Effect of GnRH treatment on the maturation and in vitro development of oocytes collected from 4- to 6-week-old Merino lambs

2007 ◽  
Vol 19 (8) ◽  
pp. 947 ◽  
Author(s):  
Jennifer M. Kelly ◽  
David O. Kleemann ◽  
W. M. Chis Maxwell ◽  
Simon K. Walker
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation ◽  
Oocyte Collection ◽  
Vitro Development

Two experiments were conducted in Merino lambs to examine the effects of gonadotrophin-releasing hormone (GnRH) treatment on the developmental competence of oocytes collected after pretreatment with follicle stimulating hormone (FSH). The first experiment examined the effects of six GnRH treatment times (control and GnRH administered 2, 4, 6, 8 and 10 h before oocyte collection) and four in vitro maturation (IVM) periods (18, 20, 22, 24 h) on the rate of oocyte nuclear maturation. The second experiment examined the effect of five GnRH treatment times (control and GnRH administered 2, 4, 6 and 8 h before oocyte collection) and three IVM periods (20, 22, 24 h) on the development of oocytes and embryos after in vitro maturation, fertilisation and culture. In Experiment 1, GnRH treatment did not influence the mean number of cumulus-oocyte-complexes (COCs) collected or COC morphology at the time of collection. However, treatment changed (P < 0.01) the distribution of follicle size and this was primarily due to a marked reduction in the number of follicles with diameters <2 mm. In addition, GnRH treatment at 6 and 8 h increased (P < 0.01) the proportion of oocytes that developed to Metaphase II (MII) (63.2 and 72.6%, respectively) compared with other treatment times (range 52.9–59.9%). Nuclear maturation was influenced by a significant (P < 0.05) interaction between GnRH treatment and IVM period due to a disproportionately greater number of oocytes at the germinal vesicle breakdown (GVBD) stage for the 2 and 4 h GnRH treatments compared with other treatments. In Experiment 2, cleavage rate (range 63.5–85.9%) was highest when GnRH was administered 8 h before collection but the percentage of cleaved oocytes that developed into blastocysts (range 10.0–35.0%) was significantly (P < 0.05) lower for the 6 and 8 h GnRH treatments compared with the control and the 2 h GnRH treatment. These results demonstrate that GnRH treatment before oocyte collection can improve nuclear maturation and cleavage rates in lamb oocytes but that these improvements are not reflected in improved rates of blastocyst development. It is speculated that this discrepancy may result from GnRH treatment either adversely affecting cytoplasmic maturation or inducing asynchrony between the maturation of the nuclear and cytoplasmic components of the oocyte.

191 Effect of the time of oocyte collection through slicing method on meiosis resumption and invitro embryo production

2020 ◽  
Vol 32 (2) ◽  
pp. 224
Author(s):  
S. Soto-Heras ◽  
A. Lorenzo ◽  
I. Menéndez-Blanco ◽  
D. Izquierdo ◽  
M. Paramio
Keyword(s):  
Acetic Acid Solution ◽  
Germinal Vesicle ◽  
Random Variable ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Oocyte Competence ◽  
Nuclear Maturation ◽  
Oocyte Collection ◽  
Group 3

