scholarly journals Kinetics of early in vitro development of bovine in vivo- and in vitro-derived zygotes produced and/or cultured in chemically defined or serum-containing media

Reproduction ◽  
2002 ◽  
pp. 553-565 ◽  
Author(s):  
P Holm ◽  
PJ Booth ◽  
H Callesen
Keyword(s):  
Embryo Development ◽  
Time Lapse ◽  
Cell Cycles ◽  
In Vitro Development ◽  
Serum Supplementation ◽  
Defined Culture ◽  
Vitro Development ◽  
Kinetics Of

The kinetics of the in vitro development of early embryos from bovine zygotes derived in vitro and in vitro were compared, investigating the effect of serum during in vitro maturation and fertilization (IVM-IVF) and in culture. Zygotes were collected from superovulated heifers or produced in vitro from immature oocytes with or without serum supplementation, and cultured subsequently in defined culture medium (SOFaaci) with or without serum supplementation. Time-lapse images were recorded every 0.5 h throughout the culture period. More in vivo- than in vitro-derived zygotes developed to the compact morula or blastocyst stages (87% versus 47-54%, respectively; P < 0.05). Embryo development was blocked predominantly at the second or fourth cell cycles (28 and 29%). However, blastomeres degenerated at all cleavage stages. Serum supplementation during IVM-IVF resulted in abnormally sized blastomeres at first cleavage (defined serum: 20-22% versus in vivo-derived: 8%, P < 0.05). The duration of the second, third and fifth cell cycles of in vivo-derived zygotes were 1-5 h shorter compared with those of in vitro-derived zygotes cultured under similar conditions (P < 0.05). However, the kinetics of embryo development was affected by serum during IVM-IVF and culture. The first and fourth cell cycles were prolonged by 4-5 h in the absence of serum during IVM-IVF, whereas the presence of serum during culture decreased the duration of the fourth cell cycle and triggered premature blastulation. The results of this study illustrate the differences and similarities between the morphology and developmental kinetics of in vivo- and in vitro-derived zygotes, and show how serum supplementation during IVM-IVF and culture can alter these parameters.

35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 124
Author(s):  
C. Feltrin ◽  
M. Machado ◽  
L. M. V. Queiroz ◽  
M. A. S. Peixer ◽  
P. F. Malard ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Aggregation State ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

249 AN EFFICIENT AND REPEATABLE IN VITRO FERTILIZATION TECHNIQUE IN THE EQUINE FOR PRESERVATION OF ENDANGERED SPECIES

2015 ◽  
Vol 27 (1) ◽  
pp. 214
Author(s):  
C. Douet ◽  
O. Parodi ◽  
F. Reigner ◽  
P. Barrière ◽  
G. Goudet
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
In Vitro Development ◽  
Vitro Development

Most wild equids are currently endangered or threatened, as mentioned in the International Union for the Conservation of Nature Red List, and several domestic horse breeds are at risk of extinction. Genome resource banking requires cryoconservation of semen, oocytes, and/or embryos. Embryo production in equids is limited in vivo because routine induction of multiple ovulation is still ineffective. Embryo production in vitro allows the production of several embryos per cycle that could easily be frozen because of their small size. Intracytoplasmic sperm injection has been widely adopted to generate horse embryos in vitro; however, intracytoplasmic sperm injection is time-consuming and requires expensive equipment and expertise in micromanipulation. Several attempts to establish an efficient IVF technique in the equine were performed, but reported IVF rates remain quite low and no repeatable equine IVF technique was available. Our objective was to develop an efficient and repeatable IVF technique in the equine. Immature cumulus-oocyte complexes (COC) were collected either from slaughtered mares in a local slaughterhouse or from our experimental mares by ovum pick up (OPU). The COC were cultured for 26 h in an in vitro maturation (IVM) medium or in preovulatory follicular fluid (FF) collected by OPU, pre-incubated for 30 min in oviducal fluid collected from slaughtered females, co-incubated for 18 h with fresh spermatozoa treated with procain, and cultured in SOF for 30 h. They were fixed and analysed either after 18 h IVF (experiment 1) or after 30 h in vitro development (experiment 2). In experiment 1, COC were collected from slaughtered mares and analysed after 18 h IVF. Zygotes with 2 pronuclei were observed. The IVF rate was similar for oocytes matured in IVM medium (22/33, 67%) or FF (24/42, 57%; chi-square test, P > 0.05). In experiment 2, COC were collected from slaughtered mares and from experimental mares and analysed after 30 h of in vitro development. We observed zygotes with 2 highly decondensed pronuclei, pronuclei decondensation being the first step of embryo development. For oocytes collected from slaughtered mares, the percentage of zygotes was similar for oocytes matured in IVM medium (8/11, 73%) or FF (10/15, 67%). For oocytes collected by ovum pickup, the percentage was similar for IVM medium (3/5, 60%) or FF (6/8, 75%). We also observed some embryonic structures with several nuclei, but the quality of these embryos was poor. In conclusion, we have established an efficient IVM-IVF technique that allows the first step of embryo development. Because we obtained similar results for 4 years, we consider that this efficient technique is repeatable. Further experiments are in progress to improve the quality of the embryos.

scholarly journals 77CLONED EMBRYO DEVELOPMENT IN VITRO: COMPARISON OF CONVENTIONAL AND INDUCED ENUCLEATION PROCEDURES FOR BOVINE CYTOPLAST PREPARATION

2004 ◽  
Vol 16 (2) ◽  
pp. 160
Author(s):  
M.-K. Wang ◽  
E.W. Overstrom
Keyword(s):  
Embryo Development ◽  
Polar Body ◽  
Blastocyst Stage ◽  
In Vitro Development ◽  
Uv Illumination ◽  
Development In Vitro ◽  
Second Polar Body ◽  
Vitro Development

