206 Effect of L-Ascorbic Acid, Polydatin, and In-Straw Rehydration of Slow-Frozen In Vitro-Produced Bovine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 243
Author(s):  
C. M. Dinndorf ◽  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
L. F. Campos-Chillon
Keyword(s):  
Reactive Oxygen Species ◽  
Ascorbic Acid ◽  
Oxygen Species ◽  
Bovine Embryos ◽  
Freezing Medium ◽  
High Lipid ◽  
Standard Procedures ◽  
Frozen Embryos ◽  
Main Effect

In vitro-produced (IVP) bovine embryos have poor cryotolerance due to high lipid content and reactive oxygen species levels that hinder post-thaw survival. We hypothesised that in-straw rehydration of slow-frozen embryos with sucrose and the addition of the antioxidant polydatin and l-ascorbic acid would increase post-thaw survival. The IVP embryos (n = 116) were generated in 7 replicates by aspirating oocytes from 2- to 8-mm follicles of abattoir ovaries, matured for 23 h, fertilized with semen from 1 of 3 bulls using standard procedures, and cultured in SCF1 medium for 7 days (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130). Stage 7 embryos were slow-frozen using 1 of 4 protocols in a 2 × 2 factorial design: embryos were equilibrated in conventional slow-freezing media for 20 min [1.5 M ethylene glycol (EG) and 0.5 M sucrose] with 1 mm l-ascorbic acid or 1 μM polydatin, and loaded in the straw adjacent to columns of freezing medium or 0.75 M EG and 0.6 M sucrose and then seeded at −6°C, cooled at 0.5°C min−1, and plunged at –32°C. Embryos were thawed in air for 10 s followed by 30 s in 32 to 35°C water bath. Once straw columns were disrupted, embryos were allowed to equilibrate for 5 min. Subsequently, embryos were washed and placed back in culture and re-expansion was assessed at 24 and 48 h. Data (Table 1) were analysed by ANOVA with means separated by Tukey’s HSD. Results indicate that there was no main effect between the 2 antioxidants or the use of rehydration columns (P < 0.05); however, there was higher (P < 0.05) re-expansion for embryos frozen with polydatin and with rehydration column than embryos frozen with l-ascorbic acid and no rehydration column. This suggests that polydatin coupled with in-straw rehydration (0.75 M EG and 0.6 M sucrose) may improve post-thaw survival of IVP bovine embryos. Table 1.Post-thaw re-expansion rates of embryos exposed to antioxidants and in-straw rehydration (± SEM)

213 EFFECT OF NOVEL SOF MEDIUM AND L-ASCORBIC ACID DURING CRYOPRESERVATION OF IN VITRO-PRODUCED JERSEY CATTLE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 215 ◽  
Author(s):  
A. R. Higginbotham ◽  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
L. F. Campos-Chillon
Keyword(s):  
Reactive Oxygen Species ◽  
Ascorbic Acid ◽  
Lipid Content ◽  
Nile Red ◽  
Reactive Oxygen ◽  
Molecular Probes ◽  
Oxygen Species ◽  
High Lipid ◽  
Blastocyst Rate

Jersey embryos have high lipid content and poor cryotolerance. High lipid and reactive oxygen species concentrations are associated with poor post-thaw survival and increased post-thaw apoptosis. It was hypothesized that culturing embryos in SOF-based medium (SCF1; SOF for conventional freezing will decrease lipid content, and adding l-ascorbic acid (l-AA) to freezing media will increase cryotolerance and decrease post-thaw apoptosis. A 2 × 2 factorial design was used to compare SOF v. SCF1 and additives in freezing media (control v. L-AA). In vitro-produced blastocysts were produced in 5 replicates by aspirating oocytes (n = 975) from 2 to 8 mm follicles of abattoir ovaries, maturing for 23 h, fertilizing with semen from 1 of 2 bulls, and culturing in SOF medium or SCF1 in 38.5°C in 5% O2, 5% CO2, and 90% N2. Randomly selected Day 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content and 300 nM Mitotracker Red CMX-Rosamine (Molecular Probes Inc., Eugene, OR, USA) for mitochondrial polarity. Remaining blastocysts were placed in 0.6 M sucrose in holding media for 2 min followed by equilibration in 1.5 M ethylene glycol and 0.5 M sucrose in holding media for 10 min with 0 or 0.1 mM l-AA. Blastocysts were thawed and assessed for re-expansion at 24 and 48 h, then stained with 4′,6-diamidino-2-phenylindole and a TUNEL assay to measure apoptosis. Ten images per stained blastocyst were acquired by confocal microscopy using a 5 µM step size at 40× magnification. Image Pro software was used to measure fluorescence of Nile Red and Mitotracker, and cells stained for TUNEL were analysed by a cell counter plug-in. Blastocyst rate, Nile Red, and Mitotracker data (Table 1) were analysed by 1-way ANOVA and means were separated by Tukey’s HSD. Post-thaw survival and apoptotic levels (Table 1) were analysed as a factorial 2 (SOF and SCF1) by 2 (0 and 0.1 mM l-AA) and means were separated by Tukey’s HSD. Results (Table 1) indicate SCF1 increased blastocyst rate and post-thaw survival and decreased lipid content (P < 0.01) with no effect on mitochondrial polarity. Post-thaw, l-AA (Table 1) increased survival (P < 0.05) but had no effect on apoptosis. The SCF1 medium increases development and lowers lipid content, whereas l-AA may lower reactive oxygen species to increase cryotolerance. Table 1. Effect of media on development, lipid content, and mitochondrial polarity (top part), and of media and l-AA on post-thaw survival and apoptosis (lower part)

