In vitro assessment of the viability of sheep zygotes after pronuclear microinjection

1990 ◽  
Vol 2 (6) ◽  
pp. 633 ◽  
Author(s):  
SK Walker ◽  
TM Heard ◽  
PJ Verma ◽  
GE Rogers ◽  
CS Bawden ◽  
...  
Keyword(s):  
Salmonella Typhimurium ◽  
In Vitro Culture ◽  
Fluid Medium ◽  
Pronuclear Microinjection ◽  
In Vivo Culture ◽  
Developmental Capacity ◽  
In Vitro Assessment ◽  
Oviduct Fluid

Microinjected sheep zygotes were cultured in synthetic oviduct fluid medium (SOFM) for either 1 or 3 days and their subsequent developmental capacity was compared with that of microinjected zygotes cultured in vivo. Two experiments were carried out, using zygotes microinjected with one of three gene constructs containing the CysE and CysM genes from Salmonella typhimurium. In Experiment 1, microinjected zygotes were allocated to one of three treatments: (1) immediate transfer to recipient ewes (in vivo culture) followed by recollection 1 or 3 days later and subsequent transfer of viable embryos to other recipient ewes, (2) culture in SOFM (in vitro culture) for either 1 or 3 days before transfer to recipient ewes, and (3) immediate transfer to recipient ewes without subsequent interference. Recipient ewes were slaughtered on Day 14 of pregnancy and the number of elongated conceptuses determined. Although fewer zygotes failed to divide during in vitro culture than during in vivo culture, there were, overall, no significant differences between treatments in the percentage of zygotes that developed into elongated conceptuses (32.6-50.0%). In Experiment 2, microinjected zygotes were transferred immediately to recipient ewes or cultured in vitro for either 1 or 3 days before transfer. The number of fetuses per ewe on Day 50 of pregnancy and the number of lambs delivered per ewe were recorded. Neither the percentage of recipient ewes that became pregnant (overall 114/166, 68.7%) nor the percentage of zygotes that developed into lambs (overall 186/803, 23.2%) was significantly influenced by the culture treatment or by the gene construct microinjected.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro culture of bovine nuclear transfer embryos in synthetic oviduct fluid medium (SOFM)

1992 ◽  
Vol 37 (1) ◽  
pp. 255
Author(s):  
K.J. McLaughlin ◽  
D.M. McLean ◽  
P.A. Lewis ◽  
L. Hicks ◽  
G. Stevens ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Fluid Medium ◽  
Oviduct Fluid

331 DEVELOPMENT OF CANINE SYNTHETIC OVIDUCT FLUID (cSOF) MEDIUM AND EFFECT OF THE cSOF IN IN VITRO NUCLEAR MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 273
Author(s):  
M. K. Kim ◽  
H. J. Oh ◽  
Y. H. Fibrianto ◽  
G. Jang ◽  
H. J. Kim ◽  
...  
Keyword(s):  
Statistical Analysis ◽  
Follicular Fluid ◽  
Total Protein ◽  
Nuclear Maturation ◽  
Surgical Methods ◽  
In Vivo Culture ◽  
Oviduct Fluid ◽  
Follicular Fluids

A bitch ovulates a primary oocyte that undergoes both maturation and fertilization within the oviduct fluid for 3 days. In an attempt to define the physiologically appropriate conditions for oocyte maturation in the bitch, in vitro conditions based upon the oviductal environment need to be established. The present study was conducted to develop canine synthetic oviduct fluid (cSOF) by investigating the composition of canine oviduct fluid, follicular fluid, and bursa fluid. The bursa and oviduct fluid were collected at Days 1 and 3 of ovulation, respectively. Before ovulation, follicles were punched and the fluid was collected by aspiration. Biochemical parameters (Ca, P, Mg, albumin, total protein, and glucose) were measured using a chromatographic enzymic method. Quantitative determination of electrolytes (Na, Cl, K) concentration in the follicular, bursa, and oviductal fluids was performed using an Electrolyte 5 Analyzer (Nora Biomedical, Waltham, MA, USA). The concentrations of sodium, potassium, and chloride were similar among oviduct (153.5, 5.2, and 121.5 mmol/L, respectively), bursa (149.5, 4.3, and 123 mmol/L, respectively), and follicular (147, 4.2, and 120.5 mmol/L, respectively) fluids. Glucose concentration was different in oviduct, bursa, and follicular fluids (1.09, 3.75, and 3.94 mmol/L, respectively). Total protein and magnesium concentrations were not different among the three fluids, but phosphorus concentration differed in oviduct, bursa, and follicular fluids (0.001, 0.044, and 0.024 g/L, respectively). The oviduct fluid showed lower concentrations of albumin and calcium (0.001 g/L and 1.372 mmol/L, respectively) compared to bursa (0.023 g/L and 2.532 mmol/L, respectively) or follicular fluid (0.025 g/L and 2.632 mmol/L, respectively). The cSOF1 and cSOF2 were developed on the basis of the oviduct and follicular fluids, respectively. Canine oocytes were recovered by slicing ovaries collected after ovariohysterectomy in bitches at follicular stages, and in vitro nuclear maturation of canine oocytes cultured in cSOF1 or cSOF2 were compared to that of intra-oviduct (in vivo) culture. For in vivo culture, the canine oocytes were transferred and cultured in intra-oviduct for 72 h and were recovered by intra-oviduct flushing using surgical methods. For in vitro culture, canine oocytes were cultured in cSOF1, cSOF2, or TCM-199 (control) for 72 h at 38.5�C in a humidified atmosphere of 5% CO2 in air. The experiment was replicated three times and statistical analysis was carried out by ANOVA with GLMs in the statistical analysis system program (SAS Institute, Inc., Cary, NC, USA). Nuclear maturation of canine oocytes to MII was not different in cSOF1, cSOF2, or intra-oviduct (2.5%, 2.5%, and 2.2%, respectively) compared to the control (1.6%). However, the degeneration rates were significantly higher in oocytes cultured in intra-oviduct (48.2%) compared to control, cSOF1, and cSOF2 (1.6%, 6.8%, and 7.5%, respectively). In conclusion, the present study analyzed the components of the oviduct, bursa, and follicular fluids and developed two canine synthetic oviduct fluids (cSOF1 and 2). In addition, the present study demonstrated that cSOFs can be used for in vitro maturation of canine oocytes.

