Replacing serum in culture medium with albumin and insulin, transferrin and selenium is the key to successful bovine embryo development in individual culture

2014 ◽  
Vol 26 (5) ◽  
pp. 717 ◽  
Author(s):  
E. Wydooghe ◽  
S. Heras ◽  
J. Dewulf ◽  
S. Piepers ◽  
E. Van den Abbeel ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Individual Factor ◽  
Group Culture ◽  
Serum Free ◽  
Individual Culture ◽  
Negative Effect ◽  
Serum Free Conditions

Individual culture of bovine embryos is usually associated with low blastocyst development. However, during preliminary experiments in our laboratory we observed high blastocyst development after individual embryo culture in a serum-free culture system. We therefore hypothesised that serum has a negative effect on embryos cultured individually whereas embryos in groups can counteract this. First, we determined whether the timing of removal of serum (during maturation or culture) had an influence on individual embryo development. The results clearly showed that removal of serum during embryo culture was the main contributing factor since high blastocyst development was observed after individual culture in synthetic oviductal fluid supplemented with bovine serum albumin (BSA) and insulin, transferrin and selenium (ITS), independent of the maturation medium. Second, we investigated whether an individual factor of the ITS supplement was essential for individual embryo development. We demonstrated that repeatable high blastocyst percentages were due to the synergistic effect of ITS. Finally, we investigated if a group-culture effect can still be observed under serum-free conditions. Group culture generated blastocysts with higher total cell numbers and less apoptosis. These data show that individual culture in serum-free conditions leads to high blastocyst development, but group culture still improves blastocyst quality.

141 BOVINE EMBRYO CULTURE IN THE MODIFIED WELL OF THE WELL SYSTEM INCREASES BLASTOCYST RATE BUT LOWERS BLASTOCYST QUALITY

2013 ◽  
Vol 25 (1) ◽  
pp. 218
Author(s):  
S. Heras ◽  
E. Wydooghe ◽  
A. Van Soom
Keyword(s):  
Embryo Culture ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Group Culture ◽  
Autocrine Factors ◽  
Individual Culture ◽  
Blastocyst Quality ◽  
Negative Effect ◽  
Blastocyst Rate

Individual culture of cattle embryos in serum-containing medium has a detrimental effect on blastocyst development. As we previously demonstrated, this effect is not caused by the individual culture itself but by some component present in serum (Heras et al. AETE Proc. 2012). The high blastocyst rates achieved with group culture in serum-containing medium can be explained by the neutralizing effect of the produced autocrine factors. Autocrine factors produced by individually cultured embryos cannot neutralize the detrimental component due to its low amount and their diffusion in the high culture volume. Using a modified well of the well (mWOW; Vajta et al. 2000 Mol. Rep. Dev.), where embryos are singly cultured in a small well, the effect of the autocrine factors produced by individual embryos can be maximized. We hypothesized that the use of mWOW for individual embryo culture in serum-containing medium, can maximize the neutralizing effect of the autocrine factors, increasing both blastocyst rate and quality. Bovine oocytes (n = 1143, 3 replicates) were matured in TCM-199 with 20% fetal bovine serum (FBS). Presumptive zygotes were cultured in groups (Gr; 25 embryos/50-µL drops), individually (Ind; 20-µL drops), or in mWOW (20-µL drops) in SOFaa with 5% FBS. Blastocyst evaluation occurred 7 and 8 days post-insemination (dpi). Hatching rate is the proportion of hatching/hatched blastocysts out of total 8-dpi blastocysts. Blastocysts were collected at 8 dpi for differential apoptotic staining, evaluating total cell number (TCN), inner cell mass (ICM) ratio, and apoptosis (ACR; Wydooghe et al. 2011 Anal. Biol.). Developmental data were analyzed using binary logistic regression; data concerning blastocyst quality were analyzed using linear mixed model analysis. Since ACR was not normally distributed, a logarithmic transformation was performed. Differences at P < 0.05 were considered significant. Regarding blastocyst development, all groups were significantly different both at 7 dpi (Gr 33.9%, Ind 10.7%, mWOW 19.6%) and at 8 dpi (Gr 37.3%, Ind 14.0%, mWOW 29.1%). Hatching rates were significantly higher in Gr (27.9%) compared to Ind (4.0%) and mWOW (4.1%). Concerning blastocyst quality, TCN was significantly higher (P < 0.001) in Gr (233.23 ± 8.39), no differences between Ind (143.77 ± 6.32) and mWOW (125.11 ± 4.30) were observed. No differences in ICM ratio were observed (Gr 0.41 ± 0.01, Ind 0.30 ± 0.02, mWOW 0.37 ± 0.02). ACR was significantly different between all groups (Gr 0.03 ± 0.002, Ind 0.04 ± 0.003, mWOW 0.08 ± 0.005). In conclusion, the mWOW had a positive effect on blastocyst quantity, increasing blastocyst rates compared to individual culture, but still lower than in group culture. Surprisingly, mWOW had a negative effect on blastocyst quality, producing the highest ACR and the lowest TCN. This negative effect can be caused by toxic products arising from the manufacture (although the wells were rigorously flushed), or by the longer manipulation time needed. This work was funded by FWO G.0210.09N and IWT g111438.

