Vitrification cryopreservation of ligaments based on zwitterionic betaine

Ligament cryopreservation enables a prolonged shelf life of allogeneic ligament grafts,which is fundamentally important to ligament reconstruction.However,conventional cryopreservation techniques fail to eliminate the damage caused by ice crystal growth and the toxicity of cryopreservation agents(CPAs).Here,we report a novel CPA vitrification formulation primarily composed of betaine for ligament cryopreservation.Comprehensive optimization was conducted on the methods for vitrification and rewarming,as well as the loading and unloading conditions,based on the critical cooling rate(CCR),critical warming rate(CWR),and permeation properties of the CPA.Using biomechanical and histological level tests,we demonstrate the superior performance of our method in ligament cryopreservation.After 30 days of vitrification cryopreservation,parameters such as the Young's modulus,tensile stress,denaturation temperature,and glycosaminoglycans content of the ligament remained essentially unchanged.This work pioneers the application of ice-free cryopreservation for ligament and holds great potential for improving the long-term storage of ligament,providing valuable insights for future cryopreservation technique 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.

Effects of Different Culture Conditions on Vitrification of Lycium barbarum L. Plantlets in Tissue Culture

The tender stems from new Lycium barbarum L. cultivar ＂Ningqi 3＂ released by Ningxia Academy of Agricultural and Forestry Sciences were regarded as explants to investigate the vitrification of Lycium barbarum plantlets in tissue culture under different concentrations of 6-BA, sucrose, agrose, culture temperature, and illumination duration with MS as basic medium. The results show that the conditions for maximal proliferation coefficient and min- imal vitrification are as following： the basic medium with 0.2 mg/L 6-BA, 3% sucrose and 0.65% agarose; culture at 25℃; 12 h/d（ daylight lamp, 2 000 lx） illumination.

Effect of Mouse Oocyte Vitrification on Mitochondrial Membrane Potential and Distribution

The effects of mouse oocyte vitrification on mitochondrial membrane potential and distribution were explored in this study. The collected mouse oocytes were randomly divided into vitrification and control groups. Ethylene glycol（EG） and dimethylsulphoxide（DMSO） were used as cryoprotectants in the vitrification group. The mitochondrial function and distribution in the oocytes were examined by using the fluorescent probes, JC-1 and Mito Tracker green. The results showed that the ratio of red to green fluorescence in mouse oocytes was significantly decreased after thawing in the vitrification group as compared with the control group（1.28 vs. 1.70, P0.05）. The percentage of polarized distribution of the mitochondria in oocytes was conspicuously reduced in the vitrification group when compared with the control group（31% vs. 63%, P0.05）. It was suggested that vitrification significantly affects the mitochondrial function and distribution in oocytes and reduces the potential of oocyte fertilization and embryo development.

Selection and Vitrification of Embryos with a Poor Morphological Score: A Proposal to Avoid Embryo Wastage

Embryos with a poor morphological score at cleavage stage are usually discarded because they are considered unsuitable for transfer and cryopreservation. This study examined the in vitro blastocyst development after extended culture of these embryos and the clinical outcomes after transfer of these blastocysts in warming cycles. A total of 597 blastocysts （24.7%） were obtained from 2421 embryos with low morphological scores after extended culture. One hundred and sixty blastocysts （6.6%） with optimal morphology were vitrified. Embryo utilization rate was increased from 30.8% to 32.6%. After warming, 61 out of 92 blastocysts （66.3%） survived and were transferred in 44 cycles. The clinical pregnancy rate and the implantation rate were 40.9% （18/44） and 32.8% （20/61） respectively. Thirteen healthy babies were born, and 5 pregnancies aborted spontaneously. Our study suggested that some blastocysts derived from embryos with a poor morphological score can be successfully vitrified and give rise to live births. Selection and vitrification of viable embryos after extended culture of embryos with a poor morphological score may constitute a proposal to avoid embryo wastage.

