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.

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.

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.

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.

53 EFFECT OF ADDITION OF L-CARNITINE AND CONJUGATED LINOLEIC ACID DURING BOVINE EMBRYO CULTURE ON CRYOSURVIVAL, LIPID CONTENT, AND GENE EXPRESSION

2015 ◽  
Vol 27 (1) ◽  
pp. 119
Author(s):  
A. Ruiz ◽  
P. J. Hansen ◽  
J. Block
Keyword(s):  
Gene Expression ◽  
Lipid Metabolism ◽  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Lipid Content ◽  
Embryo Culture ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
Bovine Embryo

The objective was to determine the effects of addition of l-carnitine (LC) and trans-10, cis-12 conjugated linoleic acid (CLA) during bovine embryo culture on cryosurvival, lipid content, and gene expression. For all experiments, embryos were produced in vitro using abattoir-derived oocytes. Following insemination, presumptive zygotes were randomly assigned in a 2 × 2 factorial to be cultured in SOF-BE1 supplemented with or without 3.03 mM LC and 100 μM CLA until Day 7. For Exp. 1, blastocyst- and expanded-blastocyst-stage embryos (n = 777) were slow-frozen in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. There was no effect of LC on post-thaw re-expansion rates, but CLA reduced (P < 0.05) and tended (P < 0.08) to reduce re-expansion rate at 24 and 48 h, respectively (76.5 ± 2.5 v. 70.4 ± 2.5 and 79.5 ± 2.2 v. 76.0 ± 2.2, respectively). Whereas hatching rate at 72 h tended (P < 0.08) to be higher for embryos cultured with LC (67.8 ± 2.5 v. 74.4 ± 2.5), treatment with CLA reduced (P < 0.05) hatching rate at 48 h (62.3 ± 2.6 v. 54.9 ± 2.6). In Exp. 2, to determine lipid content, expanded blastocyst-stage embryos (n = 263) were harvested and stained using Nile Red. Embryos were examined for fluorescence using an epifluorescence microscope, and intensity of fluorescence per unit area was quantified using ImageJ software (NIH, Bethesda, MD, USA). There was a significant interaction (P < 0.01) between LC and CLA affecting embryo lipid content. Whereas addition of CLA during culture increased lipid, treatment with LC and the combination of LC and CLA reduced lipid (22.8 ± 1.1 v. 19.1 ± 1.1 v. 28.4 ± 1.1 v. 19.2 ± 1.2 for no additive, +LC, +CLA, and +LC and CLA, respectively). For Exp. 3, the effect of LC and CLA on the relative abundance of genes involved in lipid metabolism (ELOVL6, SCD1, SQLE, HMGCS1, CYP51A1, FDPS, FDFT1, LDLR, and SC4MOL) was determined. Pools of 5 expanded blastocyst-stage embryos from each treatment were collected across 5 replicates. The RNA was purified and synthesised into cDNA for RT-qPCR analysis. The SDHA, GAPDH, and YWAZ were used as housekeeping genes. Addition of LC during culture reduced (P < 0.05) the abundance of 4 of the 9 genes analysed (SQLE, HMGCS1, CYP51A1, and FDPS) and tended (P < 0.08) to reduce a fifth (FDFT1). In addition, there was a tendency (P < 0.08) for LC to increase the abundance of SCD1. Addition of CLA during culture had minimal effects on transcript abundance. In particular, CLA treatment reduced (P < 0.01) ELOVL6 and tended (P < 0.08) to increase SCD1. In contrast to previous studies, post-thaw cryosurvival was not significantly improved by treatment with LC or CLA. Results indicate that reduced embryo lipid content caused by LC treatment is due, in part, to an alteration in the abundance of genes involved in lipid metabolism. Further research is still necessary to determine whether in vivo survival following transfer of cryopreserved embryos can be enhanced by treatment with LC or CLA.Support was provided by USDA AFRI Grant 2010–85122–20623.

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.

