99 SURVIVAL RATE AND IN VITRO DEVELOPMENT OF IN VIVO-PRODUCED AND CRYOPRESERVED DOG EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 208
Author(s):  
M. R. Luz ◽  
C. C. Holanda ◽  
J. J. Pereira ◽  
N. S. Teixeira ◽  
R. Vantini ◽  
...  
Keyword(s):  
Survival Rate ◽  
Room Temperature ◽  
Reproductive Technologies ◽  
Water Bath ◽  
Embryo Cryopreservation ◽  
In Vitro Development ◽  
Long Period ◽  
Vitro Development

Dogs have been used as an experimental model for human genetic diseases and for research applied to endangered Canidae. Moreover, application of assisted reproductive technologies (ART) by dog breeders is increasing. The aim of this study was to evaluate the survival rate and in vitro development of in vivo-produced and cryopreserved dog embryos. Seven cross-bred bitches were submitted to ovariohysterectomy 12 days after first mating or artificial insemination, and embryos were recovered by uterine horn flushing with 30 mL of PBS/horn (Nutricell, Campinas, SP, Brazil). Grade 1 and 2 morulae (MO; n = 7; IETS) and blastocysts (BL; n = 14) were frozen. For freezing, embryos were immersed in glycerol 10% (GLY; Nutricell, Campinas, SP, Brazil) or ethylene glycol 3,0 M (EG; Nutricell, Campinas, SP, Brazil) for 10 min. Straws were placed in the machine at -7.0°C (TK 3000, Uberaba, MG, Brazil), and equilibrated for 2 min. Seeding was performed at -7.0°C, and another equilibrium period of 2 min was performed. A cooling rate of -0.5°C/min until -32.0°C was used. Embryos were stored in N2L until thawing. Prior to in vitro culture, embryos were removed from N2L, kept at room temperature for 10 s, and put in a water bath at 25°C for 20 s. Embryos frozen in GLY were washed for 5 min in each thawing solution for cryoprotectant removal (0.6 M sacarose + glycerol 5%; 0.6 M sacarose + glycerol 2.5% and 0.6 M sacarose; Nutricell, Campinas, SP, Brazil). After that, embryos were washed 10 times in holding solution (Holding Plus, Bioniche, Pullman, WA, USA). Embryos frozen in EG were kept at room temperature for 10 s, put in a water bath at 25°C for 20 s, and were directly washed 10 times in holding solution. Comparison among groups was performed by ANOVA. After thawing, 9/11 (81.8%) embryos frozen in EG had rupture of zona pelucidae and 2/11 (18.2%) were intact, whereas 9/10 (90.0%) embryos frozen in GLY were intact (P < 0.05). All intact embryos (n = 11) were morphologically normal and were transferred to SOF medium (Nutricell, Campinas, SP, Brazil), cultured for 168 h at 38.3°C, and evaluated at 24-h intervals. After the last evaluation, for both cryoprotectants, hatching rate was 0.0%, but all embryos were morphologically normal. The results of this study suggest that dog embryos have a high in vitro survival rate in standard protocols used for mammalian embryo cryopreservation when glycerol 10% is used as cryoprotectant and that a long period of culture (possibly more than 10 days) is required for dog embryo hatching. On the other hand, this long period for in vitro hatching may reflect the delayed hatching and implantation that occurs in vivo in this species. Financial by FAPES/MCT/CNPq/CT-INFRA n. 19/2006; Processo n. 36600737/2007, ES- Brasil.

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.

Stage specific expression of cell cycle genes during in vivo or in vitro development of ovarian follicles in sheep

2016 ◽  
Vol 143 ◽  
pp. 1-7 ◽  
Author(s):  
V. Praveen Chakravarthi ◽  
S.S.R. Kona ◽  
A.V.N. Siva Kumar ◽  
M. Bhaskara ◽  
V.H. Rao
Keyword(s):  
Cell Cycle ◽  
Ovarian Follicles ◽  
Specific Expression ◽  
In Vitro Development ◽  
Cell Cycle Genes ◽  
Vitro Development

