Embryo Physiology Research Articles

Effects of Thermal Manipulation During the Second Half of Incubation on Embryo Physiology, Hatching Parameters, and Quality of Broiler Chickens in Tropical Climate of Togo

Chickens are sensitive to environmental challenges caused by temperature. The current study aimed to determine the effects of heat manipulation during embryonic development on the physiological responses of Goliath chickens. A total of 2000 hatching eggs from 48-week-old breeders were weighed, numbered, and randomly distributed equally into 4 incubators. Each incubator received 500 eggs (4 replicates of 125 eggs each). Eggs in two of the incubators were rotated hourly at a 45° angle and maintained at 37.8°C and 60% relative humidity (T0 groups). Between embryonic days (ED) 10 and 18 of incubation, the eggs from the other two incubators were heated to 38.5°C for 6 hours per day (T1 groups). The eggs were reweighed and candled, and viable eggs were moved to the hatching baskets at ED 18 of incubation. Hatching eggs were examined individually for hatching events every three hours during the final three days of incubation. On day 21, blood samples were collected from 12 chicks per group for hormonal and biochemical analyses. The evaluated blood parameters included Triiodothyronine (T3), T4 (thyroxine), cortisol, uric acid, lactate dehydrogenase, and total protein. At hatch, chicks were weighed and their quality (survival after hatching and performance standards) was evaluated. Data were collected on embryonic development, hatching window, hatching events, biochemical parameters, and hormonal concentrations. Results indicated that hatchability, chick’s weight, Tri-iodothyronine, and corticosterone were higher in the T1 group, compared to the control group. At hatch on day 21, the pipping muscle of chicks in the treated group (T1) was significantly heavier than that of the control group, while the embryonic mortality rate was significantly higher in the T0 group. In conclusion, applying heat treatment for 6 hours at 38.5°C from ED10-ED18 of embryogenesis increased significantly the hatching rate, the pipping muscle, and the chick’s weight in this study. Keywords: Embryonic development, Physiology, Slow-growing broiler, Thermal manipulation, Tropical climate

Per- and Polyfluoroalkyl Substances: Impacts on Morphology, Behavior and Lipid Levels in Zebrafish Embryos.

The presence of per- and polyfluoroalkyl substances (PFASs) in aquatic environments is often persistent and widespread. Understanding the potential adverse effects from this group of chemicals on aquatic communities allows for better hazard characterization. This study examines impacts on zebrafish (Danio rerio) embryo physiology, behavior, and lipid levels from exposure to perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), and heptadecafluorooctanesulfonic acid (PFOS). Embryos were exposed to lethal and sublethal levels of each chemical and monitored for alterations in physiological malformations, mortality, lipid levels, and behavior (only PFOA and PFHxS). The predicted 50% lethal concentrations for 120 hpf embryos were 528.6 ppm PFOA, 14.28 ppm PFHxS, and 2.14 ppm PFOS. Spine curvature and the inability of the 120 hpf embryos to maintain a dorsal-up orientation was significantly increased at 10.2 ppm PFHxS and 1.9 ppm PFOS exposure. All measured 120 hpf embryo behaviors were significantly altered starting at the lowest levels tested, 188 ppm PFOA and 6.4 ppm PFHxS. Lipid levels decreased at the highest PFAS levels tested (375 PFOA ppm, 14.4 PFHxS ppm, 2.42 ppm PFOS). In general, the PFAS chemicals, at the levels examined in this study, increased morphological deformities, embryo activity, and startle response time, as well as decreased lipid levels in 120 hpf zebrafish embryos.

Bovine embryo production in vitro: evolution of culture media and commercial perspectives.

In vitro produced embryos exhibit lower viability compared to their in vivo counterparts. Mammalian preimplantation embryos have the ability to reach the blastocyst stage in diverse culture media, showcasing considerable metabolic adaptability, which complicates the identification of optimal developmental conditions. Despite embryos successfully progressing to the blastocyst stage, adaptation to suboptimal culture environments may jeopardize blastocyst viability, cryotolerance, and implantation potential. Enhancing our capacity to support preimplantation embryonic development in vitro requires a deeper understanding of fundamental embryo physiology, including preferred metabolic substrates and pathways utilized by high-quality embryos. Armed with this knowledge, it becomes achievable to optimize culture conditions to support normal, in vivo-like embryo physiology, mitigate adaptive stress, and enhance viability. The objective of this review is to summarize the evolution of culture media for bovine embryos, highlighting significant milestones and remaining challenges.

