121 DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED IN SEQUENTIAL OR CONTINUOUS MEDIA

2008 ◽  
Vol 20 (1) ◽  
pp. 141
Author(s):  
L. S. Amorim ◽  
D. J. Walker ◽  
G. E. Seidel Jr
Keyword(s):  
Culture Media ◽  
Cumulus Cells ◽  
Similar Efficacy ◽  
Defined Medium ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Developmental Rates ◽  
Effects Of Media

Slaughtered bovine females have different characteristics including age, nutritional status, breed, and management system, all of which may affect the results obtained in in vitro embryo production. Another key consideration is that early embryos move from the oviduct to a slightly different environment in the uterus, which has led to development of sequential embryo culture media (e.g. Lane M et al. 2003 Theriogenology 60, 407–419). However, the benefits and importance of using sequential media are not fully known. Therefore, the aim of the present study was to compare developmental rates of oocytes obtained from slaughterhouse-derived ovaries from cows or heifers after culture in sequential media (CDM-1, CDM-2) or in a continuous medium (C-CDM). The experiment was a 3 × 2 × 2 factorial design [bulls (A, B, or C), source (cows or heifers), and medium (sequential or continuous)]. Cumulus–oocyte complexes were aspirated, within 5 h of slaughter, from 3- to 8-mm ovarian follicles of cows (1482 oocytes) and fattened heifers usually fed melengesterol acetate (2818 oocytes). Embryos were produced in vitro as described by De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596, with slight modifications. Presumptive zygotes were vortexed to remove cumulus cells and cultured for 2.5 d in C-CDM (CDM supplemented with 5.0 mm L-lactate, essential and nonessential amino acids, and 0.5% FAF-BSA, or in CDM-1 (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596) at 39°C in a humidified incubator under 5% CO2, 5% O2, and 90% N2. Cleavage was assessed after 2.5 d; 2- to 6-cell embryos were considered as cleaved, but were not cultured further. Embryos at the 7- to 8-cell stage were cultured for an additional 4.5 d in fresh C-CDM or CDM-2. The percentage blastocysts per oocyte was assessed after 7 and 8 days of culture. Data were arcsin-transformed and evaluated by ANOVA. There was a significant interaction between bull and ovary source for both 8-cell embryos and cleavage rate (P < 0.05); however, this interaction was no longer significant for blastocysts. No other interactions were significant nor a source of ovaries. Culturing embryos in CDM-C refreshed after cleavage evaluation (continuous) or culturing embryos in CDM-1 early and CDM-2 after cleavage evaluation (sequential) resulted in similar cleavage and blastocyst rates (Table 1). We conclude that bovine embryos can be produced using a single chemically defined medium (+BSA) with similar efficacy as a system using 2 sequential media. Table 1. Effects of media on embryonic development (mean ± SE)

98 EXPERIMENTAL TRANSFER OF BOVINE IVF-DERIVED 32-CELL STAGE EMBRYOS INTO THE UTERUS: ENVIRONMENTAL EFFECTS ON DEVELOPMENTAL CHARACTERISTICS

2016 ◽  
Vol 28 (2) ◽  
pp. 179
Author(s):  
M. Hoelker ◽  
D. Salilew-Wondim ◽  
F. Rings ◽  
D. Tesfaye ◽  
K. Schellander
Keyword(s):  
Culture Media ◽  
Uterine Cavity ◽  
Blastocyst Stage ◽  
Considerable Proportion ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Rates ◽  
Uterine Environment

