scholarly journals 145 COMPARISON OF DEVELOPMENT AND QUALITY OF PORCINE EMBRYOS CULTURED IN DIFFERENT OXYGEN CONCENTRATIONS

2005 ◽  
Vol 17 (2) ◽  
pp. 223 ◽  
Author(s):  
S.-A. Ock ◽  
J.-G. Kim ◽  
L.-Y. Shi ◽  
H.-F. Jin ◽  
B. Mohana Kumar ◽  
...  
Keyword(s):  
Oxygen Concentration ◽  
Cell Number ◽  
High Oxygen ◽  
Tunel Staining ◽  
Blastocyst Development ◽  
Apoptosis Index ◽  
O2 Concentration ◽  
High O2 ◽  
Oxygen Concentrations

In spite of valuable uses of pig IVP embryos, including IVF and cloned embryos for production of valuable offspring that provide recombinant protein and xenotransplantation, several problems limit the success. In cattle embryo culture, lowering the oxygen concentration resulted in increased development, quality, and cell number, and reduced apoptosis incidence. The high O2 concentration in in vitro culture caused overproduction of reactive oxygen specias (ROS), causing damage to the cell membrane or DNA. The present study, therefore, evaluated the developmental ability of porcine embryos by culturing in either low or high oxygen concentration on the rates of cleavage, development, cell number, and apoptosis. These values were then compared to parthenote embryos, which were activated with electric stimulation by 2 DC pulses of 2.0 kV/cm, 30 μs in 0.3 M mannitol containing 100 μM CaCl2 and 100 μM MgCl2 and then transferred into NCSU23 medium supplemented with 7.5 μg/mL cytochalasin B for 6 h. Cumulus-oocyte complexes from slaughterhouse ovaries were matured in TCM 199 medium supplemented with 10 ng/mL EGF, 0.5 μg/mL FSH, 0.5 μg/mL LH, 0.57 mM cysteine, and 0.91 mM Na pyruvate for 24 h and further cultured in the same medium without FSH and LH for 20 h at 38.5°C, 5% CO2 in air. IVF was carried out in mTBM for 5 h with 1 × 105 sperm/mL by following the previously reported protocols (2004 Methods Mol. Biol. 253, 227–234) with minor modifications. Zygotes were allocated two O2 concentrations; zygotes were cultured in NCSU23 medium supplemented with 0.17 mM Na pyruvate, 2.73 mM Na lactate, and 0.4% BSA for 54 h and subsequently cultured in the same medium supplemented with 5.55 mM glucose for 90 h at 38.5°C, 5% CO2, 5% O2 and 90% N2 (Treatment 1) or 5% CO2, 20% O2 in air (Treatment 2). Statistical analysis was performed with one-way ANOVA by SPSS 10.0 (P < 0.05). Most oocytes (>82%) cleaved and the rates did not differ between groups. However, the rates of blastocyst development from oocytes used as parthenote controls (5% O2, 40.0 ± 13.0 (70/162); 20% O2, 34.5 ± 11.2 (56/161)) were significantly (P < 0.05) higher than those in IVF embryos (5% O2, 27.9 ± 8.0 (70/247); 20% O2, 27.1 ± 7.2 (68/249)). Similarly cell number and apoptosis index by TUNEL staining of blastocysts in parthenote (5% O2, 42 and 15%; 20% O2, 39 and 20%) were significantly (P < 0.05) higher than those in IVF (5% O2, 32 and 13%; 20% O2, 27 and 14%, all respectively). In conclusion, no differences between low and high oxygen concentrations in culture of porcine IVF and parthenote embryos were observed on the rates of cleavages and development into blastocyst cell number and apoptosis incidence. Further research should be carried out to develop a reliable IVP system. This work was supported by grant No. R05-2004-000-10702-0 from Ministry of Science & Technology, Republic of Korea.

