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.

Modulation of lipid metabolism through multiple pathways during oocyte maturation and embryo culture in bovine

Zygote ◽  
2021 ◽  
pp. 1-9
Author(s):  
Clara Slade Oliveira ◽  
Viviane Luzia da Silva Feuchard ◽  
Sheila Costa de Souza Marques ◽  
Naiara Zoccal Saraiva
Keyword(s):  
Lipid Metabolism ◽  
Lipid Content ◽  
Embryo Culture ◽  
Lipid Accumulation ◽  
Time Course ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Multiple Pathway ◽  
Developmental Rates

Abstract Lipid accumulation occurs in cultured embryos and is associated with reduced cryotolerance. Here we report the use of a multiple pathway lipid modulator cocktail (l-carnitine, linoleic acid and forskolin) to improve cryosurvival. First, we stained oocytes and embryos with Oil Red to examine the time course of lipid accumulation during in vitro fertilization (IVF) and embryo culture. Then we evaluated the effects of the lipid modulators cocktail on lipid content, developmental rates and survival after vitrification. In our conditions, lipid accumulation was detected (P < 0.05) at the end of in vitro maturation (IVM) and after 4 days of embryo culture (D4-D5). In experiment 1, we used lipid modulator cocktail during IVM. Reduced (P < 0.05) lipid accumulation was detected in oocytes (Control: 49.9 ± 1.6, Lip. Mod. IVM: 45.0 ± 1.8) but no changes were present at blastocyst stage (Control: 62.4 ± 2.6, Lip. Mod. IVM: 66.8 ± 2.7). Treated oocytes presented decreased (P < 0.05) blastocyst rates and lower (P < 0.05) re-expansion after vitrification. In experiment 2, lipid modulators cocktail was used during embryo culture (from D4–D7 or D6–D7). Treatment had an effect on lipid metabolism, as lipid content was increased (P < 0.05) in D7 blastocysts in treated groups (Control: 52.7 ± 3.1a, D4: 65.9 ± 2.6b, D6: 78.1 ± 2.7b). However, no effect was present for cleavage, blastocyst and cryosurvival rates. No difference was detected in mean cell number comparing the three groups (Control: 78.9 ± 9.6, D4: 82.6 ± 16.5, D6: 68.3 ± 7.8), but apoptosis rate was increased (P < 0.05) in vitrified-warmed blastocysts from treated groups (Control: 14.77*, D4: 22.28, D6: 22.22). We concluded that the combined use of lipid modulators was efficient to promote changes in lipid content of oocytes and embryos in bovine, but those changes did not reflect positively on embryo development or cryosurvival.

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 The effects of conjugated linoleic acid isomers cis-9,trans-11 and trans-10,cis-12 on in vitro bovine embryo production and cryopreservation

2014 ◽  
Vol 97 (10) ◽  
pp. 6164-6176 ◽  
Author(s):  
V.A. Absalón-Medina ◽  
S.J. Bedford-Guaus ◽  
R.O. Gilbert ◽  
L.C. Siqueira ◽  
G. Esposito ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Bovine Embryo ◽  
Embryo Production

34 EFFECTS OF L-CARNITINE AND trans-10,cis-12 CONJUGATED LINOLEIC ACID SUPPLEMENTATION DURING MATURATION ON DEVELOPMENT AND CRYOTOLERANCE OF BOVINE EMBRYOS PRODUCED IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 147
Author(s):  
J. Block ◽  
A. M. Zolini ◽  
E. Carrascal-Triana ◽  
A. Ruiz ◽  
P. J. Hansen ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Maturation Medium ◽  
Fetal Bovine ◽  
Hatching Rates

The objective of the present study was to determine the effect of supplementation of maturation media with L-carnitine and trans-10,cis-12 conjugated linoleic acid (CLA) on embryo development and survival following cryopreservation. Immature bovine cumulus-oocyte complexes (n = 1796) were harvested from abattoir-derived ovaries and randomly assigned in a 2 × 2 factorial design to be matured in maturation medium [TCM-199 with Earle salts supplemented with 10% (vol/vol) bovine steer serum, 2 μg mL–1 oestradiol 17-β, 20 μg mL–1 bovine FSH, 22 μg mL–1 sodium pyruvate, 50 μg mL–1 gentamicin sulfate, and 1 mM glutamax®] supplemented with or without 100 mM CLA and with or without 3.03 mM L-carnitine for 22 to 24 h at 38.5°C in a humidified atmosphere of 5% CO2. The proportion of oocytes that cleaved was determined on Day 3 after insemination, and the proportion of oocytes developing to the blastocyst and advanced blastocysts stages (expanded, hatching, and hatched) was assessed on Day 7. Blastocyst and expanded blastocyst stage embryos (n = 270) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum and 50 μM dithiothreitol. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 5 times. There was no effect of supplementation of maturation medium with either CLA or L-carnitine on the proportion of oocytes that cleaved at Day 3 or that developed to the blastocyst and advanced blastocyst stages at Day 7 after insemination. There was no interaction between CLA and L-carnitine affecting cleavage rate or embryo development. Supplementation of maturation medium with L-carnitine did not affect post-thaw re-expansion or hatching rates. In contrast, treatment with CLA during maturation reduced (P < 0.05) post-thaw re-expansion (24 h: 75.2 ± 3.8% v. 60.3 ± 4.1%; 48 h: 82.0 ± 3.4% v. 64.9 ± 4.0%; 72 h: 78.9 ± 3.6% v. 65.9 ± 4.0%, respectively) and hatching (24 h: 33.7 ± 4.2% v. 23.5 ± 3.6%; 48 h: 61.1 ± 4.3% v. 44.0 ± 4.2%; 72 h: 62.6 ± 4.3% v. 50.2 ± 4.2%, respectively) rates at all time points. There was no interaction between CLA and L-carnitine affecting post-thaw viability. In conclusion, supplementation of maturation medium with L-carnitine did not affect embryo development or post-thaw viability. Although addition of CLA during maturation did not affect embryo development, post-thaw cryotolerance was reduced following CLA supplementation. There was no beneficial effect of supplementing maturation medium with both CLA and L-carnitine on embryo development or post-thaw cryosurvival.