Usually, in vitro-produced bovine embryos are cultured in vitro in static culture systems for 7 to 9 days in media composed according the oviducal fluid although it is well accepted that around Day 4.5–5 the bovine embryo enters the uterine cavity, providing environmental conditions different from the oviduct. Therefore, one has to raise the question whether changing culture media properties after Day 5 of culture could have beneficial effects on early development of bovine embryos. To answer that question, we transferred bovine IVF derived 32-cell stage embryos into the uterine cavity of synchronized recipients. All embryos had been matured and fertilized under routine standard conditions and were cultured in synthetic oviducal fluid supplemented with essential and nonessential amino acids (SOFaa) supplemented with either 0.3% fatty acid free bovine serum albumin (BSAfaf/Uterus) or 10% serum (serum/uterus) at 38.5°C, 5% O2, and 5% CO2 in humidified air prior transfer into the uterine environment, allowing further development to the blastocyst stage within the physiological environment prior recollection at Day 7 by routine uterine flushing followed by comparison with statically in vitro-developed embryos cultured in media supplemented with serum (serum/serum group) or BSAfaf (BSAfaf/BSAfaf group). All in all, a total of 1031 in vitro-derived 32-cell stage embryos were transferred to 21 synchronized Simmental recipient heifers. Of these, a total of 680 embryos (66%) could be recollected at Day 7. Embryos of the serum/serum group reached a higher blastocyst rate compared with embryos of the BSAfaf/BSAfaf group (68% v. 41%; P < 0.05, ANOVA, Tukey test), whereas the developmental rate to the blastocyst stage did not differ after 9 days of in vitro culture, indicating higher developmental kinetics of bovine 32-cell stage embryos when culture media is supplemented with serum. Moreover, embryos of the serum/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos of the serum/serum group (12.9% v. 68.4%). Likewise, embryos in the BSAfaf/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos in the BSAfaf/BSAfaf group (16.0% v. 40.1%). When allowed to develop for additional 48h in vitro, developmental rates to the blastocyst stage at Day 9 were still higher in BSAfaf/BSAfaf treatment compared with the BSAfaf/uterus treatment (91.4% v. 74.4%) and the serum/serum treatment compared with the serum/uterus treatment (92.5% v. 56.0%). Taken together, the results of our study demonstrate that uterine transfer of bovine 32-cell stage embryos results in reduction of developmental kinetics as well as lower developmental rates compared with embryos statically cultured in vitro. That might indicate, that a considerable proportion of bovine 32-cell stage embryos might not be able to adapt to the uterine environment.

scholarly journals 142 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS CULTURED IN KSOM, CR1AA, OR KSOM/CR1aa

2005 ◽  
Vol 17 (2) ◽  
pp. 221 ◽  
Author(s):  
M.R.B. Mello ◽  
C.E. Ferguson ◽  
A.S. Lima ◽  
M.B. Wheeler
Keyword(s):  
Embryo Culture ◽  
Cumulus Cells ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Cell Stage ◽  
In Vitro Development ◽  
Significant Difference ◽  
Developmental Rates ◽  
Vitro Development

In vitro embryo culture is an important step of in vitro production of bovine embryos. It has been shown that IVF-derived bovine embryos cultured in KSOM or CR1aa have high development rates. In our laboratory, we have observed that 8-cell embryos are morphologically superior when embryos are cultured in KSOM whereas blastocysts are morphologically superior when embryos are cultured in CR1aa. Based on these observations, we hypothesized that development of IVF-derived bovine embryos can be improved by sequential use of these media (KSOM and CR1aa). The aim of this experiment was to compare the in vitro development of bovine embryos cultured in KSOM, CR1aa or KSOM/CR1aa supplemented with BSA at Day 0 and BSA and FBS at Day 3. In order to accomplish the sequential culture, fertilized oocytes where cultured in KSOM to the 8-cell stage and then transferred to CR1aa for further development. Oocytes were purchased from Bomed (Madison, WI, USA), and after 22 hours of maturation were fertilized with frozen-thawed semen for 5 hours at 39°C in 5% CO2. After fertilization, the presumptive zygotes were denuded from cumulus cells by votexing and were randomly allotted to one of 3 treatments: (1) cultured only in KSOM (n = 110), (2) cultured only in CR1aa (n = 102), and (3) cultured in KSOM in the first 3 days and then in CR1aa from Day 3 to Day 9 (n = 110). The embryo culture was carried out in 50-μL droplets of medium that were placed in an airtight modular incubator filled with 5% CO2, 5% O2 and 90% N2. The embryos were evaluated on Days 6 to 9 post insemination. All embryo developmental rates were calculated from presumptive zygotes. The Day 6 morula rates were 52%, 40%, and 47% for KSOM, CR1aa, and KSOM/CR1aa, respectively. The Day 7 blastocyst rates for KSOM (40%), CR1aa (25%), and KSOM/CR1aa (30%) were not significantly different; however, Day 9 hatched blastocyst rates were significantly higher (P < 0.05) for KSOM (22%) compared to CR1aa (9%) but not different from KSOM/CR1aa (14%). Regarding embryo quality, Day 7 transferable embryos rates (Grade 1 and Grade 2) were 35%, 25%, and 30%, respectively for KSOM, CR1aa, and KSOM/CR1aa; however, no significant difference was observed. These results indicate that IVF-derived bovine embryos can develop in KSOM, CR1aa, or KSOM/CR1aa with no significant difference among morula, blastocyst and hatched blastocyst rates. However, the combination of KSOM and CR1aa during in vitro culture did not decrease the morula and blastocyst rates.

