Ploidy Levels and Fitness-Related Traits in Purebreds and Hybrids Originating from Sterlet (Acipenser ruthenus) and Unusual Ploidy Levels of Siberian Sturgeon (A. baerii)

The present study aimed to investigate and compare fitness-related traits and ploidy levels of purebreds and hybrids produced from sturgeon broodstock with both normal and abnormal ploidy levels. We used diploid Acipenser ruthenus and tetraploid A. baerii males and females to produce purebreds and reciprocal hybrids of normal ploidy levels. Likewise, we used diploid A. ruthenus and tetraploid A. baerii females mated to pentaploid and hexaploid A. baerii males to produce hybrids of abnormal ploidy levels. Fertilization of ova of A. ruthenus and A. baerii of normal ploidy with the sperm of pentaploid and hexaploid A. baerii produced fully viable progeny with ploidy levels that were intermediate between those of the parents as was also found in crosses of purebreds and reciprocal hybrids of normal ploidy levels. The A. ruthenus × pentaploid A. baerii and A. ruthenus × hexaploid A. baerii hybrids did not survive after 22 days post-hatch (dph). Mean body weight and cumulative survival were periodically checked at seven-time intervals. The recorded values of mean body weight were significantly higher in A. baerii × pentaploid A. baerii hybrids than other groups at three sampling points (160, 252 and 330 dph). In contrast, the highest cumulative survival was observed in A. baerii × A. ruthenus hybrids at all sampling points (14.47 ± 5.70 at 497 dph). Overall, most of the studied sturgeon hybrids displayed higher mean BW and cumulative survival compared to the purebreds. The utilization of sturgeon hybrids should be restricted to aquaculture purposes because they can pose a significant genetic threat to native populations through ecological interactions.


Introduction
Sturgeons (Acipenseridae) are one of the most unique and oldest families of fishes that are often considered to be "living fossils" because the fossil record of sturgeons dates back to Upper Cretaceous [1,2]. Evidence suggests that the sturgeon lineage diverged from the lineage that gave rise to ancient pre-Jurassic teleosts approximately 300 million years ago (MYA) according to their mitochondrial DNA analysis [2]. Sturgeons are one of the most endangered fishes in the world [3] because the sturgeon populations have experienced a dramatic decline throughout their range due to overfishing, habitat destruction and other anthropogenic causes [3][4][5]. There are 27 species of sturgeons in the world that are found along the coast of the Pacific and Atlantic oceans and in seas, inland lakes aquaculture purposes [22,32,33]. Therefore, it is interesting to evaluate the fitness-related traits of the F1 hybrids obtained by crossing sturgeon species with abnormal ploidy levels.
In the present study, we evaluated the reproductive features (fertilization rate and hatching rate), fitness-related traits such as body weight (BW), cumulative survival, growth heterogeneity (GH) and specific growth rate (SGR) of the first-generation purebreds and hybrids produced by crossing individuals of sterlet (A. ruthenus) and Siberian sturgeon (A. baerii) with normal and abnormal ploidy levels. We also analyzed the ploidy levels of the parents, purebred and hybrid groups using flow cytometry. Currently, Siberian sturgeon is the most commonly cultured sturgeon species in the world; hence we believe that this study will provide an essential insight into the global sturgeon aquaculture industry [28].

