High-efficiency Embryo Cryopreservation Technology for Mutton Sheep

Embryo cryopreservation is an important part of embryo biotechnology.It plays a key role in the development of quality animal husbandry.However,the effect of in-vivo(in-vitro)embryo freezing of cattle and sheep in China has not reached an expected level,which limits the commercial application of embryo transfer technology.The technical bottleneck that needs to be broken through is that embryos are subject to mechanical damage and protective agent toxicity during freezing and thawing.This paper summarized the successful application of plant antifreeze protein in vitrification preservation of pig embryos,which makes this technology widely used in animal husbandry production,and is of great significance in accelerating the large-scale breeding of improved livestock and saving endangered animals.

Effects of LPA on the development of sheep in vitro fertilized embryos and attempt to establish sheep embryonic stem cells

Lysophosphatidic acid(LPA)is a small molecule glycerophospholipid,which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development.In this study,sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment.At first,the maturation medium containing estrus female sheep serum and synthetic oviduct fluid(SOF)were optimized for sheep IVF,and then the effects of LPA were investigated.From 0.1 to 10μmol L^(–1),LPA had no significant effect on the cleavage rate(P>0.05),but the maturation rate and blastocyst rate increased dependently with LPA concentration(P<0.05),and the blastocyst morphology was normal.When the LPA concentration was 15μmol L^(–1),the maturation rate,cleavage rate and blastocyst rate decreased significantly(P<0.05),and the blastocyst exhibited abnormal morphology and could not develop into highquality blastocyst.Besides,the exogenous LPA increases the expression of LPAR2,LPAR4,TE-related gene CDX-2and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10μmol L^(–1).The expression of LPAR2,LPAR4,CDX-2 and OCT-4 from the LPA-0.1μmol L^(–1)to LPA-10μmol L^(–1)groups in early embryos were extremely significant(P<0.05),while the expression of these genes significantly decreased in 15μmol L^(–1)LPA-treated embryos compared with LPA-10μmol L^(–1)group(P<0.05).The inner cell mass in 15μmol L^(–1)LPA-treated embryos was also disturbed,and the blastocysts formation was abnormal.Secondly,the sheep IVF blastocysts were applied to establish embryonic stem cells.The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells.They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10μmol L^(–1),while 15μmol L^(–1)LPA decreased OCT-4 and CDX-2 expression in the derived cells.The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1μmol L^(–1)group and LPA-10μmol L^(–1)group extremely significantly increased(P<0.05),but there was significant decrease in LPA-15μmol L^(–1)group compared with LPA-10μmol L^(–1)group(P<0.05).Meanwhile,the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10μmol L^(–1)concentration.This study references the IVF embryo production and embryonic stem cell research of domestic animals.

Study on In-vitro Production Technology of Embryos from Young Dorper Sheep

This study was conducted to investigate the in-vitro production technology of embryos from young Dorper sheep, so as to provide technical support for the utilization of ovarian follicles in young Dorper sheep. Tests were conducted from the induction of Dorper sheep of 4 to 8 weeks old using follicle stimulating hormone(FSH) and pregnant horse serum(PMSG), collection of oocytes, in-vitro oocyte maturation-fertilization-zygote cultivation and 2-4-cell-stage fertilized ovum transfer. The results showed that 585 oocytes were collected from eight Dorper sheep at the age of 4 and 8 weeks, with an average of 73.13 oocytes/sheep. 346 of the 2-4-cell-stage fertilized eggs were obtained, whose cleavage rate was 59.15%. 77 in-vitro fertilized eggs at 2-4-cell stage were transplanted into 17 recipient sheep, seven of which were pregnant and gave birth to 13 "test-tube sheep" with a conception rate of 41.18%. It is indicated that the hormone induction technique, in-vitro oocyte maturation-fertilization-zygote cultivation technique and 2-4-cell-stage fertilized ovum transfer technique used in this study can serve as effective techniques for the in-vitro production of embryos from Dorper sheep of 4-8 weeks old.

