166 EFFECT OF INDIVIDUAL CULTURE SYSTEM ON IN VITRO DEVELOPMENT OF IN VITRO-MATURED - IN VITRO-FERTILIZED BOVINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 195
Author(s):  
R. Nishii ◽  
K. Imai ◽  
H. Koyama ◽  
O. Dochi
Keyword(s):  
Culture System ◽  
Calf Serum ◽  
Control Culture ◽  
Total Cell ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Culture Systems ◽  
Individual Culture ◽  
Single Culture

An individual in vitro culture system for bovine embryo needs to be developed for the study of embryo developmental competence. The objective of this study was to examine the effect of individual culture systems on the development of in vitro-matured–in vitro-fertilized bovine embryos. Two individual culture systems were compared. Cumulus–oocyte complexes (COC) were collected by aspiration of ovarian follicles (diameter, 2 to 5 mm) obtained from a local abattoir. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH. Groups of 20 COC were incubated in 100-μL droplets of IVM media at 38.5°C under an atmosphere of 5% CO2 for 20 h. After 18 h of gamete co-culture (3 × 106 sperm mL–1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% CO2, 5% O2 and 90% N2 for 9 days (fertilization = Day 0). The presumptive zygotes were randomly assigned to 1 of the following 3 treatments: single culture (1 zygote was cultured in a 5-μL droplet), well-of-the-well (WOW; Sugimura et al. 2010 Biol. Reprod. 83, 970–978) culture (25 zygotes were cultured individually in each 125-μL droplet) and control culture (25 zygotes were cultured in a 125-μL droplet). Embryo development was evaluated for cleavage and blastocyst rates, on Day 2 and Day 7 to 9 after IVF, respectively. The rates of development up to the blastocyst stage and total cell number in the blastocysts, determined by an air-drying method, were investigated. The cleavage and blastocyst rates were analysed by the chi-square test and the total cell numbers were analysed by ANOVA. The cleavage rates were significantly higher in the control and WOW groups than in the single-culture group (P < 0.01) and the blastocyst rates were significantly lower in the single-culture group than in the control culture group (P < 0.05; Table 1). The total cell numbers (mean ± s.d.) of blastocysts did not significantly differ between the single culture (154.6 ± 21.8), control culture (155.2 ± 22.5) and WOW culture (159.8 ± 27.0) groups. These results indicate that although the blastocyst rate was lower in the single-culture system than in the WOW or group culture system, in vitro-matured–in vitro-fertilized bovine embryos can be cultured using the single-culture system. Moreover, the quality of blastocysts developed by the single-culture system is the same as that of blastocysts developed using the other 2 culture systems. Table 1.Effect of different culture methods for bovine embryo development

120 EFFECT OF INDIVIDUAL CULTURE SYSTEM USING WOW OR CELL-TAK ON DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 160
Author(s):  
S. Matoba ◽  
P. Lonergan
Keyword(s):  
Embryo Culture ◽  
Culture System ◽  
Basal Medium ◽  
Bovine Embryos ◽  
Paracrine Effects ◽  
Individual Culture ◽  
Cell Adhesive ◽  
Developmental Rates ◽  
One Way Anova

