Cristina Ortega - Academia.edu (original) (raw)

Papers by Cristina Ortega

Biology of Reproduction, 2020

Some stallions yield ejaculates that do not tolerate conservation by refrigeration prior to artif... more Some stallions yield ejaculates that do not tolerate conservation by refrigeration prior to artificial insemination (AI), showing improvement after removal of most of the seminal plasma (SP) by centrifugation. In this study, the SP-proteome of 10 different stallions was defined through high-performance liquid chromatography with tandem mass spectrometry and bioinformatic analysis in relation to the ability of the ejaculates to maintain semen quality when cooled and stored at 5°C. Stallions were classified into three groups, depending on this ability: those maintaining good quality after direct extension in a commercial extender (good), stallions requiring removal of seminal plasma (RSP) to maintain seminal quality (good-RSP), and stallions, unable to maintain good semen quality even after RSP (poor). Pathway enrichment analysis of the proteins identified in whole equine SP using human orthologs was performed using g: profiler showing enriched Reactome and the Kyoto Encyclopedia of G...

Equine embryos were obtained by insemination with either fresh or frozen-thawed spermatozoa at 8,... more Equine embryos were obtained by insemination with either fresh or frozen-thawed spermatozoa at 8, 10 and 12 h post spontaneous ovulation, maintaining the pairs mare-stallion for the type of semen used. Next generation sequencing (NGS) was performed in all embryos and bioinformatic and enrichment analysis performed on the 21,058 identified transcripts. A total of 165 transcripts were downregulated in embryos obtained with cryopreserved spermatozoa respect embryos resulting from an insemination with fresh spermatozoa (p=0.021, q=0.1). The enrichment analysis using human orthologs using g:profiler on the downregulated transcripts marked an enrichment in transcription factors (TFs) in mRNAs downregulated in embryos obtained after insemination with cryopreserved spermatozoa. The 12 mRNAs (discriminant variables) most significantly downregulated in these embryos included among others, the chromatin-remodeling ATPase INO80, Lipase maturation factor 1 LMF1, the mitochondrial mRNA pseudourid...

Biology of Reproduction, 2021

The identification of stallions and or ejaculates that will provide commercially acceptable quali... more The identification of stallions and or ejaculates that will provide commercially acceptable quality post-thaw before cryopreservation is of great interest, avoiding wasting time and resources freezing ejaculates that will not achieve sufficient quality to be marketed. Our hypothesis was that after bioinformatic analysis, the study of the stallion sperm proteome can provide discriminant variables able to predict the post-thaw quality of the ejaculate. At least three ejaculates from 10 different stallions were frozen following a split sample design. Half of the ejaculate was analyzed as a fresh aliquot and the other half was frozen and then analyzed as a frozen-thawed aliquot. Computer-assisted sperm analysis and flow cytometry were used to analyze sperm quality. Detailed proteomic analysis was performed on fresh and frozen and thawed aliquots, and bioinformatic analysis was used to identify discriminant variables in fresh samples able to predict the outcome of cryopreservation. Those...

Frontiers in Veterinary Science, 2019

Data in Brief, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Redox regulation and oxidative stress have become areas of major interest in spermatology. Altera... more Redox regulation and oxidative stress have become areas of major interest in spermatology. Alteration of redox homeostasis is recognized as a significant cause of male factor infertility and is behind the damage that spermatozoa experience after freezing and thawing or conservation in a liquid state. While for a long time, oxidative stress was just considered an overproduction of ROS, nowadays it is considered as a consequence of redox deregulation. Many essential aspects of spermatozoa functionality are redox regulated, with reversible oxidation of thiols in cysteine residues of key proteins acting as an “on-off” switch controlling spermatic function. However, if deregulation occurs, these residues may experience irreversible oxidation and oxidative stress leading to spermatic malfunction and ultimately death. Stallion spermatozoa are “professional producers” of ROS due to their intense mitochondrial activity, and thus sophisticated systems to control redox homeostasis are also cha...