Origin, Maintenance and Artificial Propagation of Broodstock
Sturgeon broodstock was obtained from the Genetic Fisheries Center (GFC) at the Faculty of Fisheries and Protection of Waters, University of South Bohemia inČeské Budějovice, Czech Republic. One spontaneous pentaploid A. baerii male after cytometric examination; three males of A. ruthenus and three males of A. baerii were kept along with four females of A. baerii and six females of A. ruthenus. Fish were acclimatized in 4 m 3 hatchery tanks maintained with a water flow rate of 0.2 L/s, O 2 of 7.0 mg/L and water temperature of 15 • C for seven days ( Table 1). The final maturation was artificially induced by hormonal treatment in both males and females. The ovulated eggs were collected after micro-incision of the oviduct according to protocols from Štěch et al. [34]. The intramuscular injection of the carp pituitary powder dissolved in 0.9% (w/v) NaCl solution at 4 mg/kg body weight induced the spermiation in males artificially. After 24 h, sperm were collected from the urogenital tract using a catheter of 5 mm diameter connected to a 20 mL plastic syringe and later it was transferred into a 100 mL tissue culture flask [35]. Sperm was stored at 4 • C until the spermatozoon motility was evaluated by microscopy [36] and sperm from the males with the highest spermatozoa motility was used for further activation and fertilization. A cryopreserved sperm sample (year-old cryopreserved sample that was collected from the male from the previous year's artificial propagation) from one 15-year-old spontaneous hexaploid A. baerii male was also used in the experiment. Thawing of the sperm sample was performed in a water bath at 40 • C for 5 s, assessed for motility and was further used for fertilization [36]. Table 1. The relative volume of sperm kg −1 body mass (MB), concentration of spermatozoa (spz) and motility (mean ± SD) in sperm of sterlet (A. ruthenus), Siberian sturgeon (A. baerii), pentaploid Siberian sturgeon (A. baerii) and of the spontaneous hexaploid Siberian sturgeon (A. baerii). * denotes to multiplication.

Males
No

Fertilization and Hatching
By using a factorial mating design, eight crosses were produced, in which two were purebred crosses: A. ruthenus (St♀× St♂) and A. baerii (S♀× S♂), while the other six were hybrid crosses:  ). To produce each cross, an equal number of eggs from the females were pooled together (80 g of eggs were collected from six females of A. ruthenus and 120 g of eggs was collected from four females of A. baerii individually) and divided into plastic beakers in 40 g aliquots according to the number of males. The plastic beakers containing aliquots of eggs were then placed on an electronic shaker just before the fertilization which was set at a speed of 200 rpm and 10 mm deflection. To obtain equal genetic contribution from individual males and to avoid sperm competition between different males, each aliquot was inseminated with 1 mL sperm from one of the three males per cross, except for the pentaploid and hexaploid Siberian sturgeon and sperm were immediately activated by pouring in 200 mL dechlorinated water for fertilization. All the aliquots per cross were pooled together into a circular bowl after fertilization, and a clay suspension of 20 g L −1 was added three minutes after fertilization to remove egg stickiness and left on the electronic shaker for 50 min. Later on, the pooled aliquots were repeatedly washed with water to remove the clay remnants and then incubated in glass jar incubators in triplicates [35]. The UV sterilized re-circulating water with an O 2 saturation of 9 mg L −1 and water temperature of 15.0 • C was continuously supplied to the glass jars during the incubation period. Around 100 eggs were randomly sampled from each cross 6 h post-fertilization, in triplicate, to estimate the fertilization rate. The live embryos were counted at the 2nd or 3rd cleavage division, as previously explained [37]. The egg clumps and dead eggs were removed by siphoning the water regularly. The larvae that hatched out in 5-7 days were regularly separated from the dead larvae and shells in order to reduce the incidence of the fungal infection and consequent larval mortalities.

