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Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.

The high metabolic activity to which the dairy cattle are exposed to maintain milk production altered steroid metabolism that affects reproductive physiology and reduce oocyte competence. Our aims were (a) to characterize the competence of immature oocytes collected from dairy cattle based on the expression of genes in cumulus cells (CCs) and (b) to improve oocyte competence to support preimplantation embryo development by the supplementation of maturation medium with bone morphogenetic protein 15 (BMP15) and/or anti-mullerian hormone (AMH). Oocyte donors were identified at the moment of ovary collection and grouped by involuntarily culled dairy cows (Holstein breed) or beef cattle. The embryo development speed to blastocyst of the cull dairy cattle versus beef cattle (control group) was lower. Besides

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.

For invitro embryo production, spermatozoa with fertilizing capacity must possess optimal kinetic, morphometric, vitality, and DNA integrity characteristics. The objective of this study was to evaluate the effect of 3 sperm selection methods on sperm quality and invitro embryonic development in bovine. Frozen commercial semen (0.5 mL/straws) from one bull with known fertility was thawed at 37°C for 20s and was divided for 3 sperm selection techniques: density gradient, swim-up, and microfluidic sperm sorting. The sperm kinetic parameters (VCL=curvilinear velocity, VSL=straight line velocity, VAP=average path velocity, ALH=lateral displacement of sperm head, BCF=beat frequency cross, STR=path straightness) were assessed using computer-assisted sperm analysis (CASA). Sperm morphometric parameters were evaluated using Diff-Quick staining followed by automated analysis. To assess vitality, the sperm were stained with propidium iodide and acridine orange, then analysed under a fluorescence microscope. In addition, DNA fragmentation was assessed using sperm chromatin dispersion method. Last, the fertilizing capacity of the selected sperm was tested by fertilizing cumulus–oocyte complexes (2×106 sperm mL−1) obtained from slaughterhouse ovaries and matured invitro for 24h. A standardized invitro embryo production protocol was used with commercial medium from Vitrogen. The cleavage rate and blastocyst yield were measured on Day 2 and 7, respectively (fertilization=Day 0). The results were calculated with analysis of variance and Tukey’s test (P0.05) between the 3 sperm selection techniques: density gradient (84.0%), swim-up (75.2%), and microfluidic sorting (67.3%). However, blastocyst yield was significantly higher (P

Oocyte protection against reactive oxygen species (ROS) during invitro maturation (IVM) may play a decisive role in pre-implantation embryonic development. For instance, anthocyanins have shown greater antioxidant effects than vitamins C and E. The objective of this study was to determine the anthocyanin supplementation level that influences quantity and quality of bovine blastocysts development during IVM. Cumulus–oocyte complexes (COC) were recovered from 185 abattoir ovaries in 6 sessions and classified (Grade 1 and 2) for maturation. Oocytes were in IVM in commercial medium (Vitrogen®) supplemented with anthocyanin (pelargonidin chloride) at different concentrations: 0 (control), 1, 10, 20, and 40μM, in droplets of 70μL with 12 to 15 COC at 38.5°C, 5% CO2 and 90% humidity for 22to 24h. Sperm selection was performed by Percoll gradient method (45/90%) with centrifugation at 600×g for 6min. The final concentration for IVF was 2×106 sperm mL−1. A total of 462 oocytes were used in the experiment (6 replicates). Presumptive zygotes were invitro cultured (IVC) in commercial medium (Vitrogen) in droplets of 70µL with 12–15 zygotes at 38°C, 5% CO2, and 90% humidity until the blastocyst stage (Day 7 of culture). The cleavage (Day 2), morulae (Day 4), and blastocyst (Day 7) rates were measured during IVC. The data were processed with non-parametric tests (Kruskal–Wallis test with independent samples, P0.05). In conclusion, the supplementation of anthocyanin to the maturation medium did not affect invitro development of bovine embryos. Complementary studies at the cellular and gene expression level may be required.

