Motility analysis and energetics of the Siberian sturgeon Acipenser baerii spermatozoa

A study of motility was carried out on spermatozoa of the Siberian sturgeon ( Acipenser baerii Brandt) reared at the Cemagref experimental research station, St Seurin sur l'Isle, France. About 90% spermatozoa are activated within a few seconds after a double step dilution (final dilution 1/600) and the number of motile cells declines regularly to 10–20% after 2 mn. During the same period of time, the flagellum beat frequency remains stable at about 50 Hz for 30 sec and drops to 30 Hz after 60 sec. A morphological analysis of the movement of the flagellum shows that during the first 20 sec, waves are distributed on the entire length of the flagellum; later, they disappear progressively from the distal part within 1 mn in the case of 90% of the spermatozoa. Finally after 2 mn, most of the flagella are straight and devoid of wave. The ATP content declines very rapidly after activation from 7.58 ± 1.85 to 4.05 ± 1.51 nmol/108 spermatozoa within 5 sec (P=0.004). Spermatozoa left in the genital tract of the males over a period of 24 hrs (in vivo survival) do not show significant change in the percentage of motile cells (82 ± 14 vs 74 ± 15) nor in the ATP content (6.13 ± 2.37 vs 4.59 ± 1.42 nmoles/108 spermatozoa). An aliquot of these spermatozoa stripped from testis and stored during the same period of 24 hrs at 4°C shows a significant decrease in the percentage of motile cells from 82 ± 14 to 59 ± 30 (P<0.01) and in the ATP content from 6.15 ± 2.4 to 1.95 ± 1.37 nmoles/108 spermatozoa (P=0.001).     

西伯利亚鲟Tbx3基因的克隆和表达分析

Tbx3基因是一类重要的转录因子,在形态发生和器官形成中发挥着重要作用。该文克隆了西伯利亚鲟Tbx3基因(AbTbx3)cDNA的全长序列,该cDNA全长2908bp,包含一个2166bp的开放阅读框,编码721个氨基酸的多肽。分析表明:AbTbx3和人Tbx3的T-box结构域蛋白序列同源性达到95.2%,三维结构也具有高度的相似性。系统进化分析表明:AbTbx3与其他物种的Tbx3聚为一支,并在一个大的分支上与Tbx2聚类。半定量RT-PCR显示,AbTbx3基因从西伯利亚鲟囊胚早期即开始表达,且随着发育表达渐强,至尾芽早期表达量达到最大,随后稍有下降;在成体的眼、脑、鳃、肠、胸鳍和腹鳍中有表达,在肝、血液、心脏、肾和肌肉中均未检测到其表达。整体原位杂交表明,在37期和43期仔鱼的耳泡、后脑、松果体和后部脊索中表达量较高,同时在背鳍芽中也有表达。综上结果表明:西伯利亚鲟Tbx3与人Tbx3在结构上高度同源,在胚胎、仔鱼和成体中呈时空特异性表达。     

西伯利亚鲟仔鱼侧线系统的发育

鲟鱼属软骨硬鳞鱼,在电感受器的进化中占据着极为重要的地位。该文以光镜和扫描电镜手段研究了西伯利亚鲟侧线系统早期发育,包括侧线基板发育及感觉嵴的形成、侧线感受器的发育和侧线管道的形成。1日龄,听囊前后外胚层增厚区域出现6对侧线基板;除后侧线基板细胞向躯干侧面迁移外,其他侧线基板均形成感觉嵴结构;每一侧线基板中均有神经丘原基形成。7日龄,壶腹器官在吻部腹面两侧出现,壶腹器官的发育比神经丘晚一周左右。9日龄,神经丘下的表皮略有凹陷,侧线管道开始形成。29日龄,在吻部腹面两侧可见少数个别的壶腹器官表皮细胞覆盖壶腹器官中央区域留下3～4个小的开口;壶腹管内可见大量的微绒毛存在,在其他鲟形目鱼类、软骨鱼类中也存在类似的结构。57日龄,躯干侧线管道已完全埋于侧骨板中;壶腹器官主要分布在吻部腹面,3～4个聚集在一起,呈"梅花状",分布紧密,并且该部分皮肤表面凹陷,形成花朵状凹穴;侧线系统发育完善。     