75 EFFECT OF PHENAZINE ETHOSULFATE ON PORCINE BLASTOCYST DEVELOPMENT, APOPTOSIS, AND CRYOTOLERANCE AFTER OPEN PULLED STRAW VITRIFICATION

2008 ◽  
Vol 20 (1) ◽  
pp. 118
Author(s):  
B. Gajda ◽  
Z. Smorag ◽  
M. Bryla
Keyword(s):  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Tunel Staining ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Morula Stage ◽  
Phenazine Ethosulfate ◽  
And Control

It is possible to improve the success of cryopreservation of in vitro-produced bovine embryos by modifying the embryos with the metabolic regulator phenazine ethosulfate (PES) (Seidel 2006 Theriogenology 65, 228–235). The PES treatment increased glucose matabolism, tended to increase the pentose phosphate pathway flux of glucose, and clearly reduced accumulation of lipids in cultured bovine embryos (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607). It is known that porcine embryos have a considerably high content of lipids, and the success rates of their cryopreservation appear to be highly correlated with cytoplasmic lipid content. In our preliminary study, we observed that supplementation of NCSU-23 medium with PES has a positive effect on efficiency of pig blastocysts of good quality (Gajda et al.. 2007 Acta Biochim. Pol. 54(Suppl 1), 52 abst). In the present study, the effects of PES on pig blastocyst development, apoptosis, and survival after vitrification were investigated. In Exp. 1, porcine zygotes obtained from superovulated gilts were cultured in NCSU-23 medium supplemented with 0 (control), 0.025, 0.05, or 0.075 µm PES. The culture was performed at 39�C, with 5% CO2 in air, for 96–120 h. Embryo quality criteria were developmental competence (cleavage, morula stage, and blastocyst stage), cell number per blastocyst, and the degree of apoptosis as assessed by TUNEL staining. In Exp. 2, expanded blastocysts cultured with 0.025 µm PES were vitrified in a ethylene glycol and dimethyl sulfoxide mixture using open pulled straw (OPS) technology (Vajta et al. 1997 Acta Vet. Scand. 38, 349–352). After thawing, the blastocysts were cultured in vitro for re-expansion or transferred to synchronized recipients. Data were analyzed by chi-square test. There was a difference between the 0.025 µm PES-treated and the control group in percentage of cleaved embryos (99.0 and 91.4%, respectively; P < 0.05), between all experimental groups and control in percentage of morula stage (90.7, 87.8, 83.8, and 80.0%, respectively), and between 0.025 and 0.05 µm PES-treated and control in percentage of blastocyst rates (70.0, 75.5, and 65.7%, respectively). The number of cells and percentage of TUNEL-positive nuclei per blastocyst were lower in the PES-treated than in the control group. The survival rate of blastocysts after vitrification and thawing was enhanced in the presence of PES compared to that in the PES-free group (45.2 and 38.9%, respectively; P < 0.05). After transfer of 56 expanded blastocysts cultured with PES and vitrified into 3 recipients, two gilts were confirmed pregnant at 35 days of gestation. In conclusion, a higher blastocyst percentage with a low incidence of apoptosis was obtained in the presence of PES compared to control. These blastocysts also had an increased ability to survive cryopreservation.

Cell allocation in bovine embryos cultured in two media under two oxygen concentrations

Zygote ◽  
2002 ◽  
Vol 10 (4) ◽  
pp. 341-348 ◽  
Author(s):  
Amy Fischer-Brown ◽  
Rick Monson ◽  
John Parrish ◽  
Jack Rutledge
Keyword(s):  
Oxygen Concentration ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Development ◽  
Commercial Media ◽  
Factor Design ◽  
Medium Type ◽  
Oxygen Concentrations

Blastocyst development, total cell number and allocation to inner cell mass (ICM) and trophectoderm (TE) lineages was compared among day 9 hatched blastocysts from four culture treatments in a two-factor design. Two modified commercial media (KSOM and SOF) were used in atmospheres with two oxygen concentrations (5% and 20% O2). No significant effect of medium on development was found, but 20% O2 increased hatching (p < 0.05). There were more cells in hatched blastocysts cultured in KSOM than in SOF (181 vs 136, respectively; p < 0.0001); however, ICM/total cell ratio was not affected by medium. There was a trend suggesting that the proportion of cells allocated to ICM was lower in hatched blastocysts cultured under 5% O2 compared with 20% O2 (0.323 vs 0.380, respectively; p < 0.1). No significant interactions between medium type and oxygen concentration were found. These results indicate that culture components used in this study may affect cell proliferation without altering cell allocation, and that oxygen concentration may play a role in allocation of cells to ICM and TE lineages.

