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  • 1.
    Abe, Y
    et al.
    Tohoku University, Japan; .
    Hara, K
    Tohoku University, Japan; .
    Matsumoto, H
    Tohoku University, Japan; .
    Kobayashi-, J
    Tohoku University, Japan; .
    Sasada, H
    Tohoku University, Japan; .
    Ekwall, H
    Tohoku University, Japan; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agricultural Sciences; Sweden.
    Sato, E
    Tohoku University, Japan; .
    Feasibility of a nylon-mesh holder for vitrification of bovine germinal vesicle oocytes in subsequent production of viable blastocysts2005In: Biology of Reproduction, ISSN 0006-3363, E-ISSN 1529-7268, Vol. 72, no 6, p. 1416-1420Article in journal (Refereed)
    Abstract [en]

    To improve the feasibility of nylon-mesh holder for vitrification of bovine cumulus-oocytes complexes (GV-COCs) having germinal vesicle, this study was conducted to demonstrate effects of sugars and protocol of exposure in vitrification on subsequent in vitro maturation, ultrastructural changes, and in vitro development in bovine immature oocytes after cryopreservation using nylon mesh. Before vitrification, GV-COCs were exposed to the cryoprotectant, which was composed of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose (EFS40) or 0.3 M trehalose (EFT40), either by single step or in a stepwise way. The maturation rates in the stepwise exposure with EFS40 or EFT40 were significantly higher (P less than 0.05) compared with the corresponding rates in the single step. In the stepwise exposure, few abnormalities were observed compared with the single-step exposure, where most oocytes showed a highly vacuolated cytoplasm with many ruptured mitochondria. Cleavage rates in fertilized oocytes previously exposed stepwise to EFS40 or EFT40 were significantly higher than those exposed by the single-step procedure. The cleaved embryos derived from the stepwise exposure to EFS40 developed to blastocysts. After transfer of blastocysts derived from vitrified GV oocytes, a female calf was born. These results indicate that vitrification of large numbers of bovine GV-COCs using a nylon-mesh holder accompanied with stepwise exposure minimizes structural damage in organelles, resulting in yield of viable blastocysts following in vitro embryo production.

  • 2.
    Alkmin, Diego V.
    et al.
    University of Murcia, Spain.
    Perez-Patino, Cristina
    University of Murcia, Spain.
    Barranco, Isabel
    University of Murcia, Spain.
    Parrilla, Inmaculada
    University of Murcia, Spain.
    Vazquez, Juan M.
    University of Murcia, Spain.
    Martinez, Emilio A.
    University of Murcia, Spain.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Roca, Jordi
    University of Murcia, Spain.
    Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate2014In: Cryobiology, ISSN 0011-2240, E-ISSN 1090-2392, Vol. 69, no 2, p. 203-210Article in journal (Refereed)
    Abstract [en]

    Boar bulk ejaculates are now being collected instead of usual sperm-rich fractions (SRF) for artificial insemination purpose. The present study evaluated the influence of holding boar sperm samples before freezing surrounded in their own seminal plasma (SP), from either fractions/portions or the entire ejaculate, on post-thawing sperm quality and functionality. Ejaculates collected as bulk (BE) or as separate (first 10 mL of SRF [P1] and rest of SRF [P2]) from 10 boars were held 24 h at 15-17 degrees C and then frozen. Some bulk ejaculate samples were frozen immediately after collections as Control. In addition, epididymal sperm samples from the same 10 boars were collected post-mortem and extended in SP from P1 (EP1), P2 (EP2) and post SRF (EP3), and also held 24 h before freezing for a better understanding of the influence of SP on boar sperm cryopreservation. The sperm quality (motility, evaluated by CASA, and viability, evaluated by flow cytometry) and functionality (flow cytometry assessment of plasma membrane fluidity, mitochondrial membrane potential and intracellular generation of reactive oxygen species [ROS] in viable sperm) were evaluated at 30, 150 and 300 min post-thaw. Post-thawing sperm quality and functionality of P1 and P2 were similar but higher (p less than0.01) than BE samples. Control samples showed higher (p less than 0.01) post-thaw sperm quality and functionality than BE samples. Post-thawing sperm quality and functionality of EP1 and EP2 were similar but higher (p less than 0.05) than EP3. These results showed that boar sperm from BE are more cryosensitive than those from the SRF, particularly when held 24 h before freezing, which would be attributable to the cryonegative effects exerted by the SP from post SRF.

  • 3.
    Al-Makhzoomi, A.
    et al.
    Swedish University of Agriculture Science, Sweden .
    Lundeheim, N.
    Swedish University of Agriculture Science, Sweden .
    Haard, M.
    Svensk Avel Ek Ornsro, Sweden .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden .
    Sperm morphology and fertility of progeny-tested AI dairy bulls in Sweden2008In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 70, no 4, p. 682-691Article in journal (Refereed)
    Abstract [en]

    Use of bull semen with high levels of sperm abnormalities, reflecting genital dysfunction, is not recommended for artificial insemination (AI) since it would most likely lead to subfertility. Sperm quality, including sperm morphology, may deteriorate with increasing age of the bull thus becoming a source of concern when using older, progeny-tested AI bull sires. Although a relationship between sperm morphology and fertility after AI in progeny-tested bull sires has been reported, it is yet unclear which sperm abnormalities are most critical. This constituted the core aim of a 22-month long retrospective study in proven (aged 60-84 months at the start of the study) AI sires of the Swedish Red (SR, n = 8) and Swedish Holstein (SLB, n = 4) breeds where their semen (107 freezing batches in total, built by a single ejaculate (n = 3) or pooling two consecutive ejaculates (n = 104) collected at 1-3 months interval), were subjected to detailed morphological examinations on wet- and dry, stained smears. Attention was paid to between- and within-bull variations with regard to presence and level of sperm abnormalities. Sperm morphology differed significantly between sires and ejaculates, with 6/12 sires having ejaculates containing greater than 10% of morphologically deviating sperm head shapes, a commonly used threshold for young At bulls in Sweden. However, with the exception of pear-shaped or narrow-at-the-base anomalies, the mean values for individual defects were always within the limits expected for a normal bull sire, and were therefore considered acceptable. The percentage of morphologically normal spermatozoa was positively related to fertility, whose output differed significantly among bulls. Among sperm abnormalities, the proportion of morphologically deviating sperm head shapes were negatively correlated with fertility, pear-shaped sperm heads in particular. In conclusion, the relationship between sperm morphology and fertility after AI calls for frequent (2-3 months interval) detailed assessments of sperm morphology in AI stud bull sires. (c) 2008 Elsevier Inc. All rights reserved.

  • 4.
    Alminana, C
    et al.
    University of Murcia, Spain.
    Gil, MA
    University of Murcia, Spain.
    Cuello, C
    University of Murcia, Spain.
    Roca, J
    University of Murcia, Spain.
    Vazquez, JM
    University of Murcia, Spain.
    Rodriguez-Martinez, Heriberto
    SLU, Sweden; .
    Martinez, EA
    University of Murcia, Spain.
    Adjustments in IVF system for individual boars: Value of additives and time of sperm-oocyte co-incubation2005In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 64, no 8, p. 1783-1796Article in journal (Refereed)
    Abstract [en]

    In vitro fertilization (IVF) in pigs is still considered sub-optimal, due to the variable occurrence of polyspermy, variability mainly related to sperm differences. The present study was conducted in an attempt to increase the efficiency of the in vitro production of porcine embryos by optimizing the in vitro fertilization (IVF) protocol for individual males, with regard to the composition of the fertilization medium (experiments 1 and 2) and the length of gamete co-incubation time (experiment 3). A total of 5943 COCs were in vitro matured (IVM) and inseminated with frozen-thawed spermatozoa froth 2 boars (A and B). Experiment 1 determined the effect of additives caffeine (2 mM), hyaluronic acid (HA; [0.5 mg/mL]) and adenosine (10 mu M), alone or in combination, to the IVF-medium during sperm-oocyte co-incubation. Experiment 2 tested the addition of various HA (0, 0.5, 1.0 and 1.5 mg/ml) and adenosine (0, 10, 20 and 40 mu M) concentrations in the fertilization medium; while experiment 3 investigated the effect of two periods of sperm-oocyte co-incubation (10 thin or 6h). In the case of 10 min sperm-oocyte co-incubation, oocytes with attaching spermatozoa were further cultured in IVF-medium containing no spermatozoa until the 6 h of insemination was completed. Presumptive zygotes were cultured in embryo culture medium for 1215 h to assess fertilization parameters. In experiment 1, only caffeine significantly influenced the outcome of fertilization, albeit being a clearly boar-dependent effect. In experiment 2, similar boar differences were seen for HA supplementation while presence of exogenous adenosine did not influence fertilization parameters in either boar. The results of experiment 3 demonstrated that a short co-incubation time significantly (P less than 0.001) increased penetration rate and mean number of spermatozoa per oocyte (74.9 +/- 3.9% versus 62.7 +/- 3.9% and 1.5 +/- 3.2 versus 1.3 +/- 3.5 for 10 min or 6 h, respectively), but reduced monospermy (P less than 0.001, 57.9 +/- 2.5% versus 70.0 +/- 2.8%) when boar A was used. However, such effects were not seen with boar B, in which sperm-oocyte co-incubation time did not affect the efficiency of fertilization. In view of the present results, a preliminary screening for each individual male is required to select optimal conditions for IVF. (c) 2005 Elsevier Inc. All rights reserved.

  • 5.
    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.
    Vicente Carrillo, Alejandro
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    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.
    Hyaluronan improves neither the long-term storage nor the cryosurvival of liquid-stored CD44-bearing AI boar spermatozoa2018In: Journal of reproduction and development, ISSN 0916-8818, E-ISSN 1348-4400, Vol. 64, no 4Article in journal (Refereed)
    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™), 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 (17–20ºC) 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.

  • 6.
    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.

  • 7.
    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.
    Vicente Carrillo, Alejandro
    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.
    Correction: Conserved gene expression in sperm reservoirs between birds and mammals in response to mating (vol 18, 98, 2017)2017In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 18, article id 563Article in journal (Other academic)
    Abstract [en]

    n/a

  • 8.
    Atikuzzaman, Mohammad
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Bhai Mehta, Ratnesh
    Linköping University, Faculty of Medicine and Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science.
    Fogelholm, Jesper
    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 Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Mating induces the expression of immune- and pH-regulatory genes in the utero-vaginal junction containing mucosal sperm-storage tubuli of hens2015In: Reproduction, Vol. 150, no 6, p. 473-483Article in journal (Refereed)
    Abstract [en]

    The female chicken, as with other species with internal fertilization, can tolerate the presence of spermatozoa within specialized sperm-storage tubuli (SST) located in the mucosa of the utero-vaginal junction (UVJ) for days or weeks, without eliciting an immune response. To determine if the oviduct alters its gene expression in response to sperm entry, segments from the oviduct (UVJ, uterus, isthmus, magnum and infundibulum) of mated and unmated (control) hens, derived from an advanced inter-cross line between Red Junglefowl and White Leghorn, were explored 24 h after mating using cDNA microarray analysis. Mating shifted the expression of fifteen genes in the UVJ (53.33% immune-modulatory and 20.00% pH-regulatory) and seven genes in the uterus, none of the genes in the latter segment overlapping the former (with the differentially expressed genes themselves being less related to immune-modulatory function). The other oviductal segments did not show any significant changes. These findings suggest sperm deposition causes a shift in expression in the UVJ (containing mucosal SST) and the uterus for genes involved in immune-modulatory and pH-regulatory functions, both relevant for sperm survival in the hen's oviduct.

  • 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.
    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.

