Survival Rate Of Embryos Research Articles

Optimal conditions for cryopreservation by vitrification of largemouth bass (Micropterus salmoides) embryos

The largemouth bass (Micropterus salmoides) is one of the important freshwater aquaculture species in the world. However, due to limitations on introduction scale, high-density farming, inbreeding, and species hybridization, the germplasm resources of largemouth bass face threats such as degradation and susceptibility to diseases. Therefore, it is urgent to conduct research on the conservation of its original and good germplasm resources. We optimized the conditions of cryopreservation to vitrify and revive largemouth bass embryos, including the mixing ratio of cryoprotectants, embryo stage, equilibration step and temperature, and washing regent. The results showed that the least toxic single, binary, and ternary mixed permeating cryoprotectants were PG, PM (PG: MeOH = 2:1), and PMD (PM: DMSO = 3:1), respectively. The least toxic non-permeating cryoprotectant was 5 % glucose. The optimal vitrification solution selected was PMDG (30 % PMD + 5 % glucose) with an 80.67 % survival rate of embryos. Embryos at the heartbeat stage exhibited strong tolerance to the PMDG solution, which is the optimal embryo stage for cryopreservation. During the equilibration process, either the five-step equilibration method or pre-cooling the cryoprotectant to 4°C could reduce its toxicity. During the washing process, a 0.125 mol·L−1 sucrose solution yielded the best results. Based on the optimized conditions, 650 embryos at the heartbeat stage were subjected to cryopreservation by vitrification, resulting in a total of 350 intact transparent eggs, two of which hatched successfully. The results provide a reference for further improving the efficiency of cryopreservation by vitrification of largemouth bass and other fish species.

Ammonia exposure impairs bone mineralization in zebrafish (Danio rerio) larvae

Ammonia is a major pollutant of freshwater environments. Previous studies have indicated that ammonia exposure adversely affects the physiology of freshwater fish. However, its effect on bone mineralization in freshwater fish larvae remains unclear. In this study, zebrafish larvae were used as a model to investigate the effects of different ammonia levels (0, 2.5, 5, and 10 mM NH4Cl) on the survival rate, body length, and bone mineralization of fish. The survival rate of zebrafish embryos exposed to different NH4Cl concentrations for 8 days was not affected. In contrast, the body length and bone mineralization of zebrafish larvae at 8 days post fertilization (dpf) were significantly reduced at 5 and 10 mM NH4Cl exposure. Further investigations revealed that ammonia exposure decreased the mRNA expression of osteoblast-related genes and increased that of osteoclast-related genes. Additionally, exposure to 5 mM and 10 mM NH4Cl induced the production of reactive oxygen species (ROS). 10 mM—but not 5 mM—NH4Cl exposure reduced the calcium and phosphorus content in 8 dpf zebrafish larvae. In conclusion, ammonia exposure induces bone resorption, while decreasing the calcium and phosphorus content of the whole body and bone formation, resulting in impaired bone mineralization in fish larvae.

One-Step Organic Synthesis of 18β-Glycyrrhetinic Acid-Anthraquinone Ester Products: Exploration of Antibacterial Activity and Structure-Activity Relationship, Toxicity Evaluation in Zebrafish.

To combine the activity characteristics of 18β-glycyrrhetinic acid (18β-GA) and anthraquinone compounds (rhein and emodin), reduce toxicity, and explore the structure-activity relationship (SAR) of anthraquinones, 18β-GA-anthraquinone ester compounds were synthesized by one-step organic synthesis. The products were separated and purified by HPLC and characterized by NMR and EI-MS. It was finally determined as di-18β-GA-3-rhein ester (1, New), GA dimer (2, known), 18β-GA-3-emodin ester (3, known), and di-18β-GA-1-emodin ester (4, new). The MIC of three reactants and four products against Escherichia coli and Staphylococcus aureus were detected invitro. Its developmental toxicity and cardiotoxicity were assessed using zebrafish embryos. The experimental results showed that rhein had the best antibacterial activity against Staphylococcus aureus with MIC50 of 2.4 mM, and it was speculated that -COOH, -OH, and intramolecular hydrogen bonds in anthraquinone compounds would enhance the antibacterial effect, while the presence of-CH3 might weaken the antibacterial activity. Product 1 increased the hatching rate and survival rate of zebrafish embryos and reduced the malformation rate and cardiomyocyte apoptosis. This experiment lays the foundation for further studying the SAR of anthraquinones and providing new drug candidates.

Investigation of structural, optical, photocatalytic, and antibacterial properties of ZnO doped GO nanoparticles for environment applications.

