154 THE EFFECT OF OXYGEN TENSION ON IN VITRO DEVELOPMENT OF BOVINE EMBRYOS IN POLYDIMETHYLSILOXANE-BASED WELL OF THE WELL DISHES PREPARED UNDER ATMOSPHERIC OR REDUCED AIR PRESSURE

2010 ◽  
Vol 22 (1) ◽  
pp. 235
Author(s):  
T. Somfai ◽  
Y. Inaba ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
S. Kobayashi ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Culture Medium ◽  
Production Method ◽  
Total Cell ◽  
Air Pressure ◽  
Bovine Embryos ◽  
Cell Numbers ◽  
Significant Difference ◽  
And Control

Polydimethylsiloxane (PDMS) is a non-toxic silicon compound. Its excellent optical characteristics and easy preparation make it a good candidate material for the molding of custom-shaped dishes for embryo culture. We investigated the feasibility of PDMS-based well of the well (WOW) dishes for in vitro culture of bovine embryos under different oxygen tensions. The WOW dishes with 25 micro-wells (each of 175 μm depth and 250 μm width in diameter arranged in 5 columns and 5 rows) were molded from PDMS prepared either under atmospheric (Experiment 1) or reduced (0.1 MPa) (Experiment 2) air pressure to remove air bubbles. Presumptive zygotes obtained by the in vitro maturation and fertilization of follicular oocytes were placed and cultured for 7 days in traditional micro-drops of culture medium (Control) or in the micro-wells of PDMS-based WOW dishes (PDMS-WOW), both covered by paraffin oil. The culture medium was CR1aa supplemented with 5% calf serum. The culture drop size was 125 μL (5 μL/oocyte) in both groups. Embryo development and blastocyst cell numbers between Control and PDMS-WOW groups were compared either under 20% or 5% O2 tensions. There was no statistical difference in cleavage and blastocyst rates (ranging between 82.3-86.4% and 34.0-45.8%, respectively) between Control and PDMS-WOW embryos irrespective of oxygen tension and dish production method. In Experiment 1, the mean total cell numbers in blastocysts were lower in the PDMS-WOW group than that in Control under 20% O2 (105.0 ± 5.5 and 130.4 ± 9.9, respectively) (P < 0.05, ANOVA); however, the application of 5% O2 significantly improved the cell numbers and eliminated the difference between the PDMS-WOW and Control groups (135.4 ± 6.2 and 148.0 ± 9.0, respectively). In Experiment 2, there was no significant difference in mean total cell numbers in blastocysts between the PDMS-WOW and Control either under 20% O2 (97.2 ± 5.7 and 103.9 ± 8.9, respectively) or 5% O2 (147.5 ± 12.1 and 157.3 ± 3.9, respectively). The numbers and rates of inner cell mass and trophectoderm cells did not differ between the Control and PDMS-WOW groups, irrespective of O2 tension and production method. Our results demonstrate that bovine embryos can develop to the blastocyst stage in PDMS-based WOW dishes; however, it may express detrimental effects on embryonic cell numbers, which can be neutralized by the application of low O2 tension during culture or reduced air pressure during the PDMS preparation. This work was supported by the Research and Development Program for New Bio-Industry Initiatives.

133 EFFECTS OF FROZEN STORED CULTURE MEDIA ON PRE-IMPLANTATION DEVELOPMENT OF PARTHENOTES AND CLONED EMBRYOS IN PIGS

2009 ◽  
Vol 21 (1) ◽  
pp. 166
Author(s):  
Y. H. Zhang ◽  
H. T. Xi ◽  
Y. Liu ◽  
J. Li ◽  
A. Pederson ◽  
...  
Keyword(s):  
Culture Media ◽  
Polar Body ◽  
Frozen Storage ◽  
Test Group ◽  
Total Cell ◽  
Control Group ◽  
Cell Numbers ◽  
Significant Difference ◽  
And Control

