Transverse and vertical incisions affect the viability of in vitro-produced embryos submitted to a simplified microsurgery approach.

Integrating in vitro embryo production with embryonic microsurgery facilitates the generation of monozygotic twins. However, despite their potential benefits, these methods have not been widely adopted in commercial settings because of their substantial costs. Hence, there is a need to streamline the bisection procedure while ensuring efficient production of viable demi-embryos. In this study, we investigated the impact of different orientations of microsurgical incisions in relation to inner cell mass on embryonic development, morphology, viability, and expression of cell fate protein markers using a simplified microsurgery approach. Ovaries were transported from the slaughterhouse to the laboratory and aspirated to obtain oocytes that were selected and subjected to in vitro embryo production. The selected expanded blastocysts (n = 204) underwent microsurgery. The blastocysts were immobilized to facilitate incision using an adapted microblade, yielding demi-embryos (vertical incision) and viable embryonic fragments (transverse incision). The structures were then re-cultured for 12 h. Viability was assessed by measuring the re-expansion rate after re-culture, followed by immunofluorescence analysis of proteins (CDX2 and NANOG) and apoptosis analysis using terminal deoxynucleotyl transferase dUTP nick end-labeling (TUNEL). Microsurgically derived embryos exhibited remarkable plasticity, as evidenced by a slight reduction (P < 0.05) in the re-expansion rate (transverse 64.2 % and vertical 57.2 %) compared to that of the control group (blastocysts without microsurgery) (86.7 %). They also demonstrated the ability of morphological reconstitution after culturing. Despite the anticipated decrease (P < 0.05) in the total number of cells and embryo volume, microsurgery did not result in a significant increase (P > 0.05) in the number of apoptotic cells. Furthermore, microsurgery led to higher (P < 0.05) expression of markers associated with pluripotency, indicating its efficiency in preserving regenerative capacity. Moreover, microsurgery, whether followed by immunosurgery or not, made the isolation of embryonic cells easier. In conclusion, both transverse and vertical microsurgery incisions enabled the production of identical demi-embryos and served as tools for isolating embryonic cells without compromising the resumption of development and the apoptotic index.

To what extent are ephippia of Mexican Anomopoda (Crustacea, Cladocera) identifiable?

Diapausing embryos encased within cladoceran ephippia result from sexual reproduction and increase genetic diversity. They are also important means by which species bypass harsh environmental conditions and disperse in space and time. Once released, ephippia usually sink to the benthos and remain there until hatching. Using the Sars' method (incubating sediments to identify cladoceran hatchlings), ephippial egg bank biodiversity can be evaluated. Yet, even when samples are incubated under a variety of conditions, it is not possible to warrant that all have hatched. Few keys are available that facilitate the identification of cladocerans by using only ephippial morphology. Our goal was to analyze some cladoceran ephippia from Mexico, to develop a means to identify them using easily recognizable characteristics. Ephippia of 23 cladoceran species from waters in Aguascalientes (México) in 11 genera (Alona, Biapertura, Ceriodaphnia, Chydorus, Daphnia, Dunhevedia, Ilyocryptus, Macrothrix, Moina, Pleuroxus, and Simocephalus) were analyzed. In our analysis six morphological features were selected that permitted the identification of ephippia to species(-group) level. The results demonstrate that with a proper catalog of features, some ephippia can be identified.

Amphiphilic Single-Chain Polymer Nanoparticles as Imaging and Far-Red Photokilling Agents for Photodynamic Therapy in Zebrafish Embryo Xenografts.

