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  • 1.
    Alvarez-Rodriguez, Manuel
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences. Univ Autonoma Barcelona, Spain.
    Alvarez, M.
    Univ Leon, Spain.
    Anel-Lopez, L.
    Univ Leon, Spain.
    Guerra, C.
    Univ Leon, Spain.
    Chamorro, C. A.
    Univ Leon, Spain.
    Anel, L.
    Univ Leon, Spain.
    de Paz, P.
    Univ Leon, Spain.
    Martinez-Pastor, F.
    Univ Leon, Spain.
    Effect of length of time post-mortem on quality and freezing capacity of Cantabric chamois (Rupicapra pyrenaica parva) epididymal spermatozoa2018In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 198, p. 184-192Article in journal (Refereed)
    Abstract [en]

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

  • 2.
    Anel-López, Luis
    et al.
    Biology of Reproduction Group. National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Alvarez-Rodríguez, Manuel
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    García-Álvarez, Olga
    Biology of Reproduction Group. National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Maroto-Morales, Alejandro
    Biology of Reproduction Group. National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain.
    Garde, J Julián
    Biology of Reproduction Group. National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), Albacete, Spain.
    Martínez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain.
    Reduced glutathione and Trolox (vitamin E) as extender supplements in cryopreservation of red deer epididymal spermatozoa2012In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 135, no 1-4, p. 37-46Article in journal (Refereed)
    Abstract [en]

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

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

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

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

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

  • 5.
    Bergqvist, AS
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Killian, G
    Pennsylvania State University, USA; .
    Erikson, D
    Pennsylvania State University, USA; .
    Hoshino, Y
    Tohoku University,, Japan; .
    Bage, R
    Swedish University of Agriculture Science, Sweden; .
    Sato, E
    Tohoku University,, Japan; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Detection of Fas ligand in the bovine oviduct2005In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 86, no 1-2, p. 71-88Article in journal (Refereed)
    Abstract [en]

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

  • 6.
    Bergqvist, AS
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Sulphated glycosaminoglycans (S-GAGs) and syndecans in the bovine oviduct2006In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 93, no 1-2, p. 46-60Article in journal (Refereed)
    Abstract [en]

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

  • 7.
    Brandt, Y.
    et al.
    Swedish University of Agriculture Science SLU, Sweden .
    Einarsson, S.
    Swedish University of Agriculture Science SLU, Sweden .
    Ljung, A.
    Swedish University of Agriculture Science SLU, Sweden .
    Lundeheim, N.
    SLU, Sweden .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science SLU, Sweden .
    Madej, A.
    SLU, Sweden .
    Effects of continuous elevated cortisol concentrations during oestrus on concentrations and patterns of progesterone, oestradiol and LH in the sow2009In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 110, no 1-2, p. 172-185Article in journal (Refereed)
    Abstract [en]

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

  • 8.
    Brandt, Y
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Lang, A
    Swedish University of Agriculture Science, Sweden; .
    Madej, A
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Einarsson, S
    Swedish University of Agriculture Science, Sweden; .
    Impact of ACTH administration on the oviductal sperm reservoir in sows: The local endocrine environment and distribution of spermatozoa2006In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 92, no 1-2, p. 107-122Article in journal (Refereed)
    Abstract [en]

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

  • 9.
    Brandt, Y
    et al.
    Swedish University of Agriculture Science, Sweden; .
    Lang, A
    Swedish University of Agriculture Science, Sweden; .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science, Sweden; .
    Madej, A
    Swedish University of Agriculture Science, Sweden; .
    Einarsson, S
    Swedish University of Agriculture Science, Sweden; .
    Impact of ACTH during oestrus on the ultrastructure of the spermatozoa and their environment in the tubal reservoir of the postovulatory sow2006In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 93, no 3-4, p. 231-245Article in journal (Refereed)
    Abstract [en]

