Gradient bimetallic ion–based hydrogels for tissue microstructure reconstruction of tendon-to-bone insertion

Although gradients play an essential role in guiding the function of tissues, achieving synchronous regeneration of gradient tissue injuries remains a challenge. Here, a gradient bimetallic (Cu and Zn) ion–based hydrogel was first constructed via the one-step coordinative crosslinking of sulfhydryl groups with copper and zinc ions for the microstructure reconstruction of the tendon-to-bone insertion. In this bimetallic hydrogel system, zinc and copper ions could not only act as crosslinkers but also provide strong antibacterial effects and induce regenerative capacity in vitro. The capability of hydrogels in simultaneously promoting tenogenesis and osteogenesis was further verified in a rat rotator cuff tear model. It was found that the Cu/Zn gradient layer could induce considerable collagen and fibrocartilage arrangement and ingrowth at the tendon-to-bone interface. Overall, the gradient bimetallic ion–based hydrogel ensures accessibility and provides opportunities to regenerate inhomogeneous tissue with physiological complexity or interface tissue.

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Efficient Cryopreservation of Populus tremula by In Vitro-Grown Axillary Buds and Genetic Stability of Recovered Plants

Plants ◽

10.3390/plants10010077 ◽

2021 ◽

Vol 10 (1) ◽

pp. 77

Author(s):

Elena O. Vidyagina ◽

Nikolay N. Kharchenko ◽

Konstantin A. Shestibratov

Keyword(s):

Survival Rate ◽

Axillary Buds ◽

Long Term Storage ◽

Average Survival ◽

Transgenic Lines ◽

Ssr Loci ◽

One Step ◽

The One

Axillary buds of in vitro microshoots were successfully frozen at –196 °C by the one-step freezing method using the protective vitrification solution 2 (PVS2). Microshoots were taken from 11 transgenic lines and three wild type lines. Influence of different explant pretreatments were analyzed from the point of their influence towards recovery after cryopreservation. It was found out that the use of axillary buds as explants after removal of the apical one increases recovery on average by 8%. The cultivation on growth medium of higher density insignificantly raises the regenerants survival rate. Pretreatment of the osmotic fluid (OF) shows the greatest influence on the survival rate. It leads to the increase in survival rate by 20%. The cryopreservation technology providing regenerants average survival rate of 83% was developed. It was based on the experimental results obtained with explant pretreatment. Incubation time in liquid nitrogen did not affect the explants survival rate after thawing. After six months cryostorage of samples their genetic variability was analyzed. Six variable simple sequence repeat (SSR) loci were used to analyze genotype variability after the freezing-thawing procedure. The microsatellite analysis showed the genetic status identity of plants after cryopreservation and of the original genotypes. The presence of the recombinant gene in the transgenic lines after cryostorage were confirmed so as the interclonal variation in the growth rate under greenhouse conditions. The developed technique is recommended for long-term storage of various breeding and genetically modified lines of aspen plants, as it provides a high percentage of explants survival with no changes in genotype.

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171 Effect of progesterone and prolactin on the developmental competence of bovine oocytes during the terminal phase of in vitro maturation

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab171 ◽

2019 ◽

Vol 31 (1) ◽

pp. 210

Author(s):

G. Singina ◽

E. Shedova ◽

T. Taradajnic ◽

V. Konnova ◽

E. Tsyndrina

Keyword(s):