This work introduces rationally designed, improved amphiphilic single-chain polymer nanoparticles (SCNPs) for imaging and photodynamic therapy (PDT) in zebrafish embryo xenografts. SCNPs are ultrasmall polymeric nanoparticles with sizes similar to proteins, making them ideal for biomedical applications. Amphiphilic SCNPs result from the self-assembly in water of isolated synthetic polymeric chains through intrachain hydrophobic interactions, mimicking natural biomacromolecules and, specially, proteins (in size and when loaded with drugs, metal ions or fluorophores also in function). These ultrasmall, soft nanoparticles have various applications, including catalysis, sensing, and nanomedicine. Initial in vitro experiments with nonfunctionalized, amphiphilic SCNPs loaded with a photosensitizing Zn phthalocyanine with four nonperipheral isobutylthio substituents, ZnPc, showed promise for PDT. Herein, the preparation of improved, amphiphilic SCNPs containing ZnPc as highly efficient photosensitizer encapsulated within the nanoparticle and surrounded by anthracene units is disclosed. The amount of anthracene groups and ZnPc molecules within each single-chain nanoparticle controls the imaging and PDT properties of these nanocarriers. Critically, this work opens the way to improved PDT applications based on amphiphilic SCNPs as a first step toward ideal, long-term artificial photo-oxidases (APO).

Direct Somatic Embryogenesis in Carica papaya L. Genotypes for Genetic Modification Purposes.

This chapter presents an efficient protocol for regenerating Carica papaya plants via somatic embryogenesis from immature zygotic embryos from economically important papaya genotypes. To achieve regenerated plants from somatic embryos, in the present protocol, four induction cycles are required, followed by one multiplication cycle and one regeneration cycle. With this optimized protocol, 80% of somatic embryos can be obtained in only 3.5 months. At this stage, calli containing more than 50% globular structures can be used for transformation (via agrobacterium, biobalistics, or any other transformation method). Once transformed, calli can be transferred to the following steps (multiplication, elongation, maturation, rooting, and ex vitro acclimatization) to regenerate a transformed somatic embryo-derived full plant.

[Comparative Study of the Embryo Development and Clinical Outcomes of 3 Ovarian Stimulation Protocols in Different Age Groups]. / 不同年龄段人群三种促排卵方案胚胎发育及临床结局比较.

