Intact Embryos Research Articles

Enhancing protein signal detection in asexual and viviparous pea aphids: A guided protocol for tissue dissection and proteinase K treatment

Aphids, as hemipteran insects, reproduce via parthenogenesis and viviparity, resulting in rapid and exponential offspring production. To investigate the molecular mechanisms underlying parthenogenetic viviparity in asexual aphids, precise protein detection through immunostaining is essential. Our previous research demonstrated the need for proteinase K (PK) treatment to improve tissue permeability, enabling antibodies targeting the germ-cell marker Ap-Vas1 to access gastrulating and later-stage embryos. However, optimal PK digestion protocols have not been thoroughly explored. In this study, we propose strategies to optimize PK digestion conditions for early, middle, and late-stage pea aphid embryos, which have varying tissue thicknesses. Additionally, we extend the application of PK treatment to salivary glands, a representative somatic tissue, by optimizing conditions for antibody penetration against the salivary gland marker C002. To enhance spatial precision in signal detection, we provide a detailed protocol for tissue dissection specific to pea aphids, focusing on the preservation of tissue integrity. These comprehensive guidelines, covering tissue dissection and PK titration, are expected to improve the specificity and intensity of protein signals in pea aphids and other aphid species.•Provide aphid-specific dissection methods to obtain intact embryos and salivary glands.•Present strategies for optimizing PK treatment conditions across different tissue types.

O-172 Live imaging reveals key events in preimplantation embryo development

Abstract Preimplantation development encompasses the zygote to blastocyst stages, occurring in vivo within the Fallopian tube and in vitro within embryology labs worldwide. During these first five days of life, several key morphologic changes take place facilitating cell fate specification and resulting in blastocyst assembly containing ICM and trophectoderm lineages. These include cell polarization, embryo compaction and inner-outer segregation, which transform the collection of blastomeres into a morula. Subsequently, the blastocoel cavity forms and expands followed by hatching from the zona pellucida prior to implantation. This ability to self-organize has enabled live imaging of these events within intact embryos. Moreover, this has revealed the dynamics of early mitoses, nuclear morphology and cytoskeletal changes driving early development. Although a great deal has been learned in the mouse, events in the human embryo remain relatively unknown. Recent achievements in stem cell-based embryoid models and non-invasive live imaging now poise researchers to make significant advancements unveiling human embryo development. Furthermore, a comprehensive understanding of human embryogenesis is essential to improving IVF efficiency and outcomes. Preimplantation development in mouse and human will be reviewed with a focus on recent discoveries made possible by live imaging.

P-250 Only euploidy does not ensure optimal chances for successful live birth through single blastocyst cryotransfer

