36 The effects of E-64 on the developmental competence of porcine oocytes vitrified at the germinal vesicle stage

2019 ◽  
Vol 31 (1) ◽  
pp. 144
Author(s):  
T. Somfai ◽  
H. T. Nguyen ◽  
N. T. Men ◽  
T. Q. Dang-Nguyen ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Survival Rates ◽  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Immature Stage ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Cell Numbers

Previous studies reported the activation of the apoptotic cascade by vitrification in mature porcine oocytes (Vallorani et al. 2012 Anim. Reprod. Sci. 135, 68-74) and that the cathepsin B inhibitor E-64 improved developmental competence of bovine oocytes via an antiapoptotic effect (Balboula et al. 2013 Reproduction 146, 407-417). The present study was carried out to test whether E-64 affected the developmental competency of porcine oocytes vitrified at the germinal vesicle stage. Cumulus-enclosed porcine oocytes were vitrified in microdrops and warmed by our method (Somfai et al. 2015 J. Reprod. Dev. 61, 571-579). Then, the oocytes were subjected to in vitro maturation (IVM) for 46h in a chemically defined porcine oocyte medium supplemented with 10ng mL−1 of epidermal growth factor, 10IU mL−1 of eCG, and 10IU mL−1 of hCG and during the first 22h of IVM with 1mM dibutyryl cyclic adenosine monophosphate. Then, cumulus-oocyte complexes were fertilized in vitro and presumptive zygotes were cultured in 50-µL drops of porcine zygote medium-3 for 7 days in 6-well dishes covered by paraffin oil in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. On Day 5 (Day 0=IVF), the porcine zygote medium-3 was supplemented with 10% (vol/vol) FCS. The effects of 1.0μM of E-64 supplementation during IVM of non-vitrified and vitrified cumulus-oocyte complexes were investigated in a 2×2 factorial design. Survival rates after IVM, cleavage rates on Day 2, blastocyst rates, and total cell numbers in blastocysts on Day 7 were compared among groups. The experiment was replicated 5 times. Results were analysed by ANOVA and Tukey’s multiple comparison test. The percentages of live oocytes were statistically similar when oocytes were matured in the absence or presence of E-64 both in non-vitrified (99.2% v. 99.6%, respectively) and vitrified (94.3% v. 90.8%, respectively) groups. Similarly, IVM without or with E-64 supplementation had no effect on subsequent cleavage and blastocyst development rates in non-vitrified (67.4% v. 71.2% and 38.7% v. 43.2%, respectively) and vitrified (46.8% v. 48.8% and 14.6% v. 22.8%, respectively) oocytes. Irrespective of E-64 treatment, all survival and developmental rates in the vitrified groups were significantly lower (P<0.05) compared with those of their non-vitrified counterparts except for the blastocyst development rate in the E-64-treated vitrified group, which did not differ significantly from those of the non-vitrified groups with or without E-64 treatment. There was no statistical difference in mean blastocyst cell numbers among the groups, ranging between 86.5±15.8 and 118±10.6. In conclusion, E-64 treatment had no effect on embryo production rates, which suggests that in our system, cathepsin-mediated apoptosis during IVM might not be the factor to limit embryo production using either fresh oocytes or those vitrified at the immature stage. This work was supported by JST/JICA SATREPS.

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

207 IN VITRO MATURATION TREATMENT AFFECTS DEVELOPMENTAL COMPETENCE OF LAPAROSCOPIC OVUM PICKUP-DERIVED OOCYTES IN FOLLICLESTIMULATING HORMONE-STIMULATED GOATS

2008 ◽  
Vol 20 (1) ◽  
pp. 182 ◽  
Author(s):  
Y. Locatelli ◽  
N. Poulin ◽  
G. Baril ◽  
J.-L. Touzé ◽  
A. Fatet ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Follicular Fluid ◽  
Developmental Competence ◽  
Postmortem Changes ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Development Rates ◽  
Epidermal Growth

