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 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.

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.

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.

Attempts at in vitro fertilization and culture of in vitro matured oocytes in sei (Balaenoptera borealis) and Bryde's (B. edeni) whales

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 19-28 ◽  
Author(s):  
M. M. U. Bhuiyan ◽  
Y. Suzuki ◽  
H. Watanabe ◽  
K. Matsuoka ◽  
Y. Fujise ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Bovine Serum Albumin ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cumulus Cells ◽  
Protein Supplementation ◽  
Vitro Fertilization ◽  
Embryo Culture Medium ◽  
Morula Stage

SummaryThe cumulus–oocyte–complexes (COCs) recovery rates with respect to reproductive status per sei (Balaenoptera borealis) and Bryde's (B. edeni) whales were determined in Experiment 1. The number of COCs recovered ranged from 16.0 to 30.6 and from 6.7 to 26.8 per sei and Bryde's whales, respectively. The effects of COCs grades and protein supplementation in embryo culture medium on development of in vitro fertilized (IVF) embryos were evaluated in sei and Bryde's whales in Experiment 2. The COCs were classified into either Grade A (COCs with five or more layers of compact cumulus cells) or Grade B (COCs with less than five layers of compact or expanded cumulus cells) before being cultured for IVM. The cleavage (12.0 to 19.5%), 4-cell (8.0 to 12.0%) and 8-cell (4.0 to 8.0%) formation rates in sei whales did not vary significantly between embryos derived from either grade A or B oocytes and between embryos cultured in either fetal whale serum (FWS)- or bovine serum albumin (BSA)-supplemented medium. The cleavage (4.0 to 14.8%), 4-cell (0.0 to 7.5%) and 8-cell (0.0 to 2.6%) formation rates in Bryde's whales did not vary significantly between embryos derived from either grade A or B oocytes and between embryos cultured in either FWS- or BSA-supplemented medium. The grade B oocytes cultured in FWS-supplemented medium developed to morula stage (1.1%) in sei whales. In conclusion, the present study indicates that IVF in sei whales is possible to achieve cleaved embryos developing to morula stage. This is the first in vitro embryo production attempt in sei and Bryde's whales.

136 ADDITION OF LOW DENSITY LIPOPROTEIN TO CHEMICALLY DEFINED PORCINE EMBRYO CULTURE MEDIUM CAN PARTIALLY REPLACE BOVINE SERUM ALBUMIN

2008 ◽  
Vol 20 (1) ◽  
pp. 148
Author(s):  
L. D. Spate ◽  
K. A. Walker ◽  
C. E. McHughes ◽  
R. S. Prather
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Blastocyst Stage ◽  
Differential Staining ◽  
Low Density ◽  
Cell Stage ◽  
Defined Culture

Embryo culture media typically contain undefined biologicals such as BSA. Our goal is to develop chemically defined culture media that are based on the biology and physiology of the embryo. To that end we evaluated the presence of message in embryos at various stages of development and determined that the message for the low density lipoprotein receptor (LDLR) increased from the germinal vesicle and 4-cell stage to the blastocyst stage of porcine embryogenesis. Thus, this study was conducted to determine if the addition of low density lipoprotein (LDL) would enhance the development and quality of in vitro produced porcine embryos in an already chemically defined culture medium. Slaughterhouse ovaries were aspirated, cumulous–oocyte complexes (COC) identified, and the COC were matured for 42 h in M199 base medium supplemented with EGF, FSH, and LH. Metaphase II oocytes were then selected. Fertilization was then preformed in modified Tris buffered medium and cocultured with 0.25 � 106/mL frozen thawed porcine semen for 5 h. The presumptive zygotes were then transferred to either porcine zygote medium with 0.3% BSA or 0.1% PVA (PZM3, PZM4). After 28 h, cleaved embryos were then sorted into six treatment groups (1. PZM3, 2. PZM3 + 20 µg mL–1 LDL, 3. PZM4, 4. PZM4 + 10 µg mL–1 LDL, 5. PZM4 + 20 µg mL–1 LDL, 6. PZM4 + 50 µg mL–1 LDL). The embryos were cultured in 5%O2 5%CO2 90%N until day 7. The percentage of development to the blastocyst stage was determined and analyzed with the SAS Proc GENMOD Procedure (a–cP ≤ 0.05). The percentage blastocyst was 51.3 � 0.09a, 51.6 � 0.09a, 33.1 � 0.99c, 35.8 � 0.09c, 36.9 � 0.09c, and 41.3 � 0.06b, for treatments 1–6, respectively. Culture in PZM4 (without BSA) significantly reduced development. However, addition of 50 µg mL–1 of LDL to PZM4 improved development above PZM4 alone. We interpret these data to indicate that a high concentration of LDL in the PZM4 media did improve embryo development and that LDL could partially substitute for BSA. Differential staining was performed on the blastocysts, and preliminary results suggest that the ICM to trophectoderm ratio in the high LDL treatment group is closer to the ratio found in in vivo produced embryos. This project was supported by USDA CSREES NRI (2006-35203-17282) and Food for the 21st Century.

Effects of amino acids on development in vitro of cleavage-stage bovine embryos into blastocysts

1996 ◽  
Vol 8 (5) ◽  
pp. 835 ◽  
Author(s):  
T Pinyopummintr ◽  
BD Bavister
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Embryo Development ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Development In Vitro ◽  
Defined Culture

Effects of amino acids on early bovine embryo development in vitro were examined using a chemically-defined, protein-free culture medium. Bovine embryos produced in vitro were cultured from 18 h to 72 h post insemination in a simple medium containing lactate as the only energy source except for the amino acid treatments. Subsequently, embryos were transferred to TCM-199 supplemented with serum for blastocyst development to substantiate their developmental competence. Treatments were: (1) non-essential amino acids from TCM-199 (NEA); (2) essential amino acids from TCM-199 (EA); (3) NEA+EA; (4) Eagle's minimum essential medium amino acids (MEM AA); (5) 11 amino acids present in HECM-6 (11 AA); and (6) 0.2 mM glutamine (GLN). A higher proportion of embryos (percentage of inseminated ova) cleaved to the > or = 8-cell stage by 72 h post insemination in NEA (56.7%), EA (41.2%), 11 AA (40.3%) and GLN (51.1%) than in either NEA+EA (30.0%) or MEM AA (33.1%). However, after transfer to complex medium, embryos that had developed in EA, as well as those in MEM AA or NEA+EA, produced significantly fewer blastocysts (37.1%, 34.4% and 25.6% respectively) than those in NEA (56.7%), GLN (48.9%) or 11 AA (37.7%). The ability of blastocysts to hatch from their zonae pellucidae was also affected by amino acid treatment during cleavage stages. The present study indicated that the addition of NEA or GLN or 11 AA to a chemically-defined culture medium during the cleavage phase of bovine embryo development increases their subsequent ability to reach the blastocyst stage. These data have implications for understanding the nutritional needs of bovine embryos produced in vitro and for optimizing the composition of culture media to support their development.

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.

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