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  • 1.
    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.

  • 2.
    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.

  • 3.
    Alvarez-Rodriguez, Manuel
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Alvarez, M.
    University of Leon, Spain.
    Anel-Lopez, L.
    SaBio IREC CSIC UCLM JCCM, Spain.
    Lopez-Uruena, E.
    University of Leon, Spain.
    Manrique, P.
    University of Leon, Spain.
    Borragan, S.
    Cabarceno Pk, Spain.
    Morrell, J. M.
    Swedish University of Agriculture Science SLU, Sweden.
    de Paz, P.
    University of Leon, Spain.
    Anel, L.
    University of Leon, Spain.
    Effect of colloid (Androcoll-Bear, Percoll, and PureSperm) selection on the freezability of brown bear (Ursus arctos) sperm2016In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 85, no 6, p. 1097-1105Article in journal (Refereed)
    Abstract [en]

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

  • 4.
    Alvarez-Rodriguez, Manuel
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Gomes-Alves, S
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Borragan, Santiago
    Cabárceno Park, Cantabria, Spain.
    Martinez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Quality of frozen-thawed semen in brown bear is not affected by timing of glycerol addition.2011In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 75, no 8, p. 1561-1565Article in journal (Refereed)
    Abstract [en]

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

  • 5.
    Alvarez-Rodríguez, Manuel
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain .
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Borragan, Santiago
    Cabárceno Park, Cantabria, Spain.
    Martinez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain .
    Holt, W V
    The Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Sheffield, UK.
    Fazeli, Alireza
    The Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Sheffield, UK.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain .
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    The addition of heat shock protein HSPA8 to cryoprotective media improves the survival of brown bear (Ursus arctos) spermatozoa during chilling and after cryopreservation.2013In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 79, no 3, p. 541-550Article in journal (Refereed)
    Abstract [en]

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

  • 6.
    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.

  • 7.
    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.

  • 8.
    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.

  • 9.
    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.

  • 10.
    de Paz, Paulino
    et al.
    Department of Molecular Biology, University of León, León, Spain.
    Mata-Campuzano, María
    Department of Molecular Biology, University of León, León, Spain.
    Tizado, Emilio Jorge
    Department of Biodiversity and Environmental Management, University of León, León, Spain.
    Alvarez, Mercedes
    Department of Veterinary Medicine, Anatomy and Surgery, University of León, León, Spain.
    Alvarez-Rodríguez, Manuel
    Department of Veterinary Medicine, Anatomy and Surgery, University of León, León, Spain.
    Herraez, Paz
    Department of Molecular Biology, University of León, León, Spain.
    Anel, Luis
    Department of Veterinary Medicine, Anatomy and Surgery, University of León, León, Spain.
    The relationship between ram sperm head morphometry and fertility depends on the procedures of acquisition and analysis used.2011In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 76, no 7, p. 1313-1325Article in journal (Refereed)
    Abstract [en]

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

  • 11.
    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.

  • 12.
    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.

  • 13.
    Ekwall, Hans
    et al.
    Swedish University of Agriculture Science, Sweden.
    Hernandez, Marta
    Swedish University of Agriculture Science, Sweden.
    Saravia, Fernando
    Swedish University of Agriculture Science, Sweden.
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden.
    Cryo-scanning electron microscopy (Cryo-SEM) of boar semen frozen in medium-straws and MiniFlatPacks2007In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 67, no 9, p. 1463-1472Article in journal (Refereed)
    Abstract [en]

    In this study we demonstrate, in the frozen state, the architecture of frozen boar spermatozoa collected from the sperm-rich fraction of ejaculates (n = 13) from four fertile boars packed and split-frozen in medium-straws (MS) and MiniFlatPacks (MFP), cross-sectioned in the frozen state and evaluated by image analysis on images obtained by use of cryo-scanning electron microscopy (Cryo-SEM). The tested hypothesis was that the degree of in situ dehydration and levels of homogeneity of boar semen either frozen in MSs or MFPs packages differ between them, with MFPs allowing for a more uniform dehydration of the spermatozoa and a higher cryosurvival, monitored by computer assisted sperm analysis (CASA) as proportion of linearly motile spermatozoa, compared to semen packaged and processed in MSs. The organization and relative surface of biological material (veins; e.g., frozen extender, bound water, solutes and spermatozoa) as well as free water (lakes) was measured as the degree of dehydration of the samples. The apparent organization of lakes and veins differed between packages, with the MFPs depicting larger lakes than the MSs. The sizes of the lakes in the latter appeared, moreover, highly asymmetrical depending on their position of the section. The relative surface of these lakes per section, respectively veins differed between packages (P less than 0.05), indicating a larger amount of free-water (lakes; 81.73 +/- 2.07% vs. 77.91 +/- 1.57%) in the MFPs and, consequently, thinner veins than in MSs. In conclusion, MFPs seem to allow for a more homogenous dehydration of the spermatozoa/frozen extender compared to MSs, which might account for their somewhat better sperm quality post-thaw. (C) 2007 Elsevier Inc. All rights reserved.

  • 14.
    Gil, MA
    et al.
    University of Murcia, Sweden; .
    Roca, J
    University of Murcia, Sweden; .
    Cremades, T
    University of Murcia, Sweden; .
    Hernandez, M
    University of Murcia, Sweden; .
    Vazquez, JM
    University of Murcia, Sweden; .
    Rodriguez-Martinez, Heriberto
    University of Murcia .
    Martinez, EA
    University of Murcia, Sweden; .
    Does multivariate analysis of post-thaw sperm characteristics accurately estimate in vitro fertility of boar individual ejaculates?2005In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 64, no 2, p. 305-316Article in journal (Refereed)
    Abstract [en]

    The objective of this study was to determine if a multivariate pattern analysis of frozen-thawed sperm characteristics of boar semen of unknown fertility, thus identifying groups of ejaculates as "good" or "bad" freezers, would estimate their fertilizing potential in an in vitro embryo production (IVP) system. Frozen-thawed spermatozoa from a single ejaculate collected from 46 boars were evaluated for sperm motility and kinematic patterns, for sperm viability and for early changes in sperm membrane stability. All data generated were used for a multivariate pattern analysis (PATN; CSIRO, Canberra, Australia) which objectively classified all ejaculates within a data set in to one of two groups, categorised as "good" (n = 25) or "bad" (n = 21) according with their freezability. In vitro matured oocytes were exposed to 2000 or 4000 frozen-thawed spermatozoa per oocyte for 6 h and then cultured in embryo culture medium for either 6 h (assurance of fertilization) or 7 days (to collect data on embryo development). Rates of sperm oocyte penetration and of embryo development significantly (p less than 0.05) increased in a sperm:oocyte ratio-dependent manner. A similar pattern was observed when sperm characteristics were grouped. Indeed, ejaculates classified as "good" showed significantly (p less than 0.05) higher rates of oocyte penetration, cleavage and of blastocyst formation than those classified as "bad". However, variation was still present among individuals (ejaculates, boars) in their ability to produce blastocysts in vitro. It is therefore concluded that despite the presence of a relationship for ejaculates with good semen quality post-thaw (thus grouped as "good") to higher IVP-results, the presence of individual variation does not allow for an accurate estimation of in vitro fertility based solely on the frozen-thawed semen quality parameters of a single ejaculate from a given boar. (c) 2004 Elsevier Inc. All rights reserved.

  • 15.
    Gonzalez-Arto, Marta
    et al.
    University of Zaragoza, Spain.
    Vicente Carrillo, Alejandro
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Martinez-Pastor, Felipe
    University of Leon, Spain.
    Fernandez-Alegre, Estela
    University of Leon, Spain.
    Roca, Jordi
    University of Murcia, Spain.
    Miro, Jordi
    University of Barcelona, Spain.
    Rigau, Teresa
    University of Barcelona, Spain.
    Rodriguez-Gil, Joan E.
    University of Barcelona, Spain.
    Perez-Pe, Rosaura
    University of Zaragoza, Spain.
    Muino-Blanco, Teresa
    University of Zaragoza, Spain.
    Cebrian-Perez, Jose A.
    University of Zaragoza, Spain.
    Casao, Adriana
    University of Zaragoza, Spain.
    Melatonin receptors MT1 and MT2 are expressed in spermatozoa from several seasonal and nonseasonal breeder species2016In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 86, no 8, p. 1958-1968Article in journal (Refereed)
    Abstract [en]

    Melatonin is a ubiquitous and multipurpose molecule, and one of its roles is to regulate reproduction in some seasonal mammals. Our group has previously reported the variation in the melatonin levels in ram seminal plasma along the year and identified MT1 and MT2 receptors in ram spermatozoa. The objective of this study was to elucidate whether the presence of melatonin receptors (MT1 and MT2) in the sperm plasma membrane, and melatonin in the seminal plasma is related to seasonal breeding. For this purpose, the presence of melatonin receptors and tseasonal breeder (epididymal spermatozoa); bull as a conventional nonseasonhe levels of melatonin in seminal plasma have been examined in several species: donkey and stallion as long-day breeders; red deer as a wild, short-day, highly al breeder; boar as a seasonal breeder, under management techniques; and dog as possible a seasonal breeder not regulated by melatonin. We have detected measurable levels of melatonin in the seminal plasma of all ejaculated semen samples (from donkey, stallion, boar, bull, and dog). Also, and for the first time, we have demonstrated the presence of MT1 and MT2 melatonin receptors in the spermatozoa of all these species, regardless their type of reproduction or sperm source (ejaculated or epididymal), using indirect immunofluorescence techniques and Western blotting. Our findings suggest that melatonin and melatonin receptors may be universally distributed in the reproductive system of mammals and that the sperm melatonin receptors cells may not be necessarily related with seasonal reproduction. Furthermore, the presence of MT1 at the cytoplasmic droplet in immature ejaculated stallion spermatozoa found in one sample and epididymal red deer spermatozoa suggests that melatonin may be involved in specific functions during spermatogenesis and sperm maturation, like protecting spermatozoa from oxidative damage, this activity being mediated through these receptors. (C) 2016 Elsevier Inc. All rights reserved.

