Sturgeon Sperm Research Articles

Effect of post-thaw storage time on motility and fertility of cryopreserved beluga sturgeon (Huso huso) sperm.

The aim of this study was to test the influence of post-thaw storage time on the duration of sperm motility, percentage of motile sperm, and fertilization and hatching rates of fresh sperm and sperm stored for 0, 30 and 60 min at 4°C post-thawing. After being frozen in liquid nitrogen and then thawed, the percentage of motile sperm and duration of motility were not affected by 30 min of storage at 4°C, whereas a significant decline in these parameters was observed after 60 min of storage. Similarly, fertilization and hatching rates were significantly affected within 60 min of storage at 4°C, and the fertility of frozen-thawed sperm was significantly lower than that of fresh sperm. We conclude that cryopreserved sperm of beluga sturgeon could be stored for 30 min without the loss of sperm quality. This described procedure for beluga sturgeon cryopreservation is reliable and efficient and therefore can be recommended for hatchery practice after scaling up this technique.

The antioxidant system of sterlet seminal fluid in testes and Wolffian ducts

Oxidative stress is a possible source of spermatozoa function deterioration. Seminal fluid (SF) protects spermatozoa against reactive oxygen species (ROS) attack during development in testes and transit through the reproductive tract. Spermatozoa curvilinear velocity and percent of motile cells as well as changes in thiobarbituric acid-reactive substance (TBARS) content, superoxide dismutase, and catalase activity, and uric acid concentration in SF were evaluated in sterlet sperm collected from testes 24 h after hormone induction of spermiation and from Wolffian ducts at 12, 24, 36, and 60 h after hormone injection (HI). While testicular spermatozoa motility was not initiated in activating medium, Wolffian duct sperm showed low motility at 12 h, significant increase at 24 and 36 h, and decrease at 60 h. Testicular SF was characterized by the highest level of TBARS and activity of studied enzymes compared with SF from Wolffian duct sperm at 24 h post-HI. In fluid from Wolffian duct sperm, a significant increase in TBARS content was shown at 36-60 h post-HI. In contrast to testicular SF, in SF from Wolffian duct sperm, this increase was not counterbalanced by changes in the studied variables of antioxidant system. This may be the source of the observed decrease in spermatozoa motility parameters 60 h post-HI. The results may confirm a dual role of ROS in fish sperm physiology. The data with respect to decrease in sturgeon spermatozoa motility parameters at 60 h post-HI should be taken into account in artificial sturgeon propagation.

Motility and fertilization ability of sterlet Acipenser ruthenus testicular sperm after cryopreservation

Sturgeon spermatozoa are immotile in the testis and acquire the potential for motility after contact with urine in Wolffian duct. The present study tested if in vitro incubation of testicular sperm in seminal fluid from Wolffian duct sperm leads to the acquisition of sperm fertilization ability. Sterlet sperm was taken from the testes, matured in vitro and cryopreserved. The fertility and motility of cryopreserved semen were tested. Matured testicular sperm showed freeze–thaw survival rates similar to Wolffian duct sperm, which is commonly used in sturgeon artificial propagation. Matured testicular sperm and Wolffian duct sperm post-thaw motility rate and curvilinear velocity were not significantly different, while duration of matured testicular sperm motility was significantly shorter than that of Wolffian duct sperm. Development rates of embryos obtained with post-thaw matured testicular sperm and Wolffian duct sperm were not significantly different. In vitro maturation of sterlet testicular sperm can potentially be useful in sperm cryobanking.

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.

