96 EFFICIENT PRODUCTION OF MONOZYGOTIC TWIN BOVINE EMBRYOS USING BLASTOMERE SEPARATION TECHNIQUE WITH COMMERCIAL WELL OF THE WELL CULTURE DISH

2016 ◽  
Vol 28 (2) ◽  
pp. 178
Author(s):  
Y. Hashiyada ◽  
Y. Aikawa ◽  
S. Sugimura ◽  
H. Matsuda ◽  
M. Ohtake ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Statistical Significance ◽  
Cell Mass ◽  
Calf Serum ◽  
Monozygotic Twin ◽  
Efficient Production ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Culture Dish ◽  
Cell Stage

Monozygotic twin embryos can be produced using the technique of blastomere separation and well of the well (WOW) dish having handmade micro-wells by the needle depression (Tagawa et al. 2008). We have recently reported that developed commercial WOW dish enhances embryo competence in individual culture (Sugimura et al. 2010). The present study was conducted to evaluate the availability of commercial WOW dish for production of monozygotic twin embryos in bovine. Embryos were produced using oocytes from ovaries collected at an abattoir by IVM, IVF, and IVC. For each culture, TCM-199 supplemented with 5% calf serum (CS), Brackett-Oliphant solution supplemented with 10 mg mL–1 BSA, and CR1aa containing 5% CS were used. To evaluate the adaptability of dishes on culture of isolated blastomeres from different cell stage, 2- (n = 63), 4- (n = 94), 8- (n = 137), and 10- to 14- (n = 116) cell stages were obtained on 24–27 h, 30–36 h, 48–54 h, and 48–54 h from the beginning of fertilization, respectively. The zona pellucida was removed by exposure of 0.25% pronase, followed by gentle pipetting by inspiration and expiration in the IVC medium. Then, two halves separated from the original number of blastomeres were randomly allocated to the conical micro-wells of commercial dish (Dai Nippon Printing, Tokyo, Japan) or created micro-wells by pressing the bottom of the dish with an eyeleteer (control). The approximate diameter and depth of each 25 wells in a commercial dish was 287 and 168 μm, and each 20 wells in the control were 800 and 600 μm. The blastomeres were cultured in wells covered with a droplet of 2.5 μL well–1 IVC medium until Day 8 (IVF = Day 0). Expanded blastocysts (n = 28) derived from tetra-blastomeres of 8-cell stage were stained to determine the number of the inner cell mass (ICM) and trophectoderm (TE) in each group. Statistical significance of the blastocyst formation rates and the number of cells were analysed by the chi-square test and the Student’s t-test, respectively. In the 2-cell stage, blastocyst formation rate in commercial dish tended to be higher than that in the control (60.0% v. 46.1%). The rate of monozygotic blastocyst pairs in commercial dish was higher compared with the control (48.0% v. 26.3%, P < 0.05). In the 4-cell stage, rates of blastocyst formation (50.0% v. 33.0%, P < 0.05) and the pairs (39.5% v. 12.5%, P < 0.01) in the commercial dish, both were higher compared with the control. In the 8-cell stage, there were no differences between two groups in rates of blastocyst formation (53.1% v. 59.0%) and the pairs (41.8% v. 48.7%), similarly in the 10- to 14-cell stage (47.9% v. 56.8% and 36.2% v. 40.9%, respectively). The ICM, TE, and total cell numbers were not different between the commercial and the control dish (28.0 ± 3.2 v. 26.0 ± 3.8, 64.6 ± 4.3 v. 76.0 ± 7.9, and 92.6 ± 6.2 v. 102.0 ± 11.0, respectively). These results indicate that separated blastomeres could be developed to blastocysts efficiently and stably regardless of embryo cell stage with a commercial WOW culture dish.

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.

scholarly journals Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation

2021 ◽  
Vol 8 ◽  
Author(s):  
Yasumitsu Masuda ◽  
Ryo Hasebe ◽  
Yasushi Kuromi ◽  
Masayoshi Kobayashi ◽  
Kanako Urataki ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Three Dimensional ◽  
Preimplantation Embryos ◽  
Infrared Light ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Potential

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.

