Effect of nano-selenium and nano-zinc particles during in vitro maturation on the developmental competence of bovine oocytes

2018 ◽  
Vol 58 (11) ◽  
pp. 2021 ◽  
Author(s):  
B. R. Abdel-Halim ◽  
Nermeen A. Helmy
Keyword(s):  
Dna Damage ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Expansion Rate ◽  
Control Group ◽  
Nano Zinc ◽  
Bovine Oocytes

The objectives of the current study were to evaluate the effects of supplemental nano-selenium (NSe) and nano-zinc oxide (NZn-O) particles during in vitro maturation (IVM) on DNA damage of cumulus cells, glutathione (GSH) concentration in bovine oocytes, subsequent embryo development and re-expansion rate of vitrified warmed blastocysts. The current study was conducted on bovine ovaries obtained from a local abattoir and transported to the laboratory in sterile phosphate buffer saline with antibiotics at 37°C, within 1 h after slaughter. Ovaries were pooled, regardless of stage of the oestrous cycle of the donor. Only cumulus-intact complexes with evenly granulated cytoplasm were selected for IVM. Experimental design included the following: Experiment 1 studied the effect of addition of 1.0 µg/mL NSe or NZn-O to IVM medium on DNA damage of cumulus cells; Experiment 2 evaluated the effects of NSe or NZn-O on intracellular glutathione in oocytes and cumulus cells; in Experiment 3, the development of oocytes matured in IVM medium supplemented with 1.0 µg/mL NSe or NZn-O was investigated; and in Experiment 4, the effects of adding 1.0 µg/mL NSe and NZn-O to in vitro fertilisation media on vitrified oocytes and embryos were investigated. The DNA damage in cumulus cells decreased with supplemental NSe and NZn-O at concentration of 1 µg/mL in the IVM medium (180.2 ± 21.4, 55.8 ± 4.3 and 56.6 ± 3.9 for the control and NSe and NZn-O groups respectively). Total GSH concentrations increased following supplementation with 1 µg/mL NSe and 1 µg/mL NZn-O, compared with the control group. Re-expansion rate of vitrified warmed blastocysts in experimental media containing NSe and NZn-O with ethylene glycol was higher than that of the control. In conclusion, providing NSe and NZn-O during oocyte maturation significantly increased both intracellular GSH concentration and DNA integrity of cumulus cells. Optimal embryo development was partially dependent on the presence of NSe and NZn-O during IVM. NSe and NZn-O during oocyte maturation act as a good cryoprotective agents of vitrified, warmed blastocysts.

228 EFFECT OF INSULIN ON BOVINE OOCYTE MATURATION, CUMULUS CELL APOPTOSIS, AND EARLY EMBRYO DEVELOPMENT IN VITRO

2015 ◽  
Vol 27 (1) ◽  
pp. 203
Author(s):  
I. Lindgren ◽  
P. Humblot ◽  
D. Laskowski ◽  
Y. Sjunnesson
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Insulin Treatment ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Blastocyst Formation ◽  
Early Embryo Development ◽  
Maturation In Vitro