Abstract Study question What does a euploid blastocyst require to provide the greatest probability of successful birth? Summary answer Morphokinetic and morphological features of the inner cell mass (ICM) and trophectoderm (TE) are crucial for optimizing live birth chances in cryo-transferred euploid blastocysts. What is known already Embryo selection through preimplantation genetic testing for aneuploidy (PGT-A) alongside frozen embryo transfer strategy enhances outcomes of single-embryo transfer cycles. While euploidy is a crucial indicator, investigations on the identification of additional criteria influencing implantation and live birth rates remain imperative. Studies suggest morphokinetic and morphological quality may supplement predictive factors for euploid embryo success, with varying correlations. The impact of factors like age or gamete origin on euploid blastocyst success remains uncertain, differentiating it from clear effects on non-diagnosed embryos. Optimizing selection criteria involves navigating the intricate interplay of these variables. Study design, size, duration This retrospective study analyses the pregnancy rate (PR) in 596 single embryo cryotransfers of full euploid blastocysts (without detected mosaicism) between February 2019 and September 2023. From these, 437 had a known outcome, and the live birth rate (LBR) was calculated. The study explored the potential impact of gamete origin, oocyte provider age, recipient age, morphokinetics, and morphological quality of ICM or TE (Gardner score) on the reproductive success of the transferred embryo. Participants/materials, setting, methods All embryos were biopsied at the blastocyst stage and successively vitrified. High-resolution next-generation sequencing was used for PGT-A. Only deferred transferences of single post-thawing intact embryos were included. Morphokinetic was assessed using the KIDscoreD5 3.1. Quantitative variables (oocyte provider age, recipient age, and morphokinetic score) were compared using the t-student test, while qualitative variables (gamete origin, morphological quality of ICM and TE) were assessed using the chi-square test. Main results and the role of chance Oocytes leading to pregnancies and live births were derived from women with ages comparable to those without success (Pregnancy: 30.0±7.1 vs. 30.8±6.8, p = 0.174; Live Birth: 30.1±7.1 vs. 30.5±6.9, p = 0.499). Similar age was also observed among recipients achieving pregnancy (41.5±5.4) and live births (41.1±5.1) compared to those who did not (Pregnancy: 41.8±5.1, p = 0.506; Live Birth: 41.5±5.3, p = 0.411). The origin of oocytes and semen (own or donor) did not significantly affect PR (Oocytes: 199/273 vs. 217/323, p = 0.130; Semen: 328/473 vs. 88/123, p = 0.224) or LBR (Oocytes: 113/205 vs. 116/232, p = 0.285; Semen: 183/347 vs. 46/90, p = 0.783). Morphokinetics played a crucial role, with euploid embryos resulting in pregnancy (7.5±1.7) and live births (7.6±1.6) exhibiting significantly higher scores than those without success (6.9±1.7; 7.1±1.7; p < 0.05).199 ICM quality did not significantly impact PR (A:211/296, B:190/275, C:15/25; p = 0.469). However, higher miscarriage rates in C-quality ICM blastocysts significantly reduced the LBR (A:116/211, B:107/205, C:6/21; p < 0.05). TE quality exhibited significant differences in PR (A:221/289, B:184/289, C:11/18; p < 0.05). Since the miscarriage rate was similar among groups (p = 0.787), LBR remained higher in A-quality TE blastocysts (A:124/209, B:98/213, C:7/17; p < 0.05). Limitations, reasons for caution As a retrospective analysis, this study is inherently limited. The influence of expansion grade on success rates remains unexplored, given the use of assisted hatching on day 3 of culture. Performing assisted hatching at the time of biopsy could potentially overcome this limitation. Wider implications of the findings The pursuit of a euploid embryo often becomes the priority in IVF laboratories. However, once the milestone is achieved, morphokinetics and embryonic morphological quality must continue to be considered. This study provides valuable insights for conveying a detailed message to patients with euploid embryos about their prospects for homebirth success. Trial registration number not applicable

Human trophectoderm becomes multi-layered by internalization at the polar region

To implant in the uterus, mammalian embryos form blastocysts comprising trophectoderm (TE) surrounding an inner cell mass (ICM), confined to the polar region by the expanding blastocoel. The mode of implantation varies between species. Murine embryos maintain a single layered TE until they implant in the characteristic thick deciduum, whereas human blastocysts attach via polar TE directly to the uterine wall. Using immunofluorescence (IF) of rapidly isolated ICMs, blockade of RNA and protein synthesis in whole embryos, or 3D visualization of immunostained embryos, we provide evidence of multi-layering in human polar TE before implantation. This may be required for rapid uterine invasion to secure the developing human embryo and initiate formation of the placenta. Using sequential fluorescent labeling, we demonstrate that the majority of inner TE in human blastocysts arises from existing outer cells, with no evidence of conversion from the ICM in the context of the intact embryo.

Comparative effects of different metals on the Japanese medaka embryos and larvae.

Rapid evaluation of the toxicity of metals using fish embryo acute toxicity is facilitative to ecological risk assessment of aquatic organisms. However, this approach has seldom been utilized for the comparative study on the effects of different metals to fish. In this study, acute and sub-chronic tests were used to compare the toxicity of Se(IV) and Cd in the embryos and larvae of Japanese medaka (Oryzias latipes). The embryos with different levels of dechorionation and/or pre-exposure were also exposed to Se(IV) and Cd at various concentrations. The results showed that the LC50-144 h of Cd was 1.3-5.2 folds higher than that of Se(IV) for the embryos. In contrast, LC50-96 h of Se(IV) were 200-400 folds higher than that of Cd for the larvae. Meanwhile, dechorionated embryos were more sensitive to both Se and Cd than the intact embryos. At elevated concentrations, both Se and Cd caused mortality and deformity in the embryos and larvae. In addition, pre-exposure to Cd at the embryonic stages enhanced the resistance to Cd in the larvae. However, pre-exposure to Se(IV) at the embryonic stages did not affect the toxicity of Se(IV) to the larvae. This study has distinguished the nuance differences in effects between Se(IV) and Cd after acute and sub-chronic exposures with/without chorion. The approach might have a potential in the comparative toxicology of metals (or other pollutants) and in the assessment of their risks to aquatic ecosystems.

