86 SIMPLE METHOD FOR EMULSIFICATION OF LIPOPHILIC NUTRIENTS THAT AFFECT PRE-IMPLANTATION DEVELOPMENT OF BOVINE EMBRYOS IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 157
Author(s):  
S. Ikeda
Keyword(s):  
Culture Media ◽  
Calf Serum ◽  
Hatching Rate ◽  
Cell Stage ◽  
Simple Method ◽  
Antioxidative Vitamins ◽  
Blastocyst Rate ◽  
Development In Vitro ◽  
Additive Control

In order to investigate the effects of bioactive lipophilic nutrients on mammalian pre-implantation embryos in vitro, amphipathic vehicles are commonly used to dissolve the lipophilic substances into culture media. However, easy emulsification of these nutrients would facilitate medium preparation. We report here a simple method for emulsification of lipophilic nutrients that affect bovine pre-implantation embryonic development in vitro. We investigated the effects of emulsified oleic acid (OA) or a mixture of antioxidative vitamins – vitamin E (VE) and β-carotene (BC). Polyglyceryl-10 laurate (P10L) was used as an emulsifier and was dissolved in sterile water at 5.05% (wt/wt) in glass vials. One percent (wt/wt) of OA or a mixture of VE (α-tocopherol) and BC (VE : BC = 1000 : 1 wt/wt) was added into the vial and mixed by using a magnetic stirrer. After first exhibiting white turbidity, the solution became transparent and stabilised, indicating stable emulsification. The emulsified OA and VE+BC were designated as emOA and emVEBC, respectively. Cumulus-enclosed oocytes obtained from abattoir bovine ovaries were in vitro-matured (IVM) for 22 h in modified synthetic oviduct fluid (mSOF) supplemented with 10% (vol/vol) fetal calf serum and 0.2 IU mL–1 FSH. After IVM, the oocytes were subjected to IVF with Percoll gradient-selected sperm from a single bull in an mSOF-based medium for 20 h. After IVF, presumptive zygotes were freed from the cumulus cells and cultured in mSOF. On Day 3 (IVF = Day 0), embryos that had developed to the 8-cell stage or more (≥8-cell) were subsequently cultured in medium supplemented with 0.05% (vol/vol) of emOA or emVEBC. Blastocyst development from ≥8-cell embryos was assessed on Day 8. In the case of no-additive control and emVEBC, the hatching rate was also assessed on Day 10. All the cultures were performed at 38.5°C under 5% CO2, 5% O2, and 90% N2 and replicated 4 times with ~18 embryos per group per replicate. The development data were statistically analysed by the general linear model. The blastocyst rate in the emOA group (36.4%) was significantly (P < 0.05) lower than that in the no-additive control (54.1%). The blastocyst rate in the emVEBC group (53.9%) was similar to that in the control; however, the hatching rate was significantly higher in the emVEBC group (22.6%) than in the control (9.2%). These data suggest that emulsification of lipophilic nutrients with P10L is an easy method to allow their addition into culture media for investigating their favourable (e.g. antioxidative vitamins) or inhibitory (e.g. OA) effects on pre-implantation development in vitro.

scholarly journals 133 INSULIN, TRANSFERRIN AND SELENIUM WITH OR WITHOUT BSA IN A SERUM-FREE CULTURE SYSTEM FOR BOVINE EMBRYO, AND ITS SUITABILITY FOR EMBRYOS CULTURED IN SMALL GROUPS

2005 ◽  
Vol 17 (2) ◽  
pp. 217 ◽  
Author(s):  
C. Daniaux ◽  
B. Verhaeghe ◽  
I. Donnay
Keyword(s):  
Small Groups ◽  
Culture Medium ◽  
Culture Media ◽  
Quality Parameters ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Serum Free ◽  
Abnormal Accumulation ◽  
Development In Vitro

