Low-density colloid centrifugation removes bacteria from boar semen doses after spiking with selected species

Lacalle, Estíbaliz; Martínez-Martínez, Sonia; Fernández‐Alegre, Estela; Soriano‐Úbeda, Cristina; Morrell, Jane M.; Martínez‐Pastor, Felipe

doi:10.1016/j.rvsc.2023.03.024

Cited by 4 publications

(4 citation statements)

References 44 publications

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“…These results align with recent work linking sperm DNA fragmentation in this species with other factors, independent of oxidative damage [43]. Nevertheless, many studies have shown that pig spermatozoa could show higher levels of oxidative stress and DNA damage if obtained from stressed animals (e.g., heat stress [44]) or if submitted to harmful treatments, such as cryopreservation [45,46] or bacterial contamination [42].…”

Section: Detection Of 8-oxo-dg As a Marker Of Dna Oxidative Insult An...supporting

confidence: 89%

“…Our results concerning DNA oxidative damage must be interpreted considering the flow cytometry assessment of oxidative species. Interestingly, cytoplasmic and mitochondrial ROS levels were low (as we observed previously in unstimulated samples [36,42]), and only the latter were significantly affected by boar and storage. Therefore, it is not surprising that the correlations of these variables with the chromatin variables were non-significant or of little relevance.…”

Section: Detection Of 8-oxo-dg As a Marker Of Dna Oxidative Insult An...supporting

confidence: 78%

“…Once in the laboratory, an aliquot of each dose was processed and analyzed immediately (day 0 of the study, D0), and the remaining sample was maintained at 17 • C for 11 days and assessed (D11). These two analysis points were selected taking into account previous results [29,42,51] (good maintenance of sperm viability) and maximizing changes in the chromatin structure during the refrigerated storage (since this study also aims to select the most informative parameters; more specific time points might be assessed in subsequent studies). Analyses consisted of assessing spermatozoa functionality by flow cytometry, motility by CASA, and study of the chromatin as oxidative DNA damage, DNA fragmentation and chromatin maturity (SCSA), disulfide bonds between protamines, and chromatin protamination/condensation.…”

Section: Experimental Designmentioning

confidence: 99%

“…Flow cytometry analyses were carried out as detailed in previous works [29,42,51] A MACSQuant Analyzer 10 flow cytometer equipped with a 96-well plate processor and three lasers (violet at 405 nm, blue at 488 nm, and red at 635 nm) and controlled with MACSQuantify™ software v. 2.13 (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for all the analyses. Spermatozoa were gated using appropriate regions using FSC/SSC (forward/side scatter), and different fluorochromes for discriminating non-sperm events (debris): H342/SSC or H258/SSC cytograms (H342 − or H258 −/− events considered debris) for the sperm physiology analysis; AO in a red vs. green fluorescence cytogram (AO −/− considered debris) for SCSA; PI/SSC or H334/SSC (H342 − or PI − events considered debris) for the remaining techniques.…”

Section: Flow Cytometry Analysesmentioning

confidence: 99%

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Application of Flow Cytometry Using Advanced Chromatin Analyses for Assessing Changes in Sperm Structure and DNA Integrity in a Porcine Model

Lacalle,

Fernández-Alegre,

Gómez-Giménez

et al. 2024

IJMS

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Chromatin status is critical for sperm fertility and reflects spermatogenic success. We tested a multivariate approach for studying pig sperm chromatin structure to capture its complexity with a set of quick and simple techniques, going beyond the usual assessment of DNA damage. Sperm doses from 36 boars (3 ejaculates/boar) were stored at 17 °C and analyzed on days 0 and 11. Analyses were: CASA (motility) and flow cytometry to assess sperm functionality and chromatin structure by SCSA (%DFI, DNA fragmentation; %HDS, chromatin maturity), monobromobimane (mBBr, tiol status/disulfide bridges between protamines), chromomycin A3 (CMA3, protamination), and 8-hydroxy-2′-deoxyguanosine (8-oxo-dG, DNA oxidative damage). Data were analyzed using linear models for the effects of boar and storage, correlations, and multivariate analysis as hierarchical clustering and principal component analysis (PCA). Storage reduced sperm quality parameters, mainly motility, with no critical oxidative stress increases, while chromatin status worsened slightly (%DFI and 8-oxo-dG increased while mBBr MFI—median fluorescence intensity—and disulfide bridge levels decreased). Boar significantly affected most chromatin variables except CMA3; storage also affected most variables except %HDS. At day 0, sperm chromatin variables clustered closely, except for CMA3, and %HDS and 8-oxo-dG correlated with many variables (notably, mBBr). After storage, the relation between %HDS and 8-oxo-dG remained, but correlations among other variables disappeared, and mBBr variables clustered separately. The PCA suggested a considerable influence of mBBr on sample variance, especially regarding storage, with SCSA and 8-oxo-dG affecting between-sample variability. Overall, CMA3 was the least informative, in contrast with results in other species. The combination of DNA fragmentation, DNA oxidation, chromatin compaction, and tiol status seems a good candidate for obtaining a complete picture of pig sperm nucleus status. It raises many questions for future molecular studies and deserves further research to establish its usefulness as a fertility predictor in multivariate models. The usefulness of CMA3 should be clarified.

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Section: Detection Of 8-oxo-dg As a Marker Of Dna Oxidative Insult An...supporting

confidence: 89%

Section: Detection Of 8-oxo-dg As a Marker Of Dna Oxidative Insult An...supporting

confidence: 78%

Section: Experimental Designmentioning

confidence: 99%

Section: Flow Cytometry Analysesmentioning

confidence: 99%

See 2 more Smart Citations

Application of Flow Cytometry Using Advanced Chromatin Analyses for Assessing Changes in Sperm Structure and DNA Integrity in a Porcine Model

Lacalle,

Fernández-Alegre,

Gómez-Giménez

et al. 2024

IJMS

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Reduced bacterial load in stallion semen by modified single layer centrifugation or sperm washing

Malaluang,

Wagner,

Cojkic

et al. 2024

Theriogenology

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Antibiotics in semen extenders – a multiplicity of paradoxes

Morrell,

Cojkic,

Malaluang

et al. 2024

Reprod. Fertil. Dev.

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Addition of antibiotics to semen extenders was taken for granted for many years, from the time that commercial artificial insemination in livestock first began many decades ago. However, there is now a growing realisation that this non-therapeutic utilisation of antibacterial agents is contrary to current recommendations for prudent use that medical and veterinary professionals are advised to follow. Furthermore, antibiotics are not benign, having negative effects on sperm samples, the inseminated female, personnel and potentially the environment. The purpose of this review is three-fold: to highlight the fact that antibiotics are used in semen extenders, with the result that considerable amounts are used globally in animal breeding, to review recent studies on the negative aspects of using antibiotics for this purpose, and to look at possible alternatives. Recent changes in the legislation regarding semen extenders occurred in some, but not all, countries, leaving question marks for semen producers as to whether antibiotics should be added to semen extenders or not.

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Low-density colloid centrifugation removes bacteria from boar semen doses after spiking with selected species

Cited by 4 publications

References 44 publications

Application of Flow Cytometry Using Advanced Chromatin Analyses for Assessing Changes in Sperm Structure and DNA Integrity in a Porcine Model

Application of Flow Cytometry Using Advanced Chromatin Analyses for Assessing Changes in Sperm Structure and DNA Integrity in a Porcine Model

Reduced bacterial load in stallion semen by modified single layer centrifugation or sperm washing

Antibiotics in semen extenders – a multiplicity of paradoxes

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