Browsing by Subject "In vitro fertilization"
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- PublicationOpen AccessAldose reductase B1 in pig sperm is related to their function and fertilizing ability(2022-01-31) Mateo Otero, Yentel; Ribas Maynou, Jordi; Delgado Bermúdez, Ariadna; Llavanera, Marc; Recuero, Sandra; Barranco Cascales, Isabel; Yeste, Marc; Medicina y Cirugía AnimalAldose reductase B1 (AKR1B1) has been reported to participate in the modulation of male and female reproductive physiology in several mammalian species. In spite of this, whether or not AKR1B1 could be related to sperm quality, functionality and fertilizing ability is yet to be elucidated. The present study, therefore, aimed to investigate: i) the presence of AKR1B1 in epididymal and ejaculated sperm; ii) the relationship between the AKR1B1 present in sperm and the physiology of the male gamete; iii) the liaison between the relative content of AKR1B1 in sperm and their ability to withstand preservation for 72 h; and iv) the potential link between sperm AKR1B1 and in vitro fertility outcomes. Immunoblotting revealed that AKR1B1 is present in both epididymal and ejaculated sperm with a similar relative content. Moreover, the relative levels of AKR1B1 in sperm (36 kDa band) were found to be negatively related to several kinematic parameters and intracellular calcium levels, and positively to the percentage of sperm with distal cytoplasmic droplets after storage. Finally, AKR1B1 amounts in sperm (36 kDa band) were negatively associated to fertilization rate at two days post-fertilization and embryo development at six days post-fertilization. The results of the present work suggest that AKR1B1 in sperm is probably acquired during maturation rather than at ejaculation and could play a role in that process. Moreover, AKR1B1 seems to be related to the sperm resilience to preservation and to their fertilizing capacity, as lower levels of the 36 kDa band (putative inactive form of this protein) result in better reproductive outcomes.
- PublicationOpen AccessMorphometric study of the umbilical cord in in-vitro-derived pigs(Springer, 2022-09-15) Álvarez Martín, Úrsula; Coy, Pilar; Romar Andrés, Raquel; Párraga Ros, Ester; Seva Alcaraz, Juan; FisiologíaThe umbilical cord is the vital fetus-placenta connection and represents one of the greatest sources of precursor cells. Histologically it is a single amniotic epithelium that encloses a mucoid connective tissue, and a vein and two arteries lacking tunica adventitia. Instead, there is a special mucoid connective tissue named Wharton’s Jelly (WJ) that is divided into a perivascular and an intermediate zone, the first being the most abundant in mesenchymal stromal cells. Morphological charac-teristics of the umbilical cord and its components have been related to fetal malformations, preterm birth and low birth weight. The objective of this study was to compare the WJ and the vascular area in the umbili-cal cord of pigs born from in-vitro- and in-vivo-produced embryos (the latter born by artificial insemination of sows; AI group). In-vitro embryos (IVP) were produced after insemination in-vitro of matured oocytes and further in-vitro culture up to blastocyst stage in media sup-plemented with (RF-IVP group) or without (C-IVP group) reproductive fluids (1% porcine oviductal fluid and 1% uterine fluid). Blastocysts produced were surgically transferred at day 7 post-in-vitro fertiliza-tion. After birth, umbilical cord samples of 15 animals (5 per group) were collected and productive parameters recorded. Samples were fixed (10% buffered formaldehyde solution) and paraffin-embedded. Complete sections of 5 μm thickness were stained (hematoxylin-eosin) and digitized with a Histech MIDI II 3D scanner at 0.172 pixels/μm. The virtual microscope SlideViewer 2.5 3D Histech was used for the digital analysis of the total umbilical area, the thickness of both WJ’s zones, and each vessel’s area. Data were analyzed by one-way ANOVA (SPSS Statistics 28) and the differences were compared by Tukey’s test (P<0.05). The thickness of the WJ-perivascular zone was significantly higher in the C-IVP group (AI: 572,4 ± 39,3 μm; C-IVP: 662,2 ± 42,3 μm; RF-IVP: 501,1 ± 35,7 μm), and was significantly correlated with piglet birth weight, placental weight and placental efficiency (Pear-son <0.05). No significant differences in the WJ-intermediate zone thickness, vascular and total umbilical areas were found. These results might be related to the subsequent development of pathophysiological changes in IVP animals during their growth.
