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

Abstract

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.

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

“…CMA3 has been used to analyze sperm compaction in other species [52,53]. However, it was less informative in our study, in line with previous reports for pig spermatozoa [29,30,43]. A possible reason could be that this technique could help identify boars with altered spermatogenesis, whereas those used in this study were subjected to strict evaluation.…”

“…In contrast, the influence of the SCSA or the 8-oxo-dG levels was much lower (also shown in the models' results). The ability of mBBr analysis to find differences between treatments was not evident in previous studies [29,30] but still showed good sensitivity to incubation or storage. In any case, mBBr should be tested for discrimination power among boars and to determine if it is related to stressful events or male fertility, as is the case for SDF [15,16,38,55,56].…”

“…As a result, the analysis of chromatin maturity, compaction, and disulfide bridges has been used more sparingly in this species [29,30,39,40]. The present study proposes a combination of the SCSA, CMA3, and mBBr techniques to achieve a better understanding of the overall structure of the sperm nucleus.…”

“…The analysis of spermatozoa functionality in fresh samples was carried out by flow cytometry. The probes for the analysis were prepared in BTS-BSA as previously described by our group [30] with minor modifications: viability, apoptosis, capacitation, and acrosomal status by combining 2.7 µM H342, 37.5 nM YP, 0.7 µM M540, 2.25 µM PI, and 1.5 µM PNA; and viability, cytoplasmic and mitochondrial ROS, and mitochondrial activity by combining 4.7 µM H258, 1.5 µM CFDA, 0.7 µM MX, and 300 nM MTdr. The sperm samples were diluted to 10 6 mL −1 and incubated in the dark for 15 min at 37 • C.…”

“…It occurs as part of sperm maturation in the epididymis, mediated by nuclear glutathione peroxidase 4 (nGPX4) [26]. It is also a dynamic process, reversed at fertilization and affected by the sperm environment [27][28][29][30]. Monobromobimane (mBBr), a fluorescent reagent that covalently binds to free sulfhydryl groups, can evaluate the level of disulfide bridges in the sperm nucleus [31].…”