scholarly journals Synergistic impact of α-linolenic acid and α-tocopherol on in vitro maturation and culture of buffalo oocytes

2024 ◽  
Vol 84 ◽  
Author(s):  
A. Azam ◽  
R. Ejaz ◽  
S. Qadeer ◽  
S. Irum ◽  
A. Ul-Husna ◽  
...  
Keyword(s):  
Linolenic Acid ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Cumulus Cell ◽  
Early Embryo ◽  
Control Group ◽  
Group 5 ◽  
Group 2

Abstract The objective of the current study was to investigate the synergistic impact of α-Tocopherol and α-Linolenic acid (100 µM) on IVM and IVC of Nili Ravi buffalo oocytes. Oocytes were obtained from the ovaries of slaughtered buffaloes within two hours after slaughter and brought to laboratory. Buffalo cumulus oocyte complexes were placed randomly in the five experimental groups included; GROUP 1: Maturation media (MM) + 100 µM ALA (control), GROUP 2: MM + 100 µM ALA + 50μM α-Tocopherol, GROUP 3: MM + 100 µM ALA + 100μM α-Tocopherol, GROUP 4: MM + 100 µM ALA + 200 μM α-Tocopherol and GROUP 5: MM + 100 µM ALA + 300 μM α-Tocopherol under an atmosphere of 5% CO2 in air at 38.5 °C for 22-24 h. Cumulus expansion and nuclear maturation status was determined (Experiment 1). In experiment 2, oocytes were matured as in experiment 1. The matured oocytes were then fertilized in Tyrode’s Albumin Lactate Pyruvate (TALP) medium for about 20 h and cultured in synthetic oviductal fluid (SOF) medium to determine effect of α-Linolenic acid (100 µM) and α-Tocopherol in IVM medium on IVC of presumptive zygotes. To study the effect of α-Linolenic acid (100 µM) in IVM media and increasing concentration of α-tocopherol in the culture media on early embryo development (Experiment 3), the presumptive zygotes were randomly distributed into the five experimental groups with increasing concentration of α-tocopherol in culture media. Higher percentage of MII stage oocytes in experiment 1(65.2±2.0), embryos at morula stage in experiment 2 (30.4±1.5) and experiment 3 (22.2±2.0) were obtained. However, overall results for cumulus cell expansion, maturation of oocyte to MII stage and subsequent embryo development among treatments remain statistically similar (P > 0.05). Supplementation of α-tocopherol in maturation media having α-Linolenic acid and/or in embryo culture media did not further enhance in vitro maturation of oocyte or embryo production.

170 INFLUENCE OF OOCYTE QUALITY AND DIFFERENT MEDIA SUPPLEMENT ON IN VITRO MATURATION, CLEAVAGE, AND EMBRYO DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 199
Author(s):  
M. M. Waheed ◽  
K. H. El-Shahat ◽  
A. M. Hammam
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Oocyte Quality ◽  
Bubalus Bubalis ◽  
Poor Quality ◽  
Developmental Competence ◽  
Control Group ◽  
Excellent Quality