129 EFFECT OF SERUM SUPPLEMENTATION ON AMP-ACTIVATED PROTEIN KINASE (AMPK) ACTIVITY AND LIPID METABOLISM OF IN VITRO-CULTURED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 156
Author(s):  
S. Prastowo ◽  
F. Rings ◽  
D. S. Wondim ◽  
E. Tholen ◽  
C. Looft ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Mitochondrial Biogenesis ◽  
Reactive Oxygen ◽  
Rna Isolation ◽  
Mitochondrial Activity ◽  
Oxygen Species ◽  
Bovine Embryos ◽  
Fluorescent Intensity ◽  
Ampk Activity

A major problem of embryos cultured in vitro with serum is cytoplasmic lipid accumulation resulting in lower cryotolerance compared with those derived from in vivo or in the absence of serum. AMPK is known as a master regulator of lipid, glucose, and protein metabolism in mammalian cells. Moreover, it has been reported as controller of acetyl-CoA carboxylase α (ACC), the gene responsible for lipid synthesis, and associated with mitochondrial biogenesis and activities in response to oxidative stress. In the present study we aimed to investigate the regulation of AMPK during serum supplementation in vitro. For this, bovine embryos were produced in vitro in SOF media supplemented with oestrous cow serum or fatty acid–free BSA as a system without serum. Triplicate pools (each 10 blastocysts) from each group were used for RNA isolation using Arcturus®PicoPure®RNA Isolation Kit (Life Technologies, USA). Reverse transcription was performed using a combination of Oligo(dT)23 and random primers. Quantification of AMPK catalytic α1 (AMPKA1), ACC, peroxisome proliferator-activated receptor gamma coactivator 1 α (PGC1A), and sterol regulatory element binding transcription factor 2 (SREBP2) transcripts were performed using ABI PRISM® 7000 SDS system (Applied Biosystems, Foster City, CA, USA) using GAPDH as internal control. Normalized log-transformed transcript amount data were statistically analysed using t-test. In addition, AMPK protein was detected by immunofluorescence, mitochondrial activity by MitoTracker® Red (Invitrogen, Carlsbad, CA, USA), and reactive oxygen species by H2DCFDA molecular probe (Life Technologies, USA), and fluorescent intensity signals were visualised under confocal laser scanning microscopy LSM 710 (Carl Zeiss, Germany). Results showed that the expression of AMPKA1, PGC1A, a mitochondrial biogenesis protein, and SREBP2, a regulator of lipid oxidation, were found to be lower (0.4-, 0.2-, and 0.7-fold, respectively; P < 0.05) in blastocysts derived from cultured with serum compared to without serum. By contrast, ACC was up-regulated in blastocysts cultured with serum by 1.8-fold (P < 0.05) compared to without serum. In comparison to blastocyst cultured without serum, a reduced fluorescent intensity was observed in AMPKA1 protein and mitochondrial activity in blastocyst cultured with serum. The presence of serum was also found to be involved in increasing reactive oxygen species accumulation in embryos cultured with serum. The reduced level of AMPK leads to increased ACC and subsequently enhanced conversion of fatty acids into lipid, which is associated with reduced mitochondrial biogenesis protein, elevated reactive oxygen species level, and reduced lipid oxidation by suppression of SREBP2. In conclusion, the presence of serum in in vitro culture environment affected the AMPK activity and thereby genes associated with lipid metabolism in early bovine embryos.

