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  • 1.
    Alvarez-Rodriguez, Manuel
    University of Leon, Facultad de Ciencias Biologicas y Ambientales, Spain.
    Mejora y evolución de los protocolos de congelación de eyaculados de oso pardo (Ursus arctos)2012Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [es]

    La situación crítica del oso pardo (Ursus arctos) en España plantea la necesidad de aplicar diferentes estrategias de conservación, dentro de las cuales tiene especial relevancia la creación de un banco de recursos genéticos. La eficacia de esta herramienta depende, entre otros factores, de la adaptación específica de los protocolos de congelación espermática ya existentes y que permitan el almacenamiento eficaz de los espermatozoides del oso pardo en un banco de germoplasma. Este trabajo nos permite concluir la utilidad de la selección entre ciclos con el fin de mejorar la calidad de los espermatozoides sometidos a recongelación

  • 2.
    Alvarez-Rodriguez, Manuel
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten. Univ Autonoma Barcelona, Spain.
    Alvarez, M.
    Univ Leon, Spain.
    Anel-Lopez, L.
    Univ Leon, Spain.
    Guerra, C.
    Univ Leon, Spain.
    Chamorro, C. A.
    Univ Leon, Spain.
    Anel, L.
    Univ Leon, Spain.
    de Paz, P.
    Univ Leon, Spain.
    Martinez-Pastor, F.
    Univ Leon, Spain.
    Effect of length of time post-mortem on quality and freezing capacity of Cantabric chamois (Rupicapra pyrenaica parva) epididymal spermatozoa2018Ingår i: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 198, s. 184-192Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Genome Resource Banks are keystones in the ex-situ conservation of wild species. Post-mortem (PM) collection of epididymal spermatozoa is an opportunistic and valuable source of germplasm, the time from the death of the animal limits its use. Seeking to improve germplasm preservation strategies for the chamois (Rupicapra sp.), the effect of PM time on epididymal sperm quality and freezability was studied using the Cantabrian chamois. Samples were classified according to PM collection time, up to 216 h (refrigerated), and cryopreserved (Tris-citric acid-fructose, 430 mOsm/kg, 15% egg yolk, 8% glycerol; freezing at - 20 degrees C/min). Sperm quality was assessed after recovery and post-thawing (motility by CASA, HOS test, abnormal forms, cytoplasmic droplets, and viability and acrosomal damage by flow cytometry). The sperm mass pH and osmolality showed a positive correlation with time. Total sperm motility dropped after 2 days PM, with progressivity and sperm velocities remained similar up to 3 days PM. Sperm freezability was acceptable, with the post-thawing HOST, motility, progressivity, VAP, VCL, VSL and BCF negatively correlating with PM time. Overall, chamois epidydimal samples were not adequate for preservation after 6 days PM. Freezability capacity could make these spermatozoa suitable for specific ART even if kept refrigerated for several days PM.

  • 3.
    Alvarez-Rodriguez, Manuel
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Alvarez, M.
    University of Leon, Spain.
    Anel-Lopez, L.
    SaBio IREC CSIC UCLM JCCM, Spain.
    Lopez-Uruena, E.
    University of Leon, Spain.
    Manrique, P.
    University of Leon, Spain.
    Borragan, S.
    Cabarceno Pk, Spain.
    Morrell, J. M.
    Swedish University of Agriculture Science SLU, Sweden.
    de Paz, P.
    University of Leon, Spain.
    Anel, L.
    University of Leon, Spain.
    Effect of colloid (Androcoll-Bear, Percoll, and PureSperm) selection on the freezability of brown bear (Ursus arctos) sperm2016Ingår i: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 85, nr 6, s. 1097-1105Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The development of a species-specific conservation protocol that involves artificial insemination with frozen semen needs to validate an effective methodology for freezing semen. Colloid centrifugation has been suggested and widely applied as an effective tool for selecting animal spermatozoa for artificial breeding. The objective of the present study was to compare different methods of centrifugation, single layer using Androcoll-Bear and Percoll and double layer using PureSperm 100 (in two different discontinuous gradients 40%-80% and 45%-90%), for the selection of fresh brown bear sperm samples. In the before freezing group, all selected samples showed a higher progressive motility and viability (except Percoll for motility 43.0 +/- 5.3 [P < 0.051); all colloids except PureSperm 45/90% rendered samples with fewer damaged acrosomes. In the after thawing group, all tested centrifugation colloids showed a good capacity to decrease the number of damaged acrosomes. Furthermore, PureSperm treatment (45/90%) resulted in an increase in apoptotic-like changes not only immediately after thawing but also after the incubation test, leading us to suggest that this gradient could induce some kind of deleterious effects on the sperm samples. On the other hand, PureSperm treatment (40/80%) yielded a quality preservation capacity similar to Androcoll-Bear in number of damaged acrosomes, different relative to the control (control, 5.3 +/- 0.6; PureSperm 80, 2.0 +/- 0.3; Androcoll, 2.1 +/- 0.9 [P < 0.051) but a decrease in the number of viable spermatozoa recovered after thawing relative to the control (control, 21.2 +/- 3.1; PureSperm 80, 13.7 +/- 2.7 [P < 0.051). In conclusion, Androcoll-Bear constitutes a useful tool for handling of brown bear ejaculates owing to its simple handling and procedure with a reliable sperm selection and freezability. This colloid yielded an improvement in several sperm parameters in brown bear frozen-thawed semen; the selected spermatozoa of fresh samples with this colloid showed a better resistance to freezing compared with the control sample not only for motility but also for viability. (C) 2016 Elsevier Inc. All rights reserved.

  • 4.
    Alvarez-Rodriguez, Manuel
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Gomes-Alves, S
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Borragan, Santiago
    Cabárceno Park, Cantabria, Spain.
    Martinez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Quality of frozen-thawed semen in brown bear is not affected by timing of glycerol addition.2011Ingår i: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 75, nr 8, s. 1561-1565Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have tested several freezing protocols for brown bear semen, modifying the time when glycerol was added (before and after cooling to 5 °C). No differences were found among protocols, indicating a good tolerance of brown bear semen to glycerol. This finding indicates that freezing protocols for brown bear semen could be modified to fit practical solutions which would facilitate preparation of the seminal samples in the field with the addition of glycerol at ambient temperature.

  • 5.
    Alvarez-Rodriguez, Manuel
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Atikuzzaman, Mohammad
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Medicinska fakulteten. Department of Surgery and Theriogenology, Faculty of Veterinary Animal and Biomedical Sciences, Sylhet Agricultural University, Sylhet, Bangladesh.
    Venhoranta, Heli
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Medicinska fakulteten. University of Helsinki, Department of Production Animal Medicine, Faculty of Veterinary Medicine, Saari, Finland.
    Wright, Dominic
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biologi. Linköpings universitet, Tekniska fakulteten.
    Rodriguez-Martinez, Heriberto
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Expression of Immune Regulatory Genes in the Porcine Internal Genital Tract Is Differentially Triggered by Spermatozoa and Seminal Plasma2019Ingår i: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, nr 3, artikel-id 513Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mating or cervical deposition of spermatozoa or seminal plasma (SP) modifies the expression of genes affecting local immune defense processes at the oviductal sperm reservoir in animals with internal fertilization, frequently by down-regulation. Such responses may occur alongside sperm transport to or even beyond the reservoir. Here, immune-related gene expression was explored with cDNA microarrays on porcine cervix-to-infundibulum tissues, pre-/peri-ovulation. Samples were collected 24 h post-mating or cervical deposition of sperm-peak spermatozoa or SP (from the sperm-peak fraction or the whole ejaculate). All treatments of this interventional study affected gene expression. The concerted action of spermatozoa and SP down-regulated chemokine and cytokine (P00031), interferon-gamma signaling (P00035), and JAK/STAT (P00038) pathways in segments up to the sperm reservoir (utero-tubal junction (UTJ)/isthmus). Spermatozoa in the vanguard sperm-peak fraction (P1-AI), uniquely displayed an up-regulatory effect on these pathways in the ampulla and infundibulum. Sperm-free SP, on the other hand, did not lead to major effects on gene expression, despite the clinical notion that SP mitigates reactivity by the female immune system after mating or artificial insemination.

  • 6.
    Alvarez-Rodriguez, Manuel
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Ljunggren, Stefan
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Hjärt- och Medicincentrum, Arbets- och miljömedicin.
    Karlsson, Helen
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Hjärt- och Medicincentrum, Arbets- och miljömedicin.
    Rodriguez-Martinez, Heriberto
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Exosomes in specific fractions of the boar ejaculate contain CD44: A marker for epididymosomes?2019Ingår i: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 140, s. 143-152Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Seminal plasma (SP) is a complex fluid containing proteins, peptides, enzymes, hormones as well as extracellular vesicles (EVs). The SP interacts with spermatozoa and the inner cell lining of the female genital tract, adsorbing proteins and exosomes that modulate sperm functions and female immune responsiveness. In the present study, boar sperm-free SP was studied using flow cytometry (FC) after membrane tetraspanins (CD9, CD63 and CD81) and membrane receptor CD44 marking of non-enriched (whole SP) or gradient fractions enriched through two-step discontinuous KBr-density-gradient ultracentrifugation, in whole ejaculate or in selected ejaculate fractions. The results, evaluated by transmission electron microscopy, confirmed the presence of exosomes in all fractions of the pig SP. Noteworthy, these pig SP-exosomes were CD44-bearing when analysed by FC, with bands detected by western blotting (WB) at the expected 85 kD size. The two-step discontinuous KBr-density-gradient ultracentrifugation enriched the population of exosomes in two specific gradient fractions, indicating exosomes (either prostasomes or epididymosomes) could be separated from low-density lipoprotein (LDL) but they co-sediment with the high-density lipoprotein (HDL)-bearing fraction. The findings pave for the selective isolation of exosomes in functional studies of their function when interacting with spermatozoa, the oocyte and/or the female genitalia, including hyaluronan-CD44 interplay. (C) 2019 Elsevier Inc. All rights reserved.