Objective: The main purpose of this study is to compare the embryo development and clinical outcomes of women in different age groups undergoing in vitro fertilization (IVF) processes using gonadotrophin-releasing hormone (GnRH) antagonist protocol, GnRH agonist long protocol, and early follicular phase protocol. We aim to provide reliable reference for future clinical treatments. Methods: We conducted a detailed analysis of patients who underwent treatment between January 2021 and February 2023. 1) In the overall patient population, we comprehensively compared the basic characteristics, the embryo development, and the clinical outcomes of patients treated with three different ovarian stimulation protocols, including the GnRH antagonist protocol group (n=4173), the agonist long protocol group (n=2410), and the early follicular phase long protocol group (n=341). 2) We divided the overall population into three age groups, one group for patients under 30 years old (n=2576), one for patients aged 30-35 (n=3249), and one for patients older than 35 years old (n=1099). Then, we compared the three stimulation protocols based on the group division. We separately compared the embryo development and clinical outcomes of patients using the three stimulation protocols in the under 30 years old, the 30-35 years old, and the over 35 years old age groups. With this analysis, we aimed to explore the response of different age groups to different stimulation protocols and their impact on the success rate of IVF. Results: 1) In the overall population, we found that the average number of oocytes retrieved in the GnRH agonist long protocol group was significantly higher than that in the GnRH antagonist protocol group ([13.85±7.162] vs. [13.36±7.862], P=0.0224), as well as the early follicular phase long protocol group ([13.85±7.162] vs. [11.86±6.802], P<0.0001). Patients in the GnRH antagonist protocol group not only had a significantly lower starting dose of gonadotrophin (Gn) compared to the other two groups (P<0.05) but also had a significantly lower number of days of Gn use (P<0.05). The blastocyst formation rate in the GnRH antagonist protocol group was the highest among the three groups, significantly higher compared to the GnRH agonist long protocol group (64.91% vs. 62.35%, P<0.0001) and the early follicular phase long protocol group (64.91% vs. 61.18%, P=0.0001). However, there were no significant differences in the clinical pregnancy rates or the live birth rates among the three groups treated with different ovarian stimulation protocols (P>0.05). 2) In the <30 age group, the blastocyst formation rate in the GnRH antagonist protocol group was the highest among the three groups, significantly higher compared to the GnRH agonist long protocol group (66.12% vs. 63.33%, P<0.0001) and the early follicular phase long protocol group (66.12% vs. 62.13%, P=0.0094). In the 30-35 age group, the blastocyst formation rate in the GnRH antagonist protocol group was the highest among the three groups, significantly higher compared to the GnRH agonist long protocol group (64.88% vs. 62.93%, P=0.000 9) and the early follicular phase long protocol group (64.88% vs. 60.39%, P=0.0011). In the >35 age group, the blastocyst formation rate in the GnRH antagonist protocol group was significantly higher than that in the GnRH agonist long protocol group (59.83% vs. 56.51%, P=0.0093), while there was no significant difference compared to that of the early follicular phase long protocol group (P>0.05). In the three age groups, we found that there were no significant differences in clinical pregnancy rate, live birth rate, and neonatal outcome indicators (fetal weight and Apgar score) among the three stimulation protocols (antagonist protocol, GnRH agonist long protocol, and early follicular phase long protocol) (P>0.05). The findings showed no significant differences between clinical and neonatal outcomes in patients of all ages, regardless of the ovarian stimulation protocol, suggesting that the three ovarian stimulation protocols have similar therapeutic effects in patients of different ages. The results of this study have important implications for the selection of an appropriate ovarian stimulation protocol and the prediction of treatment outcomes. Conclusion: In the younger than 30 and 30-35 age groups, the GnRH antagonist protocol showed a more significant advantage over the GnRH agonist long protocol and the early follicular phase long protocol. This suggests that for younger and middle-aged patients, the antagonist protocol may lead to better outcomes during ovarian stimulation. In the older than 35 age group, while the antagonist protocol still outperformed the GnRH agonist long protocol, there was no significant difference compared to the early follicular phase long protocol. This may imply that with increasing age, the early follicular phase long protocol may have effects similar to the antagonist protocol to some extent. The advantages of the antagonist protocol lie in its ability to reduce stimulation duration and the dosage of GnRH, while enhancing patient compliance with treatment. This means that patients may find it easier to accept and adhere to this treatment protocol, thereby improving treatment success rates. Particularly for older patients, the use of the antagonist protocol may significantly increase the blastocyst formation rate, which is crucial for improving the success rates. Although there were no significant differences in the clinical outcomes of patients treated with the three protocols in each age group, further research is still needed to validate these findings. Future multicenter studies and increased sample sizes may help comprehensively assess the efficacy of different stimulation protocols. Additionally, prospective studies are needed to further validate these findings and determine the optimal treatment strategies.

Resveratrol intake by males increased the mitochondrial DNA copy number and telomere length of blastocysts derived from aged mice.

The present study examined whether male resveratrol intake affected mitochondrial DNA copy number (mt-cn) and telomere length (TL) in blastocysts fathered by young and aged male mice. C57BL/6N male mice supplied with water or water containing 0.1 mM resveratrol were used for embryo production at 14-23 and 48-58 weeks of age. Two-cell-stage embryos were collected from the oviducts of superovulated female mice (8-15 weeks old) and cultured for 3 days until the blastocyst stage. Mt-cn and TL levels were measured by real-time polymerase chain reaction. Resveratrol intake did not affect body weight or water consumption. Resveratrol intake increased the expression levels of SIRT1 in the liver, the antioxidative ability of serum, and extended TL in the heart, whereas there was no significant difference in mt-cn in the heart or TL in sperm. The rate of blastocyst development was significantly lower in aged male mice than in younger mice, and resveratrol intake increased the total number of blastocysts derived from both young and aged males. Resveratrol intake did not affect mt-cn or TL in blastomeres of blastocyst-stage embryos derived from young mice, but significantly increased both mt-cn and TL in blastomeres of blastocysts derived from aged fathers. In conclusion, resveratrol intake increased mt-cn and TL levels in blastocysts derived from aged male mice.

CRISPR-mediated editing of ß-lactoglobulin (BLG) gene in buffalo.

