Successful Hybridization of Acipenser Species Using Cryopreserved Sperm

Successful hybridization of sterlet (Acipenser ruthenus) × Siberian sturgeon (Acipenser baeri), sterlet × Russian sturgeon (Acipenser gueldenstaedti) and sterlet × European sturgeon (Acipenser sturio) was carried out for the first time using cryopreserved semen of sturgeon males and sterlet × sterlet crosses as controls. Sperm of all three species was diluted with a cryodiluent composed of 23.4 mM sucrose, 0.25 mM KCl, 30 mM Tris (pH 8.0) and 10% methanol. The samples were frozen in plastic straws in the vapor of liquid nitrogen at the height of 3 cm above the level of nitrogen for 3 min. Following thawing approximately 3000 sterlet eggs were fertilized with six straws of frozen-thawed sperm. The hatching rate with sterlet sperm was 30.6% while the hatching rate of A. ruthenus × A. baeri, A. ruthenus × A. gueldenstaedti and A. ruthenus × A. sturio hybrids was 50, 17.4 and 34%, respectively. Morphometric markers as well as random amplified polymorphic DNA (RAPD) assay was used to verify interspecific hybridization.     

The antioxidant system of seminal fluid during in vitro storage of sterlet <Emphasis Type="Italic">Acipenser ruthenus</Emphasis> sperm

The role of the seminal fluid antioxidant system in protection against damage to spermatozoa during in vitro sperm storage is unclear. This study investigated the effect of in vitro storage of sterlet Acipenser ruthenus spermatozoa together with seminal fluid for 36 h at 4 °C on spermatozoon motility rate and curvilinear velocity, thiobarbituric acid reactive substance level, and components of enzyme and non-enzyme antioxidant system (superoxide dismutase and catalase activity and uric acid concentration) in seminal fluid. Spermatozoon motility parameters after sperm storage were significantly decreased, while the level of thiobarbituric acid reactive substances, activity of superoxide dismutase and catalase, and uric acid concentration did not change. Our findings suggest that the antioxidant system of sterlet seminal fluid is effective in preventing oxidative stress during short-term sperm storage and prompt future investigations of changes in spermatozoon homeostasis and in spermatozoon plasma membrane structure which are other possible reasons of spermatozoon motility deterioration upon sperm storage.     

史氏鲟及小体鲟不同组织中溶菌酶水平的比较

对史氏鲟及小体鲟血清和几种主要组织的溶菌酶含量进行了测定和比较后发现,同小体鲟相比,史氏鲟的血清、鳃、肝、胃、前肠及后肠的溶菌酶量均较高,血清之间有极显著的差异,鳃、肝及后肠均有显著性的差别,胃及前肠之间无差异;小体鲟的粘液、幽门垂及脾中的溶菌酶含量较史氏鲟高,但无显著性差异,由此可见,虽然同为鲟科鲟属,溶菌酶在两种鱼之间的差别还是很显著的。     

Growth,survival rate and fatty acid composition of sterlet (Acipenser ruthenus) larvae fed fatty acid‐enriched Artemia nauplii

We evaluated the growth and survival rate of sterlet (Acipenser ruthenus) larvae fed Artemia nauplii enriched with Olioω3 or Red Pepper commercial emulsions (BernAqua NV, Belgium). Sterlet larvae, 0.022 ± 0.002 g body weight, were randomly assigned to one of three feeding regimes with two different feeding durations. After administering live feed for 7 or 14 days, larvae were weaned onto commercial food and reared to 36 days posthatching (28 days of feeding). There were no significant differences in body weight among groups at the end of the trial. A significantly higher survival rate (p < 0.05) was observed in larvae fed Artemia enriched with Red Pepper for 14 days compared to other feeding regimes. Based on the analysis of growth parameters, we can conclude that 7 days of live feeding to be sufficient for efficient rearing of sterlet larvae. And longer duration of live feeding with use of special enrichment can be recommended for a higher survival rate.     

