Effects of slow freezing and vitrification on embryo development in domestic cat

2020 ◽  
Vol 55 (10) ◽  
pp. 1328-1336
Author(s):  
Valentina I. Mokrousova ◽  
Konstantin A. Okotrub ◽  
Eugeny Y. Brusentsev ◽  
Elena A. Kizilova ◽  
Nikolai V. Surovtsev ◽  
...  
Keyword(s):  
Embryo Development ◽  
Domestic Cat ◽  
Slow Freezing

111 SLOW FREEZING AND VITRIFICATION OF IN VITRO-MATURED DOMESTIC CAT OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 173 ◽  
Author(s):  
J. Braun ◽  
C. Otzdorff ◽  
T. Tsujioka ◽  
S. Hochi
Keyword(s):  
Polar Body ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Slow Freezing ◽  
Developmental Potential ◽  
Freezing Medium ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Chi Square Analysis

The effects of slow freezing or vitrification as well as exposure to the cryoprotective media without cooling and warming of in vitro-matured domestic cat oocytes on the in vitro development to the blastocyst stage was investigated. Cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Denuded oocytes with a detectable first polar body were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. For slow freezing, oocytes were equilibrated for 20 min at ambient temperatures in PBS with 20% FCS containing either 1.5 M ethylene glycol (EG) + 0.2 M sucrose or 1.5 M EG + 0.2 M trehalose. Oocytes were loaded into 0.25-mL straws, cooled to −7°C at 2°C min, held for 5 min, seeded, cooled down to −30°C at 0.3°C min, and finally plunged into liquid nitrogen. The straws were thawed for 5 s at room temperature and for 30 s in a waterbath at 30°C. Oocytes were washed 3 times before insemination. In vitro-matured oocytes were exposed to the cryoprotective media for 30 min before they were inseminated and then they were cultured for 7 days. For vitrification (Hochi et al. 2004 Theriogenology 61, 267–275), a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice was applied. No blastocysts could be obtained after slow freezing with a cryoprotective medium containing 0.2 M sucrose. Simple exposure to the same freezing medium after in vitro maturation without cryopreservation resulted in a blastocyst rate of 7.9% (control oocytes, 10.7%; not significant (NS); chi-square analysis). Use of trehalose as an extracellular cryoprotectant resulted in the harvest of one blastocyst (0.6%) after slow freezing. Exposure to the same cryoprotective medium resulted in a blastocyst rate of 10.0% (fresh control, 10.9%; NS). After exposure of in vitro-matured oocytes to the vitrification solution, a blastocyst rate of 16.0% was observed (8/50), which was not statistically different from the blastocyst rate in fresh control oocytes (16.3%; 15/92). No blastocysts could be obtained after vitrification (0/64). The results (Table 1) demonstrate that there is no obvious toxic effect of the cryoprotectants employed here for slow freezing or vitrification on the in vitro-matured oocytes, but the developmental potential of cryopreserved oocytes to the blastocyst stage is severely impaired. Table 1. Effect of slow freezing or exposure to freezing medium of matured cat oocytes on the development to the blastocyst stage in vitro

scholarly journals 91IMPACT OF PROPYLENE GLYCOL V. ETHYLENE GLYCOL ON OOCYTE MATURATION, SPINDLE INTEGRITY, FERTILIZATION AND EMBRYO DEVELOPMENT IN VITRO IN THE DOMESTIC CAT

2004 ◽  
Vol 16 (2) ◽  
pp. 167
Author(s):  
P. Comizzoli ◽  
D.E. Wildt ◽  
B.S. Pukazhenthi
Keyword(s):  
Ethylene Glycol ◽  
Propylene Glycol ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Domestic Cat ◽  
Hoechst 33342 ◽  
Immature Oocytes ◽  
Development In Vitro ◽  
The Impact