Milk is a good source of nutrition but is also a source of allergenic proteins such as α-lactalbumin, ß-lactoglobulin (BLG), casein, and immunoglobulins. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology has the potential to edit any gene, including milk allergens. Previously, CRISPR/Cas has been successfully employed in dairy cows and goats, but buffaloes remain unexplored for any milk trait. In this study, we utilized the CRISPR/Cas9 system to edit the major milk allergen BLG gene in buffaloes. First, the editing efficiency of designed sgRNAs was tested in fibroblast cells using the T7E assay and Sanger sequencing. The most effective sgRNA was selected to generate clonal lines of BLG-edited cells. Analysis of 15 single-cell clones, through TA cloning and Sanger sequencing, revealed that 7 clones exhibited bi-allelic (-/-) heterozygous, bi-allelic (-/-) homozygous, and mono-allelic (-/+) disruptions in BLG. Bioinformatics prediction analysis confirmed that non-multiple-of-3 edited nucleotide cell clones have frame shifts and early truncation of BLG protein, while multiple-of-3 edited nucleotides resulted in slightly disoriented protein structures. Somatic cell nuclear transfer (SCNT) method was used to produce blastocyst-stage embryos that have similar developmental rates and quality with wild-type embryos. This study demonstrated the successful bi-allelic editing (-/-) of BLG in buffalo cells through CRISPR/Cas, followed by the production of BLG-edited blastocyst stage embryos using SCNT. With CRISPR and SCNT methods described herein, our long-term goal is to generate gene-edited buffaloes with BLG-free milk.

Mechanistic insights into zinc oxide nanoparticles induced embryotoxicity via H3K9me3 modulation.

The widespread application of nanoparticles (NPs) in various fields has raised health concerns, especially in reproductive health. Our research has shown zinc oxide nanoparticles (ZnONPs) exhibit the most significant toxicity to pre-implantation embryos in mice compared to other common NPs. In patients undergoing assisted reproduction technology (ART), a significant negative correlation was observed between Zn concentration and clinical outcomes. Therefore, this study explores the impact of ZnONPs exposure on pre-implantation embryonic development and its underlying mechanisms. We revealed that both in vivo and in vitro exposure to ZnONPs impairs pre-implantation embryonic development. Moreover, ZnONPs were found to reduce the pluripotency of mouse embryonic stem cells (mESCs), as evidenced by teratoma and diploid chimera assays. Employing multi-omics approaches, including RNA-Seq, CUT&Tag, and ATAC-seq, the embryotoxicity mechanisms of ZnONPs were elucidated. The findings indicate that ZnONPs elevate H3K9me3 levels, leading to increased heterochromatin and consequent inhibition of gene expression related to development and pluripotency. Notably, Chaetocin, a H3K9me3 inhibitor, sucessfully reversed the embryotoxicity effects induced by ZnONPs. Additionally, the direct interaction between ZnONPs and H3K9me3 was verified through pull-down and immunoprecipitation assays. Collectively, these findings offer new insights into the epigenetic mechanisms of ZnONPs toxicity, enhancing our understanding of their impact on human reproductive health.

The Role of Transplacental Infection in Leptospira spp. Epidemiology in Cattle in Caatinga Biome, Brazil.

