194 Extracellular vesicles derived from ampullary oviductal fluid improve developmental competence of bovine oocytes

2020 ◽  
Vol 32 (2) ◽  
pp. 225
Author(s):  
A. Asaadi ◽  
K. Pavani ◽  
N. Azari Dolatabadi ◽  
P. Van Damme ◽  
A. Van Soom
Keyword(s):  
Extracellular Vesicles ◽  
Follicular Fluid ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Control Group ◽  
Cleavage Rate ◽  
Blastocyst Quality ◽  
Oviductal Fluid ◽  
Bovine Oocytes

Invivo, oocytes mature in the presence of follicular fluid (FF) and ampullary oviductal fluid (AOF), starting from premature to final maturation stages (3-6h postovulation). Extracellular vesicles (EVs) have been identified as important mediators of gamete/embryo-maternal interactions. They carry regulatory molecules, such as microRNAs and mRNAs. So far, the functionality of EVs derived from FF and AOF has not yet been determined. The aim of this study was to evaluate the effect of EVs derived from FF and AOF during invitro oocyte maturation (IVM) on the development and quality of resulting bovine embryos. Follicular fluid of a preovulatory follicle was collected by ovum pickup, and AOF was collected from the oviducts of slaughtered cows in early luteal phase. Then, EVs from FF and AOF were isolated by size exclusion chromatography and confirmed by western blot. The concentration of EVs was determined by NanoDrop and Nanoparticle tracking analysis. Integrity and size of EVs were assessed by electron microscopy. Bovine oocytes (n=1331) were allocated at random to five groups. In the control group (C), oocytes (n=347) were cultured for 22.5h in maturation medium (TCM-199 and epidermal growth factor 20ngmL−1) (MM). In the negative control group (NC), oocytes (n=331) were cultured for 18h in MM, and then transferred into fresh MM for 4.5h. In the FF group, oocytes (n=162) were cultured in MM supplemented with FF EVs (12.5µgmL−1) for 18h, and then transferred to MM for 4.5h. In the AOF group, oocytes (n=328) were cultured for 18h in EV-free MM, and then transferred to MM supplemented with AOF EVs (1.7µgmL−1) for 4.5h. In the FF+AOF group, oocytes (n=163) were cultured in MM supplemented with FF EVs for 18h, and then transferred into MM supplemented with AOF EVs for 4.5h. At 22.5h post-incubation, mature oocytes were fertilized invitro. At 21h post-insemination, presumptive zygotes were denuded and cultured in synthetic oviductal fluid with insulin-transferrin-selenium for 8 days. Cleavage rate and blastocyst rate were recorded at 48h and 8 days post-insemination, respectively. Blastocyst quality was assessed by differential apoptotic staining. The data were analysed using one-way ANOVA. Cleavage rate was not affected by the treatment, whereas blastocyst yield in the AOF group (48.75%) was significantly higher than that in the C (42.91%) and NC (37.72%) groups. In addition, embryos produced in the AOF group showed the highest number of trophectoderm cells and inner cell mass cells, and lowest number of apoptotic cells (P<0.05). Trophectoderm and inner cell mass cells were higher and apoptotic cells were lower in FF and FF+AOF compared with the C and NC groups. In conclusion, adding AOF EVs to oocyte MM in the last 4.5h of IVM improves quantity and quality of blastocysts. In addition, although FF EVs showed no effect on blastocyst yield, they improved blastocyst quality by increasing cell number.