  • 16.
    Hallap, T
    et al.
    Swedish University of Agricultural Sciences, Sweden.
    Nagy, S
    Swedish University of Agricultural Sciences, Sweden.
    Haard, M
    Svensk Avel, Sweden.
    Jaakma, U
    Estonian Agricultural University, Estonia.
    Johannisson, A
    Swedish University of Agricultural Sciences, Sweden.
    Rodriguez-Martinez, Heriberto
    Swedish University of Agricultural Sciences, Sweden.
    Sperm chromatin stability in frozen-thawed semen is maintained over age in al bulls2005In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 63, no 6, p. 1752-1763Article in journal (Refereed)
    Abstract [en]

    n/a

  • 17.
    Hallap, T
    et al.
    Swedish University of Agricultural Sciences, Sweden.
    Nagy, S
    Swedish University of Agricultural Sciences, Sweden.
    Jaakma, U
    Estonian Agricultural University, Tartu, Estonia; .
    Johannisson, A
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Mitochondrial activity of frozen-thawed spermatozoa assessed by Mitotracker Deep Red 6332005In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 63, no 8, p. 2311-2322Article in journal (Refereed)
    Abstract [en]

    The present study was conducted to find a more objective method of evaluating sperm quality than the current subjective motility evaluations by testing the applicability of a novel fluorescent probe, Mitotracker Deep Red 633 (M-22426), for measuring the mitochondrial activity of spermatozoa both after freezing/thawing (PT) and after swim-up selection (SU), using flow cytometry (FC). The results from FC were compared to those of conventional microscopic motility evaluations and of computer-assisted sperm analysis (CASA) as well as to the fertility obtained after AI in the field. Semen from six Estonian Holstein bulls, processed when the sires were aged 3, 5, and 7 years, was included in the experiment. Sperm motility (measured either subjectively or by means of CASA) was always significantly (p less than 0.01-0.001) higher in the spermatozoa recovered by SU, for any of the age groups considered, or even when combining the age groups. Motility (measured subjectively) after SU was significantly (p less than 0.05) higher when bulls reached 7 years of age, compared to earlier collection ages, but no differences were registered between ages for CASA-assessed motility, either after SU or immediately PT. The numbers of spermatozoa. with high red fluorescence also increased after SU: p less than 0.05 (for semen from bulls aged 3 years), p less than 0.001 (5 years), p less than 0.001 (7 years), and p less than 0.001 when all age groups were combined. The proportion of spermatozoa with high mitochondrial activity as determined by Mitotracker Deep Red 633 correlated with sperm motility (p less than 0.01) both PT and after SU, but not with the fertility results. In conclusion, MitoTracker Deep Red 633 seems to be a reliable marker for frozen-thawed bovine semen viability both PT and after SU. (c) 2004 Elsevier Inc. All rights reserved.

  • 18.
    Hallap, T
    et al.
    Swedish University of Agriculture Science, Sweden.
    Nagy, S
    Swedish University of Agriculture Science, Sweden.
    Jaakma, U
    Estonian Agricultural University, Tartu, Estonia.
    Johannisson, A
    Swedish University of Agriculture Science, Sweden.
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden.
    Usefulness of a triple fluorochrome combination Merocyanine 540/Yo-Pro 1/Hoechst 33342 in assessing membrane stability of viable frozen-thawed spermatozoa from Estonian Holstein Al bulls2006In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 65, no 6, p. 1122-1136Article in journal (Refereed)
    Abstract [en]

    In a situation where technology allows for the simultaneous measurement of numerous parameters of a single sperm cell, it becomes crucial to choose those parameters which may be useful in estimating in vivo fertility. Sperm membrane destabilization is believed to occur during chilling of semen, although its effect on the post-thaw (PT) fertility of the spermatozoa has not yet been fully assessed. For this reason, we tested a new combination of fluorophores, Merocyanine 540 (M540)/Yo-Pro 1/Hoechst 33342 (H33342), to detect sperm plasma membrane destabilization in bull spermatozoa conventionally processed for artificial insemination (AI). The samples were tested by flow cytometry (FC), both immediately PT and following an in vitro swimup (SU) technique, and results were thereafter compared with conventional sperm quality Measurements (of concentration, motility, morphology, and membrane integrity), including in vivo fertility. Semen samples from six Estonian Holstein (EHF) AI bulls, frozen when the sires were aged 3, 5, and 7 years, allowed us to test the effect of bull age on quality of semen. Plasma membrane stability correlated to motility, normal head morphology (p less than 0.05), and membrane integrity (p less than 0.01). Following the SU selection, motility, membrane integrity (p less than 0.001). and membrane instability increased (p less than 0.01), as did stability (p less than 0.05). Bull age did not influence the degree of sperm membrane destabilization, except for the 3-year sample versus 7-year sample, in which the proportion of spermatozoa with destabilized plasma lemma increased PT (p less than 0.05) without affecting membrane integrity. Only parameters measured after SU, such as proportion of total motile and linearly motile spermatozoa, assessed with computer-assisted sperm analysis (CASA) (p less than 0.01), average path velocity (VAP) (p less than 0.001), and percentage of spermatozoa with unstable plasma lemma (p less than 0.05), had a significant relationship with non-return rate (NRR). The results indicate that it triple combination of the fluorophores M540/-Pro 1/H33342 is suitable for monitoring the status of membrane stability in frozen-thawed (FT) bull spermatozoa. As well, a SU preselection method seems helpful in distinguishing relationships between sperm quality and fertility among bulls in it homogenous sire population.(c) 2005 Elsevier Inc. All rights reserved.

  • 19.
    Hoflack, G.
    et al.
    University of Ghent, Belgium .
    Van den Broeck, W.
    University of Ghent, Belgium .
    Maes, D.
    University of Ghent, Belgium .
    Van Damme, K.
    University of Ghent, Belgium .
    Opsomer, G.
    University of Ghent, Belgium .
    Duchateau, L.
    University of Ghent, Belgium .
    de Kruif, A.
    University of Ghent, Belgium .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden .
    Van Soom, A.
    University of Ghent, Belgium .
    Testicular dysfunction is responsible for low sperm quality in Belgian Blue bulls2008In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 69, no 3, p. 323-332Article in journal (Refereed)
    Abstract [en]

    In a previous study, we demonstrated that Belgian Blue (BB) bulls have a higher prevalence of small scrota and poorer semen morphology compared to the Holstein Friesian (HF) breed in Belgium. The present study tested the hypothesis that the underlying reason for these BB traits negative to fertility was testicular degeneration, associated with an eventual hypoplastic background. At culling, sperm quality and testicular histology of BB bulls were assessed and compared to that of HF bulls. Besides semen quality being generally poorer in the BB breed, significantly more degenerative changes were encountered in BB compared to HF testicles (degeneration index: 37.7 +/- 11.9 versus 29.3 +/- 9.9 for BB and HF bulls, respectively; P = 0.053). These results correlated to the percentage of normal spermatozoa (r = -0.44; P = 0.024) and primary abnormalities (r = 0.38; P = 0.053). Moreover, the relative amount of collagen fibers present in the testicular interstitial connective tissue was correlated with % normal sperm (r = -0.47; P = 0.017), primary defects (0.48; P = 0.014), and the degeneration results (r = 0.63; P less than 0.001). The % testicular interstitial collagen fibers differed significantly between breeds (10.6 +/- 4.0% for the BB versus 7.6 +/- 1.9% for the HF bulls; P = 0.016). This increased amount of connective tissue in BB testes might hypothetically be responsible for the poorer sperm quality. This condition can be defined as a mild form of testicular hypoplasia, and might, in turn, be responsible for the higher sensitivity to testicular degeneration, which is encountered in the BB breed. (C) 2008 Elsevier Inc. All rights reserved.

  • 20.
    Januskauskas, A
    et al.
    Lithuanian Veterinary Academy, Lithuania.
    Lukoseviciute, K
    Lithuanian Veterinary Academy, Lithuania.
    Nagy, S
    Research Institute for Animal Breeding and Nutrition, Herceghalom, Hungary.
    Johannisson, A
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Assessment of the efficacy of Sephadex G-15 filtration of bovine spermatozoa for cryopreservation2005In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 63, no 1, p. 160-178Article in journal (Refereed)
    Abstract [en]

    Semen from five dairy AI bulls was split-filtered through a Sephadex G-15 filter and frozen in a Triscitric acid buffer egg yolk-based extender. The effect of filtration was studied morphologically for individual sperm abnormalities. Computer-assisted sperm analysis (CASA) was used for motility and sperm motion assessment. Flow cytometry was used to disclose sperm viability (SYBR- 14/PI), mitochondrial membrane potential (Mitotracker Deep Red/SYBR 14), acrosome integrity (SYBR 14/PE-PNA/PI), plasma membrane stability (Merocyanine 540/YO-PRO 1/Hoechst 333342), and chromatin stability (acridine orange staining). Filtration significantly reduced the concentration of recovered spermatozoa (P less than 0.01), but improved semen quality, reducing the number of spermatozoa with various forms of morphological defects. Filtration also affected percentages of sperm motility after equilibration and after freezing/thawing. Sperm motion characteristics were, however, not significantly affected by filtration at any stage of the cryopreservation protocol, including post-extension, equilibration, or freezing/thawing. Filtration enhanced sperm viability after thawing (P less than 0.05), but had no significant effect (P greater than 0.05) on recovery of spermatozoa with high mitochondrial potential, intact acrosomes, or preserved sperm chromatin structure. Sperm plasma membrane stability was also not affected by the filtration method used (P greater than 0.05). It can be concluded that filtration effectively separates weaken or abnormal spermatozoa in pre-freezing semen samples and therefore the procedure could be recommended to improve post-thaw sperm viability of selected, fertile sires. (c) 2004 Elsevier Inc. All rights reserved.