Leptospirosis is an infectious disease that affects domestic animals, wild animals, and humans. It represents a public health problem and has an important economic impact on livestock. This study aims to investigate the importance of genital and transplacental infection in the epidemiology of leptospirosis in cows maintained in Caatinga biome conditions, Northeastern Brazil, as well as reporting organs colonized by Leptospira spp. in embryos and fetuses. Blood, urinary tract (urine, bladder, and kidney), and reproductive tract (vaginal fluid, uterus, uterine tube, ovary, and placenta) samples were collected from 15 slaughtered pregnant cows. Two embryos and 13 fetuses were sampled. Central nervous system and choroid ovoid samples were collected from embryos. Blood, central nervous system, lung, peritoneal liquid, abomasal content, liver, spleen, urine, bladder, kidney, and reproductive system samples were collected from fetuses. Diagnostic methods included the microscopic agglutination test (MAT) using a collection of 24 serovars belonging to 17 different pathogenic serogroups of five species as antigens, as well as polymerase chain reaction (PCR). Anti-Leptospira spp. antibodies were found in 9 cows (60%), while 13 cows (86.67%) had at least one organ or urine with leptospiral DNA. No fetus was seroreactive. Among the embryos and fetuses, 13 (86.67%) presented leptospiral DNA, proving a high frequency of transplacental infection (100%). For cows, the most frequent biological materials regarding Leptospira spp. DNA detection were placenta (13 out of 15 samples; 86.7%), uterus (10 out of 15 samples; 66.7%), and vaginal fluid (5 out of 15 samples; 33.3%), while, for fetuses/embryos, the most frequent PCR-positive samples were choroid ovoid (1/2; 50%), spleen (6/13; 46.2%), kidney (5/13; 38.5%), and central nervous system (5/15; 33.3%). Sequenced samples based on the LipL32 gene presented 99% similarity with L. borgpetersenii. The results indicate that transplacental infection is an efficient way of spreading Leptospira spp. in cows maintained in Caatinga biome conditions. Therefore, prevention and control strategies must include actions that interrupt transmission through this alternative route.

Long-Term Conservation for the Safeguard of Abies nebrodensis: An Endemic and Endangered Species of Sicily.

The combined approaches between ex situ and in situ conservation are of great importance for threatened species in urgent need of protection. This study aims to develop concrete actions to preserve the relic of 30 adult trees of the Sicilian fir (Abies nebrodensis) from extinction using long-term germplasm conservation in liquid nitrogen (LN, -196 °C). Pollen grains were collected, and their moisture content (MC) was measured. Then, viability (2,3,5-tryphenyl tetrazolium chloride, TTC), in vitro germinability, and enzymatic antioxidant activity (ascorbate peroxidase, APX; catalase, CAT) were evaluated before and after cryopreservation. Seeds collected from mature cones underwent X-ray analysis, and only full seeds were used to excise the zygotic embryos (ZEs) for cryopreservation. The MC percentage of ZEs was determined, and then they were plunged in LN with (+PVS2) or without (-PVS2) Plant Vitrification Solution 2; untreated ZEs were used as a control. Viability (TTC test) and in vitro germination were assessed for all ZEs (+PVS2, -PVS2, and control). Embryogenic callus (EC) lines obtained from mature ZEs were cryopreserved applying the 'encapsulation-dehydration' technique. This study has allowed, after optimizing cryopreservation protocols for pollen, ZEs, and EC of A. nebrodensis, to establish the first cryobank of this endangered species in Polizzi Generosa (Palermo, Italy), inside the 'Madonie Regional Park'. The strategy developed for Sicilian fir conservation will pave the way for similar initiatives for other critically endangered conifer species.

Isomer-specific cardiotoxicity induced by tricresyl phosphate in zebrafish embryos/larvae.

Tricresyl phosphate (TCP) has received extensive attentions due to its potential adverse effects, while the toxicological information of TCP isomers is limited. In this study, 2 h post-fertilization zebrafish embryos were exposed to tri-o-cresyl phosphate (ToCP), tri-m-cresyl phosphate (TmCP) or tri-p-cresyl phosphate (TpCP) at concentrations of 0, 100, 300 and 600 µg/L until 120 hpf, and the cardiotoxicity and mechanism of TCP isomers in zebrafish embryos/larvae were evaluated. The results showed that ToCP or TmCP exposure induced cardiac morphological defects and dysfunction in zebrafish, characterized by increased distance between sinus venosus and bulbus arteriosis, increased atrium and pericardial sac area, trabecular defects, and decreased heart rate and blood flow velocity, while no adverse effects of TpCP on zebrafish heart were found. Transcriptomic results revealed that extracellular matrix (ECM) and motor proteins, as well as PPAR signaling pathways, were included in the cardiac morphological defects and dysfunction induced by ToCP and TmCP. Co-exposure test with D-mannitol indicated that the inhibition of energy metabolism by ToCP and TmCP affected cardiac morphology and function by decreasing osmoregulation. This study is the first to report the cardiotoxicity induced by TCP in zebrafish from an isomer perspective, providing a new insight into the toxicity of TCP isomers and highlighting the importance of evaluating the toxicity of different isomers.

