Effect of Pyruvate and Lactic Acid on the Development of Porcine Embryos

2011 ◽  
Vol 343-344 ◽  
pp. 611-617
Author(s):  
Bo Fu ◽  
Di Liu ◽  
Hong Ma ◽  
Zhong Qiu Li ◽  
Liang Wang ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Experimental Design ◽  
In Vitro Culture ◽  
Culture System ◽  
Culture Medium ◽  
Parthenogenetic Embryos

This study aimed to investigate the effects of pyruvate and lactic acid on the earlier development of porcine embryos. 5.56 mmol/L glucose in culture medium (NCSU-23) was replaced with 0.2 mmol/L pyruvate and 5.7 mmol/L lactic acid, namely mNCSU-23. Parthenogenetic embryos and nuclear transferred embryos were transferred into NCSU-23 or mNCSU-23 medium according to the experimental design. Parthenogenetic embryos and nuclear transferred embryos were evaluated for the numbers of 5-8 cells stage on Day 2. Blastocyst rates and the numbers of nuclei in the blastocyst were determined on Day 6. From the results, we observed a higher proportion of the embryos reaching 5-8 cells stage at 48h post-activation in mNCSU/NCSU treatment than control during the first part of in vitro culture(IVC) (P < 0.05). In addition, we also observed that the rates of blastocysts formation in mNCSU/NCSU treatments on Day 6 were significantly higher than other treatments, with mNCSU-23/mNCSU-23 treatment having the lowest rates of blastocysts formation on Day 6 (P < 0.05). Our results have demonstrated that replacing glucose with pyruvate and lactic acid during the first part of IVC may be beneficial to the development of the porcine embryos and stepwise culture system(mNCSU-23/NCSU-23) was optimal.

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

scholarly journals Interval in the replacement of in vitro culture medium affects the integrity and development of equine preantral follicles

2018 ◽  
Vol 38 (12) ◽  
pp. 2284-2288
Author(s):  
Camila Bizarro-Silva ◽  
Suellen M. González ◽  
Isabela Búfalo ◽  
Andressa G. Lindquist ◽  
Fabiana D. Sarapião ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture System ◽  
Culture Medium ◽  
Follicular Development ◽  
Significance Level ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Medium Exchange ◽  
Significant Difference

ABSTRACT: The efficiency of a culture system is related to the elaboration and replacement of a medium with conditions suitable for follicular development. Recent investigations suggested that in vitro culture medium should be replaced after specific time periods in various species. However, the suitable interval for the exchange of in vitro culture medium has not yet been established in equine species. The objective of this investigation was to evaluate the effect of medium exchange intervals of 24 hours (T24) or 48 hours (T48) for in vitro culture of preantral follicles at 2 or 6 days. At the end of the culture period, the fragments were processed using classical histology. Equine preantral follicles were classified according to morphological integrity and developmental stage. Data analysis was performed using Fisher’s test with a significance level of p<0.05. Out of a total of 399 follicles evaluated, 174 (43.6%) were primordial follicles, 225 (56.4%) were in development, and 63.76% were morphologically intact. In the in vitro culture performed over two days, there was no significant difference in relation to follicular integrity after medium replacement (p>0.05). Compared to the medium replacement at six days of culture, there was a statistically significant difference for T24 (68.9%, p<0.05). Therefore, we suggest changing the medium for equine species at 48 hours after the start of culture followed by subsequent daily replacements.

scholarly journals 2POSTHATCHING DEVELOPMENT SYSTEM: A NOVEL IN VITRO CULTURE OF BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 123 ◽  
Author(s):  
D.O. Brandão ◽  
G. Vajta ◽  
P. Maddox-Hyttel ◽  
D. Stringfellow ◽  
P. Lövendahl ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Bovine Serum ◽  
Culture System ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Fetal Bovine Serum ◽  
Cell Mass ◽  
Embryo Morphology ◽  
Fetal Bovine

