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.

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.

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.

Kidding after Transfer of in Vitro Produced Saanen Goat Embryos into Local Ukrainian Breed Recipients in Different Seasons

In recent years, the demand for goat products has been growing due to the fact that goat milk has a number of advantages over cow milk, for example, it is low in lactose, and is considered less allergenic and easier to digest. To increase production during both breeding and non-breeding seasons and reduce the price of dairy products, it is necessary to effectively use reproductive management and assisted reproductive technologies. In vitro embryo production makes it possible to obtain a large number of eggs from goats, which for some reason are unable to conceive, but have genetic value. Afterward in vitro produced embryos can be transferred into recipient goats of other less genetically valuable breeds, such as the Ukrainian local breed. Therefore, the aim of the present study was to investigate the effectiveness of transfers of in vitro produced embryos of Saanen goats into surrogate sires of the Ukrainian local breed in different seasons. All manipulations with animals were carried out following ethical standards (Strasbourg, 1986). Six Saanen goats were selected as the oocyte donors. After the hormonal stimulation oocytes were retrieved by laparoscopic ovum pick-up. In vitro produced embryos were transferred laparotomically into 24 recipients of Ukrainian local breed. Fifty days after embryo transfers, pregnancies were determined by ultrasound diagnostics. Although the embryo development rate in the breeding season was 20% higher than in the non-breeding season, there was no difference in pregnancy and kidding rates between seasons. In conclusion, the transfer of in vitro produced Saanen goat embryos to recipients of the Ukrainian local breed gives the opportunity to achieve pregnancy and kidding regardless of the breeding season, which will enable a faster and more efficient increase in the livestock of highly productive goats in Ukraine in the post-war period.

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.

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%.

Embryo quality and chromosomal abnormality in embryos from couples undergoing assisted reproductive technology using preimplantation genetic screening

Objective:To detect common chromosomal aneuploidy variations in embryos from couples undergoing assisted reproductive technology and preimplantation genetic screening and their possible associations with embryo quality.Methods:In this study,359 embryos from 62 couples were screened for chromosomes 13,21,18,X,and Y by fluorescence insitu hybridization.For biopsy of blastomere,a laser was used to remove a significantly smaller portion of the zona pellucida.One blastomere was gently biopsied by an aspiration pipette through the hole.After biopsy,the embryo was immediately returned to the embryo scope until transfer.Embryo integrity and blastocyst formation were assessed on day 5.Results:Totally,282 embryos from 62 couples were evaluated.The chromosomes were normal in 199(70.57%)embryos and abnormal in 83(29.43%)embryos.There was no significant association between the quality of embryos and numerical chromosomal abnormality(P=0.67).Conclusions:Embryo quality is not significantly correlated with its genetic status.Hence,the quality of embryos determined by morphological parameters is not an appropriate method for choosing embryos without these abnormalities.

Pregnancy Outcomes for Day 5 Versus Day 6 Single Frozen-thawed Blastocyst Transfer with Different Qualities of Embryos: A Large Matched-cohort Study

Objective This study aimed to determine whether the day of blastocyst expansion affects pregnancy outcomes in frozen-thawed blastocyst transfer(FBT)cycles.Methods A retrospective match-cohort study was conducted.Patients who underwent blastocyst transfer in frozen-thawed cycles at day 5 or 6 were matched for potential confounding factors.A total of 2207 matched pairs of FBT cycles were included from January 2016 to December 2019 in our Reproductive Medicine Center.Results The clinical pregnancy rate(CPR)and live birth rate(LBR)were significantly increased in day 5 blastocyst transfers when compared to day 6 blastocyst transfers,in terms of the same embryo quality.For FBT cycles with good-quality embryo,the CPR at day 5 and 6 was 61.30%and 57.56%,respectively(P=0.045),and the LBR was 44.79%and 36.16%,respectively(P<0.001).For FBT cycles with poor-quality embryo,the CPR at day 5 and 6 was 48.61%and 40.89%,respectively(P=0.006),and the LBR was 31.71%and 25.74%,respectively(P=0.019).The CPR for FBT cycles with good-quality embryo was statistically higher at day 6 than that at day 5 with poor-quality embryo transferred(57.56%vs.48.61%,P=0.001).Maternal age,anti-Müllerian hormone(AMH),endometrial thickness,embryo quality,and the day of blastocyst expansion were independently correlated with the CPR and LBR.The FBT cycles at day 5 had significantly higher CPR(adjusted odds ratio[OR]=1.246,95%confidence intervals[CI]:1.097–1.415,P=0.001)and LBR(adjusted OR=1.435,95%CI:1.258–1.637,P<0.001)than those at day 6.Conclusion The embryo quality is the primary indicator for FBT cycles.Day 5 blastocysts should be preferred when the quality of embryo at day 5 is the same as that at day 6.

