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Improvement of sperm quality with low motility by storage could ensure higher success of fertilization and maintain higher genetic diversity, especially for sturgeons, which as endangered species have limited broodstock and gametes. Sperm was collected from mature male sterlet Acipenser ruthenus and motility was evaluated using the CASA system; samples were categorized as GS 'good sperm' (80%) or BS 'bad sperm' (20%). Samples from both groups were incubated with seminal plasma from good- (GSP) and bad-quality sperm (BSP), respectively for 15 min, 6 h, 24 h and 96 h at 4 °C. Motility of BS incubated in GSP increased after different storage times compared to BS incubated in BSP, while the motility and velocity of GS incubated in BSP decreased compared to GS incubated in GSP. Fertilization rates were evaluated with samples stored for 15 min and 6 h post-stripping; fertilization and hatching rate of BS after incubation in GSP increased significantly compared to the BS incubated in BSP. Inorganic ion (Na

Made available in DSpace on 2019-10-04T12:37:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-05-01 Czech Science Foundation Ministry of Education, Youth and Sports of the Czech Republic -project CENAKVA project Biodiversity (Reproductive and genetic procedures for preserving fish biodiversity and aquaculture) All extant groups of Elasmobranches have internal fertilization and the structure of the male reproductive organs is very specific: sperm passes from the internal organs via the cloaca, but the male copulating organ (clasper) is distant from the cloaca. This suggests that sperm can contact the surrounding medium before fertilization. Because of this involvement with the environment, external signaling in sperm motility activation could occur in these species even though their fertilization mode is internal. In this case, spermatozoa of Elasmobranches should hypothetically possess a specific structure and membrane lipid composition which supports physiological functions of the sperm associated with environmental tonicity changes occurring at fertilization. Additionally, sperm motility properties in these taxa are poorly understood. The current study examined sperm lipid composition and motility under different environmental conditions for the ocellate river stingray, Potamotrygon motoro, an endemic South America freshwater species. Sperm samples were collected from six mature males during the natural spawning period. Sperm motility was examined in seminal fluid and fresh water by light video microscopy. Helical flagellar motion was observed in seminal fluid and resulted in spermatozoon progression; however, when diluted in fresh water, spermatozoa were immotile and had compromised structure. Lipid class and fatty acid (FA) composition of spermatozoa was analyzed by thin layer and gas chromatography. Spermatozoa FAs consisted of 33 +/- 1% saturated FAs, 28 +/- 1% monounsaturated FAs (MUFAs), and 41 +/- 1% polyunsaturated FAs (PUFAs), and a high content of n-6 FAs (32 +/- 2%) was measured. These results allowed us to conclude that sperm transfer from P. motoro male into female should occur without coming into contact with the hypotonic environment so as to preserve potent motility. In addition, this unusual reproductive strategy is associated with specific spermatozoa structure and lipid composition. Low level of docosahexaenoic acid and relatively low PUFA/MUFA ratio probably account for the relatively low fluidity of freshwater stingray membrane and can be the main reason for its low tolerance to hypotonicity. (C) 2019 Elsevier Inc. All rights reserved. Univ South Bohemia Ceske Budejovice, Fac Fisheries & Protect Waters, Bohemian Res Ctr Aquaculture & Biodivers Hydrocen, Zatisi 728-2, Vodnany 38925, Czech Republic Sao Paulo State Univ, Fac Engn, Dept Biol & Zootechny, Neotrop Ichthyol Lab LINEO, Moncao St 226, BR-15385000 Ilha Solteira, SP, Brazil Swedish Univ Agr Sci, Dept Mol Sci, POB 7015, S-75007 Uppsala, Sweden Univ South Bohemia Ceske Budejovice, Inst Chem, Fac Sci, Branisovska 1760, Ceske Budejovice 37005, Czech Republic Biol Ctr Czech Acad Sci, Inst Parasitol, Branisovska 31, Ceske Budejovice 37005, Czech Republic Sao Paulo State Univ, Fac Engn, Dept Biol & Zootechny, Neotrop Ichthyol Lab LINEO, Moncao St 226, BR-15385000 Ilha Solteira, SP, Brazil Czech Science Foundation: 16-03754S Ministry of Education, Youth and Sports of the Czech Republic -project CENAKVA: LM2018099 project Biodiversity (Reproductive and genetic procedures for preserving fish biodiversity and aquaculture): CZ.02.1.01./0.0/0.0/16_025/0007370