  • 21.
    Koonjaenak, S.
    et al.
    Swedish University of Agriculture Science, Sweden .
    Pongpeng, P.
    Artificial Insemination Centre (Lamphayaklang), Department of Livestock Development, Lopburi 15190, Thailand.
    Wirowuthikul, S.
    Artificial Insemination Centre (Lamphayaklang), Department of Livestock Development, Lopburi 15190, Thailand.
    Johannisson, A.
    Swedish University of Agriculture Science, Sweden; .
    Kunavongkrit, A.
    Chulalongkorn University, Bangkok 10330, Thailand.
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden.
    Seasonality affects post-thaw plasma membrane intactness and sperm velocities in spermatozoa from Thai AI swamp buffaloes (Bubalus bubalis)2007In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 67, no 9, p. 1424-1435Article in journal (Refereed)
    Abstract [en]

    Altogether 218 frozen semen Al doses, prepared between 1980 and 1989 and also between 2003 and 2005 from 18 Al Thai swamp buffalo sires, were examined to determine whether seasonality affects post-thaw viability, as plasma membrane integrity (PMI, using SYBR-14/PI), plasma membrane stability (PMS, using Annexin-V/PI), or motility (Mot, using CASA). A thermoresistance test (38 degrees C for 60 min) was used to further analyze sperm survivability in vitro. All variables were compared over 3 seasons of the year (rainy: July-October; winter: November-February; and summer: March-June), with distinct ambient temperature and humidity. PMI (% of alive spermatozoa) was higher in winter (54.6%, P less than 0.001) than in the rainy (43.5%) or summer (46.7%) seasons. Outcomes of PMS (Annexin-V/PI assay) confirmed those of PMI, the highest PMS in spermatozoa processed in winter (55.7%, P less than 0.001). Spermatozoa depicting linear Mot post-thaw ranged from 48.2% to 48.8% across seasons (ns), proportions that decreased during incubation (33.5-37.9%), albeit without seasonal differences. The mean percentages of straight linear velocity (VSL), average path velocity (VAP), or curvilinear velocity (VCL) were higher (P less than 0.05-0.001) in the rainy season than in winter or summer, while average lateral head displacement (ALH) was higher (P less than 0.05) in summer, differences maintained after incubation. In conclusion, post-thaw PMS and PMI, assessed by flow cytometry, were significantly better in sperm samples processed during winter than in samples processed during the other seasons of the year, a seasonal difference not picked up by CASA, probably due to the larger number of spermatozoa assessed. (C) 2007 Elsevier Inc. All rights reserved.

  • 22.
    Li, Junwei
    et al.
    Univ Murcia, Spain.
    Barranco, Isabel
    Univ Murcia, Spain.
    Tvarijonaviciute, Asta
    Univ Murcia, Spain.
    Molina, Manuel F.
    Univ Murcia, Spain.
    Martinez, Emilio A.
    Univ 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.
    Parrilla, Inmaculada
    Univ Murcia, Spain.
    Roca, Jordi
    Univ Murcia, Spain.
    Seminal plasma antioxidants are directly involved in boar sperm cryotolerance2018In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 107, p. 27-35Article in journal (Refereed)
    Abstract [en]

    Boar ejaculates are ejected in fractions with a specific composition in terms of sperm numbers and seminal plasma (SP), which is reflected in the varying sperm cryotolerance observed among different fractions. As boar sperm are particularly sensitive to oxidative stress, this study evaluated the role of SP antioxidants in the observed differences in sperm cryotolerance among ejaculate fractions. Ten ejaculates from five boars were manually collected in fractions: the first 10 mL of the sperm-rich fraction (SRF), the rest of the SRF and the post-SRF. Semen samples comprising the entire ejaculate (EE) were created by proportionally mixing the three fractions described above. Each of the 40 resulting semen samples was split into two aliquots: one was used for sperm cryopreservation following a standard protocol utilizing 0.5-mL straws, and the other was used to collect SP for antioxidant assessment. Frozen-thawed (FT) sperm from the SRF (the first 10 mL of the SRF and the rest of the SRF) and those from post-SRF were of the highest and worst quality, respectively, which was measured in terms of total and objective progressive motility and viability (P amp;lt; 0.01). Viable FT sperm from the post-SRF generated more reactive oxygen species and experienced more lipid peroxidation than those from the SRF (both the first 10 mL and the rest of the SRF) (P amp;lt; 0.01). The percentage of FT sperm exhibiting fragmented nuclear DNA did not differ among ejaculate fractions and the EE. Catalase, glutathione peroxidase and glutathione peroxidase 5 (GPx-5) were lowest in SP from the first 10 mL of the SRF (P amp;lt; 0.001), whereas superoxide dismutase (SOD) and paraoxonase 1 (PON-1) were highest in SP of the SRF (both the first 10 mL and the rest of the SRF) (P amp;lt; 0.01). Trolox-equivalent antioxidant capacity (TEAC) and the ferric-reducing ability of plasma (FRAP) were highest in SP from the first 10 mL of the SRF and lowest in the post-SRF (P amp;lt; 0.001), whereas cupric-reducing antioxidant capacity was lowest (P amp;lt; 0.05) in SP from the first 10 mL of the SRF. Regression analyses indicated that certain SP antioxidants had good predictive value for post-thaw recovery rates of total motility (R-2 = 54.8%, P amp;lt; 0.001; including SOD, TEAC and FRAP) and viability (R-2 = 56.1%, P amp;lt; 0.001; including SOD, PON-1, GPx-5 and TEAC). These results demonstrated that certain SP antioxidants are positively involved in boar sperm cryotolerance, minimizing the oxidative stress imposed by cryogenic handling. (C) 2017 Elsevier Inc. All rights reserved.

  • 23.
    Ljungvall, K
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Karlsson, P
    Swedish University of Agriculture Science, Sweden; .
    Hulten, F
    Swedish University of Agriculture Science, Sweden; .
    Madej, A
    Swedish University of Agriculture Science, Sweden; .
    Norrgren, L
    Swedish University of Agriculture Science, Sweden; .
    Einarsson, S
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Magnusson, U
    Swedish University of Agriculture Science, Sweden; .
    Delayed effects on plasma concentration of testosterone and testicular morphology by intramuscular low-dose di(2-ethylhexyl)phthalate or oestradiol benzoate in the prepubertal boar2005In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 64, no 5, p. 1170-1184Article in journal (Refereed)
    Abstract [en]

    The immediate and delayed effects of prepubertal exposure to di(2-ethylhexyl)phthalate (DERP) or oestradiol benzoate on the plasma concentrations of testosterone, oestradiol and LH, as well as testicular morphology were examined in prepubertal boars. In a split litter design experiment, prepubertal boars were intramuscularly exposed to DEHP, oestradiol or vehicle during five weeks, starting at six weeks of age. The dose of DEHP was 50 mg/kg of bodyweight twice weekly, which is in the same range as recently used oral doses in rodents. Oestradiol-benzoate was administered at 0.25 mg/kg of bodyweight twice weekly. One set of animals was examined immediately after the exposure, and the other set was examined at an age of 7.5 months. During the exposure period concentrations of LH in plasma were lower (p = 0.02) in the oestradiol-treated animals than in the control group. In the group exposed to oestradiol, the relative to the body weight of the testicles tended to be lower (p = 0.07) than control immediately after five weeks of exposure, and the relative to the body weight of the seminal vesicles tended to be lower (p = 0.05) than control at 7.5 months of age. In the DEHP-exposed group an elevated (p = 0.005) concentration of testosterone and increased (p = 0.04) area of the Leydig cells in the testicles compared to the control group were seen at 7.5 months of age. These data suggest that DEHP early in life causes delayed effects on the reproductive system in the adult. (c) 2005 Elsevier Inc. All rights reserved.

  • 24.
    Macias Garcia, B
    et al.
    University of Extremadura.
    Ortega Ferrusola, C
    University of Extremadura.
    Aparicio, I M
    University of Extremadura.
    Miro-Moran, A
    University of Extremadura.
    Morillo Rodriguez, A
    University of Extremadura.
    Gallardo Bolanos, J M
    University of Extremadura.
    Gonzalez Fernandez, L
    University of Extremadura.
    Balao da Silva, C M
    University of Extremadura.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Tapia, J A
    University of Extremadura.
    Pena, F J
    University of Extremadura.
    Toxicity of glycerol for the stallion spermatozoa: Effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential2012In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 77, no 7, p. 1280-1289Article in journal (Refereed)
    Abstract [en]

    Glycerol is, to date, the most widely used cryoprotectant to freeze stallion spermatozoa at concentrations between 2% and 5%. Cryoprotectant toxicity has been claimed to be the single most limiting factor for the success of cryopreservation. In order to evaluate the toxic effects of the concentrations of glycerol used in practice, stallion spermatozoa were incubated in Biggers Whitten and Whittingham (BWW) media supplemented with 0%, 0.5%, 1.5%, 2.5%, 3.5%, and 5% glycerol. In two additional experiments, a hyposmotic (75 mOsm/kg) and a hyperosmotic (900 mOsm/kg) control media were included. Sperm parameters evaluated included cell volume, membrane integrity, lipid peroxidation, caspase 3, 7, and 8 activation, mitochondrial membrane potential, and integrity of the cytoskeleton. Glycerol exerted toxicity at concentrations 3.5% and the maximal toxicity was observed at 5%. The actin cytoskeleton was especially sensitive to glycerol presence, inducing rapid F actin depolymerization at concentrations over 1.5%. The sperm membrane and the mitochondria were other structures affected. The toxicity of glycerol is apparently related to osmotic and nonosmotic effects. In view of our results the concentration of glycerol in the freezing media for stallion spermatozoa should not surpass 2.5%.