Dairy cow fertility has decreased during the last decades, and much evidence indicates that metabolic disorders are an important part of this decline. Insulin is a key factor in the metabolic challenge during the transition period that coincides with the oocyte maturation and may therefore have an impact on the early embryo development. The aim of this study was to test the effect of insulin during oocyte maturation on early embryo development by adding insulin during the oocyte maturation in vitro. In this study, abattoir-derived bovine ovaries were used and cumulus-oocyte complexes (n = 991) were in vitro matured for 22 h according to standard protocols. Insulin was added during maturation in vitro as follows: H (10 µg mL–1 of insulin), L (0.1 µg mL–1 of insulin), or Z (0 µg mL–1 of insulin). After maturation, oocytes were removed and fixed in paraformaldehyde before staining. Click-it TUNEL assay (Invitrogen, Stockholm, Sweden) was used for apoptotic staining and DRAQ5 (BioNordika, Stockholm, Sweden) for nuclear staining (n = 132). Cumulus-oocyte complexes were evaluated using laser scanning confocal microscope (Zeiss LSM 510, Zeiss, Oberkochen, Germany). Five levels of scans were used to assess oocyte maturation (MII stage) and apoptosis. Because of incomplete penetration of the TUNEL stain (3–5 layers of cumulus cells), only the outer 2 layers of the cumulus complex were investigated regarding apoptosis. Apoptotic index was calculated as apoptotic cells/total cells visualised. Remaining oocytes were fertilized and cultured in vitro until Day 8. Day 7 and Day 8 blastocyst formation was assessed as well as blastocyst stage and grade. Effect of insulin treatment on variables was analysed by ANOVA following arc sin √p transformation. Post-ANOVA comparisons between H+L group v. Z were performed by using the contrast option under GLM (Scheffé test). Results are presented as least squares means ± s.e. P-values ≤ 0.05 were considered as statistically significant. Insulin treatment during oocyte maturation in vitro had no significant effect on oocyte nuclear maturation or apoptotic index of the cumulus cells (Z: 0.052 ± 0.025, L: 0.039 ± 0.016, H: 0.077 ± 0.044, P > 0.05). No effect was seen on cleavage rates (Z: 0.85 ± 0.02, L: 0.85 ± 0.02, H: 0.89 ± 0.03, P > 0.05), but insulin treatment significantly decreased Day 7 rates from fertilized oocytes (Z: 0.19 ± 0.02, L: 0.14 ± 0.02, H: 0.12 ± 0.02, P < 0.05). This study also showed a significantly retarded developmental stage and decreased grade of blastocysts in insulin-treated groups taken together when compared with the control group (P < 0.05). In this study, no effect of insulin supplementation during in vitro maturation was seen on bovine oocyte maturation and apoptosis of cumulus cells, but blastocyst formation and development were negatively affected. Further studies are needed for understanding the relationship between the addition of insulin during maturation in vitro and impaired blastocyst formation. Insulin is a common supplement in the first phase of the first in vitro maturation medium for pig oocytes and is believed to have a beneficial effect on this species.Funding was received from Stiftelsen Nils Lagerlöfs Fond H12–0051-NLA.

315 SELECTION OF BOVINE OOCYTES BY BRILLIANT CRESYL BLUE BEFORE IN VITRO MATURATION IMPROVES BLASTOCYST DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 273 ◽  
Author(s):  
A. Sugulle ◽  
S. Katakawa ◽  
S. Yamamoto ◽  
S. Oomori ◽  
I. Itou ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Control Group ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Bovine Oocytes ◽  
Selection Of

The morphological identification of immature oocytes has commonly been used to select the bovine oocytes for IVF. However, &lt;30% of the recovered oocytes reach the blastocyst stage after fertilization, and this is probably due to the quality of the oocytes at the beginning of maturation. The brilliant cresyl blue (BCB) stain determines the activity of glucose-6-phosphate dehydrogenase, an enzyme synthesized in growing oocytes. The aim of this study was to evaluate the effect of the BCB stain on the selection of bovine oocytes and on the subsequent embryo development for in vitro production (IVP). Cumulus–oocyte complexes (COCs) were collected by the aspiration of 2- to 6-mm follicles. A total of 559 oocytes were divided into 2 groups: (1) a control group, immediately cultured, and (2) a BCB-incubated group. After 90 min of BCB staining (Pujol et al. 2004 Theriogenology 61, 735–744), the oocytes were divided into oocytes with blue cytoplasm (BCB+) and oocytes without blue cytoplasm (BCB−). The COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg mL−1 FSH at 38.5°C under an atmosphere of 5% CO2 in air. The matured COCs were inseminated with 5 × 106 sperm mL−1. After 18 h of gamete co-culture, the presumed zygotes were cultured in CR1aa supplemented with 5% CS for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (proportion of ≥5-cell stage, the total cleavage rates) and on Days 7 to 9 (blastocyst rate). The experiment was replicated 5 times, and the data were analyzed by a chi-square test and ANOVA. The results are presented in Table 1. The proportion of embryos with ≥5-cell stage was significantly higher (P &lt; 0.01) in the BCB+ group than in the BCB− group, but not in the control group. The total cleavage rate for the BCB+ embryos was significantly higher than that of either the BCB− or the control group (P &lt; 0.01). There were also significant differences (P &lt; 0.01) in the blastocyst development between the BCB+ and BCB− embryos and between the BCB− and the control embryos (P &lt; 0.05). This result showed that the selection of bovine oocytes by BCB staining before in vitro maturation may be useful for selecting oocytes that are developmentally competent up to Day 9 for IVP. Table 1.Effect of selection of oocytes by brilliant cresyl blue (BCB) staining on the subsequent embryo development of in vitro-matured/in vitro-fertilized bovine embryos

