Assisted reproductive technology (ART) has led to the birth of millions of babies. In cattle, thousands of embryos are produced annually. However, since the introduction and widespread use of ART, negative effects on embryos and offspring are starting to emerge. Knowledge so far, mostly provided by animal models, indicates that suboptimal conditions during ART can affect embryo viability and quality, and may induce embryonic stress responses. These stress responses take the form of severe gene expression alterations or modifications in critical epigenetic marks established during early developmental stages that can persist after birth. Unfortunately, while developmental plasticity allows the embryo to survive these stressful conditions, such insult may lead to adult health problems and to long-term effects on offspring that could be transmitted to subsequent generations. In this review, we describe how in mice, livestock, and humans, besides affecting the development of the embryo itself, ART stressors may also have significant repercussions on offspring health and physiology. Finally, we argue the case that better control of stressors during ART will help improve embryo quality and offspring health. K E Y W O R D S assisted reproductive technologies, bovine, embryo, human, mouse, stress

BackgroundSince the development of in vitro embryo production in cattle, different supplements have been added to culture media to support embryo development, with serum being the most popular. However, the addition of serum during embryo culture can induce high birthweights and low viability in calves (Large Offspring Syndrome). Analysis of global gene expression in bovine embryos produced under different conditions can provide valuable information to optimize culture media for in vitro embryo production.ResultsWe used RNA sequencing to examine the effect of in vitro embryo production, in either serum-containing or serum-free media, on the global gene expression pattern of individual bovine blastocysts. Compared to in vivo derived embryos, embryos produced in serum-containing medium had five times more differentially expressed genes than embryos produced in serum-free conditions (1109 vs. 207). Importantly, in vitro production in the presence of serum appeared to have a different impact on the embryos according to their sex, with male embryos having three times more genes differentially expressed than their female counterparts (1283 vs. 456). On the contrary, male and female embryos produced in serum-free conditions showed the same number (191 vs. 192) of genes expressed differentially; however, only 44 of those genes were common in both comparisons. The pathways affected by in vitro production differed depending on the type of supplementation. For example, embryos produced in serum-containing conditions had a lower expression of genes related to metabolism while embryos produced in serum-free conditions showed aberrations in genes involved in lipid metabolism.ConclusionsSerum supplementation had a major impact on the gene expression pattern of embryos, with male embryos being the most affected. The transcriptome of embryos produced in serum-free conditions showed a greater resemblance to that of in vivo derived embryos, although genes involved in lipid metabolism were altered. Male embryos appeared to be most affected by suboptimal in vitro culture, i.e. in the presence of serum.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2393-z) contains supplementary material, which is available to authorized users.

Individual culture of bovine embryos is usually associated with low blastocyst development. However, during preliminary experiments in our laboratory we observed high blastocyst development after individual embryo culture in a serum-free culture system. We therefore hypothesised that serum has a negative effect on embryos cultured individually whereas embryos in groups can counteract this. First, we determined whether the timing of removal of serum (during maturation or culture) had an influence on individual embryo development. The results clearly showed that removal of serum during embryo culture was the main contributing factor since high blastocyst development was observed after individual culture in synthetic oviductal fluid supplemented with bovine serum albumin (BSA) and insulin, transferrin and selenium (ITS), independent of the maturation medium. Second, we investigated whether an individual factor of the ITS supplement was essential for individual embryo development. We demonstrated that repeatable high blastocyst percentages were due to the synergistic effect of ITS. Finally, we investigated if a group-culture effect can still be observed under serum-free conditions. Group culture generated blastocysts with higher total cell numbers and less apoptosis. These data show that individual culture in serum-free conditions leads to high blastocyst development, but group culture still improves blastocyst quality.

In contrast to various other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. In particular, stallion spermatozoa fails to penetrate the zona pellucida, most likely due to incomplete activation of stallion spermatozoa (capacitation) under in vitro conditions. In other mammalian species, specific capacitation triggers have been described; unfortunately, none of these is able to induce full capacitation in stallion spermatozoa. Nevertheless, knowledge of capacitation pathways and their molecular triggers might improve our understanding of capacitation-related events observed in stallion sperm. When sperm cells are exposed to appropriate capacitation triggers, several molecular and biochemical changes should be induced in the sperm plasma membrane and cytoplasm. At the level of the sperm plasma membrane, (1) an increase in membrane fluidity, (2) cholesterol depletion and (3) lipid raft aggregation should occur consecutively; the cytoplasmic changes consist of protein tyrosine phosphorylation and elevated pH, cAMP and Ca 2+ concentrations. These capacitation-related events enable the switch from progressive to hyperactivated motility of the sperm cells, and the induction of the acrosome reaction. These final capacitation triggers are indispensable for sperm cells to migrate through the viscous oviductal environment, penetrate the cumulus cells and zona pellucida and, finally, fuse with the oolemma. This review will focus on molecular aspects of sperm capacitation and known triggers in various mammalian species. Similarities and differences with the horse will be highlighted to improve our understanding of equine sperm capacitation/fertilizing events.Reproduction (2019) 157 R181-R197

