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.

133 EFFECT OF PROGESTERONE SUPPLEMENTATION OF MATURATION MEDIUM ON THE DEVELOPMENT OF IN VITRO-MATURED-IN VITRO-FERTILIZED-IN VITRO-CULTURED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 180 ◽  
Author(s):  
K. Syoji ◽  
K. Imai ◽  
H. Koyama ◽  
O. Dochi
Keyword(s):  
Cell Mass ◽  
Calf Serum ◽  
Total Cell ◽  
Differential Staining ◽  
Control Group ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Maturation Medium ◽  
Significant Difference

The objective of this study was to investigate whether progesterone (P4) supplementation to in vitro maturation (IVM) medium could affect the competence of bovine oocyte to develop into blastocysts in vitro. Cumulus–oocyte complexes (COC) were collected by aspiration of ovarian follicles (2 to 6 mm in diameter) obtained from a local abattoir. The COC were matured for 20 h in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 FSH at 38.5° in an atmosphere of 5% CO2 in air. After 18 h of gamete co-culture (5 × 106 sperms mL–1), presumptive zygotes were cultured in CRlaa containing 5% calf serum at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 for 9 days (fertilization = Day 0). Progesterone was added to the IVM medium 10 h after the start of the culture (1 μg group = 1 μg mL–1 of P4; 5 μg group = 5 μg mL–1 of P4; control group = no P4). The maturation (MII) rates were investigated after 20 h of starting the IVM culture. After maturation, the COC were denuded mechanically, and a part of the oocytes were mounted on slides, fixed with aceto-alcohol (1 : 3) solution for 48 h, stained with aceto-orcein, and observed under a phase-contrast microscope to determine their nuclear status (1 μg group: n = 32; 5 μg group: n = 28; control group: n = 31). The remaining COC were used for IVF. The cleavage rates were investigated on day 2, and the blastocyst formation rates were investigated on Days 7 to 9, respectively (1 μg group: n = 264; 5 μg group: n = 274; control group: n = 277). The blastocysts from Day 7 were used for differential staining of the inner cell mass (ICM) and trophectoderm cells (TE). The total cell numbers, ICM, and TE in the blastocysts were counted (1 μg group: n = 28; 5 μg group: n = 24; control group: n = 24). The rates of MII, cleavage, and blastocyst formation were expressed and analysed by the chi-squared test. Each set of cell numbers (mean ± standard error) was analysed by the unpaired t-test. The MII rate in the control group (76.7%) was significantly lower (P < 0.05) than that in the 1 μg group (93.8%). The cleavage rate in the 1 μg group (85.6%) was significantly higher (P < 0.05) than those in the control group (74.7%) and 5 μg group (77.4%). Further, the blastocyst formation rate in the 1 μg group (47.7%) and 5 μg group (43.4%) were significantly higher (P < 0.05) than that in the control group (35.0%). The ICM numbers (mean ± s.e.) were 39.5 ± 13.8 to 36.2 ± 8.9, the TE numbers were 74.4 ± 22.4 to 66.2 ± 12.9, the total cell numbers of blastocysts were 110.6 ± 28.2 to 103.0 ± 13.8. There was no significant difference in cell numbers among the groups. These results indicate that the cleavage and blastocyst formation rates can be improved by the addition of 1 μg mL–1 of P4 to the maturation medium.

178 IMPROVED DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS BY PGI2 ANALOG TREATMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 197 ◽  
Author(s):  
B. S. Song ◽  
J. S. Kim ◽  
D. B. Koo ◽  
J. S. Park ◽  
K. K. Lee ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Developmental Rate ◽  
Treated Group ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Frozen Semen ◽  
Oviductal Fluid

