Small Embryos Research Articles

Reproductive traits of Gnathophylloides mineri Schmitt, 1933, a minute ectosymbiotic shrimp associated with the sea urchin Tripneustes ventricosus

Abstract Numerous studies on the reproduction of decapods have revealed that the female energy investment in embryo production is generally around 10% of the female weight. In decapods living together with other organisms, however, the reproductive output (RO) may be substantially higher. Here we tested the hypothesis that decapods living in association with other organisms allocate more energy in embryo production than free-living ones. We studied the shrimp Gnathophylloides mineri, which lives among the spines of the sea urchin Tripneustes ventricosus. Shrimps were collected between October 2011 and April 2013 along the southern Caribbean coast of Costa Rica. A total of 45 ovigerous females were analyzed; female size ranged from 1.44 to 2.14 mm carapace length. Females carried on average 37 ± 16 SD embryos. The embryo volume increased during the incubation period by 200% (from 0.04 ± 0.01 to 0.08 ± 0.01 mm3, mean ± SD). The RO was not related to female size, and comprised on average 27.5 ± 16.0 SD % of the dry weight of the females. In summary, G. mineri produces few and small embryos, but these embryos are relatively large when compared to similar-sized caridean species. Moreover, RO of this ectosymbiotic species was higher than in free-living species, but lower than in endosymbiotic decapods. These results support the hypothesis that energy investment in embryo production of decapods increases with the level of protection associated with the symbiosis.

P-258 Not too big, not too small: euploid blastocysts that lead to live birth have an optimal surface area, diameter and optimal pace of development

Abstract Study question Does embryo diameter and surface area at tsB and tB as determined by AI predict live birth (LB) potential amongst euploid blastocysts? Summary answer Euploid blastocysts with optimal diameter (tsB:120-129; tB:136-140μm) and surface area (tsB:11771-13577; tB:15000-15643μm2) are more likely to lead to LB than larger or smaller blastocysts. What is known already Current embryology consensus favours larger blastocysts based on Gardner grading, associating expansion with higher quality. Human assessment of embryos is subjective, and it is unknown whether this dogma holds when assessing pre-expansion blastocyst stages. The impact of blastocyst surface area and diameter at tSB and tB; and pace of development on LB remains unclear. This may help clarify whether large blastocysts are associated with accelerated embryo growth and abnormal metabolic processes. The study’s objective was to use AI to quantitatively determine the optimal embryo surface area and diameter of euploid blastocysts that lead to live birth. Study design, size, duration Retrospective study including 97 time-lapse videos of euploid embryos with known live birth outcomes from January to December 2021 Participants/materials, setting, methods Embryo diameter and surface area were measured at start of blastulation (tSB) and at time of blastulation (tB) by an AI model (CHLOE-EQ, Fairtility Ltd). Live birth (LB) outcomes were manually annotated by embryologists. An Optimal embryo surface area and diameter were determined by the highest live birth rate and the suboptimal range was determine by a decreased Live birth rate. A comparison between the optimal and suboptimal live birth rate was measured using chi-square. Main results and the role of chance Moderately sized embryos were found to have higher LB outcomes. At tSB, optimal surface area was 11771-13577 um2, while optimal embryo diameter was 120-129μm. LB rate for optimal embryo surface area was higher than for suboptimal [Optimal 80% (20/25) vs Suboptimal 55% (40/72), p < 0.001]. In addition, optimal embryo diameter had higher LB rates than embryos with suboptimal diameter [80% (16/20) vs 52% (31/60), p < 0.05). At tB, optimal embryo area was 15000-15643μm2 and optimal embryo diameter 136-140μm. LB rate for optimal embryo surface area was higher than for suboptimal 82% (14/17) vs 49% (36/73), p < 0.05]. Optimal embryo diameter had higher LB rates than embryos with suboptimal diameter (77% 20/26 vs 47% 30/64, p < 0.05). Embryos with an optimal surface area and diameter were slower than bigger suboptimal embryos: t2 (26±2 vs 25±2), t8 (59±8 vs 53±6), t9 + (71±6 vs 67±6), tM (82±7 vs 77±8), tSB (99±6 vs 93±6), tB (107±7 vs 100±6), tEB (137±2 vs 122±12); t8-t5 (12±10 vs 6±5), t8-t2 (33±8 vs 28±5), tEB-tSB (34±2 vs 23±9), p < 0.05. Optimal vs small suboptimal embryos were faster in t9 + (71±6 vs 76±4), tEB (121±11 vs 137±2, tEB-tSB (20±10 vs 34±1). But were slower in t8-t5 (12±10 vs 5±6), t8-t2 (33±8 vs 28±5), p < 0.05. Limitations, reasons for caution This is the first time a quantitative embryo surface area and diameter has been studied in its correlation with live birth. Generalisation requires the replication across diverse centers with varying demographic profiles to validate findings and establish broader applicability within the context of assisted reproductive technologies. Wider implications of the findings This data-driven approach can support the embryo selection process, correlating quantitative parameters with live birth outcomes, thereby refining the precision of the decision-making in IVF. This level of quantitative data of embryo surface area and diameter is only accessible through AI tools. Trial registration number IRB-007CITY-FL21.08.2023