As a result of their unique and novel properties, nanocomposites have found applications in a wide variety of fields. The purpose of this study is to demonstrate the ability to synthesize nanoparticles consisting of zinc oxide (ZnO) and graphene oxide (GO) via sol-gel techniques. An x-ray diffractometer (XRD) as well as a UV-visible spectrometer were used to determine the crystalline and optical characteristics of the prepared samples. A hexagonal wurtzite crystal structure was observed in both pure ZnO nanoparticles and those that contain GO based on XRD results. It was estimated that the average crystallite size is based on the broadening of x-ray lines. In comparison with pure ZnO, the antimicrobial properties were enhanced when GO was incorporated with ZnO. In addition, experiments on the absorption edge indicated the presence of a red shift as a result of the incorporation of GO. When GO is incorporated in quantitative amounts, the bandgap value of pure ZnO decreased. FTIR spectra exhibit a band of absorption at 486 cm-1, which confirms Zn-O stretching in both samples. SEM images reveal a random pattern of structural features on the surface of the prepared samples. According to the EDX spectrum, pure GO nanoparticles and those doped with ZnO contain 61%-64% zinc and 32%-34% oxygen, respectively. When annealed at a higher temperature, ZnO NPs produced more H2 with a narrower bandgap than before annealing. In addition, methyl blue (MB) was used as an example of an organic compound in order to investigate the potential photocatalytic properties of nanoparticles with ZnO doped GO. In addition to DPPH assays, ZnO nanoparticles and ZnO doped GO nanoparticles were tested for their ability to scavenge free radicals. Comparing ZnO doped GO NPs with pure ZnO, these nanoparticles showed increased antioxidant activity. Based on the increased zone of inhibition observed for pure ZnO and ZnO doped GO (5, 10, 50, and 100 mg/mL), the antibacterial activity of pure ZnO and ZnO doped GO is concentration dependent. A detailed discussion of the results of the study demonstrated that ZnO doped GO and pure ZnO are toxic in different ways depending on how long they survive in degreased Zebrafish embryos and how fast they decompose. RESEARCH HIGHLIGHTS: The scope of the manuscript was under the results of the study confirmed that both nanoparticles exhibited concentration dependent antioxidative activity. Determined that 89% of methyl orange dye can be degraded photocatalytically. ZnO nanoparticles were found to be 74.86% antioxidant at a concentration of 50 g/mL in the present study. At a concentration of 50 g/mL, ZnO doped GO NPs showed 79.1% antioxidant activity. Photocatalytic degradation mechanism scheme is implicit in the photoexcited charge carrier transportation path is observed for all the samples. Survival rate of zebrafish embryos was shown to decrease with increasing concentrations of ZnO and zinc oxide plus GO nanoparticles.

Antiviral effect of the viroporin inhibitors against Taiwan isolates of infectious bronchitis virus (IBV)

Coronaviruses (CoVs) are significant animal and human pathogens, characterized by being enveloped RNA viruses with positive-sense single-stranded RNA. The Coronaviridae family encompasses four genera, among which gammacoronaviruses pose a major threat to the poultry industry, which infectious bronchitis virus (IBV) being the most prominent of these threats. Particularly, IBV adversely affects broiler growth and egg production, causing substantial losses. The IBV strains currently circulating in Taiwan include the IBV Taiwan-I (TW-I) serotype, IBV Taiwan-II (TW-II) serotype, and vaccine strains. Therefore, ongoing efforts have focused on developing novel vaccines and discovering antiviral agents. The envelope (E) proteins of CoVs accumulate in the endoplasmic reticulum-Golgi intermediate compartment prior to virus budding. These E proteins assemble into viroporins, exhibiting ion channel activity that leads to cell membrane disruption, making them attractive targets for antiviral therapy.In this study, we investigated the E proteins of IBV H-120, as well as IBV serotypes TW-I and TW-II. E protein expression resulted in inhibited bacteria growth, increased permeability of bacteria to β-galactosidase substrates, and blocked protein synthesis of bacteria by hygromycin B (HygB). Furthermore, in the presence of E proteins, HygB also impeded protein translation in DF-1 cells and damaged their membrane integrity. Collectively, these findings confirm the viroporin activity of the E proteins from IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. Next, the viroporin inhibitors, 5-(N,N-hexamethylene) amiloride (HMA) and 4,4’-diisothiocyano stilbene-2,2’-disulphonic acid (DIDS) were used to inhibit the viroporin activities of the E proteins of IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. In chicken embryos and chickens infected with IBV serotypes TW-I and IBV TW-II, no survivors were observed at 6 and 11 days post-infection (dpi), respectively. However, treatments with both DIDS and HMA increased the survival rates in infected chicken embryos and chickens and mitigated histopathological lesions in the trachea and kidney. Additionally, a 3D pentameric structure of the IBV E protein was constructed via homology modeling. As expected, both inhibitors were found to bind to the lipid-facing surface within the transmembrane domain of the E protein, inhibiting ion conduction. Taken together, our findings provide comprehensive evidence supporting the use of viroporin inhibitors as promising antiviral agents against IBV Taiwan isolates.

Evaluating the biomedical and environmental safety of selenium nanoparticles synthesized from black pepper seed extract

In recent times, sustainable synthesis of selenium nanoparticles (Se NPs) has seen a significant rise in research, focusing primarily on microbial and plant polysaccharide-based approaches for various bioapplications. However, there are limited studies on the use of extracts from herbal spice plants as capping agents for Se NPs. Therefore, this study investigates the pioneering synthesis of Se NPs using black pepper (Piper nigrum) seed extract (PSe NPs) and assesses their antioxidant properties, hemolytic potential, antibacterial and antibiofilm activities, cytotoxicity, phytotoxicity, in vivo toxicity in zebrafish embryos, and their effectiveness in prolonging the shelf life of food materials. The green-synthesized PSe NPs were thoroughly physicochemically characterized and found to be sphere-shaped with a medium diameter of 10–100 nm. The PSe NPs exhibited substantial antibacterial effects, being more effective against S. aureus (Gram-positive)than against S. typhi (Gram-negative). Furthermore, PSe NPs significantly inhibited MRSA biofilm growth at 500 μg/mL and showed notable antioxidant activity compared to standard gallic acid. Also at 500 μg/mL, PSe NPs induced significant hemolysis, whereas inferior concentrations did not. Cytotoxicity evaluations using the MTT assay revealed that PSe NPs significantly reduced the viability of human normal embryonic kidney (HEK 293) cells and human cervical cancer (HeLa) cells after 24 hours of treatment at concentrations of 0.1–500 μg/mL. This cytotoxic effect was associated with apoptosis, as shown by acridine orange/ethidium bromide staining. In phytotoxicity assays, PSe NPs revealed significant effects on seed germination and root and shoot growth of the mung bean (Vigna radiata). The zebrafish embryotoxicity assay indicated that PSe NPs at 500 µg/mL affected embryo morphology, mortality and survival rates 96 hours after fertilization. Furthermore, the application of PSe NPs gel coatings effectively reduced weight loss and extended the shelf life of fruits such as apples and pears, maintaining their natural appearance.

