138 Ovarian factors associated with bovine in vitro embryo development and quality in an individual culture system

2022 ◽  
Vol 34 (2) ◽  
pp. 307
Author(s):  
N. Azari-Dolatabad ◽  
C. Benedetti ◽  
A. Fernandez Montoro ◽  
D. Angel Velez ◽  
G. Residwaiti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
Factors Associated ◽  
Individual Culture

35 IN VITRO BOVINE EMBRYO DEVELOPMENT AFTER NUCLEAR TRANSFER BY HANDMADE CLONING USING A MODIFIED WOW CULTURE SYSTEM

2006 ◽  
Vol 18 (2) ◽  
pp. 126 ◽  
Author(s):  
C. Feltrin ◽  
F. Forell ◽  
L. dos Santos ◽  
J. L. Rodrigues
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Inner Diameter ◽  
Group 2 ◽  
Group 1 ◽  
Handmade Cloning

The effect of the microenvironment on embryo development during in vitro culture of zona-free embryos after nuclear transfer is still unclear. The aim of this experiment was to determine the effect of the dimensions of the well (WOW; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256-264) culture system on the in vitro development of handmade cloned bovine embryos to the blastocyst stage. Appropriately ground steel needles were pressed slightly by hand to the bottom of the well of a polystyrene four-well dish (176740, Nunc, Life Technologies AS, Roskilde, Denmark). Embryos were produced by the handmade cloning (HMC) technique (Vajta et al. 2003 Biol. Reprod. 68, 571-578) with modifications, using primary cultures of skin fibroblast cells from an adult cow as nuclear donors. Cumulus-oocyte complexes were in vitro-matured in M-199 supplemented with 10% estrous cow serum (ECS), FSH, hCG, and estradiol (E2) for 17 h. After maturation, cumulus cells were removed by pipetting. Following zona pellucida removal in 0.5% protease (Sigma, Brazil), zona-free oocytes were incubated for 15 min in 5 mg/mL cytochalasin B (Sigma) and subsequently hand-bisected and screened for nuclear material under UV light after incubation in 10 mg/mL bisbenzimide (Hoechst 33342). Next, two enucleated halves and one donor cell were aggregated after a quick exposure to phytohemagglutinin (PHA) and subsequently fused by two electrical DC pulses of 1 kV/cm for 20 �s, in a BTX 453 chamber coupled to an ECM 2001 Electro Cell Manipulator System (BTX, Inc., San Diego, CA, USA), with additional exposure to brief pre- and post-fusion AC pulses of 15 V. Reconstructed embryos were chemically activated in 5 mM ionomycin (Sigma) for 5 min, followed by 2 mM 6-DMAP (Sigma) for 2.5 h. Finally, activated reconstructed cloned embryos were in vitro-cultured in one of two WOW culture systems (larger vs. smaller micro-wells) in 4-well plates containing 400 mL modified SOF medium supplemented with 10% ECS, under mineral oil, at 5% CO2, 5% O2 and 90% N2, and 39�C for 7 days. In Group 1 (large-size micro-well), embryos were cultured in individual cylindrical micro-wells with an inner diameter and depth of approximately 280 and 250 mm, respectively, whereas in Group 2 (small size micro-well), embryos were cultured in individual conical micro-wells with approximately 130 mm inner diameter and 150 mm depth. Data analysis was performed by the chi-square test. After four replicates, cleavage rates were significantly higher (P < 0.05) in Group 2 (51/63, 80.9%) than in Group 1 (43/67, 64.1%). Embryo development to the blastocyst stage was also greater (P < 0.05) in the small micro-wells (16/63, 25.3%) than in the large ones (8/67, 11.9%). In summary, these results show a significant increase in cleavage and blastocyst developmental rates in handmade cloned embryos cultured in a modified WOW system using individual small size micro-wells, suggesting that a small, tighter micro-well provides favorable in vitro conditions for embryo development.

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.

45 THE EFFECT OF MEIOTIC STAGE OF BOVINE OOCYTES ON THE SURVIVAL OF VITRIFIED CUMULUS–OOCYTE COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 135 ◽  
Author(s):  
J. R. Prentice ◽  
J. Singh ◽  
M. Anzar
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Early Embryo ◽  
Time Intervals ◽  
Early Embryo Development ◽  
In Vitro Culture System ◽  
Bovine Oocytes