The present study was designed to examine if frozen storage of porcine zygote medium (PZM3) plus 3 mg mL–1 BSA (Yoshioka et al. 2002 Bio. Reprod. 66, 112–119) is feasible to culture pig embryos produced by parthenogenetic activation and somatic nuclear transfer. Slaughterhouse-derived sow cumulus–oocyte complexes (COCs) were matured in TCM199 supplemented with 10% porcine follicle fluid, 5% cattle serum, 10 IU mL–1 eCG, 5 IU mL–1 hCG, 0.8 mm L-glutamine and 0.05 mg mL–1 gentamicin at 38.5°C, 100% humidity and 5% CO2 in air. For activation, cumulus cells were removed after 42 to 44 h of maturation, and the denuded oocytes with 1st polar body were activated with a double 160 V mm–1, 100 μs direct pulse followed by culture in PZM3. Each experiment was replicated at least three times. Data were expressed as mean ± SEM and analyzed by using chi-square module in SPSS 11.0, with P < 0.05 denoting significant difference. In Experiment 1, after preparation, liquid PZM3 was aliquoted to 50 mL falcon centrifuge tubes. Randomly, half of the tubes with PZM3 were put into –80°C freezers, and the rest were placed into 4°C refrigerator. Within one week after storage, a tube of frozen PZM3, while that stored at 4°C served as control, was warmed at 38.5°C in CO2 incubator, and more than three 4-well culture dishes were then made with 400 μL PZM3 in each well and balanced for at least 4 h in the incubator before experiment. The results showed that both cleavage (78/93, 83.9 ± 1.2% v. 87/103, 84.5 ± 1.8%, P > 0.05) and blastocyst (60/93, 65.2 ± 2.1% v. 65/103, 63.1 ± 3.8%, P > 0.05) rates were similar between frozen-warmed PZM3 and control, as was total cell numbers per blastocyst (50 ± 7 v. 47 ± 5, P > 0.05) between groups. In Experiment 2, we used somatic cloned embryos to investigate the effect of frozen-warmed PZM3 on pre-implantation development of such embryos. Our results indicated that no significant difference in rates of cleavage (68/95, 71.5 ± 5.1% v. 78/100, 78.1 ± 1.9%, P > 0.05), blastocyst formation (33/95, 34.6 ± 7.6% v. 78/100, 38.2 ± 3.5%, P > 0.05) and total cell numbers per blastocyst (40 ± 11 v. 48 ± 9, P > 0.05) was found between the test and control groups, designed the same as in Experiment 1. In Experiment 3, we tested whether PZM3 in frozen storage for 5 months was able to support in vitro development of parthenotes comparable to freshly-made ones. PZM3 after frozen storage for 5 months was warmed using the same method as Experiment 1, and the newly made PZM3 within 1 week of storage at 4°C acted as control. The results showed that although the cleavage (135/138, 97.8 ± 2.7% v. 117/129, 90.7 ± 3.1%, P > 0.05) and blastocyst (104/138, 75.4 ± 1.6% v. 84/129, 65.1 ± 2.3, P > 0.05) rates in control group were both slightly higher than that in the test group, no statistical differences was observed. We also found no significant difference in total cell numbers per blastocyst (48 ± 7 v. 46 ± 6, P > 0.05) between groups. Taken together, our results imply that frozen storage of PZM3 is feasible, and of practical value for culture pig embryos.

150 IN VITRO AND IN VIVO DEVELOPMENT OF EMBRYO FOLLOWING BIOPSY WITH DIFFERENT TECHNIQUES IN MOUSE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 223
Author(s):  
A. C. Taskin ◽  
H. Bagis ◽  
H. Sagirkaya ◽  
T. Akkoc ◽  
S. Arat
Keyword(s):  
Fetal Development ◽  
Total Cell ◽  
Trophectoderm Biopsy ◽  
Control Groups ◽  
Cell Numbers ◽  
Development Rates ◽  
Significant Difference ◽  
Implantation Sites ◽  
And Control

