Biomedical subjects

S Cseh

Publications and source records attributed to S Cseh.

At least 19 recordsLinked to original sources

PubMed2006

Endocrine characteristics of late pregnant hyperketonaemic ewes and their reproductive performance following the induction of ovarian cyclicity out of the breeding season.

Ketosis was diagnosed in a flock of Merino ewes that conceived from synchronised oestrus in the early autumn period. On day 140 of pregnancy the ewes were sampled for determination of betaOH-butyrate (BHB), AST, glucose, non-esterified fatty acids (NEFA), total cholesterol (TCH), insulin, T4, T3, cortisol, IGF-1 and leptin. The results were evaluated according to the number of fetuses born some days later and the presence of hyperketonaemia (BHB: > or = 1.60 mmol/l). In May, about 3 months after lambing, cyclic ovarian function was induced (Cronolone + eCG), and the ewes were inseminated artificially (AI) 48 h after the removal of gestagen-containing sponge. At the time of AI and 10 days later blood samples were collected again to check the plasma levels of the same constituents as previously (in samples taken at AI), and to monitor the ovarian response by assaying progesterone (in both samples). On day 140 of gestation significantly lower BHB levels were detected in dams with single (n = 41) than in those with twin (n = 57) pregnancies. Hyperketonaemia was found only in ewes bearing twins (n = 27). These animals had higher NEFA and cortisol, and lower TCH, insulin, IGF-1, leptin and T3 levels than their normoketonaemic twin-bearing flock-mates, and those with single pregnancy. The blood glucose concentrations varied within a wide range, and the means of groups did not exhibit any significant differences. The formerly hyperketonaemic individuals were characterised by lower leptin level 3 months after lambing, and they showed a poorer response to the cycle-induction procedure than the others. The non-responders had lower IGF-1 and leptin levels than those ovulated after this treatment. It was concluded that the subclinical form of ovine ketosis is characterised by complex endocrine alterations, reflecting an obvious form of negative energy balance. If attempts to induce cyclic ovarian function outside the breeding season are made soon after lambing, the ovarian response and fertility of these ewes may also be depressed.

3-Hydroxybutyric Acid↗

PubMed2004

Assisted reproductive research: laser assisted hatching and spindle detection (spindle view technique).

Animal experiments are very important for the development of new assisted reproductive techniques (ART) for use in human and animal reproductive medicine. Most technical aspects of reproductive manipulation of humans and animals are very similar, and many components of successful human ART used nowadays have been derived from animal studies. In this study we examined (1) the use of 'non-contact' laser for assisted hatching, (2) whether spindles in living mouse oocytes could safely be imaged/examined by polarisation microscope (polscope) and (3) the influence of environment (e.g. temperature, in vitro culture, etc.) on spindle detection/visualisation. The data of the study presented here show that (1) laser assisted hatching (AH) is a fast, very accurate and safe procedure without any harmful effect on embryo development and it can support very effectively the implantation of embryos, (2) the use of polscope facilitates the evaluation of oocyte quality and the selection of oocytes with spindle, (3) by monitoring the spindle position during intracytoplasmic sperm injection (ICSI), we can reduce spindle damage and increase the chance of fertilisation. Further studies are underway to test the hypothesised connection between spindle birefringence and developmental capacity of oocytes/embryos.

Animals↗

PubMed2004

Survival of mouse blastocysts after low-temperature preservation under high pressure.

Cryoinjuries are almost inevitable during the freezing of embryos. The present study examines the possibility of using high hydrostatic pressure to reduce substantially the freezing point of the embryo-holding solution, in order to preserve embryos at subzero temperatures, thus avoiding all the disadvantages of freezing. The pressure of 210 MPa lowers the phase transition temperature of water to -21 degrees C. According to the results of this study, embryos can survive in high hydrostatic pressure environment at room temperature; the time embryos spend under pressure without significant loss in their survival could be lengthened by gradual decompression. Pressurisation at 0 degrees C significantly reduced the survival capacity of the embryos; gradual decompression had no beneficial effect on survival at that stage. Based on the findings, the use of the phenomena is not applicable in this form, since pressure and low temperature together proved to be lethal to the embryos in these experiments. The application of hydrostatic pressure in embryo cryopreservation requires more detailed research, although the experience gained in this study can be applied usefully in different circumstances.

Animals↗

PubMed2003

Effect of the El Niño phenomenon on the ovarian responsiveness and embryo production of donor cows.