Preparation of PBAT microplastics and their potential toxicity to zebrafish embryos and juveniles

The extensive use of traditional non-biodegradable plastics results in the generation of microplastics (MPs), forming a new pollutant that can pose significant environmental risks. Biodegradable plastics (BP) possess degradation properties and can partially replace conventional plastics, thereby reducing pollution. However, further investigation is needed into the toxicity of biodegradable microplastics (BMPs) on aquatic organisms. This study explores the toxic effects of PBAT microplastics (PBAT-BMPs) and microplastics produced from degradable PBAT/TPS (thermoplastic starch) composite film (PBAT/TPS-BMPs) on zebrafish embryos. Our findings indicate that the presence of microplastics on the embryo's surface increases with higher BMPs concentration. Nonetheless, PBAT-BMPs tend to aggregate and are blocked by the embryonic membrane, thus diminishing their toxic effects on the embryo. Acute toxicity experiments revealed that 30 mg/L of PBAT-BMPs significantly reduced the survival rate of zebrafish embryos, whereas PBAT/TPS-BMPs had a lesser effect on survival. Both types of BMPs influenced the hatching rate of the embryos, leading to prolonged incubation periods. Additionally, both types of BMPs impacted the locomotor behavior of zebrafish larvae, causing an increase in larval locomotor speed. However, these BMPs had little impact on larval body development and heartbeat behavior. Fluorescent microplastic tracer experiments demonstrated that PBAT-BMPs persisted in juvenile fish for at least 144 h and were difficult to metabolize and excrete. Our study aims to gain a better understanding of the potential effects of BMPs on aquatic ecosystems and biological health, as well as to propose effective strategies for reducing environmental pollution and protecting organisms.

FOXM1 affects oxidative stress, mitochondrial function, and the DNA damage response by regulating p21 in aging oocytes

Fertilization capacity and embryo survival rate are decreased in postovulatory aging oocytes, which results in a reduced reproductive rate in female animals. However, the key regulatory genes and related regulatory mechanisms involved in the process of postovulatory aging in oocytes remain unclear. In this study, RNA-Seq revealed that 3237 genes were differentially expressed in porcine oocytes between the MII and aging stages (MII + 24 h). The expression level of FOXM1 was increased at the aging stage, and FOXM1 was also observed to be enriched in many key biological processes, such as cell senescence, response to oxidative stress, and transcription, during porcine oocyte aging. Previous studies have shown that FOXM1 is involved in the regulation of various biological processes, such as oxidative stress, DNA damage repair, mitochondrial function, and cellular senescence, which suggests that FOXM1 may play a crucial role in the process of postovulatory aging. Therefore, in this study, we investigated the effects and mechanisms of FOXM1 on oxidative stress, mitochondrial function, DNA damage, and apoptosis during oocyte aging. Our study revealed that aging oocytes exhibited significantly increased ROS levels and significantly decreased GSH, SOD, T-AOC, and CAT levels than did oocytes at the MII stage and that FOXM1 inhibition exacerbated the changes in these levels in aging oocytes. In addition, FOXM1 inhibition increased the levels of DNA damage, apoptosis, and cell senescence in aging oocytes. A p21 inhibitor alleviated the effects of FOXM1 inhibition on oxidative stress, mitochondrial function, and DNA damage and thus alleviated the degree of senescence in aging oocytes. These results indicate that FOXM1 plays a crucial role in porcine oocyte aging. This study contributes to the understanding of the function and mechanism of FOXM1 during porcine oocyte aging and provides a theoretical basis for preventing oocyte aging and optimizing conditions for the in vitro culture of oocytes.

Nervonic acid triggered ovarian inflammation by inducing mitochondrial oxidative stress to activate NLRP3/ IL-1β pathway

IntroductionMetabolic syndrome is a serious public health concern across the globe. However, the typical metabolites and mechanisms underlying the decreased fertility related to metabolic syndrome is still elusive. ObjectivesThe aim of the present study was to explore the typical metabolites and mechanisms underlying the decreased fertility related with metabolic syndrome. MethodsUtilizing metabolomics, a comparative analysis was conducted on fatty acid compositions in various tissues of sows with high and low reproductive performance. Additionally, serum fatty acid compositions in a metabolic syndrome model (obese mice) induced by a high-fat diet (HFD) were investigated to elucidate the lipid metabolites associated with metabolic syndrome. Furthermore, the impact of nervonic acid (NA) on ovarian function was examined using rodent animal models (rats and mice). Through biological techniques such as transcriptomics, CUT&Tag, and analysis of post-translational protein modifications, the molecular mechanisms underlying NA mediated ovarian inflammation were further elucidated based on models utilizing ovarian granulosa cells from pigs, humans, and mice. Finally, validation was performed on ovaries from patients diagnosed with polycystic ovary syndrome. ResultsIn vitro, targeted serum lipidomic analysis revealed that sows with low embryo survival rates exhibited abnormal lipid metabolism characterized by abnormal accumulation of NA in the liver, ovary, and adipose tissue. Additionally, elevated NA levels trigger ovarian inflammation to cause ovarian dysfunction in both sows and rats. Mechanistically, NA induce mitochondrial oxidative stress through inhibiting respiratory chain proteins CYTB and NDFUB8 to activate NLRP3 inflammasome, which triggers procaspase-1 into active caspase-1, and convert the cytokine precursors pro-IL-1β into biologically active IL-1β in ovarian granulosa cells. Notably, we evidenced that NA promotes IL-1β activities by increasing H3K9ac modification level of IL-1β promoter regions and regulating the expression of the transcription factor AP-1. Finally, we found that the decreased expression of CerS2 in ovaries and the increased level of chemokine CXCL14 may be the cause of abnormal NA accumulation. Surprisingly, individuals with polycystic ovary syndrome, obesity, non-alcoholic fatty liver or gestational diabetes mellitus exhibit a high level of serum NA. ConclusionCollectively, our current study suggests that NA is a typical metabolite of metabolic syndrome, which strongly influences the ovarian function and embryo survival and also provides that interfering with mitochondrial ROS production is a potential strong strategy for target solving abnormal NA accumulation.

