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 indus-try application. Adding seminal plasma (SP) could improve post-thawing quality, but its suita-bility could vary. We applied a simple cold-shock test (CST, 5 min at 0 °C) on neat semen for clas-sifying 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 kine-matic 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. This research was funded by the Ministry of Science and Innovation (Spain), grant number RTI2018-095183-B-I00 (MCI/AEI/FEDER, UE) and LE023P20, Junta de Castilla y Le-ón/Consejería de Educación/FEDER, EU.