Browsing by Author "Rizos, Dimitrios"

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Assessment of fertilizing ability of merino ram semen cold-stored up to 48h by heterologous in vitro fertilization of bovine oocytes
(CSIRO Publishing, 2019) Galarza Lucero, Diego Andrés; Ladrón de Guevara, Magdalena; Beltran Breña, Paula; Sánchez Calabuig, María Jesús; Lopez Sebastian, Antonio; Santiago Moreno, Julián; Rizos, Dimitrios; Galarza Lucero, Diego Andrés
The use of cold-stored ram semen has been applied in sheep artificial insemination programs, since it preserves its fertilizing ability similar to fresh. Besides, the heterologous in vitro fertilization (IVF) has been successfully employed to assess semen fertilizing ability in several species. Hence, we aimed to evaluate the fertilizing ability of ram semen cold-stored up to 48h at 5 ºC by assessing heterologous IVF using bovine oocytes. Fifteen pools of three normospermic Merino ram (2-7 years) ejaculates were collected using artificial vagina, diluted to 200x106 spermatozoa/ml with UHT-based extender (skim milk-6% egg yolk) and cold-stored up to 48h. In vitro matured zona-intact bovine oocytes were subjected to heterologous IVF using either fresh semen (FS, n=707), cold-stored to 24h (CS24, n=832) or cold-stored to 48h (CS48, n=611). In parallel, homologous IVF (Control, n=1356) and parthenogenesis (Parth Control non-fertilized oocytes n=334) were performed. Ram non-selected and selected (BoviPure) semen parameters were evaluated by CASA. Sperm-oocyte interaction was assessed at 2.5 hours post-insemination (hpi) by evaluating the number of bound spermatozoa whereas penetration and polyspermy were evaluated after 12 hpi. Presumptive zygotes were fixed and stained with Hoechst 33342 at 18, 20, 22, 24 and 26 hpi to assess pronuclear formation using phase contrast and confocal microscopy. Cleavage rate was evaluated in all groups at 48 hpi. Data obtained from 5 replicates was analysed using one-way ANOVA. Data was expressed as mean ± SEM. In terms of sperm storage time, non-selected semen showed a significant decrease (p<0.05) for CS24 and CS48h compared to FS on progressive motility [SPM (%): 52.30±4.1 and 36.9±5.5 vs 71.3±1.6] and straight-line velocity [VSL (mm/sec): 132.2±6.1 and 109.7±6.3 vs 176.7±4.3], respectively. However, selected semen showed a decrease (p<0.05) only for CS48h when compared to CS24h or FS on SPM (35.6±3.9 vs 56.1±6.91 and 59.3±2.6) and VSL (83.5±4.4 vs 105.3±6.5 and 110±2.0), respectively. No differences were observed between heterologous IVF groups in all parameters evaluated. Homologous IVF showed a higher percentage of penetration only when compared to heterologous FS group (44.4±6.8 vs 12.5±4.5%, p<0.01). The polyspermy was higher in heterologous CS24 group when compared to homologous IVF (11.4±3.4 vs 3.8±2.2, p<0.05). The homologous IVF group, as expected, showed the higher percentage of pronuclear formation at 18 hpi than heterologous IVF with FS (67.3±5.8 vs 35.2±5.6%), CS24 (72.1±4.5 vs 37.2±5.7%) and CS48 (63.0±6.0 vs 27.0±5.6%), respectively (p<0.001). Likewise, cleavage rate was higher in homologous group compared to heterologous IVF and parthenogenetic groups for FS (78.3±2.6.8 vs 46.3±3.2 and 7.0±2.3%), CS24 (78.4±2.6 vs 48.3±3.2 and 4.9±2.0%), and CS48 (78.4±3.3 vs 43.3±3.5 and 4.3±1.2%), respectively (p<0.001). In conclusion, Merino ram semen cold-stored up to 48h maintains its fertilization ability in the same extend as fresh and can be used for sheep crossbreeding programs
Characterization and profiling analysis of bovine oviduct and uterine extracellular vesicles and their miRNA cargo through the estrous cycle
(2021) Hamdi, Meriem; Cañon Beltrán, Karina; Cajas, Yulia N.; Mazzarella, Rosane; Verde Leal, Cláudia Lima; Gutierrez Adan, Alfonso; Gonzalez Martínez, Maria Encina; Da Silveira, Juliano; Rizos, Dimitrios
