scholarly journals Effect of oxygen tension and antioxidants on the developmental competence of buffalo oocytes cultured in vitro

2021 ◽  
Vol 14 (1) ◽  
pp. 78-84
Author(s):  
Amro M. El-Sanea ◽  
Ahmed Sabry S. Abdoon ◽  
Omaima M. Kandil ◽  
Nahed E. El-Toukhy ◽  
Amal M. Abo El-maaty ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Oxygen Tension ◽  
Cumulus Cell ◽  
Acid Group ◽  
Developmental Competence ◽  
Control Group ◽  
In Vitro Production ◽  
Nuclear Maturation ◽  
Effect Of Oxygen

Aim: Oxidative stress (OS) is one of the major disruptors of oocyte developmental competence, which appears due to the imbalance between the production and neutralization of reactive oxygen species (ROS). Materials and Methods: In Experiment 1, buffalo oocytes were in vitro matured, fertilized, and cultured at 38.5°C under 5% CO2 + 20% O2 in standard CO2 incubator (OS) or under 5% O2 + 5% CO2 + 90% N2 (Multi-gas incubator, low O2). In Experiment 2, buffalo cumulus oocytes complexes (COCs) were matured in Basic maturation medium (BMM) composed of TCM199+ 10% FCS+ 10 μg/ml FSH+ 50 μg/ml gentamicin (control group) or in BMM supplemented with 50 μM ascorbic acid (ascorbic acid group) or 3.0 mM glutathione (glutathione group) or 10-5 M melatonin (melatonin group) and cultured at 38.5°C under 20% O2 for 24 h. Matured buffalo oocytes in control, ascorbic acid, or melatonin groups were fertilized and zygotes were cultured for 8 days under the same conditions. Results: In both experiments, maturation, cleavage, and blastocyst rates were recorded. Results showed that culture of buffalo oocytes under low O2 (5% O2) significantly increased maturation, cleavage, and blastocyst rates (p<0.05). Meanwhile, under 20% O2, addition of 10-5 M melatonin or 50 μM ascorbic acid to in vitro maturation (IVM) medium significantly improved cumulus cell expansion, nuclear maturation rates of buffalo oocytes (p<0.05), and increased cleavage and blastocyst rates (p<0.05). Conclusion: About 5% O2 is the optimum condition for in vitro production of buffalo embryos, and addition of 10-5 M melatonin to IVM medium for oocytes cultured under 20% O2 could alleviate the adverse effect of high oxygen tension and increased embryo yield.

329 SUPPRESSION OF BOVINE OOCYTE IVM BY SERUM AND FSH DEPRIVATION OR BY α-AMANITIN: EFFECT ON FERTILIZATION AND EMBRYO DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 272
Author(s):  
K. Kananen-Anttila ◽  
M. Eronen ◽  
J. Matilainen ◽  
M. Kallio ◽  
J. Peippo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Embryo Cleavage ◽  
Bovine Oocytes ◽  
Sperm Aster

We have studied the effect of suppressed IVM on the developmental competence of bovine oocytes, aiming at elucidating the importance of cytoplasmic maturation in fertilization and embryo development. Six replicates of abattoir-derived oocytes were randomly divided into three IVM groups. Control (n = 950): TCM-199 with glutamax-I (Gibco, Grand Island, NY, USA), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin, 50 ng mL−1 FSH, and 10% fetal bovine serum (FBS) (Gibco); Serum+FSH-free (n = 944): same as control but without FSH and FBS; α-amanitin (n = 977): same as control but with 10 μg mL−1 α-amanitin. Nuclear maturation of oocytes was studied 24 h after the onset of IVM, the formation of sperm aster structure 10 hours post-insemination (hpi) and the formation of pronuclei 20 hpi. Sperm aster was visualized with β-tubulin antibody (modified from Navara et al. 1999 Dev. Biol. 162, 29–40). Presumptive zygotes were cultured until Day 7 in modified SOFaaci + 4 mg mL−1 fatty acid-free BSA in 5% O2. Cumulus cell expansion was seen only in the control group. The results of nuclear maturation, fertilization, and embryo development are summarized in Table 1. Serum and FSH deprivation did not have a statistically significant effect on the parameters studied (vs. control). α-amanitin exposure during IVM reduced nuclear maturation, fertilization, and Day 3 embryo cleavage vs. control, and resulted in total blockage of Day 7 blastocyst development. The treatment groups had significantly smaller mean diameters of male pronuclei (control: 14 ± 0.6 μ­m; serum+FSH-free: 12 ± 0.5 μ­m, P < 0.05; α-amanitin: 10 ± 0.6 μ­m, P < 0.001) and sperm asters (control: 86 ± 4 μ­m; serum+FSH-free: 82 ± 4 μ­m, P < 0.01; α-amanitin: 49 ± 7 μm, P < 0.001) (nonparametric Kruskall Wallis and Mann-Whitney U tests) vs. control group. Despite reduction in pronucleus and sperm aster diameter, serum and FSH deprivation during IVM did not affect in vitro developmental competence of bovine oocytes, suggesting a need for re-evaluation of the components of IVM. α-Amanitin exposure in IVM disturbed nuclear maturation, fertilization, and embryo development, indicating the essence of early transcription. Table 1. Average percentages ± (n) for nuclear maturation, fertilization (min two pronuclei), embryo cleavage, and blastocyst development