33 POST-THAW VIABILITY OF BOVINE EMBRYOS PRODUCED IN VITRO FOLLOWING TREATMENT WITH ASCORBIC ACID, DITHIOTHREITOL, AND CASPASE-3 INHIBITOR DURING CRYOPRESERVATION

2016 ◽  
Vol 28 (2) ◽  
pp. 146
Author(s):  
E. L. Carrascal-Triana ◽  
A. M. Zolini ◽  
A. Ruiz ◽  
J. M. Penitente-Filho ◽  
C. A. A. Torres ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Caspase 3 ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Freezing Medium ◽  
Fetal Bovine ◽  
Effect Of Treatment ◽  
The Mean ◽  
Hatching Rates

The aim of the present experiments was to determine whether treatment with the antioxidants ascorbic acid and dithiothreitol, or an inhibitor of caspase-3, Z-DEVD-FMK, during cryopreservation could improve the cryotolerance of bovine embryos produced in vitro. For all experiments, bovine embryos were produced in vitro using abattoir-derived ovaries. At Day 7 after insemination, blastocyst and expanded blastocyst stage embryos were harvested and subjected to controlled-rate freezing following equilibration for 8 to 10 min in freezing medium [Hepes-TALP (Parrish et al. 1986) plus 1.5 M ethylene glycol and 0.5 M sucrose] with treatments as described below. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. For Experiment 1, embryos (n = 578) were equilibrated in freezing medium containing 0, 0.1, 0.3, or 0.5 mM ascorbic acid. For Experiment 2, embryos (n = 243) were equilibrated in freezing medium containing 0, 50, 100, or 200 μM dithiothreitol. For Experiment 3, embryos (n = 227) were equilibrated in freezing medium containing 0, 50, 100, or 200 μM Z-DEVD-FMK. Embryos frozen in freezing medium containing ascorbic acid had increased (P < 0.05) re-expansion and hatching rates at 24, 48, and 72 h compared with embryos not treated with ascorbic acid (Table 1). The optimal concentration of ascorbic acid for post-thaw cryosurvival was 0.1 mM. In particular, embryos treated with 0.1 mM ascorbic acid during cryopreservation had increased (P < 0.05) re-expansion rates at 24, 48, and 72 h, as well as hatching rates at 48 and 72 h, compared with control-treated embryos (Table 1). There was no effect of treatment with dithiothreitol or Z-DEVD-FMK on re-expansion or hatching rates at 24, 48, or 72 h after thaw. In conclusion, addition of ascorbic acid to freezing medium improves the cryosurvival of bovine embryos produced in vitro. Further research is necessary to determine whether treatment with ascorbic acid can increase pregnancy rates. Table 1.Effect of ascorbic acid during cryopreservation of bovine embryos (%, means ± standard error of the mean)

scholarly journals 119CRYOPRESERVATION OF BIOPSIED BOVINE EMBRYOS PRODUCED IN VITRO USING ARABINOGALACTAN AND 1.5M ETHYLENE GLYCOL

2004 ◽  
Vol 16 (2) ◽  
pp. 182
Author(s):  
B. Shangguan ◽  
N. Yang ◽  
R. Vanderwal ◽  
M.D. Darrow
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Field Trials ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Ag Addition ◽  
Freezing Medium ◽  
Frozen Embryos

