scholarly journals Infectivity of Theileria parva sporozoites following cryopreservation in four suspension media and multiple refreezing : evaluation by in vitro titration

2006 ◽  
Vol 73 (3) ◽  
Author(s):  
V. Mbao ◽  
D. Berkvens ◽  
T. Dolan ◽  
N. Speybroeck ◽  
J. Brandt ◽  
...  
Keyword(s):  
East Coast ◽  
Calf Serum ◽  
Roswell Park Memorial Institute ◽  
Foetal Calf Serum ◽  
Theileria Parva ◽  
Minimum Essential Medium ◽  
Freezing Medium ◽  
In Vitro Technique ◽  
Phosphate Buffered Saline

Theileria parva sporozoite stabilates are used for immunizing cattle against East Coast fever and in in vitro sporozoite neutralization assays. In this study, we attempted to identify a cheaper freezing medium and quantified the infectivity loss of sporozoites due to refreezing of stabilates, using an in vitro technique. Pools of stabilates prepared using Minimum Essential Medium (MEM), Roswell Park Memorial Institute (RPMI 1640), foetal calf serum (FCS) and phosphate-buffered saline (PBS) were compared. All were supplemented with bovine serum albumin except the FCS. RPMI 1640 was as effective as MEM in maintaining sporozoite infectivity while the infectivity in PBS and FCS reached only 59 % and 67 %, respectively. In a second experiment, a stabilate based on MEM was subjected to several freeze-thaw cycles including various holding times on ice between thawing and refreezing. Refrozen stabilate gave an average sporozoite infectivity loss of 35 % per cycle. The results indicate that RPMI can be used as a cheaper freezing medium for T. parva stabilates and that refrozen stabilate doses need to be adjusted for the 35 % loss of infectivity.

Survival of rapidly frozen hatched mouse blastocysts

Zygote ◽  
2003 ◽  
Vol 11 (4) ◽  
pp. 361-366 ◽  
Author(s):  
Csaba Pribenszky ◽  
Sándor Cseh ◽  
Zsolt Abonyi-Tóth ◽  
László Solti
Keyword(s):  
Liquid Nitrogen ◽  
Zona Pellucida ◽  
Room Temperature ◽  
Calf Serum ◽  
Significant Loss ◽  
Rapid Freezing ◽  
Foetal Calf Serum ◽  
Freezing Medium

The objective of the present study was to examine the effect of rapid freezing on the in vitro and in vivo survival of zona-pellucida-free hatched mouse blastocysts. Hatched blastocysts were rapidly frozen in a freezing medium containing either ethylene glycol (EG) or glycerol (G) in 1.5 M or 3 M concentration. Prior to freezing, embryos were equilibrated in the freezing medium for 2 min, 10 min, 20 min or 30 min at room temperature. To freeze them, embryos were held in liquid nitrogen vapour [≈1 cm above the surface of the liquid nitrogen (LN2)] for 2 minutes and then immersed into LN2. After thawing, embryos were transferred either to rehydration medium (DPBS + 10% foetal calf serum + 0.5 M sucrose) for 10 minutes or rehydrated directly in DPBS supplemented with foetal calf serum. In vitro survival of embryos frozen with EG was higher than those frozen with G. The highest survival was obtained with 3 M EG and 2 min or 10 min equilibration prior to freezing, combined with direct rehydration after thawing. Frozen blastocysts developed into normal foetuses as well as unfrozen control ones did, with averages of 30% (control), 26% (EG) and 15% (G). The results show that hatching and hatched mouse blastocysts can be cryopreserved by a simple rapid freezing protocol in EG without significant loss of viability. Our data indicate that the mechanical protection of the zona pellucida is not needed during freezing in these stages.

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.

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.

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.

44 THE EFFECT OF SUCROSE CONCENTRATION FOR SINGLE-STEP DILUTION ON THE VIABILITY OF CRYOTOP-VITRIFIED IN VITRO-PRODUCED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 115
Author(s):  
S. Kondo ◽  
K. Imai ◽  
O. Dochi
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Liquid Nitrogen ◽  
Sucrose Concentration ◽  
Calf Serum ◽  
Single Step ◽  
Bovine Embryos ◽  
Vitrification Solution ◽  
Phosphate Buffered Saline