Although high blastocyst rates can be achieved in somatic cell nuclear transfer, abortions and developmental abnormalities still hamper advancement. Reliable and practical methods to evaluate early embryonic development and differentiation are required to understand and overcome the problem. Our aim was to establish an in vitro culture system for monitoring posthatching development (PHD). Slaughterhouse-derived bovine oocytes were matured in vitro, fertilized (Day 0) and cultured (Holm et al., 1999, Theriogenology, 52, 683–700). On Day 8, degenerated embryos were removed from each well and 400L of modified culture medium (SOFaaci plus 0.5% glucose and 10% fetal bovine serum) were added. At Day 11, hatched blastocysts were selected by scoring them as Quality 1 (Q1: &gt;1.0mm, clear trophoblast, compact inner cell mass), Quality 2 (Q2: 0.5mm, dark spots in the trophoblast, less compact inner cell mass), or Quality 3 (Q3: &lt;0.5mm, many dark spots in the trophoblast, spread inner cell mass). The resulting 304 blastocysts in 12 replicates were then loaded into 15mm×1.2 gel tunnels of 2.4% agarose in PBS, supplemented with either 5% (Agar5) or 10% (Agar10) fetal bovine serum, covered with the modified culture medium, and then incubated at 38.5°C in 5% CO2, 5% O2, 90% N2. Embryo morphology and length were evaluated using a stereomicroscope on Days 12, 13, 14 and 15. On Day 14, 75 embryos were removed, biopsed (1mm) for sex determination of each embryo, and processed for light and transmission electron microscopy. Qualitative and quantitative data were analyzed by χ2 test and GLM procedure of SAS, respectively, with P level of 0.05. A total of 170 embryos (56% of total) initiated elongation. This percentage was higher (LSmeansSD, n=12; P&lt;0.05) in Agar10 v. Agar5 in both Q1 (889 v. 637), Q2 (667 v. 485) and Q3 embryos (529 v. 278). Mean embryo length (mm; LSmeansSEM) on Day 13 was higher (P&lt;0.05) in Q1 (2.10.2, n=49) and Q2 (1.71.4, n=98) than Q3 (1.20.3, n=23). On Day 14, Q1 embryos (3.50.2) were longer (P&lt;0.01) than Q2 and Q3 embryos (2.70.1 and 2.00.3). On Day 15, Q1, Q2 and Q3 embryos (4.40.5, n=24, 4.00.3, n=45 and 2.90.6, n=14, respectively) had similar length, probably influenced by the low number of Q3 embryos. The percentage of males was higher (P&lt;0.001) in Q1 (95%; n=40), but similar in Q2 (39%; n=26) and Q3 (71%; n=7). Light microscopy confirmed hypoblast and epiblast formation. Ultrastructural analysis revealed that the latter had penetrated the trophoblast (Rauber’s layer), forming an embryonic disc including many degenerative cells. In conclusion, this culture system represents the first model for rapid growth, elongation, and initial differentiation of bovine posthatching embryos.

395 OVINE SPERMATOGONIA ISOLATION AND IN VITRO GROWTH ASSAY

2010 ◽  
Vol 22 (1) ◽  
pp. 354
Author(s):  
A. Qi ◽  
T. Wuliji ◽  
Y. Zhang
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Culture System ◽  
Culture Medium ◽  
Single Cells ◽  
Testicular Tissue ◽  
Basic Medium ◽  
Seminiferous Tubules ◽  
In Vitro Culture System

Spermatogonia, as adult stem cells from the male reproductive system, are attracting strong interest from those studying male reproductive gamete preservation and developing new approaches in transgenic animals. Protein gene product 9.5 (PGP9.5) is a marker of sheep spermatogonia, which has been validated by J. R. Rodriguez-Sosa et al. (2006).The objective of this study was to develop an in vitro culture system for spermatogonial growth. Twenty 2- to 3-month-old rams were randomly selected at a local slaughter house for testicular tissue collection. Two-step enzymatic digestion methods were used for spermatogonia isolation from seminiferous tubules. In brief, mechanically isolated seminiferous tubules from testicular tissue were incubated in 1:1 1 mg mL-1 collagenase and hyaluronidase with 5 μg mL-1 DNase I for 20 min at 37°C. Most of the surrounding interstitial cells will fall off from seminiferous tubules by slightly pipetting. Seminiferous tubules were from cell suspensions after natural sedimentation in PBS and then were digested in 0.25% trypsin + 0.04% EDTAfor 5 to 7 min at 37°C to disassociate tubules into single cells. Data were analyzed with ANOVA procedures. Means of 5 specimens were presented. Of the total isolated cells, 19.7 ± 5.3% were identified as PGP9.5+cells, and 23.8 ± 3.6% were identified as c-kit+ cells. C-kit, the transmembrane tyrosine kinase receptor for stem cell factor, has been identified, which is expressed and functional in differentiating A1-A4 spermatogonia but not in spermatogonial stem cells (OhtaH2000). For in vitro culture of spermatogonia, DMEM supplemented with 1X ITS (insulin, transferrin, selenium), 100 μM β-mercaptoethano, 6 mM L-Glutamine, and 1X nonessential amino acids were used as basic culture medium. We have found that in primary spermatogonia culture, cells cultured together with testis somatic cells (sertoli cells) in basic medium supplied with 2.5% fetal bovine serum (FBS) were much more efficient than culturing with a supplement of 10% FBS. In primary culture, growing round-shaped cell colonies were visible from Day 5 in basic culture medium. In subculture, colonies were enzymically digested into smaller pieces from Day 8 to 10 and then placed onto mouse embryonic fibroblasts feeder layer in 2.5% FBS basic medium with the addition of 100 ng mL-1 glial cell-derived neurotropic factor, 10 ng mL-1 leukemia inhibitory factor, and 10 ng mL-1 basic fibroblast growth factor. PGP9.5+ spermatogonia cell colonies maintained their normal round shape until 4 to 5 passages. However, in subsequent passages, the colonies became flattened and cells gradually lost their interconnection and the growth pattern present in the early passages. Also in subsequent passages, cells began expressing more C-kit than PGP9.5. In conclusion, PGP9.5+ spermatogonia were successfully isolated from 2- to 3-month-old ram testis, and PGP 9.5 cell colonies were maintained and proliferated in the in vitro culture system up to 2 months.