Technical Report: Effect of commercially available PMSG on maturation, fertilization and embryo development of buffalo oocytes in vitro

2001 ◽  
Vol 13 (6) ◽  
pp. 355 ◽  
Author(s):  
P. S. P. Gupta ◽  
S. Nandi ◽  
B. M. Ravindranatha ◽  
P. V. Sarma
Keyword(s):  
Embryonic Development ◽  
Genetic Manipulation ◽  
Culture Media ◽  
Cell Monolayer ◽  
Basic Research ◽  
Cost Effective ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
Vitro Fertilization

In vitro fertilization (IVF) technology provides an opportunity to produce embryos for genetic manipulation, embryo transfer and basic research in developmental physiology, and can be exploited for emerging biotechnologies such as transgenesis and cloning. In the present study, the effects of different concentrations of commercially available pregnant mare serum gonadotrophin (PMSG) (Folligon; Intervet, International B.V., Boxmeer, Holland) in oocyte culture media, on maturation, fertilization and embryonic development of buffalo oocytes in vitro were investigated. Oocytes aspirated from abattoir-derived ovaries were cultured in media containing TCM-199 + PMSG at 0, 2.5, 20, 30, 40 and 50 IU mL–1 in presence or absence of steer serum (10%) for 24 h in a CO2 incubator. The maturation rate was assessed on the basis of degree of expansion of cumulus cells. The matured oocytes were inseminated with 9–10 x 106 spermatozoa mL–1 in Brackett and Oliphant medium and the cleavage rate was recorded 40–42 h after insemination. Uncleaved oocytes were stained with aceto-orcein for evaluation of fertilization rates. The cleaved embryos were further cultured in TCM-199 + 10% steer serum on buffalo oviducal cell monolayer for 7 days. Maturation, fertilization, cleavage and embryonic development were significantly higher (P<0.05) in oocytes cultured in TCM-199 + 10% steer serum supplemented with 40 and 50 IU PMSG mL–1. It is concluded that commercially available PMSG can effectively be used in place of pure follicle-stimulating hormone for in vitro maturation of buffalo oocytes, making it cost effective for IVF studies.

Generation of parthenogenetic goat blastocysts: effects of different activation methods and culture media

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 327-335 ◽  
Author(s):  
Hruda Nanda Malik ◽  
Dinesh Kumar Singhal ◽  
Shrabani Saugandhika ◽  
Amit Dubey ◽  
Ayan Mukherjee ◽  
...  
Keyword(s):  
Culture Media ◽  
Combined Treatment ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Quantitative Expression ◽  
Pattern Of Variation ◽  
Better Than

SummaryThe present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts.

