Perinatal outcomes of frozen-thawed embryo transfer following blastocyst-stage embryo transfer compared to those of cleavage-stage embryo transfer:analysis of 9408 singleton newborns using propensity score analysis

Objective:Regarding frozen-thawed embryo transfer(FET),there is limited consensus on whether extending embryo culture from the cleavage stage to the blastocyst stage affects perinatal outcomes.This study aimed to compare perinatal outcomes of singletons between blastocyst-stage embryo transfer(BT)and cleavage-stage embryo transfer(CT)in FET.Methods:A total of 9408 FET cycles that met the inclusion criteria were included in this retrospective cohort study between 2019 and 2022.Blastocyst-stage embryo transfers were performed in the BT group,and cleavage-stage embryo transfers were performed in the CT group.Multivariate logistic regression analyses were performed,as well as propensity score matching(PSM)to adjust for confounders.Results:After PSM,a higher risk of pre-term birth(PTB;odds ratio[OR]:1.23,95%confidence interval[CI]:1.00-1.50,P=0.048)and being large for gestational age(LGA;OR:1.16,95%CI:1.00-1.35,P=0.050)was observed in the BT group compared to that in the CT group.After stratified PSM,in the subgroup under 35 years of age,only an increased risk of LGA was observed in the BT group compared to the CT group.Perinatal outcomes in the double-embryo transfer subgroup were similar to those in the unstratified group.However,in the subgroup beyond 35 years of age and the single embryo transfer subgroup,perinatal outcomes were not statistically different between the BT and CT groups(P>0.05).Conclusions:In FET,prolonged embryo culture to the blastocyst stage increased the risk of PTB and LGA in single fetuses.However,stratified analysis based on age and the number of transferred embryos yielded different results,necessitating further mechanistic studies.

Effect of vitrification on clinical outcomes of cleavage-stage embryos with poor quality in human embryo cryopreservation

Objective: To evaluate the effect of morphologic factors on survival rate (SR), pregnancy rate (PR), and implantation rate (IR) of human embryo vitrification following frozen embryo transfer (FET) on day 3 post-ovulation.Methods: Women undergoing FET (n = 921) with embryos cryopreserved by vitrification between 2012 and 2013 were enrolled in this retrospective cohort study.Results: Embryos with >9 blastomeres yielded the highest SR of 100%. Lower SR was observed in embryos with 5 to 6 (57.5%) and 4 blastomeres (41.4%). In terms of blastomere symmetry, the SR of embryos with equally sized blastomeres was significantly higher than that of embryos with unequally sized cells (82.5%vs. 64.6%,P < 0.05). As fragmentation increased, SR decreased from 92.1% to 20.6% (P < 0.05). Significant differences were observed among groups when analyzing PR and IR according to the 3 embryonic parameters before vitrification. Embryos with 13 to 16 blastomeres yielded the highest PR (39.5%) and IR (24.1 %). The PR and IR of embryos with blastomeres of equal size were significantly higher than those with unequally sized blastomeres (36.5%vs. 21.7%, 23.7%vs. 12.4%,P < 0.05). After warming, embryos with 13 to 16 blastomeres yielded the highest PR and IR (40.9% and 24.2%, respectively). The PR and IR were observed to grow with an increase in the percentage of intact blastomeres (23.2%-38.2%, 14.2%-23.2%).Conclusions: These results show that vitrification methods do not effectively improve survival outcomes for embryos of poor quality and it is needed to develop a comprehensive vitrification protocol that considers all the practical aspects, including the current limitation regarding cleavage-stage embryos of poor quality.

Embryo Transfer Strategies for Women with Recurrent Implantation Failure During the Frozen-thawed Embryo Transfer Cycles:Sequential Embryo Transfer or Double-blastocyst Transfer?

