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.

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.

scholarly journals ISOPEROXIDASES PATTERN OF ROBINIA PSEUDOACACIA VAR. OLTENICA IN VITRO CULTURE ON DIFFERENT MEDIA

1970 ◽  
Vol 62 ◽  
Author(s):  
Cristina Babeanu ◽  
Georgeta Ciobanu ◽  
Mihaela Corneanu ◽  
Gabriela Marinescu ◽  
C. G. Corneanu
Keyword(s):  
Thermal Stability ◽  
In Vitro Culture ◽  
Humic Acids ◽  
Culture Medium ◽  
Culture Media ◽  
Robinia Pseudoacacia ◽  
Magnetic Fluids ◽  
Electrophoretic Separation

In this work is studied the isoperoxidase pattern of the in vitro subculture of Robinia pseudoacacia var. oltenica, on various culture media, with various content of myo-inositol, humic acids and magnetic fluids. The electrophoretic separation of the isoperoxidases was performed from extracts obtained from 30 days’old vegetal material. The isoperoxidase fractions were characterized in terms of quantity and quality and a study of their thermal stability was performed. The obtained data show that the isoperoxidase activity is modulated in various ways by the presence of the studied factors into the culture medium.

168 CORRELATION OF DEVELOPMENT KINETICS AND SEX OF IN VITRO-PRODUCED BOVINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 196
Author(s):  
A. R. Buzzo ◽  
A. R. Pupulim ◽  
J. Mazucheli ◽  
F. V. Meirelles ◽  
I. P. Emanuelli
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Proteinase K ◽  
Bovine Embryos ◽  
Male And Female ◽  
Stage Of Development ◽  
Males And Females ◽  
Kinetics Of

Approaches to improve the culture medium for in vitro production (IVP) of bovine embryos have been continuous because of the high commercial demand and a portion of this attempts the production of female cattle (dairy cows and stud cattle). However, in some embryonic in vitro culture systems, the development kinetics is faster in male than in female embryos (Avery 1992 Mol. Reprod. Dev. 32, 265–70; Xu 1992 Mol. Reprod. Dev. 31, 249–50). The aim of this work was to relate the kinetics of blastocyst expansion with the production rates of male and female embryos. Cumulus–oocyte complexes (n = 917; classes I and II) of cows from a slaughterhouse were matured with TCM-199 bicarbonate and 10% FCS (38.5°C, 5% CO2) for 24 h and fertilized with frozen-thawed semen in TALP-IVF medium for 18 h. Presumptive zygotes were culture in SOF medium supplemented with 10% FSB (5% O2, 38.5°C). Seven days after IVF, embryos were divided in 2 groups according to their kinetic stage of development: nonexpanded blastocysts (n = 175), or hatched and expanded blastocysts (n = 146). Hence, embryos were individually frozen in LN and stored in cryotubes. After thawing, Proteinase K (16 mg mL–1) was added to each tube and the tubes were incubated for 60 min at 37°C. Proteinase was denatured at 98°C for 10 min and the contents of each tube were divided into 2 samples (A and B) and subjected to the PCR technique. Two pairs of primers for the specific sequence of the Y chromosome were used to amplify the sequence of 210 and 250 bp for the male bovine and 1 pair of primers was used for the autosomal bovine sequence with a 280-bp fragment. Female embryos with a 280-bp product were observed in sample A and none were observed in sample B. The presence of 2 amplicons (280 and 210 bp) in sample A and 1 amplicon of 250 bp in sample B indicated that the embryo was male. A chi-square test was used to evaluate homogeneity. An analysis of the percentage of males and females between the experimental groups was performed by logistic regression and significance was considered when P < 0.05. There was no difference in the proportions of males and females in the nonexpanded blastocyst group (49.71 and 50.29%; P > 0.05). In the hatched and expanded blastocyst group, the proportion of males (65.75%) was statistically different from the proportion of females (34.25%); that is, the chance of the embryo being male was twice as high (P < 0.0038). These results suggest that there is a difference in the kinetics of embryo development between male and female embryos and that blastocyst expansion can point that out. In vitro culture media with FCS support the development of expanded male blastocysts. Further research in culture medium modifications (FCS, the energy source, amino acids and others) are needed to respond to the trend in the production of sex-defined embryos.

