The ICSI experiments took place at the aquaculture facility of the Genetic Fisheries Center and Laboratory of Germ Cells at the Faculty of Fisheries and Protection of Waters, University of South Bohemia in Ceské Budějovice, Vodnany, Czech Republic. All experimental procedures were performed in accordance with national (reference number: 2293/2015-MZE-17214) and institutional guidelines on animal experimentation and care and approved by the Animal Research committee of the University of South Bohemia in Ceské Budějovice.

During the ICSI experiments, a mature female Russian sturgeon (23–24-year-old), Siberian sturgeon (14–15-year-old), and beluga (22–23-year-old), as well as two mature female sterlets (4–5-year-old), were used as egg-donor species. Moreover, a mature male of sterlet (5–6-year-old), Siberian sturgeon (14–15-year-old), and beluga sturgeon (22–23-year-old) were used as sperm-donor species. All fish were kept at constant temperatures of 15°C at least 1 week before collection of gametes. In order to induce ovulation, the females were injected with an intramuscular injection of carp pituitary homogenized extract (CPE) at a dose of 5 mg/kg body weight (b.w.) in two steps: first with 0.5 mg/kg b.w. and second with 4.5 mg/kg b.w. 12 h after the first injection. Egg collection in sterlets was performed 22–24 h after the second injection. Beluga, Siberian, and Russian sturgeon females were stripped 28–30 h after the second injection.

In order to induce spermiation, the males were injected once (4 mg/kg b.w.) with CPE and spermatozoa was collected 24–36 h after hormonal injection by use of a catheter from the urogenital papilla, transferred to a separate cell culture container (250 mL), and stored at 4°C until sampling (1–3 h).

Non-activated and unfertilized eggs from sterlet, Siberian sturgeon, Russian sturgeon, and beluga arrested in meiosis II stage were washed three times for 5 min with Phosphate Buffered Saline (PBS) to remove debris and somatic cells from the spawning (38). Then they were placed in a 6 cm² petri dish filled with PBS at 15°C for ICSI.

To simulate reduced motility spermatozoa with acrosomal reaction retrieved from cadavers, we pre-activated the spermatozoa before ICSI. In an Eppendorf tube (1.5 ml) a drop of fresh stripped spermatozoa (1.5 μl) from sterlet or Siberian sturgeon was diluted (1/50) in filtrated water (15°C) for its activation for 15 s. The pre-activated spermatozoa were then transferred in PBS to return in inertia and held at 4°C until its use for the ICSI experiments.

Spermatozoa cryopreservation from beluga sturgeon was performed according to Glogowski et al. (43). Fresh sperm was diluted with cryomedium in a ratio of 1:1. The composition of cryomedium was 10 % ethanol, 23.4 mM sucrose, 0.25 mM KCl, and 30 mM Tris with pH adjusted to 8.0 by using HCl. The diluted sperm with cryomedium was pipetted into 0.5 ml pejets and kept 3 cm above liquid nitrogen surface for 10 min. Afterwards, the pejets were immersed and stored in liquid nitrogen until use. Beluga sturgeon's spermatozoa thawing was performed in a 36°C water bath for 15 s.

Spermatozoon motility was evaluated visually for the percentage of motile spermatozoa after activation and total duration of motility (in seconds). Motility parameters were measured immediately after initiation of spermatozoon activation until 100 % of spermatozoa were immotile. To induce the initiation of spermatozoon motility, a 49 μl drop of medium was placed on a glass slide and then a drop of 1 μl fresh spermatozoa was diluted using a microsampler. All experiments were performed in triplicate at room temperature (17–20°C), using light microscopy under 400 × magnification. To avoid subjective bias, all measurements were carried out by the same experimenter.

In order to perform ICSI using single spermatozoa, fresh (non- or pre-activated) or cryopreserved, we placed a drop of non-diluted sperm (1.5 μl) close to the eggs. Both eggs and spermatozoa remained inactivated in a petri dish filled with PBS (15°C). To assure that the microinjection needle would aspirate only one spermatozoon at a time, the oil running through the microneedle was creating a small drop that was carefully removed before aspiration. This procedure was used to aspirate as little PBS as possible together with the single spermatozoon. ICSI using fresh single non- or pre- activated spermatozoa was performed between beluga eggs and Siberian sturgeon spermatozoa, between Siberian sturgeon eggs and sterlet spermatozoa, as well as between Russian sturgeon eggs and sterlet spermatozoa. ICSI using cryopreserved single spermatozoa was performed between sterlet eggs and beluga spermatozoa (~40 % motility, evaluation under microscope).