Effects of Genotypes and Basic Medium on Culture of Maize Mature Embryos

[Objective]This study was to screen out suitable genotypes and basic medium for the culture of maize mature embryos.[Method]Using mature embryos of nine maize genotypes as explants,the effects of genotypes and basic medium on callus induction and subculture were investigated.[Result]The genotypes performed better in callus induction and subculture were found in turn 853-35,853-209,Dan 34 and 81162.MS medium is better than N6 medium in the callus induction from maize embryos,while N6 medium is more suitable for callus subculture.[Conclusion]Our study further improved the tissue culture system in maize with mature embryos as explants.

Callus Induction from Mature Embryos of Maize

In this study we studied the factors influencing the callus induction from mature embryos of maize inbred lines Qi 319, Zhen 58, Chang 7 -2, Lx 9801 and 81162, such as genotype, combination of plant growth regulators, and low-temperature pretreatment. The results showed that the induction rate of Qi 319 was the highest among the four genotypes tested; combination of 4.0 mg/L 2,4-D ＋ 0.5 mg/L 6-BA was suitable for inducing callus from mature embryos; three days of 4℃ pretreatment can promote the callus induction significantly. The indices optimized in the present study are helpful for establishing genetic transformation system in maize without considering seasonal variation.

Study on Culturing Prunus salicina cv.Zaoshi Embryos

[Objective]The aim was to study the effective factors on culturing Prunus salicina cv.Zaoshi embryos in vitro.[Method]Different age,culture medium and GA3 which affected culturing of prumssalicina embryos were discussed.[Result]The result showed that on the part of 26-41 d,after the blooming period,the embryos remained tiny and retained endosperms and showed no signs of change after having cultured for three generations.On the part of 48 d,after the blooming period,the endosperms had disappeared,the embryos kept growing until they filled the seed cavity;when they were planted on the MS culture medium,their survival rate reached 77%,in its first generation,the response of embryos was discernible.On the part of 65 d,after the blooming period,4.5% of their embryos grew into shoots on the MS culture medium;with the age of embryos growing,the survival rate of shoots increased until it reached 26% when the fruits went into ripeness;the embryos produced calli in their first generation of culturing.On the part of 65-83 d,after the blooming period,the embryos produced calli through more than 2-3 generations.On the part of 88 d,after the blooming period,the survival rate of shoots on the WPM culturing base doubled compared with that on the MS culturing base;on the same culture medium,the embryos were inhibited from growing into shoots when BA,KT or 2,4-D was added on to the culture medium.The survival rate of shoots was increased remarkably when the seeds were treated in 1 000 mg/L GA3.[Conclusion] This study provided experimental basis for the establishment of Prunus salicina cv.Zaoshi,embryos rescue techniques and cross breading.