Bayesian modelling suggests that the sterlet (Acipenser ruthenus,Linnaeus 1758) population is ageing in the middle Danube River

1. In the past, sturgeons played an important role in commercial and recreational fisheries in the Danube River and its tributaries. Human impacts in the Danube River Basin coupled with exploitation of sturgeon stocks led to all species being either locally extinct, critically endangered or of unknown status.
2. Sterlet (Acipenser ruthenus, Linnaeus 1758) is the last known sturgeon species occurring in the upper and middle Danube; however, the population of this species is considered unbalanced and decreasing since the beginning of the twenty-first century.
3. The decline of sturgeon stocks has been noted before owing to their economic importance. With commercial fisheries being forbidden in the Slovak section of the Danube River, there is generally no information available about the status of what is considered a local population.
4. Databases containing recreational catch of sterlet (2003–2018) and historical records of commercial harvest (1961–1990) were used to describe the trend in the weight and number of sterlet caught over the following years.
5. Modelling indicated that the number of fish caught each year appears to be lower, while the average weight of each individual is increasing. This might suggest that the population is ageing.
6. Although older individuals can contribute a great deal more to spawning because they produce a greater number of eggs, several problems are apparent. The number of spawners might be decreasing as a result of bycatch or fishing, their ability to spawn might be obstructed, or annual recruitment may fail owing to unpredictable events.
7. Although restocking programmes are in place to help maintain the sterlet population in the Danube River, their efficiency seems to be drastically low. In fact, restocking could be of little value unless studies on the availability of key habitats are conducted and their protection and restoration are ensured.

     

Biochemical characterization of Siberian sturgeon Acipenser baeri and sterlet Acipenser ruthenus milt plasma and spermatozoa

This paper provides data concerning biochemical composition of milt of two sturgeon species, Siberian sturgeon bred in aquaculture facility of Inland Fisheries Institute in North Poland and sterlet (from two different populations from Danube and Odra). Milt plasma of Siberian sturgeon and sterlet, when compared to teleost fish, is characterized by much lower osmolality (up 70 to 96 mOsm kg⁻¹) and protein concentration (0.24–0.58 g l⁻¹). In spite of the presence of an acrosome and acrosin the antiproteinase activity of seminal plasma was low (12.79–25.40 U l⁻¹). Activities of arylsulfatase and β-N-acetylglucosaminidase were found in spermatozoa. This agrees with the presence of an acrosome in sturgeons sperm. Similarly to mammals, these enzymes are also present in milt plasma. We determined a range of enzymatic activities from the minimal (seminal plasma) to the maximal damage (supernatants obtained after freezing-thawing without cryoprotectant). Activities of arylsulfatase, β-N-acetylglucosaminidase, lactic dehydrogenase and acid phosphatase were released from spermatozoa after freezing-thawing. For this reason they are good potential candidates as a markers of cryoinjury to sperm acrosome and midpiece. This revised version was published online in July 2006 with corrections to the Cover Date.     

Test of Mendelian segregation among 10 microsatellite loci in the fourth generation of bester (Huso huso L. ×Acipenser ruthenus L.)

Ten microsatellite loci were tested on Mendelian segregation in the bester – hybrid of beluga, Huso huso L. and sterlet, Acipenser ruthenus L. in the fourth generation. All studied loci showed disomic inheritance and Mendelian segregation of their alleles. The molecular approach by microsatellite DNA analysis represents a reliable tool for interspecific characterization of sturgeons and their hybrids (different alleles of several loci are present in sturgeon fishes, and interspecific hybrids posesses both maternal and paternal specific alleles). This is the first report of the Mendelian segregation testing in interspecific hybrid of acipenserid fishes using microsatellite DNA markers.     

Effect of different synthetic gonadotrop-releasing hormone analogues and their combinations with an anti-dopaminergic compound on the reproduction performance of sterlet (Acipenser ruthenus L.)