The aim of this study was to test sucrose concentrations for single-step dilution on the viability of vitrified in vitro-produced bovine embryos. Blastocysts (n = 173, 7 to 8 days after fertilization) were vitrified using the Cryotop (Kitazato, Tokyo, Japan) method placement by incubating the blastocysts in Dulbecco's phosphate buffered saline supplemented with 20% calf serum, 7.5% ethylene glycol, and 7.5% dimethyl sulfoxide for 3 min and then transferring into vitrification solution (Dulbecco's phosphate buffered saline supplemented with 20% calf serum, 16.5% ethylene glycol, 16.5% dimethyl sulfoxide, and 0.5 M sucrose). Each embryo was placed on a Cryotop with minimum volume of vitrification solution, and then the Cryotop was plunged into liquid nitrogen. Total time from placement in vitrification solution to plunging into liquid nitrogen was 1 min. The blastocysts were warmed by incubation in the single-step dilution medium for 5 min [0 M sucrose (n = 42), 0.25 M sucrose (n = 44), 0.5 M sucrose (n = 43), and 1.0 M sucrose (n = 44)] at 38.0°C. After dilution, the embryos were washed in TCM-199 supplemented with 20% calf serum and 0.1 mM β-mercaptoethanol and were cultured for 72 h in the same medium at 38.5°C in an atmosphere of 5% CO2. The rates of re-expanded blastocysts and hatched blastocysts were determined at 24 and 72 h after warming, respectively. Data were analysed using the chi-squared test. The percent of re-expanded blastocysts at 24 h after warming in dilution medium supplemented with any level of sucrose was significantly higher (P < 0.05) than in blastocysts warmed without sucrose (Table 1). The hatched blastocyst rate of embryos at 72 h after warming in dilution medium with 0.5 M sucrose was significant higher than that with no sucrose. There were no differences in hatched blastocyst rates between the sucrose concentrations supplemented to the dilution medium. These results suggest that embryos vitrified by the Cryotop method can be diluted in single-step dilution using 0.25, 0.5, or 1.0 M sucrose supplemented to the medium. Table 1.The effect of sucrose concentration for single-step dilution on the viability of Cryotop vitrified in vitro-produced bovine embryos

The Factor Affecting the Spreading of Chondrocytes upon Inorganic Substrate

1973 ◽  
Vol 13 (1) ◽  
pp. 193-204
Author(s):  
M. TAKEICHI
Keyword(s):  
Conditioned Medium ◽  
Calf Serum ◽  
Fresh Medium ◽  
Foetal Calf Serum ◽  
Chick Embryos ◽  
Embryonic Cells ◽  
Mass Cultures ◽  
Rounded Form ◽  
Inorganic Substrate

The effect of conditioned medium (CM) prepared from mass-cultures of chick embryonic cells was studied on the spreading behaviour of chondrocyte derived from sterna of 16-day-old chick embryos. Freshly dissociated chondrocytes exhibited a quite rounded form, and this shape did not change when they were cultured with fresh medium (Eagle's MEM + 6% foetal calf serum) in vitro for several days. Non-dialysable material(s) in CM added into the fresh medium stimulated the formation of pseudopods of chondrocytes, without primarily affecting the synthesis of chondroitin sulphates. Such an activity of CM was not lost after boiling, but it was lost following treatment with proteases. The chondrocytes covered with newly deposited acidmucopoly-saccharides were insensitive to the effect of CM, but they became sensitive to form pseudopods after treatment of the cells with chondroitinase. These results suggest that CM contains a macromolecular material(s) to enhance the motility or adhesiveness of chondrocytes.

Cell-associated proteases affect tumour cell migration in vitro

1979 ◽  
Vol 36 (1) ◽  
pp. 241-252
Author(s):  
J. Varani ◽  
W. Orr ◽  
P.A. Ward
Keyword(s):  
Cell Migration ◽  
Human Serum ◽  
Protease Inhibitors ◽  
Trypsin Inhibitor ◽  
Calf Serum ◽  
Lima Bean ◽  
Caproic Acid ◽  
Foetal Calf Serum ◽  
Migratory Activity

The in vitro migratory activity of mouse fibrosarcoma cells in medium containing either foetal calf serum or normal human serum was studied. These 2 sera were studied because foetal calf serum contains high levels of protease inhibitor activity while human serum contains much less. The cells migrated actively in medium with foetal calf serum but migration was greatly inhibited in human serum-containing medium. When protease inhibitors such as soybean trypsin inhibitor, lima bean trypsin inhibitor and bovine pancreas trypsin inhibitor were added to human serum-containing medium cell migration was supported almost as effectively as in medium with foetal calf serum. Addition of epsilon-amino-n-caproic acid to human serum or depletion of the plasminogen from human serum did not enable it to support enhanced migration. epsilon-amino-n-caproic acid actually inhibited migration. A variant cell population with elevated levels of caseinolytic activity and elevated levels of activity against the substrate n-acetyl-DL-phenylalanine-beta-naphthyl ester (a substrate specific for chymotrypsin-like enzymes) was isolated from the parent cells. When the variant cells were compared to the parent cells regarding migratory activity in foetal calf serum or human serum-containing medium, the variant cells showed much less activity. Only a few, widely scattered variant cells migrated in the human serum-containing medium. These data suggest that a cell-associated factor interferes with the migration of the cells in medium with human serum. This factor apparently is neutralized in medium sontaining human serum to which protease inhibitors with antitrypsin activity have been added.

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