33 POST-THAW VIABILITY OF BOVINE EMBRYOS PRODUCED IN VITRO FOLLOWING TREATMENT WITH ASCORBIC ACID, DITHIOTHREITOL, AND CASPASE-3 INHIBITOR DURING CRYOPRESERVATION

2016 ◽  
Vol 28 (2) ◽  
pp. 146
Author(s):  
E. L. Carrascal-Triana ◽  
A. M. Zolini ◽  
A. Ruiz ◽  
J. M. Penitente-Filho ◽  
C. A. A. Torres ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Caspase 3 ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Freezing Medium ◽  
Fetal Bovine ◽  
Effect Of Treatment ◽  
The Mean ◽  
Hatching Rates

The aim of the present experiments was to determine whether treatment with the antioxidants ascorbic acid and dithiothreitol, or an inhibitor of caspase-3, Z-DEVD-FMK, during cryopreservation could improve the cryotolerance of bovine embryos produced in vitro. For all experiments, bovine embryos were produced in vitro using abattoir-derived ovaries. At Day 7 after insemination, blastocyst and expanded blastocyst stage embryos were harvested and subjected to controlled-rate freezing following equilibration for 8 to 10 min in freezing medium [Hepes-TALP (Parrish et al. 1986) plus 1.5 M ethylene glycol and 0.5 M sucrose] with treatments as described below. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. For Experiment 1, embryos (n = 578) were equilibrated in freezing medium containing 0, 0.1, 0.3, or 0.5 mM ascorbic acid. For Experiment 2, embryos (n = 243) were equilibrated in freezing medium containing 0, 50, 100, or 200 μM dithiothreitol. For Experiment 3, embryos (n = 227) were equilibrated in freezing medium containing 0, 50, 100, or 200 μM Z-DEVD-FMK. Embryos frozen in freezing medium containing ascorbic acid had increased (P < 0.05) re-expansion and hatching rates at 24, 48, and 72 h compared with embryos not treated with ascorbic acid (Table 1). The optimal concentration of ascorbic acid for post-thaw cryosurvival was 0.1 mM. In particular, embryos treated with 0.1 mM ascorbic acid during cryopreservation had increased (P < 0.05) re-expansion rates at 24, 48, and 72 h, as well as hatching rates at 48 and 72 h, compared with control-treated embryos (Table 1). There was no effect of treatment with dithiothreitol or Z-DEVD-FMK on re-expansion or hatching rates at 24, 48, or 72 h after thaw. In conclusion, addition of ascorbic acid to freezing medium improves the cryosurvival of bovine embryos produced in vitro. Further research is necessary to determine whether treatment with ascorbic acid can increase pregnancy rates. Table 1.Effect of ascorbic acid during cryopreservation of bovine embryos (%, means ± standard error of the mean)

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.

Effect of protein supplementation on development to the hatching and hatched blastocyst stages of cat IVF embryos

2002 ◽  
Vol 14 (5) ◽  
pp. 291 ◽  
Author(s):  
N. W. Kurniani Karja ◽  
Takeshige Otoi ◽  
Masako Murakami ◽  
Minori Yuge ◽  
Mokhamad Fahrudin ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Culture Medium ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Protein Supplementation ◽  
Vitro Fertilization ◽  
Fetal Bovine ◽  
The Mean