The aim of the present study was to assess the effect of IVM treatment on the developmental competence of oocytes recovered from repeated laparoscopic ovum pickukp (LOPU) in goats. A total of 94 LOPU sessions were performed on 33 adult goats of the Saanen and Alpine breeds. Females were synchronized (Day 0) during the nonbreeding season by inserting vaginal sponges (45 mg of fluorogestone acetate, Intervet, Boxmeer, The Netherlands). At Day 8, an i.m. injection of 50 μg of cloprostenol (Estrumate; Schering-Plough Animal Health, Pointe-Claire, Quebec, Canada) was administered. Porcine FSH (Stimufol, Merial, Brussels, Belgium, 160 mg/goat) was administered in 5 injections at 12-h intervals, starting on Day 8. The LOPU took place under general anesthesia on Day 11, and follicles ≥2 mm were aspirated with an 18-gauge needle connected to a controlled vacuum system. Vaginal sponges were removed at the time of LOPU. Treatments were repeated 2 times in a 2-week interval scheme (2 goats and 1 goat were excluded from the experiment during the second and third LOPU sessions, respectively). Cumulus–oocyte complexes were washed and evaluated for quality (graded from 1 to 3). Oocytes recovered from unstimulated slaughterhouse-derived ovaries served as a control. Cumulus–oocytes complexes from Grades 1 and 2 were submitted to IVM in TCM-199, supplemented with 100 μm of cysteamine and either 10 ng mL–1 of epidermal growth factor (EGF) or 10% follicular fluid and 100 ng mL–1 of ovine FSH (FF-FSH). Matured oocytes were then submitted to IVF and in vitro development as described by Cognié et al. (2004 Reprod. Fertil. Dev. 16, 437–445). Over the 94 LOPU sessions, 20.4 ± 0.9 follicles were aspirated (mean ± SEM), allowing the recovery of 12.3 ± 0.7 COC per goat and per session, of which 80.1% were suitable for IVM (Grades 1 and 2). Results of in vitro production are detailed in the table. The IVM treatment did not significantly affect cleavage or blastocyst development rates in oocytes derived from slaughterhouse ovaries. Cleavage rates were significantly decreased in LOPU-derived oocytes when compared with control oocytes. For LOPU-derived oocytes, cleavage and final blastocyst development rates were increased significantly and kinetics of embryo development were accelerated when FF-FSH was used during IVM as compared with EGF. The IVM with FF-FSH allowed us to produce 4.1 blatocysts per goat per LOPU session. These results demonstrate the interest in LOPU for goat embryo production once appropriate IVM treatment is used. The difference observed between LOPU and slaughterhouse oocytes in terms of response to IVM treatments may be related to FSH stimulation prior to the LOPU session or to postmortem changes in oocyte responsiveness in the slaughterhouse group. Table 1. Effects of oocyte origin [laparoscopic ovum pickukp (LOPU) or slaughterhouse derived] and maturation treatment [epidermal growth factor (EGF) or follicular fluid (FF)-FSH] on in vitro embryo production (6 replicates)

104 SURVIVAL AND APOPTOSIS RATES AFTER VITRIFICATION OF EARLY, EXPANDED, AND HATCHED CALF AND COW BLASTOCYSTS IN VITRO-PRODUCED USING CRYOTOP AS A DEVICE

2010 ◽  
Vol 22 (1) ◽  
pp. 211
Author(s):  
R. Morató ◽  
D. Izquierdo ◽  
M. T. Paramio ◽  
T. Mogas
Keyword(s):  
Cell Damage ◽  
Survival Rates ◽  
Developmental Competence ◽  
Staining Procedure ◽  
Tunel Staining ◽  
Dna Integrity ◽  
Blastocyst Development ◽  
Integrity Index ◽  
High Degree

