308 VERBASCOSIDE TREATMENT DURING IN VITRO MATURATION IMPROVES THE EMBRYO DEVELOPMENT OF PREPUBERTAL OVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 243
Author(s):  
M. E. Dell'Aquila ◽  
F. Ariu ◽  
N. A. Martino ◽  
F. Minervini ◽  
A. Cardinali ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Redox Status ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Chi Squared ◽  
Development In Vitro ◽  
Positive Effect ◽  
Olive Mill

Verbascoside (VB), a bioactive polyphenol from olive mill wastewater with known antioxidant activity, was shown to act as a pro-oxidant molecule, by impairing energy/redox status and embryo developmental competence of prepubertal ovine oocytes when added at micromolar concentrations in a continuative 24-h in vitro maturation (IVM) exposure protocol (1). The aim of the present study was to determine whether a lower (nanomolar) VB concentration and a shorter exposure time (2 v. 24 h) during IVM may improve the maturation rates of prepubertal ovine oocytes and their subsequent embryonic development in vitro. Cumulus-oocyte complexes derived from the ovaries of slaughtered 1-mo-old prepubertal sheep oocytes underwent IVM in TCM 199 with 10% oestrus sheep serum, 0.1 IU mL–1 of FSH/LH, and 100 µM cysteamine, in 5% CO2 in air at 38.5°C for 24 h. Based on our previous results (Dell'Aquila et al. 2014 Biomed. Res. Int. 2014, 878062), VB was added in the IVM medium at 1.03 nM, and 2 incubation times (24 and 2 h) were tested. In the 2-h exposure group, after 2 h of exposure to VB, oocytes were washed and cultured up to 24 h without VB. A group of oocytes were cultured in absence of VB, as controls. Matured oocytes were fertilized with frozen-thawed ram semen in SOF medium for 22 h and zygotes were cultured in vitro for 8 days. Metaphase II (MII) cleavage and blastocyst rates were analysed by Chi-squared test. Embryo quality was evaluated by staining and total cell count of the blastocyst and analysis of variance (ANOVA) was applied. Differences were considered to be significant when P < 0.05. Compared to controls, VB treatment at the concentration of 1.03 nM and 24 h of exposure had no effect on MII rates (196/268, 73% v. 226/323, 70% MII/cultured oocytes; P > 0.05). However, this treatment allowed to obtain significantly higher rates of cleaved embryos/MII oocytes (156/196, 80% v. 165/226, 73%; respectively; P < 0.05), blastocyst yield/cleaved embryos (59/156, 38% v. 45/165, 27%, respectively; P < 0.05), and total blastocyst cell numbers (108.62 ± 19.87 v. 89.61 ± 26.32, respectively; P < 0.05) compared to control oocytes. The VB treatment at the same concentration but for 2 h induced only significantly higher cleavage rate (196/210, 93% v. 165/226, 73%; P < 0.05). In conclusion, our results showed that VB treatment at 1.03 nM during 24 h of IVM exerted a positive effect on in vitro embryo development of prepubertal ovine oocytes by increasing the blastocyst yield and their quality. The hypothesis that VB at nanomolar concentrations may improve cumulus-oocyte energy/redox status is under investigation.The authors acknowledge support by the Regione Autonoma della Sardegna (LR 7, Agosto 2007, no. 7, CRP-17602). The authors thank Dr D. Bebbere and L. Falchi, Dept. Veterinary Medicine, Sassari, for statistical analysis.