The culture of embryos individually in vitro is generally associated with poorer developmental rates. However, the ability to do this successfully would greatly facilitate studies where identification of individual embryos, or the embryos from a particular donor, is necessary. The objective of this study was to examine the effect of culture system on the development of individual IVP bovine embryos. Presumptive zygotes (n = 1301, 6 replicates), produced by IVM/IVF, were used. The aim of Experiment 1 was to compare development of bovine embryos in SOF or CR1aa supplemented with 5% FCS. Zygotes were cultured in droplets under oil as follows: (i) 20/25 μL, (ii) 20/100 μL or (iii) 20/100 μL individually in the Well of the Well (WOW) system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 254–264). Twenty WOW were prepared in a 100 μL droplet of medium under oil using a sterile rod. The aim of Experiment 2 was to compare development of embryos cultured in groups but individually identifiable on the cell adhesive Cell-Tak (Stokes et al. 2005 Dev. Biol. 284, 62–71) or in the WOW system. Zygotes were cultured as follows: (i) 20/20 μL, (ii) 20/20 μL with Cell-Tak, (iii) 20/100 μL with Cell-Tak or (iv) 20/100 μL in WOW. A drop of Cell-Tak (1 μL/20 μL medium) was placed on the base of the dish, dried for 20 min, washed with sterile water and dried completely. Once dried, the area was covered with 10 μL of FCS-free medium and groups of 20 zygotes were placed on the Cell-Tak in a 5 × 4 grid formation a maximum of 160 μm apart. Then, an additional 10 μL or 90 μL medium supplemented with FCS was added to give a final volume of 20 or 100 μL. Cleavage and blastocyst rates were assessed on Day 2 and Days 7–9, respectively. Data (means ± SE) were analyzed by one way ANOVA. In Experiment 1, there were no differences between SOF and CR1aa with respect to culture of embryos individually in WOW (P > 0.05); therefore, SOF was used as the basal medium for Experiment 2. There were no differences (P > 0.05) between the cleavage and blastocyst rate among drop sizes and individual culture systems; individual culture, irrespective of the system used (Cell-Tak or WOW), resulted in the similar developmental rates to the control. In conclusion, individual embryo culture offers the opportunity to study embryo development in a more powerful manner. Furthermore, the use of the cell adhesive Cell-Tak may be more practical because it removes the potential variability associated with well dimensions in the WOW system and may improve any potential paracrine effects during embryo culture. Further studies are required to establish the viability of such embryos after transfer. Table 1.Effect of individual culture system on development of IVP bovine embryos Supported by Science Foundation Ireland.

scholarly journals Effective individual culture system for in vitro production of bovine embryos

2017 ◽  
Vol 54 (3) ◽  
pp. 209 ◽  
Author(s):  
Kelly Annes ◽  
Carlos Alexandre Soares ◽  
Camila Bruna De Lima ◽  
Marcella Pecora Milazzotto
Keyword(s):  
Culture System ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Individual Culture ◽  
Vitro Production

Estabeleceu-se um protocolo novo e eficaz de cultivo individual de embriões bovinos sem o uso de cocultivo e sem compartilhamento de meio visando à análise do metabolismo individual do embrião. Para isso, embriões foram produzidos in vitro por protocolos convencionais em três diferentes tipos de meio: KSOM, SOFaa e KSOM seguido por SOFaa no dia 2. Os zigotos presumíveis foram divididos em seis grupos: controles (cultivo em grupo – C-KSOM, C-SOFaa e C-KS) e sistema de poços individuais (W-KSOM, W-SOFaa e W-KS). As taxas de clivagem foram avaliadas nos dias 2 e 7, respectivamente. Além disso, a quantificação relativa de transcritos relacionados a importantes processos metabólicos (GLUT1, GLUT3, GSK3, SOD1, HSPD1 e G6PD) foi avaliada nos blastocistos dos grupos C-KS e W-KS. Os resultados mostram que as taxas de clivagem foram maiores apenas no grupo W-KSOM quando comparado ao grupo C-KSOM, enquanto a taxa de blastocistos diferiu apenas entre os grupos C e W-SOF. Além disso, a análise da expressão gênica mostrou que blastocistos cultivados em grupo ou em sistema de poços individuais são semelhantes quanto à expressão gênica. Assim, a conclusão obtida foi que o sistema individual proposto pode ser utilizado como um protocolo alternativo eficiente para o cultivo individual de embriões de bovino, uma vez que suas características permanecem semelhantes àquelas do sistema convencional de produção de embriões.

148 EFFECT OF FOLLISTATIN TREATMENT POST-FERTILIZATION ON TIME TO FIRST CLEAVAGE, DEVELOPMENT TO THE BLASTOCYST STAGE, AND CELL ALLOCATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 191
Author(s):  
K. B. Lee ◽  
A. Bettegowda ◽  
J. J. Ireland ◽  
G. W. Smith
Keyword(s):  
Blastocyst Stage ◽  
Total Cell ◽  
Mrna Abundance ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Trophectoderm Cells