Triphenyl phosphate-induced pericardial edema in zebrafish embryos is reversible following depuration in clean water

Triphenyl phosphate (TPHP) – a widely used organophosphate-based flame retardant – blocks cardiac looping during zebrafish development in a concentration-dependent manner, a phenotype that is dependent on disruption of embryonic osmoregulation and pericardial edema formation. However, it's currently unclear whether (1) TPHP-induced effects on osmoregulation are driven by direct TPHP-induced injury to the embryonic epidermis and (2) whether TPHP-induced pericardial edema is reversible or irreversible following cessation of exposure. Therefore, the objectives of this study were to determine whether TPHP-induced pericardial edema is reversible and whether TPHP causes injury to the embryonic epidermis by quantifying the number of DAPI-positive epidermal cells and analyzing the morphology of the yolk sac epithelium using scanning electron microscopy. First, we found that exposure to 5 μM TPHP from 24–72 h post-fertilization (hpf) did not increase prolactin – a hormone that regulates ions and water levels – in embryonic zebrafish, whereas high ionic strength exposure media was associated with elevated levels of prolactin. Second, we found that exposure to 5 μM TPHP from 24–72 hpf did not decrease DAPI-positive epidermal cells within the embryonic epithelium, and that co-exposure with 2.14 μM fenretinide – a synthetic retinoid that promotes epithelial wound repair – from 24–72 hpf did not mitigate the prevalence of TPHP-induced epidermal folds within the yolk sac epithelium when embryos were exposed within high ionic strength exposure media. Finally, we found that the pericardial area and body length of embryos exposed to 5 μM TPHP from 24–72 hpf were similar to vehicle-treated embryos at 120 hpf following transfer to clean water and depuration of TPHP from 72–120 hpf. Overall, our findings suggest that (1) the ionic strength of exposure media may influence the baseline physiology of zebrafish embryos; (2) TPHP does not cause direct injury to the embryonic epidermis; and (3) TPHP-induced effects on pericardial area and body length are reversible 48 h after transferring embryos to clean water.

A phenomics approach reveals interspecific differences in integrated developmental responses to chronic elevated temperatures.

Phenomics, high-dimensional organismal phenotyping, is advanced as a solution to quantifying complex developmental responses to elevated temperatures. 'Energy proxy traits' (EPTs) measure the phenotype as a spectrum of energy values across different temporal frequencies from pixel value fluctuations of video. Although they have proven effective in measuring the biology of complex and dynamic developing organisms, their utility in assessing environmental sensitivity of different species is untested. Using EPTs, we assess the relative thermal sensitivities of embryos of three species of freshwater snail with marked differences in their developmental event timings. Embryos of Lymnaea stagnalis, Radix balthica and Physella acuta were videoed hourly for the duration of their embryonic development at two temperatures: 20°C and 25°C. The video was used to calculate EPTs for the duration of their embryonic development, and during discrete physiological windows in development. Changes in energy spectra during development identified marked differences in thermal sensitivities between species, and suggest a relatively heightened sensitivity of gross rates of embryonic physiology and behaviour in embryos of R. balthica, developmental-window-specific thermal responses that reflect ontogenetic differences in observable physiologies, and temperature-induced changes in physiological event timing. EPTs enabled comparison of high-dimensional spectral phenotypes, providing a unique capability for assessing sensitivity continuously in developing individuals. Such integrative and scalable phenotyping is a prerequisite for improved understanding of the sensitivity of early life stages of different species.