The effect of bovine embryo culture without proteins supplements until day 4 on transcription level of hyaluronan synthases, receptors and mtDNA content

Zygote ◽  
2009 ◽  
Vol 18 (2) ◽  
pp. 121-129 ◽  
Author(s):  
A.T. Palasz ◽  
P. Beltrán Breña ◽  
J. De la Fuente ◽  
A. Gutiérrez-Adán
Keyword(s):  
Embryo Culture ◽  
Receptor Gene ◽  
Blastocyst Stage ◽  
Low Molecular Weight ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Hyaluronan Synthases ◽  
Copy Numbers ◽  
Surface Active

SummaryThe effect of bovine embryo culture on a flat surface, (without a surface-active compound) on the level of mRNA expression of hyaluronan (HA) synthases (Has1, Has2 and Has3), Ha receptors RHAMM and C44 receptors was evaluated by mitochondrial DNA concentration andin vitrodevelopment. Cultures were evaluated up to 96 h post-insemination (hpi) using SOFaa medium. Of the three Has isoforms, Has2 expression only increased in the bovine serum albumin (BSA)-only supplemented groups regardless of time of BSA addition. Expression of RHAMM receptors was highly dependent on the addition of HA, irrespective of the presence of BSA in the medium. In contrast, expression of the CD44 receptor gene was not affected by any treatment. The cleavage rates and number of embryos that developed to ≤8-cell stage by day 4 were not affected by lack of BSA in the medium, but increased numbers of blastocysts developed in medium supplemented with BSA from days 1 or 4 with or without HA than in medium that had HA only. Addition of both HA and BSA at day 4 increased mtDNA copy numbers at the blastocyst stage. Data suggest that the addition of BSA and/or HA at 96 hpi increased expression ofRHAMMandHas2genes, but notCD44,Has1orHas3genes. Higher expression levels of Has2 than Has1 and the three isoforms indicate that high- rather than low-molecular-weight HA should be used for preimplantation bovine embryo culture.

94 EFFICIENT BOVINE EMBRYO SPLITTING FOR GENE EXPRESSION AND IN VIVO DEVELOPMENT STUDIES

2014 ◽  
Vol 26 (1) ◽  
pp. 161
Author(s):  
A. Velasquez ◽  
D. Veraguas ◽  
F. O. Castro ◽  
J. F. Cox ◽  
L. l. Rodriguez-Alvarez
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Blastocyst Stage ◽  
Embryo Morphology ◽  
Bovine Embryo ◽  
Gene Markers ◽  
Developmental Potential

It is known that embryos produced in vitro are less competent than their in vivo-derived counterparts. When embryos are produced or manipulated in vitro, their developmental potential decreases significantly, which impinges upon the production of viable offspring. In bovines, embryos that will be transferred to a surrogate mother are selected at the blastocysts stage using noninvasive methods, such as their morphological features. However, many of those embryos are not able to implant or to maintain a normal pregnancy because embryo morphology does not reflect its developmental potential and a correct gene expression pattern that support a normal development. It seems that the ideal method for embryo selection would be based on the screening of gene markers that correlate with successful pregnancy after embryo transfer. In that sense, we have proposed an approach to characterise gene expression pattern of early (Day 7) bovine blastocysts and to correlate this gene expression with further developmental potential in vivo, i.e. upon elongation until Day 17. For that, it was established an efficient method to produce identical and viable hemi-embryos by splitting IVF bovine blastocysts in order to set the expression profile of certain genes in one hemi-embryo at blastocyst stage, while the counterpart embryo elongates in vivo for 10 days. A total of 129 blastocysts were split. Six groups of blastocysts were used for splitting and the results compared: 1) Day-7 early blastocysts (n = 20); 2) Day-7 expanded blastocysts (n = 25); 3) Day-7 hatched blastocysts (n = 17); 4) Day-8 early blastocysts (n = 10); 5) Day-8 expanded blastocysts (n = 12); and 6) Day-8 hatched blastocysts (n = 45). Hemi-embryos derived from day-8 grade I and well expanded blastocysts had the greatest survival rate, in vitro re-expansion (67.7%; P < 0.05) and both hemi-embryos conserved a normal morphology with a total cell number over 80 after 6 h in culture. Also both hemi-embryos at blastocyst stage showed homogeneous expression pattern of the genes OCT4, SOX2, NANOG, CDX2, ACTB, and GAPDH (P < 0.05). Finally, the in vivo survival of hemi-embryos was assessed and compared with nonsplit embryos (control) by transferring to recipient cow and collecting at Day 17 of development. For this, hemi-embryos derived from Day-8 hatched blastocyst were used. From 14 transferred hemi-embryos, 5 (35.7%) were collected, and 9 elongated from 17 controls were recovered (52.9%). Also the elongation rate was significantly lower in hemi-embryos than in control; the length of hemi-embryos had a range between 1 and 5 cm, whereas 60% of the control embryos were longer than 10 cm. Our results provide an initial approach to study the correlation among the gene expression characteristics of early bovine embryos with their further development. However, it seems that embryo splitting hampers their elongation in vivo. It might be possible that the development of split embryos is retarded because of manipulation. This work was partially supported by Fondecyt grant no. 11100082 from the Ministry of Education of Chile.

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