48 EFFECTS OF LIPID METABOLIC REGULATORS DURING BOVINE EMBRYO CULTURE ON BLASTOCYST DEVELOPMENT AND CRYOSURVIVAL

2014 ◽  
Vol 26 (1) ◽  
pp. 138 ◽  
Author(s):  
A. Ruiz ◽  
P. J. Hansen ◽  
J. Block
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Phenazine Ethosulfate ◽  
Metabolic Regulators ◽  
Hatching Rates

The overall objective was to determine the effects of addition of lipid metabolic regulators during embryo culture on blastocyst development and survival following cryopreservation. For Experiment 1, embryos produced in vitro were cultured in 5% (vol/vol) oxygen in SOF-bovine embryo 1 (SOF-BE1) medium supplemented with or without 100 μM trans-10,cis-12 conjugated linoleic acid (CLA) and 0.3 μM phenazine ethosulfate (PES). Treatment with CLA began at the initiation of culture, whereas treatment with PES began at Day 3 after insemination. At Day 7 after insemination, the proportion of oocytes that developed to the blastocyst and advanced blastocyst (expanded, hatching, or hatched) stages was recorded. Blastocysts and expanded blastocyst-stage embryos were harvested and slow frozen in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h in SOF-BE1 medium containing 10% (vol/vol) fetal bovine serum and 50 μM dithiothreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. Addition of CLA had no effect on embryo development, whereas PES reduced (P < 0.01) development to the blastocyst (26.0 ± 0.8 v. 22.1 ± 0.8%) and advanced blastocyst (19.2 ± 0.9 v. 14.4 ± 0.9%) stages. Blastocysts cultured in the presence of CLA had higher (P < 0.05) re-expansion rates at 24, 48, and 72 h (50.8 ± 3.7 v. 65.7 ± 3.7%, 57.2 ± 4.0 v. 72.0 ± 4.05%, and 57.2 ± 4.0 v. 72.0 ± 4.0%, respectively). Addition of CLA tended (P < 0.07) to increase the hatching rate at 24 h and did increase (P < 0.05) the hatching rate at 48 h (12.4 ± 1.3 v. 16.2 ± 1.3% and 39.0 ± 3.2 v. 50.0 ± 3.2%, respectively). Treatment with PES had no effect on re-expansion rates but reduced (P < 0.05) hatching rates at 24 and 48 h (18.2 ± 1.3 v. 10.3 ± 1.3 and 50.2 ± 3.2 v. 38.8 ± 3.2%, respectively). There was no interaction between CLA and PES affecting embryo development or cryosurvival. For Experiment 2, embryos were produced in vitro as in Experiment 1 and cultured in SOF-BE1 medium with or without 3.03 mM L-carnitine (LC) and 10 μM forskolin (FK). Treatment with LC began at the initiation of culture and treatment with FK began at Day 6. All other methods were as described for Experiment 1. Addition of LC did not affect development to the blastocyst stage but reduced (P < 0.05) development to the advanced blastocyst stage (21.0 ± 1.2 v. 17.1 ± 1.2%). Treatment with FK had no effect on embryo development to the blastocyst or advanced blastocyst stages. Blastocysts cultured in the presence of LC had increased (P < 0.05) re-expansion rates at 24, 48, and 72 h (60.2 ± 2.0 v. 78.0 ± 2.0%, 62.9 ± 1.2 v. 83.3 ± 1.2%, and 63.0 ± 2.4 v. 82.8 ± 2.4%, respectively) and hatching rates at 48 and 72 h (48.6 ± 4.3 v. 64.1 ± 4.3% and 59.6 ± 3.0 v. 78.5 ± 3.0%, respectively). There was no effect of FK on cryosurvival and no interaction between LC and FK affecting embryo development or cryosurvival. In conclusion, blastocyst yield was not improved by any of the lipid metabolic regulators tested. Cryosurvival was enhanced by addition of CLA and LC but FK reduced survival following freezing. There were no additive effects of either CLA and PES or LC and FK for blastocyst yield or cryosurvival.Support was provided by USDA AFRI Grant 2010-85122-20623.