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

  • 10.
    Crespo-Felez, I.
    et al.
    University of Leon, Spain.
    Castaneda-Sampedro, A.
    University of Leon, Spain.
    Sanchez, D. I.
    University of Leon, Spain.
    Fernandez-Alegre, E.
    University of Leon, Spain.
    Alvarez-Rodriguez, Manuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Dominguez, J. C.
    University of Leon, Spain.
    Morrell, J. M.
    Swedish University of Agriculture Science, Sweden.
    Martinez-Pastor, F.
    University of Leon, Spain; University of Leon, Spain.
    Effect of Single Layer Centrifugation Porcicoll (70%, 80% and 90%) or supplementation with reduced glutathione, seminal plasma and bovine serum albumin on frozen-thawed boar sperm2017In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 187, p. 167-173Article in journal (Refereed)
    Abstract [en]

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

  • 11.
    Einarsson, Stig
    et al.
    Swedish University of Agriculture Science SLU.
    Brunius, Carl
    SLU.
    Wallgren, Margareta
    Swedish University of Agriculture Science SLU.
    Lundström, Kerstin
    SLU.
    Andersson, Kristina
    SLU.
    Zamaratskaia, Galia
    SLU.
    Rodriguez-Martinez, Heriberto
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Genetics.
    Effects of early vaccination with Improvac (R) on the development and function of reproductive organs of male pigs2011In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 127, no 1-2, p. 50-55Article in journal (Refereed)
    Abstract [en]

    Gonadotropin-releasing hormone (GnRH) vaccine (Improvac (R)) is effective at diminishing boar taint by interfering with testis function. Early pre-pubertal vaccination at 10 and 14 weeks-of-age could be desirable if sufficient and sustained effects could be achieved. Crossbred male pigs (n = 24) were randomly assigned to three groups each with eight individuals: an unvaccinated control group, one group vaccinated with Improvac (R) early at ages 10 and 14 weeks, and a third group vaccinated with Improvac at the standard ages of 16 and 20 weeks. The average age at slaughter was 25 weeks. At slaughter, reductions in testes weight and bulbourethral gland length of vaccinated pigs compared with controls were observed (P andlt; 0.001), accompanied by lowered testosterone concentrations in peripheral blood (P andlt; 0.001). The diameter of tubuli seminiferi was affected: being 18% smaller in standard and 38% smaller in early vaccinated males, compared with controls (P andlt; 0.01). Leydig cells in vaccinated pigs became pycnotic, and their number decreased in early vaccinated pigs. Spermatogenesis was disrupted, evidenced by spermatocyte loss among standard vaccinated pigs to severe spermatogenic arrest among early vaccinated pigs. This histological picture was reflected in the absence of epididymal spermatozoa in 5 of 8 early vaccinated pigs and a dramatic reduction in the remaining 3 early vaccinated pigs. Among standard vaccinated pigs, 5% of the spermatozoa were morphologically normal (andgt;70% in controls, P andlt; 0.01). Early vaccination caused a more severe disruption of testicular structure and function than standard vaccination, thus providing an alternative for immunocastration of male pigs.

  • 12.
    Johannisson, A.
    et al.
    Swedish University of Agriculture Science SLU, Sweden .
    Morrell, J.M.
    Swedish University of Agriculture Science SLU, Sweden .
    Thoren, J.
    Swedish University of Agriculture Science SLU, Sweden .
    Jonsson, M.
    Swedish University of Agriculture Science SLU, Sweden .
    Dalin, A.-M.
    Swedish University of Agriculture Science SLU, Sweden .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science SLU, Sweden .
    Colloidal centrifugation with Androcoll-E (TM) prolongs stallion sperm motility, viability and chromatin integrity2009In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 116, no 1-2, p. 119-128Article, review/survey (Refereed)
    Abstract [en]