  • 25.
    Martinez, C. A.
    et al.
    University of Murcia, Spain; Institute Biomed Research Murcia IMIB Arrixaca, Spain.
    Nohalez, A.
    University of Murcia, Spain; Institute Biomed Research Murcia IMIB Arrixaca, Spain.
    Ceron, J. J.
    University of Murcia, Spain; Institute Biomed Research Murcia IMIB Arrixaca, Spain.
    Rubio, C. P.
    University of Murcia, Spain.
    Roca, J.
    University of Murcia, Spain; Institute Biomed Research Murcia IMIB Arrixaca, Spain.
    Cuello, C.
    University of Murcia, Spain; Institute Biomed Research Murcia IMIB Arrixaca, 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.
    Martinez, E. A.
    University of Murcia, Spain; Institute Biomed Research Murcia IMIB Arrixaca, Spain.
    Gil, M. A.
    University of Murcia, Spain; Institute Biomed Research Murcia IMIB Arrixaca, Spain.
    Peroxidized mineral oil increases the oxidant status of culture media and inhibits in vitro porcine embryo development2017In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 103, p. 17-23Article in journal (Refereed)
    Abstract [en]

    The use of oils with undetected alterations is a long-recognized problem for in vitro embryo production systems. Since peroxides in oils have been associated with reduced embryo production outcomes, our goals were (1) to evaluate the effects of a batch of mineral oil (MO) that was suspected to be altered on the in vitro production of pig embryos and (2) to determine oil peroxide values throughout culture and the transfer of oxidant agents from oil to culture media. Sunflower oil, which has a completely different chemical composition than MO but a higher oxidative status, and unaltered MO were used as controls. Oocyte maturation, fertilization and embryo development were affected differently depending on the oil overlay used. While the suspected MO was not able to sustain in vitro maturation and fertilization, the oocytes incubated in the presence of sunflower oil were matured and fertilized similarly to those of the unaltered MO group. Moreover, the cleavage rate of presumed zygotes cultured under the suspected MO was severely reduced compared with those cultured under the other oils, and none of the cleaved embryos developed to the blastocyst stage. Although the cleavage rates in the sunflower oil and unaltered MO groups were similar, embryos cultured under sunflower oil also failed to develop to the blastocyst stage. Our results revealed that the suspected MO and sunflower oil had similar levels of peroxides and that these levels were much higher than those of the unaltered MO. The total oxidant status was higher in media incubated under peroxidized oils than in fresh media or media incubated without an oil overlay or under unaltered MO, indicating that oxidant agents were transferred to the incubation media. However, unlike the sunflower oil group, the culture media incubated under the suspected MO had high levels of total oxidant status and low levels of hydrogen peroxide and reactive oxygen species, suggesting the presence of other unknown oxidant agents in that oil. These results indicate that a peroxidized MO overlay dramatically decreases embryo production outcomes. This decrease could be associated with the higher peroxide values of the oil but cannot be explained by the levels of hydrogen peroxide and reactive oxygen species transferred from the oil to the culture media. It is likely that different oxidant agent(s) and/or other toxic compounds present in the peroxidized MO are responsible for its damaging effects on oocytes and embryos. (C) 2017 Elsevier Inc. All rights reserved.

  • 26.
    Martinez, C. A.
    et al.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Nohalez, A.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Parrilla, I.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Lucas, X.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Sanchez-Osorio, J.
    Topigs Norsvin Espana, Spain.
    Roca, J.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Cuello, C.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, 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.
    Martinez, E. A.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Gil, M. A.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Simple storage (CO2-free) of porcine morulae for up to three days maintains the in vitro viability and developmental competence2018In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 108, p. 229-238Article in journal (Refereed)
    Abstract [en]

    The advancement of porcine embryo transfer (ET) technology is constrained by regulatory hurdles (liquid nitrogen transportation) or, more importantly, the technical obstacles of using vitrified embryos in combination with nonsurgical deep uterine ET technology. Maintaining embryos in culture during transport and prior ET collides with the need of CO2 gassing and the best choice of culture medium. In this work, we describe storage conditions for short-term embryo CO2-free storage that allowed for a majority of in vivo-derived porcine morulae to survive after 3 days of storage in a liquid state, and to develop to the blastocyst stage unhatched, a sanitary prerequisite for ET. The storage conditions included NCSU-23 medium supplemented with bovine serum albumin, where bicarbonate was partially replaced by HEPES to avoid the need for CO2 gassing, and a temperature of 37 degrees C. These conditions were able to maintain the functionality of the stored embryos (hatching capacity after exposure to conventional culture conditions) and their developmental competence after ET (normal fetuses by day 38 of pregnancy). Use of this strategy of CO2-free storage should allow the shipment of fresh embryos worldwide in the absence of liquid nitrogen. (C) 2017 Elsevier Inc. All rights reserved.

  • 27.
    Martinez, C. A.
    et al.
    University of Murcia, Spain.
    Nohalez, A.
    University of Murcia, Spain.
    Parrilla, I.
    University of Murcia, Spain.
    Vazquez, J. L.
    University of Murcia, Spain.
    Roca, J.
    University of Murcia, Spain.
    Cuello, C.
    University of 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.
    Martinez, E. A.
    University of Murcia, Spain.
    Gil, M. A.
    University of Murcia, Spain.
    Surgical embryo collection but not nonsurgical embryo transfer compromises postintervention prolificacy in sows2017In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 87, p. 316-320Article in journal (Refereed)
    Abstract [en]

    Recent advances in nonsurgical deep uterine (NsDU) embryo transfer (ET) technology allow the noninvasive transfer of porcine embryos into recipients, overcoming the most important impediment for commercial ET in this species. Although many factors in the porcine ET-field have been recently evaluated, many others remain to be explored. We investigated here the future reproductive performance of donors and recipients after artificial insemination subsequent to the default surgical embryo recovery approach and to the NsDU-ET procedure, respectively. Although surgical embryo collection did not influence subsequent farrowing rates (90.5%), litter size decreased severely (8.9 +/- 0.8 piglets) compared to presurgery (10.8 +/- 0.3 piglets) and control group (10.7 +/- 0.3 piglets). In contrast, NsDU-ETs did neither affect fertility nor prolificacy of recipients in the cycle subsequent to ET, regardless of whether they were pregnant after NsDU-ET or not. These results indicate that while the surgical embryo collection procedure compromises the reproductive future of donor sows, the NsDU-ET approach does not affect the reproductive potential of the recipients after reintroduction to the breeding stock of the farm. Further research is thus needed to improve surgical embryo collection. (C) 2016 Elsevier Inc. All rights reserved.

  • 28.
    Martínez-Pastor, Felipe
    et al.
    Department of Molecular Biology (Cell Biology), Universidad de León, Spain; INDEGSAL, Universidad de León, León, Spain.
    Álvarez, Mercedes
    INDEGSAL, Universidad de León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, Universidad de León, León, Spain.
    Guerra, Camino
    Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autònoma de Barcelona, Barcelona, Spain.
    Chamorro, César A
    Department of Medicine, Surgery and Veterinary Anatomy, Universidad de León, León, Spain; Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autònoma de Barcelona, Barcelona, Spain.
    Anel-López, Luis
    INDEGSAL, Universidad de León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, Universidad de León, León, Spain.
    de Paz, Paulino
    Department of Molecular Biology (Cell Biology), Universidad de León, Spain; INDEGSAL, Universidad de León, León, Spain.
    Anel, Luis
    INDEGSAL, Universidad de León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, Universidad de León, León, Spain.
    Alvarez-Rodriguez, Manuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences. INDEGSAL, Universidad de León, León, Spain; Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autònoma de Barcelona, Barcelona, Spain.
    Extender osmolality, glycerol and egg yolk on the cryopreservation of epididymal spermatozoa for gamete banking of the Cantabric Chamois (Rupicapra pyrenaica parva)2019In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 125, p. 109-114, article id S0093-691X(18)30573-9Article in journal (Refereed)
    Abstract [en]

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

  • 29.
    Mata-Campuzano, Maria
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Spain.
    Alvarez-Rodríguez, Manuel
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, Spain.
    del Olmo, E
    Biology of Reproduction Group, National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Fernández-Santos, M R
    Biology of Reproduction Group, National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Garde, J J
    Biology of Reproduction Group, National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Martínez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Spain.
    Quality, oxidative markers and DNA damage (DNA) fragmentation of red deer thawed spermatozoa after incubation at 37 °C in presence of several antioxidants.2012In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 78, no 5, p. 1005-1019Article in journal (Refereed)
    Abstract [en]

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

  • 30.
    Mata-Campuzano, María
    et al.
    Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
    Álvarez-Rodríguez, Manuel
    Institute for Animal Health and Cattle Development, University of León, León, Spain.
    Álvarez, Mercedes
    Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, University of León, León, Spain.
    Tamayo-Canul, Julio
    Institute for Animal Health and Cattle Development, University of León, León, Spain; Division for Research and Post-graduate Studies, Technological Institute of Conkal, Yucatán, México.
    Anel, Luis
    Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, University of León, León, Spain.
    de Paz, Paulino
    Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
    Martínez-Pastor, Felipe
    Institute for Animal Health and Cattle Development, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
    Post-thawing quality and incubation resilience of cryopreserved ram spermatozoa are affected by antioxidant supplementation and choice of extender.2015In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 83, no 4, p. 520-528Article in journal (Refereed)
    Abstract [en]

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

  • 31.
    Morrell, J.M.
    et al.
    Swedish University of Agriculture Science, Sweden .
    Johannisson, A.
    Swedish University of Agriculture Science, Sweden .
    Dalin, A.-M.
    Swedish University of Agriculture Science, Sweden .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden .
    Single-layer centrifugation with Androcoll-E can be scaled up to allow large volumes of stallion ejaculate to be processed easily2009In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 72, no 6, p. 879-884Article in journal (Refereed)
    Abstract [en]

    The objective of the current study was to optimize the volumes of Androcoll-E and sperm sample used in various sizes of centrifuge tube to scale up single-layer centrifugation (SLC) for routine use in the field. Although sperm suspensions of equivalent quality were produced using Androcoll-E in small and large tubes, the sperm yield was much lower in the latter (P less than 0.001). In contrast, in 200-mL tubes (XL), the yields were approximately 25% higher than those for the small tubes. An increased volume (4.5 mL) of extended ejaculate in small tubes (SLC-Inc) or 15 to 18 mL extended ejaculate on 15 mL of colloid of a reduced density, Androcoll-E-Large (SLC-Large), in 50-mL tubes were both found to give similar yields of motile spermatozoa as that of the SLC-Small method (SLC-Small, 49.7 +/- 18.6%; SLC-Inc, 53.3 +/- 17.1%; SLC-Large, 44.9 +/- 18.3%) and were found to be equivalent in quality (motility: 88.0 +/- 8.8%, 84.0 +/- 3.5%, 90.0 +/- 5.4%; normal morphology: 69.4 +/- 17.0%, 69.4 +/- 12.7%, 63.9 +/- 15.6%; viability: 78 +/- 16.7%, 83.8 +/- 12.5%, 80.05 +/- 14.6%; DNA fragmentation index: 14.7 +/- 10.9%, 12.8 +/- 8.1%, 11.6 +/- 7.6%, respectively). The processing of an "average" stallion Equus caballus ejaculate in approximately twenty-seven 10-mL tubes (SLC-Inc) or eight 50-mL tubes (SLC-Large) is feasible, the latter being considered more practical for on-stud use. (C) 2009 Elsevier Inc. All fights reserved.