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).

scholarly journals Zinc supplementation during in vitro embryo culture increases inner cell mass and total cell numbers in bovine blastocysts1

2019 ◽  
Vol 97 (12) ◽  
pp. 4946-4950 ◽  
Author(s):  
Lydia K Wooldridge ◽  
Madison E Nardi ◽  
Alan D Ealy
Keyword(s):  
Embryo Culture ◽  
Zinc Supplementation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Retention Rates ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cell Numbers

Abstract Deficiencies in current embryo culture media likely contribute to the poor blastocyst development rates and pregnancy retention rates for in vitro produced (IVP) bovine embryos. Of special concern is the lack of micronutrients in these media formulations. One micronutrient of interest is zinc, an essential trace element involved with various enzyme and transcription factor activities. The objective of this work was to describe whether zinc sulfate supplementation during in vitro embryo culture affects bovine embryo development and blastomere numbers. Either 0, 2, 20, or 40 µM zinc sulfate was supplemented to presumptive zygotes cultured in synthetic oviductal fluid containing AAs and bovine serum albumin for 8 d. None of the treatments affected cleavage rates. Percentage of blastocysts on days 7 and 8 postfertilization was not affected by supplementing 2 or 20 µM zinc but were reduced (P &lt; 0.05) with 40 µM zinc. In blastocysts harvested on day 8, inner cell mass (ICM) and total cell number were increased (P &lt; 0.05) with 2 µM zinc supplementation but not with the other zinc concentrations. Numbers of trophectoderm cells were not affected by zinc treatment. In conclusion, supplementing zinc during bovine embryo culture did not impact blastocyst development but improved ICM cell numbers. This improvement in ICM cell number may have implications for improved pregnancy retention rates after IVP embryo transfer as smaller ICM sizes are associated with poor pregnancy success in cattle.

scholarly journals LÍQUIDO FOLICULAR FRESCO OU CONGELADO NA PRODUÇÃO IN VITRO DE EMBRIÕES BOVINOS

2003 ◽  
Vol 8 (1) ◽  
Author(s):  
L.P. RAUBER ◽  
D.F. ALVES ◽  
F.D. MOZZAQUATRO ◽  
J.V. TESSMANN ◽  
M.L. BERNARDI ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Freezing Process ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Cumulus Oocyte Complex ◽  
Follicle Size ◽  
Blastocyst Rate ◽  
Bovine Oocytes

A manutenção dos complexos cumulus-oócitos (CCO) em líquido folicular (LF) antes da sua maturação, além de visar a capacitação, viabiliza o transporte até o laboratório por ser de baixo custo, de fácil aquisição e o congelamento do LF permite seu armazenamento para futura utilização. Neste experimento avaliou-se o efeito do congelamento do LF obtido de folículos de 2-8mm e de folículos >8mm, sobre a taxa de produção embrionária. Oócitos foram aspirados de folículos de 2 a 8mm de ovários provenientes de abatedouro. No grupo controle (n=295) os CCO foram maturados por 24h. Nos tratamentos GF (n=297) e GC (n=282), os CCO foram mantidos por 6h a 30ºC em LF fresco ou congelado, respectivamente, de folículos >8mm. Já no tratamento PF(n=278) e PC (n=281), os CCO foram mantidos em LF fresco ou congelado, respectivamente, de folículos de 2-8mm. Posteriormente, os CCO dos tratamentos GF, GC, PF e PC foram maturados por 18h. Não houve efeito negativo do congelamento do líquido folicular e nem do tamanho dos folículos sobre as taxas de clivagem e produção embrionária em D7 e D9 (P>0,05). No entanto, o congelamento do LF de folículos de 2 a 8mm resultou em redução da taxa de eclosão e do número de células dos blastocistos. A manutenção de oócitos bovinos por 6h a 30ºC, antes da maturação, pode ser efetuada em líquido folicular de folículos >8mm, fresco ou congelado. Fresh or frozen follicular fluid in vitro bovine embryo production Abstract In addition to the capacitation, the maintenance of cumulus-oocyte complex (COC) in follicular fluid (FF) before maturation, allows the transport to the laboratory, being a practical and less expensive media. The FF can be stored after freezing to future use. Oocytes aspirated from bovine slaughterhouse ovaries, were used to evaluate the effect of maintaining the oocytes in fresh or frozen bovine FF (from 2-8mm and >8mm follicles) on the blastocyst rate. In the control group (n=259) the COC were matured for 24h. On treatments GF (n=297) and GC (n=282) the COC were held for 6h at 30°C in fresh or frozen FF from >8mm follicles, respectively. In treatments PF (n=278) and PC (n=281) the COC were held in fresh or frozen FF from 2-8mm follicles, respectively. Later, the COC from GF, GC, PF and PC were matured for 18h. The freezing process as well as the follicle size had no effect on the cleavage, D7 or D9 blastocyst rates (P>0,05). Nevertheless, the frozen FF from 2-8mm follicles resulted in a reduced hatching rate and lower ICM cells. Fresh or frozen follicular fluid of >8mm follicles could be used for a 6h transport of bovine oocytes before maturation for 18h.