    The objective was to investigate the changes in stallion sperm quality (sperm motility, viability, membrane integrity and chromatin integrity) occurring during cool storage, and to study the effect of sperm selection by single layer colloidal centrifugation on these parameters of sperm quality. Spermatozoa from 3 stallions (10 ejaculates, 3-4 per stallion) were selected by centrifugation through a single layer of colloid (SLC). The resulting sperm preparations and the control samples (extended but unselected semen samples) were stored at 5 degrees C for 48 h. Assessments of sperm quality, such as sperm motility, viability (SYBR-14/PI staining), membrane stability (Annexin-V/PI staining) and chromatin integrity, were performed on aliquots of the selected sperm preparations and unselected samples on the day of collection (3 h) and after 24 and 48 h of storage. In the SLC-selected sperm samples, sperm motility, sperm viability, proportions of spermatozoa with normal morphology and with intact chromatin were significantly better than in unselected samples (motility: 77 +/- 4% vs. 64 +/- 8% at 3 h; P less than 0.001; viability: 79.5 +/- 9% vs. 64.7 +/- 9%, P less than 0.001: normal morphology 89 +/- 6% vs. 69 9%; chromatin integrity DFI 11.3 +/- 5% vs. 22.1 +/- 10%). Membrane stability, however, was not different in the SLC-selected and unselected samples (74.6 +/- 8% vs. 69.3 +/- 8%). The deterioration seen in sperm quality in the unselected samples was prevented by SLC, so that sperm viability, membrane stability and chromatin integrity were unchanged in the selected samples by 48 h compared to 3 h (Pless than0.001), whereas the unselected samples were significantly worse by 48 h (Pless than0.001). Furthermore, it should be possible to send an aliquot of a normal insemination dose (i.e. unselected spermatozoa) overnight to a reference laboratory for analysis of both plasma membrane and chromatin integrity. In conclusion, centrifugation of stallion spermatozoa through a single layer of colloid is a useful technique for selecting the best spermatozoa from an ejaculate and, moreover, sperm quality is maintained during storage. (C) 2009 Elsevier B.V. All rights reserved.

  • 13.
    Li, Junwei
    et al.
    Univ Murcia, Spain.
    Roca, Jordi
    Univ Murcia, Spain.
    Perez-Patino, Cristina
    Univ Murcia, Spain.
    Barranco, Isabel
    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.
    Is boar sperm freezability more intrinsically linked to spermatozoa than to the surrounding seminal plasma?2018In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 195, p. 30-37Article in journal (Refereed)
    Abstract [en]

    This study aimed to elucidate the effect of seminal plasma (SP) from post-SRF on boar sperm freezability and, in addition, to determine the relevance of sperm itself to sustain cryopreservation, regardless of the SP surrounding them. Twelve ejaculates from three boars were manually collected in fractions/portions, P1: the first 10 mL of the SRF, P2: the rest of the SRF and the post-SRF. Immediately, samples were centrifuged to separate spermatozoa from the surrounding SP. Spermatozoa from P1 and P2 were then incubated with its own SP or that from post-SRF, diluted in BTS (1:1, v/v) at 17 degrees C overnight before being frozen in 0.5 mL straws using a standard protocol. Sperm motility (total and progressive) deteriorated (P amp;lt; 0.05) when P1- or P2-sperm when incubated overnight in SP from post-SRF, while sperm viability differed between P1 and P2 (P amp;lt; 0.05) regardless of the SP they were incubated in. Post-thaw sperm quality and functionality differed between P1 and P2, regardless of the SP used for overnight pre-freezing incubation. Post-thaw motility (P amp;lt; 0.05) and viability (P amp;lt; 0.01), as well as plasma membrane fluidity (P amp;lt; 0.05) or lipid peroxidation values (P amp;lt; 0.01) were best in P1 sperm compared to those of P2. The protein profile of sperm from P1 and P2, analyzed by 2D-PAGE, showed qualitative differences, which suggest that sperm rather than SP would explain differences in sperm freezability between ejaculate fractions/portions. Use of P1 fraction spermatozoa seems thus optimal for cryopreservation.