Current status of the intrafollicular transfer of immature oocytes (IFIOT) in cattle: A review.

Intrafollicular Transfer of Immature Oocytes (IFIOT) has emerged as an alternative to the currently used systems for bovine embryo production. This technique associates the rapid multiplication of bovine females under a completely in vivo culture condition, eliminating the need for superstimulatory hormones in the in vivo system (IVD) and the costly laboratory setup required for in vitro embryo production (IVP). Despite being a promising technique, the results obtained to date have been unsatisfactory for commercial use. Only approximately 10 % -12 % of viable embryos are recovered from the total number of injected oocytes, which limits their use in genetic improvement programs. IFIOT problems can occur in any of the steps involved; therefore, each step must be carefully examined to identify those that have the most negative impact on the final embryo recovery. This review summarizes the different studies conducted using the IFIOT to provide a comprehensive analysis of the main factors that can influence the effectiveness of this technique.

The coculture of in vitro produced porcine embryos and oviductal epithelial cells improves blastocyst formation and modify embryo quality.

The efficiency of in vitro embryo production in mammals is influenced by variables associated with culture conditions during maturation, fertilization, and embryonic development. The embryos obtained often exhibit low quality due to suboptimal in vitro culture conditions compared to the in vivo environment. Co-culturing gametes and embryos with somatic cells has been developed to enhance in vitro culture conditions. This study aimed to assess the impact of coculturing in vitro-produced porcine embryos with porcine oviductal epithelial cells (POEC) on embryo development and quality. Firstly, a pure culture of POEC suitable for coculture systems was established. The epithelial origin of the cells was confirmed by the expression of E-cadherin and cytokeratin. The expression pattern of hormone receptors aligned with the diestrous oviduct, and POEC also secreted oviductal glycoprotein type 1 (OVGP-1). Secondly, POEC from passage 1 (POEC-1) were used to coculture with in vitro-produced porcine embryos. A successful coculture system was established without the addition of fetal bovine serum as a supplement. Coculturing POEC-1 in monolayers with in vitro-produced porcine embryos during the initial two days of culture enhanced the percentage of blastocysts and their hatching. Although the coculture did not alter the number of cells in the blastocysts or apoptosis assessed by TUNEL, it significantly reduced reactive oxygen species (ROS) levels in cleaved porcine embryos. This study represents the first report evaluating the quality of porcine embryos produced by IVF in coculture systems and assessing ROS levels in cleaved porcine embryos obtained by IVF.

Morpho-histology of co-occurrence of somatic embryos, shoots, and inflorescences within a callus of Limonium 'Misty Blue'.

This is the first attempt to report the co-occurrence of somatic embryos, shoots, and inflorescences and their sequential development from stem cell niches of an individual callus mass through morpho-histological study of any angiosperm. In the presence of a proper auxin/cytokinin combination, precambial stem cells from the middle layer of a compact callus, which was derived from the thin cell layer of the inflorescence rachis of Limonium, expressed the highest level of totipotency and pluripotency and simultaneously developed somatic embryos, shoots, and inflorescences. This study also proposed the concept of programmed cell death during bipolar somatic embryo and unipolar shoot bud pattern formation. The unique feature of this research was the stepwise histological description of in vitro racemose inflorescence development. Remarkably, during the initiation of inflorescence development, either a unipolar structure with open vascular elements or an independent bipolar structure with closed vascular elements were observed. The protocol predicted the production of 6.6 ± 0.24 and 7.4 ± 0.24 somatic embryos and shoots, respectively, from 400 mg of callus, which again multiplied, rooted, and acclimatised. The plants' ploidy level and genetic fidelity were assessed randomly before acclimatisation by flow cytometry and inter simple sequence repeats (ISSR) marker analysis. Finally, the survivability and flower quality of the regenerated plants were evaluated in the field.

Producing Human Livers From Human Stem Cells Via Blastocyst Complementation.