Seed dormancy revisited: Dormancy‐release pathways and environmental interactions

Abstract Many internal (inherent) and environmental (imposed) factors control seed dormancy and germination that we divide into three basic dormancy‐release pathways: Maternal structures and embryo physiology control inherent dormancy that is broken by various types of scarification and physiological changes, followed by imposed‐dormancy release when the prevailing environment is replaced by certain ‘standard’ conditions that stimulate germination (Pathway 1); imposed dormancy prevails even if inherent dormancy is broken or not applicable that is released when replaced by certain ‘standard’ environmental conditions which stimulate germination (Pathway 2); and release from inherent dormancy by light/dark or cold stratification is contingent on the pre‐existence of certain environmental conditions that stimulate germination (Pathway 3). On‐plant seed storage (serotiny) and frugivorous seeds are recognized here as representing special types of physical dormancy, as their properties are consistent with those of hard diaspores. Warm stratification does not require seeds to be moist as it is just a physical response. Heat may promote germination of non‐hard, as well as hard, seeds as it may increase their permeability further. Levels of germination gauge the net effect of inherent‐ and imposed‐dormancy release so that it is only possible to identify the extent of inherent‐dormancy release when conditions for germination are optimal (imposed dormancy has been annulled). While imposed dormancy may be protracted after inherent dormancy is broken by heat or chilling during the dry or cold seasons, release from both states may effectively coincide if smoke chemicals or light are received during the (wet) growing season. We suggest reserving the term secondary dormancy for seeds that return to (inherent or imposed) dormancy due to changed environmental conditions. Under seasonal climates, fluctuations in environmental conditions can lead to secondary dormancy and even dormancy cycling. We recognize four types of functional interactions between any two environmental factors that induce inherent‐dormancy release: binary interactions are ineffective, only one stimulus is effective, both are effective but non‐additive, or both are additive/synergistic. Two environmental stimuli that individually break dormancy but have no additive effect must be affecting the same process; this was demonstrated here for some interactions between heat and smoke. The three dormancy‐release pathways, together with internal, seasonal and stochastic interactions, are coordinated by the non‐dormant seed to ensure maximum germination under optimal conditions. To ignore any aspect outlined here leads to an impoverished understanding of the disparate seed ecology of species adapted to different stressful and disturbance‐prone habitats. Read the free Plain Language Summary for this article on the Journal blog.

Acid-base and hematological regulation in chicken embryos during internal progressive hypercapnic hypoxia

Development of the capacity to mitigate potential disturbances to blood physiology in bird embryos is incompletely understood. We investigated regulation of acid-base and hematology in day 15 chicken embryos exposed to graded intrinsic hypercapnic hypoxia created by varying degrees of water submersion. Metabolic acidosis with additional respiratory or metabolic acidosis occurred at 2 h according to magnitude of submersion. Acid-base disturbance was partially compensated by metabolic alkalosis at 6 h, but compensatory metabolic alkalosis was absent at 24 h. Following submersion with only air cell exposed to air, both hypercapnic respiratory acidosis and metabolic acidosis occurred within 10 min. Subsequently, both forms of acidosis created lethal levels of [HCO3-] at ∼120 min. Blood hematology showed small but significant effects associated with induced acid-base disturbance. Increased Hct occurring during partial egg submersion lasting 24 h was attributed to an increase in MCV. By day 15 of development chicken embryos are able to partially compensate for and withstand all but severe induced internal hypoxic hypercapnia.

β-hydroxybutyrate reduces blastocyst viability via trophectoderm-mediated metabolic aberrations in mice.