Engineering a computable epiblast for in silico modeling of developmental toxicity

Developmental hazard evaluation is an important part of assessing chemical risks during pregnancy. Toxicological outcomes from prenatal testing in pregnant animals result from complex chemical-biological interactions, and while New Approach Methods (NAMs) based on in vitro bioactivity profiles of human cells offer promising alternatives to animal testing, most of these assays lack cellular positional information, physical constraints, and regional organization of the intact embryo. Here, we engineered a fully computable model of the embryonic disc in the CompuCell3D.org modeling environment to simulate epithelial-mesenchymal transition (EMT) of epiblast cells and self-organization of mesodermal domains (chordamesoderm, paraxial, lateral plate, posterior/extraembryonic). Mesodermal fate is modeled by synthetic activity of the BMP4-NODAL-WNT signaling axis. Cell position in the epiblast determines timing with respect to EMT for 988 computational cells in the computer model. An autonomous homeobox (Hox) clock hidden in the epiblast is driven by WNT-FGF4-CDX signaling. Executing the model renders a quantitative cell-level computation of mesodermal fate and consequences of perturbation based on known biology. For example, synthetic perturbation of the control network rendered altered phenotypes (cybermorphs) mirroring some aspects of experimental mouse embryology, with electronic knockouts, under-activation (hypermorphs) or over-activation (hypermorphs) particularly affecting the size and specification of the posterior mesoderm. This foundational model is trained on embryology but capable of performing a wide variety of toxicological tasks conversing through anatomical simulation to integrate in vitro chemical bioactivity data with known embryology. It is amenable to quantitative simulation for probabilistic prediction of early developmental toxicity.

Differential cellular stiffness across tissues that contribute to Xenopus neural tube closure.

During the formation of the neural tube, the primordium of the vertebrate central nervous system, the actomyosin activity of cells in different regions drives neural plate bending. However, how the stiffness of the neural plate and surrounding tissues is regulated and mechanically influences neural plate bending has not been elucidated. Here, we used atomic force microscopy to reveal the relationship between the stiffness of the neural plate and the mesoderm during Xenopus neural tube formation. Measurements with intact embryos revealed that the stiffness of the neural plate was consistently higher compared with the non-neural ectoderm and that it increased in an actomyosin activity-dependent manner during neural plate bending. Interestingly, measurements of isolated tissue explants also revealed that the relationship between the stiffness of the apical and basal sides of the neural plate was reversed during bending and that the stiffness of the mesoderm was lower than that of the basal side of the neural plate. The experimental elevation of mesoderm stiffness delayed neural plate bending, suggesting that low mesoderm stiffness mechanically supports neural tube closure. This study provides an example of mechanical interactions between tissues during large-scale morphogenetic movements.

Cell-Autonomous and Non-Cell-Autonomous Mechanisms Concomitantly Regulate the Early Developmental Pattern in the Kelp Saccharina latissima Embryo.

Brown algae are multicellular organisms that have evolved independently from plants and animals. Knowledge of the mechanisms involved in their embryogenesis is available only for the Fucus, Dictyota, and Ectocarpus, which are brown algae belonging to three different orders. Here, we address the control of cell growth and cell division orientation in the embryo of Saccharina latissima, a brown alga belonging to the order Laminariales, which grows as a stack of cells through transverse cell divisions until growth is initiated along the perpendicular axis. Using laser ablation, we show that apical and basal cells have different functions in the embryogenesis of this alga, with the apical cell being involved mainly in growth and basal cells controlling the orientation of cell division by inhibiting longitudinal cell division and thereby the widening of the embryo. These functions were observed in the very early development before the embryo reached the 8-cell stage. In addition, the growth of the apical and basal regions appears to be cell-autonomous, because there was no compensation for the loss of a significant part of the embryo upon laser ablation, resulting in smaller and less elongated embryos compared with intact embryos. In contrast, the orientation of cell division in the apical region of the embryo appears to be controlled by the basal cell only, which suggests a polarised, non-cell-autonomous mechanism. Altogether, our results shed light on the early mechanisms of growth rate and growth orientation at the onset of the embryogenesis of Saccharina, in which non-cell-specific cell-autonomous and cell-specific non-cell-autonomous processes are involved. This complex control differs from the mechanisms described in the other brown algal embryos, in which the establishment of embryo polarity depends on environmental cues.

