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.

In vitro culture and non-invasive metabolic profiling of single bovine embryos

2019 ◽  
Vol 31 (2) ◽  
pp. 306
Author(s):  
Monika Nõmm ◽  
Rando Porosk ◽  
Pille Pärn ◽  
Kalle Kilk ◽  
Ursel Soomets ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Metabolic Profiling ◽  
Culture Medium ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Growth Media ◽  
Low Molecular Weight ◽  
Bovine Embryos ◽  
Non Invasive

Selecting high-quality embryos for transfer has been a difficult task when producing bovine embryos invitro. The most used non-invasive method is based on visual observation. Molecular characterisation of embryo growth media has been proposed as a complementary method. In this study we demonstrate a culture medium sampling method for identifying potential embryonic viability markers to predict normal or abnormal embryonic development. During single embryo culture, 20µL culture media was removed at Days 2, 5 and 8 after fertilisation from the same droplet (60µL). In all, 58 samples were analysed using liquid chromatography–mass spectrometry. We demonstrate that it is possible to remove samples from the same culture medium droplets and not significantly affect blastocyst rate (25.2%). Changes in any single low molecular weight compound were not predictive enough. Combining multiple low molecular weight signals made it possible to predict Day 2 and 5 embryo development to the blastocyst stage with an accuracy of 64%. Elevated concentrations of lysophosphatidylethanolamines (m/z=453, 566, 588) in the culture media of Day 8 well-developing embryos were observed. Choline (104m/z) and citrate (215m/z) concentrations were increased in embryos in which development was retarded. Metabolic profiling provides possibilities to identify well-developing embryos before transfer, thus improving pregnancy rates and the number of calves born.

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.

scholarly journals Improving In Vitro Culture of Human Male Fetal Germ Cells

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2033
Author(s):  
Myriam Martin-Inaraja ◽  
Monica Ferreira ◽  
Jasin Taelman ◽  
Cristina Eguizabal ◽  
Susana M. Chuva De Sousa Lopes
Keyword(s):  
Germ Cells ◽  
Culture Medium ◽  
Culture Media ◽  
Culture Conditions ◽  
Pronounced Difference ◽  
Coated Substrate ◽  
Male Gametes ◽  
The Media

Male human fetal germ cells (hFGCs) give rise to spermatogonial stem cells (SSCs), which are the adult precursors of the male gametes. Human SSCs are a promising (autologous) source of cells for male fertility preservation; however, in contrast to mouse SSCs, we are still unable to culture them in the long term. Here, we investigated the effect of two different culture media and four substrates (laminin, gelatin, vitronectin and matrigel) in the culture of dissociated second trimester testes, enriched for hFGCs. After 6 days in culture, we quantified the presence of POU5F1 and DDX4 expressing hFGCs. We observed a pronounced difference in hFGC number in different substrates. The combination of gelatin-coated substrate and medium containing GDNF, LIF, FGF2 and EGF resulted in the highest percentage of hFGCs (10% of the total gonadal cells) after 6 days of culture. However, the vitronectin-coated substrate resulted in a comparable percentage of hFGCs regardless of the media used (3.3% of total cells in Zhou-medium and 4.8% of total cells in Shinohara-medium). We provide evidence that not only the choices of culture medium but also choices of the adequate substrate are crucial for optimizing culture protocols for male hFGCs. Optimizing culture conditions in order to improve the expansion of hFGCs will benefit the development of gametogenesis assays in vitro.

scholarly journals Isolation and Characterization of Functionally Active Extracellular Vesicles from Culture Medium Conditioned by Bovine Embryos In Vitro

2018 ◽  
Vol 20 (1) ◽  
pp. 38 ◽  
Author(s):  
Krishna Pavani ◽  
An Hendrix ◽  
Wim Van Den Broeck ◽  
Liesbeth Couck ◽  
Katarzyna Szymanska ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Embryo Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Apoptotic Cell ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Isolation And Characterization ◽  
Embryo Culture Medium

Extracellular vesicles (EVs) play a possible role in cell–cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrepTM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25–230 nm with an average concentration of 236.5 ± 1.27 × 108 particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.