scholarly journals Individual commitment to a group effect: strengths and weaknesses of bovine embryo group culture

Reproduction ◽  
2014 ◽  
Vol 148 (5) ◽  
pp. 519-529 ◽  
Author(s):  
Eline Wydooghe ◽  
Leen Vandaele ◽  
Sofie Piepers ◽  
Jeroen Dewulf ◽  
Etienne Van den Abbeel ◽  
...  
Keyword(s):  
Classical Group ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Group Culture ◽  
Individual Culture ◽  
Better Than

Recently, new culture devices such as Corral and Primo Vision dishes have been designed for the culture of human embryos to allow the combination of group culture plus follow-up of individual embryos. Bovine inseminated oocytes were allocated to Primo Vision dishes, Corral dishes, individual culture or classical group culture. Blastocyst development in Primo Vision dishes was similar to classical group culture (34.3 and 39.0% respectively), and better than Corral dishes or individual culture (28.9 and 28.5% respectively). In Primo Vision dishes, a higher number of ‘slow’ embryos developed to the blastocyst stage compared with their individually cultured counterparts, while no differences were observed for ‘fast’ embryos. ‘Slow’ embryos in a ‘standard drop’ had a higher chance of becoming a blastocyst compared with individual culture (OR: 2.3), whereas blastulation of ‘fast’ embryos was less efficient in a ‘delayed drop’ than in individual culture (OR: 0.3). The number of non-cleaved embryos in Primo Vision dishes did not negatively influence blastocyst development. Likewise, removing non-cleaved embryos (NC removed) and regrouping the cleaved embryos afterwards (ReGR) did not affect blastocyst development and quality compared with group culture in Primo Vision dishes (CTRL, 31.6%, NC removed, 29.3% and ReGR, 29.6%). The experiments revealed that group culture of bovine embryos in Primo Vision dishes is superior to individual culture, primarily because of the higher blastocyst rate achieved by slow embryos. Non-cleaved or arrested embryos do not hamper the ability of co-cultured bovine embryos to reach the blastocyst stage in group culture.

P–227 Fatty acid regulation of Nrf2/Keap1 pathway during mouse preimplantation embryo development

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G Dionne ◽  
A J Watson ◽  
D H Betts ◽  
B A Rafea
Keyword(s):  
Oleic Acid ◽  
Fatty Acid ◽  
Palmitic Acid ◽  
Embryo Development ◽  
Embryo Culture ◽  
Preimplantation Embryo ◽  
Culture Media ◽  
Control Group ◽  
Blastocyst Development ◽  
Preimplantation Embryo Development