33 Improved Dog Cloning Efficiency Using Post-Activation with Ro-3306, a Cdk1 Inhibitor

2018 ◽  
Vol 30 (1) ◽  
pp. 156
Author(s):  
M. J. Kim ◽  
H. J. Oh ◽  
E. M. N. Setyawan ◽  
S. H. Lee ◽  
B. C. Lee
Keyword(s):  
Calcium Ionophore ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
Serum Progesterone ◽  
Significance Level ◽  
Chi Squared ◽  
Pregnancy And Delivery ◽  
Developmental Rates

Inactivation of maturation promoting factor requires proteolytic destruction of cyclin B that results in the loss of cyclin-dependent kinase 1 (Cdk1) activity and exit from metaphase. The aim of this study was to investigate that treatment of Ro-3306, a Cdk1 inhibitor, during post-activation could increase the development of somatic cell nuclear transfer (SCNT) embryos in dogs. Mixed breed female dogs aged at 1 to 5 years and weighing 20 to 35 kg were used in this study (approval number: SNU-160602-14-1). Canine cumulus–oocyte complexes were collected surgically by flushing oviducts with HEPES-buffered TCM-199 medium ~72 h after ovulation, which was determined by serum progesterone concentration. After removal of cumulus cells from oocytes by repeated pipetting in hyaluronidase, matured oocytes were selected for the following experiment. In experiment I, oocytes were activated with (1) 10 μM calcium ionophore and then post-activated with 1.9 mM DMAP (control); (2) DMAP along with 10 μM Ro-3306 (10 μM group); or (3) DMAP along with 50 μM Ro-3306 (50 μM group). Parthenotes were cultured in the synthetic oviducal fluid (SOF) medium after post-activation, and in vitro development was evaluated at 48 h (2-4 cell) and 72 h (6-8 cell). In experiment II, SCNT embryos were produced after oocyte enucleation, donor cell injection, fusion, and activation. Only fused cytoplasts were activated with (1) 1.9 mM DMAP (control) or (2) DMAP along with 50 μM Ro-3306 (50 μM group) and transferred to the oviducts of recipients. The day of embryo transfer was regarded as Day 0. Pregnancy diagnosis was performed by ultrasonography after Day 28 and cloned puppies were delivered Day 58 to 60. Embryo developmental rates in experiment I and II were analysed by one-way ANOVA and t-test, respectively, and pregnancy and delivery rate were analysed by chi-squared test using Graph Prism software (GraphPad, San Diego, CA, USA). The significance level was P < 0.05. Results in experiment I showed that cleavage rate of parthenogenetic embryos in the 50 μM group (89.3 ± 6.8%) was significantly higher than that of 10 μM group or control (50.8 ± 9.9% and 55.4 ± 18.8%, respectively). However, embryonic development to 4 cells and 6-8 cells was not different between treatments. In experiment II, pregnancy rates of recipients receiving embryos in 50 μM group (3/5, 60.0%) were significantly higher than that of control (2/6, 33.3%), but the number of healthy cloned puppies delivered in the 50 µM group (n = 6) versus the control (n = 2) was not different. In conclusion, post-activation with 50 μM Ro-3306 may enhance nuclear reprogramming of dog cloned embryos. This study was supported by RDA (#PJ010928032017), Korea IPET (#316002-05-2-SB010), Research Institute for Veterinary Science, Natural Balance Korea and the BK21 plus program.

Effects of activin A and follistatin on developmental kinetics of bovine embryos: cinematographic analysis in a chemically defined medium

Reproduction ◽  
2000 ◽  
pp. 119-125 ◽  
Author(s):  
K Yoshioka ◽  
C Suzuki ◽  
S Iwamura
Keyword(s):  
Defined Medium ◽  
Activin A ◽  
Lag Phase ◽  
Chemically Defined Medium ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Oviduct Fluid ◽  
Kinetics Of ◽  
Number Of Cells