Serum in embryo culture medium may be a potential cause of abnormal accumulation of lipid droplets, which is correlated to a higher sensitivity to cryopreservation. Moreover, serum may introduce pathogens. With the aim of developing a serum-free culture medium, we first (Experiment 1) investigated the effect of adding ITS (5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenium) as a serum substitute in SOF medium on embryos cultured in large groups (20 embryos per culture drop of 20 μL) and we then (Experiment 2) analyzed the effect of adding BSA. In this second experiment, our serum-free culture media were also tested on embryos cultured in small numbers (5 embryos per drop of 20 μL) in order to mimic ovum pickup (OPU) conditions. Embryos were obtained from slaughterhouse oocytes, matured in vitro for 24 h in a serum-free enriched 199 medium (Donnay et al. 2004 Reprod. Fertil. Dev. 16, 274) containing ITS, and fertilized for 18 h. In experiment 1, embryos were cultured in SOF (Holm et al. 1999 Theriogenology 52, 683–700) supplemented with 0.1 mg/mL polyvinylpyrrolidane (PVP) without (SOF) or with ITS (SOF-ITS), or with 5% FCS (SOF-FCS). Cavitation occurred earlier in presence of serum (Table). Adding ITS to SOF increased blastocyst rates at Day 7 and Day 8 post-insemination (p.i.) and also the hatching rate. In experiment 2, embryos were cultured in SOF-FCS, SOF-ITS, or SOF-ITS supplemented with 4 mg/mL fatty acid free BSA (SOF-ITS-BSA). Within each condition, no differences were observed for blastocyst and hatching rates between embryos cultured in large or in small groups. Adding BSA to SOF-ITS increased blastocyst rate at Day 6 p.i. and also the hatching rate. At Days 7 and 8 p.i., blastocyst rates were higher in SOF-FCS than in SOF-ITS and tended to be higher than in SOF-ITS-BSA, especially for embryos cultured in small groups. Cell numbers of the resulting embryos were unaffected. These results indicate that: (1) ITS as supplement to SOF medium promotes embryo development in vitro. (2) BSA as protein supplement to SOF-ITS medium accelerates blastulation and improves hatching rate. (3) SOF-ITS and SOF-ITS-BSA are two serum-free culture media that can sustain development of embryos, also when cultured in small number, even though SOF-FCS tended to afford better rates of development. Further studies will include evaluation of other quality parameters including resistance to cryopreservation. This work was supported by the Ministery of Agriculture of the Region wallonne de Belgique.

Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRβ) in buffalo preimplantation embryos produced in vitro: markers of successful embryo implantation

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 203-213 ◽  
Author(s):  
S. Eswari ◽  
G. Sai Kumar ◽  
G. Taru Sharma
Keyword(s):  
Culture Media ◽  
Total Cell ◽  
Inhibitory Factor ◽  
Preimplantation Embryos ◽  
Leukaemia Inhibitory Factor ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Significant Difference

SummaryThe objective of this study was to evaluate the effect of supplementation of recombinant leukaemia inhibitory factor (LIF) in culture media on blastocyst development, total cell number and blastocyst hatching rates and the reverse transcription-polymerase chain reaction analysis of preimplantation buffalo embryos to determine whether they contain the LIF-encoding mRNA and its beta receptor (LIFRβ) genes in different stages of preimplantation buffalo embryos. Cumulus–oocyte complexes retrieved from slaughterhouse buffalo ovaries were matured in vitro and fertilized using frozen buffalo semen. After 18 h of co-incubation with sperm, the presumptive zygotes were cultured in modified synthetic oviductal fluid without (control) or with rhLIF (100 ng/ml). There was no significant difference in the overall cleavage rate up to morula stage however the development of blastocysts, hatching rate and total cell numbers were significantly higher in the LIF-treated group than control. Transcripts for LIFRβ were detected from immature, in vitro-matured oocytes and in the embryos up to blastocyst stage, while transcripts for the LIF were detected from 8–16-cell stage up to blastocyst, which indicated that embryo-derived LIF can act in an autocrine manner on differentiation process and blastocyst formation. This study indicated that the addition of LIF to the embryo culture medium improved development of blastocysts, functional (hatching) and morphological (number of cells) quality of the blastocysts produced in vitro. The stage-specific expression pattern of LIF and LIFRβ mRNA transcripts in buffalo embryos indicated that LIF might play an important role in the preimplantation development and subsequent implantation of buffalo embryos.

