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

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.

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139 BOVINE PREIMPLANTATION EMBRYOS SECRETE EXTRACELLULAR VESICLES, WHICH MAY INDICATE EMBRYO COMPETENCE

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab139 ◽

2017 ◽

Vol 29 (1) ◽

pp. 178

Author(s):

E. Mellisho ◽

A. Velasquez ◽

M. J. Nuñez ◽

L. Rodriguez-Alvarez

Keyword(s):

Extracellular Vesicles ◽

Culture Media ◽

Total Cell ◽

Developmental Competence ◽

Preimplantation Embryos ◽

Lower Amount ◽

Bovine Embryos ◽

Significant Difference ◽

Blastulation Rate

Pre-implantation embryos secrete extracellular vesicles (EV) most likely to communicate with the surroundings. The objective of this study was to determine the distribution (size and concentration) of EV secreted by bovine pre-implantation embryos with different developmental competence. The IVF bovine embryos were produced from oocytes recovered from slaughterhouse ovaries. Presumptive zygotes were in vitro cultured (IVC) in groups in 4-well plates (30 zygotes per 500-µL well) using SOFaa medium at 39°C under 5% CO2, 5% O2, and 90% N2 until the morula stage (Day 5 post IVF). Morulae were cultured individually in 96 well at 39°C under until blastulation time (Day 6.5–7.5) in EV-free SOF medium. Culture medium was collected only from embryos that developed to the blastocyst stage that were classified in a group of early (Day 6.5) or late (Day 7.5) blastulation. Blastocysts were kept in culture until Day 11 to assess embryo developmental competence, considering embryo size (>350 µm) and total cell count (>500 blastomeres). For EV analysis, 4 groups were organised a posteriori: G1: Day 6.5-competent; G2: Day 6.5-not competent; G3: Day 7.5-competent; G4: Day 7.5-not competent. The EV in culture media were analysed using a nanoparticle tracking analysis (Nanosight NS300). Statistical analysis was performed using the InfoStat program (Buenos Aires, Argentina). Differences were considered significant at P < 0.05. Early blastulation rate (Day 6.5) was 40.3% (112/278), whereas late blastulation rate (Day 7.5) was 20.5% (57/278), showing a significant difference (P < 0.0001). Embryos derived from Day 6.5 blastocysts have a higher probability (39.3%: 44/112) of posthatching development [until Day 11; Day 7.5, 10.5% (6/57); P = 0.0001]. At Day 11, competent embryos (G1) derived from Day 6.5 blastocysts have a higher diameter and total cell number (447 µm; 688 cells) than those derived from Day 7.5 blastocysts (G3; 405 µm, 598 cells; P < 0.05 for both parameters). It was possible to detect EV from collected medium of individual embryos independent of their competence. Neither the EV size nor the EV concentration was statistically different between Day 6.5 and Day 7.5 blastocysts (without considering their further competence; 2.9 × 108, 147 nm; and 3.0 × 108, 149 nm, respectively). However, independent of the day of blastulation, competent embryos had a significantly lower concentration of EV (2.7 × 108 v. 3.3 × 108; P = 0.03). Moreover, competent embryos from early and late blastocysts (G1 and G3) tend to produce a lower amount of EV (G1: 2.8 × 108; G2: 3 × 108; G3: 2.6 × 108; G4: 3.5 × 108; P = 0.05). Furthermore, EV concentration was statistically different between G3 and G4 (P = 0.002). No differences in EV size were observed among groups (G1: 145 nm; G2: 148 nm; G3: 146 nm; G4: 151 nm). Our results provide an initial approach to study the EV secreted by individual pre-implantation embryos to assess their competence. From these results, we can conclude that blastulation time affects the future development of bovine embryos and a model based on blastulation time and EV secretion could be a simple noninvasive tool to improve embryo selection.

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79 MicroRNAs of extracellular vesicles secreted by embryos as an early biomarker of competence

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab79 ◽

2020 ◽

Vol 32 (2) ◽

pp. 166

Author(s):

B. Melo-Baez ◽

Y. S. Wong ◽

J. Cabezas ◽

C. J. Aguilera ◽

F. O. Castro ◽

...