STUDY QUESTIONWhat is the effect of the ketone β-hydroxybutyrate (βOHB) on preimplantation mouse embryo development, metabolism, epigenetics and post-transfer viability?SUMMARY ANSWER In vitro βOHB exposure at ketogenic diet (KD)-relevant serum concentrations significantly impaired preimplantation mouse embryo development, induced aberrant glycolytic metabolism and reduced post-transfer fetal viability in a sex-specific manner.WHAT IS KNOWN ALREADYA maternal KD in humans elevates gamete and offspring βOHB exposure during conception and gestation, and in rodents is associated with an increased time to pregnancy, and altered offspring organogenesis, post-natal growth and behaviour, suggesting a developmental programming effect. In vitro exposure to βOHB at supraphysiological concentrations (8–80 mM) perturbs preimplantation mouse embryo development.STUDY DESIGN, SIZE, DURATIONA mouse model of embryo development and viability was utilized for this laboratory-based study. Embryo culture media were supplemented with βOHB at KD-relevant concentrations, and the developmental competence, physiology, epigenetic state and post-transfer viability of in vitro cultured βOHB-exposed embryos was assessed.PARTICIPANTS/MATERIALS, SETTING, METHODSMouse embryos were cultured in vitro with or without βOHB at concentrations representing serum levels during pregnancy (0.1 mM), standard diet consumption (0.25 mM), KD consumption (2 mM) and diabetic ketoacidosis (4 mM). The impact of βOHB exposure on embryo development (blastocyst formation rate, morphokinetics and blastocyst total, inner cell mass and trophectoderm (TE) cell number), physiology (redox state, βOHB metabolism, glycolytic metabolism), epigenetic state (histone 3 lysine 27 β-hydroxybutyrylation, H3K27bhb) and post-transfer viability (implantation rate, fetal and placental development) was assessed.MAIN RESULTS AND THE ROLE OF CHANCEAll βOHB concentrations tested slowed embryo development (P < 0.05), and βOHB at KD-relevant serum levels (2 mM) delayed morphokinetic development, beginning at syngamy (P < 0.05). Compared with unexposed controls, βOHB exposure reduced blastocyst total and TE cell number (≥0.25 mM; P < 0.05), reduced blastocyst glucose consumption (2 mM; P < 0.01) and increased lactate production (0.25 mM; P < 0.05) and glycolytic flux (0.25 and 2 mM; P < 0.01). Consumption of βOHB by embryos, mediated via monocarboxylate transporters, was detected throughout preimplantation development. Supraphysiological (20 mM; P < 0.001), but not physiological (0.25–4 mM) βOHB elevated H3K27bhb levels. Preimplantation βOHB exposure at serum KD levels (2 mM) reduced post-transfer viability. Implantation and fetal development rates of βOHB-treated embryos were 50% lower than controls (P < 0.05), and resultant fetuses had a shorter crown-rump length (P < 0.01) and placental diameter (P < 0.05). A strong sex-specific effect of βOHB was detected, whereby female fetuses from βOHB-treated embryos weighed less (P < 0.05), had a shorter crown-rump length (P < 0.05), and tended to have accelerated ear development (P < 0.08) compared with female control fetuses.LIMITATIONS, REASONS FOR CAUTIONThis study only assessed embryo development, physiology and viability in a mouse model utilizing in vitro βOHB exposure; the impact of in vivo exposure was not assessed. The concentrations of βOHB utilized were modelled on blood/serum levels as the true oviduct and uterine concentrations are currently unknown.WIDER IMPLICATIONS OF THE FINDINGSThese findings indicate that the development, physiology and viability of mouse embryos is detrimentally impacted by preimplantation exposure to βOHB within a physiological range. Maternal diets which increase βOHB levels, such as a KD, may affect preimplantation embryo development and may therefore impair subsequent viability and long-term health. Consequently, our initial observations warrant follow-up studies in larger human populations. Furthermore, analysis of βOHB concentrations within human and rodent oviduct and uterine fluid under different nutritional states is also required.STUDY FUNDING/COMPETING INTEREST(S)This work was funded by the University of Melbourne and the Norma Hilda Schuster (nee Swift) Scholarship. The authors have no conflicts of interest.TRIAL REGISTRATION NUMBERN/A.

Effect of estrous cycle phases on gene expression in bovine oviduct epithelial cells.