Objective Both sequential embryo transfer(SeET)and double-blastocyst transfer(DBT)can serve as embryo transfer strategies for women with recurrent implantation failure(RIF).This study aims to compare the effects of SeET and DBT on pregnancy outcomes.Methods Totally,261 frozen-thawed embryo transfer cycles of 243 RIF women were included in this multicenter retrospective analysis.According to different embryo quality and transfer strategies,they were divided into four groups:group A,good-quality SeET(GQ-SeET,n=38 cycles);group B,poor-quality or mixed-quality SeET(PQ/MQ-SeET,n=31 cycles);group C,good-quality DBT(GQ-DBT,n=121 cycles);and group D,poor-quality or mixed-quality DBT(PQ/MQ-DBT,n=71 cycles).The main outcome,clinical pregnancy rate,was compared,and the generalized estimating equation(GEE)model was used to correct potential confounders that might impact pregnancy outcomes.Results GQ-DBT achieved a significantly higher clinical pregnancy rate(aOR 2.588,95%CI 1.267–5.284,P=0.009)and live birth rate(aOR 3.082,95%CI 1.482–6.412,P=0.003)than PQ/MQ-DBT.Similarly,the clinical pregnancy rate was significantly higher in GQ-SeET than in PQ/MQ-SeET(aOR 4.047,95%CI 1.218–13.450,P=0.023).The pregnancy outcomes of GQ-SeET were not significantly different from those of GQ-DBT,and the same results were found between PQ/MQ-SeET and PQ/MQ-DBT.Conclusion SeET relative to DBT did not seem to improve pregnancy outcomes for RIF patients if the embryo quality was comparable between the two groups.Better clinical pregnancy outcomes could be obtained by transferring good-quality embryos,no matter whether in SeET or DBT.Embryo quality plays a more important role in pregnancy outcomes for RIF patients.

Clinical validation of the early embryo viability assessment system: Analysis for the blastocyst morphology and pregnancy outcomes

Objective:To determine the relationship between the early embryo viability assessment(EEVA)and blastocyst morphological parameters and pregnancy outcomes.Methods:This retrospective cohort study was conducted on 291 intracytoplasmic sperm injection cycles including 2522 embryos with indications of prolonging embryo culture to the blastocyst stage in the Genea embryo review incubator,and 511 single vitrified-warmed blastocyst transfer cycles from January 2020 to June 2023.The EEVA system produced an EEVA score from E1(best)to E5(worse)for the potential of blastocyst formation.Blastocyst morphology was evaluated.The association between the EEVA score and each type of blastocyst morphology,implantation rate,clinical pregnancy,and ongoing pregnancy were assessed using generalized estimating equations.Results:The inner cell mass A(ICM A),trophectoderm A(TE A),blastocoele expansion degree of 3,4,5,6,7 rates were higher with lower the EEVA score.The adjusted odd ratio(aOR)(E5 vs E1)was 0.3 for ICM A,0.174 for TE A and 0.210 for BL3,4,5,6,7(all P<0.001),suggesting a significant association between lower EEVA scores and improved embryo quality.The implantation,clinical pregnancy,and ongoing pregnancy rate were also higher with lower the EEVA score.The aOR of E5 vs E1 was 0.245 for implantation,0.185 for clinical pregnancy and 0.200 for ongoing pregnancy rate(P<0.001).Conclusions:There were associations between blastocyst morphology,pregnancy outcome and EEVA scores.The good blastocyst morphology and pregnancy outcomes are higher with lower the EEVA score.

Endochondral ossification of hindlimbs in embryonic development of Japanese Quail(Coturnix japonica)

The endochondral ossification of hindlimb is essential to a bird’s ability to stand,walk and fly.Most hindlimb is ossified in the embryos before hatching in precocial birds.However,the molecular mechanisms of hindlimb ossification in birds is still unclear.Therefore,we tried to examine the process of hindlimb ossification and its molecular regulation by using an animal model—Japanese Quail(Coturnix japonica).We selected four critical stages(Embryo Day:E6,E8,E12 and E16) of skeletal development of embryonic quails for hindlimb skeleton staining to show the process of endochondral ossification and to examine the molecular regulation of endochondral osteogenesis by RNA-Seq analysis.The results showed that ossification became increased with embryonic development and most hindlimb was ossified before hatching.RNA-Seq analysis revealed that various signaling pathways were involved with endochondral ossification with thyroid hormone signaling and WNT signaling pathway particularly enriched.Moreover,the expression levels of 42 genes were continuously upregulated and 14 genes were continuously downregulated from E6 to E16.The present study might provide new insights into complex molecular mechanisms in regulation of endochondral ossification.

