343 DISTRIBUTION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE AND IN VITRO MATURATION OF PORCINE OOCYTE - CUMULUS COMPLEXES FROM SMALL AND MEDIUM FOLLICLES

2007 ◽  
Vol 19 (1) ◽  
pp. 287
Author(s):  
Y. Ishida ◽  
H. Funahashi
Keyword(s):  
In Vitro Maturation ◽  
Critical Role ◽  
Morphological Characteristics ◽  
Cumulus Cells ◽  
Pentose Phosphate ◽  
G6pd Activity ◽  
Cresyl Blue ◽  
Key Enzyme ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose metabolism through the pentose phosphate pathway (PPP) plays a critical role in meiotic maturation and fertilization. However, the relationship between the distribution of a PPP key enzyme, glucose-6-phosphate dehydrogenase (G6PD), in cumulus–oocyte complexes (COCs) and the in vitro maturation (IVM) of the oocytes is not clear. In the present study, we examined the distribution of G6PD, the morphological characteristics in OCCs derived from small (d2 mm in diameter) and medium (3 to 6 mm in diameter) follicles, and the rate of oocyte maturation. Porcine COCs were collected from small or medium follicles of slaughterhouse ovaries. The COCs were cultured in a maturation medium, BSA-free NCSU37 supplemented with 10% porcine follicular fluid, eCG, and hCG, for 20 h and then in the absence of hormones for 24 h. To determine the distribution of G6PD, the COCs were treated with 13 �M brilliant cresyl blue (BCB) in TL-HEPES-PVA for 90 min. Results from 3–6 replicates were analyzed by ANOVA and Duncan's multiple range test. The mean diameters for COCs collected from small follicles (136.7 µm for the outer zona and 103.1 µm for ooplasm) were significantly less than for those derived from medium follicles (164.1 µm and 122.0 µm, respectively). G6PD activity was detected in the cumulus cells for most of the COCs derived from medium follicles, but it was not significantly different from that of COCs derived from small follicles. In the second group of COCs, very little G6PD activity was found in both the cumulus cells and the oocytes (34.7 ± 11.5% and 18.0 ± 6.7% in COCS derived from small and medium follicles, respectively). After stimulation by eCG and hCG, the percentages of COCS in which G6PD activity was detected in the cumulus cells, but not in the oocytes, were 56.2 ± 23.8% and 72.9 ± 6.1% for small and medium follicles, respectively. The percentage of oocytes at the metaphase II stage (53% and 63.9% in oocytes from small and medium follicles, respectively) was higher for the COCs that showed higher G6PD activity in their cumulus cells. In conclusion, although no statistical differences were detected in the distribution of G6PD between COCs from small and medium follicles, due to a large variation, a higher percentage of mature oocytes seems to be the result of COCs where the G6PD activity is detected in the cumulus cells, but not in the oocyte, during IVM.

scholarly journals Selection of Ovine Oocytes by Brilliant Cresyl Blue Staining

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Liqin Wang ◽  
Jiapeng Lin ◽  
Juncheng Huang ◽  
Jing Wang ◽  
Yuncheng Zhao ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
G6pd Activity ◽  
Blue Staining ◽  
Animal Cloning ◽  
Cresyl Blue ◽  
Deep Blue ◽  
Glucose 6 Phosphate Dehydrogenase

Sheep oocytes derived from the ovaries collected from the slaughterhouse are often used for research onin vitroembryo production, animal cloning, transgenesis, embryonic stem cells, and other embryo biotechnology aspects. Improving thein vitroculture efficiency of oocytes can provide more materials for similar studies. Generally, determination of oocyte quality is mostly based on the layers of cumulus cells and cytoplasm or cytoplasm uniformity and colors. This requires considerable experience to better identify oocyte quality because of the intense subjectivity involved (Gordon (2003), Madison et al. (1992) and De Loos et al. (1992)). BCB staining is a function of glucose-6-phosphate dehydrogenase (G6PD) activity, an enzyme synthesized in developing oocytes, which decreases in activity with maturation. Therefore, unstained oocytes (BCB−) are high in G6PD activity, while the less mature oocytes stains are deep blue (BCB+) due to insuffcient G6PD activity to decolorize the BCB dye.

scholarly journals Activity of key enzymes involved in glucose and triglyceride catabolism during bovine oocyte maturation in vitro