Preantral follicles are the largest ovarian follicle population and represent an important source of potentially competent oocytes. During the lifespan of the female this large population becomes atretic during their growth. In alpacas, there are few studies that estimate the number of preantral follicles. Therefore, the objective of the present study was to compare the population and morphology of preantral follicles in the ovaries of fetal and adult alpacas. Ovaries from alpacas in fetal (fetus during the last third of gestation, n = 5) and adult stage (3–4 years, n = 5) were collected at a local slaughterhouse. The whole ovaries were individually fixed overnight at room temperature, and later dehydrated in alcohol, cleared with xylene, and embedded in paraffin. Tissue were sectioned at 7 μm with a rotating microtome. Then, sections were processed and stained with periodic acid Schiff and haematoxylin. Preantral follicles were classified for their development stage as primordial, transitional, primary, or secondary, according to the layer number and form of granulosa cells. Estimation of the number of preantral follicles was made by counting all follicles in each histological section. Only follicles in which the oocyte nucleus was visible were counted. In addition, for each follicle category (n = 30 per group), oocyte and follicle diameters were measured using Motic Images Plus 2.0 software. The population estimate and follicular diameter were compared using Kruskal–Wallis test with significance set at P ≤ 0.05 using SPSS v.2 2 software (IBM Corp.). A total of 2174 histologic sections were analysed. The results showed a higher (P = 0.045) number of preantral follicles (80 516.1 ± 3623.9) for fetal alpacas compared with adult alpacas (67 870.8 ± 2267.4). Also, primordial follicles population (31 543.4 ± 2690) and morphologically normal follicles (98.2%) were higher (P = 0.04) in fetus compared with those in the adult stage (2244.7 ± 355.37; 76.35%) respectively. On the contrary, the diameters of primordial, transitional, and primary follicles (45.34 ± 3.76; 52.38 ± 6.22; 59.79 ± 5.22 µm) from adult alpaca were greater (P = 0.04) than those of fetal preantral follicles (33.305 ± 7.2; 36.715 ± 3; 77.985 ± 15.8 µm). In conclusion, the preantral follicle population declines dramatically in adult alpaca and animals of this age show an increased percentage of degenerate primordial follicles.

In mammals, folliculogenesis begins at the fetal stage and is a complex, dynamic process that involves follicular quiescence, activation, growth, follicular migration, and cell interactions. At birth, the preantral ovarian follicular population, drastically reduced, constitutes >90% of all ovarian follicles, representing the ovarian reserve that will be used throughout the reproductive life. In alpacas, changes in follicular wave growth patterns and ovulation are different from other species; thus, it is important to know the ovarian reserve at fetal stage, as a starting point for future studies. Therefore, the aim of the present study was to establish the morphological characterisation and estimate the population of alpaca preantral follicles in the fetal stage. Ovaries from alpacas (n=5) in the fetal stage (fetus during the last third of gestation) were collected at a local slaughterhouse. Whole ovaries were individually fixed in 4% paraformaldehyde phosphate-buffered saline overnight at room temperature for routine histology. Ovaries were dehydrated in alcohol, cleared with xylene, and embedded in paraffin, and all tissue was serially sectioned at 7μm with a rotating microtome (Leica). The histological sections were mounted and stained with periodic acid-Schiff and hematoxylin. Preantral follicles were classified according to their developmental stage: primordial (one layer of flattened granulosa cells surrounding the oocyte), primary follicle transition (flattened cuboidal granulosa cells surrounding the oocyte), primary (single layer of cuboidal granulosa cells around the oocyte), or secondary (oocyte surrounded by more than one complete layer of cuboidal granulosa cells). We estimated the number of preantral follicles by counting all follicles in each histological section. Only follicles in which the oocyte nucleus was visible were counted. In addition, for each follicle category (n=40 per group), oocyte and follicle diameters were measured using an ocular micrometer. The variable means were compared using unpaired Student's t-test analysis, with significance set at P ≤ 0.05. Estimation of preantral follicular population (mean±standard deviation) was 80 516±14 575 in the ovaries of alpaca fetuses. Most of the follicles found belong to the primordial (49.2%) or primary follicle transition (39.2%) categories, followed by primary (10.8%) and secondary (0.8%) stages. Follicle and oocyte diameters of primordial (33.3±7.2; 21.5±4.6μm) and primary follicle transition stages (36.7±3.0; 23.4±2.6μm) were significantly (P