In vitro development ratios, quality evaluation, in vivo implantation, and fetal development ratios were investigated following aspiration biopsy in 8-cell mouse embryos and trophectoderm biopsy in blastocyst developed from 8-cell stage embryos in vitro. Superovulated CB6F1 hybrid female mice (5–6 weeks) were sacrificed 68 to 72 hours after hCG administration. Eight-cell embryos were flushed from oviducts of the sacrificed mouse with HTF medium supplemented with HEPES and 3 mg mL–1 BSA. Embryos were randomly divided into two groups. In the first group, embryos at 8-cell stage were used for a single cell blastomer aspiration; in the second group, embryos were cultured in vitro until blastocyst stage. Trophectoderm cells (15% of trophoblastic cells) were biopsied from developing blastocysts. There were also control groups for both groups. Biopsy procedures for both groups were applied in 50 µL drops of Ca2+/Mg2+ free HTF medium containing HEPES+3 mg mL–1 BSA+5 µg mL–1 cytochalasine B. After biopsy, embryos were cultured in Quinn’s blastocyst medium supplemented with 4 mg mL–1 BSA and incubated in 5% CO2 and 5% O2 incubator at 37°C for 48 and 24 hours for blastomer aspiration and trophectoderm biopsy groups, respectively. While some developing blastocysts were used for determining total cell number, some of them were transferred to the recipients. Results were evaluated by independent t-test and ANOVA of SPSS 17.0 statistic program (SPSS Inc., Chicago, IL, USA). In blastomere biopsy and control groups, development rates were determined as 81.02% (121/152) and 96.37% (62/63), while the total cell numbers were determined as 50 and 50, respectively. There was no significant difference between groups in terms of development ratios and total cells. In blastomer biopsy and control groups, the implantation sites and fetal development rates were found as 25% (9/36) and 26% (8/30), and 19.44% (7/36) and 20% (6/30), respectively. No significant difference was observed between groups in terms of implantation sites and fetal development rates. In trophectoderm biopsy and control groups, while the development rates were found as 86.96% (69/79) and 93.33% (23/28), the total cell numbers were 26.66 and 55.33, respectively. Although there was not any significant difference between groups in terms of development rates, there was a significant difference between groups in terms of total cell numbers (P < 0.05). In trophectoderm biopsy and control groups, the implantation sites and fetal development rates were determined as 21.88% (7/32) and 59.09% (13/22), and 0% (0/32) and 18.18% (4/22), respectively. Although there was not any significant difference between groups in terms of implantation sites, there was a significant difference between groups in terms of fetal development rates (P < 0.05). Therefore, it was concluded that biopsy applied at early stage of embryonic development does not affect embryo development negatively and biopsy procedures applied at early developmental stages have more advantages especially in embryos developing faster with low total cell numbers such as mouse species. Supported by TUBITAK KAMAG-107G027).

70 VITRIFICATION OF IMMATURE OVINE OOCYTES WITH CRYOLOOP: EFFECT OF CYTOCHALASIN B PRETREATMENT ON SUBSEQUENT DEVELOPMENT

2009 ◽  
Vol 21 (1) ◽  
pp. 135
Author(s):  
A. R. Moawad ◽  
I. Choi ◽  
J. Zhu ◽  
K. H. S. Campbell
Keyword(s):  
Cytochalasin B ◽  
Mammalian Species ◽  
Subsequent Development ◽  
Total Cell ◽  
High Survival ◽  
Control Groups ◽  
Cell Numbers ◽  
Significant Difference ◽  
And Control