Cell Number ◽

Total Cell ◽

Developmental Competence ◽

Terminal Phase ◽

Cleavage Rate ◽

Total Cell Number ◽

One Step ◽

Group 2 ◽

The One

To date, considerable progress has been achieved in in vitro production (IVP) technologies in cattle; however, developmental potentials of oocytes matured in vitro remain low compared with in vivo-matured oocytes. Thus, a better understanding of different aspects of oocyte maturation may allow us to increase the embryo development rate. Our study was aimed to assess the effects of progesterone (P4) and prolactin (PRL) on the bovine oocyte developmental competence. Bovine cumulus-enclosed oocytes (CEO) were matured using either one-step or two-step maturation conditions. For the one-step protocol, CEO were cultured for 24h in TCM-199 supplemented with 10% fetal calf serum (FCS), 10μg mL−1 porcine FSH, and 10μg mL−1 ovine LH (standard medium). For the two-step procedure, CEO were first cultured for 16h in the standard medium (n=1263) and then transferred to 1 of 3 experimental media and cultured for additional 8h in either absence or presence of either P4 (50 ng mL−1) or bovine PRL (50ng mL−1). The 3 media tested in the two-step maturation were (1) TCM-199 containing 10% FCS (group 1), (2) TCM-199 containing 3mg mL−1 BSA (group 2), or (3) Fert-TALP medium supplemented with 6mg mL−1 BSA (group 3). Fert-TALP was selected because it can potentially be used throughout maturation and fertilization. Following in vitro maturation, all oocytes underwent an IVF/in vitro culture procedure as described previously (Singina et al. 2014 Reprod. Fertil. Devel. 26, 154). The embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst rates. In addition, obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL staining. The data from 4 to 5 replicates (113-159 oocytes per treatment) were analysed by ANOVA. For oocytes matured for 24h in the one-step culture, the cleavage rate, blastocyst rate, total cell number, and apoptotic nuclei per blastocyst were 66.1±1.1, 23.7±2.0, 71.4±9.1, and 4.8±1.2%, respectively. For the two-step culture, the cleavage rate did not differ from that of the one-step culture system, ranging from 64.8 to 76.5%. Also, no effects of the two-step systems were observed on total cell number (63.0-78.8) or the proportion of apoptotic nuclei (3.3-5.3%) at the blastocyst stage. The culture of CEO in group 1 (without the supplements) had a reduced blastocyst rate (17.4±0.4%; P<0.05) compared with the standard one-step maturation group, and the addition of P4 (but not PRL) improved the blastocyst yield (P<0.05). Furthermore, when P4 (but not PRL) was added to group 2 and group 3 media, blastocyst rates increased significantly (32.9±3.1 and 32.8±2.7%, respectively) compared with those of the one-step group (P<0.05), but did not differ from those of untreated groups 2 and 3 (26.2±2.7 and 30.0±3.0%, respectively). Our data indicate that P4 supplementation during the terminal phase of two-step IVM can enhance the developmental competence of bovine oocytes and that the nature of this effect depends on the composition of IVM medium, whereas no effect of PRL supplementation was observed. The study was supported by RFBR (No. 17-29-08035).

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Ultrastructure of bovine in vitro-produced blastocysts cryopreserved by vitrification

Zygote ◽

10.1017/s0967199400005049 ◽

1998 ◽

Vol 6 (1) ◽

pp. 17-26 ◽

Cited By ~ 9

Author(s):

Shigeki Ohboshi ◽

Noboru Fujihara ◽

Tatsuyuki Yoshida ◽

Hiroshi Tomagane

Keyword(s):

Ethylene Glycol ◽

Cellular Damage ◽

Bovine Embryos ◽

Step Procedure ◽

Direct Exposure ◽

Subsequent Exposure ◽

One Step ◽

The One ◽

Serious Injuries

SummaryThe objective of this study was to examine ultrastructural aspects of bovine in vitro-produced blastocysts associated with cryopreservation by vitrification. Morphologically good embryos were used and treated with ethylene-glycol-based vitrification solution (VS). The untreated embryos had conventional fine structure. The post-warming embryos treated with direct exposure to VS (one-step procedure) showed cellular damage structurally by cryopreservation, which included loss of microvilli, disruption of the plasma membrane, mitochondrial changes and swelling of the endoplasmic reticulum. However, nuclei and junctional regions seemed to be resistant to cryoinjury. In contrast, the post-warming embryos pre-equilibra ted with 10% ethylene glycol for 5 min and subsequent exposure to VS (two-step procedure) showed less damage than those treated by the one-step procedure. Post-warming embryos treated by the two-step procedure were cultured in vitro for 18 h. Some embryos survived and their structures re-formed to the former state, while other embryos showed serious injuries and could not reconstitute the blastocoele. Three post-warming embryos treated by the two-step procedure that survived after in vitro culture were transferred to three recipients and one of these resulted in pregnancy. These results indicate that cryopreservation by vitrification can damage membranous structures of the cells of bovine embryos, the extent and nature of this damage being dependent on the vitrification procedure.