A thorough characterization of cryoprotectant (CPA) sensitivity is required to formulate a successful cryopreservation protocol for any biomaterial. The aim of this study was to characterize the toxic impact of various CPA types, concentrations, and exposure temperatures on the immature domestic cat oocyte. In Experiment 1, grade I immature oocytes (n=561) were exposed (30min; 25°C or 0°C) to 0M, 0.75M, 1.5M, or 3M of propylene glycol (PrOH) or ethylene glycol (EG) in PBS+20% fetal calf serum (v/v). After exposure, CPA was removed step-wise by subjecting oocytes to decreased CPA concentrations. Oocytes were cultured (30h; 38.5°C, 5% CO2) in IVM medium as reported previously (Wolfe and Wildt 1996 J. Reprod. Fertil. 106, 135–141). Oocytes were then fixed and stained to examine nuclear status (Hoechst 33342) and spindle integrity (FITC-labeled anti-α-tubulin antibodies; Sigma Chemical Co., St. Louis, MO). Experiment 2 was designed on the basis of Experiment 1 results to assess the impact of the spindle abnormalities on subsequent embryo development. Oocytes (n=776) were exposed to CPA conditions yielding optimal nuclear maturation with either high (0.75M or 3.0M PrOH or 1.5M EG at 25°C) or low (1.5M PrOH at 25°C) proportions of abnormal spindle. After IVM, oocytes were inseminated with thawed semen (5×105 motile sperm mL−1 ) in Ham’s F-10 (Irvine Scientific, St-Anna, CA). At 16h post-insemination, oocytes were fixed and stained (Hoechst 33342) to assess IVF success (pronuclear formation) or cultured in vitro for 7 days to assess embryo development. Data were analyzed by ANOVA and Tukey’s multiple comparison test. In Experiment 1, CPA treatment had no effect (NS) on meiotic progression to metaphase I. However, percentage of oocytes reaching metaphase II (MII) was reduced (P<0.05) in 3.0M PrOH at 0°C (29.3±8.3%; mean±SD), 3.0M EG at 25°C (33.7±8.9%), and 0°C (29.4±11.0%) compared to all other conditions examined (range, 52.0% to 62.0%). All CPA treatments also increased (P<0.05) spindle abnormalities at MII (range, 40.3% to 75.9%) compared to control (13.8±8.6%), except 1.5M PrOH at 25°C (20.7±10.1%). None of the CPA treatments in Experiment 2 influenced IVF success (range, 55% to 63%; NS). However, percentage of cleaved embryos was reduced (P<0.05) in 0.75M PrOH (32.1±4.1%), 1.5M EG (33.4±4.0%), and 3.0M PrOH (29.3±3.8%) compared to control (50.1±4.0%) or 1.5M PrOH (50.6±4.9%). Developmental competence (number of blastocysts relative to number of cleaved embryos) also was impaired (P<0.05) in 1.5M EG (16.5±7.4%) and 3.0M PrOH (14.9±7.8%) compared to the other conditions (range, 32.5% to 38.5%), including 1.5 PrOH at 25°C (32.5±7.8%). In conclusion, exposure of immature oocytes to 1.5M PrOH at 25°C does not adversely impact oocyte maturation, MII spindle, fertilization, or embryo development in vitro in the domestic cat.

scholarly journals Sucrose ‘Versus’ Trehalose Cryoprotectant Modification in Oocyte Vitrification : A Study of Embryo Development

2018 ◽  
Vol 11 (1) ◽  
pp. 97-104 ◽  
Author(s):  
Silvia W. Lestari ◽  
Khairunnisa F. Ilato ◽  
M. Iqbal A. Pratama ◽  
Nurin N. Fitriyah ◽  
Mulyoto Pangestu ◽  
...  
Keyword(s):  
Survival Rate ◽  
Embryo Development ◽  
Experimental Research ◽  
Adult Female ◽  
Light Microscope ◽  
Slow Freezing ◽  
Reverse Phase ◽  
Oocyte Vitrification ◽  
Sucrose Medium ◽  
Inverted Microscope