Arabinogalactan (AG) in combination with 1.5M ethylene glycol (EG) has been used successfully in cryopreserving biopsied in vivo bovine embryos (Darrow, 2002 Theriogenology 57(1), 531). This study was undertaken to investigate the efficiency of AG addition in a freezing medium (FM) to cryopreserve biopsied bovine embryos produced in vitro (IVP). Blastocysts of grade 1 were collected at Days 7 and 8 post-insemination. After biopsy with a small blade, embryos were transferred to CR1aa medium and cultured for 2 hours (h) before being frozen. In experiment 1, a group of unbiopsied embryos were handled in a manner similar to that used for the biopsied embryos. Embryos were frozen using either 1.5M EG+0.1M sucrose (EG+) (AB Technology, Pullman, WA, USA) or a FM containing 1.5M EG and different concentrations of AG (AG1, 2 and 3, courtesy of AB Technology). Embryos remained in FM for 10 (exp.1), 5 (exp.2), 5 and 10 (exp.3) or 5, 10, and 20 (exp.4) minutes before being loaded into a freezer and cooled down to −35°C at 0.3°C/min. Frozen embryos were thawed (35°C, 20 seconds) and cultured in CR1aa at 38.5°C for 3 days. Embryo survival rates (S%) were recorded at 24, 48 and 72h post-thawing. Data were compared with t-test or ANOVA procedures using SigmaStat 3.0. Results from exp.1 (Table) indicate that biopsied and unbiopsied embryos survived well in EG+ or AG2. While the biopsy procedure did not affect the post-thaw S% of embryos in either FM, no significant differences were observed between embryos frozen with EG+ and AG2 (P=0.055). Reducing or increasing AG concentration in FM by 2-fold (AG1 and 3, respectively) did not significantly affect the post-thaw S% at 24h (EG+, 80.0%, n=133; AG1, 83.3%, n=135; AG2, 71.4%, n=137 and AG3, 75.0%, n=135; P=0.217, exp.2). However, shortened exposure from 10 to 5 minutes to AG2 resulted in an improvement in S% at 24h, from 35.7% (n=80) to 61.4% (n=82, P&lt;0.05; exp.3). When AG1 (=0.5×AG2) was used in the FM the S% at 24h after different exposure times was not significant (5 minutes, 77.8%, n=179; 10 and 20 minutes, 66.7%, n=179 and 183; P=0.472, exp.4). This study demonstrates that addition of AG to the FM effectively sustains the viability of biopsied IVP embryos during freezing and any potential harmful impact of AG on embryo survival can be minimized by reducing AG concentration or the time of embryo exposure to AG prior to freezing. Further studies are needed to determine optimal AG concentration. Currently, field trials are underway to evaluate the ability of AG medium to promote pregnancies from frozen, biopsied IVP embryos. Table 1 Post-thaw survival rates of biopsied IVP embryos frozen in ethylene glycol with sucrose (EG+) and a FM containing arabinogalactan (AG2). Data are means±SEM

90 INTRACELLULAR REACTIVE OXYGEN SPECIES IN BOVINE EMBRYOS CULTURED IN VITRO WITH CATALASE UNDER VARIOUS OXYGEN TENSIONS

2012 ◽  
Vol 24 (1) ◽  
pp. 157 ◽  
Author(s):  
N. A. S. Rocha ◽  
B. C. S. Leão ◽  
M. F. Accorsi ◽  
G. Z. Mingoti
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Embryonic Development ◽  
Embryo Development ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Bovine Embryos ◽  
Intracellular Ros ◽  
Oxygen Tensions

The production of reactive oxygen species (ROS), such as superoxide anion (O2–), hydroxyl radical (OH–) hydrogen peroxide (H2O2) and organic peroxides, is a normal process that occurs in the cellular mitochondrial respiratory chain. The high oxygen tension in in vitro culture (IVC) conditions is believed to induce oxidative stress, as a result of increase in ROS intracellular production, that can be correlated with embryonic developmental failure. Supplementation with antioxidants during IVC appears to increase the resistance of bovine embryos to the oxidative stress and consequently improve embryo development. The aim of this study was to evaluate the effects of antioxidant (catalase) and oxygen tensions during IVC on the embryonic development and quantification of intracellular ROS. Cumulus–oocyte complexes (COC; n = 337) were in vitro matured (IVM) in TCM-199 supplemented with 0.2 mM pyruvate, 25 mM sodium bicarbonate, 75 μg mL–1 gentamicin, 10% FCS and hormones for 24 h at 38.5°C and 5% CO2 in air. Then they were fertilized and the presumptive zygotes were cultured in SOFaa medium without (control) or with 100 UI catalase (CAT) for 7 days at 38.5°C in one of 2 types of humified atmosphere: 5% CO2 in air (≈20% O2) or in gaseous mixture (7% O2, 5% CO2 and 88% N2). The cleavage rate was evaluated at 72 hours post-insemination (hpi) and the embryonic development at 168 hpi. At this time, the level of intracellular ROS was measured using the fluorescent probe 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Invitrogen, Oakville, Canada), at 5 μM (Bain et al. 2011 Reprod. Fertil. Dev. 23, 561–575). Stained embryos were imaged immediately using an inverted microscope and analysed by Q-Capture Pro image software (QImaging, Surrey, BC, Canada). The signal intensity values of embryos were subtracted by the average of backgrounds in the images. Embryo development was analysed by chi-squared test and means of the intensity of fluorescence were compared by ANOVA followed by Tukey's test (P < 0.05). The cleavage rates were 84.04%a (control 20% O2), 77.55%a (CAT 20% O2), 77.03%a (control 7% O2) and 71.83%a (CAT 7% O2). The embryonic development rates were 40.43%a (control 20% O2), 33.67%a (CAT 20% O2), 20.27%b (control 7% O2) and 16.90%b (CAT 7% O2). The fluorescent intensity were 3.9 ± 0.4a (control 20% O2), 1.8 ± 0.2b (CAT 20% O2), 2.7 ± 0.2ab (control 7% O2) and 2.8 ± 0.2ab (CAT 7% O2). Although catalase did not significantly affect blastocyst frequencies (P > 0.05), embryo development was adversely affected by reduced O2 tension (P < 0.05). H2DCFDA staining indicated a significant (P < 0.05) reduction in the levels of intracellular ROS within embryos cultured with catalase under 20% O2 compared with the control group in the same O2 tension. Additionally, a consistent but insignificant reduction in intracellular ROS within embryos cultured under 7% O2 was found. We can conclude that supplementation with catalase to IVC medium at 20% O2 is suitable for lowering intracellular ROS levels in IVP bovine embryos, without lowering the rates of blastocysts production. This finding corroborates with theory that antioxidants are beneficial to embryo quality. Alta Genetics Brazil, Deoxi Biotecnologia.

