Cryobank of Mediterranean Brown Trout Semen: Evaluation of the Use of Frozen Semen up to Six Hours Post-Collection

The aim of this study was to evaluate the effects of different cold-storage time intervals between collection and semen-freezing on both fresh and cryopreserved semen motility parameters and the post-thaw fertilizing ability of Mediterranean brown trout semen. The ejaculates were split into six aliquots and stored on ice from 1 to 6 h, until freezing. Fresh and post-thaw sperm motility was evaluated by a Computer-Assisted Sperm Analysis system, whilst the fertilizing ability was assessed by in vivo trials. In fresh semen, at 3 h of storage, a significant decrease of total motility, linear movement (STR, LIN) and beat cross frequency (BCF) was recorded, whilst the amplitude of lateral displacement of the spermatozoon head (ALH) underwent a significant increase. In frozen semen, no significant difference was observed for all the motility parameters evaluated, except for the total motility between 1 and 6 h of storage and the duration of sperm movement between 1 and 5 h. Cold-storage time did not significantly affect the percentage of live embryos following the use of frozen semen. In conclusion, our results showed that, if necessary, the Mediterranean brown trout semen can be frozen even until 6 h post-collection without losing its fertilizing ability.


Introduction
Over the last two decades, because of population reduction and extinction in many European countries, native salmonid species have been the focus of some important conservation projects [1][2][3][4][5]. The Mediterranean brown trout is one of the freshwater fish species complexes at a greater risk of extinction in the Mediterranean area; it is also listed on the Italian IUCN Red List as "critically endangered" [6] under the taxon S. cettii. The introduction of alien invasive species, such as the Atlantic strain, and their interaction with the native fauna represents a major threat to the survival of this species, as the traits of their native gene pool are altered [7][8][9][10][11][12][13].
In this context, the EU has recently funded the "LIFE" Nat.Sal.Mo project, which aims to ensure the recovery of native genetic variability and the conservation of the Mediterranean brown trout (S. macrostigma = S. cettii) inhabiting the Biferno and Volturno river basins (Molise region-Southern Italy).
The restoration of the Mediterranean brown trout's genetic integrity is among the main objectives of the project. It is being realised through the use of artificial reproduction, using frozen semen from pure wild breeders in combination with appropriate fertilization schemes, as a strategy to maximize the genetic variability within the offspring and ensure the maintenance of the fitness within self-sustaining populations. In this regard, the creation of the first European semen cryobank plays a key role for conserving extant genomic diversity of S. cettii to be used for artificial breeding activities. In order to obtain an effective freezing protocol aimed at the implementation of the first European sperm cryobank for the native Mediterranean brown trout, a series of systematic studies were performed [14][15][16][17].
Among the factors that affect the success of the cryopreservation procedure, good initial quality of semen is an indispensable prerequisite. The latter depends on the selected donor, a correct semen collection method, the appropriate conditions for transportation and the semen storage times before freezing [18]. Concerning this last factor, the time that elapses between collection and processing of semen results as an important variable in our project, since the sampling sites in the project area are not easily accessible and are distant, not only from each other but also from the laboratory. Due to this logistic problem, we have estimated that the time range that elapses between collection and semen-freezing could vary from a minimum of 1 h to a maximum of 6 h, depending on the sampling site and the time taken to capture the broodstock. As a result of these large time intervals, we questioned if the longer intervals of time prior to freezing could cause a loss in the quality of fresh semen and, consequently, adversely affect the freezability of the spermatozoa.
In light of these considerations, six possible scenarios have been simulated to evaluate the effect of different cold-storage time intervals (from 1 h to 6 h) between collection and semen-freezing on both fresh and cryopreserved semen motility parameters and post-thaw fertilizing ability of Mediterranean brown trout semen.

Effect of Storage Time Post-Semen Collection on Fresh Sperm Motility Parameters
Some motility parameters of the fresh semen were significantly affected (p < 0.05) by the cold-storage time following sperm collection ( Figure 1). Total sperm motility was not affected until 2 h, after this time-frame a significant decrease was recorded ( Figure 1A). No significant differences for kinetic parameters (VCL, VAP and VSL), during storage were recorded ( Figure 1B-D). The lowest percentage of ALH was registered at 2 h of storage, this resulted as significant in respect to the values recorded at 3, 4, 5 and 6 h ( Figure 1G).