Abstract Study question Our objective is determining whether supplementing embryo culture media with palmitic acid and/or oleic acid impacts Nrf2/Keap1 antioxidant response pathways during preimplantation mouse embryo development. Summary answer Supplementation of embryo culture media with palmitic acid increases cellular Nrf2 levels per embryo after 48-hour culture, while oleic acid reverses this effect. What is known already Obese women experience higher incidence of infertility than women with healthy BMIs. The obese reproductive tract environment supporting preimplantation embryo development is likely to include enhanced free fatty acid (FFA) levels and increased accumulation of reactive oxygen species. Exposure to palmitic acid (PA) in vitro significantly impairs mouse embryo development while increasing ER stress mRNAs. Oleic acid (OA) reverses these effects. To further define effects of FFA exposure, we are characterizing the influence of FFAs on the Nrf2–Keap1 pathway and its downstream antioxidant defense systems. We hypothesize that PA treatment induces Nrf2-Keap1 activity, while OA treatment alleviates pathway activity. Study design, size, duration Female CD–1 mice (4–6 weeks) were super-ovulated via intraperitoneal injections of PMSG, followed 48 hours later by hCG. Female mice were mated with male CD–1 mice (6–8 months) overnight. Females were euthanized using CO2 and two-cell embryos were collected by flushing oviducts. Two-cell embryos were placed into KSOMaa-based treatment groups: 1) BSA (control); 2) 100µM PA; 3) 100µM OA; 4) 100µM PA+OA, and cultured for 48 hours (37 °C; 5% O2, 5% CO2, 90% N2). Participants/materials, setting, methods After 48-hour embryo culture, developmental stages of all mouse embryos were recorded. Immunofluorescence analysis of Nrf2 and Keap1 localization was performed for embryo treatments (BSA, 100µM PA, 100µM OA & 100µM PA+OA) using rabbit polyclonal anti-Nrf2 antibody, with Rhodamine-Phalloidin and DAPI staining. Embryos were imaged using confocal microscopy and Nrf2-positive cells were counted using ImageJ. Nrf2 and Keap1 mRNA abundances were assessed after culture in each treatment condition using RT-qPCR and the delta-delta Ct method. Main results and the role of chance Inclusion of 100µM PA in embryo culture significantly decreased blastocyst development frequency from 70.06±16.38% in the BSA (control) group to 11.61±8.19% in the PA-treated group (p &lt; 0.0001). Embryo culture with 100µM OA and 100µM PA+OA co-treatment did not significantly impair blastocyst development (OA: 61.59±8.07%, p = 0.4053; PA+OA: 63.53±7.63%, p = 0.6204). Embryo culture with PA treatment significantly increased the mean percentage of Nrf2-positive cells to 56.83±30.49% compared with 21.22±15.63% in the control group (p &lt; 0.0001). Conversely, 100µM OA and 100µM PA+OA treatments did not significantly affect Nrf2-positive cell frequencies compared with the control group (OA: 33.28±21.83%, p = 0.1825; PA+OA: 34.84±12.66%, p = 0.0691). Immunofluorescence results show that treating embryos with 100µM PA for 48 hours results in increased levels of cellular Nrf2, while combining 100µM PA with 100µM OA reversed these effects. Preliminary qPCR analysis showed no significant differences in Nrf2 or Keap1 relative transcript abundance between any embryo treatment groups. Nrf2 and Keap1 mRNA levels were both higher after embryo culture with 100µM OA than all other culture groups (p = 0.6268; p = 0.3201). Notably, Keap1 relative transcript levels dropped to undetectable levels after culture with 100µM PA, which suggests an increase in Nrf2 activation.Limitations, reasons for caution: While immunofluorescence localization of Nrf2/Keap1 provides insight into how the proteins behave during preimplantation embryo development, confocal images cannot determine protein-protein interactions or activity levels. Similarly, transcript information from RT-qPCR analysis only provides information about Nrf2 and Keap1 at the transcript level. Nrf2 activity will be assessed via downstream targets. Wider implications of the findings: The Nrf2–Keap1 pathway coordinates numerous cellular defence mechanisms, and is implicated in various diseases, including cancer. Establishing an impact of free fatty acid exposure on Nrf2–Keap1 during preimplantation embryo development will provide valuable information regarding the effects of maternal obesity on outcomes for embryos produced from these patients. Trial registration number Not applicable

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

2014 ◽  
Vol 26 (1) ◽  
pp. 154 ◽  
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.

1 AUTOCRINE COMMUNICATION BETWEEN BOVINE EMBRYOS CULTURED IN Primo Vision DISHES® OUTWEIGHS POSSIBLE NEGATIVE INFLUENCES OF BAD EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 115
Author(s):  
E. Wydooghe ◽  
L. Vandaele ◽  
A. Van Soom
Keyword(s):  
Developmental Stage ◽  
Odds Ratio ◽  
Fixed Effects ◽  
Binary Logistic Regression Model ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Group Culture ◽  
Individual Culture

