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.

scholarly journals Genetic influence on the reduction in bovine embryo lipid content by L-carnitine

2016 ◽  
Vol 28 (8) ◽  
pp. 1172 ◽  
Author(s):  
Luis Baldoceda ◽  
Dominic Gagné ◽  
Christina Ramires Ferreira ◽  
Claude Robert
Keyword(s):  
Lipid Content ◽  
Culture Medium ◽  
Cell Damage ◽  
Mitochondrial Activity ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Intracellular Lipid ◽  
Embryo Culture Medium

The decreased rate of pregnancy obtained in cattle using frozen in vitro embryos compared with in vivo embryos has been associated with over-accumulation of intracellular lipid, which causes cell damage during cryopreservation. It is believed that the higher lipid content of blastomeres of bovine embryos produced in vitro results in darker-coloured cytoplasm, which could be a consequence of impaired mitochondrial function. In this study, l-carnitine was used as a treatment to reduce embryonic lipid content by increasing metabolism in cultured bovine embryos. We have observed previously that in vivo embryos of different dairy breeds collected from cows housed and fed under the same conditions differed in lipid content and metabolism. As such, breed effects between Holstein and Jersey were also examined in terms of general appearance, lipid composition, mitochondrial activity and gene expression. Adding l-carnitine to the embryo culture medium reduced the lipid content in both breeds due to increased mitochondrial activity. The response to l-carnitine was weaker in Jersey than in Holstein embryos. Our results thus show that genetics influence the response of bovine embryos to stimulation of mitochondrial metabolism.

133 EVALUATION OF OOCYTE DONOR SOURCE AND CULTURE MEDIUM ON DOMESTIC CAT EMBRYO DEVELOPMENT RATES

2011 ◽  
Vol 23 (1) ◽  
pp. 171
Author(s):  
A. J. Pearks Wilkerson ◽  
R. D. Landry ◽  
C. R. Long
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cytochalasin B ◽  
Culture Media ◽  
Domestic Cat ◽  
Bovine Embryo ◽  
Development Rates ◽  
L1 And L2

The use of assisted reproductive technology (ART), including in vitro maturation (IVM) and embryo culture, is well established in several species, including canine and feline culture systems. Embryo production conditions tend to be specific for each species and prepared in unique formulations by laboratory. However, the increasing numbers of commercially available media allows for new comparisons in companion animal systems. Therefore, a goal of this study was to compare the development rates of feline parthenotes cultured in a commercially available bovine embryo culture medium with those cultured in a published 3-step domestic cat-specific system. In addition, the source of ovaries utilised for oocyte retrieval was evaluated as a factor in development rates. Ovaries from 2 locations (L1 and L2) were collected on the same day, and harvested oocytes were held in meiotic arrest medium containing 25 μM roscovitine for 14 to 18 h. Oocytes were incubated in maturation medium for 24 h before cumulus cell removal with vigorous pipetting in 0.4% hyaluronidase, and a subset of each group was fixed and stained to determine meiotic maturation rates (n = 76 and 55 for L1 and L2, respectively). Following activation (day 0) by a single course of three 50-μs electric pulses at 1.2 kV cm–1 in 0.3 M mannitol, 0.1 mM CaCl2, and 0.1 mM MgSO4, parthenotes from each source were randomly divided to culture medium treatment of Bovine Evolve medium (Zenith Biotech, Guilford, CT, USA) with 4 mg mL–1 BSA (n = 209) or IVC-1 medium n = 269; (Pope et al. 2009 Theriogenology 71, 864–871), each containing 10 μg mL–1 cycloheximide and 7.5 μg mL–1 cytochalasin B. After a 4-h activation treatment, parthenotes were moved to culture media without cycloheximide and cytochalasin B for embryo development. All parthenotes in IVC-1 medium were moved to IVC-1a medium on day 2. On day 5, both sets of parthenotes were moved to culture media containing 10% heat-inactivated FBS instead of BSA. On day 7, all parthenotes were fixed and stained with Hoechst to determine cell number. No differences were seen in maturation rates between L1 and L2 (56.3 ± 9.5 v. 54.7 ± 9.5, respectively). However, cleavage rates tended to differ, and proportion of embryos greater than 64 cells was different (60.7 ± 5.8 v. 78.3 ± 5.8, P = 0.056 and 3.0 ± 3.1 v. 19.7 ± 3.1, P < 0.005; respectively). We hypothesised that the physical condition of the ovary donors may have affected development rates because cats from L1 tended to be feral animals, whereas cats from L2 were mostly privately owned. Bovine Evolve was similar to IVC-1 medium for cleavage, 32-cell, and 64-cell development rates (74.2 ± 6.7 v. 64.8 ± 6.7; 24.0 ± 7.5 v. 31.8 ± 7.5; 10.7 ± 4.8 v. 12.0 ± 4.8, respectively; P > 0.05). These results indicate that commercially available culture medium can support in vitro development, even if the commercial medium is developed for a different species, but that source of cat ovaries should be considered in feline ART.