Extracellular vesicles (EVs) found in various biological fluids and particularly in reproductive fluids, have gained considerable attention for their possible role in cell- to- cell communication. Among, the different bioactive molecules cargos of EVs, MicroRNAs (miRNAs) are emerging as promising diagnostic biomarkers with high clinical potential. Aiming to understand the roles of EVs in bovine reproductive tract, we intended to characterize and profile the EVs of oviduct and uterine fluids (OF-EVs, UF-EVs) and their miRNA across the estrous cycle. Nanoparticle tracking analysis and transmission electron microscopy confirmed the existence of small EV population in OF and UF at all stages, (size between 30 and 200 nm; concentration: 3.4 × 1010 EVs/ml and 6.0 × 1010 EVs/ml for OF and UF, respectively, regardless of stage). The identification of EV markers (CD9, HSP70, and ALIX proteins) was confirmed by western blot. The miRNA analysis revealed the abundance of 310 and 351 miRNAs in OF-EVs and UF-EVs, respectively. Nine miRNAs were differentially abundant in OF-EVs between stages of the cycle, eight of them displayed a progressive increase from S1 to S4 (p <.05). In UF-EVs, a total of 14 miRNAs were differentially abundant between stages. Greater differences were observed between stage 1 (S1) and stage 3 (S3), with 11 miRNAs enriched in S3 compared to S1. Functional enrichment analysis revealed the involvement of these miRNAs in relevant pathways such as cell signaling, intercellular junctions, and reproductive functions that may be implicated in oviduct and uterus modulation across the cycle, but also in their preparation for embryo/conceptus presence and development. © 2021 Federation of American Societies for Experimental Biology.
Extracellular vesicles from oviductal and uterine fluids supplementation in sequential in vitro culture improves bovine embryo quality
(2022) Gutiérrez Adán, Alfonso
Background: In vitro production of bovine embryos is a well-established technology, but the in vitro culture (IVC) system still warrants improvements, especially regarding embryo quality. This study aimed to evaluate the effect of extracellular vesicles (EVs) isolated from oviductal (OF) and uterine fluid (UF) in sequential IVC on the development and quality of bovine embryos. Zygotes were cultured in SOF supplemented with either BSA or EVs-depleted fetal calf serum (dFCS) in the presence (BSA-EV and dFCS-EV) or absence of EVs from OF (D1 to D4) and UF (D5 to D8), mimicking in vivo conditions. EVs from oviducts (early luteal phase) and uterine horns (mid-luteal phase) from slaughtered heifers were isolated by size exclusion chromatography. Blastocyst rate was recorded on days 7–8 and their quality was assessed based on lipid contents, mitochondrial activity and total cell numbers, as well as survival rate after vitrification. Relative mRNA abundance for lipid metabolism-related transcripts and levels of phosphorylated hormone-sensitive lipase (pHSL) proteins were also determined. Additionally, the expression levels of 383 miRNA in OF- and UF-EVs were assessed by qRT-PCR. Results: Blastocyst yield was lower (P < 0.05) in BSA treatments compared with dFCS treatments. Survival rates after vitrification/warming were improved in dFCS-EVs (P < 0.05). EVs increased (P < 0.05) blastocysts total cell number in dFCS-EV and BSA-EV compared with respective controls (dFCS and BSA), while lipid content was decreased in dFCS-EV (P < 0.05) and mitochondrial activity did not change (P > 0.05). Lipid metabolism transcripts were affected by EVs and showed interaction with type of protein source in medium (PPARGC1B, LDLR, CD36, FASN and PNPLA2, P < 0.05). Levels of pHSL were lower in dFCS (P < 0.05). Twenty miRNA were differentially expressed between OF- and UF-EVs and only bta-miR-148b was increased in OF-EVs (P < 0.05). Conclusions: Mimicking physiological conditions using EVs from OF and UF in sequential IVC does not affect embryo development but improves blastocyst quality regarding survival rate after vitrification/warming, total cell number, lipid content, and relative changes in expression of lipid metabolism transcripts and lipase activation. Finally, EVs miRNA contents may contribute to the observed effects.