In this study, three synthetic gonadotrop-releasing hormones (GnRH) (azagly-nafarelin; des-Gly¹⁰-( d -Ala⁶)-LH-RH; and des-Gly¹⁰-( d -Phe⁶)-LH-RH) either alone or in combination with metoclopramide were used to induce reproduction of sterlet. The GnRH analogues were applied in a single dose of 40 μg kg⁻¹ of female and 20 μg kg⁻¹ of male body weight. Metoclopramid was administered in a simultaneous injection of 10 and 5 mg kg⁻¹ of body weight for females and males respectively. There were no significant differences in the ovulatory responses of females; ovulation rates varied between 57% and 80%, and at the temperature of 15.5–16.0 °C about 30–34 h were required for final maturation, when eggs of 17.3±1.3% of body weight were stripped. However, the fertilization rates of the des-Gly¹⁰-( d -Phe⁶)-LH-RH-treated groups were significantly lower than that in the other treatment. In males, the combination of the above peptidergic hormones with metoclopramide gave significantly better results than their single application. The results demonstrate that the final stage of gamete maturation in sterlet may be achieved by several hormonal means. The possibility of using new GnRH analogues without dopamine antagonists yields new perspectives for induced breeding of sturgeons, which have particular importance in the light of meat and roe (caviar) production for human consumption.     

Meiotic gynogenesis affects the individual and gonadal development in sterlet Acipenser ruthenus

Larval growth and gonadal differentiation of gynogenetic sterlet Acipenser ruthenus were studied. The gynogenetic fish were produced by activation of albino sterlet eggs with UV‐irradiated sperm (collected from healthy coloration specimens), following by a heat shock of 34°C. Embryos were reared at 18.0 ± 0.5°C in a recirculation system and initially fed with Artemia sp. for 4 weeks and then fed with formulated feed. During the experiment, the length and weight of fish were recorded for the gynogenetic and control groups. Additionally, the analysis of gonadal sex differentiation was conducted with a 2‐week interval at 289, 303, 317, 331, 345 and 373 DPH. The mean body weight of the control group was significantly higher (p < .05) than the gynogenetic group only at 345 and 373 DPH, while the mean total body length was significantly higher at 331 and 345 DPH (p < .05). The gonad investigation was started at 289 DPH, and ~80% of females were found in the gynogenetic group during the trial period while the number of females and males was similar at 303, 331 and 373 DPH in the control group. The first symptoms of morphological differentiation appeared at 289 DPH, and the final shaping macroscopic structure of the gonad took place earlier in females than in males.     

Protective role of antifreeze proteins on sterlet (<Emphasis Type="Italic">Acipenser ruthenus</Emphasis>) sperm during cryopreservation

The loss of sperm quality in sterlet (Acipenser ruthenus) due to freeze-thaw process in cryopreservation was investigated in the present study. Two antifreeze proteins (AFPI or AFPIII) were used at different concentrations of 0.1, 1, 10, and 100 μg/mL. We compared motility, curvilinear velocity, and plasma membrane integrity of fresh, cryopreserved sperm, and sperm cryopreserved in the presence of antifreeze proteins. Fresh sperm (control) had 85?±?4% motility and 160?±?2 μm/s curvilinear velocity, respectively. After cryopreservation, the motility of frozen-thawed sperm without addition of antifreeze proteins significantly decreased (44?±?9%), compared to the control. The highest motility of frozen-thawed sperm was obtained in cryopreserved sperm with addition of 1 μg/mL of AFPIII (58?±?14%). No significant differences were observed in curvilinear velocity between fresh sperm and cryopreserved sperm with/without addition of AFPI or AFPIII. The flow cytometry analysis revealed that fresh sperm contained 94.5?±?6% live cells, while the cryopreserved sperm only contained 26.6?±?14% live cells. Supplementation of antifreeze proteins has significantly improved the percentage of live cells in frozen-thawed sperm, except 0.1 μg/ml of AFPI group. No significant difference in percentage of live cells was detected in the sperm cryopreserved with 10 μg/mL of AFPI or AFPIII, compared to fresh sperm. Thus, addition of antifreeze proteins to cryopreservation medium could be considered to improve the post-thawed sperm quality of sterlet.     

