scholarly journals 237 INCREASE OF BLASTOCYST DEVELOPMENTAL RATE IN VITRO BY SELECTION OF DEVELOPMENTALLY COMPETENT COW OOCYTES BEFORE IVM USING A STAINING TEST

2005 ◽  
Vol 17 (2) ◽  
pp. 269
Author(s):  
H. Alm ◽  
H. Torner ◽  
B. Loehrke ◽  
T. Viergutz ◽  
I. Ghoneim ◽  
...  
Keyword(s):  
Developmental Rate ◽  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
G6pdh Activity ◽  
Blastocyst Development ◽  
Brilliant Cresyl Blue ◽  
Immature Oocytes ◽  
Cresyl Blue

A large proportion of bovine oocytes fail to develop to blastocyst stage following maturation, fertilization, and culture in vitro. While suboptimal culture conditions undoubtedly contribute to this poor development, it is recognized that immature oocytes, especially from cows with reduced reproductive performance or which are slaughtered on the end of their use, are heterogeneous in quality and developmental competence (Gordon 2003). The aim of the present study was to increase the efficiency of blastocyst production from cows after IVM/IVF by oocyte selection before maturation. Immature oocytes are known to synthesize a variety of proteins (Wassarman PM 1988, Annu. Rev. Biochem. 57, 415–442), among them, glucose-6-phosphate dehydrogenase (G6PDH). This enzyme is active in the growing oocyte, but has decreased activity in oocytes that have finished their growth phase. Brilliant cresyl blue (BCB) has been used to measure G6PDH activity. The BCB test is based on the capability of the G6PDH to convert the BCB stain from blue to colorless (Erisson et al. 1993 Theriogenology 39, 214). The ovaries were obtained from a slaughterhouse and transported to the laboratory; cumulus-oocyte complexes (COCs) were recovered by slicing the surface of the ovary. Only oocytes with a compact cumulus investment were used. Oocytes were placed into three groups: (1) control – placed immediately into culture; (2) holding control – COCs kept in PBS containing 0.4% BSA for 90 min at 38.5°C before placement into culture; and (3) treatment – incubation with brilliant cresyl blue for 90 min at 38.5°C before culture. Treated oocytes were then divided into BCB− (colorless cytoplasm, increased G6PDH) and BCB+ (colored cytoplasm, low G6PDH) on their ability to metabolize the stain. Activity of G6PDH was determined via measurement of NADP reduction in control, BCB−, and BCB+ groups; activity was significantly increased in BCB− COCs in comparison to the control and BCB+ COCs. After IVM, oocytes were fertilized in vitro. Embryos were cultured to Day 8. The rate of maturation to metaphase II was significantly higher for control and BCB+ oocytes (77.1 and 72.5%, respectively) than for BCB− oocytes (58.1%). The BCB+ oocytes yielded a significantly higher proportion of blastocysts (34.1%) than either control group (18.3 and 19.2%); and both controls and BCB+ oocytes had significantly higher blastocyst development than did BCB− oocytes (3.9%). The number of nuclei in the blastocysts was comparable in BCB+ and both control groups (105.5 ± 5.8 and 117.5 ± 8.5, 101.8 ± 6.2, respectively). Blastocysts in the BCB− group had a significantly lower cell number (61.0 ± 2.6) than did controls. The results show that the staining of COCs from cows before IVM may be useful in increasing the efficiency of blastocyst production during standard IVF procedures. In addition, classification of G6PDH activity on the basis of BCB staining may be used to effectively select cow oocytes with further developmental competence. To our knowledge, this is the first study to evaluate the association between G6PDH activity in oocytes and further blastocyst development in cows.

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.