137 PYRUVATE UPTAKE DURING EARLY CLEAVAGE PREDICTS BLASTOCYST DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 216
Author(s):  
F. Guerif ◽  
P. J. McKeegan ◽  
H. J. Leese ◽  
R. G. Sturmey
Keyword(s):  
Lactate Production ◽  
Technical Difficulty ◽  
Intermediate Level ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Individual Culture ◽  
Early Embryos ◽  
Embryo Metabolism

The widely accepted picture of embryo metabolism has originated from work carried out in a few specialised laboratories due to the technical difficulty of carrying out metabolite assays on small volume samples. We have refined the noninvasive enzymatic determination of spent embryo culture medium for use in a standard fluorescence plate reader. Using this widely accessible, highly sensitive system, we have re-examined early bovine embryo metabolism. We have measured the consumption of pyruvate and glucose and formation of lactate by embryos in individual culture at all stages of preimplantation development. The consumption of pyruvate and glucose and production lactate by in vitro produced bovine embryos at the 2- to 4-cell, 8-cell, morula, and blastocyst stages was measured. Embryos were incubated individually for 24 h in 4-µL drops of medium alongside control drops. After 24 h of culture, the morphological status of each embryo was recorded and compared to the stage recorded at the beginning of individual culture. Analysis of the control and embryo samples of media was performed with a Tecan Infinite M200 spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland) using an adapted ultramicrofluorometric technique previously described (Leese and Barton, 1984). Uptake or production over the 24 h period was calculated and expressed as pmol/embryo/h. A further experiment focusing on pyruvate consumption by early embryos was carried out. On Day 2, embryos were cultured individually for pyruvate analysis. On Day 3, embryos were assigned into 3 groups (low, intermediate, and high) on the basis of their pyruvate depletion and then cultured until Day 8. Significant differences in metabolic profile (glucose, lactate, and pyruvate) were tested by Student’s t-test and one-way ANOVA followed by Fisher’s LSD test post hoc using Statview (SAS Institute Inc., Cary, NC, USA). There was no difference in glucose depletion and lactate production between embryos that progressed and those that did not at the cleavage stages. However, 2- to 4-cell-stage embryos that progressed morphologically depleted significantly more pyruvate from the medium than those that did not progress (6.19 ± 0.66 v. 3.80 ± 0.52 pmol/embryo/h, respectively; P < 0.05, n = 88). Embryos with an intermediate level (6.14 ± 0.27 pmol/embryo/h) of pyruvate uptake were associated with the highest number of cells on Day 2 (5.43 ± 0.16, n = 60) and on Day 3 (6.37 ± 0.20, n = 60) and were also associated with the highest rate of blastocyst development on Day 8 in comparison with the groups which included embryos with low and high level of pyruvate uptake (68.3% v. 13.3% and 25.0%, respectively; P < 0.05, n = 180). Embryos on Day 2 of development with an intermediate level of pyruvate uptake have between 2 and 3 times the probability of reaching the blastocyst stage in comparison with early embryos with high or low level of pyruvate uptake. Our data suggests that, at least for pyruvate consumption, there is a middle optimum range of depletion, which correlates with high viability.

Sign in / Sign up

Export Citation Format

Share Document