Embryonic thermal manipulation:a potential strategy to mitigate heat stress in broiler chickens for sustainable poultry production

Due to high environmental temperatures and climate change, heat stress is a severe concern for poultry health and production, increasing the propensity for food insecurity. With climate change causing higher temperatures and erratic weather patterns in recent years, poultry are increasingly vulnerable to this environmental stressor. To mitigate heat stress, nutritional, genetic, and managerial strategies have been implemented with some success. However, these strategies did not adequately and sustainably reduce the heat stress. Therefore, it is crucial to take proactive measures to mitigate the effects of heat stress on poultry, ensuring optimal production and promoting poultry well-being. Embryonic thermal manipulation(TM) involves manipulating the embryonic environment's temperature to enhance broilers' thermotolerance and growth performance. One of the most significant benefits of this approach is its cost-effectiveness and saving time associated with traditional management practices. Given its numerous advantages, embryonic TM is a promising strategy for enhancing broiler production and profitability in the poultry industry. TM increases the standard incubation temperature in the mid or late embryonic stage to induce epigenetic thermal adaption and embryonic metabolism. Therefore, this review aims to summarize the available literature and scientific evidence of the beneficial effect of pre-hatch thermal manipulation on broiler health and performance.

tRNA^(Glu)-derived fragments from embryonic extracellular vesicles modulate bovine embryo hatching

Transfer RNA-derived small RNAs(tsRNAs)have been shown to be involved in early embryo development and repression of endogenous retroelements in embryos and stem cells.However,it is unknown whether tsRNAs also regulate embryo hatching.In this study,we mined the sequencing data of a previous experiment in which we demonstrated that the microRNA(miRNA)cargo of preimplantation embryonic extracellular vesicles(EVs)influences embryo development.We thus profiled the tsRNA cargo of EVs secreted by blastocysts and non-blastocysts.The majority of tsRNAs was identified as tRNA halves originating from the 5'ends of tRNAs.Among the 148 differentially expressed tsRNAs,the 19 nt tRNA fragment(tRF)tDR-14:32-Glu-CTC-1 was found to be significantly up-regulated in EVs derived from non-blastocysts.RT-qPCR assays confirmed its significant up-regulation in non-blastocyst embryos and their conditioned medium compared to the blastocyst group(P<0.05).Inhibition of tDR-14:32-Glu-CTC-1 by supplementing antagomirs to the conditioned medium improved embryo hatching(P<0.05).Transcriptomic analysis of embryos treated with tDR-14:32-Glu-CTC-1 antagomirs further showed differential expression of genes that are associated with embryo hatching and implantation.In summary,tDR-14:32-Glu-CTC-1 is up-regulated in non-blastocyst embryos and their secretions,and inhibition of tDR-14:32-Glu-CTC-1 promotes embryo hatching,while influencing embryo implantation-related genes and pathways.These results indicate that embryonic EVs containing specific tRFs may regulate preimplantation embryo development.

Pre-existing orthorhombic embryos-induced hexagonal-orthorhombic martensitic transformation in MnNiSi_(1-x)(CoNiGe)_x alloy