Efficient production of pronuclear embryos in breeding and nonbreeding season for generating transgenic sheep overexpressing TLR4

Background： Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons（breeding and non-breeding season） on superovulation and the imported exogenous gene on growth.Results： In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep.Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons（P 〉 0.05）. The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group（P 〈 0.05）. The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.Conclusions： Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.

Research Progress on Technique of Frozen Embryo Transfer in Sheep

The paper introduced the research progress on the technique of frozen embryo transfer in sheep,illustrated selection of donors and receptors,superovulation,synchronization of estrus,embryo cryopreservation and embryo transplantation. Frozen embryo transfer in sheep is another breakthrough in the high-quality sheep raising,and this technique in China is in its infancy recommendation stage,but it will be comprehensively popularized in the future.

Asynchronous Embryo Transfer in Sheep:Lack of Survival in Progestinized Recipient Ewes

Synchronization of development between the embryo and uterus is required for successful pregnancy establishment. Transfer of early embryos requires synchrony with the recipient uterus of 2 days or less in sheep, because asynchrony of 3 days or more results in failure of pregnancy recognition signaling for maintenance of corpus luteum （CL） and progesterone （P4） production and/or uterine support of the embryo. The objective was to determine if P4 treatment of recipient ewes would obviate the need for pregnancy recognition signaling and maintain a uterine environment conducive to embryo survival after asynchronous transfer, thereby establishing a universal recipient. Embryos （morulae/blastocysts） were recovered on day 6 from super-ovulated donor ewes. Recipient ewes received 25 mg P4 daily from day 6 post-estrus until 60 days after embryo transfer. Embryos were transferred into recipients on day 6,9, 12,18, or 30 post-estrus. The pregnancy rate on day 22 post-transfer was 60% for synchronous transfers to day 6 ewes, 44% and 22% for asynchronous transfers to day 9 and 12 ewes, and 0% for asynchronous transfers to day 18 and 30 ewes. On day 39 posttransfer ,pregnancy rates remained 60% for day 6 ewes,33% for day 9 ewes,and 0% for day 12,18, and 30 ewes. The P4 treatment did extend the window of uterine receptivity to early embryos in ewes by one day ,but did not create a universal recipient. Available results support the idea that a window of uterine receptivity to the conceptus exists in sheep that is independent of pregnancy recognition signaling.

Use of IVF and ET in Mexican Criollo Sheep (Ovis aries): Immediate and Delayed Embryo Transfers

There is little information in the scientific literature concerning sheep pregnancy and lambing success with regard to the timeframe from when in vitro produced embryos are transported to the designated location for embryo transfer (ET). The aim of this study was to transfer in vitro produced embryos under two different conditions that could typically occur using the aforementioned assisted reproductive techniques (ARTs). Abattoir ovaries were used to procure oocytes for in vitro embryo production and subsequent transfer to synchronized ewes. The study consisted of two experiments: Experiment 1 (Exp1)—embryos taken from the laboratory to a nearby surgical room for immediate ET, and Experiment 2 (Exp2)—ET after 5 hours (h) of transport to a rural farm. Lambing in relation to detected pregnancies, births compared to pregnancies, and the proportion of twin offspring were all higher in Exp2. Notably, in both Exp1 and Exp2, there was not a significant difference (P > 0.05) between the number of embryos transferred, i.e., 3 versus 4, respectively, and the number of ewes that underwent parturition in each group. Also, in both experiments there was not a significant difference (P > 0.05) in the number of ewes that underwent parturition based on the number (i.e., ≥1) of corpora lutea present. The results of the present study demonstrate the importance of evaluating different conditions when applying ARTs, as there are many variables that can influence the outcome. Importantly, Exp2 results show that ovine ET in places located far away from the embryo production site can be useful and successful provided that embryo transport, ET, and recipient conditions are adequate.

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.