75 EFFECT OF PHENAZINE ETHOSULFATE ON PORCINE BLASTOCYST DEVELOPMENT, APOPTOSIS, AND CRYOTOLERANCE AFTER OPEN PULLED STRAW VITRIFICATION

2008 ◽  
Vol 20 (1) ◽  
pp. 118
Author(s):  
B. Gajda ◽  
Z. Smorag ◽  
M. Bryla
Keyword(s):  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Tunel Staining ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Morula Stage ◽  
Phenazine Ethosulfate ◽  
And Control

It is possible to improve the success of cryopreservation of in vitro-produced bovine embryos by modifying the embryos with the metabolic regulator phenazine ethosulfate (PES) (Seidel 2006 Theriogenology 65, 228–235). The PES treatment increased glucose matabolism, tended to increase the pentose phosphate pathway flux of glucose, and clearly reduced accumulation of lipids in cultured bovine embryos (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607). It is known that porcine embryos have a considerably high content of lipids, and the success rates of their cryopreservation appear to be highly correlated with cytoplasmic lipid content. In our preliminary study, we observed that supplementation of NCSU-23 medium with PES has a positive effect on efficiency of pig blastocysts of good quality (Gajda et al.. 2007 Acta Biochim. Pol. 54(Suppl 1), 52 abst). In the present study, the effects of PES on pig blastocyst development, apoptosis, and survival after vitrification were investigated. In Exp. 1, porcine zygotes obtained from superovulated gilts were cultured in NCSU-23 medium supplemented with 0 (control), 0.025, 0.05, or 0.075 µm PES. The culture was performed at 39�C, with 5% CO2 in air, for 96–120 h. Embryo quality criteria were developmental competence (cleavage, morula stage, and blastocyst stage), cell number per blastocyst, and the degree of apoptosis as assessed by TUNEL staining. In Exp. 2, expanded blastocysts cultured with 0.025 µm PES were vitrified in a ethylene glycol and dimethyl sulfoxide mixture using open pulled straw (OPS) technology (Vajta et al. 1997 Acta Vet. Scand. 38, 349–352). After thawing, the blastocysts were cultured in vitro for re-expansion or transferred to synchronized recipients. Data were analyzed by chi-square test. There was a difference between the 0.025 µm PES-treated and the control group in percentage of cleaved embryos (99.0 and 91.4%, respectively; P < 0.05), between all experimental groups and control in percentage of morula stage (90.7, 87.8, 83.8, and 80.0%, respectively), and between 0.025 and 0.05 µm PES-treated and control in percentage of blastocyst rates (70.0, 75.5, and 65.7%, respectively). The number of cells and percentage of TUNEL-positive nuclei per blastocyst were lower in the PES-treated than in the control group. The survival rate of blastocysts after vitrification and thawing was enhanced in the presence of PES compared to that in the PES-free group (45.2 and 38.9%, respectively; P < 0.05). After transfer of 56 expanded blastocysts cultured with PES and vitrified into 3 recipients, two gilts were confirmed pregnant at 35 days of gestation. In conclusion, a higher blastocyst percentage with a low incidence of apoptosis was obtained in the presence of PES compared to control. These blastocysts also had an increased ability to survive cryopreservation.

scholarly journals Use of oocytes selected by brilliant cresyl blue staining enhances rabbit cloned embryo development in vitro

Zygote ◽  
2019 ◽  
Vol 27 (3) ◽  
pp. 166-172 ◽  
Author(s):  
Linying Jia ◽  
Bo Ding ◽  
Chong Shen ◽  
Shiwei Luo ◽  
Yanru Zhang ◽  
...  
Keyword(s):  
Cell Mass ◽  
Developmental Competence ◽  
Blue Staining ◽  
Control Group ◽  
Control Groups ◽  
Brilliant Cresyl Blue ◽  
Positive Group ◽  
Cresyl Blue ◽  
And Control