The effects of different Temperature Humidity Index (THI) values in cold, hot and El Niño (EN) climates on superovulation and embryo production were analysed on Holstein Friesian donor cows. There were significant differences in the THI among the three climates. The average temperature in the EN period was 6 degrees C higher than in the summer period of the previous 30 years. The number of corpora lutea (CL) and embryos were log- and back-transformed, Kolmogorov-Smirnoff test was used for normality and Lilliefors test was applied for significance. In the cold season THI was 70.74 +/- 1.35 and the average number of CL was 9.84 +/- 4.37. In the hot season the THI was 73.99 +/- 0.72 and the average number of CL was 9.70 +/- 4.49. When the THI, in the EN period, increased up to 79.74 +/- 4.01, the superovulation response was significantly (P < 0.01) reduced (average number of CL = 5.22 +/- 2.53). The embryo production result showed a similar tendency. In the hot period the average number of embryos obtained was 5.87 +/- 2.98. However, in the EN period it decreased to 4.21 +/- 2.05. Higher temperature reduced embryo quality. The proportion of live embryos (%) was 59.2 +/- 37.4 in the cold and 38.2 +/- 38.5 in the EN periods of the year (P < 0.01). However, ovarian sensitiveness showed adaptation to summer environment while the heat stress, which was more severe in the EN period, negatively affected the superovulation response and embryo production.

Animals↗

PubMed2002

Superovulation using recombinant human FSH and ultrasound-guided transabdominal follicular aspiration in baboon (Papio anubis).

The response of baboon females to a modified human ovarian stimulation protocol incorporating start of pituitary suppression in the luteal phase of the cycle with a GnRH agonist (GnRHa) and recombinant human FSH (rhFSH) was studied. A long-acting GnRHa implant supplying goserelin acetate was administered s.c. to six adult female baboons experiencing regular menstrual cycles (33-34 days) on days 22-24 of the cycle. Follicular development was monitored by transabdominal ultrasonography and serum levels of E2 and progesterone (P4) and rhFSH were determined by ELISA. Menses occurred 9-10 days after GnRHa administration. Daily i.m. administration of 75 IU rhFSH commenced 9-10 days after menses and continued for 9-10 days. When most follicles were > or =5mm diameter and serum E2 had reached its maximum level, 2000 IU hCG was administered i.m. to induce follicle maturation. Transabdominal ultrasound-guided follicular aspiration of follicles > or =2mm diameter was performed 30-34h after hCG administration. One baboon did not show an adequate response to rhFSH stimulation. This animal did not receive further treatment and no data for it are presented. The number of follicles aspirated was 21+/-4 and 17.2+/-3.8 oocytes were recovered per animal with an average recovery rate of 82% (86/105). The number of oocytes collected from five animals were 14, 21, 16, 15, and 20 (n=86). Most of the oocytes recovered were in metaphase II and 3h after recovery 91% (78/86) were considered suitable for in vitro fertilization. It was concluded that recombinant human FSH can successfully induce follicular recruitment and oocyte maturation in baboon females during pituitary suppression with a GnRHa

Animals↗

PubMed2001

Substrate specificities of recombinant mannan-binding lectin-associated serine proteases-1 and -2.

Mannan-binding lectin (MBL)-associated serine proteases-1 and 2 (MASP-1 and MASP-2) are homologous modular proteases that each interact with MBL, an oligomeric serum lectin involved in innate immunity. To precisely determine their substrate specificity, human MASP-1 and MASP-2, and fragments from their catalytic regions were expressed using a baculovirus/insect cells system. Recombinant MASP-2 displayed a rather wide, C1s-like esterolytic activity, and specifically cleaved complement proteins C2 and C4, with relative efficiencies 3- and 23-fold higher, respectively, than human C1s. MASP-2 also showed very weak C3 cleaving activity. Recombinant MASP-1 had a lower and more restricted esterolytic activity. It showed marginal activity toward C2 and C3, and no activity on C4. The enzymic activity of both MASP-1 and MASP-2 was specifically titrated by C1 inhibitor, and abolished at a 1:1 C1 inhibitor:protease ratio. Taken together with previous findings, these and other data strongly support the hypothesis that MASP-2 is the protease that, in association with MBL, triggers complement activation via the MBL pathway, through combined self-activation and proteolytic properties devoted to C1r and C1s in the C1 complex. In view of the very low activity of MASP-1 on C3 and C2, our data raise questions about the implication of this protease in complement activation.