78 SUPPLEMENTATION WITH CARNOSINE DURING IN VITRO CULTURE IMPROVES THE QUALITY OF IN VITRO-PRODUCED BOVINE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 146
Author(s):  
D. Le Bourhis ◽  
M. Verachten ◽  
P. Salvetti ◽  
M. Hochet ◽  
L. Schibler
Keyword(s):  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Oxygen Species ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Blastocyst Rate

The objective of the present study was to determine the effect of supplementation of culture medium with carnosine (β-alanyl-l-histidine; Sigma, St-Quentin Fallavier, France), a reactive oxygen species scavenger, on in vitro bovine embryo development and survival following cryopreservation. Abattoir-derived bovine oocytes (4 replicates) were in vitro matured and fertilized with frozen-thawed semen of one bull, according to our standard procedures. In Experiment 1, 20 h after IVF, groups of presumptive zygotes were cultured in 30 μL of SOF BSAaa + 1% oestrus cow serum with 0 (control; n = 205) or 5 μg mL−1 of carnosine (n = 209) under humidified air with 5% CO2, 5% O2, and 88% N2. Cleavage rates were determined on Day 2, and the blastocyst rates and grade were assessed on Day 7 according to IETS classification. Day 7 grade 1 expanded blastocysts (n = 25 control and n = 27 carnosine) were frozen in 1.5 M ethylene glycol + 0.1 M sucrose. Embryos were thawed and then cultured for 72 h in SOF-BSAaa + 1% oestrus cow serum for re-expansion and hatching rate assessments at +24 h, +48 h, and +72 h post-thawing. In Experiment 2, presumed zygotes were cultured in SOF BSAaa + 1% oestrus cow serum with 0 (control; n = 48) or 5 μg mL−1 of carnosine (n = 48) in a WOW dish and observed with Time Laps Cinematography (Primo Vision®, VitroLife, Göteborg, Sweden). Images were recorded every 15 min for up to 168 h post-insemination. For embryos that reached the blastocyst stage, mean timing of the first cleavage (C1; 2-cell stage), second cleavage (C2; 4-cell stage), second cleavage to compaction (C3), and blastocoel cavity appearance (B4) were recorded. Chi-square test for Experiment 1 and Student’s t-test for Experiment 2 were used, and differences were considered significant at P < 0.05. In Experiment 1, no differences were observed in cleavage rate, blastocyst rate on Day 7, and grade 1 blastocyst rate between both control and carnosine groups (84.0 ± 4.2 v.85.2 ± 3.8, P = 0.7; 46.9 ± 7.1 v. 45.0 ± 7.5, P = 0.7; 24.1 ± 2.0 v. 24.0 ± 6.5, P = 0.6; respectively). After thawing, the re-expansion at +24 h was not different between groups (74.1 v. 48.0% for carnosine and control groups, respectively; P = 0.06). However, at +48 h and +72 h, the survival rate of carnosine treated blastocysts was significantly higher than that of blastocysts in the control group: 70.4 ± 4.5% v. 40.0 ± 3.8% and 59.3 ± 3.8% v. 24.0 ± 3.6%, respectively. Results from Experiment 2 indicated no difference between control and carnosine groups for C1 (32.1 ± 3.9 v. 33.8 ± 6.1; P = 0.3), C2 (8.2 ± 8.9 v. 8.9 ± 0.9; P = 0.07), and B4 (147.0 ± 9.5 v. 145.4 ± 11.6; P = 0.6), whereas C3 was significantly different within groups: 59.9 ± 9.6 v. 51.8 ± 6.7 (P = 0.008). In conclusion, bovine blastocysts derived from zygotes cultured in the presence of 5 μg mL−1 carnosine possess a significantly faster kinetic from 4-cell stage to compaction and show a higher post-thawing viability. However, further analyses are still needed to clarify the relationship between the reactive oxygen species intracellular levels after carnosine treatment and in vitro bovine embryo quality. This work was supported by FECUND European project (grant agreement number 312097).