125 INTRACYTOPLASMIC SPERM INJECTION (ICSI)-BASED MOUSE EMBRYO ASSAY: CHOICE OF EMBRYO CULTURE SYSTEM OUTWEIGHS THE EFFECT OF FERTILIZATION PROCEDURE ON EMBRYO DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 209
Author(s):  
C. Schwarzer ◽  
T. C. Esteves ◽  
S. Le Gac ◽  
V. Nordhoff ◽  
S. Schlatt ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Mouse Embryo ◽  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Medium ◽  
Culture Media ◽  
Blastocyst Formation ◽  
The Absolute ◽  
Mouse Embryo Assay

Human embryo culture media, intended for assisted reproductive technologies (ARTs), are released for clinical use if they pass the mouse embryo assay (MEA). This assay prescribes that at least 70% of in vivo fertilized mouse 1-cell embryos form blastocysts, in order to grant the culture medium approval. In the fertility clinic, however, human embryos undergo more manipulation than their MEA counterparts through, for example, fertilization by intracytoplasmic sperm injection (ICSI); further, only a minority of the embryos transferred to the uterus goes on to establish gestations. In this context, we asked if the results of the MEA only depend on the type of in vitro culture, or are also affected by the method of fertilization. Superovulated B6C3F1 mouse oocytes were fertilized by ICSI using C57Bl/6 sperm. Pronuclear-stage eggs were allocated to four developmental environments: two ART culture protocols (HTF/MultiBlast, Irvine Scientific; ISM1/ISM2, Origio), standard mouse culture medium (KSOM(aa), made in-house) and the oviduct of pseudopregnant CD1 mice. As control for the invasive manipulation, pronuclear-stage eggs were generated by mating (B6C3F1 × C57Bl/6) and cultured in KSOM(aa) medium. Embryos were recovered from culture or from the CD1 uterus and scored for blastocyst formation at 96 h of development (Table 1). For these blastocysts, we determined the number of total, inner cell mass (ICM), and trophectoderm (TE) cells (Table 1) by confocal immunofluorescence microscopy (Schwarzer et al. 2012 doi:10.1093/humrep/des223). Our results show that ART culture protocols applied to mouse ICSI embryos are not equivalent in supporting blastocyst formation. Based on blastocyst rates, the ranking observed here after ICSI, reflects the ranking reported by us for IVF embryos (Schwarzer et al. 2012); that is, KSOM(aa) > HTF/MultiBlast > oviduct > ISM1/2. This similarity suggests that the effect of in vitro culture on mouse development exceeds the effect of ICSI, provided gametes are of good quality. From the analysis of cell numbers, we note that while the ICM/TE ratios are not of easy interpretation, the absolute numbers of cells in the ICM draw a clear line between the environment of the oviduct and those of culture media. Irrespective of the ICM/TE ratio, only the oviduct environment secures 8 cells in the ICM (Table 1). Soriano and Jaenisch (1986 Cell 46, 19–29) reported that 8 cells of the ICM are set aside to give rise to the body of a mouse. In summary, the current MEA is a valuable assay to assess the quality of culture medium, however, its refinement is necessary to better model the adaptive properties of embryo culture when different methods of fertilization are applied. Until the MEA is extended into postimplantation development, as we advocate (Schwarzer et al. 2012), the absolute numbers of cells in the ICM may be a better gauge of embryo quality than the blastocyst rates. Table 1.Mouse embryo assay outcomes after ICSI

scholarly journals In vitro culture of Neoechinorhynchus buttnerae (Acanthocephala: Neoechinorhynchidae): Influence of temperature and culture media

2018 ◽  
Vol 27 (4) ◽  
pp. 562-569 ◽  
Author(s):  
Carinne Moreira de Souza Costa ◽  
Talissa Beatriz Costa Lima ◽  
Matheus Gomes da Cruz ◽  
Daniela Volcan Almeida ◽  
Maurício Laterça Martins ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Treatment Strategies ◽  
Infected Fish ◽  
In Vivo Studies ◽  
Colossoma Macropomum ◽  
Tank Water

Abstract Infection by the acantocephalan Neoechinorhynchus buttnerae is considered one of most important concerns for tambaqui fish (Colossoma macropomum ) production. Treatment strategies have been the focus of several in vivo studies; however, few studies have been undertaken on in vitro protocols for parasite maintenance. The aim of the present study was to develop the best in vitro culture condition for N. buttnerae to ensure its survival and adaptation out of the host to allow for the testing of substances to be used to control the parasite. To achieve this, parasites were collected from naturally infected fish and distributed in 6-well culture plates under the following treatments in triplicate: 0.9% NaCl, sterile tank water, L-15 Leibovitz culture medium, L-15 Leibovitz + agar 2% culture medium, RPMI 1640 culture medium, and RPMI 1640 + agar 2% culture medium. The plates containing the parasites were maintained at 24 °C, 28 °C, and 32 °C. The RPMI 1640 + agar 2% culture medium showed the best survival of 24 days at 24 °C. No body alterations such as swollen parasites, body deformation, dehydration and hardening were observed in the RPMI 1640 + 2% culture medium.

