| 1. |
- Bergqvist, Ann-Sofi, et al.
(författare)
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Single Layer Centrifugation of Stallion Spermatozoa through Androcoll (TM)-E does not Adversely Affect their Capacitation-Like Status, as Measured by CTC Staining
- 2011
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Ingår i: Reproduction in domestic animals. - : Blackwell Publishing. - 0936-6768 .- 1439-0531. ; 46:1, s. e74-e78
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Tidskriftsartikel (refereegranskat)abstract
- Contents This study was designed to evaluate the effect of single layer centrifugation (SLC) and subsequent cold storage on stallion sperm capacitation-like status and acrosome reaction. Three stallions were included in the study, with three ejaculates per stallion. The samples were examined 4, 24 and 72 h after collection, extension and SLC, with storage at 6 degrees C. Sperm capacitation-like status was investigated using the fluorescent dye chlortetracycline (CTC). There was no difference in capacitation-like status between colloid-selected and non-selected spermatozoa. Sperm motility decreased significantly during cold storage, whereas the proportion of apparently capacitated spermatozoa increased. There was no change in the proportion of acrosome-reacted spermatozoa. In conclusion, SLC through Androcoll (TM)-E does not adversely affect the capacitation-like status of stallion spermatozoa, although it did increase with time during cold storage.
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| 2. |
- Kumaresan, A., et al.
(författare)
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Quantification of kinetic changes in protein tyrosine phosphorylation and cytosolic Ca2+ concentration in boar spermatozoa during cryopreservation
- 2012
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Ingår i: Reproduction, Fertility and Development. - 1031-3613 .- 1448-5990. ; 24:4, s. 531-542
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Tidskriftsartikel (refereegranskat)abstract
- Protein tyrosine phosphorylation in sperm is associated with capacitation in several mammalian species. Although tyrosine phosphorylated proteins have been demonstrated in cryopreserved sperm, indicating capacitation-like changes during cryopreservation, these changes have not yet been quantified objectively. We monitored tyrosine phosphorylation, intracellular calcium and sperm kinematics throughout the cryopreservation process, and studied the relationships among them in boar spermatozoa. Sperm kinetics changed significantly during cryopreservation: curvilinear velocity, average path velocity and straight line velocity all decreased significantly (P < 0.05). While the percentage of sperm with high intracellular calcium declined (P < 0.05), global phosphorylation increased significantly (P < 0.01). Specifically, cooling to 5 degrees C induced phosphorylation in the spermatozoa. After cooling, a 32-kDa protein not observed in fresh semen appeared and was consistently present throughout the cryopreservation process. While the level of expression of this phosphoprotein decreased after addition of the second extender, frozen-thawed spermatozoa showed an increased expression. The proportion of sperm cells with phosphorylation in the acrosomal area also increased significantly (P < 0.05) during cryopreservation, indicating that phosphorylation might be associated with capacitation-like changes. These results provide the first quantitative evidence of dynamic changes in the subpopulation of boar spermatozoa undergoing tyrosine phosphorylation during cryopreservation.
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| 3. |
- Morrell, Jane, et al.
(författare)
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Effect of osmolarity and density of colloid formulations on the outcome of SLC-selection of stallion spermatozoa
- 2011
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Ingår i: ISRN Veterinary Science. - : Hindawi Limited. - 2090-4452 .- 2090-4460. ; 2011, s. 1-5
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Tidskriftsartikel (refereegranskat)abstract
- The osmolarity and density of colloids used to prepare spermatozoa for assisted reproduction may affect sperm quality in the resultant preparation. In this study, two osmolarities of Androcoll-E for single-layer or density gradient centrifugation of stallion spermatozoa were compared: “normal” (320 mOsm) or “high” (345 mOsm). Mean yields for the two centrifugation techniques did not differ between treatments or osmolarities (single layer centrifugation:30.19±16.9×106and25.8±18.5×106spermatozoa; density gradient centrifugation:31.84±19.7×106and26.46±20.0×106spermatozoa respectively for the two osmolarities). However, use of the high osmolarity colloid for single layer centrifugation increased the proportion of morphologically normal spermatozoa (P<.05). Therefore, increasing the osmolarity of the colloid formulation may be beneficial for processing ejaculates containing a high proportion of abnormal spermatozoa by SLC. Reducing the density of the colloid used for the SLC substantially increased the yield of motile spermatozoa compared to the normal density colloid (mean ± SD:72.6±28.9×106versus28.9±24.7×106), while also prolonging sperm survival by 24 hours compared to the uncentrifuged ejaculate. This increased yield may render Single Layer Centrifugation practical for use in the field.
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| 4. |
- Morrell, Jane, et al.
