236 EFFECT OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATOR REGULATORS IN ASSOCIATION WITH BMP15 ON BOVINE EMBRYO DEVELOPMENT IN VITRO

2015 ◽  
Vol 27 (1) ◽  
pp. 207
Author(s):  
M. F. Machado ◽  
M. F. G. Nogueira ◽  
R. B. Gilchrist ◽  
M. L. Sutton-McDowall ◽  
D. G. Mottershead ◽  
...  
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Oocyte Competence ◽  
Significant Difference

BMP15 is a promising peptide to improve oocyte competence; also, addition of cyclic adenosine monophosphate modulator (cAMP) regulators prevents spontaneous maturation in vitro and promotes embryo development. We aimed to assess embryo development after prematuration [pre-in vitro maturation (IVM)] with IBMX and Forskolin (FSK) and maturation in the presence or absence of a purified pro mature region of BMP15. Immature cumulus-oocyte complexes (COC) were cultured in vitroMat (IVF Vet Solutions, Adelaide, Australia) plus 4 mg mL–1 fatty acid free-BSA and rhFSH (0.1 IU mL–1), then divided into the following treatment groups: 1) spontaneous IVM: 24 h of IVM; 2) spontaneous IVM + BMP15: 24 h of IVM in the presence of BMP15 (100 ng mL–1); 3) Pre 2 h: pretreatment with IBMX (500 µM; Sigma-Aldrich) and FSK (100 µM; Sigma-Aldrich) for 2 h following 24 h maturation; and 4) Pre 2 h + BMP15: pretreatment with IBMX and FSK for 2 h following 24 h maturation in the presence of BMP15 (100 ng mL–1). After maturation, oocytes were inseminated and zygotes were cultured for 5 days in VitroCleave (IVF Vet Solutions, Adelaide, Australia) and transferred into VitroBlast (IVF Vet Solutions, Adelaide, Australia) until blastocyst assessment (Days 7 and 8). Zona-intact embryos were retrieved to assess differential staining of trophectoderm and inner cell mass. Data were transformed into a logarithm and analysed by 1-way ANOVA and post hoc least significant difference using SigmaStat software (SPSS Inc., San Jose, CA, USA; P < 0.05). There was no difference among groups on cleavage rates or blastocyst rates at Day 7; however, both Pre 2 h treatments increase hatched blastocyst rates at Day 8 of embryo development (Table 1). Supplementation with BMP15 increased total blastocyst rates at Day 8, regardless of pretreatment with IBMX+FSK (Table 1). Our data demonstrate that embryos from oocytes matured in the presence of BMP15 or pretreated with IBMX+FSK increase trophectoderm and total cell numbers; however, no differences were observed for inner cell mass. We conclude that Pre 2 h treatment or BMP15 increase embryo development; however, no effect of cAMP regulators in association with BMP15 on embryo development was observed. Table 1.Embryo development Supported by FAPESP (project numbers: 2012/1073-8; 2013/12960-9; 2013/05083-1; 2012/50533-2).

119 Short spermatozoa-oocyte co-incubation improves outcomes of IVF in sheep

2020 ◽  
Vol 32 (2) ◽  
pp. 186
Author(s):  
D. Anzalone ◽  
M. Czernik ◽  
L. Palazzese ◽  
Y. Ressaissi ◽  
P. Scapolo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Time Window ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Basic Research ◽  
Cell Number ◽  
Differential Staining ◽  
Assisted Reproductive Technique ◽  
Significant Difference

