14 EQUINE CLONING AND EMBRYO AGGREGATION: EFFECT OF BOVINE, PORCINE, FELINE AND EQUINE OOPLAST

2012 ◽  
Vol 24 (1) ◽  
pp. 118
Author(s):  
A. Gambini ◽  
J. Jarazo ◽  
A. De Stefano ◽  
F. Karlanian ◽  
D. Salamone
Keyword(s):  
Embryo Development ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Development Stage ◽  
Blastocyst Stage ◽  
Chi Square ◽  
Cell Stage ◽  
Aggregation Effect ◽  
Group I

The low number of horse slaughterhouses is one of the reasons for the limited availability of horse oocytes for research in cloning. The aim of our study was to assess the capability of equine, bovine, porcine, or feline ooplast to produce cloned embryos when equine cells are used as donor nuclei and to evaluate if embryo aggregation improves their development. Oocytes from mentioned species were collected from ovaries derived from slaughterhouses, except for cat ovaries that were obtained from ovariectomized queens. Oocytes were matured in TCM199 supplemented following standard protocols for each species. After maturation, cumulus and zona pellucida were removed. Enucleation was performed by aspiration of the metaphase plate under ultraviolet light. Donor cell and ooplast were attached by phytohemagglutinin treatment and then electrofused. Activation protocols were ionomycin for 4 min, except for porcine, which were electrically activated, followed by culture in 1.9 mM 6-DMAP for bovine, feline and porcine, except for equine: 1 mM 6-DMAP with 5 mg mL–1 of cycloheximide. Reconstructed embryos (RE) were cultured in SOF in the well of well system in 2 different groups: only one RE per well (1X) and three RE per well (3X, aggregated embryos, AE). Blastocysts derived from homospecific clones were transferred to synchronized mares. Cleavage and maximum development stage achieved of all experimental groups were assessed. In vitro development was compared using the chi-square test. In group 1X, a total of 64, 49, 38 and 145 RE were performed for porcine, bovine, feline and equine, respectively and in group 3X, 88, 48, 48 and 195 RE. Cleavage of cloned embryos ranged from 67 to 87%. Aggregated of homospecific equine clones showed the highest blastocyst rates (1X: 5.5%, 3X: 34%) and after embryo transfer (4 recipients for each group), an ongoing pregnancy (day 300, at the time of submission) was only achieved with aggregated embryo confirming the positive effect of embryo aggregation in these clones. The stages with higher developmental arrest of heterospecific nonaggregated embryos were 2 to 4 cells for porcine ooplast (23/64, 36%) and 4 to 8 cells for bovine and feline ooplast (37/49, 75% and 18/38, 47%, respectively). Blastocyst stage was only reached using feline ooplast (group I: 2/38, 5.26% and group II: 2/16, 12.5%). Heterospecific aggregated clones were able to achieve 16-cell stage, showing statistic differences compared with group 1X. As we reported previously, embryo aggregation shows benefits for homospecific equine clones, although more studies are needed to clarify if aggregation of heterospecific clones has the same effect. All heterospecific ooplasm was able to support embryo development. The stage of major developmental arrests was similar to embryonic genomic activation stage. Our results suggest that cat oocyte seems to be the best receptor to support equine cloned embryo development.

scholarly journals 244REAL TIME PCR EVALUATION OF EXPRESSION PROFILES OF SOME MATERNAL-SOURCED GENE TRANSCRIPTS IN IN VITRO PRODUCED PRE IMPLANTATION STAGE BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 242
Author(s):  
S. Mamo ◽  
S. Ponsuksili ◽  
K. Wimmers ◽  
M. Gilles ◽  
K. Schellander
Keyword(s):  
Embryo Development ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Development Stage ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cell Stage ◽  
Gene Transcripts

