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

SIMPLE SUMMARY: The pig industry routinely uses artificial insemination with refrigerated semen. Cryopreserved semen has many advantages, but it is not widely used, partly because of unreliable results. The supplementation with seminal plasma (SP) could improve these results, but this fluid also presents variability. We evaluated if a simple cold-shock test (CST) could allow to easily identify the most suitable ejaculates for obtaining SP. Therefore, we tested 63 ejaculates, obtaining SP from the 4 showing higher quality (SPr, cold-shock-resistant) and lower quality (SPs, cold-shock-sensitive) after the CST. SPs and SPr pools supplemented thawed semen (20% SP) from six different boars, incubating at 37 °C and analyzing at times 0, 2, and 4 h. SPr was able to improve post-thawing sperm motility while maintaining viability. SPs had a similar but lower effect. SP in general seemed to stimulate sperm physiology, however decreasing membrane stability, acrosomal integrity, and disulfide bridges in the chromatin. This study supports the suitability of SP for improving thawed semen, with CST-selected ejaculates as preferable for this aim. Artificial insemination trials with thawed semen supplemented with SPr and SPs must validate the practical application of CST. 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.