(författare)
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Single layer centrifugation of stallion spermatozoa improves sperm quality compared with sperm washing
- 2010
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Ingår i: Reproductive BioMedicine Online. - : Elsevier. - 1472-6483 .- 1472-6491. ; 21:3, s. 429-436
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Tidskriftsartikel (refereegranskat)abstract
- This study compared the effect on semen quality of different handling methods used in the preparation of stallion semen doses for artificial insemination. The three methods were (i) extending the ejaculate to 30-50 x 10(6)/ml, (ii) single layer centrifugation (SLC) and (iii) sperm washing (centrifugation without a colloid). An additional treatment was to add seminal plasma (SP) in various proportions to some SLC preparations. The resulting samples were evaluated for sperm motility by computer assisted sperm analysis, membrane integrity using the Nucleocounter SP-100 and chromatin integrity by the sperm chromatin structure assay. SLC samples consistently had better sperm quality than the extended samples. Sperm washing did not confer any beneficial effect compared with the extended samples and these samples had significantly worse sperm quality than the SLC samples (motility, P less than 0.01; viability, P less than 0.001). There was no evidence to suggest that adding SP to the SLC samples could enhance sperm motility for more than a few hours. Longer term cold storage of spermatozoa in the presence of small concentrations of SP resulted in a reduction in total motility and progressive motility compared with SLC alone. High concentrations of SP were detrimental to sperm survival. (C) 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
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| 5. |
- Ortega-Ferrusola, Cristina, et al.
(författare)
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Effect of Different Extenders and Seminal Plasma on the Susceptibility of Equine Spermatozoa to Lipid Peroxidation After Single-Layer Centrifugation, Through Androcoll-E
- 2011
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Ingår i: Journal of Equine Veterinary Science. - : Elsevier Science B. V., Amsterdam. - 0737-0806 .- 1542-7412. ; 31:7, s. 411-416
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Tidskriftsartikel (refereegranskat)abstract
- his study was conducted in an attempt to see whether single-layer centrifugation (SLC) increases the susceptibility of stallion spermatozoa to lipid peroxidation (LPO), in different extenders after removing all seminal plasma (SP). The susceptibility of stallion spermatozoa to LPO was studied before and after SLC. Each ejaculate was split, and aliquots extended with one of the three different extenders: INRA 96, Kenneys, or Equipro, and stored for 24 hours at 5 degrees C (i). From the extended samples, an aliquot was kept as a control and the other was subjected to SLC through Androcoll-E. The selected spermatozoa were re-suspended in the appropriate extenders, without (ii) or with (iii) addition of 50% (v/v) pooled homologous SP for 24 hours at 5 degrees C. Using ferrous sulfate as pro-oxidant, the susceptibility for LPO was flow-cytometrically assessed using the probe Bodipy(581/591)-C(11). Sperm motility, monitored with a Qualisperm motility analyzer, increased after SLC treatment (P andlt; .001). No significant correlations were found between motility and induced LPO with ferrous sulfate. The SP and extenders, per se, did not have a significant protective effect against LPO, but the interaction between SP and Kenney increased the susceptibility to LPO. However, the selected spermatozoa through Androcoll-E and the subsequent dilution in INRA had a significant protective effect against LPO (P andlt; .05), especially when the oxidative insults were higher (80 mu M).
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| 6. |
- Sharoare Hossain, Md, et al.
(författare)
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Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art
- 2011
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Ingår i: ASIAN JOURNAL OF ANDROLOGY. - : Blackwell Publishing Ltd. - 1008-682X .- 1745-7262. ; 13:3, s. 406-419
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Forskningsöversikt (refereegranskat)abstract
- Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of bench-top flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa.
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| 7. |
- Siqueira, A P, et al.
(författare)
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Quality of boar spermatozoa from the sperm-peak portion of the ejaculate after simplified freezing in MiniFlatpacks compared to the remaining spermatozoa of the sperm-rich fraction
- 2011
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Ingår i: THERIOGENOLOGY. - : Elsevier Science B.V., Amsterdam.. - 0093-691X .- 1879-3231. ; 75:7, s. 1175-1184
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Tidskriftsartikel (refereegranskat)abstract
- Boar sperm viability post-thaw differs depending on the ejaculate fraction used, with spermatozoa present in the first 10 mL of the sperm-rich fraction (SRF) (portion 1, P1, sperm-peak portion) displaying the best cryosurvival in vitro compared with that of spermatozoa from the rest of the ejaculate (portion 2 of the SRF plus the post-spermatic fraction), even when using simplified freezing routines. This viability apparently relates to the specific profile of seminal plasma in P1 (i.e., glycoprotein and bicarbonate concentrations, and pH). However, spermatozoa from PI have not been compared with spermatozoa from the rest of the SRF (SRF P1, usually 30-40 mL of the SRF), which is routinely used for freezing. We compared P1 with SRF P I in terms of sperm kinematics (using the QualiSperm (TM) system), while membrane integrity (SYBR-14/PI), acrosome integrity (FITC PNA/PI), and sperm membrane stability (Annexin-V) were explored using flow cytomety. As well, total protein concentration and the proteomics of the seminal plasma (SP) of both portions of the SRF were studied using two-dimensional electrophoresis (2DE), mass fingerprinting (MALDI-TOF), and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) on selected peptides. The SRF portions were collected weekly from four mature boars (4-5 replicates per boar, sperm concentration: P1, 1.86 +/- 0.20; SRF P1, 1.25 +/- 0.14 x 10(9) spz/mL) and processed using a quick freezing method in MiniFlatPacks. Post-thaw sperm motility reached 50%, without differences between SRF portions, but with clear inter-boar variation. Neither plasma membrane nor acrosome integrity differed (ns) between fractions. These results indicate that there are no differences in cryosurvival after quick freezing of boar spermatozoa derived from either of the two SRF portions. While P1 and SRF-P1 clearly differed in relative total protein contents, as expected, they displayed very similar protein profiles as assessed using 2DE and mass spectrometry (tryptic peptide mass fingerprint analysis and CID-MS/MS), indicating a similar emission of epididymal protein content.