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

Effects of ghrelin on activation of Akt1 and ERK1/2 pathways during in vitro maturation of bovine oocytes

Zygote ◽  
2017 ◽  
Vol 25 (2) ◽  
pp. 183-189 ◽  
Author(s):  
Thomas-Markos Chouzouris ◽  
Eleni Dovolou ◽  
Fotini Krania ◽  
Ioannis S. Pappas ◽  
Konstantinos Dafopoulos ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Treated Group ◽  
Control Group ◽  
Dependent Manner ◽  
Nuclear Maturation ◽  
Bovine Oocytes ◽  
Phosphorylation Rate ◽  
Matured Oocyte

SummaryThe purpose of this study was to investigate the possible molecular pathways through which ghrelin accelerates in vitro oocyte maturation. Bovine cumulus–oocyte complexes (COCs), after 18 or 24 h maturation in the absence or the presence of 800 pg ml–1 of acylated ghrelin were either assessed for nuclear maturation or underwent in vitro fertilization in standard media and putative zygotes were cultured in vitro for 8 days. In a subset of COCs the levels of phosphorylated Akt1 and ERK1/2 (MAPK1/3) were assessed at the 0th, 6th, 10th, 18th and 24th hours of in vitro maturation (IVM). At 18 and 24 h no difference existed in the proportion of matured oocytes in the ghrelin-treated group, while in the control group more (P < 0.05) matured oocyte were found at 24 h. Oocyte maturation for 24 h in the presence of ghrelin resulted in substantially reduced (P < 0.05) blastocyst yield(16.3%) in comparison with that obtained after 18 h (30.0%) or to both control groups (29.3% and 26.9%, for 18 and 24 h in maturation, respectively). Ghrelin-treated oocytes expressed lower Akt1 phosphorylation rate at the 10th hour of IVM, and higher ERK1/2 at the 6th and 10th hours of IVM compared with controls. In cumulus cells, at the 18th and 24th hours of IVM Akt1 phosphorylation rate was higher in ghrelin-treated oocytes. Our results imply that ghrelin acts in a different time-dependent manner on bovine oocytes and cumulus cells modulating Akt1 and ERK1/2 phosphorylation, which brings about acceleration of the oocyte maturation process.

scholarly journals Histone deacetylase inhibitor during in vitro maturation decreases developmental capacity of bovine oocytes

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0247518
Author(s):  
Thais Preisser Pontelo ◽  
Mauricio Machaim Franco ◽  
Taynan Stonoga Kawamoto ◽  
Felippe Manoel Costa Caixeta ◽  
Ligiane de Oliveira Leme ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Embryo Quality ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Nuclear Maturation ◽  
Deacetylase Inhibitor ◽  
Developmental Capacity ◽  
Bovine Oocytes

This study aimed to evaluate the effect of scriptaid during pre-maturation (PIVM) and/or maturation (IVM) on developmental competence of bovine oocytes. Cumulus-oocyte complexes (COCs) were submitted to PIVM for 6 h in the presence or absence of scriptaid. COCs were distributed into five groups: T1-IVM for 22 h, T2-PIVM for 6 h and IVM for 22 h, T3-PIVM with scriptaid for 6 h and IVM for 22 h, T4-PIVM for 6 h and IVM with scriptaid for 22 h, and T5-PIVM with scriptaid for 6 h and IVM with scriptaid for 22 h. Nuclear maturation, gene expression, cumulus cells (CCs) expansion, and embryo development and quality were evaluated. At the end of maturation, all groups presented the majority of oocytes in MII (P>0.05). Only HAT1 gene was differentially expressed (P<0.01) in oocytes with different treatments. Regarding embryo development at D7, T4 (23%) and T5 (18%) had lower blastocyst rate (P<0.05) than the other treatments (T1 = 35%, T2 = 37% and T3 = 32%). No effect was observed when scriptaid in PIVM was used in less competent oocytes (P>0.05). In conclusion, presence of scriptaid in PIVM and/or IVM did not improve developmental competence or embryo quality.