Oocyte cryopreservation is still a challenge in most mammalian species because of their extreme sensitivity to chilling injuries. Relaxation of the cytoskeleton during vitrification may improve post-thaw survival and subsequent development; however, a previous study in immature [germinal vesicle (GV) stage] oocytes from prepubertal lambs reported that pretreatment with cytochalasin B (CB) did not improve maturation (Silvestre MA et al. 2006 Anim. Reprod. Sci. 93, 176–182). We previously reported that GV oocytes from mature ewes can be vitrified using a cryoloop with high survival, maturation, and subsequent in vitro fertilization (Moawad AR and Campbell KHS 2008 Reprod. Fertil. Dev. 20, 122). The aim of this study was to evaluate the effects of CB pretreatment prior to vitrification of GV oocytes (from mature ewes) on subsequent development. Cumulus–oocyte complexes obtained at slaughter were randomly divided into 2 groups and incubated with or without 7.5 μg mL–1 CB for 60 min before vitrification. Oocytes from each group were vitrified or used as toxicity and controls. For vitrification, oocytes were equilibrated in 10% ethylene glycol (EG) and 0.25 m trehalose (T) in HEPES/TCM-199 plus 10% fetal bovine serum (BM) for 3 min. Oocytes were then exposed to vitrification solution (20% EG and 20% DMSO in BM), loaded into the cryoloop within 1 min, and immersed in liquid nitrogen. Oocytes were warmed by exposure to (1) 10% EG and 1 m T in BM, (2) 0.5 m T in BM, and (3) BM for 3 min in each solution at 39°C. Oocytes were then matured, fertilized, and cultured in vitro as previously described. The frequency of cleavage 24 and 48 h post-insemination (pi), development to morula (5 days pi), blastocyst (7 days pi), and total cell numbers of blastocyst-stage embryos were evaluated. Cleavage was significantly lower (P < 0.001) in vitrified and CB-vitrified groups at both 24 h pi (15.2 v. 14.7%) and 48 h pi (27.3 v. 23.5%) than in other groups. Development to morula stage was significantly lower (P < 0.001) in vitrified and CB-vitrified oocytes (12.1 v. 17.7%) than in toxicity, CB-control, and control groups (43.1, 42.6, and 52.8%, respectively); however, no significant difference (P > 0.05) was observed between CB-vitrified and CB-toxicity groups. There was a significant decrease (P < 0.01) in development to blastocyst in CB-vitrified (2.9%) compared with CB-control (22.9%) and control (24.5%), but this did not differ significantly (P > 0.05) from toxicity and CB-toxicity groups (9.8 v. 11.4%). No blastocysts developed from the vitrified group. Hatched blastocysts were observed only in CB-control and control groups (8.2 v. 5.7%). Total cell numbers were significantly greater (P < 0.05) in control blastocysts than in toxicity, CB-vitrified, and CB-control (143 v. 87.25, 73, and 82, respectively). However, this did not differ significantly from the CB-toxicity group (115). These results support our previous data and suggest that pretreatment of GV-stage ovine oocytes with CB prior to vitrification has a positive effect on subsequent development.

286 INFLUENCE OF REVERSIBLE MEIOSIS INHIBITION ON SWINE EMBRYOS PRODUCED BY IN VITRO FERTILIZATION AND PATHENOGENETIC ACTIVATION

2006 ◽  
Vol 18 (2) ◽  
pp. 250
Author(s):  
M. G. Marques ◽  
A. B. Nascimento ◽  
V. P. Oliveira ◽  
A. R. S. Coutinho ◽  
M. E. O. A. Assumpção ◽  
...  
Keyword(s):  
Culture Medium ◽  
Cell Number ◽  
Control Group ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Electric Pulses ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
And Control