A series of 4 factorial-arranged experiments were conducted to study the effect of oocyte quality and different in vitro maturation (IVM) media supplements on IVM, cleavage, and embryo development of buffalo (Bubalus bubalis) oocytes. Buffalo ovaries were collected at a local abattoir in a warm (32–35°C) saline (0.9% NaCl), and oocytes were aspirated using an 18-gauge needle. In experiment 1, oocytes (n = 320) were classified according to the number of cumulus cell layers and morphology of ooplasm as excellent, good, or fair. Oocytes were cultured for IVM, fertilization, and embryo culture (IVMFC) in TCM-199 + 10% FCS. In experiment 2, excellent quality oocytes (n = 237) were subjected to IVM in TCM-199 enriched with either 10% FCS or oestrous buffalo serum (EBS; 20–40 pg mL–1) and then fertilized using frozen–thawed buffalo semen capacitated in Bracket and Oliphant's (BO) medium containing heparin (0.02 mg mL–1) and sodium caffeine benzoate (3.89 mg mL–1). In experiment 3, oocytes (n = 290) were classified into 2 groups; Group 1, without gonadotropins, served as a control; Group 2, in which IVM medium was supplemented with 20 IU mL–1 equine chorionic gonadotropins (eCG). Experiment 4 was carried out to examine the suitable capacitating agent added to BO medium, either heparin or caffeine or both (n = 210 fertilized oocytes). In all experiments (multiple replicates), oocytes (2–6 mm in diameter) were kept at 39°C under 5% CO2 for IVMFC and examined several times for cleavage and embryo development (morula and blastocyst). Statistical analysis was carried out using Chi-squared test. Excellent and good quality oocytes produced a higher (P < 0.05) maturation, cleavage, and morula development rates than poor quality oocytes (70% and 65% v. 33.3%), (50% and 46.2% v. 25%), and (42.9% and 33.3% v. 10%), respectively. Blastocyst production rate was also higher (P < 0.05) for excellent compared with good quality oocytes (28.6% v. 16.7%, respectively). In experiment 2, the IVM and cleavage rates were significantly higher (P < 0.05) in IVM medium plus 10% EBS than those cultured in 10% FCS (73% v. 45% and 50% v. 33.3%, respectively). In experiment 3, the addition of eCG to maturation medium increased (P < 0.05) developmental competence of buffalo oocytes (IVMFC) compared with control medium (16% v. 4%). In experiment 4, the addition of heparin together with caffeine to BO medium produced significantly higher (P < 0.05) cleavage and embryo developmental rates compared with heparin or caffeine alone (56.3% v. 33.3% and 35.7%, respectively; 22.2% v. 10% and 8%, respectively). In conclusion, excellent quality oocytes cultured in IVM medium supplemented with either EBS or eCG and fertilized with capacitated buffalo spermatozoa in BO medium enriched with heparin and caffeine progressively enhanced developmental competence of buffalo oocytes.

P–187 Multinucleation and reverse cleavage, sings of early embryo self-repair mechanisms

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Munuer. Puigvert ◽  
V. Montalv Pallès ◽  
J Mass. Hernáez ◽  
A García-Faura ◽  
B Marquè. López-Teijón ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Media ◽  
Live Birth Rate ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Control Group ◽  
Complete Fusion ◽  
Depth Analysis ◽  
Self Repair

Abstract Study question Have multinucleation and reverse cleavage any effect on embryo development and clinical outcomes on IVF treatments? Summary answer Embryos capable of repairing dysmorphisms and developing up to blastocyst stage keep intact their ability to become healthy babies. What is known already Time-lapse systems allow IVF laboratories to perform in-depth analysis of embryo development using the continuous monitoring tool. Some events that are impossible to detect with conventional morphologic evaluation, such as reverse cleavage or multinucleation, can be detected using time-lapse. Even though the low scientific evidence, the presence of these events is considered a negative factor when the embryo quality assessment is performed. However, it has been described the possibility that embryos have self-repair intrinsic methods. Study design, size, duration Retrospective study including data from 3,577 cycles with 21,274 embryos cultured until blastocyst stage using one-step culture media in time-lapse incubators (Embryoscope, Vitrolife) up to day 5/6 between 2014 and 2019. Participants/materials, setting, methods Three embryo groups were considered: Control group, embryos without multinucleation or reverse cleavage (CG; n = 16,897); Multinucleation group, embryos with at least one blastomere multinucleated on D + 2/3 (MNC; n = 3,879) and Reverse Cleavage group, embryos undergoing complete fusion of two blastomeres on D + 2/3 (RC; n = 498). Single embryo transfer was performed on blastocyst stage. Clinical outcome rates were compared between groups and analyzed by Chi-square test. Main results and the role of chance As published by other groups, the 2.3% of our embryos showed at least one reverse cleavage event and we observed multinucleation in the 18.2% of the embryos. Blastocyst rate of dysmorphism groups was significantly lower (p &lt; 0.05) than Control group (MNC=20.0%; RC = 27.7%; CG = 58.0%). Once transferred, MNC and RC evolutive embryos showed significantly lower pregnancy (MNC=47.9%; RC = 46.8%; CG = 60.8%; p &lt; 0.05) and clinical pregnancy rates (MNC=39.4%; RC = 40.4% CG = 50.6%; p &lt; 0.05) than the Control group (p &lt; 0.05). However, during the post-implantational development the negative effect of dysmorphisms disappears, reaching values of live birth rate comparable to the Control group (MNC=28.3%; RC = 31.9% CG = 33.8%; p = 0.17). These results prove the importance of blastocyst culture and the inherent capability of the embryos to overcome some abnormal dynamics as multinucleation and reverse cleavage. Thus, these embryos showing the poor-prognosis events can be considered for transfer or vitrify. Limitations, reasons for caution There is a wide difference on sample size between groups despite the fact that the statistical analysis considers that into account. There are some ongoing pregnancies in all groups. Wider implications of the findings: When analyzing the development of embryos undergoing reverse cleavage and multinucleation, we hypothesize that these embryos could be showing a self-correction mechanism for some type of error detected. Embryos capable of repairing and developing up to blastocyst stage keep intact their ability to become healthy babies. Trial registration number Not applicable