Thermal Stability and Crystallization Processes of Pd78Au4Si18 Thin Films Visualized via In Situ TEM.

Amorphous alloys or metallic glasses (MGs) thin films have attracted extensive attention in various fields due to their unique functional properties. Here, we use in situ heating transmission electron microscopy (TEM) to investigate the thermal stability and crystallization behavior of Pd-Au-Si thin films prepared by a pulsed laser deposition (PLD) method. Upon heating treatment inside a TEM, we trace the structural changes in the Pd-Au-Si thin films through directly recording high-resolution images and diffraction patterns at different temperatures. TEM observations reveal that the Pd-Au-Si thin films started to nucleate with small crystalline embryos uniformly distributed in the glassy matrix upon approaching the glass transition temperature Tg=625K, and subsequently, the growth of crystalline nuclei into sub-10 nm Pd-Si nanocrystals commenced. Upon further increasing the temperature to 673K, the thin films transformed to micro-sized patches of stacking-faulty lamellae that further crystallized into Pd9Si2 and Pd3Si intermetallic compounds. Interestingly, with prolonged thermal heating at elevated temperatures, the Pd9Si2 transformed to Pd3Si. Simultaneously, the solute Au atoms initially dissolved in glassy alloys and eventually precipitated out of the Pd9Si2 and Pd3Si intermetallics, forming nearly spherical Au nanocrystals. Our TEM results reveal the unique thermal stability and crystallization processes of the PLD-prepared Pd-Au-Si thin films as well as demonstrate a possibility of producing a large quantity of pure nanocrystals out of amorphous solids for various applications.

First trimester maternal tryptophan metabolism and embryonic and fetal growth: the Rotterdam Periconceptional Cohort (Predict Study).

What is the association between first trimester maternal tryptophan (TRP) metabolites and embryonic and fetal growth? Higher 5-hydroxytryptophan (5-HTP) concentrations are associated with reduced embryonic growth and fetal growth and with an increased risk of small-for-gestational age (SGA), while higher kynurenine (KYN) concentrations are associated with a reduced risk of SGA. The maternal TRP metabolism is involved in many critical processes for embryonic and fetal growth, including immune modulation and regulation of vascular tone. Disturbances in TRP metabolism are associated with adverse maternal and fetal outcomes. This study was embedded within the Rotterdam Periconceptional Cohort (Predict Study), an ongoing prospective observational cohort conducted at a tertiary hospital from November 2010 onwards. A total of 1115 women were included before 11 weeks of gestation between November 2010 and December 2020. Maternal serum samples were collected between 7 and 11 weeks of gestation, and TRP metabolites (TRP, KYN, 5-HTP, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid) were determined using a validated liquid chromatography (tandem) mass spectrometry method. Serial 3D ultrasound scans were performed at 7, 9, and 11 weeks of gestation to accurately assess features of embryonic growth, including crown-rump length (CRL) and embryonic volume (EV) offline using virtual reality systems. Fetal growth parameters were retrieved from medical records and standardized according to Dutch reference curves. Mixed models were used to assess associations between maternal TRP metabolites and CRL and EV trajectories. Linear and logistic regression models were utilized to investigate associations with estimated fetal weight (EFW) and birthweight, and with SGA, respectively. All analyses were adjusted for potential confounders. Maternal 5-HTP concentrations and the maternal 5-HTP/TRP ratio were inversely associated with embryonic growth (5-HTP, √CRL: β = -0.015, 95% CI = -0.028 to -0.001; 5-HTP 3√EV: β = -0.009, 95% CI = -0.016 to -0.003). An increased maternal 5-HTP/TRP ratio was also associated with lower EFW and birthweight, and with an increased risk of SGA (odds ratio (OR) = 1.006, 95% CI = 1.00-1.013). In contrast, higher maternal KYN concentrations were associated with a reduced risk of SGA in the unadjusted models (OR = 0.548, 95% CI = 0.320-0.921). Residual confounding cannot be ruled out because of the observational design of this study. Moreover, this study was conducted in a single tertiary hospital, which assures high internal validity but may limit external validity. The novel finding that maternal 5-HTP concentrations are associated with a smaller embryo and fetus implies that disturbances of the maternal serotonin pathway in the first trimester of pregnancy are potentially involved in the pathophysiology of fetal growth restriction. The association between higher maternal KYN concentrations and a reduced risk of SGA substantiate the evidence that the KYN pathway has an important role in fetal growth. More research is needed to delve deeper into the potential role of the maternal TRP metabolism during the periconception period and pregnancy outcome for mother and offspring. This study was funded by the Department of Obstetrics and Gynecology and the Department of Clinical Chemistry of the Erasmus MC, University Medical Center, Rotterdam, the Netherlands. The authors have no competing interests to disclose. N/A.

