as well as protein modifications. To investigate these proteomic changes, raw milk was pasteurised (72°C, 15 s), then, to separate the treatment for homogenisation, heated at these different pressures and temperatures: 45°C without no pressure applied, 45°C with 35 MPa, 80°C without pressure applied and 80°C, with 35 MPa. Proteomic analysis was done after separating the milk into three fractions: whey, casein and cream. Protein modifications in each fraction were examined and we found Maillard products as well as oxidation to be of interest. The proteins were also further identified and characterised to compare protein modification sites and differences in proteins present in the cream resulting from homogenisation and/or pasteurisation. This experiment showed that both heat and pressure during homogenisation can cause increases in protein modifications as a result of oxidation or the Maillard reaction. Total cysteine oxidation and total proline oxidation differed between treatments although this was only significantly different for cysteine. It was observed that protein modifications occurred in the same location in the protein sequence rather than in random locations which we highlighted by examining α-S1-casein, lactadherin and ß-lactoglobulin.

Proteomic determinants of uterine receptivity for pregnancy in early and mid-postpartum dairy cows†.

Dairy cow subfertility is a worldwide issue arising from multiple factors. It manifests in >30% early pregnancy losses in seasonal pasture-grazed herds, especially when cows are inseminated in the early post-partum period. Most losses occur before implantation, when embryo growth depends on factors present in maternal tract fluids. Here we examined the proteomic composition of early and mid-postpartum uterine luminal fluid (ULF) in crossbred lactating dairy cows to identify molecular determinants of fertility. We also explored changes in ULF from first to third estrus cycles postpartum in individual cows, linking those changes with divergent embryo development. For this, we flushed uteri of 87 cows at Day 7 of pregnancy at first and third estrus postpartum, recovering, and grading their embryos. Out of 1563 proteins detected, 472 had not been previously reported in this fluid, and 408 were predicted to be actively secreted by bioinformatic analysis. The abundance of 18 proteins with roles in immune regulation and metabolic function (e.g. cystatin B, pyruvate kinase M2) was associated with contrasting embryo quality. Matched-paired pathway analysis indicated that, from first to third estrus postpartum, upregulation of metabolic (e.g. creatine and carbohydrate) and immune (e.g. complement regulation, antiviral defense) processes were related to poorer quality embryos in the third estrus cycle postpartum. Conversely, upregulated signal transduction and protein trafficking appeared related to improved embryo quality in third estrus. These results advance the characterization of the molecular environment of bovine ULF and may aid understanding fertility issues in other mammals, including humans.

Proteomics and metabolomics in cow fertility: a systematic review.

Cow subfertility is a multi-factorial problem in many countries which is only starting to be unravelled. Molecular biology can provide a substantial source of insight into its causes and potential solutions, particularly through large scale, untargeted omics approaches. In this systematic review, we set out to compile, assess and integrate the latest proteomic and metabolomic research on cow reproduction, specifically that on the female reproductive tract and early embryo. We herein report a general improvement in technical standards throughout the temporal span examined; however, significant methodological limitations are also identified. We propose easily actionable avenues for ameliorating these shortcomings and enhancing the reach of this field. Text mining and pathway analysis corroborate the relevance of proteins and metabolites related to the triad oxidative stress-inflammation-disease on reproductive function. We envisage a breakthrough in cattle reproductive molecular research within the next few years as in vivo sample techniques are improved, omics analysis equipment becomes more affordable and widespread, and software tools for single- and multi-omics data processing are further developed. Additional investigation of the impact of local oxidative stress and inflammation on fertility, both at the local and systemic levels, is key towards realising the full potential of this field.

Differences in peptide generation following in vitro gastrointestinal digestion of yogurt and milk from cow, sheep and goat.

Fermentation of milk is commonly used throughout the world to produce a variety of foods with different health benefits. We hypothesised that due to differences in physicochemical properties and protein sequences among milk from different species and their fermented yogurt samples, their protein digestion and resulting peptide profiles would differ. Cow, goat and sheep milk and yogurt were compared at designated timepoints throughout in vitro gastric and intestinal digestion for differences in peptide profiles and peptide bioactivities. The results showed that most proteins in all milk and yogurt samples were digested within the early phase of gastric digestion. ß-Lg and ß-CN were digested faster in yogurt than milk, which was most evident for sheep products. Regardless of species, in vitro gastric and intestinal digestion released a higher concentration of specific peptides, particularly anti-hypertensives, from yogurt compared with their milk counterparts.