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One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

International Journal of Molecular Sciences ◽

10.3390/ijms22052249 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2249

Author(s):

Fuminori Tanihara ◽

Maki Hirata ◽

Nhien Thi Nguyen ◽

Osamu Sawamoto ◽

Takeshi Kikuchi ◽

...

Keyword(s):

Gene Editing ◽

Multiple Gene ◽

Knock Out ◽

Editing Efficiency ◽

Guide Rnas ◽

Cas9 Protein ◽

One Step ◽

Step Generation ◽

The One

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

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Surface Roughness and Gloss of Actual Composites as Polished With Different Polishing Systems

Operative Dentistry ◽

10.2341/14-014l ◽

2015 ◽

Vol 40 (4) ◽

pp. 418-429 ◽

Cited By ~ 8

Author(s):

SA Rodrigues-Junior ◽

P Chemin ◽

PP Piaia ◽

JL Ferracane

Keyword(s):

Surface Roughness ◽

Small Area ◽

In Vitro Study ◽

Surface Characteristics ◽

Pairwise Comparisons ◽

Average Surface Roughness ◽

Single Operator ◽

One Step ◽

The One

SUMMARY Objective: This in vitro study evaluated the effect of polishing with different polishing systems on the surface roughness and gloss of commercial composites. Methods: One hundred disk-shaped specimens (10 mm in diameter × 2 mm thick) were made with Filtek P-90, Filtek Z350 XT, Opallis, and Grandio. The specimens were manually finished with #400 sandpaper and polished by a single operator using three multistep systems (Superfix, Diamond Pro, and Sof-lex), one two-step system (Polidores DFL), and one one-step system (Enhance), following the manufacturer's instructions. The average surface roughness (μm) was measured with a surface profilometer (TR 200 Surface Roughness Tester), and gloss was measured using a small-area glossmeter (Novo-Curve, Rhopoint Instrumentation, East Sussex, UK). Data were analyzed by two-way analysis of variance and Tukey's test (α=0.05). Results: Statistically significant differences in surface roughness were identified by varying the polishing systems (p<0.0001) and by the interaction between polishing system and composite (p<0.0001). Pairwise comparisons revealed higher surface roughness for Grandio when polished with Sof-Lex and Filtek Z250 and Opallis when polished with Enhance. Gloss was influenced by the composites (p<0.0001), the polishing systems (p<0.0001), and the interaction between them (p<0.0001). The one-step system, Enhance, produced the lowest gloss for all composites. Conclusions: Surface roughness and gloss were affected by composites and polishing systems. The interaction between both also influenced these surface characteristics, meaning that a single polishing system will not behave similarly for all composites. The multistep systems produced higher gloss, while the one-step system produced the highest surface roughness and the lowest gloss of all.

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One-step chromogenic equivalent of activated partial thromboplastin time evaluated for clinical application

Clinical Chemistry ◽

10.1093/clinchem/37.7.1235 ◽

1991 ◽

Vol 37 (7) ◽

pp. 1235-1244 ◽

Cited By ~ 4

Author(s):

Gabriëlle E Ponjee ◽

Huib L Vader ◽

Net J de Wild ◽

Ger W T Janssen ◽

Fedde van der Graaf

Keyword(s):

