scholarly journals 296 LEUKEMIA INHIBITORY FACTOR INFLUENCES SHEEP OOCYTE PARTHENOGENETIC DEVELOPMENT DURING THE TRANSITION FROM GERMINAL VESICLE TO EARLY PRONUCLEAR STAGE

2005 ◽  
Vol 17 (2) ◽  
pp. 298
Author(s):  
G. Ptak ◽  
F. Lopes ◽  
P. Loi
Keyword(s):  
Embryo Development ◽  
Leukemia Inhibitory Factor ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Inhibitory Factor ◽  
Control Group ◽  
Blastocyst Development ◽  
Human Follicular Fluid ◽  
Oocyte Competence

Leukemia inhibitory factor (LIF) is an indispensable cytokine for female fertility. The influence of LIF on embryo development and particularly implantation has been recently confirmed; however, the effect of this cytokine on the oocyte has not been studied. The presence of LIF in human follicular fluid implies its possible role in the acquisition of oocyte competence. Furthermore, the up-regulation of LIF by steroid hormones in sheep makes entirely feasible the hypothesis that ovulatory estradiol peak plays a role in the preparation of female gamete for fertilization. With this in mind, we studied the effect of LIF during in vitro development of sheep oocytes mimicking the physiological expression of LIF induced by the ovulatory peak of estradiol in mice. GV stage oocytes matured and chemically activated in the presence of LIF and anti-LIF antibody were cultured to the blastocyst stage in our standard media. To eliminate the effect of the putative presence of LIF in heat inactivated fetal calf serum used for oocyte maturation, aliquots of LIF were treated at 56°C for 30 min and added to the maturation medium. The proportion of embryos that reached the blastocyst stage in vitro was significantly higher (P < 0.001) for oocytes matured and activated with LIF (36/93; 39%) than for the group incubated with antibody against LIF (6/68; 9%). The significant effect of anti-LIF antibody (P < 0.001) was also observed when compared with blastocysts developed from the control group of oocytes matured without LIF addition (31/106; 29%). Although the beneficial influence of LIF treatment on embryo development demonstrated with those preliminary data was not confirmed statistically, due to low number of oocytes involved, the proportion of embryos reaching the blastocyst stage in vitro was about 10% higher for those incubated with LIF than for either those cultured without the cytokine or those, matured in the presence of heat-treated LIF (15/55; 27%); however, the rate of blastocyst development appeared very similar to that of the control group. This study revealed for the first time a role of LIF in determining oocyte competence. Further investigation to determine how LIF achieves its effects on the oocyte are ongoing in our laboratory. This work was supported by FIRB RBNE01HPMX, COFIN 2002074357, COFIN 2003073943 002, and British Council 2004.

182 MATURATION OF BOVINE CUMULUS-OOCYTE COMPLEXES WITH FOLLICLE FLUID VARYING IN ESTRADIOL CONTENT AFFECTS CUMULUS CELL EXPANSION WITHOUT AFFECTING SUBSEQUENT EMBRYO DEVELOPMENT IN VITRO

2017 ◽  
Vol 29 (1) ◽  
pp. 199
Author(s):  
A. W. Harl ◽  
E. L. Larimore ◽  
A. Al Naib ◽  
L. K. Wooldridge ◽  
A. D. Ealy ◽  
...  
Keyword(s):  
Cell Expansion ◽  
Cumulus Cell ◽  
Prostaglandin F2α ◽  
Blastocyst Stage ◽  
Control Group ◽  
Blastocyst Development ◽  
Cumulus Expansion ◽  
Oocyte Competence ◽  
Maturation Medium