  • 14.
    Macias Garcia, B.
    et al.
    University of Extremadura, Spain .
    Morrell, J.M.
    University of Extremadura, Spain.
    Ortega-Ferrusola, C.
    University of Extremadura, Spain .
    Gonzalez-Fernandez, L.
    University of Extremadura, Spain .
    Tapia, J.A.
    University of Extremadura, Spain .
    Rodriguez-Martinez, Heriberto
    SLU, Sweden.
    Pena, F.J.
    University of Extremadura, Spain .
    Centrifugation on a single layer of colloid selects improved quality spermatozoa from frozen-thawed stallion semen2009In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 114, no 1-3, p. 193-202Article in journal (Refereed)
    Abstract [en]

    The present study attempted to select the subpopulation of stallion spermatozoa that best survived a conventional freezing and thawing procedure, using centrifugation of post-thawed semen samples through a single layer of a glycidoxypropyltrimethoxysilane-coated silica colloid with a species-specific formulation (Androcoll-E (TM)). Sperm motility, sperm chromatin structure, membrane integrity and mitorchondrial membrane potential were studied in filtered and non-filtered spermatozoa. Single-layer centrifugation (SLC) using Androcoll-E (TM) significantly improved all the sperm parameters studied, implying SLC may be a simple approach to improve the quality of frozen-thawed (FT) spermatozoa for AI. (c) 2008 Elsevier B.V. All rights reserved.

  • 15.
    Mata-Campuzano, María
    et al.
    INDEGSAL, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
    Álvarez-Rodríguez, Manuel
    INDEGSAL, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, University of León, León, Spain; DEPI, Institute of Technology of Conkal, Conkal, Mexico.
    Tamayo-Canul, Julio
    INDEGSAL, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
    López-Urueña, Elena
    INDEGSAL, University of León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, University of León, León, Spain.
    de Paz, Paulino
    INDEGSAL, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
    Anel, Luis
    INDEGSAL, University of León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, University of León, León, Spain.
    Martínez-Pastor, Felipe
    INDEGSAL, University of León, León, Spain; Department of Molecular Biology, University of León, León, Spain.
    Álvarez, Mercedes
    INDEGSAL, University of León, León, Spain; Department of Medicine, Surgery and Veterinary Anatomy, University of León, León, Spain.
    Refrigerated storage of ram sperm in presence of Trolox and GSH antioxidants: effect of temperature, extender and storage time2014In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 151, no 3-4, p. 137-147Article in journal (Refereed)
    Abstract [en]

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

  • 16.
    Morillo Rodriguez, A
    et al.
    University of Extremadura, Cáceres, Spain.
    Ortega Ferrusola, C
    University of Extremadura, Cáceres, Spain.
    Macias Garcia, B
    University of Extremadura, Cáceres, Spain.
    Morrell, J M
    Swedish University of Agriculture Science.
    Rodriguez-Martinez, Heriberto
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Genetics.
    Tapia, J A
    University of Extremadura, Cáceres, Spain.
    Pena, F J
    University of Extremadura, Cáceres, Spain.
    Freezing stallion semen with the new Caceres extender improves post thaw sperm quality and diminishes stallion-to-stallion variability2011In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 127, no 1-2, p. 78-83Article in journal (Refereed)
    Abstract [en]

    Ejaculates from 7 stallions were split and simultaneously frozen in three different extenders, INRA 96 egg yolk glycerol, Ghent and the newly developed extender Caceres. After thawing, samples were evaluated for motility (CASA system) sperm membrane integrity and early membrane changes (YoPro-1/Eth staining), acrosome integrity (FICT-PNA), and mitochondrial membrane potential (JC-1) (flow cytometry). Samples frozen in Caceres extender consistently showed the best results in post-thaw motility (increases ranging from 11 to 17%, p andlt; 0.05) and velocity (p andlt; 0.05), membrane integrity (increases ranging from 11 to 14%, p andlt; 0.05) and mitochondrial membrane potential (p andlt; 0.05). It is concluded that this new extender should be included in a freezeability test to determine the best extender for each individual.

