scholarly journals Innovative Alternatives for Continuous In Vitro Culture of Babesia bigemina in Medium Free of Components of Animal Origin

Pathogens ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 343
Author(s):  
Jesús A. Álvarez Martínez ◽  
Julio V. Figueroa Millán ◽  
Massaro W. Ueti ◽  
Carmen Rojas-Martínez
Keyword(s):  
In Vitro Culture ◽  
Culture Media ◽  
Culture Method ◽  
Animal Origin ◽  
Acid Mixture ◽  
Babesia Bigemina ◽  
Lipid Mixture ◽  
Proliferation In Vitro ◽  
Animal Component

In this study, we report Babesia bigemina proliferation in culture medium free of components of animal origin supplemented with a lipid mixture. Babesia bigemina continuously proliferated in VP-SFM with a higher percent parasitized erythrocyte as compare to using other animal component-free culture media. Compared with Advanced DMEM/F12 (ADMEM/F12), VP-SFM had a similar percent parasitized erythrocyte (PPE). Supplementation of VP-SF with a lipid acid mixture improved B. bigemina proliferation in vitro culture, with a maximum PPE of 11.3%. Growth of B. bigemina in a perfusion bioreactor using VP-SFM medium supplemented with lipid mixture resulted in a PPE above 28%. In conclusion, we demonstrated that B. bigemina proliferated in an animal component-free medium supplemented with the fatty acid mixture. This innovation to B. bigemina in vitro culture method presented herein is an important source of biological material for live vaccine production and understanding the mechanisms and molecules involved in parasite attachment and invasion of bovine erythrocytes.

scholarly journals Comparative suitability of two culture media to the in vitro growth of embryos of three coconut types

2007 ◽  
pp. 79-90 ◽  
Author(s):  
Tessie Nuñez
Keyword(s):  
In Vitro Culture ◽  
Culture Media ◽  
Iron Concentration ◽  
Culture Method ◽  
State University ◽  
Plant Genotype ◽  
Initial Culture ◽  
Semi Solid ◽  
Two Stages

Using a suitable medium for specific plant genotype greatly improves the efficiency of the in vitro culture method. The Visayas State University (VSU)-based National Coconut Research Center -Visayas (NCRC-V) evaluated the comparative suitability of the COGENT medium and VSU-modified Y3 (mY3) as in vitro culture media for coconut embryos using Albuera Dwarf (ALD), Baybay Tall (BAYT),and the VSU-developed Coconiño x Makapuno (VMAC1) hybrid. These two media differ in vitamin components, iron concentration and state during the first two stages of culture, namely germination and first subculture. Results showed that mY3 was more suitable for the in vitro germination and development of the coconut embryos than the COGENT medium. Significantly higher germination rates were observed in BAYT, ALD, and VMAC1 cultured in the semi-solid mY3 than those in the liquid COGENT medium from the first week until the fourth week of initial culture. Germination rates of 100%, 85.8% and 84.5% were obtained from CÑO x MAC, ALD, and BAYT, respectively. Furthermore, significantly higher percentages of germinating embryos with developing shoot and root were observed in the semi-solid mY3 than in the liquid COGENT. Likewise, better growth of plantlets in liquid mY3 was noted during the fourth and fifth months of culture. Among coconut types, VMAC1 had the highest germination rates in the two media and the best growth in mY3. BAYT had better growth in the COGENT medium while ALD had better shoot development in mY3.

scholarly journals Establishment of Babesia bovis In Vitro Culture Using Medium Free of Animal Products

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 770
Author(s):  
Jesús A. Álvarez Martínez ◽  
Julio V. Figueroa Millán ◽  
Massaro W. Ueti ◽  
Carmen Rojas-Martínez
Keyword(s):  
Culture Media ◽  
Serial Passage ◽  
Live Vaccine ◽  
Babesia Bovis ◽  
Animal Origin ◽  
Perfusion Bioreactor ◽  
Bovine Babesiosis ◽  
Lipid Mixture ◽  
Commercial Mixture