Novel droplet vitrification combined with fish antifreeze protein type III enhances cryoprotection of semen in wild endangered Persian sturgeonAcipenser persicus(Borodin, 1897)

In this study, the efficiency of a novel droplet vitrification technique along with different doses of fish antifreeze protein (AFP) type III on Persian sturgeon thawed spermatozoa quality (motility duration and motility percentage) was investigated. Semen of seven male individuals was pooled in equal volumes and diluted with 4°C Tris-Hcl (100 mM), pH = 8 extenders containing 0, 5, 10, 15 μM of AFP type III in a ratio of 1:1 (semen/extenders). Treated semen was dropped into liquid nitrogen. Solidified droplets were stored for 2, 60 and 120 days and thawed by plunging them into a tube containing 5 mL Tris-Hcl (100 mM), pH=8 with 1% BSA at 37°C. Motility duration in all treatments had no significant difference comparing to fresh sperm (P > 0.05), but their motility percentage was significantly lower. Treatment with 10 μM of AFP had significantly higher motility percentage (16.11 ± 0.5%) comparing to other treatments (P < 0.05). There was no significant difference between 0, 5, 15 μM of antifreeze protein treatments (P > 0.05), suggesting that antifreeze protein effectiveness are highly dose dependent, and dose of 10 μM is appropriate in Persian sturgeon spermatozoa droplet vitrification. Besides, the present technique obtained higher quality of spermatozoa comparing to its analogue techniques.

Induction of meiotic gynogenesis in ship sturgeon Acipenser nudiventris using UV-irradiated heterologous sperm

Diploid gynogenesis was induced in ship sturgeon Acipenser nudiventris using UV-irradiated sperm from Siberian sturgeon Acipenser baerii. The optimal condition for the retention of the second polar body in ship sturgeon was determined to be 10min after activation/fertilization in experiments. The temperature of cold shock and its duration were 2.5°C and 30min, respectively. A total of 30 gynogens of known parentage from experimental treatments were screened using microsatellite DNA analysis, and uniparental transmission in meiogens was confirmed. The results show that heterologous Siberian sturgeon sperm is applicable as UV-irradiated sperm for the induction of gynogenesis in ship sturgeon. This technique may recover the critically endangered sturgeon species that are becoming extinct.

Effects of short-term storage on the motility, oxidative stress, and ATP content of Persian sturgeon (Acipenser persicus) sperm

The effective short-term storage of semen is essential when processing multiple sperm samples and when semen must be transported from collection sites to hatcheries for the fertilization of ova, or to laboratories for cryopreservation. In the present study, the spermatozoa of Persian sturgeon (Acipenser persicus) were used to evaluate the effects of short-term storage on quality parameters (the percentage of motile cells and the total period of sperm motility), oxidative stress indices, and the ATP content. Spermatozoa cells exhibited >50% motility during 6 days of storage where the average total duration of sperm motility varied from 376.42±80.86s initially to 19.28±10.96s after 6 days. No motile spermatozoa were recorded after 9 days of storage. The levels of oxidative stress indices (TBARS and CP) and antioxidant activity (SOD) increased significantly with the storage time. The ATP content also decreased significantly after 2 days of storage. The results of this study may facilitate successful reproduction management and cryopreservation protocols for this endangered fish.

Comparison of oxidant and antioxidant status of seminal plasma and spermatozoa of several fish species

Oxidant and antioxidant activity in seminal plasma was compared with that in spermatozoa of teleost (common carp Cyprinus carpio and brook trout Salvelinus fontinalis) and chondrostean (Russian sturgeon Acipenser gueldenstaedtii, Siberian sturgeon Acipenser baerii, and sterlet Acipenser ruthenus) fishes. No differences were found between seminal plasma and spermatozoa in the level of thiobarbituric-acid-reactive substance (0.24 &amp;plusmn; 0.08 to 0.33 &amp;plusmn; 0.04 nmol/mg proteins) in Russian sturgeon, Siberian sturgeon, and sterlet. Carbonyl protein concentration was significantly higher in spermatozoa than in seminal plasma of all studied species. Analyzed antioxidants included superoxide dismutase, glutathione reductase, and glutathione peroxidase activity. Significant differences (P &amp;lt; 0.05) were detected between seminal plasma and spermatozoa in total superoxide dismutase (SOD) and glutathione reductase (GR). Total glutathione peroxidase (GPx) activity was significantly higher in brook trout (12.56 &amp;plusmn; 3.23 mU/mg proteins) and Russian sturgeon (11.56 &amp;plusmn; 3.12 mU/mg proteins) spermatozoa compared to seminal plasma (6.81 &amp;plusmn; 1.56 mU/mg proteins in brook trout and 9.56 &amp;plusmn; 3.12 mU/mg proteins in Russian sturgeon). This study provides new data on oxidant and antioxidant balance between spermatozoa and seminal plasma that may be of value in the development of methods for artificial reproduction of teleost and chondrostean species. &amp;nbsp;