The objective of this work was to determine how characteristics of bovine follicle fluid (FF; especially oestradiol content) affect cumulus cell expansion and oocyte competence. In the first study, FF was collected from abattoir-derived ovaries and pooled separately for large follicles (≥10 mm) and small follicles (≤3 mm). A portion of the FF from each category was charcoal stripped. These 4 types of FF were then used as the primary ingredient (75% vol/vol) in oocyte maturation media. A separate control group lacking FF but containing BSA was included to monitor potential impacts of protein on outcomes (control; without FF). Some of the cumulus-oocyte complexes (COC; n = 250) were matured in individual drops for analysis of cumulus expansion (photographed and measured at 0 and 21 h of maturation). Other COC (n = 770) were matured in groups of 12 to 25 in the previously described media, and then subjected to IVF procedures. Cleavage rates were recorded on Day 3, and blastocyst rates were recorded on Day 8 post-fertilization. Cumulus cell expansion was greatest when COC were matured in medium containing FF from large follicles, wherein it even exceeded the controls (P < 0.02). Maturation in FF from small follicles resulted in cumulus expansion that was intermediate between large and control. Maturation in charcoal-stripped FF severely restricted cumulus cell expansion (P < 0.05) compared with those matured in untreated FF. Despite the observed improvement in cumulus cell expansion, COC that had been matured in media containing FF were less likely to cleave (P < 0.05) and also less likely to develop to the blastocyst stage (P < 0.01) than those matured in control medium. Cleavage and blastocyst rates did not differ among any of the maturation media containing FF. In the second study, oestrous cycles of 9 crossbred cows were synchronized and FF samples were collected 36 to 42 h after prostaglandin F2α injection. Samples from individual cows were categorized as having high oestradiol (>800,000 pg mL−1; H) or low oestradiol concentrations (<800,000 pg mL−1; L). The FF was retained for use in in vitro experiments, where it was added to maturation media (20% vol/vol). Cumulus-oocyte complexes (n = 1,775) were randomly distributed into treatments across 12 in vitro maturation/fertilization replicates (H and L, balanced within replicate; 4 replicates/cow). Each replicate included the following 3 control groups: maturation medium containing BSA without FF, maturation medium without BSA with abattoir-derived FF, and maturation medium without BSA and without FF. The COC were matured in their assigned medium for 21 h, and then all COC were subjected to IVF procedures. Cleavage rates were recorded on Day 3, and blastocyst rates were recorded on Day 7 and 8 post-fertilization. Oestradiol content of the FF (H v. L) did not affect oocyte cleavage nor blastocyst rates on Day 7 or 8. The results of these studies indicate that although FF improves cumulus cell expansion during maturation in vitro, it does not result in higher rates of cleavage or blastocyst development regardless of oestradiol content.

scholarly journals In Vitro Maturation with Leukemia Inhibitory Factor Prior to the Vitrification of Bovine Oocytes Improves Their Embryo Developmental Potential and Gene Expression in Oocytes and Embryos

2020 ◽  
Vol 21 (19) ◽  
pp. 7067
Author(s):  
Meritxell Vendrell-Flotats ◽  
Tania García-Martínez ◽  
Iris Martínez-Rodero ◽  
Manel Lopez-Bejar ◽  
Jonathan LaMarre ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Embryo Development ◽  
Leukemia Inhibitory Factor ◽  
In Vitro Maturation ◽  
Expression Patterns ◽  
Inhibitory Factor ◽  
Blastocyst Development ◽  
Developmental Potential

Oocyte cryopreservation has a significant impact on subsequent embryonic development. Herein, we investigated whether supplementing in vitro maturation medium with Leukemia Inhibitory Factor (LIF) prior to vitrification affects embryo development and gene expression at different embryo developmental stages. A panel of genes including maternal effect, epigenetics, apoptosis and heat stress was relatively quantified. The results show reduced cleavage rates after vitrification, regardless of the LIF treatment. Although not statistically different from control-vitrified oocytes, oocyte apoptosis and the blastocyst yield of LIF-vitrified oocytes were similar to their non-vitrified counterparts. Vitrification increased oocyte ZAR1, NPM2 and DPPA3 gene expression while its expression decreased in LIF-vitrified oocytes to similar or close levels to those of non-vitrified oocytes. With a few gene-specific exceptions, vitrification significantly increased the expression of DNMT3A, HDAC1, KAT2A, BAX and BCL2L1 in oocytes and most stages of embryo development, while comparable expression patterns for these genes were observed between LIF-vitrified and non-vitrified groups. Vitrification increased HSPA1A expression in oocytes and HSP90AA1 in 2-cell embryos. Our data suggest that vitrification triggers stage-specific changes in gene expression throughout embryonic development. However, the inclusion of LIF in the IVM medium prior to vitrification stimulates blastocyst development and several other developmental parameters and induces oocytes and embryos to demonstrate gene expression patterns similar to those derived from non-vitrified oocytes.