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Extracellular Vesicles ◽

Culture Media ◽

Blastocyst Stage ◽

Fold Change ◽

Target Cells ◽

Bovine Embryos ◽

Log2 Fold Change ◽

Transmission Electron

Extracellular vesicles (EVs), including exosomes and microvesicles, are secreted by different cell types and participate in cellular communication by carrying molecules as microRNAs (miRNAs) that can interfere with gene expression of target cells. Extracellular vesicles have become relevant as a mechanism of embryo-maternal communication. The aim of this study was to evaluate miRNA content in EVs secreted after embryonic genome activation, by bovine embryos with different developmental potential. Bovine embryos were produced invitro and cultured in group until Day 3.5 in synthetic oviductal fluid (SOF) medium. Only 8-16-cell embryos were cultured individually in EVs-depleted SOF until Day 5. The SOF was EV depleted by ultrafiltration. Culture media (CM) were collected at Day 5 and embryos continued in culture until Day 7 with fresh SOF. Collected media were conserved individually and identified with the corresponding embryo. Then, CM were classified according to capacity of its embryo to reach blastocyst stage at Day 7: G1-CM (blocked embryos in 8-16 cell) and G2-CM (embryos that reach blastocyst stage). The EV isolation was carried out using the protocol described by Mellisho et al. (2017). Recovered EVs were evaluated by nanoparticle tracking analysis (NTA), Transmission electron microscopy and the presence of surface markers (CD9, CD63, CD81, and CD40L). After NTA, individual CM were pooled to organise 3 replicates of 10CM each, for G1 and G2. The whole miRNA isolation, library preparation, and sequencing was performed by Norgen Biotek facilities (Canada). The quality of libraries was analysed using the FastQC program platform followed by Trimmomatic to remove remnant adapters. For the miRNA library it accepted reads with value above 30 Phreads and 22 to 30bp length. The reads were mapped against the reference genome ARS-UCD1.2 using Bowtie2 software and miRDeep2 mapper, and the gene counts were calculated using HTSeq. Differential expression analysis was performed in EdgeR package. To expand this information, principal component analysis, Heatmap, and Volcano plot were plotted and pathway enrichment analysis was conducted. The NTA, transmission electron microscopy, and flow cytometry confirmed the presence of exosomes and microvesicles in isolated EVs. According to NTA, the mean size of EVs was 102.1-176.2nm and concentration of 8.4×107-8.6×108 particlesmL−1 in G1 and G2, respectively. We identified 96 miRNAs significantly expressed across the samples. Only eight miRNAs in EVs were differentially expressed between groups (G2 vs. G1). The bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were up-regulated (Log2 fold-change>1), whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were down-regulated (Log2 fold-change<1). The more significant (P-value<0.01) up-regulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The EVs secreted by embryos to culture environment carry miRNAs that can reflect the molecular state of their parental cell. This lets us suggest culture media derived-EVs and their miRNA cargo as early biomarkers to select more competent bovine embryos. This research was supported by FONDECYT, Chile (1170310).

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P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

Human Reproduction ◽

10.1093/humrep/deab130.218 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Á Martíne. Moro ◽

I Lamas-Toranzo ◽

L González-Brusi ◽

A Pérez-Gómez ◽

P Bermejo-Álvarez

Keyword(s):

Cumulus Cell ◽

Cumulus Cells ◽

Oocyte Quality ◽

Blastocyst Stage ◽

Early Embryo ◽

Developmental Competence ◽

Success Rates ◽

Developmental Potential ◽

Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p > 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

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

Reproduction Fertility and Development ◽

10.1071/rd17446 ◽

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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178 IMPROVED DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS BY PGI2 ANALOG TREATMENT

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab178 ◽

2006 ◽

Vol 18 (2) ◽

pp. 197 ◽

Cited By ~ 2

Author(s):

B. S. Song ◽

J. S. Kim ◽

D. B. Koo ◽

J. S. Park ◽

K. K. Lee ◽

...