  • 17.
    Najafi, Abouzar
    et al.
    Department of Animal Science, College of Agriculture, University of Tabriz, Tabriz, Iran.
    Daghigh-Kia, Hossein
    Department of Animal Science, College of Agriculture, University of Tabriz, Tabriz, Iran.
    Dodaran, Hossein Vaseghi
    Department of Animal Science, College of Agriculture, University of Tabriz, Tabriz, Iran.
    Mehdipour, Mahdieh
    Department of Animal Science, College of Agriculture, University of Tabriz, Tabriz, Iran.
    Alvarez-Rodriguez, Manuel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Ethylene glycol, but not DMSO, could replace glycerol inclusion in soybean lecithin-based extenders in ram sperm cryopreservation.2017In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 177, p. 35-41Article in journal (Refereed)
    Abstract [en]

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

  • 18.
    Ortega-Ferrusola, C.
    et al.
    University of Extremadura, Spain .
    Macias Garcia, B.
    University of Extremadura, Spain .
    Gallardo-Bolanos, J.M.
    University of Extremadura, Spain .
    Gonzalez-Fernandez, L.
    University of Extremadura, Spain .
    Rodriguez-Martinez, Heriberto
    SLU, Sweden .
    A. Tapia, J.
    University of Extremadura, Spain .
    J. Pena, F.
    University of Extremadura, Spain .
    Apoptotic markers can be used to forecast the freezeability of stallion spermatozoa2009In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 114, no 4, p. 393-403Article in journal (Refereed)
    Abstract [en]

    In an attempt to identify valuable markers for potential freezeability of the equine spermatozoa, three ejaculates were collected from five Andalusian stallions and frozen using a standard protocol. Before freezing, three apoptotic cell markers were studied by flow cytometry (early changes in sperm membranes, mitochondrial membrane potential and caspase activity). Post-thaw, spermatozoa were again evaluated for these parameters. Sperm kinematics using CASA were also studied before and after freezing and thawing. Receiving operating system curves were used to evaluate the relative value of the apoptotic markers herein studied, as forecast for potential freezeability. From all parameters studied, the outcome of JC-1 (as proportion of spermatozoa showing simultaneously orange and green fluorescence) had the highest diagnostic power. For potentially bad freezers (less than 25% of intact spermatozoa post-thaw), the significant area under the ROC-curve was 0.985, with a 100% sensitivity and 99.8% specificity for a cut off value of 55.7. (C) 2008 Elsevier B.V. All rights reserved.

  • 19.
    Pena, F.J.
    et al.
    Division of Comparative Reproduction, Obstetrics and Udder Health, Department of Clinical Sciences, Spain.
    Saravia, F.
    Division of Comparative Reproduction, Obstetrics and Udder Health, Department of Clinical Sciences, Spain.
    Johannisson, A.
    SLU, Sweden; .
    Wallgren, M.
    Division of Comparative Reproduction, Obstetrics and Udder Health, Department of Clinical Sciences, Spain.
    Rodriguez-Martinez, Heriberto
    Division of Comparative Reproduction, Obstetrics and Udder Health, Department of Clinical Sciences, Spain.
    Detection of early changes in sperm membrane integrity pre-freezing can estimate post-thaw quality of boar spermatozoa2007In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 97, no 1-2, p. 74-83Article in journal (Refereed)
    Abstract [en]