77 Development and quality of in vitro bovine hemi embryos produced by blastomere separation and embryo bisection

2019 ◽  
Vol 31 (1) ◽  
pp. 164
Author(s):  
A. E. Ynsaurralde Rivolta ◽  
M. Suvá ◽  
V. Alberio ◽  
C. Vazquez Echegaray ◽  
A. Guberman ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Monozygotic Twin ◽  
Cell Number ◽  
Control Group ◽  
Cell Stage ◽  
Exact Test ◽  
Development Rates

Bovine monozygotic production of twins became popular in the 1980s as a technique to multiply high value genetics. Moreover, it also became a powerful model for research. Different techniques have been used on bovine embryos obtained by superovulation. In this work, we compared the development rates and quality of monozygotic twin embryos produced by blastomere separation (BS) and embryo bisection (EB) of IVF embryos. To this aim, cumulus-oocytes complexes collected from slaughterhouse ovaries were in vitro matured in TCM 199 containing 10% fetal bovine serum, 10µg mL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine, and 2% antibiotic-antimycotic for 24h, at 6.5% CO2 in humidified air and 38.5°C. The IVF was performed with 16×106 spermatozoa per mL for 5h. Afterward, presumptive zygotes were cultured in SOF medium for 7 days at 38.5°C and 5% O2. After 24h of culture, blastomeres of 2-cell stage embryos (N=114) were separated and each one was cultured individually in a microwell for 7 days. Embryo bisection (N=179) was performed manually on Day-7 blastocysts previously depleted of their zonae pellucidae, under stereoscopic microscope. Hemi embryos were cultured for 24h and then twins and single blastocyst rates were calculated. For quality assessment, diameter, total and inner cell mass (ICM) cell number of hemi embryos (BS: 6 couples; ES: 10 couples) and the control group (C: 11) were evaluated. The ICM cell number was measured by immunofluorescence staining using SOX2 antibody and the percentage of ICM and trophectoderm (TE) cells was calculated. The results were analysed using Fisher’s exact test and ANOVA with mean comparison using Tukey’s test (P=0.05). No statistical differences were found in blastocyst rates of twins and single hemi embryos produced by BS (28 and 25%) or EB (23 and 32%). Blastocyst diameter was similar between groups and control. Hemi embryos exhibited lower total and ICM cell number than control (BS: 43±18, EB: 57±14v. C: 93±35 and BS: 16±7, EB: 12±8v. C: 34±19). However, BS hemi embryos had higher ICM and lower TE percentage (40/60%) compared with the EB group (20/80%). The control group did not differ with hemi embryo treatments for ICM and TE (30/70%). Our preliminary results have indicated that although the development rates of hemi embryos produced in vitro were similar between both techniques, blastomere separation generates better quality embryos than blastocyst bisection.

28 Short equilibration improves vitrification of invitro-produced bovine embryos using VitTrans device

2020 ◽  
Vol 32 (2) ◽  
pp. 140
Author(s):  
I. Martínez-Rodero ◽  
T. García-Martínez ◽  
M. López-Béjar ◽  
T. Mogas
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Field Conditions ◽  
Bovine Embryos ◽  
Fluid Medium ◽  
Apoptosis Index ◽  
Vitrification Solution