The effects of recombinant human activin A and follistatin on the developmental kinetics of bovine presumptive zygotes matured and fertilized in vitro using time-lapse cinematography were investigated. The presumptive zygotes were cultured for 9 days in a chemically defined medium (modified synthetic oviduct fluid, control) and modified synthetic oviduct fluid supplemented with activin A or follistatin. Development under cine-recording conditions was similar to that in an incubator. Addition of activin A to modified synthetic oviduct fluid increased, while addition of follistatin decreased, the percentage of zygotes that developed to morulae and blastocysts. Follistatin significantly prolonged the timing of development to the 9-16-cell stage compared with the control and activin A media. Activin A significantly shortened the duration of the third cell cycle compared with the control, but follistatin significantly prolonged the fourth cell cycle compared with the control and activin A. Developmental arrest ('lag-phase') during the 4-8-cell stage was observed in 95% of embryos developed to more than the 9-16-cell stage in all treatments. The greater the number of cells at the onset of the lag-phase, the earlier the onset of the phase and the shorter the duration of the phase, the further embryos were able to develop by day 9 in all treatments. The number of cells at the onset of the lag-phase in the medium containing activin A was significantly higher than it was in control or follistatin-containing media. Moreover, activin A significantly shortened the duration of the lag-phase compared with follistatin. The present results indicate that activin A may enhance in vitro development of bovine embryos by improving developmental kinetics, especially by increasing the number of cells at the onset of the lag-phase and shortening the duration of this phase.

Spent culture medium analysis from individually cultured bovine embryos demonstrates metabolomic differences

Zygote ◽  
2017 ◽  
Vol 25 (6) ◽  
pp. 662-674 ◽  
Author(s):  
Kayla J. Perkel ◽  
Pavneesh Madan
Keyword(s):  
Culture Medium ◽  
Physiological State ◽  
Proton Nuclear Magnetic Resonance ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Isoleucine Leucine ◽  
H Nmr

SummarySpent culture medium can provide valuable information regarding the physiological state of a bovine preimplantation embryos through non-invasive analysis of the sum/depleted metabolite constituents. Metabolomics has become of great interest as an adjunct technique to morphological and cleavage-rate assessment, but more importantly, in improving our understanding of metabolism. In this study, in vitro produced bovine embryos developing at different rates were evaluated using proton nuclear magnetic resonance (1H NMR). Spent culture medium from individually cultured embryos (2-cell to blastocyst stage) were divided into two groups based on their cleavage rate fast growing (FG) and slow growing (SG; developmentally delayed by 12–24 h), then analyzed by a 600 MHz NMR spectrometer. Sixteen metabolites were detected and investigated for sum/depletion throughout development. Data indicate distinct differences between the 4-cell SG and FG embryos for pyruvate (P < 0.05, n = 9) and at the 16-cell stage for acetate, tryptophan, leucine/isoleucine, valine and histidine. Overall sum/depletion levels of metabolites demonstrated that embryos produced glutamate, but consumed histidine, tyrosine, glycine, methionine, tryptophan, phenylalanine, lysine, arginine, acetate, threonine, alanine, pyruvate, valine, isoleucine/leucine, and lactate with an overall trend of higher consumption of these metabolites by FG groups. Principal component analysis revealed distinct clustering of the plain medium, SG, and FG group, signifying the uniqueness of the metabolomic signatures of each of these groups. This study is the first of its kind to characterize the metabolomic profiles of SG and FG bovine embryos produced in vitro using 1H NMR. Elucidating differences between embryos of varying developmental rates could contribute to a better understanding of embryonic health and physiology.

191 ALLEVIATION OF THE TWO-CELL BLOCK OF ICR, ddY, AND AKR MOUSE EMBRYOS BY BOVINE SERUM ALBUMIN

2007 ◽  
Vol 19 (1) ◽  
pp. 212
Author(s):  
K. Ono ◽  
R. Ohishi ◽  
H. Imai ◽  
M. Yamada
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Culture Media ◽  
Mouse Embryos ◽  
Control Group ◽  
Cell Block ◽  
Cell Stage ◽  
Developmental Rates