161 EFFECT OF OVIDUCTAL FLUID ON BOVINE OOCYTE ZONA PELLUCIDA HARDENING AND SPERM BINDING, AND ON EARLY EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 210
Author(s):  
P. Hugon ◽  
J. Lamy ◽  
E. Corbin ◽  
P. Mermillod ◽  
M. Saint-Dizier
Keyword(s):  
Corpus Luteum ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Developmental Stages ◽  
Early Embryo ◽  
Developmental Competence ◽  
Control Group ◽  
Vitro Fertilization ◽  
Oviductal Fluid

This study was designed to evaluate the effects of oviductal fluid at different periovulatory times on oocyte maturation, modification of the zona pellucida (ZP), fertilization and embryo development. Bovine oviducts were collected at a slaughterhouse and classified as preovulatory (pre-ov: 1 pre-ov follicle and a regressing corpus luteum) or post-ovulatory (post-ov: a corpus haemorrhagicum or recent corpus luteum; n = 10 cows/stage). Both oviducts were flushed with 1 mL of sterile TCM-199, and oviductal flushes (OF) were aliquoted and stored at –80°C. Abattoir-derived bovine ovaries were aspirated and cumulus‐oocyte complexes (COC) with at least 3 cumulus layers and homogeneous oocyte cytoplasm were in vitro matured for 22 h in standard maturation medium (control group, n = 319) or in standard medium with 2× concentrated additives supplemented (50% v/v) with pre-ov OF (n = 255) or post-ov OF (n = 248). After in vitro maturation (IVM), subgroups of COC were denuded, and the time of digestion of the ZP by pronase 0.1% (v/v in TCM-199) was determined to evaluate ZP hardening. After IVM, COC were fertilised in vitro for 18–20 h at a final concentration of 1.106 million spermatozoa (spz)/mL. After in vitro fertilization (IVF), COC were denuded, washed twice and cultured for 8 days more under standard conditions. After IVM, IVF, and embryo culture, oocytes/embryos were fixed with ethanol, stained with Hoescht, and examined under fluorescence microscopy for determination of (1) maturation and developmental stages, (2) numbers of fertilised and polyspermic oocytes, and (3) spz bound to the ZP. Percentages were compared between groups by chi-square. Times of ZP digestion were compared by Kruskal‐Wallis test. Numbers of spz bound to the ZP were compared by ANOVA on normalised data followed by Newman-Keuls tests. Data are presented as mean ± SEM. A P < 0.05 was considered significant. Addition of OF during IVM had no effect on maturation rates compared with the control. However, the digestion time of the ZP by pronase was reduced after IVM with pre-ov OF (313 ± 21 s; n = 26) compared with post-ov OF (459 ± 23 s; n = 23) but not with the control (416 ± 30 s; n = 25). After IVF, the number of spermatozoa bound to the ZP was increased after IVM with pre-ov OF (57 ± 5 spz/oocyte; n = 67) and decreased after IVM with post-ov OF (34 ± 3 spz/oocyte; n = 76) compared with the control (42 ± 5 spz/oocyte; n = 60). Addition of OF during IVM had no effect on rates of IVF and polyspermia. However, the rate of development to the blastocyst stage was less after IVM with post-ov OF (10%, n = 97 cleaved oocytes) compared with control (24%, n = 130) and pre-ov OF (29%, n = 101). In conclusion, the OF collected before ovulation decreased the resistance of the ZP to protease digestion and increased its ability to bind spz, whereas it was the opposite for the post-ov OF. Furthermore, the post-ov OF decreased the developmental competence of fertilised oocytes.