Vitrification of MSWI Fly Ash by Thermal Plasma Melting and Fate of Heavy Metals

Municipal solid waste incinerator （MSWI） fly ash with high basicity （about 1.68） was vitrified in a thermal plasma melting furnace system. Through the thermal plasma treatment, the vitrified product （slag） with amorphous dark glassy structure was obtained, and the leachability of hazardous metals in slag was significantly reduced. Meanwhile, it was found that the cooling rate affects significantly the immobility of heavy metals in slag. The mass distribution of heavy metals （Zn, Cd, Cr, Pb, As, Hg） was investigated in residual products （slag, secondary residues and flue gas）, in order to analyze the behavior of heavy metals in thermal plasma atmosphere. Heavy metal species with low boiling points accounting for the major fraction of their input-mass were adsorbed in secondary residues by pollution abatement devices, while those with high boiling points tended to be encapsulated in slag.

Cryopreservation of Pollen by Vitrification in Brassica

The key factors affecting pollen cryopreservation by vitrification in Brassica campestris var. purpurea were investigated. The factors involved the pollen maturity, the pollen resistance to dehydration, the components of vitrification solution(PVS), and the concentration of diluent. Thus, a suitable procedure was established for pollen vitrification, the maximum relative survival rates of mature (at the day of anthesis)and nearly mature(3 days before anthesis)pollen were about 80%, 63% respectively. This procedure has been also successfully applied to two other species ( Brassica napus, Brassica chinensis ). The advantage of cryopreservation of pollen by vitrification was discussed.

The outcomes of human blastocyst cryopreservation:vitrification using cryoloop versus slow-freezing method

Objective:To compare the outcomes of vitrification using cryoloop with slow-freezing meth-od for human blastocyst cryopreservation.Methods:In IVF-ET cycles,supernumerary embryos were cultured to Day 5 or Day 6,blas-tocysts were cryopreserved by vitrification using cryoloops or slow-freezing method,then blasto-cyst survival rate and pregnant rate were compared.Results:115 vitrified blastocysts from 39 cycles were warmed,104(90.4%)blastocysts sur-vived.After the transfer of 74 blastocysts in 38 cycles,28(73.7%)women got clinically preg-nant,2(7.1%)of them suffered from miscarriage,2 healthy babies were born in 2 deliveries,and the other 24 pregnancies are ongoing.As to slow-freezing method,87 blastocysts from 21 cy-cles were thawed,37(42.5%)of them survived,28 blastoeysts were transferred in 15 cycles,6(40%)women got clinically pregnant,1 of them miscarried,3 healthy babies were born in 2 de-liveries,and the other 3 pregnancies are ongoing.Conclusion:The survival rate and pregnant rate of vitrification using cryoloop are superior totraditional slow-freezing method,and the transfer cancel rate is lower than that of slow-freezingmethod.The miscarriage rate is similar in two methods.

Controlled Freezing and Open-Pulled Straw (OPS) Vitrification of In vitro Produced Bovine Blastocysts FollowingAnalysis of ATP Content and Reactive Oxygen Species (ROS) Level

To our knowledge,no single study has systemically compared cryopreservation efficiencies of bovine blastocysts derived from in vitro fertilization （IVF）,intracytoplasmic sperm injection （ICSI） and somatic cell nuclear transfer （SCNT） by controlled freezing and vitrification.This experiment,therefore,was designed to compare the cryopreservation of these blastocysts with controlled freezing and OPS vitrification.Adenosine-5’-triphosphate （ATP） content and reactive oxygen species （ROS） level in blastocysts were also analyzed.Firstly,for each type of blastocyst （IVF,ICSI or SCNT）,significant differences were observed between the survival rates of the controlled freezing （（81.56±2.33）,（68.18±4.72） or （47.89±5.83）%） and OPS vitrification groups （（92.24±4.54）,（82.40±3.76） or （78.71±5.91）%;P〈0.05）.Secondly,for each type of blastocyst （IVF,ICSI or SCNT）,ATP content was significantly decreased after controlled freezing or vitrification,and the ATP content in the controlled freezing group （0.43±0.06）,（0.35±0.05） or （0.21±0.02） pmol） was significantly lower than that found in the OPS vitrification group （0.62±0.04）,（0.46±0.03） or （0.30±0.01） pmol;P〈0.05）.Thirdly,ROS level in fresh IVF （（47.33±3.56） c.p.s （counted photons per second）,ICSI （（36.51±2.58） c.p.s） or SCNT blastocysts （（26.44±1.49） c.p.s） was significantly lower than that found in the OPS vitrification group （（72.14±4.31）,（58.89±3.89） or （40.11±5.73） c.p.s;P〈0.05）,but higher than that of the controlled freezing group （34.41±3.32）,（23.13±1.26） or （15.46±2.45） c.p.s;P〈0.05）.The present study indicated that vitrification is more efficient in the cryopreservation of bovine blastocysts derived from IVF,ICSI or SCNT than controlled freezing.Furthermore,both vitrification and controlled freezing significantly altered the ATP content and ROS level in those blastocysts.