  • 32.
    Nagy, S
    et al.
    University of Pannonia, Hungary .
    Johannisson, A
    Swedish University of Agriculture Science SLU, Sweden .
    Wahlsten, T
    Faba Coop, Finland .
    Ijas, R
    Viking Genet Finland, Finland .
    Andersson, M
    University of Helsinki, Finland .
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Sperm chromatin structure and sperm morphology: Their association with fertility in AI-dairy Ayrshire sires2013In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 79, no 8, p. 1153-1161Article in journal (Refereed)
    Abstract [en]

    Prognostic relations between sperm variables and sire fertility are yet elusive. A retrospective analysis of sperm morphology and chromatin stability (studied using sperm chromatin structure assay [SCSA]) and their relation to fertility after AI (as proportions of 60 days of nonreturn to estrous [NRR], corrected NRR, or calving rate) was studied with preselected frozen semen doses from a group (N = 43) of AI-sires of the Finnish Ayrshire breed composed of 50% subfertile bulls (andlt;55% NRR) and 50% fertile sires (andgt;55% NRR). Fertility, indicated by all three parameters, correlated significantly only with the percentage of morphologically normal spermatozoa, a variable which negatively correlated with the percentage of DNA fragmentation at the time of SCSA, thus confirming the value of always having high numbers of morphologically normal spermatozoa in AI-doses. Proportions of major sperm defects also related to fertility but only when considering corrected NRR, not with calving rate, indicating that proportions of normal spermatozoa, a value surpassing differences between sperm laboratory screening methods, might be valuable and could be easily made routine by the industry. Though SCSA as a method is being contested for DNA- and chromatin analyses in the light of epigenetic changes, a particular parameter, the High Green fluorescence, showed the highest values for sperm doses collected from bulls having meiotic problems and containing a high proportion of diploid spermatozoa (approximately 20%) and also in bulls having a reciprocal chromosomal translocation, thus suggesting such a parameter might be useable to discriminate which bulls ought to be studied in more detail, including cytogenetic analyses.

  • 33.
    Nicolas, Manuel
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Borragán, Santiago
    Cabárceno Park, Cantabria, Spain.
    Martinez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Cell Biology, University of León, León, Spain.
    Chamorro, C A
    ITRA-ULE, INDEGSAL, University of León, León, Spain.
    Alvarez-Rodriguez, Manuel
    ITRA-ULE, INDEGSAL, University of León, León, Spain.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Cell Biology, University of León, León, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Evaluation of the qualitative and quantitative effectiveness of three media of centrifugation (Maxifreeze, Cushion Fluid Equine, and PureSperm 100) in preparation of fresh or frozen-thawed brown bear spermatozoa.2012In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 77, no 6, p. 1119-1128Article in journal (Refereed)
    Abstract [en]

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

  • 34.
    Nohalez, A.
    et al.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Martinez, C. A.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Parrilla, I.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Roca, J.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Gil, M. A.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, 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.
    Martinez, E. A.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Cuello, C.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Exogenous ascorbic acid enhances vitrification survival of porcine in vitro-developed blastocysts but fails to improve the in vitro embryo production outcomes2018In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 113, p. 113-119Article in journal (Refereed)
    Abstract [en]

    In this study, the effects of addition of the antioxidant ascorbic acid (AsA) were evaluated during porcine in vitro embryo production (IVP) and vitrification. In experiment 1, the effects of AsA supplementation during IVM, IVF and IVC were evaluated, using a total of 2744 oocytes in six replicates. The IVM, IVF and embryo IVC media were supplemented or not (control) with 50 mu g/mL AsA in all possible combinations. No significant effects of AsA were detected in any of the maturation, fertilization or embryo development parameters assessed. In experiment 2, we evaluated the effects of adding AsA to vitrification-warming media on the post-warming survival and quality of blastocysts. Day-6 in vitro-produced blastocysts (N = 588) from six replicates were randomly divided in two groups, with vitrification and warming media either supplemented with 50 mu g/mL AsA (VW + group) or un-supplemented (VW- control). Addition of AsA increased (P amp;lt; 0.05) blastocyst survival rate after vitrification compared with that of VW control embryos. Vitrification and warming increased (P amp;lt; 0.05) the production of oxygen species (ROS) and reduced (P amp;lt; 0.05) the glutathione levels in blastocysts. Although VW + blastocysts displayed higher (P amp;lt; 0.05) ROS levels than those of fresh control blastocysts, the levels were lower (P amp;lt; 0.05) than those found in VW- control blastocysts. In conclusion, under the experimental conditions, supplementation of IVM/IVF/IVC media with AsA did not improve the embryo production in vitro. By contrast, the addition of AsA to chemically defined vitrification and warming media increased the survival of in vitro-produced porcine blastocysts by decreasing ROS production. (C) 2018 Elsevier Inc. All rights reserved.

  • 35.
    Nohalez, Alicia
    et al.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Martinez, Cristina A.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Parrilla, Inmaculada
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Maside, Carolina
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Roca, Jordi
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Gil, Maria A.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, 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.
    Martinez, Emilio A.
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Cuello, Cristina
    Univ Murcia, Spain; Inst Biomed Res Murcia IMIB Arrixaca, Spain.
    Eventual re-vitrification or storage in liquid nitrogen vapor does not jeopardize the practical handling and transport of vitrified pig embryos2018In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 113, p. 229-236Article in journal (Refereed)
    Abstract [en]

    This study aimed (1) to evaluate the in vitro post-warming survival of porcine embryos after re vitrification and (2) to assess the efficacy of transport of embryos in dry shipper (DS) in maintaining the viability and quality of vitrified embryos for a 3-day period. Embryos at the compacted or cavitating morula (CCM) and unhatched blastocyst (UBL) stages were surgically obtained from weaned, crossbred sows. In the first experiment, more than 85% of the embryos survived an initial vitrification and warming and achieved comparable survival rates to those of their fresh counterparts. In contrast, those embryos subjected to a second vitrification and warming had clearly lower survival rates (60% and 64% for re vitrified embryos from the CCM and UBL groups, respectively) compared to the survival rates of the initial vitrification and fresh control groups (P amp;lt; 0.01). Hatching rates were similar in re-vitrified blastocysts derived from vitrified CCMs and fresh control groups (50.8% and 55.3%, respectively). However, differences (P amp;lt; 0.01) in hatching rates were recorded in re-vitrified blastocysts derived from vitrified UBLs and fresh control blastocysts (14.7% and 90.0%, respectively). In the second experiment, vitrified embryos were stored in a liquid nitrogen tank for one month. Then, the straws containing the embryos were transferred to a DS (DS group) or to another liquid nitrogen tank (control group) for an additional three days. Embryos from the DS and control groups had similar survival and hatching rates, regardless of the embryonic stage considered. The DS storage of CCMs and UBLs did not affect their development after culturing, including total cell numbers, compared to the control, although their apoptotic index was slightly higher (P amp;lt; 0.05), regardless of the developmental stage. In conclusion, although re-vitrification negatively affects embryo survival, this study demonstrated that amp;gt;60% of vitrified embryos could be successfully re-vitrified and re-warmed. The present study also showed the effectiveness of the DS for the storage of vitrified porcine CCMs and UBLs for at least three 3 days. (C) 2018 Elsevier Inc. All rights reserved.

  • 36.
    Ortega Ferrusola, C.
    et al.
    University of Extremadura, Spain .
    Gonzalez Fernandez, L.
    University of Extremadura, Spain .
    Salazar Sandoval, C.
    University of Extremadura, Spain .
    Macias Garcia, B.
    University of Extremadura, Spain .
    Rodriguez Martinez, H.
    Swedish University of Agriculture Science, Sweden .
    A. Tapia, J.
    University of Extremadura, Spain .
    J. Pena, F.
    University of Extremadura, Spain .
    Inhibition of the mitochondrial permeability transition pore reduces "apoptosis like" changes during cryopreservation of stallion spermatozoa2010In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 74, no 3, p. 458-465Article in journal (Refereed)
    Abstract [en]

    In order to evaluate to what extent the changes that occur during cryopreservation involve the mitochondrial permeability transition pore (PT-pore), a specific inhibitor was used during the cryopreservation process of stallion spermatozoa. Four ejaculates from each of 7 stallions were frozen using a standard protocol. Before freezing, each ejaculate was split into three subsamples. The first was supplemented with 2.5 mu M bongkrekic acid (BA) and the second with 5 mu M BA. The third subsample served as control. The BA significantly reduced the percentage of spermatozoa depicting active caspases after thawing, reduced the percentage of spermatozoa with increased membrane permeability, and increased the mitochondrial membrane potential of thawed sperm. Sperm motility was reduced as a result of the treatment. It is concluded that the mitochondrial pathway of apoptosis seems to be an important factor involved in the sublethal damage that equine spermatozoa experience after freezing and thawing, and that sperm motility in the equine species is largely dependent on mitochondrial ATP produced by oxidative phosphorylation. (C) 2010 Elsevier Inc. All rights reserved.