Two experiments were designed to determine the ability of 7 and 8 day in vitro-produced blastocysts to survive to the vitrification procedure. Embryos were classified as early blastocysts, expanded, or hatching/hatched blastocysts. Vitrification was done using cryotop devices as described Du et al. (2007). After warming, blastocysts were incubated for 3 h in SOF medium. In the first experiment, we examined the developmental competence of early blastocysts, expanded blastocysts, and hatching/hatched blastocysts after vitrification using the Cryotop method. In the second experiment, warmed blastocysts that had been vitrified on cryotops were fixed in 4% formaldehyde and incubated with TUNEL staining for detecting DNA damaged nuclei. The percentage of TUNEL positive and negative blastomeres was assessed by confocal microscopy. In all experiments cow and calf blastocysts were compared. When the results according to the developmental stage were analyzed, no differences in the survival rates after vitrification of expanded and hatched blastocysts were observed at Day 8 from cow and calf blastocysts. After warming, survival rates of 52.4 and 50% were noted in the groups of expanded and hatched blastocysts respectively from Day 8 cow blastocysts. Similar results were observed in the groups of expanded (54.5%) and hatched (59.4%) blastocysts from Day 8 calf blastocysts. When embryos were vitrified at Day 7, survival rates of 78.4 and 66.7% were observed after warming expanded and hatched blastocysts from cows. In calves, a significant increase in the survival up to 83.3 and 80% was observed after warming expanded and hatched blastocysts. The lowest survival rates were observed in early blastocysts (from 26 to 51%), particularly in those vitrified at Day 8 (≤40%). Following vitrification, cell death was monitored in blastocysts 3 h after warming by TUNEL labelling of cells with damaged DNA. The TUNEL staining procedure was undertaken on Day 7 calf (n = 23) and cow (n = 25) blastocysts, as well as on Day 8 calf (n = 22) and cow (n = 30) blastocysts. When taking into account the stage of blastocyst development, there was a trend toward higher DNA integrity index after warming of expanded and hatched blastocysts compared with early blastocysts in calf and cow groups. So, cell damage was minimal in those blastocysts vitrified at expanded and hatched stage and rates were comparable with those from control fresh blastocysts. These findings suggest that the Cryotop technique seems to be particularly useful for blastocysts presenting a high degree of expansion (expanded and hatched blastocysts), mainly those blastocysts vitrified and warmed at Day 7.

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.

167 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES DERIVED FROM SMALL- OR MEDIUM-SIZED FOLLICLES AND DENUDED OF CUMULUS CELLS BEFORE AND DURING IN VITRO MATURATION

2017 ◽  
Vol 29 (1) ◽  
pp. 192
Author(s):  
P. Ferré ◽  
K. X. Nguyen ◽  
T. Wakai ◽  
H. Funahashi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
First Polar Body

This experiment was undertaken to assess the meiotic and developmental competences of oocytes derived from different sized follicles and denuded of cumulus cells 0, 20, and 44 h after the start of culture for in vitro maturation (IVM). Groups of 60 oocyte-cumulus complexes from small- (SF; <3 mm) and medium-sized follicles (MF; 3–6 mm) were cultured for IVM in porcine oocyte medium with 50 μM β-mercaptoethanol supplemented with 1 mM dibutyryl-cyclic adenosine monophosphate, 10 IU mL−1 of eCG, and 10 IU mL−1 of hCG for 20 h at 39°C and 5% CO2 in air. Then, after washing, they continued culture in fresh β-mercaptoethanol without dibutyryl-cyclic adenosine monophosphate and gonadotropins under the same conditions for another 24 h. At 0, 20, and 44 h of IVM, cumulus cells were removed with 0.1% (wt/vol) hyaluronidase and the denuded oocytes continued IVM culture following the protocol. Mature oocytes with the first polar body were selected, parthenogenetically activated with a single electrical pulse (DC: 1.2 kV/cm, 30 µs), incubated with 4% (wt/vol) BSA and 5 μM cytochalasin B for 4 h, and cultured in porcine zygote medium for 5 days. Cleavage and blastocyst formation rates were observed on Day 2 and 5, respectively. Blastocysts were stained with 4’,6-diamidino-2-phenylindole for cell count assessment. The experiment was replicated 5 times and analysed with a 1- or 2-way ANOVA. If P < 0.05 in ANOVA, a Tukey multiple comparisons test was performed. Regardless of the time of cumulus cell removal, oocytes from MF had significantly higher in rates of maturation, cleavage, and blastocyst rates, as compared with those from SF, whereas there were no significant differences in the cell number of blastocysts between SF and MF (32 v. 34 cells, respectively). When oocytes were denuded before IVM culture, rates of oocyte maturation (37.6% in SF and 50.8% in MF), and blastocyst formation (2.7% in SF and 27.3% in MF) were significantly lower than controls (51.2% in SF and 76% in MF; 25.8% in SF and 48.5% in MF, respectively). When oocytes were denuded 20 h after the start of IVM, oocyte maturation rates were significantly increased (64.1% in SF and 82.5% in MF) as compared with controls, whereas no significant differences were observed in cleavage and blastocyst formation rates in comparison with controls. These results conclude that removing cumulus cells from oocyte-cumulus complexes 20 h after the start of IVM improves the meiotic competence of oocytes derived from both SF and MF, without any reduction of developmental competence of the oocytes following parthenogenetical activation.