Background and Aim:The oviduct environment is of particular importance because it is the site of fertilization and early embryo development. The oviduct, as a component of the reproductive system, responds to ovarian hormone (estradiol [E2] and progesterone [P4]) stimuli depending on the estrous cycle phase. This study aimed to elucidate the effect of estrous cycle phases (follicular and early and late luteal phases) on gene expression patterns in bovine oviduct epithelial cells (BOECs).Materials and Methods:Oviducts were obtained from healthy slaughterhouse animals, corresponding to ipsilateral ovaries with dominant follicles or corpus luteum during early and late luteal phases. BOECs were recovered from the isthmus (IST) and ampulla (AMP), and the expression patterns of genes related to cytokinesis and mitosis mechanisms (rho-associated coiled-coil containing protein kinase and cellular communication network factor 2 [CCN2]), growth factors (insulin-like growth factor-binding protein 3, epidermal growth factor receptor [EGFR], vascular endothelial growth factor A, and EGFR), antioxidant mechanisms (glutathione peroxidase 4 [GPX4]), apoptosis (B-cell lymphoma 2), complement component (C3), energy metabolism (aldose reductase gene family 1-member b1 [AKRIB1] and solute carrier family 2), hormone receptors (estrogen receptor 1 and luteinizing hormone/choriogonadotropin receptor), and specific glycoproteins (oviductal glycoprotein 1) were analyzed.Results:High P4 levels (late luteal phase) affected the expression of important genes related to antioxidant mechanisms (GPX4), energy metabolism (AKRIB1), growth factors (IGBP3 and EGFR), and cell growth regulation (CCN2) in the AMP. Low P4 levels (early luteal phase) affected the expression of AKR1B1, IGBP3, and CCN2. In addition, estrogen likely had an effect on OVPGP expression in the cattle oviduct.Conclusion:Differential gene expression patterns of BOECs in the AMP during the luteal phase (antioxidant mechanisms, energy metabolism, growth factors, and immunological regulators) and in the IST during the follicular phase (glycoproteins) may influence their renewal and population proportions, modulating the oviduct environment as well as gamete and embryo physiology.

P-227 Exposure to triclosan modifies oocyte development and embryo metabolism

Abstract Study question Does the endocrine disruptor, triclosan, affect the function of cumulus-oocyte-complexes (COCs), and if so, do these effects persist in early embryo developmental competence and metabolism? Summary answer Triclosan caused COCs to i) increase progesterone secretion, ii) switch from releasing to consuming pyruvate, and iii) produce fewer blastocysts that were iv) metabolically compromised. What is known already Triclosan (TCS) is used widely as an antimicrobial agent in a range of consumer healthcare products, including hand sanitiser, toothpastes, soaps, and shampoos. Its prevalent use has led to accumulation in the environment and human exposure. Indeed, TCS has been identified in a range of bodily fluids. It is classed as an endocrine-disrupting chemical and there are reports of its detrimental impact on the vertebrate reproductive system. However, the direct effects of TCS on mammalian Cumulus-Oocyte-Complexes’ (COCs) function and subsequent development of the embryo remains unknown. Study design, size, duration COCs, isolated from abattoir-derived bovine ovaries on 5 independent occasions, were exposed of two environmentally-relevant doses of TCS (10nM and 1nM) during in vitro maturation. The TCS-exposed COCs were subsequently fertilised in vitro without further TCS addition, and the cleavage and blastocyst rate were recorded on Day 2 and Day 7/Day 8, respectively. The resulting blastocysts were collected and further cultured individually for 24 hours, and depletion/release of key-metabolites quantified. Participants/materials, setting, methods In vitro production was performed for the maturation of the bovine oocytes and the embryo culture. The production of steroid hormones (17b-estradiol and progesterone) during IVM was determined by ELISA. The concentrations of glucose, pyruvate, lactate during IVM and blastocyst formation were quantified in the spent media using microfluorometric assays. Main results and the role of chance TCS at 1nM had a significant impact on steroidogenesis during COCs in vitro maturation; progesterone production was significantly higher compared to control group (4.784 ±1.69 vs 2.85 ±0.83 pg/hour/COCs, p = 0.035; n = 5), although oestrogen release was not affected. In addition, there was a dramatic shift in pyruvate metabolism. Over the course of in vitro maturation, COCs exposed to TCS depleted pyruvate from the culture media regardless of the administrated dose whilst net pyruvate concentration increased in the medium of COCs in the control groups. The cleavage rate of bovine embryos produced from TCS-exposed COCs was not significantly different to control group. However, COCs-exposed to 1nM TCS produced significantly fewer blastocysts (12.22% vs 29.11% n = 5; p = 0.02). Those blastocysts that did originate from COCs treated with TCS depleted significantly more pyruvate from the culture media compared to controls, regardless of TCS dose (19.86 ±10.54 vs 10.12 ±12.5 pmol/hour/embryo, p = 0.011 and 21.41 ±10.08 vs 10.12 ±12.5 pmol/hour/embryo, p = 0.002, TCS-10nM and TCS-1nM compared to Control, respectively). Additionally, while glucose depletion did not differ, the lactate release from blastocysts derived from COCs exposed to 10nM TCS was approximately halved compared to controls (15.20 ±14.59 vs 31.83 ±22.73 pmol/hour/embryo p = 0.0054). Limitations, reasons for caution The origin of the ovaries may be a limitation since they came from animals at different stages of estrous cycle. Furthermore, pre-exposure to TCS prior to animal slaughter cannot be discounted. Finally, these observations were made in an animal model in a laboratory setting which may differ from natural exposure. Wider implications of the findings TCS modifies oocyte maturation, developmental competence, and metabolic activity of resulting embryos. The differences in pyruvate metabolism and steroidogenesis point to altered mitochondrial function. These data indicate that TCS directly modulates oocyte and embryo physiology which may relate to fertility in domestic livestock species and may be extrapolated to humans. Trial registration number not applicable