To perform the multiple ICSI, diluted spermatozoa in PBS (0.001 %, 15°C) was filled in a microneedle and was not floated with the eggs in a petri dish. Each egg was microinjected with a single manipulation in the animal pole (~ 0.23 μl/egg). This corresponds to ~ 3827 sterlet spermatozoa per Siberian or Russian sturgeon egg and ~ 3905 Siberian sturgeon spermatozoa/beluga egg. Multiple ICSI were performed between Siberian sturgeon eggs and sterlet spermatozoa, between Russian sturgeon eggs and sterlet spermatozoa, as well as between beluga eggs and Siberian sturgeon spermatozoa.

All ICSI experiments were performed under a stereomicroscope (Leica M165 FC.) using a hydraulic injector (Cell-Tram Oil; Eppendorf, Germany) connected to a micromanipulator (MO-152; Narishige, Japan) on the animal pole of the eggs where the multiple micropyles are located. Application of a shallow injection was used during the ICSI technology, as it has been demonstrated in sturgeon microinjection techniques to result in high developmental rates (39). All transplants were incubated in PBS (15°C) for 30–40 min, washed three times with PBS to remove the floating spermatozoa in the petri dish, and then activated with filtrated water (15°C).

At the same time, in vitro fertilization of the control groups with fresh-stripped sperm was performed: beluga eggs x Siberian sturgeon spermatozoa (n = 31, fertilization rate = 90.3%), Siberian sturgeon eggs x sterlet spermatozoa (n = 80, fertilization rate = 90%), and Russian sturgeon eggs x sterlet spermatozoa (n = 87, fertilization rate = 90.8%) (Table 1). Moreover, in vitro fertilization of the control group with cryopreserved spermatozoa was performed: sterlet eggs x beluga spermatozoa (n = 583, fertilization rate = 51.5%) (Table 2). To confirm that spermatozoa were not motile nor sticky in the surface of eggs and had no ability to fertilize the eggs in the injection medium (PBS), we made a negative control group. We placed beluga eggs (n = 184) with Siberian sturgeon spermatozoa in PBS for 30 min, then we washed the eggs three times with PBS to remove the spermatozoa from the petri dish, and placed the eggs in filtrated water. Using the IBM SPSS statistic 27 software, Fisher's exact test was used to determine if there was a significant association in developing rate between the control group and ICSI experiments for each species gamete combination and developmental stage (Supplementary Figure S1). The negative control group exhibited neither fertilization nor embryonic development (Table 1).

Immediately after activation of the ICSI transplants and control groups with filtrated water, all embryos were treated with 0.01% tannic acid (SIGMA-ALDRICH^®) alternated with filtrated water for 10 min to remove the egg surface stickiness. Development was observed at the two-cell stage, corresponding to 3–4 h post activation (hpa). At 10 hpa, forceps were used to remove the outer layers of chorion for better observation of the development. Developing embryos were placed in 0.01% penicillin and 0.01% streptomycin in filtered water (15°C) for 3 days. The inner layer of the chorion was removed at 5–6 days post activation. First feeding started after yolk resorption, 25 dpa. The freshwater annelid worm tubifex sp. was given twice per day at 8:00 am and 16:00 pm. Tanks were cleaned twice per day 2 h after the meal. All embryos and larvae were held at the ambient photoperiod at a water temperature of 15°C.