The present work evaluated the reversible meiosis inhibition effect on the development of swine embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). The efficiency of PZM3 and NCSU23 embryo culture media was also evaluated. Oocytes from ovaries collected at a slaughterhouse were subjected to IVM in two different groups: CHX (cycloheximide 5 µM for 10 h) and control, both with TCM-199 + 3.05 mM glucose + 0.91 mM sodium pyruvate + 10% porcine follicular fluid (pFF) + 0.57 mM cystein + 10 ng epidermal growth factor (EGF)/mL + 10 IU eCG/mL + 10 IU hCG/mL for the initial 22 h. In the remaining period (20 h for CHX and 22 h for control), medium without hormones was utilized. After IVM, oocytes were denuded and fertilized for 6 h (IFV) or the matured oocytes were submitted to activation by electric pulses (PA) (2 DC of 1.5 kV/cm for 30 µs), incubated for 1 h in culture medium with 10 μM of CHX, and again submitted to the same electric pulses for 60 µs. Embryo development was evaluated by cleavage rate on Day 3 and blastocyst rate and blastocyst cell number on Day 7 of culture. Cleavage and blastocyst rates were analyzed by the equality-of-two-ratios test and cell number by the Kruskal-Wallis and Mann-Whitney tests (P < 0.05). In relation to IVF, the PZM3 medium was more efficient than NCSU23 for cleavage rate in the CHX group (PZM3: 68.4%, NCSU23: 44.4%) and had a better blastocyst rate in the control group (PZM3: 13.4%, NCSU23: 5.6%). With reference to PA, NCSU23 presented better cleavage and blastocyst rates than PZM3 in the CHX group (NCSU23: 89.5%, PZM3: 78.5% and NCSU23: 20.4%, PZM3: 13.0%, respectively). In the control group, only the NCSU23 blastocyst rate was higher than that for PZM3 (NCSU23: 22.5%, PZM3: 10.8%). No culture medium effect on cell number mean of IVF and PA blastocysts was observed. Maturation block improved cleavage rates in IVF groups cultured with PZM3 (68.4% and 50.6%, respectively, for CHX and control) and in PA groups cultured with NCSU23 (89.5% and 80.3%, respectively, for CHX and control), but no improvement of blastocyst rates in both groups (IVF and PA) was verified. Table 1 below shows that maturation block decreased the IVF and increased the PA blastocyst cell numbers. As older oocytes are more effectively activated, oocytes blocked with CHX achieved the maturation stage faster than the control group, therefore resulting in high-quality PA blastocysts. In conclusion, PZM3 was more efficient for IVF embryo production in contrast to NCSU23, whereas NCSU23 can be indicated for PA embryo production. Moreover, maturation blockage with CHX influenced blastocyst cell number, decreasing in IVF embryos and increasing in PA embryos. Table 1. Mean (±SD) of blastocyst cell numbers for IVF or PA groups after in vitro maturation without (control) or with cycloheximide (CHX) and cultured in NCSU23 or PZM3 medium This work was supported by FAPESP 02/10747–1.

93 PRODUCTION OF LIVE PIGLETS BY CRYOPRESERVATION OF IN VITRO-PRODUCED PORCINE ZYGOTES

2008 ◽  
Vol 20 (1) ◽  
pp. 127
Author(s):  
T. Somfai ◽  
N. Kashiwazaki ◽  
M. Ozawa ◽  
J. Noguchi ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Culture Medium ◽  
Aluminum Foil ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
High Rate ◽  
Cell Numbers ◽  
Embryo Culture Medium ◽  
Vitrification Solution ◽  
And Control

Successful cryopreservation of in vitro-produced porcine zygotes is reported in the present study. Follicular oocytes were collected from prepubertal gilts. They were matured (IVM), fertilized (IVF), and cultured (IVC) in vitro (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Ten or 23 h after IVF, the oocytes were centrifuged at 10 000g at 37�C for 20 min to permit visualization of pronuclei. Zygotes with two or three pronuclei were selected under stereomicroscope and used for solid surface vitrification (SSV). Briefly, after equilibration in 4% ethylene glycol (EG) for 15 min, zygotes were washed in vitrification solution (35% EG, 5% polyvinyl pyrrolidone, and 0.3 m trehalose), and then dropped with about 2 µL vitrification solution onto the dry surface of aluminum foil floating on the surface of liquid nitrogen (LN2). Microdroplets were transferred into cryotubes and stored in LN2. During warming, vitrified droplets were transferred in warming solution (0.4 m trehalose) at 37�C for 1 min, and then consecutively transferred for 1-min periods into 0.2 m, 0.1 m, or 0.05 m trehalose solutions. Survival of vitrified/warmed zygotes was determined by their morphology. To assess their developmental competence, vitrified (SSV), cryoprotectant-treated (CT), and untreated (control) zygotes were cultured in vitro for 6 days. There was no difference in developmental competence between control and CT zygotes in terms of cleavage rates (88.1% and 86.1%, respectively), blastocyst rates (23.2% and 20.8%, respectively), and blastocyst cell numbers (38.0 � 2.0 and 41.2 � 1.7, respectively). The rate of live zygotes after SSV and warming was similar to that of the control (93.4% and 100%, respectively). Cleavage rates (71.7% and 86.3%, respectively) and blastocyst rates (15.8% and 24.5%, respectively) of SSV were significantly reduced after vitrification compared to control (P < 0.05, ANOVA). Blastocyst cell numbers of SSV and control embryos were similar (41.2 � 3.4 and 41.6 � 3.3, respectively). There was no difference in developmental ability between zygotes cryopreserved at an early (10 h after IVF) or late (23 h after IVF) pronuclear stage. When embryo culture medium was supplemented with 1 µm of the antioxidant glutathione, development of cryopreserved zygotes to the blastocyst stage did not differ significantly from that of the control zygotes (18.6% and 22.1%, respectively). To test their ability to develop to term, 150 vitrified zygotes were transferred into a recipient, resulting in pregnancy and the production of five live piglets. These data demonstrate that a high rate of porcine zygotes could be successfully cryopreserved at the pronuclear stage, preserving their full developmental competence.