Orchid seeds are not always short lived in a conventional seed bank!

Orchid seeds are reputed to be short lived in dry, cold storage conditions, potentially limiting the use of conventional seed banks for long-term ex situ conservation. This work explores whether Cattleya seeds are long lived or not during conventional storage (predried to ~12 % relative humidity, then stored at -18 °C). We explored the possible interaction of factors influencing seed lifespan in eight species of the genus Cattleya using physiological (germination and vigour), biochemical (gas chromatography), biophysical (differential scanning calorimetry) and morphometric methods. Seeds were desiccated to ~3 % moisture content and stored at -18 °C for more than a decade, and seed quality was measured via three in vitro germination techniques. Tetrazolium staining was also used to monitor seed viability during storage. The morphometric and germination data were subjected to ANOVA and cluster analysis, and seed lifespan was subjected to probit analysis. Seeds of all Cattleya species were found to be desiccation tolerant, with predicted storage lifespans (P50y) of ~30 years for six species and much longer for two species. Cluster analysis showed that the three species with the longest-lived seeds had smaller (9-11 %) airspaces around the embryo. The post-storage germination method impacted the quality assessment; seeds equilibrated at room temperature for 24 h or in 10 % sucrose solution had improved germination, particularly for the seeds with the smallest embryos. Chromatography revealed that the seeds of all eight species were rich in linoleic acid, and differential scanning calorimetry identified a peak that might be auxiliary to selecting long-lived seeds. These findings show that not all orchids produce seeds that are short lived, and our trait analyses might help to strengthen prediction of seed longevity in diverse orchid species.

Cas9-Mediated Gene Editing Using Receptor-Mediated Ovary Transduction of Cargo (ReMOT) Control in Bombyx mori.

Lepidoptera is one of the most speciose insect orders, causing enormous damage to agricultural and forest crops. Although genome editing has been achieved in a few Lepidoptera for insect controls, most techniques are still limited. Here, by injecting female pupae of the Lepidoptera model species, Bombyx mori, gene editing was established using the Receptor-Mediated Ovary Transduction of Cargo (ReMOT) control technique. We identified a B. mori oocytes-targeting peptide ligand (BmOTP, a 29 aa of vitellogenin N-terminal of silkworms) with a highly conserved sequence in lepidopteran insects that could efficiently deliver mCherry into oocytes. When BmOTP was fused to CRISPR-associated protein 9 (Cas9) and the BmOTP-Cas9 ribonucleoprotein complex was injected into female pupae, heritable editing of the offspring was achieved in the silkworms. Compared with embryo microinjection, individual injection is more convenient and eliminates the challenge of injecting extremely small embryos. Our results will significantly facilitate the genetic manipulation of other lepidopteran insects, which is essential for advancing lepidopteran pest control.

Direct observation and stochastic analysis on thermally activated nucleation and growth of individual magnetic domain

The nucleation and subsequent growth of the reversed magnetic domain at a finite temperature are stochastic processes, which are often analyzed using an ensemble of magnetic elements. In this study, we investigate the stochastics of magnetization reversal in multiple trials for the sole magnetic domain. Specifically, we utilize the robust magnetic domain structure formed in a dual-exchange-biased Pt/Co/Au/Cr2O3/Pt thin film. Our investigation encompasses the latency of reversed domain nucleation and the subsequent domain wall motion based on time-lapse magnetic domain observations. The time evolution of the magnetic domain is observed after applying a pulsed magnetic field superimposed on a DC field. Magnetization reversal is triggered by the nucleation of a small embryo with finite latency, followed by domain wall propagation. The nucleation probability of the embryo increases exponentially with the DC field, thus indicating that the nucleation process obeys the Poisson process. An analysis of the relaxation time for nucleation provides a suitable expression for the energy barrier. The nucleated domain wall propagates with temporal stops, thus indicating creep motion. The temperature dependence of the relaxation time and domain wall creep motion reveal that the magnetic anisotropy in the antiferromagnetic layer significantly affect both the energy barrier for nucleation and the depinning potential of domain wall propagation. This study provides comprehensive understanding into the coercivity mechanism and contributes to the thermal stability of magnetic/spintronic devices.