Acute toxicity of deoxynivalenol and bioremediation of a highly effective deoxynivalenol degrading Achromobacter spanius P-9 on zebrafish embryos and adults

Deoxynivalenol (DON) is one of the mostly concerned mycotoxins and several microbes showed bioremediation effects on DON toxic effects. In this study, the acute toxicity of a new DON degrading strain Achromobacter spanius P-9 with DON on zebrafish embryos and adults were firstly performed. For zebrafish embryos, bacterial concentrations of 2.5 × 107 CFU/mL and 5.0 × 107 CFU/mL had no significant effects on growth and development. However, at 7.5 × 107 CFU/mL, some effects were observed, and at 10.0 × 107 CFU/mL, the embryo survival rate decreased to 70%, with 3.3% teratogenicity. Higher bacterial concentrations correlated with faster heart rates. DON (100 μg/mL) significantly reduced embryo survival to 36.7% in 96 h. Bacterial solutions at 7.5 × 107 CFU/mL and 10.0 × 107 CFU/mL expanded the zebrafish intestinal tissue wall, while DON at 100 μg/mL negatively impacted intestinal morphology. Liver tissue in zebrafish exposed to Achromobacter spanius P-9 showed no significant differences from the control group. However, exposure to DON solution increased liver fluorescence intensity and caused liver cell changes, including edema, vacuolization, and blurred boundaries. For adult zebrafish, the ROS and 8-OHdG contents in the exposure group increased with the increase of bacterial solution concentration, the SOD enzyme activity, CAT enzyme activity, GST enzyme activity and MDA was not significantly different with the control group. Compared with the control group, the content of ROS, GST enzyme activity, MDA and 8-OHdG after DON treatment showed an upward trend, SOD and CAT enzyme activities showed a decreasing trend. Achromobacter spanius P-9 has no obvious inhibitory effect on the growth and development of zebrafish embryos and has no obvious death and toxicity during the growth of adult fish, providing data support for the future application of this strain in the biodegradation of DON.

Prediction of reproductive performance in wintering females of Siberian sturgeon (Acipenser baerii, Brandt) based on in vitro ovulation rate of isolated follicles

Early prediction of the reproductive success of sturgeon females is important both for artificial breeding conditions in sturgeon farms and for monitoring wild fish populations approaching spawning. This study demonstrated that ovarian follicles removed from wintering Acipenser baerii females can undergo in vitro ovulation when exposed to 17α,20β-dihydroxy-4-pregnen-3-one. The research was conducted from 2015 to 2021 and involved the examination of 110 wintering Siberian sturgeon females (A. baerii), with an average weight of 8–11 kg and an age range of 8–10 years. Selection of females for spawning was based on the polarization index (PI) at the time when the river temperature dropped to +8 °C. The selected females were overwintered in concrete flow-through tanks with water from the Volga River for approximately two months. At the end of this period, oocyte samples were collected for in vitro incubation. An increase in water temperature to 15 °C, which precedes spawning, was achieved by mixing with warm effluent from a regional power station. The reproductive success of females was evaluated based on fertilization and embryonic survival rates. High reproductive success was characterized by females with fertilization rates above 70% and hatching rates above 60%. We identified a positive correlation between the percentage of in vitro ovulated follicles and the in vivo fertilization and embryo survival rates, with correlation coefficients of Rsp = 0.60 and Rsp = 0.48, respectively. Statistical analysis showed that females whose in vitro ovulation rate of follicles exceeded 30% displayed high reproductive performance compared to those with an in vitro ovulation rate below 30%. The predictive accuracy of this criterion was determined by calculating the ratio of ranks between females with high and low reproductive performance, averaging 80.9% for fertilization and 77% for embryonic survival. Therefore, we propose a new criterion to select females of A. baerii for spawning based on the in vitro oocyte ovulation rate obtained from wintering individuals.

Physicochemical properties and immune enhancement activity of oligosaccharide fraction purified from biomass of green macro-algal Ulva prolifera

Ulva prolifera (U. prolifera), as the dominant species of green macro-algal bloom, caused great damage to marine aquaculture and economy. This study was carried out to find its industrial utilization ways by determination the structure characteristics and the immune enhancement effect of oligosaccharide fractions produced from this alga. The result indicated that oligosaccharide fraction of F3 showed the best immune enhancement activity via RAW 264.7 macrophages and zebrafish assays. Chemical and structural analysis indicated that F3 with 11.14%sulfate content was composed of rhamnose, xylose, and glucose in ratio of 77.11:16.33:6.56. NMR spectroscopy showed that F3 contained (1 → 4)-α-l-rhamnopyranose-3-SO3 and α-l-rhamnopyranose-3-SO3-(1 → 4)-β-D-xylopyranose, with sulfate groups located at C-3 of rhamnopyranose. The results of immune effect in vitro demonstrated that F3 increased the production of nitric oxide (NO) and prostaglandin E2 (PGE2) with up-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. F3 also promoted the secretion of cytokines (TNF-α, IL-1β and IL-6) and activated the expression of mitogen-activated protein kinases (MAPK) and NF-κB signaling pathways. In vivo assay, F3 boosted the embryo survival rate and heart rate, as well as promoted the generation of NO and reactive oxygen species (ROS) in a dose-dependent manner. The novel structure and excellent activity in this study suggested that F3 could be the potential material for use as a natural immuno-enhancing agent in the biomedical and functional food industries.