Group culture is being used extensively for mammalian embryos, but has not been adopted so far in human embryo culture. Some doubts about its possible benefits remain because it has been hypothesised that bad quality embryos might have a negative effect on other embryos. New group culture devices have been designed allowing individual follow-up of embryos, such as Primo Vision dishes® (well of-the-well for 10 embryos in group culture; Cryo-Innovation, Budapest, Hungary). By using Primo Vision dishes®, we investigated the influence of the developmental stage of the neighbours and co-cultured embryos on the outcome at 192 h post-insemination (hpi) of a particular embryo compared with its individually cultured counterparts. Bovine presumed zygotes (n = 789; 4 replicates) produced in vitro were randomly allocated to Primo Vision dishes® or individual culture in SOF supplemented with 0.4% BSA and insulin, transferrin, selenium (Wydooghe et al. 2013 Reprod. Fertil. Dev. Epub). Cleavage rate was checked at 45 hpi: 5- to 8-cell embryos were classified as fast embryos and 2- to 4-cell embryos as slow embryos. Blastocyst development was evaluated at 192 hpi. Moreover, to evaluate which embryos benefited most from being in group (fast or slow embryos), we looked retrospectively at the influence of the developmental stage of the neighbours and the co-cultured embryos on blastocyst development. This was done separately for slow and fast embryos compared with their individually cultured counterparts. Statistical analysis was done using a binary logistic regression model, with group and replicate as fixed effects. Blastocyst development in Primo Vision dishes® was significantly better than individual culture (39.0% v. 28.5%). This beneficial outcome was mainly caused by a higher blastocyst development of slow embryos. A markedly higher percentage of slow embryos developed into a blastocyst at 192 hpi if they were surrounded by many embryos that also developed into a blastocyst, compared with individually cultured slow embryos (odds ratio: 3.0). In this study, we showed that embryos that were not cleaved at 45 hpi did not negatively affect the potential of their neighbours to become a blastocyst at 192 hpi, regardless of whether the embryo in question was a fast or a slow embryo. However, when fast embryos were in a less than favourable environment, meaning that less than 30% of their co-cultured embryos reached the blastocyst stage, blastocyst development was compromised compared with individual culture of fast embryos (odds ratio: 0.3). From our results, we clearly show that Primo Vision dishes® can combine the benefits of group culture (autocrine communication) and individual culture (individual follow-up). Taking fast embryos out of the Primo Vision dish® for further individual culture while slow embryos remain in group is a possible approach to increase total blastocyst rates.

332 EFFECT OF DIFFERENT PIG OOCYTE ACTIVATION PROTOCOLS ON EMBRYO DEVELOPMENT IN SOF AND NCSU-23

2007 ◽  
Vol 19 (1) ◽  
pp. 281 ◽  
Author(s):  
I. Lagutina ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Cell Number ◽  
Additional Treatment ◽  
Oocyte Activation ◽  
Blastocyst Development ◽  
Chi Square ◽  
Parthenogenetic Embryo ◽  
Pig Oocyte

Several factors affect nuclear transfer success. These include efficient parthenogenetic activation and embryo culture medium that should efficiently support pre-implantation development of good quality blastocysts. We investigated pig oocyte activation and embryo development in SOFaa in response to ionomycin (Io = 5 µM Io for 4 min; Io° = 15 µM Io for 20 min) and electric impulse (EL; one 30-µs pulse of DC 1.5 kV cm−1 in the presence of 50 µM Ca) in combination with 2 mM 6-DMAP or 10 µg mL−1 cycloheximide (CHX) +5 µg mL−1 cytochalasin B (CB) for 4 h. In addition, we studied the effect of elevated (1 mM) (Cheong et al. 2002 Mol. Reprod. Dev. 61, 488) in comparison with 50 µM Ca during EL activation on embryo development in SOFaa and NCSUaa-23. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon®, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng mL−1 long-EGF, 100 ng mL−1 long-IGF1, 5 ng mL−1 bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. The rates of cleavage, blastocyst formation (BL) and BL cell number on Day 7 (BL-D7) were recorded. All experiments were done with 3 replicates. The data were compared by chi-square test. There was no difference in the ability of Io (all groups) and EL + CB activated oocytes to cleave, whereas the additional treatment of EL-activated oocytes with DMAP and CHX + CB significantly increased cleavage. Io activation resulted in poor blastocyst development in comparison with all EL-activated groups (see Table 1). When calcium levels were elevated during EL activation, significantly more embryos developed in SOFaa (35.6%, n = 191 vs. 26%, n = 192; P &lt; 0.05), but no differences were observed with culture in NCSUaa-23 (about 56%). The BL rate was significantly higher in NCSUaa-23 vs. SOFaa (55.9%, n = 68 vs. 34.8%, n = 69, respectively); however, the BL total cell number was significantly higher in SOFaa (58 ± 18, n = 40 vs. 86 ± 35, n = 56, respectively; P &lt; 0.05). In conclusion, we have found that SOFaa and NCSUaa-23 differ in ability to support pig parthenogenetic embryo development. EL activation combined with elevated Ca significantly increased the embryo developmental capacity in SOFaa but not in NCSUaa-23. NCSUaa-23 was more efficient for embryo culture, whereas SOF produced BLs of higher quality. Table 1.Effect of activation protocol on the development of pig parthenogenetic embryos in SOFaa This work was supported by grants ISS-CS11 and Fondazione Cariplo.