Oocytes from juvenile goats are collected by slicing the ovary surface because the high percentage of small antral follicles limits follicular aspiration. The time of oocyte collection can impair oocyte developmental competence due to spontaneous resumption of meiosis. The aim of this study was to assess whether the time of slicing period affects oocyte meiosis and embryo development after invitro fertilization. Ovaries from juvenile goats (1-2 months old) were recovered at a local slaughterhouse. Cumulus-oocyte complexes (COCs) were collected by slicing, selected, and kept in the slicing medium at 38.5°C in humidified air with 5% CO2 until analysis or culture. The slicing medium was HEPES-buffered (25mM) TCM-199 with 2.2mgmL−1 NaHCO3 and 50mgmL−1 gentamicin. Two slicing periods were tested: T1 (1 h) and T4 (4 h). After this time, a group of oocytes were stained with 1% orcein in 45% acetic acid solution for assessing meiotic arrest and observed as the rate of germinal vesicle (GV; 61-67 oocytes/group from 5 replicates). The remaining COCs were cultured in our conventional IVM medium (TCM-199 with FSH, LH, oestradiol, sodium pyruvate, glutamine, cysteamine, epidermal growth factor, and fetal bovine serum) at 38.5°C with 5% CO2. After 24h, a sample of oocytes were stained for assessing nuclear maturation (28-29 oocytes/group, 3 replicates), and the rest were invitro fertilized with 4×106 spermmL−1 in BO-IVF medium (IVF Bioscience) for 20h and embryo cultured in BO-IVC medium for 7 days (70-81 oocytes/group, 3 replicates). Blastocysts were stained with Hoechst 33258 for determining the number of cells. Data were analysed with two-way ANOVA with RStudio version 1.2.1335. The time of slicing was set as a fixed factor and the replicate as random variable. Data presented as percentage did not follow a normal distribution and were square root arcsine transformed before analysis. At the end of slicing periods T1 and T4, oocytes at GV were 100% and 84.7±5.0%, respectively (P&lt;0.05). After 24h of IVM, the oocytes at MII were 77.0±7.1% and 88.6±7.3%, respectively, without statistical differences. However, oocytes from T1 produced a higher rate of cleaved oocytes (84.6±0.9%) and expanded blastocysts (11.03±5.2%) than T4 (49.8±7.9%, 0%, respectively; P&lt;0.05). The total blastocyst rate for T1 and T4 was 25.4±5.8% and 9.4±4.9%, respectively (P=0.068). No differences were observed in blastocyst cell number (75.9±4.0 and 67.5±10.9, respectively). In conclusion, oocytes resume meiosis before IVM during a long slicing period, even though the slicing medium is not supplemented with hormones or growth factors. The longer slicing period does not affect nuclear maturation but impairs oocyte competence, observed as lower cleavage and blastocyst development. Further experiments are needed to determine whether the use of meiotic inhibitors in the slicing medium can prevent the negative effect of the long slicing period. This study was funded by the Spanish Ministry of Science, Innovation and Universities (AGL2017-85837-R).

scholarly journals Delay of nuclear maturation and reduction in developmental competence of pig oocytes after mineral oil overlay of in vitro maturation media

Reproduction ◽  
2002 ◽  
pp. 557-564 ◽  
Author(s):  
M Shimada ◽  
N Kawano ◽  
T Terada
Keyword(s):  
Steroid Hormones ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Mineral Oil ◽  
Developmental Competence ◽  
Cdc2 Kinase ◽  
Germinal Vesicle Breakdown ◽  
High Concentrations ◽  
P34 Cdc2

Steroid hormones, such as progesterone, oestrogen, androgen and meiosis activating sterols, are secreted from cumulus cells that are stimulated by gonadotrophins during maturation of oocytes in vitro. These steroid hormones may be absorbed by mineral oil or paraffin oil; however, in vitro maturation of pig oocytes is commonly performed using medium covered by oil. In this study, high concentrations of progesterone, oestradiol and testosterone were detected in the culture medium after pig cumulus-oocyte complexes (COCs) were cultured with FSH and LH for 44 h in medium without an oil overlay. However, high concentrations of these steroid hormones were not detected in medium when COCs were cultured with the mineral oil overlay. When high concentrations of these steroid hormones were secreted by COCs, germinal vesicle breakdown (GVBD) and the activation of p34(cdc2) kinase and mitogen-activated protein (MAP) kinase in oocytes occurred earlier in comparison with oocytes cultured in medium covered with mineral oil. Moreover, a decrease in p34(cdc2) kinase activity during meiotic progression beyond metaphase I was observed in oocytes cultured in conditions under which high concentrations of steroid hormones were secreted by COCs. In addition, the rate of development to the blastocyst stage after IVF was higher in oocytes matured in medium without an oil overlay. These adverse effects of oil may be explained by absorption by the oil of cumulus-secreted steroids or by the release of toxic compounds into the medium.