Vitrification of Bovine Oocytes by Open Pulled Straw

Bovine oocytes cultured in vitro for 6 hours or 22 hours were cryopreserved in different vitrification solutions (EFS40, EFS50, EDFS30 or EDFS40) by the two-step method with OPS (open pulled straw). The best results were achieved by using EDFS30 to cryopreserve the oocytes either for in vitro fertilization or for chemical activation. The blastocyst rates were 12% and 17% in 6 hour and 22 hour cultures respectively following in vitro fertilization. If frozen-thawed oocytes were continued in culture up to 24 hours, and were activated by chemicals, the blastocyst rates were 22% and 24% in 6-hour and 22-hour groups respectively. There were no statistical differences between frozen and fresh oocytes (P >0.05).

Mixed Wastes Vitrification by Transferred Plasma

Thermal plasma technology provides a stable and long term treatment of mixed wastes through vitrification processes. In this work, a transferred plasma system was realized to vitrify mixed wastes, taking advantage of its high power density, enthalpy and chemical reactivity as well as its rapid quenching and high operation temperatures. To characterize the plasma discharge, a temperature diagnostic is realized by means of optical emission spectroscopy （OES）. To typify the morphological structure of the wastes samples~ scan- ning electron microscopy （SEM）, and X-ray diffraction （XRD） techniques were applied before and after the plasma treatment.

Effect of melatonin and/or cysteamine on development and vitrification of buffalo embryos

Objective:To assess the effects of melatonin and/or cysteamineonin vitro maturation, culturing and post-warming of buffalo embryos.Methods: Buffalo oocytes were classified into control, cysteamine (50 μM), melatonin (10 ng/mL) and cysteamine (50 μM) + melatonin (10 ng/mL) treatment groups. In experiment 1, previous treatments were added duringin vitromaturation and culturing of buffalo oocytes.Results:Cleavage and blastocyst rates were significantly (P<0.05) increased in melatonin treated group (70.5±0.9 and 12.8±1.0, respectively). However this effect was potentiated when combined with cysteamine (74.0±1.7 and 14.8±1.7, respectively). In experiment 2, the treatements were added in maturtaion, culturing as well as post-warming culture media. Embryos at 7 d were vitrified.Viability assessement directly after warming showed significant increase (P<0.05) in cysteamine, melatonin and their combination groups (76.8±2.8, 80.0±2.1 and 83.3±1.7, respectively) than control (65.8±2.4);but the viability after 24 h post-warming was the best in cysteamine + melatonin combination group (61.4±2.1).Conculsions: Enriching maturation, culturing and post-warming media of buffalo oocytes and embryos with melatonin and/or cysteamine have significantly beneficial effects on oocyte developmental competence as well as embryos vitrification procedure outcomes which in turn resulting in enhancement of commercial buffalo embryo production.

