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.

Isolation of the Endophytic Fungi of Paris polyphylla var. yunnanensis and Their Effects on the Embryo Development of P. polyphylla var. yunnanensis Seeds

[Objective] This study aimed to isolate the endophytic fungi of Paris polyphylla var. yunnanensis and investigate their effects on the embryo development of P. polyphylla var. yunnanensis seeds. [Method] The endophytic fungi of P. polyphylla were isolated and identified morphologically, and their effects on the embryo development of P. polyphylla var. yunnanensis seeds were studied by using paraffin sectioning and microphotography. [Result] Nine endophytic fungi, i.e. P. polyphylla var. yunnanensis endophytic fungi PPYEF-1, PPYEF-2, PPYEF-3, PPYEF-4, PPYEF-5, PPYEF-6, PPYEF-7, PPYEF-8 and PPYEF-9 belonging to seven genera in five families, three orders were isolated from the rhizomes. Except PPYEF-4 (Cladosporium sp.), other fungi could promote the embryo development of the P. polyphylla var. yunnanensis seeds, mostly reaching the extremely significant or significant level. PPYEF-9 (Trichoderma sp.) resulted in the highest embryo length and embryo-emerging ratio. [Conclusion] This paper could provide a reference for the application of the endophytic fungi of P. polyphylla var. yunnanensis in the dormancy-breaking of P. polyphylla var. yunnanensis seeds.

Effect of Swim-Up and Percoll Treatment on Sperm Quality and In vitro Embryo Development in Yak

This study was designed to determine the effect of different sperm preparation treatments on yak sperm quality and in vitro embryo development.Frozen-thawed semen samples were treated using swim-up or percoll gradient centrifugation methods.Sperm concentration,progressive motility,recovery of motile sperm,membrane integrity,acrosome and chromatin integrity were scored and compared in recovered samples and controls.In addition,the effects of two sperm separation treatments on embryos capable of cleavage and in vitro development to the blastocyst stage were evaluated.Swim-up separated sperm showed a higher motility,while the concentration of spermatozoa recovered and percent recovery of motile sperm were higher with percoll gradient centrifugation separation.According to the optical and electron microscopies,swim-up produced higher percentage of sperm with intact plasma membrane and acrosome than percoll gradient centrifugation separation.However,there was no difference in the percentage of sperm with intact chromatin between two treatment groups.Cell numbers in the blastocysts of two groups were not different.The blastocyst rate was similar in both groups,whereas cleavage rate was higher when swim-up was used.

Impact of Veratrum nigrum L.and Euphorbia fischeriana steud.on Embryo Development of Cattle in Sanjiang Area

It is very important to find out the reasons and the morphological changes of cattle abortion, death embryo and teratism in Sanjiang area, in order to determine the preventive measures, to improve animal quality, and to accelerate the animal industry. In the present studies, 25 cows and 25 local bos calves were investigated. The powder of Veratrum nigrum L. and Euphorbia fischeriana steud. was medicated to the animals during the 15 - 19th day of gestation. It was found that there were different poisoning reactions. When the poisoning was on the 15 - 16th day of gestation, the pregnant animals were easy to miscarriage. When the poisoning was on the 17 - 18th day of gestation, the embryos were easy to become teratism. The joint malformation bicephalus and rachischisis could take place for calves. If the poisoning was after 19th day of gestation , there were much more death embryos. The results of the studies showed that Veratrum and Euphorbia fischeriana steud. were the most poisonous plants to the animal industry of Sanjiang area. Some preventive measures were proposed.

Relationship between Different Pronuclear Patterns and Potential of Embryo Development and Pregnancy

Objective To explore the relationship between the patterns of pronucleus and embryo development and pregnancy potential in the pronuclear stageMethods According to the number and distribution of nucleolar precursor bodies, the embryos at pronuclear stage were classified into 6 pronuclear patterns from 0 to 5, 16 - 18 h after in vitro fertilization (IV F) or intracytoplasmic sperm injection (ICSI). For each, pattern, the subsequent embryonic morphology and the pregnancy rate were analyzed.Results Embryos of Pattern 0 developed to significantly more embryos with good quality and higher pregnancy potential than the embryos developing from other patterns (83. 14% and 76. 11% respectively, P<0. 05). The pregnancy rate was decreased as less embryos of Pattern 0 were transferred . The pregnancy rate of the groups of only Pattern 0, with Pattern 0, and without Pattern 0 were 48. 08% , 32. 14% and 21. 28% respectively (P<0. 05).Conclusions The pronuclear patterns are of the predictive value of embryo development and pregnancy potential, which can be used as a new tool for the selection of embryos in IVF and ICSI.

