136 BLASTOCOELE COLLAPSE IMPROVES POST-THAW SURVIVAL OF SLOW FROZEN AND VITRIFIED IN VITRO-PRODUCED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 181 ◽  
Author(s):  
J. P. Barfield ◽  
G. E. Seidel

Slow-freeze cryopreservation of in vitro-produced bovine embryos often results in poor post-thaw embryo viability. Results with vitrification tend to be better but do not always produce consistent results for a variety of reasons. Experiments with human and equine embryos have demonstrated improved post-thaw survival when blastocoele fluid is removed before vitrification. We hypothesised that removing blastocoele fluid before vitrification or slow-freeze cryopreservation would improve post-thaw survival of in vitro-produced bovine embryos. Three replicates of embryos were generated from abattoir-derived ovaries using standard techniques. Seven days post-fertilization, embryos were evaluated for quality, and grade 1 or 2 embryos were allocated to 1 of 4 treatments: slow-freeze control (n = 103); slow-freeze collapsed (n = 106); vitrified control (n = 117); vitrified collapsed (n = 99). Blastocoele fluid was removed by aspiration with a pipette advanced to the centre of the blastocoele while the embryos were stabilised with a holding pipette. After collapse, embryos were immediately slow frozen in commercial medium [1.5 M ethylene glycol (EG) + 0.1 M sucrose, Bioniche] in 0.25-mL straws or vitrified in 2 steps: 3 min in 1.5 M EG, 30 s in 7 M EG + 0.6 M galactose + 18% Ficoll in open-pulled straws. After thawing or warming, embryos were cultured in chemically defined medium-2 + 5% FCS in 5% CO2, 5% O2, 90% N2 at 38.5°C. Embryos were evaluated for survival based on re-expansion of the blastocoele after 24 h in culture. Data were analysed by GLIMMIX (SAS Institute Inc., Cary, NC, USA). There were significant replicate effects but there were no significant differences in survival between embryos that were vitrified or slow frozen. Approximately twice as many collapsed embryos survived compared to intact control embryos regardless of whether the embryos were vitrified or slow frozen (collapsed: 50.5 ± 4.1%, control: 26.3 ± 3.5%; P < 0.001). Advanced blastocysts (fully expanded and hatched blastocysts) survived better than early blastocysts (pre-expansion blastocysts and expanding blastocysts, 45.9% ± 3.9 v. 30.0% ± 4.4, respectively; P < 0.01). The only significant interaction (P < 0.05) was that vitrification was superior to freezing for early blastocysts (39.5 ± 5.9% v. 22.1 ± 5.0% survival) but not advanced blastocysts (44.4 ± 5.3% v. 47.4 ± 5.8% survival). Removal of blastocoele fluid before cryopreservation of in vitro-produced bovine embryos may be a useful technique for improving post-thaw survival regardless of cryopreservation method or blastocyst stage.

2021 ◽  
Author(s):  
Aimé Jazmín Garza Arredondo ◽  
Diana Elisa Zamora Ávila ◽  
Uziel Castillo Velásquez ◽  
Gustavo Moreno Degollado ◽  
José Fernando De La Torre Sánchez ◽  
...  

Abstract Endogenous heat shock cognate 71 kDa protein (HSC70) has a vital role in early embryonic development. This study assessed the effects of exogenous HSC70 on bovine embryo development and expression of genes associated with apoptosis. Expression analyses of HSPA1A, HSPA8, Bcl-2, and Bax genes were performed in bovine embryos in vivo on day 7 of development. Subsequently, expression of HSPA1A and HSPA8 were associated with apoptotic genes (Bcl-2 and Bax) in cultured bovine embryos in vitro that were supplemented with various concentrations (0 or control group, 50, and 100 ng) of HSC70. The results indicated that the control group (0 ng) in vitro embryos had higher expression of HSPA8, Bax, and Bcl-2 genes, compared with the vivo embryos (P < 0.01). In vitro-produced embryos supplemented with 50 ng or 100 ng HSC70 had higher expression of HSPA1A, HSC70, Bcl-2, and Bax genes, compared with the control group (P < 0.01). Embryos supplemented with 100 ng had greater expression of the HSPA8 gene compared with the control group and the group supplemented with 50 ng. However, embryos supplemented with 50 ng had better characteristics (i.e., stage of development and quality) than the control and 100-ng groups. In conclusion, supplementation of in vitro culture medium with HSC70 promoted development to the blastocyst stage and improved blastocyst quality.


2000 ◽  
Vol 8 (3) ◽  
pp. 241-287 ◽  
Author(s):  
GM Jones

The transfer of a blastocyst established the first human clinical pregnancy following in vitro fertilization (IVF). Nine years later Cohen et al. reported pregnancies resulting from the transfer of cryopreserved human blastocysts. However, it was another six years before the first report of births resulting from the transfer of human blastocysts produced in vitro appeared in the medical literature. In the intervening period clinics have opted to transfer embryos at the early cleavage stage to the uterus, despite the fact that in vivo the embryo does not enter the uterus until two to three days later at the morula to blastocyst stage of development. The viability and potential for implantation of blastocysts is high, as indicated by the finding that more than 60% of in-vivo-derived blastocysts, recovered by uterine lavage following artificial insemination of fertile donors, implant and develop into viable fetuses when transferred to recipients. This is in stark contrast to the 10–20% of in-vitro-produced embryos transferred at the early cleavage stage of development that result in a live-birth. This reduction in viability following transfer of in-vitro-derived early cleavage stage embryos may have several possible explanations: (1) a failure of implantation due to poor synchronization between the embryo and the uterine endometrium; (2) a hostile environment in the uterus for early cleavage stage embryos; (3) sub-optimal in vitro culture conditions which result in a reduction in embryo viability; (4) the assumption that all oocytes retrieved in an IVF cycle have an equal ability to develop into viable embryos; and (5) the failure to identify the most viable embryo in a cohort. Certainly, improving culture conditions and laboratory techniques for developing high quality blastocysts routinely in vitro will not only address many of the above questions but will also improve the quality and viability of earlier stages of embryo development.


