346 BOVINE EMBRYO DEVELOPMENT AFTER IVM/IVF/IVC OF OOCYTES STORED FOR 22 H IN VARIOUS MEDIA

2007 ◽  
Vol 19 (1) ◽  
pp. 288
Author(s):  
C. Kubota ◽  
T. Kojima ◽  
T. Nagai ◽  
X. Tian ◽  
X. Yang
Keyword(s):  
Subsequent Development ◽  
Industrial Applications ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Bovine Embryo ◽  
Becton Dickinson ◽  
In Vitro Storage ◽  
Bovine Oocytes

The timing of IVM–IVF–IVC is restricted by the onset of oocyte maturation, and sometimes oocytes must be treated at midnight. If we could regulate the timing of IVM of oocytes without decreasing their developmental competence, the IVM–IVF–IVC system could be a more applied technology. The present study was performed to examine the effects of in vitro storage of bovine oocytes in simple media prior to maturation culture to manipulate the start of IVM. Bovine follicular fluid (bFF), Dulbecco's PBS (PBS), M199 Earle salts (M199), and Earle salts supplemented with 5 mM NaHCO3 (M199A) were used as the fundamental media, after an addition of antibiotics, for in vitro storage of bovine cumulus–oocyte complexes (COCs) collected from ovaries obtained at the slaughterhouse. The fundamental media except for bFF were supplemented with 10% fetal bovine serum (FBS) or 1 mg mL−1 polyvinyl alcohol (PVA). COCs were collected from follicles (3–8 mm in diameter) and washed twice in each medium; then approximately 50 COCs were submerged in 1 mL of each medium in cryotubes (Falcon #2812, 2.5 mL; Becton Dickinson Labware, Lincoln, NJ, USA), which were stored in a container kept at 38.5°C for 22 h under air-closed condition (in vitro storage: IVS). Subsequently, the stored COCs were in vitro-matured (IVM) for 22 h in M199 with 10% FBS and 20 µg mL−1 estradiol, fertilized (IVF), and cultured in CR1aa (IVC) for examination of their development to the blastocyst stage (Kubota et al. 1998 Mol. Reprod. Dev. 51, 281–286). Fresh oocytes without IVS were used as controls. The nuclear status of oocytes after IVS–IVM was compared to that of control oocytes by aceto-orcein stain. Their developmental rates to the blastocyst stage after IVM–IVF–IVC were compared between experimental and control groups. The experiment was repeated more than 3 times, and results were statistically analyzed using Student's t-test. When bFF and PBS supplemented with FBS or PVA were used for IVS, the rates of survived COCs after IVS and the development to the blastocyst stage after IVM–IVF–IVC (bFF (n = 87): 0%, 0%; PBS/FBS (n = 72): 84%, 1%; and PBS/PVA (n = 81): 89%, 6%, respectively) were significantly lower than those of the control group (n = 406; 97% and 29%, respectively). On the other hand, when M199A supplemented with FBS or PVA was used for IVS, the survival rate after IVS and the developmental rate to the blastocyst stage after IVS–IVM–IVF (M199A/FBS (n = 97): 82%, 28%; and M199A/PVA (n = 111): 98%, 31%, respectively) did not differ from those of the control group. After IVS, cumulus expansion was not seen and most of the oocyte nuclei reached the GVBD stage. These results suggest that the nuclear maturation progress of bovine oocytes can be regulated for at least 22 h in M199A without any deleterious influence on the number of oocytes surviving at an immature state after the storage and their subsequent development to the blastocyst stage after IVM–IVF–IVC. The delayed maturation allows a flexible fertilization schedule which is advantageous in research and industrial applications.

scholarly journals Abilities of cumulus and granulosa cells to enhance the developmental competence of bovine oocytes during in vitro maturation period are promoted by midkine; a possible implication of its apoptosis suppressing effects

Reproduction ◽  
2006 ◽  
Vol 132 (4) ◽  
pp. 549-557 ◽  
Author(s):  
S Ikeda ◽  
K Saeki ◽  
H Imai ◽  
M Yamada
Keyword(s):  
Dna Fragmentation ◽  
Granulosa Cells ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Development ◽  
Bovine Oocytes