- PublicationOpen AccessPig in vitro fertilization: where are we and where do we go?(2019) Romar, Raquel; Cánovas, Sebastián; Matás, Carmen; Gadea, Joaquín; Coy, Pilar; FisiologíaThe pig is an important livestock animal. Biotechnological interest in this species has increased due to its use, among others, in the generation of transgenic animals for use in biomedicine based on its greater physiological proximity to the human species than other large domestic animals. This development has paralleled an improvement in Assisted Reproduction Techniques (ART) used for this species. However, the ability to generate animals from embryos produced entirely in vitro is still limited and a wide margin for improvement remains. Here we review the procedures, additives, and devices used during pig in vitro fertilization (IVF), focusing on the main points of each step that have offered the best results in terms of increased efficiency of the system. The lack of standardized protocols and consensus on the parameters to be assessed makes it difficult to compare results across different studies, but some conclusions are drawn from the literature. We anticipate that new physiological protocols will advance the field of swine IVF, including induction of prefertilization ZP hardening with oviductal fluid, sperm preparation by swim-up method, increased viscosity through the addition of inert molecules or reproductive biofluids, and the incorporation of 3D devices. Here we also reflect on the need to expand the variables on which the efficiency of pig IVF is based, providing new parameters that should be considered to supply more objective and quantitative assessment of IVF additives and protocols.
- PublicationOpen AccessSeminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism(Elsevier, 2024-02-26) Barranco, Isabel; Spinaci, Marcella; Nesci, Salvatore; Mateo Otero, Yentel; Baldassarro, Vito Antonio; Algieri, Cristina; Bucci, Diego; Roca, Jordi; Medicina y Cirugía AnimalPorcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry; albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO2 in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism.
- PublicationRestrictedSuitability and effectiveness of single layer centrifugation using Androcoll-P in the cryopreservation protocol for boar spermatozoa(Elsevier, 2013-08) Martínez Alborcia, María J.; Morrell, Jane M.; Gil Corbalán, María Antonia; Barranco Cascales, Isabel; Maside, Carolina; Alkmin, Diego V; Parrilla Riera, Inmaculada; Martínez García, Emilio; Roca Aleu, Jorge; Medicina y Cirugía AnimalThe goal of the present experiment was to evaluate the suitability and effectiveness of single layer centrifugation (SLC), using the pig-specific colloid Androcoll-P, as a routine procedure for selecting boar spermatozoa for cryopreservation. The study focuses special attention on the effectiveness of SLC for processing a whole sperm rich ejaculate fraction and the fertilizing ability of frozen-thawed (FT) sperm selected using SLC prior to freezing. Thirteen sperm rich ejaculate fractions (one per boar) were split into three aliquots. Two aliquots of 15 and 150mL were SLC-processed (500×g for 20min) using 15 and 150mL (v/v) of Androcoll-P-Large and Androcoll-P-XL, respectively. The third aliquot remained un-processed as a control. The percentages of spermatozoa that were morphologically normal and showed rapid and progressive motility (assessed by CASA) spermatozoa were higher (P<0.01) and those with fragmented nuclear DNA (sperm chromatin dispersion test) were lower (P<0.01) after SLC than control semen samples, regardless of the Androcoll-P used. The recovery rates of total, motile, viable (flow cytometric evaluated after staining with H-42, PI and FITC-PNA) and morphologically normal spermatozoa ranged between 20 and 100% and those with intact nuclear DNA ranged between 60 and 100%, irrespective of the Androcoll-P used. Thereafter, the semen samples were cryopreserved using a standard 0.5-mL straw freezing protocol. Post-thaw percentages of sperm motility (both total motility and rapid progressive motility), viability and intact nuclear DNA were higher (P<0.05) in SLC-processed than in control semen samples, irrespective of the Androcoll-P used. SLC-processing also improved the in vitro fertilizing ability of FT-sperm (679 in vitro matured oocytes inseminated with a viable sperm:oocyte ratio of 300:1 and coincubated for 6h), measured as the percentage of penetrated oocytes and the mean number of swollen sperm heads and/or male pronuclei in penetrated oocytes. However, there was no effect of SLC-processing on the in vitro ability of putative zygotes to develop to blastocysts. Overall these results indicate that SLC-processing of boar ejaculates using Androcoll-P improves the quality and fertilizing ability of cryosurvival boar sperm. However, efforts should be made to ensure continued high recovery yields before considering the inclusion of SLC as a routine procedure in the cryopreservation protocol of boar ejaculates.
- PublicationOpen AccessTiming of oviductal fluid collection, steroid concentrations and sperm preservation method affect in vitro fertilization efficiency.(2014) Ballester, L.; Soriano Ubeda, C. M.; Matas Parra, Carmen; Romar Andrés, Raquel; Coy Fuster, Pilar; Romero Aguirregomezcorta, Jon; Fisiología