The significant influence of the number and position of fish sperm sample straws in uncontrolled cooling devices on post-thaw spermatozoa parameters, such as motility and fertilizing ability, is presented in this study. The two most popular uncontrolled cooling devices were used in this study: a Styrofoam box setup with a polystyrene floating raft on liquid nitrogen and the dry shipper setup with a straw holder. We tested the effect of different quantities of straws (6 or 60) placed on the polystyrene floating raft and the position of the straws in the holder (on the periphery or in the centre). Using these cooling methods, sperm of 10 male sterlets diluted with methanol containing cryoprotective medium was frozen. All temperature changes were recorded by a thermocouple inside the straw, and the thermogram intervals were analysed. Spermatozoa motility was evaluated by video microscopy with integrated computer-assisted sperm analysis software. Fertilization trials were conducted at a 10 5 spermatozoa/egg ratio. Post-thaw spermatozoa parameters, including the percent of motile spermatozoa, curvilinear velocity, velocity according to the smoothed path, linearity of track, beat-cross frequency and fertilization rate, were significantly decreased in the 60-straw floating raft setup in comparison to all of the other cooling methods. The freezing rate between −10 °C and −30 °C was significantly decreased by up to 18.6 ± 0.61 °C/min for the 60-straw floating raft setup in comparison to the other freezing conditions. Considering the above results, efforts to standardize cryopreservation protocols using uncontrolled cooling devices should take into account the amount of straws subjected to freezing.

In the present study, for the first time in fish spermatozoa, we describe the precise chronology of motility initiation of sterlet (sturgeon) sperm from completely immotile flagella to regular full wave propagation. The successive activation steps were investigated by high-speed video microscopy, using specific experimental situation, where sperm motility initiation was delayed in time up to several seconds (10 ± 2.68 seconds). Starting from fully immotile, the flagellum shows some trembling for a brief period, soon followed by appearance of the first real bend (so-called "principal bend") with a large wave amplitude 4.28 ± 0.65 μm, then by the "reverse bend," the latter presenting a lower (P < 0.05) wave amplitude (1.14 ± 0.32 μm). This couple of first bends formed at the basal region begins to propagate toward the flagellar tip but gradually fades when reaching the midflagellum, wherein consequently the sperm cell remains nonprogressive. This behavior repeats several times until a stage where the amplitude of the reverse bend gradually reaches a value similar that of the principal bend: The larger amplitude of this couple of bends finally leads to sustain a real "takeoff" of the sperm cell characterized by a full flagellar wave propagation generating an active forward displacement similar to that occurring during regular steady state motility (several seconds after activation). Starting from the earliest stages of motility initiation, the wave propagation along the flagellum and formation of new waves proceeded in a helical manner leading to a 3-dimensional rotation of the whole spermatozoon. Eventually, we estimated that the time period needed from the activation signal (contact with fresh water) to full wave propagation ranges from 0.4 to 1.2 seconds.

The effect of cryopreservation on the protein phosphorylation/dephosphorylation pattern of common carp (Cyprinus carpio) sperm is described. Sperm was diluted in dimethyl sulfoxide (DMSO) and ethylene glycol (EG)-based extenders, followed by equilibration, freezing, and thawing. Proteins extracted from fresh and cryopreserved spermatozoa were separated on SDS-PAGE and two-dimensional gel electrophoresis, blotted on polyvinylidene difluoride membrane, and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine antibodies. For the subsequent protein identification we used matrix-associated laser desorption/ionization time-of-flight mass spectrometry. The results demonstrated that cryopreservation with either DMSO or EG extender significantly altered the phosphorylation state of sperm proteins on tyrosine or threonine residues. A dramatic decrease in tyrosine phosphorylation was detected in the cryopreservation procedures with DMSO extender. Endoplasmin, transketolase, and S-adenosylhomocysteine hydrolase were identified as proteins that play a key role in cellular stress responses and oxidation and/or reduction reactions. Results indicate that the phosphorylation and/or dephosphorylation modifications of sperm proteins that occur during cryopreservation could stimulate a series of biochemical effects interfering with spermatozoa function and leading to a loss of motility and fertilization ability. Our findings indicated that use of EG extender provided superior protein preservation during sperm storage.