  • 10.
    Awasthi, H.
    et al.
    SLU, Sweden .
    Saravia, F.
    SLU, Sweden .
    Rodriguez-Martinez, Heriberto
    SLU, Sweden .
    Bage, R.
    SLU, Sweden .
    Do Cytoplasmic Lipid Droplets Accumulate in Immature Oocytes from Over-Conditioned Repeat Breeder Dairy Heifers?2010In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 45, no 5, p. E194-E198Article in journal (Refereed)
    Abstract [en]

    One of the main sources of repeat breeding in dairy cattle, caused by fertilization failure or early embryonic death, is metabolic stress during lactation. Nutrition seems also to play a role when the condition is seen in heifers, where oocyte cytoplasmic maturation is impaired. To determine whether over conditioning affects oocyte morphology, immature oocytes were collected by ovum pick-up (OPU) twice weekly during 5 weeks from three over-conditioned repeat breeder dairy heifers (RBH) and two normal virgin heifers (VH, controls) of the Swedish Red breed, monitored by body weight and condition. Oocyte quality was assessed under stereomicroscope and further examined by transmission electron microscope for accumulation of cytoplasmic lipid deposits. After OPU, the RBH yielded more low quality oocytes (60% vs 52% for VH, p = 0.14). The relative occupancy of osmophilic lipid droplets in the cytoplasm was higher in oocytes of bad quality compared with good ones, especially in RBH (p = 0.08) but also in VH (p = 0.11). Moreover, the oocytes from over-conditioned RBH showed higher amounts of cytoplasmic lipid deposits both in good (p = 0.14) and, even more prominent, in bad quality oocytes (p = 0.06). Such accumulation of lipid droplets may imply increased sensitivity to oxidative stress, hinder cytoplasmic maturation and lead to subfertility, as accounted in over-conditioned repeat breeders of the Swedish Red breed.

  • 11.
    Balao da Silva, C. M.
    et al.
    University of Extremadura, Spain.
    Ortega Ferrusola, C.
    University of Extremadura, Spain.
    Gallardo Bolanos, J. M.
    University of Extremadura, Spain.
    Plaza Davila, M.
    University of Extremadura, Spain.
    Martin-Munoz, P.
    Swedish University of Agriculture Science, Sweden.
    Morrell, J. M.
    Swedish University of Agriculture Science, Sweden.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Pena, F. J.
    University of Extremadura, Spain.
    Effect of Overnight Staining on the Quality of Flow Cytometric Sorted Stallion Sperm: Comparison with Tradtitional Protocols2014In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 49, no 6, p. 1021-1027Article in journal (Refereed)
    Abstract [en]

    Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sorted and compared with spermatozoa from the same ejaculate that had been sexed on the previous day. All sperm parameters evaluated remained unchanged when fresh sorted and refrigerated sorted semen were compared. Pre-sorting storage at 5 degrees C did not alter sperm velocities nor kinetics, viability or membrane permeability, production of reactive oxygen species, mitochondrial membrane potential or DNA fragmentation index of the sorted sample. The findings open for the possibility of using semen from stallions housed far from the sorting facilities. Processed and stained sperm could be shipped refrigerated on the previous day, sorted and inseminated on the next day.

  • 12.
    Balao da Silva, C M.
    et al.
    University of Extremadura Caceres, Spain .
    Ortega Ferrusola, C
    University of Extremadura Caceres, Spain .
    Morillo Rodriguez, A
    University of Extremadura Caceres, Spain .
    Gallardo Bolanos, J M.
    University of Extremadura Caceres, Spain .
    Plaza Davila, M
    University of Extremadura Caceres, Spain .
    Morrell, J M.
    Swedish University of Agriculture Science, Sweden .
    Rodriguez Martinez, H
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Tapia, J A.
    University of Extremadura Caceres, Spain .
    Aparicio, I M.
    University of Extremadura Caceres, Spain .
    Pena, F -j.
    University of Extremadura Caceres, Spain .
    Sex sorting increases the permeability of the membrane of stallion spermatozoa2013In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 138, no 3-4, p. 241-251Article in journal (Refereed)
    Abstract [en]

    At present, the only repeatable means of selecting the sex of offspring is the Beltsville semen sorting technology using flow cytometry (FC). This technology has reached commercial status in the bovine industry and substantial advances have occurred recently in swine and ovine species. In the equine species, however, the technology is not as well developed. To better understand the changes induced in stallion spermatozoa during the sorting procedure, pooled sperm samples were sorted: sperm motility and kinematics were assessed using computer assisted sperm analysis, sperm membrane integrity was assessed using the YoPro-1 assay, while plasmalemmal stability and lipid architecture were assessed using Merocyanine 540/SYTOX green and Annexin-V, respectively. Lipid peroxidation was also investigated with the probe Bodipy(581/591)-C11. All assays were performed shortly after collection, after incubation and after sex sorting using FC. In order to characterize potential molecular mechanisms implicated in sperm damage, an apoptosis protein antibody dot plot array analysis was performed before and after sorting. While the percentage of total motile sperm remained unchanged, sex sorting reduced the percentages of progressive motile spermatozoa and of rapid spermatozoa as well as curvilinear velocity (VCL). Sperm membranes responded to sorting with an increase in the percentage of YoPro-1 positive cells, suggesting the sorted spermatozoa had a reduced energy status that was confirmed by measuring intracellular ATP content.

  • 13.
    Balao da Silva, C. M.
    et al.
    Vet Teaching Hospital, Spain.
    Ortega-Ferrusola, C.
    Vet Teaching Hospital, Spain.
    Morrell, J. M.
    Swedish University of Agriculture Science, Sweden.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Pena, F. J.
    Vet Teaching Hospital, Spain.
    Flow Cytometric Chromosomal Sex Sorting of Stallion Spermatozoa Induces Oxidative Stress on Mitochondria and Genomic DNA2016In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 51, no 1, p. 18-25Article in journal (Refereed)
    Abstract [en]

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

  • 14.
    Balao da Silva, C
    et al.
    University of Extremadura.
    Ortega, C
    University of Extremadura.
    Macias, B
    University of Extremadura.
    Morillo, A
    University of Extremadura.
    Gallardo, J
    University of Extremadura.
    Aparicio, I
    University of Extremadura.
    Tapia, J
    University of Extremadura.
    Morrell, J
    SLU.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Pena, J F
    University of Extremadura.
    Effect of hoechst 33342 on stallion spermatozoa incubated in KMT or modified INRA96-tyrode in REPRODUCTION IN DOMESTIC ANIMALS, vol 46, issue SI, pp 88-882011In: REPRODUCTION IN DOMESTIC ANIMALS, Blackwell Publishing , 2011, Vol. 46, no SI, p. 88-88Conference paper (Refereed)
    Abstract [en]

    n/a

  • 15.
    Balao da Silva, C
    et al.
    Swedish University of Agriculture Science, Sweden .
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Ortega-Ferrusola, C
    University of Extremadura, Spain .
    Effect of Hoechst 33342 on stallion spermatozoa incubated in KMT or Tyrodes modified INRA962012In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 131, no 3-4, p. 165-171Article in journal (Refereed)
    Abstract [en]

    The only known means of effectively separating populations of X and Y bearing sperms is the Beltsville sexing technology. The technology implies that each individual sperm is interrogated for DNA content, measuring the intensity of the fluorescence after staining the spermatozoa with Hoechst 33342. Because there are no data regarding the effect of the staining on stallion sperm, ejaculates were incubated up to 90 min in presence of 0, 4.5, 9, 22.5, 31.5, 45, 54, 67.5, 76.5 and 90 mu M of Hoechst 33342, in two media, KMT or INRA-Tyrodes. After 40 and 90 min of incubation, motility (CASA) and membrane integrity (flow cytometry after YoPro-1/Eth staining) were evaluated. In KMT extender sperm motility significantly decreased after 45 min of incubation when sperm were incubated in the presence of concentrations of Hoechst of 45 mu M or greater (P andlt; 0.05). When incubated in modified INRA96, stallion spermatozoa tolerated greater concentrations of Hoechst, because sperm motility only decreased when incubated in presence of 90 mu M (P andlt; 0.05) and membrane integrity was not affected. After 90 min of incubation the same effect was observed, but in this case at concentrations over 45 mu M the percentage of total motile sperm was also reduced although only in samples incubated in KMT. To produce this effect in samples incubated in Tyrodes modified INRA 96, Hoechst had to be present at concentrations over 67.5 mu M. Apparently, the detrimental effect of Hoechst to stallion spermatozoa varies depending on the media, and INRA modified extender may be an alternative to KMT.

  • 16.
    Ballester, J.
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Johannisson, A.
    Swedish University of Agriculture Science, Sweden; .
    Saravia, F.
    Swedish University of Agriculture Science, Sweden; .
    Haard, M.
    Swedish University of Agriculture Science, Sweden; .
    Gustafsson, H.
    Swedish University of Agriculture Science, Sweden; .
    Bajramovic, D.
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Post-thaw viability of bull AI-doses with low-sperm numbers2007In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 68, no 6, p. 934-943Article in journal (Refereed)
    Abstract [en]

    Use of AI-doses containing low-sperm numbers are increasingly been used to optimise use of elite bulls as well as to accommodate an eventual wider application of sex-sorted semen. Since spermatozoa might, however, suffer from high extension rates, thus compromising fertility, this study evaluated the post-thaw sperm quality of semen from commercial progeny-tested, high-ranked AI-sires whose semen was within acceptable limits of normality, frozen in a split-design to 15 (control, 15M) or 2 x 106 total spermatozoa (treatment, 2M) per straw. Assessment post-thaw included computer-evaluated sperm motility (CASA), membrane integrity (SYBR-14/PI), membrane stability (Annexin-V/Pl), acrosome integrity (Carboxy-SNARF-1/PI/ FITC-PSA), and chromatin integrity (AO of in situ acid-induced DNA denaturation). High extension did not affect the proportions of linearly motile spermatozoa, of membrane integrity or stability nor chromatin integrity, immediately post-thaw. However, high extension clearly affected linear sperm motility following incubation at 38 degrees C for 30 min, sperm viability when assessed by SNARF and, particularly, acrosome integrity of the otherwise viable spermatozoa. Individual sire variation was evident. Fertility was preliminarily evaluated for one of the less affected bulls in a blind field trial. A total of 109 dairy cows were randomly inseminated with 15M or 2M-straws without differences in pregnancy rate between them (47% versus 43%). This similarity in fertility rates, confirmed the in vitro methods used were appropriate for identifying cryosurvival and further suggested the site of sperm deposition was not crucial for the fertility of low-sperm AI-numbers for this particular sire. However, the inter-bull variation seen calls for caution when cryopreserving low concentrations of bull spermatozoa with conventional freezing protocols. (C) 2007 Elsevier Inc. All rights reserved.