For the successful application of vitrification technology to field conditions, the procedures for the warming and transfer of the cryopreserved bovine embryos should be as simple as possible. The device VitTrans, designed by our group, enables warming/dilution of embryos and their transfer directly to recipient females in field conditions (Morato and Mogas 2014 Cryobiology 68, 288). VitTrans vitrification protocol consists of an incubation in equilibration solution during 12min followed by an exposure of 40s to vitrification solution. However, there are other reports using similar vitrification devices where equilibration length is shorter than ours. This study aimed to improve VitTrans methodology by comparing two vitrification protocols: short equilibration (SE) and long equilibration (LE). A total of 63 invitro-produced Day 7 blastocysts (IETS stage code 7) were randomly placed in an equilibration solution with 7.5% ethylene glycol + 7.5% dimethyl sulfoxide in holding medium (tissue culture medium-199 HEPES + 20% fetal calf serum) for either 3min (SE) or 12min (LE). Then, blastocysts were transferred to vitrification solution (15% ethylene glycol + 15% dimethyl sulfoxide + 0.5M sucrose in holding medium) for 40s, loaded onto the VitTrans device, plunged into liquid nitrogen, and covered with a 0.5mL straw. Fresh nonvitrified blastocysts (n=30) were set as control. For warming, the VitTrans was quickly submerged into a water bath at 45°C, while a syringe containing 0.3mL of diluting solution (0.5M sucrose in holding medium) at 45°C was injected through the hollow of the device. Blastocysts were then transferred to synthetic oviductal fluid medium and cultured for 24h at 38.5% in a 5% CO2 and 5% O2 environment in a humidified atmosphere. Re-expansion rates were recorded 3 and 24h after warming. Blastocysts were fixed and stained with SOX2 (Invitrogen) for inner cell mass (ICM) count, TUNEL (Roche) for apoptosis index assessment, and DAPI (Vector Laboratories) for total cell count (TCC). Images were captured using a Leica TCS SP5 confocal microscope (Leica Microsystems) and examined with Imaris 9.2 software (Oxford Instruments). Blastocyst survival rates were compared between groups using chi-squared test. Blastocyst TCC, ICM count, and apoptosis indices were analysed using analysis of variance. Significance was set at P ≤ 0.05. No differences were observed in re-expansion rate at 3h postwarming (61.3 and 59.4% for SE and LE, respectively). However, significantly higher re-expansion rates were found after 24h of culture for the blastocysts of the SE group (74.2%) when compared with the blastocysts of the LE group (65.7%). Blastocysts vitrified using the LE protocol produced the lowest TCC (115±5.9; P ≤ 0.05), whereas TCC of the SE (152±9.7) and fresh control (138±8.6) treatments were similar. No differences were found in ICM count among groups. Nevertheless, apoptosis index was higher (P ≤ 0.05) in both vitrification groups when compared with fresh control. These results indicate that short equilibration vitrification not only improves VitTrans outcomes but adds efficiency by taking less time to perform. Supported by MCIU, Spain (Project AGL2016-79802-P and Grant BES-2017-081962).

95 ROLE OF POLYAMINES IN BOVINE PRE-IMPLANTATION DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 177
Author(s):  
J. Herrick ◽  
A. Greene ◽  
W. Schoolcraft ◽  
R. Krisher
Keyword(s):  
Amino Acids ◽  
Inner Cell Mass ◽  
Gradient Centrifugation ◽  
Cell Mass ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Human Albumin ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Formation