Influence of sperm filtration and the addition of glycerol to UHT skimmed milk- and TEST-based extenders on the quality and fertilizing capacity of chilled ram sperm
(2019) Galarza Lucero, Diego Andrés; Ladrón de Guevara, Magdalena; Beltran Breña, Paula; Sánchez Calabuig, María Jesús; Rizos, Dimitrios; Lopez Sebastian, Antonio; Santiago Moreno, Julián
The poor fertility of ram semen stored chilled for long periods has encouraged the development of protocols designed to improve the kinetic vigour and cervical barrier-crossing capacity of sperm. The present work evaluated the effect of sperm selection with Sephadex filtration and the supplementation of 2% glycerol (GLY) to extenders based on ultra-heat-treated skimmed milk (UHT) or Tris-Tes-Glucose (TEST) on ram sperm kinetic parameters, plasma membrane integrity, acrosome integrity, mitochondrial function and fertilizing ability, over long chilling times. The results showed that for non-filtered semen, values for progressive sperm motility (%PSM), straight line velocity (VSL, mm/s) and the percentage of sperm with an intact plasma membrane/intact acrosome/a high mitochondrial function index (%IPIAHM) at all times up to 96 h of chilling were higher when the UHT extender (P < 0.01) was used compared to TEST extender irrespective of the presence of GLY. When semen was previously filtered with Sephadex, the addition of GLY to the UHT extender improved total motility (%TM), the %PSM and the VSL at 96 h compared to all other treatments (P < 0.01). The best results of all were obtained with nonfiltered semen and UHT either with or without GLY. Heterologous IVF using zona-intact bovine oocytes was used to assess the fertilizing capacity of non-filtered fresh (FS0), chilled-for-24 h (CS24) or chilled-for-48 h (CS48) ram semen diluted in UHT extender (GLY-free). Heterologous IVF showed that ram sperm, either FS0, CS24 or CS48, were equally capable of penetrating zona pellucida intact bovine oocytes, leading to pronuclear formation and hybrid embryo cleavage (46.3 ± 3.2; 48.8 ± 3.2; and 43.3 ± 3.5, respectively). No differences were seen with respect to fresh sperm in terms of sperm binding, penetration, polyspermy, pronucleus formation or cleavage rates (P > 0.05). In conclusion, neither Sephadex filtration nor addition of glycerol provided extra benefits to ram sperm chilled up to 96 h. Chilled, non-filtered sperm extended with UHT without GLY showed better sperm functionality than did similar sperm extended with TEST extenders. Indeed, sperm diluted in UHT extender, maintained fertilizing ability up to 48 h.
