The use of multiple omics techniques (i.e., genomics, transcriptomics, proteomics, and metabolomics) is becoming increasingly popular in all facets of life science. Omics techniques provide a more holistic molecular perspective of studied biological systems compared to traditional approaches. However, due to their inherent data differences, integrating multiple omics platforms remains an ongoing challenge for many researchers. As metabolites represent the downstream products of multiple interactions between genes, transcripts, and proteins, metabolomics, the tools and approaches routinely used in this field could assist with the integration of these complex multi-omics data sets. The question is, how? Here we provide some answers (in terms of methods, software tools and databases) along with a variety of recommendations and a list of continuing challenges as identified during a peer session on multi-omics integration that was held at the recent ‘Australian and New Zealand Metabolomics Conference’ (ANZMET 2018) in Auckland, New Zealand (Sept. 2018). We envisage that this document will serve as a guide to metabolomics researchers and other members of the community wishing to perform multi-omics studies. We also believe that these ideas may allow the full promise of integrated multi-omics research and, ultimately, of systems biology to be realized.

The mammary gland is a dynamic organ that undergoes dramatic physiological adaptations during the transition from late pregnancy to lactation. Investigation of the molecular basis of mammary development and function will provide fundamental insights into tissue remodelling as well as a better understanding of milk production and mammary disease. This is important to livestock production systems and human health. Here we use RNA-seq to identify differences in gene expression in the ovine mammary gland between late pregnancy and lactation. Between late pregnancy (135 days of gestation ± 2.4 SD) and lactation (15 days post partum ± 1.27 SD) 13 % of genes in the sheep genome were differentially expressed in the ovine mammary gland. In late pregnancy, cell proliferation, beta-oxidation of fatty acids and translation were identified as key biological processes. During lactation, high levels of milk fat synthesis were mirrored by enrichment of genes associated with fatty acid biosynthesis, transport and lipogenesis. Protein processing in the endoplasmic reticulum was enriched during lactation, likely in support of active milk protein synthesis. Hormone and growth factor signalling and activation of signal transduction pathways, including the JAK-STAT and PPAR pathways, were also differently regulated, indicating key roles for these pathways in functional development of the ovine mammary gland. Changes in the expression of epigenetic regulators, particularly chromatin remodellers, indicate a possible role in coordinating the large-scale transcriptional changes that appear to be required to switch mammary processes from growth and development during late pregnancy to synthesis and secretion of milk during lactation. Coordinated transcriptional regulation of large numbers of genes is required to switch between mammary tissue establishment during late pregnancy, and activation and maintenance of milk production during lactation. Our findings indicate the remarkable plasticity of the mammary gland, and the coordinated regulation of multiple genes and pathways to begin milk production. Genes and pathways identified by the present study may be important for managing milk production and mammary development, and may inform studies of diseases affecting the mammary gland.

The mammary gland is a complex tissue consisting of multiple cell types which, over the lifetime of an animal, go through repeated cycles of development associated with pregnancy, lactation and involution. The mammary gland is also known to be sensitive to maternal programming by environmental stimuli such as nutrition. The molecular basis of these adaptations is of significant interest, but requires robust methods to measure gene expression. Reverse-transcription quantitative PCR (RT-qPCR) is commonly used to measure gene expression, and is currently the method of choice for validating genome-wide expression studies. RT-qPCR requires the selection of reference genes that are stably expressed over physiological states and treatments. In this study we identify suitable reference genes to normalize RT-qPCR data for the ovine mammary gland in two physiological states; late pregnancy and lactation. Biopsies were collected from offspring of ewes that had been subjected to different nutritional paradigms during pregnancy to examine effects of maternal programming on the mammary gland of the offspring. We evaluated eight candidate reference genes and found that two reference genes ( PRPF3 and CUL1) are required for normalising RT-qPCR data from pooled RNA samples, but five reference genes are required for analyzing gene expression in individual animals ( SENP2, EIF6, MRPL39, ATP1A1, CUL1). Using these stable reference genes, we showed that TET1, a key regulator of DNA methylation, is responsive to maternal programming and physiological state. The identification of these novel reference genes will be of utility to future studies of gene expression in the ovine mammary gland.