    A recently developed triple staining (SNARF-1/YO-PRO-1/ethidium homodimer) was used to assess early changes in boar sperm membrane integrity (MI) with the results of cryopreservation procedures and to seek for correlations among MI-spermatozoa in pre-freeze semen and its freezeability. Ejaculates from five boars were evaluated in the fresh and frozen-thawed (FT) state, and its freezeability defined as % of membrane intactness, MI% (MI% = % of FT-spermatozoa with intact membranes x 100 divided by the % of prefreeze spermatozoa with intact membranes) estimated. Significant differences were found among boars for freezeability (MI%) and motility post-thaw (%). Interestingly, significant correlations were found between the percentage of YO-PRO-1-positive spermatozoa and freezeability (R = 0.440, p less than 0.01), indicating this new triple staining can be used to safely disclose among ejaculates prior to freezing. (c) 2006 Elsevier B.V. All rights reserved.

  • 20.
    Roca, J.
    et al.
    University of Murcia, Spain.
    Parrilla, I.
    University of Murcia, Spain.
    Gil, M. A.
    University of Murcia, Spain.
    Cuello, C.
    University of Murcia, Spain.
    Martinez, E. A.
    University of Murcia, Spain.
    Rodriguez-Martinez, Heriberto
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Non-viable sperm in the ejaculate: Lethal escorts for Contemporary viable sperm2016In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 169, p. 24-31Article in journal (Refereed)
    Abstract [en]

    Non-viable sperm ("dead sperm") are present invariable numbers in mammalian ejaculates and their number increase substantially when semen is stored, particularly cryopreserved. This review comparatively highlights, with experimental data in porcine, the role-played by non-viable sperm in the outcome of semen used in assisted reproductive technologies. As well, the review discusses our current understanding of their origin and the pathways involved when their large numbers negative influence the functional lifespan of contemporary viable sperm to eventually cause irreversible dysfunction that reduces their fertility potential and their ability to develop healthy embryos. Finally, it highlights procedures currently available to mitigate these harmful effects. (C) 2016 Elsevier B.V. All rights reserved.

  • 21.
    Saravia, F.
    et al.
    Swedish University of Agriculture Science SLU, Sweden University of Concepcion, Chile .
    Wallgren, M.
    Swedish University of Agriculture Science SLU, Sweden Qual Genet, Sweden .
    Rodriguez-Martinez, Heriberto
    Swedish University of Agriculture Science SLU, Sweden .
    Freezing of boar semen can be simplified by handling a specific portion of the ejaculate with a shorter procedure and MiniFlatPack packaging2010In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 117, no 3-4, p. 279-287Article in journal (Refereed)
    Abstract [en]

    Low sperm survival post-thaw and time-consuming procedures for conventional freezing (CF) hamper the commercial application of cryopreserved boar semen. We had previously proven that boar spermatozoa in the first 10 mL of the sperm-rich fraction, SRF (the so-called PI, the sperm-peak portion of the ejaculate) sustain best handling in vitro, since they probably bathe in an aliquot of seminal plasma (SP) with specific composition. Here, we performed three experiments to determine: Exp I: the concentration of bicarbonate among portions of the ejaculate; Exp II: the effects of bicarbonate doses on sperm motility and; Exp III: the outcome of a faster, simpler freezing method (SF), handling PI-spermatozoa packed in MiniFlatPacks(TM) (MFP) vs. CF and vs. SRF-spermatozoa (2 x 2 factorial design). The bicarbonate content in SP was, among portions/fractions of the ejaculate, lowest in P1 (13.71 mM/L, P less than 0.0001, Exp 1). Boar spermatozoa require bicarbonate in the extender (to the levels present in PI) to maintain acceptable motility over a 120-h period at 16-17 degrees C (Exp II). Sperm freezing was dramatically shortened (from 8 to 3.5 h) by the SF-procedure. P1 - and SRF-spermatozoa survived equally both CF- and SF-freezing (% total motility 30 min PT; P1-CF: 65.2 +/- 5.4% and P1-SF: 68.9 +/- 2.4%; SRF-CF: 64.4 +/- 2.7%; SRF-SF: 55.8 +/- 3.1%, ns). Interestingly. in contrast to SRF, there were no significant variations in 30-min PT-survival among either ejaculates or boars when the P1 was frozen, independent of the handling method (CF or SF). in conclusion, such a faster freezing protocol of semen packed in MFP could be advantageously applied to P1-spermatozoa (P1-SF), while the rest of the ejaculated spermatozoa could still be used for production of conventional artificial insemination (AI) doses, thus allowing for a maintained routine management of commercially relevant stud boars. (C) 2009 Elsevier B.V. All rights reserved.