Fluctuations of Endogenous Hormones in Isolated Rice Embryos During Embryogenesis and Early Stages of Germination

Fluctuations of levels of several endogenous plant hormones in isolated rice ( Oryza sativa ssp. japonica) embryos during early and mid-embryogenesis and early stages of germination were studied by enzyme-linked immunosorbent assay (ELISA). Embryos were collected at different days after pollination (DAP) and different days after imbibition (DAI) of mature seeds. The contents of gibberellin(1) (GA(1)), abscisic acid (ABA), isopentenyladenine and isopentenyladenosine ( iPAs), zeatin and zeatin riboside ( ZRs) were immunochemically assayed. The GA(1) level was the highest among all hormones tested. The variations of GA(1) levels were opposite with the ABA levels, with some exceptions. During early and mid-embryogenesis, the levels of GA(1) and ABA were the highest at 4 DAP. From 8 to 18 DAP, GA(1) level declined, whereas the ABA level increased. During germination, GA(1) level increased at 2 DAI whereas simultaneously the ABA content decreased. The highest ratio of GA(1)/ABA was observed at 2 DAI The levels of iPAs and ZRs were maxima in the embryos at 4 DAP, decreased to a very low level and maintained constant thereafter. Our results provide further evidence that GA(1) plays an important role in the early stages of embryo development and germination. The balance between GA(1) and ABA, rather than their absolute contents, controls these processes throughout the development, whereas iPAs and ZRs may play important roles in early embryogenesis. The use of isolated embryos as starting material avoids the usual interferences with other tissues such as the endosperm. In addition, this is the first report dealing with the hormonal balance of early-embryos in rice.

Improvement of Plant Regeneration from Immature Embryos of Wheat Infected by Agrobacterium tumefaciens

Wheat, one of the most important food crops, has been extensively studied with respects to plant regeneration and transformation employing the immature embryos as recipient tissues. However, the transformed tissues often become severely necrotic after co-cultivation with Agrobacterium, which is one of the major obstacles in gene delivery. In this study, wheat varieties CB037, Kenong 199, Xinchun 9, Lunxuan 987, and Shi 4185 showed desirable culture potential or high infection ability in Agrobacterium-mediated transformation. Similarly, optimal regeneration conditions were determined by testing their ability to inhibit the cell necrosis and cell death phenotype. Two auxins of 2,4-dichlorophenoxyacetic acid （2,4-D） and 3,6-dichloro-o-anisic acid （dicamba） were evaluated for highly significant effect on both callus and plantlet production, although they were genotype-dependent in wheat. Substitution of 2,4-D by dicamba enhanced the growth and regeneration ability of callus from the immature embryos of most genotypes tested. The callus growth state couldn’t be modified by adding antioxidants such as ascorbic acid, cysteine, and silver nitrate or organic additives such as thiamine HCl and asparagine to the media. On the contrary, the best tissue statement and plant regeneration was achieved by employing the media containing the simplest MS （Murashige and Skoog） components and dicamba without organic components and vitamins. Thereby, our results are thought to inhibit wheat cell necrosis effectively and suggested to be used for more wheat genotypes.

Using Isolated Embryo Sacs and Early Proembryos for Localization of Calmodulin mRNA Before and After Fertilization in Nicotiana

An in situ hybridization technique for localization of calmodulin(CaM) mRNA in isolated entire embryo sacs and proembryos in Nicotiana tabacum L.cv.W38 has been developed. This technique can be applied to small amounts of materials in which a whole view of CaM mRNA distribution can be obtained. The authors revealed that CaM mRNA expression changes dramatically before and after fertilization. Especially interesting is that a prominent CaM mRNA band appears between the egg apparatus and polar nuclei temporarily during the period of pollination and fertilization. The band disappears just prior to fertilization and expands to a fan_shaped region that occupies the micropylar portion of the embryo sac. After fertilization, CaM mRNA accumulates in the elongated zygotes with higher concentration in their chalazal portion than in the micropylar portion. Such an asymmetrical pattern continues to manifest in the early proembryos. It is supposed that CaM mRNA may be involved in the early events and signaling steps associated with double fertilization and zygote polarization in higher plants.

Effects of Environmental Temperature on the Regeneration Frequency of the Immature Embryos of Wheat(Triticum aestivum L.)