  • 7.
    Alvarez-Rodriguez, Manuel
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten. Univ Autonoma Barcelona, Spain.
    Lopez-Bejar, Manel
    Univ Autonoma Barcelona, Spain.
    Rodriguez-Martinez, Heriberto
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    The risk of using monoclonal or polyclonal commercial antibodies: controversial results on porcine sperm CD44 receptor identification2019Ingår i: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 54, nr 4, s. 733-737Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Presence of the hyaluronan (hyaluronic acid, HA) receptor CD44 on spermatozoa has been difficult to pursue, mostly obeying to the use of different commercial mono- and/or polyclonal antibodies, often lacking proper controls. Here, we describe how the presence (Western blotting) and specific location (immunocytochemistry) of the CD44 receptor differs in ejaculated pig spermatozoa depending on the type of antibody and protocol used. While we were able to detect binding to spermatozoa and mark its presence in the sperm membrane, the use of blocking peptides clearly indicated that only the monoclonal antibody could confirm the specific presence and location of the CD44 receptor, whereas the polyclonal antibody was detecting multiple presumed CD44 isoforms or degraded proteins thus proving unspecific. These results call for strict protocols when attempting immunological determination of sperm membrane receptors.

    Publikationen är tillgänglig i fulltext från 2020-02-11 15:44
  • 8.
    Alvarez-Rodriguez, Manuel
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Vicente Carrillo, Alejandro
    Linköpings universitet, Medicinska fakulteten. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Evidensia Valla Djursjukhus Linkoping, Linkoping, Sweden.
    Rodriguez-Martinez, Heriberto
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Hyaluronan improves neither the long-term storage nor the cryosurvival of liquid-stored CD44-bearing Al boar spermatozoa2018Ingår i: Journal of reproduction and development, ISSN 0916-8818, E-ISSN 1348-4400, Vol. 64, nr 4, s. 351-360Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Hyaluronan (hyaluronic acid, HA) apparently improves sperm survival in vitro and in vivo (oviduct), maintaining sperm motility and inducing capacitation, but not acrosome exocytosis, either by direct action as a macromolecule or via CD44 membrane receptors. This study explored ejaculated, liquid-extended pig spermatozoa to ascertain (i) the presence (Western blotting) and specific location (immunocytochemistry) of the CD44 receptor, using a specific monoclonal commercial antibody; (ii) whether the CD44 receptor changed location when exposed to bicarbonate, a capacitating trigger, in vitro; and (iii) whether the addition of HA, of molecular size comparable to that produced in the oviduct sperm reservoir (0.0625 to 2.0 mg/ml; 0 HA: control), to semen extenders would improve sperm liquid storage in vitro or cryosurvival post freezing. Variables tested were sperm velocity and progressive motility (Qualisperm (TM)), sperm viability and acrosome status, membrane integrity and early destabilization, mitochondrial activation, and superoxide production (flow cytometry). The CD44 receptor presence in ejaculated, liquid-stored AI boar spermatozoa, as confirmed by a porcine-specific monoclonal antibody, maintained its membrane location under in vitro capacitation-inducing conditions. HA exposure to 24-, 48-, or 72-h liquid-stored (17-20 degrees C) spermatozoa lowered sperm velocity in membrane-intact spermatozoa, but increased mitochondrial superoxide production. Finally, HA addition during cooling did not improve cryosurvival but did increase mitochondrial activation and membrane destabilization in surviving cells. These results confirm the existence of a CD44 receptor in pig spermatozoa, but the usefulness of adding HA for long-term storage or cryopreservation of liquid-stored, extended boar semen remains in question, thereby warranting further non-empirical analyses of HA-sperm membrane interactions.

  • 9.
    Alvarez-Rodriguez, Manuel
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Vicente-Carrillo, Alejandro
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Rodriguez-Martinez, Heriberto
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Exogenous Individual Lecithin-Phospholipids (Phosphatidylcholine and Phosphatidylglycerol) Cannot Prevent the Oxidative Stress Imposed by Cryopreservation of Boar Sperm.2017Ingår i: Journal of veterinary medicine and surgery, ISSN 2574-2868, Vol. 1, nr 1Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Objective: Despite the use of high proportions of the chemically undefined lipoprotein/phospholipid-rich egg-yolk in extenders, boar sperm are highly sensitive to cooling, which induces ROS generation and disrupts the plasma membrane.

    Here, we studied whether replacement of hen egg-yolk by commercially defined lecithin phospholipids, derived from egg (LPGE: phosphatidyl glycerol, LPCE: phosphatidyl choline) or soybean (LPCS: phosphatidyl choline), could individually ameliorate such oxidative effects during cryopreservation of ejaculated (sperm rich fraction, SRF) or of cauda-epididymal sperm, retrieved post-mortem from the same males.

    Methods: A conventional extender (lactose buffer, with 20% egg-yolk, 0.5% OEP and 3% glycerol) was used as control. Cryodamage was assessed as loss of sperm motility, membrane and acrosome intactness, early membrane destabilization changes, mitochondrial potential, superoxide and ROS production, to finally determine lipid peroxidation (LPO) using specific probes.

    Results and conclusion: In general, the exogenous phospholipids assayed were unable of maintaining neither sperm motility nor viability post-thaw compared to controls, owing to increased ROS production and lipid peroxidation. In our study, mitochondrial superoxide production resulted in very high levels for all groups, whereas both ROS production and lipid peroxidation were reduced in the control group, containing emulsified hen egg yolk. Further studies using various dosage and combination of LPCS should be followed for their eventual protective effect.

    Keywords: Cryodamage; Sperm; Boar; Mitochondrial activation; Mitochondrial superoxide; ROS production; Lipid peroxidation

  • 10.
    Alvarez-Rodríguez, Manuel
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Anel-López, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Martínez-Rodríguez, Carmen
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Campus de Vegazana, León Spain.
    Martínez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Campus de Vegazana, León Spain.
    Borragan, Santiago
    Cabárceno Park, Cantabria, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Campus de Vegazana, León Spain.
    The antioxidant effects of soybean lecithin- or low-density lipoprotein-based extenders for the cryopreservation of brown-bear (Ursus arctos) spermatozoa.2013Ingår i: Reproduction, Fertility and Development, ISSN 1031-3613, E-ISSN 1448-5990, Vol. 25, nr 8, s. 1185-1193Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Egg yolk low-density lipoproteins (LDL) and soybean lecithin were evaluated as replacements for egg yolk in extenders used for the cryopreservation of brown-bear spermatozoa. The motility, viability and acrosomal status of post-thawed spermatozoa were analysed, and an egg-yolk extender was used as a control. The total antioxidant capacity of these extenders was tested. Soybean lecithin showed an effect that was dependent on the soybean concentration (2%, 3.5% and 5%) and source (Type A: 24% L-α-phosphatidylcholine, and Type B: 14-23% L-α-phosphatidylcholine). Only semen cryopreserved with 5% Type A soybean exhibited a sperm motility similar to that of semen cryopreserved in egg-yolk-based extender after thawing, although the sperm viability and acrosome status were not as high. Semen frozen in an extender containing LDL (10-15%) exhibited improved sperm viability in comparison with the control, but sperm motility was lower. The LDL-based extender exhibited a higher anti-oxidant activity than the egg-yolk extender and soy lecithin-based extenders. The extenders with higher anti-oxidant activity showed improvements in frozen sperm viability but lower semen motility. These results indicate that soybean lecithin did not have the same protective effect as egg yolk during the freezing of brown-bear spermatozoa but suggest that LDL (10-15%) could be a useful substitute for egg yolk in these extenders.

  • 11.
    Alvarez-Rodríguez, Manuel
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain .
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Borragan, Santiago
    Cabárceno Park, Cantabria, Spain.
    Martinez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain .
    Holt, W V
    The Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Sheffield, UK.
    Fazeli, Alireza
    The Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Sheffield, UK.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain .
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    The addition of heat shock protein HSPA8 to cryoprotective media improves the survival of brown bear (Ursus arctos) spermatozoa during chilling and after cryopreservation.2013Ingår i: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 79, nr 3, s. 541-550Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Cantabrian brown bear survives as a small remnant population in northern Spain and semen cryopreservation for future artificial insemination is one of the measures being implemented for conservation of this species. As part of this program we investigated the value of adding heat shock protein A8 (HSPA8) to media (N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid-TRIS-fructose with 20% egg yolk) used for chilling and cryopreserving the spermatozoa. Semen samples from eight brown bears were obtained by electroejaculation during the breeding season. In experiment 1, we tested three concentrations of HSPA8 (0.5, 1, and 5 μg/mL) to determine whether sperm motility (computer assisted sperm analysis system) and sperm survival could be improved during refrigeration (5 °C) up to 48 hours. Results showed that sperm viability (test with propidium iodide) was improved by the addition of 0.5 and 5 μg/mL HSPA8. In experiment 2, HSPA8 was added to the cryopreservation media (6% final glycerol concentration) before the freezing process. Though there were no differences in sperm viability immediately after thawing (analyses to 0 hours), plasma membrane permeability (test with YO-PRO-1) was significantly lower by the presence of HSPA8 (1 μg/mL) and acrosomal damage (test with peanut agglutinin-fluorescein isothiocyanate conjugate) was reduced by higher concentrations of HSPA8 (1 and 5 μg/mL) (analyses after thermal stress test incubating over 2 hours to 37 °C). In experiment 3, results of a simple progression test carried out through artificial mucus (hyaluronic acid 4 mg/mL) showed a significant decrease in the total number of sperm able to swim a distance of 0.5 to 2 cm through a capillary tube for all HSPA8-based extenders. Nevertheless, the distance traveled by the vanguard spermatozoa, which represent a highly motile subpopulation, was restored by the inclusion of 1 and 5 μg/mL HSPA8 in the cryopreservation media. Thus, the HSPA8 addition to extender improves the quality of brown bear (Ursus arctos) sperm during chilling (viability) and after cryopreservation (number of sperm with damaged acrosomes and "apoptotic-like" changes).