Numerous studies reported that vitrification, an ultra-rapid cooling technique, seems to be highly effective and could increase oocyte survival rate rather than slow freezing. The successful of oocyte vitrification depends on the proper combination of type and concentration of cryoprotectant. This study was addressed to determine the effects of the combination of type and concentration of cryoprotectants of vitrification media, notably in the embryo development. This experimental research was conducted by using oocyte obtained from thirty-two adult female Deutschland, Denken and Yoken (DDY) mice (7-8 weeks old). The MII mice oocytes were vitrified within 24 h after retrieval using the Cryotop method with cryoprotectants as follow : sucrose (16.5% EG, 16.5% DMSO, 0.5 mol/l sucrose), trehalose (16.5% EG, 16.5% DMSO, 0.5 mol/l trehalose) and Kitazato. The embryo development and morphological grading was observed at 2-cell and 8-cells under reverse phase light microscope and inverted microscope. This study demonstrated a good embryo development and morphological grading in sucrose and trehalose vitrification media. In embryo development, trehalose medium seems more superior compared to sucrose medium, even though Kitazato was the most superior compared to both. In the morphological grading, in 2-cells embryo, there were no significant differences between the three cryoprotectants, While, in 8-cells embryo, trehalose medium appeared to be superior compared to sucrose medium, even though seemed more inferior compared to Kitazato. The appropriate type and concentration of sugar as extracellular cryoprotectant was trehalose in oocyte vitrification based on embryo development, compared to sucrose.

scholarly journals Oocyte Meiotic Competence in the Domestic Cat Model: Novel Roles for Nuclear Proteins BRD2 and NPM1

2021 ◽  
Vol 9 ◽  
Author(s):  
Daniela R. Chavez ◽  
Pei-Chih Lee ◽  
Pierre Comizzoli
Keyword(s):  
Embryo Development ◽  
Germinal Vesicle ◽  
Domestic Cat ◽  
Nuclear Proteins ◽  
Protein Levels ◽  
Nuclear Factors ◽  
Chromatin Configuration ◽  
Mammalian Female ◽  
Adult Cat ◽  
Meiotic Competence

To participate in fertilization and embryo development, oocytes stored within the mammalian female ovary must resume meiosis as they are arrested in meiotic prophase I. This ability to resume meiosis, known as meiotic competence, requires the tight regulation of cellular metabolism and chromatin configuration. Previously, we identified nuclear proteins associated with the transition from the pre-antral to the antral follicular stage, the time at which oocytes gain meiotic competence. In this study, the objective was to specifically investigate three candidate nuclear factors: bromodomain containing protein 2 (BRD2), nucleophosmin 1 (NPM1), and asparaginase-like 1 (ASRGL1). Although these three factors have been implicated with folliculogenesis or reproductive pathologies, their requirement during oocyte maturation is unproven in any system. Experiments were conducted using different stages of oocytes isolated from adult cat ovaries. The presence of candidate factors in developing oocytes was confirmed by immunostaining. While BRD2 and ASRGL1 protein increased between pre-antral and the antral stages, changes in NPM1 protein levels between stages were not observed. Using protein inhibition experiments, we found that most BRD2 or NPM1-inhibited oocytes were incapable of participating in fertilization or embryo development. Further exploration revealed that inhibition of BRD2 and NPM-1 in cumulus-oocyte-complexes prevented oocytes from maturing to the metaphase II stage. Rather, they remained at the germinal vesicle stage or arrested shortly after meiotic resumption. We therefore have identified novel factors playing critical roles in domestic cat oocyte meiotic competence. The identification of these factors will contribute to improvement of domestic cat assisted reproduction and could serve as biomarkers of meiotically competent oocytes in other species.

Progestin priming before gonadotrophin stimulation and AI improves embryo development and normalises luteal function in the cat

2015 ◽  
Vol 27 (2) ◽  
pp. 360 ◽  
Author(s):  
Rosemary A. Stewart ◽  
Adrienne E. Crosier ◽  
Katharine M. Pelican ◽  
Budhan S. Pukazhenthi ◽  
Brandon D. Sitzmann ◽  
...  
Keyword(s):  
Embryo Development ◽  
Negative Influence ◽  
Domestic Cat ◽  
Corpora Lutea ◽  
Luteal Function ◽  
Progestin Treatment ◽  
Histological Characteristics ◽  
Targeted Gene Expression