The morphology of the reproductive system of acipenseriform fishes is quite different from that of teleostean species, but an associated unique physiological difference in male sturgeons was not discovered until recently; sperm of sturgeons passes through the kidneys then via Wolffian ducts into the environment rather that emptying directly through seminal ducts. The mixing of sperm with excretory products has been found to be a requisite for the capacity to be activated (maturation step) instead of being deleterious. In the current review we summarize results of studies performed in our laboratory on physiological processes involved in sturgeon sperm maturation, namely changes in: 1) ionic environment; 2) sensitivity of spermatozoa to calcium ions (Ca2+); 3) antioxidant enzymes and proteolytic activities; and 4) content in macroergic phosphates arising during this maturation process. We also discuss taxa-specific aspects of sturgeon sperm maturation in relation to hormonal regulation of spermiation, and the unusual features of sturgeon sperm maturation relative to using testicular sturgeon sperm in aquaculture.

Damage to spermatozoa during cryopreservation is regarded as a major obstacle to the expansion of sperm storage technology. The authors used two-dimensional polyacrylamide gel electrophoresis and matrix-associated laser desorption/ionization time-of-flight mass spectrometry to explore whether the protein profile of common carp (Cyprinus carpio) spermatozoa is affected by cryopreservation. Fourteen protein spots were significantly altered following cryopreservation. Eleven of these were identified: three as specific membrane proteins (N-ethylmaleimide-sensitive fusion protein attachment protein alpha, cofilin 2, and annexin A4) involved in membrane trafficking, organization, and cell movement; six as cytoplasmic enzymes (S-Adenosylhomocysteine hydrolase, Si:dkey-180p18.9 protein, lactate dehydrogenase B, phosphoglycerate kinase 1, transaldolase 1, and esterase D/formylglutathione hydrolase) involved in cell metabolism, oxidoreductase activity, and signal transduction; and two as transferrin variant C and F. Based on these findings, the authors hypothesize that transferrin in cryopreserved sperm may protect spermatozoa against oxidative damage during the freeze-thaw process. Cryopreservation caused changes in spermatozoa protein profiles that may lead to decreased spermatozoa velocity, motility, and fertilization success, and to subsequent ova hatching rate.

Northern pike (Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 microm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 microm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg(-1) (average: 283.88+/-33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media (P

As spermatozoon motility duration differs significantly among fish species, the mechanism of ATP generation-regeneration and its distribution along the flagellum may be species-dependent. The present study compared the role of creatine kinase (CK) with that of adenylate kinase (AK) in ATP regeneration during motility of demembranated spermatozoa of taxonomically distant fish species, sterlet, and common carp, allowing investigation for the presence of the creatine-phosphocreatine (PCr) shuttle in sterlet spermatozoa. The flagellar beat frequency of demembranated spermatozoa was measured in reactivating media in the presence or absence of ATP, ADP, PCr, and CK and AK inhibitors. After demembranation, AK, CK, and total ATPase activity was measured in spermatozoon extracts. Beat frequency of demembranated spermatozoa was found to be positively correlated with ATP levels in reactivating medium and to reach a plateau at 0.8 mM and 0.6 mM ATP for carp and sterlet, respectively. It was shown for the first time that sterlet axonemal dynein ATPases have a higher affinity for ATP than do those of carp. Supplementation of reactivating medium with ADP and PCr without ATP resulted in beat frequencies comparable to that measured with 0.3 to 0.5-mM ATP for both studied species. The presence of the PCr-CK phosphagen system and its essential role in ATP regeneration were first confirmed for sturgeon spermatozoa. The inhibition of CK exerted a high impact on spermatozoon energy supply in both species, whereas the inhibition of AK was more pronounced in sterlet than in carp. This was confirmed by the quantification of enzyme activity in spermatozoon extracts. We concluded that spermatozoa of these taxonomically distant species use similar systems to supply energy for flagella motility, but with different efficacy.