Embryo recovery on consecutive days from Day 6.5 to obtain small embryos for vitrification: Is it effective?

To successfully vitrify equine embryos without aspiration or puncture of the blastocoele cavity, current methodology requires embryos ≤300µm. Such embryos are obtained by flushing mares shortly after the presumed time of entry of the embryo into the uterus (6–6.5 days post ovulation). Ovulation, and hence the timing of the flush, is usually calculated from expected ovulation times following administration of an induction agent. However, recovery rates tend to be lower at 6.5 days. A precise time for ovulation helps, but its determination is management intensive, and entry into the uterus varies by a reported 12h. The aim of this study was to determine if flushing donor mares on successive days after they failed to give an embryo on Day 6.5-7 increased recovery rates of embryos ≤300µm. During successive breeding seasons 442 inseminated cycles from donor mares, aged 3-21 years, were flushed for embryos on Day 6.5-7 with the aim of obtaining embryos ≤300µm for vitrification. Mares were flushed with Ringer's Lactate in a closed system and oxytocin given before the third flush. Filter contents were placed in a Petri dish prior to rinsing the filter with 10% PVA in Ringer's Lactate. If no embryo was found flunixin meglumine was administered and the mare re-flushed 24h later. From 442 first flushes 151 (34.2%) were positiveand 168 embryos were recovered with a median diameter of 170 µm (range; 140-320µm). The 292 negative mares, and 3 double-ovulating mares that yielded only one embryo at the first flush, were re-flushed the following day with 49 (16.6%) positive flushes yielding 51 embryos (180µm; 150-400µm). A subset of the negative mares from the second recovery attempt (n=35) were flushed the following day and 7 (20.6%) gave embryos with a median diameter of 170µm (range; 150-200 µm). Combining all flushing days, 226 embryos were recovered of which only 4 (1.8%) were ≥300µm. Although approaching significance, no difference existed in the size of embryos recovered at the first, second or third flush (P=0.051). Mare age did not appear to be a factor on the likelihood of embryo recovery on the first, second or third attempt (12 vs. 14 vs. 12 y.o., respectively; P=0.130). Therefore, when flushing donor mares to recover embryos ≤300µm, re-flushing negative mares on consecutive days results in recovery of embryos suitable for vitrification without the need for blastocoele collapse. From a physiological aspect and allowing for the lack of a precise time for ovulation in the study, there appears to be a wider margin than Day 6–6.5 for entry of the embryo into the uterus.

Embryonic morphological development is delayed in pregnancies ending in a spontaneous miscarriage

Is there a difference in embryonic morphological development between ongoing pregnancies and live pregnancies ending in a miscarriage? Embryonic morphological development, assessed by the Carnegie stages, is delayed in live pregnancies ending in a miscarriage compared to ongoing pregnancies. Pregnancies ending in a miscarriage tend to have smaller embryos and slower heart rates. Between 2010 and 2018, 644 women with singleton pregnancies, in the periconception period, were enrolled in a prospective cohort study with follow up until 1 year after delivery. A miscarriage was registered as a non-viable pregnancy before 22 weeks gestational age, defined by an absent heartbeat by ultrasound for a previously reported live pregnancy. Pregnant women with live singleton pregnancies were included and serial three-dimensional transvaginal ultrasound scans were performed. Embryonic morphological development was assessed by the Carnegie developmental stages and evaluated using virtual reality techniques. The embryonic morphology was compared to clinically used growth parameters (i.e. crown-rump length (CRL) and embryonic volume (EV)). Linear mixed models were used to evaluate the association between miscarriage and the Carnegie stages. Logistic regression with generalized estimating equations was used to calculate the odds of a miscarriage after a delay in Carnegie stages. Adjustments were made for potential confounders or covariates and include age, parity, and smoking status. A total of 611 ongoing pregnancies and 33 pregnancies ending in a miscarriage were included between 7 + 0 and 10 + 3 weeks gestational age, resulting in 1127 assigned Carnegie stages for evaluation. Compared to an ongoing pregnancy, a pregnancy ending in a miscarriage is associated with a lower Carnegie stage (βCarnegie = -0.824, 95% CI -1.190; -0.458, P < 0.001). A live embryo of a pregnancy ending in a miscarriage will reach the final Carnegie stage with a delay of 4.0 days compared to an ongoing pregnancy. A pregnancy ending in a miscarriage is associated with a smaller CRL (βCRL = -0.120, 95% CI -0.240; -0.001, P = 0.049) and EV (βEV = -0.060, 95% CI -0.112; -0.007, P = 0.027). The delay in Carnegie stage increases the odds of a miscarriage by 1.5% per delayed Carnegie stage (ORCarnegie = 1.015, 95% CI 1.002; 1.028, P = 0.028). We included a relatively small number of pregnancies ending in a miscarriage from a study population that is recruited from a tertiary referral centre. Furthermore, results of genetic testing on the products of the miscarriages or information on the karyotype of the parents were not available. Embryonic morphological development, assessed by the Carnegie stages, is delayed in live pregnancies ending in a miscarriage. In the future, embryonic morphology may be used to estimate the likelihood of a pregnancy continuing to the delivery of a healthy baby. This is of crucial importance for all women but in particular for those at risk of a recurrent pregnancy loss. As part of supportive care, both women and their partners may benefit from information on the prospective outcome of the pregnancy and the timely identification of a miscarriage. The work was funded by the Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Centre, Rotterdam, The Netherlands. The authors declare no conflicts of interest. N/A.