The assisted reproductive technique IVF is routinely applied in humans and large animals, both to boost reproductive performance and also for basic research. Despite its value, IVF has seen very little progress in the last two decades and relies on established paradigms, such as overnight sperm-egg co-incubation. However, the long exposure of oocytes to spermatozoa in a dish increases the risk of polyspermy and could be detrimental for early stages of embryonic development. We identified a time window within which fertilization occurs, in order to reduce the length of sperm-egg co-incubation and optimize the procedure, comparing polyspermy rate and embryo development after short (shIVF) and overnight (o/nIVF) spermatozoa-oocyte co-incubation. A total of 666 invitro-matured sheep oocytes were co-incubated with spermatozoa in IVF medium (synthetic oviductal fluid (SOF) with 20% oestrus sheep serum and 16 µM isoproterenol). First, small batches of oocytes were collected every 30min to check for the presence of a fertilizing spermatozoon. To assess this, cumulus cells were removed and presumptive fertilized oocytes were fixed and stained with propidium iodide for nuclei and Pisum sativum agglutinin for zona pellucida (ZP) detection, respectively. Then, pronuclear formation (PN) and embryo development were evaluated after 16h (PN), 24h (2 cells), and 7 days of culture (blastocyst). The oocytes that were not cleaved at 24h were stained for DNA content with Hoechst 33342. Furthermore, we evaluated embryo quality by counting cells of 8-day blastocysts after differential staining of inner cell mass (ICM) and trophectoderm (TE). We found that spermatozoa reach the ZP no earlier than 90min from the beginning of co-incubation and achieve fertilization within 4h. Polyspermic fertilization (&gt;2PN) was lower in shIVF (6.5%) than in o/nIVF (17.8%; P=0.006). This proportion of polyspermy was maintained between groups in noncleaved oocytes at 24h from fertilization. Likewise, cleavage and blastocyst rate were higher in shIVF compared with the o/n-IVF group (2-cells: 48.3% vs. 31.6%, P=0.001; blastocyst: 29.4% vs. 20.5%, P=0.046, respectively). Differential staining of blastocysts revealed no significant difference in cell number between the blastocysts of the two groups. This work demonstrates that 4h of sperm-egg interaction are sufficient to achieve fertilization, reduce polyspermy, and improve the rate of embryos reaching blastocyst stage without compromising embryo quality.

192 PREMATURATION OF BOVINE CUMULUS-OOCYTE COMPLEXES WITH CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS AFFECTS BOTH OOCYTE AND BLASTOCYST ULTRASTRUCTURE

2016 ◽  
Vol 28 (2) ◽  
pp. 227
Author(s):  
E. Razza ◽  
H. Pedersen ◽  
L. Stroebech ◽  
M. Machado ◽  
M. Nogueira ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Smooth Endoplasmic Reticulum ◽  
Cell Mass ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Developmental Competence ◽  
Cortical Granules ◽  
Cytoplasmic Vesicles ◽  
Globally Distributed

Oocytes resume meiosis spontaneously when subjected to in vitro maturation (IVM). Cyclic adenosine monophosphate (cAMP) elevating agents have been used for artificial blocking of meiotic resumption (pre-IVM) to allow the oocyte to prepare for maturation, potentially increasing its developmental competence. However, the ultrastructural effects of this pharmacological approach on oocytes and embryos remain to be addressed. We assessed the effects of pre-IVM with cAMP modulators in oocytes (10 for each group) at the end of IVM and in blastocyst (10 for each group) after 7 days of culture. Cumulus-oocyte complexes (COC) were subjected to pre-IVM for 2 h with forskolin (Sigma, St. Louis, MO, USA; 100 μM) and 3-isobutyl-1-methylxanthine) (IBMX, Sigma, 500 μM) followed by 24 h of IVM with FSH-enriched media (IVF Vet Solutions, Adelaide, Australia). Simultaneously, another group of COC was subjected to conventional IVM (con-IVM) for 24 h (EmbryoTransBiotech, Copenhagen, Denmark) with BSA (4 mg mL–1, Sigma), gentamycin (50 mg mL–1), and FSH (0.1 IU mL–1). Matured oocytes were collected for qualitative ultrastructural analysis or followed to IVF. The morphology was carefully evaluated on serially sectioned oocytes and embryos, where each serial section (~60.2-μm section per oocyte/embryo) was analysed under light microscopy. Subsequently, the equatorial section from each oocyte and the section giving the optimal representation of the inner cell mass in each blastocyst was re-embedded and sectioned for electron microcopy as previously described (Hyttel and Madsen 1987 Acta Anat. 129, 12–14). Blastocyst rates did not differ between groups. Ultrastructural analyses revealed subtle ultrastructural differences between pre-IVM and con-IVM conditions. In both groups, oocytes had matured to metaphase II. The perivitelline space of pre-IVM oocytes was significantly narrower than con-IVM. The cytoplasmic vesicles were more abundant and globally distributed in pre-IVM oocytes, whereas at con-IVM a vesicle-free periphery of the ooplasm was frequent, except for cortical granules and clusters of mitochondria associated with smooth endoplasmic reticulum (SER). We observed typical hooded mitochondria and cortical granules either clustered in the periphery or solitarily distributed in the cortical ooplasmic region for both groups. In the blastocysts, differences were noted with respect to especially distribution of ribosomes. In pre-IVM blastocysts, ribosomes were mostly organised in free clusters (polysomes) and peripherally located in cells of the inner cell mass. Con-IVM blastocysts showed ribosomes preferentially associated with the rough ER and often associated with mitochondria. Lipid droplets and rounded mitochondria were observed in both groups as well as apically located tight junctions and desmosomes between adjacent trophectoderm (TE) cells. Pleomorphic and elongated mitochondria were abundant in the TE of pre-IVM blastocysts, whereas the mitochondrial population was more homogenous at con-IVM. These findings suggest that pre-IVM for 2 h affects oocyte and blastocyst ultrastructure. Research was supported by grants 12/50533-2 and 12/23409-9 from FAPESP.