Fish Rearing
The larvae of each cross were initially reared in a 0.3 m 3 separate indoor trough system after complete hatching. The larvae were not fed on any natural or artificial feed until the complete yolk sac absorption. The larvae were firstly fed with diced sludge worms (Tubifex tubifex) ad libitum for the initial two weeks. They were then shifted to co-feeding with a combination of diced sludge worms and formulated dry feed. The larval feeding completely turned to the dry formulated feed after four weeks of co-feeding. The various formulated feeds fed to the fishes throughout the experiment were from Alltech Coppens, the Netherlands. The larvae of each cross moved to separate 3.5 m 3 indoor tanks, which were maintained with the average temperature of 22 • C for separate group-nursing after 100 days of initial rearing. The larvae of each cross were separately stocked with an initial stocking density of 7 kg/m 3 , and they were fed ad libitum with a formulated commercial feed (Coppens ® Start Premium; Coppens International B.V., Helmond, The Netherlands) containing 54% protein, 15% fat, 1% crude fiber and 9.4% ash. The 160 dph juveniles were color-marked with subcutaneous Visible Implant Elastomers (Northwest Marine Technologies Inc., Anacortes, WA, USA) on the inward ventral side of the rostrum to indicate the origin of the fish. These color-marked fishes were periodically checked, and an equal number from each group were stocked for communal rearing in triplicates with uniform environmental conditions, such as continuous partial water exchange, aeration rate, feeding rate and photoperiod. The Individual Passive Integrated Transponder (PIT) tags (134.2 kHz; AEG Company, Ulm, Germany) were subcutaneously implanted into the fish groups on 330 dph. The juvenile fishes were transferred to 4 m 3 indoor circular tanks at 4 • C without feeding for overwintering at the stocking density of~7 kg m 3 after the second summer. After wintering, the fish were transferred to the 3.5 m 3 outdoor circular tanks, which were maintained in an average temperature of 22 • C and were daily fed on a commercial diet of 4% of total fish biomass (Coppens ® Supreme-10 containing 49% of protein, 10% of fat, 0.8% of crude fiber and 7.9% of ash).

Measurement of Fitness-Related Traits and Mid-Parental Heterosis
Fishes were periodically measured to calculate the cumulative survival rate and mean BW on 22, 65, 160, 252, 330, 424, and 497 dph. The specific growth rate (% day −1 ) was calculated as SGR = 100 × (lnW 2 − lnW 1 ) × ∆T −1 , where W 1 and W 2 are the initial and final mean body weight, and ∆T is the time interval between sampling measurements in days. Likewise, the growth heterogeneity was calculated from CV FBW /CV IBW , in which CV is the coefficient of variation (100 × SD/mean) and IBW and FBW are the initial and final mean body weight.
The mid-parental heterosis for the cumulative survival and mean BW and of hybrid crosses were calculated as mid-parental heterosis = ((F1 − MP)/MP) × 100, in which F1 = the value of F1, and MP = the mean value of the two parents (purebred groups).

Flow Cytometry and DNA Content Analysis
The ploidy analysis of the broodstock was conducted using the sample of peripheral blood that was collected into a heparinized syringe from the caudal vessel of the fish. The blood sample was mixed with physiological saline (two drops blood 1 mL −1 saline), kept at 4 • C and after that treated with a 1 mL CyStain DNA 1 step staining solution (Sysmex Europe GmbH, Norderstedt, Germany). The blood samples were investigated by flow cytometry (Partec CCA I; Partec GmbH, Münster, Germany) in order to measure the relative DNA content per cell. The calibration of the device was undertaken using ready-to-use, DNA-stained and fixed rainbow trout erythrocytes. The erythrocytes of a functionally diploid A. ruthenus with a relative DNA content of 3.74 pg DNA per cell [14] was used as the reference diploid standard and its spermatozoa was used as the reference haploid standard for comparison of the different ploidy levels.
The ploidy level of the prelarvae was estimated from the caudal fin fragment using the CyStain DNA 2 step kit (Sysmex Europe GmbH, Norderstedt, Germany). A tissue sample of around 1-2 mm −2 was minced and incubated in 500 µL of the nucleus extraction buffer to segregate and permeabilize the cells. The cells were stained using a 2 mL staining solution, which contained the fluorescent DNA dye DAPI (4 , 6-diamidino-2-phenylindol). Thirty-five prelarvae samples from each group were processed for flow cytometry.

