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

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.

Single layer centrifugation (SLC) for bacterial removal with Porcicoll positively modifies chromatin structure in boar spermatozoa.

The storage of boar semen samples at 17 °C for artificial insemination (AI) doses enables the proliferation of the bacteria, making antibiotics necessary. This can contribute to the development of antimicrobial resistance (AMR). This study tested bacterial presence and sperm chromatin structure after using a low-density colloid (Porcicoll) as an antibiotic alternative to eliminate bacteria. Ejaculates (8 boars, 3 ejaculates each) were split as control and low-density colloid centrifugation (single layer centrifugation, SLC, 20%, and 30% Porcicoll) into 500 ml tubes. Analyses were carried out at days 0, 3, and 7 (17 °C) for microbial presence and sperm chromatin structure analysis: %DFI (DNA fragmentation) and %HDS (chromatin immaturity), monobromobimane (mBBr; free thiols and disulfide bridges), and chromomycin A3 (CMA3; chromatin compaction). Besides comparing bacterial presence (7 species identified) and chromatin variables between treatments, the associations between these sets of variables were described by canonical correlation analysis (CCA). Results showed a significant decrease of some bacteria or a complete removal after SLC (especially for P30). SLC also caused a decrease of %HDS and an increase of disulfide bridges and low and medium mBBr populations, suggesting the removal of immature sperm (poor chromatin compaction). CCA showed an association pattern compatible with the degradation of sperm chromatin parameters with bacterial contamination, especially Enterobacteria, P. aeuriginosa, and K. variicola. In conclusion, bacterial contamination affects sperm chromatin beyond DNA fragmentation; SLC with low-density colloid not only removes bacteria from boar semen, but also chromatin structure is enhanced after selection.

Bos taurus and Cervus elaphus as Non-Seasonal/Seasonal Models for the Role of Melatonin Receptors in the Spermatozoon.

Melatonin is crucial in reproduction due its antioxidant, hormonal, and paracrine action. Melatonin membrane receptors (MT1/MT2) have been confirmed on spermatozoa from several species, but functionality studies are scarce. To clarify their role in ruminants as reproductive models, bull (Bos taurus, non-seasonal) and red deer (Cervus elaphus, highly seasonal) spermatozoa were analyzed after 4 h of incubation (38 °C, capacitating media) in 10 nM melatonin, MT1/MT2 agonists (phenylmelatonin and 8M-PDOT), and antagonists (luzindole and 4P-PDOT). Motility and functionality (flow cytometry: viability, intracellular calcium, capacitation status, reactive oxygen species (ROS) production, and acrosomal and mitochondrial status) were assessed. In bull, MT1 was related to sperm viability preservation, whereas MT2 could modulate cell functionality to prevent excess ROS produced by the mitochondria; this action could have a role in modulating sperm capacitation. Deer spermatozoa showed resistance to melatonin and receptor activation, possibly because the samples were of epididymal origin and collected at the breeding season's peak, with high circulating melatonin. However, receptors could be involved in mitochondrial protection. Therefore, melatonin receptors are functional in the spermatozoa from bull and deer, with different activities. These species offer models differing from traditional laboratory experimental animals on the role of melatonin in sperm biology.

Melatonin affects red deer spermatozoa motility and physiology in capacitating and non-capacitating conditions.

Melatonin affects sperm physiology, possibly through membrane receptors. Effects were tested at low concentrations (1 pM, 100 pM, 10 nM and 1 µM) in red deer epididymal spermatozoa as a model for high-seasonality species. Samples were incubated with melatonin as uncapacitated or capacitating conditions (heparin) and evaluated for motility and physiology (flow cytometry). Most effects occurred at low concentrations (nM-pM), mainly protecting from apoptosis and maintaining acrosomal integrity, suggesting a role for membrane receptors rather than a direct antioxidant effect. Intracellular calcium was not affected, differing from other studies and perhaps because of the epididymal origin. This study supports the relevance of melatonin on sperm physiology and could contribute to the application of reproductive technologies in wild ruminants.

Bicarbonate-Triggered In Vitro Capacitation of Boar Spermatozoa Conveys an Increased Relative Abundance of the Canonical Transient Receptor Potential Cation (TRPC) Channels 3, 4, 6 and 7 and of CatSper-γ Subunit mRNA Transcripts.

Sperm capacitation is a stepwise complex biochemical process towards fertilization. It includes a crucial early calcium (Ca2+) transport mediated by CatSper channels and Canonical Transient Potential Channels (TRPC). We studied the relative abundance of mRNA transcripts changes of the CatSper ß, γ and δ subunits and TRPC-channels 1, 3, 4, 6 and 7 in pig spermatozoa, after triggering in vitro capacitation by bicarbonate ions at levels present in vivo at the fertilization site. For this purpose, we analyzedfive5 ejaculate pools (from three fertile adult boars) before (control-fresh samples) and after in vitro exposure to capacitation conditions (37 mM NaHCO3, 2.25 mM CaCl2, 2 mM caffeine, 0.5% bovine serum albumin and 310 mM lactose) at 38 °C, 5% CO2 for 30 min. In vitro capacitation using bicarbonate elicits an increase in the relative abundance of mRNA transcripts of almost all studied Ca2+ channels, except CatSper-δ and TRPC1 (significantly reduced). These findings open new avenues of research to identify the specific role of each channel in boar sperm capacitation and elucidate the physiological meaning of the changes on sperm mRNA cargo.

Cold-Shock Test Is a Practical Method for Selecting Boar Ejaculates Yielding Appropriate Seminal Plasma for Post-Thawing Supplementation.

Artificial insemination (AI) with cryopreserved semen is still unreliable for extensive pig industry application. Adding seminal plasma (SP) could improve post-thawing quality, but its suitability could vary. We applied a simple cold-shock test (CST, 5 min at 0 °C) on neat semen for classifying ejaculates (n = 63) as resistant or sensitive, obtaining two SP pools (CST-resistant: SPr, sensitive: SPs). Subsequently, frozen/thawed spermatozoa from six boars were incubated (37 °C) in MR-A® extender (control), 20% SPr, or 20% SPs, and analyzed at 0, 2, and 4 h. SP improved total and progressive motility, with a higher effect for SPr and STR (p < 0.05), decreasing kinematic parameters VCL and VAP, ALH, and BCF. Sperm viability was unaffected. SP increased apoptotic and membrane disorder ratios, and acrosomal damage, not affecting the chromatin structure (DNA fragmentation and immaturity by SCSA), protamination (CMA3), or disulfide levels (mBBr). However, the proportion of spermatozoa with elevated free thiols (disulfide bridges reduction) significantly increased. Results support a stimulatory role of SP on thawed semen, with additional benefits from SPr. The effect of SP and especially SPr after AI should be tested since CST could be a practical test for selecting suitable ejaculates in AI centers.