313 PRE-IN VITRO MATURATION WITH CYCLIC AMP AND CYCLIC GMP MODULATORS IMPROVES DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 245 ◽  
Author(s):  
N. W. Santiquet ◽  
A. F. Greene ◽  
W. B. Schoolcraft ◽  
R. L. Krisher
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Nuclear Maturation ◽  
Oocyte Developmental Competence ◽  
Oocyte Collection ◽  
Short Period

In vitro maturation (IVM) of cumulus-oocyte complexes (COC) results in oocytes with reduced quality and is still not as efficient as in vivo maturation in most species. One hypothesis that could explain the low developmental competence of oocytes following IVM is that the oocytes resume meiosis too quickly after being retrieved from the follicles. Studies in mice and bovine have shown that a short period of prematuration in the presence of cAMP modulators, before IVM, enhances oocyte developmental competence. Moreover, other studies have recently demonstrated that cGMP is also a crucial molecule involved in meiotic resumption. Here, our objective was to examine the effect of a cGMP modulator in combination with a cAMP modulator during a short period of prematuration on mouse oocyte nuclear maturation and subsequent embryo development following IVF. The COC were collected (6 replicates) from 2-month-old outbred CF1 mice 48 h after PMSG (5 IU) injection in the presence (pre-IVM) or absence (control) of cGMP and cAMP modulators. Pre-IVM COC (n = 184) were then placed in prematuration medium that also contained these cGMP and cAMP modulators. After 2 h, pre-IVM COC were washed and transferred to our in-house prepared, completely defined IVM medium (Paczkowski et al. 2014 Reprod.) for the remaining 16 h of culture; 10 oocytes per 50 µL drop under oil, at 37°C in 7.5% CO2 and 6.5% O2 due to the increased altitude at our location. Control COC (n = 161) were matured in the same IVM medium under identical conditions for 18 h, without prematuration. After IVM, oocytes were fixed for assessment of nuclear maturation, or fertilized and cultured in vitro and subsequent development (96 and 112 h) was recorded (Paczkowski et al. 2014 Reprod.). Results were analysed by ANOVA. A short 2-h prematuration period in the presence of cGMP and cAMP modulators had no impact on oocyte nuclear maturation to metaphase II after IVM or on embryo cleavage after IVF. However, pre-IVM treatment improved the developmental competence of the oocyte, as demonstrated by increased embryo development. More (P < 0.02) blastocysts (96 h of culture) and hatched blastocysts (112 h of culture) developed in the pre-IVM treatment compared to control (31.0 ± 3.4 v. 19.9 ± 3.2%; 31.5 ± 3.4 v. 19.9 ± 3.2%, respectively). In conclusion, a combination of cGMP and cAMP modulators during oocyte collection and a subsequent short pre-IVM improves oocyte developmental competence and could therefore be a potential tool to improve embryo yield following IVM.

scholarly journals Development to Blastocyst Stage of Pig Oocytes Matured, Fertilized and Electroactivated In Vitro

2002 ◽  
Vol 45 (6) ◽  
pp. 547-556
Author(s):  
N. R. Mtango ◽  
M. D. Varisanga ◽  
D. Y. Juan ◽  
P. Wongrisekeao ◽  
T. Suzuki
Keyword(s):  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Activation Rates ◽  
Oviduct Fluid

Abstract. This study was designed 1) to determine the effectiveness of two in vitro maturation (IVM) media (tissue culture medium [TCM] and modified synthetic oviduct fluid supplemented with amino acids [mSOFaa]), 2) to compare the effects of two in vitro fertilization (IVF) media (modified Tris-buffered medium [mTBM] and mSOFaa) on the developmental competence of pig oocytes, and 3) to test the activation ability of IVM pig oocytes matured in TCM or mSOFaa, electroactivated and cultured in mSOFaa. The nuclear maturation rates were similar between IVM media (91.0 % vs. 89.0 %). A similar result was obtained when the activation rates were 54.2 % in TCM and 56.0 % in mSOFaa, and the blastocyst rates were 7.9 % and 6.1 %, respectively. There was no significant difference between mSOFaa and mTBM in the percentage of embryos with two pronuclei 33.2 % vs. 13.8 % or polypronuclei 5.3 % vs. 13.4 %. The cleavage rate was the same in both media. The medium mSOFaa gave a significantly higher (P< 0.05) blastocyst rate than mTBM (12.7 % vs. 3.9 %). We concluded that mSOFaa can enhance in vitro maturation, fertilization and culture of pig oocytes.