Effect of Storage Time Post-Collection on Post-Thaw Fertilization Rate
The percentage of eyed embryos obtained using fresh semen was of 79.4% ± 5.0%, confirming the good quality of the eggs used for the fertilization trial.
Cold-storage time did not significantly affect the percentage of eyed embryos following the use of frozen semen (Table 1). In fact, the average fertilization percentages ranged from 65% to 55% without significant differences among the storage times postcollection tested.  The highest values of STR, LIN and BCF were found at 2 h, these values were significantly different in comparison to those recorded at 3, 4, 5 and 6 h.
The duration of sperm movement was significantly higher at 1 h compared to the other storage times and it reached the lowest value at 2 and 6 h. At 4 h of storage, an increase in duration of sperm movement was observed; this was a significant result in respect to those found at 2 h and 6 h of storage ( Figure 1I).

Effect of Storage Time Post-Collection on Frozen Sperm Motility Parameters
The cold-storage time after sperm collection significantly decreased the total post-thaw motility between 1 and 6 h of storage ( Figure 1A) and the duration of sperm movement between 1 and 5 h ( Figure 1I), whilst no significant differences were observed for all the other parameters tested.

Effect of Storage Time Post-Collection on Post-Thaw Fertilization Rate
The percentage of eyed embryos obtained using fresh semen was of 79.4% ± 5.0%, confirming the good quality of the eggs used for the fertilization trial.
Cold-storage time did not significantly affect the percentage of eyed embryos following the use of frozen semen (Table 1). In fact, the average fertilization percentages ranged from 65% to 55% without significant differences among the storage times post-collection tested.

Discussion
In this study, we evaluated the effect of different cold-storage time intervals (from 1 h to 6 h) that 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 Salmo 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 cold-storage interval significantly influenced some motility parameters of fresh semen, but surprisingly no significant effects were observed on all post-thaw sperm motility parameters tested (except for total motility recorded at 6 h of storage) and on fertilization rates.
In accordance with the results found on rainbow trout by Lahnsteiner [19], in fresh semen, no significant difference of sperm motility parameters was recorded up until 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), whilst ALH values underwent a significant increase. Thus, our results showed that as the amount of storage time was increased, the trajectory of sperm movement tended to become increasingly circular and less progressive. Cremades et al. [20], showed that changes in boar sperm movement patterns are the results of physiological events in spermatozoon. Similar to what has been observed in mammalian sperm, the movement patterns that occur in fresh trout sperm, starting from the second hour onwards equate to those of mammalian spermatozoa in a hyperactive state. Indeed, the movement pattern of hyperactive sperm is generally characterized by low STR and LIN values, high amplitude and asymmetrical flagellar beating, representing an increased torsional force [20][21][22][23][24][25][26]. As a result, hyperactive sperm tend to swim in vigorous circles [27].
In mammals, hyperactivation usually occurs during sperm capacitation, which allows spermatozoa to penetrate the pellucida zone and fertilize the oocyte. Therefore, it is considered a critical event in the success of fertilization [28,29]. Moreover, sperm in a state of hyperactivation were observed in mammals during the cryopreservation process. This phenomenon is called "cryocapacitation" and it is triggered during the cooling process when the temperature is in the vicinity of 5 • C. This is due to the enhancement of cold shock [30,31], which provokes a pathologic influx of Ca 2+ in sperm and the hyperactivation of its motility [20,[32][33][34], thereby decreasing the life span of sperm [20,32]. Unlike mammalian spermatozoa, there is no evidence of the presence of hyperactivation and cryocapacitation phenomena in fish sperm, including in salmonids, as they do not possess acrosome. However, we can speculate that changes in the sperm motility patterns observed in our study during cold storage, may be due to mechanisms that trigger the hyperactive movement, similar to those that are found in cryocapacitated mammalian sperm.
Several studies on different fish species such as rainbow trout [35][36][37], common carp [38], Atlantic croaker [39], and sterlet sturgeon [40] have reported that calcium chan-Fishes 2021, 6, 26 5 of 10 nels play a key role in regulating sperm motility. Recently, some authors have identified the presence of CatSper-like protein in the spermatozoa of many fish, including rainbow trout [41,42]. In particular, CatSper is a Ca 2+ -specific channel of mammalian spermatozoa plasma membrane, which by mediating Ca 2+ i influx induces the initiation of the vigorous and hyperactive sperm motility prior to fertilization [43,44]. A recent study [42], demonstrated, for the first time, that this ionic channel plays a key role even in the sperm motility of Atlantic salmon. In the light of these considerations, we could assume that a cold-storage time longer than 2 h, prior to freezing, could cause cold shock injury in the plasma membrane of trout spermatozoa and an increase in membrane permeability, resulting in a pathologic influx of Ca 2+ into the cell. Consistent with our results, Labbè and Maisse [45] 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 led us to think that possible physical, biochemical and physiological changes in trout sperm begin to be triggered from the second hour of cold storage. However, further analysis to support our hypothesis, such as the measurement of concentration of the cholesterol in sperm membrane and the intracellular Ca 2+ in the cryopreserved semen samples, is needed The most interesting result that emerged in our study is that in contrast to in vitro results observed in fresh semen, the cold-storage time did not significantly affect the post-thaw sperm motility parameters, with the exception of the total motility recorded at the sixth hour of storage. Thus, we can postulate that the cold-storage time eliminates the weakest sperm population leaving those that are more cryoresistant. To be specific, two distinct sperm populations could coexist: one cool-sensitive population, which 2 h after semen collection undergoes cold shock injury, thereby losing its freezability features, and a second cryoresistant population, which survives cold injuries up to 6 h and keeps the sperm motility parameters constant 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 [46].
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 were affected 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 Table 1 show a wide inter-individual variability to 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.