Effect of vitrification procedure on chromosomal status of embryos achieved from vitrified and fresh oocytes

Background: In order to assess the chromosomal status in embryos obtained from vitrified and fresh donated oocytes, preimplantational genetic diagnostic (PGD) was performed after biopsy of one blastomere at day 3. METHODS: A total of 249 oocytes were obtained from 23 oocyte donors, 80 oocytes were used in the vitrified group and 151 oocytes were used in the fresh group. Nine chromosomes (13, 15, 16, 17, 18, 21, 22, X and Y) were investigated by fluorescence in situ hybridization (FISH) analysis in 56 and 121 embryos from vitrified and fresh group respectively. Fertilization, cleavage rate, embryo quality and chromosomal abnormality rate were compared between groups evaluated. Results: Vitrified oocytes showed a survival rate of 97.5%. There was no significant difference in the fertilization rate (82.7% and 91.4%), Day 2 cleavage rate (90.3% and 87.7%) or blastocyst formation rate (31.1% and 44.6%) for the vitrified and fresh groups respectively. Chromosomal abnormality rate (66.1% versus 71.9%), percentage of abnormal blastocysts (61.1% versus 64.8%) and percentage of abnormalities for each analyzed chromosome were similar for the vitrified group compared with the control group. Conclusions: The rates of chromosomal abnormalities in embryos from vitrified oocytes are similar to those published previously;and comparable to those observed in embryos from fresh oocytes. These results confirm that the developmental competence and chromosomal status of embryos obtained from vitrified oocytes is not affected by the vitrification procedure, and they preserve the potential to be fertilized and to develop in to blastocyst stage similar to embryos from fresh oocytes.

Effects of MTG and GSH on Human Sperm Motility and DNA Integrity during Vitrification in the Presence of Trehalose

Limited information exists about the effects of antioxidants on sperm motility and DNA integrity during vitrification in humans. This study compared the effects of reduced glutathione (GSH) and monothioglycerol (MTG) at different concentrations on post-thaw sperm motility and DNA integrity after vitrification in humans using 0.25 M trehalose as a cryoprotective agent, and found that supplementation of MTG or GSH at 0.5 mM resulted in significantly higher (P < 0.05) recovery rates of post-thaw total and progressive motilities. GSH was more powerful than MTG at the same concentration in cryoprotecting sperm motility (38.9% ± 3.6% vs 32.8% ± 2.4% compared to the control 26.8% ± 2.1% in recovery rate of progressive motility), but both had no significant influence on sperm DNA integrity during vitrification. It was concluded that sperm motility is more sensitive to oxidative stress during vitrification than sperm nuclear DNA, and supplementation of MTG or GSH in vitrification medium is beneficial in cryoprotecting sperm motility.

The application of vitrification in human blastocyst cryopreservation: Analysis of clinical outcome of 117 cycles

Objective: To summarize the clinical outcomes of 117 human vitrified blastocyst transfer cycles and to determine the impact factors.Methods: In IVF-ET cycles, supernumerary embryos were cultured to 5-Day(D5) or 6-Day(D6), blastocysts of various stages were cryopreserved by vitrification using cryoloops. Survival rate and clinical pregnancy rate were observed.Results: A total of 312 blastocysts were thawed in 117 frozen embryo transfer cycles, the survival rate was 90.7% (283/312) after thawing. After the transfer of 230 blastocysts in 115 cycles, 69.6% (80/115) of the women got clinically pregnant, and 17.5% (14/80) of them suffered from miscarriage, 39 healthy babies were born in 28 deliveries, and the other 38 pregnancies are ongoing. The implantation rate was 47.4% (109/230). In 107 transfer cycles with 2 hatched blastocysts transferred in each cycle, 72.9% (78/107) got clinically pregnant, while in 8 cycles with 1 or no hatched blastocysts in the two transferred blastocysts, the clinical pregnancy rate is 25%(2/8). The clinical pregnancy rates were not statistically different between natural (77.4%, 24/31) or artificial endometrium preparation (66.7%, 56/84) cycles. Conclusions: These findings suggest that blastocyst vitrification is effective in terms of implantation rate and pregnancy outcome. Transferring of two hatched blastocyst can achieve a higher pregnancy rate.