scholarly journals Bioinformatic and experimental findings to indicate anti-cancer targets and mechanisms of calycosin against nasopharyngeal carcinoma

2020 ◽  
Author(s):  
Fangxian Liu ◽  
Qijin Pan ◽  
Liangliang Wang ◽  
Shijiang Yi ◽  
Peng Liu ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Molecular Mechanisms ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Network Pharmacology ◽  
Tunel Staining ◽  
Pharmacological Targets ◽  
Experimental Findings

Abstract Background: Calycosin is a naturally-occurring phytoestrogen that reportedly exerts anti- nasopharyngeal carcinoma (NPC) effects. Nevertheless, the molecular mechanisms for anti-NPC using calycosin remain unrevealed. Methods: Thus, a network pharmacology was used to uncover anti-NPC pharmacological targets and mechanisms of calycosin. Additionally, validated experiments were conducted to validate the bioinformatic findings of calycosin for treating NPC. Results: As results, bioinformatic assays showed that the predictive pharmacological targets of calycosin against NPC were TP53, MAPK14, CASP8, MAPK3, CASP3, RIPK1, JUN, ESR1, respectively. And the top 20 biological processes and pharmacological mechanisms of calycosin against NPC were identified accordingly. In clinical data, NPC samples showed positive expression of MAPK14, reduced TP53, CASP8 expressions. In studies in vitro and in vivo, calycosin-dosed NPC cells resulted in reduced cell proliferation, promoted cell apoptosis. In TUNEL staining, calycosin exhibited elevated apoptotic cell number. And immunostaining assays resulted in increased TP53, CASP8 positive cells, and reduced MAPK14 expressions in calycosin-dosed NPC cells and tumor-bearing nude mice. Conclusion: Altogether, these bioinformatic findings reveal optimal pharmacological targets and mechanisms of calycosin against NPC, following with representative identification of human and preclinical experiments. Notably, some of original biotargets may be potentially used to treat NPC.

scholarly journals 160EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF DOMESTIC CAT IVF EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 202 ◽  
Author(s):  
W.F. Swanson ◽  
A.L. Manharth ◽  
J.B. Bond ◽  
H.L. Bateman ◽  
R.L. Krisher ◽  
...  
Keyword(s):  
Culture Media ◽  
Ionic Composition ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Vitro Development

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P&gt;0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P&gt;0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)

scholarly journals The FLASH effect depends on oxygen concentration

2020 ◽  
Vol 93 (1106) ◽  
pp. 20190702 ◽  
Author(s):  
Gabriel Adrian ◽  
Elise Konradsson ◽  
Michael Lempart ◽  
Sven Bäck ◽  
Crister Ceberg ◽  
...  
Keyword(s):  
Oxygen Concentration ◽  
Underlying Mechanism ◽  
Oxygen Depletion ◽  
Therapeutic Window ◽  
High Dose ◽  
Dose Rates ◽  
Conventional Dose ◽  
Oxygen Concentrations

Objective: Recent in vivo results have shown prominent tissue sparing effect of radiotherapy with ultra-high dose rates (FLASH) compared to conventional dose rates (CONV). Oxygen depletion has been proposed as the underlying mechanism, but in vitro data to support this have been lacking. The aim of the current study was to compare FLASH to CONV irradiation under different oxygen concentrations in vitro. Methods: Prostate cancer cells were irradiated at different oxygen concentrations (relative partial pressure ranging between 1.6 and 20%) with a 10 MeV electron beam at a dose rate of either 600 Gy/s (FLASH) or 14 Gy/min (CONV), using a modified clinical linear accelerator. We evaluated the surviving fraction of cells using clonogenic assays after irradiation with doses ranging from 0 to 25 Gy. Results: Under normoxic conditions, no differences between FLASH and CONV irradiation were found. For hypoxic cells (1.6%), the radiation response was similar up to a dose of about 5–10 Gy, above which increased survival was shown for FLASH compared to CONV irradiation. The increased survival was shown to be significant at 18 Gy, and the effect was shown to depend on oxygen concentration. Conclusion: The in vitro FLASH effect depends on oxygen concentration. Further studies to characterize and optimize the use of FLASH in order to widen the therapeutic window are indicated. Advances in knowledge: This paper shows in vitro evidence for the role of oxygen concentration underlying the difference between FLASH and CONV irradiation.

