Boar-sperm cryopreservation is not usually performed immediately after semen collection, but rather a holding time (HT) of 4 h-30 h at 17°C is spent before starting this procedure. Taking this into account, the aim of this study was to go further indepth into the mechanisms underlying the improving effects of HT at 17°C on boar-sperm cryotolerance by evaluating the effects of two different HTs (3 h and 24 h) on overall boar-sperm function and survival before and after cryopreservation. Given that phospho/dephosphorylation mechanisms are of utmost importance in the overall regulation of sperm function, the phosphorylation levels of serine residues (pSer) in 30 different sperm proteins after a 3 h- or 24 h-HT period were also assessed. We found that a HT of 24 h contributed to a higher sperm resistance to freeze-thawing procedures, whereas miniarray protein analyses showed that a HT of 24 h induced a significant (P<0.05) increase in pSer (from 100.0±1.8 arbitrary units in HT 3 h to 150.2±5.1 arbitrary units in HT 24 h) of HSP70 and, to a lesser extent, in protein kinases GSK3 and total TRK and in the cell-cycle regulatory protein CDC2/CDK1. In the case of HSP70, this increase was confirmed through immunoprecipation analyses. Principal component and multiple regression analyses indicated that a component explaining a percentage of variance higher than 50% in sperm cryotolerance was significantly correlated with pSer levels in HSP70. In addition, from all the parameters evaluated before freeze-thawing, only pSer levels in HSP70 resulted to be able to predict sperm cryotolerance. In conclusion, our results suggest that boar spermatozoa modulate its function during HT, at least partially, by changes in pSer levels of proteins like HSP70, and this is related to a higher cryotolerance. © 2014 Yeste et al.