77 Development and quality of in vitro bovine hemi embryos produced by blastomere separation and embryo bisection

2019 ◽  
Vol 31 (1) ◽  
pp. 164
Author(s):  
A. E. Ynsaurralde Rivolta ◽  
M. Suvá ◽  
V. Alberio ◽  
C. Vazquez Echegaray ◽  
A. Guberman ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Monozygotic Twin ◽  
Cell Number ◽  
Control Group ◽  
Cell Stage ◽  
Exact Test ◽  
Development Rates

Bovine monozygotic production of twins became popular in the 1980s as a technique to multiply high value genetics. Moreover, it also became a powerful model for research. Different techniques have been used on bovine embryos obtained by superovulation. In this work, we compared the development rates and quality of monozygotic twin embryos produced by blastomere separation (BS) and embryo bisection (EB) of IVF embryos. To this aim, cumulus-oocytes complexes collected from slaughterhouse ovaries were in vitro matured in TCM 199 containing 10% fetal bovine serum, 10µg mL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine, and 2% antibiotic-antimycotic for 24h, at 6.5% CO2 in humidified air and 38.5°C. The IVF was performed with 16×106 spermatozoa per mL for 5h. Afterward, presumptive zygotes were cultured in SOF medium for 7 days at 38.5°C and 5% O2. After 24h of culture, blastomeres of 2-cell stage embryos (N=114) were separated and each one was cultured individually in a microwell for 7 days. Embryo bisection (N=179) was performed manually on Day-7 blastocysts previously depleted of their zonae pellucidae, under stereoscopic microscope. Hemi embryos were cultured for 24h and then twins and single blastocyst rates were calculated. For quality assessment, diameter, total and inner cell mass (ICM) cell number of hemi embryos (BS: 6 couples; ES: 10 couples) and the control group (C: 11) were evaluated. The ICM cell number was measured by immunofluorescence staining using SOX2 antibody and the percentage of ICM and trophectoderm (TE) cells was calculated. The results were analysed using Fisher’s exact test and ANOVA with mean comparison using Tukey’s test (P=0.05). No statistical differences were found in blastocyst rates of twins and single hemi embryos produced by BS (28 and 25%) or EB (23 and 32%). Blastocyst diameter was similar between groups and control. Hemi embryos exhibited lower total and ICM cell number than control (BS: 43±18, EB: 57±14v. C: 93±35 and BS: 16±7, EB: 12±8v. C: 34±19). However, BS hemi embryos had higher ICM and lower TE percentage (40/60%) compared with the EB group (20/80%). The control group did not differ with hemi embryo treatments for ICM and TE (30/70%). Our preliminary results have indicated that although the development rates of hemi embryos produced in vitro were similar between both techniques, blastomere separation generates better quality embryos than blastocyst bisection.

216 EFFECT OF SPERM PRETREATMENT WITH LYSOLECITHIN AND Triton X-100 ON PRONUCLEAR FORMATION AND QUALITY OF BOVINE EMBRYOS PRODUCED BY INTRACYTOPLASMIC SPERM INJECTION

2016 ◽  
Vol 28 (2) ◽  
pp. 239
Author(s):  
F. Zambrano ◽  
M. E. Arias ◽  
T. Vargas ◽  
L. Aguila ◽  
R. Felmer
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Cell Mass ◽  
Harmful Effect ◽  
Hydrolytic Enzymes ◽  
Control Group ◽  
Cleavage Rate ◽  
Blastocyst Rate ◽  
Triton X ◽  
Pronuclear Formation

