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

334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 275
Author(s):  
H. S. Lee ◽  
Y. I. Seo ◽  
X. J. Yin ◽  
S. G. Cho ◽  
I. H. Bae ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Uv Light ◽  
Cumulus Cells ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Oviduct Fluid

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.

224 LOCALIZATION OF NITRIC OXIDE SYNTHASE ACTIVITY IN BUFFALO (BUBALUS BUBALIS) OOCYTES AND EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 211
Author(s):  
K. R. Babu ◽  
R. Sharma ◽  
K. P. Singh ◽  
A. George ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Epifluorescence Microscopy ◽  
Rt Pcr ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Nos Isoforms

Ovarian nitric oxide (NO) and that produced within the oocytes and embryos have been reported to play important roles in oocyte meiotic maturation and embryo development. Production of NO is catalyzed by NO synthase (NOS), which exists in 3 isoforms, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms and the inducible (iNOS) isoform. We have previously shown that low concentrations of NO stimulate and high concentrations inhibit embryo development, and that endogenous NO produced by iNOS is necessary for optimal embryo development in the buffalo. The present study was aimed at localizing different isoforms of NOS and examining their relative mRNA abundance in buffalo oocytes and embryos. Oocytes from slaughterhouse ovaries were subjected to in vitro maturation in 100-μL droplets (10 to 15 oocytes/droplet) of in vitro maturation medium (TCM-199 + 10% FBS + 5 μg mL–1 of pFSH + 1 μg mL–1 of oestradiol-17β + 0.81 mM sodium pyruvate + 10% buffalo follicular fluid + 50 μg mL–1 of gentamicin) for 24 h in a CO2 incubator (5% CO2 in air) at 38.5°C. In vitro fertilization was carried out by incubating in vitro-matured oocytes with 2 to 4 million spermatozoa mL–1 for 18 h. The presumed zygotes were cultured on original beds of cumulus cells in in vitro culture medium (mCR2aa + 0.6% BSA + 10% FBS) for up to 8 days post-insemination. Immature and in vitro-matured oocytes and embryos at the 2-cell, 4-cell, 8- to 16-cell, morula, and blastocyst stages were examined for the presence of NOS isoforms by indirect immunofluorescence staining using epifluorescence microscopy and RT-PCR. Each experiment was repeated in triplicate, and data were analysed using one-way ANOVA, after arcsine transformation of percentage values. Expression of all 3 NOS isoforms was detected inside the cytoplasm, in all the stages of oocytes and embryos examined, by both immunofluorescence and RT-PCR. Abundance of the iNOS transcript was significantly higher (P ≤ 0.01) in the morula and blastocyst stages compared with that in immature and in vitro-matured oocytes and in embryos at the 2-cell, 4-cell, and 8- to 16-cell stages, indicating that its expression was up-regulated at the 8- to 16-cell stage. The expression of eNOS was significantly higher (P ≤ 0.05) in the immature and mature oocytes and in 8- to 16-cell stage embryos, morulae, and blastocysts than in the early-cleavage embryos at the 2- and 4-cell stages, indicating that it was down-regulated after fertilization and was up-regulated again at the 8- to 16-cell stage. Abundance of the nNOS transcript was not significantly different among all the stages of oocytes and embryos examined. These results demonstrate that different NOS isoforms are expressed in a dynamic manner during embryonic development in the buffalo. The role of an increase in expression of iNOS and eNOS at the 8- to 16-cell stage, at which a developmental block occurs in this species, needs to be examined.

231 STAGE-DEPENDENT CHANGES OF IP3R1 PHOSPHORYLATION DURING IN VITRO MATURATION AND DEVELOPMENT IN PIG OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 195
Author(s):  
J. Ito ◽  
C. Suzukamo ◽  
T. Mochida ◽  
A. Furugaichi ◽  
N. Nakajima ◽  
...  
Keyword(s):  
Cell Cycle ◽  
In Vitro Maturation ◽  
Mammalian Species ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Cell Stage ◽  
Mouse Oocytes ◽  
M Phase ◽  
Pn Stage