Coincubating equine gametes in the presence of procaine has been reported to facilitate in vitro fertilization, with cleavage rates exceeding 60%. We report that while procaine does trigger sperm hyperactivation, it independently induces cleavage of equine oocytes. First, we found that procaine (1-5 mM) did not facilitate stallion sperm penetration of equine oocytes but instead induced sperm-independent oocyte cytokinesis in the absence of the second polar body extrusion. Indeed, 56 ± 4% of oocytes cleaved within 2.5 days of exposure to 2.5 mM procaine regardless of sperm presence. However, the cleaved oocytes did not develop beyond 8 to 16 cells, and the daughter cells either lacked nuclei or contained aberrant, condensed DNA fragments. By contrast, intracytoplasmic sperm injection (ICSI) was followed by second polar body extrusion and formation of normal blastocysts. Moreover, neither the calcium oscillations detectable using fura-2 AM staining nor the cortical granule reaction visualized by LCA-FITC staining, after oocyte activation induced by ICSI or ionomycin treatment, were detected after exposing oocytes to 2.5 mM procaine. Instead, procaine initiated an ooplasmic alkalinization, detectable by BCECF-AM staining that was not observed after other treatments. This alkalinization was followed, after an additional 18 h of incubation, by cortical F-actin depolymerization, as demonstrated by reduced actin phalloidin-FITC staining intensity, that resembled preparation for cytokinesis in ICSI-fertilized zygotes. Overall, we conclude that procaine induces cytokinesis in equine oocytes accompanied by aberrant chromatin condensation and division; this explains why embryos produced after exposing equine oocytes to procaine fail to develop beyond the 8- to 16-cell stage.

In the absence of the maternal genital tract, preimplantation embryos can develop in vitro in culture medium where all communication with the oviduct or uterus is absent. In several mammalian species, it has been observed that embryos cultured in groups thrive better than those cultured singly. Here we argue that group-cultured embryos are able to promote their own development in vitro by the production of autocrine embryotropins that putatively serve as a communication tool. The concept of effective communication implies an origin, a signalling agent, and finally a recipient that is able to decode the message. We illustrate this concept by demonstrating that preimplantation embryos are able to secrete autocrine factors in several ways, including active secretion, passive outflow, or as messengers bound to a molecular vehicle or transported within extracellular vesicles. Likewise, we broaden the traditional view that inter-embryo communication is dictated mainly by growth factors, by discussing a wide range of other biochemical messengers including proteins, lipids, neurotransmitters, saccharides, and microRNAs, all of which can be exchanged among embryos cultured in a group. Finally, we describe how different classes of messenger molecules are decoded by the embryo and influence embryo development by triggering different pathways. When autocrine embryotropins such as insulin-like growth factor-I (IGF-I) or platelet activating factor (PAF) bind to their appropriate receptor, the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathway will be activated which is important for embryo survival. On the other hand, the mitogen-activated protein kinase (MAPK) pathway is activated when compounds such as hyaluronic acid and serotonin bind to their respective receptors, thereby acting as growth factors. By activating the peroxisome-proliferator-activated receptor family (PPAR) pathway, lipophilic autocrine factors such as prostaglandins or fatty acids have both survival and anti-apoptotic functions. In conclusion, considering different types of messenger molecules simultaneously will be crucial to understanding more comprehensively how embryos communicate with each other in group-culture systems. This approach will assist in the development of novel media for single-embryo culture.

Problems caused by sulfur oxide emissions to the atmosphere and restrictive legal limitations to sulfur content in fuels have made biodesulfurization (BDS) to be proposed as a complementary technology to hydrodesulfurization (HDS). In this work, a genetically modified microorganism, Pseudomonas putida CECT 5279, is employed as a desulfurizing biocatalyst. This microorganism has the ability of carrying out the metabolic 4S pathway from Rhodococcus erythropolis IGTS8, to desulfurize dibenzothiophene (DBT), as a model compound. In previous works (

BackgroundGlobal epigenetic reprogramming is considered to be essential during embryo development to establish totipotency. In the classic model first described in the mouse, the genome-wide DNA demethylation is asymmetric between the paternal and the maternal genome. The paternal genome undergoes ten-eleven translocation (TET)-mediated active DNA demethylation, which is completed before the end of the first cell cycle. Since TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, the latter is postulated to be an intermediate stage toward DNA demethylation. The maternal genome, on the other hand, is protected from active demethylation and undergoes replication-dependent DNA demethylation. However, several species do not show the asymmetric DNA demethylation process described in this classic model, since 5-methylcytosine and 5-hydroxymethylcytosine are present during the first cell cycle in both parental genomes. In this study, global changes in the levels of 5-methylcytosine and 5-hydroxymethylcytosine throughout pronuclear development in equine zygotes produced in vitro were assessed using immunofluorescent staining.ResultsWe were able to show that 5-methylcytosine and 5-hydroxymethylcytosine both were explicitly present throughout pronuclear development, with similar intensity levels in both parental genomes, in equine zygotes produced by ICSI. The localization patterns of 5-methylcytosine and 5-hydroxymethylcytosine, however, were different, with 5-hydroxymethylcytosine homogeneously distributed in the DNA, while 5-methylcytosine tended to be clustered in certain regions. Fluorescence quantification showed increased 5-methylcytosine levels in the maternal genome from PN1 to PN2, while no differences were found in PN3 and PN4. No differences were observed in the paternal genome. Normalized levels of 5-hydroxymethylcytosine were preserved throughout all pronuclear stages in both parental genomes.ConclusionsIn conclusion, the horse does not seem to follow the classic model of asymmetric demethylation as no evidence of global DNA demethylation of the paternal pronucleus during the first cell cycle was demonstrated. Instead, both parental genomes displayed sustained and similar levels of methylation and hydroxymethylation throughout pronuclear development.