124 TAXOL™ COULD PROMOTE EMBRYO DEVELOPMENT OF BOVINE OOCYTES VITRIFIED BY OPS

2007 ◽  
Vol 19 (1) ◽  
pp. 179
Author(s):  
R. Morató ◽  
D. Izquierdo ◽  
M. J. Palomo ◽  
B. Anguita ◽  
A. R. Jiménez-Macedo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Calf Serum ◽  
Subsequent Development ◽  
Control Group ◽  
Control Groups ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Microtubule Stabilizer ◽  
Calf Oocytes

Stabilizing the cytoskeleton system during vitrification could be beneficial for improving post-thawed survival and subsequent development of vitrified oocytes. Taxol™, paclitaxel, is a microtubule stabilizer that has been found to improve development competence of vitrified mouse and human oocytes. The objective of this work was to study the effect of a Taxol pretreatment before OPS vitrification on the post-thaw cow and calf oocyte development. Oocytes were aspirated from slaughterhouse-derived ovaries and matured in TCM-199. Oocytes were randomly assigned to one of 3 experimental groups: (1) control oocytes matured in vitro for 24 h, (2) oocytes matured for 22 h and vitrified by the OPS method (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58), and (3) oocytes matured for 22 h and vitrified by OPS method with 1 µM Taxol. OPS and Taxol–OPS oocytes were transferred back into the maturation dishes and matured for 2 additional h before being subjected to fertilization. Fertilization was performed using frozen–thawed Percoll-selected sperm. At 22 h after insemination, presumptive zygotes were pipetted and then cultured in drops of 25 µL SOF medium and 5% fetal calf serum under paraffin oil at 38.5°C in 5% CO2, 5% O2, 90% N2, and maximum humidity. The Taxol–OPS group provided a significantly higher cleavage rate than the OPS group in cows (41.9% and 34.0%, respectively) or in calves (33.7% and 23.5%, respectively). However, cleavage rate in the experimental groups was significantly lower than in the control group (78.3% and 69.7% for cow and calf control groups, respectively). Blastocyst yield was also higher for the Taxol–OPS group (3.2%) than the OPS group (0%) in cow oocytes. There was no blastocyst development when calf oocytes were vitrified with or without Taxol pretreatment. As expected, cow and calf vitrification groups triggered a significantly lower blastocyst yield when compared with their control (26.7% and 14.9% for cow and calf control groups, respectively). In conclusion, this study showed that supplementation of 1 µM Taxol could promote embryo development after thawing. Further research is indicated to clarify the function of Taxol and its optimal concentration in order to improve the rate of embryo development. Table 1. Effect of Taxol pretreatment on development of cow and calf oocytes vitrified by OPS

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

scholarly journals 324SPHINGOSINE-1-PHOSPHATE PROTECTS CULTURED BOVINE OOCYTES FROM PHYSIOLOGICALLY RELEVANT THERMAL STRESS

2004 ◽  
Vol 16 (2) ◽  
pp. 282 ◽  
Author(s):  
Z. Roth ◽  
P.J. Hansen
Keyword(s):  
Thermal Stress ◽  
Heat Shock ◽  
Blastocyst Stage ◽  
Caspase Activity ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Oocyte Competence ◽  
Cell Death Pathway

