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  • 1.
    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. Univ Autonoma Barcelona, Spain.
    Lopez-Bejar, Manel
    Univ Autonoma Barcelona, 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.
    The risk of using monoclonal or polyclonal commercial antibodies: controversial results on porcine sperm CD44 receptor identification2019In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 54, no 4, p. 733-737Article in journal (Refereed)
    Abstract [en]

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

  • 2.
    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.

  • 3.
    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.

  • 4.
    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.

  • 5.
    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.

  • 6.
    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.

  • 7.
    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.

  • 8.
    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.

  • 9.
    de Paz, Paulino
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology, University of León, León, Spain.
    Alvarez-Rodriguez, Manuel
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León.
    Nicolas, Manuel Aguilar
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León.
    Chamorro, Ca
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León.
    Borragán, Santiago
    Cabárceno Park, Cantabria, Spain.
    Martinez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology, University of León, León, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León.
    Optimization of glycerol concentration and freezing rate in the cryopreservation of ejaculate from brown bear (Ursus arctos).2012In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 47, no 1, p. 105-112Article in journal (Refereed)
    Abstract [en]

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

  • 10.
    Hallap, T
    et al.
    Swedish University of Agriculture Science, Sweden .
    Jaakma, U
    Estonian Agricultural University, Tartu, Estonia.
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden.
    Changes in semen quality in Estonian Holstein AI bulls at 3, 5 and 7 years of age2006In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 41, no 3, p. 214-218Article in journal (Refereed)
    Abstract [en]

    The predictability of semen quality of mature sires from measurements at an early age is not well established. The aim of the present study was to determine age-dependent changes in the quality of bull semen from six Estonian Holstein (EHF) bulls, processed when the sires were 3, 5 and 7 years old. Fertility data such as 60-day non-return to oestrus rates (60d-NRRs) were available for 3-year-old bulls. From each batch, semen straws were analysed immediately after thawing [i.e. post-thaw (PT)] (controls) and after a swim-up (SU) procedure. The analyses comprised subjective and computerized measurements of sperm motility using computer-assisted sperm analysis (CASA) as well as estimations of sperm concentration, morphology and membrane integrity. There was a significant (p less than 0.05) increase in the percentage of sperm motility (SU), membrane integrity (PT, SU) and normal tail and acrosome morphology (SU) with an increase in the age of the sires. The percentage of total motile spermatozoa PT measured by CASA correlated between 3- and 7-, and between 5- and 7-year-old bulls (p less than 0.05). In addition, the proportion of head abnormalities tended to correlate between all three age groups both PT and after SU (p less than 0.1). The sperm parameters correlating with fertility were average path velocity (VAP) (p less than 0.001), total motility as measured by CASA (p less than 0.01) linearly motile spermatozoa (p less than 0.05) and CASA-assessed numbers of motile spermatozoa (p less than 0.05), all after SU selection. The results showed that overall semen quality examined at 3 years of age is related to the semen parameters later in bulls life. Moreover, CASA-assessed motility after SU seems to be a reliable marker for semen quality assessment as it shows correlation not only between the ages, but also to field fertility.

  • 11.
    Kareskoski, A. M.
    et al.
    University of Helsinki, Faculty of Veterinary Medicine.
    Rivera del Alamo, M. M.
    Autonomous University of Barcelona.
    Guvenc, K.
    University of Istanbul, Faculty of Veterinary Medicine.
    Reilas, T.
    MTT Agrifood Research Finland, Ypäjä.
    Calvete, J. J.
    Institute of Biomedicine, Valencia.
    Rodriguez-Martinez, Heriberto
    Swedish University of Agricultural Sciences (SLU), Division of Reproduction, Uppsala.
    Andersson, M
    Univ Helsinki, Fac Vet Med, Helsinki, Finland.
    Katila, T
    University of Helsinki, Faculty of Veterinary Medicine.
    Protein Composition of Seminal Plasma in Fractionated Stallion Ejaculates2011In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 46, no 1, p. 79-84Article in journal (Refereed)
    Abstract [en]

    Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre-sperm fluid and the first sperm-rich jets (HIGH-1), the main sperm-rich portion (HIGH-2), the jets with low sperm concentrations (LOW), and a combined whole-ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5 degrees C, 24 h) with spermatozoa from HIGH-2 and skim milk extender. Sperm motility was evaluated after storage in extender mixed with the stallion's own SP or SP from one of the other stallions (sperm from a normal stallion stored in SP from a subfertile stallion and vice versa). Protein composition was analysed using reverse-phase liquid chromatography (RP-HPLC), N-terminal sequencing and mass spectrometry. The area-under-the-curve (AUC) was used for quantitative comparison of proteins within fractions. Trial II: semen samples were collected from seven stallions. Fractions with the highest (HIGH) and lowest (LOW) sperm concentrations and WE samples were examined using SDS-PAGE and densitometry. No significant differences emerged between fractions in the AUC-values of the Horse Seminal Protein-1 (HSP-1) and HSP-2 peaks, or the peak containing HSP-3 and HSP-4 (HSP-3/4). Levels of HSP-1, HSP-2 and HSP-3/4 were not significantly correlated with total sperm motility, progressive sperm motility or average path velocity after storage. Significant differences between ejaculate fractions in the amount of different protein groups present in SP were not found in Trial I; but in Trial II, the proteins in the 60-70 kDa range were more abundant in LOW than in HIGH and WE, indicating that this band contained proteins derived mainly from the seminal vesicles, which produce most of the SP in LOW.