Animal Capture and Sperm and Eggs Collection
Specimens of autochthonous Salmo cettii were caught in the Biferno River (Molise region, latitude: 41 • 28 47.8 N and longitude: 14 • 28 40.9 E) during the spawning season (January-February 2020), by electro-fishing. Twelve individuals were first identified according to their phenotypic traits [47][48][49] and, subsequently, were crossed with genetic data to ensure that the individuals were autochthonous. These individuals (10 males and 2 females) were aged between 2+ to 5+ years, and the average total lengths of the fish were 24.9 ± 6.1 cm for males and 29.7 ± 7.3 cm for females. Sperm samples were collected by gentle abdominal massaging; abdomens and urogenital papilla were dried with special care before stripping, in order to avoid contamination of semen with urine, mucus and blood cells. The tubes containing sperm were transferred to Eggs were stripped by gentle abdominal massage into a dry metal bowl and were checked visually to ensure that those used in the fertilization experiments were wellrounded and transparent.
The experiments were conducted in accordance with the Code of Ethics of the EU Directive 2010/63/EU for animal experiments. This study took place within Nat.Sal.Mo LIFE project, which received "a positive opinion" from the Ministry of the Environment and the Protection of the Territory and the Sea. The sampling and handling of fish followed animal welfare practices as reported in the Ministerial Protocol (ISPRA). All experiments were carried out with the appropriate authorizations from the Molise Region-Dipartimento Governo del Territorio, Mobilità e Risorse Naturali cod. DP.A4.02.4N.01 (protocol number 3969, 3 August 2018), according to the current regulations on the protection of the species, biosecurity, protocols of sampling of fresh water and animal welfare.

In Vitro Experimental Design and Cryopreservation Procedure
To begin, six aliquots of equal volume from each sperm sample (n = 10 male) were split into 1.5 mL cryovials and stored on ice for 1, 2, 3, 4, 5 and 6 h respectively, until freezing (6 intervals × 10 males = 60 total aliquots). After each storage time interval, an aliquot of fresh semen was subjected to sperm motility evaluation by a Computer-Assisted Sperm Analysis (CASA) system, whilst another one was frozen using the cryopreservation procedure optimized in our previous work [17] (Figure 2). Briefly, each semen aliquot was diluted with a freezing extender to reach 0.15 M of glucose, 7.5% of methanol and sperm concentration of 3.0 × 10 9 sperm/mL. The diluted semen was charged into 0.25 mL plastic straws and equilibrated for 15 min on ice (at the height of 3 cm), lastly the straws were cryopreserved by exposure to liquid nitrogen (LN 2 ) vapor at 3 cm above the LN 2 level for 5 min and plunged into LN 2 . Frozen semen samples were tested after each post-collection storage time, and were thawed at 40 • C for 5 s and immediately evaluated using the sperm motility analysis.