Base Sequence↗

PubMed2001

Interaction properties of human mannan-binding lectin (MBL)-associated serine proteases-1 and -2, MBL-associated protein 19, and MBL.

The mannan-binding lectin (MBL) activation pathway of complement plays an important role in the innate immune defense against pathogenic microorganisms. In human serum, two MBL-associated serine proteases (MASP-1, MASP-2) and MBL-associated protein 19 (MAp19) were found to be associated with MBL. With a view to investigate the interaction properties of these proteins, human MASP-1, MASP-2, MAp19, as well as the N-terminal complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1-epidermal growth factor (CUB-EGF) segments of MASP-1 and MASP-2, were expressed in insect or human kidney cells, and MBL was isolated from human serum. Sedimentation velocity analysis indicated that the MASP-1 and MASP-2 CUB-EGF segments and the homologous protein MAp19 all behaved as homodimers (2.8-3.2 S) in the presence of Ca(2+). Although the latter two dimers were not dissociated by EDTA, their physical properties were affected. In contrast, the MASP-1 CUB-EGF homodimer was not sensitive to EDTA. The three proteins and full-length MASP-1 and MASP-2 showed no interaction with each other as judged by gel filtration and surface plasmon resonance spectroscopy. Using the latter technique, MASP-1, MASP-2, their CUB-EGF segments, and MAp19 were each shown to bind to immobilized MBL, with K:(D) values of 0.8 nM (MASP-2), 1.4 nM (MASP-1), 13.0 nM (MAp19 and MASP-2 CUB-EGF), and 25.7 nM (MASP-1 CUB-EGF). The binding was Ca(2+)-dependent and fully sensitive to EDTA in all cases. These data indicate that MASP-1, MASP-2, and MAp19 each associate as homodimers, and individually form Ca(2+)-dependent complexes with MBL through the CUB-EGF pair of each protein. This suggests that distinct MBL/MASP complexes may be involved in the activation or regulation of the MBL pathway.

Amino Acid Motifs↗

PubMed2001

Studies on factors affecting superovulation and embryo transfer in Hungarian merino ewes.

The objectives of this study were (a) to assess the ovulatory response and embryo production of Hungarian Merino ewes after superovulation, (b) to investigate the factors influencing the efficiency of embryo transfer (ET) in Hungarian Merino ewes, (c) to compare the results of two ovarian stimulation protocols (PMSG and PMSG + FSH treatment) in Hungarian Merino ewes, and (d) to study how superovulation, laparoscopic insemination and surgical embryo retrieval (ER) affect the subsequent reproduction of Hungarian Merino donor females after an ET programme. There was no significant difference between the ovarian stimulation protocols in the ratio of donor ewes responding to superovulation nor in the average number of corpora lutea. However, the number of transferable embryos recovered per donor ewe was higher in the PMSG + FSH group. The proportion of transferable embryos, unfertilized oocytes and degenerated embryos did not differ between the treatment protocols. The total pregnancy rate was 53.4% (179/335). Neither the developmental stage of the embryo nor the number of transferred embryos affect the implantation of embryos. However, the increased number of transferred embryos positively influenced the pregnancy rate. No difference was found in the pregnancy rate between synchronised and non-synchronised groups of recipients. Thirty-six out of 45 donor ewes (80%) became pregnant within one year after the ET programme, indicating that ovarian stimulation and surgical ER did not affect adversely their reproduction.

Animals↗

PubMed2000

The cleavage of two C1s subunits by a single active C1r reveals substantial flexibility of the C1s-C1r-C1r-C1s tetramer in the C1 complex.

The activation of the C1s-C1r-C1r-C1s tetramer in the C1 complex, which involves the cleavage of an Arg-Ile bond in the catalytic domains of the subcomponents, is a two-step process. First, the autolytic activation of C1r takes place, then activated C1r cleaves zymogen C1s. The Arg463Gln mutant of C1r (C1rQI) is stabilized in the zymogen form. This mutant was used to form a C1q-(C1s-C1rQI-C1r-C1s) heteropentamer to study the relative position of the C1r and C1s subunits in the C1 complex. After triggering the C1 by IgG-Sepharose, both C1s subunits are cleaved by the single proteolytically active C1r subunit in the C1s-C1rQI-C1r-C1s tetramer. This finding indicates that the tetramer is flexible enough to adopt different conformations within the C1 complex during the activation process, enabling the single active C1r to cleave both C1s, the neighboring and the sequentially distant one.