Genomic DNA was extracted from caudal fin tissue of five egg-donors (a beluga sturgeon, a Siberian sturgeon, a Russian sturgeon, and two sterlets), three sperm-donors (a Siberian sturgeon, a sterlet, and a beluga sturgeon), along with 37 ICSI transplants, and one in vitro produced control embryo using GenElute Mammalian Genomic DNA Miniprep Kit (SIGMA-ALDRICH^®) according to the manufacturer's instructions. The presence of the beluga sturgeon's genome in the respective ICSI transplants was investigated using beluga specific primer pair 153_HHp + 153_uni (44) that amplifies 153 bp fragment of beluga nuclear DNA. The presence of sterlet's genome in the respective ICSI transplants was tested by sterlet specific primer pair 247_ARp + 247_uni (44), which amplifies 247 bp fragment from sterlet nuclear DNA. The presence of Siberian or Russian sturgeon's genome in the respective ICSI transplants was tested using specific primer pair 395_ABp + 395_uni (45), which amplifies 395 bp fragment from Siberian or Russian sturgeon's nuclear DNA. All reactions were performed according to (44, 45) in two independent replicates. Together with the species-specific primers from above, the genomic constitution of the ICSI transplants was estimated by parentage-like assignment using six microsatellite markers: AciG_35 (46), AfuG_135 (47), Aox_45 (48), Spl_101, Spl_163, and Spl_173 (49). Amplification and microsatellite fragment analysis were carried out according to the protocol described by Havelka et al. (50). Genotypes were scored in GENEIOUS 8.1.9, using Microsatellite Plugin 1.4.4.

ICSI of non-activated single spermatozoa from Siberian sturgeon into beluga unfertilized eggs reconstructed 39 transplants, from which 23 (59.0 %) exhibited initial cleavages and reached the blastula stage. Seven (17.9%) reached the gastrula stage from which 6 (15.4 %) fulfilled epiboly, neurulation, hatching, and feeding until 25 dpa, upon which they were sacrificed. Pre-activated and immobilized single spermatozoa from Siberian sturgeon were injected into beluga unfertilized eggs reconstructing 26 transplants, from which 22 (84.6 %) exhibited initial cleavages and reached the blastula stage. Nine transplants (34.6 %) formed the blastopore in the gastrula stage and 4 (15.4 %) fulfilled the epiboly, the neurula stage, hatched, and fed until 25 days post activation (dpa), upon which they were sacrificed. ICSI using multiple spermatozoa with a single manipulation from Siberian sturgeon into beluga eggs resulted in reconstruction of 63 transplants from which 40 (63.5 %) exhibited initial cleavage and reached the blastula stage. Four transplants (6.3 %) entered the gastrula stage, three (4.8 %) fulfilled epiboly, and one (1.6 %) entered the neurula stage, hatched, and fed until 25 dpa, upon which it was sacrificed (Table 1).

Sterlet pre-activated and immobilized single spermatozoa were injected into Siberian sturgeon unfertilized eggs reconstructing 90 transplants, from which 73 (81.1 %) exhibited initial cleavages and reached the blastula stage. Nine transplants (10.0 %) formed the blastopore at gastrula stage and stopped development. ICSI of non-activated single spermatozoa from sterlet into Siberian sturgeon unfertilized eggs reconstructed 52 transplants, from which 30 (57.7 %) exhibited initial cleavages and reached the blastula stage. Twenty-two (42.3 %) reached the gastrula stage from which 2 (3.8 %) fulfilled epiboly, entered the neurula stage, hatched, and fed until 25 dpa, upon which they were sacrificed. ICSI using multiple spermatozoa with a single manipulation from sterlet into Siberian sturgeon eggs resulted in reconstruction of 52 transplants from which 14 (26.9 %) exhibited initial cleavage and reached the blastula stage. Five (9.6 %) reached the gastrula stage at blastopore formation and stopped development (Table 1).

Sterlet pre-activated (15 s) and immobilized single spermatozoa were injected into Russian sturgeon unfertilized eggs reconstructing 54 transplants, from which 44 (81.2 %) exhibited initial cleavages and reached the blastula stage. Two transplants (4.5 %) formed the blastopore at gastrula stage and stopped development. ICSI of non-activated single spermatozoa from sterlet into Russian sturgeon unfertilized eggs reconstructed 32 transplants, from which 20 (62.5 %) exhibited initial cleavages and reached the blastula stage. Two (6.3 %) reached the gastrula stage, the neurula stage, hatched, and fed until 25 dpa, upon which they were sacrificed. ICSI using multiple spermatozoa with a single manipulation from sterlet into Russian sturgeon eggs resulted in reconstruction of 62 transplants from which 16 (25.8 %) exhibited initial cleavage and reached the blastula stage. Fourteen (22.6 %) reached the gastrula stage at blastopore formation and 6 (9.7 %) fulfilled epiboly and stopped development (Table 1).