22 EFFECT OF OOCYTE AGE ON THE DEVELOPMENT OF MOUSE PARTHENOGENETIC EMBRYOS AND EFFECT OF OXYGEN CONCENTRATION ON EMBRYO DEVELOPMENT IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 119
Author(s):  
H. Bagis ◽  
S. Arat ◽  
H. Odaman ◽  
A. Tas
Keyword(s):  
Embryo Development ◽  
Formation Rate ◽  
Cell Number ◽  
Blastocyst Formation ◽  
Group 4 ◽  
Mouse Embryo Development ◽  
O2 Concentration ◽  
Group 3 ◽  
Development In Vitro

The objective of this study was to investigate the effects of two parameters on mouse embryo development in vitro. These parameters were the effect of oocyte age on activation and the effect of O2 concentration in culture. In the first experiment, oocytes were recovered from superovutated mice at 15 h (group 1) or 20 h (group 2) after human chorionic gonadotropin (HCG) injection. All oocytes were activated for 6 h with 10 mM Sr2+ in Ca2+ free medium in the presence of 5 �g/mL of cytochalasin B. After activation, embryos were cultured in KSOM.aa medium for 4.5-5.5 days. Zygotes from naturally bred mice were used as control. Differences in blastocyst formation rate and blastocyst cell number among treatments were analyzed by one-way ANOVA after arcsin square transformation. In the first experiment, blastocyst formation rate in the first group was higher than in the second group (62.6% vs. 47.1%; P < 0.05). In addition, blastocyst cell number was also higher in the first group than in the second one (69.4 � 3.2 vs. 52.4 � 2.2; P < 0.05). However, both values were higher in control group (80%, 76.2 � 1.2; P < 0.05) than in the experimental groups. These results showed that young oocytes were activated more effectively than aged oocytes. In the second experiment, mouse zygotes were cultured in a humidified atmosphere of 5% CO2 in air (group 3) or 5% CO2, 5% O2, and 90% N2 (group 4). Blastocyst formation rate and blastocyst cell number of zygotes cultured in low O2 concentration (group 4) for 4.5 days were higher than for group 3 (76.3% vs. 56.4 and 69.0 � 3.4 vs. 52.8 � 2.3; P < 0.05). There was a significant difference in blastocyt formation rate of embryos for 5.5 days between the two groups (25.8% for group 4 vs. 14.4% for group 3; P < 0.05). This suggests that the embryos developed more slowly in high O2 concentration. These results showed that low O2 concentration provided a more suitable environment for mouse embryo development in vitro. The same experiment was repeated with parthenogenetic embryos recently in our laboratory. This study was supported by a grant from TUBITAK, Turkey (VHAG-1022).

225 DEVELOPMENTAL COMPETENCE AND QUALITY OF PIG EMBRYOS OBTAINED FROM STANDARD IN VITRO FERTILIZATION OR INTRACYTOPLASMIC SPERM INJECTION

2013 ◽  
Vol 25 (1) ◽  
pp. 260 ◽  
Author(s):  
I. Grad-Mandryk ◽  
J. Kosenyuk ◽  
B. Gajda
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Quality Criteria ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Tunel Staining ◽  
In Vitro Production ◽  
Total Cell Number ◽  
Vitro Fertilization