Partial Thromboplastin Time ◽

Clinical Laboratory ◽

Intrinsic Factor ◽

Activated Partial Thromboplastin Time ◽

Factor Vii ◽

Factor Deficiency ◽

One Step ◽

The One

Abstract We evaluated the clinical usefulness of a recently developed semi-automated one-step chromogenic equivalent of activated partial thromboplastin time (APTT; Behring). This simple test is easily adaptable for automation. Generally, the results with this chromogenic one-step APTT were at least as precise as those obtained with comparative coagulometric methods. The chromogenic one-step APTT showed, both in vitro and in vivo, adequate sensitivity to congenital intrinsic factor deficiency but no sensitivity to Factor VII deficiency. Unlike a two-step coagulometric APTT (Dade), the one-step chromogenic APTT seemed sensitive to activation products of the contact system, which are present in immunoadsorbed factor-deficient plasma. The in vitro sensitivity of the chromogenic APTT to heparin was comparable with that of a coagulometric APTT, but the sensitivity to heparin in patients' samples differed slightly. The chromogenic APTT is relatively insensitive to anomalies in the fibrinogen-fibrin conversion. Finally, we observed discrepancies between the chromogenic and coagulometric APTT results for plasma of patients with disseminated intravascular coagulation. We conclude that this one-step chromogenic APTT warrants further evaluation for possible use as a routine test for the clinical laboratory.

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Leishmania (V.) guyanensis: isolation and characterization of glucantime-resistant cell lines

Canadian Journal of Microbiology ◽

10.1139/m96-121 ◽

1996 ◽

Vol 42 (9) ◽

pp. 944-949 ◽

Cited By ~ 13

Author(s):

K. C. Ferreira-Pinto ◽

A. L. Miranda-Vilela ◽

C. Anacleto ◽

A. P. S. M. Fernandes ◽

M. C. B. Abdo ◽

...

Keyword(s):

Cell Lines ◽

Blot Hybridization ◽

Resistant Cell ◽

Cross Resistance ◽

Isolation And Characterization ◽

Stability Of Resistance ◽

One Step ◽

The Stability ◽

The One

A glucantime sensitive Leishmania (V.) guyanensis strain was used to obtain in vitro resistant cell lines, by increments in glucantime concentrations employing both one step and stepwise protocols. Whereas the effective concentration of drug that inhibited the growth of wild type cells by 50% (EC50 value) was 0.20 mg Sbv/mL, the resistant cells were able to grow in glucantime concentrations greater than 8.0 mg/mL. The resistant cell lines were partially characterized by their in vitro response to glucantime, the stability of resistance phenotype, cross resistance to a range of drugs, and also by the analysis of total DNA fragments generated by restriction endonucleases and blot hybridization. Amplified DNA sequence similar to a P-glycoprotein analog from Leishmania tarentolae (ltpgpA gene) was observed in all the resistant cell lines obtained through the one-step protocol. These cell lines showed cross resistance to heavy metals but were sensitive to puromycin, vinblastine, and pentostam.Key words: Leishmania, glucantime resistance, pentavalent antimony, gene amplification.

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43 NEW METHOD FOR ONE-STEP WARMING/IN-STRAW CRYOPROTECTANT DILUTION FOR IN VITRO-PRODUCED BOVINE BLASTOCYSTS AFTER VITRIFICATION WITH THE CRYOLOGIC VITRIFICATION METHOD

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab43 ◽

2015 ◽

Vol 27 (1) ◽

pp. 114

Author(s):

J. N. Caamaño ◽

E. Gómez ◽

B. Trigal ◽

M. Muñoz ◽

S. Carrocera ◽

...

Keyword(s):