91 INTERACTION OF OXYGEN TENSION AND CYSTEAMINE SUPPLEMENTATION DURING IN VITRO CULTURE OF BOVINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 158
Author(s):  
L. M. Stauber ◽  
G. E. Seidel
Keyword(s):  
In Vitro Culture ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cumulus Cells ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Embryo Culture Medium ◽  
Mammalian Embryos ◽  
Defined Culture

Reactive oxygen species damage early mammalian embryos, so culture of bovine in vitro-produced (IVP) embryos at 5% O2 is clearly superior to 20% O2. The thiol compound cysteamine is an antioxidant and improves in vitro blastocyst production when added to in vitro maturation (IVM) medium. The purpose of this study was to investigate supplementation of IVP embryo culture medium with cysteamine at different oxygen tensions. Bovine ovaries from feedlot heifers were collected from a local abattoir and cumulus–oocyte complexes (COC) were aspirated from 3- to 8-mm follicles. COC were matured in maturation medium with 100μM cysteamine (Reprod. Fertil. Dev. 18, 585) for 23 h in a humidified incubator at 38.5°C in 5% CO2 in air. COC at 23 h of maturation were co-incubated with sperm for 18 h. Cumulus cells were then removed and presumed zygotes were cultured in our chemically defined culture medium system (Reprod. Fertil. Dev. 18, 585) in a 2 × 2 factorial arrangement: with or without 50 μM cysteamine × atmospheres of either 5% O2, 5% CO2, 90% N2, or 5% CO2 in air. There were no treatment differences (P > 0.01) in percentages of oocytes cleaving or reaching the 8-cell stage (Table 1). However, blastocyst production rates were lower (P < 0.01) in the group cultured without cysteamine at 20% O2 compared with all the other groups. Adding cysteamine for embryo culture at 20% O2 resulted in blastocyst rates similar to those cultured at 5% O2 with or without cysteamine. Cysteamine was not beneficial at 5% O2. Table 1.Cysteamine supplementation during in vitro culture of bovine embryos

scholarly journals Antioxidant activity of oily extract obtained from Lippia origanoides improves the quality of bovine embryos produced in vitro

2019 ◽  
Vol 71 (3) ◽  
pp. 723-731
Author(s):  
N.V. Sollecito ◽  
E.C.M. Pereira ◽  
J.G.V. Grázia ◽  
B.P. Neves ◽  
B.V.R. Couto ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Embryo Culture Medium ◽  
Vitro Production