The thermal-elastic martensitic transformation from high-temperature Ni_(2)In-type hexagonal structure to low-temperature TiNiSi-type orthorhombic structure has been widely studied in MnMX(M=Ni or Co,and X=Ge or Si)alloys.However,the answer to how the orthorhombic martensite nucleates and grows within the hexagonal parent is still unclear.In this work,the hexagonal-orthorhombic martensitic transformation in a Co and Ge co-substituted MnNiSi is investigated.One can find some orthorhombic laths embedded in the hexagonal parent at a temperature above the martensitic transformation start temperature(M_(s)).With the the sample cooing to M_(s),the laths turn broader,indicating that the martensitic transformation starts from these pre-existing orthorhombic laths.Microstructure observation suggests that these pre-existing orthorhombic laths do not originate from the hexagonal-orthorhombic martensitic transformation because of the difference between atomic occupations of doping elements in the hexagonal parent and those in the preexisting orthorhombic laths.The phenomenological crystallographic theory and experimental investigations prove that the pre-existing orthorhombic lath and generated orthorhombic martensite have the same crystallography relationship to the hexagonal parent.Therefore,the orthorhombic martensite can take these pre-existing laths as embryos and grow up.This work implies that the martensitic transformation in MnNiSi_(1-x)(CoNiGe)_(x) alloy is initiated by orthorhombic embryos.

Effect of coenzyme Q10 supplementation on post-vitrification mouse embryo development

Objective:To investigate the effects of coenzyme Q10(CoQ10)supplementation on post-vitrification embryo development and gross morphology.Methods:Balb/c mouse embryos were cultured in potassium simplex optimised medium(KSOM)with varying CoQ10 concentrations[0(control),20,40,and 60μM].The most effective CoQ10 concentration(40μM)was selected for subsequent post-vitrification morphology study.Embryos were randomly divided into four groups:Group A(non-vitrified without CoQ10),Group B(non-vitrified with CoQ10),Group C(vitrified without CoQ10),and Group D(vitrified with CoQ10),followed by vitrification at the 8-cell stage.Survival rates and development until the blastocyst stage were evaluated through morphological examinations using ASEBIR's system,distinguishing normal and abnormal embryos.Results:Supplementation of 40μM CoQ10 significantly increased blastocyst formation(95%)compared to the control group(92%),20μM(62%),and 60μM(56%)(P<0.001).Following vitrification,Group D exhibited a significant increase in blastocyst formation(92%)compared to Group C(82%)(P<0.05).Morphological assessments indicated superior embryo quality in Group B over Group D during the cleavage stage,morula,and blastocyst(P<0.05).Conclusions:CoQ10 supplementation exhibits promising potential to enhance preimplantation embryo development,increase blastocyst formation rates,and improve embryo quality post-vitrification.This offers a promising approach to mitigate oxidative stress on embryos,potentially improving overall assisted reproductive technology outcomes.

A New Micropropagation Technology of Tilia amurensis:In VitroMicropropagation of Mature Zygotic Embryos and the Establishment of a PlantRegeneration System

Tilia amurensis is an economically valuable broadleaf tree species in Northeast China.The production of highqualityT.amurensis varieties at commercial scales has been greatly limited by the low germination rates.Thereis thus a pressing need to develop an organogenesis protocol for in vitro propagation of T.amurensis to alleviate ashortage of high-quality T.amurensis seedlings.Here,we established a rapid in vitro propagation system forT.amurensis from mature zygotic embryos and analyzed the effects of plant growth regulators and culture mediain different stages.We found that Woody plant medium(WPM)was the optimal primary culture medium formature zygotic embryos.The highest callus induction percentage(68.76%)and number of axillary buds induced(3.2)were obtained in WPM+0.89μmol/L 6-benzyladenine(6-BA)+0.46μmol/L kinetin(KT)+0.25μmol/Lindole-3-butryic acid(IBA)+1.44μmol/L gibberellin A_(3)(GA_(3)).The multiple shoot bud development achievedthe highest percentage(83.32%)in the Murashige and Skoog(MS)+2.22μmol/L 6-BA+0.25μmol/L IBA+1.44μmol/L GA_(3).The rooting percentage(96.70%)was highest in 1/2 MS medium+1.48μmol/L IBA.Thesurvival percentage of transplanting plantlets was 82.22%in soil:vermiculite:perlite(5:3:1).Our study is the firstto establish an effective organogenesis protocol for T.amurensis using mature zygotic embryos.