We previously reported that when midkine (MK), a heparin-binding growth differentiation factor was used inin vitromaturation (IVM) culture of bovine cumulus-enclosed oocytes (CEOs), their developmental competence to the blastocyst stage afterin vitrofertilization (IVF) was enhanced and the effect of MK might be mediated by its action upon mural granulosa cells and cumulus cells that closely surround the oocyte. In the present study, when denuded oocytes (DOs) were matured in IVM medium with or without MK (200 ng/ml) in the presence or absence of isolated cumulus cell masses and subjected to IVF, the enhancing effects of MK on the developmental competence of DOs to the blastocyst stage after IVF were exerted only in the presence of cumulus cells. In addition, we prepared the conditioned media of granulosa cells cultured with or without 200 ng MK/ml (CMMK+ or CMMK− respectively) and examined their effects on the IVM of DOs in terms of their developmental competence to the blastocyst stage after IVF. The supplementation of CMMK+ into IVM medium at 40% (v/v) significantly enhanced the blastocyst development compared with the no additive control and the CMMK− supplemented groups. Furthermore, the effects of MK during IVM of bovine CEOs on the cumulus cell apoptosis were investigated. CEOs were cultured up to 24 h in IVM medium without (control) or with 200 ng MK/ml. The genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect the apoptotic internucleosomal DNA fragmentation. DNA fragmentation was scarcely detected at the start of IVM, whereas it increased time-dependently as the IVM culture progressed. The degree of the fragmentation was significantly lower in the MK-treatment group compared with the control group at 18 and 24 h of IVM. The apoptosis-suppressing effect of MK on cumulus cells was further confirmedin situby using TUNEL on CEOs. In conclusion, data from the present study further confirmed that MK enhances the developmental competence of bovine oocytes via cumulus and granulosa cells. It was also demonstrated that MK suppresses the apoptosis that occurs in cumulus cells during the period of IVM of bovine CEOs. The putative soluble factor(s) from cumulus cells was suggested from the experiment using CMMK+ . MK may promote the production of such factors in part by its anti-apoptotic effects on cumulus cells.

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-328
Author(s):  
Galina Singina
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P&lt;0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P&lt;0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

102 DEVELOPMENTAL COMPETENCE OF BUFFALO (BUBALUS BUBALIS) OOCYTES VITRIFIED AT DIFFERENT STAGES OF MATURATION IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 159
Author(s):  
K. Loganathasamy ◽  
R. Rajhans ◽  
G. SaiKumar ◽  
G. T. Sharma
Keyword(s):  
Expression Pattern ◽  
Glucose Transporter ◽  
Storage Period ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Control Group ◽  
Hsp 70 ◽  
Glucose Transporter 1 ◽  
Total Rna