In cattle, intracytoplasmic sperm injection (ICSI) has low efficiency and has application for sexed semen, semen of poor quality of high genetic value, or semen of endangered breeds. The acrosome content has a harmful effect within the oocyte because of the presence of hydrolytic enzymes. With the aim of removing the acrosome and destabilising the membranes, cryopreserved bovine spermatozoa were treated with lysolecithin (LL) and Triton X-100 (TX). Frozen straws of bull semen were thawed and the spermatozoa were selected by Percoll gradient. The selected spermatozoa were incubated at concentrations of 0.05% LL (ICSI-LL), 0.05% TX (ICSI-TX), and a control group (without treatment) for 1 min with vortex, and were then immediately washed by centrifugation at 400 × g in Sp-TALP medium. Following ICSI, oocytes were activated with 5 mM ionomycin and 5 µg mL–1 cycloheximide. Embryos were cultured in KSOM medium at 38.5°C, 5% CO2, 5% O2, and 90% N2, to Day 8 to record blastocyst rate. Pronuclear formation was evaluated at 19 h after activation, and the presumptive zygotes were fixed in methanol-glacial acetic acid (3 : 1), stained with Hoechst, and analysed with a fluorescence microscope. The results were evaluated by a chi-squared test with Bonferroni’s correction. Significance was set at P < 0.05. The quality of embryos was evaluated by counting the total cell numbers (T), inner cell mass cells (ICM), and trophectoderm cells (TE) after staining with Hoechst and propidium iodide. We injected 345 oocytes in the control group, 335 in ICSI-LL, 304 in ICSI-TX, and 351 were parthenogenetically activated (12 replicates for each group). A higher cleavage rate was observed in ICSI-TX (66%; 231/351) and ICSI-LL (62%; 207/335) compared the control group (53%; 183/345). In addition, a significantly higher blastocyst rate was observed in both treatments: 27% (82/304) and 27.5% (92 of 335) for ICSI-TX and ICSI-LL groups, respectively, compared with the control group (22.6%; 78/345). In the parthenogenetic activation group without ICSI, the cleavage rate was 41.9% (147/351) and blastocyst rate was 15.4% (54/351). Differences among treatments were analysed using one-way ANOVA after arcsine transformation, and significance was set at P < 0.05. No differences were observed in pronuclear formation (with male and female pronuclei and 2 polar bodies) for ICSI-LL (77%; 48/53), ICSI-TX (76.4%; 39/51), and the control group (71.6%; 38/53) with 3 replicates for each group. No significant differences were observed in the quality of the embryos, ICM cell number (control: 35.7 ± 2.8, ICSI-LL: 38 ± 4.5, and ICSI-TX: 40.8 ± 8.2), TE (control: 139 ± 31.5, ICSI-LL: 124 ± 10.4, and ICSI-TX: 129 ± 18.5), T (control: 170 ± 25.6, ICSI-LL: 156 ± 14.3, and ICSI-TX: 176 30.3), with 8 replicates for each group. In conclusion, this study proposed treatments able to improve the rate of development without affecting the quality of embryos produced by ICSI.

High hydrostatic pressure treatment improves the quality of in vitro-produced ovine blastocysts

2011 ◽  
Vol 23 (6) ◽  
pp. 809 ◽  
Author(s):  
Luisa Bogliolo ◽  
Federica Ariu ◽  
Giovanni Leoni ◽  
Stefania Uccheddu ◽  
Daniela Bebbere
Keyword(s):  
Hydrostatic Pressure ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Lower Percentage ◽  
Control Group ◽  
Hatching Rate ◽  
Embryo Survival

Exposure to sub-lethal hydrostatic pressure (HP) treatment is emerging as an approach to improve the general resistance of gametes and embryos to in vitro conditions. The present study was aimed to evaluate the effect of HP treatment on in vitro-produced ovine blastocysts. Experiment 1 was aimed to define optimal treatment parameters: two different HP treatments were applied to blastocysts and embryo survival was evaluated. In Experiment 2, HP parameters (40 MPa, 70 min, 38°C) selected in Experiment 1 were used to treat blastocysts. Embryo quality was assessed and compared with untreated controls by counting total cell number, the inner cell mass (ICM) and trophectoderm (TE) cells and by evaluating nuclear picnosis. HP-treated blastocysts were processed for gene expression analysis (AQP3, ATP1A1, BAX, CDH1, HSP90β, NANOG, OCT4 and TP53) 1, 5 h after the end of HP exposure. Results showed that the hatching rate of embryos treated at 40 MPa was significantly higher than that of the 60 MPa-treated group (P < 0.01) and similar to untreated embryos. Blastocysts exposed at 40 MPa showed higher ICM (P < 0.05) and TE (P < 0.01) cell number and a lower percentage of picnotic nuclei (P < 0.05) compared with the control group. Significantly lower abundance for BAX (P < 0.01) and OCT4 (P < 0.05) transcripts were observed in HP embryos than in the control group. In conclusion, treatment with HP improved the quality of in vitro-produced ovine blastocysts by increasing their cell number and reducing the proportion of nuclear picnosis.