SummaryRabbits play an important role in people’s lives due to their high nutritional value and high-quality hair that can be used as raw material for textiles. Furthermore, rabbits are an important animal model for human disease, as genome-edited animals are particularly valuable for studying gene functions and pathogenesis. Somatic cell nuclear transfer (SCNT) is an important technique for producing genome-edited animals and it has great value in saving endangered species and in clone stem cell therapy. However, the low efficiency of SCNT limits its application, with the selection of suitable rabbit oocytes being crucial to its success. In the present study, we collected oocytes from ovarian follicles and stained them with 26 μM brilliant cresyl blue (BCB). We then matured the oocytes in vitro and used them for SCNT. Comparison of the BCB-positive oocytes with BCB-negative oocytes and the control group showed that the BCB-positive group had a significantly higher maturation rate (81.4% vs. 48.9% and 65.3% for the negative and control groups, respectively), cleavage rate (86.6% vs. 67.9% and 77.9%), blastocyst rate (30.5% vs. 12.8% and 19.6%), total number of blastocysts (90±7.5 vs. 65.3±6.3 and 67.5±5.7), and inner cell mass (ICM)/ trophectoderm (TE) index (42.3±4.2 vs. 30.2±2.1 and 33.9±5.1) (P<0.05). The BCB-positive group had a significantly lower apoptosis index (2.1±0.6 vs. 8.2±0.9 and 6.7±1.1 for the negative and control groups, respectively) (P<0.05). These findings demonstrate that BCB-positive oocytes have a higher maturation ability and developmental competence in vitro, indicating that BCB staining is a reliable method for selecting oocytes to enhance the efficiency of SCNT.

315 SELECTION OF BOVINE OOCYTES BY BRILLIANT CRESYL BLUE BEFORE IN VITRO MATURATION IMPROVES BLASTOCYST DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 273 ◽  
Author(s):  
A. Sugulle ◽  
S. Katakawa ◽  
S. Yamamoto ◽  
S. Oomori ◽  
I. Itou ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Control Group ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Bovine Oocytes ◽  
Selection Of

The morphological identification of immature oocytes has commonly been used to select the bovine oocytes for IVF. However, &lt;30% of the recovered oocytes reach the blastocyst stage after fertilization, and this is probably due to the quality of the oocytes at the beginning of maturation. The brilliant cresyl blue (BCB) stain determines the activity of glucose-6-phosphate dehydrogenase, an enzyme synthesized in growing oocytes. The aim of this study was to evaluate the effect of the BCB stain on the selection of bovine oocytes and on the subsequent embryo development for in vitro production (IVP). Cumulus–oocyte complexes (COCs) were collected by the aspiration of 2- to 6-mm follicles. A total of 559 oocytes were divided into 2 groups: (1) a control group, immediately cultured, and (2) a BCB-incubated group. After 90 min of BCB staining (Pujol et al. 2004 Theriogenology 61, 735–744), the oocytes were divided into oocytes with blue cytoplasm (BCB+) and oocytes without blue cytoplasm (BCB−). The COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg mL−1 FSH at 38.5°C under an atmosphere of 5% CO2 in air. The matured COCs were inseminated with 5 × 106 sperm mL−1. After 18 h of gamete co-culture, the presumed zygotes were cultured in CR1aa supplemented with 5% CS for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (proportion of ≥5-cell stage, the total cleavage rates) and on Days 7 to 9 (blastocyst rate). The experiment was replicated 5 times, and the data were analyzed by a chi-square test and ANOVA. The results are presented in Table 1. The proportion of embryos with ≥5-cell stage was significantly higher (P &lt; 0.01) in the BCB+ group than in the BCB− group, but not in the control group. The total cleavage rate for the BCB+ embryos was significantly higher than that of either the BCB− or the control group (P &lt; 0.01). There were also significant differences (P &lt; 0.01) in the blastocyst development between the BCB+ and BCB− embryos and between the BCB− and the control embryos (P &lt; 0.05). This result showed that the selection of bovine oocytes by BCB staining before in vitro maturation may be useful for selecting oocytes that are developmentally competent up to Day 9 for IVP. Table 1.Effect of selection of oocytes by brilliant cresyl blue (BCB) staining on the subsequent embryo development of in vitro-matured/in vitro-fertilized bovine embryos

scholarly journals Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep

Reproduction ◽  
2011 ◽  
Vol 142 (4) ◽  
pp. 517-527 ◽  
Author(s):  
Maria Gracia Catalá ◽  
Dolors Izquierdo ◽  
Svetlana Uzbekova ◽  
Roser Morató ◽  
Montserrat Roura ◽  
...  
Keyword(s):  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Oocyte Diameter ◽  
Constitutive Function ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Maturation Promoting Factor