  • 12.
    Macias Garcia, B.
    et al.
    University of Extremadura, Spain .
    Gonzalez Fernandez, L.
    University of Extremadura, Spain .
    Morrell, J.M.
    SLU, Sweden .
    Ortega Ferrusola, C.
    University of Extremadura, Spain .
    Tapia, J.A.
    University of Extremadura, Spain .
    Rodriguez Martinez, Heriberto
    SLU, Sweden .
    Pena, F.J.
    University of Extremadura, Spain .
    Single-Layer Centrifugation Through Colloid Positively Modifies the Sperm Subpopulation Structure of Frozen-Thawed Stallion Spermatozoa2009In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 44, no 3, p. 523-526Article in journal (Refereed)
    Abstract [en]

    The present study attempted to select the subpopulation of stallion spermatozoa that best survived a conventional freezing and thawing procedure, using centrifugation of post-thawed semen samples through a single layer of a glycidoxypropyltrimethoxysilane-coated silica colloid with a species-specific formulation (Androcoll-E (TM)). After freezing and thawing, four sperm subpopulations were identified, listed as FT1 to FT4. While subpopulations FT1 and FT2 were characterized by low sperm velocity, high velocities characterized the ones called FT3 and FT4. The single-layer centrifugation (SLC)-handled sperm sample was enriched in subpopulation FT3, reaching a proportion of 82.6% of the present spermatozoa, in contrast with the non-filtered control post-thawed semen, where this sperm subpopulation only accounted for 16.3% of the total. It is concluded that in the equine industry, the SLC is a practical, easy-to-perform approach to improve the quality of equine frozen-thawed semen samples.

  • 13.
    Macias Garcia, B.
    et al.
    Veterinary Teaching Hospital, Laboratory of Spermatology, University of Extremadura, Cáceres, Spain.
    Gonzalez Fernandez, L.
    Department of Physiology, University of Extremadura, Avd de la Universidad s/n, Cáceres, Spain.
    Ortega Ferrusola, C.
    Veterinary Teaching Hospital, Laboratory of Spermatology, University of Extremadura, Cáceres, Spain.
    Salazar-Sandoval, C.
    Veterinary Teaching Hospital, Laboratory of Spermatology, University of Extremadura, Cáceres, Spain.
    Morillo Rodriguez, A.
    Veterinary Teaching Hospital, Laboratory of Spermatology, University of Extremadura, Cáceres, Spain.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Developmental Biology. Linköping University, Faculty of Health Sciences.
    Tapia, J.A.
    Department of Physiology, University of Extremadura, Avd de la Universidad s/n, Cáceres, Spain.
    Morcuende, D.
    Department of Animal Production and Food Science Faculty of Veterinary Medicine, University of Extremadura, Avd de la Universidad s/n, Cáceres, Spain.
    Pena, F.J.
    Veterinary Teaching Hospital, Laboratory of Spermatology, University of Extremadura, Cáceres, Spain.
    Membrane Lipids of the Stallion Spermatozoon in Relation to Sperm Quality and Susceptibility to Lipid Peroxidation2011In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 46, no 1, p. 141-148Article in journal (Refereed)
    Abstract [en]

    Contents: Lipids were extracted from ejaculated spermatozoa from seven individual stallions to distinguish neutral lipids (NL) and polar lipids (PL) and determine their variation among stallions and their relationship with sperm quality and sperm susceptibility to lipid peroxidation. The isolated fatty acids were correlated with sperm quality (membrane integrity, mitochondrial membrane potential (ΔΨm) and expression of active caspases) and the sensitivity of the sperm plasma membrane to LPO. The miristic (C14: 0), palmitic (C16: 0), stearic (C18: 0) and oleic (C18: 1n9) acids were predominant among the NLs. Within the phospholipid fraction, the docosapentanoic acid (C22: 5n6) was dominant, albeit varying among stallions. Surprisingly, the percentage of polyunsaturated fatty acids was positively correlated with sperm quality and a low propensity for LPO, probably because these particular fatty acids provide a higher fluidity of the plasma membrane. The stallion showing the poorest sperm membrane integrity plus a high level of LPO in his ejaculate had a lower percentage (p less than 0.05) of this fatty acid in his sperm plasma membranes. © 2010 Blackwell Verlag GmbH.

  • 14.
    Martinez, EA
    et al.
    University of Murcia, Spain; .
    Vazquez, JM
    University of Murcia, Spain; .
    Parrilla, I
    University of Murcia, Spain; .
    Cuello, C
    University of Murcia, Spain; .
    Gil, MA
    University of Murcia, Spain; .
    Rodriguez-Martinez, Heriberto
    SLU, Sweden.
    Roca, J
    University of Murcia, Spain; .
    Vazquez, JL
    University Miguel Hernandez, Elche, Spain.
    Incidence of unilateral fertilizations after low dose deep intrauterine insemination in spontaneously ovulating sows under field conditions2006In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 41, no 1, p. 41-47Article in journal (Refereed)
    Abstract [en]