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that can block the sphingomyelin cell-death pathway by suppressing ceramide-induced apoptosis. The present study was performed to test whether S1P protects oocytes from heat shock during in vitro maturation. Cumulus-oocyte complexes obtained by slicing follicles were placed in maturation medium with or without 50nM S1P and cultured at 38.5°C (CON) or 41°C (41C) for the first 12h of maturation. Incubation during the last 10h of maturation (22-h total maturation time), fertilization, and embryonic development were performed at 38.5°C and 5% (v/v) CO2. Blastocyst development was recorded at 8 days post-insemination (dpi) and activity of group II caspases in 8-day blastocysts was determined using a fluoroprobe, PhiPhiLux-G1D2 (OncoImmunin, Gaithersburg, MD, USA). Data were analysed by least-squares ANOVA with the GLM procedure of SAS. Percentage data were subjected to arcsin transformation before analysis. Exposure of oocytes to thermal stress during the first 12h of maturation reduced cleavage rate (P&lt;0.01) and the number of oocytes developing to the blastocyst stage (P&lt;0.04). There was a temperature x S1P interaction for cleavage rate (P&lt;0.03) because S1P blocked effects of thermal stress on cleavage rate. Without S1P, the percentage of oocytes that cleaved by 3 dpi were 83.6±2.7% and 65.8±2.7% for CON and 41C, respectively. In the presence of S1P, percent cleavage was 86.7±2.7% and 83.9±2.7% for CON and 41C, respectively. There was a trend (P=0.06) for a temperature x S1P interaction for percent oocytes developing to blastocyst stage because S1P blocked effects of heat shock on development. Without S1P, the percentages of oocytes that developed to the blastocyst stage were 28.7±3.0% and 15.2±3.0% for CON and 41C, respectively. In the presence of S1P, percent blastocysts were 24.3±3.4% and 23.9±3.0% for CON and 41C, respectively. When development was expressed as percentage of cleaved embryos, however, there were no effects of temperature, S1P, or temperature x S1P on percent development to the blastocyst stage. Blastocyst caspase activity was not affected by temperature or S1P. In summary, exposure to physiologically relevant thermal stress during the first 12h of maturation has a deleterious effect on oocyte competence and this effect can be reduced by S1P. The fact that heat shock reduced the percentage of oocytes but not the percentage of cleaved embryos that became blastocysts suggests that oocytes that survive effects of heat shock and cleave have normal potential to develop to the blastocyst stage. Moreover, since heat shock did not affect caspase activity, it is likely that blastocysts from heat-shocked oocytes have normal developmental potential, at least as determined by caspase activity. Support: BARD FI-330-2002 and USDA Grants 2002-35203-12664 and 2001-52101-11318.

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Group 13 ◽  
Time Lapse Imaging

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

197 DOES DURATION OF BOVINE OOCYTE MATURATION IN VITRO AFFECT THE SPEED OF EMBRYO DEVELOPMENT IN VITRO AND SEX RATIO AT THE TWO-CELL OR BLASTOCYST STAGE?

2008 ◽  
Vol 20 (1) ◽  
pp. 177
Author(s):  
P. Bermejo-Álvarez ◽  
A. Gutiérrez-Adán ◽  
P. Lonergan ◽  
D. Rizos
Keyword(s):  
Sex Ratio ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Blastocyst Development ◽  
Maturation In Vitro ◽  
Maturational Stage ◽  
Development In Vitro ◽  
Oviduct Fluid

The faster-developing blastocysts in IVC systems are generally considered more viable and better able to survive following cryopreservation or embryo transfer than those that develop more slowly. However, evidence from several species indicates that embryos that reach the blastocyst stage earliest are more likely to be males than females. The aim of this study was to determine whether the duration of maturation could affect early embryo development and, furthermore, the sex ratio of early- or late-cleaved embryos and blastocysts. Cumulus–oocyte complexes were matured in vitro for 16 h (n = 2198) or 24 h (n = 2204). Following IVF, presumptive zygotes from each group were examined every 4 h between 24 and 48 h postinsemination (hpi) for cleavage, and all embryos were cultured to Day 8 in synthetic oviduct fluid to assess blastocyst development. Two-cell embryos at each time point and blastocysts on Days 6, 7, and 8 from both groups were snap-frozen individually for sexing. Sexing was performed with a single PCR using a specific primer BRY. There was a significantly lower number of cleaved embryos from the 16-h compared with the 24-h maturation group at 28 (10.0 � 1.51 v. 28.8 � 3.57%), 32 (35.3 � 1.48 v. 57.6 � 3.33%), 36 (54.8 � 1.76 v. 67.4 � 2.81%), 40 (63.3 � 1.82 v. 72.0 � 2.54%), and 48 (70.6 � 1.78 v. 77.1 � 2.18%) hpi, respectively (mean � SEM; P d 0.05). However, the blastocyst yields on Day 6 (17.1 � 3.11 v. 16.4 � 2.11%), 7 (30.6 � 4.10 v. 34.6 � 3.51%), or 8 (34.1 � 3.90 v. 39.4 � 4.26%) were similar for both groups (mean � SEM; 16 v. 24 h, respectively). Significantly more 2-cell early cleaved embryos (up to 32 hpi) were male compared with the expected 1:1 ratio from both groups (16 h: 1.24:0.76 v. 24 h: 1.17:0.83, P ≤ 0.05); however, the overall sex ratio among 2-cell embryos was significantly different from the expected 1:1 in favor of males only for the 16-h group (1.18:0.82, P ≤ 0.05). The sex ratio of blastocysts on Day 6, 7, or 8 from both groups was not different from the expected 1:1. However, the total number of male blastocysts obtained after 8 days of culture from the 24-h group was significantly different from the expected 1:1 (1.19:0.81, P ≤ 0.05) and approached significance in the 16-h group. These results show that the maturational stage of the oocyte at the time of fertilization has an effect on the kinetics of early cleavage divisions but not on blastocyst yield. Furthermore, irrespective of the duration of maturation, the sex ratio of early-cleaving 2-cell embryos was weighted in favor of males, and this observation was maintained at the blastocyst stage.