Previous studies from our laboratory have demonstrated a positive association of follistatin mRNA abundance with oocyte competence. Follistatin mRNA is greater in germinal vesicle stage oocytes collected from prepubertal (model of poor oocyte competence) vs. adult animals. Furthermore, follistatin mRNA abundance is also greater in early-cleaving 2-cell bovine embryos (collected prior to the maternal zygotic transition and initiation of significant transcription from the embryonic genome) than their late-cleaving counterparts. Given these results and the fact that early-cleaving embryos develop to the blastocyst stage at a greater rate, we hypothesized that follistatin has a stimulatory role in early embryonic development. To begin to test this hypothesis, we determined the effects of follistatin treatment of in vitro-produced bovine embryos (during the initial 72 h post-fertilization) on time to first cleavage, development to the blastocyst stage (Day 7), and blastocyst cell allocation (quality). Cumulus–oocyte complexes (COCs) were harvested from ovaries obtained from a local abattoir, matured, and fertilized in vitro. After 20 h of co-incubation with spermatozoa, presumptive zygotes were stripped of cumulus cells and cultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10, or 100 ng mL-1 follistatin (n = 25 presumptive zygotes per treatment; n = 6 replicates). Proportions of embryos reaching the 2-cell stage within 30 h (early-cleaving), 30–36 h (late-cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Embryos at the 8–16-cell stage were separated 72 h after fertilization and cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% FBS until Day 7. The proportion of embryos reaching the blastocyst stage at Day 7 post-fertilization was recorded and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells determined by differential staining. Follistatin treatment did not increase the rate of total cleavage and the proportion of late-cleaving embryos when compared to control. However, supplementation with 1 and 10, but not 100, ng mL-1 follistatin increased the proportion of early-cleaving embryos (26.3 and 35.3% vs. 9.5%) and development to the blastocyst stage (28.6 and 31.7% vs. 18.4%) relative to controls (P &lt; 0.05). Treatment with 10 ng mL-1 follistatin increased total cell numbers (130.1 vs. 110.9) and proportion of trophectoderm cells (61.6% vs. 48.4%) and decreased the ICM/total cell ratio (38.4% vs. 51.5%) in Day 7 blastocysts relative to controls (P &lt; 0.05). The results indicate that exogenous follistatin treatment during the early stages of in vitro bovine embryo development can enhance time to first cleavage, development to the blastocyst stage, and cell allocation in favor of increased trophectoderm cells, and can support a potential functional role for follistatin in early embryogenesis.

scholarly journals Culture system and long-term storage of culture media in the in vitro production of bovine embryos

2011 ◽  
Vol 59 (1) ◽  
pp. 129-139 ◽  
Author(s):  
Santiago Varga ◽  
Carmen Diez ◽  
Lina Fernández ◽  
Jenny Álvarez ◽  
Adelino Katchicualula ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
Culture Media ◽  
Calf Serum ◽  
Significant Loss ◽  
Bovine Embryo ◽  
In Vitro Production ◽  
Fresh Culture Medium ◽  
Hatching Rates

The optimum culture system for in vitro matured and fertilised oocytes still remains to be clarified. Culture media (CM) for mammalian embryos are routinely prepared fresh for use and preserved under refrigeration during one or two weeks. The purposes of this work were (1) to compare the efficiency of a synthetic oviduct fluid (SOF) with two different bovine serum albumin (BSA) concentrations (3 and 8 g/L) for the in vitro production of bovine blastocysts, (2) to test the effect of timing on adding fetal calf serum (FCS) to the SOF, and (3) to evaluate the effects on bovine embryo development of freezing and lyophilisation as procedures for preserving the SOF. Supplementation of SOF with 3 g/L BSA increased Day-7 blastocyst expansion rates (18.3 ± 1.6 vs. 14.4 ± 0.7; P < 0.05), although no differences in hatching rates were found. Addition of FCS to SOFaa (SOF with amino acids) medium supplemented with sodium citrate (SOFaaci) at 48 and at 72 h post-insemination (PI) allowed obtaining higher Day-6 embryo development rates than when FCS was added at 18 or 96 h PI (Day-6 morulae + blastocyst rate: 30.0 ± 1.1, 40.8 ± 1.1, 43.9 ± 2.3 and 39.3 ± 0.5 for FCS addition at 18, 48, 72 and 96 h, respectively). Hatching rates were significantly improved when serum was added at 72 h PI. Finally, both refrigeration and lyophilisation appeared as useful cryopreservation procedures for SOFaaci, although a significant loss of its ability to support embryo development, compared to the control fresh culture medium, was observed.