  • 12.
    Alvarez-Rodríguez, Manuel
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    López-Urueña, Elena
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Martínez-Rodriguez, Carmen
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain.
    Borragan, Santiago
    Cabárceno Park, Cabárceno, Cantabria, Spain.
    Anel-López, Luis
    SaBio IREC (CSIC-UCLM-JCCM), Albacete, Spain.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Brown bear sperm double freezing: Effect of elapsed time and use of PureSperm(®) gradient between freeze-thaw cycles.2013Ingår i: Cryobiology, ISSN 0011-2240, E-ISSN 1090-2392, Vol. 67, nr 3, s. 339-346Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze-thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed. We carried out two experiments: (1) to assess the effects of the two freezing-thawing cycles on sperm quality and to analyze three different elapsed times between freezing-thawing cycles (30, 90 and 180 min), and (2) to analyze the use of PureSperm between freezing-thawing cycles to select a more motile and viable sperm subpopulation which better survived first freezing. The motility, viability and undamaged acrosomes were significantly reduced after the second thawing respect to first thawing into each elapsed time group, but the elapsed times did not significantly affect the viability and acrosome status although motility was damaged. Our results with the PureSperm gradient showed higher values of viability in freezability of select sample (pellet) respect to the rest of the groups and it also showed a significant decrease in the number of acrosome damaged. In summary, the double freezing of bear semen selected by gradient centrifugation is qualitatively efficient, and thus could be useful to carry out a sex-sorting of frozen-thawed bear spermatozoa before to send the cryopreserved sample to a biobank. Given the low recovery of spermatozoa after applying a selection gradient, further studies will be needed to increase the recovery rate without damaging of the cell quality.

  • 13.
    Anel-López, Luis
    et al.
    Biology of Reproduction Group. National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Alvarez-Rodríguez, Manuel
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    García-Álvarez, Olga
    Biology of Reproduction Group. National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Maroto-Morales, Alejandro
    Biology of Reproduction Group. National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain.
    Garde, J Julián
    Biology of Reproduction Group. National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Martínez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain.
    Reduced glutathione and Trolox (vitamin E) as extender supplements in cryopreservation of red deer epididymal spermatozoa2012Ingår i: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 135, nr 1-4, s. 37-46Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The use of assisted reproductive techniques in cervids is increasing as the commercial use of these species increase. We have tested the suitability of the antioxidants Trolox and reduced glutathione (GSH) for freezing red deer epididymal spermatozoa, aiming at improving post-thawing quality. Samples from 19 stags were frozen in a TES-Tris-fructose extender (20% egg yolk, 8% glycerol), with 1 or 5 mM of antioxidant. Motility (CASA), lipoperoxidation (malondialdehyde -MDA- production), membrane status, mitochondrial activity, acrosomal status (flow cytometry) and chromatin status (SCSA: %DFI and %HDS; flow cytometry) were assessed after thawing and after 6 h at 39°C. There were few differences between treatments after thawing, with Trolox reducing MDA production in a dose-response manner. After the incubation, sperm quality decreased and %DFI increased moderately, with no change for MDA. GSH improved motility, kinematic parameters and mitochondrial status, with a slight increase in %HDS. GSH 5 mM also increased moderately MDA production and %DFI, possibly due to enhanced metabolic activity and reducing power. Trolox maintained MDA low, but was detrimental to sperm quality. Trolox might not be appropriate for the cryopreservation of red deer epididymal spermatozoa, at least at the millimolar range. GSH results are promising, especially regarding motility improvement after the post-thawing incubation, and should be selected for future fertility trials.

  • 14.
    Atikuzzaman, Mohammad
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Carrillo, Alejandro Vicente
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Johnsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biologi. Linköpings universitet, Tekniska fakulteten.
    Wright, Dominic
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biologi. Linköpings universitet, Tekniska fakulteten.
    Rodriguez-Martinez, Heriberto
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Conserved gene expression in sperm reservoirs between birds and mammals in response to mating.2017Ingår i: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 18, nr 1Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BACKGROUND: Spermatozoa are stored in the oviductal functional sperm reservoir in animals with internal fertilization, including zoologically distant classes such as pigs or poultry. They are held fertile in the reservoir for times ranging from a couple of days (in pigs), to several weeks (in chickens), before they are gradually released to fertilize the newly ovulated eggs. It is currently unknown whether females from these species share conserved mechanisms to tolerate such a lengthy presence of immunologically-foreign spermatozoa. Therefore, global gene expression was assessed using cDNA microarrays on tissue collected from the avian utero-vaginal junction (UVJ), and the porcine utero-tubal junction (UTJ) to determine expression changes after mating (entire semen deposition) or in vivo cloacal/cervical infusion of sperm-free seminal fluid (SF)/seminal plasma (SP).

    RESULTS: In chickens, mating changed the expression of 303 genes and SF-infusion changed the expression of 931 genes, as compared to controls, with 68 genes being common to both treatments. In pigs, mating or SP-infusion changed the expressions of 1,722 and 1,148 genes, respectively, as compared to controls, while 592 genes were common to both treatments. The differentially expressed genes were significantly enriched for GO categories related to immune system functions (35.72-fold enrichment). The top 200 differentially expressed genes of each treatment in each animal class were analysed for gene ontology. In both pig and chicken, an excess of genes affecting local immune defence were activated, though frequently these were down-regulated. Similar genes were found in both the chicken and pig, either involved in pH-regulation (SLC16A2, SLC4A9, SLC13A1, SLC35F1, ATP8B3, ATP13A3) or immune-modulation (IFIT5, IFI16, MMP27, ADAMTS3, MMP3, MMP12).

    CONCLUSION: Despite being phylogenetically distant, chicken and pig appear to share some gene functions for the preservation of viable spermatozoa in the female reservoirs.

  • 15.
    Atikuzzaman, Mohammad
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Vicente Carrillo, Alejandro
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Johnsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biologi. Linköpings universitet, Tekniska fakulteten.
    Wright, Dominic
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biologi. Linköpings universitet, Tekniska fakulteten.
    Rodriguez-Martinez, Heriberto
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Correction: Conserved gene expression in sperm reservoirs between birds and mammals in response to mating (vol 18, 98, 2017)2017Ingår i: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 18, artikel-id 563Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    n/a

  • 16.
    Atikuzzaman, Mohammad
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Sanz, Libia
    Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain.
    Pla, Davinia
    Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Rubér, Marie
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Wright, Dominic
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biologi. Linköpings universitet, Tekniska fakulteten.
    Calvete, Juan J.
    Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain.
    Rodriguez-Martinez, Heriberto
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Selection for higher fertility reflects in the seminal fluid proteome of modern domestic chicken2017Ingår i: Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics, ISSN 1744-117X, E-ISSN 1878-0407, Vol. 21, s. 27-40Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The high egg-laying capacity of the modern domestic chicken (i.e. White Leghorn, WL) has arisen from the low egg-laying ancestor Red Junglefowl (RJF) via continuous trait selection and breeding. To investigate whether this long-term selection impacted the seminal fluid (SF)-proteome, 2DE electrophoresis-based proteomic analyses and immunoassays were conducted to map SF-proteins/cytokines in RJF, WL and a 9th generation Advanced Intercross Line (AIL) of RJF/WL-L13, including individual SF (n = 4, from each RJF, WL and AIL groups) and pools of the SF from 15 males of each group, analyzed by 2DE to determine their degree of intra-group (AIL, WL, and RJF) variability using Principal Component Analysis (PCA); respectively an inter-breed comparative analysis of intergroup fold change of specific SF protein spots intensity between breeds. The PCA clearly highlighted a clear intra-group similarity among individual roosters as well as a clear inter-group variability (e.g. between RJF, WL and AIL) validating the use of pools to minimize confounding individual variation. Protein expression varied considerably for processes related to sperm motility, nutrition, transport and survival in the female, including signaling towards immunomodulation. The major conserved SF-proteins were serum albumin and ovotransferrin. Aspartate aminotransferase, annexin A5, arginosuccinate synthase, glutathione S-transferase 2 and l-lactate dehydrogenase-A were RJF-specific. Glyceraldehyde-3-phosphate dehydrogenase appeared specific to the WL-SF while angiotensin-converting enzyme, γ-enolase, coagulation factor IX, fibrinogen α-chain, hemoglobin subunit α-D, lysozyme C, phosphoglycerate kinase, Src-substrate protein p85, tubulins and thioredoxin were AIL-specific. The RJF-SF contained fewer immune system process proteins and lower amounts of the anti-inflammatory/immunomodulatory TGF-β2 compared to WL and AIL, which had low levels- or lacked pro-inflammatory CXCL10 compared to RJF. The seminal fluid proteome differs between ancestor and modern chicken, with a clear enrichment of proteins and peptides related to immune-modulation for sperm survival in the female and fertility.

