Development in vitro of the female germ cells of the polychaete Ophryotrocha labronica

Development ◽  
1985 ◽  
Vol 85 (1) ◽  
pp. 151-161
Author(s):  
Hadar Emanuelsson ◽  
Siw Anehus
Keyword(s):  
Amino Acids ◽  
Germ Cells ◽  
Nurse Cell ◽  
Sea Water ◽  
Calf Serum ◽  
Growth Period ◽  
Foetal Calf Serum ◽  
Leading Role ◽  
Development In Vitro

In the polychaete Ophryotrocha labronica each oocyte is during its growth period associated with a single nurse cell. The fact that the oocyte-nurse cell pairs occur isolated in the female coelom makes them easily removable for analysis of their developmental ability in vitro. Using Dulbecco's modified Eagle medium supplemented with amino acids, nucleosides, foetal calf serum and sea water, we have managed to support development in vitro of germ cell pairs from early and mid-oogenesis until maturation of the oocyte, when the nurse cell degenerates and the oocyte enters meiotic metaphase. Radiolabelling of germ cells in mid-oogenesis with tritiated amino acids and uridine during the first day of incubation indicates normal development with synthesis of RNA and protein, and pulses two days later verify a continued normal protein synthesis and yolk formation. The investigation confirms autosynthesis of yolk proteins in the germ cells of this species and indicates a leading role of the nurse cell in the process.

Drug activity against Onchocerca gutturosa males in vitro: a model for chemotherapeutic research on onchocerciasis

1987 ◽  
Vol 61 (4) ◽  
pp. 271-281 ◽  
Author(s):  
Simon Townson ◽  
C. Connelly ◽  
A. Dobinson ◽  
R. Muller
Keyword(s):  
Culture System ◽  
Serum Proteins ◽  
Good Condition ◽  
Calf Serum ◽  
New Drugs ◽  
Foetal Calf Serum ◽  
Partial Recovery ◽  
Feeder Cells ◽  
Compound Identification

ABSTRACTAn in vitro system for chemotherapeutic research using adult male Onchocerca gutturosa has been developed as a model for O. volvulus. Using a culture system consisting of medium MEM+10% heat inactivated foetal calf serum (IFCS)+LLCMK2 (monkey kidney) feeder cells in an atmosphere of 5% CO2 in air, we examined the effects of a range of antiparasitic drugs on worm motility. Ivermectin, levamisole, furapyrimidone, Mel W, chloroquine, metrifonate, flubendazole, amoscanate and the Ciba-Geigy compounds CGP 6140, CGP 20′376 and CGI 17658 either immobilized or significantly reduced motility levels at a concentration of 5x10−5M or less within a 7-day period. Worms were affected at very low concentrations by ivermectin (effective conc. to reduce motility levels to 50% of controls, 3.14x10−8M), levamisole (7.95x10−8M), CGP 6140 (8.87x10−9M) and CGP 20′376 (2.78x10−8M). Difficulties were experienced in accurately repeating the immotile endpoint for levamisole due to an inconsistent partial recovery of motility. Over a 7-day period diethylcarbamazine had little effect on motility levels, while suramin caused a slight increase in activity compared to controls at some timepoints. Subsequent experiments demonstrated some differences in drug efficacy depending on the presence or absence of serum and feeder cells in the culture system probably because of drug avidly binding to serum proteins. However, serum and cells were found to be essential ingredients of the culture system to maintain worms in good condition, indicating that new drugs should be evaluated both in the presence and absence of serum and cells. Comparisons were made between the responses of O. gutturosa and Brugia pahangi to certain drugs and these species were found to significantly differ in their sensitivities to ivermectin and a novel compound (Wellcome), indicating that Onchocerca parasites should be used wherever possible for compound identification and development intended for the treatment of onchocerciasis. The in vitro system described here, using male O. gutturosa, provides a basis for further research and a practical alternative to O. volvulus.

‘Spontaneous’ sex reversal in organ cultures of the embryonic male gonad of the bird

Development ◽  
1974 ◽  
Vol 31 (3) ◽  
pp. 611-620
Author(s):  
Gregory F. Erickson
Keyword(s):  
Germ Cells ◽  
Primordial Germ Cells ◽  
Calf Serum ◽  
Sex Reversal ◽  
Biologically Active ◽  
Foetal Calf Serum ◽  
Developmental Sequence ◽  
Organ Cultures ◽  
Male Gonad ◽  
Ovarian Cortex

The left embryonic testis of the bird (4–8 days of incubation) was organ cultured in medium that contained 10% foetal calf serum. Under these conditions, the germinal epithelium (GE) of the 4-day gonad differentiates into an ovarian cortex and the male primordial germ cells (PGCs) complete a developmental sequence similar to normal oocytes, i.e. they divide mitotically, develop a Balbiani body, divide synchronously in groups of two, four, and eight germ cells, and some enter pre-leptotene. No medullary tissue develops in the 4-day explants. The pieces of 6- and 8-day gonad differentiate into true ovotestes in which the GE develops into a cortex and the medulla develops into seminiferous cords. The PGCs in the cortex differentiate as oocytes and those in the seminiferous cords differentiate as spermatogonia. The possibility that biologically active oestrogens are present in the growth medium is discussed.