Gene expression profiling data collected in a time series and quality related parameters are important for understanding the developmental mechanisms carried out in a developing embryo, and are also a source to enrich the knowledge base of embryo development. However, such data are frequently constrained by limitation and handling of the sample as well as cost associated with generating such data. Cumulatively, these factors have contributed to the existing insufficient data compared to the large need stemming from a drive to control and guide optimum embryo development. In this ongoing study, with objectives to quantify and evaluate gene transcripts identified from certain developmental stages, expression profiles of two ESTs (C256 and C112), derived from an oocyte cDNA library, were analyzed from the above perspectives to understand the change in the level of these gene transcripts throughout the pre-implantation stage of embryo development. For this analysis, pools of oocytes and embryos were prepared by balancing the amount proportional to the number of cells present. mRNA was isolated separately from each pool of matured oocytes, 2-cell, 4-cell, 8-cell, and 16-cell stages, as well as morula and blastocyst stages by using Dynal beads Oligo (dt)25 (Dynabeads, Dynal Biotech, Oslo, Norway) following the manufacturer’s recommendations. These mRNAs were checked for DNA contamination and, when proved free, first-strand cDNA was synthesised by reverse transcribtion at 42°C for 2h following standard laboratory procedures. Transcript quantification was performed by real-time PCR using gene-specific primers, equal amounts of cDNA from each sample and SYBR Green universal master mix. Following this analysis, both transcripts were found to be expressed in a wave-like manner being highly expressed in mature oocytes, declining gradually as the development stage advanced, with the lowest level at the 16-cell stage, and then reviving in level thereafter until it reached blastocyst stage. Taking the 16-cell stage as calibrator for both, C256 was 26.4, 23.2, 8.5, 1.7. 2.4 and 2.7 times more expressed in oocyte, 2-cell, 4-cell, 8-cell, morula and blastocyst stages, respectively. Similarly, C112 was 110.7, 169.2, 9.8, 2.5, 4.1 and 7.3 times more expressed in oocyte, 2-cell, 8-cell, morula and blastocyst stages, respectively. These expression patterns suggest the probable origin of these transcripts initially to be maternal. C256 is strongly similar to human retinoid X receptor beta (RXRb) gene (NM_021976.3), which is involved in transcriptional functions and in increasing DNA binding, whereas C112 is strongly similar to TATA box-binding protein-associated factor gene (AY189986.1), which is also involved in transcriptional functions. As seen from their functions, these transcripts can be vital for developing the embryo and the variations at different developmental stages shows their most probable role as part of genes contributing to developmental competence in pre-implantation development stages.

scholarly journals PREGNANCY RATE OF RECIPIENT COWS AFTER TRANSFER OF IN VITRO-PRODUCED NELLORE EMBRYOS

2019 ◽  
Vol 32 (4) ◽  
pp. 1087-1091 ◽  
Author(s):  
MÔNICA ZUCHELLI JAGUSZESKI ◽  
ADALGIZA PINTO NETO ◽  
WILLIAM DE OLIVEIRA ◽  
JONATAS CATTELAM ◽  
HELTON APARECIDO GARCIA GREGIANINI
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Embryo Development ◽  
Development Stage ◽  
Blastocyst Stage ◽  
Private Company ◽  
Chi Square ◽  
Development Stages ◽  
Advanced Development

ABSTRACT The objective of this study was to evaluate the pregnancy rate (PR) of cows of six farms in Bolivia and in the states of Acre and Rondônia in Brazil after the transfer of 4,321 in vitro Nellore embryos produced by a private company, in 2015 to 2016. The effects of the farm location, year of embryo transfer (ET), season of ET, number of previous ET per recipient, and embryo development stage on the PR of the recipient cow (chi square p<0.05) were evaluated. The PR of the six evaluated farms were similar, with an average of 45.15%. The farm location, year of ET (2015 or 2016), and season of ET (rainy or dry season) did not affect the PR (p>0.05). The PR found after embryo transfer for recipient cows that had already been used once (45.58%) or twice (43.40%) for this purpose were higher than that of recipient cows used three to eight times (29.01%) (p<0.05). ET at expanded or hatched blastocyst stage resulted in higher PR (47.35%) when compared to the ET at morula or initial blastocyst (41.06%) and at blastocyst stage (43%) (p<0.05), which did not differ from each other (p>0.05). Under the conditions of this study, the transfer of in vitro-produced Nellore embryos at advanced development stages (expanded or hatched blastocyst) results in higher PR in cows that had been used once or twice for this purpose.

