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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)2012Doctoral thesis, comprehensive summary (Other academic)
    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.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    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) sperm2016In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 85, no 6, p. 1097-1105Article in journal (Refereed)
    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.

  • 3.
    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.2011In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 75, no 8, p. 1561-1565Article in journal (Refereed)
    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.

  • 4.
    Alvarez-Rodriguez, Manuel
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Atikuzzaman, Mohammad
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Medicine and Health Sciences. Department of Surgery and Theriogenology, Faculty of Veterinary Animal and Biomedical Sciences, Sylhet Agricultural University, Sylhet, Bangladesh.
    Venhoranta, Heli
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Medicine and Health Sciences. University of Helsinki, Department of Production Animal Medicine, Faculty of Veterinary Medicine, Saari, Finland.
    Wright, Dominic
    Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Expression of Immune Regulatory Genes in the Porcine Internal Genital Tract Is Differentially Triggered by Spermatozoa and Seminal Plasma2019In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 3, article id 513Article in journal (Refereed)
    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.

  • 5.
    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.2013In: Reproduction, Fertility and Development, ISSN 1031-3613, E-ISSN 1448-5990, Vol. 25, no 8, p. 1185-1193Article in journal (Refereed)
    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.

  • 6.
    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.2013In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 79, no 3, p. 541-550Article in journal (Refereed)
    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).

  • 7.
    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.2013In: Cryobiology, ISSN 0011-2240, E-ISSN 1090-2392, Vol. 67, no 3, p. 339-346Article in journal (Refereed)
    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.

  • 8.
    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 spermatozoa2012In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 135, no 1-4, p. 37-46Article in journal (Refereed)
    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.

  • 9.
    Atikuzzaman, Mohammad
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Alvarez-Rodriguez, Manuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Carrillo, Alejandro Vicente
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Johnsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
    Wright, Dominic
    Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Conserved gene expression in sperm reservoirs between birds and mammals in response to mating.2017In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 18, no 1Article in journal (Refereed)
    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.

  • 10.
    Atikuzzaman, Mohammad
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Sanz, Libia
    Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain.
    Pla, Davinia
    Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain.
    Alvarez-Rodriguez, Manuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Rubér, Marie
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Wright, Dominic
    Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
    Calvete, Juan J.
    Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Selection for higher fertility reflects in the seminal fluid proteome of modern domestic chicken2017In: Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics, ISSN 1744-117X, E-ISSN 1878-0407, Vol. 21, p. 27-40Article in journal (Refereed)
    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.

  • 11.
    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).2012In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 47, no 1, p. 105-112Article in journal (Refereed)
    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.

  • 12.
    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.2011In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 76, no 7, p. 1313-1325Article in journal (Refereed)
    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.

  • 13.
    Fernández-Gago, Rocío
    et al.
    Department of Medicine, Surgery and Veterinary Anatomy, University of León, 24071 León, Spain.
    Alvarez-Rodriguez, Manuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    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.2017In: Reproduction, fertility, and development, ISSN 1031-3613, Vol. 29, no 8, p. 1576-1584Article in journal (Refereed)
    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.

  • 14.
    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 cytometry2010In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 45 Suppl 2, p. 67-78Article in journal (Refereed)
    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.

  • 15.
    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
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences. 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)2019In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 125, p. 109-114, article id S0093-691X(18)30573-9Article in journal (Refereed)
    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.

  • 16.
    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.2012In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 78, no 5, p. 1005-1019Article in journal (Refereed)
    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.

  • 17.
    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.2012In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 47, no 6, p. 907-914Article in journal (Refereed)
    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.

  • 18.
    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 time2014In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 151, no 3-4, p. 137-147Article in journal (Refereed)
    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.

  • 19.
    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.2015In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 83, no 4, p. 520-528Article in journal (Refereed)
    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.

  • 20.
    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
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Ethylene glycol, but not DMSO, could replace glycerol inclusion in soybean lecithin-based extenders in ram sperm cryopreservation.2017In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 177, p. 35-41Article in journal (Refereed)
    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.

  • 21.
    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.2012In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 77, no 6, p. 1119-1128Article in journal (Refereed)
    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.

  • 22.
    Rodriguez-Martinez, Heriberto
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Tienthai, P.
    Chulalongkorn University, Thailand.
    Atikuzzaman, Mohammad
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Vicente Carrillo, Alejandro
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Rubér, Marie
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Alvarez-Rodriguez, Manuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    The ubiquitous hyaluronan: Functionally implicated in the oviduct?2016In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 86, no 1, p. 182-186Article, review/survey (Refereed)
    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.

  • 23.
    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.2011In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 129, no 3-4, p. 188-199Article in journal (Refereed)
    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.

  • 24.
    Vicente Carrillo, Alejandro
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Alvarez-Rodriguez, Manuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    The mu (µ) and delta (δ) opioid receptors modulate boar sperm motility2016In: Molecular Reproduction and Development, ISSN 1040-452X, E-ISSN 1098-2795, Vol. 83, no 8, p. 724-734Article in journal (Refereed)
    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.

  • 25.
    Vicente-Carrillo, Alejandro
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Ekwall, H
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Álvarez-Rodríguez, Manuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa2016In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 51, no 5, p. 665-679Article in journal (Refereed)
    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.

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