| 1. |
- Caballero, Ignacio, et al.
(författare)
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Immunolocalization and possible functional role of PSP-I/PSP-II heterodimer in highly extended boar spermatozoa
- 2006
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Ingår i: Journal of Andrology. - : American Society of Andrology. - 0196-3635 .- 1939-4640. ; 27:6, s. 766-773
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Tidskriftsartikel (refereegranskat)abstract
- PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma which is able to preserve, in vitro, the viability, motility, and mitochondrial activity of highly extended boar spermatozoa for at least 5 hours. However, little is known about the binding pattern of the heterodimer to the sperm plasma membrane and its eventual relation with the maintenance of the sperm functionality. The present study investigated the effect of exposing highly extended boar spermatozoa (11 million/mL) to 1.5 mg/mL of PSP-I/PSP-II for 0.5, 5, and 10 hours at 38 degrees C on sperm characteristics and the changes in PSP-I/PSP-II localization as a result of both the addition of PSP-I/PSP-II to the extender and the incubation time. Exposure of the spermatozoa to PSP-I/PSP-II preserved sperm viability, motility, and mitochondrial activity when compared to nonexposed spermatozoa. This protective effect lasted for 10 hours (P less than.05). After immunolabeling of highly extended semen with rabbit monospecific polyclonal antibody against PSP-I/PSP-11, the percentage of immunopositive spermatozoa declines over time from 71% (0.5 hours) to 49% (10 hours). However, more than 80% of spermatozoa remained labeled during the 10-hour incubation period if PSP-I/PSP-11 was added. Scanning electron microscopy revealed 4 different binding patterns. The heterodimer was mainly localized to the acrosomal area, being redistributed to the postacrosomal area or lost during in vitro incubation. In conclusion, the protective effect of the heterodimer appears to be related to its adhesion to the acrosomal area, and the loss of this protective effect coincides with a stepwise redistribution of PSP-I/PSP-II during incubation.
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| 2. |
- Hernandez, Marta, et al.
(författare)
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Cryo-scanning electron microscopy (Cryo-SEM) of semen frozen in medium-straws from good and sub-standard freezer AI-boars
- 2007
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Ingår i: Cryobiology. - : Elsevier. - 0011-2240 .- 1090-2392. ; 54:1, s. 63-70
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Tidskriftsartikel (refereegranskat)abstract
- A major limiting factor for commercial cryopreservation of boar semen for artificial insemination (AI) is the large individual variation to cooling, where the degree of cell dehydration during ice (re)shaping seems to play a major role. This study investigated, in the frozen state, the degree of dehydration and ice crystal distribution in boar semen doses whose spermatozoa displayed different viability after thawing. Cross-sectioned medium-straws (0.5 mL, n = 10) from a total of 10 stud boars classified as "good" (n = 5) or sub-standard (e.g., "bad" freezers, n = 5) by conventional analyses (computer assisted motility and sperm viability) were examined by Cryo-scanning electron microscopy (Cryo-SEM) to determine whether differences between groups could be already distinguishable prior to thawing. The degree of hydration was monitored in relation to the areas of ice crystal formed extracellularly (lakes), the areas of frozen, concentrated extender (veins) where spermatozoa were located and the degree of compartmentalization (number of lakes) present. Irrespectively of the region studied, the gradient of main dehydration (as lakes) observed along the cross-section area of the straws was very irregular. Most spermatozoa were enclosed in the freezing extender matrix and no obvious signs of external membrane damage were observed. None of the Cryo-SEM variables significantly correlated with post-thaw sperm parameters (p greater than 0.05). However, we identified significant differences (p less than 0.0001) among boars for all ultrastructure variables studied, including the size of the veins, where differences in solute concentration is expected. We concluded that despite the large variability in ice crystal formation during the conventional freezing process among boars, this is unrelated to inter-boar post-thaw sperm differences. (c) 2007 Elsevier Inc. All rights reserved.
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| 3. |
- Hernandez, Marta, et al.
(författare)
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Differences in SCSA outcome among boars with different sperm freezability
- 2006
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Ingår i: International Journal of Andrology. - : Wiley-Blackwell. - 0105-6263 .- 1365-2605. ; 29:6, s. 583-591
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Tidskriftsartikel (refereegranskat)abstract
- Spermatozoa from some boars sustain the process of cryopreservation poorly and yield poor fertility after artificial insemination. Poor freezability has not been disclosed using conventional semen analyses. A defective chromatin can, if present in a substantial number of spermatozoa, affect the fertilizing ability of spermatozoa. Here we tested the hypothesis that nuclear DNA instability could explain differences in freezability among boars, and complement or supersede conventional tests for sperm quality such as sperm motility or membrane assessments. Frozen-thawed (FT) spermatozoa from a total of 44 stud boars were assessed by the sperm chromatin structure assay (SCSA), in relation to computer-assisted sperm analysis-derived sperm motility variables and sperm viability (triple fluorescent microscopic staining), including three experiments. The first trial, including 24 boars, evaluated the relationship between the sperm motility and viability with levels of DNA integrity. The SCSA showed that most spermatozoa had intact DNA [levels of DNA fragmentation index (%DFI) ranging from 0.63% to 11.85%] significantly correlated (albeit weakly) with current sperm quality variables. The second trial, on 15 boars, assessed the influence of two different thawing rates (20 s at 37 degrees C vs. 8 s at 70 degrees C) and the post-thaw incubation times (0, 30, 150 and 300 min) at 37 degrees C on FT-boar sperm quality. The highest sperm survival (p less than 0.05) and the lowest DNA damage (p less than 0.01) were achieved when thawing was carried out at 70 degrees C for 8 s, without any change during the first 150 min of incubation. Finally, the third experiment studied if differences in sperm freezability showed by stud boar semen, as good or bad freezers by conventional analyses, could be attributed to differences in chromatin structure. All SCSA parameters were low, but significantly (p less than 0.05-0.001) higher for bad freezers, showing they had less homogeneous sperm chromatin than the good freezers. The results indicate that SCSA outcome complements conventional assessment of FT-boar spermatozoa, disclosing differences in their ability to sustain freezing and thawing. However, the low overall DNA damage observed in FT spermatozoa seems to have poor biological significance.
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