354 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS AFTER NITRIC OXIDE INHIBITION

2007 ◽  
Vol 19 (1) ◽  
pp. 292
Author(s):  
K. R. L. Schwarz ◽  
T. H. C. de Bem ◽  
T. T. Zampieri ◽  
P. R. Adona ◽  
C. L. V. Leal
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Control Group ◽  
Sperm Cells ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Nitric oxide (NO) is a chemical messenger detected in several cell types such as endothelial cells, neurons, and macrophages, exerting varied functions including vasodilatation, neurotransmission, and cell death induction. NO is generated by the activity of the enzyme nitric oxide synthase (NOS), which has been detected in several organs and tissues including the reproductive system. The aim of the present study was to assess the dose-response effect of N-omega-nitro-l-arginine-methyl ester (l-NAME), an NOS inhibitor, on in vitro nuclear and cytoplasmic maturation of bovine oocytes. Slaughterhouse ovaries were collected and their follicles (2–6 mm) were aspirated to obtain cumulus–oocyte complexes (COCs). Increasing l-NAME concentrations (0, 10-7, 10-5, 10-4, and 10-3 M) were added to IVM medium (TCM-199, supplemented with 10% fetal calf serum, 0.5 �g mL-1 FSH, 5.0 �g mL-1 LH, 0.2 mM pyruvate, and 10 mg mL-1 gentamicin); oocytes were cultured for 22 h. Nuclear maturation was assessed by propidium iodide staining (10 �g mL-1). For IVF, frozen–thawed semen prepared by Percoll gradient was used. Sperm cells were co-cultured with the oocytes at a final concentration of 2 � 106 sperm cells mL-1 in TALP-IVF medium supplemented with 2 �M penicillamine, 1 �M hypotaurine, 250 �M epinephrine, and 20 �g mL-1 heparin. After 20 h, presumptive zygotes were partially denuded and transferred to IVC medium (TCM-199 supplemented with 10% fetal calf serum, 2.0 mM pyruvate, and 10 mg mL-1 gentamicin). All cultures were at 38.5�C under 5% CO2 in air and maximum humidity. Cytoplasmic maturation was assessed by blastocyst development rates on Day 7. DNA fragmentation was assessed on Day 8 embryos by TUNEL (In Situ–Cell Death Detection kit, fluorescein; Roche Diagnostica Brasil, Sao Paulo, Brazil). Data were analyzed by ANOVA using the GLM procedure (SAS Institute, Inc., Cary, NC, USA), and means were compared by Duncan test at a 5% level. After IVM, the control group (0 M l-NAME) showed a greater number of oocytes in metaphase II (MII: 95.8 � 3.7%; P &lt; 0.05), whereas the groups cultured with l-NAME had lower MII rates (78–82%; P &lt; 0.05), irrespective of concentration (P &gt; 0.05). Many oocytes remained in metaphase I (MI: 18–22%). Cleavage rates at 48 h IVC was not affected (77–88%; P &gt; 0.05). Blastocyst rates (34.0 � 7.2% to 41.5 � 4.8%; P &gt; 0.05) and total cell numbers (151 to 174) were also unaffected by NO inhibition by l-NAME. However, the number of TUNEL-positive cells was lower in the control group (1.4 � 4.7; P &lt; 0.05) than in the treated groups (2.7 � 4.8 to 4.4 � 6.4; P &gt; 0.05). In conclusion, NO synthesis inhibition in oocytes during IVM reduces nuclear maturation, particularly during MI–MII transition, and increases apoptosis in blastocysts, suggesting that NO may be involved in oocyte maturation and apoptosis protection.

scholarly journals Effect of storage tube material and resveratrol during liquid storage of matured bovine oocytes on subsequent development