scholarly journals Influence of the composition of the culture media on the development of sporofites Osmunda regalis L. in vitro

2020 ◽  
pp. 104-111
Author(s):  
V. I. Malyarovskaya ◽  
R. S. Rakhmangulov ◽  
N. G. Koninskaya
Keyword(s):  
In Vitro Culture ◽  
Ammonium Nitrate ◽  
Culture Medium ◽  
Culture Media ◽  
Salt Content ◽  
And Control

One of the important stages for the propagation of the rare endangered fern Osmunda regalis L. through spores is the regeneration of sporophytes from gametophytes at in vitro culture. The article shows that the effective formation of O. regalis sporophytes occurred earlier (after 60 days) and in a larger percentage (51.6%) on a culture medium with a lower salt content % MS, and the exclusion of ammonium nitrate and vitamins from the medium is significantly enhanced the growth of sporophytes, compared with other variants of media and control.

Some features of clonal micropropagation of decorative cultivars of Amelanchier Medik.

2020 ◽  
pp. 97-104
Author(s):  
E. N. Raeva-Bogoslovskaya ◽  
O. I. Molkanova
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Culture Conditions ◽  
Clonal Micropropagation ◽  
Morphogenetic Potential

In vitro culture conditions were optimized for representatives of the genus Amelanchier Medik. at the stages of micropropagation and rooting. A significant effect of the mineral and hormonal compositions of culture media on the morphogenetic potential of the cultivars of serviceberry has been established. The use at the stage of micropropagation of the MS culture medium with addition 1,0 mg / L 6-benzylaminopurine (6-BAP) promoted the active microshoot regeneration of the studied genotypes. For the induction of rhizogenesis the type of auxin as IB A at a concentration of 1,0 mg / L was used.

Pertumbuhan Dan Perkembangan Anggrek Dendrobium anosmum Pada Media Kultur In Vitro Dengan Beberapa Konsentrasi Air Kelapa

Agrologia ◽  
2018 ◽  
Vol 1 (1) ◽  
Author(s):  
S. Tuhuteru ◽  
Meity L Hehanussa ◽  
Simon H.T Raharjo
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Water Concentration ◽  
Medium Composition ◽  
Coconut Water ◽  
Orchid Species ◽  
Randomized Design ◽  
Wet Weight ◽  
Completely Randomized Design

Dendrobium anosmum is one of natural orchids in Indonesia. Optimization of medium composition for orchid propagation through in vitro culture is necessary to enhance propagule multiplication capabilities and quality. This study was aimed to study the influence of concentration of coconut water in culture medium on in vitro growth and development of D. anosmum orchid species and to determine the optimal coconut water concentration in culture media.  The experiment were arranged in a Completely Randomized Design with four treatments and eight replications. The treatments consisted of the addition of coconut water with concentrations: 0 ml•l -1 (control), 50 ml•l-1, 100 ml•l-1 and 150 ml•l-1. The results showed that addition of coconut water in culture medium gave different effect on shoot growth and multiplication of D. anosmum orchids.  Coconut water concentration of 100 ml•l-1 was the best concentration for growth and multiplication of D. anosmum orchids, based on both shoots and roots growth, plantlet height and wet weight.

P–218 Analysis of the occurrence of microbial contamination in IVF culture system and the effect of microorganisms on embryo development and clinical outcomes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
F Du ◽  
R Li ◽  
Q Zhang ◽  
W Wang
Keyword(s):  
Escherichia Coli ◽  
In Vitro Culture ◽  
Clinical Outcomes ◽  
Embryo Transfer ◽  
Follicular Fluid ◽  
Culture System ◽  
Microbial Contamination ◽  
Culture Medium ◽  
Ivf Et