Reproduction ◽  
2002 ◽  
pp. 675-681 ◽  
Author(s):  
P Cetica ◽  
L Pintos ◽  
G Dalvit ◽  
M Beconi
Keyword(s):  
Enzymatic Activity ◽  
Pentose Phosphate Pathway ◽  
In Vitro Maturation ◽  
Specific Activity ◽  
Lipolytic Activity ◽  
Cumulus Cells ◽  
Pentose Phosphate ◽  
Key Enzymes ◽  
Glucose 6 Phosphate Dehydrogenase

Little is known about the metabolic profile of cumulus-oocyte complexes (COCs) during maturation. The aim of this study was to determine the differential participation of enzymatic activity in cumulus cells and the oocyte during in vitro maturation of bovine oocytes, by measuring the activity of key enzymes involved in the regulation of glycolysis (phosphofructokinase), the pentose phosphate pathway (glucose-6-phosphate dehydrogenase) and lipolysis (lipase). COCs were matured in medium 199 plus 10% (v/v) steer serum for 22-24 h at 39 degrees C in 5% CO(2):95% humidified air. Phosphofructokinase, glucose-6-phosphate dehydrogenase and lipase activities were measured in immature and in vitro matured COCs, denuded oocytes and cumulus cells, respectively. Phosphofructokinase and glucose-6-phosphate dehydrogenase activities (enzymatic units) remained constant during in vitro maturation of COCs, but there was a significant decrease in lipase activity (units) (P < 0.05), as activity in cumulus cells decreased significantly (P < 0.05). For the three enzymes studied, enzyme activity (units) remained unchanged in the oocyte during in vitro maturation. Specific activity increased in the oocyte (P < 0.05) and decreased in cumulus cells as a result of maturation (P < 0.05). In cumulus cells, phosphofructokinase was the most abundant of the three enzymes followed by glucose-6-phosphate dehydrogenase and then lipase (P < 0.05), whereas in the denuded oocyte this order was reversed (P < 0.05). Thus, the metabolism of cumulus cells is adapted to control the flow of metabolites toward the oocyte, which maintains its enzymatic activity even when dissociated from cumulus cells during maturation. The high activity of phosphofructokinase in cumulus cells indicates that glucose is metabolized mainly via the glycolytic pathway in these cells. The greater relative activity of glucose-6-phosphate dehydrogenase recorded in the oocyte indicates that glucose uptake could be directed mainly toward the pentose phosphate pathway. The marked lipolytic activity concentrated in the oocyte indicates an active participation in lipid catabolism during maturation.

248 OPTIMAL IN VITRO MATURATION TIME DEPENDS ON MORPHOLOGICAL CHARACTERISTICS OF BOVINE CUMULUS - OOCYTE COMPLEXES

2008 ◽  
Vol 20 (1) ◽  
pp. 203
Author(s):  
F. Morales-Pliego ◽  
M. Barceló-Fimbres ◽  
L. S. Amorim ◽  
G. E. Seidel Jr
Keyword(s):  
Significant Interaction ◽  
In Vitro Maturation ◽  
Morphological Characteristics ◽  
Cumulus Cells ◽  
Percoll Gradient ◽  
Maturation Time ◽  
Cell Stage ◽  
Cull Cows ◽  
Cell Embryo

Oocytes with compact cumulus cells are usually chosen for bovine in vitro maturation and IVF experiments, while oocytes that do not have compact cumulus cells are often discarded. In this experiment, we studied the interaction of maturation time and cumulus characteristics. Five cumulus classifications (compact, partially expanded, expanded, denuded, dark-coagulated) were studied using 3 maturation times. A total of 1923 oocytes were aspirated from 3- to 8-mm follicles; matured for 1, 4, or 23 h; and fertilized with sperm from 3 bulls (2 replicates per bull with one replicate using ovaries of cull cows and the other, ovaries of fattened heifers). IVF was done with sperm centrifuged through a 45/90% Percoll gradient. All media were chemically defined (CDM) plus 0.5% fatty acid-free BSA. Sperm were co-incubated with oocytes for 16–18 h; presumptive zygotes were then vortexed to remove cumulus cells and cultured for 7 days (De La Torre-Sanchez et al. 2006 Mol. Reprod. Dev. 18, 585–596). Overall blastocyst production was low, partly due to differences among bulls (P < 0.01) (2.8, 4.5, and 11.3% blastocysts per oocyte for semen of bulls A, B, and C), and partly because some ovaries were from fattened heifers fed a progestin. There was a significant interaction between type of cumulus and maturation time for development to the 8-cell stage (P < 0.01) and blastocysts (P < 0.07), but not for cleavage (data not presented). From Table 1, it is clear that 1 or 4 h is insufficient maturation time for oocytes from all cumulus classifications except for expanded cumulus. For that category, 1 h maturation resulted in blastocyst production equivalent to oocytes with compact cumulus matured 23 h. Standard timing of 23 h maturation was suboptimal for oocytes with expanded cumulus. For oocytes with compact cumulus, 23 h maturation was superior to shorter times (P < 0.05); oocytes with partially expanded cumulus cells responded similarly to those with compact cumulus except for a higher percent of 8-cell embryos with 23 h maturation. While 8-cell embryo production was above 40% per oocyte for all groups with 23 h of maturation, blastocyst production was very low for oocytes with dark cumulus, and also low when oocytes were denuded or had expanded cumulus. In conclusion, while 23 h maturation was optimal for oocytes with compact or partially expanded cumulus, similar blastocyst rates were obtained with oocytes with expanded cumulus if maturation time was shortened to 1 h. Table 1. Mean percentage development of oocytes to 8-cell and blastocyst stages