Cryopreservation of unfertilized oocytes at very low temperature (-196�C) is carried out to ensure their continuous availability during different assisted reproductive techniques. However, various problems associated with the freezing of oocytes influence their developmental competence, resulting in suboptimal embryo production. The present study was planned to assess the developmental competence of buffalo oocytes vitrified at different meiotic stages of maturation. Expression profile of developmentally important genes, viz, heat shock protein 70 (HSP 70) and glucose transporter 1 (Glut1), was verified in these vitrified warmed oocytes. Buffalo cumulus-oocyte complexes were collected from slaughterhouse ovaries and divided into six groups: control (no vitrification); 0 h group (vitrified before maturation), and 6-, 12-, 18-, and 24-h groups [vitrified respectively at 6, 12, 18, and 24 h post-in vitro maturation (IVM)]. Vitrification solution consisted of propylene glycol (40% w/v), and trehalose (0.25 mol/L) in PBS + BSA (4% w/v) and vitrification was carried out by directly plunging 0.25-mL French mini-straws into liquid nitrogen. After a minimum storage period of 7 days, the straws were thawed at 37�C for 30 sec. In all groups, the oocytes completed 24-h of maturation. After 24 h maturation, a few oocytes from each of the six groups were stained with ethidium bromide to reveal their nuclear status. The remaining oocytes were co-incubated with frozen thawed buffalo semen in fertilization TALP with 6 mg/mL fatty acid free BSA and 10 �g/mL heparin sodium salt for 18 h. Presumptive zygotes were cultured in mSOF for 8 days. Vitrified warmed oocytes were subjected to total RNA isolation and RT-PCR for detection of mRNA transcripts of HSP 70 and Glut1 genes. Data were analyzed by one-way ANOVA and F-test analysis. Differences of P < 0.05 were considered significant. The percentage of oocytes recovered from all five vitrification groups varied from 89 to 92 out of which 84-91% of oocytes were morphologically normal. A higher proportion of nonvitrified control oocytes (72.8%; 40/55) reached the metaphase II stage than for the oocytes vitrified at 24 (60%; 36/60), 18 (54.4%; 31/57), 12 (42.3%; 22/52), 6 (33.3%; 20/60), and 0 (31.7%; 19/60) h of IVM. The cleavage rate of nonvitrified control oocytes was higher (36.8%) than that of oocytes vitrified at 0 (1.6%), 6 (2.0%), 12 (3.2%), 18 (5.3%), and 24 (5.2%) h of IVM. With regard to subsequent development, 0- and 6-h oocytes were blocked at 8 cells, whereas in other groups development reached the late morula (4.8%) and blastocyst (3.5%) stages, confirming that the stage of maturation at which oocytes are vitrified influenced the nuclear maturation and developmental competence. Total RNA content was 2.24 � 0.40 ng/oocyte in the control group and 2.11 � 0.22 ng/oocyte in the group vitrified after 24 h of IVM. The expression pattern of HSP 70 and Glut1 was identical in control and vitrified groups, indicating that the vitrification protocol did not alter the expression pattern of these genes.

40 TRICHOSTATIN A TREATMENT EFFECTS ON IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER CAT EMBRYOS DEPEND ON RECIPIENT CYTOPLASM SPECIES

2015 ◽  
Vol 27 (1) ◽  
pp. 113
Author(s):  
L. T. K. Do ◽  
Y. Sato ◽  
M. Taniguchi ◽  
T. Otoi
Keyword(s):  
Nuclear Transfer ◽  
Treatment Effects ◽  
Trichostatin A ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Control Group ◽  
Fluid Medium ◽  
First Polar Body ◽  
Bovine Oocytes

The developmental ability of interspecies somatic cell nuclear transfer (iSCNT) embryos decreases as the taxonomic distance between the donor and recipient species increases. Treatment of cat iSCNT embryos using bovine oocytes with 50 nM of trichostatin A (TSA) improves in vitro embryonic development (Wittayarat et al. 2013 Cell. Reprogram. 15, 301–308). This study investigated whether the TSA treatment effects differ between the development of cat iSCNT embryos reconstructed with porcine and bovine oocytes. Porcine and bovine cumulus-oocyte complexes were in vitro matured for 44 h and 24 h, respectively. After cumulus cell removal, enucleation was performed by aspiration of the metaphase II plate and the first polar body using a piezo-driven pipette. A cat fibroblast cell was then injected into cytoplasm of successfully enucleated oocyte. Reconstructed cybrids were electrically activated by a single 1.5 kV cm–1 pulse for 100 µs (pig-cat embryos), or a 2.3 kV cm–1 pulse for 30 µs (cow-cat embryos). Pig-cat and cow-cat embryos were cultured in porcine zygote medium (PZM)-5 and modified synthetic oviducal fluid medium (mSOF), respectively. After electrical activation, pig-cat and cow-cat embryos were cultured in medium supplemented with 5 µg mL–1 cytochalasin B + 50 nM TSA (TSA group) or without TSA (control group), and the cow-cat embryo medium was also supplemented with 10 µg mL–1 cycloheximide. After 2 h, TSA-treated pig-cat and cow-cat embryos were incubated in medium supplemented with TSA for 22 h, followed by 48 h incubation without TSA. Pig-cat and cow-cat control embryos were cultured in medium without TSA for 70 h after activation. Then, all pig-cat and cow-cat embryos were cultured in porcine blastocyst medium (PBM) or mSOF medium supplemented with 5% fetal bovine serum, respectively, for 5 additional days. Four to seven replicates were performed for each experiment. Data were analysed using Student's t-test. For pig-cat embryos, no difference was observed in cleavage rates between both groups, but development to the blastocyst stage was higher in the pig control group (n = 147, 8.0%) than that of pig TSA group (n = 131, 0.7%; P < 0.05). In contrast, development to the blastocyst stage in cow-cat embryos was not observed in the cow control group (n = 125, 0%), but it was observed in cow TSA group (n = 136, 3.7%). These results indicate that TSA treatment effects are species-specific, but those effects remain to be clarified.