174 THE EFFECT OF DIFFERENT OVARY TRANSPORT TEMPERATURES ON IN VITRO DEVELOPMENT AND POST-THAW SURVIVAL OF BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 203
Author(s):  
S. R. Cho ◽  
S. H. Choi ◽  
H. J. Kim ◽  
C. Y. Choe ◽  
H. J. Jin ◽  
...  
Keyword(s):  
Room Temperature ◽  
Development Rate ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Embryo Freezing ◽  
Nuclear Maturation ◽  
Vitro Development ◽  
And Control

The present study was carried out to investigate the effect of different ovary transport temperatures on in vitro development and post-thaw survivability of bovine embryos. Bovine ovaries were collected at a local slaughterhouse and transported at 4 different temperature categories to the laboratory: 7–10�C (T1), 11–17�C (T2), 18–25�C (T3), and above 26�C (control group). The cumulus–oocyte complexes (COCs) were aspirated from 2–8 mm antral follicles using a syringe with an 18 gauge needle. Selected COCs were washed in HEPES-buffered tissue culture medium (TCM-199) supplemented with 5% FBS. Sets of 50 COCs were matured for 22 h in 4-well dishes of TCM-199 supplemented with 5% FBS, 10 �g mL-1 LH, and 10 �g mL-1 FSH, that had been previously covered with mineral oil and equilibrated in an atmosphere of 5% CO2 in air at 39�C. Mature COCs were fertilized with frozen–thawed semen treated with BO medium. To evaluate nuclear maturation to the metaphase II stage, the matured COCs were fixed in 1 : 3 acetic acid–ethanol for 30 s and stained with 3% basic Fuchsin. For embryo freezing, Day 7 and 8 blastocysts were equilibrated for 15 min in 1.8 M ethylene glycol as a cryoprotectant. Embryos were loaded into 0.25-mL straws at room temperature, plunged directly into a cooling chamber, kept at -7�C for 10 min, including time for seeding, and further cooled to -35�C at -0.3�C min-1; after 2 min at this temperature, they were plunged into liquid nitrogen. Thawing was performed by keeping straws at room temperature for 10 s, followed by immersion in a water bath at 37�C. The appearance of the embryos was evaluated immediately after warming and again at 24-h intervals for at least 3 days. The development rate was assessed by the re-expansion of the blastocoel and the hatching of blastocysts. Results were compared by ANOVA. The rates of maturation (to metaphase II), cleavage, and development to blastocysts were compared among treatment groups. Furthermore, frozen–thawed blastocysts were in vitro cultured to compare the survivability among groups. The maturation rates in the T1, T2, and T3 groups (24/40, 60.0%; 25/41, 61.0%; and 30/44, 68.2%, respectively) were significantly lower than that in the control group (36/44, 81.8%; P &lt; 0.05). The cleavage rates in the T1 and T2 groups (61/116, 52.6% and 66/121, 54.5%) were significantly lower than that in the control group (112/134, 83.6%; P &lt; 0.05). However, there was no difference in the development rate to blastocysts among all groups (27.9–33.0%; P &gt; 0.05). The survivability of frozen–thawed embryos was significantly lower in the T1 group (6/13, 46.2%) than in the T2 (11/16, 68.8), T3 (13/18, 72.2%), and control groups (19/26, 73.1%; P &lt; 0.05). In conclusion, the results suggest that ovary transport at 26�C may be optimal for better in vitro development and survival of frozen–thawed embryos produced in vitro. Furthermore, exposure of ovaries to temperatures below 10�C during transport may significantly decrease both in vitro development and survivability of frozen-thawed blastocysts.

