Effects of Reactive Oxygen Species and Antioxidants during In vitro Maturation Oocytes and Embryo Development in Pigs

2017 ◽  
Vol 41 (1) ◽  
pp. 17-23
Author(s):  
Won-Hee Lee ◽  
◽  
Ji-Eun Park ◽  
Yong Hwangbo ◽  
Hwa-Young Kim ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Reactive Oxygen ◽  
Oxygen Species

scholarly journals Reactive oxygen species production and redox state in parthenogenetic and sperm-mediated bovine oocyte activation

Reproduction ◽  
2013 ◽  
Vol 145 (5) ◽  
pp. 471-478 ◽  
Author(s):  
S Morado ◽  
P Cetica ◽  
M Beconi ◽  
J G Thompson ◽  
G Dalvit
Keyword(s):  
Reactive Oxygen Species ◽  
Embryo Development ◽  
Redox State ◽  
Reactive Oxygen ◽  
Developmental Competence ◽  
Ros Production ◽  
Oxygen Species ◽  
Oocyte Activation ◽  
Parthenogenetic Activation

The knowledge concerning redox and reactive oxygen species (ROS)-mediated regulation of early embryo development is scarce and remains controversial. The aim of this work was to determine ROS production and redox state during early in vitro embryo development in sperm-mediated and parthenogenetic activation of bovine oocytes. Sperm-mediated oocyte activation was carried out in IVF-modified synthetic oviductal fluid (mSOF) with frozen–thawed semen. Parthenogenetic activation was performed in TALP plus ionomycin and then in IVF-mSOF with 6-dimethylaminopurine plus cytochalasin B. Embryos were cultured in IVF-mSOF. ROS and redox state were determined at each 2-h interval (7–24 h from activation) by 2′,7′-dichlorodihydrofluorescein diacetate and RedoxSensor Red CC-1 fluorochromes respectively. ROS levels and redox state differed between activated and non-activated oocytes (P<0.05 by ANOVA). In sperm-activated oocytes, an increase was observed between 15 and 19 h (P<0.05). Conversely, in parthenogenetically activated oocytes, we observed a decrease at 9 h (P<0.05). In sperm-activated oocytes, ROS fluctuated throughout the 24 h, presenting peaks around 7, 19, and 24 h (P<0.05), while in parthenogenetic activation, peaks were detected at 7, 11, and 17 h (P<0.05). In the present work, we found clear distinctive metabolic patterns between normal and parthenogenetic zygotes. Oxidative activity and ROS production are an integral part of bovine zygote behavior, and defining a temporal pattern of change may be linked with developmental competence.

277 EFFECTS OF ERYTHROCYTE AND ERYTHROCYTE HEMOLYSATE ON BOVINE PREIMPLANTATION EMBRYO DEVELOPMENT IN VITRO UNDER REACTIVE OXYGEN SPECIES CONDITION

2010 ◽  
Vol 22 (1) ◽  
pp. 295
Author(s):  
A. Ideta ◽  
K. Tsuchiya ◽  
Y. Nakamura ◽  
M. Urakawa ◽  
M. Murakami ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Embryo Development ◽  
Reactive Oxygen ◽  
Hemoglobin Concentration ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Oxygen Species ◽  
Preimplantation Embryo Development ◽  
Presence And Absence

Reactive oxygen species (ROS) damage preimplantation embryos by increasing DNA fragmentation, leading to early embryonic death. Erythrocytes have been shown to protect other cells and tissues against ROS. In mice, erythrocytes were recently found to improve the early development of embryos by their antioxidant effect. The purpose of the present study was to examine the effect of erythrocytes on the in vitro development of bovine IVF embryos in medium supplemented with ROS. COCs were aspirated from ovaries collected from a local slaughterhouse and were cultured for 22 h in TCM-199 containing 5% fetal bovine serum. IVF was performed using an IVF100 (Research Institute for the Functional Peptides, Yamagata, Japan) according to the manufacturer’s instructions. In experiment 1, IVF embryos were cultured in CR1aa medium supplemented with an oxidizing agent, 0.5 mM hypoxanthine and 0.01 U mL-1 xanthine oxidase (HX/XOD), in the presence and absence of erythrocytes (5 × 104, 5× 105, 5×106, and 5 × 107 erythrocytes mL-1). In experiments 2 and 3, the development of embryos under the condition without ROS was assessed in the presence and absence of erythrocytes (5 × 106 erythrocytes mL-1) or erythrocyte hemolysate (hemoglobin concentration of 1.9 g L-1), respectively. At 7 days after in vitro culture, the development to the blastocyst stage of IVF embryos was examined using a stereomicroscope. Data were analyzed using Fisher’s PLSD test and Student’s t-test In experiment 1, the presence of HX/XOD significantly inhibited embryo development to the blastocyst stage in vitro (P < 0.05). The addition of erythrocytes to medium supplemented with HX/XOD markedly improved preimplantation development (Table 1). In experiments 2 and 3, supplementation of erythrocytes or erythrocyte hemolysate promoted the development of embryos to the blastocyst stage (experiment 2: erythrocyte 42.4 ± 3.1%, control 28.5 ± 5.7%, P < 0.1; experiment 3: erythrocyte hemolysate 39.1 ± 3.3%, control 30.2 ± 1.0%, P < 0.1). In conclusion, we suggest that the addition of erythrocytes to culture medium can counteract the negative effects of ROS on embryo development and blastocyst formation. Table 1.Effect of HX/XOD and erythrocyte supplementation on embryo development to blastocyst stage

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.

Sign in / Sign up

Export Citation Format

Share Document