255 SONIC HEDGEHOG PROMOTES IN VITRO OOCYTE MATURATION AND EMBRYO DEVELOPMENT IN GOATS

2015 ◽  
Vol 27 (1) ◽  
pp. 217 ◽  
Author(s):  
W. De-Chi ◽  
H. Jan-Chi ◽  
L. Neng-Wen ◽  
C. Hsin-I ◽  
C. Lih-Ren ◽  
...  
Keyword(s):  
Embryo Development ◽  
Sonic Hedgehog ◽  
Oocyte Maturation ◽  
Survival Rates ◽  
Cumulus Cells ◽  
Cell Surface Receptor ◽  
Control Group ◽  
Embryo Survival ◽  
Receptor System

The signalling of the Hh family peptides is mediated through a cell surface receptor system consisting of 2 proteins: patched (Ptc) and smoothened (Smo). In the absence of Hh ligand, the Hh receptor Ptc represses Smo, whereas in the presence of Hh, the suppression of Smo is lifted, leading to the activation of downstream transcriptional factors (Gli1, Gli2, and Gli3) in vertebrates. Previous studies have examined Sonic hedgehog (Shh) signalling pathways in developing and adult mouse ovaries and concluded that the Shh signalling pathway may be involved in granulosa cell proliferation and oocyte maturation. We investigated the effects of Shh protein on caprine oocyte maturation, embryo development, and embryo survival rate after transfer of vitrified/thawed in vitro-produced (IVP) embryos to recipients. Cumulus-oocyte complexes (COC) were collected by slicing ovarian follicles (1–5 mm in diameter). On average, 40 to 50 oocytes were randomly allocated to each well containing 500 μL of IVM medium and supplemented with 0 (control), 0.125, 0.25, 0.5, or 1.0 μg mL–1 recombinant mouse Shh protein. After 24 h of IVM, cumulus cells were partially removed. Oocytes were washed and transferred into a droplet of 80 μL of fertilization medium and were fertilized with frozen-thawed sperm for 18 h at 38.8°C. After IVF, presumptive zygotes were cultured on goat oviduct epithelial monolayers in M199 for 9 days. The 2 frozen-thawed selected embryos were transferred to one recipient. All data were subjected to ANOVA, using the general linear model procedure in SAS (version 9), followed by Tukey's test. Embryo survival rates were compared by using the chi-square test. The RT-PCR analyses showed that the expressions of Shh, SMO, Ptch1, and Gli1 were detected in whole ovaries, granulosa cells, COC, cumulus cells, oocytes, and oviduct epithelia except for Ptch1 in cumulus cells. Supplementation of Shh (0.25 or 0.5 μg mL–1) enhanced oocyte maturation as opposed to the control group (92.4%, n = 67 and 95.0%, n = 62 v. 86.2%, n = 64, respectively, P < 0.05). This effect could be reversed by the simultaneous addition of cyclopamine (0.5–1.0 μm), a Shh inhibitor. Similar to intact COC, denuded oocytes showed enhanced maturation (72.0%, n = 94 v. 60.5%, n = 126) with Shh supplementation. For subsequent embryo development, an improved blastocyst rate (P < 0.05) was 66.3 ± 10.9 (n = 135) when embryos were derived from the oocytes matured in the presence of 0.5 μg mL–1 Shh rather than 41.4 ± 12.9 (n = 137) of the control group. After embryo transfer, the kidding and embryo survival rates of vitrified embryos derived from the Shh-supplemented group were 56 (16 recipients) and 31% (48 embryos) higher than that 38 (16 recipients) and 15% (54 embryos) without Shh supplementation (P < 0.05). The present study suggests that Shh signalling is active in caprine ovaries during folliculogenesis and beneficial to oocyte maturation and subsequent embryo development to the blastocyst stage (in vitro) and to term.