Babesia bovis, an etiological agent of bovine babesiosis, causes a significant burden to the cattle industry worldwide. The most efficient method to mitigate bovine babesiosis is a live vaccine produced by serial passage in splenectomized cattle. However, there are several concerns regarding live vaccine production, including variation between batches and the use of many animals. In this study, we report a B. bovis-SF strain continuously cultured in a medium free of components of animal origin enriched with a chemically defined lipid mixture (CD lipid mixture) and the use of a perfusion bioreactor to harvest a large amount of B. bovis. Six culture media were compared, including VP-SFM, CD-CHO, CD-Hydrolyzed, CD-CHO, SFM, and ADMEM/F12. We found that the VP-SFM medium performed the best for B. bovis growth, with a maximum percentage of parasitized erythrocytes (PPE) of 8.6%. The effect of six dilutions of a commercial mixture of CD lipids added to VP-SFM showed that the CD lipid mixture at a dilution of 1:100 had the best B. bovis growth curve, with a maximum PPE of 13.9%. Propagation of the in vitro B. bovis culture was scaled up in a perfusion bioreactor using VP-SFM with a CD lipid mixture, and the PPE reached over 32%. The continuous in vitro B. bovis culture in a medium free of animal origin components could potentially reduce and replace the use of animals to produce a reagent for diagnostics and live vaccines to control bovine babesiosis.

scholarly journals Rheological and Microstructural Features of Plant Culture Media Doped with Biopolymers: Influence on the Growth and Physiological Responses of In Vitro-Grown Shoots of Thymus lotocephalus

2021 ◽  
Vol 2 (2) ◽  
pp. 538-553
Author(s):  
Natacha Coelho ◽  
Alexandra Filipe ◽  
Bruno Medronho ◽  
Solange Magalhães ◽  
Carla Vitorino ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Average Pore Size ◽  
Physiological Responses ◽  
Gum Arabic ◽  
Shoot Elongation ◽  
Hydroxyethyl Cellulose ◽  
Plant Culture

In vitro culture is an important biotechnological tool in plant research and an appropriate culture media is a key for a successful plant development under in vitro conditions. The use of natural compounds to improve culture media has been growing and biopolymers are interesting alternatives to synthetic compounds due to their low toxicity, biodegradability, renewability, and availability. In the present study, different culture media containing one biopolymer (chitosan, gum arabic) or a biopolymer derivative [hydroxyethyl cellulose (HEC), carboxymethyl cellulose (CMC)], at 100 or 1000 mg L−1, were tested regarding their influence on the growth and physiological responses of Thymus lotocephalus in vitro culture. Cellulose-based biopolymers (HEC and CMC) and gum arabic were used for the first time in plant culture media. The results showed that CMC at 100 mg L−1 significantly improved shoot elongation while chitosan, at the highest concentration, was detrimental to T. lotocephalus. Concerning only the evaluated physiological parameters, all tested biopolymers and biopolymer derivatives are safe to plants as there was no evidence of stress-induced changes on T. lotocephalus. The rheological and microstructural features of the culture media were assessed to understand how the biopolymers and biopolymer derivatives added to the culture medium could influence shoot growth. As expected, all media presented a gel-like behaviour with minor differences in the complex viscosity at the beginning of the culture period. Most media showed increased viscosity overtime. The surface area increased with the addition of biopolymers and biopolymer derivatives to the culture media and the average pore size was considerably lower for CMC at 100 mg L−1. The smaller pores of this medium might be related to a more efficient nutrients and water uptake by T. lotocephalus shoots, leading to a significant improvement in shoot elongation. In short, this study demonstrated that the different types of biopolymers and biopolymer derivatives added to culture medium can modify their microstructure and at the right concentrations, are harmless to T. lotocephalus shoots growing in vitro, and that CMC improves shoot length.

scholarly journals A Comparative Genomic Study of Attenuated and Virulent Strains of Babesia bigemina

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 318
Author(s):  
Bernardo Sachman-Ruiz ◽  
Luis Lozano ◽  
José J. Lira ◽  
Grecia Martínez ◽  
Carmen Rojas ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Virulence Genes ◽  
Laboratory Strain ◽  
Host Cells ◽  
Comparative Genomic ◽  
Local Alignment ◽  
Host Immune System ◽  
Babesia Bigemina ◽  
Genomic Study