  • 17.
    Barranco, I.
    et al.
    University of Murcia, Spain.
    Tvarijonaviciute, A.
    University of Murcia, Spain.
    Perez-Patino, C.
    University of Murcia, Spain.
    Alkmin, D. V.
    University of Murcia, Spain.
    Ceron, J. J.
    University of Murcia, Spain.
    Martinez, E. A.
    University of Murcia, Spain.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Roca, J.
    University of Murcia, Spain.
    The activity of paraoxonase type 1 (PON-1) in boar seminal plasma and its relationship with sperm quality, functionality, and in vivo fertility2015In: Andrology, ISSN 2047-2919, E-ISSN 2047-2927, Vol. 3, no 2, p. 315-320Article in journal (Refereed)
    Abstract [en]

    Paraoxonase 1 (PON-1) is a hydrolytic enzyme present in body fluids, capable of protecting cells against oxidative stress. The hypothesis was hereby to test that PON-1, present in seminal plasma (SP), acts protecting boar spermatozoa when showing a reasonable high activity in the ejaculate. SP-PON-1 activity differed (pless than0.001) among boars (from 0.10 to 0.29IU/mL). Intra-boar variability was also observed (pless than0.05), but only in two of the 15 boars. SP-PON-1 activity differed among ejaculate portions, showing the spermatozoa-peak portion of spermatozoa-rich ejaculate fraction the highest levels (0.35 +/- 0.03IU/mL, ranging from 0.12 to 0.69) and the post-sperm ejaculate fraction the lowest levels (0.12 +/- 0.01IU/mL, ranging from 0.03 to 0.21). SP-PON-1 activity was positively correlated with the percentage of spermatozoa with rapid and progressive movement (pless than0.01) and negatively correlated with the generation of intracellular reactive oxygen species (pless than0.01) in semen samples after 72h of liquid storage. SP-PON-1 activity was highest (pless than0.01) in boars with highest farrowing rates. In conclusion, SP-PON-1 activity differed among boars and ejaculate fractions/portions. SP-PON-1 activity was positively correlated with sperm quality and functionality of liquid-stored semen samples and it evidenced a positive association with in vivo fertility.

  • 18.
    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
    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.
    Ceron, Jose J
    University of Murcia, Murcia, Spain.
    Martinez, Emilio A
    University of Murcia, Murcia, 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.
    Roca, Jordi
    University of Murcia, Murcia, Spain.
    Active paraoxonase 1 is synthesised throughout the internal boar genital organs.2017In: Reproduction (Cambridge, England), ISSN 1741-7899, Vol. 154, no 3, p. 237-243Article in journal (Refereed)
    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.

  • 19.
    Barranco, Isabel
    et al.
    University of Murcia, Spain.
    Roca, Jordi
    University of Murcia, Spain.
    Tvarijonaviciute, Asta
    University of Murcia, Spain.
    Rubér, Marie
    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 Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Atikuzzaman, Mohammad
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Ceron, Jose J.
    University of Murcia, Spain.
    Martinez, Emilio A.
    University of Murcia, Spain.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Measurement of Activity and Concentration of Paraoxonase 1 (PON-1) in Seminal Plasma and Identification of PON-2 in the Sperm of Boar Ejaculates2015In: Molecular Reproduction and Development, ISSN 1040-452X, E-ISSN 1098-2795, Vol. 82, no 1, p. 58-65Article in journal (Refereed)
    Abstract [en]

    This study revealed and characterised the presence of the antioxidant enzymes paraoxonase (PON) type 1 (PON-1, extracellular) and type 2 (PON-2, intracellular) in boar semen. To evaluate PON-1, an entire ejaculate from each of ten boars was collected and the seminal plasma was harvested after double centrifugation (1,500g for 10min). Seminal plasma was analysed for concentration as well as enzymatic activity of PON-1 and total cholesterol levels. Seminal-plasma PON-1 concentration ranged from 0.961 to 1.670ng/ml while its enzymatic activity ranged from 0.056 to 0.400 IU/ml, which represent individual variance. Seminal-plasma PON-1 concentration and enzymatic activity were negatively correlated (r=-0.763; Pless than0.01). The activity of seminal-plasma PON-1 negatively correlated with ejaculate volume (r=-0.726, Pless than0.05), but positively correlated with sperm concentration (r=0.654, Pless than0.05). Total seminal-plasma cholesterol concentration positively correlated with PON-1 activity (r=0.773; Pless than0.01), but negatively correlated with PON-1 concentration (r=-0.709; Pless than0.05). The presence of intracellular PON-2 was determined via immunocytochemistry in spermatozoa derived from artificial insemination. PON-2 localised to the post-acrosomal area of the sperm head and principal piece of the tail in membrane-intact spermatozoa. In summary, PON is present in boar semen, with PON-1 at low levels in seminal plasma and PON-2 within the spermatozoa. Further studies are needed to characterise the relationship between antioxidant PONs with sperm and other seminal-plasma parameters. Mol. Reprod. Dev. 82: 58-65, 2015. (c) 2014 Wiley Periodicals, Inc.

  • 20.
    Barranco, Isabel
    et al.
    University of Murcia, Spain.
    Rubér, Marie
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Perez-Patino, Cristina
    University of Murcia, Spain.
    Atikuzzaman, Mohammad
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Martinez, Emilio A.
    University of Murcia, Spain.
    Roca, Jordi
    University of Murcia, Spain.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    The Seminal Plasma of the Boar is Rich in Cytokines, with Significant Individual and Intra-Ejaculate Variation2015In: American Journal of Reproductive Immunology, ISSN 1046-7408, E-ISSN 1600-0897, Vol. 74, no 6, p. 523-532Article in journal (Refereed)
    Abstract [en]

    Problem The boar, as human, sequentially ejaculates sperm-rich and sperm-poor fractions. Seminal plasma (SP) spermadhesins (PSP-I/PSP-II) induce a primary endometrial inflammatory response in female sows, similar to that elicited by semen deposition in other species, including human. However, the SP is also known to mitigate such response, making it transient to allow for embryo entry to a cleansed endometrium. Although cytokine involvement has been claimed, the exploration of cytokines in different SP fractions is scarce. This study determines Th1, Th2, Th17 and Th3 cytokine profiles in specific ejaculate SP fractions from boars of proven fertility. Methods SP samples from the sperm-rich fraction (SRF) and the sperm-poor post-SRF fraction (post-SRF) of manually collected ejaculates from eight boars (four ejaculates per boar) were analysed by commercial multiplex bead assay kits (Milliplex MAP, Millipore, USA) for interferon-gamma, interferon gamma-induced protein 10, macrophage-derived chemokine, growth-regulated oncogene, granulocyte-macrophage colony-stimulating factor, monocyte chemo-attractant protein-1, interleukins (IL)-6, IL-8, IL-10, IL-15, IL-17 and transforming growth factor (TGF)-beta 1-beta 3. Results Cytokine concentrations differed between the ejaculate fractions among boars, being highest in the post-SRF. Conclusion Boar SP is rich in Th1, Th2, Th17 and Th3 cytokines, with lowest concentrations in the sperm-peak-containing fraction, indicating its main immune influence might reside in the larger, protein-rich sperm-poor post-SRF.

  • 21.
    Barranco, Isabel
    et al.
    University of Murcia, Spain.
    Tvarijonaviciute, Asta
    University of Murcia, Spain.
    Perez-Patino, Cristina
    University of Murcia, Spain.
    Parrilla, Inmaculada
    University of Murcia, Spain.
    Ceron, Jose J.
    University of Murcia, Spain.
    Martinez, Emilio A.
    University of Murcia, Spain.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Roca, Jordi
    University of Murcia, Spain.
    High total antioxidant capacity of the porcine seminal plasma (SP-TAC) relates to sperm survival and fertility2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, no 18538Article in journal (Refereed)
    Abstract [en]

    The study attempted to clarify the role of total antioxidant capacity of seminal plasma (SP-TAC) on boar sperm survival and fertility after artificial insemination (AI). SP-TAC differed (P < 0.001) among boars (no = 15) and, to a lesser degree, among ejaculates within male (4 ejaculates/boar). SP-TAC also differed (P < 0.001) among ejaculate fractions (43 ejaculates and 3 fractions per ejaculate), of which the sperm-peak portion of the sperm rich ejaculate fraction (SRF) had the highest SP-TAC. SP-TAC was not correlated with sperm quality (motility and viability) or functionality (intracellular ROS generation and lipid peroxidation) of liquid AI-semen samples stored at 17 degrees C for 72 h (90 AI-samples), but the decline in sperm quality was larger (P < 0.05) in ejaculates with low, compared with high SP-TAC (hierarchically grouped). The SP-TAC differences among ejaculate portions agree with sperm cryosurvival rates (14 ejaculates from 7 boars), showing sperm from sperm-peak portion better (P < 0.01) post-thaw quality and functionality than those from the entire ejaculate (mainly post-SRF). Boars (no = 18) with high SP-TAC (hierarchically grouped) had higher (P < 0.05) fertility outcomes (5,546 AI-sows) than those with low SP-TAC. Measurement of SP-TAC ought to be a discriminative tool to prognosis fertility in breeding boars.

  • 22.
    Barranco, Isabel
    et al.
    University of Murcia, Spain.
    Tvarijonaviciute, Asta
    University of Murcia, Spain.
    Perez-Patino, Cristina
    University of Murcia, Spain.
    Vicente Carrillo, Alejandro
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Parrilla, Inmaculada
    University of Murcia, Spain.
    Ceron, Jose J.
    University of Murcia, Spain.
    Martinez, Emilio A.
    University of Murcia, Spain.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Roca, Jordi
    University of Murcia, Spain.
    Glutathione Peroxidase 5 Is Expressed by the Entire Pig Male Genital Tract and Once in the Seminal Plasma Contributes to Sperm Survival and In Vivo Fertility2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 9, article id e0162958Article in journal (Refereed)
    Abstract [en]

    Glutathione peroxidase-5 (GPX5) is an H2O2-scavenging enzyme identified in boar seminal plasma (SP). This study attempted to clarify its origin and role on sperm survival and fertility after artificial insemination (AI). GPX5 was expressed (Western blot and immunocytochemistry using a rabbit primary polyclonal antibody) in testes, epididymis and accessory sex glands (6 boars). SP-GPX5 concentration differed among boars (11 boars, P amp;lt; 0.001), among ejaculates within boar (44 ejaculates, P amp;lt; 0.001) and among portions within ejaculate (15 ejaculates). The first 10 mL of the spermrich fraction (SRF, sperm-peak portion) had a significantly lower concentration (8.87 +/- 0.78 ng/mL) than the rest of the SRF and the post-SRF (11.66 +/- 0.79 and 12.37 +/- 0.79 ng/mL, respectively, P amp;lt; 0.005). Spermmotility of liquid-stored semen AI-doses (n = 44, at 15-17 degrees C during 72h) declined faster in AI-doses with low concentrations of SP-GPX5 compared to those with high-levels. Boars (n = 11) with high SP-GPX5 showed higher farrowing rates and litter sizes than those with low SP-GPX5 (a total of 5,275 inseminated sows). In sum, GPX5 is widely expressed in the boar genital tract and its variable presence in SP shows a positive relationship with sperm quality and fertility outcomes of liquid-stored semen AI-doses.