Unveiling how vitrification affects the porcine blastocyst: clues from a transcriptomic study

Background:Currently,there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes.To date,vitrification(VIT)is the most efficient method for pig embryo cryopreservation.Despite a high number of embryos survives in vitro after vitrification/warming procedures,the in vivo embryo survival rates after embryo transfer are variable among laboratories.So far,most studies have focused on cryoprotective agents and devices,while the VIT effects on porcine embryonic gene expression remained unclear.The few studies performed were based on vitrified/warmed embryos that were cultured in vitro(IVC)to allow them to re–expand.Thus,the specific alterations of VIT,IVC,and the cumulative effect of both remained unknown.To unveil the VIT-specific embryonic alterations,gene expression in VIT versus(vs.)IVC embryos was analyzed.Additionally,changes derived from both VIT and IVC vs.control embryos(CO)were analyzed to confirm the VIT embryonic alterations.Three groups of in vivo embryos at the blastocyst stage were analyzed by RNA–sequencing:(1)VIT embryos(vitrified/warmed and cultured in vitro),(2)IVC embryos and(3)CO embryos.Results:RNA–sequencing revealed three clearly different mRNA profiles for VIT,IVC and CO embryos.Comparative analysis of mRNA profiles between VIT and IVC identified 321,differentially expressed genes(DEG)(FDR<0.006).In VIT vs.CO and IVC vs.CO,1901 and 1519 DEG were found,respectively,with an overlap of 1045 genes.VIT-specific functional alterations were associated to response to osmotic stress,response to hormones,and developmental growth.While alterations in response to hypoxia and mitophagy were related to the sum of VIT and IVC effects.Conclusions:Our findings revealed new insights into the VIT procedure-specific alterations of embryonic gene expression by first comparing differences in VIT vs.IVC embryos and second by an integrative transcriptome analysis including in vivo control embryos.The identified VIT alterations might reflect the transcriptional signature of the embryo cryodamage but also the embryo healing process overcoming the VIT impacts.Selected validated genes were pointed as potential biomarkers that may help to improve vitrification.

Cryopreservation of Queen Honeybee (Apis mellifera carnica) Born Worker Eggs by Vitrification

Many species of insect egg can be targeted individually or （and） collectively for cryopreservation by vitrification. However, there has been no report on cryopreservation of honeybee eggs by vitrification. In an attempt to define a preliminary procedure of cryopreservation of honeybee eggs by vitrification, queen honeybee born worker eggs （worker eggs） were stored through vitrification in liquid nitrogen up to 1 h, and then post-vitrification survival of the vitrified worker eggs in vitro and their hatching rates during maturation in vivo were observed using microscopic and close visual inspections. The procedure of cryopreservation by vitrification included dechorionation with sodium hypochlorite and permeabilization with isopropyl alcohol; equilibration by addition of loading solution （i.e., 25% vitrification storage solution） and dehydration by gradual replacement of loading solution with vitrification storage solution; cooling in liquid nitrogen vapor right before droplet vitrification in liquid nitrogen; and recovery in liquid nitrogen vapor right after storage in liquid nitrogen, thawing at temperature of thawing medium （5% sucrose in TC 100-insect medium） and rehydration by gradual replacement of vitrification storage solution with rehydration solution （5% fetal bovine serum in TC 100-insect medium）. It was found that among the worker eggs experiencing cyropreservation by vitrification, 1.25% of them were successfully passed through the four life stages, viz., egg, larva, pupa, and adult. In summary, it can be inferred that although a majority of worker eggs were dead after cyropreservation by vitrification, a few of them were developed into larvae, pupae, and finally emerged as adults.

Developmental Competence of Frozen-thawed Germinal Vesical Porcine Oocytes by Vitrification Method following Maturation,Fertilization and Culture In Vitro