Energy pathways associated with sustained spermatozoon motility in the endangered Siberian sturgeon Acipenser baerii

Sturgeon spermatozoa are unique for their sustained motility. We investigated the relative importance of bioenergetic pathways in the energy supply of Siberian sturgeon Acipenser baerii spermatozoa during motile and immotile states. Spermatozoon motility and oxygen consumption rate (OCR) were analysed following exposure to inhibitors of oxidative phosphorylation (sodium azide, NaN₃), glycolysis (2-deoxy-D-glucose, DOG) and β-oxidation of fatty acids (sodium fluoride, NaF), and to an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenyl hydrazine, CCCP). No significant difference in curvilinear velocity was observed after addition of these reagents to activation medium (AM) or nonactivation medium (NAM) for incubation. Incubation of spermatozoa in NAM containing CCCP or NaN₃ resulted in significantly decreased motility duration compared to controls. The OCR of sturgeon spermatozoa in AM (11.9 ± 1.4 nmol O₂ min⁻¹ (10⁹ spz)⁻¹) was significantly higher than in NAM (8.2 ± 1.5 nmol O₂ min⁻¹ (10⁹ spz)⁻¹). The OCR significantly declined with addition of NaN₃ to AM and NAM. No significant difference in motility parameters or OCR was observed with NaF or DOG. These results suggest active oxidative phosphorylation in both immotile and motile spermatozoa. Nevertheless, mitochondrial respiration occurring during motility is not sufficient to meet the high energy demands, and the energy required for sustained motility of Siberian sturgeon spermatozoa is derived from adenosine triphosphate accumulated during the quiescent state.     

Intraperitoneal injection urocortin-3 reduces the food intake of Siberian sturgeon (Acipenser baerii)

Urocortin-3 (UCN3), one of the corticotropin releasing factor (CRF) family peptides, which was discovered in 2001, has a variety of biological functions. However, the researches of UCN3 in fish were scarce. In order to understand whether UCN3 play a role in regulating food intake in fish, we first cloned the ucn3 cDNAs sequence of Siberian sturgeon (Acipenser baerii Brandt), and investigated the ucn3 mRNA levels in 11 tissues. The Siberian sturgeon ucn3 cDNA sequence was 1044 bp, including an open reading frame (ORF) of 447 bp that encoded 148 amino acids with a mature peptide of 40 amino acids, a 5ʹ-terminal untranslated region (5ʹ-UTR) of 162 bp and a 3ʹ-terminal untranslated region (3ʹ-UTR) of 435 bp. The result of tissue distribution showed that ucn3 widely distributed in 11 tissues with highest expression in brain. We also assessed the effects of periprandial (pre- and post-feeding), fasting and re-feeding on ucn3 mRNAs abundance in brain. The results showed the expression of ucn3 mRNA in brain was significantly elevated after feeding, decreased after fasting 17 days and increased after re-feeding. To further investigate the food intake role of UCN3 in Siberian sturgeon, we performed intraperitoneal (i.p.) injection of Siberian sturgeon UCN3 (SsUCN3) with three doses (60, 120 or 240 ng/g) and recorded the food intake. Acute and chronic i.p. injection SsUCN3 reduced the food intake in a dose-dependent pattern. In conclusion, this study indicates that SsUCN3 acts as a satiety factor to inhibit the food intake of Siberian sturgeon.     

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.     