  • 23.
    Berg, Göran
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Obstetrics and gynecology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center of Paediatrics and Gynaecology and Obstetrics, Department of Gynaecology and Obstetrics in Linköping.
    Ernerudh, Jan
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Immunology and Transfusion Medicine.
    Jenmalm, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Sydsjö, Gunilla
    Linköping University, Department of Clinical and Experimental Medicine, Obstetrics and gynecology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center of Paediatrics and Gynaecology and Obstetrics, Department of Gynaecology and Obstetrics in Linköping.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Special Issue: Marcus Wallenberg International Symposium in Comparative Reproductive Immunology, "Immunology at the fetal maternal interface: Basic science and clinical applications", July 7-8th, 2011, Linkoping University, Sweden2011In: American Journal of Reproductive Immunology, ISSN 1046-7408, E-ISSN 1600-0897, Vol. 66, no Issue supplement 1, p. 1-1Article in journal (Other academic)
  • 24.
    Bergqvist, Ann-Sofi
    et al.
    Swedish University of Agriculture Science, Sweden .
    Ballester, Joan
    Swedish University of Agriculture Science, Sweden.
    Johannisson, Anders
    Swedish University of Agriculture Science, Sweden.
    Hernandez, Marta
    University of Murcia, Spain.
    Lundeheim, Nils
    Swedish University of Agriculture Science, Sweden.
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden.
    In vitro capacitation of bull spermatozoa by oviductal fluid and its components2006In: Zygote (Cambridge. Print), ISSN 0967-1994, E-ISSN 1469-8730, Vol. 14, no 3, p. 259-273Article in journal (Refereed)
    Abstract [en]

    Sperm capacitation is crucial for fertilization. However, debate continues on exactly how, where and when capacitation is elicited in the bovine female genital tract. In this study we used merocyanine-540 and the chlortetracycline (CTC) assay to test how capacitation of bull spermatozoa is affected in vitro by exposure to oviductal fluid (ODF) collected in vivo, various glycosaminoglycans (GAGs) or bicarbonate. Following different durations of exposure, spermatozoa were stained with CTC or merocyanine-540, and evaluated with epifluorescent light microscopy or flow cytometry, respectively. Incubation time did not significantly affect capacitation. Exposure (30-120 min) to ODF capacitated (p less than 0.05) bull spermatozoa as measured by either merocyanine-540 or CTC. Hyaluronan was the only GAG that induced a significant increase in B-pattern spermatozoa (capacitated; p = 0.012) compared with controls. Dermatan sulphate also induced capacitation (merocyanine-540 high fluorescence; p = 0.035). Exposure to bicarbonate-enriched media also yielded an increase in merocyanine-540 high fluorescence (p less than 0.0001). When bicarbonate was added to the other treatments (ODF or GAGs) an equal increase in merocyanine-540 high fluorescence was noted (p less than 0.0001), compared with before addition of bicarbonate and independent of the treatment before exposure. There was no significant difference in the number of B-pattern spermatozoa when bicarbonate was added, but an significant increase in spermatozoa with an acrosome-reacted (AR)-pattern (p less than 0.0001) was observed. Exposure of spermatozoa to solubilized zonae pellucidae significantly increased the AR-pattern spermatozoa (p = 0.016). In conclusion, ODF was more potent in inducing capacitation of bull spermatozoa than the individual GAGs. Our results also indicate that bicarbonate is an effector of bull sperm capacitation.

  • 25.
    Bergqvist, A.-S.
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Ballester, J.
    Swedish University of Agriculture Science, Sweden; .
    Johannisson, A.
    Swedish University of Agriculture Science, Sweden; .
    Lundeheim, N.
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Heparin and dermatan sulphate induced capacitation of frozen-thawed bull spermatozoa measured by merocyanine-5402007In: Zygote (Cambridge. Print), ISSN 0967-1994, E-ISSN 1469-8730, Vol. 15, no 3, p. 225-232Article in journal (Refereed)
    Abstract [en]

    Glycosaminoglycans (GAGs) are present in the oviduct in which the major part of sperm capacitation occurs. In this study we have tested how capacitation of frozen-thawed bull spermatozoa is effected by exposure to different GAGs detectable or possibly present in oviductal fluid; i.e. heparin, hyaluronan, heparan sulphate, dermatan sulphate and chondroitin sulphate. Following exposure of different duration, the spermatozoa were stained with either Chlortetracycline (CTC) or merocyanine-540 and evaluated with epifluorescent light microscopy or flow cytometry, respectively. Heparin elicited a significant increase in the number of alive, capacitated spermatozoa, either expressed as higher merocyanine-540 fluorescence (p less than 0.0001) or as B-pattern (p = 0.0021) in the CTC assay, during 4 h of incubation. When comparing the different GAG treatments one by one to the negative control in the flow cytometric study, only heparin and dermatan sulphate were significant (p less than 0.0001) higher than the control at 0-30 min of incubation. Duration of incubation did not affect the proportion of capacitated spermatozoa when measured as merocyanine-540 fluorescence or CTC B-pattern, but the length of the incubation did affect the number of dead (Yo-PRO 1 positive) spermatozoa (p less than 0.0001). Exposure to zona pellucida proteins significantly increased the proportion of acrosome reacted spermatozoa (p = 0.016). Both heparin and dermatan sulphate induce capacitation of frozen-thawed bull spermatozoa in vitro.

  • 26.
    Bergqvist, A-S
    et al.
    Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Johannisson, A.
    Department of Anatomy, Physiology & Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Bäckgren, L.
    Department of Anatomy, Physiology & Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Dalin, A-M
    Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Rodriguez-Martinez, Heriberto
    Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Morrell, J M.
    Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Single Layer Centrifugation of Stallion Spermatozoa through Androcoll (TM)-E does not Adversely Affect their Capacitation-Like Status, as Measured by CTC Staining2011In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 46, no 1, p. e74-e78Article in journal (Refereed)
    Abstract [en]

    Contents This study was designed to evaluate the effect of single layer centrifugation (SLC) and subsequent cold storage on stallion sperm capacitation-like status and acrosome reaction. Three stallions were included in the study, with three ejaculates per stallion. The samples were examined 4, 24 and 72 h after collection, extension and SLC, with storage at 6 degrees C. Sperm capacitation-like status was investigated using the fluorescent dye chlortetracycline (CTC). There was no difference in capacitation-like status between colloid-selected and non-selected spermatozoa. Sperm motility decreased significantly during cold storage, whereas the proportion of apparently capacitated spermatozoa increased. There was no change in the proportion of acrosome-reacted spermatozoa. In conclusion, SLC through Androcoll (TM)-E does not adversely affect the capacitation-like status of stallion spermatozoa, although it did increase with time during cold storage.

  • 27.
    Bergqvist, AS
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Killian, G
    Pennsylvania State University, USA; .
    Erikson, D
    Pennsylvania State University, USA; .
    Hoshino, Y
    Tohoku University,, Japan; .
    Bage, R
    Swedish University of Agriculture Science, Sweden; .
    Sato, E
    Tohoku University,, Japan; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Detection of Fas ligand in the bovine oviduct2005In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 86, no 1-2, p. 71-88Article in journal (Refereed)
    Abstract [en]

    Presence of a Fas-Fas ligand (FasL) system defines the immune-privileged status of certain tissues such as placenta. This study examined the fluids and tissue(s) of the bovine oviduct, where both spermatozoa and early embryos escape elimination by the female immune system, for the presence and the distribution of Fas and FasL, which might provide an explanation for the immune-privilegded site of this organ. In the present study, the immunolocalisation of FasL and Fas, as well as the gene expression of FasL, were determined in the uterotubal junction (UTJ), isthmic (I) and ampullar (A) segments of the oviduct during oestrus and the luteal phase of the oestrous cycle. The degree of apoptosis of oviductal epithelium was examined by the TUNEL method. Oviductal fluid (01317), collected chronically via indwelling catheters from the I or A segments during both non-luteal and luteal phases of the cycle, was analysed for the presence of FasL. The Fas immunostaining was scattered along the epithelium of all regions of the oviduct and cycle stages investigated, whereas FasL immunolabelling was more conspicuous in oestrous samples. This staining disappeared during the luteal phase, which was particularly evident in the sperm reservoir (UTJ and I). There were fewer TUNEL-positive cells than Fas- or FasL-positive cells in the oviductal epithelium, suggesting that tubal Fas and FasL are not directly involved in epithelia apoptosis. Western blot analyses detected FasL in ODF collected from both I and A, most conspicuously as a 24-27 kDa band but also at a 40-45 kDa band level. FasL mRNA was expressed in the epithelial cells from the sperm reservoir and A during both non-luteal and luteal phases. However, the level of expression differed significantly between segments during the luteal phase. The results provide novel evidence that the Fas-FasL system is present in the bovine oviduct and could be involved in mediating survival of spermatozoa and early embryos. (c) 2004 Elsevier B.V. All rights reserved.

  • 28.
    Bergqvist, AS
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Sulphated glycosaminoglycans (S-GAGs) and syndecans in the bovine oviduct2006In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 93, no 1-2, p. 46-60Article in journal (Refereed)
    Abstract [en]

    In vivo, bull sperm capacitation seems to occur mainly in the oviduct. Capacitation of bull spermatozoa can be triggered in vitro by exposure to heparin, a heavily sulphated glycosaminoglycan (S-GAG). We determined the concentration of S-GAGs in oviductal fluid from dairy heifers, collected over the course of several oestrous cycles via surgically implanted intraluminal catheters. We also investigated the presence of syndecans, i.e. heparan sulphate proteoglycans, in the bovine oviductal epithelium of Swedish dairy cattle during standing oestrus and the luteal phase of the oestrous cycle, using immunohistochemistry for three different polyclonal antibodies raised against human syndecan-2 and rat syndecan-1 and syndecan-2, respectively. The concentration of S-GAGs in oviductal fluid obtained from the ampullar segment of the oviduct was significantly higher (P = 0.0026) than it was in fluid from the isthmic segment during the functional period, i.e. from prooestrus to metaoestrus (73.5 +/- 10.49 mg/L in ampullar ODF, compared to 43.2 +/- 10.74 mg/L in isthmic ODF); least square mean (L.S.M.) standard error of the mean (S.E.M.). There was also a sianificantly higher concentration of S-GAGs in the fluid from the oviduct ipsilateral to the ovulation side 73.5 +/- 10.54 mg/L on the ovulation side, compared to 43.1 +/- 10.71 mg/L in the oviduct on the contralateral side (L.S.M. +/- S.E.M., P = 0.0026) during this period. Both syndecan-1 and syndecan-2 were present in the epithelial cells lining all studied segments of the bovine oviduct, i.e. the UTJ, isthmus and ampulla, during both standing oestrus and dioestrus. The syndecans and S-GAGs found may influence the gametes. while they reside in the oviduct; the amounts of S-GAGs found in the bovine oviduct seem sufficient to act as capacitating factors in vivo. (c) 2005 Elsevier B.V. All rights reserved.

  • 29.
    Bergqvist, AS
    et al.
    Swedish University of Agriculture Science, Sweden.
    Yokoo, M
    Tohoku University , Japan; .
    Bage, R
    Swedish University of Agriculture Science, Sweden.
    Sato, E
    Tohoku University, Japan; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden.
    Detection of the hyaluronan receptor CD44 in the bovine oviductal epithelium2005In: Journal of reproduction and development, ISSN 0916-8818, E-ISSN 1348-4400, Vol. 51, no 4, p. 445-453Article in journal (Refereed)
    Abstract [en]

    Hyaluronan is involved in fundamental reproductive events such as sperm storage in the female reproductive tract, fertilization, and early embryo development, these functions are presumably mediated by its major cell surface receptor, CD44. The present study was conducted to investigate the presence and localization of CD44 in the bovine oviductal epithelium, using immunohistochemical and Western blot methods on tissue sections and epithelial cell extracts collected from the uterotubal junction (UTJ), isthmus, and ampulla of animals in the oestrus or luteal phase of the oestrous cycle. While positive immunolabelling for CD44 was found on the ad-luminal surface and supra-nuclear region of epithelial cells in all tubal segments investigated, in the UTJ, there were epithelial cells in which the entire cytoplasm positively stained. We found no differences in terms of CD44-positive staining between the different stages of the oestrous cycle. Presence of CD44 was detected by Western blotting in the tubal epithelium as a single band at 200 kDa. Although it appeared in all tubal segments, the expression of CD44 protein was more accentuated in the sperm reservoir (UTJ) than in the other segments. This is the first time CD44 has been detected in the epithelium of the tubal sperm reservoir in cattle, suggesting a pathway for the action of hyaluronan in this segment.