Development in vitro of mouse embryos from the two-cell egg stage to the early somite stage

Development ◽  
1974 ◽  
Vol 31 (1) ◽  
pp. 235-245
Author(s):  
Yu-Chih Hsu ◽  
John Baskar ◽  
Leroy C. Stevens ◽  
John E. Rash
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Cell Stage ◽  
Cord Serum ◽  
Somite Stage ◽  
Development In Vitro

About 1–3% of mouse blastocysts, which had initially been cultured from the two-cell stage in chemically defined medium or about 3–5% of blastocysts which were explanted from the uterus, developed to the early somite stage when cultured in vitro on collagen. Two-cell eggs were initially cultivated in chemically defined medium to the blastocyst stage. Blastocysts were then transferred to Eagle's minimal essential medium (MEM) plus 10% heat-inactivated calf serum. Two barriers to further development were overcome. First, the formation of endoderm and ectoderm from the inner cell mass immediately after attachment to collagen. Second, formation of the embryo proper from the embryonic region. Both barriers were overcome by using heat-inactivated human cord serum after the blastocysts hatched from the zona pellucida and attached to collagen. After attachment, embryos were cultured in MEM plus 20% heat-inactivated human cord serum which was changed daily until early somite stages. Apparently normal healthy development in vitro occurred, as judged by light and electron microscopic examination.

Nobiletin-induced partial abrogation of deleterious effects of AKT inhibition on preimplantation bovine embryo development in vitro

2021 ◽  
Author(s):  
Yulia N Cajas ◽  
Karina Cañón-Beltrán ◽  
Carolina Núñez-Puente ◽  
Alfonso Gutierrez-Adán ◽  
Encina M González ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Media ◽  
Cell Number ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Developmental Effect ◽  
Promote Cell Survival ◽  
Development In Vitro

Abstract During preimplantational embryo development, PI3K/AKT regulates cell proliferation and differentiation and nobiletin modulates this pathway to promote cell survival. Therefore, we aimed to establish whether, when the AKT cascade is inhibited using inhibitors III and IV, nobiletin supplementation to in vitro culture media during the minor (2 to 8-cell stage, MNEGA) or major (8 to 16-cell stage, MJEGA) phases of EGA is able to modulate the development and quality of bovine embryos. In vitro zygotes were cultured during MNEGA or MJEGA phase in SOF + 5% FCS or supplemented with: 15 μM AKT-InhIII; 10 μM AKT-InhIV; 10 μM nobiletin; nobiletin+AKT-InhIII; nobiletin+AKT-InhIV; 0.03% DMSO. Embryo development was lower in treatments with AKT inhibitors, while combination of nobiletin with AKT inhibitors was able to recover their adverse developmental effect and also increase blastocyst cell number. The mRNA abundance of GPX1, NFE2L2, and POU5F1 was partially increased in 8- and 16-cell embryos from nobiletin with AKT inhibitors. Besides, nobiletin increased the p-rpS6 level whether or not AKT inhibitors were present. In conclusion, nobiletin promotes bovine embryo development and quality and partially recovers the adverse developmental effect of AKT inhibitors which infers that nobiletin probably uses another signalling cascade that PI3K/AKT during early embryo development in bovine.

Impact of adding 5.5 mM glucose to SOF medium on the development, metabolism and quality of in vitro produced bovine embryos from the morula to the blastocyst stage

Zygote ◽  
2002 ◽  
Vol 10 (3) ◽  
pp. 189-199 ◽  
Author(s):  
I. Donnay ◽  
J.M. Feugang ◽  
S. Bernard ◽  
J. Marchandise ◽  
S. Pampfer ◽  
...  
Keyword(s):  
Cell Mass ◽  
Calf Serum ◽  
Apoptotic Index ◽  
Hatching Rate ◽  
Fluid Medium ◽  
Lactate Release ◽  
Morula Stage ◽  
Blastocyst Rate

Although toxic for early stages of embryo development, glucose is a physiological metabolic substrate at the morula and blastocyst stages. We evaluated the effect of adding 5.5 mM glucose from the morula stage on bovine blastocyst development and quality. In vitro matured and fertilised bovine oocytes were cultured in modified Synthetic Oviduct Fluid medium containing 5% fetal calf serum, but without added glucose, up to day 5 post-insemination (pi). Morulae were selected and further cultured in the presence or absence of 5.5 mM glucose. Blastocyst and hatched blastocyst rates were recorded. Oxygen, glucose and pyruvate uptakes as well as lactate release were evaluated. The quality of the resulting blastocysts was evaluated by the cell allocation to the inner cell mass (ICM) and trophectoderm (TE) and by the apoptotic index. Adding glucose increased the blastocyst rate at day 8 pi (80% vs 65%) but had no impact on hatching rate (25% vs 28%). A 22% decrease in oxygen uptake was observed in the presence of glucose, concomitant with an increase in lactate release, although no change was observed in pyruvate uptake. A slight decrease in blastocyst cell number was observed at day 7 in the presence of glucose while neither the ICM/TE cell ratio nor the apoptotic index were affected. In conclusion, adding 5.5 mM glucose from the morula stage has a limited impact on blastocyst rate and quality although important modifications were observed in embryo metabolism. It remains to be determined whether those modifications could influence embryo viability after transfer.

