In this study we evaluated the effect of different cool storage time intervals (from 1h to 6h) elapsed between collection and semen freezing on both fresh and cryopreserved semen motility parameters and post-thaw fertilizing ability of Mediterranean brown trout semen. The rationale of this research was to understand if S. cettii sperm could be kept on ice up to a maximum of six hours storage time without losing its suitability for freezing.
The results showed that the cool storage interval significantly influenced some motility parameters of fresh semen, but surprisingly no significant effects were observed on post-thaw sperm motility and fertilization rate.
In fresh semen, in accordance with the research on rainbow trout by Lahnsteiner et al. [23]no significant difference of sperm motility parameters was recorded up to 2 h of storage on ice. After the second hour the total sperm motility, linear movement parameters (STR, LIN) and BCF showed a progressive decrease (P < 0.05), conversely the percentage of ALH underwent a significant increase. At the same time, even if not significant, visible increases for sperm velocity parameters, VCL and VAP, at 3 and 4 hours of storage were observed, while a simultaneous and progressive decrease of the VSL was recorded.
In other words, as storage time increased, the trajectory of movement became increasingly circular and less progressive. Cremades et al. [24], showed that changes in boar sperm movement patterns are the result of physiological events in spermatozoon. In agreement with what has been observed in mammalian the movement patterns of fresh trout sperm, as shown from the second storage hour onwards, equates to that of mammalian spermatozoa in the hyperactive state. Indeed, the movement pattern of hyperactive sperm is generally characterized by low VSL, STR and LIN values and an increase in ALH and VCL parameters [24–29]. The flagella of hyperactivated sperm forms deeper bends and their beating is usually asymmetric. As a result, hyperactivated sperm tends to swim vigorously in circles [30].
In mammals, hyperactivation usually occurs during sperm capacitation, allowing spermatozoa to penetrate the zona pellucida and fertilize the oocyte, therefore it is considered a critical event to the success of fertilization [31, 32]. However, also during the cryopreservation process mammalian spermatozoa in state of hyperactivation were observed. This phenomenon is called "cryocapacitation" and it triggers during the cooling process in the vicinity to 5°C due to the enhancement of cold shock [33, 34], which provokes a pathologic influx of Ca2+ in sperm and the hyperactivation of its motility [24, 35–37], decreasing the life span of sperm [24, 35].
Unlike mammalian spermatozoa, there is no mention in literature of hyperactivation and cryocapacitation phenomena of spermatozoa in fish species. However, we can speculate that changes in the fish sperm motility pattern before fertilization, and then probably even during the cooling process, may have something in common with the hyperactivated movement of mammalian spermatozoa which are about to fertilize eggs [38], or that occurs as a result of cryocapacitation [33, 34]. Our speculation can be supported by recent findings [39], in which the presence of cAMP-dependent protein kinase and CatSper-like protein in the spermatozoa of many fish, including trout, were identified. In particular, CatSper is a Ca2+-specific channel of mammalian spermatozoa plasma membrane, which by mediating Ca2 + i influx induces the initiation of the vigorous and hyperactive sperm motility prior to fertilization [40, 41].
In the light of these considerations, we assume that a cool storage time longer than 2 h, prior to freezing, could causes cold shock injury in plasma membrane of trout spermatozoa and an increase of membrane permeability, resulting in a pathologic influx of Ca2+ into the cell.
Consistent with our results, Labbè and Maisse [42] claimed that semen from rainbow trout does not undergo any cold shock when it is stored for 1 h, at 4°C just after stripping. Our data adds further evidence that the possible physical, biochemical and physiological changes in trout sperm are triggered starting from the second hour of cool storage. These changes are reflected in turn in spermatozoa swimming patterns, which exhibit similar behaviour to that of hyperactive mammalian spermatozoa [24, 29, 43].
The most interesting result that emerged in our study is that on the contrary to in vitro results observed in fresh semen, the cool storage time didn’t significantly affect the post-thaw sperm motility parameters. Although the cryopreservation process caused an overall decrease in the most of the sperm motility parameters at almost each time point tested compared to fresh semen, no significant differences was observed among post-thaw values recorded over time, both in term of fast and linear movement.
Thus, we can sustain that the cool storage time eliminates the weakest sperm leaving those cryoresilient. In this regard, we could hypothesize the presence of two distinct sperm populations: one cool-sensitive population which, after 2 h collection, underwent cold shock injury losing its freezability features and a second cryoresilient population, which survived cold injuries up to 6 hours and kept constant the average sperm motility parameters even after freezing. The cold-sensitive population could be the product of defective spermatogenesis, resulting in membrane weakness, defective enzymatic activity, low glycolytic activity and mitochondrial respiration, or a consequence of the stripping method which induces the release of non-completely mature spermatozoa [44].
The post-thaw motility results were consistent with those of post-thaw fertilization obtained from in vivo trials, using semen stored on ice for 1, 3 or 6 h prior to freezing. Although a progressive decrease in fertilization rate was observed over time, no significant differences were recorded from 1 to 6 h of storage. In agreement with values of sperm total motility and the duration of movement recorded in fresh semen, the highest percentage of fertilization was achieved at 1 h of storage, of which we assume that fewer spermatozoa are affect by cryo-injuries. In this regard, it is important to stress that the number of spermatozoa affected by cryo-injuries is always related to the individual ejaculates initial quality. Indeed, the range of the fertilization rates reported in the Table 1, show a wide inter-individual variability of the response to storage time on the post-thaw fertilization rate. However, regardless of the storage time and inter-male variability, a minimum success of fertilization rate that ranged from 40–43%, was always guaranteed.