Enhancement of porcine in vitro embryonic development through luteolin‑mediated activation of the Nrf2/Keap1 signaling pathway

Background Oxidative stress,caused by an imbalance in the production and elimination of intracellular reactive oxygen species(ROS),has been recognized for its detrimental effects on mammalian embryonic development.Luteolin(Lut)has been documented for its protective effects against oxidative stress in various studies.However,its specific role in embryonic development remains unexplored.This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism.Results After undergoing parthenogenetic activation(PA)or in vitro fertilization,embryos supplemented with 0.5μmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates,with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control.Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control.Moreover,Lut supplementation significantly augmented mitochondrial content and membrane potential.Intriguingly,activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut,leading to the upregulation of antioxidant-related gene transcription levels.To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development,we cultured PA embryos in a medium supplemented with brusatol,with or without the inclusion of Lut.The positive effects of Lut on developmental competence were negated by brusatol treatment.Conclusions Our findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence,and offers insight into the mechanisms regulating early embryonic development.

Embryo-to-lamella transition of grain boundary twins in magnesium

A combined experimental and computational analysis is performed to investigate the less commonly studied embryo-to-lamella transition of deformation twins in magnesium. This work aims to understand the structural variables controlling the embryo-to-lamella transition from grain boundaries. Statistical analysis of hundreds of early-stage twins in the lightly deformed microstructure reveals a prevailing wedge shape,with a much thicker base along the grain boundary(GB) where they originate and a thinner tip terminating in the crystal. The analysis also shows that the GB base is super thick and identifies a minimum GB twin thickness among all early-stage twins that is about one micron. A crystal plasticity-based full-field model is employed to calculate the driving forces to migrate the boundary of a three-dimensional GB twin embryo. The stress analysis, considering a full range of embryo shapes and neighboring grain orientations, indicate that the twin embryo is most likely going to form a wedge shape when it first propagates. The calculations predict that the thickness of the embryo at the GB needs to be significantly larger than its length into the crystal in order to propagate into the crystal. The analysis finds that the more aligned the twin embryo variant is with basal slip in the neighboring grain, the thinner the twin embryo needed for propagation.

Extracellular vesicles from oviductal and uterine fluids supplementation in sequential in vitro culture improves bovine embryo quality

Background:In vitro production of bovine embryos is a well-established technology,but the in vitro culture(IVC)system still warrants improvements,especially regarding embryo quality.This study aimed to evaluate the effect of extracellular vesicles(EVs)isolated from oviductal(OF)and uterine fluid(UF)in sequential IVC on the development and quality of bovine embryos.Zygotes were cultured in SOF supplemented with either BSA or EVs-depleted fetal calf serum(dFCS)in the presence(BSA-EV and dFCS-EV)or absence of EVs from OF(D1 to D4)and UF(D5 to D8),mimicking in vivo conditions.EVs from oviducts(early luteal phase)and uterine horns(mid-luteal phase)from slaughtered heifers were isolated by size exclusion chromatography.Blastocyst rate was recorded on days 7-8 and their quality was assessed based on lipid contents,mitochondrial activity and total cell numbers,as well as survival rate after vitrification.Relative mRNA abundance for lipid metabolism-related transcripts and levels of phosphorylated hormonesensitive lipase(pHSL)proteins were also determined.Additionally,the expression levels of 383 miRNA in OF-and UF-EVs were assessed by qRT-PCR.Results:Blastocyst yield was lower(P<0.05)in BSA treatments compared with dFCS treatments.Survival rates after vitrification/warming were improved in dFCS-EVs(P<0.05).EVs increased(P<0.05)blastocysts total cell number in dFCS-EV and BSA-EV compared with respective controls(dFCS and BSA),while lipid content was decreased in dFCSEV(P<0.05)and mitochondrial activity did not change(P>0.05).Lipid metabolism transcripts were affected by EVs and showed interaction with type of protein source in medium(PPARGC1B,LDLR,CD36,FASN and PNPLA2,P<0.05).Levels of pHSL were lower in dFCS(P<0.05).Twenty miRNA were differentially expressed between OF-and UF-EVs and only bta-miR-148b was increased in OF-EVs(P<0.05).Conclusions:Mimicking physiological conditions using EVs from OF and UF in sequential IVC does not affect embryo development but improves blastocyst quality regarding survival rate after vitrification/warming,total cell number,lipid content,and relative changes in expression of lipid metabolism transcripts and lipase activation.Finally,EVs miRNA contents may contribute to the observed effects.