Quality of rabbit vitrified/thawed transgenic embryos

Zygote ◽  
2011 ◽  
Vol 21 (1) ◽  
pp. 53-58 ◽  
Author(s):  
P. Chrenek ◽  
Z. Turanová ◽  
J. Slamečka ◽  
A. V. Makarevich
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Developmental Rate ◽  
Control Group ◽  
Cell Counts ◽  
Transgenic Rabbits ◽  
Significant Difference ◽  
Transgenic Embryos ◽  
Rabbit Embryos

SummaryThe aim of our study was to investigate the influence of vitrification on developmental rate and quality (total number of cells, number of blastomeres in inner cell mass (ICM) area, apoptotic index and embryo diameter) of transgenic (carrying an endogenous–hFVIII or exogenous–enhanced green fluorescent protein (EGFP) gene) rabbit embryos. EGFP-positive rabbit embryos were produced under in vitro conditions by the microinjection of foreign genes into the pronucleus of fertilized eggs. The transgenic rabbit embryos with the hFVIII gene were produced by mating homozygous transgenic rabbits and flushing at the single-cell stage. Developmental rate of vitrified/thawed transgenic embryos that reached hatching blastocyst stage (68.00% and 69.00%) and differed significantly (p < 0.001) from those in control embryos (100.00%). Significant difference (p < 0.05) was found in total cell counts between control (117.00 ± 36.00) and vitrified (141.00 ± 34.80) hFVIII-positive embryos. The higher proportion of ICM cells (32.00%) and greatest embryo diameter (130.85 ± 10.90) were found in the control group compared with the transgenic. Ratio of apoptotic cells was significantly higher (p < 0.01) in the control group (2.50%) and vitrified EGFP-positive embryos (2.90%) compared with the vitrified, hFVIII-positive group of embryos (0.70%). Our results demonstrate that neither gene microinjection itself, nor exogenous (EGFP) and endogenous (hFVIII) gene expression interferes with developmental rate and quality of rabbit embryos. However, a combination of microinjection and vitrification significantly decreases (p < 0.001) the survival rate of rabbit embryos.

Comparison of different fertilisation media for an in vitro maturation–fertilisation–culture system using flow-cytometrically sorted X chromosome-bearing spermatozoa for bovine embryo production

2016 ◽  
Vol 28 (11) ◽  
pp. 1695
Author(s):  
Luis B. Ferré ◽  
Yanina Bogliotti ◽  
James L. Chitwood ◽  
Cristóbal Fresno ◽  
Hugo H. Ortega ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Female Sex ◽  
Grade 3 ◽  
Oviductal Fluid ◽  
Hatching Rates

High demand exists among commercial cattle producers for in vitro-derived bovine embryos fertilised with female sex-sorted spermatozoa from high-value breeding stock. The aim of this study was to evaluate three fertilisation media, namely M199, synthetic oviductal fluid (SOF) and Tyrode’s albumin–lactate–pyruvate (TALP), on IVF performance using female sex-sorted spermatozoa. In all, 1143, 1220 and 1041 cumulus–oocyte complexes were fertilised in M199, SOF and TALP, respectively. There were significant differences among fertilisation media (P < 0.05) in cleavage rate (M199 = 57%, SOF = 71% and TALP = 72%), blastocyst formation (M199 = 9%, SOF = 20% and TALP = 19%), proportion of Grade 1 blastocysts (M199 = 15%, SOF = 52% and TALP = 51%), proportion of Grade 3 blastocysts (M199 = 58%, SOF = 21% and TALP = 20%) and hatching rates (M199 = 29%, SOF = 60% and TALP = 65%). The inner cell mass (ICM) and trophectoderm (TE) cells of Day 7 blastocysts were also affected by the fertilisation medium. Embryos derived from SOF and TALP fertilisation media had higher numbers of ICM, TE and total cells than those fertilised in M199. In conclusion, fertilisation media affected cleavage rate, as well as subsequent embryo development, quality and hatching ability. SOF and TALP fertilisation media produced significantly more embryos of higher quality than M199.