Comparative Evaluation of Cytotoxicity of Zinc Oxide Nanoparticles Mixed With Herbal Extract as an Intracanal Medicament: A Zebrafish Model Study.

Objective In this study, zebrafish embryos are used to study the cytotoxic effects of a novel intracanal medication (ICM) based on zinc oxide nanoparticles (ZnO NPs) loaded with polyherbal extracts (Azadirachta indica and Solanum xanthocarpum). Material and methods In the present study, a green and sustainable method was employed for the synthesis of ZnO NPs mixed with bark and seed extracts of Azadirachta indica and Solanum xanthocarpum to be used as a polyherbal ICM. Formulation of ZnO NPs was confirmed with color change in mixture produced upon dissolving zinc acetate dihydrate in distilled water followed by slow addition of sodium hydroxide solution and herbal extracts. The effects of these green synthesized ZnO NPs were evaluated through a zebrafish embryo toxicity test. Embryos were exposed to different concentrations (25, 50, and 100 µg/mL) of synthesized experimental doses of ZnO NP and compared with the control embryos. Toxicological endpoints, such as the zebrafish embryo's survival rate, hatching rate, and heart rate, were noted and described. Results A concentration-dependent increase in mortality rate and hatching delay followed by declined heart rate was observed in green synthesized ZnO NP-treated embryos. The maximum toxicity was observed with an increase in the concentration of 100 µg/mL of the experimental dose, and at a low concentration of 25 µg/mL, it does not effectively show any developmental alteration in zebrafish embryos. Conclusion A novel polyherbal ICM loaded with ZnO NPs exhibited a dose-dependent effect on the heart rate, hatching, and mortality rate of the embryos. At optimal concentrations, the medication demonstrated minimal developmental malformations and cytotoxic effects, indicating its safety for use. However, increasing concentrations of the medication resulted in severe developmental malformations.

Zygote cryobanking applied to CRISPR/Cas9 microinjection in mice.

Microinjection of CRISPR/Cas9 requires the availability of zygotes that implies animal breeding, superovulation schemes, and embryo collection. Vitrification of zygotes may allow having ready-to-use embryos and to temporally dissociate the workload of embryo production from microinjection. In this study, fresh (F group) or vitrified (V group) zygotes were microinjected with CRISPR/Cas9 system to test the hypothesis that vitrified zygotes could be a suitable source of embryos for microinjection. In Experiment 1 (in vitro evaluation), B6D2F1/J zygotes were microinjected and cultured until blastocyst stage. Embryo survival and cleavage rates after microinjection were similar between groups (~50% and ~80% respectively; P = NS). Development rate was significantly higher for F than V group (55.0% vs. 32.6%, respectively; P<0.05). Mutation rate did not show statistical differences among groups (P = NS). In Experiment 2 (in vivo evaluation), C57BL/6J zygotes were microinjected and transferred to recipient females. Embryo survival was significantly lower in fresh than in vitrified zygotes (49.2% vs. 62.7%, respectively; P<0.05). Cleavage rate did not show statistical differences (~70%; P = NS). Pregnancy rate (70.0% vs. 58.3%) and birth rate (11.9% vs. 11.2%) were not different between groups (F vs. V group; P = NS). Offspring mutation rate was higher for F than V group, in both heterodimer analysis (73.7% vs. 33.3%, respectively; P = 0.015) and Sanger sequencing (89.5% vs. 41.7%, respectively; P = 0.006). In conclusion, vitrified-warmed zygotes present a viable alternative source for CRISPR/Cas9 microinjection when the production of fresh embryos is impeded by limited technical support. The possibility of zygote cryobanking to perform microinjection sessions on demand seems to be a suitable alternative to avoid the breeding and maintenance of animals all over the year, enhancing the implementation of CRISPR technology.

P-253 Development of embryos from aged mothers transferred to the uterus of young mice

Abstract Study question Is the development of embryos from advanced maternal age (AMA) mice, similar to those from young maternal age (YMA) mice when transferred to young uteri? Summary answer AMA mouse embryos are characterized by altered organ morphology, particularly of the brain, which may predispose to neurodevelopmental disorders. What is known already Human and animal studies have linked AMA pregnancy to elevated risk of neurodevelopmental disorders and behavioural alterations in offspring, primarily attributed to compromised uterine (maternal) conditions. In human, it was also suggested that postnatal care of older mother may influence offspring behaviour. Animal studies revealed that behavioural alterations of AMA offspring are programmed during pregnancy and so they persist in litters fostered by young mouse. It remains unclear whether an embryo of older mother will undergo normal development after embryo transfer to younger female’s uterus. Study design, size, duration Embryos for Advanced Maternal Age, AMA (n = 105) and control Young Maternal Age, YMA (n = 44) groups were collected from thirty females 8-14 months old and six females 3-5 months old, respectively and transferred to pseudo-pregnant mice for further development. At embryonic day 16 (E16) foetuses were collected, fixed for histology and evaluated using Theiler and Kaufman’s classification of mouse development, where E16 corresponds to Theiler stage 24 (TS24) Participants/materials, setting, methods CBA x C57BL6 hybrid mice were used throughout the experiment. At E16, embryo survival rate (number of foetuses collected / number of transferred embryos) was scored for both groups. Foetuses were assessed for visible abnormalities and underwent histological evaluation of organs accordingly to TS 24. Parameters such as size/diameter, signs of vacuolization, cell density and layers of cerebral cortex in brain of foetuses were analysed. Values were considered different when p &amp;lt; 0.05, Mean±SD; Mann-Whitney test. Main results and the role of chance At E16, the survival of AMA embryos did not differ significantly from YMA control (39/105; 37% vs. 20/44; 45%, respectively) however, 5% of AMA foetuses (2/39) displayed oedema or cerebral haemorrhage. Histological analysis revealed alterations in the head/brain of AMA in comparison to YMA foetuses, such as smaller head size (head area in mid-sagittal section: 25.25±4.87 vs. 31.39±3.79 mm2, p &amp;lt; 0.05), thinner cerebral cortex (435.78±12.95 vs. 515.22±17.78µm, p &amp;lt; 0.005), reduced ratio of cortical plate (CP) to cerebral cortex thickness (0.20±0.03 vs. 0.26±0.009, p &amp;lt; 0.05) and an increased ratio of ventricular zone (VZ) to cerebral cortex thickness (0.29±0.002 vs. 0.24±0.002, p &amp;lt; 0.05). Moreover, decreased cellular density in CP layer (10378.86 vs. 12066.40 cells/mm2, P &amp;lt; 0.05) was noted. AMA brains were characterised by high degree of cellular vacuolisation, particularly in medulla oblongata, suggestive for spongiosis (1550.27 vs. 571.92 vacuoles/mm2, p &amp;lt; 0.05). These brain alterations may cause neurodevelopmental disorders in offspring. AMA foetuses had: smaller bronchial diameter (31.52 vs. 36.62µm, p &amp;lt; 0.05), no ossification centres and reduced skin wrinkling, corresponding to TS23 (that is previous developmental stage). These observations may suggest a delayed development of AMA foetuses. Limitations, reasons for caution The results of controlled animal experiments play a key role in developing conceptual models and formulating hypothesis about target species. However, extrapolation of these results to humans requires caution because species-specific developmental features may differ. Wider implications of the findings Our study demonstrates that advanced maternal age (AMA) impacts oocyte/embryo programming in mice. Morphological changes in AMA-conceived foetuses suggest potential risks for neurodevelopmental and other health issues in offspring, even when carried in the uterus of a young female, emphasizing the far-reaching effects of AMA on progeny health. Trial registration number not applicable