Effects of background colour on growth indices and stress of young sterlet (Acipenser ruthenus) in a closed circulated system

In this study, the physiological response and growth performances of Acipenser ruthenus were investigated after a long‐term background colour adaptation (12 weeks). Twelve groups of 10 individuals with initial mean body weight of 183 g were reared in black, dark blue, grey and white tanks (three replicates for each colour). At the end of the experiments, growth (initial body weight, final body weight, weight gain per cent, food efficiency ratio, protein efficiency ratio, specific growth rate), blood (cortisol, glucose, pO_2, pCO₂, pH, haematocrit, osmolality, triglycerides, cholesterol, total lipids) and liver (hepatosomatic index, total lipids, glycogen) parameters were analyzed. Plasma cortisol in the dark‐adapted sterlet (21.95 ± 3.9 ng mL^?1) was significantly lower than those in white‐adapted fish (39.44 ± 6.5 ng mL^?1), whereas there were no significant differences in cortisol levels between the grey‐adapted fish (23.05 ± 4.1 ng mL^?1) and dark blue‐adapted fish (24.2 ± 3.6 ng mL^?1). A remarkable increase in mean of body weight (%) was detected in dark‐adapted sterlet (45.2 ± 3.2) being 27.67%, 12.1% and 11.8% higher than the white, grey and dark blue‐adapted fish respectively. The obtained results verified that different background colours lead to different growth performances and physiological responses of starlet, depending on rearing conditions.     

Using machine learning algorithms to analyse the scute structure and sex identification of sterlet Acipenser ruthenus (Acipenseridae)

Sturgeons are valued as specialty black caviar, which is very expensive. Only females are used in the technology of caviar aquaculture. Universal method of sex determination has not yet been developed. Most of known methods are not sufficiently accurate, or used at a relatively late age, or difficult to use. Perfect early determination of sex is considered to be impossible. Because of the dark colour of most sturgeons and important morphological differences, which fish of almost all ages have, were overlooked. We first found that the scute structure of sterlet sturgeon depends on the sex. The found dependencies with the help of machine learning algorithms open a possibility for creation of sex determination equipment using the artificial intelligence. Our results open a perspective for creation of sex determination methods for other 23 sturgeon species, which can increase the efficiency of caviar aquaculture and restoration of sturgeons in natural waters.     

Effect of thiamine injection on growth performance,hematology and germinal vesicle migration in sterlet sturgeon Acipenser ruthenus L.

The present study aimed to investigate the effect of thiamine on growth, hematological, egg thiamine content and oocyte nucleus migration indices in sterlet sturgeon (Acipenser ruthenus). A total of 45 female fish (698.6 ± 8.9 g) were distributed in nine fiberglass tanks with three treatments (each in three replicates). Experimental fish were fed once a day with practical diet supplemented with 1 g kg^?1 amprolium hydrochloride (as the antithiamine) for 5 months before spawning. Thiamine hydrochloride was injected to fish with one of three doses at 0, 5 and 50 mg kg^?1 body weight (BW) at three different stages. At the end of each month, fish were weighed and growth parameters such as weight gain, specific growth rate, feed efficiency and final weight were determined. At the end of the 5-month period, hematological parameters and egg thiamine content were measured. The results showed no significant differences in terms of growth performance. Hemoglobin, number of red blood cells and white blood cells were not significantly different among the treatments, but hematocrit was significantly higher in fish injected with 5 mg kg^?1 BW thiamine. The mean corpuscular volume and mean corpuscular hemoglobin were not significantly different, but lower value of mean corpuscular hemoglobin concentration was observed in fish injected with 5 mg thiamine. Percentage of lymphocytes, neutrophils, eosinophils and monocytes were not significantly different among treatments. The results of the germinal vesicle migration index at different stages showed no significant differences in different groups. Results showed that free thiamine, thiamine pyrophosphate and total thiamine in eggs significantly increased at 50 mg kg^?1 injection dose compared with the control, but no significant different was found in thiamine monophosphate. The results revealed that thiamine injection to sterlet broodstocks has positive accumulation in egg thiamine content and some hematological parameters but have no effect on growth and ovarian maturation. We conclude that thiamine can affect some physiological features of broodstock, which needs to be specified in further studies.     