332 GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) ENHANCES CUMULUS CELLS EXPANSION OF IN VITRO-MATURED BOVINE CUMULUS - OOCYTE COMPLEXES IN A CHEMICALLY DEFINED MEDIUM

2010 ◽  
Vol 22 (1) ◽  
pp. 322
Author(s):  
D. D. Bücher ◽  
M. A. Castro ◽  
M. E. Silva ◽  
M. A. Berland ◽  
I. I. Concha ◽  
...  
Keyword(s):  
Cell Viability ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Cell Number ◽  
Control Group ◽  
Cumulus Expansion ◽  
Gm Csf ◽  
Nuclear Stage

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine that stimulates proliferation, differentiation and function in different cells types. We have previously demonstrated (Bücher DD et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 146 abst.) that both subunits of GM-CSF receptor are expressed in granulosa cells from antral follicles in bovine ovaries. Also, we determined that the cytokine enhances glucose uptake through facilitative hexose transporters in granulosa cells in primary culture. The goals of the present study were to characterize the expression of GM-CSF receptor in cumulus cells and oocytes from bovine antral follicles and to determine its effects on in vitro-matured bovine COCs in a chemically defined medium. To determine the presence of a and |5 subunits of GM-CSF receptor, COCs were aspirated from follicles <8 mm in diameter, fixed, and submitted to immunocytochemistry. To study the effect of GM-CSF on in vitro maturation of oocytes, COCs (n =481) were cultured using serum-free medium (SOF) containing 0, 1, 10, and 100 ng mL-1 of human recombinant GM-CSF (R&D Systems, Inc., Minneapolis, MN, USA) for 22 h at 39°C, 5% CO2 in humidified air. Nuclear stage, cumulus expansion, cumulus cell number, and viability were analyzed after in vitro maturation. Cumulus expansion was assessed using the cumulus expansion index (CEI) (Fagbohun C and Down S 1990 Biol. Reprod. 42, 413-423). Nuclear stage was evaluated using aceto-orcein stain. To determine cumulus cell viability and number, COCs (n = 10-12 per group) were transferred into an Eppendorf tube and cumulus cells were removed by vortexing for 3 min, stained with trypan blue and counted with a hemocytometer. The study was conducted in 6 replicates. Data from cumulus expansion and cell number were analyzed by Kruskal-Wallis analysis. Data for nuclear stage and cell viability were analyzed by chi-square analysis and one way ANOVA, respectively. Both receptor subunits were present in cumulus cells and oocytes from COCs. COCs cultured in 10 and 100 ng mL-1 GM-CSF had CEI scores (0.8 and 1.22, respectively) greater (P < 0.01) than controls (0.2), but the proportion of COCs displaying second metaphase did not differ (P = 0.5) among treatment groups. GM-CSF at a concentration of 100 ng mL-1 increased (P < 0.01) cumulus cell viability by more than 20% compared to the control group. Similarly, GM-CSF at concentrations of 10 and 100 ng mL-1 increased (P < 0.05) cumulus cell number by more than 20% and 45%, respectively, from the control group. The use of a specific inhibitor of PI3 kinase (Ly294002; 10 and 100 μM) blocked the stimulatory effect of GM-CSF on cumulus expansion, cell viability, and cell number. In conclusion, the results of the study suggest a plausible modulator role of GM-CSF in the metabolism and function of cumulus cells and oocytes during in vitro maturation. Funding from Faculty of Veterinary Sciences, Universidad Austral de Chile, MECESUP AUS-0005, AUS-0601, and DID D-2006-24 and from Universidad Católica de Temuco, research grant 2007 DGI-CDA-04.