Statistical Analyses
Data analysis was performed with Statistica 13 (STATISTICA advanced, module STATISTICA Multivariate Exploratory Technique; Statsoft). The data were initially analyzed for normal distribution using the Kolmogorov-Smirnov test. Multiple comparisons of the fertilization rate, hatching rate, mean BW, specific growth rate and growth heterogeneity between different crosses were carried out by using the analysis of variance. Initially, the assumptions of ANOVA were checked, and when they were not respected, the Kruskal-Wallis test was performed. Later on, Tukey's post hoc (for ANOVA) or Dunn's post hoc (for Kruskal-Wallis test) tests were performed to evaluate the significant differences between the crosses. The differences in cumulative survival were determined using Pearson's Chi-square test on the surface significance of α = 0.05. Statistical significance of the differences within and between the individual groups was tested by analysis of variance ANOVA with the significance of α = 0.05. The histograms for the relative DNA content in the parents and f1 generation were combined together by overlaying raw data in the *.fcs format using CYTO-SW 0.3 software (Wolf & Danniel s.r.o., Mlada Boleslav, Czech Republic).

Confirmation of Ploidy Levels of Parents with Normal and Abnormal Ploidy
The results from the flow cytometry analysis of the broodstock revealed the relative DNA content of the three males of the A. ruthenus standard to be diploid ( Figure 1, blue peak 2n), three males of A. baerii to be tetraploid ( Figure 1, blue peak 4n) and one hexaploid specimen of A. baerii to be 3-fold of the diploid A. ruthenus ( Figure 1, blue peak 6n). Likewise, the sperm of the analyzed males had an average relative DNA content equivalent to haploidy ( Figure 1, red peak 1n), diploidy ( Figure 1, red peak 2n) and triploidy ( Figure 1, red peak 3n) in diploid A. ruthenus, tetraploid A. baerii and hexaploid A. baerii, respectively. Likewise, one pentaploid male of A. baerii ( Figure 2, blue peak 5n) was around 2.5-fold that of the reference diploid sterlet ( Figure 2, red peak 2n). The sperm of the analyzed pentaploid A. baerii (Figure 2, blue peak~2.5n) had a 2.5-fold higher average relative DNA content than the A. ruthenus sperm ( Figure 2, red peak 1n). The coefficient of variation (C V ) in both the sperm and erythrocyte relative DNA content was equal to or lower than 3% for all specimens.

Detection of Ploidy Levels in the F1 Generation Hybrids
The relative DNA contents of the reference diploid A. ruthenus and tetraploid A. baerii purebreds were 50.89 ± 2.14 and 101.56 ± 4.50, respectively ( Table 2). The ploidy allotment of all the abnormal ploidy was done by comparing the relative DNA content with the reference diploid A. ruthenus and tetraploid A. baerii purebreds. The ploidy level of the purebred A. ruthenus and A. baerii were 100% diploid (Table 2; Figure 3, red and blue peak 2n) and tetraploid (Table 2; Figure 3, green peak 4n), respectively, whereas the reciprocal hybrids produced from A. ruthenus and normal A. baerii (S × St and St × S) were 100% triploids (Table 2; Figure 3, green peak 3n). The ploidy level of the hybrid between diploid A. ruthenus and pentaploid A. baerii (St × S 5n) was~3.5n (Table 2; Figure 3, red peak~3.5n), whereas the hybrids of diploid A. ruthenus and hexaploid A. baerii (St × S 6n) were 4n (Table 2; Figure 3, green peak 4n). Likewise, the ploidy level of the hybrid between tetraploid A. baerii and pentaploid A. baerii (S × S 5n) was~4.5n (Table 2; Figure 3, blue peak~4.5n), whereas the hybrids of tetraploid A. baerii and hexaploid A. baerii (S × S 6n) were 5n ( Table 2).

Fertilization and Hatching
The highest fertilization rate (76.54 ± 2.3) was recorded in the St × S hybrid, which was not significantly different from the sterlet purebred, Siberian sturgeon purebred, St × S 5n hybrid and S × S 5n hybrid. The lowest fertilization rate (11.16 ± 0.74) was observed in the St × S 6n hybrid, which was significantly different than the other crosses ( Table 3). The highest hatching rate (67.93 ± 2.60) was observed in the Siberian sturgeon purebred, which was not significantly different from the majority of the crosses except for St × S 6n, S × S 6n and S × S 5n. The lowest hatching rate (6.57 ± 2.35) was observed in the St × S 6n thawed hybrid, which was significantly different than the other crosses (Table 3). Table 3. Fertilization and hatching rates among the produced hybrid and purebred crosses of different ploidy levels of Siberian sturgeon (A. baerii) and sterlet (A. ruthenus). Columns with the same alphabetic superscript did not differ significantly at p < 0.05. St: diploid sterlet; S: tetraploid Siberian sturgeon; S 5n: pentaploid Siberian sturgeon; S 6n: hexaploid Siberian sturgeon.