94 USE OF β-MERCAPTOETHANOL AND CYSTEINE FOR IN VITRO MATURATION AND CULTURE OF SHEEP EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 159
Author(s):  
J. Pradiee ◽  
L. L. Viana ◽  
E. C. S. Santos ◽  
A. Gonçalves ◽  
R. G. Mondadori ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
In Vitro Production ◽  
Embryo Viability ◽  
Negative Effects ◽  
Cleavage Rate ◽  
Chi Squared ◽  
Antibiotic Solution ◽  
The Media

During in vitro production (IVP), embryos are sensitive to suffering negative effects from catabolites, such as reactive oxygen species (ROS). Under physiological conditions, the action of the ROS is blocked by antioxidants such as glutathione, but glutathione's concentration is reduced during the main steps of the IVP process. The objective of the present study is to evaluate the effect of the supplementation of the media for in vitro maturation (IVM) and in vitro culture (IVC) with β-mercaptoethanol and cysteine on the rates of embryo development and viability after vitrification in open pulled straws (OPS). Ten IVP routines were conducted for IVP, using ovaries form pubertal sheep collected in a slaughterhouse. The ovaries were kept in a saline/antibiotic solution at 30°C during transport to the laboratory. The cumulus oophurus–oocytes complexes (COC) selected for IVM were allocated to 2 treatments: T1 (control), including no antioxidants in the IVM and IVC media (n = 676); and T2, including 50 μM β-mercaptoethanol and 600 μM cysteine, in the IVM and IVC media (n = 729). The IVM was conducted using the TCM 199 medium including oestradiol, FSH, LH, pyruvate, heat inactivated sheep serum and antibiotics, for 22 to 24 h. Sperm selection was conducted by swim-up in medium with tris-glucose-citric acid with fresh semen. For IVF, conducted for 18 to 22 h, 1 × 106 spermatozoa per mL were used in SOF medium including 2% heat-inactivated sheep serum. Both IVM and IVF were conducted with incubation with 5% CO2 at 39°C with saturated humidity. After IVF, the probable zygotes were denuded and cultured for 8 days in SOF medium with 0.4% BSA, at 39°C, in bags with 3 gases (5% CO2, 90% N2 and 5% O2). The criteria considered for embryo viability were: cleavage rate at Day 2 (cleaved/inseminated), embryo development at Day 7 (blastocysts/cleaved) and the reexpansion rate 24 h post-vitrification. Such frequencies were compared between treatments by the chi-squared test. The cleavage rate did not differ (P > 0.05) for T1 (60.3%) and T2 (64.3%). The rate of embryro development at Day 7 was also similar (P > 0.05) for T1 (33.6%) and T2 (36.6%). The reexpansion rate for T1 (76.9%) and T2 (54.1%) were also similar (P > 0.05). Thus, supplementation of IVM and IVC media with β-mercaptoethanol and cysteine presented no effect in the development and viability of vitrified sheep embryos. CAPES, MARFRIG Group.

129 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES AFTER IN VITRO MATURATION AND IN VITRO CULTURE UNDER COMPARATIVE OXYGEN TENSION

2011 ◽  
Vol 23 (1) ◽  
pp. 169
Author(s):  
J. T. Kang ◽  
M. Atikuzzaman ◽  
D. K. Kwon ◽  
S. J. Park ◽  
S. J. Kim ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Oxygen Tension ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Mrna Abundance ◽  
Developmental Competence ◽  
Multiple Genes ◽  
Oxygen Concentrations

