scholarly journals STUDY OF CELL FREE MITOCHONDRIAL DNA CONTENT OF EMBRYO CULTURE MEDIUM AT DAY 3 AND BLASTOCYST DEVELOPMENT AND QUALITY DURING INTRACYTOPLASMIC SPERM INJECTION CYCLES.

2016 ◽  
Vol 4 (8) ◽  
pp. 1867-1872
Author(s):  
AzzaA.Abdel Razik ◽  
◽  
MagdEldinM. Mohamed ◽  
ManalO. ElHamshary ◽  
OmimaA. Khamiss ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Dna Content ◽  
Culture Medium ◽  
Blastocyst Development ◽  
Mitochondrial Dna Content ◽  
Embryo Culture Medium

71 Removal of hypotaurine from porcine embryo culture medium decreases message for pro-apoptotic genes but does not affect development at low oxygen tension

2019 ◽  
Vol 31 (1) ◽  
pp. 161
Author(s):  
P. R. Chen ◽  
E. C. Leffeler ◽  
L. D. Spate ◽  
R. S. Prather
Keyword(s):  
Oxidative Stress ◽  
Oxygen Tension ◽  
Embryo Culture ◽  
Culture Medium ◽  
Atmospheric Oxygen ◽  
Tunel Staining ◽  
Blastocyst Development ◽  
Embryo Culture Medium ◽  
Porcine Embryo ◽  
High O2

Hypotaurine (HT) is a routine additive to embryo culture medium, acting primarily as an oxygen radical scavenger. However, the practice of culturing embryos at ~5% O2 has been widely adopted because this is more physiologically relevant to the oxygen tensions detected in the oviduct and uterus. Thus, the utility of HT may be diminished as fewer oxygen radicals are produced during culture at 5% O2. The objective here was to determine the effects of removing HT from our porcine embryo culture medium (MU2) on the development of embryos incubated at a lower oxygen tension (5% O2) compared with atmospheric oxygen (~20% O2). Porcine cumulus-oocyte complexes were aspirated, matured, and fertilized with standard procedures from our laboratory. Presumptive zygotes were cultured in 1 of 4 conditions: MU2 with 5mM HT at 5% O2 (low O2 +HT; control), MU2 without HT at 5% O2 (low O2 −HT), MU2 with 5mM HT at 20% O2 (high O2 +HT), or MU2 without HT at 20% O2 (high O2 −HT). The percentage of presumptive zygotes that developed to the blastocyst stage on Day 6 and total number of nuclei in the blastocysts were determined. The RNA was extracted from pools of 30 blastocysts, and cDNA was synthesised for quantitative PCR for genes associated with HT synthesis, oxidative stress, and apoptosis. Damage to DNA was assessed by TUNEL staining of Day 6 blastocysts. Data were analysed for normality by using a Shapiro-Wilk test, and differences between means were detected by using 2-way ANOVA followed by Tukey’s honest significant difference test with P<0.05 considered significant. Embryos cultured with high O2 −HT had significantly decreased blastocyst development compared with all other groups (26.2±2.5% v. 36.9-41.7±3.3-4.3%). Embryos cultured with low O2 −HT had significantly more nuclei than those cultured with high O2 +HT (50.5±1.5v. 45.5±1.2). Notably, differences in blastocyst development (41.7±3.3% v. 36.9±3.3%) or total number of nuclei (50.0±1.8v. 50.5±1.5) were not detected between embryos cultured with low O2 +HT or low O2 −HT. The abundance of messages for genes involved in HT synthesis (cysteine sulfinic acid decarboxylase [CSAD]) and oxidative stress (superoxide dismutase 1 [SOD1] and glutathione peroxidase 6 [GPX6]) did not differ between groups. Contrarily, messages for 2 pro-apoptotic markers (BCL2 associated agonist of cell death [BAD] and caspase 3 [CASP3]) were significantly increased in embryos cultured with +HT regardless of oxygen tension; however, percentages of DNA-damaged nuclei in blastocysts after TUNEL staining were not different between groups (5.4-6.5±0.5-1.0%). These results indicate that HT is not necessary for porcine pre-implantation development at 5% O2 but is beneficial at atmospheric oxygen tension. Further investigation is required to confirm if HT promotes apoptosis in pre-implantation embryos. This research was supported by Food for the 21st Century at the University of Missouri and R01 HD080636.