The need for organ transplants exceeds donor organ availability. In the quest to solve this shortage, the most remarkable area of advancement is organ production through the use of chimeric embryos, commonly known as blastocyst complementation. This technique involves the combination of different species to generate chimeras, where the extent of donor cell contribution to the desired tissue or organ can be regulated. However, ethical concerns arise with the use of brain tissue in such chimeras. Furthermore, the ratio of contributed cells to host animal cells in the chimeric system is low in the production of chimeras associated with cell apoptosis. This review discusses the latest innovations in blastocyst complementation and highlights the progress made in creating organs for transplant.

Noise accelerates embryonic development in a key crab species: Morphological and physiological carryover effects on early life stages.

Anthropogenic noise is considered one important global pollutant. The impact of noise on marine invertebrates has been less assessed. The present study evaluated the chronic effect of the motorboat noise obtained from a lagoon's soundscape, the natural habitat of the key crab Neohelice granulata, on its whole embryonic development, considering morphological and physiological carryover effects on embryos and hatched larvae. Results demonstrated that embryonic development was shortened under noise exposure. The effects on advanced embryos, larvae and adult females were: increased heartbeats and non-viable eggs, and decreased fecundity. Biochemical responses showed lipid peroxidation in embryos while antioxidant enzymes were activated in larvae and adults, indicating a counteracting effect related to the life stage. The negative effects on fitness offspring may imply ecological consequences at the population level. Results are discussed in terms of the ecosystem engineer species studied and the habitat, a MAB UNESCO Reserve lagoon, suggesting the urgent need to develop mitigation plans.

Toxicity of different-sized cobalt ferrite (CoFe2O4) nanoparticles to Oncorhynchus mykiss at early development stages.

As innovative and versatile agents with potential applications in a wide range of fields including medicine, electronics, wastewater treatment, cosmetics, and energy storage devices, magnetic nanoparticles (NPs) are significant attention. However, our knowledge of the harmful effects of different-sized NPs, particularly of their effects on aquatic animals, is limited. In this study, we evaluated the impact of different-sized (sub-2, 5, and 15 nm) cobalt ferrite (CoFe2O4) NPs on the biological parameters of rainbow trout (Oncorhynchus mykiss) embryos and larvae. The NPs were characterized using techniques such as high-resolution transmission electron microscopy (HRTEM) for imaging, X-ray diffraction (XRD) for crystallographic analysis, and energy-dispersive X-ray spectroscopy (EDX) for elemental analysis, and were tested for impact through a series of toxicity, genotoxicity, and biochemical assays at a concentration of 100 mg/L. The obtained results showed that toxicity of CoFe2O4 NPs depended on the size of NPs and the developmental stage of the fish. Our results, which revealed significant changes in biological parameters of O. mykiss under exposure to CoFe2O4 NPs, imply that these NPs may be not environmentally safe. The hierarchical cluster analysis showed that embryos of the control group were clearly separated from those exposed to NPs of various sizes. However, in the exposed larvae, the effects of control and the smallest-sized NPs (sub-2 nm) differed from those induced by larger NPs (5 nm and 15 nm). Additional research is necessary to comprehend the mechanisms underlying the observed variations, which would be advantageous for both managing the risk of NPs to humans and advancing the field of aquatic nanotoxicology.

Thermal stressors during embryo incubation have limited ontogenic carryover effects in brook trout.