Keyword(s):

Inner Cell Mass ◽

Culture Media ◽

Cell Mass ◽

Developmental Rate ◽

Treated Group ◽

Developmental Competence ◽

Bovine Embryos ◽

Frozen Semen ◽

Oviductal Fluid

The microenvironment of the follopian tube, in which the oviductal fluid contains a variety of cytokines and growth factors, affects pre-implantation development of fertilized embryos in mammals. Prostaglandin I2 (PGI2, prostacyclin) exists in oviductal fluid and is synthesized from arachidonic acid by prostacyclin synthetase. PGI2 also enhances the implantation rate of mouse embryos. In this study, the effect of PGI2 analog on the development of bovine embryos was examined. Bovine cumulus oocytes complexes (COCs) were matured in TCM-199 medium supplemented with 10 IU/mL pregnant mare serum gonadotropin (PMSG), 10 IU/mL hCG, and 10 ng/mL epidermal growth factor (EGF) at 39�C, 5% CO2 in air for 20-22 h. Following in vitro maturation, COCs were fertilized in Fert-TALP medium containing 0.6% BSA using frozen semen. Also, oocytes matured in vitro were enucleated, individually reconstructed with bESF cells, fused, and then activated by treatment with 5 �M ionomycin for 5 min and 2 mM 6-DMAP for 4 h. In vitro-fertilized (IVF) and nuclear-transferred (NT) eggs were cultured in 50 ��L drops of CR1-aa medium supplemented with 0.3% BSA in the absence or presence of 1 �M PGI2 analog at 39�C, 5% CO2 in air, respectively. At 3 days of culture, cleaved embryos were further cultured in the same culture media supplemented with 10% FBS for 4 days. Allocations of blastocysts to inner cell mass (ICM) and trophoblast (TE) cells were investigated to assess embryo quality. All experiments were repeated more than three times. All data were analyzed by using the Duncan test of ANOVA by the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA) and numbers of nuclei in blastocysts were expressed as mean � SE. No difference was detected in the cleaved rate of the eggs between the treated- and nontreated groups. IVF zygotes treated with PGI2 analog represented a higher developmental rate (33%, 122/418) to the blastocyst stage than nontreated controls (24%, 107/456) (P < 0.05). Among IVF-derived blastocysts, interestingly, the proportion (46%, 84/181) of expanded blastocysts was significantly higher in the PGI2 analog-treated group compared with that in the nontreated group (28%, 46/164). The number of nuclei in (165 � 6.1, n = 15) in blastocysts in the PGI2 analog-treated group was higher than that (146.12 � 5.7, n = 18) in the nontreated group (P < 0.05). No difference was detected in the ratio of ICM to total cells between PGI2 analog-treated (42.0 � 3.0%) and nontreated groups (41.9 � 2.9%). Like the IVF embryos, NT embryos in the PGI2 analog-treated group showed a higher in vitro developmental rate (33.6%, 43/128) than the nontreated embryos (24.2%, 32/132) (P < 0.05). Our results indicate that PGI2 analog improves the kinetics of embryo development in cattle.

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58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab58 ◽

2009 ◽

Vol 21 (1) ◽

pp. 129

Author(s):

J. G. Zhao ◽

J. W. Ross ◽

Y. H. Hao ◽

D. M. Wax ◽

L. D. Spate ◽

...

Keyword(s):

Histone Acetylation ◽

Histone Deacetylases ◽

Nuclear Transfer ◽

Blastocyst Stage ◽

Untreated Group ◽

Developmental Competence ◽

Developmental Potential ◽

Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

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Generation of parthenogenetic goat blastocysts: effects of different activation methods and culture media

Zygote ◽

10.1017/s0967199413000580 ◽

2014 ◽

Vol 23 (3) ◽

pp. 327-335 ◽

Cited By ~ 5

Author(s):

Hruda Nanda Malik ◽

Dinesh Kumar Singhal ◽

Shrabani Saugandhika ◽

Amit Dubey ◽

Ayan Mukherjee ◽

...

Keyword(s):

Culture Media ◽

Combined Treatment ◽

Blastocyst Stage ◽

Developmental Competence ◽

Cleavage Rate ◽

Cell Stage ◽

Quantitative Expression ◽

Pattern Of Variation ◽

Better Than

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.