    A new procedure for non-surgical deep intrauterine insemination (DUI) in unrestrained sows hormonally induced to ovulate, has been reported. In comparison with standard artificial insemination (AI), with this procedure, the sperm numbers inseminated can be reduced 20-fold without reducing the reproductive performance of these hormonally treated sows. The present study evaluated, using two experiments, the reproductive performance applying 20-fold different sperm numbers per AI dose using DUI or standard AI in spontaneously ovulating sows, under field conditions. In experiment 1, AI was applied to crossbred sows at 12, 24 and 36 h after onset of spontaneous oestrus using one of the following two regimes: (i) DUI (treatment) with 0.15 x 10(9) fresh boar spermatozoa in 5 ml of Beltsville thawing solution (BTS) extender (n = 95), and (ii) standard cervical AI (control) with 2.85 x 10(9) fresh spermatozoa in 95 ml of BTS extender (n = 95). The farrowing rates of the two groups of sows were statistically similar (NS). However, a decrease (p less than 0.002) in litter size and the total number of pigs born alive was observed in sows inseminated with the DUI procedure. In experiment 2, 42 post-weaned oestrus sows were inseminated following the same design described for experiment 1 during spontaneous oestrus. On day 6 after onset of oestrus, the proximal segment of the uterine horns of the sows were flushed under surgery to retrieve eventual embryos and evaluate the success of fertilization per cornua (e.g. occurrence of effective uni- vs bilateral sperm transport rendering uni- or bilateral, complete or partial fertilization). Retrieved embryos were assessed for cleavage and number of accessory spermatozoa. Although identical overall pregnancy rates were achieved in both insemination groups, the percentage of sows with partial bilateral fertilization and unilateral fertilization was markedly higher (p less than 0.05) in the DUI group (35%) compared with the control (standard AI) group (5%), with a consequent lower (p less than 0.001) percentage of viable early embryos after DUI. The number of accessory spermatozoa in the zona pellucida of the embryos was highly variable, but higher (p less than 0.001) in control animals than in DUI-AI. No accessory spermatozoa were found in oocytes retrieved from sows depicting unilateral fertilization. In conclusion, DUI in spontaneously ovulating sows with 0.15 x 10(9) spermatozoa renders similar farrowing rates but a lower litter size compared with use of standard AI with a 20-fold higher sperm dose. The lower litter size ought to be related to a decreased distribution of spermatozoa after DUI leading to a higher incidence of partial bilateral and unilateral fertilization.

  • 15.
    Martínez-Pastor, Felipe
    et al.
    ITRA-ULE, INDEGSAL, Departments of Molecular Biology (Cell Biology), University of León, León, Spain.
    Mata-Campuzano, Maria
    ITRA-ULE, INDEGSAL, Departments of Molecular Biology (Cell Biology), University of León, León, Spain.
    Alvarez-Rodriguez, Manuel
    ITRA-ULE, INDEGSAL, Animal Reproduction and Obstetrics, University of León, León, Spain.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, Animal Reproduction and Obstetrics, University of León, León, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, Animal Reproduction and Obstetrics, University of León, León, Spain.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, Departments of Molecular Biology (Cell Biology), University of León, León, Spain.
    Probes and techniques for sperm evaluation by flow cytometry2010In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 45 Suppl 2, p. 67-78Article in journal (Refereed)
    Abstract [en]

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

  • 16.
    Mata-Campuzano, María
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Spain.
    Alvarez-Rodríguez, Manuel
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, Spain.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, Spain.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Spain.
    Garde, Julian
    Biology of Reproduction Group, National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Martínez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Spain.
    Effect of several antioxidants on thawed ram spermatozoa submitted to 37°C up to four hours.2012In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 47, no 6, p. 907-914Article in journal (Refereed)
    Abstract [en]

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

  • 17.
    Mekasha, Y.
    et al.
    Swedish University of Agriculture Science, Sweden Alemaya University, Ethiopia .
    Tegegne, A.
    ILRI, Ethiopia .
    Abera, A.
    Department Livestock Resources Dev, Ethiopia .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden .
    Body size and testicular traits of tropically-adapted bucks raised under extensive husbandry in Ethiopia2008In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 43, no 2, p. 196-206Article in journal (Refereed)
    Abstract [en]

    Five tropically adapted buck breeds extensively managed in Ethiopia were studied to determine possible effects of breed and age on body size and testicular traits. In all, 177 bucks were selected following stratified random sampling, and evaluated in three age groups: less than 14 months (young), 14-19.5 months (intermediate) and 19.6-24 months (old). The breeds studied were Arsi-Bale (AB; N = 35), Central Highlands (CH; N = 33), Afar (N = 35), Boran (N = 36) and Woito-Guji (WG; N = 38). In all the three age groups, Boran and CH bucks were the heaviest (p less than 0.05), Afar were the lightest, and AB and WG were in between. The highest body weight (BW) was achieved in the intermediate age group for Afar, but in the oldest age group for the other breeds. In the youngest age group, scrotal circumference (SC) was the widest (p less than 0.05) for Boran and CH and the narrowest for Afar, AB and WG, while in the intermediate and the oldest age groups, Boran showed the widest SC. Boran, WG and CH had higher (p less than 0.05) testicular weight (TW) than Afar and AB in the youngest age group. Boran retained the highest (p less than 0.05) TW in the intermediate and the oldest age groups, while in the oldest age group WG and AB medium TW and Afar had the lowest TW. However, Afar had the highest TW expressed as percentage of BW. SC was well correlated with TW (p less than 0.001; r = 0.74) and BW (p less than 0.001; r = 0.61), indicating a linear, positive association between BW and TW (p less than 0.001; r = 0.51). In conclusion, body size and testicular traits of Ethiopian bucks under an extensive management system are influenced differently by breed and age group.