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.

scholarly journals Nobiletin enhances the development and quality of bovine embryos in vitro during two key periods of embryonic genome activation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karina Cañón-Beltrán ◽  
Yulia N. Cajas ◽  
Serafín Peréz-Cerezales ◽  
Claudia L. V. Leal ◽  
Ekaitz Agirregoitia ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Signaling Pathway ◽  
Akt Signaling ◽  
Mitochondrial Activity ◽  
Bovine Embryos ◽  
Akt Signaling Pathway ◽  
Cell Stage ◽  
Development In Vitro

AbstractIn vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.

scholarly journals Metabolites Secreted by Bovine Embryos In Vitro Predict Pregnancies That the Recipient Plasma Metabolome Cannot, and Vice Versa

Metabolites ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 162
Author(s):  
Enrique Gomez ◽  
Nuria Canela ◽  
Pol Herrero ◽  
Adrià Cereto ◽  
Isabel Gimeno ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Stearic Acid ◽  
Palmitic Acid ◽  
Hippuric Acid ◽  
Culture Media ◽  
Capric Acid ◽  
Glyceryl Monostearate ◽  
Invasive Approach ◽  
Hydrocinnamic Acid

This work describes the use of mass spectrometry-based metabolomics as a non-invasive approach to accurately predict birth prior to embryo transfer (ET) starting from embryo culture media and plasma recipient. Metabolomics was used here as a predictive platform. Day-6 in vitro produced embryos developed singly in modified synthetic oviduct fluid culture medium (CM) drops for 24 h were vitrified as Day-7 blastocysts and transferred to recipients. Day-0 and Day-7 recipient plasma (N = 36 × 2) and CM (N = 36) were analyzed by gas chromatography coupled to the quadrupole time of flight mass spectrometry (GC-qTOF). Metabolites quantified in CM and plasma were analyzed as a function to predict pregnancy at Day-40, Day-62, and birth (univariate and multivariate statistics). Subsequently, a Boolean matrix (F1 score) was constructed with metabolite pairs (one from the embryo, and one from the recipient) to combine the predictive power of embryos and recipients. Validation was performed in independent cohorts of ETs analyzed. Embryos that did not reach birth released more stearic acid, capric acid, palmitic acid, and glyceryl monostearate in CM (i.e., (p < 0.05, FDR < 0.05, Receiver Operator Characteristic—area under curve (ROC-AUC)> 0.669). Within Holstein recipients, hydrocinnamic acid, alanine, and lysine predicted birth (ROC-AUC > 0.778). Asturiana de los Valles recipients that reached birth showed lower concentrations of 6-methyl-5-hepten-2-one, stearic acid, palmitic acid, and hippuric acid (ROC-AUC > 0.832). Embryonal capric acid and glyceryl-monostearate formed F1 scores generally >0.900, with metabolites found both to differ (e.g., hippuric acid, hydrocinnamic acid) or not (e.g., heptadecanoic acid, citric acid) with pregnancy in plasmas, as hypothesized. Efficient lipid metabolism in the embryo and the recipient can allow pregnancy to proceed. Changes in phenolics from plasma suggest that microbiota and liver metabolism influence the pregnancy establishment in cattle.

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 Viability of ‘Candidatus Liberibacter asiaticus’ Prolonged by Addition of Citrus Juice to Culture Medium

2014 ◽  
Vol 104 (1) ◽  
pp. 15-26 ◽  
Author(s):  
Jennifer K. Parker ◽  
Sarah R. Wisotsky ◽  
Evan G. Johnson ◽  
Faraj M. Hijaz ◽  
Nabil Killiny ◽  
...  
Keyword(s):  
Grapefruit Juice ◽  
Culture Medium ◽  
Quantitative Polymerase Chain Reaction ◽  
Chemical Characterization ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Greening Disease ◽  
Candidatus Liberibacter ◽  
The Media ◽  
Liberibacter Asiaticus

Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’. Infection with ‘Ca. L. asiaticus’ is incurable; therefore, knowledge regarding ‘Ca. L. asiaticus’ biology and pathogenesis is essential to develop a treatment. However, ‘Ca. L. asiaticus’ cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of ‘Ca. L. asiaticus’ in vitro, ‘Ca. L. asiaticus’ inoculum obtained from seed of fruit from infected pomelo trees (Citrus maxima ‘Mato Buntan’) was added to different media, and cell viability was monitored for up to 2 months using quantitative polymerase chain reaction in conjunction with ethidium monoazide. Media tested included one-third King's B (K), K with 50% juice from the infected fruit, K with 50% commercially available grapefruit juice, and 100% commercially available grapefruit juice. Results show that juice-containing media dramatically prolong viability compared with K in experiments reproduced during 2 years using different juice sources. Furthermore, biofilm formed at the air–liquid interface of juice cultures contained ‘Ca. L. asiaticus’ cells, though next-generation sequencing indicated that other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining ‘Ca. L. asiaticus’ viability in vitro, which will contribute to future development of a culture medium for ‘Ca. L. asiaticus’.

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