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The effect of antifreeze proteins on sterlet, Acipenser ruthenus sperm motility variables and fertilization rate were investigated after cryopreservation. Two types of antifreeze proteins (AFPI or AFPIII) were used at concentrations of 0.1, 1, 10 and 100 μg/mL. The motility variables of fresh and cryopreserved sperm with and without addition of antifreeze proteins were evaluated by the Computer Assisted Semen Analyzer (CASA). The fertilization rate using about 200,000 spermatozoa per egg was evaluated after 54 h incubation at 17 °C during the early stage of organogenesis. The motility, curvilinear velocity and straight-line velocity of fresh sperm was 93 ± 5%, 128 ± 13 μm/s and 89 ± 9 μm/s, respectively. There was a significant decrease of sperm motility rate between fresh sperm and cryopreserved sperm with/without addition of antifreeze proteins. The greatest motility among thawed samples was in the sperm cryopreserved with 10 μg/mL of AFPI (56 ± 20%), however, these data were not different compared to the sperm without antifreeze proteins (49 ± 14%). No statistical variations were detected in curvilinear velocity nor straight-line velocity. The fertilization rate with fresh sperm was 67 ± 7%. No significant differences were detected in fertilization rate between fresh and cryopreserved spermatozoa with/without addition of antifreeze proteins, except the sperm cryopreserved with 100 μg/mL of AFPIII (39 ± 14%). Thus, it is concluded that addition of antifreeze proteins to cryopreservation medium do not improve nor have toxicity effects on the quality and fertilization capacity of sterlet sperm after thawing.

Fish sperm cryopreservation is a well-established technique allowing for artificial insemination on a commercial scale. The extent of proteome alterations in seminal plasma and sperm due to cryopreservation, however, is not known. This study was conducted to evaluate the effect of cryopreservation on motility variables of sterlet Acipenser ruthenus sperm and to detect the differences in protein profiles of fresh and cryopreserved sterlet sperm and seminal plasma. Fresh sperm had 89 ± 3% motility and 160 ± 14 μm/s curvilinear velocity at 15 s post-activation. The motility rate of cryopreserved sperm (37 ± 5%) was less at 15 s post-activation. No difference (ANOVA; P > 0.05) in mean curvilinear velocity of fresh and cryopreserved sperm was detected. The protein profiles of seminal plasma and sperm were characterized using comparative proteomics to determine the influence of cryopreservation. Six altered protein spots in seminal plasma and thirteen altered spots in sperm were detected in fresh and thawed sperm. Subsequent protein characterization suggested that the proteins identified were involved in sperm metabolism, cytoskeleton, and stress response. The results broaden the understanding of the effects of cryopreservation and identify the proteins associated with cryo-injury. These data may help to determine the function of altered proteins and provide new insights into improving sperm cryopreservation.

Investigation of the heterogametic sex in sterlet Acipenser ruthenus L. was performed using meiotic gynogenesis and gonadal histology. Eggs from the albino females were activated by UV irradiated sperm of wild colored males and exposed to a heat shock. The resultant fish were all albino and exhibited exclusively maternal inheritance of the microsatellite DNA markers. Cytogenetic analysis indicated that gynogenetic progeny were diploids with 120 chromosomes. Based on the histological analysis, more than 86% of the gynogenetic individuals were found to be females. Moreover, some males (7%), sterile speciemens (3.5%) and fish with unidentified gonads (3.5%) were observed among the gynogenetic fish. Presence of both females and males in the gynogenetic offspring is indicative that the heterogametic sex in sterlets is female.

Sterlet Acipenser ruthenus was used to assess egg and embryo development when incubated at 17 °C in Petri dishes placed in a hatchery tank (300 L recirculating dechlorinated water) with incubation occurring in a static tabletop system in an air-conditioned laboratory, or in a 700 L Q-cell incubator. Eggs in each dish were placed in a plastic box with 300 mL dechlorinated water. Separated eggs from three individual females were fertilized using pooled sperm from four males with there being four replicates. There were no differences (P > 0.05) in mean percentages of neurulation and embryos undergoing cleavage for eggs incubated in the hatchery tank and with use of the static tabletop system. Furthermore, there were no differences (P > 0.05) in percentage of embryos undergoing cleavage, neurulation and hatching for each female when eggs were incubated using the two systems. Results indicate a Petri dish placed in a small plastic box with 300 mL of dechlorinated water was adequate for incubation of sterlet eggs. Results of the study also indicate that with the static system: 1) eggs should be fertilized from each female to retain individual identity; 2) eggs should be dispersed in Petri dishes to avoid clumping; 3) water should be changed at 24 h, but not at 48 h (neurulation) post-fertilization; and 4) embryos that do not optimally develop should be removed the day after neurulation (72 h of post-fertilization period) and water should be exchanged every day subsequent to the 48 h time-point post-fertilization.

Species may be prevented from interspecific hybridization by a number of different reproductive barriers that operate precopulatory and postcopulatory. In situation, when natural precopulatory reproductive barriers are affected by anthropogenic factors, postcopulatory reproductive barriers may be important for maintaining gametic isolation and hence preventing interspecific hybridization. This is highly topical in sturgeon (order Acipenseriformes) which exhibits remarkable ease of interspecific hybridization. The objectives of the present study were to evaluate the fertilization success of Acipenser ruthenus and Acipenser baerii spermatozoa under the interspecific competitive conditions and assessed, whether their spermatozoa tend to differentially fertilize eggs of conspecifics. We set up several in vitro fertilization experiments: (i) pooled eggs of both species were fertilized by sperm of each species separately; (ii) eggs of each species were fertilized by pooled sperm; (iii) pooled eggs were fertilized by pooled sperm and (iv) purebred and hybrid control groups. Using parental assignment by molecular markers, we found that when these species competed in pooled sperm, 78.9% of progeny were sired by A. ruthenus and 21.1% by A. baerii, demonstrating higher fertilization success for the former, irrespective of conspecificity of fertilized eggs. When pooled eggs were inseminated by A. ruthenus or A. baerii sperm separately, progeny almost equally comprised hybrid and purebred individuals. Hence, neither A. ruthenus nor A. baerii eggs showed a tendency to biased fertilization by spermatozoa of conspecific males. These findings together show that there may not be postcopulatory mechanisms preventing hybridization between A. ruthenus and A. baerii.

