The Effect of Cryopreserved Sperm on the Early Development, Survival, and Growth of Intergeneric Sterbel Hybrids (Acipenser ruthenus × Huso huso).

The aim of this study was to analyze the survival and growth of intergeneric (Acispenser ruthenus × Huso huso L.) sterbel hybrids obtained by fertilizing sterlet eggs with cryopreserved beluga semen. The rate of embryonic development did not differ between sterbel hybrids (experimental groups) and sterlets (control groups), and the hatching period was identical in all groups. The survival rate of hybrid larvae was higher in the experimental groups than in the control groups. Body weight and body length measurements revealed that sterbel hybrids grew at a faster rate than the control group sterlets. The hybrid origin of sterbels produced with the use of cryopreserved beluga semen was confirmed in a genetic analysis based on species-specific DNA fragments. To the best of the authors' knowledge, this is the first study to analyze the growth of sterbel hybrids derived from cryopreserved semen. The research findings indicate that this type of intergeneric hybridization delivers satisfactory results and can be applied in sturgeon aquaculture.

Effect of temperature and spawning agents on wild female asp (Leuciscus aspius) reproductive efficiency under controlled conditions.

Artificial reproduction is a bottleneck to produce stocking material for many species of freshwater fish. One of these species is the asp, Leuciscus aspius. Research in the field of artificial reproduction of this species is very scarce and often incomplete. There are no breeding protocols specifying optimal environmental conditions and hormonal stimulation for many species of rheophilic cyprinids, including asp. Since the number of natural asp populations is constantly decreasing, it is important to support natural stocks by restocking with high quality stocking material. For this reason, optimized protocols are needed to breed this species under controlled conditions to produce stocking material with high biodiversity and good health. Such an approach will make it possible to maintain the population of natural asp at a constant level. The aim of this study was to develop the protocol of asp artificial reproduction using optimized thermal conditions and appropriate hormonal stimulation. In experiment I, the influence of constant temperature (10.0, 12.0 and 14.0 °C) on the effectiveness of artificial reproduction of asp. In experiment II, the effectiveness of asp reproduction was checked after the application of spawning agents: Ovopel, Ovaprim or a combination of these two agents The obtained results indicate that for the final maturation of oocytes (FOM) and artificial reproduction of asp in controlled conditions, water temperatures of 10-12 °C are the most useful. Under these thermal conditions, the highest percentages of female's ovulation and embryo survival, as well as the percentage of hatching, were obtained. Hormone injections are necessary to perform final oocyte maturation (FOM) in female asp in captivity. All spawning agents used were especially useful for artificial reproduction of asp, however, the best values of the studied indices, such as ovulation rate and embryo survival, were obtained after the application of Ovaprim or the combination of Ovopel and Ovaprim in water temperature at a range of 10-12 °C. It was found that the pH of ovarian fluid may be a preliminary indicator of the biological quality of eggs in asps. The optimal pH value is 8.0-8.4. At pH below 7.4, no viable embryos were observed.

Effect of anti-adhesive supplement doses on sperm motility characteristics in the European whitefish (Coregonus lavaretus).

Sperm adhering to glass slides is one of the main problems during fish sperm motility analyses with CASA systems. To mitigate this, albumin is the supplement added most frequently to activating solutions. However, there is no data on the use of supplements other than albumin (in various concentrations) in analyses of European whitefish (Coregonus lavaretus) sperm motility. This issue was investigated in the presented research using three anti-adhesive supplements (albumin, casein, Pluronic F-127) that were added to Billard solution (BS: 20 mM Tris, 1 mM CaCl2, 154 mM NaCl, 30 mM glycine at pH 9.0) at different concentrations (0.0; 0.1; 0.2; 0.5; 1.0; 2.0%). It was noted that the addition of the lowest concentration (0.1%) of albumin, casein, or the pluronic to BS had a significant effect on the motility and kinetic parameters of whitefish sperm compared to pure BS. BS supplemented with 0.2-0.5% albumin was the most appropriate variant used for whitefish sperm motility activation in the present experiment. BS supplemented with the pluronic at 1.0-2.0% concentrations resulted in significantly higher values of almost all CASA parameters compared to casein at the same concentrations. Moreover, CASA parameters determined in this variant of the pluronic (1.0-2.0%) were similar to those when BS was supplemented with the same albumin concentrations. This indicated that instead of albumin, the pluronic at higher concentrations in BS might be used to analyze whitefish sperm motility.