337 EFFECTS OF CANINE SYNTHETIC OVIDUCT FLUID MEDIUM SUPPLEMENTED WITH THE VARIOUS ENERGY SUBSTRATES ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 276
Author(s):  
H. J. Oh ◽  
M. K. Kim ◽  
Y. H . Fibrianto ◽  
G. Jang ◽  
H. J. Kim ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
The Other ◽  
Germinal Vesicle Breakdown ◽  
Energy Substrates ◽  
Fluid Medium ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Oviduct Fluid

In most mammals, maturation occurs within the ovarian follicle, and preovulatory oocytes are ovulated and ready for fertilization within the oviduct. In contrast, bitch ovulate primary oocytes, over a three day period, undergo both maturation and fertilization within the oviduct. The present study was conducted to evaluate the effects of canine synthetic oviduct fluid (cSOF) supplemented with the various energy substrates on in vitro maturation of canine oocytes. Oocytes were recovered by mincing ovaries collected after ovariohysterectomy in bitches at the follicular stage. Only oocytes with more than two layers of cumulus cells and with homogeneous cytoplasm >100 mm in diameter were selected. Then, oocytes cultured in tissue culture medium (TCM)-199 (control) or cSOF supplemented with various concentrations of glucose (0, 1.11, 3.89, or 5.56 mM, Exp. 1) or fructose (0, 1.11, 3.89, or 5.56 mM, Exp. 1), pyruvate (0, 0.1, 0.25, or 0.5 mM, Exp. 2) or lactate (0, 0.5, 1.0, or 5.0 mM, Exp. 3). In Exp. 4, the combined effects of glucose (1.11 mM), pyruvate (0.5 mM) and lactate (5.0 mM) on nuclear maturation of canine oocytes were investigated. A total of 2990 canine oocytes from 205 ovaries were used for experiments with replication at least three times. The oocytes were cultured for 72 h at 38.5�C in a humidified atmosphere of 5% CO2 in air. After 72 h, the oocytes were stained with 1.9 �g/mL Hoechst 33342 in glycerol and then evaluated under UV light to determine the stage of meiosis as follows: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII) with first polar body. The results of Exp. 1 showed that maturation of canine oocytes to MII was significantly higher (P < 0.05) in medium supplemented with 1.11 mM glucose (4.8%) than for the control (1.8%) and the other glucose-supplemented groups (0 to 1.8%). In Exp. 2, oocytes cultured in cSOF supplemented with 0.5 mM pyruvate showed a significantly higher (P < 0.05) maturation rate to MII (6.3%) than did the other pyruvate-supplemented (0, 0.8, or 2.5%) groups or the control (2.4%). In Exp. 3, more oocytes were matured to the MII stage in cSOF supplemented with 5.0 mM lactate (7.3%) than were the other lactate-supplemented groups (0 to 2.4%) or the control (2.5%). Results of Exp. 4 showed more oocytes progressed to MII in cSOF supplemented with 0.5 mM pyruvate (8.2%), 1.11 mM glucose + 0.5 mM pyruvate (7.4%), or 1.11 mM glucose + 0.5 mM pyruvate 0.5 + 5.0 mM lactate (7.3%) than did the other combination groups (2.2 to 5.2%). In conclusion, the present study demonstrated that supplementing cSOF with 1.11 mM glucose, 0.5 mM pyruvate, or 5.0 mM lactate significantly increased the maturation of canine oocytes to MII, and the combined supplementation of 1.11 mM glucose, 0.5 mM pyruvate, and 5.0 mM lactate further promoted oocyte nuclear maturation compared to 1.11 mM glucose alone and the control. This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-01-00).

171 IN VIVO AND IN VITRO MATURATION OF WOOD BISON (BISON BISON ATHABASCAE) CUMULUS–OOCYTE COMPLEXES DURING THE OVULATORY SEASON

2014 ◽  
Vol 26 (1) ◽  
pp. 199
Author(s):  
M. P. Cervantes ◽  
M. Anzar ◽  
R. J. Mapletoft ◽  
J. M. Palomino ◽  
G. P. Adams
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Bison Bison ◽  
Germinal Vesicle Breakdown ◽  
Cumulus Expansion ◽  
Nuclear Maturation ◽  
Wood Bison