The aim of this study was to test the Brilliant Cresyl Blue (BCB) stain to select prepubertal sheep oocytes for in vitro blastocyst production. Oocyte diameter, mitochondrial activity, maturation-promoting factor (MPF) activity and mRNA relative expression (RE) of genes related to metabolism (ATPase Na+/K+ transporting α 1 (ATP1A1) and cytochrome c oxidase subunit 1 (COX1)) and constitutive function of the cell (cytoplasmic polyadenylation-element-binding protein (CPEB) and S100A10) were assessed. Immature oocytes were exposed to different BCB concentrations (13, 26, 39 and 52 μM) and classified according to their cytoplasm colouration as grown BCB+ (blue cytoplasm) and growing BCB− (colourless cytoplasm). Staining oocytes with 13 μM BCB during 60 min allows selection of (BCB+) the largest (123.66 μm) and most competent oocytes to develop to the blastocyst stage (21%) with a higher number of cells (69.71±6.19 s.e.m.) compared with non-stained BCB− oocytes (106.82 μm, 9% and 45.91±3.35 s.e.m. respectively). Mitochondrial activity, assessed by MitoTracker Orange CMTMRos probe, was significantly higher in BCB+ than in BCB− oocytes after in vitro maturation (3369 and 1565 AU respectively). MPF activity was assessed by CDC2 kinase activity assay showing significantly higher activity at metaphase II stage in BCB+ than in BCB− oocytes (1.479±0.09 and 1.184±0.05 optical density respectively). The genes analysed in this work, ATP1A1, COX1, CPEB and S100A10, did not show significant effect in mRNA RE between BCB selected oocytes. In conclusion, BCB stains larger and more competent oocytes to develop to the blastocyst stage with more active mitochondria and MPF activity and higher blastocyst cell number.

scholarly journals Cathepsin B activity has a crucial role in the developmental competence of bovine cumulus–oocyte complexes exposed to heat shock during in vitro maturation

Reproduction ◽  
2013 ◽  
Vol 146 (4) ◽  
pp. 407-417 ◽  
Author(s):  
A Z Balboula ◽  
K Yamanaka ◽  
M Sakatani ◽  
M Kawahara ◽  
A O Hegab ◽  
...  
Keyword(s):  
Heat Shock ◽  
Cathepsin B ◽  
Caspase 3 ◽  
Developmental Rate ◽  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Tunel Staining

Cathepsin B was found to be correlated inversely with the quality of bovine oocytes and embryos. The aims of this study were to evaluate i) the relationship between heat shock during in vitro maturation (IVM) of bovine cumulus–oocyte complexes (COCs) and cathepsin B activity in relation to apoptosis and ii) the effect of supplementation of cathepsin B inhibitor (E-64) during IVM of heat-shocked COCs on embryonic development. After IVM at 38.5 °C for 22 h (control group) or at 38.5 °C for 5 h followed by 41 °C for 17 h (heat shock group) either with or without 1 μM E-64, activities and protein expression of cathepsin B and caspase 3 were evaluated as well as TUNEL staining. After IVF, developmental rate, total cell number, and the percentage of apoptotic cells in blastocysts were evaluated on day 8 (day 0, IVF day). Heat-shocked IVM COCs showed significantly high activities and expressions of both cathepsin B, and caspase 3 accompanied by a significant increase in number of TUNEL-positive cells. Addition of E-64 significantly decreased the activities of cathepsin B and caspase 3, and TUNEL-positive cells in heat-shocked IVM COCs. Moreover, addition of 1 μM E-64 during IVM under heat shock conditions significantly improved both developmental competence and quality of the produced embryos. These results indicate that heat shock induction of cathepsin B is associated with apoptosis of COCs, and inhibition of cathepsin B activity can improve the developmental competence of heat-shocked COCs during IVM.