87 EVALUATION OF THE NUMERICAL CHROMOSOMAL ABNORMALITIES ON IN VITRO EARLY BOVINE EMBRYOS: EFFECT OF THE CELL CO-CULTURE WITH GRANULOSA CELLS

2014 ◽  
Vol 26 (1) ◽  
pp. 157
Author(s):  
S. Demyda-Peyrás ◽  
M. Hidalgo ◽  
J. Dorado ◽  
M. Moreno-Millan
Keyword(s):  
Embryo Development ◽  
Granulosa Cells ◽  
Chromosomal Abnormalities ◽  
Culture Media ◽  
Gradient Centrifugation ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Humid Atmosphere ◽  
Early Embryos

Chromosomal numerical abnormalities (CNA) were described as a major cause of developmental failures in in vitro-produced (IVP) embryos. It has been described that CNA are influenced by the post-fertilization culture environment of the embryo. Furthermore, it was demonstrated that the use of different culture media affects the CNA rates. The addition of granulosa cells during early embryo development is a well-known procedure to simplify the culture of bovine IVP and cloned embryos. This technique avoids the use of culture environments saturated with N2 (tri-gas chambers). The aim of this study was to determine the effect of the addition of granulosa cells in the chromosomal abnormalities of IVP cattle embryos. Cumulus–oocyte complexes (COC) were matured in TCM-199 medium, supplemented with glutamine, sodium pyruvate, FSH, LH, oestradiol, and gentamicin during 20 h at 38.5°C in a 5% CO2 humid atmosphere. Subsequently, matured oocytes were fertilized in IVF-TALP medium using 1 × 106 spermatozoa mL–1, selected through a Percoll gradient centrifugation. After fertilization, zygotes were divided in 2 groups and cultured in TCM-199 medium for 48 h, with (TCM-GC) or without (TCM) the addition of 1 × 106 granulosa cells. These cells were obtained by centrifuging and washing the follicular fluid remaining from searching dishes and adjusted to the working concentration. After culture, a total of 106 early embryos (72 hpi) were cytogenetically evaluated following our standard laboratory techniques. Embryos showing normal development were individually fixed onto a slide, disaggregated into blastomeres with acetic acid, and stained with Giemsa solution. Chromosomal numerical abnormalities were evaluated by direct observation at 1250× magnification in a brightfield microscope. Percentage of normal diploid embryos (D) and abnormal haploid (H), polyploid (P), or aneuploid (A) embryos were determined. Results were statistically compared between treatments using a Z test for proportions. Results were: D = 81.4%, H = 7.2%, P = 7.2%. and A = 3.6% in TCM and D = 84.3%, H = 3.9%, P = 9.8%, and A = 1.9% in TCM-GC. No significant differences (P > 0.05) were found between culture media in the chromosomal abnormality rates. According to our results, the use of somatic cells in co-culture during embryo development did not influence the appearance of abnormal complements in the produced embryos. This would allow the use of GC as a potential complement to simplify the techniques used in the culture of bovine embryos until Day 3.