Chicken Incubation Conditions: Role in Embryo Development, Physiology and Adaptation to the Post-Hatch Environment.

The chicken hatching egg is a self-contained life-supporting system for the developing embryo. However, the post-hatch performance of birds depends on several factors, including the breeder management and age, egg storage conditions and duration before incubation, and the incubation conditions. Studies have determined the effect of incubation factors on chick post-hatch growth potential. Therefore, chick physical quality at hatch is receiving increasing attention. Indeed, although incubation temperature, humidity, turning and ventilation are widely investigated, the effects of several variables such as exposure of the embryo to high or low levels, time of exposure, the amplitude of variations and stage exposures on embryo development and post-hatch performance remain poorly understood. This review paper focuses on chick quality and post-hatch performance as affected by incubation conditions. Also, chick physical quality parameters are discussed in the context of the parameters for determining chick quality and the factors that may affect it. These include incubation factors such as relative humidity, temperature, turning requirements, ventilation, in ovo feeding and delay in feed access. All these factors affect chick embryo physiology and development trajectory and consequently the quality of the hatched chicks and post-hatch performance. The potential application of adapted incubation conditions for improvement of post-hatch performance up to slaughter age is also discussed. It is concluded that incubation conditions affect embryo parameters and consequently post-hatch growth differentially according to exposure time and stage of exposure. Therefore, classical physical conditions are required to improve hatchability, chick quality and post-hatch growth.

Illustrating the past, present and future perspective of Human Embryo Culture Media

Culture media has an important role in invitro embryo development and in determining the success rate of assisted reproduction technologies. Extensive research during the last few decades have brought about major advancements in the IVF culture media. Studies on the composition of the mammlian oviductal fluids and the uterine fluids have improved our understanding on the evolving metabolic needs of the preimplantation embryo. This has led to the gradual development of human IVF media, from simple salt solutions to highly complex defined media. Regarding ideal composition of IVF culture media two different philosophies have come up: “back-to-nature” or sequential culture principle and “let-the-embryo-choose” or one-step culture principle. Sequential culture closely mimics the physiological conditions of female reproductive tract, while the mono culture system employs simultaneous use of all components in an optimal concentration. Besides role in viability maintenance, culture media can also take up a new role as a therapeutic agent in future. The improvised media apart from maintaining the viability may work on improving the quality of gametes/embryos obtained from poor prognosis patients. This review would discuss the progressive evolution of embryo culture media based on studies on pre-implantation embryo physiology and lastly will hypothesize the future of this technology.

Human Trophectoderm Spheroid Derived from Human Embryonic Stem Cells.

The use of human embryos for studying the early implantation processes and trophoblast is restricted by ethical concerns. The development of models mimicking the peri-implantation embryos is critical for understanding the physiology of human embryos and many pathophysiological disorders including recurrent implantation failure and miscarriage. Three-dimensional (3D) models of trophoblastic spheroids have been successfully derived from human embryonic stem cells (hESC). Simultaneous activation of the BMP pathway and blockage of the Activin/Nodal pathway favor the differentiation of hESC into trophoblast. Here we describe a 3D trophectoderm differentiation protocol with the use of BAP (BMP4, A83-01, and PD173074) to generate hESC-derived trophectoderm spheroids (BAP-EB). BAP-EB is highly reproducible and exhibits morphological and transcriptomic similarities to human early blastocysts.