  • 37.
    Payan-Carreira, R.
    et al.
    University of Tras Os Montes and Alto Douro, Portugal.
    Pires, M. A.
    University of Tras Os Montes and Alto Douro, Portugal.
    Santos, C.
    University of Tras Os Montes and Alto Douro, Portugal.
    Strom Holst, B.
    Swedish University of Agriculture Science SLU, Sweden.
    Colaco, J.
    University of Tras Os Montes and Alto Douro, Portugal.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Immunolocalization of E-cadherin and beta-catenin in the cyclic and early pregnant canine endometrium2016In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 86, no 4, p. 1092-1101Article in journal (Refereed)
    Abstract [en]

    Putative changes in E-cadherin and beta-catenin during implantation in dogs are of interest to study, as they are relevant proteins for epithelial integrity. E-cadherin and beta-catenin were immunolocalized in the canine endometrium during the estrous cycle and early pregnancy, using monoclonal antibodies. Both proteins were detected in all types of endometrial epithelia (surface epithelium [SE], superficial glandular, and deep glandular epithelia) at all stages of the estrous cycle and in early placental structures. E-cadherin depicted a gradient of intensity apparently being lowest in the SE to progressively increase toward the deepness of the endometrial glands, regardless of the stage of estrous cycle. The overall immunostaining was, however, weaker at diestrus. In pregnant samples, the trophoblast was conspicuously immunolabeled compared with the endometrial surface lining epithelium. In the latter, the cytoplasmic pattern predominated over the membrane bound, as was also seen in the decidual cells of the placental labyrinth. In the early placenta, only trophoblast cells and lacunae retained membrane signals. beta-Catenin membrane labeling appeared relatively constant throughout the cycle, although a tendency toward a decrease in intensity was detected at the secretory stages. In addition, a dislocation of the immunoreaction from membrane to the cytoplasm was observed in both the SE and the glandular epithelia at particular stages of the cycle. In early pregnancy, a loss of the membranous pattern was observed in the SE and labyrinth, but neither on trophoblast nor in lacunae. The results show the existence of a softening of the adherens junctional complex in progestagen-dominated stages favoring embryo-maternal interactions and endometrial invasion during canine implantation. (C) 2016 Elsevier Inc. All rights reserved.

  • 38.
    Payan-Carreira, R
    et al.
    University of Tras Os Montes and Alto.
    Santana, I
    University of Tras Os Montes and Alto.
    Pires, M A
    University of Tras Os Montes and Alto.
    Strom Holst, B
    SLU.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Localization of tumor necrosis factor in the canine testis, epididymis and spermatozoa2012In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 77, no 8, p. 1540-1548Article in journal (Refereed)
    Abstract [en]

    Tumor necrosis factor (TNF), formerly known as Tumor necrosis factor alpha is now regarded as a natural component of the mammalian seminal plasma (SP). Although not completely clarified, its functions in the SP have been associated with paradoxal roles, such as sperm survival in the female genital tract, while at high levels negatively affect sperm survival and fertility potential. Recently, it has been discovered that canine inseminated spermatozoa display a strong immunoreaction for TNF when lining the female endometrium. As a continuation of this finding, the present work aimed at documenting TNF localization in the canine testes and epididymis and in freshly ejaculated spermatozoa (SPZ) through immunohisto- or cytochemistry. less thanbrgreater than less thanbrgreater thanImmunoreaction for TNF was found in all samples used. In the dog testis, TNF immunoexpression was limited to the seminiferous tubules, where late round spermatids (SPD) showed weak intensity of immunostaining, while elongating and elongated SPD evidenced moderate and the residual bodies a strong intensity. In the epididymis, a gradual progressive increase of TNF immunolabelling was found throughout the epididymal regions, ranging from a weak intensity at the caput epididymis to a moderate intensity at the cauda. TNF immunolabelling was found in mature SPZ during the epididymal transit and also in freshly ejaculated SPZ, which showed a strong midpiece immunolabelling. Data presented here provide important information on expression of TNF in spermatozoa, which is acquired by the SPZ during their formation at the testis. It further provides the basis for subsequent studies on the physiological importance of cytokines in sperm function.

  • 39.
    Roca, J.
    et al.
    University of Murcia, Spain.
    Parrilla, I.
    University of Murcia, Spain.
    Bolarin, A.
    Topigs Norsvin, 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.
    Will AI in pigs become more efficient?2016In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 86, no 1, p. 187-193Article, review/survey (Refereed)
    Abstract [en]

    AI is commercially applied worldwide to breed pigs, yielding fertility outcomes similar to those of natural mating. However, it is not fully efficient, as only liquid-stored semen is used, with a single boar inseminating about 2000 sows yearly. The use of liquid semen, moreover, constrains international trade and slows genetic improvement. Research efforts, reviewed hereby, are underway to reverse this inefficient scenario. Special attention is paid to studies intended to decrease the number of sperm used per pregnant sow, facilitating the practical use of sexed frozen-thawed semen in swine commercial insemination programs. (C) 2015 Elsevier Inc. All rights reserved.

  • 40.
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Role of the oviduct in sperm capacitation2007In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 68, p. S138-S146Article in journal (Refereed)
    Abstract [en]

    Following insemination of spermatozoa pre-ovulation, the mammalian oviduct ensures, by the formation of a functional sperm reservoir (SR), that suitable (low) numbers of viable and potentially fertile spermatozoa are available for fertilization at the ampullary isthmic junction (AIJ). As ovulation approaches, a proportion of the SR-stored spermatozoa is continuously distributed towards the AIJ and individually activated leading to step-wise capacitation and the attainment of hyperactivated motility. This paper reviews in vivo changes in the intra-luminal milieu of the oviduct of pigs and cows, in particular the SR and the AIJ which relate to the modulation of sperm capacitation around spontaneous ovulation. In vivo, most viable spermatozoa in the pre-ovulatory SR are uncapacitated. Capacitation rates significantly increase after ovulation, apparently not massively but concurrent with the individual, continuous sperm dislocation from the SR. Bicarbonate, whose levels differ between the SR and the AIJ, appears as the common primary effector of the membrane destabilizing changes that encompasses the first stages of capacitation. Sperm activation can be delayed or even reversed by co-incubation with membrane proteins of the tubal lining, isthmic fluid or specific tubal glycosaminoglycans, such as hyaluronan. Although the pattern of response to in vitro induction of sperm activation-capacitation in particular-is similar for all spermatozoa, the capacity and speed of the response is very individual. Such diversity in responsiveness among spermatozoa insures full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation at the AIJ, thus maximizing the chances of normal fertilization. (C) 2007 Elsevier Inc. All rights reserved.

  • 41.
    Rodriguez-Martinez, Heriberto
    et al.
    Swedish University of Agriculture Science SLU, Sweden .
    Saravia, F.
    [Rodriguez-Martinez, Spain; .
    Wallgren, M.
    Qual Genet, Sweden .
    Roca, J.
    Fac Vet Med, Spain .
    J. Pena, F.
    Fac Vet Med, Spain .
    Influence of seminal plasma on the kinematics of boar spermatozoa during freezing2008In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 70, no 8, p. 1242-1250Article in journal (Refereed)
    Abstract [en]

    Sperm motolity is, for its relation to cell viability and fertility, a central component of the spermiogram, where consideration of motion patterns allows discrimination of sub-populations among boar spermatozoa. Extension and cryo-preservation imposes changes in these patterns in connection to handling, additives, temperature changes and the removal of boar seminal plasma (BSP) which apparently makes spermatozao susceptible to xodative stress, thus affecting survival and motility post-thaw. Detailed kinematic analyses during sperm cooling are sparse, particularly when considering the instrumentation and settings used for analyses, the effect of extenders, and of the BSP the processed spermatozoa are exposed to. Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction of the ejaculate (portion 1. P1-BSP), have shown an increased ability to sustain motility during and after cryo-preservation than spermatozoa immersed in the rest of the ejaculate (portion 2, P2). When P2-spermatozoa were cleansed from their BSP and exposed for 60 min to pooled P1-BSP, their motility post-thaw increased to similar levels as P1-spermatozoa. This BSP-influence is sire-dependent, presumably related to the protein concentration in the different ejaculate poriton,s andapparently unrealted to changes in membrane integrity or membrane stability through conventiona, controlled cooling. (C) 2008 Elsevier Inc. All rights reserved.

  • 42.
    Rodriguez-Martinez, Heriberto
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Saravia, F
    Swedish University of Agriculture Science, Sweden; .
    Wallgren, M
    Swedish University of Agriculture Science, Sweden; .
    Tienthai, P
    Swedish University of Agriculture Science, Sweden; .
    Johannisson, A
    Swedish University of Agriculture Science, Sweden; .
    Vazquez, JM
    University of Murcia, Murcia, Spain.
    Martinez, E
    University of Murcia, Murcia, Spain.
    Roca, J
    University of Murcia, Murcia, Spain.
    Sanz, L
    Institute of Biomedicine, CSIC, Valencia, Spain.
    Calvete, JJ
    Institute of Biomedicine, CSIC, Valencia, Spain.
    Boar spermatozoa in the oviduct2005In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 63, no 2, p. 514-535Article in journal (Refereed)
    Abstract [en]

    In the pig, a functional tubal sperm reservoir (SR) is established before ovulation to ensure availability of suitable numbers of viable spermatozoa for fertilization. The boars large ejaculate is split: most spermatozoa are delivered in a sperm-rich fraction (SRF) followed by a post-SRF fraction containing increasing amounts of the spermadhesin PSP-I/PSP-II-rich seminal vesicle secretion. This heterodimer acts as leukocyte chemoattractant both in vitro and in vivo, contributing to the phagocytosis of those spermatozoa not reaching the SR. Sequential ejaculate deposition of marked spermatozoa and SR screening showed that most spermatozoa in the SR arose from the fortuitous PSP-poor, first portion of the SRF fraction, escaping phagocytosis and replenishing the SR within 23 h. The SR-sperm numbers diminish gradually in relation to ovulation, spermatozoa. being continuously redistributed toward the upper isthmus. In vitro, only uncapacitated spermatozoa bind to epithelial explants, suggesting that the SR influences sperm capacitation. In vivo, most viable spermatozoa - usually harbored in the deep furrows in the pre- or peri-ovulatory SR during spontaneous standing estrus - are uncapacitated, but capacitation significantly increases after ovulation. Pre-/peri-ovulatory SR spermatozoa promptly capacitate in vitro when exposed to the effector bicarbonate, an influence that can be reversed by co-incubation with SR fluid or its component hyaluronan. Fluid collected from the ampullar segment (rich in bicarbonate) induces capacitation in vitro. In conclusion, the lack of massive sperm capacitation in the SR and the diverse individual response to capacitation shown by tubal spermatozoa would relate both to the insurance of full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation in the upper oviduct, thus maximizing the chances of normal fertilization. (C) 2004 Elsevier Inc. All rights reserved.