212 EFFECT OF CANINE OVIDUCT CELLS AND CUMULUS CELLS CO-CULTURE ON IN VITRO MATURATION OF PORCINE OOCYTES AND EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 237
Author(s):  
S. H. Lee ◽  
H. J. Oh ◽  
G. A. Kim ◽  
M. J. Kim ◽  
Y. B. Choi ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cells ◽  
In Vitro Development ◽  
First Polar Body ◽  
Vitro Development ◽  
And Control

In oestrus stage, canine oocytes surrounded by cumulus cells undergo maturation in oviduct for 3 days after ovulation. We hypothesised that canine cumulus cells (cCC) and canine oviduct cells (cOC) in oestrus stage might affect the maturation of oocyte and embryo development. Therefore, the present study was aimed to compare the effects of cCC and cOC co-culture system on oocyte in vitro maturation and embryo in vitro development. cCC were separated from cumulus‐oocyte complex (COC) in ovary from bitches in oestrus phase. cOC were collected from oviduct flushing of bitches in oestrus phase. Both cCC and cOC were cultured and cryopreserved until use for co-culture. In the first experiment, the effect of co-culture using cCC and cOC on porcine oocyte in vitro maturation (IVM) were investigated. The porcine COC were randomly cultured in different co-culture groups as follows: 1) co-culturing with cCC for 42 h, 2) co-culturing with cOC for 42 h, and 3) culturing in absence of cCC or cOC. After IVM, extrusion of the first polar body was observed under a microscope. In the second experiment, the matured oocytes with the first polar body derived from each group were activated with electrical stimulus. Parthenotes were cultured in porcine zygote medium-5 (PZM-5) for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The embryo developmental competence was estimated by assessing the in vitro development under microscope. The third experiment was to evaluate the reactive oxygen species (ROS) levels in each supernatant medium obtained from cCC and cOC co-culture group after IVM using a OxiselectTM ROS ELISA Assay kit. Last, analysis of genes (MAPK1/3, SMAD2/3, GDF9 and BMP15) expression in cCC and cOC co-cultured with porcine COC using real-time PCR is in progress. As results, IVM rate of cOC group (91.19 ± 0.45%) was significantly higher than that of cCC and control group (86.50 ± 0.61% and 79.81 ± 0.82%; P < 0.05). Also, cOC groups expressed the highest efficiency in cleavage rate, blastocyst formation rate, and the total cell number in blastocyst (P < 0.05). In ROS levels, cOC group (555 ± 7.77 nM) were significantly lower than cCC and control groups (596.8 ± 8.52 nM and 657.8 ± 11.34 nM). The present study demonstrated that co-culture with cOC improved the in vitro oocyte maturation and the in vitro development rate of porcine embryos. The ROS level decreased in cOC co-culture would have beneficial influence on oocytes maturation. For further study, we will investigate the relation between gene expression related to oocyte maturation and the co-culture results. This research was supported by a global PhD Fellowship Program through NRF funded by the Ministry of Education (NRF-20142A1021187), RDA (#PJ010928032015), IPET (#311011–05–4-SB010, #311062–04–3-SB010), Research Institute for Veterinary Science, and the BK21 plus program.

36 EFFECT OF cAMP MODULATORS DURING OOCYTE IN VITRO MATURATION ON GAP JUNCTIONAL ACTIVITY OF VITRIFIED BOVINE OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 148
Author(s):  
C. A. S. Monteiro ◽  
G. R. Leal ◽  
H. F. R. A. Saraiva ◽  
A. J. R. Camargo ◽  
P. M. S. Rosa ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Fluorescence Intensity ◽  
In Vitro Maturation ◽  
Staining Pattern ◽  
Control Group ◽  
Immature Oocytes ◽  
Junctional Activity ◽  
Gap Junctional ◽  
Bovine Oocytes