Effect of royal jelly on in vitro fertilization and early embryo development following nicotine treatment in adult female rats

Objective:To scrutinize the protective role of royal jelly as an antioxidant on nicotine-induced changes in malondialdehyde(MDA)level,p53 expression,in vitro fertilization(IVF)rate,and early embryo development in adult female rats.Methods:A total of 56 adult female Wistar rats were divided into 8 groups(n=7 in each group).Group 1 served as an untreated control group,group 2,3 and 4 received nicotine at a dose of 0.50,1.00 and 2.00 mg/kg respectively,group 5 received royal jelly at a dose of 100.00 mg/kg,and group 6,7 and 8 received 0.50,1.00 and 2.00 mg/kg nicotine,respectively,with 100.00 mg/kg body weight royal jelly.Nicotine and royal jelly were administered daily for 49 days in the experimental groups intra-peritoneally and orally,respectively.At the end of the experimental period,p53 expression,IVF rate and early embryo development as well as MDA concentration were measured.Results:The IVF rate,number of cumulus oocytes,two-cell embryos and blastocysts decreased in the nicotine-treated groups in a dose-dependent manner.In addition,p53 mRNA expression and MDA levels increased in the nicotine-treated groups.Royal jelly co-administration led to partial improvement in the aforementioned parameters.Conclusions:Royal jelly may have a repro-protective effect in nicotine-administered female rats in terms of its anti-oxidant and anti-apoptotic properties.

The Expresion of Quox-1 Gene in the Embryo Development of Andrias davianus

Two set of Andrias davidanus fertilized eggs and its seedlings were got by means of artificial re production.The sections of the embryo of Andrias davidianus were made by histological method The expres sion of homologous sequences of Quox 1 gene has been studied by immunohistochemical method with special antibody.Results show that the expression of homologous sequences of Quox 1 gene was detected in the epi dermis of blastula and gastula.The highly expression was investigated in the epidermis at the neural tube phrase and the tail bud stage.

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.

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.

Observation on the Embryonic Development in Citrus after Cross Pollination

Embryonic development was studied in six cross combinations ofCitrus sinensis x C. tangerina, C. sinensis x C. reticulata, C. sinensis x (C. tangerina + C.reticulata), C. sinensis x Poncirus trifoliate, C.reticulata x C grandis and C. grandis xPoncirus trifoliate. The results showed that on the 30th day after pollination thezygote remained a single cell. On the 40th day, the zygote began to divide. On the50th day, zygotic embryo became globular-shaped while nucellar embryos had notinvaded the embryo sac. On the 55th day, a few nucellar embryos began to invadethe embryo sac. On the 60th day, the zygotic embryo became heart-shaped, and atthe same time, a large number of nucellar embryos invaded the embryo sac. On the80th day after pollination, the zygotic embryo was surrounded by nucellar embryosand it was not easy to distinguish these embryos. The cross combination affected thedevelopments of zygotic embryos, ovules and fruits, which were mainly determined bythe cross parents. As compared with interspecies crossing, the zygotic division ofintergenus crossing began later, the zygotic embryos developed slowlier and theinvading time of nucellar embryos was also delayed.

Arabidopsis Squalene Epoxidase 3 （SQE3） Complements SQE1 and Is Important for Embryo Development and Bulk Squalene Epoxidase Activity

The existence of multigenic families in the mevalonate pathway suggests divergent functional roles for pathway components involved in the biosynthesis of plant sterols. Squalene epoxidases （SQEs） are key components of this pathway, and Squalene Epoxidase 1 （SQE1） has been identified as a fundamental enzyme in this biosynthetic step. In the present work, we extended the characterization of the remaining SQE family members, phylogenetically resolving between true SQEs and a subfamily of SQE-like proteins that is exclusive to Brassicaceae. Functional characterization of true SQE family members, Squalene Epox- idase 2 （SQE2） and Squalene Epoxidase 3 （SQE3）, indicates that SQE3, but not SQE2, contributes to the bulk SQE activity in Arabidopsis, with sqe3-1 mutants accumulating squalene and displaying sensitivity to ter- binafine. We genetically demonstrated that SQE3 seems to play a particularly significant role in embryo development. Also, SQE1 and SQE3 both localize in the endoplasmic reticulum, and SQE3 can functionally complement SQEI. Thus, SQE1 and SQE3 seem to be two functionally unequal redundant genes in the pro- motion of plant SQE activity in Arabidopsis.