Embryo Transfer ◽

Survival Rates ◽

Field Conditions ◽

Control Group ◽

Embryo Survival ◽

Grant Support ◽

One Step ◽

The One ◽

New System

Vitrification is considered an alternative to slow-rate freezing to cryopreserve in vitro-produced (IVP) bovine embryos. However, the use of vitrified IVP embryos for embryo transfer under field conditions is difficult because of the requirements of the current thawing protocols. The objective of this study was to develop a simple one-step warming/in-straw cryoprotectant dilution procedure for IVP bovine blastocysts that were vitrified using the cryologic vitrification method. In this study, 109 Day-7 IVP blastocysts were subjected to vitrification using the conventional fibreplugs (groups of 5 embryos were loaded in 3 mL of vitrification medium). Warming was performed in one-step in MS1 (0.25 M sucrose in BV = TCM 199-Hepes + 20% FCS) either using a 4-well plate for 5 min (control group) or in a new system that allowed in-straw cryoprotectant dilution designed to avoid losses of embryos and to maintain the temperature required during this procedure. This new system is composed of an adaptor with a wider opening that is coupled to the French straw and a heated metal chamber to protect and keep the straw at 41°C. Warmed embryos were washed and subsequently cultured in mSOFaaci + 6 gL–1 BSA + 10% FCS for 48 h. Re-expansion (at 2, 24, and 48 h) and hatching rates (at 24 and 48 h) were recorded. Data were analysed by ANOVA and are presented as LSM ± standard error. Embryo survival rates of embryos warmed by the one-step warming/in-straw cryoprotectant dilution procedure did not differ from the control group (see Table 1). These results suggest that the cryologic vitrification method combined with our warming system for in-straw cryoprotectant dilution may be used for direct embryo transfer under field conditions. Table 1.Embryo survival rates of in vitro-produced embryos vitrified by the cryologic vitrification method and warmed by the new one-step warming/in-straw cryoprotectant dilution procedure This study received grant support: INIA-RTA 2011–0090 and FEDER. M. Muñoz was supported by grant MICINN-RYC08-03454, and B. Trigal by a grant from Cajastur. The authors are members of the COST Action FA1201 Epiconcept.

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106 THE WARMING PROCEDURE: A FIRST STEP FOR IMPROVING THE NONSURGICAL DEEP INTRAUTERINE TRANSFER OF SOPS-VITRIFIED PORCINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab106 ◽

2012 ◽

Vol 24 (1) ◽

pp. 165

Author(s):

J. Gomis ◽

C. Cuello ◽

J. Sanchez-Osorio ◽

M. A. Gil ◽

I. Parrilla ◽

...

Keyword(s):

Reproductive Performance ◽

Petri Dish ◽

Control Group ◽

Hatching Rate ◽

Embryo Viability ◽

Step Method ◽

One Step ◽

The One

We previously reported successful nonsurgical deep intrauterine embryo transfer (ET) of fresh in vivo–derived porcine embryos. However, several trials from our laboratory demonstrated that when this procedure was used in combination with vitrified/warmed (VW) embryos, its efficiency was very low. Recently, we have shown that the one-step warming method in syringe, which was used in the earlier trials, compromises the in vitro embryo viability. The aim of this study was to confirm the negative effect of the direct warming in syringe and to evaluate the effect of alternative warming procedures on the in vivo development of VW embryos after nonsurgical ET. In Experiment 1, morulae and early blastocysts were collected on Days 5 to 6 (Day 0: onset of oestrus) and assigned to one of the following groups: 1) syringe group: vitrified embryos (n = 88) were warmed by the one-step method directly in a 1-mL syringe containing 300 μL of warming medium, which was connected to the ET catheter and then transferred to recipients (n = 6); 2) dish group: vitrified embryos (n = 194) were warmed with one-step warming method in a Petri dish containing 1 mL of warming medium, loaded into a Tom Cat catheter and transferred to recipients (n = 13); and 3) control group: fresh embryos (n = 129) were loaded in a 1-mL syringe in 100 μL of transfer medium and transferred to 9 recipients. An average of 15 embryos were transferred to each recipient on Day 4 or 5. Embryos were surgically recovered 24 h after ET. Data were analysed by ANOVA. The embryo recovery rate was similar among groups (range: 70.7 ± 4.8% to 77.2 ± 6.5%). The embryo survival (ES) and the hatching rate (HR) from the control group (94.0 ± 2.1% and 33.4 ± 7.6%, respectively) were higher (P ≤ 0.05) than those from the dish group (80.4 ± 4.6% and 14.5 ± 4.1%, respectively). All embryos from the syringe group were degenerated. Some viable recovered embryos (n = 135) were cultured for 48 h to evaluate their subsequent in vitro development. No differences were observed in ES between the control and the dish group (100.0 ± 0.0% vs 98.9 ± 1.0%). The HR in the control group (71.5 ± 2.1%) was higher (P ≤ 0.01) than that of the dish group (42.7 ± 6.1%). In experiment 2 we evaluated the reproductive performance of naturally cycling recipients after nonsurgical ET of vitrified embryos warmed with the one-step method in a Petri dish. An average of 35 VW morulae and blastocysts were transferred to each recipient (n = 10) on Days 4 to 6. Four recipients returned to oestrus at Days 21 to 22. The remaining 6 recipients were diagnosed pregnant by ultrasonography on Day 26. At Days 50 to 55, 5 recipients remained pregnant. In conclusion, the one-step in syringe warming method for vitrified porcine embryos had a completely adverse effect on the vivo viability, whereas nonsurgical ET of embryos warmed in a Petri dish allowed us to obtain promising reproductive performance. Supported by MICINN (AGL2009-12091) and SENECA (GERM 04543/07).