Extracellular vesicles (EV) are currently considered a mechanism of cell communication. These are secreted from different cell types, including embryos, to serve as mediators of short and long distance signals. EV can be identified in vivo in different biological fluids, as well as in vitro embryo culture medium. Usually, media used for embryo in vitro culture are supplemented with serum or other protein sources that favour cell proliferation and development. Serum and protein sources contain EV, including microvesicles and exosomes that in principle can be internalized by embryonic cells. The aim of this study was to evaluate if serum-derived EV are internalized by the embryo at different stages of the early development, and if EV from the serum are required for in vitro bovine embryo development. For that, it was first evaluated if EV depleted culture media affect embryo development up to the blastocyst stage; oocytes were in vitro matured for 22 to 23h and in vitro fertilized for 18h. Posteriorly, presumptive zygotes were in vitro cultured in groups (25 embryos/well in 4-well plates) in SOF or SOF depleted of EV for 8 days. To evaluate EV internalization, culture media was supplemented with labelled EV and confocal imaging was performed. The EV were obtained by ultrafiltration (centrifugal filter devices 100 kDa, Amicon; Millipore, Billerica, MA, USA) for 15min at 3000 rpm. Then, EV were stained with PKH67 dye and washed 3 times with PBS by ultrafiltration to remove excess dye. The EV labelled with PKH67 were resuspended in SOFaa depleted of EV (3×109 particles per 500µL) and supplemented for 24h at the 1-cell stage (Day 1 post IVF), 16 cells (Day 4 post IVF), and early blastocyst (Day 6 post IVF) in 5% CO2, 5% O2, and 90% N2. PBS with PKH67 dye was used as a control treatment. Hoechst 33343 was used to label the nuclei before washing with PBS and fixation with 0.4% paraformaldehyde. Images were acquired on a Zeiss (Zeiss, Jena, Germany) LSM 780 confocal microscope. There were no statistical differences on blastocyst rate at Day 8 between embryos cultured in SOF depleted of EV (19.5%) and control group (SOF; 22.7%; P>0.05). We observed punctuated green fluorescence near the embryo nuclei in the 3 stages studied in embryos supplemented with EV but not in the control treatment, which indicates that EV from serum are uptaken by embryonic cells in early development. Therefore, we demonstrated uptake of EV from fetal calf serum added to culture media, although its absence does not affect embryo development. Research was supported by FONDECYT, Chile (1170310).

Earlier we successfully isolated and characterised endometrial (eMSC) and adipose (aMSC) mesenchymal stem cells from the same donors. Mesenchymal stem cells share biological traits but display different surface marker phenotype and migration ability. Here we extended our research to their mRNA signature using next-generation sequencing. The RNA from cells (3 biological replicates from each cell type and 3 technical replicates) at 90% confluence was extracted using a total RNA extraction kit and sent for mRNA-Seq (Norgen, Ontario, Canada; Illumina Sequencing Platform NextSEqn 500). Raw 76-bp single-end reads were aligned against the EquCab3 genome using RNA-STAR aligner. Counts were filtrated at a minimum of 5. Pairwise comparisons between the cell types were the input for gene ontology enrichment analysis. Only genes differentially expressed (DE) with 5 folds change (FC; P50×: 8 and 13; >20×, 10×, 5×, 2×