Induced enucleation (IE) of oocytes with demecolcine produces competent ooplasts for SCNT as demonstrated previously in mouse, goat, cow and pig. Whether bovine IE cytoplasts are more or less competent than conventionally enucleated MII oocytes to support nuclear reprogramming of somatic chromatin and embryo development in vitro is not known. This study compared in vitro development of cloned bovine embryos produced by conventional and IE enucleation methods. Three experimental groups were: (1) Parthenogenetic controls. In vitro-matured, MII-arrested bovine oocytes were activated by a single (1×Act, 10μM ionomycin in Tyrodes-HEPES, 5min) or double activation (2×Act; 1×Act, wash 5min, 10μgmL−1 cycloheximide [CHX] 20min, repeat 1×Act) followed by incubation in CHX and 5μgmL−1 cytochalasin B (CB) for 6h, and then culture (BARC medium) for 7 days. (2) Conventional SCNT. MII oocytes were enucleated by micromanipulation in HEPES-buffered enucleation medium (BARC containing 7.5μgmL−1 CB, 5μgmL−1 Hoechst 33342, 10% FBS) under UV illumination (3–5s). Donor cells (fibroblasts, passage 7–9) were inserted into the perivitelline space, and the reconstructed couplets activated (1×Act). Reconstructed couplets were then electrofused, placed in BARC medium containing 10μgmL−1 CHX and 5μgmL−1 CB (6h), and then cultured for 7 days. (3) IE SCNT. MII oocytes were activated (1×Act), placed into BARC-5% FBS containing 0.4μgmL−1 demecolcine (DEME), 10μgmL−1 CHX, 2μgmL−1 cytochalasin D for 20min, then 20min without DEME, then returned to DEME. At 1–1.5h post-activation, the extruding second polar body (PB2) containing nuclear chromatin was removed by micromanipulation, couplets were reconstructed and fused as above, and additionally activated (two pulses, 20–30V/mm, 20μs). Embryos were cultured in 10μgmL−1 CHX and 5μgmL−1 CB medium for 4–5 hour, then BARC for 7 days. The results (Table 1) reveal that 2×Act increases embryo development at Day 2, but not Day 7. Further, there are no significant differences in embryo development rates between conventional and IE SCNT protocols. Respectively, 46%, 32% and 21% of cleaved control (1×Act), conventional and IE embryos developed to 16 cells on Day 7. In vitro development of cleavage embryos to the blastocyst stage was greater in controls (25–32%) than in conventional (22%) and IE (17%) SCNT groups on Day 7. Further comparisons of in vivo development between conventional and IE SCNT methods following embryo transfer are warranted. Supported by ACT, Cyagra and USDA NRI \#2001-35205-09966. Table 1 Embryo development: Conventional v. induced enucleation

scholarly journals Necrostatin-1 Supplementation to Islet Tissue Culture Enhances the In-Vitro Development and Graft Function of Young Porcine Islets

2021 ◽  
Vol 22 (16) ◽  
pp. 8367
Author(s):  
Hien Lau ◽  
Shiri Li ◽  
Nicole Corrales ◽  
Samuel Rodriguez ◽  
Mohammadreza Mohammadi ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Oxygen Consumption ◽  
Insulin Secretion ◽  
Oxygen Consumption Rate ◽  
Consumption Rate ◽  
In Vitro Development ◽  
Porcine Islets ◽  
Vitro Development

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.

scholarly journals Single Cell Analysis Reveals That IL-4 Receptor/Stat6 Signaling Is Not Required for the In Vivo or In Vitro Development of CD4+Lymphocytes with a Th2 Cytokine Profile

2000 ◽  
Vol 164 (6) ◽  
pp. 3047-3055 ◽  
Author(s):  
Dragana Jankovic ◽  
Marika C. Kullberg ◽  
Nancy Noben-Trauth ◽  
Patricia Caspar ◽  
William E. Paul ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Cytokine Profile ◽  
Cell Analysis ◽  
In Vitro Development ◽  
Th2 Cytokine ◽  
Vitro Development ◽  
Cd4 Lymphocytes

scholarly journals 64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

2004 ◽  
Vol 16 (2) ◽  
pp. 154
Author(s):  
H.S. Park ◽  
M.Y. Lee ◽  
S.P. Hong ◽  
J.I. Jin ◽  
J.K. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P&lt;0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P&lt;0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P&lt;0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

scholarly journals Bioreactors as physiologicallike in vitro models

2020 ◽  
Author(s):  
Sara Mantero ◽  
Federica Boschetti
Keyword(s):  
Culture Conditions ◽  
In Vitro Models ◽  
In Vivo Models ◽  
In Vitro Development ◽  
Culture Cells ◽  
Vitro Development ◽  
Tissue Models ◽  
Mechanical Stretching

Bioreactors are powerful tools for in vitro development of engineered substitutes through controlled biological, physical, and mechanical culture conditions: bioreactor technology allows a closer in vitro replication of native tissues. One of bioreactors applications is the design of in vitro 3D tissue models as a bridge between 2D and in vivo models, allowing the application of 3R (replacement, reduction, refinement) principle. To this aim, bioreactors can be used to culture cells seeded on engineered scaffolds under in vivo-like conditions. Another key use of bioreactors is for perfusion decellularization of tissues and organs to be used as scaffolds. This contribution describes a dynamic stretching. bioreactor, imposing a mechanical stretching to the cultured constructs, allowing the development of skeletal muscle engineered constructs, and a decellularization bioreactor, designed for decellularization of blood vessels.

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