99 In Vitro Development of Alpaca Embryos Obtained by Bisection

2018 ◽  
Vol 30 (1) ◽  
pp. 189
Author(s):  
L. Landeo ◽  
R. S. Molina ◽  
M. E. Zuñiga ◽  
T. R. Gastelu ◽  
C. Sotacuro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Streptomyces Griseus ◽  
Petri Dish ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Morula Stage

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.

scholarly journals Effect of storage tube material and resveratrol during liquid storage of matured bovine oocytes on subsequent development

2017 ◽  
Vol 65 (4) ◽  
pp. 546-555
Author(s):  
Tayita Suttirojpattana ◽  
Tamás Somfai ◽  
Satoko Matoba ◽  
Takashi Nagai ◽  
Rangsun Parnpai ◽  
...  
Keyword(s):  
Embryo Development ◽  
Subsequent Development ◽  
Control Group ◽  
Tube Material ◽  
Blastocyst Development ◽  
Liquid Storage ◽  
Glass Tubes ◽  
Bovine Oocytes

This study determined the optimum storage vessel and the effects of resveratrol for the storage of in vitro matured (IVM) bovine oocytes. After IVM, the oocytes were kept in a Hepes-buffered medium at 25 °C for 20 h in different containers including Eppendorf tubes (ET) made of polypropylene (PP) and polystyrene (PS), and tissue culture tubes (TCT) made of PP, PS, and glass. Then oocytes were subjected to IVF and subsequent in vitro embryo development was compared among the groups and to that of a control group without storage. The percentage of blastocyst development in the control group was significantly higher than in the stored groups (P < 0.05). Among oocytes stored in TCT, the percentage of blastocyst development of oocytes stored in glass TCT was significantly higher than that of oocytes stored in PP and PS TCT (P < 0.05); however, it did not differ from that of oocytes stored in ET. The quality of blastocysts did not differ among the control and stored groups. Embryo development was not affected when 0.1, 1 or 10 μM resveratrol was added to the medium during oocyte storage. In conclusion, glass tubes were optimal for oocyte storage and resveratrol did not improve the development of stored oocytes.

133 VITRIFICATION CAUSES DAMAGE OF THE ANTIOXIDANT DEFENSE SYSTEM IN IVM PORCINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 184 ◽  
Author(s):  
T. Somfai ◽  
M. Ozawa ◽  
J. Noguchi ◽  
H. Kaneko ◽  
K. Ohnuma ◽  
...  
Keyword(s):  
Polar Body ◽  
Antioxidant Defense System ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Cleavage Rate ◽  
Sperm Penetration ◽  
Second Polar Body ◽  
Polar Body Extrusion