The purpose of this study was to evaluate the viability and subsequent developmental ability of porcine germinal vesicle（GV） oocytes vitrified step-wise exposure to cryoprotectants. Oocytes were transferred to a vitrification solution composed of 10% ethylene glycol（EG）,10% dimethyl sulfoxide（DMSO）, 300 g/L-1 Ficoll and 0.5 mol/L-1 sucrose（EDFS40） in a direct manner （non-preequilibrium） or in step-wise manner（ single- and two-step preequilibrium）. After vitrification and storage in liquid nitrogen, the oocytes were thawed,washed and in vitro maturation, fertilization and culture. In the non-preequilibrium group, the rates of post-thawed oocytes surviving, maturing to metaphase-Ⅱ, cleavage rate and blastocysts rate was significantly lower than that of sigle- and two-step preequilubrium groups（P<0.05）. In the single- and two- step groups, the rates of metaphase-Ⅱ stage were 46.8%, 42.7% and 49.7%, respectively, the rates which developed to blastocysts were 10.5%,11.1% and 14.8%, respectivaly. In the non-vitrified control group,the rates of oocytes maturing to metaphase-Ⅱ, developing to blastocysts was significantly higher than that vitrified groups（P<0.05）. The present study shows that the vitrification of porcine GV oocytes by a step-wise method involving two-steps preequilibrium may have advantage in maintaining the viability and subsequent production of blastocysts.

DNA Methylation Pattern in Pronuclear-Stage Mouse Embryos:Effect of Oocyte Vitrification

This study was conducted to investi- gate the pattern of DNA methylation in pronuclearstage mouse embryos derived from vitrified-warmed oocytes. Mouse oocytes at metaphase II （MII） stage of meiosis were allocated randomly into three groups： 1） untreated （control）; 2 ） exposed to vitrification solution without being plunged into liquid nitrogen （toxicity）; and 3 ） vitrified by open-pulled straw （OPS） method （vitrification）. Oocytes were fertilized in vitro （IVF）. The level of DNA methylation was examined at 8 hpf （hours post-fertilization） by immunofluorescence using an anti-5-methylcytosine （5-MeC） monoclonal antibody and fluorescein isothiocyanate （FITC）-conjugated goat anti-mouse IgG. After IVF, rates of 2-cell embryos （ 51.39% ） and blastocysts （35.82%） for vitrified-warmed oocytes were lower （P〈0.01） than that for control （70.83%, 47.82% ） or vitrification solution treated （64.80%, 46.29% ） oocytes. At 8 hpf, there were more （P 〈 0.05） pronuclear-stage oocytes in which syngamy of pronuclei had not occurred in the vitrifica- tion group. In addition, 5-MeC fluorescent intensities for the female pronucleus and zygote were lower in the vitrification group （P 〈0.01 ） compared to pronuclear-stage embryos in the control and toxicity groups. In conclusion, oocyte vitrification causes a reduction in global genomic methylation in the female pronucleus and zygote, resulting in delayed fusion of pronuclei and compromising the developmental potential of mouse zygotes and embryos.

Study on the developing potentiality of mouse morula produced in vitro or in vivo after vitrification

Objective: To assess the developing potentiality of mouse morula produced in vitro or in the after vitrification and to evaluate the effect of one-step and two-step vitrification methods. Method: Mouse morula produced in for and in the were vitrified in the solution containing ethylene glycol, Ficoll and sucrose (EFS solution) with one-step and two-step methods. The developing potential and status of the pellucid zona in vitified mouse morula were assessed. Results: The percentages of morula developed into blastocyst stage were 81. 8% and 82.4%, 97. 3% and 98.4%, respectively, after one-step and two-step exposure of in vitro morula or in vivo morula to EFS solution alone, which did not show significant difference compared to their controls (P > 0. 05). The percentage of in vitro morula developed into blastocyst vitrified by onestep method was significantly lower than that by two-step method and coned (P < 0.05, 70.6% vs 81 .3%; 70.6% vs 83 .6%, respectively). However, there was no significant difference between blastocyst rates of in vivo morula vitrified by one-step and two-step methods (P>0.05, 93. 1% us 95.7%). No rupture of pellucid zona was observed in all thawed morula after one-step and two-step vitrification, irrespective of in vitro morula or in vivo morula. Conclusion: Morula produced in vitro and in vivo after vitrification may maintain high survival rate and developing potential. Two-step vitrification method with EFS solution is suitable for in vitro morula, which can improve the developing potential of in vitro morula. Onestep and two-step vitrification method have no detrimennd effect on the developing potential of in vivo morula.