Effect of oxygen tension and serum during IVM on developmental competence of bovine oocytes

2010 ◽  
Vol 22 (7) ◽  
pp. 1074 ◽  
Author(s):  
Michele M. Pereira ◽  
Marco A. Machado ◽  
Fernanda Q. Costa ◽  
Raquel V. Serapiao ◽  
Joao H. M. Viana ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Total Cell Number ◽  
Effect Of Oxygen ◽  
Vitro Production ◽  
Bovine Oocytes

With an aim to improve the in vitro production of bovine embryos, the present study investigated the effect of serum and oxygen tension during IVM on oocyte developmental competence. Four experimental groups were evaluated: G1, 10% oestrus cow serum (OCS) with 20% O2; G2, 0.1% polyvinyl alcohol (PVA) with 20% O2; G3, 10% OCS with 5% O2; and G4, 0.1% PVA with 5% O2. The proportion of MII oocytes, blastocyst rates and total cell number were not affected (P > 0.05) when the OCS was replaced with PVA under 5% O2, whereas a higher (P < 0.05) blastocyst rate and total cell number were found with OCS compared with PVA under 20% O2. The apoptosis index was lower in blastocysts from oocytes matured with PVA under 5% O2 (G4) compared with other groups (G1, G2 and G3), but no differences (P > 0.05) were found in maturation and blastocyst rates. Significant differences were found in the amount of specific transcripts in oocytes matured under different conditions. In conclusion maturation with PVA and 5% O2 provides an efficient in vitro culture condition for the maturation of bovine oocytes.

scholarly journals The influence of ascorbic acid on in vitro maturation of canine oocytes

2016 ◽  
Vol 73 (2) ◽  
pp. 230
Author(s):  
Anamaria Jeni Pernes ◽  
Ileana Miclea ◽  
Marius Zahan ◽  
Vasile Miclea ◽  
Delia Orlovschi ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Meiotic Maturation ◽  
Control Group ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Oocyte Meiotic Maturation ◽  
Metaphase I

Abstract It is known that L-ascorbic acid (vitamin C) can modulate many biochemical processes intracellularly or extracellularly as antioxidant. The aim of the present study was to examine the effects of media supplementation with ascorbic acid on canine oocyte meiotic maturation, viability and the cumulus cell expansion. Various concentrations of ascorbic acid supplemented in in vitro maturation (IVM) media were tested. Canine oocyte was exposed to different levels of ascorbic acid (0, 50, 150, 250, 500, 750µM). Cumulus expansion, meiotic maturation and degeneration of oocytes were assessed 72 h after in vitro culture. As results, on the group treated with 250µM ascorbic acid was a significant difference compared to the control group on nuclear maturation in stages metaphase I (MI) and metaphase II (MII) (26.98% vs. 6.00%). The groups treated with 50, 150, 250, 500µM had an increase in stage (GVBD), and a significant decrease of degenerate-undefined oocytes compared with the control (23.31%, 18.85%, 13.41% vs 40.80). Concentration 750µM had similar effect to that in the control group. The groups treated with 50, 150, 250, 500µM had an increase in meiosis resumption(GVBD), metaphase I (MI) and metaphase II (MII) with the best result in the group treated with 250 µM ascorbic acid.