The Effect of the BCO2 Genotype on the Expression of Genes Related to Carotenoid, Retinol, and α-Tocopherol Metabolism in Rabbits Fed a Diet with Aztec Marigold Flower Extract.

This study investigated the effect of the BCO2 genotype and the addition of Aztec marigold flower extract to rabbit diets on the expression of BCO1, BCO2, LRAT, and TTPA genes in the liver. The levels of lutein, zeaxanthin, ß-carotene, retinol, and α-tocopherol in the liver and blood serum of rabbits, as well as plasma biochemical parameters and serum antioxidant enzyme activities were also determined. Sixty male Termond White growing rabbits were divided into three groups based on their genotype at codon 248 of the BCO2 gene (ins/ins, ins/del and del/del); each group was divided into two subgroups: one subgroup received a standard diet, and the other subgroup was fed a diet supplemented with 6 g/kg of marigold flower extract. The obtained results indicate that the BCO2 genotype may affect the expression levels of BCO1 and BCO2 genes in rabbits. Moreover, the addition of marigold extract to the diet of BCO2 del/del rabbits may increase the expression level of the BCO2 gene. Finally, an increase in the amount of lutein in the diet of rabbits with the BCO2 del/del genotype contributes to its increased accumulation in the liver and blood of animals without compromising their health status or liver function.

An efficient protocol for chromosome isolation from sterlet (A. ruthenus) embryos and larvae.

In the present manuscript an efficient and feasible protocol for chromosome preparation from sterlet (A. ruthenus) embryos and larvae was developed that can be used as routine molecular diagnostic tool of the species genome manipulation procedures verification. For this end, a multi-dimensional experimental system was conceived that enabled for the establishment of chromosome preparation protocol, which characterized the mean efficiency of chromosome extraction ranged from 70% to 100% and the average number of recorded metaphases per slide ranged between 9 and 15. The developed under the current study protocol can be used as a fast and reliable tool, alternative for flow cytometry techniques that enable for the molecular verification of genome manipulations effectiveness. As genome engineering is presently the fundamental biotechnology tool that enables for effective and sustainable aquaculture of fish, including sturgeons that are known as one of the most valuable economically and ecologically fish group, the development of easy and fast methods for the verification of genome manipulation effects is especially important. Thus, the established chromosome preparation protocol contributes to genome and reproduction studies of sturgeons, including taxonomy, inter-species hybridization, genome aberrations, sex determination system and selection. Moreover, the application of cytogenetic techniques also contributes to the development of new or improvement existing techniques of genome engineering utilized in sturgeon aquaculture.

How Cold Shock Affects Ploidy Level and Early Ontogenetic Development of the Sterlet, A. ruthenus L.

The objective of the present research was to study the effect of cold shock (3 °C and 6 °C) on fertilized eggs of the sterlet, Acipenser ruthenus L. Cold shock was applied for various durations (30, 60 and 90 min) and the ploidy levels, survival, and genotypes of the treated embryos/larvae were recorded. Analysis of ploidy levels confirmed the presence of diploid, triploid, and mosaic (1n/2n, 2n/3n, and 1n/2n/3n) genotypes in experimental groups, while it was strictly diploid in control groups. Microsatellite genotyping confirmed both the incidence of polyspermy and retention of the 2nd polar body in experimental groups. However, patterns of inheritance in all diploid offspring in experimental and control groups revealed classical Mendelian disomic inheritance. Interestingly, the observed mosaic sterlets had normal morphology and were alive. However, some larvae had abnormal morphology which may be due to haploid syndrome. In all treatment groups (treatments: 3 °C-30 min; 3 °C-60 min; 3 °C-90 min; 6 °C-60 min), where the percentage of polyploid/mosaic larvae were high, the mortality was also high. Whereas, in the control groups (where there were only diploid (2n) larvae), the mortality was relatively low.