Optimization of sperm irradiation protocol for induced gynogenesis in Siberian sturgeon, Acipenser baerii

The great diversity of optimal UV irradiation doses are used for DNA inactivation in fish sperm forcing authors to repeat optimization of irradiation treatment every time. Analysis of sperm UV irradiation protocol for induction of gynogenesis showed the importance of sperm UV light absorption estimations. The UV absorption investigation in Siberian sturgeon sperm showed average extinction coefficient 7.69 × 10−8 ± 1.04 × 10−8 cm2. It is resulted in high heterogeneity of UV irradiation of undiluted sperm samples. Therefore, it is strongly suggested to specify doses only with defined concentration of spermatozoa; otherwise, the difference in absorbance level between samples can bring a significant error to optimal UV dose estimation. This was confirmed by UV-irradiated sperm motility investigation. Results of motility investigation of UV-irradiated sperm revealed high sensitivity of Siberian sturgeon spermatozoa motion mechanisms to UV irradiation, with complete loss of motility after homogeneous UV irradiation at doses above 2,000 J/m2. Partial gynogenesis was conducted using diluted and undiluted sperm. Ploidy level of hatched larvae was estimated by flow cytometry. Percentage of haploid hatched larvae revealed sperm DNA inactivation efficiency. The highest percentage of haploid putative gynogenotes 19.67 ± 4.19 % was obtained at UV irradiation dose 200 J/m2 with sperm diluted to 1:4.

Effect of short-term storage on quality parameters, DNA integrity, and oxidative stress in Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeon sperm

The sperm of Russian sturgeon (A. gueldenstaedtii) and Siberian sturgeon (A. baerii) was used to evaluate the effects of short-term (liquid) storage on functional parameters (spermatozoa motility and velocity), DNA integrity and oxidative stress indices. Spermatozoa showed >50% motility during 6 days of storage with an average velocity of 133.12±15.4 to 87.9±11.23μm s−1 in both species. No motile spermatozoa were recorded after nine days of storage. Analysis of Russian sturgeon sperm showed no significant differences in DNA damage expressed as percent tail DNA and Olive Tail Moment for first three days of storage. In Siberian sturgeon significant differences in DNA damage were detected after two days of storage. The level of oxidative stress indices (TBARS, CP) and antioxidant activity (SOD) increased significantly with storage time in both species. Results of this study can be utilized for successful reproduction management and cryopreservation protocols of these endangered species.

Motility of sturgeon spermatozoa can sustain successive activations episodes

Here we report for the first time the possibility of sequential sperm motility activation in sturgeon (sterlet, Acipenser ruthenus), a fish with external fertilization, through changes either in osmolality (global solute concentration) or in the Ca2+ concentration of the medium surrounding the spermatozoa. Sperm motility was initiated in any of three solutions containing buffer and sucrose at 80, or 40 or 10mM (called S80, S40, S10, respectively); S80 is hypertonic relative to sterlet seminal fluid, while S40 is isotonic and S10 is hypotonic. After cessation of sperm movement at the end of this first motility period, a second and then a third, subsequent motile phase were observed. The second motility period was induced at cessation of motility in S80 by imposing a two-fold decrease in osmolality. After arrest of motility in this half-diluted S80, a third motility period could be initiated by addition of CaCl2 to 1mM final concentration. At the end of a first motility period in either S40 or S10, subsequent motility re-activation episodes were achieved only by addition of 1mM CaCl2. Depending on conditions in which sperm samples were activated, significant differences in curvilinear velocity, percent motile spermatozoa, motility duration time, and specific external features of spermatozoa flagella were observed. Altogether, these observations on the ability of sturgeon spermatozoa to sustain sequential activation episodes by experimental adjustment of their environmental conditions represent a potent model for deeper investigations on the sperm motility activation mechanisms.