The immature embryos （IEs） of wheat are the most widely used tissues for in vitro culture and genetic transformation due to its high regeneration competency. However, this explant can only be maintained in 4℃ daily cooler for a short period time for its use in plant tissue culture or transformation experiments. This study aimed to investigate the effects of environmental temperature, cryopreservation storage temperature, and heat shock culture （HSC） temperature on the regeneration frequency of wheat IEs. Results indicated that environmental temperature significantly affected the induction of embryonic calli. The optimum total accumulated temperature （TAT） during the time of anthesis and sampling for regeneration of these tissues was around 280℃ for spring wheat type cv. CB037 and approximately 300℃ for winter wheat type cv. Kenong 199. Regeneration ability obviously declined when the highest environmental temperature was over 35℃ for 1 d or a high temperature between 30 and 33℃ lasted for 5 d during anthesis and sampling. This finding was verified by culturing the freshly isolated IEs under different temperatures from 29 to 37℃ in different controlled growth incubators for 5 d; the IEs almost completely lost regeneration ability when the temperature rose to 37℃. Cryopreservation of-20℃ caused the wheat samples lost ability of producing callus or embryonic callus in a few days, and cryopreservation of-10℃ more than 10 d made the regeneration potential of the tissues dramatically declined. Comparatively, the temperature that best maintained high regeneration ability was -5℃, at which the materials can be maintained for around 1 mon. In addition, the preservation of the immature samples at -5 or -10℃ inhibited the direct germination of the IEs, avoiding the embryo axis removing process. Our results are useful for ensuring that field collection and cryopreservation of the wheat IEs are done correctly to enable tissue culture and genetic transformation.

Studies on Enhancing Yield of Embryos in Microspore Culture of Brassica napus L.

In ecological zone of Chengdu, Sichuan, microspore culture was carried out in Brassica napus L. to study the influencing factors on microspore culture. The results showed that the temperature on microspore formation stage, day and night temperature, disinfection solution of buds, cultivation concentration on microspore and strain-age were both important influencing factors on microspore culture. At a temperature below 5 ℃ or above 20 ℃, the material had a much lower embryo producing rate or even could not produce any embryo, but at the optimum temperature of 10 -15 ℃ the embryo yield was up to 300 pieces per bud; the best cultivation effect appeared when 0. 1% HgCl2 was used for disin- fection; the best density of microspore was 3 -4 buds per dish; In 2009, while strain-age was from 125 d to 150 d, the microspore embryo yield increased as strain-age increased. When stain-age was 150 days, the microspore embryo yield was up to the highest, but the yield declined after 150 days.

Toxicity of Multi-Walled Carbon Nanotubes,Graphene Oxide,and Reduced Graphene Oxide to Zebrafish Embryos

Objective This study was aimed to investigate the toxic effects of 3 nanomaterials, i.e. multi-walled carbon nanotubes （MWCNTs）, graphene oxide （GO）, and reduced graphene oxide （RGO）, on zebrafish embryos. Methods The 2-h post-fertilization （hpf） zebrafish embryos were exposed to MWCNTs, GO, and RGO at different concentrations （1, 5, 10, 50, 100 mg/L） for 96 h. Afterwards, the effects of the 3 nanomateria on spontaneous movement, heart rate, hatching rate, length of larvae, mortality, and malformations Is were evaluated. Results Statistical analysis indicated that RGO significantly inhibited the hatching of zebrafish embryos. Furthermore, RGO and MWCNTs decreased the length of the hatched larvae at 96 hpf. No obvious morphological malformation or mortality was observed in the zebrafish embryos after exposure to the three nanomaterials. Conclusion MWCNTs, GO, and RGO were all toxic to zebrafish embryos to influence embryos hatching and larvae length. Although no obvious morphological malformation and mortality were observed in exposed zebrafish embryos, further studies on the toxicity of the three nanomaterials are still needed.