  • 17.
    Barranco, Isabel
    et al.
    University of Murcia, Murcia, Spain.
    Perez-Patiño, Cristina
    University of Murcia, Murcia, Spain.
    Tvarijonaviciute, Asta
    University of Murcia, Murcia, Spain.
    Parrilla, Inmaculada
    University of Murcia, Murcia, Spain.
    Vicente-Carrillo, Alejandro
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Ceron, Jose J
    University of Murcia, Murcia, Spain.
    Martinez, Emilio A
    University of Murcia, Murcia, Spain.
    Rodriguez-Martinez, Heriberto
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Roca, Jordi
    University of Murcia, Murcia, Spain.
    Active paraoxonase 1 is synthesised throughout the internal boar genital organs.2017Ingår i: Reproduction (Cambridge, England), ISSN 1741-7899, Vol. 154, nr 3, s. 237-243Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The paraoxonase type 1 (PON1) is an enzyme with antioxidant properties recently identified in the seminal plasma (SP) of several species, including the porcine. The aims of the present study were to (1) describe the immunohistochemical localisation of PON1 in the genital organs of fertile boars and (2) evaluate the relationship among PON1 activity and high-density lipoprotein cholesterol (HDL-C) concentration in fluids of the boar genital organs. Immunohistochemical analysis demonstrated that PON1 was present in testis (specifically in Leydig cells, blood vessels, spermatogonia and elongated spermatids), epididymis (specifically in the cytoplasm of the principal epithelial cells, luminal secretion and in the surrounding smooth muscle) and the lining epithelia of the accessory sexual glands (cytoplasmic location in the prostate and membranous in the seminal vesicle and bulbourethral glands). The Western blotting analysis confirmed the presence of PON1 in all boar genital organs, showing in all of them a band of 51 kDa and an extra band of 45 kDa only in seminal vesicles. PON1 showed higher activity levels in epididymal fluid than those in SP of the entire ejaculate or of specific ejaculate portions. A highly positive relationship between PON1 activity and HDL-C concentration was found in all genital fluids. In sum, all boar genital organs contributing to sperm-accompanying fluid/s were able to express PON1, whose activity in these genital fluids is highly dependent on the variable HDL-C concentration present.

  • 18.
    Crespo-Felez, I.
    et al.
    University of Leon, Spain.
    Castaneda-Sampedro, A.
    University of Leon, Spain.
    Sanchez, D. I.
    University of Leon, Spain.
    Fernandez-Alegre, E.
    University of Leon, Spain.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Dominguez, J. C.
    University of Leon, Spain.
    Morrell, J. M.
    Swedish University of Agriculture Science, Sweden.
    Martinez-Pastor, F.
    University of Leon, Spain; University of Leon, Spain.
    Effect of Single Layer Centrifugation Porcicoll (70%, 80% and 90%) or supplementation with reduced glutathione, seminal plasma and bovine serum albumin on frozen-thawed boar sperm2017Ingår i: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 187, s. 167-173Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Selecting the optimal sperm population is essential for success with reproductive techniques. Porcicoll (formerly Androcoll-P) is a colloid formulation for selection of high-quality boar spermatozoa by single layer centrifugation (SLC). To date, most studies have been carried out with fresh semen and large volumes. We carried out 2 experiments to test the use of Porcicoll for thawed boar semen in small volumes. In Experiment 1, cryopreserved semen doses were thawed, split in 200-pL aliquots and layered on 1 mL of Porcicoll 70%, 80% or 90%, or buffer without colloid. We assessed sperm recovery (the proportion of the loading dose that appeared in the pellet, %), and the physiology of the selected spermatozoa (flow cytometry: Viability, apoptotic changes, capacitation, mitochondrial activity, intracellular reactive oxygen species). The most suitable proportion was Porcicoll 80%, allowing acceptable sperm recovery (16.9 4.2%, compared to 70% (35.4% 3.0, p amp;lt; 0.001) and 90% (8.2% 3.0, P = 0.001), and improved quality (mitochondrial activity: Porcicoll 80%: 77.7 1% vs Control: 60.3 0.7%, P amp;lt; 0.05). In Experiment 2, we compared 3 supplements to Porcicoll 80%: 500 mM reduced glutathione (GSH), 20% seminal plasma (SP) and 0.5% bovine serum albumin (BSA). Supplementation with GSH or BSA did not cause relevant changes relative to Control. In contrast, SP induced membrane and acrosomal changes resembling capacitation, which might preclude its use in some applications, and decreased recovery (5.5% 1.9 vs. 24.3% 1.2 Control; P amp;lt; 0.001). However, it could be useful prior to other applications such as in vitro fertilisation. Overall, Porcicoll is an effective colloid for isolating a high-quality population from thawed boar sperm, 80% being a balanced option for good recovery and high quality. Supplements could be useful depending on the proposed use of the spermatozoa.

  • 19.
    de Paz, Paulino
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology, University of León, León, Spain.
    Alvarez-Rodriguez, Manuel
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León.
    Nicolas, Manuel Aguilar
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León.
    Chamorro, Ca
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León.
    Borragán, Santiago
    Cabárceno Park, Cantabria, Spain.
    Martinez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology, University of León, León, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León.
    Optimization of glycerol concentration and freezing rate in the cryopreservation of ejaculate from brown bear (Ursus arctos).2012Ingår i: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 47, nr 1, s. 105-112Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In order to establish a semen bank for the endangered Cantabrian brown bear, we tested five glycerol concentrations and three freezing rates for electroejaculated semen. Electroejaculation was performed on nine males. Semen was diluted in TES-Tris-Fructose (20% egg yolk, 2% EDTA, 1% Equex) with 2%, 4%, 6%, 8% or 10% glycerol and frozen at -10, -20 or -40°C/min. Before and after cryopreservation, samples were analysed for motility (CASA), viability and acrosomal status (flow cytometry). Pre-freezing results showed that glycerol concentration had no significant effect on total motility or progressive motility, but it significantly decreased VCL, ALH, viability and acrosomal status (p < 0.05). After thawing, sperm motility was higher at extender with 4%, 6% and 8% glycerol, but only at 4% and 6% glycerol for viability and acrosomal status. For 4% and 6% glycerol, freezing rates did not have significant effects. The curve fitting gave an estimate of the optimal glycerol concentration, with all the optimal values for every parameter between 6% and 7% glycerol falling. We propose using 6% glycerol and a freezing velocity of -20°C/min for freezing brown bear ejaculated spermatozoa.

  • 20.
    de Paz, Paulino
    et al.
    Department of Molecular Biology, University of León, León, Spain.
    Mata-Campuzano, María
    Department of Molecular Biology, University of León, León, Spain.
    Tizado, Emilio Jorge
    Department of Biodiversity and Environmental Management, University of León, León, Spain.
    Alvarez, Mercedes
    Department of Veterinary Medicine, Anatomy and Surgery, University of León, León, Spain.
    Alvarez-Rodríguez, Manuel
    Department of Veterinary Medicine, Anatomy and Surgery, University of León, León, Spain.
    Herraez, Paz
    Department of Molecular Biology, University of León, León, Spain.
    Anel, Luis
    Department of Veterinary Medicine, Anatomy and Surgery, University of León, León, Spain.
    The relationship between ram sperm head morphometry and fertility depends on the procedures of acquisition and analysis used.2011Ingår i: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 76, nr 7, s. 1313-1325Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Sperm head morphometry is a parameter in the evaluation of semen that has been associated with fertility in two ways: comparing morphometric measures between predefined groups of fertility; or analyzing morphometric data by multivariate techniques to identify cell populations. We analyzed the morphometry of ram sperm head by three procedures and checked its relationship with male fertility. A Computer-Aided Sperm Morphometric Assessment procedure (CASMA), an image analysis software (NIS-Elements) in combination with an optical microscope (MO-NIS) and this image analysis software in combination with a scanning electron microscope (SEM-NIS) were used. Eight morphometric parameters were assessed: length, width, area, perimeter, ellipticity, form factor, elongation and regularity. We observed significant differences between the morphometric data of sperm head obtained with three study procedures. The CASMA procedure shows the highest values for all parameters and the SEM-NIS procedure the lowest. The analysis of a semen sample, when only the mean of morphometric parameters is used to describe the cell population, is too limited to interpret their fertilizing capacity. It is essential to analyze the complex structure of the samples by defining subpopulations by multivariate methods. With few exceptions, the means of each morphometric parameter differ between the three subpopulations analyzed in each procedure. Only the subpopulations obtained with the MO-NIS procedure showed a significant correlation with male fertility. In short, it is necessary to establish an instrumental standard for the analysis of sperm morphometry to obtain reliable results and we believe that the MO-NIS system presents these basic requirements.

  • 21.
    Fernández-Gago, Rocío
    et al.
    Department of Medicine, Surgery and Veterinary Anatomy, University of León, 24071 León, Spain.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Alonso, Marta E
    Department of Animal Production, University of León, 24071 León, Spain.
    González, J Ramiro
    Department of Medicine, Surgery and Veterinary Anatomy, University of León, 24071 León, Spain.
    Alegre, Beatriz
    Department of Medicine, Surgery and Veterinary Anatomy, University of León, 24071 León, Spain.
    Domínguez, Juan C
    Department of Medicine, Surgery and Veterinary Anatomy, University of León, 24071 León, Spain.
    Martínez-Pastor, Felipe
    INDEGSAL (Institute for Animal Health and Cattle Development), University of León, 24071 León, Spain /Molecular Biology (Cell Biology), University of León, 24071 León, Spain/ .
    Thawing boar semen in the presence of seminal plasma improves motility, modifies subpopulation patterns and reduces chromatin alterations.2017Ingår i: Reproduction, fertility, and development, ISSN 1031-3613, Vol. 29, nr 8, s. 1576-1584Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Seminal plasma could have positive effects on boar semen after thawing. In the present study we investigated changes in the motility and chromatin structure in spermatozoa over 4h incubation (37°C) of boar semen thawed in the presence of 0%, 10% or 50% seminal plasma from good-fertility boars. Cryopreserved doses were used from seven males, three of which were identified as susceptible to post-thawing chromatin alterations. Motility was analysed by computer-aided sperm analysis every hour, and data were used in a two-step clustering, yielding three subpopulations of spermatozoa (slow non-linear, fast non-linear, fast linear). Chromatin structure was analysed using a sperm chromatin structure assay and flow cytometry to determine the DNA fragmentation index (%DFI) as a percentage, the standard deviation of the DFI (SD-DFI) and the percentage of high DNA stainability (%HDS), indicating chromatin compaction. Thawing without seminal plasma resulted in a rapid loss of motility, whereas seminal plasma helped maintain motility throughout the incubation period and preserved the subpopulation comprising fast and linear spermatozoa. The incidence of chromatin alterations was very low in samples from non-susceptible males, whereas samples from males susceptible to post-thawing chromatin alterations exhibited marked alterations in%DFI and%HDS. Seminal plasma partly prevented these alterations in samples from susceptible males. Overall, 50% seminal plasma was the most efficient concentration to protect motility and chromatin. Some changes were concomitant with physiological events reported previously (e.g., semen thawed with 50% seminal plasma increased the production of reactive oxygen species and yielded higher fertility after AI). Thawing in the presence of seminal plasma could be particularly useful in the case of samples susceptible to post-thawing chromatin damage.