2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  

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.


2004 ◽  
Vol 16 (2) ◽  
pp. 202 ◽  
Author(s):  
W.F. Swanson ◽  
A.L. Manharth ◽  
J.B. Bond ◽  
H.L. Bateman ◽  
R.L. Krisher ◽  
...  

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P&gt;0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P&gt;0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)


2020 ◽  
Vol 21 (23) ◽  
pp. 8888
Author(s):  
Bárbara Melo-Baez ◽  
Yat S. Wong ◽  
Constanza J. Aguilera ◽  
Joel Cabezas ◽  
Ana C. F. Mançanares ◽  
...  

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.


2019 ◽  
Vol 31 (2) ◽  
pp. 306
Author(s):  
Monika Nõmm ◽  
Rando Porosk ◽  
Pille Pärn ◽  
Kalle Kilk ◽  
Ursel Soomets ◽  
...  

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.


2010 ◽  
Vol 22 (1) ◽  
pp. 303
Author(s):  
D. M. Paschoal ◽  
M. J. Sudano ◽  
L. C. O. Magalhães ◽  
L. F. Crocomo ◽  
F. C. Landim-Alvarenga

The increased storage of lipid granules in in vitro-produced (IVP) bovine embryos seems to be related to the presence and concentration of fetal calf serum (FCS) during culture. The presence of high concentration of lipids on embryos reduces their viability after cryopreservation, which has been one of the main obstacles for the success of vitrification of IVP bovine embryos (Moore et al. 2007 Theriogenology 68, 1316-1325). The present experiment aimed to induce cytoplasmic lipolysis in IVP bovine embryos using forskolin (Sigma-Aldrich, St. Louis, MO, USA), which raises the levels of intracellular cAMP (Seamon et al. 1981 Proc. Natl. Acad. Sci. USA, 78, 3363-3367). Nelore oocytes were matured in TCM-199 + 10% FCS, FSH, and LH in 5% CO2 in air atmosphere, at 38.5°C. After 24 h of maturation, oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the same conditions. Presumptive zygotes were cultured in 2 concentrations of FCS: Control 0% (SOFaa + 5 mg mL-1 BSA; basic medium, BM), and Control 2.5% (BM supplemented with 2.5% FCS). On Day 6 of culture embryos were divided into 2 additional treatments: Forskolin 0% (BM + 10 μM forskolin; and Forskolin 2.5% (BM supplemented with 2.5% FCS and 10 μM forskolin). All embryos were cultured in a 5% CO2, 5%O2, and 90% N2 atmosphere at 38.5°C for 7 days, when blastocyst formation rate was evaluated. Embryo viability was also checked by staining the embryos with Hoechst 33342 and propidium iodide. Data were analyzed by ANOVA followed by Tukey’s test, using a 5% significance level. No statistical differences were observed among treatments on cleavage rates, evaluated on Day 3 of culture, or on blastocyst formation rates. Although no statistical differences was observed between treatments on percentage of viable cells, embryos cultured with 0% FCS, independently of the presence of forskolin, presented significantly more damaged cells than embryos cultured with 2.5% FCS (P < 0.05). The results indicate that the presence of FCS is important to reduce degeneration of blastomeres during culture. Moreover, the presence of forskolin on Day 6 of culture did not influence embryo development, indicating that this drug could be a good alternative to reduce embryo lipid content in bovine IVP embryos produced in presence of FCS. Table 1.Effect of fetal calf serum and forskolin on embryo culture Acknowledgments: FAPESP 07/53505-1.


2019 ◽  
Vol 31 (5) ◽  
pp. 837 ◽  
Author(s):  
V. H. Do ◽  
S. Catt ◽  
J. E. Kinder ◽  
S. Walton ◽  
A. W. Taylor-Robinson

Bovine invitro fertilisation technology has been widely exploited in commercial settings. The majority of invitro-derived cattle embryos are transferred into recipient cows as recently collected (i.e. ‘fresh’) embryos due to the lack of a reliable cryopreservation method that results in favourable pregnancy rates following transfer of thawed embryos. This is a primary reason for the poor industry uptake of this extreme temperature freezing process. Numerous investigations into vitrification have revealed the importance of rapid cooling and warming rates, enhancing embryo viability after cryopreservation compared with conventional slow freezing. Those studies spawned a considerable assortment of cryovessels and diversity of procedures, delivering variable rates of success, which makes performing vitrification consistently a practical challenge. Hence, further research is required in order to both optimise and standardise vitrification methodology and to design a cryovessel that enables direct transfer of vitrified embryos to recipients after warming. In parallel with improvements in vitrification, it is important to continue to raise the quality of invitro-derived cattle embryos through modifications in laboratory culture techniques. The twin goals of methodology refinement and standardisation, leading to embryo quality enhancement, are each imperative if invitro fertilisation technology is to be adopted in the field.


Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 327-335 ◽  
Author(s):  
Hruda Nanda Malik ◽  
Dinesh Kumar Singhal ◽  
Shrabani Saugandhika ◽  
Amit Dubey ◽  
Ayan Mukherjee ◽  
...  

SummaryThe present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts.


2007 ◽  
Vol 74 (8) ◽  
pp. 972-977 ◽  
Author(s):  
Deirdre Corcoran ◽  
Dimitrios Rizos ◽  
Trudee Fair ◽  
Alex C.O. Evans ◽  
Patrick Lonergan

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