西伯利亚鲟致病性嗜水气单胞菌外膜蛋白及其抗原性分析

采用十二烷基肌氨酸钠(Sarkosyl)法提取西伯利亚鲟嗜水气单胞菌(Aeromonas hydrophila)外膜蛋白,电泳显示所提取的主要外膜蛋白分子量为26～120 kDa;为比较该菌株与气单胞菌菌属其他细菌外膜蛋白组分及抗原性异同,以致病性豚鼠气单胞菌(A.caviae)、温和气单胞菌(A.sobria)和无致病力的嗜水气单胞菌为对照,电泳图谱显示4种气单胞菌外膜蛋白的分子量主要集中在26～120 kDa之间;利用抗西伯利亚鲟嗜水气单胞菌血清的免疫印迹试验表明该菌株外膜蛋白中分子量为75 kDa、52 kDa、43 kDa、40 kDa、34 kDa、28 kDa的蛋白条带呈现阳性反应,其他3种气单胞菌外膜蛋白中均有与该抗血清反应的条带,且分子量为28 kDa、34 kDa的反应条带为4株菌共有;43 kDa与75 kDa反应条带为部分菌株共有.为进一步筛选和研究致病性气单胞菌的共同保护抗原提供参考.     

Evidence of mitochondrial DNA clones of Siberian sturgeon, Acipenser baerii, within Russian sturgeon, Acipenser gueldenstaedtii, caught in the River Volga

Eleven of 34 sturgeons caught in the River Volga classified morphologically as Acipenser gueldenstaedtii were identified as Acipenser baerii from sequence analysis of the mitochondrial cytochrome- b gene. The Caspian Sea and its tributaries including the Volga are not native habitats of A. baerii . No A. baerii haplotype was observed in A. gueldenstaedtii from the Sea of Azov or the South Caspian Sea. Genetic contamination of A. gueldenstaedtii with A. baerii or A. baerii hybrids has occurred in the Volga. Crosses and backcrosses of these specimens with native A. gueldenstaedtii resulted in the loss of the morphological diagnostic A. baerii features. These findings are of special concern for conservation and management programmes, as well as for specimen identification for caviar trading control.     

Behavioural modifications in the early life stages of Siberian sturgeon (Acipenser baerii, Brandt)

Behavioural modifications of Acipenser baeri were studied during early ontogeny at 18 °C by means of specific tests and general observations. During A. baeri ontogeny, several different periods could be clearly distinguished by behavioural patterns. From hatching to 3 days post hatch (p.h.), specimens exhibited swimming-up and drift behaviour. A transition from a pelagic to benthic behaviour occurred between 4–5 days p.h. and specimens were positively rheotactic. At 7–8 days p.h., prelarvae aggregated into schools of different shapes. During prelarval development, specimens were positively phototactic, preferred white bottoms and did not show any preference for bottom concealment. Between 9–10 days p.h. coinciding with the onset of the exogenous feeding, larvae were active bottom swimmers, schooling behaviour disappeared and larvae dispersed. During early ontogeny, changes in body coloration were observed among those specimens who swam over bottoms of different brightness and during the day/night cycle. From 9 to 12 days p.h., larvae were positively phototactic, denied bottom cover and showed preference for white substrates. At 13 days p.h., the percentage of fish exhibiting positive phototactism, preferring open to concealed bottom habitats and white bottoms started to decrease, while at 20 days p.h. specimens showed a clear preference for bottom concealment and black and greyish bottoms. The significance, use and evolution of the former behavioural patterns is deeply discussed.     