Analysis of Human Faecal Host Proteins: Responsiveness to 10-Week Dietary Intervention Modifying Dietary Protein Intake in Elderly Males.

Faecal proteomics targeting biomarkers of immunity and inflammation have demonstrated clinical application for the identification of changes in gastrointestinal function. However, there are limited comprehensive analyses of the host faecal proteome and how it may be influenced by dietary factors. To examine this, the Homo sapiens post-diet proteome of older males was analysed at the completion of a 10-week dietary intervention, either meeting the minimum dietary protein recommendations (RDA; n = 9) or twice the recommended dietary allowance (2RDA, n = 10). The host faecal proteome differed markedly between individuals, with only a small subset of proteins present in ≥ 60% of subjects (14 and 44 proteins, RDA and 2RDA, respectively, with only 7 common to both groups). No differences were observed between the diet groups on the profiles of host faecal proteins. Faecal proteins were detected from a wide range of protein classes, with high inter-individual variation and absence of obvious impact in response to diets with markedly different protein intake. This suggests that well-matched whole food diets with two-fold variation in protein intake maintained for 10 weeks have minimal impact on human faecal host proteins.

Evidence for the antagonistic form of CXC-motif chemokine CXCL10 in serous epithelial ovarian tumours.

Patients with high-grade, serous epithelial ovarian carcinoma (HGSOC) are generally diagnosed with extensive peritoneal metastases, and exhibit 5-year survival rates <30%. A subset of these tumours, defined as "immunoreactive," overexpress mRNA encoding the T-cell-recruiting chemokine CXCL10 (10-kDa interferon gamma-induced protein; C-X-C motif chemokine 10). Tumour-infiltrating CD4(+) CD8(+) T-cells are a well-documented, positive prognostic indicator for HGSOC patients; paradoxically, however, patients diagnosed with HGSOC (overexpressing CXCL10 and therefore theorised to recruit T-cells) typically exhibit poor survival. Recently, an "antagonistic" CXCL10 variant was identified that inhibited leucocyte recruitment to inflamed liver in vivo (Casrouge et al., J Clin Invest 2011;121:308-17). We hypothesised that "immunoreactive" HGSOC might also express antagonistic CXCL10, interfering with leucocyte recruitment and contributing to poor patient prognosis. CXCL10 expression was analysed in HGSOC tissues grouped according to pathology, grade and FIGO stage at diagnosis, and its localisation and association with T-cells established by immunohistochemical staining in tissue microarrays. CXCL10 expression was increased in a subset of serous epithelial tumour samples; however, it did not correlate well with CD45-positive tumour infiltrate. Immunoprecipitation and de novo sequence analysis of CXCL10 identified the N-terminally cleaved, "antagonistic" variant of CXCL10 specifically in malignant tumours, and not in benign ovarian disease. The data demonstrate the presence of the antagonistic form of CXCL10 in HGSOC for the first time, and provide a partial explanation for reduced leucocyte infiltration observed in these tumours. We suggest that CXCL10 cleavage and subsequent antagonism of immune cell recruitment may be a feature of the "immunoreactive" HGSOC subtype, leading to early impairment of the immune response and subsequently worsening patient prognosis.

Varietal characterization of hop (Humulus lupulus L.) by GC-MS analysis of hop cone extracts.

An approach is described for use in the varietal characterization of hop (Humulus lupulus L.) varieties. The study focuses on commercial hop varieties and was timed to coincide with the 2008 commercial hop harvest in Tasmania, Australia. Analysis of hop extracts was performed using GC-MS. A 60 m capillary column was employed to increase efficiency to permit the use of a quadrupole mass spectrometer in place of a time of flight mass spectrometer that is more commonly used for this type of analysis. A set of characterization functions were derived from discriminant analysis which were highly suitable for varietal characterization of the eight commercial varieties included in the study, namely Willamette, Victoria, Pride of Ringwood, Cascade, Southern Hallertau, Millennium, Southern Saaz, and Super Pride.