Haematology of gynogenetic diploids of Siberian sturgeon Acipenser baerii Brandt

Haematological analyses were conducted in gynogenetic diploids and control diploids of Siberian sturgeon (Acipenser baerii). Gynogenetic diploid fish were obtained from Polish aquaculture after induction of meiotic gynogenesis based on application of UV-irradiated hybrid Siberian sturgeon × Russian sturgeon (A. gueldenstaedtii) sperm (for eggs activation) and heat shock for inhibiting the second polar body retention. A total of fifteen 30-month-old specimens of Siberian sturgeon from the gynogenetic diploid group and 12 specimens from the control diploid group were randomly sampled. There were no differences in the number of erythrocytes and haematocrit values between gynogenetic diploids and normal diploids of Siberian sturgeon. However, the percentage of normal erythrocytes was lower in the gynogenetic group of fish and the higher percentage of the atypical red blood cell forms (microcytes, sphaerocytes, erythrocytes with displaced nuclei and with divided nuclei) were observed in this group of fish. Additionally all the haemoglobin indices (haemoglobin concentration, the mean corpuscular haemoglobin and the mean corpuscular haemoglobin concentration) were significantly reduced in gynogenetic diploids of Siberian sturgeon. These results could suggest depletion of oxygenation capacity of the blood of gynogenetic sturgeons. This is the first report of a haematological study in gynogenetic diploids of sturgeon fish.

Fins improve the swimming performance of fish sperm: A hydrodynamic analysis of the Siberian sturgeon Acipenser baerii

The flagella of sturgeon sperm have an ultrastructure comprising paddle-like fins extending along most of their length. These fins are seen in several other marine and freshwater fish. The sperm of these fish are fast swimmers and are relatively short lived: it is therefore tempting to think of these fins as having evolved for hydrodynamic advantage, but the actual advantage they impart, at such a small length scale and slow speed, is unclear. The phrase "the fins improve hydrodynamic efficiency" is commonly found in biological literature, yet little hydrodynamic analysis has previously been used to support such conjectures. In this paper, we examine various hydrodynamic models of sturgeon sperm and investigate both swimming velocity and energy expenditure. All of the models indicate a modest hydrodynamic advantage of finned sperm, in both straight line swimming speed and a hydrodynamic efficiency measure. We find a hydrodynamic advantage for a flagellum with fins, over one without fins, of the order of 15-20% in straight line propulsive velocity and 10-15% in a hydrodynamic efficiency measure.

Isolation and characteristics ofβ-N-acetylglucosaminidase present in rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baerii) milt

Summary The aim of this study was to isolate and characterise the enzyme s-N-acetylglucosaminidase (β-NAGase) in rainbow trout and Siberian sturgeon milt, in order to find similarities or differences in its involvement in the fertilization process of these two species. While sturgeon sperm possess an acrosome, teleosts do not. Applying ion exchanging chromatography to β-NAGase of rainbow trout sperm, two peaks of β-NAGase activity were obtained from the plasma material and from the sperm. The molecular weight was 74 kDa for both milt plasma forms and 127 kDa for rainbow trout spermatozoa peak. Optimum pH of purified rainbow trout enzyme peaks ranged from 4.6 to 5.0. Michaelis–Menten constants were 11.59 × 10−4 and 11.48 × 10−4 m for I and II milt plasma peaks. Incubation of purified peaks I and II at 56°C resulted in a 45 to 50% decrease in activity of both forms. Km value for rainbow trout spermatozoa β-NAGase was 5.4 × 10−4 m. Enzyme incubation tests at high temperature proved this form of the enzyme to be most resistant to high temperatures, since its activity after 20 min of incubation at 56°C decreased only by 25%. The molecular weight of the enzyme originating from Siberian sturgeon milt plasma was about 265 and 113 kDa for I and II milt plasma peaks, respectively, and 271 kDa for the sperm extract. Optimum pH for all sturgeon purified peaks ranged from 3.8 to 5.0. The Km constant was 5.12 × 10−4, 5.28 × 10−4 and 5.0 × 10−4 m for I and II milt plasma and spermatozoa peaks, respectively. The loss of s-NAGase activity at 56°C was 58% for both milt plasma peaks and 35% for the sturgeon spermatozoa peak. The kinetic parameters, especially thermostability of s-NAGase was similar to the homologous enzymes observed in mammalian semen.