140 EFFECT OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 217
Author(s):  
R. F. Gonçalves ◽  
C. Figueiredo ◽  
M. A. Achilles
Keyword(s):  
Culture Medium ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Total Cell ◽  
Cell Numbers ◽  
Group 2 ◽  
Group 1 ◽  
Hatching Rates

There are still immense differences in the quality of in vitro-produced embryos compared to their in vivo-generated counterparts. These differences include a higher sensitivity of in vitro-produced embryos towards cryopreservation. The quality of such embryos has been evaluated using various parameters like morphological examination, assessment of total cell numbers, or pregnancy rates after transfer. In the present study, the effects of glycine, alanine, taurine, and glutamine addition to SOF (Achilles Genetics culture medium, Achilles Genetics®, Garça, SP, Brazil) on the in vitro development (cleavage and blastocyst rates) and quality (total cell and apoptotic cell numbers) of bovine embryos were determined. Ovaries of Nelore cows were obtained from a slaughterhouse. Cumulus–oocyte complexes (COC) were collected from follicles ≥4 mm in diameter, matured in TCM-199, and fertilized with frozen–thawed Nelore bull semen (IVF = Day 0). On Day 1, presumptive zygotes were cultured in SOF supplemented with fetal bovine serum (FBS, group 1, n = 550) or in Achilles Genetics culture medium (SOF supplemented with Achilles Mixture and FBS, group 2, n = 557) at 38.5°C and 5% CO2 in air until Day 9. Embryos were evaluated during culture: at Day 3 cleavage rates, at Day 7 blastocyst rates, and on Day 9 hatching rates. Experiments were replicated 5 times, analysed using ANOVA, followed by a comparison of means by Tukey test (P ≤ 0.05). Blastocysts at Day 8 from Group 1 (n = 75) and Group 2 (n = 75) were fixed and permeabilized for TUNEL assay (DeadEndTM Florimetric TUNEL System, Promega, Madison, WI, USA), according to the manufacturer instructions. Total cell number, apoptotic cell number, and apoptotic cell index (calculated by dividing the apoptotic cell number by total cell number) were analyzed by analysis of variance and means were compared by Student Newman Keuls test. The threshold of significance was set at P ≤ 0.05. Cleavage rates were 79.2 ± 2.5 for group 1 and 91.0 ± 2.5 for group 2. Blastocyst and hatching rates (calculated on the total of zygotes) for group 2 (47.4 ± 2.8; 82.1 ± 1.5) were significantly greater than for group 1 (39.8 ± 2.8; 74.3 ± 1.5). The total cell numbers were not different (P > 0.05) between group 1 (112.7 ± 2.9) and group 2 (111.1 ± 2.7). Blastocysts from group 2 showed lower (P < 0.05) number of apoptotic cells (10.7 ± 1.2) than those from group 1 (20.9 ± 1.2). These results indicate that the addition of glycine, alanine, taurine, and glutamine to SOF (Achilles Mixture) may be an important energy source for the bovine blastocyst and could act synergistically to enhance embryo development to the hatching stage and embryo quality. Financial support from CNPq and FAPESP.