131 EFFECTS OF HIGH MOLECULAR WEIGHT HYALURONAN ON SHEEP EMBRYO DEVELOPMENT AND SURVIVAL AFTER CRYOPRESERVATION

2013 ◽  
Vol 25 (1) ◽  
pp. 213
Author(s):  
V. Ghaffarilaleh ◽  
F. Ghafari ◽  
M. Teresa-Paramio ◽  
A. Fouladi-Nashta
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Apoptotic Cell ◽  
Cell Mass ◽  
Differential Staining ◽  
Embryo Viability ◽  
Fluid Medium ◽  
Vitro Fertilization ◽  
Large Size

Hyaluronan (HA), a component of extracellular matrix in mammalian tissues including that of the reproductive system, has been shown to support embryo development. HA is produced in various sizes with distinct physiological functions. Cleaved sheep embryos produced after in vitro maturation and in vitro fertilization were cultured in serum free synthetic oviduct fluid medium supplemented with increasing concentrations (0, 0.25, 0.5, and 1 mg mL–1) of large size HA (Healon; 6 × 106 Da). Development to blastocyst stage was recorded at Day 7 when a group of the embryos were fixed and stained by differential staining combined with TUNEL labelling to analyse embryo quality. The remainder of the blastocysts from each treatment/repeat were vitrified in open pulled straws and then cultured for an extra period of 48 h to analyse their survival rate and quality after cryopreservation. SPSS version 20 software (SPSS Inc., Chicago, IL, USA) was used for analyzing the data with generalized linear model. Healon did not change blastocyst (33 ± 5.7, 32 ± 6.0, 35 ± 5.5; P ≥ 0.05) or survival rates (63 ± 17.1, 83 ± 15.2, 58 ± 14.2; P ≥ 0.05) as compared to the respective controls (25 ± 5.2, 38 ± 17.1). It increased the total cell (TC) number (83.6 ± 4.6, 100.7 ± 3.8, 97.2 ± 3.7, 105.0 ± 3.9; P ≤ 0.05) and trophectoderm cells (TE) (58.4 ± 3.8, 74.2 ± 3.2, 75.6 ± 3.3, 80.1 ± 3.4; P ≤ 0.05) but had no effect on the number of inner cell mass (ICM) and apoptotic cells. The ICM : TE ratio was not affected (0.45 ± 0.04, 0.36 ± 0.03, 0.33 ± 0.03, 0.30 ± 0.03; P ≥ 0.05). Surviving embryos had higher TC (63.2 ± 3.7, 130.8 ± 3.6, 113.9 ± 5.2, 149.8 ± 5.4; P ≤ 0.05), TE (42.9 ± 3.0, 96.7 ± 3.1, 85.2 ± 4.5, 111.9 ± 4.7; P ≤ 0.05) and ICM (20.3 ± 2.2, 32.9 ± 1.8, 27.7 ± 2.6, 36.5 ± 2.7; P ≤ 0.05). The apoptotic cell numbers and ICM : TE ratio of the survived embryos after cryopreservation were not affected by HA supplementation. The results indicate that large size HA improves the embryo viability and quality, which may have implication for improving embryo transfer.