  • 22.
    Gardela, Jaume
    et al.
    Univ Autonoma Barcelona, Spain.
    Ruiz-Conca, Mateo
    Univ Autonoma Barcelona, Spain.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten. Univ Autonoma Barcelona, Spain.
    Mogas, Teresa
    Univ Autonoma Barcelona, Spain.
    Lopez-Bejar, Manel
    Univ Autonoma Barcelona, Spain.
    Induction of CIRBP expression by cold shock on bovine cumulus-oocyte complexes2019Ingår i: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 54, s. 82-85Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The aim of this study was to induce the cold-inducible RNA-binding protein (CIRBP) expression on cumulus-oocyte complexes (COCs) through exposure to a sub-lethal cold shock and determine the effects of hypothermic temperatures during the in vitro maturation of bovine oocytes. Nuclear maturation, cortical granule redistribution and identification of cold-inducible RNA-binding protein (CIRBP) were assessed after 24 hr of in vitro maturation of control (38.5 degrees C) and cold-stressed oocytes (33.5 degrees C). The presence of CIRBP was assessed by Western blot in COCs or denuded oocytes and their respective cumulus cells. Based on the odds ratio, cold-stressed oocytes presented higher abnormal cytoplasmic distribution of cortical granules and nuclear maturation than the control group. Although CIRBP was detected in both control and cold-stressed groups, cold-stressed COCs had 2.17 times more expression of CIRBP than control COCs. However, when denuded oocytes and cumulus cells were assessed separately, CIRBP only was detected in cumulus cells in both groups. In conclusion, cold shock induced CIRBP expression, but it negatively affected nuclear maturation and cortical granule distribution of bovine oocytes. Moreover, the expression of CIRBP was only identified in cumulus cells but not in oocytes.

  • 23.
    Iso-Touru, Terhi
    et al.
    Nat Resources Inst Finland Luke, Finland.
    Wurmser, Christine
    Tech Univ Munich, Germany.
    Venhoranta, Heli
    Univ Helsinki, Finland.
    Hiltpold, Maya
    Swiss Fed Inst Technol, Switzerland.
    Savolainen, Tujia
    Univ Helsinki, Finland.
    Sironen, Anu
    Nat Resources Inst Finland Luke, Finland.
    Fischer, Konrad
    Tech Univ Munich, Germany.
    Flisikowski, Krzysztof
    Tech Univ Munich, Germany.
    Fries, Ruedi
    Tech Univ Munich, Germany.
    Vicente Carrillo, Alejandro
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Medicinska fakulteten.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Nagy, Szabolcs
    Univ Pannonia, Hungary.
    Mutikainen, Mervi
    Nat Resources Inst Finland Luke, Finland.
    Peippo, Jaana
    Nat Resources Inst Finland Luke, Finland.
    Taponen, Juhani
    Univ Helsinki, Finland.
    Sahana, Goutam
    Aarhus Univ, Denmark.
    Guldbrandtsen, Bernt
    Aarhus Univ, Denmark.
    Simonen, Henri
    VikingGenet, Finland.
    Rodriguez-Martinez, Heriberto
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Andersson, Magnus
    Univ Helsinki, Finland.
    Pausch, Hubert
    Swiss Fed Inst Technol, Switzerland.
    A splice donor variant in CCDC189 is associated with asthenospermia in Nordic Red dairy cattle2019Ingår i: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 20, nr 1, artikel-id 286Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background

    Cattle populations are highly amenable to the genetic mapping of male reproductive traits because longitudinal data on ejaculate quality and dense microarray-derived genotypes are available for thousands of artificial insemination bulls. Two young Nordic Red bulls delivered sperm with low progressive motility (i.e., asthenospermia) during a semen collection period of more than four months. The bulls were related through a common ancestor on both their paternal and maternal ancestry. Thus, a recessive mode of inheritance of asthenospermia was suspected.

    Results

    Both bulls were genotyped at 54,001 SNPs using the Illumina BovineSNP50 Bead chip. A scan for autozygosity revealed that they were identical by descent for a 2.98 Mb segment located on bovine chromosome 25. This haplotype was not found in the homozygous state in 8557 fertile bulls although five homozygous haplotype carriers were expected (P = 0.018). Whole genome-sequencing uncovered that both asthenospermic bulls were homozygous for a mutation that disrupts a canonical 5′ splice donor site of CCDC189 encoding the coiled-coil domain containing protein 189. Transcription analysis showed that the derived allele activates a cryptic splice site resulting in a frameshift and premature termination of translation. The mutated CCDC189 protein is truncated by more than 40%, thus lacking the flagellar C1a complex subunit C1a-32 that is supposed to modulate the physiological movement of the sperm flagella. The mutant allele occurs at a frequency of 2.5% in Nordic Red cattle.

    Conclusions

    Our study in cattle uncovered that CCDC189 is required for physiological movement of sperm flagella thus enabling active progression of spermatozoa and fertilization. A direct gene test may be implemented to monitor the asthenospermia-associated allele and prevent the birth of homozygous bulls that are infertile. Our results have been integrated in the Online Mendelian Inheritance in Animals (OMIA) database (https://omia.org/OMIA002167/9913/).

  • 24.
    Liffner, Susanne
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Barn- och kvinnocentrum, Kvinnokliniken i Linköping.
    Pehrson, Isabelle
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Barn- och kvinnocentrum, Kvinnokliniken i Linköping.
    Garcia-Calvo, Laura
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Barn- och kvinnocentrum, Kvinnokliniken i Linköping.
    Nedstrand, Elisabeth
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Barn- och kvinnocentrum, Kvinnokliniken i Linköping.
    Zalavary, Stefan
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Medicinska fakulteten.
    Hammar, Mats
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Barn- och kvinnocentrum, Kvinnokliniken i Linköping.
    Rodriguez-Martinez, Heriberto
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Diagnostics of DNA fragmentation in human spermatozoa: Are sperm chromatin structure analysis and sperm chromatin dispersion tests (SCD-HaloSpermG2 (R)) comparable?2019Ingår i: Andrologia, ISSN 0303-4569, E-ISSN 1439-0272, Vol. 51, nr 8, artikel-id e13316Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Men affected with idiopathic infertility often display basic spermiogramme values similar to fertile individuals, questioning the diagnostic impact of the World Health Organization (WHO) thresholds used. This study explored sperm DNA fragmentation in single ejaculates from 14 fertile donors and 42 patients with idiopathic infertility providing semen for assisted reproductive techniques in a university fertility clinic. Each ejaculate was simultaneously studied for sperm DNA fragmentation by the flow cytometer-based sperm chromatin structure analysis (SCSA) and the new light-microscopy-based sperm chromatin dispersion assay (SCD-HaloSpermG2 (R)), before and after sperm selection for in vitro fertilisation with a colloid discontinuous gradient. The WHO semen variables did not differ between groups, but DNA fragmentation after SCSA (DFI) or SCD (SDF) was significantly (p amp;lt; 0.05) higher in patients (DFI: 40.2% +/- 3.0 vs. SDF: 40.3% +/- 1.4) than in fertile donors (DFI: 17.1% +/- 2.1 vs. SDF: 20.9% +/- 2.5). Sperm selection led to lower proportions of DNA-fragmented spermatozoa (DFI: 11.9 +/- 1.7 vs. SCD: 10.0 +/- 0.9, p amp;lt; 0.05). The techniques output correlated highly and significantly (r(2) = 0.82). DNA fragmentation is confirmed as a relevant variable for scrutinising patients with idiopathic infertility, beyond the evidently insufficient WHO semen analyses. Since both techniques yielded similar results, the reduced necessity of complex equipment when running SCD ought to be considered for a clinical setting.

    Publikationen är tillgänglig i fulltext från 2020-05-17 10:44
  • 25.
    Martínez-Pastor, Felipe
    et al.
    ITRA-ULE, INDEGSAL, Departments of Molecular Biology (Cell Biology), University of León, León, Spain.
    Mata-Campuzano, Maria
    ITRA-ULE, INDEGSAL, Departments of Molecular Biology (Cell Biology), University of León, León, Spain.
    Alvarez-Rodriguez, Manuel
    ITRA-ULE, INDEGSAL, Animal Reproduction and Obstetrics, University of León, León, Spain.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, Animal Reproduction and Obstetrics, University of León, León, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, Animal Reproduction and Obstetrics, University of León, León, Spain.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, Departments of Molecular Biology (Cell Biology), University of León, León, Spain.
    Probes and techniques for sperm evaluation by flow cytometry2010Ingår i: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 45 Suppl 2, s. 67-78Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    CONTENTS: Flow cytometry has become an important technique in sperm evaluation and is increasingly used both for routine assessment and for research in veterinary science. We have revised the literature, describing fluorescent probes that have been used for analysing spermatozoa by flow cytometry, regarding: viability, acrosomal status, capacitation, mitochondrial status, apoptotic markers, oxidative stress markers, DNA damage, sperm counting and sperm sizing. Details and problems of some techniques are reviewed, with special attention to the occurrence of non-sperm particles in the samples ('debris'). New and promising aspects of flow cytometry, such as sperm sorting using viability markers as selection criteria, are highlighted. The relationship between flow cytometry analyses and fertility and their future improvements are considered.