Abstract Study question what is the source, prevalence, and influence of microbial contamination on in vitro fertilization (IVF) and embryo transfer (ET) cycles? Summary answer Microbial contamination mainly occurs on Day 2, most caused by Escherichia coli carried with semen. ICSI could prevent contamination effectively and get good clinical outcomes. What is known already Microbial contamination occurs in IVF-ET system occasionally, which is hard to stop happening. The IVF culture system and laboratory environment, the patients’ follicular fluid and semen are not absolutely sterile, while the antibiotics in culture medium isn’t effective for all microbe types, and the artificial operations may bring in microbes. Generally, microbial contamination leads to degradation of embryos, reduction the number of embryos available, and infection of female reproductive tract, which would increase the cost of patients’ time, money, and bring psychological damages. A better understanding of embryo contamination in IVF culture system is of added value. Study design, size, duration A total of 29583 IVF-ET cycles were enrolled in this prospective observational study, from January 2010 to December 2020, included 70 microbial contamination cycles discovered in Day1-Day3 (D1-D3) of in vitro culture. Follicular fluid and semen saved on oocyte retrieval day, and culture medium contaminated were examined and identified for microorganisms at each contamination cycle. Participants/materials, setting, methods Compared the contamination rate of different insemination methods (IVF/ICSI/IVF+ICSI), different in vitro culture days (D1-D3), and different samples examination (follicular fluid, semen, culture medium) respectively, identified the source of microorganism types, compared the IVF culture outcomes and clinical outcomes between total contamination group (TC group, 42 cases) and partial contamination group (PC group, 28 cases). Main results and the role of chance A total of 70 microbial contamination cases occurred in 29583 oocyte retrieving cycles (0.24%), and it was observed only in IVF embryos but never in ICSI (Intracytoplasmic sperm injection) embryos. 38 contamination cases occurred on D2 with a highest ratio (54.3%) compared to D1 (32.9%) and D3(12.9%); Compared with follicular fluid, semen was the main cause inducing contamination from D1 to D3, and Escherichia coli in semen and culture medium, Enterococcus faecalis in follicular fluid proved to be the most common sources. Compared with TC group, the PC group showed a lower rate of No-available embryos (21.4% vs 81.0%) and a higher rate of blastocyst formation (41.2% vs 28.6%), In addition, the clinical pregnancy rate of PC group was higher than that of TC group in both fresh and frozen-thawed embryo transfer cycles (31.3% vs 16.7%, 38.5% vs 0.0%). Limitations, reasons for caution Further study is still necessary to better understand the sources that induce microbial contamination embryos, and more efficient methods are required to remove the microbes on these contaminated embryos so as better develop and manage a sterile micro-environment for successful embryo growth. Wider implications of the findings: The differential embryonic microbe types associated to different IVF culture and clinical outcomes in patients undergoing IVF-ET might have profound implications for understanding the microbial sources and making a better management of IVF culture system. Trial registration number Not applicable

Biochemical markers for pregnancy in the spent culture medium of in vitro produced bovine embryos

2021 ◽  
Author(s):  
Gabriela de Oliveira Fernandes ◽  
Marcella Pecora Milazzotto ◽  
Andrei Antonioni Guedes Fidelis ◽  
Taynan Stonoga Kawamoto ◽  
Ligiane de Oliveira Leme ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Biochemical Markers ◽  
Culture Medium ◽  
Culture Media ◽  
Ionization Mass ◽  
Metabolic Profiles ◽  
Bovine Embryos ◽  
The Media

Abstract The present study aimed to identify biomarkers to assess the quality of in vitro produced (IVP) bovine embryos in the culture media. IVP embryos on Day (D) 5 of development were transferred to individual drops, where they were maintained for the last 48 h of culture. Thereafter, the medium was collected and the embryos were transferred to the recipients. After pregnancy diagnosis, the media were grouped into the pregnant and nonpregnant groups. The metabolic profiles of the media were analyzed via electrospray ionization mass spectrometry, and the concentrations of pyruvate, lactate, and glutamate were assessed using fluorimetry. The spectrometric profile revealed that the media from embryos from the pregnant group presented a higher signal intensity compared to that of the nonpregnant group; the ions 156.13 Da [M + H]+, 444.33 Da [M + H]+, and 305.97 Da [M + H]+ were identified as biomarkers. Spent culture medium from expanded blastocysts (Bx) that established pregnancy had a greater concentration of pyruvate (p = 0.0174) and lesser concentration of lactate (p = 0.042) than spent culture medium from Bx that did not establish pregnancy. Moreover, pyruvate in the culture media of Bx can predict pregnancy with 90.9% sensitivity and 75% specificity. In conclusion, we identified markers in the culture media that helped in assessing the most viable IVP embryos with a greater potential to establish pregnancy.

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