Arabidopsis OBG-Like GTPase （AtOBGL）Is Localized in Chloroplasts and Has an Essential Function in Embryo Development

OBG-like GTPases, a subfamily of P-loop GTPases, have divers and important functions in bacteria, including initiation of sporulation, DNA replication, and protein translation. Homologs of the Bacillus subtilis spo0B GTP-binding protein （OBG） can be found in plants and algae but their specific function in these organisms has not yet been elucidated. Here, it is shown that ATSG18570 encodes an Arabidopsis thaliana OBG-like protein （AtOBGL） that is localized in chlor- oplasts. In contrast to the bacterial members of this protein family, AtOBGL and other OBG-like proteins from green algae and plants possess an additional N-terminal domain, indicating functional adaptation. Disruption of the gene locus of ATOBGL by TDNA insertion resulted in an embryo-lethal phenotype and light microscopy using Normarski optics revealed that embryo maturation in the atobgl mutant is arrested at the late globular stage before development of a green embryo. Expression of 35S：：ATOBGL within the atobgl mutant background could rescue the mutant phenotype, confirming that embryo-lethality is caused by the loss of AtOBGL. Together, the data show that the bacterial-derived OBG-like GTPases have retained an essential role in chloroplasts of plants and algae. They furthermore corroborate the significance of chloroplast functions for embryo development -- an important stage within the Arabidopsis lifecycle.

Acetylglutamate kinase is required for both gametophyte function and embryo development in Arabidopsis thaliana

The specific functions of the genes encoding arginine biosynthesis enzymes in plants are not well characterized. We report the isolation and characterization of Arabidopsis thaliana N-acetylglutamate kinase （NAGK）, which catalyzes the second step of arginine biosynthesis. NAGK is a plastid-localized protein and is expressed during most developmental processes in Arabidopsis. Heterologous expression of the Arabidopsis NAGK gene in a NAGK-deficient Escherichia coli strain fully restores bacterial growth on arginine-deficient medium, nagk mutant pollen tubes grow more slowly than wild type pollen tubes and the phenotype is restored by either specifically through complementation by NAGK in pollen, or exogenous supplementation of arginine, nagk female gametophytes are defective in micropylar pollen tube guidance due to the fact that female gametophyte cell fate specification was specifically affected. Expression of NAGK in synergid cells rescues the defect of nagk female gametophytes. Loss- of-function of NAGK results in Arabidopsis embryos not developing beyond the four-celled embryo stage. The embryo-defective phenotype in nagk/NAGK plants cannot be rescued by watering nagk/NAGK plants with arginine or ornithine supplementation. In conclusion, our results reveal a novel role of NAGK and arginine in regulating gametophyte function and embryo development, and provide valuable insights into arginine transport during embryo development.

Mitochondrial replacement techniques or therapies (MRTs) to improve embryo development and to prevent mitochondrial disease transmission

The mitochondrion which contains its own double-stranded circular DNA is a semi-independent organelle that plays critical roles in cell activity. Mitochondrial DNA （mtDNA） is maternally inherited through several mechanisms that have been proposed （Luo et al., 2013） and, if mitochondrial mutations are inherited to the offspring, it is possible to cause mitochondrial diseases such as neuropathy, cardiomyopathy, myopathy, and liver failure.

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.

Transcriptome profiles of embryos before and after cleavage in Eriocheir sinensis:identification of developmental genes at the earliest stages

In crab, embryogenesis is a complicated developmental program marked by a series of critical events. RNA-Sequencing technology offers developmental biologists a way to identify many more developmental genes than ever before. Here, we present a comprehensive analysis of the transcriptomes of Eriocheir sinensis oosperms （Os） and embryos at the 2-4, cell stage （Cs）, which are separated by a cleavage event. Atotal of 18 923 unigenes were identified, and 403 genes matched with gene ontology （GO） terms related to developmental processes. In total, 432 differentially expressed genes （DEGs） were detected between the two stages. Nine DEGs were specifically expressed at only one stage. These DEGs may be relevant to stage-specific molecular events during development. A number of DEGs related to ＇hedgehog signaling pathway＇, ＇Wnt signaling pathway＇ ＇germplasm＇, ＇nervous system＇, ＇sensory perception＇ and ＇segment polarity＇ were identified as being up-regulated at the Cs stage. The results suggest that these embryonic developmental events begin before the early cleavage event in crabs, and that many of the genes expressed in the two transeriptomes might be maternal genes. Our study provides ample information for further research on the molecular mechanisms underlying crab development.

Alteration of ERβ gene Rsal polymorphism may contribute to reduced fertilization rate and embryonic developmental competence

This paper aims to determine the possible role of estrogen receptor-β （ERβ） gene Rsal polymorphism on sperm fertility and early embryonic development in humans. Three groups of Chinese men were recruited： in vitro fertilization （IVF） group, including 374 couples who underwent conventional IVF; intracytoplasmic sperm injection （ICSI） group, including 294 couples who underwent an ICSI procedure using ejaculated sperm; and azoospermic group, consisting of 197 couples who underwent ICSI using either testis or epididymis sperm. Rsal polymorphism in the ERβ gene was detected by polymerase chain reaction （PCR）-restriction fragment length polymorphism technique; fertilization and high-quality embryo rates were evaluated for each group. In each group, no significant differences were found in the overall rates of fertilization and high-quality embryos among GG, AG and AA genotypes. However, the proportion of cycles possessing a satisfactory high-quality embryo rate with the AA genotype was significantly lower than that in the wild-type GG genotype from each group. These results demonstrated that sperm possessing the ERβ RsalA genotype may have reduced fertilization ability and decreased early embryonic developmental potential, which could directly or indirectly contribute to the low fertilization rate and early embryonic developmental arrest in some cases.