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| 8. |
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| 9. |
- Jansson, Anna, et al.
(författare)
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Plasma aldosterone concentration and cardiovascular response to low sodium intake in horses in training
- 2010
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Ingår i: Equine Veterinary Journal. - : Wiley. - 0425-1644 .- 2042-3306. ; 42, s. 329-334
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Tidskriftsartikel (refereegranskat)abstract
- P>Reason for performing the study:Horses in training lose large amounts of sodium but little is known about the cardiovascular response to low sodium intake.Objectives:To investigate the effect of low sodium intake on plasma aldosterone (pAldo) concentrations and the cardiovascular system of athletic horses, and to identify markers of low sodium intake.Methods:Seven Standardbred geldings in training (trained twice a week) were randomly offered a standardised diet supplemented (NaS, 58 mg Na/kg bwt) and not supplemented (NaN, 3 mg Na/kg bwt) with NaCl for 5 weeks in a changeover design. Blood samples were taken once a week and in Week 5, before and following an exercise test until 22.30 h and analysed for blood sodium (bNa), total plasma protein (TPP), pAldo, troponin I and packed cell volume (PCV). Blood pressure (BP) was measured and pulse wave recorded at rest with high definition oscillometric-technique (HDO). ECG and echocardiography were recorded. Water intake was measured before and on the day of exercise and voluntary saline intake was measured for 2 days after each period. Faecal samples were taken weekly and analysed for sodium and potassium content.Results:The pAldo and the PCV was higher in NaN compared to NaS. There were no differences between diets in BP, ECG, plasma troponin I and echocardiogram but HDO pulse amplitude tended to be smaller on diet NaN. Water intake was lower on diet NaN and saline intake higher. The response to exercise in bNa, pAldo, PCV and TPP was different on the 2 diets. Faecal potassium/sodium ratio was higher on NaN than on NaS.Conclusion:This study shows that 5 weeks of low sodium intake increased plasma aldosterone concentration and PCV but no alterations in heart function was observed. Faecal potassium/sodium ratio could be used to assess sodium status in horses.
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| 10. |
- Johannisson, Anders
(författare)
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Bronchial microdialysis of cytokines in the epithelial lining fluid in experimental intestinal ischemia and reperfusion before onset of manifest lung injury
- 2010
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Ingår i: Shock. - 1073-2322 .- 1540-0514. ; 34, s. 517-524
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Tidskriftsartikel (refereegranskat)abstract
- Today, there is no continuous monitoring of the bronchial epithelial lining fluid. This study used microdialysis as a method of continuous monitoring of early lung cytokine response secondary to intestinal ischemia-reperfusion in pigs. The authors aimed to examine bronchial microdialysis for continuous monitoring of IL-1 beta, TNF-alpha, IL-8, and fluorescein isothiocyanate Dextran 4,000 Da (FD-4). The superior mesenteric artery was cross-clamped for 120 min followed by 240 min of reperfusion (ischemia group, n = 8). Four sham-operated pigs served as controls. The pigs were anesthetized and normoventilated (peak inspiratory pressure, <20 cm H(2)O; positive end-expiratory pressure, 7 cm H(2)O). Samples from bronchial and luminal intestinal and arterial microdialysis catheters (flow-rate of 1 mu L/min) were collected during reperfusion in 60-min fractions. Samples were analyzed for TNF-alpha, IL-1 beta, IL-8, and FD-4. Data are presented as median (interquartile range). A lung biopsy was collected at the end of the experiment. During reperfusion, there was an increase in bronchial concentrations of both IL-8 (3.70 [1.47-8.93] ng/mL per h vs. controls, 0.61 [0.47-0.91] ng/mL per h; P < 0.001) and IL-1 beta (0.32 [0.05-0.56] ng/mL per h vs. controls, 0.07 [0.04-0.10] ng/mL per h; P = 0.008). In the intestinal lumen, IL-8 was increased in the ischemia group (6.33 [3.13-9.23] ng/mL per h vs. controls, 0.89 [0.21-1.86] ng/mL per h; P < 0.001). The FD-4 did not differ between groups. Pulmonary vascular resistance and pulmonary shunt increased versus controls. During reperfusion, PaO(2)/FiO(2) ratio decreased in the ischemia group. Histology was normal in both groups. Bronchial microdialysis detects altered levels of cytokines in the epithelial lining fluid and can be used for continuous monitoring of the immediate local lung cytokine response secondary to intestinal ischemia-reperfusion.
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