A novel automated method for the simultaneous detection of breathing frequency and amplitude in zebrafish (Danio rerio) embryos and larvae

Stress responses of fish to disruption of oxygen homeostasis include adjusted oxygen consumption rate (MO2) as well as the hyperventilation consisting of changes in breathing frequency (fv) and amplitude (fampl). However, studying the HVR in very small organisms such as zebrafish (Danio rerio) embryos and larvae is challenging, and breathing movements (i.e., fv) are usually manually counted, which is time- and human resource-intense, error-prone and does not provide information on the amplitude of breathing movements of the response, the breathing amplitude (fampl). Hence, in the present study, a new automated method was developed to simultaneously measure fv and fampl in small zebrafish embryos and larvae with the computer software DanioScope™. To compare HVR strategies at different life-stages of zebrafish and the physiologically linked MO2, hatched 4 d old embryos and early gill-breathing 12 d old larvae were treated with the HVR-inducing neurotoxic compound lindane (γ-hexachlorocyclohexane; γ-HCH) as a model substance. Comparison of manually counted fv with fv data measured by DanioScope™ at both life-stages showed high to moderate agreement between the two methods with respect to fv in control fish and in fish treated with lower lindane concentrations (3 - 18% deviation at 25 µg/L γ-HCH). With increasing lindane concentrations (100 and 400 µg/L γ-HCH), however, manual counts showed an average underestimation of fv by up to 30%, mainly due to very fast, rapidly successive, and indistinct movements of the fish, which cannot be properly detected by manual counts. Automated measurement thus proved significantly more sensitive, although several pre- and post-processing steps are needed. The improved automated detection of fv and the first reliable estimation of fampl in small fish embryos and larvae, as well as the inclusion of MO2, may provide new insights into different respiratory strategies and may, thus, represent a tool to lower the detection limit for reactions of different life-stages of fish to environmental stressors. In the present study, this became evident, as early gill-breathing 12 d old zebrafish larvae showed symptoms of respiratory failure (i.e., increase in fv, fampl and MO2, followed by subsequent lethargy) after exposure to lindane, whereas skin-breathing in 4 d old embryos proved mainly insensitive to the paralytic effects of lindane.

A novel method for measuring acute thermal tolerance in fish embryos.

Aquatic ectotherms are vulnerable to thermal stress, with embryos predicted to be more sensitive than juveniles and adults. When examining the vulnerability of species and life stages to warming, comparable methodology must be used to obtain robust conclusions. Critical thermal methodology is commonly used to characterize acute thermal tolerances in fishes, with critical thermal maximum (CTmax) referring to the acute upper thermal tolerance limit. At this temperature, fish exhibit loss of controlled locomotion due to a temperature-induced collapse of vital physiological functions. While it is relatively easy to monitor behavioural responses and measure CTmax in larval and adult fish, this is more challenging in embryos, leading to a lack of data on this life stage, or that studies rely on potentially incomparable metrics. Here, we present a novel method for measuring CTmax in fish embryos, defined by the temperature at which embryos stop moving. Additionally, we compare this measurement with the temperature of the embryos' last heartbeat, which has previously been proposed as a method for measuring embryonic CTmax. We found that, like other life stages, late-stage embryos exhibited a period of increased activity, peaking approximately 2-3°C before CTmax. Measurements of CTmax based on last movement are more conservative and easier to record in later developmental stages than measurements based on last heartbeat, and they also work well with large and small embryos. Importantly, CTmax measurements based on last movement in embryos are similar to measurements from larvae and adults based on loss of locomotory control. Using last heartbeat as CTmax in embryos likely overestimates acute thermal tolerance, as the heart is still beating when loss of response/equilibrium is reached in larvae/adults. The last movement technique described here allows for comparisons of acute thermal tolerance of embryos between species and across life stages, and as a response variable to treatments.