2017 ◽  
Vol 65 (4) ◽  
pp. 546-555
Author(s):  
Tayita Suttirojpattana ◽  
Tamás Somfai ◽  
Satoko Matoba ◽  
Takashi Nagai ◽  
Rangsun Parnpai ◽  
...  
Keyword(s):  
Embryo Development ◽  
Subsequent Development ◽  
Control Group ◽  
Tube Material ◽  
Blastocyst Development ◽  
Liquid Storage ◽  
Glass Tubes ◽  
Bovine Oocytes

This study determined the optimum storage vessel and the effects of resveratrol for the storage of in vitro matured (IVM) bovine oocytes. After IVM, the oocytes were kept in a Hepes-buffered medium at 25 °C for 20 h in different containers including Eppendorf tubes (ET) made of polypropylene (PP) and polystyrene (PS), and tissue culture tubes (TCT) made of PP, PS, and glass. Then oocytes were subjected to IVF and subsequent in vitro embryo development was compared among the groups and to that of a control group without storage. The percentage of blastocyst development in the control group was significantly higher than in the stored groups (P < 0.05). Among oocytes stored in TCT, the percentage of blastocyst development of oocytes stored in glass TCT was significantly higher than that of oocytes stored in PP and PS TCT (P < 0.05); however, it did not differ from that of oocytes stored in ET. The quality of blastocysts did not differ among the control and stored groups. Embryo development was not affected when 0.1, 1 or 10 μM resveratrol was added to the medium during oocyte storage. In conclusion, glass tubes were optimal for oocyte storage and resveratrol did not improve the development of stored oocytes.

329 SUPPRESSION OF BOVINE OOCYTE IVM BY SERUM AND FSH DEPRIVATION OR BY α-AMANITIN: EFFECT ON FERTILIZATION AND EMBRYO DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 272
Author(s):  
K. Kananen-Anttila ◽  
M. Eronen ◽  
J. Matilainen ◽  
M. Kallio ◽  
J. Peippo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Embryo Cleavage ◽  
Bovine Oocytes ◽  
Sperm Aster

We have studied the effect of suppressed IVM on the developmental competence of bovine oocytes, aiming at elucidating the importance of cytoplasmic maturation in fertilization and embryo development. Six replicates of abattoir-derived oocytes were randomly divided into three IVM groups. Control (n = 950): TCM-199 with glutamax-I (Gibco, Grand Island, NY, USA), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin, 50 ng mL−1 FSH, and 10% fetal bovine serum (FBS) (Gibco); Serum+FSH-free (n = 944): same as control but without FSH and FBS; α-amanitin (n = 977): same as control but with 10 μg mL−1 α-amanitin. Nuclear maturation of oocytes was studied 24 h after the onset of IVM, the formation of sperm aster structure 10 hours post-insemination (hpi) and the formation of pronuclei 20 hpi. Sperm aster was visualized with β-tubulin antibody (modified from Navara et al. 1999 Dev. Biol. 162, 29–40). Presumptive zygotes were cultured until Day 7 in modified SOFaaci + 4 mg mL−1 fatty acid-free BSA in 5% O2. Cumulus cell expansion was seen only in the control group. The results of nuclear maturation, fertilization, and embryo development are summarized in Table 1. Serum and FSH deprivation did not have a statistically significant effect on the parameters studied (vs. control). α-amanitin exposure during IVM reduced nuclear maturation, fertilization, and Day 3 embryo cleavage vs. control, and resulted in total blockage of Day 7 blastocyst development. The treatment groups had significantly smaller mean diameters of male pronuclei (control: 14 ± 0.6 μ­m; serum+FSH-free: 12 ± 0.5 μ­m, P < 0.05; α-amanitin: 10 ± 0.6 μ­m, P < 0.001) and sperm asters (control: 86 ± 4 μ­m; serum+FSH-free: 82 ± 4 μ­m, P < 0.01; α-amanitin: 49 ± 7 μm, P < 0.001) (nonparametric Kruskall Wallis and Mann-Whitney U tests) vs. control group. Despite reduction in pronucleus and sperm aster diameter, serum and FSH deprivation during IVM did not affect in vitro developmental competence of bovine oocytes, suggesting a need for re-evaluation of the components of IVM. α-Amanitin exposure in IVM disturbed nuclear maturation, fertilization, and embryo development, indicating the essence of early transcription. Table 1. Average percentages ± (n) for nuclear maturation, fertilization (min two pronuclei), embryo cleavage, and blastocyst development

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