In vitro production of porcine embryos is still relatively inefficient. The main reasons for this limited performance are polyspermy after IVF and the poor developmental ability of obtained zygotes. Intracytoplasmic sperm injection (ICSI) is one possible solution to eliminate polyspermy. The aim of this study was to compare the developmental competence of pig zygotes, total cell number, and DNA fragmentation of pig blastocysts derived from IVF or ICSI. Cumulus–oocyte complexes were obtained by aspiration from antral follicles of ovaries collected from slaughtered gilts. The oocytes were then cultured in modified TC-199 medium to metaphase II for 42 h. Semen for IVF was incubated in modified capacitation medium (M199) for 1 h. The sperm fraction (1 × 106 cells mL–1) was introduced into droplets containing oocytes, and then gametes were co-incubated for 4 h in modified TC-199 medium. Intracytoplasmic sperm injection was performed using a mechanical micromanipulator (Research Instruments Limited, Cornwall, UK). Micromanipulation was carried out in modified NCSU-37 medium. The tails of spermatozoa were broken, and then single spermatozoa were aspirated into the injection pipette. The oocyte was fixed by a holding pipette, and the sperm head was then introduced into the oocyte cytoplasm. Presumptive zygotes were cultured in vitro for 144 h in NCSU-23 medium. The embryo quality criteria were developmental competence (morula and blastocyst rates), total cell number per blastocyst, and degree of apoptosis assessed by TUNEL staining. Data were analysed by chi-squared test. The experiment was performed on 136 zygotes (6 replicates) obtained after IVF and 83 zygotes (4 replicates) obtained after ICSI. Percentages of embryos developed to the morula and blastocyst stages were 42.3 ± 6.1 and 28.8 ± 4.7 after IVF, respectively, and 51.7 ± 15.4 and 34.5 ± 18.9 after ICSI, respectively (no differences were observed). Significant differences were noticed in total number of cells per blastocyst between embryos after IVF and ICSI (33.7 ± 5.39 v. 22.8 ± 3.22; P < 0.01). However, there was no difference in the degree of apoptosis between IVF and ICSI embryos (5.14 ± 3.49 and 6.14 ± 4.88, respectively). Our preliminary studies demonstrated a higher proportion of cell numbers in IVF-derived embryos compared with those produced by ICSI, but the developmental competence and degree of apoptosis, as evaluated by the TUNEL method, in both groups were comparable. This study was funded by project N N311 516140 by the NCN, Poland.

125 EXAMINING CRITERIA FOR EXTENDING BOVINE BLASTOCYST SURVIVAL IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 143
Author(s):  
F. N. T. Cooke ◽  
T. M. Rodina ◽  
P. J. Hansen ◽  
A. D. Ealy
Keyword(s):  
Conditioned Medium ◽  
Culture System ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Interferon Tau ◽  
Cell Numbers ◽  
Bovine Blastocyst

Most of the current culture procedures used for bovine in vitro embryo production terminates at the blastocyst stage. Developing procedures for extending embryo lifespan beyond this phase will provide a valuable tool for understanding events that occur during the second week of pregnancy in cattle. The overall objective of the present studies was to identify culture conditions required to support bovine blastocyst development beyond its initial formation. In the first study, individual day 8 blastocysts (day 0 = day of IVF) were cultured until day 11 in 30 µL microdrops of Potassium Simplex Optimized Medium-Bovine Embryo 2 containing 0.1 mm non-essential amino acids or Tissue Culture Medium 199 (M199). Both media were supplemented with 5% [v/v] fetal bovine serum (FBS) and incubations were in an atmosphere of either 5 or 21% (v/v) oxygen. A medium by oxygen interaction (P = 0.007) occurred when assessing cell number on day 11. Blastocysts cultured in M199 and in a 5% O2 environment had greater (P < 0.002) cell numbers (536 � 49) than blastocysts incubated in other conditions (339 � 28). Conditioned medium from blastocysts incubated in 21% O2 contained greater (P < 0.05) concentrations of bioactive interferon-tau (IFNT) than blastocysts incubated in 5% O2 regardless of medium type (70.5 � 28 v. 17.2 � 2.6 ng mL–1). In a follow-up study, blastocysts could remain morphologically viable through day 11 in M199 containing at least 2.5% FBS. To examine whether oxidative stress was responsible for the increase in IFNT production under 21% O2, blastocysts were incubated under a 5% O2 atmosphere in M199 containing 2.5% FBS and increasing concentrations of tert-butylhydroperoxide (tBH), a membrane-permeable oxidative agent. Addition of e3 nm tBH decreased cell numbers but did not increase IFNT concentrations in conditioned medium. To examine whether blastocysts could survive beyond day 11 in culture, day 11 blastocysts were transferred to 400 �L of M199 with 20% FBS under 5% oxygen and cultured from day 11 to 20–21 post-IVF. Half of the medium was replaced every 2–3 days. On day 13–14, 16.6 � 6.1% of blastocysts showed initial signs of degeneration. A portion of blastocysts (32.9 � 9.6%) began attaching to plates on days 13–15 and produced outgrowths that appeared viable on days 20–21. All of the non-attached blastocysts degenerated by day 17–18. No blastocyst elongation was detected. In conclusion, a culture system was developed that sustains blastocyst viability and IFNT production in vitro to day 11. Although this culture system allowed blastocyst survival until day 14, normal conceptus development (i.e. elongation/filamentation) was not achieved. Nonetheless, the culture system provides a useful tool for examining the initial stages of blastocyst development and IFNT production from individual bovine embryos.

