Extracts of forage plants affect the developmental competence of ovine oocytes in vitro

2019 ◽  
Vol 59 (10) ◽  
pp. 1814 ◽  
Author(s):  
Anna Aryani Amir ◽  
Jennifer M. Kelly ◽  
David O. Kleemann ◽  
Zoey Durmic ◽  
Dominique Blache ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
The Other ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Reproductive Process ◽  
Forage Plants ◽  
Methanolic Extracts ◽  
Blastocyst Rate

Forage plants may contain secondary compounds that disrupt reproduction in ruminants so, as ‘duty of care’, proposed new forage species need to be tested for harmful effects on reproduction before industrial release. We evaluated the effects of Bituminaria bituminosa, Medicago sativa, Chicorium intybus, Trifolium subterraneum, Trifolium pratense, Biserrula pelecinus and Eremophila glabra, on the in vitro developmental competence of ovine oocytes. Crude methanolic extracts of each plant were added to the medium (final concentrations: 0, 50 or 100 μg dry extract per mL) used for in vitro maturation of cumulus-oocyte complexes derived from abattoir-sourced adult ewe ovaries. After in vitro fertilisation, we quantified cleavage rate, blastocyst rate, hatching rate, blastocyst efficiency, and total blastocyst cell number (TCN). Extract from B. pelecinus, at 50 μg/mL concentration, increased cleavage rate at (P < 0.05), and at 100 μg/mL, increased blastocyst rate and efficiency (P < 0.05). The other plant extracts did not affect these measures. TCN was affected by stage of development and treatment, but not by the interaction between stage and treatment. Within treatments, TCN was increased by C. intybus (at both 50 and 100 μg/mL) but decreased by M. sativa (at both 50 and 100 μg/mL; P < 0.05). We conclude that methanolic extracts of forage plants, present during in vitro oocyte maturation, did not disrupt subsequent fertilisation and embryo development until the blastocyst stage. On the contrary, B. pelecinus appears to improve fertilisation and embryo development. Overall, these observations suggest that these plants will not disrupt in vivo oocyte maturation but further testing is still required, especially for the other stages of the reproductive process.

74 Treatment with Melatonin During In Vitro Culture Enhances Porcine Parthenogenetically Activated Embryo Development

2018 ◽  
Vol 30 (1) ◽  
pp. 175
Author(s):  
G. A. Kim ◽  
J.-X. Jin ◽  
S. Lee ◽  
A. Taweechaipaisankul ◽  
B. C. Lee
Keyword(s):  
Oxidative Stress ◽  
Embryo Development ◽  
Developmental Competence ◽  
Free Radical Scavengers ◽  
Control Group ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Significant Difference ◽  
Blastocyst Rate

Melatonin and its metabolites are powerful antioxidants and free radical scavengers. Because porcine embryos are vulnerable to oxidative stress in vitro, the addition of various protective chemicals to the culture medium, including melatonin, has been explored. The aim of this study was to investigate the effect of melatonin on in vitro developmental competence of porcine parthenogenetically activated (PA) embryos. Immature cumulus–oocyte complexes (COC) were collected and cultured in medium comprising TCM-199 supplemented with 10 ng mL−1 epidermal growth factor, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μL mL−1 insulin, transferrin selenium solution 100×, 10% porcine follicular fluid, 10 IU mL−1 eCG, and 10 IU mL−1 hCG for 44 h. Then, COC were denuded and PA with electrical stimulation, and PA embryos were cultured in porcine zygote medium 5 (PZM-5) supplemented with melatonin at increased concentrations (10−9, 10−7, 10−5 M) at 39°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 7 days. Subsequent embryo development, including cleavage rate, blastocyst rate, and blastocyst cell numbers, was compared between groups (mean no. of embryos; control, 27.14; 10−9 M, 28.86; 10−7 M, 27.71; 10−5 M, 26.43). The experiments were repeated 7 times for each treatment group. Statistical analyses of all data were performed using one-way ANOVA with Dunn’s multiple comparison test. Results are expressed as the mean ± SEM and all differences were considered significant at P < 0.05. No apparent effect on cleavage rate of melatonin treatment of various concentrations was noted. Blastocyst cell number did not show any significant difference between groups. However, the potential of PA oocytes to develop into blastocysts was significantly higher in the group supplemented with 10−9 M melatonin compared with the control group (35.44 ± 3.84 v. 24.71 ± 1.59) and other melatonin treated groups (10−5 M, 21.35 ± 2.82; 10−7 M, 24.01 ± 2.31; P < 0.05). These indicated that treatment with 10−9 M melatonin in embryo culture might reduce the oxidative stress properly compared with other concentrations, which results in improvement of blastocyst rate formation. In conclusion, treatment with 10−9 M melatonin positively promoted the blastocyst formation rate of porcine PA embryos with no beneficial effects on their blastocyst cell numbers or cleavage rate. This study was supported by the National Research Foundation (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science and the BK21 PLUS Program.