Maturation of porcine oocytes for 33 or 44 hours in vitro in the presence or absence of serum

1998 ◽  
Vol 1998 ◽  
pp. 180-180
Author(s):  
K. Rust ◽  
M.E. Staines ◽  
GJ. McCallum ◽  
N.S. Prathalingam ◽  
S.A. Edwards ◽  
...  
Keyword(s):  
Disease Transmission ◽  
Calf Serum ◽  
International Markets ◽  
Foetal Calf Serum ◽  
Maturation Time ◽  
Nuclear Maturation ◽  
Defined Media ◽  
Porcine Embryo ◽  
Disease Risks

Porcine embryo production in vitrois providing the impetus for the development of cryopreservation strategies aimed at welfare-friendly domestic and international marketing and movement of stock in a manner that minimises risks of disease transmission. In the context of disease risks, defined media, which avoid the use of serum and other biohazardous products, are likely to become essential in the production of embryos for international markets. In preparing for this situation, the present comparative study investigated in vitronuclear maturation of porcine oocytes in the presence of either foetal calf serum (FCS) or polyvinyl alcohol (PVA). In addition, the effect of restricting the maturation time to 33 rather than 44 hours was examined.

Oesophagostomum radiatum: inhibition of in vitro development by newborn calf serum

1990 ◽  
Vol 64 (1) ◽  
pp. 9-14
Author(s):  
I. J. East ◽  
C. J. Fitzgerald
Keyword(s):  
Inhibitory Action ◽  
Natural Infection ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Newborn Calf Serum ◽  
In Vitro Development ◽  
Specific Antibodies ◽  
Serum Inhibition ◽  
Vitro Development

ABSTRACTOesophagostomum radiatum developed to fourth stage larvae after 14 days in in vitro culture. However, development was totally inhibited if the standard 50% foetal calf serum in the medium was replaced by newborn calf serum. Inhibition did not occur with serum from cattle immune to O. radiatum through natural infection or experimental vaccination irrespective of the titre of specific antibodies to O. radiatum in each serum. The inhibitory action of NCS could be abolished by heat treatment at 56°C for 1 h but not by dialysis or repeated freeze-thawing. The inhibition was not consistent with observed differences in the activity of 19 enzymes in the various sera or the absence of various thiol-containing stimulants of worm development.

Trans-tegumental absorption of L-alanine and L-leucine by a monogenean, Diclidophora merlangi

Parasitology ◽  
1978 ◽  
Vol 76 (1) ◽  
pp. 29-37 ◽  
Author(s):  
D. W. Halton
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Sea Water ◽  
Low Molecular Weight ◽  
Acid Uptake ◽  
Neutral Amino Acids ◽  
Freeze Dried ◽  
Significant Difference

SummaryAn in vitro investigation has been made of the relative roles of the gut and tegument in the absorption of the neutral amino acids L-alanine and L-leucine by a marine fish-gill parasite, Diclidophora merlangi. The use of ligatures to preclude oral ingestion of trace-labelled medium has proved inadequate, invariably damaging the tegument, as revealed by stereoscan electron microscopy, and resulting in artifactual levels of absorption. Three alternative procedures have given consistently reliable data on the route of entry of low molecular weight substrates. (1) Ultrastructural examination of worms previously incubated in electron-dense cationic tracers has shown that, in vitro, there is no oral intake of sea water. (2) The suspending of worms in trace-labelled medium with the mouth out of the medium and comparing amino acid uptake with that of worms totally immersed in medium has revealed no statistically significant difference in the absorption levels. (3) Application of section (freeze-dried) auto-radiography to detect diffusible isotope has demonstrated directly transtegumental absorption of a neutral amino acid. It is concluded from these experiments that Diclidophora has a tegumental transport system for absorbing certain neutral amino acids, and whilst, clearly, the worm is sanguinivorous and digests blood in a well-developed gut, it may also be capable of supplementing this diet with low molecular weight organic nutrient absorbed directly from sea water via the tegument.

Effects of amino acids on development in vitro of cleavage-stage bovine embryos into blastocysts

1996 ◽  
Vol 8 (5) ◽  
pp. 835 ◽  
Author(s):  
T Pinyopummintr ◽  
BD Bavister
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Embryo Development ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Development In Vitro ◽  
Defined Culture