Exogenous gonadotrophins administered before AI can adversely alter endocrine dynamics and inhibit embryo development in felids. In the present study, we tested the hypothesis that priming the domestic cat ovary with progestin mitigates the negative influence of gonadotrophin therapy by normalising early embryogenesis and luteal function. Queens were given either: (1) progestin pretreatment plus chorionic gonadotrophins (n = 8; primed); or (2) gonadotrophins only (n = 8; unprimed). Ovulatory response was assessed laparoscopically, and cats with fresh corpora lutea (CL) were inseminated in utero. Ovariohysterectomy was performed 3 days later to recover intra-oviductal embryos for in vitro culture; one ovary was prepared for histology, and CL from the remaining ovary were excised and assessed for progesterone content and targeted gene expression. Of the six primed and seven unprimed queens inseminated, embryo(s) were recovered from five individuals per group. Embryos from progestin-primed donors more closely simulated normal stage in vivo development (P < 0.05). No 2- or 4-cell embryos from either group developed beyond 16-cells in vitro; however, 50% of unprimed and 66.7% of primed (P > 0.05) 5–16-cell embryos progressed to morulae or blastocysts by Day 4 of culture. Although histological characteristics were unaffected by progestin priming (P > 0.05), luteal progesterone was unusually high (P < 0.05) in unprimed compared with primed cats (72.4 ± 5.8 vs 52.2 ± 5.5 ng mg–1, respectively). Two genes associated with progesterone biosynthesis (luteinising hormone receptor and 3β-hydroxysteroid dehydrogenase) were upregulated in unprimed versus primed individuals (P = 0.05 and P < 0.05, respectively), indicating potential mechanistic pathways for the protective influence of pre-emptive progestin treatment. Building on earlier findings that progestin priming prevents spontaneous ovulation, increases ovarian sensitivity to gonadotrophins and ensures a normative endocrine environment, the present study demonstrates that pretreatment with this steroid also benefits embryo development and normalisation of early luteal function.

354 POOR EMBRYO DEVELOPMENT AFTER ICSI WITH DOMESTIC CAT TESTICULAR SPERM IS OVERCOME BY CENTROSOME AND MIDPIECE REPLACEMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 284
Author(s):  
P. Comizzoli ◽  
D. Wildt ◽  
B. Pukazhenthi
Keyword(s):  
Cell Cycle ◽  
Embryo Development ◽  
Polar Body ◽  
Domestic Cat ◽  
Testicular Sperm ◽  
Developmental Potential ◽  
Kinetics Of ◽  
Sperm Aster ◽  
Fisher Scientific

Testicular sperm from the cat can be used via intracytoplasmic sperm injections (ICSI) to produce embryos in vitro, but the proportion of morulae and blastocysts is less than that obtained using ejaculated sperm. Compromised embryo development has been linked to the inability of the cat testicular sperm centrosome to form a normal sperm aster during the first cell cycle post-ICSI. The aim of the present study was to improve embryo development after ICSI with a testicular spermatozoon by centrosome/midpiece replacement from an ejaculated spermatozoon. Sperm suspensions used for ICSI (fresh testicular sperm from one adult testis vs. frozen-thawed sperm from one ejaculate in each replicate; four replicates) were sonicated for 3 s at setting 3 using a 60 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA, USA) to separate heads from centrosome/midpieces. Control immunostaining with monoclonal centrin antibodies revealed that centrosomes were attached to midpieces and were separated from sperm heads after sonication. A single sperm head (testicular, Th; ejaculated, Eh) was carefully positioned proximal to a single centrosome/midpiece (testicular, Tc; ejaculated, Ec) before injection into an oocyte with a visible polar body (in each replicate, n = 18 oocytes injected with each ThEc, EhEc, EhTc, or ThTc combination; four replicates). Injected oocytes then were activated with 7% ethanol and cultured in vitro in Ham's F10 (38.5�C, 5% CO2 in air). Percentages of first cleavages were recorded from 20 to 32 h post-activation (hpa). Nuclear status of uncleaved oocytes was evaluated at 48 hpa, and embryo development was assessed after 7 days of in vitro culture. Values were expressed as mean � standard deviation and analyzed by ANOVA. None of the uncleaved oocytes were activated. Percentages of cleaved oocytes (relative to the total number of injected oocytes) were not different (range, 61-65%; P > 0.05) among combinations of sperm heads and centrosome/midpieces. The mean times of the first cleavage, however, were earlier (P < 0.05) after combinations with ejaculated centrosome/midpieces (ThEc, 25.1 � 1.1 h; EhEc, 25.0 � 0.9 h) than with testicular centrosome/midpieces (EhTc, 29.2 � 0.8 h; ThTc, 29.3 � 1.2 h). Percentages of morulae produced were higher (P < 0.05) after combinations with ejaculated centrosome/midpieces (ThEc, 15.3 � 2.7%; EhEc, 16.7 � 2.1%) than with testicular centrosome/midpieces (EhTc, 8.3 � 3.1%; ThTc, 6.9 � 2.2%). Likewise, percentages of blastocysts were higher (P < 0.05) after combinations with ejaculated centrosome/midpieces (ThEc, 12.5 � 3.0%; EhEc, 13.9 � 2.8%) than with testicular centrosome/midpieces (EhTc, 5.6 � 3.3%; ThTc, 6.9 � 2.9%). These results demonstrated that: (1) kinetics of the first cell cycle and the success of embryonic development were determined by centrosome/midpiece source; and (2) poor developmental potential of domestic cat testicular sperm could be circumvented by centrosome/midpiece replacement from an ejaculated spermatozoon.