Technologies are being developed to conserve the genetic diversity of wood bison. Knowledge of the characteristics of in vivo and in vitro maturation of the cumulus–oocyte complex (COC) are needed in wood bison to design efficient in vitro embryo production protocols. The objectives were to (1) determine the optimal interval after hCG treatment for in vivo maturation of COC in superstimulated wood bison, and (2) compare the characteristics of COC after in vitro and in vivo maturation. Ovarian synchronization was induced in 25 bison during October and November by giving a luteolytic dose of prostaglandin followed 8 days later by follicular ablation (Day –1). Ovarian superstimulation was induced with FSH (Folltropin-V) given i.m. on Day 0 (300 mg) and Day 2 (100 mg). A second luteolytic dose of prostaglandin was given on Day 3. Bison were assigned randomly to 5 groups (n = 5/group). The COC were collected by transvaginal follicle aspiration on Day 4 and were either assessed immediately (0 h, control), or matured in vitro for 24 or 30 h (in vitro maturation), or collected on Day 5 (in vivo maturation), 24 or 30 h after bison were given 2000 IU of hCG i.m. on Day 4. In vitro maturation was done in TCM-199 with 5% calf serum, 5 μg mL–1 LH, 0.5 μg mL–1 FSH, and 0.05 μg mL–1 gentamicin, at 38.5°C and in a 5% CO2 humidified atmosphere. Nuclear maturation was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), or metaphase II (MII) with anti-lamin AC/DAPI staining. Groups were compared by analysis of variance and Fisher's exact test (Table 1). A mean (±s.e.m.) of 7.3 ± 1.7 COC were collected per bison, with no difference among groups. The COC in the control (0 h) group were at the nonexpanded GV stage. Cumulus cells were more expanded after in vivo than in vitro maturation, and the percentage of fully expanded COC was the highest in the 30-h in vivo maturation group (87%; P < 0.05). The greatest number of oocytes reached MII stage after 24 h of in vitro maturation, and 30 h of in vivo maturation. In conclusion, nuclear maturation occurred more quickly in vitro compared with in vivo, but the degree and incidence of cumulus expansion was greater after in vivo maturation. The competence of oocytes to undergo fertilization and develop into embryos remains to be investigated. Table 1.Cumulus expansion and nuclear maturation of wood bison oocytes

Early germinal vesicle breakdown is a predictor of high preimplantation developmental competent oocytes in mice

Zygote ◽  
2016 ◽  
Vol 25 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Shogo Higaki ◽  
Masao Kishi ◽  
Keisuke Koyama ◽  
Masashi Nagano ◽  
Seiji Katagiri ◽  
...  
Keyword(s):  
Time Course ◽  
Evaluation Method ◽  
Germinal Vesicle ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation ◽  
Non Invasive ◽  
Germinal Vesicle Break Down

SummaryThe preselection of highly developmentally competent oocytes for in vitro maturation (IVM) is crucial for improving assisted reproductive technology. Although several intrinsic markers of oocyte quality are known to be closely related to the onset of nuclear maturation (germinal vesicle break down, GVBD), a direct comparison between GVBD timing and oocyte quality has never been reported. In this study, we established a non-invasive oocyte evaluation method based on GVBD timing for preselecting more developmental competent oocytes in mice. Because the O2 concentration during IVM may affect the nuclear kinetics, all experiments were performed under two distinct O2 concentrations: 20% and 5% O2. First, we determined the time course of changes in nuclear maturation and preimplantation developmental competence of in vitro-matured oocytes to estimate GVBD timing in high developmental competent oocytes. Two-thirds of oocytes that underwent GVBD in early IVM seemed to mainly contribute to the blastocyst yield. To confirm this result, we compared the preimplantation developmental competence of the early and late GVBD oocytes. Cleavage and blastocyst formation rates of early GVBD oocytes (80.2% and 52.7% under 20% O2, respectively, and 67.6% and 47.3% under 5% O2, respectively) were almost double those of late GVBD oocytes (44.8% and 26.0% under 20% O2, respectively, and 40.4% and 17.9% under 5% O2, respectively). With no observable alterations by checking the timing of GVBD in preimplantation developmental competence, oocyte evaluation based on GVBD timing can be used as an efficient and non-invasive preselection method for high developmental competent oocytes.