PLOS ONE, 2019

Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technolog... more Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-ovulation on successive cycles with either fresh sperm or frozen-thawed sperm from the same stallion, providing matched embryo pairs at each day. RNA was isolated from two matched pairs (4 embryos) for each day, and cDNA libraries were built and sequenced. Significant differences in transcripts per kilobase million (TPM) were determined using (i) genes for which the expression difference between treatments was higher than 99% of that in the random case (P < 0.01), and (ii) genes for which the fold change was � 2, to avoid expression bias in selection of the candidate genes. Molecular pathways were explored using the DAVID webserver, followed by network analyses using STRING, with a threshold of 0.700 for positive interactions. The transcriptional profile of embryos obtained with frozen-thawed sperm differed significantly from that for embryos derived from fresh sperm on all days, showing significant down-regulation of genes involved in biological pathways related to oxidative phosphorylation, DNA binding, DNA replication, and immune response. Many genes with reduced expression were orthologs of genes known to be embryonic lethal in mice. This study, for the first time, provides evidence of altered transcription in embryos resulting from fertilization with cryopreserved spermatozoa in any species. As sperm cryopreservation is

Biology of Reproduction, 2019

Oxidative stress is considered a major mechanism causing sperm damage during cryopreservation and... more Oxidative stress is considered a major mechanism causing sperm damage during cryopreservation and storage, and underlies male factor infertility. Currently, oxidative stress is no longer believed to be caused only by the overproduction of reactive oxygen species, but rather by the deregulation of redox signaling and control mechanisms. With this concept in mind, here, we describe for the first time the presence of the soluble carrier family 7 member 11 (SLC7A11) antiporter, which exchanges extracellular cystine (Cyss) for intracellular glutamate, in stallion spermatozoa, as well as its impact on sperm function using the specific inhibitor sulfasalazine. Spermatozoa incubated with Cyss exhibited an increased intracellular GSH content compared with controls (P < 0.01): 50% in fresh extended stallion spermatozoa and 30% in frozen-thawed spermatozoa. This effect was prevented by the addition of sulfasalazine to the media. Cystine supplementation also reduced the oxidation–reduction p...

Background The population of stallion spermatozoa surviving thawing, experience, among other chan... more Background The population of stallion spermatozoa surviving thawing, experience, among other changes, compromised mitochondrial functionality and accelerated senescence. It is known that the stallion spermatozoa show very active oxidative phosphorylation that may accelerate sperm senescence through increased production of reactive oxygen species. Rosiglitazone has proven to enhance the glycolytic capability of stallion spermatozoa maintained in refrigeration. Objectives Thus, we hypothesized that thawed sperm may also benefit from rosiglitazone supplementation. Material and Methods Thawed sperm doses were washed and re-suspended in Tyrodes media, split sampled and supplemented with 0 or 75 μM rosiglitazone. After 1 and two hours of incubation, mitochondrial functionality, Akt phosphorylation and caspase 3 activity were evaluated. Further samples were incubated in presence of the Akt1/2 inhibitor, compound C (AMPK inhibitor) and GW9662 (antagonist of the PPARγ receptor). Results Rosi...

Biology of Reproduction, 2018

We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation o... more We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menadione and DMNQ induced damage to sperm membranes after 1 h (P < 0.001; P < 0.05) and after 3 h of incubation (P < 0.001, P < 0.05). Pre-incubation with cysteine ameliorated both the menadione-and DMNQ-induced increase in 4-hydroxynonenal (P < 0.001). As cysteine is a precursor of GSH, we hypothesized that stallion spermatozoa are able to synthesize this tripeptide using 1090

Reproduction in domestic animals = Zuchthygiene, 2018

Flow cytometry is increasingly used in research and also in clinical andrology. Recent developmen... more Flow cytometry is increasingly used in research and also in clinical andrology. Recent developments in instrumentation, availability of probes and bioinformatics expand the possibilities of flow cytometry well beyond the classical two parametric analyses in use. In this paper, an overview of recent developments in flow cytometry will be presented under the perspective of the authors; aspects such a multicolor assays and computational cytometry will be discussed as well.

