Effect of supplementation of different growth factors in embryo culture medium with a small number of bovine embryos on in vitro embryo development and quality

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

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

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/1678-4162-10323 ◽

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.

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Supplementation with cysteamine during maturation and embryo culture on embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue test

Zygote ◽

10.1017/s0967199403002405 ◽

2003 ◽

Vol 11 (4) ◽

pp. 347-354 ◽

Cited By ~ 17

Author(s):

Aixa Urdaneta ◽

Ana-Raquel Jiménez-Macedo ◽

Dolors Izquierdo ◽

Maria-Teresa Paramio

Keyword(s):

Embryo Development ◽

Embryo Culture ◽

Culture Medium ◽

Blastocyst Development ◽

Brilliant Cresyl Blue ◽

Cresyl Blue ◽

Development Rates ◽

Embryo Culture Medium ◽

Normal Fertilization

Our previous studies have shown that the addition of 100 μM cysteamine to the in vitro maturation (IVM) medium increased the embryo development of prepubertal goat oocytes. The aim of the present study was to evaluate the effect of adding different concentrations of cysteamine to the IVM medium and to the in vitro embryo culture medium (IVC) on the embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue (BCB) test. Oocytes were exposed to BCB and classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) or oocytes without blue cytoplasm or growing oocytes (BCB−). In Experiment 1, oocytes were matured in a conventional IVM medium supplemented with 100 μM, 200 μM or 400 μM cysteamine. In Experiment 2, oocytes were matured with 400 μM cysteamine and following in vitro fertilization (IVF) were cultured in SOF medium supplemented with 50 μM and 100 μM cysteamine. In Experiment 1, BCB+ oocytes matured with 100 μM and 200 μM cysteamine showed higher normal fertilization and embryo development rates than BCB− oocytes. Oocytes matured with 400 μM cysteamine did not present these differences between BCB+ and BCB− oocytes. In Experiment 2, the addition of 50 μM and 100 μM cysteamine to culture medium did not affect the proportion of total embryos obtained from BCB+ oocytes (35.89% and 38.29%, respectively) but was significantly different in BCB− oocytes (34.23% and 29.04%, respectively, P<0.05). In conclusion, the addition of 400 μM cysteamine to the IVM improved normal fertilization and embryo development of BCB− oocytes at the same rates as those obtained from BCB+ oocytes. The proportions of morulae plus blastocyst development were not affected by the treatments.

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

International Journal of Molecular Sciences ◽

10.3390/ijms20010038 ◽

2018 ◽

Vol 20 (1) ◽

pp. 38 ◽

Cited By ~ 15

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.

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Effect of early addition of bone morphogenetic protein 5 (BMP5) to embryo culture medium on in vitro development and expression of developmentally important genes in bovine preimplantation embryos

Theriogenology ◽

10.1016/j.theriogenology.2015.04.018 ◽

2015 ◽

Vol 84 (4) ◽

pp. 589-599 ◽

Cited By ~ 8

Author(s):

Elina V. García ◽

Dora C. Miceli ◽

Gabriela Rizo ◽

Pablo A. Valdecantos ◽

Antonio D. Barrera

Keyword(s):

Bone Morphogenetic Protein ◽

Embryo Culture ◽

Culture Medium ◽

Preimplantation Embryos ◽

In Vitro Development ◽

Morphogenetic Protein ◽

Embryo Culture Medium ◽

Vitro Development

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Effects of changing culture medium on preimplantation embryo development in rabbit

Zygote ◽

10.1017/s0967199421000721 ◽

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.

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80 DEVELOPMENT OF A SYNTHETIC MEDIUM FOR THE IN VITRO CULTURE OF BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab80 ◽

2014 ◽

Vol 26 (1) ◽

pp. 154 ◽

Cited By ~ 1

Author(s):

D. Moreno ◽

A. Neira ◽

L. Dubreil ◽

L. Liegeois ◽

S. Destrumelle ◽

...