scholarly journals Nobiletin enhances the development and quality of bovine embryos in vitro during two key periods of embryonic genome activation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karina Cañón-Beltrán ◽  
Yulia N. Cajas ◽  
Serafín Peréz-Cerezales ◽  
Claudia L. V. Leal ◽  
Ekaitz Agirregoitia ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Signaling Pathway ◽  
Akt Signaling ◽  
Mitochondrial Activity ◽  
Bovine Embryos ◽  
Akt Signaling Pathway ◽  
Cell Stage ◽  
Development In Vitro

AbstractIn vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.

scholarly journals A simple method of suspending implant wear particles for more physiological modelling of cell-particle interactions in vitro

2021 ◽  
Author(s):  
Christine Poon
Keyword(s):  
Wear Debris ◽  
Culture Media ◽  
Polymeric Materials ◽  
Wear Particle ◽  
Physicochemical Characteristics ◽  
Wear Particles ◽  
Simple Method ◽  
Implant Wear

AbstractArthroplasty implants e.g. hip, knee, spinal disc sustain relatively high compressive loading and friction wear, which lead to the formation of wear particles or debris between articulating surfaces. Despite advances in orthopaedic materials and surface treatments, the production of wear debris from any part of a joint arthroplasty implant is currently unavoidable. Implant wear debris induces host immune responses and inflammation, which causes patient pain and ultimately implant failure through progressive inflammation-mediated osteolysis and implant loosening, where the severity and rate of periprosthetic osteolysis depends on the material and physicochemical characteristics of the wear particles. Evaluating the cytotoxicity of implant wear particles is important for regulatory approved clinical application of arthroplasty implants, as is the study of cell-particle response pathways. However, the wear particles of polymeric materials commonly used for arthroplasty implants tend to float when placed in culture media, which limits their contact with cell cultures. This study reports a simple means of suspending wear particles in liquid medium using sodium carboxymethyl cellulose (NaCMC) to provide a more realistic proxy of the interaction between cells and tissues to wear particles in vivo, which are free-floating in synovial fluid within the joint cavity. Low concentrations of NaCMC dissolved in culture medium were found to be effective for suspending polymeric wear particles. Such suspensions may be used as more physiologically-relevant means for testing cellular responses to implant wear debris, as well as studying the combinative effects of shear and wear particle abrasion on cells in a dynamic culture environments such as perfused tissue-on-chip devices.

scholarly journals Resveratrol-supplemented holding or re-culture media improves viability of fresh or vitrified-warmed in vitro-derived bovine embryos

2021 ◽  
Vol 10 (14) ◽  
pp. e367101422097
Author(s):  
Arianny Rafaela Neto Silva ◽  
Thaisa Campos Marques ◽  
Elisa Caroline Silva Santos ◽  
Tiago Omar Diesel ◽  
Isabelle Matos Macedo ◽  
...  
Keyword(s):  
Culture Media ◽  
Cell Number ◽  
Time Dependent ◽  
Expansion Rate ◽  
Ros Generation ◽  
Hatching Rate ◽  
Protective Effects ◽  
Bovine Embryos ◽  
In Vitro Production