P-391 Association between meteorological season at the time of embryo transfer and pregnancy outcomes in freeze-all cycles: lessons from seasonal reproductive mammals

Abstract Study question Is there any effect of meteorological season at the time of embryo transfer on clinical outcomes of freeze-all cycles? Summary answer Optimal conditions for implantation appear to be associated with spring and summer. Embryo transfers performed in autumn or winter are associated with reduced ongoing pregnancy. What is known already Extensive research has consistently shown that most mammals are seasonal breeders, and as day length increases, gonadotropin secretion increases, to anticipate reproduction in the spring and summer months. There is intriguing evidence that there is a seasonal variance in human reproduction, but the data are inconclusive. Therefore, the goal for the present study was to evaluate the effect the meteorological season at the time of embryo transfer on clinical outcomes of freeze-all cycles. Study design, size, duration This historical cohort study was performed in a private university-affiliated IVF center between February 2018 and December 2022. A total of 4952 frozen-thawed embryos transferred to 3725 cycles in a single clinic were analyzed. Participants/materials, setting, methods Both vitrification and the warming procedures were performed using the Cryotop method. Embryos survival and clinical outcomes were analyzed by the season, at the time of embryo transfer, using generalized linear models adjusted for potential confounders. Main results and the role of chance The mean maternal age was 37.0 ± 4.7 years. Embryos were cryopreserved for a mean of 8.1 ± 17.3 months. Autumn was designated as the reference season for all analyses. No significant differences were observed in embryo survival rates when the seasons were compared, however, lowest implantation and pregnancy rates were observed when embryo transfers occurred in autumn (44.6%), followed by winter (45.8%, OR: 1.049, CI: 0.876 – 1.257, p = 0.603) and summer (49.0%, OR: 1.196, CI: 1.010 – 1.415, p = 0.038), while spring embryo transfers had a 30% increased odds of pregnancy (51.2%, OR: 1.302, CI: 1.100 – 1.541, p = 0.002). An increased miscarriage rate was observed when embryo transfers occurred in autumn (13.9%), followed by winter (11.4%, OR: 0.510, CI: 0.323 – 0.805, p = 0.004) and summer (10.4%, OR: 0.461, CI: 0.292 – 0.727, p = 0.001), while spring embryo transfers had a 63% decreased odds of miscarriage (5.6%, OR: 0.369, CI: 0.230 – 0.592, p &amp;lt; 0.001). Limitations, reasons for caution The use of historical cohort groups is a drawback. Despite the eligibility criteria for inclusion in the analysis, potential differences in the baseline characteristics cannot be ruled out. Wider implications of the findings In seasonal breeding mammals, melatonin increases the gonadotropins’ release during the transition period. Melatonin rise favors embryo implantation and protects offspring by increasing progesterone levels. Our evidence suggests that human reproductive potential may be influenced by climatic variations. Trial registration number N/A

P-539 Chaotic blastocysts in preimplantation genetic testing for aneuploidies: prevalence, characterization and re-biopsy results