  • 18.
    Mekasha, Y.
    et al.
    Swedish University of Agriculture Science, Sweden Haramaya University, Ethiopia .
    Tegegne, A.
    Int Livestock Research Institute, Ethiopia .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden .
    Feed intake and sperm morphology in Ogaden bucks supplemented with either agro-industrial by-products or khat (Catha edulis) leftover2008In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 43, no 4, p. 437-444Article in journal (Refereed)
    Abstract [en]

    In this study, feed intake and sperm morphology were evaluated in Ogaden bucks supplemented with either agro-industrial by-products or khat leftover. Thirty-five bucks at about 1 year of age, and 15 +/- 1.5 kg initial body weight, were involved in a 12-week feeding programme that had four diet groups. The control (C) diet comprised grass hay alone, fed ad libitum; treatment 1 (T1) comprised grass hay ad libitum supplemented with a mix of agro-industrial by-products at 1% of body weight (BW); treatment 2 (T2) comprised grass hay ad libitum supplemented with khat leftovers at 1% of BW; and treatment 3 (T3) comprised khat leftover alone, fed ad libitum. Bucks in T1-T3 consumed the highest (p less than 0.001) dry matter (DM), crude protein (CP), organic matter (OM) and gross energy (GE) compared with control. Among the treatment groups, DM, OM and GE intakes were higher (p less than 0.05) in T3 and T2, while CP intake was highest (p less than 0.05) in T1. T1 and T3 improved (p less than 0.001) the percentage of morphologically-normal spermatozoa in comparison with C, with bucks in T3 being best. The proportion of total abnormal sperm head shapes and proximal cytoplasmic droplets was lowest (p less than 0.01) in T1 and T3 and highest in C. Although feeding with T1 improved feed intake and sperm morphology, feeding with T3 showed the highest response. Thus, we conclude that T3 and T1 could be utilized as feedstuff to improve sperm morphology in goats under the smallholder farming system in Ethiopia.

  • 19.
    Mohamad, K
    et al.
    Bogor Agriculture University.
    Olsson, M
    Swedish University of Agriculture Science.
    Andersson, G
    Swedish University of Agriculture Science.
    Purwantara, B
    Bogor Agriculture University.
    van Tol, H T A
    University of Utrecht.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Colenbrander, B
    University of Utrecht.
    Lenstra, J A
    University of Utrecht.
    The Origin of Indonesian Cattle and Conservation Genetics of the Bali Cattle Breed2012In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 47, no SI, p. 18-20Article in journal (Refereed)
    Abstract [en]

    Both Bos indicus (zebu) and Bos javanicus (banteng) contribute to the Indonesian indigenous livestock, which is supposedly of a mixed species origin, not by direct breeding but by secondary cross-breeding. Here, the analysis of mitochondrial, Y-chromosomal and microsatellite DNA showed banteng introgression of 1016% in Indonesian zebu breeds with East-Javanese Madura and Galekan cattle having higher levels of autosomal banteng introgression (2030%) and combine a zebu paternal lineage with a predominant (Madura) or even complete (Galekan) maternal banteng origin. Two Madura bulls carried taurine Y-chromosomal haplotypes, presumably of French Limousin origin. There was no evidence for zebu introgression in five populations of the Bali cattle, a domestic form of the banteng.

  • 20.
    Morrell, J.M.
    et al.
    Swedish University of Agriculture Science SLU, Sweden .
    Johannisson, A.
    Department Anat Physiol and Biochem, Sweden .
    Dalin, A-M
    Swedish University of Agriculture Science SLU, Sweden .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science SLU, Sweden .
    Morphology and Chromatin Integrity of Stallion Spermatozoa Prepared by Density Gradient and Single Layer Centrifugation Through Silica Colloids2009In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 44, no 3, p. 512-517Article in journal (Refereed)
    Abstract [en]

    The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically normal spermatozoa varied between stallions (p less than 0.001) and was increased by both methods of colloidal centrifugation (median value before centrifugation 67.5%; after SLC 78%; after DGC 77%; p less than 0.001). The incidence of certain abnormalities was reduced, e.g. proximal cytoplasmic droplets were reduced from 12.9% to 8.8% (p less than 0.001), and mid-piece defects from 5.3% to 1.4% (p less than 0.05). Similarly, sperm motility and chromatin integrity were significantly improved (p less than 0.001), with no difference between the two centrifugation methods. Centrifugation through colloids can enrich the proportions of stallion spermatozoa with normal morphology and normal chromatin structure in sperm preparations. The new method, SLC, was as effective as DGC in selecting motile stallion spermatozoa with normal morphology and intact chromatin. SLC, being simpler to use than DGC, would be appropriate for routine use by stud personnel to improve stallion sperm quality in insemination doses.

  • 21.
    Morrell, J.M.
    et al.
    Swedish University Agriculture Science.
    Mari, G.
    University of Bologna.
    Kutvolgyi, G.
    Aleback Stud Farm, Lidkoping.
    Meurling, S.
    Flyinge AB.
    Mislei, B.
    University of Bologna.
    Iacono, E.
    University of Bologna.
    Rodriguez-Martinez, Heriberto
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Genetics.
    Pregnancies Following Artificial Insemination with Spermatozoa from Problem Stallion Ejaculates Processed by Single Layer Centrifugation with Androcoll-E2011In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 46, no 4, p. 642-645Article in journal (Refereed)
    Abstract [en]

    Some stallions produce ejaculates of low quality and/or low fertility when used for artificial insemination (AI). The purpose of these five case studies was to use Single Layer Centrifugation (SLC) to select the best spermatozoa from problem ejaculates for subsequent use in AI. Sperm quality, in terms of motility, morphology and chromatin integrity, was improved in the SLC-selected samples compared to the corresponding uncentrifuged samples, with the exception of one stallion thought to have ampullary stasis. In this stallion, neither the incidence of spermatozoa with detached heads nor the proportion of damaged chromatin was decreased by SLC, in contrast to previous results. Pregnancies were obtained after using SLC-selected spermatozoa from the five stallions for AI, indicating that the spermatozoa were functional after SLC. Overall, the results suggest that SLC may be useful when preparing AI doses from some problem ejaculates.