Effects of amino acids on early bovine embryo development in vitro were examined using a chemically-defined, protein-free culture medium. Bovine embryos produced in vitro were cultured from 18 h to 72 h post insemination in a simple medium containing lactate as the only energy source except for the amino acid treatments. Subsequently, embryos were transferred to TCM-199 supplemented with serum for blastocyst development to substantiate their developmental competence. Treatments were: (1) non-essential amino acids from TCM-199 (NEA); (2) essential amino acids from TCM-199 (EA); (3) NEA+EA; (4) Eagle's minimum essential medium amino acids (MEM AA); (5) 11 amino acids present in HECM-6 (11 AA); and (6) 0.2 mM glutamine (GLN). A higher proportion of embryos (percentage of inseminated ova) cleaved to the > or = 8-cell stage by 72 h post insemination in NEA (56.7%), EA (41.2%), 11 AA (40.3%) and GLN (51.1%) than in either NEA+EA (30.0%) or MEM AA (33.1%). However, after transfer to complex medium, embryos that had developed in EA, as well as those in MEM AA or NEA+EA, produced significantly fewer blastocysts (37.1%, 34.4% and 25.6% respectively) than those in NEA (56.7%), GLN (48.9%) or 11 AA (37.7%). The ability of blastocysts to hatch from their zonae pellucidae was also affected by amino acid treatment during cleavage stages. The present study indicated that the addition of NEA or GLN or 11 AA to a chemically-defined culture medium during the cleavage phase of bovine embryo development increases their subsequent ability to reach the blastocyst stage. These data have implications for understanding the nutritional needs of bovine embryos produced in vitro and for optimizing the composition of culture media to support their development.

Echinococcus granulosus: observations on strobilar development in in vitro monophasic culture

1995 ◽  
Vol 69 (2) ◽  
pp. 173-175 ◽  
Author(s):  
F. Ponce Gordo ◽  
C. Cuesta Bandera
Keyword(s):  
Liquid Medium ◽  
Echinococcus Granulosus ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Stages Of Development ◽  
Protein Substrate ◽  
Cultivation Technique ◽  
Biphasic Medium

AbstractThe in vitro cultivation technique of Echinococcus granulosus protoscoleces usually states the necessity of a biphasic medium with a solid protein substrate for strobilar development to take place; otherwise, in a monophasic medium, protoscoleces follow a vesicular development. However, in some monocphasic cultures, the development of several strobilate individuals (in different quantities and stages of development, depending on the culture) were observed. The only known diference form cultures made previously and snice, where the development was vesicular, was the batch of foetal calf serum used in the constitution of the liquid medium, and this is presumed to be the cause of this unexpected strobilar development.

86 SIMPLE METHOD FOR EMULSIFICATION OF LIPOPHILIC NUTRIENTS THAT AFFECT PRE-IMPLANTATION DEVELOPMENT OF BOVINE EMBRYOS IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 157
Author(s):  
S. Ikeda
Keyword(s):  
Culture Media ◽  
Calf Serum ◽  
Hatching Rate ◽  
Cell Stage ◽  
Simple Method ◽  
Antioxidative Vitamins ◽  
Blastocyst Rate ◽  
Development In Vitro ◽  
Additive Control

In order to investigate the effects of bioactive lipophilic nutrients on mammalian pre-implantation embryos in vitro, amphipathic vehicles are commonly used to dissolve the lipophilic substances into culture media. However, easy emulsification of these nutrients would facilitate medium preparation. We report here a simple method for emulsification of lipophilic nutrients that affect bovine pre-implantation embryonic development in vitro. We investigated the effects of emulsified oleic acid (OA) or a mixture of antioxidative vitamins – vitamin E (VE) and β-carotene (BC). Polyglyceryl-10 laurate (P10L) was used as an emulsifier and was dissolved in sterile water at 5.05% (wt/wt) in glass vials. One percent (wt/wt) of OA or a mixture of VE (α-tocopherol) and BC (VE : BC = 1000 : 1 wt/wt) was added into the vial and mixed by using a magnetic stirrer. After first exhibiting white turbidity, the solution became transparent and stabilised, indicating stable emulsification. The emulsified OA and VE+BC were designated as emOA and emVEBC, respectively. Cumulus-enclosed oocytes obtained from abattoir bovine ovaries were in vitro-matured (IVM) for 22 h in modified synthetic oviduct fluid (mSOF) supplemented with 10% (vol/vol) fetal calf serum and 0.2 IU mL–1 FSH. After IVM, the oocytes were subjected to IVF with Percoll gradient-selected sperm from a single bull in an mSOF-based medium for 20 h. After IVF, presumptive zygotes were freed from the cumulus cells and cultured in mSOF. On Day 3 (IVF = Day 0), embryos that had developed to the 8-cell stage or more (≥8-cell) were subsequently cultured in medium supplemented with 0.05% (vol/vol) of emOA or emVEBC. Blastocyst development from ≥8-cell embryos was assessed on Day 8. In the case of no-additive control and emVEBC, the hatching rate was also assessed on Day 10. All the cultures were performed at 38.5°C under 5% CO2, 5% O2, and 90% N2 and replicated 4 times with ~18 embryos per group per replicate. The development data were statistically analysed by the general linear model. The blastocyst rate in the emOA group (36.4%) was significantly (P < 0.05) lower than that in the no-additive control (54.1%). The blastocyst rate in the emVEBC group (53.9%) was similar to that in the control; however, the hatching rate was significantly higher in the emVEBC group (22.6%) than in the control (9.2%). These data suggest that emulsification of lipophilic nutrients with P10L is an easy method to allow their addition into culture media for investigating their favourable (e.g. antioxidative vitamins) or inhibitory (e.g. OA) effects on pre-implantation development in vitro.

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