The microenvironment of the follopian tube, in which the oviductal fluid contains a variety of cytokines and growth factors, affects pre-implantation development of fertilized embryos in mammals. Prostaglandin I2 (PGI2, prostacyclin) exists in oviductal fluid and is synthesized from arachidonic acid by prostacyclin synthetase. PGI2 also enhances the implantation rate of mouse embryos. In this study, the effect of PGI2 analog on the development of bovine embryos was examined. Bovine cumulus oocytes complexes (COCs) were matured in TCM-199 medium supplemented with 10 IU/mL pregnant mare serum gonadotropin (PMSG), 10 IU/mL hCG, and 10 ng/mL epidermal growth factor (EGF) at 39�C, 5% CO2 in air for 20-22 h. Following in vitro maturation, COCs were fertilized in Fert-TALP medium containing 0.6% BSA using frozen semen. Also, oocytes matured in vitro were enucleated, individually reconstructed with bESF cells, fused, and then activated by treatment with 5 �M ionomycin for 5 min and 2 mM 6-DMAP for 4 h. In vitro-fertilized (IVF) and nuclear-transferred (NT) eggs were cultured in 50 ��L drops of CR1-aa medium supplemented with 0.3% BSA in the absence or presence of 1 �M PGI2 analog at 39�C, 5% CO2 in air, respectively. At 3 days of culture, cleaved embryos were further cultured in the same culture media supplemented with 10% FBS for 4 days. Allocations of blastocysts to inner cell mass (ICM) and trophoblast (TE) cells were investigated to assess embryo quality. All experiments were repeated more than three times. All data were analyzed by using the Duncan test of ANOVA by the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA) and numbers of nuclei in blastocysts were expressed as mean � SE. No difference was detected in the cleaved rate of the eggs between the treated- and nontreated groups. IVF zygotes treated with PGI2 analog represented a higher developmental rate (33%, 122/418) to the blastocyst stage than nontreated controls (24%, 107/456) (P < 0.05). Among IVF-derived blastocysts, interestingly, the proportion (46%, 84/181) of expanded blastocysts was significantly higher in the PGI2 analog-treated group compared with that in the nontreated group (28%, 46/164). The number of nuclei in (165 � 6.1, n = 15) in blastocysts in the PGI2 analog-treated group was higher than that (146.12 � 5.7, n = 18) in the nontreated group (P < 0.05). No difference was detected in the ratio of ICM to total cells between PGI2 analog-treated (42.0 � 3.0%) and nontreated groups (41.9 � 2.9%). Like the IVF embryos, NT embryos in the PGI2 analog-treated group showed a higher in vitro developmental rate (33.6%, 43/128) than the nontreated embryos (24.2%, 32/132) (P < 0.05). Our results indicate that PGI2 analog improves the kinetics of embryo development in cattle.

scholarly journals Effect of cysteamine supplementation during in vitro culture of early stage bovine embryos on blastocyst rate and quality

2012 ◽  
Vol 81 (3) ◽  
pp. 229-234 ◽  
Author(s):  
Martina Lojkic ◽  
Iva Getz ◽  
Marko Samardžija ◽  
Mario Matkovic ◽  
Goran Bacic ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Oviductal Fluid

The aim of this study was to evaluate whether the addition of cysteamine to the in vitro culture media enhances the yield, hatching rate, total cell number and inner cell mass/total cell number ratio of bovine embryos. A total of 933 bovine oocytes collected from ovaries of 60 slaughtered donors were subjected to in vitro maturation and in vitro fertilization. Following fertilization, embryos were cultured in synthetic oviductal fluid without glucose. After 24 h embryos were transferred into synthetic oviductal fluid with 1.5 mM glucose and 0 (control), 50, 100 and 200 µM of cysteamine. After 48 h, the embryos were transferred into synthetic oviductal fluid with glucose but without cysteamine and cultured until Day 9. The number of cleaved embryos on Day 2, the total number of blastocysts on Day 7 and the number of hatched blastocysts on Day 9 were calculated. Differential staining of inner cell mass and trophectoderm cells of blastocysts were performed on Day 7 and Day 9 of in vitro culture. Supplementation of in vitro culture media with 100 µM cysteamine increased the blastocyst yield (P < 0.05) without affecting the hatching rate. Furthermore, the embryos cultured in the presence of 100 µM cysteamine had significantly higher number of inner cell mass cells (P < 0.05) and the proportion of inner cell mass cells (P < 0.05) compared with the controls. The results of the present study demonstrated that the addition of 100 µM cysteamine to the in vitro culture media improved blastocyst production rate and enhance embryo quality, which could lead to the improvement of the in vitro culture system for bovine embryos.

scholarly journals Interleukin-6 requires JAK to stimulate inner cell mass expansion in bovine embryos

