scholarly journals 314EFFECT OF MACROMOLECULE SUPPLEMENTATION DURING IN VITRO MATURATION ON THE DEVELOPMENTAL COMPETENCE OF GOAT OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 276
Author(s):  
J.R. Herrick ◽  
E. Behboodi ◽  
E. Memili ◽  
S. Blash ◽  
Y Echelard ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Maturation ◽  
Mixed Model ◽  
Linear Mixed Model ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Similar Proportion ◽  
Developmental Potential ◽  
Cell Counts

In vitro maturation of goat oocytes has traditionally involved the use of serum or BSA. However, these products introduce variability and complicate evaluation of the effects of other medium components. The objective of this study was to examine the effects of citrate and hyaluronate in the absence or presence of BSA during IVM on the developmental competence of goat oocytes. Abattoir-derived, cumulus-oocyte complexes (COC) were matured for 20–22h (6.0% CO2 in air, 38.7°C) in modified SOF medium (1.5mM glucose, 3.0mM L-lactate, 0.1mM pyruvate, 1.0mM glutamine, 0.1mM taurine) supplemented with 1×MEM nonessential amino acids, 0.5×MEM essential amino acids, 1×MEM vitamins, 0.1mM cysteamine, 5μg mL−1 insulin, 5μgmL−1 transferrin, 5ng mL−1 selenium, 50ngmL−1 EGF, 0.01U mL−1 LH and FSH, and 50μgmL−1 gentamicin. Treatments were: (1) 1mgmL−1 PVA (protein-free, defined); (2) 4mgmL−1 BSA (semi-defined); (3) 0.5mM citrate and 0.5mgmL−1 hylauronate (C+H, defined); and (4) 0.5mM citrate and 0.5mgmL−1 hylauronate with 4mgmL−1 BSA (C+H+BSA, semi-defined). At the end of IVM, COC were transferred to modified Brackett and Oliphant’s medium with 7.7mM Ca-(l)-lactate and 20% FCS for IVF. Frozen-thawed sperm were processed through a 45%:90% Percoll gradient and added to IVF drops (50μL) containing COC at a final concentration of 14–15×106 spermmL−1. Gametes were coincubated in the presence of heparin (25μgmL−1) for 22–24h in 7% CO2 in air at 38.7°C. After coincubation, cumulus cells were removed and zygotes were cultured (6% CO2, 5% O2, 89% N2, 38.7°C) in G1 v.3 for 3 days followed by 4 days in G2 v.3. Cleavage was evaluated when embryos were moved to G2, and development to the blastocyst stage was assessed at the end of culture. All blastocysts were fixed and stained with Hoechst 33342 for total cell counts. Analysis of variance was performed using the general linear mixed model macro of SAS. Means are presented ±SEM and probability values P<0.05 were considered significant. The use of BSA did not improve (P>0.05) the developmental potential of goat oocytes (Table 1). Furthermore, a similar proportion (P>0.05) of oocytes developed to the blastocyst and hatching blastocyst stage after maturation under defined conditions compared to oocytes matured with BSA. In conclusion, developmentally competent goat oocytes can be produced by IVM under defined conditions. Table 1 Development of goat oocytes following IVM with different macromolecules.

130 PREDICTION OF PARTHENOGENETIC DEVELOPMENTAL POTENTIAL BY POLAR BODY EXTRUSION AND FIRST CLEAVAGE ON IN VITRO MATURATION AND DEVELOPMENT OF PORCINE FOLLICULAR OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 145
Author(s):  
H. J. Kim ◽  
S. R. Cho ◽  
C. Y. Choe ◽  
S. H. Choi ◽  
D. S. Son ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Non Invasive ◽  
First Polar Body ◽  
Polar Body Extrusion

