Ionizing Radiation Effects In Vitro Study; Using The Rat Whole Embryo Culture Model

2021 ◽  
Vol 6 (13) ◽  
pp. 91-100
Author(s):  
Esra Balcioglu ◽  
Munevver Baran ◽  
Mehtap Nisari ◽  
Ozge Goktepe ◽  
Pinar Bilgici ◽  
...  
Keyword(s):  
Ionizing Radiation ◽  
Embryo Culture ◽  
Radiation Effects ◽  
Cell Number ◽  
Yolk Sac ◽  
Culture Period ◽  
Histological Evaluation ◽  
Ionizing Irradiation ◽  
Whole Embryo Culture

Background: Ionizing radiation poses a threat to the early embryo possibly leading to prenatal death, growth retardation, organ malformation, or mental retardation. It is important, assessment of any adverse effects of radiation upon the embryo. This study aimed to evaluate the outcomes of embryos irradiated with 1Gy doses in vitro and investigate hematopoiesis in the yolk sac of the irradiated embryos. Materials and methods: In the study, the experimental group of rats was be exposed to total body ionizing radiation on days 8.5th of gestation. All embryos in the control and radiation group cultured from gestation day 9.5 to 11.5 were alive at the end of the culture period. After 48 hours culture period, the embryos from each group were harvested and analyzed morphologically. Histological evaluation of the vWF+ cell number was performed in vivo. Results: The results showed that the embryonic growth and development during organogenesis decreased in the radiation exposed embryos when compared to control embryos. Additionally, the immunofluorescent examination showed that the vWF+ cell number reduced in the yolk sac of embryos exposed to ionizing radiation. Conclusion: Consequently, these findings support the conclusion that 1 Gy ionizing irradiation may increase prenatal death, intrauterine growth restriction on embryonic development when ionizing irradiation decreases the vWF+ cell number in the yolk sac compared to control embryos. This research related to radiation was the first study using the in vitro embryo culture technique; thus, future studies that will be performed by using different doses of radiation will contribute to the literature.

scholarly journals Znanstvena istraživanja u 21. stoljeću - etički aspekti uporabe laboratorijskih životinja i alternativni modeli

2022 ◽  
Vol 53 (5) ◽  
Author(s):  
Ivana Kmetič ◽  
Monika Roller ◽  
Marina Miletić ◽  
Teuta Murati
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Embryo Culture ◽  
Embryonic Stem ◽  
Whole Embryo Culture ◽  
Cell Test

U toksikološkim istraživanjima uz uporabu klasičnih (in vivo) istraživanja, primjenjuju se alternativni test sustavi. Korištenje laboratorijskih životinja, embrija, humanog i animalnog tkiva, kultura stanica i fetalnog seruma u istraživanjima smatra se etički problematičnim te se ograničava zakonima, pravilnicima i praksom. Razmatranjem načina kojima bi se neetičnost mogla izbjeći, došlo je do razvoja “3R” načela (akronim za tri pristupa koja bi se trebala provoditi pri istraživanjima na laboratorijskim životinjama), a to su: smanjenje/racionalizacija uporabe laboratorijskih životinja (engl. Reduction), načelo njihove zamjene (engl. Replacement) i poboljšanje uvjeta uzgoja, smještaja i skrbi za životinje (engl. Refinement). Većina je alternativnih testova toksičnosti još uvijek u postupku validacije. Pojedini in vitro testovi za istraživanja embriotoksičnosti (etički posebno osjetljivo područje) koja su priznala nadležna regulatorna tijela, su EST (engl. Embryonic Stem cell Test), WEC (engl. Whole- Embryo Culture) i MM (engl. MicroMass) test. Standardizacija protokola i uvođenje novih in vitro modela predstavlja važan segment napretka u toksikološkim istraživanjima. Znanstvena budućnost tu vidi mogućnost razvoja i implementacije načela etičnosti u istraživanja primjenjujući sustave koji će promišljeno i bez korištenja živih organizama dijelom nadomjestiti metode u biomedicini, veterinarskoj medicini, biotehnologiji i užem smislu - toksikologiji i farmakologiji.