Extracellular vesicles (EV) secreted by blastocysts might be relevant to predict competence of embryos produced in vitro. The aim of this study was to develop a model to select competent embryos that combines blastocyst morphokinetics data and morphological parameters of EV secreted during blastulation (Days 5-7.5). Embryos were cultured in groups up to Day 5; morulae were selected and individually cultured in SOFaa depleted of EV until Day 7.5 after IVF. Embryo competence was determined by in vitro post-hatching development up to Day 11. A retrospective classification of blastocyst and culture media was performed based on blastulation time [early (EB) or late (LB)] and competence at Day 11 [competent (C) or non-competent (NC)]. The EV were isolated from culture media of individual embryos, their properties determined by nanoparticle tracking analysis. The model was based on a binary logistic regression to describe the dichotomous-dependent variable of the blastocyst (C=1 and NC=0). A set of independent variables of blastocyst morphokinetics (blastulation time, blastocyst stage, blastocyst quality and blastocyst diameter at Day 7.5) and EV morphological parameters [mean size (ME), mode size (MO) and particle concentration (CO)] were analysed with multiple regression. The analysis generated the coefficients and their standard errors and significance level of an equation to calculate a probability, where values between 0.5 and 1 predict competent embryos. To verify the predictive power of the algorithm, the following indicators were used: the receiver operating characteristic with the determination of area under the curve, percentage correct predictions, and Omnibus tests. Statistical significance was determined at the P

Extracellular vesicles (EVs) have been identified within different body fluids and cell culture media. However, there is very little information on the secretion of these vesicles during early embryonic development. The aims of this work were first to demonstrate the secretion of extracellular vesicles by pre-implantation bovine embryos and second to identify and characterize the population of EVs secreted by bovine blastocysts during the period from day seven to nine of embryo culture and its correlation with further embryo development up to day 11. Bovine embryos were produced by in vitro fertilization (IVF) or parthenogenetic activation (PA) and cultured until blastocyst stage. Blastocyst selection was performed at day 7 post IVF/PA considering two variables: stage of development and quality of embryos. Selected blastocysts were cultured in vitro for 48 hours in groups (exp. 1) or individually (exp. 2) in SOF media depleted of exosomes. At day 9 post IVF/PA the media was collected and EVs isolated by ultracentrifugation. Transmission electron microscopy revealed the presence of heterogeneous vesicles of different sizes and population: microvesicles (MVs) and exosomes (EXs) of rounded shape, enclosed by a lipid bi-layer and ranging from 30 to 385 nm of diameter. Flow cytometry analysis allowed identifying CD63 and CD9 proteins as exosome markers. Nanoparticle tracking analysis generated a large number of variables, which required the use of multivariate statistics. The results indicated that the concentration of vesicles is higher in those blastocysts with arrested development from day 9 up to day 11 of in vitro development (6.7 x 108 particles/ml) derived from IVF (p

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

In alpacas, improvement of reproductive efficiency of male camelids is limited by the small size of the testes, extended period of ejaculation, and low quality of semen. This study was designed to determine the effect of 2 sperm preparation treatments before IVF on the cleavage rate. The sperm was obtained by slicing the head of the epididymis of slaughtered male alpacas (n = 8), diluting in Tris-yolk-glycerol, and freezing with the slow-cooling method. Frozen semen straws per each male were thawed in a water bath at 37°C for 15 s and evaluated for percentage of progressive motility (32 ± 8.6%) and concentration (66.5 ± 24 × 106 sperm mL–1) post-thawing. Sperm selection by the swim-up method was performed by centrifugation at 1077 × g for 5 min with washing sperm medium eliminating the supernatant; sperm were settled in inclined tube with fertilization medium (without capacitating agent) for 60 min, after which 100 μL from the surface was recovered for use in IVF. The washing method consisted in repeated washing (twice) of sperm in washing sperm medium and fertilization medium by centrifugation at 1077 × g for 5 and 3 min, respectively, and recovery of 50 μL from the bottom of the tube for use in IVF. Sperm selected by swim-up or washing methods had similar characteristics of progressive motility (18 and 23%); however, the concentration was higher for the washing v. swim-up method (52 v. 14 × 106 sperm mL–1, respectively). Cumulus-oocyte complexes (COC) were recovered from 278 ovaries of alpacas killed at abattoirs and classified (Grade 1 and 2) for in vitro maturation (38.5°C at 5% CO2 in air for 27 h in 50 μL of 10 COC per drop). A total of 839 oocytes cultured for 27 h in maturation medium were partially stripped out of cumulus cells by gentle aspiration with a pipette. Sperm suspensions in Fert TALP medium (5 μL) from each treatment group were added to each fertilization drop with 10 oocytes per drop of 45 μL obtaining a final concentration of 10 × 106 sperm mL–1 and cultivated for 72 h until their evaluation. The data for the 13 repetitions of the rate of cleavage (2 to 8 cells) were converted to angular values (angle = arcsin √%) with the object of normalizing the distribution of the data; the analysis of variance was performed (complete randomised design with sub-sampling, P