ABSTRACT The aim of this study was to evaluate the supplementation of embryo culture medium with antioxidant obtained from oily extract of Lippia origanoides on in vitro blastocyst development and quality. Oocytes collected from slaughterhouse ovaries were matured and fertilized in vitro following standard laboratory procedures. Zygotes were cultured in SOF medium supplemented according to the following treatments: T1 embryo culture medium without antioxidant supplementation; T2)50μM/mL Cysteamine; T3)2.5μg/mL; T4)5.0μg/mL and T5)10.0μg/mL of antioxidant obtained from oily extract of Lippia origanoides. On the seventh day of culture, the blastocysts were fixed and evaluated for apoptosis rates, number of total cell and inner cell mass cells by means of the TUNEL Test. The use of antioxidants during cultivation did not increase (P> 0.05) the final blastocyst production rate. The treatments T2, T3, T4 and T5 had the lowest (P< 0.05) apoptotic indexes (4.5±1.1%, 8.4±2.5%, 3.4±1.1% and 5.5±0.9%, respectively) when compared to T1 treatment (10.0±1.4%). The number of inner cell mass did not differ (P> 0.05) among embryos from different treatments. The addition of antioxidant obtained from oily extract of Lippia origanoides reduces the apoptosis rate and improves the quality without increasing the total in vitro production of bovine embryos.

Oxygen tension modulates extracellular vesicles and its miRNA contents in bovine embryo culture medium

2019 ◽  
Vol 86 (8) ◽  
pp. 1067-1080 ◽  
Author(s):  
Gabriella Mamede Andrade ◽  
Monalisa Medrado Bomfim ◽  
Maite del Collado ◽  
Flávio Vieira Meirelles ◽  
Felipe Perecin ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Extracellular Vesicles ◽  
Embryo Culture ◽  
Culture Medium ◽  
Bovine Embryo ◽  
Embryo Culture Medium

P–146 Differential impact of three embryo culture media for IVF on in vitro development and perinatal outcome: a single-center RCT

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
M Murakami ◽  
K Tanaka ◽  
H Otsubo ◽  
S Mizumoto ◽  
Y Nagao ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Embryo Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Cleavage Stage ◽  
Embryo Culture Medium ◽  
Cleavage Stage Embryo ◽  
Stage Embryo

Abstract Study question This report provides updated data from an RCT determining which embryo culture medium yields optimal IVF outcomes. Summary answer Embryo culture systems used for IVF differentially affected preimplantation development and resultant obstetric and perinatal outcomes, including birthweights of live-born singletons. What is known already Currently, multiple embryo culture medium systems are in use for IVF, raising questions regarding which is optimal. However, the ability of a medium to yield preimplantation embryos is not necessarily indicative of embryo viability. For example, supplementation of medium with serum was commonly used to increase animal blastocyst yields, but this impaired embryonic, fetal, and offspring health. In humans, medium composition and culture duration can influence IVF efficacy and offspring phenotype. Given the importance of culture systems in determining clinical outcomes, existing data regarding differential culture system impacts are insufficient and additional well-designed studies are required. Study design, size, duration Between February 2016 and August 2017, 795 couples undergoing their first autologous clinical IVF cycle and freeze-all strategy were recruited. Participants were randomized via computer-generated tables into three groups. Following standard oocyte retrieval and IVF/ICSI procedures, embryos were cultured using three different culture media, G1 Plus/G2 Plus (G1/G2; Vitrolife), Global Total (GT; LifeGlobal), or Sequential Cleav/Sequential Blast (SC/SB; Origio). Thirty-eight patients exhibiting no 2PN oocytes following insemination or those undergoing fresh embryo transfers were excluded. Participants/materials, setting, methods For patients yielding a single good-quality cleavage-stage (day–2 or day–3) embryo, that cleavage-stage embryo was vitrified. For patients yielding two or more good-quality cleavage-stage embryos, two or less good-quality cleavage-stage embryos were vitrified. The culture period of the remaining embryos was extended, and all good-quality blastocyst-stage (day–5 or day–6) embryos were vitrified. This report presents data for vitrified embryo transfer performed until the end of December 2020. Main results and the role of chance The mean per-cycle vitrified embryo yield (± SD) was comparable between groups for cleavage-stage embryos, but significantly different for blastocyst-stage embryos (G1/G2: 1.69 ± 2.2, GT: 2.53 ± 3.01, SC/SB: 2.04 ± 2.42; P = 0.001). Following vitrified cleavage- or blastocyst-stage embryo transfers, biochemical pregnancy rates were significantly different between groups (G1/G2: 55.6%, GT: 59.1%, SC/SB: 46.2%; P = 0.011). Furthermore, a between-group trend towards different live birth rates was observed (G1/G2: 41.7%, GT: 42.1%, SC/SB: 33.1%; P = 0.063). Of 382 live births, data for first-borns (n = 323; 295 singletons and 14 twin-pairs) are reported here. Perinatal data did not differ significantly between groups for both cleavage- and blastocyst-stage embryo transfers, including gestational age- and gender-adjusted singleton birthweight (z-score). Following multiple linear regression (including selected covariates), adjusted mean singleton birthweights were significantly lower in the G1/G2 and GT groups than in the SC/SB group (by 131 g; P = 0.011 and 110 g; P = 0.032, respectively) and tended to be lower for cleavage-stage embryo transfers than for blastocyst-stage embryo transfers (by 102 g; P = 0.053). Limitations, reasons for caution A larger cohort size and longer-term follow-up are required to verify and further elucidate the impact of embryo culture methods on child health. Such studies will raise awareness regarding the sensitivity of in vitro-cultured human embryos to their environment, ultimately resulting in practices that decrease IVF risks to offspring. Wider implications of the findings: Pregnancy outcome of the medium yielding fewer blastocysts was comparable or superior to that of other media, highlighting the importance of differentiating between the ability to support preimplantation development versus the ability to yield viable embryos. Embryo culture medium had a greater impact than embryo transfer stage on live birthweight. Trial registration number UMIN000020910

