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).

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.

Effect of bovine oviductal fluid on development and quality of bovine embryos produced in vitro

2017 ◽  
Vol 29 (3) ◽  
pp. 621 ◽  
Author(s):  
Ricaurte Lopera-Vasquez ◽  
Meriem Hamdi ◽  
Veronica Maillo ◽  
Valeriano Lloreda ◽  
Pilar Coy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Cell Mass ◽  
Water Channel ◽  
Calf Serum ◽  
Cell Counting ◽  
Bovine Embryos ◽  
Oviductal Fluid

To evaluate the effect of bovine oviductal fluid (OF) supplementation during in vitro culture of bovine embryos on their development and quality, in vitro-produced zygotes were cultured in synthetic oviductal fluid (SOF; negative control; C–) supplemented with OF or 5% fetal calf serum (positive control; C+). Embryo development was recorded on Days 7–9 after insemination and blastocyst quality was assessed through cryotolerance, differential cell counting of the inner cell mass and trophectoderm, and gene expression. OF was added to the culture medium at concentrations ranging from 0.625% to 25%. The higher OF concentrations (5%, 10% and 25%) had a detrimental effect on embryo development. Lower OF concentrations (1.25% and 0.625%) supported embryo development until Day 9 (27.5%) and produced higher-quality blastocysts, as reflected by their cryotolerance (53.6% and 57.7% survival at 72 h, respectively, vs 25.9% in C+) and total cell number (mean (± s.e.m.) 165.1 ± 4.7 and 156.2 ± 4.2, respectively, vs 127.7 ± 4.9 in C– and 143.1 ± 4.9 in C+). Consistent with these data, upregulation of the water channel aquaporin 3 (AQP3) mRNA was observed in blastocysts supplemented with 1.25% OF compared with C– and C+. Serum supplementation resulted in a reduction in the expression of glucose and lipid metabolism-related genes and downregulation of the epigenetic-related genes DNA methyltransferase 3A (DNMT3A) and insulin-like growth factor 2 receptor (IGF2R). In conclusion, in vitro culture with low concentrations of OF has a positive effect on the development and quality of bovine embryos.

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.

137 PHENAZINE ETHOSULFATE AND FETAL CALF SERUM EFFECTS ON THE DEVELOPMENT AND APOPTOSIS OF IN VITRO PRODUCED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 173
Author(s):  
M. J. Sudano ◽  
D. M. Paschoal ◽  
T. S. Rascado ◽  
L. C. O. Magalhães ◽  
L. F. Crocomo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Fetal Calf Serum ◽  
Bos Indicus ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Blastocyst Quality ◽  
Phenazine Ethosulfate

Phenazine ethosulfate (PES) is a metabolic regulator that inhibits fatty acid synthesis and favours the pentose-phosphate pathway. Supplementation of fetal calf serum (FCS) during culture has been correlated with the reduction of quality of in vitro produced bovine embryos (IVPE). The aim of the present study was to evaluate embryo development and apoptosis in blastocysts after the supplementation of PES and FCS in culture medium of IVPE. Oocytes (N = 4320) were matured and fertilized in vitro (Day 0). The zygotes (Bos indicus) were cultured in SOFaa medium with 4 concentrations of FCS (0, 2.5, 5, and 10%) and with the use or not of 0.3 μM PES from Day 4 (after 96 h of embryo culture). Embryo development was evaluated after 7 days of culture. Apoptosis in blastocysts (N = 60–80) was accessed through TUNEL reaction. Embryos (Bos indicus) recovered from superstimulated cows were used as in vivo control (n = 15). Data were analysed by ANOVA followed by LSD using PROC GLIMMIX (SAS; SAS Institute Inc., Cary, NC, USA) means ± SEM. Increasing FCS concentration in the culture media did not change cleavage (86.7 ± 1.7, 82.3 ± 1.6, 86.3 ± 1.4, 87.0 ± 1.5, P > 0.05) and augmented blastocyst production (30.5 ± 2.5a, 41.8 ± 2.4b, 40.5 ± 2.6b, 47.2 ± 2.8b, P < 0.05), respectively, for 0, 2.5, 5, and 10%. Additionally, increasing FCS concentration increased apoptosis in blastocysts (13.8 ± 1.2b, 19.1 ± 1.8b, 20.7 ± 1.9bc, 28.4 ± 2.3c, P < 0.05, respectively, for 0, 2.5, 5, and 10%). The addition of PES from Day 4 in the culture medium did not affect (P > 0.05) cleavage (87.0 ± 1.3 and 84.4 ± 1.3), blastocyst production (42.0 ± 2.8 and 43.0 ± 2.0), and apoptosis in blastocysts (20.7 ± 2.0b and 18.9 ± 2.1b), respectively, for control and PES Day 4 groups. Independent of FCS withdrawal or PES addition to culture medium, the in vivo control group presented the lowest apoptosis rate (6.3 ± 1.1a). Therefore, increasing FCS concentration augmented embryo development and reduced blastocyst quality. However, the addition of 2.5% of FCS in the culture medium increased the embryo development without the reduction of blastocyst quality. Moreover, the PES supplementation from Day 4 did not affect embryo development and blastocyst quality. São Paulo Research Foundation – FAPESP.

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.

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.