  • 26.
    Martínez-Pastor, Felipe
    et al.
    Department of Molecular Biology (Cell Biology), Universidad de León, Spain; INDEGSAL, Universidad de León, León, Spain.
    Álvarez, Mercedes
    INDEGSAL, Universidad de León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, Universidad de León, León, Spain.
    Guerra, Camino
    Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autònoma de Barcelona, Barcelona, Spain.
    Chamorro, César A
    Department of Medicine, Surgery and Veterinary Anatomy, Universidad de León, León, Spain; Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autònoma de Barcelona, Barcelona, Spain.
    Anel-López, Luis
    INDEGSAL, Universidad de León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, Universidad de León, León, Spain.
    de Paz, Paulino
    Department of Molecular Biology (Cell Biology), Universidad de León, Spain; INDEGSAL, Universidad de León, León, Spain.
    Anel, Luis
    INDEGSAL, Universidad de León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, Universidad de León, León, Spain.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten. INDEGSAL, Universidad de León, León, Spain; Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autònoma de Barcelona, Barcelona, Spain.
    Extender osmolality, glycerol and egg yolk on the cryopreservation of epididymal spermatozoa for gamete banking of the Cantabric Chamois (Rupicapra pyrenaica parva)2019Ingår i: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 125, s. 109-114, artikel-id S0093-691X(18)30573-9Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Germplasm banking is a key technology enabling the ex-situ conservation of wild species. However, cryopreservation protocols must be tested to assure the applicability of the banked material. The objective of this study was defining a range of parameters for the composition of a semen extender for Cantabrian chamois epididymal spermatozoa (post-mortem collection). The freezing extender was based in a TES-Tris-fructose buffer, modifying its composition in three experiments: Osmolality of the buffer (320, 380 or 430 mOsm/kg, 8% glycerol, 15% egg yolk), glycerol (4 or 8%, 430 mOsm/kg, 15% egg yolk), egg yolk (10 or 15%, 430 mOsm/kg, 4% glycerol). Sperm was extended at 100 mill. spermatozoa/ml, cooled at 5 °C and frozen at -20 °C/min. Sperm quality was assessed pre and post-thawing (CASA, HOS test, abnormal forms, cytoplasmic droplets, and viability and acrosomal damage by flow cytometry). Freezability was good overall, with total motility of 65.5% ± 2.4 initial and 55.8% ± 2.4 post-thawing. The extenders affected the post-thaw sperm quality marginally. Whereas osmolalities and glycerol concentrations seemed not to differ, 430 mOsm/kg and 4% glycerol might be preferred. Egg yolk concentrations only differed on sperm velocity (VCL: 84.0 ± 6.7 μm/s in 10% vs. 70.7 ± 6.2 μm/s in 15%, P < 0.05). Our results suggest a good cryotolerance of chamois epididymal spermatozoa, with a preferred extender composition of hyperosmotic buffer, glycerol in the 4% range and lower egg yolk (10% range) than other ruminants.

  • 27.
    Mata-Campuzano, Maria
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Spain.
    Alvarez-Rodríguez, Manuel
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, Spain.
    del Olmo, E
    Biology of Reproduction Group, National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Fernández-Santos, M R
    Biology of Reproduction Group, National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Garde, J J
    Biology of Reproduction Group, National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Martínez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Spain.
    Quality, oxidative markers and DNA damage (DNA) fragmentation of red deer thawed spermatozoa after incubation at 37 °C in presence of several antioxidants.2012Ingår i: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 78, nr 5, s. 1005-1019Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Antioxidants may be useful for supplementing sperm extenders. We have tested dehydroascorbic acid (DHA), TEMPOL, N-acetyl-cysteine (NAC) and rutin on epididymal spermatozoa from red deer, during incubation at 37 °C. Cryopreserved spermatozoa were thawed, washed and incubated with 1 mM or 0.1 mM of each antioxidant, including oxidative stress (Fe(2+)/ascorbate). Motility (CASA and clustering of subpopulations), viability, mitochondrial membrane potential, and acrosomal status were assessed at 2 and 4 h. Lipoperoxidation, intracellular reactive oxygen species (ROS) and DNA damage (DNA) status (TUNEL) were checked at 4 h. Oxidative stress increased ROS, lipoperoxidation and DNA damage. Overall, antioxidants negatively affected motility and physiological parameters. Only DHA 1 mm protected motility, increasing the fast and progressive subpopulation. However, it had a detrimental effect on acrosomal and DNA status, in absence of oxidative stress. Tempol and rutin efficiently reduced lipoperoxidation, ROS, and DNA damage in presence of oxidative stress. NAC was not as efficient as TEMPOL or rutin reducing lipoperoxidation or protecting DNA, and did not reduce ROS, but its negative effects were lower than the other antioxidants when used at 1 mm, increasing the subpopulation of hyperactivated-like spermatozoa at 2 h. Our results show that these antioxidants have mixed effects when spermatozoa are incubated at physiological temperatures. DHA may not be suitable because of prooxidant effects, but TEMPOL, NAC and rutin may be considered for cryopreservation trials. In general, exposure of red deer spermatozoa to these antioxidants should be limited to low temperatures, when only protective effects may develop.

  • 28.
    Mata-Campuzano, María
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Spain.
    Alvarez-Rodríguez, Manuel
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, Spain.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, Spain.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Spain.
    Garde, Julian
    Biology of Reproduction Group, National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Martínez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Spain.
    Effect of several antioxidants on thawed ram spermatozoa submitted to 37°C up to four hours.2012Ingår i: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 47, nr 6, s. 907-914Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Thawed ram spermatozoa were incubated at 37°C in the presence of dehydroascorbic acid (DHA), TEMPOL (TPL), N-acetyl-cysteine (NAC) and rutin (RUT), at 0.1 and 1 mm, in order to test their effects on sperm physiology. Cryopreserved spermatozoa from four rams were thawed, pooled, washed and incubated in TALP-Hepes with 1 mm or 0.1 mm of each antioxidant, performing a replicate with induced oxidative stress (Fe(2+) /ascorbate). Motility (CASA), viability and mitochondrial membrane potential (flow cytometry) were analysed at 2 and 4 h. Lipoperoxidation (MDA production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were analysed at 4 h. Antioxidants, except DHA 0.1 mm, decreased motility and kinematic parameters, but had little effect on viability or mitochondrial activity. Except 1 mm DHA, the antioxidants reduced ROS at 4 h. Moreover, NAC 1 mm, rutin and TEMPOL reduced ROS and DNA damage in the presence of oxidative stress. N-acetyl-cysteine, rutin 1 mm and TEMPOL reduced lipoperoxidation in the presence of oxidative stress. However, DHA did not affect lipoperoxidation. At 1 mm, DHA increased DNA damage in the absence of oxidative stress. Dehydroascorbic acid effects could arise from spermatozoa having a low capacity for reducing it to ascorbic acid, and it may be tested in the presence of other antioxidants or reducing power. Future research should focus in testing whether the inhibition of motility observed for NAC, rutin and TEMPOL is reversible. These antioxidants might be useful at lower temperatures (refrigerated storage or cryopreservation) when their protective effects could be advantageous.

  • 29.
    Mata-Campuzano, María
    et al.
    INDEGSAL, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
    Álvarez-Rodríguez, Manuel
    INDEGSAL, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, University of León, León, Spain; DEPI, Institute of Technology of Conkal, Conkal, Mexico.
    Tamayo-Canul, Julio
    INDEGSAL, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
    López-Urueña, Elena
    INDEGSAL, University of León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, University of León, León, Spain.
    de Paz, Paulino
    INDEGSAL, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
    Anel, Luis
    INDEGSAL, University of León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, University of León, León, Spain.
    Martínez-Pastor, Felipe
    INDEGSAL, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
    Álvarez, Mercedes
    INDEGSAL, University of León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, University of León, León, Spain.
    Refrigerated storage of ram sperm in presence of Trolox and GSH antioxidants: effect of temperature, extender and storage time2014Ingår i: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 151, nr 3-4, s. 137-147Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Antioxidants have a potential to improve the quality and fertility of refrigerated-stored ram semen. Reduced glutathione (GSH) and Trolox (0.2, 1 and 5mM) were evaluated in ram semen preserved at 15 and 5°C up to 48 and 96h, respectively. Extenders were also evaluated (15°C: Tris-citrate-fructose, TCF, without lipids, and TES-Tris-fructose 10% egg yolk, TTF-EY; 5°C: TTF-EY and 3.5% soybean lecithin, TTF-SL; INRA96 at both temperatures). Storage at 5°C resulted in poorer quality than 15°C up to 48h, while allowing acceptable quality at 96h. Antioxidants had few effects on sperm quality, with use of Trolox resulting in reduced motility and viability in TCF. Storage at 15°C in the TCF extender resulted in decreased motility, viability and mitochondrial activity compared with use of TTF-EY. Sperm quality when storage was at 5°C was similar, but storage in TTF-SL resulted in decreased motility and mitochondrial activity. Acrosomal status was only slightly affected by extender and antioxidant. Mitochondrial activity was improved by antioxidants in TTF-SL, and GSH at 5mM when semen was stored at 5°C in TTF-EY. A preliminary artificial insemination trial indicated that supplementation with GSH has the potential for improving lambing (P<0.1). In conclusion, use of antioxidants resulted in lesser effects than extender composition or storage time on quality of ram semen. Use of Trolox negatively impacted sperm quality and GSH had some positive impacts. The use of soybean lecithin requires further research to assess its impact on mitochondria.