Natural nest substrates influence squamate embryo physiology but have little effect on hatchling phenotypes.

Vertebrate embryos require access to water; however, many species nest in terrestrial habitats that vary considerably in moisture content. Oviparous, non-avian reptiles have served as models to understand how environmental factors, like moisture availability, influence development because eggs are often exposed to prevailing environments in the absence of parental care. Though much research demonstrates the importance of water absorption by eggs, many ecological factors that influence moisture availability in natural nests have received little attention. For example, the type of substrate in which nests are constructed is understudied. We experimentally incubated eggs of the brown anole lizard (Anolis sagrei) in 2 naturally occurring nest substrates that were treated with varying amounts of water to determine how natural substrates influence development at different moisture concentrations. One substrate consisted of sand and crushed seashells and the other was mostly organic material (i.e. decayed plant material). Both are common nesting substrates at our field site. When controlling for water uptake by eggs, we found that egg survival and hatchling phenotypes were similar between substrates; however, embryos developed more quickly in the sand/shell substrate than the organic substrate, indicating substrate-specific effects on embryo physiology. These results demonstrate that different natural substrates can result in similar developmental outcomes if the water available to eggs is comparable; however, some aspects of development, like developmental rate, are affected by the type of substrate, independent of water availability. Further study is required to determine how natural substrates influence embryo physiology independent of water content.

Physiological and environmental control of seed germination timing in Mediterranean mountain populations of Gundelia tournefortii

Fruit and seed morphology interact with embryo physiology and environmental conditions to control seed germination timing. This interaction plays a pivotal role in ecosystems with narrow windows for seedling establishment, such as the Mediterranean mountains. In this study, we investigated the germination responses of the secondary capitula (disseminules) of Gundelia tournefortii from East Mediterranean mountain populations. When incubated at 15 °C, intact capitula did not reach 20% of final germination, with or without the addition in the germination substrate of GA3 (250 mg L−1), while extracted fruits reached 50% of germination, which increased to ca. 70% when treated with GA3. Cold stratification enhanced final germination of the capitula at 15 °C to ca. 65%, although almost half of the initially sown capitula germinated during the second month of stratification at 5 °C. During the stratification at 5 °C, peak puncture force needed to pierce the basal part of the capitula decreased linearly and capitula started germinating after one month, which corresponded to a peak puncture force of 0.41–0.35 N. These findings highlight the presence of mechanical and hormonal components of physiological seed dormancy. The morphology of the disseminules controls seed germination timing, by interacting with cold winter temperatures and starting seed germination only in early winter. These findings not only provide new insights on the reproduction from seeds of this plant, but by highlighting high germination of cold-stratified intact capitula, can also support plant propagation programmes for this key wild edible species, very important for food security and the livelihoods of local communities in the East Mediterranean region.

Developmental and lifelong dioxin exposure induces measurable changes in cardiac structure and function in adulthood

Congenital heart disease (CHD) is the most common congenital abnormality. A precise etiology for CHD remains elusive, but likely results from interactions between genetic and environmental factors during development, when the heart adapts to physiological and pathophysiological conditions. Further, it has become clearer that early exposure to toxins that do not result in overt CHD may be associated with adverse cardiac outcomes that are not manifested until later life. Previously, interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR), either by gene ablation or by in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AHR ligand, was shown to cause structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. Here, we show that continuous exposure to TCDD from fertilization throughout adulthood caused male mice to underperform at exercise tolerance tests compared to their control and female counterparts, confirming previous observations of a sexually dimorphic phenotype. Renin-angiotensin stimulation by angiotensin II (Ang II) caused measurable increases in blood pressure and left ventricle mass, along with decreased end diastolic volume and preserved ejection fraction. Interestingly, TCDD exposure caused measurable reductions in the myocardial hypertrophic effects of Ang II, suggesting that endogenous AHR signaling present in adulthood may play a role in the pathogenesis of hypertrophy. Overall, the findings reported in this pilot study highlight the complex systems underlying TCDD exposure in the development of cardiac dysfunction in later life.