340 EFFECT OF GLUCOSE ON THE MORPHOLOGY OF GERMINAL VESICLE AND MEIOTIC PROGRESSION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM

2007 ◽  
Vol 19 (1) ◽  
pp. 285
Author(s):  
H. Funahashi ◽  
T. Koike
Keyword(s):  
Critical Role ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Defined Medium ◽  
Pentose Phosphate ◽  
Chemically Defined Medium ◽  
Dibutyryl Camp ◽  
Germinal Vesicle Breakdown ◽  
Maturation Medium ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose metabolism through the pentose phosphate pathway (PPP) seems to play a critical role in meiotic resumption in mouse oocytes (Downs et al. 1998 Biol. Reprod. 58, 1084–1094). However, the role is not clear in porcine oocytes. In the present study, we examined whether glucose affects morphological change of germinal vesicles and the resumption of meiosis in porcine oocytes in a chemically defined medium. In the first experiment, porcine cumulus–oocyte complexes (COCs) were collected from 3–6-mm follicles of slaughterhouse ovaries and cultured in a chemically defined medium, mNCSU37-PVA with/without 5.55 mM glucose in the presence of eCG, hCG, and dibutyryl cAMP for 20–22 h and then in the absence of eCG, hCG, and dibutyryl cAMP for 24 h. In the second experiment, 5.55 mM glucose in the maturation medium was replaced with the same concentration of Na pyruvate. In the third experiment, the PPP inhibitor 6-aminonicotinamide (6-AN) was added to the maturation medium at various concentrations (0, 10, 50, and 100 �M). To determine the activity of glucose-6-phosphate dehydrogenase (G6PD), OCCs were fixed, blocked, and treated with anti-G6PD polyclonal antibody and the secondary antibody labeling a fluorescent material. Results from 3–5 replicates were analyzed by ANOVA and Duncan&apos;s multiple range test. When OCCs were cultured in glucose-free chemically defined maturation media, regardless of the presence of hormones and dibutyryl cAMP, germinal vesicle breakdown (GVBD) of oocytes was inhibited (10.0&ndash;21.3&percnt;), as compared with OCCs cultured in the presence of glucose and hormones (91.4&ndash;92.0&percnt;). In a majority of oocytes in which GVBD was inhibited, the arrest occurred at the GV-I stage. When OCCs were cultured in maturation media in which glucose was replaced with Na pyruvate, GVBD was not inhibited any more than in control samples that were cultured in the presence of glucose (97.4&percnt; vs. 97.1&percnt;). However, the incidence of oocytes developing to the metaphase II stage was significantly lower in this condition than in controls (4.8&percnt; vs. 49.9&percnt;, respectively). A majority of the oocytes were at the metaphase I stage (86.0&percnt; vs. 45.5&percnt; in controls). The presence of 6-AN in maturation media significantly inhibited GVBD of oocytes (77.3, 29.0, 7.4, and 8.4&percnt; at 0, 10, 50, and 100 &micro;M, respectively) and arrested the oocytes at the GV-I stage. Immunocytochemistry with anti-G6PD demonstrated the activity of G6PD in cumulus cells of OCCs. In conclusion, these results demonstrate that glucose plays a critical role in the release of porcine oocytes arrested at the GV-I stage, probably through PPP of cumulus cells. The current results also suggest the possibility of gluconeogenesis in porcine OCCs when glucose in maturation media was replaced with Na pyruvate.

scholarly journals In vitro maturation of collared peccary (Pecari tajacu) oocytes after different incubation times

2018 ◽  
Vol 38 (9) ◽  
pp. 1863-1868 ◽  
Author(s):  
Alana A. Borges ◽  
Maria V.O. Santos ◽  
Luiza B. Queiroz Neta ◽  
Moacir F. Oliveira ◽  
Alexandre R. Silva ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Morphological Characteristics ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Fisher Exact Test ◽  
First Polar Body ◽  
Collared Peccary ◽  
Culture Environment