Bovine serum albumin (BSA) is an embryotrophic macromolecule used in embryo culture media and improves embryo development in vitro. However, when 1-cell embryos from some strains of mouse were cultured in traditional medium, even with BSA, developmental arrest occurred at the 2-cell stage, termed '2-cell block'. The developmental block is known to be alleviated by adding EDTA to the medium for ICR and ddY strains, and deleting phosphate from the medium for the AKR strain. Recently, our preliminary experiments revealed that the 2-cell block is relieved by adding deionized BSA (d-BSA) to the medium for the ICR strain. Thus, in the present study, we investigated whether d-BSA could rescue the embryos from ICR, ddY, and AKR strains from the 2-cell block. Fertilized 1-cell embryos were collected 20 h post-hCG from superovulated ICR, ddY, and AKR females (8-week-old) that had been mated with the ICR strain of males. Stock solutions (15%) of commercially available fraction V BSA, ovalbumin (ova), and γ-globulin (γG) were deionized over a mixed-bed ion adsorption resin. Embryos were cultured in EDTA-depleted KSOM medium with or without these deionized or non-deionized proteins at 37�C under 5% CO2 in air for 4 days. Experiments were done in at least 3 replicates, and the statistical analyses of the data were done by ANOVA and Fisher&apos;s PLDS test. To observe the distribution of BSA in the embryos from the 1-cell to the blastocyst stages, immunofluorescence study was performed using anti-BSA antibody with a laser confocal microscope. The developmental rates to the 4-cell stage of 1-cell embryos cultured in medium without (control group) or with BSA at 0.3% in ICR and 0.6% in ddY and AKR (BSA group) were very low (ICR: 10% (4/38) and 37% (17/47); ddY: 9% (7/73) and 23% (9/37); AKR: 0% (0/60) and 0% (18/30), respectively). However, when embryos were cultured with d-BSA at 0.3% in ICR and 0.6% in ddY and AKR, the rates to the 4-cell stage significantly increased (ICR: 91% (51/56), ddY: 82% (61/76), AKR: 82% (50/60) vs. control group or BSA group: P &lt; 0.05), and development to the blastocyst stage was observed (ICR: 79% (44/56), ddY: 65% (47/76), AKR: 63% (38/60)). When ICR embryos were cultured with 0.3% deionized-ova or deionized-�G, no significant increase was observed in developmental rates to the 4-cell stage (25% (10/40) and 24% (10/42), respectively). We next examined the critical culture period for the beneficial effects of d-BSA and intracellular distribution of BSA using ddY mouse embryos. It was found that exposure to d-BSA during the late 1-cell (24 h post-hCG) and early 2-cell stages (42 h post-hCG) promoted the development beyond the 2-cell stage. The distribution of BSA in the cytoplasm of embryos at any stage was observed. Interestingly, BSA localized in the nuclei of embryos during the late 1-cell and early 2-cell stages. In conclusion, our results suggest that BSA itself has a potential to remove the 2-cell block in ICR, ddY, and AKR strains. In addition, nuclear localization of BSA may play a key role in regulating the development beyond the 2-cell stage in the mouse embryos.

scholarly journals 248 EMBRYO DEVELOPMENT IN MICRODROPS AND MICROCHANNELS: COMPARISON OF NCSU23 SEQUENTIAL AND NON-SEQUENTIAL CULTURE MEDIUM

2005 ◽  
Vol 17 (2) ◽  
pp. 274
Author(s):  
A.S. Lima ◽  
C.E. Ferguson ◽  
M.B. Wheeler
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Culture Media ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
The Other ◽  
Cell Stage ◽  
Development Rates ◽  
Hatching Rates