Evaluation of blastocyst re-expansion, quality in relation to storage temperature, and sexing using blastocoel fluid after manual perforation with a hand-held needle involving in vivo produced equine embryos

The present study was designed to evaluate equine blastocyst re-expansion rate, quality, and sex following perforation of the blastocoel, collection of blastocoel fluid (BF), and PCR amplification of free DNA. Experiment 1 tested the feasibility of the BF sample collection with a hand-held, small-gauged needle (26g) and subsequent PCR amplification of the TSP-Y gene for males and AMEL-Y gene for males and AMEL-X gene for females. Experiment 2 tested the application of the technique. Equine embryos were collected via uterine flushes 8d after ovulation. Thereafter, embryos (n = 19) were initially assessed and transferred to a 50 μL droplet of holding medium in which the blastocoel was manually perforated as in Experiment 1. Within 1 min of detecting a diameter decrease or collapse, the entire volume of each droplet of medium was collected and stored at −20 °C until PCR. In Experiment 1, amplification of the TSP-Y gene was positive for males at 60% (9/15) and negative for females at 40% (6/15). In Experiment 2, a total of 42 embryos were randomly assigned to a collapsed embryo (CE) or intact embryo (IE) groups and stored at room temperature (RT, 25 °C) or cold temperature (CT, 5 °C) for 24h as follows: 1) CERT, n = 11; 2) CECT n = 11; 3) IERT, n = 10; and 4) IECT, n = 10. After 24h, embryo diameter and quality were reassessed. For all collapsed embryos (n = 19), blastocoel fluid was subjected to double PCR amplification of the TSPY gene with blood from adult male and female horses as controls. Positive gene amplification indicated 57.9% (11/19) of embryos were male and negative amplification indicated 31.6% (6/19) of embryos were female. Relative to the least diameter (0%) after perforation of collapsed embryos or fullest diameter (100%) of intact embryos at T0, percentage change in diameter and quality Grade 1 or 2 embryos after 24h of storage for all groups were, respectively: 31.2% and 54% for CERT group, 28.2% and 0% for CECT group, 25.9% and 100% for IERT group, 4.3% and 80% for IECT group, respectively. Thus, needle-induced leakage and collapse of the blastocoel at T0 resulted in a high rate of blastocyst re-expansion (69%) with many embryos (54%) achieving good quality at T24 with potential for transfer as either male or female embryos. For both collapsed and intact embryos, it was observed that storage for 24h at room temperature (25 °C) was associated with improved embryo growth and morphological quality compared to storage at cold temperature (5 °C).

Robust axis elongation by Nodal-dependent restriction of BMP signaling.

Embryogenesis results from the coordinated activities of different signaling pathways controlling cell fate specification and morphogenesis. In vertebrate gastrulation, both Nodal and BMP signaling play key roles in germ layer specification and morphogenesis, yet their interplay to coordinate embryo patterning with morphogenesis is still insufficiently understood. Here, we took a reductionist approach using zebrafish embryonic explants to study the coordination of Nodal and BMP signaling for embryo patterning and morphogenesis. We show that Nodal signaling triggers explant elongation by inducing mesendodermal progenitors but also suppressing BMP signaling activity at the site of mesendoderm induction. Consistent with this, ectopic BMP signaling in the mesendoderm blocks cell alignment and oriented mesendoderm intercalations, key processes during explant elongation. Translating these ex vivo observations to the intact embryo showed that, similar to explants, Nodal signaling suppresses the effect of BMP signaling on cell intercalations in the dorsal domain, thus allowing robust embryonic axis elongation. These findings suggest a dual function of Nodal signaling in embryonic axis elongation by both inducing mesendoderm and suppressing BMP effects in the dorsal portion of the mesendoderm.

Clinical and neonatal outcomes of complete zona pellucida removal by laser-assisted hatching after single vitrified-warmed blastocyst transfer.