Embryonic Development and Survival of Siberian Sturgeon × Russian Sturgeon (Acipenser baerii × Acipenser gueldenstaedtii) Hybrids Cultured in a RAS System.

The aim of this study was to describe the selected stages of embryonic development in Siberian sturgeon (Acipenser baerii) and hybrids of Siberian sturgeons and Russian sturgeons (Acipenser baerii × Acipenser gueldenstaedtii). For this purpose, embryos representing nine distinct developmental stages (stage 1-2.0 hpf, stage 2-5.5 hpf, stage 3-13.0 hpf, stage 4-20.0 hpf, stage 5-24.0 hpf, stage 6-26.0 hpf, stage 7-35.0 hpf, stage 8-55.0 hpf, and, stage 9-160.0 hpf; hpf-hours postfertilization) were sampled from each group (group A, group B, and group C) during incubation. Stages of embryogenesis were identified based on a 30-point scale of embryonic development in sturgeons. A total of 13 developmental stages were identified, including early cleavage, blastula formation, early and late gastrulation, onset of neurulation, beginning of organogenesis, and prelarvae. During gastrulation, the survival of hybrid embryos was highest in group B (93.8%) and lowest in group A (86.7%). Embryonic deformation was not observed during experimental incubation. The archived data relating to the embryonic development of Siberian sturgeon × Russian sturgeon hybrids could be applied to identify the individual stages of embryogenesis in hybrid sturgeons during egg incubation.

Domestication affected stress and immune response markers in Perca fluviatilis in the early larval stage.

It is already known that domestication modifies stress and immune responses in juveniles and adults of several fish species. However, there is a lack of information on whether these modulations result from adaptability along the life cycle or if they are pre-determined in very early developmental stages. To shed light on mechanisms that help to explain the process of domestication, a study was conducted to analyze comparatively Eurasian perch larval performance, stress, and immune status between wild and domesticated specimens. Eurasian perch larvae obtained from wild and domesticated (generation F5 reared in recirculating aquaculture systems) spawners were reared in the same conditions during the main rearing trial (MRT) and also subjected to a thermal challenge (TC). During the study, larval performance (including survival, growth performance, swim bladder inflation effectiveness, deformity rate), the expression of genes involved in immune and stress response, and the specific activity of oxidative stress enzymes (during MRT only) were analyzed. No significant differences in hatching rate, deformity rate, or swim bladder inflation effectiveness between wild and domesticated larvae were found, whereas specific growth rate, final total length, and wet body weight were significantly lower in wild larvae. Higher mortality was also observed in wild larvae during both MRT and TC. The data obtained in this study clearly indicated that during domestication, significant modifications in stress and immune response, such as complement component c3, were noted as early as just after hatching. Generally, domesticated fish were characterized by a lower stress response and improved immune response in comparison to the wild fish. This probably resulted from the domesticated larvae being better adapted to the conditions of artificial aquaculture. The data obtained provided information on how domestication affects fish in aquaculture, and they contribute to the development of efficient selective breeding programs of Eurasian perch and other freshwater teleosts.

Germline cysts and asymmetry in early previtellogenic ovarian follicles in cultured albino females of sterlet Acipenser ruthenus L. 1758 (Chondrostei, Acipenseriformes).