Improvement of the developmental competence of canine oocyte using caffeine supplementation during IVM at different maturation time

Zygote ◽  
2018 ◽  
Vol 26 (2) ◽  
pp. 162-167 ◽  
Author(s):  
Mohamed Fathi ◽  
A. Salama ◽  
Magdy R. Badr
Keyword(s):  
Developmental Competence ◽  
Control Group ◽  
Free Medium ◽  
Maturation Time ◽  
Fluid Medium ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Oviductal Fluid

SummaryThe aim of the current study was to investigate the effect of caffeine supplementation during in vitro maturation (IVM) for different maturation times on the developmental potential of canine oocytes recovered from ovariohysterectomized bitches. The recovered cumulus–oocytes complexes were in vitro matured for 72 h. Here, 10 mM caffeine was added to the maturation medium for different incubation times (caffeine from 0–72 h maturation, caffeine for the first 24 h of maturation only, caffeine addition from 24 to 48 h maturation time, caffeine addition from 48 to 72 h maturation or in caffeine-free medium, control group). The matured oocytes were in vitro fertilized using frozen–thawed spermatozoa. The presumptive zygotes were in vitro cultured in synthetic oviductal fluid medium for 5 days. The results showed that both maturation and fertilization rates were significantly higher (P ˂ 0.05) using caffeine-treated medium for the first 24 h of maturation compared with the control and other two groups of caffeine treatment (from 24 to 48 h and from 48 to 72 h), whereas use of caffeine-treated medium for a 0–72 h incubation time did not affect these rates (P > 0.05). Interestingly, the matured oocytes in caffeine-supplemented medium for the first 24 h or from 0–72 h showed a significant (P ˂ 0.05) increase in the total number of cleaved embryos compared with the control group. In conclusion, supplementation of the maturation medium with 10 mM caffeine for the first 24 h of maturation or during the whole maturation time (0–72 h) improved nuclear maturation and subsequent embryo development preimplantation following in vitro fertilization.

247 EFFECTS OF CYSTEINE IN IVM MEDIA ON IN VITRO MATURATION UNDER LOW OXYGEN TENSION, IN VITRO FERTILIZATION, AND IN VITRO CULTURE OF PORCINE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 203
Author(s):  
N. V. Linh ◽  
D. N. Q. Thanh ◽  
M. Ozawa ◽  
B. X. Nguyen ◽  
K. Kikuchi ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Blastocyst Stage ◽  
Low Oxygen ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Low Oxygen Tension ◽  
High O2 ◽  
Group 1

