| 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. |
- Johannisson, Anders, et al.
(författare)
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Colloidal centrifugation with Androcoll-E (TM) prolongs stallion sperm motility, viability and chromatin integrity
- 2009
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Ingår i: Animal Reproduction Science. - : Elsevier Masson. - 0378-4320 .- 1873-2232. ; 116:1-2, s. 119-128
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 3. |
- 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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| 4. |
- Morrell, Jane, et al.
(författare)
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Colloidal Centrifugation of Stallion Semen: Changes in Sperm Motility, Velocity, and Chromatin Integrity during Storage
- 2009
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Ingår i: Journal of Equine Veterinary Science. - : WB Saunders. - 0737-0806 .- 1542-7412. ; 29:1, s. 24-32
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Tidskriftsartikel (refereegranskat)abstract
- The current study investigated the changes in sperm quality (motility, velocity, and chromatin integrity) occurring during storage at room temperature or 5 degrees C for up to 48 hours in spermatozoa after extension or single-layer centrifugation (SLC) throught Androcoll-E. In unselected samples, all parameters of sperm quality deteriorated significantly during storage (P less than .01), although the deterioration was faster at room temperature (22-30 degrees C) than for cool storage (P less than .01). The SLC-selected spermatozoa had higher motility, velocity, and chromatin integrity than the overall unselected population (motility: selected 85 +/- 10%, unselected 56 +/- 13%; P less than .001; velocity: selected 85.1 +/- 13 mu m/second, unselected 63.5 +/- 15 mu m/second; P less than .001; and DFI selected 12.2 +/- 4.8 mu m/second; unselected 23.6 +/- 7.4 mu m/second; P less than .001). Furthermore, sperm quality did not deteriorate with storage in the SLC-selected samples, either at room temperature (22-30 degrees C for 24 hours) or cooled to 4 degrees C (for at least 48 hours), whereas a significant deterioration in sperm quality was observed in the unselected sperm samples (P less than .01). Thus, room temperature storage of SLC-selected spermatozoa may be an option for insemination doses from stallions whose spermatozoa do not tolerate cooling. In addition, a new sperm analyzer, the Qualisperm, showed good correlation with subjective motility assessment (r = 0.8, P less than .001), was user-friendly, and provided a reasonable volume of data. This instrument may be a useful adjunct to sperm quality assessment at the study.
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| 5. |
- 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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| 6. |
- Morrell, Jane, et al.
(författare)
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Morphology and chromatin integrity of stallion spermatozoa prepared by density gradient and single layer centrifugation through silica colloids
- 2009
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Ingår i: Reproduction in Domestic Animals. - : Wiley. - 0936-6768 .- 1439-0531. ; 44, s. 512-517
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Tidskriftsartikel (refereegranskat)abstract
- The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically normal spermatozoa varied between stallions (p < 0.001) and was increased by both methods of colloidal centrifugation (median value before centrifugation 67.5%; after SLC 78%; after DGC 77%; p < 0.001). The incidence of certain abnormalities was reduced, e.g. proximal cytoplasmic droplets were reduced from 12.9% to 8.8% (p < 0.001), and mid-piece defects from 5.3% to 1.4% (p < 0.05). Similarly, sperm motility and chromatin integrity were significantly improved (p < 0.001), with no difference between the two centrifugation methods. Centrifugation through colloids can enrich the proportions of stallion spermatozoa with normal morphology and normal chromatin structure in sperm preparations. The new method, SLC, was as effective as DGC in selecting motile stallion spermatozoa with normal morphology and intact chromatin. SLC, being simpler to use than DGC, would be appropriate for routine use by stud personnel to improve stallion sperm quality in insemination doses.
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| 7. |
- 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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| 8. |
- Morrell, Jane, et al.
(författare)
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Single-layer centrifugation with Androcoll-E can be scaled up to allow large volumes of stallion ejaculate to be processed easily
- 2009
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Ingår i: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 72:6, s. 879-884
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- 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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| 10. |
- Revay, T., et al.
(författare)
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Macrocephaly in Bull Spermatozoa Is Associated with Nuclear Vacuoles, Diploidy and Alteration of Chromatin Condensation
- 2009
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Ingår i: Cytogenetic and Genome Research. - : S. Karger. - 1424-8581 .- 1424-859X. ; 126:1-2, s. 202-209
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Tidskriftsartikel (refereegranskat)abstract
- Spermatozoa from 2 dairy AI (artificial insemination) bulls (A and B), identified by their abnormal spermiogram with cells depicting frequent macrocephaly, double tails and nuclear vacuoles, were case-investigated and compared to normal spermatozoa from a control AI sire (C). Head sizes were measured and morphological abnormalities scored using brightfield and differential interference contrast microscopy. The degree of sperm maturation and of resistance to acid-induced DNA denaturation in situ were determined after uploading of acridine orange using flow cytometry of 5,000 cells/sample. Nuclear fragmentation, i.e. the ratio of red to total (red + green) fluorescence, reached 7.1% and 31% in bulls A and B, compared to 2% in bull C. The proportion of immature spermatozoa, i.e. those with incomplete histone-protamine exchange and depicting higher green fluorescence compared to the main population of the control bull, reached 9.54% in A and 7.75% in B, compared to only 0.47% in the control. In the second part of this study the previously unknown chromosomal constitution of large-headed spermatozoa of bull A was investigated by fluorescence in situ hybridization using an X-Y painting probe set. The 7.5% XY-bearing cells and the presence of diploid spermatozoa detected by flow cytometry indicate a meiotic arrest in the first division in bull A, becoming the first proven case of association of macrocephaly and M1 diploidy. The diverse approaches used for the investigation of spermatozoal DNA provide insights into the etiology of macrocephaly. Copyright (C) 2009 S. Karger AG, Basel
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