188 THE IMPACT OF OIL SOURCE AND TYPE OF GLUTAMINE ON MEDIA EQUILIBRATION TIMES AND EMBRYO DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 210
Author(s):  
D. W. Linck ◽  
D. K. Gardner
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Cell Numbers ◽  
Oil Source ◽  
The Impact

Recently, there has been much debate involving the time necessary to effectively equilibrate an embryo culture system, i.e. dishes, media, and oil. Glutamine present in the culture medium spontaneously deaminates at 37�C to release embryo-toxic ammonium. Additionally, the source of the oil overlay can impact the culture environment. A sub-optimal oil overlay, combined with free glutamine (Gln), could effectively become embryo-toxic over a short time period (&lt;48 h). Therefore, the aim of this study was to determine how times of media equilibration, using various combinations of oil source and type of Gln, affect embryo development. Zygotes were collected from 4-week-old CF1 outbred female mice following superovulation and mating. Embryos were cultured in groups of 10 in 20-�L drops of medium G1.2. Initially, embryos were cultured in one of 4 treatment media. In this 2 � 2 factorial design, the culture medium was pre-equilibrated 18 h prior to embryo retrieval and contained free Gln or the heat-stable dipeptide alanyl-glutamine (AlaGln), combined with an oil source of either Sigma mineral oil (Sigma-Aldrich Corp., St Louis, MO, USA) or Ovoil paraffin oil (Vitrolife, Inc., Englewood, CO, USA). The initial study was then repeated using only the best and worst case groups to determine the effect of incubation time as a variable (either 2 h or 18 h). Blastocyst development and total cell numbers were analyzed after 72 h of culture, and differences between treatments were assessed using Fisher&apos;s exact test and Student&apos;s t-test. After 18 h of pre-equilibration (n e 300 embryos/treatment), blastocyst development in Ovoil + AlaGln (38.6%) was significantly greater when compared to: Ovoil + Gln: 25.5% (P &lt; 0.01), Sigma + AlaGln: 12.8% (P &lt; 0.01), and Sigma + Gln: 11.9% (P &lt; 0.01). Additionally, the total cell numbers in comparison to Ovoil + AlaGln (44.6 � 10) were significantly decreased: 35.5 � 7 (P &lt; 0.001), 34.9 � 9 (P &lt; 0.001), and 29.9 � 9 (P &lt; 0.001), respectively. In the second experiment, blastocyst development and total cell number between Ovoil + AlaGln (n = 224) and Sigma + Gln (n = 264) after 18 h of pre-equilibration were: 40.4% vs. 9.9% (P &lt; 0.01) and 46.6 � 9 vs. 29.4 � 9 P &lt; 0.001), respectively. However, after 2 h of pre-equilibration, the results between Ovoil + AlaGln (n = 260) and Sigma + Gln (n = 284) were: 42.3% vs. 18.3% (P &lt; 0.01) and 46.9 � 10 vs. 33.6 � 6 (P &lt; 0.001), respectively. Therefore, when comparing blastocyst development and total cell number between pre-equilibration times (2 h vs. 18 h), the Ovoil + AlaGln group, 42.3% vs. 40.4% and 46.9 � 10 vs. 46.6 � 9, showed no significant differences, respectively. In contrast, the Sigma + Gln group produced significant differences for both blastocyst development, 18.3% vs. 9.9% (P &lt; 0.01), and total cell number, 33.6 � 6 vs. 29.4 � 9 (P &lt; 0.05), between pre-equilibration times (2 h vs. 18 h), respectively. Data presented confirm the need for an alternative source of glutamine in embryo culture media. The data also indicate that the source of oil has a profound effect on the experimental outcome. Using the appropriate oil and form of Gln means that media can be safely equilibrated for 18 h.

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