Rocky Histories: The Effect of High Excitations on the Formation of Rocky Planets

Rocky planets both in and outside of our solar system are observed to have a range of core-mass fractions (CMFs). Imperfect collisions can preferentially strip mantle material from a planet, changing its CMF, and are therefore thought to be the most likely cause of this observed CMF variation. However, previous work that implements these collisions into N-body simulations of planet formation has struggled to reliably form high CMF super-Earths. In this work, we specify our initial conditions and simulation parameters to maximize the prevalence of high-energy, CMF-changing collisions in order to form planets with highly diverse CMFs. High-energy collisions have a large v imp/v esc ratio, so we maximize this ratio by starting simulations with high eccentricity and inclination disks to increase the difference in their orbital velocities, maximizing v imp. Additionally, we minimize v esc by starting with small embryos. The final planets undergo more high-energy, debris-producing collisions, and experience significant CMF change over their formation. However, we find that a number of processes work together to average out the CMF of a planet over time; therefore, we do not consistently form high CMF, high-mass planets. We do form high CMF planets below 0.5 M ⊕. Additionally, we find, in these highly eccentric environments, loss of debris mass due to collisional grinding has a significant effect on final planet masses and CMFs, resulting in smaller planets and a higher average planet CMF. This work highlights the importance of improving measurements of high-density planets to better constrain their CMFs.

Dynamic regulation of the transcriptome and proteome of the equine embryo during maternal recognition of pregnancy.

During initial maternal recognition of pregnancy (MRP), the equine embryo displays a series of unique events characterized by rapid blastocyst expansion, secretion of a diverse array of molecules, and transuterine migration to interact with the uterine surface. Up to date, the intricate transcriptome and proteome changes of the embryo underlying these events have not been critically studied in horses. Thus, the objective of this study was to perform an integrative transcriptomic (including mRNA, miRNAs, and other small non-coding RNAs) and proteomic analysis of embryos collected from days 10 to 13 of gestation. The results revealed dynamic transcriptome profiles with a total of 1311 differentially expressed genes, including 18 microRNAs (miRNAs). Two main profiles for mRNAs and miRNAs were identified, one with higher expression in embryos ≤5 mm and the second with higher expression in embryos ≥7 mm. At the protein level, similar results were obtained, with 259 differentially abundant proteins between small and large embryos. Overall, the findings demonstrated fine-tuned transcriptomic and proteomic regulations in the developing embryo associated with embryo growth. The identification of specific regulation of mRNAs, proteins, and miRNAs on days 12 and 13 of gestation suggested these molecules as pivotal for embryo development and as involved in MRP, and in establishment of pregnancy in general. In addition, the results revealed new insights into prostaglandin synthesis by the equine embryo, miRNAs and genes potentially involved in modulation of the maternal immune response, regulation of endometrial receptivity and of late implantation in the mare.

Topographical relationships of the yolk sac remnant and vitelline vessels with the midgut loop in the extra-embryonic coelom of human embryos.

The yolk sac is supplied by the vitelline artery and vein (VA, VV), which run through the yolk stalk in combination with the omphaloenteric duct. Moreover, the VV takes a free posterior course outside the midgut mesentery containing the secondarily-developed superior mesenteric vein (SMV). However, the regression process of these structures has not been demonstrated photographically. The present study evaluated serial histological sections from 20 embryos of stages 15–19 or crown-rump length (CRL) 7.5–20 mm. All specimens carried the SMV as sequential tissue slits. However, an omphaloenteric duct with epithelia continuous with the midgut loop was not observed. In smaller embryos (CRL <13 mm) the VA extended distally or anteriorly from the midgut apex in the extra-embryonic coelom, whereas in larger embryos (CRL 16–20 mm) the artery was absent from the distal side of the apex. The entire course or part of the VV outside the mesentery was always seen, but four larger embryos lacked the venous terminal near the duodenum. A vacuole-like remnant of the yolk sac was present in all smaller embryos (CRL <10 mm), but was absent from 7 of the 11 larger embryos. The size of the remnant was equal to the thickness of the VA or VV, with the remnant being sandwiched between the VA and VV. Moreover, the regressing yolk sac often communicated with or opened to the VV. Consequently, the yolk sac regressed first, followed by the regression of the VA until 6 weeks. The yolk stalk was clearly observed until 5 weeks.

