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Abstract Background Subclinical mastitis, the inflammation of the mammary gland lacking clinical symptoms, is one of the most prevalent and costly diseases in dairy farming worldwide. Milk microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EVs analysis regarding sampling time-point and natural infections. To estimate the reliability of EVs measurements in raw bovine milk, we first evaluated changes in EVs size and concentration using Tunable Resistive Pulse Sensing (TRPS) during three consecutive days of sampling. Then, we analysed daily differences in miRNA cargo using small RNA-seq. Finally, we compared milk EVs differences from naturally infected udder quarters with their healthy adjacent quarters and quarters from uninfected udders, respectively. Results We found that the milk EV miRNA cargo was very stable over the course of three days regardless of the health status of the quarter, and that infected quarters did not induce relevant changes in milk EVs of adjacent healthy quarters. Chronic subclinical mastitis induced changes in milk EV miRNA cargo, but neither in EVs size nor concentration. We observed that the changes in immunoregulatory miRNAs in quarters with chronic subclinical mastitis were cow-individual, however, the most upregulated miRNA was bta-miR-223-3p across all individuals. Conclusions Our results showed that the miRNA profile and particle size characteristics remained constant throughout consecutive days, suggesting that miRNAs packed in EVs are physiological state-specific. In addition, infected quarters were solely affected while adjacent healthy quarters remained unaffected. Finally, the cow-individual miRNA changes pointed towards infection-specific alterations.

Abstract Background The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts. Results In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14–3-3-mediated signaling, xenobiotic metabolism general signaling pathway, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts further were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts. Conclusions Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

Abstract Background During the preimplantation phase in the pig, the conceptus trophoblast elongates into a filamentous form and secretes estrogens, interleukin 1 beta 2, interferons, and other signaling molecules before attaching to the uterine epithelium. The processes in the uterine endometrium in response to conceptus signaling are complex. Thus, the objective of this study was to characterize transcriptome changes in porcine endometrium during the time of conceptus attachment considering the specific localization in different endometrial cell types. Results Low-input RNA-sequencing was conducted for the main endometrial compartments, luminal epithelium (LE), glandular epithelium (GE), blood vessels (BV), and stroma. Samples were isolated from endometria collected on Day 14 of pregnancy and the estrous cycle (each group n = 4) by laser capture microdissection. The expression of 12,000, 11,903, 11,094, and 11,933 genes was detectable in LE, GE, BV, and stroma, respectively. Differential expression analysis was performed between the pregnant and cyclic group for each cell type as well as for a corresponding dataset for complete endometrium tissue samples. The highest number of differentially expressed genes (DEGs) was found for LE (1410) compared to GE, BV, and stroma (800, 1216, and 384). For the complete tissue, 3262 DEGs were obtained. The DEGs were assigned to Gene Ontology (GO) terms to find overrepresented functional categories and pathways specific for the individual endometrial compartments. GO classification revealed that DEGs in LE were involved in ‘biosynthetic processes’, ‘related to ion transport’, and ‘apoptotic processes’, whereas ‘cell migration’, ‘cell growth’, ‘signaling’, and ‘metabolic/biosynthetic processes’ categories were enriched for GE. For blood vessels, categories such as ‘focal adhesion’, ‘actin cytoskeleton’, ‘cell junction’, ‘cell differentiation and development’ were found as overrepresented, while for stromal samples, most DEGs were assigned to ‘extracellular matrix’, ‘gap junction’, and ‘ER to Golgi vesicles’. Conclusions The localization of differential gene expression to different endometrial cell types provided a significantly improved view on the regulation of biological processes involved in conceptus implantation, such as the control of uterine fluid secretion, trophoblast attachment, growth regulation by Wnt signaling and other signaling pathways, as well as the modulation of the maternal immune system.