133 EVALUATION OF OOCYTE DONOR SOURCE AND CULTURE MEDIUM ON DOMESTIC CAT EMBRYO DEVELOPMENT RATES

2011 ◽  
Vol 23 (1) ◽  
pp. 171
Author(s):  
A. J. Pearks Wilkerson ◽  
R. D. Landry ◽  
C. R. Long
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cytochalasin B ◽  
Culture Media ◽  
Domestic Cat ◽  
Bovine Embryo ◽  
Development Rates ◽  
L1 And L2

The use of assisted reproductive technology (ART), including in vitro maturation (IVM) and embryo culture, is well established in several species, including canine and feline culture systems. Embryo production conditions tend to be specific for each species and prepared in unique formulations by laboratory. However, the increasing numbers of commercially available media allows for new comparisons in companion animal systems. Therefore, a goal of this study was to compare the development rates of feline parthenotes cultured in a commercially available bovine embryo culture medium with those cultured in a published 3-step domestic cat-specific system. In addition, the source of ovaries utilised for oocyte retrieval was evaluated as a factor in development rates. Ovaries from 2 locations (L1 and L2) were collected on the same day, and harvested oocytes were held in meiotic arrest medium containing 25 μM roscovitine for 14 to 18 h. Oocytes were incubated in maturation medium for 24 h before cumulus cell removal with vigorous pipetting in 0.4% hyaluronidase, and a subset of each group was fixed and stained to determine meiotic maturation rates (n = 76 and 55 for L1 and L2, respectively). Following activation (day 0) by a single course of three 50-μs electric pulses at 1.2 kV cm–1 in 0.3 M mannitol, 0.1 mM CaCl2, and 0.1 mM MgSO4, parthenotes from each source were randomly divided to culture medium treatment of Bovine Evolve medium (Zenith Biotech, Guilford, CT, USA) with 4 mg mL–1 BSA (n = 209) or IVC-1 medium n = 269; (Pope et al. 2009 Theriogenology 71, 864–871), each containing 10 μg mL–1 cycloheximide and 7.5 μg mL–1 cytochalasin B. After a 4-h activation treatment, parthenotes were moved to culture media without cycloheximide and cytochalasin B for embryo development. All parthenotes in IVC-1 medium were moved to IVC-1a medium on day 2. On day 5, both sets of parthenotes were moved to culture media containing 10% heat-inactivated FBS instead of BSA. On day 7, all parthenotes were fixed and stained with Hoechst to determine cell number. No differences were seen in maturation rates between L1 and L2 (56.3 ± 9.5 v. 54.7 ± 9.5, respectively). However, cleavage rates tended to differ, and proportion of embryos greater than 64 cells was different (60.7 ± 5.8 v. 78.3 ± 5.8, P = 0.056 and 3.0 ± 3.1 v. 19.7 ± 3.1, P < 0.005; respectively). We hypothesised that the physical condition of the ovary donors may have affected development rates because cats from L1 tended to be feral animals, whereas cats from L2 were mostly privately owned. Bovine Evolve was similar to IVC-1 medium for cleavage, 32-cell, and 64-cell development rates (74.2 ± 6.7 v. 64.8 ± 6.7; 24.0 ± 7.5 v. 31.8 ± 7.5; 10.7 ± 4.8 v. 12.0 ± 4.8, respectively; P > 0.05). These results indicate that commercially available culture medium can support in vitro development, even if the commercial medium is developed for a different species, but that source of cat ovaries should be considered in feline ART.