The in vitro developmental abilities of porcine oocytes are generally increasing steadily at a similar ratio to those of in vivo embryos. However, it has been suggested that the in vitro culture system for the development of porcine embryos is not optimal. In this study, we investigated the effect of 2 oxygen concentrations (5 and 20%) on porcine embryo development during in vitro maturation and in vitro culture and analyzed differences in gene expression of resulting blastocysts. Oocytes were recovered by aspiration of slaughterhouse ovaries and then matured in tissue culture medium (TCM) 199 supplemented with 10% porcine follicular fluid (pFF), epidermal growth factor (EGF), insulin, pyruvate, cystine, and gonadotropin. Matured oocytes were then activated parthenogenetically, cultured in PZM-3 media for 7 days. In vitro maturation (M group) of oocytes was carried out under two oxygen concentration (5 and 20%) in terms of nuclear maturation (polar body extrusion; Exp. 1). The developmental differences between 5% oxygen culture group and 20% oxygen culture group during in vitro culture (C group) of embryos after parthenogenetic activation was investigated in terms of first cleavage and blastocyst formation (Exp. 2). Relative mRNA abundance of multiple genes in blastocysts was analyzed for transcript abundance of genes related with metabolism (GLUT1, LDHA), oxidative response (MnSOD, GPX1), apoptosis (BAX, Bcl2), and developmental competence (CCNB1, IGF2R; Exp. 3). The results show there were no significant differences in maturation rate between 2 oxygen concentrations during in vitro maturation (83 v. 86%). It was thought that cumulus cells surrounding oocytes might have attenuated oxidative stress, but number of resulting blastocysts were (P < 0.05) increased in 5% IVC group when compared with 20% IVC group (18.67 v. 14.09%, respectively). Moreover, the M20C5 group (23.01%) had a beneficial effect on in vitro culture compared with M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. Total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each group altered the expression of genes in various patterns. Therefore, it could be concluded that high oxygen tension during in vitro maturation and low oxygen tension during in vitro culture might alter the expression of multiple genes related to oocyte competence and improve (P < 0.05) embryo development, but not blastocyst quality. This study was supported by MKE (#2009-67-10033839, #2009-67-10033805), NRF (#M10625030005-508-10N25), BK21 for Veterinary Science, IPET (#109023-05-1-CG000), and Hanhwa L&C.

167 INHIBITION OF HEAT SHOCK PROTEIN 90 REDUCES DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 197
Author(s):  
E. D. Souza ◽  
F. B. E. Paula ◽  
C. C. R. Quintao ◽  
J. H. M. Viana ◽  
L. T. Iguma ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
In Vitro Maturation ◽  
Hsp90 Inhibitor ◽  
Stress Conditions ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Cleavage Rate ◽  
Bovine Oocytes

The 90-kDa heat shock protein (HSP90) is a chaperone that is important for maintaing protein homeostasis under stress conditions. HSP90 seems also to be required for maturation of Xenopus oocytes (Fisher et al. 2000 EMBO J. 19, 1516) and first cleavage of mouse zygotes (Audouard et al. 2011 PloS One 6, e17109). This study aimed to evaluate the effect of inhibition of HSP90 by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma St. Louis, MO, USA) during in vitro maturation (IVM) on bovine oocyte developmental competence. Immature cumulus–oocyte complexes (COC) were randomly allocated in 3 treatments during IVM: T0 (control; n = 240), no HSP90 inhibitor; T1: 2 μM HSP90 inhibitor (17AAG; n = 250) for the first 12 h of IVM; and T2: 2 μM HSP90 inhibitor (n = 188) for 24 h of IVM. In vitro maturation was performed in Nunc plates containing 400 μL of TCM-199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2, 95% humidity, and 38.5°C for 24 h. Oocytes were in vitro fertilized for 20 h and incubated under the same IVM conditions. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) an IVF performed with 2 × 106 spermatozoa mL–1. Presumptive zygotes were completely denuded in a PBS solution with hyaluronidase and then cultured in wells with 500 μL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h post-fertilization and blastocyst rates were evaluated at Day 7 and Day 8. Data from 6 repetitions were analysed by generalized linear model procedure of SAS software (version 9.1; SAS Institute Inc., Cary, NC, USA), and means were compared by Student-Newman-Keuls test. Values are shown as mean ± s.e.m. There was a tendency (P = 0.08) for a lower cleavage rate in T2 (52.6 ± 5.8%) than in T0 (control; 74.2 ± 4.1%). Inhibition of HSP90 by 17AAG for 12 h and 24 h of IVM (T1 and T2, respectively) decreased blastocyst rates at Day 7 (20.4 ± 3.0% and 14.3 ± 2.6%, respectively; P < 0.01) and Day 8 (22.6 ± 4.1% and 16.9 ± 2.7%, respectively; P < 0.05) when compared with control (T0 = 31.8 ± 2.5% and 34.1 ± 2.9% for Day 7 and Day 8, respectively). In addition, the inhibition of HSP90 for 24 h decreased (P < 0.05) the proportion of hatched blastocysts at Day 8 (9.5 ± 5.0% for T2, respectively) when compared with control (T0 = 35.8 ± 3.9%), indicating a reduction on embryo quality. In conclusion, inhibition of HSP90 by 17AAG during IVM results in lower developmental competence, suggesting that this protein is also important for bovine oocytes. Further studies are required to investigate if the role of HSP90 on developmental competence of bovine oocyte is affected when under stress conditions. The authors acknowledge CNPq 473484/2011-0, FAPEMIG and FAPES for financial support.