Polyamines are involved in trophectoderm attachment and outgrowth, but little is known about their role in earlier stages of development. The objective of this study was to evaluate the effects of an inhibitor of polyamine synthesis (difluoromethylornithine, DFMO) on development (blastocyst formation and hatching) and cell allocation to the trophectoderm (TE, CDX2-positive) and inner cell mass (ICM, SOX2-positive) in the bovine embryo. Cumulus-oocyte complexes (COCs) were recovered from slaughterhouse ovaries and matured for 24 h in a defined maturation medium (5.0 mM glucose, 0.6 mM cysteine, 0.5 mM cysteamine, 0.1 IU mL–1 FSH, 50 ng mL–1 EGF, and 2.5 mg mL–1 recombinant human albumin). Frozen-thawed spermatozoa were processed by gradient centrifugation and co-incubated (2 × 106 mL–1) with COC [10 COC/50 µL; 7.5 µg mL–1 heparin, 2 mM caffeine, and 8.0 mg mL–1 fatty-acid free (FAF) BSA] for 20 to 22 h. After removing cumulus cells, zygotes were cultured (10 embryos/20 µL) in a medium for cleavage stage bovine embryos (0.5 mM glucose, 0.3 mM pyruvate, 6.0 mM lactate, 0.25 mM citrate, 1.0 mM alanyl-glutamine, 0.25 × MEM nonessential and essential amino acids, 5 µM EDTA, and 8.0 mg mL–1 FAF BSA). After 72 h, embryos with >4 cells were randomly allocated (5 embryos/20 µL) to a culture medium for compaction and blastocyst formation (3.0 mM fructose, 0.1 mM pyruvate, 6.0 mM lactate, 0.5 mM citrate, 1.0 mM alanyl-glutamine, 1× MEM nonessential amino acids, 0.5× MEM essential amino acids, 0.075 mM myo-inositol, and 8.0 mg mL–1 FAF BSA) containing 0 (control), 5, or 10 mM DFMO. Embryonic development was evaluated at 192 h post-insemination (96 h in the second medium containing DFMO treatments), and hatching or hatched blastocysts were fixed for analysis of cell allocation. All data were analysed by ANOVA and P < 0.05 was considered significant. Blastocyst formation and hatching (% of embryos cultured in the presence of treatments) were both inhibited (P < 0.05) when embryos (n = 157/treatment) were cultured with 5 (39.5 ± 3.9%, 14.6 ± 2.8%) or 10 (39.5 ± 3.9%, 14.0 ± 2.8%) mM DFMO compared with embryos cultured without DFMO (53.5 ± 4.0%, 26.1 ± 3.5%). The number of TE cells was also reduced (P < 0.05) in the presence of 5 (121.4 ± 7.2) and 10 (123.6 ± 6.7) mM DFMO compared with embryos cultured without DFMO (152.4 ± 9.7), but the number of ICM cells (45.2 to 54.0) and the total number of cells (TE+ICM, 168.8 to 201.1) were not affected (P > 0.05). In a second experiment (n = 163 to 165/treatment), the negative effects of DFMO on hatching (17.0 ± 2.9%; P < 0.05, v. control, 30.7 ± 3.6%) could be partially reversed when embryos were cultured with both 10 mM DFMO and an exogenous polyamine (100 µM putrescine, 23.0 ± 3.3% DFMO+Put; P > 0.05 v. control). The number of TE cells for embryos cultured with DFMO+Put (153.9 ± 8.7) was intermediate between embryos cultured with (138.0 ± 6.9) or without DFMO (control, 161.6 ± 8.7), but these differences were not significant (P > 0.05). These results provide the first evidence of a role for polyamines during blastocyst formation and hatching of bovine embryos, with specific effects on trophectoderm proliferation and hatching.

179 RELATIONSHIP BETWEEN GENE EXPRESSION IN INDIVIDUAL BLASTOMERES OF 2-CELL STAGE BOVINE EMBRYOS AND THE NORMALITY OF FIRST CLEAVAGE

2016 ◽  
Vol 28 (2) ◽  
pp. 220
Author(s):  
S. Matoba ◽  
M. Kaneda ◽  
T. Somfai ◽  
T. Nagai ◽  
M. Geshi
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Calf Serum ◽  
Transcript Abundance ◽  
Pcr Primers ◽  
Developmental Competence ◽  
Cleavage Pattern ◽  
Bovine Embryos ◽  
Culture Dish ◽  
Cell Stage