  • 30.
    Mata-Campuzano, María
    et al.
    Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
    Álvarez-Rodríguez, Manuel
    Institute for Animal Health and Cattle Development, University of León, León, Spain.
    Álvarez, Mercedes
    Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, University of León, León, Spain.
    Tamayo-Canul, Julio
    Institute for Animal Health and Cattle Development, University of León, León, Spain; Division for Research and Post-graduate Studies, Technological Institute of Conkal, Yucatán, México.
    Anel, Luis
    Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, University of León, León, Spain.
    de Paz, Paulino
    Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
    Martínez-Pastor, Felipe
    Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
    Post-thawing quality and incubation resilience of cryopreserved ram spermatozoa are affected by antioxidant supplementation and choice of extender.2015Ingår i: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 83, nr 4, s. 520-528Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The performance of cryopreserved semen in ovine artificial insemination still needs improvement. Some antioxidants have been tested, with variable success. We cryopreserved semen from Churra rams using TES-Tris-fructose with 4% glycerol and 10% egg yolk (EY) or 3.5% soybean lecithin (SL), with 1 mM of reduced glutathione (GSH), Trolox, crocin, or cysteamine. Samples were analyzed after thawing and incubation (6 hours, 38 °C) for motility (computer-assisted sperm analysis [CASA]), viability, acrosomal integrity, apoptosis, mitochondrial activity, chromatin status, and lipoperoxidation (malondialdehyde production). Interactions (antioxidant/extender/incubation) were significant for most variables. Extenders yielded similar results, although SL depressed mitochondrial activity and linearity (P < 0.001), it improved motility (P < 0.05), DNA fragmentation (P < 0.05), and acrosomal damage (P < 0.001). The control, GSH, and Trolox showed greater viability with SL (P < 0.01). Cysteamine depressed motility (0 hours: 51.6 ± 2.0% vs. 32.3 ± 4.3%; 6 hours: no motility vs. 32.5 ± 1.9%; P < 0.001), but improved viability when using EY (P = 0.004). Crocin increased acrosomal damage (P = 0.022) but improved linearity-related parameters after thawing (P = 0.014). Trolox considerably reduced malondialdehyde production in both extenders (8.6 ± 0.4 nmol per 10(8) cells vs. 14.2 ± 0.3 in EY and 20 ± 0.6 in SL; P < 0.001). Interestingly, thiol antioxidants (cysteamine and GSH) increased DNA fragmentation (percentage of DNA fragmentation index), whereas crocin reduced it (P < 0.05). Interactions between extender and antioxidant must be taken into account for improving sperm cryopreservation. Soybean lecithin seems to be a suitable replacement for EY, but its effect on mitochondria must be investigated. Trolox and crocin might be useful for ram semen freezing.

  • 31.
    Najafi, Abouzar
    et al.
    Department of Animal Science, College of Agriculture, University of Tabriz, Tabriz, Iran.
    Daghigh-Kia, Hossein
    Department of Animal Science, College of Agriculture, University of Tabriz, Tabriz, Iran.
    Dodaran, Hossein Vaseghi
    Department of Animal Science, College of Agriculture, University of Tabriz, Tabriz, Iran.
    Mehdipour, Mahdieh
    Department of Animal Science, College of Agriculture, University of Tabriz, Tabriz, Iran.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Ethylene glycol, but not DMSO, could replace glycerol inclusion in soybean lecithin-based extenders in ram sperm cryopreservation.2017Ingår i: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 177, s. 35-41Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The aim of this study was to evaluate the effects of glycerol, ethylene glycol or DMSO in a soybean lecithin extender for freezing ram semen. In this study, 20 ejaculates were collected from four Ghezel rams and diluted with soybean lecithin extender with glycerol (7%), ethylene glycol (3%, 5% and 7%) or DMSO (3%, 5% and 7%). Sperm motility (CASA), membrane integrity (HOS test), viability, total abnormality, mitochondrial activity (Rhodamine 123) and apoptotic features (Annexin V/Propidium iodide) were assessed after thawing. There was no significant difference between glycerol and ethylene glycol at different concentrations (3% and 5%) regarding sperm total and progressive motility, viability, and membrane integrity. The least percentages of mitochondrial functionality were observed in samples frozen with all different DMSO concentrations tested (P<0.05). Moreover, the percentage of post-thawed dead sperm was the greatest for all the DMSO concentrations compared with other groups (P<0.05). Thus, DMSO had an adverse effect on the post thaw ram sperm parameters. In contrast, ethylene glycol could be a desirable substitute of glycerol in the freezing extender, in view of similar results obtained in post-thaw quality of ram semen cryopreserved in a soybean lecithin extender. We propose that glycerol in a soybean lecithin based extender could be replaced by ethylene glycol at 3% or 5% concentrations.

  • 32.
    Najafi, Davood
    et al.
    Baqiyatallah Univ Med Sci, Iran; Univ Tabriz, Iran.
    Taheri, Ramezan Ali
    Baqiyatallah Univ Med Sci, Iran.
    Najafi, Abouzar
    Baqiyatallah Univ Med Sci, Iran; Univ Tabriz, Iran.
    Rouhollahi, Abbas Abbas
    Univ Tehran, Iran.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten. Univ Autonoma Barcelona, Spain.
    Effect of Achillea millefolium-loaded nanophytosome in the post-thawing sperm quality and oxidative status of rooster semen2018Ingår i: Cryobiology, ISSN 0011-2240, E-ISSN 1090-2392, Vol. 82, s. 37-42Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The aim of this study was to compare the effectiveness of antioxidants including Achillea millefolium extract (AmE) (n0t1.5: 1.5, n0t3: 3 and n0t4.5: 4.5 mg/L) and AmE loaded in nano phytosome (n1t1.5: 1.5, n1t3: 3 and n1t4.5: 4.5 mg/L) in the freezing of Ross 308 rooster semen. Sperm motility (CASA), membrane integrity (HOS test), viability, total abnormality and enzymatic parameters (SOD, CAT and GPx) were assessed after thawing. AmE-loaded nano phytosome at a concentration of 3 mg/l resulted in significantly (P amp;lt; 0.05) higher total motility (MOT) (73.78 +/- 2.92) and at concentrations of 1.5 mg/L and 3 mg/L in progressive motility (PROG) (14.12 +/- 0.38, 16.78 +/- 0.38) in comparison with the control group (MOT: 58.48 +/- 2.92; PROG: 9.08 +/- 0.38). Sperm viability (Vi) was higher (P amp;lt; 0.05) in n1t3 (74.62 +/- 1.55) and membrane integrity (Mi) in n0t3 and n1t3 groups (65.91 +/- 1.91, 63.73 +/- 1.91, respectively) compared to the control groups (Vi: 66.85 +/- 1.55; Mi: 53.18 +/- 1.91). Moreover, the lowest percentage of MDA was measured in n1t3 group (1.31 +/- 0.31). There was no significant difference for SOD and CAT values with the use of various extenders. In conclusion, we suggest that AmE loaded in nano phytosome at 3 mg/l dose can be added to basic extender for improving rooster sperm motility, viability and oxidative stress values during the freezing procedure.

  • 33.
    Nicolas, Manuel
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Borragán, Santiago
    Cabárceno Park, Cantabria, Spain.
    Martinez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Cell Biology, University of León, León, Spain.
    Chamorro, C A
    ITRA-ULE, INDEGSAL, University of León, León, Spain.
    Alvarez-Rodriguez, Manuel
    ITRA-ULE, INDEGSAL, University of León, León, Spain.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Cell Biology, University of León, León, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Evaluation of the qualitative and quantitative effectiveness of three media of centrifugation (Maxifreeze, Cushion Fluid Equine, and PureSperm 100) in preparation of fresh or frozen-thawed brown bear spermatozoa.2012Ingår i: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 77, nr 6, s. 1119-1128Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Centrifugation is a crucial procedure in sperm cryopreservation protocols of brown bear (Ursus arctos), because the semen must be processed to increase sperm concentration and/or clean urine-contaminated samples. The efficacy of three media for centrifugation (Maxifreeze [IMV technologies, L'Aigle, France], Cushion Fluid Equine (Minitübe, Tiefenbach, Germany), and PureSperm [Nidacon, Gothenburg, Sweden]) on the quality of bear spermatozoa was evaluated. In experiment one, two cushioned media used for protecting against mechanical stress during centrifugation were analyzed. In experiment two, a density gradient based on PureSperm was assessed in relation to the maximum retrieval and the quality of fresh spermatozoa, and the freezability of the spermatozoa selected in this density gradient was studied in experiment three. Finally, the selection of frozen-thawed sperm using PureSperm was analyzed in experiment four. Our results indicate that the use of dense isotonic cushion solutions (Maxifreeze, Cushion Fluid Equine) in centrifugation did not improve the quality of recovered spermatozoa compared with standard centrifugation. However, a density gradient prepared with PureSperm improved the quality of spermatozoa in fresh semen and frozen-thawed semen, but the spermatozoa selected from the fresh sample with this density gradient did not show a better resistance to freezing with this density gradient in comparison with the control sample.