Motility and fertilizing capacity of frozen/thawed sperm of Siberian sturgeon after a short-time exposure of fresh semen to mercury and cadmium

Summary The objective of this study was to analyze whether frozen/thawed sperm quality, in the Siberian sturgeon sperm (Acipenser baerii, Brandt, 1869), in particular if motility parameters, viability and fertilization capacity would be affected following a 4 h exposure of fresh semen to mercury and cadmium (0–100 mg L−1) prior to cryopreservation. The percentage of motile sperm was reduced at 1 mg Hg2+ L−1 and 10 mg Cd2+ L−1. The sperm's ability to fertilize was significantly lower at 10 mg Hg2+ L−1. Moreover, results showed that the negative impact of heavy metals on the sperm motility and ability to fertilize did not result from disturbances to sperm membrane integrity. The percentage of motile sperm seems to be a more useful indicator than sperm velocity for evaluating fertilization impairment in Siberian sturgeon sperm when exposed to mercury. Mercury appeared to be more detrimental than cadmium in terms of ability to fertilize. Our results suggest that the sperm of acipenserid fish may be characterized by a higher resistance to cadmium ions compared to that of the teleost fish.

Sperm biology and control of reproduction in sturgeon: (II) sperm morphology, acrosome reaction, motility and cryopreservation

In the present review, sperm morphology, acrosome reaction, motility, short-term storage and cryopreservation are summarized and discussed in sturgeon (Chondrostei, Acipenseriformes). The elongated head of spermatozoon comprises an acrosome with 8–12 posterolateral projections. Usually three endonuclear canals are observed in the nucleus. Proximal and distal centrioles and 3–6 mitochondria are located in the midpiece region. The flagellum consists of an axoneme with a typical “9 + 2” structure of microtubules and presents a ribon-like structure due to two lateral membranous fins. Egg water, Ca2+ and Mg2+ can trigger acrosome reaction. Trypsin- and chymotrypsin-like activities are reported in sturgeon sperm. These physiological properties of sturgeon sperm are identified as serine activity with 33 kDa molecular mass and can be inhibited by their respective inhibitors. The K+ prevents sperm activation in seminal plasma, and hypo-osmolality or decrease of extracellular K+ triggers sperm activation. Extracellular Ca2+ is involved in flagellar beating pattern and sperm velocity. After activation, sperm motility, velocity, and flagellar beating frequency, wavelength and amplitude decrease, while number of waves and curvature increase. Sturgeon sperm can be stored for several days at 4 °C; however it is better to add K+ into the immobilizing medium because it prevents sperm activation during incubation. Regarding sperm cryopreservation, methanol is a better cryoprotectant than DMSO. Either short-term storage or cryopreservation of sperm generates damage to spermatozoa that lead to reduction of sperm motility performance. Some studies suggest using an activation medium containing Ca2+ for enhancing sperm motility performance of incubated or frozen-thawed sperm.

Regulation of spermatozoa motility in response to cations in Russian sturgeon Acipenser gueldenstaedtii

The aim of this study was to investigate the response of Russian sturgeon (Acipenser gueldenstaedtii) sperm to external cations (Na+, K+, Ca2+, and Mg2+) and their susceptibility on the induction of motility and swimming behavior. An in vitro spermatozoa motility assay was used by a computer-aided Motion-Analysis system. Sperm motility was inhibited by 60 mm NaCl (∼140 mOsm/kg) and 0.7 mm KCl solutions (∼ 21.4 mOsm/kg). The Ca2+ and Mg2+ ions were not able to inhibit spermatozoa motility. By contrast, Na+ within a limited concentration range (between 45 and 55 mm) was able to reverse the inhibitory effect of K+ at the critical concentration (0.7 mm). Ca2+ and Mg2+ were also able to reverse the K+-mediated spermatozoa motility restriction at concentrations starting at 0.01 and 0.1 mm, respectively. These results provide evidence for the role of K+ in suppressing spermatozoa motility, and suggest that Ca2+, Mg2+, and possibly Na+ trigger motility in Russian sturgeon sperm.