P–132 Centralized versus local embryologists scoring of blastocyst quality obtained in a large European multicenter clinical trial

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
M Montag ◽  
E Va. de. Abbeel ◽  
T Ebner ◽  
P Larsson ◽  
B Mannaerts
Keyword(s):  
Quality Assessment ◽  
Data Storage ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Time Lapse ◽  
Ongoing Pregnancy Rate ◽  
Quality Classification ◽  
Blastocyst Quality ◽  
Selection Of ◽  
Local Assessment

Abstract Study question Does blastocyst quality scoring by central assessment deviate from local assessment and potentially lead to the selection of a different single blastocyst for transfer? Summary answer Central and local assessment provided the same quality classification (poor / good / top) in 69% of all blastocysts and 63% of all transferred blastocysts. What is known already Blastocyst quality is scored most frequently by three morphological parameters, namely expansion and hatching (EH) status, inner cell mass (ICM) grading and trophectoderm (TE) grading. The score is used to define the quality classification (poor / good / top) which determines which embryo is to be transferred or cryopreserved. Blastocyst scoring and grading can be highly subjective, which does influence the choice for transfer and cryopreservation. Time-lapse imaging technology captures additional input about embryo development as well as enables centralized data storage and sharing for independent central assessments. Study design, size, duration Pooled embryo analysis from a prospective, randomized, multicenter trial (RAINBOW) of 619 women undergoing ovarian stimulation with an individualized dose of follitropin delta in a long GnRH agonist protocol between May 2018 and January 2020. Blastocysts were centrally assessed using time-lapse images by two independent assessors and one adjudicator . Selection of the blastocyst for transfer by local assessment was based on morphological scoring and not on morphokinetic time-lapse parameters. Participants/materials, setting, methods Oocytes were fertilized by ICSI and cultured in the Embryoscopeâ (Vitrolife) up to day 5 for transfer or day 5/6 for cryopreservation. Embryos were assessed as either non-blastocyst or blastocyst. Blastocysts were graded centrally and locally at 116 hrs of development, based on EH status (1–6), ICM (A-D) and TE grading (A-D). Central assessors were blinded to local assessment and embryo transfer selection. Main results and the role of chance In total 4282 embryos were assessed centrally, of which 2046 day 5 embryos (48%) were adjudicated due to a scoring difference of at least one parameter between the two central assessors. In total 38% of day 5 embryos were judged as non-blastocysts and 62% as blastocysts of which 61% (i.e. 38% of all embryos) were determined to be of good or top quality. Identical results in terms of quality classification (poor / good / top) were obtained for 69% of blastocysts between local and central assessment and in 78%, between the two central assessors. Moreover, central and local scoring were identical in 62% for EH status, 53% for ICM grading and 57% for TE grading. For all transferred blastocysts (n = 508), central and local quality assessment was aligned for 63%. The ongoing pregnancy rate following single blastocyst transfer (SBT) was 41% (202/489), and similar to when considering only the transfers for which the central assessment had the same or a higher classification than the local assessment (166/411=40%). In 16% of all SBT, central quality assessment gave a lower score for the transferred blastocyst than the central assessment. This discrepancy could potentially have led to transfer of a different blastocyst. Limitations, reasons for caution This trial included assessments made by embryologists from 20 IVF centres. Some centres has limited experience with time-lapse technology for morphological blastocyst scoring. Scoring could therefore have been affected by differences in focal planes, magnification and contrast compared to inverted microscopy, with potential influence on blastocyst scores and quality classification. Wider implications of the findings: Local and central blastocyst quality classification based on morphology aligns well but remains subjective. Embryo assessment may benefit from using tools like artificial intelligence-based algorithms for a more objective analysis. Trial registration number NCT03564509

scholarly journals Does Blastocyst Morphology Influence Live Birth Rate after Frozen-thawed Single Euploid Embryo Transfer?