The objective of this study was to examine the selection effects of in vitro matured porcine follicular oocytes with polar body extrusion and early cleavage as a non-invasive marker to know the developmental competence in advance. Porcine oocytes matured for 48 h and then examined for polar body extrusion. The examined oocytes were matured for an additional 16–18 h, activated with 7% ethanol, and cultured in 5 µg mL–1 cytochalasin B for 5 h for diploid formation. The treated oocytes were examined for cleavage after 48 h and continued culturing for 5 days. Each treatment was replicated by 3–4 times. Oocytes of 21.9% (70/320) were discarded in morphological selection, and 32.1% (167/520) oocytes were discarded by failure of first polar body extrusion. The selected oocytes were matured and activated, and after 48 h, the cleavage rate was examined. In morphologically selected oocytes, 15.8% (30/190) were not cleaved, 52.6% (100/190) were normally cleaved (consisted of 2–7 cells), and 31.6% (60/190) were hyper-cleaved (consisted of 8 cells or more) at 48 h after activation. However, in the first polar body extruded oocytes, 7.1% (18/253) were not cleaved, 73.1% (185/253) were normally cleaved, and 19.8% (50/253) were hyper-cleaved. From the morphologically selected oocytes, 16.7% (10/60) were developed up to blastocyst stage from those in which cleavage selection was not performed and 31.7% (19/60) from those in which cleavage selection was performed. From the polar body extruded oocytes, 39.0% (39/100) were developed up to blastocyst stage from those in which cleavage selection was not performed and 49.0% (49/100) from those in which cleavage selection was performed. Cleavage was examined within 12 h interval after activation (0 = time of activation) up to 48 h. At 0–12, 12–24, 24–36, and 36–48 h intervals, 4.1% (9/220), 68.6% (151/220), 19.1% (42/220), and 2.3% (5/220) oocytes were cleaved, respectively, and 5.9% (13/220) oocytes were not cleaved at 48 h after activation. The cleaved embryos in each interval were cultured and developed up to blastocyst with 0 (0/9), 39.1 (59/151), 9.5 (4/42), and 0% (0/5), respectively. This result suggests that the polar body extruded and cleaved at 12–36 h embryo has higher developmental potential than the others.

scholarly journals Pregnancies and improved early embryonic development with bovine oocytes matured in vitro with 9-cis-retinoic acid

Reproduction ◽  
2003 ◽  
pp. 409-416 ◽  
Author(s):  
CO Hidalgo ◽  
C Diez ◽  
P Duque ◽  
N Facal ◽  
E Gomez
Keyword(s):  
Retinoic Acid ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
Cell Counts ◽  
Bovine Oocytes ◽  
Number Of Cells

Retinoids have an important role in cell growth, morphogenesis and differentiation. In the present study the developmental potential of bovine oocytes was examined after in vitro maturation in the presence of 9-cis-retinoic acid, a vitamin A metabolite, at 5 nmol l(-1) in chemically defined conditions. Experiments studied early in vitro development, blastocyst differential cell counts and the capacity of embryos to establish pregnancy after transfer to recipients. After in vitro fertilization and culture in simple medium, blastocyst development and hatching rates increased in oocytes matured with 9-cis-retinoic acid. Addition of ethanol (used as a solvent for 9-cis-retinoic acid) resulted in higher cell counts and proportions of cells in the inner mass of day 7 blastocysts. Day 8 blastocysts represented most differences observed in the number of cells. In these embryos, ethanol and 9-cis-retinoic acid increased both the number of cells and proportions in the inner mass. However, while ethanol treatment reduced the number of cells in the trophectoderm, 9-cis-retinoic acid did not. The total number of cells was unaffected by treatment within 1 day, although untreated oocytes lead to day 8 blastocysts with reduced total cell counts. Once transferred to recipients, both fresh and vitrified-warmed blastocysts derived from oocytes matured with 9-cis-retinoic acid yielded more pregnancies at day 60. Modifications of retinoid metabolism affect development and trophectoderm differentiation, and in vitro maturation with 9-cis-retinoic acid increased the developmental competence of the oocyte.