  • 43.
    Rodriguez-Martinez, Heriberto
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Tienthai, P.
    Chulalongkorn University, Thailand.
    Atikuzzaman, Mohammad
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Vicente Carrillo, Alejandro
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Rubér, Marie
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Alvarez-Rodriguez, Manuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    The ubiquitous hyaluronan: Functionally implicated in the oviduct?2016In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 86, no 1, p. 182-186Article, review/survey (Refereed)
    Abstract [en]

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

  • 44.
    Sanchez-Osorio, J.
    et al.
    University of Murcia, Spain .
    Cuello, C.
    University of Murcia, Spain .
    Gil, M.A.
    University of Murcia, Spain .
    Parrilla, I.
    University of Murcia, Spain .
    Alminana, C.
    University of Murcia, Spain .
    Caballero, I.
    University of Murcia, Spain .
    Roca, J.
    University of Murcia, Spain .
    Vazquez, J.M.
    University of Murcia, Spain .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science SLU, Sweden .
    Martinez, E.A.
    University of Murcia, Spain .
    In vitro postwarming viability of vitrified porcine embryos: Effect of cryostorage length2010In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 74, no 3, p. 486-490Article in journal (Refereed)
    Abstract [en]

    Porcine embryos, which had been vitrified and stored in liquid nitrogen for up to three yr, were retrospectively analyzed to evaluate the influence of duration of storage on their in vitro viability post-warming. All embryos were vitrified (OPS or SOPS) and warmed (three-step or direct warming) using procedures that resulted in the same in vitro survival, hatching rates, and numbers of cells. Therefore, embryo data obtained using the different procedures were pooled according to their developmental stage as morulae (n = 571) or blastocysts (n = 797) and to the length of their storage in liquid nitrogen: a) 1-9 d; b) 10-30 d; c) 31-90 d; d) 1-3 yr. Non-vitrified embryos of corresponding developmental stages were used as a fresh control group (n = 280). Survival and hatching rates were evaluated after in vitro culture to assess embryo viability. The total number of cells was counted in the resulting viable blastocysts as an indicator of quality. A total of 1,648 fresh and vitrified embryos were analyzed. In vitro survival and hatching rates, but not the number of cells, differed significantly between vitrified morulae and their fresh counterparts irrespective of the duration of cryostorage. Length of storage in liquid nitrogen (LN(2)) did not influence in vitro viability among different groups of vitrified/warmed morulae nor embryos at the blastocyst stage. In conclusion, duration of storage in LN2 has no effect on the post-warming viability of porcine embryos vitrified at morula or blastocyst stage. (C) 2010 Elsevier Inc. All rights reserved.

  • 45.
    Saravia, F.
    et al.
    Avenida Universidad s.n. Campus Universitario 10071, Cáceres, Spain.
    Nunez-Martinez, I.
    Avenida Universidad s.n. Campus Universitario 10071, Cáceres, Spain.
    M. Moran, J.
    Avenida Universidad s.n. Campus Universitario 10071, Cáceres, Spain.
    Soler, C.
    Avenida Universidad s.n. Campus Universitario 10071, Cáceres, Spain .
    Muriel, A.
    Avenida Universidad s.n. Campus Universitario 10071, Cáceres, Spain.
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    J. Pena, F.
    Avenida Universidad s.n. Campus Universitario 10071, Cáceres, Spain.
    Differences in boar sperm head shape and dimensions recorded by computer-assisted sperm morphometry are not related to chromatin integrity2007In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 68, no 2, p. 196-203Article in journal (Refereed)
    Abstract [en]

    Although sperm head shape and relative dimensions are considered reliable indicators of sperm quality, their quantification is most often operator-driven, e.g., subjective. Artificial insemination semen doses from 35 mature stud boars of known fertility and belonging to three breeds and two hybrid breeds (Duroc, Large White, Landrace, respectively, Yorker and Risco) were used in this study. Sperm samples were extended to 100 x 10(6) Cells per mL and 10 mu L of the sperm suspension used to made smears which, stained, were examined using phase contrast microscopy interfaced with an automated sperm morphology analyzer (ASMA, 2 ISAS (R)). Each sperm head was measured for four primary parameters [area (A) mu m(2), perimeter (P) mu m, length (L) mu m, width (M mu m], and four derived parameters of head shape [(L/W, (4 pi A/P-2), ((L - W/(L + M), (pi LW/4A)]. Definition of head size was statistically performed. The threshold for each class was established on the basis of the area values, considering the 25th percentile as small and the 75th percentile as large spermatozoa. In a second step, sperm head shape was determined as normal, elliptic, abnormal (rugose) contour, long or irregular and percentiles set as above to define spermatozoa with normal values for each shape parameter. Significant differences were found among breeds in the size of morphologically normal spermatozoa, which were significantly larger and more elliptic (P less than 0.001) in the Duroc breed. Sperm chromatin integrity was studied using the SCSA-assay, with significant differences observed in the degree of fragmentation intensity (DFI) although this value was consistently low in all animals studied. The hereby-validated ASMA was able to determine significant differences in sperm shape and dimensions among breeds, which were not accompanied by deviations in chromatin structure neither within nor between fertile AI-boars. (c) 2007 Elsevier Inc. All rights reserved.

  • 46.
    Saravia, F.
    et al.
    Swedish University of Agriculture Science SLU, Sweden .
    Wallgren, M.
    Swedish University of Agriculture Science SLU, Sweden Qual Genet HB, Sweden .
    Johannisson, A.
    Swedish University of Agriculture Science SLU, Sweden .
    Calvete, J.J.
    CSIC, Spain .
    Sanz, L.
    CSIC, Spain .
    Pena, F.J.
    Fac Vet Med, Spain .
    Roca, J.
    University of Murcia, Spain .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science SLU, Sweden .
    Exposure to the seminal plasma of different portions of the boar ejaculate modulates the survival of spermatozoa cryopreserved in MiniFlatPacks2009In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 71, no 4, p. 662-675Article in journal (Refereed)
    Abstract [en]

    Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction (SRF) of the boar ejaculate (portion 1, PI I have higher documented viability during and after cryopreservation than spermatozoa in the rest of the ejaculate (portion 2, P2). probably in relation to different features of the surrounding seminal plasma (SP). In the present study. We investigated whether the SP from these ejaculate portions (SP1 or SP2) was able to differently influence sperm viability and chromatin structure of the P1- or P2-contained spermatozoa from individual boars primarily or secondarily exposed (e.g., following cleansing and re-exposure) to pooled SP1 or SP2 from the same males during 60 min. Spermatozoa were subjected to controlled cooling and thawing in MiniFlatPacks (MFPs) and examined for motility (using computer-assisted sperm analysis, CASA) at selected stages of processing Moreover, sperm plasma membrane intactness (investigated using, SYBR-14/propidium iodide, PI), plasma membrane architecture (examined using Annexin-V-PI staining), and chromatin (deoxyribonucleic acid, DNA) integrity (tested using sperm chromatin structure assay, SCSA) were assessed post-thaw (PT). A higher proportion of PI spermatozoa than of P2 spermatozoa incubated in their native SP portion were confirmed to be motile from collection to PT. When P1 spermatozoa were cleansed from their original SP and re-exposed to pooled P2-SP. sperm kinematics deteriorated from extension to PT. By contrast, cleansed P2 spermatozoa increased motility to P1 levels, especially PT when re-exposed to pooled P1-SP. Such differential effects on motility were not clearly accompanied by biologically related modifications of sperm membrane or chromatin structure. This influence of the SP on sperm kinematics was not sire-dependent and it Was presumably related to different concentrations or either SP proteins or bicarbonate in the different ejaculate portions. (C) 2009 Elsevier Inc. All rights reserved.

  • 47.
    Saravia, F
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Wallgren, M
    Swedish University of Agriculture Science, Sweden; .
    Nagy, S
    Swedish University of Agriculture Science, Sweden; .
    Johannisson, A
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Deep freezing of concentrated boar semen for intra-uterine insemination: effects on sperm viability2005In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 63, no 5, p. 1320-1333Article in journal (Refereed)
    Abstract [en]

    The use of deep-frozen boar semen for artificial insemination (AI) is constrained by the need for high sperm numbers per dose, yielding few doses per ejaculate. With the advancement of new, intrauterine insemination strategies, there is an opportunity for freezing small volumes containing high sperm numbers, provided the spermatozoa properly sustain cryopreservation. The present study aimed to concentrate (2 x 10(9) spz/mL) and freeze boar spermatozoa packed in a 0.5 mL volume plastic medium straw (MS) or a multiple FlatPack (MFP) (four 0.7 mL volume segments of a single FlatPack [SFP]) intended as AI doses for intra-uterine AI. A single freezing protocol was used, with a conventional FlatPack (SFP, 5 x 10(9) spz/5 mL volume) as control. Sperm viability post-thaw was monitored as sperm motility (measured by computer-assisted sperm analysis, CASA), as plasma membrane integrity (PMI, assessed either by SYBR-14/PI, combined with flow cytometry, or a rapid hypo-osmotic swelling test [sHOST]). Sperm motility did not differ statistically (NS) between test-packages and control, neither in terms of overall sperm motility (range of means: 37-46%) nor sperm velocity. The percentages of linearly motile spermatozoa were, however, significantly higher in controls (SFP) than in the test packages. Spermatozoa frozen in the SFP (control) and MFP depicted the highest PMI (54 and 49%, respectively) compared to MS (38%, P less than 0.05) when assessed with flow cytometry. In absolute numbers, more viable spermatozoa post-thaw were present in the MFP dose than in the MS (P less than 0.05). Inter-boar variation was present, albeit only significant for MS (sperm motility) and SFP (PMI). In conclusion, the results indicate that boar spermatozoa can be successfully frozen when concentrated in a small volume. (C) 2004 Elsevier Inc. All rights reserved.