55 EFFECT OF FUSION-ACTIVATION INTERVAL AND EMBRYO AGGREGATION ON IN VITRO AND IN VIVO DEVELOPMENT OF CLONED BOVINE EMBRYOS PRODUCED BY HANDMADE CLONING

2010 ◽  
Vol 22 (1) ◽  
pp. 185
Author(s):  
R. P. C. Gerger ◽  
F. Forell ◽  
J. C. Mezzalira ◽  
F. Zago ◽  
F. K. Vieira ◽  
...  
Keyword(s):  
Somatic Cell ◽  
High Rate ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Cloned Bovine ◽  
Vitro Development ◽  
Handmade Cloning ◽  
Activation Intervals

Despite the apparent success of cloning by somatic cell nuclear transfer (SCNT), the efficiency in development to term remains low, with a high rate of losses occurring throughout pregnancy due to faulty reprogramming and conceptus abnormalities. As the ideal fusion-activation interval for optimal nuclear reprogramming after cloning is still ill-defined, the aim of this study was to determine the effect of 2 distinct fusion-activation intervals and embryo aggregation on in vitro development of cloned bovine embryos. Bovine COCs from slaughterhouse ovaries were used after IVM for the production of cloned embryos by handmade cloning, according to our established procedures (Ribeiro et al. 2009 Cloning Stem Cells, in press). Following cumulus and zona removal, oocytes were manually bisected, with hemi-cytoplasts selected by DNA staining. Two hemi-cytoplasts and an adult skin somatic cell were attached and fused with a 15V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1 DC pulse for 20 μs. Reconstructed embryos were activated in ionomycin exactly at 2 or 4 h post-fusion (2 hpf or 4 hpf), followed by an incubation in 6-DMAP for 4 h. Cloned embryos from both fusion-activation intervals were in vitro-cultured in the well of the well (WOW) system for 7 days, allocating one (1 × 100%) or two (2 × 100%) cloned embryos per WOW. Grade 1 Day-7 blastocysts were transferred to synchronous recipients. Cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, and pregnancy (Days 30 and 150) rates were compared using the chi-square or the Fisher test, with results from 9 replications summarized in Table 1. Increasing the fusion-activation interval to 4 h decreased cleavage but not blastocyst rates in 1 × 100% embryos. Also, blastocyst rates were lower in 1 × 100% embryos activated 2 h post-fusion. In general, cleavage and blastocysts rates for 2 × 100% embryos (91.5 and 46.0%) were higher than for 1 × 100% embryo counterparts (74.4 and 31.3%), respectively, regardless of the activation time. In addition, blastocyst rates for 4 hpf-activated embryos (50.3%), based on cleavage, were higher than for 2 hpf-activated embryos (38.3%), irrespective of the aggregation scheme. Nonetheless, despite differences in in vitro development, pregnancy rates and conceptus development in the first half of pregnancy were similar between groups. A longer fusion-activation interval (4 hpf) or embryo aggregation (2 × 100%) increased blastocyst yield but did not improve in vivo development and pregnancy maintenance following the transfer to female recipients in cattle. Table 1.In vitro and in vivo development of cloned bovine embryos This study was supported by FAPESP and CAPES, Brazil.

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

In vivo and in vitro development of in vitro fertilized bovine follicular oocytes obtained by laparoscopy

1985 ◽  
Vol 23 (1) ◽  
pp. 230 ◽  
Author(s):  
M.A. Sirard ◽  
R.D. Lambert ◽  
P. Guay ◽  
D.P. Ménard ◽  
M. Bedoya
Keyword(s):  
In Vitro Development ◽  
Vitro Development

Fasciation d'extrémités caulinaires du Celosia cristata (Amarantacées) cultivées in vitro

1981 ◽  
Vol 59 (8) ◽  
pp. 1367-1372 ◽  
Author(s):  
D. Driss-Ecole
Keyword(s):  
Indoleacetic Acid ◽  
Leaf Primordia ◽  
Root Initiation ◽  
Salt Mixture ◽  
Shoot Apices ◽  
In Vitro Development ◽  
Long Period ◽  
Celosia Cristata ◽  
Vitro Development

In vitro development of fasciation was achieved from the excised shoot apices of Celosia cristata in a 16-h photoperiod. An 8-h photoperiod produced no development. The explants consisted of the meristematic dome with two leaf primordia. The best results were obtained with a nutrient medium composed of Murashige and Skoog salt mixture, 30 g/L sucrose, 5 g/L agar, and 1 mg/L indoleacetic acid (IAA). Isolated shoot apices developed callus at the bases of the explants and numerous leaves on one, two, or three flattened and fasciated meristems. Root initiation occurred sometimes but only after a long period of culture.

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