Cysteine is considered to promote male pronuclear (MPN) formation in porcine through oocyte glutathione (GSH) synthesis (Yoshida et al. 1993 Biol. Reprod. 49, 89–94). The GSH has an important role in providing cells with a redox state and in acting to protect cells from toxic effects of oxidative damage (Meister et al. 1976 AM Rev. Biochem. 45, 559–604). However, such previous investigations were carried out under high O2 tension (20% O2) incubation conditions. Here we simply study IVM-IVF-IVC competence of porcine oocytes matured in IVM media supplemented with cysteine of different concentrations under low oxygen tension (5% O2). Cumulus–oocyte complexes (COCs) from prepubertal gilts were collected, matured, and fertilized in vitro according to Kikuchi et al. (2000 Biol. Reprod. 66, 1033–1041). COCs were cultured in IVM medium supplemented with 0 (Group 1; control), 0.05 (Group 2), 0.1 (Group 3), 0.2 (Group 4), and 0.6 mm (Group 5) cysteine under low oxygen tension. Nuclear maturation of oocytes, fertilization status, and number of cells in resultant embryos were assessed with orcein staining; also, the GSH content of IVM oocytes was measured by the method described by Ozawa et al. (2002 Reproduction 124, 683–689). Maturation rates of Groups 1–5 were 68.2 � 3.2, 70.6 � 7.7, 69.7 � 15.9, 75.9 � 7.7, and 68.8 � 8.0%, respectively, indicating no difference in maturation competence among the groups (P > 0.05 by ANOVA). The rates of sperm penetration, MPN formation (95.9 � 2.4, 100 � 0, 92.8 � 4.7, 94.0 � 4.1, and 92.4 � 2.7%, respectively), monospermy, and even blastocyst rates after 6 days of IVC were not different among the groups (P > 0.05 by ANOVA). Moreover, the cell numbers of blastomeres in blastocysts (38.68 � 3.5, 40.1 � 3.1, 37.5 � 3.0, 36.2 � 3.3, and 43.8 � 4.0, respectively) were uniformly the same among the groups (P > 0.05 by ANOVA). However, GSH content of IVM oocytes increased significantly (P < 0.05 by ANOVA) as the concentration of cysteine increased (12.2 � 0.6, 14 � 0.8, 15.1 � 0.5, 16.4 � 0.4, and 16.4 � 0.5 pmol/oocyte, respectively). The GSH level of oocytes in Group 1 (control) seems to be higher than that reported by Aberydeera et al. (1998 Biol. Reprod. 58, 213–218), who matured porcine oocytes under high O2 tension. This may reflect the effect of low O2 tension and explain the same developmental rate to the blastocyst stage as that of oocytes matured in the media supplemented with cysteine in this study. In conclusion, an addition of 0.05–0.6 mm cysteine during IVM, under 5% O2 tension, of porcine oocytes significantly increased intracellular GSH synthesis according to its concentration. However, it had no promoting effects on nuclear maturation, fertilization, male pronucleus formation, and subsequent embryonic development to the blastocyst stage. Thus, O2 tension during IVM of oocytes is suggested to be important for the in vitro production of porcine blastocysts.

282 DIFFERENT CONCENTRATIONS OF FORSKOLIN FOR MEIOSIS BLOCK AND TO IMPROVE IN VITRO PRODUCTION OF BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 230
Author(s):  
D. Paschoal ◽  
R. Maziero ◽  
M. Sudano ◽  
M. Guastali ◽  
L. Crocomo ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Least Squares ◽  
Control Group ◽  
In Vitro Production ◽  
Intact Cells ◽  
Significance Level ◽  
Nuclear Maturation ◽  
Oocyte Meiosis ◽  
Group Control

The inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. It was suggested that the inhibition of spontaneous nuclear IVM might allow for more time to accumulate the molecules important for embryonic development. The objective of this work was to evaluate blocking oocyte meiosis with the addition of forskolin. Slaughterhouse-derived bovine Zebu ovaries were collected and carried to the laboratory. Oocytes (n = 584) with at least 3 intact layers of cumulus cells and homogeneous cytoplasm were selected for IVM. The oocytes were transferred to drops of TCM 199 plus 10% FCS and hormones. The oocytes remained in IVM medium in 3 different concentrations of forskolin (6886), 0.1, 0.05, 0.025 mM, and a control group (withouth forskolin), for 6 h. Then they were maturated for an additional 18 h in forskolin-free medium. The first period above was an attempt to block (Block) and the second to resume (Res) the oocyte meiosis. The oocytes were incubated in a humidified atmosphere with 5% CO2 at 38.5°C in an air incubator. The oocytes were assessed for the stage of nuclear maturation, to see if they were in M II. Then oocytes were in vitro fertilized (IVF) with frozen Nelore bull semen (Bos taurus indicus). Presumptive zygotes (20–30/group) were cultured in SOFaa (synthetic oviducal fluid) supplemented with 5 mg mL–1 of BSA; the embryos were kept in an incubator with 5% CO2, 5% O2, and 90% N2 at 38.5°C and absolute humidity. On Day 7 (Day 0 = IVF) the blastocyst, the number of viable cells, and apoptosis rate (terminal deoxynucleotide transferase uridine nick-end labelling) were observed. Data were analysed with ANOVA using SAS PROC GLM (SAS Inst. Inc., Cary, NC, USA). Sources of variation in the model, including treatment and replication, were respectively considered fixed and random effects. If ANOVA was significant, the contrasts of means were performed using the least-squares difference. Data are presented as the mean and the standard error of least-squares. For all analyses, we used a significance level of 5%. No differences were observed for the stage of nuclear maturation of the oocyte (N = 336; control: 67.7 ± 8.3; F 0.025 mM, Block/Res: 67.7 ± 8.9; F 0.05 mM, Block/Res: 65.9 ± 9.8; F 0.1 mM, Block/Res: 50.2 ± 8.9), the blastocyst rate (N = 584; Control: 36.7 ± 3.7; F0.025 mM, Block/Res: 32.6 ± 3.7; F0.05 mM, Block/Res: 29.2 ± 3.7; F0.1 mM, Block/Res: 25.1 ± 3.7), and total number of intact cells (N = 10–15 embryos/group; Control:140.1 ± 13.0; F0.025 mM, Block/Res: 129.9 ± 13.0; F0.05 mM, Block/Res: 139.0 ± 13.0; F0.1 mM, Block/Res: 104.4 ± 13.0; P > 0.05). However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (N = 10–15 embryos/group): Control: 12.1 ± 2.5a; F 0.025 mM, Block/Res: 12.9 ± 2.5a; F0.05 mM, Block/Res: 13.5 ± 2.5a; F 0.1 mM, Block/Res: 30.2 ± 2.5b (P < 0.05). We conclude that all the experimental groups reached the stage of M II after the addition of forskolin and the highest concentration of forskolin caused cellular degeneration without harming embryo production on the seventh day.