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Group 13 ◽  
Time Lapse Imaging

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

221 SOMATIC CELL GOAT CLONING USING ALLOGENEIC OR SYNGENEIC TRANSGENIC CELL LINES

2014 ◽  
Vol 26 (1) ◽  
pp. 224
Author(s):  
L. T. Martins ◽  
L. H. Aguiar ◽  
C. E. M. Calderón ◽  
S. G. Neto ◽  
K. C. S. Tavares ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Donor Cell ◽  
Serum Starvation ◽  
Chi Square ◽  
Cell Stage ◽  
Primary Fibroblast ◽  
Skin Cell ◽  
Standard Procedures

The aim of this study was to compare the efficiency of goat cloning by using cell lineages from distinct transgenic backgrounds. Primary fibroblast skin cell cultures from 2 females (allogeneic), transgenic for the human lysozyme gene (hLZ), were established following standard procedures. Cells from one hLZ genotype were used for the establishment of 2 double transgenic syngeneic cell lines by cell transfection (Nucleofector®, Lonza, Germany) with transgene cassettes containing either the human glucocerebrosidase gene (hGC) and neomycin resistance gene, or the human lactoferrin gene (hLF) with no selection gene. The hGC-transfected hLZ cells were antibiotic-selected (G418, Sigma-Aldrich, St. Louis, MO, USA) until the isolation of positive cell colonies, whereas hLF-transfected hLZ cells were seeded onto 100-mm culture plates (100 cells/plate) to allow colony outgrowth from individual cells. Isolated colonies were screened by PCR using specific primers for each transgene (hGC or hLF) and for hLZ and GAPDH (controls). Positive cells from one hLZ-hGC and one hLZ-hLF colony were used for cloning at passage 9, whereas hLZ cells from the other genotype were at passage 4. Cells were synchronized by high confluence and 24 h of serum starvation. Goat cloning was performed according to standard procedures (Feltrin et al. 2012 Reprod. Fertil. Dev. 25, 163). Briefly, cumulus-oocyte complexes from abattoir ovaries were in vitro-matured for 20 h. Oocyte enucleation and hLZ, hLZ-hGC, or hLZ-hLF donor cell insertion were done by micromanipulation. Reconstructed structures were fused by two 1.2-KV cm–1 DC pulses for 20 μs. Cloned embryos were cultured for 1 h in cytochalasin B and then activated in ionomycin/6-DMAP. After 12 h of in vitro culture in G-1™ medium (Vitrolife, USA), 1-cell stage embryos were transferred into the oviduct of synchronous females (Keefer et al. 2002 Biol. Reprod. 66, 199-203). Pregnancy diagnosis was performed by ultrasonography on Day 30, with weekly monitoring afterwards. Preliminary data from 6 replicates were analysed by the chi-square test (P < 0.05). Maturation rate and survival after enucleation were 42.8% (610/1425) and 72.9% (291/399), respectively. A total of 271 structures were reconstructed using the 3 donor cell lines. Fusion rates did not differ between hLZ (59.5%), hLZ-hGC (47.5%), and hLZ-hLF (48.5%) groups. A total of 68 hLZ, 92 hLZ-hGC, and 39 hLZ-hLF-derived embryos were transferred to 5, 7, and 3 recipients, respectively. No pregnancies were detected with the use of hLZ and hLZ-hLF cells. However, 3 pregnancies (one nonviable) were detected on Day 30 with hLZ-hGC cells (42.9%), with both viable pregnancies lost on Days 40 and 130 of gestation. Molecular analyses confirmed both concepti as transgenic clones from the hLZ-hGC cell line. In summary, antibiotic selection of positive colonies was effective at maintaining cell viability, with a positive response when used for cloning. Replications are in progress to evaluate the effect of cell colony isolation from individual cells (e.g. hLZ-hLF cells) on cell viability over time and on cloning outcome.

53 EFFECT OF THE TIME INTERVAL BETWEEN OVARY COLLECTION AND OOCYTE IN VITRO MATURATION ON EQUINE CLONED EMBRYO DEVELOPMENT

2010 ◽  
Vol 22 (1) ◽  
pp. 184
Author(s):  
A. Gambini ◽  
J. Jarazo ◽  
R. Olivera ◽  
D. Salamone
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Limiting Factor ◽  
Time Interval ◽  
Activation Process ◽  
Blastocyst Development ◽  
Sodium Pyruvate ◽  
Chi Square ◽  
Group I