Effects of changing culture medium on preimplantation embryo development in rabbit

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
Haixia Wang ◽  
Wenbin Cao ◽  
Huizhong Hu ◽  
Chenglong Zhou ◽  
Ziyi Wang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cell Number ◽  
Apoptotic Index ◽  
Total Cell ◽  
Toxic Substances ◽  
Total Cell Number ◽  
Preimplantation Embryo Development

Summary Many studies have focused on the optimization of the composition of embryo culture medium; however, there are few studies involving the effect of a culture medium changing procedure on the preimplantation development of embryos. In this study, three groups were designed: a non-renewal group, a renewal group and a half-renewal group. The levels of reactive oxygen species (ROS), apoptotic index, blastocyst ratio and blastocyst total cell number were analyzed in each group. The results showed that the ROS level and the apoptotic index of blastocyst in the non-renewal group were significantly higher than in the renewal group and the half-renewal group (P < 0.05). The blastocyst ratio and blastocyst total cell number were significantly higher in the half-renewal group than that in non-renewal group and the renewal group (P < 0.05). These results demonstrated that the procedure of changing the culture medium influenced ROS level, apoptotic index, blastocyst ratio and total cell number of blastocysts. In addition, the result suggested that changing the culture medium may lead to a loss of important regulatory factors for embryos, while not changing the culture medium may lead to the accumulation of toxic substances. Half-renewal can alleviate the defects of both no renewal and renewal, and benefit embryo development. This study will be of high value as a reference for the optimization of embryo culture in vitro, and is very significant for assisted reproduction.

155 QUALITY OF PORCINE EMBRYO PRODUCED IN TWO EMBRYO CULTURE MEDIA AS ASSESSED BY TOTAL CELL NUMBER AND TIME COURSE OF BLASTOCYST HATCHING

2006 ◽  
Vol 18 (2) ◽  
pp. 185 ◽  
Author(s):  
Y. Agca ◽  
H. Men ◽  
S. F. Mullen ◽  
L. K. Riley ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Time Course ◽  
Culture Media ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Blastocyst Hatching ◽  
Hatching Rates

The ability to produce porcine embryos of good quality will have a significant impact on a number of porcine assisted reproductive technologies, such as cloning, intracytoplasmic sperm injection, and embryo cryopreservation. However, porcine embryos resulting from current serum-free embryo culture systems differ significantly both structurally and functionally from those derived in vivo (Wang et al. 1999 Mol. Reprod. Dev. 53, 99-107). In this experiment, the quality of porcine embryos produced by North Carolina State University (NCSU)-23 medium (Petters and Wells 1993 J. Reprod. Fertil. Suppl. 1993, 48, 61-73) and porcine zygote medium (PZM)-1 (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) were compared by assessing the total cell number and the time course of in vitro blastocyst hatching. Porcine embryos were produced by in vitro maturation and fertilization using serum-free systems. After fertilization, presumptive zygotes were randomly allocated to either PZM-1 or NCSU-23 for subsequent development. On Day 4 of culture, the embryo culture media were supplemented with 10% fetal bovine serum (FBS). Day 6 blastocysts from each group were counted and the blastocysts were subsequently fixed in 4% formalin for counting the total cell number. The cell number in each embryo was determined by counting the nuclei after staining with bisbenzimide (Hoechst 33342). To assess the hatching ability of blastocysts, Day 6 blastocysts were cultured until Day 9 and hatched blastocysts were counted daily. Day 6 blastocyst rates (ratio of blastocysts to oocytes) and total cell number count were replicated three times. The time course of blastocyst hatching experiment was repeated four times. The data were analyzed using a chi-square test, Fisher's exact test, or Student's t-test. The blastocyst rate from culture in PZM-3 was 19.4 � 0.96% (mean � SEM), which was similar to that (16.7 � 3.2%) resulting from culture in NCSU-23 (P > 0.05). However, the total cell number in Day 6 blastocysts cultured in PZM-3 was significantly higher than for blastocysts cultured in NCSU-23 (57 � 3.1 vs. 46 � 1.7; P < 0.01). The total hatching rates (ratio of hatched blastocysts to total blastocysts) by Day 9 were similar between the two culture systems (50.1 � 9.1% vs. 50.7 � 4.1%; P > 0.05). However, on Day 6, 2.1% of blastocysts from PZM-3 culture hatched whereas no blastocysts from NCSU-23 culture hatched. The cumulative hatching rates from PZM-3 culture on Day 7 were significantly higher than those from NCSU-23 culture (15.1 � 3.8% vs. 2.6 � 1.1%; P < 0.01). In conclusion, these data suggest that blastocysts produced in PZM-3 medium have better quality than blastocysts produced in the NCSU-23 culture system as assessed by the total cell number and the time course of blastocyst hatching. This project was supported by a grant from the National Institutes of Health (U42 RR 018877).