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87ONE-STEP VERSUS TWO-STEP VITRIFICATION AND IN-STRAW DILUTION OF IN VITRO-PRODUCED BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab87 ◽

2004 ◽

Vol 16 (2) ◽

pp. 165

Author(s):

L.F. Campos-Chillon ◽

D.J. Walker ◽

J.F. De La Torre-Sanchez

Keyword(s):

Least Square ◽

Bovine Embryos ◽

Step Method ◽

Slow Cooling ◽

Standard Procedures ◽

Step Procedure ◽

Main Effect ◽

One Step ◽

The One

Slow-cooling techniques are widely used in cryopreservation of bovine embryos. We have previously developed a simple, two-step vitrification technique for direct transfer in the field; however, simplification to one-step vitrification would be attractive. Therefore, factorial combinations of two techniques (one-step and two-step) and two post-thaw temperatures until culture (24 and 37°C) were studied. Blastocysts (n=220) sired by two bulls were obtained in vitro in four replicates. Briefly, oocytes were aspirated from 2–8-mm follicles of ovaries obtained at a slaughterhouse, matured, fertilized and cultured in vitro with standard procedures using chemically defined media (CDM1/2 or G1/2). Two-step embryos were transferred in 1μL into 1mL of V1-CDM (5M ethylene glycol (EG) in HEPES-buffered holding medium (HCDM2)), and one-step embryos into a 7-μL droplet of V2-CDM (7M EG, 0.5M galactose and 18% w/v Ficoll 70 in HCDM2) for 3min at 24°C. Next, embryos for the two-step method were moved in 1μL into a 7μL droplet of V2-CDM at 24°C. Droplets containing embryos (one or two-step) were loaded into 0.25-mL straws preloaded with a 1-cm column of D-HCDM (0.5M galactose in HCDM2), then 0.5cm air, and then 7cm of D-HCDM followed by 0.5cm air. The column containing the embryos (0.5cm (7μL)) was followed with 0.5cm air and 1cm of D-HCDM. Straws were heat-sealed and plunged vertically, sealed end first, into liquid nitrogen just covering the embryo, and the rest of the straw was then slowly immersed. The time from loading to plunging was 40–50s. Straws were thawed in air (24°C) for 10s and then in water horizontally at 37°C until ice disappeared. Straws were gently shaken to mix the columns; then, after 5min at 24 or 37°C, embryos were expelled and cultured in CDM2+5% FCS. Re-expansion and hatching rates were evaluated 48 h post thaw. Data (Table 1) were calculated as a percentage of non-vitrified controls for respective replicates (control means: re-expansion 87%; hatching 74%) and analyzed by ANOVA. There were no main effects of post-thawing temperature (P>0.1), indicating that, after thawing, embryos can be kept at room or body temperature. Also, main effect means for re-expansion and hatching for one-step or two-step addition of cryoprotectant were similar (P>0.1), but there was a tendency for higher survival for the two-step procedure. Further refinements of the one-step technique including EG concentrations, embryological stages and equilibration times should be studied. Table 1 Main effect means (least-square means±SEM) of vitrified embryos (% of non-vitrified controls)

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