The availability of viable equine oocytes is a limiting factor on in vitro embryo production; therefore, it is necessary to assess some of the variables that affect oocyte viability. The aim of our study was to evaluate one of those variables: the effect of time between the collection of the ovary and oocyte in vitro maturation. Ovaries of slaughtered mares were collected during the breeding season (Argentine, Southern hemisphere). They were separated in bags every half hour and treated separately after arriving at the laboratory. COCs were recovered by a combination of scraping and washing of all visible follicles with a syringe filled with DMEM supplemented with 1 mM sodium pyruvate and 15 IU mL-1 heparin. COCs were matured for 24 to 26 h in 3 groups, according to time interval: 4 to 7 (group I), 7 to 10 (II), and 10 to 12 (III) hours. The medium for maturation was TCM-199 supplemented with 10% fetal bovine serum (FBS), 1 μL mL-1 insulin-transferrin-selenium, 1 mM sodium pyruvate, 100 mM cysteamine, and 0.1 mg mL-1 of FSH at 39°C in a humidified atmosphere of 5% CO2 in air. The cumulus was removed by a trypsin treatment and vortexing in hyaluronidase (1 mg mL-1). Cloning and fusion procedures were performed following the zona-free technique described by Lagutina et al. (2007 Theriogenology 67, 90-98). Two experiments were carried out by using different activation protocols. In experiment 1, the activation process was 22 mM ionomycin in H-TALP for 4 min followed by 3h culture in 1.9 mM 6-DMAP in SOF, whereas in experiment 2, we used 8.7 mM ionomycin in H-TALP for 4 min followed by 4 h culture in 1 mM 6-DMAP and 10 mg mL-1 cycloheximide in SOF. Embryos were cultured in wells of well (WOW) system. Half of the medium was renewed on Day 3 with fresh SOF and on Day 5 with DMEM/F12 with 10% FBS. Cleavage was assessed 48 h after activation; the rate of blastocyst formation was recorded at Days 8 and 9. Results were compared using chi-square test (P < 0.05). In experiment 1, maturation rates were significantly different between group I (n = 135, 54.1%) and III (n = 94, 40.4%), group II did not differ from them (n = 138, 53%). Cleavage rates differed statistically between II (n = 44, 75%) and III (n = 27, 40.7%), but not with group I (n = 53, 98%). No significant differences were found in blastocyst development; however, we observed a certain tendency towards an increase in the blastocyst rate as the time interval was lower (I: 3/53, 5.7%; II: 1/44, 2.3%; III: 0/27, 0%). In experiment 2, there were no significant differences between group I and II in rates of maturation (n = 56, 59% v. n = 111, 44.5%), cleavage (n = 22, 91% v. n = 34, 82%) or blastocyst rates (1/22, 4.5% v. 7/34, 20.6%). We conclude that cloned equine embryo development, using the two activation protocols tested, is not affected when the time interval between ovary collection and oocyte IVM is within 4 to 10 h.

90 TRANSCRIPTION OF USP9Y AND ZFY IN DEVELOPING AND ARRESTED BOVINE EMBRYOS IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 193
Author(s):  
J. Caudle ◽  
C. K. Hamilton ◽  
F. A. Ashkar ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Male Embryo ◽  
Male Development ◽  
Embryonic Genome ◽  
Genome Activation

Sexual dimorphisms such as differences in growth rate and metabolism have been observed in the early embryo, suggesting that sex chromosome-linked gene expression may play an active role in early embryo development. Furthermore, in vitro sex ratios are often skewed toward males, indicating that Y-linked genes may benefit development. While little attention has been paid to the Y chromosome, expression of some Y-linked genes such as SRY and ZFY has been identified in the early embryo, and only a few studies have systematically examined early stages. Identification of transcripts of Y-linked genes in the early embryo may provide insights into male development and provide markers of embryonic genome activation in male embryos. The objectives of this study were i) to examine the timing of transcription of 2 Y chromosome-linked genes involved with sperm production and male development, ubiquitin-specific peptidase 9 (USP9Y) and zinc finger protein (ZFY), in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst stage and ii) to determine if USP9Y and ZFY transcripts are present in in vitro-produced embryos arrested at the 2- to 8-cell stages. To examine the chronology of transcription of these genes, pools of 30 embryos for each developmental stage, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst, were produced by bovine standard in vitro embryo production (Ashkar et al. 2010 Hum. Reprod. 252, 334–344) using semen from a single bull. Pools of 30 were used to balance sex ratios and to account for naturally arresting embryos. Embryos for each developmental stage were harvested and snap frozen. Total RNA was extracted from each pool, reverse transcribed to cDNA and by using PCR, and transcripts of USP9Y and ZFY were detected as positive or negative. In addition pools of 30 embryos arrested at the 2- to 8-cell stage harvested 7 days after IVF were processed and analysed in the same way to determine if transcripts from the Y chromosomes are present in developmentally arrested embryos. Transcripts of USP9Y and ZFY were detected in the pooled embryos from the 8-cell stage through to the blastocyst stage, but none were detected in the 2-cell or 4-cell pools. Transcripts of ZFY were detected in the arrested 2- to 8-cell embryo pool, but transcripts of USP9Y were not detected. Given that these Y genes begin expression at the 8-cell stage, coincident with embryonic genome activation, it was concluded that these genes may be important for early male embryo development. Furthermore, the results suggest that arrested embryos that have stopped cleaving before the major activation of the embryonic genome are still capable of transcribing at least some of these genes. The absence of USP9Y transcripts in the arrested embryos suggests that it may be important for early male embryo development. Funding was provided by NSERC, the CRC program, and the OVC scholarship program.