  • 22.
    Morrell, J.M.
    et al.
    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.
    Andersson, M.
    University of Helsinki, Finland .
    Colloid Centrifugation Selects Normal Spermatozoa from Polymorphic Bull Ejaculates: A Case Study2014In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 49, no 2, p. 281-284Article in journal (Refereed)
    Abstract [en]

    Contents Semen from a Western Finncattle bull exhibiting a highly polymorphic spermiogram was processed by colloid centrifugation using Androcoll-B, a species-specific silane-coated silica colloid. In the first experiment, Single Layer Centrifugation (SLC) was used to identify which density colloids were needed to separate different cell populations. Colloids of the two chosen densities were then used in a density gradient resulting in two sperm subpopulations, one containing nearly all normally sized spermatozoa and the other enriched for the macrocephalic spermatozoa. Microcephalic spermatozoa did not appear in either of the selected subpopulations. Using a combination of SLC and DGC with this species-specific colloid, it was possible to separate the spermatozoa into different subpopulations, that is, a subpopulation containing nearly all normally sized spermatozoa, and another one enriched for the macrocephalic spermatozoa. Thus, colloid centrifugation could be used to select sufficient normal spermatozoa from a highly polymorphic ejaculate for AI, if desired.

  • 23.
    Munoz, P. Martin
    et al.
    University of Extremadura, Spain.
    Anel-Lopez, L.
    University of Leon, Spain.
    Ortiz-Rodriguez, J. M.
    University of Extremadura, Spain.
    Alvarez, M.
    University of Leon, Spain.
    de Paz, P.
    University of Leon, Spain.
    Balao da Silva, C.
    Super Agriculture School Elvas, Portugal.
    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.
    Gil, M. C.
    University of Extremadura, Spain.
    Anel, L.
    University of Leon, Spain.
    Pena, F. J.
    University of Extremadura, Spain.
    Ortega Ferrusola, C.
    University of Leon, Spain.
    Redox cycling induces spermptosis and necrosis in stallion spermatozoa while the hydroxyl radical (OH center dot) only induces spermptosis2018In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 53, no 1, p. 54-67Article in journal (Refereed)
    Abstract [en]

    Oxidative stress is a major factor explaining sperm dysfunction of spermatozoa surviving freezing and thawing and is also considered a major inducer of a special form of apoptosis, visible after thawing, in cryopreserved spermatozoa. To obtain further insights into the link between oxidative stress and the induction of apoptotic changes, stallion spermatozoa were induced to oxidative stress through redox cycling after exposure to 2-methyl-1,4-naphthoquinone (menadione), or hydroxyl radical formation after FeSO4 exposure. Either exposure induced significant increases (p amp;lt; 0.05) in two markers of lipid peroxidation: 8-iso-PGF(2) and 4-hydroxynonenal (4-HNE). While both treatments induced changes indicative of spermptosis (caspase-3 activation and decreased mitochondrial membrane potential) (p amp;lt; 0.01), menadione induced sperm necrosis and a dramatic reduction in motility and thiol content in stallion spermatozoa. Thus, we provided evidence that oxidative stress underlies spermptosis, and thiol content is a key factor for stallion sperm function.

  • 24.
    Ortega-Ferrusola, C.
    et al.
    University of Leon, Spain.
    Gil, M. C.
    University of Extremadura, 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.
    Anel, L.
    University of Leon, Spain.
    Pena, F. J.
    University of Extremadura, Spain.
    Martin-Munoz, P.
    University of Extremadura, Spain.
    Flow cytometry in Spermatology: A bright future ahead2017In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 52, no 6, p. 921-931Article, review/survey (Refereed)
    Abstract [en]

    Techniques such as mass spectrometry have led to unprecedented knowledge of the proteins that are present in the spermatozoa of humans and other mammals. However, in spite of their high-throughput and fractioning techniques, most of the techniques in use only offer average values for the entire sperm population. Yet, ejaculate is very heterogeneous, and average values may mask relevant biological information. The application of flow cytometry may overcome this disadvantage, allowing proteomic analysis at the single-cell level. Moreover, recent advances in cytometry, allowing multiple analyses within a single cell combined with powerful statistical tools, as an expanding subfield in spermatology, are described. The increased use of advanced flow cytometers in andrology laboratories will allow the rapid development of multiparametric, multicolour flow cytometry in andrology that will expand the clinical applications and research possibilities of flow cytometry-based proteomic approaches, especially in the subfields of clinical andrology and sperm biotechnology.

  • 25.
    Ortega-Ferrusola, C.
    et al.
    University of Extremadura, Spain .
    Gonzalez-Fernandez, L.
    University of Extremadura, Spain .
    Muriel, A.
    University of Extremadura, Spain .
    Macias-Garcia, B.
    University of Extremadura, Spain .
    Rodriguez-Martinez, Heriberto
    SLU, Sweden .
    A. Tapia, J.
    University of Extremadura, Spain .
    M. Alonso, J.
    University of Extremadura, Spain .
    Pena, F.J.
    University of Extremadura, Spain .
    Does the Microbial Flora in the Ejaculate Affect the Freezeability of Stallion Sperm?2009In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 44, no 3, p. 518-522Article in journal (Refereed)
    Abstract [en]