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Extracellular vesicles secreted during blastulation show viability of bovine embryos

Reproduction ◽

10.1530/rep-19-0233 ◽

2019 ◽

Vol 158 (6) ◽

pp. 477-492 ◽

Cited By ~ 7

Author(s):

Edwin A Mellisho ◽

Mario A Briones ◽

Alejandra E Velásquez ◽

Joel Cabezas ◽

Fidel O Castro ◽

...

Keyword(s):

Extracellular Vesicles ◽

Culture Media ◽

Differential Expression Analysis ◽

Binary Logistic Regression ◽

Small Rna Sequencing ◽

Bovine Embryos ◽

Embryo Viability ◽

Group V ◽

Mean Size

Extracellular vesicles (EVs) secreted by blastocysts may be clinically relevant, as indicator of embryo viability on in vitro fertilization. We tested if the characteristics of EVs secreted during blastulation are related to embryo viability. Morulae were individually cultured in SOF media depleted of EVs until day 7.5 post IVF. Viable embryos were determined by a system of extended in vitro culture of bovine embryos until day 11 (post-hatching development). Afterward, a retrospective classification of blastocyst and culture media was performed based on blastulation time (early blastulation (EB) or late blastulation (LB)) and post-hatching development at day 11 (viable (V) or non-viable embryo (NV)). A total of 254 blastocysts and their culture media were classified in four groups (V-EB, NV-EB, V-LB, NV-LB). Group V-EB had a larger blastocyst diameter (170.8 μm), higher proportion of good-quality blastocysts (77%) and larger mean size of population of EVs (122.9 nm), although the highest concentration of EVs (5.75 × 109 particles/mL) were in group NV-EB. Furthermore, small RNA sequencing detected two biotypes, miRNA (86–91%) and snoRNA (9–14%), with a total of 182 and 32 respectively. In differential expression analysis of miRNAs between V versus NV blastocysts, there were 12 miRNAs upregulated and 15 miRNAs downregulated. Binary logistic regression was used to construct a non-invasive novel model to select viable embryos, based on a combination of variables of blastocyst morphokinetics and EVs characteristics, the ROC-AUC was 0.853. We concluded that characteristics of EVs secreted during blastulation vary depending on embryo quality.

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314EFFECT OF MACROMOLECULE SUPPLEMENTATION DURING IN VITRO MATURATION ON THE DEVELOPMENTAL COMPETENCE OF GOAT OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab314 ◽

2004 ◽

Vol 16 (2) ◽

pp. 276

Author(s):

J.R. Herrick ◽

E. Behboodi ◽

E. Memili ◽

S. Blash ◽

Y Echelard ◽

...

Keyword(s):