258 ROLE OF PROGESTERONE AND ITS RECEPTORS ON DEVELOPMENTAL COMPETENCE OF OOCYTES IN CATTLE

2011 ◽  
Vol 23 (1) ◽  
pp. 227
Author(s):  
I. M. Aparicio ◽  
M. Garcia-Herreros ◽  
L. C. O'Shea ◽  
C. Hensey ◽  
P. Lonergan ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Intracellular Signaling ◽  
Developmental Competence ◽  
Ovulatory Cycle ◽  
Cleavage Rate ◽  
Ru 486 ◽  
Day 3 Embryos

Several studies have demonstrated the importance of progesterone (P4) through its receptors (PR) in the regulation of the ovulatory cycle, but its participation in oocyte maturation in mammals has not yet been clarified. Previous results in our group showed changes in the protein expression of genomic (nPR-A/B) and nongenomic (mPRα/β) PR in bovine cumulus–oocyte complexes (COC) during in vitro maturation (IVM), indicating a possible function for these receptors on bovine oocyte maturation. Therefore, we aimed to study the role of P4 and PR in oocyte developmental competence. Good-quality immature COC were placed in maturation medium [TCM-199 supplemented with 10% (vol/vol) FCS and 10 ng mL–1 of epidermal growth factor] and cultured at 39°C for 22 h in a humidified atmosphere containing 5% CO2, in the presence or absence of trilostane (0.001, 0.1, and 10 μM), P4 (50 and 100 ng mL–1), promegestone (50 and 100 ng mL–1), RU 486 (0.1, 1, and 10 μM), or antibodies against mPRα or mPRβ. Matured COC were washed and placed in wells containing 250 μL of fertilization medium (25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mL–1 fatty acid-free BSA, and 10 mg mL–1 heparin). In vitro fertilization was performed with 250 μL of frozen–thawed semen at a final concentration of 1 × 106 spermatozoa mL–1 at 39°C under 5% CO2 during 20 h. Presumptive zygotes were denuded, washed, and transferred to 25-μL culture droplets (SOF + 5% FCS) at 39°C under 5% CO2, 90% of N2, and 5% O2 atmosphere with maximum humidity. Number of embryos was recorded at day 2, 3, 7, and 8 (Day 0 = day of IVF). Data were analysed using ANOVA analysis using SPSS v. 15.0 software package. Inhibition of P4 production by 10 μM of trilostane during IVM reduced progesterone production in the media, cumulus cells expansion, but had no effect on meiotic resumption in Day 2 and Day 3 embryos; however, there was a significant decrease in the percentage of blastocysts at Day 7 (12 ± 3.5) and Day 8 (14 ± 3.3) compared with the control (30 ± 4.5 and 42 ± 2.5). This reduction on embryo development was overcome by the addition of 50 or 100 ng mL–1 of P4 at Day 8 (33 ± 5.7 and 32 ± 4.1). The same results were obtained with nonmetabolizable P4, where the reduction on blastocysts with trilostane at Day 8 (20 ± 2.0) was completely overcome by 50 or 100 ng mL–1 of promegestone (41 ± 5.1 and 40 ± 6.7). Specific inhibition nPR-A/B with 10 μM of RU 486 produced a significant reduction on blastocysts at Day 7 (24 ± 4.3) and Day 8 (29 ± 5.5) compared with the control (44 ± 2.6 and 47 ± 3.0). Inhibition of mPRα reduced cleavage rate and Day 3 embryos, whereas the inhibition of mPRβ had no effect on meiotic resumption or embryo development. In conclusion, the intracellular signaling of P4 on developmental competence of oocytes in cattle seems to be mainly mediated by nPR-A/B receptors and could be associated with cytoplasmic maturation.