2021 ◽  
Author(s):  
Na Li ◽  
Yichun Guan ◽  
Bingnan Ren ◽  
Yuchao Zhang ◽  
Yulin Du ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Live Birth ◽  
Birth Rate ◽  
Inner Cell Mass ◽  
Medical Center ◽  
Cohort Analysis ◽  
Cell Mass ◽  
Live Birth Rate ◽  
Poor Quality ◽  
Blastocyst Quality

Abstract Objective To investigate whether the morphologic parameters of euploid blastocyst influence the live birth rate (LBR) following single frozen-thawed embryo transfer (FET) cycles? Methods A retrospective cohort analysis involving autologous single FET cycles after preimplantation genetic testing for aneuploidy (PGT-A) through next generation sequencing (NGS) by a university-based reproductive medical center that was performed from June 2017 to September 2019.Women were divided into three age groups (< 30, 30–34 and ≥ 35 years). The primary outcome measure was LBR. Outcomes were compared to determine the association between different blastocyst quality (Good, Average and Poor), inner cell mass (ICM) grade (A and B), and trophectoderm (TE) grade (A, B and C) and LBR. Results We included 232 single FET cycles for analysis, the total LBR was 48.48%. In the youngest group (< 30 years, n = 86), LBR were compared between cycles with various blastocyst quality (72.22% for good quality, 54.55% for average quality and 34.78% for poor quality; P = 0.019), ICM grade (70.59% for grade A and 42.03% for grade B; P = 0.035) and TE grade (85.71% for grade A,57.58% for grade B and 34.78 for grade C; P = 0.015). Similarly, in the 30–34 years group, LBR ranged from 50.00% for good quality to 45.45% for poor quality (P = 0.870), from 35.29% for ICM grade A to 51.22% for ICM grade B (P = 0.291), from 60.00% for TE grade A to 45.45% for TE grade C (P = 0.634). Likewise, in the oldest group (≥ 35years, n = 47), LBR were also comparable between these subgroups, but no significant differences were seen in blastocyst morphologic parameters and LBR (P > 0.05). Conclusion After single FET cycles, the LBR was associated with morphologic parameters of euploid blastocysts, especially in women < 30 years old. However, these differences were not found in women older than 30 years. We suggested that for older women whose embryos undergoing PGT-A with NGS to be euploid have the same development potential regardless of their blastocyst morphology.

PSX-B-13 Effect of epidermal growth factor and prolactin on the quality of bovine oocytes aging in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-327
Author(s):  
Ekaterina Shedova ◽  
Galina Singina ◽  
Irina Y Lebedeva ◽  
Aleksandr Lopukhov
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Control Group ◽  
Tunel Staining ◽  
Apoptosis Resistance ◽  
Prolonged Culture ◽  
Epidermal Growth ◽  
Bovine Oocytes

Abstract The evaluation of factors responsible for the protection of the oocytes attained the metaphase-II stage from aging is importance for successful in vitro embryo reproduction. The aim of the present research was to study dose-dependent effects of epidermal growth factor (EGF) and prolactin (PRL) on the quality of bovine oocytes after their aging in vitro. Bovine cumulus-enclosed oocytes (CEOs) were matured in vitro for 20 h in TCM 199 containing 0.2 mM sodium pyruvate, 10% fetal calf serum (FCS), 10 μg/ml FSH and LH. At the end of in vitro maturation, oocytes were transferred to TCM 199 supplemented with 10% FCS (aging medium) and cultured for additional 24 h in the absence (Control) and in presence of EGF (10 and 50 ng/ml) and PRL (20 and 50 ng/ml). After prolonged culture oocytes were used for apoptosis detection (TUNEL staining, n=251) and the state of chromosomes evaluation (Tarkowski’s cytogenetic method, n=359). The data from 3–4 replicates were analyzed by ANOVA. At the end of prolonged culture (24 h) the rate of apoptotic oocytes in the Control group was 47.4±8.5%. EGF at concentration of 10 ng/ml and PRL at both doses decreased this rate to 15.0–22.1% (p &lt; 0.05). Furthermore, PRL (not EGF) reduced the frequency of abnormal chromosome modifications (decondensation, adherence, clumping) at concentrations of 20–50 ng/ml from 58.7±2.1% (Control) to 41.2±1.9 and 45.6±2.7% respectively (p &lt; 0.01). Thus, EGF and PRL is able to maintain the apoptosis resistance of bovine oocytes during their prolonged in vitro culture as well as PRL have the decelerating effect on abnormal modifications of M-II chromosomes. The research was supported by RFBR (17-29-08035) and the Ministry of Science and Higher Education of Russia.