Previously early first and second cleavages after IVF associated with even blastomeres without fragments or protrusions were found to be a potent criterion for the selection of embryos with high development competence (Sugimura et al. 2012 PLOS One 7, e36627). The aim of this study was to examine the relationship between an early normal first cleavage pattern and the transcript abundance in each blastomere in 2-cell stage bovine embryos. IVF-derived bovine embryos were cultured individually in microwells culture dish in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL–1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2. First cleavage and cleavage patterns were categorised as being either normal (the first cleavage within 28 h after IVF with 2 even blastomeres without fragment or protrusion) or abnormal (2 uneven blastomeres, with/without fragment/protrusion and/or later than 28 h after IVF at the first cleavage). Next, cleaved embryos were placed in 0.5% actinase-E in Ca- and Mg-free PBS and blastomeres were separated by pipetting. Individual blastomeres (n = 71, 10 replicates) were analysed for gene expression by quantitative RT-PCR. Primers were designed for 19 target genes related to pluripotency, cell cycle, metabolism, pregnancy reorganization, placentation and fetal growth (NANOG, OCT4, PLAC8, ATP1A1, CCNB1801, CDH1, COX1, CTNNB1, G6PDH, Glut8, MNSOD-3end, SOX2, DYNLL1, IGF1R, IGF2, IGF2R, IGFBP2, IGFBP3, and PMSB1) and a reference gene (PPIA). Transcript abundance of target genes in both of individual blastomeres of cleaved embryos was examined in embryos that cleaved early with a normal cleavage pattern and in those that showed abnormal cleavage pattern. Values were normalised to the average values of the reference genes and means were compared by the student t-test. Transcript abundance of OCT4, ATP1A1, CCNB1801, CDH1, COX1, CTNNB1, MNSOD-3end, IGF2R, and IGFBP2 was significantly higher in blastomeres associated with all categorised abnormal blastomeres compared towith an early normal cleavage (P < 0.05). Furthermore, the expression of PLAC8, IGF1R, and PMSB1 in embryos having 2 uneven blastomeres, Glut8 and SOX2 in 2 uneven blastomeres with fragment/protrusion was higher than that in normal cleavage (P < 0.05). However, the level of G6PDH was lower in embryos having 2 uneven blastomeres than that in those showing normal cleavage (P < 0.05). Our results reveal blastomere gene expression in bovine embryos at the first cleavage may correlated with oocyte developmental competence. This study was supported by JSPS KAKENHI (26450388).

Analysis of Cyclin E1 Functions in Porcine Preimplantation Embryonic Development by Fluorescence Microscopy

2017 ◽  
Vol 23 (1) ◽  
pp. 69-76
Author(s):  
Jing Guo ◽  
Kyung-Tae Shin ◽  
Xiang-Shun Cui
Keyword(s):  
Cell Cycle ◽  
Embryonic Development ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Formation ◽  
Cellular Functions ◽  
Cell Stage ◽  
Double Stranded Rna ◽  
Cyclin E1 ◽  
Cycle Regulation

AbstractCyclin E1 (CCNE1) is a core component of cell cycle regulation that drives the transition into the S phase. CCNE1 plays critical roles in cell cycle, cell proliferation, and cellular functions. However, the function of CCNE1 in early embryonic development is limited. In the present study, the function and expression of Ccne1 in porcine early parthenotes were examined. Immunostaining experiments showed that CCNE1 localized in the nucleus, starting at the four-cell stage. Knockdown of Ccne1 by double-stranded RNA resulted in the failure of blastocyst formation and induced blastocyst apoptosis. Ccne1 depletion increased expression of the pro-apoptotic gene Bax, and decreased the expression of Oct4 and the rate of inner cell mass (ICM)/trophectoderm formation. The results indicated that CCNE1 affects blastocyst formation by inducing cell apoptosis and ICM formation during porcine embryonic development.

75 Culture of isolated blastomeres supplemented with L-ascorbic acid 2-phosphate in a well-of-the-well culture dish

2019 ◽  
Vol 31 (1) ◽  
pp. 163
Author(s):  
Y. Hasiyada ◽  
H. Matsuda ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
T. Yamanouchi
Keyword(s):  
Formation Rate ◽  
Calf Serum ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Culture Dish ◽  
Cell Stage ◽  
Quality Grade