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 Changes in the relative abundance of mRNA transcripts for insulin-like growth factor (IGF-I and IGF-II) ligands and their receptors (IGF-IR/IGF-IIR) in preimplantation bovine embryos derived from different in vitro systems

Reproduction ◽  
2001 ◽  
pp. 601-610 ◽  
Author(s):  
MA Yaseen ◽  
C Wrenzycki ◽  
D Herrmann ◽  
JW Carnwath ◽  
H Niemann
Keyword(s):  
Growth Factor ◽  
Polyvinyl Alcohol ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Culture Systems ◽  
Igf I ◽  
Oviduct Fluid

The aim of this study was to determine the relative abundance of mRNAs for the insulin-like growth factor I (IGF-I) and IGF-II ligands, and for the IGF receptors (IGF-IR and IGF-IIR) in in vitro preimplantation bovine embryos from the oocyte to the hatched blastocyst stage using two different culture systems: TCM-199 supplemented with oestrous cow serum, or synthetic oviduct fluid supplemented with polyvinyl alcohol. Development to the two- to four-cell stage and blastocyst stage was significantly higher (P < or = 0.05) in embryos cultured in TCM supplemented with oestrous cow serum than in those cultured in synthetic oviduct fluid supplemented with polyvinyl alcohol (61 and 25% versus 55 and 17%, respectively). A semi-quantitative RT-PCR assay did not detect IGF-I transcripts at any stage of preimplantation bovine development, including the hatched blastocyst stage. In both culture systems, IGF-IR, IGF-II and IGF-IIR were expressed throughout preimplantation development up to the hatched blastocyst stage in a varying pattern. The expression patterns of IGF-IR, IGF-II and IGF-IIR in embryos generated in the two culture systems were not significantly different, except at the expanded blastocyst stage, at which significantly higher amounts of IGF-IIR were observed in the TCM system than in the synthetic oviduct fluid system. These results indicate that transcripts of IGF-IR and IGF-IIR follow the standard pattern in which maternal stores of mRNA in the oocyte are slowly depleted up to the 16-cell stage and then re-established at the onset of embryonic expression of these genes. The lack of detectable IGF-I transcripts in the bovine embryo indicates a predominantly paracrine mode of action. The bovine embryo is capable of producing IGF-II, IGF-IIR and IGF-IR in large amounts, particularly after hatching, which may be important for the formation of the filamentous conceptus. Results indicate an autocrine mechanism for IGF-II and modulation of IGF family expression by culture conditions.

138 TIME-LAPSE CINEMATOGRAPHY-COMPATIBLE INJECTION-MOLDED MICROWELL CULTURE SYSTEM FOR TRACKING THE DEVELOPMENT OF INDIVIDUAL BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 173
Author(s):  
S. Sugimura ◽  
T. Akai ◽  
T. Somfai ◽  
M. Hirayama ◽  
Y. Aikawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Culture System ◽  
Cell Mass ◽  
Calf Serum ◽  
Time Lapse ◽  
Pregnancy Rates ◽  
Cleavage Pattern ◽  
Bovine Embryos ◽  
Physiological Level

We have developed a polystyrene-based well of-the-well system (WOW) using injection moulding to track individual embryos throughout culture using time-lapse cinematography (TLC). The WOW-cultured bovine embryos following in vitro fertilization (IVF) were compared with conventional droplet (control)-cultured embryos on in vitro and in vivo development. Twenty-five of zygotes were cultured in each culture system containing 125 μL of CR1aa medium supplemented with 5% calf serum for 168 h after IVF. No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality, inner cell mass (ICM), and trophectoderm (TE) cell numbers and post-vitrification survival rates were not different between control- and WOW-derived blastocysts; however, incidence of apoptosis in the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at days 30 and 60 after embryo transfer (P < 0.05). The TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pick-up; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of 2 blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers. This work was supported by the Research and Developmental Program for New Bio-Industry Initiatives.