Abstract Study question Are we excluding blastocysts with favorable reproductive potential when embryos are designated as chaotic by Preimplantation Genetic Testing for Aneuploidies (PGT-A)? Summary answer The re-biopsy of chaotic blastocysts can rescue embryos with favorable reproductive potential only when there are ≥5 chromosomes displaying intermediate copy number values (CNV). What is known already An embryo is deemed as chaotic when multiple aneuploidies (5, 6 or more) are detected in the biopsied cells of the trophectoderm. Such embryos are classified as aneuploid, therefore their transfer is not recommended. There is evidence that chaotic embryos may result from the biopsy of apoptotic cells, cellular fragments and, more broadly, DNA degraded by multiple factors. Studies involving the re-biopsy and re-testing of a limited number of chaotic embryos have found a relevant percentage of euploid results. Moreover, there has been published a case report of a healthy live birth after the transfer of a chaotic embryo. Study design, size, duration This is a retrospective study of 1,442 cycles of PGT-A performed in twenty-two in vitro fertilization (IVF) clinics from November 2017 to December 2023. A total of 5,801 blastocysts were biopsied and analyzed in a single reference laboratory using Next-Generation Sequencing. Ninety-three chaotic blastocysts donated from patients who provided informed consent were thawed for re-biopsy in two IVF clinics. The subsequent re-analysis was performed in the same PGT laboratory using the same methodology. Participants/materials, setting, methods This study assessed the prevalence of chaotic embryos in all included cycles. Chaotic embryos were considered those with ≥5 chromosomes showing CNV below 1.75 or above 2.25. We sub-classified these embryos into pure chaotic (≥5 uniform aneuploidies), mixed chaotic (≥5 alterations considering uniform aneuploidies and chromosomes with intermediate CNV) and mosaic chaotic (≥5 chromosomes with intermediate CNV). Re-biopsies of 69 chaotic blastocysts were compared to the initial PGT-A results. Main results and the role of chance The prevalence of chaotic embryos was 4.3% (251/5,801); 13.5% (n = 34) pure, 53.0% (n = 133) mixed and 33.5% (n = 84) mosaic. After thawing 93 embryos, the survival rate was 74.2%. The 69 embryos that survived underwent re-biopsy yielding the following PGT-A results: 24.6% (n = 17) euploid, 37.7% (n = 26) chaotic, 26.1% (n = 18) aneuploid non-chaotic, 7.2% (n = 5) mosaic and 4.4% (n = 3) non informative. Nevertheless, outcomes varied substantially when the analysis was conducted based on the sub-category of chaotic embryos. In the pure category (n = 11), 72.7% (n = 8) showed identical results to the initial biopsy while 27.3% (n = 3) were aneuploid non-chaotic confirming at least one aneuploidy identified at the initial biopsy. Among the mixed category (n = 24), 54.2% (n = 13) were also mixed chaotic, and 45.8% (n = 11) were aneuploidy non-chaotic. All re-biopsies in this category also manifested at least one common aneuploidy with the initial biopsy or its opposite (trisomy/monosomy). In the mosaic category (n = 31), 54.8% (n = 17) were euploid, 16.1% (n = 5) were mosaic chaotic, 16.1% (n = 5) were mosaic non-chaotic and 12.9% (n = 4) were aneuploid non-chaotic. The concordance with the initial biopsy in terms of aneuploid result was 100% in the pure and mixed chaotic categories. However, over half of the embryos initially categorized as mosaic chaotic were, in fact, euploid. Limitations, reasons for caution Implantation, miscarriage and live birth rates are not explored. These outcomes are crucial for understanding the clinical significance of euploid embryos initially deemed as chaotic. The survival rate of chaotic embryos should be considered as it is lower than the survival rate of vitrified blastocysts reported in most IVF laboratories. Wider implications of the findings Previous research has suggested that re-biopsy can rescue embryos from PGT-A cycles given that a chaotic result has reduced predictive value. However, our current study reveals that not all chaotic embryos are suitable for re-biopsy, as 100% of pure and mixed chaotic blastocysts were aneuploid when re-tested. Trial registration number Not applicable

O-298 Embryo long-term storage does not affect ART outcome: analysis of 58,001 vitrified blastocysts

Abstract Study question Does duration of storage affect clinical outcome in frozen embryo transfer (FET) cycles? Summary answer Long-term storage does not affect live birth rate (LBR) in elective and non-elective FET cycles. What is known already Despite the success rates provided by the embryo vitrification programs, some studies found an association between the duration of cryo-storage and decreased clinical outcome, raising concern about the potential negative effect that prolonged storage may have on embryos. Study design, size, duration This was a retrospective multicenter study including a total of 58001 vitrified/warmed day-5 blastocysts from two different populations according to the reason for FET: elective FET which involved patients undergoing freeze all cycles (N = 16,615 blastocysts and 16,615 patients); and non-elective FET including 41,386 embryos from 25,571 patients. FETs were performed in the period between January 2010 and December 2020. Participants/materials, setting, methods Main outcome was live birth rate (LBR) and secondary outcomes were embryo survival, miscarriage, clinical and ongoing pregnancy rates. The impact of storage time and the potential confounding variables was assessed by univariate analyses. Those covariables found to significantly modify the LBR were included in a multivariable logistic regression. GEE (generalized estimated equation) was performed to allow the analysis of repeated measurements. Odds ratios (OR) and 95% confidence intervals (CI) were provided. Main results and the role of chance The blastocysts sample was clustered into deciles which provided specific storage duration categories that ranked from ≤0.67 to ≥ 4.34 and ≤1.8 to ≥ 34.81 months in freeze-all and non-elective FET, respectively. In the freeze-all group, maximum storage time was 34.8 months (2.9 years), while it was 121.2 months (10.1 years) in the non-elective FET group. Embryo survival did not show statistical differences across the categories of storage time in freeze-all and non-elective FET groups. In the freeze-all group, statistical differences were found for LBR across some, but not all the subgroups of storage duration. Similarly, in the non-elective FET group, LBR among the deciles showed statistical differences in some comparisons but not in all. However, after adjusting for confounding variables, the multivariate analysis showed no association between storage time and LBR in both groups. Blastocyst quality, body mass index (BMI), number of retrieved oocytes, endometrial preparation, male factor and uterine factor were related to the drop in the LBR in the freeze-all group (p &amp;lt; 0.05). In the non-elective FET group, the variables that showed significant association with the LBR were age at retrieval and at FET, number of embryos transferred, number of retrieved oocytes, BMI, endometrial preparation, sperm origin and female factor. Limitations, reasons for caution The retrospective design of the study represents its main limitation. Wider implications of the findings Our results demonstrate the safety of long-term cryo-storage. This is reassuring for both IVF practitioners and patients especially considering the exponential increase in FET cycles observed in the last decades. Trial registration number Not applicable