  • 30.
    Bergqvist, AS
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Yokoo, M
    Tohuku University, Japan; .
    Heldin, P
    Uppsala University, Sweden; .
    Frendin, J
    Swedish University of Agriculture Science, Sweden; .
    Sato, E
    Tohuku University, Japan; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Hyaluronan and its binding proteins in the epithelium and intraluminal fluid of the bovine oviduct2005In: Zygote (Cambridge. Print), ISSN 0967-1994, E-ISSN 1469-8730, Vol. 13, no 3, p. 207-218Article in journal (Refereed)
    Abstract [en]

    Hyaluronan (HA) is involved in several important steps of sperm storage and of fertilization. This study investigates the presence and concentration of HA in oviductal fluid (ODF), together with the localization of HA and the presence of hyaluronan-binding proteins (HABPs) in the oviductal epithelium of normally cycling dairy heifers and cows. The concentration and amount of HA in ODF, collected over the course of several oestrous cycles via catheters placed in the isthmic and ampullar tubal segments, were measured using an ELISA. The concentration and amount of HA in ODF did not vary significantly between these anatomical regions, nor between the stages of the oestrous cycle (p greater than 0.05), although the amount of HA seemed to peak during oestrous. The most HA per day (2.9 +/- 0.64 mu g, least square mean +/- SEM) was produced on the day of ovulation, whereas the lowest amount (1.25 +/- 0.68 mu g) was produced 4 days before ovulation. To investigate the localization of HA, tissue samples were retrieved at well-defined stages of the oestrous cycle and from corresponding regions of the oviduct. Sections and protein extracts from the tissue samples were studied histochemically using biotinylated HABP and immunoblotted with fluorescein isothiocyanate (FITC)-HA, respectively. Presence of HA labelling in the oviductal epithelium was restricted to the sperm reservoir, a localization that seemed to be cycle-independent. The immunoblotting of samples from the lining epithelium revealed seven bands of HABPs. We confirm that the bovine oviduct produces HA and its binding proteins, and that HA is mainly localized to the epithelium of the sperm reservoir.

  • 31.
    Bierla, Joanna B.
    et al.
    Warsaw Agricultural University, Warsaw, Poland.
    Gizejewski, Zygmunt
    Polish Academy of Sciences, Olsztyn, Poland.
    Leigh, Christopher M.
    The University of Adelaide, SA 5005, Australia.
    Ekwall, Hans
    Swedish University of Agricultural Sciences, Uppsala S-75007, Sweden.
    Soderquist, Lennart
    Swedish University of Agricultural Sciences, Uppsala S-75007, Sweden.
    Rodriguez-Martinez, Heriberto
    Swedish University of Agricultural Sciences, Uppsala S-75007, Sweden.
    Zalewski, Kazimierz
    University of Warmia and Mazury, Olsztyn, Poland.
    Breed, William G.
    The University of Adelaide, SA 5005, Australia.
    Sperm morphology of the Eurasian beaver, Castor fiber: An example of a species of rodent with highly derived and pleiomorphic sperm populations2007In: Journal of morphology (1931. Print), ISSN 0362-2525, E-ISSN 1097-4687, Vol. 268, no 8, p. 683-689Article in journal (Refereed)
    Abstract [en]

    The structural organization of the spermatozoon from the Eurasian beaver, Castor fiber (Family: Castoridae), was determined and compared to that of other sciuromorph rodents. The beaver spermatozoon has a head, which is variable in form but usually paddle-shaped, with a small nucleus and very large acrosome, and a tail that is relatively short compared to that of most other rodents. Transmission electron microscopy indicates that in most testicular spermatozoa the acrosome projects apically, although in a few it becomes partly flexed. During the final stages of maturation, however, the acrosome becomes highly folded so that the apical segment comes to lie alongside part of the acrosome that occurs lateral to the nucleus, with, in some cases, fusion taking place between the outer acrosomal membranes. The sperm nucleus is wedge-shaped, being broader basally and narrowing apically with an occasional large nuclear vacuole occurring. This spermatozoon structure is markedly different from that found in the other species of Geomyoidea, which is the sister group of the Castoridae. The findings thus emphasize the highly divergent nature of the beaver spermatozoon and demonstrate that, within the proposed Infraorder Castorimorpha, very large differences in sperm structure have evolved.

  • 32.
    Bolarin, A.
    et al.
    University of Murcia, Spain .
    Hernandez, M.
    University of Murcia, Spain .
    Vazquez, J.M.
    University of Murcia, Spain .
    Rodriguez-Martinez, Heriberto
    SLU, Sweden .
    A. Martinez, E.
    University of Murcia, Spain .
    Roca, J.
    University of Murcia, Spain .
    Use of frozen-thawed semen aggravates the summer-autumn infertility of artificially inseminated weaned sows in the Mediterranean region2009In: Journal of Animal Science, ISSN 0021-8812, E-ISSN 1525-3163, Vol. 87, no 12, p. 3967-3975Article in journal (Refereed)
    Abstract [en]

    Improvement of farrowing rate (FR) and litter size (LS) of sows that are AI with frozen-thawed (FT) semen can hardly be reached without identification of the factors behind the high variability seen among trials. Three experiments using weaned (4-d wean-to-estrus interval) multiparous (parity 2 to 7) sows were conducted to evaluate the effect of period of the year on FR and LS of FT-inseminated sows in southern Spain. Sows were grouped into 2 periods of the year: winter-spring (November to April; WS) and summer-autumn (May to October; SA). Ovarian status was monitored by transrectal ultrasonography to record how long before or after ovulation AI was performed (pre-, peri-, or postovulatory AI) and to determine the onset of estrus-to-ovulation interval (EOI). Inseminations were performed using deep intrauterine AI with 1.5 x 109 FT sperm per dose. The first experiment was designed to determine the influence of the period of the year on FR and LS of FT semen. Sows (116 in WS and 100 in SA) were AI at 33 and 39 h after the onset of estrus. The period of the year influenced the FR and LS (P less than 0.01). Farrowing rate and LS were least in SA (P less than 0.05). This pattern of annual variation was similar to that shown by sows on the same farm currently undergoing AI with liquid semen (cervical AI at 12 and 36 h after the onset of estrus with 3 x 109 sperm per dose). However, the FR reduction in SA respect to WS was more substantial in sows artificially inseminated with FT (77.6 vs. 50%, P less than 0.001) than those artificially inseminated with liquid semen (83.9 vs. 71.8%, P less than 0.05). More pre- and less periovulatory AI were performed in SA sows than in WS sows (P = 0.05). Experiment 2 was designed to evaluate whether the period of the year influenced EOI. Ovarian status was transrectal ultrasonography scanned every 6 h after the onset of estrus until the end of ovulation (WS: 30; SA: 31 sows). There were more sows with long EOI (greater than48 h) in SA than in WS (P = 0.05). Experiment 3 aimed to improve the reduced FR and LS recorded in SA sows when using FT semen (Exp. 1) by inducing ovulation with eCG + hCG. A single AI with FT semen was performed 5 h before the expected ovulation (55 sows). As a control, spontaneously ovulating sows (n = 53) were FT-inseminated as in Exp. 1. Hormonal induction of ovulation did not improve FR and LS (P greater than 0.05). In the Spanish Mediterranean area, a longer EOI during SA negatively influenced the FR and LS of weaned sows after AI. This effect was particularly evident when FT semen was used. These findings were not ameliorated by hormonal induction of ovulation.

  • 33.
    Bolarin, A
    et al.
    University of Murcia, Spain.
    Roca, J
    University of Murcia, Spain.
    Rodriguez-Martinez, Heriberto
    SLU, Sweden; .
    Hernandez, M
    University of Murcia, Spain.
    Vazquez, JM
    University of Murcia, Spain.
    Martinez, EA
    University of Murcia, Spain.
    Dissimilarities in sows ovarian status at the insemination time could explain differences in fertility between farms when frozen-thawed semen is used2006In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 65, no 3, p. 669-680Article in journal (Refereed)
    Abstract [en]

    Deep intrauterine insemination (DUI) offers a suitable alternative for the commercial use of frozen-thawed boar semen. The present study evaluated how the ovarian status at DUIs of frozen-thawed spermatozoa. (1 x 10(9) spz/dose, two DUls, 30-31 and 36-37 h after detection of oestrus) in 179 sows would explain differences in fertility between two farms with similar, but not equal, reproductive management (experiment 1). A further experiment investigated whether an increase in sperm number per AI-dose (1 versus 2 x 10(9) spz/dose, two DUls, 30-31 and 36-37 h after detection of oestrus, on 228 sows) could minimize this effect (experiment 2). Ovaries were checked by transrectal ultrasonography at the time of DUI and sows were classified into three categories: F-: ovarian pre-ovulatory follicles were visible during two examinations; O-: ovulation visible during one examination; and C-sows: corpora hemorragica visible during the two examinations. Overall farrowing rates differed (P less than 0.01) between farms (70.1 versus 51.2%, farms A and B, respectively). Distribution of sows among ultrasonography categories also differed (P less than 0.05) between farms (17.5, 72.2 and 10.3% were classified as F, O- and C-sows in farm A, versus 40.2, 29.3 and 30.5% in farm B). Nevertheless, farrowing rates and litter sizes within categories did not vary between farms (P greater than 0.05). In addition, a two-fold increase in the number of spermatozoa per DUI improved (P less than 0.05) fertility in F- and C-sows, but not in O-sows. In conclusion, the interval DUI-to-ovulation provides a major explanation for fertility differences between farms when frozen-thawed spermatozoa are used. (c) 2005 Elsevier Inc. All rights reserved.

  • 34.
    Brandt, Y.
    et al.
    Swedish University of Agriculture Science SLU, Sweden .
    Einarsson, S.
    Swedish University of Agriculture Science SLU, Sweden .
    Ljung, A.
    Swedish University of Agriculture Science SLU, Sweden .
    Lundeheim, N.
    SLU, Sweden .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science SLU, Sweden .
    Madej, A.
    SLU, Sweden .
    Effects of continuous elevated cortisol concentrations during oestrus on concentrations and patterns of progesterone, oestradiol and LH in the sow2009In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 110, no 1-2, p. 172-185Article in journal (Refereed)
    Abstract [en]

    This study investigated the effect of continuous elevated cortisol concentrations during standing oestrus on time of ovulation and patterns Of progesterone. oestradiol and luteinising, hormone (LH) in sows. The elevation of cortisol concentrations was achieved through repeated intravenous injections of synthetic adrenocorticotropic hormone (ACTH) every 2 It for approximately 48 h, from the onset of the second standing oestrus alter weaning. Treatment was terminated when ovulation was detected (monitored by transrectal ultrasonography every 4h) or when (lie sow had received a maximum of 24 injections. The close of ACTH (2.5 mu g/kg) was chosen to mimic the cortisol concentrations seen during mixing of unfamiliar SOWS. The sows (n = 14) Were surgically fitted with jugular vein catheters and randomly divided into a control (C group) where only NaCl solution were injected) or an ACTH group. Blood samples were collected every 2 h. In parallel with the blood sampling, saliva samples for cortisol analyses were taken from eight sows before onset of treatment and from four of the sows during treatment. There was no difference in time from onset of standing, oestrus to ovulation between the two groups. The interval between the peaks of oestradiol and LH to ovulation was prolonged in the ACTH group compared to the C group (p less than 0.05). with a tendency towards all earlier decline of oestradiol in the ACTH group. Cortisol and progesterone Concentrations were significantly elevated during treatment in the ACTH group (p less than 0.001). with cortisol peak concentrations occurring between 40 and 80 min after each ACTH injection. Cortisol concentrations in saliva and Plasma were highly correlated (p less than 0.001). In conclusion, elevated cortisol concentrations from the onset of standing oestrus increase progesterone concentrations and prolong the interval between oestradiol and LH peaks to ovulation, the latter possible due to an early decline in oestradiol concentrations and a change of the LH peak outline. the effect these hormonal changes have on reproductive performance need to be further investigated. (C) 2008 Elsevier B.V. All rights reserved.