Reproduction ◽  
2019 ◽  
Vol 158 (4) ◽  
pp. 303-312 ◽  
Author(s):  
Lydia K Wooldridge ◽  
Sally E Johnson ◽  
Rebecca R Cockrum ◽  
Alan D Ealy
Keyword(s):  
Interleukin 6 ◽  
Nuclear Translocation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Janus Kinase ◽  
Bovine Embryos ◽  
Cell Numbers ◽  
Stat3 Phosphorylation

Supplementing interleukin-6 (IL6) to in vitro-produced bovine embryos increases inner cell mass (ICM) cell numbers in blastocysts. A series of studies were completed to further dissect this effect. Treatment with IL6 increased ICM cell numbers in early, regular and expanded blastocysts but had no effect on morulae total cell number. Treatment with IL6 for 30 min induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and nuclear translocation in all blastomeres in early morulae and specifically within the ICM in blastocysts. Also, IL6 supplementation increased SOCS3 mRNA abundance, a STAT3-responsive gene, in blastocysts. Chemical inhibition of Janus kinase (JAK) activity from day 5 to day 8 prevented STAT3 activation and the IL6-induced ICM cell number increase. Global transcriptome analysis of blastocysts found that transcripts for IL6 and its receptor subunits (IL6R and IL6ST) were the most abundantly expressed IL6 family ligand and receptors. These results indicate that IL6 increases ICM cell numbers as the ICM lineage emerges at the early blastocyst stage through a STAT3-dependent mechanism. Also, IL6 appears to be the primary IL6 cytokine family member utilized by bovine blastocysts to control ICM cell numbers.

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.

The effect of extracellular matrix molecules on mouse preimplantation embryo development in vitro

2002 ◽  
Vol 14 (7) ◽  
pp. 443 ◽  
Author(s):  
F. Figueiredo ◽  
G. M. Jones ◽  
G. A. Thouas ◽  
A. O. Trounson
Keyword(s):  
Culture Medium ◽  
Preimplantation Embryo ◽  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Sulfate Proteoglycan ◽  
Preimplantation Embryo Development ◽  
Cell Numbers ◽  
Mouse Preimplantation Embryo

The extracellular matrix (ECM) molecules, laminin (LN), chondroitin sulfate (CS), fibronectin (FN), hyaluronic acid (HA), mucin (MUC) and heparan sulfate proteoglycan (HS), were investigated as supplements to culture medium to improve the in vitro development of mouse 1-cell zygotes to blastocysts. Development was also compared with that in medium supplemented with bovine serum albumin (BSA) to determine the potential for ECM molecules as suitable alternatives to serum albumin in culture medium. Supplementation of sequential culture media with LN at all concentrations examined failed to result in more than 70% of zygotes developing to blastocysts; therefore, LN was considered unsuitable as a replacement for BSA and was not examined further. The optimal concentration of the remaining ECM molecules was used to supplement sequential culture media and the effect on blastocyst quality was assessed by determining the differential cell numbers of blastocysts grown in BSA-supplemented medium. Development to blastocyst was similar, regardless of the macromolecule used. The number of inner cell mass cells was significantly higher in HS-supplemented medium compared with controls. Trophectoderm cell numbers were similar to control values for all ECM molecules examined except CS for which there were fewer trophectoderm cells. It is concluded that ECM molecules, FN, HA, MUC and HS may be used as substitutes for serum protein supplementation of culture media EG0/G2 for mouse preimplantation embryo development. Heparan sulfate proteoglycan increases inner cell mass numbers and this may be due to interactions with the growth factors fibroblast growth factor 4 (FGF-4) and granulocyte–macrophage colony-stimulating factor.

scholarly journals Stereomicroscopic and histological examination of bovine embryos following extended in vitro culture

2005 ◽  
Vol 17 (8) ◽  
pp. 799 ◽  
Author(s):  
Natalie I. Alexopoulos ◽  
Gábor Vajta ◽  
Poul Maddox-Hyttel ◽  
Andrew J. French ◽  
Alan O. Trounson
Keyword(s):  
In Vitro Culture ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Collagen Gel ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Term Survival