Cross
Fertilization

Mean Body Weight and Cumulative Survival
The swim-up larvae of the St × S 5n hybrid showed 100% mortality after five days of hatching. Likewise, the hatched individuals of the St × S 6n hybrid showed 100% mortality after the first sampling point (22 dph). The observed mean BW and cumulative survival of the crosses during all the sampling periods are shown in Figures 4 and 5. The highest mean BW (277.79 ± 127.79) was recorded in the St × S hybrid on 497 dph (Figure 4). The mean BW in the St × S hybrid was not significantly different from the S × S 5n hybrid on 497 dph. The recorded values of the mean BW in the S × S 5n hybrid was found to be significantly higher than the other groups on three sampling points (160, 252 and 330 dph). The highest cumulative survival (14.47 ± 5.70) was observed in the S × St hybrid on 497 dph ( Figure 5). The S × St hybrid displayed a significantly higher cumulative survival than the other crosses on all the sampling points. The significant differences between the groups in the cumulative survival were not estimated for the first three sampling points since the groups were separately reared in different tanks till 160 dph; they were also not reared in replicate tanks. The sterlet purebred and S × S 5n hybrid displayed comparatively lower cumulative survival than other crosses at most sampling points.

Mid-Parental Heterosis, Specific Growth Rate and Growth Heterogeneity
The mid-parental heterosis recorded in the S × St hybrid for cumulative survival was found to be the highest and positive for all the sampling points, whereas the S × S 5n hybrid displayed negative heterosis for cumulative survival at all the sampling time points (Figure 6). The observed values of SGR differed significantly among groups, from 22-65 dph, with the sterlet purebred (8.21 ± 0.68) exhibiting the highest SGR and S × S 6n hybrid (6.58 ± 0.41) the lowest. There were no significant between-group differences in SGR at later sampling points. The observed values of growth heterogeneity differed significantly among groups from 22-65 dph, with the S × S 5n hybrid exhibiting the highest GH and the S × S 6n hybrid the lowest. However, no significant GH differences were recorded between the groups at the later sampling points (Table 4).

Mean Body Weight and Cumulative Survival
The swim-up larvae of the St × S 5n hybrid showed 100% mortality after five days of hatching. Likewise, the hatched individuals of the St × S 6n hybrid showed 100% mortality after the first sampling point (22 dph). The observed mean BW and cumulative survival of the crosses during all the sampling periods are shown in Table 4. The highest mean BW (277.79 ± 127.79) was recorded in the St × S hybrid on 497 dph (Figure 4). The mean BW in the St × S hybrid was not significantly different from the S × S 5n hybrid on 497 dph. The recorded values of the mean BW in the S × S 5n hybrid was found to be significantly higher than the other groups on three sampling points (160, 252 and 330 dph). The highest cumulative survival (14.47 ± 5.70) was observed in the S × St hybrid on 497 dph ( Figure 5). The S × St hybrid displayed a significantly higher cumulative survival than the other crosses on all the sampling points. The significant differences between the groups in the cumulative survival were not estimated for the first three sampling points since the groups were separately reared in different tanks till 160 dph; they were also not reared in replicate tanks. The sterlet purebred and S × S 5n hybrid displayed comparatively lower cumulative survival than other crosses at most sampling points.