  • 35.
    Brandt, Y
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Lang, A
    Swedish University of Agriculture Science, Sweden; .
    Madej, A
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Einarsson, S
    Swedish University of Agriculture Science, Sweden; .
    Impact of ACTH administration on the oviductal sperm reservoir in sows: The local endocrine environment and distribution of spermatozoa2006In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 92, no 1-2, p. 107-122Article in journal (Refereed)
    Abstract [en]

    The objective of the study was to investigate if short-term stress in sows (simulated by injections of synthetic adrenocorticotrophic hormone (ACTH)) during standing oestrus had a negative effect on the local environment in the utero-tubal junction (UTJ) and isthmus and the distribution of spermatozoa in these segments. Fourteen sows were monitored for ovulation using ultrasonography in two consecutive oestruses. The sows were fitted with jugular catheters and, from onset of the second oestrus, blood samples were collected every second hour. In the 2nd oestrus, seven sows were given ACTH every second hour, from the onset of standing oestrus until the sow ovulated (ACTH-group), whereas the other seven sows remained as controls (C-group) and were given NaCl solution. The sows were artificially inseminated 16-18 h before expected ovulation. Six hours after ovulation the sows were anaesthetised, and blood samples were repeatedly taken from veins draining the uterus and the UTJ-isthmus, respectively. This oviduct was thereafter removed and divided in four adjacent sections consisting of: (i) the UTJ, (ii) the first, and (iii) the second isthmus segment prior to (iv), the ampullary-isthmic junction (AIJ) and the ampulla. The three first-mentioned segments were flushed to retrieve spermatozoa, whereas the last one was flushed to collect oocytes/ova. The number of spermatozoa attached to the zona pellucida was counted. The concentrations of cortisol in jugular blood of the ACTH-group sows during the time of ACTH-injections were significantly higher than of the C-group sows (p less than 0.05), as were the levels of progesterone (p less than 0.001). Progesterone and cortisol concentrations measured in the blood samples draining the UTJ-isthmic region 6 h after ovulation did not significantly differ between the groups, but the C-group displayed significantly higher concentrations of progesterone in the UTJ-isthmic region compared with the levels measured in parallel samples taken of jugular blood (P less than 0.01). The C-group, but not the ACTH-group, also displayed a significant elevation in progesterone concentration 6 h after ovulation compared with the basal levels before ovulation (p less than 0.01). Numbers of retrieved spermatozoa were not significantly different between the C-group and the ACTH-group. However, there was a tendency for a larger number of spermatozoa among sows in the ACTH-group, especially in the isthmic segment adjacent to the AIJ. In conclusion, simulated stress induced by injections of ACTH during standing oestrus results in elevated concentrations of progesterone before ovulation and may interfere with the rise of progesterone after ovulation. However, ACTH-injections appeared to augment transport of spermatozoa through the female genital tract of pigs. (c) 2005 Elsevier B.V. All rights reserved.

  • 36.
    Brandt, Y
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Lang, A
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Madej, A
    Swedish University of Agriculture Science, Sweden; .
    Einarsson, S
    Swedish University of Agriculture Science, Sweden; .
    Impact of ACTH during oestrus on the ultrastructure of the spermatozoa and their environment in the tubal reservoir of the postovulatory sow2006In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 93, no 3-4, p. 231-245Article in journal (Refereed)
    Abstract [en]

    This study investigated whether injections of synthetic ACTH (simulating short-term stress) in sows during standing oestrus have a negative effect on spermatozoa and the local intraluminal environment in the utero-tubal junction (UTJ) and isthmus. Seven of the 14 sows were given ACTH through a jugular catheter every 2 h from the onset of standing oestrus until the sow ovulated (ACTH-group), while the other seven sows were given NaCl solution (C-group). All sows were artificially inseminated before ovulation. Six hours after ovulation (detected with transrectal ultrasonography) the sows were anaesthetised, the right oviduct was fixed in toto by vascular perfusion with glutaraldehyde, and the UTJ and specimens from the isthmus were prepared for scanning electron microscopy (SEM). SEM revealed that a seemingly viable population of spermatozoa remained in the UTJ 6 It after ovulation. A majority of sows in the ACTH-group had moderately to exaggerated amounts of mucus in the intraluminal environment of the sperm reservoir. In conclusion, stress simulated by exogenous ACTH in sows may alter the intraluminal environment of the sperm reservoir. (c) 2005 Elsevier B.V. All rights reserved.

  • 37.
    Brandt, Y.
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Lundeheim, N.
    Swedish University of Agriculture Science, Sweden; .
    Madej, A.
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Einarsson, S.
    Swedish University of Agriculture Science, Sweden; .
    Effects of ACTH injections during estrus on concentrations and patterns of progesterone, estradiol, LH, and inhibin alpha and time of ovulation in the sow2007In: Domestic Animal Endocrinology, ISSN 0739-7240, E-ISSN 1879-0054, Vol. 32, no 2, p. 122-137Article in journal (Refereed)
    Abstract [en]

    This study investigated whether injections of ACTH for 48 h, from the onset of the second standing estrus after weaning, had any impact on time of ovulation and patterns of progesterone, estradiol, luteinizing hormone (LH), and inhibin a. The studied sows (n = 15) were fitted with jugular vein catheters and randomly divided into a control (C group) and an ACTH group. From the onset of standing estrus, the sows were injected (NaCl or synthetic ACTH, 5 mu g/kg) every 4 h; blood samples were collected immediately before and 45 min after each injection. Ovulation was monitored using ultrasonography. The ACTH-group sows stopped displaying signs of standing estrus sooner after ovulation in their second estrus, but no impact was found on time of ovulation. There were no significant differences in the intervals between LH peak, estradiol peak, and the onset of standing estrus between the C and ACTH groups. The cortisol and progesterone concentrations were significantly elevated (p less than 0.001) in samples taken 45 min after ACTH injection. There were minor differences in estradiol and LH concentrations between the groups. Overall inhibin a concentrations were significantly higher during the treatment period in the ACTH than in the C group, but there were no significant differences between samples taken either 45 min or 4h after injection. In conclusion, injections of synthetic ACTH during estrus in the sow apparently disturb the duration of signs of standing estrus and the hormonal pattern of progesterone, and possibly of inhibin ot, estradiol and LH. (c) 2006 Elsevier Inc. All rights reserved.

  • 38.
    Brandt, Y.
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Madej, A.
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Einarsson, S.
    Swedish University of Agriculture Science, Sweden; .
    Effects of exogenous ACTH during oestrus on early embryo development and oviductal transport in the sow2007In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 42, no 2, p. 118-125Article in journal (Refereed)
    Abstract [en]

    This study was conducted to assess the effects of ACTH injections on the early development of embryos and their transportation to the uterus. Fifteen sows were monitored for ovulation using transrectal ultrasonography during the first two oestrous periods after weaning. The sows were randomly divided into a control group (C group, n = 8) and an ACTH-treated group (ACTH group, n = 7), and were all surgically fitted with intra-jugular catheters. From the onset of the second standing oestrus after weaning, the sows were injected (NaCl/synthetic ACTH) every 4 h. Blood samples were collected immediately before and 45 min after each injection. All sows were inseminated once 10-33 h before ovulation in their second oestrus after weaning. At 48 (n = 4) or 60 (n = 11) h after ovulation during their second oestrus, the sows were killed and the embryos retrieved from the oviduct and uterus. The embryos were counted and compared with the number of corpora lutea, cleavage rate was noted and, finally, the embryos were prepared for confocal laser scanning microscopy and transmission electron microscopy. There was no difference between the groups regarding cleavage rate, the cytoskeleton, or the number of active nucleoli. However, the ACTH group had significantly (p less than 0.05) fewer ova/embryos retrieved (51%) than the C group (81%), and there was a tendency towards faster transportation to the uterus in the ACTH group, possibly because of high progesterone concentrations during treatment. To conclude, administration of ACTH every 4 h from onset of oestrus to 48 h caused significant loss of oocytes or embryos, and possibly faster transportation through the oviduct.

  • 39.
    Bruessow, K.-P.
    et al.
    FBN Research Institute Biol Farm Anim, Germany .
    Ratky, J.
    Research Institute Anim Breeding and Nutr, Hungary .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science SLU, Sweden .
    Fertilization and early embryonic development in the porcine fallopian tube2008In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 43, p. 245-251Article in journal (Refereed)
    Abstract [en]

    Fertilization and early embryo development relies on a complex interplay between the Fallopian tube and the gametes before and after fertilization. Thereby the oviduct, as a dynamic reproductive organ, enables reception, transport and maturation of male and female gametes, their fusion, and supports early embryo development. This paper reviews current knowledge regarding physiological processes behind the transport of boar spermatozoa, their storage in and release from the functional sperm reservoir (SR), and of the interactions that newly ovulated oocytes play within the tube during their transport to the site of fertilization. Experimental evidence of an ovarian control on sperm release from the SR is highlighted. Furthermore, the impact of oviductal secretion on sperm capacitation, oocyte maturation, fertilization and early embryo development is stressed.

  • 40.
    Buranaamnuay, K.
    et al.
    Chulalongkorn University, Thailand .
    Tummaruk, P.
    Chulalongkorn University, Thailand .
    Singlor, J.
    Chulalongkorn University, Thailand .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science SLU, Sweden .
    Techakumphu, M.
    Chulalongkorn University, Thailand .
    Effects of Straw Volume and Equex-STM (R) on Boar Sperm Quality after Cryopreservation2009In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 44, no 1, p. 69-73Article in journal (Refereed)
    Abstract [en]

    The present experiments were designed to study the effect of adding the detergent Equex-STM (R) to freezing extender, and of straw volume (0.25 ml vs 0.5 ml), on boar sperm quality after cryopreservation. Three ejaculates from each of four purebred boars (three Landrace and one Yorkshire) were collected and frozen with a lactose-egg yolk extender containing glycerol with or without 1.5% Equex-STM (R). The extended semen was loaded into either 0.25- or 0.5-ml straws. The straws were placed in liquid nitrogen (LN2) vapour approximately 3 cm above the level of LN2 for 20 min and then were plunged into LN2. Thawing was achieved in warm water at 50 degrees C for 12 s and then was incubated in a 38 degrees C water-bath for 30 min before evaluating sperm quality. Results showed that the individual motility, viability and acrosomal normal apical ridge (NAR) were improved (p less than 0.001) when Equex-STM (R) was added to the freezing extender. There was no difference (p = 0.48) in sperm motility between 0.25- and 0.5-ml straws when Equex-STM (R) was added. The percentages of viable and of NAR sperm in 0.5-ml straws were higher than those in 0.25-ml straws (p = 0.02, p = 0.0003 respectively). The percentages of membrane intact sperm evaluated using the short hypo-osmotic swelling test were not affected by straw volume or the adding of Equex-STM (R) (p greater than 0.05). The results of these investigations suggested that Equex-STM (R) exerts a beneficial effect on the quality of cryopreserved boar semen and this cryopreservation protocol was favourable for a 0.5-ml straw.