Sperm Analysis
The fresh semen concentration was measured with a Neubauer chamber. The samples were diluted 1:1000 (v:v) with 3% NaCl (w:v), and sperm counts were carried out in duplicate, at a magnification of 400× and expressed as ×10 9 /mL. The average sperm concentration was 15.3 ± 4.9 × 10 9 sperm/mL.
The sperm motility parameters were determined with the use of a CASA system coupled to a phase contrast microscope (Nikon model Ci-L) using the Sperm Class Analyser (SCA) software (VET Edition, Barcelona, Spain). For the sperm motility activation, fresh and frozen spermatozoa were diluted as reported in our previous paper [16], reaching a concentration of 6.0 × 10 7 sperm/mL. After, an aliquot of 3 µL was loaded onto a 20 micron Leja slide (Leja Standard Count, Nieuw Vennep The Netherlands) and

Sperm Analysis
The fresh semen concentration was measured with a Neubauer chamber. The samples were diluted 1:1000 (v:v) with 3% NaCl (w:v), and sperm counts were carried out in duplicate, at a magnification of 400× and expressed as ×10 9 /mL. The average sperm concentration was 15.3 ± 4.9 × 10 9 sperm/mL. The sperm motility parameters were determined with the use of a CASA system coupled to a phase contrast microscope (Nikon model Ci-L) using the Sperm Class Analyser (SCA) software (VET Edition, Barcelona, Spain). For the sperm motility activation, fresh and frozen spermatozoa were diluted as reported in our previous paper [16], reaching a concentration of 6.0 × 10 7 sperm/mL. After, an aliquot of 3 µL was loaded onto a 20 micron Leja slide (Leja Standard Count, Nieuw Vennep The Netherlands) and the following sperm motility parameters were evaluated: The duration of sperm movement was evaluated using a chronometer. Three replicates were performed for sperm motility evaluation of each fresh and frozen semen sample.

Fertilizing Ability Trials of Cryopreserved Semen
The fertilization trial was performed in February 2020. Pooled eggs from two females were divided into batches of 80 ± 9 eggs, using 36 glass laboratory jars: the eggs in six of the jars were fertilized using excess fresh semen at the beginning and at the end of the fertilization trial (control groups), in order to test the quality of the eggs, whilst the eggs in the remaining 30 jars were divided into three treatment groups. Each treatment group was fertilized using the semen of individual males (n = 10) frozen/thawed after a cold-storage interval of 1, 3 or 6 h, using a spermatozoa-to-egg ratio of 4.5 × 10 5 :1. Before adding the semen, 5 mL of fertilization solution D532 (20 mM Tris, 30 mM glycine, 125 mM NaCl, pH 9.0 [50]) was added to the eggs. The sperm was gently mixed with the eggs for 10 s, and then about 20 mL of hatchery water was added. After 2 min, the eggs were washed with hatchery water and placed into an incubator with running water at about 10 • C.
Unfertilized and dead eggs were counted and removed every day during the incubation. After 25-30 days, the eggs reached the eyed-egg stage. The fertilization success was established by calculating the percentage of embryos at the eyed stage, using the initial number of eggs (number of eyed eggs × initial egg number −1 × 100).

Statistical Analysis
Values are expressed as mean ± standard error (SE). The statistical analysis was conducted with the software package SPSS (SPSS 15.0 for Windows, 2006; SPSS, Chicago, IL, USA). Sperm motility parameters and fertilization rates measured across the different storage time intervals were compared by one-way analysis of variance (one-way ANOVA) followed by Duncan's comparison test. The level of significance for all statistical tests was set to 5% (p < 0.05).

Conclusions
In conclusion, our results showed that although it is recommendable to freeze semen of S. cettii within 1-2 h of collection, good fertilization rates are achievable even 6 h after semen collection from wild breeders. This last scenario is a valid expedient for us when the sampling sites are far away from the laboratory. These results provide an important contribution to improving the sampling management in the case of our wild specimen. Finally, further studies are needed to understand the biological mechanisms involved in the trout sperm movement pattern, and if these patterns are similar to the hyperactivity triggered following the cooling steps.