BMC veterinary research, Jan 26, 2017

Sperm selection methods such as Single Layer Centrifugation (SLC) have been demonstrated to be a ... more Sperm selection methods such as Single Layer Centrifugation (SLC) have been demonstrated to be a useful tool to improve the quality of sperm samples and therefore to increase the efficiency of other artificial reproductive techniques in several species. This procedure could help to improve the quality of genetic resource banks, which is essential for endangered species. In contrast, these sperm selection methods are optimized and focused on farm animals, where the recovery task is not as important as in endangered species because of their higher sperm availability. The aim of this study was to evaluate two centrifugation methods (300 x g/20 min and 600 x g/10 min) and three concentrations of SLC media (Androcoll-Bear -80, 65 and 50%) to optimise the procedure in order to recover as many sperm with the highest quality as possible. Sperm morphology could be important in the hydrodynamic relationship between the cell and centrifugation medium and thus the effect of sperm head morphomet...

PloS one, 2017

Testicular function is particularly susceptible to vascular insult, resulting in a negative impac... more Testicular function is particularly susceptible to vascular insult, resulting in a negative impact on sperm production and quality of the ejaculate. A prompt diagnosis of testicular dysfunction enables implementation of appropriate treatment, hence improving fertility forecasts for stallions. The present research aims to: (1) assess if Doppler ultrasonography is a good tool to diagnose stallions with testicular dysfunction; (2) to study the relationship between Doppler parameters of the testicular artery and those of sperm quality assessed by flow cytometry and (3) to establish cut off values to differentiate fertile stallions from those with pathologies causing testicular dysfunction. A total of 10 stallions (n: 7 healthy stallions and n: 3 sub-fertile stallions) were used in this study. Two ejaculates per stallion were collected and preserved at 5°C in a commercial extender. The semen was evaluated at T0, T24 and T48h by flow cytometry. Integrity and viability of sperm (YoPro®-1/E...

Open biology, Dec 1, 2016

Previous studies suggested that the aryl hydrocarbon receptor (AhR) contributes to mice reproduct... more Previous studies suggested that the aryl hydrocarbon receptor (AhR) contributes to mice reproduction and fertility. However, the mechanisms involved remain mostly unknown. Retrotransposon silencing by Piwi-interacting RNAs (piRNAs) is essential for germ cell maturation and, remarkably, AhR has been identified as a regulator of murine B1-SINE retrotransposons. Here, using littermate AhR(+/+) and AhR(-/-) mice, we report that AhR regulates the general course of spermatogenesis and oogenesis by a mechanism likely to be associated with piRNA-associated proteins, piRNAs and retrotransposons. piRNA-associated proteins MVH and Miwi are upregulated in leptotene to pachytene spermatocytes with a more precocious timing in AhR(-/-) than in AhR(+/+) testes. piRNAs and transcripts from B1-SINE, LINE-1 and IAP retrotransposons increased at these meiotic stages in AhR-null testes. Moreover, B1-SINE transcripts colocalize with MVH and Miwi in leptonema and pachynema spermatocytes. Unexpectedly, AhR...

Biology of reproduction, Sep 14, 2016

Stallion-to-stallion variability in the quality of cryopreserved ejaculates post-thaw affects the... more Stallion-to-stallion variability in the quality of cryopreserved ejaculates post-thaw affects the commercial acceptability of frozen semen and thus is a major constraint for the equine industry. In recent years, the molecular mechanisms associated with sperm damage during cryopreservation have become better understood. Identification of the freezability of the ejaculates before the freezing process is initiated will have a major impact on the equine industry. We studied three markers of oxidative stress in sperm, including 8-iso-PGF2alpha, 8-OH guanosine, and 4-hydroxynonenal (4-HNE); the presence of active caspase 3; and their changes after sperm cryopreservation. While 4-HNE levels increased after cryopreservation (7 to 33% p<0.001), 8-OH-guanosine and 8-ISO-PGF2alpha levels decreased after cryopreservation (130 to 35 arbitrary fluorescence units, p<0.01 and 1280 to 1233,…

Reproduction in Domestic Animals, 2015

To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville s... more To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville sorting technology using flow cytometry. Improvement of this technology in the equine species requires increasing awareness of the modifications that the sorting procedure induces on sperm intactness. Oxidative stress is regarded as the major damaging phenomenon, and increasing evidence regards handling of spermatozoa-including sex sorting-as basic ground for oxidative damage. The aim of this study was to disclose whether the flow cytometric sorting procedure increases the production of reactive oxygen species (ROS), and to identify if ROS production relates to DNA damage in sorted spermatozoa using specific flow cytometry-based assays. After sorting, oxidative stress increased from 26% to 33% in pre-and post-incubation controls, to 46% after sex sorting (p<0.05). Proportions of DNA fragmentation index post-sorting was approximately 10% higher (31.3%); an effect apparently conduced via oxidative DNA damage as revealed by the oxyDNA assay. The probable origin of this increased oxidative stress owes the removal of enough seminal plasma due to the unphysiological sperm extension, alongside a deleterious effect of high pressure on mitochondria during the sorting procedure.