15 HISTONE ACETYLATION PROFILE OF PORCINE EMBRYOS PRODUCED BY 2 CLONING METHODS WITH OR WITHOUT IN VITRO CULTURE

2016 ◽  
Vol 28 (2) ◽  
pp. 137
Author(s):  
Y. Liu ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
R. Li ◽  
D. Hermann ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Histone Acetylation ◽  
Mrna Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Cell Numbers ◽  
Cloning Method

Conventional “Dolly”-based cloned (CNT) embryos maintain zona pellucida and can be transferred early in development. Handmade cloned (HMC) embryos are zona free and are cultured to later stages for transfer. We have shown differences between HMC and CNT embryos (Rep. Fert. Dev. 26, 123), and both in vitro culture and cloning method (NT) are associated with alterations in histone acetylation. More studies are needed to clarify whether CNT and HMC embryos differ in epigenetic profiles due to NT method or culture condition. Here we investigated histone acetylation profile of NT embryos produced by CNT or HMC with or without 5 to 6 days in vitro culture, emphasising quality and gene expression in resulting embryos. Both NT methods were performed on Day 0 (D0) with same oocyte batch, donor cells, and culture medium (CNT in group, HMC in well of well). On D0, 5, and 6 after CNT (Clon. Stem Cells 10, 355) or HMC (Zygote 20, 61), all developed embryos of all morphological qualities were collected for immunostaining of H3K18ac, and on D0 and 6 for mRNA expression of the genes KAT2A/2B, EP300, HDAC1/2, DNMT1o/s, and GAPDH. Embryo quality was evaluated normal (clear inner cell mass, high cell number, no fragments) or bad (no clear inner cell mass, low cell number, fragments). Cell numbers per blastocyst were counted on D5 and 6. Differences in cell number and H3K18ac level between different groups and days were analysed by ANOVA; gene expression data were analysed by GLM (SAS version 9.3, SAS Institute Inc., Cary, NC, USA). Embryo development rates of both NT methods were reported previously (Rep. Fert. Dev. 26, 123). On D5 and 6, all HMC embryos were evaluated as normal, but the CNT group contained both normal and bad embryos. Regarding cell numbers (Table 1), on D5 there was no difference between normal CNT and HMC embryos, but numbers were lower in CNT bad embryos. On D6 the blastocyst cell number was lower in both normal and bad CNT embryos compared with HMC. Regarding H3K18ac levels (Table 1), no differences were found on D5 between normal CNT and HMC embryos, but on D6 both CNT normal and bad embryos had higher H3K18ac level compared with HMC. On D0, no difference was found in mRNA expression of all 8 genes. On D6, KAT2A expression was slight increased (1.8-fold) in CNT compared with HMC embryos (P < 0.05). In conclusion, no differences were found between CNT and HMC embryos after completed NT procedure (D0) or after 5 days in vitro culture. However, differences in quality (cell number and H3K18ac) and gene expression between the 2 NT methods were observed when blastocyst expansion was initiated (D6). Thus, the 2 NT methods seem to produce embryos of similar quality, which is maintained over 5 days in vitro culture, but thereafter gene expression and histone acetylation are more active in CNT embryos. Table 1.Cell number and H3K18ac level1

294 USE OF FORSKOLIN TO PRODUCE IN VITRO BOVINE EMBRYOS UNDER TWO CONCENTRATIONS OF FETAL CALF SERUM

2010 ◽  
Vol 22 (1) ◽  
pp. 303
Author(s):  
D. M. Paschoal ◽  
M. J. Sudano ◽  
L. C. O. Magalhães ◽  
L. F. Crocomo ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Fetal Calf Serum ◽  
Formation Rate ◽  
Calf Serum ◽  
Basic Medium ◽  
Intracellular Camp ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Embryo Viability ◽  
High Concentration

The increased storage of lipid granules in in vitro-produced (IVP) bovine embryos seems to be related to the presence and concentration of fetal calf serum (FCS) during culture. The presence of high concentration of lipids on embryos reduces their viability after cryopreservation, which has been one of the main obstacles for the success of vitrification of IVP bovine embryos (Moore et al. 2007 Theriogenology 68, 1316-1325). The present experiment aimed to induce cytoplasmic lipolysis in IVP bovine embryos using forskolin (Sigma-Aldrich, St. Louis, MO, USA), which raises the levels of intracellular cAMP (Seamon et al. 1981 Proc. Natl. Acad. Sci. USA, 78, 3363-3367). Nelore oocytes were matured in TCM-199 + 10% FCS, FSH, and LH in 5% CO2 in air atmosphere, at 38.5°C. After 24 h of maturation, oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the same conditions. Presumptive zygotes were cultured in 2 concentrations of FCS: Control 0% (SOFaa + 5 mg mL-1 BSA; basic medium, BM), and Control 2.5% (BM supplemented with 2.5% FCS). On Day 6 of culture embryos were divided into 2 additional treatments: Forskolin 0% (BM + 10 μM forskolin; and Forskolin 2.5% (BM supplemented with 2.5% FCS and 10 μM forskolin). All embryos were cultured in a 5% CO2, 5%O2, and 90% N2 atmosphere at 38.5°C for 7 days, when blastocyst formation rate was evaluated. Embryo viability was also checked by staining the embryos with Hoechst 33342 and propidium iodide. Data were analyzed by ANOVA followed by Tukey’s test, using a 5% significance level. No statistical differences were observed among treatments on cleavage rates, evaluated on Day 3 of culture, or on blastocyst formation rates. Although no statistical differences was observed between treatments on percentage of viable cells, embryos cultured with 0% FCS, independently of the presence of forskolin, presented significantly more damaged cells than embryos cultured with 2.5% FCS (P < 0.05). The results indicate that the presence of FCS is important to reduce degeneration of blastomeres during culture. Moreover, the presence of forskolin on Day 6 of culture did not influence embryo development, indicating that this drug could be a good alternative to reduce embryo lipid content in bovine IVP embryos produced in presence of FCS. Table 1.Effect of fetal calf serum and forskolin on embryo culture Acknowledgments: FAPESP 07/53505-1.

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