It is a first report on the structure of germline cells in ovaries of albino sterlet Acipenser ruthenus L. 1758. Ovarian nests, follicles, and germinal epithelium have been examined in gynogenetic and control specimens of this species. The structure of oogonia (named the cystoblasts) and of germline cysts in the nests has been described in detail. Also, the asymmetry in the cytoplasm and early growth of cystocytes in the cysts and of early previtellogenic oocytes has been described. In the cytoplasm of cystoblasts and in all cystocytes, a precursor of granular cytoplasm (Balbiani cytoplasm) is present and defines future vegetal region in the oocytes. Interestingly, the nuclei in cystoblasts comprise a large dense body that contains deoxyribonucleic acid (DNA). The role of this body in formation of multiple nucleoli has been explained. During the zygotene and pachytene stages, massive extrachromosomal amplification of DNA begins in the nucleoplasm of all cystocytes. As a result of the accumulation of extra DNA, an irregularly shaped DNA-body is formed. Multiple nucleoli arise in this DNA-body and around fragments of dense bodies. The asymmetry of the early previtellogenic oocyte cytoplasm is well marked by the presence of the granular cytoplasm. Moreover, the cisternae of the rough endoplasmic reticulum, dictyosomes, mitochondria, complexes of mitochondria with cement, nuage accumulations, and lipid droplets are located in specific zones in the granular cytoplasm. The follicular epithelium is composed of two subpopulations of somatic follicular cells (FCs): the main body cells and future micropylar cells.

Reproduction of endangered river lamprey (Lampetra fluviatilis) in controlled conditions.

The research reported focuses on reproduction of the river lamprey Lampetra fluviatilis,(Linnaeus, 1758) in controlled conditions. There was specific emphasis on fish harvesting dates (autumn and spring), holding conditions and reproduction in a controlled environment. Attempts were also made to synchronize the time of ovulation among river lampreys, egg and sperm collections. Hormonal stimulation was conducted using carp pituitary homogenate (CPH) at a total dose of 4 mg/kg which allowed for shortening of the egg-laying period from 2 to 3 weeks to a few days while sustaining embryo survival rates and larvae quality. River lamprey males were found to not require hormonal treatment to yield good-quality sperm, as measured using the CASA system. River lamprey broodstocks adapted well to different manipulations in hatchery conditions when harvested in the autumn and spring. The results of the present study may be used to restore endangered natural populations of the river lamprey (egg and sperm collection, fertilization or gamete preservation) because ovulation and spermiation synchronization is very difficult to achieve without hormonal treatment in controlled conditions.

Presence of gynogenetic males suggests a female heterogamety in sterlet Acipenser ruthenus L.

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.

Ovarian nests in cultured females of the Siberian sturgeon Acipenser baerii (Chondrostei, Acipenseriformes).

Ovaries of Acipenser baerii are of an alimentary type and probably are meroistic. They contain ovarian nests, individual follicles, inner germinal ovarian epithelium, and fat tissue. Nests comprise cystoblasts, germline cysts, numerous early previtellogenic oocytes, and somatic cells. Cysts are composed of cystocytes, which are connected by intercellular bridges and are in the pachytene stage of the first meiotic prophase. They contain bivalents, finely granular, medium electron dense material, and nucleoli in the nucleoplasm. Many cystocytes degenerate. Oocytes differ in size and structure. Most oocytes are in the pachytene and early diplotene stages and are referred to as the PACH oocytes. Oocytes in more advanced diplotene stage are referred to as the DIP oocytes. Nuclei in the PACH oocytes contain bivalents and irregularly shaped accumulation of DNA (DNA-body), most probably corresponding to the rDNA-body. The DNA-body is composed of loose, fine granular material, and comprises multiple nucleoli. At peripheries, it is fragmented into blocks that remain in contact with the inner nuclear membrane. In the ooplasm, there is the rough endoplasmic reticulum, Golgi complexes, free ribosomes, complexes of mitochondria with cement, fine fibrillar material containing granules, and lipid droplets. The organelles and material of nuclear origin form a distinct accumulation (a granular ooplasm) in the vicinity of the nucleus. Some of the PACH oocytes are surrounded by flat somatic cells. There are lampbrush chromosomes and multiple nucleoli present (early diplotene stage) in the nucleoplasm. These PACH oocytes and neighboring somatic cells have initiated the formation of ovarian follicles. The remaining PACH oocytes transform to the DIP oocytes. The DIP oocytes contain lampbrush chromosomes and a DNA-body is absent in nuclei. Multiple nucleoli are numerous in the nucleoplasm and granular ooplasm is present at the vegetal region of the oocyte.