    In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze, three ejaculates from five stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The isolated microorganisms were: Staphylococcus spp. and Micrococcus spp. (in all the stallions), beta-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp. (in stallions 1, 3-5), Rhodococcus spp. (in stallion number 2), Pseudomonas spp. (in stallion number 1) and Klebsiella spp. (in stallions 1, 3 and 5). The presence and richness of Klebsiella and beta-haemolytic Streptococcus in the ejaculate were related to two sperm variables post-thaw, namely the proportion of dead spermatozoa (ethidium+ cells; r = 0.55, p less than 0.05) and the amplitude of lateral displacement of the sperm head (ALH, mu m; r = -0.56, p less than 0.05), respectively. The degree of growth of Corynebacterium spp. in the ejaculate was positively correlated with the percentage of spermatozoa showing high caspase activity post-thaw (r = 0.62, p less than 0.05). The presence and number of colonies of beta-haemolytic Streptococcus were negatively correlated (r = -0.55, p less than 0.05) with low sperm caspase activity. It is concluded that the microbial flora of the equine ejaculate may be responsible for some of the sublethal damage experimented by the spermatozoa during cryopreservation.

  • 26.
    Ortega-Ferrusola, C.
    et al.
    University of Extremadura, Spain .
    Macias Garcia, B.
    University of Extremadura, Spain .
    Suarez Rama, V.
    University of Extremadura, Spain .
    M. Gallardo-Bolanos, J.
    University of Extremadura, Spain .
    Gonzalez-Fernandez, L.
    University of Extremadura, Spain .
    Tapia, J.A.
    University of Extremadura, Spain .
    Rodriguez-Martinez, Heriberto
    SLU, Sweden .
    J. Pena, F.
    [Gonzalez-Fernandez, Sweden; .
    Identification of Sperm Subpopulations in Stallion Ejaculates: Changes after Cryopreservation and Comparison with Traditional Statistics2009In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 44, no 3, p. 419-423Article in journal (Refereed)
    Abstract [en]

    In an attempt to improve the information obtained after computer-assisted sperm analysis (CASA), data from five stallions (three ejaculates from each) were analysed before (fresh, extended semen) and after cryopreservation using traditional statistics as well as a cluster analysis. The data matrix consisted of 13 987 observations of individual spermatozoa for fresh, extended semen, and 8305 for frozen-thawed samples. As expected, freezing and thawing resulted in a marked decrease of CASA-derived variables of sperm kinematics. All sperm velocities were significantly lower in frozen-thawed samples than in samples before cooling. Using sperm velocities, six sperm subpopulations were identified in fresh semen (S1-S6). As such, subpopulations S1 and S2 were characterized by low sperm velocities, subpopulations S3 and S4 corresponded to spermatozoa depicting medium speed values, and finally, subpopulations S5 and S6 were those depicting the highest velocities. After freezing and thawing, four sperm subpopulations were identified, listed as nr FT1 to FT4. While subpopulations FT1-FT3 were characterized by low sperm velocities, and thus corresponded speed-wise to those listed as S1-S4 for fresh, extended semen, the one called number FT4 in frozen semen was characterized by high velocities, of the same range as that of the subpopulations S5 and S6 for fresh spermatozoa. The sperm subpopulation structure varied among stallions, but the cluster analysis hereby assayed was able to provide valuable information about the freezability of the samples that the customary statistics did not reveal.

  • 27.
    Pena, F.J.
    et al.
    University of Extremadura, Spain .
    Rodriguez Martinez, Heriberto
    Swedish University of Agriculture Science SLU, Sweden .
    Tapia, J.A.
    University of Extremadura, Spain .
    Orteag Ferrusola, C.
    University of Extremadura, Spain .
    Gonzalez Fernandez, L.
    University of Extremadura, Spain .
    Macias Garcia, B.
    University of Extremadura, Spain .
    Mitochondria in Mammalian Sperm Physiology and Pathology: A Review2009In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 44, no 2, p. 345-349Article, review/survey (Refereed)
    Abstract [en]

    While, for a long time, the role of mitochondria in sperm physiology and pathology has been largely ignored, recent research points out the mitochondria as a major organelle with key roles in sperm function both under physiological and biotechnological conditions. This paper briefly reviews these novel findings regarding the role of mitochondria in sperm, paying special attention to the most practical, readily applicable, aspects of the topic such as their role as a major source of the sublethal damage that sperm experiments after cryopreservation.

  • 28.
    Purwantara, B
    et al.
    Bogor Agriculture University of IPB.
    Noor, R R
    Bogor Agriculture University.
    Andersson, G
    Swedish University of Agriculture Science.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Banteng and Bali Cattle in Indonesia: Status and Forecasts2012In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 47, no SI, p. 2-6Article in journal (Refereed)
    Abstract [en]

    Bali cattle still represents 27% of the total cattle population in Indonesia, and it is considered the pillar breed for small farmers. Moreover, it is a breed of evolutionary importance regarding its direct ancestry from Banteng. However, there is a need for the establishment of a rational system for the evaluation of breeding soundness for indigenous Bali bulls to be used as sires for artificial insemination breeding programmes. Moreover, there is a need for cryobanking of well-identified genetic resources pertaining their use in evolutionary research and application as essential germplasm in breeding programmes.

  • 29.
    Roca, J.
    et al.
    University of Murcia, Spain.
    Broekhuijse, M. L. W. J.
    Topigs Norsvin Research Centre BV, Netherlands.
    Parrilla, I.
    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.
    Martinez, E. A.
    University of Murcia, Spain.
    Bolarin, A.
    Topigs Norsvin, Spain.
    Boar Differences In Artificial Insemination Outcomes: Can They Be Minimized?2015In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 50, p. 48-55Article, review/survey (Refereed)
    Abstract [en]

    In Western countries, where pig breeding and production are intensive, there is a documented variability in fertility between farms with boar-related parameters only accounting to 6% of this total variation of invivo fertility. Such low boar effect could be a result of the rigorous control of sires and ejaculates yielding AI-doses exerted by the highly specialized AI-centres that monopolize the market. However, some subfertile boars pass through these rigorous controls and consequently reach the AI-programmes. Here, we discuss why testing young boars for chromosomal defects, sperm nuclear chromatin integrity and invitro fertilizing ability can be discriminative and economically sound for removing these less fertile boars. Alongside, we discuss why boars differ in the ability of their sperm to tolerate cryopreservation or sex sorting.