Cattle babesiosis is a socio-economically important tick-borne disease caused by Apicomplexa protozoa of the genus Babesia that are obligate intraerythrocytic parasites. The pathogenicity of Babesia parasites for cattle is determined by the interaction with the host immune system and the presence of the parasite’s virulence genes. A Babesia bigemina strain that has been maintained under a microaerophilic stationary phase in in vitro culture conditions for several years in the laboratory lost virulence for the bovine host and the capacity for being transmitted by the tick vector. In this study, we compared the virulome of the in vitro culture attenuated Babesia bigemina strain (S) and the virulent tick transmitted parental Mexican B. bigemina strain (M). Preliminary results obtained by using the Basic Local Alignment Search Tool (BLAST) showed that out of 27 virulence genes described and analyzed in the B. bigemina virulent tick transmitted strain, only five were fully identified in the attenuated laboratory strain. In all cases, the identity and coverture of the identified genes of the wildtype strain were higher than those of the laboratory strain. This finding is putatively associated with the continuous partial loss of virulence genes in the laboratory strain after several passages of the parasite population under optimal in vitro growth conditions. The loss of virulence factors might be reflected in the absence of symptoms of the disease in cattle inoculated with the attenuated strain despite the presence of infection in the bovine host cells.

scholarly journals In vitro culture as an aid to conservation of indigenous ferns: Diplazium proliferum

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Zubeir M. Golamaully ◽  
Vishwakalyan Bhoyroo ◽  
Nadeem Nazurally ◽  
Vineshwar Gopal
Keyword(s):  
Growth Rate ◽  
In Vitro Culture ◽  
Mercuric Chloride ◽  
Rare Species ◽  
Vascular Plants ◽  
Culture Media ◽  
Fungal Contamination ◽  
Increase Growth Rate ◽  
Growing Population

With the ever growing population and economic needs of Mauritius, the flora of Mauritius has never been in more danger and one group of vascular plants is even more in peril; ferns.<em> Diplazium proliferum</em> is indigenous to the Mascarene region and is considered as a rare species in Mauritius. The need to develop a tested <em>in vitro</em> propagation protocol is a must to protect the biodiversity of Mauritius. This experiment was geared towards the establishment of a proper sterilization technique and the effect of 6-benzylaminopurine (BAP) and light on <em>in vitro</em> culture of this fern. Sterilization with 0.05% Mercuric chloride was effective to eliminate fungal contamination and allow germination of spores. Culture media supplemented with BAP did not significantly increase growth rate of both gametophytes and sporophytes of<em> D. proliferum</em>. Present results suggest efficient sterilization methods to be a crucial stage for successful<em> in vitro r</em>egeneration of ferns. The established protocol will be used as an optimized baseline protocol for the propagation of other indigenous ferns.

The effect of osmolarity on mouse parthenogenesis

Development ◽  
1974 ◽  
Vol 31 (2) ◽  
pp. 513-526
Author(s):  
M. H. Kaufman ◽  
M. A. H. Surani
Keyword(s):  
Protein Synthesis ◽  
Culture Medium ◽  
Culture Media ◽  
Polar Body ◽  
Amino Acid Mixture ◽  
Culture Conditions ◽  
Follicle Cells ◽  
Acid Mixture ◽  
Second Polar Body

Eggs from (C57B1 × A2G)F1 mice were activated by treatment with hyaluronidase, which removed the follicle cells, and cultured in vitro. Observations were made 6–8 h after hyaluronidase treatment to determine the frequency of activation and the types of parthenogenones induced. Cumulus-free eggs resulting from hyaluronidase treatment were incubated for 2¼ h in culture media of various osmolarities. The frequency of activation was found to be dependent on the postovulatory age of oocytes, while the types of parthenogenones induced were dependent on the osmolarity of the in vitro culture medium and their postovulatory age. Culture in low osmolar medium suppressed the extrusion of the second polar body (2PB). This decreased the incidence of haploid eggs with a single pronucleus and 2PB and immediately cleaved eggs from 97·5% to 42·3% of the activated population. Where 2PB extrusion had been suppressed, 97·4% of parthenogenones contained two haploid pronuclei. Very few were observed with a single and presumably diploid pronucleus. Serial observations from 11 to 18 h after hyaluronidase treatment were made on populations of activated eggs as they entered the first cleavage mitosis after 2¼ h incubation in medium either of normal (0·287 osmol) or low (0·168 osmol) osmolarity. A delay in the time of entry into the first cleavage mitosis similar to the duration of incubation in low osmolar medium was observed. Further, eggs were incubated in control and low osmolar culture media containing uniformly labelled [U-14C]amino acid mixture to examine the extent of protein synthesis in recently activated eggs subjected to these culture conditions. An hypothesis is presented to explain the effect of incubation in low osmolar culture medium in delaying the first cleavage mitosis.

scholarly journals Combining Medicinal Plant In Vitro Culture with Machine Learning Technologies for Maximizing the Production of Phenolic Compounds