More than a simple egg:Underlying mechanisms of cold tolerance in avian embryos

Avian embryos,which develop within eggs,exhibit remarkable tolerance to extremely low temperatures.Despite being a common trait among all birds,the mechanisms underlying this cold tolerance in avian embryos remain largely unknown.To gain a better understanding of this phenomenon and the coping mechanisms involved,we reviewed the literature on severe cold tolerance in embryos of both wild and domestic birds.We found that embryos of different bird orders exhibit tolerance to severe cold during their development.In response to cold stress,embryos slow down their heartbeat rates and metabolism.In severe cold temperatures,embryos can suspend these processes,entering a torpid-like state of cardiac arrest.To compensate for these developmental delays,embryos extend their regular incubation periods.Depending on their embryonic age,embryos of all bird species can tolerate acute severe cold regimes;only a few tolerate chronic severe cold regimes.We also discussed various extrinsic and intrinsic factors that affect the tolerance of bird embryos to low temperatures before and after incubation.Cold tolerance appears to be a heritable trait shared by wild and domestic embryos of all bird classes,regardless of egg size or development(altricial/precocial).Driven by environmental variability,cold tolerance in avian embryos is an optimal physiological and ecological strategy to mitigate the adverse effects of cold conditions on their development in response to fluctuating environmental temperatures.

Anethole improves the developmental competence of porcine embryos by reducing oxidative stress via the sonic hedgehog signaling pathway

Background Anethole(AN)is an organic antioxidant compound with a benzene ring and is expected to have a positive impact on early embryogenesis in mammals.However,no study has examined the effect of AN on porcine embryonic development.Therefore,we investigated the effect of AN on the development of porcine embryos and the underlying mechanism.Results We cultured porcine in vitro-fertilized embryos in medium with AN(0,0.3,0.5,and 1 mg/mL)for 6 d.AN at 0.5 mg/mL significantly increased the blastocyst formation rate,trophectoderm cell number,and cellular survival rate compared to the control.AN-supplemented embryos exhibited significantly lower reactive oxygen species levels and higher glutathione levels than the control.Moreover,AN significantly improved the quantity of mitochondria and mitochondrial membrane potential,and increased the lipid droplet,fatty acid,and ATP levels.Interestingly,the levels of proteins and genes related to the sonic hedgehog(SHH)signaling pathway were significantly increased by AN.Conclusions These results revealed that AN improved the developmental competence of porcine preimplantation embryos by activating SHH signaling against oxidative stress and could be used for large-scale production of high-quality porcine embryos.

Multi-protective effects of wheat embryo globulin on D-gal-induced aging mice

Wheat embryo globulin(WEG)has been proven to possess multiple biological activities,including antioxidative properties,immunomodulatory,and so on.Aged mouse model were established by subcutaneous injection of D-galactose(D-gal),and the effects of WEG on learning,memory,and antioxidant capacity in aging mice were explored through behavioural tests and antioxidant enzyme activities determination.Compared with the Model group,WEG improved the percentage of the platform quadrant,increased the number of crossing platforms,and enhanced the identification indexs.WEG also increased total antioxidant capacity(T-AOC),superoxide dismutase(SOD),and glutathione peroxidase(GSH-Px)activities in the liver and brains of aging mice,and reduced malondialdehyde(MDA)content.Pathological observations indicated that WEG protected against damage to brain in D-gal-induced aging mice.These results effectively revealed that WEG not only improved the abilities of learning and memory,and the cognitive impairment,but also delayed the aging process of the D-gal-induced mice.