39 THE EFFECTS OF RESVERATROL DURING IN VITRO MATURATION ON THE DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES VITRIFIED AT THE IMMATURE STAGE

2017 ◽  
Vol 29 (1) ◽  
pp. 127 ◽  
Author(s):  
E. C. S. Santos ◽  
T. Somfai ◽  
R. Appeltant ◽  
T. Q. Dang-Nguyen ◽  
H. Kaneko ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Developmental Rate ◽  
Immature Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Dibutyryl Camp ◽  
Cleavage Rate ◽  
Nuclear Maturation

Previously, live offspring have been produced from porcine oocytes vitrified at the immature stage (Somfai et al. 2014 PLoS One 9, e97731); however, their embryo developmental rates remain low. The aim of our current research was to test the effects of resveratrol, an antioxidant and anti-apoptotic agent on the developmental competence of immature vitrified oocytes during in vitro maturation (IVM) after warming. Follicular porcine cumulus-oocyte complexes (COC) were vitrified on Cryotop® sheets (Kitazato Corp. Shizuoka, Japan) using the cryoprotectant treatment and warming method of Somfai et al. (2015 J. Reprod. Dev. 61, 571–579). After warming, the oocytes were subjected to IVM for 46 h in a chemically defined porcine oocyte medium (POM) enriched with 10 ng mL−1 epidermal growth factor, 10 IU mL−1 eCG, and 10 IU mL−1 hCG. During the first 22 h of IVM, the medium was supplemented with 1 mM dibutyryl cAMP. The following 24 h of IVM was performed in POM without dibutyryl cAMP. Vitrified/warmed COC (vitrified group) and freshly collected COC (control group) were matured either in the absence or presence of 2 µM resveratrol (RES− and RES+, respectively) throughout the entire IVM. At the end of IVM, oocytes were denuded and their survival was evaluated. Then, those with 1 polar body (PB1+) were selected for parthenogenetic activation (Day 0). Activated oocytes were cultured for 7 days in PZM-3. Survival, nuclear maturation, cleavage, and blastocyst rates were assessed. The experiment was replicated 5 times. Results were analysed by one-way ANOVA and Tukey’s multiple comparison test. Vitrification reduced the percentage of live oocytes after IVM both in RES− and RES+ groups in a similar manner (47.9 and 51.8%, respectively) compared with control RES− and RES+ groups (99.4 and 100%, respectively; P < 0.05) There was no statistical difference among groups in the percentage of PB1+ oocytes (ranging between 76.1 and 90.2%). On Day 2, the cleavage rate in vitrified RES− group was lower than those in control RES− and RES+ groups (55.9 v. 78.5% and 79.2%, respectively) whereas the vitrified RES+ group did not differ from the others (72.1%). The blastocyst developmental rate calculated from total cultured oocytes on Day 7 in vitrified RES+ group was significantly higher (P < 0.05) than that in the vitrified RES− group (26.2% v. 6.9%, respectively) and did not differ significantly from those of control RES− and RES+ groups (32.1 and 36.0%, respectively). Blastocyst rates in control RES− and RES+ groups were significantly higher (P < 0.05) than that in vitrified RES− group but did not differ from one another. In conclusion, supplementation of IVM medium with resveratrol improved the developmental competence of vitrified, but not freshly collected oocytes. This work was supported by JSPS KAKENHI (Grant Number: 26870839) and JST/JICA SATREPS. E.C.S. Santos was supported by a CNPq-Brasil fellowship.

scholarly journals The effect of interaction between macromolecule supplement and oxygen tension on bovine oocytes and embryos cultured in vitro

Zygote ◽  
2009 ◽  
Vol 17 (4) ◽  
pp. 321-328 ◽  
Author(s):  
G.Z. Mingoti ◽  
V.S.D. Caiado Castro ◽  
S.C. Méo ◽  
L.S.S. Barretto ◽  
J.M. Garcia
Keyword(s):  
Oxygen Tension ◽  
Embryo Development ◽  
Calf Serum ◽  
Atmospheric Condition ◽  
Germinal Vesicle ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation

SummaryAiming to improve in vitro production of bovine embryos and to obtain supplements to replace serum for in vitro maturation (IVM), this study evaluated the effects of macromolecular supplementation of IMV medium (bovine serum albumin – BSA, polyvinyl alcohol – PVA, polyvinyl pyrrolidone – PVP, Ficoll, KnockoutSR, or fetal calf serum – FCS) and oxygen tension [5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2 (5% O2)] on oocyte maturation and embryo development. Nuclear progression to germinal vesicle breakdown, metaphase I and metaphase II stages were evaluated and overall results revealed that undefined (FCS) and semi-defined (BSA) media gave better results at 20% O2 and defined media (PVA, PVP and Ficoll) at 5% O2. Independent of macromolecule supplement, IVM at 20% O2 was considered optimal for nuclear maturation. To evaluate embryo development, oocytes matured in the previously described conditions were fertilized and cultured at the same oxygen tension used for IVM and assessed for cleavage (43.0 to 74.8%) and development to morulae (16.4 to 33.8%), blastocyst (7.7 to 52.9%) and hatched blastocyst (9.6 to 48.1%). Apart from oxygen tension, all treatments, except Knockout (22.7%), gave similar results for blastocyst development (26.5 to 38.7%). Independently of macromolecule supplement, higher development rates were obtained in an oxygen tension of 20% O2 (67.4% cleavage, 29.2% morulae, 40.8% blastocyst and 34.0% hatched blastocyst) when compared with 5% O2 (52.5, 21.8, 18.2 and 15.6%, respectively). This study indicates that BSA, PVA, PVP and Ficoll can replace serum during IVM and that the optimal atmospheric condition for in vitro production of bovine embryos is 5% CO2 and 20% O2.

scholarly journals 132 BLASTOCYST DEVELOPMENT OF EQUINE OOCYTES WITH LOW MEIOTIC COMPETENCE HELD IN ROSCOVITINE BEFORE IN VITRO MATURATION

2005 ◽  
Vol 17 (2) ◽  
pp. 216
Author(s):  
Y.H. Choi ◽  
L.B. Love ◽  
D.D. Varner ◽  
K. Hinrichs
Keyword(s):  
Epithelial Cells ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Exact Test ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Chromatin Configuration ◽  
Meiotic Competence

At the time of recovery, immature equine oocytes may be separated into those with either expanded cumuli (Ex) or compact cumuli (Cp). The Cp oocytes originate from viable follicles but are largely juvenile, with low meiotic competence (20 to 30% maturation to MII), and possibly reduced developmental competence. We previously found that in Cp oocytes recovered immediately after slaughter, suppression of meiosis with roscovitine for 24 h before maturation increased embryo development at 4 days after intracytoplasmic sperm injection (ICSI; Franz et al. 2003 Reproduction 125, 693–700). The present study was conducted to evaluate the effect of roscovitine suppression on nuclear maturation and blastocyst formation of Cp oocytes recovered after transport of ovaries from the abattoir (i.e. recovered 5–9 h after slaughter). Compact oocytes recovered from transported ovaries were cultured in M199 with 10% FBS containing 66 μM roscovitine with or without an oil cover. After 16–18 or 24 h, oocytes were fixed to examine the chromatin configuration. Treatment for 16–18 h without oil resulted in the lowest rate of meiotic resumption (0%); thus this treatment was utilized in further studies. Resumption in other treatments ranged from 3 to 6%. Following roscovitine suppression, oocytes were cultured for 30 h in M199 with 10% FBS and 5 μU mL−1 FSH for maturation; control oocytes were cultured for 30 h in the same medium immediately after recovery. Mature oocytes were subjected to ICSI, then cultured in DMEM/F-12 with 10% FBS with or without co-culture with equine oviductal epithelial cells under mineral oil in 5% CO2 in air at 38.2°C, and then evaluated at 7.5 days. Progression to MII (82/376, 22%) after maturation of roscovitine-treated oocytes was similar to that for control oocytes (74/395, 19%). There was no significant difference in cleavage rates after ICSI (72–78%) among treatments. Development to blastocyst was highest in roscovitine-treated oocytes in DMEM/F-12 with co-culture (11/30, 37%); this was significantly higher than that of non-treated oocytes in DMEM/F-12 alone (5/36, 14%), but similar to that of non-treated/DMEM/F-12/co-culture (10/37, 27%) and roscovitine/DMEM/F-12 alone (8/39, 21%). These data indicate that roscovitine induces a fully reversible meiotic suppression in Cp equine oocytes recovered 5–9 h after slaughter, and that this suppression does not harm subsequent developmental competence. This treatment may be used to manipulate the time of onset of maturation of equine oocytes for ease of subsequent procedures. Co-culture with oviductal epithelial cells tended to increase blastocyst rate (P = 0.1, Fisher's exact test) in contrast to our previous findings with embryos from Ex oocytes (Choi et al. 2004 Biol. Reprod. 70, 1231–1238). Further work is needed to determine whether this is related to differences in intrinsic developmental competence between oocyte types. This work was supported by the Link Equine Research Endowment Fund (Texas A&M University).

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