The aim of this study was to investigate the response of Russian sturgeon ( Acipenser gueldenstaedtii ) sperm to external cations (Na + , K + , Ca 2+ , and Mg 2+ ) 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 Ca 2+ and Mg 2+ 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). Ca 2+ and Mg 2+ 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 Ca 2+ , Mg 2+ , and possibly Na + trigger motility in Russian sturgeon sperm.

We attempted to select a fraction of common carp, Cyprinus carpio spermatozoa that best survived a conventional freeze/thaw procedure, by centrifugation of frozen/thawed sperm through a Percoll gradient (45% and 90%). The proportion of motile spermatozoa (65.81 ± 5.19%), their velocity (77.58 ± 31.07 μm/sec), and membrane integrity (83.66 ± 4.38% intact) were significantly higher in separated sperm than in whole samples (motility 23.36 ± 2.98%, velocity 55.55 ± 19.03 μm/sec, and membrane integrity 57.92 ± 4.65%). Our results demonstrated that Percoll gradient centrifugation shows promise as a technique for selecting high quality cryopreserved fish spermatozoa, which could be useful for cryobiological research. Further studies are needed to evaluate the potentially higher fertilizing ability of the separated spermatozoa.

The objectives of the present study were to characterize sperm volume and density, seminal plasma indices (ionic contents and osmolality) and to study the effects of dilution ratio, ions and osmolality on sperm motility parameters (percentage of motile sperm and sperm velocity) in farmed European perch (Perca fluviatilis L.). The means of sperm volume (ml), sperm density (x10(9)spermml(-1)) and total number of sperm (volumexdensity) per fish were 2.75+/-0.51, 29.19+/-3.15 and 82.19+/-15.26. The seminal plasma osmolality (mOsmkg(-1)), sodium, chloride, potassium and calcium ions concentrations (mM) were measured to be 298.07+/-5.09, 130.97+/-2.19, 106.75+/-2.37, 10.70+/-0.61 and 2.41+/-0.09, respectively. At 15s post-activation of stripped sperm, the percentage of motile sperm (%) and sperm velocity (mums(-1)) were 91.90+/-1.27 and 115.54+/-1.25, respectively, and decreased significantly following sperm activation (P

Culture of bovine embryos with insulin-like growth factor-1 (IGF-1) can improve development to the blastocyst stage and embryo survival following transfer to heat-stressed, lactating dairy cows. Two experiments were conducted to determine whether IGF-1 could improve embryo survival and development at Day 14 after ovulation. In Experiment 1, non-lactating Holstein cows (n = 58) were selected as recipients following synchronization for timed-embryo transfer. Embryos were produced in vitro and cultured with or without 100 ng/mL IGF-1. At Day 7 after expected ovulation (Day 0), groups of 7–12 embryos were randomly transferred to each recipient. Embryos were recovered at Day 14. Embryo length and the presence or absence of an embryonic disc was recorded. Recovered embryos were cultured individually for 24 h to determine interferon-t (IFN-t) secretion. There was no effect of IGF-1 on embryo recovery rate, embryo length or IFN-t secretion. In Experiment 2, non-lactating (n = 56) and lactating (n = 35) Holstein cows were selected as recipients following synchronization for timed-embryo transfer. Embryos were produced as described in Experiment 1. At Day 7 after expected ovulation (Day 0), a single embryo was randomly transferred to each recipient. Embryos were recovered at Day 14. Embryo length and IFN-t secretion were determined as in Experiment 1. Recovery rate at Day 14 tended (P = 0.1) to be higher for recipients that received IGF-1 treated embryos compared to control embryos (43.2% versus 26.1%, respectively). There was no effect of IGF-1 on embryo length or IFN-t secretion. In conclusion, results suggest that exposure to IGF-1 through Days 7–8 of development does not enhance capacity of embryos to prevent luteolysis. Results of the single embryo-transfer experiment suggested that IGF-1 treatment might affect embryo survival post-transfer as early as Day 14 after ovulation. Further experimentation is warranted to verify this finding.

Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion–specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)–biotin–dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-μM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants at 21 days after fertilization. Moreover, the body cavities of MO-treated and nontreated fish were examined by histology and in situ hybridization, showing gonads which had no germ cells in morphants at various stages (60, 150, and 210 days after fertilization). Taken together, these results report the first known and functional method of sturgeon sterilization.