Animals↗

PubMed2000

Economical and ecological importance of indigenous livestock and the application of assisted reroduction to their preservation.

Among the many mammalian species that are threatened as the result of habitat destruction are numerous species of rare or little-known native livestock that possess features that render them ideally adapted to their environment. Because of the vital and valuable role many of these species play both to the ecology and economy of their native countries, attention is being directed towards initiating breeding programs that might insure their continued survival. This review introduces and highlights the importance of some of these indigenous species and outlines efforts currently underway to apply assisted reproductive technologies to their conservation.

Animals↗

PubMed2000

Ovarian consequences of low dose peroral Fusarium (T-2) toxin in a ewe and heifer model.

The effect of low dose peroral Fusarium produced T-2 toxin intake upon the ovarian function was evaluated in ewes (n = 30; Trial 1) and heifers (n = 7; Trial 2). Half of the ewes and all of the heifers were fed rich, acidosis-inducing concentrate. The 30 ewes were divided into 6 groups of 5 animals each. They were given 0, 0.3 or 0.9 mg/day (0, 5 or 15 ug/kg) purified T-2 toxin per os for 21 days (3x2 factorial design). Four of the 7 heifers were fed 9 mg/day (25 ug/kg) of the same purified T-2 toxin for 20 days while 3 remained untreated. The estrus cycles in all animals were synchronized prior to the trials and the T-2 exposure was started in the mid-luteal phase. The acidic condition in the rumen was estimated by the determination of urinary net acid-base excretion. The ovarian activity was followed with blood sampling for progesterone on alternate days (Trial 1) or with ultrasonography and sampling for progesterone daily (Trial 2). All of the heifers and concentrate-fed ewes showed a compensated acidosis, during first two thirds of T-2 exposure. In Trial 1, ovarian malfunction manifested as lower P4 peak concentration in the midluteal phase, shortening of the CL lifespan and prolonged follicular phases. These malfunctions were detected in 3 and 3 ewes fed concentrate and 0.3 mg and 0.9 mg T-2 toxin. Lower P4 peak concentration was observed in 1 ewe fed regular diet and 0.9 mg T-2 toxin. None of the control and acidotic groups (0 mg T-2), or ewes fed regular diet with 0.3 mg T-2 showed any ovarian malfunction. In Trial 2, after PGF2, administration the ovulation occured later and the plasma progesterone level remained low (< 3 nmol/l) for a longer period in T-2 treated heifers, than their untreated control mates (5.0+/-0.7 vs 3.7+/-0.5 d, P<0.05 and 8.3+/-0.4 vs 6.3+/-0.9 d, P<0.01, respectively). These results show that the peroral T-2 intake can significantly retard the folliculus maturation and ovulation and perhaps the subsequent luteinisation also in ruminants kept on concentrate-rich diet.

Acidosis↗

PubMed2000

Electroejaculated baboon (Papio anubis) sperm requires a higher dosage of pentoxifylline to enhance motility.

PURPOSE: Sperm collected by electroejaculation often show poor motility. The objective was to determine whether the addition of the phosphodiesterase inhibitor, pentoxifylline, would stimulate electroejaculated baboon sperm motility. METHODS: Electroejaculation was performed on several occasions on a male baboon and sperm collected after familiarization. Pentoxifylline was tested at the standard concentration (1 mg/ml) and at twice the concentration. Sperm parameters were evaluated using a sperm motility analyzer, as well as acrosome and DNA integrity techniques. RESULTS: Sperm exposed to 2 mg/ml pentoxifylline had higher total motility when compared with the control and 1 mg/ml treatment. Rapid progression and velocities were higher after pentoxifylline. The acridine orange DNA normality test showed that over 90% of collected sperm had intact unfragmented DNA. About half the sperm population had normal morphology and intact acrosomes. A low percentage had cytoplasmic droplets. CONCLUSIONS: Sperm collected by rectal probe electroejaculation required a higher concentration (2 mg/ml) of pentoxifylline for enhanced total motility, rapid progression, and higher velocity. This suggested differences in membrane properties or phosphodisterase activity in electrojeaculated sperm. The electroejaculation procedure did not denature sperm DNA at the acridine orange assay level nor were the acrosomes disrupted. The present study also documented unique information on baboon kinematic parameters.

Acridine Orange↗

PubMed2000

Analysis of papillomavirus consensus L1 gene in a closed colony of baboons (Papio anubis).