Keyword(s):

Growth Factors ◽

Embryo Culture ◽

Disease Transmission ◽

Culture Medium ◽

Culture Media ◽

Synthetic Medium ◽

Wilcoxon Test ◽

Blastocyst Development ◽

Negative Effect

In the majority of media for embryo culture, 2 of typical components used are FCS or BSA; however, the presence of FCS in the culture medium has been shown to have a negative effect on embryo quality and the use of animal-derived proteins in culture media increases the risks of disease transmission through in vitro embryo production. The aim of this study was to develop an in vitro embryo culture medium free from FCS and BSA, but with the addition of various growth factors and cytokines (GF-CYK: IGF-I, IGF-II, bFGF, LIF, GM-CSF) 50 ng mL–1 and (TGF-β1) 100 ng mL–1 supplemented with hyaluronan (HA) and recombinant albumin (RA). Bovine oocytes (n = 1043, 6 replicates) from abattoir ovaries were matured in TCM-199 medium with 60 μg mL–1 penicillin, 60 μg mL–1 streptomycin, and 10 ng mL–1 EGF for 24 h at 39°C and 5% CO2 in humidified air. Afterward, the oocytes were fertilized in IVF-TALP medium with 6 mg mL–1 fatty acid-free BSA and 1.7 IU mL–1 heparin for 18 h under the same conditions. After fertilization, presumptive zygotes were divided into two groups and cultured in 30 μL droplets of SOF supplemented with (1) 0.4% BSA + 5 μg mL–1 insulin, 5 μg mL–1 transferrin, and 5 ng mL–1 selenium (ITS) as a control; or (2) GF-CYK + 0.5 mg mL–1 HA + 0.15% RA (M1). Droplets were preserved under mineral oil in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. Blastocyst development and blastocyst diameter was observed at 7 and 8 days post-fertilization (dpf). Developmental and diameter data were analysed using the Wilcoxon test by using R software. The blastocyst rates were not significantly different between the control and M1 medium: at 7 dpf (22.9% ± 4.8 and 30.2% ± 3.0), and at 8 dpf (29.6% ± 5.1 and 37.4% ± 2.0 respectively; P > 0.05). The blastocyst diameter obtained with the M1 medium was significantly greater (P < 0.05) than that of the control at 7 dpf (173.3 μm ± 4.9 and 157.2 μm ± 4.1, respectively); however, no significant differences were observed at 8 dpf (190.3 μm ± 5.2 and 179.7 μm ± 5.3, respectively). In conclusion, the FCS- and BSA-free medium with GF-CYK, HA, and RA (M1) showed a comparable development rate to the control medium at 7 and 8 dpf. These growth factors and cytokines in association with hyaluronan and recombinant albumin have a synergistic action by promoting an increase in the blastocyst diameter at 7 dpf. This is fully synthetic method of embryo culture; it presents a valuable tool to reduce the risks of disease transmission via embryo transfer.

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Cytokines and embryo/endometrial interactions

Reproductive Medicine Review ◽

10.1017/s0962279900001095 ◽

1995 ◽

Vol 4 (2) ◽

pp. 87-100 ◽

Cited By ~ 6

Author(s):

Andrew Sharkey

Keyword(s):

Growth Factors ◽

Animal Models ◽

Embryo Culture ◽

Culture Medium ◽

Dramatic Reduction ◽

Rt Pcr ◽

Embryo Viability ◽

Experimental Animal Models ◽

Implantation Rates

Experimental animal models have shown that the in vitro embryo culture involved in many treatments for infertility results in a dramatic reduction in embryo viability. Recent advances in methodology such as RT-PCR for localization and quantitation of cytokines and their receptors, are revealing the role that this group of growth factors plays in the basic physiology of embryo development and the process of implantation itself. These studies offer the likelihood of dramatically improving in vitro embryo culture in humans and other species by supplementation of culture medium with growth factors or antagonists to improve embryo viability and hence implantation rates.

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