Single-Step Genome Editing of Small Ruminant Embryos by Electroporation.

We investigated the possibility of single-step genome editing in small ruminants by CRISPR-Cas9 zygote electroporation. We targeted SOCS2 and PDX1 in sheep embryos and OTX2 in goat embryos, utilizing a dual sgRNA approach. Gene editing efficiency was compared between microinjection and three different electroporation settings performed at four different times of embryo development. Electroporation of sheep zygotes 6 h after fertilization with settings that included short high-voltage (poring) and long low-voltage (transfer) pulses was efficient at producing SOCS2 knock-out blastocysts. The mutation rate after CRISPR/Cas9 electroporation was 95.6% ± 8%, including 95.4% ± 9% biallelic mutations; which compared favorably to 82.3% ± 8% and 25% ± 10%, respectively, when using microinjection. We also successfully disrupted the PDX1 gene in sheep and the OTX2 gene in goat embryos. The biallelic mutation rate was 81 ± 5% for PDX1 and 85% ± 6% for OTX2. In conclusion, using single-step CRISPR-Cas9 zygote electroporation, we successfully introduced biallelic deletions in the genome of small ruminant embryos.

Floral characteristics, pollen morphology, and viability of sugarcane hybrids (Saccharum × officinarum) and the neotropical wild relative, S. villosum

Interspecific hybridization occurs naturally only under restricted conditions and is a phenomenon with evolutionary and agricultural implications. Production of viable pollen grains is required in at least one of the species involved, provided the other parental species is female-fertile. In this study, we assessed pollen viability in the South American native Saccharum villosum and in S. x officinarum (sugarcane), which are partly sympatric. Sex distribution in sugarcane hybrids and reproductive morphology of S. villosum were analyzed to verify potential conditions for interspecific hybridization. Five of the sugarcane hybrids were shown to be exclusive hermaphrodites, two of them protogynous and one protandrous, and three hybrids were shown to be gynomonoecious, all potentially able to receive and donate pollen. All hybrids have viable pollen grains, but at different degrees (ca. 4% to 62%). S. villosum produces more viable pollen than sugarcane in southeastern Brazil. Pollen grains in S. villosum are middle-sized (32-39 μm in diameter), monoporate, monadic, prolato-spheroidal, isopolar, with exine ornamentation consisting of ungrouped granules, being distinguishable from sugarcane pollen. Pollen grains in S. villosum are highly variable for the five internal traits measured, especially for nexine and tectum thicknesses. Seed development of S. villosum occurs before panicle emergence. A morpho-anatomical analysis of the germinated seed revealed that S. villosum has traits considered as derived within Poaceae, such as the lack of epiblast. However, this species is an early-divergent Saccharum member, based on a small relative embryo size. In conclusion, the absence of natural hybrids between sugarcane and S. villosum is more probably associated with either autogamy or agamospermy in the wild species rather than with pollen inviability or absence of pollen production in sugarcane.

Improvement of In Vitro Seed Germination and Micropropagation of Amomum tsao-ko (Zingiberaceae Lindl.)

Black cardamom (Amomum tsao-ko Crevost &amp; Lemarié) is a spice plant of great commercial value in Vietnam, but with limited propagation ability. Its seeds are characterized by a thick and hard seed coat, a small endosperm, and a small embryo, which are the causes of the physical dormancy of the seeds and low germination. Attempts in this study to improve the germination rate and achieve uniform germination included mechanical scarification, immersion in hot or cold water, acid scarification, and the application of plant growth regulators. Although immersion of seeds in cold water and application of plant growth regulators (PGRs) (gibberellic acid (GA3) and 1-naphtaleneacetic acid (NAA)) showed positive effects on seed germination and subsequent seedling growth, mechanical scarification provided the highest germination rate of black cardamom seeds (68.0%) and significantly shortened germination time (53.7 days) compared to control (16.0% and 74.7 days). On the other hand, an efficient micropropagation protocol has been established using shoot tip explants derived from in-vitro-grown seedlings. Murashige and Skoog (MS) medium supplemented with 4.0 mg/L 6-benzylaminopurine (BAP) + 0.5 mg/L NAA proved to be most suitable for rapid multiplication and rooting, providing a mean of 5.4 shoots per explant, 6.8 cm shoot length, and 16.2 roots per explant after 7 weeks of culture. Well-rooted black cardamom plantlets have been successfully adapted to ex vitro conditions. “Fasco” bio-soil was more suitable for acclimatization, with a 48.9% survival rate, 23.3 cm plant length, and 5.7 leaves per plant after 3 months of planting. Improved germination and multiplication protocols can be used to improve propagation performances and to develop elite of black cardamom planting material.