OBJECTIVE: The purpose of this study was to analyze cervical specimens and semen from a closed colony of baboons for the presence of the papillomavirus consensus L1 gene. STUDY DESIGN: Cervical swabs were collected from lightly anesthetized female baboons. Semen was collected from a male baboon by standard electroejaculation techniques. Deoxyribonucleic acid was extracted from the cells by two different methods and analyzed by polymerase chain reaction targeting the L1 consensus gene common for >25 genital papillomaviruses. RESULTS: Analyses of the polymerase chain reaction-amplified products did not reveal bands for the papillomavirus in either the cervical specimens or the semen. CONCLUSIONS: The hypothesis of a linkage between primates with papillomavirus as a common factor is not supported by the results of this study. This information is also important in assisting clinicians in setting up specific pathogen-free colonies of baboons.

Animals↗

PubMed2000

Importance of assisted reproductive technologies in the conservation of wild, rare or indigenous ungulates: review article.

Biodiversity is increasingly threatened by intensive agriculture, environmental pollution, extinction of natural habitats and several other factors. Several mammalian species including ungulates have disappeared or are threatened by extinction. However, ungulates play an important role both in the ecosystem and in the economy. In general, species or breeds are considered endangered if their population does not exceed 1,000 individuals. In these cases conservation programmes should be initiated in order to maintain or even increase their number. This review deals with the possibilities and limitations of assisted reproductive technologies (ART) in the conservation of ecologically valuable wild, rare and indigenous ungulates. The methods discussed here are artificial insemination, cryopreservation of semen and embryos, embryo recovery and transfer, in vitro production of embryos, as well as micromanipulation techniques including sperm injection, assisted hatching and cloning. Some of these procedures are already being exploited in the breeding of farm ungulates, but more basic information about the reproductive patterns of wild, rare and indigenous animal species is needed before the routine use of ARTs.

Animals↗

PubMed1999

Vitrification of mouse embryos in two cryoprotectant solutions.

The objective of this study was to compare the efficiency of 2 media on the vitrification of mouse compacted morulae, early blastocysts and expanded blastocysts after equilibration at room temperature of 4 degrees C. Embryos were equilibrated for 10 min in either 25% VS3 (Rall Equilibration Medium, REM) or 10% glycerol + 20% propylene glycol (Massip Equilibration Medium, MEM) in DPBS at 20 degrees C or 4 degrees C. For vitrification either 100% VS3 (Rall Vitrification Medium, RVM) or 25% glycerol + 25% propylene glycol (Massip Vitrification Medium, MVM) in DPBS was used. Embryos equilibrated at room temperature were loaded in 20 microL of vitrification media into 250 microL straws and then immediately (30 sec) plunged into liquid nitrogen (LN2). After equilibration at 4 degrees C the embryos were put into straws with 20 microL of precooled vitrification medium, and after 20 min at 4 degrees C they were plunged into LN2. Embryos from both groups were thawed in a 20 degrees C water bath for 20 sec, transferred to 1.0 M sucrose in DPBS for 5 min and then cultured for 24 to 48 h in Whitten's medium at 37 degrees C in 5% CO2 in air. In the groups of embryos prepared for vitrification at room temperature the survival rate of compact morulae vitrified in RVM was higher than those vitrified in MVM (65/70, 93% vs 49/74, 66%; P < 0.01). No difference was found in the survival rate of early blastocysts and expanded blastocysts vitrified in RVM or MVM (30/83, 36% vs 25/75, 33% and 4/66, 6% vs 4/76, 5%). No difference was found between the survival rate of compact morulae after equilibration with RVM or MVM at 4 degrees C (62/75, 83% vs 52/74, 70%). Both the early blastocysts and expanded blastocysts equilibrated at 4 degrees C MVM yielded a higher survival rate than RVM (28/74, 38% and 40/70, 57% vs 4/75, 5% and 4/77, 5%; P < 0.01). We conclude that, of the 3 developmental stages, compact morulae withstand the vitrification process best, and reduction of the temperature prior to plunging into LN2 is not required. A 10-fold increase in the survival rate of expanded blastocysts can be achieved using low temperature equilibration (4 degrees C) and MVM.

Animals↗

PubMed1999

One active C1r subunit is sufficient for the activity of the complement C1 complex: stabilization of C1r in the zymogen form by point mutations.