scholarly journals Porcine follicular fluid derived from > 8 mm sized follicles improves oocyte maturation and embryo development during in vitro maturation of pigs

Zygote ◽  
2020 ◽  
pp. 1-6
Author(s):  
Ji-Eun Park ◽  
Sang-Hee Lee ◽  
Yong Hwangbo ◽  
Choon-Keun Park
Keyword(s):  
Embryonic Development ◽  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Control Group ◽  
Cumulus Expansion ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Summary The aim of the present study was to investigate the effects of porcine follicular fluid (pFF) from large-sized (LFF; >8 mm in diameter) and medium-sized (MFF; 3–6 mm in diameter) follicles on the maturation and developmental competence of porcine oocytes. Cumulus–oocyte complexes (COCs) were collected from follicles 3–6 mm in diameter. The collected COCs were incubated for 22 h with LFF or MFF (in vitro maturation (IVM)-I stage) and were incubated subsequently for 22 h with LFF or MFF (IVM-II stage). Cumulus expansion was confirmed after the IVM-I stage and nuclear maturation was evaluated after the IVM-II stage. Intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured and embryonic development was evaluated. Relative cumulus expansion and GSH levels were higher in the LFF group compared with in the MFF group after the IVM-I stage (P < 0.05). After the IVM-II stage, the numbers of oocytes in metaphase-II were increased in the LFF group and GSH content was higher in all of the LFF treatment groups compared with in the MFF treatment groups during both IVM stages (P < 0.05). ROS levels were reduced by LFF treatment regardless of IVM stage (P < 0.05). Blastocyst formation and the total numbers of cells in blastocysts were increased in all LFF treatment groups compared with the control group (P < 0.05). These results suggested that pFF from large follicles at the IVM stage could improve nucleic and cytoplasmic maturation status and further embryonic development through reducing ROS levels and enhancing responsiveness to gonadotropins.

355 DEVELOPMENTAL COMPETENCE OF IMMATURE BUBALINE CUMULUS - OOCYTE COMPLEXES VITRIFIED BY THE OPEN PULLED STRAW AND CONVENTIONAL STRAW METHODS

2007 ◽  
Vol 19 (1) ◽  
pp. 293
Author(s):  
A. Sharma ◽  
G. N. Purohit
Keyword(s):  
Morphological Changes ◽  
Developmental Competence ◽  
Control Group ◽  
Equal Concentration ◽  
Nuclear Maturation ◽  
Developmental Capacity ◽  
Phosphate Buffered Saline ◽  
Dose Dependent Increase ◽  
Dose Dependent