In vitro embryo culture in the production of identical merino lambs by nuclear transplantation

1990 ◽  
Vol 2 (6) ◽  
pp. 619 ◽  
Author(s):  
KJ McLaughlin ◽  
L Davies ◽  
RF Seamark
Keyword(s):  
Nuclear Transplantation ◽  
Cell Stage ◽  
Fluid Medium ◽  
Stage Of Development ◽  
Simple Medium ◽  
Pregnancy Status ◽  
Oviduct Fluid ◽  
Similar Stage

This study examined the viability of embryos developed in vitro from 8- to 16-cell stage blastomeres fused with enucleated oocytes. Of 209 blastomeres recovered and subjected to manipulation and electrofusion procedures, 190 (91%) fused successfully, with 86 (45%) of those undergoing cleavage up to the 4- to 16-cell stage when cultured for 66 h in a synthetic oviduct fluid medium. The viability of the embryos was examined by transferring them to recipient ewes and determining the ewes' pregnancy status by ultrasound on Day 45. Of 86 embryos transferred, 14 developed to fetuses in 8 of the 36 recipients, including four sets of identical twins and one set of quads. In contrast, with uncultured and unmanipulated embryos, 15 fetuses developed from 19 embryos transferred at a similar stage of development. The viability of embryos derived from manipulated zygotes cultured in vitro was comparable to that previously reported for studies employing in vivo culture, indicating the potential of in vitro culture systems based on a simple medium for nuclear-transplantation embryos.

Effect of hexoses and gonadotrophin supplementation on bovine oocyte nuclear maturation during in vitro maturation in a synthetic follicle fluid medium

2005 ◽  
Vol 17 (4) ◽  
pp. 407 ◽  
Author(s):  
Melanie L. Sutton-McDowall ◽  
Robert B. Gilchrist ◽  
Jeremy G. Thompson
Keyword(s):  
Polar Body ◽  
Culture Conditions ◽  
Fluid Medium ◽  
Nuclear Maturation ◽  
Meiotic Progression ◽  
Developmental Capacity ◽  
Polar Body Formation ◽  
Bovine Oocytes

In vitro oocyte maturation (IVM) culture conditions have been relatively unchanged over the past few decades and remain suboptimal. In contrast, studies of the in vivo environment have led to significant improvements to in vitro embryo culture technologies. The aim of the present study was to determine the effect of maturing bovine cumulus–oocyte complexes (COCs) in medium based on the composition of bovine follicular fluid (Bovine VitroMat; Cook Australia, Eight Mile Plain, Qld, Australia). In particular, the effect of different glucose concentrations and glucosamine supplementation on meiotic maturation was determined. Culturing COCs in the presence of gonadotrophins in Bovine VitroMat, containing either physiological glucose concentrations (2.3 mm) or 5.6 mm (equivalent to levels in Tissue Culture Medium 199 (TCM199)) supplemented with glucosamine resulted in comparable cumulus expansion to COCs cultured in TCM199 plus gonadotrophins. However, nuclear maturation was 1.3-fold lower in Bovine VitroMat cultures containing 2.3 mm glucose compared with 5.6 mm glucose and this effect was independent of glucosamine supplementation. Investigations into the effects of different glucose concentrations and gonadotrophin supplementation during the initial 6 h of maturation demonstrated that COCs cultured in Bovine VitroMat with 5.6 mm glucose without gonadotrophins had a twofold acceleration of the rate of meiotic resumption, yet the rate of polar body formation was decreased by approximately 20% compared with cultures in 2.3 mm glucose and TCM199. However, this effect was not seen when COCs were cultured for the initial 16 h in Bovine VitroMat + 5.6 mm minus gonadotrophins or in Bovine VitroMat + 2.3 mm glucose ± gonadotrophins. These data demonstrate that glucose concentrations and the timing of the introduction of gonadotrophin during IVM have variable effects on nuclear maturation. Manipulation of glucose concentrations may be a mechanism to influence oocyte meiotic progression and may lead to the development of improved IVM systems, allowing for an increased developmental capacity of bovine oocytes.

