Abstract
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 in-depth 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 mini-array 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.
Highlights
Mammalian sperm cryopreservation is a stressful event that generates damaged spermatozoa through mechanisms such as oxidative stresses and cold-shock (See Rath et al [1], for a review)
Our results confirm that holding time (HT) for 24 h has an improving effect on boar-sperm cryotolerance
We have shown that this improving effect is concomitant with changes in phosphorylation levels of serine residues (pSer) levels of proteins like HSP70
Summary
Mammalian sperm cryopreservation is a stressful event that generates damaged spermatozoa through mechanisms such as oxidative stresses and cold-shock (See Rath et al [1], for a review). In the case of porcine spermatozoa, cryopreservation is usually performed after sperm has been stored for up to a 24-h period (Holding Time, HT) at 17uC after collection, as this period in contact with seminal plasma has been reported to yield higher tolerance to low temperatures [2,3,4]. While Kotzias-Bandeira et al [5] and Eriksson et al [6] found that a longer HT, rather than a shorter one, was beneficial for post-thaw sperm viability, Guthrie and Welch [7] found no significant HT effect on post-thaw sperm survival These discrepancies can be related to the alternate sperm processing procedures utilised by each investigation. More information is needed to reach a consensus on this point
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