113 EFFECT OF PLANT PROTEIN ON DEVELOPMENT AND QUALITY OF CULTURED IN VITRO PORCINE ZYGOTES

2009 ◽  
Vol 21 (1) ◽  
pp. 157 ◽  
Author(s):  
B. Gajda ◽  
I. Grad ◽  
Z. Smorag
Keyword(s):  
Serum Albumin ◽  
Culture Media ◽  
Quality Criteria ◽  
Cell Number ◽  
Developmental Competence ◽  
Plant Protein ◽  
Control Group ◽  
Group 1

Basic culture media are usually supplemented with serum albumin or serum, which contain amino acids that play an important role as energy sources, osmoregulators, and pH stabilizers. However, the presence of undefined serum in culture media introduces a variation from batch to batch and increases viral or prion contamination risk. The aim of the present study was to investigate the possibility of using plant protein substitute (PP) in place of bovine serum albumin (BSA) during in vitro culture of porcine zygotes. The PP is a mixture of several plant proteins and soya lecithin prepared using a high pressure homogenization process. The experiment was done on pig zygotes obtained surgically from superovulated gilts at 24–26 h after insemination. Morphologically normal zygotes were cultured in vitro in 5% CO2 in air at 39° in NCSU-23 medium supplemented with: 0.002 g mL–1 (group 1), 0.004 g mL–1 (group 2), 0.008 g mL–1 (group 3) PP or 0.004 g mL–1 BSA (control group). Embryo quality criteria were developmental competence (cleavage, morula and blastocyst rates), total cell number per blastocyst and degree of apoptosis as assessed by TUNEL method. Results were analyzed by Chi-square test. There were no differences in cleavage rates on Day 2 between zygotes cultured in NCSU-23 medium supplemented with PP (86.0, 88.0, 84.8; group 1 to 3, respectively) and BSA (91.0%, control group). Culture with 0.008 g mL–1 PP increased morula (85.7%) and blastocyst (69.2%) production as compared with control (75.0% and 56.3%, respectively; P < 0.05) and 0.002 g mL–1 PP (79.5% and 51.8%, respectively; P < 0.05). The mean number of cells in Day 7 blastocysts cultured in NCSU-23 medium + 0.004 g mL–1 BSA was lower (P < 0.05) than in NCSU-23 + 0.004 g mL–1 PP (39.1 v. 43.7, respectively). The blastocysts cultured in NCSU-23 medium + 0.002 g mL–1 PP had higher average number of apoptotic nuclei (13.0) as compared with the control (6.5) and 0.004 g mL–1 PP (6.9). In conclusion, this study suggest the positive effect of PP on development in vitro of porcine zygotes to the morula/blastocyst stage. However, further studies are required to determine the quality of the embryos cultured with PP. This study was supported by Scientific Net of Animal Reproduction Biotechnology.

Meiotic progression, mitochondrial features and fertilisation characteristics of porcine oocytes with different G6PDH activities

2010 ◽  
Vol 22 (5) ◽  
pp. 830 ◽  
Author(s):  
István Egerszegi ◽  
Hannelore Alm ◽  
József Rátky ◽  
Bassiouni Heleil ◽  
Klaus-Peter Brüssow ◽  
...  
Keyword(s):  
Developmental Competence ◽  
Mitochondrial Activity ◽  
G6pdh Activity ◽  
Cresyl Blue ◽  
Meiotic Progression ◽  
Germinal Vesicles ◽  
Nuclear Development ◽  
Chromatin Configuration ◽  
Glucose 6 Phosphate Dehydrogenase