Vitrification is a rapid freezing method in which cells/tissues are frozen in a glass state without ice crystal formation. However, vitrification of bovine oocytes is challenging due to their complex structure and sensitivity to chilling. Oocytes at the germinal vesicle (GV) stage of maturation are thought to be less prone to chromosomal and microtubular damage during cryopreservation because no spindle is present and genetic material is contained within the nucleus. However, immature oocytes are thought to be more sensitive to osmotic stress and have lower cell membrane stability than mature, metaphase II (MII) stage oocytes. The present studies aimed to validate the in vitro culture system used in our laboratory and to evaluate the effect of vitrification of bovine cumulus-oocyte complexes (COC) at different meiotic stages on their in vitro maturation (IVM), cleavage and early embryo development. Analyses were conducted on each dataset with PROC GLIMMIX in SAS using binary distribution (for yes/no response variable) and considering replicate as a random factor. In Experiment 1, meiotic progression of oocytes was evaluated at different time intervals during IVM. The following COC stages were predominantly found at different IVM time intervals: GV (89%) at 0 h, GV (47%) and germinal vesicle breakdown (GVBD; 44%) at 6 h, metaphase I (MI; 90%) at 12 h and MII (84%) at 22 h (n > 62 oocytes at each time group). In Experiment 2, bovine COC at 0, 6, 12 and 22 h of IVM were exposed to vitrification solution (15% dimethyl sulfoxide + 15% ethylene glycol + 0.5 M sucrose + 20% CS in TCM-199), loaded onto a cryotop device and vitrified by plunging in liquid nitrogen. Following warming (1 min in 0.5 M sucrose + 20% CS in TCM-199), COC completed 22 h of IVM and the nuclear stage was evaluated with lamin A/C-4′6-diamidino-2-phenylindole staining. Upon completion of 22 h of IVM, 23, 23, 35 and 89% of oocytes from 0-, 6-, 12- and 22-h groups, respectively were detected at MII (P < 0.0001). In Experiment 3, cleavage and embryo development of oocytes vitrified at 0, 12 and 22 h of IVM were evaluated. The cleavage rate did not differ among vitrification groups (i.e. 14% at 0 h, 17% at 12 h and 14% at 22 h; P = 0.825). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified (control) group than in vitrified groups (i.e. 73 vs 15% and 22 vs 0.3%, respectively). In conclusion, the maturation kinetics validated our in vitro culture system and vitrification adversely affected the ability of bovine oocytes to undergo in vitro maturation to the MII stage, in vitro fertilization and early embryo development. Vitrification of oocytes at GV, MI and MII stages of nuclear maturation did not differ in their subsequent survivability. This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.

scholarly journals Effect of various co-culture systems on embryo development in ovine

2013 ◽  
Vol 58 (No. 10) ◽  
pp. 443-452 ◽  
Author(s):  
B. Heidari ◽  
A. Shirazi ◽  
M.-M. Naderi ◽  
M.-M. Akhondi ◽  
H. Hassanpour ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
Cell Number ◽  
Culture Conditions ◽  
The Other ◽  
Culture Period ◽  
Hatching Rate ◽  
Embryonic Fibroblast ◽  
One Way Anova

Considering the advent of mesenchymal stem cells (MSCs) as a new source of somatic cells in embryo co-culture system, the current study was aimed to compare in vitro embryo development using embryonic MSCs monolayer with embryonic fibroblast cells (EFCs), oviductal epithelial cells (OECs), and cell-free culture system. The IVM/IVF presumptive sheep zygotes were randomly cultured in different culture conditions as follows: (1) SOFaaBSA medium for the whole culture period (SOF, n = 371), (2) SOFaaBSA medium for the first 3 days followed by co-culturing with MSCs for the next 5 days (SOF-MSCs, n = 120), (3) co-culturing with MSCs for the first 3 days followed by culture in SOFaaBSA medium for the next 5 days (MSCs-SOF, n = 133), (4) co-culturing with MSCs for the whole culture period (MSCs, n = 212), (5) SOFaaBSA medium for the first 3 days followed by co-culturing with EFCs for the next 5 days (SOF-EFCs, n = 132), (6) co-culturing with EFCs for the first 3 days followed by culture in SOFaaBSA medium for the next 5 days (EFCs-SOF, n = 165), (7) co-culturing with EFCs for the whole culture period (EFCs, n = 236), and (8) co-culturing with OECs for the whole culture period (OECs, n = 255). One-Way ANOVA by multiple pairwise comparisons using Tukey&rsquo;s test was performed. Co-culturing in MSCs group had no superiority over EFCs and OECs groups. Though, when co-culturing with MSCs and EFCs was limited to the first 3 days of culture, the embryo development indices were improved compared to the other co-cultured groups. Considering both the hatching rate and total cell number, the application of MSCs for the first 3 days of culture (MSCs-SOF) was superior to the other co-culture and SOF groups. &nbsp;

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.

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.

Sealed culture system for supporting mouse preimplantation embryo development in vitro

2020 ◽  
Vol 32 (9) ◽  
pp. 879
Author(s):  
Jie Liu ◽  
Zhao Wang ◽  
Zhen Gao ◽  
Hui Zhang ◽  
Jianfeng Gu ◽  
...  
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Culture System ◽  
Transforming Growth Factor ◽  
Expression Patterns ◽  
Control Group ◽  
Cell Stage ◽  
Preimplantation Embryo Development ◽  
Transforming Growth Factor Α

This study investigated the possibility of a sealed culture system in polymerase chain reaction (PCR) tubes to maintain embryo development. The embryo density that could support the development of 2-cell stage mouse embryos to the hatching stage was determined. At an embryo density of 1:2 (100 embryos cultured in 200μL CZB medium that had been pretreated with a reference gas containing 5% O2), the developmental rate was higher and fewer embryos exhibited reactive oxygen species- or hypoxia-induced injury compared with other sealed culture groups. Expression of growth factors (insulin-like growth factor (IGF) 1, IGF2, epidermal growth factor and transforming growth factor-α) and their receptors was evaluated, with similar expression patterns seen for embryos in sealed culture (5% O2, embryo density of 1:2) compared with the control group (embryos cultured in microdrops and placed in a 37°C, 5% CO2 water-jacketed incubator; P&gt;0.05). After transfer of blastocysts generated by the sealed culture into recipients, there were no obvious differences in the rate of normal live pups births between the sealed culture and control groups (P&gt;0.05). Thus, the sealed embryo culture system in PCR tubes is feasible for use in situations which cannot use a traditional incubator, such as in space and during the transport of embryos.

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