P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Á Martíne. Moro ◽  
I Lamas-Toranzo ◽  
L González-Brusi ◽  
A Pérez-Gómez ◽  
P Bermejo-Álvarez
Keyword(s):  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Developmental Competence ◽  
Success Rates ◽  
Developmental Potential ◽  
Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p > 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

scholarly journals MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence

2020 ◽  
Vol 21 (23) ◽  
pp. 8888
Author(s):  
Bárbara Melo-Baez ◽  
Yat S. Wong ◽  
Constanza J. Aguilera ◽  
Joel Cabezas ◽  
Ana C. F. Mançanares ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Fatty Acid Biosynthesis ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Target Cells ◽  
Bovine Embryos ◽  
Developmental Potential ◽  
Maternal Communication

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.

327 OOCYTE-SECRETED FACTORS DIRECTLY AFFECT OOCYTE DEVELOPMENTAL COMPETENCE DURING IN VITRO MATURATION OF THE BOVINE CUMULUS - OOCYTE COMPLEX

2006 ◽  
Vol 18 (2) ◽  
pp. 271 ◽  
Author(s):  
T. S. Hussein ◽  
R. B. Gilchrist ◽  
J. G. Thompson
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Paracrine Factors ◽  
Cell Functions ◽  
Oocyte Developmental Competence ◽  
Secreted Factors

Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions including proliferation, differentiation, and apoptosis. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this study was to determine if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were obtained by aspiration of >3-mm follicles from abattoir-derived ovaries. IVM was conducted in Bovine VitroMat (Cook Australia, Eight Mile Plains, Brisbane, Australia) supplemented with 0.1 IU/mL rhFSH for 24 h under 6% CO2 in air at 38.5�C. In the first experiment, COCs were co-cultured with denuded oocytes (DOs, 5/COC in 10 �L) beginning at either 0 or 9-h of IVM. To generate the 9-h DO group, COCs were first cultured intact for 9-h and then denuded. In the second experiment, specific OSFs, recombinant bone morphogenetic protein-15 (BMP-15) and growth differentiation factor 9 (GDF-9), were prepared as partially purified supernatants of transfected 293H cells, and used as 10% v/v supplements in Bovine VitroMat. Treatments were: (1) control (no supplement), (2) BMP-15, (3) GDF-9, (4) BMP-15 and GDF-9, and (5) untransfected 293H control. Following maturation, in vitro production of embryos was performed using the Bovine Vitro system (Cook Australia) and blastocysts were examined on Day 8 for development. Developmental data were arcsine-transformed and analyzed by ANOVA, followed by Tukey's test. Cell numbers were analyzed by ANOVA. Co-culturing intact COCs with DOs from 0 or 9 h did not affect cleavage rate, but increased (P < 0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination (50.6 � 1.9 and 61.3 � 1.9%, respectively), compared to COCs cultured alone (40.7 � 1.4%). Therefore, paracrine factors secreted by DOs increased the developmental competence of oocytes matured as COCs. OSFs also improved embryo quality, as co-culture of COCs with DOs (0 or 9 h) significantly increased total cell (156.1 � 1.3 and 159.1 � 1.3, respectively) and trophectoderm (105.7 � 1.3 and 109.8 � 0.4, respectively) numbers, compared to control COCs (total = 148 � 1.2, trophectoderm = 98.2 � 0.8, P < 0.001). BMP-15 alone or with GDF-9 also significantly (P < 0.001) increased the proportion of oocytes that reached the blastocyst stage post insemination (57.5 � 2.4% and 55.1 � 4.5%, respectively), compared to control (41.0 � 0.9%) and 293H-treated (27.1 � 3.1%) COCs. GDF-9 also increased blastocyst yield (49.5 � 3.9%) but not significantly. These results are the first to demonstrate that OSFs, and particularly BMP-15 and GDF-9, directly affect bovine oocyte developmental competence. These results have far-reaching implications for improving the efficiency of IVM in domestic species and human infertility treatment, and support the role of OSF production by oocytes as a diagnostic marker for developmental competence.