233. Oxygen concentration during in vitro maturation of murine oocytes affects blastocyst cell lineage

2005 ◽  
Vol 17 (9) ◽  
pp. 91
Author(s):  
K. M. Banwell ◽  
M. Lane ◽  
D. L. Russell ◽  
K. L. Kind ◽  
J. G. Thompson
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Cell Lineage ◽  
Developmental Competence ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
Treatment Groups ◽  
Significant Difference ◽  
O2 Concentration

Follicular antral oxygen tension is thought to influence subsequent oocyte developmental competence. Despite this, in vitro maturation (IVM) is routinely performed in either 5 or 20% O2 and while low O2 has been shown to be beneficial to embryo development in many species, the effect of altering O2 concentration during IVM has not been adequately investigated. Here we investigated the effects of a range of O2 concentrations during IVM on meiotic maturation and subsequent embryo development after IVF. Ovaries from eCG-stimulated CBA F1 female mice (21 days) were collected and intact cumulus oocyte complexes (COCs) cultured for 17–18 h under 2, 5, 10 or 20% O2 (6% CO2 and balance of N2). Matured COCs were denuded of cumulus cells, fixed and stained (1% aceto-orcein) for visualisation of maturation status. No significant difference in maturation rates between treatment groups was observed. Following IVF (performed under 5% O2, 6% CO2 and balance of N2), no difference in fertilisation rates between treatment groups was observed in a randomly selected cohort 7 h post-fertilisation. There was also no significant difference in cleavage rates after 24 h or ability to reach blastocyst stage after 96 h, with a tendency (P = 0.079) for more blastocysts in 2% O2. However there was a significant increase in the number of trophectoderm cells present in the resulting blastocysts (P < 0.05) in the 2% O2 group (35 ± 2.1) compared to 20% O2 (25 ± 2.8). Our data suggests that O2 concentration during IVM does not influence nuclear maturation or subsequent fertilisation, cleavage and blastocyst development rates. However, maturation in 2% O2 significantly alters subsequent cell lineage within blastocysts to favour trophectoderm development. Such skewed trophectoderm cell number may influence embryo viability. Funded by NHMRC and NIH.

scholarly journals 205.The effect of FSH concentration during IVM and gamete co-incubation length during IVF on the development of unstimulated prepubertal ewe oocytes

2004 ◽  
Vol 16 (9) ◽  
pp. 205 ◽  
Author(s):  
K. M. Morton ◽  
W. M. C. Maxwell ◽  
G. Evans
Keyword(s):  
Blastocyst Stage ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Oocyte Collection ◽  
Chi Squared ◽  
Development In Vitro ◽  
Stage 1 ◽  
Vitro Development