Amino Acids ◽

In Vitro Maturation ◽

Mixed Model ◽

Linear Mixed Model ◽

Blastocyst Stage ◽

Developmental Competence ◽

Similar Proportion ◽

Developmental Potential ◽

Cell Counts

In vitro maturation of goat oocytes has traditionally involved the use of serum or BSA. However, these products introduce variability and complicate evaluation of the effects of other medium components. The objective of this study was to examine the effects of citrate and hyaluronate in the absence or presence of BSA during IVM on the developmental competence of goat oocytes. Abattoir-derived, cumulus-oocyte complexes (COC) were matured for 20–22h (6.0% CO2 in air, 38.7°C) in modified SOF medium (1.5mM glucose, 3.0mM L-lactate, 0.1mM pyruvate, 1.0mM glutamine, 0.1mM taurine) supplemented with 1×MEM nonessential amino acids, 0.5×MEM essential amino acids, 1×MEM vitamins, 0.1mM cysteamine, 5μg mL−1 insulin, 5μgmL−1 transferrin, 5ng mL−1 selenium, 50ngmL−1 EGF, 0.01U mL−1 LH and FSH, and 50μgmL−1 gentamicin. Treatments were: (1) 1mgmL−1 PVA (protein-free, defined); (2) 4mgmL−1 BSA (semi-defined); (3) 0.5mM citrate and 0.5mgmL−1 hylauronate (C+H, defined); and (4) 0.5mM citrate and 0.5mgmL−1 hylauronate with 4mgmL−1 BSA (C+H+BSA, semi-defined). At the end of IVM, COC were transferred to modified Brackett and Oliphant’s medium with 7.7mM Ca-(l)-lactate and 20% FCS for IVF. Frozen-thawed sperm were processed through a 45%:90% Percoll gradient and added to IVF drops (50μL) containing COC at a final concentration of 14–15×106 spermmL−1. Gametes were coincubated in the presence of heparin (25μgmL−1) for 22–24h in 7% CO2 in air at 38.7°C. After coincubation, cumulus cells were removed and zygotes were cultured (6% CO2, 5% O2, 89% N2, 38.7°C) in G1 v.3 for 3 days followed by 4 days in G2 v.3. Cleavage was evaluated when embryos were moved to G2, and development to the blastocyst stage was assessed at the end of culture. All blastocysts were fixed and stained with Hoechst 33342 for total cell counts. Analysis of variance was performed using the general linear mixed model macro of SAS. Means are presented ±SEM and probability values P<0.05 were considered significant. The use of BSA did not improve (P>0.05) the developmental potential of goat oocytes (Table 1). Furthermore, a similar proportion (P>0.05) of oocytes developed to the blastocyst and hatching blastocyst stage after maturation under defined conditions compared to oocytes matured with BSA. In conclusion, developmentally competent goat oocytes can be produced by IVM under defined conditions. Table 1 Development of goat oocytes following IVM with different macromolecules.

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160 In vitro maturation of pre-pubertal goat oocytes and their development after chemical activation

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab160 ◽

2019 ◽

Vol 31 (1) ◽

pp. 205

Author(s):

N. A. Wani ◽

S.-B. Hong

Keyword(s):

In Vitro Maturation ◽

Culture Media ◽

Chemical Activation ◽

Blastocyst Stage ◽

Cleavage Rate ◽

Developmental Potential ◽

Epidermal Growth ◽

Humidified Air ◽

Activation Status

Experiments were conducted to study in vitro maturation of pre-pubertal goat oocytes and their developmental potential after chemical activation. Cumulus-oocyte complexes (n=1170) collected from the ovaries of pre-pubertal goats slaughtered at a local abattoir were matured in TCM-199 supplemented with 0.15mg mL−1 l-glutamine, 0.25mM sodium pyruvate, 0.1mM l-cysteine, 20ng mL−1 epidermal growth factor, 10mg mL−1 FSH, 10mg mL−1 LH, 1μg mL−1 oestradiol and 10% FCS for 24h at 39°C under 5% CO2 in humidified air. In Experiment 1, matured oocytes were activated (r=6) with either 5mM ionomycin (n=85) or 7% ethanol (n=91) followed by culture in 6-DMAP for 4h. All the activated oocytes were then cultured in KSOM supplemented with 3mg mL−1 BSA and were fixed and stained with Hoechst 33342 after 18h of culture to evaluate their activation status. In Experiment 2, oocytes activated with 5mM ionomycin and 6-DMAP were cultured for 7 days (r=6) in 1 of the 4 different culture media [Charles Rosenkrans medium (CR-1), modified TCM-199, KSOM and SOF] to study their developmental potential. All media were supplemented with 3.0mg mL−1 BSA for the first 3 days and 10% FCS for the subsequent 4 days. Of these pre-pubertal oocytes, 59% reached metaphase II stage, and 83% of these oocytes were classified as activated in the group using ionomycin in comparison with 69% in the group using ethanol as an activating agent (P<0.05). No difference was observed in the cleavage rate of activated oocytes cultured in any of the 4 culture media (65.7v. 55.0v. 61.0v. 56.2%, respectively). However, the development to blastocyst stage was observed in only KSOM (16%) and SOF (5%) media. In conclusion, the present study demonstrates that pre-pubertal goat oocytes can mature in vitro and can be activated with 5mM ionomycin, and KSOM, and to a lesser extent SOF, supports development to the blastocyst stage. We plan to use these oocytes as a cytoplast source for interspecies somatic cell NT; however, before that, more studies are needed to evaluate their requirements in culture media to enhance their development to the blastocyst stage.

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