To evaluate the effects of thiamine on physiological changes and spawning performance of Sterlet sturgeon Acipenser ruthenus, 45 farmed female fish (698.6±8.9g) were randomly distributed in 9 tanks (1000L) and fed a diet with 1g/kg of an anti-thiamine drug. This was provided for 5 months prior to spawning. Thiamine hydrochloride was intraperitoneally injected to fish at three different doses: 0 (T0, as control), 5 (T5) and 50 (T50) mg/kg body weight at days 30, 90 and 150 after the experiment started. After five months, the results showed no significant differences in weight gain and hemoglobin level, but hematocrit significantly increased in T5 group. There was no significant difference in glucose-6-phosphate dehydrogenase and estradiol-17β, but testosterone was significantly increased in the T50 group. Total thiamine concentration in the eggs was significantly higher in T50 than that detected in the control group. Fecundity and larval mortality at 6day post hatch (dph) showed no significant differences among treatments, while the number of eggs per gram was significantly lower in T0 than that observed in T50. Larval weights at 1 (11.6mg) and 6 (23.1mg) dph and larval lengths at 6 (15.6mm) dph were significantly affected by the treatment with the highest level of thiamine injection (T50). Diseases symptoms such as yolk sac deformation, erratic pattern of swimming, and loss of equilibrium were observed at 4 dph in T0 and T5 groups. The overall results revealed that thiamine injection has positive effects on reproductive performance in the sturgeon and the negative impacts of anti-thiamine in the offspring can be reduced by the injection of this vitamin to the broodstock.

This study reported facultative parthenogenetic cleavage development of sterlet sturgeon Acipenser ruthenus eggs and quantified the percentage of parthenogenetically developed eggs in relation to the fertilization ability of different females. When eggs were activated in freshwater, 5.1–13.7% of eggs developed parthenogenetically, while among those activated eggs 3.6–9.4% developed to 2 cells, 0.4–4.5% developed to 4 cells, and 0–0.8% developed to 8 cells. The mean percentage of fertilized and parthenogenetically activated eggs among the females was negatively correlated (R 2 =0.71, p=0.036), which indicates that parthenogenetic activation rate of sterlet eggs depends on the quality of eggs in terms of fertilization rate.

Standardization of fertilization protocols for sterlet Acipenser ruthenus is crucial for improving reproductive techniques and for conservation purposes. Our objectives were to determine the number of sperm (tested 430,000:1, 43,000:1, 4300:1, 430:1 sperm to egg) required to fertilize eggs and explore how pre-incubation of eggs in freshwater for 0min, 0.5min, 1min, and 10min interacts with different sperm ratios. Fertilization success ranged from 29.7% at 430:1 to 84.2% at 430,000:1. Pre-incubation time had no effect on fertilization success at 430,000:1 and 43,000:1 sperm to egg ratios, while it was significant at the 4300:1 and 430:1 ratios. The use of adequate experimental suboptimal sperm to egg ratio revealed a positive effect of pre-incubation time, such that at the 430:1 ratio, 0.5min pre-incubation increased the fertilization rate than 10min. At 0min pre-incubation the proportion of fertilized eggs increased at the 430,000:1 ratio, while at 1min fertilization increased at the 4300:1 ratio. At the 10min pre-incubation time, fertilization increased at the 43,000:1 ratio. Moreover, at the 0.5min pre-incubation time, the 43,000:1 ratio increased the fertilization rate than the 430:1 ratio. Generally, for 430:1 ratio, the fertilization rate is lower than in control. Transmission electron microscopy showed that pre-incubation of eggs in water for

Ultrastructure of sterlet Acipenser ruthenus L. 1758 sperm was examined by scanning and transmission electron microscopy, which allowed us to use various methods for visualizations of different parts of sterlet spermatozoa. Sperm cells possess a head with a distinct acrosome, a midpiece and a single flagellum surrounded by the flagellar plasma membrane. The average length of the head including the acrosome and the midpiece was estimated as 5.14 ± 0.42 μm. Nine to 10 posterolateral projections were derived from the acrosome. Three inter-twining endonuclear canals bounded by membranes traversed the nucleus in its whole length from the acrosome to the implantation fossa. Acrosin was located in all the three parts (acrosome, endonuclear canals and implantation fossa). The proximal and distal centrioles located in the midpiece compacted of nine peripheral triplets of microtubules. One cut of the midpiece contained from two to six mitochondria with area of 215 ± 85 nm2 in average. The flagellum was 42.47 ± 1.89 μm in length with typical eukaryotic organization of one central pair and nine peripheral pairs of microtubules. It passed through a cytoplasmic channel in the midpiece, which was formed by an invagination at the plasmalemma. The flagellum gradually developed two lateral extensions of its plasma membrane, so-called “fins”. Detected morphological variation can be described by four principal component axes corresponding to groups of individual morphometric characters defined on the sperm structures. Correlations among the characters indicate that the sperms are variable in their shape rather than size. Significant variation among examined fish individuals was found only in flagellum and nucleus length. Comparison between the present and previous studies of morphology of sturgeon spermatozoa confirmed large inter- and/or intra-specific differences that could be of substantial taxonomic value.