In alpacas, the male has a low reproductive performance due to the small size of the testes, extended period of ejaculation, and low quality of semen. This work had an objective to evaluate 2 methods of cryopreservation on sperm viability of spermatozoa obtained from the cauda epididymis of male alpacas. Testes from 19 male alpacas (>3 years old) were obtained from the slaughterhouse of Huancavelica and transported to the laboratory in isothermal conditions within 3 h of slaughter. The spermatozoa were obtained by slicing the head of the epididymis, diluting in tris-yolk-glycerol at environment temperature, and then refrigerating for 2.5 h at 5°C. The freezing process was carried out by 2 methods, slow cooling and rapid cooling, the results for percentage of progressive motility, vital staining (eosin nigrosin staining), and hypoosmotic swelling test for each method were evaluated. Cryopreservation of spermatozoa by slow cooling was using 0.25-mL straws immediately after the addition of the extenders and sealed with polyvinyl alcohol. The freezing procedure consisted of placing a metallic rack in a polystyrene foam box of 25 × 20 × 15 cm (length × width × height) and pouring LN (–196°C) within the box, keeping the level of LN below the surface of the metallic rack by 6 cm. The straws were placed onto the metallic rack exposing them to the vapors of the LN and closing the box hermetically by 10 min to freeze and then store by immersion in LN. The cryopreservation of spermatozoa by rapid cooling was carried out in pellets of 0.25 mL immediately finishing the addition of the extenders a final concentration of 60 × 106 sperm mL–1. The freezing process consisted of placing the suspension of spermatozoa in holes made on the surface of a block of solid carbon dioxide (dry ice, –79°C) with a micropipette placing aliquots of 0.25 cc quickly and successively, trying to not let the time between the first pellet formed and the last exceed a minute, and then stored by immersion in LN. Semen was thawed at 37°C in hot bath for 1 to 2 min for pellets and 30 s for straws. Percentage of progressive motility, vital staining, and hypoosmotic swelling test were analysed statistically using ANOVA at a significance level of P

The Peruvian Paso Horse has become a flagship product of Peru, for which it is very important to work on promoting and preserving their genetic material through semen. The objective of this study was to evaluate the thermoresistance of epididymal sperm of stallion frozen with 2 cryoprotectants (dimethylformamide and glycerol). The testes with the epididymides (n = 16) of 8 adult stallions in good health condition were obtained in the slaughter house and transport at isothermal conditions (30°C). Spermatozoa from the epididymis were recovered and incubated with lactated ringer's solution (Hartman solution) for 30 min, centrifuged at 2000 RPM for 15 min, and resuspended with an extender based on sucrose (7%) and egg yolk (3%) with different levels of cryoprotectants: dimethylformamide-DMF (T4%), glycerol-GLI (4%), and a combination of dimethylformamide-DMF (2%) + glycerol-GLI (2%) with an equilibrium time of 20 min at 5°C. The final dilution was 160 million motile spermatozoa placed on 0.5-mL straws. These straws were kept over liquid nitrogen fumes for 15 min over a tripod stand. After cryopreservation, the straws were stored in liquid nitrogen tanks (–196°C). The thawing process of the straws was initiated at 75°C for 7 s, immediately followed by an evaluation of sperm motility at 4 times 0, 30, 60, and 90 min in which sperm was incubated on water bath at 37°C (thermoresistance test). The data was evaluated (ANOVA) with the SAS 9.12 program (SAS Institute Inc., Cary, NC, USA) and, for the media, Tukey's test was carried out. The results showed an effect of the treatment on the motility of the frozen-thawed semen; dimethylformamide, the cryoprotectant, had the best performance. Table 1.Motility of frozen-thawed stallion epididymal sperm (thermoresistance sperm test)1

[This corrects the article DOI: 10.1371/journal.pone.0289751.]., (Copyright: © 2024 Huayhua et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)