How different rearing temperatures affect growth and stress status of Siberian sturgeon Acipenser baerii larvae

Environmental temperature is one of the critical factors affecting fish development. The aim of this study was to examine the impact of three different rearing temperatures (16, 19 and 22°C) throughout the endogenous feeding phase of the Siberian sturgeon Acipenser baerii. This was performed by assessing (a) larval survival and growth; (b) immunofluorescence localization and expression of genes involved in muscle development and growth – myog and Igf1; and (c) stress status through the expression of thermal stress genes – Hsp70, Hsp90α and Hsp90β – and whole body cortisol. Overall survival rate and larval weight did not differ significantly across temperatures. Larvae subjected to 22°C showed faster absorption of the yolk-sac than larvae subjected to 19 or 16°C. Both at schooling and at the end of the trial, larvae reared at 16°C showed significantly lower levels of cortisol than those reared at 19 or 22°C. IGF-1 immunopositivity was particularly evident in red muscle at schooling stage in all temperatures. The expression of all Hsps as well as the myog and Igf1 genes was statistically higher in larvae reared at 16°C but limited to the schooling stage. Cortisol levels were higher in larvae at 22°C, probably because of the higher metabolism demand rather than a stress response. The observed apparent incongruity between Hsps gene expression and cortisol levels could be due to the lack of a mature system. Further studies are necessary, especially regarding the exogenous feeding phase, in order to better understand if this species is actually sensitive to thermal stress.     

Identification of a new mineralized tissue in the notochord of reared Siberian sturgeon (Acipenser baerii)

In a study aiming to improve knowledge on the mineralization of the axial skeleton in reared Siberian sturgeon (Acipenser baerii Brandt, 1869), we discovered a new mineralized tissue within the notochord. To our knowledge, such a structure has never been reported in any vertebrate species with the exception of the pathological mineralization of the notochord remains in degenerative intervertebral disks of mammals. Here, we describe this enigmatic tissue using X‐ray microtomography, histological analyses and solid state NMR‐spectroscopy. We also performed a 1‐year monitoring of the mineral content (MC) of the notochord in relation with seasonal variations of temperature. In all specimens studied from 2‐year‐old juveniles onwards, this mineralized structure was found within a particular region of the notochord called funiculus . This feature first appears in the abdominal region then extends posteriorly with ageing, while the notochord MC also increases. The mineral phase is mainly composed of amorphous calcium phosphate, a small amount of which changes into hydroxyapatite with ageing. The putative role of this structure is discussed as either a store of minerals available for the phosphocalcic metabolism, or a mechanical support in a species with a poorly mineralized axial skeleton. A pathological feature putatively related to rearing conditions is also discussed.     

Development of the skull and pectoral girdle in Siberian sturgeon,Acipenser baerii,and Russian sturgeon,Acipenser gueldenstaedtii (Acipenseriformes: Acipenseridae)

The head is considered the major novelty of the vertebrates and directly linked to their evolutionary success. Its form and development as well as its function, for example in feeding, is of major interest for evolutionary biologists. In this study, we describe the skeletal development of the cranium and pectoral girdle in Siberian (Acipenser baerii ) and Russian sturgeon (A. gueldenstaedtii ), two species that are commonly farmed in aquaculture and increasingly important in developmental studies. This study comprises the development of the neuro‐, viscero‐ and dermatocranium and the dermal and chondral components of the pectoral girdle, from first condensation of chondrocytes in prehatchlings to the early juvenile stage and reveals a clear pattern in formation. The otic capsules, the parachordal cartilages, and the trabeculae cranii are the first centers of chondrification, at 8.4mm TL. These are followed by the mandibular, then the hyoid, and later the branchial arches. Teeth form early on the dentary, dermopalatine, and palatopterygoid, and then appear later in the buccal cavity as dorsal and ventral toothplates. With ongoing chondrification in the neurocranium a capsule around the brain and a strong rostrum are formed. Dermal ossifications start to form before closure of the dorsal neurocranial fenestrae. Perichondral ossification of cartilage bones occurs much later in ontogeny. Our results contribute data bearing on the homology of elements such as the lateral rostral canal bone that we regard homologous to the antorbital of other actinopterygians based on its sequence of formation, position and form. We further raise doubts on the homology of the posterior ceratobranchial among Actinopteri based on the formation of the hyoid arch elements. We also investigate the basibranchials and the closely associated unidentified gill‐arch elements and show that they are not homologous. J. Morphol. 278:418–442, 2017. © 2017 Wiley Periodicals, Inc.     