249 FOLLICLE-STIMULATING HORMONE AND DIBUTYRYL cAMP TREATMENTS DURING IN VITRO MATURATION IMPROVE THE DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 204
Author(s):  
R. Oishi ◽  
Y. Isaji ◽  
H. Imai ◽  
M. Yamada
Keyword(s):  
In Vitro Maturation ◽  
Basal Medium ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Medium ◽  
Dibutyryl Camp ◽  
Mouse Oocytes ◽  
Junctional Communication

The high level of cyclic adenosine monophosphate (cAMP), which is provided to the oocytes from cumulus cells via gap junctional complexes in cumulus-enclosed oocytes (CEOs), is known to contribute to meiotic arrest at the germinal vesicle (GV) stage of CEOs. However, whether intraoocyte cAMP during the period of in vitro maturation (IVM) affects postfertilization developmental competence of mouse oocytes still remains unclear. The aim of this study was to examine the effects of FSH or dibutyryl cAMP (dbcAMP) treatment during IVM on in vitro development of mouse oocytes after in vitro fertilization (IVF). Whether a junctional association between cumulus cells and the oocyte would be essential for a cytoplasmic maturation-promoting effect was also examined. CEOs were isolated from and eCG-primed 3-week-old ICR mouse by rupturing preovulatory follicles with needles in M16 medium with 5% FCS and essential and nonessential amino acids (basal medium). IVM media used were basal medium without (control) or with 100 µm dbcAMP or 1 IU mL–1 FSH. Carbenoxolone (100 µm, CBX), an inhibitor of gap junction, was used to inhibit a junctional association between cumulus cells and the oocyte. Denuded oocytes (DOs) were prepared by repeatedly pipetting in basal medium with 0.2% hyaluronidase. CEOs and DOs were cultured in IVM media at 37�C under 5% CO2 in air for 16.5 h, and then transferred to TYH medium (a modified Krebs-Ringer bicarbonate medium) containing 0.4% BSA, followed by insemination with capacitated sperm. After 6 h of IVF, inseminated oocytes were cultured in KSOM medium with 0.3% BSA. Development to the 2-cell and blastocyst stages was estimated at 24 h and 120 h after IVF, respectively. All experiments were done in 3 replicates, and the statistical analysis was carried out by ANOVA and Fisher's protected least-squares difference (PLSD) test. When CEOs were matured in IVM media, the rates of postfertilization development to the 2-cell and blastocyst stages of oocytes matured in the control medium were very low(29% and 13%, respectively), whereas those of oocytes matured with FSH or dbcAMP significantly increased (FSH: 61% and 52%, dbcAMP: 63 and 57%, respectively, v. control; P < 0.05). Next, when CEOs were matured in basal medium with 1 IU mL–1 FSH and 100 µm CBX, the developmental rate to the 2-cell stage (56%) was similar to that in medium with FSH alone (61%) but the rate to the blastocyst stage (40%) was little lower compared with that in medium with FSH alone (52%), although not significantly different (P > 0.05). Furthermore, when DOs were matured in IVM media, the developmental rates to the blastocyst stage after IVF of the oocytes matured with FSH or dbcAMP significantly increased (FSH: 25%, dbcAMP: 15%; P < 0.05) compared with those in control medium (7%). Taken together, it is suggested that increasing the concentration of intraoocyte cAMP during the IVM period is important to improve the developmental competence after IVF of mouse oocytes, and that the competence is acquired in part in a cumulus-oocyte junctional communication-independent manner.

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

99 In Vitro Development of Alpaca Embryos Obtained by Bisection

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

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

scholarly journals cAMP Modulators before In Vitro Maturation Decrease DNA Damage and Boost Developmental Potential of Sheep Oocytes

Animals ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 2512
Author(s):  
Daniela-Alejandra Medina-Chávez ◽  
Irene Sánchez-Ajofrín ◽  
Patricia Peris-Frau ◽  
Carolina Maside ◽  
Vidal Montoro ◽  
...  
Keyword(s):  
Dna Damage ◽  
In Vitro Maturation ◽  
Phosphodiesterase Inhibitor ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Assisted Reproduction Technologies ◽  
Underlying Mechanisms