Previtellogenic and vitellogenic oocytes in ovarian follicles of cultured siberian sturgeon Acipenser baerii (Chondrostei, Acipenseriformes).

Previtellogenic and vitellogenic oocytes in ovarian follicles from cultured Siberian sturgeon Acipenser baerii were examined. In previtellogenic oocytes, granular and homogeneous zones in the cytoplasm (the ooplasm) are distinguished. Material of nuclear origin, rough endoplasmic reticulum, Golgi complexes, complexes of mitochondria with cement and round bodies are numerous in the granular ooplasm. In vitellogenic oocytes, the ooplasm comprises three zones: perinuclear area, endoplasm and periplasm. The endoplasm contains yolk platelets, lipid droplets, and aggregations of mitochondria and granules immersed in amorphous material. In the nucleoplasm, lampbrush chromosomes, nucleoli, and two types of nuclear bodies are present. The first type of nuclear bodies is initially composed of fibrillar threads only. Their ultrastructure subsequently changes and they contain threads and medium electron dense material. The second type of nuclear bodies is only composed of electron dense particles. All nuclear bodies impregnate with silver, stain with propidium iodide, and are DAPI-negative. Their possible role is discussed. All oocytes are surrounded by follicular cells and a basal lamina which is covered by thecal cells. Egg envelopes are not present in previtellogenic oocytes. In vitellogenic oocytes, the plasma membrane (the oolemma) is covered by three envelopes: vitelline envelope, chorion, and extrachorion. Vitelline envelope comprises four sublayers: filamentous layer, trabecular layer 2 (t2), homogeneous layer, and trabecular layer 1 (t1). In the chorion, porous layer 1 and porous layer 2 are distinguished in most voluminous examined oocytes. Three micropylar cells that are necessary for the formation of micropyles are present between follicular cells at the animal hemisphere. J. Morphol. 278:50-61, 2017. ©© 2016 Wiley Periodicals,Inc.

Microsatellite genotyping of cryopreserved spermatozoa for the improvement of whitefish semen cryobanking.

The cryobanking of semen is recognized as an emerging tool for the conservation of fish biodiversity. Microsatellite analysis of the DNA of cryopreserved sperm would facilitate the assessment of genetic variability of cryobanked semen specimens. The aim of this study was to compare microsatellite profiles of DNA extracted from adipose fins and cryopreserved semen collected from eleven male whitefish (Coregonus lavaretus L.). The following microsatellite loci were employed: Cocl-Lav-8, Cocl-Lav-18, Cocl-Lav-28, Cocl-Lav-80, Str-73 and Sfo-292. The chelex 100 method was used for the successful isolation of DNA from somatic tissue, and the DNeasy method with additional modifications was used for the successful isolation of DNA from sperm. Genotyping was possible with the use of a very low number of spermatozoa (5 × 106 which is less than 0.1% of spermatozoa in standard 250 µL straw). The results of the DNA analysis from both the adipose tissue and spermatozoa were identical. Therefore, microsatellite analysis of cryopreserved spermatozoa can be recommended for future whitefish sperm banking.

Induction of diploid gynogenesis in Wels catfish (Silurus glanis) using UV-irradiated grass carp (Ctenopharyngodon idella) sperm.

This article describes the first successful induction of meiotic gynogenesis in Wels catfish (Silurus glanis) using heterologous sperm of grass carp (Ctenopharyngodon idella) with DNA inactivated by means of UV-irradiation at 4800 J/m(2). The cold shock at 6 degrees C (duration of 20 min) started in the nineth minute after fertilization was found as efficient in retaining the second polar body and producing meiotic diploids. Successful induction of meiotic gynogenesis was confirmed by the microsatellite DNA analysis, which revealed only maternal inheritance in gynogenetic offspring. Ploidy of gynogenetic offspring was verified by the application of active nucleoli counting analysis.