192 PREMATURATION OF BOVINE CUMULUS-OOCYTE COMPLEXES WITH CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS AFFECTS BOTH OOCYTE AND BLASTOCYST ULTRASTRUCTURE

2016 ◽  
Vol 28 (2) ◽  
pp. 227
Author(s):  
E. Razza ◽  
H. Pedersen ◽  
L. Stroebech ◽  
M. Machado ◽  
M. Nogueira ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Smooth Endoplasmic Reticulum ◽  
Cell Mass ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Developmental Competence ◽  
Cortical Granules ◽  
Cytoplasmic Vesicles ◽  
Globally Distributed

Oocytes resume meiosis spontaneously when subjected to in vitro maturation (IVM). Cyclic adenosine monophosphate (cAMP) elevating agents have been used for artificial blocking of meiotic resumption (pre-IVM) to allow the oocyte to prepare for maturation, potentially increasing its developmental competence. However, the ultrastructural effects of this pharmacological approach on oocytes and embryos remain to be addressed. We assessed the effects of pre-IVM with cAMP modulators in oocytes (10 for each group) at the end of IVM and in blastocyst (10 for each group) after 7 days of culture. Cumulus-oocyte complexes (COC) were subjected to pre-IVM for 2 h with forskolin (Sigma, St. Louis, MO, USA; 100 μM) and 3-isobutyl-1-methylxanthine) (IBMX, Sigma, 500 μM) followed by 24 h of IVM with FSH-enriched media (IVF Vet Solutions, Adelaide, Australia). Simultaneously, another group of COC was subjected to conventional IVM (con-IVM) for 24 h (EmbryoTransBiotech, Copenhagen, Denmark) with BSA (4 mg mL–1, Sigma), gentamycin (50 mg mL–1), and FSH (0.1 IU mL–1). Matured oocytes were collected for qualitative ultrastructural analysis or followed to IVF. The morphology was carefully evaluated on serially sectioned oocytes and embryos, where each serial section (~60.2-μm section per oocyte/embryo) was analysed under light microscopy. Subsequently, the equatorial section from each oocyte and the section giving the optimal representation of the inner cell mass in each blastocyst was re-embedded and sectioned for electron microcopy as previously described (Hyttel and Madsen 1987 Acta Anat. 129, 12–14). Blastocyst rates did not differ between groups. Ultrastructural analyses revealed subtle ultrastructural differences between pre-IVM and con-IVM conditions. In both groups, oocytes had matured to metaphase II. The perivitelline space of pre-IVM oocytes was significantly narrower than con-IVM. The cytoplasmic vesicles were more abundant and globally distributed in pre-IVM oocytes, whereas at con-IVM a vesicle-free periphery of the ooplasm was frequent, except for cortical granules and clusters of mitochondria associated with smooth endoplasmic reticulum (SER). We observed typical hooded mitochondria and cortical granules either clustered in the periphery or solitarily distributed in the cortical ooplasmic region for both groups. In the blastocysts, differences were noted with respect to especially distribution of ribosomes. In pre-IVM blastocysts, ribosomes were mostly organised in free clusters (polysomes) and peripherally located in cells of the inner cell mass. Con-IVM blastocysts showed ribosomes preferentially associated with the rough ER and often associated with mitochondria. Lipid droplets and rounded mitochondria were observed in both groups as well as apically located tight junctions and desmosomes between adjacent trophectoderm (TE) cells. Pleomorphic and elongated mitochondria were abundant in the TE of pre-IVM blastocysts, whereas the mitochondrial population was more homogenous at con-IVM. These findings suggest that pre-IVM for 2 h affects oocyte and blastocyst ultrastructure. Research was supported by grants 12/50533-2 and 12/23409-9 from FAPESP.

Effect of dibutyryl cyclic adenosine monophosphate on reactive oxygen species and glutathione of porcine oocytes, apoptosis of cumulus cells, and embryonic development

Zygote ◽  
2012 ◽  
Vol 21 (3) ◽  
pp. 305-313 ◽  
Author(s):  
Sang-Hyoun Park ◽  
Il-Jeoung Yu
Keyword(s):  
Reactive Oxygen Species ◽  
Embryonic Development ◽  
Reactive Oxygen ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Oxygen Species ◽  
Cell Numbers