scholarly journals LÍQUIDO FOLICULAR EQÜINO NA MATURAÇÃO IN VITRO DE OÓCITOS BOVINOS

2004 ◽  
Vol 9 (1) ◽  
Author(s):  
M.G.L. PINTO ◽  
M.I.B. RUBIN ◽  
C.A.M. SILVA ◽  
T.F. HILGERT ◽  
M.F. SÁ FILHO ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Mineral Oil ◽  
Control Group ◽  
Sodium Pyruvate ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Bovine Oocytes

O desenvolvimento embrionário de oócitos bovinos maturados in vitro (MIV) foi avaliado em meio suplementado com líquido folicular eqüino (Lfe). Foram distribuídos 1045 oócitos em 11 repetições formando três grupos tratamentos (T1, T2, T3) e um controle (C). O meio de maturação utilizado foi o TCM-199 acrescido de piruvato de sódio, hormônio folículo estimulante recombinante (rFSHh) e hormônio luteinizante equino (LHe). Suplementou-se esse meio com 10% de soro de égua em estro para o grupo controle e para T1, T2 e T3, o meio foi suplementado com 5, 10, e 20% de LFe, respectivamente. Os oócitos foram maturados in vitro (MIV) por 24h. A fecundação in vitro (FIV) foi realizada em meio Talp-Fert. A MIV e a FIV foram realizadas em estufa a 39ºC com 5% de CO2 em ar e umidade saturada. Os zigotos foram cultivados em meio SOFaaci, sob óleo mineral no interior de bolsas plásticas gaseificadas. As taxas de clivagem e de blastocistos foram observadas diariamente (D), e em D7, foram superiores (P0,05) às do grupo controle. Em D9, a taxa de blastocistos do T2 foi superior (P0,05). O LFe, na concentração de 10% pode ser utilizado, em substituição ao soro de égua em estro para suplementar o meio de MIV de oócitos bovinos. Equine follicular fluid on in vitro maturation of bovine oocytes Abstract Embryo development of bovine oocytes was evaluated using maturation medium supplemented with equine follicular fluid (eFF). One thousand and forty five (1045) oocytes were distributed in 11 replications forming three treatment groups (T1, T2 e T3) and one Control (C). TCM-199 added with sodium pyruvate, rFSHh and LHe was used as maturation medium. This medium was supplemented with 10% estrous mare serum for Control group, and 5, 10, and 20% eFF, respectively, for T1, T2 e T3 groups. In vitro maturation (IVM) of all groups was performed during 24h. In vitro fertilization (IVF) was performed in TALP-FERT medium. IVM and IVF were carried out in an incubator at 39ºC with 5% CO2 in air and saturated humidity. Zygotes were cultured in SOFaaci medium, under mineral oil in gasified bags. Cleavage and blastocyst rates were daily observed (D), and at D7, were higher (P0.05) for those from control group. At D9, blastocyst rate of T2 was higher (P0.05). The eFF, at a 10% concentration, can replace the use of estrous mare serum to supplement the IVM medium of bovine oocytes.

scholarly journals Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 861-874 ◽  
Author(s):  
Korakot Nganvongpanit ◽  
Heike Müller ◽  
Franca Rings ◽  
Michael Hoelker ◽  
Danyel Jennen ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Polar Body ◽  
Cell Mass ◽  
Free Water ◽  
Double Stranded Rna ◽  
Immature Oocytes ◽  
Selective Degradation ◽  
First Polar Body ◽  
Bovine Oocytes

RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2,in vitroproduced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P<0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P<0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C-mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis.

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