Oocyte cryopreservation is a strategic tool for in vitro embryo production, but low rates of cryosurvival are reported for bovine oocytes. Simulated physiological oocyte maturation system (Albuz et al. 2010 Hum. Reprod. 25, 12) uses cAMP modulators to increase oocyte competence by the extension of meiosis block and gap junctional communications activity. The aim of this study was to investigate the effect of simulated physiological oocyte maturation system on gap junctional activity of vitrified bovine oocytes. Oocytes from slaughterhouse ovaries were divided into 4 groups: C (control: fresh immature oocytes); V (vitrified immature oocytes); PM-V (vitrified oocytes after a 2-h pre-in vitro maturation phase – in the presence of AMPc modulators, 100 μM Forskolin, and 500 μM IBMX); and PM (fresh immature oocytes subjected to pre-in vitro maturation). Viable oocytes (n = 404 obtained from 4 replicates) were stained with Calcein-AM using the protocol of Thomas et al. (2004 Biol. Reprod. 71(4), 1142–1149) in order to measure gap junctions activity. Images were captured in fluorescence microscope, and fluorescence intensity was analysed with ImageJ software. Mean fluorescence intensity of each group was normalized to control group to obtain relative intensity value. Means were compared by Kruskal-Wallis test and Dunn post-test. A second analysis was performed considering the percentage of each staining pattern (low, middle, and high intensity) for each group. Results were analysed using Fisher exact test. All statistical analysis were performed in GraphPad Instat program with 5% significance level. Results demonstrated that all treatments induced an increase (P < 0.05) in fluorescence intensity (V: 1.76 ± 1.13; PM-V: 1.58 ± 0.98; PM: 1.38 ± 0.94) compared with control (C: 1.00 ± 0.48). Regarding the staining patterns analyses, immature vitrified oocytes (V group) differed from control group in middle and low patterns (G1, calibrator – high: 11.2%ab, middle: 43.8%a, low: 44.9%a; G2 – high: 8.2%ab, middle: 63.9%b, low: 27.9%b; G3 – high: 16.3%a, middle: 42.3%a, low: 41.3%a; G4 – high: 6.7%b, middle: 53.9%ab, low: 39.3a). In conclusion, unexpectedly, vitrification also increased gap junctional activity, as was found for pre-in vitro maturation group. However, staining pattern analysis results showed only vitrified group was different from control, suggesting vitrified and pre-in vitro maturation groups could have gap activity affected by different ways. This research was supported by FAPERJ (E26/111.61/2013) and CAPES.

Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

2012 ◽  
Vol 92 (1) ◽  
pp. 124-127 ◽  
Author(s):  
L. Takada ◽  
A. Martins Junior ◽  
G.Z. Mingoti ◽  
J.C.C. Balieiro ◽  
J. Cipolla-Neto ◽  
...  
Keyword(s):  
Dna Damage ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Cumulus Cells

Effect of cyanocobalamin on oocyte maturation, in vitro fertilization, and embryo development in mice

Zygote ◽  
2020 ◽  
pp. 1-8
Author(s):  
Tamana Rostami ◽  
Fardin Fathi ◽  
Vahideh Assadollahi ◽  
Javad Hosseini ◽  
Mohamad Bagher Khadem Erfan ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Control Group ◽  
Vitro Fertilization ◽  
Maturation In Vitro

Summary The aim of this study was to investigate the effect of cyanocobalamin supplementation on in vitro maturation (IVM), in vitro fertilization (IVF), and subsequent embryonic development competence to the blastocyst stage, and in vitro development of mouse 2-cell embryos. Cumulus cells were prepared from mouse cumulus–oocyte complexes (COCs) and incubated for 24 h in an in vitro culture (IVC) medium that contained different concentrations of cyanocobalamin (100, 200, 300 or 500 pM). We collected 2-cell embryos from superovulated NMRI mice and cultured them in the same concentrations of cyanocobalamin (100, 200, 300 or 500 pM). After 42 h of IVM, we observed significantly increased oocyte maturation in the 200 pM cyanocobalamin-treated group compared with the control group (P < 0.0001). Mature oocytes cultured in 200 pM cyanocobalamin were fertilized and cultured in IVC medium with cyanocobalamin (100, 200, 300 or 500 pM) during early embryogenesis. The matured oocytes that were cultured in 200 pM cyanocobalamin had significantly higher 2-cell development rates compared with the control oocytes (P < 0.01). Embryos obtained from in vitro mature oocytes and in vivo fertilized oocytes that were cultured in 200 pM cyanocobalamin had significantly greater frequencies of development to the blastocyst stage and a significant reduction in 2-cell blocked and degenerated embryos compared with the control embryos (P < 0.0001). Embryos derived from oocytes fertilized in vivo with 200 pM cyanocobalamin had a higher percentage of blastocyst embryos compared with those derived from matured oocytes cultured in vitro (P < 0.0001). These finding demonstrated that the effects of cyanocobalamin on oocyte maturation, fertilization, and embryo development in mice depend on the concentration used in IVC medium.

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