The present study investigated the ability of in vitro-matured (IVM) porcine oocytes to be fertilized in vitro after vitrification. Oocytes matured in vitro for 46 h according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) were cryopreserved by solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) or subjected to the steps of SSV without cooling (toxicity control, TC). Oocyte viability was assessed 2 h after treatment by morphology and fluorescein diacetate staining. Live oocytes were in vitro-fertilized (IVF) and cultured (IVC) for 6 days according to Kikuchi et al. (2002). Fertilization and pronuclear development of oocytes were assessed 10 h after IVF by aceto-orcein staining. Cleavage and blastocyst rates were recorded during IVC. Glutathione (GSH) and hydrogen peroxide levels in oocytes were analyzed by DTNB-glutathione disulfide reductase recycling assay and 20,70-dichlorofluorescein fluorescence assay, respectively. Data were analyzed by ANOVA and paired t-test. The rate of live oocytes after SSV was lower compared to the control and the TC groups (54.4%, 100%, and 100%, respectively; P &lt; 0.05). Sperm penetration rates of SSV oocytes were lower than those of the control group (51.9% and 67.8%, respectively; P &lt; 0.05). Significantly fewer penetrated oocytes in the SSV group formed male pronuclei than those in the control and the TC groups (66.7%, 96.5%, and 98.5%, respectively; P &lt; 0.05). There were no differences in second polar body extrusion and monospermy rates between the treatment groups. The cleavage rate of SSV oocytes was significantly lower than that of the control and the TC groups (13.3%, 46.6%, and 47.7%, respectively; P &lt; 0.05). Blastocyst rates of control and TC oocytes were similar (20.7% and 23.6%, respectively), whereas only a single embryo developed to the blastocyst stage in the SSV group. GSH content of SSV oocytes was significantly lower than that of the control oocytes (7.3 pM and 10.5 pM, respectively), whereas the peroxide level was higher in SSV oocytes than in the control oocytes (59.0 and 50.5 FIU, respectively; P &lt; 0.05). Our results reveal a cryopreservation-related drop of intracellular GSH level in oocytes, which may cause their decreased ability to form a male pronucleus and their increased sensitivity to oxidative stress. These factors might contribute to the low developmental competence of vitrified oocytes. This work was supported by a grant-in-aid for the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers (P05648) and the Bilateral Scientific and Technological Collaboration Grant between Hungary and Japan (TET, no. JAP-11/02).

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

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

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

scholarly journals Developmental competence and expression of the MATER and ZAR1 genes in immature bovine oocytes selected by brilliant cresyl blue

Zygote ◽  
2009 ◽  
Vol 18 (3) ◽  
pp. 209-216 ◽  
Author(s):  
Gustavo Bruno Mota ◽  
Ribrio Ivan Tavares Pereira Batista ◽  
Raquel Varella Serapião ◽  
Mariana Cortes Boité ◽  
João Henrique Moreira Viana ◽  
...  
Keyword(s):  
Developmental Competence ◽  
Control Group ◽  
Blue Dye ◽  
Brilliant Cresyl Blue ◽  
Immature Oocytes ◽  
Relative Expression ◽  
Cresyl Blue ◽  
Blastocyst Rate ◽  
Bovine Oocytes

SummaryThe objective of this work was to evaluate the selection of immature bovine oocytes by brilliant cresyl blue dye (BCB) and expression of transcripts MATER and ZAR1. Cumulus–oocyte complexes (COCs) from slaughterhouse ovaries were exposed to BCB diluted in mDPBS and incubated for 60 min at 38.5 °C in humidified air. After exposure those COCs were distributed in two groups, according to their cytoplasm colour: BCB+ (coloured cytoplasm) or BCB− (colourless cytoplasm). The control group was submitted to in vitro maturation (IVM) immediately after morphological selection and holding control group COCs were exposed to mDPBS without BCB but in the same incubation conditions of BCB+ and BCB− group. The COCs of all groups were submitted to IVM, in vitro fertilization (IVF) and in vitro culture (IVC). Cleavage rate (72 h post-insemination) was similar between control (65.3%) and BCB+ (64.4%) groups, but greater than (p < 0.05) holding control (49.8%) and BCB− (51.3%) groups. Blastocyst rate (192 h post-insemination) was not different between BCB+ (18.5%) and control (16.3%) groups, but greater (p < 0.05) than BCB− (8.4%) group. No difference was found for blastocyst rate between holding control group (14.2%), control and BCB+ groups. The relative expression of MATER and ZAR1 genes was evaluated by real-time PCR in immature oocytes collected from the control, holding control, BCB+ and BCB− groups. Despite the relative expression of MATER in holding control, BCB+ and BCB− were down regulated in comparison to control group there was no statistical difference (p > 0.05) in the relative expression of MATER and ZAR1 transcripts among groups. The results indicate that the BCB dye detects immature oocyte populations with different developmental competence, although no improvement in in vitro embryo production using oocytes exposed or not to BCB was observed. Development competence of immature oocytes exposed to BCB does not seem to be associated with variations in the expression of MATER and ZAR1 transcripts.

Sign in / Sign up

Export Citation Format

Share Document