The aim of the present study was to investigate the developmental competence, mitochondrial characteristics and chromatin status of immature follicular porcine oocytes selected for their glucose-6-phosphate dehydrogenase (G6PDH) activity by brilliant cresyl blue (BCB) staining. In Experiment 1, the oocyte parameters were determined in parallel right after BCB staining (T0), after 22 h of in vitro maturation (IVM) (T22) and after 44 h of IVM (T44) (n = 496). BCB-stained oocytes (BCB+) at T0 were characterised by fibrillated chromatin filaments in their germinal vesicles (GV) and diakinesis stages whereas unstained (BCB–) oocytes at T0 contained in their GV mainly condensed stages of chromatin (P < 0.05). After 22 h of IVM BCB+ oocytes showed a prominent chromatin configuration of metaphase I and after 44 h the majority developed a M II nuclear configuration in contrast to the BCB– group (P < 0.0001). Differences were also observed between the two oocyte populations in their mitochondrial activity (P < 0.05). At the beginning of IVM BCB+ oocytes were characterised by high mitochondrial activity in their cytoplasm. The BCB+ oocytes showed clear visible homogenous distributions of mitochondria (P < 0.005) and contained more aggregated clusters of mitochondria in contrast to BCB– oocytes (P < 0.005). In Experiment 2, 318 oocytes were tested for their G6PDH activity and introduced to IVM and IVF. Only oocytes from the BCB+ group, which were matured after 44 h up to the stage of M II (81.6%) were fertilised (17.4%), penetrated (46%) or activated (15.6%) after IVF. These results indicate a relationship between the G6PDH activity of porcine oocytes before IVM and their subsequent nuclear development, mitochondrial activity and aggregation.

Relationship between follicle size and oocyte developmental competence in prepubertal and adult pigs

2007 ◽  
Vol 19 (7) ◽  
pp. 797 ◽  
Author(s):  
Melanie A. Bagg ◽  
Mark B. Nottle ◽  
David T. Armstrong ◽  
Christopher G. Grupen
Keyword(s):  
In Vitro Maturation ◽  
Fold Increase ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Parthenogenetic Activation ◽  
Follicle Size ◽  
Low Efficiency ◽  
The Relationship

The present study compared the distribution and steroid composition of 3-, 4- and 5–8-mm follicles on the surface of prepubertal and adult ovaries, and determined the relationship between follicle size and developmental competence of oocytes following parthenogenetic activation. The effect of 1 mm dibutyryl cAMP (dbcAMP) for the first 22 h of in vitro maturation (IVM) on the embryo development of prepubertal oocytes from the three follicle size cohorts was also determined. Compared with adult, prepubertal ovaries contained a higher proportion of 3-mm follicles (46 v. 72%, respectively), but a lower proportion of 4-mm (33 v. 22%, respectively) and 5–8-mm follicles (21 v. 6%, respectively). Adult follicular fluid (FF) contained 11-fold higher levels of progesterone (P4) than prepubertal FF, with similar levels observed between all adult follicle sizes. In prepubertal FF, the P4 concentration increased with follicle size from 3 to 4 to 5–8 mm. Rates of blastocyst development following parthenogenetic activation of adult oocytes from all three follicles sizes were similar (approximately 55%), whereas rates from prepubertal oocytes increased with increasing follicle size from 3 (17%) to 4 (36%) to 5–8 mm (55%). Treatment with dbcAMP for the first 22 h of IVM led to a 1.5-fold increase in the rate of blastocyst development for prepubertal oocytes from 3-mm follicles, but had no effect on prepubertal oocytes from the 4 and 5–8 mm classes. Mean blastocyst cell number increased with follicle size in prepubertal ovaries and was similar for all follicle sizes in adult ovaries. The present study demonstrates that the low efficiency of in vitro embryo production observed using prepubertal compared with adult pig oocytes is due to a greater proportion of 3-mm follicles on prepubertal ovaries, which contain oocytes of inferior developmental competence.

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

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