西伯利亚鲟(Acipenser baerii)致病性维氏气单胞菌的分离鉴定

摘要：【目的】本研究旨在寻找引起养殖西伯利亚鲟鱼(Acipenser baerii)病害的致病因子。【方法】从北京地区自然患病的西伯利亚鲟鱼体内分离到致病菌株X-1-06909,采用生理生化鉴定结合16S rRNA基因序列的系统发育学分析确定该菌株的系统发育地位。同时采用琼脂扩散法对抗菌类药物的敏感性进行测定。【结果】菌株X-1-06909与Aeromonas veronii ATCC 35624T的16S rRNA基因序列相似性达99.6％;结合形态特征与生理生化测定结果,革兰氏阴性杆菌,具极生单鞭毛     

Comparative coelomic fluid composition of sterlet sturgeon Acipenser ruthenus Linnaeus, 1758, Siberian sturgeon Acipenser baerii Brandt, 1869, and Russian sturgeon Acipenser gueldenstaedtii Brandt & Ratzeburg, 1833

The objectives of this study were to compare the physicochemical properties of coelomic fluid (CF) in three sturgeon species, sterlet sturgeon Acipenser ruthenus (age 5–8 years), Siberian sturgeon Acipenser baerii (age 15–20 years), and Russian sturgeon Acipenser gueldenstaedtii (age 13–18 years). For the study, CF was collected by plastic pipette from the eggs of mature female sterlet sturgeon (N = 10), Siberian sturgeon (N = 7) and Russian sturgeon (N = 4); osmolality, pH, ionic composition (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?), glucose, cholesterol and total protein of the CF were then analyzed. Mean concentration of K⁺ (4.4–6.11 mmol/L), Cl^? (89.8–98.0 mmol/L), Ca²⁺ (0.87–0.96 mmol/L), Mg²⁺ (0.57–0.67 mmol/L), cholesterol (0.13–0.16 mmol/L), total protein (2.41–3.57 g/L), pH (7.92–7.98), and osmolality (190–213 mOsm/kg) of CF were not significantly different among these three species. However, the mean concentration of Na⁺ was significantly lower in sterlet sturgeon (104.6 ± 7.7 mmol/L; p < .05) than in the Siberian (126.4 ± 6.2) and Russian (123.0 ± 5.9) sturgeon. For these three species, Na⁺, K⁺, Cl^?, and Mg²⁺ were the dominating ions; several significant correlations were observed among these ions and other physiochemical properties of CF. This is the first report on the ionic and biochemical composition of the CF of sterlet, Siberian and Russian sturgeon, which can be used as a reference point for further development of artificial media for the short‐term storage of unfertilized sturgeon eggs as well as for the standardization of the fertilization protocol for these species in controlled reproduction.     

The evidence of apelin has the bidirectional effects on feeding regulation in Siberian sturgeon (Acipenser baerii)