The developmental competence of prepubertal oocytes can be increased by the administration of gonadotrophins prior to oocyte collection (1); but this is not possible with abattoir-sourced oocytes, and modifications to the IVP system may increase in vitro development. Experiments were conducted to determine the effects of FSH concentration (10, 20 or 60 μg mL-1) during IVM (5 replicates) and gamete co-incubation length (short: 2-3 h, long: 18-20 h) during IVF (6 replicates) on subsequent embryonic development. For both experiments ovaries were collected from prepubertal lambs (16-24 weeks) slaughtered at an abattoir and embryos produced in vitro (1). Data were analysed by chi-squared test. Oocyte cleavage at 48 hours post-insemination (hpi) was higher for oocytes matured in medium containing 20 (60/77; 77.9%) and 60 (56/73; 76.7%) than 10 μg mL-1 (40/67; 59.7%) FSH. Blastocyst formation (% cultured oocytes) on Day 7 (Day 0 = IVF) was higher for oocytes matured with 20 (31/77; 40.3%) than 10 (16/67; 23.9%) or 60 μg mL-1 (20/73; 27.4%). Oocyte cleavage at 48 hpi was reduced for short (36/57; 63.2%) compared with long (49/55; 89.1%) co-incubation, although blastocyst formation (% cultured oocytes; Day 7) did not differ between groups (22/57; 38.6% and 23/55; 41.8%, respectively). These results demonstrate that increasing the FSH concentration above normal levels during IVM of prepubertal lamb oocytes improves development in vitro. Gamete co-incubation length did not influence the proportion of oocytes progressing to the blastocyst stage. (1) Morton et al. (2003) Proc. Soc. Reprod. Fert. P18.

scholarly journals Antioxidant Nobiletin Enhances Oocyte Maturation and Subsequent Embryo Development and Quality

2020 ◽  
Vol 21 (15) ◽  
pp. 5340
Author(s):  
Yulia N. Cajas ◽  
Karina Cañón-Beltrán ◽  
Magdalena Ladrón de Guevara ◽  
María G. Millán de la Blanca ◽  
Priscila Ramos-Ibeas ◽  
...  
Keyword(s):  
Embryo Development ◽  
Biological Effects ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Cortical Granules ◽  
Cleavage Rate ◽  
Cytoplasmic Maturation

Nobiletin is a polymethoxylated flavonoid isolated from citrus fruits with wide biological effects, including inhibition of reactive oxygen species (ROS) production and cell cycle regulation, important factors for oocyte in vitro maturation (IVM). Therefore, the objective of the present study was to evaluate the antioxidant activity of nobiletin during IVM on matured bovine oocyte quality (nuclear and cytoplasmic maturation; oocyte mitochondrial activity; intracellular ROS and glutathione (GSH) levels) and their developmental competence, steroidogenesis of granulosa cells after maturation, as well as quantitative changes of gene expression in matured oocytes, their cumulus cells, and resulting blastocysts. Bovine cumulus-oocyte complexes were in vitro matured in TCM-199 +10% fetal calf serum (FCS) and 10 ng/mL epidermal growth factor (EGF) (Control) supplemented with 10, 25, 50, or 100 μM of nobiletin (Nob10, Nob25, Nob50, and Nob100, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for nobiletin dilution). A significantly higher percentage of matured oocytes in metaphase II was observed in Nob25 and Nob50 compared to other groups. Similarly, cleavage rate and cumulative blastocyst yield on Days 7 and 8 were significantly higher for Nob25 and Nob50 groups. Oocytes matured with 25 and 50 μM nobiletin showed a higher rate of migration of cortical granules and mitochondrial activity and a reduction in the ROS and GSH content in comparison with all other groups. This was linked to a modulation in the expression of genes related to metabolism (CYP51A1), communication (GJA1), apoptosis (BCL2), maturation (BMP15 and MAPK1), and oxidative stress (SOD2 and CLIC1). In conclusion, nobiletin offers a novel alternative for counteracting the effects of the increase in the production of ROS during IVM, improves oocyte nuclear and cytoplasmic maturation, and subsequent embryo development and quality in cattle.

scholarly journals Quercetin improves developmental competence of mouse oocytes by reducing oxidative stress during in vitro maturation

2018 ◽  
Vol 18 (1) ◽  
pp. 87-98
Author(s):  
Seyede Zahra Banihosseini ◽  
Marefat Ghaffari Novin ◽  
Hamid Nazarian ◽  
Abbas Piryaei ◽  
Siavash Parvardeh ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Mature Oocyte ◽  
Developmental Competence ◽  
Nuclear Maturation ◽  
Mouse Oocytes ◽  
Blastocyst Rate ◽  
And Control