82 EARLY PORCINE EMBRYO ENERGY PREFERENCE AND SUBSEQUENT DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 170
Author(s):  
L. D. Spate ◽  
B. K. Redel ◽  
R. S. Prather
Keyword(s):  
Energy Source ◽  
Subsequent Development ◽  
Cell Number ◽  
Total Cell ◽  
Control Medium ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Cell Stage ◽  
Porcine Embryo

Early porcine embryo metabolism in vitro is not completely understood. It has been suggested that before embryo genome activation (4-cell stage), the preferred energy source of the embryo is pyruvate. In our porcine zygote culture medium (MU1), the energy sources are 0.2 mM pyruvate and 2.0 mM calcium lactate. Three experiments were performed with in vitro-matured and IVF embryos to examine the effect on blastocyst development after withholding pyruvate and/or lactate during the first 48 h of culture. In Experiment 1, embryos were cultured without lactate for 48 and then cultured to Day 6 in control medium containing lactate. Control embryos were cultured in medium with lactate starting after fertilization to Day 6. All data were analysed by using SAS 9.3 with a GENMOD procedure used for the blastocyst data and a GLM procedure used for the cell number data. On Day 6, the percentage of embryos that formed blastocysts was 30.2% for control and 26.5% for embryos cultured for 48 h without lactate (n = 490, 4 replications). The difference was not significant P > 0.05. In Experiment 2, embryos were cultured without pyruvate for 48 and then cultured to Day 6 in control medium containing pyruvate. Control embryos were cultured in medium with pyruvate starting after fertilization to Day 6. On Day 6, the percentage of embryos that formed blastocysts was 31.1% for control and 30.5% for embryos cultured for 48 h without pyruvate (n = 385, 3 replications). In Experiment 3, embryos were cultured in control medium for the first 48 h and then cultured to Day 6 in medium without pyruvate, thus forcing the embryos to use lactate instead of pyruvate. On Day 6, the percentage of embryos that formed blastocysts in the pyruvate free medium increased from 28.6%a ± 1.0 to 33.9%b ± 1.0; P ≤ 0.05 (n = 490, 4 replications) compared with the control and total cell number increased from 30.7a ± 1.5 to 41.3b ± 1.8 cells, respectively; P ≤ 0.05 (n = 65, 4 replications). The results from Experiments 2 and 3 were unanticipated as it was believed that the embryo would be more dependent on pyruvate for energy up to the blastocyst stage. We believed in Experiment 2 that from zygote to 4 cells the embryos were not as capable of using lactate and that removing the pyruvate would hinder further development. In Experiment 3, forcing the embryo to use lactate from Day 2 to Day 6 significantly improved blastocyst development and total cell number, suggesting that the embryo is not dependent on a specific energy source or that there are adequate pyruvate stores in the oocyte to 4-cell stage, to promote development to blastocyst. Funding was provided by Food for the 21st Century, the University of Missouri, and the National Institutes of Health (OD011140).

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