The effect of fish and rapeseed oils on growth performance,egg fatty acid composition and offspring quality of sterlet sturgeon (Acipenser ruthenus)

The aim of this study was to evaluate the long‐term effects (7‐month experiment) of diets consisting of fish oil (Kilka fish) and vegetable oil (rapeseed) on the reproductive performance of sterlet sturgeon (Acipenser ruthenus) broodstock. Forty‐five broodstock (990.3 ± 20.05 g) were randomly allocated to three different diet treatments. Three experimental diets were formulated with graded levels of fish oil (100% FO), vegetable oil (100% VO), and a combination of fish and vegetable oil (50% FO + 50% VO). At the end of the 7‐month feeding trial period, the weight gain and final weight were changed significantly different between the treatments (p < 0.05). Broodstock fed the FO + VO diet had higher growth than those fed the only FO or VO diets (p < 0.05). The highest germinal vesicle migration percentage was observed in FO + VO treatment (p < 0.05). The DHA/EPA, DHA/ARA and EPA/ARA ratios in oocyte exhibited a significant difference in the different treatments (p < 0.05). This study indicates that nutrition of broodstock with diet including FO + VO (p < 0.05) can positively affect the growth performance of larvae compared with only FO or VO diets. Furthermore, the high levels of 18:1n‐9, AL and ALA contents in oocytes from broodstock fed VO and the lowest ALA content in oocytes from broodstock fed FO underlined the important role of broodstock diets in the reproductive process and embryonic and/or larval developments of sterlet.     

Studies on the semen biology and sperm cryopreservation in the sterlet, Acipenser ruthenus L.

The present study investigated motility, acrosome reaction, fertility and cryobiological parameters of the semen of the sterlet, Acipenser ruthenus L. Sperm motility persisted for about 4 min in water, and the main swimming type was the linear motion. Motility was prolonged at osmolalities of 12.5 mosmol kg^?1 and in the presence of magnesium ions, while calcium had no effect. Also a pH in the range of 7.0–9.0 had no effect on ` motility. At osmolalities of 25–50 mosmol kg^?1 the sperm motility was partly inhibited, at osmolalities of 100 mosmol kg^?1, completely and irreversibly. In 50 mosmol kg^?1 solutions with 2.5–5 mM L^?1 KCl the motility inhibition was total, but reversible. The acrosome reaction was not induced by one of the described solutions, but the percentage of spermatozoa with reacted acrosomes was low (<20%) and highly variable in all experiments. The optimal extender base for cryopreservation was a solution consisting of 50 mM L^?1 NaCl, 5 mM L^?1 KCl, 10 mM L^?1 Tris (pH 8.5). From the tested cryoprotectants only dimethylsulphoxide (DMSO) and methanol provided sufficient cryoprotection. After freezing and thawing, the motility rates and swimming velocities were higher with DMSO than with methanol. However, the fertility was very significantly reduced with DMSO (10.3±0.5%) while with methanol fertilization rates in a similar range (32.7±4.4%) as with fresh semen (33.90±0.8%) could be obtained. Optimal freezing conditions for sterlet semen were in the vapour of liquid nitrogen 3–5 cm (?95°C to ?85°C) above its surface, the optimal thawing conditions at 25°C for 30 s. The acrosome reaction was not induced by these cryopreservation protocols.     

小体鲟胚胎发育特征观察

观察了水温(18±1)℃条件下,小体鲟(Acipenser ruthenus Linnaeus)的胚胎发育特征。结果显示:成熟小体鲟卵圆球形、黑色、不透明,为粘性卵,卵径2.2～2.8 mm。水温(18±1)℃条件下,小体鲟受精卵历时110 h孵化出膜,所需总积温为2023～2308℃·h。小体鲟受精卵的卵裂方式为特殊的辐射状卵裂;原肠中期时动物极细胞覆盖胚胎三分之二;心跳期,心脏呈C型;尾端接近心脏时,眼囊内已经有色素沉着。根据胚胎发育过程的形态特征,可将小体鲟胚胎发育过程分为受精卵、卵裂、囊胚、原肠、神经胚、器官发生和出膜7个连续阶段,34个时期。另外,本研究还将小体鲟胚胎发育和鲟形目其他鱼类胚胎发育特征进行了对比分析。     

Successful sterlet, Acipenser ruthenus L., propagation with synthetic LH-RH hormone

Abstract. The synthetic LH-RH (luliberine) has proved to be successful in inducing the ovulation of sterlet, Acipenser ruthenus L. Three dosages of LH-RH 50, along with carp pituitary, and 100 and 300 μg/kg of body weight were administered, and 100 and 300 μg of LH-RH/kg body weight were found to be successful in ovulation. Spawners were kept at low temperature (9–12°C). Forty-two hours after treatment the eggs entered ovulation in the ovary and became suitable for stripping. The ripening ratio for spawners varied between 75 and 83%. The eggs that could be obtained by opening the abdominal wall were fertilized with milt diluted with pond water to avoid polyspermism. The fertility of the stripped eggs was 89–90%. Despite a reduced number of subjects (12 treated, 2 control females), as a result of the favourable fertilization percentage, approximately 55 000 fingerlings were reared in tanks to approximately 5–7cm in size.     