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 793-803 ◽  
Author(s):  
V.E. Papaioannou ◽  
K.M. Ebert
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Staining Technique ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Morphological Development ◽  
Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

scholarly journals A shorter equilibration period in the VitTrans in-straw bovine embryo vitrification method improves post-warming outcomes

2020 ◽  
Author(s):  
Iris Martínez-Rodero ◽  
Tania García-Martínez ◽  
Erika Alina Ordóñez-León ◽  
Meritxell Vendrell-Flotats ◽  
Carlos Olegario-Hidalgo ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Field Conditions ◽  
Bovine Embryo ◽  
Direct Transfer ◽  
Treatment Groups ◽  
Cell Counts ◽  
Hatching Rates

Abstract Background VitTrans is a device that enables the vitrification and warming/dilution of in vitro produced bovine embryos followed by their direct transfer to recipient females in field conditions. This study sought to improve the VitTrans method by comparing two equilibration times: short (SE: 3 min) and long (LE: 12 min). Outcome measures recorded in vitrified D7 and D8 expanded blastocysts were survival and hatching rates, differential cell counts, apoptosis rate and gene expression. Results While survival rates at 3 h and 24 h post-warming were reduced (P < 0.05) after vitrification, hatching rates of D7 embryos vitrified after SE were similar to those obtained in fresh non-vitrified blastocysts. Hatching rates of vitrified D8 blastocysts were lower (P < 0.05) than of fresh controls, regardless of treatment. Total cell counts, and inner cell mass and trophectoderm cell numbers were similar in hatched blastocysts derived from D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values, regardless of treatment. The rate of apoptotic cells was significantly higher in both treatment groups when compared to fresh controls, although apoptosis rates were lower using the SE than LE protocol. No differences emerged in expression of the genes BAX, AQP3, CX43 and IFNτ between blastocysts vitrified after SE or LE, whereas a significantly higher abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE compared to LE. Conclusions The VitTrans device combined with a shorter exposure to the equilibration medium improves vitrification/warming outcomes facilitating the direct transfer of vitrified embryos under field conditions.

scholarly journals PTPN11 (SHP2) Is Indispensable for Growth Factors and Cytokine Signal Transduction During Bovine Oocyte Maturation and Blastocyst Development

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1272 ◽  
Author(s):  
Muhammad Idrees ◽  
Lianguang Xu ◽  
Seok-Hwan Song ◽  
Myeong-Don Joo ◽  
Kyeong-Lim Lee ◽  
...  
Keyword(s):  
Growth Factors ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Specific Inhibitor ◽  
Mrna Translation ◽  
Bovine Oocyte ◽  
Blastocyst Development

This study was aimed to investigate the role of SHP2 (Src-homology-2-containing phosphotyrosine phosphatase) in intricate signaling networks invoked by bovine oocyte to achieve maturation and blastocyst development. PTPN11 (Protein Tyrosine Phosphatase, non-receptor type 11) encoding protein SHP2, a positive transducer of RTKs (Receptor Tyrosine Kinases) and cytokine receptors, can play a significant role in bovine oocyte maturation and embryo development, but this phenomenon has not yet been explored. Here, we used different growth factors, cytokines, selective activator, and a specific inhibitor of SHP2 to ascertain its role in bovine oocyte developmental stages in vitro. We found that SHP2 became activated by growth factors and cytokines treatment and was highly involved in the activation of oocyte maturation and embryo development pathways. Activation of SHP2 triggered MAPK (mitogen-activated protein kinases) and PI3K/AKT (Phosphoinositide 3-kinase/Protein kinase B) signaling cascades, which is not only important for GVBD (germinal vesical breakdown) induction but also for maternal mRNA translation. Inhibition of phosphatase activity of SHP2 with PHPS1 (Phenylhydrazonopyrazolone sulfonate 1) reduced oocytes maturation as well as bovine blastocyst ICM (inner cell mass) volume. Supplementation of LIF (Leukemia Inhibitory Factor) to embryos showed an unconventional direct relation between p-SHP2 and p-STAT3 (Signal transducer and activator of transcription 3) for blastocyst ICM development. Other than growth factors and cytokines, cisplatin was used to activate SHP2. Cisplatin activated SHP2 modulate growth factors effect and combine treatment significantly enhanced quality and rate of developed blastocysts.