The effects of protein supplementation in culture medium on development to the hatching and hatched blastocyst stages of cat in vitro-fertilized embryos were investigated. In the first experiment, presumptive zygotes derived from in vitro maturation and in vitro fertilization (IVF) were cultured in modified Earle's balanced salt solution (MK-1) supplemented with 0.4% bovine serum albumin (BSA) or 5% fetal bovine serum (FBS) for 9 days. There were no significant differences between the BSA and FBS groups with respect to the proportion of cleavage and development to the morula and blastocyst stages of zygotes. However, the presence of FBS in the medium enhanced development to the hatching blastocyst stage of zygotes compared with the BSA group (31.4% v. 7.8%). Moreover, 2.9% of zygotes cultured with FBS developed to the hatched blastocyst stage. The mean cell number of blastocysts derived from zygotes cultured with FBS was significantly higher (P&lt;0.01) than that from zygotes cultured with BSA (136.6 v.101.5). In the second experiment, embryos at the morula or blastocyst stage, which were produced by culturing in MK-1 supplemented with 0.4% BSA after IVF, were subsequently cultured in MK-1 with 0.4% BSA or 5% FBS. Significantly more morulae developed to the blastocyst (P&lt;0.05) and hatching blastocyst stages (P&lt;0.01) in the FBS group than in the BSA group (71.5% and 53.6% v. 44.9% and 6.0%, respectively). Although none of the morulae cultured with BSA developed to the hatched blastocyst stage, 11.5% of morulae cultured with FBS developed to the hatched blastocyst stage. Moreover, the proportion of development to the hatching blastocyst stage of blastocysts was significantly higher (P&lt;0.01) in the FBS group than in the BSA group (68.7% v. 9.8%). None of the blastocysts cultured with BSA developed to the hatched blastocyst stage, whereas 7.3% of blastocysts cultured with FBS developed to the hatched blastocyst stage. The results of the present study indicate that supplementation with FBS at different stages of early embryo development promotes development to the hatching and hatched blastocyst stages of cat IVF embryos.

206 Effect of L-Ascorbic Acid, Polydatin, and In-Straw Rehydration of Slow-Frozen In Vitro-Produced Bovine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 243
Author(s):  
C. M. Dinndorf ◽  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
L. F. Campos-Chillon
Keyword(s):  
Reactive Oxygen Species ◽  
Ascorbic Acid ◽  
Oxygen Species ◽  
Bovine Embryos ◽  
Freezing Medium ◽  
High Lipid ◽  
Standard Procedures ◽  
Frozen Embryos ◽  
Main Effect

In vitro-produced (IVP) bovine embryos have poor cryotolerance due to high lipid content and reactive oxygen species levels that hinder post-thaw survival. We hypothesised that in-straw rehydration of slow-frozen embryos with sucrose and the addition of the antioxidant polydatin and l-ascorbic acid would increase post-thaw survival. The IVP embryos (n = 116) were generated in 7 replicates by aspirating oocytes from 2- to 8-mm follicles of abattoir ovaries, matured for 23 h, fertilized with semen from 1 of 3 bulls using standard procedures, and cultured in SCF1 medium for 7 days (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130). Stage 7 embryos were slow-frozen using 1 of 4 protocols in a 2 × 2 factorial design: embryos were equilibrated in conventional slow-freezing media for 20 min [1.5 M ethylene glycol (EG) and 0.5 M sucrose] with 1 mm l-ascorbic acid or 1 μM polydatin, and loaded in the straw adjacent to columns of freezing medium or 0.75 M EG and 0.6 M sucrose and then seeded at −6°C, cooled at 0.5°C min−1, and plunged at –32°C. Embryos were thawed in air for 10 s followed by 30 s in 32 to 35°C water bath. Once straw columns were disrupted, embryos were allowed to equilibrate for 5 min. Subsequently, embryos were washed and placed back in culture and re-expansion was assessed at 24 and 48 h. Data (Table 1) were analysed by ANOVA with means separated by Tukey’s HSD. Results indicate that there was no main effect between the 2 antioxidants or the use of rehydration columns (P < 0.05); however, there was higher (P < 0.05) re-expansion for embryos frozen with polydatin and with rehydration column than embryos frozen with l-ascorbic acid and no rehydration column. This suggests that polydatin coupled with in-straw rehydration (0.75 M EG and 0.6 M sucrose) may improve post-thaw survival of IVP bovine embryos. Table 1.Post-thaw re-expansion rates of embryos exposed to antioxidants and in-straw rehydration (± SEM)