Winter climate is changing rapidly in northern latitudes, and these temperature events have effects on salmonid thermal biology. Stressors during winter egg incubation could reduce hatching success and physiological performance of fall-spawning fishes. Here we quantified the potential for ontogenic carryover effects from embryonic thermal stress in multiple wild and hatchery-origin populations of brook trout (Salvelinus fontinalis), a temperate ectotherm native to northeastern North America. Fertilized eggs from four populations were incubated over the winter in the laboratory in four differing thermal regimes: ambient stream-fed water, chronic warming (+2 °C), ambient with a mid-winter cold-shock, and short-term warming late during embryogenesis (to stimulate an early spring). We examined body size and upper thermal tolerance at the embryonic, fry (10 weeks post-hatch and 27-30 weeks post-hatch) and gravid adult (age 2+) life stages (overall N = 1482). In a separate experiment, we exposed developing embryos to acute seven-day heat stress events immediately following fertilization and at the eyed-egg stage, and then assessed upper thermal tolerance (CTmax) 37 weeks post-hatch. In all cases, fish were raised in common garden conditions after hatch (i.e., same temperatures). Our thermal treatments during incubation had effects that varied by life stage, with incubation temperature and life stage both affecting body size and thermal tolerance. Embryos incubated in warmer treatment groups had higher thermal tolerance; there was no effect of the mid-winter melt event on embryo CTmax. Ten weeks after hatch, fry from the ambient and cold-shock treatment groups had higher and less variable thermal tolerance than did the warmer treatment groups. At 27-30 post-hatch and beyond, differences in thermal tolerance among treatment groups were negligible. Collectively, our study suggests that brook trout only exhibit short-term carryover effects from thermal stressors during embryo incubation, with no lasting effects on phenotype beyond the first few months after hatch.

Developmental effects and lipid disturbances of zebrafish embryos exposed to three newly recognized bisphenol A analogues.

Bisphenol G (BPG), bisphenol M (BPM) and bisphenol TMC (BPTMC), are newly recognized analogues of bisphenol A (BPA), which have been detected in multiple environmental media. However, the understanding of their negative impacts on environmental health is limited. In this study, zebrafish embryos were exposed to BPA and the three analogues (0.1, 10, and 1000 µg/L) to identify their developmental toxic effects. According to our results, all of the three analogues induced significant developmental disorders on zebrafish embryos including inhibited yolk sac absorption, altered heart rate, and teratogenic effects. Oil Red O staining indicated lipid accumulation in the yolk sac region of zebrafish after bisphenol analogues exposure, which was consistent with the delayed yolk uptake. Untargeted lipidomic analysis indicated the abundance of triacylglycerols, ceramides and fatty acids was significantly altered by the three analogues. The combined analysis of lipidomics and transcriptomics results indicated BPG and BPM affected lipid metabolism by disrupting peroxisome proliferator-activated receptor pathway and interfering with lipid homeostasis and transport. This partly explained the morphological changes of embryos after bisphenol exposure. In conclusion, our study reveals that BPG, BPM and BPTMC possess acute and developmental toxicity toward zebrafish, and the developmental abnormalities are associated with the disturbances in lipid metabolism.

Enhancing antioxidant levels and mitochondrial function in porcine oocyte maturation and embryonic development through notoginsenoside R1 supplementation.

This study examines the impact of Notoginsenoside R1 (NGR1), a compound from Panax notoginseng, on the maturation of porcine oocytes and their embryonic development, focusing on its effects on antioxidant levels and mitochondrial function. This study demonstrates that supplementing in vitro maturation (IVM) medium with NGR1 significantly enhances several biochemical parameters. These include elevated levels of glutathione (GSH), nuclear factor erythrocyte 2-related factor 2 (NRF2) and mRNA expression of catalase (CAT) and GPX. Concurrently, we observed a decrease in reactive oxygen species (ROS) levels and an increase in JC-1 immunofluorescence, mitochondrial distribution, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) and nuclear NRF2 mRNA levels. Additionally, there was an increase in ATP production and lipid droplets (LDs) immunofluorescence. These biochemical improvements correlate with enhanced embryonic outcomes, including a higher blastocyst rate, increased total cell count, enhanced proliferative capacity and elevated octamer-binding transcription factor 4 (Oct4) and superoxide dismutase 2 (Sod2) gene expression. Furthermore, NGR1 supplementation resulted in decreased apoptosis, reduced caspase 3 (Cas3) and BCL2-Associated X (Bax) mRNA levels and decreased glucose-regulated protein 78 kD (GRP78) immunofluorescence in porcine oocytes undergoing in vitro maturation. These findings suggest that NGR1 plays a crucial role in promoting porcine oocyte maturation and subsequent embryonic development by providing antioxidant levels and mitochondrial protection.