PSV-5 Relationship between sire conception rate, sperm motility, sperm SERPINA5 relative concentration, and in vitro produced embryos in dairy bulls

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 310-310
Author(s):  
Saulo Menegatti Zoca ◽  
Julie Walker ◽  
Taylor Andrews ◽  
Adalaide C Kline ◽  
Jerica J Rich ◽  
...  
Keyword(s):  
Embryo Development ◽  
Sperm Motility ◽  
Relative Concentration ◽  
Early Embryo ◽  
Conception Rate ◽  
Cleavage Rate ◽  
Blastocyst Rate ◽  
Positive Correlations ◽  
Sire Conception Rate

Abstract Sire conception rate (SCR) is a field measure of fertility among bulls, but it can be influenced by several factors (Sperm transport, sperm-egg binding, early embryo development, etc). The objective of this study was to evaluate the relationship between SCR, sperm motility, SERPINA5 concentrations, and in vitro embryo development. Measurements were performed in 19 bulls with SCR values ranging from -7.7 to 4.45. For each bull, an aliquot of frozen-thawed semen was used for analyses of total (TMOT) and progressive (PROG) motility. Remaining semen was fixed with 2% formaldehyde, and concentration of SERPINA5 was determined by immunolocalization (antibody SERPINA5/Dylight405; PA5-79976-Invitrogen / ab201798-Abcam). Mean fluorescence intensity was determined in ~200 sperm heads/bull. Approximately 149 oocytes/bull were fertilized in vitro for embryo development analysis (cleavage and blastocyst rates). Statistical procedures were performed in SAS (9.4) using the procedures CORR for correlations (SCR, TMOT, PROG, SERPINA5, cleavage and blastocyst) and GLIMMIX for comparison of “field-fertility” (SCR divided in HIGH or LOW) and “field-embryo-fertility” (LOW-SCR sires were divided based on blastocyst rate (HIGH or LOW) resulting in two classifications; LOW-HIGH≥31% and LOW-LOW≤26%, respectively). There were positive correlations (P &lt; 0.05) between cleavage-blastocyst (r=0.50), SERPINA5-cleavage (r=0.48), and TMOT-PROG (r=0.76). Sire SCR was not associated with SERPINA5, TMOT, PROG, cleavage and blastocyst rate (P &gt; 0.52). Among LOW-SCR sires, LOW-LOW sires (-4.83±0.60) tended to have a better SCR score than LOW-HIGH (-6.18±0.42) sires (P = 0.08), but there were no differences (P &gt; 0.43) between LOW-HIGH, LOW-LOW, and HIGH sires for SERPINA5, TMOT, PROG, and cleavage. In conclusion, some LOW SCR sires have good embryo development indicating a different mechanism for their low SCR; however, these differences in SCR could not be explained by TMOT, PROG, SERPINA5, cleavage and blastocyst. There were, however, positive correlations between cleavage-blastocyst rate, and SERPINA5-cleavage rate.

scholarly journals Maturation of human oocytes in vitro and their developmental competence

Reproduction ◽  
2001 ◽  
pp. 51-75 ◽  
Author(s):  
A Trounson ◽  
C Anderiesz ◽  
G Jones
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Growth Phase ◽  
Developmental Competence ◽  
Minimum Size ◽  
Success Rates ◽  
Human Oocytes ◽  
Maturation In Vitro

Complete maturation of oocytes is essential for the developmental competence of embryos. Any interventions in the growth phase of the oocyte and the follicle in the ovary will affect oocyte maturation, fertilization and subsequent embryo development. Oocyte size is associated with maturation and embryo development in most species examined and this may indicate that a certain size is necessary to initiate the molecular cascade of normal nuclear and cytoplasmic maturation. The minimum size of follicle required for developmental competence in humans is 5-7 mm in diameter. Maturation in vitro can be accomplished in humans, but is associated with a loss of developmental competence unless the oocyte is near completion of its preovulatory growth phase. This loss of developmental competence is associated with the absence of specific proteins in oocytes cultured to metaphase II in vitro. The composition of culture medium used successfully for maturation of human oocytes is surprisingly similar to that originally developed for maturation of oocytes in follicle culture in vitro. The presence of follicle support cells in culture is necessary for the gonadotrophin-mediated response required to mature oocytes in vitro. Gonadotrophin concentration and the sequence of FSH and FSH-LH exposure may be important for human oocytes, particularly those not exposed to the gonadotrophin surge in vivo. More research is needed to describe the molecular and cellular events, the presence of checkpoints and the role of gene expression, translation and protein uptake on completing oocyte maturation in vitro and in vivo. In the meantime, there are very clear applications for maturing oocytes in human reproductive medicine and the success rates achieved in some of these special applications are clinically valuable.