The effect of resveratrol supplementation on fresh (E1) or vitrified/warmed (E2) in vitro produced bovine embryos was investigated by evaluating the time-dependent response. After in vitro production, resveratrol (0.5 µM) was added to the incubation media and after two incubation periods with or without resveratrol, blastocysts were re-cultured for 24h. The rates of re-expansion, hatching, total cell number (TCN), apoptotic cells (ACN), reactive oxygen species (ROS) and intracellular glutathione (GSH) content were evaluated. For E1, the re-expansion rate differed at 6 and 10h within and between treatments (P<0.05), as did the re-expansion rate after 24h (P<0.01). The hatching rate increased after 10h with resveratrol (P<0.01) with differences within (P<0.05), but not between treatments after 24h of re-cultivation. At E2, hatching rate differed between treatments at 24h (P<0.01), with higher TCN in resveratrol-treated blastocysts after 10h (P<0.01). Resveratrol supplementation reduced ROS generation in E1 and E2 after 10h of incubation and increased GSH content (P<0.01). These results indicate that supplementation of holding re-cultivation medium with resveratrol for treatment of fresh or vitrified/warmed in vitro produced bovine embryos has a positive and time-dependent effect. The reduction of ROS content, the increase of GSH and the anti-apoptotic ability of resveratrol are responsible for its protective effects, allowing an extension of embryo storage time before transfer to recipients.

scholarly journals 63 IMPROVED IN VITRO DEVELOPMENT OF PORCINE EMBRYOS PRODUCED BY NUCLEAR TRANSFER, IVF AND PARTHENOGENESIS

2005 ◽  
Vol 17 (2) ◽  
pp. 181 ◽  
Author(s):  
D. Sage ◽  
P. Hassel ◽  
B. Petersen ◽  
W. Mysegades ◽  
P. Westermann ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Media ◽  
Cumulus Cells ◽  
Cell Number ◽  
Foster Mother ◽  
Chi Square ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate ◽  
Parthenogenetic Embryos

Porcine nuclear transfer (NT) is an inefficient process and it is necessary to use as many as 120 NT embryos for each foster mother to obtain small litters of live piglets. In these experiments, we evaluated the effects of culture atmosphere and medium on the development of NT embryos by monitoring blastocyst rate and cell number of Day 6 blastocysts. Age matched IVF and parthenogenetic embryos were also evaluated for comparison. For all experiments a pool of oocytes was aspirated from ovaries collected in a local abattoir. Following aspiration, oocytes were allowed to mature for 40 h in North Carolina State University (NCSU)-37 medium (supplemented with cAMP and hCG/eCG for the first 22 h). After removal of the cumulus cells, denuded oocytes with polar bodies were selected for NT, enucleated, fused with fetal fibroblasts, and sequentially activated electrically and chemically by 3 h of treatment with 6-dimethylaminopurine (6-DMAP). A second group of oocytes from the same denuded pool were maintained in TL-HEPES medium and activated in parallel with the NT group to produce parthenogenetic embryos. A third group was fertilized with frozen-thawed epididymal semen and co-cultured for ∼12 h to give IVF embryos. All three treatment groups were subdivided into a control subgroup and an experimental subgroup. In the first experiment, we compared the effects of atmosphere (20% vs. 5% oxygen) on in vitro embryonic development in NCSU-23 medium. In the second experiment, we used only the 5% oxygen concentration and compared different culture media. One subgroup was maintained in standard NCSU-23 medium and the second subgroup was cultured in a two-step system for the first 58 h in modified NCSU-23 (without glucose but supplemented with 2.0 mM lactate and 0.2 mM pyruvate), followed by addition of glucose to give a final concentration of 5.55 mM. Data were statistically analyzed by analysis of variance and chi square test. Blastocyst rate and mean cell number in all three embryo groups were improved under 5% oxygen. The most dramatic effect was observed in the NT group, in which the blastocyst rate increased significantly (P < 0.001) from 6.7% ± 5.9 (n = 279) to 19.6% ± 8.9 (n = 250) and mean cell number increased from 17.7 ± 12.1 to 25.8 ± 10.3 cells per blastocyst. With 5% oxygen there was also an increase of blastocyst rates and mean cell numbers in both IVF and parthenogenetic groups. In the second experiment, blastocyst rate for NT embryos increased significantly (P < 0.05) from 21.8% ± 7.6 (n = 242) in conventional NCSU-23 to 31.5% ± 11.0 (n = 271) in the modified system whereas there was almost no difference in the mean cell number of both groups (29.2 ± 4.3 vs. 31.5 ± 5.3). In the groups of IVF and parthenogenetic embryos no difference was found. These results indicate that both the reduced oxygen and the modified culture medium are important for pre-implantation development of porcine nuclear transfer embryos.

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