168 INHIBITION OF BOVINE OOCYTE CUMULUS CELL PROGESTERONE SYNTHESIS DURING IN VITRO MATURATION AFFECTS CHROMOSOME ALIGNMENT AND SPINDLE INTEGRITY IN FERTILIZED EGGS AND EARLY EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 198
Author(s):  
E. Daly ◽  
A. G. Fahey ◽  
M. M. Herlihy ◽  
T. Fair
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Spindle Formation ◽  
Vitro Fertilization ◽  
Time Treatment

We have previously demonstrated the importance of progesterone (P4) synthesis by cumulus cells during oocyte maturation in vitro (IVM) for bovine oocyte acquisition of developmental competence and subsequent embryo development (Aparicio et al. 2011 Biol. Reprod. 84). The aim of this study was to identify key processes that may be deregulated by the inhibition of P4 signalling in the cumulus–oocyte complex (COC) during IVM. To this end, good quality immature COC were placed in IVM medium [TCM-199 supplemented with 10% (vol/vol) FCS and 10 ng mL–1 epidermal growth factor] and cultured at 39°C for 22 h in a humidified atmosphere containing 5% CO2, in the presence or absence of 10 μM trilostane (which blocks P4 synthesis by inhibiting 3 β-hydroxysteroid dehydrogenase; Stegram Pharmaceuticals Ltd., Surrey, UK). Matured COC were washed and placed in 250 μL of fertilization medium (25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mL–1 fatty acid-free BSA, and 10 mg mL–1 heparin). In vitro fertilization (IVF) was performed with 250 μL of frozen–thawed semen at a final concentration of 1 × 106 spermatozoa mL–1 at 39°C under 5% CO2 during 20 h. Presumptive zygotes were denuded, washed, and transferred to 25-μL culture droplets (SOF + 5% FCS) at 39°C under 5% CO2, 90% of N2, and 5% O2 atmosphere with maximum humidity. Subsets of presumptive fertilized eggs and developing embryos were recovered at 6, 72, 120, and 192 h postinsemination (hpi) and processed for confocal whole-mount immunocytochemistry. The meiotic and mitotic spindles and chromosomes were visualised by immunofluorescent labelling of α-tubulin and 4′,6-diamindino-2-phenylindole (DAPI), respectively, and classified as normal if the chromosomes were correctly aligned or appropriately segregated, or abnormal if lagging chromosomes or abnormal chromosome segregation were observed. Samples were collected from 5 replicates (n = 50 zygotes/embryos per treatment, per timepoint) and a total of 157 spindles were observed. Logistic regression analysis was conducted to determine the probability of abnormal spindle formation. The incidence of spindle abnormality was regressed on time, treatment, and treatment by time. For all time points, there was significant reduction in the odds of abnormal spindle formation in control samples versus trilostane-treated samples (P < 0.001). In conclusion, our data imply a role for P4 signalling in maintaining spindle integrity during oocyte meiotic maturation and progression through the initial mitotic divisions of early embryo development in cattle.

45 THE EFFECT OF MEIOTIC STAGE OF BOVINE OOCYTES ON THE SURVIVAL OF VITRIFIED CUMULUS–OOCYTE COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 135 ◽  
Author(s):  
J. R. Prentice ◽  
J. Singh ◽  
M. Anzar
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Early Embryo ◽  
Time Intervals ◽  
Early Embryo Development ◽  
In Vitro Culture System ◽  
Bovine Oocytes