Mid-Parental Heterosis, Specific Growth Rate and Growth Heterogeneity
The mid-parental heterosis recorded in the S × St hybrid for cumulative survival was found to be the highest and positive for all the sampling points, whereas the S × S 5n hybrid displayed negative heterosis for cumulative survival at all the sampling time points (Figure 6). The observed values of SGR differed significantly among groups, from 22-65 dph, with the sterlet purebred (8.21 ± 0.68) exhibiting the highest SGR and S × S 6n hybrid (6.58 ± 0.41) the lowest. There were no significant between-group differences in SGR at later sampling points. The observed values of growth heterogeneity differed significantly among groups from 22-65 dph, with the S × S 5n hybrid exhibiting the highest GH and the S × S 6n hybrid the lowest. However, no significant GH differences were recorded between the groups at the later sampling points (Table 4).  same alphabetic superscript did not differ significantly at p < 0.05. St: diploid sterlet; S: tetraploid Siberian sturgeon; S 5n: pentaploid Siberian sturgeon; S 6n: hexaploid Siberian sturgeon.

Discussion
It is well known in the literature that sturgeons are highly prone for hybridization as well as for spontaneous autopolyploidization [7,9,10,13]. Although the most probable hybrid origin of polyploid sturgeon species has been confirmed by various data [24], the bases of their evolutionary success of abnormal ploidy levels and exceptional genome plasticity are still poorly understood. Additionally, the scope for expansion of sturgeon aquaculture has constantly been increasing these days due to the high commercial importance of black caviar. Notably, a considerable amount of aquaculture caviar production directly comes from sturgeon hybrids [31]. This led to increased production of various sturgeon hybrids formed between two species that originated from different parental ploidy levels in commercial aquaculture farms for better vigor of the hybrids [18,28,38].
In this study, by using flow cytometry analysis, we initially confirmed the presence of the abnormal ploidy levels of sturgeon broodstock in Genetic Fisheries Center, Vodňany. The results from the flow cytometry analysis revealed that the erythrocyte relative DNA content of the A. ruthenus standards were diploid (2n), A. baerii were tetraploid (4n) and one suspected A. baerii male with abnormal ploidy was pentaploid (5n). The origin of the hexaploid and pentaploid A. baerii is probably due to a spontaneous polyploidy phenomenon. Spontaneous polyploidy already has been reported in several cultured fish species [13]. The presence of an additional set of chromosomes in diploid (2n) species gives rise to triploid (3n) individuals that are infertile or sub-sterile. In case of tetraploid (4n) sturgeon species, spontaneous polyploidization gives rise to fertile hexaploid (6n) individuals that are already reported in A. baerii [22], A. mikadoi [39] and A. transmontanus [23]. Ploidy levels of the sterlet and Siberian sturgeon broodstock used in the experiment corresponded to 2n and 4n as previously reported for these species [13,22]. Likewise, some scientists discussed the possible fertility of sturgeon hybrids having an intermediate DNA content referring to recent triploids (3n) or evolutionary hexaploids (6n) [19]. Using flow cytometry, these authors detected evolutionary pentaploid (5n) and evolutionary heptaploid (7n) individuals among sturgeon aquaculture stocks, and suggested that these individuals might have originated from hybridization of an evolutionary hexaploid specimen (6n) with an evolutionary tetraploid specimen (4n), and an evolutionary hexaploid specimen (6n) with an evolutionary octoploid specimen (8n), respectively.
We then produced the hybrids using the pentaploid A. baerii male with A. ruthenus and tetraploid A. baerii females. As mentioned before, the cryopreserved sperm sample of one hexaploid (6n) A. baerii confirmed with flow cytometry analysis from the previous year was also used for the production of hybrids with abnormal ploidy levels. The cryopreserved sperm sample of the hexaploid (6n~368 chromosomes) A. baerii male that we used in our experiment was confirmed to be of spontaneous polyploid origin [13]. Fertilization of ova of A. ruthenus and A. baerii with normal ploidy with the sperm of pentaploid and hexaploid A. baerii produced fully viable progeny whose ploidy levels were found intermediate to those of the parents. We did not observe any evident rise in the percent of malformed non-viable larvae in any specific hybrid cross. Finally, we can conclude that the ploidy levels of the studied F1 generation hybrids were in accordance with the widely accepted assumption that the hybrids obtained from the breeding between parents with different ploidy levels are considered to have ploidy level intermediate to those of parental individuals [40].