The Effect of Accretion Rate and Composition on the Structure of Ice-rich Super-Earths

It is reasonable to assume that the structure of a planet and the interior distribution of its components are determined by its formation history. We thus follow the growth of a planet from a small embryo through its subsequent evolution. We estimate the accretion rate range based on a protoplanetary disk model at a large-enough distance from the central star for water ice to be a major component. We assume the accreted material to be a mixture of silicate rock and ice, with no H–He envelope, as the accretion timescale is much longer than the time required for the nebular gas to dissipate. We adopt a thermal evolution model that includes accretional heating, radioactive energy release, and separation of ice and rock. Taking the Safronov parameter and the ice-to-rock ratio as free parameters, we compute growth and evolutionary sequences for different parameter combinations, for 4.6 Gyr. We find the final structure to depend significantly on both parameters. Low initial ice-to-rock ratios and high accretion rates, each resulting in an increased heating rate, lead to the formation of extended rocky cores, while the opposite conditions leave the composition almost unchanged and result in relatively low internal temperatures. When rocky cores form, the ice-rich outer mantles still contain rock mixed with the ice. We find that a considerable fraction of the ice evaporates upon accretion, depending on parameters, and assume it is lost, thus the final surface composition and bulk density of the planet do not necessarily reflect the protoplanetary disk composition.

In Situ Electrochemical Derivation of Sodium-Tin Alloy as Sodium-Ion Energy Storage Devices Anode with Overall Electrochemical Characteristics

Inspired by the fermentation of multiple small bread embryos to form large bread embryos, in this study, the expansion of tin foil inlaid with sodium rings in the process of repeated sodium inlaid and removal was utilized to maximum extent to realize the formation of sodium-tin alloy anode and the improvement of sodium storage characteristics. The special design of Sn foil inlaid with Na ring realized the in-situ electrochemical formation of fluffy porous sodium-tin alloy, effectively alleviated the volume expansion and shrinkage of non-electrochemical active Sn metal, and inhibited the generation of sodium dendrites. The abundance of sodium ions provided by the Na metal ring compensated for the active sodium components consumed during the repeated formation of SEI. When sodium-tin alloy in situ derived by Sn foil inlaid with Na ring was used as negative electrodes matched with SCDC and Na0.91MnO2 hexagonal tablets (NMO HTs) positive electrodes, the as-assembled sodium-ion energy storage devices present high specific capacity and excellent cycle stability.

Polyembryony in citrus: does the largest embryo in the seed develop a nucellar seedling?

A generalized concept in the Citrus genus is that highly polyembryonic varieties produce only a small number of hybrids. Small zygotic embryos congregate primarily near the micropyle, while nucellar embryos organize near or away from the micropyle. In the present study, the authors determined the number of polyembryonic seeds, embryos per seed, and the largest embryo (LE) position in five citrus cultivars: C35 citrange, Volkamer lemon, Amblycarpa mandarin, Minneola tangelo, and Valencia orange. The percentage of nucellar seedlings obtained exclusively from the LE per seed was then calculated. The polyembryony percentage varied largely between genotypes, from 65 to 98 %, and the mean number of embryos ranged from 2.9 to 4.6. The chalaza contained up to 87 % of the LE. Out of 30 primers, 17 Simple sequence repeats [SSRs] (AG14, ATC09, CAT01, CCSM147, CCSM18, CCSM13, CCSM4, F2, F4, F6, GT03, TAA41, TAA45, TAA1, F7, F11 and TAA52) identified nucellar plants identical to the female parent (genetic similarity index [GSI] value ≥ 0.95). This study establishes for the first time the relationship between the sexual or asexual origin of seedlings derived from LE embryos isolated from seeds and the SSR primers described above. While the five citrus cultivars had high polyembryony levels, 30 % of the resulting plants differed from the female parent in C35 Citrange, 45 % in Volkamer lemon, 15 % in Amblycarpa mandarin, 15 % in Valencia orange, and 45 % in Minneola tangelo. The largest seedling is not always nucellar: in the five citrus species studied, the LE produces 55 to 85 % of the nucellar embryos.