5-Lipid peroxidation, assessed with BODIPY-C11, increases after cryopreservation of stallion sper... more 5-Lipid peroxidation, assessed with BODIPY-C11, increases after cryopreservation of stallion spermatozoa, is stallion-dependent and is related to apoptotic-like changes. Ortega Ferrusola C, González

Biology of reproduction, Jan 4, 2015

Oxidative stress has been linked to sperm death and the accelerated senescence of cryopreserved s... more Oxidative stress has been linked to sperm death and the accelerated senescence of cryopreserved spermatozoa. However, the molecular mechanisms behind this phenomenon remain poorly understood. Reactive oxygen species (ROS) are considered relevant signaling molecules for sperm function, only becoming detrimental when ROS homeostasis is lost. We hereby hypothesize that a major component of the alteration of ROS homeostasis in cryopreserved spermatozoa is the exhaustion of intrinsic antioxidant defence mechanisms. To test this hypothesis, semen from seven stallions was frozen using a standard technique. The parameters of sperm quality (motility velocity, membrane integrity) and markers of sperm senescence (caspase 3, 4-hydroxynonenal, mitochondrial membrane potential) were assessed before and after cryopreservation. Moreover, changes in the intracellular thiol content were also monitored. Cryopreservation caused significant increases in senescence markers as well as a dramatic depletion...

PloS one, 2015

Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells a... more Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37°C in the presence of rotenone, antimycin-A or control vehicle. Incubation in the presence of these inhibitors reduced sperm motility and velocity (CASA analysis) (p<0.01), but the effect was more evident in the presence of rotenone (a complex I inhibitor). These inhibitors also decreased ATP content. The inhibition of complexes I and III decreased the production of reactive oxygen species (p<0.01) as assessed by flow cytometry after staining with CellRox deep re...

Theriogenology, 2015

Acquired inguinal herniation is a very common condition in stallions, usually leading to unilater... more Acquired inguinal herniation is a very common condition in stallions, usually leading to unilateral or bilateral castration to prevent future recurrence. Recently, several surgical techniques such as the standing laparoscopic peritoneal flap hernioplasty (SLPFH) have been developed to avoid herniation recurrence and also preserve the breeding activity of high economic value stallions. However, studies on SLPFH lack more comprehensive and systematic data about reproductive-related adverse effects and outcomes. The aim of this study was to evaluate whether SLPFH of the internal inguinal rings produces changes in the testicular blood flow in a 1-year follow-up. For that purpose, six healthy stallions were used and testicular blood flow was assessed before, 3, 6, and 12 months (T0, T3, T6, and T12) after the procedure. Blood flow was evaluated ultrasonographically, using the pulsed-wave color Doppler mode. Peak systolic velocity, end-diastolic velocity, the time-averaged maximum velocity, and the derived indexes (resistive index) and pulsatility index) of the testicular artery were measured in two localizations: in the spermatic cord and on the caudal epididymal edge of the testicle. On the spermatic cord, the peak systolic velocity of the testicular artery increased significantly at T12. However, on the epididymal edge location of the artery, the pulsatility and resistive indexes were decreased at T12 (P < 0.05). This pattern of blood flow was related to a hyperemic process. Furthermore, SLPFH might have compressed the spermatic cord, causing a slight occlusion of the testicular artery and triggering a compensatory hyperemia to compensate the deficit of blood flow that supplies the testes. The SLPFH of the internal inguinal ring affected the testicular perfusion in stallions in a 1 year follow-up, although there was no effect on sperm production during this time. The spectral Doppler ultrasound is a useful tool to asses the testicular perfusion after reproductive surgical procedure and provides information which anticipates vascular supply compromise of the stallion testicles.