139 BOVINE PREIMPLANTATION EMBRYOS SECRETE EXTRACELLULAR VESICLES, WHICH MAY INDICATE EMBRYO COMPETENCE

2017 ◽  
Vol 29 (1) ◽  
pp. 178
Author(s):  
E. Mellisho ◽  
A. Velasquez ◽  
M. J. Nuñez ◽  
L. Rodriguez-Alvarez
Keyword(s):  
Extracellular Vesicles ◽  
Culture Media ◽  
Total Cell ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Lower Amount ◽  
Bovine Embryos ◽  
Significant Difference ◽  
Blastulation Rate

Pre-implantation embryos secrete extracellular vesicles (EV) most likely to communicate with the surroundings. The objective of this study was to determine the distribution (size and concentration) of EV secreted by bovine pre-implantation embryos with different developmental competence. The IVF bovine embryos were produced from oocytes recovered from slaughterhouse ovaries. Presumptive zygotes were in vitro cultured (IVC) in groups in 4-well plates (30 zygotes per 500-µL well) using SOFaa medium at 39°C under 5% CO2, 5% O2, and 90% N2 until the morula stage (Day 5 post IVF). Morulae were cultured individually in 96 well at 39°C under until blastulation time (Day 6.5–7.5) in EV-free SOF medium. Culture medium was collected only from embryos that developed to the blastocyst stage that were classified in a group of early (Day 6.5) or late (Day 7.5) blastulation. Blastocysts were kept in culture until Day 11 to assess embryo developmental competence, considering embryo size (>350 µm) and total cell count (>500 blastomeres). For EV analysis, 4 groups were organised a posteriori: G1: Day 6.5-competent; G2: Day 6.5-not competent; G3: Day 7.5-competent; G4: Day 7.5-not competent. The EV in culture media were analysed using a nanoparticle tracking analysis (Nanosight NS300). Statistical analysis was performed using the InfoStat program (Buenos Aires, Argentina). Differences were considered significant at P < 0.05. Early blastulation rate (Day 6.5) was 40.3% (112/278), whereas late blastulation rate (Day 7.5) was 20.5% (57/278), showing a significant difference (P < 0.0001). Embryos derived from Day 6.5 blastocysts have a higher probability (39.3%: 44/112) of posthatching development [until Day 11; Day 7.5, 10.5% (6/57); P = 0.0001]. At Day 11, competent embryos (G1) derived from Day 6.5 blastocysts have a higher diameter and total cell number (447 µm; 688 cells) than those derived from Day 7.5 blastocysts (G3; 405 µm, 598 cells; P < 0.05 for both parameters). It was possible to detect EV from collected medium of individual embryos independent of their competence. Neither the EV size nor the EV concentration was statistically different between Day 6.5 and Day 7.5 blastocysts (without considering their further competence; 2.9 × 108, 147 nm; and 3.0 × 108, 149 nm, respectively). However, independent of the day of blastulation, competent embryos had a significantly lower concentration of EV (2.7 × 108 v. 3.3 × 108; P = 0.03). Moreover, competent embryos from early and late blastocysts (G1 and G3) tend to produce a lower amount of EV (G1: 2.8 × 108; G2: 3 × 108; G3: 2.6 × 108; G4: 3.5 × 108; P = 0.05). Furthermore, EV concentration was statistically different between G3 and G4 (P = 0.002). No differences in EV size were observed among groups (G1: 145 nm; G2: 148 nm; G3: 146 nm; G4: 151 nm). Our results provide an initial approach to study the EV secreted by individual pre-implantation embryos to assess their competence. From these results, we can conclude that blastulation time affects the future development of bovine embryos and a model based on blastulation time and EV secretion could be a simple noninvasive tool to improve embryo selection.