The aim of the present study was to elaborate cryopreservation methods for ex situ conservation of tench. Success of cryopreservation was tested during two series of experiments. The first set of experiments studied the effects of two types of cryoprotectants (DMSO and a combination of DMSO with propanediol at ratio 1:1) at concentrations of 8 and 10% and three different equilibration times in two different immobilization solutions (IS) (Kurokura 180 and Kurokura) before freezing (0.0, 2.0 and 4.0h after T(0)). The K4 cooling programme was used to freeze 1ml of cryoextended sperm using 1.8ml cryotubes. Main monitored parameter was hatching rate after using of cryopreserved sperm. The second set of experiments studied the volume effect of 0.5, 1 and 5ml straws and compared these with 1.8ml cryotubes as well as the effect of the cooling programme (K4 and L1). Following the results of the first study, a combination of DMSO and propanediol (ratio 1:1) at concentration of 10% was added to extended sperm in Kurokura 180 IS. Main monitored parameter was hatching rate after using cryopreserved sperm, supplementary parameters were sperm velocity and motility percentage assessed at 10s post-activation. Sperm was collected directly into IS and stored at 4 degrees C for 2.5h. Thereafter were sperm samples pooled, equlibred in IS (first set of experiments) or directly mixed with cryoprotectants (DMSO or a mixture of DMSO with propanediol at ratio 1:1) and transferred to 1.8ml cryotubes or straws (0.5, 1 and 5ml). Then the cryotubes/straws were directly transferred to pre-programmed PLANER Kryo 10 series III and cooled using two different cooling programmes including a slow cooling programme (a) named K4 (from +4 to -9 degrees C at a rate of 4 degrees Cmin(-1) and then from -9 to -80 degrees C at a rate of 11 degrees Cmin(-1)) and a rapid cooling programme (b) named L1 (directly from +4 to -80 degrees C at a rate of 20 degrees Cmin(-1)). Both slow (K4) and rapid (L1) cooled samples were held 6min at -80 degrees C. Finally, samples were transferred into liquid N(2). The frozen spermatozoa were thawed in a water bath (40 degrees C) according to the frozen volume and checked for fertilization and hatching rates. Percentage of sperm motility and sperm velocity were measured using video recorded frames. ANOVA showed a significant influence of frozen and fresh sperm in all treatments. The hatching rates of 33.8% were obtained when sperm was equilibrated for 0h before freezing in IS of Kurokura 180 and frozen with a 10% of mixture 1:1 of DMSO and propanediol into straws of 5ml and cooled using program L1. The velocity of frozen-thawed spermatozoa ranged from 31 to 46microms(-1) and in post-thawed sperm was not significantly different according to frozen sperm volume, but a higher velocity was obtained when sperm was fast frozen using programme L1. A large volume of frozen sperm could reveal the best procedure for freezing, but also for simulating methods of artificial propagation for future practical use of frozen tench sperm at a large scale.

Structure of tench (Tinca tinca L.) spermatozoa was investigated by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Spermatozoa of 26.1+/-3.8 microm total length possessed typical primitive simple structure, called "aqua sperm", without acrosomal head structures. It was probably the smallest spermatozoon described among cyprinid fishes. Heads were mostly composed of dense and slightly granular material, which appeared to be fairly homogeneous except for the occasional appearance of vacuoles. The midpiece remained separated from the flagellum by the cytoplasmic channel; it was cylindric/cone-shaped, 0.86+/-0.27 microm in length and 1.17+/-0.24 microm in width at proximal part. The proximal centriole was located in the "implantation fossa". The distal centriole appeared almost tangential to the nucleus and it functioned as a basal body for the flagellum. It had an orientation of 140 degrees with respect to the distal centriole. The sperm flagellum with 25.45+/-2.47 microm of total length had no any fin. The diameter of the flagellum perpendicular to the plane of the doublet of central microtubules was 173.67+/-20.45 nm and horizontal plane of the central microtubules was 200.71+/-20.45 nm. Peripheral doublets and the central doublet of microtubules measured 23.39+/-3.18 and 35.88+/-4.44 nm in width, respectively. The diameter of a microtubule was only 9.14+/-2.97 nm. A vesicle was attached to the most basal region of the flagellum and located just under plasma membrane of the flagellum.