scholarly journals Sonic hedgehog supplementation of oocyte and embryo culture media enhances development of IVF porcine embryos

Reproduction ◽  
2011 ◽  
Vol 142 (1) ◽  
pp. 87-97 ◽  
Author(s):  
Ngoc Tan Nguyen ◽  
Neng-Wen Lo ◽  
Sing-Ping Chuang ◽  
Ya-Lan Jian ◽  
Jyh-Cherng Ju
Keyword(s):  
Embryonic Development ◽  
Sonic Hedgehog ◽  
Embryo Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Total Cell ◽  
Cell Numbers ◽  
Histone 3 ◽  
Embryo Culture Medium

We investigated the expression of sonic hedgehog (SHH) receptor PTCH1 and its co-receptor smoothened (SMO) in fertilized porcine embryos. Effects of exogenous SHH on embryonic development and expressions of survival- and pluripotency-related genes were also determined. We found that PTCH1 and SMO are expressed from two-cell to blastocyst embryos. When oocytes or fertilized embryos were respectively cultured in the maturation or embryo culture medium supplemented with SHH (0.5 μg/ml), their blastocyst rates and total cell numbers increased (P<0.05) compared with the untreated control. When cultured simultaneously in thein vitromaturation (IVM) andin vitroculture (IVC) media supplemented with SHH, the oocytes gained increased blastocyst rates and total cell numbers in an additive manner, with reduced apoptotic indices (P<0.05). Interestingly, SHH treatment did not affect the expression of theBCL2L1(BCL-XL) gene, yet reducedBAXexpression. Blastocysts cultured with various SHH regimes had similar pluripotency-related gene (POU5F1(OCT-4) andCDX2) expression levels, but blastocysts derived from SHH treatment during IVM had higherZPF42(REX01) expression (P<0.05). The highestZPF42expression was observed in the blastocysts derived from SHH-supplemented IVC and from dual IVM and IVC treatments. The levels of acetylated histone 3 (AcH3K9/K14) increased in the two-cell and the four-cell embryos when IVM and/or IVC media were supplemented with SHH (P<0.05). Our findings indicate that SHH conferred a beneficial effect on preimplantation development of porcine embryos, particularly when both IVM and IVC media were supplemented with SHH, and the effects may be further carried over from IVM to the subsequent embryonic development.

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