168 EFFECT OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS DURING OOCYTE IN VITRO MATURATION ON BOVINE EMBRYOS (Gyr×HOLSTEIN) QUALITY

2016 ◽  
Vol 28 (2) ◽  
pp. 214
Author(s):  
G. R. Leal ◽  
C. A. S. Monteiro ◽  
H. F. R. A. Saraiva ◽  
A. J. R. Camargo ◽  
P. M. S. Rosa ◽  
...  
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Bovine Embryos ◽  
Tropical Conditions ◽  
Significant Difference ◽  
Number Of Cells

In vitro embryo production (IVP) is an important tool for cattle breeding. Brazilian dairy systems are based on Gyr × Holstein crossbreds, which integrates adaptability to tropical conditions and milk production. Quality determines the oocyte proportion that will develop to blastocyst stage, and although the lipid content is important in oocyte development, a high concentration in embryos is associated with cryotolerance reduction, making this a relevant issue for IVP systems. The in vitro maturation system (IVM) simulated physiological oocyte maturation (SPOM) mimics the physiological maturation events by using cyclic adenosine monophosphate (cAMP) modulators, which promote the increase of oocyte competence. Among the modulators, Forskolin has lipolytic properties. The aim of this study was to evaluate the effect of the SPOM system (Albuz 2010 Hum. Reprod. 25, 12) on bovine embryos (Gyr × Holstein) regarding their total number of cells (TNC) and lipid content. Oocytes were obtained by ovum pick-up from Gyr cows in 5 replications. After selection, they were randomly divided into 2 groups: SPOM (S) and control (C). The IVM lasted 24 h for group C (TCM 199 medium without FBS) in culture oven at 38.5°C, 5% CO2 in atmospheric air and high humidity. In the SPOM system, oocytes were in pre-IVM [TCM 199 medium + 100 µM Forskolin + 500 µM 3-isobutyl-1-methylxanthine (IBMX)] for 2 h and followed for extended IVM (TCM 199 medium + 20 µM cilostamide) for 28 h under the same conditions as control group. After IVM, oocytes were fertilised with semen from a single Holstein bull that was prepared by Percoll gradient method in Fert-TALP medium (Bioklone® Animal Reproduction, São Paulo, Brazil) for 22 h and transfered to culture droplets, where they remained for 7 days (n = 10–13 per group). The lipid content analysis was performed by staining with Oil red and the stained area fraction of each embryo was measured using software ImageJ (NIH, Bethesda, MD, USA). The TNC was measured after being stained with Hoechst 33342 and results were analysed by Student's t-test in Instat GraphPad program, with a 5% significance level. There was no significant difference (P > 0.05) between embryos from both groups on TNC (group S: 88.9 ± 28.0A; group C: 101.6 ± 29.1a) and lipid content (group S: 0.93 ± 12:18A; group C: ±0.15 to 0.96) analysis. Some studies have shown there is a beneficial effect on embryo quality when using this system; however, our results demonstrated that there was no effect on total number of cells using our conditions. Some authors have also demonstrated a reduction in embryo lipid content using Forskolin during in vitro culture. Our results suggest that the time of Forskolin exposure was not enough to ensure lipolytic action on the structures produced from oocytes (Gyr) treated in pre-IVM. It was concluded that the SPOM system had no effect on TNC and lipid content of Gyr/Holstein embryos. Financial support from FAPERJ and CAPES is acknowledged.