To date, the underlying mechanisms by which cAMP modulators act during in vitro maturation to improve oocyte developmental competence are poorly understood. Here, we sought to fill this knowledge gap by evaluating the use of phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and adenylyl cyclase activator forskolin during a culture period of 2 h before in vitro maturation (pre-IVM) on the nuclear and cytoplasmic maturation features in essential organelles, cumulus cells activity, and in vitro developmental potential of sheep oocytes. Results showed that pre-IVM treatment significantly decreased (p < 0.05) the DNA damage of mature oocytes (pre-IVM = 2.08% ± 3.51% vs. control = 20.58% ± 3.51%) and increased (p ≤ 0.05) expanded blastocyst rates compared to the control (from the total of oocytes: pre-IVM = 23.89% ± 1.47% vs. control = 18.22% ± 1.47%, and from the cleaved embryos: pre-IVM = 45.16% ± 1.73% vs. control = 32.88% ± 1.73%). Considering that oocytes are highly vulnerable to the accumulation of DNA damage because of exposure to in vitro culture conditions, our results suggest that the modulation of intra-oocyte cAMP levels with forskolin and IBMX before IVM might afford oocytes a more effective DNA repair mechanism to overcome damage obstacles and ultimately improve developmental competence. This previously unappreciated action of cAMP modulators could help to develop improved methods for assisted reproduction technologies in animal and clinical research.

160 In vitro maturation of pre-pubertal goat oocytes and their development after chemical activation

2019 ◽  
Vol 31 (1) ◽  
pp. 205
Author(s):  
N. A. Wani ◽  
S.-B. Hong
Keyword(s):  
In Vitro Maturation ◽  
Culture Media ◽  
Chemical Activation ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Epidermal Growth ◽  
Humidified Air ◽  
Activation Status

Experiments were conducted to study in vitro maturation of pre-pubertal goat oocytes and their developmental potential after chemical activation. Cumulus-oocyte complexes (n=1170) collected from the ovaries of pre-pubertal goats slaughtered at a local abattoir were matured in TCM-199 supplemented with 0.15mg mL−1 l-glutamine, 0.25mM sodium pyruvate, 0.1mM l-cysteine, 20ng mL−1 epidermal growth factor, 10mg mL−1 FSH, 10mg mL−1 LH, 1μg mL−1 oestradiol and 10% FCS for 24h at 39°C under 5% CO2 in humidified air. In Experiment 1, matured oocytes were activated (r=6) with either 5mM ionomycin (n=85) or 7% ethanol (n=91) followed by culture in 6-DMAP for 4h. All the activated oocytes were then cultured in KSOM supplemented with 3mg mL−1 BSA and were fixed and stained with Hoechst 33342 after 18h of culture to evaluate their activation status. In Experiment 2, oocytes activated with 5mM ionomycin and 6-DMAP were cultured for 7 days (r=6) in 1 of the 4 different culture media [Charles Rosenkrans medium (CR-1), modified TCM-199, KSOM and SOF] to study their developmental potential. All media were supplemented with 3.0mg mL−1 BSA for the first 3 days and 10% FCS for the subsequent 4 days. Of these pre-pubertal oocytes, 59% reached metaphase II stage, and 83% of these oocytes were classified as activated in the group using ionomycin in comparison with 69% in the group using ethanol as an activating agent (P&lt;0.05). No difference was observed in the cleavage rate of activated oocytes cultured in any of the 4 culture media (65.7v. 55.0v. 61.0v. 56.2%, respectively). However, the development to blastocyst stage was observed in only KSOM (16%) and SOF (5%) media. In conclusion, the present study demonstrates that pre-pubertal goat oocytes can mature in vitro and can be activated with 5mM ionomycin, and KSOM, and to a lesser extent SOF, supports development to the blastocyst stage. We plan to use these oocytes as a cytoplast source for interspecies somatic cell NT; however, before that, more studies are needed to evaluate their requirements in culture media to enhance their development to the blastocyst stage.

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