Abstract Quercetin is a natural flavonoid with strong antioxidant activity. In the present study, we evaluate the influence of different concentrations of quercetin (QT) on intracytoplasmic oxidative stress and glutathione (GSH) concentration, during in vitro maturation (IVM) and fertilization in mouse oocytes. IVM was carried out in the presence of control (QT0), 5 (QT5), 10 (QT10), and 20 (QT20) μg/mL of QT. Nuclear maturation, intracellular GSH and ROS content were evaluated following the IVM. In these oocytes, we subsequently evaluated the effect of QT supplementation on embryo development, including 2-cell, 8-cell, and blastocyst rate. The results of the present study showed that the supplementation of 10 μg/mL QT in maturation medium increased the number of MII oocytes. In addition, fertilization and blastocyst rate in QT10 treatment group were significantly higher in comparison to the other groups, and elevated the amount of intracellular GSH content compared to other QT concentrations and control groups. The intracellular ROS level was the lowest among oocytes matured in Q5 and Q10 treatment groups. This result suggested that quercetin dose-dependently improves nuclear maturation and embryo development, via reducing intracytoplasmic oxidative stress in mature oocyte.

21 EFFECT OF SEVERAL PARAMETERS ON PARTHENOGENETIC BOVINE EMBRYO DEVELOPMENT IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 119
Author(s):  
S. Arat ◽  
H. Bagis ◽  
A. Tas ◽  
T. Akkoc
Keyword(s):  
Embryo Development ◽  
Development Rate ◽  
Electrical Pulse ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
The Third ◽  
Parthenogenetic Embryo ◽  
Development In Vitro ◽  
Parthenogenetic Embryos

The activation of oocytes is one of the most important steps for a successful cloning and has great importance on embryo development in vitro. The objective of this study was to examine the different parameters affecting parthenogenetic embryo development in vitro. In the first experiment, two activation protocols were compared to examine the effect of electrical pulse on activation. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM-199 supplemented with fetal bovine serum (FBS), sodium pyruvate, penicillin/streptomycin, rat insulin-like growth factor (rIGF-1), bovine follicle-stimulating hormone (bFSH), and bovine luteinizing hormone (bLH). A group of oocytes was exposed to a DC pulse of 133 V/500 �m for 25 �s, and then activated by calcium ionophore (5 �M) for 10 min, cytochalasin D (CD) (2.5 �g/mL) + cycloheximide (CHX, 10 �g/mL) for 1 h, and CHX alone for 5 h (Group 1). Another group of oocytes was activated only by chemicals without electrical pulse. Activated oocytes were cultured for 72 h in G1-3 and then 4-6 days in G2-3 medium. In the second experiment, oocytes activated by electrical pulse and chemicals were cultured in Barc medium for 7-9 days or 72 h in G1-3 and then 4-6 days in G2-3 medium. In the third experiment, oocytes activated by electrical pulse and chemicals were cultured for 48 h or 72 h in G1-3 and then 5-7 days or 4-6 days in G2-3 medium. The differences among groups were analyzed by one-way ANOVA after arcsin square transformation. In the first experiment, cleavage rate (75.6%), development rate (37.3%), and blastocyst cell number (78.4 � 3.2) of oocytes activated by electrical pulse was higher than for the group without electrical pulse (28.7%, 8.0%, 59.5 � 4.3, respectively; P < 0.05). This result showed that activation was started more effectively by electrical pulse than by chemicals. In the second experiment, there was no significant difference on cleavage rate between the two groups (66.6%, 65.0%, respectively), and the blastocyst development rate of parthenogenetic embryos cultured in G1-3/G2-3 (36.6%) was higher than in the Barc medium group (16.6%; P < 0.05). This result showed that G1-3/G2-3 medium was more effective for parthenogenetic embryo development than Barc medium. In the third experiment, although significant differences could not be found between the two groups in the development rate of parthenogenetic embryos cultured for a total of 7-9 days (30.8%, 39.2%, respectively), the development rate of embryos cultured for 72 h in G1-3 was higher (26.4%) than for the 48-h group (15%; P < 0.05) on Day 7. This result showed that embryos developed more slowly when cultured for a shorter time in G1-3 medium before transfer to G2-3 medium. This study was supported by a grant from TUBITAK, Turkey (VHAG-1022).