The in vitro culture systems used to produce pig embryos generally result in few embryos developing to the blastocyst stage. The use of pyruvate (pyr) and lactate (lac) during the culture of zygotes to the 8-cell stage followed by glucose (glu) supplementation replacing pyr and lac appears to be beneficial for embryo development in the pig. The aim of this study was to compare the embryo development rates from pig oocytes fertilized with and without cumulus cells in 100-μL microdrops (MD) and cultured in 100-μL MD or microchannels (MC), using NCSU23 containing 8 mg/mL of BSA and supplemented with (1) glu or (2) pyr/lac or (3) pyr/lac for the first three days and then with just glu for the remainder of culture period (pyr/lac-glu). Sow oocytes were matured in TCM199 supplemented with gonadotropins for the first 22 h, and for an additional 22 h without hormones. After 44 h of maturation, oocytes were placed in MD of modified tris-buffered medium to be fertilized using 3 × 105 sperm/mL. Oocytes were divided into two groups for fertilization: with and without cumulus cells. Following 6 h of fertilization, all inseminated oocytes were washed, divided into groups of 15, allotted to the three culture media treatment groups as described above, and incubated in either MD or MC. With the exception of one treatment there were no significant differences in development rates among embryos cultured in MD or MC, hence data were pooled from these two culture devices. Only oocytes fertilized without cumulus cells and cultured in pyr/lac in MC appeared to have lower rates of blastocyst formation (11.67%) than those cultured in MD (26.67%) in the same culture medium. When the six treatments were compared, oocytes fertilized with cumulus cells and cultured in glu had significantly higher (P < 0.05) blastocyst rates and hatching rates compared with the other treatments, with the exception of those fertilized without cumulus cells and cultured in pyr/lac-glu. There were no significant differences among other treatments in Day 7 blastocyst or in Day 9 hatching rates. In conclusion, both culture devices can be used to reach similar blastocyst rates with different treatments. In this experiment, the removal of cumulus cells before fertilization appeared to enhance embryo development in vitro when sequential media are used. On the other hand, the presence of cumulus cells before fertilization seems to enhance embryo development when non-sequential glu medium is used. Table 1. Embryo development rates on Day 9 for three different culture treatments

84 THE EFFECT OF β-MERCAPTOETHANOL ON CLEAVAGE RATES, DEVELOPMENTAL COMPETENCE AND QUALITY OF IN VITRO PRODUCED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 156 ◽  
Author(s):  
E. R. Lliteras ◽  
M. Chong ◽  
S. Andries ◽  
E. Merckx ◽  
E. P. A. Jorssen ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Media ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Dapi Staining ◽  
Total Cell Number

The production of excessive levels of reactive oxygen species can be a major problem during in vitro embryo culture. Although studies have shown that supplementation with exogenous antioxidants can improve embryo quality, the results are controversial among researchers. In this study, we examined the effects of different concentrations of β-mercaptoethanol (β-ME) added to the culture media, on cleavage rates, the quality and developmental competence of in vitro-produced bovine embryos. The embryos were produced in vitro as described previously (Van Hoeck et al., 2013). Briefly, in total, 753 grade I cumulus–oocyte complexes (COC) from 2- to 6-mm-diameter follicles were matured in groups of 50 in 500 μL of TCM with 20 ng mL–1 EGF for 24 h, fertilized in groups of 100 in 500 μL of fertilization medium for 20 h (5% CO2, 38.5°C). Presumptive zygotes were denuded and randomly assigned to 4 treatments with different concentrations of β-ME: 0 μM (control), 50 μM, 100 μM, and 150 μM. They were cultured in groups of  ±25 in 50 μL of SOF supplemented with ITS (10 μg mL–1 insulin; 5.5 μg mL–1 transferrin; 6.7 ng mL–1 selenium) and 2% BSA and covered with mineral oil (5% O2, 5% CO2, 38.5°C). At 48 h post-insemination (p.i.), cleavage rate was evaluated and expressed as the number of cleaved embryos on total number of oocytes. At Day 7 p.i., blastocyst rate was determined (number of blastocysts on total number of oocytes), blastocysts were fixed in 4% paraformaldehyde, and total cell number was determined by DAPI staining. Data were analysed by ANOVA and post hoc test. Comparable cleavage rates were obtained in treated groups: control (80.8%), 50 μM (77.7%), 100 μM (77.9%), and 150 μM (73.6%; P > 0.05). Also, no significant effect of treatment could be found on blastocyst rates: control (36%), 50 μM (36.5%), 100 μM (38.4%), and 150 μM (30.4%). The total cell number per blastocyst increased significantly (P < 0.05) using 100 μM of β-ME compared with the controls (158.0 ± 24.3 v. 123.2 ± 9.72, respectively). These results suggest that the inclusion of 100 μM β-ME during in vitro embryo culture could be used for production of high quality bovine blastocysts.

Sign in / Sign up

Export Citation Format

Share Document