  • 22.
    Sharoare Hossain, Md.
    et al.
    Swedish University of Agriculture Science SLU.
    Johannisson, Anders
    Swedish University of Agriculture Science SLU.
    Pimenta Siqueira, Amanda
    Swedish University of Agriculture Science SLU.
    Wallgren, Margareta
    Swedish University of Agriculture Science SLU.
    Rodriguez-Martinez, Heriberto
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Genetics.
    Spermatozoa in the sperm-peak-fraction of the boar ejaculate show a lower flow of Ca(2+) under capacitation conditions post-thaw which might account for their higher membrane stability after cryopreservation2011In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 128, no 1-4, p. 37-44Article in journal (Refereed)
    Abstract [en]

    Boar spermatozoa collected in the ejaculate sperm peak-portion (P1, first 10 mL of the sperm-rich fraction, SRF), had shown a higher resilience to freezing and thawing compared to spermatozoa from the rest of the ejaculate (2nd portion of the SRF plus the post-sperm-rich fraction, PSRF), even when using a simplified freezing technique, as long as spermatozoa were incubated in their own seminal plasma (SP). This experiment studied the stability of P1- and SRF-P1 boar spermatozoa frozen in MiniFlatPacks (MFP), post-thaw, using flow cytometry. Since spermatozoa from either portion showed similar cryosurvival and low proportions of unstable membranes (less than3%, annexin-V/propidium iodide staining), and only a tendency for SRF-P1 live spermatozoa to depict acrosome exocytosis (FITC-PNA/PI/H33342); they were explored for Ca(2+) contents using a Fluo-4 probe under in vitro capacitating conditions (mBO+ medium), as well they were tested for their ability to sustain a short Ca(2+)-ionophore (A23187) in vitro challenge. The proportions of live spermatozoa depicting high Ca(2+)-levels were initially less than2% but increased over incubation time, particularly in SRF-P1(P less than 0.05), while proportions of live spermatozoa with low Ca(2+)-levels were basically constant over incubation time (similar to 11-14%), for either portion. Incubation in capacitation medium did not modify the proportions of low-Ca(2+) but dramatically increased the proportions of high-Ca(2+) spermatozoa (P less than 0.001) already after 15 min exposure, highest for SRF-P1 spermatozoa. While the proportion of live spermatozoa with intact acrosome was significantly decreased among SRF-P1 (P less than 0.001), that of P1-spermatozoa remained unchanged, probably owing to the lowest relative content of cytosolic Ca(2+). The results suggest that spermatozoa in the P1-portion are more resilient to express acrosome exocytosis post-thaw compared to those bathing in the rest of the SRF-fraction when cryopreserved using a simplified technique, in MFPs.

  • 23.
    Tamayo-Canul, Julio
    et al.
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Alvarez, Mercedes
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Mata-Campuzano, Maria
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology, University of León, León, Spain.
    Alvarez-Rodríguez, Manuel
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    de Paz, Paulino
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology, University of León, León, Spain.
    Anel, Luis
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
    Martínez-Pastor, Felipe
    ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology, University of León, León, Spain.
    Effect of storage method and extender osmolality in the quality of cryopreserved epididymal ram spermatozoa.2011In: Animal Reproduction Science, ISSN 0378-4320, E-ISSN 1873-2232, Vol. 129, no 3-4, p. 188-199Article in journal (Refereed)
    Abstract [en]

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

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