We have reported monozygotic twin calves that can be produced efficiently by blastomere separation of 2-cell stage embryos and by the use of a commercially provided well-of-the-well culture dish (Hashiyada 2017 J. Reprod. Dev.). The present study was conducted to evaluate the effect of a culture supplement, l-ascorbic acid 2-phosphate (AA-2P), a sustained antioxidant substance that reduces reactive oxygen species. Embryos were produced using oocytes from ovaries collected at an abattoir by in vitro maturation, IVF, and in vitro culture (IVC). TCM199 supplemented with 5% calf serum, Brackett-Oliphant solution supplemented with 10mg mL−1 BSA, and CR1aa containing 5% calf serum were used for each culture step. Two-cell stage embryos were obtained 24 to 27h post-insemination (hpi). Zonae pellucidae were removed by exposure to 0.25% pronase. Then, embryos were separated into each blastomere by gentle pipetting in IVC medium. Each blastomere was introduced into a single conical micro-well of 25 wells, each having a diameter and depth of ~287 and 168µm (Dai Nippon Printing, Tokyo, Japan). Culture of blastomeres in wells was performed covered with a droplet of 2.5 µL/well IVC medium supplemented with 0 (n=212), 250 (n=214), 500 (n=206), and 750 µM (n=204) of AA-2P. The blastocyst formation rate at Day 8 after IVF, the quality of blastocysts assessed by morphological observation, and the cell numbers were compared among each concentration of AA-2P. In addition, the developmental speed to the blastocyst stage was analysed using time-lapse cinematography for 0 and 500 µM of AA-2P (n=40, respectively). Statistical analysis was performed using Fisher’s exact test and ANOVA. The blastocyst formation rate (32-40%), the total cell number (108-114), and inner cell mass cell number (26-28) did not differ among groups. The time to reach the 4-cell stage was significantly shorter in media supplemented with 0 µM (43 hpi) than with 500 µM (52 hpi); however, the time to reach the blastocyst stage did not differ (150 and 155 hpi, respectively). Regarding the proportion of quality grade 1 to 3 blastocysts and the developmental speed to the blastocyst stage, high-quality grade 1 embryos were significantly faster than those of middle and low-quality grade 2 and 3 ones in 0 (145 v. 154 hpi; P&lt;0.05) and 500 µM (150v. 158 hpi; P&lt;0.05) supplemented medium. In this experiment, no effect of AA-2P was observed for the culture of isolated blastomeres from 2-cell stage embryos, although it was suggested that blastomeres with high developmental competence reach the blastocyst stage faster, which might reflect the quality of the embryos.

Development in vitro of mouse embryos from the two-cell egg stage to the early somite stage

Development ◽  
1974 ◽  
Vol 31 (1) ◽  
pp. 235-245
Author(s):  
Yu-Chih Hsu ◽  
John Baskar ◽  
Leroy C. Stevens ◽  
John E. Rash
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Cell Stage ◽  
Cord Serum ◽  
Somite Stage ◽  
Development In Vitro

About 1–3% of mouse blastocysts, which had initially been cultured from the two-cell stage in chemically defined medium or about 3–5% of blastocysts which were explanted from the uterus, developed to the early somite stage when cultured in vitro on collagen. Two-cell eggs were initially cultivated in chemically defined medium to the blastocyst stage. Blastocysts were then transferred to Eagle's minimal essential medium (MEM) plus 10% heat-inactivated calf serum. Two barriers to further development were overcome. First, the formation of endoderm and ectoderm from the inner cell mass immediately after attachment to collagen. Second, formation of the embryo proper from the embryonic region. Both barriers were overcome by using heat-inactivated human cord serum after the blastocysts hatched from the zona pellucida and attached to collagen. After attachment, embryos were cultured in MEM plus 20% heat-inactivated human cord serum which was changed daily until early somite stages. Apparently normal healthy development in vitro occurred, as judged by light and electron microscopic examination.