  • 34.
    Peris-Frau, Patricia
    et al.
    SaBio IREC CSIC UCLM JCCM, Spain.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Martin-Maestro, Alicia
    SaBio IREC CSIC UCLM JCCM, Spain.
    Iniesta-Cuerda, Maria
    SaBio IREC CSIC UCLM JCCM, Spain.
    Sanchez-Ajofrin, Irene
    SaBio IREC CSIC UCLM JCCM, Spain.
    Julian Garde, Jose
    SaBio IREC CSIC UCLM JCCM, Spain.
    Rodriguez-Martinez, Heriberto
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten.
    Josefa Soler, Ana
    SaBio IREC CSIC UCLM JCCM, Spain.
    Comparative evaluation of DNA integrity using sperm chromatin structure assay and Sperm-Ovis-Halomax during in vitro capacitation of cryopreserved ram spermatozoa2019Ingår i: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 54, s. 46-49Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    This study aimed to compare the ability of sperm chromatin structure assay (SCSA (R)) and Sperm-Ovis-Halomax (R) to detect DNA fragmentation in frozen-thawed ram spermatozoa incubated under capacitating conditions in synthetic oviductal fluid (SOF) supplemented with oestrous sheep serum (SOF-ESS) at multiple time points (0-240 min). Incubation in SOF-ESS had no significant effects on SCSA (R) parameters while the percentage of spermatozoa with fragmented DNA measured by Sperm-Ovis-Halomax (R) increased after 180 min of incubation. In addition, no correlation or agreement was found between the techniques, suggesting that SCSA (R) and Sperm-Ovis-Halomax (R) may quantify different types of DNA damage in ram spermatozoa under these experimental conditions.

  • 35.
    Rodriguez-Martinez, Heriberto
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Tienthai, P.
    Chulalongkorn University, Thailand.
    Atikuzzaman, Mohammad
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Vicente Carrillo, Alejandro
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Rubér, Marie
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    The ubiquitous hyaluronan: Functionally implicated in the oviduct?2016Ingår i: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 86, nr 1, s. 182-186Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Hyaluronan (hyaluronic acid) is a simple, nonantigenic, nonsulfated glycosaminoglycan present everywhere in the extracellular compartments of the body. Noteworthy, it is highly conserved phylogenetically, from sauropsida to mammals; and plays a plethora of roles from embryonic/fetal development to adult physiological and pathological events, including tumor development. In reproduction, hyaluronan has proven related to initial events as sperm survival, buildup of the sperm reservoir in the oviduct, regulation of sperm capacitation, and prefertilization to later participate in embryo, fetal, and placental development. Synthesis, binding (via the CD44 membrane receptor), and degradation of hyaluronan occur in male and female genital organs, the oviduct being no exception. This review discusses our current knowledge on roles of this ubiquitous glycosaminoglycan on the survival of immunologically foreign spermatozoa in the pig oviduct, a relevant event for fertility. During preovulatory storage in the functional tubal sperm reservoir, spermatozoa are entrapped in a mucus-like tubal fluid. This fluid contains fluctuating levels of hyaluronan, which is synthesized by the lining epithelium by hyaluronan synthase 3. Both hyaluronan and its CD44 receptor are particularly evident in the deep mucosal furrows of the sperm reservoir, in which most spermatozoa are embedded in; kept alive, uncapacitated but also undetected by the immune system of the female. Hyaluronan is also present in the seminal plasma, and evidence points toward an involvement of hyaluronan and its receptor in the local (tubal and possibly uterine) production of antiinflammatory cytokines, such as interleulcin-10, pertaining maternal immune tolerance of these foreign cells. (C) 2015 Elsevier Inc. All rights reserved.

  • 36.
    Tamayo-Canul, Julio
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Mata-Campuzano, Maria
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology, University of León, León, Spain.
    Alvarez-Rodríguez, Manuel
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology, University of León, León, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Martínez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology, University of León, León, Spain.
    Effect of storage method and extender osmolality in the quality of cryopreserved epididymal ram spermatozoa.2011Ingår i: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 129, nr 3-4, s. 188-199Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Post-mortem sperm recovery and cryopreservation could be a complement to germplasm banking in sheep, especially for endangered breeds. This study is an attempt to identify factors for improving the success of cryopreserving ram epididymal spermatozoa, considering the decrease of sperm quality with post-mortem time. Epididymal spermatozoa from 9 rams were kept at 5°C using three storage methods: within the epididymes, undiluted sperm mass, and diluted in extenders of different osmolality (TES-Tris-fructose at 320, 370 or 420 mOsm/kg, 20% egg yolk, 8% glycerol). At 0, 24, 48 and 72h, spermatozoa were cryopreserved using each extender. Samples were analyzed before and after cryopreservation by CASA (motility) and flow cytometry (viability and acrosomal status). Post-mortem time decreased pre-freezing and post-thawing sperm quality. Some storage x extender combinations improved the effect of post-mortem time on sperm quality. Both epididymis storage combined with the 420 extender, and storing the spermatozoa diluted in the 320 extender improved post-thawing quality, especially at long post-mortem times. Storing the spermatozoa diluted in the 370 extender was detrimental for the acrosomal status. These findings have practical applications. The simplest storage method (within the epididymes) seems to be adequate if hyperosmotic extenders were used for freezing. An alternative method could be storing the spermatozoa diluted in a hypoosmotic extender. These recommendations are limited to the osmolalities tested in this study (420 mOsm/kg and 320 mOsm/kg); other osmolalities should be tested.

  • 37.
    Vicente Carrillo, Alejandro
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Alvarez-Rodriguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Rodriguez-Martinez, Heriberto
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    The mu (µ) and delta (δ) opioid receptors modulate boar sperm motility2016Ingår i: Molecular Reproduction and Development, ISSN 1040-452X, E-ISSN 1098-2795, Vol. 83, nr 8, s. 724-734Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Endogenous and exogenous opioids modulate reproductive functions in target cells via opioid receptors (µ, δ, and κ). Sperm motility is a metric of gamete functionality, and serves as a suitable parameter for in vitro drug-induced toxicity assays. This study identifies the presence and location of opioid receptors in pig spermatozoa as well as their functional response after in vitro challenge with known agonists (morphine [µ]; [D-Pen 2,5]-enkephanile [δ]; and U 50488 [κ]) and antagonists (naloxone [µ]; naltrindole [δ]; and nor-binaltrorphimine [κ]). Only the µ- and δ-opioid receptors were present in the sperm plasma membrane, overlying the acrosome, neck, and principal piece. Challenge experiments with agonists and antagonists identified both µ- and δ-opioid receptors as regulators of sperm kinematics, wherein µ maintains or increases sperm movement whereas δ decreases sperm motility over time. This article is protected by copyright. All rights reserved.

  • 38.
    Vicente-Carrillo, Alejandro
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Ekwall, H
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Álvarez-Rodríguez, Manuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Rodriguez-Martinez, Heriberto
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa2016Ingår i: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 51, nr 5, s. 665-679Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Freezing of boar spermatozoa includes the cryoprotectant glycerol, but renders low cryosurvival, owing to major changes in osmolarity during freezing/thawing. We hypothesize that aquaporins (AQPs) 7 and 9 adapt their membrane domain location to these osmotic challenges, thus maintaining sperm homeostasis. Western blotting (WB) and immunocytochemistry (ICC) at light and electron microscope levels with several commercial primary antibodies and protocols explored AQP location on cauda epididymal and ejaculated spermatozoa (from different fractions of the ejaculate), unprocessed, extended, chilled and frozen-thawed. Although differences in WB and ICC labelling were seen among antibodies, AQP-7 was conspicuously located in the entire tail and cytoplasmic droplet in caudal spermatozoa, being restricted to the mid-piece and principal piece domains in ejaculated spermatozoa. AQP-9 was mainly localized in the sperm head in both caudal and ejaculated spermatozoa. While unaffected by chilling (+5°C), freezing and thawing of ejaculated spermatozoa clearly relocated the head labelling of AQP-7, but not that of AQP-9. In vitro mimicking of cell membrane expansion during quick thawing maintained the localization of AQP-9 but relocated AQP-7 towards the acrosome. AQP-7, but not AQP-9, appears as a relevant marker for non-empirical studies of sperm handling.

  • 39.
    Vicente-Carrillo, Alejandro
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Álvarez-Rodríguez, Manuel
    Linköpings universitet, Medicinska fakulteten. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa.
    Rodríguez-Martínez, Heriberto
    Linköpings universitet, Medicinska fakulteten. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa.
    The CatSper channel modulates boar sperm motility during capacitation.2017Ingår i: Reproductive biology, ISSN 2300-732X, Vol. 17, nr 1, s. 69-78Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The cation channel of sperm (CatSper) comprises four transmembrane subunits specifically expressed in human, equine, murine and ovine spermatozoa, apparently implicated in capacitation, hyperactivation and acrosome exocytosis. Western blotting and immunocytochemistry showed hereby that CatSper subunits are also present in boar spermatozoa, primarily over the sperm neck, tail and cytoplasmic droplets; albeit CatSper -1 presented in addition some distribution over the membrane of the acrosome and CatSper -2 and -4 over the membrane of the post-acrosome. The role of the Catsper channel in boar spermatozoa was investigated by extending the spermatozoa in media containing different calcium (Ca(2+)) availability and exposure to the capacitation-trigger bicarbonate, to progesterone or CatSper inhibitors (Mibefradil and NNC 55-0396), separately or sequentially, at physiological and toxicological doses. Extracellular Ca(2+) availability, combined with bicarbonate exposure (capacitation-inducing conditions) decreased sperm motility, similarly to when spermatozoa incubated in capacitation-inducing conditions was exposed to Mibefradil and NNC 55-0396. Exposure of these spermatozoa to progesterone did not cause significant changes in sperm motility and nor did it revert its decrease induced by CatSper antagonists. In conclusion, the CatSper channel regulates sperm motility during porcine capacitation-related events in vitro.

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