Proteomic profile of pre-implantational ovine embryos produced in vivo.

The present study was conducted to decipher the proteome of in vivo-produced pre-implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC-MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan (http://www.targetscan.org) and mIRBase (http://www.mirbase.org) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre-implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa-mir-106b-5p; hsa-mir-30-5p; hsa-mir-103a-5p and hsa-mir-106a-5p) with potential interactions with embryo-expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF-beta, p53 and Toll-like receptor. The current study describes the first major proteomic profile of 6-day-old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species.

Metabolic activity of human blastocysts correlates with their morphokinetics, morphological grade, KIDScore and artificial intelligence ranking.

Is there a relationship between blastocyst metabolism and biomarkers of embryo viability? Blastocysts with higher developmental potential and a higher probability of resulting in a viable pregnancy consume higher levels of glucose and exhibit distinct amino acid profiles. Morphological and morphokinetic analyses utilized in embryo selection provide insight into developmental potential, but alone are unable to provide a direct measure of embryo physiology and inherent health. Glucose uptake is a physiological biomarker of viability and amino acid utilization is different between embryos of varying qualities. Two hundred and nine human preimplantation embryos from 50 patients were cultured in a time-lapse incubator system in both freeze all and fresh transfer cycles. A retrospective analysis of morphokinetics, morphology (Gardner grade), KIDScore, artificial intelligence grade (EmbryoScore), glucose and amino acid metabolism, and clinical pregnancies was conducted. ICSI was conducted in all patients, who were aged ≤37 years and previously had no more than two IVF cycles. Embryos were individually cultured in a time-lapse incubator system, and those reaching the blastocyst stage had their morphokinetics annotated and were each assigned a Gardner grade, KIDScore and EmbryoScore. Glucose and amino acid metabolism were measured. Clinical pregnancies were confirmed by the presence of a fetal heartbeat at 6 weeks of gestation. Glucose consumption was at least 40% higher in blastocysts deemed of high developmental potential using either the Gardner grade (P < 0.01, n = 209), KIDScore (P < 0.05, n = 207) or EmbryoScore (P < 0.05, n = 184), compared to less viable blastocysts and in blastocysts that resulted in a clinical pregnancy compared to those that failed to implant (P < 0.05, n = 37). Additionally, duration of cavitation was inversely related to glucose consumption (P < 0.05, n = 200). Total amino acid consumption was significantly higher in blastocysts with an EmbryoScore higher than the cohort median score (P < 0.01, n = 185). Furthermore, the production of amino acids was significantly lower in blastocysts with a high Gardner grade (P < 0.05, n = 209), KIDScore (P < 0.05, n = 207) and EmbryoScore (P < 0.01, n = 184). Samples were collected from patients who had ICSI treatment and from only one clinic. These results confirm that metabolites, such as glucose and amino acids, are valid biomarkers of embryo viability and could therefore be used in conjunction with other systems to aid in the selection of a healthy embryo. Work was supported by Virtus Health. D.K.G is contracted with Virtus Health. The other authors have no conflict of interest to declare. N/A.

Embryonic substances induce alarm response in adult zebrafish (Danio rerio).

Many aquatic animals rely on chemicals released by injured individuals of the same species to assess predation risk. Among these chemical cues, alarm substances released from the injured skin of ostariophysan fishes have been extensively examined. In most fish species examined, these cues appear to be released by all injured individuals (including larvae, juveniles and adults) and elicit alarm responses in conspecifics. Adult alarm cues also affect development and physiology of embryos. Nonetheless, whether embryos produce alarm cues that affect adults is not known. This study reports that extracts of zebrafish (Danio rerio) embryos at 36 h post-fertilization or later induce antipredator behaviours reminiscent of those induced by skin alarm substances. At an equivalent of 10-6 g embryo per millilitre, the extract induced bottom-dwelling and freezing in adults. These behaviours are consistent with those induced by adult alarm substances. This study concludes that zebrafish embryos produce alarm substances.

In memory of Michelle Lane: 1970 - 2020.

In memory of Michelle Lane: 1970 - 2020.