Protein glycation:a wolf in sweet sheep’s clothing behind neurodegeneration

At the beginning of the 16th century,Paracelsus coined the maxim:“the dose makes the poison”.This principle can be applied to all living organisms,including organs and cells.The brain and its glial and neuronal cells are no exception.Even small compounds that are essential for the life of brain cells can become truly toxic when overdosed.

Supplementation of Lycium barbarum residue increases the growth rate of Tan sheep by enhancing their feed intake and regulating their rumen microbiome and metabolome

Lycium barbarum residue(LBR),a by-product of L.barbarum processing,is packed with bioactive components and can be potentially utilized as a feed additive in animal husbandry.However,the fundamental understanding of its effectiveness on livestock animals is still lacking,particularly in ruminants.To explore the effects of LBR on the growth performance,rumen fermentation parameters,ruminal microbes and metabolites of Tan sheep,sixteen fattening rams(aged 4 mon)were fed a basal diet(CON,n=8)or a basal diet supplemented with 5%LBR(LBR,n=8).The experiment lasted for 70 d,with 10 d adaptation period and 60 d treatment period.The results showed that the LBR enhanced the average daily feed intake,average daily gain(P<0.05),and ruminal total volatile fatty acids(P<0.01)while decreasing ammonia-nitrogen concentration and rumen pH value(P<0.05).Additionally,the LBR improved the relative abundances of Prevotella,Succiniclasticum,Ruminococcus,Coprococcus,Selenomonas,and Butyrivibrio(P<0.05)and reduced the relative abundances of Oscillospira and Succinivibrio(P<0.05).The LBR altered the ruminal metabolome(P<0.01)by increasing the abundances of ruminal metabolites involved in amino acids(e.g.,L-proline,L-phenylalanine,L-lysine,and L-tyrosine),pyrimidine metabolism(e.g.,uridine,uracil,and thymidine),and microbial protein synthesis(e.g.,xanthine and hypoxanthine).In conclusion,LBR had positive effects on the growth rate of Tan sheep as well as on rumen fermentation parameters,rumen microbiome and rumen metabolome.

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.

Etiological Agent, Pathogenesis, Diagnosis, Treatment, Measures for Prevention and Control of Caseous Lymphadenitis Disease in the Small Ruminants with Special Reference to Sheep

Caseous lymphadenitis (CL) is a chronic infectious disease caused by Corynebacterium pseudotuberculosis (C. pseudotuberculosis) that is transmitted mainly by contact with contaminated exudate wounds. Caseous lymphadenitis affects different species of animals and is considered an occupational zoonotic disease. CL is responsible for important economic losses in the small ruminant industry, which include decreased production, damaged quality of milk and wool, reproductive disorders, total or partial confiscation of carcasses, and depreciation of the skin. Caseous lymphadenitis disease can present in two clinical pictures: a skin or superficial picture and a visceral or deep picture. The presumptive diagnosis of CL in the skin is based on the exploration of superficial lymph nodes. These have little value in diagnosing visceral CL, where the main sign is emaciation. The prevention and control of CL involve identifying the skin condition and debriding the abscesses. In addition to the treatment of superficial lesions, the animal facilities must be repaired and adapted. To avoid exposure of negative herds to CL, new animals must be subjected to observation to identify any of the clinical manifestations. Another form of control is vaccination, though only some countries have commercial vaccines. General information on the etiological agent and its characteristics can be used to improve the understanding of this disease.

Cysticercus Tenuicollis in Visceral Organs of Goats and Sheep in Uganda: A Case Study in Lira Municipal Abattoir