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

2010 ◽  
Vol 58 (4) ◽  
pp. 465-474 ◽  
Author(s):  
Tamás Somfai ◽  
Yasushi Inaba ◽  
Yoshio Aikawa ◽  
Masaki Ohtake ◽  
Shuji Kobayashi ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Culture Systems ◽  
Significant Difference

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O 2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O 2 compared to 5% O 2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O 2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O 2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O 2 tension, whereas IVD101 supported blastocyst formation only under low O 2 levels but enhanced the proliferation of ICM cells.

132 EFFECTS OF OXYGEN TENSION, MEDIUM, AND WELL OF THE WELL ON IN VITRO DEVELOPMENT OF THE MOUSE EMBRYO

2011 ◽  
Vol 23 (1) ◽  
pp. 170
Author(s):  
Y. S. Li ◽  
Z. B. Cao ◽  
Y. Liu ◽  
H. G. Cao ◽  
Y. Tao ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Culture System ◽  
Total Cell ◽  
Mouse Embryos ◽  
In Vitro Development ◽  
Cell Numbers ◽  
Gas Phases ◽  
Blastocyst Rate ◽  
Vitro Development

The objectives of the present research were to investigate whether embryo culture media have preferences for oxygen tension, to explore the feasibility of using physical lung air to support the in vitro development of mouse embryos, and to evaluate the effect of well of the well (WOW) culture on in vitro preimplantational development of mouse embryos. The results are as follows. First, cleavage rate and blastocyst rate were not significantly different between medium CZB and mKSOM regardless of using 3 gas phases: 4% CO2 + 16% O2 + 78% N2 + 2% H2O (lung air), 5% CO2 + 5% O2 + 90% N2 (5% O2, low oxygen), and 5% CO2 + 95% air (20% O2, high oxygen; P > 0.05), but mean total cell numbers per blastocyst cultured in CZB medium were higher than those in mKSOM when the lung air was used (P < 0.05). Second, when mKSOM was used as the basic medium, the blastocyst rate (22.6%) in the 5% O2 gas phase was notably higher than that in other 2 gas phases (P < 0.05). Third, for the CZB medium, the blastocyst rate was not different significantly among 3 gas environments (P > 0.05). Fourth, both the blastocyst rate (74.6 ± 5.1%) and the mean total cell numbers per blastocyst (76 ± 2) cultured in the WOW system were obviously higher than those in the group culture system (38.2 ± 6.6% and 58 ± 4). Taken together, our results indicate that mKSOM and CZB have preferences for oxygen tension during in vitro culture of mouse embryos, the lung air was reaffirmed to be able to effectively support in vitro preimplantation development of mouse embryos, and the WOW culture system can apparently enhance in vitro developmental competence and blastocyst quality of mouse embryos. L. YS, C. ZB: equal contribution; supported by NSFC (30700574), 863 (2008AA101003).

Glycine and methionine transport by bovine embryos

Zygote ◽  
1997 ◽  
Vol 5 (3) ◽  
pp. 273-276 ◽  
Author(s):  
Catherine Guyader-Joly ◽  
Chaqué Khatchadourian ◽  
Yves Ménézo
Keyword(s):  
Female Genital Tract ◽  
Bovine Embryos ◽  
Mouse Blastocyst ◽  
Cell Numbers ◽  
Significant Difference ◽  
Methionine Uptake ◽  
Methionine Transport ◽  
Female Genital

SummaryAs glycine is one of the most concentrated amino acids in the female genital tract, we investigated its uptake by bovine in vitro matured/in vitro fertilised blastocysts in the presence of increasing concentrations of radiolabelled glycine. We also determined methionine uptake by in vitro and in vivo produced embryos. In our study, the hypothesis of more than one site of enzyme activity for glycine substrate was not validated. We determined a Vmax of 23.4fmol/min per embryo and a Km value of 13.3μM. No significant difference was observed either between in vivo and in vitro derived embryos or between grade 1 and grade 2 embryos for methionine uptake. The methionine and glycine uptake of a day 7 bovine was similar to that of a day 4 mouse blastocyst. This is rather low if we consider the relative cell numbers.

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