Relationships between sex steroids of the seminal fluid and milt quality parameters of Persian sturgeon, Acipenser persicus Borodin, 1897

To investigate the role of sex steroids of the seminal fluid on sperm quality, the relationships between sex steroids and milt quality parameters (sperm motility and sperm production) were investigated in the Persian sturgeon. The seminal fluid levels of 17α,20β,21-trihydroxy-4-pregnen-3-one (20βs), and 11-ketotestosterone (11-KT) had positive relationships with sperm motility characteristics (percentage and duration of motility) and sperm density. Also, no relationships were found between other sex steroids including: Testosterone (T), progesterone (P), 17α-hydroxyprogesterone (OHP), and milt quality parameters. The good correlation of 20βs and 11-KT of the seminal fluid with sperm motility and sperm density suggests that these steroids may be important hormones involving in final maturation of the Persian sturgeon spermatozoa.

Cryopreservation of sterlet (Acipenser ruthenus) spermatozoa using different cryoprotectants

Summary The present study examined the effectiveness of different cryoprotectan uses for cryopreservation of sterlet (Acipenser ruthenus) sperm. Four different cryoprotectans [dimethyl-sulfoxide (DMSO), dimethylacetamide (DMA), ethyleneglycol (EG) and methanol (MET)] in concentrations of 5 and 10% in the extender media (30 mm sucrose, 1 mm KCl, 25 mm Tris–HCl pH 8.5) were used for the cryopreservation of sperm from five sterlet males. Percentages of motility, velocity, fertilization and hatching rate were measured. The highest post-thawing motility was observed in sperm samples cryopreserved with 10% MET (46 ± 19%), 10% DMA (47 ± 18%) and 10% DMSO (45 ± 7%). Fertilization rate in the control (fresh sperm) was 69 ± 9% and the hatching rate 61 ± 8%. A higher hatching rate after cryopreservation was obtained with 10% MET (32 ± 17%) and 5% DMA (23 ± 3%) when compared to data obtained with 10% DMA (9 ± 5%), 5% MET and 5 and 10% DMSO, wherein the hatching rate was around zero. Our results demonstrate that DMA can be used for cryopreservation of sturgeon spermatozoa.

Roles of osmolality, calcium — Potassium antagonist and calcium in activation and flagellar beating pattern of sturgeon sperm

The present study shows the roles of osmolality, calcium (Ca2+)–potassium (K+) antagonist and Ca2+ in sperm activation and flagellar beating of a sturgeon species, sterlet (Acipenser ruthenus). Sperm motility was activated at hypoosmolality relative to seminal plasma and suppressed at 175 mOsmol kg−1. Sperm activation was totally suppressed by 0.35mM K+, but Ca2+ could fully reverse K+ inhibitory effect at Ca2+: K+ ratio of 0.25. Neither EGTA (a chelator of Ca2+ ions) nor nifedipine (a Ca2+ channel blocker) prevented sperm activation. But, sperm motility and velocity were significantly decreased by EGTA, nifedipine and an inhibitor for Ca2+/calmodulin activated phosphodiesterase (w-7) that suggest role of Ca2+ signaling after triggering sperm activation through hypoosmolality. Symmetric flagellar beating was also turned to asymmetric after activation in w-7, which is an evidence for modulation of Ca2+-binding proteins activity. Sturgeon sperm, similar to salmonids, is immotile in seminal plasma due to high K+ concentrations, but the mechanism of sperm activation seems to be closer to other fish species where osmolality prohibits sperm activation in seminal plasma. In these species, hypoosmolality is the primary signal for sperm Ca2+-dependent signaling of axonemal beating.