scholarly journals Nuclear and mitochondrial DNA in blastocoele fluid and embryo culture medium: evidence and potential clinical use

2016 ◽  
Vol 31 (8) ◽  
pp. 1653-1661 ◽  
Author(s):  
Elizabeth R. Hammond ◽  
Andrew N. Shelling ◽  
Lynsey M. Cree
Keyword(s):  
Mitochondrial Dna ◽  
Embryo Culture ◽  
Culture Medium ◽  
Clinical Use ◽  
Embryo Culture Medium

scholarly journals Mitochondrial DNA in Day 3 embryo culture medium is a novel, non-invasive biomarker of blastocyst potential and implantation outcome

2014 ◽  
Vol 20 (12) ◽  
pp. 1238-1246 ◽  
Author(s):  
S. Stigliani ◽  
L. Persico ◽  
C. Lagazio ◽  
P. Anserini ◽  
P.L. Venturini ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Embryo Culture ◽  
Culture Medium ◽  
Non Invasive ◽  
Embryo Culture Medium ◽  
Day 3 Embryo

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.

251 FACTORS INFLUENCING THE SUCCESS OF EQUINE INTRACYTOPLASMIC SPERM INJECTION IN A CLINICAL PROGRAM

2016 ◽  
Vol 28 (2) ◽  
pp. 258
Author(s):  
K. Hinrichs ◽  
Y. H. Choi
Keyword(s):  
Pregnancy Rate ◽  
Density Gradient ◽  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Medium ◽  
Ongoing Pregnancy ◽  
Ongoing Pregnancy Rate ◽  
Immature Oocytes ◽  
Embryo Culture Medium ◽  
Blastocyst Rate

Intracytoplasmic sperm injection (ICSI) is currently being used clinically in horses, but little information is available on factors affecting its success. We have conducted research trials and evaluated data from our clinical ICSI caseload, currently over 450 cases per year, to provide information in this area. In the following summaries, blastocyst data are reported per injected oocyte; clinical data are from 2013 and 2014. Clinically, when immature follicles were aspirated, the number of follicles aspirated per mare decreased significantly with mare age, from 16.2 at age 12–15 to 8.9 at age 24–25; however, maturation and blastocyst rates of recovered oocytes did not differ significantly. Immature oocytes shipped to the laboratory by referring veterinarians yielded a significantly higher blastocyst rate than that for immature oocytes aspirated at the laboratory (27 v. 21%, respectively). Shipped oocytes recovered from stimulated preovulatory follicles yielded a higher blastocyst rate per oocyte than did shipped immature oocytes (39 v. 27%, respectively), but provided fewer mature oocytes per aspiration (1.0 v. 4.5 for immature). From research data, administration of gentamicin and ampicillin to mares before immature oocyte aspiration did not affect blastocyst rate. Holding the aspirate for ~1.5 h at ambient temperature (26 to 33°C) was associated with a blastocyst rate of 32%; however, holding for 2 h at 32°C yielded only 16% blastocysts v. 23% for control. Blastocysts (18%) were obtained from oocytes recovered in the nonbreeding season (December and January). Holding oocytes at room temperature overnight before maturation did not affect blastocyst rate (25 to 34%), nor did inclusion of zinc in the maturation medium (18 to 31%). Diluting and refreezing semen to increase doses available for ICSI did not affect blastocyst rate (23 to 27%); blastocysts (13%) were also obtained after injection of nonmotile sperm. Significant differences in cleavage and blastocyst rates were identified among stallions. For one stallion, use of density gradient plus swim-up before ICSI increased cleavage (49 v. 18%) and blastocyst rates (11 v. 0%) compared to density gradient alone. Blastocyst production was not affected by the amount of glucose added to a human embryo culture medium (0 or 5 mM added glucose on Days 0–5, then 10 or 20 mM added glucose; 31 to 46% blastocysts). Replacement of 10% FBS in embryo culture medium with a mixture of FBS, human serum replacement, and equine follicular fluid lowered blastocyst rate (15 v. 37% for FBS alone). Clinically, embryos were vitrified or were shipped to embryo transfer centers for transfer. There was no significant difference in ongoing pregnancy rate for embryos from shipped immature v. preovulatory oocytes (54 and 69%). For immature oocytes, embryos developing to blastocyst on Day 10 or 11 had a lower ongoing pregnancy rate after transfer (40 and 0%) than did those developing on Days 7 to 9 (51 to 75%). This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by the Clinical Equine ICSI Program, Texas A&M University.