Vitrification of bovine embryos followed by in vitro hatching and expansion

Zygote ◽  
2017 ◽  
Vol 26 (1) ◽  
pp. 99-103 ◽  
Author(s):  
J.F. Souza ◽  
C.M. Oliveira ◽  
L.L. Lienou ◽  
T.V. Cavalcante ◽  
E. Alexandrino ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Added Sugar ◽  
Glucose Addition ◽  
Hatching Rates

SummaryThe objective of this study was to assess the effects of bovine embryo vitrification by applying three different vitrification solutions containing ethylene glycol (EG) and dimethylsulphoxide (DMSO) at different concentrations (10, 20 or 25% each) combined with 1.0 M glucose or 1.0 M sucrose, on the in vitro hatching and expansion rates. Healthy oocytes were selected for in vitro maturation and fertilization from 200 bovine ovaries, and subsequently cultured up to the blastocyst stage (n = 800). Control (n = 200) and vitrified cells (n = 100 per treatment; 600 in total) were cultured for an extra 24 or 48 h to evaluate hatching and expansion, respectively. Vitrification significantly decreased embryonic re-expansion and hatching rates independently of the tested solution when compared with control embryos, but solutions with 25% EG + 25% DMSO resulted in the highest re-expansion (75%) and hatching (70%) rates, independently of the added sugar. The addition of sucrose resulted in higher rates of re-expanded and hatched embryos when compared with glucose addition. We concluded that the combination of 25% EG + 25% DMSO and 1.0 M sucrose allowed hatching and expansion of vitrified-warmed bovine embryos produced in vitro.

163 EFFECTS OF SERUM AND L-CARNITINE ON DEVELOPMENT AND CRYOTOLERANCE OF BOVINE EMBRYOS PRODUCED IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 211
Author(s):  
A. Zolini ◽  
E. L. Carrascal-Triana ◽  
A. Ruiz ◽  
J. M. Penitente-Filho ◽  
P. J. Hansen ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Beneficial Effects ◽  
Serum Supplementation ◽  
Fetal Bovine ◽  
Concentration Table ◽  
Hatching Rates

Cryotolerance of bovine embryos produced in vitro (PIV) can be improved by l-carnitine. The objective of the present study was to determine whether the optimal concentration of l-carnitine is dependent on serum. Bovine embryos were produced in vitro with abattoir-derived oocytes. After fertilization (Day 0), oocytes (n = 2768) were randomly assigned in a 2 × 4 factorial design to culture in SOF-BE1 medium supplemented with or without 5% fetal bovine serum and l-carnitine at concentrations of 0.0, 0.75, 1.5, and 3.03 mM at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. The proportion of oocytes that cleaved was assessed on Day 3, and the proportion of oocytes that developed to the blastocyst and advanced blastocyst (expanded, hatching, and hatched) stages was determined on Day 7. Blastocysts and expanded blastocysts (n = 466) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. After thaw, embryos were cultured for 72 h in SOF-BE1 supplemented with 10% (v/v) fetal bovine serum and 50 mM dithiothreitol at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 9 times, and data were analysed by logistic regression. There was no interaction between serum and l-carnitine, at any of the concentrations tested, on embryo development or cryotolerance. Cleavage rates were not affected by serum or l-carnitine. Addition of serum during culture increased (P < 0.05) development to the blastocyst (19.7 ± 1.1% v. 25.3 ± 1.4%) and advanced blastocyst (9.1 ± 0.8% v. 12.4 ± 1.2%) stages. While l-carnitine did not affect blastocyst development, advanced blastocyst development was reduced (P < 0.05) for l-carnitine at 3.03 mM (0 mM: 10.9 ± 1.2%, 0.75 mM: 12.2 ± 1.4%, 1.5 mM: 13.5 ± 1.5%, 3.03 mM: 7.0 ± 1.0%). Serum reduced (P < 0.01) re-expansion (78.1 ± 3.4% v. 65.5 ± 3.1%, 81.0 ± 3.0% v. 68.4 ± 2.7%, 78.4 ± 3.4% v. 65.8 ± 3.1%, for 24, 48, and 72 h, respectively) and hatching (52.0 ± 4.0% v. 39.8 ± 3.6%, 61.2 ± 4.1% v. 45.4 ± 3.8%, 61.2 ± 4.1% v. 45.4 ± 3.8%, for 24, 48, and 72 h, respectively) rates at all time points. In contrast, treatment of embryos with l-carnitine during culture increased (P < 0.05) post-thaw re-expansion rates at 24 and 48 h, regardless of concentration (Table 1). In conclusion, post-thaw viability of bovine embryos PIV can be improved by the addition of l-carnitine during culture. Moreover, the beneficial effects of l-carnitine on cryosurvival are not dependent on serum supplementation. Table 1.Effect of addition of l-carnitine during culture on post-thaw re-expansion and hatching rates