In total, 24 ICSI transplants were analyzed for the presence of DNA of both egg- and sperm-donor species (Table 2).

The contribution of Siberian sturgeon sperm-donor DNA in developing embryos was studied in 14 specimens (Figure 1). We combined two markers: 395_AB (45) and 153_HH (44). The marker 395_AB amplifies 395 bp band only if a sample contains Siberian's and/or Russian's sturgeon genomic DNA while the marker 153_HH amplifies 153 bp band only if a sample contains genomic DNA of beluga. Ten transplants coming from the group ICSI with pre-activated single spermatozoa (n = 5), from the group ICSI with non-activated single spermatozoa (n = 3), and from the group of ICSI with multiple spermatozoa (n = 2) showed presence of DNA from both parents, thus confirming successful incorporation of sperm-donor DNA in the transplants (71.4 %). One transplant from the group ICSI using non-activated single spermatozoa showed contribution of only sperm-donor's DNA without any contribution from the beluga egg-donor (7.1 %). Three transplants showed amplification of DNA only from females (21.4 %) from the group ICSI with multiple spermatozoa (n = 1) and from the group ICSI with non-activated spermatozoa (n = 2) (Figure 1).

Efficiency of ICSI was studied in 10 transplants (Figure 2), by species specific markers. We combined two markers: 395_AB (45) and 247_AR (44). The marker 247_AR amplifies 247 bp band only if a sample contains genomic DNA of sterlet. Consequently, 9 specimens (90 %) showed presence of DNA from both parents, from the group ICSI using non-activated single sterlet spermatozoa into Siberian sturgeon's eggs (n = 2), the group ICSI using non-activated single sterlet spermatozoa into Russian sturgeon's eggs (n = 2), and the group ICSI using multiple sterlet spermatozoa into Russian sturgeon's eggs (n = 5). One specimen showed development of only egg-donor's DNA (10 %) in the group ICSI using multiple spermatozoa from sterlet into Russian sturgeon's eggs (Figure 2).

Microsatellite analysis of the ICSI transplants from Table 2 showed that 19 transplants (N°1–3, 7, 9–21, 23, and 24) contained both egg- and sperm-donor's DNA. One transplant (N°21) contained two haploid genomes from the sperm-donor according to the microsatellite markers AfuG_135 and Spl_163. Furthermore, one transplant (N°5) contained only sperm-donor's DNA, without any genomic contribution from the egg-donor (Table 3).

Non-activated single cryopreserved spermatozoa from beluga sturgeon (~ 40 % motility) were injected into sterlet unfertilized and non-activated eggs. Ninety-seven transplants reconstructed, from which 22 (22.7 %) exhibited initial cleavages and reached the blastula stage. Thirteen transplants (9.3 %) formed the blastopore, 10 (6.2 %) fulfilled the epiboly, and 4 (4.1 %) reached the neurula stage (Table 4).

In total, 13 ICSI transplants were analyzed for the presence of DNA of both egg- and sperm-donor species (numbers 25–33 and 35–38) and one control embryo (number 34) that was produced after in vitro fertilization of beluga cryopreserved spermatozoa to sterlet eggs. The transplants were created after ICSI using single cryopreserved spermatozoa from beluga into sterlet eggs. Numbers 25–27 arrested at gastrula stage in blastopore formation, 28–33 arrested at gastrula stage at 2/3 epiboly, and 35–38 arrested at neurula stage. The control embryo (number 34) arrested at neurula stage (Table 5).

Molecular analysis of 14 embryos has been performed by species specific markers (Figure 3, Table 6). We combined two markers: 247_AR and 153_HH (44). Eight ICSI transplants (61.5 %) showed presence of DNA from both parents, while 5 (38.5 %) showed development of only sterlet egg-donor's DNA without contribution of beluga sperm-donor's DNA (Figure 3, Table 6).

All resultant embryos and larvae followed a normal developmental pattern (38, 39, 51) without visible abnormalities in shape.