SummaryThe present study was conducted to investigate the effect of dibutyryl cyclic adenosine monophosphate (dbcAMP) supplemented into porcine maturation medium on reactive oxygen species (ROS) and glutathione (GSH) levels of oocytes, and apoptosis of cumulus cells (CC). In addition, the effect of dbcAMP on embryonic development following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Cumulus–oocyte complexes (COCs) were cultured in 0 mM (control), 0.5 mM, 1 mM, 5 mM, or 10 mM dbcAMP-supplemented medium for 22 h, then for another 22 h without dbcAMP. GSH and ROS levels of oocytes were assessed at 44 h of culture by dichlorohydrofluorescein diacetate or 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin staining, respectively. Additionally, COCs were cultured in 0.5 mM or 1 mM dbcAMP and then fertilized in vitro or activated parthenogenetically. Embryonic development and blastocyst cell numbers and apoptosis levels on day 8 of culture were investigated. CC apoptosis at 44 h of culture and blastocyst apoptosis were assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. GSH levels in the 0.5 mM dbcAMP and control groups were increased (P < 0.05), while levels of oocyte ROS and CC apoptosis in the control, 0.5 mM, and 1 mM dbcAMP groups were significantly lower than the levels in other groups. Cleavage and blastocyst rates, cell numbers, and apoptosis levels were not significantly different in embryos derived by either IVF or PA among the groups, with the exception of significantly increased apoptotic levels in IVF blastocysts produced from oocytes treated with 1 mM dbcAMP. In conclusion, dbcAMP treatment during in vitro maturation (IVM) did not improve embryonic development under our study's parameters compared with control conditions, although 0.5 mM dbcAMP showed significantly higher GSH levels and lower blastocyst apoptotic levels compared with 1 mM dbcAMP.

230 EVALUATION OF APOPTOSIS IN IN VITRO-PRODUCED BOVINE EMBRYOS MATURED WITH FORSKOLIN

2013 ◽  
Vol 25 (1) ◽  
pp. 263
Author(s):  
D. M. Paschoal ◽  
M. J. Sudano ◽  
R. R. D. Maziero ◽  
M. D. Guastali ◽  
L. F. Crocomo ◽  
...  
Keyword(s):  
Linear Models ◽  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
High Dose ◽  
Bovine Embryos ◽  
Nuclear Maturation ◽  
Tunel Reaction ◽  
Level Of Significance

The maintenance of oocytes in the germinal vesicle stage for a few hours could result in more competent oocytes for use in biotechnology. Related to this, forskolin (Sigma-Aldrich, St. Louis, MO, USA) is an efficient inhibitor of nuclear maturation because of its ability to increase the levels of intracellular cyclic adenosine monophosphate. This study aimed to show whether the use of forskolin would be able to inhibit maturation in bovine oocytes, producing a higher rate of in vitro embryos. Nellore oocytes from a slaughterhouse (n = 960) were matured in TCM-199 with Earle’s salt + 10% FCS, FSH, and LH, in a 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 h in medium with forskolin at 3 different concentrations, 0. 1 mM (n = 240), 0.05 mM (n = 240), and 0.025 mM (n = 240), whereas untreated oocytes acted as controls (n = 240). The oocytes were then cultured for 18 h in agent-free medium to resume meiosis, completing 24 h of maturation. After 24 h (Day 0) of maturation, oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the same conditions as described above. Semen was selected through Percoll gradient, and the concentration was adjusted to 2 × 106 sperm mL–1. The presumed zygotes were cultured in 90-µL droplets of SOFaa + 0.6% BSA + 2.5% FCS in a 5% CO2, 5% O2, and 90% N2 atmosphere until Day 7, when blastocysts were evaluated. Apoptosis in blastocysts was accessed through TUNEL reaction. Data were analysed by ANOVA, followed by Tukey’s test using the general linear models procedure (PROC GLM) of SAS (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. No statistical differences were observed in blastocyst production rate (n = 297): control (n = 88): 36.7% ± 3.7; 0.1 mM forskolin (n = 61): 25.1% ± 3.7; 0.05 mM forskolin (n = 70): 29.2% ± 3.7; 0.025 mM forskolin (n = 78): 32.6% ± 3.7 (P > 0.05). However, when we analysed the apoptosis rates, differences were found among groups: control: 6.0% ± 6.3a; 0.1 mM forskolin: 33.4% ± 6.3b; 0.05 mM forskolin: 27.2% ± 6.3ab; 0.025 mM forskolin: 10.0% ± 6.3ab (P < 0.05). Although there was no difference in blastocyst rate, the TUNEL technique allowed us to identify that a high dose of forskolin was detrimental for in vitro-produced bovine embryos. FAPESP: 10/50410-2.

Sign in / Sign up

Export Citation Format

Share Document