The in vitro maturation (IVM), fertilization (IVF), and morphological changes in buffalo cumulus–oocyte complexes (COCs) cryopreserved by ultrarapid freezing using conventional (CON) and open pulled staw (OPS) methods were tested. COCs were cryopreserved using a vitrification solution comprised of Dulbecco&apos;s phosphate-buffered saline+0.5 M sucrose+0.4% BSA and two concentrations (4.5 or 5.5 M) of each cryoprotectant ethylene glycol (EG) and dimethylsulfoxide (DMSO) by either the CON or the OPS method. Vitrified COCs were stored in LN for 7 days and then thawed; morphologically normal COCs were used for IVM (n = 1070) and IVF (n = 933) in 2 separate experiments to record morphological damage of COCs due to vitrification, nuclear maturation 24 h after culture (9 replicates), and fertilization 24 h after insemination (10 replicates). The COCs were matured in vitro in TCM-199 media with hormone supplements and fertilized using TALP-BSA as described previously (Purohit et al. 2005 Anim. Reprod. Sci. 87, 229–239). Freshly collected COCs were separately used for IVM (n = 110) and IVF (n = 130) and kept as controls. The arcsin transformed data of the proportions of oocytes matured or fertilized was compared by Duncan&apos;s new multiple range test. The highest proportion of morphologically normal oocytes was seen in 5.5 M EG with the CON method (94.5%) and the lowest was seen in 4.5 M DMSO with the OPS method (82.4%). At the end of experiment 1, it was apparent that IVM in all vitrification groups was significantly lower (P &lt; 0.05) compared to the control group (66.4%). Among the various vitrification treatments, the highest IVM occurred in 5.5 M EG with the OPS method (39.2%) and the lowest in 4.5 DMSO with the CON method (19.3%). Comparison of both concentrations of EG and DMSO showed that the proportion of COCs attaining Metaphase-II (M-II) increased with increasing concentration of both of the cryoprotectants. However, at equal concentration of EG and DMSO, the proportion of COCs attaining M-II was significantly higher in the OPS method compared to the CON method. In experiment 2, a significantly higher (P &lt; 0.05) IVF was seen for fresh COCs (45.4%) compared to vitrified COCs. Among the vitrification treatments, the highest fertilization was seen in 5.5 M EG with the OPS method (33.6%) and the lowest in 4.5 M DMSO with the CON method (15.17%). A dose-dependent increase in the proportion of oocytes fertilized was seen with increasing concentration of both EG and DMSO [CON: 4.5 M (15.2%), 5.5 M (25.6%); OPS: 4.5 M (21.3%), 5.5 M (27.5%)] in both CON and OPS methods. Comparison of the 2 cryoprotectants revealed that EG was better compared to DMSO.At equal concentrations of EG or DMSO, a significantly higher (P &lt; 0.05) proportion of fertilized oocytes was seen in the OPS method compared to the CON method. It was concluded that vitrification results in some damage to oocytes, with decrease in their subsequent IVM and IVF. Developmental capacity of vitrified buffalo oocytes can be improved by using OPS instead of conventional straws.

49 Developmental Potential of Dromedary Camel Oocytes Vitrified at the Germinal Vesicle Stage: Effects of Different Cryoprotectant Combinations and Cryo-Carriers

2018 ◽  
Vol 30 (1) ◽  
pp. 164
Author(s):  
M. Fathi ◽  
A. R. Moawad ◽  
M. R. Badr
Keyword(s):  
Survival Rates ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Dromedary Camel ◽  
In Vitro Production ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
And Control ◽  
Vitro Production