During fertilization in mammalian species, a sperm-induced intracellular Ca2+ signal [Ca2+] is well suited to mediate the highly specialized spatiotemporal patterns of [Ca2+]i responses that underlie fertilization. Recently, we demonstrated that the expression pattern of inositol 1,4,5- triphosphate receptor type 1 (IP3R1) did not change during in vitro maturation and parthenogenetic activation in mouse oocytes; however, the phosphorylation status of IP3R1 depended on the cell cycle during meiosis. Moreover, it was shown that IP3R1 phosphorylation played a crucial role in the induction of [Ca2+]i oscillations (Lee et al. 2006 Development 133, 4355–4365). In other species, expression of IP3R1, especially phosphorylation levels of IP3R1 during meiosis, has not been examined. The aim of this study was to examine the kinetics of IP3R1 expression and phosphorylation during in vitro maturation and activation in pig oocytes. Immature oocytes at the germinal vesicle (GV) stage were collected from ovaries and cultured in modified NCSU37 up to 48 h. After culture, cumulus cells were removed and oocytes were parthenogenetically activated by 25 µm Ca2+ ionophore for 3 min and 2 mm 6-DMAP for 6 h. After activation, oocytes were further cultured up to the 2-cell stage. Groups of 30 oocytes were collected at each culture period for detection of IP3R1. According to our previous report in the mouse, IP3R1s were detected by western blotting using MPM-2 and Rbt03 antibody for detecting IP3R1 phosphorylation and total IP3R1 expression, respectively (Lee et al. 2006). In pig oocytes, IP3R1 was abundantly expressed at the GV stage. The total level of IP3R1 expression did not change during in vitro maturation or after activation. However, phosphorylated IP3R1 levels increased by 24 h although they were undetectable at the start of culture. Phosphorylation of IP3R1 reached maximal levels at 36 h. After activation, phosphorylation levels decreased progressively until the pronuclear (PN) stage. Phosphorylation of IP3R1 was observed at mitosis I to some extent. From these results, we detected for the first time IP3R1 expression and phosphorylation in pig oocytes. Moreover, our data suggest that phosphorylation of IP3R1 is dependent on cell cycle at least during meiosis, especially M-phase, as already shown for mouse oocytes. In vitro kinase assays for p34cdc2 kinase and MAPK will be carried out to clarify the relationship between IP3R1 phosphorylation and M-phase kinase(s).

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&apos;s multiple range test. The mean diameters for COCs collected from small follicles (136.7 &micro;m for the outer zona and 103.1 &micro;m for ooplasm) were significantly less than for those derived from medium follicles (164.1 &micro;m and 122.0 &micro;m, 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 &plusmn; 11.5&percnt; and 18.0 &plusmn; 6.7&percnt; 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 &plusmn; 23.8&percnt; and 72.9 &plusmn; 6.1&percnt; for small and medium follicles, respectively. The percentage of oocytes at the metaphase II stage (53&percnt; and 63.9&percnt; 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 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.

230 IN VITRO MATURATION OF OOCYTES FROM ENDANGERED DORCAS GAZELLE (GAZELLA DORCAS NEGLECTA)

2006 ◽  
Vol 18 (2) ◽  
pp. 223 ◽  
Author(s):  
E. R. S. Roldan ◽  
F. Berlinguer ◽  
S. Succu ◽  
R. Gonzalez ◽  
A. del Olmo ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Captive Breeding ◽  
Polar Body ◽  
Calf Serum ◽  
Genetic Material ◽  
Cumulus Cells ◽  
Ministry Of Education ◽  
Cell Stage ◽  
Captive Breeding Program