scholarly journals 105VITRIFICATION OF BOVINE EMBRYOS USING THE CLV METHOD

2004 ◽  
Vol 16 (2) ◽  
pp. 174 ◽  
Author(s):  
W. Lindemans ◽  
L. Sangalli ◽  
A. Kick ◽  
C.R. Earl ◽  
R.C. Fry
Keyword(s):  
Glass Bead ◽  
Membrane Integrity ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Developmental Potential ◽  
Stage Of Development ◽  
Solid Liquid ◽  
Metal Block ◽  
Hatching Rates

Vitrification has become the preferred method for cryopreserving in vitro-produced bovine embryos (IVP). Here we introduce a technique for vitrification developed at CryoLogic (the CLV Method), in conjunction with a study comparing the post-thaw viability of IVP embryos frozen by the widely used open pulled straw method (OPS—Vajta et al., 1997 Cryo-Letters 18, 191) and the new CLV Method. Vitrification on thin metal surfaces has been explored and demonstrated previously (Le Gal &amp; Massip 1999, Cryobiology 38, 290), and Dinnyes presented a Solid Surface Vitrification (SSV) (Dinnyes et al., 2000, Biol. Reprod. 63, 513). The CLV Method utilizes vitrification on the surface of a solid metal block. This surface has been custom shaped and treated to enhance vitreous formation. The method also includes handling, storage and thawing protocols designed to avoid damage from crystallization of the unstable glass. Briefly, the block is precooled in LN2 to −196°C. Up to 10 embryos are collected into a droplet of medium (3μL), on the end of a fibre carrier attached to a handle. The droplet is presented to the vitrification surface, where it is converted into a glass bead by cooling rates comparable to that of plunging into solid/liquid phase nitrogen (−210°C) (Arav et al., 2001 Theriogenology 55, 313). The glass bead, fibre and handle are transferred quickly into a half-sealed, precooled straw, and the handle seals the open end. A bead is thawed very rapidly by removing the handle, fibre and bead from the straw and transferring the bead into washing medium. COCs collected from bovine ovaries obtained from abattoirs were matured, fertilized and cultured for 6 days (Fry et al., 2003 Theriogenology 59, 446). Embryos reaching the blastocyst or expanding blastocyst stage of development were graded (Grades 1, 2, and 3), equilibrated for 5min in HEPES-199 medium with 20% FCS (HFm), placed in HFm with 10% EG, and 10% DMSO (VS1) for 2min, and then transferred to HFm with 20% EG, 20% DMSO (VS2) for 30s (Vajta). Between 5 and 10 IVP embryos were processed and collected for vitrification, either in an OPS plunged into LN2, or in a 3μL droplet vitrified by the CLV Method. The two sets of specimens were stored in LN2, and later thawed. Both OPS tips and beads were thawed in 0.5mL of HFm with 0.2M sucrose at 39°C. Embryos were maintained at 39°C, examined after 5min for contraction, and again after 6 h for re-expansion. They were then transferred to culture medium, incubated and examined at 24 and 48h to assess hatching. As shown in Table 1, the CLV method appears to be satisfactory for maintaining membrane integrity (expansion) and developmental potential (hatching) for even poorer grade embryos, that might be more sensitive to the stresses of cryopreservation. Table 1 Re-expansion and hatching rates of graded thawed bovine embryos vitrified by OPS or CLV methods