Attempts to support survival of mammalian embryos after hatching have met with limited success, although some mouse studies have reported growth at the post-implantation stage. The aim of the present research was to establish and characterise an in vitro culture system that could support extended growth and differentiation of bovine embryos. Abattoir-derived oocytes were matured and fertilised in vitro. Presumptive zygotes were cultured in modified synthetic oviduct fluid (SOFaaci) medium supplemented with 5% cow serum (CS). On Day 9, single hatched blastocysts (n = 160) were randomly allocated to SOFaaci supplemented with either 5% bovine serum albumin, 5% CS, 5% fetal calf serum (FCS) or SOF only and cultured on a collagen gel substrate for up to 45 days. Embryos were evaluated at various time-points until complete disaggregation or the total disappearance of embryonic cells. Blastocyst viability post hatching was severely compromised in protein-free SOFaaci medium. Addition of FCS generated increased embryonic growth for the longest time period (Day 45) when compared to the other groups. Long-term survival of embryonic cells was observed stereomicroscopically by the proliferation and development of three-dimensional tubular structures to 85% confluence in culture. Haematoxylin and eosin staining of morphological structures obtained from all treatment groups revealed embryos displaying trophoblast, inner cell mass and hypoblast development to varying degrees. Regardless of treatment, extended in vitro culture did not result in development comparable with that described for in vivo embryos. In the present work, however, there was evidence of extended culture of bovine embryos beyond that achieved previously. However, further research is required to identify the exact requirements for extended in vitro culture for bovine embryos.

140 PRODUCTION OF RECONSTRUCTED IN VITRO PRODUCED BOVINE EMBRYOS BY INNER CELL MASS AND TROPHECTODERM AGGREGATION IN VITRO

2011 ◽  
Vol 23 (1) ◽  
pp. 174
Author(s):  
I. P. Emanuelli ◽  
E. Razza ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
In Vitro Culture ◽  
Fetal Calf Serum ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
Calf Serum ◽  
Cell Aggregation ◽  
Bovine Embryos ◽  
Advanced Stages

The efficiency of embryonic chimerism tends to decrease when embryos in advanced stages of development, such as morulae and blastocysts, are used. To perform the inner cell mass (ICM) transfer to a trophectoderm (TE) receptor, it is essential to use embryos at an advanced stage and blastocoel presence. This method of embryo reconstruction has been performed only by the micromanipulator microinjection method (Zheng et al. 2005 Zygote 1, 73–71; Loi et al. 2007 Trends Biotechnol. 25, 195–200; Roth et al. 1989 Biol. Reprod. 41, 675–682; and Murakami et al. 2006 Cloning Stem Cells 8, 51–60). This study aimed to validate a manual procedure to reconstruct embryos using the method of ICM and TE approximation in the presence of phytohemagglutinin. Bos indicus ovaries from the abattoir were used to obtain 230 cumulus–oocyte complexes (COC; quality I and II). The COC were matured in 90-μL drops of TCM-199 bicarbonate supplemented with 10% fetal calf serum (FCS) and incubated in vitro for 22 to 24 h. Fertilization occurred in TALP-IVF medium, and the COC were incubated for 18 h. Presumptive zygotes were transferred to SOF medium to in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. In vitro produced (IVP) embryos 8.5 days after fertilization were used for the experiment. Ninety-three hatching or hatched blastocysts were put into 3-μL microdrops of protein-free HEPES-buffered SOF (HSOF) medium to hold the embryos on the dish bottom and to allow handmade sections of ICM and TE. The section was performed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under a stereomicroscope (35× magnification). Seventy half-structures from 35 different blastocysts were obtained to form pairs (ICM+TE). Each pair was transferred to drops with 500 μg mL–1 of phytohemagglutinin-L (3 min) before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264) to in vitro culture (38.5°C; 5% O2 and 5% CO2). The aggregation rate was 25.7% (9/35) and all the reconstructed blastocysts by aggregation (24 h) re-expanded after 48 h of culture. The technique of handmade ICM and TE section and posterior aggregation in the presence of an agglutinating agent was feasible for the structural and functional reconstruction and re-expansion of the blastocyst produced. Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).

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.

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system

2005 ◽  
Vol 17 (8) ◽  
pp. 751 ◽  
Author(s):  
Mona E. Pedersen ◽  
Øzen Banu Øzdas ◽  
Wenche Farstad ◽  
Aage Tverdal ◽  
Ingrid Olsaker
Keyword(s):  
Gene Expression ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Glut 1 ◽  
Significant Difference ◽  
Oviduct Fluid

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

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