97 SUPPLEMENTATION WITH FOLATE IN VITRO INCREASES TROPHECTODERM AND TOTAL CELL NUMBER IN IN VITRO-DERIVED PORCINE BLASTOCYSTS

2012 ◽  
Vol 24 (1) ◽  
pp. 161 ◽  
Author(s):  
B. K. Redel ◽  
L. D. Spate ◽  
A. N. Brown ◽  
R. S. Prather
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Transcriptional Profiling ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Total Cell Number ◽  
Culture Systems

It is vital that improvements are made to current culture environments because in vitro culture systems are suboptimal compared with in vivo. A previous transcriptional profiling endeavour conducted by Bauer et al. (2010 Biol. Reprod. 83, 791–798) identified hundreds of mRNA transcripts that were mis-expressed in porcine embryos fertilized in vivo and then cultured in vitro to Day 6 compared with in vivo Day-6 embryos. Enriched in the downregulated transcripts were 4 genes involved with the one carbon pool by folate KEGG pathway. This downregulation of genes involved with folate metabolism may illustrate an impaired folate homeostasis in embryos cultured in the current culture environment. The objective of this study was to determine the effects folate had on embryo development of in vitro fertilized embryos. Porcine cumulus–oocyte complexes were matured for 44 h in M199 supplemented with epidermal growth factor (EGF), FSH and LH. Oocytes with a visible polar body were selected and fertilized in modified tris buffered medium for 5 h and then placed into porcine zygote medium 3 with 0 mM, 0.2 mM, 0.4 mM and 0.8 mM folate to find the optimal concentration of folate. Twenty-eight hours post-fertilization, cleaved embryos were selected and moved into 25-μL drops of respective culture medium and cultured to Day 6 in a water-saturated atmosphere of 5% CO2, 5% O2, 90% N2, at 38.5°C. To determine the effect folate had on development, the blastocyst rate for each treatment group was measured. Results were log-transformed and analysed by using PROC GLM in SAS (SAS Institute Inc., Cary, NC). A least-significant difference post-test comparison was completed to determine if significant differences existed between treatment groups. The percentage of cleaved embryos on Day 6 that developed to blastocyst was 56.2%, 55.9%, 66.9% and 61.8% (n = 133, 149, 135 and 135) in 0 mM, 0.2 mM folate, 0.4 mM folate and 0.8 mM, respectively. The 0.4 mM folate group tended (P = 0.07) to have a higher number of cleaved embryos that developed to the blastocyst stage. Consequently, this concentration was used for all further embryo culture experiments. Differential staining was completed to compare the number of trophectoderm and inner cell mass nuclei for embryos cultured in 0 mM or 0.4 mM folate concentrations. Staining revealed that embryos cultured with folate had an increase in number of trophectoderm (29.7 ± 1.5 vs 24.4 ± 1.4 cells; P = 0.0058) and total cell (36.9 ± 1.0 vs 31.7 ± 1.0; P = 0.0007) numbers compared with embryos cultured without folate. These results illustrate that the addition of folate to current culture medium doesn't hinder development to blastocyst and by increasing trophectoderm and total cell number may give rise to better-quality in vitro-derived embryos. It is evident that using transcriptional profiling can be a great method of identifying ways to improve embryo culture systems and, in this case, supplementing with folate. Funded by Food for the 21st Century.

scholarly journals Effect of various co-culture systems on embryo development in ovine