Apelin is a peptide, mainly produced in the brain, which participates in several physiologic effects. However, knowledge about the mechanism of appetite regulation in teleosts, including the role of apelin is not well understood. The aim of this study is to explore the effect of feeding status on the expression of apelin mRNA in the whole brain and the effects of injection of apelin on food intake in Siberian sturgeon (Acipenser baerii). In this study, we first cloned the apelin cDNA sequence of the Siberian sturgeon. We obtained a 1046-bp cDNA fragment, including a 237-bp open reading frame (ORF) that encoded 78 amino acids. Apelin was widely distributed in 11 tissues related to feeding regulation, with the highest expression in thewhole brain, followed by the spleen and trunk kidney. In addition, we measured the effects of periprandial (preprandial and postprandial) change, fasting and re-feeding on apelin mRNA expression in whole brain. The level of apelin mRNA was significantly decreased 1 h after feeding. The results of the fasting experiment showed that the expression of apelin mRNA in the brain was significantly reduced after 1 day of fasting but consistently increased throughout the 15-day food deprivation period. When the 15-day fasted fish were re-fed, apelin mRNA expression in the brain was significantly increased as compared to that of the control. These results suggest that apelin may play a bidirectional role in the regulation of food intake in the Siberian sturgeon. In order to further examine the effect of apelin on feeding regulation in Siberian sturgeons, acute and chronic intraperitoneal (i.p.) injection experiments were performed and food intakes were recorded. Results showed that acute i.p. injection of apelin-13 reduced food intake, however, chronic i.p. injection apelin-13 increased the food intake for 7 days in Siberian sturgeons. In conclusion, our results show that apelin has a bidirectional effect on feeding regulation in Siberian sturgeons by acting as a satiety factor in short-term feeding regulation and a starvation factor in long-term feeding regulation.     

Morphology and ultrastructure of Siberian sturgeon (Acipenser baerii) spermatozoa using scanning and transmission electron microscopy

BACKGROUND INFORMATION: Available data concerning the sperm morphology of teleost fishes demonstrate wide variation. In the present study, the spermatozoa of Siberian sturgeon (Acipenser baerii Brandt, 1869), a chondrostean fish, was investigated. In contrast with teleost fish, chondrostean spermatozoa have a head with a distinct acrosome, whereas other structures, such as a midpiece and a single flagellum, are present in spermatozoa of most species. RESULTS: The average length of the head including the acrosome and the midpiece was 7.01+/-0.83 microm. Ten posterolateral projections derived from the acrosome were present on a subacrosomal region, with mean lengths of 0.94+/-0.15 microm and widths of 0.93+/-0.11 microm. The nucleus consisted of electrodense homogeneous nuclear chromatin. Three intertwining endonuclear canals, bound by membranes, traversed the nucleus longitudinally from the acrosomal end to the basal nuclear fossa region. There were between three and six mitochondria, two types of centrioles (proximal and distal) in the midpiece and two vacuoles composed of lipid droplets. The flagellum (44.75+/-4.93 microm in length), originating from the centriolar apparatus, had a typical 9+2 eukaryotic flagellar organization. In addition, there was an extracellular cytoplasm canal between the cytoplasmic sheath and the flagellum. CONCLUSIONS: A principal components analysis explained the individual morphological variation fairly well. Of the total accumulated variance, 41.45% was accounted for by parameters related to the head and midpiece of the sperm and the length of the flagellum. Comparing the present study with previous studies of morphology of sturgeon spermatozoa, there were large inter- or intra-specific differences that could be valuable taxonomically.     

Ontogenetic behavior of Siberian sturgeon, Acipenser baerii: A synthesis between laboratory tests and field data

We have reviewed field data and studies on the behavior and development of Siberian sturgeon at early-life intervals and related them to different ecologically relevant environmental factors that may play a role in the distribution, recruitment, and survival of young fish. Four behavioral phases (swimming-up, rheotactism, shoaling, and foraging) are observed from hatching to the juvenile phase. Each behavior is associated with an early-life interval and might allow fish to occupy different river habitats, directly influencing their distribution, survival, and recruitment. River current intensity, substrate typology, food resources, and predation pressure seem to be the most important factors affecting the distribution of Siberian sturgeon free embryos and larvae, while juveniles and adult fish disperse and migrate according to food abundance and reproduction. Mechanisms involved in regulating downstream migration during Siberian sturgeon early life stages are different than those observed in anadromous sturgeon species. In all large Siberian rivers, with the exception of the Lake Baikal, the Siberian sturgeon is represented by population continuums, and in many cases the foraging range also includes the spawning areas. Ontogenetic changes in Siberian sturgeon behavior could be interpreted as a species-specific mechanism to maintain the population continuums described in this species without significant mixture of local populations within the river.