Laser-assisted hatching (LAH) is a widely used and unavoidable technique in assisted reproductive technology after frozen embryo transfer (FET). We aimed to investigate the safety and efficacy of completely ZP (zona pellucida) removed by an assisted laser group compared to an intact group after FET. This retrospective study involved 320 SVBT single vitrified-warmed blastocyst transfers of 213 patients who underwent clomiphene citrate (CC) based minimal stimulation protocol. This study compared 160 SVBT with ZP that were completely removed using laser-assisted (laser beams of 10-15, a wavelength of 1.48μm, and duration of 1.8-2.2ms) to 160 cases of intact SVBT. Fisher's exact test, chi-squared test, and logistic regression are used for statistical analysis. The women's age, cause of infertility, BMI, transferred embryo day, insemination methods, and blastocyst grade were not significantly different between two groups. The implantation rate (66.2% vs. 51.2%, P < 0.01), the clinical pregnancy rate (CPR, 52.5% vs. 39.3%, P = 0.01), and the live birth rate (LBR, 43.7% vs. 29.3%, P < 0.01) were significantly higher in the completely ZP removed than the intact group. In logistic regression analysis, the laser-assisted hatching group showed about 2 times higher implantation rate and CPR and LBR than the control group. But miscarriage, gestational weeks, gender, birth weight, and twin births were insignificant between two groups. Our study suggests ZP free embryo transfer by assisted laser is more effective and safer than intact embryo transfer.

Analysis of Mid- to Late-Gestation Phenotypes in Mice.

Mid- to late gestation is characterized by tissue differentiation, maturation, organogenesis, and growth, and many mutant genes have detrimental effects during this phase of development. The outcome may be lethal before birth or may be compatible with life but result in birth defects. Some of the common causes of death during late gestation are hematopoietic defects, cardiovascular problems, and placental insufficiency. Many morphological abnormalities, lethal or not, can be investigated with gross and histological analyses or by visualization of the developing skeleton. Molecular characterization of mutant phenotypes, guided by the expression pattern of the mutant gene, can reveal disruptions in gene expression patterns of known developmental genes. Cell proliferation and cell death assays will reveal disruptions in cellular dynamics. Various modalities of 3D imaging of intact embryos can provide volumetric information about mutant phenotypes.

Improved methodology for fixation and preparation of Aedes aegypti embryos.

The yellow fever mosquito Aedes aegypti is a major disease vector and an increasingly popular emerging model research organism. We present here an improved protocol for the collection, fixation, and preparation of A. aegypti embryos for immunohistochemical and in situ hybridization studies. The processing of A. aegypti embryos for such studies is complicated by the inability to easily remove the vitelline membrane, which prevents the reagents needed for staining from reaching their targets, and which furthermore obscures visualization of the embryo since the membrane is highly sclerotized. Previously described protocols for removal of the vitelline membrane are very low throughput, limiting the capacity of work that can be accomplished in a reasonable timeframe. Our adapted protocol increases the throughput capacity of embryos by an individual user, with experienced users able to prepare an average of 100-150 embryos per hour. The protocol provides high-quality intact embryos that can be used for morphological, immunohistochemical, and in situ hybridization studies. The protocol has been successfully tested on embryos of ages ranging from 14h after egg laying (AEL) at 27°C through to 55h AEL. Critical to the success of the optimized protocol is the selection, fabrication, and description of the tools required. To this end, a video-demonstrated protocol has been placed at protocols.io to clarify the protocol and provide easy access and training to anyone interested in the preparation of A. aegypti embryos for biological studies.