ABSTRACT: Oocyte in vitro maturation (IVM) is the first step of the in vitro reproductive technologies that enables mature oocytes to be generated ex vivo and after used for embryo production. In this sense, the establishment of culture environment, as oocyte incubation time, is essential for the success of the IVM. Therefore, the study was carried out to investigate the relationship between the meiotic potential and the IVM times of collared peccary oocytes, wild mammals of great commercial and ecological interest. Thus, ovaries were collected of females derived from captivity and transported to the laboratory within 1 hour of slaughtering. The oocytes derived from follicles (3-6mm in diameter) were recovered by aspirated and sliced. Good quality oocytes (evenly granulated cytoplasm with a least one layer of surrounding cumulus cells) were selected and subjected to culture in TCM 199 supplemented with 10µg/mL FSH, 10% FBS and 100µM cysteamine at 38.5°C, 5% CO2 and maximum humidity for 24 or 48 hours. After the incubation period, the nuclear status, the presence of first polar body and the expansion of cumulus cells of oocytes were assessed. The data obtained were analyzed by Fisher exact test (P<0.05). A total of four sessions (2-3 females per session) were performed, resulting in eighteen aspirated and sliced ovaries with normal morphological characteristics. An oocyte recovery rate of about 83.1% (59/71) was obtained with 3.3 oocytes/ovary and 2.3 viable oocytes/ovary. After different incubation times, differences (P<0.05) were observed in 24 and 48 hours for expansion of the cumulus cells (38.1% vs. 100%), presence of first polar body (52.4% vs. 90.5%) and nuclear status in second metaphase (19.0% vs. 76.2%), respectively. In conclusion, 48 hours is suitable time for the in vitro maturation of oocytes derived from collared peccaries when compared to the time of 24 hours, according to the meiotic potential observed. Additional studies should be conducted to improve the quality of the oocyte culture environment, as medium composition, aiming to obtain viable mature oocytes for other in vitro biotechnologies.

194 IN VITRO MATURATION AND RNA CONTENT AND DISTRIBUTION OF PORCINE OOCYTES DERIVED FROM SMALL AND MEDIUM FOLLICLES AND CLASSIFIED BY BRILLIANT CRESYL BLUE ASSAY

2012 ◽  
Vol 24 (1) ◽  
pp. 209 ◽  
Author(s):  
L. T. Ngoc Thanh ◽  
H. Funahashi
Keyword(s):  
In Vitro Maturation ◽  
Cell Mass ◽  
Cumulus Cell ◽  
Total Content ◽  
Cumulus Cells ◽  
Brilliant Cresyl Blue ◽  
Total Rna ◽  
Cresyl Blue ◽  
Rna Content

Oocytes collected from the surface of slaughterhouse ovaries clearly have heterogeneous quality. The objective was to characterize a change or difference in RNA distribution and the content of porcine oocytes that were collected from small (SF; 1–2 mm in diameter) and medium follicles (MF; 3–6 mm in diameter) and assessed for glucose-6-phosphate dehydrogenase (G6PD) activity by brilliant cresyl blue (BCB) assay. Following BCB staining, porcine cumulus–oocyte complexes (COC) with dark blue (DB; G6PD-inactive, suggesting good quality) and light blue (LB; G6PD-active, suggesting immaturity) ooplasm were then separately cultured in vitro to evaluate nuclear maturation and analyze the characteristics of the RNA aspect. RNA distributions in cumulus cell mass and ooplasm were labeled with fluorescence, SYTO RNA select green and then examined at different periods of culture for in vitro maturation (IVM; with gonadotropins and dbcAMP for 20 h and then without those for 24 h). Total RNA content of oocytes and cumulus cell mass were measured by using a manufacture's kit (Invitrogen Quant-iT RNA assay kit). Statistical analyses of results from 3 to 5 replicated trials were performed by ANOVA with a Bonferroni/Dunn post-hoc test (significance, P < 0.05). When oocytes were classified by BCB assay, the percentage of oocytes with DB cytoplasm was much higher (P < 0.05) in oocytes from MF (72.5% of 208) compared with those of SF (53.6% of 352). Regardless of the origin of oocytes (SF vs MF), the incidence of mature oocytes following culture was higher in DB than LB (64.0% of 147 and 72.1% of 133 vs 50.9% of 108 and 52.8% of 54, respectively). Twenty hours after the start of IVM, the DB oocytes had a higher proportion of RNA-free zone inside the germinal vesicle compared to the LB oocytes (66.1% of 193 and 26.8% of 159 in MF; 47.9% of 185 and 17.9% of 184 in SF; P < 0.05). When the total content of RNA was examined during IVM, both oocytes from SF and MF contained higher levels of total RNA at the beginning of IVM as compared with 20 and 44 h after the start of IVM, whereas the content (40 denuded oocytes/sample) was higher in oocytes with DB cytoplasm from MF than those from SF (P < 0.05). Regardless of the origin of oocytes from SF and MF, the total content of RNA in oocytes with LB cytoplasm was significantly lower than in oocytes with DB cytoplasm (P < 0.05) before the start of IVM. The total RNA content of cumulus cells (40 COC/sample) before IVM culture was also higher in the cell mass surrounding the DB oocytes than the LB ones and in the cell mass surrounding the oocytes from MF rather than SF (P < 0.05). These results demonstrate a possibility that differences in RNA distribution and content in oocytes, as well as the content in cumulus cells, reflect in the ability of porcine oocytes to mature in vitro.