P-245 Evaluating the impact of embryos undergoing multiple freeze-thaw cycles in preimplantation genetic testing for aneuploidy (PGT-A) on treatment outcomes in IVF

Abstract Study question Do cryopreserved embryos subjected to thaw, biopsy, re-freeze (TBR) exhibit inferior pregnancy outcomes compared to embryos undergoing standard PGT-A with a single vitrification-warming cycle? Summary answer Transfer of twice-thawed euploid embryos demonstrated comparable clinical pregnancy outcomes, yet elevated miscarriage rates following a repeated vitrification-warming cycle warrants further scientific inquiry. What is known already There is often hesitancy to test cryopreserved embryos that were not initially PGT-A tested due to concerns about embryo survival during the additional freeze-thaw cycle those embryos will endure. However, the TBR technique has shown efficacy, particularly for IVF patients with recurrent failed attempts using fresh, untested embryos. Previous studies on TBR consistently report comparable embryo survival rates to fresh cycles. Nevertheless, there is no consensus on whether transferring euploid TBR embryos adversely affects pregnancy outcomes compared to euploid fresh biopsy embryos, potentially due to the relatively low number of transferred TBR embryos in such studies. Study design, size, duration This study retrospectively analysed euploid freeze-thaw single embryo transfer (SET) data from embryos that underwent PGT-A at a large fertility clinic in London, between January 2019 and June 2022. Pregnancy outcomes were compared between freshly biopsied embryos (control group, n = 349) and cryopreserved embryos that underwent thaw and biopsy before freeze-thaw SET (twice-thawed group, n = 35). Embryos previously biopsied with PGT-A, subsequently cryopreserved, and then subjected to thaw and re-biopsy were classified in the twice-thawed group. Participants/materials, setting, methods Blastocysts cultured from fresh embryos (control group) or derived from frozen-thawed blastocysts (twice-thawed group) underwent PGT-A using standard protocols before freeze-thaw SET. The study assessed biopsy rates, post first thaw and survival rates post second thaw in the twice-thawed group, comparing to the control group. The clinical outcomes evaluated were Implantation rate (IR), Live Birth Rate (LBR), and Miscarriage Rate (MR). Statistical analysis included one-way ANOVA for continuous and Pearson’s chi-squared test for categorical variables Main results and the role of chance Blastocyst characteristics, including morphological quality (characterised into ‘GOOD’, ‘FAIR’ and ‘POOR’) and day of biopsy (5 or 6), did not significantly differ between blastocyst transfers of the twice-thawed and control group. Furthermore, maternal factors including age, number of previous PGT-A attempts and endometrial thickness did not significantly differ between groups. However, compared with the control group, the number of eggs collected was significantly higher in the twice-thawed group (15.8±8.20 vs. 25.8±18.7, p = &amp;lt;0.001). The biopsy rate of previously cryopreserved embryos that underwent TBR was 79%. However, there was no significant difference between the survival rate after the second thaw and before SET between the twice-thawed and control groups. There was no difference in IRs (71.1% vs. 74.3%, p = 0.687) and LBRs (56.4% vs. 40.0%, p = 0.062) between groups. Although, compared to the control, MRs were significantly higher in the twice-thawed group (20.6% vs. 46.2% p = 0.003). Limitations, reasons for caution The twice-thawed group includes patients with diminished ovarian reserve, previously undergoing single freeze-thaw SET resulting in recurrent miscarriages. Thus, further research is essential to clarify whether elevated miscarriage rates result from repeated freeze-thaw cycles. Additionally, as single freeze-thaw SET is preferred, small sample size in the twice-thawed group limits conclusions. Wider implications of the findings This study suggests employing TBR on previously frozen blastocysts yields non-inferior IRs and LBRs, with an additional freeze-thaw cycle not adversely affecting embryo survival before SET. Therefore, such techniques should be considered in previously frozen IVF embryos to reduce wastage, mitigate unforeseen fertility complications, and concurrently achieve favourable pregnancy outcomes. Trial registration number Not applicable

Cryopreservation of date palm tissue cultures by vitrification and assessment of genetic stability using ISSR analysis

In this work, a successful cryopreservation method of date palm tissue cultures by vitrification was established. Callus and somatic embryos of three date palm cultivars were exposed to dimethylsulfoxide (DMSO) and Plant Vitrification Solution 2 (PVS2) for different durations before plunging into liquid nitrogen for cryoprotection. After freezing phase, the cryopreserved cultures were thawed and cultured on recovery media. Among different times of exposure to DMSO (10%), 60 min gave best results of survival and regrowth of the date palm tissue cultures. Regarding recovery, ‘Zaghlool’ cultivar gave the highest values of callus fresh mass (0.5 g) and growth value (1.00). Also, the highest differentiation percentages (75%) and number of proliferated shootlets (8.50) were recorded with ‘Zaghlool’ cultivar. Concerning PVS2, survival and regrowth gradually increased as exposure time increasing till 30 min and then decreased. Unlike of callus cultures, the survival and regrowth rates of somatic embryos were relatively higher at all PVS2 exposure time. With regard to recovery, the genotype effect took same trend in cryopreservation of callus cultures, since ‘Zaghlool’ was superior in growth parameters in comparison to the other two cultivars. On the other hand, genetic stability of the cryopreserved date palm tissue cultures was assessed using ISSR markers. Among four screened primers, three gave monomorphic bands; all of these banding profiles were similar to those of non-cryopreserved cultures. Generally, the results of ISSR showed high genetic similarity suggesting that cryopreservation using vitrification does not affect genetic stability of date palm tissue cultures. The results of this study demonstrated PVS2 was more effective in cryoprotection of date palm tissue cultures compared with DMSO and cryopreservation by vitrification is a suitable tool for conservation of genetic stable date palm germplasm.