Microsurgical separation of deconserved embryos

At the current level of development of cell engineering methods in cattle breeding, the accelerated production of monozygotic twins is acquiring important scientific and economic significance. This will make it possible to determine the effectiveness of breeding methods in assessing genetic progress in a population over a number of generations. The method of embryonic cloning by microsurgical separation of embryos is close to solving this problem. Of particular interest is the long-term preservation of demi-embryos to obtain genetic analogues. But the possibilities of preserving demi-embryos at low temperatures are limited, since existing methods are multi-stage and extremely difficult to implement in practice. The search for applied methods of microsurgery in combination with long-term preservation of demi-embryos is an urgent task. Early studies have reported micromanipulation of frozen-thawed embryos. In 1984, in experiments by S. Willadsen &amp; R. Godke, after microsurgery of deconserved embryos, the survival rate was 54.2%. Data on survival rate are not provided. No other data was found, and therefore we conducted studies to determine the influence of the processes of cryo- and deconservation, and dissection of embryos on the survival and ability of demi-embryos to develop totipotently after transplantation. The research was carried out in 2022 at the Regional Research and Production Center for the Reproduction of Farm Animals of Kalmyk State University named after B.B. Gorodovikov. The efficiency of dissection of intact embryos was 86.1% - after separation of 61 embryos, 105 suitable demi-embryos were obtained out of 122 possible, which was 5.7% higher than the same figure for dividing deconserved embryos - 80.4% (37 suitable demi-embryos out of 46 possible after separation 23 embryos). The survival rate of demi-embryos as the ability to nidate in the recipient's uterus in intact and depreserved demi-embryos differed within acceptable limits - 52.2% when transplanting 1 intact demi-embryo (12 pregnant out of 23 recipients) and 65.9% when transplanting 2 demi-embryos (27 pregnant from 41). Similar indicators for transplantation of deconserved demi-embryos were respectively: 42.8 (3 pregnant recipients out of 7 transplanted) and 60.0% (9 pregnant recipients out of 15).

Mounting of Embryos, Larvae, and Pupae for Live Drosophila Dendritic Arborization Neuron Imaging.

Live imaging approaches are essential for monitoring how neurons go through a coordinated series of differentiation steps in their native mechanical and chemical environment. These imaging approaches also allow the study of dynamic subcellular processes such as cytoskeleton remodeling and the movement of organelles. Drosophila dendritic arborization (da) neurons are a powerful experimental system for studying the dendrite arbor in live animals. da neurons are located on the internal surface of the body wall and, therefore, are easily accessible for imaging. Moreover, many genetic tools target da neurons to disrupt genes or proteins of interest and allow the investigator to visualize fluorescent markers and endogenously tagged proteins in the neurons. This protocol introduces methods for preparing and mounting intact Drosophila embryos, larvae, and pupae, allowing live imaging of dynamic cellular processes in da neurons.

Ventral Nerve Cord Dissection and Microscopy of Drosophila Embryos.

The technique of visualizing axon pathways in the embryonic ventral nerve cord using antibody labeling has been fundamental to our understanding of the genetic and developmental mechanisms underlying nervous system wiring in Drosophila. High-resolution microscopic examination of the ventral nerve cord remains an essential component of many experiments in Drosophila developmental neuroscience. Although it is possible to examine the ventral nerve cord in intact whole-mount embryos, to collect the highest-quality images it is often useful to isolate the nervous system away from the other embryonic tissues through embryo dissection. This protocol describes methods for dissecting ventral nerve cords from Drosophila embryos that have been fixed and stained via immunofluorescence or horseradish peroxidase (HRP) immunohistochemistry. The process of making fine dissection needles for this purpose from electrolytically sharpened tungsten wire is also described. Dissected and mounted ventral nerve cords can be examined and imaged using a variety of microscopy techniques including differential interference contrast (DIC) optics, epifluorescence, or confocal microscopy.

High-Resolution Magic Angle Spinning (HRMAS) NMR Identifies Oxidative Stress and Impairment of Energy Metabolism by Zearalenone in Embryonic Stages of Zebrafish (Danio rerio), Olive Flounder (Paralichthys olivaceus) and Yellowtail Snapper (Ocyurus chrysurus).

Zearalenone (ZEA) is a mycotoxin, commonly found in agricultural products, linked to adverse health impacts in humans and livestock. However, less is known regarding effects on fish as both ecological receptors and economically relevant "receptors" through contamination of aquaculture feeds. In the present study, a metabolomics approach utilizing high-resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) was applied to intact embryos of zebrafish (Danio rerio), and two marine fish species, olive flounder (Paralichthys olivaceus) and yellowtail snapper (Ocyurus chrysurus), to investigate the biochemical pathways altered by ZEA exposure. Following the assessment of embryotoxicity, metabolic profiling of embryos exposed to sub-lethal concentrations showed significant overlap between the three species and, specifically, identified metabolites linked to hepatocytes, oxidative stress, membrane disruption, mitochondrial dysfunction, and impaired energy metabolism. These findings were further supported by analyses of tissue-specific production of reactive oxygen species (ROS) and lipidomics profiling and enabled an integrated model of ZEA toxicity in the early life stages of marine and freshwater fish species. The metabolic pathways and targets identified may, furthermore, serve as potential biomarkers for monitoring ZEA exposure and effects in fish in relation to ecotoxicology and aquaculture.