239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 209
Author(s):  
T. Fanti ◽  
N. M. Ortega ◽  
R. Garaguso ◽  
M. J. Franco ◽  
C. Herrera ◽  
...  
Keyword(s):  
Phosphodiesterase Inhibitor ◽  
Basal Medium ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Oocyte Competence

In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P > 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P < 0.01). Blastocyst hatching rate was significant (P < 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos. Table 1.Effect of treatment on maturation, cleavage, and cryotolerance

148 EFFECTS OF CAFFEINE SUPPLEMENTATION ON BOVINE OOCYTE DEVELOPMENTAL CAPACITY

2017 ◽  
Vol 29 (1) ◽  
pp. 182
Author(s):  
S. M. Bernal-Ulloa ◽  
A. Lucas-Hahn ◽  
P. Aldag ◽  
D. Herrmann ◽  
U. Baulain ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Mammalian Species ◽  
Cell Mass ◽  
Phosphodiesterase Inhibitor ◽  
Developmental Competence ◽  
Differential Staining ◽  
Final Phase ◽  
Cell Numbers ◽  
Developmental Capacity

Oocyte culture in the presence of the nonspecific competitive phosphodiesterase inhibitor caffeine has been reported to increase developmental capacity of oocytes in different mammalian species. Here, we evaluated the effects of caffeine supplementation during the final phase of in vitro maturation (IVM) on developmental rates and blastocyst cell numbers. Bovine ovaries were collected from a local abattoir. A total of 1142 cumulus-oocyte-complexes were obtained by slicing. Cumulus-oocyte complexes were either in vitro matured for 24 h (Standard) or matured for 20 h followed by additional culture for 6 h in fresh IVM medium supplemented with 10 mM caffeine (Caffeine 6 h). In vitro fertilization was performed for 19 h using frozen-thawed sperm from 2 different bulls. After IVF, presumptive zygotes were cultured in vitro for 8 days until the blastocyst stage. Cleavage and blastocyst rates were evaluated 3 and 8 days after IVF, respectively. Expanded blastocysts from the different treatments were submitted to differential staining. SAS/STAT software (SAS Institute Inc., Cary, NC, USA) was used to evaluate cleavage and blastocyst rates using the Glimmix procedure and blastocyst cell numbers were compared using the linear model procedure. Cleavage rates were lower using caffeine for bull B and blastocyst production decreased for bull A. Caffeine treatment increased inner cell mass (ICM) number for bull B and decreased trophectoderm (TE) and total cell numbers for bull A. However, similar TE and total cells were obtained for bull B (Table 1; P < 0.05). Results show that developmental competence can be affected by caffeine supplementation at the final phase of IVM probably due to oocyte-sperm interaction changes. Table 1. In vitro developmental competence of oocytes cultured with caffeine at the end of IVM

scholarly journals Initiation of X Chromosome Inactivation during Bovine Embryo Development

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1016 ◽  
Author(s):  
Bo Yu ◽  
Helena T. A. van Tol ◽  
Tom A.E. Stout ◽  
Bernard A. J. Roelen
Keyword(s):  
Embryo Development ◽  
X Chromosome ◽  
Developmental Process ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Morula Stage

X-chromosome inactivation (XCI) is a developmental process that aims to equalize the dosage of X-linked gene products between XY males and XX females in eutherian mammals. In female mouse embryos, paternal XCI is initiated at the 4-cell stage; however, the X chromosome is reactivated in the inner cell mass cells of blastocysts, and random XCI is subsequently initiated in epiblast cells. However, recent findings show that the patterns of XCI are not conserved among mammals. In this study, we used quantitative RT-PCR and RNA in situ hybridization combined with immunofluorescence to investigate the pattern of XCI during bovine embryo development. Expression of XIST (X-inactive specific transcript) RNA was significantly upregulated at the morula stage. For the first time, we demonstrate that XIST accumulation in bovine embryos starts in nuclei of female morulae, but its colocalization with histone H3 lysine 27 trimethylation was first detected in day 7 blastocysts. Both in the inner cell mass and in putative epiblast precursors, we observed a proportion of cells with XIST RNA and H3K27me3 colocalization. Surprisingly, the onset of XCI did not lead to a global downregulation of X-linked genes, even in day 9 blastocysts. Together, our findings confirm that diverse patterns of XCI initiation exist among developing mammalian embryos.

Sign in / Sign up

Export Citation Format

Share Document