| 1. |
- Balao da Silva, C M., et al.
(författare)
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Sex sorting increases the permeability of the membrane of stallion spermatozoa
- 2013
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Ingår i: Animal Reproduction Science. - : Elsevier Masson. - 0378-4320 .- 1873-2232. ; 138:3-4, s. 241-251
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Balao da Silva, C. M., et al.
(författare)
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Effect of Overnight Staining on the Quality of Flow Cytometric Sorted Stallion Sperm: Comparison with Tradtitional Protocols
- 2014
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Ingår i: Reproduction in domestic animals. - : Wiley. - 0936-6768 .- 1439-0531. ; 49:6, s. 1021-1027
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Tidskriftsartikel (refereegranskat)abstract
- Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sorted and compared with spermatozoa from the same ejaculate that had been sexed on the previous day. All sperm parameters evaluated remained unchanged when fresh sorted and refrigerated sorted semen were compared. Pre-sorting storage at 5 degrees C did not alter sperm velocities nor kinetics, viability or membrane permeability, production of reactive oxygen species, mitochondrial membrane potential or DNA fragmentation index of the sorted sample. The findings open for the possibility of using semen from stallions housed far from the sorting facilities. Processed and stained sperm could be shipped refrigerated on the previous day, sorted and inseminated on the next day.
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| 3. |
- Agmon-Levin, Nancy, et al.
(författare)
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International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies
- 2014
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Ingår i: Annals of the Rheumatic Diseases. - : BMJ. - 0003-4967 .- 1468-2060. ; 73:1, s. 17-23
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Tidskriftsartikel (refereegranskat)abstract
- Anti-nuclear antibodies (ANA) are fundamental for the diagnosis of autoimmune diseases, and have been determined by indirect immunofluorescence assay (IIFA) for decades. As the demand for ANA testing increased, alternative techniques were developed challenging the classic IIFA. These alternative platforms differ in their antigen profiles, sensitivity and specificity, raising uncertainties regarding standardisation and interpretation of incongruent results. Therefore, an international group of experts has created recommendations for ANA testing by different methods. Two groups of experts participated in this initiative. The European autoimmunity standardization initiative representing 15 European countries and the International Union of Immunologic Societies/World Health Organization/Arthritis Foundation/Centers for Disease Control and Prevention autoantibody standardising committee. A three-step process followed by a Delphi exercise with closed voting was applied. Twenty-five recommendations for determining ANA (1-13), anti-double stranded DNA antibodies (14-18), specific antibodies (19-23) and validation of methods (24-25) were created. Significant differences between experts were observed regarding recommendations 24-25 (p<0.03). Here, we formulated recommendations for the assessment and interpretation of ANA and associated antibodies. Notably, the roles of IIFA as a reference method, and the importance of defining nuclear and cytoplasmic staining, were emphasised, while the need to incorporate alternative automated methods was acknowledged. Various approaches to overcome discrepancies between methods were suggested of which an improved bench-to-bedside communication is of the utmost importance. These recommendations are based on current knowledge and can enable harmonisation of local algorithms for testing and evaluation of ANA and related autoantibodies. Last but not least, new more appropriate terminologies have been suggested.
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| 4. |
- Cema, Grzegorz, et al.
(författare)
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Dissolved oxygen as a factor influencing nitrogen removal rates in a one-stage system with partial nitritation and Anammox process
- 2011
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Ingår i: Water Science and Technology. - : IWA Publishing. - 0273-1223 .- 1996-9732. ; 64:5, s. 1009-1015
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Tidskriftsartikel (refereegranskat)abstract
- A biofilm system with Kaldnes biofilm carrier was used in these studies to cultivate bacteria responsible for both partial nitritation and Anammox processes. Due to co-existence of oxygen and oxygen-free zones within the biofilm depth, both processes can occur in a single reactor. Oxygen that inhibits the Anammox process is consumed in the outer layer of the biofilm and in this way Anammox bacteria are protected from oxygen. The impact of oxygen concentration on nitrogen removal rates was investigated in the pilot plant (2.1 m(3)), supplied with reject water from the Himmerfjarden Waste Water Treatment Plant. The results of batch tests showed that the highest nitrogen removal rates were obtained for a dissolved oxygen (DO) concentration around 3 g O(2) m(-3). At a DO concentration of 4 g O(2) m(-3), an increase of nitrite and nitrate nitrogen concentrations in the batch reactor were observed. The average nitrogen removal rate in the pilot plant during a whole operating period oscillated around 1.3 g N m(-2)d(-1) (0.3 +/- 0.1 kg N m(-3)d(-1)) at the average dissolved oxygen concentration of 2.3 g O(2) m(-3). The maximum value of a nitrogen removal rate amounted to 1.9 g N m(-2)d(-1) (0.47 kg N m(-3)d(-1)) and was observed for a DO concentration equal to 2.5 g O(2) m(-3). It was observed that increase of biofilm thickness during the operational period, had no influence on nitrogen removal rates in the pilot plant.
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| 5. |
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| 6. |
- Gallardo Bolanos, Juan M., et al.
(författare)
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Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7
- 2014
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Ingår i: Reproduction. - : Bioscientifica. - 1470-1626 .- 1476-3990. ; 148:2, s. 221-235
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Tidskriftsartikel (refereegranskat)abstract
- AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers-Whitten-Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 mu M SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, with inhibition reaching a maximum after 1 h of incubation. This decrease in phosphorylation was attributable to either dephosphorylation or suppression of the active phosphorylation pathway. Stallion spermatozoa spontaneously dephosphorylated during in vitro incubation, resulting in a lack of a difference in AKT phosphorylation between the SH5-treated sperm and the control after 4 h of incubation. AKT inhibition decreased the proportion of motile spermatozoa (total and progressive) and the sperm velocity. Similarly, AKT inhibition reduced membrane integrity, leading to increased membrane permeability and reduced the mitochondrial membrane potential concomitantly with activation of caspases 3 and 7. However, the percentage of spermatozoa exhibiting oxidative stress, the production of mitochondrial superoxide radicals, DNA oxidation and DNA fragmentation were not affected by AKT inhibition. It is concluded that AKT maintains the membrane integrity of ejaculated stallion spermatozoa, presumably by inhibiting caspases 3 and 7, which prevents the progression of spermatozoa to an incomplete form of apoptosis.
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