Direct force measurement and loading on developing tissues in intact avian embryos.

Developmental morphogenesis is driven by tissue stresses acting on tissue rheology. Direct measurements of forces in small tissues (100 µm-1 mm) in situ, such as in early embryos, require high spatial precision and minimal invasiveness. Here, we introduce a control-based approach, tissue force microscopy (TiFM), that integrates a mechanical cantilever probe and live imaging with closed-loop feedback control of mechanical loading in early chicken embryos. By testing previously qualitatively characterized force-producing tissues in the elongating body axis, we show that TiFM quantitatively captures stress dynamics with high sensitivity. TiFM also provides the means to apply stable, minimally invasive and physiologically relevant loads to drive tissue deformation and to follow the resulting morphogenetic progression associated with large-scale cell movements. Together, TiFM allows us to control tissue force measurement and manipulation in small developing embryos, and promises to contribute to the quantitative understanding of complex multi-tissue mechanics during development.

A mechanistic understanding of the effects of polyethylene terephthalate nanoplastics in the zebrafish (Danio rerio) embryo

Plastic pollution, especially by nanoplastics (NPs), has become an emerging topic due to the widespread existence and accumulation in the environment. The research on bioaccumulation and toxicity mechanism of NPs from polyethylene terephthalate (PET), which is widely used for packaging material, have been poorly investigated. Herein, we report the first use of high-resolution magic-angle spinning (HRMAS) NMR based metabolomics in combination with toxicity assay and behavioural end points to get systems-level understanding of toxicity mechanism of PET NPs in intact zebrafish embryos. PET NPs exhibited significant alterations on hatching and survival rate. Accumulation of PET NPs in larvae were observed in liver, intestine, and kidney, which coincide with localization of reactive oxygen species in these areas. HRMAS NMR data reveal that PET NPs cause: (1) significant alteration of metabolites related to targeting of the liver and pathways associated with detoxification and oxidative stress; (2) impairment of mitochondrial membrane integrity as reflected by elevated levels of polar head groups of phospholipids; (3) cellular bioenergetics as evidenced by changes in numerous metabolites associated with interrelated pathways of energy metabolism. Taken together, this work provides for the first time a comprehensive system level understanding of toxicity mechanism of PET NPs exposure in intact larvae.

Transmission of Mycoplasma bovis infection in bovine in vitro embryo production

Mycoplasma bovis (M. bovis) causes several costly diseases in cattle and has a negative effect on cattle welfare. There is no effective commercial vaccine, and antimicrobial resistance is common. Maintaining a closed herd is the best method to minimize the risk of introduction of M. bovis. Assisted reproduction is crucial in a closed herd to make genetic improvements. M. bovis has been found in commercial semen, and contaminated semen has been the source of disease in naïve dairy herds. The objective of this study was to evaluate M. bovis transmission in bovine in vitro embryo production (IVP) using several possible exposure routes. We used a wild-type M. bovis strain isolated from semen at a final concentration of 106 CFU/mL to infect cumulus–oocyte complexes, spermatozoa, and 5-day-old embryos. We also used naturally contaminated semen in fertilization. Blastocysts were collected on day 7–8 and zona pellucida (ZP)-intact embryos were either washed 12 times, including trypsin washes as recommended by the International Embryo Technology Society (IETS), or left unwashed. Washed and unwashed embryos, follicular fluids, maturation medium, cumulus cells, fertilization medium, and G1 and G2 culture media, as well as all wash media were analyzed using enrichment culture followed by real-time PCR detection of M. bovis. Altogether, 76 pools containing 363 unwashed embryos and 52 pools containing 261 IETS washed embryos were analyzed after oocytes, spermatozoa, or 5-day-old embryos were infected with M. bovis or naturally contaminated semen was used in fertilization. We could not detect M. bovis in any of the embryo pools. M. bovis was not found in any of 12 wash media from different exposure experiments. M. bovis did not affect the blastocyst rate, except when using experimentally infected semen. Contrary to an earlier study, which used a cell co-culture system, we could not demonstrate M. bovis in embryo wash media or tight adherence of M. bovis to ZP-intact embryos. Naturally infected semen did not transmit M. bovis to embryos. We conclude that by using our IVP system, the risk of M. bovis transmission via IVP embryos to recipient cows is very low.