Coming to the fitness-related traits, we observed a composite pattern for both heterosis and outbreeding depression in interspecific hybrids of sturgeons with abnormal ploidy levels. The swim-up larvae of St × S 5n hybrid did not survive after hatching, and the St × S 6n hybrid showed 100% mortality by reaching 65 dph. This might be due to extreme genetic incompatibility between their genomes and chromosome numbers. However, other hybrids, like the S × S 5n and St × S hybrids, displayed a significantly higher mean BW in most sampling points. Interestingly, the S × S 5n hybrid, which grew well, showed the lowest survival in the majority of the sampling points. The observed heterosis levels in the F1 generation hybrids changed depending on the trait or the time period considered in the present study. However, these levels were quite comparable with other studies dealing with heterosis in sturgeons [29,30]. The perspectives on the influence of hybridization on evolution have changed from time to time. Some authors suggested that hybrids are the "raw materials" of evolution and a creative source of functional adoption [41][42][43], whereas a few studies concluded hybridization as an evolutionary dead-end of a species [44]. Overall, in this study, the hybrids displayed better cumulative survival and mean BW in all the sampling points. However, the incidence of females with abnormal ploidy levels have not to be reported yet. It would be interesting to study the gamete interaction between sturgeon females and males with normal and abnormal ploidy levels. The differences in the growth rate, reproductive features, survivability, disease resistance and metabolic processes in polyploid sturgeons are the key considerations from a scientific and an applied point of view.
The significant effect of polyploidization on wild sturgeon populations has not been intensively investigated. Spontaneous polyploidization has been reported in wild populations of fishes like cobitids and cyprinids [45][46][47][48]. There is no reported evidence on the incidence of spontaneous polyploidization in the natural waters for sturgeons as far [13]. However, currently, climate change and global warming are the two biggest hurdles in the natural habitat that can expose sturgeon gametes to rapidly fluctuating temperature stresses that might affect the meiotic or fertilization disorders, resulting in an increased rate of autopolyploidization. Additionally, spontaneous polyploid individuals are already reported in farmed conditions and can be assigned to logistic delays during artificial propagation [48]. Accidental escapees from hatchery conditions to the wild environment can pose a potential threat to genetic diversity and disturb the ploidy level of the native species. Presently, restocking is the most widely applied management strategy to enhance declining fish populations affected by environmental degradation or overharvesting with considerable interest. Meanwhile, the reintroduction of A. transmontanus with an abnormal ploidy level into the wild waters, resulting in delayed reproductive maturation in the F2 generation and subsequent generations, are already reported in North America [49]. Such reintroduction programs without a proper genetic screening can easily contaminate the gene pool of the native stocks. As reported in the captive populations of sturgeons in the aquaculture farms, the fertile spontaneous polyploid individuals might spawn in natural waters, and they can result in progeny with reduced fertility. In addition to that, due to the growth properties, hybrids could be more prominent in food competition and can have access to limited food resources easily in the wild. This may generate F1 generation individuals with reduced fitness, and thus can have a harmful effect on the original net population genetic site and on the productivity and survival of the overall native population. Yet, despite the recent progress in the field of molecular genetics, we still have a lot to discover when it comes to the molecular mechanisms that are involved in chromosome duplication and elimination in early germ cells in cell cycle control in polyploid sturgeons, which need to be addressed in future studies.

Conclusions
This is the first study that describes the comparative performance evaluation of the F1 generation hybrids with the purebreds produced from normal and abnormal ploidy levels of Siberian sturgeon and sterlet. Most of the produced hybrids displayed a better growth rate and survivability compared to the purebreds, except the St × S 5n and St × S 6n hybrids. With the given importance of polyploid sturgeons in aquaculture, further studies on their performance in terms of reproduction, growth, stress, immune responses and metabolic processes should be studied, to expand our knowledge on the physiological and immunological differences in sturgeon hybrids of abnormal ploidy levels.

Conflicts of Interest:
The authors stated that they have no conflict of interest.