| 1. |
- Ortega Ferrusola, C., et al.
(författare)
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Effect of Cryopreservation on Nitric Oxide Production by Stallion Spermatozoa
- 2009
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Ingår i: Biology of Reproduction. - : Society for the Study of Reproduction. - 0006-3363 .- 1529-7268. ; 81:6, s. 1106-1111
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Tidskriftsartikel (refereegranskat)abstract
- The ability of stallion spermatozoa to produce nitric oxide (NO) before (fresh) and after freezing and thawing (FT) was evaluated by means of flow cytometry after loading the sperm suspension with the probe, 4,5-diaminofluorescenin diacetate. The presence of NO synthase (NOS) was investigated by Western blotting using anti-NOS1, anti-NOS3, or anti-universal NOS antibodies (Abs). While NO was detected both in fresh and FT sperm suspensions, its production increased after cryopreservation only when egg yolk was removed from the extender. Anti-NOS1 Ab intensively labeled a single band with an apparent molecular mass of approximately 83 kDa. On the other hand, the Ab developed against the NOS3 showed a band of approximately 96 kDa in fresh and FT sperm lysates. NO production was positively correlated with sperm motility and velocity after thaw, suggesting an NO role for the functionality of cryopreserved stallion spermatozoa; but the production of NO is compromised in egg yolk-containing extenders.
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| 2. |
- Ortega Ferrusola, C., et al.
(författare)
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Lipid peroxidation, assessed with BODIPY-C-11, increases after cryopreservation of stallion spermatozoa, is stallion-dependent and is related to apoptotic-like changes
- 2009
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Ingår i: Reproduction. - : BioScientifica. - 1470-1626 .- 1476-3990. ; 138:1, s. 55-63
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Tidskriftsartikel (refereegranskat)abstract
- Lipid peroxidation (LPO) of stallion spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in the susceptibility to LPO and its possible relationship with freezability. Innate levels of LPO were very low in fresh spermatozoa but increased after thawing, a change that was largely stallion-dependent. The level of LPO in fresh spermatozoa was not correlated with that of the thawed spermatozoa. Negative correlations existed between LPO and intact membranes post-thaw (r= -0.789, Pless than0.001), and also between LPO and spermatozoa with high mitochondrial membrane potential (Delta psi m) post-thaw (r= -0.689, Pless than0.001). LPO was also highly and significantly correlated with caspase activity. The correlation between caspase activity in ethidium positive cells and LPO was r=0.772, Pless than0.001. This LPO is unlikely to represent, per se, a sign of cryopreservation-induced injury, but it is apparently capable of triggering apoptotic-like changes that could result in the sub-lethal cryodamage often seen among surviving spermatozoa. Reproduction (2009) 138 55-63
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| 3. |
- Macias Garcia, B., et al.
(författare)
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Centrifugation on a single layer of colloid selects improved quality spermatozoa from frozen-thawed stallion semen
- 2009
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Ingår i: Animal Reproduction Science. - : Elsevier Masson. - 0378-4320 .- 1873-2232. ; 114:1-3, s. 193-202
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Macias Garcia, B., et al.
(författare)
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Single-Layer Centrifugation Through Colloid Positively Modifies the Sperm Subpopulation Structure of Frozen-Thawed Stallion Spermatozoa
- 2009
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Ingår i: Reproduction in domestic animals. - : Wiley-Blackwell. - 0936-6768 .- 1439-0531. ; 44:3, s. 523-526
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Tidskriftsartikel (refereegranskat)abstract
- 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)). After freezing and thawing, four sperm subpopulations were identified, listed as FT1 to FT4. While subpopulations FT1 and FT2 were characterized by low sperm velocity, high velocities characterized the ones called FT3 and FT4. The single-layer centrifugation (SLC)-handled sperm sample was enriched in subpopulation FT3, reaching a proportion of 82.6% of the present spermatozoa, in contrast with the non-filtered control post-thawed semen, where this sperm subpopulation only accounted for 16.3% of the total. It is concluded that in the equine industry, the SLC is a practical, easy-to-perform approach to improve the quality of equine frozen-thawed semen samples.