Studies using sheep models indicate that the fetal mammary gland is sensitive to maternal nutrition during gestation; however, results have been inconsistent and do not identify critical feeding periods. This study aimed to clarify previous findings by partitioning the period of maternal nutritional manipulation into 2 stages: early and mid-to-late pregnancy. Sixty-six twin-born, twin-bearing ewes, born to dams that were fed either submaintenance, maintenance, or ad libitum during early pregnancy (d 21 to 50 of pregnancy; SmP21–50, MP21–50, or AdP21–50, respectively) and then either maintenance or ad libitum during mid-to-late pregnancy (d 50 to 140 of pregnancy; MP50–140 or AdP50–140, respectively) were milked once a week, starting from d 7 ± 1 postpartum, for 7 subsequent weeks to enable estimation of daily milk yield and composition. Their lambs were weighed weekly. Ewes born to dams fed MP21–50 tended to have greater accumulated milk (P = 0.10), fat (P = 0.07), and NE (P = 0.06) yields over 50 d compared with ewes born to dams fed SmP21–50 and AdP21–50. In contrast, ewes born to dams fed AdP50–140 tended to have greater accumulated milk (P = 0.10) and lactose (P = 0.09) yields compared with ewes born to dams fed MP50–140. Grandoffspring birth weights were unaffected by granddam nutrition during pregnancy. Ewes born to dams fed AdP21–50 weaned lighter lambs (P = 0.05) than ewes born to dams fed AmP21–50 and tended to wean lighter lambs (P = 0.07) than ewes born to dams fed MP21–50 whereas there were no differences between the weaning weights of lambs (P = 0.43) from ewes born to dams fed AdP50–140 and MP50–140. Maintenance nutrition of dams during early pregnancy appears to be associated with an improved lactation performance of ewe offspring. Higher levels of nutrition during mid-to-late pregnancy also appears to improve the first-lactation performance of ewe offspring. Interestingly, although grandoffspring birth weights were unaffected, weaning weight appears to be influenced by granddam early pregnancy nutrition in a manner discordant with the lactational performance of their dam. Results from this study indicate that dam nutrition during early pregnancy can influence the lactational performance of ewe offspring and the BW at weaning of their grandoffspring, which may ultimately affect farmer profits. This highlights the importance of nutritional management of breeding ewes during this period. Copyright © 2013. American Society of Animal Science .

Understanding the cumulative risk of chemical mixtures at environmentally realistic concentrations is a key challenge in honey bee ecotoxicology. Ecotoxicogenomics, including transcriptomics, measures responses in individual organisms at the molecular level which can provide insights into the mechanisms underlying phenotypic responses induced by one or more stressors and link impacts on individuals to populations. Here, fifth instar honey bee larvae were sampled from a previously reported field experiment exploring the phenotypic impacts of environmentally realistic chronic exposures of the pesticide imidacloprid (5 μg.kg^-1 for six weeks) and the acaricide thymol (250 g.kg^-1 applied via Apiguard gel in-hive for four weeks), both separately and in combination. RNA-seq was used to discover individual and interactive chemical effects on larval gene expression and to uncover molecular mechanisms linked to reported adult and colony phenotypes. The separate and combined treatments had distinct gene expression profiles which represented differentially affected signaling and metabolic pathways. The molecular signature of the mixture was characterised by additive interactions in canonical stress responses associated with oxidative stress and detoxification, and non-additive interactions in secondary responses including developmental, neurological, and immune pathways. Novel emergent impacts on eye development genes correlated with long-term defects in visual learning performance as adults. This is consistent with these chemicals working through independent modes of action that combine to impact common downstream pathways, and highlights the importance of establishing mechanistic links between molecular and phenotypic responses when predicting effects of chemical mixtures on ecologically relevant population outcomes.

The foetal mammary gland is sensitive to maternal weight and nutrition during gestation, which could affect offspring milk production. It has previously been shown that ewes born to dams offered maintenance nutrition during pregnancy (day 21 to 140 of gestation) produced greater milk, lactose and CP yields in their first lactation when compared with ewes born to dams offered ad libitum nutrition. In addition, ewes born to heavier dams produced greater milk and lactose yields when compared with ewes born to lighter dams. The objective of this study was to analyse and compare the 5-year lactation performance of the previously mentioned ewes, born to heavy or light dams that were offered maintenance or ad libitum pregnancy nutrition. Ewes were milked once per week, for the first 6 weeks of their lactation, for 5 years. Using milk yield and composition data, accumulated yields were calculated over a 42-day period for each year for milk, milk fat, CP, true protein, casein and lactose using a Legendre orthogonal polynomial model. Over the 5-year period, ewes born to heavy dams produced greater average milk (P=0.04), lactose (P=0.01) and CP (P=0.04) yields than offspring born to light dams. In contrast, over the 5-year period dam nutrition during pregnancy did not affect average (P>0.05) offspring milk yields or composition, but did increase milk and lactose accumulated yield (P=0.03 and 0.01, respectively) in the first lactation. These results indicate that maternal gestational nutrition appears to only affect the first lactational performance of ewe offspring. Neither dam nutrition nor size affected grand-offspring live weight gain to, or live weight at weaning (P>0.05). Combined these data indicate that under the conditions of the present study, manipulating dam weight or nutrition in pregnancy can have some effects of offspring lactational performance, however, these effects are not large enough to alter grand-offspring growth to weaning. Therefore, such manipulations are not a viable management tool for farmers to influence lamb growth to weaning.