  • 30.
    Roca, J
    et al.
    University of Murcia.
    Parrilla, I
    University of Murcia.
    Rodriguez-Martinez, Heriberto
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Genetics.
    Gil, M A
    University of Murcia.
    Cuello, C
    University of Murcia.
    Vazquez, J M
    University of Murcia.
    Martinez, E A
    University of Murcia.
    Approaches Towards Efficient Use of Boar Semen in the Pig Industry2011In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 46, no SI, p. 79-83Article in journal (Refereed)
    Abstract [en]

    The current cervical artificial insemination (CAI) procedure, involving deposition of excessive sperm numbers, is uneconomical for pig industry. The most obvious alternative requires uterine deposition in combination with fixed-time AI, which would reduce the number of sperm required per pregnant sow, thus allowing the best use of valuable boars and, ultimately, the commercial integration of frozen-thawed and sexed sperm. This review depicts possible best ways to implement an efficient use of liquid-stored, frozen-thawed and sexed sperm by the pig industry.

  • 31.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Assisted Reproductive Techniques for Cattle Breeding in Developing Countries: A Critical Appraisal of Their Value and Limitations2012In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 47, no SI, p. 21-26Article in journal (Refereed)
    Abstract [en]

    Commercialization of animal biotechnologies, including those related to reproduction [also known as assisted reproductive techniques (ARTS)], is an increasing reality in developing countries, following the enormous flow of information around us and the increasing global commercial interests in areas where cattle production has its major assets. The present review discusses the achievements of various biotechnological tools for reproduction in cattle including semen handling for artificial insemination (AI), superovulation and embryo transfer (MOET), in vitro handling of oocytes and production of embryos, reproductive cloning and emerging technologies (sex selection, gene targeting and nuclear transfer for livestock transgenesis, genomics for marker-assisted selection, etc.). The application of these technologies for cattle breeding is critically discussed in relation to their impact in the improvement of the efficiency of dairy and beef production in developed and particularly in developing countries, which ultimately rule the possibilities of a competitive and sound production of food for human consumption. Despite the remarkable progress made and the punctual importance of some of the above-mentioned technologies, AI remains the most important assisted reproductive technology (ART) in developing countries. Any attempt to gain widespread of any other ART under the predominant economical conditions in developing countries ought to match the simplicity and the success of AI as a breeding tool.

  • 32.
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Can we increase the estimative value of semen assessment?2006In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 41Article in journal (Refereed)
    Abstract [en]

    Estimating the fertility of a semen sample or of the male from where it has been collected by simple assessment of in vitro sperm characteristics is still difficult, owing to the variable correlations that laboratory results achieve with in vivo fertility. A major reason behind these variations is the fact that the ejaculate and the artificial insemination (AI)-doses it generates are composed of a diverse sperm population. Such heterogeneity is reflected both in differences of intactness of attributes needed for fertilization, such as motility or morphology, but also in the relative ability of spermatozoa to prevail fertile over time, handling and exposure to different stimuli, all of which account for innate variations in fertilizing ability among doses, ejaculates and sires. However, methods are already available to select sub-populations of intact spermatozoa which can be tested for their degree of competence for fertilization and whose estimative power is promising, allowing the elimination of cases of sub-fertility, particularly in bovine. Examples of these methods are the separation of viable spermatozoa by swim-up or discontinuous gradient centrifugation, followed by testing the ability of the selected spermatozoa to dose-response/time sustain capacitation and acrosome reaction induction. Finding how large a sperm population with non-compensable attributes for fertilization and ability to display and sustain stimuli is, perhaps by a quick screening of membrane integrity and stability by multi-parametric methods, would allow, provided the particular male produces this sub-population in a repeatable manner, for a better estimation of fertility.

  • 33.
    Thys, M.
    et al.
    University of Ghent, Belgium .
    Vandaele, L.
    University of Ghent, Belgium .
    Morrell, J.M.
    Swedish University of Agriculture Science, Sweden .
    Mestach, J.
    University of Ghent, Belgium .
    Van Soom, A.
    University of Ghent, Belgium .
    Hoogewijs, M.
    University of Ghent, Belgium .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden .
    In vitro Fertilizing Capacity of Frozen-thawed Bull Spermatozoa Selected by Single-layer (Glycidoxypropyltrimethoxysilane) Silane-coated Silica Colloidal Centrifugation2009In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 44, no 3, p. 390-394Article in journal (Refereed)
    Abstract [en]