Role of reproductive fluids and extracellular vesicles in embryo-maternal interaction during early pregnancy in cattle
(2021) Sánchez Gómez, José María
The coordinated interaction between the developing embryo and the maternal reproductive tract is essential for the establishment and maintenance of pregnancy in mammals. An early cross-talk is established between the oviduct/uterus and the gametes and embryo. This dialogue will shape the microenvironment in which gamete transport, fertilisation, and early embryonic development occur. Due to the small size of the gametes and the early embryo relative to the volume of the oviductal and uterine lumina, collection of tissue and fluid adjacent to these cells is challenging in cattle. Thus, the combination of in vivo and in vitro models seems to be the most appropriate approach to better understand this fine dialogue. In this respect, the aim of this review is to summarise the recent findings in relation to gamete/embryo-maternal interaction during the pre-elongation period
Sperm selection by density-gradient centrifugation of merino ram semen cold-stored up to 48 h improves viability and membrane integrity
(2018) Galarza Lucero, Diego Andrés; Rizos, Dimitrios; Lopez Sebastian, Antonio; Santiago Moreno, Julián; Ladrón de Guevara, Magdalena; Beltran Breña, Paula; Galarza Lucero, Diego Andrés
Liquid ram semen stored at 5ºC would be more competent than frozen/thawed for sheep crossbreeding programs. The aim was to evaluate the kinetics and membrane integrity of Merino ram semen cold-stored up to 48h at 5ºC before and after density-gradient centrifugation (DGC) selection. Pools of 3 normospermic Merino ram (2-7 years) ejaculates were collected by artificial vagina in fifteen sessions (45 ejaculates), diluted to 200x106 spermatozoa/ml with skim milk-based extender contained 6% egg yolk and cold-stored up to 48h at 5ºC. Motile spermatozoa were separated by BoviPure DGC (Galarza et al., 2018, Anim Reprod Sci 192: 261-270) using 250ul of fresh (n = 30) and cold-stored semen (24h: n = 10 and 48h: n = 10). The final pellet of 300ul was used to assess semen quality. The kinetic parameters were evaluated by computer-assisted sperm analysis (CASA) while plasma, acrosomal and mitochondrial membrane status was analyzed by PI/FITC PNA/Mitotracker fluorescence. The effects of storage time (fresh, 24 & 48h) and sperm selection process were analysed by univariant ANOVA and Bonferroni´s test (P < 0.05). In terms of sperm storage time, CASA analysis of non-selected semen samples showed a significant decrease after storage for 24 and 48h compared to fresh samples with regard to progressive motility [SPM (%): 52.30 ± 4.1 and 36.9 ± 5.5 vs 71.3 ± 1.6], straight line velocity [VSL (um/sec): VSL 132.2 ± 6.1 and 109.7 ± 6.3 vs 176.7 ± 4.3], linearity [LIN (%): 69.2 ± 3.5 and 59.0 ± 5.0 vs 82.0 ± 1.2], and straightness [STR (%): 75.7 ± 3.3 and 66.0 ± 4.3 vs 86.9 ± 0.9], respectively. However, analysis of DGC-selected semen showed a decrease only at storage for 48h when compared to 24h or fresh samples with regards to SPM (35.6 ± 3.9 vs 56.1 ± 6.91 and 59.3 ± 2.6), VSL (83.5 ± 4.4 vs 105.3 ± 6.5 and 110 ± 2.0) and LIN (63.9 ± 3.4 vs 75.0 ± 3.7 and 80.7 ± 2.4), respectively. A comparison between DGCselected and non-selected samples showed a significant lower total motility [TM (%): 94.4 ± 0.8 vs 85.4 ± 1.90], VSL (176.7 ± 4.2 vs 110.0 ± 2.0) and wobble [WOB (%): 94.2 ± 0.6 vs 88.5 ± 1.5] only for fresh semen. Fluorescence analysis evidenced a decrease only in 24h cold-stored non-selected compared with fresh semen with regard to plasma membrane integrity [PMI (%): 64.8 ± 2.9 vs 80.1 ± 1.7], high mitochondrial function [HMF (%): 88.2 ± 1.6 vs 93.9 ± 1.0] and total intact plasma/intact acrosome/high mitochondrial function [IPIAHM (%): 61.8 ± 3.1vs 78.7 ± 2.0]. In contrast, no differences were observed between fresh and cold-stored DGC-selected semen. A comparison between selected and non-selected semen showed a significant increase of PMI (64.8 ± 3.14 to 89.4 ± 2.32), HMF (88.2 ± 1.26 to 96.0 ± 1.26) and IPIAHM (61.8 ± 3.14 to 87.6 ± 2.04) only for 24h. These results suggest that kinetic activity of cold-stored and DGC-selected ram spermatozoa is maintained and the selection process results in improved viability and membrane integrity. Therefore, liquid storage combined with DGC selection might become a good alternative to fresh or frozen non-selected semen to be used for artificial insemination in sheep crossbreeding programs.