298 CYCLIC AMP AND CYCLIC GMP CONTENTS IN PORCINE OOCYTE–CUMULUS COMPLEXES, DENUDED OOCYTES, AND CUMULUS CELLS DERIVED FROM SMALL AND MIDDLE FOLLICLES DURING IN VITRO MATURATION

2015 ◽  
Vol 27 (1) ◽  
pp. 238
Author(s):  
Y. Okudaira ◽  
H. Funahashi
Keyword(s):  
Cyclic Amp ◽  
In Vitro Maturation ◽  
Critical Role ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Intracellular Camp ◽  
Camp Content ◽  
Porcine Oocyte ◽  
Camp And Cgmp

Drastic changes in intracellular cAMP and cGMP levels play a critical role in the regulation of meiotic resumption. The objective of this study was to compare cAMP and cGMP contents in cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell masses (CC) derived from small (SF) and middle follicles (MF) during in vitro maturation (IVM). The COC were aspirated from SF (1–3 mm in diameter) or MF (3–6 mm in diameter) of prepubertal gilt ovaries. The COC were cultured in modified porcine oocyte medium (mPOM) with eCG, hCG, and dibutyryl cAMP for 20 h and then in fresh mPOM without those supplements for 4 h in an atmosphere of 5% CO2 in air at 39°C. At 0, 10, 20, and 24 h of IVM, COC, DO, and CC were collected. The DO were prepared by removal of cumulus cells and zona pellucida. The CC were prepared by puncturing ooplasm by using 18-gauge needle. Intracellular contents of cAMP and cGMP were determined by direct enzyme immunoassay kits. Statistical analyses of 3 to 7 replicated data were performed by ANOVA. There were no significant differences in contents of cAMP and cGMP between DO from SF and MF in all observation points (P > 0.05). Cyclic AMP contents in COC and CC derived from MF were higher than those from SF at 20 h of IVM (MF 33.0 ± 0.5 fmol/COC v. SF 28.4 ± 1.0 fmol/COC, MF 20.9 ± 0.9 fmol/CC v. SF 14.6 ± 0.8 fmol/CC; P < 0.05), whereas there were no significant differences between origins of those (SF v. MF, P > 0.05) at 0, 10, and 24 h of IVM. Furthermore, although cAMP content in CC from MF was not significantly different between 10 and 20 h of IVM (25.4 ± 1.7 and 20.9 ± 0.9 fmol/CC, respectively; P > 0.05), the content in CC from SF significantly decreased between 10 and 20 h (23.1 ± 1.2 , and 14.6 ± 0.8 fmol/CC, respectively; P < 0.05). At 0 and 10 h of IVM, cGMP contents in COC and CC from MF were significantly higher than those from SF (0 h: 81.8 ± 4.5 fmol/COC from MF v. 41.7 ± 10.6 fmol/COC from SF and 82.7 ± 7.5 fmol/CC from MF v. 10.7 ± 2.7 fmol/CC from SF; 10 h: 64.8 ± 8.4 fmol/COC from MF v. 24.8 ± 8.2 fmol/COC from SF, 49.3 ± 14.9 fmol/CC from MF v. 13.5 ± 4.8 fmol/CC from SF; P < 0.05). However, there were no significant differences in cGMP contents in COC and CC between the origins (MF v. SF) at 20 and 24 h of IVM (P > 0.05). From these results, we conclude that cAMP and cGMP contents in cumulus cells are significantly differences between the origins (MF v. SF) during IVM.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

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