scholarly journals Expression Profile of New Marker Genes Involved in Differentiation of Canine Adipose-Derived Stem Cells into Osteoblasts

2021 ◽  
Vol 22 (13) ◽  
pp. 6663
Author(s):  
Maurycy Jankowski ◽  
Mariusz Kaczmarek ◽  
Grzegorz Wąsiatycz ◽  
Claudia Dompe ◽  
Paul Mozdziak ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Osteogenic Differentiation ◽  
Molecular Mechanisms ◽  
Marker Genes ◽  
P Value ◽  
Illumina Platform

Next-generation sequencing (RNAseq) analysis of gene expression changes during the long-term in vitro culture and osteogenic differentiation of ASCs remains to be important, as the analysis provides important clues toward employing stem cells as a therapeutic intervention. In this study, the cells were isolated from adipose tissue obtained during routine surgical procedures and subjected to 14-day in vitro culture and differentiation. The mRNA transcript levels were evaluated using the Illumina platform, resulting in the detection of 19,856 gene transcripts. The most differentially expressed genes (fold change >|2|, adjusted p value < 0.05), between day 1, day 14 and differentiated cell cultures were extracted and subjected to bioinformatical analysis based on the R programming language. The results of this study provide molecular insight into the processes that occur during long-term in vitro culture and osteogenic differentiation of ASCs, allowing the re-evaluation of the roles of some genes in MSC progression towards a range of lineages. The results improve the knowledge of the molecular mechanisms associated with long-term in vitro culture and differentiation of ASCs, as well as providing a point of reference for potential in vivo and clinical studies regarding these cells’ application in regenerative medicine.

In vitro assessment of food-derived-glucose bioaccessibility and bioavailability in bicameral cell culture system

2020 ◽  
Vol 45 (5) ◽  
pp. 631-637
Author(s):  
Cansu Ozel-Tasci ◽  
Gozde Pilatin ◽  
Ozgur Edeer ◽  
Sukru Gulec
Keyword(s):  
Cell Culture ◽  
Culture System ◽  
Metabolic Diseases ◽  
Enzymatic Digestion ◽  
Cell Culture System ◽  
Culture Studies ◽  
Experimental Approaches ◽  
In Vitro Assessment

AbstractBackgroundFunctional foods can help prevent metabolic diseases, and it is essential to evaluate functional characteristics of foods through in vitro and in vivo experimental approaches.ObjectiveWe aimed to use the bicameral cell culture system combined with the in vitro digestion to evaluate glucose bioavailability.Materials and methodsCake, almond paste, and pudding were modified by adding fiber and replacing sugar with sweeteners and polyols. Digestion process was modeled in test tubes. Rat enterocyte cells (IEC-6) were grown in a bicameral cell culture system to mimic the physiological characteristics of the human intestine. The glucose bioaccessibility and cellular glucose efflux were measured by glucose oxidase assay.Results and discussionThe glucose bioaccessibilities of modified foods were significantly lower (cake: 2.6 fold, almond paste: 9.2 fold, pudding 2.8 fold) than the controls. Cellular glucose effluxes also decreased in the modified cake, almond paste, and pudding by 2.2, 4, and 2 fold respectively compared to their controls.ConclusionOur results suggest that combining in vitro enzymatic digestion with cell culture studies can be a practical way to test in vitro glucose bioaccessibility and bioavailability in functional food development.

In vivo survival of transferred sheep embryos following puncture of the zona pellucida and in vitro culture

1994 ◽  
Vol 35 (1-2) ◽  
pp. 81-89 ◽  
Author(s):  
P.A. Pugh ◽  
J.G. Thompson ◽  
K. Logan ◽  
H.R. Tervit
Keyword(s):  
In Vitro Culture ◽  
Zona Pellucida

scholarly journals In Vivo and In Vitro Characterizations of Nanobodies Raised Against the Melibiose Permease melB of Salmonella Typhimurium

2021 ◽  
Vol 120 (3) ◽  
pp. 74a
Author(s):  
Satoshi Katsube ◽  
Katleen Willibal ◽  
Elena B. Tikhonova ◽  
Hariharan Parameswaran ◽  
Sangama Vemulapally ◽  
...  
Keyword(s):  
Salmonella Typhimurium
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