    Barriers to the use of density gradient centrifugation for preparing animal spermatozoa for artificial insemination (AI) include the scarcity of animal-specific formulations and the daunting prospect of processing large volumes of ejaculate in small aliquots (1.5 ml extended semen). Recently, new colloid formulations have been tested in vitro in a modified procedure, centrifugation on a single layer of colloid. The present study investigated the fertilizing ability during in vitro fertilization (IVF) of frozen-thawed bovine spermatozoa following centrifugation through a single layer of glycerolpropylsilane (GS)-coated silica colloid with a species-specific formulation (patent applied for; treatment, T). Controls (C) included centrifugation through gradients of either the same colloid (C1) or Percoll (TM) (C2). Sperm recovery surpassed 50% for both C1-C2 and T (n.s.). Mean values of various parameters of computerized analysis of sperm motility did not differ between T and C1 (n.s.), and only the proportions of path straightness and linearity were lower in T vs C2 (p less than 0.05). In T, the mean (+/- SD) percentages of fertilization rate, blastocyst development rate and the total number of blastomeres were 58.1 +/- 23.3%, 24.5 +/- 14.3% and 94.6 +/- 23.4%, respectively. The proportions did not differ significantly from controls (C1/C2). Therefore, centrifugation through a single layer of colloid offers an alternative method to density gradient centrifugation for selection of viable, potentially fertile frozen-thawed bull spermatozoa. This single-layer technique is gentle, versatile and convenient because it facilitates scaling-up the process of sperm preparation to allow larger numbers of spermatozoa (for instance, whole ejaculates) to be processed for AI.

  • 34.
    Vicente-Carrillo, Alejandro
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Ekwall, H
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Álvarez-Rodríguez, Manuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa2016In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 51, no 5, p. 665-679Article in journal (Refereed)
    Abstract [en]

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

  • 35.
    Wright, Dominic
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Rubin, C
    Uppsala University.
    Schutz, K
    AgResearch Ltd.
    Kerje, S
    Uppsala University.
    Kindmark, A
    University of Uppsala Hospital.
    Brandstrom, H
    University of Uppsala Hospital.
    Andersson, L
    Uppsala University.
    Pizzari, T
    University of Oxford.
    Jensen, Per
    Linköping University, Department of Physics, Chemistry and Biology, Zoology. Linköping University, The Institute of Technology.
    Onset of Sexual Maturity in Female Chickens is Genetically Linked to Loci Associated with Fecundity and a Sexual Ornament2012In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 47, no SI, p. 31-36Article in journal (Refereed)
    Abstract [en]

    Onset of sexual maturation is a trait of extreme importance both evolutionarily and economically. Unsurprisingly therefore, domestication has acted to reduce the time to sexual maturation in a variety of animals, including the chicken. In comparison with wild progenitor chickens [the Red Junglefowl (RJF)], domestic layer hens attain maturity approximately 20% earlier. In addition, domestic layers also possess larger combs (a sexual ornament), produce more eggs and have denser bones. A large quantitative trait loci (QTL) analysis (n = 377) was performed using an F2 intercross between a White Leghorn layer breed and a RJF population, with onset of sexual maturity measured and mapped to three separate loci. This cross has already been analysed for comb mass, egg production and bone allocation. Onset of sexual maturity significantly correlated with comb mass, whilst the genetic architecture for sexual maturity and comb mass overlapped at all three loci. For two of these loci, the QTL for sexual maturity and comb mass were statistically indistinguishable from pleiotropy, suggesting that the alleles that increase comb mass also decrease onset of sexual maturity.

  • 36.
    Yindee, M.
    et al.
    Chulalongkorn University, Bangkok.
    Techakumphu, M.
    Chulalongkorn University, Bangkok.
    Lohachit, C.
    Chulalongkorn University, Bangkok.
    Sirivaidyapong, S.
    Chulalongkorn University, Bangkok.
    Na-Chiangmai, A.
    Buffalo Research and Development Group, Bangkok.
    Rodriguez-Martinez, Heriberto
    Division of Reproduction, Swedish University of Agricultural Sciences (SLU), Uppsala.
    van der Weyden, G. C.
    Utrecht University, the Netherlands.
    Colenbrander, B.
    Utrecht University, the Netherlands.
    Follicular Dynamics and Oestrous Detection in Thai Postpartum Swamp Buffaloes (Bubalus bubalis)2011In: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 46, no 1, p. e91-e96Article in journal (Refereed)
    Abstract [en]

    This study characterized follicular activity and oestrous behaviour from 5 to 9 days post-calving up to the 4th ovulation postpartum (pp) in 16 multiparous (range 2-7 parities) Thai swamp buffalo cows (Bubalus bubalis), aged 4-12 years and weighing from 432 to 676 kg. Ovarian follicular activity was examined by transrectal ultrasonography (TUS) every morning. Oestrous detection was performed twice daily by direct personal observation of behaviour and for presence of clear cervical mucus discharge and indirectly by video camera recording during 21 h/day. A follicular wave-like pattern was present before the 1st ovulation leading to short oestrous cycles. Growth rates and maximum diameters of the ovulatory follicles did not differ between the 1st and 4th ovulations. However, growth rate for non-ovulatory dominant follicles (DF) before the 1st ovulation was lower than for the ovulatory follicle (p < 0.05). In addition, the diameter of all ovulatory follicles (14.3 +/- 0.46 mm, n = 39) was significantly larger (p < 0.01) than those of the preceding last but one non-ovulatory DF (10.8 +/- 0.20 mm, n = 5), but similar to the last preceding non-ovulatory DF diameter (12.92 +/- 0.96 mm, n = 14). Short oestrous cycles were most common between the 1st and 2nd ovulations (93.75%, 15/16 cows, 10.2 +/- 0.38 days) decreasing in prevalence thereafter (50%, 3/6 buffaloes, 12.0 +/- 1.53 days). Oestrous signs were relatively vague around the 1st ovulation pp to become more easily detectable thereafter. This study suggests that properly fed swamp buffaloes could be mated successfully within 2 months pp, at their 2nd spontaneous ovulation, provided oestrous detection is at least performed daily at 06:00-08:00 hour.

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