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

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&gt;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&gt;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)

Effect of pterostilbene on development, equatorial lipid accumulation and reactive oxygen species production of in vitro‐produced bovine embryos

2020 ◽  
Vol 55 (11) ◽  
pp. 1490-1500
Author(s):  
Froylan Sosa ◽  
Salvador Romo ◽  
Michael E. Kjelland ◽  
Horacio Álvarez‐Gallardo ◽  
Sandra Pérez‐Reynozo ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Lipid Accumulation ◽  
Reactive Oxygen Species Production ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Bovine Embryos ◽  
Species Production

Effects of gaseous atmosphere and antioxidants on the development and cryotolerance of bovine embryos at different periods of in vitro culture

Zygote ◽  
2013 ◽  
Vol 23 (2) ◽  
pp. 159-168 ◽  
Author(s):  
Nathália Alves de Souza Rocha-Frigoni ◽  
Beatriz Caetano da Silva Leão ◽  
Ériklis Nogueira ◽  
Mônica Ferreira Accorsi ◽  
Gisele Zoccal Mingoti
Keyword(s):  
Reactive Oxygen Species ◽  
Embryo Development ◽  
Gaseous Mixture ◽  
Intracellular Reactive Oxygen Species ◽  
Oxygen Species ◽  
Bovine Embryos ◽  
Antioxidant Supplementation ◽  
Antioxidant Treatment ◽  
Intracellular Ros

SummaryThis study examined the effects of antioxidant supplementation and O2 tension on embryo development, cryotolerance and intracellular reactive oxygen species (ROS) levels. The antioxidant supplementation consisted of 0.6 mM cysteine (CYST); 0.6 mM cysteine + 100 μM cysteamine (C+C); 100 IU catalase (CAT) or 100 μM β-mercaptoethanol (β-ME) for 3 or 7 days of in vitro culture (IVC). Two O2 tensions (20% O2 [5% CO2 in air] or 7% O2, 5% CO2 and 88% N2 [gaseous mixture]) were examined. After 7 days of antioxidant supplementation, the blastocyst frequencies were adversely affected (P < 0.05) by CYST (11.2%) and C+C (1.44%), as well as by low O2 tension (17.2% and 11.11% for 20% and 7% O2, respectively) compared with the control (26.6%). The blastocyst re-expansion rates were not affected (P > 0.05) by the treatments (range, 66–100%). After 3 days of antioxidant supplementation, the blastocyst frequencies were not affected (P > 0.05) by any of the antioxidants (range, 43.6–48.5%), but they were reduced by low O2 tension (P < 0.05) (52.1% and 38.4% for 20% and 7% O2, respectively). The intracellular ROS levels, demonstrated as arbitrary fluorescence units, were not affected (P > 0.05) by antioxidant treatment (range, 0.78 to 0.95) or by O2 tension (0.86 and 0.88 for 20% and 7% O2, respectively). The re-expansion rates were not affected (P > 0.05) by any of the treatments (range, 63.6–93.3%). In conclusion, intracellular antioxidant supplementation and low O2 tension throughout the entire IVC period were deleterious to embryo development. However, antioxidant supplementation up to day 3 of IVC did not affect the blastocyst frequencies or intracellular ROS levels.

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