82 HOW LOW CAN YOU GO? DEFINING THE MINIMAL NUTRIENT REQUIREMENTS FOR BOVINE EMBRYOS IN CULTURE

2017 ◽  
Vol 29 (1) ◽  
pp. 148
Author(s):  
J. R. Herrick ◽  
A. F. Greene ◽  
J. Becker ◽  
W. B. Schoolcraft ◽  
R. L. Krisher
Keyword(s):  
Fatty Acid ◽  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Nutrient Concentrations ◽  
Acid Oxidation ◽  
Control Medium ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Blastocyst Development

Recent metabolomic studies in our laboratory have indicated that bovine embryos only use a small amount of the nutrients provided to them in culture media. Our objective was to determine the effects of reducing nutrient concentrations in our media to 75, 50, and 25% (experiment 1) or 25, 12.5, and 6.25% (experiment 2) of those in our control medium (100%) on the development of in vitro-matured/IVF bovine embryos in a serum-free medium. Cumulus–oocyte complexes were matured in a defined maturation medium (0.1 IU mL−1 recombinant human FSH, 50 ng mL−1 recombinant mouse epidermal growth factor, and 2.5 mg mL−1 recombinant human serum albumin) and co-incubated with frozen–thawed spermatozoa (2 × 106 mL−1, 20 h). Embryos were cultured (7.5% CO2, 6.5% O2, 38.7°C) in a sequential media system (0–72 h and 72–168 h). Concentrations of salts, bicarbonate, and protein (2.5 mg mL−1 fatty acid–free BSA) were the same in all treatments. All nutrients (glucose, lactate, pyruvate, amino acids, and vitamins) were diluted to the same extent (e.g. 25%) relative to the control medium for each culture period. Blastocyst formation and hatching (per cleaved embryo) were evaluated on Day 7 of culture. Hatching blastocysts were stained to determine the number of inner cell mass (ICM; SOX2+), trophectoderm (TE; CDX2+), and total cells (ICM+TE) in the embryo. All data were analysed by ANOVA. The proportion of zygotes that cleaved on Day 3 was not affected (P > 0.05) by the concentration of nutrients present. In experiment 1, dilution of nutrients to 25% did not affect (P > 0.05) blastocyst development (40.1 ± 3.7%) or hatching (16.3 ± 2.8%) compared with 100% (45.2 ± 3.8% blastocyst and 24.9 ± 3.3% hatching). In experiment 2, dilution of nutrients to 12.5% tended (P = 0.08) to reduce hatching (12.9 ± 2.6%) compared with 100% (20.0 ± 3.1%) but did not affect (P > 0.05) blastocysts formation (12.5%, 41.7 ± 3.9% v. 100%, 40.0 ± 3.8%). It was not until nutrient concentrations were reduced to 6.25% that blastocyst formation (18.3 ± 3.0%) and hatching (3.0 ± 1.3%) were inhibited (P < 0.05). Hatching blastocysts cultured with 25 or 12.5% nutrients had fewer total (P < 0.05; 150.7 ± 9.7 and 121.6 ± 7.6, respectively), TE (P < 0.05; 124.1 ± 8.5 and 90.5 ± 7.1), and ICM (P = 0.06; 26.6 ± 3.4 and 30.7 ± 4.0) cells compared with control embryos (195.2 ± 15.9 total, 156.1 ± 14.1 TE, and 39.1 ± 4.0 ICM). To determine whether the embryo’s ability to develop with reduced concentrations of nutrients was dependent on lipid metabolism, embryos were cultured with 50, 25, 12.5, and 6.25% nutrients in the presence or absence of an inhibitor of fatty acid oxidation (50 μM etomoxir). The presence of etomoxir reduced (P < 0.05) blastocyst development at all nutrient concentrations, but this effect was more pronounced when nutrients were limited (≤25% nutrients, 28.7 to 40.9% reduction) compared with 50% (12.5% reduction). Although blastocyst cell numbers decrease when nutrient concentrations are reduced to 25% of those in control media, the proportion of embryos reaching the blastocyst stage is not affected until nutrients are reduced to 6.25%. The ability to develop under nutrient-restricted conditions appears to be related to fatty acid metabolism.

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