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| 5. |
- Ortega-Ferrusola, C., et al.
(författare)
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Apoptotic markers can be used to forecast the freezeability of stallion spermatozoa
- 2009
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Ingår i: Animal Reproduction Science. - : Elsevier Masson. - 0378-4320 .- 1873-2232. ; 114:4, s. 393-403
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Ortega-Ferrusola, C., et al.
(författare)
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Does the Microbial Flora in the Ejaculate Affect the Freezeability of Stallion Sperm?
- 2009
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Ingår i: Reproduction in domestic animals. - : Wiley-Blackwell. - 0936-6768 .- 1439-0531. ; 44:3, s. 518-522
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Tidskriftsartikel (refereegranskat)abstract
- In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze, three ejaculates from five stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The isolated microorganisms were: Staphylococcus spp. and Micrococcus spp. (in all the stallions), beta-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp. (in stallions 1, 3-5), Rhodococcus spp. (in stallion number 2), Pseudomonas spp. (in stallion number 1) and Klebsiella spp. (in stallions 1, 3 and 5). The presence and richness of Klebsiella and beta-haemolytic Streptococcus in the ejaculate were related to two sperm variables post-thaw, namely the proportion of dead spermatozoa (ethidium+ cells; r = 0.55, p less than 0.05) and the amplitude of lateral displacement of the sperm head (ALH, mu m; r = -0.56, p less than 0.05), respectively. The degree of growth of Corynebacterium spp. in the ejaculate was positively correlated with the percentage of spermatozoa showing high caspase activity post-thaw (r = 0.62, p less than 0.05). The presence and number of colonies of beta-haemolytic Streptococcus were negatively correlated (r = -0.55, p less than 0.05) with low sperm caspase activity. It is concluded that the microbial flora of the equine ejaculate may be responsible for some of the sublethal damage experimented by the spermatozoa during cryopreservation.
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| 7. |
- Ortega-Ferrusola, C., et al.
(författare)
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Identification of Sperm Subpopulations in Stallion Ejaculates: Changes after Cryopreservation and Comparison with Traditional Statistics
- 2009
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Ingår i: Reproduction in domestic animals. - : Wiley-Blackwell. - 0936-6768 .- 1439-0531. ; 44:3, s. 419-423
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Tidskriftsartikel (refereegranskat)abstract
- In an attempt to improve the information obtained after computer-assisted sperm analysis (CASA), data from five stallions (three ejaculates from each) were analysed before (fresh, extended semen) and after cryopreservation using traditional statistics as well as a cluster analysis. The data matrix consisted of 13 987 observations of individual spermatozoa for fresh, extended semen, and 8305 for frozen-thawed samples. As expected, freezing and thawing resulted in a marked decrease of CASA-derived variables of sperm kinematics. All sperm velocities were significantly lower in frozen-thawed samples than in samples before cooling. Using sperm velocities, six sperm subpopulations were identified in fresh semen (S1-S6). As such, subpopulations S1 and S2 were characterized by low sperm velocities, subpopulations S3 and S4 corresponded to spermatozoa depicting medium speed values, and finally, subpopulations S5 and S6 were those depicting the highest velocities. After freezing and thawing, four sperm subpopulations were identified, listed as nr FT1 to FT4. While subpopulations FT1-FT3 were characterized by low sperm velocities, and thus corresponded speed-wise to those listed as S1-S4 for fresh, extended semen, the one called number FT4 in frozen semen was characterized by high velocities, of the same range as that of the subpopulations S5 and S6 for fresh spermatozoa. The sperm subpopulation structure varied among stallions, but the cluster analysis hereby assayed was able to provide valuable information about the freezability of the samples that the customary statistics did not reveal.
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