Abstract Background Porcine embryos undergo rapid differentiation and expansion between Days 8 and 12 before attaching to the maternal uterine epithelial surface after Day 13. It is known that maternal recognition of pregnancy and successful implantation are driven by mutual interactions between the elongated conceptus and the maternal endometrium. While most of the genes involved in regulation of embryo development are located on autosomal chromosomes, gene expression on sex chromosomes is modulating development through sex-specific transcription. To gain more insights into the dynamic transcriptome of preimplantation embryos at the onset of elongation and into X-linked gene expression, RNA-seq analyses were performed for single female and male porcine embryos collected on Days 8, 10, and 12 of pregnancy. Results A high number of genes were differentially expressed across the developmental stages (2174 and 3275 for Days 8 vs 10, and 10 vs 12, respectively). The majority of differentially expressed genes (DEGs) were involved in embryo elongation, development, and embryo-maternal interaction. Interestingly, a number of DEGs was found with respect to embryo sex (137, 37, and 56 on Days 8, 10 and 12, respectively). At Day 8, most of these DEGs were X-linked (96). Strikingly, the number of DEGs encoded on the X chromosome dramatically decreased from Day 10 to Day 12. Conclusions The obtained results deepen the understanding about temporary transcriptomic changes in porcine embryos during the phase of conceptus elongation, meanwhile reveal dynamic compensation of X chromosome in the female and distinct transcriptional differences between female and male embryos.

Abstract Background The expression of microRNAs (miRNAs) is essential for the proper development of the mammalian embryo. A maternal exposure to endocrine disrupting chemicals during preimplantation bears the potential for transgenerational inheritance of disease through the epigenetic perturbation of the developing embryo. A comprehensive assembly of embryo-specific miRNAs and respective isoforms (isomiR) is lacking to date. We aimed at revealing the sex-specific miRNA expression profile of single porcine blastocysts developing in gilts orally exposed to exogenous estradiol-17 β (E2). Therefore we analyzed the miRNA profile specifically focusing on isomiRs and potentially embryo-specific miRNAs. Results Deep sequencing of small RNA (small RNA-seq) result in the detection of miRNA sequences mapping to known and predicted porcine miRNAs as well as novel miRNAs highly conserved in human and cattle. A set of highly abundant miRNAs and a large number of rarely expressed miRNAs were identified by using a small RNA analysis pipeline, which was integrated into a novel Galaxy workflow specifically benefits incompletely annotated species. In particular, orthologue species information increased the total number of annotated miRNAs, while mapping to other non-coding RNAs avoided falsely annotated miRNAs. Neither the low nor the high dose of E2 treatment (10 and 1000 µE2/kg body weight daily, respectively) affected the miRNA profile in blastocysts despite the distinct differential mRNA expression and DNA methylation found in previous studies. The high number of generated sequence reads enabled a comprehensive analysis of the isomiR repertoire showing various templated and non-templated modifications. Furthermore, potentially blastocyst-specific miRNAs were identified. Conclusions In pre-implantation embryos, numerous distinct isomiRs were discovered indicating a high complexity of miRNA expression. Neither the sex of the embryo nor a maternal E2 exposure affected the miRNA expression profile of developing porcine blastocysts. The adaptation to the continuous duration of the E2 treatment might explain the lack of an effect.

Abstract Background Along with trophoblast elongation (Days 10 to 12), estradiol is secreted in increasing amounts for recognition of pregnancy. Endometrial secretions driven by ovarian progesterone and conceptus signals are essential for conceptus growth and development. Results of transcriptome analyses of whole endometrial tissue samples in the pig indicated the need for cell type-specific endometrial gene expression analysis for a better understanding of transcriptome changes associated with establishment of pregnancy. Results The most distinct transcriptome profile and the majority of differentially expressed genes (DEGs) were identified in luminal epithelium (LE). Many DEGs were found only in the cell type-specific analysis. The functional classification of DEGs identified in specific endometrial cell types revealed various distinct functions and pathways. Genes related to immune activation, estrogen signaling pathway, embryo development, and cell proliferation were upregulated in LE of pregnant gilts. Genes involved in sterol biosynthetic and metabolic processes and extracellular matrix were upregulated in stroma. Genes associated with cell communication such as via exosomes and vesicles were found as differential in LE, glandular epithelium (GE), and stroma (S). Conclusions This study revealed that conceptus signals induce different transcriptomic regulations in the endometrial compartments/cell types related to their specific function during recognition and establishment of pregnancy.

Abstract We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array™ on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.