88 EFFECT OF BICARBONATE/CO2 LEVEL DURING EMBRYO CULTURE ON EQUINE BLASTOCYST RATE AFTER INTRACYTOPLASMIC SPERM INJECTION

2017 ◽  
Vol 29 (1) ◽  
pp. 151
Author(s):  
Y. H. Choi ◽  
P. Tinetti ◽  
J. G. Brom-de-Luna ◽  
K. Hinrichs
Keyword(s):  
Standard Method ◽  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
High Glucose ◽  
Culture Medium ◽  
Loss Rate ◽  
Second Step ◽  
Mixed Gas ◽  
Blastocyst Development

Equine embryos appear to require a high glucose concentration for development to the blastocyst stage. The complete cell-culture medium, DMEM/F-12 (DM), which contains 17 mM glucose, has been widely used for equine embryo culture; however, in other species, high glucose during the early stages of embryo development is detrimental. To avoid this, we initiated a 2-step system using a low-glucose human embryo culture medium (Global) from Days 0 to 5 [Day 0 = day of intracytoplasmic sperm injection (ICSI)], with glucose (20 mM) added to the medium in the second step (Days 5 to 10; Choi et al. 2015 Reproduction 150, 31–41). We noted a high pregnancy loss rate (20%) in our clinical ICSI program (Hinrichs et al. 2014 J. Equine Vet. Sci. 34, 176), which used this 2-step Global system. Limited data are available on pregnancy with DM-produced embryos, but in one study, the loss rate was 1/13 (7.7%; Choi et al. 2011 Reproduction 142, 529–538). It is possible that use of DM in the second step of culture would better support normal blastocyst development than does Global with added glucose. However, DM is typically used at 5% CO2, and Global at 6% CO2, so use of both media would necessitate 2 sets of incubators. In the present study, we explored the use of DM in the second step of a two-step equine embryo culture system, under different CO2 environments. Oocytes were collected from research mares via follicle aspiration and were held overnight before being matured in vitro for 30 h. All media included 10% fetal bovine serum. On Days 0 to 5 after ICSI, all embryos were cultured in Global under 6% CO2 in mixed gas (5% O2 and remainder N2) at 38.2°C. In Experiment 1, on Day 5, embryos were transferred to DM prepared according to our standard method, with 14.3 mM NaHCO3 and 5 mM NaOH, and were cultured in mixed gas at either 5% CO2 or 6% CO2. Five replicates were performed. In Experiment 2, DM was prepared by our standard method, or with 24.2 mM bicarbonate and no NaOH. When pH was measured using a pH meter after media were equilibrated overnight, this higher bicarbonate provided the same pH at 6% CO2 (pH 7.3), as was achieved with the standard DM preparation at 5% CO2. Six replicates were performed. In both experiments, blastocyst development was assessed on Days 7 to 10, and blastocyst rates were compared between treatments by Fisher’s exact test. In Experiment 1, blastocyst rates were 43%, 13/30 and 27%, 8/30 for the standard DM preparation in 5% and 6% CO2, respectively (P > 0.05). In Experiment 2, the blastocyst rates were 34%, 14/44 for the standard DM preparation at 5% CO2 and 43%, 19/44 for the high-bicarbonate DM at 6% CO2 (P > 0.05). We conclude that a 2-step Global-DM system can support equine blastocyst production under a consistent CO2 environment (6%) if DM bicarbonate levels are adjusted to balance the increased CO2. This work was supported by the Clinical Equine ICSI Program, Texas A&M University, and by the Link Equine Research Endowment Fund, Texas A&M University.

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

Zygote ◽  
2003 ◽  
Vol 11 (4) ◽  
pp. 347-354 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document