Antioxidants ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 210 ◽  
Author(s):  
Pascual García-Pérez ◽  
Eva Lozano-Milo ◽  
Mariana Landín ◽  
Pedro Pablo Gallego
Keyword(s):  
Machine Learning ◽  
Phenolic Compounds ◽  
In Vitro Culture ◽  
Culture Media ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Total Phenolic ◽  
Starting Point ◽  
Plant In Vitro Culture

We combined machine learning and plant in vitro culture methodologies as a novel approach for unraveling the phytochemical potential of unexploited medicinal plants. In order to induce phenolic compound biosynthesis, the in vitro culture of three different species of Bryophyllum under nutritional stress was established. To optimize phenolic extraction, four solvents with different MeOH proportions were used, and total phenolic content (TPC), flavonoid content (FC) and radical-scavenging activity (RSA) were determined. All results were subjected to data modeling with the application of artificial neural networks to provide insight into the significant factors that influence such multifactorial processes. Our findings suggest that aerial parts accumulate a higher proportion of phenolic compounds and flavonoids in comparison to roots. TPC was increased under ammonium concentrations below 15 mM, and their extraction was maximum when using solvents with intermediate methanol proportions (55–85%). The same behavior was reported for RSA, and, conversely, FC was independent of culture media composition, and their extraction was enhanced using solvents with high methanol proportions (>85%). These findings confer a wide perspective about the relationship between abiotic stress and secondary metabolism and could serve as the starting point for the optimization of bioactive compound production at a biotechnological scale.

474 Effect of four assisted hatching techniques and two in vitro culture media on embryo hatching rate

2017 ◽  
Vol 95 (suppl_4) ◽  
pp. 231-231
Author(s):  
N. C. Negota ◽  
L. P. Nethenzheni ◽  
N. R. Serota
Keyword(s):  
In Vitro Culture ◽  
Culture Media ◽  
Assisted Hatching ◽  
Hatching Rate

160 COMPARATIVE STUDY OF IN VITRO CULTURE MEDIA AND ASSISTED HATCHING TECHNIQUES ON MOUSE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 188
Author(s):  
N. C. Negota ◽  
L. P. Nethenzheni ◽  
M. L. Mphaphathi ◽  
D. M. Barry ◽  
T. L. Nedambale
Keyword(s):  
In Vitro Culture ◽  
Chorionic Gonadotropin ◽  
Culture Medium ◽  
Culture Media ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Significant Difference ◽  
Equine Chorionic Gonadotropin ◽  
F1 Generation

The in vitro culture media and assisted hatching techniques remain challenging obstacles to be utilised widely. Mechanical, chemical, enzymatic thinning, and laser-assisted techniques have been used previously but information is still lacking on its application in livestock. The aim of this study was to compare the effect of 2 in vitro culture media (Hamster F10 and TMC-199) and 4 (mechanical, chemical, enzymatic, and laser) assisted hatching techniques on blastocyst formation and hatching rate using murine embryos as a model. The C57/b and Balb/c breeds were raised until they reached maturity and bred naturally to produce F1 generation. The light in the breeding house was controlled at 14 h light and 10 h dark. Feed and water were provided ad libitum for the mice. Superovulation of females were stimulated using equine chorionic gonadotropin and human chorionic gonadotropin. The F1 generation was used for the collection of the 400 blastocysts and randomly allocated into 4 assisted hatching techniques. Blastocysts were paired into a group of 10 and replicated 4 times for each assisted hatching technique. The general linear model of SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) was used to analyse the data. Assisted hatching techniques of laser, mechanical, enzymatic, and chemical yielded 46.9 ± 37.1, 51.1 ± 40.2, 39.1 ± 35.8, and 33.3 ± 4.5%, respectively, under in vitro culture of Hamster F10. The TCM-199, laser, mechanical, enzymatic, and chemical assisted hatching techniques yielded 56.3 ± 43.3, 52.6 ± 35.5, 49.2 ± 37.5, and 33.9 ± 35.5%, respectively, with a significant difference. There was no significant difference observed in assisted hatching techniques and Hamster F10 culture medium. However, the hatching rate of embryos for all techniques was higher when in vitro cultured in TCM than cultured in Hamster F10. Hatching rate of blastocysts increased from chemical, enzymatic, mechanical, and laser with response to Hamster F10 and TCM; thus, laser is a suitable assisted hatching technique with TCM-199.

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