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

scholarly journals MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence

2020 ◽  
Vol 21 (23) ◽  
pp. 8888
Author(s):  
Bárbara Melo-Baez ◽  
Yat S. Wong ◽  
Constanza J. Aguilera ◽  
Joel Cabezas ◽  
Ana C. F. Mançanares ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Fatty Acid Biosynthesis ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Target Cells ◽  
Bovine Embryos ◽  
Developmental Potential ◽  
Maternal Communication

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.

scholarly journals Optimization of culture and analysis methods for enhancing long-term Brugia malayi survival, molting and motility in vitro

2018 ◽  
Vol 4 ◽  
Author(s):  
I. M. Lu ◽  
T. Kassis ◽  
A. M. Rogers ◽  
A. Schudel ◽  
J. Weil ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Fetal Bovine Serum ◽  
Brugia Malayi ◽  
Mosquito Vector ◽  
Third Stage ◽  
Impact On Survival ◽  
Fetal Bovine ◽  
Parasite Biology

Abstract Lymphatic filariasis is a neglected tropical disease caused by roundworm parasites such as Brugia malayi that spread via a mosquito vector. In vitro culture of these parasites provides controlled conditions to understand parasite biology and provides a cheaper way to screen potential micro- and macrofilaricides. Published studies have used a wide array of approaches and metrics regarding in vitro cultures of B. malayi; as a result, drawing comparisons and identifying the reasons why inability to reproduce outcomes are difficult. This study sought to determine conditions that ensure reproducible outcomes and used evaluation metrics that are easily measured and can be automated to ensure objectivity. We found culturing B. malayi third-stage larvae (L3) in endothelial basal media supplemented with 20% fetal bovine serum and 75 µ m ascorbic acid in a temperature- and humidity-controlled incubator produced better survival and molting rates as well as longer and more motile parasites than previously reported. The benefit of ascorbic acid seemed to be unique to L3 parasites, as the addition of ascorbic acid to adult parasites had no significant impact on survival or motility. The methods reported in this study will help in designing experiments for both parasite behaviour studies and drug screening applications for disease eradication.

In vitro culture and non-invasive metabolic profiling of single bovine embryos

2019 ◽  
Vol 31 (2) ◽  
pp. 306
Author(s):  
Monika Nõmm ◽  
Rando Porosk ◽  
Pille Pärn ◽  
Kalle Kilk ◽  
Ursel Soomets ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Metabolic Profiling ◽  
Culture Medium ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Growth Media ◽  
Low Molecular Weight ◽  
Bovine Embryos ◽  
Non Invasive

Selecting high-quality embryos for transfer has been a difficult task when producing bovine embryos invitro. The most used non-invasive method is based on visual observation. Molecular characterisation of embryo growth media has been proposed as a complementary method. In this study we demonstrate a culture medium sampling method for identifying potential embryonic viability markers to predict normal or abnormal embryonic development. During single embryo culture, 20µL culture media was removed at Days 2, 5 and 8 after fertilisation from the same droplet (60µL). In all, 58 samples were analysed using liquid chromatography–mass spectrometry. We demonstrate that it is possible to remove samples from the same culture medium droplets and not significantly affect blastocyst rate (25.2%). Changes in any single low molecular weight compound were not predictive enough. Combining multiple low molecular weight signals made it possible to predict Day 2 and 5 embryo development to the blastocyst stage with an accuracy of 64%. Elevated concentrations of lysophosphatidylethanolamines (m/z=453, 566, 588) in the culture media of Day 8 well-developing embryos were observed. Choline (104m/z) and citrate (215m/z) concentrations were increased in embryos in which development was retarded. Metabolic profiling provides possibilities to identify well-developing embryos before transfer, thus improving pregnancy rates and the number of calves born.

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