131 GENE SILENCING IN BOVINE ZYGOTES: SIRNA TRANSFECTION v. MICROINJECTION

2010 ◽  
Vol 22 (1) ◽  
pp. 224 ◽  
Author(s):  
C. M. O'Meara ◽  
J. D. Murray ◽  
J. F. Roche ◽  
S. Mamo ◽  
E. Gallagher ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Embryo Development ◽  
Relative Abundance ◽  
Gene Function ◽  
Developmental Stages ◽  
Analysis Data ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
E Cadherin

Ribonucleic acid interference (RNAi) has become an effective tool for studying gene function in a variety of cells. The objective of this study was to compare the efficiency of gene silencing when siRNA were introduced into bovine zygotes by microinjection (as done previously; Tesfaye D et al. 2007 Mol. Reprod. Dev. 74, 978-988) v. a novel method of transfection in terms of gene knockdown and embryo development. For microin-jection, in vitro-produced bovine zygotes (16 h post insemination) were randomly assigned to 1 of 3 groups over 2 experiments. In Experiment 1, E-cadherin siRNA was injected at 100 μM (n = 168) and compared with PBS-injected (n = 180) and noninjected controls (n = 152). In Experiment 2, E-cadherin siRNA was injected at 375 μM (n = 154) and compared with PBS-injected (n = 136) and noninjected controls (n = 151). Embryos were subsequently cultured in vitro until Day 7 (day of IVF = Day 0). For transfection, the zona pellucida was removed from in vitro-produced zygotes. Zona-free zygotes were randomly assigned to 1 of 4 groups (i) GAPDH (n = 67), (ii) scrambled (n = 66), (iii) E-cadherin (n = 69) siRNA treatments at 100 nM or (iv) nontransfected controls (n = 66). Zygotes were incubated in transfection medium with siRNA for 1 h at 39°C, cultured individually in the well-of-the-well system to Day 7. The proportion of zygotes undergoing cleavage and developing to the blastocyst stage was recorded, and Day 7 embryos were frozen individually for mRNA analysis. Data for mRNA expression were fitted to a general linear model, and developmental stages were tested using ANOVA. Microinjection of 100 μM E-cadherin siRNA had no effect on phenotype (P > 0.05). Injection of PBS or 375 μM E-cadherin siRNA resulted in a decrease in the number of embryos reaching the 8-cell stage (51.5%, 45.5%, and 62.9%, respectively) and blastocyst stage (39.0%, 32.5%, and 45%, respectively) compared with noninjected controls (P < 0.05). The mRNA abundance of the target gene was suppressed by 36 and 46% when siRNA targeting E-cadherin was injected at 100 μM and 375 μM compared with control and PBS-injected groups (P < 0.05). Transfection with E-cadherin siRNA decreased development of 8-cell embryos (20.3 v. 53.0%, respectively) and blastocysts (7.2 v. 18.2%, respectively) compared with controls (P < 0.05). The mRNA relative abundance was not different between controls (nontransfected, or transfected with GAPDH or scrambled siRNA). However, transfection of zygotes with 100nM E-cadherin siRNA led to a 70% reduction in E-cadherin mRNA relative abundance in Day 7 blastocysts compared with controls (P < 0.05). Zona removal and transfection resulted in decreased embryo development compared with microinjection (P < 0.05). However, transfection yielded more efficient gene silencing of E-cadherin mRNA with reduced embryo development compared with microinjection. This technique of gene silencing could improve the efficiency of gene function studies in early bovine embryogenesis. Supported by Science Foundation Ireland.