The binding of C1 (the first component of complement) to immune complexes leads to the autoactivation of C1r through the cleavage of the Arg463-Ile464 bond in the catalytic domain. Spontaneous activation of C1r (and C1) also occurs in the fluid phase, preventing the characterization of the zymogen form of C1r. To overcome this difficulty, the zymogen form of human C1r was stabilized by mutating the Arg in the Arg463-Ile464 bond to Gln. This mutant was designated as mutant QI. Recombinant C1r (wild type (wt) or mutant) was expressed in insect cells using serum-free medium in functionally pure form; therefore, the cell culture supernatant was suitable to reconstruct C1 for the hemolytic assay. Mutant QI was a stable, nonactivable zymogen and showed no hemolytic activity in reconstituted C1. However, this stable zymogen C1r mutant could form an active mixed dimer with the wt C1r, indicating that one active C1r subunit in the C1 complex is sufficient for the full activity of the entire complex. Our experiments also showed that the exchange of C1r monomers between the C1r dimers is completed in less than 16 h even at pH 7 and 4 degrees C. Two other mutants were also constructed by changing Arg463 to Lys, or Ile464 to Phe, and were designated as mutants KI and RF, respectively. Although these substitutions did increase the stability of the proenzyme in the cell culture supernatant, the mutant proteins retained their ability to autoactivate, and both had a wt-like hemolytic activity.

Animals↗

PubMed1998

Isolation of human alpha 2HS-glycoprotein synthesized by Sf9 cells.

The human alpha 2HS-glycoprotein is a negative acute phase protein synthesized by hepatocytes. Because of its fragility and the difficulty of its purification, we used recombinant DNA techniques to produce the protein in order to investigate its biological effects. The cDNA coding for the whole alpha 2HS-glycoprotein from human liver library had been cloned into the baculovirus expression vector system using the pVL 1392 transfer vector and Sf9 cells. The recombinant protein was synthesized in the Sf9 cells and was isolated on a hydroxiapatite column from the culture medium. Western blot analysis indicated that the cells synthesized large quantities of the recombinant human protein. The molecular mass of the recombinant AHSG was the same as that of the protein in human plasma but was slightly lower than that of AHSG in the cell culture supernatants of HepG2 and higher than that of AHSG from Hep3B cell cultures, respectively.

Acute-Phase Proteins↗

PubMed1997

The effect of quick-freezing in ethylene glycol on morphological survival and in vitro development of mouse embryos frozen at different preimplantation stages.

This study was conducted to examine the effect of a quick-freezing protocol on morphological survival and in vitro development of mouse embryos cryopreserved in ethylene glycol (EG) at different preimplantation stages. One-cell embryos were harvested from 6-to 8-wk-old CB6F1 superovulated mice, 20 to 23 h after pairing with males of the same strain and hCG injection. The embryos were cultured in human tubal fluid (HTF) containing 4 mg/ml BSA under mineral oil at 37 degrees C in 5% CO(2) plus 95% room air at maximal humidity. Twenty-four to 96 h after collection, the embryos were removed from culture and frozen at the 2 cell, 4 to 8-cell, compact morula, early blastocyst, expanding blastocyst and expanded blastocyst stages. To perform the quick-freeze procedure, embryos were equilibrated in Dulbecco's phosphate buffered saline (DPBS) + 10 % fetal bovine serum (FBS) + 0.25 M sucrose + 3 M ethylene glycol (freeze medium) for 20 min at room temperature (22 to 26 degrees C) and loaded in a single column of freeze medium into 0.25-ml straws (4 to 5 embryos per straw). The straws were held in liquid nitrogen vapor for 2 min and immersed in liquid nitrogen. Embryos were thawed by gentle agitation in a 37 degrees C water bath for 20 sec and transferred to DPBS + 10 % FBS + 0.5 M sucrose (re-hydration medium) for 10 min at room temperature, rinsed 2 times in HTF plus 4 mg/ml BSA and then cultured for 24 to 96 h. Survival of embryos was based on their general morphological appearance after thawing and their ability to continue development upon subsequent culture in vitro. Survival of blastocysts after thawing also required expansion or reexpansion of the blastocoel after several hours in culture. Significant differences were found in the survival and development of mouse embryos at different developmental stages quick-frozen in ethylene glycol and sucrose: 2-cell embryos 43/84 (51%), 4 to 8-cell embryos 44/94 (47%), morulae and early blastocysts 56/70 (80%; P</=0.05), expanding and expanded blastocysts 10/59 (17%; P</=0.05). Our data indicate that the developmental stage in which mouse embryos are subjected to this quick-freeze protocol affects survival and development in vitro and that most (80%) morula and early blastocyst stage embryos survive the procedure.

Journal Article↗