Growth and maturation in sterlet <Emphasis Type="Italic">Acipenser ruthenus</Emphasis> under high concentrations of dissolved oxygen

This study investigates the effect of high dissolved oxygen concentrations (DO) on sterlet Acipenser ruthenus maturation. Two groups, each comprising 246 individuals of 8-month-old juvenile sterlets, were reared for 37 months under high DO (group H, mean DO of 11.2 mg/L) or normal DO (group C, mean DO of 7.3 mg/L). Significant differences in body weight were observed between group H (638.7?±?191.9 g) and group C (572.2?±?151.9 g) in 12 months (P?<?0.05). Growth until 12 months occurred at a specific growth rate of 0.6%/day for group H and at 0.5%/day for group C. The food conversion rate of groups H and C was 5.2 and 5.8, respectively. The gonadosomatic index of group H was 3.1?±?1.1 for females and 3.1?±?9.7 for males, and showed no significant difference with group C (3.0?±?0.7 for females and 2.8?±?8.9 for males, P?>?0.05) in 24 months. Significant differences in follicle diameter in mature females were also observed. Group H included 68 mature females, with an average follicle diameter of 2318.0?±?175.3 µm, whereas group C included 56 mature females, with a smaller average follicle diameter of 2152.3?±?287.0 µm (P?<?0.001). However, the polarization index in group H (0.194?±?0.049) was higher than that in group C (0.178?±?0.042) (P?<?0.05). These results suggest that, for sterlet, rearing conditions with high DO can affect growth of both the body and follicle diameter of fish compared to normal rearing conditions.     

Impact of feed ration on growth and the results of sterlet,Acipenser ruthenus L., artificial reproduction

In the study the effects of artificial reproduction of sterlet (Acipenser ruthenus) fed different feed ration were compared. Three doses of feed were used: 1.0%, 0.75% and 0.5% of the fish biomass per day. The results indicate that the feed rations for fish used for caviar production or breeding material should differ. The optimal ration for the quality of sex products and progeny was 0.75% of the fish biomass, whereas in view of the number of maturing fish, the better ration was that of 1% of the fish biomass. In group fed 0.75% of the fish biomass per day the highest GSI value, the largest eggs and hatch and the highest survival to the hatch were noted. The feed ration of 0.5% of the fish biomass was too low, as indicated by the low parameters of eggs obtained, their survival and the smallest number of mature and ovulating females. These results can provide direction for the creation and exploitation of spawner stocks as well as for other sturgeon species cultured under controlled conditions.     

Protease in sturgeon sperm and the effects of protease inhibitors on sperm motility and velocity

In mammals, proteases are present in sperm acrosome and play key role in fertilization. Sturgeon sperm has an acrosome, but its physiology, biochemistry, and potential role in fertilization are unknown. In the present study, we have observed high protease activity in acidic extract of intact sperm compared to that of seminal plasma in sterlet (Acipenser ruthenus). The protease activity was decreased and increased in acidic extract of motility-activated sperm and in the activation medium, respectively. Molecular analysis revealed total protease and serine (acrosin) protease activities in sperm acidic extract which was accumulated in a protein band with relative molecular mass of 35 kDa. Immunoelectron microscopy using an affinity-purified polyclonal antibody for boar acrosin localized the protease at the acrosome region. Moreover, initiation of sperm motility was inhibited after activation in the presence of inhibitors for both trypsin-like and chymotrypsin-like proteases, while the effects of protease inhibitors on sperm velocity were uncertain. Our results indicate similarities in physiology and biochemistry of acrosome between sturgeon and mammals and suggest potential role of protease in the initiation of sperm motility in sturgeon.