Messenger RNAs in metaphase II oocytes correlate with successful embryo development to the blastocyst stage

Zygote ◽  
2012 ◽  
Vol 22 (1) ◽  
pp. 69-79 ◽  
Author(s):  
Fernando Henrique Biase ◽  
Robin Edward Everts ◽  
Rosane Oliveira ◽  
Weruska Karyna Freitas Santos-Biase ◽  
Giovana Krempel Fonseca Merighe ◽  
...  
Keyword(s):  
Embryo Development ◽  
Rna Binding ◽  
Blastocyst Stage ◽  
Messenger Rnas ◽  
Cell Stage ◽  
Maternal Mrna ◽  
Expression Of Genes ◽  
Oocyte Cytoplasm ◽  
Metaphase Ii Oocytes

SummaryThe mRNAs accumulated in oocytes provide support for embryo development until embryo genomic activation. We hypothesized that the maternal mRNA stock present in bovine oocytes is associated with embryo development until the blastocyst stage. To test our hypothesis, we analyzed the transcriptome of the oocyte and correlated the results with the embryo development. Our goal was to identify genes expressed in the oocyte that correlate with its ability to develop to the blastocyst stage. A fraction of oocyte cytoplasm was biopsied using micro-aspiration and stored for further expression analysis. Oocytes were activated chemically, cultured individually and classified according to their capacity to develop in vitro to the blastocyst stage. Microarray analysis was performed on mRNA extracted from the oocyte cytoplasm fractions and correlated with its ability to develop to the blastocyst stage (good quality oocyte) or arrest at the 8–16-cell stage (bad quality oocyte). The expression of 4320 annotated genes was detected in the fractions of cytoplasm that had been collected from oocytes matured in vitro. Gene ontology classification revealed that enriched gene expression of genes was associated with certain biological processes: ‘RNA processing’, ‘translation’ and ‘mRNA metabolic process’. Genes that are important to the molecular functions of ‘RNA binding’ and ‘translation factor activity, RNA binding’ were also enriched in oocytes. We identified 29 genes with differential expression between the two groups of oocytes compared (good versus bad quality). The content of mRNAs expressed in metaphase II oocytes influences the activation of the embryonic genome and enables further develop to the blastocyst stage.

scholarly journals 282MATURATION MEDIUM EXCHANGE IS EFFECTIVE ON BOVINE EMBRYO DEVELOPMENT IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 261
Author(s):  
Y.S. Park ◽  
S.H. Choi ◽  
H.D. Park ◽  
M.D. Byun
Keyword(s):  
Embryo Development ◽  
Total Protein ◽  
Harmful Effect ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Chi Square ◽  
Medium Exchange ◽  
Chi Square Test

In vitro embryo development is strongly influenced by IVM conditions. Increased duration of IVM may cause aging of the oocytes, which has a harmful effect on the embryo development. Oocyte maturation depends upon the synthesis of several proteins that may play important roles in the cytoplasmic maturation. These experiments were conducted to determine the effect of IVM duration(18-h or 24-h) and medium exchange (at 18h) on embryo development, and to investigate the protein quantities in IVM medium. Korean Native Cow (KNC) ovaries were obtained from a local slaughterhouse, and cumulus-oocyte complexes (COCs) were aspirated from 2- to 8-mm follicles. Groups of 15 COCs were matured in 50-μL drops of TCM-199 supplemented with 10% fetal calf serum (FBS), 1μgmL−1 MFSH, 10μgmLLH and 1μgmL−1 Estradiol-17β for 18h or 24h. In vitro-matured oocytes were fertilized using frozen-thawed percoll separated spermatozoa (Day 0) in fer-TALP medium for 20h and cultured in CR1aa medium supplemented with 0.3% BSA (before Day 3) or 10% FBS (After Day 3). All types of cultures were carried out in an incubator at 39°C, 5% CO2 in air. The total protein quantity in IVM medium at 18h or 24h were compared by 2-dimensional gel electrophoresis using a 10–15% polyacrylamide gradient gels. Data from three replicates were analyzed by chi-square test. The proportions of oocytes reaching the blastocyst stage was significantly higher in 18h IVM group than 24h IVM group (Table 1). However, there was no difference detected in blastocyst rate between 18h IVM group and 18h medium exchange group. Total protein quantity was reduced between 18h and 24h in IVM medium. There were 299 protein spots identified in IVM medium;; there was an increase at 10 spots in the IVM medium analyzed at 18h and a decrease of 20 spots at 24h. This study suggests that duration of IVM affects subsequent embryo development. The total protein quantity was decreased between 18h and 24h in IVM medium. These proteins may be absorbed into the oocytes and reduce development to the blastocyst stage. However, this may be overcome by IVM medium exchange. Table 1 Effects of duration of IVM and medium exchange on embryo development of KNC oocytes