344 EFFECTS OF BMP4 AND ITS INHIBITOR, NOGGIN, ON OOCYTE MATURATION AND DEVELOPMENT OF BOVINE PREIMPLANTING EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 328
Author(s):  
I. La Rosa ◽  
R. Fernandez y Martín ◽  
D. A. Paz ◽  
D. F. Salamone
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cell Number ◽  
Bmp Signaling ◽  
Control Group ◽  
Cleavage Rate ◽  
Exact Test ◽  
Student’S T

BMP4 regulates different events during development in all vertebrates and Noggin is one of its powerful inhibitors that blocks BMP4 interaction with its receptors (Groppe et al. 2002). In this work, the effect of these factors on bovine oocyte maturation and subsequent embryo development has been investigated. COCs were aspirated from abattoir ovaries and in vitro-matured for 22 h or 24 h in a 5% CO2 humidified atmosphere at 39°C in TCM containing 0.6% BSA, 2 mM FSH, 10 mM cysteamine, 1% antibiotic and 1% pyruvate, control group (C), plus 100 ng mL-1 of BMP4 (B), or 100 ngmL-1 of Noggin (NOG). Oocytes were stained with Hoechst 33342 and classified by their nuclear stage. Effects on embryo development were investigated for embryos produced by parthenogenic activation (PA) and IVF For PA, denuded oocytes were chemically activated in 5 μM ionomycine for 4 min, and immediately incubated in 1.9 mM of 6-dimethilaminopurine for 3 h. For IVF, frozen-thawed semen was centrifuged and resuspended in Bracket and Oliphant (BO) solution and incubated with 22 h matured COCs for 5 h. Embryos were cultured in CR2 medium free of serum and co-culture. Cleavage and blastocyst formation were registered at Day 2 and 9 respectively. Fischer’s exact test was used and P ≤ 0.05 was considered significant. Nuclear progression was not affected by maturation treatments [% of MII: 79.4(C, n = 102), 72.4 (B, n = 98), 80.9 (NOG, n = 89)]. For PA, both factors significantly increased cleavage rates [%: 51.7 (C, n = 284), 65 (B, n = 186), 62.1 (NOG, n = 198)] while blastocyst rates were not affected [%: 8.8 (C), 7.5 (B), and 8.6 (NOG)]. On the other hand, for IVF, cleavage rate was statistically lower for Noggin group [%: 70.7 (C, n = 140), 71.3 (B, n = 157), 64 (NOG, n = 159)] while blastocyst rates were similar between groups [%: 15.7 (C), 13.4 (B), 14.5 (NOG)]. Any of the added factors affected cell number of the embryos at Day 2. Blastocysts did not differ in the number of cells at Day 9 (Student’s t-test was used) neither for PA [mean ± SD: 100 ± 33 (C, n = 9), 88 ± 14 (B, n = 3) and 68 ± 8,(NOG, n = 3)] nor for IVF [mean ± SD: 90 ± 24 (C, n = 9), 132 ± 18 (B, n =4) and 99 ± 8 (NOG, n = 3)]. It is noticeable that addition of these factors during in vitro maturation showed different effects on subsequent embryo development depending on whether the embryos were PA or IVF. Probably, these responses represent differences in the BMP signaling system between these embryos which could be associated with different imprinting pattern. Further experiments are needed to elucidate clearly the mechanisms implicated. To our knowledge, this is the first work to study BMP4 inhibition during bovine in vitro maturation. To “Merlo” and “Nueva Escocia” Slaughterhouses

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