Comparative study of embryo development and apoptosis vitrification vs. slow freezing of biopsied mouse embryo

2007 ◽  
Vol 88 ◽  
pp. S345
Author(s):  
Y.J. Jung ◽  
J.H. Lee ◽  
M.J. Kook ◽  
K.W. Choi ◽  
S.J. Lee ◽  
...  
Keyword(s):  
Comparative Study ◽  
Embryo Development ◽  
Mouse Embryo ◽  
Slow Freezing

127 IN VITRO DEVELOPMENT OF IVF CRYOPRESERVED BOVINE EMBRYOS SUPPORTED BY TCM-199 OR SOFaa CULTURE MEDIUM

2007 ◽  
Vol 19 (1) ◽  
pp. 181
Author(s):  
A. C. Nicacio ◽  
W. B. Feitosa ◽  
M. Rovegno ◽  
R. Simões ◽  
J. S. de A. Gonçalves ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Embryo Development ◽  
Culture Medium ◽  
Cell Monolayer ◽  
Slow Freezing ◽  
Bovine Embryo ◽  
Quick Freezing ◽  
Slow Group ◽  
Hatching Rates

In vitro bovine embryo production is commercially applied around the world. However, the cryopreservation of these embryos is not yet possible, which raises difficulties for the expansion of this biotechnology. The aim of this work was to evaluate the influence of the culture media on embryo development after cryopreservation. Cumulus–oocyte complexes obtained from slaughterhouse bovine ovaries were in vitro-matured, fertilized, and cultured. A total of 600 expanded blastocysts (between 7 and 9 days of culture) were cryopreserved by slow freezing, quick freezing, or vitrification methods. For slow freezing (slow group), the embryos were exposed to 10% ethylene glycol (EG) for 10 min and cryopreserved at 1.2�C per minute. For quick freezing (quick group), the embryos were exposed to 10% EG for 10 min and to 20% EG + 20% glycerol (Gly) for 30 s. For vitrification (vitrification group), the embryos were exposed to 10% EG for 10 min and to 25% EG + 25% Gly for 30 s. The straws (quick and vitrification groups) were placed in nitrogen vapor (0.8 cm over the liquid nitrogen) for 2 min and then immersed in liquid nitrogen. The embryos were thawed in air for 10 s and in a water bath at 25�C for 20 s. For warming, embryos were washed in PBS + 0.2% BSA + 0.3 M sucrose for 3 min and in PBS + 0.2% BSA for 3 min. To evaluate development after thawing, the embryos were cultured on a granulosa cell monolayer with TCM-199 or SOFaa for 4 days. The embryos of the slow group showed re-expansion rates of 55.55% (55/99) and 29.00% (29/100), respectively, for TCM-199 and SOFaa. The quick group showed re-expansion rates of 4.85% (5/103) and 7.22% (7/97), and the vitrification group 10.89% (11/101) and 14.00% (14/100), respectively, for TCM-199 and SOFaa. The slow group showed hatching rates of 47.47% (47/99) and 11.00% (11/100), respectively; the quick group did not show hatching rates in either medium. The vitrification group showed hatching rates of 7.92% (8/101) and 6.00% (6/100), respectively, for TCM-199 and SOFaa. The results were analyzed by chi-square test, and all values were significant at P &lt; 0.05. The slow group showed difference in re-expansion and hatching rates when the different media were compared. The quick and vitrification groups did not show differences in re-expansion and hatching rates when the different media were compared. The slow group showed higher re-expansion and hatching rates than the quick and vitrification groups. In conclusion, the culture medium influences embryo development after slow freezing, and the TCM-199 is more appropriate than SOFaa. This work was supported by FAPESP 04/05335-1

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