18 DEVELOPMENTAL COMPETENCE OF CLONED PORCINE EMBRYOS PRODUCED WITH DIFFERENT CLONING PROCEDURES

2014 ◽  
Vol 26 (1) ◽  
pp. 123
Author(s):  
Y. Liu ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
R. Li ◽  
P. Hassel ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
Handmade Cloning

Two nuclear transfer (NT) techniques are routinely used to produce cloned animals, traditional cloning (TC) and handmade cloning (HMC). The TC embryos keep their zona and can be transferred at early stages, whereas HMC embryos are zona-free and must be cultured to the morula/blastocyst stage before transfer. Some studies have shown that in vitro culture reduces embryo development and quality, but it is not known whether embryos produced by TC or HMC differ because of the NT method or the in vitro culture. Therefore, we investigated the developmental competence and histone acetylation (H3K18ac) of porcine NT embryos produced by TC and HMC with (Day 5 and 6) or without (Day 0) in vitro culture. Nuclear transfer experiments were performed on same day (Day 0), using same batch of porcine oocytes and donor cells and same in vitro culture conditions. Cloning procedures were previously described (TC : Cloning Stem Cells 10 : 355; HMC : Zygote 20 : 61). Parthenogenetically activated embryos (PA) were used as control of activation and culture conditions. Embryos from all groups were collected for immunostaining of H3K18ac on Days 0, 5, and 6. The normalized H3K18ac level was calculated as previously described (Epigenetics 6 : 177). Cell numbers per blastocyst in each group were counted on Days 5 and 6. The cleavage rate (Day 2) and blastocyst rates (Days 5 and 6) between groups were analysed by Chi-squared test, whereas cell number per blastocysts and H3K18ac level between groups and days were analysed by ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Cleavage rate of HMC embryos was lower than that of TC embryos, but blastocyst rate and cell number per blastocyst were higher in the HMC group compared with TC (Table 1). Differences of H3K18ac level between HMC, TC, and PA groups were only observed on Day 6 but not on Day 0 or Day 5. Within HMC and TC groups, there was no difference in H3K18ac level between Day 0 and Day 5, but the level was lower on Day 6 compared with Day 5 in the HMC group, whereas the TC group displayed the opposite pattern. In conclusion, NT embryos produced by HMC show higher blastocyst rate and cell number per blastocyst compared with TC embryos. Both in vitro culture and the NT method result in differences of the normalized H3K18ac levels. Further study is needed to investigate putative differences between NT embryos produced by HMC and TC compared to in vivo embryos also after transfer to recipients. Table 1.Cleavage and blastocyst rate, cell numbers, and normalized H3K18ac level for handmade cloning (HMC), traditional cloning (TC), and parthenogenetically activated (PA) embryos1

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.

60 EFFECT OF CYTOPLASMIC VOLUME ON DEVELOPMENTAL COMPETENCE OF HAND-GUIDED CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 135
Author(s):  
S. K. Panda ◽  
A. George ◽  
A. P. Saha ◽  
R. Sharma ◽  
N. M. Kamble ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryonic Stem ◽  
Cell Types ◽  
Bubalus Bubalis ◽  
Developmental Competence ◽  
Cloning Efficiency ◽  
Cleavage Rate ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate

Despite recent successes in the birth of buffalo calves cloned through SCNT or hand-guided cloning (HGC), the cloning efficiency is very low in this species because of lack of information on factors that influence it. The goal of this study was to examine the effects of cytoplasmic volume on the developmental competence of cloned buffalo embryos produced by HGC. In vitro matured oocytes were stripped of their cumulus investment and zona pellucida using hyaluronidase and pronase, respectively. Protrusion cone-guided bisection of zona-free oocytes was performed to remove the nucleus. For reconstructing control HGC embryos, 2 enucleated oocytes (demi-cytoplasts) were fused with a single somatic cell. For reconstruction of embryos with lower or higher cytoplasmic volume, 1 or 3 demi-cytoplasts were fused, respectively, with the donor somatic cell. 2 different cell types, i.e. buffalo fetal fibroblasts (BFF) between passage 10 and 15 and buffalo embryonic stem cell (ESC)-like cells between passage 22 and 25 were used as nuclear donors in 2 different experiments. Data were analysed by 1-way ANOVA after arcsine transformation of percentage values. For BFF, the blastocyst rate for doublet and triplet embryos were significantly higher (P ≤ 0.01) than that for singlet embryos despite the cleavage rate for the 3 groups being similar. For the ESC-like cells, the cleavage and the blastocyst rate were significantly lower (P ≤ 0.01) for the singlet than that for the doublet embryos. The pregnancies were established only in doublet and triplet embryo groups using BFF cells and in the doublet embryo group using ESC-like cells. These results indicate that increasing the cytoplasmic volume could be helpful in improving cloning efficiency in terms of blastocyst production rate in buffaloes. Table 1.Effect of cytoplasmic volume on the developmental competence of cloned buffalo embryos This work was funded by NAIP grant C 2-1-(5)/2007 to SKS.

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.

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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