2013 ◽  
Vol 58 (No. 10) ◽  
pp. 443-452 ◽  
Author(s):  
B. Heidari ◽  
A. Shirazi ◽  
M.-M. Naderi ◽  
M.-M. Akhondi ◽  
H. Hassanpour ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
Cell Number ◽  
Culture Conditions ◽  
The Other ◽  
Culture Period ◽  
Hatching Rate ◽  
Embryonic Fibroblast ◽  
One Way Anova

Considering the advent of mesenchymal stem cells (MSCs) as a new source of somatic cells in embryo co-culture system, the current study was aimed to compare in vitro embryo development using embryonic MSCs monolayer with embryonic fibroblast cells (EFCs), oviductal epithelial cells (OECs), and cell-free culture system. The IVM/IVF presumptive sheep zygotes were randomly cultured in different culture conditions as follows: (1) SOFaaBSA medium for the whole culture period (SOF, n = 371), (2) SOFaaBSA medium for the first 3 days followed by co-culturing with MSCs for the next 5 days (SOF-MSCs, n = 120), (3) co-culturing with MSCs for the first 3 days followed by culture in SOFaaBSA medium for the next 5 days (MSCs-SOF, n = 133), (4) co-culturing with MSCs for the whole culture period (MSCs, n = 212), (5) SOFaaBSA medium for the first 3 days followed by co-culturing with EFCs for the next 5 days (SOF-EFCs, n = 132), (6) co-culturing with EFCs for the first 3 days followed by culture in SOFaaBSA medium for the next 5 days (EFCs-SOF, n = 165), (7) co-culturing with EFCs for the whole culture period (EFCs, n = 236), and (8) co-culturing with OECs for the whole culture period (OECs, n = 255). One-Way ANOVA by multiple pairwise comparisons using Tukey&rsquo;s test was performed. Co-culturing in MSCs group had no superiority over EFCs and OECs groups. Though, when co-culturing with MSCs and EFCs was limited to the first 3 days of culture, the embryo development indices were improved compared to the other co-cultured groups. Considering both the hatching rate and total cell number, the application of MSCs for the first 3 days of culture (MSCs-SOF) was superior to the other co-culture and SOF groups. &nbsp;

Synergism between photochemical and ionizing radiation effects in MCF-7 cells in vitro

1992 ◽  
Author(s):  
Gulshan Ara ◽  
Terence S. Herman ◽  
Archana Varshney ◽  
Timothy Korbut ◽  
Beverly A. Teicher
Keyword(s):  
Ionizing Radiation ◽  
Radiation Effects ◽  
Mcf 7

Valproic acid-induced gene expression responses in rat whole embryo culture and comparison across in vitro developmental and non-developmental models

2013 ◽  
Vol 41 ◽  
pp. 57-66 ◽  
Author(s):  
Elisa C.M. Tonk ◽  
Joshua F. Robinson ◽  
Aart Verhoef ◽  
Peter T. Theunissen ◽  
Jeroen L.A. Pennings ◽  
...  
Keyword(s):  
Gene Expression ◽  
Valproic Acid ◽  
Embryo Culture ◽  
Whole Embryo Culture ◽  
Developmental Models

scholarly journals 152EFFECT OF REPLACEMENT OF PYRUVATE/LACTATE IN CULTURE MEDIUM WITH GLUCOSE ON PREIMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 198
Author(s):  
N.W.K. Karja ◽  
S. Medvedev ◽  
D. Fuchimoto ◽  
A. Onishi ◽  
M. Iwamoto ◽  
...  
Keyword(s):  
Energy Source ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Culture Period ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Energy Substrates ◽  
Anova Test

Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) reported that replacement of pyruvate and lactate with glucose, as energy substrates, at 48h of culture in IVC medium enhanced the quality of IVP porcine blastocysts. However, the exact time during early cleavage stages when the utilization of glucose as an energy source is optimal has not yet been determined. The purpose of this study was to examine the effects of glucose supplementation at different times of culture on the developmental competence of IVP porcine embryos. Porcine cumulus-oocytes complexes were matured in modified NCSU-37 solution and fertilized in vitro according to Kikuchi et al. All cultures were performed at 38.5°C, 5% O2, 5% CO2, and 90% N2. In experiment 1, after being fertilized (Day 0), putative zygotes (1158 in 6 trials) were cultured in NCSU-37 supplemented with 0.4% BSA, 0.17mM sodium pyruvate, and 2.73mM sodium lactate (IVC-pyr/lac). Embryos (30–50 in each group) were then transferred into NCSU-37 supplemented with 0.4% BSA and 5.55mM D-glucose (IVC-glu) at 24, 48, 72, 96, or 118h of culture. As control groups, putative zygotes (391) were cultured in IVC-pyr/lac or IVC-glu for the whole culture period. In experiment 2, after being fertilized, putative zygotes (543 in 4 trials, 30–50 in each group) were cultured in IVC-pyr/lac, and then were transferred into IVC-glu at 48h, 53h, 58h, or 63h of culture, because glycolytic activity of in vitro-derived porcine embryos was reported to increase around the 8-cell stage, and some embryos develop to that stage before 72h of culture in experiment 1. All embryos were cultured for 6 days, and then development to the blastocyst stage and number of cells per blastocyst were assessed. When IVF embryos were cultured in IVC pyr/lac for 24h or 48h and subsequently in IVC-glu until day 6 in experiment 1, the rates of blastocyst formation were significantly higher (P&lt;0.05, ANOVA test) than those of embryos cultured in IVC-pyr/lac for the whole culture period (24.4% and 23.0% v. 14.5%, respectively). However, when IVC pyr/lac was replaced with IVC-glu, there were no significant differences between the energy source replacement groups and the glucose-only group in terms of the proportions of cleavage, development to the blastocyst stage and mean cell number per blastocyst (P&gt;0.05, ANOVA test) (15.2%–24.4%, and 16.8%, respectively). Replacement of pyruvate and lactate with glucose at 58h of culture in experiment 2 significantly enhanced the rate of blastocyst formation (P&lt;0.05, ANOVA test) but not the mean cell number compared with zygotes in which the replacement was done at 48, 53, and 63h of culture (31.3% v. 20.6%, 20.8%, and 21.1%, respectively) (P&lt;0.05, ANOVA test). In conclusion, replacement of pyruvate and lactate with glucose as energy substrates was optimal at 58h of culture for the in vitro development of pig embryos to the blastocyst stage.

scholarly journals Effect of N-acetyl cysteine on the quality of blastocyst formation rate using cultured vitrified murine embryos

2020 ◽  
Vol 71 (3) ◽  
pp. 2315
Author(s):  
S. SIGÜENZA ◽  
I.S. ÁLVAREZ ◽  
E. MATILLA
Keyword(s):  
Embryo Culture ◽  
Formation Rate ◽  
Cell Number ◽  
Embryo Cryopreservation ◽  
Radical Scavenger ◽  
Murine Embryos ◽  
And Control

Vitrification is the best method for embryo cryopreservation although it increases endogenous reactive oxygen species (ROS) production. N-acetylcysteine (NAC) a free radical scavenger may be used for reducing ROS toxic effects. The aim of the present study is to investigate potential beneficial effects of NAC on the developmental embryo competence applying different culture conditions in vitrified-warmed 2-cell embryos derived in vivo or in vitro. Thus, 2-cell embryos were vitrified or cultured fresh in presence or absence of 1 mM of NAC during: a) the entire embryo culture, b) for 24 hours with NAC at days 1.5 (G1) or 2.5 (G2) and returned to basal embryo culture (KSOM) or c) cultured in the presence of NAC for 12 hours at day 3.5 (G3). Despite NAC addition to fresh or vitrified embryos produced in vivo or by IVF, blastocyst rates remained unchanged. In vitrified-warmed IU or IVF-derived embryos, total cell number varied when NAC was added at day 1.5 although differences were not significant (60.1 ± 1.9 vs. 59.4 ± 1.3 for IU G1 and control respectively; and 59.3 ± 1.6 and 52.6 ± 3.0 IVF G1 and control respectively; mean cell number ± SEM, p > 0.05). It seems that the embryo culture medium supplementation with 1 mM of NAC in the first day after vitrification of development improves blastocyst quality of murine embryos and does not exert any beneficial effect at oyher culture points.

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