Reactive oxygen and nitrogen species regulate porcine embryo development during pre-implantation period:A mini-review

Significant porcine embryonic loss occurs during conceptus morphological elongation and attachment from d 10 to 20 of pregnancy,which directly decreases the reproductive efficiency of sows.A successful establishment of pregnancy mainly depends on the endometrium receptivity,embryo quality,and utero-placental microenvironment,which requires complex cross-talk between the conceptus and uterus.The understanding of the molecular mechanism regulating the uterine-conceptus communication during porcine conceptus elongation and attachment has developed in the past decades.Reactive oxygen and nitrogen species,which are intracellular reactive metabolites that regulate cell fate decisions and alter their biological functions,have recently reportedly been involved in porcine conceptus elongation and attachment.This mini-review will mainly focus on the recent researches about the role of reactive ox-ygen and nitrogen species in regulating porcine embryo development during the pre-implantation period.

Bioinformatic analysis of embryo development related small heat shock protein Hsp26 in Artemia species

Artemia embryos can endure extreme temperature, long-term anoxia, desiccation and other wide variety of stressful conditions. How the embryos survive these stresses is a very interesting and unsolved subject. To solve this question we analyzed the nucleotide and deduced protein sequence for Hsp26, a molecular chaperone specific to Artemia embryo development, cDNAs of Hsp26 were sequenced from eight Artemia species and deduced Hsp26 amino acid sequences were analyzed. Computer-assisted analysis indicated that the 5＇-untranslated region and all the 3 introns contain many putative cis-acting elements for Hsp26 gene expression during development, including heat shock elements （HSEs）, Dfd, dl, CF2-II, Hb and AP-1 binding sites. Secondary structure of the Hsp26 3＇-untranslated terminator contains the basic structure basis for transcriptional termination. Hsp26 shares sequence similarity with sHSPs （small heat shock protein） from other organisms. The physico-chemical properties of the deduced protein, such as theoretical molecular weight, protein extinction coefficient, isoelectric point and antigenic sites were also obtained. One seven-peptide nuclear localization signals （NLS） ＂PFRRRMM＂ was found, which suggested that the Hsp26 protein was hypothesized to be located inside the nucleus. The numbers of phosphorylation sites of serine, threonine and tyrosine and kinase specific phosphorylation sites are also located in Hsp26 protein sequence. These studies will help us achieve a better understanding of Hsp26 and broad implications for sHSPs function in crustacean embryo development.

Effects of Feeding OmniGen-AF^®during Superovulation on <i>in Vitro</i>Development of Embryos Recovered from Donor Beef Cows

Embryo quality is crucial when selecting embryos for transfer. Variation in quality may be attributed to poor oocytes, semen, stress, inflammation, and potential immune system dysregulation. OmniGen-AF® (OG) feeding supports immune system function and animal health. Our laboratory recently reported lower percent degenerate embryos recovered and increased plasma progesterone in beef cattle donors fed OG during superovulation. In vitro development of embryos recovered from donor cows fed OG prior to collection is presented here. Embryos were recovered from 24 beef cows assigned to four treatment groups: 0 g OG/hd/d and 200 mg Folltropin®-V (FSH) (0/200);0 g OG/hd/d and 400 mg FSH (0/400), 56 g OG/hd/d, 200 mg FSH (56/200) and 56 g OG/hd/d and 400 mg FSH (56/400). Good to excellent quality early blastocysts were cultured for 8 d. and development through hatching, embryonic volume and plasminogen activator (PA) production were quantified. The complete protocol was repeated 90 - 120 d later as Replicate 2. Optimal development was observed by embryos recovered from 0/200 cows where percent blastocysts hatching was greater (P < 0.05) compared to 56/200 and 0/400 cows and embryonic volume was greatest (P < 0.05) in Replicate 1. However, percent blastocysts hatching from 0/200 cows was similar (P > 0.10) to 56/400 cows and embryos recovered from 56/400 cows in Replicate 1 produced more (P < 0.05) PA compared to all other groups. For cows superovulated with the standard 400-mg FSH dose, feeding OG supported in vitro embryo development similar to that observed for 0/200 cows.