  • 41.
    Caballero, I
    et al.
    University of Murcia, Spain; .
    Vazquez, JM
    University of Murcia, Spain; .
    Rodriguez-Martinez, Heriberto
    University of Murcia, Spain; .
    Gil, MA
    University of Murcia, Spain; .
    Calvete, JJ
    University of Murcia, Spain; .
    Sanz, L
    Institute of Biomedicine, CSIC, Valencia, Spain; .
    Garcia, EM
    Institute of Biomedicine, CSIC, Valencia, Spain; .
    Roca, J
    University of Murcia, Spain; .
    Martinez, EA
    University of Murcia, Spain; .
    Influence of seminal plasma PSP-I/PSP-II spermadhesin on pig gamete interaction2005In: Zygote (Cambridge. Print), ISSN 0967-1994, E-ISSN 1469-8730, Vol. 13, no 1, p. 11-16Article in journal (Refereed)
    Abstract [en]

    The seminal plasma PSP-I/PSP-II spermadhesin is able to preserve, in vitro, the viability of highly extended boar spermatozoa, suggesting it might be used as a suitable ameliorator for the damaging effects of sperm handling, including in vitro fertilization. However, little is known about the ligand capability of PSP-I/PSP-II as regards the zona pellucida (ZP) or its possible role in gamete interaction. The present study evaluated the effect of the presence of PSP-I/PSP-II (1.5 mg/ml) during in vitro oocyte maturation and also during co-incubation of frozen-thawed boar spermatozoa with either immature (IM) or in vitro matured (IVM) oocytes, either enclosed by cumulus cells or denuded. Exposure of the gametes to the heterodimer during in vitro gamete co-incubation showed a significant blocking effect of sperm penetration rates and a decreased number of spermatozoa per oocyte in both IM and IVM denuded oocytes. Such an effect was not present in cumulus-enclosed oocytes, suggesting the effect could be mediated by exposed ZP receptors. In addition, when PSP-I/PSP-II was added to the IVM medium, oocyte maturation rates were significantly reduced. In conclusion, the results suggest that PSP-I/PSP-II, when present in vitro, blocks sperm-ZP binding.

  • 42.
    Caballero, Ignacio
    et al.
    University of Murcia, Murcia, Spain .
    M. Vazquez, Juan
    University of Murcia, Murcia, Spain.
    M. Garcia, Eva
    University of Murcia, Murcia, Spain.
    Roca, Jordi
    University of Murcia, Murcia, Spain.
    A. Martinez, Emilio
    University of Murcia, Murcia, Spain .
    J. Calvete, Juan
    Institute of Biomedicine, C.S.I.C., Valencia, Spain.
    Sanz, Libia
    Institute of Biomedicine, C.S.I.C., Valencia, Spain.
    Ekwall, Hans
    Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
    Rodriguez-Martinez, Heriberto
    Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
    Immunolocalization and possible functional role of PSP-I/PSP-II heterodimer in highly extended boar spermatozoa2006In: Journal of Andrology, ISSN 0196-3635, E-ISSN 1939-4640, Vol. 27, no 6, p. 766-773Article in journal (Refereed)
    Abstract [en]

    PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma which is able to preserve, in vitro, the viability, motility, and mitochondrial activity of highly extended boar spermatozoa for at least 5 hours. However, little is known about the binding pattern of the heterodimer to the sperm plasma membrane and its eventual relation with the maintenance of the sperm functionality. The present study investigated the effect of exposing highly extended boar spermatozoa (11 million/mL) to 1.5 mg/mL of PSP-I/PSP-II for 0.5, 5, and 10 hours at 38 degrees C on sperm characteristics and the changes in PSP-I/PSP-II localization as a result of both the addition of PSP-I/PSP-II to the extender and the incubation time. Exposure of the spermatozoa to PSP-I/PSP-II preserved sperm viability, motility, and mitochondrial activity when compared to nonexposed spermatozoa. This protective effect lasted for 10 hours (P less than.05). After immunolabeling of highly extended semen with rabbit monospecific polyclonal antibody against PSP-I/PSP-11, the percentage of immunopositive spermatozoa declines over time from 71% (0.5 hours) to 49% (10 hours). However, more than 80% of spermatozoa remained labeled during the 10-hour incubation period if PSP-I/PSP-11 was added. Scanning electron microscopy revealed 4 different binding patterns. The heterodimer was mainly localized to the acrosomal area, being redistributed to the postacrosomal area or lost during in vitro incubation. In conclusion, the protective effect of the heterodimer appears to be related to its adhesion to the acrosomal area, and the loss of this protective effect coincides with a stepwise redistribution of PSP-I/PSP-II during incubation.

  • 43.
    Colleoni, Silvia
    et al.
    AVANTEA.
    Lagutina, Irina
    AVANTEA.
    Lazzari, Giovanna
    AVANTEA.
    Rodriguez-Martinez, Heriberto
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Genetics.
    Galli, Cesare
    AVANTEA.
    Morrell, Jane M
    Swedish University Agriculture Science.
    New Methods for Selecting Stallion Spermatozoa for Assisted Reproduction2011In: Journal of Equine Veterinary Science, ISSN 0737-0806, E-ISSN 1542-7412, Vol. 31, no 9, p. 536-541Article in journal (Refereed)
    Abstract [en]

    Improved sperm selection techniques are needed to increase the efficiency of equine-assisted reproduction. Single layer centrifugation (SLC) of spermatozoa has been shown to improve the quality of stallion sperm samples. In this study, the functionality of selected stallion spermatozoa was tested by intracytoplasmic sperm injection of equine oocytes after selection by SLC through Androcoll-E or by discontinuous density gradient centrifugation (DGC) through Redigrad and Tyrodes medium with added albumin, lactate, and pyruvate. The mean cleavage rates of the injected oocytes from SLC- and DGC-selected spermatozoa were 67% and 66%, respectively, whereas the proportion of blastocysts developing from cleaved oocytes was 28% and 22%, respectively (P andgt; .05, not significant). An incidental finding was that there was a tendency for SLC-selected spermatozoa to have a higher percentage of spermatozoa with normal morphology than DGC (70% +/- 22% vs. 58% +/- 38%) and for more blastocysts to be obtained from subfertile ejaculates (21 [19.6%] vs. 15 [14.4%], respectively). In further experiments, stallion spermatozoa bound to hyaluronan, although binding may depend on the semen extender and sperm treatment as well as incubation time. In conclusion, SLC-selected stallion spermatozoa function normally when injected into oocytes. SLC may potentially be better than DGC at selecting spermatozoa from subfertile ejaculates, but this effect needs rigorous investigation with a much larger sample size. Use of the hyaluronan-binding assay for assessing the potential fertility of stallion spermatozoa may be useful but requires further evaluation.

  • 44.
    Cox, J.F.
    et al.
    University of Concepcion, Chile.
    Alfaro, V.
    University of Concepcion, Chile.
    Montenegro, V.
    University of Concepcion, Chile.
    Rodriguez-Martinez, Heriberto
    SLU, Sweden; .
    Computer-assisted analysis of sperm motion in goats and its relationship with sperm migration in cervical mucus2006In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 66, no 4, p. 860-867Article in journal (Refereed)
    Abstract [en]

    In vitro sperm migration in cervical mucus relates to sperm concentration at the utero-tubal junction and to in vivo fertilization performance in goats. The present study aimed to characterize, using Computer-Assisted Sperm Analysis (CASA), motility patterns depicted by buck sperm and their relation to the migration efficiency in homologous (goat) and heterologous (heifer) cervical mucus in vitro. Semen was collected from 23 sexually mature bucks from three breeds by artificial vagina and sperm were assessed for motility parameters with a Hobson Sperm analyzer following extension in Sperm Analysis Medium (SAM). To study the relationship between kinematics parameters and the ability of sperm to migrate in cervical mucus, in a first experiment, motility performance of buck sperm suspended in SAM was compared against seminal plasma. In a second experiment, kinematics parameters of sperm were characterized. In a third experiment, bucks with sperm that differed in specific motion parameters were compared for the ability of their sperm to migrate through goat and bovine cervical mucus collected at estrus. In a fourth experiment, ejaculates that were compared in their migration ability and were assessed simultaneously for their motility parameters. Overall, sperm suspended in SAM medium had better velocity and similar linearity and lateral head displacement than those suspended in seminal plasma; furthermore, caprine sperm swam relatively fast (relative to bovine and ovine sperm), following a very linear trajectory. Under the conditions used, velocity parameters, linearity and lateral head displacement seemed to be related to sperm migration efficiency in homologous mucus but not in bovine cervical mucus. (c) 2006 Elsevier Inc. All rights reserved.

  • 45.
    Cremades, T
    et al.
    University of Murcia, Spain; .
    Roca, J
    University of Murcia, Spain; .
    Rodriguez-Martinez, Heriberto
    SLU, Sweden.
    Abaigar, T
    Estacion Experimental de Zonas Aridas, Consejo Superior de Investigaciones Cientificas (CSIC), Almeria, Spain.
    Vazquez, JM
    University of Murcia, Spain; .
    Martinez, EA
    University of Murcia, Spain; .
    Kinematic changes during the cryopreservation of boar spermatozoa2005In: Journal of Andrology, ISSN 0196-3635, E-ISSN 1939-4640, Vol. 26, no 5, p. 610-618Article in journal (Refereed)
    Abstract [en]

    The present study evaluates the effect that various steps of a conventional cycle of cryopreservation have on the patterns of movement exhibited by boar spermatozoa. Sperm-rich ejaculate fractions collected from 24 mature fertile boars (1 ejaculate per boar) were cryopreserved following a standard freeze-thaw procedure with 0.5-mL plastic straws. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in 5 steps of the cryopreservation procedure. These steps were as follows: 1) at the time that the fresh semen was extended, 2) at 17 degrees C, after sperm concentration by centrifugation and re-extension of the pellet with lactose-egg yolk extender; 3) at 5 degrees C, after added freezing extender; 4) at the time that thawed semen was held in a water bath at 37 degrees C for 30 minutes; and 5) at the time that thawed semen was held in a water bath at 37 degrees C for 150 minutes. Data from individual motile spermatozoa, defined by 7 kinematic parameters (curvilinear velocity [VCL], straight-line velocity [VSL], average path velocity [VAP], linearity [LIN], straightness [STR], mean amplitude of lateral head displacement [ALH], and beat cross frequency [BCF]), were analyzed using a pattern analysis technique (PATN) to identify and quantify populations and subpopulations of motile sperm within the semen samples. After the first cluster analysis, 3 motile sperm populations (P) were identified (P1: progressive and/or vigorous cells [90.4%], P2: poorly progressive cells [8.3%], and P3: nonprogressive cells [1.3%]). These populations remained constant (P greater than .05) throughout the 5-step cryopreservation procedure. A second PATN was carried out within the P1 sperm population, which identified 3 sperm subpopulations (sP) (eg, sP1: cells with progressive and vigorous movement [58.7%], sP2: progressive cells only [24.6%], and sP3: vigorous cells only, hyperactive-like [16.7%]). Although the relative frequency of these 3 subpopulations varied among ejaculates (boars), there was no interaction with any cryopreservation step we examined. Whereas sP1 remained constant (P greater than .05), sP2 and sP3 varied significantly (P less than .05) through the cryopreservation procedure, with the increase in sP3 after centrifugation at 17 degrees C and during cooling at 5 degrees C considered particularly relevant. In conclusion, the present study confirms the heterogeneity of sperm movement patterns in boar semen, patterns that vary through the cryopreservation procedure, especially after removal of the seminal plasma by centrifugation and subsequent extension at 17 degrees C and after the slow cooling at 5 degrees C, when obvious increases in hyperactivated movement appeared. The vast majority of spermatozoa, those exhibiting progressive and vigorous movement, remained constant during the cryopreservation procedure, although the proportion differed among boars.