140 THE EFFECTS OF INTERVALS OF MECHANICAL VIBRATION DURING IN VITRO CULTURE ON THE DEVELOPMENT OF BOVINE EMBRYO DERIVED FROM LOW-QUALITY OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 183
Author(s):  
M. Takehisa ◽  
S. Kondo ◽  
K. Imai ◽  
O. Dochi ◽  
H. Koyama
Keyword(s):  
In Vitro Culture ◽  
Vibration Control ◽  
Mechanical Vibration ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cleavage Rate

Mechanical vibration enhances the cytoplyasmic maturation of in vitro-matured (IVM) pig oocytes (Mizobe et al. 2010 J. Reprod. Dev. 56, 285–290), as well as the development of in vitro-cultured (IVC) bovine embryos (Fujita et al. 2010 Rakuno Gakuen University Graduation thesis,1–36). In this study, the effects of intervals of mechanical vibration during in vitro culture, after IVF, on the development of embryos derived from low-quality oocytes were examined. Cumulus-oocyte complexes (COC) were collected by aspiration of ovarian follicles (diameter = 2 to 6 mm) obtained from a local abattoir. In this experiment, only grade 3 oocytes (i.e. those with one layer or partially remaining cumulus cells and normal cytoplasm) were used. Groups of 20 COC were matured in 100-μL droplets of in vitro TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH at 38.5°C under an atmosphere of 5% CO2 in air for 20 h. Matured COC were inseminated with 5 × 106 sperms mL–1 for 18 h. After 18 h of gamete co-culture, the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% O2, 5% CO2, and 90% N2 for 9 days (fertilization = Day 0). Presumptive zygotes were cultured in vitro without mechanical vibration (control; n = 467) and with mechanical vibration for 5 s at 5 min (n = 180), 10 min (n = 180), 15 min (n = 180), and 60 min (n = 200) for 9 days. Embryo development was evaluated for cleavage and blastocyst rates, on Days 3 and 7 to 9 after IVF, respectively. The cleavage and blastocyst formation rates were analysed by the chi-squared test. Vibration at 15-min intervals increased (P < 0.05) cleavage rate compared to 5 min, 60 min, and control (control: 66.2 ± 22.1%; 5 min: 49.4 ± 10.2%; 10 min: 70.0 ± 7.7%; 15 min: 86.2 ± 6.6%; and 60 min: 64.0 ± 8.5%).The highest (P < 0.05) blastocyst rate among the experimental groups was found with 15-min intervals for vibration (control: 21.6 ± 9.2%; 5 min: 15.0 ± 5.3%; 10 min: 22.8 ± 1.8%; 60 min: 21.5 ± 5.0%). These results indicated that the cleavage and blastocyst formation rates of IVM-IVF-IVC bovine embryos derived from low-quality oocytes can be improved by physical stimulus during IVC. In addition, it was shown that 15-min intervals of mechanical vibration elicited the highest benefit for the development of embryos.

279 IN VITRO DEVELOPMENT IN THREE CULTURE SYSTEMS OF BOVINE OOCYTES FERTILIZED WITH SEX-SORTED SPERM

2011 ◽  
Vol 23 (1) ◽  
pp. 237
Author(s):  
B. Trigal ◽  
E. Gomez ◽  
M. Muñoz ◽  
J.N. Caamaño ◽  
S. Carrocera ◽  
...  
Keyword(s):  
Culture System ◽  
Final Analysis ◽  
Vero Cells ◽  
Bovine Embryos ◽  
Vitro Fertilization ◽  
In Vitro Development ◽  
Culture Systems ◽  
Cell Counts ◽  
Vitro Development