scholarly journals 160 IN VIVO-CULTURE OF BOVINE EMBRYOS: TRANSFER OF SEMEN PRE-INCUBATED OOCYTES, ZYGOTES AND 4 TO 8 CELL STAGE EMBRYOS INTO THE BOVINE OVIDUCT

2005 ◽  
Vol 17 (2) ◽  
pp. 231
Author(s):  
V. Havlicek ◽  
F. Wetscher ◽  
T. Huber ◽  
M. Gilles ◽  
D. Tesfaye ◽  
...  
Keyword(s):  
Embryo Development ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Group Iii ◽  
Group I ◽  
In Vivo Culture ◽  
Group Ii

Oviduct as well as oocyte and embryo development are subject to developmental changes which have crucial effects on the application of in vivo culture. The present study aimed at optimizing in vivo culture of IVP bovine embryos at different developmental stages in the bovine oviduct. Cumulus oocyte complexes (COC) were collected from slaughterhouse ovaries, matured in vitro for 22 h and assigned to four groups. In groups I and II, oocytes were pre-incubated for 3 to 4 h with 5 × 106 sperm/mL, and then immediately transferred to recipients, which had just completed ovulation (group I), or kept in vitro for a further 12 to 18 h and transferred to Day 1 synchronized recipients (group II). In groups III and IV, COC were subjected to standard IVF/IVC; then embryos were either transferred at the 4- to 8-cell stage on Day 3 into the oviducts of Day 3-synchronized recipients (group III) or kept in vitro for a further 4 to 5 days (group IV). Thirty-four 18- to 30-month-old temporary recipients were synchronized using a standard Ovsynch protocol. COC and embryos were transferred and re-collected by transvaginal endoscopy. COC or embryos were loaded into a 180° curved glass capillary, which was inserted via the infundibulum 5 to 8 cm deep into the ampulla ipsilateral to the CL. On recipient Day 7, a 90° curved metal canula served for tubal flushing prior to conventional uterine embryo flushing. Sixty mL of PBS containing 1% fetal calf serum were rinsed through the oviduct into the uterus and a further 400 mL of medium were finally used for flushing of the uterine horn and collected via an embryo filter. Embryo development was evaluated directly after flushing (Day 7) and on Day 8. For statistical analysis (ANOVA), the blastocyst rates (Days 7 and 8) in group III were related to COC corrected by the collection rate. In group I, 575 COC were transferred to 11 recipients and 420 (73%) were re-collected as oocytes or embryos. The blastocyst yields on Day 7 and Day 8 were 23% (97) and 25% (104), respectively. In group II, the transfer of 489 presumptive zygotes into 13 heifers resulted in only 175 re-collected (36%), of which 15% developed into blastocysts (Day 7: 26; Day 8: 27). Ten heifers (group III) served for in vivo culture of 643 embryos at the 4- to 8-cell stage. On Day 7, 568 (88%) embryos were flushed and 171 (30%) reached the blastocyst stage. A further 24 h culture in vitro finally resulted in 244 (42%) blastocysts. The complete in vitro production system delivered 13% (63/477) blastocysts on Day 7 and 34% (161/477) blastocysts on Day 8. The collection rates (P < 0.001) and the blastocyst rates on Day 7 (P < 0.05) and Day 8 (P < 0.001) differed significantly in all groups. The present data demonstrate that the developmental stage of transferred complexes has an influence on embryo recovery as well as an embryo development. This work was supported by Austrian BMBWK and BMLFUW (#1227).

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