Vitrification is a rapid freezing method in which cells/tissues are frozen in a glass state without ice crystal formation. However, vitrification of bovine oocytes is challenging due to their complex structure and sensitivity to chilling. Oocytes at the germinal vesicle (GV) stage of maturation are thought to be less prone to chromosomal and microtubular damage during cryopreservation because no spindle is present and genetic material is contained within the nucleus. However, immature oocytes are thought to be more sensitive to osmotic stress and have lower cell membrane stability than mature, metaphase II (MII) stage oocytes. The present studies aimed to validate the in vitro culture system used in our laboratory and to evaluate the effect of vitrification of bovine cumulus-oocyte complexes (COC) at different meiotic stages on their in vitro maturation (IVM), cleavage and early embryo development. Analyses were conducted on each dataset with PROC GLIMMIX in SAS using binary distribution (for yes/no response variable) and considering replicate as a random factor. In Experiment 1, meiotic progression of oocytes was evaluated at different time intervals during IVM. The following COC stages were predominantly found at different IVM time intervals: GV (89%) at 0 h, GV (47%) and germinal vesicle breakdown (GVBD; 44%) at 6 h, metaphase I (MI; 90%) at 12 h and MII (84%) at 22 h (n > 62 oocytes at each time group). In Experiment 2, bovine COC at 0, 6, 12 and 22 h of IVM were exposed to vitrification solution (15% dimethyl sulfoxide + 15% ethylene glycol + 0.5 M sucrose + 20% CS in TCM-199), loaded onto a cryotop device and vitrified by plunging in liquid nitrogen. Following warming (1 min in 0.5 M sucrose + 20% CS in TCM-199), COC completed 22 h of IVM and the nuclear stage was evaluated with lamin A/C-4′6-diamidino-2-phenylindole staining. Upon completion of 22 h of IVM, 23, 23, 35 and 89% of oocytes from 0-, 6-, 12- and 22-h groups, respectively were detected at MII (P < 0.0001). In Experiment 3, cleavage and embryo development of oocytes vitrified at 0, 12 and 22 h of IVM were evaluated. The cleavage rate did not differ among vitrification groups (i.e. 14% at 0 h, 17% at 12 h and 14% at 22 h; P = 0.825). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified (control) group than in vitrified groups (i.e. 73 vs 15% and 22 vs 0.3%, respectively). In conclusion, the maturation kinetics validated our in vitro culture system and vitrification adversely affected the ability of bovine oocytes to undergo in vitro maturation to the MII stage, in vitro fertilization and early embryo development. Vitrification of oocytes at GV, MI and MII stages of nuclear maturation did not differ in their subsequent survivability. This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.

scholarly journals Deadly decisions: the role of genes regulating programmed cell death in human preimplantation embryo development

Reproduction ◽  
2004 ◽  
Vol 128 (3) ◽  
pp. 281-291 ◽  
Author(s):  
Andrea Jurisicova ◽  
Beth M Acton
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Cell Fate ◽  
Embryo Development ◽  
Preimplantation Embryo ◽  
Culture Media ◽  
Culture Conditions ◽  
Early Embryo ◽  
Preimplantation Embryo Development

Human preimplantation embryo development is prone to high rates of early embryo wastage, particularly under currentin vitroculture conditions. There are many possible underlying causes for embryo demise, including DNA damage, poor embryo metabolism and the effect of suboptimal culture media, all of which could result in an imbalance in gene expression and the failed execution of basic embryonic decisions. In view of the complex interactions involved in embryo development, a thorough understanding of these parameters is essential to improving embryo quality. An increasing body of evidence indicates that cell fate (i.e. survival/differentiation or death) is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins, many of which are expressed during oocyte and preimplantation embryo development. The recent availability of mutant mice lacking expression of various genes involved in the regulation of cell survival has enabled rapid progress towards identifying those molecules that are functionally important for normal oocyte and preimplantation embryo development. In this review we will discuss the current understanding of the regulation of cell death gene expression during preimplantation embryo development, with a focus on human embryology and a discussion of animal models where appropriate.

scholarly journals Effect of oocyte vitrification before and after in vitro maturation towards Bcl-2, Bax and Bcl-2/Bax ratio expression

2017 ◽  
Vol 24 (2) ◽  
pp. 56
Author(s):  
Zakiyatul Faizah ◽  
Haryanto Aswin ◽  
Hamdani Lunardhi
Keyword(s):  
Treatment Group ◽  
Ethylene Glycol ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Basic Medium ◽  
Control Group ◽  
Oocyte Vitrification ◽  
Expression Ratio ◽  
Before And After