In vitro maturation of oocytes recovered from dead animals provides an opportunity for rescuing genetic material for biodiversity conservation. The dorcas gazelle (Gazella dorcas) is regarded by the World Conservation Union (IUCN) as ‘vulnerable’ but the subspecies G. dorcas neglecta is thought to be endangered due to excessive hunting. A captive breeding program for dorcas gazelles has been developed at the Estacion Experimental de Zonas Aridas (CSIC) in the South of Spain where efforts have so far concentrated on natural breeding and on the development of sperm cryopreservation protocols. The aim of the present study was to explore the possibility of recovering and maturing in vitro healthy oocytes from animals that die suddenly for the establishment of a program to rescue female gametes. Ovaries of a dorcas female that died unexpectedly were collected about 7 h after death of the animal. Cumulus–oocyte complexes (COCs) were recovered by slicing the ovaries. Collection and washing of COCs were performed in warmed TCM-199-HEPES with antibiotics and polyvinyl alcohol. Degenerated oocytes or those with expanded cumulus cells were removed. A total of 15 COCs were cultured in TCM-199 with 10% heat-treated fetal calf serum, 10 μg/mL ovine FSH/LH, 1 µg/mL estradiol, and 0.1 mg/mL glutamine at 38.5°C under 5% CO2/air with high humidity. After 24 h of culture, matured oocytes, as revealed by the presence of a polar body, were activated with 7% ethanol for 10 min and further incubation for 3 h. Meiotic progression and activation were evaluated by staining with Hoechst 33342 and propidium iodide (1 μg/mL each) and visualization under a fluorescence microscope. Results at the end of incubations showed that 4/15 oocytes were degenerated, 4/15 were arrested at the MI stage, and 7/15 (46.7%) progressed to the MII stage. One oocyte was found to be at the 2-cell stage but it could not be established whether this was the result of the activation method used. These results demonstrate that it is possible to recover viable oocytes several hours after death and rescue them for subsequent in vitro maturation and fertilization. More studies are needed to characterize suitable conditions for oocyte maturation, fertilization, and culture in the dorcas gazelle. This would, in turn, help in the effort to rescue biomaterials from wildlife for generating offspring. This work was supported by the Spanish Ministry of Education and Science (REN 2003–01587) and Acciones Integradas (HI20030336).

scholarly journals In vitro production of zygote from slaughterhouse driven buffalo oocyte

2020 ◽  
pp. 127-132
Author(s):  
GK Deb ◽  
MFH Miraz ◽  
SMJ Hossain ◽  
MF Afroz ◽  
MA Kabir ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Meat Production ◽  
In Vitro Production ◽  
Embryo Production ◽  
Cell Stage

Buffalo is a highly potential animal species in terms of milk and meat production but traditionally they are regarded as poor breeder. In vitro embryos production technology has been introduced in many countries to improve reproductive efficiency of buffalo. Considering the above fact, the present study was undertaken aiming to produce in vitro buffalo embryo in the laboratory. Ovaries of slaughtered buffaloes were collected from abattoir and transported to the laboratory within 4 to 5 hr of slaughter. Cumulusoocyte- complexes (COCs) possessing an even cytoplasm and covered with minimum 3 layers of compact cumulus cells was selected for in vitro maturation (IVM) for 24 hr (5% CO2 in air at 38.5°C with maximum humidity). After IVM, the presumptive matured COCs were co-cultured with capacitated fresh spermatozoa for 18 hr After IVF, the presumptive zygote were denuded, washed and transferred in to in vitro culture medium (IVC 1) for 3 days. After three days cleavage were recorded and 4 cell embryos were transferred in to in vitro culture media II for next 2 days. The development of embryos was evaluated on day 6. A total of 227 buffalo ovaries were collected from the slaughterhouse and categorized into 2 groups based on presence (n=83) or absence (n=144) of corpus luteum (CL). A total of 1464 follicles were counted on the ovarian surface, 1066 being from CL absent and 398 from CL-containing ovaries. A significantly higher (P<0.01) number of follicles, aspirated follicles, normal COCs and total COCs (7.4 ± 0.21, 5 ± 0.00, 1.98 ± 0.77 and 2.98 ± 0.16 respectively) were observed in CL-absent ovaries than those aspirated from CL-containing ovaries (4.80 ± 0.17, 3.92 ± 0.95, 0.88 ± 0.60 and 1.88 ± 0.16 respectively). Total 358 normal COCs were set for in vitro maturation and underwent for IVF and IVC. Results showed that cleavage rates were 56.42%. Among the cleaved embryos, 137 were at 2-cell stage and 65 were at 4-cell stage. Therefore, development rate to 2 cell and 4-cell stage was 38.27% and 18.15% respectively. No embryo developed beyond 4-cell stage. This result indicates that follicle and oocyte numbers and oocyte quality are associated with CL of ovaries and current culture system support in vitro embryo production upto 4-cell stage. The in vitro culture condition may be improved for increasing efficiency of embryo production. Bangladesh J. of Livestock Res. 21-25: 127-132, 2018

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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