Evaluating in Vitro Culture Medium of Gut Microbiome with Orthogonal Experimental Design and a Metaproteomics Approach

2017 ◽  
Vol 17 (1) ◽  
pp. 154-163 ◽  
Author(s):  
Leyuan Li ◽  
Xu Zhang ◽  
Zhibin Ning ◽  
Janice Mayne ◽  
Jasmine I. Moore ◽  
...  
Keyword(s):  
Experimental Design ◽  
In Vitro Culture ◽  
Gut Microbiome ◽  
Culture Medium ◽  
Orthogonal Experimental Design

239 CANINE OOCYTES CAN BE SPONTANEOUSLY PARTHENOGENERATED AND DEVELOPED TO 2-CELL TO 8-CELL STAGE PARTHENOGENERATED EMBRYOS IN COCULTURE SYSTEM WHICH THEY WERE COCULTURED WITH OVIDUCT CELLS OR OVIDUCT EXTRACTS

2009 ◽  
Vol 21 (1) ◽  
pp. 217
Author(s):  
L. X. Wang ◽  
S. Wang ◽  
J. Hou ◽  
Y. F. Chen ◽  
R. L. Hu ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Cell Volume ◽  
Granulosa Cells ◽  
Culture System ◽  
Culture Medium ◽  
Cell Stage ◽  
Culture Systems ◽  
System A ◽  
Coculture System

The objective of this study was to coculture canine oocytes with oviduct cells or oviduct extracts in modified M199 culture medium to improve the MII rate. The canine oocytes were collected from the local slaughterhouse and were cultured in three culture systems while in the same culture medium. In culture system A, canine oocytes were cocultured with ‘small-round’ oviduct cells and ‘big-round’ oviductal cells which based on cell volume and morphology for 96 h. In culture system B, canine oocytes were cultured with in the modified M199 medium with 10% canine follicle fluid (in the CEF group) or without canine follicle fluid (in the compared group) for the first 6 h; following this, they were cocultured with ‘small-round’ oviductal cells for another 90 h, which were preblanced at least 2 h. In the culture system C, the canine oocytes were cocultured with canine estrous oviduct extracts for 96 h (in the compared group) or were cocultured with granulosa cells together with canine estrous oviduct extracts (in the G + CEOE group) for 96 h. These results demonstrated that, in culture system A, one 2-cell and one 8-cell stage parthenogenerated embryo in the small-round group and one 2-cell stage parthenogenerated embryo in the compared group were achieved after in vitro culture for 96 h. Meanwhile, the MII rates were greater in small-round group (47.1%) than that in big-round group (36.1%) and the compared group (32.4%). In the culture system B, one 3-cell stage parthenogenerated embryo was detected in CEF group. The MII rates were almost equal in CEF group (28.0%) and in the compared group (26.1%). In culture system C, one 2-cell stage parthenogenerated embryo was found in the compared group. The MII rate were almost equal in CEOE group (25.8%) and in the G + CEOE group (23.5%). It is concluded that some unidentified factors secreted by the oviductal cells or oviduct extracts may promote the development of canine oocytes in vitro and parthenogenerate the canine oocytes beyond 2-cell stage parthenogenerated embryos. The culture system in which the canine oocytes were cocultured with oviducts or oviductal cells has been improved to date.

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 In-vitro culture system for mesenchymal progenitor cells derived from waste human ovarian follicular fluid

2014 ◽  
Vol 29 (4) ◽  
pp. 457-469 ◽  
Author(s):  
Federica Riva ◽  
Claudia Omes ◽  
Roberto Bassani ◽  
Rossella E Nappi ◽  
Giuliano Mazzini ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Progenitor Cells ◽  
Follicular Fluid ◽  
Culture System ◽  
Mesenchymal Progenitor Cells ◽  
In Vitro Culture System ◽  
Ovarian Follicular Fluid

Synthesis of RNA in oocytes of Xenopus laevis during culture in vitro

Development ◽  
1974 ◽  
Vol 32 (2) ◽  
pp. 515-532
Author(s):  
A. Colman
Keyword(s):  
Xenopus Laevis ◽  
In Vitro Culture ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Salt Medium ◽  
Complex Media ◽  
18S Ribosomal Rna ◽  
Rna Precursor

RNA synthesis can be maintained in large oocytes of Xenopus laevis during periods of in vitro culture of at least 10 days. A simple salt medium, modified Barth's solution, is found to be as effective a culture medium for these oocytes as several other complex media. The newly synthesized RNA is characterized electrophoretically and shown to consist predominantly of ribosomal RNA precursor, 28S and 18S ribosomal RNA, and 4S RNA. The distribution of this RNA within the oocyte is detected autoradiographically, where it is found to be greatly concentrated over the nucleoli. No qualitative alterations in either of these parameters are found during culture, within the limits of sensitivity of the assay procedures.

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