Cryopreservation of oocyte would be an alternative to overcome the limited availability of dromedary camel oocytes and allow improvements in in vitro production in this species. Our aim was to develop a protocol for vitrification of dromedary camel oocytes at the germinal vesicle (GV) stage using various cryoprotectant combinations and cryo-carriers. In experiment 1, cumulus–ppcyte complexes (COC) obtained at slaughter were equilibrated in a solution composed of 10% ethylene glycol (EG) and 0.25 M trehalose. The oocytes were then exposed for 60 s to vitrification solutions (VS) composed of 20% EG and 20% dimethyl sulfoxide (DMSO; VS1) or 25% EG plus 25% DMSO (VS2) or 25% EG and 25% glycerol (VS3). The COC were then transferred into decreasing concentration of trehalose solution (toxicity test). In experiment 2, COC were randomly divided into 4 groups and vitrified by using straw or open pulled-straw (OPS) or solid surface vitrification (SSV) or cryotop in VS1 or VS2. Following vitrification and warming viable oocytes were matured in vitro for 30 h at 39°C in 5% CO2 in air. Matured oocytes were fertilized in vitro by epididymal spermatozoa of mature male camels and then cultured in modified KSOMaa medium for 7 days. Oocyte viability, maturation, fertilization, and embryo development were evaluated. Data were analysed using one-way ANOVA and t-test. Viability and nuclear maturation rates were significantly lower (P ≤ 0.05) in oocytes exposed to VS3 (44.8% and 34.0%) than those exposed to VS1 (68.2% and 48.0%) and VS2 (79.3% and 56.9%). Although recovery rates were significantly lower (P ≤ 0.05) in oocytes vitrified using SSV or cryotop in either VS1 or VS2 solutions (66.9% to 71.1%) than those vitrified by straws using VS1 or VS2 solutions (86.3% to 91.0%), survival rates were higher in SSV and cryotop groups (90.7% to 94.8%) than straw and OPS (68.2% to 86.5%) groups. Among vitrified groups, maturation and fertilization rates (51.8% and 39.2%, respectively) were the highest in the cryotop-VS2 group. Those values were comparable to those seen in the controls (59.2% and 44.6%, respectively). Cleavage (22.5% to 27.9%), morula (13.2% to 14.5%), and blastocyst (6.4% to 8.5%) rates were significantly higher (P ≤ 0.05) in SSV and cryotop groups than in straws. No significant differences were observed in these parameters between cryotop and control groups. Together, the results show that both vitrification solution and cryodevice affect viability and developmental competence of vitrified/warmed dromedary camel oocytes. We report for the first time that dromedary camel oocytes vitrified at the GV stage have the ability to be matured, fertilized, and subsequently develop in vitro to produce blastocyst embryos at frequencies comparable to those obtained using fresh oocytes.

The effects of cysteine addition during in vitro maturation on the developmental competence, ROS, GSH and apoptosis level of bovine oocytes exposed to heat stress

Zygote ◽  
2011 ◽  
Vol 20 (3) ◽  
pp. 249-259 ◽  
Author(s):  
Hisashi Nabenishi ◽  
Hiroshi Ohta ◽  
Toshihumi Nishimoto ◽  
Tetsuo Morita ◽  
Koji Ashizawa ◽  
...  
Keyword(s):  
Heat Stress ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Formation ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Bovine Oocytes

SummaryIn the present study, we investigated the effects of various concentrations of cysteine (0.0, 0.6, 1.2 and 1.8 mM) added to the maturation medium on nuclear maturation and subsequent embryonic development of bovine oocytes exposed to heat stress (HS: set at 39.5 °C for 5 h, 40.0 °C for 5 h, 40.5 °C for 6 h, and 40.0 °C for 4 h versus 38.5 °C for 20 h as the control group). This regime mimicked the circadian rhythm of the vaginal temperature of lactating dairy cows during the summer season in southwestern Japan. Moreover, we also evaluated the oocyte's reactive oxygen species (ROS) and glutathione (GSH) levels and the apoptosis levels of the oocytes and cumulus cells in the presence or absence of 1.2 mM cysteine. As a result, HS in the without-cysteine group significantly suppressed (p < 0.05) both the nuclear maturation rate up to the metaphase (M)II stage and the blastocyst formation rate compared with that of the control group. In addition, this group showed significantly higher (p < 0.05) ROS levels and significantly lower (p < 0.05) GSH levels than those of the control group. Moreover, the level of TdT-mediated dUTP nick end labelling (TUNEL)-positive cumulus cells in the HS without-cysteine group was significantly higher (p < 0.05) than that of the control group. However, the addition of 1.2 mM cysteine to the maturation medium restored not only the nuclear maturation, blastocyst formation rates and GSH contents, but also increased the ROS and TUNEL-positive levels of the cumulus cells, but not oocytes, to that of the control group. These results indicate that the addition of 1.2 mM cysteine during in vitro maturation (IVM) may alleviate the influence of heat stress for oocyte developmental competence by increasing GSH content and inhibiting the production of oocyte ROS followed by apoptosis of cumulus cells.

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