Objectives: to compare the expression of Bcl-2, Bax and Bcl-2/Bax ratio in cumulus cell and oocyte between vitrified oocyte pre and post in vitro maturation.Materials and Methods: Maturation was operated in medium TC 100 µl for 24 hours. Vitrification begins with washing oocyte in PBS basic medium supplemented of 20% serum for 1-2 minutes, followed by equilibration medium PBS + 20% serum + 10% ethylene glycol for 10-14 minutes, then transferred to 20% serum + PBS + 0.5 M sucrose + 15% ethylene glycol + PROH 15% for 25-30 seconds. Thawing is processed by submerging the oocytes in the media: 1). PBS + 20% serum + 0.5 M sucrose, 2). PBS + 20% serum + 0.25 M sucrose, and 3). PBS + 20% serum + 0.1 M sucrose. Imunocytochemistry observed the expression of Bcl-2, bax and Bcl-2/bax ratio.Results: Bcl-2 expression on oocyte in control group differed significantly with treatment group, Bcl-2 expression on cumulus in control group differed significantly with treatment 1 group. Bax expression on oocyte in control group differed significantly with treatment group. Bax expression on cumulus in control group differed significantly with treatment group. Bcl-2/Bax expression ratio on oocyte and cumulus did not differ significantly in all groupConclusion: No difference Bcl-2/Bax expression ratio on oocyte and cumulus between vitrified oocyte pre and post in vitro maturation.

116 EFFECTS OF l-GLUTAMINE ON CRYOPRESERVATION OF IMMATURE BOVINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 167
Author(s):  
C. Yamada ◽  
M. D. Goissis ◽  
H. V. A. Caetano ◽  
A. R. S. Coutinho ◽  
M. E. O. A. Assumpção ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Maturation ◽  
Complex Structure ◽  
Cumulus Cell ◽  
Epifluorescence Microscopy ◽  
Control Group ◽  
Maturation Medium ◽  
Cell Layers ◽  
Bovine Oocytes

The cryopreservation of bovine oocytes remains a challenge despite significant reported progress. Immature bovine oocytes have a complex structure and the conventional cryoprotectants (penetrating cryoprotectants, sugars, and macromolecules) appear to be not sufficient to preserve them efficiently during freezing. Studies on semen and fibroblast cryopreservation indicate that amino acids, particularly l-glutamine, protect enzymes during freezing and increase the post-thaw viability. Therefore, the amino acids may optimize oocyte cryopreservation when associated with conventional cryoprotectants. This work evaluated the effect of l-glutamine on cryopreservation of immature bovine oocytes after in vitro maturation. Oocytes with homogeneous cytoplasm and several cumulus cell layers from slaughterhouse ovaries were distributed randomly in three groups: non-vitrified control, vitrified control, and vitrified with l-glutamine. Oocytes from vitrified groups were exposed for 10 min to PBS + 10% FCS + 10% ethylene glycol (EG) + 0.25 m trehalose (T), and for 30 s to PBS + 10% FCS + 25% EG + 25% dimethylsulfoxide + 0.5 m T at room temperature, adding 80 mm l-glutamine for the third group. Oocytes were loaded into OPS and plunged in liquid nitrogen. For thawing, OPS were immersed in PBS + 10% FCS + 10% EG + 1 m T for three min. Oocytes werethen placed in PBS + 10% FCS + 0.5 m T and in PBS + 10% FCS, remaining three min in each solution. For in vitro maturation, oocytes were washed three times on holding medium (TCM-HEPES + FCS + pyruvate + gentamycin), washed three times in maturation medium (TCM-bicarbonate + FCS + pyruvate + gentamycin + hCG + FSH + estradiol), and cultured in microdrops (90 μL) of maturation medium covered with mineral oil at 38.5°C under 5% CO2 in air and high humidity for 24 h. Oocytes were denuded, fixed in paraformaldehyde and triton, stained with Hoechst 33342, and evaluated under epifluorescence microscopy. Oocytes at metaphase II were considered matured. The group vitrified with l-glutamine had a significantly higher maturation rate than the group vitrified without l-glutamine; however, both had significantly lower maturation rates than the non-vitrified control group. In conclusion, l-glutamine improved the viability of vitrified oocytes. Table 1. Oocyte maturation rates of non-vitrified control, vitrified control, and vitrified with glutamine groups This work was supported by FAPESP 03/08543-1.

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