Following in vitro fertilization and culture, the timing of both embryonic cleavage and appearance of blastocysts has been reported to be altered when using sorted spermatozoa (Lu et al. 1999 Theriogenology 52, 1393–1405). To optimize an in vitro culture system for bovine embryos produced with sexed sperm, sorted and unsorted (control) semen samples of 5 bulls (semen samples provided by XY Inc., Fort Collins, CO, USA) were separated using a Percoll gradient followed by a washing step with Fert-TALP medium (Trigal et al. 2010 Reprod. Domest. Anim. 45, 83). The obtained pellet was used for fertilizing in vitro matured cumulus–oocyte complexes from slaughterhouse ovaries. Zygotes were cultured in 1) Vero cells monolayer + B2 + 10% FCS; 2) SOFaaci + 10% FCS; or 3) SOFaaci + 6 g L–1 BSA. A total of 1980 oocytes were in vitro matured and fertilized. Development was recorded on Days 3 and Days 6 to 8, and analysed by ANOVA and Duncan test. Bull, replicate, and culture effects were nonsignificant, and consequently were not considered in the final analysis. Data are percentages of development related to the zygotes cultured, and are expressed as LSM ± SE (see Table 1). The use of sexed sperm significantly reduced development rates compared with unsorted sperm. Thus, sorted sperm showed reduced 5- to 8-cell, Day-6 morulae, and Day-8 blastocyst rates over unsorted sperm within the 3 culture systems analysed. Culture in SOF + BSA was the least efficient embryo culture system in terms of Day-7 blastocyst rates, both with unsorted and sorted sperm. Day-7 blastocyst rates produced with unsorted sperm after culture in Vero cells or SOF + FCS were significantly higher than those obtained after culture in SOF + BSA. Day-7 blastocyst rates obtained with sorted sperm and cultured in Vero cells or SOF + FCS were comparable to those obtained with unsorted sperm in SOF + BSA. No differences were detected between male- and female-sorted sperm (data not shown). Sorted sperm is an effective tool for producing sex-known embryos. Quality assessment (differential cell counts and cryosurvival) of embryos produced with sorted sperm under different conditions are being undertaken. Table 1.In vitro development of bovine embryos fertilized with either untreated or sorted sperm and cultured in Vero cells, SOF + FCS, or SOF + BSA Supported by INIA RTA2008.#0082. M. Muñoz, B. Trigal and D. Martin are sponsored by RYC08-03454, Cajastur and PTA2007-#0268-I. We acknowledge Sexing Technologies for collaboration.

148 EFFECTS OF Wnt3A SUPPLEMENTATION ON BOVINE BLASTOCYST CELL NUMBER AND ALLOCATION

2010 ◽  
Vol 22 (1) ◽  
pp. 232
Author(s):  
M. D. Goissis ◽  
P. J. Ross ◽  
J. B. Cibelli
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Cell Counts ◽  
Esc Derivation ◽  
Hatching Rates

Derivation of true bovine embryonic stem cells (ESC), as defined by their capacity to form robust teratomas and/or contribute to the germ line in chimeras, has not been achieved despite several attempts. It is possible that failures to derive bonafide bovine ESC are due to the inability of bovine embryonic cells to adapt to in vitro culture conditions that favor ESC derivation. Wnt pathways are involved in pluripotency and self-renewal of mouse and human ESC. Wnt signaling is also required for implantation competence in mouse blastocysts. Given the shared developmental potential between inner cell mass (ICM) and ESC, we hypothesized that Wnt could act on the ICM of bovine embryos increasing its proliferation potential. The objective of this study was to evaluate the effect of post-embryonic genome activation Wnt3A supplementation on blastocyst formation and cell allocation to ICM and trophectoderm (TE). In vitro fertilized bovine embryos at Day 4 of culture in KSOM medium were divided into 3 treatments: Control, no co-culture; co-culture with regular mouse embryonic fibroblasts (MEF); and co-culture with mouse L fibroblasts overexpressing Wnt3A protein (L-Wnt3A, Willert et al. 2003 Nature 423, 448-452). Embryos were cultured until Day 8 when blastocyst and hatching rates were recorded. Then, embryos were submitted to differential staining of ICM and TE by brief exposure to 0.25% Triton X-100 in PBS and staining with bisbenzimide and propidium iodide. Six IVF replications were performed and a total of 39 embryos were counted: 11 for Control, 16 for MEF, and 12 for L-Wnt3A. Only intact embryos after processing were used for cell count. Statistical analysis was performed by ANOVA using PROC MIXED of SAS software (SAS Institute Inc., Cary, NC, USA) in which each IVF was considered as a block with Tukey’s adjustment for mean comparison of rates and Bonferroni adjustments for mean comparison of cell counts. Results for blastocyst rate, hatching rate, ICM, TE, and total cell number are presented in the table below. Different superscript letters within columns indicate significant statistical difference (P < 0.05). These results indicate that L-Wnt3A fibroblast co-culture exerts a positive effect on bovine embryo cell number, resulting in a larger number of ICM cells in bovine embryos, which could be beneficial for ESC derivation attempts. Table 1.Blastocyst and hatching rates, ICM, TE, and total cell number results

Sign in / Sign up

Export Citation Format

Share Document