The fate of surplus embryos in the setting of assisted reproductive technology:A scoping review

Objective:To identify the attitudes of infertile couples toward their surplus frozen embryos.Methods:This study was according to PRISMA-ScR as the guideline for scoping review.Studies that assessed the attitudes of patients or infertile couples who had surplus embryos were included.We conducted systematic searches in English studies from April 2011-April 2021 using 7 databases:PubMed,Science Direct,EBSCO,Scopus,the Cochrane Library,Sage Journals,and Google Scholar.Data were charted based on author,year of publication,country,purpose,data collection,key findings,and research focus/domain.Results:A total of 37 research articles were included in the analysis.Their attitudes encompassed:supporting the donation of the surplus embryos for both research and reproductive purposes,continuing to store the frozen embryos,and disposing of the surplus embryos.Conclusions:Most of the infertile patients support donating their surplus embryos for research and reproductive purposes.

Thermal conditioning of quail embryos has transgenerational and reversible long‑term effects

Background In the current context of global warming,thermal manipulation of avian embryos has received increasing attention as a strategy to promote heat tolerance in avian species by simply increasing the egg incubation temperature.However,because of their likely epigenetic origin,thermal manipulation effects may last more than one generation with consequences for the poultry industry.In this work,a multigenerational and transgenerational analysis of thermal manipulation during embryogenesis was performed to uncover the long-term effects of such procedure.Results Thermal manipulation repeated during 4 generations had an effect on hatchability,body weight,and weight of eggs laid in Japanese quails,with some effects increasing in importance over generations.Moreover,the effects on body weight and egg weight could be transmitted transgenerationally,suggesting non-genetic inheritance mechanisms.This hypothesis is reinforced by the observed reversion of the effect on growth after five unexposed generations.Interestingly,a beneficial effect of thermal manipulation on heat tolerance was observed a few days after hatching,but this effect was not transgenerational.Conclusions Our multigenerational study showed that thermal conditioning of quail embryos has a beneficial effect on post-hatch heat tolerance hampered by transgenerational but reversible defects on growth.Assuming that no genetic variability underlies these changes,this study provides the first demonstration of epigenetic inheritance of traits induced by environmental temperature modification associated with long-term impacts in an avian species.

Transcriptomic and proteomic studies of condylar ossification of the temporomandibular joint in porcine embryos

Background:The ossification mechanism of the temporomandibular joint(TMJ)condyle remains unclear in human embryo.The size and structure of TMJ,shape of articular disc and the characteristics of omnivorous chewing in the pig are similar to those of humans.The pig is an ideal animal for studying the mechanism of ossification of the TMJ condyle during the embryonic period.Method:In a previous study by our group,it was found that there was no condylar ossification on embryonic day(E)45,but the ossification of condyle occurred between E75 and E90.In this study,a total of 12 miniature pig embryos on E45 and E85 were used.Six embryos were used for tissue sections(3 in each group).The remaining six embryos were used for transcriptomic and proteomic studies to find differential genes and proteins.The differentially expressed genes in transcriptome and proteomic analysis were verified by QPCR.Results:In total,1592 differential genes comprising 1086 up-regulated genes and 506 down-regulated genes were screened for fold changes of≥2 to≤0.5 between E45 and E85.In the total of 4613 proteins detected by proteomic analysis,there were 419 differential proteins including 313 up-regulated proteins and 106 down-regulated proteins screened for fold changes of≥2 to≤0.5 between E45 and E85.A total of 36 differential genes differing in both transcriptome and proteome analysis were found.QPCR analysis showed that 14 of 15 selected genes were consistent with transcriptome analysis.Conclusion:Condylar transcriptome and proteomic analysis during the development of TMJ in miniature pigs revealed the regulatory genes/proteins of condylar ossification.

High-throughput sorting of two-color fluorescent-labeled zebrafish embryos

The zebrafish embryos were widely employed in genetics,development and drug discovery studies as miniatured animal models.Sorting of two-color fluorescent embryos is often required in large-scale experiments but it is challenging to manually sort with high efficiency.Here,we reported a high-throughput sorting system for two-color fluorescent zebraflsh embryos.The embryos can be automatically loaded from a sample pool and sorted based on the average fluorescent intensity.The two-color fluorescent signals were split into two lines and detected by an area array camera.The system achieves the sorting of 100 embryos in less than 10 min with an accuracy of greater than 95%.