Cysticercus tenuicollis is a metacestode stage of Taenia hydatigena residing in the visceral organs of small ruminants and its effects cause economic losses due to condemnation of visceral organs in slaughterhouses. This study reports on the prevalence of Cysticercus tenuicollis in small ruminants slaughtered in Lira Municipal Abattoir. A total of 70 sheep and 70 goats were sampled and examined after slaughter for the presence of Cysticercus tenuicollis in the visceral organs using standard meat inspection procedures. An abattoir data collection sheet was used to record data, which was then analysed in SPSS version 20. The study revealed 51.4% of goats and 68.6% of sheep to be infested with Cysticercus tenuicollis. Following the district of origin for the small ruminants, Otuke district had the highest prevalence of the parasite (82.6%), followed by Kole (72.4%), Alebtong (66.7%), Apac (59.4%) and Lira (31.4%). Cysticercus tenuicollis was more frequently seen in the intestines (57.8%) of sheep and goats than in any other visceral organs. The liver had a prevalence of 20.7%, the lungs 17.8%, the kidneys 3.5% and the fetal sac;(15.7%) in goats and (12.9%) in sheep. Cysticercus tenuicollis was detected in 68.8% of females and 58.8% of male small ruminants. It is recommended to routinely deworm goats and sheep by farmers most especially in districts with the highest prevalence of Cysticercus tenuicollis infestation. The sale of the condemned parts of sheep and goats and the feeding of dogs on these contaminated materials should be stopped forthwith. It is also being suggested that a national study be carried out to determine the prevalence and economic impact of Cysticercus tenuicollis in small ruminants.

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.

Genomic signatures of selection,local adaptation and production type characterisation of East Adriatic sheep breeds

Background The importance of sheep breeding in the Mediterranean part of the eastern Adriatic has a long tradition since its arrival during the Neolithic migrations.Sheep production system is extensive and generally carried out in traditional systems without intensive systematic breeding programmes for high uniform trait production(carcass,wool and milk yield).Therefore,eight indigenous Croatian sheep breeds from eastern Adriatic treated here as metapopulation(EAS),are generally considered as multipurpose breeds(milk,meat and wool),not specialised for a particular type of production,but known for their robustness and resistance to certain environmental conditions.Our objective was to identify genomic regions and genes that exhibit patterns of positive selection signatures,decipher their biological and productive functionality,and provide a"genomic"characterization of EAS adaptation and determine its production type.Results We identified positive selection signatures in EAS using several methods based on reduced local variation,linkage disequilibrium and site frequency spectrum(eROHi,iHS,nSL and CLR).Our analyses identified numerous genomic regions and genes(e.g.,desmosomal cadherin and desmoglein gene families)associated with environmental adaptation and economically important traits.Most candidate genes were related to meat/production and health/immune response traits,while some of the candidate genes discovered were important for domestication and evolutionary processes(e.g.,HOXa gene family and FSIP2).These results were also confirmed by GO and QTL enrichment analysis.Conclusions Our results contribute to a better understanding of the unique adaptive genetic architecture of EAS and define its productive type,ultimately providing a new opportunity for future breeding programmes.At the same time,the numerous genes identified will improve our understanding of ruminant(sheep)robustness and resistance in the harsh and specific Mediterranean environment.

Research and Application of Compressed Fermented Chestnut Leaf Feed in Fattening Mutton Sheep

Chestnut leaves and bran were evenly mixed according to different mass ratios,and the mixtures were sprayed and inoculated with a certain amount of prepared EM microbial liquid,and then compressed into 70 cm×40 cm×30 cm blocks by a fully automatic yellow-storage block compression and packaging machine.The obtained blocks were packaged and sealed with plastic film,and placed in a freely ventilated place for more than 15 d of anaerobic fermentation,so as to obtain compressed fermented chestnut leaf block feed.[Results]Through the detection and analysis of nutritional components in the compressed fermented chestnut leaf block feed in the laboratory,the protein content was equal to or greater than 10.2%,which was 44.68%higher than that of unfermented chestnut leaf feed;the tannin content was equal to or greater than 638 mg/kg,which was 18.41%lower than that of the original feed;and the crude fiber content was equal to or greater than 19.5%,which was 14.09%lower than that of the original feed.[Conclusions]This study improves the palatability of chestnut leaf feed,increases the use efficiency of feed and reduces feeding cost.It is worth popularizing.

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.

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.

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.