  • 46.
    Cuello, C.
    et al.
    University of Murcia, E-30071 Murica, Spain.
    A. Gil, M.
    University of Murcia, E-30071 Murica, Spain.
    Alminana, C.
    University of Murcia, E-30071 Murica, Spain.
    Sanchez-Osorio, J.
    University of Murcia, E-30071 Murica, Spain.
    Parrilla, I.
    University of Murcia, E-30071 Murica, Spain.
    Caballero, I.
    University of Murcia, E-30071 Murica, Spain.
    M. Vazquez, J.
    University of Murcia, E-30071 Murica, Spain .
    Roca, J.
    University of Murcia, E-30071 Murica, Spain.
    Rodriguez-Martinez, Heriberto
    SLU, Sweden; .
    A. Martinez, E.
    University of Murcia, E-30071 Murica, Spain.
    Vitrification of in vitro cultured porcine two-to-four cell embryos2007In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 68, no 2, p. 258-264Article in journal (Refereed)
    Abstract [en]

    The objective of this experiment was to evaluate the effect of a 5-day period of in vitro culture of two-to-four cell porcine embryos up to the blastocyst stage on their ability to survive vitrification and warming. In order to increase the cooling rate, superfine open pulled straws and Vit-Master (R) technology were used for vitrification. Two-to-four cell embryos were collected from weaned sows (n = 11) on day 2 (DO = onset of estrus). Some embryos (N = 63) were vitrified within 3 It after collection, warmed and cultured for 120 h (Group V2). Additionally, 81 two-to-four cell embryos were cultured for 96 It in order to obtain blastocysts; these were then vitrified, warmed and cultured for 24 h (Group VB; N = 65). The remaining two-to-four cell embryos were used as controls and thus not vitrified (control embryos; N = 70) but were cultured in vitro for 120 h. The V2, VB and control embryos were evaluated for their developmental progression and morphology during culture. All embryos (V2, VB and controls) were fixed on the same day of development in order to assess the total number of blastomeres. The survival and blastocyst formation rates obtained from V2 embryos were very poor (9.6 +/- 0.7% and 3.2 +/- 0.5%, respectively). The survival and hatching rates of VB embryos (75.0 +/- 0.69% and 33.6 +/- 0.13%) were lower (p less than 0.001) than those obtained with control embryos (89.1 +/- 0.8% and 47.5 +/- 0.12%). Hatched VB embryos had a lower (p less than 0.01) total cell number than hatched control embryos (70.3 +/- 4.5 versus 90.6 +/- 3.2, respectively). There was no difference between expanded VB and control blastocysts. In conclusion, blastocysts derived from in vitro culture of two-to-four cell pig embryos could be successfully vitrified using SOPS straws and Vit-Master (R). (c) 2007 Elsevier Inc. All rights reserved.

  • 47.
    Cuello, C.
    et al.
    University of Murcia, Spain Swedish University of Agriculture Science, Sweden .
    Sanchez-Osorio, J.
    University of Murcia, Spain .
    Alminana, C.
    University of Murcia, Spain .
    Gil, M.A.
    University of Murcia, Spain .
    Parrilla, I.
    University of Murcia, Spain .
    Roca, J.
    University of Murcia, Spain .
    Vazquez, J.M.
    University of Murcia, Spain .
    Martinez, E.A.
    University of Murcia, Spain .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden .
    Superfine open pulled straws vitrification of porcine blastocysts does not require pretreatment with cytochalasin B and/or centrifugation2010In: Reproduction, Fertility and Development, ISSN 1031-3613, E-ISSN 1448-5990, Vol. 22, no 5, p. 808-817Article in journal (Refereed)
    Abstract [en]

    The present study investigated the in vitro development of and cytoskeletal disruption suffered by in vivo-derived porcine blastocysts subjected to superfine open pulled straws (SOPS) vitrification. Blastocysts were either untreated prior to SOPS vitrification or were subjected to one of the following three pretreatment protocols: (1) centrifugation (12 min, 13 000g); (2) 25 min equilibration with 7.5 mu g mL(-1) cytochalasin B; or (3) equilibration with cytochalasin B followed by centrifugation. After 24 h culture, fresh (n = 32) and vitrified-warmed (n = 188) blastocysts were evaluated by stereomicroscopy, with survival and hatching rates recorded. Some blastocysts were stained with 4,6-diamidino2- phenylindole and processed for cytoskeletal evaluation. Three cytoskeletal patterns were identified: Grade I, intact cytoskeleton; Grade II, gross maintenance of integrity, but with some clumps of actin within the cytoplasm; and Grade III, a highly disrupted cytoskeleton. There were no differences in the survival, hatching and cell death rats, total cell number or cytoskeletal integrity between the different vitrification groups. Cell death was greater for vitrified blastocysts than for fresh blastocysts (3.6 +/- 0.4% v. 0.4 +/- 0.7%, respectively; P less than 0.05) and the percentage of blastocysts with a Grade I cytoskeletal pattern was lower for vitrified compared with fresh blastocysts (60.8% v. 92%, respectively; P less than 0.05). The vitrified-warmed blastocysts that hatched during culture exhibited a Grade I cytoskeletal pattern. In conclusion, successful SOPS vitrification of porcine blastocysts does not require pretreatment with cytochalasin B and/or centrifugation.

  • 48.
    De Ambrogi, Marco
    et al.
    Swedish University of Agriculture Science, Sweden.
    Ballester, Juan
    Swedish University of Agriculture Science, Sweden.
    Saravia, Fernando
    Swedish University of Agriculture Science, Sweden.
    Caballero, Ignacio
    Swedish University of Agriculture Science, Sweden.
    Johannisson, Anders
    Swedish University of Agriculture Science, Sweden.
    Wallgren, Margareta
    Swedish University of Agriculture Science, Sweden.
    Andersson, Magnus
    University of Helsinki, Mäntsälä, Finland.
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden.
    Effect of storage in short- and long-term commercial semen extenders on the motility, plasma membrane and chromatin integrity of boar spermatozoa2006In: International Journal of Andrology, ISSN 0105-6263, E-ISSN 1365-2605, Vol. 29, no 5, p. 543-552Article in journal (Refereed)
    Abstract [en]

    For artificial insemination (AI) in pigs, preservation of liquid boar semen at 16-20 degrees C is still common practice as sperm cryopreservation remains suboptimal in this species. To meet the different needs of the swine industry, several extenders have been developed to preserve semen in liquid form for short- and long-term storage. In the present study, three different commercial extenders devised for short-term (BTS+) or long-term preservation (MR-A and X-Cell), were used to test whether storage of semen from four mature, fertile boars at 17 degrees C for 96 h would affect sperm characteristics relevant for fertility, such as motility, membrane integrity and chromatin stability. Computer-assisted sperm analysis, and stainings with the acylated membrane dye SYBR-14/propidium iodide, and acridine orange in connection with flow cytometry were used to evaluate these variables. Percentages of total motile spermatozoa decreased slightly, but significantly, after 72-96 h. While membrane integrity values varied during the period of study, no significant changes in either membrane integrity or chromatin stability were, however, registered. This suggests a customary 96-day storage at 17 degrees C in these extenders was too short an interval to cause losses of integrity in nuclear DNA in the boar population studied.

  • 49.
    Einarsson, S.
    et al.
    Swedish University of Agriculture Science, Sweden .
    Andersson, K.
    Swedish University of Agriculture Science, Sweden .
    Wallgren, M.
    Swedish University of Agriculture Science, Sweden Qual Genet, Sweden .
    Lundstrom, K.
    Swedish University of Agriculture Science, Sweden .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden .
    Short- and long-term effects of immunization against gonadotropin-releasing hormone, using Improvac (TM), on sexual maturity, reproductive organs and sperm morphology in male pigs2009In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 71, no 2, p. 302-310Article in journal (Refereed)
    Abstract [en]

    The objective of this study was to determine the short and long term effects of a gonadotropin-releasing hormone (GnRH) vaccine (Improvac (TM) Pfizer Ltd.), on sexual maturity, development of the reproductive organs, and the morphology of caudal epididymal spermatozoa in non-castrated male pigs. The pigs were slaughtered 4, 16 or 22 weeks after the second Improvac (TM) vaccination. A total of 80 crossbred non-castrated male pigs were included in this study comprising two experiments, a short-effect (Experiment 1) and a long-effect (Experiment 2). The first experiment included 56 pigs, 24 of them were maintained as controls and 32 were vaccinated twice, and slaughtered 4 weeks after the second vaccination. The second experiment included 24 pigs, 12 controls and 12 vaccinated twice, and slaughtered either 16 weeks (n = 6) or 22 weeks (n = 6) after the second vaccination. None of the immunized pigs was sexually mature at slaughter, i.e. 4, 16 or 22 weeks after second vaccination. Corresponding results of the control pigs showed that 50% had reached sexual maturity at the age corresponding to 4 weeks after the second vaccination. and 100% at slaughter 16, respectively, 22 weeks after vaccination. At 4, 16 and 22 weeks after second vaccination both testes weight and bulbourethral length were significantly reduced (p less than 0.001). The percentages of proximal droplets and abnormal heads were significantly lower in the control pigs than in the immunized pigs at slaughter 4 weeks after vaccination, whereas distal droplets were higher. For the other morphological parameters no significant differences were seen, but all mean values except for acrosome defects were numerically lower in the control pigs compared with the immunized pigs. For pigs slaughtered 16 or 22 weeks after vaccination, the vaccination effect was significant for percentages of proximal droplets, distal droplets, acrosome defects, acrosome abnormality and abnormal heads (p = 0.017-0.001). The immunization clearly disrupted the number and morphology of the interstitial Leydig cells, lasting throughout the study period (4-22 weeks after vaccination). Spermatogenesis was also clearly affected in the immunized pigs, to various degrees, from mild disruption (spermatocyte loss, decrease of the normal number of layers of germ cells) to severe loss of germ cells including tubuli with Sertoli cells-only (complete disappearance of germ cells), also covering the entire study period. The results indicated that the effect of immunization persisted for at least 22 weeks after the second vaccination. (c) 2009 Elsevier Inc. All rights reserved.

  • 50.
    Einarsson, S.
    et al.
    Swedish University of Agriculture Science, Sweden .
    Brandt, Y.
    Swedish University of Agriculture Science, Sweden .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden .
    Madej, A.
    Swedish University of Agriculture Science, Sweden .
    Conference Lecture: Influence of stress on estrus, gametes and early embryo development in the sow2008In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 70, no 8, p. 1197-1201Article in journal (Refereed)
    Abstract [en]

    Systems with loose-housed sows have become common. Regrouping, which is commonly done after weaning and may coincide with many important reproductive events, causes stressful situations with elevated blood cortisol concentrations. Depending on group size, approximately 2-7 d are required for a new group of sows to become relatively stable. In a series of studies, the social stress after regrouping was simulated with repeated adrenocorticortriphic hormone (ACTH) treatments for approximately 48 h. Sows were allocated into control and experimental groups, fitted with jugular catheters, and blood samples were collected every 2 or 4 h. Follicular development and ovulation were monitored by transrectal ultrasonography every 4 h. Simulated stress during proestrus prolonged estrus and disturbed the follicular growth and ovulation. Giving ACTH during estrus elevated concentrations of cortisol and progesterone, and chagned the intraluminal environment, including exaggerated amounts of mucus in the UTJ and isthmus. Although ACTH had no effect on the time of ovluation (relative to onset of standing estrus), or on embryo development, fewer oocytes/embryos were retrieved from the ACTH group than from the control group (51% vs. 81%, P less than 0.05), and there was a tendency towards faster embryo transportation to the uterus. Short-term fasting after ovulation had an unfavourable effect on sperm numbers in UTJ/isthmus, cleavage rate of fertlized ova, as well as ova transport through the isthmic part of the oviduct. Treatment with ACTH after ovulation redcued numbers of spermatozoa at the zona pellucida and retarded cleavage rate of fertilized ova. Therefore, the timing of stress seemed to be an important factor regarding effects on reproductive events. (C) 2008 Elsevier Inc. All rights reserved.

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