  • 48.
    Spjuth, L.
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Johannisson, A.
    Swedish University of Agriculture Science, Sweden; .
    Lundeheim, N.
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, H.
    Swedish University of Agriculture Science, Sweden; .
    Early pre-pubertal exposure to low-dose oral di(2-ethylhexyl) phthalate does not affect sperm plasma membrane stability, acrosomal integrity or chromatin structure in the post-pubertal2007In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 68, no 2, p. 186-195Article in journal (Refereed)
    Abstract [en]

    This study aimed to determine whether pre-pubertal exposure in boars to di(2-ethylhexyl) phthalate (DEHP), a plasticizer reported to have toxic effects on rodent reproduction, would affect the sperm ability to undergo capacitation and acrosome reaction (AR) in vitro or give rise to a higher degree of chromatin instability associated with acid-induced denaturation. Spermatozoa were collected from 16 boars (n = 8/group) 8-9 months of age, exposed to 300 mg/kg body weight of DEHP or placebo per os three times a week, from 3 to 7 weeks of age. The spermatozoa were cryopreserved and examined post-thaw by flow cytometry for their ability to capacitate in vitro when exposed to the effector bicarbonate and to acrosome-react when exposed to calcium ionophores, using the lipid stain Merocyanine-540 (m-540), and peanut agglutinin-fluorescein isothiocyanate, respectively, as probes. The ability of the DNA to sustain denaturation in vitro was tested using a sperm chromatin structure assay (SCSA). No significant differences between the DEHP-exposed group and controls were found for any of the sperm attributes examined. Frozen-thawed spermatozoa showed similar rates of non-capacitated cells between groups, and were capacitated at similar rates. Rates of induced ARs were also similar. Values of DNA denaturation were low and showed no differences between groups. In conclusion, pre-pubertal exposure to DEHP does not seem, under the conditions of the present experiment, to affect the ability of frozen-thawed spermatozoa collected post-puberty to capacitate or acrosome-react (the main requisites for fertilization) or to present damage in their nuclear genome. (c) 2007 Elsevier Inc. All rights reserved.

  • 49.
    Tejerina, F.
    et al.
    Swedish University of Agriculture Science, Sweden University of Leon, Spain .
    Buranaamnuay, K.
    Swedish University of Agriculture Science, Sweden Chulalongkorn University, Thailand .
    Saravia, F.
    Swedish University of Agriculture Science, Sweden .
    Wallgren, M.
    Swedish University of Agriculture Science, Sweden .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden .
    Assessment of motility of ejaculated, liquid-stored boar spermatozoa using computerized instruments2008In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 69, no 9, p. 1129-1138Article in journal (Refereed)
    Abstract [en]

    Visual-motility assessment is a tool used to determine the quality of boar ejaculates. This method is subjective by nature, and consequently, computer-assisted sperm analysis (CASA), with different software designs, has been developed for more objective assessment using conventional image analysis or particle counting. In the present study, we compared the results of sperm analysis using a conventional CASA system (Cell Motion Analyzer, SM-CMA (TM)), with those using a novel software (QualiSperm (TM)) and those of visual assessment performed by two operators. Ejaculates were collected weekly from four Swedish Landrace boars for 4 weeks. Each ejaculate was divided into three aliquots of different sperm concentration (300, 125, and 40 million spermatozoa/mL) and stored at similar to 17 degrees C for 96 h. Only samples at 40 million spermatozoa/mL concentration were analyzed using both automated systems; for the remaining concentrations, the SM-CMA (TM) was not used due to its inability to examine higher sperm concentrations. The number of spermatozoa analyzed was highest for the QualiSperm (TM) (similar to 300-5000 spermatozoa), followed by the SM-CMA (TM) (similar to 200 spermatozoa), and lastly, by subjective motility evaluation (similar to 100 spermatozoa). There was a time-course decrease in motility of the liquid-stored semen, detectable by either computerized method. Although the percentage of motile spermatozoa measured by the two automated systems correlated well (r greater than= 0.75), there was disagreement between operators. In conclusion, because of the lower degree of variation, the numbers of spermatozoa analyzed, and the speed of analysis (similar to 1 min per sample), QualiSperm (TM) appears to be a suitable instrument for routine work, provided it maintains stability and is available at an affordable price. (C) 2008 Elsevier Inc. All rights reserved.

  • 50.
    Wongtawan, T
    et al.
    Swedish University of Agriculture Science, Sweden.
    Saravia, F
    Swedish University of Agriculture Science, Sweden.
    Wallgren, M
    Swedish University of Agriculture Science, Sweden.
    Caballero, I
    Swedish University of Agriculture Science, Sweden.
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden.
    Fertility after deep intra-uterine artificial insemination of concentrated low-volume boar semen doses2006In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 65, no 4, p. 773-787Article in journal (Refereed)
    Abstract [en]

    Boar semen can be successfully frozen - highly packed - in small containers (medium-straw, MS or MiniFlatPack, MFP). The use of deep intra-uterine artificial insemination (DIU-Al) can make possible the deposition of small volumes of this thawed, non re-extended semen deeply intra-Uterine, close to the sperm reservoir. The present experiments studied the fertility achieved after single or double DIU-Al per oestrus, with special attention to the interval between AI and spontaneous ovulation. Semen from two boars of proven fertility was frozen in MS or MFP holding 1 x 109 total spermatozoa. Multiparous (2-5 parity, n = 42) crossbred sows were checked for oestrous behaviour after weaning and the occurrence of spontaneous ovulation was checked with transrectal ultrasonography (TUS) to establish the mean interval between onset of oestrus (OO) and ovulation which was found to be when approximately 2/3 of the oestrus period has passed. The sows were, in the following standing oestrus, Subjected to DIU-Al using thawed semen from either MS (n = 20) or MFP (n = 22), inseminated Without further re-extension. The sows were randomly allotted to one of three groups: (1) single DIU-Al 8 h before expected ovulation (control group, n = 19); (2) single DIU-AI 4 h before expected ovulation (treatment group S, n = 15); and (3) double DIU-AI 12 and 4 h before expected ovulation (treatment group D, n 8). Occurrence of spontaneous ovulation was confirmed by TUS, performed as during the first oestrous period and used to determine the real interval of DIU-AI and ovulation. Pregnancy was also confirmed by TUS 28 days after 00 in those sows not returning to oestrus. These sows were slaughtered (30-45 days of pregnancy), and the appearance of the reproductive tract and ovaries, the number of live and dead foetuses, of implantation sites and of corpora lutea (CL) were recorded. Sows (n = 9) returning to oestrus ("open") were re-inseminated (either once [n = 4] or twice [n = 51) the following oestrus with either MFP (n = 5) or MS (n = 4) and slaughtered 12-14 h post-ovulation for recovery of tubal oocytes and of spermatozoa from the uterotubal junctions (sperm reservoir), to assess the degree of effectiveness of sperm transport. Post-thaw sperm motility was 44.3 +/- 3.21% in MFP and 42.8 +/- 0.72% for MS (LSmean +/- S.E.M., n.s.), and did not significantly change from thawing to AI. The DIU-AI could be performed in all sows, but insertion was difficult (slow greater than 5 min) in 5/42 sows. Four of these sows returned to oestrus. Pregnancy rate averaged 35% (group D: 25%, group S: 40%, control: 36%, n.s.). The interval between DIU-AIs 13 to -3 h for group C, for group S from and spontaneous ovulation varied largely, ranging from 0 -11 to +3 h and for group D from -17 to -4 h. Pregnancy rates were clearly related to the interval DIU-AI and ovulation, being highest (60%, 12/20) when AI occurred between 8 and 4 h before spontaneous (not expected) ovulation. The number of implantation sites ranged 6-22 (n.s. among groups.), and the number of alive foetuses 2-11 (n.s. among groups). Implantation rate (total number of implantations/CL) ranged 48.0-69.7% being highest in the D-group (P less than 0.05). The examination of the "open" sows slaughtered 12-14 h post-ovulation revealed few recovered oocytes were fertilized (approximately 10%). Only 40% of oocytes had spermatozoa bound to the zona pellucida, not more than two spermatozoa per oocyte. Moreover, low sperm numbers (approximately 4000) were found in the sperm reservoirs (UTJs), irrespective of using single or doube DIU-AI (n.s.). The highest values (P less than 0.05) for these variables were recorded when DIU-AI (either single or double [second AI]) occurred 4-8 h before Ovulation, especially when MFP-semen was used (P less than 0.05). In conclusion: (1) DIU-AI can be easily performed in most sows; (2) pregnancies can be obtained by the DIU-Al of low volumes of highly concentrated frozen-thawed boar semen, once or twice during oestrus, but fertility is still low, probably owing to an unsatisfactory sperm transport when expected and real ovulation differ; and (3) fertility is related to the interval DIU-AI and ovulation which should be - 8 to -4 h of spontaneous ovulation and to the package, MFP having shown better results in vivo. The results stress the need for careful, and frequent, control of oestrus signs. (c) 2005 Elsevier Inc. All rights reserved.

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